Nitric Oxide (NO) and Cancer: Prognosis, Prevention, and Therapy (Cancer Drug Discovery and Development)

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Cancer Drug Discovery and Development

Series Editor Beverly A. Teicher Genzyme Corporation, Framington, MA, USA

For other titles published in this series, go to www.springer.com/series/7625

Benjamin Bonavida Editor

Nitric Oxide (NO) and Cancer Prognosis, Prevention, and Therapy

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Editor Benjamin Bonavida Department of Microbiology, Immunology & Molecular Genetics David Geffen School of Medicine at UCLA University of California at Los Angeles Los Angeles, CA [email protected]

ISBN 978-1-4419-1431-6 e-ISBN 978-1-4419-1432-3 DOI 10.1007/978-1-4419-1432-3 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010925377 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

The Nobel Price in Physiology and Medicine was awarded in 1998 to Drs. Furchgott, Ignarro, and Murad for their discoveries concerning the “Nitric oxide as a signaling molecule in the cardio-vascular system.” Nitric oxide (NO) is a short-lived, endogenously produced gas that acts as a signaling molecule in the body. NO-induced signaling events within the cell producing it and its diffusibility to other cells has led to the discovery of many other physiological functions in many other types of cells including cancer cells. Noteworthy, nitroglycerin, invented by Alfred Nobel, has been used for the treatment of chest pain and associated cardiovascular diseases and has now started to be used in clinical studies including cancer (see below). Several reports have addressed the roles of tumor-expressing iNOS and NO donors on tumor behavior in vivo. Data reported demonstrated the contrasting roles of NO mediating either tumor promotion or tumor regression. In an effort to sort out the roles of iNOS and NO in cancer, the “ First International Conference on Nitric Oxide and Cancer” was held in Paris, France, November 26–28, 2007. This conference was attended by leaders in the field and Dr. Wink and collaborators presented convincing data demonstrating that the levels of NO dictate the outcome of tumor cell response. This conference resulted in the publication of a special issue “Nitric Oxide and Cancer: Clinical and Therapeutic Implications” (Nitric Oxide, Vol 8/19, September 2008). The rapid advances made in the field of nitric oxide and cancer were the impetus to develop this special volume summarizing the current status of NO and cancer to be published by Springer and contains over 25 chapters that have been contributed by leaders in this field. This volume was divided arbitrarily into seven parts, namely (I) General overview, (II) Nitric Oxide and the pathogenesis of cancer, (III) Dual roles of NO in protecting against or inducing cell death, (IV) Role of NO in metastasis, (V) Nitric oxide as a sensitizing agent for chem-radio-immunotherapy, (VI) Prognostic applications of iNOS in various cancers, and (VII) The application of nitric oxide as a therapeutic. Briefly below, I will discuss the highlights presented by each contributor in each section. In Part I, Drs. Harris, Wink, and colleagues from the National Cancer Institute present an overview of the field and provide chemical and molecular analyses

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underlying the pleiotropic activities observed with NO in cancer. They also discuss the roles of NO in the modulation of host immune responses and suggest novel strategies to develop new applications of NO as a therapeutic drug. In Part II, Drs. Counter, Chadhuri, and Masini describe the role of NO in the pathogenesis of cancer. Dr. Counter discusses the role of eNOS in tumorigenesis through the activation of the Ras family of proteins. Several experimental models are presented to corroborate their findings. They also raise several questions that need to be explored regarding the underlying mechanism(s) of the eNOS/Raf interaction in tumors. They also suggest novel targets for therapeutic interventions and show an inhibitor of eNOS inducing an anti-tumor activity. Dr. Chadhuri also describes the multifaceted roles of NO in cancer, namely, in cell growth and apoptosis and highlights several other gene products that are regulated by NO. Dr. Masini discusses the dual roles of NO in cancer as well. In addition, the coordinate expression of iNOS, COX2, and VEGF in certain tumor cells are shown to promote new blood vessel formation and tumor growth. In addition, contrasting findings are presented and overall the iNOS/COX2 pathway is considered as target for cancer treatment. In Part III, five contributors review the dual roles of NO in protecting or inducing cell death. Dr. Rojanasakul and colleagues describe the role of S-nitrosylation by NO in cell death. In addition, they review the involvement of NO in the tumor microenvironment and mechanisms of tumor progression toward metastasis. The role of NO-mediated cysteine nitrosylation on carcinogenesis is also discussed. Dr. Soma reviews the recent literature and presents several schematic and illustrative diagrams summarizing the various pathways that regulate the various proand anti-apoptotic roles of NO. Dr. Chung and colleagues review the reported studies and theirs on the effects of NO concentrations, sources, half-life, chemical interactions, and the microenvironment all of which would influence the outcome. Dr. Weinberg reviews the dual roles of NO and emphasizes the role of iNOS overexpression in hematological malignancies, particularly CLL. In these tumor cells the overexpression of iNOS is protective and inhibition of iNOS results in significant cell death in CLL. It is suggested that overexpression of iNOS results in NO-induced inhibition of caspases which leads to resistance to apoptotic stimuli. It is suggested that the use of specific inhibitors targeting iNOS in this cancer may be therapeutic. Dr. Kolb and colleagues also describe the anti-apoptotic role of NO in CLL. The overexpression of iNOS in CLL is regulated by the toll-like receptor 7 (TLR-7). While NO exerts contrasting effects on apoptosis in many tumor cells, however, in CLL it is protective against apoptosis. In Part IV, Drs. Estrala and Baritaki describe the role of NO in metastasis. Dr. Estrala reviews the intravascular origin of metastasis, the cytotoxic effect of NO derived from the vascular endothelials in the tumor microenvironment and tumor survival. Also, the role of anti-apoptotic gene products including NOS in the regulation of metastasis as well as the role of NO in the regulation of angiogenic factors is reviewed. Dr. Baritaki, in contrast, describes a novel mechanism of tumor cell inhibition of metastasis by NO donors. Treatment of metastatic human prostate cancer cell lines with the use of high levels of the NO donor, DETANONOate, results in the inhibition of constitutive survival pathway such as NF-kB and downstream the metastasis-inducer transcription factor,

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Snail, and inhibition of the epithelial–mesenchymal transition (EMT). Also, in this chapter, she describes NO-induced expression of the metastasis suppressor gene product, RKIP. This study suggests the potential therapeutic application of NO donors in the regulation of metastasis. In Part V, four contributors discuss the role of NO as a sensitizing agent to reverse tumor cell resistance to cytotoxic therapies. Dr. Siejo discusses the role of NO as a sensitizing agent for radio-chemo- and immuno-therapy. Irradiated tumor cells result in the release of NO and ROS that potentiate the cytotoxic effect of radiation. Likewise, the addition of NO donors to irradiated tumor cells potentiates the cytotoxic effect. Similar findings are discussed with respect to tumor therapy and NO. Dr. Jeannin discusses the primary role of NO as an enhancer for cancer therapy. In this review, he also discusses the mechanism by which NO exerts its sensitizing effect to both chemo- and immunotherapy. Dr. Effert reports on the resistance induced in cancer cells by hypoxia and how NO inhibits the transcription factor, HIF-1alpha. The inhibition of HIF-1alpha results in the inhibition of many resistance gene products and, hence, the tumor cells become sensitized to chemotherapeutic drug-induced apoptosis. Dr. Garbán reviews the role of NO in reversing tumor cell resistance to cytotoxic drugs. He postulates that NO induces oxygenation of tumor cells by increasing blood flow and resulting in the increase of the delivery of the cytotoxic drug to the tumor. Further, NO modulates the host immune response by regulating the expression of death receptor on the tumor cells and, thus, potentiating their sensitivity to hostimmune cytotoxic lymphocytes. In addition, Dr. Garbán presents his expert opinion on the above chapters. In Part VI, four contributors review the prognostic significance of iNOS expression in various cancers. Drs. Ekmekcioglu and Grimm present the prognostic significance of iNOS in human melanoma. Their findings demonstrate that iNOS overexpression in melanoma is an independent prognostic factor for Stage III melanoma. Drs. Pascale and Feo review the molecular increase in the alteration of iNOS and NO and, in particular, the high level of iNOS in a subgroup of hepatocellular carcinoma and correlation with poor prognosis. Drs. Matsumoto and colleagues describe the prognostic significance of iNOS in nasopharyngeal cancer. They demonstrate that overexpression of iNOS is associated with p53 overexpression but not associated with prognosis. They suggest that iNOS contributes to tumorigenesis but not to tumor progression. Drs. Hiraku and Kawanishi discuss the overexpression of 8-nitroguanine. In patients with nasopharyngeal carcinoma infected with EBV, the prognostic significance of 8nitroguanine is reported. They also found that in patients with soft tissue tumors strong 8-nitroguanine formation was associated with poor prognosis. In Part VII, the therapeutic application of NO in cancer is reviewed by six contributors. Drs. Thatcher and Anand describe the therapeutic potential and cancer prevention of nitric oxide-releasing molecules. They review the activities of various classes of NO donors, and in particular, the new generations of NORMs, nitric oxide redox molecules. Drs. Hirst and Robson discuss the anti-tumor properties of NO donors in both experimental and clinical trials in patients. They also describe the physiological effects as single agents or in combination with other agents. They summarize in a table format all the reported studies undertaken to-date by nitric oxide against

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tumor cells, both in vitro and in vivo. Drs. Bonavida and associates describe the role of various NO donors and their mechanisms of action on the reversal of tumor cell resistance to various cytotoxics. They discuss the beneficial effects of NO donors as sensitizing agents and that they can be considered as universal sensitizing agents when compared to specific targeted agents. NO donors perturb many survival antiapoptotic pathways in the tumor cells and act upstream to various pathways in contrast to various specific inhibitors which act downstream. Drs. Nicoletti and colleagues describe an NO donor compound, GTT-27NO, as a tumor-specific cytotoxic drug and its ability to reverse resistance of tumor cells. They also discuss the induction by GTT-27NO of ROS, RNS, and nitration of tyrosine residues. They suggest the potential application of this drug in the clinic. Drs. Yasuda and colleagues describe the therapeutic application of NO in in vitro models and in humans. They describe the current clinical trials of the use of nitroglycerin in combination with cytotoxic drugs in patients with non-small cell lung carcinoma. Both randomized and non-randomized studies are described. Dr. Rustum describes his opinion of the preceding chapters in Part VII and also describes his own studies with the compound SE-methyl selenium and its ability to inhibit HIF-1alpha.

Acknowledgments

I wish to acknowledge the assistance of several individuals who have contributed in the preparation of this volume. My assistants at UCLA, namely, Erica Keng, Tiffany Chin, Kerry Choy, and Anna Sahakyan, were patient and I am grateful for their help in revising the manuscripts as per guidelines. In addition, I am indebted to Rachel Warren and Brian Halm from Springer for advice and coordination throughout the development of this volume. I also acknowledge the assistance of the UCLA Johnson Comprehensive Cancer Center and their staff. The support of my wife and two sons and their sacrifice are greatly acknowledged during the preparation of this volume. Los Angeles, CA, USA

Benjamin Bonavida

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Contents

Part I

General Overview

1 Nitric Oxide and Cancer: An Overview . . . . . . . . . . . . . . . Robert Cheng, Lisa A. Ridnour, Sharon A. Glynn, Christopher H. Switzer, Wilmarie Flores-Santana, Perwez Hussain, Douglas D. Thomas, Stefan Ambs, Curtis C. Harris, and David A.Wink Part II

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Nitric Oxide and the Pathogenesis of Cancer

2 A Role for eNOS in Oncogenic Ras-Driven Cancer . . . . . . . . . David F. Kashatus and Christopher M. Counter

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3 Dual Role of Nitric Oxide in Cancer Biology . . . . . . . . . . . . Shehla Pervin, Rajan Singh, Suvajit Sen, and Gautam Chaudhuri

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4 Nitric Oxide Expression in Cancer . . . . . . . . . . . . . . . . . . Emanuela Masini, Fabio Cianchi, Rosanna Mastroianni, and Salvatore Cuzzocrea

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Part III Dual Roles of Nitric Oxide in Protecting or Inducing Cell Death 5 S-Nitrosylation – How Cancer Cells Say NO to Cell Death . . . . Anand Krishnan V. Iyer, Neelam Azad, Liying Wang, and Yon Rojanasakul

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6 Cytotoxic and Protective Activity of Nitric Oxide in Cancers . . . Gen-Ichiro Soma, Chie Kohchi, and Hiroyuki Inagawa

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7 Cytotoxic/Protective Activity of Nitric Oxide in Cancer . . . . . . Eun-Kyeong Jo, Hyun-Ock Pae, Yong Chul Lee, and Hun-Taeg Chung

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8 Nitric Oxide and Life or Death of Human Leukemia Cells . . . . J. Brice Weinberg

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9 Inhibition of Apoptosis by Endogenous Nitric Oxide in Chronic Lymphocytic Leukaemia . . . . . . . . . . . . . . . . . Christian Billard, Claire Quiney, and Jean-Pierre Kolb Part IV

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Role of Nitric Oxide in Metastasis

10 Nitric Oxide: A Rate-Limiting Factor for Metastases Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angel Ortega, Salvador Mena, and José M. Estrela

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11 Nitric Oxide Inhibits Tumor Cell Metastasis via Dysregulation of the NF-κB/Snail/RKIP Loop . . . . . . . . . . . Stavroula Baritaki and Benjamin Bonavida

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Part V

Nitric Oxide as a Sensitizing Agent for Chem-RadioImmunotherapy

12 Sensitizing Effect of Nitric Oxide to Cytotoxic Stimuli . . . . . . . Peter Siesjö

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13 Nitric Oxide Is a Promising Enhancer for Cancer Therapy . . . . Marion Cortier, Lissbeth Leon, Néjia Sassi, Catherine Paul, Jean-François Jeannin, and Ali Bettaieb

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14 Role of Nitric Oxide for Modulation of Cancer Therapy Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas Efferth 15 Breaking Resistance: Role of Nitric Oxide in the Sensitization of Cancer Cells to Chemo- and immunotherapy . . . . . . . . . . . . . . . . . . . Hermes J. Garbán Part VI

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Prognostic Significance of NOS and NO

16 Prognostic Significance of iNOS in Human Melanoma . . . . . . . Suhendan Ekmekcioglu and Elizabeth A. Grimm

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17 Prognostic Significance of iNOS in Hepatocellular Carcinoma . . Rosa M. Pascale, M. Frau, and Francesco Feo

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18 Prognostic Significance of iNOS in Esophageal Cancer . . . . . . Manabu Matsumoto, Yuji Ohtsuki, and Mutsuo Furihata

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19 Prognostic Significance of Nitrative DNA Damage in Infection- and Inflammation-Related Carcinogenesis . . . . . . Yusuke Hiraku and Shosuke Kawanishi

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Part VII Therapeutic Applications of Nitric Oxide 20 Nitric Oxide-Releasing Molecules for Cancer Therapy and Chemoprevention . . . . . . . . . . . . . . . . . . . . . . . . . S. Anand and Gregory R.J. Thatcher

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21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? . . . . . . . . . . . . . . . . . . . . . . . . . . David G. Hirst and Tracy Robson

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22 Therapeutic Applications of Nitric Oxide for Malignant Tumor in Animal Models and Human Studies . . . . . . . . . . . Hiroyasu Yasuda, Kazuhiro Yanagihara, Katsutoshi Nakayama, Tadashi Mio, Takahiko Sasaki, Masanori Asada, Mutsuo Yamaya, and Masanori Fukushima 23 (S,R)-3-Phenyl-4,5-dihydro-5-isoxazole acetic acid–Nitric Oxide (GIT-27NO) – New Dress for Nitric Oxide Mission . . . . . Sanja Mijatovic, Danijela Maksimovic-Ivanic, Marco Donia, Stanislava Stosic-Grujicic, Gianni Garotta, Yousef Al-Abed, and Ferdinando Nicoletti 24 Nitric Oxide Donors Are a New Class of Anti-cancer Therapeutics for the Reversal of Resistance and Inhibition of Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benjamin Bonavida, Stavroula Baritaki, Sara Huerta-Yepez, Mario I. Vega, Ali R. Jazirehi, and James Berenson 25 Role of Inducible Nitric Oxide Synthase (iNOS) in Regulation of Nitric Oxide (NO) Production and Stabilization of HIF-1α: Potential Role of Se-Methylselenocysteine (MSC), an Antioxidant Multi-targeted Small Molecule . . . . . . . . . . . . . . . . . . . . Sreenivasulu Chintala, Shousong Cao, and Youcef M. Rustum Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors

Yousef Al-Abed Laboratory of Medicinal Chemistry, North Shore Long Island Jewish Health System, New Hyde Park, NY, USA Stefan Ambs Radiation Biology Branch National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA S. Anand Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA Masanori Asada Department of Geriatrics and Gerontology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan Neelam Azad Department of Pharmaceutical Sciences, Hampton University, Hampton, VA 23668, USA Stavroula Baritaki Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at University of California at Los Angeles, Le Conte Avenue, Los Angeles, CA, 90095–1747, USA James Berenson Hematology/Oncology, Institute for Myeloma & Bone Cancer Research, West Hollywood, CA, USA Ali Bettaieb Laboratory of Cancer Immunology and Immunotherapy, Université de Bourgogne, EPHE/INSERM U866, 7 bd Jeanne d’Arc, Dijon 21079, France Christian Billard Centre de Recherche des Cordeliers, UMRS 872 INSERM/University Pierre et Marie Curie/University Paris Descartes, Paris, France Benjamin Bonavida Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, University of California at Los Angeles, Le Conte Avenue, Los Angeles, CA, 90095-1747, USA Shousong Cao Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

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Contributors

Gautam Chaudhuri Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA Robert Cheng Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Sreenivasulu Chintala Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA Hun-Taeg Chung School of Biological Sciences, Ulsan University College of Natural Sciences, Daehackrho 102, Namgu, Ulsan 680–749, Republic of Korea Fabio Cianchi Department of General Surgery, Medical School, University of Florence, Florence, Italy Marion Cortier Laboratoire d’immunologie et immunothérapie des cancers, Université de Bourgogne, EPHE/Inserm, U866, Dijon F-21000, France Christopher M. Counter Department of Pharmacology and Cancer Biology, Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA Salvatore Cuzzocrea Institute of Pharmacology, University of Messina, Messina, Italy Marco Donia Department of Biomedical Sciences, University of Catania, Catania, Italy Thomas Efferth Department of Phatmaceutical Biology, Institute of Pharmacy and Biochemisty, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany Suhendan Ekmekcioglu Department of Experimental Therapeutics, Unit 362, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA José M. Estrela Department of Physiology, University of Valencia, 15 Av. Blasco Ibañez, Valencia, 46010, Spain Francesco Feo Division of Experimental Pathology and Oncology, Department of Biomedical Sciences, University of Sassari, Sassari 07100, Italy Wilmarie Flores-Santana Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA M. Frau Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy Masanori Fukushima Outpatient Oncology Unit, Kyoto University Hospital, Kyoto 606-8507, Japan Mutsuo Furihata Department of Pathology, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan

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Hermes J. Garbán Division of Dermatology, Department of Medicine, LA BioMed Research Institute at Harbor-UCLA Medical Center, “David Geffen” School of Medicine at the University of California, Los Angeles, CA 90502, USA; UCLA’s Jonsson Comprehensive Cancer Center, Los Angeles, CA 90502, USA Gianni Garotta GaNiAl Immunotherapeutics Inc., Wilmington, DE, USA Sharon A. Glynn Laboratory of Human Carcinogenesis, Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Elizabeth A. Grimm Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Curtis C. Harris Radiation Biology Branch National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Yusuke Hiraku Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507, Japan; Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan David G. Hirst School of Pharmacy, Medical Biology Centre, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK Sara Huerta-Yepez Unidad de Invastigacion en Enfarmeda des Oncologicas, Hospital Infantil de Mexico, Federico Gomez, Mexico City, Mexico Perwez Hussain Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Hiroyuki Inagawa Department of Integrated and Holistic Immunology, Faculty of Medicine, Kagawa University, 1750-1 Mikicho, Kida-gun, Kagawa 761-0793, Japan; Macrophi Inc., Hayashi-cho, Takamatsu-shi, Kagawa 761-0301, Japan Anand Krishnan V. Iyer Department of Pharmaceutical Sciences, Hampton University, Hampton, VA, 23668, USA Ali R. Jazirehi Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, University of California at Los Angeles, Le Conte Avenue, Los Angeles, CA, 90095-1747, USA Jean-François Jeannin Laboratory of Cancer Immunology and Immunotherapy, Université de Bourgogne, EPHE/INSERM U866, 7 bd Jeanne d’Arc, Dijon 21079, France Eun-Kyeong Jo Department of Microbiology, Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon, 301–747, Republic of Korea

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David F. Kashatus Department of Pharmacology and Cancer Biology, Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA Shosuke Kawanishi Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie, 514-8507, Japan; Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan Jean-Pierre Kolb Centre de Recherche des Cordeliers, UMRS 872 INSERM/University Pierre et Marie Curie/University Paris Descartes, 15 rue de l’Ecole de Médecine, Paris cedex 06 75270, France Chie Kohchi Department of Integrated and Holistic Immunology, Faculty of Medicine, Kagawa University, 1750-1 Mikicho, Kida-gun, Kagawa 761-0793, Japan; Macrophi Inc., Hayashi-cho, Takamatsu-shi, Kagawa 761-0301, Japan Yong Chul Lee Department of Internal Medicine and Airway Remodeling Laboratory, Chonbuk National University Medical School, Jeonju 570-752, Republic of Korea Lissbeth Leon Laboratoire d’immunologie et immunothérapie des cancers, Université de Bourgogne, EPHE/Inserm, U866, Dijon F-21000, France Danijela Maksimovic-Ivanic Department of Immunology, Institute for Biological Research “Sinisa Stankovic,” Belgrade University, Belgrade, Serbia Emanuela Masini Department of Preclinical and Clinical Pharmacology, Medical School, University of Florence, Florence, Italy Rosanna Mastroianni Department of Preclinical and Clinical Pharmacology, Medical School, University of Florence, Florence, Italy Manabu Matsumoto Laboratory of Diagnostic Pathology, Kochi Medical School Hospital, Nankoku, Kochi 783-8505, Japan Salvador Mena Department of Physiology, University of Valencia, Valencia, Spain Sanja Mijatovic Department of Immunology, Institute for Biological Research “Sinisa Stankovic,” Belgrade University, Belgrade, Serbia Tadashi Moi Department of Multidisciplinary Cancer Treatment, Kyoto University School of Medicine, Kyoto 606-8507, Japan Katsutoshi Nakayama Department of Respiratory Medicine, Jikei University School of Medicine, Tokyo 105-8461, Japan Ferdinando Nicoletti Department of Biomedical Sciences, University of Catania„ Via Androne, 83, Catania 95124, Italy Yuji Ohtsuki Division of Pathology, Matsuyama-shimin Hospital, Matsuyama, Ehime, Japan

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Angel Ortega Department of Physiology, University of Valencia, Valencia, Spain Hyun-Ock Pae Department of Microbiology and Immunology, Wonkwang University School of Medicine, Iksan, 570-749, Republic of Korea Rosa M. Pascale Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Sassari, 07100, Italy Catherine Paul Laboratoire d’immunologie et immunothérapie des cancers, Université de Bourgogne, EPHE/Inserm, U866, Dijon, F-21000, France Shehla Pervin Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Internal Medicine, Charles Drew University of Medicine and Science, Los Angeles, CA, USA Claire Quiney Tumor Biology Labs, Biochemistry Department, UCC, Cork, Ireland Lisa A. Ridnour Radiation Biology Branch National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Tracy Robson School of Pharmacy, Medical Biology Centre, Queen’s University Belfast, Belfast, BT9 7BL, UK Yon Rojanasakul Department of Pharmaceutical and Pharmacological Sciences, West Virginia University, Morgantown, WV 26506, USA Youcef M. Rustum, Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA Takahiko Sasaki Department of Respiratory Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan Néjia Sassi Laboratoire d’immunologie et immunothérapie des cancers, Université de Bourgogne, EPHE/Inserm, U866, Dijon F-21000, France Suvajit Sen Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA Peter Siesjö The Rausing Laboratory, Section of Neurosurgery, Department of Clinical Sciences, University of Lund, Lund, Sweden; Department of Neurosurgery, University of Lund, Lund, Sweden Rajan Singh Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Internal Medicine, Charles Drew University of Medicine and Science, Los Angeles, CA, USA Gen-Ichiro Soma Department of Integrated and Holistic Immunology, Faculty of Medicine, Kagawa University, 1750–1 Mikicho, Kida-gun, Kagawa 761–0793, Japan; Macrophi Inc., Hayashi-cho, Takamatsu-shi, Kagawa 761–0301, Japan;

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Institute for Health and Science, Tokushima Bunri University, Nishihama, Yamashirocho, Tokushima, 770–8514, Japan Stanislava Stosic-Grujicic Department of Immunology, Institute for Biological Research “Sinisa Stankovic,” Belgrade University, Belgrade, Serbia Christopher H. Switzer Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Gregory R.J. Thatcher Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA Douglas D. Thomas Department of Medicinal Chemistry and Pharmacognosy. College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA Mario I. Vega Unidad de Investigacion Medica En Inmunologia e Infactologia, Hospital de Infectologia. CMN “LA Caza” IMSS, Mexico City, Mexico Liying Wang Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA J. Brice Weinberg Departments of Medicine and Immunology, Duke University and Veterans Affairs Medical Centers, 508 Fulton Street, Durham, NC, USA David A. Wink Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Mutsuo Yamaya Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University School of Medicine, Sendai 980-8574, Japan Kazuhiro Yanagihara Outpatient Oncology Unit, Kyoto University Hospital, Kyoto 606-8507, Japan Hiroyasu Yasuda Department of Clinical Application, Translational Research Center, Tohoku University, Sendai 980-8574, Japan; Outpatient Oncology Unit, Kyoto University Hospital, Kyoto 606-8507, Japan

Part I

General Overview

Chapter 1

Nitric Oxide and Cancer: An Overview Robert Cheng, Lisa A. Ridnour, Sharon A. Glynn, Christopher H. Switzer, Wilmarie Flores-Santana, Perwez Hussain, Douglas D. Thomas, Stefan Ambs, Curtis C. Harris, and David A. Wink

Abstract An involvement of nitric oxide, a diatomic radical, has been described for numerous areas from environmental pollution to cardiovascular disease, carcinogenesis, tumor progression, genotoxicity, and angiogenesis. Previously, it has been demonstrated that NO may perform different functions dependent on NO levels achieved in a particular microenvironment. Furthermore, researchers also have discovered and identified the various sources of NO, which can elicit different biological responses of NO. In order to better understand the biological consequences of NO responses, one must first understand the chemical biology of NO. Since the first discussions during the early 1990s, it became widely accepted that NO chemical biology can be classified into two classes: direct interaction and indirect interaction. These two classes provided us with the means to understand the basic chemical toxicological effects of NO and its resulting reactive nitrogen species (RNS). NO has been reported to be involved in several steps of carcinogenesis, including interactions with p53 at both the genetic and the protein level and through regulation of the apoptotic pathways and DNA repair mechanisms. Recently, NO has also been linked to various immune and inflammation responses, especially in cancer development and wound healing process. Tumors are known to alter the immune response and tissue vascularization which involves NO. Therefore, a better understanding of the roles of NO in immune response modulation and wound healing would allow us to design a better treatment plan and improve NO drug efficacy. Keywords Nitric oxide · Cancer · Oxidative–nitrosative stress Nitricoxide (NO) is a simple diatomic radical that has been associated with air pollution and cigarette smoke for several decades (Schwartz and White 1983). This

D.A. Wink (B) Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA e-mail: [email protected]

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_1, C Springer Science+Business Media, LLC 2010

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diatomic molecule is relatively unreactive, only reacting with metal and highly reactive radicals (Wink and Mitchell 1998). However, under aerobic conditions, NO can also react with oxygen, forming reactive nitrogen oxides, NO2 and N2 O3 (Wink and Mitchell 1998). Nitrogen dioxide is a major component of the brownish air, or smog, that is seen over many major cities. At less than 1 ppm, NO2 already can induce pulmonary toxicity. In the early 1980s, Furchgott and coworkers showed that a substance generated by endothelial cells mediated the relaxation of vascular smooth muscle (Furchgott and Zawadzki 1980). This became known as the endothelial-derived relaxation factor or EDRF. In a series of critical experiments, it was found that nitric oxide could activate guanylyl cyclase (GC) in tissue (Arnold et al.1977) and that the active component of nitrovasodilators was NO (Ignarro et al. 1980a, b, 1981). The identification of NO in endothelial beds as EDRF was seminal finding that launched this area of research (Ignarro et al. 1987; Palmer et al. 1987). During the same time there was a separate line of research in the field of immunology. Hibbs and coworkers have showed that there was a substance that was generated by macrophage and is critical in the antitumor and anti-pathogen response (Hibbs et al. 1977, 1984, 1987; Drapier and Hibbs 1986). They found by increasing the concentration of this substance, they could inhibit respiration and modulate iron metabolism. They also found that this substance was dependent on arginine and eventually confirmed that the substance is NO. Subsequently, several groups have shown that leukemic cells were killed by NO derived from macrophages (Hibbs et al. 1988; Stuehr and Nathan 1989). Tannenbaum and coworkers have shown that infections in human could lead to increase nitrate and nitrite (Green et al. 1981). A few years later, macrophages were identified as a source for nitrite and nitrate (Stuehr and Marletta 1985). The same authors later found that there was an increase in nitrosoproline which suggested the possibility that nitric oxide release leads to an increase in nitrosamines (Stuehr and Marletta 1985). Several studies have since shown that nitrite and nitrosamines are released from activated macrophages and hepatocytes (Marletta, 1988; Liu et al. 1991, 1992; Ohshima et al. 1991). These data indicated that nitrosamine and nitrite are produced as part of the immune response under infectious conditions. In 1990, the Fe-NO EPR signal was discovered, and it was demonstrated that dinitroso iron complexes (DNIC) were formed during the inflammation process (Lancaster and Hibbs 1990). These findings suggested that NO has many diverse functions and that different endogenous concentrations of this molecule could result in very different biological responses. Another important observation was that L-NG-monomethyl arginine (L-NMMA) could inhibit the formation of NO (Hibbs et al. 1987). This discovery of NOS inhibitors provided a novel tool to uncover the myriad of processes that NO is participating in, including damaging and protective mechanisms. While many speculated that NO may play the role of an enhancer in different pathophysiological processes due to its potentially toxic nature (Moncada et al. 1991), NO was also found to protect cells from oxidative stress and damage under ischemia reperfusion (Kubes et al. 1991; Ma et al. 1993; Wink et al. 1993). This dichotomous property of NO has been

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at the heart of many debates as to the dominant effect of NO in pathophysiology and diseases. Part of the answer lies in the understanding of the chemistry of NO and its related intermediates. The primary determinant for the role of NO and the resulting reactive nitrogen species (RNS) is the surrounding redox environment. The chemical biology of NO and its dependency on the redox environment was first described in the 1990s, when there was a first discussion of the different chemical reactions for NO in the context of kinetic parameters and the description of those reactions are likely to happen in vivo (Wink et al. 1996; Wink and Mitchell 1998). The resulting chemical biology of NO provides a guide to our current understanding of NO effects in living cells and describes chemical toxicological effects of NO and the resulting RNS. The reactions of NO can be divided into two different types: direct and indirect. The direct reactions are where NO directly reacts with their biological targets, including metals and other highly reactive radicals (e.g., lipid radicals). In contrast, the indirect reactions are those that result from the reaction with either O2 or O2 − . These result in the generation of N2 O3 , NO2 , and ONOO− , which can react with different biomacromolecules and alter their functions. The indirect mechanism can be further subdivided into nitrosative and oxidative stress. N2 O3 is the primary initiator of nitrosative stress and can generate nitrosamines as discussed above. Though ONOO− is a powerful oxidant, it is rapidly converted to NO2 which is the primary oxidant (Fig. 1.1) and can lead to lipid oxidation and create products such as 4-hydroxynonenal that can alter DNA, as well as inducing signal transduction cascades (Cline et al. 2004; West et al. 2004). Thus, it was anticipated that high NO levels such as those created by an activated macrophage or hepatocyte would lead to nitrosative or oxidative stress. In 1991, it was found that in addition to nitrosamine formation, NO and RNS could also modify DNA and resulting in deamination via nitrosation (Wink et al. 1991; Nguyen et al. 1992). These results suggested that N2 O3 and nitrosative stress can lead to deamination of purines and cytosine/methylcytosine. There are a number of chemical mechanisms such as deamination (hydrolysis), depurination,

Fig. 1.1 Nitric oxide in mechanisms of genotoxicity

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tautomerism, which explain the spontaneous mutations that occur on daily basis in DNA, and NO was proposed as another mutation source. DNA shuttle vector assays that determined the ability to repair lesion in bacteria and human cells have showed that a transition mutation was the most common base mutation induced by NO (Routledge et al. 1993, 1994a, b). In contrast, transversion are the primary lesions in DNA when challenged with oxidants and alkylating agents (Marmenout et al. 1984; Richardson et al. 1987; Loeb et al. 1988; Wood et al. 1992). Nitrosamine modification of DNA results in a mix of transversions and transitions. However, a transition is the primary observed mutation after NO exposure (Hidaka et al. 1978). From these experiments it can be concluded that nitrosative stress tends to cause transitions while oxidative stress favors transversions. NO does modify not only DNA directly but also proteins that maintain the integrity of this macromolecule. Specific DNA repair proteins such as alkyltransferase, ligase, and Fpg function differentially under nitrosative stress conditions (Laval et al. 1996). In addition, cells that are exposed to NO have different responses to various other cyto- and genotoxic agents than cells that are not exposed to NO. Alkyltransferase is inhibited by nitrosation resulting in cells becoming more sensitive to agents such as l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) (Laval and Wink 1994). It was further shown that conditions of nitrosation by N2 O3 react with zinc thiolates and alter the zinc finger quaternary structure rendering the protein unable to bind to DNA (Wink and Laval 1994). Since many DNA interacting proteins, such as PARP, contain zinc fingers, this suggests that conditions of nitrosative stress result in less efficient DNA repair (Sidorkina et al. 2003). In addition, the blockade of caspase and PARP prevents cells with compromised DNA from undergoing apoptosis, leading to increase survival in cells with altered DNA. This in turn would increase the rate of mutation. Nitrosative stress has been shown to inhibit ligase by causing a deamination of lysine to homocysteine rendering it inactive (Graziewicz et al. 1996). This prevents the DNA from being coiled after translation and would be a mechanism that could lead to increased DNA strand breaks. The inhibition of specific DNA repair proteins indicates that conditions of nitrosative stress would result in specific lesion and mutation as well as increasing the overall mutation rate. Studies of p53 point mutations in human cancer provided some understanding of the lesions that are associated with NO exposure (Felley-Bosco et al. 1995). Several studies have shown that chronic inflammation increases both p53 and NOS2 expression (Hofseth et al. 2003). Furthermore, a majority of solid cancers have a disruption of the p53 tumor suppressor signaling pathway cascade. There is an important interrelationship between p53 and NO signaling. NO increases p53 activity by inducing phosphorylation and acetylation (Hofseth et al. 2003). In turn, p53 can inhibit the activity of NOS2 by decreasing its expression in a negative feedback loop (Forrester et al. 1996). It was found that inflammation-induced NOS2 is modulated by p53 in vivo and p53 null animals have a super and sustained induction of NOS2 (Ambs et al. 1998b). This finding has important consequences for apoptotic and DNA repair mechanisms. p53 mutated cells are less susceptible to apoptosis and may tolerate higher NO levels which can lead to a higher rate of mutation, and neoplastic transformation or cancer progression. NO-induced apoptosis can be p53 dependent, so

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that mutant p53 cells can have a clonal expansion advantage On the other hand, cancer cells that are p53 wild type are susceptible to macrophage-derived NO and are more likely to undergo apoptosis than mutant p53 cells (Wang et al. 2003). Therefore, NO ability for tumorigenicity and DNA repair are influenced by the p53 status. However, inp53−/− and NOS2−/− animals have a dramatic increase in leukemia as compared to p53−/− NOS2+/+ indicating that NO has important role in the immune surveillance response beyond p53 (Hussain et al. 2008). In colorectal cancer, point mutations in the p53 gene were found to be positively associated with NOS2 expression (Ambs et al. 1999). In ulcerative colitis, it has been shown that p53 mutations are linked to increased risk of colorectal cancer. The predominant type of mutations identified was transition mutations, suggesting that nitrosative stress rather than oxidative stress is the major contributor to p53 mutation in the inflamed colon. The proportion of G to T transversion or G to A transition mutations did not differ in frequency in both adenocarcinoma (AC) and squamous cell carcinoma (SCC) of lung cancers. Interestingly, mutations at a CpG hotspot in SCC were all C to T transitions (Hussain et al. 2001a, b). Both tumor types demonstrated preferential mutation of the non-transcribed strand (100% of all G to T transversions and 55% of the G to A transitions). These results suggest that p53 mutations in both types of lung cancers may arise from adduction by both PAHs and nitrosamines (Hussain et al. 2001a, b). In addition, tar which increases oxidative stress and lipid peroxidation may also increase the proportion of transversion mutation. In contrast, the primary p53 mutations in nonsmokers under the study are transition mutation. In patients with hemochromatosis there is evidence of increased p53 mutation rate of transversion relative to transition, which also associated with the increased NOS2 expression (Hussain et al. 2007). Based on these findings, the heme-rich environment iron and Fenton chemistry may play a role in p53–NO interactions. Besides genotoxicity, NO has also been shown to play a role in angiogenesis, proliferation, and metastasis. Early studies provided a link between NOS, inflammation and cancer. In the mid-1990s, two studies employing NOS2 overexpression in colon tumor cells found increased tumorigenicity and aggressiveness, while transfection of NOS2 into melanoma cells lead to inhibition of proliferation and increased cell death (Jenkins et al. 1994, 1995; Thomsen et al. 1994, 1995; Xie et al. 1995, 1996). There is also evidence that NOS2 can enhance the secretion of angiogenic factors that promote tumor growth. These early findings indicate that different levels of NOS, NO, and RNS play specific roles culminating in different tumor phenotypes. While a small number of studies have demonstrated a favorable prognosis associated with high NOS2 expression in ovarian (Anttila et al. 2007) and lung cancers (Puhakka et al. 2003), the results of the majority of the clinical studies indicate that poor survival was associated with elevated NOS2 in a variety of cancer type (Chen et al. 2005; Wang et al. 2005; Lee et al. 2007; Tanriover et al. 2008) including breast (Bulut et al. 2005), colon (Goodman et al. 2004), gastric (Shen et al. 2004; Li and Xu 2005; Chen et al. 2006), esophageal (Matsumoto et al. 2003), prostate (Aaltoma et al. 2001), melanoma (Ekmekcioglu et al. 2000, 2006, cervical (Chen et al. 2005), squamous cell carcinoma (Shigyo et al. 2007), hepatocellular carcinoma (Sun et al.

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2005), and leukemia (Levesque et al. 2006). Moreover, in several studies a link was observed between the inflammatory biomarkers NOS2 and COX2 and resistance to conventional cancer treatments and the development of a more aggressive phenotype (Jadeski et al. 2002; Ekmekcioglu et al. 2006; Grimm et al. 2008). Inflammation is a hallmark of numerous cancers at every stage of the disease (Hussain and Harris 2007). Moreover, 1/3 of all cancers are thought to have arisen from chronic inflammatory conditions (Ames et al. 1995). Two mediators of inflammation, NOS2 and COX2, are associated with cancer progression and poor prognosis in several tumor types (Turini and DuBois 2002; Ekmekcioglu et al. 2005). Inflammation can be classified into acute and chronic. Though, there are profound temporal differences, there are some similarities between the processes. One of the best examples of the acute inflammation response is the major tissue restoration processes that occur after ischemia reperfusion injury, while classic examples of chronic inflammation include ulcerative colitis or neurological degeneration diseases such as Alzheimer’s disease. In the case of cancer, it has been linked to a wound that does not heal (Riss et al. 2006). In solid tumors, hypoxia is one of the determinants of patient outcome (Brizel et al. 1997). It has been proposed that transient hypoxia reperfusion occurs in the tumor. This is in part due to the poorly developed tumor vasculature and lack of unidirectional flow. Thus, in the tumor environment cells are constantly being challenged and stressed. This leads to increased inflammation and proliferation, resulting in the induction of proteins such as NOS2 and COX2. These in turn promote proliferation and migration and may lead to a more invasive phenotype. Inflammation contains several basic processes (Fig. 1.2). The initiation, events such as loss of blood flow, bacterial or viral infection would trigger the inflammatory process, followed by the increase of neutrophil patrolling the lesion and the

Fig. 1.2 Nitric oxide, cancer, and wound healing

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activation of platelets occurs. Such systemic changes would increase the production of Th1 cytokines, such as IFN-γ,• TNF-α, and IL-1β, and lead to the recruitment of monocytes and their subsequent polarization to macrophage in M1. Those cytokines are able to mediate a tumoricidial and antibacterial immunological responses. In response, macrophage would initiate phagocytosis in order to remove the cellular debris at the lesion. At the same time, increased expressions of NFκB and AP1 at the lesion would lead to the increase of NOS2 and COX2 expressions. NOS2 producing high levels of NO would eventually inhibit COX2 expression (Stadler et al. 1993). Thus, initially there is an increase in NO levels that could induce nitrosative stress. These conditions resulted in P-p53 that inhibits NOS2 activity (Ambs et al. 1998), increases in activated TGFβ• (Vodovotz et al. 1999) and IL-10 (Niedbala et al. 2007) which in turn decreases NO production leading to M2 polarized macrophage. When the level of NO decreases, COX2 activity would increase, generating PGE2. As discussed below, when NO levels at 50–100 nM, COX2 would generate PGE2 that will activate MAPK and Akt pathways and forming a complimentary signal for the proliferation and apoptotic resistant phenotypes. In a normal wound healing model, these genes are downregulated in order to restoring the tissue. However, in tumors, the M2 polarized macrophages are perpetually activated and do not undergo the resolution. Tumor-associated macrophages (TAMs) are M2 polarized, implying that a proliferative state is present in the tumor. This simple model may in part explain why COX2 and NOS2 became poor prognostic markers in some tumors. One of the major insights regarding NO’s paradoxical nature is that its biochemical and physiological effects greatly depends on its microenvironment concentration and temporal profile. The influence of steady-state NO concentration and temporaldependent regulation of signal transduction has also been evaluated (Ridnour et al. 2008; Thomas et al. 2008). In MCF-7 breast adenocarcinoma cells, specific signaling pathways were regulated by distinct fluxes of NO. Levels below 50 nM NO were associated with increased cGMP-mediated ERK phosphorylation; intermediate levels (>100 nM) lead to HIF-1α stabilization; and high NO levels (>300 nM) were associated with phosphorylation of p53 at ser(-)15, which persisted even after dissipation of NO (summarized in Thomas et al. 2008). These phenotypic responses favor a pro-growth and anti-apoptotic paradigm at steady-state NO levels at or below 100 nM. However, the pro-survival effects of NO are lost at concentrations above 400 nM, which is signified by increases in phosphorylation and acetylation of p53 and the induction of p53 tumor suppressor activity (Hofseth et al. 2003). Other proteins including MKP-1, a phosphatase that regulates pERK, are also increased at or above 400 nM steady-state NO (Pervin et al. 2003; Ridnour et al. 2005). This signaling profile was also mimicked by activated macrophages which co-cultured with MCF-7 cells at varying ratios, which suggests that tumor cell response(s) in vivo can be regulated by NO released from tumor-associated macrophages (Thomas et al. 2004). Indeed, tumor phagocyte density and aberrant p53 expression correlated significantly with the phosphorylation of Akt at ser(-)473 in human breast cancer tissue (Prueitt et al. 2007). Similarly, a strong correlation between p53-P-(ser(-)15) and NOS2 protein expression in human samples of ulcerative colitis provided

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further evidence that NO induces a p53 pathway activation in vivo (Hofseth et al. 2003). Another important signaling aspect involves the temporal properties of NO. Though NO is short lived, the sustained NO flux generated by NOS can vary in duration from seconds to days. NO-mediated HIF-1α stabilization correlates directly with concentration and time, (Thomas et al. 2008). While pERK increases immediately in response to NO, it also transiently decreases despite the maintenance of steady-state NO levels. Moreover, NO-induced p53 phosphorylation remains stably elevated even after the dissipation of steady-state NO. Thus, signaling responses to NO are temporally and spatially defined (Thomas et al. 2008). Several endogenous sources of NO exist in the immune system. Macrophages perform a variety of functions from fighting bacteria and tumors, to coordinating wound healing, and tissue remodeling. Murine macrophages can generate a number of different levels of NO that mediate a wide range of functions. For instance, low steady-state NO released by NOS3 (eNOS) activates guanylyl cyclase in the resting macrophage, which is critical during cytokine activation (Connelly et al. 2003). Moreover, macrophage-activating agents demonstrate significant variation in NO output. For example, IFN-γ-pretreated macrophages that are stimulated with TNF-α or IL-1β release nearly 10 times less NO than those stimulated by Toll-like receptor activation (e.g., LPS, PIPC, and Listeria) (Espey et al. 2000). Taken together, macrophages provide an example of a cell type with large variations in NO output dependent on the stimulatory microenvironment. Superoxide and ROS have a role in the regulation of NO-mediated signal transduction. For instance, when MCF-7 cells were exposed to high levels of NO (> 500 nM), the presence of a constant flux of superoxide would attenuate p53 phosphorylation and HIF-1α stabilization (Thomas et al. 2006). Similarly, if superoxide fluxes were held constant and NO levels were increased, phosphorylation of p53 and HIF1α signaling was again attenuated. Since NO is freely diffusible, the intracellular or extracellular generation of superoxide would have had the same effect. These results demonstrated how ROS and NO mutually regulate each other’s signaling cascades. Although NO elicits many unique cellular responses in vitro, the following question remains: Are these NO concentrations and conditions actually happening in vivo? Several studies have looked at in vivo concentrations of NO under different biological conditions by different methods (Tarpey et al. 2004; Zhang 2004). One method to address this question is to compare the cellular response to NO generated from NO donor compounds with responses from NO generated from NOS. Experiment with NO donor compounds has shown that each NO-specific target has different concentration sensitivity to NO. By measuring biological effects of NO and correlating them with measured NO concentrations in vitro, we can approximate the in vivo NO concentrations necessary to elicit specific cellular responses. Using this “molecular signature” approach for various concentration levels of NO, the redox environment can be determined under specific biological conditions. For example, MCF-7 cells co-culture with activated NO-producing ANA-1 murine macrophages resulting in an increase in the phosphorylation of Akt and p53, as well as the stabilization of HIF-1α• (Hofseth et al. 2003; Thomas et al. 2004; Prueitt et al. 2007). As the number of ANA-1 cells was increased, there was a proportional increase

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in the measurable NO in bulk solution (Thomas et al. 2004; Prueitt et al. 2007). When the ratio of MCF-7 and ANA-1 was increased from 1:1 to 1:8, first there was an increase in Akt-P (Prueitt et al. 2007) followed by elevated HIF-1α and then P-p53 at the highest macrophages concentration (Thomas et al. 2004). The measured NO concentration from NO donors in the media would induce p53 phosphorylation when NO > 400 nM was used, but only > 160 nM NO is needed when the source was from activated macrophages (Thomas et al. 2004). This discrepancy can be rectified by considering that NO generated from NO donors is uniformly distributed throughout the media, whereas the enzymatic production of NO from ANA-1 cells generates a concentration gradient emanating from the point of origin. Due to the close proximity of the macrophages to the MCF-7 cells in this example, the actual local concentration of NO is substantially greater than 160 nM and at least as much as 400 nM (Laurent et al. 1996; Wang et al. 2003; Thomas et al. 2004), which is the minimum amount of NO necessary to cause p53 phosphorylation (Thomas et al. 2004). Macrophages can generate different levels of NO that can serve different functions (Espey et al. 2000). For instance, NOS3 stimulation of guanylyl cyclase in non-activated macrophages is required for the fully activated LPS-induced NOS2 in activated macrophages (Connelly et al. 2003). Interestingly, the amount of NO produced form activated macrophages is dependent on the manner by how they are stimulated. When cultured macrophages are IFN-γ pretreated followed by activation with TNF-α or IL-1β• the amount of NO measured into solution is approximately 10 times less than when they are stimulated with IFN-γ + LPS (PIPC and Listeria) even though there is only a modest difference in NOS activity (Espey et al. 2000; Ogawa et al. 2001). In addition, the amount of NO-mediated nitrosation from TNFα or IL-1β stimulation was 30 times less than those stimulated with LPS or PIPC (Espey et al. 2000). This suggests that NO generated from cytokine stimulation leads to considerably lower NO concentrations in the surrounding microenvironment than those agents that activate through the Toll-like receptors. These examples demonstrated that the murine macrophages are capable of producing wide range of potential NO concentrations but depending on the source of stimulus. In contrast, endothelial cells are very sensitive to NO exposure. It has been shown that at < 1 nM NO levels will suppress the TSP-1, the anti-angiogenic agent (Ridnour et al. 2005). In addition, this concentration of NO will induce proliferation of endothelial cells. This phenomenon was shown to be associated with membranebound NOS3, and the local microenvironment is associated. This requirement also indicates that endothelial cells exist in the presence of RBCs. Garthwaite has suggested that neurons with increased cGMP level only require 100 pM amount of NO (Hall and Garthwaite 2006). These observations suggested that NO signaling can occur at very low concentration of NO produced endogenously. It has been shown that a wide range of NO concentrations that mediate discrete biological responses are all depend on the factors of concentration and temporary profile of NO (Thomas et al. 2004; Thomas et al. 2006). The concentration range from sub nanomolar to micromolar indicates that NO can elicit totally different responses over a 1000-fold different concentration range. These simple concepts can explain much of the complexity of the biological response. However, how can

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these different concentrations of NO be achieved and what are the factors that enable these different environments to occur remaining unknown. In fact, the NO signaling can be antagonized by ROS. This implies that NOX and other ROS-generating enzyme can readily influence the redox environment, thus fine-tuning the biological response. The NO’s wide range of responses, reactivity, and rapid chemistry makes it ideal and versatile signal transduction agents. Various cancer types acquired different molecular mechanisms for the growth, their exposure to radiotherapy, surgical, and/or chemotherapy would enhance their susceptibility to cell death, then followed by the onset of inflammatory and wound healing response. These processes are similar to that of a wound healing response in normal tissue where different inflammatory cells infiltrate the wound followed by chemotactic attraction of fibroblasts, lymphocytes, and endothelial cells. These cell types participate in matrix remodeling and neovascularization processes for the restoration of tissue architecture and perfusion capabilities (Crowther et al. 2001). As mentioned before, tumor-associated macrophages (TAMs) recognize tumors as persistent, nonhealing wounds (Whalen 1990). Moreover, tumor growth is an analogous to wound repair as both involve the formation of new tissue and angiogenesis in response to similar local signals including cytokines and pro-angiogenic factors (Whalen 1990). Because the NOS knockout animals demonstrated an impaired wound response, NO modulation via NOS inhibition coupled with radiotherapy and/or drugs aimed at specific targets may be therapeutically beneficial. Modulation of these basic properties in particular antigenic response has also been proposed (Ridnour et al. 2006). Several recently published studies have shown that patients’ 5-year survival would decrease with elevated NOS2 expression and inflammation response (Ekmekcioglu et al. 2006; Prueitt et al. 2007). These studies suggest a link between inflammation, COX2, and NOS2 expression. As stated above the wound healing process is considerably impaired in the NOS2 knockout when compared to wild type (Schwentker et al. 2002). It has been suggested that NO levels that can increase HIF-1α would ultimately increase VEGF after ionizing radiation (Li et al. 2007). This would lead to elevated angiogenesis and increased tumor regrowth. Thus, the application of NOS inhibitors may be in part beneficial for inhibition of macrophage-associated factors that stimulate tumor growth. Macrophages coordinate many immune processes during inflammation and wound healing response. In a Th1 environment, increased production of Th1 cytokines would increase tumor inflammatory processes that sterilize the region via removal of debris. The expressions of both NOS2 and COX2 are also enhanced during this period. Increased NOS2 will increase HIF-1α and VEGF (Griffiths et al. 2005), where PGE2 will increase VEGF via cAMP (Sonoshita et al. 2001) in some tumor models. Also, high levels of NO will increase IL-10 production from Treg cells via activation of p53 as well as activate latent TGFβ•, all necessary to downregulate Th1 to facilitate tissue restoration (Vodovotz et al. 1999; Niedbala et al. 2007). The occurrence of post-therapeutic tumor regrowth is similar to that of a wound healing response following normal tissue injury. Current research into

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conventional therapeutic approaches including radiotherapy, chemotherapy, and surgery has focused on the post-therapeutic involvement of angiogenesis during tumor regrowth. The observation of impaired wound healing response in NOS2 knockout mice suggests the importance of NOS2 during wound healing (Schwentker et al. 2002). This may be due to the fact that NO has a multidimensional role in the regulation of cellular responses as they occur in angiogenesis. As discussed above, NO donors and NOS2 transfection would increase angiogenic response in tumors, which can ablate by L-NAME (Gallo et al. 1998). A potential mediator of NO-induced angiogenic response in tumor may involve the regulation of MMPs, including MMP-9, which is associated with the progression of several types of cancer and can be induced by factors including hypoxia and pro-inflammatory cytokines. Two independent reports have demonstrated that the NO regulation of MMP-9 in epithelial and macrophage cells. Bove et al. reported that NO generated by DETA/NO (∼17 nM steady-state flux) mediated an increase in MMP-9 expression (Bove et al. 2007). In addition, a macrophage model demonstrated NO (∼50 nM steady state) regulation of MMP-9 by cGMP-dependent TIMP-1 suppression (Ridnour et al. 2007). In both cases, NO-mediated MMP-9 regulation was a critical factor during wound response. An important area of NO functionality is the formation of blood vessels via angiogenic processes. Enhanced angiogenesis prior to radiotherapy or drug treatment can increase the therapeutic efficacy via enhanced oxygen effect or drug delivery. In contrast, inhibition of post-treatment angiogenesis can delay tumor regrowth through suppression of oxygen levels and nutrient delivery. Several reports have shown that NOS3 phosphorylation by different pro-angiogenic factors will generate a prolonged flux of NO, which leads to increased cGMP-mediated angiogenic response (Roberts et al. 2007). Many anti-angiogenic factors inhibit NOS3 phosphorylation by increasing PP2A level, the phosphatase for NOS3 serine-1179 (Roberts et al. 2007). Interestingly, the anti-angiogenic factor TSP-1 targets cGMP production and its subsequent down stream targets and is downregulated by NO (Roberts et al. 2007). Toward this end, TSP-1 expression is suppressed by NO at concentrations that are at or below our ability to detect (Ridnour et al. 2005). NOmediated TSP-1 suppression requires ERK phosphorylation and is inhibited by the MEK1/2 inhibitor. Thus NO/cGMP suppression of TSP-1 involves the pro-growth ERK signaling pathway. However, as steady-state NO levels continue to increase, TSP-1 begins to re-accumulate in the media. This is associated, at least in part, with increased MKP-1-mediated dephosphorylation of ERK and is consistent with the demonstration of growth inhibition or cytostasis by higher steady-state levels of NO. Overall, Nitric oxide and nitrogen oxides play a wide variety of roles ranging from positive and beneficial effects to increasing carcinogenesis. As it is said “the devil is in the details” and with NO the mechanism and related biological effects is important to understand in the context of the biology. The understanding of these mechanisms is important in helping us recognize new aspects of cancer that provide new insights into methods for prevention and treatment of this disease.

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Sun, M.H., Han, X.C., Jia, M.K., Jiang, W.D., Wang, M., Zhang, H., Han, G., Jiang, Y. (2005). Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma. World J. Gastroenterol. 11, 5931–5937. Tanriover, N., Ulu, M.O., Isler, C., Durak, H., Oz, B., Uzan, M., Akar, Z. (2008). Neuronal nitric oxide synthase expression in glial tumors: correlation with malignancy and tumor proliferation. Neurol. Res. 30, 940–944. Tarpey, M.M., Wink, D.A., Grisham, M.B. (2004). Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R431–44. Thomas, D.D., Espey, M.G., Ridnour, L.A., Hofseth, L.J., Mancardi, D., Harris, C.C., Wink, D.A. (2004). Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc Natl. Acad. Sci. USA 101, 8894–8899. Thomas, D.D., Ridnour, L.A., Espey, M.G., Donzelli, S., Ambs, S., Hussain, S.P., Harris, C.C., DeGraff, W., Roberts, D.D., Mitchell, J.B., Wink, D.A. (2006). Superoxide fluxes limit nitric oxide-induced signaling. J. Biol. Chem. 281, 25984–25993. Thomas, D.D., Ridnour, L.A., Isenberg, J.S., Flores-Santana, W., Switzer, C.H., Donzelli, S., Hussain, P., Vecoli, C., Paolocci, N., Ambs, S., Colton, C.A., Harris, C.C., Roberts, D.D., Wink, D.A. (2008). The chemical biology of nitric oxide: implications in cellular signaling. Free Radic. Biol. Med. 45, 18–31. Thomsen, L.L., Lawton, F.G., Knowles, R.G., Beesley, J.E., Riveros-Moreno, V., Moncada, S. (1994). Nitric oxide synthase activity in human gynecological cancer. Cancer Res. 54, 1352–1354. Thomsen, L.L., Miles, D.W., Happerfield, L., Bobrow, L.G., Knowles, R.G., Moncada, S. (1995). Nitric oxide synthase activity in human breast cancer. Br. J. Cancer 72, 41–44. Turini, M.E., DuBois, R.N. (2002). Cyclooxygenase-2: a therapeutic target. Annu. Rev. Med. 53, 35–57. Wang, C., Trudel, L.J., Wogan, G.N., Deen, W.M. (2003). Thresholds of nitric oxide-mediated toxicity in human lymphoblastoid cells. Chem. Res. Toxicol. 16, 1004–1013. Wang, L., Shi, G.G., Yao, J.C., Gong, W., Wei, D., Wu, T.T., Ajani, J.A., Huang, S., Xie, K. (2005). Expression of endothelial nitric oxide synthase correlates with the angiogenic phenotype of and predicts poor prognosis in human gastric cancer. Gastric Cancer 8,18–28. West, J.D., Ji, C., Duncan, S.T., Amarnath, V., Schneider, C., Rizzo, C.J., Brash, A.R., Marnett, L.J. (2004). Induction of apoptosis in colorectal carcinoma cells treated with 4-hydroxy-2nonenal and structurally related aldehydic products of lipid peroxidation. Chem. Res. Toxicol. 17, 453–462. Whalen, G.F. (1990). Solid tumours and wounds: transformed cells misunderstood as injured tissue? Lancet 336, 1489–1492. Wink, D.A., Hanbauer, I., Grisham, M.B., Laval, F., Nims, R.W., Laval, J., Cook, J.C., Pacelli, R., Liebmann, J., Krishna, M.C., Ford, M.C., JB, M. (1996). The Chemical Biology of NO. Insights into Regulation, Protective and Toxic Mechanisms of Nitric Oxide. Curr.Top. Cell. Regul. 34, 159–187. Wink, D.A., Hanbauer, I., Krishna, M.C., DeGraff, W., Gamson, J., Mitchell, J.B. (1993). Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl. Acad. Sci. USA 90, 9813–9817. Wink, D.A., Kasprzak, K.S., Maragos, C.M., Elespuru, R.K., Misra, M., Dunams, T.M., Cebula, T.A., Koch, W.H., Andrews, A.W., Allen, J.S., Keefer, L.K. (1991). DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 254, 1001–1003. Wink, D.A., Laval, J. (1994). The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo. Carcinogenesis 15, 2125–2129. Wink, D.A., Mitchell, J.B. (1998). The Chemical Biology of Nitric Oxide: Insights into Regulatory, Cytotoxic and Cytoprotective Mechanisms of Nitric Oxide. Free Rad. Biol. Med. 25, 434–456.

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Vodovotz, Y., Chesler, L., Chong, H., Kim, S.J., Simpson, J.T., DeGraff, W., Cox, G.W., Roberts, A.B., Wink, D.A., Barcellos-Hoff, M.H. (1999). Regulation of transforming growth factor beta1 by nitric oxide. Cancer Res. 59, 2142–2149. Wood, M.L., Esteve, A., Morningstar, M.L., Kuziemko, G.M., Essigmann, J.M. (1992). Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli. Nucleic Acids Res. 20, 6023–6032. Xie, K., Dong, Z., Fidler, I.J. (1996). Activation of nitric oxide synthase gene for inhibition of cancer metastasis. J. Leukoc. Biol. 59, 797–803. Xie, K., Huang, S., Dong, Z., Juang, S.H., Gutman, M., Xi, e. Q.W., Nathan, C., Fidler, I.J. (1995). Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. J. Exp. Med. 181, 1333–1343. Zhang, X. (2004). Real time and in vivo monitoring of nitric oxide by electrochemical sensors– from dream to reality. Front Biosci. 9, 3434–3446.

Part II

Nitric Oxide and the Pathogenesis of Cancer

Chapter 2

A Role for eNOS in Oncogenic Ras-Driven Cancer David F. Kashatus and Christopher M. Counter

Abstract Nitric oxide (NO) is a highly diffusible gas that is generated by the family of nitric oxide synthases and is increasingly associated with tumorigenesis. While both pro- and anti-tumorigenic properties have been ascribed to NO signaling, recent evidence suggests that eNOS or endothelial nitric oxide synthase promotes tumor formation through its effects on proliferation, cell survival, and angiogenesis. In this chapter we discuss recent evidence that eNOS promotes tumorigenic growth through the activation of the Ras family of proteins. Keywords Nitric oxide · eNOS · Tumorigenesis · Pancreatic cancer · Ras

eNOS Endothelial nitric oxide synthase (eNOS) is a member of the nitric oxide synthase (NOS) family of enzymes comprised of eNOS, neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). NOS enzymes catalyze the conversion of L-arginine and oxygen to citruline and the highly diffusible gas nitric oxide (NO) (Knowles and Moncada 1994). eNOS differs from the other two NOS family members in regard to its tissue distribution, intracellular localization, regulation by intracellular signaling pathways, and the relative levels of NO generated. Specifically, eNOS is expressed in endothelial cells, but it has been detected at lower levels in other tissues, whereas nNOS concentrates in neuronal cells and iNOS is induced in a wide variety of cell types (Alderton et al. 2001). eNOS is localized to the caveolae of plasma membranes and Golgi through myristoylation at an N-terminal glycine residue and palmitoylation at two N-terminal cysteine residues (Liu et al. 1995; Liu et al. 1997; Michel 1999). nNOS is also localized to the plasma membrane, but through protein–protein interactions vis-á-vis a unique C.M. Counter (B) Department of Pharmacology and Cancer Biology, Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_2, C Springer Science+Business Media, LLC 2010

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N-terminal PDZ domain, whereas iNOS is a cytosolic protein (Brenman et al. 1996; Alderton et al. 2001). eNOS is activated by Ca2+ release, but, as discussed below, also by phosphorylation at serine 1177 (in human eNOS) by the kinase AKT (PKB). Although nNOS is also activated by Ca2+ , it is not activated by AKT, whereas iNOS activity is independent of Ca2+ signaling (Bredt and Snyder 1990; Abu-Soud et al. 1994; Gachhui et al. 1996; Salerno et al. 1997; Gachhui et al. 1998). Lastly, both eNOS and nNOS typically generate nanomolar levels of NO while iNOS can produce micromolar levels (Xie et al. 1992; Xie and Nathan 1994). Thus, eNOS is distinct from nNOS and iNOS by a number of criteria.

eNOS Activation by AKT Recently, it was discovered that eNOS is regulated independent of elevated Ca2+ in endothelial cells via phosphorylation of S1177 by the kinase AKT. Specifically, AKT co-immunoprecipitates with eNOS (Dimmeler et al. 1999; Michell et al. 1999) and phosphorylates the bovine eNOS at the amino acid’s corresponding to the human protein, S633 and S1177 (Dimmeler et al. 1999; Fulton et al. 1999; Michell et al. 1999), leading to eNOS activation as measured by an increase in NO production (Dimmeler et al. 1999; Fulton et al. 1999; Michell et al. 1999). However, only activating (S→D) and inactivating (S→A) mutation at S1177 (and not S633) enhance (Dimmeler et al. 1999) and abolish (Dimmeler et al. 1999; Fulton et al. 1999) catalytic activity, respectively, indicating that S1177 is the target of AKT signaling. Activation depends on AKT kinase activity (Fulton et al. 1999) and is inhibited with a dominant-negative AKT protein (Dimmeler et al. 1999) or the PI3K inhibitor wortmannin (Dimmeler et al. 1999; Michell et al. 1999). eNOS must associate with the plasma or Golgi membranes to be phosphorylated by AKT, as S1177 phosphorylation is reduced by mutating the palmytolation (Fulton et al. 1999; Gonzalez et al. 2002) or myristoylation (Gonzalez et al. 2002) sites of eNOS, and this effect is rescued if eNOS is artificially re-targeted to membranes (Fulton et al. 2004). AKT phosphorylation does not change the subcellular distribution of eNOS (Fulton et al. 2002), but instead appears to both enhance the electron flux during catalysis and reduce Ca2+ dependency (Dimmeler et al. 1999; Michell et al. 1999; McCabe et al. 2000). Thus, AKT phosphorylates S1177 of membrane-bound eNOS, leading to generation of NO.

PI3K–AKT: Signaling and Cancer The PI3K–AKT signal is inappropriately activated in many cancers to promote tumorigenesis (Luo et al. 2003; Engelman et al. 2006). Activation of the canonical p110/p85 (class IA) family of phosphatidylinositol 3-kinase (PI3K) enzymes begins with engagement of growth factors with receptor tyrosine kinases. The activated receptor activates PI3K either by recruiting the regulatory p85 subunit, via direct binding to autophosphorylation sites on the receptor or an adaptor protein, or through activation of the small GTPase Ras, which when in the active

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GTP-bound state binds directly to the Ras-binding domain of the p110 subunit. Once recruited to the membrane, the p85/p110 PI3K complex, hereafter referred to simply as PI3K, phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2), generating PIP3. PIP3, in turn, recruits other proteins such as the PDK1 kinase and the family of AKT kinases composed of AKT1, AKT2, and AKT3. Recruitment of AKT kinases and phosphorylation by PDK1 activates AKT, which in turn phosphorylates a large number of diverse proteins, including eNOS. This pathway is, in turn, negatively regulated by conversion of PIP3 to PIP2 by the phosphoinositide phosphatase PTEN, a tumor suppressor (Fig. 2.1) (Luo et al. 2003; Engelman et al. 2006). The PI3K pathway can be illegitimately activated via activating mutations or amplification of AKT1, AKT2, p85, PDK1, or more often, PI3K3CA in a subset of cancers (Samuels and Velculescu 2004; Hennessy et al. 2005; Parsons et al. 2005; Karakas et al 2006). More commonly, an upstream activator of AKT, Ras, is mutated to remain in the oncogenic active GTP state in one-third of human cancers (Bos 1989), and loss of expression of the negative regulator of PI3K signaling, PTEN, occurs at a frequency second only to p53 (Eng 2003). Activating mutations in Ras are mutually exclusive with loss of PTEN expression in experimental tumor models (Mao et al. 2004) and human cancers (Ikeda et al. 2000; Tsao et al. 2000; Mizoguchi et al. 2004). Thus, together alterations in Ras or PTEN account for a large amount of PI3K–AKT activation in human cancer (Fig. 2.1) that promotes tumor growth (Hennessy et al. 2005).

Fig. 2.1 PI3K–AKT signaling to eNOS. Simplified diagram of receptor tyrosine kinase (RTK) activation of PI3K, leading to activation of the AKT family of kinases, and in turn phosphorylation of eNOS. Oncogenic mutations to Ras, loss of PTEN expression, and amplifications or mutations to PI3K (PIK3CA) represent the most common events leading to AKT activation in cancer. P: phosphorylated amino acid

Links Between eNOS and Cancer While the role of NO in cancer is controversial and conflicting – NO has been found to both promote and inhibit tumor growth depending on the experimental design and setting (Lechner et al. 2005; Fukumura et al. 2006) – mounting

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circumstantial evidence suggests that eNOS may play some role in cancer by affecting cell proliferation, apoptosis, and angiogenesis. In regard to the link of eNOS with cell proliferation, a phenotype of cancer cells (Vogelstein and Kinzler 1993), eNOS has been detected in tumor cells. Specifically, eNOS-positive tumor cells have been reported in colorectal (Yagihashi et al. 2000), breast (Mortensen et al. 1999a; Martin et al. 2000; Loibl et al. 2002; Tse et al. 2005), brain (Colasanti et al. 1997; Broholm et al. 2001; Broholm et al. 2003), pancreatic (Nussler et al. 1998), Karposi sarcoma (Weninger et al. 1998), and melanoma (Tu et al. 2006) tumors, as well as in mammary carcinoma (Zeillinger et al. 1996; Mortensen et al. 1999b), pancreatic adenocarcinoma (Nussler et al. 1998), and choriocarcinoma (Kiss et al. 1998) cancer cell lines. Moreover, eNOS−/− mice show reduced keratinocyte proliferation resulting in reduced wound margin epithelia (Stallmeyer et al. 2002). Additionally, exposure to NO donors such as GSNO, SNAP, and DEA-NO has been shown to increase DNA synthesis, cell proliferation, and migration of endothelial cells in a number of cell culture models including rabbit aortic endothelial cells (RAECs) (Oliveira et al. 2003), bovine aortic endothelial cells (BAECs) (Zaragoza et al. 2002; Kawasaki et al. 2003), and both human saphenous vein endothelial cells (HSVECs) and human aortic endothelial cells (HAECs) (Kawasaki et al. 2003). Similarly, inhibition of the eNOS pathway with the inhibitor L -NAME has been shown to inhibit proliferation in the oral squamous cancer cell line TSCCa, as measured by the MTT assay and crystal violet staining (Shang et al. 2006). eNOS has also been found to play a pro-survival role in some settings, which has relevance to cancer as evading apoptosis is a hallmark of cancer cells (Vogelstein and Kinzler 1993). Specifically, in the prostate cancer cell line PC-3, ectopic expression of wild-type or the activated S1177D form of eNOS reduced tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)-induced apoptosis by 50 and 75%, respectively (Tong and Li 2004). Additionally, in rat Nb2 lymphoma cells, which express eNOS, but not iNOS or nNOS, addition of exogenous NO through treatment with L-arginine or the NO donor DEA-NO, or generation of endogenous NO, through an FGF-induced increase in eNOS expression, provided protection against apoptosis through the upregulation of Bcl-2 (Dodd et al. 2000; Murphy et al. 2001). In CCD 1106 KERTr keratinocytes, treatment with the NOS inhibitor L-NAME or the sGC inhibitor ODQ led to an increase in apoptosis whereas treatment with the NO donor SNAP led to protection against UV-B-induced apoptosis (Weller et al. 2003). eNOS has also been shown to have a role in the modulation of apoptosis in the breast cancer cell line MCF-7, where blocking eNOS-generated NO using the scavenger PTIO leads to an increase in apoptosis, whereas low concentrations (but not high concentrations) of the NO donor sodium nitroprusside are protective (Mortensen et al. 1999). Finally, relatively low levels of NO, consistent with the amounts produced by eNOS, but not high levels of NO more commonly associated with iNOS, result in the activation of the pro-survival and pro-proliferative protein kinase C (PKC), extracellular signal-related protein kinase (ERK) and Jun in endothelial cells (Jones et al. 2004; Ridnour et al. 2005). The most established role of eNOS in cancer is in regard to angiogenesis, the process by which tumors become vascularized (Duda et al. 2004; Ying and Hofseth

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2007). First, eNOS is detected by immunohistochemistry (IHC) in endothelial cells of tumor stroma from a wide spectrum of cancers (Cobbs et al. 1995; Tschugguel et al. 1996; Takahashi et al. 1997; Nussler et al. 1998; Weninger et al. 1998; Doi et al. 1999; Iwata et al. 1999; Klotz et al. 1999; Mortensen et al. 1999; Yagihashi et al. 2000; Broholm et al. 2001; Kruse et al. 2002; Broholm et al. 2003; Lin et al. 2003; Tse et al. 2005; Wang et al. 2005; Tu et al. 2006), and in some (Takahashi et al. 1997; Nussler et al. 1998; Iwata et al. 1999; Klotz et al. 1999; Yagihashi et al. 2000; Broholm et al. 2001; Kruse et al. 2002; Broholm et al. 2003; Tse et al. 2005; Wang et al. 2005), but not all cases (Rajnakova et al. 1997; Doi et al. 1999; Mortensen et al. 1999; Lin et al. 2003), at elevated levels compared to normal tissue. Second, pro-angiogenic molecules such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2 ) are known to activate eNOS through a calcium–calmodulin-dependent mechanism or through activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which results in the Akt-mediated phosphorylation of eNOS (Duda et al. 2004; Namkoong et al. 2005). Third, loss of eNOS function inhibits angiogenesis, as eNOS−/− mice are defective in angiogenesis following tissue ischemia (Murohara et al. 1998) and display a ∼2-fold reduction in vessel density in implanted collagen gels (Fukumura et al. 2001). A similar level of reduction of angiogenesis has also been found in tumor xenografts derived from B16 murine melanoma (Kashiwagi et al. 2005) and Lewis lung carcinoma (LLC) (Gratton et al. 2003) cell lines when implanted into eNOS−/− mice. Fifth, loss of eNOS in tumor stroma inhibits tumor growth as eNOS−/− mice exhibit a ∼2-fold reduction in tumor growth of xenografts of B16 (Kashiwagi et al. 2005) or LLC (Gratton et al. 2003) cell lines. A similar reduction of LLC and human HepG2 hepatocellular carcinoma tumor cell growth was also observed in wild-type mice treated with a peptide inhibitor of eNOS, an effect that was ascribed to inhibiting stromal eNOS activity (Gratton et al. 2003).

Hypothesis: AKT Promotes Tumorigenesis by Activating eNOS in Cancer Cells Because (1) AKT is commonly activated in human cancers to promote tumorigenesis, (2) eNOS is activated by AKT, at least in endothelial cells, (3) eNOS has been detected in cancer cells, and (4) perturbing eNOS expression in a number of settings disrupts tumor phenotypes, such as cell proliferation, apoptosis, and angiogenesis, we propose that illegitimate activation of AKT in cancer cells may act through eNOS to promote tumorigenesis (Fig. 2.1).

eNOS Activation in Pancreatic Cancer As mentioned, Ras is mutated to remain in the active oncogenic state in one-third of human cancers, and 90% of pancreatic cancers (Bos 1989), and it is well established that oncogenic Ras activates the PI3K–AKT pathway to promote cancer

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(Campbell and Der 2004). AKT, in turn, has been shown to activate eNOS through phosphorylation of S1177 in endothelial cells. We thus tested whether eNOS is phosphorylated and thereby activated at the AKT site S1177 in the cancer most characterized by oncogenic Ras mutations, human pancreatic cancer (Bos 1989). A panel of nine pancreatic cancer cell lines and two normal pancreatic tissue specimens were immunoblotted with an α-phospho(S1177)-eNOS antibody (Chen et al. 1999; Michell et al. 1999), with the finding that S1177 phosphorylation of eNOS was elevated in a subset of the tumor cell lines samples compared to the normal tissue control (Fig. 2.2a and (Lim et al. 2008)). We extended these results to actual pancreatic cancer tumor specimens, only this time finding an increase in S1177 phosphorylation in all tumor samples compared to the matched and unmatched normal controls (Fig. 2.2b and (Lim et al. 2008)). eNOS is thus phosphorylated at the AKT site S1177 in Ras-driven human pancreatic cancer specimens.

Fig. 2.2 eNOS is phosphorylated at S1177 in oncogenic KRas-driven tumors. Immunoblot of (a) indicated human pancreatic cancer cell lines or (b) pancreatic biopsies of non-malignant (N) and tumor (T) tissue with an α-phospho(S1177)-eNOS antibody. Actin and tubulin: loading controls. Adapted from (Lim et al. 2008) and used with permission from the publisher

Phosphorylated eNOS is Required for Ras-Mediated Tumor Growth To assess the biological consequence of oncogenic Ras-mediated phosphorylation of the AKT site of eNOS during tumorigenesis, we tested whether inhibiting AKT phosphorylation of eNOS impeded tumor growth of Ras-transformed cells. For this analysis we employed the genetically defined HEK-TtH cell line engineered to express oncogenic Ras (RasG12V ). Such RasG12V -TtH cells were chosen because they are human, an important consideration as Ras oncogenesis can differ between human and murine cells (Hamad et al. 2002; Rangarajan et al. 2004), the cells have a defined genetic background, thereby simplifying analysis (O’Hayer and Counter 2006), and are absolutely dependent on RasG12V for tumor growth (Hahn et al. 1999). RasG12V -TtH cells were stably infected with a retrovirus encoding no transgene (vector) or eNOS shRNA, and appropriate knockdown of eNOS expression was confirmed by immunoblot (Fig. 2.3a). To assess whether phosphorylation of eNOS was required for tumor growth, the latter cells were stably infected with a retrovirus encoding human eNOS engineered to be resistant to shRNA by introduction of silent point mutations (eNOSR ) in either the S1177A mutant or wild-type

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Fig. 2.3 eNOS phosphorylation is required for tumor growth. RasG12V-TtH cells expressing the indicated transgenes or shRNAs were (a) immunoblotted with an α-phospho(S1177)-eNOS Ab to detect phosphorylated endogenous (P-eNOS) and ectopic HA-tagged (P-eNOSR ) eNOS, and (b) injected into the flanks of four mice, of which representative mice and excised tumors are shown. Adapted from Lim et al. (2008) and used with permission from the publisher

(WT) configuration, and expression validated by immunoblot (Fig. 2.3a). All four cell lines were then each injected into the flanks of four immunocompromised mice and tumor growth monitored. The first important observation of these studies was that knockdown of eNOS clearly reduced the tumor growth of the cells compared to scramble control (Fig. 2.3b), arguing that in addition to a role for eNOS in stromal tissue, the enzyme is also required in cancerous cells for Ras-mediated tumor growth. This loss of tumor growth was rescued by expressing wild-type eNOSR , ruling out any offtarget effects of the eNOS shRNA, but not by the S1177A mutant version of eNOSR (Fig. 2.3b). Thus, not only is eNOS required for Ras-driven tumor growth, but eNOS must be phosphorylated at the AKT site S1177 to promote tumor growth. These results were validated in two different model systems. First, stable shRNAmediated knockdown of eNOS reduced the tumorigenic growth potential of the two pancreatic cancer cell lines characterized by oncogenic KRas mutations, MiaPaCa2 and CFPac1 (Lim et al. 2008), when injected into immunocompromised mice. Second, the polycyclic aromatic hydrocarbon carcinogen 9,10-dimethyl-1,2benzanthracene (DMBA) followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) was topically applied on eNOS−/− mice and, as a control, eNOS+/+ mice (Iversen 1991; Stern and Conti 1996). This approach has been extensively used to induce benign skin papillomas characterized by HRas oncogenic mutations that progress to adenocarcinomas (Balmain et al. 1984; Quintanilla et al. 1986; Kemp et al. 1994; Stern and Conti 1996). We found that there was a ∼3-fold reduction in the number

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of tumors per mouse in eNOS−/− compared to control eNOS+/+ mice (Lim et al. 2008). Thus, in three different model systems, loss of eNOS expression reduced oncogenic Ras-driven tumor growth.

How Does eNOS Promote Tumorigenesis? Although eNOS plays a role in oncogenic Ras-driven tumorigenesis, the mechanism of action remains to be resolved. Presumably eNOS promotes tumorigenesis by generating NO, as the S1177A mutant of eNOS, which cannot be phosphorylated by AKT but is an otherwise wild-type protein, cannot rescue the loss of tumor growth upon knockdown of endogenous eNOS, whereas wild-type eNOS can rescue this effect (Lim et al. 2008). NO produced by eNOS could have many effects, as NO can be converted to a number of other molecules depending on the chemical environment in which it is generated (Alderton et al. 2001). One possibility is that NO activates the canonical target of NO signaling, sGC. This enzyme could very well be activated in the stromal tissue, especially in regard to angiogenesis. However, knockdown of eNOS can reduce proliferation of cancer cell lines (Zaragoza et al. 2002; Kawasaki et al. 2003; Oliveira et al. 2003; Shang et al. 2006), suggesting a cell autonomous defect. Thus, perhaps eNOS targets sGC in cancer cells, or even more intriguingly, it may target other molecules aside from sGC to promote tumor phenotypes. One attractive substrate for NO in cancer is the Ras protein itself. The Ras family of small GTPases is composed of HRas, NRas, and KRas. All three Ras family members contain a conserved cysteine at position 118. Aside from the wellestablished effect of NO with redox active metal centers, as exemplified in the case of sGC (Davis et al. 2001), NO can also lead to modification of the thiol group of cysteines. Derived reactive nitrogen species from NO, primarily •NO2 and ONOO–, can lead to S-nitrosylation, S-glutathionylation (in the presence of glutathione), and disulfides or sulfenic, sulfinic, or sulfenic acid derivatives of the thiol group of cysteines (Cooper et al. 2002; Hess et al. 2005). At least in the case of HRas, C118 has been shown to be S-nitrosylated and S-glutathionylated in vitro and in vivo, which leads to an increase in the active GTP-bound state. Consistent with this, mutating C118 to serine in HRas, a minor change that exchanges the sulfur atom for oxygen, abolishes both modifications and leaves the protein in an inactive GDP-bound state or reduced Ras signaling (Lander et al. 1996; Ji et al. 1999; Clavreul et al. 2006). Thus, we indirectly measured S-nitrosylation of HRas using the biotin switch assay (Jaffrey and Snyder 2001) in immortalized human epithelial cells and confirmed that HRas is nitrosylated and that it is specific for C118 (Fig. 2.4a). Additionally, we directly measured the GTP loading of HRas by capture of active Ras via the RBD of Raf1 followed by immunoblot with an HRas-specific antibody (Taylor et al. 2001) in two independent pancreatic cancer cell lines (MiaPaCa2 and CFPac1) in which eNOS expression was reduced by shRNA. We found that reducing eNOS

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Fig. 2.4 Nitrosylation leads to an increase in GTP-bound HRas. (a) Immortalized human embryonic kidney cells expressing either wild type or C118S mutant flag-tagged HRas were analyzed by the biotin switch method to determine the levels of nitrosylated HRas. Tubulin: loading control. (b) CFPac-1 cells expressing shRNA specific for either eNOS (eNOSi) or a scrambled control sequence (scram) were assayed for levels of GTP-bound and total HRas. Tubulin: loading control. Adapted from Lim et al. (2008) and used with permission from the publisher

expression in these cell lines led to a reduction of GTP-bound HRas (Fig. 2.4b). Similar results were found when wild-type NRas was also assayed (Lim et al. 2008). Taken together, these results suggest that the modification detected on C118 of HRas is most consistent with S-nitrosylation, and further, that the eNOS-dependent modification of this cysteine activates HRas and NRas. To assess the biological impact of eNOS-dependent activation of HRas (and NRas) on tumorigenesis, HRas was stably knocked down in oncogenic KRas mutation-positive CFPac1 pancreatic cancer cells and complemented with (i) RNAiresistant HRas as a positive control to rescue the loss of HRas expression, (ii) no transgene as a negative control, and lastly, (iii) RNAi-resistant HRas in the C118S mutant configuration (Fig. 2.5a). The four cell lines were then each injected into the flanks of four immunocompromised mice and tumor growth monitored over time. We found that knockdown of wild-type HRas in the oncogenic KRas-driven tumor cell line CFPac1 reduced tumor growth, and this was rescued by the wildtype HRas, but not the C118S mutant (Fig. 2.5b). Similar results were observed in another cancer cell line, and when NRas was similarly knocked down and complimented with wild-type or C118S mutant versions of RNAi-resistant NRas (Lim et al. 2008). These results argue that the eNOS-dependent modification detected on C118 by the biotin switch assay, most consistent with S-nitrosylation, is required for tumor growth, which in turn suggests that HRas and NRas act downstream of eNOS in tumorigenesis.

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Fig. 2.5 C→S mutation on C118 of wild-type HRas inhibits tumor growth. (a) Immunoblot analysis of HRas expression and (b) excised tumors from CFPac1 cells stably infected with retrovirus encoding a scramble sequence or eNOS shRNA sequence and complemented with no transgene or RNAi-resistant HRas in a wild type or C118S mutant configuration. Adapted from Lim et al. (2008) and used with permission from the publisher

More Questions than Answers These experiments have only just begun to shed light on what is surely to be a much more complex picture for how eNOS promotes tumorigenesis. First, the nature of the eNOS-dependent chemical modification at C118 of HRas in vivo remains to be resolved. Specifically, while detection of this modification by the biotin switch assay in cells (Raines et al. 2006; Lim et al. 2008), and mass spectrometric identification of S-nitrosylation of this site in vitro (Lander et al. 1997) suggest that C118 is modified by S-nitrosylation, this remains to be independently validated in vivo. Second, production of NO by eNOS could have widespread consequences, and hence it remains to be determined if Ras proteins are the only targets of NO in cancer cells. In this regard, proteins known to be modified by NO such as sGC (Denninger and Marletta 1999) and caspases (Mannick et al. 2001) are interesting candidates. Third, why a cancer cell that already has one Ras family member activated vis-á-vis an oncogenic mutation would rely on activation of the other Ras family members remains a mystery. This is especially confusing insofar as the catalytic and effector-binding domains of HRas, NRas, and KRas are nearly identical at the amino acid level. Fourth, AKT is activated by a variety of changes in cancer cells, not just by oncogenic Ras mutations, and hence it will be interesting to test whether other changes, for example, loss of PTEN, also rely on eNOS for tumor growth. Fifth, whether these studies can be translated into therapies remain to be determined, but it is encouraging that cavtratin, a peptide derived from caveolin-1, inhibits eNOS and displays anti-tumor activity, and general NOS inhibitors such as L -NAME and L -NMMA have been tested in clinical trials for other diseases. Lastly, the exact nature of a requirement for eNOS in tumor growth is unknown, and since eNOS can effect cell proliferation, apoptosis, and angiogenesis, there is surely more here than meets the eye.

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Chapter 3

Dual Role of Nitric Oxide in Cancer Biology Shehla Pervin, Rajan Singh, Suvajit Sen, and Gautam Chaudhuri

Abstract Extensive research work performed over the past decades has focused on understanding the role of nitric oxide (NO) in both promoting and preventing cancer. The precise role of NO in tumor biology has been the cause of intense debate. Experimental evidences available in the literature highlight contrasting proand anti-tumor effects of NO. It is now becoming clear that concentration- and time-dependent regulation of NO leads to tumor growth, cytostasis, or cell death. It is known that NO participates in various signaling pathways including Ras, extracellular signal-regulated kinases (ERKs), Akt, cyclin D1/retinoblastoma (Rb) as mammalian target of rapamycin (mTOR) that are crucial for tumor cells. NO mediated post-translational modification of key proteins including S-nitrosylation of caspases, tyrosine nitration of mitochondrial manganese superoxide dismutase (MnSOD), or cytochrome c. Various attempts toward developing NO-based cancer therapy are still in primitive stages, and a clear understanding of the levels of NOS expression, its timing, and the concentrations of NO produced in the tumor microenvironment is key to the development of novel strategies for tumor treatment and prevention. Keywords Nitric oxide · Cancer · Mammalian target of rapamycin · MAP kinase phosphatase-1 · Nω -Hydroxy-L-arginine

Introduction Nitric oxide (NO) is a potent bioactive molecule that plays a critical role in various physiological and pathophysiological processes (Gong et al. 2004; Torreilles 2001; Schindler and Bogdan 2001). Its diffusible gaseous molecule is synthesized from S. Pervin (B) Division of Endocrinology, Charles Drew School of Medicine and Science, 3084A Hawkins Building, Los Angeles, CA, USA e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_3, C Springer Science+Business Media, LLC 2010

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L -arginine by NO synthase (NOS) enzymes (Moncada and Higgs 1993; Knowles and Moncada 1994; Ignarro 1989, 1996). There are three isoforms of NOS: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). nNOS and eNOS are expressed predominantly in neuronal and endothelial cells, respectively, and are referred to as constitutive NOS (cNOS). The activity of cNOS is calcium dependent and is regulated by intracellular localization, phosphorylation (Dimmeler et al. 1999), and interaction with regulatory molecules (Sessa 2004). On the other hand, iNOS is regulated transcriptionally and is induced by inflammatory cytokines, oxidative stress, aging hypoxia, and various endotoxins (Nathan and Xie 1994; Michel and Feron 1997; Kleinert et al. 2003; Dachs and Tozer 2000; Ganster et al. 2001). Intracellular levels of calcium do not regulate iNOS. Induction of iNOS leads to the production of high concentrations of NO, usually in the micromolar range during pathophysiological conditions (Kolios et al. 2004; Goligorsky et al. 2004). Many of the physiological processes promoted by NO are mediated by NO– cGMP signaling pathway (Murad 1994; McDonald and Murad 1995). Posttranscriptional modifications of proteins via S-nitrosylation of cysteine thiol residues have emerged as one important signaling pathway that mediates various physiological functions (Stamler et al. 2001; Marshall et al. 2000). The cytotoxic effects of NO mediated via NO or its metabolites lead to DNA damage and loss of protein function resulting in necrosis and apoptosis (Hussain et al. 2003). The role of NO in cancer biology has been extensively investigated in the last decade with conflicting reports, suggesting the complexity of interactions between the levels of NO and various aspects of tumor development and progression. The fundamental question whether NO promotes or inhibits cancer progression remains unanswered. Several earlier studies reported that NO is a critical component of immune response of macrophages, and transfection of iNOS leads to the suppression of various murine and human cancers (Moilanen and Vapaatalo 1995; Kawabe et al. 1992). On the other hand, contrasting reports suggest a role of iNOS in eliciting different physiologic and pathologic effects (Xie et al. 1995, 1997). This paradoxical dichotomy presents a clear challenge to determine the effects of NO in cancer biology and define a therapeutic role of NO-based tumoricidal strategies.

Expression of NOS in Tumors Several investigators have reported the expression of iNOS within the tumor microenvironment. Thomsen et al. suggested that iNOS activity is higher in less-differentiated breast carcinoma and is primarily detected in tumor-infiltrating macrophages (Thomsen et al. 1995). iNOS is also expressed in approximately 60% of human colon adenomas and 25% of colon carcinoma, while its expression is either low or absent in the surrounding tissues (Ambs et al. 1998a; Nosho et al. 2005). In human ovarian cancer, iNOS activity was detected in tumor cells but not in normal ovarian tissue (Thomsen et al. 1994; Massi et al. 2001). Measurement of eNOS and iNOS expression in benign and malignant lesions of breast revealed

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that eNOS is expressed in 6% tumor samples and positively correlates with the expression of iNOS (Loibl et al. 2002). Several groups subsequently demonstrated breast carcinoma cells to express iNOS (Wilson et al. 1998; Ambs et al. 1998a; Klotz et al. 1998; Swana et al. 1999; Hajri et al. 1998; Cobbs et al. 1995). The expression of iNOS was positively correlated with angiogenesis (and high VEGF expression), microvascular density, tumor size, and poor survival (Ambs et al. 1998b). Histological examinations reveal a positive co-relationship between high angiogenic activity and iNOS expression in human brain, breast, colon, head and neck, and lung tumors (Fukumura et al. 2006). Increased NOS activity and elevated NO production have been found in lung cancer patients and in the inflamed colonic mucosa from patients with ulcerative colitis (Liu et al. 1998). In malignant neoplasm of the central nervous system, a high level of NOS activity suggests that NO and its related nitrogen species could be associated with pathophysiological processes important to the tumor (Fiore et al. 2006). These findings suggest that NO produced by tumor cells mediates tumor angiogenesis, invasion, and growth. A positive correlation between iNOS expression and poor clinical outcome of patients affected with different types of cancer suggests that NO should be considered as a potential mediator of tumor development and progression (Ekmekcioglu et al. 2000; Raspollini et al. 2004; Gallo et al. 1998). However, there have been several studies that offer conflicting observations regarding the role of NO in tumor progression and development. The histological examination of several human cancers does not reveal any correlation of iNOS expression with tumor progression. On the other hand, it inversely correlated with tumor stage, grade, and progression and positively correlated with apoptosis and patient survival (Brennan et al. 2000; Kitano et al. 1999; Ambs et al. 1999). Reduced tumor growth was observed in p53-positive tumors after induction of iNOS (Ambs et al. 1998c). A positive role of NO in inhibiting tumor angiogenesis came from studies by Nunakawa demonstrating that high NO production resulting from iNOS induction can inhibit proliferation of endothelial cells and vascular smooth cells (Nunokawa and Tanaka 1992). It has been shown that transduction of iNOS in tumor cells inhibits tumor growth and metastasis in several mouse tumor models and human colon and renal carcinoma cells (Xie et al. 1995; Juang et al. 1998; Le et al. 2005). An inverse relationship has been reported between tumor cell-derived NO and platelet aggregation in metastatic human colorectal carcinoma cell line (Gasic et al. 1968). Since platelets store angiogenic factors and stimulate vessel growth leading to the retention of metastasizing tumor cells in blood vessels, it may indicate that iNOS-derived NO has anti-tumorigenic role.

Nitric Oxide Regulation of Apoptotic Pathways Programmed cell death is mediated via activation of a family of cysteine proteases called caspases, which are involved in both death receptor (extrinsic)- and mitochondrial (intrinsic)-dependent apoptotic pathways.

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The extrinsic pathway involves binding of specific death ligands to their respective cell surface receptors, such as Fas, tumor necrosis factor alpha (TNF-α) receptor, and TRAIL and activates downstream signaling pathways via adapter molecules. Adapter molecules subsequently recruit and initiate cysteine protease (e.g., caspase-8) which, in turn, cleaves downstream effector caspases, such as caspase-9 and caspase-3, leading to the activation of DNA degradation. The intrinsic pathway is dependent on the release of cytochrome c from the mitochondria and other pro-apoptotic molecules, Smac/DIABLO, into the cytoplasm. Its association with Apaf1 activates caspase-9 and caspase-3 to trigger apoptosis (Olson and Garbán 2008). It has been reported that NO can have both pro- and anti-apoptotic properties (Li and Billiar 1999; Weller et al. 2002; Liu et al. 1998; Pervin et al. 2001a, b, 2003a, b). Inhibition of apoptosis by NO is reported in endothelial cells, hepatocytes, and other tumor cells (Li and Billiar 1999; Weller et al. 2002; Liu and Stamler 1999). NO induces S-nitrosylation of active site cysteines in caspases and other related proteins leading to inhibition of apoptosis and the formation of S-nitrosothiols leading to the oxidation of thiol proteins, which might act as switches in cell survival and apoptotic signaling pathway (Mannick et al. 2001; Azad et al. 2006; Marshall et al. 2000). NO can promote the formation of a highly carcinogenic substance nitrosamine. Nitrosylation of nucleic acid bases leads to the semi-conversion of cytosine to uracil and guanine (Lechner et al. 2005). NO can inactivate several DNA repair enzymes such as DNA alkyltransferase, xeroderma pigmentosum A (XPA) protein, and 8-oxoguanine glycosylase-1 (OGC1) (Wink et al. 1998; Morita et al. 1996; Jaiswal et al. 2001). It has been suggested that NO-mediated inhibition of DNA repair processes might be one of the key elements favoring the process of carcinogenesis during inflammatory diseases. NO is known to stimulate anti-apoptotic Akt pathway via activation of Ras (Pervin et al. 2007). NO can protect human colon cancer cells from apoptosis by scavenging mitochondrial superoxide in vitro (Wenzel et al. 2003). In human neuroblastoma cells, NO upregulates NAD(P)H quinone oxidoreductase 1 (Nqo1) and NF-E2-related factor-2 (Nrf2) (Dhakshinamoorthy and Porter 2004). On the other hand, several reports suggest that NO can also exert pro-apoptotic effects. p53, a tumor suppressor gene is accumulated as a consequence of NOmediated DNA damage leading to growth arrest and apoptosis (Hussain et al. 2003). A negative feedback loop is formed between NO generation and p53 accumulation which may constitute part of a physiological mechanism of response to DNA damage by endogenously produced NO (Hussain et al. 2003). NO inhibits the activity of NF-κB, a major anti-apoptotic pathway often dysregulated in tumor cells (Reynaert et al. 2004). It is suggested that NO stabilized the NF-κB inhibitor Iκ Bα, by preventing its degradation from NF-κB. NO also increased the mRNA expression of Iκ Bα (Peng et al. 1995). It is also shown that NF-κB can be inhibited directly by NO via S-nitrosylation of p50 subunit, thus inhibiting its binding to the target DNA site (Matthews et al. 1996). Using ovarian carcinoma exogenous NO donor, SNAP has been reported to inhibit growth (Cantuaria et al. 2000). NO sensitizes ovarian tumor

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cells to Fas-induced apoptosis by the disruption of repressor binding to the silencer region of the Fas promoter site (Garbán and Bonavida 1999). NO induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), a serine threonine phosphatase, leading to the dephosphorylation of extracellular signal-regulated kinase (ERK), which is the initial and essential step that commits breast cancer cells to apoptosis (Pervin et al. 2003a). Inactivation of ERK preceded the dephosphorylation of Akt and apoptosis, and specific inhibition of MKP-1 either by pharmacological inhibitors or by anti-sense oligonucleotides abolished NO-induced apoptosis in breast cancer cells. Other breast cancer cells, where NO did not induce MKP-1 and there was no ERK inactivation, showed no apoptosis (Pervin et al. 2003a). Our group also reported that NO induced BAX integration into the mitochondrial membrane leading to the induction of apoptosis in MDA-MB-468 cells (Pervin et al. 2003b). NO-induced activation of caspase-3 and caspase-9 was detected as early as 8 h but reached a maximum at 36 h, which coincided with the maximum release of cytochrome c from the mitochondria. NO-induced release of cytochrome c was independent of changes in mitochondrial membrane potential (MMP) but was dependent on BAX integration into the mitochondrial membrane. The migration of BAX to the mitochondria was suggested to be caused by reduction in both total and phosphorylated form of Akt (Pervin et al. 2003b). Apoptosis can also be promoted by NO via downregulation of anti-apoptotic proteins survivin in human lung carcinoma cells (Chao et al. 2004). NO-downregulated survivin in breast cancer cells precedes MKP-1 induction and a possible inverse relationship has been suggested (Wen et al. 2006). It is reported that IGF-1 and pro-inflammatory environment induced phosphorylation of Akt (Prueitt et al. 2007). Furthermore, moderate to high levels of phosphorylated Akt, BAD, and caspase-9 were found to be expressed in breast tumor specimens. Phosphorylation of Akt, BAD, and caspase-9 in a sequence activates a survival pathway and raises the threshold of apoptosis as BAD and caspase-9 phosphorylation is known to inhibit their pro-apoptotic functions (Prueitt et al. 2007). It is likely that low concentration of NO produces similar effects to that of IGF-1. Some breast cancer cells express high levels of arginase, an enzyme that converts L -arginine to L -ornithine, which is the precursor of polyamines, essential components of cell proliferation (Singh et al. 2000) (Fig. 3.1). N∞ -Hydroxy-L-arginine (NOHA), an intermediate in the NO biosynthetic pathways, which is generated up to 30% of the total amount of NO during high-throughput NO production, is reported to selectively inhibit cell proliferation and induce apoptosis in high arginase-expressing MDA-MB-468 cells (Singh et al. 2000) (Fig. 3.2). After 48 h NOHA arrested these cells in the S phase of the cell cycle, increased the expression of p21, and reduced spermine content. In contrast, the ZR-75-30 cell line maintained its viability and its L-ornithine and spermine levels in the presence of NOHA, suggesting that NOHA has anti-proliferative and apoptotic actions on arginaseexpressing human breast cancer cells that are independent of NO. NOHA-induced apoptosis was associated with activation of caspase-8 leading to truncation of proapoptotic molecule called BID. Truncated BID (tBID) was associated with release

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Fig. 3.1 Expression of NOS and arginase activities in various human breast cancer cell lines. (a) Enzyme activity of calcium-dependent and calcium-independent NOS. (b) Enzyme activity of arginase. (c) Reverse transcription polymerase analysis of various NOS and arginase isoforms. Adapted from Singh et al. (2000) and used with permission from the publisher

of cytochrome c from the mitochondria in the absence of mitochondrial membrane depolarization that led to the activation of caspase-3 and finally cleavage of PARP and induction of apoptosis (Singh et al. 2002). These apoptotic effects of NOHA in high arginase-expressing cells were completely abolished in the presence of exogenous L-ornithine. However, L-ornithine was able to block the effects of NOHA as the level of mitochondria. A detailed study suggested that L-ornithine was able to block NOHA-induced apoptosis by blocking cytochrome c release and activation of caspase-9 and caspase-3 and cleavage of PARP, without blocking caspase-8 induction and cleavage of BID. These experiments suggested that mitochondria are the main target of NOHA-induced apoptosis in arginase-rich breast cancer cells. A proteomics approach is being undertaken in order to identify key mitochondrial targets, where NOHA would induce apoptosisrelated proteins present exclusively in the mitochondria and would be antagonized with a simultaneous treatment of L-ornithine (Singh et al. 2007).

Nitric Oxide Regulation of Translation Deregulation of proliferation pathways and protein synthesis has been strongly implicated in the pathogenesis of cancer and metastasis (Pervin et al. 2008a).

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Fig. 3.2 Induction of apoptosis in MDA-MB-468 cells with arginase inhibitor, Nω -hydroxy-Larginine (NOHA), and its inhibition in the presence of exogenous L-ornithine. (a) Time course of caspase-3 enzyme activation with NOHA (1 mM) and its inhibition by L-ornithine (1 mM). (b) Time course of proteolytic cleavage of caspase-3 with NOHA and its inhibition by L-ornithine. (c) Time course of PARP cleavage with NOHA and its inhibition by L-ornithine. Adapted from Singh et al. (2002) and used with permission from the publisher

Translational control has been predicted for many proliferation-related proteins after sequence comparison of their 5 -untranslated region (UTR) with other mRNA species (Parsa and Holland 2004). Studies have identified cyclin D1 and ODC as two integral cell cycle transit proteins whose cap-dependent transcripts are subjected to strong translational control (Parsa and Holland 2004). In Ras overexpressed cells, considerable increase in ODC activity was observed due to upregulation of ODC translation. Both cyclin D1 and ODC mRNA contain highly structured 5 -UTRs, rendering them to be less efficiently translated in normal cells (Shantz 2004). Cyclin D1 is overexpressed in 80% of in situ ductal carcinoma whereas it is low or absent in normal breast tissue, suggesting that cyclin D1 may play an important role during transition from a benign state to carcinoma, and unregulated overexpression of cyclin D1 may be common early even in mammary carcinomas (Fantl et al. 1993). Using highly undifferentiated MDA-MB-231 cells, it was reported that exogenous NO induced cell cycle arrest, which was associated with downregulation of cyclin D1 protein that precedes the decrease of its mRNA and was associated

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with hypophosphorylation of retinoblastoma protein (Pervin et al. 2001a). This suggests that the effect of NO on cancer cells may be regulated at translational level. Dependence of cancer cells on cyclin D1, a short half-life protein, for proliferation reinforces the emerging importance of the translational machinery to maintain oncogenesis, and therefore, therapeutic drugs may be designed to specifically target translational machinery. NO has also been found to inhibit the initiation steps of protein synthesis in a variety of cell types by phosphorylation and inactivating eukaryotic initiation factor 2-α (eIF2-α) (Kim et al. 1998). Using DETA-NONOate, a long half-life NO donor, we have reported that low and high oxidative stress (as induced by 1 mM and 2 mM DETA-NONOate) inhibits the initiation steps of protein synthesis (Pervin et al. 2008a). Breast cancer cells with low NO stress induced a small upregulation of phosphorylated eIF2-α (peIF2-α) levels, which led to the decline in short half-life proteins, whereas high NO stress induced a prominent increase in peIF2-α and induced decline of both short and long halflife proteins. In contrast, human mammary epithelial (HME) Ras-transfected HME (MCF-10AneoN) cells were less susceptible to NO-induced inhibition of protein synthesis and apoptosis. Low NO stress induced heme-regulated inhibitor (HRI) activation, which facilitated gradual decline in short half-life proteins. On the other hand, high NO stress induced HRI and protein kinase R (PKR) activation, which facilitated gradual decline in short half-life protein. PKR overexpression in breast cancer cells induces suppression of protein synthesis and possibly induction of apoptosis (Pervin et al. 2008a) (Fig. 3.3).

Fig. 3.3 Flow charts of differential NO stress leading to inhibition of protein synthesis and cytostasis in human breast cancer cells. HRI, heme-regulated inhibitor; PKR, protein kinase R. Adapted from Pervin et al. (2008a) with permission from the publisher

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NO (nM concentration) has been reported to activate Ras post-translational modification via S-nitrosylation of critical cys118 residue which stimulates guanine nucleotide exchange (Gallo et al. 1998). We have reported that NO (60 nM) treatment of MDA-MB-231 cells led to Ras-mediated increase in PI-3 kinase/Akt and Raf/MEK/ERK signal transduction pathways which was independent of cGMP. It was shown that pre-treatment of the cells by farnesyl transferase inhibitors (FTIs), which block the post-translational modification of Ras protein, decreased the basal levels of pERK1/2, pAKT, and cyclin D1 to induce cytostasis (Pervin et al. 2007). Growth factor receptor binding protein 2, which complexes with adapter proteins involved in Ras signaling, is overexpressed in breast cancer cells (Janes et al. 1994; Pandey et al. 1995). It is possible that low concentrations of NO could amplify Ras signaling by inducing conformational changes of the membrane-bound Ras protein. There is a possibility that NO might be modulating Rheb/mTOR activity to promote guanine nucleotide exchange on Ras to promote Ras-GTP levels.

Concentration-Dependent Effects of NO The signaling pathways in cancer biology induced by NO are reported to be diverse and often associated with opposing biological activities, which are associated with enormous variety of chemical reactions and biological properties associated with the molecule. Specific NO concentrations are reported to elicit precise cellular responses including apoptosis, senescence, and angiogenesis in various cancer cells. It is believed that the therapeutic outcome is likely dependent on different levels of NO present or artificially induced within the tumor microenvironment. It has been implicated that low physiological concentrations of NO regulate different processes of tumorigenesis. Clinical and experimental studies support a positive relationship between tumor malignancy and NOS activity in certain tumors (Wink et al. 1998; Thomsen and Miles 1998). Solid tumors have been found to produce sustained levels of NO, which is produced by tumor cells themselves or macrophages that infiltrate these tumors (Gratton et al. 2003). Thomas et al. (2008) exposed MCF-7 cells to NO at various concentrations for defined periods of time and observed that various proteins were sensitive to distinct concentrations of NO. At sustained levels of NO between 10 and 30 nM, extracellular signal-regulated kinase (ERK) was phosphorylated through cGMP-dependent mechanism. Akt was found to be phosphorylated at 30–60 nM NO (Pervin et al. 2007, Prueitt et al. 2007; Thomas et al. 2004). At a threshold concentration of 100 nM NO, hypoxia-inducible factor-α (HIF-α) was found to be stabilized, whereas at 400 nM NO, p53 was found to be phosphorylated and acetylated (Thomas et al. 2004). At NO concentrations close to 1 μM, nitrosation of critical proteins such as PARP and caspases was reported (Chong et al. 2005). These studies provide insight regarding specific mechanisms of action of NO and highlight its dichotomous nature under various biological conditions and have suggested that NO responses are dependent on context and levels of NO (Fig. 3.4). It is now well accepted that low doses

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Fig. 3.4 Effect of different concentrations of NO on thymidine uptake in MDA-MB-231 cells. Cells were plated on 96-well plates and allowed to grow overnight. Cells were treated with 3 Hthymidine for 3 h with various concentrations of DETA-NONOate; cells were harvested and 3 H-thymidine incorporation was measured. Data are presented as percentage change compared to control cells. Adapted from Pervin et al. (2008b) with permission from the publisher

of NO (<1–30 nM) can modulate proliferation and protective effects. In a recent study, it has been demonstrated that low nanomolar (2–50 nM) concentrations of NO increased total protein synthesis in MDA-MB-231 and MCF-7 cells, which was associated with increased proliferation (Pervin et al. 2007). The levels of cell cycle proteins involved in G1–S progression suggested that nanomolar concentrations of NO increased the rate of synthesis of cyclin D1 and ornithine decarboxylase (ODC) in these cells. The enzymatic activity of ODC was also found to be increased with nanomolar concentrations of NO. This effect of NO was cGMP independent, indicating that low concentrations of NO can act by both a cGMP-independent and cGMP-dependent manners. NO-induced activation of Ras was followed by activation of the downstream signaling cascade, the MAP kinase pathway. The involvement of Ras in the NO-mediated increase in breast cancer cell proliferation was confirmed by using either FTI or small-interfering RNA targeting Ras, where a significant attenuation of NO induced increase in cell proliferation, cyclin D1, and ODC translation (Pervin et al. 2007). The critical components of the translation machinery like mammalian target of rapamycin (mTOR) and its downstream targets like phosphorylated eukaryotic translation initiation factor-4E (peIF-4E) and p70S6 kinase were upregulated following low nanomolar concentrations of NO (Pervin et al. 2007). Relatively higher levels of NO phosphorylated Akt at serine 472 (Pervin et al. 2003a, b, 2007; Prueitt et al. 2007) which is known to be protective against apoptosis by inducing phosphorylation of Bad and caspase-6 (Chong et al. 2005). Therefore, as a general rule, low relative concentrations of NO tend to favor growth and anti-apoptotic responses, whereas higher levels of NO favor pathways that induce cell cycle arrest, cytostasis, or apoptosis (Pervin et al. 2001a, 2003a, b; Wink et al. 1998; Pervin et al.

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2007; Ridnour et al. 2008). Low steady-state NO levels (∼50 nM) increase matrix metalloproteinase (MMP) activity secreted from macrophages, which are important mediators of angiogenesis and cancer via cGMP-dependent pathways (Ridnour et al. 2007). Studies from animal models where iNOS levels could be regulated have demonstrated that low and high iNOS expressions are associated with tumor progression and regression, respectively (Wang et al. 2003).

NO-Based Cancer Therapy Our current knowledge of the role of NO in carcinogenesis has come to a general consensus that NO plays a paradoxical role in cancer biology. Cells can counterbalance antioxidant mechanisms with pro-oxidant agents during the process of tumorigenesis. The final outcome depends on the experimental model, the surrounding microenvironment, the overall levels of NO, and the activity and localization of NOS isoforms. The central role played by NO in the process of accelerated and sustained production of ROS/RNS, inhibition of DNA repair process, and cell apoptosis to ultimately favor carcinogenesis has prompted the development of therapeutic approaches to target NO production/activity (Hofseth et al. 2003; Hobbs et al. 1999). These approaches include inhibition of NOS enzymatic activity and expression by drugs or RNA interference or using scavengers of NO (Beeharry et al. 2004). Among NO scavengers, cPTIO has been shown to reduce cancer-related vascular permeability (Wu et al. 1998), ebselen inhibitors matrix metalloproteinase in malignant breast cancer cells (Zhang et al. 2002). In colon cancer in vivo models, curcumin has been shown to inhibit cancer development through a mechanism that scavenges NO and inhibits iNOS expression by inhibiting NF-κB activation (Surh et al. 2001). L -Arginine structural analogues have been used in several studies as antiangiogenic agents to selectively inhibit tumor growth and proliferation (Thomsen and Miles 1998; Hofseth et al. 2003; Lu and Schroit 2005). Using murine breast cancer model, it has been shown that L-NAME significantly reduced the total mass of stroma and tumor cells and neo-vascularization compared to those treated with inactive enantiomer D-NAME. This suggests that NO is a key mediator of tumor-induced angiogenesis, and the anti-tumor ability of L-NAME is, at least in part, due to its ability to interfere with the angiogenic pathway (Jadeski and Lala 1999; Jadeski et al. 2000). Using KHM-4, a highly vascularized plasmacytoma cell line, derived from chemotherapy-resistant multiple myeloma, it has been demonstrated that a significant reduction in tumor size and decreased angiogenesis and VEGF expression were observed with L-NAME in SCID mice (Uneda et al. 2003). Selective inhibitors of iNOS resulted in inhibition of NO-mediated prostaglandin E2 (PGE2) production and chemical-induced esophageal carcinogenesis model (Chen et al. 2004). The rate of development of colon carcinoma and adenocarcinoma models (Schleiffer et al. 2000; Kawamori et al. 2000) and adenomatous polyps (Ahn and Ohshima 2001) was significantly decreased with L-NAME and iNOS-specific

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inhibitors. On the other hand, in studies using L-NAME and iNOS knockout models, conflicting results have been reported (Schleiffer et al. 2000; Kawamori et al. 2000; Yamamoto et al. 1998). It has been shown in both normal and malignant cells that administration of NO prior to or together with TNF-α can modulate the anti-neoplastic effects of TNF-α (Mocellin et al. 2005). It has also been shown that co-administration of TNF-α and L-NAME significantly increases the tumor regression rate in sarcoma-bearing animals compared to TNF-α alone (de Wilt et al. 2000). On the other hand, in contrast to the above findings, there is evidence that NO also acts as anti-neoplastic agent, and therapeutic strategies have been developed to deliver different NO donors or use of gene therapy using NOS (Park and Wei 2003; Xie and Huang 2003; Garbán and Bonavida 2001). DETA-NO, an NO donor, has been shown to prevent dendritic cell apoptosis in animal models of immunemediated tumor rejection by inhibiting changes in the expression of pro- and anti-apoptotic proteins and activation of caspase-9 (Perrotta et al. 2004). This effect was mediated via cGMP. An NO mimetic compound YC1 developed for circulatory disorders that inhibits platelet aggregation and vascular contraction has been shown to elicit significant anti-angiogenic and pro-apoptotic effects in preclinical in vitro and in vivo tumor models (Pan et al. 2005; Yeo et al. 2003; Liu et al. 2001). The anti-tumor activity of cytokine IL-13 was decreased by NOS inhibitor L-NMMA, suggesting that host production of NO is required for optimal therapeutic effects (Kawakami et al. 2004). Using gene therapy approach, it was demonstrated that iNOS transfection suppresses tumorigenicity and abrogates metastasis in murine melanoma cells (Xie et al. 1995, 1997). Post-translational modification of proteins by the addition of a farnesyl group has emerged as an important event for the function of a variety of proteins including Ras. During farnesylation, Ras protein undergoes C-terminal modification events that facilitate Ras association with the membrane and cell transformation (Tamanoi et al. 2001). Recently, farnesyl transferase inhibitors (FTIs), a class of anti-neoplastic agents, has been developed that inhibit the function of Ras oncogene by inhibiting its anchorage to the membrane (Selleri et al. 2003). A variety of animal studies have demonstrated the ability of FTIs to block or even regress the growth of tumor cells; however, after some preclinical studies, it was concluded that FTI treatment may require long-time administration with increased side effects and development of resistance, and a clear need for combination therapy using FTIs and other drugs was suggested. Mitochondria play a key role in mediating apoptotic stimulus in situations of non-receptor-stimulated apoptosis (Costantini et al. 2000). Since NO (micromolar concentrations)-mediated changes in the mitochondrial membrane potential were reported to induce cytochrome c release and caspase activation, it was explored whether low nanomolar concentrations of NO would synergize with FTIs to induce apoptosis in breast cancer cells. A combination of small dose of NO released by DETA-NONOate was able to significantly increase the apoptotic activity of FTIs in human breast cancer cell lines within 24 h by increasing cytochrome c release and caspase-9 and caspase-3 activation with minimal toxicity to the normal breast epithelial cell lines (Pervin et al. 2001b). Treatment of prostate carcinoma

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cell lines with another shorter half-life NO donor DETA-NO sensitized malignant cells to TRAIL via inhibition of NF-κB activity and Bcl-XL expression leading to the activation of apoptosis (Huerta-Yepez et al. 2004). Although the dual role of NO in tumor biology has been established, technically we are not yet well equipped to harvest its beneficial effects in a therapeutic setting. The residual low deleterious effects of NO still could be threatening, if not quenched from a therapeutic high concentration delivery to cancer cells. Effective future strategies need to be developed before we can solidify the benefits of NO-based therapy in a cancer microenvironment.

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Weller, R., Billiar, T., and Vodovotz, Y. (2002). Pro- and anti-apoptotic effects of nitric oxide in irradiated keratinocytes: the role of superoxide. Skin Pharmacol. Appl. Skin Physiol. 15(5), 348–352. Wen, J.C., Chaudhuri, G., and Pervin, S. (2006). An inverse interaction between survivin and MAP kinase phosphatase 1 regulates nitric oxide-induced apoptosis. J. Invest Med. 54, S145–S145 (abstract). Wenzel, U., Kuntz, S., De Sousa, U.J., and Daniel, H. (2003). Nitric oxide suppresses apoptosis in human colon cancer cells by scavenging mitochondrial superoxide anions. Int. J. Cancer. 106(5), 666–675. Wilson, K.T., Fu, S., Ramanujam, K.S., and Meltzer, S.J. (1998). Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in Barrett’s esophagus and associated adenocarcinomas. Cancer Res. 58(14), 2929–2934. Wink, D.A., Vodovotz, Y., Laval, J., Laval, F., Dewhirst, M.W., and Mitchell, J.B. (1998). The multifaceted roles of nitric oxide in cancer. Carcinogenesis 19(5), 711–721. Wu, J., Akaike, T., and Maeda, H. (1998). Modulation of enhanced vascular permeability in tumors by a bradykinin antagonist, a cyclooxygenase inhibitor, and a nitric oxide scavenger. Cancer Res. 58(1), 159–165. Xie, K., Huang, S., Dong, Z., Juang, S.H., Gutman, M., Xie, Q.W., Nathan, C., and Fidler, I.J. (1995). Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. J. Exp. Med. 181(4), 1333–1343. Xie, K., Huang, S., Dong, Z., Juang, S.H., Wang, Y., and Fidler, I.J. (1997). Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene. J. Natl. Cancer Inst. 89(6), 421–427. Xie, K. and Huang, S. (2003). Contribution of nitric oxide-mediated apoptosis to cancer metastasis inefficiency. Free Radic. Biol. Med. 34(8), 969–986. Yamamoto, T., Terada, N., Seiyama, A., Nishizawa, Y., Akedo, H., and Kosaka, H. (1998). Increase in experimental pulmonary metastasis in mice by L-arginine under inhibition of nitric oxide production by NG-nitro-L-arginine methyl ester. Int. J. Cancer 75(1), 140–144. Yeo, E.J., Chun, Y.S., Cho, Y.S., Kim, J., Lee, J.C., Kim, M.S., and Park, J.W. (2003) YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1. J. Natl. Cancer Inst. 95(7), 516–525. Zhang, H.J., Zhao, W., Venkataraman, S., Robbins, M.E., Buettner, G.R., Kregel, K.C., and Oberley, L.W. (2002). Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involves reactive oxygen species. J. Biol. Chem. 277(23), 20919–20926.

Chapter 4

Nitric Oxide Expression in Cancer Emanuela Masini, Fabio Cianchi, Rosanna Mastroianni, and Salvatore Cuzzocrea

Abstract Nitric oxide (NO) is an inorganic, colorless gas, with good stability in water. NO is generated by a family of enzymes, termed NO synthases (NOS) and the distribution of the different NOS isoforms is largely related to their respective functions. Vascular endothelial NOS (eNOS) is important for cardiovascular homeostasis, vessel remodeling and angiogenesis; neuronal NOS (nNOS) is expressed in neurons, primarly in the cerebellum and hippocampus and implicated in glutamatergic neurotransmission. Inducible NOS (iNOS) is believed to be of fundamental importance to inflammatory processes. An increased activity of iNOS isoform has been found in several tumors; however, the role of NO in cell proliferation and apoptosis is still not fully elucidated. In fact, the actions of NO on cancer are dichotomous in that effects consistent with cancer promotion and prevention or reversion have been reported. Moreover, iNOS and COX-2 have been found to be co-expressed within the same tumor cells and involved in the regulation of tumor growth. In conclusion, iNOS and COX-2 products may represent a common final pathway controlling different tumorigenic mechanism. Keywords L-arginine-NO pathway · eNOS · nNOS · iNOS · Cell proliferation · Apoptosis · NO-NSAIDs · NOS-COX-2 interaction

Biosynthesis of Nitric Oxide Nitric oxide (NO) is an inorganic, colorless gas with good solubility in water. The half-life of NO in water is considerably longer, about 3 s, than would be expected for other free radicals. This is, in part, due to the reluctance of NO to dimerism and to the third-order kinetic of its reaction with oxygen. However, NO reacts rapidly E. Masini (B) Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy e-mail: [email protected]

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with superoxide anions to form peroxynitrite, a relatively stable product (Beckman et al. 1990). In the gas phase, NO reacts with oxygen to form nitrogen dioxide (•NO2 ); under aqueous, aerobic conditions, NO spontaneously oxidizes to its inactive, stable products nitrite and nitrate, at a half-life of 6–10 s. This property explains the previously puzzling observation that mammals excrete more nitrate than they ingest, especially when inflammation is present (Wagner et al. 1983). In vivo, nitrite reacts with various biologically active species (e.g., oxyhemoglobin) and the stable end-product that can be measured in plasma is nitrate (Butler and Williams 1993; Feldman et al. 1993; Ignarro et al. 1993). Nitric oxide can react with thiols to form S-nitrosothiols, such as S-nitrosocysteine and S-nitrosoglutathione. Some Snitrosothiols and protein thiols may have significant stability (Feldman et al. 1993; Stamler et al. 1992; Nathan 1992) and it is possible that NO circulates in plasma as an S-nitroso adduct of serum albumin (Stamler et al. 1992). Nitric oxide has a high affinity for both heme and non-heme iron atoms present in the prosthetic groups of proteins (Lancaster and Hibbs 1990), and it can form complexes with hemoglobin, myoglobin, cytochrome c, and guanylyl cyclase. Furthermore, NO can interact with iron–sulfur centers in various enzymes, such as aconitase and complexes I and II of the mitochondrial respiratory chain, altering their biological activity (Feldman et al. 1993; Nathan 1992). Ribonucleotide reductase is another iron-containing enzyme inhibited by NO (Lepoivre et al. 1990). By blocking this enzyme, NO impairs DNA synthesis and cell division; it damages DNA through nitrosylative and deaminative reactions causing strand breaks (Wink et al. 1991). Peroxynitrite, but not NO, nitrosylates tyrosine residues of iron, manganese, and copper–zinc superoxide dismutases, as well as other copper-containing proteins (Ischiropoulos et al. 1992). The role of NO in normal physiologic homeostasis became apparent when two groups of researchers identified it as the “endothelium-derived relaxing factor” (EDRF), which helps to regulate blood pressure (Ignarro et al. 1987; Palmer et al. 1987). After the discovery that a variety of cell types synthesize the free radical NO, research focusing on this simple diatomic molecule has led to a formidable number of publications, determining that NO• plays significant roles in most fields of life sciences (Nathan and Xie 1994). However, a number of questions regarding NO• biology still remain unanswered, the most challenging and confusing problem being set by the ambivalent character of NO. While being a critical signaling messenger involved in the regulation of a wide range of physiologic processes, NO• also has the ability to turn into a major cytotoxic effector molecule that is involved in a number of pathophysiologic conditions and in the pathogenesis of a growing list of human diseases (Walia et al. 2003). The double fate of NO• is particularly troublesome when one considers manipulating NO• availability as a potential therapeutic option in different pathologic conditions. Reducing or increasing NO• availability in a given circumstance may inevitably be associated with both beneficial effects and deleterious consequences. In addition, further adding to an already complex situation, some theoretical misconceptions have also contributed to the confusion surrounding the perplexing biological functions of NO•. For instance, the proper effects of NO have often, and abusively, been assimilated to those of a family of NO-derived molecules,

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collectively termed reactive nitrogen species (RNS), which all possess their unique biochemical characteristics. Another frequent misconception is that NO, as a free radical, is a highly reactive molecule, with a very short lifetime. Although the free radical nature of NO constitutes the chemical basis of its biological activity, its reactivity is relatively weak and basically NO interacts only with transition metals, oxygen, and other free radicals (Beckman and Koppenol 1996). This low reactivity, combined with a high lipophilicity, confers to NO the potential to diffuse away from the point of origin, and thereby to carry out its function as a messenger molecule. The direct effects of NO prevail in conditions of low and brief NO production and mainly support protective and signaling functions, which are consistent with the chemical biology of NO encountered under normal, physiologic conditions (Wink and Mitchell 1998). In contrast, indirect effects occur under high and sustained flux of NO, as noted under pathophysiologic circumstances, and will essentially result in toxic consequences, which include oxidation, nitrosation (adjunction of NO+ ), and nitration (adjunction of NO2 + ) reactions (Wink and Mitchell 1998; Grisham et al. 1999). It appears, then, that the type of NO chemistry prevailing at a particular moment in time is the key feature that determines its biological actions. Nitric oxide is generated by a family of enzymes termed nitric oxide synthase (NOS; EC 1.14.23) via a five-electron oxidation of the terminal guanidinium nitrogen of the amino acid L-arginine. The reaction is both oxygen- and NADPHdependent and yields L-citrulline in addition to NO, in a 1:1 stoichiometry: molecular oxygen serves as electron acceptor (Kwon et al. 1990). The enzyme is stereospecific for the L-isomer of arginine since D-arginine is not a substrate. The distribution of different isoforms of NOS is largely related to their respective functions. Although the L-arginine–NO pathway has been identified in many species including fish, birds, and bacteria, NOS has been best studied in mammals (Knowles and Moncada 1994; Nathan 1992). Based upon several criteria including cellular localization, regulation of activity, and substrate/inhibitor profiles, three isoforms of NOS enzymes have been described and subsequently cloned. Molecular cloning has shown these to share 50–60% homology. First, a constitutive form, whose activity is regulated by Ca2+ and calmodulin was found in neuronal tissue, both centrally and peripherally, neuronal NOS (nNOS, or type I). Neuronal NOS is also present in the epithelium of rat trachea and human bronchi (Kobzik et al. 1993) and in human skeletal muscle (Nakane et al. 1993). A second Ca2+ /calmodulin requiring constitutive enzyme is present in vascular endothelial cells, endothelial NOS (eNOS), or type III, and an inducible Ca2+ /independent isoform, inducible NOS (iNOS), or type II which can be isolated from macrophages and from a number of cell types following induction with inflammatory mediators and bacterial products. Depending on the source, these enzymes are active as monomers or homodimers with monomeric molecular weights of 125–155 kDa; only the dimeric forms exhibit catalytic activity. All forms of NOS characterized thus far are flavoproteins with the rare property of containing both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); the reduced forms of nicotinamide adenine dinucleotide phosphate (NADPH+ ) and

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tetrahydrobiopterin (BH4 ) are required as cofactors. The C-terminal half of the NOS protein bears remarkable resemblance to only one other mammalian protein, cytochrome P450 reductase (CPR) and appears to possess the same cofactor-binding sites, this is often referred to as the reductase domain. Nitric oxide synthase, unlike CPR, is a self-sufficient enzyme; the oxygenation of substrate, L-arginine, occurs at the heme site in the N-terminal portion (oxygenase domain) of the protein. Stoichiometric amounts of heme are present in NOS and are required for the catalytic activity (Stuehr and Ikeda-Saito1992; White and Marletta 1992). Heme coordination is thought to be provided by Cys-415 (nNOS) based on homology to cytochrome P450 and heme incorporation following site-directed mutagenesis. Indeed, this Cys residue is conserved in all NOS isoforms across differing species and corresponds to Cys-200 in human iNOS and Cys-184 in human eNOS. Close to the heme, the catalytic site is the binding-site for the substrate, L -arginine. All forms of NOS contain four prosthetic groups: flavin adenine dinucleotide; flavin mononucleotide; tetrahydrobiopterin (BH4 ); and a heme complex, iron protoporphyrin IX. Zinc has been recently identified as an additional prosthetic group of NOS. The mechanism of NOS-catalyzed oxidation of L-arginine to NO• proceeds in at least two distinct steps. The initial reaction involves N-hydroxylation of the guanidinium nitrogen to form N-hydroxy-L-arginine. The reaction utilizes one equivalent of NADPH+ and O2 to conduct a simple two-electron oxidation of nitrogen. This reaction mimics classical P450-like hydroxylation (Cho et al. 1972). The subsequent step in the conversion of N-hydroxy-L-arginine to NO• and Lcitrulline is unclear. Recent studies have shown that nitroxyl (HNO) and not nitric oxide is the preferred nitrogen oxide product (Fukuto et al. 1993). If NO were to be the product, nitric oxide synthase has to facilitate an odd-electron oxidation; but such reaction is difficult to reconcile with P450 chemistry (2e− transfer). In this way, HNO would be the expected two-electron oxidation product from N-hydroxyl guanidines. Therefore, NOS generates HNO from L-arginine, in a four-electron process, consistent with the enzymology of cytochrome P450, and a subsequent one-electron oxidation of this product yields nitric oxide as proposed by Hobbs and co-workers in 1994. All of the NOS isoforms can be inhibited to a variable degree with NG -substituted L-arginine analogs, e.g., NG -monomethyl-L-arginine (L-NMMA) (Hobbs et al. 1999).

Endothelial Cell NOS (eNOS) Vascular endothelial NOS (eNOS) is a calcium–calmodulin-dependent enzyme, with a monomeric molecular weight of 133 kDa that is bound to the cell membrane with a myristoylate bridge linked to the N-terminal glycine of the enzyme (Nathan 1992; Sessa et al. 1992). It is now well appreciated that eNOS is important for cardiovascular homeostasis, vessel remodeling, and angiogenesis (Nadaud et al. 2000; Chiou 2001).

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In contrast to nNOS and iNOS, which are cytosolic, eNOS is particulate and not cytosolic – posttranslational modification at these sites is necessary for its membrane association to occur preferentially at the caveoli (Fulton et al. 2001). Such a localization favors high local concentrations of NO• in the vicinity of circulating blood cells and vascular smooth muscle. Little is known about the turnover and the regulation of eNOS, although it has been established that tumor necrosis factor alpha (TNF-α) down-regulates eNOS by enhancing the degradation of its messenger ribonucleic acid (mRNA) (Yoshizumi et al. 1993). Agents that increase intracellular calcium by activating influx of extracellular calcium or releasing calcium from intracellular stores cause endothelium- and NO-dependent relaxation in many blood vessels in vitro and in vivo. Important from a physiologic point of view is the finding that pulsatile pressure, visible light, and electrical field stimulation also release this labile mediator with a half-life counted in seconds. Veins seem to produce less NO• than do arteries, both basally and upon stimulation. Furthermore, NO• derived from eNOS has numerous effects on the vessel wall, including vasodilation (Rees et al. 1989; Vallance et al. 1989), inhibition of platelet aggregation (Sneddon and Vane 1988), inhibition of the production of monocyte chemoattractant protein-1 and macrophage-colony stimulating factor (De Caterina et al. 1995), changes in vascular permeability (Kurose et al. 1993), inhibition of smooth muscle cell proliferation (Garg and Hassid 1989), and inhibition of leukocyte–endothelium interaction (Kubes et al. 1991; Kurose et al.1993). Nitric oxide is therefore cytoprotective and beneficial in various experimental models of vascular pathology. Inhibition of the basal release of NO• by NOS inhibitors results in a rapid, prolonged, and L-arginine-reversible increase in blood pressure (Rees et al. 1989). Administration of NOS inhibitors produces a reduction in blood flow to most organs, including the brain, heart, lung, and kidney. Nitric oxide synthase inhibitors reduce the hypotensive effect of the “endothelium-dependent vasodilator substances” (Aisaka et al. 1989). Moreover, eNOS knock-out mice are hypertensive (Whittle et al. 1989) and show impaired wound healing and angiogenesis (Lee et al. 1999). Acute release of NO from the endothelium may be involved in some forms of reactive hyperemia, such as postischemic or exercise-induced hyperemic response (Gilligan et al. 1994). In this scenario, eNOS is responsible for momentto-moment changes in vascular tone that form the basis for responses to ischemia and to exercise. Vasodilation is an important component of angiogenesis. Endothelial NOS contributes to the pro-angiogenic program of capillary endothelium by triggering cell growth and proliferation throughout a cyclic GMP-dependent protein kinase (PKG) and mitogen-activated protein kinase (MAPK) cascade (Hood and Granger 1998) leading to membrane-linked signals to the nuclear level. Moreover, the eNOS pathway controls the balance between metalloproteinases (MMP-2 and MMP-9) and their inhibitors (Donnini et al. 2004). Additional stimuli, such as vascular endothelial growth factor (VEGF), estrogen, sphingosine 1-phosphate (S1P), and bradykinin, can bind to their receptors, stimulate PI3K/Akt, and lead to eNOS activation via phosphorylation of serine residues (Sessa 2004).

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Two autoinhibitory control elements (ACE-I and ACE-II) impede eNOS activation and influence the calcium/CaM sensitivity of the enzyme (Lane and Gross 2002). Endothelial NOS can interact with various proteins in its “less active” and “more active” states. N-Myristoylated and palmitoylated membrane-bound eNOS associates with the caveolae coat protein caveolin-1 (Cav-1) and with heat shock protein 90 (Hsp90). The C-terminal Hsp70-interacting protein interacts with Hsp90 and Hsp70 and negatively regulates eNOS trafficking into the Golgi complex. By contrast, NOS-interacting protein and NOS-traffic inducer can negatively regulate eNOS in the plasma membrane (Zabel et al. 2002). Once NO is produced by the endothelium, it can regulate several aspects of vascular function via the activation of the primary NO receptor, sGC, or initiate nitrosylation of iron–sulphur-centered proteins or proteins with reactive thiols (S-nitrosylation) (Sessa 2004). In the vascular system, NO-dependent relaxation of vascular smooth muscle is sGC and protein kinase G (PKG) dependent, whereas the anti-proliferative action and ion channel modulation is PKG or via nitrosation reaction (Miranda et al. 2003). Nitrosylation of caspase-3 and caspase-8 inactivates the protein, thus leading to inhibition of apoptosis (Stamler et al. 2001). Chronic inhibition of eNOS causes renal morphologic changes such as glomerular damage (Baylis et al. 1992). Endothelial NOS is also expressed in cardiac myocytes (Feron et al. 1996), where NO• has a paracrine action on cardiac contractility and oxygen consumption (Loke et al. 1999).

Neuronal NOS (nNOS) There is no doubt that nitric oxide from the neuronal enzyme plays a critical role as a neurotransmitter and neuromodulator in the nervous system. However, unlike classical neurotransmitters, NO is not stored in synaptic vesicles, nor is it released by exocytosis, nor is its activity terminated by re-uptake or enzymatic degradation – rather it is synthesized on demand, diffuses from nerve terminals, and its actions terminated post-inactivation with a substrate. The NOS isoform present in the central and peripheral nervous system [nNOS (NOS I)] is a Ca2+ –calmodulin-dependent enzyme with a molecular weight of 166 kDa that is present in dimeric form (Calver et al. 1993). The primary structure of neuronal NOS, revealed by molecular cloning, indicates that the protein has an α-helical, calmodulin-binding consensus sequence and a cAMP-dependent protein kinase phosphorylation sequence (referred to nNOSα). Four splice variants have been identified to date – nNOSβ, nNOSγ, nNOSμ, and nNOS-2 (Nakane et al. 1993; Gibson et al. 2001). They each appear to have distinct cellular and tissue locations. All nNOS-positive neurons have the α-nicotinamide adenine dinucleotide phosphate (NADPH+ ) diaphorase activity which has been used as a histochemical marker of nitrergic neurons. Although the main NOS found in the nervous system is nNOS, eNOS and iNOS have been found. Neuronal NOS is expressed in neurons in many parts of the brain, primarily in the cerebellum and hippocampus (Bredt and Snyder 1990); in other areas such as

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the cerebral cortex or the stratum, NOS I-positive neurons comprise about 1–2% of the total neuronal population. The modulation of various physiological functions by NO has been proposed; among them, pain perception, sleep, feeding behavior or thermoregulation, and, in particular, regulation of microcirculation (Szabò 1996). Neuronal NOS is targeted to proteins of the postsynaptic density in close association with N-methyl D-aspartate (NMDA) receptor (Garthwaite and Boulton 1995). Calcium-dependent stimulation of NOS I activity has been implicated in excitatory glutamatergic neurotransmission, long-term potentiation, and long-term depression (Garthwait and Garthwait 1991; Iadecola et al. 1997). Thus, NOS I may be involved in phenomena based on synaptic plasticity such as learning and memory formation, tasks which are supported, and in NOS I deficient neurons may be taken over, by NOS III. In turn, NO has been implicated in excitotoxicity after excessive stimulation of neurons by glutamate, as occurs in stroke, although the underlying mechanisms of neuronal cell death as well as of the resistance of NOS I-containing neurons to NO• are still a matter of debate. Nevertheless, excessive generation of neuronal NO• appears to be responsible for ischemia reperfusion and traumatic injury of the brain which can only in part be overcome by the beneficial effect of endothelial cell-derived NO on blood flow. Evidence is increasing that neurons are able to express iNOS in response to proper stimulation and that endogenously produced NO leads to cell death by necrotic or apoptotic mechanisms (Heneka et al. 1998). Transcriptional activation of the iNOS gene in astrocytes occurs upon incubation with several pro-inflammatory cytokines, either alone or in various combinations (Simmons and Murphy 1994). Other activators include HIV coat protein (Hori et al. 1999) and β-amyloid peptide (Rossi and Bianchini 1996) or the increase of intraocular pressure (Liu and Neufeld 2000). In excess, NO• is toxic to neurons: this toxicity is mediated largely by an interaction with the superoxide anion, presumably through the generation of the oxidant, peroxynitrite. The toxic effect of NO• may occur through a variety of mechanisms, including inhibition of the mitochondrial electron transport chain, inhibition of ribonucleotide reductase, inhibition of cis-aconitase, and enhanced adenosine diphosphate (ADP) ribosylation of glutaryl-aldehyde-phosphate-dehydrogenase (GADPH). Nitric oxide- or peroxynitrite-mediated neuronal injury involves damages to DNA with the subsequent activation of the nuclear protein, poly(ADPribose)synthetase (PARS) (Zhang et al. 1994). Evidence is also growing that nNOS is also present in skeletal muscle where it is involved in the regulation of metabolism and muscle contractility.

Inducible NOS (iNOS) In comparison to constitutive NOS isoenzymes, it is inducible NOS (iNOS) which is thought to mediate the vast majority of pathophysiological effects attributed to NO• and, consequently, this isoform is believed to be of fundamental importance to inflammatory processes.

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Under physiologic conditions, unlike eNOS and nNOS, iNOS is not expressed constitutively in mammalian cells, but rather is induced by pro-inflammatory stimuli, such as bacterial lipopolysaccharide (LPS), or the cytokines TNF-α, IL1β, or interferon-γ (IFN-y), individually, or in combination. Inducible NOS possesses tightly bound calmodulin in a non-covalent manner and is calcium independent. Therefore, once expressed, iNOS continues to synthesize NO• in large amounts for a prolonged period of time. Inducible NOS activity is regulated by protein expression rather than functional modulation (Lee et al. 2003). Inducible NOS is active as a homodimer of approximately 260 kDa and only the dimeric forms exhibit catalytic activity. The expression of iNOS is regulated both at the level of transcription and at the level of iNOS mRNA stability. The mechanism of iNOS induction involves de novo transcription and biosynthesis of new protein. The 5 -flanking region of murine and human iNOS shares a 66% homology and contains conserved consensus sequences for nuclear factor kappa-B (NF-κB) as well as both INF-γ-/TNF-responsive elements (Chartrain et al. 1994). Despite their tight similarities, the transcriptional control of iNOS in murine and humans differs in that in the former but not in the latter, a 1.6-kb 5 -flanking region has the necessary promoter sequences to induce full gene expression. These differences might help shed light as to why it has been so difficult in inducing iNOS in human cells (Weinberg et al. 1995). Agents known to interfere with NF-κB activity seem to modulate the induction of iNOS. Many antioxidants including pyrrolidine-dithiocarbamate and diethydithicarbamate inhibit iNOS expression in cultured cells (Sherman et al. 1993; Mulsch et al. 1993) in addition to nonselective protease inhibitors. Several other distinct classes of agents have been demonstrated to prevent expression of iNOS via inhibition of the NF-κB transduction system. Glucocorticoids such as dexamethasone interfere with iNOS expression in many cell types (Di Rosa et al. 1990); moreover, thrombin, macrophage deactivation factor, tumor growth factor β (TGFβ), platelet-derived growth factor (PDGF), IL-4, IL-8, and IL-10 inhibit iNOS induction. The same immunological stimuli that induce iNOS also induce guanosine triphosphate (GTP) cyclohydrolase, an enzyme that produces BH4 and thus supplies iNOS with its cofactor (Salvemini and Masferrer 1996). Induction of iNOS is associated with the induction of arginosuccinate synthetase, which may supply iNOS with its substrate from intracellular sources by turning on the “re-cycling” of L-arginine from L-citrulline (Salvemini et al. 1995). In some cell types, the same stimuli that induce iNOS also upregulate the membrane transport system for L-arginine, thereby supplementing intracellular L-arginine from extracellular sources. Some NOS inhibitors (e.g., NG -monomethyl-L-arginine) also inhibit the Y+ cationic transporter system responsible for L-arginine and other cationic amino acid transport into the cells, whereas others (e.g., nitro-L-arginine) do not affect it (Bogle et al. 1992). Induction of iNOS may have either toxic or protective effects. Factors that appear to dictate the consequences of iNOS expression include the type of insult, the tissue type, the level and duration of NOS expression, and probably the redox status of the tissue. Much attention has focused on the toxic effects of iNOS. For example, induction of iNOS in endothelial cells produces endothelial injury; moreover,

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induction of iNOS has been shown to inhibit cellular respiration in macrophages and vascular smooth muscle cells; these processes can lead to cell dysfunction and cell death. Such processes, when occurring within vascular smooth muscle cells, play a key role in the same cell where the activation of iNOS, in turn, can act as NO• donors, activating guanylyl cyclase (Moore et al. 1994; Connor et al. 1995). Such a mechanism has been proposed in relation to bradykinin-induced cell signaling in pulmonary blood vessels (Zingarelli et al. 1997). The generation of large amount of NO• following iNOS induction in activated macrophages accounts for the antimicrobial effects of these cells. In various pathophysiological conditions iNOS not only is expressed in macrophages but can also be induced in various other cells, including fibroblasts, Kupffer’s cells, hepatocytes, neutrophils, mesangial cells, chondrocytes, pancreatic islet cells, microglia, epithelial and endothelial cells, smooth muscle cells, cardiac myocytes, and megakaryocytes. Since NO• is a radical, it reacts with other radicals; this is exemplified by the interaction of NO• with superoxide yielding peroxynitrite (ONOO− ). Indeed, this reaction proceeds three times more rapidly than the disproportion of superoxide by superoxide dismutase (SOD). The combination of these two radicals yielding peroxynitrite has received considerable attention over the past few years since it has been suggested that peroxynitrite may represent an important mediator of cytotoxicity and cytostasis (Radi et al. 1991). Currently, little information is available regarding the “physiological” roles of peroxynitrite, while the evidence for its roles in pathophysiological conditions is expanding. Although there are a number of experimental difficulties related to delineation of the actual role of peroxynitrite in many pathophysiological conditions, theoretical considerations strongly favor the production of peroxynitrite when NO and superoxide are produced simultaneously, because the reaction of these two species is nearly diffusion-controlled. In fact, the reaction of superoxide with NO is the only reaction that outcompetes the reaction of superoxide with SOD (Corbett et al. 1993). The finding that peroxynitrite is produced during inflammation and shock is not surprising, in light of the previous evidence for the overproduction of oxygen-derived free radicals. Nitrotyrosine formation, and its detection by immunostaining, was initially proposed as a relatively specific means for detecting the “footprint” of peroxynitrite. Recent evidence, however, indicates that certain other reactions can also induce tyrosine nitration; for example, the reaction of nitrite with hypochlorous acid and also the reaction of myeloperoxidase, and other peroxidases, with hydrogen peroxide can lead to the formation of nitrotyrosine (Tsai et al. 1994; Egan et al. 1976). The physiopathological relevance of this reaction remains to be further elucidated. More recent reviews take an increased nitrotyrosine staining as an indication of “increased nitrosative stress” rather than a specific marker of peroxynitrite (Jijon et al. 2000). The formation of nitrotyrosine has recently been demonstrated in inflammation, ischemia-reperfusion, and shock; the staining was accomplished by treatment of the animals with iNOS inhibitors and peroxynitrite scavengers (Cuzzocrea et al. 1998, 2000, 2001). Several evidences strongly suggest that peroxynitrite is produced in shock and inflammation. Specific peroxynitrite scavengers that could help to further elucidate the role of peroxynitrite in pathological situations are not available.

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Glutathione, melatonin, and uric acid, a putative scavenger of peroxynitrite, are sometimes used as a probe for peroxynitrite (Inoue et al. 1993; Landino et al. 1996; Upmacis et al. 1999); however, uric acid can interfere with a number of other oxidants and caution should therefore be applied in the interpretation of these results (Notoya et al. 2000). Therefore, the evidence implicating the role of peroxynitrite in a given pathophysiological condition can only be indirect. A simultaneous protective effect of superoxide-neutralizing strategies and NO synthesis inhibition, coupled with the demonstration of peroxynitrite in the particular pathophysiological condition, can be taken as a strong indication for the role of peroxynitrite. However, it is likely that additional interactions of oxygen- and nitrogen-derived free radicals also contribute to cell injury.

Nitric Oxide, Tumor Cell Proliferation, and Apoptosis The role of NO in cell proliferation and apoptosis is still not fully elucidated. In general, reported effects of NO on cancer are dichotomous, in that effects consistent with cancer promotion and cancer prevention or reversion have been reported. Inducible NOS is up-regulated in several tumors. An increase in iNOS activity has been found in head and neck cancer, associated with elevated cGMP levels (Gallo et al. 1998). The expression of iNOS was significantly correlated with lymph node metastases and the degree of tumor angiogenesis evaluated as microvessel density. The same authors have reported data about the possibility of a close regulation of iNOS activity by the tumor suppressor gene p53. They found that their tumor samples expressing a mutated p53 protein released an increased level on nitrite/nitrate, suggesting a key role of p53 mutation in the up-regulation of iNOS (Gallo et al. 2003). This hypothesis has been confirmed by in vitro studies where the restoration of wild-type p53 phenotype in A431 cancer cell line results in down-regulation of iNOS mRNA and protein expression. Moreover, a correlation between metalloproteinase (MMP) overexpression, activity of iNOS pathway, p53 status, and angiogenesis in patients with head and neck squamous cell carcinoma (HNSCC) has been shown (Franchi et al. 2002). MMPs are a family of proteolytic enzymes involved in the degradation of extracellular matrix components that play a relevant role in several steps of tumor progression, including invasion, angiogenesis, and metastasis. The involvement of the p53 tumor suppressor gene in the regulation of iNOS, MMP expression, and angiogenesis raises the possibility that the p53 mutation, which is frequently present in patients with HNSCC, may result in increased angiogenesis and invasiveness related to increased NO and MMP production by tumor cells, contributing to tumor progression. These results indicate that iNOS and MMP are p53 target genes subjected to p53 repression and might have important implications for the potential use of p53 gene therapy in HNSCC. On the other hand, recent results demonstrate that conditions favoring low NO levels conferred resistance against cisplatin/taxol-induced apoptosis in HNSCC cell lines (Fetz et al. 2009). In fact, low doses of the NO

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donors S-nitroso N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) enhance the expression of survivin, an antiapoptotic protein, and this phenomenon is correlated with chemoresistance against taxol- and cisplatin-induced cell death. Moreover, the depletion of survivin with RNA interference neutralized the cytoprotective effect of NO and sensitized HNSCC cells against drug-induced apoptosis. Nitric oxide induction of survivin expression is mediated by phosphorylation of PI3K/Akt, although NO signaling may activate additional antiapoptotic pathways involving Bcl-2 or p53 (Salvucci et al. 2001) and/or act by direct inactivation of caspases by S-nitrosylation (Fukumura et al. 2006). Currently, survivin is vigorously pursued as a cancer drug target by multiple strategies (Altieri 2008a). Supported by a favorable profile, the first survivin antisense oligonucleotide is undergoing phase I/II trials in patients with advanced cancers (Altieri 2008b). Observations indicate that iNOS expression is increased in fresh tumor tissue from gynecologic and brain neoplasms (Thomsen et al. 1998; Cobbs et al. 1995), as well as in colorectal, esophageal, and gastric cancer (Cianchi et al. 2005). The role of iNOS in ovarian tumors and tumor-associated macrophages was investigated by Klimp et al. (2001). An overexpression of both iNOS and COX-2 was found not only in adenocarcinomas but also in borderline and benign ovarian tumors, such as cystadenomas, indicating the involvement of the two enzymes in ovarian tumorigenesis. On the contrary, only few samples of tumor-associated macrophages were found to express iNOS and were in an activated state against tumor cell proliferation. A possible explanation of this feature could be that ovarian tumors can release mediators that can suppress iNOS expression in tumor-associated macrophages and thus their tumoricidal activity. Immunohistochemical positivity for iNOS was demonstrated in 78 stage III patients of ovarian cancer associated with a poor prognosis after surgical and chemotherapeutic treatments. The same study showed that both iNOS and COX-2 negative ovarian carcinomas were correlated with a complete clinical response to the first-line chemotherapy (Raspollini et al. 2004). Angiogenesis plays a key role in the development of brain cancer, such as astrocytic gliomas. The expression of COX-2, iNOS, and vascular endothelial growth factor (VEGF) was examined in 51 high-grade astrocytomas including 31 glioblastomas (grade IV) and 20 anaplastic astrocytomas (grade III), 49 lowgrade astrocytomas (grade II), and 43 reactive astrogliosis specimens (Hara and Okayasu 2004). A stepwise increase of COX-2, iNOS, and VEGF expression was found from astrogliosis through low-grade to high-grade astrocytoma, and COX-2 expression was significantly correlated with iNOS, VEGF, and microvessel density, whereas iNOS expression was weakly correlated with the degree of angiogenesis. The authors concluded that iNOS/COX-2 interaction could contribute to astrocytic tumorigenesis by promoting new vessel formation. Nitric oxide synthase activity is up-regulated in melanoma cell lines, and NO release was considered to encourage metastasis and tumor growth by maintaining the vasodilator tone in vessels around the melanoma (Joshi et al. 1996). Murine melanoma cells can be induced to express iNOS with a combination of cytokines and did not metastasize when the cells were injected into syngenic mice, whereas

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metastasizing melanocytes cannot be induced to express iNOS (Dong et al. 1994). Transfection of a malignant melanoma cell line with an active iNOS gene reduced metastasis after injection into syngenic mice compared with cells transfected with an inactive iNOS gene (Xie et al. 1995) and transfected cells underwent apoptosis. It has been also observed that NO-exposed melanocytes become less adherent to extracellular matrix components and this action might represent a mechanism by which NO exposure reduces tumorigenicity. In non-small cell lung cancer iNOS expression and activity significantly correlate with VEGF expression and microvessel density suggesting that stimulation of angiogenesis is one of the most important mechanisms involved in iNOS-mediated carcinogenesis (Marrogi et al. 2000). In Barrett’s metaplasia and in esophageal adenocarcinoma an increase in mRNA expression of iNOS was found in 76% of tissues and it was significantly correlated with the expression of transforming growth factor-α (TGF-α). Up-regulation of both iNOS mRNA and protein levels was also found in esophageal adenocarcinoma arising in Barrett’s mucosa when compared with normal adjacent esophagus. These findings clearly indicate that up-regulation of iNOS is involved in Barrett’s associated neoplastic progression. The possible molecular mechanisms underlying the involvement of iNOS in Barrett’s metaplasia–dysplasia–carcinoma sequence is that iNOS switches to an antiapoptotic phenotype because of increased Bcl-xl expression and decreased Bax expression (Van der Woude et al. 2002); however, at the moment no clear correlation can be established between iNOS expression and activation of proapoptotic and antiapoptotic genes. The pathogenesis of gastric lymphomas from mucosa-associated lymphoid tissue (MALT) has been demonstrated to be linked to chronic infection with Helicobacter pylori (H. pylori). It has been demonstrated that not only COX-2 but also iNOS is potentially involved in H. pylori-induced gastric mucosa alterations and development of this type of lymphoma; in fact a high positive immunostaining rate for iNOS and COX-2 has been demonstrated (Fu et al. 1999; Li et al. 2004). In 32 gastric MALT lymphomas, iNOS expression was significantly correlated with COX-2 expression, tumor cell proliferation, assessed by Ki-67 labeling index, and p53 accumulation state, indicating a role for iNOS and COX-2 in the evolution of H. pylori-associated gastritis to gastric MALT lymphoma (Li et al. 2004). In regard to gastric adenocarcinoma, Son et al. (2001) investigated iNOS gene up-regulation in 23 tumor samples obtained from patients who underwent gastrectomy. They found iNOS mRNA significantly higher in gastric cancer tissues than in adjacent normal gastric mucosa while no association was found among iNOS mRNA level, inflammation, and H. pylori infection. Another study demonstrated a correlation between iNOS expression and tumor stage. In the same tumors, the immunohistochemical accumulation of p53, which is an indicator for a loss of p53 tumor suppressor function, was found to correlate with iNOS, suggesting that tumor-associated production of NO may provide a selective growth advantage to tumor cells with mutant p53 (Rajnakova et al. 2001).

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The expression of iNOS was evaluated in diffuse and intestinal adenocarcinoma tumor according to Lauren’s gastric cancer classification. Gastric carcinomas of the diffuse type are associated with a poor prognosis compared with tumors of the intestinal type; high iNOS expression was found but no difference existed between the two types of tumors. Moreover the expression of Fas, Bcl-xl, Bcl-2, Bax, active caspase-3, and Ki-67 was investigated but no significant correlation was found between these apoptosis-related proteins and iNOS expression (Van der Woude et al. 2002). It is well known that inflammation is a significant factor in the development of pancreatic cancer, and both hereditary and sporadic forms of chronic pancreatitis may be associated with increased cancer risk (Whitcomb 2004). Inducible NOS has been demonstrated to be overexpressed in chronic pancreatitis and in pancreatic cancer and a significant correlation with COX-2 expression was observed, providing evidence of a link between inflammation and tumor development. Other authors have shown a co-expression of the two inducible enzymes in pancreatic adenocarcinoma (Kong et al. 2002), but the activities of the two enzymes have been found to counteract each other: COX-2 overexpression positively correlated with high Ki-67 expression and tumor proliferation, and high iNOS expression was significantly associated with a high apoptotic index. Although the involvement of prostaglandins and nitric oxide in the development of pancreatic cancer is not completely defined, COX-2 up-regulation in pancreatic cancer might be an antagonistic pathway of an iNOS-induced apoptotic system and NO-related apoptosis might be a result of various tumorigenic effects of NO, such as DNA damage, p53 mutation, and poly(ADP-ribose) polymerase-1 (PARP-1) activation. The role of iNOS enzyme in colorectal tumor development and the molecular carcinogenetic mechanisms have been extensively investigated (Rao et al. 2002; Rao 2004). The effects of SC-51, a selective inhibitor of iNOS, administered alone or in combination with the COX-2 selective inhibitor, celecoxib, were investigated against azoxymethane (AOM)-induced formation of aberrant crypt foci (ACF) in rat colonic mucosa. Both iNOS and COX-2 activities were selectively inhibited by SC51, but the administration of both drugs together was more effective in inhibiting AOM-induced ACF formation than was administration of these drugs individually. These data suggest that suppression of iNOS activity may lead to a downregulation of COX-2 activity in colonic mucosa. Overexpression of iNOS has also been correlated with advanced stages of disease in humans; however, few data exist regarding the possible correlation between NO and prostaglandins in promoting colorectal cancer progression. Our group has recently demonstrated that iNOS and COX-2 are co-expressed within the same cancer cells and iNOS activity is significantly correlated with PGE2 production (Cianchi et al. 2004). In vitro data on the stimulatory effect of both endogenous and exogenous NO on COX-2 activity in HCT116 and HT29 colon cancer cell lines confirmed our hypothesis. A co-induction of iNOS and COX-2 activities in response to epidermal growth factor (EGF) or Escherichia coli lipopolysaccharide (LPS) treatment was demonstrated in iNOS-positive/COX-2 negative HCT 116

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cells. The selective inhibition of iNOS activity by 1400 W significantly reduced both LPS- and EGF-induced PGE2 production. The stimulated production of PGE2 was also inhibited by celecoxib. These findings suggest that COX-2 is the main source of the endogenous NO-stimulated increase in PGE2 production after LPS or EGF treatment. Moreover, the administration of sodium nitroprusside, a NO donor, to iNOS-negative/COX-2 positive HT29 cells determined an increase in PGE2 production. In conclusion, NO seems to be correlated with increased COX-2 activity in colon cancer cells. Prostaglandin production significantly correlated with microvessel density and VEGF expression, whereas the products of iNOS pathway did not correlate with any angiogenic markers investigated. The ability of NO to induce COX-2 in colorectal cancer was evaluated by other authors (Liu et al. 2003). Their results clearly indicate that the NO donor, S-nitroglutathione increases both COX2 protein expression and PGE2 production in a dose- and time-dependent manner in the HCA7, HT29, and HCT116 human colon cancer cells. The same author has recently proposed an elegant model to elucidate the molecular mechanisms of NO-mediated COX-2 induction in both murine colonic epithelial cells and human colorectal cancer cells. Nitric oxide causes activation of MMPs which leads to the degradation of E-cadherin. This effect causes a cytosolic accumulation of β-catenin and nuclear formation of the transcription complex between β-catenin and TCF/LEF. The authors found that NO through the above-mentioned β-catenin pathway stimulates the expression of the transcription factor polyoma enhancer activator 3 (PEA3) and its binding to DNA. PEA3 has been shown to stimulate the activity of the promoter of COX-2 and, thus, to increase the transcription of COX-2 gene (Liu et al. 2004). Tumor cells develop molecular mechanisms that dysregulate apoptotic pathways resulting in resistance to the current armamentarium of cytotoxic agents against this malignancy. Thus, novel therapeutic interventions are needed to increase the survival rate in patients with advanced cancer and/or to improve the efficacy of the current chemotherapeutic treatments while minimizing drug-induced toxicities. Recent results have shown that tumor tissues from patients with advanced colon cancer presented a significant down-regulation of the apoptosis-inducing factor (AIF) (Huerta et al. 2009), and substantial evidence of the potential role of AIF in carcinogenesis has been suggested in several malignancies (Millan and Huerta 2009). Apoptosis-inducing factor induction has been noted in several tumors by treatment with several cytotoxic agents (Pratt et al. 2006; Soto-Cerrato et al. 2005; Ambrose et al. 2006). Recently it has been shown that the NO donor DETANONOate sensitizes SW620 metastatic colon cancer cells to several proapoptotic treatment and this effect was correlated with the induction of AIF gene and protein (Huerta et al. 2009). Moreover, both exogenous and endogenous NO can induce apoptosis via a mitochondria-dependent mechanism (Wu et al. 2007). The NO donor, sodium nitroprusside (SNP), induced events characteristic of apoptosis, including increases in caspase-3 and caspase-7 expression and down-regulating Bcl-2 expression. Altogether, these results indicate that NO donors can be used as chemosensitizing agents in patients who respond poorly to current chemotherapeutic agents or suffer from drug-induced toxicities. The development of NO-drug

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hybrids, whereby an NO moiety is attached to currently available drugs, such as NO-NSAIDs, provides additive effects to the NO released by the hybrid. NO-ASA, NO-ibuprofen, and NO-sulindac have been studied on the growth of cultured human colon cancer cell in vitro (Williams et al. 2001). Existing data indicate that NONSAIDs are superior to traditional NSAIDs in colon cancer prevention. The exact mechanism of their action and the reasons underlying their enhanced effectiveness are still not completely understood. Several mechanisms can be considered as potentially participating in these effects. NO-NSAIDs have a profound effect on colon cancer kinetics, inhibiting both proliferation and enhancing cell death. This is accompanied by a dramatic effect on cell cycle, arresting the progression of the cell to the S phase (Williams et al. 2001). Moreover, NO-NSAIDs affect several important pathways and these effects may contribute to their chemopreventive effect; they include the eicosanoid pathway, the NO pathway, and the signaling system involving NF-κB pathway (Fig. 4.1). The study of Wallace’s group (Bak et al. 1998) indicates that NO-ASA failed to affect COX isoenzymes and prostaglandin levels in rat colon tissue. This finding, combined with the observation that NO-NSAIDs do not require the presence of COX for their effect on cancer cell kinetics, indicated that this new class of drugs acts on targets beyond COX. Several NO-NSAIDs inhibit the induction of iNOS and the inhibition of iNOS expression may explain their inhibitory action on colon cancer cell proliferation, as iNOS activity is correlated with p53 mutation and VEGF production (Cianchi et al. 2004). It is well known that the NF-κB family of transcription factors is critical to several cellular responses. In nonstimulated cells, NF-κB dimers are maintained in the cytoplasm through their interaction with inhibitory proteins, the IκBs. Nitric oxide enhances the expression and prevents the degradation of cytoplasmic NF-κB inhibitor, IκBα, thereby inhibiting the activation of NF-κB and its subsequent translocation to the nucleus where it activates the transcription of several genes. This action might augment the inhibitory effect of ASA on NF-κB (Peng et al. 1995). In conclusion, all the available evidence suggests the potential use of NO-NSAIDs in cancer as (a) chemopreventing agents, (b) against already developed cancers (chemotherapy), and (c) for the control of cancer symptoms, notably cancer pain.

Fig. 4.1 Effects of NO-NSAIDs on COX and NOS nuclear factor κB (NF-κB) pathways. NO-NSAIDs inhibit proliferation and induce cell death reducing tumor mass

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Inducible NOS/COX-2 Pathway Interaction: A Target for Cancer Treatment Inducible NOS shares significant features with COX-2 in terms of tissue distribution, regulatory function, and participation in pathophysiological phenomena. As reported previously, NO is synthesized by a family of three NOS isoenzymes. The eNOS and nNOS are Ca2+ -dependent and calmodulin-dependent isoforms and are constitutively expressed and responsible for low levels of NO production for short periods. The inducible NOS is Ca2+ independent and requires induction in response to immunological and inflammatory stimuli in every cell type. It can produce large quantities of NO over an extended period of time. Two different isoforms of COX are present: COX-1, constitutively expressed in most tissues and appears to be responsible for the production of PGs that control physiological functions, and COX-2, undetectable in most normal tissues, is rapidly induced by both growth factors and inflammatory stimuli. Inducible NOS and COX-2 have been found to be frequently co-expressed within the same type of cells and under the same experimental circumstances, including inflammation and exposure to several cytokines such as IL-1, TNF-α, and INF-γ (Mollace et al. 2005). Several studies have shown that NO can exert a stimulatory effect on COX-2 catalytic activity in various in vitro and in vivo systems. The molecular mechanisms of this activation have not been elucidated yet. Some authors have shown that NO enhances COX-2 activity through a GMP-independent mechanism and this effect could be mediated by peroxynitrite, the product of NO and O2 − • interaction, possibly through the interaction with the heme group of COX. Another possibility is that lipid peroxidation initiated by peroxynitrite liberates arachidonic acid from the cell membranes which in turn activates COX-2 (Cianchi et al. 2005). Interestingly, not only does NO modulate the activity of COX-2, but NO can stimulate the COX-2 gene and protein expression. The interaction between NO and COX has been studied in several models of inflammation; it has been reported that NO modulates the signal-transduction cascades leading to COX-2 expression via the cGMP-dependent stimulation of tyrosine phosphatase activity or activation of JNK and p38 MAPK. Among these transcriptional factors, NF-κB and activator protein 1 (AP-1) appear of primary importance (Mollace et al. 2005). Interestingly, the demonstration of the NO-mediated up-regulation of COX-2 may provide insight into the link between chronic inflammation and carcinogenesis. Exogenous NO increases the expression of COX-2 at both mRNA and protein levels in the mouse colonic epithelial cells, this could be a result of NOmediated accumulation of free-soluble β-catenin in the cytoplasm and the formation of β-catenin/T-cell factor-lymphocyte enhancing factor (TCF-LEF) DNA-binding complex in the nucleus. Moreover, NO-mediated stimulation of COX-2 mRNA and protein expression through a p38 MAPK and JNK1/2-mediated pathway has been also demonstrated in the mouse cholangiocyte cell line 603B (Ishimura et al. 2004). It is well known that products of iNOS and COX-2 pathways are involved in the regulation of several processes responsible for tumor growth. Both genes and protein overexpression of the two enzymes have been demonstrated in several experimental

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and human tumors (Koki et al. 2003). In synthesis, we can say that iNOS activity has been associated with DNA damage, stimulation of angiogenesis, activation of transcription factors, and induction of oncogene expression; COX-2 activity has been linked to stimulation of angiogenesis and cell proliferation, inhibition of apoptosis and immune surveillance, as well as activation of metalloproteinase (Cianchi et al. 2005, for a review). In conclusion, iNOS and COX-2 have been frequently found to be co-expressed within the same tumor cells. These findings support the hypothesis of a causal relationship between the activity of the two enzymes even in tumor cells. Although the nature of the cross talk between iNOS and COX-2 is complex, most evidence points to a stimulatory effect of NO on both COX-2 expression and activity, and from the majority of the studies present in literature, we can say that iNOS and COX-2 products may represent a common final pathway controlling different tumorigenic mechanisms. Among these, stimulation of tumor angiogenesis appears to be the most frequently involved. This observation suggests that the possible angiogenic effect of NO is mediated by inducing COX-2 activity and prostaglandin production. Figure 4.2 summarized the possible model of signaling pathways for iNOS-mediated COX-2 induction in tumor cells.

Fig. 4.2 Pro-inflammatory and tumor-promoting agents stimulate the activation of both inducible nitric oxide synthase (iNOS) and COX-2. iNOS produces NO that enhances both activity and expression (via a transductional and transcriptional regulation) of COX-2, which leads to the production of large amounts of tumor-promoting prostaglandin E2 (PGE2 ). AP-1, activator protein-1; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; PKC, protein kinase C; TCF-LEF, T-cell factor-lymphocyte enhancing factor. + denotes stimulation and ↑ denotes increase

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Part III

Dual Roles of Nitric Oxide in Protecting or Inducing Cell Death

Chapter 5

S-Nitrosylation – How Cancer Cells Say NO to Cell Death Anand Krishnan V. Iyer, Neelam Azad, Liying Wang, and Yon Rojanasakul

Abstract Nitric oxide (NO) is a highly reactive gaseous free radical that regulates various physiological and pathological processes. Of these functions, the role of NO in the transformation, growth, and metastasis of cancers has generated particular interest. However, research describing the role of NO in cancers seems to be conflicting – some studies describe the suppressive role of NO in cancers, while numerous other studies describe the positive role of NO in tumor survival and progression. In this review, we shall examine the various roles of NO with a focus on how NO regulates tumor microenvironment and the various phases of tumor progression from neoplastic evolution, tumor growth, and metastasis. We will also discuss how NO-mediated S-nitrosylation, or covalent attachment of NO to protein cysteine thiols, regulates carcinogenesis by modulating proteins involved in the different phases of cancer development. Keywords Nitric oxide · S-Nitrosylation · Cancer · Apoptosis · Angiogenesis · Metastasis

Abbreviations NO: NOS: SNO: RNOS: Bcl-2: c-FLIP: DISC: TNF-α:

nitric oxide NO synthases S-nitrosothiol reactive nitrogen–oxygen species B-cell lymphoma-2 cellular FLICE-inhibitory protein death-inducing signaling complex tumor necrosis factor-α

Y. Rojanasakul (B) Department of Pharmaceutical and Pharmacological Sciences, West Virginia University, Morgantown, WV 26506, USA e-mail: [email protected]

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_5, C Springer Science+Business Media, LLC 2010

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Fas ligand c-Jun N-terminal kinase mitogen-activated protein kinase apoptosis signal-regulating kinase-1 vascular endothelial growth factor hypoxia-inducible factor-1α von Hippel–Lindau matrix metalloproteinases

Introduction Nitric oxide (NO, formula N=O) is an inorganic gaseous molecule and an extremely reactive free radical with a very short half-life (Palmer et al. 1987). First described as endothelium-derived relaxing factor (EDRF) in 1978, NO has since been shown to exert its action in a broad range of physiological and pathological processes (Ignarro et al. 1987; Ignarro 1989). NO is released when various NADPH-dependent enzymes called nitric oxide synthases (NOS), together with co-factors such as tetrahydrobiopterine (BH4), oxygen, and protoporphyrin IX, act on the terminal guanidino nitrogen atom of L-arginine, converting it to L-citrulline (Knowles and Moncada 1994; Moncada 1993). To date, three isoforms of NOS have been characterized – NOS1 (neuronal or nNOS), NOS2 (endothelial or eNOS), and NOS3 (inducible or iNOS) (Knowles and Moncada 1994; Knowles 1996). NOS1 and NOS2 are constitutively expressed predominantly in neuronal cells and endothelial cells, respectively, and are collectively referred to as constitutive NOS (cNOS). The activity of cNOS is dependent on the concentration of intracellular calcium levels, which increase under various physiologic stimuli. Inducible NOS, on the other hand, is calcium-independent, is much more ubiquitous, and produces much higher levels of NO as compared to cNOS (Nathan and Xie 1994). However, differential expression of all NOS species has been detected in several established tumors. The physiological actions of NO include inhibition of platelet aggregation, regulation of vascular tone, macrophage activity against bacteria and parasites during injury, and activity during angiogenesis (Brennan et al. 1999; Chiarugi et al. 1998; Lala and Orucevic 1998; Nathan and Hibbs, Jr. 1991; Snyder and Bredt 1992). However, there has been considerable interest over the past two decades in assessing the pathological implications of NO activity, particularly in tumorigenesis and cancer progression in various tissues (Fukumura et al. 2006). Intriguing is the fact that NO has been shown to exert both protective and cytotoxic responses in tumors (Lancaster, Jr. and Xie 2006; Lechner et al. 2005; Li et al. 2004). The dichotomous nature of NO signaling in cancer arises from the fact that individual cells respond differently to NO exposure. Also, different tissues express varied levels of the different forms of NOS, ultimately adding to the complexity of NO signaling (Fukumura et al. 2006).

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As mentioned earlier, the generation of NO has multifarious effects inside the cell, mainly due to the strong reactivity of NO with oxygen and its reactive intermediates such as superoxide anions, leading to the formation of a number of reactive nitrogen–oxygen species (RNOS). Thereafter, RNOS such as peroxynitrite and dinitrogen trioxide are free to exert direct and indirect effects, leading to oxidative, nitrosative, and nitrative stress inside the cell (Beckman et al. 1992; Beckman and Koppenol 1996; Padmaja and Huie 1993). Important antioxidant enzymes such as superoxide dismutase (SOD), ascorbate, and glutathione (GSH) are constitutively produced by cells that combat the reactivity of these NO intermediaries. In addition to the functions of various RNOS, NO itself can directly nitrosate biological molecules such as proteins and peptides, and such post-translational modifications may lead to both altered signaling responses and conformational changes in the substrate. NO can nitrosate the reactive amine of tyrosine residues, leading to the formation of nitrotyrosines, which can potentially prolong the activity of NO, either by acting as second messengers or as reservoirs that slowly release NO. However, another important modification caused by NO, known as S-nitrosylation (the coupling of NO to the reactive thiol moiety of cysteine residues) has garnered increasing attention over the past decade (Gow et al. 2002; Stamler et al. 1992; Stamler et al. 2001). Several protein substrates for S-nitrosylation have been reported in the literature over the past few years (Broillet 1999; Hess et al. 2005). Any irregularities or dysregualtion of proteins in the signaling pathways associated with S-nitrosylation have been shown to cause disease (Foster et al. 2003). There is growing interest amongst researchers in the cancer field due to increasing evidence of S-nitrosylation of substrates that play key roles in mediating tumorigenesis and metastatic potential (Gow et al. 2002). In this chapter, we focus on the implications for NO signaling during the various phases of cancer progression, starting with the initial transformation of cells to form the tumor, proliferation and vascularization of the tumor finally leading to its metastatic growth and dissemination. We specifically address the role of S-nitrosylation in tumors by discussing important substrates that can have putative effects on cancer progression and metastasis.

Nitric Oxide and Cancer One of the first steps in cancer is the neoplastic transformation of normal cells, which typically occurs due to either chronic inflammation of tissues in response to stress, mutations in somatic cells due to genotoxic effects from carcinogen exposure, or both. Either forms of insult lead to phenotypic changes in cellular morphology and activation of oncogenes, ultimately initiating tumorigenesis. Once neoplastic transformation is initiated, multiple cellular processes are put into motion, including proliferation and differentiation, inhibition of apoptosis, increase in vascular permeability of the surrounding tissue through increased angiogenesis, and increase in migratory and invasive potential of the tumor, collectively leading to malignancy.

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Fig. 5.1 Phases of cancer progression. When normal cells or tissues are subjected to genotoxic, cytotoxic, or carcinogenic insult, they may undergo transformation in a nitric-oxide-dependent manner. This process is characterized by activation of signaling cascades that result in increased proliferation and inhibition of apoptosis, causing neoplastic evolution in the process, which is accompanied by various biophysical and biochemical changes. Once the tumor is established, the release of angiogenic mediators is stimulated, leading to increased blood vessel formation, accompanied with increased vascularization of the tumor. Finally, in the metastatic phase, the tumors develop migratory and invasive properties that facilitate dissemination of the tumor and metastasis to secondary sites

These various phases of tumor progression are summarized in Fig. 5.1. Interestingly, all of these stages have been shown to involve either direct or indirect regulation through NO, which is mediated by NOS (Lirk et al. 2002). However, the role that NO plays in the tumor environment remains very unclear because while many studies have shown that NO can promote tumor growth and metastasis, others have shown NO to be tumor suppressive (Lancaster, Jr. and Xie 2006; Ridnour et al. 2006; Wink et al. 1998). In summing the results that have been obtained over the past few years as to the actual role of NO in tumors, it would be safe to surmise that, in addition to cell/tissue-specific responses, the levels of NO determine the outcome of its signaling. The cytoprotective role of NO is observed typically at relatively low but sustained levels of NO, whereas the tumoricidal effects of NO are seen at extremely high and acute concentrations (Hirst and Robson 2007; Lala and Chakraborty 2001; Mocellin et al. 2007).

Proliferative and Anti-apoptotic Effects Recent evidences indicate that depending on its expression levels NO can exert either pro- or anti-apoptotic effects. NO can induce cell death in a number of cancers including breast, colon, and pancreatic cancer cells (Gansauge et al. 1998; Kwak et al. 2000; Mortensen et al. 1999). Melanoma and sarcoma cells undergo extensive apoptosis when exposed to NO (Xie et al. 1995a, b). Also, transfection of iNOS

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into metastatic tumors resulted in a marked decrease in the metastatic potential of these cells (Dong et al. 1994). On the other hand, there have been a number of studies demonstrating the promoting role of NO in tumor progression. High levels of both inducible and constitutive forms of NOS have been detected in several human tumor specimens including head and neck (Park et al. 2003; Rosbe et al. 1995), breast (Thomsen et al. 1995), cervical (Thomsen et al. 1994), lung (Ambs et al. 1998a), prostate (Klotz et al. 1998), pancreas (Hajri et al. 1998), brain (Cobbs et al. 1995), and colon cancers (Radomski et al. 1991). Human and murine carcinomas expressing iNOS were found to be extremely aggressive when implanted into mice (Jenkins et al. 1995). Furthermore, there seems to be a direct correlation between the expression of NOS and the tumor grade, suggesting a causative role for NO in promoting metastasis (Jenkins et al. 1995). The pro-proliferative/survival effects of NO are mediated through the regulation of both pro- and anti-apoptotic proteins that regulate cell growth. Thus, NO can promote tumor proliferation and growth, by both down-regulating anti-apoptotic proteins and up-regulating pro-survival proteins. A number of pro-apoptotic proteins such as caspases (Torok et al. 2002), p53 (Ambs et al. 1998b), and poly-ADP ribose polymerase (PARP) (Sidorkina et al. 2003) are either directly or indirectly inhibited by NO. On the other hand, pro-proliferative proteins such as p21Ras , Akt1, B-cell lymphoma-2 (Bcl-2), cellular FLICE-like inhibitory protein (c-FLIP), and other associated proteins are either stabilized or up-regulated in response to NO stimulation (Dhakshinamoorthy and Porter 2004; Park et al. 2004). NO also downregulates or inhibits the activity of a number of DNA repair enzymes such as DNA alkyl transferase (Wink et al. 1998) and xeroderma pigmentosum-A (XPA) (Morita et al. 1996). Finally, studies show that NOS expression levels correlate with poor clinical outcomes for patients afflicted with different types of cancers, supporting the role of NO in growth and survival (Ekmekcioglu et al. 2006; Raspollini et al. 2004).

Angiogenesis and Vascular Permeability Angiogenesis or neovascularization is an important event for tumor growth and metastasis. Enhanced angiogenesis can lead to faster tumor growth and has a direct correlation with tumor metastatic potential (Carmeliet and Jain 2000). Angiogenesis is regulated by a number of soluble factors, the most important of them being vascular endothelial growth factor (VEGF). These soluble factors bind cell surface receptors in the cells of the vascular endothelium, stimulating the formation of a scaffolding matrix on which endothelial cells migrate to form new blood vessels. This process is potentiated by other cytokines such as basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and transforming growth factor-α (TGF-α), all of which are regulated by NO (Montrucchio et al. 1997). There have been several studies illustrating the positive role of NO in mediating angiogenesis. NO can up-regulate interleukin-8 (IL-8) levels in melanomas and colon cancer cells. Increased VEGF production was observed in liver cancer cells exposed to NO donors; conversely, administration of NOS inhibitors led

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to a decrease in both VEGF production and vessel density in human gastric cancer xenografts and murine melanomas (Kashiwagi et al. 2005; Wang et al. 2005). NO can also up-regulate hypoxia inducible factor-1α (HIF-1α), which is a key regulator of VEGF (Kimura et al. 2000, 2001). In addition, NO has also been shown to mediate the down-regulation of major inhibitors of angiogenesis such as angiostatin and thrombospondin-1 (TSP-1) (Deininger et al. 2003). In order to gain additional traction from the increased angiogenesis, tumors also increase their vascular permeability so as to maximize perfusion through the inadequate vasculature and also to potentially allow higher uptake of nutrients, thus bolstering the growth of the tumor (Doi et al. 1996). This increase in permeability is shown to be mediated by NO which is produced by the tumors themselves (Nakano et al. 1996). In addition, NO also increases the production of prostaglandins such as prostaglandin E2 (PGE2), which plays an important role in increasing the leakiness of the tumor vasculature (Davel et al. 2002).

Metastasis Metastasis involves the translocation of a tumor to a secondary site or organ from its host environment. This is achieved by first detaching from its primary site and escaping into either the vascular or the lymphatic system and docking onto a favorable target organ. During the initiation of this process, signaling cascades regulating migratory and invasive functions are activated and have been shown to occur in a NO-dependent manner (Siegert et al. 2002; Williams and Djamgoz 2005). For example, mammary tumors that have been exposed to NOS inhibitors exhibit decreased migratory and invasive potential (Jadeski et al. 2000, 2003). Also, NO stimulates the release of various matrix metalloproteinases (MMPs) that facilitate the invasion process by degradation of the extracellular matrix (Ishii et al. 2003). As alluded to earlier, a number of processes that dictate tumorigenesis and metastasis involves the modulation of several proteins by NO. This may be achieved either by second messengers such as RNOS or through direct post-translational modifications by NO. One such post-translational modification is S-nitrosylation. Over the past several years, S-nitrosylation of a number of proteins involved in the proliferative and angiogenic phases of tumor development has been documented, and some of them are discussed below.

Protein S-Nitrosylation – Implications for Tumor Survival S-Nitrosylation is the reversible coupling of a nitroso moiety to a reactive cysteine thiol, which leads to the formation of S-nitrosothiol (SNO) (Stamler 1994; Stamler et al. 1997). As mentioned earlier, NO regulates a wide range of proteins through S-nitrosylation, and the principal target of cellular NO is the thiol group of cysteine residues contained in signature motifs (Hess et al. 2005; Stamler et al. 2001). The source of the coupled NO moiety could be provided by NO itself or by other RNOS, metal–NO complexes, NOS, or other SNOs. However, which cysteine residue

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gets nitrosylated or de-nitrosylated depends upon factors such as hydrophobicity, electrostatic environment, orientation of aromatic residues and proximity of target thiols to redox centers, and protein–protein interactions (Hess et al. 2005; Lane et al. 2001). Several agents have been identified that play an important role in catalysis of this process, although specific enzymes that exclusively mediate S-nitrosylation have not yet been characterized (Gaston et al. 2003). S-Nitrosylation has been shown to regulate a number of physiological processes and has been described to occur for a number of proteins including transcription factors, G-coupled kinases, and receptors (Hess et al. 2005). However, discussing these proteins is beyond the scope of this review (for an extensive list of proteins that undergo S-nitrosylation, see Stamler et al. 2001). We focus here on proteins involved in cancer that, when S-nitrosylated, tilt the scales toward increased tumorigenesis and metastasis. These proteins along with their cellular functions, specific cysteine S-nitrosylation and the NOS involved, as well as their effects on cell growth and malignancy are summarized in Table 5.1. Some of these proteins and their role in carcinogenesis regulation through protein S-nitrosylation are further discussed below.

Table 5.1 S-Nitrosylation of protein targets Protein

Cellular function

S-NO site

NOS Functional species effect

Effect on activity Reference Negative Mannick et al. 1999, 2001; Hoffmann et al. 2001; Haendeler et al. 1997 Negative Kim et al. 2000

Caspase-3

Increased apoptosis

Cys-163

iNOS

Decreased enzymatic activity

Caspase-8

Apoptosis Cys-287 activation of caspase-3

iNOS

Caspase-9

Apoptosis, Cys-287 iNOS caspase activation Inhibition of Cys-158 iNOS cytochrome c and Cys-229

Decreased enzymatic activity and apoptosis Decreased enzymatic activity Inhibits Bcl-2 degradation

Bcl-2

c-FLIP

Caspase-8 inhibitor

TRX

Redox regulation, apoptosis

Cys-254 iNOS and Cys-259 Cys-69 iNOS

Negative Mannick et al. 1999 Positive

Inhibits c-FLIP Positive degradation Decreased apoptosis, increase in ROS

Azad et al. 2006; Chanvorachote et al. 2006 Chanvorachote et al. 2005

Negative Haendeler et al. 2002

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Protein

Cellular function

S-NO site

NOS Functional species effect

ASK1

Apoptosis

Cys-869

iNOS

Effect on activity Reference

Inhibits ASK1 Negative activity and binding to MKK-3 and -6 Src Proto-oncogene Unknown iNOS Increased auto- Positive phosphorylation p21Ras GTPase Cys-118 nNOS Increased Positive cellular GTPase activity PTP-1B Phosphatase Unknown iNOS Increased EGFR Negative phosphorylation PTEN Phosphatase Unknown iNOS Increased Negative stability and activity of HIF-1α HIF-1α Angiogenesis Cys-533 iNOS Increased Positive and stability and Cys-800 transcriptional activity MMP-9 Proteolysis of Unknown iNOS Increased Positive ECM migration and invasion of cells Dynamin-2 GTPase Cys-86 and iNOS Increased PKG Positive Cys-607 activation and survival Albumin SNO reservoir Cys-410 iNOS Incrased cyto- Positive preotective and anti-bacterial effect Negative Cys-340 iNOS Increased GRK2 Negative activity regulator of of β-AR β-AR signaling Negative NOS Multiple effects Cys-99 eNOS Inhibition of NOS activity and NO production Glucokinase Glucose Cys-371 nNOS Increase in GK Positive metabolism activity Negative JNK1 Apoptosis Cys-116 iNOS Decreased activation of c-Jun, increased apoptosis

Park et al. 2004

Akhand et al. 1999 Lander et al. 1995; Williams et al. 2003 Li and Whorton 2003 Carver et al. 2007

Li et al. 2007; Yasinska and Sumbayev 2003 Harris et al. 2008

Kang-Decker et al. 2007 Ishima et al. 2008

Whalen et al. 2007

Ravi et al. 2004

Rizzo and Piston 2003 Park et al. 2000

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Table 5.1 (continued) Protein

Cellular function

S-NO site

NOS Functional species effect

AP-1

Transcription factor

NF-κB p65

Transcription factor Transcriptional Catalytic iNOS regulator cysteines

Estrogen receptor

Cys-154 iNOS (c-Fos) Cys-7272 (c-Jun) Cys-38 iNOS

Effect on activity Reference

Inhibits binding Negative delaTorre et al. of c-Fos and 1998; c-Jun Nikitovic et al. 1998 Decreased iNOS Negative Kelleher et al. transcription 2007 Decreased tran- Negative Garban et al. scriptional 2005 activity

This table summarizes some of the proteins involved in tumorigenesis that undergo S-nitrosylation and have been discussed in this chapter. A few other notable proteins that also undergo S-nitrosylation and have been identified in recent years have also been included (italicized entries)

Growth/Survival and Apoptosis-Regulatory Proteins Apoptosis, characterized by cell shrinkage, membrane blebbing, and DNA fragmentation, is a highly regulated process leading to cell death (Wyllie et al. 1980). One of the principal regulators of this process is the caspase family of cysteine proteases. Initiation of apoptotic signaling through death-inducing ligands such as Fas ligand (FasL) and tumor necrosis factor-α (TNF-α) occurs through the extrinsic pathway of cell death, leading to the recruitment of procaspase-8 to the death-inducing signaling complex (DISC) where it is cleaved to its active form (Salvesen and Dixit 1997; Wallach et al. 1999; Yuan 1997). Activated caspase-8 can then cause apoptosis through effector caspase-3 either directly or indirectly through caspase-9 in intrinsic death pathway. In the intrinsic pathway, pro-apoptotic Bcl family of proteins that normally reside in the cytosol translocates to the mitochondria, where they promote the release of cytochrome c. This leads to the formation of apoptosome, where procaspase-9 is cleaved to its active form (Reed 1997; Green and Reed 1998). Caspase-9 can subsequently activate executor caspases such as caspase-3 leading to apoptosis. Since caspases share active cysteines in their catalytic sites, they were investigated for evidence of S-nitrosylation. Indeed, almost all members of the caspase family have shown the potential to be S-nitrosylated at their active cysteines, leading to inhibition of enzyme activity (Mannick et al. 2001). Interestingly, procaspase3 and caspase-9 were found to be constitutively S-nitrosylated in resting cells and de-nitrosylated following treatment with death ligands such as FasL and TNF-α (Matsumoto et al. 2003). Kim et al. demonstrated that S-nitrosylation of procaspase-9 prevents its cleavage and subsequent activation, thereby protecting cells from caspase-dependent apoptosis in human colon adenocarcinoma cells (Kim and Tannenbaum 2004).

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Other key regulators of apoptosis are Bcl-2, which inhibits the intrinsic pathway of apoptosis, and c-FLIP, which regulates the extrinsic pathway by hetero-dimerizing with caspase-8 and preventing its degradation (Irmler et al. 1997; Klas et al. 1993; Nagata, 1997; Oltvai et al. 1993; Yang et al. 1997). Over-expression of Bcl-2 and c-FLIP renders cells resistant to apoptotic cell death by various DNAdamaging agents, an important feature of malignant cells (Ben-Ezra et al. 1994; Ikegaki et al. 1994; Jiang et al. 1995). Previously, our group has investigated the role of NO in modulating the apoptotic pathway. We demonstrated that both Bcl-2 and c-FLIP are S-nitrosylated, but found no evidence of such posttranslational modification affecting the activity of these enzymes or their substrates (Azad et al. 2006; Chanvorachote et al. 2005, 2006). Instead, S-nitrosylation of both Bcl-2 and c-FLIP inhibited ubiquitin-mediated proteasomal degradation of these proteins. Thus, sustained levels of these caspase inhibitors have a cumulative negative effect on apoptosis, leading to survival. Another important anti-apoptotic protein that is subjected to regulation by S-nitrosylation is thioredoxin-like oxidoreductase (TRX). S-Nitrosylation of TRX was found to be important for its repressive effect on apoptosis signal-regulating kinase-1 (ASK1) in endothelial cells (Haendeler et al. 2002). ASK1 regulates both Jun N-terminal kinase (JNK) and p38/mitogen-activated protein kinase (p38 MAPK), and both are required for ligand-induced apoptosis (Ichijo et al. 1997). Interestingly, there is evidence for direct repression of both ASK1 and JNK by S-nitrosylation of critical cysteines, which could result in increased survival and decreased apoptosis of these cells (Park et al. 2000, 2004). Therefore, S-nitrosylation of proteins in the apoptotic pathway has clear implications for cancer, where repression of pro-apoptotic signals would provide impetus for neoplastic transformation and growth (Iyer et al. 2008). On the other hand, S-nitrosylation has also been shown to increase the basal activity of a few pro-survival factors, one of which is Src, a non-receptor protein tyrosine kinase. Src is a proto-oncoprotein whose activity is up-regulated in a number of cancers (Dehm and Bonham 2004). Exposure of cells to NO donors led to an S-nitrosylation-dependent increase in the phosphorylation and subsequent activation of Src (Akhand et al. 1999). Active Src can in turn activate a number of genes encoding for pro-survival proteins including p21Ras , extracellular signal-regulated kinase (ERK) MAPK, phosphatidylinositol-3 kinase (PI3K), and Akt. In addition to Src, p21Ras can also be S-nitrosylated, leading to increased activity and potentially higher stimulation of pro-survival genes (Lander et al. 1995; Williams et al. 2003).

Regulators of Vascularization and Metastatic Potential One of the most important mediators of angiogenesis is hypoxia-inducible factor1α (HIF-1α), which is an important regulator of cellular oxygen homeostasis and is up-regulated under hypoxic conditions. HIF-1α regulates the transcription of several genes through binding hypoxia-responsive elements, and one of the most important proteins regulated by HIF-1α is VEGF. Therefore, modulation of HIF-1α

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levels or its activity has direct implications for angiogenesis under both physiologic and pathologic conditions (Pugh and Ratcliffe 2003). Li et al. showed increased S-nitrosylation of HIF-1α in breast tumors when exposed to radiation, which led to increased stability and activation of HIF-1α (Li et al. 2007). Sumbayev et al. demonstrated that HIF-1α can be S-nitrosylated at Cys800 and that S-nitrosylation of HIF-1α is required for its binding to co-factors such as cyclic-AMP-responsive element-binding protein (CREB)/p300 protein downstream of HIF-1α (Sumbayev et al. 2003; Yasinska and Sumbayev 2003). S-Nitrosylation of HIF-1α can have an effect on transcriptional activity and stability of the protein. Moreover, induction of NO under normoxic conditions, where HIF-1α is rapidly degraded via the ubiquitin–proteasome pathway mediated by von Hippel–Lindau (VHL) protein, led to decreased association of HIF-1α and VHL, thus increasing HIF-1α activity (Metzen et al. 2003). Such up-regulation of HIF-1α may lead to increased VEGF production, ultimately leading to increased angiogenesis in tumors. Another class of proteins that is important for cancer metastasis is MMPs, proteases that can cleave extracellular matrix components. Studies have shown that S-nitrosylation of both MMP-1 and MMP-9 can occur in the presence of NOS, and this leads to increased activity of MMPs in both normal and cancer cells (Harris et al. 2008; Ishii et al. 2003). Additionally, the activity of other protein classes such as protein phosphatases may also be modulated by S-nitrosylation. For example, enzymes such as protein tyrosine phosphatase 1B (PTP1B), Src homology-2 (SH2) domain-containing PTPases 1 (SHP1) and 2 (SHP2) can be S-nitrosylated at their active cysteine residues, leading to their inactivation (Caselli et al. 1995; Li and Whorton 2003; Mikkelsen and Wardman 2003). Also, Carver et al. demonstrated that S-nitrosylation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) inactivates the enzyme, promoting the activation of HIF-1α by Akt (Carver et al. 2007). Such loss of phosphatase function may result in the hyper-phosphorylation of protein kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), leading to potentially sustained survival signaling from proteins, which would otherwise be attenuated by the action of these phosphatases (Callsen et al. 1999).

Summary Protein S-nitrosylation has emerged as an important regulatory mechanism that controls the activity and function of a number of important proteins. Dysregulation in this process may contribute to various pathologies that are typically associated with NO exposure. NO plays an essential role in regulating various functions in normal cells including growth, development, and immunity. However, the role of NO in cancer is multi-factorial and its effects may vary based on its temporal and spatial levels. There have been a number of cancer drugs that involve either NO donors or NO scavengers – an excellent review by Mocellin et al. covers this in exhaustive detail (Mocellin et al. 2007). However, NO itself may be tumorigenic or tumoricidal based on the tumor type, the concentration of NO, and the presence of other

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co-factors. This fact suggests that a broad-spectrum drug that acts as an activator or inhibitor against NO for the treatment of cancers may not be effective. A more effective approach would be to target the NO-targeted putative proteins that are dysregulated in cancer. One such strategy is to target S-nitrosylated proteins that contribute to tumorigenesis, which will lead to a more specific therapeutic effect and reduced side effect. Acknowledgment This work was supported by the National Institutes of Health Grant R01HL076340.

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Chapter 6

Cytotoxic and Protective Activity of Nitric Oxide in Cancers Gen-Ichiro Soma, Chie Kohchi, and Hiroyuki Inagawa

Abstract Nitric oxide (NO), synthesized from L-arginine by NO synthases, is a small, lipophilic, diffusible, highly reactive molecule with dichotomous regulatory roles in many biological events under physiological and pathological conditions. NO promotes apoptosis in some tumor cells, but provokes anti-apoptotic activity in other tumor cells. For this reason, conflicting viewpoints have arisen as to whether nitric oxide is cytotoxic or protective in cancer cells. Part of the complexity of NO concentrations in tumor cells or tissues can be attributed to cell death and the formation of an anti-apoptotic cascade with nitrosylation of biological molecules from substances such as metal ions, thiol, the amino acid tyrosine, and reactive oxygen species. During the last 5 years, there have been many excellent reviews concerning the role of NO in cancer therapy, tumor apoptosis, and metastases. Here, the recent knowledge of cytotoxic (apoptotic) and cytoprotective (anti-apoptotic) activity of NO in cancer will be reviewed. Keywords Apoptosis · Anti-apoptosis · Caspase · Bcl · Mitochondria · TNF · Fas · TRAIL · NF-κB · S-nitrosylation · Proteasome Nitric oxide (NO) is a low weight reactive free radical gas synthesized from L-arginine by NO synthase (NOS). NO exerts nitrosylation of a wide range of proteins to induce a pleiotropic functions involved in a growing tumor and immune cell as a double-edged sword, inasmuch as it can exert opposite effects depending on its concentration and the cell type. There are many excellent reviews concerning the role NO in cancer therapy, tumor apoptosis and metastases during last 5 years (Bonavida et al. 2008; Bonavida et al. 2006; Coulter et al. 2008; Fukuzawa et al. 2004; Hirst and Robson. 2007; Hofseth. 2008; Huerta et al. 2008; Jeannin et al. 2008; Kashfi and Rigas. 2005; Lechner et al. 2005; Mocellin et al. 2007; Olson and

G.-I. Soma (B) Department of Integrated and Holistic Immunology, Faculty of Medicine, Kagawa University, 1750-1 Mikicho, Kida-gun, Kagawa 761-0793, Japan; Macrophi Inc., Hayashi-cho, Takamatsushi, Kagawa 761-0301, Japan; Institute for Health and Science, Tokushima Bunri University, Nishihama, Yamashirocho, Tokushima, 770-8514, Japan e-mail: [email protected]; [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_6, C Springer Science+Business Media, LLC 2010

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Garban. 2008; Ridnour et al. 2006; Rigas. 2007; Rigas and Kashfi. 2004; Weigert and Brune. 2008; Wink et al. 2008). In this chapter, we introduce the new research concerning the role of NO in cancer therapy, tumor apoptosis and metastases. Moreover, the knowledge of NO functions in the tumor microenvironment should be helpful for better understanding of tumor therapy with NO.

Direct Role of NO Cytotoxicity One of the mechanisms that cause cytotoxicity (apoptosis) of tumor cells by nitric oxide is the nitrosative stress which occurs via S-nitrosylation by the interaction of NO with the biological thiols of proteins. Nitrosative stress can promote apoptosis because of the activation of mitochondrial apoptotic pathways, such as the release of cytochrome c, an apoptosis-inducing factor, and endonuclease G from mitochondria. Another mechanism of NO-induced apoptosis is the nitrosylation of NF-κB which causes the downstream inhibition of several anti-apoptotic proteins. These NO functions facilitate the activation of apoptotic pathways with both chemotherapy and immunotherapy. In this chapter we review the mechanisms whereby S-nitrosylation and nitrosative stress regulate the apoptotic signal cascade. The cytotoxic effects of NO in this section are illustrated in Figs. 6.1 and 6.2.

Fig. 6.1 Cytotoxic (apoptotic) cascade by NO in tumor cells

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Fig. 6.2 Cytotoxic (apoptotic) cascade by NO in tumor cells

New Mechanism of NO Cytotoxicity GAPDH Is a New NO Target The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been associated with transcriptional regulation of apoptosis by NO. S-Nitrosylation of GAPDH by NO-enhanced apoptosis occurs in association with Siah1, an E3 ubiquitin ligase. These effects were blocked by an inhibitor of iNOS and nNOS in knockout mice. Depletion of either GAPDH or Siah1 from wild-type cells with siRNA inhibited apoptosis. Thus, these data suggest that NO-S-nitrosylationGAPDH-Siah1 is a novel apoptotic route of NO (Hara et al. 2005). Denitrosylation by Thioredoxins Causes Apoptosis S-Nitrosylation of caspase-3 on their catalytic-site cysteine by endogenous NO production inhibits the apoptotic pathway. Reverse denitrosylation reactions cause apoptosis in S-nitrosylated cells. Benhar et al. reported that thioredoxin-1 is rate limiting for denitrosylation of cytosolic S-nitrosylated caspase-3 in several types of cells. Thioredoxin has been found to be a hydrogen donor for ribonucleotide reductase for DNA replication (Holmgren 1989). The thioredoxin-1 actively denitrosylates S-nitrosylated caspase-3 and increases cytotoxicity of NO. Thioredoxin system also mediates Fas-induced denitrosylation (Benhar et al. 2008).

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Inhibition of Anti-apoptotic Protein Survivin by NO Survivin, which increases in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase, is one of a number of inhibitors of apoptosis proteins. Survivin is present in most transformed cell lines and has been shown to inhibit caspase directly and by apoptosis (LaCasse et al. 1998). Overexpression of this protein is frequently found in many tumor cells and correlates with malignancy (Chandele et al. 2004). NO-treated tumor cells displayed a preferential loss of survivin protein expression via a proteasome-dependent pathway. Dendritic cell-mediated killing was associated with the accelerated turnover of survivin (Huang et al. 2005). In context with cell cycle-dependent responses to chemotherapy, the data in this chapter suggest the possibility that the survivin pathway can be explored to induce apoptosis in tumor cells by dendritic cells. Inhibition of Yin-Yang1 Upregulation of Apoptosis Signal by NO Some tumors expressed significantly lower sensitivity to Fas ligand compared to normal cells (Basolo et al. 2000). NO upregulated Fas receptor expression in many tumor cell lines (Garban and Bonavida 1999; Lim et al. 2009). Bonavida et al. found a new cascade of this regulation by Yin-Yang1 protein, a transcription repressor that binds the silencer region of the Fas, interferon-γ •(IFN-γ)•• (Ye et al. 1996a), IL-3 (Ye et al. 1999), and GM-CSF (Ye et al. 1996b) gene promoters. Yin-Yang1 plays a pivotal role in the upregulation of Fas gene expression in human tumor cells by nitric oxide (Garban and Bonavida 2001). Moreover, a recent report suggests that TRAILresistant tumor cells are also sensitized to TRAIL-induced apoptosis concomitantly with DR5 upregulation by NO. TRAIL, tumor necrosis factor alpha (TNF-α)-related apoptosis-inducing ligand, is a member of the TNF family of cytokines that promotes apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells (Suliman et al. 2001). The mechanism of sensitization was the inhibition of the Yin-Yang1 that binds to a DR5 promoter and negatively regulates DR5 transcription (Bonavida et al. 2008; Engels et al. 2008; Huerta-Yepez et al. 2009). Thus, application of NO donors as sensitizing agents in combination with TRAIL/DR4 or DR5 mAbs is a therapy for TRAIL-resistant tumors. NO Inhibits Tumor Cell Proliferation by Inhibiting EGFR Tyrosine Kinase Activity NO not only induces the apoptosis of tumor cells but also inhibits tumor cell proliferation. Growth inhibition of tumor cells can be elicited either by exogenous NO or by endogenous NO or in tumor cells or in their neighboring cells (Estrada et al. 1997; Sordella et al. 2004; Williams et al. 2003). One of the mechanisms of NO growth inhibition associates epidermal growth factor (EGF) and EGF receptors (EGFR) which are an important growth promoting system in some tumors (Mendelsohn and Baselga 2000). Also, gefitinib, a tyrosine kinase inhibitor of EGFR, induces significant growth inhibition in clinical therapy (Ono and Kuwano 2006). NO inhibits

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human epithelial carcinoma (A431) cells or human neuroblastoma (NB69) growth by tyrosine phosphorylation of epidermal growth factor receptors (EGFR) with tyrosine kinase inhibition (Gonzalez-Fernandez et al. 2008; Ruano et al. 2003). NO Downregulation of Cdc25A (Anti-apoptotic Protein) Nitrosative stress-induced suppression of Cdc25A primed cells for apoptosis signalregulating kinase-1 (ASK-1)-dependent apoptosis. Cdc25A is overexpressed in numerous human cancers and possesses anti-apoptotic activities (Ray and Kiyokawa 2007). ASK-1 is a mitogen-activated protein 3 kinase (MAP3K) that is involved in the cellular apoptotic response resulting from oxidative stress or TNF-α exposure and is also involved in the control of cellular proliferation, differentiation, and survival (Kherrouche et al. 2006). NO was sufficient to suppress Cdc25A expression, consistent with its role in mediating nitrosative stress. Nitrosative stress decreased the Cdc25A-bound fraction of ASK-1 and sensitized cells to apoptosis induced by the ASK-1-activating chemotherapeutic cisplatin (CDDP) (Tomko and Lazo 2008).

Endogenous NO Nitric oxide (NO) is a multifunctional low weight molecule synthesized from L -arginine by NO synthase (NOS). The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS or NOS2), endothelial NOS (eNOS or NOS3), and neuronal NOS (nNOS or NOS1) (Yasuda 2008). eNOS and nNOS are calcium dependent and there is constitutive production of picomolar to nanomolar NO in neuronal tissue and endothelium. By contrast, iNOS produces high amounts of NO in macrophages up to the micromolar range. NO is a major effector molecule for the destruction of tumor cells by genotoxicity. The high concentration of NO which is induced by activated macrophages may be cytostatic or cytotoxic for tumor cells. Lipopolysaccharide (LPS) or some cytokines such as TNF-α and IFN-γ can efficiently regulate iNOS transcription by activation of nuclear factors such as NF-κB and AP-1. Thus, macrophage activation to induce high concentration of NO by LPS or some cyotokines is a candidate for anti-tumor therapy. Th1 and M1 Cytokines Induce iNOS in Tumors Th1 and M1 type cytokines induce significant amounts of NO in tumor cells that correlate with the rate of apoptosis. Interferon-γ (IFN-γ), interleukin-1β (IL-1β), and TNF-α induced the iNOS gene in ovarian carcinomas (OVCAR-3, HOC-7, and 2774) (Rieder et al. 2001). The iNOS inhibitor (aminoguanidine) significantly suppressed the apoptosis of these tumors. The neuroblastoma (NB-39-nu) cell line expressed iNOS mRNA following treatment with a combination of IFN-γ and cisplatin (Ogura et al. 1997); rat glioma cell line (C-6) was induced to express iNOS gene and NO by leptin with IFN-γ, TNF-α, or IFN-γ plus IL-1β (Mattace Raso

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et al. 2006). Melanoma cells (K-1735) were induced by IL-1α and IFN-γ (Xie et al. 1997). Therefore, localization of Th1 and M1 type cytokines may be capable of inducing apoptosis by endogenous NO production in tumor cells. In fact, using the technique of isolation perfusion, combination therapy with TNF-α, IFN-γ, and melphalan induced complete regression in more than 70% of limb melanomas and soft tissue sarcomas (Verhoef et al. 2007). Endogenous NO Enhances Fas Expression The Fas ligand (Fas-L) is an essential effector molecule of cytotoxic T lymphocytes, natural killer cells, and cytotoxic macrophages (Ben-Hur et al. 2000; Hahn and Erb 1999; Medvedev et al. 1997). The sensitivity of ovarian carcinoma cells (A2780 and AD10) to Fas-L-mediated cytotoxicities can be regulated by the induction of endogenous NO. Th1 type cytokines, M1 type cytokines, and immunostimulators (IFN-γ, IL-12, TNF-α, IL-1β, LPS, etc.) induce iNOS gene in ovarian carcinoma (Garban and Bonavida 1999). Endogenously produced NO resulted in the upregulation of Fas in these tumor cells. iNOS Gene Transfer Therapy Gene transfer is not yet a practical technique, but experimental results indicate significant tumor suppression (McCarthy et al. 2008). It is reported that strong expression of the iNOS gene significantly increased levels of NO. This was observed by CMV/iNOS plasmid gene transfer in human prostate (DU145 and PC3) and human colon (HT29) cancer cell lines (Adams et al. 2009). These tumor cells showed enhanced cytotoxicity to cisplatin. In vivo transfer of CMV/iNOS by direct injection into established radiation-induced fibrosarcoma-1 (RIF-1 tumor) caused a significant delay in tumor growth. Significant chemosensitization of cisplatin cytotoxicity was observed in the presence of NO. Colorectal tumor cells inducing iNOS gene showed higher sensitivity with radiation therapy in a caspase-dependent manner (Chung et al. 2003). Moreover, IFN-γ gene therapy in human and mouse prostate cancer cells inhibited tumor growth with induction of the iNOS gene and NO activity (Olson et al. 2006). iNOS Induction Using a Plant Extract Evodiamine is an extract of the traditional Chinese herb, Evodia rutaecarpa, and is a bioactive anti-inflammatory alkaloid (Liao et al. 2005). Evodiamine upregulated the iNOS gene, generated NO, and exhibited cytotoxic activities on cervical cancer, melanoma, lung carcinoma, and colon carcinoma cells (Fei et al. 2003; Ogasawara and Suzuki 2004). Furthermore, it was shown that evodiamine generated NO-induced apoptosis, cell cycle arrest, and was linked to the activation of p53 and p21. It was concluded that p38 was critical to the NO producing system, which contributed greatly to the apoptosis and cell cycle arrest in evodiamine-incubated cells (Yang et al. 2008a; Yang et al. 2008b).

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Statins Induce iNOS Gene in Tumor Cells The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) reduce serum cholesterol levels and cardiovascular morbidity and mortality. Statins inhibit cancer cell proliferation in in vivo tumor growth in animal models (Campbell et al. 2006; Kusama et al. 2002; Paragh et al. 2003; Sivaprasad et al. 2006). Moreover, they increased iNOS mRNA and protein expression in the human breast adenocarcinoma cell line (MCF-7). NO cytotoxicity and tumor cell cytotoxicity were inhibited by iNOS inhibitors (Kotamraju et al. 2007). Based on these data, statins which are well known as a class of hyperlipidemic blockbuster drugs and are routinely used for lowering serum cholesterol levels are potential cancer drugs for use as NO inducers.

Synthetic Retinoid Induces iNOS Gene in Tumor Cells The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) produced high levels of NO via the induction of iNOS. The 4-HPR exhibited apoptotic and antiinvasive effects against bone metastatic breast cancer cells (Simeone et al. 2002) and showed inhibition of the invasion of ovarian cancer (Um et al. 2001), prostate cancer (Kim et al. 1995; Quader et al. 2001; Sharp et al. 2001; Webber et al. 1999), and Kaposi’s sarcoma (Ferrari et al. 2003; Simeone et al. 2006).

Polyphenols in Dark Grapes Induce eNOS in Vascular Endothelium Cells Trans-pterostilbene (t-PTER; 3,5-dimethoxy-4 -hydroxystilbene) and quercetin (QUER; 3,3 ,4 ,5,6-pentahydroxyflavone) are structurally related and are naturally occurring polyphenols in dark grapes. t-PTER and QUER sensitize vascular endothelium cytotoxicity by induction of eNOS genes and inhibit metastatic growth of highly malignant B16 melanoma F10 (B16M-F10) cells (Ferrer et al. 2007). Orally administered polyphenol conjugates may be part of new cancer therapy which would be reliant on the release of free polyphenols by hydrolysis.

Exogenous NO NO is a free radical gas that exerts nitrosylation of a wide range of proteins and modification of biological functions of the cell. Thus, NO donors are candidates for a new approach to drugs that inhibit tumor cell survival and anti-apoptotic pathways by conventional cancer therapy. NO donors continuously produce NO gas after various time periods (seconds to days). A large variety of nitric oxide donors are available as tools for tumor treatments. The details of NO donors are well documented in recent reviews (Bonavida et al. 2008; Huerta et al. 2008; Yamamoto and Bing 2000). In this section, we introduce a third category of NO donors and their anti-tumor functions. These types of cytotoxic effects of NO are illustrated in Figs. 6.1 and 6.2.

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Organic Nitrate Types of NO Donors This class of NO donors, such as GTN (glyceryl trinitrate, nitroglycerin), ISDN (isosorbide dinitrate), and ISMN (mononitrate), has been used as vasodilators for angina pectoris therapy for a long time. Intravenous GTN and ISDN have a halflife of 3–5 min and 24 min, respectively. ISMN possesses a longer half-life (5 h) (Prakash and Markham 1999). In the blood circulation system, NO was released from organic nitrates with non-enzymatic and enzymatic pathways (Noble 1999). For example, GTN released NO following exposure to mitochondrial aldehyde dehydrogenase. In the case of both non-enzymatic and enzymatic activation of organic nitrates, it has been suggested that thiol groups are involved. S-Nitrosothiol Type of NO Donors This class of NO donors (the use of S-nitrosothiols) is organic compounds or functional groups containing a nitroso group attached to the sulfur atom of a thiol. They have an inherent disadvantage of instability, but this provides a mechanism for releasing NO over a long period. Examples of this class are SNAP (S-nitroso-N-acetylpenicillamine), GSNO (S-nitrosoglutathion), and glyco-SNAP-1 (N-(b-d-glucopyranosyl)-N2 -acetyl-S-nitroso-D, L-penicillaminamide). SNAP has a somewhat higher stability compared to other nitroso compounds. Some of these produce NO naturally and/or following reactions with xanthine oxidase, superoxide dismutase, protein disulphide isomerase, and various hydrogenase enzymes. The half-life in an aqueous medium of SNAP is 4.6 h (Ignarro et al. 1981). In the presence of cells, the SNAP half-life is about 1 h, and the GSNO half-life is 48 min. Glyco-SNAPs are more stable analogs of SNAP and the release of NO can be monitored over a period of 24–30 h. Their decomposition is catalyzed by Cu+ ions, which themselves can be formed by reduction of Cu2+ ions by thiols (Butler et al. 1998). Diazeniumdiolates (NONOates) Type of NO Donors Diazeniumdiolates (NONOates) are a prodrug class that is stable in neutral aqueous media but releases bioactive nitric oxide (NO) when metabolized by esterases. Moreover, they release NO spontaneously in a pH-dependent fashion (i.e., at a neutral or acidic pH), with no requirement of redox or light activation. This prodrug class may potentially be useful as cytotoxic agents in various tumors (Saavedra et al. 2000). They stabilize NO during storage in a solid form, are highly soluble in water, and can release NO at rates that can be reliably adjusted over a wide range with judicious choice of the carrier nucleophile. Examples of this class of molecule include DETA/NO (1-substituted diazen-1-ium-1,2-diolates), DEA/NO (1-diethyl-2-hydroxy-2-nitroso-hydrazine), PAPA/NO ((Z)-1-[N-(3ammoniopropyl)-N-(n-propyl) amino]diazen-1-ium-1,2-diolate), JS-K (O2 -(2,4dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate), and SPER/NO (spermine NONOate, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine. DETA/NO has a half-life of 20 h and releases NO in

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RAW 264.7 cells (Guastadisegni et al. 2002; Keefer et al. 1996). The half-life of DEA/NO in 0.1 M phosphate buffer (pH 7.4) is 2 min, 37ºC. The half-life of PAPA/NO in RAW 264.7 cells is 15 min (Guastadisegni et al. 2002). JS-K is designed to be stable at a physiological pH, but releases NO upon reaction with glutathione. Glutathione/glutathione S-transferase activation has potent anti-tumor effects (Shami et al. 2003). In the absence of GSH, JS-K is stable in aqueous buffers; the half-life is over a week, but in the presence of glutathione, the half-life is less than 30 min (Chakrapani et al. 2008). The half-life of SPER/NO is 37 min (Ferrero et al. 1999). SPER/NO has a long half-life providing controlled biological release of NO in vivo (Kuntscher et al. 2002). NONO/AM (diethylamine NONOate/AM) is designed to generate NO spontaneously in aqueous media at physiological pH and temperature. DPTA/NONO spontaneously dissociates in a pH-dependent, first-order process with a half-life of 3 h and 5 h at 37◦ C and 22–25◦ C, respectively (pH 7.4) and liberates 2 moles of NO per mole of parent compound (Keefer et al. 1996). Hybrid Type of NO Donors A promising novel class of drugs is nitric oxide NSAIDs (NO-NSAIDs) that have been designed to conjugate a NO-donating moiety and have been found to be more active than classical NSAIDs. NCX 4040 showed a striking cytocidal activity in three human pancreatic adenocarcinoma cell lines (Capan-2, MIA PaCa-2, and T3M4) (Rosetti et al. 2006). NO-ASA (nitric oxide-donating aspirin, NCX4016, nitroaspirin, benzoic acid 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester) is a promising agent for the prevention of colon and other cancers. The half-life of NO-ASA is less than 15 min in rat liver (Dunlap et al. 2008; Gao et al. 2005). These substances may be developed into new pharmacologically active molecules of potential use in various therapeutic areas. Cytotoxic Effect of NO Donors NO donors have the potential to be tumor cytotoxic agents. Thus, nitric oxide donors have the dual functions of both sensitizing tumor cells to chemotherapy and immunotherapy and are also involved in the regulation and inhibition of metastasis. NO donors constitute a promising new class of tumor cytotoxic agents. In this section, we introduce some examples of the cytotoxic characteristic that have been reported. Organic Nitrate Type of NO Donors GTN sensitizes the tumor cells cytotoxicity to Fas-mediated cell death by increasing the expression of Fas and decreasing the expression of several inhibitors of apoptosis (IAPs) in colon cancer cells (Millet et al. 2002). ISMN and ISDN have the potential to inhibit growth and metastatic properties of Lewis lung carcinoma (LLC) in mice by suppressing angiogenesis. Both ISMN and ISDN have an angiogenesis inhibition

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effect (chick chorioallantoic membrane study) in a dose-dependent manner. Two weeks of daily intraperitoneal administration of ISMN significantly decreased the mass of the intramuscularly inoculated LLC tumor and metastatic foci of the lungs, while there is no growth inhibition in the cultures of LLC cells (Pipili-Synetos et al. 1995). S-Nitrosothiol Type of NO Donors GSNO induced apoptosis in two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells (Jeon et al. 2005). GSNO inhibits ornithine decarboxylase by an oxygen-independent mechanism. Nitric oxide regulates ornithine decarboxylase activity which is the essential enzyme for polyamine synthesis in mammalian cells (Bauer et al. 2001). SNAP conjugated to glucose (2-gluSNAP) can be readily transported across the membrane by GLUT1 transporters. Cytotoxicity of SNAP and 2-gluSNAP was demonstrated on a GLUT1-rich glioblastoma cell line T98G. Differential expression of Glut1 mRNA and protein levels correlates with increased sensitivity to the glyco-conjugated nitric oxide donor (2-glu-SNAP) in different tumor cell types (Subbarayan et al. 2008). Cytotoxic Effect of Diazeniumdiolate Type of NO Donors DEA/NO and PAPA/NO resulted in the production of highly reactive nitrogen species, which subsequently sensitized cells to cisplatin treatment. NO donors such as cisplatin improve chemotherapeutic agent cytotoxicity in tumor cells. Pretreatment with DETA/NO resulted in almost a threefold reduction of the LD50 of cisplatin in a head and neck squamous cell carcinoma (HNSCC) cell line (Azizzadeh et al. 2001). Sensitization by DETA/NO was shown to inhibit the transcription repressor Yin Yang1 which was shown to regulate resistance to both Fas ligand and TRAIL (details of Yin-Yang1 were described in Section “Inhibition of Yin-Yang1 Upregulation of Apoptosis Signal by NO”). Moreover, DETA/NO upregulated cytotoxicity by inhibition of NF-κB, which induces several anti-apoptotic gene products (Bonavida et al. 2008). NONO-AM exhibits apoptotic and anti-invasive effects against bone metastatic breast cancer cells. The NONO-AM, which releases NO upon activation by intracellular esterases, has been shown to induce apoptosis in leukemia cells (Saavedra et al. 2000; Simeone et al. 2006). SPER/NO showed a biphasic effect on the proliferation of human salivary gland neoplastic (HSG) cells. SPER/NO at more than 100 μM concentrations inhibits cell proliferation (Bonavida et al. 2008). JS-K releases NO following glutathione S-transferase activity and induces apoptosis in vitro and in vivo in human multiple myeloma cells by DNA double-strand breaks (Kiziltepe et al. 2007). JS-K is a potent anti-tumor agent against human prostate cancer, liver cancer, and multiple myeloma xenografts in mice. JS-K showed significant cytotoxicity in both conventional therapy-sensitive and therapy-resistant multiple myeloma cell lines. This cytotoxicity was associated with caspase-8 and caspase-9 cleavage, increased Fas/CD95 expression, and Bcl-2 phosphorylation (Udupi et al. 2006). JS-K induces cytotoxicity of tumor cells in

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vitro and in vivo, indicating that it may be a promising new therapeutic for cancer (Vasilev 2008). Hybrid Type of NO Donors Non-steroidal anti-inflammatory drugs (NSAIDs) have repeatedly been shown to be effective in tumor prevention, but important side effects limit their wide clinical use. NO-releasing NSAIDs derivatives (NO-NSAIDs) are a promising class of compounds synthesized by combining a classic NSAID molecule with an NO-releasing moiety to counteract side effects (Kashfi and Rigas 2005; Rigas and Kashfi 2004). NCX 4040 (a novel NSAIDs) is a cytotoxic agent in human pancreatic adenocarcinoma (Capan-2, MIA PaCa-2 and T3M4), colon (LoVo, LoVo Dx, WiDr, LRWZ), bladder (HT1376, MCR), and pancreatic (Capan-2, MIA PaCa-2, T3M4) cell lines. NO is the primary active molecule in NCX 4040 that exerts drug activity (Dunlap et al. 2008; Rosetti et al. 2006; Tesei et al. 2008). The cytotoxic effect is induced by a mitochondria-dependent pathway. Anti-tumor activity of NCX 4040 was indicated in xenografted colon cancer in mice (Tesei et al. 2008). NCX 4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies (Bratasz et al. 2008; Bratasz et al. 2006). NO-ASA is a promising agent for the prevention of HT-29 in human colon adenocarcinoma, Hepa 1c1c7 mouse liver adenocarcinoma cells (Gao et al. 2006), pancreatic cancer (Ouyang et al. 2006; Rosetti et al. 2006; Tesei et al. 2008), and human ovarian cancer cells (HOCCs) (Bratasz et al. 2006). Moreover, NO-ASA has chemopreventative properties against azoxymethane-induced colon cancer in rats (Rao et al. 2006).

Tumor Microenvironment In order to grow beyond 1–2 m3 in size, tumor cells stimulate the development of blood vessels that infiltrate the tumor, in a process called angiogenesis. Various kinds of angiogenetic cytokines are produced in tumor tissues such as vascularderived endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), the matrix metalloproteinases MMP-9, and MMP-2 (Boudreau and Myers 2003; Carmeliet and Jain 2000). Many types of stromal cells, such as fibroblasts and macrophages, reside in the tumor microenvironment. The macrophages that migrate into tumor tissue, termed tumor-associated macrophages (TAMs), are recruited to tumor tissues by several growth factors and chemokines, which are often produced by the tumor cells (Shieh et al. 2009). TAMs have pivotal roles in the suppression, progression, and metastasis of tumors. In early stages of tumor progression, TAMs which produce NO exhibited tumor suppression. In contrast, after the tumor is established, they provide angiogenetic cytokines (VEGF, bFGF) which develop new blood vessels in the hypoxic tumor tissues. These molecules proliferate and migrate to endothelial cells where they cause matrix remodeling to form new blood vessels.

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Tumor progression is regulated through paracrine interactions between tumor cells and stromal cells in the microenvironment. Correlation of the number of TAMs with poor prognosis is well documented by many reports. Breast, prostate, ovarian, and cervical cancers had especially poor prognoses, whereas we found that the abundant presence of TAMs was associated with good prognosis in endometric and gastric cancer patients (Ohno et al. 2003a, b, 2005, 2008). Moreover, TAMs induce iNOS and release NO which likely results in vasodilation and increased vascular flow (MacMicking et al. 1997). In this section, we introduce the recent findings of the cytotoxic roles of NOS and NO in tumor microenvironments. Induction of iNOS Gene and NO by Tumor-Infiltrating Dendritic Cells LPS or IFN-γ activated rat dendritic cells, which produce high amounts of IL-12 and TNF-α, kill several tumor cells (colon carcinoma) but requires cell-to-cell contact, and depends on NO production in vitro. Moreover, intratumoral injection of LPS induces an increase of iNOS expression in tumor-infiltrating DCs and a significant arrest of tumor growth in vivo (Nicolas et al. 2007). NO Production by Irradiation-Activated Macrophages Radiation therapy is the medical use of ionizing radiation as part of cancer treatment to control malignant cells. Irradiated macrophages (RAW 264.7 cells) or bovine aortic endothelial cells (BAECs) showed an upregulation of iNOS or eNOS genes. Interestingly, LPS-stimulated and -irradiated RAW 264.7 cells induced an upregulation of NF-κB and iNOS genes and increased the DNA damage in bystander EL-4 cells. Treatment of RAW 264.7 cells with L-NAME (N-nitro-L-arginine methyl ester) significantly reduced the induction of gene expression and DNA damage in the bystander EL-4 cells (Ghosh et al. 2008). Hepatoma HepG2 cells co-cultured with irradiated BAECs showed apoptosis in the presence of L-arginine, but apoptosis was prevented by L-NAME (Hirakawa et al. 2002). NO Upregulates Fas Expression Nitric oxide is a mediator of IFN-γ-induced sensitization of human ovarian carcinoma cell lines (A2780 and AD10) to Fas-mediated apoptosis. Treatment of quiescent A2780 and AD10 ovarian carcinoma cells with IFN-γ alone induced the expression of iNOS mRNA as examined by RT-PCR. Endogenously produced NO, by IFN-γ pretreatment or exogenous nitrodonors, resulted in the upregulation of the Fas receptor mRNA and protein expression (Garban and Bonavida 1999). NO Downregulates MMP by Stromal Cells Nitric oxide (NO) is a key molecule in the regulation of tumor–microenvironment interactions. One of the NO functions is the regulation of tumor cell cross talk with stromal cells. Because tumor cell anoikis and invasion are both regulated by

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the myofibroblast-derived matrix, eNOS-dependent downregulation of the matrix protease cathepsin B and silencing of cathepsin B attenuated the tumor cell invasive capacity (Decker et al. 2008).

NO Production from Nitrite in Tumor Environments Hypoxia and acidity are characteristics of the tumor microenvironment. Thus, nitrite can preferentially produce NO in tumor tissues. Frerart et al. demonstrated that a low dose of NO could sensitize tumors to radiotherapy, leading to a significant growth delay and an increase in mouse survival. A sensitive environment favors tumor-selective production of NO in response to nitrite systemic injection. This work opens new perspectives for the use of nitrite as a safe and clinically applicable radiosensitizing modality (Frerart et al. 2008).

Protective Effect of NO from Cytotoxic Stimuli NO is a pleiotropic mediator and signaling molecule that has been found to be involved in a growing number of cell functions. It has been described as a double-edged sword, inasmuch as it can exert opposite effects depending on its concentration and the cell type. Apoptotic cell death is inhibited by S-nitrosylation of the redox-sensitive thiol in the catalytic site of several apoptosis-regulating proteins such as caspases. NO probably has a pathophysiological role in carcinogenesis because cancer cells frequently express NOS. Anti-apoptotic effects are associated with low levels (10 nM–1 mM) of exposure from the activation of endogenous NO synthases and slow release rates from NO donors. Chronic inflammation, which is accompanied by activation of iNOS and generation of NO, is associated with cancer development in a variety of gastrointestinal diseases. Some reactive nitrogen species such as NO or peroxynitrate ONOO– cause nitrosation of sulfur-containing biological target molecules that interact readily with proteins or nucleic acids. Low levels of endogenous or exogenous NO by donors have been reported to prevent apoptosis in various tumor cells. Anti-apoptotic mechanisms for NO have been proposed as (1) inhibition of caspase-8, -9, and -3 by S-nitrosylation (Kim et al. 2000; Kim et al. 1997; Torok et al. 2002); (2) upregulation of anti-apoptotic proteins (such as heat shock protein 70, Bcl-2) (Azad et al. 2006; Choi et al. 2002); and (3) downregulation of cytochrome c, apoptosis-inducing factor (AIF), SMAC (Diablo), and endonuclease G (Estrada et al. 1997; Kim et al. 2002; LaCasse et al. 1998). There are several excellent reviews of the anti-apoptotic role of NO in cancer cell proliferation (Beltz et al. 2006; Brune et al. 1998; Choi et al. 2002; Ekmekcioglu et al. 2005; Kim et al. 2001; Kolb 2000; Mocellin et al. 2007; Rivoltini et al. 2002; Tarr et al. 2006). In this chapter, we review the anti-apoptotic effect of NO against spontaneous or induced tumor cytotoxicity (apoptosis) (Figs. 6.3 and 6.4).

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Fig. 6.3 Protective (anti-apoptotic) cascade by NO in tumor cells

Fig. 6.4 Protective (anti-apoptotic) cascade by NO in tumor cells

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Mechanism of Protective (Anti-apoptotic) Effect of NO There are numerous studies on the possible involvement of nitric oxide in multiple cancer types. These studies cover every step of cancer development including carcinogenesis, progression, invasion, metastasis, angiogenesis, escape from immune surveillance, and modulation of therapeutic response. Inhibition of Mitochondrial Permeability Transition Pores Mitochondrial cytochrome c release is regulated by the Bcl-2 family of proteins (Tsujimoto and Shimizu 2000), which are targeted at the mitochondrial permeability transition pore (PTP) whose multisubunit protein complex includes the mitochondrial voltage-dependent anion channel. NO inhibits the opening of PTP. Thus, NO exposure results in significantly lower cytochrome c release for the same degree of PTP opening in apoptotic cells (Brookes et al. 2000). Inhibition of the Caspase Family Caspase inhibition has been observed after exposure to NO in normal and tumor cells by chemical NO donors or NO generated by endogenous NOS. NO inhibited caspase-3, -8, and -9 activity by S-nitrosation in the catalytic site of the enzyme in cells. Moreover, transfection of the iNOS gene inhibited TNF-α-induced activation of caspase in cells (Torok et al. 2002). Inhibition of the Ceramide Pathway The ceramide–sphingomyelin pathway is an evolutionarily conserved signaling pathway. Ceramide potentiated TNF-α-induced TRADD recruitment and caspase-8 activity. NO inhibits TNF-α-induced U937 cells apoptosis by reducing the generation of ceramide. Recent evidence indicates that the cross talk of sphingomyelinases and their product ceramide with NO and its generating enzyme eNOS plays prominent roles during key pathophysiological processes such as inflammation, proliferation, death, and differentiation (De Nadai et al. 2000; Perrotta et al. 2005). Inhibition of Fas Signal Pathway Fas–Fas ligand ligation induces apoptosis pathways in tumor cells by cytotoxic T lymphocytes, NK cells, and macrophages. Fas downregulates the FLICE inhibitory protein (FLIP) as well as caspase-8 activation and apoptosis. NO inhibited all apoptosis in Fas signaling without significant effect on Fas and Fas-associated death domain (FADD) adapter protein levels. Downregulation of FLIP is mediated by a ubiquitin–proteasome pathway that is negatively regulated by NO. Therefore, S-nitrosylation of FLIP is an important mechanism which renders FLIP resistant to ubiquitination and proteasomal degradation by Fas-L (Chanvorachote et al. 2005).

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Endogenous NO Interestingly, various studies have shown that eNOS, nNOS, and iNOS can be involved in promoting or inhibiting the pathophysiological role of cancer. NOS activity has been detected in tumor cells of various histogenetic origins and has been associated with tumor grade, proliferation rate, and expression of important signaling components associated with cancer development such as the estrogen receptor. Increased NO generation in a cell may select for mutant p53 cells and contribute to tumor angiogenesis by upregulating VEGF. Inhibition of the Fas Signal Pathway NO also protected cells through the selective nitrosylation, and inhibition, of the protein kinase Cε (PKCε). PKCε interacted and phosphorylated the anti-apoptotic protein cFLIP. Inhibition of cFLIP recruitment to the DISC led to increased activation of caspase-8 and subsequently to apoptosis. Inhibition of PKCε using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis, suggesting that NO-mediated inhibition of PKCε plays an important role in the regulation of Fas-induced apoptosis (Dash et al. 2007). iNOS Inhibition by AMG iNOS was always expressed in melanoma cells from metastatic lesions. Inhibition of endogenous NO synthesis by aminoguanidine (AMG) led to downregulation of Bcl-2 protein levels and cell death by apoptosis. Inhibition of NO synthesis led to upregulation of mRNA levels of genes involved in the apoptosis pathway such as Bax, caspase-1, caspase-3, caspase-6, gadd45beta (a family of genes in the regulation of cell cycle progression and apoptosis, Zhang et al. 2005), mdm2 (an oncoprotein to inhibit p53-mediated growth arrest and apoptosis, Dilla et al. 2000), and TRAIL. By contrast, iNOS inhibition by AMG did not promote apoptosis in normal adult melanocytes (Salvucci et al. 2001). VEGF Promotes iNOS Expression Vascular endothelial growth factor (VEGF) significantly promoted the growth of breast carcinoma cells, particularly at the early stages of tumor development (Yoshiji et al. 1997). Increased nitric oxide synthase (NOS) activity was also observed in the VEGF (+) tumors. VEGF contributed to tumor growth through inhibition of apoptosis and increased NOS activity (Ambs et al. 1998; Harris et al. 2002). Inhibition of Proteasomal Degradation of Bcl-2 Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway. It is dependent on posttranslational modifications, such as ubiquitination and proteasomal degradation (Dimmeler et al. 1999). NO S-nitrosylates Bcl-2 and inhibits

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ubiquitin–proteasomal degradation of Bcl-2. Inhibition of NO increased ubiquitination of Bcl-2 S-nitrosylation and promoted apoptotic cell death. Fas ligand and buthionine sulfoxide also induced Bcl-2 S-nitrosylation. This general phenomenon regulates Bcl-2 stability and functions under various stress conditions. These findings indicate a novel function of NO and its regulation of Bcl-2, which provides a key mechanism for the control of apoptotic cell death and cancer development (Azad et al. 2006). Inhibition of IFN-Induced Apoptosis by NO IFN-γ •induces iNOS mRNA and protein and anti-apoptosis in human hepatoma cells. The iNOS inhibitor, L-NAME, sensitized hepatoma cells (HuH7, Hep3B) to IFN-γ (Vadrot et al. 2006). Anti-apoptotic Effect of Survivin iNOS and NO signaling in head and neck squamous cell carcinoma (HNSCC) and epithelial ovarian cancer (EOC) can both promote or inhibit tumor progression. RNAi-mediated depletion of survivin blocked NO anti-apoptotic effects and low NO levels induced resistance against cisplatin/taxol-induced apoptosis in HNSCC and EOC cell lines. These NO-dependent cytoprotections were mediated by survivin. The iNOS/survivin axis appears to also be of clinical relevance since immunohistochemistry revealed that iNOS expression correlated with enhanced survivin levels in HNSCC specimens. In contrast, high NO concentrations suppressed survivin levels in HNSCC and EOC, resulting in apoptosis. NOS and survivin expressions were associated with increased risk for disease progression. Pharmacogenetic iNOS/survivin-targeting approaches are being pursued as potential therapeutic strategies in HNSCC and EOC (Engels et al. 2008; Fetz et al. 2008).

Exogenous NO Anti-apoptotic Effect of NO Donors by Heme Oxygenase-1 Heme oxygenase-1 (HO-1) is a 32-kDa stress-induced enzyme that degrades heme to carbon monoxide (CO). Recent evidence suggests that HO-1 is an important cellular target of NO. Induction of HO-1 functions as an anti-apoptotic defense of the tumor against oxidative stress induced by NO. NO donor SNP induced HO-1 expression, and preincubation with SNP suppressed apoptosis triggered by anti-Fas antibodies. Treatment of rat hepatoma (AH 136B) cells with SNAP and P/NONO (propylamine NONOate) resulted in strong upregulation of HO-1 expression, and treatment of rat glioma cells (C-6) with SPER/NO also resulted in upregulation of HO-1. AH136B cells treated with the HO-1 inhibitor resulted in extensive changes in apoptosis of tumor cells both in vivo and in vitro (Pae et al. 2004; Tanaka et al. 2003).

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Anti-apoptotic Effect of NO Donors by Survivin The dual role of survivin as an apoptosis inhibitor and mitotic regulator has been associated in ovarian cancer. High amounts of the NO donors SNAP and SNP were able to suppress survivin levels via the p38MAPK pathway and were able to trigger apoptosis in neck squamous cell carcinoma (HNSCC) and epithelial ovarian cancer (EOC). Importantly, low NO concentrations conferred resistance against carboplatin/paclitaxel-induced apoptosis. Survivin-mediated cytoprotection was upregulated by low doses of the NO donors SNAP and SNP in HNSCC and EOC. In contrast, high NO concentrations suppressed survivin levels in HNSCC and EOC (Engels et al. 2008; Fetz et al. 2008).

Inhibition of Proteasomal Degradation of Bcl-2 Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway whose function is dependent on its expression levels. NO prevented Bcl-2 cleavage and suppressed cytochrome c release in TNF-α and actinomycin D-treated adenocarcinoma (MCF-7) cells exposed to SNAP (Kim et al. 1998). This level is regulated by a ubiqutination–proteasome degradation system. There was inhibition of NO production by the NO scavenger. The NO donors DPTA/NONO and sodium nitroprusside effectively upregulated Bcl-2 S-nitrosylation, decreased its ubiquitination, and inhibited apoptotic cell death induced by chromium. The effect of NO on Bcl-2 stability was shown to be independent of its dephosphorylation (Azad et al. 2006).

Inhibition of Apoptosis by Scavenging of Superoxide Anions In cells lacking a functional p53 tumor suppressor protein, the endogenous free radical NO inhibits apoptosis and promotes growth of cancer cells. SNP or SNAP inhibited apoptosis in HT-29 human colon carcinoma cells initiated by flavone. NO effectively inhibits apoptosis by scavenging superoxide anions generated in the mitochondria of p53 mutant cells and thereby prevents the downregulation of the anti-apoptotic factor Bcl-X(L), which controls the mitochondrial apoptosis pathway (Wenzel et al. 2003).

Inhibition of Apoptosis by Pleiotrophin Pleiotrophin is an 18-kDa heparin-binding growth factor named for its pleiotrophic effects. The described biological consequences for pleiotrophin-mediated signaling include neurite outgrowth, angiogenesis, and mitogenesis of fibroblast, endothelial, epithelial, and some tumor cell lines. SNP, through activation of soluble guanylate cyclase, significantly and concentration dependently increased expression of pleiotrophin (Polytarchou et al. 2009).

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Inhibition of Apoptosis by Caspase-9 Nitrosylation SNAP inhibits activation of caspase-9 in etoposide-treated cells. Extracted procaspase-9 from SNAP-treated cells by immunoprecipitation showed sixfold more than nitrosylation estimated by the Saveille reaction. Thus, the cellular procaspase-9 is susceptible to nitrosylation by NO (Torok et al. 2002). Inhibition of Apoptosis by the Ceramide Pathway The ceramide–sphingomyelin pathway is an evolutionarily conserved signal. Ceramide potentiated TNF-α-induced TRADD recruitment and caspase-8 activity. Interactions of NO and sphingolipids have been shown to involve regulation of the sphingolipid metabolic pathways. SNAP inhibits TNF-α-induced U937 cell apoptosis by reducing the generation of ceramide (De Nadai et al. 2000; Perrotta et al. 2005). Inhibition of Apoptosis by Fas Signal Pathway SNP and DPTA/NONO inhibited all apoptotic process in Fas signaling without a significant effect on Fas and FADD adapter protein levels. Fas-L-induced downregulation of FLIP is mediated by a ubiquitin–proteasome pathway that is negatively regulated by NO. S-Nitrosylation of FLIP is an important mechanism that renders FLIP resistant to ubiquitination and proteasomal degradation by Fas-L (Chanvorachote et al. 2005).

Tumor Microenvironment To better understand tumor growth mechanisms, the knowledge of NO functions in tumor microenvironment should be helpful. The tumor microenvironment is an essential element in developing tumor malignancy. Macrophage reprogramming shifts responses from a M1 (the pro-inflammatory and killing phenotype) to a M2 (the anti-inflammatory and pro-angiogenic phenotype). Decreased oxygen availability is a typical characteristic of tumor tissues and evokes adaptive responses to cause hypoxia-inducible factor-1 (HIF-1). Hypoxic cores of a growing tumor cell mass generate apoptotic tumor cells which release sphingosine-1-phosphate (S1P). This reprograms macrophages to the M2 phenotype. M2 macrophages produce EGVF, FGF, TGF-β and act as a healer in tumor tissues. NO and hypoxia share the ability to stabilize HIF-1α and mimic hypoxia. In this section, we described the functions of NO in promoting tumor growth in the tumor microenvironment. Macrophage Reprogramming to M2 Phenotype by S1P The M1 phenotype is typified by IFN-γ• primed macrophages and is characterized by increased MHC class II, IL-12, and iNOS expression. M2 macrophages are

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characterized by an elevated expression of anti-inflammatory cytokines such as IL-10 (Gordon 2003). The M2 phenotype is promoted by Th2 cytokines such as IL-4 and IL-13. Generally, M2 macrophages are considered as anti-inflammatory cells, whereas M1 macrophages are pro-inflammatory. In tumor tissues, tumorassociated macrophages (TAM) are the major stromal cells, comprising up to 50% of the cell mass in several solid tumors (Condeelis and Pollard 2006; Sica et al. 2006). Tumors start to develop TAM and exhibit an immunosuppressive M2 phenotype. TAM shows defective expression of inflammatory cytokines, iNOS, and a consequent decrease in tumoricidal activity. Recently, IKKβ has been shown to inhibit the “classically” activated (M1) macrophage phenotype during infection through negative cross talk with the Stat1 pathway (Hagemann et al. 2008). Moreover, apoptotic cells release factors such as sphingosine-1-phosphate (S1P) that also reprograms macrophage phenotypes. S1P is known for its role of inducing transformation, cancer cell growth, or angiogenesis (Saddoughi et al. 2008). M1 macrophages are reprogrammed to the M2 phenotype, which is anti-inflammatory and pro-angiogenic (Weigert and Brune 2008). Hagemann et al. demonstrated that depletion of IKKβ signaling in TAMs promotes regression of advanced tumors in vivo by induction of macrophage tumoricidal activity with upregulation of iNOS gene and NO (Hagemann et al. 2008). This stimulates innate immune responses. TAM activation by NF-κB or S1P targeting causes iNOS induction and NO release and represents a powerful therapeutic tool in cancer therapy. Upregulation of Chemoresistant Future of Tumor by Stromal Fibroblast iNOS When the chemosensitive human pancreatic carcinoma cell lines T3M4 and PT45P1 were kept in coculture with fibroblasts, both cell lines became much less sensitive toward treatment with etoposide. The chemoresistant future of T3M4 and PT45P1 cells was increased by the NO production from neighboring fibroblasts which exhibited significant iNOS expression and NO secretion. This suggests that there were tumor–stromal fibroblast interactions in the chemoresistance of pancreatic carcinoma due to NO production (Muerkoster et al. 2004). Shedding of MHC Class I by NO Under Hypoxic Tumor Conditions Tumor hypoxia is associated with poor clinical outcomes for cancer patients and contributes to tumor cell shedding of MHC class I chain through a mechanism involving impaired nitric oxide (NO) signaling. The molecules play important roles in tumor immune surveillance through their interaction with the NKG2D receptors on natural killer and cytotoxic T cells. Pharmacologic inhibition of endogenous NO signaling increased MIC shedding. GTN attenuated the growth of xenografted MHC class I chain related-expressing human prostate tumors. The hypoxic tumor microenvironment contributes to the resistance to immune surveillance, suggesting that activation of NO signaling is of potential use in cancer immunotherapy (Kashiwagi et al. 2008).

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Normalization of Tumor Vasculature Is an Emerging Strategy to Improve Cytotoxic Therapies Eliminating nitric oxide (NO) production from tumor cells via nNOS silencing or inhibition establishes perivascular gradients of NO in human glioma xenografts in mice and normalizes the tumor vasculature. This results in an improved tumor oxygenation and response to radiation treatment. Creation of perivascular NO gradients may be an effective strategy for normalizing abnormal vasculature (Kashiwagi et al. 2008).

Concluding Remarks and Future Directions This chapter has introduced the new research that has addressed the mechanisms of NO-induced cytotoxicity and cytoprotection (apoptosis and anti-apoptosis). The current studies on NO cytotoxicity (apoptosis) suggest new mechanisms for tumor cells apoptosis. New anti-tumor drugs may be developed using new NO donors, especially NONOate. Conversely, while there has been much excellent research on NO cytoprotection (anti-apoptosis) during the 1995–2000 period, there has been a decrease in this type of research in the past several years. There are many discussions on the specificity for cytotoxicity reactions and cytoprotective effects of NO. The behavior of NO has opposing aspects and is like a double-edged sword. The physical structure of the NO gas causes non-specific nitrosylation of target proteins. Most nitrosylated proteins showed hypofunction. Thus the anti-apoptosis function of NO is caused by nitrosylation of apoptosis-inducing proteins. In addition to the cytotoxicity functions, NO also causes nitrosylation of apoptosis inhibitory proteins. Thus, when NO is present, it is difficult to predict the cellular fate. The biological plasticity appears to have potential value and deserves further investigation. It is currently difficult to regulate localization of NO production by the systemic administration of NO donors. The manipulation of NO donors in tumor tissues also requires research. There is a large variety of potential NO donors that may allow for localization in tumor tissues. It can be anticipated that there will be a new era for anti-tumor therapy that employs NO in combination therapies with radiation, chemotherapeutic agents, and cytokines for conventional therapy-resistant tumors.

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Chapter 7

Cytotoxic/Protective Activity of Nitric Oxide in Cancer Eun-Kyeong Jo, Hyun-Ock Pae, Yong Chul Lee, and Hun-Taeg Chung

Abstract Nitric oxide (NO) is an important signaling molecule that plays a significant role in the regulation of cancer biological functions. Accumulating evidences have reported two facets of NO as a possible mediator of cancer development and anti-cancer therapeutics. The paradoxical action of NO in cancer biology depends on concentration, chemical variability, cancer microenvironment, etc. As an anti-cancer therapeutic, NO may be useful as in vivo chemo-sensitizers or radiosensitizers to target chemo- and radioresistant tumors. However, continuous exposure to NO is associated with neoplastic transformation and metastasis through induction of cancer proliferation, invasion, and expression of angiogenic factors. Moreover, recent advances suggest that the expression of iNOS can serve as a prognostic marker. Therefore, an approach to modulate NO production and regulation of signaling pathways for iNOS expression may open up new avenues of research by allowing a potential approach to effective treatment of cancer. Keywords Nitric oxide · Tumorigenesis · Cytotoxicity · Tumor microenvironment

Cytotoxic/Protective Activity of Nitric Oxide in Cancer Nitric oxide (NO) is a hydrophobic and a soluble gaseous molecule that plays multiple physiological functions, including vasodilatation, neuronal transmission, smooth muscle relaxation, and immunity. NO is synthesized from L-arginine and oxygen by four major isoforms of NO synthase (NOS) family: neuronal NOS, endothelial NOS (eNOS), inducible NOS (iNOS), and, more recently, mitochondrial NOS (Chartrain et al. 1994; Marsden et al. 1993; Ying and Hofseth 2007). Both nNOS and eNOS are constitutively expressed in selected tissues and are transiently activated by an increase in intracellular Ca2+ (Kavya et al. 2006; Li and Wogan 2005). In contrast, iNOS expression is induced by various stimuli, including interferon (IFN)-γ, tumor H.-T. Chung (B) School of Biological Sciences, Ulsan University College of Natural Sciences, Daehackrho 102, Namgu, Ulsan 680-749, Republic of Korea e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_7, C Springer Science+Business Media, LLC 2010

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necrosis factor (TNF)-α, and interleukin (IL)-1β, endotoxin (e.g., lipopolysaccharide), and the hypoxia-inducible factor (HIF)-1-mediated pathway (Geller et al. 1993). The iNOS expression is regulated independently of free Ca2+ and produces NO that can persist for several days (Alderton et al. 2001). Low levels of NO can function as a signaling molecule in many physiological processes (Ying and Hofseth 2007). In contrast, the potential toxic effects of this soluble gas have been reported in the infection or inflammations through the induction of iNOS (Ying and Hofseth 2007). If the iNOS floods the tissue and increases NO concentrations to toxic levels, NO diffuses to neighboring cells, bacteria, and viruses and damages foreign molecules or modified self (McCann et al. 2005). It is now thought that low concentrations of endogenous NO may increase tumor cell proliferation, whereas high concentrations of inducible origin NO are associated with tumor cell cytotoxicity and apoptosis (Rao 2004). The two facets of NO as an inhibitor or enhancer of tumor development have been continuously defined and reported. The principal concept required for understanding of paradoxical actions of NO in the tumor biology seems to be that of heterogeneity. As well as its concentration, chemical variability and microenvironment associated with cellular adaptation/selection to the cytotoxic actions of NO may determine the complicated cellular effect of NO. In this chapter, both cytotoxic and protective roles of NO will be discussed.

Direct Role in Cytotoxicity Endogenous NO In the past decades, macrophage-mediated tumor cell killing and its mechanisms have been extensively studied. Fully activated macrophages (MacKay and Russell 1986) can provide a defense line against microbial invasion and tumor cells in a direct manner. This process involves the production of oxygen radicals, TNF-α, and NO that are harmful to microorganisms and/or cancer cells. Several clinical trials have been performed to accomplish antitumor therapeutics through activated macrophages or dendritic cells (Klimp et al. 2002). Macrophage-mediated antitumor effects through NO in rodents have been well established (Cui et al. 1994; Farias-Eisner et al. 1994; Keller et al. 1990), although the roles of NO remain controversial. The iNOS gene is maximally induced through two signals: IFN-γ and other stimulus such as an endotoxin, TNF-α, or IL-1β (Fitzpatrick et al. 2008; Kamijo et al. 1994; Martin et al. 1994; Saura et al. 1999). The interaction between two transcription factors nuclear factor (NF)-κB and interferon regulatory factor (IRF)-1 may lead to modifications in the physical structure of the NOS 5 flanking region, thus contributing to the synergism in the activation of iNOS expression in macrophages (Saura et al. 1999). In tumors which have often hypoxic conditions in the central areas, the molecular interaction between HIF-1α and IRF-1 could induce iNOS expression in macrophages, leading to NO-mediated apoptosis of tumor cells (Tendler et al. 2001).

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The conditions or secretion pathways for NO production in human monocytes/ macrophages are different from rodent system. In addition, the secretion of NO and NO metabolites by human cells was substantially lower than that by rodent macrophages (Albina 1995). When human monocytes were stimulated with various cytokines such as IFN-γ and TNF-α –similar conditions for rodent cells resulted in release of large amounts of NO (Zembala et al. 1994) – they did not induce NO secretion. On the other hand, NO was secreted in human monocytes, upon coincubation with tumor cells (Zembala et al. 1994).

Exogenous NO Nitric Oxide Donors The emerging compounds, NO-donating non-steroidal anti-inflammatory drugs (NO-NSAIDs), are known to be efficacious for treatment and prevention of colorectal and other forms of cancer, although they did not show the same effect on all given cell lines (Rigas and Kashfi 2004). NO-NSAIDs have several key features, including NO effects on the enhancement of potency of NSAIDs for inhibiting growth of cancer cells from various tissue origins (Rigas and Kashfi 2004). The development of NO-NSAIDs was based on a structural coupling of a NO-donating moiety of an NSAID (del Soldato et al. 1999; Rigas and Kashfi 2004), resulting in a NO release to the site of gastric damage induced by an NSAID so as to overcome the limitations of traditional NSAIDs (Kaza et al. 2002). To date, NO-aspirin (NO-ASA) derivatives have been reported as the most potent chemopreventive agents among the NO-NSAIDs studied (Rigas and Kashfi 2004; Rigas and Kashfi 2005). In several experiments, NO-ASA derivatives seem to be at least 100- to 1000-fold more active in inhibiting the colon cancer cell growth than other NO-NSAIDs (Yeh et al. 2004). They may be synthesized as one of three different positional isomers, ortho-, meta-, or para-, depending on where the nitrogen-donating (CH2 ONO2 ) group is attached to the benzene spacer (McIlhatton et al. 2007). In the last decades, in vitro NO-donating NSAIDs have been widely studied about, which are more effective than conventional NSAIDs in induction of apoptosis and inhibiting tumor cell proliferation (Tesei et al. 2005; Williams et al. 2001). This effect is thought to be attributable to an additional NO group. Indeed, NO-releasing derivatives, such as NCX 4040, but not its parental compound aspirin, showed an in vivo anti-cancer effect on the human colon cancer xenografts (Tesei et al. 2005). In addition, much effort has been made to find promising lead compounds in the search for new classes of NO-releasing antineoplastic agents. O2 (2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active prodrug that can target NO to the leukemia cells (Kitagaki et al. 2008). This antiproliferative agent had an effect against HL-60 cells, human myeloid leukemia cell line, at relatively low concentrations (for example, an IC50 of 0.006 mM for a cell-permeant prodrug that is activated for NO release by intracellular esterases) (Saavedra et al. 2000; Shami et al. 2003). Recent studies have

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revealed the novel mechanisms of apoptotic cell death by NO prodrug JS-K through inactivation of the ubiquitin system by blocking E1 (ubiquitin-activating enzyme) activity via inhibition of the ubiquitin-E1 thioester formation (Kitagaki et al. 2008). HIF-1, an essential factor of many hypoxia-regulated genes, promotes carcinogenesis independently of histogenetical origin (Acs et al. 2003; Griffiths et al. 2007). NO is able to inhibit the HIF-1 activity (Sogawa et al. 1998) and to stimulate the expression of vascular endothelial growth factor (VEGF) by stabilizing HIF-1α expression and activation of HIF-1 complex (Kimura et al. 2000). However, there is a contradiction among effects of various NO donors. It was also reported that the NO donors including S-nitroso-N-acetyl-penicillamine (SNAP), S-nitroso-glutathione (GSNO), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18) up-regulated the VEGF reporter gene activity and also induced HIF-1α accumulation and HIF-1 DNA-binding activity (Kimura et al. 2000). Therefore, the role of NO donors in the HIF-1 regulation has not been exactly clarified.

Tumor Microenvironment NO is a multifunctional signaling molecule in the regulation of tumor microenvironment interactions, because of its potential generation from eNOS within endothelial cells, which are associated with hyperdynamic circulation and angiogenesis during cancer environment. Although the precise role of NO has not been completely understood, recent studies have suggested that a NO gradient within the tumor microenvironment affects a progression of cancer through orchestrated controlling of the molecular interactions between cancer cells and stroma. For example, a recent study by Decker et al. (2008) has shown that the mice with an eNOS deficiency showed higher frequency of tumorigenesis in response to carcinogens and associated with a greater cumulative tumor burden, when compared with control animals. Mechanistically, this was achieved by NO-dependent down-regulation of the matrix protease cathepsin B, and silencing of cathepsin B attenuated tumor cell invasive capacity (Decker et al. 2008). Hypoxia, a major signature of the tumor microenvironment, is a principal cause of clinical radioresistance and local failure in tumor microenvironment. Hypoxia in solid tumor promotes activation of HIF-1α in cancer cells and results in an acidic extracellular milieu which contributes to tumor growth, invasion, and development (Swietach et al. 2007). HIF-1α is an essential transcriptional factor encoding numerous genes involved in erythropoiesis, angiogenesis, and induction of glycolytic enzymes in tumor tissues (Yasuda 2008). Hypoxia and accumulation of HIF-1α have been reported to modulate cancer cell cycle, cancer proliferation, and tumor metastasis by activation of VEGF and glycolytic enzymes (Semenza et al. 1994; Yasuda 2008). Therefore, HIF-1α has been considered to be a potentially promising target for tumor radiosensitization (Moeller and Dewhirst 2006). Recently, NO and iNOS are considered to be an alternative strategy that takes advantage of the microenvironment in a variety of solid tumors for tumor-specific radiosensitization (De Ridder et al. 2008b). The local production of NO at high

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rates inside tumor cells efficiently radiosensitizes the hypoxic tumor cells at nontoxic extracellular concentrations of NO (De Ridder et al. 2008a). Chronic hypoxia up-regulated the mRNA and protein expression of iNOS, and cooperation with cytokines, such as IFN-γ and IL-1β, is required to achieve full iNOS expression (Berge et al. 2001). It was suggested that both macrophages and tumor cells are affected by paracrine IFN-γ loop, resulting in potentiating the release of NO (Martin et al. 1994; Onier et al. 1999). Numerous studies have reported that hypoxic tumor cells were efficiently radiosensitized by NO, released either from chemical NO donors or synthesized inside the tumor cells through the iNOS/l-arginine pathway (Berge et al. 2001; De Ridder et al. 2004; Janssens et al. 1998; Janssens et al. 1999; Mitchell et al. 1993; Verovski et al. 1996). Although the picture of the complex interaction between tumor cells and macrophages is far from being complete, macrophages may play a role in an endogenous source of TNF-α and NO, thus providing immunostimulatory and radiosensitizing effects. Therefore, it was proposed that the pro-inflammatory infiltrate in tumor environment may contribute to radiosensitizing strategies through two mechanisms: (1) tumor-infiltrating immune cells (macrophages, T/NK-cells) are a major source of cytokines in the induction of iNOS/NO pathway inside tumor cells; (2) tumor-associated macrophages themselves can produce the massive amounts of NO that can radiosensitize bystander tumor cells. Ongoing research will clarify the exact role of immune infiltrates in tumor environment in terms of immunostimulatory and radiosensitizing strategies.

Protective Effect of NO from Cytotoxic Stimuli Targeting iNOS, an enzyme with high NO output, has been extensively studied and reviewed for cancer prevention and treatment. However, the exact role of iNOS is complex and remains unclear because numerous studies showing the paradoxical outcomes for the antitumor and pro-tumor consequences of iNOS expression or inhibition (Lancaster and Xie 2006). When the level of iNOS activity is lower than the levels associated with cytotoxicity and apoptosis, NO signals and iNOS expression in the intratumoral environment may lead to monocyte/macrophage recruitment and provide a positive growth signal within a tumor microenvironment (Thomsen and Miles 1998). Prolonged activation of inflammatory cells is able to damage host DNA and tissues through production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), thus contributing to carcinogenesis (Ohshima et al. 2003). In addition, several lines of evidence suggest a pro-tumor action of NO, when released at a very low level. Physiologic, low (nanomolar, nmol/L) concentrations of NO are consistently detected in tumor microenvironment and have been found to promote tumorigenesis (Ekmekcioglu et al. 2005; Siegert et al. 2002; Stepnik, 2002). NO production, probably by eNOS, can promote precancerous condition, tumor migration, and endothelial cell proliferation and differentiation (Ohshima et al. 2003; Ying and Hofseth 2007). Specifically, eNOS is a NOS isoform with

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low levels of production of NO. Although still controversial, it seems that eNOS plays a pro-tumorigenic role (Ohshima et al. 2003). Endogenous NO Chronic inflammation is associated with a risk factor for tumorigenesis. During inflammation, activated inflammatory cells can actively release RNS, ROS, lytic enzymes, and cytokines. Free radicals including RNS are ubiquitous in our body and are well known to induce inflammation and tumorigenesis in target tissues (Ibuki and Goto 1997). NO and iNOS have been recognized as a strong mediator for inflammation and subsequent damage of DNA and protein modification, which might be involved in pro-tumor effects (Fukumura et al. 2006; Lancaster and Xie 2006). Specifically, human iNOS gene is transcriptionally regulated by cytokine mixtures such as IFN-γ, TNF-α, IL-1β, and IL-6, thereby resulting in enhanced NO production (de Vera et al. 1996). Pro-inflammatory cytokines play a dual role during carcinogenesis, promoting or inhibiting neoplastic progression dependent on the inflammatory microenvironment (Ren et al. 2008). The inflammatory cytokines crucially function in the initiation and maintenance of cellular senescence, which can be induced prematurely in both normal and transformed cells by a variety of stress challenges, and are highly associated with cancer development (Ren et al. 2008). Numerous inflammatory mediators produced by senescent cells are known to enhance angiogenesis and oncogenic proliferation, invasion, and cancer metastasis (Ren et al. 2008). IL-1, a pluripotent inflammatory cytokine, is highly expressed in various human tumor specimens including non-small-cell lung carcinoma, colorectal adenocarcinoma, and melanoma tumor samples. IL-1 gene is frequently expressed in metastases from patients with several types of human cancers (Elaraj et al. 2006). Recent studies using mouse models of human cancer indicate that smoldering and polarized inflammation are involved in both tumor promotion and tumor progression (Balkwill et al. 2005; Karin, 2006; Karin and Greten 2005). TNF-α is a potent inflammatory cytokine, produced by LPS stimulation in chronic inflammatory diseases (Lin and Yeh 2005), and is suggested of its tumor-promoting effects by some pre-clinical studies (Mocellin et al. 2005). Moreover, NO can activate cyclooxygenase-2 (COX-2), which is highly associated with the progression of a variety of cancers through induction of prostaglandin synthesis (Goodwin et al. 1999; Mei et al. 2000). Recent studies have shown that treatment with Corynebacterium parvum induced inflammation, increased iNOS expression, and accelerated tumorigenesis, mostly T-cell lymphoma, in p53-deficient mice with intact iNOS (Hussain et al. 2008). C. parvum-treated p53−/− iNOS+/+ mice showed an increased number of splenocytes and Foxp3-positive regulatory T cells, which may contribute to a pro-tumor shift of the immune environment favoring an accelerated tumor development (Hussain et al. 2008). These studies indicate that a role of NO is associated with acceleration of tumor development in an inflammatory microenvironment. These recent insights might pave the way to further investigation of a novel approach to cancer therapy by modulating cytokines and NO.

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Exogenous NO NO Donors Recent study by Pervin et al. (2007) has shown that NO (nmol/L) induced by DETA-NONOate, a long acting NO donor, dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 cells and also increased cell proliferation. Following NO donor treatment, cellular targets including translation of cyclin D1 and ornithine decarboxylase (ODC) were increased; several signaling machineries were up-regulated including phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were also up-regulated in these cells (Pervin et al. 2007). Furthermore, the same study has shown that Ras activation is involved in the NO-induced increase in proliferation signaling and cyclin D1 and ODC translation (Pervin et al. 2007). Metastasis to regional lymph nodes is a common step in tumor progression. Earlier study showed a contributory role of NO in tumor metastasis using experimental tumor models (Edwards et al. 1996). Recent evidence by Yasuoka et al. showed that incubation of K1 and B-CPAP PTC cells with an NO donor resulted in induction of functional CXCR4 expression in a NO-dependent manner (Yasuoka et al. 2008). Tumor Microenvironment The involvement of NO in tumorigenesis may depend on the quantity of NO and the cellular microenvironment in tumor development. NO and NOS are ubiquitously detected in malignant tumor cells. The effect of NO on tumor progression depends on its source (tumor cells or stromal cells), type, and activity of NOSs (Fukumura et al. 2006). Although it depends on tumor cell type and stage, cancer cells often express iNOS and in some cases eNOS and nNOS. The iNOS is mainly expressed in tumor-associated stromal fibroblasts and immune cells. The positive or negative effects of NO during tumor progression depend on its source, type, and activity of NOSs (Fukumura et al. 2006). Although studies on eNOS in carcinogenesis are just starting, evidence is accumulating that low, but pathologically relevant, levels of NO from eNOS might act in carcinogenesis (Ying and Hofseth 2007). In addition, eNOS is predominantly expressed in tumor vascular endothelial cells or surrounding stromal cells and therefore has been focused in angiogenesis (Fukumura et al. 2006; Ying and Hofseth 2007). The expression levels and activities of the different NOSs are upregulated in many tumors, compared with corresponding normal tissues (Fukumura et al. 2006). Radiation therapy induced the expression and activation of eNOS in a dose-dependent manner, suggesting a potential application of the coordinated use of antiangiogenic strategies and radiotherapy in clinical practice (Sonveaux et al. 2003). Future studies using eNOS−/− animal models and gene transfer or small interfering RNA (siRNA) against eNOS into specific tissues or cells can pave a way to research on the assessment of carcinogenesis (Ying and Hofseth 2007).

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Notably, a series of studies have indicated that NO produced by iNOS can also initiate and/or promote tumorigenesis (Crowell et al. 2003; Hofseth et al. 2003). NO-derived RNS and NO promote DNA damage in the cells and inhibition of DNA repair enzymes (such as alkyl transferase and DNA ligase) (Lala and Chakraborty 2001; Wink et al. 1998). Regardless of its sources, continuous exposure to NO is thought to promote neoplastic transformation and metastasis by direct induction of tumor cell proliferation, migration, and invasion and indirectly through the expression of angiogenic and lymphangiogenic factors in tumor cells. It was reported that high levels of NOS expression were found in malignant neoplasms of the central nervous system and endothelial cells (Cobbs et al. 1995). In addition, the expression of iNOS was closely related to tumor angiogenesis and high incidence of metastasis and poor prognosis in gastric and colorectal carcinomas (Lagares-Garcia et al. 2001; Song et al. 2002). It has been reported that the expression of iNOS may serve a prognostic marker for its risk of metastasis of gastrointestinal tumors, which could provide a new approach to tumor therapy. In addition, modulation of NO production and/or its downstream signaling pathways may provide potential strategies for cancer treatment (Fukumura et al. 2006). A summarized model for cytotoxic/protective roles of NO is shown in Fig. 1.

Fig. 7.1 Schematic representation of the cytotoxic/protective roles of NO in cancer. The complicated outcome of cytotoxic/protective activity of NO depends on the concentration of NO and microenvironment with tumor cells and tumor-associated macrophages. High concentrations of NO, principally induced by iNOS, may induce anti-cancer effects in tumor cells, whereas low, but continuous, concentrations of NO, mainly induced by eNOS, may result in pro-cancer effects. NO has also the potential to enhance both radiotherapy and chemotherapy

Concluding Remarks and Future Directions NO is a ubiquitous molecule that exerts a variety of biological effects. NO can play an important role as a new onco-preventive agent in cancer development and in

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tumor therapeutics. A variety of NO donors may serve as a tool of investigating the anti- or pro-neoplastic properties of NO in cancer. Accumulating evidence has indicated that NO functions as a novel therapeutic when used as a single agent or when used in combination as a sensitizing agent to target radio- and chemo-resistant hypoxic tumor cells. However, the quantity of NO and the tumor microenvironment may influence the role of NO in tumor development. Indeed, NO is involved in both cytotoxic and anti-apoptotic effects on tumor cells. Therefore, the balance between NO-mediated cellular proliferation and cytotoxicity will determine the outcome of tumorigenesis or antitumor effects. High, but physiologically relevant, concentrations of NO may direct cancer cells to be apoptotic, whereas lower concentrations of NO can cause cancer cells to be anti-apoptotic. It is also conceivable that its ultimate manifestation may result from complicated interactions depending on the tumor cell types studied and/or its sensitivity to NO. Given the role of anti-cancer effect of NO, one may consider its clinical application as a chemo-sensitizing or immunosensitizing agent which might present new challenges in cancer therapy. On the other hand, the procarcinogenic role of NO may raise a possibility of targeted blocking of NOS (e.g., eNOS–siRNA) as a useful chemotherapeutic or chemopreventive strategy against cancer.

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Chapter 8

Nitric Oxide and Life or Death of Human Leukemia Cells J. Brice Weinberg

Abstract Nitric oxide (NO) has many actions that affect physiologic and pathologic processes. These effects are important in maintenance of appropriate smooth muscle tone, learning and memory, cell signaling, and defense from infection and neoplasia. NO is an important regulator of apoptosis and death of both normal and malignant cells, and it can serve as a mediator of inflammation, mutagenesis, and carcinogenesis. NO produced by bone marrow “stromal” cells (e.g., fibroblasts, macrophages, fat cells, and endothelial cells) can influence growth and differentiation of normal hematopoietic cells. In addition, hematopoietic cells themselves can produce NO that might influence these processes. As the importance of NO was being noted in vascular biology in the early 1980s, NO was also being recognized as the key mediator of macrophage-mediated neoplastic cell stasis and death. NO from cells or from NO pro-drugs can induce apoptosis and death of many neoplastic cells in vitro in an immunologically nonspecific manner. NO can also inhibit apoptosis and death of tumor cells by blocking activity of caspases. Malignant cells may express NO synthase (NOS), and NOS inhibitors induce apoptosis of these cells in vitro under certain conditions. High levels of NO (micromolar) cause apoptosis and death of normal bone marrow hematopoietic cells in vitro. Likewise, high levels of exogenous NO mediate cytotoxicity for acute leukemia cells and chronic lymphocytic leukemia (CLL) cells in vitro. CLL cells overexpress inducible NOS (NOS2) and neuronal NOS (NOS1), and a variety of NOS inhibitors effectively kill CLL cells in vitro. NOS1 inhibitors are the most potent at inducing cytotoxicity in CLL cells in vitro. The cytotoxicity may be caused by prevention of an NO-mediated block in caspase-mediated apoptosis of the leukemia cells. Investigators are working to move results of preclinical studies into clinical trials of NOS inhibitors or novel NO pro-drugs for treatment of human leukemias.

J.B. Weinberg (B) Departments of Medicine and Immunology, Duke University and Veterans Affairs Medical Centers, 508 Fulton Street, Durham, NC, USA e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_8, C Springer Science+Business Media, LLC 2010

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Keywords Nitric oxide · Apoptosis · Caspase · Leukemia · Chronic lymphocytic leukemia Abbreviations AR-17477 7-Nitro Vinyl-L-NIO 1,3 PB-ITU L-NIL

N-(4-(2-((3-chlorophenylmethyl) amino) ethyl) phenyl)-2thiophe-carboxamidine dihydrochloride 7-nitroindazole N 5 -(1-Imino-3-butenyl)-l-ornithine S,S -(1,3-Phenylene-bis(1,2-ethanediyl))bis-isothiourea ·2HBr L-N 6 -(1-Iminoethyl)-lysine

Introduction Macrophage-mediated cytotoxicity for tumor cells (an antibody-independent, immunologically nonspecific stasis and killing of cancer cells) was studied intensely in the 1970s. Researchers in the 1970s and 1980s gained information that demonstrated (in retrospect) that the effector molecule(s) had the characteristics of nitric oxide (NO). Hibbs and others studied macrophage-mediated tumor cell cytotoxicity in detail, showing that animals with chronic infection with intracellular organisms such as bacillus Calmette-Guerin (BCG), Listeria monocytogenes, and Toxoplasma gondii had macrophages that were static and lytic for tumor cells in vitro and in vivo (Hibbs 1973, 1974; Hibbs et al. 1978; Hibbs et al. 1972; Hibbs et al. 1977). The killing was dependent on close contact between the macrophage and the target tumor cell. Many different neoplastic target cells (including the mouse T cell leukemia line L1210) were killed. Researchers were unable to identify or purify a soluble cytotoxic mediator. The killing did not appear to be mediated by reactive oxygen species. Anti-tumor activity paralleled anti-microbial activity (Nathan and Hibbs 1991), and the cytotoxicity was inhibited by erythrocytes, hemoglobin, and iron (Weinberg and Hibbs 1977). Malignant cells exposed to cytotoxic, activated macrophages had diminished cell proliferation, blebbing of the cytoplasm (similar to that seen in apoptosis), inhibition of energy generation, inhibition of respiration and the mitochondrial electron transport enzymes, and death (Drapier and Hibbs 1986; Granger and Lehninger 1982; Granger et al. 1980). The cytotoxic activity for tumor cells was dependent on L-arginine, and L-arginine analogues markedly inhibited macrophage-mediated tumor cell cytotoxicity (Hibbs et al. 1987). Hibbs and co-workers eventually identified the (or a) cytotoxic factor as NO (Hibbs et al. 1988). Separate methodical and detailed work by Nathan and colleagues almost simultaneously confirmed and subsequently extended these observations (Macmicking et al. 1997; Stuehr and Nathan 1989). Since this early work, many investigators have studied the anti-cancer/antileukemia actions of NO and have also characterized pro-cancer/pro-leukemia actions of NO. This review summarizes topics related to NO and leukemia.

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NO Generalities NO is a lipid soluble, gaseous free radical produced with enzymatic conversion of Larginine to L-citrulline. NO is unstable within cells with a half-life measured in milliseconds to seconds. The short NO half-life results from its reactions with oxygen, transition metal ions, and thiols (Stamler et al. 2000; Thomas et al. 2008). Reaction of NO with oxygen converts NO to nitrite and nitrate ions, relatively stable catabolites that can be readily measured as surrogate markers of NO production (Granger et al. 1999). NO also reacts with superoxide, and superoxide dismutase (SOD) prolongs NO life by eliminating superoxide. NO reaction with free sulfhydryls may generate nitrosothiols (Foster et al. 2003). NO binds with high affinity to iron in heme groups of proteins such as hemoglobin (Hb), myoglobin (Mb), and guanylyl cyclase. The binding to iron (and other metals) quenches NO action. NO forms peroxynitrite on reacting with superoxide. Peroxynitrite is a very reactive and toxic molecule (Pacher et al. 2007). NO quenchers/scavengers inhibit the actions of NO in a variety of systems (Fricker 1999; Harbrecht 2006). Effective quenchers include heme-containing proteins (e.g., Hb and Mb), iron-containing complexes (e.g., iron– diethylenetriaminepentaacetic acid, iron ferrioxamine B complexes, or ruthenium complexes), and cobalt-containing compounds (e.g., hydroxocobalamin) (Brouwer et al. 1996; Fricker 1999; Harbrecht 2006).

NO Synthases NO is produced from L-arginine by three NOS isoforms in humans that are encoded by separate genes. NOS1 (“neural” NOS) and NOS3 (“endothelial” NOS) generally produce low levels of NO and are constitutively expressed, while inducible NOS (NOS2) is induced by cytokines and microbial factors. In human cells, NOS2 produces NO in response to several stimuli including IFN-α, IFN-γ, IL-1, TNF-α, IL-6, and endotoxin (LPS) (Thomas et al. 2008; Weinberg 1998). NOS3 is constitutively expressed, and it plays a major role in regulating vascular tone. NOS2 is seen in many cell types, but is prototypically noted in macrophages, hepatocytes, and chondrocytes. It is regulated transcriptionally. NOS2 can produce very high levels of NO (micromolar amounts). NOS1 is found in nervous tissue cells, muscle cells, and testicular cells. It is expressed constitutively, and its activity (like NOS3) is tightly regulated by calcium and calmodulin. NOS1 produces very small amounts of NO (low nanomolar amounts), levels capable of acting in signaling, for example, but generally not high enough for other functions such as cytotoxicity. While NOS1 and NOS3 are thought of as constitutive enzymes, both can also be regulated at the level of transcription. Regulation of NOS2 occurs primarily transcriptionally, but regulation can occur at multiple steps (Nathan and Xie 1994) including mRNA transcription, mRNA stability, and mRNA translation. Both NOS2 and NOS3 have been detected in human B lymphocytes (Levesque et al. 1998; Levesque et al. 2003; Mannick et al. 1994; Mannick et al. 1997; Reiling et al. 1996; Zhao et al. 1998).

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NOS3 mRNA and protein (RT-PCR and histochemistry) have been found in tonsilderived B cells and in the Daudi and Raji B cell lines (Reiling et al. 1996 ). NOS2 mRNA and protein have been detected in EBV-negative and EBV-positive human B lymphoma cell lines (Mannick et al. 1994; Mannick et al. 1999; Mannick et al. 1997). NOS2 in these cell lines is functional as evidenced by its ability to produce NO that inhibits reactivation of latent Epstein-Barr virus infection and blocks Fas-mediated apoptosis (Mannick et al. 1994). The human NOS1 isoform of NOS is expressed from a very complex 240 kb locus at 12q24.2 composed of 19 exons (Christopherson and Bredt 1997; Hall et al. 1994; Wang et al. 1999a). Although initially described in neural tissues, other tissues and cell types may express NOS1. These include central and peripheral nervous tissue, muscle, and Leydig cells of the testis (Christopherson and Bredt 1997; Wang et al. 1999a; Wang et al. 2002). Expression of NOS1 mRNA and protein in different cell types is influenced transcriptionally by different transcription start sites and promoters, alternative splicing, and translationally by alternative splice sites in the 5 UTR (Brenman et al. 1997; Newton et al. 2003; Wang et al. 1999b). The amino terminal PDZ domain of NOS1 serves to localize the enzyme to critical regions of the cell (e.g., the NMDA receptor in nerve tissue and alpha-syntrophin in myoctes). PIN (protein inhibitor of nNOS) is a small protein of 89 amino acids initially described as a light chain subunit of dynein and as an inhibitor of NOS1 (Ahmed and Van Den Oord 1999). In vitro, PIN binds to a unique NOS1 domain encompassing amino acids 163–245. PIN blocks the formation of the active NOS1 dimer and thus NOS1 activity. While NOS1 is considered a “constitutive” enzyme, NOS1 mRNA transcription is also regulated by physical factors and chemical and biological agents such as ischemia-reperfusion injury and cytokines (Chesler et al. 2004; Forstermann et al. 1998; Schmidt et al. 1995). Levesque and colleagues reported expression of NOS1 in CLL cells and that NOS1 inhibitors of NOS1 induce apoptosis and death in freshly isolated CLL cells in vitro (Levesque et al. 2008). This is discussed in more detail below. Some nonHodgkin’s, small lymphocytic lymphoma, and multiple myeloma cells also express NOS1 (Mendes et al. 2001).

NOS Inhibitors Most NOS inhibitors bind to the oxygenase domain of NOS, with the guanidinium group of the inhibitor binding to NOS glutamate. Hibbs et al. described the importance of arginine in macrophage-mediated cytotoxicity, and demonstrated for the first time that arginine analogues such as NG -monomethylarginine (NMMA) could inhibit cytotoxicity [a function they later described as being related to NO production (Hibbs et al. 1987)]. Subsequently, a variety of NOS inhibitors have been described (Alderton et al. 2001; Babu and Griffith 1998). NOS oxidase inhibitors (e.g., diphenyleneiodonium which also inhibits NADPH oxidase) inhibit NO formation, and inhibitors of NOS dimer formation [e.g., various pyrimidine imidazoles

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(Blasko et al. 2002)] block NO formation by NOS. NOS2-specific inhibitors have been targeted for use in a variety of conditions, most prominently septic and cardiac shock, and arthritis. NOS1-specific inhibitors have been targeted for use in psychiatric diseases such as depression and anxiety and for neurodegenerative diseases such as Alzheimer’s disease and amyotrophic sclerosis (Chabrier et al. 1999). Some NOS inhibitors have been used in research in humans. The nonselective inhibitor NMMA has been used in normals and in trials for septic shock (Bakker et al. 2004) and for migraine headache (Tepper et al. 2001). A phase II randomized, double-blind trial of NMMA to treat patients with shock associated with acute myocardial infarction revealed no beneficial or possibly deleterious effects (Alexander et al. 2007). In a different trial, a pro-drug for the NOS2-specific inhibitor L-NIL was administered orally to normal individuals and to those with asthma. The drug reduced exhaled NO and had no effects on blood pressure, pulse, and respiratory function (Hansel et al. 2003). There have been numerous preclinical studies in non-human animals of a variety of nonselective and selective inhibitors. Several NOS1-specific inhibitors have been studied in animal models of amyotrophic lateral sclerosis, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, depression, and anxiety (Chabrier et al. 1999).

NO and Apoptosis The role of NO in apoptosis is complex (Kolb et al. 2003; Li and Wogan 2005). Overall, it appears that high level NO from extracellular sources induces apoptosis and cell death by several mechanisms including direct membrane damage, inhibition of ribonucleotide reductase, and inhibition of cellular ATP generation by mitochondrial electron transport enzymes, aconitase, and GAPDH (Adams et al. 2001; Fehsel et al. 1995; Hibbs et al. 1988; Li and Wogan 2005; Mannick et al. 1994; Mannick et al. 1997; Nicotera et al. 1997; Shami et al. 1998) (Fig. 8.1). However, endogenous or low level NO can also inhibit apoptosis by nitrosylating caspases and by increasing Bcl-2 expression (Genaro et al. 1995; Li and Wogan 2005). Delivery of NO from NO pro-drugs in vitro (micromolar to millimolar concentrations) to cultures of acute nonlymphocytic leukemia (ANLL cells) (cell line cells and freshly isolated, primary cells) and freshly isolated chronic lymphocytic leukemia (CLL) cells causes apoptosis and death (Adams et al. 2001; Magrinat et al. 1992; Shami et al. 1995; Shami et al. 1998). The degree of toxicity is indirectly related to the rate of NO delivery from the pro-drug (higher kill with slower, chronic release rates) (Adams et al. 2001; Shami et al. 1998). NO killing versus protection for cells may also be determined by the origin of the NO (exogenously supplied and endogenously generated), the rate of production/delivery, and the target cell type (Mohr et al. 1998; Thomas et al. 2008). Apoptosis can be triggered by a variety of mechanisms via the “mitochondrial pathway” (endogenous) and via the “death receptor” (exogenous) pathway. In a

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Fig. 8.1 Nitric oxide and apoptosis. High level NO from extracellular sources can lead to apoptosis and death of cells by several mechanisms including nitrosylation and inhibition of the actions of the transcription factor YY1 (noted at the upper left). Extracellular NO can also mediate direct membrane damage, inhibition of ribonucleotide reductase, and inhibition of cellular generation of ATP by the electron transport enzymes, aconitase, and GAPDH (bottom right). Caspases participate as mediators of apoptosis. NO and several proteins (including Bcl-2 and Bcl-xL) can inhibit apoptosis. Endogenous NO inactivates caspases and YY1 by nitrosylation. NF-kB enhances transcription of a variety of genes including YY1 and anti-apoptotic factors such as Bcl-2 and Bcl-xL. NO blocks activity of NF-kB and enhances YY1, Bcl-2, and Bcl-xL expression. The transcription factor YYI normally suppresses transcription of CD95 (Fas) and DR5 (death receptor 5). NO nitrosylates YY1, and this negates the YY1-mediated suppression of CD95 and DR5 expression, resulting in increased levels of these death receptors. NOS inhibitors and NO quenchers may facilitate apoptosis by blocking the NO-mediated caspase inactivation and the NO-mediated inhibition of NF-kB activation. On the other hand, NO may enhance apoptosis in certain settings by decreasing activity of YY1; this diminished YY1 activity increases levels of CD95 and DR5 Abbreviations: l-arg: L-arginine; NOS: NO synthase; Casp: caspase; DR: death receptor; NF-kB: nuclear factor-kB; XRT: x-ray therapy; uv: ultraviolet light; Cyt C: cytochrome C; Mitoch: mitochondrium; Ribo Reduc: ribonucleotide reductase

simplified view (Fig. 8.1), apoptosis is mediated through activation of intracellular cysteine–aspartate proteinases (caspases). Anti-apoptotic proteins include those of the Bcl-2 family (Yip and Reed 2008) NO binds to and inhibits the active site of many of the human caspase family members including caspases 3, 8, and 9 (Li et al. 1997). In CLL, there are a variety of caspases and apoptosis inhibitor proteins that may be important in determining spontaneous and drug-induced apoptosis and response to therapy (Chiorazzi et al. 2005; King et al. 1998; Kitada et al. 1998). Also, in resting, normal B lymphocytes, the active site cysteine of caspase 3 is nitrosylated (and inhibited by this nitrosylation), and it undergoes denitrosylation upon Fas activation and apoptosis (Mannick et al. 1999). By nitrosylating the p53 subunit of NF-kB, NO inhibits NF-kB activity (Marshall and Stamler 2001). NO generally suppresses activation of NF-kB, and NF-kB activation generally increases expression of members of the Bcl-2 and Bcl-xL. Thus, NO leads to increased expression of the anti-apoptotic factors Bcl-2 and Bcl-xL, (Bonavida et al. 2008; Jeannin et al. 2008), and this increased expression results in decreased apoptosis. This has been

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noted in malignant cells and in normal mouse splenic B cells in vitro (Bonavida et al. 2008; Genaro et al. 1995; Jeannin et al. 2008). Ying-Yang 1 protein (YY1) is a transcription factor that represses transcription of several genes including TNF receptor family members [e.g., Fas (CD95) and death receptor 5 (DR5)]. Bonavida and co-workers showed that NO sensitizes tumor cells to the toxicity mediated by certain chemotherapy drugs, and that this sensitization is accompanied by increased levels of CD95 and DR5. These researchers found that NO nitrosylates a cysteine in YY1 and destroys its inhibitory activity, with subsequent increases in CD95 and DR5 expression (Bonavida et al. 2008; Hongo et al. 2005). NO produced by NOS1 is an important modulator of nervous tissue cell apoptosis. Andoh and colleagues noted that NOS1 influences Bcl-2 and other apoptosis regulators, and accounts for some of the neural cell resistance to apoptosis of preconditioning stress (Andoh et al. 2000). Others showed increased NOS1 in dorsal root ganglion neurons, as well as an NO inhibition of Bax, caspases, and apoptosis (Thippeswamy et al. 2001). NOS1 is found in normal hematopoietic cells, and it influences proliferation and differentiation of these cells (see below) (Enikolopov et al. 1999; Krasnov et al. 2008; Michurina et al. 2004). NOS1 is expressed in human CLL cells, and NOS1 inhibitors induce apoptosis and death in these cells (see below for further discussion of this) (Levesque et al. 2008).

NO and Normal Hematopoiesis Hematopoietic cells and nonhematopoietic “stromal” cells (e.g., fibroblasts, adipocytes, and endothelial cells) can produce NO (Fig. 8.2). Punjabi and coworkers noted mouse bone marrow NO production in vitro with treatment with IFN-γ and endotoxin (Punjabi et al. 1992). The production stimulated by IFN-γ and endotoxin was synergistically enhanced by GM-CSF, IL-3, and TNF, and it could be inhibited by TGF-beta. NOS inhibitors blocked the NO production. Stimulation of NO production was accompanied by decreased bone marrow colony formation in vitro, and this inhibition could be blocked by inhibiting NO production with a NOS inhibitor (Punjabi et al. 1992). Maciejewski et al. evaluated the effects of NO on the growth of normal human bone marrow hematopoietic cell colonies (Maciejewski et al. 1995). The NO donor DETA-NO decreased marrow myeloid and erythroid colony formation in vitro. The decrease paralleled the induction of marrow cell apoptosis. They noted [like Punjabi and colleagues who studied mouse bone marrow cells (Punjabi et al. 1992)] that treatment of human bone marrow cells (or purified CD34 cells) with IFN-γ and TNF-induced NOS2 mRNA and protein caused apoptosis of the bone marrow cells and diminished bone marrow colony formation. They also found that an inhibitor of NOS blocked the apoptosis and decreased the inhibition of colony formation (Maciejewski et al. 1995). Shami and Weinberg also studied the effects of NO on the growth and differentiation of normal human BM cells (Shami and Weinberg 1996). NO delivered

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Fig. 8.2 Nitric oxide and hematopoietic cell proliferation and differentiation. Differentiation and proliferation of hematopoietic cells are influenced in a paracrine fashion by factors (both protein and non-protein) elaborated by “stromal” cells. These stromal cells include endothelial cells, macrophages, fibroblasts, lymphocytes, natural killer (NK) cells, platelets, and adipocytes. NO can be made by most of these cells, and in an autocrine and paracrine fashion, it can influence their differentiation and proliferation, and inhibit or enhance their survival Abbreviations: CFU: colony forming unit; GEMM: granulocyte, erythroid, megakaryocyte, monocyte; BFU: burst forming unit; E: erythroid; Ly: lymphocyte; NO: nitric oxide; IL: interleukin; PDGF: platelet-derived growth factor; IFN: interferon; TGF: transforming growth factor; SCF: stem cell factor; LPS: lipopolysaccharide; CpG: phosphate bond-linked cytosines and guanosines

from the drugs nitroprusside, SIN-1, or SNAP inhibited development of marrow colonies when cells were cultured in methylcellulose with erythropoietin and colony stimulating factors. NO reduced formation of BFU-E, CFU-E, CFU-GM, and CFUM. Using purified CD34+ cells, they showed that the NO most likely affected the hematopoietic precursor cells and not adherent “stromal” cells (Shami and Weinberg 1996). When using isolated CD34+ cells, both erythroid and myeloid (more so for erythroid) colonies were inhibited by SNAP, while SNP inhibited BFU-E and increased CFU-GM (Shami and Weinberg 1996). The effects of NO in enhancing CFU-GM formation may be related to NO inhibition of apoptosis in hematopoietic cells [e.g., by blocking of caspases (Li et al. 1997) or induction of anti-apoptotic factors such as Bcl-2 (Bonavida et al. 2008; Genaro et al. 1995; Jeannin et al. 2008)]. None of the noted inhibitions appeared to be related to cGMP (Shami and Weinberg 1996).

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Enikolopov et al. studied drosophila and found that NO generally inhibits cell proliferation (Enikolopov et al. 1999), that NO reduces hematopoietic stem cell formation, and that NOS inhibitors increase blood cell formation (Michurina et al. 2004). The NO can arise from hematopoietic cells (autocrine) or from adjacent nonhematopoietic cells (paracrine). Krasnov and co-workers demonstrated in mouse studies that NOS1 is expressed in bone marrow cells and in fetal stromal stem cells and that this NOS1 contributes to regulation of hematopoietic cell formation (Krasnov et al. 2008). They noted that all three NOS isoforms are found in mouse bone marrow, with NOS1 being especially prominent (greater in stromal cells than hematopoietic cells). NOS inhibitor treatment causes increased hematopoietic progenitors, while NO donors decrease these progenitors (Krasnov et al. 2008). They postulated that while NOS1 and NO expressed by both hematopoietic cells and stromal cells modulate hematopoiesis, the stromal cell contribution is the greater. Studies in mice also showed that a NOS inhibitor used in vivo caused increased numbers of stem cells and increased blood neutrophil counts after radiation and after bone marrow transplant (Michurina et al. 2004).

NO and Acute Non-lymphoid Leukemia As work studying NO and macrophages progressed in the late 1980s, investigators explored the hypothesis that NO produced in the BM would be a modulator of leukemic hematopoiesis. Magrinat and co-workers noted that NO [delivered as NO-saturated buffer, or from the drugs nitroprusside, 6-morpholino-sydnonimine (SIN-1), or S-nitrosoacetylpenicillamine (SNAP)] potently inhibited the growth of HL-60 myeloblastic leukemia cells, and induced monocytic differentiation of these cells (Magrinat et al. 1992). This differentiation was associated with modulation of gene expression – NO treatment reduced expression of c-myc and c-myb mRNAs and increased transcription of mRNA for IL-1 and TNF. The differentiated cells were vacuolated and had increased expression of nonspecific esterase, CD11b, and CD14. These workers then analyzed freshly isolated leukemia cells from patients with ANLL for their responses to NO in vitro (Shami et al. 1995). It was important to do this, since cells of leukemia cell lines may not accurately reflect the physiology of human leukemia cells in vivo. Freshly isolated cells all responded to NO treatment (decreased viability and induction of monocytic differentiation in the remaining cells), but overall their responses were less consistent than that noted with the more uniform cell line HL-60. Cells of monocytic phenotype ANLL (M4 and M5) were the most responsive to NO treatment (Shami et al. 1995). Shami and co-workers showed that NO-induced growth inhibition of ANLL cells was associated with apoptosis in a rate- and concentration-dependent fashion, and that NO donors with the longest half times of NO delivery (DETA-NO) were the most potent inhibitors of leukemia cell and colony growth (Shami et al. 1998). Shami, Keefer, and colleagues explored ways to specifically deliver NO to leukemia cells. An esterase-sensitive diazeniumdiolate delivered the NO pro-drug

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to human leukemia cells relatively selectively where it was then cleaved by cellular esterases resulting in death of the cells (Saavedra et al. 2000). However, plasma esterases also caused release of NO from the pro-drug. Subsequently, other derivatives were synthesized and tested (Chakrapani et al. 2008; Kiziltepe et al. 2007; Shami et al. 2006; Shami et al. 2003). The compound JS-K does not release NO well unless acted upon by a glutathione S-transferase (GST) and it does not cause systemic hypotension in experimental animals. Since malignant cells express more GST than do normal cells, they reasoned that it would be relatively tumor specific. They found that JS-K induces apoptosis and death of cells of the ANLL line HL-60 with an ED50 of 0.2–0.5 μM in vitro (Shami et al. 2003; Udupi et al. 2006) and induces necrosis of established tumors of HL-60 and prostate cancer (PPC-1) in vivo in a NOD/SCID xenograft model (Shami et al. 2003). In vitro, HL-60 cells treated with JS-K differentiate to monocyte-like cells (Shami et al. 2003) in ways comparable to that noted earlier with in vitro treatment with other NO sources (Magrinat et al. 1992). JS-K also is cytotoxic in vitro for other malignant cells: human monocytic leukemia (U937), human prostate cancer (PPC-1), human colon cancer (DLD-1), mouse sarcoma (Meth A) (Shami et al. 2003), and human multiple myeloma (Kiziltepe et al. 2007). For multiple myeloma, JS-K is active in vitro and in vivo in a xenograft model (Kiziltepe et al. 2007). JS-K activates apoptosis through both the death receptor path and the mitochondrial path and it results in DNA double strand breaks (Kiziltepe et al. 2007). The activity is mediated through JNK activation and its antiproliferative/cytotoxic actions are dependent on JNK (Kiziltepe et al. 2007; Ren et al. 2003). Attempts are underway to test the efficacy of this agent in clinical trials in humans with leukemia and multiple myeloma. NO donors may play some role as novel cancer therapeutics when used alone or with existing cancer chemotherapy agents (Huerta et al. 2008). NO donors available or in development include organic nitrates, metal–NO complexes, S-nitrosothiols, sydnonimines (which produce both NO and superoxide, and hence peroxynitrite), diazeniumdiolates (“NONOates”), and certain “NO-hybrids” (NO linked to active drugs of other classes) (Huerta et al. 2008). Examples of NO-hybrids include NO-aspirin, other NO-nonsteroidal anti-inflammatory drugs, and NO-statins.

Expression of NOS by Leukemia Cells and Myelodysplastic Syndrome (MDS) Cells NOS2 and NOS3 have been detected in human hematopoietic cell line cells, either at basal state or after stimulation with a variety of factors (Weinberg 1999 for review). Human cell lines displaying NOS include the myeloblastic line HL-60; the monoblastic lines THP-1, U937, and Mono-Mac 6 (Weinberg 1999); EB virus positive (Akata, B3HR1, and LCL 1.7) and EB virus negative (BJAB, 2F7, 10C9, and BL-41) B cell lines (Mannick et al. 1994; Mannick et al. 1997; Reiling et al. 1996); the hairy cell leukemia line ESKOL (Eigler et al. 1998; Roman et al. 2000); the T cell line Jurkat (Mannick et al. 1994; Mannick et al. 1997; Reiling et al. 1996);

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and the HTLV-1-infected T cell lines MT-1, SLB-1, C5/MJ (Mori et al. 1999), and SKT1B, ED, K3T, MT2, YY, and K3T (Sonoki et al. 1999). NOS has been detected in freshly explanted leukemia cells also. These include CLL cells (Kolb et al. 2003; Levesque et al. 1998; Levesque et al. 2003; Mendes et al. 2001; Zhao, et al. 1998); hairy cell leukemia (Roman et al. 2000); HTLV-1 associated T cell leukemia (Mori et al. 1999; Sonoki et al. 1999); acute myeloblastic leukemia (Brandao et al. 2001); and myelodysplastic syndrome (Kitagawa et al. 1999). Most evidence suggests that endogenously produced NO in these leukemia cells would be anti-apoptotic, but it is possible that if enough were generated, the NO could also cause apoptosis of the leukemia cells. Heller contends that high-level NO generated by tumors can act to induce tumor cell apoptosis and death and inhibit tumor metastasis, and that inability to express adequate levels of NOS and to produce enough NO contributes to tumor growth and metastasis (Heller 2008). Erythrocytes (and hemoglobin) in a tumor might quench/scavenge NO levels and reduce NO bioavailability. Also, limitation of the NOS substrate arginine [secondary to excess arginase in tumors (produced by tumor cells or tumor-infiltrating macrophages or other myeloid cells)] would restrict NO production, and possibly reduce anti-tumor NO actions and modify host immune responses (Ochoa et al. 2007). Furthermore, endogenous NOS inhibitors generated within the tumor might block NO generation by NOS (Heller 2008). For example, TGF-beta 1 may be produced within tumors and inhibit NOS transcription and translation (Vodovotz et al. 1993). Also chlorotaurine produced by myeloperoxidase from neutrophils and bromotaurine produced by eosinophil-derived peroxidase in tumors can inhibit NOS (Heller 2008; Kim and Kim 2005; Liu et al. 1998)

NO and Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is one of the most common forms of leukemia in North America and Europe, accounting for approximately 30% of all leukemia cases. This leukemia is characterized by CD5+ B lymphocytes with defective apoptosis and slow (but higher than normal B cell) proliferation, and resultant accumulation of the malignant cells (Chiorazzi et al. 2005; Keating et al. 2003; Messmer et al. 2005). Although treatments exist for CLL, it is essentially an incurable malignancy. The groups of Kolb and Weinberg initially found that human CLL cells constitutively express high levels of NOS2 mRNA and protein and have high NOS enzyme activity (Levesque et al. 1998; Zhao et al. 1998). When CLL cells are cultured with NOS inhibitors, there is high-level cell killing associated with leukemia cell apoptosis. Recent research has shown that of the NOS inhibitors, NOS1-specific inhibitors are clearly the most effective, and that CLL cells express NOS1 protein and mRNA (in addition to NOS2 protein and mRNA (Levesque et al. 2008). Since some NOS inhibitors are being tested in humans for non-malignant diseases, and they have low toxicity, investigators are attempting to move agents of this class into phase II studies with CLL patients.

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CLL cells differ in many ways from ANLL cells and normal hematopoietic cells. In addition to their different lineage, they are unique in that they generally have a very low growth fraction, exist primarily in the G0 phase of the cell cycle, and have defective apoptosis. In ways comparable to what had been done with ANLL cells, the effects of exogenous NO donors on the freshly isolated CLL cells were examined (Adams et al. 2001). CLL cell apoptosis and death were induced by the pure NO donors DETA-NO, PAPA-NO, and MAMA-NO. The agents’ potencies for CLL cytotoxicity are comparable to those for ANLL, with the cytotoxic effect being inversely related to the NO release rates of the donors (Shami et al. 1998). DETA-NO acted synergistically with fludarabine to kill CLL cells. NO also synergized with the ara-guanosine pro-drug 2-amino-9-β-D-6-methoxy ara-guanine (also called 506U78). However, the NO–drug interactions were restricted; DETA-NO did not enhance the activity of several other chemotherapy agents (5-fluorouracil, gemcitabine, doxorubicin, chlorambucil, or the CPT-11 metabolite SN-38) (Adams et al. 2001). Although most think of mononuclear phagocytes when they consider NOS2, normal T and B lymphocytes contain NOS2 and NOS3 (see above). CD5+ B lymphocytes share many features with macrophages (Borrello and Phipps 1996). Thus, it was postulated that the CD5+ B lymphocytes of CLL would express functional NOS2. CLL cells were found to have increased NOS activity and to overexpress NOS2 (Levesque et al. 1998; Zhao et al. 1998). NOS enzyme activity in CLL cell samples was increased compared to that of blood mononuclear cells from normal individuals (Levesque et al. 1998; Levesque et al. 2003). Immunoblot analysis detected NOS2 in most of the CLL samples. Levesque et al. did detect NOS2 or NOS3 by immunoblot analysis of purified B cell from normal controls. With RTPCR, NOS1 and NOS2 (but not NOS3) mRNA were noted in cells from CLL patients, while NOS2 and NOS3 mRNA were absent in cells from normal controls (Levesque et al. 1998; Levesque et al. 2003). The proteins of both NOS1 and NOS2 are present in CLL cells. Kolb and co-workers have carefully studied NOS and NO in leukemia (Kolb et al. 2003 for review). They noted that ligation of the low affinity IgE receptor (CD23) reduced proliferation and induced differentiation of monocytic leukemia U937 cells in vitro, that these effects were blocked by a NOS inhibitor (Ouaaz et al. 1994), and that it induced expression of NOS3 mRNA (Roman et al. 1997). They showed that CLL cells overexpressed NOS2 and overproduced NO, and that the nonspecific NOS inhibitor NMMA caused apoptosis and death of the cells in vitro (Zhao et al. 1998). Kolb and colleagues found that flavopiridol (an effective drug for treating refractory and poor risk CLL) reduced expression of CLL cell NOS2. They postulated that this was important in its fludarabine anti-leukemia cell activity (Billard et al. 2003). In further work, they have shown that a variety of agents decrease CLL cell NOS2 expression, and that this reduction accompanies induction of CLL cell apoptosis. The agents include resveratrol, certain polyphenols and heterocyclic agents, and hyperforin compounds (Billard et al. 2003; Menasria et al. 2008; Quiney et al. 2004; Roman et al. 2002). Certain toll-like receptor (TLR) agonists have been noted to improve viability and cause proliferation of CLL cells in vitro (Decker et al.

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2000; Grandjenette et al. 2007; Jahrsdorfer et al. 2005; Spaner et al. 2006). Kolb et al. have recently noted that CLL cells express receptors for TLR-7 and TLR-9, and that engagement of TLR-7 increases their resistance to apoptosis (Hammadi et al. 2008). This increased resistance to apoptosis is associated with increased expression of NOS2 and NO production, and NF-kB activation. And finally, these processes are prevented by inhibition of NOS (Hammadi et al. 2008). Levesque et al. investigated the effects of different cytokines and growth factors on the viability of CLL cells in vitro, NOS2 expression, and spontaneous and NOS-inhibitor induced cell death (Levesque et al. 2003). Culture of cells with IL-4 or IFN-γ (but not TNF-α, IL-2, IL-6, IL-8, G-CSF, nerve growth factor, or IFN-α) increased NOS activity. IL-4 increased NOS2 mRNA and protein. Apoptosis was induced by high doses of NMMA, and incubating cells with IL-4 or IFN-γ reduced apoptosis. This suggested that cytokine-induced NO prevents NMMA-induced apoptosis. Despite these findings, these investigators could not detect NO production by the CLL cells (Levesque et al. 2003). Other researchers have noted comparable difficulty demonstrating NO production in vitro, even when NOS inhibitors produced dramatic biologic consequences (Mannick et al. 1994; Mannick et al. 1997; Reiling et al. 1996). This is especially true with the constitutive isoforms NOS1 and NOS3, since they result in biologically significant changes despite producing only low nanomolar amounts of NO (Thomas et al. 2008). Since IL-4 and IFN-γ induce NOS2 and modulate CD38 expression in CLL cells in vitro, Levesque and colleagues sought to determine if CLL patients had elevated levels of these cytokines and if the levels related to CD38 expression by the leukemia cells (Levesque et al. 2006). Analysis of 170 serum samples from 64 different patients showed that serum IL-4 levels were significantly elevated in CLL patients, and that there was an association of IL-4 levels with the absence of CD38 expression and increased NOS2 expression (Levesque et al. 2006). In a study of numerous nonspecific and specific NOS inhibitors, Levesque and co-workers found that NOS1 inhibitors were generally the most effective at inducing apoptosis and death of CLL cells in vitro (Levesque et al. 2008). NOS1 protein was found by immunoblot in all of the CLL samples tested, and NOS1 mRNA (by PCR) was noted in approximately one-third of samples analyzed for NOS1 mRNA by RTPCR (Levesque et al. 2008). NOS1 protein was not noted (or noted only in scant amounts) in PBMC samples from normal individuals. When CLL cells were cultured with a variety of NOS inhibitors, there was dose-dependent killing of cells; this was apparent as early as 12–14 h. Cytotoxicity was of high level (up to 100% dead cells). Figure 8.3 shows examples of cytotoxicity induced by some of the NO inhibitors in CLL cells. These inhibitors induced apoptosis within 4–8 h as measured by annexin V positivity and appearance of caspase 3 activity in the treated CLL cells. Non-specific NOS inhibitors and NOS2-specific inhibitors either did not induce CLL cell death or induced CLL cell death with EC50 values over 2000 μM. In contrast, NOS1-specific inhibitors induced CLL cell death at much lower concentrations. In general, the lower the Kd for inhibiting recombinant purified human NOS1, the more likely the compound was to kill CLL cells (Levesque et al. 2008).

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Fig. 8.3 Cytotoxicity of NOS inhibitors for CLL cells in vitro. Curves show the mean ± SEM percent cytotoxicity for inhibitors. The NOS1 inhibitor AR-17477 had the lowest ED50, while the NOS2 inhibitor L-NIL had the highest

There were no NOS3-specific inhibitors available to test. The NO binders and quenchers carboxy-PTIO and hydroxocobalamin exhibited no cytotoxicity for CLL cells. As determined by NOS enzyme assays using recombinant human NOS1 and NOS2, ETPI [S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea] is a highly specific NOS1 (versus NOS2) inhibitor, and ARL-14744 [N-[4-(2-[[(3-chlorophenyl) methyl] amino] ethyl)phenyl]-2-thiophenecarboximide]] is a very potent NOS1 inhibitor (ID50 of 280 nM) with high specificity for NOS1 relative to NOS2 (Levesque et al. 2008). Thus, the findings demonstrate that of NOS inhibitors, NOS1 inhibitors are the most effective at mediating CLL cell cytotoxicity in vitro. This suggests that NOS1 is the most important target in these cells. The effects of NOS2 and NOS1 inhibitors on viability of normal human blood B lymphocytes were also examined. Generally, the NOS1 inhibitors’ cell toxicity is relatively specific for CLL versus normal human lymphocytes (Levesque et al. 2008). While some work shows that exogenous NO in high amounts may be proapoptotic for CLL cells (Adams et al. 2001), NO produced endogenously effectively inhibits CLL cell apoptosis and cell death (Kolb et al. 2003; Levesque et al. 1998; Levesque et al. 2008; Zhao et al. 1998). Furthermore, inhibiting NOS1 with a variety of inhibitors induces CLL cell apoptosis and cell death. NOS and NO appear to be important treatment targets in CLL. The ability to use isoform-specific NOS inhibitors for CLL treatment is very desirable, since one would expect that these agents would not be toxic for the bone marrow and that overall side effects would be minimal.

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Summary and Conclusions NO made by hematopoietic cells and by non-hematopoietic cells can modulate the differentiation and survival of normal and leukemic blood cells. Depending on the source of NO (extrinsic versus intrinsic to the cell), the rate of production/delivery, the concentration, and most likely the specific target cell, NO can either mediate cytotoxicity (stasis, lysis, apoptosis, death) or cytoprotection. Treatments aimed at directing delivery of high-dose NO to leukemia cells using cell type-specific NO pro-drugs may be useful in the treatment of certain leukemias. Likewise, certain NOS inhibitors reproducibly induce apoptosis and death of leukemia cells, and these drugs may be useful in the treatment of certain leukemias.

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Chapter 9

Inhibition of Apoptosis by Endogenous Nitric Oxide in Chronic Lymphocytic Leukaemia Christian Billard, Claire Quiney, and Jean-Pierre Kolb

Abstract Chronic lymphocytic leukaemia (CLL) is a malignant lymphoproliferative disease characterized by a dramatic resistance to spontaneous and drug-induced apoptosis. In the present review the role of endogenous nitric oxide (NO) in this resistance has been analysed. Although a contribution of NOS3 cannot be excluded, NO is mainly produced by an inducible NOS (NOS2) that is constitutively expressed by the leukaemia cells, at variance with normal B lymphocytes. The expression of this enzyme in the tumour cells appears to be regulated by engagement of the tolllike receptor-7 (TLR-7) and is modulated by the ligation of the low-affinity IgE receptor/CD23 and by various cytokines such as interleukin-4 (IL-4) and IFN-γ. According to its concentration, flux, cell type and redox state, NO exerts contrasting effects on apoptosis, activating transduction pathways leading to apoptosis, whereas in other cases protecting cells against spontaneous or induced apoptosis. In CLL cells, the level of endogenous NO is correlated with the abundance of mitochondria and resistance to apoptosis. NO inactivates caspase(s) through oxidation and S-nitrosylation of a cysteine present in their active site, providing an efficient means to block apoptosis. Conversely, a caspase-sensitive down-regulation of iNOS expression and of NO production appears to be associated with the induction of apoptosis by a variety of reagents. Other protective effects of NO on apoptosis probably rely on the modulation through S-nitrosylation-dependent and S-nitrosylation-independent pathways of members of the Bcl-2/Bax family that control the release of apoptogenic factors by mitochondria. If appropriately targeted, NOS inhibitors would provide an efficient mean to reinduce apoptosis in CLL cells and to allow the development of a new therapeutic approach. Keywords CLL · Apoptosis · NOS · Caspases · Bcl-2 family

J.-P. Kolb (B) Centre de Recherche des Cordeliers, UMRS 872 INSERM/University Pierre et Marie Curie/University Paris Descartes, 15 rue de l’Ecole de Médecine, 75270 Paris cedex 06, France e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_9, C Springer Science+Business Media, LLC 2010

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Introduction Chronic lymphocytic leukaemia (CLL) is the most frequent leukaemia in Western countries and still remains an incurable disease, despite recent progress (CaligarisCappio and Ghia 2008). It affects mostly people over 60 years and leads to the accumulation of a mono- or oligoclonal population of CD5+ B lymphocytes in the blood and lymphoid organs. About 80% of the patients exhibit various cytogenetic alterations, yet CLL cells do not express a typical translocation. A small pool of highly proliferating cells has been identified in the bone marrow and lymph nodes that feed the blood compartment (Messmer et al. 2005). The latter consists in anergic and non-dividing small B lymphocytes, 95–98% of them being arrested in the G0 /G1 phase of the cell cycle. Although these cells are highly resistant to apoptosis in vivo, they become sensitive when cultured ex vivo and die rather rapidly, unless they are incubated in the presence of stromal cells that can rescue them from programmed cell death. This suggests that their micro-environment protect them from apoptosis induction in vivo and therefore CLL is a disease of both proliferation and accumulation. A hallmark of CLL cells resides in their dramatic resistance to both spontaneous and drug-induced apoptosis. The latter relies on multiple mechanisms that prevent the leukaemia cells or rescue them from entering the process of programmed cell death. Years ago, our group established that NO released by an endogenous nitric oxide synthase, NOS, markedly contributed to this resistance. This review will therefore be focused on the regulation of NOS expression and activity in CLL cells in correlation with the induction of apoptosis in these cells.

Detection of NOS in CLL Cells Normal resting B lymphocytes do not express any of the NOS isoforms. However, their activation by polyclonal mitogens results in NO production that is inhibited by aminoguanidine, a relatively specific inhibitor of iNOS; this inhibition is accompanied by a stimulation of the apoptotic process in the activated cells (Hortelano and Bosca 1997). Furthermore, ex vivo mature B cells cultured with NO donors display a delayed PCD and a rescue from antigen-induced apoptosis. The mechanism of this protection involves a cGMP-dependent process and the sustained expression of the proto-oncogene Bcl-2 indicating that a pathway links NO signalling with Bcl-2 expression (Genaro et al. 1995). In addition, a low constitutive expression of iNOS was reported in EBV-transformed human B lymphocytes and Burkitt’s lymphoma cell lines and the NO released maintained EBV latency and inhibited apoptosis through cGMP-independent mechanisms (Mannick et al. 1994). The above results suggested that NO could play an anti-apoptotic role in human B lymphocytes, at least in some situations. Inasmuch as CLL cells exhibit a marked resistance to apoptosis in vivo and frequently display elevated levels of Bcl-2 (and

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related anti-apoptotic members of the Bcl-2 family), this prompted us to investigate the presence of NOS isoforms in these leukaemia cells. Indeed, whereas the presence of eNOS (NOS3) was quite undetectable, our group demonstrated the existence of iNOS (NOS2) in leukaemia cells, freshly isolated from the quasi-totality of the untreated CLL patients tested. This was evidenced by the detection of iNOS mRNA by RT-PCR and that of the iNOS protein as shown by flow cytometry and Western blotting. This iNOS is functional, as attested by the conversion of 3H- (or 14C-) labelled arginine into 3H- (or 14C-) labelled citrulline by cytoplasmic extracts from CLL cells. Most importantly, the addition of specific iNOS inhibitors to ex vivo cultures of CLL cells resulted in a marked increase of apoptosis. In contrast, we were unable to detect the presence of eNOS (type 3 NOS) mRNA and protein in these cells (Zhao et al. 1998). Of note, we found similar results, i.e. the existence of a functional iNOS endowed with an anti-apoptotic role, in hairy cell leukaemia, a closely related haematological malignancy (Roman et al. 2000). These results were confirmed by Levesque et al. (2003) who similarly found that CLL cells expressed NOS enzyme activity, NOS2 protein and mRNA and that NOS inhibitors elicited cell death. More recently, the same group determined the half-maximal concentration of various NOS inhibitors that induced CLL cell death in vitro; these authors found a correlation of the NOS1 dissociation constant and the hydrophobicity partitioning coefficient of each NOS inhibitor and its ED50 (Levesque et al. 2008). This suggested that hydrophobic NOS inhibitors putatively binding tightly to NOS1 could induce CLL cell death. The expression of NOS1 was detected in CLL cells, but the presence of a calcium-dependent NOS catalytic activity in CLL cells remains to be demonstrated. Histopathologic studies also concluded to the presence of NOS1 protein in 5/10 cases of multiple myeloma (MM) and 15/16 cases of non-Hodgkin’s lymphoma (NHL), including CLL; NOS2 was detected in all cases of MM and in 14/16 cases of NHL, whereas NOS3 was positive in 3/10 of MM and in only 1/16 cases of NHL (Mendes et al. 2001). Conflicting results have been reported regarding the presence of products of NO oxidation, NO2 and NO3, in the serum of CLL patients. Bakan et al. (2003) showed that serum NO concentrations were higher in CLL patients compared with the control group whereas another group did not observe such an increase (Levesque et al. 2003). Our own group occasionally observed that some patients displayed small amounts of NO2 /NO3 in their serum, but no correlation could be established with clinical and biological parameters. Together, these observations indicate that NO, produced by iNOS/NOS2 and eventually by NOS3 in CLL cells, contributes to the resistance of these leukaemia cells to apoptosis and that relevant NOS inhibitors may be of interest for therapeutic purposes. The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) displays anti-tumour activity against a variety of cultured tumour cell lines and induces apoptosis of malignant human B cells ex vivo, including CLL cells. This compound and its more efficient imidazolide derivative are also active in a mouse model of CLL and analysis of blood cells recovered from treated mice showed that they are potent inducers of tumour cell death in vivo (Kress et al. 2007). Since CDDO is an

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inhibitor of iNOS (Couch et al. 2005), its therapeutic action could be related to this suppressive effect.

Regulation of iNOS Expression in CLL Cells Freshly collected CLL cells do not or very rarely display characteristic features of apoptosis, suggesting that cells undergoing programmed cell death are rapidly cleared from the blood by phagocytes. In vitro, leukaemia CLL B cells, but not normal B cells, are rescued from spontaneous apoptosis by contact with normal bone marrow stromal cells. This indicates that the latter cells provide in vivo signals that counteract the normal process of apoptosis, thus conferring to the tumour cells a selective advantage. One can thus speculate that in vivo leukaemia cells receive a signal that leads to the induction of iNOS and contributes, via the NO released, to protect these cells from the normal apoptotic process. Inasmuch as the normal counterpart of CLL cells, the small B lymphocytes, do not spontaneously express any NOS isoform, the question arose of the factor(s) that initiate and regulate the expression of iNOS in the leukaemia cells and that contribute to trigger apoptosis resistance. In our first report of a functional iNOS in CLL cells, we observed that ligation of CD23, the low-affinity IgE receptor and hallmark of these leukaemia cells, markedly increased iNOS expression and activity and simultaneously decreased the percentage of cells undergoing apoptosis (Zhao et al. 1998). This was reminiscent of what we observed previously with human IL-4-activated human monocytes (Paul-Eugene et al. 1995; Dugas et al. 1995). IL-4 and IFN-γ were also found to increase NOS2 enzyme activity and protein expression in cultured CLL cells, respectively, at the transcriptional and post-transcriptional levels, and these treatments diminished NOS inhibitor-induced B-CLL cell death (Levesque et al. 2003). Moreover, serum IL-4 levels are higher in CLL patients than in normal individuals and are associated with CD38 (a marker of unfavourable outcome) and NOS2 expression in these patients (Levesque et al. 2006). Inasmuch as the monoclonal immunoglobulins of the B cell receptors expressed by the leukemic cells present a restricted use of some VH sub-groups, it has been suggested that the latter have encountered specific auto-antigens and/or microbialderived antigens. The binding of these antigens to the BCR (B cell receptor) provides an activation signal resulting in enhanced survival, hence could be involved in the aetiology of the disease. At the interface of innate and cognate immunity, tolllike receptors, TLR, recognize PAMPs (pathogen-associated molecular patterns) expressed by various bacteria and viruses as well as some self-antigens. We, thus, hypothesized that TLR were involved in the early steps of B-CLL oncogenesis, notably apoptosis resistance through the induction of iNOS expression and the production of NO. Our recent results showed that CLL cells indeed expressed TLR-7 and TLR-9. Moreover, incubation of these leukaemia cells with TLR-7 agonists

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was found to enhance their viability and to increase their resistance to apoptosis that was reverted with the NOS inhibitor L-NMMA. This indicates that the signalling through TLR-7 protects CLL cells from spontaneous apoptosis and suggests the involvement of NO in the protection afforded. This hypothesis was also supported by the observation that caspase 3 activity in these cells was markedly reduced following their incubation with resiquimod, a TLR-7 ligand, in agreement with the fact that the activity of caspases is impaired by NO (see below). Indeed, the TLR7-driven resistance to apoptosis was associated with enhanced iNOS expression (protein and mRNA) and NO release, stimulation of both the canonical and the alternative pathways of NF-κB activation (notably the p65/RelA and p52 components), phosphorylation of IκBα, all these events being suppressed with wedelolactone or Bay 11-7085, two inhibitors of IκBα phosphorylation (Hammadi et al. 2008). The latter step is necessary for the ubiquitination of the inhibitor IκBα and its degradation by the proteasome, leading to the migration of NF-κB to the nucleus, its binding to κB responsive elements and transcriptional activation of various genes, including iNOS. In addition, TLR-7 ligation induces the activation of several members of the family of AP-1 transcription factors and notably increases the phosphorylation of c-Jun. Furthermore, the protection afforded by TLR-7 ligation is significantly reduced by an inhibitor of the c-Jun kinase (Hammadi et al. 2009). The human iNOS gene promoter contains several κB responsive elements (de Vera et al. 1996) and is transcriptionally regulated by NF-κB and AP-1 (Nunokawa et al. 1996; Marks-Konczalik et al. 1998). Together, our data thus indicate that TLR-7 signalling stimulates apoptosis resistance, notably through NF-κB- and AP-1-dependent activation of the NO pathway. A tentative and simplified schema depicting the possible signalling events leading from the engagement of TLR-7 to the transcriptional activation of the iNOS gene is depicted in Fig. 9.1, based on the review by Banerjee and Gerondakis (2007). Since single-strand RNA (ssRNA) is the physiological ligand of TLR-7 (Heil et al. 2004; Diebold et al. 2004), we suggest that CLL cells or their precursors could be triggered by ssRNA present in their micro-environment, either as components of virus (PAMPs) or as degradation products of self-origin. Through the induction of NO, this would provide to these cells an increased resistance to apoptosis and a selective survival advantage. This hypothesis is compatible with the sequences of monoclonal immunoglobulins of the BCR of CLL cells that, for many of them, present a bias suggestive of an encounter with type 2 T-independent antigens such as some auto-antigens and antigens of microbial origin. It also fits with the recent demonstration that many CLL immunoglobulins effectively bind and recognize antigens representing molecular motifs exposed on apoptotic cells and bacteria, this binding resulting in an enhanced survival (Lanemo Myhrinder et al. 2008). In addition to TLR-7 (and TLR-9), it has been reported recently that CLL cells also express other functional toll-like receptors, such as TLR-1, TLR-2, TLR-6, TLR-10, and the closely related NOD1 and NOD2. The engagement of these receptors with their specific ligands leads to activation of NF-κB, enhanced survival and decreased apoptosis (Muzio et al. 2008). It is therefore possible that these

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Fig. 9.1 Induction of iNOS expression through TLR-7 signalling. Adapted and modified from Banerjee and Gerondakis (2007). BCR (B cell receptor) can bind peculiar antigens (Ag) coexpressing PAMP (pathogen-associated molecular pattern). The internalization of this complex allows the recognition and binding of PAMP by TLR-7 expressed at the level of endosomes. Through the adaptor MyD88 (myeloid differentiation antigen 88) this results in the sequential activation/phosphorylation of IRAK4 and IRAK1 (IL-1 receptor-activated kinase), then of TRAF6 (TNF receptor-activated factor 6) and of the MAP3Ks (mitogen-activated protein kinase kinase kinase) TAK1 (TGF-β associated kinase 1) and Tpl2 (tumor progression locus 2, also known as Cot/MAP3K8). TAK1 activates the IKKα/β kinases (IκB kinases α/β) through the regulatory NEMO (NF-κB essential modulator)/IKKγ subunit, in turn leading to the phosphorylation of IκB (inhibitor of κB). The latter is then ubiquitinated and degraded by the proteasome, allowing the liberation of the members of the NF-κB family p50 and p65, their migration to the nucleus, binding to the κB responsive elements present in the promoter of the iNOS gene, thus contributing to its transcriptional activation. TAK1 can also activate the stress-activated kinases JNK (c-Jun N-terminal kinase) and p38 through the MKK3,6 (MAP kinase kinase 3,6). In addition, the engagement of TLR-7 can eventually lead to the activation of another MAP3K, Tpl2, normally bound to p105, the precursor of the NF-κB1 complex. This would result in the sequential activation of MKK1,2 and downstream of ERK1,2 (extracellular signal-regulated kinase). This would ultimately result in the activation of the transcription factor Jun/Fos that, through their binding to an AP-1 site on the iNOS promoter, would amplify the transcription of this gene in synergy with NF-κB

receptors are also able to trigger the induction of iNOS, but this remains to be demonstrated. Other factors contributing to the survival of CLL cells and their resistance to spontaneous and drug-induced apoptosis are the molecules BAFF (B cell-activating

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factor of the TNF family) and APRIL (a proliferation-inducing ligand), two members of the TNF superfamily. We have shown a few years ago that these two factors contribute to rescue CLL cells from programmed cell death through autocrine and paracrine mechanisms (Kern et al. 2004). This protection towards apoptosis involves the stimulation of both the classical and the noncanonical pathway of NF-κB activation (Kern et al. 2008; Haiat et al. 2006). This prompted us to test whether BAFF and APRIL were able to stimulate the production of NO in CLL cells. Indeed, we found that the addition of BAFF or APRIL to cultures of CLL cells resulted, for about 25% of the patients tested, in a significant increase in the production of NO by these cells (Hammadi et al. 2009). Interestingly, the cells responding to BAFF and APRIL by an augmentation of the release of NO were those which presented the lowest spontaneous NO production. It is therefore likely that BAFF and APRIL provide an amplification signal only for CLL cells that have not reached their optimum production of nitric oxide.

Endogenous NO Is Anti-apoptotic in CLL Cells Since our original description of the existence of an iNOS in CLL cells and of the inhibitory role of the endogenously released nitric oxide in the resistance of these cells to spontaneous apoptosis, NO has also been described as a potent suppressor of drug-induced apoptosis in these leukaemia cells. For instance, Carew et al. (2004) have reported that CLL cells contain significantly more mitochondria than lymphocytes from normal donors. Moreover, they showed the existence in CLL of a correlation between the level of endogenous NO and the mitochondrial mass. This increased number of mitochondria was associated with an elevated mRNA expression of two mitochondrial biogenesis factors, namely NRF-1 and its downstream target TFAM, in comparison with normal lymphocytes. The role of NO in this increased mitochondria biogenesis was demonstrated by experiments showing that addition of exogenous NO to B cell lines results in an increased mitochondrial mass, as estimated with a fluorescent mitotracker-specific probe. This role of NO in mitochondrial biogenesis has been confirmed in various cells and is mediated by the induction of peroxisome proliferator-activated receptor γ coactivator 1α (Nisoli et al. 2003; Nisoli et al. 2004). Of note, there was a significant correlation between the mitochondrial mass of CLL and their sensitivity to fludarabine in vitro, the leukaemia cells with higher mitochondrial mass and NO levels being less sensitive to fludarabine (Carew et al. 2004). These results indicate that NO is a key mediator of mitochondrial biogenesis in CLL cells and that the latter modulation may alter cellular sensitivity to fludarabine. They confirm previous work reporting a protective role of endogenous NO against apoptosis in CLL. In addition, they shed new light on the action of NO through its capacity to stimulate mitochondrial biogenesis and indicate a potential link between the oxidative and nitrosative stress and the integrative function of mitochondria in different cell functions, such as control of energy and apoptosis.

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Down-regulation of iNOS Expression and NO Production: Cause or Consequence of Apoptosis Induction? A down-regulation of iNOS expression at the transcriptional level was reported for CLL cells treated with TRAIL or chlorambucil (Secchiero et al. 2007). Interestingly, the expression of iNOS appears to differ, both in normal B lymphocytes and CLL cells, according on pro- or anti-apoptotic conditions. Indeed, Tiscornia et al. (2004) have identified a new splice variant characterized by a complete deletion of exon 14 which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. They have detected another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (exons 13–16) that was correlated to a decreased CLL cell viability. In addition, treatment of leukaemia cells with fludarabine resulted in an induction of this variant transcript (Tiscornia et al. 2004). We have also observed this alternative splicing in CLL cells treated by some, but not all, pro-apoptotic molecules (unpublished data). The observed reduction of NO production in these cells could thus be due to an impaired enzymatic activity of the resulting iNOS protein, due to the partial deletion of the flavodoxin region. During the work of our laboratory aimed at elucidating the mechanism of action of several drugs already recognized or newly discovered as pro-apoptotic agents for CLL cells, we made the observation that most of these derivatives elicited a marked decrease of iNOS expression and NO production by the leukaemia cells. This was true for resveratrol, a grape-derived compound, its dimer, -viniferin, and the acetate derivatives of these two polyphenols. These agents, which are potent inducers of CLL cells apoptosis ex vivo (Roman et al. 2002; Billard et al. 2002), were found to inhibit the production of endogenous NO through a down-regulation of iNOS. The same results were observed with a synthetic diaminomethoxyflavone, both in leukaemia B cell lines and leukaemia cells freshly collected from CLL patients (Quiney et al. 2004). For some CLL patients, cross-linking of CD5 was found to elicit apoptosis that was accompanied by a down-regulation of several anti-apoptotic proteins, such as Bcl-2, Mcl-1 and iNOS (Cioca and Kitano 2002). Similarly, ex vivo treatment of leukaemia cells from CLL patients with flavopiridol, an inhibitor of cyclin-dependent kinases and potent apoptosis inducer, resulted in the inhibition of iNOS expression, as determined by immunofluorescence and Western blotting, and in a marked inhibition of NO production measured with a specific fluorescent probe. These effects were accompanied by membrane, mitochondrial and nuclear events of apoptosis. Furthermore, flavopiridol-promoted apoptosis was partially reverted by the addition of a chemical NO donor, suggesting that inhibition of the NO pathway could participate in the apoptotic effects of flavopiridol on the leukaemia cells (Billard et al. 2003a; Billard et al. 2003b; Lin and Porcu 2004). Moreover, the lack of additivity of the apoptotic effects of flavopiridol and of L-NIL, a specific iNOS inhibitor, also suggests that both are proceeding through the same mechanism, inhibition of NO production. In addition, low concentrations of an NO donor can partially reverse flavopiridol-elicited

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apoptosis. Although differing from flavopiridol by its effect on members of the Bcl-2 family (Zaher et al. 2009), the phloroglucinol hyperforin was also found to trigger apoptosis and to decrease NO production in CLL cells (Quiney et al. 2006). Thus, inhibition of the NO pathway during apoptosis of leukaemia B cells appears to be a common mechanism for several compounds belonging to two distinct families of molecules. The flavopiridol-induced down-regulation of iNOS expression associated with apoptosis induction was further analysed. An impairment of iNOS transcription can be excluded, inasmuch as iNOS mRNA levels in CLL cells were not affected by flavopiridol treatment, as estimated by semi-quantitative RT-PCR. In addition, the splice variant reported by Tiscornia et al. (2004) was not detected in cells treated by flavopiridol, although its presence was evidenced in CLL cells triggered by other apoptogenic stimuli. Of importance, the flavopiridol-driven iNOS reduction was found to be caspase dependent since it was reverted in the presence of the general caspase inhibitor Z-VAD-fmk (Billard et al. 2008a). Moreover, experiments carried out with the Eskol cell line derived from hairy cell leukaemia, that shares with CLL cells iNOS overexpression (Roman et al. 2000) and sensitivity to the apoptotic effects of flavopiridol (Quiney et al. 2004), show that flavopiridol elicited the degradation of the 135 kDa iNOS protein into a shorter fragment of 115 kDa. The possibility that human iNOS could be a substrate of caspase(s), despite the absence in its sequence of canonical sites of cleavage by these enzymes was thus tested. Inasmuch as caspase 3 activity is markedly stimulated by flavopiridol, we therefore analysed by Western blot and ELISA whether recombinant human iNOS could be cleaved by purified caspase 3. However, we were unable to detect significant degradation of iNOS by this enzyme (unpublished data). For these reasons, we favour the hypothesis that degradation of iNOS is a consequence of its cleavage by a protease-activated downstream of caspases. A proteolytic cleavage of iNOS by calpain I has been reported by Walker et al. (2001). In addition, Z-VAD-fmk was shown to block the activation of calpain through its inhibitory effect on caspase and a caspase-dependent activation of calpain was observed during drug-induced apoptosis (Wood and Newcomb 1999). It is therefore possible that, in CLL cells, some pro-apoptotic drugs elicit an activation of calpain, downstream of that of caspase(s), leading to a proteolysis of iNOS and a decrease in NO production, resulting in turn in an enhanced caspase activity and thus providing an amplification loop of the apoptotic process. The catabolism of iNOS is also largely mediated by the ubiquitination– proteasome pathway (Kolodziejski et al. 2002; Musial and Eissa 2001). It is therefore possible that the caspase-sensitive step of flavopiridol-driven iNOS downregulation occurs upstream the ubiquitination–proteasome pathway. Interestingly, a cell cycle-dependent caspase-like activity, termed KIPase, which is able to cleave the cdk inhibitor p27kip1 (Medina-Palazon et al. 2004), was recently identified as the β1 subunit of the 20S proteasome (Tambyrajah et al. 2007). If iNOS down-regulation was an initial event by which flavopiridol induces apoptosis of CLL cells, it would occur prior to caspase activation. The observation that

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the inhibitory effect of flavopiridol on iNOS expression is caspase dependent indicates it is not by itself responsible for triggering the apoptotic process. Therefore, the inhibition of NO pathway seems rather to be one of the mechanisms involved in the apoptotic machinery subsequent to the interaction of flavopiridol with its primary target. This mechanism, although not an initial event, seems to play an important role in the re-establishment of apoptosis. However, it must be noted that, although very frequent, a decrease in iNOS expression is not always associated with apoptosis induction, as exemplified by 4-arylcoumarines; these analogues of combretastatin are potent inducers of the classical caspase-dependent apoptotic pathway, yet they are unable to elicit a significant reduction in iNOS expression (Billard et al. 2008b).

Mechanisms of NO Anti-apoptotic Action in CLL Cells The possible mechanisms involved in the pro- and anti-apoptotic role of NO in human leukaemia, including CLL, have been reviewed previously (Kolb 2000), therefore, only some points concerning the protective effect of NO will be discussed. Whereas the cGMP pathway does not seem to be involved in most instances, the main protective action of NO appears to result from its capacity to induce Snitrosylation of various molecules implicated at different steps of the apoptotic process. Among them, caspases are inactivated by exposure to NO (either from chemical donors or released by endogenous NOS) through S-nitrosylation of the cysteine in the active site and/or oxidation (Dimmeler et al. 1997; Mohr et al. 1997; Li et al. 1997; Li et al. 1999; Rossig et al. 1999; Kim, Talanian and Billiar 1997). Mass spectrometry analysis revealed that several cysteines in caspase 3 were modified by S-nitrosylation but that only the Cys 163 present in the catalytic site was resistant to transnitrosation in the presence of glutathione (Zech et al. 1999). Inhibition of caspase activity is thus likely to play an essential role if the resistance towards apoptosis is conferred by endogenous NO to CLL cells. Indeed, we have shown that caspase 3 activity in these leukaemia cells is markedly enhanced following their incubation with a specific iNOS inhibitor and this is associated with an increased apoptosis. Conversely, caspase 3 activity is greatly reduced in the presence of a chemical NO donor, in parallel with a marked diminution of apoptosis. NO has also been reported to inhibit Fas-induced apoptosis through Snitrosylation and inactivation of caspase 8 (Dimmeler et al. 1998) and of the downstream effector, caspase 3 (Mannick et al. 1997). This could explain, in part, why CLL cells are relatively resistant to Fas-mediated cell death and why there is a potentiation of L-NAME-induced apoptosis by co-stimulation of the Fas pathway (Zhao et al. 1998). Conversely, ligation of Fas stimulates the denitrosylation of caspase 3, leading to its activation (Mannick et al. 1999). This reciprocal regulation of caspase activity by NO and Fas through S-nitrosylation/denitrosylation of the cysteine in the active site, therefore, provides a fine tuning of the apoptotic process in CLL cells. Recently, Benhar et al. (2008) found that thioredoxin and thioredoxin reductase together act as a principal denitrosylation mechanism. In human cells,

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thioredoxin 1 and 2 are the cytosol-nuclear and mitochondrial isoforms, respectively, and the products of two separate genes; the same is true for thioredoxin reductase. Thioredoxin together with thioredoxin reductase control the amount of basal S-nitrosylation of caspases in response to Fas activation. Benhar et al. showed that both thioredoxin 2 in the mitochondria and thioredoxin 1 in the cytosol and nucleus are denitrosylases in the context of apoptosis. The existence of a dynamic equilibrium between nitrosylation and denitrosylation would therefore finely control the activity of various molecules, notably caspases, involved in the apoptotic pathway. It has been also suggested that thioredoxin could control NOS activity through a denitrosylation of the synthase which would stimulate NO production (Holmgren 2008). On the other hand, stromal cells present in proliferation centres (pseudofollicles) in the CLL lymph nodes were found to express and secrete high levels of thioredoxin that significantly protected the CLL cells from undergoing apoptosis (Backman et al. 2007). The links among thioredoxin, protein nitrosylation, redoxcontrolled signalling and apoptosis resistance are thus complex and the unravelling of the mechanisms involved is only at its beginning (Iyer et al. 2008).

NO, Bcl-2 Family and the Control of Mitochondrial Apoptosis A major cause of defective apoptosis in CLL is a translocation-independent overexpression of the anti-apoptotic protein Bcl-2 (Gottardi et al. 1995). More generally, the expression pattern of Bcl-2 gene family is shifted towards prevention from apoptosis, with an overexpression of those members (Bcl-2, Bcl-XL, Mcl-1) regulating negatively the release by mitochondria of pro-apoptotic mediators (Gottardi et al. 1996). CLL cells with low Bcl-2/Bax ratios are more drug sensitive when compared to cells with intermediate to high ratios. Several factors have been proposed to explain the overexpression of Bcl-2, notably an hypomethylation of the gene and a lack of negative regulation by the mIR15a–mIR16a microRNAs in the case of CLL patients with a 13q14 deletion (Cimmino et al. 2005). In addition, an inhibition of the cleavage of Bcl-2 by NO has been observed in B lymphocytes leading to an increased half-life of this protein, thus favouring its anti-apoptotic effect (Genaro et al. 1995). This results from the S-nitrosylation of Bcl-2 that prevents its degradation by the ubiquitin–proteasome pathway (Azad et al. 2006). In support of this proposal, we have regularly observed that CLL cells treated with various apoptosis inducers display a reduced expression of Bcl-2 in parallel with that of iNOS, suggesting that the mechanism described above is active in CLL. Similarly, the induction of apoptosis in CLL patients by CD5 cross-linking is accompanied by a simultaneous down-regulation of Bcl-2, Mcl-1 and iNOS (Cioca and Kitano 2002). Sanz et al. (2004) similarly observed that cell death of CLL cells resulting from in vitro culture was associated with decreased Bcl-2, Bcl-w, Bfl-1, Mcl-1, Bak, Bax and Bcl-2/Bax expression, confirming that several Bcl-2 family genes are regulated during CLL spontaneous apoptosis and that their relative levels may contribute to in vivo progression of the disease.

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However, the resistance of CLL cells to chemotherapeutic agents is better correlated with an overexpression of Mcl-1 than of Bcl-2. For instance, the level of Mcl-1 was correlated with failure to achieve remission after therapy with fludarabine or chlorambucil (Kitada et al. 1998). The high expression of Mcl-1 has been explained, in part, by the existence in CLL cells of 6 and/or 18 base pair insertions in the promoter of the Mcl-1 gene that are associated with increased Mcl-1 RNA and protein expression, poor response to therapy and overall survival (Moshynska et al. 2004), a result, however, challenged by Coenen et al. (2005). As an alternative explanation, NO could contribute to the overexpression of Mcl-1. First, NO elicits the transcription of this suppressor of apoptosis in chondrocytes (Nikolaev et al. 2002) which, although not demonstrated, could also be the case in CLL cells. Second, the expression of Mcl-1 is reduced in parallel with that of iNOS following treatment of CLL cells with some apoptosis inducers, such as flavopiridol, suggesting, as for Bcl-2, a possible S-nitrosylation of the protein (containing one putative cysteine target) preventing its degradation. This hypothesis is, however, unlikely, inasmuch as we were unable to detect any S-nitrosylation of Mcl-1, both in healthy and apoptotic CLL cells (unpublished data).

Concluding Remarks The endogenous release of NO by CLL cells clearly endows the leukemic cells with a survival advantage due to the anti-apoptotic action of this mediator. Although the mechanisms responsible for the “constitutive” expression of iNOS in CLL cells are not fully elucidated, they involve signals provided by the micro-environment, such as selected cytokines and the ligation of specific receptors. Among these receptors, TLR appear to be of paramount importance, due to their capacity to activate a set of factors necessary for the transcription of the iNOS gene. A coordinate recognition of specific antigens by the BCR and of PAMPs by the TLR(s) expressed by CLL leukemic cells or their precursors would favour the resistance of these cells to physiological apoptosis, notably by eliciting the production of NO. Due to their capacity to recirculate between blood and lymphoid organs, these cells would increase their chance to be (re)stimulated by antigens and PAMPs displayed by microorganisms and/or dying cells. This would provide a persistent stimulus responsible for the apparent “constitutive” expression of iNOS by CLL cells. This expression could be further amplified by various cytokines, such as IL-4, IFN-γ, BAFF and APRIL. The possibility of reinducing a normal apoptotic process in the leukemic cells by a targeted inhibition of the production of endogenous NO can thus be envisaged for therapeutic purposes. In this respect, it should be recalled that, due to its well-known biphasic effects on apoptosis (Mocellin et al. 2007), supraphysiological concentrations of nitric oxide, such as provided by chemical NO donors, can, on the contrary, elicit apoptosis in CLL cells. As an example, high concentrations of NONOates were found to stimulate apoptosis and to synergize with fludarabine (Adams et al. 2001).

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The identification of the mechanisms involved in the anti-apoptotic action of endogenous NO should also provide new possible targets for the treatment of CLL. For instance, NO-induced S-nitrosylation is known to elicit not only caspase inactivation, but also to prevent the down-regulation of Bcl-2 by impairing its degradation by the proteasome. Several other molecules that play various roles during the completion of the apoptotic process have been reported to be affected by S-nitrosylation in various cell types (Iyer et al. 2008). Further studies are needed to evaluate their implication in the control of CLL survival and apoptosis and their potential use for new therapeutic approach. Acknowledgments This work was supported by INSERM, University Pierre et Marie Curie and Canceropole Ile-de-France.

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Part IV

Role of Nitric Oxide in Metastasis

Chapter 10

Nitric Oxide: A Rate-Limiting Factor for Metastases Development Angel Ortega, Salvador Mena, and José M. Estrela

Abstract Genomic and phenotypic instability associates with cancer cell heterogeneity. Although it has been argued that metastatic/invasive phenotypes are already present in primary tumors, highly aggressive and resistant cancer cell subsets may develop during in vivo growth and/or as a consequence of therapy. Moreover, factors such as the attack of our immune system or organ-specific microenvironments also affect cancer cell behavior and the subsequent response to drugs and/or other therapeutic agents. Interaction of cancer and endothelial cells in capillary beds initiates a cascade of molecular events that involve cytokines, growth factors, bioactive lipids, and reactive nitrogen and oxygen species (RNS and ROS) produced by either the cancer or the endothelial cells. Vascular endothelium-derived NO and H2 O2 are not only cytotoxic for the cancer cells but also help to identify some critical molecular targets that appear essential for survival of invasive cells. Growing metastatic cells may keep adapting for survival in a sequence of molecular events where RNS play a key role. Keywords Nitric oxide · Metastases · Metastatic microenvironment · Tumor resistance

Introduction The role of NO in the metastatic process is only partially understood. The available literature suggests positive and negative effects of NO at different key steps, including arrest within the microvessels, the host inflammatory response, the interaction between cancer and endothelial cells, the survival and growth of invasive cells, angiogenesis promotion, and the regulation of tumor blood flow, or the adaptation J.M. Estrela (B) Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, 15 Av. Blasco Ibañez, Valencia 46010, Spain e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_10, C Springer Science+Business Media, LLC 2010

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of growing metastatic cells to a new organ microenvironment. These controversial effects may depend on the NO generated by the metastatic cells and on the NO present in the metastatic microenvironment. Thus, activity and localization of the different NOS isoforms and duration of NO exposure are also important factors to keep in mind. This chapter offers a general perspective of how NO is involved in regulating the metastatic cascade. Nevertheless, our present knowledge is limited and still fully ignores critical issues such as the malignant phenotype and potential. We will focus on the molecular events that take place after the arrest of circulating cancer cells within the microvascular system of an organ.

Interaction Between Cancer and Endothelial Cells: The Intravascular Origin of Metastasis Cancer Cell Arrest Within the Microcirculation and Organ Selectivity Metastases formation involves interactions between tumor cells and a changing microenvironment that influences cell proliferation, migration, invasion, and colonization, as well as cell survival (Fidler 2003; Sahai 2007). Tumor cells that survive the harsh circulatory stream and reach distant organs/tissues still need to extravasate to begin secondary colonization. How this process occurs is critical in the metastatic cascade and two potential mechanisms have been proposed (Chambers et al. 2002). One mechanism is tumor cell arrest in microvessels. Using fluorescence-tagged tumor cell and video-capturing image techniques, Weiss et al. found that many tumor cells injected into mice intraperitoneally were arrested in capillaries (Weiss et al. 1992). Tumor cells often aggregate with platelets and due to the size of their mass can be found physically trapped in the capillaries. These arrested tumor cells may either remain inactive or start growing and eventually extravasate by secreting proteolytic enzymes and rupturing the blood vessel (Al-Mehdi et al. 2000). However, most cancer cells arrested in the microcirculation appear to die, at least in part, due to deformation and surface-membrane rupture (Weiss et al. 1992). NO was found to inhibit the aggregation of platelets via a cGMP-dependent mechanism (Radomski et al. 1990). In fact, although the ability of metastatic cells to form aggregates with platelets correlates with their metastatic potential, it is inversely proportional to NO generation (Radomski et al. 1991). The other mechanism of extravasation mimics the infiltration of leukocytes to the inflammatory site, a process which requires adhesion of tumor and endothelial cells. When leukocytes are attracted to the site of inflammation via chemokine gradient, their extravasation occurs in four steps (Rao et al. 2007). In the first step, leukocytes adhere to the vessel wall via selectin molecules and roll along the endothelium surface (a reversible process). The second step is tight adhesion of leukocytes to the endothelium through other adhesion molecules on the endothelial cells such as

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integrins, intercellular adhesion molecule 1, and platelet/endothelial cell adhesion molecule. The third step is diapedesis and leukocytes start extravasating or crossing the endothelial wall. This step also involves adhesion molecules such as leukocyte functional antigen 1 and macrophage receptor 1, and this interaction enables the leukocytes to migrate through the endothelial cell junction or squeeze their bodies through capillary pores (Engelhardt and Wolburg 2004). The fourth step is the migration to the inflammatory site following a mechanism of attraction by different types of chemokines. Because many cancer cells express adhesion molecules similar to those expressed on the migrating leukocytes, it is generally accepted that metastatic cells use a similar strategy for adhesion to the endothelial cells during metastatic preinvasion. Which mechanism prevails in the metastatic process is still under discussion. Nevertheless, it is essential to remark that the enigma of tissue specificity observed in metastatic colonization of tumor cells has finally began to unfold itself more than a century after Paget developed the theory of seed and soil (Fokas et al. 2007). Cancer metastasis results from a non-random process, in which organ selectivity by the tumor cells is largely determined by factors that are expressed at the remote organs that eventually turn into preferred sites of metastasis formation. These factors support the consecutive steps required for metastasis formation, including tumor cell adhesion to microvessel walls, extravasation into target tissue, and migration. In this sense, chemokines are important constituents of the tumor microenvironment at metastatic sites, dictating directionality of chemokine receptor-expressing tumor cells, facilitating their adhesion and extravasation, and eventually contributing to organ selectivity (Ben-Baruch 2008). Therefore, although mechanical trapping at the capillaries is indeed observed, the biochemical organ microenvironment presumably plays a critical role in attracting circulating tumor cells to specific microcirculatory areas. The available information suggests that NO signalling regulates cancer cell adhesion to the vascular endothelium either positively or negatively (Williams and Djamgoz 2005). This is probably linked to the observed apparent variability in the NO concentration in tissues, either within cells or at the extracellular compartments. NO has been shown to inhibit adhesion to extracellular matrix components of many cell types including eosinophils (Ferreira et al. 2004) and neutrophils (Clancy et al. 1995). N-nitro-l-arginine (a general NOS inhibitor) or aminoguanidine (a specific iNOS inhibitor) increased neutrophil adhesion to endothelium (Dal Secco et al. 2003). A similar effect was observed in adhesion of eosinophils from rats administered with the NOS inhibitor N-nitro-l-arginine methyl ester hydrochloride (l-NAME). Whereas in another study, NO was shown to inhibit expression of cell adhesion molecules (e.g., ICAM-1) (Ozturk et al. 2003). Nevertheless, NO has also been found to have pro-adhesive effects. Using rat brain microvascular endothelial cells, expression of ICAM-1 was upregulated synergistically by VEGF and NO (Radisavljevic et al. 2000). This process was mediated through a phosphatidylinositol-3-OH-kinase (PI3K)/AKT pathway: VEGF caused phosphorylation of AKT by PI3K leading to production of NO (Radisavljevic et al. 2000).

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In metastatic invasion the motile cancer cells will adhere and migrate into secondary tumor sites. Cellular adhesion molecules are involved both directly in the adherence detachment and indirectly in motility. Thus NO fluxes in the metastatic microenvironment may play a critical role in determining metastatic cell dynamics and, consequently, the success or failure of extravasation. Indeed, NO has been positively correlated with migration in mammary adenocarcinoma cells via sequential stimulation of NOS, guanylate cyclase, and mitogen-activated protein kinase (Jadeski et al. 2003). The liver is a paradigmatic example of a common site for metastasis development. It has been shown that under experimental conditions, a significant percentage of circulating cancer cells are mechanically trapped in the liver microvasculature (Weiss 1990). Interaction of metastatic cells with the hepatic sinusoidal endothelium (HSE) and Kupffer cells activates local release of proinflammatory cytokines, which can then act as molecular signals promoting cancer cell adhesion, invasion, and proliferation (Anasagasti et al. 1997a; Bayon et al. 1996; Li et al. 1991). Selectins, integrins, cadherins, and immunoglobulins, as well as unclassified molecules, have been demonstrated to control the adhesive interactions between metastatic cells and the vascular endothelium (Borsig et al. 2002; Koukoulis et al. 1998). This process has common features with the recruitment of leukocytes onto the vascular endothelium before extravasation to injured tissues and may partially explain the link between inflammation and metastasis (Kobayashi et al. 2007; Wilson and Balkwill 2002). The integrin family of cell adhesion receptors has been shown to play a critical role in all of these processes (consistent with their extracellular matrix binding properties) and may indeed promote various stages of metastasis by modulating the effects of growth factor receptors, extracellular proteases, and chemotactic molecules (Guo and Giancotti 2004). Other cell adhesion molecules such as E-selectin have been found strongly upregulated in the liver, thereby facilitating the arrest of further incoming cancer cells (Khatib et al. 1999). E-selectin expression by the HSE was shown to be only part of the proinflammatory response of the host-organ microenvironment to arrested cancer cells, which includes the release of TNF-α by Kupffer cells and the subsequent P-selectin, VCAM-1, and ICAM-1 expression by the HSE (Auguste 2007). This process is one of the first steps leading to the creation of a favorable metastatic niche. Initial contact between metastatic cells and the endothelium (docking) is weak and transient and likely mediated by carbohydrate–carbohydrate recognition (Orr et al. 2000). In the case of the interaction between murine B16 melanoma (B16M) and HSE, the mechanism includes mannose receptor-mediated melanoma cell attachment to the HSE, which subsequently causes proinflammatory cytokine release (TNF-α, IL-1β, and IL-18) and VCAM-1-dependent adherence (reinforcing or locking the initial intercellular binding). Some metastatic cells expressed high levels of the integrin VLA-4, the ligand for VCAM-1 on activated endothelial cells (Garofalo et al. 1995; Klemke et al. 2007). Release of endogenous NO from tumor cells may also have indirect effects on their motility by disrupting cellular barriers. In fact, monolayer permeability of enterocytes was increased by S-nitroso-N-acetyl-DL-penicillamine (SNAP) and this

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enhanced bacterial translocation (Xu et al. 2002) possibly by dilation of tight junctions between enterocytes (Salzman et al. 1995). Thus, endogenous generation of NO during migration could facilitate, directly or indirectly, cancer cell migration to secondary sites. Hepatic zonal heterogeneity has also been shown to play a significant role influencing the patterns of intercellular adhesion molecule expression. With a lower expression around the terminal portal venule regions (acinar zone 1) under basal conditions, ICAM-1 was significantly upregulated across the entire acinus following lipopolysaccharide (LPS) administration. On the other hand, VCAM-1 and E-selectin were minimally or not expressed in unstimulated livers, although they were highly expressed in acinar zone 1 than in other zones after LPS stimulation (Wang et al. 1999). LPS stimulation also increased the number of arrested B16MF1 (low metastatic potential) melanoma cells in the liver, particularly in the terminal portal venule region likely by increased expression of ICAM-1 and VCAM-1 (Wang et al. 2002).

Endothelium-Induced Cancer Cytotoxicity Recent in vivo and in vitro experimental evidence from different research groups indicates that during the interactions between endothelial cells and intravascular tumor cells, NO plays a key role as a cytotoxic natural defensive effector, produced by the vascular endothelium, exerting toxic effects on potential invader tumor cells and interacting with intercellular adhesion molecules and regulating the subsequent metastatic tumor formation in the secondary organ (Carretero et al. 2001; Mena et al. 2007; Wang et al. 2005a; Xie and Huang 2003). Direct in vitro lysis of metastatic tumor cells by cytokine-activated murine vascular endothelial cells has also been shown (Weiss 1990). In the B16M model, HSE releases large amounts of ROS in response to endotoxins and IL-1. Such proinflammatory mediators have been shown to promote cancer cell adhesion, invasion, and proliferation. In fact, VCAM-1 gene expression in HSE is coupled to an oxidative stress-dependent mechanism (Bayon et al. 1996; Marui et al. 1993). Rolling and early adhesion of B16M cells to the HSE, IL-1-dependent endothelial release of H2 O2 through IL-18, and late adhesion of surviving melanoma cells are sequential steps during B16M cell attachment to the HSE that occur in a short period (3–6 h) and enhance melanoma cell adhesion. These mechanisms would likely compensate for ROS-induced direct cytotoxic effects on adherent vulnerable melanoma cells and lead to the metastatic progression of H2 O2 -resistant melanoma cells (Anasagasti et al. 1997b; Mendoza et al. 1998). Wang et al. identified a natural defense mechanism against metastatic cells whereby their arrest in the HSE induces endothelial NO release, leading to sinusoidal cancer-cell killing and reduced hepatic metastasis formation (Wang et al. 2000). However, within the tumor microenvironment NO can be produced by several cells (tumor cells, macrophages, endothelial, or stromal cells). In fact, tumor-derived and host-derived NO differentially regulates breast carcinoma metastasis to the

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lungs (Gauthier et al. 2004). Moreover, in many different types of cancer, expression of NO synthases, mainly the inducible (iNOS) isoform, has been positively correlated with tumor invasion and angiogenesis (Jadeski et al. 2003; Tu et al. 2006; Wang et al. 2005b). In fact, NO signalling has been involved in regulating proliferation, apoptosis, adhesion, migration, invasion, and angiogenesis (Fukumura et al. 2006). In agreement with these concepts, transfection with the iNOS gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells (Xie et al. 1995). Previous reports have also shown cooperative cytotoxic actions of NO and H2 O2 in Escherichia coli (Pacelli et al. 1995) and Fu5 rat hepatoma cells (Ioannidis and de Groot 1993). Interestingly ROS and RNS formed during hepatic ischemiareperfusion have been involved in the killing of weakly but not highly metastatic colorectal cancer cells (Jessup et al. 1999). In agreement with this idea, it has been demonstrated that endothelial NO and H2 O2 and their derived reactive species show synergistic cytotoxic effects on metastatic cells (Carretero et al. 2001). Isolated HSE cells treated in vitro with TNF-α and IFN-γ (a maneuver that mimics the proinflammatory scenario of the metastatic microenvironment) release measurable amounts of NO and H2 O2 into the culture medium in a time-dependent fashion (Carretero et al. 2001). Coculture of these endothelial cells with B16M cells showed that during the adhesion process, most of the NO and H2 O2 were generated by the HSE. Inhibition of NO production using HSE cells isolated from eNOS-deficient (eNOS−/− ) mice (which abolished eNOS-dependent NO production) or l-NAME showed that H2 O2 released by the HSE did not induce tumor cytotoxicity. However, NO was particularly tumoricidal in the presence of the H2 O2 because addition of exogenous catalase that eliminates H2 O2 released to the extracellular medium decreased tumor cytotoxicity significantly (Carretero et al. 2001). These findings are in agreement with later reports showing inhibition of B16M lung metastasis by local release of eNOS-derived NO (Qiu et al. 2003). When we explored the chemical mechanisms by which NO and H2 O2 are cytotoxic, we found that a major part of the effect requires the presence of trace metals capable of generating highly oxidant radicals, likely ·OH and − OONO (Carretero et al. 2001; Jessup et al. 1999). Despite the fact that HSE-derived NO and H2 O2 cause B16M cytotoxicity (Carretero et al. 2001), and although findings in other cell systems support this cooperative action (Chiang et al. 2000; Farias-Eisner et al. 1996; Ioannidis and de Groot 1993; Pacelli et al. 1995), others have suggested that NO protects against ROS (Gupta et al. 1997; Wink et al. 1993). A plausible explanation for this apparent paradox is that both NO and H2 O2 can show very different effects depending on their concentrations. Indeed, experiments in isolated mitochondria showed that NO reversibly inhibited permeability transition pore (PTP) opening with an IC50 of 11 nM; however, at higher concentrations (>2 μM) NO accelerated pore opening (Gupta et al. 1997). Identically, low levels of H2 O2 (3–15 μM) may cause a significant mitogenic response in mammalian cells, whereas higher concentrations (>100 μM) may cause growth arrest and cell damage (Davies 1999). Therefore, considering the amounts of NO and H2 O2 released by the HSE during the process of cancer cell adhesion, tumor cytotoxicity must be expected as it indeed occurs (Carretero et al. 2001).

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Molecular Determinants of Metastatic Cell Survival Since some metastatic cells survive after interacting with the vascular endothelium, it is plausible that reactive oxygen and nitrogen species (ROS and RNS) have a limited toxicity against them or that some of these cells able to extravasate and proliferate are ROS/RNS-resistant due to, e.g., overexpression of their intracellular antioxidant machinery. In this respect, it has been shown that high levels of intracellular glutathione (GSH, the most prevalent non-protein thiol in mammalian cells) protect cancer cells against ROS/RNS-induced cytotoxicity (Carretero et al. 2001; Obrador et al. 1997). A high percentage of tumor cells with high GSH content survive the combined nitrosative and oxidative attack and thus may represent the main task force in the metastatic invasion (Carretero et al. 2001). B16M-F10 (high metastatic potential) cells cultured to low density (LD) with high GSH content were more resistant to NO and H2 O2 than B16M cultured to high density (HD, with ∼25% of the GSH content found in LD cells) (Carretero et al. 2001). NO- and H2 O2 -dependent cytotoxicity in B16M-F10 cells attached to cytokine (TNF-α and IFN-γ)-activated HSE was of ∼18% in LD B16M-F10 cells and of ∼78% in HD B16M-F10 cells (Carretero et al. 2001). HSE-induced tumor cytotoxicity in l-buthionine (S,R)-sulfoximine (BSO, a specific GSH synthesis inhibitor)-treated LD B16M cells was similar to that found in HD B16M cells (Carretero et al. 2001), which suggested a direct involvement of GSH in protecting B16M cells against HSE-induced cytotoxicity. In addition, it was shown that metastatic growth can be implemented in B16M-F1 cells (low metastatic activity) by using GSH ester, which directly increases their GSH content (Obrador et al. 2002). Nevertheless, even after BSO-induced GSH depletion, a significant amount of LD B16M-F10 cells (∼32%) survived during in vitro interaction with the HSE (Carretero et al. 2001). This is a critical fact since highly resistant metastatic cell subsets may be responsible for the explosive metastatic growth that follows tissue invasion under in vivo conditions. Therefore, although high GSH content status is an important parameter for metastasis progression in metastases cells, other factor(s) must necessarily contribute to the survival of some cell subsets with high metastatic potential. The expression of genes known to affect apoptosis (e.g., Bcl-2, p53, fas, NO synthetases) may affect tumor growth and possibly metastatic inefficiency (Lowe and Lin 2000). Takaoka et al. observed that Bcl-2 overexpression in B16M cells enhanced pulmonary metastasis (Takaoka et al. 1997). Similarly, melanoma cells resistant to fas-mediated apoptosis were found more susceptible to metastasis (Owen-Schaub et al. 1998). Furthermore, although apoptotic Hras and v-myc transformed metastatic fibroblasts labeled with green fluorescent protein were observed in the lungs, in vitro-induced Bcl-2 overexpression in these cells conferred resistance to apoptosis 24–48 h after inoculation (Wong et al. 2001). Thus, it is plausible that regulation of cell death mechanisms influences metastatic growth, at least in the early stages after attachment to the vascular endothelium. The proto-oncogene Bcl-2 and its anti-apoptotic homologs are mitochondrial membrane permeabilization inhibitors (Gross 2001) and participate in development of chemoresistance (Reed 1997), whereas expression of pro-death genes such as

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Bax or Bak is often reduced in cancer cells (Hickman 2002). The thiol redox status (controlled by GSH) is one of the physiological effectors involved in regulating the mitochondrial permeability transition pore complex (Kroemer and Reed 2000). The importance of GSH in regulating Bcl-2’s ability to prevent apoptosis was first detected in GT1-7 neuronal cells where Bcl-2-induced suppression of apoptosis required GSH (Kane et al. 1993). Later, different reports indicated that GSH depletion may have therapeutic use in sensitizing Bcl-2-overexpressing cells to apoptotic cell death (Armstrong and Jones 2002; Mirkovic et al. 1997; Rudin et al. 2003; Vahrmeijer et al. 2000). Analysis of a Bcl-2 family of genes revealed that B16MF10 cells, as compared with B16M-F1 cells, preferentially overexpressed Bcl-2 (∼5.7-fold) (Ortega et al. 2003a). HSE-induced B16M-F10 cytotoxicity in vitro increased from ∼19% (controls) to ∼97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS) (Ortega et al. 2003a). BSO-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver (Ortega et al. 2003a). However, the combination of BSO and Bcl-2-AS abolished metastatic invasion (Ortega et al. 2003a). Bcl-2 overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared to controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of control values. However, this critical threshold increased to ∼60% of control values in Bcl2-AS-treated B16M-F10 cells (Ortega et al. 2003a). Thus, GSH-dependent cancer cell survival within the microvasculature and Bcl-2-dependent cell death regulation may be closely related mechanisms which cooperate to favor survival of highly metastatic cell subsets. However, the mitochondrial system for GSH uptake is impaired in iB16M cells [invasive cells that survive after interaction with the vascular endothelium (Ortega et al. 2003b)]. This is important since mtGSH depletion may facilitate mitochondrial membrane permeabilization, permeability transition pore (PTP) opening, and the release of apoptosis-inducing molecular signals (Obrador et al. 2001). Endotheliumderived NO and H2 O2 damage the high- and the low-affinity components of this system. This fact can challenge iB16M cells to maintain their physiological mtGSH levels under conditions of low cytosolic GSH levels (<1 mM). Thus maintenance of mtGSH homeostasis may be a limiting factor for the survival of metastatic cells in the immediate period following intrasinusoidal arrest and interaction with activated vascular endothelial cells. Mitochondrial dysfunction is a common event in the mechanisms leading to cell death (Costantini et al. 2000), and recently it has been found to be an essential step for killing of non-small cell lung carcinomas resistant to conventional treatments (Joseph et al. 2002). Mitochondrial permeability transition (MPT) is critical in the process leading to apoptosis and it is linked to the opening of the PTP complex (Kroemer and Reed 2000). This molecular gate is regulated by many endogenous

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factors, including divalent cations (e.g., Ca2+ and Mg2+ ), protons, the mitochondrial membrane potential (MMP), the concentration of adenine nucleotides, the thiol (controlled by GSH) and the pyrimidine redox state, the rate of ROS and NO generation, the concentration of lipoids (e.g., ceramide), the concentration of certain peptides targeting proteins for mitochondrial import, and the function of different pro- and anti-apoptotic proteins (Kroemer and Reed 2000). GSH, which is not synthesized by mitochondria but taken up from the cytosol through a multicomponent transport system (Martensson et al. 1990), is the only defense against peroxides generated from the electron transport chain (Arai et al. 1999) and may be an important regulator of the MPT and PTP opening (Hall 1999; Kroemer and Reed 2000). Thus, impairment of GSH uptake by mitochondria may enhance effectiveness of molecular effectors (e.g., oxidative stress inducers such as TNF-α) capable of activating the mitochondrion-based death mechanism in growing metastatic cells.

Extravasation and Metastatic Growth Migration and Limited Survival of Early Micrometastases The metastatic process is inefficient since very few of the tumor cells released into the circulation develop into metastases (Weiss 1990). Different studies have shown that only ∼0.01% of all cancer cells injected into the circulation form metastatic foci (Fidler 1970). However, although it is uncertain at which steps in the process cells are lost, it has generally been accepted that most cancer cells are rapidly destroyed in the circulation by different mechanisms: lytic action of immunocompetent lymphocytes and macrophages, mechanical trauma produced by blood flow, locally released ROS/RNS, and their inability to withstand deformation (Orr et al. 2000). In addition, the ability of cancer cells to extravasate into the surrounding tissue by degrading basement membrane and extracellular matrix has been considered another major rate-limiting step in metastasis (Liotta et al. 1991). Indeed, metastatic cells that survive after interacting with the endothelium release a variety of molecules including proteases, growth factors, and cytokines. Although there is little evidence for involvement of NO in regulating tumor secretion, NO has been shown to promote cancer cell migratory ability, invasion, and angiogenesis in mammary adenocarcinoma metastases (Jadeski et al. 2000; Williams and Djamgoz 2005). Furthermore, release of NO from metastatic cells may also have effects on their motility by damaging cellular barriers. In fact, monolayer permeability of enterocytes was increased by NO donors such as SNAP (Xu et al. 2002). A study in which B16M-F1 melanoma cells were injected intraportally found that >80% of the injected cells survived and had extravasated by day 3. However, few extravasated cells began to grow with only 1 in 40 forming micrometastases (4–16 cells) by day 3. Furthermore, few micrometastases continued to grow, with only 1 in 100 progressing to form macroscopic tumors by day 13 (Luzzi et al. 1998). Surprisingly, 36% of injected cells remained by day 13 as solitary cancer cells,

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95% of which were shown to be dormant; in contrast, within macroscopic tumors, only 3% of cells were dormant (Luzzi et al. 1998). Thus, in the B16M-F1 model, metastatic inefficiency appears to be determined by the failure of solitary cells to initiate growth and the failure of early micrometastases to continue growth into macroscopic tumors. iNOS expression/activity caused human colorectal adenocarcinoma cell line HRT-18 to be more invasive as compared to the iNOS-lacking HT-29 cell line counterpart (Siegert et al. 2002). Mammary adenocarcinoma cells (expressing eNOS but not iNOS) with high metastatic potential (C3L5) produce more NO than their weakly metastatic counterpart (C10), and this could be correlated with higher invasiveness (Jadeski et al. 2000). Therefore, the available literature strongly suggests a positive relationship between pathophysiological NO levels in the metastatic microenvironment and invasion.

Angiogenesis and Regulation of Tumor Blood Flow NO, by regulating vasodilatation, platelet aggregation, angiogenesis, production of prostaglandins, leukocyte proliferation, or direct tumor cytotoxicity (Wink et al. 1998), can affect tumor cell arrest in capillaries and secondary metastatic growth. NO reversibly binds to cytochrome c oxidase and inhibits tumor cell respiration, an effect that can be suppressed by GSH (Nishikawa et al. 1998). Interacting with metal ions or forming RNS, NO can affect the activity of different proteins including the NMDA receptor, hemoglobin, and transcription factors such as NF-κB and SoxR. In addition, by inducing S-nitrosylation of caspases and tissue transglutaminase, NO may regulate the balance between apoptosis and necrosis (Melino et al. 1997). Angiogenesis is necessary for metastases progression. However, tumors contain not only tumor cells but also non-tumor cells such as vascular cells, fibroblasts, and immune cells. In many cases, iNOS upregulation in fibroblasts and immune cells associated with a similar finding in tumor cells (Fukumura et al. 2006). Isolated tumor-associated macrophages or cytokine-activated fibroblasts have been shown to have tumoricidal activity through iNOS-derived NO (Fukumura et al. 2006). Paradoxically, Konopka et al. showed lower VEGF expression and slower growth of B16 melanoma in iNOS−/− mice, which indicates that iNOS in tumor stroma promotes tumor growth through induction of VEGF expression and angiogenesis (Konopka et al. 2001). Moreover, NO may also increase endothelial cell proliferation and migration through S-nitrosylation and the sGC–cGMP pathway (Kawasaki et al. 2003; Zaragoza et al. 2002). Findings reflect different NO levels associated with different tumor microenvironments, different regulatory inputs (local or systemic), and perhaps different NO sensitivity among types of cancer cells. NO has been shown to mediate the function of different angiogenic factors. VEGF, sphingosine-1-phosphate, angiopoietins, oestrogen, shear stress, and metabolic stress activate eNOS through phospholipase-C–Ca2+ –calmodulin binding and PI3K–AKT-induced and adenylate cyclase–protein kinase-A-induced

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phosphorylation (Fukumura et al. 2006). In agreement with these facts, antiangiogenic factors exert their effects through inhibition of eNOS–NO signaling. Endostatin dephosphorylates eNOS at serine 1177 through protein phosphatase 2A and an NO donor reverses the inhibitory effect of endostatin on endothelial cell migration (Urbich et al. 2002). Inhibition of eNOS using a pharmacological or a genetic approach inhibits tumor angiogenesis (e.g., Gratton et al. 2003). Furthermore, NO donors can also induce VEGF expression and coronary vein endothelial cell proliferation through the expression of basic fibroblast growth factor (Chin et al. 1997). NO activates the transcription factor hypoxia-inducible factor 1α (HIF1α), which in turn upregulates VEGF, thereby promoting angiogenesis (Xie and Huang 2003). Nevertheless, NO has also been shown to inhibit hypoxia-dependent HIF1α activation and VEGF expression (Sogawa et al. 1998). Again, this paradox may be explained by differences in NO exposure (time and concentration). NO is also involved in tumor vessel maturation. In the B16M model, a decrease in the perivascular cell (pericytes and smooth muscle cells) coverage was found in cells with low NOS expression (Kashiwagi et al. 2005). In particular, eNOSderived NO, as shown using eNOS−/− mice, is the main promoter of perivascular cell recruitment (Kashiwagi et al. 2005). NO, acting as the classical endothelium-derived relaxing factor, can also regulate tumor blood flow. However, the vascular responses to NO inhibitors and/or donors may be very variable (Fukumura et al. 2006), thus suggesting a heterogeneous NOS distribution. Finally, and downregulation of tumor vascular permeability by NO have been also described (Fukumura et al. 2006; Kubes 1995). Increased microvessel protein leakage was observed in normal tissues in the presence of NOS inhibitors. NOS inhibition prevents or significantly decreases vascular permeability induction by a variety of factors including different cytokines and growth factors (Fukumura et al. 2006). In fact, the available literature strongly suggests that low levels of NO preserve physiological blood vessel integrity, whereas high NO levels generated under pathological conditions might increase vascular permeability.

Adaptive Response Toward Higher Resistance Microarray-based gene expression analysis revealed changes in 2000 transcripts in the response of HL60 cells to low H2 O2 concentrations. The biological mediators overrepresented are key factors in carcinogenesis, such as NF-κB activation or DNA methylation, genes for cytokine and chemokine ligands and receptors, the redox regulator thioredoxin interacting protein, the histone deacetylase sirtuin, heat-shock proteins (HSP40 and HSP70), and the AP-1 transcription factor components Fos and FosB (Fratelli et al. 2005). Moreover, it has been shown that ROS regulate HIF-1 and VEGF in ovarian cancer cells and that elevated levels of endogenous ROS are required for inducing angiogenesis and tumor growth (Xie and Huang 2003).

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Low NO concentrations act on several signaling pathways activating transcription factors, such as NF-κB or AP-1, and hence influence gene expression. In contrast, upon inflammation high NO levels are released for prolonged periods of time. The resulting nitrosative stress directly affects redox-sensitive transcription factors implied in carcinogenesis, including NF-κB, AP-1, or c-Myb. Moreover nitrosative stress, indirectly, may modulate activity or stability of HIF-1 or NF-κB or accessibility of promoters via increased DNA methylation and histone deacetylation (Kroncke 2003). NO and H2 O2 , at the high levels released by the vascular endothelium, may counteract signaling pathways and cause direct molecular damages and cell death (Hirst and Robson 2007). Oxidative and nitrosative stress often function as a double-edged sword, inducing cell death when in excess or protecting cells against various apoptotic or necrotic insults when present at more physiological levels. Stress can induce genetic and epigenetic alterations leading to expansion of resistant metastatic cell subsets (Xie and Huang 2003). In B16M cells interacting with the vascular endothelium, H2 O2 - and NO-induced adaptations include cell adhesion molecule expression in both endothelial and cancer cells (see above), activation of the early growth response-1 transcription factor gene, activation of cancer, and endothelial cell metalloproteinases, increase of antioxidant enzymes such as Mn-containing superoxide dismutase and catalase (Benlloch et al. 2005), and induction of key invasive growth-related molecules such as VEGF-A, HIF-1, and protein 8 (Mena et al. 2007). A growing body of evidence suggests that many cellular responses to oxidative and nitrosative stress are indeed regulated at the transcriptional level (Marshall et al. 2000). Nitrosylation or oxidation of critical Cys residues in the DNA-binding domains or at allosteric sites may regulate transcription of target genes (Marshall et al. 2000), although molecular mechanisms underlying redox control of mammalian gene expression have not been elucidated in any well-defined cellular system. Therefore, the net result of pro- and anti-metastatic ROS and RNS effects may determine the progression of metastatic cells within a tissue. Availability of large-scale multigene expression analysis might represent a powerful approach to better understand RNS- and ROS-induced adaptive responses in selected invasive cells and may help to explain the mechanisms by which they become highly resistant to conventional therapies.

Concluding Remarks NO plays an important role as a cell-signalling molecule, but its biological effects are complex and dependent upon many regulatory factors. Further research is necessary to improve our understanding of the complex mechanisms that regulate the roles of NO in tumor biology. The presence of activated oncogenes and/or inactivated tumor suppressor genes may result in activation of multiple transcription factors. At advanced stages, uncontrolled tumor growth and the development of a stress microenvironment, such

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as hypoxia, acidosis, and free radical overproduction, may alter the activity of these transcription factors. These events may cause aberrant expression of multiple metastasis-related proteins and confer survival and growth advantages to metastatic cells. Indeed, NO affects multiple targets that allow regulation of gene expression. Although there is no evidence for direct NO-responsive DNA elements within promoter regions of eukaryotic genes, numerous indirect signalling pathways exist to explain NO-regulated gene expression. Thus, further understanding of these molecular mechanisms is necessary. Cancer cells exposed to low levels of NO, or capable of resisting NO-mediated injury, undergo a clonal selection that favors their survival. In addition they also use NO-mediated mechanisms for promotion of growth, invasion, and metastasis: (a) stimulation of tumor cell invasiveness; (b) promotion of tumor angiogenesis and blood flow; and (c) suppression of host anti-tumor defense. Tumor-derived NO has been shown to promote tumor cell invasiveness and angiogenesis. The invasionstimulating effects of NO are due to an upregulation of matrix metalloproteases and a downregulation of their natural inhibitors. Thus, treatment of tumor-bearing mice with NO-blocking agents may reduce the growth and vascularity of primary tumors and, likely, their spontaneous metastases. The metabolic fate of NO gives rise to a further series of compounds, collectively known as the reactive nitrogen species (RNS). However, the consequences of many of the chemical reactions involving RNS in vivo and, particularly, in growing tumors are not known. It is also of particular interest to further investigate if NO and/or other endothelium- or immune cell-derived cytotoxic/signalling molecules reveal other key targets involved in metastatic cell escape mechanisms. However, it is essential to remark the importance of studying biological effects of NO in the presence of in vivo relevant NO concentrations. Finally, the effects of NO and its synergy with members of the TNF family, with cytotoxic drugs, and with ionizing radiations also appear to be worthy of further investigation. Nevertheless, considering metastases as the most severe stage for cancer patients, the more critical question remains to be answered: will manipulation of NO, either as a stand-alone therapy or in combination with conventional treatments, lead to a significant improvement of patient survival?

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Chapter 11

Nitric Oxide Inhibits Tumor Cell Metastasis via Dysregulation of the NF-κB/Snail/RKIP Loop Stavroula Baritaki and Benjamin Bonavida

Abstract The major cause of cancer-related death is metastasis. A clearer understanding of the underlying mechanisms of metastasis should improve current therapies following the design of new treatment modalities. In this chapter, we discuss the therapeutic potential of endogenous nitric oxide (NO) or NO donors in the inhibition of both the tumor progression and the induction of metastasis through inhibition of the epithelial to mesenchymal transition (EMT). Briefly, the findings show that NO donors’ treatment of metastatic human cancer cell lines results in several modifications of cell survival signaling pathways that regulate metastasis and particularly EMT. NO donors inhibit NF-κB activity and downstream the metastasis inducer transcription factor, Snail. In addition, NO donors trigger the metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) via inhibition of the RKIP transcription repressor Snail. NO-induced inhibition of NF-κB and Snail and induction of RKIP established the NO-induced dysregulation of the NF-κB/Snail/RKIP circuitry in the regulation of metastasis. These findings suggest the therapeutic role of NO donors in the inhibition of metastasis. Keywords Nitric oxide · metastasis · EMT · NF-κB · Snail · RKIP

General Features of the Metastatic Process Metastatic disease is the primary cause of death for most cancer patients. Metastasis is a process that allows many tumors to expand to distant areas of the body from the primary tumor localization. The dissemination of tumor cells to those areas requires tumor cell migration and invasion into the extracellular matrix and then into the blood and lymphatic organs (Stetler-Stevenson, Aznavoorian and Liotta 1993; Steeg 2006; Pantel, Brakenhoff and Brandt 2008). The epithelial to mesenchymal transition (EMT) process is the principal mean through which metastasis occurs, namely, S. Baritaki (B) Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA 90095, USA e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_11, C Springer Science+Business Media, LLC 2010

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beginning with a disruption of intercellular contacts and the enhancement of cell motility and, thereby, resulting in the release of cells from the parent epithelial tissue. Epithelial cells, therefore, lose their association with epithelial cell sheets and acquire, instead, many of the attributes of mesenchymal cells including acquisition of increased invasiveness and resistance to apoptosis (Condeelis and Pollard 2006; Shook and Keller 2003). This trans-differentiation program is regulated by distinct pleiotropically acting transcription factors such as Snail, Twist, Slug, and Goosecoid (Thiery and Sleeman 2006; LaBonne and Bronner-Fraser 2000). At the biochemical level, the EMT program involves the downregulation of epithelial protein expression, notably E-cadherin and cytokeratins, and the induction of mesenchymal protein expression, including vimentin, N-cadherin, fibronectin, platelet-derived growth factor receptor (PDGFR), and matrix metalloproteinases, and acquisition of motility and invasiveness (Thiery and Sleeman 2006). When active in cancer cells, the EMT program enables the cells to complete the initial steps of invasiveness in the metastatic cascade, specifically local invasion, intravasation, survival in the circulation, and extravasation. Although surgery and radiation therapy effectively control many cancers at the primary site, the development of metastasis signals a poor prognosis. Most metastatic lesions are not treated by surgery, as the presence of one lesion often signals wider systemic disease. Chemotherapy, hormonal therapy, and radiation serve palliative purposes in the metastatic setting, and some offer a modest but statistically significant extension of survival. However, morbidity and mortality arising from metastatic disease can result from direct organ damage by the growing lesions, paraneoplastic syndromes, or from both the complications and/or the inefficacy of the currently available anti-metastatic treatments (Steeg 2006). Relatively few components of the metastatic process have been successfully developed as therapeutic targets; thus, the underlying mechanisms of the development of metastasis and its regulation should facilitate the design of more efficient therapies and improve patients’ outcome.

Molecular Mechanisms Regulating Metastasis Implication of the NF-κ B Survival Pathway in Tumor Metastasis NF-κB, a member of the Rel transcription factor family, participates in the mediation of many biological activities such as inflammation, immune response, cell survival, and programmed cell death. Its role in tumorigenesis is well established since it is in a hyperactivated state in several tumors and malignant tissues. NF-κB contributes to the development and/or progression of malignancy by regulating the expression of genes involved in cell growth and proliferation, apoptosis, and angiogenesis. NF-κB in its inactive form normally resides in the cytoplasm in association with its inhibitor IκBα. Through non-covalent association, IκBα masks the nuclear localization signal of NF-κB, thereby preventing NF-κB nuclear translocation and its activation. IκBα is phosphorylated by the IκBα kinase complex (IKK)

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which leads to IκBα poly-ubiquitination and degradation in the proteasome, thereby NF-κB is translocated into the nucleus where it participates in transcriptional regulation of a wide array of genes with diverse biological activities. TAK1 and NIK kinases, which belong to the MAPKKK family, are located upstream of IKK and activate IKK by phosphorylation. NF-κB is activated in response to a variety of stimuli such as cytokines and inflammatory responses and controls the transcription of various genes such as cytokines, adhesion molecules, and anti-apoptotic gene products (Rothwarf and Karin 1999). For instance, NF-κB regulates the activation of the inflammatory cytokines IL-1, IL-6, IL-8, and TNF-α, the anti-apoptotic c-IAP (e.g., c-IAP-1, -2) and Bcl-2 family members (e.g., Bcl-2, Bcl-xL , Mcl-1, Bfl-1/A1), and those that negatively regulate the NF-κB itself (e.g., IκBα) (Li and Stark 2002). Thus, NF-κB can alter the expression of pro- and anti-apoptotic genes leading to various biological outcomes including tumor resistance to chemotherapy or immunotherapy (Karin et al. 2002). Despite the essential role of NF-κB in the regulation of tumor cell survival, progression, and resistance to apoptosis induced by conventional therapeutics, the involvement of NF-κB in tumor metastasis is less studied. NF-κB regulates adhesion molecules and matrix proteases such as VLA-4 and its ligands, VCAM and vimentin (reviewed by Min et al. 2008). NF-κB-mediated expression of genes involved in angiogenesis (e.g., IL-8, VEGF), invasion, and metastasis (e.g., MMP9, uPA, uPA receptor) contributes to the progression and metastasis of tumor cells (Suh and Rabson 2004). In cells deficient in NF-κB signaling the expression of VLA-4, VLA-5, ICAM, and VCAM was dramatically decreased (Tozawa et al. 1995). Inhibition of NF-κB activity reduced the invasive phenotype of 7, 12-dimethylbenz(a)anthracene (DMBA) carcinogen-transformed mammary tumor cells driven by the NF-κB c-Rel subunit (Shin et al. 2006). In addition, inhibition of NF-κB in Ras-transformed epithelial cells (EpRas cells) led to a 10-fold reduction in metastases to the lungs following tail vein injection into nude mice and to a 3-fold decrease in tumor weight in a mammary fat pad model (Huber et al. 2004). Knockdown of NF-κB by siRNA reversed the mesenchymal-like phenotype and suppressed motility and invasion capacity of the bladder metastatic cell line BLS. The cells acquired a rounded and less elongated shape, E-cadherin expression, and significantly downregulated the expression of the mesenchymal marker vimentin. In vitro motility and invasion assays showed that both the motility and the invasive potential of p65 siRNA-transformed cells were significantly reduced (Zhang, Chen and Li 2008). Thus, NF-κB regulates, in part, the metastatic potential of several tumors and the inhibition of its activity could result in reduced tumor metastasis.

Implication of Raf-1 Kinase Inhibitor Protein (RKIP) in the Regulation of Tumor Metastasis Raf-1 kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine binding protein (PEBP) family, a highly conserved group of proteins found in a

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variety of organisms from plants to Drosophila and mammals. Analysis of database for proteins having homology to PEBP has revealed no significant sequence similarity with other proteins suggesting the unique characteristics of this family of proteins (Banfield et al. 1998). PEBPs are 21–23 kDa (187 amino acids in human PEBP) basic cytosolic proteins, which were originally purified from bovine brain (Schoentgen and Jolles 1995). Expression of PEBP mRNA has been detected in all mammalian tissues tested with high levels in spermatids, brain oligodendrocytes, Purkinje cells, and specific cortical and hippocampal neuronal cell layers. PEBP is believed to be the precursor of the hippocampal neurostimulating peptide (HCNP) that is involved in the differentiation of neurons in the medial septal nucleus, enhancing the synthesis of choline acetyltransferase (Seddiqi et al. 1996). Mammalian PEBPs have been found to regulate serine proteases by selectively inhibiting their activities. Serine proteases are involved in many processes in the nervous system where they play important roles in development and tissue homeostasis. Binding studies have shown that PEBPs have an affinity for phosphatidylethanolamine, nucleotides like GTP, GDP, and small GTP binding proteins, as well as for other hydrophobic ligands. Investigators evaluated the possible involvement of human PBP (hPBP) in GTP binding of G proteins and found a stimulatory effect of hPEBP on GTP binding to the cellular membranes. PEBPs have been shown to associate with cellular membranes and thus could participate in G protein-dependent signaling in a membrane-dependent fashion (Banfield et al. 1998). The high affinity of PEBP for phospholipids as well as its high expression levels in growing cells confirms a possible role of PEBP in membrane organization and biogenesis (Schoentgen and Jolles 1995). Furthermore, RKIP has homology to rat PEBP-3 (Simister, Banfield and Brady 2002) with a molecular weight of 23 kDa. Immunohistochemical studies revealed a cytoplasmic localization of RKIP; yet, under different tissue culture conditions the localization is not always restricted to the cytoplasm or the inner leaflet of the plasma membrane but it can be also found in the nucleus (unpublished data). Further, RKIP is found to be a hydrophilic protein and with time unfolds at the air and water interface (Vallee et al. 2001). Fu et al. (2003, 2006) used gene array analysis to identify more genes whose expression change during the transition from the non-metastatic LNCaP prostate carcinoma cells to the metastatic prostate cancer cells derived from LNCaP, C4-2B. They found that one gene, RKIP, was lower in the metastatic than the non-metastatic cell lines. The expression of RKIP was confirmed by RT-PCR and western blot analysis whereby C4-2B had four to five times less levels of RKIP than the parental LNCaP. These investigators suggested that RKIP functions as a suppressor of metastasis. RKIP is the thirteen metastasis suppressor gene product described in the literature (reviewed by Granovsky and Rosner 2008). Metastatic suppressors may be independent prognostic markers. There are suggestions that metastatic suppressor genes are not mutated but, instead, are differentially expressed at the protein translational level, due to gene multiplication, histone acetylation, and mRNA or protein stability. Recent studies by Fu et al. (2006) have demonstrated that the low levels of RKIP mRNA and protein are correlated with the metastatic potential of human C4-2B prostate cancer cells when compared with parental non-metastatic

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LNCaP cells. Moreover, overexpression of RKIP in C4-2B cells decreased cell invasion in vitro and progression of lung metastases in vivo. In addition, increased levels of RKIP were associated with decreased vascular invasion in the primary tumor with no effect on primary tumor growth in mice. Other studies have also identified RKIP as an invasion suppressor protein in nasopharyngeal carcinomas (Chen et al. 2008). These results suggest that RKIP does not affect the tumorigenic properties of cancer cells but may represent a clinically significant suppressor of metastasis by decreasing vascular invasion. Besides prostate cancer, the expression of RKIP has been associated with the progression and clinical outcome of other cancers. We and others have shown that RKIP levels are significantly decreased or completely absent in several highly metastatic tumors including hepatocellular carcinoma (Schuierer et al. 2004), breast (Hagan et al. 2005), colon (Minoo et al. 2007), gastric (Chatterjee et al. 2008), nasopharyngeal carcinoma (Chen et al. 2008), and rectal cancer (Zlobec et al. 2008). Therefore, RKIP has been considered to be a good prognostic marker of the pathogenesis of human prostate and colorectal cancer survival and disease (Fu et al. 2006; Minoo et al. 2007). Our preliminary findings have also shown reduced expression of RKIP in lung tumors as determined by tissue microarray analysis (unpublished data).

Implication of the Transcription Repressor Snail in the Regulation of Metastasis Snail family members encode transcription factors of the zinc-finger type. They all share a similar organization, being composed of a highly conserved carboxyterminal region, which contains from four to six zinc fingers and a much more divergent amino-terminal region. The fingers correspond to the C2H2 type 22 and function as sequence-specific DNA-binding motifs. Upon binding to the E-boxes of target promoters, Snail family members are thought to act as transcriptional repressors. The repressor activity depends not only on the finger region but also on at least two different motifs that are found in the amino-terminal region (reviewed by Nieto 2002). The first member of the Snail family, Snail, was initially described in Drosophila melanogaster, where it was shown to be essential for the formation of the mesoderm (Alberga et al. 1991). In this regard, the Snail family of zinc-finger transcription factors occupies a central role in morphogenesis in several organisms from flies to mammals (Hammerschmidt and Nüsslein-Volhard 1993). In humans, Snail has been identified as a key modulator of normal and neoplastic EMT program via strong inhibition of the metastasis suppressor gene product E-cad transcription (Cano et al. 2000; Batlle et al. 2000). Overexpression of Snail contributes directly to EMT, accelerating tumor survival, migration, and bad prognosis. These have been shown in different cancer cell lines and tumor biopsies including breast cancer (Blanco et al. 2002), gastric cancer (Rosivatz et al. 2002), hepatocellular carcinomas (Jiao et al. 2002), ovarian carcinoma (Elloul et al. 2006), oral squamous cell carcinoma (Yokoyama et al. 2001), and head and neck cancer (Yang et al. 2007).

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The biological significance of specific and stable Snail interference has been tested in two murine carcinoma cell lines, HaCa4 and CarB, showing endogenous expression of Snail and Slug and very aggressive tumorigenic, invasive, and metastatic behavior (Navarro et al. 1991; Llorens et al. 1998; Cano et al. 2000). Stable interference of Snail expression in HaCa4 and CarB cells led to significant changes in the expression of several markers associated with EMT, namely, activation of the E-cad promoter, decreased expression of vimentin and fibronectin at both the protein and the mRNA levels, and downregulation of MMP-9 expression and secreted activity. Importantly, Snail blockade leads to a significant decrease in in vitro invasiveness of both HaCa4 and CarB cells (Olmeda et al. 2007). Therefore, Snail silencing interferes with biological properties of carcinoma cells related to inhibition of metastasis. Julien et al. (2007) have recently reported that activation of NF-κB by upregulation of AKT results in downstream upregulation of Snail expression leading to induction of EMT, thus suggesting that the Snail promoter is regulated positively by NF-κB p65. Besides the regulation of Snail at the transcriptional level, Snail is also regulated post-transcriptionally. Zhou et al. (2004) proposed that GSK-3β binds and phosphorylates Snail (at motif 2) and induces its nuclear export. Subsequent phosphorylation by GSK-3β (motif 1) results in the association of Snail with β-Trcp leading to its degradation. In cancer cells GSK-3β is regulated by many oncogenic ligands, such as PI3K/Akt, MAPK, and Wnt, leading to inhibition of GSK-3β and therefore to stabilization and nuclear localization of Snail in order to trigger cell migration and EMT. A variant of Snail (Snail-6SA), which abolishes the GSK-3β phosphorylation sites, is much more stable and resides exclusively in the nucleus to induce EMT (Beach et al. 2008). In contrast, normal epithelial cells express negligible levels of Snail RNA.

Inhibition of Metastasis by High NO Concentrations The reported role of NO in cancer has been controversial. Various studies have shown that NO can both promote and inhibit tumor progression and metastasis depending on concentration and duration of NO exposure, tumor microenvironment, activity and localization of the enzymes catalyzing NO production, and the cell resistance to NO toxic effects (Williams and Djamgoz 2005; Fukumura, Kashiwagi and Jain 2006; Ridnour et al. 2006; Wink et al. 1998). Although tumor cells synthesize NO during metastasis, the interaction between tumor and host cells leads to additional production of NO. Abundant evidence in the literature suggests that the levels and distribution of NO regulate positively or negatively not only the tumor cell survival but also influence cancer metastasis in a manner depending on the level and duration of NO exposure as well as the tumor responsiveness to NO (Xie and Huang 2003; Williams and Djamgoz 2005). Several events participating in the metastatic process, including EMT, invasion, and angiogenesis, are differentially regulated by NO at both the transcriptional and the post-transcriptional stages.

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NO has been reported to promote tumor progression and metastasis by induction of tumor cell proliferation, migration, and invasion. In tumor cells NO acts indirectly through the expression of angiogenic and lymphangiogenic factors. In contrast, the cytotoxic effects of NO, typically induced by high levels, promote DNA damage, protein dysfunction by S-nitrosylation, gene mutation, and tumor cell death, which contribute to tumor regression and inhibition of metastasis. However, gene mutation and/or transformation together with cell death of wild-type cells could contribute to clonal selection of adapted cells and the acquisition of apoptosis resistance and therefore promote tumor progression (reviewed by Fukumura et al. 2006). Several experimental models have shown that NO derived from tumor cells and host stromal cells (such as tumor-associated macrophages or cytokine-activated fibroblasts), which is mainly synthesized by iNOS, promotes tumor growth, angiogenesis, and invasion (Kisley et al. 2002; Rao et al. 2002; Ellies et al. 2003; Nam et al. 2004). In contrast, silencing of iNOS by anti-sense oligonucleotides or in vivo deletion of iNOS in knockdown mice has resulted in decreased tumor growth and spread (Dong et al. 1995; Jenkins et al. 1995; Ambs et al. 1998; Liu et al. 1998; Ahn and Ohshima 2001; Kisley et al. 2002; Yamaguchi et al. 2002; Ellies et al. 2003; Muerkoster et al. 2004; Nam et al. 2004). Wei et al. (2003) have shown in iNOS−/− or wild-type mice bearing iNOS-deficient tumor cells that host iNOS-suppressed growth and metastasis of methylcholanthrene-induced murine fibroblastomas. These findings suggest that NO produced by tumor-associated host stromal cells has the same tumorigenic activity like the NO produced by tumor cells and that the host might suppress tumor growth and metastasis. According to the reported functions of NO in cancer, modulation of the endogenous NO signaling might be useful for the treatment of tumors. The literature supports both decreasing and increasing NO signaling as potential strategies. However, the type and stage of tumor, as well as the activity and distribution of NOSs together with the source of NO production (tumor, stromal, or vascular endothelial cells) are considered as critical determinants of the effect of the endogenously produced NO on tumor progression and metastasis. Pre-clinical studies in different experimental models have shown that NO downregulation by selective inhibitors of iNOS or eNOS reduced tumor angiogenesis, blood flow, tumor growth, as well as the vessel density and the microvascular permeability (Gallo et al. 1998; Swaroop et al. 2000; Gratton et al. 2003; Kashiwagi et al. 2005). Reduction of endogenous NO has also been reported to be useful in chemoprevention (Crowell et al. 2003; Hofseth et al. 2003). On the other hand, increase of NO signaling via different approaches including either iNOS gene or iNOS expressing cell delivery or induction of iNOS by inflammatory cytokines has revealed beneficial effects on induction of tumor cell apoptosis, decreased angiogenesis, and cell proliferation resulting in tumor regression and inhibition of metastasis (Xie et al. 1995; Edwards et al. 1996; Xu et al. 2002). However, there are a number of limitations in the use of iNOS transduced cells in vitro and in vivo which are related with the short time of tumor exposure to the NO effect, inadequate supply of NOS substrate, induction of NO resistance, or discrepancy of tumor cell responses to NO in vitro and in vivo (Edwards et al. 1996; Singh et al. 2000; Xie and Huang 2003). Specifically, iNOS

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transduced cells might undergo apoptosis quickly and produce low levels of NO and for a limited time. Consequently, relatively low levels of NO might induce NO resistance and promote cell growth rather than tumor cell killing (Xie and Huang 2003). The use of different NO-releasing agents for the regulation of tumor metastasis, in in vitro and in vivo studies, has generated conflicting findings regarding the impact of the above agents on the metastatic behavior of tumor cells due to the inconsistency of the obtained results. The challenge regarding the use of NOgenerating agents is to deliver NO in a sustained and controlled manner considering that, like iNOs induction, low NO release promotes tumor growth, invasion, and metastasis whereas high NO release has the opposite effect. The reported inhibitory effects of NO-releasing agents on the metastatic tumor cell behavior seem to be sufficiently established and have gained continuous recognition during the latest years. About 14 years ago, Pipili-Synetos et al. (1995) first reported that NO-producing nitrovasodilators such as isosorbide mononitrate (ISMN) and isosorbide dinitrate (ISDN) could inhibit angiogenesis in an in vivo angiogenesis model of the chick chorioallantoic membrane (CAB) and suppress growth and metastatic properties of the Lewis lung carcinoma (LLC) in mice. Similar anti-metastatic effects have been shown for NO donors used alone or in combination with other agents in different tumor models. In vitro analysis indicated that the expression of bFGF and TGF-beta1 in prostate tumor cells could be suppressed by the NO donor sodium nitroprusside and it could mimic the effects of IFN-beta transduction on suppressing orthotopic prostate tumors and the development of metastasis in mice (Cao et al. 2001). In a different in vitro study S-nitroso-N-acetylpenicillamine suppressed the attachment and invasion of human hepatoma cells transfected with the hepatomaassociated antigen CD147, via NO/cGMP-mediated regulation of store-operated calcium mobilization (Jiang et al. 2001). SNAP also has been reported to possess an additive anti-metastatic effect in combination with lecithinized superoxide dismutase (PC-SOD) in a pulmonary metastatic mice model. This was achieved through prevention of excessive formation of oxygen radicals and peroxynitrate (ONOO− ) which cause cell damage and facilitate tumor metastasis (Takenaga et al. 1999). Recently, NO prodrugs such as JS-K or NONO-AM (reported to release high levels of NO) at doses which were not cytotoxic were shown to exhibit apoptotic and antiinvasive effects against metastatic breast cancer cells, through induction of TMP production and inhibition of p38 (Simeone et al. 2006, 2008). We are currently investigating the potential anti-metastatic properties of the NO donor DEATANONOate in vitro and in vivo in a human prostate tumor cell line PC-3 that is characterized by high metastatic potential. Our preliminary findings show that DETANONOate at 1000 μM concentration is able to inhibit in vitro the expression of metastasis-related mesenchymal gene products such as vimentin and fibronectin and to upregulate epithelial markers such as E-cadherin. In mice bearing PC-3 xenografts and treated with DETANONOate, we observed suppressed development of metastasis at distant organs compared to untreated mice. Tumor biopsies from primary sites revealed reduced expression of mesenchymal markers such as vimentin and fibronectin and increased levels of epithelial markers such as

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E-cadherin. In PC-3-bearing mice, the combination then of DETANONOate and CDDP had an additive suppressing effect on metastasis development and in the downregulation of the mesenchymal markers (unpublished data).

Underlying Molecular Mechanisms of NO-Mediated Inhibition of EMT Metastasis is regulated by several complex mechanisms. Both RKIP and Snail levels of expression in tumor cells dictate the metastatic behavior of tumor cells; hence, overexpression of RKIP is able to inhibit metastasis and, in contrast, overexpression of Snail induces EMT and metastasis (Cano et al. 2000; Fu et al. 2003). According to our recent findings on the anti-metastatic effect of DETANONOate in prostate tumors, we anticipated that NO might target the above gene products via different mechanisms affecting, therefore, the induction of EMT. Since Snail is transcriptionally regulated, in part, by NF-κB and NO inhibits NF-B activity, we hypothesized that NF-κB might act as a key modulator of NO-mediated regulation of the metastatic cascade via regulation of Snail and RKIP expression. In the following sections we discuss the critical role of the above gene products in the induction of EMT as potential targets of NO.

NO-Mediated Inhibition of Metastasis via Inhibition of NF-κB We and others have shown that NO released by NO donors can serve as an efficient inhibitor of NF-κB activity at multiple levels (Fig. 11.1a). We have shown that treatment of the highly metastatic PC-3 or DU145 prostate tumor cell lines as well as the Ramos Non-Hodgkin’s B cell lymphoma line with the NO donor DETANONOate results in inhibition of NF-κB promoter activity and reduced NF-κB DNA-binding activity as assessed by a Luc reporter system and EMSA assays, respectively (Huerta-Yepez et al. 2004, 2009; Vega et al. 2005; unpublished data). The above findings were confirmed by using an NF-κB inhibitor, Bay-17805, which in addition to decrease on NF-κB DNA binding and promoter activities also resulted in suppression of NF-κB-dependent anti-apoptotic gene products including Bcl-xL, Bax, and survivin. Analysis of the activation status of NF-κB in several tumor cell lines upon treatment with a range of different NO donors has confirmed the inhibitory role of NO on NF-κB activation (Santos-Silva, Sampaio de Freitas, and Assreuy 2001; Santos-Silva, Freitas, and Assreuy 2006; Sun and Rigas 2008). In human ovarian cancer cell lines treated with TNF-α, the addition of the NO donor, S-nitroso-N-acetyl-d,l-penicillamine (SNAP), mediated disruption of NF-κB translocation and activation by interacting with O2 and reducing the generation of H2 O2 , a potent NF-κB activator (Garban and Bonavida 2001). We have also shown that DETANONOate induces S-nitrosylation of the p50 NF-κB subunit resulting in NF-κB inactivation (Huerta-Yepez et al. 2004). Park and Wei (2003) have proposed

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Fig. 11.1 NO suppresses NF-κB and Snail transcription factors and upregulates RKIP expression. (a) NO has been shown to inactivate NF-κB via different mechanisms including S-nitrosylation of p50 subunit, inhibition of its promoter activity, and reduction of its DNA-binding activity. NF-κB activation levels are considered as crucial determinants for tumor progression and development of metastatic lesions as well as for tumor resistance to chemo- and immunotherapy. (b) NO suppresses the Snail transcriptional repressor activity at both the mRNA and the protein levels. Snail has an active role in the initiation of EMT via regulating the expression of mesenchymal markers and suppressing the expression of the metastasis suppressor gene, E-cad. (c) NO regulates positively the transcription and translation of RKIP, known for its anti-metastatic function via inhibition of NF-κB and Raf-1/MEK/ERK survival pathways

an indirect way of loss of NF-κB transcriptional activity on target genes by NO. They demonstrated that NO released by sodium nitroprusside (SNP) inhibits the activation of the c-myc gene promoter in P19 mouse embryonal carcinoma cells, by dissociating the active form of NF-κB and replacing it with a repressive NFκB complex (p50/p50 homodimer complex) and correlated with the recruitment of gene-silencing histone deacetylases. In light of the findings that NF-κB stimulates tumor growth and metastasis, the above observations suggest that NO is a potent NF-κB inhibitor and should be investigated as a prospective therapeutic anti-cancer agent.

NO-Mediated Inhibition of Metastasis via Induction of RKIP Yeung et al. (1999) first hypothesized that the complexity of the regulation of the Ras/Raf-1/MEK/ERK module, a pathway known to be hyperactivated in tumor progression, may include associations with scaffolding and regulatory proteins (Moodie et al. 1993). To isolate such proteins, the Raf-1 kinase domain, BXB, was used as bait in a yeast two-hybrid screen. Using this two-hybrid system, Yeung et al. (1999) have identified both in vitro and in vivo, RKIP binding to Raf-1, MEK, and ERK.

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RKIP interfered with the activation of the ERK1/2 signaling pathway: RKIP overexpression suppressed the ERK pathway whereas downregulation of RKIP had the opposite effect. Considerable progress has also been achieved in the identification of kinases that activate the IKK complexes, known for their role on NF-κB activation and tumor development and progression. However, little was known until recently about negative regulators that may interfere with these pathways (Karin et al. 2002). Using both in vitro and in vivo assays, Yeung et al. (2001) have shown that RKIP inhibits the NF-κB pathway through its interaction with upstream kinases TAK1, NIK, and IKK. RKIP physically interacts with and blocks TAK1 and NIK, but not MEKK1 or NAK1, and thus inhibits the activation of these kinases leading to inactivation of NF-κB. Further, they showed that RKIP reduces the TNF-α-mediated activation of NF-κB. TAK1 is implicated in IL-1β signaling immediately upstream of NIK. RKIP physically interacts with the α and β subunits of IKK and inhibits the phosphorylation of IκBα, thus inhibiting the NF-κB pathway. These studies suggest that RKIP acts as a break on TNF-α and IL-1β signaling by antagonizing the activation of IKKs by NIK and TAK1 as well as by directly downmodulating the activity of the IKKs. We have confirmed the above observations showing that RKIP overexpression in prostate cell lines inhibits NIK expression and downregulated NF-κB promoter activity (Baritaki et al. 2007b). Based on the findings that RKIP interferes with the Raf-1/MEK/ERK and NF-κB survival pathways and the fact that most drugs interfere with the same cascades to exert their cytotoxic effects, it is plausible that RKIP regulates drug and/or immune-mediated apoptosis and also mediates the NO anti-metastatic properties. As mentioned above, studies with various cancer cell lines have shown an upregulation of RKIP upon treatment with an array of drugs suggesting that one potential mechanism by which various drugs induce apoptosis might be through the induction of RKIP (Chatterjee et al. 2004; Baritaki et al. 2009a). Similarly, we have shown that treatment of various cancer cell lines including prostate and NHL tumor cell lines with DETANONOate results in significant potentiation of RKIP mRNA and protein levels (Fig. 11.1b). We also showed that tumor biopsies of SCID mice bearing PC-3 xenografts and treated with DETANONOate present increased levels of RKIP expression and reduced tumor growth, compared to the corresponding biopsies from untreated mice (unpublished data). Previous findings on the human prostate carcinoma cell line DU145 have shown extensive apoptosis upon treatment with the topoisomerase I inhibitor 9-nitrocamptothecin (9NC) (Chatterjee et al. 2004). Yet treatment of the DU145 drug-resistant cell line RC1 with 9NC did not result in apoptosis but did result in the robust induction of NF-κB activity. Given the association between RKIP and NF-κB activity it was shown that there was a correlation between the levels of RKIP in the 9NC-sensitive and RC1-resistant cell lines. Accordingly, 9NC triggered significant induction of RKIP in the parental DU145 cells but not in RC1 cells. Thus, induction of RKIP may be necessary for these cells to undergo 9NC-triggered apoptosis. This hypothesis was confirmed when blocking RKIP (by anti-sense siRNA approaches) abrogated 9NC-induced apoptosis in DU145 cells. Moreover, ectopic expression of RKIP in RC1 cells sensitized these cells to 9NC.

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9NC induced significant increase in RKIP expression in the androgen-independent PC-3 cell line as well. In addition, exposure to other genotoxic stimuli such as etoposide (VP-16) or cisplatin (CDDP) induced RKIP expression in the DU145 model. Similar results were found in a drug-sensitive and drug-resistant breast carcinoma model system. The molecular mechanism(s) responsible for the induction of RKIP protein by the various cytotoxic agents is elusive. In addition, pre-treatment of PC-3 or DU145 cells with DETANONOate sensitizes the treated cells to drug-induced apoptosis, via NO-mediated RKIP upregulation (unpublished data). Overall, the direct correlation between RKIP induction and sensitivity to drugs suggests that RKIP overexpression or induction may be necessary for apoptosis in tumor cells after exposure to clinically relevant cytotoxic agents. Consistently with Fu et al. (2003), overexpression of RKIP in our systems of PC-3 and DU-145 highly metastatic prostate cell lines not only increased cell sensitivity to both chemo- and immunotoxic stimuli (Baritaki et al. 2007b; 2009a) but also resulted in reversal of the mesenchymal tumor phenotype by decreasing the levels of vimentin, fibronectin, and Snail and increasing the expression of E-cadherin and cytokeratin 18. These modifications were reflected by decreased tumor cell migratory and invasiveness properties (Baritaki et al. 2009b).

Underlying Mechanisms of RKIP Upregulation by NO In view of the metastasis suppression function of RKIP, its downregulation in human metastasized cancer cells, and its upregulation following chemotherapeutic drug or NO treatment, it is important to understand how RKIP expression is regulated at the molecular level. Hence, the transcriptional, translational, and the post-translational regulation of RKIP expression in different cell lines are important determinants of the tumor metastatic behavior and the outcome of tumor cells’ fate in response to noxious stimuli. Therefore, the expression of RKIP must be tightly regulated at these levels. The mechanism by which RKIP expression is regulated at different levels in different cell types and in different stages of differentiation is not completely clear, as well as how NO regulates RKIP expression. In concordance with clinical tumor studies, we observed that the expression levels of RKIP proteins are progressively decreasing in breast and prostate cancer cell lines of increasing metastatic potential. qRT-PCR analysis demonstrated the same trend of expression in different cancer cell lines. These results, therefore, suggest that the expression of RKIP is regulated at the level of RNA. As a first step, in an effort to determine molecularly how the expression of RKIP is regulated at the level of transcription, the human RKIP gene was isolated and characterized. Multiple human tissue Northern blots revealed the presence of a single mRNA of 1.8 kb in different tissues (Seddiqi et al. 1996). Analysis of annotated human sequence database revealed that the human RKIP gene is located on chromosome 12 and contains four exons. The putative transcription start site was assigned to 120 base pairs upstream from the translation start site ATG by cDNA sequence, size of the mRNA,

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and EST analyses. A 2.2 kb DNA fragment upstream from the ATG was isolated and used to direct the synthesis of a luciferase (Luc) reporter gene. Transfection of the RKIP reporter construct resulted in a 2- to 3-fold increase in Luc activity in DU145 24 h after 9NC treatment. The expression of RKIP in cancer cell lines perfectly recapitulates the RKIP expression pattern in both prostate and breast cancers in vivo. Computer database analysis (TESS master analysis) of the promoter sequence revealed putative transcription factors which might regulate RKIP expression including AP-1, clusters of SP-1, Ying, and Yang-1 (YY1), and Snail consensus binding sites. Dissection of the roles of each of these factors in the overall transcriptional regulation of RKIP is a complex mechanism. However, our and the collaborative laboratories have begun to dissect the role of the above factors in the regulation of RKIP transcription in different cell types. The transcriptional regulation of RKIP might be influenced by the activity of transcriptional activators and/or repressors. Different mechanisms as well as different expression levels govern the activity of the activators and the repressors. Thus, it is possible that, for instance, overexpression of the transcription repressor YY1 may dominate over the transcriptional activators by minimizing the expression of RKIP. The nuclear protein YY1 (δ, NF-E1, UCRBP, CF1) is a highly conserved 68-kDa zinc-finger transcription factor (located on 14q32) that acts as both a transcriptional repressor and an activator (reviewed by Gordon et al. 2006). Theoretically, YY1 binding to RKIP could repress the transcription of RKIP; thus, inhibition of YY1 DNA binding would allow RKIP to be expressed. Treatment of prostate and NHL cells with NO or other agents inhibits YY1 activity and induces RKIP expression consistent with the role of YY1 in the regulation of RKIP (Jazirehi et al. 2004; Huerta-Yepez et al. 2009; Bonavida et al. 2008). Removal of this inhibitory effect should theoretically enhance RKIP expression. Indeed, treatment of the prostate carcinoma cell lines with inhibitors for NF-κB, which positively regulates YY1 transcription (Wang et al. 2007), results in upregulation of RKIP expression (Baritaki et al. 2007a, 2007b; Huerta-Yepez et al. 2009). In addition, co-transfection of the prostate cell line DU145 with siRNA against YY1 and an RKIP-luciferase construct resulted in upregulation of RKIP promoter activity (unpublished data). However, ChiP analysis is needed to confirm the direct transcriptional regulation of RKIP by YY1. Among the transcription factors mentioned above to have putative binding sites in the RKIP promoter, Snail is the only transcription factor that has recently been identified to be a direct suppressor of RKIP transcription in prostate and breast tumors (Beach et al. 2008). In light of the downregulation of RKIP expression in metastatic breast, prostate, and other cancer cell lines, it was hypothesized that Snail may act upstream of RKIP. Indeed, introduction of Snail into three non-metastatic cancer cell lines with comparatively high levels of RKIP expression resulted in the repression of RKIP expression, both at the protein and at the RNA levels. Importantly, in metastatic prostate cancer cells with low RKIP expression, the expression level of RKIP was increased when the expression of Snail was knocked down by specific siRNA (Baritaki et al. 2009b). Inspection of the RKIP promoter reveals the presence of at least four potential Snail binding

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consensus sites (E-box, CANNTG) clustered in two locations in the proximal RKIP promoter. To determine whether Snail regulates the expression of RKIP in an Ebox-dependent manner, the effect of overexpressing Snail on the activity of the RKIP promoter driven luciferase reporters in MCF7 breast cells was examined. MCF7 cells were chosen because the cells are comparatively easy to be transfected. Importantly, MCF7 cells have a low level of Snail expression. Three different RKIP promoter luciferase reporters containing all four, one, and no E-box binding sites were used. Consistent with the observation that ectopic expression of Snail downregulated RKIP, forced expression of Snail repressed RKIP reporters that contained one or all four of the E-box cis-elements. The effect of Snail was found dependent on the E-box elements as an RKIP reporter lacking all the E-box or an E-cad promoter with a mutated E-Box failed to respond to Snail repression. In addition, Snail interaction with the RKIP promoter directly was performed using a CHiP analysis with purified cross-linked chromatin prepared from the prostate cell line LNCaP (it expresses relatively higher levels of RKIP compared to other metastatic prostate tumor cell lines) stably transfected with a flag-tagged Snail-S6A expression vector. In support of the regulatory role of Snail in RKIP expression, a detectable amount of Snail was found associated with the RKIP promoter at E-box 2, but less at E-box 1 but not at exon 3 of the RKIP gene locus. These results confirmed the transient reporter assay, which showed that the proximal E-box is sufficient for Snail-mediated repression of RKIP promoter (Beach et al. 2008). Based on the findings regarding the direct transcriptional repression of RKIP by Snail, the regulation of Snail by NF-κB and the inhibitory effect of NO on NF-κB activity, we hypothesized that the NO-mediated RKIP induction might be triggered via suppression of Snail resulting in inhibition of EMT. The Snail repressive activity on RKIP expression and its role in NO-induced inhibition of metastasis are further discussed below.

Pivotal Role of NO-Mediated Inhibition of Snail in the Induction of RKIP and Inhibition of Metastasis It has been reported that in primary and metastatic prostate tumors Snail expression is inversely correlated with RKIP and E-cadherin levels (Beach et al. 2008). In addition, our recent observations (Baritaki et al. 2009b) have revealed that overexpression of the stable Snail form, Snail-6SA, in the non-metastatic prostate cell line LNCaP results in RKIP downregulation and acquisition of the EMT phenotype with expression of vimentin and fibronectin, gene products previously shown to be indirectly regulated by Snail (Nieto 2002). This is in agreement with previous studies showing that, in addition to E-cadherin suppression, Snail transfectants downregulate other epithelial markers such as mucin1 and cytokeratin 18 (Batlle et al. 2000), and upregulate and redistribute mesenchymal markers such as vimentin and fibronectin (Cano et al. 2000). In contrast, the shift of EMT markers and

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consequently the EMT phenotype was reversed by siRNA-mediated repression of Snail expression or RKIP induction by a CMV-RKIP expression vector in DU145 (Baritaki et al. 2009b). This finding corroborates previous reports by Yang et al. (2007) and Olmeda et al. (2007), indicating suppression of tumor growth and invasiveness by Snail silencing. Indeed, we showed that NO has a significant inhibitory effect on Snail expression, and this inhibition was directly associated with reexpression of RKIP and E-cadherin, inhibition of mesenchymal gene markers and induction of an epithelial cell phenotype as assessed by Snail silencing (Baritaki et al. 2009b). In addition, we recently identified an additional mechanism by which NO might deactivate Snail transcriptional activity in prostate tumor cells and this is via NO-mediated S-nitrosylation (Baritaki et al. 2009b) (Fig. 11.1c). Overall, the above findings suggest that inhibition of Snail may be pivotal for NO-mediated RKIP and E-cadherin induction resulting in EMT inhibition. Thus these observations suggest that Snail might be an appropriate therapeutic target to interfere with the EMT process and, in turn, to block tumor invasion.

NO-Mediated Inhibition of the NF-κB/Snail/RKIP Loop Results in Inhibition of Metastasis The intra-relationship between RKIP and Snail in metastasis has been recently characterized by an inverse correlation, through the direct transcriptional repressive effect of Snail on RKIP promoter activity. Our novel findings also demonstrate the key role of NF-κB as modulator of the NO-mediated regulation of the metastatic cascade via regulation of Snail/EMT network during EMT. Thus, the NF-κB/Snail/RKIP cross talk is addressed for the first time as a critical signaling network that interferes not only with tumor cell resistance to apoptotic stimuli but also with initiation of the metastatic cascade. Besides the reported involvement of each of the gene products that participates in the network in the regulation of tumor cell progression and metastasis, we have revealed a connecting link among them. Here, we elucidate a strong NF-κB/Snail/RKIP cross talk in the tumor metastatic process and propose that this network might be considered as a putative therapeutic target to regulate anti-metastatic responses by using agents that are able to modify it. Our findings suggest a new mechanism by which NO may inhibit metastasis via inhibition of Snail and induction of the metastasis suppressor gene products RKIP and E-cadherin. We further show that RKIP induction attenuates directly a feedback loop of Snail suppression via NF-κB inhibition in addition to suppression mediated directly by NO. We also demonstrate that Snail suppression by NO not only induces metastasis suppressor and epithelial genes expression, but also represses the expression of Snail-regulated mesenchymal markers. These modifications result in the reversal of the mesenchymal cell phenotype and inhibition of the migratory and invasive properties of the tumor cells (Fig. 11.2). However, more studies should be addressed in order to elucidate the complete mechanism of NO-induced inhibition of metastasis by challenging the NF-κB/Snail/RKIP network.

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Fig. 11.2 NO inhibits tumor cell metastasis via regulation of the NF-κB/Snail/RKIP loopNO mediates its biological effect mainly by inhibiting the NF-κB pathway and consequently the expression of NF-κB-regulated gene products. The Snail transcription factor, an essential initiator of EMT, is under the positive regulation of NF-κB and it inhibits the expression of metastasis suppressor genes such as RKIP and E-cadherin, while it induces directly and/or indirectly the expression of mesenchymal markers, resulting in the acquisition of a metastatic phenotype by the tumor cells. We show that, in addition to the direct NO-induced NF-κB inhibition, NF-κB could also be inhibited by NO-mediated RKIP induction resulting in the modulation of tumor cell metastatic potential. RKIP induction by NO may result from downregulation of its transcriptional repressor Snail via inhibition of its upstream activator NF-κB by NO (feedback loop). The NO-mediated regulation of the NF-κB/Snail/RKIP loop via the above mechanism results in reversal of the mesenchymal cell phenotype (mesenchymal to epithelial transition, MET) and inhibition of the migratory and invasive properties of the tumor cells. Solid lines correspond to the NO-mediated effects on the indicated gene products, while dotted lines correspond to the constitutive basil levels in tumor cells in the absence of NO

Potential Use of NO-Releasing Agents in the Management of Tumor Metastasis Exogenous NO sources constitute a powerful way to supplement NO when the body cannot generate sufficient NO for normal biological functions. Delivery of low concentrations of exogenous NO is an attractive therapeutic option for treatment of several conditions including cardiovascular disorders. In contrast, delivery of high concentrations might have completely different therapeutic targets and act via the cytotoxic actions of NO, such as in the case of tumor suppression. The chemical versatility of NO has led to the synthesis of a wide range of NO donors, each with different modes and rate of NO release. Worth mentioning are the families of S-nitrosothiols, diazeniumdiolates (formerly NONOates), and hybrid NO donor drugs (NO-donating non-steroidal anti-inflammatory drugs, NO-NSAIDs), which have proved extremely popular in various types of experimental studies related to cancer management (Rigas et al. 2007; Miller and Megson 2007). NO donors that spontaneously generate large amounts of NO independent of iNOS induction are activated at physiological pH and can induce NO-mediated systemic hypotension. NO prodrugs are another type of iNOS-independent NO-releasing agents. NO prodrugs do not release NO spontaneously, but rather can be activated to generate high levels of NO by intracellular enzyme targets such as glutathione S-transferases (GSTs).

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A range of NONOates has now been described with half-lives varying from seconds to hours (Morley and Keefer 1993). At present, NONOates are not used clinically, although they have been tested frequently in different experimental models. An attractive feature of this class of compounds is that their decomposition is not catalyzed by thiols or biological tissue, unless specifically designed to, and because NO release follows simple first-order kinetics (Kavdia and Lewis 2003). The rate of NO release can be accurately predicted. This can be achieved via specific modifications of the NONOate structure which can stabilize the drug in solution and potentially engender a selective NO release in different organs, vascular beds, or specific cell types. Subsequently, the biological activities, such as vasodilatation, inhibition of platelet aggregation, blood coagulation, and cell proliferation (Diodati et al. 1993; Nielsen 2001), closely correlate with the amount of NO generated in vitro. Additionally, the lack of tissue requirement for NO release is most likely responsible for the apparent lack of tolerance experienced with these compounds (Brilli et al. 1997). The use of NONOates adducts has received interest from the viewpoint of targeting delivery of high concentrations of NO specifically in tumor cells. Tang et al. (2001) have reported that coupled PYRRO/NO to a chain of amino acids is recognized to be a substrate for prostate-specific antigen (PSA). PSA is inactive in blood plasma, but upregulated within prostate cancer metastases. This conjugated NONOate did not release NO in solution in the absence of PSA, offering the possibility that NO release will occur exclusively within cancer cells, especially the highly metastatic tumor cells, rather than in the blood. A variety of other conjugated NONOates have been also reported by other groups to engender greater and specific cytotoxicity on metastatic cancer cells carrying the recognized antigen (Cai et al. 2003). Overall, NONOate members have experimentally served as a valuable scientific tool for researching NO physiology in tumor metastasis and seem to hold a great deal of promise for a potential clinical application of NONOates in management of tumor progression and metastasis. We are currently aware of only a single clinical trial in humans with respiratory distress syndrome using NONOates (Lam et al. 2002). The toxicity of NONOates’ by-products needs to be more fully confirmed, especially as subsequent reactions between decomposition products could lead to the formation of carcinogenic nitrosamines. At present, conjugated NONOates have been widely introduced as potential experimental tools for the treatment of certain cancers, although further characterization of these drugs is essential. However, the predictable nature of NO release by NONOates, especially at concentrations high enough to inhibit tumor progression and metastasis, will undoubtedly lead to further clinical investigations, once long-term safety has been established.

Conclusive Remarks and Future Perspectives Although surgery, chemotherapy, immunotherapy, and radiation therapies effectively control many cancers at the primary site, the development of metastatic disease signals a poor prognosis with increased morbidity and mortality. Relatively

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few factors of the metastatic process have been successfully developed as therapeutic targets. New insights into the initial steps of the metastatic process were revealed in developmental genetics by a set of regulators that induce the transdifferentiation EMT program. The reported regulation of cell survival, apoptotic pathways, and tumor cell metastatic behavior by RKIP-mediated inhibitory effects on survival pathways, including NF-κB, suggests that this protein may play a master regulatory role in tumor progression and may represent a novel prognostic marker of several tumor-promoting processes including metastasis. The lack and/or low levels of RKIP expression resulting from strong suppression by Snail in tumor cells would theoretically allow the progression of the disease and resistance to apoptosisinducing stimuli. This is consistent with the role of RKIP in the regulation of cell survival. Thus, cells that overexpress RKIP are more susceptible to apoptotic stimuli. Cells that express moderate levels of RKIP (for example, early stage tumor cells) will be prone to cell survival and less susceptible to apoptotic stimuli. In contrast, cells that lack RKIP (advanced cancer and metastatic tumors) will be highly resistant to apoptotic stimuli and maintain their metastatic behavior. Thus, identifying the critical role of RKIP in the regulation of tumor progression and metastasis we could determine it as a good potential therapeutic target. Despite the conflicting reports on the role of NO in cancer progression and metastasis, it seems that relatively low levels of NO might induce NO resistance and promote cell growth and angiogenesis rather than tumor killing, while high concentrations might have completely different therapeutic targets and act via the cytotoxic actions of NO. In contrast to limitations in strategies aimed to induce endogenous NO, exogenous administrating NO donors are capable of delivering NO into tissues and the bloodstream in a sustained and controlled manner. The anti-angiogenic and anti-invasiveness properties of high NO concentrations are mediated by different mechanisms; however, most of them have not been elucidated with respect to the gene products targeted by NO. Our recent findings on the anti-metastatic properties of NO in prostate tumors introduce the modulation of the NF-κB/Snail/RKIP circuit by NO as a key player in the regulation of the initiation of the metastatic process. We have shown that the inhibitory effects of DETANONOate in metastasis are via increase of the ratio between metastasis suppressor factors (RKIP, E-cadherin, epithelial markers) and metastasis inducer factors (Snail, NF-κB, mesenchymal markers). Thus, we propose the therapeutic application of NO in the management of metastatic tumors. The clinical relevance and significance of inhibiting Snail and restoring RKIP expression by NO might correlate with a favorable clinical outcome accompanied by diminution of tumor progression and spread. Moreover, RKIP induction by NO may improve the efficacy of anti-tumor therapies, especially if they are combined with conventional immunotherapeutics and/or chemotherapeutics, as well as the host immune surveillance against cancer as reported by us (Baritaki et al. 2007b). Also, we propose that RKIP and Snail expression profiles in tumors could be used as potential prognostic biomarkers.

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Acknowledgments We wish to acknowledge all the members of our laboratory and collaborators for their valuable contributions. In addition, we wish to thank Dr. Kam Yeung for helping us in both the conceptual and the execution of the various studies presented in this chapter. We also acknowledge the Jonsson Comprehensive Cancer Center at UCLA for their continuous support. We appreciate the technical assistance of Erica Keng and Tiffany Chin in the preparation of the chapter.

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Part V

Nitric Oxide as a Sensitizing Agent for Chem-Radio-Immunotherapy

Chapter 12

Sensitizing Effect of Nitric Oxide to Cytotoxic Stimuli Peter Siesjö

Abstract During irradiation of tumor tissue NO is released by myeloid cells, tumor cells, endothelial cells, and other stromal cells. By reacting with oxygen radicals NO will inflict tumor cell damage that will be added to the direct effect of DNA damage. The relative role of the NO secreting cells during radiotherapy is not well studied and further knowledge in this field could help optimize dose and timing in order to achieve maximal tumor cell death. The use of NO donors during radiotherapy could possibly further potentiate these effects. Release of nitric oxide and other cytotoxic molecules has been shown to mediate some of the secondary effects of chemotherapeutic agents. It is also obvious that release of nitric oxide can potentiate the cytotoxic effects of chemotherapeutic agents either by direct synergistic cytotoxic effects or by increase of blood supply and vascular permeability. The induction of cytotoxicity by NO in vivo can boost T-cell responses by partially degrading tumor cells and thus facilitate antigen presentation of APC. Furthermore NO is essential in the early stages of T-cell activation. However prolonged secretion of NO can also induce tolerance and/or immunosuppression, which will dampen the anti-tumor immunity. Consequently, the combination of immunotherapy with NO-modulating approaches has to be specifically tailored, considering the tumor type, immunization timetable, and the suppressive network present in the tumor tissue. Keywords NO · Cytotoxicity · Radiotherapy · Chemotherapy · Immunotherapy

P. Siesjö (B) The Rausing Laboratory, Section of Neurosurgery, Department of Clinical Sciences, University of Lund, 22185 Lund, Sweden e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_12, C Springer Science+Business Media, LLC 2010

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Radiotherapy Preclinical Studies During the last decade it has become increasingly evident that the cytotoxic effects of radiotherapy on tumors are not only the result of the DNA damage inflicted to the tumor cells. Radiation can induce the release of several factors, with direct or indirect cytotoxic capacity, not only from tumor cells but also from different bystander cells that can trigger death of tumor cells. Radiosensitization on the other hand can be defined as an external manipulation of tumor cells or tissues that will increase their susceptibility to radiation in a mode that is not simply additive, e.g., represents a synergistic effect. NO has been shown to be involved in both the above-described mechanisms: the effect of NO released by irradiation itself and the effects on radiation after different measures to increase intratumoral NO production during radiation. In the former group different sources of NO after irradiation can be discussed as tumor cells, endothelial cells, other stromal cells, or resident alternatively invading immune cells. These different cellular sources are closely linked to different isotypes of the NOS enzyme as nNOS, eNOS, and iNOS. One early study of the effect of in vivo radiation could not verify any increase of iNOS in the brain after radiation although NO-inducing cytokines such as TNF-α and IL-1β were up-regulated (Hong et al. 1995). However, in vitro induction of NO in tumor cells by IFN-γ treatment as well as by irradiation itself was shown to substantially increase the radiosensitivity of not only these cells but also bystander cells (Janssens et al. 1998; Matsumoto et al. 2000). NO release after eNOS upregulation in endothelial cells after irradiation of both experimental and human tumor tissues has also been demonstrated to have indirect potentiating effects by increasing tumor blood flow and thereby decreasing tumor hypoxia by vasodilation (Sonveaux et al. 2002, 2003). As macrophages are highly radioresistant they will often survive radiation but will become activated and produce large amounts of NO and other toxic molecules. It is now increasingly recognized that NO released by these mechanisms accounts for a large part of the effects ascribed to ionizing radiation itself. On the other hand also the side effects of irradiation of normal tissues are also related to NO release and inhibitors of NOS have been shown to dampen such side effects (Liebmann et al. 1994). In this context Chen et al. have reported a correlation between the risk for radiotoxicity and polymorphisms in genes related to oxidative stress in combination with high body mass index (Ahn et al. 2006). The increased release of NO after irradiation of MO seems to be mediated by increased signaling of the mitogen protein kinase (MAPK) family (Narang and Krishna 2008). One could argue that NO release after radiation is only an epiphenomenon but does not really potentiate radiation-induced cell death. However, several reports have shown a clear senzitizing effect using combinations of radiotherapy and NO donors as isosorbide dinitrate (Jordan et al. 2003) and nitrite (Frerart et al. 2008).

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NO released by intratumoral macrophages can, together with other reactive species, also affect the activity of HIF-1 in a complex pattern with both tumor enhancing and tumor inhibiting effects as reviewed by Bonavida and Yasuda (Bonavida et al. 2008; Yasuda, 2008; Brennan et al. 2005). NO released after irradiation can induce apoptosis in tumor cells by several mechanisms as activation of p53 not only in neuroblastoma cells (Wang et al. 2003) but also in normal lung tissue (Davis et al. 2000). The bystander effect after irradiation of tumor cells, defined as killing of larger proportions of cells than those targeted, has been shown to depend on both NO and TGF-β1 production as reported by Shao et al. (2004, 2005, 2008).

Clinical Studies Jayasura et al. attempted to correlate the iNOS expression in head and neck tumors with outcome after radiotherapy and found that iNOS expression was linked to worse outcome (Jayasurya et al. 2003) and this was confirmed by Chen et al. in human squamous cell carcinoma (Chen et al. 2005). Contrary to this Oka et al. found no correlation in patients with cervical carcinoma treated with radiotherapy (Oka et al. 2003). These contradictory results could be due to the fact that iNOS expression was assessed before radiation when the enzyme could be predicted to upregulate during and after irradiation.

Concluding Remarks and Future Directions The cytotoxic effects of radiotherapy are only partially the result of radiationinduced DNA damage in tumor cells. By release of NO and other reactive species not only from myeloid-derived cells but also from stromal cells, tumor cells, and endothelial cells, secondary cytotoxicity will evolve. Conversely the administration of NO donors to irradiated tissue could further increase the effects of radiation and this might be tailored in order to avoid collateral damage to normal surrounding tissues. Further research should aim at exploring ways to increase NO release intratumorally at time points which will yield maximal synergistic effects. The role of macrophages and monocytes during irradiation is only superficially studied and future research should aim at understanding the basic mechanisms underlying macrophage responses to irradiation. In this way signals that guide macrophage reactivity during irradiation can be identified and used in therapeutic settings.

Chemotherapy The effects of chemotherapy in neoplastic disease do not only rely on direct cytotoxic effects on the tumor cells. Also, stromal and endothelial cells can be targets of chemotherapeutic agents, which can lead to secondary reduction of tumor

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cells through bystander effects and depletion of oxygen and nutrients (Chabner and Roberts, Jr. 2005). Nitric oxide has recently been implicated in several mechanisms of cytotoxicity by chemotherapeutic agents as recently reviewed by Bonavida and Yasuda (Bonavida et al. 2008; Yasuda 2008). First some chemotherapeutic agents have been reported to exert some of their cytotoxic effects via the release of NO mainly from cells of myeloid origin; second the cytotoxic effects of certain chemotherapeutic agents have been demonstrated to be strengthened by the delivery of nitric oxide from NO donors; and third by the release of NO, vessel permeability can increase thus facilitating the delivery of chemotherapeutic agents to the tumor tissue.

Preclinical Studies Several lines of evidence in experimental tumor models have shown that nitric oxide can increase the cytotoxic effects of chemotherapeutic agents. Shinohara et al. could demonstrate that the increased effect of a bacterial lipopeptide JBT on chemotherapy of experimental colon tumors by irinotecan was dependent on NO and IL-15 release from macrophages (Shinohara et al. 2000). Nitric oxide donors in the form of organic nitrite could furthermore increase the cytotoxic capacity of subtherapeutic doses of cyclophosphamide against systemic and intracerebral leukemias and B16 melanomas in mice (Konovalova et al. 2003). Also taxol combined with the immunomodulator AS101 had a clear synergistic effect on the spontaneous mouse lung carcinoma. The effect, which could be reverted by inhibitors of NO, was shown to be mediated by activation of macrophages and release of nitric oxide (Kalechman et al. 1996). An indirect mechanism of cytotoxic potentiation by nitric oxide was proposed by Perotta et al. where a combined therapy of melanoma in mice with dendritic cells and low doses of cisplatinum was dependent on the pretreatment of DC with nitric oxide donors. By pretreating DC with NO donors these were protected from induction of apoptosis by cisplatinum (Perrotta et al. 2007). However, one study has reported a lack of evidence for a role of nitric oxide in the effects of a combined therapy with IL-12 and doxorubicin (Zagozdzon et al. 1999). Due to the blood–brain barrier tumors are less sensitive to chemotherapy and different strategies have been launched in order to increase the permeability to BBB during chemotherapy. Weyerbrock et al. used a short-acting NO donor, Proli/NO, to disrupt the BBB during delivery of carboplatin in the rat C6 glioma model and found a strong synergistic effect by the combined therapy (Weyerbrock et al. 2003). However the C6 glioma was raised in an outbred rat strain, thus mechanisms of immune rejection in this model could have accounted for some of the effects. Likewise, in the 9L rat glioma model, nitric oxide levels and opening of BBB could be increased by treatment with L-arginine and hydroxyurea, both of which act as substrates for NO production. In this model the specific increase of eNOS in tumor vessels was stated to mediate the effects of both compounds. In contrast to compounds such as RMP-7 and bradykinin, which also open the BBB, L-arginine and hydroxyurea had a longer duration of BBB opening (Yin et al. 2008).

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Clinical Studies In a xenograft model of human tumor cells Blumental et al. could show a differential response of radio-immunotherapy which could be ascribed to different levels of nitric oxide and delivery of arginine could boost the effect of the combined therapy (Blumenthal et al. 1997). In studies of a human breast carcinoma cell line and a mouse melanoma cell line resistant to the chemotherapeutic agents 5-fluorouracil and doxorubicin increased not only by increasing hypoxia but also after inhibition of iNOS. Consequently, the authors hypothesized that some of the effects of hypoxia could be lack of nitric oxide in the tumor tissue (Matthews et al. 2001). The role of NO in maintaining tumor blood supply is double-edged and both increasing and decreasing intratumoral levels of nitric oxide could have anti-tumor effects. A phase I study aiming at reducing tumor blood supply by the administration of inhibitors of iNOS has been launched with clear evidence of proof of concept as reduction of tumor blood flow (Ng et al. 2007).

Concluding Remarks and Future Directions As for radiotherapy some of the cytotoxic effects of chemotherapy are not the effect of direct cytotoxicity but rather the result of indirect mechanisms. Release of nitric oxide and other cytotoxic molecules have been shown to mediate some of the secondary effects of chemotherapeutic agents. It is also obvious that release of nitric oxide can potentiate the cytotoxic effects of chemotherapeutic agents either by direct synergistic cytotoxic effects or by increase of blood supply and vascular permeability. Practically either NO donors or inhibitors of NOS can be evaluated for the potentiating effects on chemotherapy of tumors. Given the dual effects of nitric oxide in combination with chemotherapeutic agents increased, respectively, decreased levels of nitric oxide could be envisaged to have different effects in different tumor types and during different therapeutic conditions. Therefore, clinical trials will have to address these issues by evaluating manipulation of levels of nitric oxide in close relation to tumor type and therapeutic settings.

Immunotherapy Introduction Immunotherapy has been widely investigated for its potential use in cancer therapy. In both human and experimental animal neoplastic pathology spontaneous responses to tumor-associated antigens (TAAs) have been reported (Drake et al. 2005; Valmori et al. 2000). However, the expression of TAAs by tumor cells is

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generally weak and has been shown to rather induce T-cell anergy than effective anti-tumor responses. The successful cure of experimental tumors has not been able to translate into effective clinical immunotherapy where response rates are around 10% or below (Mocellin et al. 2005; Rosenberg et al. 2004). The limited clinical efficacy is partly due to immune evasion by tumor cells, e.g., down-regulation of expression or presentation of tumor antigens. In addition to that, in the last decade, accumulating evidence has demonstrated that failure of immunotherapy could be the result of the development of a wide range of immunosuppressive mechanisms, triggered by tumors or tumor-associated stroma (Peschos et al. 2006; Handsley and Edwards 2005). These suppressive mechanisms also involve the induction of nitric oxide synthase (iNOS) (reviewed elsewhere) with ensuing release of NO and formation of peroxynitrate in a subset of immature myeloid leukocytes recently re-named myeloid-derived suppressor cells (MDSCs). However, one should point out that not all the effects mediated by NO will down-regulate immune reactivity against tumors. NO has also been reported to act as an effector molecule inducing apoptosis in tumor cells. Thus, to be able to improve immunotherapy by manipulation of the NO networks, we need to understand how NO interacts and affects the cells of the immune system during different immune responses against tumor cells.

The Dual Role of NO in Immune Cell Responses In order to understand how NO can be used to manipulate anti-tumor immune responses knowledge of the role of the immune system has to be pursued. NO plays an important and diverse role during the regulation of immune responses (Bogdan et al. 2000a, b). This diversity is partly depending not only on the levels of NO produced but also of cellular targets and timing of release. Overall, it is believed that high levels of NO induce immune suppression, while low levels are required for functional immunity (Niedbala et al. 1999). Since the initial finding that IFN-γ secretion from activated T cells can induce indirect cytotoxicity by release of reactive species (later identified as NO and peroxynitrite) from macrophages (Nathan et al. 1983; Nathan et al. 1984) an increasing amount of data on the role of NO in the immune and inflammatory systems have accumulated. NO has been shown to influence different immune functions both during innate and adaptive immune responses, including T-cell activation and proliferation, cytokine production, APC expansion and maturation, central and peripheral tolerance, Th cell differentiation, as well as T-cell apoptosis (Brito et al. 1999; MacMicking et al. 1997). In addition to its effects on immunological memory, NO has been implicated in regulation of central and peripheral tolerance (Moulian et al. 2001; Virag et al. 1998). In central tolerance, antigen-presenting cells (APCs) in thymus, including dendritic cells (DCs) and macrophages (MOs), were shown to trigger the selection process both to self- and to allo-Ag by releasing NO (Aiello et al. 2000). During peripheral tolerance on the other hand, regulatory T cells have been shown to induce

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NO production from APCs, to control other pathogenic T cells (Chen et al. 2006). Recently NO was also implicated in the direct induction of CD+CD25+Foxp3 regulatory T cells via p53, IL-2, and CD40 release (Niedbala et al. 2007).

Regulation of T-Cell Priming by NO It has since long been established that NO production is initiated during or shortly after the priming phase of an immune response as a consequence of antigen encounter by T cells. NO production in CD4 T-cell-DC co-cultures was not initiated unless co-stimulatory molecules were up-regulated and IFN-γ was produced (Hoffman et al. 2002). The crucial role for NO in the initiation of an immune response was supported by experiments showing that NO inhibition during the priming phase rather inhibited T-cell proliferation (Niedbala et al. 1999), which in its turn was potentiated by low amounts of NO. The T-cell inhibitory effect exerted by histamine was also recently ascribed to the reduction of NO production by histamine confirming the role of NO in T-cell priming (Koncz et al. 2007). Recently Ibiza et al. showed that eNOS is induced in T cells shortly after antigen binding and is involved in the regulation of TCR signaling by activating N-Ras (Ibiza et al. 2006; Ibiza et al. 2008). However the immune stimulatory capacity of NO during T-cell priming is probably tightly coupled to the amount of NO that is released and by increasing NO levels immune suppression can be evoked. Initial reports showed that following Ag presentation, NO produced from macrophages could also inhibit Th cell proliferation (van der Veen et al. 2000). The suppressive role of NO at priming has then been demonstrated in various situations such as DCs exposed to apoptotic cells (Ren et al. 2008), anti-CD28-induced immune tolerance (Dugast et al. 2008), and T-cell suppression by mesenchymal stromal cells (Sato et al. 2007). Thus NO release is coupled to normal T-cell responses but NO can also act to suppress primary immune responses during physiological circumstances as processing of apoptotic cells. NO has been reported to regulate both T-cell subsets equally, or preferentially induce Th1- but not Th2 cell differentiation. High concentrations of NO are regarded to be toxic and inhibit both Th1 cell proliferation and IFN-γ production. Thus low concentrations of NO seem to selectively induce Th1 differentiation by upregulating IL-12 receptor β2 expression (Niedbala et al. 2002). These studies have highlighted how different concentrations of NO can affect the outcome of immune reactions.

The Immune Suppressive Role of NO In addition to its direct inhibitory effects, NO can react with other oxidants such as super oxide, and form other reactive nitrogen species such as peroxynitrite. Peroxynitrite is immunosuppressive and was shown to inhibit proliferation of T lymphocytes in a dose-dependent manner via nitration of tyrosine residues thereby

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blocking the activation-induced protein phosphorylation and by inducing apoptosis in T cells (Brito et al. 1999). Furthermore, NO and peroxynitrite seems to regulate both T-cell memory and survival, since iNOS−/− mice or mice treated with inhibitors of peroxynitrite displayed increased CD4+ and CD8+ memory T cells following immunization (Vig et al. 2004).

Growth Promoting Effects of NO in Tumor Cells Various tumor cells have been reported to express both iNOS and nNOS in vitro but proof of in vivo expression is more difficult to find due to the existence of infiltrating macrophages and monocytes in tumor tissue. NO released from tumor cells have been suggested to both promote and inhibit tumor growth by direct and indirect mechanisms (Shinoda and Whittle 2001). The critical role of iNOS during tumor progression was assessed using iNOS KO C6 glioma cells, which resulted in a significant reduction of the tumor volume compared to control cells (Yamaguchi et al. 2002). However, from these studies it is difficult to say whether the in vivo effects were only the result of diminished tumor cell growth and not from diminished immune suppression (especially as the C6 glioma was induced in outbred animals).

The Role of NO in the Regulation of Cytotoxic Stimuli in Immunotherapy Preclinical Findings Potentiation of Immune-Derived Cytotoxicity Against Tumor Cells Initial reports by Nathan et al. showed that activated lymphocytes can induce a cytotoxic capacity in monocytes and macrophages by release of reactive species via secretion of IFN-γ (Stuehr et al. 1989; Stuehr and Nathan 1989). After these sentinel discoveries several different mechanisms of NO-mediated cytotoxicity against tumor cells have been proposed. In response to IL-2, iNOS was induced in rat NK cells and was suggested to be responsible for its cytotoxic functions (Cifone et al. 1999). Furthermore, it has been shown that the effector function of NK cells during AK-5 tumor rejection was mediated by overproduction of NO, and that NK cells induced tumor cell lysis through NO-mediated cytotoxicity (Jyothi and Khar 1999). Another possibility to enhance the anti-tumor immune responses is to skew the suppressive property of tumor-associated macrophages (TAM) to a classically activated type 1 macrophages producing high levels of NO, by targeting the NF-κB signaling pathway (Hagemann et al. 2008). Intracranial administration of LPS and IFN-γ significantly prolonged the survival of B16F10 murine melanoma-bearing animals, which seemed to be dependent on NO generation, because the survival was

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decreased using the NOS inhibitor L-NAME. These data show that NO by itself can inhibit tumor progression (Samdani et al. 2004). Mechanistically NO has been shown to potentiate apoptotic cell death by synergy with members of the TNF family as TRAIL, FASL, and TNF-α even in primary resistant tumor cells as reviewed by Jeannin et al. (2008) and Bonavida et al. (2008). NO-Mediated Immune Suppression in Tumor-Bearing Hosts During tumor progression in experimental mouse models in addition to DCs and MO, immature Gr-1+ myeloid cells exert part of their suppression via NO-mediated pathways (Bronte et al. 2005; Serafini et al. 2006). Gr-1+ myeloid cells derived from tumor-bearing animals have been shown to inhibit T-cell activation induced through CD3/CD28 co-stimulation, via peroxynitrite-dependent mechanisms (Bronte et al. 2005; Kusmartsev et al. 2000). This effect was mediated via IFN-γ production by inducing NO in the myeloid cells. The Gr-1+ myeloid cell inhibition of lymphocyte proliferation required cell–cell contact to achieve sufficient levels of NO and seemed to be partly mediated by the T-cell protein tyrosine phosphatase (TC-PTP) (Dupuis et al. 2003). These results were supported by the finding that CD11b+ Gr1+ MSCs isolated from iNOS null mice did not inhibit T-cell proliferation, indicating that NO indeed is an essential component of myeloid cell-mediated immune suppression. NO blocks signaling through the IL-2 receptor expressed by T lymphocytes by impeding phosphorylation of the intracellular-signaling proteins STAT5, AKT, and ERK (Mazzoni et al. 2002). Co-stimulatory-related ligands have also been shown to participate in the NO-mediated suppression and blockade of B7-H1 on MOs (Yamazaki et al. 2005). iNOS and subsequent NO production expression can also be induced by immunotherapy per se, as observed in a rat glioma model (Johansson et al. 2002), where iNOS expression was only observed in tumors from animals immunized with tumor cells transfected with IFNγ, but not in animals immunized with wild-type tumor cells. Thus only tumor-infiltrating NK cells or lymphocytes can secrete sufficient amounts of interferon-gamma for the induction of iNOS in tumor-infiltrating macrophages (van der Veen et al. 2003).

Boosting Anti-tumor Immunity by Modulation of NO-Mediated Immunosuppression The use of NO donors, which when released intracellularly can inhibit iNOS, has been reported to be successful in different malignancies. NO-releasing aspirin was able to improve the effects of a GM-CSF-based cancer vaccine in a murine colon cancer model, through inhibition of iNOS and other suppressive enzymes (De et al. 2005). NO release can also protect against immune suppression by other mechanisms as in the B16 melanoma mouse model, where DCs treated ex vivo with NO donors

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became resistant to tumor-induced apoptosis, due to prevention of activation of the down-stream proapoptotic events including Bcl-2, Bax, and caspase-9 (Perrotta et al. 2004). However, the major observed effects of NO during anti-tumor immunotherapy tend to be suppressive. In our earlier studies we showed that the systemic immunosuppression induced by large tumor burdens in tumor-bearing hosts was mediated by NO production (Hegardt et al. 2000, 2001). Immunotherapy that directly or indirectly leads to increased levels of activated T cells or NK cells can lead to immunosuppression due to an increased release of NO. Koblish et al. demonstrated that an IL-12-based experimental immunotherapy was unsuccessful due to an NO-mediated down-regulation of the immune response but this could be reversed by unspecific inhibitors of NOS (Koblish et al. 1998). Down-regulation of T lymphocyte responses induced by administration of polyclonal stimulation also involves NO release and we could show that the selective iNOS inhibitor L-NIL was superior in reversing this than the unspecific inhibitor L -NAME (Badn et al. 2007). In this report the in vivo combination of L -NIL and immunotherapy with IFN-γ secreting tumor cells gave a slight prolongation of survival. These results were markedly improved when the inhibitor of both iNOS and COX, mercaptoethylguanidine (MEG), was used in the same experimental model and 40% of rats bearing intracerebral tumors could be cured (Badn et al. 2007). These effects were strictly dependent on the timing by which the iNOS inhibitor was administered in relation to the time of immunization, pointing out the important feature of modulating NO during anti-tumor immune responses. This study also demonstrated that systemic levels of NO were actually increased during immunotherapy above the levels of tumor bearers underscoring the fact that immunotherapy per se can induce further immunosuppression, in this case by release of NO. The reports of successful immunotherapy combined with inhibition of NO have in common that the immunotherapies have directly or indirectly been linked to release of IFN-γ, the major trigger of NO release. This is also the case of the combined immunotherapy with IL-12- and IL-18-secreting tumor cells in an intrahepatic rat colon cancer model. Due to the abundance of NO-producing myeloid cells in the liver immunotherapy per se did not have any affect but the combination of L-NAME and low doses of an anti-angiogenic compound, combretastatin, significantly prolonged the survival (Badn et al. 2006). Thus to obtain a functional immunotherapy we need to understand which are the suppressive mechanisms presented in a particular tumor type. However in our experience inhibition of NOS or iNOS during immunizations could abrogate the effects and underscore the importance of certain levels of NO for T-cell priming. By combining inhibitors of iNOS and COX-2 by separate drugs we have preliminarily shown that there is a synergistic effect of the compounds and that iNOS inhibition boosts memory responses. Following treatment with the iNOS inhibitor L -NIL and/or cycle oxygenase 2 (COX-2) inhibitor parecoxib, in combination with

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an IFN-γ-based anti-tumor immunotherapy, animals receiving the iNOS inhibitor survived more often after rechallenges (Badn and Eberstal, unpublished data).

Clinical Studies The combination of immunotherapy with NO-modulating approaches has not yet been explored in patients. However, there are a couple of ex vivo studies indicating the involvement of NO in the tumor-induced immunosuppression. In prostate organ cultures treatment with the specific iNOS inhibitor L-NMMA was able to restore the suppressed functions of tumor-infiltrating T lymphocytes (Bronte et al. 2005). Induction of apoptosis was obtained after administration of NO donors on human colon carcinoma cells in vitro (Millet et al. 2002) and on tumor growth and metastasis of B16F10 melanoma in vivo (Postovit et al. 2004). NO–NSAIDs such as NO–aspirin, NO–ibuprofen, and NO–sulindac have been shown to induce apoptosis in human colon cancer cells in vitro (Rigas and Williams 2002). A derivative of NO–aspirin NCX-4016 has been tested in healthy volunteers and with gastrointestinal safety, proposing future use in combination with other therapies in cancer patients.

Concluding Remarks and Future Directions Due to the dual effects of NO on the immune system immunotherapeutic approaches should be designed to achieve a positive net effect of NO, either using NO donors or inhibitors, depending on the type of the tumor as well as the type of the immunotherapy. It is important to consider the fact that the manipulation of the immune system using different immunotherapeutic approaches might itself induce tolerance and/or immunosuppression. Although NO donors have shown increased induction of apoptosis in vitro it is still unclear whether they actually inhibit iNOS activity in vivo in some circumstances, The induction of cytotoxicity by NO in vivo could also boost T-cell responses by partially degrading tumor cells and thus facilitating antigen presentation of APC. Thus, the combination of immunotherapy with NO-modulating approaches has to be specifically tailored, considering the tumor type, immunization timetable, and the suppressive network presented in that tumor.

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Chapter 13

Nitric Oxide Is a Promising Enhancer for Cancer Therapy Marion Cortier, Lissbeth Leon, Néjia Sassi, Catherine Paul, Jean-François Jeannin, and Ali Bettaieb

Abstract This report summarizes the present state of our knowledge pertaining to the nitric oxide (NO)-induced sensitization of tumor cell death. The effects of NO and its synergy with ionizing radiations, with members of the TNF family, and with chemotherapy have been investigated. The effect of NO-induced sensitization and the underlying molecular mechanisms are discussed. Keywords Cytokine · Chemotherapy · Ionizing radiation · Apoptosis · Cell death receptors

Introduction Nitric oxide (NO) is a ubiquitous molecule that plays important roles in vasodilation (Palmer et al. 1987), neurotransmission (Moncada and Bolanos 2006), and immune responses (Liew and Cox 1991). In cancer, NO has been associated with cancer progression and metastasis, cancer angiogenesis, cancer cell apoptosis, cancer chemoprevention, and a modulator for chemo-radio-immuno-therapy. The opposite effects of NO are probably contradictory only at the first sight as reported by Gauthier et al. in the murine mammary carcinoma model, who showed that the NO produced by host cells mainly promotes tumoral growth, whereas the NO produced by tumoral cells essentially inhibits its growth (Gauthier et al. 2004). This study confirmed prior results obtained in the rat colon carcinoma model showing that

J.-F. Jeannin (B) Laboratory of Cancer Immunology and Immunotherapy, EPHE/INSERM U866, 7 bd Jeanne d’Arc, Dijon 21079, France e-mail: [email protected]

Université

de

Bourgogne,

Financial Support: Our group is supported by grants from Nièvre, Haute-Marne, Saône et Loire and Côte d’Or committees of the Ligue Nationale Contre le Cancer and from the Conseil Régional de Bourgogne.

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_13, C Springer Science+Business Media, LLC 2010

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peritoneal tumors induced spleen macrophage to produce NO which was responsible for the inhibition of T cell proliferation (Lejeune et al. 1994) and the adaptive immune response. Also, TGF-β1 produced by tumor cells inhibited the expression of the inducible NO synthase (NOS II) and promoted tumor growth (Lagadec et al. 1999). Opposite effects were found at the cellular level whereby NO can inhibit or promote tumor cell death. The opposite effects of NO reported in the literature have been interpreted as contradictory because of the lack of knowledge underlying the great complexity of the NO biology and the great complexity of tumorigenesis and the unlimited growth of tumor cells. This report summarizes the present state of our knowledge regarding the NO-induced sensitization to death in tumor cells.

Synergy of NO with Ionizing Radiations Preclinical Studies Ionizing radiations are used as first-line therapy in malignancies including colorectal, breast, lung, prostate, and esophageal cancers. Hypoxic cells are a characteristic of tumors that are known to limit the success of conventional treatments and have recently been implicated in the promotion of neoplastic progression and metastasis (Brown 2000). In an effort to circumvent this resistance mechanism, classes of drugs known as hypoxic cell radiosensitizers are in development such as tirapazamine (Marcu and Olver 2006) and NO (Yasuda 2008). The radiosensitivity of NO has been first reported by Howard-Flanders, who demonstrated that a low concentration of NO gas could efficiently radiosensitize hypoxic bacterial cells to ionizing radiations (Howard-Flanders 1957). This effect was confirmed on mammalian tumor cells (Dewey 1960; Gray et al. 1958). Recently, Wardman et al. (2007) showed that NO (1% v/v NO/N2 ) caused significant sensitization of various tumor cells (rodent and human) to low-radiation doses. This effect is correlated with stable NO/base adducts formation with uracil radicals. Since NO gas can damage lung tissue, a series of compounds as NO donors have been tested to evaluate their radiosensitizing potential. Mitchell et al. (1993) showed that the NO-releasing agent DEA/NO2 results in radiosensitization of hypoxic Chinese hamster V79 lung cells. Similar effects were observed in other cell types and with other NO donors. Indeed, Verovski et al. showed that NO released by SNP or GSNO3 was responsible for the increase in radiosensitivity of human pancreatic tumor cells grown in hypoxic conditions in vitro. This radiosensitizing activity was correlated with the enhancement of single-strand DNA breakage caused by radiation (Verovski et al. 1996). It was confirmed that DEA/NO or SPER/NO4 (Griffin et al. 1996) 1 TGF:

tumor growth factor. diethylamine nitric oxide. 3 GSNO: S-nitrosoglutathione. 4 SPERNO: spermine-NONOate. 2 DEA/NO:

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and SNAP or PAPA/NO were efficient in radiosensitization in murine mammary SCK carcinoma cells [16] and in mammary murine EMT-6 tumor cells (Janssens et al. 1999) grown in hypoxic conditions. Similarly, Worthington et al. showed that transfection of murine RIF-1 fibrosarcoma in vitro with iNOS constructs eliminated the radioresistance observed under hypoxic conditions. In vivo iNOS-expressing tumor cells injection resulted in a dramatic delay in tumor growth (Worthington et al. 2002). Meanwhile, an enhancement of the cytotoxicity induced by ionizing radiations was found with NO donors (SNAP or SNP and SNAP or DETANO) in glioma cells5 and colon cancer cells6 in normoxic conditions (Kurimoto et al. 1999; Wang et al. 2004). It was confirmed in colon cancer cells7 overexpressing NOS II (Wang et al. 2004). In this latter work, the radiosensitizing effects of an adenoviral gene transfer of iNOS (AdiNOS) were efficient at low infection (4% of tumor infected), indicating a significant bystander effect. Furthermore, intratumoral injections of AdiNOS-induced larger vascularization of tumors allowed a better oxygenation of tumor cells which enhanced tumor growth delay compared to radiation alone (Wang et al. 2004). Wood et al. linked the in vivo effect of the NO donor SIN-1,8 administered immediately before irradiation of murine sarcomas,9 to an increased oxygenation within the tumors (Wood et al. 1996). Similar observations were done with isosorbide dinitrate in another model of murine sarcoma,10 in which NO increased tumor blood flow and so tumor cell oxygenation, which may explain the increased delay in tumor regrowth after irradiation (Jordan et al. 2003). Several mechanisms have been involved in NO-induced radiosensitization. Indeed, activation of the transcription factor p53 was shown to play a role since the synergy between NO and radiation was decreased in tumors induced by p53-mutated cells. Furthermore, the transfer of p53 gene into tumor cells lacking functional p53 enhanced the synergy (Cook et al. 2004). The synergy was due to the phosphorylation of p53 on serine 15. Synergy was obtained in vivo when tumor cells transduced with an adenoviral vector carrying the NOS II gene were injected in mice. It was also obtained in vitro in transduced tumor cells or tumor cells treated with SNAP (Cook et al. 2004). The role of p53 phosphorylation was established in a subsequent report. It was shown that GSNO enhanced markedly the ability of low-dose ionizing radiation to elicit apoptotic killing of human neuroblastoma cells expressing cytoplasmic wild-type p53. NO induced the phosphorylation of p53 on serine 15 in these cells via the kinase ATR11 and promoted p53 nuclear retention. Blocking the kinase activity of ATR effectively abolished the ability of NO to cause p53 nuclear retention. These findings imply that through augmenting p53 nuclear retention NO can

5 Human

T98G or U87 and rat C6 cells. cancer cells. 7 SNU-1040 cancer cells. 8 SIN-1: 3-morpholinosydnonimine. 9 SCCVII/Ha. 10 FSaII fibrosarcoma. 11 ATR: ATM and Rad3-related. 6 HCT-116

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increase the ionizing radiation-induced apoptosis (Wang et al. 2003). However, NO also stimulates DNA-dependent protein kinase and p38 mitogen-activated protein kinase, both of which may enhance the phosphorylation of serine 15. The combination of ionizing radiation and SNAP potentiated Fas-induced apoptosis of human HeLa cancer cells. Suboptimal dose of radiations and SNAP upregulated the cell surface Fas antigen, detected by FACScan. This sensitization was completely abrogated by anti-Fas neutralizing antibody. During the synergistic apoptosis, mitochondria permeabilization, cytochrome c release, and DNA fragmentation were detected (Park et al. 2003). Furthermore, the cytotoxic synergy shown in human colon carcinoma cells treated with SNAP or transduced with the NOS II gene and exposed to ionizing radiations was caspase dependent (Chung et al. 2003). Another mechanism used by NO to sensitize radiation-induced cell death was its capability to increase tumor vascularization and tumor oxygenation in mice treated with ionizing radiations, which implies an increase of angiogenesis and thus a control of related genes. Although NOS II has been implicated in VEGF12 gene activation, gene activation was reported as responsible for NO and radiation synergistic cytotoxicity.

Synergy of NO with Members of the TNF Family Death receptors such as CD95 (APO-1/Fas) and TRAIL13,14 DR4/DR5 are important inducers of the extrinsic apoptosis of many tumor cells. In vivo, tumor eradication by the immune response involves tumor cell killing especially by the ligands of these receptors, FasL15 and TRAIL. Changes in the expression of these receptors or their respective ligands are frequently found in human cancer. Death receptors downregulation or mutation has been proposed as a mechanism by which cancer cells escape destruction by the immune system through reduced apoptosis sensitivity. Thus, every strategy leading to the upregulation of these receptors may result in a direct induction of cell death and/or sensitization of cancer cells to cytotoxic stimuli. It was shown by Garban and Bonavida that NO, produced by exogenous nitrodonors or endogenously by IFN-γ16 pretreatment, upregulated the Fas surface cell expression and sensitized FasL-resistant human ovarian carcinoma cells (AD10, A2780) to apoptosis induced by the agonistic FasL mAb CH-1117 (Garban and Bonavida 1999). NO-mediated upregulation of Fas expression was the result of the inhibition of a Fas transcription repressor factor, Yin-Yang-1 (YY-1) (Garban and Bonavida 2001). The same group has reported that NO inhibited 12 VEGF:

vascular endothelial growth factor. tumor necrosis factor. 14 TRAIL: tumor necrosis factor-related apoptosis-inducing ligand. 15 FasL: ligand of Fas (CD95/APO-1 receptor). 16 IFN: interferon. 17 CH11: agonist anti-Fas antibody. 13 TNF:

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YY1 DNA-binding activity through S-nitrosylation which resulted in Fas expression increase and tumor cell sensitization to FasL cytotoxicity (Hongo et al. 2005). We confirmed these results with human colon carcinoma cells (SW480 and HCT116) showing that the NO donor GTN18 sensitizes tumor cells to FasL-induced apoptosis by increasing cell surface Fas and decreasing IAP19 expression (Millet et al. 2002). Using human colon carcinoma CX-1 cells and the NO donor SNP,20 Lee et al. reported that SNP sensitizes the TRAIL-resistant CX-1 cells to TRAIL killing via a mitochondria-dependent pathway since overexpression of Bcl-2 completely blocked the enhancing effect of NO. The combined treatment caused an increase in cytochrome c release, caspase-3 activation, and PARP cleavage. Further, TRAIL interacts with two death receptors DR4 and DR5, which trigger the death signal, but the combination of TRAIL plus SNP did not increase the level of DR4 and DR5 and did not decrease decoy receptors (Lee et al. 2001). Another NO-releasing component, nitrosylcobalamin21 (NO–vitamin B12), synergized with interferon beta to induce eradication of the human tumor xenograft ovarian cancer, the NIH-OVCAR-3 (Bauer et al. 2002). This antitumor effect is related to the capacity of nitrosylcobalamin to induce the mRNA expression of FasL, DR4, DR5, and TRAIL. In addition, this NO donor increased mRNA level and the activity of the initiator caspase-8, as well as the mRNA levels of the effector caspases, caspase-3, caspase-6, and caspase-7 (Bauer et al. 2002). The same group has demonstrated that nitrosylcobalamin promotes cell death of human ovarian carcinoma cells via S-nitrosylation of DR4 at cysteine 336. Cells which express DR4 C336 mutation lacked S-nitrosylation, exhibited reduced caspase activity, and were more resistant to NO- and TRAIL-induced apoptosis than other mutants. Furthermore, in these models, sensitization to apoptosis was increased by overexpression of wild-type DR4 (Tang et al. 2006). In another type of cancer, prostate cancer cells (CaP22 cells), neither TRAIL alone nor DETANO alone, were able to provoke apoptosis. However, the combination of both was synergistic in apoptosis. DETANO induced the mitochondrial membrane depolarization and release of cytochrome c and Smac/diablo, NF-κB activity inhibition, and the downregulation of Bcl-xL expression (Huerta-Yepez et al. 2004), but failed to provoke caspase activation. This step was achieved by the combination which activated caspases-9 and -3 resulting in apoptosis (Huerta-Yepez et al. 2004). Analogous results were obtained by Chawla-Sarkar et al. and Huerta-Yepez et al. using the human melanoma tumor cell line A375 and nitrosylcobalamin, and the human prostate cancer cells (CaP cells) and DETANO (Chawla-Sarkar et al.

18 GTN:

glyceryl trinitrate. inhibitor of apoptosis. 20 SNP: sodium nitroprusside. 21 Nitrosylcobalamin, prodrug relatively tumor-specific due to higher transcobalamin receptor expression in tumor cells compared with normal tissue [32] that releases NO inside the cell. 22 Cap cells: DU145, PC-3, CL-1 and LNCaP cell lines. 19 IAP:

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2003; Huerta-Yepez et al. 2004). The NO donors sensitize cancer cells to TRAILmediated death via inhibition of NF-κB and XIAP23 activities or Bcl-xL expression (Secchiero et al. 2001). In a recent paper, Huerta-Yepez et al. reported that the NO-dependent sensitization of prostate carcinoma cells to TRAIL-induced apoptosis occurred via the inhibition of YY-1 that binds and negatively regulates TRAIL receptor DR5 transcription and expression and this was correlated with resistance to TRAIL-induced apoptosis (Huerta-Yepez et al. 2009). NO also sensitizes tumor cells to TNF-α-mediated cytotoxicity via inhibition of NF-κB activation, as shown with the AD10 human ovarian carcinoma cells. The sensitivity of AD10 cells to TNF-α was induced by IFN-γ and inhibited in the presence of IFN-γ by NOS inhibitors; IFN-γ could be replaced by the NO donor SNAP. By reducing the generation of H2 O2 , NO disrupts NF-κB activation and sensitizes tumor cells to TNF-α-mediated cytotoxicity (Garban and Bonavida 2001; Huang et al. 2005). It has also been reported that the NO donor PAPANO,24 via its capacity to induce loss of survivin protein cell expression, sensitized lymphoma cells to murine bone marrow-derived dendritic cell (DC)-mediated cytotoxicity in vitro in a Fas/FasLdependent mechanism (Huang et al. 2005). Perrotta et al. also reported that DC pretreated with NO donors (DETA/NO or isosorbide dinitrate) enhanced cisplatininduced tumor regression and animal survival in the B16 mouse model of melanoma (Perrotta et al. 2007).

Synergy of NO with Cytotoxic Drugs There is also evidence that the antitumor effect of certain cancer chemotherapy agents might be enhanced by NO. Wink et al. reported that Chinese hamster V79 lung fibroblasts cells treated with NO gas or DEANO or PAPANO were sensitized to cisplatin cytotoxicity (Wink et al. 1997). These latter NO donors also enhanced the melphalan-induced cytotoxicity in V79 and human MCF-7 breast cancer cells (Cook et al. 1997). This synergetic effect seemed to involve several pathways, including possibly inhibition of DNA repair (Cook et al. 1997). DEANO and DETANO were also reported to increase the cytotoxicity of fludarabine in human B-CLL25 cells (Adams et al. 2001). Other reports dealt with doxorubicin and cisplatin. DEANO and NO gas increased the cytotoxicity of doxorubicin to human breast carcinoma MCF-7 cells, as did NOS II overexpression (Evig et al. 2004). Human breast carcinoma MDA-MB-231 cells cultured as multicellular aggregates (spheroids) are doxorubicin-resistant; however, DETANO or GTN decreased by 33–50% their resistance (Muir et al. 2006). The same effect was obtained in human head and neck

23 XIAP,

X-linked inhibitor of apoptosis. 3-(2-hydroxy-2-nitroso-1-propyl hydrazino)-1-propanamine. 25 B-CLL: B-cell chronic lymphocytic leukemia. 24 PAPANO:

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squamous cell carcinoma with DETANO (Azizzadeh et al. 2001). Reverting doxorubicin resistance of colon cancer cells was also obtained by the expression and activation of NOS II by GTPase RhoA silencing. This effect was correlated with the tyrosine nitration of the multidrug resistance protein 3 transporters (MRP3) and the decrease of the ATPase activity of P-glycoprotein (Pgp) that contributed to a reduced doxorubicin efflux (Doublier et al. 2008). Similarly, it has been reported that the synergy between NO and cytotoxic drugs on cancer cell death can be due to the accumulation of drugs including doxorubicin, cisplatin, and arsenic in human lung, liver, colorectal carcinoma cells, and in leukemic cells (Chawla-Sarkar et al. 2003; Riganti et al. 2005). This resulted from inhibition by SNAP of MDR proteins26 in human colon carcinoma cells HT29 (Riganti et al. 2005). New NO donors, NO-releasing diazeniumdiolates,27 JS-K and CB-3-100, increased cisplatin and arsenic cytotoxicity in rat liver carcinoma CAsE cells (Liu et al. 2004). The enhancement of cytotoxicity with these NO donors was accompanied by increased accumulation of arsenic and cisplatin within cells and by enhanced activation of mitogen-activated protein kinase JNK and ERK (Liu et al. 2004). Recently, Kitagaki et al. have reported that the NO prodrug JS-K could function by inhibition of ubiquitin E1 leading to a decrease in total cellular ubiquitination and an increase in cellular p53 levels and cancer cell death (Kitagaki et al. 2009). In vivo, in mice bearing syngeneic ovarian carcinoma, chemosensitization was obtained between cisplatin treatment and IFN-γ by direct injection into tumors of liposomes containing an expression vector for IFN-γ. This treatment suppressed tumor growth and increased the long-term survival of animals, but was ineffective in iNOS (−/−) mice indicating that NO was a direct mediator of chemosensitization (Son and Hall 2000). In a recent preclinical study, Frederiksen et al. have reported that in PC-3 human prostate tumor xenografts combination of doxorubicin and continuous transdermal GTN delivery resulted in a 40% decrease in the median rate of tumor growth and a 55% decrease in the mean rate of tumor growth (Frederiksen et al. 2007).

Clinical Studies A noteworthy report in the field of chemosensitization of cancer cells by NO was recently reported by Yasuda et al. (Yasuda et al. 2006). These authors investigated, in a randomized phase II trial, the efficacy and safety of GTN plus vinorelbine and cisplatin in 120 patients with previously untreated stage IIIB/IV non-small-cell lung cancer. They demonstrated that treatment with GTN improved the response rate, time to disease progression, and survival in patients with advanced cancer, without major adverse effects. A prospective randomized phase III trial to evaluate GTN plus

26 MDR:

multidrug resistance, MDR proteins, the main extrusion pump for hydrophobic drugs.

27 Substrates of glutathione-S-transferase that catalyze the conjugation of xenobiotics with reduced

glutathione.

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vinorelbine and cisplatin is underway in patients with stage IIIB/IV non-small-cell lung cancer.

Conclusion NO production by NOS II is not high enough to kill tumor cells, but in cells able to express NOS II, NO concentration is high enough to sensitize tumor cells to the cytotoxic effect of ligands of the TNF family, of some cytotoxic drugs, or of ionizing radiations. It is probable that sensitization is mostly due to S-nitrosylation of either transcription factors (NF-κB, p53, YY1) or proteins which regulate the apoptotic pathways. When treatments fail to eradicate tumor, generally due to their inability to induce NOS II expression or due to the selection of resistant tumor cells, we are led to force NO production. Thus, one challenge is to target specifically the tumor with NO donors or NOS II gene. NO donors producing NO spontaneously in aqueous media have to be protected before reaching tumor site; NO donors producing NO after biotransformation in the cells by their enzymatic machinery have to be targeted into tumor cells. These could be achieved using nanovesicles or using gene therapy.

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Lee, Y.J., Lee, K.H., Kim, H.R., Jessup, J.M., Seol, D.W., Kim, T.H., Billiar, T.R., and Song, Y.K. (2001). Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondriadependent pathway in human colorectal carcinoma CX-1 cells. Oncogene 20, 1476–1485. Lejeune, P., Lagadec, P., Onier, N., Pinard, D., Ohshima, H., and Jeannin, J.F. (1994). Nitric oxide involvement in tumor-induced immunosuppression. J. Immunol. 152, 5077–5083. Liew, F.Y. and Cox, F.E. (1991). Nonspecific defence mechanism: the role of nitric oxide. Immunol. Today 12, A17–A21. Liu, J., Li, C., Qu, W., Leslie, E., Bonifant, C.L., Buzard, G.S., Saavedra, J.E., Keefer, L.K., and Waalkes, M.P. (2004). Nitric oxide prodrugs and metallochemotherapeutics: JS-K and CB3-100 enhance arsenic and cisplatin cytolethality by increasing cellular accumulation. Mol. Cancer Ther. 3, 709–714. Marcu, L. and Olver, I. (2006). Tirapazamine: from bench to clinical trials. Curr. Clin. Pharmacol. 1, 71–79. Millet, A., Bettaieb, A., Renaud, F., Prevotat, L., Hammann, A., Solary, E., Mignotte, B., and Jeannin, J.F. (2002). Influence of the nitric oxide donor glyceryl trinitrate on apoptotic pathways in human colon cancer cells. Gastroenterology 123, 235–246. Mitchell, J.B., Wink, D.A., DeGraff, W., Gamson, J., Keefer, L.K., and Krishna, M.C. (1993). Hypoxic mammalian cell radiosensitization by nitric oxide. Cancer Res. 53, 5845–5848. Moncada, S. and Bolanos, J.P. (2006). Nitric oxide, cell bioenergetics and neurodegeneration. J. Neurochem. 97, 1676–1689. Muir, C.P., Adams, M.A., and Graham, C.H. (2006). Nitric oxide attenuates resistance to doxorubicin in three-dimensional aggregates of human breast carcinoma cells. Breast Cancer Res. Treat. 96, 169–176. Palmer, R.M., Ferrige, A.G., and Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524–526. Park, I.C., Woo, S.H., Park, M.J., Lee, H.C., Lee, S.J., Hong, Y.J., Lee, S.H., Hong, S.I., and Rhee, C.H. (2003). Ionizing radiation and nitric oxide donor sensitize Fas-induced apoptosis via up-regulation of Fas in human cervical cancer cells. Oncol. Rep. 10, 629–633. Perrotta, C., Bizzozero, L., Falcone, S., Rovere-Querini, P., Prinetti, A., Schuchman, E.H., Sonnino, S., Manfredi, A.A., and Clementi, E. (2007). Nitric oxide boosts chemoimmunotherapy via inhibition of acid sphingomyelinase in a mouse model of melanoma. Cancer Res. 67, 7559–7564. Riganti, C., Miraglia, E., Viarisio, D., Costamagna, C., Pescarmona, G., Ghigo, D., and Bosia, A. (2005). Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux. Cancer Res. 65, 516–525. Secchiero, P., Gonelli, A., Celeghini, C., Mirandola, P., Guidotti, L., Visani, G., Capitani, S., and Zauli, G. (2001). Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies. Blood 98, 2220–2228. Son, K.K. and Hall, K.J. (2000). Nitric oxide-mediated tumor cell killing of cisplatin-based interferon-gamma gene therapy in murine ovarian carcinoma. Cancer Gene Ther. 7, 1324–1328. Tang, Z., Bauer, J.A., Morrison, B., and Lindner, D.J. (2006). Nitrosylcobalamin promotes cell death via S nitrosylation of Apo2L/TRAIL receptor DR4. Mol. Cell Biol. 26, 5588–5594. Verovski, V.N., Van den Berge, D.L., Soete, G.A., Bols, B.L., and Storme, G.A. (1996). Intrinsic radiosensitivity of human pancreatic tumour cells and the radiosensitising potency of the nitric oxide donor sodium nitroprusside. Br. J. Cancer 74, 1734–1742. Wang, X., Zalcenstein, A., and Oren, M. (2003). Nitric oxide promotes p53 nuclear retention and sensitizes neuroblastoma cells to apoptosis by ionizing radiation. Cell Death Differ. 10, 468–476. Wang, Z., Cook, T., Alber, S., Liu, K., Kovesdi, I., Watkins, S.K., Vodovotz, Y., Billiar, T.R., and Blumberg, D. (2004). Adenoviral gene transfer of the human inducible nitric oxide synthase gene enhances the radiation response of human colorectal cancer associated with alterations in tumor vascularity. Cancer Res. 64, 1386–1395.

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Wardman, P., Rothkamm, K., Folkes, L.K., Woodcock, M., and Johnston, P.J. (2007). Radiosensitization by nitric oxide at low radiation doses. Radiat. Res. 167, 475–484. Wink, D.A., Cook, J.A., Christodoulou, D., Krishna, M.C., Pacelli, R., Kim, S., DeGraff, W., Gamson, J., Vodovotz, Y., Russo, A., and Mitchell, J.B. (1997). Nitric oxide and some nitric oxide donor compounds enhance the cytotoxicity of cisplatin. Nitric Oxide 1, 88–94. Wood, P.J., Sansom, J.M., Stratford, I.J., Adams, G.E., Szabo, C., Thiemermann, C., and Vane, J.R. (1996). Changes in energy metabolism and X-ray sensitivity in murine tumours by the nitric oxide donor SIN-1. Br. J. Cancer Suppl. 27, S177–S180. Worthington, J., Robson, T., O Keeffe, M., and Hirst, D.G. (2002). Tumour cell radiosensitization using constitutive (CMV) and radiation inducible (WAF1) promoters to drive the iNOS gene: a novel suicide gene therapy. Gene Ther. 9, 263–269. Yasuda, H. (2008). Solid tumor physiology and hypoxia-induced chemo/radio-resistance: novel strategy for cancer therapy: nitric oxide donor as a therapeutic enhancer. Nitric Oxide 19, 205–216. Yasuda, H., Yamaya, M., Nakayama, K., Sasaki, T., Ebihara, S., Kanda, A., Asada, M., Inoue, D., Suzuki, T., Okazaki, T., Takahashi, H., Yoshida, M., Kaneta, T., Ishizawa, K., Yamanda, S., Tomita, N., Yamasaki, M., Kikuchi, A., Kubo, H., and Sasaki, H. (2006). Randomized phase II trial comparing nitroglycerin plus vinorelbine and cisplatin with vinorelbine and cisplatin alone in previously untreated stage IIIB/IV non-small-cell lung cancer. J. Clin. Oncol. 24, 688–694.

Chapter 14

Role of Nitric Oxide for Modulation of Cancer Therapy Resistance Thomas Efferth

Abstract Reactive nitrogen species (RNS) act as central second messengers in a balanced cellular network. While the complexity of nitric oxide (NO) signaling is far from being understood, and many controversial data can be found in the literature, there is evidence for NO as a major player of modulation of resistance to anticancer drugs and radiotherapy. Hypoxia in cancer tissues causes therapy resistance, and the hypoxia-inducing factor-1 (HIF-1) plays a predominant role in hypoxia-induced resistance. NO and NO-donating compounds sensitize tumor cells by inhibiting HIF-1 mediated transcription in hypoxic cells. Among a plethora of other genes, HIF-1-induced the transcription of the multidrug resistance gene 1, MDR1, and the angiogenesis-inducing vascular endothelial growth factor gene (VEGF). NOmediated down-regulation of glutathione and antioxidant stress response genes as well as the inhibition of DNA repair proteins also contributes to sensitivity of tumors to chemo- and radiotherapy. Hypoxic tumors tend to accumulate cells with mutations in the tumor suppressor gene, TP53. NO activates wild-type p53 protein by peroxynitrite-mediated DNA damage and exerts resistance-modulating effects on wild-type, but not on mutant p53. Furthermore, the antiapoptotic transcription factor, NF-κB, is inhibited by NO and NO donors. Thereby, NO not only enhances susceptibility to anticancer drugs and radiotherapy but also suppresses the NF-κB-mediated transcription of metastasis-regulating genes. Keywords Angiogenesis · Cancer · Drug resistance · HIF-1 · Hypoxia · NF-κB · P53 · Radio-resistance

T. Efferth (B) Department of Phatmaceutical Biology, Institute of Pharmacy and Biochemisty, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany e-mail: [email protected]

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_14, C Springer Science+Business Media, LLC 2010

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Introduction Nitric oxide belongs to the oldest molecules on earth and has been developed in the primitive atmosphere of the cooling planet (Moncada and Martin 1993). Due to its high reactivity with other molecules, its half-life time in biological systems is only 0.1–5 seconds (Gaston et al. 1994). The development of an oxygen-containing atmosphere forced the evolution to metabolize and detoxify oxygen. Whereas oxygen and its reaction products can be toxic on the one hand, they foster the generation of DNA mutations on the other hand, which represent the engines of evolution. This ambivalent function has been termed “the paradox of oxygen.” Keeping the balance of the cellular redox state was a requirement for life on earth. Any imbalance of this redox state could cause diseases. Reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) act as central second messengers in this balanced network. ROS can bind to cysteine residues of proteins, thereby altering the proteins’ conformation and function in a manner comparable to tyrosine phosphorylation of proteins. Therefore, altering the cysteine oxidation of cancerrelated target proteins as well as interfering with RNS-mediated signaling routes could provide a basis for the development of novel treatment modalities.

The Biochemistry of ROS and RNS Oxygen can be reduced to superoxide radical anion, hydrogen peroxide, and hydroxyl radical. The conversion of hydrogen peroxide to hydroxyl radical is facilitated by ferrous iron (Fe2+ ), the so-called Fenton reaction. ROS are generated at three major sites in the body: 1. Complexes I and III of the mitochondrial respiratory chain transfer electrons to oxygen resulting in superoxide. This process is dependent on the mitochondrial membrane potential. With increasing mitochondrial membrane potential, the respiration rates decrease. 2. The nuclear electron transport chain consists of NADH oxidase, NADH cytochrome C reductase, and cytochrome C oxidase. This cascade does not only provide ATP needed for RNA synthesis but also produces superoxide and peroxide. 3. Cytochrome P450 oxidases, cytochrome b5, and NADPH cytochrome P450 reductase are connected with the electron transport chain of the endoplasmic reticulum. This transport chain also represents an important ROS generator. Other enzymes such as NADPH oxidoreductases or xanthine oxidases also form ROS, albeit their function and signaling networks are incompletely understood. The generation of RNS is tightly linked to the ROS pathways. Nitric oxide (NO) is formed by three nitric oxide synthases, the endothelial (eNOS), neuronal (nNOS),

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and inducible (iNOS) isoforms. Both eNOS and nNOS produce NO at low amounts and are calcium- and calmodulin-dependent, while iNOS is a high-level NO producer and is calcium- and calmodulin-independent. With the help of these enzymes, arginine is oxidized forming citrulline and NO, which requires NADPH as electron source. Under physiological conditions, only low NO amounts are produced by eNOS and nNOS. Upon activation, e.g., by cytokines, high NO amounts are generated by iNOS. NO inhibits the mitochondrial respiratory chain complex IV, which in turn increases mitochondrial ROS generation. NO can react with superoxide forming peroxynitrite, a labile product reacting with carbon dioxide to generate nitrosoperoxycarbonate (ONOOCO2 − ). This in turn decomposes into carbon dioxide and nitrogen dioxide radicals. Within this reaction balance between ROS and RNS, each system regulates the other one. On the other hand, exogenous and endogenous antioxidants greatly influence the redox state of a cell and, thereby, affect ROS/RNS-dependent cysteine redox transformations and signaling pathways. One of the most important redox buffers in the cell represents reduced glutathione (GSH). Oxidation causes dimerization to glutathione disulfate (GSSG), a reaction which can be reversed by glutathione reductase using NADPH as electron source. Superoxide dismutase converts superoxide to peroxide and oxygen, and catalase forms oxygen and water from peroxide. Glutathione reductase and 1- and 2-cysteine peroxiredoxins detoxify peroxides. Furthermore, thioredoxin and glutaredoxin reduce protein thiols. Thereby, thioredoxin and glutaredoxin are oxidized. Thioredoxin reductase reduces thioredoxin using NADPH as electron source, while glutaredoxin uses GSH or glutathione reductase as electron donor (Giles 2006).

ROS-Independent Signaling Pathways of RNS in Cancer The anticancer activity of NO was first shown as a component of the macrophage activity toward leukemia cells (Hibbs et al. 1987). Only recently, over-expression of iNOS was found to be associated with tumor development and progression independently of macrophages (Le et al. 2005). Elevated NO levels and over-expression of iNOS have been observed in inflammatory conditions, which are frequently linked to predisposition for carcinogenesis (Farrow et al. 2004). NO further fosters tumor development by interfering with DNA repair (Rao 2004) and immunosuppression (MacMicking et al. 1997; Eisenstein 2001; Halliday et al. 2004). Sustained production of NO endows macrophages as a part of the innate immune system with cytostatic or cytotoxic activity toward viruses, bacteria, fungi, protozoa, helminths, and tumor cells. On the other hand, inhibition of NO is associated with cytotoxicity toward macrophages (Konkimalla et al. 2008). Increased NOS expression is found in many tumor types and is linked with tumor progression (Weigert and Brüne 2008).

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Independent of ROS signaling, NO acts through the cyclic guanosine monophosphate (cGMP) signaling route. NO binds guanylate cyclase forming cGMP. This molecule activates protein kinase C, an important enzyme for downstream phosphorylation of caspases in tumor biology (Gomez et al. 1999; Hofmann 2004; Redig and Platanias 2008; Van Buren II et al. 2006). Somewhat confusing at first sight is that NO can act in a contradictory manner. It elicits both cytoprotective and cytotoxic effects (Hibbs et al. 1987; Lechner et al. 2005). It has been attempted to reconcile these modes of action by postulating a biphasic response of NO in tumor biology (Goodman et al. 2004; Ridnour et al. 2006). According to this hypothesis, low NO levels are required for tumor promotion and growth. Cell cycle arrest or programmed cell death does not occur at low NO concentrations. Tumor cell subpopulations with activated oncogenes (e.g., BCL2) or mutated tumor suppressor genes (e.g., TP53) possess a selective survival advantage and reveal an NO-resistant phenotype (Goodman et al. 2004; Hussain et al. 2004; Ekmekcioglu et al. 2005). In contrast, high amounts of NO are cytotoxic toward cancer cells. NO affects the transcription factors, p53 and HIF-1α (Zhou and Brüne 2005). Independent of cGMP signaling, NO induces apoptosis, cell cycle arrest by p53 stabilization and up-regulation, caspase activation, and modulation of Bcl-2 family members (Brüne et al. 1998). HIF-1α is activated by mild hypoxia. Both NO and hypoxia lead to a stabilization and accumulation of HIF-1α (Metzen et al. 2003; Schmid et al. 2004; Zurer et al. 2004).

Modulation of Chemo-resistance and Radio-resistance by NO-Mimetic Agents NO and NO-donating drugs act as sensitizing compounds both for radiotherapy (Verovski et al. 1996; De Ridder et al. 2008) and chemotherapy (Matthews et al. 2001; Konovalova et al. 2003; Frederiksen et al. 2007). An important mechanism of therapy resistance of tumors in vivo represents hypoxia. Hypoxic regions are preferentially localized in the central parts of a tumor and contain much less oxygen than peripheral tumor areas or normal tissues (Brown and Wilson 2004). Hypoxia develops, when the proliferative rate of tumor cells exceeds the diffusion distance. This induces tumor neo-angiogenesis. However, tumor blood vessels frequently show aberrant structures and permeability leading to malfunction and insufficient blood supply of inner tumor areas (Harris 2002; Jain 2005). If hypoxia is followed by re-oxygenation periods, the excess molecular oxygen causes the generation of reactive oxygen species (ROS) affecting DNA, proteins, and lipids. Repeated hypoxia/regeneration cycles represent selective pressures for tumor cell subpopulations that can foster the invasive and metastatic behavior of a tumor to escape the unfavorable conditions in their environment (Sullivan and Graham 2007). Therefore, hypoxia is a prognostic factor for worse outcome of tumor diseases (Brizel et al. 1996; Brizel et al. 1997; Hockel et al. 1996; Movsas et al. 2000). During radiation therapy, oxygen is required for cellular damage due to ROS formation. Hence,

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hypoxic tumor areas resist radiotherapy (Moeller et al. 2007). Several chemotherapeutics, e.g., doxorubicin, cisplatin, melphalan, 5-fluorouracil, cytarabin, also need oxygen for their action (Pham and Hedley 2001; Pelicano et al. 2004; Hwang et al. 2007) leading to drug resistance in hypoxic tumors. The molecular signaling routes of oxygen sensing are still incompletely understood, but include the MAPK- and PI3K signal transduction pathways as well as transcription factors such as HIF-1, AP-1, NF-κB, p53, Sp1, and CREB (Harris et al. 2002; Semenza 2003; Pelicano et al. 2004; Pennington et al. 2005; Sullivan and Graham 2007), all of which are involved in the response of tumor cells to radio- and chemotherapy. As the activity of NOS is oxygen dependent, NO production is reduced under hypoxic conditions (Whorton et al. 1997; McCormick et al. 2000). Inhibition of NOS activity and NO signaling induces drug resistance (Matthews et al. 2001; Frederiksen et al. 2007). Hence, hypoxia, oxidative stress, and impaired NO signaling are different sides of the same coin contributing to treatment refractoriness. This represents the rational basis for the concept that increasing NO levels by pharmacological interventions should improve the response of tumor cells to radioand chemotherapy. As a consequence, NO-mimetic agents such as glyceryl trinitrate (GTN), diethylenetriamine NO (DETA/NO), and isosorbide dinitrate (ISDN) have been investigated for their effects on drug resistance. Indeed, they inhibit hypoxiainduced resistance to doxorubicin, paclitaxel, and 5-fluorouracil (Matthews et al. 2001; Frederiksen et al. 2003, 2007). This is observable not only in monolayer cell cultures but also in cells grown as spheroids, which reflect much better the in vivo situation (Santini and Rainaldi 1999; Muir et al. 2006). Furthermore, drug-resistant cell lines selected by continued exposure to anticancer agents can also be modulated by augmentation of NO levels as shown for NCX-4016 (an NO-releasing derivative of aspirin) in cisplatin-resistant tumor cells (Bratasz et al. 2006). NMO (3,3-bis(nitroxymethyl)oxetane) sensitizes tumors in vivo to cisplatin, doxorubicin, and cyclophosphamide (Konovalova et al. 2003).

Molecular Mechanisms of Modulation of Drug Resistance by NO-Mimetic Agents While the complexity of NO signaling is far from being understood, and many controversial data can be found in the literature, there is evidence for NO as major player of modulation of therapy resistance (Fig. 14.1).

Hypoxia-Inducing Factor 1α Hypoxia-inducing factors (HIF) play a predominant role in hypoxia-induced therapy resistance (Yasuda 2008). During hypoxia, HIF-1α protein expression is stabilized leading to HIF-1-mediated gene transcription (Harris 2002; Hofer et al. 2002;

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Fig. 14.1 Molecular mechanisms of nitric oxide and nitric oxide-donating agents

Semenza 2003). HIF-1α stabilization results from insufficient oxygen levels necessary for inactivating the enzyme activity of HIF prolyl hydroxylases. HIF-1α binds to HIF-1β, forms a functional heterodimer, and acts as a transcription factor for downstream genes. Alternatively, HIF-1α can also bind to HIF-2α. While HIF-1α is oxygen-regulated, HIF-1β is constitutively expressed (Wang et al. 1995; Elvidge et al. 2006; Semenza 2007). Dimerized HIF-1α/HIF-1β translocates to the nucleus and attaches to CBP (Arany et al. 1996). The HIF-1/CBP complex binds to a 47 bp hypoxia response element (HRE) in promoter regions necessary for activation of transcription of target genes (Melillo 2007). HIF-regulated genes with HREs are the multidrug resistance-conferring transporter genes ABCB1/MDR1 and ABCG2/BCRP (Zhong et al. 1999; Talks et al. 2000), the genes coding for erythropoietin, VEGF, and glycolytic enzymes, all of which support adaptation of cancer cells to hypoxic conditions (Semenza and Wang 1992; Semenza et al. 1994; Forsythe et al. 1996). The pro-apoptotic BID gene is down-regulated by HIF-binding to an HRE in the BID promoter suppressing apoptosis and, hence, inducing drug resistance (Erler et al. 2004). Silencing HIF-1α by RNA interference increases sensitivity of hypoxic tumor cells to chemotherapy providing evidence for a causative role of HIF for drug resistance (Yasuda et al. 2006a). Hence, HIF-1α represents an attractive target protein for the development of small molecule inhibitors (Brown et al. 2006). NO-mimetic drugs such as sodium nitroprusside (SNP), SIN-1, and Snitrosoglutathione (GSNO) inhibit HIF-1-mediated transcription in hypoxic cells (Sogawa et al. 1998; Huang et al. 1999; Wang et al. 2002) due to protein destabilization (Wang et al. 2002; Callapina et al. 2005).

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HIF-Dependent Genes (P-gp, VEGF) ABC Transporters ATP-binding cassette (ABC) transporters such as ABCB1/MDR1/P-gp, ABCC1/MRP1, or ABCG2/BCRP confer multidrug resistance toward a wide rage of anticancer drugs (anthracyclines, taxanes, Vinca alkaloids, epipodophyllotoxins, camptothecins, methotrexate, cisplatin, etc.). They act as ATP-consuming drug efflux pumps that extrude drug molecules out of the cell (Efferth 2001; Gillet et al. 2007). Over-expression of ABC transporters has been shown to be associated with worse prognosis for response of tumors toward chemotherapy and survival of patients (Schaich et al. 2005). The promoters of both ABCB1/MDR1 and ABCG2/BCRP contain HREs and are under the transcriptional regulation of HIF-1 under hypoxic conditions (Comerford et al. 2002; Krishnamurthy et al. 2004). Treatment of colon cancer cell lines over-expressing ABCB1/MDR1/P-gp and another transporter, ABCC3/MRP3, with atorvastatin increases NOS activity resulting in increased cellular doxorubicin accumulation, decreased numbers of functional transporters, and increased cytotoxicity (Riganti et al. 2005). NO antagonists and NO scavengers reverse this effect, whereas NO-mimicking agents such as Snitroso-N-acetyl-d,l-penicillamine (SNAP), GSNO, or SNP show the same effect as atorvastatin. Furthermore, the nitrotyrosine residues of the ABCC3/MRP3 protein are reduced. Comparable results were observed with mevastatin, simvastatin, or SNAP in mesothelioma cells (Riganti et al. 2006) Vascular Endothelial Growth Factor (VEGF) VEGF is released from hypoxic tumor cells and induce angiogenesis and vascular permeability (Epstein 2007). HIF-1 and HIF-2 regulate VEGF expression (Forsythe et al. 1996; Shinojima et al. 2007). On the other hand, VEGF induces HIF-1α and represses apoptosis leading to survival of cancer cells under hypoxic conditions (Calvani et al. 2008). This signaling route probably also contributes to drug resistance and metastasis, which are conferred by VEGF. Anti-VEGF treatment by antibodies or small molecule inhibitors, therefore, increases sensitivity toward anticancer drugs (Kerbel 2006; Grothey et al. 2008).

Angiogenesis and Blood Flow Inner tumor areas are not only hypoxic due to an undersupply by blood vessels. Neo-angiogenesis in tumors frequently is also associated with malformed, tortuous, and leaky vessel formation (Harris 2002; Jain 2005). Malfunctioning blood flow affects the success of chemotherapy, as sufficient drug concentrations cannot reach the tumor tissue (Minchinton and Tannock 2006). Low oxygenation in tumors is accompanied by decreased NO production (Fukumura and Jain 1998). Indeed, administration of No-mimeticsubstances such as spermine NO (SNO), ISDN, GTN, or SNP dilates blood vessels and increases perfusion and oxygenation (Fukumura

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and Jain 1998; Jordan et al. 2000). Sudden oxygenation provokes increased ROS production. Therefore, exogenous supply of NO should improve blood flow and chemosensitivity and support anticancer drugs killing of cancer cells (Pelicano et al. 2004). These observations on blood flow and drug resistance are substantiated by results with radiotherapy. Increasing tumor blood flow by carbogen, pentoxifylline, or calcium channel blockers increases radiosensitivity in mice (Griffin et al. 1999; Muruganandham et al. 1999; Bennewith and Durand 2001). Similarly, NO-donating agents, including isosorbide dinitrate, SNP, or nitroglycerin, increase blood flow in tumors (Kruuv et al. 1967; Jordan et al. 2000). Interestingly, enhanced blood flow is associated with inhibition of the HIF-1-mediated pathway, which leads to downregulation of ABCB1/MDR1/P-gp and VEGF. As a consequence, chemosensitivity of tumors toward taxanes was improved (Yasuda et al. 2006a, b).

Oxidative Stress As outlined above, NO interacts with ROS and ROS-detoxifying mechanisms such as the glutathione redox cycle and antioxidant enzymes. NO reduces superoxide levels by formation of peroxynitrite. In normal tissues and under physiological conditions, peroxynitrite does not exert detrimental effects. Under pathological conditions with unbalanced redox status, peroxynitrite is cytotoxic, although the published data are controversial (Thomas et al. 2006). Increased ROS levels in hypoxic tumors provokes cellular adaptation by upregulation of glutathione (GSH) and antioxidant enzymes. Increased antioxidant enzyme activity and an up-regulated GSH redox system are associated with drug resistance (Volm et al. 1993; Volm et al. 2002; Efferth and Volm 1993, 2005; Bracht et al. 2007). NO-mediated down-regulation of GSH and other defense mechanisms, therefore, increases sensitivity of tumors to chemo- and radiotherapy. This has been shown for 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) (Ji et al. 2002), GSNO (Clancy et al. 1994; Keese et al. 1997), and reactive nitrogen species generated by peroxynitrite and tetranitromethane (Wong et al. 2001), which inhibit glutathione S-transferases. Dinitrosyl iron complexes (DNICs) inhibit various GST isoenzymes as well as glutathione reductase (Keese et al. 1997; de Maria et al. 2003). NCX-4016 depletes GSH levels (Bratasz et al. 2006). The relevance of these in vitro data needs to be corroborated in animal experiments and in the clinical setting.

DNA Damage and Repair ROS generated by hypoxia/re-oxygenation cycles induce oxidative DNA damage (Konovalova et al. 2003; Bindra et al. 2007; Gajewski et al. 2007) that lead to intrastrand cross-links or DNA–protein cross-links. If oxidative damage is not prevented by GSH and antioxidant enzymes, specific DNA repair enzymes may be activated.

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Oxidative damage is mainly repaired by the base excision repair (BER) pathway. Oxidative damage leading to double-strand breaks may be repaired by enzymes of the homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. Data in the literature are rather sparse and inconclusive pointing to the complexity of hypoxia and DNA repair. Hypoxia in mouse fibroblasts is associated with decreased repair of an UV-damaged reporter plasmid (Yuan et al. 2000). In contrast, HIF-1α induces improved repair of reporter constructs in mouse embryonic fibroblasts leading to resistance toward carboplatin, etoposide, and ionizing radiation (Unruh et al. 2003). Hypoxia increased the activity of the APE/Ref-1/HAP1 protein, whose expression is increased in repair of apurinic/apyrimidinic sides of DNA, whereas inhibition of this enzyme sensitizes cervical carcinoma cells toward alkylating agents and oxidative stressors (Walker et al. 1994). Furthermore, the ATM/ATR damage response pathway is activated by hypoxia/re-oxygenation (Bindra et al. 2007), while chronic hypoxia suppresses DNA mismatch repair and HR (Kondo et al. 2001; Rodríguez-Jiménez et al. 2008). NO donors modulate DNA repair and reverse drug resistance. DEA/NO inhibits the repair of BCNU-induced O6 chloroethyl guanine DNA lesions by MGMT (Laval and Wink 1994). Thereby, cellular BCNU sensitivity is enhanced. DEA/NO also blocks other enzymes involved in DNA repair, e.g., PARP, DNA ligase, or Fapy-DNA-glycosylase (Wink and Laval 1994; Graziewicz et al. 1996; Sidorkina et al. 2003).

The Tumor Suppressor p53 Mutations in the tumor suppressor gene, TP53, do not only play a role for carcinogenesis, they also cause drug resistance (Lowe et al. 1993) and radio-resistance (Lee and Bernstein 1993). Moreover, mutant p53 is tightly related to hypoxia. Low oxygen induces apoptosis in minimally transformed mouse embryo fibroblasts depending on the mutational status of TP53 (Gräeber et al. 1996). If these cells are grown as solid tumors in mice, apoptosis occurs in hypoxic regions derived from p53 wild-type mice. In tumors derived from p53 knock-out cells much less apoptosis and no co-localization with hypoxia are observed. This implicates that hypoxia selects for cell populations carrying mutant p53. Hence, hypoxia predisposes tumors to a more malignant phenotype and to drug- and radioresistance. This is supported by the observation that mutant, but not wild-type p53, induces VEGF expression (Yuan et al. 2002), which fosters tumor progression and distant metastasis by angiogenesis (Epstein 2007). Hypoxia-associated p53 mutations in solid tumors (Fels and Koumenis 2005) are important factors for worse treatment outcome and poor prognosis. Interestingly, NO activates wild-type p53 by peroxynitrite-mediated DNA damage (Schneiderhan et al. 2003). The NO donors, spermine NO and nitroglycerin activate and phosphorylate p53 increasing tumor sensitivity to cisplatin or vinorelbine in vitro and in vivo (Lowe et al. 1993). Importantly, a combination regimen of nitroglycerin, cisplatin, and vinorelbine improves response rate and delays time to

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relapse in lung cancer patients (Yasuda et al. 2006a, b). NO can not only be provided by chemical NO donors. Gene therapy with an iNOS adenoviral vector also activates p53 by phosphorylation in colorectal xenograft tumors (Cook et al. 2004). NO exerts its resistance-modulating effects on wild-type, but not on mutant p53. In isogenic colon carcinoma p53 knock-out cells, the effect of NO on ionizing radiation is much less than in corresponding p53 wild-type cells (Cook et al. 2004). This indicates that the resistance-reversing effect in hypoxic tumors, which have already acquired a high portion of p53 mutated cells, is limited.

NF-κB Among a plethora of other functions, NF-κB acts in an antiapoptotic manner and, therefore, confers drug- and radio-resistance (Ahmed and Li 2008; Nakanishi and Toi 2005). NF-κB is a complex consisting of p50, p65, and I-κBα (Aggarwal 2003). Phosphorylation and degradation of the inhibitory subunit, I-κBα, activate the complex, which then translocates from the cytosol into the nucleus (Kumar et al. 2004). As transcription factor, NF-κB induces the expression of many genes, including antiapoptotic ones, such as survivin, TRAF, cFLIP, Bcl-2, and Bcl-xL. Since NO donors inhibit p50 phosphorylation by S-nitrosylation (Marshall and Stamler 2001), NO overcomes NF-κB-mediated therapy resistance. The NO-donating agents, SNAP and diethylenetriamine NONOate (DETA NONOate), mimic the effect of interferon-γ by up-regulation of the death receptor, FAS, and sensitization of tumor cells to the extrinsic, receptor-driven pathway of apoptosis (Garban and Bonavida 1999). This effect is mediated by inhibiting NF-κB and by repression of the transcription factor, YY1 (Garban and Bonavida 2001; Hongo et al. 2005), the former inactivating Fas expression, the latter one activating it. Comparable results have been found for other death receptors (TNF-R, TRAIL) and their ligands (TNF-α, DR4, DR5) (Garban and Bonavida 2001; HuertaYepez et al. 2004). Inhibiting the molecular brakes for apoptosis, NF-κB and YY1, sensitizes tumor cells to cisplatin (Jazirehi and Bonavida 2005). Interestingly, NO donors do not only sensitize tumor cells to chemotherapy. They do also affect the metastatic process (Bonavida et al. 2008). Inhibition of NF-κB by DETA NONOate leads to down-regulation of Snail and RKIP. Snail induces the epithelial–mesenchymal transition (Batlle et al. 2000; Cano et al. 2000). Changes in the expression of adhesion molecules represent one precondition for dissociation of single cells from the bulk tumor, invasion into the surrounding tissue, and metastasizing (Leber and Efferth 2009). RKIP is a putative metastatic tumor suppressor, which is down-regulated in metastatic tumor disease (Granovsky and Rosner 2008).

Conclusion and Perspective Interfering with NO-related pathways represents an attractive and appealing novel concept for the improvement of cancer treatment. In all likelihood, every novel

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anticancer agents will clinically be applied in combination with other established treatment modalities. Therefore, exploring the full range of chemo- and radiosensitizing effects of NO donors is of utmost importance. Apart from the resistance-modulating effects, it must not be overseen that NO donors exert antiproliferative and cytotoxic activity toward cancer cells if applied as single drugs (Coulter et al. 2008; Huerta et al. 2008; Wink et al. 2008). This is a desirable effect which supports tumor cells eradication. Drugs sharing both features can be looked upon as “two-in-one” drugs. On the other hand, this poses the question of unwanted side effects of manipulation of NO levels on normal tissues (Di Napoli and Papa 2003; Ng et al. 2007).

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Chapter 15

Breaking Resistance: Role of Nitric Oxide in the Sensitization of Cancer Cells to Chemo- and immunotherapy Hermes J. Garbán

Abstract Nitric oxide (NO) has recently joined the clinical area of cancer therapy. There has been an increasing amount of preclinical data supporting the specific role of NO in the sensitization of resistant cancerous cells to radio-, chemo-, and immunotherapy. Hypoxia has been long related to the survival and resistance of tumor cells to either the host immune response or therapeutic interventions. Although some times controversial, NO has been demonstrated to improve the levels of oxygenation in the tumor tissue by directly increasing blood flow (by “normalization” of the tumor vasculature) and by the regulation of many factors that will counteract the hypoxic environment of the tumor. By increasing the levels of oxygen in the malignant tissue, NO will increase the effectiveness of anticancer chemotherapeutic drugs, thereby decreasing their potential secondary effects. In addition, NO can also act in the modulation of the immune system on the enhancement of tumor-specific immune response and also in the sensitization of resistant tumor cells to immune-related effector mechanisms by regulating the expression of apoptosis-related genes including those belonging to the TNF receptor family. There are promising clinical data pointing toward the potential use of NO-releasing compounds for the sensitization of resistant tumor cells to conventional chemo- and immunotherapy. Herein, the role of NO will be discussed in the sensitization of tumor cells to radio-, chemo-, and immunotherapy in light of the most recent reports included in this volume by Thomas Efferth, Peter Siesjö, and Marion Cortier and collaborators. Keywords Chemosensitization · Radiosensitization · Immunosensitization · Apoptosis · Therapy · Synergy · Immunomodulation · Reactive oxygen species (ROS) · Reactive nitrogen species (RNS) · Hypoxia · Chemotherapy · Radiotherapy · Immunotherapy · Clinical Trials

H.J. Garbán (B) Division of Dermatology, Department of Medicine, LA BioMed Research Institute at HarborUCLA Medical Center, “David Geffen” School of Medicine at the University of California, 1124 West Carson St., Torrance, CA 90502, USA e-mail: [email protected], [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_15, C Springer Science+Business Media, LLC 2010

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Introduction One of the most recurrent problems in the treatment and management of malignancies is the prevalence and/or appearance of resistance mechanisms in the cancerous cells. These can be intrinsically established or acquired by genetic or epigenetic changes during the development of the tumor cells. Recently, many research and clinical efforts have been focused on the understanding of the nature of the mechanisms involved in the resistance of tumor cells to either the patient’s immune system or chemo-, radio-, and immunotherapeutic approaches. An emerging concept that could reverse the status of resistance of tumor cells to multiple therapeutic interventions is the use of nitric oxide (NO). The specific role of nitric oxide signaling in tumor biology and cancer has remained elusive and many controversial data found in the literature contribute to the difficulty in delineating the specific role of NO as an anticancer therapy. A broad spectrum of activities has been assigned to either the physiology or the pathophysiology of NO in tumor cells (for a review, see Moncada et al. 1991). The first distinction we can make is related to the amount and sources of NO being generated. Low output of NO has been correlated with increased blood flow and new blood vessels (angiogenesis) feeding the tumor area (Jenkins et al. 1995). In addition, the generation of NO by tumor cells may inhibit the activation and proliferation or increase apoptosis of surrounding lymphocytes that can account for the immune suppression observed that accompanies tumor growth. Furthermore, high intratumoral output of NO could inhibit the activation of caspases and therefore antagonizes the pro-apoptotic signals (Liu et al. 2000; Liu and Stamler 1999). However, the opposite effect also has been observed in many other systems whereby the generation of high output of NO, either by iNOS induction or by the use of NO donors, inhibits tumor growth and metastasis (Garban and Bonavida 2001a; Hibbs et al. 1987; Shi et al. 1997; Garban and Bonavida 1999). Therefore, the final outcome of NO-mediated signaling will be determined by many factors including the local concentration and sources of NO in the tissue and the presence of reactive molecules that might redirect the redox status in the cell with the potential to synergize with other anticancer therapeutic modalities. Herein, all these elements will be discussed that relate to the role of NO in the sensitization of tumor cells to radio-, chemo-, and immunotherapy in light of the most recent reports included in this volume by Thomas Efferth, Peter Siesjö, and Marion Cortier and collaborators.

Basic Concepts About NO Biology Nitric oxide (NO) is a diatomic molecule that plays important roles as the smallest pleiotropic signaling messenger in mammalian cells (Nathan 1992). NO has an unpaired electron; it rapidly reacts with other molecules and easily diffuses through the plasma membranes to reach target proteins within the cell due to its lipophilic

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nature. NO is biologically synthesized by NO synthases (NOS). NOS catalyze the oxidation of L-arginine resulting in the formation of NO and L-citrulline. NO is produced by three different NOS, two of which are generally constitutively expressed, primarily in neurons (nNOS or Type I) and endothelial cells (eNOS or Type III), respectively (Knowles and Moncada 1994; Mayer and Hemmens 1997; Nathan and Xie 1994). An inducible isoform (iNOS or Type II) can be upregulated considerably in immune cells and many other tissues (Schmidt and Walter 1994; Xie and Nathan 1994). It has been shown that IFN-γ alone or in combination with TNF-α, interleukin 1β (IL-1β), and bacterial lipopolysaccharide (LPS) can induce the expression of iNOS in a wide variety of tissue organs and in some tumor cell lines (Geller et al. 1993; Sherman et al. 1993). The inducible type of NO synthase (iNOS) is considered to be a central protein in the regulation of the immune response against tumor cells (Hokari et al. 1994; Langrehr et al. 1993).

NO and Radiotherapy The therapeutic effect of ionizing radiations is not only achieved by the specific damage inflicted to the cellular DNA either by direct or indirect generation of reactive oxygen species (ROS). There are many other mechanisms involved including the regulation of critical gene products that would determine the final outcome in the process of DNA repair or induction of apoptosis. Hypoxia has been long related to the survival and resistance of tumor cells to either the host immune response or therapeutic interventions. As highlighted by Thomas Efferth in his chapter and the studies cited therein, repeated hypoxia/regeneration cycles represent selective pressures for tumor cell subpopulations that can foster the invasive and metastatic behavior of a tumor to escape the unfavorable conditions in their environment. Therefore, hypoxia is a prognostic factor for worse outcome of tumor diseases. During radiation therapy, oxygen is required for cellular damage due to ROS formation. Hence, hypoxic tumor areas resist radiotherapy. Marion Cortier and collaborators summarized in their chapter a set of observations that lead to the definition of NO as radiosensitizer molecule in bacteria and mammalian tumor cells. The effect of NO in the sensitization of tumor cell to radiotherapy has been demonstrated in preclinical and clinical studies. These effects can be either direct, by the cellular induction or generation of NO by the target tissue (NOS-mediated induction or activation) as revealed by Peter Siesjö in his chapter, or indirect by the use of NO-releasing compounds such as NO donors extensively discussed in these chapters. Overall, the action of NO in the sensitization of resistant tumor cells to radiotherapy seems to be linked to the “normalization” of the tumor vasculature and the re-oxygenation of hypoxic areas of the tumor and increased better access of immune-related cells and pro-apoptotic stimuli to the tumor and its intrinsic sensitization as well. The specific role of NO in the regulation of critical factors in the tumor cells and the tumor microenvironment such as HIF-1, VEGF, TGF-ß,

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apoptosis-related genes that might contribute to the synergistic effect observed in experimental radiotherapy has been discussed. From a clinical perspective, the data connecting NO and radiotherapy are controversial and merely phenomenological confronting correlations with iNOS expression and prognostic and clinical outcomes without considering timing of expression and/or actual generation of NO by NOS (Chen et al. 2005; Oka et al. 2003; Shao et al. 2008). Despite the general trend in the observations supporting the sensitizing role of NO to radiotherapy, there are plenty of unresolved issues that require the attention of more basic and detail studies to elucidate the mechanisms of action of either endogenously generated NO or therapeutically supplied NO in combination with radiotherapy in order to control cancer cells in a clinical setting.

NO and Chemotherapy The effect of most of the chemotherapeutic modalities against solid tumors relies on the availability of oxygen by the malignant tissue. Here again, as mentioned above, hypoxia in tumor tissues might represent a selection pressure that would promote the development of resistant cancerous cells to chemotherapy. NO has been recently implicated in the sensitization of resistant tumor cells to chemotherapy. In addition, NO has been demonstrated to modify and improve the blood flow in the tumor site promoting a better distribution of oxygen, immune-related cells, and chemotherapeutic agents. Further, endogenous or exogenous sources of NO have been associated with the modification of a milieu of apoptosis-related genes that might affect the sensitivity of tumor cells to chemotherapy (Olson and Garban 2008). The effect of chemotherapy in malignant cells is not only restricted to the direct cytotoxic effect on tumor cells. As discussed by Peter Siesjö in his chapter, stromal and endothelial tissues are also targeted by chemotherapeutic agents. This can influence the reduction of tumor cells through a bystander effect. Induction and activation of NOS and its subsequent generation of NO in the tumor site by macrophages and other cell types have been correlated with increase in effectiveness of the chemotherapeutic action of drugs such as taxol, cyclophosphamide, cisplatin, doxorubicin, and others. However, the exact mechanisms by which NO synergize with these chemotherapeutic agents remain elusive. Several studies support the sensitizing role of NO to chemotherapy. As delineated by Marion Cortier and collaborators in their chapter, there are multiple evidences supporting the anti-tumor effect of cancer chemotherapeutic agents that are enhanced by the use of NO-releasing compounds. They examined this premise from two perspectives: first, from the experimental point of view (preclinical) and the use of “clean” NO donors such as DETA/NO, DEA/NO, PAPA/NO and the modification of sensitivity of different cancer cells to chemotherapeutic drugs such as cisplatin, doxorubicin, melphalan; second, the use of “dirty” NO donors but with

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clinical and pharmacological relevance (preclinical and clinical) such as sodium nitroprusside (SNP), nitroglycerin (GTN), diazeniumdiolates in combination with chemotherapeutic regimes in vivo such as the use of vinorelbine and/or cisplatin in combination or each drug alone. Noteworthy, a randomized phase II clinical trial has been conducted to test the efficacy of GTN plus vinorelbine and cisplatin and vinorelbine and cisplatin alone in previously untreated stage IIIB/IV non-small-cell lung cancer (NSCLC) (Yasuda et al. 2006). In this study it is concluded that the use of GTN combined with vinorelbine and cisplatin may improve overall response and time to progression (TTP) in patients with stage IIIB/IV NSCLC. They discussed the possible effect of NO-donating drugs such as GTN in reducing the resistance of tumor cells to chemotherapy via improvement of hypoxic conditions, reduced HIF-1 stabilization, direct effect of NO cancer cells, increase in p53 protein and via an increase in drug delivery in tumor tissue. All these elements were reviewed in detail by Thomas Efferth in his chapter. In summary, we can say that despite many conflicting data presented in the literature, there is sufficient evidence to support the role of NO in the sensitization of tumor cells to chemotherapy. Its effect might vary from the direct cytotoxic activity of NO and RNS generated to the subtle modulation of apoptosis-related and resistance genes and the modification of drug accessibility, regulation of hypoxia conditions, and “normalization” of tumor vasculature. There are increasing preclinical and clinical data to support further clinical trials using NO-releasing compounds in combination with established chemotherapeutic drugs.

NO and Immunotherapy One of the most productive and dynamic areas of research in the field of NO biology and cancer has been the understanding of the role of NO in the sensitization of tumor cells to immuno-related stimuli. An early seminal observation reported by Hibbs et al. in 1987, where cytokine-activated macrophages were able to kill tumor cells via NO generation, started a series of concurrent studies supporting the key role of NO in the direct and indirect elimination of tumor cells. As stated by Peter Siesjö in his chapter, NO plays an important and diverse role during the regulation of the immune responses. This diversity is partly dependent on the source and levels not only of NO generated but also of cellular targets and timing of release. In concordance with the previous two sections, the specific role of NO in the immune responses and immunotherapy does not escape from controversy. There are confounding data that can mislead the possible role of NO in this system against cancerous cells. On the one hand, we have the possible role of NO-inducing (intrinsic or exogenously generated) suppression of the immune system by increasing the killing of tumor reactive T cells, activating suppressive mechanisms, or inducing the proliferation of T regulatory cells (Brito et al. 1999; Chen et al. 2006; Niedbala et al. 2007). On the other hand, we have the direct generation of NO and RNS by activated

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macrophages and the sensitization of tumor cells to TNF receptor family-mediated apoptosis by the NO-dependent regulation of apoptosis-related genes (including the expression of the receptors per se) (Bonavida et al. 2008; Jeannin et al. 2008; Olson and Garban 2008). Endogenous NO generation by cytokine induction in immunerelated cells and exogenous NO, provided locally by NO-releasing compounds, have been demonstrated to be essential for the priming of the immune response (T cell priming) against specific antigens and some tumor-associated antigens (TAAs) (Koncz et al. 2007; Niedbala et al. 1999). As mentioned above, one of the best understood mechanism by which NO sensitizes tumor cells to immune-related stimuli is the role NO has in the regulation of the expression of apoptosis-related genes including the majority of the TNF receptor family members (Fas/APO-1, TNFRs, and TRAILR/DR5-DR4). As discussed by Marion Cortier and collaborators and expanded by Thomas Efferth in their respective chapters, NO can influence the sensitivity of tumor cells to immunerelated apoptotic stimuli. The NO-donating agents, SNAP and DETA/NO, mimic the effect of interferon gamma (IFN-γ) by upregulation of the death receptor, FAS, and subsequent sensitization of tumor cells to the extrinsic, receptor-driven pathway of apoptosis (Garban and Bonavida 1999). This effect is mediated by inhibiting NF-κB and by repression of the transcription factor, YY1 (Garban and Bonavida 2001b; Hongo et al. 2005). Comparable results have been found for other death receptors (TNFR, TRAIL) and their ligands (TNF-α, DR4, DR5) (Garban and Bonavida 2001a; Huerta-Yepez et al. 2004). Inhibiting the molecular brakes for apoptosis, NF-κB and YY1, sensitizes tumor cells to cisplatin (Jazirehi and Bonavida 2005). Interestingly, NO donors do not only sensitize tumor cells to chemotherapy. They do also affect the metastatic process (Bonavida et al. 2008). In summary, NO can exert its effect on the immune system and on the sensitization of tumor cells to immunotherapeutic approaches in a direct or indirect way. NO might contribute to the direct killing of tumor cells as part of the endogenous generation of NO by macrophages or other immune-related cells. In addition, NO has been demonstrated to control the sensitivity of tumor cells by the regulation of apoptosisrelated genes that can synergize or potentiate the effect of specific tumor-targeted immunotherapy.

Concluding Remarks Although the apparent controversy due to conflicting data, the specific role of NO in the sensitization of tumor cells to radio-, chemo-, and immunotherapy seems to be gaining more support from the scientific community involved in the development of a better understanding of the biology of NO in cancer. Thus, the biological activity of NO or related molecules depends on the source of NO, local concentration, and the presence of other reactive molecules that can direct the function of NO. NO can act directly as a cytotoxic element triggered by ionizing radiation, anticancer chemotherapeutic drugs, and immunological stimuli. In addition, NO has

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been demonstrated to synergize with the cytotoxic action of ionizing radiation, anticancer chemotherapeutic drugs, and immunological stimuli. These two areas of action of NO frequently overlap. Hypoxia has been identified as one of the most critical element in the development of resistance in tumor cells. NO has been shown to, somehow, restore or improve the levels of hypoxia in the tumor tissue by directly increasing blood flow (by “normalization” of the tumor vasculature) and by the regulation of many factors that will counteract the hypoxic environment of the tumor. By increasing the levels of oxygenation in the malignant tissue, NO will increase the effectiveness of anticancer chemotherapeutic drugs and decrease their potential secondary effects. There are promising clinical data pointing toward the potential use of NOreleasing compounds for the sensitization of resistant tumor cells to conventional chemotherapy. NO can also act in the modulation of the immune system on the enhancement of tumor-specific immune responses and also in the sensitization of resistant tumor cells to immune-related effector mechanisms by regulating the expression of apoptosis-related genes including those bellowing to the TNF receptor family. Overall, NO represents a promising intervention for the radio-, chemo-, and immunosensitization of tumor cells. However, a better understanding of the molecular mechanisms involved in the NO signaling in cancer will help in designing more effective NO-based therapeutic strategies. Acknowledgments I am indebted to Dr. Bejamin Bonavida and Dr. Louis J. Ignarro for the academic and research support and for the extensive opportunity to play in the NO field. I would also like to acknowledge the laboratory and administrative assistance of Mr. Samuel Y. Olson, and Dr. Diana C. Márquez for her unconditional support and critical revision of this chapter.

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Part VI

Prognostic Significance of NOS and NO

Chapter 16

Prognostic Significance of iNOS in Human Melanoma Suhendan Ekmekcioglu and Elizabeth A. Grimm

Abstract Melanoma is the leading cause of death from skin cancer, particularly in young adults. Precise staging of melanoma progression is highly critical as even relatively small melanomas can metastasize and it is extremely difficult to treat once the tumor has metastasized beyond the locoregional area. Clinical and histological variables such as primary tumor invasion, ulceration, and lymph node status might be unsuccessful to identify early stage that will eventually progress to further stages of disease. Therefore, there is an urgent need to develop biomarkers that might help to identify patients with early-stage melanoma who are likely to develop advanced disease and would benefit from additional therapies. In recent years, molecular biology and the identification of molecular markers in melanoma have been a major focus for cancer researchers. Thus, for human melanoma, a growing number of molecular markers, including transcription factors, oncogene products, and loss of tumor suppressor molecules, have been proposed. Most human melanoma tumor cells are known to express the enzyme, inducible nitric oxide synthase (iNOS), which is responsible for cytokine-induced nitric oxide (NO) production in macrophages during immune responses. This constitutive expression of iNOS in many patients’ tumor cells, as well as its strong association with poor patient survival, has led to the consideration of iNOS as a molecular marker of poor prognosis, as well as a possible target for therapy. The mechanisms underlying the expression of iNOS expression and the molecular pathways affected by NO are currently active areas of research. Keywords Nitric Oxide · iNOS · Melanoma · Prognostic Markers · Inflammation

S. Ekmekcioglu (B) Department of Experimental Therapeutics, Unit 362, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA e-mail: [email protected]

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_16, C Springer Science+Business Media, LLC 2010

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Introduction Our understanding of prognostic factors in melanoma has evolved significantly over the last decade. Despite the fact that known clinicopathological factors such as tumor thickness, lymph node involvement, distant metastasis, ulceration, mitotic rate, and age are the most reliable prognostic factors currently available, none can satisfactorily predict the long-term survival outcome on a single patient basis. As the malignant phenotype is sustained by the complex molecular arrangements of tumor cells, it is logical to expect that advancements in molecular pathology will provide clinicians with novel and more effective prognostic tools. In recent years, molecular biology and the identification of molecular markers in melanoma have been a major focus for cancer researchers. Thus, for human melanoma, a growing number of molecular markers, including transcription factors, oncogene products, and loss of tumor suppressor molecules, have been proposed. These include markers such as Akt, MITF, PTEN, Bcl-2, iNOS, for all of which a prognostic value has been claimed (see Cerroni et al. 1995; Mooy et al. 1995; Grover and Wilson 1996; Ekmekcioglu et al. 2000, 2006; Salti et al. 2000; Vlaykova et al. 2002; Miller et al. 2004; Dai et al. 2005; Mikhail et al. 2005; Bosserhoff 2006; Fecker et al. 2006; Stark and Hayward 2007; Ugurel et al. 2007). The discovery of potential prognosis indicators has been recently accelerated by the implementation of high-throughput technologies, such as gene microarray. However, the statistical significance and validation of molecular markers, compared to traditional clinicopathological factors, have not been demonstrated in all series, which are often small and heterogeneous. More importantly, these results are rarely confirmed in different series or outside cohorts which are necessary for adequate validation. Besides the potential prognostic value of these molecular factors, their value for being predictive of response to therapy is another active and exciting field of research. The predictive markers would be useful for selecting patients who most benefit from a given anticancer agent, which ultimately would lead to increase the therapeutic index of available treatments. Prognostic markers predict the natural course of an individual patient with different risks of outcome and help clinicians to decide which patient to treat and how aggressive the treatment will be. The current american joint committee on cancer (AJCC) staging system for melanoma incorporates only some of the molecular prognostic factors of proven significance (i.e., LDH) but considerable efforts have been made in exploring molecular prognostic markers of malignant melanoma to detect relevant gene expression, serum markers, and genetic/molecular markers. However, this field of research is still in its infancy for melanoma. Our future efforts should be well balanced in both the prognostic molecular marker discoveries and their predictive use in the clinic. Most human melanoma tumors cells are known to express the enzyme, inducible nitric oxide synthase (iNOS), which is responsible for cytokine-induced nitric oxide (NO) production in macrophages during immune responses (Grimm et al. 2008). Molecular analysis supports the hypothesis that iNOS-produced NO in melanoma can be responsible for driving proliferation as well as regulating resistance to apoptosis; this hypothesis is supported by results of experiments in which

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chemical quenching of NO resulted in G2 growth arrest followed by a gain of cisplatin-induced apoptosis (Tang and Grimm 2004). An increasing series of publications support the hypothesis that NO is the product of aberrant constitutive expression of iNOS as detected by immunohistochemistry (IHC) (Ekmekcioglu et al. 2000, 2006). This constitutive expression of iNOS in many patients’ tumor cells and its strong association with poor patient survival have led to the consideration of iNOS as a molecular marker of poor prognosis as well as a possible target for therapy. The expression of iNOS in patients’ tumors was also found to associate with nitrotyrosine, COX-2, pSTAT-3, and arginase. The mechanisms underlying the expression of iNOS expression and the molecular pathways affected by NO are currently active areas of research. Interestingly, examples of key molecules involved in melanoma growth promotion and apoptosis resistance that are regulated by NO-mediated post-translational modifications (e.g., p53 and N-ras) are currently being pursued. In addition, efforts to identify specific nitrated and S-nitrosated proteins in melanoma as a hypothesis-generating approach for future targeting of growth and survival pathways are in progress.

iNOS Expression in Primary Melanomas Published data on iNOS expression in primary cutaneous melanomas revealed that the expression of this protein can exist at very early stages of malignancy (Massi et al. 2001). In this study, Massi et al. showed a high iNOS content in metastatic melanomas, whereas benign melanocytic nevi were iNOS negative, with an upregulation of iNOS expression during melanoma progression (in situ, invasive, metastastic). They have specifically shown that melanocytic nevi never expressed iNOS. Their finding implied important practical applications, since iNOS, in this setting, could be potentially employed as an additional IHC marker to discriminate malignant melanoma from benign melanocytic nevi. Therefore, they concluded that iNOS expression shows parallel characteristics with tumor progression and may play a role in the malignant transformation of melanocytes and in tumor growth. However, a controversial study by Ahmed and Van Den Oord (2000) showed that virtually all nevi express iNOS protein in their cytoplasm but very few expressed nitrotyrosine, indicating either that iNOS in nevi is functionally inactive, or that nevus cells lack reactive oxygen radicals, and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In their melanoma samples, iNOS was most frequently expressed in the radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in rare cases; moreover, in these latest phases of tumor progression, iNOS staining was weak and focal. As a result, they conclude that iNOS is expressed de novo in most benign pigment cell lesions and the iNOSgenerated NO appears to play an important part in the early steps of invasion, where it may stimulate neo-angiogenesis and may be a prerequisite for further tumor progression. The overall conclusion from these few studies suggest that iNOS

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expression exist in the benign phase of this disease but most likely it is inactive. However, its expression in early phases of melanoma progression is highly controversial and its prognostic role in primary melanomas has not yet been explored. Our study employing primary melanomas for iNOS expression (Fig. 16.1) and its prognostic significance is currently in progress. An interim statistical analysis was performed in only a part of a targeted group of primary cutaneous tumor samples from patients with newly diagnosed melanoma for their iNOS protein expression immunohistochemistry. For the set used for the interim analysis, we have examined 257 primary cutaneous tumor samples from patients with newly diagnosed melanoma for their iNOS protein expression. The median follow-up duration in these patients is now 1.82 years (range, 0.08–3.87 years), and 12 patients have died of their disease. An additional 34 patients have had a subsequent disease event.

Fig. 16.1 IHC of iNOS in human primary melanoma Table 16.1 Overall survival – hazard ratios for iNOS number (n = 257) iNOS number

n

# Deaths

HR

95% CI

p-value

0 1 2 3

135 69 38 15

4 2 5 1

1.00 0.88 5.26 2.24

(0.16, 4.83) (1.40, 19.82) (0.25, 20.09)

0.89 0.014 0.47

HR: hazard ratio; CI: confidence interval Table 16.2 Disease-free survival – hazard ratios for iNOS number (n = 257) iNOS number

n

# Events

HR

95% CI

p-value

0 1 2 3

135 69 38 15

17 7 8 2

1.00 0.64 1.66 1.06

(0.26, 1.54) (0.72, 3.86) (0.24, 4.59)

0.32 0.24 0.94

HR: hazard ratio; CI: confidence interval

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Thus, at this time, we consider 40 patients (15% of targeted group) to have had disease events. Statistically, significant associations were found between the percentage iNOS-positive cells and intensity of labeling (p < 0.0001). The median disease-free survival (DFS) was 2.80 years (95% CI: not attained) in the high number of iNOS expressing group while the median was not attained in the low number of iNOS nonexpressing group. The median overall survival (OS) duration has yet to be attained; however, univariate analysis of the various subgroups indicates significant differences in that the medium iNOS intensity and percentage positive group resulted in a hazard ratio (HR) more than five times higher than that for the iNOS-negative group (Tables 16.1 and 16.2). Our group’s data are maturing, and clinically relevant events continue to occur.

iNOS Expression in Metastatic Melanomas We have previously reported the iNOS expression in melanoma cells in two different patient settings of metastatic melanoma (Ekmekcioglu et al. 2000, 2006). The data which we have previously published demonstrate the utility of tumor iNOS determination as a marker of poor survival in stage III melanoma. Univariate analysis reveals a strong effect of iNOS expression on the hazard ratio of death from melanoma. Multivariate analysis suggests that iNOS, as a predictor of survival, may be independent of the other known prognostic factors. These data extend our original report of iNOS in melanoma, which included only 20 patients who had then received investigational neoadjuvant therapy prior to surgical excision of tumor, raising the possibility that iNOS expression in the tumor was a result of treatment (Ekmekcioglu et al. 2000). Our later study eliminates this confounding factor and is more relevant to the typical stage III patient for whom tumor samples are normally obtained for diagnostic purposes prior to the administration of any therapy (Ekmekcioglu et al. 2006). Overall, we conclude that a significant association exists between tumor iNOS expression and shortened survival in untreated stage III melanoma patients. Therefore, the ability of iNOS to predict outcomes for these patients may be independent of other known prognostic factors, providing a new molecular marker with significant potential for clinical utility. Subsequent studies by other groups have shown similar characteristics in stage III malignant melanomas. Most recently, Johansson et al. (2008) confirmed that iNOS is an independent prognostic factor for OS in stage III malignant cutaneous melanoma. This study was designed to compare the prognostic value of iNOS to that of COX-2, as well as the presence of activating BRAF/NRAS mutations in metastatic lymph nodes from stage III cutaneous melanoma patients. They report that iNOS expression correlates significantly with the presence of BRAF mutations, and independently and significantly predicts a shortened survival in these patients with a higher odds ratio (OR) than that of COX-2. They have further described that iNOS increased its OR and was always higher than that of COX-2 in the multivariate logistic regression analysis together with stage IIIB/C, ulceration, metastatic lymph nodes, and Breslow tumor thickness. This study suggested that iNOS is an independent and stronger adverse

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prognostic factor for OS than COX-2 in stage III cutaneous melanoma patients. A previous report by our group has also shown the association of pERK and iNOS expression based on the fact that both ERK activation and iNOS expression enhance melanoma tumor survival (Ellerhorst et al. 2006). Thus, we hypothesized that the iNOS gene lies downstream of the p44/42 MAPK pathway in human melanoma, thus connecting activating mutations of NRAS and BRAF with constitutive iNOS expression and aggressive tumor behavior. In this study, we have explored this hypothesis in cultured human melanoma cells and preserved human melanoma tumor samples and validated our hypothesis by demonstrating that melanoma iNOS expression is regulated by the p44/42 MAPK pathway.

Regulatory Effects on Melanoma Progression The production of NO within the tumor environment is believed to promote melanoma growth. Tumor-cell-derived NO has been proposed to be an important mediator of tumor angiogenesis and metastasis formation by directly inducing vessel dilation, promotion of blood flow and vascular permeability, and endothelial cell proliferation through up-regulation of the vascular endothelial growth factor (VEGF) and the basic fibroblastic growth factor (Joshi et al. 1996; Fukumura and Jain 1998). In addition, more recent observations indicate that NO is involved in the regulation of lymphatic vessels permeability and flow (Ohhashi et al. 2005). Most recently, it has been demonstrated that iNOS activity correlated with lymphangiogenesis and spread to lymph nodes in head and neck squamous cell carcinoma and melanoma (Franchi et al. 2006; Massi et al. 2009). Massi et al. (2009) demonstrated a significant direct correlation between iNOS expression in melanocytic tumor cells and the density of lymphatic vessels and the density of peritumoral blood microvessels. Their findings support the notion that iNOS is implicated not only in the blood, but also in the lymphatic vascular neoformation in melanoma. It is possible that in the tumor microenvironment NO stimulates angiogenesis and/or lymphangiogenesis cooperatively with other pro-angiogenic and lymphangiogenic growth factors. Another potential mechanism in which melanoma progression could be affected is the stem cell factor (SCF)-regulated cytokine expression in the tumor environment. In a study by Prignano et al., c-kit-positive metastatic melanoma cells from human melanoma metastases and c-kit-positive human melanoma cell lines showed a definite reduction of cytokines normally up-regulated during melanoma progression after SCF stimulation (Prignano et al. 2006). SCF was also able to maintain all metastatic melanoma cells and melanoma cell lines in a well-differentiated status with an increase in organellogenesis and in particular of melanosomes in various degrees of differentiation. This increase of melanosomes matched an increase of tyrosinase production. SCF enhanced the expression of HLA-DR molecules on the metastatic melanoma cell membranes. It is possible that IFNs-induced HLA-DR overexpression leads indirectly to iNOS expression in melanoma cells. Overall, their data stress the biological activity of SCF as a cytokine which is able to maintain

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metastatic melanoma cells in a well-differentiated status and suggest the involvement of a complex cytokine signaling with possible effects of SCF on melanoma cells. Our group has also shown another cytokine, IL-24 (originally named as MDA7), is responsible for the inhibition of melanoma progression by modulating iNOS expression in melanoma cells (Ekmekcioglu et al. 2003). Our in vitro studies revealed that iNOS expression in melanoma cell lines is lost in a dose-dependent fashion after treatment with an adenoviral vector encoding the mda-7 gene (Ad-mda7) or with rhMDA-7/IL-24 protein, demonstrating that MDA-7/IL-24 downregulates iNOS expression. Furthermore, we demonstrate that the STAT-3modulated expression of IFN regulatory factors 1 and 2 is regulated by MDA7/IL-24, which may alter iNOS gene expression. These studies demonstrate that expression of the MDA-7/IL-24 tumor suppressor can negatively regulate iNOS expression in malignant melanoma cell lines. Thus, we conclude that altering the balance between the tumor-progressive iNOS and tumor-suppressive MDA-7/IL24 may open new avenues to controlling tumor progression in melanoma and potentially other tumor types. Another signaling pathway in melanoma progression which involves iNOS regulation is NF-κB-mediated mitogen-activated protein kinase (MAPK) pathway. Our group has recently published data which suggest that the constitutively activated melanoma MAPK pathway stimulates the activation of NF-κB hetero- and homodimers, which, in turn, drive iNOS expression and support melanoma tumorigenesis (Uffort et al. 2009). The Ras/Raf/MEK/ERK pathway is a major signaling pathway involved in tumor cell growth and survival. As human melanoma is driven by constitutive activation of this pathway, the elucidation of downstream effector molecules is critical to the understanding of melanoma pathobiology. We have originally reported the positive regulation of melanoma iNOS expression by the MAPK pathway and the profoundly negative clinical implications of tumor iNOS expression in this malignancy (Ekmekcioglu et al. 2006; Ellerhorst et al. 2006) and recently showed that NF-κB mediates this regulatory event (Uffort et al. 2009). This study demonstrates that the inhibition of MAPK signaling with the MEK inhibitor U0126 is accompanied by diminished nuclear NF-κB DNA binding. Furthermore, this is the first study to show that NF-κB regulates the expression of iNOS in melanoma cells. Notably, the MAPK/NFκB/iNOS pathway was demonstrated in both BRAF mutant and wild-type cells, suggesting that the source of MAPK activation does not alter these downstream events. Hypoxia-inducible factor-1 (HIF-1) is an oxygen-regulated transcriptional activator that plays a central role in tumor angiogenesis and is also known to increase the expression of several angiogenesis-related genes, including iNOS (Semenza 2003). In response to this signal and in a positive feedback circuit, NO can also activate HIF-1 (Kimura et al. 2000). Furthermore, NO induces HIF-1 expression under nonhypoxic conditions (Kasuno et al. 2004). It has been suggested that NO within the tumor microenvironment might have the same effect as hypoxia in inducing angiogenesis (Semenza 2001). The majority of the studies, however, that investigated the association of HIF-1α and iNOS were conducted in non-melanoma models. They

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also show that the expression of HIF-1α is related to VEGF expression and is a poor prognostic marker in nodular malignant melanomas of the skin (Giatromanolaki et al. 2003). Lastly, NO may also favor tumor progression by enhancing tissue invasion (Lala and Chakraborty 2001). Studies examining the signaling mechanisms underlying this phenomenon show that both tumor and endothelial cell migration is reduced by NOS inhibition or NOS knockdown (Siegert et al. 2002; Jadeski et al. 2003). The migratory capacity of malignant cells is also reduced by the MAPK-kinase (MEK) inhibitor UO126, demonstrating that the MAPK pathway is essential for endogenous NO-mediated migratory responses (Jadeski et al. 2003). The MAPK pathway appears to be involved in NO-mediated induction of matrix-metalloproteinase (MMP) activation/overexpression in melanoma (Ishii et al. 2003) which are key features of cancer invasiveness. These findings suggest that increased expression of MMPs in response to NO may be associated with melanoma progression under inflammation and is involved in the melanoma metastasis.

NO, Inflammation, and Melanoma Although many cancers arise de novo without an identifiable predisposing disease, it is now believed that chronic inflammation is a strong supportive factor in tumor development and associated with a high cancer risk. At the molecular level, free radicals and aldehydes, produced during chronic inflammation, can induce lethal gene mutations and post-translational modifications of key cancer-related proteins. Other products of inflammation, including cytokines, growth factors, and transcription factors such as nuclear factor kappa B (NF-κB), control the expression of cancer genes and key inflammatory enzymes such as iNOS and COX-2. These enzymes, in turn, directly influence reactive oxygen species (ROS). Once the inflammation-associated tumors are formed, iNOS expression may be steadily stimulated by cytokines and NF-κB that are prevalent within the tumor inflammatory microenvironment (Li and Verma 2002). NO by itself or in collaboration with other inflammatory factors may also regulate angiogenesis, leukocyte adhesion and infiltration, and metastasis (Rao 2004). A recent population-based study found that specific polymorphisms in the promoter region of the iNOS gene led to higher promoter activities correlated with a higher incidence of gastric cancer in Japanese women (Tatemichi et al. 2005). This study also suggested that, due to the higher promoter activities of iNOS, excess NO may be produced and cause chronic inflammation, which contributes to the Helicobacter pylori-induced gastric cancer. Recent studies using a wide range of in vitro and in vivo models show that iNOS/NO signaling can also induce COX2, which itself is a promising link between inflammation and cancer (Rao 2004). The pro-cancerous outcome of chronic inflammation is increased DNA damage, increased DNA synthesis, cellular proliferation, disruption of DNA repair pathways, inhibition of apoptosis, and promotion of angiogenesis and invasion. Chronic inflammation is also associated with immunosuppression, which is another risk factor for cancer.

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Tumor cells produce various cytokines and chemokines that attract leukocytes. The inflammatory component of a developing neoplasm includes a distinct leukocyte population (neutrophils, dendritic cells, macrophages, mast cells, and others), all of which are capable of producing a group of cytokines, cytotoxic mediators including ROS, matrix metalloproteinases (MMPs), and soluble mediators of cell killing, such as TNF-α, interleukins (ILs), and interferons (IFNs) (Wahl and Kleinman 1998; Kuper et al. 2000). The balance of cytokines in any given tumor is critical for regulating the type and extent of the inflammatory infiltrate that formed. Production of an abundance of pro-inflammatory cytokines can lead to a level of inflammation that potentiates angiogenesis, thus favoring neoplastic growth. Tumor-associated macrophages (TAMs) are a significant component of inflammatory infiltrates in neoplastic tissues and are derived from monocytes that have a dual role in neoplasms. Although they may kill neoplastic cells following activation by IL-2, IFN, and IL-12 (Brigati et al. 2002; Tsung et al. 2002), TAMs produce a number of potent angiogenic and lymphangiogenic growth factors, cytokines, and proteases, all of which are mediators that potentiate neoplastic progression (Schoppmann et al. 2002). During development of melanoma, activated macrophages produce TGF-β, TNF-α, IL-1α, and extracellular proteases (Torisu et al. 2000). Indeed, macrophage infiltration is closely associated with the depth of invasion of primary melanoma due to macrophage-regulated tumorassociated angiogenesis (Ono et al. 1999). In addition to altering the local balance of pro-angiogenic factors during melanoma development, TAMs play a role in the formation of lymphatic vessels and lymphatic metastases (Schoppmann et al. 2002). By placing TAMs at the center of the recruitment and response to angiogenic and lymphangiogenic stimuli, they may foster the spread of tumors. Another important feature of TAM in melanoma is their involvement in iNOS and COX-2 expressions which may lead to tumor progression. Although there is a great body of publications showing iNOS and COX-2 expression in TAM and their role in cancer metastasis, their values as prognostic molecular markers in cancer still remain unclear. Moreover, the association of iNOS expression in tumor cells and TAM is an open research area of considerable interest. Our preliminary data in primary melanoma tumor samples show that iNOS expression in TAMs is statistically associated with intensity of iNOS expression in tumor cells. However, its significance in survival and prognostic value need to be explored.

Localization of Protein Expression An increasing body of cancer research literature indicates iNOS protein expression in gynecologic cancers (Thomsen et al. 1994), breast cancer (Thomsen et al. 1995), brain tumors (Cobbs et al. 1995), and colon cancer (Ambs et al. 1998) and our results in melanoma (Ekmekcioglu et al. 2000, 2006). These original reports are specific for the expression of the protein in tumor cells. Researchers have proposed that iNOS expression and levels of NO produced are not only species-specific but also

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cell-specific. The expression of iNOS and its functional end products, such as NT, in various tumor cells has been a major focus for researchers in the last decade. Our research provides strong evidence that the presence of iNOS protein in melanoma cytoplasm predicts poor overall as well as disease-specific survival in melanoma patients (Ekmekcioglu et al. 2000, 2006). Specifically, we show that tumor expression of iNOS is an independent predictor of disease-specific survival in patients with stage III melanoma. We first observed this in patients who received systemic treatment (Ekmekcioglu et al. 2000), and we found consistent results from tumors of patients who had not received systemic therapy (Ekmekcioglu et al. 2006). Our data indicate that the overall median survival duration in the cohort was approximately 5 years reflecting the M.D. Anderson experience. The patients with iNOS-positive tumors had a median survival of 2 years, whereas those with iNOS-negative tumors had a median survival of >10 years. Thus, we concluded that iNOS is a powerful predictor of outcome in this heterogeneous patient population. Our studies indicated that iNOS protein in melanoma cells did not result from immunotherapy or chemotherapy but reflected the natural biology of the disease in these patients. Other important sources of iNOS-produced NO in the tumor microenvironment are endothelial cells and stromal cells, such as TAMs. These macrophages are derived from peripheral blood monocytes recruited into the tumor mass. In the past decade, TAMs have been extensively studied and proposed as a major contributor to tumor progression (Bingle et al. 2002; Murdoch et al. 2004; Murdoch and Lewis 2005). In case of activation, the TAMs can release a variety of growth factors, cytokines, inflammatory mediators, and proteolytic enzymes. Many of these factors are key agents in tumor progression. It has been recently revealed that TAMs of early melanomas show an activated phenotype in which iNOS prevails on arginase (Massi et al. 2009). However, in an in vitro model, the release of high NO levels by TAMs required that the tumor microenvironment contains activated lymphocytes or natural killer cells producing IFN-γ (Massi et al. 2007). Thus, it may be possible that iNOS expressed by non-melanoma cells contributes to lymphangiogenesis. Regardless of the function of iNOS expression in these cells, now it is believed that the protein expression in TAMs contributes to melanoma progression. The role of TAMs in angiogenesis, as well as lymphatic microvessel formation, and potential prognostic significance of the iNOS expression are innovative research areas to be explored in melanoma.

Therapeutic Opportunities for Melanoma Most patients with thin local (stage I) melanoma can be cured by local excision and do not require adjuvant therapy. However, melanoma is an extremely aggressive disease with high metastatic potential and a notoriously high resistance to cytotoxic agents. There are several approved postoperative adjuvant therapies for malignant melanoma. Interferon-α (IFN-α) is the most commonly used adjuvant immunotherapy for advanced melanoma. High-dose interleukin-2 (IL-2) has

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also been approved with low response rates and high toxicity. Finally, dacarbazine (DTIC) is the reference-approved chemotherapeutic agent for the treatment of advanced melanoma, and drugs such as carmustine, paclitaxel, temozolomide, and cisplatin have shown single-agent activity in metastatic disease (Tarhini and Agarwala 2006). Therefore, we have an urgent need to differentiate the patient tumor characteristics and develop targeted therapies. The effective translation of candidate agents from the laboratory to the clinic requires a preclinical strategy that allows for selection of appropriate patients for each therapy. In addition, the correlation of specific molecular events with resistance to a therapy may suggest rational combinatorial approaches to improve efficacy. Our finding that iNOS expression predicted shortened survival in both treated as well as untreated stage III melanoma patients supports the value of this enzyme as a marker and further suggests the investigation of whether iNOS-produced NO could actually be inhibited and used as a novel targeted therapy option in melanoma. However, the selectivity for specific enzyme, poor cellular and tissue penetration, and significant toxicities has been proposed as problems in developing clinically useful iNOS inhibitors (Alderton et al. 2001). Selectivity is important for therapeutic utility in light of the important homeostatic functions of NO (Alcaraz and Guillen 2002). Equally, total chronic inhibition of iNOS may also be detrimental, given the apparent physiological functions of this isoform. Tissue- or cell-specific inhibitors may prove to be the most clinically useful (Vallance and Leiper 2002). Recently, in addition to the regulation at the transcriptional level, many translational and post-translational modifications of iNOS have been demonstrated. These regulatory steps may be important points for inhibition of overexpression of iNOS and iNOS-produced NO, depending on the cell type and other related pathways, in the treatment of melanoma and for the design of new drugs for melanoma, especially for the patients whose tumor express iNOS. Thus, our research should focus on the control of physiological levels of NO and for the design of new drugs that inhibit iNOS to prevent low and steady levels of NO production for the treatment of melanoma.

Concluding Remarks Melanoma is a complex and biologically heterogeneous disease which is determined by numerous progressive pathways affecting growth control, differentiation, cell adhesion, and metastasis, as well as survival. Melanoma, as all other human cancers, undergoes a continuous development from benign to malignant states, from benign nevi to metastatic melanoma. Although a tremendous amount of research has been conducted on the discovery of molecular prognostic markers, as of today, none of the new marker molecules was shown to be superior to conventional histological classification in larger studies. Clark level and tumor thickness are still the factors with best prognostic significance. However, biomarkers for defined problems in analysis of malignant melanoma have to be discovered; in order to differentiate the malignant lesions from benign, define the prognosis of the disease and finally propose

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novel therapeutic targets. Recent developments on detailed analysis by using new high-throughput techniques like multiple tissue arrays will accelerate the establishment of such markers. As the biomarker research progresses toward establishing the essentials of personalized medicine, the future of drug discovery and biomarker development will identify the unique population that would benefit from a specific drug. The development of novel biomarkers in specific diseases will also serve as surrogate end points and diagnostic indicators for disease screening. Moreover, they will provide us valuable information on monitoring disease progression and treatment efficacy and in evaluating patient outcome or identifying potential side effects such as in toxicity. In the near future, biomarker research will be heavily focused on transitioning biomarkers from the development and validation phases to their clinical applications incorporated into novel drug trials for particular diseases. As for the role of NO and iNOS expression, their significance in carcinogenesis is still complicated and in many ways contradictory. Enhanced antioxidant mechanisms in tumor cells in vivo have been implicated in chemoresistance and lead to poor prognosis, whereas many in vitro studies have reported tumor-suppressing properties of these enzymes. However, elevated expression or activity of iNOS has been described in many tumors compared to normal counterpart, including melanoma. Our experience in the prognostic value of iNOS expression strongly suggests that iNOS is an independent prognostic marker for stage III melanomas (Ekmekcioglu et al. 2000, 2006). Therefore, based on many other studies in different histological types of cancer, NOS expression is likely to play an active role in tumor biology. However, the precise function(s) of NO in cancer prognosis remains unclear; its presence in either tumor or stromal cells at the tumor site is likely to contribute to the altered biology of cancers. The field of NO therapeutics is now entering a crucial and exciting stage. Selective inhibitors of iNOS have been identified and will enter clinical trials shortly. The range of effects of iNOS inhibitors is likely to be broad and whether this inhibition might be beneficial is unlikely to become clear until after some years of their use to explore their therapeutic effect. Novel approaches to achieving isoform and cell or tissue selectivity of NOS inhibition will lead to more successful therapies in the near future.

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Chapter 17

Prognostic Significance of iNOS in Hepatocellular Carcinoma Rosa M. Pascale, M. Frau, and Francesco Feo

Abstract Epidemiological research indicates a contribution of chronic inflammatory liver diseases to development of hepatocellular carcinoma (HCC). Mounting evidence shows a carcinogenic role of NO• produced by inflammatory or cancer cells. The calcium-independent inducible isoform, iNOS, produces under inflammatory stimulation large amounts of NO• through the conversion of l-arginine to l-citrulline. NO• and NO•-derived oxidants can oxidize biomolecules, causing DNA damage. NO• interferes with the oxidative metabolism at different levels. It upregulates the AMP-activated protein kinase, thus switching off the ATP-consuming pathways such as lipogenesis or gluconeogenesis, while switching on the ATPproducing pathways such as fatty acid and glucose oxidation. Moreover, increase in mitochondrial NO• above physiological levels affects the oxygen-binding site of cytochrome c oxidase and induces pyruvate dehydrogenase kinase-1, an inhibitor of pyruvate dehydrogenase complex, thus inhibiting electron transport and oxygen consumption. NO• may also interact with different signaling pathways in hepatocarcinogenesis, including COX2, inhibitor of κB kinase (IKK)/nuclear factor B (NF-κB), and RAS/extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling. NO• stimulates COX-2 activity, and COX-2 inhibitors block NO• production in HCC cells. Furthermore, NO• and its derivatives may influence prostaglandin production by inducing lipid peroxidation and arachidonic acid release from cell membranes. Stimulation of EP2 receptor by PGE2 induces the association of the α subunit of the regulator G protein signaling and AXIN. This leads to the inactivation of glycogen synthase kinase-3β (GSK-3β) with consequent nuclear accumulation of β-catenin and increase in its transcriptional targets, c-Myc, c-Jun, and cyclin D1. PGE2 can also stimulate cell growth through the activation of several tyrosine kinase receptors, including EGFR and the PI3K/Akt pathways. Recent research on the interplay between iNOS and IKK/NF-κB and RAS/ERK pathways in HCC

F. Feo (B) Division of Experimental Pathology and Oncology, Department of Biomedical Sciences, University of Sassari, Sassari 07100, Italy e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_17, C Springer Science+Business Media, LLC 2010

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showed that these interactions are highest in the highly aggressive preneoplastic and neoplastic liver lesions of genetically susceptible rats and c-Myc/Tgf-α transgenic mice. The determination of iNOS expression in human HCC showed highest values in a subtype with poorer prognosis. Interestingly, iNOS levels are directly correlated with genomic instability, proliferation rate, and microvessel density of human HCC and inversely correlated with apoptosis and patients’ survival. These observations suggest that iNOS upregulation and changes in iNOS/NF-κB and iNOS/H-RAS/ERK cross talks are prognostic markers for HCC. Moreover, the block of iNOS signaling by a specific inhibitor such as aminoguanidine leads to a consistent decrease in HCC growth in c-Myc/TGF-α transgenic mice, decrease in growth and increase in apoptosis in human HCC cell lines, suggesting that the key components of iNOS signaling could represent therapeutic targets. Keywords Hepatocarcinogenesis · iNOS · Glycolysis · Mitochondrial activity · Prostaglandins · Signal transduction · S-adenosylmethionine · DNA methylation Hepatocellular carcinoma (HCC) is one of the most frequent human cancers, with ∼1 million of newly diagnosed cases each year. The highest frequencies are found in sub-Saharan Africa and far eastern Asia, where hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are endemic, and in regions where food contaminated with aflatoxin B1 is consumed (Thorgeirsson and Grisham 2002; Bruix et al. 2004; Farazi and DePinho 2006). Other risk factors associated with the development of HCC include alcoholic steatohepatitis, high dose of androgen steroids, type 2 diabetes, and various genetic disorders such as hemochromatosis, glycogen storage disease (types 1 and 2), a1-antitrypsin deficiency, Wilson’s disease, and environmental agents (cycasin, pyrrolizidine alkaloids, etc.). HCC incidence is rising, even in countries with relatively low incidence (Tanaka et al. 2002). HCC is a rapidly fatal disease, with a life expectancy of about 6 months from the time of diagnosis. Partial liver resection or liver transplantation is potentially curative, but only a minority of cases are amenable to these treatments. Hepatocarcinogenesis is a complex multistep process involving the accumulation of genetic and epigenetic events such as point mutations, chromosomal rearrangements, oncogene activation, and oncosuppressor gene inactivation (Feitelson et al. 2002). Epidemiological evidence has shown a striking contribution of chronic inflammatory liver diseases, including hepatitis B virus or hepatitis C virus (HCV) infection, and alcoholic steatohepatitis, to HCC development through inflammationrelated mechanisms (Feitelson et al. 2002). It has been shown that persistent liver injury due to increase in nitrative and oxidative DNA damage enhances carcinogenesis (Biegon et al. 2002; Iwai et al. 2002; Jüngst et al. 2004; Kawanishi et al. 2006). Liver infiltration by phagocytes, during liver injury, provides an important source of reactive oxygen species (ROS) which cause damage to DNA, proteins, and lipids when their generation exceeds the ability of the antioxidant systems to remove them. 8-Hydroxy-2 -deoxyguanosine (8-OH-dG) was first reported as a major form of oxidative DNA damage product which preferentially mispairs with adenine during DNA replication, resulting in GC → TA transversion (Shibutani et al. 1991).

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In recent years, increasing attention has been devoted to the role of reactive nitrogen species in carcinogenesis. Nitric oxide (NO•) participation in numerous biologic processes, including vasodilation, bronchodilation, inhibition of phagocyte, and platelet aggregation, has been well documented (Moncada et al. 1991). Excessive production of NO• during inflammation can have detrimental effects. However, mounting evidence indicates a carcinogenic role of inflammatory NO•, as well as its production by cancer cells, and interference with signal transduction pathways involved in cancer cell growth.

Production and Metabolic Effects of Reactive Nitrogen Species and Hepatocarcinogenesis NO• is a product of the conversion by nitric oxide synthase (NOS) of l-arginine to l-citrulline (Fig. 17.1). Three different classes of NOS can be found in the liver, the neuronal isoform (nNOS), present in the peribiliary plexus, the calciumdependent endothelial (eNOS) in endothelial cells, and the calcium-independent

Fig. 17.1 Schematic representation of the production of nitric oxide and peroxynitrite anion and their possible effects on hepatocarcinogenesis. HCC, hepatocellular carcinoma

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inducible isoform (iNOS) in hepatocytes, Kupffer and stellate cells, and cholangiocytes. In general, iNOS is not expressed at a significant level in normal cells (Beckman and Koppenol 1996; Wu and Morris 1998). When induced by inflammatory/immunological stimuli (including inflammatory cytokines or bacterial endotoxin), iNOS is highly expressed in many cell types and produces a large amount of NO• (Wu and Morris 1998). During inflammation, NO•-derived oxidants are formed by inflammatory cells. Stimulation of these cells results in activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to form O2 – and secretion of myeloperoxidase into the phagosomes or the extracellular space. Simultaneous production of NO• either by inflammatory cells or by adjacent parenchymal cells causes formation of peroxynitrite anion (ONOO– ) which can act as a substrate for myeloperoxidase with formation of nitrogen dioxide (NO2 ; Beckman and Koppenol 1996). NO• and NO•-derived oxidants can oxidize biomolecules (including proteins, fatty acids, and DNA), thereby damaging cell membranes, inhibiting vital biochemical reactions (e.g., the mitochondrial Krebs cycle and respiratory chain), and even causing cell death (Demple and Harrison 1994; Fang et al. 2002). NO• causes a variety of DNA damages, including DNA strand breaks and oxidation, and inhibition of DNA repair (Fig. 17.1). All of these alterations may be responsible for liver necrosis. However, NO• at lower concentrations may favor HCC development. Notably, DNA mutations in hepatocytes surviving to oxidative stress may induce cell transformation, in the absence of sufficient DNA repair, and liver necrosis, when not extended to the majority of liver mass, could enhance liver regeneration and tumor promotion (Fig. 17.1). Furthermore, NO• causes vasodilatation depending on its reaction with the ferrous iron, in the heme prosthetic group of the soluble guanylate cyclase, that increases the concentration of guanosine-3 ,5 -cyclic monophosphate (cGMP) within the respective target cell, thus mediating its relaxation (Pannen 2002). Vasodilatation can favor HCC development by providing tumor cells with sufficient metabolites and oxygen. Free radicals at physiological levels are signaling molecules involved in metabolic regulation (Wu et al. 2004). Their overproduction leads to metabolic alterations which could modulate cancer development. AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. AMPK activation by increased [AMP]:[ATP] ratio occurs via the phosphorylation by the serine–threonine protein kinase LKB1. The overall effect of AMPK activation is to switch off the ATPconsuming pathways such as lipogenesis or gluconeogenesis while switching on the ATP-producing pathways such as fatty acid and glucose oxidation. Recently, NO• has been found to be an endogenous AMPK activator (Zhang et al. 2008), and low concentrations of peroxynitrite anion activate AMPK through a c-Srcmediated and phosphatidylinositol 3-kinase (PI3K)-dependent pathway in cultured bovine aortic endothelial cells and in mouse aorta and heart (Zou et al. 2003). Accordingly, NO• donors were found to inhibit the conversion of lactate and pyruvate into glucose in rat hepatocytes (Horton et al. 1994). It has been also reported that in hepatocytes NO• inhibits glycogen synthase and glycogen synthesis from glucose (Sprangers et al. 1998). On the other hand, NO• decreases the activities of key

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glycolytic enzymes such as glucokinase (Monti et al. 2000) and glyceraldehyde3-phosphate dehydrogenase (Stadler et al. 1995). Thus, the net effect of NO• on hepatic glucose metabolism likely could depend on the balance between two opposing effects: AMPK-mediated inhibition of gluconeogenesis and reduced glucose utilization. In 1926, Warburg (1956) observed that cancer cells produce most of their ATP through glycolysis, even under aerobic conditions. This phenomenon, subsequently denominated “Warburg effect,” has been the object of several studies showing that even if mitochondria isolated from HCC may efficiently respire and produce ATP (Feo et al. 1973), intact HCC cells use prevalently glycolytic ATP for protein synthesis (Terranova et al. 1964), and there is a correlation between glycolytic ATP production and aggressiveness of the tumor cells (Kim and Dang 2006). Thus, the Warburg effect could be considered as a positive modifier of cancer, such that it may not be causative but rather facilitates tumor progression (Kim and Dang 2006). NO• may modulate mitochondrial activity by different mechanisms. Physiological NO• levels may increase the supply of metabolic substrates and oxygen to mitochondria by regulating blood flow. Moreover, high NO• levels may directly modulate the activity of the mitochondrial electron transport system (Brown 2001). Exogenous NO• concentrations, that are likely to increase NO• in mitochondria above physiological levels, reversibly bind the oxygen-binding site of cytochrome c oxidase, thus inhibiting electron transport and oxygen consumption (Fig. 17.2). This results in a reduced supply of ATP from substrate oxidation and enhanced glycolysis as a significant alternative ATP-producing pathway (Brown 2001; Nisoli et al. 2004). This situation, which should favor ADP consuming reactions of glycolysis and glucose consumption for ATP synthesis (Fig. 17.2), apparently contrasts with the above-reported inhibitory effect of NO• on some key glycolytic enzymes. It should be noted, in this respect, that upregulation of the genes encoding glycolytic enzymes (see below) could overcome the enzyme activity restriction by NO•, and thus NO• effects on glucose metabolism could play an important role in adaptation to low-oxygen conditions which may characterize HCC (Wu et al. 2007). The hypoxia-inducible factor 1 (HIF-1) consists of an oxygen-sensitive HIF-1α subunit that heterodimerizes with HIF-1β to bind DNA. In high oxygen tension, HIF-1α is hydroxylated by prolyl hydroxylases (PHD; Kim and Dang 2006). The hydroxylated HIF-1α subunit is recognized by the von Hippel–Lindau (VHL) protein and designated for degradation by the proteasome. Hypoxia is a pathophysiologic stimulus of anaerobic glycolysis through stabilization of HIF-1 and its direct transactivation of glycolytic enzyme genes (Kim and Dang 2006). iNOS is a target of HIF-1 (Harris 2002). Conversely, NO• induces H-RAS activation in HCC (Calvisi et al. 2008a; Fig. 17.2), and activated H-RAS (H-RAS-GTP) has been found to increase the level of HIF-1 (Chen et al. 2001) and PI3K signaling may stabilize HIF1 (Semenza 2003). HIF-1 stabilization may induce angiogenesis via activation of the VEGF-A gene. HIF-1 also induces pyruvate dehydrogenase kinase-1 (PDK-1), an inhibitor of pyruvate dehydrogenase complex (PDH; Fig. 17.2). The consequent block of pyruvate decarboxylation inhibits the Krebs cycle and the mitochondrial respiratory chain.

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Fig. 17.2 Possible effects of nitric oxide on the mitochondrial respiratory chain, Krebs cycle, and glycolysis. NO· induces H-RAS activation and activated H-RAS (RAS-GTP) increases HIF-1 level. HIF-1 induces pyruvate dehydrogenase kinase-1 (PDK-1), which inhibits the pyruvate dehydrogenase complex (PDH). The consequent block of pyruvate decarboxylation inhibits the Krebs cycle and mitochondrial respiratory chain. The mitochondrial respiratory chain may be also inhibited by mutated P53, through inhibition of synthesis of cytochrome c oxidase 2 (SCO-2), required for the assembly of cytochrome oxidase subunit II into the cytochrome c oxidase complex. HIF-1 and AKT oncogenes enhance glucose uptake and activate hexokinase 2 (HK2) to phosphorylate and trap intracellular glucose. The MYC transcription factor activates virtually all glycolytic enzyme genes. High MYC expression can result in high mitochondrial ROS and NO· production, which may result in mitochondrial DNA (mtDNA) mutations. HIF-1 activates lactate dehydrogenase (LDH), thus favoring the reduction of pyruvate to lactate

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The mitochondrial respiratory chain may be also inhibited by mutated P53, through inhibition of synthesis of cytochrome c oxidase 2 (SCO-2; Fig. 17.2). SCO-2 is required for the assembly of the COXII subunit (cytochrome oxidase subunit II) into the cytochrome c oxidase complex, which is integral to the respiratory chain (Leary et al. 2007). Notably, NO• was shown to induce accumulation of wildtype p53 protein in colon cancer (Hussain and Harris 2006). This could inhibit cell proliferation, thus favoring the selection of tumor cells with mutated P53. In P53 mutants iNOS increases VEGF expression and promotes tumor growth, suggesting that the activity of NO• on tumor cells may be influenced by the P53 status of the tumor (Wink et al. 2008). These observations are of particular interest for HCC in which frequent P53 mutations occur as an early event in areas with AFB1 exposure and as a late event in HCC associated with viral infections of other etiologies (Teufel et al. 2007). Several oncogenes have been implicated in the Warburg effect (Fig. 17.2). The AKT oncogene, encoding a protein serine–threonine kinase, enhances glucose uptake and activates hexokinase 2 (HK2) to phosphorylate and trap intracellular glucose (Elstrom et al. 2004; Fig. 17.2). Overexpression of AKT is associated with glycolytic flux without affecting mitochondrial oxidative phosphorylation, thereby presumably contributing to the Warburg effect. The MYC oncogene, which is activated in several cancer types, including HCC (Calvisi et al. 2007a), encodes a transcription factor which activates virtually all glycolytic enzyme genes and directly binds numerous glycolytic genes, including those encoding HK2, enolase, and lactate dehydrogenate-A (Kim and Dang 2005). MYC is involved in mitochondrial biogenesis which, when sustained by high MYC levels, can result in high mitochondrial ROS and NO• production, which may induce mtDNA mutations that in turn contribute to dysfunctional mitochondria (Kim and Dang 2006; Fig. 17.2). So far, the relationships between NO• availability, cell metabolism, and cancer progression have been prevalently evaluated in extrahepatic tumors. On the basis of these studies and of the observation that HCCs overexpress iNOS, MYC, AKT, and HIF-1 (see below), it may be reasonably suggested that NO• overproduction, in different stages of hepatocarcinogenesis, is likely to contribute to the progressive utilization of glycolysis as a major energy source, which may facilitate the progression of HCC cells (Weber et al. 1977).

iNOS and Signal Transduction Pathways Although the role of NO• has been studied in depth in inflammatory cells, chronic inflammation, oxidative damage, usually associated with viral hepatitis and HCC, and elevated NO• plasma levels are present in patients with cirrhosis and HCC (Moriyama et al. 2000). The regulation of NO• production and interactions of iNOS with signaling pathways in hepatocarcinogenesis remain poorly investigated. Cyclooxygenase type 2 (COX2) is an important mediator of inflammation involved in prostaglandin synthesis (Fig. 17.3). Arachidonic acid, released from

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Fig. 17.3 Interplay between COX2 and iNOS. Various signals can activate COX2 and iNOS expression. Prostaglandins (PGs) and NO contribute to the inflammatory processes. dPGJ2 (through PPAR-γ) and PGE2 decrease iNOS expression. NO may increase COX2 activity and favors PG production by inducing the release of arachidonic acid as a consequence of membrane lipid peroxidation. Nevertheless, possible decrease in COX2 by NO has also been reported. PGs favor cell growth by activating growth factor receptors, such as EGFR and MAPK signaling

membrane phospholipids by phospholipase A2, is metabolized by cyclooxygenases to prostaglandin H2 (PGH2) that is subsequently converted to various prostanoids by specific synthases (Weinberg 2000; Wu 2006). PGE2, the major prostaglandin in hepatocytes, after release in the extracellular space binds the membrane receptors EP1, EP2, EP3, and EP4, coupled with G proteins, on the same and neighboring cells. dPGJ2 (through PPAR-γ) and PGE2 induce a decrease in iNOS expression, and NO• may either increase or decrease COX2 activity and prostaglandin production (Weinberg 2000). These findings indicate the existence of a cross talk between COX-2 and iNOS pathways in hepatocarcinogenesis, as also supported by the observation that COX-2 inhibitors block NO• production in HCC cells (Fantappiè et al. 2002). Furthermore, NO• and its derivatives may influence prostaglandin production by inducing lipid peroxidation and arachidonic acid release from cell membranes. Stimulation of the EP2 receptor by PGE2 induces the association of the α subunit of the regulator G protein signaling and AXIN (Castellone et al. 2005). This leads to the inactivation of glycogen synthase kinase-3β (GSK-3β), a downstream effector of the WNT pathway, with consequent nuclear accumulation of β-catenin and increase in its transcriptional targets c-MYC, C-JUN, and CYCLIN D1. PGE2 can also stimulate cell growth through the activation of several tyrosine kinase receptors, including EGFR. This results in GRB2 and SOS phosphorylation and RAS activation, which drives the mitogen-activated protein kinase (MAPK) pathway leading to extracellular signal-regulated kinases 1 and 2 (ERK1/2; Pai et al. 2002). Finally,

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PGE2 can induce actin polymerization and epithelial mesenchymal transition, leading to increase in cancer metastasis through the activation of the PI3K/Akt pathway (Sheng et al. 2001). The mechanisms described above, involved in tumor progression and formation of metastases, have been mainly explored in gastrointestinal cancer. However, Rahman et al. (2001) showed, by immunohistochemical analysis, that the expression of both COX-2 and iNOS are significantly higher in hepatitis C virus (HCV)positive HCCs and the COX-2 expression level was significantly correlated with iNOS expression and microvascular density (MVD). The combined negative expression of iNOS and COX-2 had a significant impact on patient survival, suggesting an important role in the prognosis of HCV-positive HCC patients. In addition, EGFR is expressed in human HCC and is a target of anticancer compounds (Hung et al. 1993; Furuse 2008). EGFR activation upregulates COX-2 expression, thereby enhancing PGE2 production (Han et al. 2006). On the other hand, PGE2 transactivates EGFR in human HCC cells, and this effect is mediated by the EP1 receptor and involves the c-Src protein. These findings and the observation of iNOS upregulation in HCC (Simile et al. 2005; Calvisi et al. 2008a) envisage the existence of a scenario in HCC implying a contribution of NO• overproduction to the growth and progression of this tumor. Other signaling pathways are involved in cross talks with iNOS, during hepatocarcinogenesis. iNOS, nuclear factor-κB (NF-κB), RAS, and ERK are upregulated in preneoplastic rat liver lesions (Simile et al. 2005; Calvisi et al. 2008a), dysplastic and neoplastic liver from c-Myc-TGF-α transgenic mice (Calvisi et al. 2004b), and human HCCs (Ikeguchi et al. 2002; Sun et al. 2005). Recent research in our laboratory (Calvisi et al. 2008a) showed the existence of an interplay between the iNOS and the inhibitor of κB kinase (IKK)/NF-κB and RAS/ERK pathways in HCC (Fig. 17.4). The interactions are always highest in the most aggressive preneoplastic and neoplastic liver lesions of the genetically susceptible F344 rats, compared to the resistant BN rats, and of c-Myc-TGF-α transgenic mice much prone to hepatocarcinogenesis than TGF-α transgenic mice. Furthermore, the determination of iNOS expression in human HCC showed highest values in a subtype with poorer prognosis (based on the length of survival after partial liver resection) compared to a subtype with better prognosis. The suppression of iNOS signaling by aminoguanidine (Misko et al. 1993) in c-Myc/TGF-α mice and human HCC cell lines resulted in significant reduction in HCC growth and NF-κB and RAS/ERK expression and increase in apoptosis (Calvisi et al. 2008a). In contrast, NO• production by glyco-S-nitroso-N-acetylpenicillamine 2 (Glyco-Snap-2) inhibited apoptosis of in vitro growing human HCC cells. Conversely, the block of NF-κB signaling by sulfasalazine (Favata et al. 1998) or siRNA, or ERK signaling by the MAPK kinase (MEK) inhibitor UO126 (Weber et al. 2000), caused iNOS downregulation in HCC cell lines. In transgenic mice and human HCC cell lines, iNOS antiapoptotic effect seems to be mediated by the NF-κB cascade. The latter induces various antiapoptotic proteins, such as Bcl-2-related protein, the long isoform (BCL-xL), the inhibitor of apoptosis, X-linked (XIAP), and the inhibitor of apoptosis protein 1 (cIAP-1), and inhibits the proapoptotic Jun NH2 terminal kinase (JNK; Nakano et al.

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Fig. 17.4 Schematic representation of the iNOS interplay with IKK/NF-κB and Ha-RAS/ERK signaling cascades. iNOS activates IKK which allows proteasomal degradation of the NF-κB inhibitor, IkB-α, resulting in NF-κB activation. iNOS also activates H-RAS, thus triggering the MAPK cascade which leads to ERK activation. The activation of NF-κB by active ERK may occur through AURKA, which inhibits IκB-α. Thus, iNOS interplay with IKK/NF-κB and HaRAS/ERK cascades results in activation of genes controlling cell growth and angiogenesis and inhibiting apoptosis. Pointed and blunt arrows indicate activation and inhibition, respectively. The dotted arrow indicate hypothetical activation

2006). Accordingly, iNOS suppression by aminoguanidine caused downregulation of NF-κB and antiapoptotic proteins, and upregulation of pJNK, in c-Myc/TGF-α and HCC cell lines (Calvisi et al. 2008a). However, these findings cannot exclude the contribution of other mechanisms to the antiapoptotic action of iNOS. These observations assign a role to iNOS upregulation in the control of the proliferative phenotype of preneoplastic and neoplastic liver cells through the activation of the IKK/NF-κB axis (Fig. 17.4). They also imply a cross talk between iNOS and H-RAS/ERK. The mechanism of NF-κB regulation by pERK1/2 is still unclear. The pERK1/2 contribution to NF-κB upregulation through direct activation of iNOS is still unproved. According to recent observations, pERK1/2 activates AURORA-A (AURKA), which in turn may activate NF-κB through inhibition of the inhibitor of κB (IKB-α) (Briassouli et al. 2007; Fig. 17.4). Interestingly, correlative experiments showed that iNOS levels are directly correlated with genomic instability, assessed by random amplified polymorphic DNA analysis methodology, proliferation rate, and MVD of HCC, and inversely correlated with apoptosis and patients’ survival (Calvisi et al. 2008a). These observations and the presence of highest expression of iNOS and its downstream targets in more aggressive rodents and human HCC suggest that iNOS upregulation and changes in iNOS/NF-κB and iNOS/H-RAS/ERK cross talks are prognostic markers for HCC. These results agree with the observation of a significant association of iNOS and

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metalloproteinase-9 expression with HCC recurrence (Sun et al. 2005), and iNOS overexpression with poor prognosis for gastric cancer (Li and Xu 2005), adenoid cystic carcinoma of salivary glands (Zhang et al. 2005), fibrous histiocytoma (Hoki et al. 2007), and colorectal cancer (Cianchi et al. 2004). These iNOS effects could at least, in part, depend on its angiogenic properties and are intensified by COX2 upregulation (Rahman et al. 2001; Cianchi et al. 2004). However, no correlation between iNOS overexpression and prognosis has been reported for pancreatic and ovarian tumors (Kong et al. 2002; Ozel et al. 2006). Furthermore, iNOS ablation did not prevent hepatocarcinogenesis induced by a choline-deficient, l-amino aciddeficient diet in mice (Denda et al. 2007), suggesting a relatively minor role of iNOS signaling. In this model of hepatocarcinogenesis, high production of lipid peroxides in hepatocyte nuclei (Ghoshal 1995) may cause DNA damage and contribute to HCC development via generation of genomic instability in a iNOS-independent manner. The contribution of iNOS overexpression to growth deregulation in preneoplastic and neoplastic liver cells through a cross talk with Ha-RAS/ERK and IKK-NF-κB axis does not exclude per se the activation of iNOS signaling by other mechanisms, such as inflammatory cytokines or the Wnt/β-catenin signaling (Du et al. 2006). Indeed, IL-1b, IL-6, TNF-α, and IFN-γ expressions are elevated in the dysplastic and neoplastic livers of transgenic mice, with highest values for TNFα and IFN-γ, in double-transgenic HCC. Rise in cytokine expression also occurs in human HCC and surrounding non-tumorous liver, with highest values in HCC with poorer prognosis, for IL-1b, IL-6, and TNF-α (Calvisi et al. 2008a). However, the role of Wnt/β-catenin signaling in iNOS upregulation seems to be unlikely due to the observation of equal β-catenin activation (nuclear localization) in HCCs from both F344 and BN rats (Frau, unpublished data) expressing sharply different iNOS mRNA levels. β-catenin activation also occurs in a lower percentage of HCC cells from c-Myc/TGF-α than TGF-α transgenics (12 vs 30%; Calvisi et al. 2004a), although highest iNOS expression occurs in HCC from double transgenic mice. According to recent research (Ying et al. 2007), NO• induces pRb hyperphosphorylation apparently through the soluble guanylyl cyclase/cGMP/cGMP-dependent protein kinase (sGC/cGMP/PKG) signaling pathway, during chronic inflammation in the mouse colitis model. Some evidence was presented indicating that the effect of sGC/cGMP on pRb phosphorylation is dependent on the MEK/ERK and PI3K/AKT pathways. These results reveal a role of sGC/cGMP/PKG signaling in cell cycle control, through downstream MEK/ERK and PI3K/AKT pathways. The links between sGC/cGMP/PKG signaling and MEK/ERK and PI3K/AKT signaling are not completely clear and should be the object of future work. pRb hyperphosphorylation occurs in preneoplastic and neoplastic rat and human liver lesions and is correlated to HCC aggressiveness (Pascale et al. 2002; Pascale et al. 2005). Moreover, the MEK/ERK and PI3K/AKT pathways are upregulated in HCCs (Tanaka et al. 2006). However, the interplay between NO• production and the sGC/cGMP/PKG pathway has not been explored in hepatocarcinogenesis and the possibility that other mechanisms are responsible for the changes in the pRb phosphorylation and the MEK/ERK and PI3K/AKT pathways in HCC cannot be excluded.

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NO Interference with SAM Synthesis and DNA Methylation Early appearance of global DNA hypomethylation, associated with promoter hypermethylation and inactivation of oncosuppressor genes, is a common feature of hepatic carcinogenesis in rodents (Pascale et al. 1991; Teufel et al. 2007). This abnormality could induce genomic instability in cells throughout the different steps of hepatic carcinogenesis and facilitate the occurrence of irreversible gene changes (Shen et al. 1994; Kim et al. 1997; Kanai et al. 1999). A body of evidence indicates the existence in preneoplastic and neoplastic rat liver lesions and human cirrhosis and HCC of marked decrease in S-adenosyl-l-methionine (SAM) and SAM: S-adenosylhomocysteine (SAH) ratio, associated with global DNA hypomethylation (Garcea et al. 1989; Pascale et al. 1992; Wainfan and Poirier 1992). Decrease in liver SAM content has been attributed to changes in the methionine adenosyltransferase (MAT) isozyme pattern. In mammals, the MAT1A gene, expressed only in liver, encodes MATI/III isozymes, whereas widely expressed MAT2A encodes MATII isozyme (Avila et al. 2000). In response to liver injury or during reparative growth, MAT1A is down-regulated whereas the Mat2A, induced by NF-κB, is switched on (Huang et al. 1998; Yang et al. 2003). Fall in MAT1A expression with concomitant upregulation of MAT2A also occurs in hepatoma cell lines and rodent HCC as well as in human liver cirrhosis and HCC (Cai et al. 1998; Mato et al. 2002). Mice lacking Mat1A show reduced SAM liver content and spontaneously develop HCC (Lu et al. 2002). Differential expression of MAT1A and MAT2A genes influences DNA methylation and growth of human HCC (Cai et al. 1998; Avila et al. 2000; Calvisi et al. 2007b). There is evidence that SAM interferes with the activity of various genes and proteins. SAM treatment inhibits the expression of c-myc and c-H-Ras of neoplastic liver nodules in rats (Garcea et al. 1989) and lipopolysaccharide-induced TNF-α and iNOS expression in rat liver and RAW 264.7 cell line (Majano et al. 2001; Veal et al. 2004), and SAM binding to cystathionine β-synthase stabilizes this protein against degradation (Prudova et al. 2006). MAT1A downregulation in precancerous cells may contribute to maintain active NF-κB (Fig. 17.5). Since SAM enhances the synthesis of the NF-κB inhibitor, IκB-α, probably by targeting IkB-α gene (Majano et al. 2001; Simile et al. 2005), low MatI/III activity and SAM content in precancerous liver should contribute to the observed relatively low levels of NF-κB/IkB-α complex as well as to NF-κB activation and overexpression of genes targeted by the nuclear factor, such as c-myc, cyclin D1, iNos, and Vegf-A (Simile et al. 2005; Calvisi et al. 2008a). On the other hand, transactivation of iNos by NF-κB (Majano et al. 2001) and iNos over-activity in preneoplastic lesions (Calvisi et al. 2008a) should result in NO• overproduction that may inhibit hepatocyte MatI/III and SAM production (Martinez-Chantar et al. 2002). Furthermore, NO• activates IκB kinase with consequent IκB-α phosphorylation and ubiquitination (Zingarelli et al. 2002). Thus, NO• overproduction can contribute, by modulating SAM level and IκB kinase activity, to decrease in MatI/III activity and increase in NF-κB level (Fig. 17.5). NO• may also interfere with SAM metabolism by inhibiting 5,10-methyltetrahydrofolate reductase (Danishpajooh et al. 2001), the enzyme that catalyzes the synthesis of methionine from homocysteine and

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Fig. 17.5 Effect of nitric oxide on the synthesis of methionine and S-adenosylmethionine and methylation reactions. NO inhibits methyltetrahydrofolate reductase (MTR). This results in a decrease in tetrahydrofolate (FH4 ) and methionine. Additional reduction in the FH4 level may occur by the NO-induced oxidation of ferritin, a compound that inhibits the proteasomal degradation of FH4 . NO affects SAM synthesis not only by inducing a decrease in methionine synthesis but also by directly inhibiting the liver-specific methyl-thioadenosyltransferase I/III (MATI/III) isozymes. The fall in SAM level cannot be fully compensated by an increase in the extrahepatic isozyme MATII, since this enzyme is inhibited by its reaction product. The reduction in homocysteine utilization for methionine synthesis may result in homocysteine accumulation. This probably does not lead to a consistent rise in cystathionine and reduced glutathione synthesis, due to a reduced stabilization of cystathionine β-synthase (CBS) by SAM. Consequently, an increase in SAH, associated with a decrease in the SAM/SAH ratio, inhibits methyltransferases (MT) and DNA methylation. The reduction in SAM level may decrease IκBα activation, thus favoring NF-κB activity

5-methyltetrahydrofolate (Fig. 17.5). This may result in decrease in methionine and SAM levels, homocysteine accumulation, and reduction in the SAM/SAH ratio. Altogether, the above observations are of particular interest in light of the recent findings indicating that the extent of DNA hypomethylation and genomic instability are prognostic markers for human HCC (Calvisi et al. 2007b).

Concluding Remarks The specific roles of NO• in carcinogenesis have not yet been completely understood. This, at least, in part, depends on the fact that NO• effects are largely influenced by its concentration, interaction with other free radicals, metal ions and proteins, and cell type and genetic background (Hussain and Harris 2006). Although the results of studies on iNOS regulation and implication in hepatocarcinogenesis are still fragmentary, they depict a complex, although incomplete, scenario in which

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the production of NO• and its derivatives insufficient to kill initiated hepatocytes is likely to affect HCC development and progression. A body of evidence indicates that NO• and its by-products may influence the oxidative metabolism of cancer cells, by inhibiting some key reactions of the respiratory chain, and contributing to the progressive adaptation of cancer cells to prevalent production of glycolytic ATP and survival in hypoxic conditions. This situation could favor the progression of transformed hepatocytes (Kim and Dang 2006). The interplay between iNOS and signal transduction pathways is an important aspect of the role of NO• in carcinogenesis. In vivo studies in both rodents and humans have discovered situations characterized by NF-κB and ERK activation, prostaglandin overproduction, and interference with SAM metabolism, which may favor tumor cell growth and progression. Notably, highest deregulation of the interplay of iNOS with signaling pathways involved in cell growth control occurs in more aggressive human and experimental HCCs. Accordingly, a strong, direct correlation of iNOS expression with proliferation rate, MVD, and genomic instability has been found in human HCCs (Calvisi et al. 2008a). An inverse correlation occurs between iNOS expression and apoptosis and patients’ survival after partial liver resection. These clearly indicate that iNOS expression is potentially a prognostic marker for human HCC. The association of the block of iNOS signaling by a specific inhibitor such as aminoguanidine with a consistent decrease in HCC growth in c-Myc/TGF-α transgenic mice, and decrease in growth and increase in apoptosis in human HCC cell lines (Calvisi et al. 2008a), suggests that the key components of this pathway could represent therapeutic targets that may contribute to create networked biological therapies. Selective iNOS inhibitors have chemopreventive effects in rodent models of colorectal and esophageal carcinogenesis (Rao et al. 2002; Hagos et al. 2007; Stoner et al. 2007). Combined utilization of the iNOS inhibitor SC-51 and the COX-2 inhibitor celecoxib appears more effective than either agent alone in suppressing colonic aberrant crypt foci formation in the azoxymethane rat model of colon carcinogenesis (Rao et al. 2002). The effect of these inhibitors, alone or in combination, has not been evaluated in hepatic carcinogenesis. Another important aspect of the deregulation of iNOS signaling in hepatocarcinogenesis is the demonstration that the susceptibility to hepatocarcinogenesis, either dependent on susceptibility genes or genomic engineering, influences the iNOS-linked signaling in liver preneoplastic and neoplastic lesions (Calvisi et al. 2008a). The genetically resistant phenotype is characterized by the incapacity of early preneoplastic lesions to acquire autonomous growth and progress to HCC (Pascale et al. 2005). Autonomous growth of the lesions is supported, in susceptible F344 rats, by the deregulation of the cell cycle (Pascale et al. 2002; Pascale et al. 2005) and the Ras/Erk pathway (Calvisi et al. 2008b; Calvisi et al. 2008c). These alterations are limited or absent in the lesions of BN-resistant rats. iNOS upregulation occurs in early stages of hepatocarcinogenesis in both F344 and BN rats. However, while iNOS protein expression sharply and progressively increases in nodules and HCC, it undergoes lower changes in slow-growing nodules and HCC of BN rats. A link between fast growth and signaling deregulation characterizes

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human HCC with poor prognosis, whereas HCC with better prognosis behaves as the lesions of resistant rats (Pascale et al. 2005; Calvisi et al. 2008a; Calvisi et al. 2008c). This does not necessarily imply a genetic regulation of signaling pathways in humans like that found in rodents, in which polygenic inheritance with several low-penetrance genes and a main gene regulates the genetic predisposition to HCC (Feo et al. 2006). Even if a genetic model, similar to that of rodents, can influence human hepatocarcinogenesis, further studies are needed to clarify the influence of susceptibility genes on signaling pathways supporting tumor growth and progression in humans.

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Chapter 18

Prognostic Significance of iNOS in Esophageal Cancer Manabu Matsumoto, Yuji Ohtsuki, and Mutsuo Furihata

Abstract Inducible nitric oxide synthase (iNOS) is an enzyme responsible for the production of nitric oxide and has been suggested to play an important role in tumor biology. However, because of the complexity of iNOS biology, conflicting findings have been obtained, e.g., iNOS expression has been found to be associated with poor prognosis or better prognosis in different types of tumors, while in some cases no significant relationship has been found between iNOS expression and prognosis. In our study of esophageal squamous cell carcinoma (SCC), iNOS was expressed at relatively high frequency and was positively associated with p53 overexpression but not associated with prognosis, suggesting that iNOS might be associated with p53 alteration and contribute to tumorigenesis rather than tumor progression in esophageal SCC. Keywords Esophageal squamous cell carcinoma · Inducible nitric oxide synthase · p53 · Immunohistochemistry

Introduction Esophageal carcinoma is a common malignant neoplasm worldwide, with a variable geographic distribution, which might be due to genetic and/or epidemiological risk factors such as cigarette smoking, alcohol consumption, or nitrosamine-containing diet (Blot 1994; Ribeiro et al. 1996). A recent study of esophageal squamous cell carcinoma (SCC) found that such epidemiological risk factors can induce nitric oxide (NO) production and contribute to tumor progression (Kato et al. 2000). NO is a free radical gas, which mediates several biological activities including angiogenesis, neural transmission, and cytotoxicity (Schmidt and Walter 1994). M. Matsumoto (B) Laboratory of Diagnostic Pathology, Kochi Medical School Hospital, Nankoku, Kochi 783-8505, Japan e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_18, C Springer Science+Business Media, LLC 2010

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It is synthesized from l-arginine by nitric oxide synthase (NOS) (Moncada et al. 1991). There are two classes of NOS, constitutive NOS (neural type and endothelial type) and inducible NOS (iNOS). Generally, neural NOS and endothelial NOS, which are known to generate NO at low concentrations, are expressed constitutively in neurons and endothelial cells, respectively, and their activity depends on elevated intracytoplasmic calcium/calmodulin levels. On the other hand, iNOS, which generates large amounts of NO for prolonged periods of time, is produced by neutrophils, macrophages, epithelial cells, and many other cell types, and induction of it requires bacterial products or inflammation-associated cytokines independent of calcium/calmodulin concentration (Nathan and Xie 1994; Knowles and Moncada 1994). In normal conditions, constitutively expressed NO is a very important intercellular messenger molecule (Schmidt and Walter 1994). However, NO can also have genotoxic effects and long-term exposure of cells to high NO concentrations could play an active role in carcinogenesis (Xu et al. 2002). The genotoxic effects of NO are mediated through several mechanisms including DNA damage resulting from nitrosative deamination, DNA strand breakage, and DNA modifications (Wink et al. 1998; Felley-Bosco 1998). Since prolonged, high concentrations of NO result from activation of iNOS, much cancer research has been directed toward this isoenzyme (Nathan and Xie 1994). Many recent studies have shown that iNOS is frequently expressed in many types of carcinomas and is correlated with clinicopathological factors such as histological grade, vascular invasion, high pT classification, and prognosis (Yagihashi et al. 2000; Vakkala et al. 2000; Wolf et al. 2000; Tanaka et al. 1999; Thomsen et al. 1994; Thomsen et al. 1995; Aaltomaa et al. 2000). However, conflicting findings exist suggesting a role for iNOS in either suppression or promotion of tumor progression (Wink et al. 1998). In this chapter, we mainly review the possibility of relationship between iNOS expression and tumor behavior in esophageal cancer.

Epidemiology and Etiology of Esophageal Cancer Esophageal cancer is common throughout the world and is the sixth most frequent cause of cancer death (Parkin et al. 1999). The overall survival rate of patients with esophageal carcinoma is relatively poor because of rapid continuous tumor extension beyond the esophageal wall: only 14% of these patients live 5 years or longer after diagnosis (Enzinger and Mayer 2003). There are two main types of esophageal cancer with distinct etiological and pathological characteristics, squamous cell carcinoma and adenocarcinoma (Stoner et al. 2000). These two types of tumor are responsible for more than 90% of all esophageal carcinomas (Daly et al. 2000). The incidence of esophageal SCC varies markedly in geographic distribution and is very high in certain regions such as China, Iran, and South Africa (Stoner et al. 2000). China has the highest rate, with ∼250,000 new cases diagnosed yearly, accounting for half of the world’s new cases (Daly et al. 2000). On the other hand, nearly 10,000 cases of SCC of the esophagus are diagnosed in the United

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States annually, accounting for nearly 2% of all cancers and 7% of gastrointestinal malignancies in that country (Herskovic et al. 1992; LoCicero 1993; Forastiere 1992). One of the most notable changes in esophageal cancer is the increasing incidence of adenocarcinoma in many Western countries in the past two decades. For instance, in the United States, the incidence of adenocarcinoma has increased about 450% among white men and 50% among black men during the past 20 years, while the rate of esophageal SCC has decreased by 35% (Devesa et al. 1998; Ji and Hemminki 2006; Freedman et al. 2007). There are several risk factors for esophageal cancer, including cigarette smoking, alcohol ingestion, betel nut chewing, nitrosamines, mycotoxin-containing diet, diet low in micronutrients or anti-oxidants, and a high-starch diet without fruits and vegetables (Blot 1994; Kato et al. 2000; Stoner et al. 2000; Phukan et al. 2001; Sepehr et al. 2001; Layke and Lopez 2006; Kollarova et al. 2007). The geographic variation in the incidence of esophageal cancer might stem, in part, from these epidemiological risk factors. For example, available findings indicate higher and more frequent nitrosamine content in Asian foods than in Western foods (Hotchkiss 1989). Other research has provided evidence that some nitrosamines contaminate the salted fish traditionally consumed in southern China (Huang et al. 1978). It is generally accepted that such environmental carcinogens can affect the genetic status of host cells, including aberrant regulation of multiple genes in esophageal cells leading to uncontrolled growth and, ultimately, esophageal cancer (Stoner et al. 2000). A recent study summarized genetic changes observed in esophageal SCCs as follows: (i) alterations in tumor-suppressor genes leading to altered DNA repair, cell proliferation, and apoptosis; (ii) disruption of the G1/S cell cycle checkpoint and loss of cell cycle control; and (iii) alterations in oncogene function leading to deregulation of cell signaling pathways (Stoner and Gupta 2001). The genetic alterations most commonly associated with esophageal SCCs include p53 tumor-suppressor gene mutation (Hollstein et al. 1991). In response to DNA damage, levels of short-lived p53 protein rapidly increase, principally through stabilization of this protein, and DNA binding of p53 is activated (Levine et al. 1991; Lowe et al. 1993; Cox et al. 1995). Activation of p53 induces or inhibits the expression of more than 150 genes that mediate cell cycle arrest, apoptosis, and DNA repair processes (El-Deiry 1998; Vousden 2000; Bode and Dong 2004). Oncogenic studies have revealed that p53 mutations, most of which occur in the DNA-binding domain, are among the most common genetic alterations in human cancers (El-Deiry et al. 1992; Cho et al. 1994). Cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors play important roles in controlling major checkpoints in the mammalian cell cycle (Matsumoto et al. 1999). In the cell cycle, the transition from G1 phase to S phase is believed to be the most important checkpoint (Hunter and Pines 1994). We previously found that in esophageal SCCs, G1 phase cyclin, cyclin D1, its catalytic partner, CDK4, and CDK inhibitors such as p27 and p57 could contribute to tumor progression (Matsumoto et al. 1999; Ishikawa et al. 1998; Anayama et al. 1998; Matsumoto et al. 2000).

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Other alterations of oncogene function in esophageal cancer include amplification of c-erbB1, c-erbB 2, c-myc, c-ras, Int-2/hst-1, and EGFR and elevations in hTERT, BMP-6, iNOS, COX-2, and β-catenin levels (Stoner et al. 2000; McCabe and Dlamini 2005). One or more of these genetic alterations undoubtedly contribute to the carcinogenesis, growth, and invasive and metastatic potential of esophageal cancer.

iNOS Expression in Esophageal Cancer Although iNOS was originally identified in murine macrophages, it has also been detected in tumor cells (Tanaka et al. 1999). Previous in vitro studies demonstrated that several tumor cell lines express iNOS, albeit at different levels (Massi et al. 2001). Many in vivo studies also revealed that iNOS is frequently expressed in many types of carcinomas, including gastric cancer, colon cancer, breast cancer, and hepatocellular carcinoma (Chen et al. 2006; Yagihashi et al. 2000; Vakkala et al. 2000; Sun et al. 2005). Inducible NOS expression in esophageal SCC was first described by Tanaka et al. (1999), who detected iNOS expression in 87.7% of human surgical specimens examined. In our immunohistochemical study of 105 cases of esophageal SCC, 56 (53.3%) tumors exhibited cytoplasmic staining for iNOS (Fig. 18.1), including 17 (16.2%) with homogeneous and intense immunostaining and 39 (37.1%) with heterogeneous staining (Matsumoto et al. 2003). This discrepancy in iNOS positivity of tumors between the study by Tanaka et al. and our study might be due to differences in methods of scoring of iNOS expression. In non-neoplastic esophageal epithelium, iNOS is weakly observed in basal and parabasal cells adjacent to cancer (Matsumoto et al. 2003). In breast, both malignant and benign lesions exhibited iNOS immunoreactivity at high incidence, though both the intensity and the quantity of iNOS expression were significantly higher in the samples of cancer when compared with benign lesions (Bulut et al. 2005). Inducible

Fig. 18.1 Tumor cells exhibited cytoplasmic staining for iNOS

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NOS has also been shown to be markedly expressed in 60% of human adenomas and in 20–25% of colon carcinomas, while expression of it was either low or absent in surrounding normal tissues (Ambs et al. 1998; Chhatwal et al. 1994). Moreover, in ovarian cancer, iNOS activity has been found to be confined to tumor cells and not present in normal tissue (Thomsen et al. 1994). These findings suggest a possible role of iNOS in malignancy.

Association Between iNOS and p53 in Esophageal Cancer Previous studies have revealed a positive correlation between iNOS and p53 status in various types of tumors including gastric adenocarcinoma (Wang et al. 2005), head and neck cancer (Gallo et al. 2003), colorectal cancer (Ambs et al. 1999), and endometrial carcinoma (Cinel et al. 2002). In the case of esophageal SCC, we found that of 62 p53 protein-positive tumors, 40 (63.5%) were positive for iNOS, and of 43 p53 protein-negative tumors, 27 (62.8%) tumors were negative for iNOS, revealing a significant relationship between iNOS immunoreactivity and p53 protein overexpression (p = 0.0058) (Matsumoto et al. 2003). Various explanations of the association between expression of iNOS and p53 may be possible. Forrester et al. (1996) demonstrated that expression of wild-type p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of iNOS expression through the binding of p53 protein to a promoter site on the iNOS gene and inhibition of promoter activity. These findings are consistent with the hypothesis of the existence of a negative feedback loop in which high concentrations of NO produced by iNOS cause DNA damage, resulting in induction of p53 accumulation to minimize DNA damage and p53-mediated transrepression of iNOS gene expression (Ambs et al. 1997). This is one possible explanation for the p53 staining representing wild-type p53 protein accumulation. On the other hand, other findings suggest that NO may promote carcinogenesis by inactivating the tumor-suppressor p53. Although cells containing wild-type p53, when exposed to excess NO, accumulate p53 protein, excess NO also modifies p53 protein through nitration of tyrosine residues in it, so that its specific DNA binding and subsequent biological activity are lost (Calmels et al. 1997; Chazotte-Aubert et al. 2000). However, in human cancer, mutation of the p53 tumor-suppressor gene is the most common genetic alteration (Matsumoto et al. 2006). The finding that esophageal SCCs with a mutated p53 gene had significantly more frequent p53 protein overexpression than those with wild-type p53 in our previous study suggests that overexpressed p53 protein in esophageal SCC is a mutant form in a comparatively large number of cases (Matsumoto et al. 2003). We also found a significant relationship between iNOS expression and p53 gene status. Of 34 iNOS-positive tumors, 23 (67.6%) carried a p53 gene mutation, and of 17 iNOS-negative tumors, 12 (70.6%) had a wild-type p53 gene. Fisher’s exact probability test revealed a significant relationship between iNOS immunoreactivity and p53 mutation frequency (p = 0.0163). Because loss of p53 function with mutation of the p53 gene may result

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in failure to suppress iNOS expression, resulting in DNA damage (Nguyen et al. 1992), with induction of angiogenesis (Montrucchio et al. 1997) and suppression of immune responses (Lejeune et al. 1994), tumor-associated nitric oxide production appears to promote cancer progression by providing a selective growth advantage to tumor cells expressing mutant p53. Moreover, further mutation of p53 may occur in the presence of high NO levels (Lala and Chakraborty 2001). A positive correlation between total NOS activity and frequency of p53 mutation was reported for colorectal cancer and lung adenocarcinoma; the predominant mutation was G:C to T:A transversion at CpG dinucleotides in the former study, and the G:C to A:T transition in the latter (Ambs et al. 1999; Fujimoto et al. 1998). In esophageal SCC, however, we failed to find a significant relationship between iNOS expression and type of p53 mutation (Matsumoto et al. 2003). Although it has been debated whether iNOS is overexpressed due to p53 mutation or expressed iNOS induces p53 mutation, it is clear that iNOS activity might be associated with p53 alteration and contribute to tumorigenesis.

iNOS Expression and Prognosis The prognostic significance of iNOS expression in cancer is controversial. Earlier studies of various malignancies found iNOS positivity to be related to better prognosis, poor prognosis, or no relation to prognosis at all (Anttila et al. 2007). Dueñas-Gonzalez et al. (1997) found a very strong correlation between the presence of iNOS and axillary lymph node metastasis of breast cancer, suggesting that NO synthesis and the resulting increase in vascularity in tumor play a role in facilitating metastasis. In addition, in an immunohistochemical study of 111 cases of breast cancer, a strong association was found between iNOS positivity of stromal cells and/or tumor cells and local microvascular density and apoptotic indices, both of which are indicators of poor prognosis (Vakkala et al. 2000). In hepatocellular carcinoma (HCC), NO produced by iNOS could modulate MMP-9 production and contribute to tumor cell angiogenesis and invasion and metastasis (Sun et al. 2005). Strong expression of iNOS and MMP-9 in HCC may thus be helpful in evaluating recurrence of HCC and be predictive of poor prognosis. In vivo tumor models have provided more convincing evidence for a direct role of NO in tumor growth. Growth of iNOS-19 tumor, a clone derived from human DLD-1 colon adenocarcinoma that has been transfected to express iNOS constitutively, was abrogated by treatment with a selective iNOS inhibitor, 1400 W (Thomsen et al. 1997). More recently, Takahashi et al. (2008) found that ONO-1714, an iNOS-selective inhibitor, can suppress the development of invasive cancer expressing iNOS in the N-nitrosobis(2-oxopropyl)amine-treated hamster pancreas. On the other hand, other reports have suggested an inhibitory effect of iNOS on tumor progression or a significant association between iNOS expression and improvement of survival. Dong et al. (1994) were one of the first groups to

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demonstrate a significant anti-tumor effect of iNOS. They found that, in murine K-1735 melanoma, iNOS expression is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis. In breast carcinoma, apoptosis appeared to be associated with iNOS expression (Vakkala et al. 2000). Stimulation of iNOS and enhanced NO activity in pancreatic tumors also led to apoptosis and inhibition of tumor growth (Kong et al. 2001). Study of nasopharyngeal carcinoma revealed that patients with low iNOS expression in tumor samples had higher rates of recurrence and distant metastasis after radiation therapy (Jayasurya et al. 2003). In vitro studies have suggested that, in the case of colorectal and non-small cell lung cancer, patients with tumors exhibiting high expression of iNOS and other NOS isozymes had better survival rates (Puhakka et al. 2003; Ropponen et al. 2000). To the best of our knowledge, only one study, our own, has shown a relationship between iNOS protein expression and prognosis in the case of esophageal SCCs (Matsumoto et al. 2003). In that study, we immunohistochemically examined 82 cases of esophageal SCC and calculated cumulative survival rates of patients with iNOS-positive SCC and iNOS-negative SCC by the Kaplan–Meier method. The cumulative survival curves for patients with esophageal SCC are shown in Fig. 18.2. The cumulative 5-year survival rates for cases with and without iNOS expression were 33.7 ± 7.8 and 15.9 ± 7.0%, respectively. With the log-rank test, however, iNOS protein status was not found to be a potential prognostic marker (p = 0.2012). Our results are in line with those presented in other reports, in which no significant associations between expression of iNOS and prognostic value were found in pancreatic, pharyngeal, and cervical carcinomas (Kong et al. 2002; Oka et al. 2003; Pukkila et al. 2002). The positive association between

Fig. 18.2 Cumulative Kaplan–Meier survival curves for patients with esophageal SCCs divided by iNOS immunoreactivity

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p53 and iNOS described above suggests that iNOS expression might affect carcinogenesis rather than tumor progression, in association with alteration of p53 in esophageal SCC. Some of the discrepancies among these findings might reflect differences in experimental protocols used or might be a consequence of cell type-specific differences and differences in the genetic makeup of cells. It is also clear that these discrepancies stem, in part, from conflicting dual roles of NO in tumor progression. For example, high levels of NOS expression may be cytotoxic for tumor cells, mediating NO-induced apoptosis, whereas low levels of expression can have the opposite effect and promote tumor growth (Lala 1998). When transfected with a functional iNOS, the highly metastatic murine melanoma cell line K-1735, which expresses low levels of iNOS, exhibits poor growth and survival in vitro as well as in vivo (Xie et al. 1995). On the other hand, another study showed that a human colonic adenocarcinoma cell line engineered to generate NO led to continuous increase in growth and vascularity of tumors when transplanted in nude mice (Jenkins et al. 1995). It is likely that iNOS overexpression in the melanoma cell line in the former study led to much greater NO production than that in the human colonic adenocarcinoma cell line examined in the latter study. In addition, the presence of other regulators, such as p53, might dictate the role of NO in tumor biology (Lala 1998). As noted above, cytostatic effects of NO are observed in tumors with wild-type p53 because of NO-mediated accumulation of p53 protein, while tumors with mutant p53 exhibit resistance to NO-mediated injury and promotion of tumor growth in the presence of endogenous NO.

Conclusion Inducible NOS expression in esophageal SCC might not itself affect tumor progression and not be useful as an indicator of prognosis. However, it appears that iNOS might be associated with p53 alteration and contribute to carcinogenesis rather than tumor progression. Additional studies involving a large number of tumors and experimental tumor models are needed to confirm the present findings.

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Chapter 19

Prognostic Significance of Nitrative DNA Damage in Infection- and Inflammation-Related Carcinogenesis Yusuke Hiraku and Shosuke Kawanishi

Abstract Chronic infection and inflammation considerably contribute to environmental carcinogenesis. Reactive oxygen and nitrogen species generated from inflammatory and epithelial cells may play an important role in inflammation-related carcinogenesis by causing DNA damage. We demonstrated that 8-nitroguanine, a potentially mutagenic nitrative DNA lesion, was formed at the sites of carcinogenesis in various clinical specimens and animal models. 8-Nitroguanine was formed in bile duct epithelium of animals and patients infected with the liver fluke Opisthorchis viverrini, which causes cholangiocarcinoma. 8-Nitroguanine formation was also observed in gastric gland epithelial cells of patients with Helicobacter pylori infection and in hepatocytes of patients with chronic hepatitis C. The formation of this DNA lesion in atypical cells of patients with cervical intraepithelial neoplasia, caused by human papilloma virus, was increased with its grade. 8-Nitroguanine formation in cancer cells of patients with nasopharyngeal carcinoma (NPC), associated with Epstein-Barr virus (EBV) infection, was significantly stronger than that in nasopharyngeal epithelium of EBV-mediated nasopharyngitis. Moreover, in patients with soft tissue tumor, strong 8-nitroguanine formation was closely associated with a poor prognosis. On the basis of these findings, we have proposed that 8-nitroguanine can be used as a biomarker to evaluate the risk of infection- and inflammation-related carcinogenesis and the prognosis of cancer patients. In this chapter, we discuss the significance of 8-nitroguanine in inflammation-related carcinogenesis and tumor development. Keywords Carcinogenesis · DNA damage · Inducible nitric oxide synthase · Inflammation · 8-Nitroguanine

S. Kawanishi (B) Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan e-mail: [email protected]

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_19, C Springer Science+Business Media, LLC 2010

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Introduction Epidemiological and experimental studies have provided evidence indicating that a variety of infectious agents constitute one of the main causes of cancer (Coussens and Werb 2002; IARC 2003a). The International Agency for Research on Cancer (IARC) has estimated that approximately 18% of cancer cases worldwide is attributable to infectious diseases (IARC 2003a). Viruses are the principal infectious agents, which are associated with particular tumor types. Certain parasites and bacteria are also involved in carcinogenesis. These infectious agents can cause chronic inflammation and contribute to 1.6 million cases of infection-related malignancies per year (Table 19.1). In addition to infection, many other physical, chemical, and immunological factors induce chronic inflammation (Coussens and Werb 2002; Ohshima et al. 2003). Many malignancies arise from areas of infection and inflammation (Balkwill and Mantovani 2001: Coussens and Werb 2002). In a recent review, chronic inflammation is estimated to account for approximately 25% of human cancers (Hussain and Harris 2007).

Table 19.1 The burden of cancer caused by infectious agents worldwide [Adapted and modified from IARC “World Cancer Report” (IARC 2003)] Infectious agents

IARC classificationa

Cancer site

Bacterial infection H. pylori

1

Stomach

490,000

5.4

Viral infection HPV High-risk types Low-risk types HBV, HCV EBV

Cervix and other sites

550,000

6.1

1 2B 1 1

390,000 99,000

4.3 1.1

HHV-8 HTLV-1

2A 1

Liver Lymphoma Nasopharyngeal carcinoma Kaposi sarcoma Leukemia

54,000 9,000

0.6 0.1

1

Bladder

2,700

0.1

1

Intra- and extrahepatic bile ducts

Parasitic infection Schistosoma haematobium Liver flukes Opisthorchis viverrini Clonorchis sinensis

% of cancer cases worldwide

800

2A Total infectionrelated cancers Total cancers in 1995

a Group

Number of cancer cases

1,600,000 9,000,000

17.7 100

1, carcinogenic to humans; group 2A, probably carcinogenic to humans; group 2B, possibly carcinogenic to humans

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Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are capable of causing damage to various cellular constituents, such as nucleic acids, proteins, and lipids. These reactive species are considered to play an important role in carcinogenesis by causing oxidative and nitrative DNA damage (Hussain et al. 2003; Kawanishi and Hiraku 2006; Kawanishi et al. 2006). ROS can induce the formation of oxidative DNA lesions, including 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG) (Evans et al. 2004; Kawanishi et al. 2001, 2002; Wiseman and Halliwell 1996). Because misincorporation of adenine occurs opposite 8-oxodG during DNA synthesis and leads to G → T transversions (Bruner et al. 2000; Shibutani et al. 1991), this DNA lesion is considered to be potentially mutagenic. Unlike RNS, ROS are generated from multiple sources, including not only inflammatory cells but also carcinogenic chemicals and their metabolites and electron transport chains in mitochondria (Kawanishi and Hiraku 2006). Nitric oxide (NO) is generated particularly during inflammation via the expression of inducible nitric oxide synthase (iNOS) in inflammatory and epithelial cells (Fig. 19.1). Excess NO production plays a crucial role in an enormous variety of pathological processes including cancer (Hussain et al. 2003; Ohshima et al. 2003). NO reacts with superoxide (O2 •– ) to form highly reactive peroxynitrite (ONOO– ), capable of causing nitrative and oxidative DNA damage. Incubation of DNA fragments with ONOO− induced the formation of 8-oxodG (Inoue and Kawanishi 1995). Yermilov et al. have demonstrated that 8-nitroguanine, a nitrative

Fig. 19.1 Proposed mechanism of mutation via 8-nitroguanine formation mediated by chronic inflammation

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DNA lesion, is formed via the interaction of ONOO− with guanine in an in vitro experimental system (Yermilov et al. 1995a, b). 8-Nitroguanine formed in DNA is chemically unstable, and thus can be spontaneously released, resulting in the formation of an apurinic site (Yermilov et al. 1995b). The apurinic site forms a pair preferably with adenine during DNA synthesis, leading to G → T transversions (Loeb and Preston 1986) (Fig. 19.1). Cells deficient in subunits of DNA polymerase ζ were hypersensitive to NO, and translesion DNA synthesis past apurinic site mediated by this polymerase might contribute to extensive point mutations (Wu et al. 2006). It has also been reported that adenine is preferentially incorporated opposite 8-nitroguanine during DNA synthesis mediated by the DNA polymerases η and κ (Suzuki et al. 2005). In the ONOO− -treated supF shuttle vector plasmid, which was then replicated in Escherichia coli, the majority of mutations occurred at G:C base pairs, predominantly involving G → T transversions (Juedes and Wogan 1996; Kim et al. 2005). Therefore, 8-nitroguanine is a potentially mutagenic DNA lesion leading to carcinogenesis as well as 8-oxodG. In an in vivo experimental system, Akaike et al. have shown that 8-nitroguanine is formed via inflammation in the lung tissues of mice with viral pneumonia (Akaike et al. 2003). We focused on the importance of 8-nitroguanine formation in infection- and inflammation-related carcinogenesis, and first demonstrated that this DNA lesion was formed at the site of carcinogenesis in various animal models and clinical specimens of patients with cancer-prone inflammatory diseases (Kawanishi and Hiraku 2006; Kawanishi et al. 2006). 8-Nitroguanine was formed in bile duct epithelial cells and inflammatory cells in the liver of hamsters infected with the liver fluke Opisthorchis viverrini (OV) (Pinlaor et al. 2003). In clinical specimens, 8-nitroguanine formation was observed in gastric gland epithelial cells of patients with Helicobacter pylori infection (Ma et al. 2004). In chronic hepatitis C patients, 8-nitroguanine was formed in hepatocytes, and this DNA lesion was markedly decreased after interferon therapy (Horiike et al. 2005). 8-Nitroguanine was also formed in oral epithelium of patients with premalignant and inflammatory diseases, oral lichen planus (OLP) (Chaiyarit et al. 2005) and leukoplakia (Ma et al. 2006). Moreover, we have recently demonstrated that 8-nitroguanine participates not only in the onset of carcinogenesis but also in tumor progression and poor prognosis of cancer patients. Here, we review our recent studies on 8-nitroguanine formation during inflammation-related carcinogenesis and discuss its prognostic significance.

Liver Fluke Infection and Cholangiocarcinoma Infection with the liver fluke Opisthorchis viverrini (OV) is a major risk factor of cholangiocarcinoma especially in the north-eastern region of Thailand (Haswell-Elkins et al. 1994; IARC 1994). Although the incidence of intrahepatic cholangiocarcinoma is generally low, approximately 70% of OV-induced cholangiocarcinoma occurs in the intrahepatic bile ducts (Uttararvichen et al. 1996). We investigated DNA damage in the liver of hamsters with OV infection as

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a model of inflammation-related carcinogenesis. OV infection induced the formation of 8-nitroguanine in the bile duct epithelial cells (Pinlaor et al. 2003). This is the first in vivo study demonstrating that 8-nitroguanine is formed at the sites of inflammation-related carcinogenesis. Double immunofluorescence staining revealed that the immunoreactivities of 8-nitroguanine and 8-oxodG were prominently observed in inflammatory cells in the acute phase (days 21–30), whereas these DNA lesions remained in bile duct epithelial cells in the chronic phase (day 180) (Pinlaor et al. 2004a). Repeated OV infection augmented the formation of these DNA lesions and iNOS expression in the epithelium of bile ducts compared with single infection (Pinlaor et al. 2004b). Moreover, the treatment of OV-infected hamsters with the antiparasitic drug praziquantel dramatically reduced DNA damage and iNOS expression (Pinlaor et al. 2006). On the basis of these findings, we have proposed that 8-nitroguanine can be used as a biomarker to evaluate the risk of inflammation-related carcinogenesis and the efficacy of drug treatment. In parasitic infection, the local parasite-specific inflammatory response may participate in carcinogenesis. OV antigen was distributed in bile duct epithelial cells of the liver, and the presence of the antigen was associated with inflammatory cell infiltration in OV-infected hamsters (Sripa and Kaewkes 2000). Relevantly, the antibody level against OV antigen was associated with the severity of hepatobiliary disease and cholangiocarcinoma (Akai et al. 1994). Our in vitro study using the RAW 264.7 macrophage cell line and in vivo study using OV-infected hamsters have revealed that the OV antigen induces an inflammatory response through the Toll-like receptor (TLR)-2-mediated pathway leading to the expression of iNOS and cyclooxygenase2 (COX-2) via activation of nuclear factor-κB (NF-κB) (Pinlaor et al. 2005b, 2006). TLRs activate homologous signal transduction pathways leading to nuclear localization of NF-κB/Rel-type transcription factors (Kawai and Akira 2007; O’Neill and Greene 1998) and subsequently participate in inflammation-related carcinogenesis (Rakoff-Nahoum and Medzhitov 2009). NF-κB is a key player in inflammation that regulates the expression of various genes involved in controlling inflammatory responses including iNOS expression (Karin and Greten 2005; Kundu and Surh 2008). Noteworthy, NF-κB participates in the promotion and progression of inflammation-related cancer (Karin 2006; Pikarsky et al. 2004). Recently, it has been reported that extracellular proteoglycans upregulated in carcinoma tissues activate myeloid cells through TLR2 to stimulate metastasis (Kim et al. 2009). Therefore, TLR-mediated inflammatory responses may participate in tumor progression, and the molecules involved in this process could be potential therapeutic targets for inflammation-related cancer.

Human Papilloma Virus and Cervical Cancer Cervical cancer is the second most common cancer among women worldwide, being the most common among women in many regions of developing countries (IARC 2003b). A wealth of evidence has led to the conclusion that virtually all cases of

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cervical cancer are attributable to persistent infection by a subset of human papilloma virus (HPV) (Bosch et al. 2002; Chen and Hunter 2005; IARC 1995; Munoz et al. 2003; Sisk and Robertson 2002; Tindle 2002). In 2008, Dr. Harald zur Hausen was awarded the Nobel Prize for the discovery of HPV. IARC previously determined that HPV-16 and HPV-18 are carcinogenic to humans (group 1) (IARC 1995). Recently, other high-risk types of HPV (HPV-31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66) have also been evaluated as group 1 carcinogens, although these HPV types can differ by an order of magnitude in risk for cervical cancer (IARC 2007). Low-risk HPV types (HPV-6 and HPV-11), capable of causing condyloma acuminatum, have been evaluated to be possibly carcinogenic (group 2B) (IARC 2007). HPV infection is a necessary event preceding the development of premalignant lesions in the cervical epithelium, referred to as cervical intraepithelial neoplasia (CIN), which can partially progress to cancer (Castle and Giuliano 2003). At the molecular level, the HPV oncoproteins E6 and E7 are known to participate in HPVinduced cervical carcinogenesis by inactivating the tumor suppressor gene products, p53 and Rb, respectively (Chen and Hunter 2005; von Knebel Doeberitz 2002). However, several studies have demonstrated that these oncoproteins are not sufficient to transform human cells, because additional cellular events are required for cervical carcinogenesis (Duensing and Munger 2004). Recently, it has been proposed that inflammation plays an integral role in HPV-mediated cervical carcinogenesis. Epidemiological studies have revealed that cervical inflammation in women infected with HPV is associated with cervical neoplasia (Castle et al. 2001; Castle and Giuliano 2003), although it is still unclear whether HPV infection alone induces the inflammatory states. There are reports showing that co-infection with HPV and other pathogens increases the risk of cervical cancer. Among the HPV DNA-positive women, seropositivity of herpes simplex virus-2 was associated with increased risks of invasive cervical carcinoma (Smith et al. 2002). Molecular epidemiological studies have revealed the overexpression of cyclooxygenase-2 (COX-2) in cervical cancer (Kim et al. 2004; Kulkarni et al. 2001), which mediates cancer development via various pathogenic events, including inflammatory responses, inhibition of apoptosis and angiogenesis (Kundu and Surh 2008; Warner and Mitchell 2004; Williams et al. 2000). Therefore, chronic inflammation may play an important role in cervical carcinogenesis. We examined 8-nitroguanine formation in biopsy specimens of patients with CIN and condyloma acuminatum. Double immunofluorescence labeling revealed that 8-nitroguanine was formed in atypical epithelial cells (Fig. 19.2). Samples from patients with high-grade CIN (CIN2 and 3), most of whom were infected with highrisk HPV subtypes, exhibited significantly more intense staining for 8-nitroguanine than those with condyloma acuminatum (Hiraku et al. 2007). p16 overexpression has been observed in patients with CIN and cervical cancer, and proposed as a biomarker of cervical neoplasia (Klaes et al. 2001; Sano et al. 1998; Wang et al. 2004). The HPV E7 oncoprotein binds to Rb, leading to the release of the transcription factor E2F (von Knebel Doeberitz 2002), which induces the expression of p16-related transcripts (Khleif et al. 1996). We observed the overexpression of p16 in cervical epithelial cells from samples of patients with both CIN and

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Fig. 19.2 Formation of 8-nitroguanine and 8-oxodG in biopsy specimens of patients with cervical dysplasia, assessed by double immunofluorescence staining. Paraffin sections were incubated with the primary antibodies (rabbit polyclonal anti-8-nitroguanine and mouse monoclonal anti-8oxodG antibodies), and then with the secondary antibodies (Alexa 594-labeled goat anti-rabbit IgG and Alexa 488-labeled goat anti-mouse IgG antibodies). Sections were examined using an inverted laser scan microscope. 8-Nitroguanine (red) and 8-oxodG (green) colocalized in the nuclei of atypical epithelial cells (yellow). In the patients with condyloma acuminatum, little or no DNA damage occurred. Scale bar = 50 μm

condyloma acuminatum. In contrast, samples from patients with condyloma acuminatum exhibited little or no 8-nitroguanine formation (Hiraku et al. 2007). These results suggest that high-risk HPV types participate in the formation of nitrative DNA lesions, which leads to dysplastic changes and carcinogenesis; in contrast, p16 appears to be merely a marker of HPV infection regardless of the HPV type. Thus, 8-nitroguanine is a more suitable and promising biomarker for evaluating the risk of inflammation-mediated cervical carcinogenesis than p16. Thus, inflammationmediated DNA damage, which precedes the genomic abnormalities caused by HPV oncoproteins, may play an important role in cervical carcinogenesis.

Epstein-Barr Virus and Nasopharyngeal Carcinoma Nasopharyngeal carcinoma (NPC) and Burkitt lymphoma are strongly associated with Epstein-Barr virus (EBV) infection (IARC 1997) and account for approximately 1% of world cancer cases (IARC 2003a). NPC is an epithelial tumor with a high prevalence in southern China, where the incidence rate is about 25–50 per 100,000 people-year and 100-fold higher than that in the Western world (Jeannel et al. 1999; McDermott et al. 2001). Latent EBV infection is detected in cancer cells of virtually all cases of undifferentiated NPC in endemic regions (IARC 2003a; Tsai

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et al. 1998). Among viral genes, the latent membrane protein 1 (LMP1) may play the most important role in EBV-mediated NPC. In addition to EBV infection, environmental and dietary factors are considered to contribute to NPC carcinogenesis. The traditional foods of southern China, such as salted fish and other preserved food containing volatile nitrosoamines, are important carcinogenic factors of NPC (Lo et al. 2004). Moreover, Chinese-style salted fish contains substances capable of causing activation of latently infected EBV (IARC 1997). IARC has evaluated that Chinese-style salted fish is a group 1 carcinogen (IARC 1993). An epidemiological study has revealed that herbal medicine use increases the risk of NPC, probably through reactivation of EBV or a direct promoting effect on EBV-transformed cells (Hildesheim et al. 1992). A phorbol diester, an EBV-activating substance, was identified in the soil collected from under Sapium sebiferum (Takeda et al. 1991). We obtained biopsy specimens of nasopharyngeal tissues from patients with nasopharyngitis and NPC in southern China, and examined 8-nitroguanine formation. 8-Nitroguanine formation and iNOS expression were observed in epithelial cells of EBV-positive patients with chronic nasopharyngitis, and interestingly their intensities were significantly stronger in cancer cells in NPC patients (Ma et al. 2008). Strong 8-nitroguanine formation also occurred in inflammatory cells in

Fig. 19.3 Proposed mechanism of 8-nitroguanine formation mediated by EBV infection

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stroma of NPC patients. In EBV-negative subjects, no or little DNA damage was observed. We have examined the mechanism by which EBV infection causes 8nitroguanine formation in nasopharyngeal epithelial cells. Lo et al. have demonstrated that EGFR interacts with STAT3 in the nucleus, leading to transcriptional activation of iNOS (Lo et al. 2005). EGFR and phosphorylated STAT3 were strongly expressed in cancer cells of NPC patients, suggesting that a STAT3dependent mechanism is important for NPC carcinogenesis. IL-6 was expressed mainly in inflammatory cells, mainly macrophages, of nasopharyngeal tissues of EBV-infected patients. EBV-encoded RNAs (EBERs) and LMP1 were detected in cancer cells from all EBV-infected patients. In LMP1-expressing cultured cells, EGFR was accumulated in the nucleus, and IL-6 treatment induced the expression of phosphorylated STAT3 and iNOS and the formation of 8-nitroguanine (Ma et al. 2008). These data suggest that nuclear accumulation of EGFR and STAT3 activation by IL-6 plays the key role in iNOS expression and resultant DNA damage, leading to EBV-mediated NPC. The proposed mechanism of EBV-induced carcinogenesis in nasopharyngeal epithelial cells is shown in Fig. 19.3.

DNA Damage and Prognosis of Patients with Soft Tissue Sarcoma The involvement of nitrative DNA damage in tumor development and prognosis has attracted our interest. Malignant fibrous histiocytoma (MFH) is one of the most common soft tissue sarcomas occurring in adult patients (Weiss and Goldbum 2002). MFH has been proposed to be a lesion accompanied with inflammatory responses. The expression of cytokines in inflammatory MFH may account for the local inflammatory infiltrate and the aggressive nature observed in malignant cells (Melhem et al. 1993). In the early phase of experimentally induced rat sarcoma, an inflammatory reaction characterized by an infiltration of lymphocytes, monocytes, and macrophages occurred (Richter et al. 1999). These findings raised the possibility of inflammatory responses playing a role in the pathogenesis of MFH. We examined the distribution of DNA lesions and the expression of inflammation-related molecules in surgical specimens from MFH patients. Immunohistochemical staining revealed that the formation of 8-nitroguanine occurred in MFH tissue specimens, and iNOS, NF-κB, COX-2, and hypoxiainducible factor (HIF)-1α were colocalized with 8-nitroguanine in MFH tissues (Hoki et al. 2007a,b). On the other hand, little or no immunoreactivity of 8nitroguanine and these proteins was observed in adjacent non-tumor tissues. It is noteworthy that the statistical analysis using the Kaplan–Meier method demonstrated strong 8-nitroguanine staining to be associated with a poor prognosis (Hoki et al. 2007a). Tumor cells adapt to hypoxia by increasing the synthesis of HIF-1αmediated transcription of various genes, including iNOS (Harris 2002). On the other

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hand, an increase in NO production through iNOS expression induces the accumulation and activation of HIF-1α (Mateo et al. 2003; Thomas et al. 2004). Therefore, reciprocal activation between HIF-1α and iNOS during tumor growth mediates persistent DNA damage, which may contribute to poor prognosis of cancer patients. A recent study has demonstrated that IκB kinase (IKK)-β, which is involved in NF-κB activation, is required for HIF-1α protein accumulation under hypoxia in cultured cells and animals (Rius et al. 2008), whereas NF-κB has been reported to be regulated under hypoxia in an HIF-1α-dependent manner (Walmsley et al. 2005). Thus, reciprocal activation of HIF-1α and NF-κB may be also involved in DNA damage and carcinogenesis. Noteworthy, we have demonstrated that in cholangiocarcinoma patients, 8-nitroguanine and 8-oxodG are formed in cancerous tissues to a much greater extent than in the adjacent non-cancerous tissues. Moreover, these DNA lesions in cancerous and adjacent tissues were associated with neural and lymphatic tumor invasion (Pinlaor et al. 2005a). Therefore, 8-nitroguanine may participate not only in the onset of carcinogenesis but also in tumor progression, and could be used as a promising biomarker to evaluate the prognosis of cancer patients.

Conclusion and Future Perspectives In relation to inflammation-related carcinogenesis, we examined nitrative DNA damage in experimental animal models and clinical specimens. We have first demonstrated that 8-nitroguanine was specifically induced at sites of carcinogenesis under various inflammatory conditions. Experimental evidence has suggested that 8-nitroguanine is a mutagenic DNA lesion, which preferentially leads to G → T transversions (Suzuki et al. 2005; Yermilov et al. 1995b). Indeed, this type of mutation has been observed in vivo in the ras gene (Bos 1988) and the p53 tumor suppressor gene in lung and liver cancer (Hsu et al. 1991; Takahashi et al. 1989). These findings imply that DNA damage mediated by ROS and RNS may participate in carcinogenesis via activation of protooncogenes and inactivation of tumor suppressor genes. Moreover, 8-nitroguanosine is a highly redox-active molecule that strongly stimulated O2 •− generation (Sawa et al. 2003). 3-Nitrotyrosine, a biomarker of inflammation, is capable of inducing oxidative DNA damage via redox reaction (Murata and Kawanishi 2004). Therefore, such inflammation-derived products may serve as mutagens for host tissues and participate in carcinogenesis via additional oxidative stress. We summarized the proposed mechanism of DNA damage and carcinogenesis mediated by chronic inflammation in Fig. 19.4. Various pathogenic agents, including bacteria, viruses, parasites, and other environmental factors, induce various inflammatory responses and the production of reactive oxygen and nitrogen species from inflammatory and epithelial cells. iNOS expression is regulated by transcription factors, including NF-κB, STAT, and HIF-1α, and NO can activate these transcription factors. HIF-1α is upregulated in a hypoxic environment during tumor growth and participates in tumor progression. Collectively, various molecular events

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Fig. 19.4 Proposed mechanism of 8-nitroguanine formation and tumor development

converge to nitrative stress, and resulting DNA damage contributes to accumulation of genetic alterations in tissues throughout the carcinogenic process. Particularly, 8-nitroguanine formation would participate in inflammation-related carcinogenesis as the common mechanism and can be used as a potential biomarker. Recently, it has been reported that 8-nitro-cGMP, formed by the reaction of RNS with cGMP, regulates the redox-sensor signaling proteins, via S-guanylation of cysteine sulphydryls, and mediates an adaptive response to oxidative and nitrative stress (Sawa et al. 2007). S-Nitrosylated proteins, formed via reaction of thiols with NO or its reactive metabolites, possess cytoprotective properties (Ishima et al. 2007). In inflammation-related carcinogenesis, nitrated or nitrosylated molecules may participate in protection of initiated cells bearing genetic alterations due to oxidative and nitrative DNA damage and contribute to tumor development. Recently, we measured the levels of 8-oxodG in the urine of OV-infected subjects and cholangiocarcinoma patients using an electrochemical detector coupled to high-performance liquid chromatography (HPLC-ECD). Urinary 8-oxodG levels were significantly increased in the order of healthy subjects < OV-infected patients < cholangiocarcinoma patients. The urinary 8-oxodG levels in OV-infected patients significantly decreased to comparable levels to those in healthy subjects after praziquantel treatment (Thanan et al. 2008). Establishment of the methods for quantitative analysis of 8-nitroguanine in biological or clinical specimens could be useful to evaluate the risk of inflammation-related carcinogenesis. However, 8-nitroguanine formed in DNA is chemically unstable and thus this characteristic

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may hamper its quantitative analysis. Therefore, free 8-nitroguanine released from DNA in urine might be available for quantitative analysis as a biomarker. Urinary 8-nitroguanine was measured using an HPLC-ECD coupled with immunoaffinity purification, and the amount of 8-nitroguanine excreted in urine was associated with cigarette smoking (Sawa et al. 2006). Development of quantitative analysis of 8-nitroguanine by liquid chromatography with mass spectrometry and glyoxal derivatization has been reported (Ishii et al. 2007). Establishment of quantitative analysis of 8-nitroguanine in biological samples, such as blood and urine, would be useful for prediction of carcinogenic risks derived from chronic inflammation and contribute to cancer prevention and improvement of prognosis of cancer patients. Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare of Japan.

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Part VII

Therapeutic Applications of Nitric Oxide

Chapter 20

Nitric Oxide-Releasing Molecules for Cancer Therapy and Chemoprevention S. Anand and Gregory R.J. Thatcher

Abstract The Nobel Prize for Medicine and Physiology in 1998 to Furchgott, Ignarro, and Murad provided a new impetus to research in prodrugs of the messenger molecule, nitric oxide (NO). Although more famously known for its cardiovascular roles in the treatment of angina (nitrates) and more recently erectile dysfunction R (Viagra ), NO has been extensively researched within the realm of cancer biology. This review briefly highlights the various chemical classes of NO donors with potential utility in cancer chemotherapy and chemoprevention. These molecules release NO upon bioactivation and thus engender the therapeutic rationale of using them in a clinical setting. Bearing these factors in mind, and in the light of current research findings, special emphasis is given to a newer generation of NORMs (nitric oxide-releasing molecules), viz., NONOates, NO-NSAIDs, and furoxans. Keywords Cancer biology · Cancer chemoprevention · Furoxans · Nitric oxide releasing molecule (NORM) · NONOate · NO-NSAID · Quinone methide

Introduction Cancer has been the scourge of mankind for centuries and remains a leading cause of death, despite man’s achievements in biomedical research, chemotherapy, radiation therapy, and surgical methods. In the United States, cancer, second only to heart disease, is the largest killer. A total of 1,437,180 new cancer cases and 565,650 deaths from cancer are projected to occur in the United States in 2008 (Jemal et al. 2008). It is estimated that there will be more than 12 million new cancer cases in 2007 worldwide, and estimates for total cancer deaths in 2007 are 7.6 million (about 20,000 cancer deaths a day) (Parkin et al. 2005). By 2050, the global burden

G.R.J. Thatcher (B) Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_20, C Springer Science+Business Media, LLC 2010

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is expected to grow to 27 million new cancer cases and 17.5 million cancer deaths, simply due to the growth and aging of the population. The need to find both new therapies and chemopreventive agents is as urgent as ever. Given the dichotomous role of nitric oxide (NO) in cancer and cell biology, it is perhaps not surprising that NO donors are in development for both chemoprevention and chemotherapy. NO is a messenger molecule that mediates diverse physiological processes such as vasodilation, smooth muscle relaxation, neurotransmission, platelet aggregation, and immune response (Furchgott 1999; Ignarro 1999; Murad 1999). The mechanisms governing the biological activity of NO are yet unclear, but the physiological effects of NO are directly related to local concentration in cells and the length of exposure (Wink and Mitchell 2003; Wink et al. 1998). In the cardiovascular system (CVS), NO helps maintain vascular homeostasis through several mechanisms, which include vasodilation, inhibition of platelet aggregation, and modulation of leukocyte adhesion to the endothelium. NO plays a key role in learning and memory formation in the central nervous system (CNS) and acts to mediate neurotransmitter stimuli in the peripheral nervous system (PNS). In these areas, NO is thought to be produced in nanomolar (nM) concentrations and triggers its effects by activating soluble guanylyl cyclase (sGC). NO can be produced in micromolar (μM) concentrations in macrophages and other immunomodulatory cell types, as part

Fig. 20.1 Chemical classes of NORMs. It should be noted that several classes have been reported to provide “NO bioactivity” or to release nitrogen oxides in a variety of redox states equivalent to NO, NO+ , and NO¯. Most classes generate inorganic nitrite as a major primary or secondary product

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of pathogen defense systems, exerting cytotoxic effects by formation of reactive nitrogen species (RNS). The evidence supporting the role of NO in promoting cancer development and progression, countered by evidence supporting a diametrically opposite role, presents challenges to researchers in evaluating the therapeutic applicability of strategies based upon NO-releasing molecules (NORMs): first, to assess the role of NO in various aspects of cancer (e.g., angiogenesis, metastasis, immune response) and, second, to predict from in vitro data, the effects of NO on carcinogenesis in vivo. The fact that these challenges have not been largely overcome is exacerbated by the complex chemistry of these agents and significant differences existing within the various chemical classes of NORMs. There are a variety of NORMs that may be useful in either chemoprevention or chemotherapy (Fig. 20.1). However, it is the structural variation in these molecules that modulates the chemical reactivity and consequently dictates both the type of nitrogen oxide released and the kinetics of release.

The Status of NORMs in Cancer Treatment The past decade has seen increasing interest in NORMs, particularly in chemoprevention using the nitrate and furoxan classes, and in chemotherapy using the diazeniumdiolate class. NORMs are in development for cancer treatment for exploiting three general properties: (1) Chemotherapy: The act of NO release through apoptotic, genotoxic, or other cell death pathways can be harnessed selectively toward malignant neoplastic cells or tissues in chemotherapy, or used to enhance chemo/radiotherapy, or to attenuate resistance to such therapy. (2) Chemoprevention: The act of NO release induces chemopreventive, cytoprotective, and anti-oncogenic mechanisms in normal cells or inhibits cancer promotion and malignant transformation, or is anti-proliferative toward pre-cancerous cells. (3) Side effect attenuation: The act of NO release attenuates a side effect of a drug that is acting as the direct chemotherapeutic or chemopreventive agent. Of all such agents reported, the NO-NSAIDs (nitric oxide non-steroidal antiinflammatory drugs) have progressed the furthest clinically, having been the focus of a large NIH-supported phase II chemoprevention clinical trial for colorectal cancer (CRC). The NO-ASA (nitric oxide-acetyl salicylic acid) in question, NCX-4016, was subsequently found to be genotoxic in vitro, and therefore the trial was terminated before yielding any data (Di Napoli and Papa 2003; Rigas 2007). NO-ASA is a hybrid NORM, i.e., the NO-releasing moiety is conjugated to a known drug (ASA) via a linker. Hybrid NORMs, in which the NO-releasing moiety is conjugated to a bioactive carrier, represent the dominant contemporary approach in this area of drug discovery and development and therefore hold the greatest prospect of success.

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Hybrid NORMs and Prodrugs Hybrid NORMs come closest to the classic definition of a prodrug in medicinal chemistry (Albert 1958): a drug or lead molecule chemically modified to correct a flaw in the molecule. Chronic use of NSAIDs has fatal side effects owing to severe gastrointestinal (GI) damage. The NO-releasing chemical modification of ASA and other NSAIDs was intended to mollify the GI toxicity associated with NSAIDs and later was also argued to be beneficial in attenuating NSAID cardiotoxicity (Becker et al. 2004; Davies et al. 1997; Wallace et al. 1995). NO-NSAIDs are thus classic prodrugs: NSAIDs with an improved side effect profile. NO-NSAIDs were designed for use in arthritis and other pain treatments, but extension to CRC chemoprevention was logical, given the promise of NSAIDs, such as ASA, in CRC clinical trials (Rosenberg et al. 1998). NO-NSAIDs were conceived as prodrugs to utilize NO release for side effect attenuation. However, growing evidence for the chemotherapeutic and chemopreventive attributes of NO release has led researchers to attempt to harness these properties. The NO releaser in these hybrid NORMs is, in the parlance of chemotherapeutics, a drug “warhead.” Hybrid NORMs have come to dominate the field; therefore, before reviewing the individual classes of NORMs, it is important to examine the concept of the hybrid NORM. Six classes of hybrid NORMs are depicted in (Fig. 20.2), incorporating three classes of NO-releasing warheads: NONOate, nitrate, and furoxan. Despite the very different chemistry of NO release from these three warheads, and the diverse structures of the hybrid NORMs, there are many common features. All five hybrids presented (A–C, E, F) contain a salicylate NSAID as the bioactive carrier. In four of these (A–C, F) a labile linker connects to the warhead, liberating the bioactive carrier by esterase bioactivation. In the fifth (E) the labile linker is a disulfide that can be bioactivated by thiol–disulfide exchange. At least three of these five hybrid NORMs have reported anti-inflammatory activity in the ability to inhibit cytokine release independent of the bioactive NSAID carrier: B8 (Turnbull et al. 2008); GT-094 (Hagos et al. 2008); NCX-4016 (Fiorucci et al. 2000), although this is queried by Turnbull et al. (2008). Two of these five are apoptotic cytotoxins, viz., NCX-4040 (Fabbri et al. 2005) and GIT-27NO (Mijatovic et al. 2008), as is the sixth hybrid NORM, JS-K (Kitagaki et al. 2008; Kiziltepe et al. 2007). JS-K may seem the odd man out among the depicted hybrid NORMs since the dinitrophenolate carrier is designed to engender selective bioactivation by GST; this “targeted” carrier is in fact a bioactive carrier, comparable to the GST inhibitor, 2,4-dinitrochlorobenzene (CDNB). Molecular deconstruction of the hybrid NORMs, GT-094, NCX-4040, and NCX4016 (Dunlap et al. 2007, 2008; Hulsman et al. 2007), demonstrated that the chemopreventive and chemotherapeutic properties were reproduced in appropriate control compounds that did not possess an NO-release warhead. Similarly, the biological activity of JS-K has been proposed to result in part from NO-independent actions of the carrier (Ren et al. 2003). In all these cases of NO-independent activity, the hybrid NORMs have a common feature, the provision of a thiophilic electrophile

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Fig. 20.2 Six representative hybrid NORMs containing furoxan, nitrate, and NONOate warheads. Each molecule incorporates at least two component parts: a bioactive carrier and a warhead. All examples shown have labile linkers that are bioactivated to release bioactive molecules including the warhead. In most cases the warhead requires further bioactivation to release NO bioactivity. The figure illustrates the uniform dependence on reactions with biological thiols and products that will contribute to varied levels of cellular oxidative stress

able to deplete GSH and to modify biomolecules including redox sensor proteins. Figure 20.2 illustrates that all six representative hybrid NORMs, including furoxans, are likely to modify biological thiols and to induce some level of oxidative stress. The prospects and obstacles for hybrid NORMs in cancer treatment may be summarized thus:

(1) Hybrid NORMs require one or more bioactivation process which may be utilized to enhance selectivity for cells and tissues; multiplicity of bioactivation processes and the diffusibility of NO are not compatible with specificity. (2) The majority of hybrid NORMs provides NO bioactivity without acting as pure NO donors; the exception being the NONOates, which are true NO donors. NO release is rarely measured for NORMs; the common use of surrogates (NO2− , oxyHb→metHb) has often produced confusion in the field and is not compatible with the drive toward agents that release specific nitrogen oxides, such as NO itself or nitroxyl (NO)− (King 2005; Miranda et al. 2005).

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(3) Hybrid NORMs are expected to induce cellular oxidative stress by release of NOx , thiophilic electrophiles, and through depletion of bioactivating species. High levels of oxidative stress and NO release are compatible with induction of apoptosis, DNA damage, and a role in chemotherapy, while lower levels of oxidative stress and lower rates of NO release are commensurate with induction of multiple chemopreventive pathways. (4) Despite uncertainty over mechanisms of bioactivation to nitrogen oxide species, the chemistry of NORMs can be controlled to provide agents with predictable properties, as either chemopreventive or chemotherapeutic agents. The rigorous use of appropriate control compounds is essential to define a role for the NO release warhead. (5) The importance of bioactivation for all hybrid NORMs requires care in extrapolating results obtained in vitro (where bioactivation may be at very low levels) to predict properties in vivo. For example, despite the demonstrated in vitro impotence of the warheads of GT-094 and NO-ASA, the GI protective effects of the nitrate warheads will be important in vivo. (6) The comparison of a hybrid NORM drug versus a comparable combination therapy has rarely been studied in vivo; for example, a cocktail of NSAID + ISDN (isosorbide dinitrate) compared to an NO-NSAID. In one case where a comparison was made with NCX 4016 the cocktail was equi-efficacious (Brzozowska et al. 2004). In several hybrid NORM designs, such as JS-K and GT-094, bioactivation to release the warhead bioactivity is contingent upon the presence of the linker (Shami et al. 2003; Zavorin et al. 2001). (7) Drugs that are FDA approved (classical nitrates, nitrites, molsidomine) or have completed early phase clinical trials in other indications (NO-NSAIDS) have, at present, the best prospects for clinical use. The in vitro genotoxicity of several examples of most classes of NORMs is a dampener to use in cancer chemoprevention, even in the absence of any evidence of carcinogenicity.

Individual Classes of NORMs Classical Organic Nitrates and Nitrites The oldest class of clinically used NO donors are the organic nitrates (R-ONO2 ) (Fig. 20.3), which are nitric acid esters of mono and polyhydric alcohols, including glyceryl trinitrate (GTN; A.1), pentaerithrityl tetranitrate (PETN; A.2), isosorbide dinitrate (ISDN; A.3), and isosorbide-5-mononitrate (ISMN; A.4). Metabolites of GTN, viz., glyceryl mononitrate (GMN) and glyceryl dinitrate (GDN), are pharmacologically active, but less potent than GTN. Organic nitrates are indicated in the treatment of angina, myocardial infarction, congestive heart failure, and the control of blood pressure. The release of NO from these compounds is dependent upon bioactivation by a number of mechanisms, reviewed fully elsewhere (Thatcher 2007; Thatcher et al. 2004). Several

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Fig. 20.3 Classical nitrate and nitrite drugs

metabolic enzymes, including aldehyde dehydrogenase 2 (ALDH2), xanthine oxidoreductase (XOR), glutathione-S-transferase (GST), cytochrome P450 oxidase (CYP450 oxidase), and cytochrome P450 reductase (CYP450 reductase), are capable of de-nitrative metabolism of nitrates, yielding predominantly inorganic nitrite. No enzyme, at physiological amounts, has been demonstrated to catalyze the threeelectron reduction of a classical nitrate, at pharmacological concentrations, to NO. Indeed, there is evidence in tissues that GTN does not itself release NO (Kleschyov et al. 2003; Núñez et al. 2005), and therefore it is imprudent to consider organic nitrates as simple NO donors. The phenomenon of nitrate tolerance has been raised as a deterrent to development of chronic nitrate therapies (Keefer 2005); nevertheless, the hybrid nitrates (NO-NSAIDs) have been studied in several clinical trials without comment on the observation of nitrate tolerance. To some extent, clinical nitrate tolerance is a red herring, because it is readily controlled by dosing, and is easily avoided for mononitrates, such as ISMN. However, the importance of formulations and dosing must be considered early in any nitrate drug development program (Manabe et al. 2001; Stauch et al. 1990; Thadani et al. 1994). The question of cross-tolerance with other NO-release warheads is unclear. For example, crosstolerance with furoxans is reported in some (Feelisch et al. 1992) but not in other cases (Bohn et al. 1995; Civelli et al. 1996). Organic nitrites, such as iso-amyl nitrite (IAN; A.5), can be prepared by esterifying alcohols using nitrous acid or other nitrosating agents such as nitrosonium salts or alkyl nitrites. Like their nitrate cousins, these compounds are also used in clinical settings as potent vasodilators to manage angina or hypertension. NO release from nitrites simply requires homolysis or single electron reduction, whereas protein nitrosation (associated directly with NO bioactivity) is directly accomplished by nitrites. Upon hydrolysis, nitrites release nitrite ion.

N-Nitrosamines Nitrosating agents such as nitrous acid, dinitrogen trioxide, dinitrogen tetraoxide, nitrosyl chloride, and alkyl nitrites are sources of NO+ (nitrosonium ion), which react with secondary amines to yield the corresponding N-nitrosamines. Similarly, secondary amides yield their corresponding N-nitrosoamides, ureas yield N-nitrosoureas, and carbamates, the N-nitrosocarbamates.

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Fig. 20.4 Representative set of the two classes of N-nitrosamines

N-nitrosamines have been divided into two classes (Wang et al. 2002), as shown in Fig. 20.4: Class I includes dialkyl, alkyl aryl, and diaryl derivatives, and Class II includes the N-nitrosamines, N-nitrosoureas, N-nitrosocarbamates, N-nitrosoguanidines, and N-acyl-N-nitroso compounds. Class II nitrosamines act as trans-nitrosating agents toward thiol nucleophiles, readily yielding nitrosothiols (Oh and Williams 1989). N-nitrosamines are potential NO•/NO+ donors via cleavage of the N−NO bond, N-aryl-N-nitrosamines being viewed as more prone to release of NO. As S-nitrosating agents, N-nitrosamines have been reported to be inhibitors of cysteine containing enzymes. For example, N-methyl-N-nitrosoanilines, such as dephostatin, inhibit protein tyrosine phosphatases, papain, and caspases (Guo et al. 1998, 2001). Some N-nitrosoureas, e.g., 2-chloroethylnitrosoureas, have been used as alkylating agents; their DNA alkylating abilities as well as potential genotoxicity and mutagenicity have restricted their use in a clinical setting (Lucas et al. 1999, 2001).

N-Hydroxy-N-nitrosamines These compounds offer a significant advantage over their non-hydroxylated cousins, as they do not decompose into carcinogenic nitrosamines. Three important examples

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Fig. 20.5 N-hydroxy-N-nitrosamines as potential drugs

of N-hydroxyamines (Fig. 20.5) are cupferron (C.1), alanosine (C.2), and dopastatin (C.3), while Keefer has synthesized N-oxy-N-nitrosamines (Hrabie and Keefer 1999; Lehmann 2000) that are thermodynamically stable and are slow NO donors (C.4). Cupferron and its derivatives have a NONOate moiety (NONO group) attached directly to carbon, instead of oxygen or nitrogen; many of these analogs release NO via enzymatic oxidation (Alston et al. 1985). Tuning the NO donor scaffold allows for the preparation of donors with potential for selective targeting (Hrabie and Keefer 2002). The instability of cupferron and its derivatives can be a liability in delivery of NO, and this led to the synthesis of O-alkylated derivatives of cupferron and neocupferron (Hou et al. 2000a, b). These derivatives are more stable than their parent compounds and can be used to deliver NO in a more controlled manner by chemical, enzymatic, or photochemical liberation of the O-protecting group.

Diazeniumdiolates As shown in Fig. 20.6, a diazeniumdiolate (D.1) contains a structural unit R-N(O)==NOR1 , where R and R1 are substituents, the R–N bond is covalent and the O−R1 linkage is either covalent or ionic (Keefer 2005). Diazeniumdiolates, where the R group is an amine nitrogen, are commonly referred to as NONOates. Structurally cognate with the N-hydroxy N-nitrosamines, the role of their protecting groups (R1 ) (Saavedra et al. 1992) at the O2 oxygen is to increase stability and to exert some control over NO release rates (D.2). These diazeniumdiolates are perhaps the only chemical class among the NORMs that act as true NO donors, the vast majority releasing close to the theoretical yield of 2 mol equivalents of NO. The substituent R moderates prodrug stability and the rate of NO release, while the substituent R1 functions as a labile carrier in a tripartite double prodrug (Wermuth 2003). By altering the nature of the amine substituent R, both electronically and sterically, varying NO release rates can be obtained from the diazeniumdiolates (Keefer et al. 1996). The compounds SPER/NO (D.5) and DEA/NO (D.6) are widely used as rapid- and medium-release rate NO donors. Both compounds are genotoxic in the AMES test (Maragos et al. 1991, 1993; Martínez et al. 2001), although the mechanism of DNA damage is not fully defined (Schmutte et al. 1994). Potential

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Fig. 20.6 Diazeniumdiolates. (a) Formation of diazeniumdiolates. (b) Representative set of “NONOates” with varying NO-releasing rates. (c) Structure-based modification of JS-K leads to PABA/NO. (d) 5-Fluorouracil and three derivatives bearing the NO-releasing group(s)

advantages of diazeniumdiolates as NORMs include (1) stability; (2) the potential for site selective release of NO; (3) alterable release rates; and (4) control of the duration of exposure (Keefer 2003). Thus, diazeniumdiolates represent viable drug leads for NORM-based chemotherapy.

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The P1 isoform of the phase II detoxification enzyme glutathione-S-transferase (GST-P1) is often expressed at higher levels in certain tumor cells (Tew and Gate 2001; Townsend and Tew 2003b). GST binds glutathione (GSH) catalyzing its reaction with aromatic substrates such as 1-chloro-2,4-dinitrobenzene (CDNB), forming a stable product that inhibits the enzyme (Armstrong 1991; Armstrong 1997). The nucleofugality of a diazeniumdiolate is comparable to the chloride of CDNB (Saavedra et al. 2001) and this has been exploited for drug design. By installing an aryl group similar to CDNB as the R1 substituent, the O2 -arylated diazeniumdiolate JS-K (D.7) becomes an excellent substrate for GST, releasing the NONOate group, and thereby NO, upon activation (Shami et al. 2003). While this may be one of the pathways by which JS-K is cytotoxic, results from in vitro studies (Shami et al. 2003; Udupi et al. 2006) that have been conducted on the anti-tumor effects of JS-K point to the existence of multiple modes of action (Ren et al. 2003) including GST inhibition and GSH depletion. Despite the lack of clear-cut evidence about its mode of action, JS-K is a worthy lead in anti-cancer drug discovery (Shami et al. 2006). GST-P1 is a widely studied member of the GST family because overexpression in tumor tissues may contribute to resistance to several anti-cancer drugs (O’Brien et al. 2000; Townsend and Tew 2003a). Based upon the JS-K scaffold, the synthesis of PABA-NO (D.8) was designed to increase relative GST-P1 selectivity. Addition of bulk in the form of an aryl ester was designed to reduce binding of PABA/NO to GST-α, the predominant liver isoform and increase selectivity for GST-P1 (Ji et al. 2008), thus resulting in increased selectivity for tumor cells while minimizing NO release and GST inhibition in the liver. Overexpression of GST-P1 results in higher levels of cytolytic NO from PABA/NO (Findlay et al. 2004). PABA-NO was reported to be carcinostatic toward human ovarian cancer xenografts in mice (Findlay et al. 2004). 5-Fluorouracil (5-FU, D.9) remains even now one of the most used anti-tumor agents for treating solid tumors, such as breast and colorectal cancers. However, it is toxic and poorly tumor selective, causing adverse events in bone marrow, GI tract, central nerve system, and skin. Novel prodrugs of 5-FU possessing a broader spectrum of anti-tumor activity and less toxicity have been sought (Malet-Martino et al. 2002), including three 5-FU-NONOate hybrid NORMs (Cai et al. 2003). In the DU-145 human prostate cancer cell line, compounds D.10, D.11, and D.12 were found to be 2×, 3×, and 3× more potent than D.9, respectively. In the HeLa cell line, compound D.12 held similar potency as D.9, while D.10 and D.11 were 2× and 5× more toxic than 5-FU.

Nitrosothiols (RSNOs) Many biological functions associated with NO bioactivity are directly associated with endogenous formation of nitrosothiols (or alkylthionitrites), which are represented by the general formula RSNO. The biological activities of RSNOs were well studied even before the physiological functions of NO were realized and received greater attention when the role of NO in biological systems was established

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(Al-Kaabi et al. 1982; Ignarro and Gruetter 1980; Williams 1985). Application of the cell permeable nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP; E.1), resulted in a decrease in the invasive phenotype in DU-145, PC-3, and RWPE1derived human prostate cancer cell lines in vitro (Chaiswing et al. 2008). At a pH between 6.0 and 8.0 at 37◦ C, in the presence of transition metal chelators, SNAP has a half-life of about 6 h (Singh et al. 1996). As SNAPs represent a unique type of NO donor molecule, distinct from the endogenous nitrosothiol, S-nitrosoglutathione (GSNO), research has focused on the design and synthesis of novel analogs with promising pharmacological properties. Wang and co-workers designed sugar-SNAPs (Fig. 20.7; E.2–E.7) on the basis of the known active transport by the glucose transporter family of transmembrane proteins of monosaccharides bearing homology to glucose (Bell et al. 1993; Mellanen et al. 1994). Sugar-SNAPs comprise an NO-release aglycone warhead (nitrosothiol) and a carbohydrate bioactive carrier that accentuates the aqueous solubility, cell penetration, and potentially allows targeting of cells with upregulated glucose transport, such as cells with increased energy demand for glucose. Theoretically, these attributes may increase selective NO cytotoxicity. Sugar-SNAPs were also reported to show increased half-life and a more prolonged NO release compared to SNAPs (Hou et al. 1999b; Ramirez et al. 1996). The cytotoxicity of glucose-2-SNAP (E.2) and fructose-2-SNAP (E.4) were evaluated and compared against E.1 using DU-145 human prostate cells in vitro. E.2 was found to be 4× more cytotoxic than E.1, while E.4 was found to be 13× more cytotoxic than E.1 (Hou et al. 1999a).

Furoxans and Benzofuroxans While the wide spectrum of biological activities for compounds containing the furoxan or benzofuroxan heterocycle were reported over three decades ago (Ghosh

Fig. 20.7 S-nitroso-N-acetylpenicillamine (SNAP) and representative set of sugar-SNAPs

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et al. 1981; Stuart 1975), there has been a recent renewed interest among medicinal chemists in these compounds that bear the 1, 2,5-oxadiazole-2-oxide warhead, on account of emerging reports of bioactivity against different targets. Cerecetto and González have produced an excellent review of these molecules, but the few examples in cancer treatment reflect the relative lack of attention to this indication (Cerecetto and González 2007). Early work by Ghosh and Whitehouse (Ghosh and Whitehouse 1968) reported the anti-leukemic properties of certain benzofuroxans and benzofurazans, with nitrobenzofuroxans described as “thiol-neutralizing agents” (Whitehouse and Ghosh 1968). This led to the critical evaluation of 1,2,5-oxadiazoles (furoxans), benzo-1,2,5-oxadiazoles (benzofuroxans), and their N-oxide derivatives as potential anti-cancer agents. However, the majority of benzofuroxans are not expected to be NO-release warheads on the basis of a thorough study by Medana and coworkers using appropriate analytical tools to measure separately NO2 ¯ , N2 O, and NO release (Medana et al. 1999). In the presence and absence of thiols, polyfuroxan derivatives such as BF.1 released NOx (NO 20%; N2 O 11%), but benzofuroxans did not. However, benzofuroxans were reported to react directly with oxyHb and possibly directly to activate sGC. A recent example of a pro-apoptotic benzofurazan GST inhibitor, and potential anti-cancer agent, is BF.2 (Turella et al. 2005). Although it has been speculated that furazans are oxidized to furoxans in vivo, there is little evidence that furazan activity is caused by NO release. However, both furazans and furoxans are thiophilic and it is likely that these warheads share NO-independent anti-neoplastic effects through modification of biological thiols, a mechanism speculated on in 1992 (Feelisch et al. 1992). Furoxans demonstrate thermal and acid stability but are susceptible to break down in basic conditions. It has been proposed that nucleophiles, in particular thiolate anions, attack furoxans to form either NO or nitrosothiols (Feelisch et al. 1992; Medana et al. 1994; Sako et al. 1998). A detailed mechanism was speculated upon (Feelisch et al. 1992), but the only products rigorously identified from a reaction of impramidil (F.8) with thiol were nitrosothiol, nitrite, nitrate, and a bis-oxime (F.9) (Fig. 20.8). Feelisch’s early paper is seminal in the field, but the speculative NO release mechanism presented has not been greatly expanded upon. Although there is no doubt that furoxans are NO-release warheads, there is only one report of direct detection of NO by Buonsanti and co-workers (Buonsanti et al. 2007). The identification of a second organic product, 4-thio-5-aminoisoxazole, led to the speculation that NO is released subsequent to nitrosothiol formation (Medana et al. 1994). Degradation of a large series of furoxans to inorganic nitrite in the presence of 5 mM cysteine showed reasonable correlation with vascular tissue relaxation, but with a number of outliers (Ferioli et al. 1995). Further work has identified an intramolecular acid catalyst of furoxan degradation that allows some control of reactivity and consequently activity (Sorba et al. 1997). Gasco has pioneered the development of NO-release furoxan warheads (Gasco et al. 2004), evaluated for their anti-trypanosomal (Cerecetto et al. 1999) and antiinfective properties (Calvino et al. 1980). Compounds F.1 and F.2 were found to possess interesting anti-infective properties, but with mutagenic effects, while

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Fig. 20.8 Structures of various furoxans and nitric oxide releasing molecules (NORMs)–I

the corresponding furazan, F.3, retained the mutagenicity, though not the antiinfective property. F.4 was found to possess anti-microbial activity. The closely related furoxan vasodilator CHF-2363 (F.6) and the water-soluble analog, (F.7), are genotoxic, mutagenic, and cytotoxic at micromolar concentrations, whereas for CAS-1609 (F.5) these effects were seen only at much higher concentrations (Balbo et al. 2008). The furazan analogs were neither genotoxic nor cytotoxic. The linkage of potential anti-cancer effects of furazans to the release of genotoxic levels of NO was made by Feelisch and others. Furoxan sulfonylhydrazones (F.10 and F.11) (Fig. 20.9) were developed and shown to inhibit colony formation by HeLa cells (Fruttero et al. 1989). Compounds F.12, F.13, and F.14 showed moderate cytotoxicity, though the phenyl furoxan

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Fig. 20.9 Structures of various furoxans and nitric oxide releasing molecules (NORMs)–II

derivatives displayed greater activity than methyl furoxan derivatives. Chemical modifications of these structures yielded F.15, which is a hybrid NORM incorporating a DNA-intercalator (Cerecetto et al. 2000), and F.16, which was the most cytotoxic agent reported (Boiani et al. 2001). The DNA-intercalating bioactive carrier failed to enhance the toxicity of the warhead. The pro-apoptotic, anti-proliferative hybrid NORM, GIT-27NO, has already been introduced [Fig. 20.2 (f)] (Mijatovic et al. 2008). This potential anti-cancer agent also significantly inhibited the expression of the transcription repressor and apoptotic-resistant factor YY1 and amplified the p53 anti-oncogene. GIT-27NO, like the hybrid NORM B.8 introduced earlier [Fig. 20.2 (c)], incorporates antiinflammatory bioactive carriers. Hybrid furoxans incorporating a 2 -deoxyuridine bioactive carrier were reported to be highly toxic toward a battery of cancer cell lines in addition to normal human fibroblasts (Moharram et al. 2004). NO release was not measured, but degradation to inorganic nitrite in the presence of 18 mM cysteine did not correlate with toxicity. Importantly, the hybrid NORMs did not show higher toxicity than the furoxan warhead alone and it was proposed that DNA alkylation, rather than NO release, was responsible. Hybrid furoxan NORMs containing oleanolic acid as a putative hepatoprotective carrier have been reported to be toxic at sub-micromolar concentrations toward the frequently studied HepG2 human hepatocarcinoma cell line (Chen et al. 2008). This chapter reported positive data in animal models for compound F.17. Perplexingly, the warhead in these hybrids was 1-phenylsulfonylfuroxan, commonly studied because of its known NO-release properties; yet zero release of inorganic nitrite and nitrate was reported in all cell lines with the exception of HepG2 and Hep3b.

Diazetine Dioxides Diazetine dioxides (Fig. 20.10), or cyclic azo dioxides, are four-membered heterocycles prepared by the peroxide-mediated oxidation of the corresponding bis-hydroxy

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Fig. 20.10 Diazetine dioxides: general structures, including resonance hybrids

amines (Greene and Gilbert 1975). They can also be considered to be highly strained intramolecular C-NO dimers that decompose in aqueous solutions to liberate two moles of NO per mole of compound, at physiological temperature and pH. Intracellularly, the rate of NO release from these compounds or their halogenated derivatives depends upon the concentration of thiolate anions (Kirilyuk et al. 1998). Diazetine dioxides exhibit strong vasorelaxant and anti-aggregative properties, attributable to the activation of sGC to release NO (Severina et al. 1994, 1996). C-Nitroso NORMs developed by Toone and co-workers (Chakrapani et al. 2009; Gooden et al. 2005) bear some similarity in that these compounds exist in equilibrium between intermolecular C-NO dimers and the monomeric form. These compounds are interesting in that they may release NO without the need for bioactivation, although the reaction products may be more complex than those arising from diazeniumdiolates.

Sydnonimines The sydnonimines (Fig. 20.11) are a class of meso-ionic heterocycles and an important class of NO donors. Molsidomine (H.4) and N-acyl derivatives of sydnonimines (H.1; R = acyl groups) are stable solid compounds, which can be stored away from light at room temperature. Molsidomine has been used in Europe to treat angina, and one of its main advantages over GTN is the absence of tolerance. Molsidomine is a prodrug that is readily deacetylated in the liver and elsewhere to give an active metabolite SIN-1 (H.5), a potent vasorelaxant and anti-platelet agent owing to its ability to release NO, essentially spontaneously, owing to base catalysis in aqueous

Fig. 20.11 Sydnonimines: general structures, including resonance hybrids. Also depicted are Molsidomine and its active metabolite SIN-1

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solution. Curiously, SIN-1 is used almost exclusively in biomedical research as a peroxynitrite donor, because its breakdown in normoxic aqueous solution leads to both NO release and generation of an electron that is captured by O2 ; the concomitant production of NO and O2¯ yields ONOO¯. Thus in vitro (1) SIN-1 induces lipid peroxidation in contrast to NO, which is an inhibitor of lipid peroxidation (Nicolescu et al. 2006; Thatcher et al. 2001), and (2) SIN-1 was determined to be an oxidative mutagen giving a response differing spectrum of responses compared to the true NO donors, DEA/NO and SPER/NO (Martínez et al. 2001). In accord with these findings, SIN-1 was reported to induce mitochondrial membrane depolarization, DNA strand breaks, and apoptosis in human lymphoblastoid cell lines (Li et al. 2002). Nevertheless, there has been little activity toward developing sydnonimines for cancer therapy.

NO-NSAIDs Combining two pharmacophores with distinct yet complementary biological activities into a single molecule is a well-known approach to drug design (Baldwin 1987; Christiaans and Timmerman 1996; Nicolaus 1983). Two NO-ASA hybrid NORMs that attempt to exploit this design principle are introduced above (Bolla et al. 2005). NSAIDs elicit their pharmacological effects by the inhibition of prostaglandin (PG) synthesis, which includes inhibition of the cyclo-oxygenase (COX), COX-1 and COX-2 isoforms. However, the long-term use of NSAIDs results in an increased risk of GI bleeding and ulcerogenesis. NO has been clearly shown to mimic many properties of PGs; literature evidence suggests that NO and PGs are both involved in stimulation of mucosal secretion and healing mucosal injury (Kitagawa et al. 1990; Lopez-Belmonte et al. 1993; MacNaughton et al. 1989). Among their adverse effects, NSAIDs have been demonstrated to cause a reduction in mucosal blood flow and neutrophil adhesion to the vascular endothelium. NO has been shown to attenuate both these effects. Colorectal cancer (CRC) is the second largest type of cancer prevalent in the United States. In the 1970s, Jaffe (Jaffe 1974) and Bennett (Bennett et al. 1977) reported that PG E2 was found in higher concentration in colorectal tumor tissue, which led to the hypothesis that NSAIDs could be employed in its treatment. Extensive studies demonstrated that COX-2 inhibitors produced a marked inhibition of carcinogenesis in rodents. NO-NSAIDs are comparable to regular NSAIDs in their ability to inhibit PG synthesis (Mitchell et al. 1993). Three NONSAIDs, NO-ASA, NO-sulindac, and NO-ibuprofen were shown to reduce the growth of cultured HT-29 colon adenocarcinoma cells much more effectively than the corresponding NSAIDs (Williams et al. 2001). The metabolic steps by which NO-NSAIDs produce NO have not yet been established (Govoni et al. 2006). However, since the “linker” between the NSAID and NO-release warhead was assumed to be inert, the superior pharmacological properties of NO-NSAIDs, compared to the parent NSAID, were ascribed to NO (Burgaud et al. 2002a, b; Wolfe et al. 1999).

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The NO-ASA family of NO-NSAIDs, notably NCX 4016 [Fig. 20.2(a)] and NCX 4040 [Fig. 20.2(b)], has been extensively researched. These two isomeric hybrid NORMs differ only in the substitution of the linker group. Hulsman et al. was the first to comment that in NCX-4040 the “presumed invisible” linker is in fact solely responsible for the anti-tumor effect of the molecule and that both the NOrelease warhead and the ASA are passive bystanders (Hulsman, Medema et al. 2007). The linker warhead moiety is efficiently bioactivated to a quinone methide thiophilic electrophile, in simile with much earlier literature reports (Myers and Widlanski 1993). Molecules in which the nitrate group of NO-ASA was replaced by a comparably good leaving group (termed X-ASA; Fig. 20.12) showed very similar properties in vitro with respect to activity: (1) cytotoxic/genotoxic; (2) antiproliferative; (3) chemopreventive (ARE activation – phase II enzyme induction); and (4) the anti-inflammatory (Dunlap et al. 2007, 2008).

Fig. 20.12 Comparison of hybrid NORMs and X-ASA molecules; the nucleofugality and isomeric location determines the formation of the reactive quinone methide, a thiophilic electrophile

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The biological activity of NCX-4040 and the corresponding X-ASA cognates was accounted for by bioactivation to a quinone methide, which was shown to trap GSH and inferred to react with other quinone targets such as redox sensor proteins and DNA. Similarly, but perhaps more surprisingly, the biological activity of NCX4016 was replicated by X-ASA, that possessed neither the NO-releasing warhead nor the ability to be bioactivated to a quinone methide. It was concluded that all NO-NSAIDs containing a hydroxybenzyl linker would manifest biological activity resulting from bioactivation to electrophilic metabolites. Rigas has recently confirmed the work of Hulsman and others (Dunlap et al. 2007, 2008; Hulsman et al. 2007) that an X-ASA derivative demonstrates similar anti-proliferative activity to the parent NO-ASA in a variety of cell lines (Zhao et al. 2009). However, it should be strongly emphasized that conclusions on NCX-4016 and NCX-4040 do not extend to NO-NSAIDs that contained different linkers and moreover that care should be taken in extrapolating observations to activity in vivo. Nevertheless, Rigas has recently reported (Rigas and Kozoni 2008; Rigas and Williams 2008; Sun et al. 2009) that the balance between deleterious effects and chemopreventive potential of NO-ASA and X-ASA with electrophile-release warheads can be struck by combining our understanding of metabolic bioactivation and chemical structure toward the design clinical candidates.

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Tew, K.D. and Gate, L. (2001). Glutathione S-transferases as emerging therapeutic targets. Expert Opin. Ther. Targets 5, 477–489. Thadani, U., Maranda, C.R., Amsterdam, E., Spaccavento, L., Friedman, R.G., Chernoff, R., Zellner, S., Gorwit, J., and Hinderaker, P.H. (1994). Lack of pharmacologic tolerance and rebound angina pectoris during twice-daily therapy with isosorbide-5-mononitrate. Ann. Intern. Med. 120, 353–359. Thatcher, G.R.J. (2007). Organic nitrates and nitrites as stores of No, radicals for life: The various forms of nitric oxide. In van Fassen, E., and Vanin, A. (ed.). Amsterdam, Elsevier. Thatcher, G.R.J., Nicolescu, A., Reynolds, J.N., Zavorin, S.S., and Toader, V. (2001). Inhibition of lipid peroxidation by nitrates and nitrites. 222nd ACS National Meeting. Chicago, IL, United States, August 26–30, 2001. Abstracts of Papers TOX-022. Thatcher, G.R.J., Nicolescu, A.C., Bennett, B.M., and Toader, V. (2004). Nitrates and No release: Contemporary aspects in biological and medicinal chemistry. Free Radical Biol. Med. 37, 1122–1143. Townsend, D.M. and Tew, K.D. (2003a). Cancer drugs, genetic variation and the glutathione-Stransferase gene family. Am. J. Pharmacogeno. 3, 157–172. Townsend, D.M. and Tew, K.D. (2003b). The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 22, 7369–7375. Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M.R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A.M. (2005). Proapoptotic activity of new glutathione S-transferase inhibitors. Cancer Res. 65, 3751–3761. Turnbull, C., Marcarino, P., Sheldrake, T., Lazzarato, L., Cena, C., Fruttero, R., Gasco, A., Fox, S., Megson, I., and Rossi, A. (2008). A novel hybrid aspirin-No-releasing compound inhibits Tnf-a release from Lps-activated human monocytes and macrophages. J. Inflamm. 5, 12. Udupi, V., Yu, M., Malaviya, S., Saavedra, J.E., and Shami, P.J. (2006). Js-K, a nitric oxide prodrug, induces cytochrome C release and caspase activation in Hl-60 myeloid leukemia cells. Leuk. Res. 30, 1279–1283. Wallace, J.L., McKnight, W., Soldato, P.D., Baydoun, A.R., and Cirino, G. (1995). Anti-thrombotic effects of a nitric oxide-releasing, gastric-sparing aspirin derivative. J. Clin. Invest. 96, 2711–2718. Wang, P.G., Xian, M., Tang, X., Wu, X., Wen, Z., Cai, T., and Janczuk, A.J. (2002). Nitric oxide donors: Chemical activities and biological applications. Chem. Rev. 102, 1091–1134. Wermuth, C.G. (ed.) (2003). Designing Prodrugs and Bioprecursors. Practice of Medicinal Chemistry. Academic, London. Whitehouse, M.W. and Ghosh, P.B. (1968). 4-nitrobenzofurazans and 4-nitrobenzofuroxans: A new class of thiol-neutralising agents and potent inhibitors of nucleic acid synthesis in leucocytes. Biochem. Pharmacol. 17, 158–161. Williams, D.L.H. (1985). S-nitrosation and the reactions of S-nitroso compounds. Chem. Soc. Rev. 14, 171–196. Williams, J.L., Borgo, S., Hasan, I., Castillo, E., Traganos, F., and Rigas, B. (2001). Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (Nsaids) alter the kinetics of human colon cancer cell lines more effectively than traditional Nsaids: Implications for colon cancer chemoprevention. Cancer Res. 61, 3285–3289. Wink, D.A. and Mitchell, J.B. (2003). Nitric oxide and cancer: An introduction. Free Radical Biol. Med. 34, 951–954. Wink, D.A., Vodovotz, Y., Laval, J., Laval, F., Dewhirst, M.W., and Mitchell, J.B. (1998). The multifaceted roles of nitric oxide in cancer. Carcinogenesis 19, 711–721. Wolfe, M.M., Lichtenstein, D.R., and Singh, G. (1999). Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs. N. Engl. J. Med. 340, 1888–1899. Zavorin, S.I., Artz, J.D., Dumitrascu, A., Nicolescu, A., Scutaru, D., Smith, S.V., and Thatcher, G.R.J. (2001). Nitrate esters as nitric oxide donors: Ss-nitrates. Org. Lett. 3, 1113–1116. Zhao, W., Mackenzie, G.G., Murray, O.T., Zhang, Z., and Rigas, B. (2009). Phosphoaspirin (Mdc-43), a novel benzyl ester of aspirin, inhibits the growth of human cancer cell lines more potently than aspirin: A redox-dependent effect. Carcinogenesis 30, 512–519.

Chapter 21

Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? David G. Hirst and Tracy Robson

Abstract Nitric oxide is a small molecule with enormous untapped potential in cancer therapy. It is involved in regulating many of the pathways that define the malignant phenotype, and its expression within tumours has now been shown to affect the growth of tumours and their response to conventional therapies. NO has clear anticancer potential as a single agent in colon, liver and thyroid tumours growing as xenografts whether delivered by donor drugs or gene therapy. It is also showing promise, as a single agent in a clinical trial in prostate cancer. However, there is considerable evidence that NO alters the way that cells repair DNA damage, so it is not surprising that it has shown efficacy in combination with chemotherapy and radiotherapy. Significant enhancement has been reported in experimental models of human colon, prostate and ovarian cancer, and a clinical trial in lung cancer in combination with cisplatin and vinorelbine has shown an impressive prolongation of survival. NO has low toxicity to normal tissues and can easily be administered using donor agents, characteristics that make it well suited to cancer therapy in an adjuvant setting. Keywords Nitric oxide donors · Nitric oxide synthase · Gene therapy · Radiotherapy · Chemotherapy

Introduction The role of NO in cancer biology and the mechanisms by which it exerts its effects have been the subjects of numerous recent reviews (Lechner et al. 2005; Hirst and Robson 2007; Bonavida et al. 2006; Mocellin et al. 2007; Miller and Megson 2007; McCarthy et al. 2008). The available data implicate the generation of reactive nitrogen intermediates, such as peroxynitrite (Pacher et al. 2007), in a profusion of pathways that may provide some specificity for tumours compared D.G. Hirst (B) School of Pharmacy, Medical Biology Centre, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK e-mail: [email protected] B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_21, C Springer Science+Business Media, LLC 2010

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with normal tissues. Involvement in these pathways suggests that NO may in the future play a role in combination with novel biological therapies or immunotherapy (Bonavida et al. 2008); however, the aim of this chapter is to focus very specifically on the application of NO-generating strategies to cancer therapy and its interaction with existing conventional treatments. We believe that the available data show convincingly that many of these strategies are safe, practical and effective, both as stand-alone therapies and in combination with conventional cytotoxic chemotherapy and radiotherapy. Clinical trials have been carried out with impressive results. NO has been recognised for decades as an irritant component of atmospheric pollution (see Aranda and Pearl 2000 for review), but it was not until the late 1980s that its specific role as a cytotoxic effector molecule was recognised. The contribution of nitrosative stress to the “respiratory burst” by which macrophages kill pathogens was shown to be arginine dependent (Iyengar et al. 1987), and the following year NO was specifically identified as a cytotoxic effector in macrophages (Hibbs et al. 1988). Other cell types including endothelial cells can also be activated by cytokines and this has been shown to lead to bystander killing of tumour cells (Li et al. 1991). This group soon recognised the therapeutic potential of this effect as a means of targeting circulating, potentially metastatic tumour cells and went on to show that tumour cells could be killed (as determined by a cell lysis assay), by co-culturing them with endothelial cells that had been activated by cytokines (Li et al. 1991). This cytotoxic effect could be entirely blocked by the addition of the NOS inhibitor NMA, confirming the key role of NO. This result was consistent with the previous observation that authentic NO gas at a concentration of 1.25 mM had a cytostatic effect, as determined by the rate of incorporation of [H3 ]TdR, on lymphoma cells (Stuehr and Nathan 1989). The ability of a variety of NO/nucleophile complexes to affect the survival and growth of A375 human melanoma cells has since been investigated (Maragos et al. 1993). Exposure of the cells to these NO donors for 8 h had little effect on their immediate survival (as determined by trypan blue exclusion), but most had a marked effect on proliferation as determined by [H3 ]TdR incorporation. An early indication of the damaging action of NO was revealed by Wink et al. (1991) who showed that NO induced deamination of deoxynucleosides, deoxynucleotides and intact DNA under physiological conditions.

Monotherapy Since these early observations, cancer therapy with NO has been considered mainly as a means of sensitising malignant cells to conventional treatments such as radio and chemotherapy; studies of these interactions will be discussed later in this chapter. However, a number of investigators have demonstrated that NO, on its own, can exhibit potent anticancer properties both in vitro and in vivo, generally without significant toxicity (Table 21.1). The methods that have been used to generate therapeutic levels of NO in cancer cells both in vitro and in vivo are the

Source of NO

NONOates

NONOates

NONOates

NONOate and GTN

NO-NSAIDS

Reference

Maragos et al. (1993)

Shami et al. (1998)

Adams et al. (2001)

Postovit et al. (2004)

Williams et al. (2001)

NO monotherapy

Human colon cancer (HT2)

−

Human leukaemia (B-CLL)

Human leukaemia (HL-60 and U937)

Human melanoma (A375)

Cell line

In vitro

−

−

Proliferation (PCNA); apoptosis (morphology and flow cytometry) Lowest IC50 : 1 μM (NCX 4040)

−

−

−

Exposure in vitro to GTN (20 pM) or DETA-NO (1 fM) reversed hypoxia-enhanced metastasis −

−

−

[3 H]TdR uptake reduced by <95%; lowest IC50 : 24 μM (SPER-NO) Clonogenicity reduced; dependent on half-time of NO release MTS cell proliferation dependent on half-time of NO release Lowest IC50 : 274 μM (DETA-NO) − Murine melanoma (B16F10)

Tumour inhibition

Cell line

Assay/cytotoxic effect

In vivo

Table 21.1 Cytotoxicity of NO against tumour cells in vitro and in vivo

21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? 389

Source of NO

NO-NSAIDS

NO-NSAIDS

NO-NSAID

NO-NSAIDS

Aryl diazeniumdiolate (ADD)

Reference

Kashfi et al. (2002)

Royle et al. (2004)

Gao et al. (2005)

Tessei et al. (2005)

Ren et al. (2003)

NO monotherapy

Human hepatoma (Hep 3B)

Human colon (LoVo, LoVo Dx, WiDr and LRWZ)

Human colon adenocarcinoma (SW48, HCT-15 and LoVo)

Human pancreatic (PaCa-2), prostate (LNCap), lung (A549), colon (HT-29 and HCT-15) and tongue (SCC-25) cancers Human prostate (LNCap and PC-3)

Cell line

In vitro

−

−

40% reduction in tumour weight after NCX 4040 (10 mg/kg) 5 times/week for 6 weeks −

−

−

Human colon cancer cell lines (LoVo, LoVo Dx, WiDr)

Cell number (dye exclusion) Complete growth inhibition for 72 h by JS-K (10 μM); IC50 : 8 μM

−

−

−

Cell growth curves Lowest IC50 : 1 μM (NO aspirin)

Cell viability (MTT) and apoptosis (TUNEL) <80% growth inhibition by NO aspirin (NCX 4060) and <90% apoptotic cells Cell growth (luciferase assay) inhibition; Lowest IC50 : 34 μM (NCX 4040 for 48 h) Cell growth (SRB assay) and apoptosis (TUNEL) Lowest IC50 : 10 μM (NCX 4040)

Tumour inhibition

Cell line

In vivo Assay/cytotoxic effect

Table 21.1 (continued)

390 D.G. Hirst and T. Robson

Source of NO

ADD

ADD

NO-linoleic acid derivatives

Gene therapy: naked CMViNOS DNA

Reference

Kiziltepe et al. (2008)

Simeone et al. (2008)

Chen et al. (2008)

Soler et al. (2000)

NO monotherapy

−

Human breast cancer (MDA-MB-231, MDA-MB-231/F10 and MCF7 Human hepatoma (HepG2)

Human multiple myeloma (MM.1S)

Cell line

In vitro

−

Cell death (by LDH assay) Lowest IC50 : ∼0.03 μM (8b)

Cell invasion through Matrigel Invasion reduced by ∼70% by JS-K (10 μM)

Cell viability (MTT) and apoptosis (Annexin V/PI) 95% cell death after JS-K (48 h 1 μM)

Assay/cytotoxic effect

Table 21.1 (continued)

Mouse orthotopic thyroid cancer (rMTC 6-23)

Human hepatoma xenograft in NOD/SCID mice

−

Human multiple myeloma (OPM1)

Cell line

In vivo

Tumour growth reduced to 25% of control by 15 mg/kg (8b) daily for 35 days Tumour size reduced by 50% after intra-tumoural injection

Tumour growth to 4 times treatment volume prolonged from 12 to 39 d by JS-K (4 μmol/kg i.v. 3 times/week) −

Tumour inhibition

21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? 391

Source of NO

Gene therapy: CMViNOS plasmid in liposome

Gene therapy: AdiNOS

Gene therapy: pE9iNOS plasmid in liposome

Gene transfer: hOCiNOS plasmid in liposome

Reference

Worthington et al. (2002)

Wang et al. (2004)

Worthington et al. (2005)

McCarthy et al. (2007)

NO monotherapy

Assay/cytotoxic effect −

−

−

Clonogenic cell survival reduced by 80–90%

Cell line

−

−

−

Human prostate cancer (PC-3 and DU145)

In vitro

Table 21.1 (continued)

−

Mouse fibrosarcoma (RIF-1)

Human colon xenograft (HCT-116)

Mouse fibrosarcoma (RIF-1)

Cell line

In vivo

Single intra-tumoural injection of CMViNOS (25 μg) slowed tumour growth by 50%. Intra-tumoural injection of AdiNOS slowed tumour growth by ∼20% Single intra-tumoural injection of pE9iNOS (25 μg) slowed tumour growth by >60%. −

Tumour inhibition

392 D.G. Hirst and T. Robson

Source of NO

Gene therapy: Tf-PEG-PEICMViNOS plasmid in liposome

Reference

Coulter et al. (2008)

NO monotherapy

Assay/cytotoxic effect −

Cell line

−

In vitro

Table 21.1 (continued)

Mouse fibrosarcoma (RIF-1)

Cell line

In vivo

Single intra-venous injection of Tf-PEG-PEICMViNOS (30 μg) slowed tumour growth by 50%.

Tumour inhibition

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following: NO donor drugs, iNOS induction by cytokines and iNOS gene therapy, and the published data will be reviewed under these categories.

Nitric Oxide Donor Drugs Many nitrogen-containing compounds will release nitric oxide by a variety of mechanisms (see Miller and Megson 2007 for review). These include rapid bioactivation, such as by mitochondrial aldehyde dehydrogenase 2 in the case of glyceryl trinitrate (Munzel 2008; Thatcher et al. 2004); or by slow degradation of NONOates such as DEA/NO and PAPA/NO (Wink et al. 1997). Other classes of drug such as the NO/NSAIDS have attractive NO delivery characteristics (Miller and Megson 2007), while glutathione S-transferase-activated agents such as JS-K (Shami et al. 1998) or diolate-modified NONOates (Butler and Russell 2005) may offer characteristics that favour NO delivery to the tumour microenvironment. Can nitric oxide donor drugs generate sufficient NO to affect growth of tumour cells? The earliest studies using NO donors, including the NONOates, as anticancer agents were aimed at determining their radiosensitising and chemosensitising potency (Mitchell et al. 1993; Wink et al. 1997). These studies will be discussed later, but unfortunately the data were presented in a way that does not allow the effects of the NO donors alone on cancer cells to be determined. Early studies examined the cytotoxicity of a range of NONOates (MAMA-NO, PAPA-NO and DETA-NO) against acute nonlymphocytic leukaemia cells (Shami et al. 1998) and chronic lymphocytic leukaemia (B-CCL) cells and compared the IC50 of each with their NO release kinetics (Adams et al. 2001). The most cytotoxic (IC50 274 μM) was DETA-NO with a release half-time of 20 h whereas MAMA-NO with a release half-time of ∼1 min was the least toxic (IC50 1.6 mM). A linear relationship was established between IC50 and the log of the release half-time. The anticancer activity of NO-releasing non-steroidal anti-inflammatory drugs (NO-NSAID) has been investigated in a number of studies, with the emphasis on colon cancer. Williams et al. (2001) showed that NO aspirin (NCX 4040), NO ibuprofen (NCX 2210) and NO sulindac (NCX1102) inhibited the growth of colon cancer cell lines (HT-29 and HCT-15) with IC50 values of 1 μM, 42 μM and 150 μM, respectively, after 24 h exposure to the drugs. The same group then expanded their study to include tumours of the lung, tongue, prostate and pancreas (Kashfi et al. 2002), all of which showed sensitivity to NO aspirin, NO sulindac and NO ibuprofen. Royle et al. (2004) investigated the cytostatic and pro-apoptotic effects of other NO-ibuprofen (NCX 2111) and NO-aspirin (NCX 4060) compounds in hormone-dependent (LNCap) and hormone-independent (PC3) prostate cancer cell lines. Both compounds were potent inducers of apoptosis, as determined by flow cytometric labelling and TUNEL staining, at concentrations greater than 10 μM. These effects were accompanied by increased expression of caspase 3. The ability of an NO aspirin to inhibit growth of SW480 colon cancer cells and induce extensive apoptosis was also reported by Gao et al. (2007). A concentration of 120 μM

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caused complete cessation of tumour cell growth and induced apoptosis in ∼80% of the cells. In a similar study, another NO-NSAID, NCX 4040, inhibited the growth of a panel of colon cancer cell lines and at higher concentrations (10 μM) it induced apoptosis in 90% of the cells. Furthermore, when the colon cancer cells were grown as xenografts in nude mice, NCX 4040 (given orally at 10 mg/kg five times per week for 6 weeks) caused a marked slowing of tumour growth (Tesei et al. 2005). The potency and clinical potential of a targeted nitric oxide generator, JS-K, against multiple myeloma cells has recently been demonstrated both in vitro and in vivo (Kiziltepe et al. 2007). In that study, human OPM1 cells were grown as xenografts in mice; when the tumours reached ∼200 mm3 , intravenous injections of 4 μmol/kg were given three times per week for 9 weeks. Tumour growth rate was dramatically reduced (to ∼20% of control). Examination of the tumours by flow cytometry and immunohistochemistry revealed extensive apoptosis, a characteristic of generation of NO at high concentrations. Furthermore, there was no significant toxicity, including no systemic hypertension. This class of NO donor, the O2 -aryl diazeniumdiolates and particularly JS-K shows considerable promise. A recent study demonstrated its efficacy against orthotopic hepatomas in rats via a mechanism that was dependent on both NO and MAP kinase (Ren et al. 2003). However, other NO donors have also been shown in another recent study to inhibit growth of liver tumours in vivo (Chen et al. 2008). Treatment (15 mg/kg, i.p., daily for 35 days) of SCID mice bearing subcutaneous human hepatocellular carcinomas as xenografts, with two furoxan-based NO donor derivatives of oleanolic acid resulted in a reduction in growth rate by about 70% compared with controls and again there was no liver-specific or systemic toxicity. While the data reviewed here point clearly to a role for NO therapy in controlling the rate of tumour cell growth both in vitro and in vivo, there is also exciting evidence that NO generation can also strongly inhibit metastasis. The NO donors GTN and DETA-NO caused marked and significant inhibition of the development of melanoma nodules in the lungs of mice given i.v. injections of B16F10 melanoma cells (Postovit et al. 2004). The NO donor KS-K was also shown to inhibit the invasion of several breast cancer cell lines through a matrigel membrane (by 80–90%) by decreasing phosphorylation of p38, a negative upstream regulator of a tissue inhibitor of matrix metalloproteinases (TIMP-2) (Simeone et al. 2008).

iNOS Gene Therapy A prerequisite for NO-based cancer therapeutics is the targeted generation of NO in malignant tissue; gene therapy using the high-output isoform of NO synthase (inducible nitric oxide synthase, iNOS) has the potential to do this. The first demonstration of the efficacy of this strategy was by Soler et al. (2000). They injected naked iNOS plasmid DNA directly into experimental thyroid tumours in mice and showed that this approximately halved the growth rate of the tumours. More complex vector systems have since been used by other groups. Cationic lipid vectors

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were used to deliver the iNOS gene, driven by the strong constitutive promoter CMV, to mouse and human tumour cells, both in vitro and in vivo (Worthington et al. 2002). Direct intra-tumoural injection was used in vivo. This treatment caused extensive apoptosis and necrosis in mouse tumours 5 days after transfection, and this was reflected in a halving of the growth rate compared to untreated tumours. Even larger antitumour effects were observed with direct liposomal delivery of a construct incorporating the E9 promoter driving iNOS (Worthington et al. 2005). This promoter proved to be stronger than CMV and resulted in an 8-fold increase in NO levels at 3–5 days after transfection compared with controls and in a reduction in tumour growth rate by 62%. The same group has since shown that iNOS gene therapy can also be delivered successfully by the intravenous route (Coulter et al. 2008b). Mice bearing RIF-1 sarcomas were given a single injection of 30 μg of a transferrin–polyethyleneglycol– polyethylenimine complex (Kursa et al. 2003) incorporating DNA-encoding iNOS via the tail vein. Tumours in mice treated with systemic iNOS gene therapy grew 30% more slowly than those in untreated mice. Adenoviral delivery can also be an efficient method of infecting cells in vivo with a gene payload. Direct injection of AdiNOS was used by one group to achieve increased iNOS expression and NO generation in a model of human colorectal cancer (HCT-116) growing as a xenograft in nude mice (Wang et al. 2004). A modest increase in apoptosis and a modest delay in tumour growth (∼20%) were achieved compared with untreated control tumours. All studies that have so far been reported demonstrate the potency of nitric oxide therapy against cancer cells and solid tumours in vivo, however, there are no reports of significant toxicity to normal tissues. A compelling explanation for this has been proposed by the group in Freiburg (Heigold et al. 2002). They demonstrated that the NO donors, sodium nitroprusside and spermine NONOate, were potent inducers of apoptosis in transformed cells, but not in untransformed cells. This toxic action could be totally abrogated by exposing the cells to exogenous superoxide dismutase (SOD); O2 – has been shown to be generated by transformed fibroblasts (Irani et al. 1997) and was present in their study. Furthermore, supplementation of non-transformed cells with a superoxide anion-generating system allowed peroxynitrite to be formed and sensitised them to apoptosis induction by NO donors. These data strongly support the view that the tumour specificity of NO therapy is a consequence of the preferential formation of the potent apoptosis inducer peroxynitrite in malignant cells, but not in normal cells (reviewed in Hirst and Robson, 2007). Given the clear antiapoptotic and antimetastatic properties of NO donors, together with their low-toxicity profile, it is perhaps surprising that few clinical trials have been initiated to date. One study is currently under way, in which the NO donor GTN was administered via slow release dermal patch to men with a PSA recurrence after primary therapy for localised prostate cancer (Seimens et al. 2007); PSA levels are used as a biomarker for efficacy. GTN markedly and significantly increased PSA doubling time to 32 months compared with 13 months in the control group, in the absence of any serious adverse events.

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Radiosensitisation The role of oxygen in determining the biological consequences of irradiation with X-rays has been a recurrent theme in radiobiology research over the past 7 decades. Its importance in radiotherapy outcomes has also been widely reported (see Overgaard 2007 for review). Much less well known is the fact that NO was first identified as a potent radiosensitiser at around the same time as the oxygen effect was demonstrated (Howard-Flanders 1957) albeit in bacteria. However, while radiosensitisation by NO was of a similar magnitude (SER of ∼2 at 15 μM) it was considered something of a curiosity, because at that time the fundamental importance of NO in mammalian physiology was not known. However, when the biological significance of NO was finally recognised 30 years later, several groups began to investigate how the generation of NO could alter radio-responsiveness of cancer cells and considered its implications for therapy (Table 21.2). The first of these studies examined the effect of adding either authentic NO gas (103 –105 ppm) or the NO-releasing agent DEA/NO (0.1–2.0 mM) prior to irradiation of hypoxic V79 fibroblasts with 4 MeV X-rays (Mitchell et al. 1993). The highest concentrations gave SERs of 3.2 and 2.9 respectively, at least as high as that achieved with oxygen; however, on a molar basis O2 was four times more efficient as a radiosensitiser than NO. These data lead the group to conclude that if a means of delivering high concentrations of NO to tumours could be found, it would be an effective means of treating radioresistant hypoxic cells. Very similar data were later obtained for radiosensitisation of mouse mammary carcinoma cells exposed to either DEA/NO or the slower releasing donor SPER/NO (Griffin et al. 1996). SERs of 2.8–3.0 were achieved at drug concentrations of 2 mM. Several other classes of NO donor have been investigated for their radiosensitising properties. S-Nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) at mM concentrations gave similar enhancement ratios to that of oxygen (Mitchell et al. 1996). In a similar study, three glioma cell lines were sensitised (SER, 1.4) to radiation by exposure to the donors SNP and SNAP at a concentration of 100 μM (Kurimoto et al. 1999). Radiosensitisation of neuroblastoma cells (IMR32) by GSNO and DETA/NO has also been reported using p53-dependent apoptosis induction as the endpoint (Wang et al. 2003). In these cells low to moderate radiation doses produced little apoptosis, but the combination with GSNO (1 mM) enhanced apoptosis induction 5- to 15-fold at different times after exposure to the combination of agents. More complex reactions can also be used to generate NO. Addition of the nitroxyl (NO− ) donor to cells (V79 fibroblasts) in combination with the electron acceptor agents, ferricyanide or tempol, resulted in generation of low μM concentrations of NO, but that was sufficient to give an enhancement ratio of ∼2.0 when the cells were irradiated with γ-rays (Mitchell et al. 1998). This emphasises that NO levels achieved by NO donors are inevitably lower than the concentrations of the parent drug. While various donor drugs are clearly capable of generating effective radiosensitising concentrations of NO, many cell types, including cancer cells have the

NO gas or NONOate

NONOates

Angeli’s salt + electron acceptors SNAP or SNP

Mitchell et al. (1993)

Griffin et al. (1996)

Mitchell et al. (1998)

NO-NSAIDS, SNAP or iNOS induced by IFN-γ iNOS induced by IFN-γ + IL-1β

NO-NSAID or GSNO

Van den Berge et al. (2001)

Wang et al. (2003)

Jannsens et al. (1998)

Kurimoto et al. (1999)

Source of NO

Reference

NO radiosensitisation

Human neuroblastoma (IMR32)

Mouse mammary carcinoma (EMT-6)

Mouse mammary carcinoma (EMT-6)

Human glioma (T98G and U87)

Hamster lung fibroblasts (V79)

Human mammary carcinoma (SCK)

Hamster lung fibroblasts (V79)

Cell line

In vitro

Clonogenic survival SER <3.0 in 105 ppm NO; SER 2.9 at 2 mM DEA-NO Clonogenic survival SER 3.0 in 2 mM DEA-NO; SER 2.8 at 2 mM SPER-NO Clonogenic survival SER 2.5 in 100 μM Angeli’s salt + 1 mM ferricyanide Cell growth curves SERs 1.8–1.9 in 100 μM SNP Clonogenic survival SER 2.4 in 10 units/ml IFN-γ or in 300 μM PAPA-NO Clonogenic survival SER 2.1 in 3 units/ml IFN-γ + IL-1β Apoptosis (flow cytometry, sub G1 ) 10-fold (1 mM GSNO) or 2.5-fold (DETA-NO) enhancement in radiation (2 Gy) induced apoptosis

Assay/cytotoxic effect

Tumour inhibition − − − − −

− −

Cell line − − − − −

− −

In vivo

Table 21.2 Radiosensitisation of tumour cells by NO in vitro and in vivo

398 D.G. Hirst and T. Robson

Clonogenic survival SER 1.6–1.7 (gene transfer)

−

Human colon (HCT-116 and SNU-1040)

−

Gene therapy: AdiNOS NONOate or SNAP

Gene therapy: AdiNOS

Wang et al. (2004)

Cook et al. (2004)

Clonogenic survival SER 2.0

Mouse fibrosarcoma (RIF-1)

Gene therapy: CMViNOS or WAF1iNOS plasmids in liposome

Worthington et al. (2002)

Assay/cytotoxic effect

Cell line

Source of NO

In vitro

Reference

NO radiosensitisation

Table 21.2 (continued)

Human colon xenograft (HCT-116)

Human colon xenograft (HCT-116)

Mouse fibrosarcoma (RIF-1)

Cell line

In vivo

Growth delay Single intra-tumoural injection of CMViNOS (25 μg) gave SER of >2.0 at 2 Gy Intra-tumoural AdiNOS + X-rays (3 × 2 Gy) doubled growth delay compared to X-rays alone Growth delay intra-tumoural AdiNOS + X-rays (3 × 2 Gy) gave SERs of 1.3 (p53 knockout)–1.9 (p53 WT)

Tumour inhibition

21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? 399

Source of NO

Gene therapy: WAF1iNOS plasmid in liposome

NO gas

Gene transfer: WAF1iNOS plasmid in liposome

Reference

Worthington et al. (2004)

Wardman et al. (2007)

Worthington et al. (2007)

NO radiosensitisation

−

Clonogenic survival: >2.0 in 1000 ppm NO γ-H2AX: SERs 2.0 in 1% NO −

−

−

Hamster lung fibroblasts (V79), human breast cancer (MCF7)

Assay/cytotoxic effect

Cell line

In vitro

Table 21.2 (continued)

Mouse fibrosarcoma (RIF-1) and human colon cancer (HT29)

Mouse fibrosarcoma (RIF-1) and human colon cancer (HT29)

Cell line

In vivo

Intra-tumoural injections (2 over 3 days) of WAF1iNOS (25 μg) enhanced fractionated irradiation (10 fractions in 5 days)

Single intra-tumoural injection of WAF1iNOS (25 μg) gave SERs of >2.0 (RIF-1) or 2.0 (HT29)

Tumour inhibition

400 D.G. Hirst and T. Robson

Source of NO

Gene therapy: pE9iNOS plasmid in liposome

Reference

McCarthy et al. (2008)

NO radiosensitisation

Mouse fibrosarcoma (RIF-1)

Cell line

In vitro

Clonogenic survival SER <2.0

Assay/cytotoxic effect

Table 21.2 (continued)

Mouse fibrosarcoma (RIF-1)

Cell line

In vivo

Single intra-tumoural injection of pE9iNOS (25 μg) + X-rays (10 Gy) gave 50% increase in tumour growth delay compared with X-rays alone

Tumour inhibition

21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? 401

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D.G. Hirst and T. Robson

necessary intra-cellular machinery required to generate very high concentrations endogenously by upregulation of the inducible, high-output isoform of nitric oxide synthase (see de Ridder et al. 2008 for review). The principal role of the iNOS system is to respond to pathogens; IFN-γ is therefore an efficient inducer of iNOS at the transcriptional level and is capable of stimulating NO generation. The first study to apply this as a radiosensitising strategy showed that while the NO concentrations that could be generated were considerably lower than those achieved with donor drugs (SNAP and PAPA/NO), they were still sufficient to give SERs of up to 2.4 after exposure to 10 units /ml of IFN-γ (Janssens et al. 1998). This suggests either that NO generated endogenously is more efficient at radiosensitising cells or more likely that the intracellular concentrations of NO generated by iNOS are higher than the equilibrium concentration reached in the extracellular medium. Whatever the mechanism, there is also evidence that cytokine induction of iNOS and subsequent NO generation is much more efficient at low oxygen tensions, as existing in extensive areas of most solid tumours (Berge et al. 2001). Exposure of EMT-6 mouse tumour cells to 1 unit/ml of both IFN-γ and IL-1β resulted in a significant greater induction of iNOS message, a dramatic increase in iNOS protein and a ∼10-fold higher concentration of NO in the medium of cells under 1% oxygen compared with cells under 21% oxygen; this resulted in a 1.8-fold radioenhancement of hypoxic cells. Thus, the hypoxic dependency confers considerable specificity for the microenvironment of tumours. In both of these studies using cytokine induction of iNOS to radiosensitise hypoxic cells, the effect could be totally abrogated by the addition of a NOS inhibitor, confirming the key role of NO. As described earlier, a more targeted approach to increasing iNOS levels in tumour cells is to use a gene therapy strategy with iNOS under the control of a promoter that is preferentially activated in tumours undergoing radiotherapy. We have pioneered this approach using radiation-inducible promoters. One potential candidate is the WAF1/p21 promoter (el-Deiry et al. 1993). This promoter was used successfully to drive iNOS expression and so to generate NO in a time-dependent and radiation dose-dependent manner in isolated mammalian blood vessels, suggesting that WAF1/iNOS gene therapy might be used to relax the blood vessels supplying tumours and so reducing the number of radioresistant hypoxic cells (Worthington et al. 2000). However, NO is also a potent radiosensitiser in its own right (see above) and this property could be demonstrated as a consequence of iNOS gene transfer to tumour cells in vitro (Worthington et al. 2002). Transfection of RIF1 mouse fibrosarcoma cells with a plasmid construct incorporating iNOS under control of the WAF-1 promoter (activated by a priming dose of 4 Gy) resulted in a sensitiser enhancement ratio of ∼2.0 when the cells were irradiated under hypoxia such that they were almost as radiosensitive as cells irradiated in air. The sensitisation by NO could be completely abrogated by incubation of the cells with the non-specific NOS inhibitor L-NMMA for 1 h prior to irradiation. WAF-1/iNOS gene therapy was then extended to isogenic mouse tumours (RIF-1) and to human colon tumour (HT29) xenografts growing in SCID mice. Following a single intratumoural injection of the WAF-1/iNOS plasmid in a cationic lipid vector, plasmid sequence was found to be dispersed to most tissues in the body, confirming that gene

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therapy does not remain localised to the injected tissue; however, increased iNOS protein expression was found only when tissues were irradiated, i.e. the tumour itself and the immediately surrounding normal tissue. Furthermore, this targeted expression of iNOS lead to marked radiosensitisation of ∼2.0, exemplified by the fact that WAF-1/iNOS gene therapy plus 10 Gy of X-irradiation caused a similar delay in tumour growth to that caused by 20 Gy of X-rays alone. An additional benefit of the WAF-1 promoter is that it is also inducible by hypoxia and may therefore be activated in hypoxic regions of tumours. We have shown that exposure of human tumour cells to a 2 h period of hypoxia increased the expression of a WAF-1 driven reporter gene (pEGFP) resulting in a >4-fold increase in GFP protein level compared with normoxic control (Worthington et al. 2002) and that the increase persisted for at least 24 h. It should be noted, however, that oxygen binding to the ferrous haeme of NOS enzymes is essential for both steps of catalysis of L-arginine and hence generation of NO (Berka et al. 2004). Thus, while tumour cells that can activate the WAF-1 promoter as a consequence of low pO2 may synthesise high levels of iNOS, they may be compromised in their ability to catalyse the NO-generating reaction. The detailed oxygen dependency of WAF-1 promoter induction has not been established, but we do know that the Km for NO synthesis (measured as nitrite) by iNOS (in macrophages, not tumour cells) is 10.8% (McCormick et al. 2000). In terms of tumour oxygenation and radiosensitisation, this is a high value and cells would be fully radiosensitised at that level (Denekamp 1989) and so additional sensitisation by NO would be minimal. At the Km for the oxygen effect (∼2%) NO generation would be 20% of maximal; whether or not this is of radiobiological significance depends on the relative potency of NO and O2 as radiosensitisers, and recent evidence suggests that the efficiency of NO may even exceed that of O2 at radiation doses that are relevant to radiotherapy practice (Wardman et al. 2007). However, it is also well established that hypoxia in tumours is not a static phenomenon and oxygen tension can fluctuate depending on variations in perfusion (see Brown 2007 for review). On this basis, it is highly likely that a given cell could be at one time sufficiently hypoxic to induce the WAF-1 promoter with a resultant prolonged expression of iNOS and then later become sufficiently toxic to generate NO. Only if hypoxia was then re-established would radiosensitisation of that cell occur, but of course, the highly diffusible nature of NO (Lancaster 1997) will undoubtedly also lead to killing of bystander cells. The concept of restricting the expression of iNOS to the tumour volume is conceptually attractive, but it may not be strictly necessary. Impressive radiosensitisation has been demonstrated using strong constitutive promoters such as CMV (Worthington et al. 2002) and pE9 (Coulter et al. 2008), driving iNOS. These effects were achieved in vivo without significant systemic toxicity. Other investigators have used a similar approach, but with adenoviral expression vectors. Colorectal cancer cell lines (HCT-116 and SNU-1040) were infected with AdiNOS and were markedly more radiosensitive (SERs ∼ 1.6) than cells infected with a control vector (AdLacZ)(Wang et al. 2004); this was accompanied by a 20- to 40-fold increase in nitrite levels in the tumour cells 48 h after infection. In one of these cell lines (HCT-116), radiosensitisation was even more impressive when ADiNOS treatment

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D.G. Hirst and T. Robson

was given either as a single intra-tumoural injection followed by a single 2 Gy dose or three 2 Gy doses. In each case, the delay in tumour growth was approximately three times greater in tumours treated with iNOS gene therapy plus 2 Gy compared with tumours receiving 2 Gy only. This group also measured the distribution of iNOS throughout the tumour using immunohistochemistry and showed that only 4% of the tumour cells expressed it. They concluded that the degree of radiosensitisation could be explained only if there was a large bystander effect, with NO diffusing to most of the cells within the tumour. This is entirely consistent with what is known about NO’s diffusibility (Lancaster 1997) and emphasises one of the highly desirable therapeutic characteristics of the molecule. Most early studies of radiosensitisation by NO have used single radiation doses that are relatively high compared with those currently used in therapy (Mitchell et al. 1993) and the data obtained indicate that on a molar basis NO is a rather less (∼3-fold) efficient radiosensitiser than O2 . A more recent study using much lower X-ray doses in the range 1–5 Gy, however, reached a dramatically different conclusion. An enhancement ratio of 2.0, for clonogenic cell killing of V79-379A fibroblast-like cells, was achieved at an NO concentration of 80 ppm whereas a concentration of 1000 ppm of oxygen was required to produce the same effect (Wardman et al. 2007a). While the exact nature of the mechanism by which O2 interacts with DNA radicals is not fully clarified, an acknowledged characteristic of the “oxygen effect” is that it is remarkably consistent between widely diverse cell types including animals, plants and bacteria and this has been attributed to the common role of DNA in all cells and to the fundamental nature of the radical interactions involved (Wardman 2007). Little is known, however, about the mechanism by which NO radiosensitises cells, but it is likely that the mechanism is very different from that of O2 radiosensitisation. As recently pointed out by Wardman (2007b), while NO and O2 share free radical properties and are highly reactive with other radicals, NO forms spin paired bonds and not other radicals as is the case with oxygen. It is generally accepted that the component of low LET radiation damage that is modifiable by oxygen results from the generation of OH radicals from water and that these highly reactive species then interact with macromolecules in the cell, including DNA where they form DNA radicals. Wardman et al. (2007) showed that DNA radicals formed in this way are highly reactive towards NO. However, with the information that is available we cannot determine if NO radiosensitisation is as universal as the “oxygen effect”; given the potential of NO to enhance radiotherapy, this area of research is worthy of much more attention. Nevertheless, there is no doubt that NO is a potent chemical radiosensitiser, but it is also clear that in vivo there are additional physiological actions that could lead to radiosensitisation. Jordan et al. (2002) administered insulin to tumour-bearing mice, which caused an NO-dependent (i.e. could be blocked by the NOS inhibitor L-NAME) radiosensitisation together with a concomitant decrease in tumour oxygen consumption, an increase in tumour pO2 and an increase in tumour blood flow. In a more recent study, mice bearing liver tumours implanted into the leg muscle were injected intravenously with nitrite solution (blood concentration 100 μmol/l), and various tumour parameters were measured (Frerart et al. 2008). Tumour pO2 , as measured using an EPR oxymetry probe, increased significantly between 5 and

21

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20 min after i.v. nitrite, but there was no concomitant increase in tumour blood flow. It is likely, therefore, that the enhanced oxygenation was a consequence of reduced oxygen consumption by tumour cells, a concept that was first demonstrated experimentally over 30 years ago (Durand and Biaglow 1977). This response to exogenous nitrite was shown to occur only at lower pH (6.7), as is often found within solid tumours (Gerweck and Seetharaman 1996). Recent evidence also suggests that low oxygen tensions in the tumour microenvironment are permissive for the generation of NO from nitrite by enzymic reduction by xanthine oxidase (Zhang et al. 1998) or NOS (Gautier et al. 2006;Vanin et al. 2007), so nitrite is probably acting as an NO donor in this context. Whatever the mechanisms, however, i.v. nitrite, while ineffective on its own, caused significant slowing of tumour growth and prolongation of survival when given 15 min prior to 6 Gy of X-irradiation (Frerart et al. 2008). Thus, NO exhibits many physical, physiological and pharmacological properties that confer the ability to sensitise solid tumours to radiation at clinically relevant doses and in a manner that does not cause systemic or local toxicity. However, we are aware of no studies to date that permit a true therapeutic gain to be calculated, because no studies of normal tissue radiosensitisation have been carried out in vivo.

Chemosensitisation It has long been recognised that the therapeutic ratios of most conventional chemotherapy drugs are simply too low to allow eradication of most disseminated solid tumours. Many investigators have therefore sought to enhance this ratio by combining these cytotoxic drugs with agents that have radiosensitising properties; the nitroimidazoles are probably the most widely studied (see Brown 1982, for review). However, more recently, NO delivery strategies have shown considerable promise when combined with cytotoxic chemotherapy (Table 21.3).

Nitrosourea Shortly after their demonstration of radiosensitisation of mammalian cells by NO donor drugs (Mitchell et al. 1993), investigators at the NCI carried out a series of similar experiments combining NO donors with cytotoxics. The first agent to be studied was the nitrosourea BCNU (Laval and Wink 1994). Rat hepatoma cells were exposed in vitro to 1.5 mM DEA/NO for 30 min prior to and for 30 min during exposure to a range of concentrations of BCNU. This procedure gave a sensitiser enhancement ratio of ∼1.4 compared with BCNU alone. Sensitisation was attributed to inhibition of the DNA repair protein O6 -methylguanine-DNA-methyltransferase as a result of S-nitrosylation by NO or its reaction products.

Cisplatin Sensitisation of cells to other DNA-damaging agents has also been attributed to the formation of products formed by the reaction of NO with O2 . Wink et al. (1997)

NONOates/ cisplatin

NMO /cisplatin

Konovalova et al. (2003)

NO gas or NONOate/ cisplatin

NONOate/ BCNU

Source of NO/chemotherapy

Azizzadeh et al. (2001)

Laval & Wink (1994) Wink et al. (1997)

Reference

NO chemosensitisation

Assay/cytotoxic effect

Cell line

In vivo

Clonogenic survival SER 1.4 in 1.5 mM DEA-NO Hamster lung fibroblasts Clonogenic survival − (V79) 50-fold reduction in cell survival in NO saturated medium >1000-fold reduction in 1 mM DEA-NO or PAPA-NO Laryngeal SCC Cell viability (MTT) − (CCL23) LD50 reduced 2.7-fold by pre-exposure to DETA-NO (100 μM) − − Mouse leukaemia (P388) i.p.

Rat hepatoma (H4)

Cell line

In vitro

Table 21.3 Sensitisation of tumour cells to cytotoxic chemotherapy by NO in vitro and in vivo

Animal survival increased from 30 to 80% by co-injection of NMO (76 mg/kg×3)

−

−

Tumour inhibition

406 D.G. Hirst and T. Robson

Gene therapy: CMViNOS plasmid in liposome/cisplatin

Adams et al. (2008)

Human prostate (DU-145, PC33) and colon (HT29, HCT116)

Human ovarian cancer (A2780 and cisplatin-resistant clone, A2780 cDDP )

NO-NSAID/ cisplatin

Bratasz et al. (2008)

Cell line

In vitro

Human colon cancer (WiDr, LoVo, LoVo Dx, LRWZ)

Source of NO/chemotherapy

Leonetti et al. (2006) NO-NSAID/ oxaliplatin

Reference

NO chemosensitisation

Cell line

In vivo

Clonogenic survival SER 1.5–2.0

Mouse fibrosarcoma (RIF-1)

Cell viability (MTT) Human ovarian Reduced to 25% (NCX 4040, 25 cancer xenograft μM + cisplatin) compared with (A2780 cDDP) 80% for cisplatin alone

Cell viability (sulphorhodamine B) Human colon cancer SERs 3.7–4.0 in NCX 4040 (WiDr, LoVo, (10 μM) LoVo Dx)

Assay/cytotoxic effect

Table 21.3 (continued)

Growth delay greater than 40 d compared with oxaliplatin alone (NCX 4040, 10 mg/kg, p.o., 5 times /week for 4 weeks) Growth delayed by 5 days compared with cisplatin alone (NCX 4040, 5 mg/kg, i.p. daily) Growth delayed by 5 days compared with cisplatin alone (additive enhancement) (CMV iNOS)

Tumour inhibition

21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? 407

Source of NO/chemotherapy

NONOate/cisplatin

NONOate or GTN/ doxorubicin (DOX)

GTN/DOX

NO gas or gene transfer (AdiNOS) or NONOate

Reference

Huerta et al. (2009)

Matthews et al. (2001)

Frederiksen et al. (2003)

Evig et al., 2004

NO chemosensitisation

Apoptosis (TUNEL) 1 mM DETANONOate increased >2-fold compared with cisplatin alone

Assay/cytotoxic effect

Clonogenic survival GTN or DETA-NO (1 μM) reverse hypoxia-induced resistance to DOX Human (PC3-3) and Clonogenic survival mouse prostate cancer GTN (0.1 nM) reversed (TRAMP-C2) hypoxia-induced resistance to DOX Human breast cancer Clonogenic survival (MCF-7) SER 2.0 (ADiNOS); clonogenic survival reduced by 60–80% (NO gas or DEA-NO)

Human breast cancer (MDA-MB-231)

Human colon cancer (SW480 and SW620)

Cell line

In vitro

Table 21.3 (continued)

−

−

−

−

−

Growth delayed by ∼2 days compared with cisplatin alone No effect of DETANONOate alone (0.4 mg/kg) every 2 days for 2 weeks) −

Tumour inhibition

Human colon cancer (SW620)

Cell line

In vivo

408 D.G. Hirst and T. Robson

Source of NO/chemotherapy

NO-pegylated epirubicin

GTN or ISDN/DOX

NONOate/ melphalan

HMO or NMO/ cyclophosphamide (CPA)

Reference

Santucci et al. (2006)

Frederiksen et al. (2007)

Cook et al. (1997)

Konovalova et al. (2003)

NO chemosensitisation

Assay/cytotoxic effect

Apoptosis (Annexin V flow cytometry) Increased 1.8-fold (@5 μM) compared with epirubicin Human prostate cancer Clonogenic survival (DU145) GTN (0.1–1000 nM) or ISDN 0.1 nM) weak reversal of hypoxia-induced resistance to DOX Hamster lung fibroblasts Clonogenic survival (V79) and human SER (DEANO, 1 mM): 3.6 breast cancer (V79) and 4.3 (MCF-7). (MCF-7) − −

Human colon cancer (Caco-2)

Cell line

In vitro

Table 21.3 (continued)

Growth delayed by 4 days compared with control (GTN, transdermal patch)

Human prostate cancer xenograft (PC3-3)

Animal survival increased from 60% to 100% by co-injection of NMO (1 mg) Tumour regrowth completely inhibited for 40 d in 50% of animals (NMO 1 mg/kg x 3).

−

−

Mouse leukaemia (p388) Mouse lung carcinoma (LL)

Tumour inhibition

Cell line

In vivo

21 Nitric Oxide: Monotherapy or Sensitiser to Conventional Cancer Treatments? 409

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D.G. Hirst and T. Robson

examined the effect of combining either NO gas or the NO donor drugs DEA/NO or PAPA/NO with cisplatin. All three sources of NO caused massive enhancement of V79 cell killing compared with that from exposure to cisplatin alone. Enhancement (SER = 2.8) of cisplatin cytotoxicity was also reported in a head and neck squamous cell carinoma cell line (CCL23) (Azizzadeh et al. 2001). The effect was restricted to the longer acting NONOate DETA/NO, the shorter acting agents, DPTA/NO and PAPA/NO having no sensitising effect. Platinum compounds are one of the most widely used classes of agent in cancer therapy (Hartmann and Lipp 2003), so the ability to translate the chemoenhancing action of NO to the in vivo setting and eventually to the clinic would be very attractive. NO delivery in vivo has been tested in combination with all three of the leading platinum compounds: cisplatin, carboplatin and oxaliplatin. In one study (Konovalova et al. 2003), survival of mice injected intraperitoneally with L1210 leukaemia cells was increased by administration of 1 mg/kg cisplatin, but a much larger effect was obtained by combining cisplatin with an NO donor (3,3-bis[hydroxymethyl]oxetane; HMO). A super-additive interaction between the NO/NSAID NCX 4040 and oxaliplatin was demonstrated in a panel of colon cancer cell lines (Leonetti et al. 2006). They also investigated the effect of the combination against the cell lines growing as xenografts in vivo. In each case, oxaliplatin alone (daily for 6 days) had little or no effect on tumour growth, whereas NCX 4040 alone (daily for 28 days) delayed growth by between 8 and 20 days; however, the combination of the two agents caused >20 days growth delay in all cell lines and caused almost complete cessation of growth of one cell line (LoVo) for up to 50 days. These effects were reflected in a massive increase in the apoptotic index after combination therapy. Very similar results were also obtained in a recent study combining NCX 4040 with cisplatin in human ovarian cancer xenografts (Bratasz et al. 2008). A cisplatin-resistant cell line (A2780 cDDP) was significantly sensitised by daily i.p. injection of 5 mg/kg NCX 4040. Again enhancement of apoptosis was implicated. Another recent study also showed sensitisation to cisplatin by an NO donor (DETANONOate) in a human tumour xenograft model of colon cancer (Huerta et al. 2009) and also provided strong evidence for a role for apoptosis inducing factor (AIF), an inter-membrane flavoprotein capable of inducing caspase-independent chromatin condensation and DNA fragmentation, in NO-mediated cisplatin chemosensitisation. Gene therapy with iNOS also offers the opportunity to enhance the antitumour effect of cisplatin, but with the advantage that even a single injection of the construct allows prolonged expression of iNOS protein and generation of NO within the tumour. In a recent study with a mouse sarcoma model, cisplatin (8 mg/kg i.p.) or CMViNOS gene therapy each produced significant growth delay, and the combination gave an additive effect (Adams et al. 2008). Undoubtedly, the most exciting development of NO/platinum therapy, however, is its extension to clinical trial. In one study (Yasuda et al. 2006b), non-small cell lung cancer patients with inoperable disease were divided into two groups and all given vinorelbine/cisplatin combination therapy. One group also received NO therapy via a nitroglycerin transdermal patch replaced daily. Tumour size was followed using CT, and this revealed a highly significant increase in response rate in the

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group receiving the combination therapy with NO. More importantly, progressionfree survival and overall survival were both significantly extended; medial survival was increased by about 250 days without any increase in severe neutropoenia. The same group also concluded in a preliminary study that nitroglycerin could be used to enhance chemosensitivity to docetaxel plus carboplatin in patients with inoperable adenocarcinoma of the lung (Yasuda et al. 2006a).

Alkylating Agents NO chemosensitisation to alkylating agents has also been investigated. Melphalan cytotoxicity against both V97 fibroblasts and MCF-7 breast cancer cells was shown to be enhanced for several hours after exposure to DEA/NO (Cook et al. 1997). Possible mechanisms including cell cycle redistribution, glutathione depletion and melphalan uptake were investigated and while changes in these parameters were seen they were not consistent in each cell line. The authors concluded that complex and cell line-dependent mechanisms must be responsible for NO-dependent chemosensitisation to melphalan. Konovalova et al. (2003) investigated the effect of the NO donor HMO on the efficacy of cyclophosphamide (CPA) against growth of leukaemia cells (P388) in mice. Survival of the animals was dramatically increased by the combination of NMO (10 mg/kg i.p.) with CPA (60 mg/kg) compared with CPA alone. NMO also inhibited the development of resistance to CPA in this cell line in vitro. At the lower concentration of 1 mg/kg, it also enhanced the ability of CPA to inhibit the development of metastases from subcutaneously implanted B16 melanomas. The NMO/CPA combination was also very effective at slowing the growth of intramuscularly implanted LL carcinomas in mice, though curiously there appeared to be a separation into responding tumours and non-responding tumours, and no explanation for this was offered.

Anthracyclines It has been known for some time that doxorubicin (DOX) can inhibit the production of NO in colorectal cancer cells, in a manner that is dependent on iNOS transcription (Jung et al. 2002) and that the inhibition of NOS (by the non-isoform-specific L-NMMA) can lead to a dramatic increase in the resistance of breast cancer cells (MDA-MB-231) to DOX in a manner that was similar to hypoxia-induced DOX resistance (Matthews et al. 2001). Addition of the NO donors DETA/NO or GTN partially reversed the hypoxia-induced resistance to DOX. Very similar data were also obtained in DU-145 prostate cancer cells (Frederiksen et al. 2003). In vitro, hypoxia caused resistance to DOX, but that could be partially reversed by exposure of the cells to even low concentrations of the NO donors GTN or isosorbide dinitrate. The chemosensitising effect of GTN, administered via a transdermal patch, was also demonstrated in PC3 xenografts in nude mice (Frederiksen et al. 2007). DOX (4 mg/kg biweekly, i.p.) alone had little effect on tumour growth, but there

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was a significant growth delay when this treatment was combined with the GTN patch. This group speculated that chemosensitisation by NO could be related to the intercalating action of DOX, but there is also convincing evidence for another mechanism relating to multidrug resistance (MDR). Cells that have been exposed to DOX (amongst several other agents) develop resistance through an enhanced capability for drug efflux (Gottesman et al. 2002) resulting in reduced intracellular concentrations. However, exposure of HT-29 colon cancer cells to NO donors (GSNO, SNAP or SNP) dramatically increased intracellular DOX levels and eliminated the differential resistance between naïve cells and those that had previously been exposed to DOX (Riganti et al. 2005). These effects were directly correlated to NO-induced reductions in drug efflux. Some selectivity of NO-mediated chemosensitisation to anthracyclines for tumour versus normal cells is suggested in a comparative study with breast cancer cells (MCF-7) and cardiac myoblasts (H9c2) (Evig et al. 2004). Highly significant sensitisation of MCF-7 cells to DOX was seen when they were exposed to NO gas or were transduced with the iNOS gene, but there was no effect on the sensitivity of the H9c2 cells. In another study, the addition of an NO-releasing moiety to a pegylated derivative of epirubicin resulted in sparing of normal cell lines (HUVEC, H9c2 embryonic rat heart derived cells and adult mouse cardiomyocytes) from drug toxicity, but enhanced apoptosis in a human colon adenocarcinoma cell line (Caco-2), giving a double enhancement to the potential therapeutic gain (Santucci et al. 2006).

5-Fluorouracil We are aware of only one published study of the interaction between an NO donor and 5-FU (Leonetti et al. 2006). The two agents were tested in a range of colon adenocarcinoma cell lines in vitro and in vivo and in no case was there evidence of any effective interaction.

Conclusion and Therapeutic Implications There is now abundant evidence that the generation of NO using a variety of donor drugs or gene therapy has the potential to inhibit the growth of tumours and to sensitise them to radio and chemotherapy without enhanced toxicity to normal tissues. The lack of commercial incentive has meant that progress has been slow, but, taken together, the body of evidence is impressive and reveals the efficacy of NO therapies against many of the most common cancers including colon, liver, prostate and ovary. Most impressive of all are the early demonstrations of efficacy in clinical trials in recurrent prostate (Seimens et al. 2007) and primary lung cancer (Yasuda et al. 2006a), which suggest strongly that there will be a role for NO therapy in the future management of these diseases.

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Chapter 22

Therapeutic Applications of Nitric Oxide for Malignant Tumor in Animal Models and Human Studies Hiroyasu Yasuda, Kazuhiro Yanagihara, Katsutoshi Nakayama, Tadashi Mio, Takahiko Sasaki, Masanori Asada, Mutsuo Yamaya, and Masanori Fukushima

Abstract In cancer science, nitric oxide (NO) has been mainly discussed as an oncogenic molecule over the past decades. However, NO has recently been noted in cancer biology to be associated with cancer cell apoptosis, cancer cell cycle, cancer progression and metastasis, cancer angiogenesis, cancer chemoprevention, and a modulator for chemo/radio/immunotherapy. NO is produced and released from three different isoforms of NO synthase (NOS) and from exogenously administered NO donors in vivo. Over-expression of inducible NOS (iNOS) in cancer tissues is associated with an increase in microvascular density in tumor tissues and poor prognosis in patients with cancers. Recently, NO donors and iNOS transfer have been demonstrated to enhance the effects of cancer therapy including chemotherapy, radiotherapy, and immunotherapy for solid cancers, resulting in a prolonged survival time. In this chapter, we discuss the recent animal experiments and clinical trials to develop these investigations for clinical applications. Keywords Nitric oxide · Nitric oxide synthase · Cancer biology · Cancer progression · Metastasis · Apoptosis · Cell cycle · Proliferation · Chemoprevention · Chemotherapy · Radiotherapy · Immunotherapy · Animal experiment · Clinical trial Abbreviations AIF eNOS HIF-1α iNOS LLC

apoptosis-inducing factor endothelial NOS hypoxia-inducible factor-1 alpha inducible NOS Lewis lung carcinoma

H. Yasuda (B) Department of Clinical Application, Translational Research Center, Tohoku University, Sendai, 980-8574, Japan e-mail: [email protected] Dr. H. Yasuda wrote the draft of the manuscript. All co-authors contributed to our ongoing clinical researches. Dr. Yasuda had final responsibility for the decision to submit for publication.

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_22, C Springer Science+Business Media, LLC 2010

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NO NOS NSAIDS NSCLC TNF TRAIL VEGF YY1

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nitric oxide NO synthase non-steroidal anti-inflammatory drug non-small cell lung cancer tumor necrosis factor TNF-related apoptosis-inducing ligand vascular endothelial growth factor Yin–Yang 1

Animal Models There are three checkpoints before estimating the results of experiments associated with nitric oxide (NO) in cancer science. First, in an effort to demonstrate the effects of NO on cancer cells, we must recognize that NO behaves as a double-edged sword. NO can act as a cytotoxic factor (Millet et al. 2002) as well as a cytoprotective factor (Fukunaga-Takenaka et al. 2003) to cancer cells according to the environment (redox status, i.e., anoxia, hypoxia, normoxia, or hyperoxia) or condition (low or high NO concentration) (Chinje and Stratford 1997). Differences in experimental conditions, especially in NO-related studies, often bring about contrasting results such as an increase or decrease in protein expression. For example, NO donors such as (Z)-1[2-aminoethyl-N-(2-ammmonioethyl)amino]diazen-1-ium-1,2-diolate (DETANO), nitroglycerin, sodium nitroprusside, and isosorbide dinitrite were reported to inhibit the accumulation of hypoxia-inducible factor-1α (HIF-1α) in malignant tumor cells under hypoxic conditions, in vitro (Huang et al. 1999; Takabuchi et al. 2004; Hagen et al. 2003; Stewart et al. 2009; Brüne and Zhou 2007) and in vivo (Yasuda et al. 2006; Yasuda et al. 2007). On the other hand, NO donors such as DETANO and supermine NONOate were reported to promote accumulation of HIF-1 in human breast carcinoma (MCF7) under normoxic conditions (Thomas et al. 2004; Brüne and Zhou 2007). These results suggest that we should pay attention to the experimental conditions before generalizing results. Second, hypoxic conditions have been demonstrated to exist in malignant solid tumors but not in normal tissues due to abnormal tumor vasculature (Wang et al. 2004; Yasuda et al. 2006; Yasuda et al. 2007; Vaupel and Mayer 2007; Yasuda 2008). The hypoxia in malignant tumor tissues has been reported to associate with poor prognosis and resistance to cancer therapies (Swinson et al. 2004; Lau et al. 2007; Vaupel and Mayer 2007; Yasuda 2008). Hypoxic conditions result in chemoresistance to anti-cancer drugs in cancer cells in vitro (Matthews et al. 2001; Frederiksen et al. 2007) as well as in vivo (Yasuda et al. 2007; Frederiksen et al. 2007). Several reports have suggested that hypoxia-induced resistance to radiotherapy (Jordan et al. 2003; Wang et al. 2004; Cook et al. 2004) or chemotherapy (Matthews et al. 2001; Frederiksen et al. 2003; Frederiksen et al. 2007) may be reversed by NO donors

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or inducible NO synthase (NOS) transfer in vitro (Matthews et al. 2001; Bratasz et al. 2006; Frederiksen et al. 2007) or in vivo (Jordan et al. 2003; Wang et al. 2004; Cook et al. 2004; Frederiksen et al. 2007). Furthermore, NO donors were reported to restore hypoxia-induced decrease in the immune response to malignant cells (Siemens et al. 2008) and reported to augment anti-cancer immunity (Garbán and Bonavida 1999; Garbán and Bonavida 2001; Huerta-Yepez et al. 2004, 42; Huerta-Yepez et al. 2004; Perrotta et al. 2007). Therefore, it is empirical to perform studies in hypoxic conditions in order to demonstrate the effects of NO on cancer cells. Many reports, however, conclude results regarding the effects of NO on cancer cells from experiments with normoxic conditions as if they held true in tumor tissues. Third, malignant tumor tissues are composed of parenchymal and stromal elements (Zalatnai 2006). Vasculature (Vaupel and Mayer 2007; Yasuda 2008), blood perfusion (Vaupel and Mayer 2007; Trédan et al. 2007; Yasuda 2008), bystander cells (Xie et al. 1997), interstitial pressure between cancer cells (Yasuda 2008; Heldin et al. 2004), and immunological cells such as tumor-associated macrophages (Talks et al. 2000) all behave differently when observed in vivo versus in vitro, even if the hypoxic condition can be arranged by a hypoxic chamber. Cancer cells crosstalk with bystander cells and stromal cells (Zalatnai 2006; Decker et al. 2008). These circumstances suggest that the complete imitation of intratumor microenvironment cannot be reproduced by in vitro experiments. Furthermore, Edwards et al. reported that lipopolysaccharide/interferon-stimulated NO production in murine breast cancer (EMT-6) tumor cells inhibits tumor cell growth in vitro, yet paradoxically augments tumor growth and metastasis in vivo (Edwards et al. 1996). Therefore, results from in vitro experiments do not always hold true from those derived from in vivo, especially in the field of NO research. Therefore, in vivo animal experiments are needed to demonstrate the effects of NO on cancer cells in tumor tissues for their development for clinical applications. In this section, I would introduce the results of animal experiments and early stage clinical trials in the development of NO-associated research into their clinical applications via translational research.

Role of NO in Prostate Cancer NO Donor Tumor growth curve after treatment with doxorubicin and with nitroglycerin patch was significantly inhibited compared with tumors treated with doxorubicin alone in a human prostate cancer cells’ (PC-3 cells) xenograft solid tumor model. These investigators concluded that the cGMP signaling pathway enhanced by nitroglycerin was able to attenuate hypoxia-induced drug resistance to doxorubicin in PC-3 in vivo (Frederiksen et al. 2007). In this study, nitroglycerin was

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R administered as a transdermal patch (Minitran nitroglycerin transdermal patch 3 M Pharmaceuticals, London, Ontario, Canada; 7.5 μg/h, changed daily) to attenuate hypoxia-induced chemoresistance (Frederiksen et al. 2007), the same patch as in our previous studies and ongoing clinical trials. This method seems to be appropriate because it can effectively and consistently keep the blood nitroglycerin concentration at least for half a day (Frederiksen et al. 2007). Therefore, it can significantly reduce HIF-1 protein, VEGF, and P-glycoprotein in tumor tissues in vivo (Yasuda et al. 2006b). Mice bearing PC-3 tumor xenografts were injected intratumorally with DETANONOate and systematically with cisplatin (Bonavida et al. 2008). Significant tumor growth inhibition was achieved by the combined treatment of DETANONOate and cisplatin, whereas there were no effects observed when the diluent control; cisplatin alone or DETANONOate alone was administered (Bonavida et al. 2008). In vivo, tissues from nude mice bearing PC-3 xenografts treated with DETANONOate showed an inhibition of a transcriptional factor, Yin–Yang 1 (YY1), and up-regulation of a TNF-related apoptosis-inducing ligand (TRAIL) receptor, DR5. YY1 negatively regulates DR5 transcription and expression which is responsible for TRAIL-induced apoptosis. DETANONOate inhibits both NF-κB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis (Huerta-Yepez et al. 2009). The major histocompatibility complex (MHC) class I chain-related (MIC) molecules play important roles in tumor immune surveillance through their interaction with the NKG2D receptor on natural killer (NK) cell and cytotoxic T lymphocyte (CTL) (Groh et al. 2002; Doubrovina et al. 2003). Hypoxia in tumor tissues increased tumor cell shedding of MHC class I chain-related molecules, MICA and MICB (Siemens et al. 2008). Therefore, cytotoxic activities of NK cells and CTL (cancer immune) are inhibited by hypoxic conditions in tumor tissues (Siemens et al. 2008). A nude mouse model bearing PC-3 treated with a transdermal patch delivering low concentrations of nitroglycerin (7.3 μg/h) showed significant delayed tumor growth compared with mice treated with a placebo patch (Siemens et al. 2008). These data suggest that the use of nitroglycerin patch may enhance cancer immunity via increase in NK cells and CTL activities in patients with cancer.

NOS Induction or Inhibition The murine macrophage iNOS (Ad.iNOS) gene and the EGFP gene (Ad.EGFP) used as a control were transferred with adenoviral vectors into the PC-3 M cells (Le et al. 2005), which were then inoculated into prostate tissues in nude BALB/c mice (Le et al. 2005). Tumor growth of PC-3 M cells transfected with Ad.iNOS was significantly inhibited compared to that of Ad.EGFP-transfected cells (Le et al. 2005). This result suggests that iNOS over-expression may inhibit tumor growth of the PC-3 M cells.

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Lymphoma NOS Induction or Inhibition Lymphoma developed more rapidly in p53 and iNOS double knockout mice (p53−/−iNOS−/−) or p53−/−iNOS+/− mice than in p53−/−iNOS+/+ mice that were crossbred into C57BL/6 mice (Hussain et al. 2004). Likewise, sarcoma and lymphomas developed faster in p53+/−iNOS−/− or p53+/−iNOS+/− mice than in p53+/−iNOS+/+ mice (Hussain et al. 2004). p53−/−iNOS+/+ mice, compared with p53−/−iNOS−/− mice, showed a higher apoptotic index, decreased proliferation index, and an increased expression of CD95L, TRAIL, and the cell cycle checkpoint protein p21waf1 in the spleen and thymus before tumor development (Hussain et al. 2004). Anti-inflammatory interleukin-10 was produced in higher amounts in p53−/−iNOS−/− mice compared with in p53−/−iNOS+/+ mice (Hussain et al. 2004). These findings indicate that NO from iNOS can suppress tumorigenesis. Homozygous disruption of iNOS (iNOS−/−) decreased the incidence of thymic lymphomas by almost 40 and 90% in p53−/− and p53+/− mice, respectively, compared to the respective iNOS wild-type mice but significantly increased the development of nonthymic lymphomas in Trp53−/− and Trp53+/− mice (Wang et al. 2005).

Other Cancers Breast Cancer NOS Induction or Inhibition Lind et al. reported that in vitro, EMT-6 cells treated with adriamycin (ADR) showed apoptosis in a dose-dependent manner with ADR and released nitrite in proportion to the degree of apoptosis (Lind et al. 1997). Apoptotic EMT-6 cells treated with ADR exhibited over-expression of NOS, and the NO derived from NOS promoted apoptosis of the EMT-6 cells in vitro (Lind et al. 1997). Furthermore, NO from activated NOS in cancer cells by ADR treatment was reported to correlate with chemosensitivity to ADR in the EMT-6 tumor bearing mice in vivo (Lind et al. 1997). In contrast, additional use of a NOS inhibitor, aminoguanidine with ADR significantly promoted tumor growth. There was a decrease in chemosensitivity to ADR compared with those treated with ADR alone in the EMT-6 tumor-bearing murine model (Lind et al. 1997). Therefore, iNOS induction after exposition to ADR may promote chemosensitivity to ADR in murine breast cancer cells. Wister-MS rats were exposed to whole-body irradiation with γ-rays (1.5 Gy) immediately after weaning and then treated with diethylstilbestrol as an irradiated control. The tumor incidence (85%) in irradiated rats increased 7.6-fold compared with the non-irradiated control (11%) (Inano and Onoda 2005). Furthermore,

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the tumor incidence declined to 28.6% in the rats injected intraperitoneally with phenyl-N-tert-butylnitrone (PBN, 160 mg/kg), an inhibitor of iNOS (Inano and Onoda 2005). The tumor incidence (25%) in rats orally administered with N-(3(aminomethyl)-benzyl)-acetamide (1400W, 2.30.1 mg/day), a selective inhibitor of iNOS, for 3 days after irradiation was less than one-third of the irradiated controls (Inano and Onoda 2005). Many of the mammary tumors developed in the irradiated rats were positive for the estrogen receptor (ER). On the other hand, ER was not detected in the tumors derived from irradiated rats administered with PBN or 1400W (Inano and Onoda 2005). These results indicate that NO from activated iNOS after irradiation may contribute to the formation of estrogen-dependent mammary adenocarcinoma. The murine macrophage iNOS (Ad.iNOS) gene and EGFP gene (Ad.EGFP) as control were infected with adenovirus into the human breast cancer cell line (MDA-MB-453 cells), which were then inoculated into mammary gland tissues in nude BALB/c mice. Tumor growth of MDA-MB-453 cells with Ad.iNOS was significantly inhibited compared to that with Ad.EGFP (Le et al. 2005). This result suggests that iNOS over-expression may inhibit tumor growth in MDA-MB-453 cells. Inhibition of iNOS activity and NO production in macrophages with a NOS inhibitor, NG -monomethyl-l-arginine (NGMMA), was reported to augment the activity of the CTL response to the anti-tumor response of the murine lymphoblastlike mastocytoma cell line, P815 cells, in vitro and in vivo due to NO inhibition of CTL clonal expansion (Medot-Pirenne et al. 1999).

Gastric Cancer NOS Induction or Inhibition The murine macrophage iNOS (Ad.iNOS) gene and the EGFP gene (Ad.EGFP) as control were transfected with adenovirus into the human gastric cancer cell line (AGS cells). Then, they were inoculated into stomach tissues in nude BALB/c mice. Tumor growth of AGS cells with Ad.iNOS was significantly inhibited compared with that tested with Ad.EGFP (Le et al. 2005). These results indicate that iNOS over-expression may inhibit tumor growth in AGS cells. iNOS-deficient mice (iNOS−/−) and wild-type littermates were subjected to combination treatment comprising N-methyl-N-nitrosourea administration and Helicobacter pylori infection (Nam et al. 2004). The overall incidence of gastric cancer at week 50 was significantly lower in iNOS−/− compared with iNOS wildtype mice. Immunoblotting analyses showed that iNOS and 3-nitrotyrosine were also expressed in both adenoma and adenocarcinoma tissues from iNOS wild-type mice. Furthermore, iNOS and 3-nitrotyrosine expression was higher in tumor tissues than in non-tumor tissues. These findings suggest that iNOS contributes to H. pylori-associated gastric carcinogenesis in mice.

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Colorectal Cancer NO Donor Nitroglycerin injection twice a week (0.02 mg/kg or 0.2 mg/kg/per injection) combined with cisplatin significantly inhibited tumor growth compared with cisplatin alone in an animal model of murine colon cancer (colon-26) inoculated into BALB/c mice (Yasuda et al. 2007b). The CDDP-resistant metastatic human colon cancer cell line (SW620), which had lower expression of apotosis-inducing factor (AIF) than that in the nonmetastatic SW480 cells, was sensitized by DETANONOate (500 and 1000 μM) for 6 h to CDDP-induced apoptosis through up-regulation of AIF (Huerta et al. 2008). The SW480 colon cancer cell line was established from a primary stage II lesion while the SW620 cell line was obtained from the same patient a year later from a colon cancer lymph node metastasis (stage III). Metastatic SW620 colon cancer cells have undergone multiple gene product modifications resulting in resistance to CDDP-induced apoptosis (Huerta et al. 2003, 2007). Furthermore, tumor growth in the murine model bearing SW620 was significantly inhibited by additional treatment with DETANONOate compared with mice treated with CDDP alone (Huerta et al. 2008). Nitroglycerin induced tyrosine nitration of β-catenin, along with its dephosphorylation on serine 33, 37, and 45 and threonine 41. Furthermore, nitroglycerin promoted β-catenin degradation and down-regulated its transcriptional activity in colon cancer cells, resulting in tumor regression in vivo (Prévotat et al. 2006).

NOS Induction or Inhibition The additional use of interferon-gamma (IFN-gamma and IL-1beta) in tumors made of a colon adenocarcinoma chemically induced in a BD IX rat (DHD/K12-PROb) enhanced the anti-tumor effects during lipid A treatment by increasing iNOS activity and NO concentration in tumor tissues (Onier et al. 1999). The murine macrophage iNOS (Ad.iNOS) gene and EGFP gene (Ad.EGFP) as control were transfected into the human colon cancer cell lines, DLD-1 cells, which were then inoculated into colon tissues in nude BALB/c mice. Tumor growth of the DLD-1 cells with Ad.iNOS was significantly inhibited compared with that transfected with Ad.EGFP (Le et al. 2005). This result suggests that iNOS over-expression may inhibit tumor growth of the DLD-1 cells. The adenoviral gene transfer of the human iNOS gene enhances the radiation response of human colorectal cancer (HCT-116) associated with alterations in tumor vascularity (Wang et al. 2004). Furthermore, adenoviral gene transfer of human iNOS gene and concurrent irradiation to solid tumor synergistically increased activated p53 compared with adenoviral empty control vector transfer in a xenograft murine model of human colorectal cancer (HCT-116) with wild-type p53 (Cook et al. 2004). NO was reported to activate p53 in cancer cells through DNA damage

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by peroxynitrite (Schneiderhan et al. 2003). Activated p53 was reported to regulate the apoptotic response to DNA damages induced by NO, ionizing irradiation (Unger et al. 1999), and p53-dependent anti-cancer drugs including cisplatin (Lowe et al. 1993). On the other hand, disruption of p53 is known to induce resistances to chemotherapy (Lowe et al. 1993) and radiotherapy (Lee and Bernstein 1993). These findings suggest that iNOS gene transfer to cancer cells may increase response to ionizing radiation and/or anti-cancer drugs including cisplatin or oxaliplatin through the activation of p53 in cancer cells such as colorectal cancer. An ApcMin/+ mouse is a model mouse of familial adenomatous polyposis. ApcMin/+ Nos2−/− mice developed significantly more intestinal adenomas than ApcMin/+ Nos2+/+ littermates. Epithelial cell iNOS mRNA expression was decreased in adenomas compared with histologically normal ApcMin/+ Nos2+/+ intestine. There was no significant difference in Cox-2 expression or activity in either intestine or bone marrow-derived macrophages from ApcMin/+ Nos2+/+ and ApcMin/+ Nos2−/− animals (Scott et al. 2001). Therefore, iNOS plays an anti-neoplastic role in the ApcMin/+ mouse. L -Arginine is metabolized either to polyamines through arginase and ornithine decarboxylase (ODC) activities or to citrulline and NO through the NOS pathway. Azoxymethane, a chemical carcinogen, induced the formation of colonic aberrant crypt foci (ACF) in adult male Wistar rats. In rats treated with l-NAME, a NOS inhibitor, the number of ACF was higher than in controls by 47% and ODC activity was enhanced by 11-fold (Schleiffer et al. 2000). This data suggests that l-NAME promotes carcinogen-induced preneoplastic changes in the colon by inhibiting NOS activity and by stimulating polyamine biosynthesis in vivo. iNOS gene knockout Apc (Min/+) mice (Apc (Min/+) iNOS(−/−) or Apc (Min/+) iNOS (−/+)) were reported to develop significantly fewer adenomas in both small and large intestines than Apc (Min/+) iNOS (+/+) mice (Ahn and Ohshima 2001).

NO-Donating Non-steroidal Anti-inflammatory Drugs (NSAIDs) GT-094, a novel nitrate containing an NSAID, significantly reduced azoxymethane (carcinogen)-induced aberrant crypt foci (ACF) in male Fisher rats. Furthermore, GT-094 reduced proliferation of crypt cells and increased p27 expression in azoxymethane-treated colons in the rats (Hagos et al. 2007). NO-donating aspirin inhibits angiogenesis by suppressing vascular endothelial growth factor (VEGF) expression in human colon cancer (HT-29) mouse xenografts (Ouyang et al. 2008). NCX 4040, {2-(acetyloxy)benzoic acid 4-(nitrooxy-methyl)phenyl ester}, combined with either oxaliplatin or 5-FU significantly inhibited tumor growth, prolonged delays in tumor growth, and increased apoptotic indexes compared with those treated with oxaliplatin alone in xenograft models using the three human colon cancer cell lines (WiDr, LoVo, and LoVoDx) (Leonetti et al. 2006).

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Lung Cancer NO Donor Concomitant use of nitroglycerin injection intraperitoneally with cisplatin significantly inhibited tumor growth compared with cisplatin alone in an animal model of murine lung adenocarcinoma, Lewis lung carcinoma (LLC), inoculated into C57BL/6 mice (Yasuda et al. 2007b).

NOS Induction or Inhibition Newcastle disease virus (NDV), an agent with interesting immune stimulatory and anti-tumor activities, was investigated for its capacity to activate anti-tumor activity in murine macrophages in vitro and in vivo. Repeated intravenous transfer of NDV activated macrophages in which iNOS was highly expressed and the NO produced exerted a significant suppressive effect on pulmonary metastases in a mammary carcinoma tumor model as well as in a lung carcinoma model in vivo (Schirrmacher et al. 2000). Male B6/129P-Nos2tm1Lau (iNOS−/−) and B6/129P-F2 (wild-type, iNOS+/+) mice were injected intraperitoneally with 1 g/kg urethane once weekly for seven consecutive weeks and tumors were enumerated and sized 16 weeks after the initial urethane injection. iNOS-deficient (iNOS−/−) mice developed fewer tumors than wild-type mice. iNOS deficiency did not affect tumor incidence and tumor size. Tumors from iNOS-deficient mice contained less VEGF protein than tumors from wild-type mice. These results strongly support examining iNOS-specific inhibitors in potential lung cancer chemopreventive agents.

Renal Cellular Carcinoma NOS Induction or Inhibition The highly metastatic human renal cellular carcinoma, SN12PM6 cell line, was transfected with the control or murine macrophage iNOS retroviral vectors, pLXSN. The cells were then injected into the kidney subcapsule of Balb/c nude mice (Juang et al. 1998). Non-transfected or control cells produced larger tumors in the kidney and a larger number of experimental lung metastasis than those in the iNOStransfected cells in the mice (Juang et al. 1998). These findings suggest that NO from iNOS shows anti-tumor cytotoxicity and abrogation of metastasis. The murine macrophage iNOS (Ad.iNOS) gene and EGFP gene (Ad.EGFP) as control were transferred with adenovirus into the SN12PM6 cells. Then, these cells were inoculated into kidney tissues in nude Balb/c mice. Tumor growth of SN12PM6 cells with Ad.iNOS was significantly inhibited compared with that with Ad.EGFP (Le et al. 2005). This result indicates that iNOS over-expression inhibits tumor growth in SN12PM6 cells.

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Hepatic Cellular Carcinoma NO Donor Intravenous injection of isosorbide dinitrite, an organic NO donor (0.2 mg/kg), increased partial oxygen pressure in tumor tissues by 79% via increase in tumor perfusion compared with treatment with vehicle in the transplantable murine liver tumor model (Jordan et al. 2000). These findings demonstrated that increase in oxygenation by NO may enhance radiosensitivity.

Malignant Melanoma NO Donor Chemoimmunotherapy with cisplatin in dendritic cells pretreated with NO released by DETA-NO or isosorbide dinitrite was significantly more efficacious than cisplatin alone in tumor regression and animal survival in the B16 mouse model of melanoma. These findings demonstrated that the pretreatment of dendritic cells with NO protected the dendritic cells from cisplatin-induced apoptosis via inhibition of acid sphingomyelinase, ceramide generation, caspase-3 and 9 activation, and mitochondrial depolarization (Perrotta et al. 2007). NOS Induction or Inhibition iNOS knockout (iNOS−/−) and wild-type (iNOS+/+) mice were inoculated subcutaneously with the murine tumorigenic melanoma cells (B16-F1 cells). The median tumor weight of B16-F1 cells in iNOS knockout mice was significantly heavier than that of wild-type mice. VEGF mRNA and immunoreactive VEGF protein in tumor tissues in the iNOS knockout mice were significantly higher than tumor tissues in wild-type mice (Konopka et al. 2001). These investigations concluded the dependence of tumor growth on iNOS activity and reinforce the importance of iNOS as a therapeutic target in cancer.

Other Malignant Tumors NO Donor JS-K is a pro-drug designed to release NO following reaction with glutathione. JS-K treatment significantly inhibited tumor (human multiple myeloma, MM, cell line: OPM1 cells in NIH III mice) growth compared with mice treated with vehicle (control). Furthermore, JS-K significantly prolonged survival of the murine MM models compared with control mice. JS-K induced apoptosis in MM cells via caspase-8 and 9 cleavages, increase in Fas/CD95 expression, Mcl-1 cleavage, and

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Bcl-2 phosphorylation as well as cytochrome c, AIF, and endonuclease G release (Kiziltepe et al. 2007). NOS Induction or Inhibition Three iNOS knockout (iNOS−/−) fibrosarcoma cell lines, KX-dw1, LX-dw4, and KX-dw7 cells, were established. These cells were subcutaneously implanted or intravenously injected into iNOS+/+ and iNOS−/− C57BL/6 mice. The KX-dw1, LX-dw4, and KX-dw7 cells grew much faster and produced many more lung metastases in the iNOS knockout mice (Wei et al. 2003). These findings indicate that NO from iNOS may directly inhibit tumor growth and metastasis. In vivo transfer of constitutively expressed CMV/iNOS plasmid DNA by direct injection into established radiation-induced fibrosarcoma-1 tumors treated with cisplatin caused a significant delay in tumor growth compared with that of vacant plasmid DNA with cisplatin (Adams et al. 2009). Because most solid tumors contain hypoxic areas, a certain number of solid tumors show a resistance to radiotherapy (De Ridder et al. 2008). NO from endothelial NOS (eNOS) significantly inhibited liver metastasis and significantly prolonged survival after implantation of L3.6 pancreatic cancer cells transfected with L3.6-eNOS cDNA via portal vein injection compared with those transfected with L3.6-LacZ (control) cDNA in SCID mice (Decker et al. 2008). NO from eNOS was demonstrated to result in down-regulation of the matrix protease cathepsin B which is responsible for invasive capacity of cancer cells (Decker et al. 2008). The iNOS gene was cloned into a vector containing nine CArG element repeats (pE9/iNOS) (Coulter et al. 2008). The specific transcriptional control (CArG) elements were identified within the Egr-1 gene that are up-regulated in response to ionizing radiation (Datta et al. 1993). An ex vivo rat tail artery treated with pE9/iNOS showed rapid and more pronounced vasodilation compared to CMV/iNOS (Worthington et al. 2005). The CMV/iNOS, pE9/iNOS, and pE9/GFP controls were transfected in the murine sarcoma cell line (RIF-1) and the cells were treated with a radiation dose of 4 Gy 16 h after transfer. Transfection with CMV/iNOS and pE9/iNOS plasmids significantly reduced clonogenic cell survival when compared with pE9/iNOS-transfected cells. Furthermore, the RIF-1 cells were inoculated into female C3H mice and CMV/iNOS, pE9/iNOS, and pE9/GFP plasmids were injected intratumorally and 4 Gy of irradiation was performed. In conclusion, the pE9/GFP treatment inhibited the most (Coulter et al. 2008). The murine macrophage iNOS (Ad.iNOS) gene and EGFP gene (Ad.EGFP) as control were transfected with adenovirus into the human pancreas cell line (L3.3 cells), the human ovarian cancer cell line (SKOV3), the human urinary cyst cancer cell line (253J BV cells) and the human fibrosarcoma cell line (HT-1080 cells). These cells were then inoculated into nude Balb/c mice. Tumor growth of the L3.3 cells, the SKOV3 cells, the 253J BV cells, and the HT-1080 cells that were transfected with Ad.iNOS was significantly inhibited compared with those transfected with Ad.EGFP (Le et al. 2005). This result suggests that iNOS over-expression may

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inhibit tumor growth in the L3.3, the SKOV3, 253J BV, and the HT-1080 cells. Furthermore, the distant metastases, regional lymph node metastases, and microvascular vascular density (MVD) (by immunohistochemical study of CD31 in tumor tissues) by iNOS gene transfer were examined in the mice in which the L3.3 cells were inoculated into pancreas tissues. The distant metastases to the liver and/or regional metastases to lymph nodes were also inhibited in Ad.iNOS-produced NO level-dependent manners in the mice. On the other hand, there was no increase or decrease in MVD in tumors formed by cells transduced with the iNOS gene (Le et al. 2005). These findings suggest that NO from iNOS has cytotoxic effects on cancer cells in tumor tissues depending on the level of NO production. They also suggest that NO production did not significantly alter tumor angiogenesis in vivo. Chronic inflammation caused by subcutaneous implantation of plastic plates accelerated sarcoma development in heterozygous p53-deficient (Trp53+/−) mice. Oxidative and nitrative damage caused by inflammatory cells infiltrating around the implant may be implicated in sarcoma development, likely, through the induction of p53 loss of heterozygosity (Tazawa et al. 2007). Treatment of 7,12-dimethylbenz[a]anthracene-initiated 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted mice with nitroglycerin increased the latency period, decreased the tumor incidence by 32%, and decreased tumor yield by twofold as compared with the TPA-alone treated group by 20 weeks in murine skin (Trikha et al. 2001). Electrical stimulation at 5 Hz with 0.2 ms duration pulses was reported to increase tumor partial oxygen pressure by 161% as well as increase in NO concentration by 40% through endogenous NO production by activation of eNOS in tumor tissues of the syngeneic FSaII tumor model in C3H mice (Jordan et al. 2003). These findings suggest that increase in oxygenation in tumor tissues by activation of NOS may be a strategy for enhancement of radiosensitivity in vivo.

Concluding Remarks and Future Directions Several reports suggest that NO from NO-donating drugs or NOS may have potent positive or negative influences on carcinogenesis, tumor growth, and metastasis in vivo. The conclusions have been controversial. Experimental conditions including the dosage of NO concentration, the way of administration of NO-donating agents or NOS gene transfer, the type of cancer cell lines, and redox status in cancer cells may affect these results. Appropriate conditions and methods for good outcomes for clinical application in patients with malignant diseases should be developed. To demonstrate the effects of iNOS expression on cancer cell apoptosis, tumor growth, metastasis, and response to cancer therapy, we should consider whether NO itself from iNOS or genetic backgrounds such as p53-deficient cancer cells really determines the experimental results. Endogenous NO from NOSs may have different outcomes than exogenous NO from NO donors. NO promotes p53 activation via DNA damage by peroxynitrite (Schneiderhan et al. 2003; Wang et al. 2004; Cook

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et al. 2004; Yasuda 2008) and brings about down-regulation of iNOS expression by negative feedback (Chang et al. 2004; Yasuda 2008). On the other hand, iNOS expression depends upon p53 activity, HIF-1α accumulation, and NO concentration as described below. Chronic hypoxia promotes accumulation of HIF-1α and aberrant p53 in cancer cells (Fels and Koumenis 2005; Yasuda 2008). Activated HIF-1α promotes transcription of target genes including VEGF and P-glycoprotein which are known to be chemoresistant factors (Semenza et al 1994; Hicklin and Ellis 2005; Yasuda et al. 2007; Yasuda 2008), resulting in the promotion of tumor growth and metastasis and poor prognosis (Nakamura et al. 2006; Trastour et al. 2007; Yasuda 2008). Accumulation of aberrant p53 is associated with chemo- (Lowe et al. 1993) and/or radio-resistance (Wang et al. 2004; Cook et al. 2004). Furthermore, accumulation of inactive p53 was positively correlated with iNOS expression in cancer cells in tumor tissues in vivo (Wang et al. 2005). These findings suggest that in addition to the over-expression of iNOS, the accumulation of aberrant p53 may promote production of VEGF and increase microvascular density in tumor tissues, resulting in chemoradioresistance and promotion of metastasis and poor prognosis. Strict design of animal experiments is needed to develop NO-related research for clinical applications.

Human Studies There are several clinical studies related to NO and cancers including nonrandomized studies and randomized studies to study the effects of NO therapeutic applications in patients with cancer.

Non-randomized Studies There are two reports regarding the combined use of nitroglycerin with cytotoxic anti-cancer drugs in patients with non-small cell lung cancer (NSCLC) (Yasuda et al. 2006b, 2007a). Of the two clinical reports, one is a phase II trial in untreated patients with advanced NSCLC (Yasuda et al. 2006b) and the other is a case report in patients with refractory and recurrent NSCLC (Yasuda et al. 2007a). Furthermore, two nonrandomized clinical trials with NO-associated drugs without use of anti-cancer drugs were reported. Of the two studies, one is a phase I/II trial in patients with prostate cancer (Siemens et al. 2007) and the other is a phase I trial in patients with several kinds of cancer (Ng et al. 2007). Now, we are conducting a non-randomized clinical trial of phase II study with nitroglycerin plus amrubicin in patients with refractory and recurrent advanced NSCLC as a third-line regimen. Moreover, we are now planning another non-randomized phase II trial with nitroglycerin plus docetaxel in elderly untreated patients with advanced NSCLC. A non-randomized study in patients with operable lung adenocarcinoma was performed to study the effects of continuous use of nitroglycerin patch (25 mg/patient,

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daily) due to ischemic heart diseases before operation. The expressions of protein in tumor tissues were performed to study the effects of nitroglycerin (25 mg/patient, daily for 3 days) before the start of chemotherapy with docetaxel (60 mg/m2 , on day 1) and carboplatin (AUC 5, on day 1). Plasma VEGF concentration which is regulated by activity of HIF-1α was reported to associate with clinical response to chemotherapy in untreated stage IIIB/IV NSCLC (Yasuda et al. 2006b). The study showed that the continuous use of nitroglycerin patch significantly decreased the rates of expression of HIF-1α, VEGF, and P-glycoprotein in resected tumor tissues in patients with operable lung adenocarcinoma. Furthermore, the use of nitroglycerin patch for 3 days before the start of administration of anti-cancer drugs significantly decreased the plasma VEGF levels and there was a significant relationship between decrease levels in plasma VEGF and response rate to chemotherapy (Yasuda et al. 2006b). Nitroglycerin treatment has a tendency to reduce plasma VEGF levels in patients with NSCLC, but not always reduced its levels in some individuals (Yasuda et al. 2006b). The use of nitroglycerin patch was suggested to increase intratumor perfusion and oxygenation and brought about subsequent decrease in HIF-1α in tumor tissues, resulting in decreases in VEGF and P-glycoprotein in tumor tissues (Yasuda et al. 2006b, 2007b). Therefore, it was thought that nitroglycerin might augment chemosensitivity to docetaxel through decrease in P-glycoprotein in tumor tissues only in patients with lung adenocarcinoma in whose tumor tissues nitroglycerin could enhance oxygenation via increase in intratumor perfusion by dilatation of arterioles (Yasuda et al. 2006b, 2007b). The results of the clinical study (Yasuda et al. 2006b) were consistent with those in animal studies (Yasuda et al. 2007b). These findings suggest that the effect of nitroglycerin treatment on the increase in response to chemotherapy may depend on the pattern of intratumor vascularization and that the combined use of nitroglycerin with anti-cancer drugs may be more effective than anti-cancer drugs alone in patients whose plasma VEGF levels can be significantly decreased (more than 16 pg/ml) by pretreatment with nitroglycerin for 3 days before the start of administration of anti-cancer drugs. In a case report, we reported two patients who achieved partial response to R , 10 mg/day for 5 days/cycle) plus amrubicin (60% dose nitroglycerin (Millistape 2 intensity, 27 mg/m on day 1, 2, and 3, every 4 weeks) regimen in the fourth-line regimen and fifth-line regimen, respectively (Yasuda et al. 2007a). Amrubicin is a novel synthetic 9-aminoanthracycline derivative and has been developed in Japan (Sugiura et al. 2005). Recently, amrubicin was approved in Japan for the treatment of small cell lung cancer and NSCLC. The maximum tolerated dose and recommended dose in Japan were determined to be 50 and 45 mg/m2 /day, respectively (Sugiura et al. 2005). Amrubicin is a potent inhibitor of topoisomerase II (Ohe et al. 1989) and has more potent anti-tumor activity against various tumor tissues in in vivo experiments than other anthracyclines and with less heart damages (Morisada et al. 1989). The combined use of nitroglycerin may increase response to amrubicin as well as doxorubicin, anthracycline anti-cancer drugs, and docetaxel through decrease in P-glycoprotein in solid cancers (Yasuda et al. 2007b; Frederiksen et al. 2007). Therefore, amrubicin effect or resistance and the effects of nitroglycerin

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on chemosensitivity to amrubicin should be studied in cancer cells, animal experiments, and clinical trials. We are now performing a non-randomized phase II trial using nitroglycerin plus amrubicin in patients with refractory and recurrent NSCLC as a third-line chemotherapy, described below. Two non-randomized clinical studies with NO-related drug but without anticancer drugs have been reported. Siemens et al. (2007) reported that treatment with sustained NO-releasing nitroglycerin patch without anti-cancer drugs prolonged doubling time in the tumor marker of prostate cancer, prostate-specific antigen, in patients with clinically recurrent prostatic cancer after initial treatment in a phase I/II trial. They demonstrated beneficial effects of nitroglycerin patches without anti-cancer drugs on recurrent prostate cancer (Siemens et al. 2007). Randomized phase III trials in such patients should be performed in the future. On the other hand, a NOS inhibitor, N-nitro-l-arginine (L-NNA), was demonstrated in 7 women and 11 men (12 with non-small cell lung cancer, 5 prostate cancer, and 1 cervical cancer) in a phase I trial (Ng et al. 2007). In the 18 patients, toxic effects were self-limiting cardiovascular changes: 3 patients had Common Toxicity Criteria version 2.0 grade 1 hypertension; 2 had grade 1 sinus bradycardia; and 1 had grade 1 palpitation. There was a significant correlation between L-NNA plasma area under the curve and the reduction in tumor blood volume at 24 h after L-NNA (r=0.83; p=0.010) (Ng et al. 2007). They concluded that nitric oxide has a role in maintaining tumor blood supply. A non-randomized phase II study in patients with refractory and recurrent R , 10 mg/day for 5 days/cycle) plus amruNSCLC using nitroglycerin (Millistape 2 bicin (80% dose intensity, 36 mg/m on days 1, 2, and 3, every 4 weeks) as a thirdline chemotherapy is now being performed (KUOPLC0701) at Kyoto University, Tohoku University, Showa University, National Cancer Center at Kashiwa, and R , 5 mg/patch, Shinshu University (Yasuda et al.). In this study, the millistape is used as nitroglycerin patch because it has low possibility to bring about clinical tolerance according to its characteristic in pharmacokinetics. It has a peak level in its blood concentration at about 6 h after the start of administration and its blood concentration is reported to return to the baseline about 12 h after the start of use. Therefore, it is used twice a day (10 mg/ day). The reason for the decrease in the amrubicin dose (20% decrease) in this study is due to potent myelosupressive effects. Because this study is designed as a third-line chemotherapy, neutrophil number and platelet number in peripheral blood tend to be promptly decreased and are difficult to return to the baseline before the start of amrubicin administration in enrolled patients due to myeloid fatigue. The data center of this trial is the Department of Clinical Trial Design and Management, Translational Research Center, Kyoto University Hospital, Kyoto, Japan. The primary end point is progression-free survival and the secondary end points are response rate, disease control rate, toxicity, overall survival, and the relationship between change of plasma VEGF levels and clinical response to the chemotherapy. The target number of enrolled patients calculated by sample-size analysis in this study is 60 patients. Taxanes including docetaxel are approved for second-line treatment of advanced NSCLC in the USA and European Union (Shepherd et al. 2000; Fossella et al. 2000).

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However, there is no approved cytotoxic drug for third-line treatment of relapsed or recurrent NSCLC (Massarelli et al. 2003). The aim and design to perform this study was on the basis of a case report, as described previously (Yasuda et al. 2007a). If excellent results will be shown in this phase II trial, we will perform a three-armed, randomized, phase III trial comparing nitroglycerin plus amrubicin with amrubicin alone with standard regimen at that time as a third-line chemotherapy in patients with refractory and recurrent NSCLC. We are now preparing another non-randomized phase II trial using nitroglycerin and docetaxel in previously untreated and elderly patients with stage IIIB/IV NSCLC for the next phase III trial. The clinical trial has been designed by the results of a randomized phase III trial, WJTOG 9904 (Kudoh et al. 2006), in which single use of docetaxel has shown the higher response rate and prolonged progression-free survival with feasible adverse effects compared with those treated with vinorelbine alone in untreated elderly patients with NSCLC. The primary end point is response rate and the secondary end points are progression-free survival, toxicity, overall survival, and the relationship between change of plasma VEGF levels and clinical response to the chemotherapy in this study. The target number of enrolled patients calculated by sample-size analysis in this study is 40 patients. If this phase II trial with docetaxel and nitroglycerin would show an excellent response rate and progression-free survival more than those treated with docetaxel alone or equivalent to those with standard platinum-doublet regimens as a first-line chemotherapy, we will perform the next randomized phase III trial comparing the docetaxel and nitroglycerin regimen with a standard platinum-doublet regimen such as cisplatin and docetaxel in previous untreated elderly patients with NSCLC.

Randomized Studies According to Internet survey, three randomized trials in untreated stage IIIB/IV patients with NSCLC regarding a nitric oxide-donating drug, nitroglycerin, have been detected. Of the three trials, one randomized trial had been completed and two randomized trials have been performed in Japan and in Australia. Furthermore, a randomized phase I in patients at high risk of colorectal cancer has been completed. We have demonstrated that a combinational use of nitroglycerin plus vinorelbine and cisplatin (VC) regimen improved the response rate of 72% and median time to progression of 11 months compared with response rate of 42% and median time to progression of 4.2 months in VC alone in previously untreated stage IIIB/IV patients with NSCLC in a randomized phase II trial, as previously described (Yasuda et al. 2006a). Twenty-five milligrams per patch per day of nitroglycerin was used for 5 days per one cycle between 3 days before the start of VC administration and 2 days after the start of VC regimen. Twenty-five milligrams per square meter of vinorelbine on days 1 and 8 and 80 mg/m2 of cisplatin on day 1 every 3 weeks for the maximum of four cycles were administered (Schiller et al. 2002). Characteristic

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effects in the group treated with nitroglycerin plus VC were in Common Toxicity Criteria for adverse events version 2.0 (Trotti et al. 2000). Grade 1 headache and grade 1 hypotension which occurred significantly higher than those in VC alone in the trial (Yasuda et al. 2006a). These findings suggest that the combined use of nitroglycerin with VC regimen in previously untreated advanced NSCLC may have clinical benefits without additional severe adverse effects. A largerscale randomized clinical trial comparing nitroglycerin plus VC with VC alone should be performed to ascertain the additional effects of nitroglycerin on clinical responses. We are now performing another randomized phase II trial (TRILC0702, R ) and pacliNCT00616031) comparing nitroglycerin plus carboplatin (paraplatin R taxel (taxol ) (TJ) regimen with TJ alone in previously untreated stage IIIB/IV patients with NSCLC for the next phase III trial at Kyoto University, Tohoku University, Kumamoto University, Jikei University, Juntendo University, National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka Police Hospital, and Shiga Medical Center for adults, in Japan (Yasuda et al.). Ten milligrams per R ) has been used for 5 days per two patches per day of nitroglycerin (Millistape one cycle between 3 days before the start of TJ administration and 2 days after the start of TJ regimen. Two hundred milligrams per square meter of paclitaxel on day 1 and area under the curve 6 of carboplatin on day 1 every 3 weeks for the maximum of six cycles have been administered (Kelly et al. 2001). A primary end point in this trial is response rate. The secondary end points are progression-free survival, toxicity, overall survival, the relationship between change of VEGF levels during 3 days using nitroglycerin before the start of TJ administration at first course and therapeutic effects such as response rate, and the effects of nitroglycerin on pharmacokinetics of plasma paclitaxel concentration. The target number of enrolled patients calculated by sample-size analysis in this study is 150 patients (75 patients in each group). The data center is the Translational Research Informatics (TRI) Center, Kobe, Japan. Furthermore, case report forms are collected by the means of Electronic Data Capturing (EDC) system via Internet developed by TRI. Randomization is performed via Internet at any time in 24 h. This study was started on January 1, 2008, and we are now recruiting patients. A randomized, double-blind, phase I multiple-dose safety, pharmacokinetics and pharmacodynamic clinical study of the nitric oxide-releasing acetylsalicylic acid derivative (NCX4016) in patients at high risk of colorectal cancer has been completed (NCI). This trial was performed to evaluate the side effects and best dose of NCX4016 in preventing colorectal cancer in patients at high risk of colorectal cancer. The primary end point was the effects of NCX4016 on aberrant cryptic foci (ACF) multiplicity after the second dose at 6 months (NCI). The secondary end points were (1) pharmacokinetic profile by blood, urine, and colon tissue sampling; (2) incidence of ACF as measured by magnification chromoendoscopy; (3) assessment of biomarkers expressed in colon tissue, including PGE2, COX-1, COX-2, NF-kB, and β-catenin (measured by immunohistochemistry) at baseline, and at the final visit; (4) data on C-reactive protein as a marker for inflammation; and (5) safety and tolerability of long-term oral administration of NCX4016 as measured

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by NCI-CTCAEv3.0 (NCI). Estimated enrollment was 240 patients. The results of this study have not been reported yet. We can detect a randomized phase III trial of adding nitroglycerin to first-line chemotherapy in patients with advanced NSCLC performed by Dr. M. Boyer at the University of Sydney by Internet (Boyer et al.). However, the details of the trial are not available.

Concluding Remarks and Future Directions Now, several clinical trials, mainly phase II trials, regarding NO and cancer have been conducted around the world. Many principal investigators have paid attention to the significance of the role of NO on cancer cells and to the clinical applications of NO to cancer therapies such as chemotherapy, radiotherapy, and immunotherapy. At first, we must establish some concepts regarding the effects of NO on cancer cells from in vitro studies, in vivo experiments, or clinical experiences. In spite of extensive research performed by many investigators for cancer therapy, the therapeutic effects have not always been satisfied by the cancer patients as yet. Furthermore, the cost for cancer therapy has risen due to prevalence of expensive drugs such as molecular targeting agents. Many patients in the world cannot receive current cancer therapy due to its expensive cost. We, researchers, are now requested to develop inexpensive, safety, and effective cancer therapies. To deal with these problems, we effectively make a use of abandoned or neglected old drugs such as thalidomide for multiple myeloma. We can use nitroglycerin as an NO donor inexpensively, safely, at any time, and anywhere in the world. If the proof of concept of nitroglycerin for its enhancement of anticancer agents is proven, cancer patients will be satisfied. Therefore, we must proceed to the next phase III trials in patients with cancer. Acknowledgment The chapter was supervised by Dr. K. Yanagihara and Prof. M. Fukushima. Dr. H. Yasuda was supported by a Grant-In-Aid for Scientific Research from the Ministry of Education, Science and Culture (17790524, 19689018) of the Japanese government and The Kanae Foundation for the Promotion of Medical Science.

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Scott, D.J., Hull, M.A., Cartwright, E.J., et al. (2001). Lack of inducible nitric oxide synthase promotes intestinal tumorigenesis in the Apc(Mic/+) mouse. Gastroenterology 121, 889–899. Semenza, G.L., Roth, P.H., Fang, H.L., et al. (1994). Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem. 269, 23757–23763. Shepherd, F.A., Dancey, J., Ramlau, R., et al. (2000). Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy. J. Clin. Oncol. 18, 2095–2103. Siemens, D.R., Heaton, J., Adams, M., et al. (2007). A phase I/II pilot trial of low-dose, sustainedrelease GTN for prostate cancer patients with recurrence after primary therapy. Nitric Oxide 17, S15, A26. Siemens, D.R., Hu, N., Sheikhi, A.K., et al. (2008). Hypoxia increases tumor cell shedding of MHC class I chain-related molecule: role of nitric oxide. Cancer Res. 68, 4746–4753. Stewart GD, Nanda J, Brown DJ, et al. (2009). NO-sulindac inhibits the hypoxia response of PC-3 prostate cancer cells via the Akt signalling pathway. Int. J. Cancer 124, 223–232. Sugiura, T., Ariyoshi, Y., Negoro, S., et al. (2005). Phase I/II study of amrubicin, a novel 9-aminoanthracycline, in patients with advanced non-small-cell lung cancer. Invest. New Drugs 23, 331–337. Swinson, D.E., Jones, J.L., Cox, G., et al. (2004). Hypoxia-inducible factor-1 alpha in non small cell lung cancer: relation to growth factor, protease and apoptosis pathways. Int. J. Cancer 111, 43–50. Takabuchi, S., Hirota, K., Nishi, K., et al. (2004). The inhibitory effect of sodium nitroprusside on HIF-1 activation is not dependent on nitric oxide-soluble guanylyl cyclase pathway. Biochem. Bioph. Res. Co. 324, 417–423. Talks, K.L., Turley, H., Gatter, K.C., et al. (2000). The expression and distribution of the hypoxiainducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumorassociated macrophages. Am. J. Pathol. 157, 411–421. Tazawa, H., Tatemichi, M., Sawa, T., et al. (2007). Oxidative and nitrative stress caused by subcutaneous implantation of a foreign body accelerates sarcoma development in Trp53+/- mice. Carcinogenesis 28, 191–198. Thomas, D.D., Espey, M.G., Ridnour, L.A., et al. (2004). Hypoxic inducible factor 1 alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc. Natl. Acad. Sci. USA. 101, 8894–8899. Trastour, C., Benizri, F., Ettore, F., et al. (2007). HIF-1alpha and CA IX staining in invasive breast carcinomas: prognosis and treatment outcome. Int. J. Cancer 120, 1451–1458. Trédan, O., Galmarini, C.M., Patel, K., et al. (2007). Drug resistance and the solid tumor microenviroment. J. Natl. Cancer Inst. 99, 1441–1454. Trikha, P., Sharma, N., Athar, M. (2001). Nitroglycerin: a NO donor inhibits TPA-mediated tumor promotion in murine skin. Carcinogenesis 22, 1207–1211. Trotti, A., Byhardt, R., Stetz, J., et al. (2000). Common toxicity criteria: Version 2.0-An improved reference for grading the acute effects of cancer treatment: Impact on radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 47, 13–47. Unger, T., Sionov, R.V., Moallem, E., et al. (1999). Mutations in serines 15 and 20 of human p53 impair its apoptotic activity. Oncogene 18, 3205–3212. Vaupel, P., Mayer, A. (2007). Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev. 26, 225–239. Wang, Y.Z., Cao, Y.Q., Wu, J.N., et al. (2005). Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA. World J. Gastroenterol. 11, 46–50. Wang, Z., Cook, T., Alber, S., et al. (2004). Adenoviral gene transfer of the human inducible nitric oxide synthase gene enhances the radiation response of human colorectal cancer associated with alterations in tumor vascularity. Cancer Res. 64, 1386–1395. Wei, D., Richardson, E.L., Zhu, K., et al. (2003). Direct demonstration of negative regulation of tumor growth and metastasis by host-inducible nitric oxide synthase. Cancer Res. 63, 3855–3859.

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Worthington, J., Robson, T., Scott, S., et al. (2005). Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer. Gene Ther. 12, 1417–1423. Xie, K., Huang, S., Dong, Z., et al. (1997). Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene. J. Natl. Cancer Inst. 89, 421–427. Yasuda, H. (2008). Solid tumor physiology and hypoxia-induced chemo/radio-resistance: Novel strategy for cancer therapy: Nitric oxide as a therapeutic enhancer. Nitric Oxide 19, 205–216. Yasuda, H., Yamaya M., Nakayama K., et al. (2006a). Randomized phase II trial comparing nitroglycerin plus vinorelbine and cisplatin with vinorelbine and cisplatin alone in previously untreated stage IIIB/IV non-small cell lung cancer. J. Clin. Oncol. 24, 688–694. Yasuda, H., Nakayama, K., Watanabe M., et al. (2006b). Nitroglycerin treatment may enhance chemosensitivity to docetaxel and carboplatin in patients with lung adenocarcinoma. Clin. Cancer Res. 12, 6748–6757. Yasuda, H., Kaneta, T., Takai, Y., et al. (2007). Tumor hypoxia imaging with [F-18] fluoronitroimidazole in non-small-cell lung cancer, J. Am. Geriatr. Soc. 55, 1142–1144. Yasuda, H., Nakayama, K., Watanabe, M., et al. (2007a). Nitroglycerin may increase response to anti-cancer drugs in non-small cell lung cancer via reduction of HIF-1α pathway. Nitric Oxide 17, S15, A24. Yasuda, H., Nakayama, K., Sasaki, T., et al. (2007b). Partial response by nitroglycerin plus amrubicin regimen in patients with refractory and recurrent advanced non-small cell lung cancer who had received at least third-line chemotherapy. Cancer Ther. 5, 451–456. Yasuda, H., Yanagihara, K., Mio,T., et al. http://clinicaltrials.gov/ct2/show/NCT00616031 Yasuda, H., Yanagihara, K., Mio, T., et al. https://center.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi? function=brows&action=brows&recptno=R000000973&type=summary&language=E Zalatnai, A. (2006). Molecular aspects of stromal-parenchymal interactions in malignant neoplasms. Curr. Mol. Med. 6, 685–693.

Chapter 23

(S,R)-3-Phenyl-4,5-dihydro-5-isoxazole acetic acid–Nitric Oxide (GIT-27NO) – New Dress for Nitric Oxide Mission Sanja Mijatovic, Danijela Maksimovic-Ivanic, Marco Donia, Stanislava Stosic-Grujicic, Gianni Garotta, Yousef Al-Abed, and Ferdinando Nicoletti

Abstract Nonsteroidal-anti-inflammatory drugs modified by covalent attachment of nitric oxide (NO) have been recognized as compounds with antitumor properties. By adopting this approach the new compound GIT-27NO was synthesized at GaNiAl Immunotherapeutics Inc. (Wilmington, Delaware, USA) on the basis of the anti-inflammatory isoxazoline derivative VGX-1027. In contrast to the usual modification, i.e., connection via a spacer molecule, GIT-27NO was generated by direct addition of a releasing NO moiety. Contrary to the parental compound which is completely inefficient as an antitumor drug, the modified compound acquired strong anticancer potential. The drug reduced the growth of various cell lines in vitro as well as some solid localized and even metastatic tumors in vivo. Decreased viability of tumor cells was caused by induction of different types of programmed cell death whereas accidental cell death was a secondary event. The outcome of the drug treatment was independent of the type of intracellular response, since the absence or inactivation of key executive mediators of apoptosis, like p53 or caspases, did not affect the death signal triggered by GIT-27NO. Furthermore, cells made resistant to apoptotic stimuli are sensitive to GIT-27NO as well. Although the drug efficacy is explicitly related to NO liberation, GIT-27NO did not function as a simple exogenous donor. Signal for NO release came from cells, and further events included the generation of ROS, RNS and subsequent nitration of tyrosine residues, caspase inhibition, or decreased activity of the YY1 repressor. The drug effect on the MAP signaling pathway was heterogeneous and defined by the cell specificity, the plasticity of the agent’s action, its high efficacy, and low toxicity and suggests that GIT-27NO is a candidate for anticancer drug of the future. Keywords Cancer · Cell death · Nitric oxide · MAP kinases · GIT-27NO

F. Nicoletti (B) Department of Biomedical Sciences, University of Catania, Via Androne, 83, Catania 95124, Italy e-mail: [email protected]

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3_23, C Springer Science+Business Media, LLC 2010

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Introduction Treatment of pain, inflammation, and fever most frequently implies administration of nonsteroidal anti-inflammatory drugs (NSAIDs). Besides their primary role in the treatment of inflammation, recent epidemiologic studies, as well as preclinical and clinical studies, have shown that some NSAIDs are effective in cancer prevention (Puntoni et al. 2008). This effect is particularly well documented in relation to colorectal and gastric cancers (Wang et al. 2003; Gao, Liu and Rigas 2005; Dube et al. 2007). Several anticancer properties of NSAIDs have been proposed to play important roles in cancerogenesis prevention: stimulation of apoptosis, cell growth suppression, inhibition of angiogenesis, and metastasis prevention (Chan et al. 1998; Chan 2002). Furthermore, overexpression of COX-2 (a subtype of COX) has been reported in various tumor cells and tissues (Soydan et al. 1997; Lim et al. 2000; Scartozzi et al. 2004). However, the inhibition of cyclooxygenase (COX) by NSAIDs may not be the unique explanation for their chemopreventive effect. In fact, several lines of evidence based on conserved anticancer properties of NSAIDs in COX-negative cells suggested the relevance of COX-independent mechanisms in their antitumor action (Kopp and Ghosh 1994; Grilli et al. 1996; Bak et al. 1998; Yin et al. 1998; Kashfi et al. 2002). It has been recently demonstrated that aspirin and several other NSAIDs can promote apoptosis through the inhibition of NF-κB activity, activation of mitochondrial pathways by cytochrome c release and activation of caspase-9 and extrinsic pathways by activation of caspase-8, induction of oxidative stress, and inhibition of proteasome functions (Jana 2008). Unfortunately, serious side effects limited the application of those above drugs. The most common adverse events of NSAID are the development of ulcers and subsequent bleeding in the upper gastrointestinal tract and different renal side effects, such as acute renal failure, acute interstitial nephritis, worsening of chronic kidney disease (CKD), salt and water retention, and hypertension (Rigas and Kashfi 2004; Wallace and Vong 2008; House et al. 2007). A chemical modification of these drugs based on covalent attachment of nitric oxide (NO) has been proposed to overcome the most common NSAID-associated adverse events (Lanas 2008). This approach was supported by an idea that NO shares similar properties with prostaglandins (PGs) with regard to the capacity of PGs to influence local blood flow (Rigas and Kashfi 2004). NO attached to the drug through the spacer molecule might be delivered to the damaged site, thereby decreasing gastric toxicity induced by diminished PG levels (Rigas and Kashfi 2004; Lanas 2008). However, designed hybrid nitrates presented a new active molecule with possible application in different therapeutic areas (Keeble and Moore 2002). It was shown that drugs with this modification have hundred to thousand folds augmented antitumoral potential in comparison to original drugs (Rigas and Kashfi 2004; Huguenin et al. 2005; Ouyang et al. 2006; Kashfi and Rigas 2007; Rigas and Williams 2008). This stronger effect is not a simple consequence of NO release and NO–NSAIDs did not behave explicitly like NO donors. The conjugation of the NSAIDs with the NO moiety employed enzymatic cleavable of ester bonds in

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order to perform the NO release from the functional NSAID in the biological milieu (Dunlap 2008). For that purpose various chemical fragments bearing the nitrate ester group have been reported. Moreover, aliphatic or aromatic molecules known as “spacer” or “linker” present in NO-NSAIDs, possess their own antitumor feature and contribute to a net effect of the compound. (Kashfi and Rigas 2007; Fu, et al. 2008; Rigas and Williams 2008).

VGX-1027 Versus Its NO-Modified Derivative, GIT-27NO In the field of research for more efficient therapeutic approaches in cancer, the nitric oxide-donating technology led to the development of new chemical entities with a significantly different pharmacological profile than the parental compound (Rigas and Williams 2008). Accordingly, we synthesized an isoxazoline compound with such a structural modification that has been named GIT-27NO (Maksimovic-Ivanic et al. 2008). GIT-27NO is composed of VGX-1027 (previously GIT-27) and an NO-donating group. VGX-1027 is an established immunomodulator in several animal models of autoimmune and inflammatory diseases (Stojanovic et al. 2007; Mangano et al. 2008a, b; Stosic-Grujicic et al. 2007). The NO-donating group was synthesized in few steps and then coupled to GIT-27 to yield GIT-27NO as described previously (Maksimovic-Ivanic et al. 2008), and, importantly, it is covalently attached to the parental compound without including any spacer. The original drug, VGX-1027, was shown to express anti-inflammatory activity in several animal models of acute and chronic immunoinflammatory diseases. Thus, VGX-1027 markedly ameliorated carrageenan-induced pleurisy, LPSinduced lethality, type II collagen-induced arthritis, endotoxin-induced uveitis, immunoinflammatory colitis, as well as immunoinflammatory and autoimmune forms of diabetes (Mangano et al. 2008a, b; Stojanovic et al. 2007; Stosic-Grujicic et al. 2007). Suppression of inflammation was related to reduced production of pro-inflammatory mediators such as TNF-α, IL-1β, MIF, and iNOS. In contrast to VGX-1027, the NO-modified compound GIT-27NO did not have any therapeutical effect in the model of LPS-induced sepsis (Nicoletti et al. unpublished data), since lethality of mice was unchanged in comparison to control animals. The findings indicated that the new drug looses its anti-inflammatory potential for the original drug and did not affect the production of pro-inflammatory mediators which were observed after the treatment with VGX-1027 (Nicoletti et al. unpublished data). However, the capability of the NO-modified compound to prevent ConA-induced hepatitis in mice raises concerns on the conclusion that the modified drug does not possess an immunomodulatory potential (Donia et al. 2009). In detail, in animals treated with GIT-27NO before the induction of disease with ConA, the level of transaminases was significantly lower in comparison to untreated control and damage of the liver and the presence of infiltrates were significantly reduced. It is

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tempting to speculate that caspase inhibition by the drug could be the base of hepatitis prevention. Besides, it was shown that this mechanism was responsible for prophylactic effects of NO-modified aspirin and either prophylactic or therapeutic effects of molsidomine that is metabolized into SIN-1 by the liver and subsequently generates NO in ConA-induced hepatitis (Fiorucci et al. 2000; Ding et al. 2004). Thus, due to NO–NSAID’s ability to inhibit caspase activity, apoptosis triggered by pro-inflammatory cytokines could be disabled and, consequently, reduce the liver damage mediated by the immune cells’ action in the local area. However, the precise role of NO as well as NO-modified anti-inflammatory drugs on experimental models of hepatitis needs to be further investigated. In fact, at least in regard to NO, there are discordant results in the literature about its capacity to ameliorate or exacerbate the course of experimental models of hepatitis (Laskin et al. 2001; Hasselblatt et al. 2007; Ding et al. 2004). It has been also previously hypothesized that, whether or not nitric oxide or secondary oxidants generated from nitric oxide act as mediators of hepatic injury or protect against toxicity, the end results depend on the precise targets of these reactive nitrogen intermediates, as well as the levels of superoxide anion present and the extent to which tissue injury can be mediated by reactive oxygen intermediates (Laskin et al. 2001).

The Effects of GIT-27NO on Tumor Cell Growth In Vitro and In Vivo It is obvious that the modification performed on VGX-1027 resulted in significant changes in its primary mode of action but also generated one completely new chemical entity. While the parental anti-inflammatory drug did not show antitumor effects in a broad range of doses tested (Maksimovic-Ivanic et al. 2008), the modified compound acquired powerful antitumor potential confirmed in vitro, as well as in vivo with several models of solid or metastatic tumors. Structural modification of the original drug by direct covalent attachment of NO resulted in strong dose-dependent reduction of cell viability in numerous tumor cell lines in vitro (Table 23.1). The new compound acted unselectively with equal efficiency in lines from different embryonal origin, type of primary tissue from which they were generated, or species specificity (Maksimovic-Ivanic et al. 2008; Mijatovic et al. 2008; Table 23.1 Cell lines sensitive to GIT-27NO treatment Rodent cell lines Mouse fibrosarcoma L929 Mouse melanoma B16 Mouse cisplatin-resistant B16 Mouse breast carcinoma TA3H Rat astrocytoma C6

Human cell lines Glioblastoma U251 Prostate cancer PC3 Prostate cancer LnCap Colon cancer LS174 Adenocarcinoma HeLa Breast cancer BT20 Melanoma A375

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Mijatovic et al. 2010, Donia et al. 2009). In brief, several cell lines that differ in their specificity related to intracellular signaling involved in malignant transformation, in defect or amplified expression of molecules engaged by cell proliferation, death, or differentiation and even in the activation of mechanisms related to cisplatin resistance, did not present any limitation to the drug efficacy. In vitro data were confirmed in vivo in syngeneic, allogeneic, and xenogenic tumor graft models such as solid melanoma (Maksimovic-Ivanic et al. 2008; Mijatovic et al. 2008), hormonalindependent prostate carcinoma (Donia et al. 2009), and a metastatic model of breast cancer (Mijatovic et al. 2010). It is important to mention that in these tumor models, cancer treatment started at a later stage of disease and the end effect could be described as therapeutical rather than preventive. Treatment of C57BL/6 mice with GIT-27NO, in which subcutaneous melanoma was induced by the application of B16 cells, started from day 10 and lasted for additional 14 days. A significantly reduced volume of tumors was observed in animals receiving the drug in comparison to the vehicle-treated group (Maksimovic-Ivanic et al. 2008). In the model of solid prostate cancer induced by the androgen-independent p53-deficient PC3 cell line, treatment with GIT-27NO started from day 7 after cell implantation in immunodeficient nude mice (Donia et al. 2009). The outcome of the treatment, similar to the one observed in the melanoma model, clearly indicated reduced growth of tumors in comparison to control animals. Efficacy of the GIT-27NO in several solid tumor models was further confirmed in a metastatic model of breast carcinoma (Mijatovic et al. 2010). In the latest, the beneficial effect of applied therapy was evaluated through survival rate as well as body variation index in animals with disseminated peritoneal carcinosis induced by the TA3H cells inoculum. The mortality of animals that received GIT-27NO from day 5 after cells implantation was significantly decreased in comparison to control- or vehicle-treated groups. Considering that treatments for advanced metastatic cancer, in general, are restricted and this stage of the disease is mainly responsible for the uppermost number of cancer deaths, previous data are of primary scientific interest (Vogel 2000).

Subacute and Acute Toxicity of GIT-27NO Drug administration was not followed by acute or subacute toxicity manifestations (Maksimovic-Ivanic et al. 2008). Injection of a single dose in a range of 5–80 mg/mouse showed that the first dose of GIT-27NO capable to induce death from 24 to 72 h was 80 mg/mouse. This dose was exactly 80 or 160 times higher (depending on the model) than effective daily dose used in vivo. On the other hand, a continuous treatment lasting for four consecutive weeks with significantly higher dose than the dose with the best anticancer in vivo activity (2.5 mg/mouse/day) was not accompanied with the clinical appearance of toxicity and body weight loss. Blood values of glucose, cholesterol, urea, GOT, and triglycerides were similar to control- or vehicle-treated animals.

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Possible Mechanisms of GIT-27NO Action Induction of Cell Death In order to evaluate the mechanisms responsible for the capacity of GIT-27NO to reduce the tumor cell growth, further analyses were performed to establish whether the drug can act with a cytostatic and/or cytotoxic action. The former is related to cell cycle arrest in different phases of the cell cycle and the final outcome could not be necessarily related to the induction of cell death, but could be an indication of cells entering the stage of terminal differentiation or senescence (Zhang et al. 1995; Roninson, Broude and Chang 2001; Mijatovic et al. 2005). On the other hand, the cytotoxic activity includes the induction of accidental or programmed cell death. Programmed cell death (PCD) is often associated with apoptosis, although it is known that a group of genetically determined types of cell death substantially differ from apoptosis in both the mechanisms and morphological aspects (Fiers et al. 1999; Abraham and Shaham 2004; Fink and Cookson 2005; Levine and Yuan 2005; Bras et al. 2005; Scripture and Figg 2006). For instance, there is the lysosomal enzyme-mediated cell death known as autophagic cell death recognizable as “selfcannibalism” (de Bruin and Medema 2008 ). It is the evolutionary, very old process, that represents one of the important mechanisms of maintaining homeostasis during embryogenesis. Paradoxically, autophagy functions as a prosurvival pathway and as a key process in the elimination of damaged cellular structures and, in some circumstances, it is opposed to apoptotic signals (Levine and Yuan 2005). Under the conditions which are probably defined by the level of damage or cell specificity, autophagy promotes cell destruction instead of prevention. Induction of cell death by autophagy is one of the most promising approaches in current research for therapy of cancer resistant to induction of apoptosis (Bilir et al. 2001; Paglin et al. 2001; Kanzawa et al. 2003, 2004; Gozuacik and Kimchi 2004; Gorka et al. 2005; de Bruin and Medema 2008). GIT-27NO treatment resulted in suppression of tumor cell growth caused mainly through induction of cell death (Table 23.2). The contribution of proliferation inhibition as a mechanism that could be responsible for decreased viability was

Table 23.2 Different types of cell death triggered by GIT-27NO Cell line

Apoptosis

Necrosis

Autophagic cell death

Astrocytoma C6 Fibrosarcoma L929 Melanoma B16 Breast carcinoma TA3H Melanoma A375 Prostate cancer PC3 Prostate cancer LnCap

− + − + + + +

− − −/+ − − + +

+ − + − − − −

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minimal (Maksimovic-Ivanic et al. 2008; Mijatovic et al. 2008). Results of CFSE staining clearly indicated that the rate of cell division was not significantly changed, whereas the accumulation of hypodiploid cells, as revealed by the cell cycle distribution analysis, confirmed the presence of fragmented nucleic acid substrates as a sign of programmed or accidental cell death (Okada and Mak 2004; Zhivotovsky 2004; Krysko et al. 2008). Delayed LDH release, detected not before 18 h of cell incubation in the presence of higher concentrations of GIT-27NO, demonstrated the irrelevance of necrotic cell death as a primary mechanism of drug action. However, the drug was equally efficient in cisplatin-sensitive cells as well as their resistant subclones. Precisely, continuous culture of mouse melanoma B16 cells in the presence of low concentrations of cisplatin resulted in the selection of resistant clones which did not respond to cisplatin treatment but, however, retained the sensitivity to GIT-27NO quite similar to that observed in the maternal line (Maksimovic-Ivanic et al. 2008). At the end of the apoptotic cascade, genetic material is fragmented. This phenomenon is mainly mediated by caspases, a group of proteases responsible for activation of DNases and subsequent cleavage of DNA (Liu et al. 1997; Oliveri et al. 2001). An original characteristic of GIT-27NO mechanism of action is the capacity to induce apoptosis with all morphological features but associated with strong inhibition of caspase activity at both the protein and transcriptional levels (Mijatovic et al. 2010). In cells such as human melanoma A375 or the mouse epithelial breast cancer TA3H, type I PCD or apoptotic cell death induced by GIT-27NO was marked by typical signs of early and late apoptotic processes and engaged the p53 tumor suppressor as its official mediator (Mijatovic et al. 2008; Mijatovic et al. 2010). Surprisingly, even in p53-deficient PC3 cell line, GIT-27NO triggered the apoptotic process with the same efficiency as observed in the androgen-dependent LnCa P (Donia et al. 2009). In some other types of cell lines whose growth was reduced by GIT-27NO, but in which no classical apoptotic signs have been demonstrated, high levels of autophagosomes in the cytoplasm were observed (Maksimovic-Ivanic et al. 2008). Moreover, inhibition of autophagosome formations resulted in significant recovering from drug’s toxicity, showing that these cells died through the induction of type II PCD or autophagic cell death. One of the leading problems of contemporary cancer therapy is related to the capacity of malignant cells to develop resistance to treatment with chemotherapeutic drugs or radiation through clonal selection (Nadkar et al. 2006; Lage 2008). The mechanisms involved in drug resistance are often connected to overexpression of protective signals as well as with defective responses to apoptotic stimuli (Gabriel et al. 2003; Kim et al. 2006). Novel pharmacological compounds such as GIT-27NO could present a significant advantage in the field of cancer therapy due to their plasticity manifested through their capacity to overcome resistance to a most common type of malignant cell death and their ability to induce different types of cell death and to perform its toxic action independently of key molecules of the apoptotic process (p53 and caspases).

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NO as the Mediator of Tumoricidal Action of GIT-27NO In normal conditions, mammalian cells grow at a low level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) environment. Antioxidant systems, including the superoxide dismutases (SOD) enzymes, E, A, and C vitamins, and glutathione (GSH), buffer the oxidative action of ROS/RNS and prevent cell damage. Rapid influx of free NO can dramatically alter this oxidant/antioxidant balance at different levels. Although it is an uncharged molecule, its reactivity arises from the presence of an unpaired electron in the outermost orbital, permitting it to function as oxidant (electron donor) or antioxidant (electron acceptor) (Bonavida et al. 2006; Blaise et al. 2005). Thus, NO affects the function of inorganic molecules, DNA structure, prosthetic groups, and proteins through S-nitrosylation of thiol groups, nitration of tyrosine residues or interruption of metal–sulfide groups like zinc-finger domains or iron–sulfide complexes (Wiseman and Halliwell 1996; Bogdan 2001; Leon et al. 2008). Together with superoxide anion (O2 ), NO creates nitrogen oxide radical (NO), nitrogen dioxide radical (NO2 ), peroxynitrite (ONOO– ), and other molecules of the RNS family, all in charge for protein oxidation (Tuteja et al. 2004; Leon, et al. 2008). Furthermore, the addition of NO group to the thiol side chain of cysteine residues of proteins (S-nitrosylation) strongly affected intracellular signal transduction (Martínez-Ruiz and Lamas 2004; Iyer et al. 2008). The balance between NO and superoxide anion concentration defines the ratio ONOO– / N2 O3 . N2 O3 – mainly generated in situations when the concentration of NO multiply overlaps the quantity of O2 , is thought to be crucial for the S-nitrosylation process in the intracellular compartment (Handy and Loscalzo 2006). It was of interest to define the role of NO in GIT-27NO activity against tumor cells both in vitro and in vivo and to establish whether the chemical modification by the covalent attachment of NO to the VGX-1027 created another conventional exogenous NO donor or a new compound with unique and more complex pharmacological activity than classical molecules. The complex role of this highly reactive free radical was the subject of intensive research and has been recently described in the literature. NO is a molecule with extreme destructive and, at the same time, protective potential (Fukumura et al. 2006; Bonavida et al. 2002; Iyer et al. 2008; Bonavida et al. 2006). What will be the outcome of its action in the cell, death or prevention of death triggered by some other stimuli, depends on numerous parameters such as concentration, dynamic of release, and the cell specificity defined by the efficacy of repair mechanisms, stress protective signals, antioxidative action (Bogdan 2001; Bonavida et al. 2002). GIT-27NO did not release NO spontaneously in the media, even after 48 h. However, in the presence of the cells, nitrite accumulation, as an indirect measurement of NO release determined in cell culture supernatant, was significant and time dependent (Maksimovic-Ivanic et al. 2008). More interestingly, incubation of the cells in the presence of both the drug and the extracellular scavenger hemoglobin completely neutralized the deleterious effect of the drug on cell viability, thus indicating the dominant role of drugs liberated NO in its anticancer mission (Maksimovic-Ivanic et al. 2008). However, the

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NO release may not be the only mechanism by which GIT-27NO exerts its antitumoral properties; in fact, unlike the classical NO donor DETANONOate that sensitizes TRAIL-resistant PC-3 and LnCAP cell lines for TRAIL-mediated apoptosis without modifying their viability per se (Huerta-Yepez et al. 2009), GIT-27NO treatment alone inhibited viability of both cell lines. These more powerful effects of GIT-27NO can probably be ascribed to the different chemical characteristics and pharmacological profile of the parental compound VGX-1027. VGX-1027 exhibits per se potentially anti-inflammatory properties that could favor the antitumor activity of GIT-27NO including NF-κB inhibition that could synergize with the antitumor effects due to NO release from GIT-27NO (Stojanovic et al. 2007). Furthermore, the same set of experiments revealed that GIT-27NO received crucial stimuli for NO liberation from the extracellular compartment. Similar effect was detected when the drug was added to cell-conditioned medium obtained from the cells upon 24 h of incubation, clearly indicating that the possible trigger for NO release could be one or more soluble products, and/or cell membrane molecules (Maksimovic-Ivanic et al. 2008). Moreover, lower sensitivity of primary cells indicated that those cells are possibly restricted producers of the NO triggering molecules. The exact activator of the drug is still unknown, but it is speculated that the thiol functional groups at the intracellular and/or elevated acidity in the conditioned medium of tumor cells could be a specific trigger for NO generation. However, changes of pH, except in extreme values, did not affect the rate and dynamic of NO liberation. Uptake of released NO is rapid and is highly an intensive process. In treated cells, independently of the cell type, intracellular amount of NO was detectable even after 2 h of incubation, whereas elevated ROS production was determined only 5 min upon exposure to the drug (Maksimovic-Ivanic et al. 2008). The interplay between RNS and ROS resulted in extreme nitration of tyrosine residues. Considering that this amino acid plays a crucial role in the regulation of protein activity and delivering the life and death signals inside the cells through phosphorylation or dephosphorylation, significant changes observed in the MAP kinase pathway activity upon the GIT-27NO could be expected. There are numerous intracellular targets of NO. Besides tyrosine, the consequence of NO reaction with cysteine residues also dramatically affects the cell physiology. Inhibition of caspase activity and decreased level of active caspase-3 was observed in breast cancer cells treated with the drug. In addition, gene expression of caspases 3, 8, and 9 is also modified by NO (Mijatovic et al. 2010). These data were in concordance with the well-defined sensitivity of caspase promoters to NO (Wang et al.1999; Liedtke et al. 2003). As a consequence of NO reactivity, numerous transcriptional factors were targeted. One of them, the YY1 repressor, was markedly reduced in human melanoma cells treated with the GIT27NO (Mijatovic et al. 2008), as well as in many different other cell lines by other NO donors like DETANONOate (Bonavida et al. 2006; Huerta-Yepez et al. 2009). More precisely, YY1 is ubiquitous and multifunctional zinc-finger transcription factor with fundamental roles in different biological processes such as differentiation and embryogenesis (Gordon et al. 2006). The expression of YY1 is highly elevated in different tumor cells (Gordon et al. 2006). Furthermore, the role of this

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transcriptional factor in repression of Fas- and TRAIL-mediated apoptosis is well described in the literature. Nitric oxide donors as well as nitric oxide synthase gene therapy showed the ability to sensitize tumor cells to chemo- and immunotherapy (Bonavida et al. 2006; Adams et al. 2008). The last one is a consequence of the potential of NO to downregulate YY1 repressor activity (Garba’n and Bonavida 2001) to DNA sequences important in regulation of death receptor expression. Therefore, the capacity of GIT-27NO to downregulate YY1 expression as well as activity presents an important aspect of drug’s intracellular action and amplifying the possibilities of drug utilization. In parallel with reduced tumor cell viability as a consequence of decreased YY1 engagement, the same effect can influence the sensitivity of the cells to induction of apoptosis through stimulation of death receptors from the TNF receptor family (Vega et al. 2005; Garba’n and Bonavida 2001). This effect could be successfully exploited by combining GIT-27NO to immunotherapy. Also, according to the in vitro data generated by Adams et al. (Adams et al. 2008), NO-based therapy could also be usefully combined to traditional chemotherapeutics like cisplatin. Altogether, drug efficacy has to be evaluated in light of the direct effects on the physiology of malignant cells, but also through indirectly promoting upregulation of natural immune cell-mediated antitumor response or chemosensitization to conventional chemotherapeutics.

The Influence of GIT-27NO on MAP Kinase Activity Intracellular mediators of physiological processes are regulated through an extensive network of interactive signal transduction pathways such as mitogen-activated protein kinase (MAPK) family (Seger and Krebs 1995; Arbabi and Maier 2002). MAPKs mediate diverse processes ranging from the transcription of protooncogenes to the programming of cell death. Their role is crucial in making a cell decision to divide, proliferate, or die. Three major members in the MAPK family are extracellular signal-mediated kinase – ERK, p38, and Jun N-terminal kinase (JNK) (Boutros et al. 2008). All of them can be activated through a process of dual phosphorylation on both threonine and tyrosine residues that are separated by a single amino acid, thus forming a tripeptide motif (Davis 2000). An activation of MAPKs cascade is rapid and enables cell to respond to environmental changes and different stimuli in a prompt and regulated fashion. The MAPKs response to the GIT-27NO treatment is heterogeneous and conducted by cell specificity. Whereas in the mouse fibrosarcoma L929 cells GIT-27NO induced time-dependent upregulation of ERK1/2 and p38, treatment of the mouse melanoma B16 cells resulted in strong phosphorylation of JNK and p38. In C6 rat glioma cells, GIT-27NO simultaneously upregulated JNK and ERK, while the activity of p38 was not affected. (Maksimovic-Ivanic et al. 2008). Despite differentially affected MAPKs by the drug, in several tumors, all tumor cells responded to the treatment by a similar sensitivity. However, the differences in the MAPK response could define the type of cell death, but did not have any repercussion to the success of the GIT-27NO treatment.

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Concluding Remarks and Future Directions A major challenge for designing novel antineoplastic drugs is the generation of compounds with improved efficacy, lower side effects, and potential synergism with currently available antitumor agents. In spite of extensive research to develop new pharmacotherapeutic approaches to prevent or cure the disease, successful anticancer therapy is still not found. The major problem in this field arises from the intrinsic (before therapy) and acquired (caused by therapy) drug resistance. In light of this, the discovery of a compound with the potential to adapt its mode of action to cellular specificity and be “bright enough” to overcome the eventual barriers, such as nonfunctional apoptotic mediators or over functional protective signals, is one of the most desirable event. Different from the most cytostatic drugs, the intracellular response to GIT-27NO treatment is dictated by cell specificity, but not by the drug alone. Independently from this, the compound nonselectively downregulated the growth of a large spectrum of different types of tumors, apoptotic sensitive or resistant, p53 deficient or wild-type counterpart, and even in caspase-inhibited conditions promoted by itself. These data warrant further studies to evaluate the possible translation of these findings to the clinical settings. Acknowledgments This work was supported by the Serbian Ministry of Science (Grant 143029).

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Chapter 24

Nitric Oxide Donors Are a New Class of Anti-cancer Therapeutics for the Reversal of Resistance and Inhibition of Metastasis Benjamin Bonavida, Stavroula Baritaki, Sara Huerta-Yepez, Mario I. Vega, Ali R. Jazirehi, and James Berenson

Abstract Several novel therapeutic strategies are currently being explored for the treatment of tumors that are refractory to conventional cytotoxic therapies. Further, therapies aimed at the prevention and treatment of metastasis are also being investigated in pre-clinical and clinical studies. Most of these novel therapeutics are aimed at targeting gene products that regulate resistance and metastasis and have yielded several FDA-approved drugs/antibodies for the treatment of specific cancers. Nitric oxide donors, such as nitroglycerine which has been approved for the treatment of cardiovascular diseases, have been examined for their potential role in the treatment of cancer. NO donor-induced anti-tumor activities have been documented in several in vitro and in vivo animal studies. In addition, nitroglycerine therapeutic application in cancer patients has been initiated. Depending on the level of NO released and sustained, it is well documented that high levels of NO are anti-tumorigenic due to their complex activities, such as inhibiting constitutively hyperactivated cell survival/anti-apoptotic pathways and leading to their ability to sensitize drug/immune-resistant tumor cells to apoptosis by cytotoxic drugs. In addition, inhibition of such survival pathways also leads to the regulation of gene products that participate in the epithelial to mesenchymal transition (EMT) and metastasis and result in the inhibition of metastasis. Hence, NO donors can exert simultaneously a multitude of anti-cancer activities, including enhancement of apoptotic stimuli, inhibition of metastasis, inhibition of angiogenesis, and inhibition of hypoxia, depending on the concentration of the NO donor and on the cancer type and stage. Therefore, while novel therapeutics are being tested for specific anti-tumor targeting effects, we suggest that activation of endogenous iNOS or exogenous NO donors can substitute a large number of specific agents and may

B. Bonavida (B) Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at University of California at Los Angeles, Le Conte Avenue, Los Angeles, CA 90095-1747, USA e-mail: [email protected]

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be considered as universal anti-cancer therapeutics when used alone or in combination with subtoxic cytotoxic drugs. Thus, the development of a new generation of NO donors with minimal toxicity and higher anti-tumor efficacy is warranted for investigation in clinical trials in cancer patients

Introduction Several advances have been made in the treatment of cancer, beginning with the introduction of cytotoxic chemotherapeutic drugs. This was followed by the introduction of other modalities, such as radiation, hormonal therapy, and immunotherapy. Such treatments are effective initially and result in significant objective clinical responses and prolongation of survival. However, many patients develop resistance to further treatments and such patients succumb to the disease. Hence, there is an urgent need to determine the underlying molecular basis of tumor resistance and develop targeted therapies to reverse the resistance. It became apparent that the development of resistance to cytotoxic therapies emanates from the findings that most cytotoxic drugs exert their lethal effect by inducing apoptosis in the tumor cells. Resistant tumor cells, thereby, develop mechanisms to evade apoptosis and thus become refractory to the majority of cytotoxic apoptotic therapeutics, including chemotherapy, radiation, and immunotherapy (Ng and Bonavida 2002). Current approaches to override the development of tumor cell resistance to apoptosis are based on the development of targeted therapies directed against gene products that regulate the apoptotic pathways. Two major pathways involved in apoptosis have been characterized, namely, the extrinsic (Type I) and intrinsic (Type II) apoptotic pathways. The extrinsic pathway is induced by cytotoxic lymphocytes (or ligands) expressed on the surface and consists of ligands of the TNF-α family, such as TNF-α, FasL, and TRAIL. The interaction between the cytotoxic lymphocytes and the target cells results in the activation of the cytotoxic lymphocytes, resulting in the release of granzymes and activating the corresponding receptors on the target cells (TNF-R1, Fas, DR4, and DR5) and induction of apoptosis. Apoptosis is a result of the activation of caspase 8 through DISC formation and downstream activation of the effector caspase 3, PARP cleavage, DNA fragmentation, and apoptosis. Stress, radiation, and chemotherapeutic agents activate the intrinsic pathway by activating the mitochondria to release cytochrome c and Smac/DIABLO into the cytosol. These lead to the formation of the apoptosome and activate caspases 9, 7, and 3, leading to downstream PARP cleavage, DNA fragmentation, and apoptosis. Hence, there is a convergence of both Type I and Type II at the effector caspase 3 step. These two pathways are regulated by gene products (pro- and anti-apoptotic) whose expression levels will regulate the outcome of the apoptotic stimulus (Ng and Bonavida 2002; Micheau and Tschopp 2003). The development of tumor cell resistance can emanate from the dysregulation of gene products involved in the apoptotic pathways. For instance, tumor cells may overexpress anti-apoptotic gene products and exhibit mutations with inhibitory activity and deletions (Reed 1995). Therefore, identification of the mechanisms involved in

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resistance and characterization of gene products that regulate resistance have been the hallmarks in the development of novel therapeutics to reverse resistance. Most tumor cells exhibit constitutively hyperactivated cell survival anti-apoptotic pathways that regulate cell proliferation, anti-apoptotic gene products and gene products that regulate metastasis. One predominant highly activated pathway in cancers, for example, is the NF-κB pathway. The NF-κB pathway plays a pivotal role in tumorigenesis, immunomodulation, angiogenesis, and tumor remodeling (Aggarwal 2004). Under resting conditions, NF-κB consists of a heterotrimer of p50, p65, and IκBα protein complex in the cytoplasm. The activity of NF-κB consists of the phosphorylation, ubiquitination, and degradation of IκBα and phosphorylation of p65, which in turn leads to the translocation of NF-κB from the cytoplasm to the nucleus where it binds to specific response elements in the DNA (Kumar et al. 2004). NF-κB has been shown to regulate the expression of a number of genes whose products are involved in tumorigenesis. These include anti-apoptotic genes (examples are cIAPs, survivin, TRAF, c-FLIP, Bcl-2, Bcl-xL, and XIAP), genes encoding adhesion molecules and invasion (examples are COX-2, MMP-9, and VEGF), genes encoding chemokines and inflammatory cytokines, and cell cycle regulatory genes (examples are cyclin D1 and c-myc). Hence, many new therapeutics are aimed at the selective inhibition of NF-κB such as those mediated by proteasome inhibitors (Adams 2004). Bortezomib is the first FDA-approved proteasome inhibitor for the treatment of multiple myeloma (Adams 2002). Nitric oxide (endogenous or exogeneous) has been reported to have contrasting effects on tumor cells, namely it can act as an anti-tumor agent or can enhance tumorigenesis. These dual effects appear to be the result of the levels of NO: low levels are tumorigenic and higher levels are anti-tumorigenic (Wink et al. 2008). High levels of NO inhibit NF-κB activity by several mechanisms. For instance, the NO donor DETANONOate inhibits the phosphorylation and subsequent degradation of IκBα which prevents nuclear localization of NF-κB (Katsuyama et al. 1998). Also, NO may generate reactive oxygen species that are responsible for the activation of NF-κB (Garban and Bonavida 2001a). Further, NO induces the S-nitrosylation of NF-κB p50 and reduces its DNA-binding activity (Connelly et al. 2001; Marshall and Stamler 2001). Inhibition of NF-κB activity and S-nitrosylation of p50 by NO was corroborated by us using the NO donor DETANONOate (Huerta-Yepez et al. 2004). These findings demonstrated that high levels of NO inhibit NF-κB activity and prompted us to examine the effect of NO on the reversal of resistance in tumor cells by both cytotoxic chemotherapeutic and immunotherapeutic drugs. These studies revealed that NO donors can sensitize tumor cells to various apoptotic stimuli and reverse their resistance. Noteworthy, the underlying mechanisms regulating the sensitization by NO donors were investigated in these studies (Garban and Bonavida 2001a, b; Huerta-Yepez et al. 2004, 2009; Hongo et al. 2005; Baritaki et al. 2007; Huerta et al. 2008;). Further, the in vivo effect of DETANONOate in tumor regression in combination with drugs was reported recently in a colon cancer xenograft model in mice (Huerta et al. 2008). Several novel therapeutics are aimed at inhibiting NF-κB activities and/or selectively inhibiting gene products regulated by NF-κB. Since our findings showed that NO donors can inhibit NF-κB activity, we have hypothesized that a single

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therapeutic agent like NO donors should mimic a variety of distinct therapeutic drugs in both the reversal of resistance and inhibition of metastasis. In this chapter, we will present our current findings demonstrating that NO donors, like DETANONOate, can substitute for many cytotoxic agents that are currently being investigated in the reversal of resistance and inhibition of metastasis (see overall scheme in Fig. 24.1 of NO-mediated anti-tumorigenic effects).

Fig. 24.1 This scheme represents tumor cells that exhibit constitutively hyperactivated survival anti-apoptotic pathways such as the NF-κB and the Raf-1/MEK1/2/ ERK1/2 pathways. These pathways have been shown to regulate the transcription of many genes that regulate apoptosis and resulting in tumor cells expressing the resistant phenotype and respond poorly to cytotoxic stimuli (at doses non-toxic to normal tissues). The hyperactivation of these survival pathways results in the development of resistance to chemotherapy, radiotherapy, and immunotherapy. Several sensitizing agents have been reported that have the ability to modify intrinsically these pathways at different sites and result in the modification of gene products that regulate the apoptotic pathways. Hence, treatment with these agents results in tumor cells expressing lower thresholds of intrinsic resistance and upon exposure to cytotoxic drug respond effectively through the activation of the apoptotic pathways resulting in tumor cell death. Thus, resistance in tumor cells can be reversed by two complimentary signals derived from the sensitizing agents and from the cytotoxic agents (see Ng and Bonavida 2002)

NO and Cancer Nitric oxide (NO) is a small, unstable, highly diffusible molecule that crosses cell membranes and is produced by a group of enzymes termed nitric oxide synthases (NOS). Three isoforms are well known, namely endothelial NOS (eNOS), neuronal

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NOS (nNOS), and inducible NOS (iNOS). Both eNOS and nNOS are calciumdependent and generate low levels of NO, whereas iNOS is calcium-independent and produces high levels (100- to 1000-fold greater than constitutive NOS) (Huerta et al. 2008). Conflicting data have been consistently reported in the literature regarding the pro-tumor effect or anti-tumor effect of NO (Wink et al. 2008). To date, such conflicting data may be resolved by the findings that elevated levels of endogenous NO in tumor tissues are pro-tumorigenic since NOS inhibits tumor growth. In contrast, overexpression of NO by several orders of magnitude generally activates cell death pathways (Gauthier et al. 2004). The pro-tumorigenic effect of NO has been recently reviewed in several articles and will not be discussed in this chapter (Wink et al. 2008; Ridnour et al. 2008; Grimm et al. 2008; Coulter et al. 2008; Yasuda 2008). The anti-tumorigenic effect of NO has been recognized several years ago, though its relevance and impact in tumors have only recently been recognized. Overexpression of NO results in inhibition of angiogenesis and tumor growth (Jones et al. 2004). Also, overexpression of iNOS results in anti-tumor activity and inhibition of metastasis (Dong et al. 1994; Sarih et al. 1993). Other effects of NO were revealed by the use of NO donors or transfection of functional iNOS gene (Coulter et al. 2008). NO-producing tumor cells did not form tumors or metastases in ectopic or orthotopic xenograft mouse models due to NO-mediated apoptosis (Le et al. 2005). NO-donating non-steroidal anti-inflammatory drugs (NSAIDs), organic nitrates conjugated via labile linkers to NSAIDs such as aspirin, have been reported to inhibit colon cancer cell growth (Rigas and Kashfi 2004). Hypoxia in solid tumor promotes activation and stabilization of HIF-1α in cancer cells. The accumulation of HIF-1α in cancer cells promotes chemoresistance, radioresistance, tumor growth, and metastasis through the active transcription of VEGF, glycolytic enzymes, and ABC transporters. Further, overexpression of HIF-1α in tumor tissue is a bad prognosis. NO donors were reported to inhibit HIF-1α accumulation and activation in hypoxic malignant cells both in vitro and in vivo (Huang et al. 1999; Yasuda et al. 2006a). NO has been shown to act as a chemosensitizer (Matthews et al. 2001), a radiosensitizer (Wang et al. 2004), and an immunesensitizer (Garban and Bonavida 1999, 2001a, b; Bonavida 2008).

NO Is a New Class of Therapeutics with Multivalent-Targeted Sensitizing Activity: Role of Reversal of Tumor Cell Resistance to Chemotherapeutic Drugs and Immunotherapy Current approaches to circumvent tumor cell resistance to cytotoxic drugs are based primarily on identifying gene targets that regulate resistance and develop targeted agents to modify such gene products. Many of the gene products that regulate resistance to apoptotic stimuli are transcriptionally regulated by constitutively hyperactivated cell survival pathways in cancer cells (the NF-κB, the MAPK, and the Akt pathways). There have been many reports on the characterization of many agents that can sensitize tumor cells to apoptosis by chemotherapeutic drugs,

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immunotherapy, and radiation. Several of these have been summarized in a recent volume (Bonavida 2008). Below, we will focus on studies performed in our laboratory with the use of several sensitizing agents and discuss how NO mimics closely these agents and can substitute for their activities. Hence, we propose that NO can serve as a multivalent sensitizing agent for the reversal of tumor cell resistance. In this vain, we will discuss briefly the interrelationship between NO and the following examples of sensitizing agents as models, e.g., chemotherapeutic drugs, rituximab (anti-CD20 Ab), NF-κB inhibitors, and proteasome inhibitors.

NO Mimics Chemotherapeutic Drugs as Sensitizing Agents Tumor cell resistance to immunotherapy (e.g., FasL, TRAIL)-induced apoptosis was reversed by treatment with subtoxic concentrations of chemotherapeutic drugs (Mizutani et al. 2001; Uslu et al. 1997; Morimoto et al. 1993). For FasL-induced sensitization, the sensitizing drugs upregulated Fas expression in the cells through inhibition of NF-κB. Likewise, treatment of FasL-resistant tumor cells through either induction of iNOS or treatment with NO donors sensitizes cells to FasLinduced apoptosis. For instance, treatment of tumor cells with pro-inflammatory cytokines (i.e., IFN-γ, TNF-α, IL-1) stimulates the induction of iNOS and generates high levels of NO. Through treatment of tumor cells with IFN-γ or the NO donor SNAP, we have shown that the FasL-resistant tumor cells were sensitized to the FasL agonist monoclonal antibody, CH-11, and synergy in apoptosis was achieved (Garban and Bonavida 2001b). The mechanism by which NO sensitizes cells to FasL and upregulates Fas expression was found to be due to NO-mediated inhibition of the Fas transcription repressor Yin Yang 1 (YY1). Inhibition of YY1 by NO was the result of NO-induced inhibition of NF-κB upstream of YY1 and also to NOmediated S-nitrosylation of YY1 and inhibiting its DNA-binding activity (Hongo et al. 2005). These findings demonstrate that both iNOS induction and NO donors mimic drug-induced sensitization of FasL-resistant tumor cells to FasL-induced apoptosis. Treatment of drug/immune-resistant tumor cells with subtoxic concentrations of chemotherapeutic drugs sensitizes TRAIL-resistant tumor cells to TRAIL-induced apoptosis. TRAIL has been shown to be selectively cytotoxic to tumor cells with minimum toxicity to normal tissues (Ashkenazi et al. 1999). Therefore, TRAIL has currently been examined clinically for its therapeutic efficacy in patients bearing refractory tumors. However, most cancers are resistant to TRAIL and require a sensitizing agent to be used in combination with TRAIL to reverse resistance. We have initially examined whether the resistance of human bladder cancer cells to TRAIL and drugs may be overcome by the combination of TRAIL and drugs used clinically in bladder cancer such as cisplatin and carboplatin. This study demonstrated, clearly, that treatment of resistant bladder cancer cells with the combination resulted in significant potentiation of cytotoxicity and apoptosis and synergy was achieved. The findings were corroborated with freshly derived patient bladder cancer cells.

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These findings demonstrated that drug-resistant tumor cells can still be responsive to the same drugs by possibly reducing the resistance threshold and, thus, allowing TRAIL to mediate its apoptotic activity (Mizutani et al. 2001). Another study by Zisman et al. (2001) reported that treatment of TRAIL/drug-resistant human prostate cancer cells with subtoxic concentrations of various chemotherapeutic drugs (actinomycin D, paclitaxel, cisplatin, gemcitabine) and radiation sensitized tumor cells to TNF-α, Fas ligand, and TRAIL-mediated apoptosis. A follow-up study by Ng et al. (2002) examined the potential underlying mechanism of druginduced sensitization of prostate cancer cells to TRAIL-induced apoptosis. The findings demonstrated that treatment of prostate cancer cells with subtoxic concentrations of actinomycin D followed by treatment with TRAIL resulted in synergistic apoptosis as a result of the activation of caspases 8, 9, and 3. Treatment with TRAIL alone, although insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and the release of cytochrome c in the cytoplasm from the mitochondria and in the absence of activation of caspases, suggesting a downstream resistant factor. Treatment with actinomycin D inhibited the anti-apoptotic X-linked inhibitor of apoptosis (XIAP). The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited the inhibitors of apoptosis (cIAPs, XIAP) and sensitized cells to TRAIL apoptosis. This finding suggested that inhibition of XIAP by actinomycin D (Signal 1) and TRAIL-induced release of cytochrome c (Signal 2) resulted in complementation and reversal of resistance. The drug-induced sensitization to TRAIL was also achieved with the same drug to which the tumor cells developed resistance. This was corroborated by the use of adriamycin-resistant variant of the multiple myeloma cell line RPMI-8226, namely RPMI-8226/Dox40. Treatment with subtoxic concentrations of adriamycin resulted in synergistic cytotoxicity and apoptosis in both the parental and resistant cell lines. Treatment with adriamycin had no effect on several gene products although pro-caspase 9 and Apaf-1 were upregulated. The combination treatment with TRAIL and adriamycin resulted in significant depolarization of the mitochondrial membrane, activation of caspases 9 and 3, and apoptosis (Jazirehi et al. 2001). Similar to the findings above with drug-induced sensitization to TRAIL apoptosis, we have found that NO donors, such as DETANONOate, also sensitized tumor cells to TRAIL-induced apoptosis. The rationale of this study was based on the demonstration that drug-induced sensitization of TRAIL-resistant cells resulted from drug-induced downregulation of anti-apoptotic gene products regulated by NF-κB such as XIAP, Bcl-2, and Bcl-xL. Like drugs, NO donors were shown to inhibit constitutively activated NF-κB and thus, inhibited downstream various anti-apoptotic gene products including XIAP, Bcl-2, and Bcl-xL. Thus, treatment with DETANONOate of resistant prostate cancer cells followed by treatment with TRAIL resulted in significant potentiation of apoptosis and synergy was achieved. Treatment with DETANONOate resulted in the inhibition of NF-κB (through Snitrosylation of p50) and downstream inhibition of anti-apoptotic Bcl-2 family members. We have found that overexpression of Bcl-xL was responsible, in large part, for resistance and its inhibition by DETANONOate reversed resistance. The

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direct role of Bcl-xL inhibition by DETANONOate was corroborated by the use of the chemical inhibitor 2-methoxy antimycin A3, which mimicked DETANONOate for TRAIL sensitization. Sensitization by DETANONOate was the result of depolarization of the mitochondrial membrane potential and the release of modest levels of cytochrome c and Smac/DIABLO in the absence of downstream activation of caspases 9 and 3. The combination treatment, however, resulted in the activation of caspases 9 and 3, leading to apoptosis. Further analysis of the mechanism of NOmediated sensitization to TRAIL apoptosis was examined and was recently reported by Huerta-Yepez et al. (2009). The drug-induced sensitization to TRAIL apoptosis did not address the possible effect of the drug in the regulation of TRAIL death receptors. Several reports in the literature showed that treatment of cancer cells with certain chemotherapeutic drugs resulted in the sensitization to TRAIL apoptosis concomitantly with the upregulation of DR4, DR5, or both and suggested that such upregulation may be responsible for sensitization (Shigeno et al. 2003; LaVallee et al. 2003); however, the molecular mechanism was not known. In the study by Huerta-Yepez et al. (2009), we showed that treatment with DETANONOate resulted in upregulation of DR5 expression concomitantly with inhibition of NF-κB and downstream the transcription factor YY1. A direct role of YY1 in DETANONOate-mediated upregulation of DR5 and sensitization to TRAIL was demonstrated in cells transfected with YY1 siRNA. In that study, YY1 was also shown to directly inhibit the transcription of DR5 and delineated the YY1-binding site on the DR5 promoter. These findings demonstrated NO-mediated inhibition of NF-κB and YY1, which resulted in the upregulation of DR5 and apoptosis achieved by combination with TRAIL. The combination resulted in modification of several anti-apoptotic gene products responsible for apoptosis.

NO Mimics Rituximab-Induced Sensitization of Resistant Tumor Cells Sensitization to FasL and TRAIL-Induced Apoptosis Rituximab (chimeric anti-CD20 monoclonal antibody) is the first FDA-approved antibody for cancer treatment and has been used for the treatment of B-NonHodgkin’s lymphoma (B-NHL) and also for B-cell-mediated autoimmune diseases like rheumatoid arthritis. Like most drugs, not all patients respond to rituximab treatment and a subset becomes refractory to further treatment. The mechanisms governing resistance are not clear though several potential mechanisms have been revealed from studies performed in vitro. We have reported that treatment of drug/immune resistant B-NHL cell lines with rituximab sensitizes the cells to apoptosis by various chemotherapeutic and immunotherapeutic drugs (Jazirehi et al. 2005; Jazirehi and Bonavida 2005). Treatment of B-NHL with rituximab inhibited several constitutively activated survival pathways including the NF-κB, MAPK, Raf-1/MEK-1/2/ERK-1/2, and AKT. Rituximab-induced inhibition of those pathways resulted downstream in the inhibition of selective anti-apoptotic gene products

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such as Bcl-2 and Bcl-xL and these were shown to be responsible, in large part, for resistance. The direct role of Bcl-2 and Bcl-xL in rituximab-induced sensitization was corroborated by the use of specific inhibitors. Rituximab-mediated sensitization to FasL was shown to result through inhibition of NF-κB and downstream inhibition of the Fas transcription repressor YY1 (Vega et al. 2005a, b). Rituximab-mediated inhibition of NF-κB and YY1 was mimicked by treatment with NO donors. We also demonstrated that treatment of B-NHL with DETANONOate sensitizes cells to TRAIL apoptosis, like rituximab, via inhibition of NF-κB and YY1 (unpublished).

Sensitization to Chemotherapeutic Drug-Induced Apoptosis We have reported that treatment of drug-resistant B-NHL cells with rituximab sensitizes tumor cells to apoptosis by various chemotherapeutic drugs. The mechanism of rituximab-induced chemosensitization was examined and the findings revealed that rituximab inhibited survival/anti-apoptotic pathways and resulting in the inhibition of anti-apoptotic gene products and the reversal of resistance in the presence of low concentrations of chemotherapeutic drugs. The anti-apoptotic gene products Bcl-2 and Bcl-xL were primarily involved in resistance and their inhibition by rituximab sensitized the cells to Type II mitochondrial-mediated apoptosis pathway (Jazirehi et al. 2005). Similar findings to those achieved by rituximab were found following treatment of B-NHL cells with the NO donor DETANONOate. Treatment with DETANONOate inhibited NF-κB activity and downstream Bcl-2 and Bcl-xL expression. Further, DETANONOate induced the expression of Raf kinase inhibitor protein (RKIP), which plays a role in the direct inhibition of NF-κB. The role of DETANONOate in chemosensitization was corroborated by the use of siRNAs and specific chemical inhibitors (unpublished).

NO Mimics NF-κB Inhibitors in Tumor Cell Sensitization to Cytotoxic Drugs Tumor cells develop resistance to apoptotic stimuli induced by cytotoxic drugs and the resistance is due, in large part, to the constitutive hyperactivation of cell survival/anti-apoptotic pathways. Such hyperactivated pathways regulate downstream various gene products involved in the apoptotic pathways. The NF-κB signaling pathway plays an important role in the development of resistance by transcribing several anti-apoptotic gene products, such as Bcl-2, Bcl-xL, Mcl-1, cIAPs, XIAP, and survivin. Such gene products regulate the intrinsic mitochondrial apoptotic pathway induced by chemotherapeutic drugs as well as immunotherapy. For example, overexpression of the anti-apoptotic gene products Bcl-2 and Bcl-xL via NF-κB activation blocks the release of cytochrome c from the mitochondria and thereby blocking downstream the activation of caspases 9 and 3 and inhibiting apoptosis. Inhibitors of NF-κB have been shown to reverse tumor cell

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resistance to both chemotherapy and immunotherapy. We have demonstrated that the NF-κB inhibitor DHMEQ can sensitize tumor cells to FasL, TRAIL, and various chemotherapeutic drugs (Katsman et al. 2007). Likewise, DETANONOate mimicked DHMEQ in sensitization to these cytotoxic agents (Baritaki et al. 2007). The NF-κB signaling pathway plays an important role in the development of chemoresistance through the transcription of several anti-apoptotic gene products such as Bcl-2, Bcl-xL, Mcl-1, cIAPS, XIAP, and survivin. Such gene products regulate the intrinsic mitochondrial apoptotic pathway induced by chemotherapeutic drugs and also by immunotherapy. The overexpression of the anti-apoptotic gene products such as Bcl-2 and Bcl-xL, for instance, via NF-κB transcriptional activity, blocks the release of cytochrome c from the mitochondria and, thereby, blocking downstream activation of caspases 9, 7, and 3 and blocking apoptosis (Shabbits et al. 2003). The NF-κB-mediated effect has been correlated with decreased sensitivity to a variety of chemotherapeutic drugs and the emergence of chemoresistance (Del Poeta et al. 2008). NF-κB also exerts its pro-survival activity through the anti-apoptotic proteins described above (Karin and Lin 2002).

NO Mimics Proteasome Inhibitors in the Reversal of Tumor Cell Resistance The proteasome has been shown to play a major role in the regulation of tumor cell survival and proliferation as well as metastasis. Hence, proteasome inhibitors have been developed with the aim of inhibiting cell survival (for example, via inhibition of NF-κB activity) and enhance tumor cell death by cytotoxic drugs. Bortezomib (PS-341, Velcade) is the first FDA-approved proteasome inhibitor for the treatment of multiple myeloma (MM). We have recently reported that treatment of tumor cells with the novel proteasome inhibitor, NPI-0052 (Salinosporide A), sensitized tumor cells to TRAIL-induced apoptosis. NPI-0052 inhibited NF-κB activity and downstream the TRAIL DR5 transcription repressor YY1 and resulting in upregulation of DR5 and apoptosis by TRAIL (Baritaki et al. 2008). Noteworthy, treatment of tumor cells with DETANONOate mimicked NPI-0052 in sensitization to TRAILinduced apoptosis. Both NPI-0052 and DETANONOate inhibited NF-κB and YY1, upregulated DR5 expression and in combination with TRAIL, induced Type II mitochondrial pathway of apoptosis (Huerta-Yepez et al. 2009). We have also found that treatment of tumor cells with NPI-0052 resulted in the induction of RKIP via inhibition of NF-κB and the RKIP transcription repressor Snail (Baritaki et al. 2009). These modifications by NPI-0052 were also found to be the case for cells treated with DETANONOate. The roles of each of the regulatory factors in the loop of NF-κB-Snail-RKIP in both chemo and immune resistance were corroborated by the use of siRNA or overexpression (Baritaki et al. in preparation). Hence, both DETANONOate and NPI-0052 utilize common mechanisms of chemoimmunosensitization of drug-resistant tumor cells.

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NO Mimics Radiosensitizing Agents Tumor cell response to radiation is dependent upon oxygen and oxygen must be present during or within milliseconds after radiation. Thus, oxygen supply to the tumor may dictate the response to radiation. Cells lying near the capillaries are well oxygenated and radiosensitive. In contrast, cells lying at the edge and beyond the oxygen diffusion distance are hypoxic and radioresistant (Thomlinson and Gray 1955). Also, closing of blood vessels may prevent oxygen supply. Several strategies have been adopted to eliminate hypoxia-induced radioresistance, including patients breathing high oxygen content gas under hyperbolic conditions with significant clinical results. Also, nicotinamide, a vitamin B3 analog, is an efficient radiosensitizer in murine models. Nicotinamide is commonly being evaluated in a phase III clinical trial in combination with accelerated radiotherapy and carbogen breathing (ARCON) in laryngeal cancer (Kaanders et al. 2002). Adams and Cooke (1969) introduced the concept of hypoxic cell radiosensitizers, chemicals that mimic oxygen and enhance radiation damage. Selective killing of hypoxic cells can be achieved with bioreductive drugs. Most promising are the organic nitroxides, of which terapazamine is the lead compound. It is currently being evaluated in phase III trial in patients with stages IB–IVA carcinoma of the cervix (Denny and Wilson 2000). While NO from NONOates is an important radiosensitizer, NONOates are not tumor selective and high levels in the circulation may cause vasoactive complications (septic-like shock). Alterations to decrease NO in the circulation were evaluated by de Ridder et al. (2008). These investigators explored the tumor microenvironment for the selective generation of NO by bioreduction. Bioreduction of sodium nitroprusside (SNP) and N-niroso-N-acetylpenicillamine (SNAP) resulted in the generation of NO and radiosensitization of hypoxic mouse and human cancer cells. Also, endogenous induction of iNOS resulted in significant radiosensitization. iNOS activated by pro-inflammatory cytokines is capable of radiosensitizing tumor cells through endogenous production of NO at non-toxic concentrations. Tumorassociated immune cells may also contribute to iNOS-mediated radiosensitization by the generation of pro-inflammatory cytokines and NO, which may diffuse toward bystander cells.

NO Inhibits Metastasis and Mimics Other Inhibitors of Metastasis Inhibition of EMT The above findings in Section “NO and Cancer” demonstrated that NPI-0052 and DETANONOate inhibit the RKIP transcriptional repressor Snail. Snail has been shown to be involved in the induction of the initiation of metastasis via epithelial to mesenchymal transition (EMT). EMT is the major pathway by which metastasis

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is initiated and it involves the disruption of intercellular contacts and enhancement of cell motility from the primary site. Epithelial cells, thus, acquire many of the features of mesenchymal cells, including invasiveness and resistance to apoptosis (Shook and Keller 2003; Condeelis and Pollard 2006). The transdifferentiation program is regulated by specific transcription factors, such as Snail, Twists, Slug, and Goosecoid (Thiery and Sleeman 2006). One of the hallmarks of EMT is the functional loss of E-cadherin, considered to be a suppressor of invasion. In addition to E-cadherin, Snail downregulates the expression of tight junction components such as claudins and occludins and epithelial markers mucin-1 and cytokeratin-18 (Poser et al., 2001). Snail also upregulates the expression of the mesenchymal markers vimentin and fibronectin involved in cancer metastasis and metalloproteases 2 and 9 and the transcription factors ZEB-1 and LEF-1 (Cano et al. 2000; DeCraene et al. 2005). Our findings demonstrated clearly that treatment of metastatic tumor cells with either NPI-0052 or DETANONOate resulted in inhibition of EMT via inhibition of Snail and its regulated gene products involved in EMT. Overall, NO donors mimic proteasome inhibitors in the inhibition of the metastatic cascade. In addition to the above proteasome inhibitors, NF-κB inhibitors such as DHMEQ have also been shown to inhibit metastasis (Katsman et al. 2007). We have found that DHMEQ behaves like NPI-0052 in the inhibition of EMT and hence these findings demonstrate that NO donors also mimic NF-κB inhibitors in the regulation of metastasis (Baritaki et al. 2009).

Inhibition of Hypoxia by NO Hypoxia in tumors can cause resistance to chemotherapy, radiotherapy, and also leads to metastasis. Oxygen levels decrease in hypoxia due to distance from the vasculature. Hypoxia contributes to drug resistance by upregulation of HIF-1-mediated genes that confer drug resistance (Comerford et al. 2002) or by altering cellular metabolism that detoxifies the drug molecules (Teicher 1994). Downregulation of the pro-apoptotic proteins occurs in hypoxia leading to resistance (Erler et al. 2004). For example, tirapazamine (TPZ) is one of the first compounds to show specific hypoxic cytotoxicity (Zeman et al. 1986) and demonstrates anti-tumor activity in clinical trials (Brown 1993). Increase of iNOS activity corresponds with an increase in TPZ metabolism and toxicity under hypoxic conditions (Saunders et al. 2000). NOS/NO can be used in combination with bioreductive drugs such as TPZ to target radio- and chemoresistant hypoxic tumor cells. iNOS has also the ability to produce high concentrations of NO, which is a potent radio- and chemosensitizing agent as well as being directly cytotoxic. Hypoxia in tumor tissues promotes stabilization and activation of HIF-1α. HIF-1α/β activates the transcription of many genes that code for proteins involved in erythropoiesis, glycolysis, angiogenesis, cell growth, and metastasis (Yasuda 2008). The associations between high levels of gene and protein expression of HIF-1α in tumor tissue have had bad prognosis in many cancers. The accumulation of HIF-1α as well as hypoxia in solid tumor tissues may be

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associated with the promotion of malignant diseases and poor prognosis in patients with malignancies. HIF-1α-targeting anti-cancer therapies have been considered as novel approaches (Yeo et al. 2003). NO donors including nitroglycerine, sodium nitroprusside, and isosorbic dinitrite were also reported to inhibit HIF-1α accumulation and activation in hypoxic malignant cells in vitro (Takabuchi et al. 2004) and in vivo (Yasuda et al. 2006a).

NO Mimics Anti-angiogenic Drugs VEGF is produced from hypoxic malignant cells and is associated with the promotion of angiogenesis, increased vascular permeability, increase in paracrine/autocrine growth factor release, tumor growth, enhancement of cell motility, promotion of metastasis, inhibition of apoptosis, and chemoresistance (Epstein 2007; Hicklin and Ellis 2005; Gallo et al. 1998). The VEGF family of polypeptide growth factors regulates a family of VEGF receptor (VEGFR) tyrosine kinases with pleiotropic downstream effectors. A humanized VEGF antibody, bevacizumab, is able to sensitize a wide spectrum of tumor types to anti-cancer drugs (Giantonio et al. 2007). Hypoxia regulates VEGF expression in hypoxic cells via an HIF1-dependent pathway as well as HIF-2 (Semenza 2007). Both bevacizumab and avastin have been approved by the FDA for several cancers due to their ability to inhibit VEGF-mediated effects in the promotion of tumor growth. NO has been shown to inhibit HIF-1α accumulation and activation in hypoxic cells, in vitro and in vivo (Huang et al. 1999; Yasuda et al. 2006a, b). Through the inhibition of HIF1α/β and downstream VEGF, NO, therefore, mimics bevacizumab and avastin in the inhibition of angiogenesis and tumor progression.

NO Donors in the Treatment of Cancer Patients The therapeutic efficacy of NO donors in the treatment of cancer patients will be described in more detail in this volume by Dr. Yasuda and colleagues. Briefly, below are highlights on the novel applications of NO donors in the clinic and their potential anti-tumor efficacy. Yasuda et al. (2006a, b) have reported that the combination of nitroglycerine and vinorelbine plus cisplatin regimen improved the response rate of 72% of the patients and the median time to progression of 11 months in comparison with vinorelbine and cisplatin alone in previously untreated stage IIIB/IV patients with NSCLC in a phase II trial. These investigators are currently conducting different clinical trials with various combinations of nitroglycerine and chemotherapeutic drugs. Siemens et al. (2007) reported that a treatment with low dose and sustained release of nitroglycerine with combination of anti-cancer drugs significantly prolonged the doubling time of tumor marker prostate-specific antigen (PSA) compared with no treatment group in patients with biochemical recurrence of prostate cancer after primary treatment in an open label phase I/II trial.

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NO Donors: Designs for the Future It is recognized nowadays that NO at higher levels acts as an important anti-cancer agent and promotes cell death, as well as inhibits angiogenesis and metastasis. These effects have been observed in in vitro studies against a variety of cancer types and also corroborated in pre-clinical animal models. Thus, while the approved nitroglycerine donors show promise in cancer patients as discussed in Section “NO Donors: Designs for the Future”, novel NO donors alone or in conjugates are being explored for better therapeutic indices and reduced toxicity. Rigas (2007) and Rigas and Williams (2008) have developed and studied various NO-donating non-steriodal anti-inflammatory drugs (NO-NSAIDs) for anti-tumor efficacy. These compounds exert potent chemopreventive properties against a variety of cancers in pre-clinical models. Their use in humans awaits their efficacy. Fitzpatrick et al. (2008) have explored the therapeutic efficacy of delivering of iNOS by gene therapy or by exploring the two oxygenase domains and its reductase domain for cancer therapeutics. Tesei et al. (2008) have examined NCX-4040, an NO-donating acetylsalicylic acid derivative, and its efficacy in cancer cell models. Huerta et al. (2008) and Bonavida et al. (2008) have reviewed recently the potential anti-tumor effects of NO donors. There are currently many NO donors available for analysis both in vitro and in vivo in animals for both chemoprevention and therapeutics. The safety of these agents such as nitroglycerine has already been established for the management of coronary artery disease and its role as a chemosensitizing agent in NSCLC (Yasuda 2008).

Concluding Remarks This chapter described, briefly, the important role NO may play in the treatment of tumor cell resistance and malignancy. Endogenous NO production by iNOS and exogenous NO donors (high levels) induce a multitude of anti-tumor activities encompassing tumor prevention, sensitization to chemotherapeutic drugs (Bonavida et al. 2008; Evig et al. 2008), immunotherapy, radiation, and inhibition of the metastatic cascade. These effects are the result of NO-mediated chemical reactions that affect cell survival/anti-apoptotic pathways, DNA replication, mitochondrial respiration, proteasome apparatus, etc. Hence, depending on the tumor type and stage, as well as the level of NO, one may tune up the system for best anti-tumor activities. In this case, NO may substitute for a variety of currently explored specific therapeutics and can be, therefore, classified as a universal general therapeutic agent or sensitizing agent when used in combination with subtoxic doses of cytotoxic therapies. The dual roles of NO in the reversal of resistance and inhibition of metastasis endow this molecule with a substantial and superior therapeutic index. The clinical application of NO (activation of endogenous iNOS and NO donors or NO-SAIDs) in patients, however, must be explored with extreme caution due to the underlying toxic effects of high levels of NO in the circulation. Thus, agents that induce NO selectively or within the tumor

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microenvironment may reduce toxicity of exogenous NO donors. Alternatively, synthesis of novel NO donors with selective anti-tumor effects and reduced toxicity awaits their potential therapeutic activity in clinical trials. It is reassuring that the use of nitroglycerine, either in patch or systemically, in cancer patients has resulted in significant clinical responses and these early studies indicate the importance of the continuous exploration of NO in the treatment of cancer patients. Acknowledgments The author acknowledges the various associates in his laboratory and collaborators for their valuable contributions and whose works were an integral part of this manuscript. The author is also grateful for the assistance of Katherine Wu, Erica Keng, and Tiffany Chin in the preparation of this manuscript. The continuous assistance of the UCLA Jonsson Comprehensive Cancer Center is also acknowledged.

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Chapter 25

Role of Inducible Nitric Oxide Synthase (iNOS) in Regulation of Nitric Oxide (NO) Production and Stabilization of HIF-1α: Potential Role of Se-Methylselenocysteine (MSC), an Antioxidant Multi-targeted Small Molecule Sreenivasulu Chintala, Shousong Cao, and Youcef M. Rustum Abstract The authors of chapters presented in Part VII dealt with the therapeutic application of nitric oxide (NO). The authors extensively reviewed the field demonstrating the potential therapeutic value of NO donors alone and in combination with cytotoxic therapy in the treatment of various solid tumor types. The authors indicate that while the mechanisms of activity of these agents may vary according to tumor type and treatment conditions, the fact that these agents have minimal toxicity profile and therefore potentially selective in their effects provide the scientific rationale for their continued development at the basic level and more importantly, the need to validate the clinical potential of this interesting class of relatively non-toxic agents with significant biological and therapeutic potential. This opinion is offered relevant to the data presented in Part VII and offers new and novel approaches on the potential role of Se-methylselenocysteine (MSC) on the expression levels of hypoxia-inducible factor 1α (HIF-1α) and its transcriptionally regulated genes, including vascular endothelial growth factor (VEGF), in therapy of squamous cell carcinoma of the head and neck. The use of MSC as a multi-targeted, non-toxic inhibitor of iNOS and other biomarkers such as COX-2, HIF-1α, and VEGF as therapeutic modulators of anticancer drugs against established tumors in xenografts is discussed. Keywords MSC · Inhibitor of iNOS and HIF-1α · Cancer therapy

Introduction The chapters in Part VII and other parts clearly documented the potential value of NO donor molecules alone and in combination with chemo/radiotherapy in therapy of solid tumor malignancies. With all the supportive evidence generated to date, Y.M. Rustum (B) Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA e-mail: youcef [email protected]

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however, limited clinical evaluation of this important concept has been systematically evaluated. The basis for the need for clinical validation of this concept is based in part on that fact that NO donors have limited toxicity against normal tissues and yet maintain their antitumor activity, a desired clinical property. The opinion offered is to add additional focus, mainly the potential therapeutic benefit that can be derived from cascade regulation of the inducible nitric oxide synthase (iNOS), NO, cyclooxygenase (COX-2), and hypoxia-inducible factor 1α (HIF-1α) by non-toxic but a biologically effective and selective small multi-targeted molecule, selenium, with considerable antioxidant properties. Human tumors are heterogeneous, composed of normal cells and tumors of different types, stages of differentiation, genetic makeup, and histology. Growth and metastasis, as well as response to chemotherapy of tumor cells, represent a balance input of multiple factors – including immunological, genetic, and microenvironmental. Unstable tumor microenvironment could hinder selective delivery and distribution of cytotoxic treatment to the tumor, in particular to the hypoxic region with poor vasculature, and may provide tumor sanctuary amenable to rapid tumor relapse (Bhattacharya, Seshadri, Oven et al. 2008; Bhattacharya, Toth, Durrani et al. 2008). Furthermore, advanced tumors which are hypoxic are associated with overexpression of a molecular marker HIF-1α. HIF-1α is known to transcriptionally regulate over 200 genes, including VEGF among others, involved in angiogenesis. Furthermore, HIF-1α expression is associated with resistance to chemo/radiotherapy.

Tumor Tissues Heterogeneity: A Basis for Multi-targeted Drug Development Human tumors are generally heterogeneous composed of normal cells, invading tumor cells at various phases of the cell cycle, genetically unstable and consisting of multiple histological characteristics. Thus, it is unlikely that a molecule that targets a specific and single target will yield the desired treatment outcome. It is likely, however, that molecules that target multiple sites could produce significant cascade modifications of multiple targets involved in tumor growth, metastasis, and resistance. Recent examples of VEGFR and EGFR inhibitors demonstrated their impact on treatment outcome only when combined with standard chemotherapy. It is important to point out, however, that the dose and sequence of administration of these biological agents relative to chemo/radiotherapy should be optimized. To this end, our laboratory identified a new therapeutic use of selenium-containing molecules, Se-methylselenocysteine (MSC) and selenomethionine (SLM), as highly selective, effective multi-targeted molecules in combination with anticancer drug in the therapy of solid tumor xenografts and inhibition of iNOS, COX-2, and HIF-1α (Yin et al. 2006; Chintala et al. 2010; Bhattacharya et al. 2009; Bhattacharya et al. 2008; Bhattacharya et al. 2008; Cao et al. 2004).

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Selenium and Cancer Almost half of all adults with newly diagnosed cancer use mineral and vitamin supplements, with very few studies directly confirming if any benefit is achieved (Bernstein and Grasso 2001). Selenium (Se), an essential dietary trace element and a normal component of mammalian physiology (Goldhaber 2003; Thomson 2004), has been in use as an antioxidant and also in cancer prevention. Selenium supplements are consumed worldwide with up to 9% of adults in the United States using these supplements (Institute of Medicine 2000). Selenium, as part of selenoproteins and antioxidant enzymes, helps prevent damage from free radicals generated by oxygen metabolism (Combs and Gray 1998). Daily Se supplementation at a dose of 400 μg/d taken orally was found to protect from oxidative stress in radical radiotherapy done in stage III human oral squamous cell carcinoma patients (Elango et al. 2006). An inverse correlation exists between selenium and cancer mortality (Combs et al. 1997; Clark et al. 1991), with the serum selenium concentration being a significant prognostic marker for different cancers including non-Hodgkin’s lymphoma (Last et al. 2003). A daily supplementation of 200 μg of Se was reported to reduce the recurrence of a number of cancer types (Fleet 1997; Clark et al. 1996). However, recent results from a prevention trial using low doses of selenomethionine (SLM) (200 μg/d) alone and in combination with vitamin E (400 IU/d) did not prevent prostate cancer (Lippman et al. 2009). The SLM dose used in this trial was equivalent to 2.5 mg/kg/d, achieving total plasma selenium concentrations of 2–3 μM – concentrations comparable to those found in plasma of individuals on a standard American diet. In contrast, the dose of MSC defined for optimal therapeutic synergy in xenografts in our laboratory was 10 mg/kg, achieving total plasma selenium concentrations of 20–30 μM. To determine whether these high concentrations can be achieved clinically without toxicity, results of a clinical phase I trial at Roswell Park Cancer Institute demonstrated 7200 μg/d achieving 20–30 μmol plasma selenium can be administered to patients with advanced cancer (Fakih et al. 2008). The novelty of the approach proposed is (1) in the use of MSC as a highly effective modulator of the therapeutic efficacy of anticancer drugs against a variety of established human tumor xenografts without host toxicity and (2) being a highly potent inhibitor of HIF-1α, ROS, and VEGF levels. The demonstrated effective and sustained inhibition of HIF-1α by MSC against hypoxic cells in vitro and established tumors in vivo is novel and new. Unlike many HIF-1α inhibitors under development, MSC, using well-defined and characterized doses and schedules, is cost effective, orally bioavailable, non-toxic, but biologically effective. Furthermore, documentation that quality of response to chemotherapy is dependent on the pre-existing level of selenium in tumor tissues could provide the basis for defining the optimal selenium supplementation dose for individualized therapy. Thus, the data generated in our preclinical models should define and provide mechanistic endpoints that should be integrated into the design of phase II clinical trials of MSC in combination with chemotherapy. This will assure appropriate translation of laboratory data into the design of clinical trials and provide for definitive verification of the clinical potential of this new and novel approach.

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Hypoxia-Inducible Factor (HIF-1α) and Prolylhydroxylases (PHDs) In most tumors, hypoxia develops during tumor growth and also as a consequence of chemo/radiotherapy. As a response to limited availability of oxygen, HIF-1α is overexpressed and transcriptionally regulates multiple genes associated with tumor growth and angiogenesis. Published reports clearly demonstrated that HIF-1α is a poor prognostic molecular marker associated with drug resistance (Berra et al. 2006; Lopez-Lazaro 2006; Patiar and Harris. 2006). HIF-1α is a heterodimeric complex consisting of HIF-1α and HIF-1β subunits (Maxwell 2005). Under normoxic conditions, cellular levels of HIF-1α are very low to non-detectable due to its rapid degradation by the VHL tumor suppressor gene. For degradation to occur, however, hydroxylation of two proline residues of HIF-1α by PHDs and oxygen, iron, 2-oxoglutarate, and ascorbate are required (Patiar and Harris 2006). PHDs belong to a superfamily of iron-dependent and 2-oxoglutarate-dependent dioxygenases needing O2 and 2-oxoglutarate as co-substrates in the hydroxylation reaction where one oxygen atom is added to a peptidyl proline to form hydroxyproline while the other is used in a coupled decarboxylation reaction that converts 2-oxoglutarate to succinate (Berra et al. 2006). Fe2+ and ascorbate are used as cofactors with Fe2+ required for activating O2 and as a template for the orderly binding of reactants. Ascorbate acts by reducing Fe3+ that binds to the active site of the enzyme after the decarboxylation reaction and is thus needed for its re-activation (Berra et al. 2006). Increase in oxidative stress and mitochondrial ROS during hypoxia regulate activation of HIF-1α by inhibiting PHDs via decreasing Fe2+ availability. Intracellular HIF-1α protein accumulation occurs around 5% O2 concentration and increases as the oxygen level approaches anoxia. ROS inhibits PHDs by oxidizing Fe2+ to Fe3+ resulting in hypoxic stabilization of HIF-1α (Gerald et al. 2004; Brunelle et al. 2005; Chandel et al. 2000; Mansfield et al. 2005). In the absence of mitochondrial ROS, HIF-1α continues to be degraded (Mansfield et al. 2005). Under hypoxia, HIF-1α transactivates a feedback loop that controls HIF-1α accumulation and is independent of PHDs, VHL and occurs only when HIF-1α is accumulated (Demidenko et al. 2005). Se, by reducing ROS generation, could lead to stabilization of PHDs resulting in enhanced hydroxylation of HIF-1α and its degradation by VHL under hypoxia (Chintala et al. 2010). Various redox genes including antioxidants, GPx1, 2, and 3, Se P, and GSH, could play a critical role in the regulation of ROS (Brunelle et al. 2005; Chandel et al. 2000; Mazur et al. 1996; Papp et al. 2007). Hence, an agent like MSC that can interfere with HIF-1α, a key player in cancer biology, is likely to increase tumor response to treatment with anticancer drugs. Preliminary results indicate that a noncytotoxic concentration of MSA is an effective upregulator of GPx2 and Se P but not GPx1, GPx3, SOD, and GSH (Chintala unpublished results).

HIF-1α Transcriptionally Regulates VEGF and Glut-1 Genes HIF-1α is a transcriptional factor that is activated in response to hypoxia and plays a key role in adaptation of tumor cells by activating the transcription of

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several genes including angiogenesis, cell survival, and glucose metabolism (Patiar and Harris 2006; Semenza 2003). Antioxidant MSC has been reported as the regulator of HIF-1α and its transcriptional regulated down-stream marker VEGF which is involved in angiogenesis in FaDu tumors (Yin et al. 2006; Chintala et al. 2010). Our results demonstrated that HIF-1α degradation by MSA under hypoxic conditions was further evidenced by downregulating the VEGF expression in the hypoxic cells (Chintala et al. 2010). We have recently reported the MSC treatment resulted in mature tumor vasculature by decreasing microvessel density (Bhattacharya, Seshadri, Oven et al. 2008). HIF-1α upregulation of VEGF under hypoxic conditions has been reported as the key regulator of angiogenesis (Liao and Johnson 2007; Adamski et al. 2008). The role of VEGF as a pro-survival marker in cancer cells (Dias, Shmelkov, Lam et al. 2002; Dias, Choy, Alitalo et al. 2002) indicates that VEGF is involved in cancer cell survival. The glucose transporter (Glut-1) is overexpressed in hypoxic tumor cells in order to increase glucose transport for anaerobic glycolysis (Patiar and Harris 2006; Wincewicz et al. 2007; Sulkowska et al. 2009) and its expression was correlated with HIF-1α in colorectal cancer patients (Wincewicz et al. 2007). Glut-1 is highly expressed in different solid tumors including HSCCHN in which the combined assessment of carbonic anhydrase (CAIX), a hypoxia marker, and Glut-1 independently correlated with chemo±radiotherapy treatment in patients (Goldhaber 2003). CAIX is an HIF-1αtranscriptionally regulated gene, expressed in hypoxic tumors and involved in pH regulation (Patiar and Harris 2006; Pastorekova et al. 2008) and is widely used as an hypoxia marker in tumors (Bhattacharya et al. 2004). Since VEGF and Glut-1 are overexpressed in hypoxic tumors overexpressing HIF-1α, it is reasonable to propose that downregulation of VEGF and Glut-1 levels, as a consequence of HIF-1α inhibition by MSC, represents important mechanisms of MSC action against HSCCHN tumor cells.

Regulation of Angiogenic and Molecular Marker of Resistance by Modulating iNOS Recent data generated by Lee and colleagues (Lee et al. 2008) demonstrated that activation of iNOS by radiation results in NO production and activation of epidermal growth factor receptor (EGFR), a marker associated with drug resistance (Schema 25.1). Furthermore, clinical results strongly implicate the role of EGFR status (wild vs. mutated) in response to erbitux or panitum mAbs. Whether agents that activate iNO will alter the status of EGFR expression is an area worth investigation. Quintero and his colleagues (Quintero et al. 2006) demonstrate that “nitric oxide is a factor in stabilization of HIF-1α in cancer by a mechanism dependent on free radicals.” A schematic representation of the consequence of activation of nitric oxide synthase (NOS) and resulting cascade activation of a number of molecules leads to stabilization of HIF-1α increases levels of angiogenic activity under conditions of innate alter treatment-induced hypoxia, such as chemo/radiotherapy (Fig. 25.1). Based on data generated in our laboratory (Yin et al. 2006), treatment of human squamous cell carcinoma of the head and neck (FaDu) xenografts with a defined

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Schema 25.1 Regulation of HIF-1α and angiogenesis by iNOS

dose (10 mg/kg/d) and schedule (oral 28 days) of MSC in combination with the optimal schedule and dose of irinotecan (100 mg/kg/wk × 4) resulted in downregulation of nitric oxide, as the result of inhibition of the upregulated iNOS by irinotecan, COX-2, and VEGF. These effects induced by MSC resulted in the reduction of angiogenesis and vessel maturation (Yin et al. 2006; Bhattacharya, Seshadri, Oven et al. 2008). As outlined in Schema 25.1, increased NO results in the activation of COX-2, leading to increased PGE2, HIF-1α, and angiogenic markers including VEGF. Studies reported by Hussein and coworkers (Hussain et al. 2008) demonstrated that NO is a key regulator of tumor growth. Furthermore, in studies reported by Lee and coworkers (Lee et al. 2008), radiation treatment of lung carcinoma tumor cells, A549, resulted in the activation of iNOS, induction of NO, and these effects resulted in activation of the epidermal growth factor receptor (EGFR), a biomarker associated with drug resistance. Most solid tumors are associated with distinct regions of cellular hypoxia, and cytotoxic treatment has the potential to increase the level of hypoxia. Furthermore, hypoxic cells are associated with increased levels of HIF-1α regulated in part by iNOS, which in turn transcriptionally regulates angiogenic molecules. Targeting iNOS could have cascaded down-stream inhibition of HIF-1α. Data generated in our laboratory (Yin et al. 2006) clearly demonstrated that a non-toxic dose of MSC is a highly effective inhibitor of iNOS, COX-2, PGE-2, HIF-1α, and VEGF. It is not known, however, whether the observed therapeutic synergy and selectivity of anticancer drug by MSC is associated with inhibition of a specific marker such as iNOS or the cascade inhibition leading to inhibited HIF-1α and VEGF are sufficient predictors.

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Fig. 25.1 Inhibition of COX-2 leads to enhanced antitimor activity of irinotecan against FaDu xenografts

Data generated in our laboratory (Fakih and Rustum 2009; Cao et al. 2004) demonstrated that irinotecan, a topoisomerase I poison, active in advanced colorectal cancer in combination with celecoxib, an inhibitor of COX-2 biomarker regulated by NO, resulted in synergistic antitumor activity against xenografts FaDu squamous cell carcinoma of the head and neck. While the cure rate with irinotecan was 30%, the MSC or celecoxib in combination with irinotecan resulted in 100 and 70% cures respectively, as shown in Fig. 25.1. The increase in cure rate was not associated with increase in toxicity against normal tissues. The fact that MSC in combination with irinotecan yielded a significantly higher cure rate than the combination of celecoxib with irinotecan indicates that MSC being a multi-targeted agent affecting a number of molecules associated with induction of HIF-1α supports the need to develop agents that alter the profile of molecular heterogeneity of solid tumor if one is to make a significant impact on the therapeutic outcome and selectivity.

Concluding Remarks It has been demonstrated, mostly in preclinical models, that NO donor molecules are selective and efficacious agents alone and in combination with cytotoxic therapy in a variety of solid tumor malignancies. Recent data (Lee et al. 2008) indicate that NO levels can be altered by activation of iNOS which in turn activate multiple targets such as EGFR, COX-2, HIF-1α, and VEGF. These molecules that inhibit iNOS could have the potential for wide and selective alteration of multiple targets associated with tumor growth, metastasis, and resistance. Quintero and his

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colleagues demonstrated that “nitric oxide is a factor in stabilization of HIF-1α in cancer by mechanisms dependent on free radical” (Quintero et al. 2006). Our laboratory identified selenium-containing molecules which are highly effective and selective inhibitors of iNOS, COX-2, HIF-1α, and VEGF levels in tumor xenografts. Collectively, these effects contribute significantly to the therapeutic synergy with anticancer drugs such as irinotecan, a topoisomerase I poison, active in a variety of solid tumor malignancies. The mechanism of HIF-1α regulation by selenium is under intensive investigation in our laboratory. The potential clinical value of selenium in combination with chemotherapy is also under investigation at Roswell Park Cancer Institute.

References Adamski, J.K., Estlin, E.J., Makin, G.W. (2008). The cellular adaptations to hypoxia as novel therapeutic targets in childhood cancer. Cancer Treat. Rev. 34(3), 231–246. Bernstein, B.J., Grasso, T. (2001). Prevalence of complementary and alternative medicine use in cancer patients. Oncology 15(10), 1267–1272. Berra, E., Ginouves, A., Pouyssegur, J. (2006). The hypoxia-inducible-factor hydroxylases bring fresh air into hypoxia signaling. EMBO Rep. 7(1), 41–45. Bhattacharya, A., Toth, K., Mazurchuk, R., et al. (2004). Lack of microvessels in welldifferentiated regions of human head and neck squamous cell carcinoma A253 associated with functional magnetic resonance imaging detectable hypoxia, limited drug delivery, and resistance to irinotecan therapy. Clin. Cancer Res. 10(23), 8005–8017. Bhattacharya, A., Seshadri, M., Oven, S., Toth, K., Vaughan, M., Rustum, Y.M. (2008). Tumor vascular maturation and improved drug delivery induced by Methylselenocysteine leads to therapeutic synergy with anticancer drugs. Clin. Cancer Res. 14(12), 3926–3932. Bhattacharya, A., Toth, K., Durrani, F.A., Cao, S., Slocum, H.K., Chintala, S., Rustum, Y.M. (2008). Hypoxia specific drug tirapazamine does not abrogate hypoxic tumor cells in combination therapy with irinotecan and Methylselenocysteine in well-differentiated human head and neck squamous cell carcinoma A253 xenografts. Neoplasia 10(8), 857–865. Bhattacharya, A., Toth, K., Sen, A., et al. (2009). Inhibition of colon cancer growth by methylselenocysteine-induced angiogenic chemomodulation is influenced by histologic characteristics of the tumor. Clin. Colorectal Cancer. 8(3), 155–162. Brunelle, J.K., Bell, E.L., Quesada, N.M., et al. (2005). Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab. 1(6), 409–414. Cao, S., Durrani, F.A., and Rustum, Y.M. (2004). Selective modulation of the therapeutic efficacy of anticancer drugs by selenium containing compounds against human tumor xenografts. Clin. Cancer Res. 10, 2561–2569. Chandel, N.S., McClintock, D.S., Feliciano, C.E., et al. (2000). Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J. Biol. Chem. 275(33), 25130–25138. Chintala, S., Toth, K., Cao, S., et al. (2010). Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1alpha. Cancer Chemother. Pharmacol. [Epub ahead of print] DOI 10.1007/s00280-009-1238-8. Clark, L.C., Cantor, K.P., Allaway, W.H. (1991). Selenium in forage crops and cancer mortality in U.S. counties. Arch. Environ. Health 46(1), 37–42. Clark, L.C., Combs, G.F. Jr., Turnbull, B.W., et al. (1996). Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 276(24), 1957–1963. Combs, G.F. Jr., Clark, L.C., Turnbull, B.W. (1997). Reduction of cancer mortality and incidence by selenium supplementation. Med. Klin. (Munich) 92 (Suppl 3), 42–45.

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Combs, G.F. Jr., Gray, W.P. (1998). Chemopreventive agents: selenium. Pharmacol. Ther. 79(3), 179–192. Demidenko, Z.N., Rapisarda, A., Garayoa, M., Giannakakou, P., Melillo, G., Blagosklonny, M.V. (2005). Accumulation of hypoxia-inducible factor-1alpha is limited by transcription-dependent depletion. Oncogene 24(30), 4829–4838. De Schutter, H., Landuyt, W., Verbeken, E., Goethals, L., Hermans, R., Nuyts, S. (2005). The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy +/– chemotherapy. BMC Cancer 5, 42, doi:10.1186/1471-2407-5-42. Dias, S., Shmelkov, S.V., Lam, G., Rafii, S. (2002). VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition. Blood 99(7), 2532–2540. Dias, S., Choy, M., Alitalo, K., Rafii, S. (2002). Vascular endothelial growth factor (VEGF)-C signaling through FLT-4 (VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to chemotherapy. Blood 99(6), 2179–2184. Elango, N., Samuel, S., Chinnakkannu, P. (2006). Enzymatic and non-enzymatic antioxidant status in stage (III) human oral squamous cell carcinoma and treated with radical radio therapy: influence of selenium supplementation. Clin. Chim. Acta 373(1–2), 92–98. Fakih, M.G., Pendyala, L., Brady, W., et al. (2008). A Phase I and pharmacokinetic study of selenomethionine in combination with a fixed dose of irinotecan in solid tumors. Cancer Chemother. Pharmacol. 62(3), 499–508. Fakih, M.G., Rustum, Y. M. (2009). Does celecoxib have a role in the treatment of patients with colorectal cancer? Clinical Colorectal Cancer 8(1), 11–14. Fleet, J.C. (1997). Dietary selenium repletion may reduce cancer incidence in people at high risk who live in areas with low soil selenium. Nutr. Rev. 55(7), 277–279. Gerald, D., Berra, E., Frapart, Y.M., et al. (2004). JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 118(6), 781–794. Goldhaber, S.B. (2003). Trace element risk assessment: essentiality vs. toxicity. Regul. Toxicol. Pharmacol. 38(2), 232–242. Hussain, S. Perwez, H.E. Peijun, H., Subleski, J., Hofseth, L., et al. (2008). Nitric oxide is a key component in inflammation-accelerated tumorigenesis. Cancer Res. 68(17), 7130–7136. Institute of Medicine. (2000). Dietary reference intakes for vitamin C, vitamin E, selenium and carotenoids (58–72). National Academies Press, Washington, DC. Last, K.W., Cornelius, V., Delves, T., et al. (2003). Presentation serum selenium predicts for overall survival, dose delivery, and first treatment response in aggressive non-Hodgkin’s lymphoma. J. Clin. Oncol. 21(12), 2335–2341. Lee, H., An, S., Lee, H., Woo, S., et al. (2008). Activation of epidermal growth factor receptor and its downstream signaling pathway by nitric oxide in response to ionizing radiation. Mol. Cancer Res. 6, 996–1002. Liao, D., Johnson, R.S. (2007). Hypoxia: a key regulator of angiogenesis in cancer. Cancer Metastasis Rev. 26(2), 281–290. Lippman, S.M., Klein, E.A., Goodman, P.J., et al. (2009). Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 301(1), 39–51. Lopez-Lazaro, M. (2006). Hypoxia-inducible factor 1 as a possible target for cancer chemoprevention. Cancer Epidemiol. Biomarkers Prev. 15(12), 2332–2335. Mansfield, K.D., Guzy, R.D., Pan, Y., et al. (2005). Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation. Cell Metab. 1(6), 393–399. Mazur, A., Nassir, F., Gueux, E., et al. (1996). Diets deficient in selenium and vitamin E affect plasma lipoprotein and apolipoprotein concentrations in the rat. Br. J. Nutr. 76(6),899–907. Maxwell, P.H. (2005). Hypoxia-inducible factor as a physiological regulator. Exp. Physiol. 90(6), 791–797

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Papp, L.V., Lu, J., Holmgren, A., Khanna, K.K. (2007). From selenium to selenoproteins: synthesis, identity, and their role in human health. Antioxid. Redox. Signal 9(7), 775–806. Pastorekova, S., Ratcliffe, P.J., Pastorek, J. (2008). Molecular mechanisms of carbonic anhydrase IX-mediated pH regulation under hypoxia. BJU international 101(Suppl 4), 8–15. Patiar, S., Harris, AL. (2006). Role of hypoxia-inducible factor-1alpha as a cancer therapy target. Endocrine-related cancer 13(Suppl 1), S61–S75. Quintero, M., Brennan, P., Thomas, G., and Moncada, S. (2006). Nitric Oxide Is a Factor in the Stabilization of Hypoxia-Inducible Factor-1 in Cancer: Role of Free Radical Formation. Cancer Res. 66, 770–774. Semenza, G.L. (2003). Targeting HIF-1 for cancer therapy. Nature reviews 3(10), 721–732. Sulkowska, M., Wincewicz, A., Sulkowski, S., Koda, M., Kanczuga-Koda, L. (2009). Relations of TGF-beta1 with HIF-1alpha, GLUT-1 and longer survival of colorectal cancer patients. Pathology 41(3), 254–260. Thomson, C.D. (2004). Assessment of requirements for selenium and adequacy of selenium status: a review. Eur. J. Clin. Nutr. 58(3), 391–402. Wincewicz, A., Sulkowska, M., Koda, M., Kanczuga-Koda, L., Witkowska, E., Sulkowski, S. (2007). Significant coexpression of GLUT-1, Bcl-xL, and Bax in colorectal cancer. Ann. N Y Acad. Sci. 1095, 53–61. Wincewicz, A., Sulkowska, M., Koda, M., Sulkowski, S. (2007). Clinicopathological significance and linkage of the distribution of HIF-1alpha and GLUT-1 in human primary colorectal cancer. Pathol. Oncol. Res. 13(1), 15–20. Yin, M.B., Li, Z.R., Toth, K., et al. (2006). Potentiation of irinotecan sensitivity by Semethylselenocysteine in an in vivo tumor model is associated with downregulation of cyclooxygenase-2, inducible nitric oxide synthase, and hypoxia-inducible factor 1alpha expression, resulting in reduced angiogenesis. Oncogene 25(17), 2509–2519.

Subject Index

Note: The letters ‘t’ and ‘f’ following the locators refer to tables and figures respectively.

A ABC transporters, see ATP-binding cassette (ABC) transporters AC, see Adenocarcinoma (AC) Accelerated radiotherapy and carbogen breathing (ARCON), 469 ACE-I/ACE-II, autoinhibitory control elements, 64 Action mechanisms, GIT-27NO induction of cell death autophagic cell death, 448 cell death triggered by GIT-27NO, 448t cytostatic/cytotoxic action, 448 PCD, 448 NO, mediator of tumoricidal action of action in intracellular/extracellular compartment, 450–451 DETANONOate treatment, 451 function as oxidant/antioxidant, 450 protective/destructive signals, parameters, 450 See also Cell death by GIT-27NO Acute inflammation, 8 Acute nonlymphocytic leukemia (ANLL), 151, 155–156, 158 Adenocarcinoma (AC), 7, 29, 49, 330–331 colorectal, 138, 198 esophageal, 70 human breast, 109 human colon, 113, 334, 336 intestinal gastric, 71 lung, 334 pancreatic, 26, 71, 113 Adenoviral gene transfer of iNOS (AdiNOS), 255, 392t, 396, 399t, 403, 408t AdiNOS, see Adenoviral gene transfer of iNOS (AdiNOS)

Alanosine, 369, 369f Alkylating agents, 411 Alzheimer’s disease, 8, 151 Aminoguanidine, 107, 118, 170, 191, 310, 317, 322, 423 AMP-activated protein kinase (AMPK), 312 AMPK, see AMP-activated protein kinase (AMPK) Angiogenesis, 13 and blood flow, 271–272 and regulation of tumor blood flow iNOS-derived NO, tumoricidal activity, 198 NO, role in inhibition/promotion of angiogenesis, 198 NO, role in tumor cell respiration, apoptosis and necrosis, 198 NO as classical endothelium-derived relaxing factor, 199 NO downregulation of tumor vascular permeability, 199 and vascular permeability, role of NO down-regulation of angiogenesis inhibitors, 90 HIF-1α upregulation, 90 IL-8 upregulation, 89 increase in production of PGs, effects, 90 VEGF, enhanced angiogenesis, 89 ANLL, see Acute nonlymphocytic leukemia (ANLL) Anthracyclines, 271, 411–412, 432 Anti-apoptotic effect of NO donors by heme oxygenase-1, 119 of NO donors by survivin, 120 Anti-apoptotic properties, NO carcinogenesis promotion DNA repair process, inhibition of, 42 inhibition of apoptosis by NO, 42

B. Bonavida (ed.), Nitric Oxide (NO) and Cancer, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-1432-3, C Springer Science+Business Media, LLC 2010

489

490 Anti-apoptotic (cont.) NOHA-induced apoptosis, 43–44, 48f L-ornithine, effects of, 44 survivin downregulation in lung/breast cancer cells, 43 Anticancer drugs, 271, 481–482, 486 Anti-metastatic treatments, 210 Anti-tumor therapy, 107, 123 macrophage activation, 107 Apoptosis, see Apoptosis and NO Apoptosis and NO, 152f apoptosis, causing mechanisms, 151 bone marrow NO production CD34+ cells, role, 154 DETA-NO, effects on marrow formation, 153 treatment with IFN-γ and endotoxin, 153 treatment with IFN-γ and TNF-induced NOS2 mRNA, 153 drosophila study (Enikolopov), 155 mitochondrial pathway/death receptor pathway, 151–152 NOS1, modulator of nervous tissue cell apoptosis, 153 NOS1expression in mouse study (Krasnov), 155 YY1, role, 153 Apoptosis inhibition by caspase-9 nitrosylation, 121 by the ceramide pathway, 121 by Fas signal pathway, 121 by pleiotrophin, 120 by scavenging of superoxide anions, 120 Apoptosis-regulatory proteins, 93–94 and tumor survival Bcl-2, inhibition of intrinsic pathway of apoptosis, 94 caspases, evidence of S-nitrosylation, 93 extrinsic pathway of cell death, 93 Src, pro-survival factors, 94 TRX, anti-apoptotic protein, 94 Apoptotic pathways, NO regulation of by caspases, extrinsic/intrinsic pathways, 41–42 pro and anti- apoptotic properties, 42 Arachidonic acid, 74, 315–316, 316f ARCON, see Accelerated radiotherapy and carbogen breathing (ARCON) Arginase, 43 Aspirin, 111, 135, 156, 245, 247, 269, 390t, 394, 426, 444, 446, 463

Subject Index Astrocytic gliomas, 69 ATP-binding cassette (ABC) transporters, 271 Autophagic cell death, 448 Autophagy, 448 B Bacillus Calmette-Guerin (BCG), 148 BAECs, see Bovine aortic endothelial cells (BAECs) BAFF/APRIL, autocrine/paracrine mechanisms, 174–175 Barrett’s metaplasia, 70 Base excision repair (BER) pathway, 273 Bax integration, 43 B cell receptor (BCR), 172, 174f Bcl-2 protein, 94, 118–120, 151–153, 152f, 195–196, 211, 246, 257, 268, 274, 294, 461, 465, 467–468 proteasomal degradation, inhibition of, 118–119 BCNU, see L,3-bis(2-chloroethyl)-lnitrosourea (BCNU) BCR, see B cell receptor (BCR) Benzofuroxans and furoxans, 372–375 BER pathway, see Base excision repair (BER) pathway BID, 43–44 Biotin switch assay, 30–32 B16M and HSE, interaction, 192 Bone marrow NO production, 153 Bovine aortic endothelial cells (BAECs), 26, 114, 312 Bradykinin, 63, 67, 240 Breast cancer, 9, 26, 43–51, 109, 112, 213, 216, 221, 258, 301, 332, 334, 391t, 395, 400t, 408t–409t, 411–412, 423–424, 447, 449, 451 NOS induction/inhibition, 424 Burkitt lymphoma, 347 Bystander effect, tumor cells, 239, 255, 286, 404 C CAB, see Chorioallantoic membrane (CAB) Cancer, NO expression in biosynthesis of nitric oxide affinity to iron atoms, 60 eNOS, 62–64 half-life of NO in water, 59 iNOS, 65–68 nitrate and nitrite formation, 60 nNOS, 64–65 NO as EDRF, 60 NO reaction with thiols, 60

Subject Index NOS isoforms, molecular cloning of, 62 inducible NOS/COX-2 interaction, target for cancer treatment, 74–75 iNOS-mediated COX-2 induction in tumor cells, signaling pathways, 75f nitric oxide, tumor cell proliferation, and apoptosis angiogenesis, brain cancer development, 68 apoptosis-inducing factor induction by cytotoxic agents, 72 colorectal tumor development, role of iNOS, 71 HNSCC patients, study, 69 iNOS activity in Barrett’s metaplasia and in esophageal adenocarcinoma, 70 iNOS activity in gastric adenocarcinoma, 70 iNOS activity in pancreatic cancers, 71 iNOS expression and tumor stage, correlation, 70 NO-NSAIDs, chemopreventive effects, 73, 73f NO-NSAIDs, study of Wallace’s group, 73 ovarian tumors/tumor-associated macrophages, role of iNOS in, 69 PGE2 production, NO on COX-2 activity in, 71 tumor angiogenesis/supression by iNOS activity, study, 68 Cancer and endothelial cells, interaction of cancer cell arrest within microcirculation infiltration of leukocytes to the inflammatory site, 190 inflammation and metastasis, link, 191–192 organ selectivity, importance, 190–191 Paget’s theory of seed and soil, 191 tumor cell adhesion to vasular endothelium, inhibition/ promotion, 191 tumor cell arrest in microvessels, mechanism, 190 endothelium-induced cancer cytotoxicity NO and H2 O2, cytotoxic actions of, 193–194 in vitro lysis of metastatic tumor cells (Weiss), 193 Wang’s mechanism, 193 molecular determinants of metastatic cell survival

491 Bcl-2/its anti-apoptotic homologs, permeabilization inhibitors, 195 Bcl-2 overexpression in B16M cells, 195 GSH regulation of Bcl-2, importance, 195–196 high GSH content, parameter, 195 HSE-induced cytotoxicity, 193–194 mitochondrial dysfunction/MPT, death mechanisms, 196 ROS/RNS toxicity, 194 Cancer and nitric oxide COX2 and NOS2, prognostic markers in tumors, 9 inflammation, cause of cancers, 8 transient hypoxia reperfusion, 8 and wound healing, 8f NO-mediated MMP-9 regulation, 13 See also Nitric oxide (NO) Cancer and selenium daily Se supplements intake 200 μg/d, effcets, 481 400 μg/d, effcets, 481 daily Se supplements intake, effcets, 481 MSC optimal dose, defined, 481 Se concentrations, clinical phase I trial novelty of approach, 481 SLM/vitamin E, prevention trials, 481 Cancer therapy and chemoprevention cancer deaths in US/worldwide, estimation, 361–362 hybrid NORMs and prodrugs, 364–366 See also Hybrid NORMs NO, physiological effects, 362 NO, role in CNS/PNS, 362 NO, role in CVS, 362 NO-NSAIDs, 377–379 NORMs, classes of, 362f classical organic nitrates and nitrites, see Nitrates and nitrites (organic) diazeniumdiolates, 369–371 diazetine dioxides, 375 furoxans and benzofuroxans, 372–375 N-hydroxy-N-nitrosamines, 368–369 nitrosothiols (RSNOs), 371–372 N-nitrosamines, 367–368 sydnonimines, 376 NORMs therapeutic applications, evaluation, 363 status of NORMs in cancer treatment chemoprevention, 363 chemotherapy, 363 side effect attenuation, 363

492 Cancer therapy resistance, NO modulation of NO development in oxygen-containing atmosphere “the paradox of oxygen,” 266 NO-mimetic agents, chemo/ radio-resistance modulation anticancer agents modulation, role in drug resistance, 269 chemotherapeutics, use in drugresistance, 269 hypoxia, tumor therapy resiatance, 268 MAPK- and PI3K signal transduction pathways, 269 radiation therapy effects on hypoxia, 268–269 NO-mimetic agents, drug resistance modulation mechanisms angiogenesis and blood flow, 271–272 DNA damage and repair, 272–273 HIF-dependent genes (P-gp, VEGF), 271 hypoxia-inducing factor 1α, 269–270 NF-κB, 274 nitric oxide/nitric oxide-donating agents, molecular mechanisms, 270f oxidative stress, 272 p53, tumor suppressor, 273–274 ROS and RNS, biochemistry of Fenton reaction, 266 mitochondrial respiratory chain inhibition, effects, 267 reaction balance between ROS/RNS, 267 sites of generation, 266 thioredoxin and glutaredoxin, role, 267 ROS-independent signaling pathways of RNS in cancer, 267–269 NO cytoprotective/cytotoxic effects, contradiction, 268 Cancer treatments, conventional chemosensitisation alkylating agents, 411 anthracyclines, 411–412 cisplatin, 405–411 5-FU, 412 nitrosourea, 405 sensitisation of tumour cells to cytotoxic chemotherapy by NO in vitro/ in vivo, 406t–409t monotherapy cytotoxicity of NO against tumour cells in vitro/in vivo, 389t–393t

Subject Index iNOS gene therapy, 395–396 NO donor drugs, 394–395 NO, cytotoxic effector in macrophages, 388 NO/nucleophile complexes, growth/survival of A375 human melanoma cells, 388 radiosensitisation, 397–405 therapeutic implications, 412 Carcinogenesis, 13, 42, 49–50, 70, 72, 74, 91, 115, 117, 137–139, 199, 267, 273, 304, 320–323, 330, 332–333, 336, 341–352, 377, 430 Cardiovascular system (CVS), 362 Cationic lipid vectors, 395–396 Cdc25A, NO downregulation of, 107 CD34+ cells, 154 CDDO, see 2-cyano-3,12-dioxooleana-1,9dien-28-oic acid (CDDO) CDKs, see Cyclin-dependent kinases (CDKs) Cell death by GIT-27NO, 448t caspases mediation of nucleic acid fragmentation, 449 cisplatin activity, inhibition of, 449 drug resistance, mechanisms/problems, 449 necrotic cell death, 449 type I PCD/apoptotic cell death in breast cancer, 449 Cell death receptors, 256 Central nervous system (CNS), 41, 140, 362 Cervical cancer common among women, 345 formation of 8-nitroguanine/8-oxodG in biopsy specimens, assessment double immunofluorescence staining, 345–347, 347f HPV infection, cause CIN, 346 high-risk types of HPV, 346 HPV-16 and HPV-18, carcinogenic to humans, 346 HPV oncoproteins, role, 346 low-risk HPV types, 346 inflammation, role in HPV-mediated cervical carcinogenesis, 346 Cervical intraepithelial neoplasia (CIN), 346 cGMP-dependent/independent mechanisms, 170 Chemokines, 113, 190–191, 461 Chemo-resistance, 268–269 Chemosensitisation alkylating agents, 411 anthracyclines, 411–412

Subject Index cisplatin, 405–411 5-FU, 412 nitrosourea, 405 Chemotherapeutics, use in drug-resistance, 269 Chemotherapy clinical studies, 240–241 cytotoxicity effects by chemotherapeutic agents on NO, 239–240 future directions, 239 preclinical studies blood–brain barriers, 240 C6 glioma model/9L rat glioma model, study, 240 cytotoxic potentiation by NO, mechanism, 240 Chemotherapy and NO chemotherapeutic agents, anti-tumor effect perspectives clean/dirty NO donors, use, 286–287 GTN, randomized phase II clinical trial study, 287 improved blood flow/better distribution of oxygen, 286 “normalization” of tumor vasculature, 287 stromal/endothelial tissues, chemotherapeutic targets bystander effect, 286 Cholangiocarcinoma, 344–345, 350–351 Chorioallantoic membrane (CAB), 112, 216 Chronic inflammation, 8 gene mutations/modifications of cancer-related proteins, 300– Helicobacter pylori-induced gastric cancer, cause, 300 immunosuppression, associated with, 300 ROS, influence on, 300 Chronic lymphocytic leukemia (CLL), 151, 153, 157–160, 169–181 Chronic nitrate therapies, 367 CIN, see Cervical intraepithelial neoplasia (CIN) Cisplatin, 68–69, 107–108, 112–113, 119, 220, 258–260, 269, 271, 273–274, 286–288, 295, 303, 405–410, 406t–409t, 422, 425–429, 434, 447, 449, 452, 464–465, 471 and carboplatin, drugs used in bladder cancer, 464 CLL, see Chronic lymphocytic leukemia (CLL) CLL, apoptosis inhibition by NO (endogenous) common in Western countries, 170

493 detection of NOS in CLL cells, see NOS detection in CLL cells disease of proliferation and accumulation, 170 down-regulation of iNOS expression and NO production caspase 3 activity, Western blot/ELISA analysis, 177 flavopiridol-promoted apoptosis, 176–177 proteolytic cleavage of iNOS by calpain I (Walker), 177 TRAIL or chlorambucil treatnent, 176 ubiquitination–proteasome pathway, 177 NO, Bcl-2 family and control of mitochondrial apoptosis, 179–180 NO (endogenous), anti-apoptotic in CLL cells mechanisms, 178–179 mitochondria biogenesis, role of NO in, 175 regulation of iNOS expression BAFF/APRIL, CLL protection by autocrine/paracrine mechanisms, 174–175 increased iNOS expression, factors, see INOS expression, increase in TLR-7 agonists, role, 172–173, 174f in vivo leukaemia cells, role, 172 cNOS, see Constitutive NOS (cNOS) CNS, see Central nervous system (CNS) Colon cancer cells, 42, 71–73, 111–112, 247, 255, 259, 395, 410, 425 Colorectal adenocarcinoma, 138, 198 Colorectal cancer (CRC), 7, 71–72, 194, 213, 319, 333–334, 363, 371, 377, 396, 403, 411, 425–426, 434–435, 483–484 NO donor, 425 NOS induction/inhibition, 425–426 NSAIDs, 426 Condyloma acuminatum, 346–347, 347f “Constitutive” enzyme, see Neural NOS (NOS1) Constitutive NOS (cNOS), 40, 65, 86, 330, 463 Conventional cancer treatments chemosensitisation alkylating agents, 411 anthracyclines, 411–412 cisplatin, 405–411 5-FU, 412 nitrosourea, 405

494 Conventional cancer (cont.) tumour cells sensitisation to chemotherapy by NO in vitro/ in vivo, 406t–409t monotherapy cytotoxicity of NO against tumour cells in vitro/in vivo, 389t–393t iNOS gene therapy, 394–396 NO donor drugs, 394–395 NO, cytotoxic effector in macrophages, 388 NO/nucleophile complexes, growth/survival of A375 human melanoma cells, 388 radiosensitisation, 397–405 therapeutic implications, 412 Corynebacterium parvum, 138 COX2, see Cyclooxygenase type 2 (COX2) CPR, see Cytochrome P450 reductase (CPR) CRC, see Colorectal cancer (CRC) Cupferron, 369, 369f Curcumin, 49 CVS, see Cardiovascular system (CVS) 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), 171 Cyclin-dependent kinases (CDKs), 176, 331 Cyclooxygenase type 2 (COX2), 315–316, 316f Cytochrome c, 104 Cytochrome P450 reductase (CPR), 62, 266, 367 Cytotoxic effects of endogenous NO anti-tumor therapy macrophage activation, 107 eNOS induction in vascular cells by polyphenols, 109 Fas expression, enhancement, 108 iNOS gene transfer therapy, 108 iNOS induction in tumor cells by synthetic retinoid, 109 by Th1 and M1 cytokines, 107–108 using plant extract, 108 using statins, 109 Cytotoxic effects of exogenous NO cytotoxic effect of NO donors diazeniumdiolate type of NO donors, 112–113 hybrid type NO donors, 113 organic nitrate type of NO donors, 111–112 S-nitrosothiol type of NO donors, 112 diazeniumdiolates (NONOates) type of NO donors, 110–111

Subject Index hybrid type of NO donors, 111 organic nitrate types of NO donors, 110 S-nitrosothiol type of NO donors, 110 Cytotoxic/protective activity of NO in cancer, 140f direct role in cytotoxicity endogenous NO, see Endogenous NO, cytotoxic effects exogenous NO, see Exogenous NO, cytotoxic effects high/low NO concentrations, effects on tumor cells, 134 NO synthesis by NOS isoforms, 137–138 nNOS/eNOS vs. iNOS, 133–134 protective effect of NO from cytotoxic stimuli endogenous NO, see Endogenous NO, protective effects eNOS, pro-tumorigenic role, 138 exogenous NO, see Exogenous NO, protective effects Cytotoxic stimuli regulation in immunotherapy, preclinical findings NO-mediated immune suppression in tumor-bearing hosts, 245–247 potentiation of immune-derived cytotoxicity against tumor cells, 244 D Death-inducing signaling complex (DISC), 93 Dephostatin, 368 DETA/NO, see Diethylenetriamine NO (DETA/NO) DFS, see Disease-free survival (DFS) Diazeniumdiolates, 370f advantages as NORMs, 370 5-FU, anti-tumor agent, 371 GST-P1, anti-cancer drug, 371 JS-K, anti-cancer drug, 371 NONOates, NO donors, 110–113 SPER/NO and DEA/NO, rapid- and medium-release rate NO donors, 369 Diazetine dioxides, 375 Diethylenetriamine NO (DETA/NO), 269, 274 Differential NO stress, flow charts, 46f Dinitroso iron complexes (DNIC), 4 DISC, see Death-inducing signaling complex (DISC) Disease-free survival (DFS), 296 DNA damage, 341–352

Subject Index DNA damage and repair oxidative damage, repair mechanisms HR enzymes, 273 NHEJ mechanism, 273 DNA repair enzymes DNA alkyltransferases, 42 OGC1, 42 XPA protein, 42 DNA repair proteins, 6 DNIC, see Dinitroso iron complexes (DNIC) Dopastatin, 369, 369f Dr. Harald zur Hausen, 346 E EBERs, see EBV-encoded RNAs (EBERs) EBV-encoded RNAs (EBERs), 349 EBV infection, see Epstein-Barr virus (EBV) infection E-cad transcription, 213 EDRF, see Endothelium-derived relaxing factor (EDRF) EGF, see Epidermal growth factor (EGF) EMT process, see Epithelial to mesenchymal transition (EMT) process Endogenous NO, cytotoxic effects anti-tumor therapy macrophage activation, 107 eNOS induction in vascular cells by polyphenols, 109 Fas expression, enhancement, 108 iNOS gene induction, signals, 134 iNOS gene transfer therapy, 108 iNOS induction in tumor cells by synthetic retinoid, 109 by Th1 and M1 cytokines, 107–108 using plant extract, 108 using statins, 109 macrophage-mediated tumor cell killing, 134 macrophages, antitumor effects, 134 NO production in humans/rodent, distinction, 135 Endogenous NO, protective effects anti-apoptotic effect of survivin, 119 chronic inflammation, risk factor, 138 Fas signal pathway, inhibition of, 118 IFN-induced apoptosis by NO, inhibition of, 119 IL-1 gene expression, study, 138 iNOS inhibition by AMG, 118 pro-inflammatory cytokines, role, 138 proteasomal degradation of Bcl-2, inhibition of, 118–119

495 treatment with Corynebacterium parvum, effects, 138 VEGF promotes iNOS expression, 118 Endonuclease G, 104 Endothelial nitric oxide synthase (eNOS), 23–32, 61, 62–64 activation by AKT, 24 activation in pancreatic cancer, 27–28 angiogenesis, role in, 63 chronic inhibition of, effects, 64 member of NOS family, 23 post-translational modification, 63 protein interactions in less active/more active states, 64 role in tumorigenesis C118 mutations of wild-type HRas, 31, 32f eNOS-dependent activation of HRas, impact, 31 nitrosylated GTP-bound HRas levels, determination, 30–31, 31f NO production, effects, 30 sGC, target in cancer cells, 30 vascular pathology NO derived from eNOS, effects on vessel wall, 63 NOS inhibitors, role, 63 vs. nNOS/iNOS, 23–24 Endothelial NOS (eNOS), 40 Endothelium-derived relaxing factor (EDRF), 4, 60, 86, 199 eNOS, see Endothelial nitric oxide synthase (eNOS); Endothelial NOS (eNOS) eNOS induction in vascular cells by polyphenols, 109 Epidermal growth factor (EGF), 71 Epithelial to mesenchymal transition (EMT) process, 209–210, 469–470 Epstein-Barr virus (EBV) infection, 150, 347–349 ERK, see Extra-cellular signal-regulated kinase (ERK) E-selectin, 192–193 Esophageal adenocarcinoma, 70 Esophageal cancer, prognostic significance of iNOS in epidemiology and etiology CDK inhibitors, role in tumor progression, 331 esophageal SCC study, inference, 329 iNOS and p53, association, 333–334 iNOS expression and prognosis anti-tumor effect of iNOS, 335

496 Esophageal cancer (cont.) esophageal SCC, cumulative Kaplan–Meier survival curves, 335f iNOS-19 tumor, growth of, 334 ONO-1714, invasive cancer inhibitor, 334 tumor cell angiogenesis in HCC by MMP-9 modulation, 334 wild-type/mutant p53, tumor biology, 336 iNOS expression in in non-neoplastic esophageal epithelium, 332 in ovarian cancer, 333 tumors exhibited cytoplasmic staining for iNOS, 332, 332f in vitro/in vivo study, 332 NO, genotoxic effects, 330 NOS, classes constitutive NOS, 330 iNOS, 330 OS rate of patients, 330 risk factors, 329, 331 types adenocarcinoma, 330 SCC, 330 Esophageal SCC genetic changes caused by carcinogens p53 tumor-suppressor gene mutation, 331 incidence in China, US, 330–331 Estrogen, 63, 93t, 118, 198, 424 Exogenous NO, cytotoxic effects cytotoxic effect of NO donors diazeniumdiolate type of NO donors, 112–113 hybrid type NO donors, 113 organic nitrate type of NO donors, 111–112 S-nitrosothiol type of NO donors, 112 diazeniumdiolates (NONOates) type of NO donors, 110–111 hybrid type of NO donors, 111 NO donors JS-K, role, 135–136 NCX 4040, in vivo anti-cancer effect, 135 NO and HIF-1 activity, 136 NO-aspirin, chemopreventive agents, 135 NSAIDs, role in treatment of cancers, 135 organic nitrate types of NO donors, 110

Subject Index S-nitrosothiol type of NO donors, 110 tumor microenvironment hypoxia, 136 mice with an eNOS deficiency, study results, 136 pro-inflammatory infiltrate in, radiosensitizing strategies, 137 Exogenous NO, protective effects anti-apoptotic effect of NO donors by heme oxygenase-1, 119 anti-apoptotic effect of NO donors by survivin, 120 inhibition of apoptosis by caspase-9 nitrosylation, 121 by the ceramide pathway, 121 by Fas signal pathway, 121 by pleiotrophin, 120 by scavenging of superoxide anions, 120 NO donors, 139 proteasomal degradation of Bcl-2, inhibition of, 120 tumor microenvironment, 139–140 Extra-cellular signal-regulated kinase (ERK), 9, 13, 26, 43, 47, 94, 218f, 245, 259, 298, 309–310, 317, 318f, 319, 322, 452, 466 F FAD, see Flavin adenine dinucleotide (FAD) Farnesyl transferase inhibitors (FTIs), 47, 50 Fas expression enhancement, 108 upregulation by NO, 114 Fas signal pathway, inhibition of, 118 Fe-NO EPR signal, 4 Fenton reaction, 266 Flavin adenine dinucleotide (FAD), 61 Flavin mononucleotide (FMN), 61 Flavopiridol, 158, 176–178 Fluorescence-tagged tumor cell and video-capturing image techniques, 190 5-Fluorouracil (5-FU), 158, 241, 269, 370f, 371, 412, 426 FMN, see Flavin mononucleotide (FMN) FTIs, see Farnesyl transferase inhibitors (FTIs) 5-FU, see 5-Fluorouracil (5-FU) Furoxans and benzofuroxans, 372–375 G Gastric adenocarcinoma, 70, 333 Gastric cancer, 69, 70–71, 90, 114, 213, 300, 319, 332, 424, 444

Subject Index Lauren’s classification, 71 NOS induction/inhibition, 422 GC, see Guanylyl cyclase (GC) GDN, see Glyceryl dinitrate (GDN) GIT-27NO action mechanisms induction of cell death, 448–449 NO as mediator of tumoricidal action of, 450–452 See also Action mechanisms, GIT-27NO aspirin and NSAIDs, role in apoptosis, 444 limitations, 444 modification of drugs, outcomes, 444–445 design of novel antineoplastic drugs, aim, 453 effects on tumor cell growth in vitro/in vivo cell lines sensitive to GIT-27NO treatment, 446t treatment of androgen-independent p53-deficient PC3 cell line, 447 treatment of C57BL/6 mice with GIT-27NO, efficacy, 447 influence on MAP kinase activity, 452 NSAIDs, anticancer properties of, 444 subacute/acute toxicity of, 447 VGX-1027 vs. its NO-modified derivative, GIT-27NO composition of GIT-27NO, 445 ConA-induced hepatitis, findings, 445–446 Glucose transporter (Glut-1), 372, 483 Glut-1, see Glucose transporter (Glut-1) Glutaredoxin, 267 Glutathione disulfate (GSSG), 267 Glyceryl dinitrate (GDN), 366 Glyceryl mononitrate (GMN), 366 Glyceryl trinitrate (GTN), 269, 366 Glycolysis, 313, 314f, 315, 470, 483 GMN, see Glyceryl mononitrate (GMN) GST-P1, see P1 isoform of the phase II detoxification enzyme glutathioneS-transferase (GST-P1) GTN, see Glyceryl trinitrate (GTN) Guanylyl cyclase (GC), 4, 10–11, 60, 67, 149, 319 H HAECs, see Human aortic endothelial cells (HAECs) Hairy cell leukaemia, 171 Hazard ratio (HR), 297

497 HCC, see Hepatocellular carcinoma (HCC) HCC, prognostic significance of iNOS in hepatocarcinogenesis, 310 iNOS and signal transduction pathways COX2 and iNOS, cross talk, 316, 316f EGFR activation, effects, 317 HCV-positive HCC patients, study, 317 iNOS interplay with IKK/NF-κB and Ha-RAS/ERK pathways, 317–318, 318f PGE2, role in cancer metastasis, 317 PGs, role in inflammatory processes, 316f sGC/cGMP/PKG signaling pathway, mouse colitis model, 317 liver infiltration, DNA damage, 310 NO and peroxynitrite production, effects on HCC, 311f NO interference with SAM synthesis and DNA methylation synthesis of methionine/Sadenosylmethionine and methylation reactions, 321f partial liver resection/liver transplantation, treatments, 310 risk factors, 310 RNS, role in carcinogenesis, 311 RNS and hepatocarcinogenesis, production/metabolic effects AMPK activation, effects, 312 hepatic glucose metabolism, NO effects, 313 mitochondrial respiratory chain/Krebs cycle/glycolysis, NO effects, 313, 314f NO-mediated DNA repair inhibition/ vasodilation, HCC cause, 312 NOS isoforms in liver, 311 Warburg effect, 313 HCNP, see Hippocampal neurostimulating peptide (HCNP) Head and neck squamous cell carcinoma (HNSCC), 68, 112, 119, 298 Helicobacter pylori (H. pylori), 70 -induced gastric cancer, 300 infection, 344 Hematopoiesis, 153–155 Hematopoietic/nonhematopoietic cells, 153 Heme-regulated inhibitor (HRI), 46, 46f Hepatic sinusoidal endothelium (HSE), 192 Hepatic zonal heterogeneity, 193 Hepatitis B virus (HBV), 310

498 Hepatitis C virus (HCV), 310 Hepatocarcinogenesis, 310 and RNS AMPK activation, effects, 312 hepatic glucose metabolism, NO effects, 313 mitochondrial respiratory chain/Krebs cycle/glycolysis, NO effects, 313, 314f NO-mediated DNA repair inhibition/vasodilation, HCC cause, 312 NOS isoforms in liver, 311 Warburg effect, 313 Hepatocellular carcinoma (HCC), 309–323 NO donor, 428 See also HCC, prognostic significance of iNOS in HIF, see Hypoxia-inducible factor (HIF-1α) HIF-dependent genes (P-gp, VEGF), 271 ABC transporters, 271 VEGF, 271 Hippocampal neurostimulating peptide (HCNP), 212 HME, see Human mammary epithelial (HME) cells HNSCC, see Head and neck squamous cell carcinoma (HNSCC) Homologous recombination (HR), 273 Hormonal therapy, 210, 460 4-HPR, see N -(4-hydroxyphenyl) retinamide (4-HPR) HPV, see Human papilloma virus (HPV) HPV oncoproteins, 346 HR, see Hazard ratio (HR); Homologous recombination (HR) HRI, see Heme-regulated inhibitor (HRI) HSE, see Hepatic sinusoidal endothelium (HSE) HSVECs, see Human saphenous vein endothelial cells (HSVECs) Human aortic endothelial cells (HAECs), 26 Human breast adenocarcinoma, 109 Human colon adenocarcinoma, 93, 113, 390t, 412 Human HeLa cancer cells, 256 Human leukemia cells and NO expression of NOS by leukemia/MDS cells, 156–157 macrophage-mediated cytotoxicity, research study, 148 NO and acute non-lymphoid leukemia, 155–156

Subject Index NO and apoptosis, 151–153 NO and CLL, 157–160 NO and normal hematopoiesis, 153–155 NO generalities, 149 NOS inhibitors, 150–151 NO synthases NO production from NOS isoforms, 149–150 Human mammary epithelial (HME) cells, 46 Human melanoma, prognostic significance of iNOS in gene microarray technology, 294 iNOS expression in metastatic melanomas, 297–298 iNOS expression in primary melanomas, see Primary melanomas, iNOS expression in iNOS-produced by NO, molecular analysis, 294 localization of protein expression, 301–302 melanoma, therapeutics for DTIC, 303 IFN-γ, 302 IL-2, 302-303 iNOS inhibitors, selectivity, 303 iNOS-produced NO, novel targeted therapy, 303 melanoma progression, regulatory effects HIF-1, role in tumor angiogenesis, 299 inhibition of, by IL-24, 299 iNOS expression and lymphangiogenesis, correlation, 297–298 MAPK pathway, 298 Ras/Raf/MEK/ERK pathway, 299 SCF-regulated cytokine expression, 298 tissue invasion, role of NO in, 300 tumor-cell-derived NO, cause, 298 NO, inflammation, and melanoma chronic inflammation, high cancer risk, 300 Helicobacter pylori-induced gastric cancer, 300 immunosuppression, cancer risk, 300 iNOS gene, gastric cancer in Japanese women, 300 pro-inflammatory cytokines, neoplastic growth effects, 301 TAM, role in cancer metastasis, 301 prognostic factors available, 294 prognostic markers, significance, 294 AJCC staging system, 294

Subject Index Human ovarian cancer, 40 Human papilloma virus (HPV), 345–347 cervical cancer, cause, 345 discovery of, Dr. Harald zur Hausen, 345 high-risk types of HPV, 345 HPV-16 and HPV-18, carcinogenic to humans, 345 Human saphenous vein endothelial cells (HSVECs), 26 Hybrid NORMs, 364–366 classes of, 364, 365f anti-inflammatory activity, 364 NO-releasing warheads/apoptotic cytotoxins, 364 NSAID, bioactive carrier, 364 definition, 364 GI damage, chronic use of NSAIDs, 364 molecular deconstruction of, 364–365 NO-ASA, 363 prospects and obstacles, 365–366 Hybrid type of NO donors, 111, 113 Hydrophobic NOS inhibitors, 171 Hypoxia, 268 Hypoxia-inducible factor (HIF-1α), 94, 121, 134, 199, 269–270, 299, 313, 420, 480, 482 Hypoxic cell radiosensitizers, 254, 469 I IAN, see Iso-amyl nitrite (IAN) IARC, see The International Agency for Research on Cancer (IARC) IFN-induced apoptosis by NO, inhibition of, 119 IIIB/IV non-small-cell lung cancer, 259–260 IκBα phosphorylation, inhibitors of, 173 Immunotherapy dual role of NO in immune cell responses NO, influence on immune functions, 242 NO regulation of central/peripheral tolerance, 242 growth promoting effects of NO in tumor cells, 244 immune suppressive role of NO, 243–244 immunosuppressive mechanisms, 242 regulation of T-Cell priming by NO, 243 TAA responses, study, 241–242 Immunotherapy and NO cytokine-activated macrophages, role, 287 direct/indirect elimination of tumor cells, 287–288 direct/indirect immunotherapeutic approaches, 288

499 expression of apoptosis-related genes, regulation of, 288 NO donors, sensitization of tumor cells/metastatic process, 288 Inducible nitric oxide synthase (iNOS), 23, 61, 65–68 in Barrett’s metaplasia and in esophageal adenocarcinoma, 70 calcium independent, 66 expression, inhibition of, 66–67 in gastric adenocarcinoma, 70 induced by pro-inflammatory stimuli, 65 induction mechanism, 65 NO reaction with radicals, 67 peroxynitrite, physiological/pathophysiological roles, 67–68 toxic/protective effects, 66 inflammatory processes, importance in, 65 in pancreatic cancers, 71 and signal transduction pathways COX2 and iNOS, cross talk, 315–316, 316f EGFR activation, effects, 317 HCV-positive HCC patients, study, 317 iNOS interplay with IKK/NF-κB and Ha-RAS/ERK pathways, 317–318, 318f PGE2, role in cancer metastasis, 316–317 PGs, role in inflammatory processes, 316, 316f sGC/cGMP/PKG signaling pathway, mouse colitis model, 319 Inducible NOS (iNOS), 40 Infection/inflammation-related carcinogenesis, prognostic significance of nitrative DNA damage cancer caused by infectious agents worldwide, 342t DNA damage and prognosis of patients with soft tissue sarcoma, 349–350 EBV and NPC, 347–349 human papilloma virus and cervical cancer, 345–347 liver fluke infection and cholangiocarcinoma, see OV infection, risk of cholangiocarcinoma 8-nitroguanine formation by chronic inflammation, 343f, 344 See also 8-Nitroguanine ROS/RNS, cause of oxidative/nitrative DNA damage, 343

500 Infiltration of leukocytes to the inflammatory site, process, 190 Inflammation basic processes, 8f cause of cancers, 8 and metastasis, link, 192 B16M and HSE, interaction, 192 carbohydrate–carbohydrate recognition, 192 E-selectin, role, 192 liver, common site for metastasis development, 192 types acute/chronic, 8 Inflammatory mediators, 138 Inorganic nitrites, formation of, 367 iNOS, see Inducible nitric oxide synthase (iNOS); Inducible NOS (iNOS) iNOS expression, increase in factors IL-4 and IFN-γ, 172 ligation of CD23, 172 low-affinity IgE receptor, 172 iNOS expression by VEGF, 118 iNOS gene/NO induction by tumor-infiltrating dendritic cells, 114 iNOS gene transfer therapy, 108 iNOS induction in tumor cells by synthetic retinoid, 109 by Th1 and M1 cytokines, 107–108 using plant extract, 108 using statins, 109 iNOS inhibition by AMG, 118 iNOS-19 tumor, 334 Interaction of cancer and endothelial cells cancer cell arrest within microcirculation infiltration of leukocytes to the inflammatory site, 190 inflammation and metastasis, link, 192 organ selectivity, importance, 190–191 Paget’s theory of seed and soil, 191 tumor cell adhesion to vascular endothelium, inhibition/ promotion, 191 tumor cell arrest in microvessels, mechanism, 190 endothelium-induced cancer cytotoxicity NO and H2 O2, cytotoxic actions of, 194 in vitro lysis of metastatic tumor cells (Weiss), 193 Wang’s mechanism, 193

Subject Index molecular determinants of metastatic cell survival Bcl-2/its anti-apoptotic homologs, permeabilization inhibitors, 195 Bcl-2 overexpression in B16M cells, 195 GSH regulation of Bcl-2, importance, 196 high GSH content, parameter, 195 HSE-induced cytotoxicity, 195 mitochondrial dysfunction/MPT, death mechanisms, 196 ROS/RNS toxicity, 195 The International Agency for Research on Cancer (IARC), 342 Intestinal adenocarcinoma, 71 Intrahepatic cholangiocarcinoma, 344 Intra-tumoural injection, 396 Ionizing radiations, 254–256 ISDN, see Isosorbide dinitrate (ISDN) Iso-amyl nitrite (IAN), 367 Isosorbide dinitrate (ISDN), 110, 216, 238, 255, 258, 269, 272, 366, 411 K Kaplan–Meier method, 349 KIPase, 177 Krebs cycle, 313, 314f Kupffer cells, 192 L Lauren’s gastric cancer classification, 71 L,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), 6 Leukoplakia, 344 Lewis lung carcinoma (LLC), 27, 111, 216, 427 Lipopolysaccharide (LPS), 66, 71, 107, 134, 154f, 193, 285, 320, 421 Listeria monocytogenes, 148 Liver, common site for metastasis development, 192 Liver fluke infection, 344–345 LLC, see Lewis lung carcinoma (LLC) L-NG-monomethyl arginine (L-NMMA), 4 L-NIL, 151 L-NMMA, see L-NG-monomethyl arginine (L-NMMA) LPS, see Lipopolysaccharide (LPS) Lung adenocarcinoma, 334 Lung cancer NO donor, 427 NOS induction/inhibition, 427

Subject Index Lymphoma Burkitt, 170, 347 gastric, 70 lymphocytic, 150 non-Hodgkin’s, 171, 217, 466, 481 NOS induction/inhibition, 423 M Macrophage reprogramming by S1P, 121–122 Malignant fibrous histiocytoma (MFH), 349 Kaplan–Meier method, statistical analysis, 349 Malignant melanoma NO donor, 428 NOS induction/inhibition, 428 Malignant tumor, therapeutic applications of NO animal models, 420–421 breast cancer NOS induction/inhibition, 423–424 colorectal cancer NO donor, 425 NOS induction/inhibition, 425–426 NSAIDs, 426 gastric cancer NOS induction/inhibition, 424 hepatic cellular carcinoma NO donor, 428 human studies non-randomized studies, 431–434 randomized studies, 434–436 lung cancer NO donor, 427 NOS induction/inhibition, 427 lymphoma NOS induction/inhibition, 423 malignant melanoma NO donor, 428 NOS induction/inhibition, 428 other malignant tumors NO donor, 428–429 NOS induction/inhibition, 429–430 renal cellular carcinoma NOS induction/inhibition, 427 role of NO in prostate cancer NO donor, 421–422 NOS induction/inhibition, 422 MALT, see Mucosa-associated lymphoid tissue (MALT) MAPK, see Mitogen-activated protein kinase (MAPK) MDSCs, see Myeloid-derived suppressor cells (MDSCs)

501 Mechanism of NO cytotoxicity apoptosis, denitrosylation by thioredoxins, 105 EGFR tyrosine kinase activity inhibition, 106–107 GAPDH, NO target, 105 NO downregulation of Cdc25A, 107 survivin by NO, inhibition of, 106 Yin-Yang1 apoptosis upregulation by NO, inhibition of, 106 Mechanism of NO protection from cytotoxins inhibition of ceramide pathway, 117 inhibition of Fas signal pathway, 117 inhibition of mitochondrial permeability transition pores, 117 inhibition of the caspase family, 117 Metastasis, definition, 209 Metastatic melanomas, iNOS expression in iNOS/COX-2, prognostic value, 297 NRAS and BRAF mutations, 297 patients treated by therapy/prior to therapy, study, 297 univariate/multivariate analysis, 297 MFH, see Malignant fibrous histiocytoma (MFH) MHC Class I shedding under hypoxic tumor conditions, 122 Microenvironment of tumor Fas expression upregulation, NO, 114 iNOS gene/NO induction by tumorinfiltrating dendritic cells, 114 MMP downregulation by stromal cells, 114–115 NO production by irradiation-activated macrophages, 114 NO production from nitrite, 115 Micrometastases, migration/limited survival of, 197–198 Mitochondria biogenesis, role of NO in, 175 Mitochondrial membrane potential (MMP), 43, 197 Mitochondrial permeability transition (MPT), 196 Mitochondrial respiratory chain, 313–315 Mitogen-activated protein kinase (MAPK), 299 Mitogen-activated protein kinase MAPK phosphatase-1 (MKP-1), 43 MKP-1, see Mitogen-activated protein kinase MAPK phosphatase-1 (MKP-1) MM, see Multiple myeloma (MM)

502 MMP, see Mitochondrial membrane potential (MMP) MMP downregulation by stromal cells, 114–115 Molecular cloning, 61 “Molecular signature” approach, 10 Monotherapy iNOS gene therapy adenoviral delivery, 396 cationic lipid vectors/direct intra-tumoural injection, use of, 395–396 delivered via intravenous route, 396 E9 promoter, antitumor effects, 396 GTN administration for localised prostate cancer, 396 Soler’s strategy, 395 NO donor drugs inhibition of liver tumours, study, 395 JS-K, in vitro/in vivo study in myeloma cells, 395 NCX 4040, study, 395 NO-ibuprofen/aspirin, study of cytostatic/pro-apoptotic effects, 394 NONOates, radio-/chemo-sensitising potency assessment, 394 NO-NSAID, anticancer activity determination, 394 NO release by nitrogen-containing compounds, mechanisms, 394 MPT, see Mitochondrial permeability transition (MPT) MSC, see Se-methylselenocysteine (MSC) MSC, potential role angiogenic regulation/molecular resistance by iNOS modulation EGFR, drug resistance marker, 483 FaDu xenografts of MSC/irinotecan, iNOS expression, 483–484 inhibition of COX-2, results, 485f regulation of HIF-1α and angiogenesis by iNOS, 484f HIF-1α and PHDs hydroxylation of proline residues of, 482 selenium and cancer, 481 tumor tissues heterogeneity, 480 VEGF/Glut-1, HIF-1α transcriptionally regulated genes HIF-1α and Glut-1, colorectal cancer patients study, 483 HIF-1α degradation by MSA, 483 HIF-1α upregulation of VEGF, 483

Subject Index Mucosa-associated lymphoid tissue (MALT), 70 Multifaceted role of NO in cancer biology concentration-dependent effects of NO, 47–49 NOS expression/arginase activities, human breast cancer cells, 46f thymidine uptake in MDA-MB-231 cells, effects, 48f expression of NOS in tumors, see NOS expression in tumors NO-based cancer therapy, 49–51 NO in physiological processes, signaling pathways, 40 NO regulation of apoptotic pathways, see Apoptotic pathways, NO regulation of NO regulation of translation cyclin D1, role, 45 DETA-NONOate, protein synthesis inhibition, 46 oncogenesis maintenance/therapeutic drug design, importance, 46 Multiple myeloma (MM), 171 Myeloid-derived suppressor cells (MDSCs), 242 N Nasopharyngeal carcinoma (NPC) EBV infection, cause, 347 LMP1, role in, 348 environmental and dietary factors, risk of herbal medicine, increased risk, 348 phorbol diester, increased risk, 348 higher incidence in southern China, 347 mechanism of 8-nitroguanine formation by EBV infection, 348–349, 348f EBER/LMP1, detection, 349 IL-6, role in iNOS expression, 349 NCX-4016, 363 Neural NOS (NOS1), 149 expression, 150 mRNA transcription, regulation, 150 Neuronal nitric oxide synthase (nNOS), 23, 61, 64–65 excitotoxicity of NO to neurons, 65 expression sites of brain, 64 nitric oxide- or peroxynitrite-mediated neuronal injury, 65 NO as neurotransmitter/ neuromodulator, 64

Subject Index NOS I, Ca2+ –calmodulin-dependent enzyme calcium-dependent stimulation of, effects, 65 synaptic plasticity, 65 structure upon molecular cloning, 64 Neuronal NOS (nNOS), 40 NF-κB role in tumor metastasis, 210–211 regulation of tumor cell survival/metastasis, 211 transcriptional regulation of genes, 211 N G -monomethylarginine (NMMA), 150 acute myocardial infarction, treatment of, 151 migraine headache, inhibitor for, 151 NHEJ, see Non-homologous end joining (NHEJ) NHL, see Non-Hodgkin’s lymphoma (NHL) N -hydroxy L-arginine (NOHA), 43 N-hydroxy-N-nitrosamines as potential drugs alanosine, 369, 369f cupferron, 369, 369f dopastatin, 369, 369f N -(4-hydroxyphenyl) retinamide (4-HPR), 109 NIH-OVCAR-3, 257 Nitrates and nitrites (organic), 367f inorganic nitrite, formation, 367 nitrate tolerance chronic nitrate therapies, 367 NO donors, organic nitrates, 366 Nitrate tolerance/cross-tolerance, 367 Nitric oxide-acetyl salicylic acid (NO-ASA), 363 Nitric oxide/nitric oxide-donating agents, molecular mechanisms, 270f Nitric oxide (NO) biochemical and physiological effects p53 pathway activation in vivo, 9–10 temporal properties, 10 dichotomous property, 4–5 DNIC formation, 4 GC activation, 4 identification in endothelial cells/macrophages, 4 in mechanisms of genotoxicity, 5f nitrovasodilators, active component of, 4 NOS2 expression in tumor cells, study, 7–8 pathophysiological processes, role in, 4–5 post-therapeutic tumor regrowth, role in, 12–13

503 p53 point mutations in human cancer, study, 6–7 reactions, types direct reactions, 5 indirect reactions, 5 signaling pathways application of NOS inhibitors, 12 macrophages, role in mediation of NO functions, 10, 11 superoxide and ROS, role, 10 in vivo study “molecular signature” approach, example, 10–11 and wound healing, 8f Nitric oxide non-steroidal antiinflammatory drugs (NO-NSAIDs), 363 classic prodrugs, 364 phase II chemoprevention clinical trial for CRC, 363 use in arthritis/pain treatments/CRC, 364 Nitric oxide- or peroxynitrite-mediated neuronal injury, 65 Nitric oxide-releasing molecules (NORMs), 361–379 Nitric oxide synthase (NOS), 61–63 family, iNOS/eNOS/nNOS, 23 NOS-catalyzed oxidation of L-arginine to NO, steps, 62 prosthetic groups, 62 vs. CPR, 62 Nitrites, 4 Nitrogen dioxide, 4 8-Nitroguanine, 343f, 344 apurinic site, formation of, 344 carcinogenesis/8-oxodG, cause, 344 formation of in chronic hepatitis C patients, interferon therapy, 344 formation mechanism and tumor development, 351f formation mechanism by chronic inflammation, 343f formation mechanism by EBV infection, 348–349, 348f in liver infected with liver fluke OV, 344 in patients with cancer-prone inflammatory diseases, 344 in patients with Helicobacter pylori infection, 344 in premalignant and inflammatory diseases/OLP/leukoplakia, 344 Nitrosamine, 42 Nitrosamines, 4

504 Nitrosating agents, 367 Nitrosative stress, 5–6, 104 transition mutations, 7 Nitrosoproline, 4 Nitrosothiols (RSNOs), 371–372 Nitrosourea, 405 Nitrosylation, 31f Nitrovasodilators, 4 N -methyl-N -nitrosoanilines, see Dephostatin NMMA, see N G -monomethylarginine (NMMA) N -nitrosamines classes of, 368, 368f cysteine inhibitors, example, 368 formation of, 367 N -nitrosoamides, 367 N -nitrosocarbamates, 367 N -nitrosoureas, 367 alkylating agents, 368 NNOS, see Neuronal nitric oxide synthase (nNOS); Neuronal NOS (nNOS) NO, anti-apoptotic role in B lymphocytes addition of iNOS inhibitors, effects, 171 hydrophobic NOS inhibitors, CLL cell death, 171 NOS isoforms in leukaemia cells, detection, 171 NO, enhancer for cancer therapy clinical studies, 259–260 NO produced by tumor cells/host cells, models, 253–254 synergy of NO with cytotoxic drugs, 258–259 synergy of NO with ionizing radiations preclinical studies, 254–256 See also NO synergy with ionizing radiations synergy of NO with members of TNF family FasL and TRAIL, tumor cell killing receptors, 256 GTN, tumor cell sensitized to FasL-induced apoptosis, 257 nitrosylcobalamin, sensitization to apoptosis, 257 PAPANO sensitization of lymphoma cells, 258 SNP/TRAIL, 256–257 TNF-α-mediated cytotoxicity, NO sensitization of, 258 NO, general reactions, 149 NO, pro-adhesive effects, 191

Subject Index NO, rate-limiting factor for metastases development cancer and endothelial cells, interaction, see Interaction of cancer and endothelial cells extravasation and metastatic growth adaptive response toward higher resistance, 199–200 angiogenesis and regulation of tumor blood flow, 198–199 migration and limited survival of early micrometastases, 197–198 NO, sensitization of cancer cells to chemo-immunotherapy NO and chemotherapy, 286–287 NO and immunotherapy, 287–288 NO and radiotherapy, 285–286 NO biology, concepts iNOS, immune response regulation against tumor cells, 285 smallest pleiotropic signaling messenger, 284 NO, sensitizing effect to cytotoxic stimuli boosting anti-tumor immunity by modulation of NO-mediated immunosuppression aspirin, GM-CSF-based cancer vaccine, 245 clinical studies, 247 iNOS and COX-2 inhibitors, treatment, 246–247 NO release, anti-tumor immune responses, 245–246 chemotherapy clinical studies, 241 future directions, 241 preclinical studies, 240 cytotoxic stimuli regulation in immunotherapy, preclinical findings NO-mediated immune suppression in tumor-bearing hosts, 245 potentiation of immune-derived cytotoxicity against tumor cells, 244–245 immunotherapy dual role of NO in immune cell responses, 242–243 growth promoting effects of NO in tumor cells, 244 immune suppressive role of NO, 243–244

Subject Index regulation of T-Cell priming by NO, 243 radiotherapy clinical studies, 239 further research, 239 preclinical studies, 238–239 NO, tumor cell metastasis inhibition inhibition of metastasis by high NO concentrations anti-metastatic properties of DEATANONOate, detection, 216–217 NO derived from tumor cells synthesized by iNOS, effects, 215 NO-releasing agents/generating agents, impact, 216 NO signal modulation, treatment of tumors, 215 silencing of iNOS by anti-sense oligonucleotides, effects, 215 metastatic process, features EMT process, cause of metastasis, 209–210 transcription factors, 210 molecular mechanisms regulating metastasis NF-κB survival pathway, implication of, 210–211 RKIP in regulation of tumor metastasis, 211–213 SNAIL in regulation of tumor metastasis, 213–214 NO-mediated inhibition of EMT, molecular mechanisms of inhibition of snail in induction of RKIP, 222–223 metastasis via induction of RKIP, inhibition, 218–220 metastasis via inhibition of NF-κB, inhibition, 217–218, 218f RKIP upregulation by NO, mechanisms, 220–222 NO-mediated inhibition of NFκB/snail/RKIP loop results, 223–224 NO-releasing agents, management of tumor metastasis, 224–225 NO and acute non-lymphoid leukemia, 155–156 NO and apoptosis, 152f apoptosis, causing mechanisms, 151 bone marrow NO production CD34+ cells, role, 154

505 DETA-NO, effects on marrow formation, 153 treatment with IFN-γ and endotoxin, 153 treatment with IFN-γ and TNF-induced NOS2 mRNA, 153 drosophila study (Enikolopov), 155 mitochondrial pathway/death receptor pathway, 151–152 NOS1, modulator of nervous tissue cell apoptosis, 153 NOS1expression in mouse study (Krasnov), 155 YY1, role, 153 NO and chemotherapy chemotherapeutic agents, anti-tumor effect perspectives clean/dirty NO donors, use, 286–287 GTN, randomized phase II clinical trial study, 287 improved blood flow/better distribution of oxygen, 286 “normalization” of tumor vasculature, 287 stromal/endothelial tissues, chemotherapeutic targets bystander effect, 286 NO and immunotherapy cytokine-activated macrophages, role, 287 direct/indirect elimination of tumor cells, 287–286 direct/indirect immunotherapeutic approaches, 288 expression of apoptosis-related genes, regulation of, 288 NO donors, sensitization of tumor cells/metastatic process, 288 NO and life/death of human leukemia cells expression of NOS by leukemia/MDS cells, 156–157 macrophage-mediated cytotoxicity, research study, 148 NO and acute non-lymphoid leukemia, 155–156 NO and apoptosis, 151–153 NO and CLL, 157–160 NO and normal hematopoiesis, 153–155 NO generalities, 149 NOS inhibitors, 150–151 NO synthases NO production from NOS isoforms, 149–150 NO and normal hematopoiesis cell proliferation/differentiation, 154f

506 NO and peroxynitrite production, effects on HCC, 311f NO and radiotherapy hypoxia, prognostic factor for tumor outcomes, 285 NO as radiosensitizer, 285 NO sensitization of tumor cells to radiotherapy controversy/issues, 286 NO-ASA, see Nitric oxide-acetyl salicylic acid (NO-ASA) NO-aspirin (NO-ASA), 135 NO-based cancer therapy DETA-NO/YC1, pro- and anti-apoptotic effects, 50 gene therapy approach, 50 inhibition of NOS enzymatic activity, approach, 49 cPTIO, 49 curcumin, 49 ebselen inhibitors, 49 NO as anti-neoplastic agent, 50 post-translational modification of proteins farnesylation of Ras protein, 50 FTI, tumor cell growth inhibition, 50 mitochondria, role in apoptosis, 50–51 tumor growth and proliferation, inhibition L-arginine, use of, 49 L-NAME/D-NAME, treatment with, 49 use of KHM-4, effects, 49–49 NO-cGMP signaling pathway, 40 NO cytotoxicity, mechanism of apoptosis, denitrosylation by thioredoxins, 105 EGFR tyrosine kinase activity inhibition, 106–107 GAPDH, NO target, 105 NO downregulation of Cdc25A, 107 survivin by NO, inhibition of, 106 Yin-Yang1 apoptosis upregulation by NO, inhibition of, 106 NO donors, anti-cancer therapeutics designs for future, 472 extrinsic (Type I)/intrinsic (Type II) apoptotic pathways, 460 metastasis inhibition and mimic of other inhibitors of metastasis inhibition of EMT, 469–470 inhibition of hypoxia by NO, 470–471 NO mimics anti-angiogenic drugs, 471 multivalent-targeted sensitizing activity mimic of chemotherapeutic drugs as sensitizing agents, 464–466

Subject Index mimic of NF-κB inhibitors in tumor cell sensitization to cytotoxic drugs, 467–468 mimic of proteasome inhibitors in reversal of tumor cell resistance, 468 mimic of rituximab-induced sensitization of resistant tumor cells, 466–467 NF-κB and Raf-1/MEK1/2/ ERK1/2, anti-apoptotic pathways, 461, 462f NO, dual effects, 461 NO and cancer NO, chemo-/radio-/immunesensitizer, 463 NO, pro-tumor/anti-tumor effect, 463 NO-producing tumor cells/NSAIDs, role, 463 overexpression of NO, effects, 463 NO donors, treatment of cancer patients, 471 NO mimics radiosensitizing agents, 469 NO donors, cytotoxic effects diazeniumdiolate type, 112–113 hybrid type, 113 organic nitrate type, 111–112 S-nitrosothiol type, 112 NO donors by heme oxygenase-1, anti-apoptotic effects, 119 NO donors by survivin, anti-apoptotic effects, 120 NO (endogenous), cytotoxic effects of anti-tumor therapy macrophage activation, 107 eNOS induction in vascular cells by polyphenols, 109 Fas expression, enhancement, 108 iNOS gene transfer therapy, 108 iNOS induction in tumor cells by synthetic retinoid, 109 by Th1 and M1 cytokines, 107–108 using plant extract, 108 using statins, 109 NO (endogenous), protective effects anti-apoptotic effect of survivin, 119 Fas signal pathway, inhibition of, 118 IFN-induced apoptosis by NO, inhibition of, 119 iNOS inhibition by AMG, 118 proteasomal degradation of Bcl-2, inhibition of, 118–119 VEGF promotes iNOS expression, 118

Subject Index NO (exogenous), cytotoxic effects of cytotoxic effect of NO donors diazeniumdiolate type of NO donors, 112–113 hybrid type NO donors, 113 organic nitrate type of NO donors, 111–112 S-nitrosothiol type of NO donors, 112 diazeniumdiolates (NONOates) type of NO donors, 110–111 hybrid type of NO donors, 111 organic nitrate types of NO donors, 110 S-nitrosothiol type of NO donors, 110 NO (exogenous), protective effects anti-apoptotic effect of NO donors by heme oxygenase-1, 119 anti-apoptotic effect of NO donors by survivin, 120 inhibition of apoptosis by caspase-9 nitrosylation, 121 by the ceramide pathway, 121 by Fas signal pathway, 121 by pleiotrophin, 120 by scavenging of superoxide anions, 120 proteasomal degradation of Bcl-2, inhibition of, 120 NO expression in cancer biosynthesis of nitric oxide affinity to iron atoms, 60 eNOS, 62–64 half-life of NO in water, 59 iNOS, 65–68 nitrate and nitrite formation, 60 nNOS, 64–65 NO as EDRF, 60 NO reaction with thiols, 60 NOS isoforms, molecular cloning of, 61 inducible NOS/COX-2 interaction, target for cancer treatment, 74–75 iNOS-mediated COX-2 induction in tumor cells, signaling pathways, 75f nitric oxide, tumor cell proliferation, and apoptosis angiogenesis, brain cancer development, 69 apoptosis-inducing factor induction by cytotoxic agents, 72 colorectal tumor development, role of iNOS, 71 HNSCC patients, study, 68

507 iNOS activity in Barrett’s metaplasia and in esophageal adenocarcinoma, 70 iNOS activity in gastric adenocarcinoma, 70 iNOS activity in pancreatic cancers, 71 iNOS expression and tumor stage, correlation, 70 NO-NSAIDs, chemopreventive effects, 73, 73f NO-NSAIDs, study of Wallace’s group, 73 ovarian tumors/tumor-associated macrophages, role of iNOS in, 69 PGE2 production, NO on COX-2 activity in, 71 tumor angiogenesis/supression by iNOS activity, study, 68 NOHA, see N -hydroxy L-arginine (NOHA) NO in cancers, cytotoxic/protective activity cytotoxic (apoptotic) effects, 104f, 105f endogenous NO, see Endogenous NO, cytotoxic effects exogenous NO, see Exogenous NO, cytotoxic effects mechanism of NO cytotoxicity, see NO cytotoxicity, mechanism of nitrosative stress/S-nitrosylation, cause, 104 tumor microenvironment, see Microenvironment of tumor protective (anti-apoptotic) effects, 116f endogenous NO, see Endogenous NO, protective effects exogenous NO, see Exogenous NO, protective effects mechanism of protective (anti-apoptotic) effect of NO, see Protective effect of NO, mechanism tumor microenvironment, see Tumor microenvironment, protection from cytotoxins NO interference with SAM synthesis and DNA methylation, 320–321 synthesis of methionine/ S-adenosylmethionine and methylation reactions, 321f NO-mediated inhibition of EMT, molecular mechanisms inhibition of snail in induction of RKIP, 222–223 metastasis via induction of RKIP, inhibition, 218–220

508 NO-mediated inhibition (cont.) metastasis via inhibition of NF-κB, inhibition, 217–218, 218f RKIP upregulation by NO, mechanisms, 220–222 NO-mimetic agents chemo/radio-resistance modulation anticancer agents modulation, role in drug resistance, 269 chemotherapeutics, use in drug-resistance, 269 hypoxia, tumor therapy resistance, 269 MAPK- and PI3K signal transduction pathways, 269 radiation therapy effects on hypoxia, 268–269 drug resistance DETA/NO, 269 GTN, 269 ISDN, 269 See also NO-mimetic agents, drug resistance modulation mechanisms NO-mimetic agents, drug resistance modulation mechanisms angiogenesis and blood flow, 271–272 DNA damage and repair oxidative damage, BER pathway, 273 oxidative damage, repair mechanisms, 273 HIF-dependent genes (P-gp, VEGF), 271 hypoxia-inducing factor 1α, 269–270 NF-κB, 274 nitric oxide/nitric oxide-donating agents, molecular mechanisms, 270f oxidative stress ROS-detoxifying mechanisms, 272 p53, tumor suppressor, 273–274 NO mimics chemotherapeutic drugs as sensitizing agents FasL-induced sensitization, 464 NO-mediated inhibition of YY1, 464 TRAIL-induced apoptosis, sensitization cisplatin and carboplatin, drugs used in bladder cancer, 464–465 combination of TRAIL and adriamycin, treatment results, 465 DETANONOate, reversed resistance, 465–466 XIAP, role, 465 NO mimics NF-κB inhibitors in tumor cell sensitization to cytotoxic drugs anti-apoptotic gene products, apoptosis inhibition, 467

Subject Index DHMEQ, NF-κB inhibitor, 468 NO mimics proteasome inhibitors in reversal of tumor cell resistance, 468 NO mimics radiosensitizing agents hypoxic cell radiosensitizers, 469 nicotinamide/terapazamine, study, 469 NONOates, vasoactive complications, 469 oxygen supply, importance, 469 NO mimics rituximab-induced sensitization of resistant tumor cells sensitization to chemotherapeutic drug-induced apoptosis, 467 sensitization to FasL and TRAIL-induced apoptosis, 466–467 Non-Hodgkin’s lymphoma (NHL), 171 Non-homologous end joining (NHEJ), 273 NONOates, 369 NO-NSAIDs, see Nitric oxide non-steroidal antiinflammatory drugs (NO-NSAIDs) NO-producing nitrovasodilators, 216 NO production by irradiation-activated macrophages, 114 from nitrite, 115 NO quenchers/scavengers, 149 NO regulation of translation cyclin D1, role, 45 low/high NO stress, impact, 46 DETA-NONOate, protein synthesis inhibition, 44–46 differential NO stress, flow charts, 46f NO treatment of MDA-MB-231 cells, 45 oncogenesis maintenance/therapeutic drug design, importance, 46 pre-treatment with FTI, effects, 47 NO-releasing agents, 224–225 Normal hematopoiesis and NO, 153–155 Normalization of tumor vasculature, cytotoxic therapy, 123 NORMs, see Nitric oxide-releasing molecules (NORMs) NORMs, classes of classical organic nitrates and nitrites, 366–367 diazeniumdiolates, 369–371 diazetine dioxides, 375 furoxans and benzofuroxans, 372–375 N-hydroxy-N-nitrosamines, 368–369 nitrosothiols (RSNOs), 371–372 N-nitrosamines, 367–368 sydnonimines, 376–377 NOS, see Nitric oxide synthase (NOS) NOS, isoforms, 40

Subject Index NOS2, inducible NOS, 149 NOS3, endothelial NOS, 149 NOS/COX-2 interaction astrocytic tumorigenesis, cause, 69 target for cancer treatment, 74–75 NOS detection in CLL cells CDDO, role in apoptosis of CLL, 171–172 iNOS expression in B lymphocytes cGMP-dependent/independent mechanisms, 170 MM/NHL, histopsthologic study, 171 NO, anti-apoptotic role, 170–171 NOS dimer formation, inhibitors of, 150–151 NOS expression by leukemia/MDS cells, 156–157 NOS expression in tumors NO, anti-tumorigenic role, 41 reduced tumor growth, Nunakawa study, 41 tumor development and progression, study association with angiogenesis, 41 increased activity in lung cancer/ ulcerative colitis patients, 41 iNOS activity in breast carcinoma, 40 iNOS activity in human ovarian cancer, 40 NOS I, Ca2+ –calmodulin-dependent enzyme, 64 NOS inhibitors NMMA, cytotoxicity inhibition, 150 NOS dimer formation, inhibitors of, 150–151 NOS oxidase inhibitors, 150 NOS1-specific inhibitors, 151 NOS2-specific inhibitors, 151 NOS oxidase inhibitors, 150 NOS1-specific inhibitors, 151 study in animal models, 151 NOS2-specific inhibitors, 151 L-NIL, oral drug for asthma, 151 NO synergy with ionizing radiations preclinical studies activation of p53, effects, 255 cytotoxic synergy in human colon carcinoma cells, SNAP treated, 256 gene activation, 256 hypoxic cell radiosensitizers, 252 NO donors/releasing agents, sensitization effects, 254–255 NO synthases, 149–150 NPC, see Nasopharyngeal carcinoma (NPC)

509 O OGC1, see 8-oxoguanine glycosylase-1 (OGC1) OLP, see Oral lichen planus (OLP) Oncogenic Ras-driven cancer eNOS, role, 23–24 eNOS activation by AKT, 24 eNOS activation in pancreatic cancer, 27–28 S1177 phosphorylation of eNOS, 28f eNOS and cancer, links angiogenesis, role in, 26–27 apoptosis evasion, role, 26 phosphorylated eNOS in Ras-mediated tumor growth, 28–30, 29f model systems, validity of results, 29–30 PI3K–AKT signaling and cancer, 24–25 PI3K–AKT signaling to eNOS, 25f PI3K pathway, activation of, 25 tumorigenesis promotion by AKT, 27 role of eNOS, 30–32 ONO-1714, invasive cancer inhibitor, 334 Opisthorchis viverrini (OV), 344 Oral lichen planus (OLP), 344 Organic nitrates (R-ONO2) GDN, 366 GMN, 366 GTN, 366 ISDN, 366 ISMN, 366 PETN, 366 treatments, use in, 366–367 Organic nitrate types of NO donors, 110–112 Organic nitrites IAN, preparation, 367 protein nitrosation mediation, 367 OV, see Opisthorchis viverrini (OV) Ovarian cancer, 40, 69, 109, 113, 119–20, 199, 217, 257, 333, 371, 407t, 410, 429 OV infection, risk of cholangiocarcinoma formation of 8-nitroguanine, in vivo study double immunofluorescence staining in acute/chronic phase of inflammatory cells, 345 parasitic infection OV antibody level, severity of hepatobiliary disease/ cholangiocarcinoma, 345 TLR-mediated inflammatory responses, 345

510 OV infection (cont.) in vitro study with RAW264.7, 345 in in vivo study with OV-infected hamsters, 345 praziquantel, treatment with, 345 Oxidative–nitrosative stress, 5–6 Oxidative stress, 5–7, 272 transversions, 6–7 8-oxodG, 343–345, 347f, 350–351 8-oxoguanine glycosylase-1 (OGC1), 42 “Oxygen effect” in DNA, 404 P Paget’s theory of seed and soil, 191 PAMPs, see Pathogen-associated molecular patterns (PAMPs) Pancreatic adenocarcinoma, 26, 71, 111, 113 Partial liver resection/liver transplantation, treatments, 310 Pathogen-associated molecular patterns (PAMPs), 174 PCD, see Programmed cell death (PCD) PDGFR, see Platelet-derived growth factor receptor (PDGFR) PEA3, see Polyoma enhancer activator 3 (PEA3) PEBP, see Phosphatidylethanolamine binding protein (PEBP) PeIF2-α, see Phosphorylated eIF2-α (peIF2-α) Pentaerithrityl tetranitrate (PETN), 366 Peripheral nervous system (PNS), 64, 362 Peroxynitrite, 60, 65, 67, 74, 87, 149, 156, 242–243, 245, 267, 272–273, 295, 311f, 312, 343, 377, 387, 396, 426, 450 PETN, see Pentaerithrityl tetranitrate (PETN) PGH2, see Prostaglandin H2 (PGH2) PGs, see Prostaglandins (PGs) Phases of tumor progression metastatic phase, 295 RGP, 295 VGP, 295 PHDs, see Prolyhydroxylase (PHDs) Phorbol diester, 348 Phosphatidylethanolamine binding protein (PEBP), 211–212 affinity to phospholipids, effects, 212 characteristics, 212 gene array analysis (Fu), 212 HCNP, choline acetyltransferase synthesis, 212

Subject Index Phosphatidylinositol 3-kinase (PI3K), 24, 27, 312 Phosphorylated eIF2-α (peIF2-α), 46 PI3K, see Phosphatidylinositol 3-kinase (PI3K) PIN, see Protein inhibitor of nNOS (PIN) P1 isoform of the phase II detoxification enzyme glutathione-S-transferase (GST-P1), 371 PKR, see Protein kinase R (PKR) Platelet-derived growth factor receptor (PDGFR), 95, 210 PNS, see Peripheral nervous system (PNS) Polyoma enhancer activator 3 (PEA3), 72 Polyphenols, naturally occurring, see Quercetin (QUER); Trans-pterostilbene (t-PTER) p53 (protein 53) mutations, 7, 273–274, 331 Praziquantel, antiparasitic drug, 345 Primary melanomas, iNOS expression in IHC of iNOS in human primary melanoma, 296f malignant transformation of melanocytes, role, 295 phases of tumor progression, 295 primary cutaneous tumor samples, interim statistical analysis DFS/OS HR estimation, 296t Pro-apoptotic properties, NO NF-κB activity, inhibition of, 42 NO-induced apoptosis in breast cancer cells ERK inactivation/MKP-1 inhibition, 43 p53 accumulation, 42 Programmed cell death (PCD), 448 Pro-inflammatory cytokines, 138 Prolyhydroxylase (PHDs), 482 Prostaglandin H2 (PGH2), 316 Prostaglandins (PGs), 71–72, 90, 198, 315–316, 316f, 444 production, 316 Prostanoids, 316 Prostate cancer, role of NO NO donor, 421–422 NOS induction/inhibition, 422 Proteasomal degradation of Bcl-2, inhibition of, 120 Protective effect of NO, mechanism inhibition of ceramide pathway, 117 inhibition of Fas signal pathway, 117 inhibition of mitochondrial permeability transition pores, 117 inhibition of the caspase family, 117

Subject Index Protective effects of endogenous NO from cytotoxins anti-apoptotic effect of survivin, 119 Fas signal pathway, inhibition of, 118 IFN-induced apoptosis by NO, inhibition of, 119 iNOS inhibition by AMG, 118 proteasomal degradation of Bcl-2, inhibition of, 118–119 VEGF promotes iNOS expression, 118 Protective effects of exogenous NO from cytotoxins anti-apoptotic effect of NO donors by heme oxygenase-1, 119 anti-apoptotic effect of NO donors by survivin, 120 inhibition of apoptosis by caspase-9 nitrosylation, 121 by the ceramide pathway, 121 by Fas signal pathway, 121 by pleiotrophin, 120 by scavenging of superoxide anions, 120 proteasomal degradation of Bcl-2, inhibition of, 120 Protein inhibitor of nNOS (PIN), 150 Protein kinase R (PKR), 46, 46f Protein nitrosation, 367 Q QUER, see Quercetin (QUER) Quercetin (QUER), 109 R Rabbit aortic endothelial cells (RAECs), 26 Radial growth phase (RGP), 295 Radiation therapy, 108, 114, 139, 210, 268, 285, 335, 361 See also Radiotherapy Radio-resistance, 268–269, 273–274, 431 Radiosensitisation, cancer treatments insulin administration to tumour-bearing mice, results, 404 mice bearing liver tumours implanted into leg muscle, study, 404–405 radiation-inducible promoters, use of ADiNOS treatment, 403–404 CMV and pE9 promoters, 403 WAF1/p21 promoter, 402 single radiation doses/X-ray doses, use of “oxygen effect” in DNA, 404 of tumour cells by NO in vitro/in vivo, 398t–401t Radiosensitization, definition, 238

511 Radiotherapy clinical studies, 239 further research, aim, 239 preclinical studies bystander effect of tumor cells, 239 NO released by intratumoral macrophages, tumor effects, 238 radioresistant macrophages, role, 238 in vitro/in vivo induction of NO in tumor cells, effects, 238 Radiotherapy and NO hypoxia, prognostic factor for tumor outcomes, 285 NO as radiosensitizer, 285 NO sensitization of tumor cells to radiotherapy controversy/issues, 286 RAECs, see Rabbit aortic endothelial cells (RAECs) Raf-1 kinase inhibitor protein (RKIP), 211–213 nasopharyngeal carcinomas, invasion suppressor protein, 213 PEBP family, member of, 211 reduced expression of, tissue microarray analysis, 213 Ras signaling NO treatment of MDA-MB-231 cells, 45 pre-treatment with FTIs, 47 Reactive nitrogen species (RNS), 5, 30, 61, 112, 115, 137, 201, 243, 266, 272, 311–315, 343, 450 See also ROS and RNS, biochemistry of Receptor tyrosine kinase (RTK), 24, 25f Redox sensor proteins, 365, 379 Renal cellular carcinoma, 427 NOS induction/inhibition, 427 Resiquimod, 173 Retinoblastoma protein, 46 Ribonucleotide reductase, 60, 65, 105, 151, 152f RKIP, see Raf-1 kinase inhibitor protein (RKIP) RKIP upregulation by NO, mechanisms, 220–222 RNS, see Reactive nitrogen species (RNS) RNS, role in carcinogenesis, 310–311 RNS and hepatocarcinogenesis, production/ metabolic effects AMPK activation, effects, 312 hepatic glucose metabolism, NO effects, 313

512 RNS and hepatocarcinogenesis (cont.) mitochondrial respiratory chain/Krebs cycle/glycolysis, NO effects, 313, 314f NO-mediated DNA repair inhibition/ vasodilation, HCC cause, 312 NOS isoforms in liver, 311 Warburg effect, 313 ROS and RNS, biochemistry of Fenton reaction, 266 mitochondrial respiratory chain inhibition, effects, 267 reaction balance between ROS/RNS, 267 sites of generation, 266 thioredoxin and glutaredoxin, role, 267 ROS-detoxifying mechanisms, 272 Roswell Park Cancer Institute, 481 RTK, see Receptor tyrosine kinase (RTK) S S1177, phosphorylation of, 24, 28, 28f SAM synthesis and DNA methylation, 320–321 Sapium sebiferum, 348 SCC, see Squamous cell carcinoma (SCC) SCF, see Stem cell factor (SCF) Selenium and cancer daily Se supplements intake 200 μg/d, effcets, 481 400 μg/d, effcets, 481 daily Se supplements intake, effcets, 481 MSC optimal dose, defined, 481 Se concentrations, clinical phase I trial novelty of approach, 481 SLM/vitamin E, prevention trials, 481 Selenomethionine (SLM), 480–481 “Self cannibalism,” see Autophagic cell death Se-methylselenocysteine (MSC), 479–486 See also MSC, potential role Serine proteases, 212 SGC/cGMP/PKG, see Soluble guanylyl cyclase/cGMP/cGMP-dependent protein kinase (sGC/cGMP/PKG) SLM, see Selenomethionine (SLM) Smac DIABLO, 42, 257, 460, 465–466 SNAIL in regulation of tumor metastasis post-transcriptional regulation GSK-3β phosphorylation, 214 Snail interference in HaCa4 and CarB, 214 as transcriptional repressors E-cad transcription, 213

Subject Index zinc-finger type encoding, 213 S-nitrosothiol (SNO), 42, 60, 90, 110, 112, 156, 224 S-nitrosylation nitric oxide and cancer angiogenesis and vascular permeability, 89–90 metastasis, 90 NO, cytoprotective/tumoricidal effects, 88 phases of cancer progression, 88f proliferative and anti-apoptotic effects, 88–89 protein S-nitrosylation, tumor survival growth/survival and apoptosisregulatory proteins, 93–94 S-nitrosylation of protein targets, 91t–93t vascularization and metastatic potential, regulators of, 94–95 SNO, see S-nitrosothiol (SNO) SOD, see Superoxide dismutase (SOD) Soft tissue sarcoma, 349–350 See also Malignant fibrous histiocytoma (MFH) Soluble guanylyl cyclase/cGMP/cGMPdependent protein kinase (sGC/cGMP/PKG), 319 “Spacer” or “linker,” 445 Sphingosine 1-phosphate (S1P), 63, 121–122, 198 Squamous cell carcinoma (SCC), 7, 213, 239, 259, 298, 329–330, 481, 483, 485 See also Head and neck squamous cell carcinoma (HNSCC) Src, proto-oncoprotein, 94 Stem cell factor (SCF), 154f, 296 Stromal fibroblast iNOS, chemoresistant upregulation of tumor by, 122 Superoxide dismutase (SOD), 60, 67, 87, 110, 149, 200, 216, 267, 396, 450 Survivin, 43, 69, 106, 119–120, 217, 258, 461, 467–468 anti-apoptotic effect of, 119 Sydnonimines, 156, 376, 376f T TAAs, see Tumor-associated antigens (TAAs) TAM, see Tumor-associated macrophage (TAM) TBID, see Truncated BID (tBID) T-cell priming, 243, 246

Subject Index Terapazamine, 469 “The paradox of oxygen,” 266 Thiol proteins, oxidation of, 42 Thioredoxin, 94, 105, 178–179, 199, 267 Thioredoxin-like oxidoreductase (TRX), 91t, 94 TIMP-2, see Tissue inhibitor of matrix metalloproteinases (TIMP-2) Tissue inhibitor of matrix metalloproteinases (TIMP-2), 395 Toxoplasma gondii, 148 T-PTER, see Trans-pterostilbene (t-PTER) Transferrin–polyethyleneglycol– polyethylenimine complex, 396 Trans-pterostilbene (t-PTER), 109 Truncated BID (tBID), 43 TRX, see Thioredoxin-like oxidoreductase (TRX) Tumor-associated antigens (TAAs), 241, 288 Tumor-associated macrophage (TAM), 9, 12, 113–114, 122, 244, 301–302 Tumor cell adhesion to vasular endothelium, inhibition/promotion, 191 hepatic zonal heterogeneity, 193 Tumor cell arrest in microvessels, mechanism, 190 Tumor-infiltrating dendritic cells, 114 Tumor microenvironment, protection from cytotoxins chemoresistant upregulation of tumor by stromal fibroblast iNOS, 122 macrophage reprogramming to M2 phenotype by S1P, 121–122 MHC Class I shedding under hypoxic tumor conditions, 122 normalization of tumor vasculature, cytotoxic therapy, 123 Tumor tissues heterogeneity, 480 MSC/SLM, multi-targeted molecules, 480

513 V Vascular endothelial growth factor (VEGF), 12, 27, 41, 49, 63, 69, 72–73, 89–90, 94, 113, 118, 136, 191, 198–199, 211, 256, 271, 272–273, 298, 299–300, 313, 315, 320, 422, 426–428, 431–455, 461, 463, 471, 480, 484 Vascularization and metastatic potential, regulators of, 94–95 Vasodilation, 63, 114, 238, 253, 311, 362, 429 VEGF, see Vascular endothelial growth factor (VEGF) Vertical growth phase (VGP), 295 Vessel remodeling, 62 VGP, see Vertical growth phase (VGP) VGX-1027 vs. GIT-27NO, 445–446 W WAF-1/iNOS gene therapy, 402–403 WAF1/p21 promoter, 402 Warburg effect, 313, 314f, 315 overexpression of the AKT oncogene, cause, 313 “Warhead,” 364 Wortmannin, PI3K inhibitor, 24 Wound healing model, 9 X Xeroderma pigmentosum A (XPA), 42, 89 XIAP, see X-linked inhibitor of apoptosis (XIAP) X-linked inhibitor of apoptosis (XIAP), 258, 317, 461, 465, 467–468 XPA, see Xeroderma pigmentosum A (XPA) Y Ying-Yang 1 protein (YY1), 152f, 153, 221, 257, 274, 288, 375, 422, 451–452, 464, 466–468 YY1, see Ying-Yang 1 protein (YY1)