COX-2 Inhibitor Research

COX-2 INHIBITOR RESEARCH No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

COX-2 INHIBITOR RESEARCH

MAYNARD J. HOWARDELL EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2006 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data COX-2 inhibitor research / Maynard J. Howardell, editor. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61668-114-2 (E-Book) 1. Nonsteroidal anti-inflammatory agents. 2. Arthritis--Chemotherapy. 3. Anti-inflammatory agents. [DNLM: 1. Anti-Inflammatory Agents, Non-Steroidal--therapeutic use. 2. Cyclooxygenase 2 Inhibitors--therapeutic use. 3. Cyclooxygenase 2 Inhibitors--pharmacology. QV 95 C8767 2006] I. Howardell, Maynard J. RM405.C69 2006 615'.7--dc22 2006000899

Published by Nova Science Publishers, Inc. New York

Contents Preface

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Chapter I

Cyclooxygenase-2 Inhibitor and Gastric Cancer Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

Chapter II

Prevention and Chemoprevention of Gastric Cancer: Dietary Habits, Helicobacter Pylori and COX-2 Inhibitors Gerardo Nardone and Alba Rocco

Chapter III

Cyclooxygenases in Cancer Daniela Foderà, Nadia Lampiasi, Antonella Cusimano and Melchiorre Cervello

Chapter IV

Nephrotoxicity of Nonsteroidal Anti-Inflammatory Drugs: Focus on Selective Cyclooxygenase-2 (COX-2) Inhibitors Steven G. Coca and Mark A. Perazella

Chapter V

Chapter VI

Index

Are COX-2 Inhibitors Active on Intracellular Oxidative Processes? A Study on In Vitro and Cellular Models Ange Mouithys-Mickalad, Ginette Deby-Dupont, Carol Deby, Thierry Franck, Didier Serteyn and Maurice Lamy Theoretical Mechanism Studies on Dual Inhibition of Human Cyclooxygenase-2 and 5-Lipoxygenase by Diaryl-Pyrrolizine Derivatives R. Pouplana, C. Pérez and J. Ruiz

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209 237

Preface COX-2 Inhibitors are newly developed drugs for inflammation that selectively block the COX-2 enzyme. Blocking this enzyme impedes the production of the chemical messengers (prostaglandins) that cause the pain and swelling of arthritis inflammation. Cox-2 inhibitors are a new class of nonsteroidal anti-inflammatory drugs (NSAIDS). Because they selectively block the Cox-2 enzyme and not the Cox-1 enzyme, these drugs are uniquely different from traditional NSAIDS. This new book explores new research in this field. Cyclooxygenase-2 (COX-2) is the crucial enzyme in conversion of arachidonic acid to prostaglandins, and is inducible by various agents such as growth factors and tumor promoters. As COX-2 is frequently overexpressed in various tumors, it is being intensively evaluated as a pharmacologic target for both the prevention and treatment of cancer. The contribution of COX-2 to carcinogenesis and the malignant phenotype of tumor cells have been thought to be related to its abilities to: increase production of prostaglandins; convert procarcinogens to carcinogens; inhibit apoptosis; promote proliferation and angiogenesis; modulate inflammation and immune function as well as increase tumor cell invasiveness etc. Various possible direct mechanisms for COX-2 implication in carcinogenesis have been studied in order to pursue a target to block tumor growth. Epidemiological and experimental studies have demonstrated the effect of non-steroidal anti-inflammatory drugs (NSAIDs) in the prevention of human cancers. NSAIDs block endogenous prostaglandin synthesis through inhibition of cyclooxygenase enzymatic activity. However, the exact mechanisms that account for the anti-proliferative effects of NSAIDs in COX-2 deficient tumors are still controversial as to whether or not these effects are mediated predominantly through the inhibition of COX-2 activity and prostaglandin synthesis. Therefore, intense interest has recently been focused on COX-2-independent effects of NSAIDs. Selective COX-2 inhibitors possess more potent anti cancer effect and less side effects than traditional NSAIDs. A number of studies have investigated the relationship between COX-2 inhibitors and Helicobacter Pylori-associated gastric cancer. Moreover, a wide spectrum of studies continue to be undertaken in both laboratory and clinical settings to elucidate the mechanisms underlying these anti-tumor effects of COX-2 inhibitors, to find out new functions of COX, and to investigate the efficacy and safety of COX-2 inhibitors in the clinical application for cancer chemoprevention and therapy. Chapter I will review the various pathophysiological mechanisms and current status of COX-2 inhibitors in the prevention and treatment of gastric

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cancer and other tumors under the following headings. 1) COX-2 and gastric cancer; 2) COX-2 inhibitors and gastric cancer; 3) Mechanism underlying anti-tumorigenesis of COX-2 inhibitors; 4) COX-2 inhibitors for gastrointestinal lesion; 5) Current status and future perspective of COX-2 inhibitors for cancer. COX-2 inhibitors will play a promising role in the prevention and treatment of gastric cancer. Despite the decrease in incidence, gastric cancer remains the second leading cause of cancer-related death worldwide. Prevention is likely to be the most effective means not of only reducing the incidence but also mortality from this disease. The term chemoprevention, has been referred to the prevention of cancer using specific agents to suppress or reverse the carcinogenic process. In recent years, attention has been focused on the anticancer properties of nonsteroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori (H. pylori)eradication therapy and dietary habits. In vitro and in vivo studies show that widespread and long-term use of NSAIDs may be used in the healthy population for gastric chemoprevention. Albeit, enthusiasm has been thwarted by the potential toxic effects, i.e., risk of peptic ulcer disease. As reported in chapter II, the new NSAIDs, selective cyclo-oxygenase-2 (COX-2) inhibitors, causing less injury to the mucosa of the upper gastrointestinal tract may be a valid alternative. However, the mechanisms of the anti-tumoral action of the COX-2 inhibitors still remain to be defined and may vary from agent to agent. In vitro studies have shown a variety of COX-related mechanisms in controlling proliferation and apoptosis balance. Experimental studies are often performed with much higher pharmacological doses than those used in clinical studies. Human observational studies are prevalently of the case-control type and often suffer from inadequate sample size to avoid a type II statistical error. Furthermore, due to the high cost of these new agents, cost-effectiveness analyses must be carried out to optimize the allocation of resources. The cumulative probability of developing a lesion from birth to 80 years of age is less than 4% thus, in the general population, more than 95% of those treated prophylactically with COX-2 inhibitors will not benefit. Therefore, chemoprevention with selective COX-2 inhibitors may be a worthwhile goal only in those subjects known to be at an increased risk of gastric cancer. However, also in these subjects, fundamental aspects such as safety, efficacy, mechanisms of action, optimal treatment regimens need to be defined. Although epidemiological studies have clearly established that H. pylori infection is associated with gastric cancer, there are, so far, no definitive prospective studies showing that eradication treatment significantly reduces the development of neoplasia. Prospective studies are hampered by the long period of time elapsing between infection and cancer development. Cost-effect analyses suggest that only a subgroup of H. pylori-infected patients may present beneficial changes following eradication therapy i.e., individuals living in high risk areas, relatives of gastric cancer patients, patients with gastric atrophy or intestinal metaplasia. Diet plays an important role in the pathogenesis of gastric cancer by either increasing the risk or protecting against cancer development. Thus, a reasonable suggestion for the general population is a natural chemoprevention based on lifestyle “eat to live, not live to eat”. Chapter III reviews the features of COX enzymes, the role of expression of COX isoforms in carcinogenesis and mechanisms by which they contribute to cancer, the pharmacological properties of COX-2 selective inhibitors, the antitumor effects of COX inhibitors, and the rationale and feasibility of COX-2 inhibitors for treatment of cancer.

Preface

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Traditional (non-selective) NSAIDs cause nephrotoxicity through inhibition of cyclooxygenase (COX) activity and prostaglandin formation in the kidney. Patients with prostaglandin-dependent disease states are the group at most risk for this adverse effect. It has become apparent that the COX-2 enzyme isoform is constitutively expressed and upregulated in the human kidney during states of renal stress. COX-2 derived prostaglandins importantly modulate renal blood flow and glomerular filtration rate as well as sodium, potassium and water excretion by the kidney. As described in chapter IV, clinical renal syndromes induced by the selective COX-2 inhibitors are quite similar to those described with the traditional NSAIDs, suggesting that COX-2 derived prostaglandins are important in maintaining normal renal function. Inhibition of prostaglandins causes a reduction in renal blood flow and acute renal failure in patients with predisposing conditions. These include true volume depletion from nausea/vomiting, diarrhea and excessive diuretic therapy. Effective volume depletion from clinical disease states such as heart failure, cirrhosis, and nephrotic syndrome as well as diseases such as chronic kidney disease and renal artery stenosis also portend risk of acute renal failure from prostaglandin inhibition. Prostaglandins also modulate renal potassium excretion through stimulation of the renin-angiotensin-aldosterone system. Inhibition of prostaglandins can result in hyperkalemia when co-existent conditions such as renal failure, diabetes mellitus and therapy with certain medications (ACE inhibitors, angiotensin receptor blockers, potassium-sparing diuretics) are also present. The classic syndrome of hyporeninemic hypoaldosteronism with a type-4 renal tubular acidosis (RTA) picture (hyperkalemic metabolic acidosis) can be observed when selective COX-2 inhibitor therapy is superimposed. Inhibition of prostaglandins is associated with decreased renal sodium and water excretion and all NSAIDs, including the selective COX-2 inhibitors cause some degree of sodium retention. All patients suffer from this effect, but only patients with certain clinical conditions develop obvious edema, hypertension or heart failure. Patients with underlying hypertension (especially those on antihypertensive medications), heart disease and other saltretentive disease states (cirrhosis, nephrosis, renal failure) are at highest risk for these complications. Hypertension is a particularly important complication of these drugs as small changes in blood pressure are associated with increased cardiovascular events. Hyponatremia from impaired water excretion also complicates therapy. Less commonly, acute interstitial nephritis (with or without a glomerulopathy) has been described with these drugs. To reduce adverse renal effects from NSAIDs, including all of available the selective COX-2 inhibitors, identification of patients with renal risk should be undertaken. Defining patient risk profiles based on level of kidney function (stage of chronic kidney disease) as well as on the presence of certain co-morbidities (hypertension, heart failure, diabetes mellitus, liver disease/cirrhosis, electrolyte imbalance, old age, certain medications) is one simple approach that can be taken. Based on the renal risk, recommendations for therapy and monitoring can be utilized in a rational fashion. In the last years, there has been an increasing interest of using cyclooxygenase-2 (COX2) inhibitors to treat the inflammatory pain and chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. The beneficial effects were to avoid the secondary adverse effects such as bleeding and gastric irritation, generally observed with aspirin and conventional NSAIDs. COX-1 is constitutively expressed in most tissues and involved in the regulation of normal homeostatic functions, while COX-2 is not detected in most tissues but

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induced by inflammatory stimuli. These outcomes motivated the commercial development of selective COX-2 inhibitors. Recent data suggested that the COX-2 enzyme can be expressed within atherosclerotic lesions and could play a crucial role in various types of cancers, by the way of its activity on the ROS production, gene transcription and prostaglandin (PGE2) production. Consequently, the COX-2 enzyme has become a real target for the study of various classes of compounds and specially the possible additional properties as COX-2 inhibitors. The authors of chapter V and other groups have already investigated the pro or antioxidant profile of conventional NSAIDs and some COX-2 inhibitors. With the recent withdrawal of two compounds of the coxib’s family (rofecoxib and celecoxib), for adverse cardiovascular events, concerns regarding the safety of all COX-2 inhibitors have been raised. To answer to these concerns, different approaches were developed by studying on in vitro models, the potential inhibiting-or-stimulating activities on oxidative phenomena of new drugs with already recognized therapeutic effects. Preliminary data obtained with COX-2 inhibitors showed a moderate inhibiting effect on the intracellular oxidant processes and others a stimulating activity. New hypotheses for the treatment of inflammation are now suggested for compounds like nimesulide and its analogous, which are selective towards COX-2 with little activity on COX-1. Chapter V reports the in vitro effects of some COX-2 inhibitors, in comparison with traditional drugs (ibuprofen, diclofenac and aceclofenac), by using two cellular models: a human lung type II alveolar cell line (A549) and a human promonocyte cell line (THP-1). The direct interactions between the drugs and ROS were also investigated in cell-free systems. Chapter VI modelled the active site of the human 5-LOX on the basis of the X-ray coordinates obtained for the rabbit 15-lipoxygenase and introduced in a dynamic approach the diaryl-pyrrolizine inhibitor compounds. Also, modelled is the binding mode for these compounds in the active site of the human COX-2. The binding mode on the COX-2 proposed for 6-7-diaryl-2,3—dihydropyrrolizine derivatives compounds have been shown a major anchor point defined by residues Tyr-355, Val-523, His-90, Gln-192, and Arg-513. Another mode of interaction for Licofelone inside the COX-2 active site was the polar moiety carboxylic group lying in the proximity of Tyr-385 and Ser-530. The binding mode on 5LOX proposed for these compounds inserts the “COX fragment” deep in the cavity with the methylsulfonyl moiety at the bottom, interacting with Gln-413, Lys-423 and Asn-425. The “5-LOX part” fills the entrance of the active site interacting with Phe-421, Leu-414 and Gln363 and also forms a salt bridge with the carboxylic oxygen (licofelone) and Lys-423 and Gln-413. All of these drugs do not present a selective COX-2 inhibition and the future clinical data of compounds, such as licofelone and 6-7-diaryl-2,3-dihydropyrrolizine derivatives, could point out the interest of a balanced inhibition of the two COX isoforms, associated with the blockade of the 5-LOX pathway.

In: COX-2 Inhibitor Research Editor: Maynard J. Howardell, pp. 1-83

ISBN 1-59454-994-X © 2006 Nova Science Publishers, Inc.

Chapter I

Cyclooxygenase-2 Inhibitor and Gastric Cancer

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2

Yumin Li1, 2, Raaj K. Praseedom2, Andrew Butler2, Ligang Zhou3 and Yonghong Yang4

Department of Surgery, First Hospital, Lanzhou University, China Department of Surgery, Addenbrooke’s Hospital, University of Cambridge, UK 3 Department of Clinical Biochemistry, University of Cambridge, UK 4 Health Sciences Center, University of Oklahoma, USA

Abstract Cyclooxygenase-2 (COX-2) is the crucial enzyme in conversion of arachidonic acid to prostaglandins, and is inducible by various agents such as growth factors and tumor promoters. As COX-2 is frequently overexpressed in various tumors, it is being intensively evaluated as a pharmacologic target for both the prevention and treatment of cancer. The contribution of COX-2 to carcinogenesis and the malignant phenotype of tumor cells have been thought to be related to its abilities to: increase production of prostaglandins; convert procarcinogens to carcinogens; inhibit apoptosis; promote proliferation and angiogenesis; modulate inflammation and immune function as well as increase tumor cell invasiveness etc. Various possible direct mechanisms for COX-2 implication in carcinogenesis have been studied in order to pursue a target to block tumor growth. Epidemiological and experimental studies have demonstrated the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the prevention of human cancers. NSAIDs block endogenous prostaglandin synthesis through inhibition of cyclooxygenase enzymatic activity. However, the exact mechanisms that account for the anti-proliferative effects of NSAIDs in COX-2 deficient tumors are still controversial as to whether or not these effects are mediated predominantly through the inhibition of COX-2 activity and *

Correspondance to: Yumin Li E-mail: [email protected] (current –February, 2006) work at: Department of Surgery, BOX 202, Level E9, Addenbrook’s Hospital, Hills Road, University of Cambridge, CB2 2QQ ,UK. (After February, 2006 ) Work at: Department of Surgery, First Hospital, Lanzhou University, 1 Donggang west Road ,Lanzhou 730000, China.

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang prostaglandin synthesis. Therefore, intense interest has recently been focused on COX-2independent effects of NSAIDs. Selective COX-2 inhibitors possess more potent anti cancer effect and less side effects than traditional NSAIDs. A number of studies have investigated the relationship between COX-2 inhibitors and Helicobacter Pyloriassociated gastric cancer. Moreover, a wide spectrum of studies continue to be undertaken in both laboratory and clinical settings to elucidate the mechanisms underlying these anti-tumor effects of COX-2 inhibitors, to find out new functions of COX, and to investigate the efficacy and safety of COX-2 inhibitors in the clinical application for cancer chemoprevention and therapy. The present paper will review the various pathophysiological mechanisms and current status of COX-2 inhibitors in the prevention and treatment of gastric cancer and other tumors under the following headings. 1) COX-2 and gastric cancer; 2) COX-2 inhibitors and gastric cancer; 3) Mechanism underlying anti-tumorigenesis of COX-2 inhibitors; 4) COX-2 inhibitors for gastrointestinal lesion; 5) Current status and future perspective of COX-2 inhibitors for cancer. COX-2 inhibitors will play a promising role in the prevention and treatment of gastric cancer.

Key Words: COX-2 inhibitor, prostaglandin, gastric cancer, tumor carcinogenesis.

Introduction Gastric cancer is one of the leading causes of death from malignant tumors in the whole world. The main treatment of gastric carcinoma is surgery. However, long-term outcome of surgical treatment is often dismal. Early detection is difficult, and metastasis has been shown to be present in over half of patients at diagnosis (Chen et al., 2001). Understanding the mechanisms involved in the development and metastasis of gastric cancer could further provide insights for rationally designed new therapeutic strategies for prevention and treatment of gastric cancer (Karamouzis et al., 2004). Carcinogenesis is a multistep process of long-term accumulation of genetic and epigenetic aberrations at the molecular level; therefore, appropriate modifications may avoid formation of tumor (Moran, 2002). Effective chemoprevention agents may reduce the risk of cancer by inhibiting the initiation stage of carcinoma through induction of apoptosis or DNA repair in cells harboring mutations, or act to prevent promotion of tumor growth. Similarly, chemoprevention may entail blocking cancer progression to an invasive phenotype (Stratton and Alberts, 2002). A key issue relating to the prevention of cancer is the identification of a central molecular target which could then blocked to prevent carcinogenesis. A large number of studies conducted in experimental animal models for many human cancers, including those of lung, skin, mammary gland, urinary bladder, colon, and pancreas, have demonstrated that carcinogenesis often may be inhibited by the administration of a highly diverse group of biologic and chemical agents (Subongkot et al., 2003). In recent years, studies have shown that the discovery and elucidation of prostaglandin pathways, in particular the molecular and clinical role of cyclooxygenase (COX)-2 functions, has had important applications to neoplasms (Pruthi et al., 2003). Epidemiological evidences suggest that chronic use of aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) might be associated with a reduced risk of gastrointestinal cancers, including gastric cancer (Jiang

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and Wong, 2003; Saukkonen et al., 2003; Karamouzis and Papavassiliou, 2004), This is chiefly due to NSAIDs inhibiting COX activity, thus blocking the endogenous prostaglandin production (Karamouzis and Papavassiliou, 2004). The COX enzymatic system includes two isoenzymes, COX-1 and COX-2, which convert arachidonic acid to prostaglandins. COX-1 is constitutively expressed and synthesizes cytoprotective prostaglandins in the gastrointestinal tract. COX-2 is a crucial isoenzyme in this biochemical cascade and is induced by various oncogenic stimuli and other cytokines. It is over-expressed in human cancer cells in which it stimulates cellular division angiogenesis and inhibits apoptosis (Moran, 2002). A large volume of research data has shown that COX-2 is often upregulated in many malignant tumours, rendering it an attractive candidate target for cancer therapeutics and prevention. Many studies have demonstrated that COX-2 is associated with gastric carcinogenesis. Expression of COX-2 is elevated in gastric adenocarcinomas and is correlated with several clinicopathological parameters, including depth of invasion and lymph node metastasis. This suggests that COX-2-derived prostanoids promote aggressive behavior of adenocarcinomas of the stomach. COX-2 expression is especially prominent in intestinal-type gastric carcinoma and it is already present in dysplastic precursor lesions of this disease, which suggests that COX-2 contributes to gastric carcinogenesis already at the preinvasive stage. Such observations implicating COX-2 in many of the basic processes of tumor development have suggested that targeting COX-2 with specific inhibitors may be an effective strategy for cancer treatment (Uchida et al., 2005). Taken together these data support the efforts to initiate clinical studies to investigate the effect of COX-2 inhibitors as chemotherapeutic agents and as adjuvant treatment modalities against gastric neoplasias (Saukkonen et al., 2003). The exact mechanisms that account for the anti-proliferative effects of NSAIDs in COX-2 deficient tumors are still controversial as to whether or not these effects are mediated predominantly through the inhibition of COX-2 activity and prostaglandin synthesis. Therefore, recent studies have been focused on COX-2-independent effects of NSAIDs. In the meantime, new research has also implicated COX-2 inhibition in the prevention of Helicobacter Pylori-associated gastric cancer. Aspirin and other non elective nonsteroidal anti-inflammatory drugs have been commercially available for decades, and their ability to reduce pain and inflammation are well known (Moyad, 2001). However, these agents have a potential for adverse gastrointestinal (GI) effects such as bleeding and ulcers, particularly in the presence of risk factors such as older age, history of peptic ulcer disease, and concomitant use of corticosteroids and anticoagulants. COX-2 selective inhibitors are newer drugs launched in late 1998 (celecoxib) and mid-1999 (rofecoxib) that have a GI safety advantage over traditional NSAIDs (Jalpa et al., 2004). Recent stuay data have shown that COX-2 selective inhibitor, celecoxib, reduced the size of the adenomas in mice (Saukkonen et al., 2003). It has also been documented the ability of some of these agents also reduce a primary or secondary cardiovascular event has also been documented. There has been much interest recently in using COX-2 inhibitors along with conventional anticancer therapy based on the idea that many of the COX-2 regulated genes that contribute to tumor progression may also be determinants of tumor chemo or radiosensitivity (Uchida et al., 2005). With development of new NSAIDs and intensive research in field of COX-2 inhibition, more and more novel theories, application approaches and pronounced clinical effects have continuously emerged

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

in anti-tumor rersearch. These observations and studies of COX-2 inhibitors and gastric cancer endeavor to elucidate the mechanisms by which the COX-2 inhibitors for prevent and treat gastric cancer so that the mortality and morbidity from gastric carcinoma can be reduced.

COX 2 and Gastric Cancer 1. Prostaglandin Synthesis and COX COX, as prostaglandin endoperoxide synthase, is the key enzyme required for the conversion of arachidonic acid to prostaglandins (Turini and DuBois, 2002). Namely, Arachidonic acid metabolism by cyclooxygenase results in the generation of such eicosanoid products as prostaglandins and thromboxanes. The functions of these bioactive lipid molecules include processes such as inflammation, ovulation, mitogenesis, and differentiation. These effects result from modulation of a number of signaling pathways that control distinct physiologic activities. The first step in this pathway is the liberation of arachidonic acid from membrane phospholipids as a result of phospholipase activity. The key step in prostaglandin synthesis is believed to occur at the conversion of arachidonic acid to prostaglandin H2 (PGH2). PGH2 then serves as a substrate for terminal PG synthases— specific PGs (Harris et al., 2002; Smith and Langenbach, 2001), namely, PGH2 is the immediate substrate for a number of cell-specific prostaglandin synthesis that ultimately generate such prostanoids as PGE2, PGD2, PGF1α, prostacyclin (PGI2), as well as thromboxane A2 (TXA2). The cellular expression pattern of the synthases determines the PG repertoire produced by individual cells. Thus, if a cell expresses the PGE and PGD synthases, it is likely to produce PGE2 and PGD2 (Phipps et al., 2004). Whereas, an intricate system of talented enzymes, including phospholipases, cytochrome P450, COX, lipoxygenases (LOX) and the so-called“terminal enzymes”, i.e., those converting endoperoxides to end products, generates an array of biologically active eicosanoids from polyunsaturated fatty acids such as arachidonic and linoleic acids. At times these eicosanoids have antithetic functions ( Rigas and Kashfi,2005). There is an outline of prostaglandin sythesis in Figure1,2,8,9. Overview of prostaglandin synthesis. Arachidonic acid is metabolized by at least 3 different pathways: the cyclooxygenase pathway, the lipoxygenase pathway, and the cytochrome P-450 monooxygenase pathway. COX-2 is induced under a variety of pathologic conditions, and subsequent prostaglandin production is thought to mediate downstream effects via receptor-mediated signaling pathways(Wang et al., 2005). PG endoperoxide synthase/COX is one of the rate limiting enzymes of PG synthesis from arachidonic acids. Two COX isoforms have been identified a constitutively COX-1 and an inducible COX-2 (Tatsuguchi et al., 2000). COX-1, purified to homogeneity from bovine vesicular glands in 1976 (Williams et al., 1999), is often referred to as the constitutive cyclooxygenase, inasmuch as COX-1 mRNA and protein are present at relatively stable levels in most tissues, and produces prostaglandins involved in maintenance of the gastric mucosa, regulation of renal blood flow, and platelet aggregation (Dannenberg et al, 2001). As its cellular level of expression remain constant and as such is sometimes thought of as a

Cyclooxygenase-2 Inhibitor and Gastric Cancer

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housekeeping gene product (Smith and Langenbach, 2001; Harris et al., 2002). It is widely believed that COX-1 contributes to the production of prostaglandins that are important in normal homeostatic functions. For example, it is believed that cytoprotective prostaglandins in the gastric mucosa, such as prostacyclin, are produced predominately via COX-1 (Williams et al., 1999).

Figure 1. (Wang et al., 2005 modified)

In contrast, COX-2 can be rapidly up-regulated by microbial products and certain cytokines and classified as an “immediate-early” response gene (Phipps et al., 2004). In 1989, an inducible form of cyclooxygenase (COX-2) was identified (Simmons et al., 1989). This 70 kDa cyclooxygenase isoform was independently identified by differential screening of a phorbol ester stimulated Swiss 3T3 fibroblast cDNA library (Williams et al., 1999). COX-2 is often referred to as the inducible cyclooxygenase, because COX-2 expression COX-2 mRNA and protein are influenced by a wide range of extracellular and intracellular stimuli, including in macrophages by lipopolysaccharide (Tatsuguchi et al., 2000), in fibroblasts by platelet derived growth factor (Tatsuguchi et al., 2000), and in epithelial cells by epidermal growth factor (EGF) family peptides (Nakano et al., 1995), forskolin, IL-1, tumor necrosis factor, IFN-r retinoic acid, and endothelin (Williams et al., 1999). In many cell lines mitogenic stimulation induces the formation of prostaglandins, and increased prostaglandin levels closely coincide with a significant increase in COX-2 protein. A recent study indicated that COX-2 also has a protective function in the gastrointestinal tract (Bertolini et al., 2002; Tanaka et al., 2002; Meyer-Kirchrath et al., 2000). Other studies found a third isoform, called COX-3, which is thought to produce anti-inflammatory prostanoids (Gilroy et al., 1999; Willoughby et al., 2000). It was reported that the resolution of inflammation of coxibs and non-steroidal anti-inflammatory drugs (NSAIDs) on carrageenan-

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

induced pleurisy in rats for 48 h after the injection of the irritant. Near the time of complete resolution of inflammation, they found a second peak of increased COX-2, associated with anti-inflammatory prostaglandins (PGD2 and PGF2α) and a member of the cyclopentenone family (15-deoxy -12,14-PGJ2). They suggested that the expression of this protein may be the third isoform COX-3 (Gilroy et al., 1998). A third COX isozyme may be as a product of COX-1 gene that retains intron 1 in the mRNA (Chandrasekharan et al., 2002). Until now, there has been no evident relation between this COX-1 variant and COX-3. The effect of the postulated isoform in both inflammation and tumorigenesis is controversial and needs further investigation (Gasparini et al., 2003) (Figure 2). Recently, a large number of studies in both laboratory and clinic on COX-2 have shown that COX-2 was involved with tumorogenesis and development of tumor. Therefore, study has focused on COX-2 that is a targed gene for treating and preventing to cancer.

Figure 2. Arachidonic acid pathway

Arachidonic acid may be metabolized by either of two (or three) isoforms of the enzyme cyclooxygenase (COX). When metabolized by the COX-1 isoform, the resulting prostaglandins are active in a variety of “housekeeping” functions, including the maintenance of the gastric mucosa and homeostasis. When metabolized by the COX-2 isoform, the resulting prostaglandins are active in a number of proinflammatory functions. Increased COX-2 activity is observed in a number of tumors. The new study find that COX-2 isoform may probably contributes to tissue protection and is even constitutively expressed in healthy human stomach mucosa.

2. COX and Gastritis and Gastric Ulcer Cyclooxygenase exists in two isoenzymatic forms, COX-1 and COX-2. COX-1 appears to be constitutively expressed in many tissues and produces prostaglandins, which regulate normal cellular functions. However, COX-2 activity is induced by proinflammatory cytokines

Cyclooxygenase-2 Inhibitor and Gastric Cancer

7

and produces prostaglandins that mediate the inflammatory response and pain signaling transmission (Urban, 2000). Human gastric mucosa, however, normally expresses barely detectable level of COX-2 protein (Murata et al., 1999). COX-2 expression in the human stomach are induced by gastritis or ulceration. Recent studies have shown that COX gene expression and enzyme activity are regulated in the gastric mucosa bearing erosions or ulcers (Schmassmann et al., 1998; Takahashi et al., 1998). A study with 54 gastric ulcer (associated with H pylori infected) and 15 healthy individual has demonstrated that COX-2 was found to be strongly expression in macrophages and fibroblasts exclusively localized between granulation and necrotic tissues of and around ulcer beds in the human stomach. The output of COX-2 expressing cells was significantly higher in samples obtained from the ulcer margin than in samples from either the ulcer scar or gastritis mucosa away from the ulcer margin. Moreover, the study shown that COX-2 can up-regulate the level of interleukin 1 (an inflammatory cytokine), hepatocyte growth factor, and basic fibroblast growth factor (bFGF) etc. It suggested that COX-2 may promote angiogenesis and accelerate restitution in the ulcer bed and be involved in gastric ulcer healing (Tatsuguchi et al., 2000). The percentage of COX-2 expressing cells in a mucosa away from the ulcer margin in H.Pylori positive than in H.pylori negative subjects, the study shown that H.pylori related gastricits alone induces COX-2 protein expressing in gastric mucosa. The distribution of COX-2 immunoreactivity in gastritis is apparently different from that colitis. Therefore, the difference in the extent of inflammatory reaction of the mucosa may be the reason for differences in the intensity of COX-2 protein expression between colitis and gastritis epithelial cells. In mice (Oshima et al., 1996), the strong and persistent COX-2 expression in colonic epithelium of inflammatory bowel diseases may be involved in the increased risk of carcinogenesis in the colonic epithelium (Singer et al., 1998). Whereas, COX-2 protein would not be directly related to the risk of gastric cancer noted in patients with H.pylori related gastritis. The study also showed the expression of COX-1 immunoreactivity in lamina propria mononuclear cells, and suggested that COX-1 plays a major role in protecting the mucosa against injury in the intact stomach. The difference between the role of COX-1 and COX-2 proteins in human stomach may be related to the difference in their distribution in the gastric mucosa rather than subcellular localisa (Tatsuguchi et al., 2000). Furthermore, COX-1, but not COX-2, is expressed in the intact stomach without lesions. COX-1 is involved as local physiological mediators or modulators of gastric mucosal function (Whittle, 2000). Prostaglandins of the E and I series, PGE2 and prostacyclin, respectively, are formed by gastric mucosal tissue (Whittle, 2000). These prostanoids can inhibit gastric acid secretion; stimulate gastric bicarbonate and mucus secretion, as well as affecting sodium and chloride ionic flux across the injured mucosa. In addition, these prostanoids induce vasodilatation in the mucosal microcirculation, as well as preventing the leucocyte endothelial adhesion and vascular stasis induced by damaging agents (Whittle, 2000). Whereas, expression of COX-2 mRNA, but not that of COX-1 mRNA, is increased in gastric mucosal lesions induced experimentally in animals by intragastric administration of acidified ethanol or the ischemia reperfusion technique (Takahashi et al, 1998). In all, COX, enzymes are induced by inflammatory factors to be higher expressing in the human stomach for H.pylori infection, gastric ulcer, and gastritis.

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

3. COX-2 and Gastric Cancer Carcinogenesis is a multistep process of long-term accumulation of genetic and epigenetic aberrations at the molecular level (Karamouzis and Papavassiliou, 2004). Therefore, recently a wide spectrum of studies concentrate to tumorigenesis in order to focus on targeted tumor gene for treatment and prevention of tumor. It is well known that COX-2, which is induced by a variety of cytokines, hormones, and tumor promoter, leading to more PGs producing, has associated with colorectal cancer, and other tumor (Wu et al., 2001; Chan et al., 1999; Zimmermann et al., 1999; Jang et al., 2000). COX enzymes may not be playing simply as oncogenes in tumor development (Simmons et al., 2004). Several studies have indicated that they promote tumorigenesis. A study showed that deletion of COX-2 decreased significantly the number of intestinal tumors in Apc 716 mice (Oshima et al., 1996) (although deletion of COX-1 also attenuated tumor formation in the same mice (Chulada et al., 2000)). Virgin female transgenic mice which may overexpress the human COX-2 gene in the mammary glands demonstrated a greatly incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors (Liu et al., 2001). Deletion of COX-2 decreased significantly the number of intestinal tumors in Apc 716 mice. The clear implication from many data is that enhanced COX-2 expression is sufficient to induce mammary gland tumorigenesis (Rigas and Kashfi, 2005). Therefore, a series of studies have confirmed that COX-2 levels elevated in colorectal carcinoma, overexpression of COX-2 in colorectal cancer was associated with carcinogenesis, development (Rao et al., 2002; Oshima et al., 2001; Leahy et al., 2002). Some studies indicated that pression of COX-2 also is correlated with poor prognosis in colorectal cancer (Sheehan et al., 1999; Fosslien, 2001). But, what is COX-2 for gastric cancer? Gastric cancer is one of the most common malignancies of human beings and is one of the most frequent and lethal malignancies worldwide, and the 5-year survival rate is only about 20% (Stadtländer and Waterbor, 1999), with a 5-year mortality risk of 79% (Greenlee et al., 2001). The incidence of gastric cancer is typically high in China and as a result, more than 170 000 people die of it each year (Fu et al., 2004). In the U.S., gastrointestinal (GI) carcinoma accounts for approximately one in five malignancies. In addition, one in four cancer deaths will be secondary to tumors arising from the GI tract. Five of the top 10 fatal malignancies among men, and 3 of the top 10 among women, are GI malignancies (Greenlee et al., 2001). Approximately 22,000 new cases of gastric carcinoma will occur in the U.S. this year (Greenlee et al., 2001). The primary histologic type of gastric carcinoma is adenocarcinoma. The etiological background of stomach cancer is complex and a combination of environmental factors (diet), host factors (including H. pylori infection, partial gastrectomy and gastric adenomas) and genetic factors (hereditary non-polyposis colorectal cancer syndrome and mutations of E-cadherin) play a role in gastric carcinogenesis. In few years, a large volume of research data has shown that COX-2 is often up-regulated in gastric carcinoma (Ohno et al., 2001; Uefuji et al., 2000; Saukkonen et al., 2001; Murata et al., 1999), precancerous gastric lesions and gastric cancers contain high levels of COX-2 (Ristimaki et al., 1997; Walker, 2002). To highlight study on expression of COX-2 in gastric cancer is an intensively interesting subject, particularly, to elucidate mechanism underlying the tumorigenesis of COX-2 is

Cyclooxygenase-2 Inhibitor and Gastric Cancer

9

significant for the treatment and prevention of gastric cancinoma. COX-2 contributes to human carcinogenesis by removing excess arachidonic acid, by producing prostanoids, and by metabolizing other compounds. Under experimental conditions many of the effects of COX-2 can be facilitated by PGE2, including inhibition of apoptosis, promotion of invasion and metastasis, stimulation of angiogenesis and induction of immunosuppression (Dannenberg, 2001; Gupta and Dubois, 2001). Gastric cancer tissues release much higher levels of PGE2 when compared with nonneoplastic mucosa (Uefuji et al., 2000). The EP4 receptor is expressed on mucosal CD3 T lymphocytes in the lumenal third of the gastric mucosa, whereas EP2, EP3, and EP4 are found predominantly on epithelium lining gastric pits (Takafuji et al., 2002). Expression of COX-2 mRNA and protein is elevated in gastric adenocarcinoma and in (dysplastic) precursor lesions of this disease (Saukkonen et al., 2001, van Rees et al., 2002). Whereas, trefoil peptide TFF1 (named previously pS2) is expressed in normal epithelium of the stomach, where it protects the gastrointestinal mucus membrane from injuries, including those caused by nonsteroid anti-inflammatory drugs (Ribieras et al., 1998), So, TFF1 is a tumor suppressor gene. Trefoil factor 1 (TFF1) deficient mice develop hyperplasia of the antral glands at 3 weeks of age, and at the age of 5 months all TFF1 knockout mice have an adenoma with dysplasia in the antropyloric region . At this later time point 30% of these mice also have foci of invasive carcinoma (Lefebvre et al., 1996). It showed that COX-2 is expressed in TFF1 knockout adenomas (Saukkonen et al., 2003). The central role of PGE2 in tumorigenesis has been further emphasized using homozygous deletion of one of the PGE2 receptors EP2, which leads to reduced number and size of intestinal polyps in the Apc 716 mouse model (Sonoshita et al., 2001) and others studies have demonstrated increased invasion and metastasis in gastric cancer (Murata et al., 1999; Xue et al., 2003; Uefuji et al., 2001; Ohno et al., 2001; Uefuji et al., 2000; Han et al., 2003; Yamamoto et al., 1999; Lee et al., 2001; Leung et al., 2001). Many studies in human gastric carcinoma have demonstrated that expression of COX-2 is elevated in gastric adenocarcinomas, which is correlated with several clinicopathological parameters, including depth of invasion, metastasis of lymph node, tumor stage and implicating poorly prognosis. A study with reference to the expression of β-actin gene, COX2 mRNA level were examined in cancerous tissues and adjacent noncancerous mucosa from 33 patients by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). This study result demonstrated that the β-actin mRNA expressed constitutively in all tissues, including normal gastric mucosa and tumor tissues. COX-2 mRNA expressed in 29 0f 33(87.88%) human gastric cancer specimens, over-expression was in 26 of 33(78.79%) cases. COX-2 index in gastric carcinoma was significantly higher than that in normal mucosa. Significantly higher expression of COX-2 mRNA was also observed in patients with lymph node involvement than that in those without. Furthermore, the staging in the UICC TNM classification(1985) significantly correlated with COX-2 overexpression COX-2 index in stages III and IV was significantly higher than those in stages I and II .COX-2 index showed no correlation with patient’s age, sex, blood group, tumor location, gross typing, depth of invasion, differentiation, and the greatest tumor dimension(Xue et al., 2003). Another investigation showed that increased expression of COX-2 protein was present in the cytoplasmic region of cancer cell in gastric adenocarcinoma (33/34) as compared with that of

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

normal control group. There was a positive correlation between tumor histology or metastasis and COX-2 protein expression. COX-2 was also observed in some interstitial cells, metaplastic glandular cells and nearly all fundic glandular cells. COX-1 protein expression was present in some interstitial cells but rarely seen in the epithelial cells (Lu et al., 2002). Western-blotting used by a study with 15 gastric carcinoma tissue specimens and ac companying adjacent mucosa specimens obtained from surgical resections (Murata et al., 1999). The result showed that compared with paired non cancerous specimens, COX-2 was overexpressed in 10 of 15 carcinoma tissue specimens (66.7%). Overall, COX-2 levels in carcinoma tissue were significantly higher. Two early carcinomas (confined to the mucosa and submucosa) and 3 of 13 advanced carcinomas (extended below the submucosa into the muscular wall) had weak or similar COX-2 expression in paired tissue specimens. COX-2 overexpression in tumors significantly correlated with wall and metastasis to the lymph nodes. Furthermore, the stage grouping in the TNM classification significantly correlated with COX-2 overexpression. In contrast, COX-2 overexpression did not correlate with histopathological grading, surface size, and venous vessel invasion of the tumors. COX-1 levels were similar between paired tissues. This study concluded that COX-2 overexpression might enhance lymphatic invasion and metastasis in patients with gastric carcinoma, implicating a poor prognosis and involved in tumor TNM classification (Murata et al., 1999). The staging in the UICC TNM classification was significantly correlated with COX-2 overexpression, although several investigators (Ohno et al., 2001) reported that the COX-2 level was not associated with UICC TNM stage, but majority COX-2 may contribute to progression of tumor in human gastric adenocarcinoma. The COX-2 level in Stage III and IV was significantly higher than in Stage I and II; but the difference of COX-2 level between Stages III and IV showed no statistical significance (Xue et al., 2003). Moreover, another study with 37 gastric canceres supported that expression of COX-2 was higher in patients with metastasis of lymphoid node than without metastasis (Uefuji et al., 2001). Interestingly, COX-2 is associated with vascular endothelial cell growth factor (VEGF) in gastric cancer. An investigation showed that expressions of COX-2 and VEGF were 77.8% and 75.56% in gastric cancer , respectively. It suggested that COX-2 and VEGF were positively correlated with the growth, invasion, metastasis, and development of gastric cancer. COX-2 may induce the expression of VEGF (Li et al, 2003 ). In vitro studies have shown that over-expression of COX-2 in both gastric cells and primary gastric cancers produces high levels of PGE2, which up-regulates VEGF expression and increases angiogenesis (Leung et al., 2003) PGE2 can mediate its effects by transactivating EGFR in gastric epithelial cells and in rat gastric mucosa (Pai et al., 2002). COX-2 can induce cell growth through a JNK-AP-1 signaling pathway in vitro (Wong et al., 2004). Blocking MAPK (ERK2) kinase signaling can inhibit proliferation and growth of human gastric cancer cells in vitro (Husain et al., 2001). This is because there is a positive correlation between COX-2 expression, VEGF, and angiogenesis in human gastric adenocarcinomas (Li et al., 2003) COX-2-mediated angiogenesis provides a likely mechanism by which COX-2 promotes tumor growth and invasion (Wang et al., 2005).

Cyclooxygenase-2 Inhibitor and Gastric Cancer Table 1. COX-2 expression in gastric adenocarcinoma delineated by different authors (Saukkonen et al., 2003 modified) Method

Comments regarding COX-2 expression (other comments) 9-fold higher in cancer specimens versus controls. (COX-1 expression was not elevated in the tumors.)

Reference

NB

COX-2-positive tumors (n) 73% (11)

RT-PCR

51% (37)

Correlated with depth of invasion and size of the tumor.

Uefuji et al. 2001

1.6-fold (33)

Correlated with depth of invasion.

Ohno et al. 2001

76% (50)

Correlated with depth of invasion, size, LN metastasis, stage and intestinal type. Correlated with LN metastasis and stage.

Han et al. 2003

Correlated with LNmetastasis and stage (Expression of cPLA2 and COX-1 was not elevated in the tumors.) 4.5-fold higher in cancer specimens versus controls.

Wu et al 2004 Soydan et al. 1997

Correlated with LN metastasis and stage. Less fre- quent expression in tumors with microsatellite insta bility. Correlated with LN metastasis and stage. (COX-1 expression was elevated in 1/15 tumors.) Correlated with LN metastasis and microvessel density. (PGE2 levels correlated with COX-2 expression.) Correlated with H. pylori cagA+ infection.

Yamamoto et al. 1999

More frequent in corpus cancer than in cardia cancer. Was also detected in (dysplastic) adenomas.

Ratnasinghe et al. 1999

No correlation with prognosis. H.pylori eradication reduced COX-2 expression in patients with meta plasia. Correlated with vascular invasion, H.pylori infection and prognosis. (COX-1 did not correlate with any of the clinicopathological variables.) Correlated with depth of invasion. Less frequent expression in tumors with microsatellite instability. Trend for association with prognosis. Correlated with LN metastasis and p53 mutations. Trend for association with prognosis.

Sung et al. 2000

87.88% (33 WB

72.30% 47 73% (6) 83% (23) 70% (100)

67% (15)

74% (42)

FC

84% (32)

IHC

47% (50) 100% (104) 56% (25)

68% (71) IHC

64% (109)

49% (33)

Ristimäki et al. 1997

Xue et al. 2003

Uefuji et al. 1998

Murata et al. 1999

Uefuji et al. 2000

Guo et al. 2003

Lim et al. 2000

Chen et al. 2001

Lee et al. 2001

Leung et al. 2001

11

12

Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang Table 1. Continued

Method

COX-2-positive tumors (n) 71% (31)

43% (61)

61% (140) 73% (91) 62% (53)

61% (33) 63.8%(47) 77.78%(45)

78%(50)

Comments regarding COX-2 expression (other comments) Correlated with size, stage, metastasis and p53 immunoreactivity. (COX-1 correlated with small size and low stage.) More frequent in intestinal-type than in diffuse-type cancers. Was also detected in gastric dysplasias.

Reference

Correlated with depth of invasion. No correlation with prognosis. Correlated with p53 immunoreactivity.

Joo et al. 2002

Similar frequency of positivity in stump and conven tional carcinomas. Increased during progression from non-neoplastic epithelium to dysplasia. More frequent in intestinal-type than in diffuse-type cancers. Correlated with TNM stage, metastasis, and H.pylori infection. Correlated with the growth, invasion, metastasis and development of gastric cancer. COX-2 may induce expression of VEGF. Correlated with early stage, H.pylori infection and trace element.

van Rees et al. 2002

Rajnakova et al. 2001

Saukkonen et al. 2001

Kawabe et al. 2002

Yamagata et al.2002 Li YM et al 2003 Li et al.2004

Li YM et al.2004.

NB=Northern blot; RT-PCR=reverse transcriptase-polymerase chain reaction; WB=Western blot; FC=flow cytometry; IHC=immunohistochemistry; cPLA2=cytosolic phospholipase A2; LN=lymph node; PGE2=prostaglandin E2.

These investigations suggested that COX-2 may influence lymphatic involvement by the way of increasing tumor invasiveness in patients with gastric carcinoma. Furthermore, some studies have found that over expression of COX-2 decreased the expression of both E cadherin and the transforming growth factor-β receptor, which has been linked to enhancing tumorigenic potential and increasing tumor invasiveness (Zhang and DuBois, 2001; Rowland, 2001; Sheng et al., 1999; Shao et al., 1999). Meantime, the overexpression of the COX-2 promotes invasiveness in gastric cancer through the induction of metalloproteinase-2 and membrane-type metalloproteinase (Murata et al., 1999; Rao et al., 2002). COX-2 expression may be associated with the carcinogenesis of the intestinal type gastric cancer and, speculatively, inhibition of COX-2 might have preventative effects on the intestinal type gastric cancer (Yamagata et al., 2002). There are a lot of studies concerning COX-2 expression in gastric cancer in Table 1. We could find that normal gastric mucosa expresses COX-1, but COX-2 expression is low or below the detection limit in Table 1. Expression of COX-2 is elevated in gastric adenocarcinomas as compared to the non-neoplastic mucosa. COX-2 mRNA is expressed in 51–87.88% of the tumors as detected by Northern blot or RT-PCR analysis. Immunoblotting

Cyclooxygenase-2 Inhibitor and Gastric Cancer

13

data show that COX-2 protein levels are elevated in 67–83% of the cases. Those studies by immunohistochemistry demonstrate that COX-2 immunoreactivity can be measured in 43– 100% of the cancer tissues. Several investigators found that the COX-2 signal was localized al most exclusively to the neoplastic epithelial cells (Saukkonen et al., 2003). Others investigations found strong immunopositivity in stromal cells (Rajnakova, et al., 2001; van Rees et al., 2002). Most consistently COX-2 has been connected with depth of invasion, lymph node metastasis and ad vanced stage. Some investigators have also found a correlation with in creased size of the tumor (Rowland et al., 2001) and microvessel density (Saukkonen et al., 2003). COX-2-derived prostanoids promote aggressive behavior of adenocarcinomas of the stomach. It means that overexpression of COX-2 may induce invasive and metastatic processes that can lead to a more aggressive behavior of the disease (Saukkonen et al., 2003). Whereas, some different researches suggest that COX-2 is predominantly expressed in intestinal-type gastric carcinomas expressed in intestinal-type gastric carcinomas and in precursor lesions of this disease, and it is already present in dysplastic precursor lesions of this disease (Saukkonen et al., 2001). A study found that some non-malignant hyperplasic gastric glands that may represent premalignant lesions were stained for the COX-2 protein (Ristimaki et al., 1997). COX-2 protein expression was found in 58% (25/43) of intestinaltype carcinomas and in 44% (4/9) of definitive dysplasias, but only in one of 18 diffuse-type tumors (6%) as detected by im unohistochemistry (Saukkonen et al., 2003). The intestinal type of gastric cancer has certain precursor lesions that lead to invasive carcinoma, which include chronic atrophic gastritis, intestinal metaplasia and dysplasia. It suggests that Cox-2 contributes to gastric carcinogenesis already at the preinvasive stage (Saukkonen et al., 2003). In a word, a wide spectrum of studies have clearly shown that COX-2 protein is overexpressed in early stage and development stage of gastric cancer.

4. COX-2 and H.Pylori Infected Gastric Cancer In the human stomach, Helicobacter pylori (H.pylori) infection is associated with active gastritis and ulcer disease, and is correlated with gastric adenocarcinoma, including gastric mucosa-associated lymphoid tissue lymphoma (Blaser et al., 1995). Generally, gastric adenocarcinoma develops through multistep process from normal gastric mucosa to chronic active gastritis, to gastric atrophy and intestinal metaplasia, and finally to dysplasia and neoplasia (Nam et al., 2004). H. pylori infection is associated with a 6-fold increased risk of gastric carcinoma (Murata et al, 1999). Furthermore, On the basis of epidemiologic data, WHO/IARC classified H.pylori as group carcinogen (Nam et al., 2004).The mechanism of H.polyri for promoting development and metastasis of tumor, however, has yet to be elucidated unclearly. A large number of researches both in laboratory and clinic on H.pylori has clarified that H. pylori infection is correlated with tumorigenesis for gastric cancinoma. Whereas, debate still exists as to whether H.pylori is really carcinogen or cancer promoter, and whether eradication of H. pylori is considered as a means of prevention for gastric cancer to be

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

beneficial to people free of gastric tumors (Webb et al., 1996). Therefore, clinical study of H. pylori is intensively regarded, whose study have gained markedly progression, particularly, in study on relationship H.pylori and COX-2 with gastric cancer. A number of studies have shown that the link between H.pylori infection and gastric carcinogenesis is that H. pylori infection raised COX-2 mRNA/protein levels, and stimulated release of prostaglandin E2 in H. pylori associated premalignant and malignant gastric lesions (Tatsuguchi et al., 2000; Saukkonen et al., 2001; Xiao et al., 2001). There is strong evidence that COX-2 is causally involved in gastrointestinal cancer ( Zimmermann et al., 1999; Ristimaki et al., 1997; DuBois et al., 1996). COX-2 expression was also found in Helicobacter pylori–associated premalignant and malignant gastric lesions (Sung et al., 2000; Sheu et al., 2003). Interestingly, it has been observed that COX-2 expression was reduced following successful eradication of H pylori in this study (Wang et al., 2005). Li YM et al reported a research with 47 patients and 16 healthy control group involved in COX-2 and H.pylori (Li YM et al., 2003). In gastric cancer group samples there were tubular adenocancinoma 10, papillary adenocarcinoma 9, poorly differentiated adenocarcinoma 23, muc-adenocarcinoma 3,signet-ring cell carcinoma 2,having metastasis in lymph node 31, and according to TNM stage: stage14, stage 7, stage 16, stage 10, and according to location of cancinoma ; cardia-fundus 9,corpus 13, antrum 25. Regard as control group 16 were originated apart from tumor tissues 5 cm of distance .The investigation shown that normal gastric tissues had no expression of COX-2, whereas , rate of expression of COX-2 in gastric tumor tissues was 63.8%, which were mainly expressed in tumor cells, vascularendotheliocyte surrounding tumor,and small amounts of expression in fibrocyte. Furthermore, the expression of COX-2 were correlated with TNM stage , metastasis in lymphoid node,but there were no statistically significance with age, sex, location of tumor ,as well as histological types. Interestingly, positive rate of H. pylori infection in gastric cancer tissues was 61.75(29/47), but expression of COX-2 in gastric cancer with positive outcome of H. pylori stained by Giemsa was 72.4 %(21/29), which was statistically significance compared to negative outcome of H.pylori stained by Gimsa. It suggested there are interaction between COX-2 and H .pylori in gastric acncer (Li YM et al., 2003). In order to elucidate the role of COX-2 in gastric carcinogenesis, thirty-three early gastric cancers obtained from 30 patients infected with H.pylori were studied (Yamagata et al., 2002). It suggested that COX-2 expression may be associated with the carcinogenesis of the intestinal type gastric cancer and, speculatively, inhibition of COX-2 might have preventative effects on the intestinal type gastric cancer (Yamagata et al., 2002). In early gastric cancer and in intestinal metaplasia the expression of COX-2 in patients infected by H.pylori is increased in intestinal type compared to diffuse type gastric cancer and in intestinal metaplasia. In tumours of mixed type, COX-2 is also increased in the intestinal component compared to the diffuse component (Walker, 2002). The relationship COX-2 and H.pylori in gastric carcinogenesis was reported by a research involved with trace elements (Li YM et al., 2004). The study investigated 50 blood samples from the gastric cancer patients, as a control with another 50 blood samples from healthy volunteers .This study detected the level of trace elements, the rate of H.pylori infection, and the expression of COX-2 in gastric tissue. The results showed the levels of Cu/Zn, Fe in the serum of the gastric cancer group were higher than those of the control

Cyclooxygenase-2 Inhibitor and Gastric Cancer

15

group respectively, and the levels of Zn, Mn were lower than those of the control group respectively. The data on Zn were submitted to multi-variatenon-conditional logistic analysis, it was markedly statistically significance. The rate of H.pylori infection and the positive expression of COX-2 were 88% and 78% in the gastric cancer group, respectively, 42.0% and 0 in the control group. These findings suggest that the decrease of Zn in serum may be a precancerous factor of gastric cancer development which induces H.pylori infection and the higher expression of COX-2 and hence may lead to the development of gastric cancer. Detecting the trace elements could enhance the diagnosis ratio of gastric cancer. Regulating the level of trace element in the patient can be an effective chemoprevention for gastric cancer. H.pylori may promote and up-regulate COX-2 expression in stomach hence contribute to cancinogenesis (Table 2, 3). Table 2. Comparison of H.pylori, COX-2 between healthy people and gastric cancer patients

Group (n) Control Research P value

HP infection case positive negative + 50 21 50 44 0.01

COX-2 expression rate

%

29 6

42 88

positive + 0 39

negative 50 11 0.01

rate

% 0 78

Table 3. Relationship between COX-2 expression and H.pylori infection in gastric cancer

COX-2 expression P value

Positive (+) negative (-)

H.pylori infection Positive (+) Negative (-) 38 1 6 5 0.01

H.pylori is one of important initiator to gastric carcinogenesis, H.pylori infection may cause activation of proto-oncogene and anti-oncogene inactivation,particularly which is prone to lead to mutation at site 12 in C-Ha-ras, oncogene, activation of C-Ha-ras is intimately associated with carcinogenesis, also , correlated with C-erbB-2,APC,DCC,Cmet,Bcl-2,P53,K-ras (Akhtr et al., 2001) and INOS to promote tumorigenesis (Wison et al., 1996). Although H.pylori is regarded as an important carcinogenesis factor, what is relationship between H.pylori and COX-2 in gastric cancer? There is apparently dependablity from Table 3, which are 38 cases of positive outcome and 5 cases of negative outcome both COX-2 and H.pylori in gastric cancer, 6 cases with positive H.pylori infection and negative COX-2 expression, 1 case with negative H.pylori infection and positive COX-2 expression (P
16

Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

2002). Whereas, H.pylori produces vacuolus–toxin, VacA and cytotoxin associated protein cagA, which were correlated with severely inflammatory reaction in gastric epithelial tissues, moreover, produced a series of cytokines, such as interleukin 8, lead to remarkable inflammatory reaction due to its stronger neutrophil chemotaxis. In addition, H.pylori may induce production of nitric oxide (NO) through to draw assistance from INOS (Jackson et al., 2000). Described above the factors facilitate over-expression of COX-2, which mediate inflammatory process and stimulate carcinogenesis and development of gastric carcer. H.pylori increased release of prostaglandin E2 and COX-2, which was over-expressed in most metaplastic and adenomatous tissues, as well as in gastric adenocarcinoma (Ristimaki et al., 1997; Lim et al., 2000). H.pylori up-regulated COX-mRNA expression and stimulated release of prostaglandin E2 in gastric cancer cell line (Lim et al., 2000) as well as in the gastric mucosa of animal models and in humans (Xiao et al., 2001). Overexpression of COX-2 was property shared by both intestinal and diffuse gastric carcinomas. It seems that COX-2 might play an important role during the early stage of gastric carcinogenesis (Romano et al., 1998). H.pylori infection is a major risk factor for gastric carcinoma. Therefore, high levels of H.pylori infection might up-regulate COXexpression, which, in turn, could lead to the development of gastric carcinogenesis (Lim et al., 2000). Experimentally, COX-2 was directly involved in hyperplastic changes in mice infected with H. pylori (Xiao et al., 2001). Pharmacological inhibition of COX-2 overexpression may be useful against H.pylori associated gastric cancer development and progression (Nam et al., 2004). In fact, Helicobacter pylori may play a role of “two edges sword” for gastric tumorigenesis , It means that H.pylori not only cause carcinogenesis as a tumor initiator, but also it may up-regulate expression of COX-2 to lead to carcinoma( Figure 3).

Figure 3. Intereaction of H.pylori, COX-2 and trace element in gastric cancer

Cyclooxygenase-2 Inhibitor and Gastric Cancer

17

5. Mechanism Underlying COX-2 Caused Gastric Cancer Although there are a number of studies for mechanisms of COX-2 promoting gastric carcinogenesis in a few years, but in order to elucidate the exact mechanisms of COX-2 inducing carcinogenesis present paper will have generally discussion combinated with research of other tumors. It is clear that COX-2 plays an important role in tumor and endothelial cell biology (Gately and Kerbel, 2003). This is because COX-2, a key isoenzyme in conversion of arachidonic acid to prostaglandins, is inducible by various agents inflammatory factors and tumor promoters, such as growth factors (epidermal growth factor(EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)) and cytokines (tumour necrosis factor α (TNFα), interleukins 1α and 1β) (Gasparini et al., 2003). Certain mutations COX-2 (eg, v-src, v-Ha-ras, HER-2/neu, and Wnt) also up-regulate (Araki et al., 2003; Vadlamudi et al., 1999). Moreove, there are lots COX-2 associated with tumorigenesis genes, thus, inactivation of tumor suppressor genes and activation of oncogenes (for example, amplification of HER-2) may induce COX-2 expression in human gastric adenocarcinomas (Saukkonen et al., 2003). The contribution of COX-2 to carcinogenesis and the malignant phenotype of tumor cells has been thought to be related to its abilities to: increase production of prostaglandins; convert procarcinogens to carcinogens; promote angiogesis; inhibit apoptosis; modulate inflammation and immune function; and increase tumor cell invasiveness (Gasparini et al., 2003), although some studies indicated that NSAIDs have COX-2-independent effects (Xu, 2002).

Figure 4 (Gasparini et al., 2003 modified). The pathways that stimulate tumour growth through COX-2 and the mechanisms of action of coxibs

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

5.1. Mechanisms of COX-2 Regulation There are several pathways for regulation of COX-2 in human cancers, which is indicated in Figure 5 (Gasparini et al., 2003 modified).

Figure 5. (Gasparini et al., 2003 modified)

Increased expression of COX-2 in human cancers is likely to occur via several pathways: mitogen-activated protein kinases (MAPKs), protein kinase Cζ(PKCζ), c-Jun N-terminal kinase (JNK), p38, and protein kinase A (PKA), that induce cAMP synthesis and activation of NF-κB and NF-IL6, as well as the CRE promoter site. COX2 gene transcription is induced through NF-κB by an extracellular signal related kinase (ERK2), p38, and JNK, through NFIL6 via p38, and through CRE via ERK2 and JNK pathways. PKCζ seems to mediate COX-2 transcription through all the three promoter sites. COX-2 is transcriptionally downregulated by APC and upregulated by c-Myb, and nuclear accumulation of β-catenin, through the Wntsignalling pathway, in human colon and liver carcinogenesis, whereas K-ras induces COX2 mRNA stabilisation. DR, death receptor; FADD, Fas-associated death domain protein (Gasparini et al., 2003). The COX-2 gene has been shown to be induced in p53-defective cells and down-regulated by wild-type p53 (Subbaramaiah et al., 1999). Therefore, inactivation of tumor suppressor genes and activation of oncogenes (for example, amplification of HER-2) may induce COX-2 expression in human gastric adenocarcinomas (Saukkonen et al., 2003). 5.2. Angiogenesis Several studies have shown a relation between angiogenesis and COX-2 expression (Gasparini et al., 2003). Actually, to neoplastic epithelium, COX-2 is highly expressed in the tumor microvasculature in a wide variety of human tumors (Fosslien et al., 2000; Masferrer et al., 2000). Increased expression of COX-2 occurs in multiple cells within the tumor microenvironment that can impact on angiogenesis (Crosby et al., 2003). COX-2 plays a key role in the release and activity of proangiogenic proteins. COX-2 induces proangiogenic factors such as VEGF, inducible nitric oxide synthase, interleukins 6 and 8, and TIE2 (angiopoietins 1-2 receptor) (Dannenberg et al., 2001; Tsujii et al., 1998), COX-2 results in

Cyclooxygenase-2 Inhibitor and Gastric Cancer

19

the production of eicosanoid products TXA2, PGI2, PGE2 that directly stimulate endothelial cell migration and angiogenesis in vivo. COX-2 produces prostaglandins that have both autocrine and paracrine effects on proliferation and migration of endothelial cells in vitro.and is overexpressed in “activated” tumour endothelial cells, and whereas COX-1 is expressed in normal endothelial cells (Leahy et al., 2002). In endothelial cells in vitro, FGF2 and VEGF increased by three to five times the synthesis of thromboxane A2 and increased the migration activity of the cells (Gasparini et al., 2003). Thromboxane A2 induces migration of endothelial cells and FGF-induced corneal angiogenesis; All results in enhanced tumor cell, and possibly, vascular endothelial cell survival by upregulation of the antiapoptotic proteins Bcl-2 and/or activation of PI3K-Akt (Gately and Kerbel, 2003). Another important mechanism is to promote angiogenesis due to increased expression of COX-2 occurs in multiple cells within the tumor microenvironment that can impact on angiogenesis (Gately and Kerbel, 2003). A significant association of COX-2 with tumour vascularisation, microvessel density, and VEGF has been reported in human head and neck cancer as well as gastric adenocarcinoma (Uefuji et al., 2000; Leahy et al., 2002). COX-2-derived PGE2 may also support tumor growth through angiogenic endothelial cell growth and blood vessel formation (Ben et al., 2002). COX-2 plays a key role in the release and activity of proangiogenic proteins; it results in the production of eicosanoid products TXA2, PGI2, PGE2 that directly stimulate endothelial cell migration and angiogenesis in vivo, and results in enhanced tumor cell, and possibly, vascular endothelial cell survival by upregulation of the antiapoptotic proteins Bcl-2 and/or activation of PI3K-Akt (Crosby et al., 2003). Angiogenesis of COX-2 for gastric cancer was delineated in Figure 6 (Gasparini et al., 2003 modified)

Figure 6. (Gasparini et al., 2003 modified)

20

Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

COX-2 is overexpressed in several cell types, such as macrophages, synoviocytes, fibroblasts, osteoblasts, tumour endothelial cells, and “activated” endothelial cells, it may contribute to tumour growth through several mechanisms: COX-2-dependent -prostaglandins can stimulate intracellular receptors (intracrine mechanism), self-prostaglandin membrane receptors (autocrine mechanism), and prostaglandin membrane receptors of different cells, such as endothelial cells, with proangiogenic effects (paracrine or landscaping effect) (Gasparini et al., 2003). 5.3. Stimulation of Proliferation Tumor growth is dependent on disrupting the normal balance of cell proliferation and apoptosis (Wang et al., 2005). COX-2-derived PGE2 in colorectal car cinoma cells stimulates cell proliferation through transactivation of the epidermal growth factor receptor (EGFR) (Yoshimoto et al. 2002). And, it has been demonstrated that the EGFR signaling pathway is involved in many different types of cancer (Kelloff et al., 1996). HER-2/neu over-expression appears to be correlated with colorectal carcinogenesis in certain situations and is associated with approximately one fourth of all gastrointestinal malignancies (Ross and McKenna, 2001) blocking both the COX-2 and EGF-like HER-2/neu pathways synergistically reduced tumor xenograft growth in HCA-cells (Mann et al., 2001). 5.4. Aromatase Modulation COX-2-derived PGE2 modulates aromatase. PGE2 stimulates aromatase transcription, leading to increased concentrations of oestrogens (Harris et al., 1999). Coexpression of cytochrome P450 enzyme aromatase (CYP 19) and COX-2 in human breast cancer, with a significant association with gene expression of both (Figure 1, Figure 9). Hereby, COX-2 may be the cause of progression of oestrogen-dependent breast cancer by autocrine and oestrogen-dependent breast cancer by autocrine and proliferation, or by indirect upregulation of aromatase activity (Gasparini et al., 2003). 5.5. Enhanced Tumour Cell Invasiveness Distant metastases are the major cause of death for tumor patients. In general, metastases are depended on the primary tumor stage; liver metastases can occur in 20% to 70% of patients and lung metastases in 10% to 20% (Wang et al., 2005). COX-2 may contribute to the growth of metastatic cells by autocrine, paracrine, or intracrine mechanisms, or a combination: PGE2 stimulates osteoblasts and COX-2 is over-expressed in bone neovasculature (Prescott, 2000). Migration is an essential step in tumor cell invasion and metastasis. In vitro studies demonstrated that over-expression of COX-2 promotes colorectal tumor cell invasiveness (Tsujii et al., 1997). The mechanism probably is that PGE2 has been shown to transactivate the EGFR in CaCo-colon cancer cells based on probed the regulation of cell motility downstream of COX-2-derived PGE2 (Pai et al., 2002) LS-174T CRC cell migration through an EP4-EGFR PI3K-Akt pathway (Buchanan et al., 2003). It is suggested that PGE2 may trigger growth factor receptor-mediated signaling pathways due to expression of EGFR directly associates with the ability of human CRC cells to metastasize to the liver (Wang et al., 2005). Moreover, Tumor cell invasiveness was associated with lower tumour

Cyclooxygenase-2 Inhibitor and Gastric Cancer

21

concentrations of β-catenin, cyclin D1, VEGF, MMP2, MMP9, and interleukin 10, but higher amounts of interleukin 12, which is a naturally occurring inhibitor of angiogenesis (Crosby and DuBois, 2003). PGE2 and PGI2 are involved in activation of the small GTPases CDC 42 and RAC, as the result of engagement of integrin ανβ3 with its substrate. NSAIDs suppress integrin ανβ3 dependent activation of the small GTPases and inhibit endothelial cell spreading and migration in vitro as well as FGF2-induced angiogenesis in vivo (Gasparini et al., 2003). 5.6. Apoptosis Delayed cell survival through inhibition of apoptosis can promote the accumulation of successive genetic mutations, and facilitate tumor progression. Over-expression of COX-2 in a transformed rat epithelial cell line was found to decrease the rate of cell apoptosis (Shaheen et al., 2002). In sporadic adenomas, colonic carcinomas and the colorectal mucosa of FAP patients have been found reduction in the rate of cells undergoing apoptosis (Bedi et al., 1995). In 50% of adenomas and 90% of sporadic colon carcinomas were found COX-2 overexpression. Increased expression of COX-2 in rat intestinal epithelial (RIE) cells promoted their resistance to butyrate induced apoptosis and result in increased Bcl-2 protein expression (Wang et al., 2005). COX-2-derived PGE2 may increase resistance to programmed cell death (Wang et al., 2005). PGE2 may regulate programmed cell death and reduce the basal apoptotic rate by increasing levels of antiapoptotic proteins such as Bcl-2 (Sheng et al., 1998) or other members of the Bcl gene family such as Mcl-1. COX-2-derived PGE2 inhibits apoptosis induced by nonselective NSAIDs and the COX-2 selective inhibitor SC-58125 in human colon carcinoma cells (Sheng et al., 1998). PGE2 may involve induction of NFκВ(p65/Rel A) transcriptional activity( an antiapoptotic signaling pathway) (Poligone and Baldwin, 2001). Elevated COX-2 expression and high PGE2 production may increase resistance to therapy by giving cells survival advantage. Molecular evidence that COX-2derived PGE2 regulates the apoptotic rate of tumor cells supports that the COX-2 pathway plays key role in preventing apoptosis in CRCs (Wang et al., 2005). 5.7. Modulation of Immunosuppression Tumor growth can be modulated by host immune response, and prostanoids have been shown to act as immunosuppressive agents (Dannenberg et al., 2001). COX-2 inhibitor may regulate immunity for anti-tumor. It was founded that in all celecoxib-treated adenomas, heavy infiltration of chronic inflammatory cells were observed and it is possible that this inflammatory reaction was induced by the drug therapy (Saukkonen et al., 2003). In general, immune responses to tumor divide into cell-mediated immunity (CMI) and humoral immunity (HI). Expression of COX-2 in dendritic cells (DCs) suppresses important DC functions; DCs serve as professional antigen-presenting cells and are important for appropriate activation of the host immune response to tumor antigens. PGE2 suppresses DC differentiation and function through EP2 receptor signaling, which leads to diminished CMI mediated antitumor immune responses in vivo (Sharma et al., 2003; Yang et al., 2003). Mechanistically these immune response can depend on the cytokine microenvironment, since COX-2-derived prostanoids can alter release of IL-10 and IL-12 from lymphocytes and macrophages, resulting in repression of host immunity (Stolina et al., 2000). COX-2-derived PGE2 has been demonstrated to inhibite antitumor activity of natural killer cells and

22

Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

macrophages; Also, PGE2 affects the ability of lymphocytes to produce antitumor Th1 cytokines (TNF-α, IFN-r and IL-2). Whereas, PGE2 up-regulates the production of Th2 cytokines: IL-4 in keratinocytes, IL-10 in peripheral blood lymphocytes and macrophages, and IL-6 in lymphocytes. The shift to Th2-dominant response from Th1 in malignant disease results in the down-regulation and inhibition of cell-mediated antitumor immune responses. Therefore, PGE2 on the immune system may enable neoplastic cells to evade attack (Wang et al., 2005). 5.8. Increased Mutagenesis The cyclooxygenase enzymes may be mediated by peroxidase activity through oxidation of variety of procarcinogens. Some of these oxidized substrates are carcinogenic and lead to the production of mutagens. Mutagens malondialdehyde can be produced as side products of the prostaglandin biosynthetic pathway (Figure 1) (Wang et al., 2005).

COX-2 Inhibitor and Gastric Cancer 1. Development and Pharmacology of COX-2 Inhibitor In 1971, John Vane reported the seminal observation that two different nonsteroidal antiinflammatory drugs (NSAIDs), sodium salicylate and indomethacin, inhibited prostaglandin production and that prostaglandin blockade likely accounted for their therapeutic effects (Williams et al., 1999). NSAIDs are compounds with a nonsteroid-like structure that possess one or more of several anti-inflammatory properties such as analgesia, anti-pyretic, and edema-reducing effects. Long-term use of aspirin and other NSAIDs has shown to reduce the risk of cancer of the colon and other gastrointestinal organs as well as of cancer of the breast, prostate, lung, and skin. Mammary gland, urinary bladder, and pancreas (Gasparini et al., 2003; Shaheen et al., 2002; Rao and Reddy, 2004; Eschwege et al., 2001; Lynch, 2001; Anderson et al., 2003; Crosby and DuBois, 2003; Evans and Kar , 2004; Moyad, 2001; Lipsky, 1999; Stratton, 1999; Tustin, 1999; Subongkot et al., 2003; Jacoby et al., 2000; Raz, 2002). Several epidemiological, clinical and experimental studies established NSAIDs as promising cancer chemopreventive agents. NSAIDs nonselectively inhibit both the constitutive COX-1 associated with side effects and the desired therapeutic target COX-2, which is induced in inflammation and neoplasia. Whereas, recent year many COX-2 selective inhibitors have become so successful within the same year of their launch attests to the perceived need for novel agents that can control the signs and symptoms of inflammatory diseases, but with minimal risk of gastrointestinal side effects (Whittle, 2000). Because NSAIDs have proven efficacy in treating arthritis and pain yet can also cause deleterious side effects, a major goal of the pharmaceutical industry was to design an anti-inflammatory drug with a wider therapeutic window that lacked the serious side effects of non-selective NSAIDs (Marco et al., 2002). In fact, the development of the scientific rationale for the efficacy and safety of NSAIDs and COX-2 inhibitors covers some 30 years, since beginning with the identification of cyclooxygenase inhibition by aspirin and the other classical non-steroidal anti-inflammatory

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drugs, as a mechanism of both their anti-inflammator analgesic actions and their side effects on the gut (Whittle, 2000). This was followed in 10 years later, it was identified that site selective inhibition of COX was a rational basis for the development of less gut injurious anti-inflammatory agents (Whittle, 2000). In early 1990s found the molecular identification and current focus for selective inhibitors of the COX-2 isoform is based (Whittle, 2000). A decade later, the initial regulatory approval of celecoxib (Celebrex; Searle), the first specially designed selective COX-2 inhibitor, was granted in the USA, followed rapidly by its launch onto the market in early 1999. It has subsequently obtained marketing authorization in many different territories, with approval in the UK in May 2000. Another selective COX-2 inhibitor, rofecoxib (Vioxx; Merck), was approved and launched in the USA, and was similarly approved in the UK in June 1999 (Whittle, 2000). A number of assay systems have developed to evaluate the ability of NSAIDs to inhibit COX. These include purified enzyme assays, microsomal membrane preparations from cells expressing COXs, disrupted cells, whole cells, and whole blood, to name a few. The selectivity for each isoform can be expressed as the COX-2/COX-1 IC50 ratio. Inasmuch as the absolute IC50's and selectivity ratios will vary from assay to assay, it is impossible to directly compare IC50's. However, rank order of selective inhibitors can be compared in different models (Williams et al., 1999). In general, there are 4 kinds of cox-2 inhibitors according to different selective ability to COX:1).nonselective COX inhibitor, such as Indomethacin, Sulindac, Ketoprofen; 2). COX-1 specific inhibitors, such as Aspirin, SC-560; 3). Selective COX-2 inhibitors, such as NS398, Aceclofenac etc; 4). COX-2 specific inhibitors, such as Celecoxb, Refecoxib, Nimesulide, Dup697, CGP28238, etc. However, other three classes of COX inhibitors have been developed: aspirin; indomethacin and other NSAIDs; and coxibs (Celecoxib, Rofecoxib, Valdecoxib, Etoricoxib, etc, (FitzGerald and Patrono, 2001). Selectivity for COX-2 may be evaluated by whole blood assays in vitro or in vivo based on the production of thromboxane B2 during blood clotting (an index of platelet COX-1 activity) and the production of PGE2 by bacterial lipopolysaccharide in whole blood (an index of monocyte COX-2 activity). Genetic variability in the target or metabolizing enzymes, drug interactions, and the clinical characteristics of the patient can affect both efficacy and toxicity of coxibs (Gasparini et al., 2003). The selectivity depends on plasma drug concentrations. Celecoxib is oxidised by cytochrome P450 2C9, 3A4 and interacts with inhibitors of this enzyme and warfarin, resulting in higher drug plasma concentrations and possible hemorrhagic events (Gasparini et al., 2003). On the basis of results of phase III studies, the FDA approved celecoxib for the treatment of osteoarthritis and rheumatoid arthritis and rofecoxib for osteoarthritis and acute musculoskeletal pain. The Vioxx Gastrointestinal Outcomes Research (VIGOR) (Gasparini et al., 2003) and the Celecoxib Long-term Arthritis Safety Study (CLASS) assessed the safety and efficacy of coxibs for clinlc (Gasparini et al., 2003). VIGOR study demonstrated absolute and relative reduction of the risk of gastrointestinal tract ulcer or bleeding (2.4% and 50%, respectively) in the rofecoxib group (Gasparini et al., 2003). Whereas, that study showed a higher rate of cardiovascular events in patients treated with rofecoxib (Gasparini et al., 2003). A number of studies have shown that the COX-2 gene is over-expressed in reflux oesophagitis, Barrett's oesophagus, gastric and colon cancer, familial adenomatous polyposis, pancreatic cancer, hepatocellular carcinoma, hepatotoxicity, cirrhosis, and inflammatory

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

bowel disease. NSAIDs are the most widely used therapeutic agents in the treatment of pain, inflammation and fever. They also have a role in the management of cancer prevention, Alzheimer's disease and prophylaxis against cardiovascular disease. These drugs act primarily by inhibiting cyclooxygenase enzyme, which has two isoforms, COX-1 and COX2. NSAIDs are sometimes stated that all “traditional” NSAIDs inhibit both COX-1 and COX2. However, this generalization is not accurate since there are several compounds that are classified and as NSAIDs yet do not inhibit either of the COX enzames (sulindac sulfone and salicylic acid are two prime example).It is therefore imperative to divide them. When evaluating the cellular mechanisms mediating the anti-neoplastic of NSAIDs (Raz, 2002). NSAIDs have been postulated to inhibit carcinogeneisi by both COX-2 dependent and COX2-independent mechanism (Gupta and DuBois, 2000; DuBois, 2000). It is more important that specific COX-2 inhibitors have been tried experimentally and clinically and found effective. Here a Medline search from January 1980 to January 2002, with English-language experimental studies and controlled clinical trials were performed and relevant citations were noted (Jos Grover et al., 2003). It is indicated that eview of available literature shows 1). Sulindac and COX-2 inhibitors are effective in preventing as well as regressing familial adenomatous polyposis. Whereas, they have not been demonstrated to prevent cancer in these patients. 2). NSAIDs and COX-2 inhibitors in carcinogen-induced and genetically manipulated animal models of various cancers have been promising especially in conditions such as Barrett's oesophagus, oesophageal and hepatocellular carcinoma and pancreatic cancer.3). COX-2 inhibitors may be of value in the treatment of reflux oesophagitis, pancreatitis and hepatitis, although carefully planned randomized controlled clinical trials demonstrating their efficacy need to be conducted. 4) Currently, NSAIDs and COX-2 inhibitors cannot be recommended for average-risk individuals or for those with sporadic colorectal neoplasia (or other forms of cancers) as chemo-preventive agents.5).COX-2 inhibitors may open up a new therapeutic era in which these drugs can be used for chemo-prophylaxis.6). COX-2 selective inhibitors retain renal adverse effects of the non-selective inhibitors and the concern regarding the pro-thrombotic potential of COX-2 inhibitors will limit their value as chemo-preventive agents (Jos Grover et al., 2003). Importantly, selective COX-2 inhibitors do not inhibit platelet function and cause fewer gastrointestinal side effects (peptic ulcer disease) than traditional nonsteroidal antiinflammatory drugs. Selective COX-2 inhibitors provide potent anti-inflammatory and analgesic effects without the side effects of gastric and renal toxicity and inhibition of platelet function. For example, Celecoxib is a potent COX-2 inhibitor being developed for the treatment of rheumatoid arthritis and osteoarthritis (Kısmet et al., 2004). Two specific COX-2 inhibitors, namely, rofecoxib (Vioxx) and celecoxib (Celebrex), both oral agents and FDA approved, have been shown preclinically and clinically to have efficacy comparable to that of NSAIDs for relief of pain and inflammation in osteoarthritis, with decreased risk of gastrointestinal damage. Significant preclinical evidence strongly supports the potential role for these inhibitors for the treatment of cancer. On June 29, 2001, the Radiation Therapy Oncology Group, a National Cancer Institutesponsored cooperative group, held a symposium focusing on the potential role of inhibitors of COX-2 in the treatment of cancer (Dicker, 2003).

Cyclooxygenase-2 Inhibitor and Gastric Cancer

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A number of studies have demonstrated that the administration of NSAIDs can actually prevent cancer. The epidemiological studies reported to date, describing collectively results on more than one million subjects, have pointed out the protective effect of NSAIDs against colon esophageal, gastric, breast (estrogen receptor positive) and perhaps pancreatic and ovarian cancers (Rigas and Kashfi, 2005). Several COX-2 inhibitors, with the potential for less toxicity than that associated with traditional NSAIDs, are under development (Lynch, 2001). Clinical trials are warranted to define the role of selective COX-2 inhibitors in the prevention and treatment of cancer.

2. COX-2 Inhibitor and Gastric Cancer 2.1. COX-2 Inhibitor for Tumor Since the discovery of the two isoforms of COX, the therapeutic effects of NSAIDs can be distinguished from their adverse effects linked to the inhibition of the constitutional form via selective inhibition of the inducible form. A large number of studies have demonstrated that carcinogenesis often may be inhibited by the administration of NSAIDs for many years. The results of COX-2 inhibitor for gut strongly suggest that COX-2 contributes to the development of intestinal tumors and that inhibition of COX is chemopreventative (Williams et al., 2001). Selective pharmacologic inhibition of COX-2 represents a viable therapeutic option for the treatment of malignancies (Crosby et al., 2003). Agents that selectively inhibit COX-2 appear to be safe, and well tolerated suggesting that chronic treatment for angiogenesis inhibition is feasible (Crosby et al., 2003).One of the strongest evidence that NSAIDs has the capability of chemoprevention and treatment of colorectal cancer was the obvious effect of sulindac in treatment of FAP (Wu et al., 2001). A population based investigation for a period of five years or more, of 104, 277 patients aged 65 years or over, indicated that long term NSAIDs use halved the risk of colon cancer (Smalley et al., 1999). Another large-scale survey has demonstrated that users of NSAIDs were a significantly reduced risk of oesophageal and gastric carcinoma (Farrow et al., 1998). In FAP patients in December 1999 approved by FDA, celecoxib has become the first approved drug for this indication based on a 28% reduction in polyp number in a double blind study in 83 patients compared with 5% reduction with placebo. The efficacy of celecoxib in treating colon cancer is being evaluated in a phase III study while its effect in a number of other cancer types, including Barrett’s oesophagus and sporadic adenomatous colonic polyps, is also being explored. Similar studies with polyps, is also being explored. Similar studies with rofecoxib are known to be underway (Whittle, 2000). Jacoby Another study (Jacoby et al., 2000) used the adenomatous polyposis coli (Apc) mutant Min mouse model to determine whether the selective COX-2 inhibitor celecoxib is effective for adenoma prevention and/or regression, and whether it might be safer than the nonselective NSAID previously shown to be most effective in this model, piroxicam. To distinguish prevention from regression effects, groups were treated either "early" (before adenomas develop) or "late" (after most adenomas are established). Celecoxib caused dramatic reductions in both the multiplicity and size of tumors in a dose-dependent manner. Early treatment of celecoxib was effective for prevention, decreasing tumor multiplicity to 29% and tumor size to only 17% of controls. Late treatment

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

demonstrated regression effects, reducing tumor multiplicity and size by about half. In contrast to the significant toxicity of piroxicam, which caused ulcers complicated by perforation and bleeding, celecoxib caused no gastrointestinal side effects and did not inhibit platelet thromboxane B2 at plasma drug levels similar to those obtained in early clinical trials in humans. These results provide the first evidence that selective inhibitors of COX-2 are safe and effective for the prevention and regression of adenomas in a mouse model of adenomatous polyposis and strongly support ongoing clinical trials in humans with the same syndrome. The broader population of patients with common sporadic adenomas that have somatic mutations of the same gene (APC) may also benefit from this treatment approach (Jacoby et al., 2000). Whereas, how is COX-2 inhibitor for gastric cancer? 2.2. In Experiment Studies A study involved with the effects of JTE-522, a newly developed COX-2-specific inhibitor, on gastric cancer cell lines (MKN28 and MKN45) have been reported (Uefuji et al., 2000). The investigation showed that the baseline levels of COX-2 expression were higher in MKN45 than in MKN28. JTE-522 obviously suppressed the levels of COX-2 mRNA, COX-2 protein and PGE2 at a dose of 250 microM in both cancer cells. Apoptosis was induced at 24 hours after treatment with JTE-522 (250 microM) in both cancer cells. Additionally, it indicated that these effects of JTE-522 were more dramatic in MKN45 than in MKN28. This study suggested that JTE-522 might serve as a chemopreventive agent to suppress strongly cell growth by inducing apoptosis in gastric cancer cell lines (Uefuji et al., 2000). Another similar research investigated the effects of NSAIDs, which are specific and nonspecific inhibitors of COX-2, on proliferation of the gastric cancer cell lines KATOIII, MKN28, and MKN45 (Sawaoka et al., 1998). The protein level of COX-2 was examined in these cell lines by Western analysis, and mRNA levels of COX-1/2 by Northern analysis. These cell lines expressed comparable levels of COX-1 mRNA. Whereas, expression level of COX-2 were more dramatic in MKN45 than KATOIII and MKN28. It was observed that the effects of NS-398 and indomethacin, specific and nonspecific inhibitors of COX-2 for these cell lines.It showed that NS-398 and indomethacin suppressed proliferation of MKN45 cells that overexpressed COX-2, although they exerted minimal effects on proliferation of KATOIII and MKN28, which expressed lower levels of COX-2, which was same to the result described above (Uefuji et al., 2000). It was proposed that COX-2 plays an important role in development of gastric cancer cells in vitro. It was more important that NSAIDs may exert antiproliferative activity against gastric adenocarcinomas that overexpress COX-2 (Sawaoka et al., 1998 ;). Other study was undertaken to analyze the effect of sulindac in two gastric cancer cell lines as compared with two HCC cell lines, the human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG2 and SMMC7721. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygen ase-2 (COX-2) and Bcl-2 were detected by Western dot blotting (Wu et al., 2001). Various concentrations of sulindac were incubated with cells for 24h and 48h. Cell survival was determined by MTT assay. Result indicated that sulindac could inhibit the growth of gastric cancer cells and HCC cells in a dose-and time-dependent manner. Sulindac showed a more potent effect in reducing HepG2 cells’ growth as compared with SMM C77

Cyclooxygenase-2 Inhibitor and Gastric Cancer

27

21, MKN45 and MKN28 cells. The cell death rate was more obvious in MKN45 cells than in MKN28. After 24 hours incubation with sulinda c at 2mmol·L-1 and 4mmol·L-1, the level of COX-2 and Bcl-2 protein in were lowered in MKN45, SMMC7721 and HepG2 cells but not in MKN28 cells (Wu et al., 2001). The result demonstrated that sulindac, a COX-2 nonspecific inhibitors, which had little effect in renal prostanoid synthesis and provides additional advantage for its use in clinical trials, could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2 and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater than that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells (Wu et al., 2001). In vitro NSAIDs and COX-2 inhibitors play a role of obviously inhibited and regressed gastric carcinoma. 2.3. In Animal Studies Animal data regarding chemoprevention of gastric carcinoma data regarding chemoprevention of gastric carcinoma data regarding chemoprevention of gastric carcinoma induced neoplasms. Again, results with respect to the effect of NSAIDs on these experimentally induced tumors are mixed. In one study of mice induced to develop gastric neoplasia by the tobacco specific carcinogen NNK, the administration of sulindac and ibuprofen led to a decrease in tumor size and number, whereas the administration of piroxicam did not (Shaheen et al., 2002). In a second study, experimentally induced gastric tumors in a rat model actually were greater in number in those animals treated concurrently with flurbiprofen than in controls (Shaheen et al., 2002). A study involved with effect of celecoxib in gastric adenomas of trefoil factor 1-deficient ﹡

mice have performed (Saukkonen et al., 2003). Expression of TFF1 is lost in 50% of human gastric adenocarcinomas, and TFF1 knockout mice develop dysplastic gastric adenomas with ﹡

full penetrance, which can further develop to adenocarcinomas (Saukkonen et al., 2003). TFF1 can be considered as a tumor suppressor gene. In this vivo expression of COX-2 was －／－

first studied in adenomas derived from TFF1

mice (n 10) and in pyloric tissues derived －／－

from wild-type mice (n 2) as detected by immunohistochemistry. Also, two TFF1 mice adenomas and two wild-type mice pyloric regions were processed for in situ hybridization as described below. Drug treatment trial consisted of four groups of animals: The weight of the mice was measured weekly. After treatments for 3 months, the mice were sacrificed, and conducted analysis of Blood Plasma for Celecoxib. －／－

mice developed a dysplastic pyloric adenoma (n The result showed: all pretrial TFF1 10), whereas the pylorus of the wild-type mice was of normal histology. COX-2 mRNA and protein were strongly expressed in the pyloric adenomas of the TFF1

－／－

mice as detected by in situ hybridization and immunohistochemistry. Nonneoplastic －／－

gastrointestinal tissues of wild-type or TFF1 mice expressed low or nondetectable levels of COX-2. Celecoxib caused ulceration and inflammation of the adenoma in all treated TFF1

－／－

mice. This effect of the drug was adenoma specific, because no histological

alterations were observed in the non-neoplastic gastric or intestinal tissues in the TFF1

－／－

or

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

wild-type mice receiving the drug treatment. All untreated TFF1

－／－

mice had an adenoma, ﹡

but none demonstrated the combination of ulceration and inflammation (Saukkonen et al., 2003). These data strongly show that COX-2 is expressed in gastric adenomas of the －／－

mice and suggest that inhibition of COX-2 disturbs the integrity of the adenoma by TFF1 promoting ulceration and in flammation. It is possible that COX-2 expression is first induced in stromal cells and later in neoplastic epithelial cells. At least in mice, this stromal COX-2 expression may promote growth of carcinoma cells (Williams et al., 2000). Selective COX-2 inhibitors impairs healing of experimentally induced gastric ulcers in rodents, and inhibition ﹡

of angiogenesis was suggested as one mechanism of action (Saukkonen et al., 2003) It is possible that the adenoma-specific ulcerogenic effect of celecoxib in the TFF1 knockout ﹡

model is, at least partially, attributable to inhibition of angiogenesis (Saukkonen et al., 2003). These findings support the effort to initiate clinical studies to investigate the effect of ﹡

Cox-2 inhibitors as chemotherapeutic modality for dysplasias of the stomach (Saukkonen et al., 2003). In order to observe the anti-cancer effects of COX-2 inhibitors and investigate the relationship between COX-2 inhibitors and angiogenesis, infiltration or metastasis, a study with cancer xenografts was performed in vivo (Fu et al., 2004). Thirty athymic mice xenograft models with human stomach cancer cell SGC7901, human moderately differentiated gastric cancer cell line, were established and divided randomly into 3 groups of 10 each. Sulindac, one non-specific COX inhibitor belonging to non-steroidal antiinflammatory drugs (a series of COX inhibitors known as NSAIDs) and celecoxib, one selective COX-2 inhibitor (known as SCIs) were orally administered to mice of treatment groups. Immunohistochemistry was used to examine the expression of PCNA, CD44v6 and microvessel density (MVD). Apoptosis was detected by using TUNEL assay. During the experiment, the growth, diet, activity, etc. of mice were carefully observed, no hematuresis and hematochezia were shown during experiment. The growth of xenografts in treatment groups was significantly suppressed compared with the controls, but there was no difference between two treatment groups. Tumors in sulindac and celecoxib groups were significantly smaller than those in control group from the second week after drug administration (P<0.01). In treatment group, the cell proliferation index was lower (P<0.05) and apoptosis index was higher (P<0.05) than those in control groups. Immunohistochemical staining of CD34 revealed that celecoxib and sulindac could suppress angiogenesis of SGC7901 xenografts. Microvessel density (MVD) in sulindac and celecoxib groups was apparently lower than that in the control. Although it was lower in celecoxib group, the difference is not notable. Membranes of tumor cells were stained brown by CD44v6 staining. By analysis of staining intensity and quantity of positive cells with Image-Pro Plus software, the expression of CD44v6 was markedly weakened by the treatment with sulindac and celecoxib, but there was no apparent difference between sulindac and celecoxib group. This study inoculated athymic mice with SGC7901 to observe the effects of sulindac and celecoxib, a clinically applied selective COX-2 inhibitor, on in vivo tumor by establishing animal models of gastric cancer. The results showed that both drugs had a notable inhibition on gastric cancer growth. Although the effect of celecoxib was better than of sulindac, no statistical difference was shown. The conclusion suggested that COX-2

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inhibitors have anticancer effects on gastric cancer. They play important roles in angiogenesis and infiltration or metastasis of stomach carcinoma. The anticancer effects of COX-2 inhibitors may include inducing apoptosis, suppressing proliferation, reducing angiogenesis and weakening invasiveness (Fu et al., 2004). A study investigated the effects of NS-398, a specific COX-2 inhibitor, or indomethacin, a non-specific COX-2 inhibitor, on the growth of gastric cancer in vivo. Both drugs reduced the tumor volume significantly by inducing apoptosis in cancer cells in a dose-dependent manner and suppressing replication of tumor cells. There was a significant negative correlation between tumor volume and apoptotic cell number within the tumor. The data indicated that COX-2 expression plays an important role in the growth of gastric cancer xenografts in vivo by inhibiting apoptosis and maintaining proliferation of gastric cancer cells (Kısmet et al., 2004). Animal xenograft models have confirmed the tumor suppressing effects of COX-2 inhibitors (Jiang and Wong, 2003). 2.4. In Human Observational Studies A number of studies have indicated that the incidence of gastric carcinoma have been suppressed due to effect of NSAIDs and epidemiologic investigations have demonstrated that the risk of gastric cancer is reduced in correlated with the administration of Aspirin (Saukkonen et al., 2003; Shaheen et al., 2002). Observational studies of several case control and cohort studies of NSAID use in gastric carcinoma have shown a chemoprotective effect of NSAIDs. A case-control of study found that regular NSAIDs use can inhibited the risk of gastric carcinoma in a hospital based case control study of 254 patients, 5952 controls, at least 4 days/week, (OR = 0.3; 95% CI, 0.1– 0.6 ; OR=odds ratio; CI=confidence interval) (Coogan et al., 2000). A case control study with 448 cases and 610 controls showed OR= 0.60, 95% CI, 0.41-0.90 (Zaridze et al., 1999); Langman et al reported a cases control study with 12174 cases and 34934 controls were OR=0.51 95%CI, 0.33-0.79 (in this study endpoint is incidence); and the study reported that as pirin reduced risk only for non-cardia gastric cancer (Langman et al., 2000). Another case control study of observation incidence showed OR=0.70 95%CI, 0.60-10, in which there were 567 cases and 1165 controls (Akre et al., 2001). While in this study the risk reduction was apparent for both cardia and non cardia tumors. Interestingly, in this latter study it was observed that reduction of cancer risk was associated with intestinal-type tumors, but was uncertain for diffuse-type carcinomas (Akre et al., 2001). Farrow etal, reported a case-control study there were 612 carcinoma cases (255 adenocarcinomas of the cardia and 357 adenocarnomas elsewhere in the stomach) and 687 controls in this study , the results were apparent incuding current aspirin user elsewhere in the stomach (OR=0.46;95% CI, 0.31-0.68), current NSAIDs user elsewhere in the stomach ( OR=0.23; 95% CI,0.11-0.34) (Farrow et al., 1998). In a large cohort study of 635,031 U.S.residents participanting in American Cancer Society (ACS) study) followed over 6 years, whose endpoint is mortality, ACS pronounced that regular (≥16 times/mo) exposure to aspirin exerted a protective effect against gastric carcinoma, who were found to have approximately 50% the risk of gastric carcinoma compared with nonusers (OR = 0.53; 95% CI, 0.34 – 0.81) (Thun et al., 1993). Another cohort study with 12668, endpoint is incidence, showed OR=0.93,95% CI, 0.49-0.174; (Schreinemachers and Everson, 1994). A cohort study of 11683 patients with rheumatoid arthritis presumably using NSAID indicated that gastric

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carcinoma risk was decreased compared with similar patients without arthritis in the same geographic area (standardized incidence ratio [SIR] = 0.63; 95% CI, 0.50.9) (Shaheen et al., 2002). Although the data concerning a potential chemoprotective effect of NSAIDs in gastric cancer is less convincing and less numerous than those with respect to colorectal carcinoma, a growing body of evidence suggests that NSAIDs may have some profiting effect. Although the results from animal studies are conflicting, questions regarding the applicability of these models to humans limit their utility. Human studies, although again not universally supportive of a profiting effect, are for the most part suggestive of chemoprevention (Shaheen et al., 2002). Human studies are underway to examine the use of COX-2 inhibitor in the treatment of pre-cancerous lesions. COX-2 inhibitors have a promising role in the prevention and treatment of gastric cancer (Jiang and Wong, 2003). In the clinic research the influence of rofecoxib (Vioxx), a specific COX-2 inhibitor, were observed for twenty-four gastric cancer (GC) patients. For comparison, 48 age- and sexmatched healthy controls and 24 similarly matched Helicobacter pylori (Hp)-positive subjects were enrolled and treated with Vioxx as GC patients (Konturek et al., 2003). Once before and then following a 14-day treatment with Vioxx at a dose of 25 mg twice daily. The study demonstrated that inhibition of COX-2 activity by Vioxx resulted in a significant reduction in serum and tumor levels of progastrin and gastrin and serum IL-8 and TNF-alpha levels, while inhibiting the expression of Bcl-2 and surviving. The result indicated that COX-2 inhibitor exert chemoprevention against gastric cancer (Konturek et al., 2003). One clinical study demonstrated that the nonselective NSAID indomethacin increased survival and reduced pain in end-stage metastatic patients with primary cancers of the stomach and colon (Wang et al., 2005). 2.5 COX-2 Inhibitor and Helicobacter Pylori Related with Gastric Cancer and Gastritis

2.5.1 COX-2 Inhibitor Regress H.Pylori Associated Gastric Cancer Epidemiological studies have found that H.pylor infection is an important risk factor for gastric cancer (Huang et al., 1998). It is well known that H.pylori infection can lead to gastric cancer, and COX-2 is overexpressed in the stomach during H. pylori infection. But it remains unknown as to whether COX-2 may be target for chemoprevention of H.pylori-associated with gastric carcinogenesis Therefore, a study used H.pylori-associated gastric cancer mouse model to investigate the preventive effects of the selective COX-2 inhibitor nimesulide, and studied the mechanisms underlying nimesulide-induced chemoprevention with AGS human gastric cancer cell line (Nam et al., 2004). C57BL/6 mice were treated with the carcinogen Nmethyl-N-nitrosourea (MNU) and/or H. pylori. Mice were randomized into five groups( group 1, H. pylori alone; group 2, MNU alone; group 3, five weeks’ MNU administration followed by H.pylori infection; group 4, H. pylori infection followed by continuous nimesulide via diet; and group 5, five weeks’ MNU administration followed by H. pylori infection and nimesulide treatment ). In vitro experiments with the human gastric cancer cell line AGS were also performed to determine the effect of COX-2 inhibition, nimesulide was mixed with feed pellets and administered for the duration of the experiment. Histopathology,

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immunohistochemistry, and Western blotting for COX-2, Bax and Bcl-2 were performed in stomach tissues. The result showed that the incidence of tumors at sacrifice was 0% (0/13) in group 1, 10% (1/10) in group 2, and 68.8% (11/16) in group 3. Group showed significantly higher incidence of gastric tumor compared with groups and (0.0001 for both comparisons)signifying that the mice were somewhat resistant to carcinogen-stimulated gastric tumorigenesis, and that H. pylori infection promoted, rather than initiated, gastric tumorigenesis (Nam et al., 2004). These findings indicate that H. pylori promotes carcinogeninduced gastric tumorigenesis.This is similar to another report that GC patients show significantly higher H.pylori and CagA seropositivity than controls, but not H.pylori-positive subjects, indicating that infection with cytotoxic H.pylori is linked to GC (Konturek et al., 2003). Nimesulide significantly suppressed H. pylori associated gastric carcinogenesis (Nam et al., 2004) the incidence of gastric adenocarcinoma was markedly reduced in group 5 compared with group 3(5.6% (1/18) versus 43.8% (7/16)); these data indicate nimesulide inhibited either the development of H. pylori-associated gastric tumorigenesis or the process of gastric carcinogenesis (Nam et al., 2004). Whereas, substantial inductions of apoptosis were observed in treated Mice. The mean expressions of proapoptotic Bax and antiapoptotic Bcl-2 were measured in mixed homogenates of stomach tissues according to each group. Compared with group (MNU alone)the mean expression of Bax in group (H.pylori alone) was markedly increased, and the mean expression of Bcl-2 was rather decreased after H. pylori infection than in group 2, suggesting that significant apoptotic-prone tendency was induced after H. pylori infection. Interestingly, the expression of Bcl-2 in group (MNU→H. pylori) was significantly increased, but the expression of Bax was attenuated, resulting in significant decreased in the mean ratio of Bax/Bcl-2 intensity. Significant increases in the expression of Bax were observed in groups treated with nimesulide signifying that the apoptotic actions of nimesulide might be the reason why carcinogenesis was ameliorated in animal groups treated with nimesulide. Significant attenuation in cell survivals was noted in cells treated with either celecoxib or nimesulide alone. These changes were more evident in cells cotreated with a COX-2 inhibitor and H. pylori, suggesting that cell death processes were more activated than with either COX-2 inhibitor or H. pylori alone (Nam et al., 2004). Therefore, the combination of COX-2 inhibitor and H. pylori provoked and augmented apoptosis were more evidenced with Western blot of caspase-and PARP and with flow cytometry analyses. Active caspase-3, cleavage of PARP, and increased fractions of positive annexin V observed after COX-2 inhibitor treatment were more apparently increased after the cotreatment of COX inhibitor and H. pylori (Nam et al., 2004). The study demonstrated that nimesulide and H. pylori when combined acted synergistically to induce more apoptosis than either alone in vitro. Nimesulide, preferred selective inhibitor of COX-2 belonging to the sulfon amide class, has been used clinically as an anti-inflammatory drug with less ulcerogenic effects in the gastrointestinal tract than classical NSAIDs. The study found that gastric tumorigenesis was significantly attenuated by long-term administration of the COX-2 inhibitor, nimesulide, in an H.pylori-associated gastric cancer mouse model, and propose that selective COX-2 inhibitors may be clinically useful in protecting against gastric cancer development (Nam et al., 2004).

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2.5.2 Effect of COX-2 Inhibitor on H.Pylori Infected Gastritis H.pylori and NSAIDs are two well-known important causative factors of gastric damage through apparently different mechanisms. Moreever, H.pylori increase overexpression of COX-2 and prostaglandin production, It also increases apoptosis of gastric epithelial cells and is an important factor in peptic ulcer and gastric cancer. NSAIDs and COX-2 inhibitors reduce prostaglandins and mucosal proliferation in infected mucosa and may induce cell apoptosis and have antineoplastic effects and reduce gastric cancer risk. However, their interactions remain unclear. Particularlly, NSAIDs and H. pylori have opposite effects on COX and PG synthesis. So that, it is still a point of controversy whether Helicobacter pyloriinfected patients are more likely to develop mucosal damage while taking NSADIs. Selective cyclooxygenase (COX-2) inhibitors may be associated with less severe gastric mucosal damage than conventional NSAIDs, but this association is undefined in H. pylori-induced gastritis (Bhang et al., 2002). Recently, several studies on COX-2 inhibitor and H.pylori infection for gastric mucosa have reported. (Scheiman et al., 2003; Kim et al., 2001; Bhang et al., 2002; Gu et al., 2004; Cryer and Feldman, 1999). The effects of NSAIDs and selective COX-2 inhibitor on the apoptosis and proliferation of gastric epithelial cells and gastric inflammation in H. pyloriinfected mice has investigated (Kim et al., 2001). C57BL/6 mice were sacrificed 8 weeks after H. pylori SS1 inoculation. Indomethacin or NS-398 was administered subcutaneously once daily for 10 days before sacrifice. Gastric inflammatory activity, gastric COX protein expression by Western blotting; gastric prostaglandin E2 levels by enzyme immunoassay, apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and cell proliferation by Ki67 immunostaining were analyzed. H. pylori infection and/or NSAID treatment increased COX-1 and COX-2 protein expression. Gastric prostaglandin E2 levels, apoptotic index, cell proliferation index, neutrophil activity, and the degree of chronic inflammation were all increased by H. pylori infection, and these effects were significantly decreased by indomethacin treatment. However, NS-398 treatment after H. pylori infection did not show a significant reduction. The results indicate that NSAIDs can reverse the increased apoptosis and proliferation of epithelial cells and inflammatory activity in the stomachs of H. pylori-infected mice and that, like COX-2 activation, COX-1 induction promote the change of gastric mucosal cell turnover and inflammation induced by H. pylori infection (Kim et al., 2001; Scheiman et al., 2003). Another study evaluated gastric mucosal damage, inflammatory cell infiltration and prostaglandin E2 concentration in H.pyloriinfected and uninfected Mongolian gerbils administrated with indomethacin, NS-398 or vehicle alone. In H. pylori-uninfected groups, the indomethacin treated group showed the highest mucosal damage score and the lowest PGE2 concentration. There was no difference in mucosal damage scores, irrespective of the type of drugs administered. Gastric neutrophil and monocyte infiltration scores were higher in H.pylori-infected groups than in uninfected groups. However, there was no difference in these scores according to the type of drugs administered, within H. pylori-infected or uninfected groups. COX-2 protein expression was observed in H.pylori-infected Mongolian gerbils but not in uninfected ones. PG concentration was increased by COX-2 expression, reducing NSAID-induced gastric mucosal damage in H.pylori-induced gastritis. Selective COX-2 inhibitors, instead of conventional NSAIDs, are expected to reduce gastric mucosal damage in H. pylori uninfected stomachs, whereas these

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selective COX-2 inhibitors will not have such effect in H. pylori-induced gastritis (Bhang et al., 2002). On the other hand, a clinic study with twenty patients infected H. pylori and six uninfected healthy volunteers received rofecoxib showed that the COX-2 inhibitor did not significantly affect PGE2 levels, gastritis scores or proliferation indices in the body or antrum in the H. pylori-positive or negative subjects (Scheiman et al., 2003). However, rofecoxib was lack of effect on gastric prostaglandin levels and proliferation in H. pylori-infected mucosa, and the absence of an increased ulcer risk, among COX-2 inhibitor users with H. pylori infection raises uncertainty regarding the potential of specific COX-2 inhibitors for chemoprevention of gastric cancer (Scheiman et al., 2003). In order to determine the effects of COX-2 specific inhibitor and NSAIDs on the growth, urease activity and antimicrobial susceptibility of H. pylori, three H. pylori reference strains, and 18 clinical isolates were treated with SC-236 or indometa cin for 24 and 48 h. Growth, urease activity and susceptibility to clarithromycin and metronidazole of the bacteria were assessed by viable colony counting, spectrophotometry and E-test respectively. Both SC-236 and indometacin suppressed the growth and urease activity of H. pylori in a dose dependent manner, and increased its susceptibility to the antibiotics (Gu et al., 2004).

2.5.3 Mechanism Underlying Different and Independent Helicobacter pylori infection and the use of NSAIDs are the two most common causes of gastric damage or ulcers, but their pathogenic mechanisms are different and independent. H. pylori induces gastric mucosal damage via direct mechanism (Smoot, 1997) and indirect inflammatory response associated with increased production of cytokines (Ernst and Crowe; 1997). H. pylori infection may induce strong mucosal inflammation, stimulate cytokine release, and provoke apoptosis. H. pylori had been known to be responsible for significant apoptosis, by which gastric ulcerations or gastric atrophy (significant apoptotic cell death of gastric stem cells or parietal cells) can be developed. For this, several cytotoxins of CagA or VacA, ammonia generated from urease actions, chemokines, or cytokines like interleukin (IL)-8, interferon-γ, IL-12, tumor necrosis factor α ,oxidative stress and HNP (helicobacter neutrophil-activating peptide)and so forth, had been known to be responsible for apoptosis after H. pylori infection (Nam et al., 2004). On the other hand, the major pathogenic mechanism of gastric mucosal damage caused by NSAIDs is interference with‘cytoprotective’ prostaglandin (PG) synthesis by inhibiting COX (Bhang et al., 2002). NSAIDs may inhibit mucosal prostaglandin synthesis, leading to weakening in the gastric mucosal barrier, and impaired resistance to acid injury (Nam et al., 2004). NSAIDs alone also contributed to apoptosis either by COX-2 inhibition or by direct activation of other cellular targets such as peroxisome proliferator-activated receptor. It is suggested that gastric defense mechanisms operated well in the groups exposed to both H.pylori and COX-2 inhibitor and nimesulide administered to the H.pylori-infected mice must have blocked the effects of COX-2 on cellular proliferation, release of inflammatory mediators, and cell adhesion to matrix, which cause increased gastric inflammation. Furthermore, H.pylori-alone provoked cellular apoptosis (Katori and Majima, 2000; Nam et al., 2004).

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NSAIDs decrease COX expression and PG synthesis whereas H. pylori increases COX-2 expression, and PG synthesis in the gastric mucosa, indicating that H.pylori infection may not significantly accelerate mucosal injury caused by NSAIDs. The combination NSAIDs and H.pylori infection in gastric mucosa may enhance apoptosis (Gu et al., 2004; Cryer and Feldman, 1999). Recently, two large, randomized, multi center trials suggested that NSAID users with H. pylori infection had higher rates of ulcer healing and less frequent recurrence than those without H. pylori infection (Yeomans, 1998; Grösch et al., 2001). 2.6. Summary A wide spetrum of studies in animal models, epidemiologic studies, and in treatment of patients have shown that chemoprevention and tumor regression of NSAIDs and others COX2 inhibitors (Moore and Simmons, 2000). COX-2 inhibitor is the use of pharmacological or natural agents to prevent, suppress, interrupt or reverse the process of gastric carcinogenesis. The exact biochemical and cellular mechanisms underlying each of these phenomena is only partially understood. Processes that have been recently implicated as being important include the inhibition of tumor cell growth, prevention of angiogenesis, and induction of apoptosis in neoplastic cellsdiscuss (next chapter will discuss further). Clinically it will be important to determine whether selective COX-2 inhibitors deliver safer or at least as effective anticancer properties as compared to nonselective NSAIDs. In addition, subtypes of premalignant lesions and invasive cancers (based on histology, stage and/or genotype) that are especially sensitive to these drugs should be recognized. Finally, it will be important to perform biomarker studies using selective COX-2 inhibitors in a neoadjuvant setting, integrate these drugs into already existing protocols as adjuvant treatments, and at the same time continue research on alternative and hopefully less toxic combination treatments for gastric cancer. All this should help to target the use of COX-2 selective drugs to those cancer patients who are most likely to benefit from this treatment (Saukkonen et al., 2003). Taken together these data COX-2 inhibitors may be as chemoprevention agents and as adjuvant treatment modalities against gastric neoplasias. Similar to CRC, the combination of COX-2 selective inhibitor with another therapeutic modality may have additive effects that reduce gastric tumor growth. (Wang et al., 2005).

Mechanism Underlying COX-2 Inhibitor for Therapeutics and Prophylaxia of Carcigenesis 1. Introduction Several lines of evidence suggest that the cyclooxygenase enzymes (specifically COX-2) might be an important molecular target associated with gastriointestinal cancer, including gastric cancer. Therefore, targeted inhibition of COX , especially the COX-2 isoform, seems to be one of the most promising pathways to achieve chemopreventive and anticancer effects at both early and late stages of some cancers (Jiang and Wong, 2003; Masmoudi et al., 2004;

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Anderson et al., 2003; Dang et al., 2002; Dermond and Ruegg, 2001). Indeed, Increased expression of COX-2 occurs in multiple cells within the tumor microenvironment that can impact on angiogenesis. COX-2 appears to: (a) play a key role in the release and activity of proangiogenic proteins; (b) result in the production of eicosanoid products TXA2, PGI2, PGE2 that directly stimulate endothelial cell migration and angiogenesis in vivo. Moreover, COX-2 overexpression also contributes to carcinogenesis and the malignant phenotype of tumor cells to be related by different mechanisms including (i) increase production of prostaglandins, (ii) convert procarcinogens to carcinogens, (iii) inhibit apoptosis, (iv) modulate inflammation and immune function, and (v) increase tumor cell invasiveness (Xu, 2002; Masmoudi et al., 2004; Crosby et al., 2003; Dermond and Ruegg, 2001). COX-2 is overexpressed during carcinogenesis, and appears to have a role in both tumour initiation and promotion and is amenable to intervention (Anderson et al., 2003). Therefore, a large number of active researches have been conducted to unravel further the cellular mechanisms mediating the anti-tumorigenic effects of NSAIDs and their association with COX inhibition. Particularly, with the development of COX-2 specific inhibitors, numerous laboratory and clinical studies are underway to help advanced understand the role of COX-2 in cancer and the potential use of COX-2 selective inhibitors for cancer treatment or prevention (Crosby and DuBois, 2003). Whereas, there arise a important controversy wheather mechanism of COX-2 inhibitor anticarcigenesis is exerted through COX-2 dependent pathway or COX-2 independent as well as other pathway. (Xu, 2002) (Jiang XH, Wong, 2003; Masmoudi et al., 2004; Dermond and Ruegg, 2001; Crosby and DuBois, 2003; Dang et al., 2002). Because the mechanism behind COX-2 inhibition suppression cancer are a extraordinary complex process, Thereby here analysis and delineating mechanish concerning anticarcinogenesis of COX-2 inhibitor will be not only limited in gastric cancer, but also combinated with other tumor researches, based on the studies of tumor common characteristics, to pursue more and exact explaination the mechanism for COX-2 inhibition to block tumor growth.

2. Mechanism Underlying COX-2 Dependent Pathway for Anticarcinogenesis 2.1. Inhibite Activity of COX-2 and PGs to Suppress Tumor A number of studies demonstrate that expression of COX-2 are elevated in many gastriointestinal tumors. Moreover, transfection of colon carcinoma cells with a vector expressing COX-2 was found to stimulate the metastatic potential of these cells in vivo (Tsujii et al., 1997). Another study in colon tumor showed that Cultured cells which were derived from intestinal epithelium and programmed to express COX-2 showed several phenotypic changes in favor of carcinogenesis, including resistance to apoptosis and enhancement of cell proliferation, angiogenesis, and invasion (Tsujii, 2001). Tumor growth implanted in COX-2 null mice was significantly attenuated, but not in COX-1 null or wild type mice, suggesting that COX-2 in stroma also has an important role in tumor growth. Moreover, PGE2, one of COX-2 metabolites, reversed these antitumor effects, indicating that inhibition of PGE2 production has a pivotal role in tumor suppression (Tsujii, 2001). It is

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further elucidated that COX-2 dependent pathway is main mechanism. The newest clinical research further support COX-2 dependent pathway based on intratumoral COX-2 gene expression is a predictive factor for colorectal cancer response to fluoropyrimidine-based chemotherapy (Uchida et al., 2005). The mechanism of PGs-dependent, which is mediated by COX-2, PGs plays a role in multiple cell-regulatory functions, including DNA synthesis and cell division (Shaheen et al., 2002). PG expression has been found to be increased in a number of experimental tumors, as well as in sporadically occuring large bowel neoplasms in human (Shaheen et al., 2002). whereas, the suppression of PG production may attenuate DNA production and proliferation to suppress carcinogenesis (Shaheen et al., 2002). There are different accesses in mechanism underlying COX-2 dependent, several prostaglandins, such as PGE2, suppress immunosurveillance through downregulation of lymphokines, T-cell and B-cell proliferation, cytotoxi activity of natural killer cells, and secretion of TNFα and interleukin-10 (Stolina et al., 2000). The study showed a close link between PGE2 and EGF-receptor signaling systems. PGE2 induces the activation of metalloproteinases MMP2 and MMP9, increases expression of TGFα, transactivates EGF receptor, and initiates mitogenic signaling in gastric epithelial and colon cancer cells as well as in rat gastric mucosa in vivo (Pai et al., 2002). This suggested how PGE2 exerts its trophic action on gastriointestinal mucosa, causing hypertrophy and colon cancers. NSAIDs and coxibs significantly inhibit chemically induced carcinogenesis in rodents (Gasparini et al., 2003). Moreover, NSAIDs may inhibit carcinogen-DNA adducts generated by COX enzymes (Levy, 1997). Hepatocyte growth factor (HGF) is strongly associated with tumor invasion and metastasis and the activities of HGF are mediated by HGF receptor (c-Met) in gastric tumor cells (Liu et al., 1997). It is more important that overexpression of c-Met and elevation of serum HGF level is involved in more advanced tumor or metastasis (Han et al., 1999). c-Met is over-expressed in the majority of gastric cancer patients with poor prognosis (Chen et al., 2001). HGF was able to trigger activation of the COX-2 gene in rat gastric mucosa cells through phosphorylation of c-Met/HGF receptor and activation of the extracellular signal kinase (ERK2) signaling pathway (Jones et al., 1999). It is very likely that HGF mediates autocrine or paracrine stimulation in the development and progression of gastric cancer by interacting with c-Met which then leads to ERK2 activation, up-regulation of COX-2 expression, and increase synthesis of PGE2 he HGF-COX-2-PGE loop is functional in gastric cancer cells remains to be elucidated (Chen et al., 2001). These results together suggest that the signaling pathway of HGF and c-Met may be mediated through ERK2 activation, upregulation of COX-2 and increased production of PGE2 in gastric cancer cells. COX-2 specific inhibitors may provide beneficial effects in these patients (Chen et al., 2001) Furthermore, tht combined treatment with nonselective NSAID plus an EGFR tyrosine kinase inhibitor significantly inhibites polyp formation in Apc Min mice (Torrance et al., 2000). The combination of COX selective inhibitor, an EGFR tyrosine kinase inhibitor, and protein kinase antisense construct markedly inhibited proliferation and angiogenesis of human colon and breast cancer cells in soft agar and xenograft studies (Tortora et al., 2003). One such novel treatment option could be a combination of NSAIDs with inhibitors of the Erb/ HER family of growth factor receptors (Saukkonen et al., 2003).

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2.2. Inhibite Angiogenesis Recent studies made in a number of laboratories have provided strong evidence that the anti-tumor activity of NSAIDs is associated with suppression of tumor angiogenesis. Hence, inhibition of angiogenesis by NSAIDs emerged an important mechanism by which NSAIDs prevent tumor progression (Dermond and Ruegg, 2001). The experimental data suggest that the established antitumour mechanisms of action of coxibs include blockage of angiogenesis, modulation of aromatase, and proapoptotic activity (Gasparini et al., 2003). Celecoxib inhibits basic fibroblast growth factor induced angiogenesis in vivo (Saukkonen et al., 2003), and that colon cancer angiogenesis in vivo , which overexpress COX-2, produce angiogenic factors in vitro (Saukkonen et al., 2003). It is thus possible that the adenoma specific ulcerogenic effect of celecoxib in the TFF1 knockout model is, at least partially, due to inhibition of angiogenesis, which is an important property of ulcer healing (Saukkonen et al., 2003). It is clear that carcinogenesis is a multistep process involving the activation of oncogenes and inactivation of tumor suppressor genes giving rise to a cell capable of uncontrolled proliferation and survival (Compagni and Christofori, 2000). The formation of a vascular network in the tumor stroma also implicated to as tumor angiogenesis, play a cricial role in tumor progression and metastasis formation (Carmeliet and Jain, 2000). In recent years, tumor angiogenesis promoted tumor progress and many of the cellular and molecular events of tumor angiogenesis have been uncovered (Yancoppoulos et al., 2000). Masferrer et al examined more than 150 samples of different types of human cancers and found COX-2 expression in most cancer tissues;. Moreover, they clearly detected the presence of COX-2 in the angiogenic vasculature in most of the tumors analyzed. Another important finding was the detection of COX-2 in the angiogenic blood vessels present in livers from patients with metastatic colon carcinoma (Masferrer et al., 2000). One studies have shown that overexpression of COX-2 in both gastric cells and primary gastric cancers produces high levels of PGE2, which up-regulates VEGF expression and increases angiogenesis in vitro (Leung et al., 2003). COX-2 controls tumor angiogeneis by modulating three critical events in angiogenesis: production of vessle endothelial growth factor (VEGF); biological response to VEGF integrin-depengent signalling (Dermond and Ruegg, 2001). A large number of researches indicated that inhibition of COX-2 by NSAIDs reduced tumor growth and this effect was associated with suppressed tumor angiogenesis (Kısmet et al., 2004; Dermond and Ruegg, 2001) and in human cancer, inhibition of COX-2 might prove effective in two ways: by blunting the tumor cell production of angiogenic growth factors and by inhibiting the growth of the neovascular cells themselves (Kısmet et al., 2004).

2.2.1 COX-2 Regulates Expressing and Function of VEGF (Figure7) COX-2 is implicated VEGF expression. A study showed that in mice treatment of wildtype fibroblastes with a selective COX-2 inhibitor reduced VEGF production by over 90% (Williams et al., 2000). COX-2 inhibitor NS-398 also suppressed FGF-2-induced expression of VEGF in a rat sponge implant model (Tsujii et al., 1998). A study demonstrated that deletion of COX-2 gene in mice caused 94% inhibition of VEGF production fibroblasts (Williams et al., 2000). Consistent with the ideal that COX-2 regulates VEGF expression, it was shown that PGs stimulate VEGF production in many different cells (Hoper et al., 1997;

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

Ben-Av et al., 1995). PGE2 supports tumor growth is the induction of the angiogenesis necessary to supply oxygen and nutrients to tumors more than 2 mm in diameter (Kısmet et al., 2004). PGE2 can mediate its effects by transactivating EGFR in gastric epithelial cells and in rat gastric mucosa (Wang et al., 2005). Inhibition of COX-2 by NS-398 prevented VEGF/VEGF-r2-mediated MAPK activation (Dormond et al., 2001). Another report showed that both COX selective inhibitor, NS-398, and nonselective NSAID indomethacin, inhibit proliferation and growth of human gastric cancer cells by blocking MAPK (ERK2) kinase signaling in vitro (Wang et al., 2005). Because there is positive correlation between COX-2 expression, VEGF, and angiogenesis in human gastric adenocarcinomas (Wang et al., 2005). COX-2-mediated angiogenesis provides likely mechanism by which COX-2 promotes tumor growth and invasion (Wang et al., 2005). VEGF contributes to sustained endothelial cell proliferation and vascular permeability, two key features of the tumor vasculature (Ferrara, 1999; Dvorak et al., 1999). VEGF is upregulated early during tumorigenesis as a direct consequence of the activation of oncogenes or loss of tumor suppress genes (Kerbel et al., 1998). Whereas, Expression of angiogenic factors like VEGF and basic fibroblast growth factor (bFGF) are up-regulated in colorectal cancer cells engineered to overexpress COX-2 (Tsujii et al, 1998). The combination of an NSAID with an EGFR kinase inhibitor remarkably inhibited polyp formation in Apc Min mice; it is possible that combinations of inhibitors using both EGFR tyrosine kinase inhibitors and COX-2 selective inhibitors at lower doses may yield additive effects that could completely block metastatic spread of disease. Hence, it will be essential to evaluate COX-2 selective inhibitors for their use as adjuvant therapy for early-stage disease and their combined use with EGFR tyrosine kinase inhibitors in clinical trials (Wang et al., 2005).

2.2.2. COX-2 Control Integrin-Mediated Endothelial Cell Migration (Daniel Et Al, 1999) This is because angiogenesis strongly depends on adhesive interactions between vascular cell and ECM proteins mediated by selected members of the integrin family of adhesion receptors (Eliceiri and Cheresh, 2001). COX-2 and its products PGs and TXA2 regulate angiogenesis, at least in part, by controlling intracellular cAMP levels, and prostaglandings induce VEGF expresson through an elevation of the cAMP concentration (Hoper et al., 1997). Thus, there is a rationale for clinical coadministration of thromboxane-receptor antagonists and coxibs (Gasparini et al., 2003). During angiogenesis endothelial cell cAMP levels must be tightly controlled and that COX-2 metabolites promote this regulation (Dermond and Ruegg, 2001). TXAs and vascular integrin, ανβζ play a critically regulation effect in endothelial cell migration and mediating tumor angiogenesis. (Daniel et al, 1999; Dormond et al., 2001). NS-398 blocked ανβζ dependent endothelial cell spreading and migration in vitro and FGF-2 induced angiogenesis in vivo, as was associated with the inhibition of ανβζ dependent activation of Cdc42 and Rac, Which are two members of the Rho family of GTPases and able to regulate cytoskeletal organization and migration (Dermond and Ruegg, 2001). These studies indicated that another important mechanism underlying COX-2 inhibitor suppressing tumor angiogenesis is associated with inhibition of ανβζ mediated Rac activation. Moreover, the inhibitio of ανβζ-mediated Rac activation by NS398 correlates with a decrease in cAMP concentration and of a cell permeable cAMP analog

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prevented this inhibition (Dermond and Ruegg, 2001). COX-2 and PGs result in enhanced tumor cell, and possibly, vascular endothelial cell survival by upregulation of the antiapoptotic proteins Bcl-2 and/or activation of PI3K-Akt (Crosby et al., 2003) On the other hand, the study found that COX-2 modulates the production of angiogenic factors by tumour cells, whereas COX-1 regulates angiogenesis of endothelial cells in normal tissues (Tsujii et al, 1998). It means that COX-1 activity in endothelial cells played an important role in the modulation of angiogenesis and might be a relevant target for cancer prevention or treatment in tumors lacking COX-2 expression (Kısmet et al., 2004). Two mechanisms of NSAID-angiogenesis inhibition were suggested: inhibition of COX2 activity in colon carcinoma cells by downregulation of angiogenic factors, and inhibition of COX-1 activity in endothelial cells (Gasparini et al., 2003). Because observation of that aspirin treatment or inhibition of COX-1 synthesis by antisense oligonucleotide treatment markedly inhibited expression of Ets-1, a transcription factor shown to be involved in angiogenesis. It might be that PGs produced by COX-1 in endothelial cells could be important in regulating genes required for endothelial tube formation (Kısmet et al., 2004). In a word, a primary effect of COX-2 inhibition on tumor growth may be to starve the tumor’s blood supply (Shaheen et al., 2002).

Figure 7. (Wang et al., 2005 modified)

PGE2 induces angiogenesis through a positive-feedback loop. COX-2-derived PGE2 induces VEGF and bFGF, which have been shown to induce COX-2 expression and further increase production of PGE2. In addition, both nonselective and selective NSAIDs block the production of angiogenic factors and inhibit the proliferation, migration, and tube formation of vascular endothelial cells (Wang et al., 2005). 2.3. Anti-Tumor Invasiveness COX-2 expression and PGE2 treatment induced the expression of glycosyltransferases and type I sialyl Lewis antigens, leading to greater tumour-cell adhesion to endothelial cells, formation of metastasis. COX-2 promotes MMP1 and MMP2 (Tsujii et al., 1997). COX-2 is associated with increased expression of CD44, the cell surface receptor of hyaluronate, which

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contributes to tumour-cell invasion (Dohadwala et al., 2001). Coxibs blocked these changes (Gasparini et al., 2003). It was indicated that COX-2 inhibitor was involved in lower tumour concentrations of β-catenin, cyclin D1, VEGF, MMP2, MMP9, and interleukin 10, but higher amounts of interleukin 12, which is a naturally occurring inhibitor of angiogenesis (Gasparini et al., 2003). In addition, PGE2 and PGI2 are referred to activation of the small GTPases CDC 42 and RAC, as the result of engagement of integrin ανβζwith its substrate (Gasparini et al., 2003). NSAIDs inhibit integrin ανβζ-dependent activation of the small GTPases and suppress endothelial-cell spreading and migration in vitro as well as FGF2-induced angiogenesis in vivo (Gasparini et al., 2003).There are other mechanisms in Figure 8. 2.4. Promote Apoptosis There was a report that 1483 human head and neck xenograft tumors express COX-2 similar to the pattern observed in human solid tumors and that COX-2 activity produces high levels of prostaglandin E2 (PGE2) (Ben et al., 2002). Inhibition of COX-2 by celecoxib resulted in loss of intratumor PGE2 levels and reduced tumor growth in a dose-dependent manner. In contrast, a selective COX-1 inhibitor, SC-560, did not measurably reduce tumor prostaglandin levels or tumor growth despite the presence of COX-1 in the host and tumor cells. Celecoxib-treated tumors showed reduced proliferation and increased apoptosis of both tumor and stromal cells compared with vehicle controls. Specific inhibition of PGE2 activity by a neutralizing antibody mimicked the reduced tumor growth observed after celecoxib treatment, suggesting growth is PGE2 mediated. These data indicate that a major antitumor mechanism of action of celecoxib is inhibition of COX-2-derived prostaglandins, particularly PGE2, and suggest celecoxib as a novel therapeutic agent for human head and neck cancer (Ben et al., 2002). COX-2 promoted cell survival by up-regulating the level of Mcl-1 by activating the PI3K/Akt-dependent pathway. This investigation revealed that either overexpression of COX-2 or exposure to PGE2 could increase the apoptosis threshold in human lung adenocarcinoma cells by up-regulating the mcl-1 gene. Treatment of COX-2 overexpressed cells with NS-398 and celecoxib caused an effective reduction of the increased level of mcl-1 (Lin et al., 2001). The nonselective NSAIDs can inhibit the growth of gastric cancer cells via induction of apoptosis associated with reduced COX-2 and Bcl-2 levels (Wu et al., 2001). The COX-2 selective inhibitor NS-398 induced apoptosis in 15 CRC cell lines, with more significant effects on cells expressing COX-2, which suggests that NSAIDs can induce apoptosis in tumor cells by inhibiting the COX-2 pathway (Li et al., 2001). The molecular evidences supporting role for COX-2-derived PGE2 in the inhibition of apoptosis has stimulated PGE2 in the inhibition of apoptosis has stimulated great interest in combination therapies employing NSAIDs with conventional anticancer agents such as cisplatin. This approach may harbor particular promise because chemotherapeutic agents and radiation therapy enhance COX-2 protein expression as well as PGE2 synthesis in human cancer cells (Wang et al., 2005). However, COX-2 independent effects of NSAIDs-induced apoptosis have also been reported (Wang et al., 2005). Perhaps, COX-2 inhibitor result apoptosis mediates through both COX-2 activity dependent and independent mechanisms (Kısmet et al., 2004).

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2.5. COX-2 Inhibitor Contribute to Stimulate Immunological Ability This may depend on the cytokine microenvironment, since COX-2-derived prostanoids can alter release of IL-10 and IL-12 from lymphocytes and macrophages, resulting in repression of host immunity (Saukkonen et al., 2003). In addition, in vivo a heavy infiltration of chronic inflammatory cells was observed in all celecoxib treated adenomas, and it is possible that this inflammatory reaction was induced by the drug therapy (Saukkonen et al., 2003). 2.6. Delayed K Channel ﹢ COX-2 inhibitor were associated with delayed K channel in oncogenesis of human gastric cancer cell (Wu et al., 2002). Human COX-2 encoding gene was cloned with PR-PCR strategy and its antisense recombinant eukaryotic expression vector was constructed. COX-2 highly expressed human gastric cancer cell line SGC7901 was stably transfected with the antisense vector. The whole cell recording technique of perforated patch clamp was ﹢

﹢

employed to observe the change of delayed rectifier K current (Ik) of SGC7901 after gene transfer or treatment with COX-2 inhibitor indomethacin.MMT was also performed to detemine the effect of dlayed rectifier K+ channel inhibitors on cell growth.This study showed that delayed rectifier K+ channel, existing in human gastric cancer cell line SGC7901, is related to the growth of the cell. The highly expressed COX-2 may affect the biological behaviour of gastric cancer cell by regulating this ion channel. COX-2 inhibitor ﹢

may inhibit delayed K channel (Wu et al., 2002). All in all , there is a singlificant mechanisn underlying COX-2-dependent for anti-tumor of COX-2 inhibitor in Figure 8 (Wang et al., 2005 modified).

Figure 8. (Wang et al., 2005 modified)

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

Roles of PGE2 in carcinogenesis. PGE2 mediates many of the molecular mechanisms contributing to pathology downstream of COX-2. These include proliferation, angiogenesis, invasion, reduction of apoptosis, immune responses and delayed K channel, etc.

3. Mechanism Underlying COX-2 Independent for Anti-Carcinogenes The regular use of various nonsteroidal NSAIDs was shown to decrease the incidence of gastrointestinal cancers. A similar protective effect has been demonstrated by the specific COX-2 inhibitors. (Jiang et al., 2002; Grösch et al., 2001). This effect is thought to be caused predominantly by inhibition of COX-2 and, subsequently, prostaglandin synthesis. However, NSAIDs show antitumor effects in cancer cells lacking COX-1 or COX-2 expression, and some derivatives lacking the ability to suppress COX activity. For example, NSAIDs induced suppression of proliferation and soft agar colony formation and induction of apoptosis in fibroblasts deficient for both of COX-1 and COX-2 (Zhang et al., 1999). FGN-1, a sulfone metabolite of sulindac that lacks COX-2-blocking activity inhibited tumor growth in mice (Skopinska-Rozewska et al., 1998). The first study which provided a potential mechanism for the chemopreventive effect of COX-2 specific NSAIDs was performed by Elder et al. in 1997 and the effect was not dependent on the expression of COX-2 protein (Elder et al., 1997). Also, NS-389, a selective COX-2 inhibitor, had the ability to induce apoptosis, and was chemopreventive by COX independent mechanisms. These data are also consistent with the observations that, nimesulide and SC-58635 inhibit the formation of aberrant crypt foci, the cells of which are unlikely to express COX-2 (Kısmet et al., 2004). The COX-2 inhibitor nimesulide induces apoptosis in gastric adenocarcinoma cells (Li JY et al., 2003). There are a lot of evidences that the ability of COX-2 inhibitors to promote apoptosis may be dissociated from the enzyme activity of COX-2. Firstly, there is a discrepancy by nearly three orders of magnitude between the concentration of celecoxib and other COX-2 inhibitors needed to inhibit COX-2 activity and that required to induce apoptosis. Secondly, different COX-2 inhibitors with similar IC50 values display different degree of efficacy in the induction of apoptosis; and the addition of exogenous PGE2 does not provide appreciable protection against celecoxib-induced apoptosis. Thirdly, although constitutive expression of COX-2 enhances the metastatic potential of intestinal epithelial cells, ectopic expression of COX-2 in endothelial cells results in growth disadvantage and increased apoptotic death (Kısmet et al., 2004). A number of studies have indicated that NSAIDs-induced apoptosis in a variety of cancers, including those of colon, stomach and prostate (Kısmet et al., 2004) . All of the in vitro and in vivo data indicate that the antitumor effects of celecoxib probably are mediated through COX-2 independent mechanisms and are not restricted to COX-2 over-expressing tumors (Grösch et al., 2001). All described above strongly suggest that NSAIDs exert some of their anti-tumor effects independent of inhibition of COX-2 activity (Shiff et al., 1999; Piazza et al., 1997; Malkinson et al., 1998; Thompson et al., 1997; Shiff et al., 1997). For the development of novel and effective therapies, it is required to elucidate mechanisms underlying antitumor effects of NSAIDs and COX-2 inhibitors (Tsujii, 2001; Jiang et al., 2002; Grösch et al., 2001). One

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potential mechanism for the chemopreventive effect of NSAIDs independent-COX-2 is through induction of apoptosis and inhibition of proliferation (Brown et al., 2001). The mechanism of NS-398 induced xenograft growth suppression was suggested to be in creased apoptosis and reduced angiogenesis (Saukkonen et al., 2003; Han et al., 1999). 3.1. Induce Apoptosis Some distinct mechanisms underlying independent COX-2 inhibitor-induced apoptosis have been proposed although other mechanisms remain unknown (Kısmet et al., 2004). 3.1.1. Several reports have confirmed COX-2 independent effects of celecoxib, at relatively high concentrations (50 microM), where apoptosis is stimulated in cells that are lack of both COX-1 and COX-2 (Figure 8). Moreover, celecoxib of structural modifications were no association between the COX-2 inhibitory and proapoptotic activities of celecoxib (Crosby et al., 2003). And some studies showed that celecoxib induced apoptosis in different cancer types (Kern et al., 2002; Wu et al., 2003). A study with anti-tumorigenic effects in vivo in a parallel dose-dependent manner has been demonstrated that NSAIDs blood plasma concentrations of 0.1-5 microM in vivo exerted antitumor effect (Raz, 2002). Significantly, the same compounds tested at the same concentrations in incubations with cultured tumor cells in vitro similarly inhibited COX activities, but are devoid of anti-proliferative activity. Yet, at much higher concentrations (100-20,000 microM), these same NSAIDs do exhibited anti-proliferative effects in vitro due to apparent non-specific toxic effects, as evidenced by disruption of ion transport and mitochondrial oxidation in some cells (Raz, 2002). High doses of NSAIDs can inhibit tumor cell growth and induc tumor cell apoptosis by regulating genes other than COX (Williams, et al., 2000; Janne and Mayer, 2000; Piazza et al., 1995), including 15-LOX-1 (Shureiqi et al., 2000), proapoptotic Par-4 (Zhang and DuBois, 2000), Many of the above studies employed concentrations of NSAIDs much higher than those required to inhibit COX-2 activity in vivo without significant toxicity. At therapeutic concentrations of NSAIDs, however, the best-characterized biochemical target of NSAIDs remain the cyclooxygenase enzymes (Wang et al., 2005). 3.1.2. Traditional NSAIDs sulindac, sulindac sulfone and salicylic acid, which lacks the ability to inhibit COX-2, inhibited tumorigenesis by COX-2 independent and PGEindependent mechanism (Raz, 2002) during the apoptosis signaling (Piazza et al., 1995; Hanif et al., 1996). But which can reduce cellular and intercellular level of PGE2 by inhibiting phospholipase-mediated release of arachidonic acid from phospholipids leading to depressed synthesis of prostanoids (Raz, 2002). One study showed that NSAIDs treatment of colon tumor cells resulted in an increase in arachidonic acid by stimulation of sphingomyelinase (which converts sphingomyelin to ceramide, a potent inducer of apoptosis (Chan et al., 2002), or, a known death signal, by activating neutral sphingomyelinase (Chan et al., 2002). Unesterified arachidonic acid modulates mitochondrial permeability and causes release of cytochrome C, resulting in apoptosis (Scorranoet et al., 2001). 3.1.3 Some of the COX-2 independent mechanisms for NSAIDs and selective COX-2 inhibitors include activation of protein kinase G (Crosby et al., 2003). NSAIDs suppressed expression of the antiapoptotic gene Bclxl, thereby increasing the cellular ratio of Bax to Bclxl. Bax / cells are resistant to NSAID-induced apoptosis (Zhang et al., 2000). An increase of c-myc protein and a decrease of Bcl-2 protein were observed in both cells treated ﹣﹣

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

with JTE-522 (Uefuji et al., 2000). JTE-522 could induce apoptosis by enhancing c-myc expression and diminishing Bcl-2 expression as well as suppressed proliferation activity in both cell lines (Uefuji et al., 2000). Mechanisms COX-2 independent involved may be a decrease of Bcl-2 expression (Huang et al., 2005). The COX-2 selective inhibitors were reported to sensitize colon and prostate cancer cells to apoptosis by down-regulating Bcl-2 (Sheng et al., 1998; Liu et al., 1998; Zhang et al., 2000). 3.1.4. Another mechanism underlying anti-tumor is inactivating peroxisome proliferators-activated receptor PPAR delta, an anti-apoptosis protein expressed in colon cancer cells with a mutated APC gene (He et al., 1999; Crosby et al., 2003) and activation of PPAR gamma (Crosby et al., 2003). 3.1.5 SC-236 induced apoptosis in gastric cancer cells was not dependent on COX-2 inhibition. SC-236 down-regulated the protein expression and kinase activity of PKC-beta (1), increased the expression of PKCdelta and PKCeta, but did not alter the expression of other PKC isoforms in AGS cells. Moreover, exogenous prostaglandins or PGE (2) receptor antagonists could not reverse the inhibition effect on PKCbeta (1) by SC-236, which demonstrated that this effect occurred through a mechanism independent of cyclooxygenase activity and prostaglandin synthesis. Over-expression of PKCbeta (1) attenuated the apoptotic response of AGS cells to SC-236 and was associated with overexpression of p21 (waf1/cip1). Inhibition of PKCbeta (1)-mediated over-expression of p21 (waf1/cip1) partially reduced the anti-apoptotic effect of PKCbeta (1). The down-regulation of PKCbeta (1) provides an explanation for COX-independent apoptotic effects of specific COX-2 inhibitor in cultured gastric cancer cells. It is suggested that PKCbeta (1) act as survival mediator in gastric cancer, and its down-regulation by COX-2 inhibitor SC-236 may provide new target for future treatment of gastric cancer (Jiang et al., 2002). 3.1.6. Mechanisms COX-2 independent involved may be accumulation of quiescent G0/G1 phase (Huang et al., 2005). A study showed that induction of apoptosis and alteration of cell cycle regulatory proteins were major mechanisms accounting for the anti-proliferative effects of celecoxib. The study assessed the effects of celecoxib (selective COX-2 inhibitor) and SC560 (selective COX-1 inhibitor) on cell survival, cell cycle distribution, and apoptosis in three colon cancer cell lines that either express COX-2 (HT-29 and Caco-2) or are COX-2 deficient (HCT-15). Both drugs induced a G0/G1 phase block and reduced cell survival independent of whether or not the cells expressed COX-2. Celecoxib was more potent than SC560. The G0/G1 block caused by celecoxib could be attributed to a decreased expression of cyclin A, cyclin B1, and cyclin-dependent kinase-1 and an increased expression of the cell cycle inhibitory proteins p21Waf1 and p27Kip1. In addition, celecoxib and SC560 induced apoptosis through independent of the COX-2 expression of the cells. In vivo, celecoxib as well as SC560 reduced the proliferation of HCT15 (COX-2 deficient) colon cancer xenografts in nude mice, but both substances had no significant effect on HT-29 tumors, which express COX-2 constitutively (Grösch et al., 2001). The time course of the cell cycle and the apoptosis-related protein levels were examined in order to determine the mechanisms of apoptosis induction by JTE-522. The result demonstrated that an increase in the G1 phase and a decrease in the S phase were observed

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prior to apoptosis (Uefuji et al., 2000). The study suggested that JTE-522 induced apoptosis by blocking the cell cycle and suppressed proliferation activity (Uefuji et al., 2000). 3.1.7. Celecoxib-induced apoptosis, in part, by blocking the activation of the antiapoptotic kinase Akt (protein kinase B) in human prostate cancer cells and induction of apoptosis by celecoxib was independent of Bcl-2, an antiapoptotic protein. The independence of Bcl-2 function suggests the potential use of celecoxib in the treatment of metastatic prostate cancer. In contrast, overexpression of constitutively active Akt protected prostate cancer cells from celecoxib-induced apoptosis (Kısmet et al., 2004). 3.1.8 Sodium salicylate, sulindac and two sulfated sulindac metabolites inhibit activation of I-kB kinase B(IKKB) in many tumor cell lines, resulting in inhibition of antiapoptotic NFκB. Activation and reduced tumor cell survival (Yamamoto et al., 1999; Grilli et al., 1996). 3.1.9. A study investigated the distribution of catenin in 1,2-dimethylhydrazine-induced rodent colorectal tumors from animals treated with NSAIDs. The frequency of nuclear βcatenin staining correlated directly with the volume of tumor and inversely with the rate of apoptosis. NSAID treatment resulted in significantly less nuclear β-catenin staining; however, the cytoplasmic staining for β-catenin was unchanged. The result indicated that inhibition of nuclear β-catenin translocation might cause reduced transcription of target genes that were associated with apoptosis and cell proliferation (Kısmet et al., 2004; Brown et al., 2001). 3.1.10. celecoxib induced apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phospho-inositide-dependent kinase-1 activity (Arico et al., 2002). 3.1.11. In addition, COX-2 inhibitor reduce tumor gowth may have no PG involving (Han et al., 1999). The inhibition of COX-2 down-regulated production of PGs, cellular arachidonic acid (the metabolic precursor of PG) may be incraesed. The effect of increased pool of arachidonic acid may be shunted down other metabolic pathways, such that there is increaseed production of other metabolites that suppress cellular division. This suggests that COX-2 takes part in other cellular reactions beyond the production of PGs. NSAIDs are postulated to shift the metabolite profile from COX-2 metabolites to lipoxygenase (LOX)-derived metabolites, resulting in the accumulation of substrate for LOXderived products (Shureiqi and Lippman, 2001) and the lipoxygenase pathway functions as a regulator of apoptosis (Tang et al., 1996). Inhibition of lipooxygenases leads also to cell death through increased cytoplasmic concentrations of arachidonic acid. 3.1.12. Another possible mechanism of apoptosis regulation is the effect of celecoxib on intracellular calcium concentration. The study found that exposure of prostate cancer cells to celecoxib stimulated an immediate intracellular calcium concentration rise in a dose-and time-dependent manner (Johnson et al., 2002). This might represent part of the signalling mechanism that celecoxib used to trigger rapid apoptotic death in cancer cells (Hsu et al., 2000). 3.2 Inhibite Angiogenesis Inhibition of angiogenesis by NSAIDs might also occur through COX-2 independent mechanisms. This possibility is suggested by the observation that exogenous PGE2 or PGI2 did not reverse the suppression of ανβζ mediated cell migtion and Rac activation in response to indomethacin (Dormond et al., 2001). Moreover, exogenous PGE2 or PGI2 failed a: so to

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

prevent the inhibition of VEGF- induced MAPK activation in indomethacin-treated cells (Jones et al., 1999). One or more of these COX-2 independent effects could contribute to the antiangiogenic properties of NSAIDs and selective COX-2 inhibitors (Crosby et al., 2003). These studies show that antiangiogenic activity of cele coxib may be an important mechanism of action in inhibiting tumor growth and metastasis (Kısmet et al., 2004). 3.3. Suppress Proliferotion SC-236, COX-2 selective inhibitor, blocked JNK-c-Jun/AP-activation and suppressed gastric cancer cell growth in vitro, suggesting that COX-2 can induce cell growth through JNK-AP-signaling pathway (Wong et al., 2004).

4. Mechanism Both (Combinated) of COX-2 Dependent and Independent Pathway 4.1. COX-2, insulin-like growth factor (IGF) II, and IGF-I receptor (IGF-IR) are upregulated in tumor and associated with tumor growth and invasion. This study investigated the effects of COX-2 inhibitor and drugs blocking the biological activities of angiotensin II, angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs), on IGF-IR expression and tumor growth, and also studied the effects of PGE(2), a major COX-2 product, in cancer cells and the effects of angiotensin II on IGF-IR expression,in vivo with Colon 26 cells inoculated into BALB/c mice. On the other hand, in vitro study, three colon cancer cell lines (Colon 26, HCA-7, and LS174T)were examinated the effects of NSAIDs on IGF-IR expression. The results showed that NSAIDs reduced IGF-IR expression in a dose-dependent manner in all three cell lines. NSAIDs also inhibited IGF-II-stimulated growth and invasion in a dose-dependent manner. PGE(2) or angiotensin II treatment reversed the NSAID-induced down-regulation of IGF-IR expression, growth, and invasion in vitro. It is suggested that PGE(2) and angiotensin II induced Akt phosphorylation, and LY294002 or wortmannin inhibited PGE(2) or angiotensin II-induced IGF-IR expression, indicating that PGE(2) and angiotensin II both regulate IGF-IR expression by the same Akt/phosphatidylinositol-3 pathway. Thus, combination therapy with NSAIDs and ACE inhibitors targeting IGF-IR might be a novel and potentially promising strategy for the chemoprevention of cancer (Yasumaru et al., 2003). 4.2. Another type of non-COX inhibitors are the R-isomers of NSAIDs, based on the structure of 2-arylpropionic acid. Comparison of the anti-tumoriogenic activities of R-flurbiprofen (non-COX-inhibition) and S-flurbiprofen (COX inhibition) in an experimental mouse tumor model found that the R isomer,at 10 mg/kg per day, significantely improved several tumor and biological endopoints (number of tumors, weight gain) as well as prolonged survival (Raz, 2002). R-Flurbiprofen was also found to be effective in inhibiting the progression of prostate cancer in a mouse prostate cancer model (Raz, 2002). These results strongly demonstrate involvement of a COX-2 independent mechanism in this process. This finding should be tested for a possibility of R to S inversion in vivo that would render the R-isomer effect as being due to the S-isomer

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generated in vivo from it, as such inversion were demonstrated to occur to varied extents in several species (Raz, 2002). The exact mechanism of action underlying the anti-proliferative effect of the P-isomer is unknown, althrough a partial but significant inversion in vivo to the COX-inhibiting S-isomer is probably when administrating NSAIDs (Raz, 2002). 4.3. In order to elucidate relationship between COX-2 expression and certain growth hormones in gastric carcinogenesis whether specific blockade of this enzyme has any influence on tumor growth and progression, the influence of rofecoxib (Vioxx), a specific COX-2 inhibitor, for twenty-four gastric cancer (GC) patients were examined twice with serum and tumor levels of gastrin and its precursor, progastrin, as well as on tumor gene expression of COX-2, peroxisome proliferator-activated receptor gamma (PPARgamma), and apoptosis-related proteins (Bax and Bcl-2, caspase-3, and surviving; Konturek et al., 2003). Treatment of GC patients with Vioxx resulted in a significant decrease in plasma and tumor contents of both progastrin and gastrin and this was accompanied by the increment in tumor expression of COX-2, PPARy, Bax, and caspase-3 with a concomitant reduction in Bcl-2 and survivin expression (Konturek et al., 2003). It is suggested that serum levels of progastrin, gastrin, IL-8 and TNF-alpha were significantly higher in GC patients than in matched controls, as confirming that both gastrins may be implicated in gastric carcinogenesis and more widespread gastritis in GC. COX-2, PPARgamma, Bcl-2, and survivin were overexpressed in gastric cancer, which was showed that gastrin and proinflammatory cytokines could mediate the up-regulation of COX-2 in gastric cancerogenesis. The inhibition of COX-2 activity by Vioxx resulted in a significant reduction in serum and tumor levels of progastrin and gastrin and serum IL-8 and TNF-alpha levels (Konturek et al., 2003). It also enhanced expression of COX-2, PPARy, Bax, and caspase-3, while inhibiting the expression of Bcl-2 and surviving. The result indicated that COX-2 inhibitor exert chemoprevention against gastric cancer possibly is due to enhancement of the PPARy and proapoptotic proteins-dependent apoptosis and the reduction in progastrin/gastrin-induced promotion of tumor growth (Konturek et al., 2003).

COX-2 Inhibitor for Gastrointestinal Lesion Introduction NSAIDs are still the most commonly used remedies for rheumatic diseases. But, NSAIDs have been shown to cause mucosal injury in the gastrointestinal tract as a side effect, for example, gastric ulceration and renal damage, occasionally turning out to be severe complications such as bleeding and perforation (Bertolini et al., 2002; Sakamoto, 2003). The discovery of COX-2 has provided the rationale for the development of a new class of NSAIDs, the selective COX-2 inhibitors (denominated coxibs), with the aim of reducing the gastrointestinal (GI) toxicity associated with the administration of NSAIDs by virtue of COX-1 sparing (Capone et al., 2003). It means that they appear to be devoid of gastrointestinal toxicity, in that they spare mucosal prostaglandin synthesis (Bertolini et al.,

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Yumin Li, Raaj K. Praseedom, Andrew Butler, Ligang Zhou and Yonghong Yang

2002). A number of researches involved with COX-2 inhibitor for gastrointestinal lesion have reported to benefit from the drugs with anti-inflammatory, analgesia and anti-tumorigenesis.

1. Search for New Agents It is well known that a major clinical problem encountered with the use of classical NSAIDs, is gastrointestinal complications. However, in a large clinical trial, COX-2 inhibitors have demonstrated to halve the incidence of serious upper GI events vs a nonselective NSAID (Capone et al., 2003). The potential clinical benefit of COX-2 inhibitors is significant due to the number of patients chronically treated with NSAIDs and the three to ten fold higher risk of gastrointestinal injury and death associated with traditional NSAIDs. Recently, a class of anti-inflammatory medications has developed, which primarily inhibits COX-2 while sparing the enzymatic activity of COX-1 at therapeutic dosages (Urban, 2000). Particularlly, specifically, selective inhibitors of COX-2 were developed in order to improve the anti-inflammatory and analgetic specificity and potency of the compounds and to reduce side effects in the GI tract (Meyer-Kirchrath and Schror, 2000). The development of coxibs was based on the notion that inhibition of COX-2, the induced isoform of COX, will diminish the proinflammatory activities of COX, whereas sparing COX-1, the constitutive isoform of COX, will diminish the gastrointestinal and other side effects of NSAIDs (Simmons et al., 2004). A number of available experimental studies and clinical trials of selective COX-2 inhibitors have been conducted over the past ten years that generally support the favourable side effect profile of COX-2 preferential compounds. It suggests improved gastric tolerance as compared to conventional, non-selective NSAIDs (Meyer-Kirchrath and Schror, 2000). For examples, a meta analysis of controlled trials with nimesulide indicated that it had a better risk-benefit ratio than the standard NSAIDs (Wober, 1999). In a number of studies, etodolac showed reduced side effects on the gut compared with standard NSAIDs (Schnitzer and Constantine, 1997). In another comparative study, comparable efficacy and safety were also reported with nabumentone and the clinically used agent aceclofenac (Gijon, 1997). In the MELISSA trial in 10,000 patients with osteoarthritis and the SELECT trial involving 9286 patients with osteoarthritis, a significantly reduced incidence of side effects was noted in the meloxicam group compared with the comparators diclofenac and piroxicam, respectively (Whittle, 2000). Therefore, these clinical effects were indeed upheld by the selective COX-2 inhibitors. Celecoxib was approved for the treatment of rheumatoid arthritis, osteoarthritis and for relief of acute pain by the FDA, based on submitted findings from 5285 patients in controlled trials. This is more important that celecoxib has become the first approved drug for phaseIII studies on regression and reduction of colorectal polyps in familial adenomatous polypopsis (FAP), (a precancerous condition, this disease is a precursor of colonic cancer in these people) in December 1999, and approval was based on a 28% reduction in polyp number in a double blind study in 83 patients compared with 5% reduction with placebo. The efficacy of celecoxib in treating colon cancer is being evaluated in a phase III study while its usefulness in a number of other cancer types, including Barrett’s oesophagus and sporadic adenomatous

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colonic polyps, is also being explored (Whittle, 2000). In a study in 665 patients with rheumatoid arritis over a 24 weeks period, gastroduodenal ulceration was detected in only 4% of patientsreceiving celecoxib in contrast with 15% in the diclofenac treatment because of gastrointestinal side effects (Emery et al., 1999). In a 12 weeks study in osteoarthritic patients, celecoxib and naproxen had equal efficacy and were both well tolerated (Bensen et al., 1999). In addition to anti-inflammatory utilities in osteoarthritis, the COX-2 selective rofecoxib has been approved by the FDA for the treatment of acute pain in adults, dysmenorrhoea, and osteoarthritis as well as chemotherapeutic control of cancer, based on preclinical efficacy and findings from clinical studies (Whittle, 2000). In a comparison of eight studies involving 5435 patients with osteoarthritis, rofecoxib was associated with an overall significantly lower incidence of upper gastrointestinal tract bleeding than the comparator NSAIDs, including diclofenac and ibuprofen (Langman et al., 1999). Rofecoxib has been shown to spare COX-1 activity ex vivo, in platelets and gastric mucosa, when administered at therapeutic doses (Capone et al., 2003). Recently, based on a hypothesized with the peculiar chemical feature may lead to an enhanced concentration in inflammatory sites that may translate into an improved clinical efficacy, other selective COX-2 inhibitors with different COX-1/COX-2 selectivity and pharmacokinetic features have been developed, for instance, valdecoxib, parecoxib, etoricoxib and lumiracoxib. The improved biochemical selectivity of valdecoxib vs celecoxib in vitro (COX-1/COX-2 ratio: 60 vs 30, respectively) may result in clinically relevant an improved GI safety. Especially, parecoxib, a pro-drug of valdecoxib, is the only injectable coxib. Etoricoxib, showing only a slightly higher COX-2 selectivity than rofecoxib in vitro (COX-1/COX-2 ratio: 344 vs 272, respectively), has been reported to cause a similar specific COX-2 inhibition ex vivo that should translate into comparable GI safety. Lumiracoxib, the most selective COX-2 inhibitor in vitro (COX-1/COX-2 ratio: 400), is the only acidic coxib. The results of clinical trials have shown that coxibs have a improving clinical efficacy (Capone et al., 2003). Additionally, several strategies have been adopted in order to avoid gastrointestinal toxicity induced by NSAIDs associated with gastroprotective agents that counteract the damaging effects of prostaglandin synthesis suppression. An incorporation of a nitric oxide (NO)-generating moiety into the molecule of several NSAIDs was shown to greatly attenuate their ulcerogenic activity (Bertolini et al., 2002). However, a combination therapy introduces problems of pharmacokinetics, toxicity, and patient’s compliance etc (Bertolini et al., 2002). Moreover, COX-2-selective compounds at anti-inflammatory doses might have other sideeffects, namely, COX-2 selective NSAIDs might have lost the cardiovascular protective effects of non-selective NSAIDs, effects which are mediated through COX-1 inhibition (in addition, COX-2 has a role in sustaining vascular prostacyclin production) (Bertolini et al., 2002; Meyer-Kirchrath and Schror, 2000). Conversely, COX-1 (not only COX-2) may also be induced at sites of inflammation. Besides prostaglandins, it also is another important inflammatory mediator, leukotrienes. This is because products generated by the 5lipoxygenase pathway (leukotrienes) are particularly important in inflammation: indeed, leukotrienes increase microvascular permeability and are potent chemotactic agents; moreover, inhibition of 5-lipoxygenase indirectly reduces the expression of TNF-alpha (a

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cytokine that plays a key role in inflammation). This indicates the efforts to obtain drugs able to inhibit both 5-lipoxygenase and cyclooxygenases: Such compounds retain the activity of classical NSAIDs, while avoiding their main drawbacks, in that curtailed production of gastroprotective prostaglandins is associated with a concurrent curtailed production of the gastro-damaging and bronchoconstrictive leukotrienes. Furthermore, combination drugs could not merely alleviate symptoms of rheumatic diseases, but might also satisfy, at least in part, the criteria of curative drugs. Indeed, leukotrienes are pro-inflammatory, increase microvascular permeability, are potent chemotactic agents and attract eosinophils, neutrophils and monocytes into the synovium (Bertolini et al., 2002). Therefore, indeed, multi-pathway inhibition may be the newly promising way in chemoprevention. Such considerations have led to the development of dual inhibitors of both COX and 5-LOX. Licofelone, a compound blocking both a LOX and COX is already in clinical trials (Figure 9 Rigas and Kashfi, 2005). These new inhibitors are called dual acting anti-inflammatory drugs (Ulbrich et al., 2005). A study investigated the effect of the LOX/COX inhibitor licofelone for contribution of mechanisms other than the reduction of inflammatory prostaglandins and leukotrienes to the anti-inflammatory. The study showed that the novel 5-LOX/COX inhibitor licofelone possesses anti-inflammatory activity that, in addition to COX/LOX inhibition, involves effects on leukocyte-endothelial interactions (Ulbrich et al., 2005). Recent data strongly suggest that dual inhibitors may have specific protective activity also in neurodegeneration (Bertolini et al., 2002).

Figure 9. (Rigas and Kashfi, 2005). Overvew of arachidonic metabolism and blocking approaches of various drugs

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2. Mechanism Underlying Gastrointestinal Lesion of Nsaids and COX-2 Inhibitor A number of recent studies raised serious questions about the two central tenets that support this approach, namely that the prostaglandins that mediate inflammation and pain are produced solely via COX-2 and that the prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1 (Bertolini et al., 2002). Inhibition of prostaglandin biosynthesis via inhibition of the fatty acid COX is the mechanism of action of NSAIDs. This results in an inhibition of the inflammatory and painproducing activities of prostaglandins at a site of tissue injury but also in inhibition of prostaglandin production in the gastrointestinal tract (GI) and platelets, i.e. sites where endogenous prostaglandins are possibly involved in control of physiological functions (Meyer-Kirchrath and Schror, 2000). According to conventional concept, NSAIDs-caused mucosal injury was attributed to their inhibitory effects on the activity of COX-1 which is expressed and shown to play a crucial role for the mucosal protection via producing prostaglandin E2 in the stomach. However, a recent progress of the understanding about COX physiology has revealed that NSAIDs cause gastric mucosal injury by inhibiting not only COX-1 but also COX-2 in the stomach (Sakamoto, 2003). A study relevant mechanism of COX-2 inhibitor generated gut tract events was investigated in vivo with measuring gastric damage, PGE2 content, mucosal permeability, myeloperoxidase (MPO) activity as well as gastric motility were examined (Tanaka et al., 2002). The results demonstrated that nonselective COX inhibitor indomethacin inhibited PGE2 production, enhanced gastric motility, and provoked severe lesions in the stomach, with an increase in mucosal permeability and MPO activity. Whereas, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach and had no effect on mucosal PGE2 content. Similarly, the selective COX-1 inhibitor SC-560 also caused no gastric damage, despite inhibiting PGE2 production. But, the combined administration of SC-560 and rofecoxib, stimulated gross damage in the gastric mucosa, in a dose-dependent manner for each drug. SC-560, but not rofecoxib, caused marked gastric hypermotility and an increase in mucosal permeability, although an increase in MPO activity was observed only when rofecoxib was coadministered. The normal gastric mucosa expressed COX-1 mRNA and not COX-2 mRNA, but COX-2 mRNA was expressed in the stomach after administration of SC-560 as well as indomethacin but not rofecoxib. It is suggested that the gastric ulcerogenic properties of NSAIDs are not accounted for solely by COX-1 inhibition, but require the inhibition of both COX-1 and COX-2 (Bertolini et al., 2002; Tanaka et al., 2002; Brzozowski et al., 2001). A study involved in COX-2 inhibitor during healing of chronic gastric ulcers was reported in order to elucidate the role for expression of COX-2 and its products such as PGE2 and cytokines ,including interleukin (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in ulcer healing in Wistar rats (Brzozowski et al., 2001). In this study there were 6 groups: vehicle (saline); NS-398 and Vioxx, both, highly specific COX-2 inhibitors; meloxicam, a preferential inhibitor of COX-2; resveratrol, a specific COX-1 inhibitor; indomethacin; and aspirin, non-selective inhibitors of both COX-1 and COX-2. The area of gastric ulcers was determined by planimetry and histology, gastric blood flow (GBF) at ulcer base and margin

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was measured by H2 clearance technique, and blood was withdrawn for measurement of plasma IL-1beta and TNFalpha levels. The mucosal biopsy samples were taken for the determination of PGE2 generation by RIA and expression of COX-1, COX-2, IL-1beta, and TNFalpha mRNA by RT-PCR. In vehicle-treated rats, gastric ulcers healed progressively and at day 14 the healing was completed, accompanied by a significant rise in the GBF at ulcer margin. The IL-1beta, TNFalpha, and COX-1 mRNA were detected in intact and ulcerated gastric mucosa, whereas COX-2 mRNA were upregulated only in ulcerated mucosa with peak observed at day 3 after ulcer induction. The plasma IL-1beta level was significantly increased at day 3 and 7 but then declined at day 14 to that measured in vehicle-controls. Indomethacin and ASA, which suppressed PGE2 generation both in the non-ulcerated and ulcerated gastric mucosa, significantly delayed the rate of ulcer healing and this was accompanied by the fall in GBF at ulcer margin and further elevation of plasma IL-1beta and TNFalpha levels, which was sustained up to the end of the study. Treatment with NS-398 and Vioxx, which caused only a moderate decrease in the PGE2 generation in the non-ulcerated gastric mucosa, delayed ulcer healing and attenuated significantly the GBF at ulcer margin and PGE2 generation in the ulcerated tissue, while raising the plasma IL-1beta and TNFalpha similarly to those observed in indomethacin and ASA treated rats. Resveratrol, which suppressed the PGE2 generation in both non-ulcerated and ulcerated gastric mucosa, prolonged ulcer healing and this was accompanied by the fall in the GBF at the ulcer margin and a significant increase in plasma IL-1beta and TNFalpha levels (Brzozowski et al., 2001). The result demonstrated that classic NSAIDs delay ulcer healing due to suppression of endogenous PG, impairment in GBF at ulcer area, and excessive cytokine expression and release, and this adverse effect of classic NSAIDs on the healing of pre-existing ulcers can be reproduced by selective COX-1 and COX-2 inhibitors (Brzozowski et al., 2001). The mechanism is probably that the inhibition of COX-1 up-regulates COX-2 expression, and COX-2/PGs may, in turn, counteract the deleterious affects of gastric hypermotility due to COX-1 inhibition. Therefore, evidence is accumulating that COX-2 might not only be considered as a putatively detrimental enzyme but rather a highly regulated enzyme that also contributes to tissue protection and is even constitutively expressed in healthy human stomach mucosa (Meyer-Kirchrath and Schror, 2000). It plays a physiological role in several body functions (Bertolini et al., 2002). On the other hand, a different study with NSAIDs induce both necrosis and apoptosis in vitro and the COX dependency of this cytotoxic effect of NSAIDs and its involvement in NSAIDs induced gastric lesions was investigated (Tomisato et al., 2004). This result showed that all selective COX-2 inhibitors except rofecoxib had necrosis and apoptosis by NSAIDs and was not inhibited by exogenously added prostaglandin E2, It was indicated that cytotoxicity of NSAIDs seems to be independent of the inhibition of COX. Orally administered selective COX-2 inhibitors, which did not inhibit COX at gastric mucosa, also did not produce gastric lesions. Intravenously administered indomethacin, which completely inhibited COX activity at gastric mucosa, did not produce gastric lesions. But, a combination of the oral administration of each of all selective COX-2 inhibitors except rofecoxib with the intravenous administration of indomethacin clearly produced gastric lesions. These results demonstrated that in addition to COX inhibition by NSAIDs, direct cytotoxicity of NSAIDs may be involved in NSAIDs induced gastric lesions (Tomisato et al., 2004).

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3. Summary Recent study demonstrated that inhibition of both COX-1 and COX-2 is required for the development of NSAIDs induced gastric lesions. The mechanism is probably that the inhibition of COX-1 up-regulates COX-2 expression. COX-2 might not only be considered as a putatively detrimental enzyme, but also contributes to tissue protection and is even constitutively expressed in healthy human stomach mucosa; Yet NSAIDs induced gastric lesions induced by NSAIDs may result from direct cytotoxicity of NSAIDs

Current Status and Perspective of COX-2 Inhibitor for Anti-Tumor Epidemiological, clinical and experimental studies established NSAIDs as promising cancer chemopreventive agents. Long-term use of aspirin and other NSAIDs has been shown to reduce the risk of cancer of the colon and other gastrointestinal organs as well as of cancer of the breast, prostate, lung, and skin (Rao and Reddy, 2004). Understanding the action of NSAIDs provides substantial insights into the mechanisms by which these unique agents regulate tumor cell growth and enable better strategies for prevention and treatment. Discovery of an isoform of COX- 1, the inducible COX-2, has made it possible to avoid some side effects of non-specific COX inhibitors (Grover et al., 2003). A number of studies have been shown that specific COX-2 inhibitors exert experimentally and clinically ptontial chemoprevention role for many tumors. Whereas, efficacy, safety, cost and ease of administration will likely be the main criteria for candidate chemoprevention agents and the balance of their risk/benefit relationship will need to be assessed (Rigas and Kashfi, 2005).

1. Currently Unclear and/or Unsolved Issues for COX-2 Inhibitor as Clinical Agent 1.1. Influence to Gut Proof of principle has come from a range of experimental models and clinical studies which demonstrate the clinical efficacy of DSAIDs as anti-inflammatory and analgesic agents as well as anti-tumorigenesis (Whittle, 2000). Whereas, a numbers are available for the incidence of the side effects of NSAIDs, As many as 25% of individuals using NSAIDs experience some side effect and up to 5% develop serious health consequences (Marco et al., 2002). Another report showed that among patients using NSAIDs, up to 4% per year suffer serious gastrointestinal complications (Rigas and Kashfi, 2005). The issue of safety is clearly underscored by the report that in 1998 in the US the number of deaths from NASAIDinduced gastrointestinal complications was virtually equal to the number of deaths from AIDS (Rigas and Kashfi, 2005). Particularly as these agents could delay the healing of duodenal ulcers and interfere with several COX-2-induced physiological functions (Blain et al., 2000).

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Although COX-2 inhibitors have more advantages in gut compared with traditional NSAIDs, it is clear, however, that these COX-2 selective agents cannot be classed as “super aspirins” as their therapeutic actions as anti-inflammatory analgesics demonstrated so far do not surpass those of aspirin or the classical NSAIDs (Whittle, 2000). It is not known how the selective COX-2 inhibitor drugs will behave on more prolonged high dose administration over several years, especially under the conditions prevailing in patients with chronic inflammatory conditions or in the elderly (Whittle, 2000). In addition, whether such agents, in common with other NSAIDs, would be counterindicated in patients with inflammatory bowel diseases has not been established in appropriate clinical studies. Exacerbation with a range of COX-2 selective inhibitors in experimental colonic inflammation has been reported, and such agents do not appear to offer anti-inflammatory benefit in colitic models (Lesch et al., 1999). It is not yet known whether such high specificity in novel COX-2 inhibitors will be associated with greater clinical efficacy or a superior side effect profile than the existing COX-2 inhibitors. It is possible that any newer more selective agents will receive the endorsement, so far withheld by regulatory agencies such as the FDA for the two launched products, that selective COX-2 inhibiting agents represent a new class of anti-inflammatory agent (Whittle, 2000). 1.2. Influence of Cardiovascular System COX-2 inhibitors may open up a new therapeutic era in which these drugs can be used for chemo-prophylaxis. However, COX-2 selective inhibitors retain renal adverse effects of the non-selective inhibitors and the concern regarding the pro-thrombotic potential of COX-2 inhibitors will limit their value as chemo-preventive agents (Grover et al., 2003). COX-2 inhibitors would not be suitable for some key indications for which aspirin is used, particularly in the prevention of platelet aggregation and cardiovascular disease, a fact that must be emphasised to the prescribing community (Whittle, 2000). Another problem of COX-2 inhibitor is cardiovascular side effects of coxibs. This may be particularly relevant to chemoprevention, in which a chemopreventive agent against cancer will be administered on a long-term basis to older subjects, i.e., those likely to have atheromatous lesions. Now rofecoxib has been withdrawed from trial. This is because two thousand six hundred subjects with previously removed colorectal polyps were randomly assigned to receive rofecoxib or placebo. The data showed that 3.5% of rofecoxib recipients and 1.9% of placebo recipients suffered myocardial infarctions or strokes during the trial. These outcomes of adverseness prompted the termination of this and all related trials and the permanent withdrawal of rofecoxib. This has been a episode to the use of COX-2 inhibitors to prevent colon and perhaps other cancers. (Rigas and Kashfi, 2005). The mechanism of the increased cardiovascular risk is not yet clear. It has been suggested that since COX-2 is the principal enzyme involved in the production of PGI2 (prostacyclin), its inhibition by COX-2 inhibitors could increase cardiovascular risk by tipping the balance toward platelet aggregation and vasoconstriction (Fitzgerald et al., 2004). Remarkably, a recent study demonstrated that COX-2 derived prostacyclin possesses atheroprotection on female mice, suggesting that chronic treatment of patients with selective inhibitors of COX-2 could undermine protection from cardiovascular disease in premenopausal females (Egan et al., 2004).

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1.3. The Clinical Effect of COX-2 Inhibitor The limited clinical efficacy: A Medline search was performed of English-language experimental studies and controlled clinical trials from January 1980 to January 2002, and relevant citations were noted. Results show that review of available literature shows that sulindac and COX-2 inhibitors are effective in preventing as well as regressing familial adenomatous polyposis. However, they have not been shown to prevent cancer in these patients (Grover et al., 2003). At present NSAIDs and COX-2 inhibitor cannot be recommended for average-risk individuals or for those with sporadic colorectal neoplasia (or other forms of cancers) as chemo-preventive agents (Grover et al., 2003). Although NSAIDs, particularly selective COX-2 inhibitors such as celecoxib, have been shown to inhibit angiogenesis in cell culture and in rodent models of angiogenesis, whereas, exploration of the multistep process of carcinogenesis has provided substantial insights into the mechanisms by which NSAIDs modulate these events (Rao and Reddy, 2004). Moreover, unresolved questions with regard to safety, efficacy, optimal treatment regimen, and mechanism of action currently limit the clinical application of these drugs to the prevention of polyposis in FAP patients (Rao and Reddy, 2004). The limited clinical efficacy of coxibs When the COX-2 inhibitor celecoxib was used in FAP, suppression of the neoplastic process was modest. After six months, FAP patients receiving 400 mg of celecoxib twice daily had a 28.0% reduction in the mean number of colorectal polyps and a 30.7% reduction in the polyp diameter, as compared with reductions of 4.5% and 4.9%, respectively, in the placebo group (Steinbach et al., 2000). Furthermore, taking together a global overview of the status of the duodenum, all patients administration of celecoxib 400 mg twice a day for 6 months had a 14.5% reduction in involved areas compared with a 1.4% for placebo .But, patients with clinically significant disease at baseline (greater than 5% covered by polyps) showed a 31% reduction in involved areas with celecoxib 400 mg twice daily compared with 8% on placebo (Rigas and Kashfi, 2005). These results could be interpreted in one of two ways: either COX-2 is not central to the neoplastic process or celecoxib is not a sufficiently strong COX-2 inhibitor (Rigas and Kashfi, 2005). Whereas, all Sulindac had a potential effect on colorectal polyps in FAP patients. A study with statistically significant decrease in number of polyps and their diameter occurred in patients treated with sulindac (150 mg orally twice a day), as compared with placebo. When treatment was stopped at nine months, compared to baseline values, the number of polyps had decreased by 56% and the diameter of the polyps by 65% (Rigas and Kashfi, 2005). Other studies have shown that long-term use of sulindac is effective in reducing polyp number and preventing recurrence of higher-grade adenomas in the retained rectal segment of most FAP patients (Cruz-Correa et al., 2002). Whereas, what these studies have failed to provide is the details of the big picture: which one of the nearly 30 NSAIDs is the most effective, what is the optimal (Rigas and Kashfi, 2005). Therefore, COX-2 inhibition alone is sufficient to arrest carcinogenesis, may be wrong. In fact, it would not be altogether surprising if it is COX-2 that is the “collateral target” in cancer prevention (Rigas and Kashfi, 2005)

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1.4. Disadvantages of COX-2 as a Target for Cancer Prevention Epidemiological and experimental studies have demonstrated the effect of NSAIDs in the prevention of human cancers (Xu, 2002). NSAIDs block endogenous prostaglandin synthesis through inhibition of cyclooxygenase COX enzymatic activity. COX-2, a key isoenzyme in conversion of arachidonic acid to prostaglandins, is inducible by various agents such as growth factors and tumor promoters, and is frequently overexpressed in various tumors. The contribution of COX-2 to carcinogenesis and the malignant phenotype of tumor cells has been mainly considered to be related to its abilities of increase production of prostaglandins; convert procarcinogens to carcinogens; inhibit apoptosis; promote angiogenesis; modulate inflammation and immune function, as well as increase tumor cell invasiveness (Xu, 2002). Overexpression of COX-2 has been demonstrated for several human cancers, besides colorectal adenocarcinomas, including gastric, breast, lung, esophageal and hepatocellular carcinomas (Turini and DuBois, 2002). However, there are a few disadvantages of COX-2 as a target for prevention and treatment of carcinogenesis, or, there are lots different pointviews.

1.4.1. Not Concordant Results In case these studies do not provide compelling results or COX-2 colon carcinomas (Rigas and Kashfi, 2005). A potentially significance of discovery, a recent study indicated that single nucleotide COX-2 polymorphisms may be associated with disease or with individual responses to drug therapies. A study has indicated reduced an association between the COX-2 V511A response to NSAIDs, appears to be supported. Polymorphisms in other genes of the eicosanoid pathway may account for inter-individual differences in cancer susceptibility or response to NSAIDs (Lin et al., 2002).

1.4.2. Shift Arachidonic Acid to the LOX Pathway COX-2 inhibition may modulate alternative eicosanoid pathways in a manner that promotes carcinogenesis (Rigas and Kashfi, 2005). The concept of the role of COX-2 in cancer should be viewed against the accumulating appreciation of the role of the eicosanoid pathways in carcinogenesis (Rigas and Kashfi, 2005). Thus a case can be considered where inhibition of COX-2 could shift arachidonic acid to the LOX pathway thereby suppressing apoptosis. Lipoxygenase (LOX) products may also play a role in carcinogenesis (Shureiqi and Lippman, 2001). A recent human study suggested that oral celecoxib increased LTB4 production (Rigas and Kashfi, 2005). That is not a desirable effect in cancer prevention (Figure 1, Figure 9).

1.4.3. Effect of COX-1 Deletion of COX-1 also attenuated tumor formation in Apc 716 mice (Chulada et al., 2000). It means that COX-1 (as well as COX-2) plays a key role in intestinal tumorigenesis; therefore, it may also be a chemotherapeutic target for NSAIDs (Chulada et al., 2000).

1.4.4. Model that COX-2 Is Central to Carcinogenesis? The pattern of COX-2 expression during colon carcinogenesis does not entirely fit the model that COX-2 is central to carcinogenesis. There is expression in aberrant crypt foci

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would be highly advantageous, if not a requirement, as chemoprevention would be easiest at this stage of lower complexity. Moreover, the expression of COX-2 commences only at the adenoma stage and in less than half of them, increasing as the transition to cancer progresses and becoming positive in 85% of them (Eberhart et al., 1994).

1.4.5. NSAIDs and Coxs Inhibit Tumor Through COX-2-Independent Pathway Whether COX-2 as a central role in carcinogenesis is the controversial concept. Originally study was that NSAIDs do not require the presence of COX-2 to prevent cancer (Hanif et al., 1996). This was because in vitro NSAIDs exert effects compatible with cancer prevention such as inhibition of cell proliferation, induction of apoptosis, inhibition of angiogenesis and many others, in the deficience of COX-1 or COX-2. Later, newly reported by many studies pointing to an array of molecular targets affected by NSAIDs (Shiff and Rigas, 1999). Most studies using cell culture systems have to be terminated by 72 to 96 hours and thus high concentrations of an agent are at times necessary to detect an effect. In contrast in vivo chemoprevention studies comparatively lower doses of an agent are administered for much longer periods of time and the effect of the agent may thus become apparent. The “low dose - long duration” or “high dose - short duration” balance may be considered potentially useful agents (Rigas and Kashfi, 2005). Furthermore, there has been extensive evidence which account for coxibs have several COX-2 independent activities to regress tumor.

1.4.6. Too Sparse and Inconsistent for Pancreatic and Hepatic Malignancies Although most evidence suggests that chronic NSAIDs use may diminish the risk of esophageal and gastric carcinomas. Data assessing the effects of NSAIDs on the incidence of pancreatic and hepatic malignancies currently are too sparse and inconsistent to draw any conclusions (Shaheen et al., 2000). Recently, a selective COX-2 inhibitor in COX-2 positive pancreatic cancer reduced tumor growth and angiogenesis, the opposite effect was observed in COX-2 negative pancreatic cancer. That means, the selective COX-2 inhibitor increased angiogenesis and tumor growth (Eibl et al., 2005).

1.4.7. The Expression of COX-2 Is Not Restricted to Tumor Cells While COX-2 is undetectable in most tissues in the absence of stimulation, it is induced in a limited repertoire of cells, notably in monocytes, macrophages, neutrophils, and endothelial cells (Simmons et al., 2004). Among the stimulants of COX-2 induction are bacterial lipopolysaccharides (LPS), growth factors, cytokines, and phorbol esters (Rigas and Kashfi, 2005). High levels of COX-2 are detected in activated and proliferating vascular tissues, such as angiogenic microvessels and atherosclerotic tissues. Atheromatous lesions contain both COX-1 and COX-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery, whereas, smooth muscle cells show lower levels (Schonbeck et al., 1999).

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2. Currently Status and Future Prospect Of COX-2 Inhibitor for Anti-Tumor 2.1. Current Status

2.1.1. COX-2 as a Important Melocular Target for Tumor It is well known that about 70% of cancer cases are due to environmental, dietary, or lifestyle factors. Accordingly, these cases maybe avoided by appropriate modifications. It means that that prevention of tumor is much more important than therapy of tumor. So that, active chemoprevention has become a major interventional approach following the epidemiological observation of a beneficial effect of NSAIDs in colon cancer prevention. This is chiefly due to the inhibition of the COX enzymes (Moran, 2002). COX-2 is inducible by the oncogenes and cytokines; A large volume of research datas has shown that COX-2 is often upregulated in human cancer cells (Moran, 2002; Karamouzis and Papavassiliou, 2004; Moyad, 2001; Lynch, 2001; Stratton and Alberts, 2002; Blain et al., 2000; Subongkot et al., 2003) at both early and late stages of some cancers (Anderson et al., 2003), it stimulates cellular division and angiogenesis and inhibits apoptosis (Moran, 2002). One more direct causal link between COX-2 expression and malignancy was shown in studies of COX-2overexpressing transgenic mice, which displayed mammary gland hyperplasia and transformation in breast tissue overexpressing COX-2 (Liu et al., 2001). The relationship between COX-2 and colon cancer is further confirmed by studies using the murine models of adenomatous polyposis coli, in which NSAIDs and gene knockouts reduce the number of spontaneously developing intestinal polyps, supporting a causative role of COX-2 in tumorigenesis (Nam et al., 2004; Kawai et al., 2002). Studies of human colorectal tumors revealed that COX-2 is overexpressed in more than 80% of carcinomas and in at least 50% of premalignant adenomas (Sano et al., 1995). COX-2 expression in intestinal epithelial cells increases resistance to apoptosis, promotes tumor angiogenesis, and enhances invasion and metastasis. COX-2 expression in stromal cells appears to have a role in tumor angiogenesis because tumor growth is attenuated when colon cancer cells are implanted in COX-2 knockout mice due to a decreased vascular supply to the tumors (Kawai et al., 2002). Recent study ascertained if COX-2 gene expression is associated with tumor response in the clinical treatment of colorectal cancer with the fluoropyrimidine-based therapy S-1, the result found a significant association between high COX-2 expression in colorectal tumors and less favorable clinical outcome for patients treated with S-1 chemotherapy, both in terms of tumor response and patients’ survival (Uchida et al., 2005). Therefore, COX-2 might be an important molecular target for the intervention of tumor (Moran, 2002; Karamouzis and Papavassiliou, 2004; Moyad, 2001; Evans and Kargman, 2004; Stratton and Alberts, 2002; Blain et al., 2000; Anderson et al., 2003). SO, NSAIDs and selective COX-2 inibitor may act via COX-2 to inhibit cancer growth (Kawai et al., 2002; Evans and Kargman, 2004; Shaheen et al., 2000; Subongkot et al., 2003). There have been numerous animal studies using specific COX-2 inhibitors providing support for the concept that COX-2 inhibition both prevents and regresses tumors arising from a variety of tissues (Rigas and Kashfi, 2005), including colon, stomach, lung, breast, pancreas, prostate, skin and others due to be attributed to the inhibition of the enzyme in the tumor as well as in stromal cells, resulting in reduction of carcinogen production, anti-

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proliferative and pro-apoptopic actions as well as anti-angiogenic and pro-immune (Evans and Kargman, 2004). The most remarkable result was that dietary administration of celecoxib inhibited both the incidence an multiplicity of colon tumors by about 93% and 97%, respectively. It also suppressed the overall colon tumor burden by more than 87% (Rigas and Kashfi, 2005). Taken together a spectrum of data for the role of COX-2 in carcinogenesis, ranging from the compelling (breast carcinogenesis) to the rather weak and controversial (skin carcinogenesis), Overall, the evidence from animal studies suggests strongly that COX enzymes, particularly COX-2 participate in a significant way in carcinogenesis (Bjorkman, 1999). Whereas, the regressive effects of sulindac on foci of aberrant crypts in the colon (considered to be precursors of adenoma), and on adenocarcinoma of the colon, are of particular interest because this NSAID does not have an inhibitory effect on COX. This may support the view that the antineoplastic effect of NSAIDs may also be due to a mechanism other than COX-2 inhibition, a intense interest has recently been focused on COX-2independent effects of NSAIDs. ( Karamouzis and Papavassiliou, 2004; Moran, 2002; Kawai et al., 2002; Shaheen et al., 2002). Therefore, pathway independent COX-2 is another important mechanism of COX-2 inhibitor and NSAIDs anti-tumor.

2.1.2. Effect of COX-2 Inhibitor for Tumor The development of successful chemoprevention agents should be to require a strategy that not only generates mechanistic insights in vivo, but also advances them rapidly to their clinical evaluation (Rigas and Kashfi, 2005). Particularlly, the development of selective COX-2 inhibitors, as chemoprotective agents, resulted in better clinical tolerance than that associated with traditional NSAIDs in general, with the potential for less toxicity known to occur after the inhibition of COX-1 (Moran, 2002; Lynch, 2001; Shaheen et al., 2002). Encouraging results have been obtained with these new agents in familial adenomatous polyposis, colon, breast, and prostate cancer (Moran, 2002). COX-2 specific inhibitors, for example cerecoxib, are potentially interesting for human therapy for chemoprevention of epithelial cancer or as medical treatment, alone or in combination with other anticancer treatments (Crosby and DuBois, 2003; Kawai et al., 2002; Stratton and Alberts, 2002; Eschwege et al., 2001). Colorectal cancer is an excellent model for studying cancer prevention by means of secondary (e.g., polypectomy to remove a precursor adenoma) and primary (chemoprevention) strategies (Lynch, 2001). Result of a study has shown a significant reduction in adenoma burden in familial adenomatous polyposis patients who received the selective COX-2 inhibitor celecoxib (Celebrex) and the design of other ongoing or planned clinical trials (Lynch, 2001). In multiple studies of colorectal carcinoma, chronic NSAIDs use has shown a protective effect, with the majority of studies demonstrating a 30-70% risk reduction associated with NSAID use (Shaheen et al., 2002). In breast cancer, large cohort studies reported a 40 to 50% reduced risk of developing cancer, a smaller size of the primary tumor, and a reduction in the number of involved axillary lymph nodes. Similar findings have been reported in the esophagus and stomach, but not in gastric cardia adenocarcinoma (Moran, 2002). Whereas, one clinical trial, indicated

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little benefit of the COX-2 selective inhibitor rofecoxib in treatment of metastatic CRC (Fenwick et al., 2003). Invesigations of liver metastasis model demonstrated that rofecoxib attenuated the growth and metastatic potential of colorectal carcinomas in mice (Yao et al., 2003; Yamauchi et al., 2003). Therefore, COX-2 has contributed to initiation of clinical trials testing COX-2 inhibitors for the chemoprevention of a wide variety of cancers (Kawai et al., 2002; Stratton and Alberts, 2002). In addition, recently, metronomic dosing regimens of standard chemotherapeutic agents without extended rest periods were shown to target the microvasculature in experimental animal models and result in significant antitumor activity. This antiangiogenic chemotherapy regimen could be enhanced by the concurrent administration of an angiogenesis inhibitor (Crosby et al., 2003). There is not doubt that studies of COX-2 in various cancers has provided a strong stimulus in the last decade to elucidate many pathways likely related to cancer pathogenesis and intensively interesting the focus on cancer prevention as a realistic option. However, in present, there are lots problems that need to resolve for example, theoretically mechanism (Figure 1-9), adverse effect, clinical application etc. 2.2. Future Prospect An important principle for chemoprevention is referred to overcome two prohibitive limitations concerning their safety and efficacy.The ideal chemopreventive agent should posses an efficacy approaching 100% and a safety profile that is nearly perfect. Of course, perfection is, probably unavailable. But, there are a few strategies considered to be hopeful ways for cancer prevention, including further research theoretically knowledge, designing and improving new drug; combined with chemotherapy ,etc.

2.2.1. Explore Targets beyond COX-2 It should be focused that NSAIDs and COX-2 specific inhibitors modulate targets other than COX-2, and to be factored in when analyzing their effects on cancer prevention based on their mechanisms underlying anti-tumor (Rigas and Kashfi, 2005). Several reports have confirmed COX-2 independent effects of celecoxib, at relatively high concentrations (50 microM), where apoptosis is stimulated in cells that lack both COX-1 and COX-2 (Crosby et al., 2003). A study described structural modifications to celecoxib that revealed no association between the COX-2 inhibitory and proapoptotic activities of celecoxib (Crosby et al., 2003). COX-2 specific inhibitors besides inhibit COX-2, also require the contribution of their non-COX-2 effects for their(probably suboptimal) effect on cancer prevention; Conventional NSAIDs prevent colon and other cancers most likely by modulating several molecular targets; NSAIDs act on multiple molecular targets, of which COX-2 is but one. It is crucial to evaluate the mechanistic relevance of each pathway. Focusing exclusively on COX-2 inhibition may not be an effective strategy for cancer prevention; exploring targets beyond COX-2 is likely to yield a productive approach to cancer prevention (Rigas and Kashfi, 2005). It is clear that the study of the non inherent in this approach is the need to resolve issues of mechanistic dominance, cooperation and redundancy and then make informed choices for drug development (Rigas and Kashfi, 2005). To discovery newly promising drug is necessary for improving efficacy and safety. But, it is important to resolve the issues of dominance

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versus cooperation versus redundancy of pathways. Design of reasoning should underscore the need to explore targets beyond COX-2, particularly, in viewpoint of emerging limitations of the COX-2 approach (Rigas and Kashfi, 2005). A few studies have actively pursued the idea that regulating targets other than COX-2 can prevent cancer (Niitsu et al., 2004, Nobuoka et al., 2004). These results have demonstrted that the phase II enzyme glutathioneS-transferase P1-1 may be an appropriate target for chemoprevention.

2.2.2. COX-2 Inhibitor Combinated with Chemotherapy or/and Radiation It should be carried out a number of clinical trials using COX-2 inhibitor and find out new approach, from which will increase our understanding of COX-2 inhibition in both cancer treatment and prevention (Xu, 2002). The most pressing need in cancer chemoprevention is to identify agents or combinations of agents that combine high efficacy with minimal toxicity (Rigas and Kashfi, 2005). The findings of preclinical studies coupled with the overexpression of COX-2 observed in advanced human tumours are the basis for new therapeutic anticancer strategies based on combinations of coxibs with other anticancer treatment modalities (Gasparini et al., 2003). COX-2 inhibitor combinated with chemotherapy or/and radiation. It will be important to determine whether patients treated with combinations of chemotherapy or/and radiation therapy with long-term NSAID use respond better than those not treated with NSAIDs. Early clinical studies have documented the feasibility, good tolerability, and promising activity of coxibs combined with particular focus on the opportunities that have emerged for treatment of cancer (Gasparini et al., 2003). The combination of COX-2 inhibitors with radiation or/and chemotherapy may reduce their side effects in future cancer prevention and treatment. Recent studies reported progress in the treatment and prevention of cancers of the colon, esophagus, lung, bladder, breast and prostate with NSAIDs, especially COX-2 inhibitors (Xu, 2002). Preliminary evidence suggests that COX-2 selective inhibitors potentiate the effects of radiotherapy (Wang et al., 2005). Trials that will evaluate continuous low dose cyclophosphamide in combination with celecoxib are underway in patients with metastatic renal cancer, and non-Hodgkin's lymphoma (Crosby et al., 2003). Given the safety and tolerability of the selective COX-2 inhibitors, and the potent antiangiogenic properties of these agents, the combination of antiangiogenic chemotherapy with a COX-2 inhibitor warrants clinical evaluation (Crosby et al., 2003). They should have at least additive benefit in combination with standard chemotherapy and radiation therapy . In preclinical models, a selective inhibitor of COX-2 showed to potentiate the beneficial antitumor effects of ionizing radiation with no increase in normal tissue cytotoxicity (Crosby et al., 2003). Recent study demonstrated that combined treatment with nonselective NSAID plus an EGFR tyrosine kinase inhibitor significantly inhibites polyp formation in Apc Min mice supports the notion that combinations of different agents for cancer prevention and treatment may be more effective than single agent therapy alone, targeted to one molecule (Wang et al., 2005). Preclinical experiments support that the combination of COX-2 selective inhibitor JTE-522 and a chemotherapeutic agent, cisplatin, in which elicited a synergistic antitumor effect in gastric cancer cells as well as an animal xenograft model (Wang et al., 2005). The availability of COX-2-specific NSAIDs opens the possibility of using this drug class as antiangiogenic agents in combination with chemotheapy or radiotherapy and other new

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molecularly-targeted compounds for the treatment of human cancer, with promising results (Dermond and Ruegg, 2001; Karamouzis et al., 2004). The combination of COX-2 inhibitor with standard cancer chemotherapeutic and/or radiation may provide additional therapeutic paradigms in the treatment of various human cancers (Evans and Kargman, 2004). Therefore, it is necessary to search new therapeutic anticancer strategies in clinic fir COX-2.

2.2.3. Emerging Dual Drug The development of safe and effective NSAIDs for chemoprevention is complicated by the potential that rare, serious toxicity may offset the benefit of treatment with these drugs given to healthy individuals who have a low risk of developing the disease (Rao and Reddy, 2004). To targets beyond COX-2 will provide alternative or complementary approaches, the latter representing mechanism-driven drug combinations (Rigas and Kashfi, 2005). Growing knowledge in this area has brought about innovative approaches using combine actions of NSAIDs with other agents that have different modes of action. It has also led to the development of nitric oxide-releasing NSAIDs, which induce tumor cell apoptosis and compensate for COX function, as a means of increasing efficacy and minimizing toxicity. Multi-pathway inhibition may be the newly promising way in chemoprevention. Such considerations have led to the development of dual inhibitors of both COX and 5-LOX. Licofelone (Rigas and Kashfi, 2005). (Figure 9). These new inhibitors are called dual acting anti-inflammatory drugs (Ulbrich et al., 2005). There is growing optimism for the view that full exploration of the role of NSAIDs in the prevention and treatment of epithelial cancers will serve towards reducing of mortality and morbidity from various cancers (Rao and Reddy, 2004).

2.2.4. Further Evaluate Clinic Effects On the basis of COX-2 expression in Alzheimer's disease and colon cancer, COX-2specific inhibitors may find clinical utility in the prevention or treatment of these conditions (Lipsky, 1999; Blain et al., 2000). Despite this apparently optimistic outlook for future uses of COX-2 inhibitors, most of the findings supporting this perspective are based on in vitro and in vivo models, but must be rigorously corroborated in human studies. some of which are already planned (Lipsky, 1999) and randomized clinical trials are needed, and because numerous individuals are currently using COX-2 inhibitors, a large volume of data should make at least retrospective studies more plausible further evaluation those effects (Moyad, 2001). Therefore, the challenge for researchers and clinicians is to further understand which NSAIDs, COX-2 inhibitor and what dosage and duration may provide the optimal benefit (if any), and to accurately construe the available current data on these agents for patients inquiring about these compounds (Moyad, 2001).

2.2.5. Further Investigate the Protective Function of COX-2 Recent study demonstrated that COX-2 might not only be considered as a putatively detrimental enzyme, but also contributes to tissue protection and is even constitutively expressed in healthy human stomach mucosa (Bertolini et al., 2002; Meyer-Kirchrath and Schror, 2000; Brzozowski et al., 2001).

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2.2.6. Continue to Study on COX-3 So far, the effect of the postulated COX-3 in both inflammation and tumorigenesis is controversial and need further investigation (Gasparini et al., 2003; Rigas and Kashfi, 2005). (Figure 2, 9)

2.2.7. Emphasize to Study on COX-1 It is well known that COX-1 is constitutive isoform. But, deletion of COX-1 also attenuated tumor formation in Apc 716 mice (Chulada et al., 2000). It means that COX-1 (as well as COX-2) plays a key role in intestinal tumorigenesis; therefore, it may also be a chemotherapeutic target for NSAIDs (Chulada et al., 2000).

2.2.8. Further Study on Inversion Mechanism For example, one type of non-COX inhibitors are the R-isomers of NSAIDs, which are based on the structure of 2-arylpropionic acid. This finding should be tested for a possibility of R (R-flurbiprofen, non-COX-inhibition) to S (S-fluurbiprofen (COX inhibition) inversion in vivo that would render the R-isomer effect as being due to the S-isomer generated in vivo from it, as such inversion were demonstrated to occur to varied extents in several species (Raz, 2002).

2.2.9. Continue to Explore Newmarker of Prognosis and Diagnosis for Tumor For example, to determine whether intratumoral COX-2 expression is a predictive factor for tumor response to fluoropyrimidine-based chemotherapy (Uchida, et al., 2005). This hypothesis is based on the above discussion would be that high COX-2 levels should render the tumors less responsive to the drugs (Uchida, et al., 2005). Patients with advanced (stage IV) colorectal cancer were treated with S-1 twice daily based on the patient’s body surface area for 28 days followed by a 2-week period rest. The overall response rate in a group of 44 patients treated with S-1was 40.9%. Sufficient tumor tissue was available from 40 of these patients for COX-2 mRNA quantitation. COX-2 gene expression was significantly lower in the responding tumors compared with the nonresponders. Patients with COX-2 high expression had a significantly shorter survival than those with COX-2 gene low expressions (Uchida, et al., 2005). COX-2 expression may be a determinant of drug efficacy for fluoropyrimidine therapy of colorectal cancer, and coadministration of COX-2 inhibitors may improve the clinical outcome of conventional chemo/radiotherapy, but the beneficial effects of using COX-2 inhibitors probably based on the initial intratumoral COX-2 expression level. Intratumoral COX-2 gene expression is associated with likelihood of response to chemotherapy with S-1and is a prognostic factor for survival of patients after the start of S-1 chemotherapy (Uchida, et al., 2005).

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Conclusion It is beyond doubt that over the past decade in vitro, vivo, preclinical, and clinical data have supported that COX-2 plays a important role in oncogenesis and that treatments with COX-2 inhibitors offer an effective chemoprevention strategy. A wide spectrum of studies in epidemiology , experiment have strongly demonstrated that NSAIDs and COX-2 inhibitors exert potential effect of anti-gastric carcinogenesis and other anti-tummorigenesis. Primary clinic efficacy have indicated that COX-2 inhibitors may suppress gastric carcinoma and other cancer. Therefore，COX-2 inhibitors may be a promising agent for prevention and treatment of gastric carcinoma as well as other tumors . Whereas, the mechanism underlying COX-2 inhibitors anti-tumorigenesis are based on COX-2 dependent and independent pathway, and also remain some mechanisms unknown. Long term administration of NSAIDs and COX-2 inhibitors may be associated with some side effects. The concept of COX-2 as a central target for cancer prevention should be further investigated. Moreover, COX-2 also play a role of protective function for human. COX-3 will need to further study to be confirmed. There exist a lots unsolved clinic issues, for instance, exact dosage and period with administration of COX-2 inhibitors, and application combined with others, as well as how to promote their effects, etc. In order to further enhance the clinic efficacy and safety of COX-2 inhibitor for chemoprevention and therapy of carcinoma, it is necessary that COX-2 inhibitors should be improved in many aspects, including: 1). target other than COX-2; 2). emphasize study of mechanism for carcinogenesis and anti-carcinogenesis; 3). Search for new drug, for example, Dual adent; 4). Promote application combinated with chemotherapy or/and radiation .5). Study on suitable clinical application approaches including indications, method, time, etc. It is firmly believed that COX-2 inhibitors would be a novel development and bright outlook for treatment and prevention of tumor，Along with the development of COX-2 specific inhibitors based on numerous laboratory and clinical studies.

Acknowledgement This work is supported by department of surgery, Addenbrooke’s Hospital, University of Cambridge. Particularly, we would like to acknowledge Professor Andrew Bradley, who give authors a lot of instructions, also acknowledge Mrs. Rita Pickering, Mr. Paul smith and Mr. Zhenquan Wei, who have many helps for the process of writing the paper.

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In: COX-2 Inhibitor Research Editor: Maynard J. Howardell, pp. 85-114

ISBN 1-59454-994-X © 2006 Nova Science Publishers, Inc.

Chapter II

Prevention and Chemoprevention of Gastric Cancer: Dietary Habits, Helicobacter Pylori and COX-2 Inhibitors Gerardo Nardone2 and Alba Rocco Department of Clinical and Experimental Medicine, Gastroenterology Unit, University “Federico II”, Naples, Italy

Abstract Despite the decrease in incidence, gastric cancer remains the second leading cause of cancer-related death worldwide. Prevention is likely to be the most effective means not of only reducing the incidence but also mortality from this disease. The term chemoprevention, has been referred to the prevention of cancer using specific agents to suppress or reverse the carcinogenic process. In recent years, attention has been focused on the anticancer properties of nonsteroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori (H. pylori)-eradication therapy and dietary habits. In vitro and in vivo studies show that widespread and long-term use of NSAIDs may be used in the healthy population for gastric chemoprevention. Albeit, enthusiasm has been thwarted by the potential toxic effects, i.e., risk of peptic ulcer disease. The new NSAIDs, selective cyclo-oxygenase-2 (COX-2) inhibitors, causing less injury to the mucosa of the upper gastrointestinal tract may be a valid alternative. However, the mechanisms of the antitumoral action of the COX-2 inhibitors still remain to be defined and may vary from agent to agent. In vitro studies have shown a variety of COX-related mechanisms in controlling proliferation and apoptosis balance. Experimental studies are often performed with much higher pharmacological doses than those used in clinical studies. Human observational studies are prevalently of the case-control type and often suffer from inadequate sample size to avoid a type II statistical error. Furthermore, due to the high * Gerardo Nardone, MD, Dipartimento di Medicina Clinica e Sperimentale, Gastroenterologia, Università “Federico II” Via S. Pansini 5, 80131 Napoli, Italia. Tel./Fax. 0039 0817464293 ; e-mail: [email protected]

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Gerardo Nardone and Alba Rocco cost of these new agents, cost-effectiveness analyses must be carried out to optimize the allocation of resources. The cumulative probability of developing a lesion from birth to 80 years of age is less than 4% thus, in the general population, more than 95% of those treated prophylactically with COX-2 inhibitors will not benefit. Therefore, chemoprevention with selective COX-2 inhibitors may be a worthwhile goal only in those subjects known to be at an increased risk of gastric cancer. However, also in these subjects, fundamental aspects such as safety, efficacy, mechanisms of action, optimal treatment regimens need to be defined. Although epidemiological studies have clearly established that H. pylori infection is associated with gastric cancer, there are, so far, no definitive prospective studies showing that eradication treatment significantly reduces the development of neoplasia. Prospective studies are hampered by the long period of time elapsing between infection and cancer development. Cost-effect analyses suggest that only a subgroup of H. pyloriinfected patients may present beneficial changes following eradication therapy i.e., individuals living in high risk areas, relatives of gastric cancer patients, patients with gastric atrophy or intestinal metaplasia. Diet plays an important role in the pathogenesis of gastric cancer by either increasing the risk or protecting against cancer development. Thus, a reasonable suggestion for the general population is a natural chemoprevention based on life-style “eat to live, not live to eat”.

Keywords: gastric cancer, chemoprevention, H. pylori, COX-2 inhibitors. Despite the decreasing incidence and mortality rates observed over the last 50 years, gastric cancer still ranks as one of the most frequent and lethal types of cancer worldwide [1]. Today, it is the fourth leading cancer type in incidence, accounting for 8.6% of the all cancers. Close to a million new gastric cancer cases are diagnosed annually (934 000 cases as reported by the International Agency for the Research on Cancer (IARC) in 2002) [2]. Gastric cancers are often resistant to radio- and chemotherapy, and, surgery is the only form of treatment with a curative potential albeit, two-thirds of Western gastric cancer patients are still diagnosed in advanced stages, when surgery can only be palliative [3]. Thus, the 5-year-relative survival rate remains < 20% [1,2]. At present, prevention or chemoprevention are likely to be the most effective means of reducing not only the incidence of this disease but also the mortality rate [4]. However, to be successful, this strategy depends upon a better understanding of the etiologic factors and pathogenetic mechanisms involved in gastric carcinogenesis. Topographically, gastric cancer may arise in the cardia of the stomach or more distally (non-cardia cancer) [5]. Epidemiological data show that the etiological factors and possibly the pathogenesis of these two types of cancer are completely different [6]. Cardia cancer appears to be closely associated with gastroesophageal reflux while non-cardia cancer results from the interaction of several factors, including environmental factors, and individual genetic susceptibility [7]. In the present chapter, evidence on the association between non-cardia gastric cancer and environmental factors is presented with special reference to prevention and chemoprevention strategies. The incidence of gastric cancer varies considerably worldwide, occurring more frequently in developing than in industrialized countries, with a tendency to strike primarily

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the urban and lower socioeconomic groups [6,7]. The highest incidence rates occur in Costa Rica, Japan, South America, Central and Eastern Europe, while the lowest rates occur in the United States, parts of Africa (Uganda) and Southeast Asia [6,7]. The distinct variations in the incidence, over time, between and even within countries, in differing socioeconomic groups, suggest that environmental factors, more than genetic susceptibility play a key role in the carcinogenetic process. Moreover, evidence that this variation is due mainly to environmental factors, comes from observations on immigrants: first generation migrants tend to maintain the homeland risk while subsequent generations have risk levels similar to those of the host country [8]. Based on these data and experimental studies on animals, the World Health Organization (Food and Agriculture Organization) concluded that of the various environmental factors, eating habit was the main factor involved in gastric cancer risk. Thus, the change in gastric cancer risk is due mainly to the degree of which western diet has been adopted. The relationship between diet and cancer was first clearly demonstrated in the 1930s, in a series of classical experimental studies in which severe caloric restriction markedly reduced the occurrence of cancer in rodents [9]. However, the relationship between diet and gastric cancer is much more complex and conflicting results have been reported in the literature. Thus, to better understand the role of diet in gastric carcinogenesis, we have analyzed the evidence for and against reported focusing on retrospective and prospective observational studies and interventional trials.

Diet and Gastric Cancer: Observational Epidemiology Studies Several epidemiology studies, aimed at evaluating the role of diet in gastric carcinogenesis, have been carried out both in high- and low-risk geographic areas [10-49] (Tables 1-3). Despite the lack of homogeneity concerning age, ethnicity, socio-economic status of the populations studied as well as the different methodological approaches used to evaluate diet, one of the most remarkable features emerging from the results of these studies is that a high intake of salted, pickled or smoked foods, as well as dried fish and meat, significantly increases the risk of developing gastric cancer while fiber, fresh vegetables and fruits were found to be inversely associated with gastric cancer risk [11,18,21,28]. Focusing on single groups of food components, high consumption of refined carbohydrates has been shown to be associated with a significantly increased risk of developing gastric cancer with an estimated odds ratio (OR) ranging from 1.5/100 mg [10] to OR 8.73/100 mg of daily intake [20]. However, the increased trend in risk appeared particularly high in females (OR highest quartile of consumption frequency [Q4] vs. lowest quartile [Q1] 14.8) [27]. Moreover, high consumption of animal protein, saturated fat and cholesterol enhanced the risk of gastric cancer in males (OR Q4 vs. Q1 10.3 for animal protein; ORQ4 vs. Q1 3.24 for saturated fat and ORQ4 vs. Q1 2.76 for cholesterol, respectively) [16,27].

Table 1. Epidemiological studies (population-based case-control) on dietary factors and gastric cancer Author

Year

Geographic area

Risch HA[10] Buiatti E[11] Graham S[12] Ramon JM[13] Li D[14] Kaaks R[15] Lopez-Carrillo L[16]

1985 1990 1990 1993 1995 1998 1999

Canada Italy USA Spain China Belgium Mexico

Case/Control n. 246/146 1016/1159 293/293 117/234 88/-301/2851 220/752

Mathew A[17]

2000

India

194/305

Palli D[18]

2001

Italy

382/561

Mayne ST19

2001

USA

352/687

Jedrychowski W[20] Hamada GS[21] Chen H[22] Hara M[23] Nomura AM[24] Sipetic S[25] Lagiou P[26]

2001 2002 2002 2003 2003 2003 2004

Poland Brazil Nebraska Japan Hawaii Serbia Greece

80/-97/192 124/449 149/287 300/446 131/131 110/100

Qiu JL[27]

2004

China

103/133

De Stefani E[28]

2004

Uruguay

240/960

Increased risk

Decreased risk

Nitrite, chocolate, carbohydrates Nitrites, protein Sodium, fat, retinol Carbohydrates Vit. A, Vit. B12, mono, disaccharides Protein, saturated fat, cholesterol Rice, spicy foods, chili, high-temperature food Protein, nitrite, sodium Animal protein, cholesterol, Vit. B12, nitrite Carbohydrates Beef Saturated fat -Processed meat, bacon High fat milk, meat, sugar, salted foods -Animal protein, cholesterol, saturated fat, salt Salted-stewed meat, rice, tuber

Fiber , Vit. C Vit. C, carotene, α-tocopherol, vegetable fat Carotene, raw vegetables, onions, cucumbers Vit. A, Vit. C Vit. A, Vit. C Polyunsaturated fat, Vit. C/B1-B2-B6, C/A Polyunsaturated fat, fiber, Vit. E -Vit. C/B6, β-carotene, α-tocopherol, nitrates Fiber, β-carotene, folate, Vit. C Vit. E, β-carotene Fruits Fiber, Vit. C Cruciferous vegetables, mushrooms β-carotene, Vit. C, Vit. E, folate Margarine, fish Flavanone Vit. A, Vit. C Vegetables, legumes, fruit, black tea

Table 2. Epidemiological prospective cohort studies on association between dietary factors and gastric cancer (1990-1999) Author

Year

Geographic area

Subjects n.

FU yrs

Increased risk

Decreased risk

No effect

Chyou PH [29]

1990

USA (Hawaii)

8,006

18

--

Green/ cruciferous vegetables, fruit

--

Kneller RW [30]

1991

USA

17,633

20

--

--

Kato I [31] Nomura A [32] Dorant E [33] Goldbohm RA[34]

1992 1995 1996 1996

Japan USA (Hawaii) The Netherlands The Netherlands

9,753 8,006 120,852 120,852

6 25 3.3 4.3

Carbohydrates, salted-fish, bacon, cooked cereals, milk Alcohol, broiling meat ----

-Alcohol Leek, garlic Black tea

Ocke MC[35]

1998

The Netherlands

12,763

25

--

Terry P[36]

1998

Sweden

11,946

25

--

Fruit Fruit, vegetables Onions -Vegetables, fruit, fiber-rich cereals Fruit, vegetables

Galanis DJ[37]

1998

USA (Hawai)

11,907

14.8

Coffee

Fruit, raw vegetables

Knekt P[38]

1999

Finland

9,985

24

--

--

Jansen MC[39]

1999

Netherlands

12,000

25

Refined grains

Fruit

#

FU: follow-up; * effect limited to women; effect limited to men; NDMA: N-nitrosodimethylamine

--Pickled vegetables, dried/salted fish Nitrates, nitrites, NDMA Vegetables Whole grain

Table 3. Epidemiological prospective cohort studies on association between dietary factors and gastric cancer (2000-2004) Author

Year

Geographic area

Subjects n.

FU yrs

Increased risk

Decreased risk

Botterweck AA[40]

2000

The Netherlands

120,852

6.3

Retinol, β-carotene

Vit. C

Tsubono Y[41]

2001

Japan

26,311

8

--

McCullough ML[42]

2001

USA

1,200,000

14

Vegetables*

Nagata C[43]

2002

Japan

33,304

7

--

Ngoan LT[44]

2002

Japan

13,000

10

Processed meat, cooking oil, pickled food, soup

Kobayashi M[45]

2002

Japan

39,993

10

Masaki M[46]

2003

Japan

5,765

10

Khan MM[47] Kim MK[48] Sasazuki S[49]

2004 2004 2004

Japan Japan Japan

3,158 42,112 72,743

18 10 11

-Vegetables, citrus fruit, whole grain# Soy products Green/yellow vegetables, fruit, cuttle-fish, tofu, potatoes Fruit, vegetables Vegetable and fruit pattern Western breakfast pattern Miso soup# Healthy dietary pattern* Green tea*

-Meat pattern Rice/snack pattern Carbonated drink/juice* Traditional dietary pattern --

No effect Folate, Vit. E, α−carotene, lycopene, fibers, Vit.A, BHA, BHT Green tea -----

FU: follow-up; * effect limited to women; # effect limited to men; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene (cooking fats, oils, mayonnaise, creamy salad dressing, dried soup)

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The World Cancer Research Fund and the American Institute for Cancer Research analyzing 16 case-control studies, reported a close association between salt and salted food and the risk of gastric cancer [50]. However, focusing on the overall dietary salt or sodium intake, only four studies showed a marked, statistically significant, increase in gastric cancer risk for the highest intake level (OR from 2.1 to 5.0). Similar conflicting results have emerged on the role of the dietary micronutrients (Vitamin C, Vitamin E, carotenoids, fiber, flavonoids and selenium) commonly held to be responsible for the presumed protective role of fresh fruit and vegetables. While evidence on the protective effect of beta-carotene has been very consistent, the ≅ 50% reduction in risk associated with Vitamin C intake, reported in some studies (OR ranging from 0.3; 95% CI 0.1-0.8 to 0.60; CI 0.41-088) [13,15,18,19,27] has not been confirmed in others [10,24,26]. Bias in the epidemiological approach of the case-control design could, in part, account for these contrasting results. Since observational case-control studies are based on the retrospective assessment of eating habits, the data collected may be affected by the so-called “recall-bias”. Onset of symptoms affects the dietary habit and this is difficult to establish following the diagnosis of cancer. Prospective observational cohort studies, in which the evaluation of diet is unaffected by symptoms, should ideally provide much more reliable evidence. Analysis of the data obtained in 21 studies involving a total of 1651231 individuals, followed for periods ranging between 3.3 and 25 years [29-49], substantially confirmed the significantly increased risk of developing gastric cancer due to high intake of total carbohydrates, salted fish, processed meat, refined grains and saturated fat [30,39,44]. Two Japanese studies based on the analysis of dietary pattern demonstrated a significant increased risk of gastric cancer in middle-aged males with a “meat” or “rice” prevalent diet (relative risk [RR] 1.00; 95% CI 0.55-1.10 and RR 1.00; 95% CI 0.52-1.19, respectively) [46] while the “traditional pattern” was a risk factor for both genders (RR 2.88; 95% CI 1.76-4.72 for males and RR 2.40; 95% CI 1.324.35 for females) [49]. It is likely that the greatest part of this geographic variation in gastric cancer, not only in Japan but also worldwide, can be explained, at population level, by the variability in daily salt intake levels. However, very few prospective studies regarding the role of salt in gastric cancer risk have appeared and in some instance show conflicting results. In a study on 13,000 Japanese and 116 gastric cancer deaths with a 10 year follow-up, a greater consumption of pickled food and traditional soups was associated with an increased gastric cancer risk, however, results were not statistically significant [44]. The Netherlands Cohort Study comprising 120,852 adults and 282 incident gastric cancer cases confirmed during 6.3 year’s follow-up [51], demonstrated that the intake of dietary salt and various types of cured meat showed only a weakly positive association with gastric cancer risk. Tsugane et al. conducted a population based prospective study in four different areas in Japan on a total of 18,684 males and 20,381 females age range 40-59 years. They documented gastric cancer in 358 men and in 128 women during 12 year’s follow-up [52]. Salt intake was dose-dependently associated with the risk of gastric cancer in males while the trend was not clear in females. Furthermore, in another study, no association was found with the intake of table salt or soy sauce, whereas salted fish intake was associated with an increased risk of gastric cancer in white American males, (RR = 1.9 for the highest intake level) [53]. Sodium excretion level in 24-h urine samples is considered to be one of the most reliable estimates for

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evaluating daily salt intake, particularly at population level [54]. Large amounts of salts disrupt the mucin layer lining and protecting the gastric epithelium and further damage epithelial cells with the generation of a high osmotic pressure. This, in turn, stimulates the proliferation of stem cells of the gastric epithelium providing favorable conditions for onset of mutation. Prolonged lesions result in chronic atrophic gastritis and intestinal metaplasia, both of which are held to be precursor lesions for intestinal type gastric cancer [55]. The worldwide decline in the incidence of gastric cancer observed during the last 50 years might be due to the change in food preservation, from salting methods to refrigeration. Furthermore, the use of refrigeration has increased the consumption of fresh fruit and vegetables. In the Swedish Twin Registry, comprising 11,546 individuals, the lowest compared to the highest fruit and vegetable intake showed a RR 5.5 (95% CI 1.7-18.3) with a statistically significant dose-risk trend (p < 0.05) [36]. The Japan-Hawaii Cancer Study on 8,006 Hawaiian males of Japanese ancestry reported that all types of vegetables were protective against gastric cancer. Subjects in the group of highest vegetable consumption (≥ 80 g/day) had a RR of developing gastric cancer of 0.6 (95% CI 0.3-0.9) compared to non-consumers [29,32]. Green and yellow vegetables showed the highest protective effect against gastric cancer (RR 0.4; 95% CI 0.2-0.9 and 0.64; 95% CI 0.45-0.92, respectively) [44,45]. In a recent large population-based prospective study with a 10-year follow-up, vegetable and fruit intake, even in low amounts, was associated with a lower risk of gastric cancer [45]. The protective role of diet appeared to be mainly due to the anti-oxidant potential of the micronutrients. Indeed, using the total radical-trapping antioxidant potential (TRAP) of different plant foods to convert food frequency intake into antioxidant potential, the intake of antioxidant equivalents was inversely related with the risk of gastric cancer (OR 0.65; 95% CI 0.48-0.89) [56]. A prospective cohort study on 30,304 Japanese people followed for 7 years, showed that intake of soy isoflavones reduces the risk of death from gastric cancer [43]. In contrast, the Seven Countries Study Research Group found no association between total vegetable intake and gastric cancer risk [39] and the Cancer Prevention Study on a cohort of 1.2 million United States individuals demonstrated a reduced risk in males (RR 0.79; 95% CI 0.67-0.93) and an unexpected increased risk in females (RR 1.25; 95% CI 0.991.58) [42]. Recently, research has focused on tea. Tea is one of the most popular beverages in the world and the consumption of tea has been associated with a decreased risk of stomach cancer [57]. The beneficial health effect has been attributed to the catechinis in tea: epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate [58]. The biological benefits are due to their strong antioxidant and anti-angiogenic activity as well as their potential to inhibit cell proliferation and modulate carcinogen metabolism [59].

Interventional Dietary Trials for Prevention of Gastric Cancer Randomized clinical trials provide one of the most scientifically rigorous approaches for testing hypotheses emerging from epidemiological and experimental studies and are the ideal strategic approach by which to evaluate inhibition of the carcinogenic process induced by preventive measures.

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Fruit and vegetable intake is inversely related to gastric cancer risk. These foods contain phytochemicals endowed with anti-cancer and anti-inflammatory properties offering many health benefits. These substances are rich, among other nutrients and bioactive substances, in ascorbic acid, beta carotene, and other carotenoids. Dietary interventional trials for stomach cancer prevention have, therefore, been based mainly on long-term supplementation with anti-oxidant micronutrients given alone or in combination (beta-carotene, Vitamin A, Vitamin C, Vitamin E, selenium) [60-64] (Table 4). However, all interventional studies but one failed to demonstrate any significant change in the risk of gastric cancer in subjects receiving anti-oxidant supplementation [60-62,64,65]. The most important trial, the “General Population Trial” involving 29,584 subjects living in Linxian, China, and followed for > 5 years, demonstrated no statistically significant reduction in the prevalence of gastric cancer for any of the interventional arms, even though, a reduction in total mortality, total cancer mortality and stomach cancer mortality was found among those receiving beta-carotene, Vitamin E and selenium [60]. Similar results were obtained in the Alpha-Tocopherol BetaCarotene (ATBC) Cancer Prevention Study conducted in Southwest Finland and involving 29,133 middle-aged male smokers observed between 1985 and 1993 [61,62]. Long-term supplementation with alpha-tocopherol (50 mg/day) and/or beta-carotene (20 mg/day), both at five- and six-year follow-up, had no significant effect on the overall incidence of gastric cancer (RR 1.21, 95% CI 0.85-1.74 for alpha-tocopherol and RR 1.26, 95% CI 0.88-1.80 for beta-carotene). Paradoxically, a subgroup analysis, according to histological type, suggested an increased risk for beta-carotene on intestinal type gastric cancer (RR 1.59, 95% CI 0.992.56) [61,62]. Finally, another study involving 216 atrophic gastritis patients treated with folic acid and/or beta-carotene supplementation, and, followed for a period of 8 years, failed to demonstrate any significant reduction in the incidence of gastric cancer. However, folic acid significantly improved gastric mucosa lesions by reversing gastric atrophy, inflammation, intestinal metaplasia and dysplasia at the end of follow-up [63]. Overall, these observations suggest that vitamin supplementation alone is not sufficient to prevent gastric cancer. On the other hand, a double-blinded interventional study involving 2,526 subjects at risk of developing gastric cancer and 2,507 controls from a Chinese province, demonstrated, in the first five years of follow-up, a significant reduction in the morbidity rates of malignant gastric tumors in the intervention group treated with large doses of synthetic allitridum associated with microdoses of selenium for a period of 3 years (RR 0.48; 95% CI 0.21-1.06 and RR 0.36; 95% CI 0.14-0.92 for the entire population and male group, respectively) [64]. A consistent inverse association between gastric cancer and raw garlic consumption has been reported following a large meta-analysis carried out between January 1996 and August 1999 (RR 0.53; 95% CI 0.31-0.92) [66]. Garlic extracts and synthetic allitridum on gastric cell culture can kill tumor cells [67-69], inhibit tumor growth by 50%, repair DNA synthesis, induce apoptosis and have an antimutagenic effect [70]; furthermore, they inhibit the synthetic nitrosamine in gastric fluid, have an antioxidant effect [71] and inhibit growth of Helicobacter pylori (H. pylori) in vitro [64]. However, the dose of allitridum for an adult to obtain these effects may be equivalent to eating 100-200 g of raw garlic each day.

Table 4. Randomized controlled dietary intervention trials for prevention of stomach cancer Author

Year

Geographic area

Subjects n.

Wang GQ[60]

1994

China

29,584

Varis K[61] Malila N[62]

1998 2002

Finland Finland

29,133 29,133

Zhu S[63]

2003

China

216

Li H[64]

2004

China

2526

FU: follow-up

Dietary intervention Retinol/zinc; riboflavin/niacin; Vit. C/molybdenum; carotene/Vit. E/selenium α-tocopherol 50 mg/day; β-carotene 20 mg/day; α-tocopherol 50 mg/day; β-carotene 20 mg/day Folate 20 mg/day + Vit. B12 1 mg/month Natural β-carotene 30 mg/day Synthetic β-carotene 30 mg/day Synthetic allitridum 200 mg + selenium 100 µg

Intervention (yrs)

FU (yrs)

Results

5.25

5.25

↓ gastric cancer mortality

5 5-8

5 8

= gastric cancer incidence = gastric cancer incidence

2

8

= gastric cancer incidence ↓ precancerous lesions

2

5

= gastric cancer incidence

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Dietary Factors and Mechanisms of Gastric Carcinogenesis The mechanisms by which dietary factors affect the cancer process are extremely complex. Indeed, foods and food components either induce or inhibit the carcinogenic process. Carcinogen compounds such as heterocyclic amines, polycyclic aromatic hydrocarbons and N-nitroso compounds found in cooked and cured foods, may directly damage cellular DNA contributing to changes in the genetic make-up of the cell in the initial phases of the carcinogenic process [72]. Furthermore, binding to nuclear proteins, i.e., histones and acidic proteins, which play a pivotal role in the control of gene expression, result in the induction or repression of proteins controlling cell replication, growth and differentiation [72]. Finally, dietary compounds can influence “epigenetic mechanisms” such as DNA methylation which may result in abnormal gene expression [73]. By way of these mechanisms, food components and related factors (food processing and/or storage methods) take part in all the stages of the cancer process. Food components can, on the other hand, inhibit carcinogenesis in different ways: a) modifying carcinogen activation; b) modifying carcinogen detoxification; c) scavenging DNA reactive agents, and d) suppressing the abnormal proliferation of early, preneoplastic lesions.

a) Food Components that Modify Carcinogen Activation Fruit and vegetables provide an array of chemicals able to stimulate or attenuate P450mediated reactions. Cruciferous vegetables which include cauliflower, broccoli, and cabbage, modulate the metabolism of a number of nitrosamine-carcinogens. Other phytochemicals known to modify the metabolic activation of procarcinogens are organsulfur compounds from garlic and onion, certain flavoinoids, and polyphenolic antioxidants [74].

b) Food Components that Modify Carcinogen Detoxification Detoxification of chemical carcinogens through stimulation of glutathione-S-transferases (GST) and UDP-glucuronyl transferases are two of the biochemical pathways both enhanced by dietary phytochemicals. Among the more active agents, in this regard, are the phytochemicals found in garlic and onion, cruciferous vegetables, and a few spices [75].

c) Food Components that Intercept DNA Reactive Species An increasing number of phytochemicals have antioxidant, or, radical scavenging effects. Perhaps the greatest concentration of these agents are the phenolic antioxidants found ubiquitously in the plant kingdom, but of special interest are the polyphenolic agents found in green tea [76]. Green tea releases, upon steeping in boiling water, polyphenolic agents.

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Interestingly, green tea, in concentrations consumed by humans, has been reported to inhibit a variety of chemically induced cancers. Other polyphenolic compounds inhibiting cancer are curcumin, the chemical that gives the spice tumeric its yellow colour, and ellagic acid, a phenolic acid found in a variety of fruits and nuts [77]. Moreover, this molecule is able to interfere with arachidonic acid conversion of prostanoids through its influence both on cyclooxygenases and on the lipooxygenase pathway [77].

d) Food Components that Reverse Abnormal Proliferation Calcium is cytostatic for most normal cells; calcium supplementation, in animal diets, effectively reduces the hyperproliferation associated with ingestion of high fat diets. Carotenoids and retinoids not only affect cell proliferation, but are also powerful differentiating agents in cell culture and in animals [74].

Helicobacter Pylori and Gastric Cancer The other important environmental factor involved in gastric carcinogenesis is H. pylori infection. In 1994, just 10 years after the incidental discovery by Warren and Marshall, the International Agency for Research on Cancer declared H. pylori to be a group I human carcinogen for gastric adenocarcinoma [78]. The postulated relationship between H. pylori and gastric cancer has been based mainly on epidemiological investigations albeit, the exact prevalence of H. pylori infection in gastric cancer patients still remains to be estimated, since, due to the modified gastric micro-environment, the infection can be lost locally, and even serology becomes negative [79]. Nonetheless, many epidemiological studies have shown a close correlation between H. pylori seropositivity and gastric cancer. The EUROGAST study, on 17 populations from 13 different countries (Unites States, Japan and 11 European countries), demonstrated a six-fold increased risk of gastric cancer in H. pylori-infected patients compared with uninfected subjects [80]. A meta-analysis of cohort and case-control studies revealed that H. pylori infection was associated with a two-fold increased risk for developing gastric adenocarcinoma [81]. The RR for gastric cancer was greatest in younger patients (9.29 at age < 29 years), suggesting that H. pylori infection during childhood is an important prerequisite condition. A recent nested controlled study combining 12 studies (6 from Europe, 4 from Asia, 2 from the United States), and involving 1228 gastric cancer cases and 3406 controls, revealed that the association of H. pylori infection with gastric cancer was restricted to non-cardia cancers (OR 2.97; 95% CI 2.3-3.7), and was stronger when blood samples for H. pylori serology were collected ten years or more before cancer diagnosis (OR 5.9; 95% CI 3.4-10.3) [82]. However, the most powerful evidence comes from a prospective study on 1526 Japanese patients followed for approximately 7.8 years [83]. Gastric cancer developed in 36 H. pylori-positive patients (2.9%) in contrast to none of the 280 non-infected subjects. The close relationship between H. pylori infection and gastric cancer leads to the critical question of whether antimicrobial therapy can be considered for gastric cancer chemoprevention. So far, only one prospective, randomized, placebo-controlled, population

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study has been carried out in a high-risk area of China which involves 1630 subjects observed from 1994 to 2002. A comparable incidence of gastric cancer was found in the subjects receiving H. pylori eradication treatment and those receiving placebo while, eradication of H. pylori significantly decreased the development of gastric cancer in a subgroup of H. pylori carriers not presenting precancerous lesions [84]. Interventional studies in which cancer diagnosis is the primary end-point are not easy to perform since they require follow-up of a large number of individuals for several decades. An effective alternative could be smaller and short-term trials focusing on intermediate steps or precancerous lesions i.e., atrophy, intestinal metaplasia and dysplasia. Many studies have focused on this issue but, the results are still controversial even if more data were obtained showing regression of precancerous lesions following H pylori eradication (Table 5) [85-91]. Table 5. Histopathological changes in atrophy and intestinal metaplasia following H. pylori eradication: review of 12 years (1992-2004)

Atrophy IM

Reports N. 34 34

Patients N. 1905 1961

Follow-up Range (mos) 1-84 1-84

Significant improvement 18 6

No significant change 15 27

Significant deterioration 1 1

IM: intestinal metaplasia

Interplay between H. Pylori and Diet A synergistic interaction between H. pylori infection and diet in gastric cancer has been suggested [92]. One possible mechanism by which H. pylori exerts its “carcinogenic” potential is the greater likelihood of malignant transformation due to the inflammatory response of the gastric epithelium. The generation of reactive oxygen species (ROS) and the increased level of nitric oxide (NO) synthase associated with the mucosal colonization by H. pylori cause DNA mutations which may be the initial step in the genetic alterations of gastric epithelial cells [91,93,94]. Another possible explanation is that the H. pylori-related inflammation induces morphological changes in the gastric mucosa, such as atrophy and intestinal metaplasia [95]. These latter conditions decrease the acidity in the stomach, increasing the endogenous formation of nitrosamides, the main subset of N-nitroso compounds [96]. Nitrosamides, spontaneously formed in the stomach from the nitrite and amides depend on the presence of nitrites and are favored by a high pH. Thus, the theory of “N-nitroso compounds-mediated gastric cancer risk” links with that of the “H. pylori-related gastric cancer risk” suggesting an integrated model of gastric carcinogenesis [92]. So far, only a few epidemiological studies have simultaneously evaluated the role of H. pylori infection and dietary habits in relation to gastric cancer risk but results remain inconclusive (Table 6) [97-100]. A study evaluating the role of H. pylori infection and capsaicin consumption on the risk of gastric cancer demonstrated an increased risk (OR 1.71; 95% CI: 0.76-3.88) in high-level consumers of capsaicin (90-200 mg/day) as compared to low-consumers (0-29.9 mg/day). However, this effect was independent of H. pylori status and was higher for diffuse type

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gastric cancer (OR 3.64; 95% CI: 1.09-12.2) compared to the intestinal type (OR 1.36; 95% CI: 0.31-5.89) [99]. Lastly, Machida-Montani et al. found a close correlation between gastric cancer and H. pylori infection (OR 8.2; 95% CI 3.7-18.2), frequent intake of fermented soy bean soup (OR 2.1; 95% CI 0.9-5.1), and rice (OR 2.5; 95% CI 1.0-6.1), but no significant interaction between diet and H. pylori infection [100]. In contrast, in a Korean hospital-based case-control study, patients with H. pylori infection and high salt intake had a 10-fold higher risk of developing gastric cancer than those without H. pylori infection and low salt intake (p = 0.047) [98]. In an Italian study, administration of ascorbic acid together with H. pylori eradication led to a significant improvement in intestinal metaplasia of the gastric mucosa [101]. Likewise, in Columbia, anti-H. pylori treatment and dietary supplementation with antioxidant micronutrients induced regression of cancer precursor lesions [102]. Finally, the prevalence of gastric cancer, caused by a combination of H. pylori and salted foods, has been shown to be lower in a tea-drinking population compared to non-tea-drinking controls [103].

Gastric Cancer and Chemoprevention The term “chemoprevention”, first introduced by Sporn in 1976, has been referred to the prevention of cancer using specific agents to suppress or reverse the carcinogenic process [4]. In 1998, the Physician’s Health Study showed that use of aspirin may reduce the risk of colorectal cancer [104]. Recently, attention has been focused on the anticancer properties of nonsteroidal anti-inflammatory drugs (NSAIDs) in gastric cancer. The main target of NSAIDs is the cyclo-oxygenase (COX) enzyme which catalyses the conversion of arachidonic acid to prostaglandins (PG) [105]. Since 1991, two distinct isoforms of COX have been recognised: COX-1 and COX-2 sharing > 60% identity at amino acid level and a comparable enzymatic activity [106]. COX1 is constitutively expressed in many tissues where it regulates housekeeping cellular functions, while COX-2, usually low or undetectable, is up-regulated by hormones, proinflammatory cytokines and tumor promoters [107]. The induction of COX-2 is associated with inhibition of apoptosis, promotion of neoangiogenesis and increase in metastatic potential [108]. COX-2 expression is up-regulated in gastric cancer as well as in precancerous lesions and in H. pylori induced inflammation [109-114]. Thus, the relatively early role of COX-2 in gastric carcinogenesis makes it an attractive target for cancer chemoprevention.

COX-2 Inhibitors and Gastric Cancer: Experimental in Vitro Studies Several studies have analyzed the effect of the selective and non-selective COX-2 inhibitors on gastric cell lines focusing on cell proliferation and apoptosis [115-134]. Cellular hyperproliferation and inhibition of apoptosis are considered to be important mechanisms in human carcinogenesis [135]. COX-2 plays a role in controlling apoptosis by way of two possible mechanisms: removal of the substrate arachidonic acid via COX-catalytic activity or

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generation of PG products. In addition, COX-2 and the COX-2 product PGE2 are involved in the apoptosis pathway by upregulating p53, p21, c-myc, bcl-2 and bcl-xl and down-regulating bax or bak [136]. Regardless of the cancer cell lines used and gene markers analyzed, all in vitro studies [115-134] showed inhibition of cell proliferation and induction of apoptosis (Table 7). The MKN45 and CACO-2 cell lines which abundantly express COX-2, showed a reduction not only in COX-2 mRNA and protein expression but also in the cell proliferation rate when exposed to selective and nonselective COX-2 inhibitors, NS-398 and indomethacin [115,116,118]. Furthermore, selective and nonselective COX-2 inhibitors both exerted minimal effects on proliferation of Kato III and MKN28 which express significantly lower levels of COX-2 [115,116,118,131,132]. The COX-2 specific inhibitor JTE-522 induced apoptosis and suppressed cell-proliferation in MKN28 and MKN45 cell lines by upregulation of c-myc and down-regulation of bcl-2 protein expression [117]. In the SGC7901 gastric cancer cell line, nimesulide, a selective COX-2 inhibitor, suppressed proliferation and cell viability in a time- and dose-dependent fashion by reducing PGE2 release and telomerase activity [120]. Furthermore, Leung et al. demonstrated that treatment with NS398 reduced VEGF expression in Kato III cell lines transfected with a COX-2 expressing vector [126]. Lastly, conditioned media, obtained from the SGC7901 cell line transfected with antisense COX-2 cDNA or treated with a selective COX-2 inhibitor, suppressed proliferation, migration and tube formation of human umbilical vein endothelial cells, thus, inhibiting neoangiogenesis [134].

COX-2 and Gastric Cancer: Animal Models or Experimental In Vivo Studies Experimental studies in vivo would undoubtedly led to a better understanding of the mechanism of tumor suppression by COX-2 inhibitors, before use in human protocols. Animal models involve the use of cancer-inducing agents such as MNNG (N-methyl-N1Nitro-N-Nitrosoguanidine), NSEE (N-Nitrososarcosine ethyl ester), NNK (4Methylnitrosamino-1-3-pyridyl-1-butanone) and MNU (N-methyl-N-nitrosourea). Several NSAIDs have been studied in experimentally-induced gastric cancer in rodents (Table 8), however, the results are controversial [137-145]. Lehnert et al, in two different studies, found an increase in gastric tumor incidence in the rodent model of MNNG-induced gastric cancer treated with a powerful COX-inhibitor flurbiprofen [137,139]. In two other studies, indomethacin, sulindac or ibuprofen treatment of rats exposed to NSEE or NNK led to a decrease in tumor size and number, whereas the administration of piroxicam did not produce the same results [138,140]. Finally, recent studies have demonstrated that treatment with COX-2 selective inhibitors NS-398, rofecoxib or celecoxib, suppress the growth and implant rate of a gastric cancer xenograft in nude mice through stimulation of apoptosis and inhibition of proliferation and neoangiogenesis [140142,145]. Interestingly, nimesulide prevented H. pylori-associated gastric carcinogenesis in C57BL/6 mice treated with the carcinogen MNU and infected with H. pylori, by inducing cell apoptosis [145].

Table 6. Epidemiological studies (hospital-based case-control) on association between dietary factors and H. pylori infection and gastric cancer risk Author

Year 2002

Geographic Area Thailand

Case/Control n. 131/262

Sriamporn S[97] Lee SA[98]

2003

Korea

69/199

Lopez-Carrillo L[99] Machida-Montani A[100]

2003 2004

Mexico Japan

234/468 122/235

H. pylori: Helicobacter pylori

Increased risk

Decreased risk

H. pylori risk

Salt, fermented foods Salt, kimchi, salt-fermented fish Capsaicin Fermented soy bean, rice

Vegetables, fruit Vegetables, fruit, soybean curds, broth ---

Independent Increased Independent Independent

Table 7. In vitro experimental studies of COX-inhibitors in gastric cancer lines Author Tsuji S115 Sawaoka H[116] Uefuji K[117] Husain SS[118] Zhou XM[119] Li JY[120] Jiang XH[121] Wong BC[122] Wu J[123] Liu C[124] Wang C[125] Leung WK[126] Hu GY[127] Baoping Y[128] Power JJ[129] Gu Q[130] Honjo S[131] Honjo S[132] Ma L[133] Fu YG[134]

Year 1996 1998 2000 2001 2001 2002 2002 2003 2003 2003 2003 2003 2004 2004 2004 2005 2005 2005 2005 2005

Cultured cell lines Kato III; MKN28; MKN45 Kato III; MKN28; MKN45 MKN28; MKN45 MKN28 AGS; MKN28 SGC7901 AGS AGS; MKN28 AGS; MKN45 ; MKN28 SGC7901 SGC7901 Kato III SGC7901 MKN45 AGS AGS, MKN45 MKN45, KATO III MKN45, KATO III SGC7901 SGC7901

Intervention NS-398; Indomethacin NS-398; Indomethacin JTE-522 NS-398; Indomethacin Aspirin; Indomethacin Nimesulide SC236 SC236 SC236 Melecoxicam; rofecoxib Aspirin NS398 Nimesulide Nimesulide Aspirin Aspirin NS-398 NS-398 Sulindac NS-398

Molecular mechanism ↓ COX-2 mRNA ↓ COX-2 mRNA ↑ c-myc; ↓ bcl-2 ↓ MAPK (ERK2) ↑ bax; ↑ bak; ↑ caspase 3 ↑ P27kip1 ↓ PKC-beta ↓ NF-Kb ↑ 15-LOX-1 ↓ COX-2 mRNA ↓ COX-2 mRNA; ↓ fos ↓ COX-2 ↓ TERT; ↓ PKB ↓ TERT; ↓ Akt/PKB ↑ caspase 8, -9, -3, -6, -7 ↑ caspase 8/Bid, BAX ↑ P27/Kip1; ↓ COX-2, cyclin D1, Skp2 ↑ Bad, PTEN, ↓ Akt ↓ COX-2, Bcl-2 ↓ COX-2

Results ↓ proliferation ↓ proliferation ↓ proliferation ↑ apoptosis ↓ proliferation ↑ apoptosis ↓ proliferation ↑ apoptosis ↓ proliferation ↑ apoptosis ↑ apoptosis ↑ apoptosis ↑ apoptosis ↓ proliferation ↑ apoptosis ↓ proliferation ↓ VEGF proliferation ↓ proliferation ↓ proliferation ↑ apoptosis ↑ apoptosis ↓ proliferation ↓ proliferation ↑ apoptosis ↓ proliferation ↑ apoptosis ↓ angiogenesis

Table 8. In vivo experimental studies of COX-inhibitors in gastric cancer Author Lehnert T[137] Bespalov VG[138] Lehnert T[139] Jalbert G[140] Sawaoka H[141] Liu C[124] Fu SL[142] Hu PJ[143] Nam KT[144] Yu J[145]

Year 1987 1989 1990 1992 1998 2003 2004 2004 2005 2005

Animal model Rat Rat Rat Mouse Nude mouse Nude mouse Nude mouse Rat C57BL/6 mice Wistar rat

Triggering factor MNNG NSEE MNNG NNK MKN45 SGC7901 SGC7901 SGC7901 MNU +/- H. pylori MNNG

Drug tested Flurbiprofen Indomethacin + Dexamethasone Flurbiprofen Sulindac; ibuprofen; piroxicam; naproxen NS-398; Indomethacin Rofecoxib Sulindac; Celecoxib Indomethacin; Rofecoxib Nimesulide Indomethacin; Celecoxib

Result ↑ Tumor incidence ↓ Tumor incidence ↑ Tumor incidence ↓ Tumor number ↓ Xenograft tumor volume ↓ Xenograft tumor implant ↓ Xenograft tumor volume ↓ Tumor incidence and growth ↓ Tumor incidence ↓ Tumor incidence

MNNG: N-methyl-Ni-nitro-N-nitrosoguanidine; NSEE: N-nitrososarcosine ethyl ester; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; MNU: Nmethyl-N-nitrosourea; H. pylori: Helicobacter pylori

Table 9. Epidemiological studies on COX-inhibitors in prevention of gastric cancer Author Thun MJ[146 ] Farrow DC[147]* Zaridze D[148]*§ Coogan PF[149] Langman MJ[150] Akre K[151]* Sorensen HT[152]

Year 1993 1998 1999 2000 2000 2001 2003

Protocol study Cohort Case-control Case-control Case-control Case-control Case-control Cohort

Population 635,031 629 448 254 613 567 172,057

Drug Aspirin Aspirin or NSAIDs Aspirin or NSAIDs NSAIDs NSAIDs Aspirin NSAIDs

Duration ≥ 10 yrs -2 days/wk for 6 mos 4 days/wk for 3 mos 7 times/last 13-36 mos >30 tablets/mo >10 prescriptions

OR 0.53 0.46 0.60 0.30 0.51 0.70 0.70#

* Data refer to non-cardia gastric cancer; § Reduction of risk limited to H. pylori-positive patients; # SIR: Standardized incidence ratio

95% CI 0.34-0.81 0.31-0.68 0.41-0.90 0.10-0.60 0.33-0.79 0.60-1.00 0.40-1.10

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COX-2 and Gastric Cancer: Human Studies A growing body of evidence suggests that COX-2 inhibitors may have a beneficial effect, as gastric cancer chemoprevention, even if data in the literature (Table 9) are still limited to case-control or cohort studies [146-152]. Initial reports come from record linkage studies performed in Finland and Sweden on patients with rheumatoid arthritis. In a large cohort study, supported by the American Cancer Society, on 653031 participants observed at follow-up for approximately 10 years, Thun et al. demonstrated that regular use of aspirin exerted a protective effect against gastric cancer. In that study, those patients reporting use of aspirin for more than 16 times per month showed a reduction of approximately 50% in the risk of gastric cancer when compared with nonusers [146]. Analyzing data from the population-based North Jutland prescription database and the Danish Cancer Registry, comprising 172057 individuals, a reduced risk was found for gastric cancer among non-aspirin NSAIDs users, over a 9-year study period [152]. Coogan et al. found that regular NSAIDs use (at least 4 days a week for > 3 months) reduced the risk of gastric cancer in a hospital-based case-control study of 254 patients [149]. The protective effect was more pronounced in those patients using NSAIDs continously for >5 years (OR 0.2; 95% CI 0.10.7) than in those using NSAIDs for < 5 years (OR 0.4 95% CI 0.1-0.9). In a case-control study from the United Kingdom, Langman et al. found a lower risk of gastric cancer in subjects who had used NSAIDs for 13-16 months before cancer diagnosis [150]. Results of two different case control studies, revealed that users of aspirin, compared to non users, were at decreased risk of non- cardia gastric adenocarcinoma but not of gastric cardia adenocarcinoma [147,151]. Moreover, Zaridze reported that only H. pylori-infected patients using NSAIDs were at decreased risk of gastric cancer [148].

Conclusions Gastric cancer remains a major health concern and prevention and chemoprevention are the only valid alternatives for control of the disease. Widespread and long-term use of NSAIDs has been advocated, in the healthy population, for gastric chemoprevention. Albeit, enthusiasm has been thwarted by the potential toxic effects, i.e., risk of peptic ulcer disease. Selective COX-2 inhibitors, causing less injury to the mucosa of the upper gastrointestinal tract, may be a valid alternative. However, the mechanisms of the anti-tumoral action of COX-2 inhibitors still remains to be defined and may vary from agent to agent and from tumor to tumor. In vitro studies have shown a mixture of COX-related mechanisms in controlling proliferation and apoptosis balance. Studies on animal models are often performed with much higher pharmacological doses than those clinically feasible. Human observational studies are prevalently of the case-control type and often suffer from inadequate sample size to avoid a type II statistical error. Furthermore, due to the high cost of these new agents, costeffectiveness analyses must be carried out to optimize the allocation of resources. The cumulative probability of developing a lesion from birth to 80 years of age is less than 4% thus, in the general population, more than 95% of people treated prophylactically with COX-

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2 inhibitors will reap no benefit. Therefore, chemoprevention with selective COX-2 inhibitors may be worthwhile only in those subjects known to be at an increased risk of gastric cancer. However, also in these subjects, fundamental questions such as safety, efficacy, mechanisms of actions, optimal treatment regimens need to be defined. Very recently, COX-2 inhibitor rofecoxib has been withdrawn from the market due to the high risk of inducing a coronary heart attack. Although epidemiological studies have clearly established that H. pylori infection is associated with gastric cancer, there are, so far, no definitive prospective studies showing that eradication treatment significantly reduces the development of this neoplasm. Prospective studies are hampered by the long period of time elapsing between infection and cancer development. Cost-effect analyses suggest that only a subgroup of H. pylori-infected patients may present beneficial effects following eradication therapy e.g., people living in high risk areas, relatives of gastric cancer patients, patients with gastric atrophy and intestinal metaplasia. Although significant advances have been made in the understanding of dietary, environmental, and genetic factors involved in the process of carcinogenesis, use of this knowledge has still not been useful to conduct successful intervention trials. At present, “diet for cancer prevention” (Table 10) can be proposed as a general rule of well-being and may represent the basis for a rational health policy. A reasonable suggestion for the general population is a natural chemoprevention based on life-style “eat to live, not live to eat”. Table 10. Dietary advice to reduce cancer risk -

Eat plenty of fruit and vegetables (at least five portion a day) Eat plenty of cereal foods mainly in an unprocessed form (as a source of non-starch polysaccharides) Maintain ideal body weight (body mass index 20-25) Avoid fatty foods Eat red meat and processed meat in moderation (no more than 140 g/day) Avoid high doses of vitamin supplements Alcohol in moderation (a maximum of two units a day for women and three units a day for men) Avoid highly salted and mouldy foods

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Helicobacter pylori positivity and gastric cancer in Mexico. Int J Cancer. 2003;106(2):277-82. [100] Machida-Montani, A; Sasazuki, S; Inoue, M; Natsukawa, S; Shaura, K; Koizumi, Y; Kasuga, Y; Hanaoka, T; Tsugane, S. Association of Helicobacter pylori infection and environmental factors in non-cardia gastric cancer in Japan. Gastric Cancer. 2004;7(1):46-53. [101] Zullo, A; Rinaldi, V; Hassan, C; Diana, F; Winn, S; Castagna, G; Attili, AF. Ascorbic acid and intestinal metaplasia in the stomach: a prospective, randomised study. Aliment Pharmacol Ther 2000;14:1303–9. [102] Correa, P; Fontham, ET; Bravo, JC; Bravo, LE; Ruiz, B; Zarama, G; Realpe, JL; Malcom, GT; Li, D; Johnson, WD; Mera, R. Chemoprevention of gastric dysplasia: randomized trial of antioxidant supplements and anti-Helicobacter pylori therapy. J Natl Cancer Inst 2000;92:1881–8. [103] Weisburger, JH; Chung, FL; Weisburger, JH; Chung, FL. Mechanisms of chronic disease causation by nutritional factors and tobacco products and their prevention by tea polyphenols. Food Chem Toxicol 2002;40:1145–54. [104] Sturmer, T; Glynn, RJ; Lee, IM; Manson, JE; Buring, JE; Hennekens, CH. Aspirin use and colorectal cancer: post-trial follow-up data from the Physician’s Health Study. Ann Intern Med 1998;128:713-20. [105] Vane, JR; Bakhle, YS; Botting, RM. Cyclo-oxygenase 1 and 2. Annu Rev Pharmacol Toxicol 1998;38:97-120. [106] Funk, CD; Funk, LB; Kennedy, ME; Pong, AS; Fitzgerald, GA. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment. FASEB J 1991;5(9):2304-12. [107] Smith, WL; Garavito, RM; DeWitt, DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996;271(52):33157-60. [108] Dubois, RN; Abramson, SB; Crofford, L; Gupta, RA; Simon, LS; Van De Putte, LB; Lipsky, PE. Cyclooxygenase in biology and disease. FASEB J. 1998;12:1063-1073. [109] Tatsuguchi, A; Sakamoto, C; Wada, K; Akamatsu, T; Tsukui, T; Miyake, K; Futagami, S; Kishida, T; Fukuda, Y; Yamanaka, N; Kobayashi, M. Localisation of cyclooxygenase 1 and cyclooxygenase 2 in Helicobacter pylori related gastritis and gastric ulcer tissues in humans. Gut 2000;46(6):782-9. [110] Nardone, G; Rocco, A; Vaira, D; Staibano, S; Budillon, A; Tatangelo, F; Scialli, MG; Perna, F; Salvatore, G; Di Benedetto, M; De Rosa, G; Patrignani, P. Expression of COX-2, mPGE synthase 1, MDR-1 (P-GP) and BCL-XL: a molecular pathway of H. pylori-related gastric carcinogenesis. J Pathol 2004: 202(3):305-12. [111] Sung, JJ; Leung, WK; Go, MY; To, KF; Cheng, AS; Ng, EK; Chan, FK. Cyclooxygenase-2 expression in Helicobacter pylori-associated premalignant and malignant gastric lesions. Am J Pathol 2000;157(3):729-35. [112] Ristimaki, A; Honkanen, N; Jankala, H; Sipponen, P; Harkonen, M. Expression of cyclooxygenase-2 in human gastric carcinoma. Cancer Res 1997;57(7):1276-80. [113] Saukkonen, K; Nieminen, O; van Rees, B; Vilkki, S; Harkonen, M; Juhola, M; Mecklin, JP; Sipponen, P; Ristimaki, A. Expression of cyclooxygenase-2 in dysplasia

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of the stomach and in intestinal-type gastric adenocarcinoma. Clin Cancer Res 2001;7(7):1923-31. [114] Yamagata, R; Shimoyama, T; Fukuda, S; Yoshimura, T; Tanaka, M; Munakata, A. Cyclooxygenase-2 expression is increased in early intestinal-type gastric cancer and gastric mucosa with intestinal metaplasia. Eur J Gastroenterol Hepatol 2002;14(4):359-63. [115] Tsuji, S; Kawano, S; Sawaoka, H; Takei, Y; Kobayashi, I; Nagano, K; Fusamoto, H; Kamada, T. Evidences for involvement of cyclooxygenase-2 in proliferation of two gastrointestinal cancer cell lines. Prostaglandins Leukot Essent Fatty Acids 1996;55(3):179-83. [116] Sawaoka, H; Kawano, S; Tsuji, S; Tsujii, M; Murata, H; Hori, M. Effects of NSAIDs on proliferation of gastric cancer cells in vitro: possible implication of cyclooxygenase2 in cancer development. J Clin Gastroenterol 1998;27 Suppl 1:S47-52. [117] Uefuji, K; Ichikura, T; Shinomiya, N; Mochizuki, H. Induction of apoptosis by JTE522, a specific cyclooxygenase-2 inhibitor, in human gastric cancer cell lines. Anticancer Res 2000;20(6B):4279-84. [118] Husain, SS; Szabo, IL; Pai, R; Soreghan, B; Jones, MK; Tarnawski, AS. MAPK (ERK2) kinase--a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth. Life Sci 2001;69(25-26):3045-54. [119] Zhou, XM; Wong, BC; Fan, XM; Zhang, HB; Lin, MC; Kung, HF; Fan, DM; Lam, SK. Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak. Carcinogenesis 2001;22(9):1393-7. [120] Li, JY; Wang, XZ; Chen, FL; Yu, JP; Luo, HS. Nimesulide inhibits proliferation via induction of apoptosis and cell cycle arrest in human gastric adenocarcinoma cell line. World J Gastroenterol 2003;9(5):915-20. [121] Jiang, XH; Lam, SK; Lin, MC; Jiang, SH; Kung, HF; Slosberg, ED; Soh, JW; Weinstein, IB; Wong, BC. Novel target for induction of apoptosis by cyclo-oxygenase2 inhibitor SC-236 through a protein kinase C-beta(1)-dependent pathway. Oncogene. 2002;21(39):6113-22. [122] Wong, BC; Jiang, X; Fan, XM; Lin, MC; Jiang, SH; Lam, SK; Kung, HF. Suppression of RelA/p65 nuclear translocation independent of IkappaB-alpha degradation by cyclooxygenase-2 inhibitor in gastric cancer. Oncogene 2003;22(8):1189-97. [123] Wu, J; Xia, HH; Tu, SP; Fan, DM; Lin, MC; Kung, HF; Lam, SK; Wong, BC. 15Lipoxygenase-1 mediates cyclooxygenase-2 inhibitor-induced apoptosis in gastric cancer. Carcinogenesis 2003;24(2):243-7. [124] Liu, C; Tang, C; Wan, X; Wang, C; Zhou, X. The effects of selective cyclooxygenase-2 inhibitors on the growth of gastric adenocarcinoma. Sichuan Da Xue Xue Bao Yi Xue Ban. 2003;34(3):480-3. [125] Wang, C; Tang, C. Inhibition effect of aspirin on the growth of gastric cancer and the mechanism there-in involved. Sichuan Da Xue Xue Bao Yi Xue Ban. 2003;34(3):46871. [126] Leung, WK; To, KF; Go, MY; Chan, KK; Chan, FK; Ng, EK; Chung, SC; Sung, JJ. Cyclooxygenase-2 upregulates vascular endothelial growth factor expression and angiogenesis in human gastric carcinoma. Int J Oncol. 2003;23(5):1317-22.

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[127] Hu, GY; Yu, BP; Yu, JP; Ran, ZX; Luo, HS. Nimesulide, a selective cyclooxygenase-2 inhibitor inhibits telomerase activity by blocking activation of PKB in gastric cancer cell line. Zhonghua Zhong Liu Za Zhi. 2004;26(4):209-12. [128] Baoping, Y; Guoyong, H; Jieping, Y; Zongxue, R; Hesheng, L. Cyclooxygenase-2 inhibitor nimesulide suppresses telomerase activity by blocking Akt/PKB activation in gastric cancer cell line. Dig Dis Sci 2004;49(6):948-53. [129] Power, JJ; Dennis, MS; Redlak, MJ; Miller, TA. Aspirin-induced mucosal cell death in human gastric cells: evidence supporting an apoptotic mechanism. Dig Dis Sci 2004;49(9):1518-25. [130] Gu, Q; Wang, JD; Xia, HH; Lin, MC; He, H; Zou, B; Tu, SP; Yang, Y; Liu, XG; Lam, SK; Wong, WM; Chan, AO; Yuen, MF; Kung, HF; Wong, BC. Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer. Carcinogenesis 2005;26(3):541-6. [131] Honjo, S; Kase, S; Osaki, M; Ardyanto, TD; Kaibara, N; Ito, H. COX-2 correlates with F-box protein, Skp2 expression and prognosis in human gastric carcinoma. Int J Oncol 2005;26(2):353-60. [132] Honjo, S; Osaki, M; Ardyanto, TD; Hiramatsu, T; Maeta, N; Ito, H. COX-2 inhibitor, NS398, enhances Fas-mediated apoptosis via modulation of the PTEN-Akt pathway in human gastric carcinoma cell lines. DNA Cell Biol 2005;24(3):141-7. [133] Ma, L; Xie, YL; Yu, Y; Zhang, QN. Apoptosis of human gastric cancer SGC-7901 cells induced by mitomycin combined with sulindac. World J Gastroenterol 2005;11(12):1829-32. [134] Fu, YG; Sung, JJ; Wu, KC; Wu, HP; Yu, J; Chan, M; Chan, VY; Chan, KK; Fan, DM; Leung, WK. Inhibition of gastric cancer-associated angiogenesis by antisense COX-2 transfectants. Cancer Lett 2005;224(2):243-52. [135] Moss, SF. Cellular markers in the gastric precancerous process. Aliment Pharmacol Ther. 1998;12 Suppl 1:91-109. [136] Dannenberg, AJ; Altorki, NK; Boyle, JO; Dang, C; Howe, LR; Weksler, BB; Subbaramaiah, K. Cyclo-oxygenase 2: a pharmacological target for the prevention of cancer. Lancet Oncol. 2001;2(9):544-51. [137] Lehnert, T; Deschner, EE; Karmali, RA; DeCosse, JJ. Effect of flurbiprofen and 16,16dimethyl-prostaglandin E2 on gastrointestinal tumorigenesis induced by N-methyl-N'nitro-N-nitrosoguanidine in rats. I. Squamous epithelium and mesenchymal tissue. J Natl Cancer Inst 1987;78(5):923-9. [138] Bespalov, VG; Troian, DN; Petrov, AS; Morozov, VG; Khavinson, VKh. Inhibiting effect of thymogen on the development of tumors of the esophagus and forestomach induced by N-nitrososarcosine ethyl ester in rats. Eksp Onkol 1989;11(4):23-6. [139] Lehnert, T; Deschner, EE; Karmali, RA; DeCosse, JJ. Effect of flurbiprofen and 16,16dimethyl prostaglandin E2 on gastrointestinal tumorigenesis induced by N-methyl-N'nitro-N-nitrosoguanidine in rats: glandular epithelium of stomach and duodenum. Cancer Res 1990;50(2):381-4. [140] Jalbert, G; Castonguay, A. Effects of NSAIDs on NNK-induced pulmonary and gastric tumorigenesis in A/J mice. Cancer Lett 1992;66(1):21-8.

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[141] Sawaoka, H; Kawano, S; Tsuji, S; Tsujii, M; Gunawan, ES; Takei, Y; Nagano, K; Hori, M. Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice. Am J Physiol 1998;274(6 Pt 1):G1061-7. [142] Fu, SL; Wu, YL; Zhang, YP; Qiao, MM; Chen, Y. Anti-cancer effects of COX-2 inhibitors and their correlation with angiogenesis and invasion in gastric cancer. World J Gastroenterol. 2004;10(13):1971-4. [143] Hu, PJ; Yu, J; Zeng, ZR; Leung, WK; Lin, HL; Tang, BD; Bai, AH; Sung, JJ. Chemoprevention of gastric cancer by celecoxib in rats. Gut. 2004;53(2):195-200. [144] Nam, KT; Hahm, KB; Oh, SY; Yeo, M; Han, SU; Ahn, B; Kim, YB; Kang, JS; Jang, DD; Yang, KH; Kim, DY. The selective cyclooxygenase-2 inhibitor nimesulide prevents Helicobacter pylori-associated gastric cancer development in a mouse model. Clin Cancer Res 2004;10(23):8105-13. [145] Yu, J; Tang, BD; Leung, WK; To, KF; Bai, AH; Zeng, ZR; Ma, PK; Go, MY; Hu, PJ; Sung, JJ. Different cell kinetic changes in rat stomach cancer after treatment with celecoxib or indomethacin: implications on chemoprevention. World J Gastroenterol 2005;11(1):41-5. [146] Thun, MJ; Namboodiri, MM; Calle, EE; Flanders, D; Heath, CW. Aspirin use and risk of fatal cancer. Cancer Res. 1993(53):1322-27. [147] Farrow, DC; Vaughan, TL; Hansten, PD; Stanford, JL; Risch, HA; Gammon, MD; Chow, WH; Dubrow, R; Ahsan, H; Mayne, ST; Schoenberg, JB; West, AB; Rotterdam, H; Fraumeni, JF Jr; Blot, WJ. Use of aspirin and other nonsteroidal anti-inflammatory drugs and risk of esophageal and gastric cancer. Cancer Epidemiol Biomarkers Prev. 1998;7(2):97-102. [148] Zaridze, D; Borisova, E; Maximovitch, D; Chkhikvadze, V. Aspirin protects against gastric cancer: results of a case-control study from Moscow, Russia. Int J Cancer. 1999;82(4):473-6. [149] Coogan, PF; Rosenberg, L; Palmer, JR; Strom, BL; Zauber, AG; Stolley, PD; Shapiro, S. Nonsteroidal anti-inflammatory drugs and risk of digestive cancers at sites other than the large bowel. Cancer Epidemiol Biomarkers Prev. 2000;9(1):119-23. [150] Langman, MJ; Cheng, KK; Gilman, EA; Lancashire, RJ. Effect of anti-inflammatory drugs on overall risk of common cancer: case-control study in general practice research database. BMJ. 2000;320(7250):1642-6. [151] Akre, K; Ekstrom, AM; Signorello, LB; Hansson, LE; Nyren, O. Aspirin and risk for gastric cancer: a population-based case-control study in Sweden. Br J Cancer. 2001;84(7):965-8. [152] Sorensen, HT; Friis, S; Norgard, B; Mellemkjaer, L; Blot, WJ; McLaughlin, JK; Ekbom, A; Baron, JA. Risk of cancer in a large cohort of nonaspirin NSAID users: a population-based study. Br J Cancer. 2003;88(11):1687-92.

In: COX-2 Inhibitor Research Editor: Maynard J. Howardell, pp. 115-143

ISBN 1-59454-994-X © 2006 Nova Science Publishers, Inc.

Chapter III

Cyclooxygenases in Cancer

1

Daniela Foderà1, Nadia Lampiasi1, Antonella Cusimano2 and Melchiorre Cervello1∗ Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, Consiglio Nazionale delle Ricerche, Palermo, Italy 2 Dipartimento di Medicina Clinica e Patologie Emergenti, Università di Palermo, Palermo, Italy

Abstract Here we review the features of COX enzymes, the role of expression of COX isoforms in carcinogenesis and mechanisms by which they contribute to cancer, the pharmacological properties of COX-2 selective inhibitors, the antitumor effects of COX inhibitors, and the rationale and feasibility of COX-2 inhibitors for treatment of cancer.

Introduction Several lines of evidence indicate that cyclooxygenase-2 (COX-2) is an important molecular target for anticancer therapies. Many epidemiological studies demonstrate that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) reduce the incidence and mortality of a wide range of tumors. However, conventional NSAIDs inhibit non-selectively both the “constitutive” form COX-1, and the “inducible” form COX-2. It is now wellestablished that COX-2 is chronically overexpressed in many premalignant, malignant, and metastastic cancers, and the levels of its overexpression have been shown to significantly correlate to increased invasiveness, poorer prognosis and reduced survival in some cancers.

∗

Correspondence to: Melchiorre Cervello, Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, C.N.R., Via Ugo La Malfa 153, 90146 Palermo, Italy. Fax: 39-091-6809548; Telephone: 39-091-6809534; email: [email protected]

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Although less attention has been drawn to the potential role of the constitutive COX-1 enzyme in carcinogenesis, recent evidence supports its implication in some types of tumors. In tumors, overexpression of COX-2 leads to an increase in prostaglandins (PGs) levels, which affect many mechanisms involved in carcinogenesis, such as angiogenesis, inhibition of apoptosis, stimulation of cell growth as well as invasiveness and metastatic potential of tumor cells. PGE2 is the prostaglandin most abundantly found in tumors and performs its biological function by binding to EP receptors. Recent studies have attempted to shed light on the role of the EP receptors in carcinogenesis. The introduction of novel agents, which inhibit selectively COX-2 isoenzyme, have contributed to clarify the role of this molecule, at least in some tumor types. However, the key mechanism by which COX inhibitors affect tumor cell growth is still not clear. Increasing evidence suggests the involvement of molecular targets other than COX-2 in the antiproliferative effects of COX-2 selective inhibitors, including among others the nuclear factor-κB (NF-κB) protein, the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the peroxisome proliferator-associated receptors (PPARs). Therefore, COX-inhibitors may use both COX-2 and non-COX-2 targets to mediate their antitumor activities, although their relative contribution toward the in vivo effects remains undefined. The involvement of COX-2 in endocannabinoids metabolism and in the generation of novel lipids that are structurally related to prostaglandins has been also suggested. Here we review the features of COX enzymes, the role of expression of COX isoforms in carcinogenesis and mechanisms by which they contribute to cancer, the pharmacological properties of COX-2 selective inhibitors, the antitumor effects of COX inhibitors, and the rationale and feasibility of COX-2 inhibitors for treatment of cancer.

Functions of Cyclooxygenases Cyclooxygenase (COX) enzymes also referred to as prostaglandin H synthases or prostaglandin endoperoxide synthases, catalyze the rate limiting steps in prostaglandin (PG) and thromboxane (TX) synthesis. Two distinct COX enzymes exist, COX-1 and COX-2. COX-1 was first purified and characterized in the 1970s and the gene was isolated in 1988 [1-3], whereas COX-2 was cloned in 1993 [4]. Substrates for the enzymatic COX activities are 20 carbon polyunsaturated fatty acids, most often arachidonic acid (AA), released from membrane-bound phospholipids, usually by the action of phospholipase enzymes, primarily phospholipase A2 (Figure 1). Subsequent to its release, free arachidonic acid is presented to the endoplasmic reticulum and nuclear membrane, where the COX enzymes catalyze the first step for prostaglandins synthesis. This step begins with the rate-limiting abstraction of the (13S)-hydrogen from arachidonate to yield an arachidonyl radical [5]. This is followed by sequential oxygen additions at C-11 and C-15 to yield PGG2. Since this results in cyclization of the fatty acid this is referred to as the cyclooxygenase activity. The 15-hydroperoxide group of PGG2 is then converted to an alcohol forming PGH2 by the peroxidase activity of the enzyme [6]. PGH2 is subsequently converted to other PGs (PGD2, PGE2, PGF2α, PGI2) or

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thromboxanes (TXA2). The array of PGs produced varies depending on the downstream enzymatic machinery present in a particular cell type (Figure 1).

Membrane phospholipids

Diverse physical, chemical, inflammatory and mitogenic stimuli

Phospholipase A2

COOH H

2O2

COX activity

COX-1 COX-2

Arachidonic acid

PGG2

O O

OOH

2 e-

HOX activity

PGH2

O O OH

Tissue-specific isomerases

Prostanoids

PGI2

TXA2

PGE2

PGD2

PGF2

Receptors

IP

TPα, TPβ

EP1-4

DP1,DP2

FPα, FPβ

Figure 1. Prostanoids biosynthetic pathway

Cyclooxygenase Genes Separate genes located on different human chromosomes encode COX-1 and COX-2. The gene for COX-1 enzyme is located on chromosome 9 (9q32-9q33.3) and is approximately 40 kilobase (kb) pairs, contains 11 exons and its mRNA is 2.8 kb [7]. The gene encoding COX-2 is located on chromosome 1 (1q25.2-25.3), contains 10 exons and is approximately 8.3 kb with a 4.5 kb transcript [8]. Despite the difference in genomic structure

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and transcript size, the proteins of both COX enzymes are highly similar in structure and enzymatic activity, and show a molecular weight of about 68 kilodaltons (kDa) in unmodified conditions that increases to 72-74 kDa after post-translation glycosilation [9]. COX-1 gene exhibits the features of a housekeeping gene, which lacks a TATA box [10], and it is generally not subject to transcriptional induction, but it is constitutively expressed with near constant levels and activity in most tissues and cell types. COX-2 is an inducible or early-response gene. Its expression is undetectable in most normal tissues, and in many cell type is highly induced in response to a broad spectrum of mediators involved in inflammation, such as bacterial lipopolysaccharide (LPS) [11], proinflammatory cytokines-interleukin-1β (IL-1β) [12], transforming growth factor-β (TGF-β) [13] and tumor necrosis factor-α (TNF-α) [14]. Nucleotide sequence analysis of the 5'flanking region of the human COX-2 gene promoter has shown the presence of several potential transcription regulatory sequences, including a TATA box, a C/EBP motif, two AP2 sites, three SP1 sites, two NF-kappa B sites, a CRE motif and an Ets-1 site [15]. The presence of these elements explains, at least in part, its inducibility by hormones, growth factors, phorbol esters, cyclic adenosine monophasphate (cAMP), inflammatory factors and cytokines. COX-2 gene expression is also subject to negative regulation. However, glucocorticoids, IL-4, IL-13 and the anti-inflammatory cytokine IL-10 have been reported to inhibit expression of COX-2 (16-18). COX-2 expression is also regulated at posttranscriptional levels in tumors. The 3’ untranslated region (3’-UTR) of the COX-2 mRNA contains multiple copies of the motif AUUUA that control both mRNA stability and protein translation. Such motifs represent potential targets by which different agents stabilize or destabilize the COX-2 message, thus promoting elevated or decreased levels of enzymatic activity. It was shown that HuR, an RNA binding protein, by binding to the COX-2 AU rich element prolongs the half-life of COX-2 mRNA and ultimately leads to COX-2 overexpression [19, 20]. By contrast other proteins such as tristetraprolin [21] and AUF1 [22] that bind also to the 3’-UTR can decrease the levels of the COX-2 mRNA. The different expression pattern and the different regulation of two enzymes have led to the notion that COX-1 is the constitutive form responsible for generation of PGs which mediate homeostatic or “housekeeping” function, such as maintenance of vascular tone and cytoprotection of the stomach, while COX-2 is the inducible form responsible for the generation of prostanoids involved in pathological processes, such as acute and chronic inflammatory states. However, this notion is probably an oversimplification because COX-2 is expressed constitutively in brain [23], seminal vesicles [24], kidney [25] and tracheal epithelia [26], while COX-1 levels change during development [27] and its expression can be down-regulated by heparin-binding (acidic fibroblast) growth factor-1 in endothelial cells [28] and up-regulated in mast cells exposed to cytokines and glucocorticoids [29]. Recently, it has been suggested that there is another COX enzyme formed as a splice variant of COX-1 [30], referred as COX-3. Since COX-3 is made from COX-1 gene but retains intron 1 in its mRNA, may be more appropriate to name it COX-1b. Its expression was initially reported in canine cerebral cortex and in lesser amounts in other tissues analyzed [30]. However, COX-3 might not be relevant to humans [31], as there is one nucleotide difference in intron 1 between human and canine genes thus shifting the human coding

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sequence out of frame. This would make impossible to have the expression of a full-length protein, therefore catalytically active form of COX-3 might not exist in humans.

Cyclooxygenases and Cancer Epidemiological Studies Numerous epidemiological studies have shown that long-term use of conventional NSAIDs markedly reduced the incidence and mortality of colorectal cancer [32-34]. In addition, other studies also reported a reduction in the risk of developing esophageal, gastric, breast, lung, prostate and ovarian cancer among regular users of NSAIDs [35, 36]. However, most NSAIDs, beside COX-2, inhibit also the constitutive isoform COX-1. Therefore, the prolonged use of NSAIDs has several side effects, such as dyspepsia, gastrointestinal bleeding and ulceration [37]. More recently, the availability of new drugs, which selectively inhibit COX-2, has made it possible to reduce gastrointestinal toxicity and to better clarify the role of this cyclooxygenase isoform in different tumor types.

Genetic Models The best evidence that strongly support the connection between COX-2 expression and carcinogenesis comes from genetic studies. The number and size of intestinal polyps in the APC∆716 mice, a murine model of human familial adenomatous polyposis coli (FAP), were reduced in animals that were engineered to be also COX-2 deficient [38]. In a separate study, homozygous deficiency of COX-2 reduced skin tumorigenesis in a multistage mouse skin model [39]. On the contrary, overexpression of COX-2 was sufficient to induce tumorigenesis in transgenic mice. Multiparous but not virgin females exhibited a high frequency of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors [40]. Moreover, transgenic mice that simultaneously expressed COX-2 and microsomal prostaglandin E synthase (mPGES)-1 in the gastric epithelial cells developed hyperplastic gastric tumors [41]. Whereas, COX-2 overexpression was insufficient to induce skin tumor but dramatically sensitized the tissue for genotoxic carcinogens, suggesting that COX-2 overexpression is involved in skin tumor promotion [42]. While most of the studies are focused on the role of COX-2 in carcinogenesis, increasing evidence indicates that COX-1 plays an essential role also in skin [39] and intestinal tumorigenesis [43-46]. Genetic disruption of the COX-1 gene decreased the number of intestinal polyps in the multiple intestinal neoplasia (Min) mice model by around 80% [43]. Up-regulation of COX-1 expression has been also shown in human breast [47], prostate [48], cervical [49] and ovarian cancers [50, 51]. In addition, it has been shown that cooperation between COX-1 and COX-2 is essential for intestinal polyp formation [45].

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COX Inhibitors and Experiments in Animal Models In addition to genetic evidence, pharmacological studies using NSAIDs implicate COX-2 in tumorigenesis, and suggest that COX may represent a therapeutic target for cancer prevention and treatment. At present, all the NSAIDs are defined as the drugs that have the same three beneficial effects, analgesic, anti-pyretic and anti-inflammatory, but differ in their therapeutic potency, potential for gastrointestinal damage and COX inhibition ratio. NSAIDs cover a wide range in the ratio of inhibitory potencies (i.e. selectivity) towards COX-1 and COX-2. There are NSAIDs favoring COX-1 inhibition (e.g., ketorolac, flurbiprofen, ketoprofen, piroxicam), others that are more evenly balanced inhibiting at the same time both COX isoforms (dual inhibitors; e.g. indomethacin, aspirin, naproxen, ibuprofen), NSAIDs that display some level of COX-2 selectivity (e.g. sulindac, nimesulide etodolac, meloxicam), and finally the newest highly selective for COX-2 (COXIB; e.g. celecoxib, rofecoxib, lumiracoxib, valdecoxib, etoricoxib) (Table 1). Although COXIB shares similar mechanism of action there are important differences in their chemical structure. In addition, the pharmacokinetic and metabolism of each COXIB are unique (Table 1) [52, 53]. Table 1. Pharmacologycal features of coxibs Brand: Generic: Chemistry:

Celebrex Celecoxib Sulphonamide

Metabolism

Pharmacokinetics

COX-1/COX-2 ratio 30 Oral 22-40 bioavailability (%)

Vioxx Rofecoxib Sulphonyl

Bextra Arcoxia Valdecoxib Etoricoxib Sulphonamide Sulphonyl

Prexige Lumiracoxib Phenylacetic acid

276 92-93

261 83

344 100

433 74

Tmax (h)

2-4

2-3

2.3

1

2-3

Half-life (h)

11

10-17

8-11

22

3-6

Vol.Dist. (liters)

455

86-91

86

120

9

Plasma protein binding (%)

97

87

98

92

>98

Main pathway

Oxydation CyP450 (2C9, 3A4) 29

Cytosolic reduction

Oxydation CyP450 (2C9, 3A4) 70

Oxydation CyP450 (3A4) 60

Oxydation CyP450 (2C9) 54

Urinary excretion (%)

72

The effects of NSAIDs have been studied in three different types of animal models of colorectal cancer: (i) in the APC∆716 mice with intestinal tumor [54-59]; (ii) in rats treated

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with the carcinogen azoxymethane (AOM) [60-64]; (iii) in the nude mouse subjected to tumor xenografts [65-67]. Using these three different approaches conventional NSAIDs have been shown to markedly inhibit tumor growth and reduce the number and size of tumors. Treatment with selective COX-2 inhibitors also reduces the formation of gastric [68, 69], skin [70, 71], lung [72, 73], bladder [74], esophageal [75], liver [76], and breast [77, 78] tumors in animals. However, tumor regression has rarely been seen. Finally, since COX-1 and COX-2 enzymes have only minor differences in their catalytic activity, catalyze identical reactions and have the same intracellular localization, it is possible that each COX isoform may compensate for the loss of the activity of the other. Therefore, inhibition of both isoforms may overcome such compensation. Accordingly, recent studies have shown that combination of both COX-1 and COX-2 selective inhibitors suppress more effectively polyp formation in the intestinal tumorigenesis of the Apc knockout mouse model than each inhibitor used alone [46].

Prostanoids Receptors Prostanoids produced by COX enzymes activity modulate different physiological processes (Table 2). PGs regulate renal blood flow, modulate many aspects of reproductive biology, regulate the processes of bone formation and resorption, and affect immune response. Moreover, PGs are of vital importance for the maintenance of mucosal integrity in the gastrointestinal tract and, also, are key-regulator of motility and secretion. However, prostaglandins have been also implicated in some pathological conditions, such as inflammatory diseases, cardiovascular diseases and cancer. Prostanoids exert their effects both in autocrine and paracrine fashion by binding to specific membrane-bound receptors belong to the family of G-protein-coupled rhodopsintype receptors containing seven transmembrane domains. They are designated as EP (EP1, EP2, EP3, EP4), FP, DP, IP and TP corresponding to each of the COX metabolites PGE2, PGF2α, PGD2, PGI2 and TXA2, respectively (Figure 1). Each receptor is linked to a different transduction pathway [79]. Activation of DP, IP, EP2 and EP4 results in increased levels of intracellular cAMP, whereas signaling via EP1, FP and TP results in intracellular Ca2+ mobilization. Finally the EP3 receptor signals through decrease intracellular cAMP levels. Recently it was shown that certain PGs are also ligands for a class of receptors named peroxisome proliferator-associated receptors (PPARs). PPARs are members of the nuclear hormone receptor superfamily and are expressed in a variety of tissues. They act as liganddependent transcription factors which heterodimerize with retinoid X receptors to allow binding to and activation of PPAR responsive genes. Through this mechanism, PPAR ligands can control a wide range of physiological processes [80]. Among the different PGs, PGJ2, which is a PGD2 dehydration product, is a ligand for the nuclear receptor PPARγ [81, 82], whereas PGI2 modulates transcription of specific genes via interaction with PPARδ [83, 84]. Genetic studies with EP receptor knockout mice and pharmacological studies with selective EP receptor agonists and antagonists have contributed to clarify their potential involvement in tumorigenesis. Genetic knockout of the EP1 and EP4 receptors suppressed the formation of aberrant crypt foci in mice treated with AOM, a known colon carcinogen [85,

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86]. The same effect was obtained by treatment with selective EP1 and EP4 receptor antagonists [85, 86]. Knockout of EP2 receptor caused a reduction in the number and size of intestinal polyps in Apc∆716 mice [87], and inhibited tumor angiogenesis through a reduction in the levels of the vascular endothelial growth factor (VEGF) [88]. Finally, also EP3-/receptor mice exhibited a decrease in tumor growth associated to a reduced angiogenesis [89]. Table 2. Main functions of prostanoids PGE Vasodilation

PGF2a Vasoconstriction

PGI2 Vasodilation

PGD2 Vasodilation

Increase vascular permeability Diuresis

Diuresis

Inhibition of platelet aggregation Renin release

Mast cell activation Bronchoconstriction Sleep-wake cycle

Natriuresis

Natriuresis Fever Fever Inflammatory erythema Uterine contractions Hyperalgesia Fever Embryo implantation Reduction of gastric acid secretion Uterine contactions Labour Stimulation of gastric mucus and fluid secretion Hyperalgesia Stimulation of duodenal bicarbonate secretion Renin release Reduction of gastric acid secretion Stimulation of gastric mucus and fluid secretion Stimulation of duodenal bicarbonate secretion Insomnia Inhibition of Lymphokine production Inhibition of O2 release

TXA2 Platelet activator and aggregant Vasoconstriction Bronchoconstriction

Molecular Mechanisms by which COX-2 Contributes to Cancer It is believed that the involvement of COX-2 in carcinogenesis is primarily mediated through its influence on cell proliferation, apoptosis, angiogenesis and cell invasiveness [90] (Figure 2).

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Cell proliferation

123

Angiogenesis

COX-1 (?) COX-2

Apoptosis

Invasiveness

Figure 2. Effects of COX enzymes on different cellular dynamics

COX-2 and Cell Proliferation The capability of COX-2 to stimulate cell proliferation can be attributed to the production of prostaglandins. Indeed, evidence indicates that PGs promote cell proliferation, and conversely the growth inhibitory effects of COX inhibitors can be reversed by exogenous addition of PGs. It has been demonstrated that PGE2 and PGI2, through EP1 receptor signaling, increase DNA synthesis and cell growth in primary cultures of rat hepatocytes [91]. In a different study, activation of EP1 receptor by specific agonists induced secretion of transforming growth factor-α (TGF-α) that, in turn, acted as a complete mitogen leading to cell proliferation, through activation of the MAPKs [92]. In colorectal carcinoma cells PGE2 stimulates cell proliferation through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway mediated by activation of EP4 receptor [93]. On the other hand, it has been demonstrated that COX-2 inhibitors are able to suppress cell proliferation in several type of cancers [94-96]. Their antiproliferative effect was more efficient in cells that express high level of COX-2, while they are less effective to suppress the proliferation of cells with lower level of COX-2 expression [65, 97].

COX-2 and Apoptosis Another major action of COX-2 in tumorigenesis is the prevention of apoptosis. This has been attributed mainly to two possible mechanisms: (i) removal of AA, and (ii) production of PGs. It has been shown that high levels of intracellular free AA can promote apoptosis [98101]. Chan et al., have found that inhibition of COX by NSAIDs results in accumulation of the PG precursor AA [100]. This stimulates the conversion of sphingomyelin to ceramide, a lipid that is known to initiate apoptosis. On the other hand, PGs are reported to inhibit apoptosis. As reported by Sheng et al. PGE2 prevents apoptosis in cancer cells by inducing expression of the anti-apoptotic protein Bcl-2 [102]. Furthermore, Ushio et al. reported that a possible mechanism of the anti-

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apoptotic effect of PGE2-mediated signaling is the induction of the anti-apoptotic protein BclxL [103]. Activation of cAMP signaling by PGE2, and other prostaglandins, can also suppress cell death [104].

COX-2 and Angiogenesis COX-2 enzyme promotes angiogenesis, the sprouting of capillaries from pre-existing vasculature, mainly through the synthesis of prostanoids, which can induce tumor angiogenesis in an autocrine and/or paracrine fashion by stimulating the expression of proangiogenic factors [105, 106]. However, the precise role of each prostanoid remains largely unknown. Some studies have shown that PGE2 stimulates VEGF and basic fibroblast growth factor (bFGF) expression in many cell types [107, 108] and by different mechanisms. In particular, in prostate and colon cancer cells PGE2 stimulates VEGF expression through stabilization of its transcriptional activator hypoxia-inducible factor-1α (HIF-1α) protein [109, 110]. Whereas, in endothelial cells PGE2 increases VEGF expression through activation of MAPK pathway [108]. The effects of several NSAIDs in different experimental models of angiogenesis have been also reported [111]. Inhibition of COX-2 by both nonselective and selective NSAIDs can block the production of angiogenic factors, and the proliferation and the migration of endothelial cells [105, 112-114]. The antiangiogenic activity of NSAIDs can be reverted by addition of exogenous PGE2 [115]. Using a different approach, immunohistochemical studies have shown a positive correlation between COX-2 expression and angiogenetic factors expression and neovascularization in some tumor types [116-119]. A significant association of COX-2 with tumor microvessels density has been reported in human head and neck cancer [120], gastric adenocarcinoma [119] and liver cancer [121]. Further evidence for the role of COX-2 in angiogenesis comes from experiments with colon carcinoma cells (CRC) co-cultured with endothelial cells. CRC overexpressing COX-2 promoted the migration of endothelial cells and capillary tube formation, and these events could be reverted by treatment with selective COX-2 inhibitors [105, 122]. Recent studies suggest that also COX-1, and not only COX-2, might be involved in the regulation of angiogenesis. Kim et al. [123] found that COX-1 expression was associated with VEGF expression in primary cervical cancer and at sites of metastasis to lymph nodes. Moreover, ovarian cancer expressing high levels of COX-1 had also elevated levels of proangiogenic proteins [123]. According to data reported by Gupta et al. [124] selective inhibition of COX-1 is also able to block the AA-stimulated production of VEGF, and this effect can be reversed by co-treatment with PGE2. Other studies have shown that treatment of endothelial cells with aspirin or COX-1 antisense oligonucleotides prevents their migration and capillary tube formation [105, 122].

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COX-2 and Invasiveness The link between COX-2 expression and invasiveness has been observed in several human malignancies [125, 126]. Colon cancer cells that constitutively expressed COX-2 acquired increased metastatic potential that could be reversed by treatment with COX inhibitors [127]. This phenotypic change was associated to increased expression and activation of metalloproteinase-2 (MMP-2) [127]. Indeed, PGE2 induces MMP-2 expression and activation in different types of cancer cells, including pancreatic cancer cells [128] and hepatocellular carcinoma cells [129]. However, PGE2 can promote cell invasiveness also via activation of major intracellular signal transduction pathways, such as the PI3K/PKB pathway [130] and the hyaluronate cell surface receptor (CD44)-dependent pathway [131].

COX-2 and Aromatase Activity Aromatase is the cytochrome P450 enzyme complex that catalyzes the final step of estrogen biosynthesis [132]. The activity of aromatase present in the tumor tissue, and in the adjacent tissue, could be a growth-stimulating system in estrogen-sensitive tumors [133]. Regulation of aromatase gene expression in human tissues is quite complex, and it involves alternative promoter sites that provide tissue-specific control. In normal breast tissue the 1.4 promoter directs aromatase expression, while in the malignant and surrounding tissue the major promoters directing aromatase expression are 1.3 and II [134-136]. It is though that PGE2 is a possible factor involved in switching of aromatase promoters. The role of PGE2 in the stimulation of aromatase activity is supported by the observation that agonists of EP1 and EP2 receptors induced aromatase expression and activity, while selective antagonists abolished these effects [137]. Moreover, a positive correlation between COX-2 expression and aromatase expression has been observed in breast tumors [138], and, in addition, experiments have shown that treatment with NSAIDs decreased in a dose-dependent manner the aromatase activity in breast cancer cells [139, 140].

Multidrug Resistance Accumulating evidence indicates that COX-2 overexpression can up-regulate the expression of the Multidrug Resistance 1 (MDR1) gene and the levels of its product, the multidrug efflux pump P-glycoprotein (P-gp) [141, 142]. Recently, Nardone et al. [143] reported that in patients with gastric cancer, high levels of COX-2 were associated with enhanced expression of P-gp and Bcl-xL and in addition, the MDR phenotype has been associated with the COX-2 overexpression also in liver cancer cells [144]. COX-2 could therefore contribute to the development of resistance to pharmacological treatment by the tumor cells [141, 142]. It could be speculated that a selective inhibition of COX-2 activity could reinforce the anti-tumor action of conventional chemotherapy by acting on the expression of P-gp and/or of anti-apoptotic proteins, such as Bcl-xL. The rationale behind the

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possible combination of traditional chemotherapy with selective COX-2 inhibitors is further supported by the fact that chemotherapy itself induces COX-2 expression [145].

Cox-2-Independent Effects The antineoplastic effect of NSAIDs might not be mediated only by COX-2 inhibition, but NSAIDs might act on different molecular targets as well [146] (Figure 3). The hypothesis of the existence of COX-independent mechanisms of NSAIDs action is supported by the evidence that their antineoplastic effects are observed with concentrations which are greater than those necessary to inhibit fully the synthesis of PGs, and by the observation that they inhibit cell proliferation of both COX-2 negative and positive cells [147, 148]. Treatment with sulindac caused an inhibition of cell proliferation and induction of apoptosis in COX-2 deficient human colon cancer cells [149], and celecoxib exhibits antiproliferative effects in COX-2 negative hematopoietic and epithelial cell lines [148]. In addition sulindac sulfone can inhibit AOM-induced colon carcinogenesis in rats without interfering with PGs production [150]. These data suggest that significant antitumor effects of NSAIDs may be seen in vitro and in vivo in the absence of COX-2, therefore probably antineoplastic activities of COX-2 inhibitors are not restricted to COX-2-expressing tumor. After the first report of Kopp and Ghosh [151] that aspirin inhibits the activation of nuclear factor-κB (NF-κB), several studies have investigated the potential role of this transcription factor as a target for certain NSAIDs [152-155].

COX1/2 PPARs

Akt /PKB

NSAIDs

15-LOX-1

MAPKs

PAR-4

NF- k B

Figure 3. Molecular targets of NSAIDs

Moreover, COX inhibitors are reported to induce expression of the two pro-apoptotic genes 15-lipoxygenase-1 [156, 157] and Par-4 [158], and to inhibit the expression of Bcl-XL [159]. Apoptosis induced by NSAIDs can be mediated also by the activation of p38 MAPK [160, 161], extracellular signal-regulated kinases (ERKs) [161-163], and c-Jun NH2-terminal

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kinase (JNK) [164, 165] pathways activity. Whereas, celecoxib was recently shown to induce apoptosis in a variety of cancer cells by inhibiting the phosphorylation of PKB/AKT, thereby blocking its anti-apoptotic activity [166-169]. COX inhibitors can suppress the process of angiogenesis also by COX-independent effects. Treatment with both nonselective and COX-2-selective NSAIDs inhibited angiogenesis by increasing the expression of the von Hippel-Lindau (VHL) tumor suppressor, leading to reduced accumulation of HIF-1α and, as a result, reducing VEGF production [170]. In addition NSAIDs can suppress angiogenesis also by inhibiting the activity of the MAPK ERK2 [122]. Moreover, both aspirin and COX-2-selective NSAIDs inhibited in a COX-independent manner HGF-induced invasiveness of human hepatoma cells through suppression of ERK1/2 activity and MMP-9 expression [171]. Finally, some of the antitumor effects of NSAIDs can be explained in part by their action on PPARs [172-174], which are ligand-activated transcription factors of the nuclear receptor superfamily that have been implicated in carcinogenesis [83, 172, 175-180]. COX-inhibitors have been shown to act as agonists for PPARα and γ [181, 182], and as antagonists for PPARδ [183].

COX-2 and Endocannabinoids Cannabinoids are a class of compounds that are currently used in the treatment of chemotherapy-induced nausea and vomiting, and in the stimulation of appetite. However, there is accumulating evidence that these compounds could also be useful for the inhibition of tumor cell growth in culture and animal models by modulating key survival signaling pathways [184]. Endocannabinoids are endogenous substances with cannabimimetic properties being capable to bind and functionally activate the cannabinoid receptors, CB1 receptor mainly localized in brain [185], and CB2 receptor localized in immune tissues [186]. Two compounds with these properties are the neutral ethanolamide derivative of arachidonic acid anandamide (AEA), and the monoacylglycerol derivate of arachidonic acid 2arachidonylglycerol (2-AG). There is increasing evidence that indicates the involvement of endocannabinoids in the regulation of different physiological and pathological processes. For example, at level of nervous system they modulate function such as pain perception, motor function and memory, at peripheral level they regulate the reproductive function and the immune response [187]. Several investigations have suggested that these substances may also be antitumoral agents. It was shown, for example, that the 2-AG is able to reduce the growth of colon cancer [188], to inhibit the proliferation of glioma cells [189] and to inhibit the invasiveness of prostate cancer cells [190]. Recently, it was identified a distinct biochemical role for COX-2. It is now evident that COX-2 is involved in the oxidative metabolism of endocannabinoids. COX-2 enzyme selectively oxygenates AEA and 2-AG [191, 192]. The oxygenation of these endocannabinoids generates a new family of prostaglandin-like lipids: prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) (or prostamides). These

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metabolites are less able than the corresponding endocannabinoid and PGs to bind and to activate cannabinoid and prostanoid receptors [193-195]. Prostamides are more stable than PGs so they can be subjected to further metabolism. Therefore, AEA and 2-AG oxygenation by COX-2 may represent a way to inactivate partially or completely their “cannabinergic” signal, and to produce compounds that being more stable metabolically can act on other molecular targets causing a switch from a type of signaling pathway to another [196, 197].

Conclusion There is compelling evidence that COX-2, but also COX-1, has a role in carcinogenesis, but many questions remain unanswered. There are a number of studies that have shown several mechanisms for the anticancer effects of NSAIDs, but the main mechanism remains unclear. The effects of NSAIDs on tumor growth are most likely to be multifactorial. COX-inhibitors may use both COX-2 and non-COX-2 targets to mediate their antitumor activities, however their relative contribution toward the in vivo effects remain undefined. Consequently, a better understanding of the COX-2-dependent and COX-2-independent pathways may help to optimize the chemotherapeutic potential of COX-2 inhibitors. The use of COX-2 inhibitors may enhance accumulation of chemotherapy agents and decrease resistance of tumors to chemotherapeutic drugs. Indeed, several clinical trials are underway based on combinations of COXIBs with conventional anticancer treatments (chemotherapy or radiotherapy) and with novel molecular targeting compounds [198]. Using combination therapy with agents that specifically modulate relevant biochemical targets of COX-2 inhibitors may take advantage of synergistic growth inhibitory effects against cancer cells and could reduce toxicity associated with intake of COX-2 inhibitors. Recently, concerns have been raised about the cardiovascular safety of the selective COX-2 inhibitor Rofecoxib [199, 200], and as a consequence it was withdrawn form the USA market (September 30, 2004) by Merck and Co. Further investigation is required to define the safety profile of selective COX-2 inhibitors especially when they are used at high doses and for long periods of time. On the other hand, since experimental studies provide evidence that PGs are the molecules that mediate the effects of COX overexpression, research interest is shifting towards specific PGs receptors and/or PGs synthases that may represent novel targets for prevention and treatment of certain types of cancers.

Acknowledgements We are grateful to Antonina Azzolina for her assistance in manuscript preparation. This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC).

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independently of cyclooxygenase-2 in colon cancer cells. Cancer Res. 2000; 60, 68466850. [157] Shureiqi I; Chen D; Lee JJ; Yang P; Newman RA; Brenner DE; Lotan R; Fischer SM; Lippman SM. 15-LOX-1: a novel molecular target of nonsteroidal anti-inflammatory drug-induced apoptosis in colorectal cancer cells. J Natl Cancer Inst. 2000; 92, 11361142. [158] Zhang Z; DuBois RN. Par-4; a proapoptotic gene; is regulated by NSAIDs in human colon carcinoma cells. Gastroenterology. 2000; 118, 1012-1017. [159] Zhang L; Yu J; Park BH; Kinzler KW; Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science. 2000; 290, 989-992. [160] Schwenger P; Bellosta P; Vietor I; Basilico C; Skolnik EY; Vilcek J. Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal kinase/stress-activated protein kinase activation. Proc Natl Acad Sci U S A. 1997; 94, 2869-2873. [161] Sun Y; Sinicrope FA. Selective inhibitors of MEK1/ERK44/42 and p38 mitogenactivated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells. Mol Cancer Ther. 2005; 4, 51-59. [162] Elder DJ; Halton DE; Playle LC; Paraskeva C. The MEK/ERK pathway mediates COX-2-selective NSAID-induced apoptosis and induced COX-2 protein expression in colorectal carcinoma cells.Int J Cancer. 2002; 99, 323-327. [163] Yip-Schneider MT; Schmidt CM. MEK inhibition of pancreatic carcinoma cells by U0126 and its effect in combination with sulindac. Pancreas. 2003; 27, 337-344. [164] Soh JW; Mao Y; Kim MG; Pamukcu R; Li H; Piazza GA; Thompson WJ; Weinstein IB. Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of cJun NH2-terminal kinase 1.Clin Cancer Res. 2000; 6, 4136-4141 Soh JW; Mao Y; Kim MG; Pamukcu R; Li H; Piazza GA; Thompson WJ; Weinstein IB. Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of c-Jun NH2terminal kinase 1.Clin Cancer Res. 2000; 6, 4136-4141. [165] Wong BC; Jiang XH; Lin MC; Tu SP; Cui JT; Jiang SH; Wong WM; Yuen MF; Lam SK; Kung HF. Cyclooxygenase-2 inhibitor (SC-236) suppresses activator protein-1 through c-Jun NH2-terminal kinase. Gastroenterology. 2004; 126, 136-147. Wong BC; Jiang XH; Lin MC; Tu SP; Cui JT; Jiang SH; Wong WM; Yuen MF; Lam SK; Kung HF. Cyclooxygenase-2 inhibitor (SC-236) suppresses activator protein-1 through c-Jun NH2-terminal kinase. Gastroenterology. 2004; 126, 136-147. [166] Leng J; Han C; Demetris AJ; Michalopoulos GK; Wu T. Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis. Hepatology. 2003; 38, 756-768. [167] Wu T; Leng J; Han C; Demetris AJ. The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells. Mol Cancer Ther. 2004; 3, 299-307. [168] Hsu AL; Ching TT; Wang DS; Song X; Rangnekar VM; Chen CS. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem. 2000; 275, 1139711403.

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[169] Kulp SK; Yang YT; Hung CC; Chen KF; Lai JP; Tseng PH; Fowble JW; Ward PJ; Chen CS. 3-phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells. Cancer Res. 2004; 64, 1444-1451. [170] Jones MK; Szabo IL; Kawanaka H; Husain SS; Tarnawski AS. von Hippel Lindau tumor suppressor and HIF-1alpha: new targets of NSAIDs inhibition of hypoxiainduced angiogenesis. FASEB J. 2002; 16, 264-266. [171] Abiru S; Nakao K; Ichikawa T; Migita K; Shigeno M; Sakamoto M; Ishikawa H; Hamasaki K; Nakata K; Eguchi K. Aspirin and NS-398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology. 2002; 35, 11171124. [172] Yu J; Leung WK; Chen J; Ebert MP; Malfertheiner P; Sung JJ. Expression of peroxisome proliferator-activated receptor delta in human gastric cancer and its response to specific COX-2 inhibitor. Cancer Lett. 2005; 223, 11-17. [173] Nikitakis NG; Hebert C; Lopes MA; Reynolds MA; Sauk JJ. PPARgamma-mediated antineoplastic effect of NSAID sulindac on human oral squamous carcinoma cells. Int J Cancer. 2002; 98, 817-823. [174] Wick M; Hurteau G; Dessev C; Chan D; Geraci MW; Winn RA; Heasley LE; Nemenoff RA. Peroxisome proliferator-activated receptor-gamma is a target of nonsteroidal anti-inflammatory drugs mediating cyclooxygenase-independent inhibition of lung cancer cell growth. Mol Pharmacol. 2002; 62, 1207-1214. [175] Sarraf P; Mueller E; Jones D; King FJ; DeAngelo DJ; Partridge JB; Holden SA; Chen LB; Singer S; Fletcher C; Spiegelman BM. Differentiation and reversal of malignant changes in colon cancer through PPARgamma.Nat Med. 1998; 4, 1046-1052. [176] Mueller E; Sarraf P; Tontonoz P; Evans RM; Martin KJ; Zhang M; Fletcher C; Singer S; Spiegelman BM. Terminal differentiation of human breast cancer through PPAR gamma.Mol Cell. 1998; 1, 465-470. [177] Kubota T; Koshizuka K; Williamson EA; Asou H; Said JW; Holden S; Miyoshi I; Koeffler HP.Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo.Cancer Res. 1998; 58, 3344-3352. [178] Clay CE; Namen AM; Atsumi G; Willingham MC; High KP; Kute TE; Trimboli AJ; Fonteh AN; Dawson PA; Chilton FH. Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells. Carcinogenesis. 1999; 20, 1905-1911. [179] Glinghammar B; Skogsberg J; Hamsten A; Ehrenborg E. PPARdelta activation induces COX-2 gene expression and cell proliferation in human hepatocellular carcinoma cells Biochem Biophys Res Commun. 2003; 308, 361-368. [180] Stephen RL; Gustafsson MC; Jarvis M; Tatoud R; Marshall BR; Knight D; Ehrenborg E; Harris AL; Wolf CR; Palmer CN. Activation of peroxisome proliferator-activated receptor delta stimulates the proliferation of human breast and prostate cancer cell lines. Cancer Res. 2004; 64, 3162-3170. [181] Lehmann JM; Lenhard JM; Oliver BB; Ringold GM; Kliewer SA. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J Biol Chem. 1997; 272, 3406-3410.

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[182] Jaradat MS; Wongsud B; Phornchirasilp S; Rangwala SM; Shams G; Sutton M; Romstedt KJ; Noonan DJ; Feller DR. Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen; naproxen, and indomethacin.Biochem Pharmacol. 2001; 62, 1587-1595. [183] He TC; Chan TA; Vogelstein B; Kinzler KW. PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell. 1999; 99, 335-345. [184] Guzman M. Cannabinoids: potential anticancer agents. Nat Rev Cancer. 2003; 3, 745755. [185] Herkenham; M. Cannabinoid receptors. Pertwee R. (Ed), Academic Press, London, 1995, 145-166 [186] Howlett AC. The CB1 cannabinoid receptor in the brain. Neurobiol Dis. 1998; 5, 405416. [187] Piomelli D. The molecular logic of endocannabinoid signalling. Nat Rev Neurosci. 2003; 4, 873-884. [188] Ligresti A; Bisogno T; Matias I; De Petrocellis L; Cascio MG; Cosenza V; D'argenio G; Scaglione G; Bifulco M; Sorrentini I; Di Marzo V. Possible endocannabinoid control of colorectal cancer growth. Gastroenterology 2003; 125, 677-687. [189] Jacobsson SO; Wallin T; Fowler CJ. Inhibition of rat C6 glioma cell proliferation by endogenous and synthetic cannabinoids. Relative involvement of cannabinoid and vanilloid receptors. J Pharmacol Exp Ther 2001; 299, 951-959. [190] Nithipatikom K; Endsley MP; Isbell MA; Falck JR; Iwamoto Y; Hillard CJ; Campbell WB. 2-arachidonoylglycerol: a novel inhibitor of androgen-independent prostate cancer cell invasion. Cancer Res. 2004; 64, 8826-8830. [191] Yu M; Ives D; Ramesha CS.Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2. J Biol Chem. 1997; 272, 21181-21186. [192] Kozak KR; Rowlinson SW; Marnett LJ.Oxygenation of the endocannabinoid, 2arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2. J Biol Chem. 2000; 275, 33744-33749. [193] Woodward DF; Krauss AH; Chen J; Lai RK; Spada CS; Burk RM; Andrews SW; Shi L; Liang Y; Kedzie KM; Chen R; Gil DW; Kharlamb A; Archeampong A; Ling J; Madhu C; Ni J; Rix P; Usansky J; Usansky H; Weber A; Welty D; Yang W; Tang-Liu DD; Garst ME; Brar B; Wheeler LA; Kaplan LJ. The pharmacology of bimatoprost (Lumigan). Surv Ophthalmol. 2001; 45, S337-345. [194] Woodward DF; Krauss AH; Chen J; Liang Y; Li C; Protzman CE; Bogardus A; Chen R; Kedzie KM; Krauss HA; Gil DW; Kharlamb A; Wheeler LA; Babusis D; Welty D; Tang-Liu DD; Cherukury M; Andrews SW; Burk RM; Garst ME. Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024). J Pharmacol Exp Ther. 2003; 305, 772-785. [195] Ross RA; Craib SJ; Stevenson LA; Pertwee RG; Henderson A; Toole J; Ellington HC. Pharmacological characterization of the anandamide cyclooxygenase metabolite: prostaglandin E2 ethanolamide. J Pharmacol Exp Ther. 2002; 301, 900-907. [196] Matias I; Chen J; De Petrocellis L; Bisogno T; Ligresti A; Fezza F; Krauss AH; Shi L; Protzman CE; Li C; Liang Y; Nieves AL; Kedzie KM; Burk RM; Di Marzo V;

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Woodward DF. Prostaglandin ethanolamides (prostamides): in vitro pharmacology and metabolism. J Pharmacol Exp Ther. 2004; 309, 745-757. [197] De Petrocellis L; Cascio MG; Di Marzo V. The endocannabinoid system: a general view and latest additions. Br J Pharmacol. 2004; 141, 765-774. [198] Gasparini G; Longo R; Sarmiento R; Morabito A. Inhibitors of cyclo-oxygenase 2: a new class of anticancer agents? Lancet Oncol. 2003; 4, 605-615. [199] Bombardier C; Laine L; Reicin A; Shapiro D; Burgos-Vargas R; Davis B; Day R; Ferraz MB; Hawkey CJ; Hochberg MC; Kvien TK; Schnitzer TJ; VIGOR Study Group.Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med. 2000; 343, 1520-1528. [200] Mukherjee D; Nissen SE; Topol EJ. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA. 2001; 286, 954-959.

In: COX-2 Inhibitor Research Editor: Maynard J. Howardell, pp. 145-180

ISBN 1-59454-994-X © 2006 Nova Science Publishers, Inc.

Chapter IV

Nephrotoxicity of Nonsteroidal Anti-Inflammatory Drugs: Focus on Selective Cyclooxygenase-2 (COX-2) Inhibitors Steven G. Coca1 and Mark A. Perazella2∗ 1

Fellow in Nephrology, Section of Nephrology Department of Medicine, Yale University School of Medicine 2 Associate Professor of Medicine, Director, Renal Fellowship Program Director, Acute Dialysis Services Section of Nephrology , Department of Medicine Yale University School of Medicine

Abstract Traditional (non-selective) NSAIDs cause nephrotoxicity through inhibition of cyclooxygenase (COX) activity and prostaglandin formation in the kidney. Patients with prostaglandin-dependent disease states are the group at most risk for this adverse effect. It has become apparent that the COX-2 enzyme isoform is constitutively expressed and upregulated in the human kidney during states of renal stress. COX-2 derived prostaglandins importantly modulate renal blood flow and glomerular filtration rate as well as sodium, potassium and water excretion by the kidney. As a result, clinical renal syndromes induced by the selective COX-2 inhibitors are quite similar to those described with the traditional NSAIDs, suggesting that COX-2 derived prostaglandins are important in maintaining normal renal function. Inhibition of prostaglandins causes a reduction in renal blood flow and acute renal failure in patients with predisposing conditions. These include true volume depletion from nausea/vomiting, diarrhea and excessive diuretic therapy. Effective volume depletion from clinical disease states such ∗ Mark A. Perazella, , MD, FACP. Associate Professor of Medicine, Director, Renal Fellowship Program, Director, Acute Dialysis Services, Section of Nephrology, Department of Medicine, Yale University School of Medicine,FMP 107 333 Cedar Street, New Haven, CT 06520-8029

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Steven G. Coca and Mark A. Perazella as heart failure, cirrhosis, and nephrotic syndrome as well as diseases such as chronic kidney disease and renal artery stenosis also portend risk of acute renal failure from prostaglandin inhibition. Prostaglandins also modulate renal potassium excretion through stimulation of the renin-angiotensin-aldosterone system. Inhibition of prostaglandins can result in hyperkalemia when co-existent conditions such as renal failure, diabetes mellitus and therapy with certain medications (ACE inhibitors, angiotensin receptor blockers, potassium-sparing diuretics) are also present. The classic syndrome of hyporeninemic hypoaldosteronism with a type-4 renal tubular acidosis (RTA) picture (hyperkalemic metabolic acidosis) can be observed when selective COX-2 inhibitor therapy is superimposed. Inhibition of prostaglandins is associated with decreased renal sodium and water excretion and all NSAIDs, including the selective COX-2 inhibitors cause some degree of sodium retention. All patients suffer from this effect, but only patients with certain clinical conditions develop obvious edema, hypertension or heart failure. Patients with underlying hypertension (especially those on antihypertensive medications), heart disease and other salt-retentive disease states (cirrhosis, nephrosis, renal failure) are at highest risk for these complications. Hypertension is a particularly important complication of these drugs as small changes in blood pressure are associated with increased cardiovascular events. Hyponatremia from impaired water excretion also complicates therapy. Less commonly, acute interstitial nephritis (with or without a glomerulopathy) has been described with these drugs. To reduce adverse renal effects from NSAIDs, including all of available the selective COX-2 inhibitors, identification of patients with renal risk should be undertaken. Defining patient risk profiles based on level of kidney function (stage of chronic kidney disease) as well as on the presence of certain comorbidities (hypertension, heart failure, diabetes mellitus, liver disease/cirrhosis, electrolyte imbalance, old age, certain medications) is one simple approach that can be taken. Based on the renal risk, recommendations for therapy and monitoring can be utilized in a rational fashion.

Key words: cyclooxygenase (COX), selective COX-2 inhibitors, nonsteroidal antiinflammatory drugs (NSAIDs), acute renal failure, hyponatremia, hyperkalemia, hypertension, edema, acute interstitial nephritis.

Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) have been prescribed for several decades to reduce fever, pain and inflammation. In addition to prescription NSAIDs, the general population consumes a large number of over-the-counter NSAIDs. Over 60 million people ingest these medications on a regular basis. The price paid for these beneficial therapeutic effects include gastrointestinal (GI) complications and, to a lesser extent, adverse kidney effects. In response to this problem, a new class of selective NSAIDs was designed, based on the discovery of two cyclooxygenase (COX) isoforms (COX-1, COX-2), to reduce drug-related toxicity [1,2]. The selective COX-2 inhibitors, celecoxib and rofecoxib, were introduced into clinical practice for this purpose in 1999 [3,4]. These new drugs have documented therapeutic efficacy while also significantly reducing GI toxicity and platelet dysfunction [3,4].

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In addition to improved GI safety, a reduction in traditional (non-selective) NSAIDassociated adverse renal effects was another goal of this new pharmacological class of selective COX-2 inhibitors. Based on data that suggested that COX-2 is not constitutively expressed, but can be rapidly induced in response to inflammatory stimuli, it was speculated that the functions of the COX isoforms were mutually exclusive. The paradigm that COX-1 maintains the normal physiologic functions of the kidney and that COX-2 is involved primarily in inflammatory processes was attractive. Furthermore, compartmentalization of the COX isoforms in the kidney would reduce NSAID-associated nephrotoxicity. However, the original paradigm describing the biologic activity of COX-1 and COX-2 is not completely accurate. While it is true that COX-2 is induced at sites of inflammation and plays a major role in the production of prostaglandin (PG) E2 and other arachidonic acid metabolites produced at inflammatory sites, COX-1 may also contribute to inflammatory responses (1,2). Importantly, a critical role of COX-2 in physiological processes, and namely in the maintenance of renal function has come to light. Cyclooxygenase-2 dependent renal physiological effects are inferred from observations that demonstrate constitutive expression and upregulation of this isoform in the kidney. As such, COX-2 enzyme may have an important role in the synthesis of prostanoids integral to the regulation of renal perfusion, salt and water handling and renin release.

Prostaglandins in the Kidneys Prostaglandin Synthesis Prostaglandins (PG) are the major products of COX enzyme metabolism [5-7]. These prostanoids are produced throughout the body and act locally in an autocrine and paracrine fashion [5-7]. Synthesis of prostaglandins commences with liberation of the second messenger, arachidonic acid from cell membrane phospholipids in a reaction mediated by the enzyme phospholipase A2. A number of factors trigger the activation of phospholipase A2, serving to modulate subsequent prostaglandin production through the provision of arachidonic acid substrate. Arachidonic acid is then bound by prostaglandin endoperoxide synthase, a complex of cyclooxygenase and peroxidase, which subsequently catalyzes the redox reaction. Further regulation of prostaglandin synthesis occurs through expression and activation of COX enzyme. A short-lived, unstable intermediary cyclic endoperoxide called PGG2-PGH2 is synthesized and rapidly metabolized to more stable prostaglandins by cell specific enzymes [5-7]. Following synthesis, prostaglandins promptly exit the cell via facilitated diffusion to bind prostaglandin receptors found on parent or neighbouring cells, thereby modulating cellular functions [5-7]. Figure 1 demonstrates the pathway of prostaglandin synthesis within cells and local action at the cell level.

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PGR

PGR

PL PLase AA

COX

PG

Figure 1. The pathway of prostaglandin synthesis within cells and local action at the cellular level

Cyclooxygenase Function Synthesis of prostaglandins from the arachidonic acid substrate is catalyzed by one of two isomers of cyclooxygenase, COX-1 or COX-2. These enzymes are approximately 65% identical in their amino acid sequence and nearly identical at their catalytic site [2,7]. Conservation of their structures at the catalytic site allows these isoforms to carry out similar enzymatic functions and produce similar prostaglandins. Downstream cell-specific enzymes further modify these prostaglandins to form their final prostaglandin product (TXA2, PGE2, PGI2, PGD2, PGF2α, etc). Cyclooxygenase-1 is a 22-kilobase gene located on human chromosome 9, while the 8-kilobase COX-2 gene resides on chromosome 1 [2,7]. The COX isoforms can also be distinguished by their respective patterns of gene transcription. The COX-2 DNA sequence identifies it as an “inducible” gene that is expressed chiefly in response to a variety of stimuli. The COX-2 gene has a number of sites that links its transcription to the presence of appropriate protein triggers such as cytokines, growth factors or hormones [2,7]. In contrast, the gene sequence of COX-1 lacks the sites that are required to facilitate rapid protein transcription in response to stimuli, consistent with a gene that expresses its constitutive product protein without any prerequisite signal. It is more likely that provision of arachidonic acid substrate influences COX-1 enzyme activity and subsequent prostaglandin production. The above-noted differences in gene regulation between the cyclooxygenase isomers provide a molecular basis for their purported roles as “constitutive” (COX-1) and “inducible” (COX-2) enzymes. In general, these labels accurately describe the synthesis of cyclooxygenase in most tissues, where COX-1, but not COX-2 is expressed in appreciable levels at baseline [2,7,8]. A prime example of COX-1 predominance is the GI tract, in

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particular the stomach mucosa. In contrast, abundant expression of COX-2 is demonstrated in macrophages and other cell types in response to inflammatory mediators [2,7,8]. It is these characteristics of the COX isoforms that initially suggested that inflammation, fever and pain could be targeted while homeostatic cellular functions could be spared. As will be discussed later, this paradigm is flawed by the fact that COX-2 is also constitutively expressed and upregulated in the kidney and importantly modulates renal physiology [2,9-11,12]. Table 1 lists the differences between the two COX enzyme isoforms. Table 1. Characteristics of COX-1 and COX-2 Enzymes COX-1 Enzyme Chromosome 9 22 kilobase gene Constitutive Tissue Expression GI Tract Kidney Brain Vasculature Platelets Unchanged by Glucocorticoids Instantaneous Inhibition by NSAIDs Abbreviations: COX, gastrointestinal

cyclooxygenase;

COX-2 Enzyme Chromosome 1 8 kilobase gene Inducible Tissue Expression Macrophages Synoviocytes Cartilage Kidney Brain Blocked by Glucocorticoids Time-Dependent Inhibition by NSAIDs NSAIDs,

nonsteroidal

anti-inflammatory

drugs;

GI,

Prostaglandins in Renal Physiology In healthy individuals with normal intravascular volume status, prostaglandin synthesis is of minimal or no importance in the kidney. As such, prostaglandins are not primary regulators of renal function. Rather, these eicosanoids locally modulate the effects of both systemic and locally produced vasoconstrictor hormones, contribute to sodium and water balance, and play a protective role in the renal medulla [6]. A variety of prostaglandins are synthesized within distinct anatomic locations in the kidney, including PGI2, PGE2, thromboxane A2 (TXA2), and PGF2α (Table 2). In general, PGI2 and PGE2 are the predominant mediators of physiologic activity in the kidney. PGI2 is most abundantly produced in the renal cortex by cortical arterioles and glomeruli, whereas PGE2 is synthesized in juxtamedullary glomeruli, in medullary interstitial cells, and in the medullary portion of the collecting duct [5,7,13-15]. However, there exists considerable overlap of prostaglandin synthesis at these renal sites. Functionally, PGI2 and PGE2 induce vasodilatation in interlobular arteries, afferent and efferent arterioles, and glomeruli. Prostaglandin-mediated vasodilatation increases renal perfusion in inner cortical and medullary regions and increases sodium excretion through post-glomerular perfusion [5,7,1315].

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Table 2. Renal Sites of Prostaglandin Action Eicosanoid PGI2

PGE2

Site Glomeruli, arterioles Loop of Henle Juxtaglomerular apparatus macula densa) Glomeruli, arterioles Loop of Henle

Action Vasodilatation Maintain GFR Renin Release, TG feedback Vasodilatation, Maintain GFR Renin Release Excretion of NaCl and H20 TG feedback

Juxtaglomerular apparatus (macula densa) Glomerular podocytes and Mesangial cells Medullary interstitial cells and Tubular cells

TXA2 PGF2α

Vasoconstriction Excretion of NaCl and H2O

Abbreviations: GFR, glomerular filtration rate; TG, tubuloglomerular Afferent Arteriole

Glomerulus

Efferent Arteriole

RBF

1

Distal Tubule

PG

PG Proximal Tubule

Na+

4

Na+

2

Na+

5

PG

Na+

Na+

Effects of Prostaglandins in the Nephron: 1) Dilates Afferent Arteriole 2) Decreases Na+ reabsorption in the proximal tubule 3) Decreases Na+ reabsorption in the loop of Henle 4) Decreases Na+ reabsorption in the distal tubule 5) Decrease reabsorption in the collecting duct

PG

Na+

K+

PG Thick Ascending Limb of Henle

Collecting Duct

3 Medullary Interstitial Cells

Figure 2. Site of prostaglandin action in the nephron to reduce sodium chloride reabsorption

In the loop of Henle and distal nephron (Figure 2), PGE2 decreases cellular transport of sodium chloride in thick ascending limb cells, distal tubular and collecting duct cells, respectively [5,7,13-15]. An increase in renal sodium excretion and a decrease in medullary tonicity are the direct results of PGE2 action in these nephron segments [5,7,13-15]. PGE2 and PGI2 also stimulate renin secretion in the juxtaglomerular apparatus. Renin cleaves both circulating and local angiotensinogen into angiotensin I, which is then converted to angiotensin II. Subsequently, this hormone stimulates adrenal aldosterone release, enhancing sodium retention and potassium excretion in the distal nephron. Finally, PGE2 and PGI2 also inhibit cAMP synthesis and interfere with the action of antidiuretic hormone (ADH) to increase water absorption by reducing aquaporin channels in the apical membrane [5,7,13-

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15]. This promotes a water diuresis and helps maintain normal serum osmolality and sodium concentration.

Prostaglandins in Pathophysiologic States Prostaglandins have their major role in the preservation of renal function when pathologic states supervene and compromise physiologic processes in the kidney. The development of intravascular volume depletion, as seen with vomiting, diarrhea and diuretic therapy, stimulates COX enzyme activity and prostaglandin synthesis to optimize renal blood flow (RBF) and maintain normal glomerular filtration rate [5,6,14]. In addition, effective decreases in RBF as seen with congestive heart failure (CHF), cirrhosis and nephrotic syndrome also enhance compensatory prostaglandin production [5,6,14]. PGI2 and PGE2 antagonize the local effects of circulating angiotensin II, endothelin, vasopressin and catecholamines that would normally maintain systemic blood pressure at the expense of the renal circulation [5,6,14]. Specifically, these eicosanoids preserve glomerular filtration rate (GFR) by antagonizing afferent arteriolar vasoconstriction and blunting mesangial and podocyte contraction induced by these endogenous vasopressors [5-7]. Prostaglandin production is also increased in chronic kidney disease (CKD) [16]. Upregulation of prostaglandin synthesis in CKD is induced by intrarenal mechanisms activated to increase perfusion of remnant nephrons [16]. Studies examining the effect of prostaglandins on remnant renal function substantiate the prostaglandin dependence of CKD [13,16-18]. As a result, impairment of prostaglandin production in this setting is associated with acute reductions in RBF and GFR, resulting in hemodynamic acute renal failure (ARF). Renal prostaglandins also importantly modulate salt and water homeostasis. In response to volume overload and salt loading, prostaglandin inhibition of tubular sodium chloride (NaCl) reabsorption increases salt excretion [5,6,14]. This effect modulates the blood pressure raising effect as well the tendency to form edema that would occur with sodium retention in disease states (underlying hypertension, cirrhosis, nephrosis, CKD) dependent on prostaglandin function in the kidney. Antagonism of vasopressin effect on water channels by these autacoids also facilitates excretion of a water load in patients with nonosmotic-induced elevations in vasopressin associated with cirrhosis and nephrosis [5,6,14]. In addition, the regulation of medullary blood flow by PGE2 contributes to the kidney’s ability to modify renal solute excretion [5,6,14]. Taken together, these modulating effects ensure the kidney’s regulation of salt and water through their appropriate retention or excretion. Ultimately, intravascular volume status and plasma osmolality are maintained in a more physiologic state.

Traditional NSAID-Associated Nephrotoxicity To better understand selective COX-2 inhibitor nephrotoxicity, the adverse effects of traditional NSAIDs on the kidney will be reviewed. While disruption of COX enzyme by NSAIDs produces therapeutic drug effects, inhibition of COX enzyme function produces kidney dysfunction. Several well-described clinical syndromes of NSAID-associated

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nephrotoxicity have been noted (Table 3). They include hemodynamic acute renal failure, disorders of sodium balance (edema and hypertension), disturbances in water homeostasis (hyponatremia) and abnormal renal potassium handling (hyperkalemia, type-4 RTA). Other adverse renal effects include acute interstitial nephritis with or without an associated glomerulopathy. These nephrotoxic effects will be briefly reviewed in the following sections. Table 3. NSAID-Associated Renal Syndromes • •

• •

• •

• •

Acute Renal Failure Disorders of Sodium Balance Edema Hypertension Diuretic Resistance Disorders of Water Balance Hyponatremia Disorders of Potassium Homeostasis Hyperkalemia Metabolic Acidosis Acute Interstitial Nephritis Nephrotic Syndrome Minimal Change Disease Membranous Glomerulonephritis Acute Papillary Necrosis Chronic Tubulointerstitial Nephritis Analgesic Nephropathy Chronic Papillary Sclerosis

Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs

Renal Blood Flow/Glomerular Filtration Rate Hemodynamic ARF It has been estimated that anywhere from 1 to 5% of patients who ingest NSAIDs will develop some form of nephrotoxicity [19]. Some calculations approximate that 500,000 persons are likely to develop some form of NSAID-associated adverse renal impairment. Administration of NSAIDs to patients with prostaglandin-dependent disease states has been shown to consistently precipitate ARF [5,6,14,17]. As outlined previously, patients with states of vasoconstrictor excess depend on renal prostaglandin synthesis to ensure sufficient renal perfusion. In the absence of the counterbalancing effects of prostaglandins, unopposed vasoconstriction induced by NSAIDs leads to a decrease in RBF and a decline in GFR [5,6,14] (Figure 3). Similarly, in patients with underlying CKD, NSAID administration can precipitate reversible ARF [13,16-22]. Since NSAIDs are highly protein bound, hypoalbuminemia also portends risk for renal impairment. Increased concentrations of free drug in the circulation, due to loss of sufficient protein binding of the NSAID, account for the

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adverse renal effect [6]. Although NSAID-associated ARF is probably both dose- and duration-dependent, even small doses given for short courses may induce renal impairment in patients with multiple risk factors.

Afferent Arteriole

Glomerulus

Efferent Arteriole

RBF

VASOCONSTRICTORS Catecholamines Endothelin Angiotensin II Vasopressin

VASODILATORS Prostaglandins Nitric Oxide

Normal GFR

VASOCONSTRICTOR Angiotensin II

ACE-I ARBs

NSAIDs ↓ RBF

Decreased GFR Figure 3. Effect of NSAIDs to induce vasoconstriction with an associated decrease in RBF and GFR in patients dependent on prostaglandins to maintain renal prefusion

Exposure to NSAIDs has been noted to double the risk of hospitalization for ARF in patients with CKD [23]. Similar rates of ARF with NSAIDs have been described in the elderly, those with cardiac disease and patients receiving ACE inhibitors. Several studies in patients in the general population documented adjusted relative risks of clinical ARF of two to four-fold higher in NSAID users versus nonusers [24-26]. Patients with a history of heart failure and hypertension, as well as those treated with diuretics exhibited the greatest risk (adjusted relative risk = 11.6) when treated with an NSAID [26]. The effect also appeared to be dose-related with ARF occurring with higher NSAID dose. Hospitalized patients have even greater risk, as a higher percentage of the cases of drug-induced ARF that develop in the hospital are due to NSAID therapy [27]. In general, renal failure reverses within 2 to 5 days following discontinuation of the NSAID [5,6]. However, restoration of baseline renal

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function may be prolonged in high-risk patients [5,6]. Rarely, acute dialytic intervention may be required for severe uremia, CHF or life-threatening electrolyte and acid-base disturbances.

Sodium Balance Edema Edema formation and volume overload may also complicate treatment with NSAIDs as they blunt the natriuretic effect of prostaglandins on the nephron. Sodium retention and mild lower extremity edema are frequently observed (25%) following therapy with these drugs [28]. Patients with underlying sodium avid states, such as cirrhosis and CHF, are particularly at risk to suffer from the sodium-retaining properties of NSAIDs. As a result, severe and symptomatic edema, such as CHF and anasarca may develop in some patients [28]. Two separate studies in elderly patients demonstrate that NSAIDs were associated with an increased risk for the development of volume overload and CHF [29,30]. One of these publications was a large cohort study in over 10, 500 patients [29]. A doubling of the risk for hospitalization with CHF was observed in patients taking diuretics and NSAIDs as compared with those ingesting diuretics alone [29]. A case control study similarly documented a twofold risk for first admission to the hospital with CHF in patients treated with NSAIDs as compared with patients who did not receive these drugs [30]. In this study, the effect was related to higher drug dosage and longer drug half-life. The presence of underlying heart disease significantly increased risk for CHF in NSAID treated patients as noted by an odds ratio for CHF admission of 10.5 in these patients [30]. An extreme example of this NSAID effect includes the case of a 70-year old patient who developed anasarca characterized by a fluid gain of 15 kg during a 17-day course of ibuprofen [31]. In addition to sodium retention with edema formation, NSAIDs also attenuate the natriuretic and aquaretic effects of diuretics. In fact, diuretic resistance has been noted to complicate therapy with these drugs, especially in patients with underlying salt-retentive states [28]. The underlying mechanism of diuretic resistance is due to multiple NSAID effects including 1) inhibition of the increase in renal blood flow that attends diuretic therapy, decreasing the filtered load of sodium available for excretion [32], 2) inhibition of natriuretic prostaglandin formation in the thick ascending limb of Henle typically stimulated by loop diuretics [33], 3) competition of NSAIDs with diuretic drugs at the tubular secretory level, thereby reducing the amount of drug reaching its site of action, and 4) enhanced ADH effect with resultant water retention [34].

Hypertension Since sodium retention is associated with increases in blood pressure, the inhibitory effect of NSAIDs on prostaglandin-induced natriuresis raises concerns that normal blood pressure regulation might be impaired by these drugs. In fact, development of new onset hypertension and exacerbation of previously controlled hypertension has been observed in

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patients treated with NSAIDs [28,35,36]. In a study performed in approximately 20,000 elderly Medicaid enrollees, NSAID therapy within 2 months of study initiation was associated with a two-fold risk of requiring antihypertensive therapy [35]. Average increases in mean arterial pressure of 5 to 10 mmHg were noted in patients treated with NSAIDs [3638]. This effect was most marked in hypertensive patients on blood pressure lowering medications, while normotensive patients did not develop hypertension. It appears that certain drugs are more likely to lose efficacy in lowering blood pressure when NSAIDs are administered. These include β-adrenergic blockers, ACE inhibitors, and as noted above, diuretics. In contrast, direct vasodilators such as the calcium channel blockers continue to maintain efficacy despite NSAID therapy. Patients with low renin hypertension, such as blacks and the elderly also appear more susceptible to the hypertensive effects of these drugs. The mechanism of NSAID-associated hypertension is largely related to the sodium retention that occurs, but there is likely a contribution from vasoconstriction of resistance vessels induced by inhibition of local vasodilatory prostaglandins (PGE2, prostacyclin) in the endothelium of vessels. The effect of NSAIDs on blood pressure is a particularly vexing problem since an estimated 12 million Americans receive concurrent therapy with antihypertensive drugs and NSAIDs [28].

Water Balance Hyponatremia Mild to moderate levels of hyponatremia may also develop in patients treated with NSAIDs [5,6]. This disturbance in water balance results from two major effects. First, prostaglandin deficiency induced by NSAIDs magnifies the medullary interstitial osmotic gradient and leads to greater resorption of water in the distal nephron via aquaporins, which are apical membrane water channels in collecting duct cells. Second, since prostaglandins normally blunt the antidiuretic effects of vasopressin, suppression of prostaglandin synthesis with NSAIDs allows increased entry of water into tubular cells of the collecting duct. PGE2 has been shown to reduce water reabsorption by retrieving aquaporin-2 from the apical membrane. A recent study demonstrated that indomethacin increased the shuttling of aquaporin-2 to the apical plasma membrane of collecting duct cells [39]. This effect was unrelated to changes in ADH levels and likely was a direct effect of indomethacin on adenylyl cyclase complex. These effects impair solute-free water excretion and can lead to total body water excess and hyponatremia [5,6].

Potassium Homeostasis Hyperkalemia Hyperkalemia is another potential complication of treatment with NSAIDs [5,6,40,41]. NSAIDs can result in the development of potentially life-threatening hyperkalemia when

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prescribed to patients with underlying renal insufficiency or to those ingesting other potassium-altering medications [40,41]. These medications include the widely prescribed ACE inhibitors, ARBs, aldosterone antagonists (spironolactone, eplerenone), trimethoprim, heparin, and calcineurin inhibitors. The initial description of hyperkalemia associated with an NSAID, which also provided insight into the mechanism of disturbed potassium homeostasis, was published by Tan and colleagues [41]. The patient, who had underlying proteinuric kidney disease, developed severe hyperkalemia (serum K+ = 6.2 mEq/L) and a reduction in baseline urinary prostaglandin excretion (PGE2 = 598 ng/24 hours decreased to PGE2 < 10 ng/24 hours) following therapy with indomethacin. These effects developed in association with a decline in plasma renin activity, plasma aldosterone concentration and urinary aldosterone excretion, consistent with drug-induced hyporeninemic hypoaldosteronism. All of these parameters reversed to normal following discontinuation of indomethacin. A series of five cases of hyperkalemia was also published further incriminating NSAIDs as a cause of disturbed renal potassium excretion and hyperkalemia [43]. Serum potassium concentrations increased in all patients (baseline serum creatinine concentrations in the 1.0 to 2.2 mg/dl range) treated with indomethacin and declined following drug discontinuation. It appears that this cation disorder develops from NSAID-induced reductions in the synthesis of renin and aldosterone, inducing a state of hyporeninemic hypoaldosteronism [5,6,40,41]. Since aldosterone stimulates renal potassium excretion via the creation of a favorable electrochemical gradient for potassium transport into the urinary space, it is not surprising that reduced aldosterone production by NSAIDs is the most prominent cause of hyperkalemia [41]. NSAID-associated reductions in the delivery of NaCl and water to the distal tubule also contribute to hyperkalemia. Decreased delivery of NaCl and water limits potassium secretion by diminishing the intraluminal sodium available for sodium-potassium exchange and reducing the favorable concentration gradient for potassium diffusion into the urinary space [41].

Acute Interstitial Nephritis Acute interstitial nephritis (AIN) following NSAID therapy maintains important differences from that noted with β-lactam antibiotics, which are the most frequent therapeutic agents implicated in AIN, and other culprit drugs. Acute interstitial nephritis that occurs in the setting of NSAID administration typically develops over a longer period of exposure. Treatment is often greater than one year. This contrasts development of AIN in a mean of approximately 2 weeks with β-lactam antibiotics. There is also a much lower incidence of fever, rash, and eosinophilia with NSAIDS as compared with other drugs [44]. Proteinuria is a common finding in patients with AIN from NSAIDs due to concomitant glomerular disease (minimal change disease and membranous nephropathy). On histologic evaluation, NSAIDassociated AIN is similar to other forms of drug-induced AIN except that a lesser degree of interstitial inflammation, tubulitis, and eosinophilic infiltration is present. With blockade of the cyclooxygenase enzyme, shunting of arachidonic acid into the lipoxygenase pathway may underlie the development of AIN from the pro-inflammatory leukotrienes that are synthesized via this alternative pathway. The unique clinical and pathologic characteristics of AIN

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following treatment with NSAIDs may be explained in part by the anti-inflammatory properties of this class of drugs [45]. The similar patterns of nephrotoxicity seen with a wide range of NSAIDs suggests that injury in the kidney may relate not only to the chemical structure and immunogenicity of the agent but also to the physiologic effects of COX inhibition and possibly to the shunting of arachadonic acid metabolites into these alternative pathways that modify immune function. This has been termed the “shunting” hypothesis.

Renal Localization and Function of COX-2 Enzyme One strategy to predict the nephrotoxic potential of selective COX-2 inhibitors is to examine the presence and importance of both COX-1 and COX-2 enzymes in the kidney. Not only is the absolute presence of the COX-2 enzyme important, but its localization in various renal compartments and its response to normal and pathologic physiologic stress are integral to understanding this issue. The next section looks into these very issues. Table 4 notes the localization of COX-2 in the kidney of various animals and humans. Table 4. Renal Localization of Cyclooxygenase-2 Species Mouse Rat Rabbit Dog Monkey Human

Vasc

Glom X

X X X

X X

MD X X

TAL

MIC

X

X X X

X X X

X

X

X

CD X

X

Abbreviations: Vasc, vasculature; Glom, glomerulus; MD, macula densa; TAL, thick ascending limb; MIC, medullary interstitial cells; CD, collecting duct

Animal Studies There are several animal studies that demonstrate the presence of COX-2 enzyme in renal tissue and the importance of prostaglandins derived from COX-2 in the modulation of renal physiology. First, the COX-2 enzyme is clearly localized in discrete areas of the normal kidney. In cultured rat mesangial cells, endothelin-1 (ET-1) induced expression of COX-2 mRNA and stimulated COX-2 protein accumulation, leading to increased production of PGE2 [46]. COX-2 has also been isolated from rat medullary thick ascending limb (MTAL) cells [12,47]. In these animals, upregulation of the COX-2 isoform and increased PGE2 synthesis (5-fold) were noted following exposure to angiotensin II [47]. Angiotensin-II acted to increase COX-2 mRNA upregulation through stimulation of these cells to produce tumor necrosis factor. In contrast, COX-1 mRNA was not upregulated by tumor necrosis factor exposure. In this same light, angiotensin-II markedly enhanced the expression of COX-2 mRNA in cultured rat vascular smooth muscle cells, providing another example of the role of

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COX-2 enzyme in the upregulation of prostaglandin synthesis to modulate renal vasoconstrictors and maintain GFR [48]. In this model, angiotensin-II increased COX-2 expression by suppressing COX-2 mRNA degradation rather than increasing gene transcription. Importantly, the COX-2 specific inhibitor NS-398 attenuated the angiotensin-II driven expression of COX-2 in these cells [48]. COX-2 has also been localized to the macula densa of the juxtaglomerular apparatus, the adjacent thick cortical ascending limb cells, and to the medullary interstitial cells at the papilla in adult rats [11,49]. Furthermore, volume contraction induced by chronic salt restriction increased COX-2 expression in the renal cortex and increased the number of COX2 producing cells in both the macula densa and the adjacent thick ascending limb [11,49]. In this study, salt restriction did not increase COX-2 production in papillary cells nor was COX1 detectable in cells of the macula densa. COX-2 expression in the macula densa is augmented by dietary salt restriction and renal artery constriction, two conditions in which the renin gene expression is increased [50,51]. This COX-2 localization also suggests that it has an important role in the regulation of glomerular hemodynamics through the tubuloglomerular feedback (TGF) mechanism. It is notable that administration of COX-2 inhibitors prevents TGF adaptation in the Wistar rat [52]. Absence of TGF adaptation may lead to a reduction in GFR and NaCl retention following selective COX-2 inhibitor therapy. Constitutive COX-2 expression has also been identified in rabbit medullary interstitial cells [53]. Treatment of medullary interstitial cells with COX-2 antisense downregulated COX-2 production and induced medullary cell death in vitro [53]. In a second set of experiments in rabbits, Hao and colleagues treated medullary interstitial cells with selective COX-1 inhibitors, selective COX-2 inhibitors, and traditional NSAIDs (sulindac, ibuprofen, indomethacin). All cells underwent apoptosis, however, the selective COX-2 inhibitors were 1000 times more potent than selective COX-1 inhibitors in promoting cell death. Mouse studies localize COX- and COX-2 enzyme synthesis to the cortical collecting duct [54,55]. Immunohistochemical studies demonstrate the presence of COX-1 and COX-2 enzymes in intercalated cells of the cortical collecting duct of mice kidneys [54,55]. In contrast, neither enzyme localizes to the cortical principal cells. It appears that COX-2 is the predominant enzyme in the synthesis of prostaglandins in this nephron segment. Specifically, intercalated cells exposed to selective COX-2 inhibitors had impaired prostaglandin synthesis as compared with cells exposed to selective COX-1 inhibitors [55]. Notably, PGE2 is an important mediator controlling salt and water reabsorption by the cortical and medullary collecting duct. Abassi and others have also shown that experimental congestive heart failure is associated with selective overexpression of COX-2 in the rat kidney medulla, a finding probably representing several important renal effects [56]. Medullary expression of COX-2 in this model maintains efficient diuresis and natriuresis despite the salt retentive state triggered by heart failure, and defends medullary blood flow in the face of decreased effective renal perfusion. Inhibition of COX-2 in the rat renal medulla also promotes sustained hypertension in rats fed a high sodium diet [57]. The effect of COX-2 inhibition on inducing hypertension was most potent with infusion of a selective inhibitor directly into the medulla as compared with intravenous administration, supporting the importance of sodium retention in the generation of hypertension. Thus the pattern of COX-2 expression and function in the kidney

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suggests that this isoform plays a significant role in the regulation of sodium, water and other electrolyte metabolism in both physiologic and pathophysiologic conditions.

Human Studies Studies have also disclosed the expression of COX-2 in glomerular podocytes and arteriolar smooth muscle cells in the normal human kidney obtained from surgical nephrectomy specimens [9]. The COX-2 enzyme was identified in these renal locations using polyclonal antibodies, reverse transcription-polymerase chain reaction, and c-RNA probes. In contrast, COX-1 was expressed primarily in endothelial cells of the afferent arteriole and cells of the cortical collecting duct. COX-2 has also been detected in the macula densa of adult human kidneys. Localization of COX-2 in the macula densa was demonstrated in subjects older than 60 years of age [58] and in patients with underlying hyperreninemic states (patients with congestive heart failure) [59]. COX-2 immunoreactivity was also noted in afferent arterioles and medullary interstitial cells, with limited staining observed in the thick loops of Henle in the specimens procured from the elderly subjects [58]. These findings suggest that COX-2 derived prostaglandins contribute to the regulation of renal hemodynamics probably via production of PGI2 (inducing arteriolar vasodilatation) as well as PGH2, which is subsequently metabolized to thromboxane (TxA2). However, the role of the COX-2 isoform in renal hemodynamics is probably manifest only in specific conditions that are associated with increased dependence of renal function on prostaglandins. These include decreased sodium intake, intravascular volume depletion, critical renal artery stenosis, lupus nephritis, partial renal ablation, therapy with renin-angiotensin-aldosterone system inhibitors, cirrhosis, nephrosis and congestive heart failure. Although normal regulation of renal blood flow does not depend on prostaglandins, it is possible that COX-1 has some role in normal renal hemodynamics. This function may explain the observation of a few reported cases of acute renal failure induced by NSAIDs in apparently healthy subjects [17], and the studies showing that selective COX-2 inhibitors have no effect on GFR whereas traditional NSAIDs may slightly reduce GFR in sodium-replete healthy subjects [60,61]. In contrast, the observation that selective COX-2 inhibitors reduce GFR to the same degree as non-selective NSAIDs in sodium restricted subjects and in patients with chronic stable kidney disease supports the above paradigm [62,63,64]. Thus, observations on the localization of COX-2 in the kidney, and on the increased expression of renal COX-2 under conditions known to be associated with increased renal prostaglandin dependence demonstrate the importance of constitutively expressed and upregulated COX-2 in the kidney [50,51,58,59]. Therefore, the role of COX-2 in the production of prostaglandins necessary to maintain renal hemodynamics (RBF and GFR), renin secretion and tubular function (sodium, water and potassium homeostasis) questions the earlier belief that the new class of selective COX-2 inhibitors was benign. To summarize, it is clear that animal models demonstrate the strategic location of COX-2 expression in the kidney and intimate a key role for COX-2 enzyme in modulating renal function in patients with certain disease states. In addition, localization of COX-2 to vascular tissue and glomeruli in humans suggests a role for this isozyme in the modulation of renal

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hemodynamics, clearly one of the primary cyclooxygenase-mediated functions, which is interrupted by NSAIDs. Furthermore, COX-2 expression in human macula densa and medullary interstitial cells implies an integral role of this enzyme in salt and water balance. The sites of COX-2 expression suggest that selective COX-2 inhibitors may adversely affect kidney function in patients with prostaglandin-dependent disease states.

Selective COX-2 Inhibitor-Associated Nephrotoxicity Data recently published suggest that selective COX-2 inhibitors have a renal risk profile similar to traditional NSAIDs. This is particularly true in patients at risk of adverse renal effects related to the use of NSAIDs, including patients with hypertension, cirrhosis, nephrosis and renal insufficiency. As with traditional NSAIDs, a number of clinical renal syndromes are being described with these drugs including hemodynamic ARF, edema and hypertension, and hyponatremia and hyperkalemia. Both AIN and acute papillary necrosis have also been noted with the selective COX-2 inhibitors.

Renal Blood Flow/Glomerular Filtration Rate Hemodynamic ARF A reduction of GFR is common with NSAID therapy, but clinically important acute renal failure occurs rarely following administration of NSAIDs. Generally, acute renal failure develops only in susceptible patients such as those with underlying volume depletion, kidney disease, congestive heart failure, diabetic nephropathy, nephrosis, cirrhosis and old age. Current evidence suggests that selective COX-2 inhibitors have a risk profile similar to traditional NSAIDs in their potential to reduce GFR in susceptible patients. The CLASS trial noted that the incidence of increased creatinine levels was in the same range observed with NSAIDs (~ 1% of patients), although at statistically significant lower values in the celecoxib arm [3]. However, it is important to note that patients with risk factors for the development of adverse renal events with NSAIDs and selective COX-2 inhibitors are rarely included in clinical studies of these agents. Indeed, different results were obtained in studies on patients that more closely resemble a “susceptible” population. These included patients with conditions associated with increased dependence on prostaglandins to preserve renal function such as a sodium-restricted diet and chronic kidney disease. The pertinent studies will be reviewed in the following sections. Evaluation of the effects of the selective COX-2 inhibitors on kidney function in human subjects has clarified the role of the COX-2 isoform in the human kidney and provided insight into the renal effects of these drugs. As will be seen, most studies examine the effects of these drugs in patients with minimal or no risk for hemodynamic ARF. Healthy elderly patients were studied to evaluate the effect of selective COX-2 inhibition on renal hemodynamics and solute excretion [60]. After exclusion for hypertension, diabetes mellitus, or renal impairment, thirty-six patients maintained an a high sodium diet (200 mEq/day) were

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randomized to receive rofecoxib 50 mg/day, indomethacin 50 mg three times/day, or placebo. As compared with control, both rofecoxib and indomethacin induced a transient but significant decrease in urinary sodium excretion, which returned to baseline after approximately 72 hours of drug therapy. Urinary prostaglandins were inhibited by both rofecoxib and indomethacin (compared with placebo), whereas GFR decreased significantly in the indomethacin group only. This study demonstrates that transient sodium retention occurs with both types of NSAIDs. However, it appears that healthy elderly patients with well-preserved renal function (GFR > 80 ml/min/1.73 m2) are at low risk to develop acute renal insufficiency with a selective COX-2 inhibitor. The study results suggested that prostaglandins produced by the COX-1 isoform regulated RBF and GFR while COX-2 prostaglandins were more important for modulation of sodium excretion. The renal effects of celecoxib and naproxen were examined in 24 healthy elderly subjects without underlying kidney disease (GFR > 80 ml/min/1.73 m2) who were maintained on an unrestricted diet [61]. Subjects received either five days of celecoxib 200 mg twice/day followed by an increased dose of 400 mg twice/day for another five days, or they received naproxen 500 mg twice/day for ten days. Subjects were subsequently crossed over to receive the other drug regimen. Urinary PGE2 and 6-keto-PGF1α were significantly decreased with celecoxib and naproxen. A small but statistically significant decrease in GFR (-7.5 ml/min/1.73 m2) developed only in the naproxen group. Urinary sodium excretion was reduced following celecoxib (-30%) or naproxen (-38%) administration, but returned to near baseline levels by 72 hours of treatment with either drug. Thus, as in the previous study, healthy subjects who remain volume replete (ie. not sodium restricted) maintained a preserved GFR despite therapy with a selective COX-2 inhibitor. Sodium retention, however, was at least a transient problem with both selective and non-selective COX inhibitors. Selective COX-2 and non-selective COX inhibition was examined in 40 healthy male volunteers (GFR > 100 ml/min/1.73 m2) who were salt restricted and received a dose of diuretic [62]. To examine renal effects, subjects were entered into four groups to receive celecoxib 200 mg twice/day, celecoxib 400 mg twice/day, naproxen 500 mg twice/day or placebo for 7 days. Transient but clinically significant decreases in RBF (≈ -100 ml/min) and GFR (≈ -20 ml/min) were noted after 1 day of therapy only in the celecoxib 400 mg twice/day group (Figure 4). Renal Blood flow and GFR were insignificantly decreased for both celecoxib and naproxen on day 7. Urine output as well as urine sodium and potassium excretion on day 1 was significantly decreased with both doses of celecoxib and naproxen. After 7 days of therapy, urine output and urine sodium and potassium excretion were significantly depressed with both doses of celecoxib, whereas only urine potassium excretion was significantly decreased with naproxen. These data suggest that salt-depleted patients are at risk to develop a reduced GFR and impaired solute excretion when treated with a selective COX-2 inhibitor, reminiscent of traditional NSAID renal toxicity. Multiple doses of rofecoxib and indomethacin were studied in a group of salt-restricted elderly patients to examine effects on renal function [63]. Sixty patients with stage 2-3 CKD (creatinine clearances, 30-80 ml/min/1.73 m2), were randomized into one of 4 groups that received rofecoxib 12.5 mg/day, rofecoxib 25 mg/day, indomethacin 50 mg three times/day, or placebo for six days. Rofecoxib 12.5 mg and 25 mg doses significantly decreased glomerular filtration rate by 10.2 ml/min/1.73 m2 and 9.6 ml/min/1.73 m2, respectively, while

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indomethacin similarly decreased GFR by 7.8 ml/min (Figure 5). Neither drug produced consistent reductions in urinary sodium nor potassium excretion while indomethacin significantly increased serum potassium concentration. This study further implies that a volume depleted state from salt restriction and mild to moderate CKD creates prostaglandindependent renal function that requires COX-2 to synthesize compensatory renal prostaglandins.

Changes in GFR (mll/min)

10 0 -10 *

-20 -30

Placebo Naproxen 500 mg Celecoxib 200 mg Celecoxib 400 mg

0

60

120

180

Time (min) *P<0.05 versus baseline. Figure 4. Effects of celecoxib 200 mg („) and 400 mg (z), naproxen 500 mg (V), or placebo (‹) on changes in glomerular filtration rate (GFR) over the first 3 hours in subjects on a sodium restricted diet (<50 mEq/day). Values are mean ± SE. [From ref. [62], with permission].

To summarize, in a salt restricted setting, both rofecoxib and celecoxib, reduced GFR to a similar degree as the traditional NSAIDs. Other potential states of renal stress have been studied. In patients with underlying kidney disease, celecoxib 200 mg twice/day was compared with both naproxen 500 mg twice/day and placebo for 7 days of therapy [64]. A significant reduction of GFR, similar to naproxen was noted with celecoxib. In contrast, celecoxib (400 mg, single dose) administered with irbesartan (150 mg) to healthy volunteers with slight volume depletion had no acute adverse affect on renal hemodynamics or renal salt handling [65]. It is important to recall that this was a short-term study carried out in a very limited number of healthy subjects, and therefore it cannot be used to claim renal safety for chronic therapy.

Changes in GFR (mL/min)

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5

163

*P<.03 vs placebo.

1.8 ± 2.4

0 10.2 ± 2.4

-5

9.6 ± 2.4

7.8 ± 2.4

Rofecoxib 25 mg QD

Indomethacin 50 mg TID

-10 -15

Placebo

Rofecoxib 12.5 mg QD

*

Figure 5. Effect of rofecoxib, indomethacin, or placebo on glomerular filtration rate (ml/min) in patients with mild chronic kidney disease (stages 2 and 3) on a sodium restricted diet. Measurements shown are the maximum decline in GFR (using iothalamate clearance) from baseline at day 6 of drug therapy. Bar graphs represent mean ± SE. [From ref. [63], with permission]

Selective COX-2 inhibitor therapy has also been observed to impair renal hemodynamics in several animal models. The selective COX-2 inhibitor, nimesulide significantly reduced renal blood flow and GFR in sodium-restricted dogs [66]. Another selective COX-2 inhibitor (SC-236) decreased both RBF and GFR in cirrhotic rats, although to a slightly lesser degree than ketorolac [67]. Subsequent studies suggest a lesser effect of celecoxib as compared with traditional NSAIDs on GFR in both animal and humans with compensated cirrhosis [68,69]. Clinically relevant cases of nephrotoxicity from COX-2 selective inhibitors have been published [70-76]. Three patients who developed acute renal failure and hyperkalemia following therapy with these medications (2 celecoxib, 1 rofecoxib) were the first reported by Perazella and Eras [70]. Notably, all of these patients had multiple risk factors for NSAIDinduced nephrotoxicity, including factors such as underlying chronic renal failure and true or “effective” volume depletion. The acute deterioration in renal function reversed to baseline following discontinuation of COX-2 inhibitor and treatment of the associated intravascular volume disturbance. One patient required hemodialysis for severe hyperkalemia. Since this series of cases, several other reports [71-76] of renal impairment and/or electrolyte disorders have been described (Table 5). Taken together, these cases suggest that acute renal failure, hyperkalemia and sodium retention can occur with selective COX-2 inhibitor therapy in highrisk patients with prostaglandin dependent disease states. It has been suggested that differences in renal-based adverse events between the available selective COX-2 inhibitors might reflect a peculiar difference between the agents rather than a “class”-related COX-2 pharmacologic effect. Available information, however, suggests a similar nephrotoxic potential of the selective COX-2 inhibitors represents a class effect and there are no overwhelming data to conclude in favour of a major difference in these drugs. More comparative data are necessary to demonstrate if any differences exist

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between the selective COX-2 inhibitors in regards to their potential to cause hemodynamic ARF. Until then, it is reasonable to conclude that the selective COX-2 inhibitors have the potential to cause acute renal failure in a manner similar to traditional NSAIDs. Table 5. Cases of Acute Renal Failure associated with the Selective COX-2 Inhibitors Medication Type and Dosage Celecoxib 200 mg bid Celecoxib 200 mg bid Rofecoxib 25 mg qd Celecoxib (n=2) Rofecoxib (n=3) Celecoxib 100 mg bi Celecoxib 200 mg qd (n=3) Rofecoxib 25-50mg qd (n=2) Celecoxib 400 mg qd Celecoxib 200 mg qd Rofecoxib 25 mg qd (n=2) Celecoxib 200 mg bid(n=1)

Baseline Patient Characteristics Age-63, CKD, HTN Cr=2.8 CAD, LVH Age-68, CKD, HTN, Cr=3.5 DM, CMP Age-73, CKD, DM Cr=2.2 CAD, PVD Mean Age-63 Mean GFR=60cc/min Loop diuretics (n=5) HTN (n=2), CHF (n=2) Age-52, Cirrhosis, Cr=1.1 Gout ACE inhibitors (n=2) Diuretics (n=3) HTN (n=3), CMP (n=4) CAD (n=3), CRF (n=5)

Age-57 Osteoarthritis Cr=1.0 Age-55, Pleural effusion Cr=0.7 Age-77, 78, DM, HTN, ACE Inhibitor (n=2) Cr=1.5, 1.9 Age=19, transplant on sirolomus

COX-2 Inhibitor Treatment: Data Cr=4.9 CHF K+=5.1 Edema Cr=5.6 CHF K+=5.4 Edema Cr=9.0 Volume K+=8.5 depleted Mean Max BUN=84 Mean Max Cr=2.8 Serum K+=5.1-6.4 Mean HCO3=19 Cr=9.7 GI Bleed Mean increase Cr=2.4 Serum K+=5.6-8.5 Serum HCO3=12-18 Serum Na+=126-132 CHF (n=3) Cr=3.0 HTN Edema Cr=0.8, CHF, Edema Weight gain=4.54 kg Cr=2.8, 3.6, Edema K+=5.5 (n=1) Serum HCO3=18, 22 ARF (Cr=?), Edema

Outcome After Stop COX-2 Inhibitor Recovered within 5 days (Cr=2.9) Recovered within 7 days (Cr=3.3) Recovered within 8 days (Cr=2.0) All patients reported to recover to baseline within 2-3 weeks Recovered within 3 days to baseline All recovered to baseline within 2 to 8 days, 3 patients required dialysis for hyperkalemia or severe CHF Recovered within 7 days to baseline Recovered to baseline (+ furosemide) All 3 patients recovered within 10 to 20 days to baseline

Abbreviations: Cr, creatinine (mg/dl); CKD, chronic kidney disease; CAD, coronary artery disease; HTN, hypertension; LVH, left ventricular hypertrophy; CMP, cardiomyopathy; DM, diabetes mellitus,; PVD, peripheral vascular disease; CHF, congestive heart failure; GFR, glomerular filtration rate; K+, potassium (mEq/L); BUN, blood urea nitrogen (mg/dl); HCO3, bicarbonate (mEq/L); ARF, acute renal failure. References[70-76]

Sodium Balance Edema Fluid retention is the most common NSAID-related renal effect; it has been reported to occur in approximately 5% of treated patients [28] and is due to decreased sodium excretion and mild sodium retention that occur from inhibition of renal PGE2 synthesis. As with traditional NSAIDs, the COX-2 selective drugs can cause peripheral edema. This is not a surprising occurrence since both rofecoxib and celecoxib are capable of inducing renal sodium retention. In healthy people, during either normal or restricted sodium intake

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conditions, both rofecoxib and celecoxib, in separate studies, have been demonstrated to transiently reduce urinary sodium excretion rates similar to the comparator conventional NSAIDs [28,60,61,63]. In osteoarthritis studies that included approximately 6000 patients, lower extremity edema was noted in 3.6 and 3.8% of patients treated with rofecoxib at 12.5 mg and 25 mg doses, respectively [77]. These rates were similar to those observed with the comparator NSAIDs ibuprofen 800 mg three times daily (3.8%) and diclofenac 50 mg three times daily (3.4%), but higher than placebo (1.1%). In a similar fashion, celecoxib was also observed to precipitate peripheral edema formation in the large osteoarthritis trials that comprised approximately 5,200 patients [78]. The incidence of peripheral edema in patients treated with 100 mg and 200 mg twice daily of celecoxib was 1.6 and 3.0%, respectively. The edema rates were 1.3% for placebo and 2.2% for the NSAID comparator. The edema rate for celecoxib at 100 or 200 mg twice daily in data pooled from patients with osteoarthritis and rheumatoid arthritis was 2.5%, similar to the edema rate seen with naproxen (2.2%). It is probable that the slightly lower edema rates seen for both the traditional NSAIDs and celecoxib in these trials (as compared with the rofecoxib trials) reflects a different study population and/or a different edema definition. Edema, generally in the lower extremities, is commonly reported in the selective COX-2 inhibitor clinical studies as it is during therapy with traditional NSAIDs. Edema formation with selective COX-2 inhibitors is dose-dependent and, similar to non-selective NSAIDs is associated with clinically significant weight gain in 30 to 50% of cases [79]. Although edema was a rare cause for study drop-out in the large clinical trials VIGOR and CLASS, a recent post-marketing survey noted that the development of edema led to discontinuation of the COX-2 inhibitor in 58-82% of cases [80]. Direct comparison of edema formation between rofecoxib and celecoxib has also been performed in the SUCCESS VI study [81]. Twice as many rofecoxib- compared with celecoxib-treated patients experienced edema, and a robust association emerged between the development of edema and the destabilization of hypertension control. This study and a subsequent study [82] contrasts all other studies that have demonstrated identical edema formation rates between the selective COX-2 inhibitors and traditional NSAIDs, raising questions about reliability of edema measurement in the various studies. The effect of drug half-life on edema formation (longer half-life of rofecoxib versus once daily celecoxib) also needs to be considered, since this may lead to more sodium retention over the 24-hour period. Taken together, data suggest that edema develops with both of the two approved selective COX-2 inhibitors, as seen with the traditional NSAIDs. The newer, more COX-2 selective inhibitors, such as etoricoxib do not appear to differ in respect to rofecoxib and celecoxib in terms of lower extremity edema adverse events [83]. The risk of congestive heart failure, the most serious complication of fluid retention, has been reported uncommonly in large clinical trials with selective COX-2 inhibitors, and not significantly different in respect to comparator NSAIDs. It is of interest that rofecoxib has been noted in the rat to blunt the diuretic-induced increase in urinary prostaglandin excretion, and to attenuate dose-dependently diuresis and natriuresis, as well as the stimulation of the renin-angiotensin system induced by furosemide and thiazides [84]. This likely explains the edema formation and diuretic resistance seen in humans treated with these drugs.

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Hypertension Based on blood pressure data generated from traditional NSAIDs, one might also expect that the COX-2 selective drugs would also induce or exacerbate hypertension in predisposed patients (treated and borderline hypertensive patients). As noted, a number of mechanisms have been proposed to explain the increased blood pressure associated with NSAID treatment. Inhibition of renal prostaglandin synthesis causes sodium and water retention, secondarily expanding plasma volume. Inhibition of prostacyclin synthesis by NSAIDs may increase peripheral resistance by blunting the potent vasodilator effect of prostacyclin. Negating the counter-regulatory role of vasodilator prostaglandins may unfavourably derange the balance achieved with reflex-mediated pressor mechanisms induced by initiation of antihypertensive therapy [85]. Finally, loss of the inhibitory action of prostaglandins on the renal synthesis of endothelin-1 may lead to both salt and water retention as well as increased peripheral vascular resistance [86]. The relative contributions of the COX-1 and COX-2 isoforms in the production of vasodilatory prostaglandins, and possibly of endothelin-1 are not currently known. Thus, the potential differences of the selective COX-2 inhibitors as compared with traditional NSAIDs in the ability to induce hypertension cannot be predicted in the absence of direct comparisons. Current data suggest that selective COX-2 inhibitors have a blood pressure raising profile similar to traditional NSAIDs. The effect of these drugs to reduce prostaglandin production and renal sodium excretion in patients as described in the previous studies [60-63] suggests that elevation of blood pressure could occur in “salt-sensitive” patients. Although the large osteoarthritis trials that studied rofecoxib and celecoxib demonstrated only small increments in blood pressure with both of these drugs, the populations examined in these trials were relatively healthy and possessed minimal risk for the development of hypertension. As such, the absence of severe hypertension in the selective COX-2 trials is not an unexpected finding, since the normotensive patients in the studies that evaluated the hypertensive effects of traditional NSAIDs also did not develop significant elevations in blood pressure [36-38]. To support the possibility of a blood pressure-raising effect of the selective COX-2 inhibitors, a few studies in experimental animals provide insight into the pressor effect of these drugs. Hocherl and coworkers confirmed that the COX-2 isoform may be implicated in these hypertensive mechanisms [87]. They demonstrated that rofecoxib increased systolic blood pressure in rats through inhibition of prostacyclin synthesis, and that this alteration can be prevented by salt deprivation (supporting the notion that hypertension can occur in salt sensitive groups). This latter finding confirms previous observations that hypertension is exacerbated by selective COX-2 inhibitors therapy in rats with salt-dependent hypertension [88]. Celecoxib reduced prostacyclin formation, induced weight gain and raised blood pressure (as compared with vehicle) in both normotensive and hypertensive rats [89]. Selective inhibition of COX-2 in rat renal medulla also causes hypertension in rats fed a high sodium diet [57]. Interestingly, these observations have their clinical counterpart in a study conducted in humans [81]. Hypertension and edema developed concurrently during selective COX-2 inhibitor therapy (celecoxib and rofecoxib), thus suggesting that appropriate diuretic therapy to reduce sodium retention may be an important component in re-establishing blood pressure control.

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Clinically, the data on the hypertensive effects of COX-2 inhibitors are abundant but heterogenous. It does appear that treatment with rofecoxib (12.5, 25, 50 mg once/day) causes a dose-related increase in the incidence of hypertension [90]. At therapeutic doses, the incidence of hypertension is similar to those observed with comparator NSAIDs. Furthermore, in clinical trials with celecoxib, hypertension occurred in the same range observed with conventional NSAIDs (~ 2% of patients), although the incidence of hypertension was statistically significantly lower than in the the conventional NSAID group [3]. These results need to be analyzed carefully because the doses of selective COX-2 inhibitors and comparator NSAIDs utilized in the studies were not equivalent, the use of ACE inhibitors was unbalanced between arms, and hypertension was reported by investigators rather than confirmed by objective measures. Differences in the pharmacokinetics of selective COX-2 inhibitors and traditional NSAIDs were not taken into account in reference to the time when blood pressure was measured. Additionally, there was no direct comparison between the available selective COX-2 inhibitors. Subsequent studies were performed to address some of these residual questions on the hypertensive properties of various COX-2 inihibitors. The SUCCESS VI Study, a randomized, double-blind trial in hypertensive, osteoarthritic patients older than 65 treated with either celecoxib (200 mg/day) or rofecoxib (25 mg/day) demonstrated a small, but statistically significant increase in systolic hypertension with rofecoxib as compared with celecoxib [81]. However, a few caveats from this study should be noted. First, the two arms differed in terms of numbers of patients on ACE inhibitor therapy (more frequent in the celecoxib group). It is unclear how the imbalance in ACE inhibitor use might influence the results (negatively or positively) and therefore makes the results difficult to interpret. Second, the doses of drugs employed may not have been equivalent in regards to efficacy as suggested in some studies [91]. Finally, blood pressure measurements were aimed to target trough effect, which favoured the drug with the shorter half-life (celecoxib). Measurement of blood pressure throughout the 24-hour period would have provided more accurate information about drug-associated hypertensive effect. A similar 6-week study of the blood pressure effects of celecoxib (200 mg/day) and rofecoxib (25 mg/day) was undertaken in osteoarthritic patients, the majority of which were hypertensive [92]. The major difference between this study and the previously described study was that blood pressure was measured at peak (within hours of drug administration). The development of hypertension was not different between the drug groups. In a double-blind, placebo controlled study on 67 healthy elders maintained on a 200 mEq/day sodium diet (documented sodium balance with 24 hour urine collections), equipotent doses of rofecoxib, celecoxib and naproxen were examined to assess the effect on renal sodium excretion and blood pressure [93]. Sodium balance was achieved and documented with daily 24-hour urinary sodium measurements for 8 to 13 days prior to entry into the study. All treatment arms experienced an increase in systolic blood pressure (rofecoxib, 3.4 mmHg ± 2.0 mmHg; celecoxib, 4.3 mmHg ± 2.0 mmHg; naproxen, 3.1 mmHg ± 2.0 mmHg) as compared with placebo (-1.4 mmHg ± 2.0 mmHg). No significant difference on renal sodium excretion between agents was observed, although sodium retention was increased with all three medications as compared with the placebo group. It is important to note that these were not hypertensive patients on blood pressure lowering medications. It is possible that a different effect may have occurred in hypertensive patients.

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Investigators have sought to examine the effects of COX-2 inhibitors on diurnal blood pressure variations. In a study of hypertensive osteoarthritis patients with controlled blood pressure, subjects received either 25 mg rofecoxib once daily or nabumetone (a non-selective NSAID) 2000 mg once daily for 1 week of treatment and 1000 mg for the following 3 weeks [94]. Twenty-four hour arterial blood pressure monitoring was performed. Rofecoxib treatment did not change arterial blood pressure during daytime, however, there was a distinct increase in night-time systolic and diastolic blood pressure leading to a disappearance of the physiological diurnal variation. Nabumetone, on the other hand, caused a moderate increase of day and night blood pressure, without changes in biological diurnal variation. Another clinically pertinent question is whether COX-2 inhibitors interfere with the efficiacy of antihypertensive agents. White and colleagues studied 91 patients with controlled hypertension on linsinopril (10 to 40 mg/day) and randomized them to placebo or celecoxib (200 mg bid) and monitored them with 24-hour ambulatory blood pressure recordings over 4 weeks [95]. Celecoxib had no significant effect on the mean changes of the 24-hour systolic and diastolic blood pressures during therapy with the ACE inhibitor. Similarly, a recent review of this issue in data from rofecoxib phase IIb/III osteoarthritis database containing over 5000 patients noted that there was no change in blood pressure control when rofecoxib therapy was commenced in patients on ACE inhibitors [79]. Furthermore, none of the recorded classes of antihypertensive agents (ACE inhibitors, beta-blockers, diuretics, calcium channel blockers) posed an increased risk of having a hypertension-related adverse event with rofecoxib therapy. While most of the studies reviewed thus far reveal that selective COX-2 inhibitors increase blood pressure to only a small degree in some patients, an obvious difference between the various COX-2 inhibitors has not yet been appreciated. An fair amount of more recent data, however, suggest there are some differences in the hypertensive properties of various COX-2 inhibitors, with rofecoxib possessing greater hypertensive potency. For example, a large study comprising 404 diabetic patients with controlled hypertension and osteoarthritis, examined the effects of celecoxib (200 mg daily), rofecoxib (25 mg daily) or naproxen (500 mg twice daily) on 24-hour ambulatory blood pressure [96]. Baseline antihypertensive therapy was similar among all treatment groups. Patients treated with rofecoxib for 6 weeks had higher systolic blood pressures (approximately 4 mm Hg), than patients treated with celecoxib and traditional NSAIDs. After 12 weeks, the differences remained between rofecoxib and celecoxib, but not between rofecoxib and naproxen, as this latter group manifested similar increases in systolic blood pressure at this time period. Rofecoxib also increased the proportion of patients (30%) with controlled hypertension (24hour systolic blood pressure <135 mm Hg) who developed ambulatory hypertension by week 6 to a greater extent than celecoxib (16%) or naproxen (19%). Unlike the findings of a previous study [94], diurnal variation of blood pressure was not affected by any of the agents. Three other reports similarly suggest that rofecoxib induces greater elevations in BP than celecoxib. A case-control study by Solomon and colleagues, involving 17,844 patients of 65 years of age or greater, found that rofecoxib users had an increased relative risk of developing new onset hypertension compared with those taking celecoxib (odds ratio [OR] 1.6), non-specific NSAIDs (OR 1.4) or no NSAID (OR 1.6) [97]. These risks were similarly elevated for doses of rofecoxib ≤ 25 mg daily or > 25 mg daily. In contrast, even those

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patients taking higher doses of celecoxib (> 200 mg daily) did not have an increased risk of hypertension. Another retrospective analysis examined changes in blood pressure in 121 Native Americans patients who switched selective COX-2 inhibitor therapy from celecoxib to rofecoxib [98]. Systolic blood pressure was 2.9 mm Hg higher and mean diastolic blood pressure was 1.5 mm Hg higher on rofecoxib compared with celecoxib. These differences were even greater in hypertensive patients (4.8 and 2.0 mm Hg, respectively). Twelve of the 121 patients experienced a systolic blood pressure rise of > 20 mm Hg after the switch. Unfortunately, no analysis was performed of a contrary pharmacologic switch (rofecoxib to celecoxib). Finally, a retrospective analysis of spontaneous adverse events in the US FDA database revealed approximately a 4-fold greater frequency of hospitalization for acute blood pressure elevation with rofecoxib use compared with celecoxib use [99]. This occurred despite a higher prevalence of chronic hypertension in the celecoxib group (88 vs. 67%). The median time to hospitalization after initiation of use was 10 days, and mean time was approximately 30 days. Most of the blood pressure events occurred on 25 mg daily or less of rofecoxib, while only 4 of the 26 patients were taking greater than 25 mg. Perhaps the most convincing data that there indeed differences amongst the selective COX-2 inhibitors in regards to their hypertension-inducing properties are garnered from the largest randomized, controlled trial directly comparing celecoxib and rofecoxib in 1,095 elderly, osteoarthritic patients with treated and controlled hypertension [82]. After 6 weeks of therapy, more patients in the rofecoxib group compared with the celecoxib group had a higher incidence of clinically significant systolic blood pressure elevations (change > 20 mm Hg plus absolute value > 140 mm Hg) at any time point (14.9% versus 6.9%, p<0.01). The rofecoxib-treated patients experienced a mean increase in SBP of approximately 3 mm Hg, while mean SBP in the celecoxib group did not change. As in the SUCCESS VI study [81] approximately twice as many rofecoxib- compared with celecoxib-treated patients experienced edema (7.7% vs 4.7%, p= 0.045). Interestingly, the rofecoxib-induced BP differences were only statistically significant in those patients treated with ACE inhibitors and beta-blockers alone or in combination with a diuretic. In comparison, patients treated at baseline with calcium channel blockers or diuretics alone did not experience significant blood pressure changes. Presuming that the increases in blood pressure are volume dependent (likely, given the sodium retentive properties of rofecoxib and the higher propensity to edema formation in the rofecoxib group in this study), then it would intuitively follow that ACE inhibitors and beta-blocking agents would be less effective antihypertensive agents in this setting [100]. In contrast, calcium channel antagonists, and of course, diuretics, are excellent antihypertensive agents in the setting of volume expansion. Finally, a recent meta-analysis has attempted to assess the pooled effect of selective COX-2 inhibitors on blood pressure. Nineteen randomized controlled trials consisting of a total of 45,451 patients were analyzed. Selective COX-2 inhibitors, as a whole, caused a weighted mean difference point estimate increase in systolic and diastolic blood pressure compared with placebo (3.85/1.06 mm Hg) and non-selective NSAIDs (2.83/1.34) [101]. Selective COX-2 inhibitors, as a general class again, resulted in a non-significant trend towards increases in de novo hypertension versus both placebo and non-selective NSAIDs.

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When investigating the hypertensive propensity for the specific selective COX-2 inhibitors, rofecoxib resulted in elevations in systolic blood pressure compared with celecoxib and nonselective NSAIDs (Table 6). Celecoxib induced only minimal increases in systolic and diastolic blood pressure when compared with placebo, but no increase when compared with non-selective NSAIDs (Table 7). Table 6. Effect of Rofecoxib on BP or HTN in comparison with other NSAIDs or other Selective COX-2 Inhibitors Study

Comparison

Number of Pts

Type of Study

81 82 92 93 93 94 96 96 97 97 98 99 116 117

Rof vs. Cel Rof vs. Cel Rof vs. Cel Rof vs. Cel Rof vs. NSAID Rof vs. Name Rof vs. Cel Rof vs. Nap Rof vs. Cel Rof vs. NSAID Rof vs. Cel Rof vs. Cel Rof vs. Nap Rof vs. Nap

810 1092 N/A 34 34 N/A 274 268 1264 1255 121 N/A 439 5557

RC RC RC RC RC RC RC RC CC CC Retrospective Retrospective* RC RC

Difference in BP or HTN + + + + + + + + + -

Abbreviations: BP, blood pressure; HTN, hypertension; Rof, rofecoxib; Nap, naproxen; NSAID, nonsteroidal anti-inflammatory drug; Name, namebutone; RC, randomized, controlled trial; CC, case-control study. *Analysis of FDA database examining cases of hypertensive urgencies induced by selective COX-2 inhibitors

Table 7. Effect of Celecoxib on BP or HTN in comparison with other NSAIDs or Selective COX-2 Inhibitors Study

Comparison

Number of Patients

Type of Study

3 96

Cel vs. NSAID Cel vs. Nap

655 266

RC RC

Difference in BP or HTN -* -

97 118 119

Cel vs. NSAID Cel vs. Diclof Cel vs. Nap

1747 655 918

CC RC RC

-

Abbreviations: BP, blood pressure; HTN, hypertension; Cel, celecoxib; Nap, naproxen; NSAID, nonsteroidal anti-inflammatory drug; diclof, dicofenac; RC, randomized, controlled trial; CC, casecontrol study * BP statistically significantly lower in celecoxib group

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In conclusion, hypertension appears to be a dose-related class effect of selective COX-2 inhibitors, similar to conventional NSAIDs. At this point in time, one can conservatively conclude that while both of these drugs cause little hypertension in the general population, both are capable of significantly elevating blood pressure in patients with underlying hypertension. Animal studies and initial human studies have suggested a relative equivalence among different selective COX-2 inhibitors in regard to their hypertensive effects. However, the preponderance of evidence seems to now indicate that rofecoxib possesses greater hypertensive potency than other selective COX-2 inhibitors (Table 6). The reason for this apparent difference is not well understood. One potential hypothesis is that rofecoxib results in reductions in aldosterone metabolism, as has been suggested by some authors, however, in Wistar-Kyoto rats and spontaneously hypertensive rats treated with rofecoxib, plasma aldosterone concentrations were unaffected [102]. A second hypothesis for the difference in the hypertensive properties of rofecoxib and celecoxib is that celecoxib, through inhibition of carbonic anhydrase, results in a modest natriuresis that is enough to compensate for the alterations in renal blood flow and prostaglandin-dependent renal sodium handling. However, clinically, acetazolamide, the prototypical carbonic anhydrase inhibitor, is not an effective hypertensive agent, simply because of the lack of any significant diuretic action of the agent. Thus, hypertensive patients treated with selective COX-2 inhibitors, and in particular rofecoxib, as with NSAIDs, should be carefully monitored in the initial phase of treatment for loss of blood pressure control. If hypertension worsens, the doses of anti-hypertensive agents should be accordingly modified. Further, it should be clearly recognized that changes in blood pressure are small if expressed as group averages, but they can underestimate individual changes [104]. Nevertheless, the importance of relatively small variations in blood pressure can be great due to its relevant public health implications. In many large clinical trials, sustained increases in systolic pressure of only 3 mmHg explained a 10 to 20% increase in congestive heart failure [105], a 15 to 20% increase in stroke risk (49), and a 12% increase in angina risk [106]. These findings have meaningful clinical implications for the maintenance of adequate blood pressure control, since a small but persistent loss of adequate blood pressure control can lead to an increase in adverse cardiovascular events.

Water and Potassium Balance Hyponatremia and Hyperkalemia The effect of the selective COX-2 inhibitors on water and potassium metabolism has not been specifically investigated in the large clinical trials that preceded their introduction on the market. Hyponatremia may occurs from water retention induced by the selective COX-2 [73]. Four out of five patients developed hyponatremia (serum Na+ concentrations 126 mEq/L-132 mEq/L) following therapy with these drugs. In addition, selective COX-2 inhibitors decrease urinary potassium excretion in healthy subjects and patients with other risk factors for hyperkalemia [62,63]. Not surprisingly, several cases were reported that raise concern about the risk of hyponatremia, severe hyperkalemia and type-4 renal tubular acidosis in particular clinical settings with the selective COX-inhibitors [70,73]. In nine out of 14 cases where serum potassium concentration was measured, hyperkalemia (serum K+

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concentration 5.1 mEq/L-8.5 mEq/l) occurred following therapy with a selective COX-2 inhibitor [73]. In the cases where hyperkalemia developed, contemporaneous use of ACE inhibitors, angiotensin receptor blockers, spironolactone, potassium supplements, as well as concomitant diabetes mellitus with hyporeninemic hypoaldosteronism, and renal failure were present. It is interesting that the hyperkalemic effects of the selective COX-2 inhibitors are indistinguishable from traditional NSAIDs.

AIN from Selective COX-2 Inhibitors As noted previously, traditional NSAIDs are a well-known cause of AIN. Recently, several cases of AIN following treatment with the selective COX-2 inhibitors, rofecoxib and celecoxib, were reported [106-114]. At least 9 cases of well-documented AIN from selective COX-2 inhibitors have been described (celecoxib, n=6, rofecoxib, n=3). The details are noted in Table 8. There is no obvious pattern to the clinical presentation as one case of AIN developed within 3 days, four cases occurred after 3-4 weeks of therapy, two developed at 6 to 7 months, and the two others developed after a year or greater of consumption. Eosinophilia was present in only 2/7 cases, while all cases had some level of proteinuria. The urine sediment was active in 8/9 cases with pyuria and hematuria the predominant cellular elements. Peak serum creatinine concentration following drug treatment ranged between 1.6 mg/dl and 13.6 mg/dl. Hemodialysis was required in 3/9 patients, 6 patients received corticosteroids, and all recovered renal function with serum creatinine concentrations ranging from 0.9 to 1.4 mg/dl. One of the cases of celecoxib-associated AIN also had concurrent minimal change disease, while two cases had membranous glomerulonephritis on renal biopsy, findings that have also been described with traditional NSAIDs [110,111]. One case of minimal change disease associated with acute tubular necrosis (without AIN) was noted to be a complication of celecoxib therapy [115]. Table 8. Cases of Acute Interstitial Nephritis associated with the Selective COX-2 Inhibitors Drug Celecoxib Celecoxib Celecoxib Celecoxib Celecoxib Rofecoxib

Dose 400 mg/day 200 mg/day 200 mg/day 400 mg/day 400 mg/day 25 mg/day

Peak SCr 1.6 mg/dl 4.5 mg/dl 2.1 mg/dl 11.8 mg/dl 5.9 mg/dl 8.7 mg/dl

Rofecoxib Rofecoxib

25 mg/day 25 mg/day

13.6 mg/dl 2.5 mg/day

Abbreviations: SCr, serum creatinine concentration. References [99-105]

Treatment Steroids Steroids Steroids Hemodialysis Steroids Hemodialysis Steroids Hemodialysis Steroids

Final SCr 0.9 mg/dl 1.2 mg/dl 1.1 mg/dl 1.1 mg/dl 1.2 mg/dl 1.4 mg/dl 1.3 mg/dl Baseline

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Conclusion Based on data presented in this chapter, selective COX-2 inhibitors should be considered to have equivalent nephrotoxicity as traditional NSAIDs. Therefore, patients considered at high risk for adverse renal events, such as those treated with diuretics and ACE inhibitors or angiotensin receptor blockers, volume depleted patients, the elderly, and patients with congestive heart failure, diabetic nephropathy, lupus nephritis, nephrosis, acute and chronic kidney disease and cirrhosis should be treated cautiously with selective COX-2 inhibitors. Treatment with these drugs should prompt careful monitoring for edema, hypertension, hyperkalemia, hyponatremia and acute renal failure. Finally, since selective COX-2 inhibitors have better gastrointestinal tolerance, patients may be more likely to consume doses greater than prescribed for longer periods of time, increasing risk for adverse renal events. Since the hypertensive and renal effects of selective COX-2 inhibitors are dose-dependent, patients should be alerted to strictly adhere to the prescribed regimen and contact their physician if edema, hypertension or other problems develop. As with traditional NSAIDs, both AIN and glomerular disease (membranous glomerulonephritis and minimal change disease) may complicate therapy with the selective COX-2 inhibitors.

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hypertension, osteoarthritis, and type 2 diabetes mellitus. Arch Intern Med 2005;165:161-168. [97] Solomon DH, Schneeweiss S, Levin R, Avron J. Relationship between cox-2 specific inhibitors and hypertension. Hypertension 2004;44:140-145. [98] Fredy J, Diggins DA, Morrill GB. Blood pressure in Native Americans switched from celecoxib to rofecoxib. Ann Pharmacother 2005;39:797-802. [99] Brinker A, Goldkind L, Bonnel R, Beitz J. Spontaneous reports of hypertension leading to hospitalizations in association with rofecoxib, celecoxib, nabumetone, and oxaprozin. Drugs Aging 2004;21:479-484. [100] Weir MR. The role of combination antihypertensive therapy in the prevention and treatment of chronic kidney disease. Am J Hypertension 2005; 18:100S-105S. [101] Aw TJ, Haas SJ, Liew D, Krum H. Meta-analysis of cyclooxygenase-2 inhibitors and their effects on blood pressure. Arch Intern Med 2005;165:490-496. [102] Hocherl K, Endemann D, Kammerl MC, Grobecker HF, Kurtz A. Cyclo-oxygenase-2 inhibition increases blood pressure in rats. Br J Pharm 2002;135:1117-1126. [103] Fitzgerald GA, Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med 2001;345:433-442. [104] Graves JW, Hunder IA. Worsening of hypertension by cyclo-oxygenase-2 inhibitors. J Clin Hypertens 2000;2:396-398. [105] ALLHAT Collaborative Research Group: Major cardiovascular events in hypertensive patients randomized to doxazosin vs chlortalidone: the antihypertensive and lipidlowering tratment to prevent heart attack trial (ALLHAT). JAMA 2000;283:1967-1975. [106] Collins R, Peto R, MacMahon S, et al. Blood pressure, stroke, and coronary heart disease. Part 2: short-term reductions in blood pressure: overview of randomised drug trials in their epidemiological context. Lancet 1990;335:827-838. [107] Hypertension Detection and Follow-up Program Cooperative Group: Effect of stepped care treatment on the incidence of myocardial infarction and angina pectoris: 5-year findings of the Hypertension Detection and Follow-up Program. Hypertension 1984;6:1198-1206. [108] Rocha JL, Fernandez-Alonso J. Acute tubulointerstitial nephritis associated with the selective COX-2 enzyme inhibitor, rofecoxib. Lancet 2001;357:1946-1947. [109] Alper AB, Meleg-Smith S, Krane NK. Nephrotic syndrome and interstitial nephritis associated with celecoxib. Am J Kidney Dis 2002;40:1086-1090. [110] Henao J, Hisamuddin I, Nzerue CM, Vasadani G, Hewan-Lowe K. Celecoxib-induced acute interstitial nephritis. Am J Kidney Dis 2002;39:1313-1317. [111] Markowitz GS, Falkowitz DC, Isom R, et al. Membranous glomerulopathy and acute interstitial nephritis following treatment with celecoxib. Clin Nephrol 2003;59:137142. [112] Brewster UC, Perazella MA. Acute tubulointerstitial nephritis associated with celecoxib. Nephrol Dial Transplant 2004;19:1017-1018. [113] Chow KM, Szeto CC, Li P, Lai FM. Acute interstitial nephritis and COX-2 inhibition. Hosp Med 2003;64:429.

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[114] Esteve JB, Launay-Vachar V, Brocheriou I, Grimaldi A, Izzedine H. COX-2 inhibitors and acute interstitial nephritis: case report and review of the literature. Clin Nephrol 2005;63:385-389. [115] Almansori M, Kovithavongs T, Quarri MU. Cyclooxygenase-2 inhibitor-associated minimal change disease. Clin Nephrol 2005;63:381-384. [116] Hawkey CJ, Laine L, Simon T, Quan H, Shingo S, Evans J. Incidence of gastroduodenal ulcers in patients with rheumatoid arthritis after 12 weeks of rofecoxib, naproxen, or placebo: a multicentre, randomized, double blind study. Gut 2003;52:820826. [117] Lisse JR, Perlman M, Johansson G, et al. Gastrointestinal tolerability and effectiveness of rofecoxib versus naproxen in the treatment of osteoarthritis: a randomized, controlled trial. Ann Intern Med 2003;139:539-546. [118] Emery P, Zeidler H, Kvien TK, et al. Celecoxib versus diclofenac in long-term management of rheumatoid arthritis: randomized double-blind comparison. Lancet 1999;354:2106-2111. [119] Simon LS, Weaver AL, Graham DY, et al. Anti-inflammatory and upper gastrointestinal effects of celecoxib in rheumatoid arthritis: a randomized controlled trial. JAMA 1999;282:1921-1928.

In: COX-2 Inhibitor Research Editor: Maynard J. Howardell, pp. 181-207

ISBN 1-59454-994-X © 2006 Nova Science Publishers, Inc.

Chapter V

Are COX-2 Inhibitors Active on Intracellular Oxidative Processes? A Study on In Vitro and Cellular Models

1

Ange Mouithys-Mickalad1∗, Ginette Deby-Dupont1,2, Carol Deby1, Thierry Franck1,3, Didier Serteyn1,3 and Maurice Lamy1,2

Centre for Oxygen, Research and Development (C.O.R.D.), Institute of Chemistry, B6a. 2 Department of Anaesthesia and Intensive Care Medicine, B37 3 Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine B41. University of Liège, Domaine du Sart-Tilman, 4000 Liège, Belgium.

Abstract In the last years, there has been an increasing interest of using cyclooxygenase-2 (COX-2) inhibitors to treat the inflammatory pain and chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. The beneficial effects were to avoid the secondary adverse effects such as bleeding and gastric irritation, generally observed with aspirin and conventional NSAIDs. COX-1 is constitutively expressed in most tissues and involved in the regulation of normal homeostatic functions, while COX-2 is not detected in most tissues but induced by inflammatory stimuli. These outcomes motivated the commercial development of selective COX-2 inhibitors. Recent data suggested that the COX-2 enzyme can be expressed within atherosclerotic lesions and could play a crucial role in various types of cancers, by the way of its activity on the ROS production, gene transcription and prostaglandin (PGE2) production. Consequently, the COX-2 enzyme has become a real target for the study of various classes of compounds and specially the possible additional properties as COX-2 inhibitors. We and other groups have already investigated the pro or antioxidant profile of conventional NSAIDs and some COX-2 inhibitors. With the recent withdrawal of two compounds of the coxib’s family (rofecoxib and celecoxib), for adverse cardiovascular events, concerns regarding the safety of all COX-2 inhibitors have been raised. To answer to these concerns, different approaches were ∗

Corresponding author. Fax: +32 4 366 28 66; E-mail: [email protected]

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developed by studying on in vitro models, the potential inhibiting-or-stimulating activities on oxidative phenomena of new drugs with already recognized therapeutic effects. Preliminary data obtained with COX-2 inhibitors showed a moderate inhibiting effect on the intracellular oxidant processes and others a stimulating activity. New hypotheses for the treatment of inflammation are now suggested for compounds like nimesulide and its analogous, which are selective towards COX-2 with little activity on COX-1. Here, we reported the in vitro effects of some COX-2 inhibitors, in comparison with traditional drugs (ibuprofen, diclofenac and aceclofenac), by using two cellular models: a human lung type II alveolar cell line (A549) and a human promonocyte cell line (THP-1). The direct interactions between the drugs and ROS were also investigated in cell-free systems.

Abbreviations COX-2: COX-1: NSAID: PGE2 : PGD2 : TxA2 : NOS2 : PMA : LPS : LOX : HRP: MPO: ABTS: Cp: A549: THP-1: ROS:

Cyclooxygenase-2; Cyclooxygenase-1; Non steroidal anti-inflammatory drug; Prostaglandins E2; Prostaglandins D2; Thromboxane A2: Nitric Oxide Synthase-2; Phorbol myristate acetate; lipopolysaccharide; Lipoxygenase; Horseradish peroxidase; Myeloperoxidase; 2,2’-Azinobis-benzothiazoline-6-sulfonate; Chlamydia pneumoniae; Human lung type II alveolar cell line; human promonocyte cell line; Reactive oxygen species.

1. Introduction Six years ago, the scientific community celebrated the centenary (1899) of the discovery of acetylsalicylic acid (aspirin), as the first drug for the treatment of rheumatic diseases. Several years after this important discovery, dozens of non-steroidal anti-inflammatory drugs (NSAIDs) have been developed and launched, including indomethacin (1963) and ibuprofen (1969). But the understanding of their mechanism of action remained unknown until the landmark work by Vane in 1971, which identified the enzyme cyclooxygenase (COX) as the common molecular target of all these compounds (Vane, 1971). It is well established now that NSAIDs are of huge therapeutic benefit in the treatment of rheumatic diseases. Nevertheless they are associated with several adverse effects, of which the gastro-intestinal

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side effects such as bleeding and ulcerations are the most frequent (Wallace, 1994; Dubois et al., 1998; Akarca, 2005). The management of pain and inflammation is mainly based on the use of steroids and NSAIDs, which act on the metabolism of arachidonic acid (AA) (Vane and Botting, 1998). Steroids work at the upstream of the AA cascade by inhibiting the release of arachidonic acid from membrane phospholipids (Fig. 1) and are thought to be by far more efficient than the other anti-inflammatory drugs. But they do not have any efficacy on the fever and their sustained use has long lasting undesirable effects. In contrast, NSAIDs act at the downstream of the AA cascade, not only on the COX pathways but also on the lipoxygenase ones. These findings led to a real hope in the treatment of chronic pain by using traditional NSAIDs, especially aspirin.

Figure 1. The different pathways involved in the metabolism of arachidonic acid

Arachidonic Acid Metabolism There are two pathways that involve arachidonic acid: the lipoxygenase (LOX) pathway that gives various lipoxygenase products (15-LOX-2, 12-LOX, 8-LOX and 5-LOX) and the cyclooxygenase (COX) enzyme (Fig. 1). COX is also called the PGH synthase pathway and produces, through the PGH synthase, the unstable intermediate PGG2, which is rapidly converted into PGH2 by the peroxidase activity of PGH2 synthase. Specific isomerases convert PGH2 to various prostaglandins (PGs) and thromboxane (TxA2). Linoleic acid and cytochrome P450 epoxygenase are also involved in the metabolism of AA. There are at least two forms of the COX enzyme: (i) The constitutive COX-1 is involved in the maintenance of the tissue homeostasis and is expressed in most tissues (housekeeping roles). It is responsible for platelet aggregation, systemic and renal blood flow regulation and maintenance of gastric mucosa (Takeuchi et al., 2001; Isakson, 1995). COX-1 is inhibited by traditional NSAIDs (Fig. 1) either reversibly or irreversibly, depending on the drug. The most representative of these drugs is aspirin that acts on the enzyme in an irreversible manner by covalently binding

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to a serine amino-acid residue of the enzyme. (ii) The COX-2 is an inducible form found in inflamed and neoplasic tissues (Seibert et al., 1994) and activated not only by various inflammatory (IL-1β, TNF-alpha, LPS), and mitogenic (phorbol esters) stimuli but also by growth factors (PDGF, EGF). However, recent data suggest a widespread localization of the COX-2 enzyme in the body (Watkins et al., 1999; Zimmermann et al., 1998; Komhoff et al., 1997), what cancels current opinions on the regulation of COX-2 activity. The COX-2 is expressed constitutively in human kidney and brain (Patrignani et al., 2005; Komhoff et al., 1997) and is inhibited by selective COX-2 inhibitors. Recently, a third form of the enzyme, COX-3, has been identified located mainly in the brain and the spinal cord (Chandrasekharan et al., 2002) and inhibited by acetaminophen. But there is still controversy regarding the real difference between COX-1 and COX-3, since the latter form is considered as a variant of COX-1 enzyme (Berenbaum, 2004). The two COX enzymes (COX-1 and COX-2) have about 80% homology and demonstrate the same affinity and capacity to convert AA to PGH2. Importantly, the amino acid residues for this conversion are conserved in both structures (Famaey, 1977). It should be noted that all NSAIDs target both COX enzymes at the same extent, implying that the clinical use of NSAIDs, in patients suffering of chronic or acute inflammation, will induce further adverse effects responsible of morbidity (Lanas, 2005). These findings led to the development of new drugs targeting the COX-2 enzyme with specificity and high selectivity. Several COX-2 inhibitors have been described (de Leval et al., 2000; Dannhardt and Laufer, 2000). They differ by the nature of the heterocyles and also by structural requirements needed for COX-2 selective inhibition.

Figure 2. Chemical structures of traditional NSAIDs and a polyphenolic antioxidant compound

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Chemical Structures of COX-2 Inhibitors Based on the in vitro and in vivo pharmacological data, nimesulide was thought to be a relatively selective COX-2 inhibitor (Famaey, 1997; Taniguchi et al., 1995; Glaser, 1995). It belongs to the sulphonamide family in which several other compounds (e.g. NS-398), structurally related were demonstrated to be selective COX-2 inhibitors (Fig. 3). These compounds also possess a methyl sulfonyl group that seems to be the hallmark of the selectivity (Dannhardt and Laufer, 2000; Famaey, 1997). The most attractive selective COX2 inhibitors that have been launched to treat rheumatic arthritis were the pyrazole derivative celecoxib (Celebrex®) and the furanone compound rofecoxib (Vioxx®) (for review see Lednicer, 2002; Kiefer and Dannhardt 2004). The changes in the chemical structure at the level of the carbocyclic or heterocyclic moiety with vicinal phenyl rings substituted by a methylsulfonylamino or an aminosulfonyl function have been thought to be essential pharmacophores for selective COX-2 inhibition as shown in the nimesulide’s family (Fig. 3). It rapidly appeared that the coxib’s family displayed a very high selectivity over the COX-2 form without any affinity for COX-1. The lead compound of this family is rofecoxib (or Vioxx®), launched by Merck in 1998 to treat rheumatoid arthritis. The other compounds, celecoxib (Celebrex®) and valdecoxib (Bextra®), were developed and introduced by Pfizer (Fig. 3). Up to now, other classes of molecules have been described as selective COX-2 inhibitors, including lumiracoxib and etoricoxib, which remain under consideration by the FDA (Fig. 3). The role of template is important for the efficacy in the COX-2 inhibitors’ family and is represented by heterocycles (pyrazole, furanone, oxazole).

Figure 3. Chemical structures of COX-2 inhibitors

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COX-2 and Intracellular Redox Processes There is a growing interest in the role of COX-2 inhibition in the inflammatory process of various diseases such as osteoarthritis, cancer and atherosclerosis (Hasegawa et al., 2005; Wang et al., 2005; Saha et al., 2003). During inflammation, there is an activation of different enzymes (NOS2, COX, LOX) and a production of inflammatory mediators such as prostaglandins (PGs), cytokines and reactive oxygen species (ROS). New insight on the activity of COX-2 is that the enzyme is involved in various oxidative processes that might cause pathological events through the redox signalling mechanisms. In this context, carcinogenis-associated events such as NF-kappaB activation and ROS production that were found to be triggered by cytokines were also related to an increased expression of COX-2 in a specific type of lung cancer (adenocarcinoma). NSAIDs and specifically COX-2 inhibitors have been described to modulate the intracellular redox processes either by a COX-2dependent or independent pathway: they are also implicated in cancer diseases by acting on the signalling pathways of Wnt and NF-kappaB pathways and on the LOX and NOS-2 enzymes.

COX-2 Inhibitors and Chemopreventive Actions A substantial literature exists demonstrating the elevation of COX-2 gene expression both in colon tumours and in epithelial cells adjacent to these sites of tumour initiation, suggesting that COX-2 misregulation plays a role in colon cancer initiation and progression (Hida et al., 2000; Kawamori et al., 1998). It has also been reported that PGE2 levels increased in colon cancer compared to normal mucosa (Rigas et al, 1993; Bennet et al, 1987). In recent studies, celecoxib was reported to induce apoptosis in prostate cancer cells by interfering with multiple signalling targets, including Akt, extracellular-regulation kinase-2 (ERK2) (Hsu et al., 2000). Celecoxib was also found to be efficient in the treatment of colorectal cancer, but acting by a COX-2-independent pathway. COX-2 is overexpressed in 45% of colon adenomas and 85% of colon carcinomas (Kawamori et al., 1998; Eberhart et al., 1994), but the COX-2 overexpression is not a dominant event in colon carcinogenesis. Furthermore, the possibility that the drug might inhibit targets other than COX-2 may have contributed to this effect (Eberhart et al., 1994). In addition, pharmacological studies suggest that anti-angiogenic effects may play a major role in the efficacy of COX-2 inhibitors on the prevention of colon and cancer lung progression (Masferrer et al., 2000), but Chuluda et al. demonstrated that both COX isoforms play similar roles in colon tumour development (Chuluda et al., 2000). The role of COX-2 in tumours and the efficacy of some COX-2 inhibitors as adjuvant chemotherapy constitute now a major investigative areas, even if there is still a debate to know whether the in vivo anticancer properties of COX-2 inhibitors are prostaglandin-mediated, PG-independent or both.

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COX-2 Inhibitors and Chronic Inflammatory Diseases: The Case of Atherosclerosis In these last ten years, COX-2 enzyme was implicated in inflammatory diseases, including atherosclerosis, which is now recognized as a chronic inflammation, and a major contributor to mortality and morbidity in the developed countries (Steinberg, 2002; Lusis, 2000; Baker et al., 1999; Ross, 1999). It has been reported that COX-2 was expressed within the atherosclerotic lesions in an animal model and that the COX-2 inhibitors stabilized the atherosclerotic plaque evolution (Hernandez-Presa et al., 2002; Burleigh et al., 2002). Macrophages play a crucial role in the formation of the foam cells, the first step in the formation of the plaque. According to the classical hypothesis, high plasma concentrations of cholesterol, particularly low-density lipoprotein (LDL) cholesterol, are recognized as one of the principal risk factors of atherosclerosis (Fruchart and Duriez, 2001; Chisolm and Steinberg, 2000; Ross, 1999; Steinberg, 1997). However, despite the changes in lifestyle and the use of pharmacological approaches to lower the plasma cholesterol, cardiovascular diseases still remain a huge public health problem. Beyond the commonly associated risk factors, infection agents have been proposed as potential triggers for the development and progression of atherosclerosis (Becker et al., 2001; Kowalski, 2001; Speir et al., 1998; Danesh et al., 1997). Among the infectious agents, Chlamydia pneumoniae (Cp) is largely studied, and an association was made between the presence of Cp antibodies, chronic coronary disease, and acute myocardial infarction on the basis of seroepidemiological studies (Saikku et al., 1988; Kalayoglu et al., 1999; Saikku, 2000). On the other hand, Cp was also reported as a possible contributing factor in asthma even if the mechanisms responsible for the persistence of this inflammatory response are only partially known (MacDowell and Bacharier, 2005). Treatment by macrolides to eradicate the Cp is still questioned in patients suffering of chronic asthma (Weinberger, 2004). The mechanism of action of Cp on the intracellular redox process is the subject of important research, not only for its priming action on the monocytes or other cells, but also for the possible induction of morphological changes (Yang et al., 2003). Recently, our group showed that the Cp-induced differentiation of THP-1 cells was accompanied by the triggering of respiratory burst, depending on the activity of the enzyme NADPH-oxidase (Mouithys-Mickalad et al., 2001). One year after, Yamaguchi et al. reported that Cp increased the enzymatic activity of human monocytes (Yamaguchi et al., 2002). In this report, we studied the in vitro effects of COX-2 inhibitors, in comparison with traditional NSAIDs (ibuprofen, diclofenac, aceclofenac) on two cellular models: a human lung type II alveolar cell line (A549) and a human promonocyte cell line (THP-1). We also investigated whether the Chlamydia pneumoniae-induced PGE2 release by A549 cells was inhibited in the presence of two COX-2 inhibitors (nimesulide and NS-398) or a well-known antioxidant resveratrol or both (combined effect). Taking into account that NSAIDs and especially COX-2 inhibitors may have other molecular targets than COX-2, we were interested to assess their potential capacity of modulating the enzyme activity. To reach this objective, the effects of the drugs on the activity of the myeloperoxidase (MPO) and on an enzymatic system (HRP/H2O2/ABTS), that generates the ABTS·+ radical cation, was investigated.

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2. Materials and Methods 2.1. Reagents Reagents were dissolved in 50 mM phosphate buffer saline (PBS, pH 7.4) or in analytical grade ethanol or DMSO (Merck, Belgium) for the NSAIDs. Analytical grade phosphate salts were from Merck (Belgium). The chemiluminescence’s enhancer lucigenin (2-Nmethylacridinium nitrate) and 2’,7’-dichlorofluorescin diacetate (DCFH-DA) were purchased from Aldrich (Belgium). NS-398 [N-(2) cyclohexyloxy-4-nitrophenyl) methanesulfonamide] was from Cayman Chemical Company (Ann Arbor, USA), and nimesulide [N-(2- phenyloxy4-nitrophenyl) methanesulfonamide] was graciously provided by Therabel-Belgium. The coxibs (celecoxib [(4-([5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1yl]benzenesulfonamide)], and rofecoxib [4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H) furanone]) and the traditional NSAIDs ibuprofen [ -methyl-4-(2-methylpropyl) benzeneacetic acid], diclofenac [2-(2,6-dichlorophenyl)amino benzeneacetic acid)], aceclofenac [(2[2,6-dichlorophenyl)amino] benzeneacetic acid carboxymethyl ester)] were generously provided by Professor B. Pirotte (Natural and Synthetic Drug Research Centre, University of Liege-Belgium). Aspirin (acetylsalicylic acid, ASA) and resveratrol (3,4’,5trihydroxystilbene) were from Aldrich (Belgium). Glucose, mercaptoethanol, HEPES, sodium pyruvate and horseradish peroxidase (HRP) were from Calbiochem (Germany); the HRP working solution was prepared daily. Antibiotics were obtained from Sigma (Belgium). Culture medium MEM and RPMI were from Gibco (Invitrogen-Life Technologies, Belgium). Foetal bovine serum and glutamine were from Cambrex BioSciences (Verviers, Belgium). Phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and 2,2’-Azinobis-(3ethylbenzothiazoline-6-sulfonate (ABTS) were purchased from Aldrich (Belgium). Amplex red (10-acetyl-3,7-dihydroxyphenoxazine) was from Molecular Probes (Belgium). All chemicals were of analytical grade. For the ELISA and SIEFED technique of equine myeloperoxidase (MPO), the coating buffer was 20 mM phosphate buffer (pH 7.4), the diluting buffer was the coating buffer with 5 g/l BSA and 0.1% Tween 20 and the washing buffer was a solution of 154 mM NaCl with 0.1% Tween 20. Equine MPO was purified from equine neutrophils as previously described (Franck et al., 2005).

2.2. Cell Culture 2.2.1. Chlamydia Pneumoniae Culture Chlamydia pneumoniae (Cp) strain (TW-183) were obtained after a 92 h culture in MacCoy cells (BioWhittaker, Europe) as previously described (Mouithys-Mickalad et al., 2001). After infection and 92 h culture, Mac-Coy cells were split in fresh culture medium, and after centrifugation, the supernatant containing the Cp was conserved in sucrose phosphate buffer in liquid nitrogen.

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2.2.2. Human Monocyte Cells (THP-1) Culture The human promonocytic cell line THP-1 (American Type Culture Collection; Rockville, MD, USA) was cultured in RPMI 1640 supplemented with 5% foetal bovine serum, 2 mM Lglutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 0.25% glucose (2.38 mg/ml), 1.1 µg/ml sodium puryvate, 36 µl of pure mercaptoethanol and 5 ml HEPES (10 mM), at 37°C in a 5% CO2 incubator. For assays, the monocyte culture was centrifuged (450 g, 10 min), the supernatant was decanted and cells were counted and distributed in culture medium. For fluorescence assay, 1x106 cells/assay were put in multi-well plates. THP-1 cells differentiation into macrophages was achieved after an incubation period of 19 h with either C. pneumoniae (1.5 to 2 pg endotoxin/106 cells) or 10-8 M PMA. The preincubation/differentiation of the cells was performed in the absence or presence of the drugs (NS-398, nimesulide, resveratrol, recofecoxib, celecoxib, ibuprofen acetyl salicylic acid, aceclofenac and diclofenac) at the final concentration of 10 and 100 µM. After preincubation, the macrophages were stimulated with 5.10-7 M PMA. The potential toxicity of a 19 h incubation of the drugs together with cells was assessed by light microscopy counting of the viable cells after staining with trypan blue. 2.2.3. Human Epithelial Alveolar Cells (A549) Culture Epithelial lung adenocarcinoma A549 cell line (ATCC Rockville, MD, USA) was cultured in MEM supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 µg/mL) and amphotericin (0.1 µg/mL). This cell line has the characteristics of the type II pneumocytes. Confluent cells were used after cell growth on plastic dishes in a humidified atmosphere (5% CO2, and 95% air) at 37°C. For the experimental assays of PGE2 release with or without stimulation with LPS, A549 cells were distributed in the wells of the six-well microplates and incubated for overnight with Cp with or without the tested drugs. After this incubation, the supernatant was collected for PGE2 measurement.

2.3. Neutrophils Isolation Equine neutrophils were isolated from citrated (5.26 g/l sodium citrate) fresh blood drawn from the jugular vein of healthy horses, by centrifugation at room temperature on a discontinuous density gradient (polymorphoprep, Axis-Shield PoC AS, Oslo, Norway). The cells were re-suspended in 20 mM PBS (pH 7.4).

2.4. Measurement of ROS Production in Cellular Systems 2.4.1. Fluorescence Assays on THP-1 Cells The assay is based on the DCFH oxidation, induced by oxidant species produced within the cells: DCFH-DA is known to cross the cell membrane and to generate DCFH that can be oxidized into dichlorofluorescein (DCF) by intracellular oxidant species (LeBel et al., 1992). The DCFH-DA stock solutions were prepared by dissolving 2 mg of DCFH-DA probe in 2

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ml ethanol, aliquoted and kept at –20 °C (Silveira et al., 2003). THP-1 cells were cultured on the 24-well microplates. After pre-incubation/differentiation with or without drug addition, centrifugation and supernatant removal, the cells (1x106 cells/assay) were added with 20 µl DCFH-DA (40 µM), 15µl HRP (4.5 µg/ml) and stimulated with 5.10-7 M PMA in phosphate buffer at pH 7.4 (final volume of 1 ml). The excitation wavelength was 555 nm and the fluorescence was monitored at 485 nm, at 37 °C for 30 min with a Fluoroskan Ascent device (Thermolab Systems, Belgium). 2.4.2. Chemiluminescence (CL) Assay on Equine Neutrophils The CL assay was performed according to the method previously described by Benbarek et al, with minor modifications (Benbarek et al., 1998). The chemiluminescence assay, with lucigenin as enhancer, was measured with the Fluoroskan Ascent (Thermo Labsystems) equipped with scintillation and luminescence counter. After neutrophil isolation, the cell suspension (106 cells/200 µl PBS) was incubated with the drug for 10 min, at the final concentrations of 10-4, 10-5 or 10-6 M, directly in the 96wells micro-plate. To this suspension were added, 25 µl CaCl2 (7,5 µM), and 2µl lucigenin (5µM). The CL measurement started with the addition of 10 µl PMA (5x10-7 M final concentration) in 200 µl PBS buffer. The temperature was kept at 37°C. The CL response was expressed in arbitrary units (either as the peak value or as the integral value of the total emission of CL).

2.5. Measurement of PGE2 Produced by A549 Cells A549 cells (5x105 cells/assay) were cultured in 6-well culture plates, incubated during 30 min with the tested drug at the final concentration of 10-5 M and further incubated with C. pneumoniae for 19 hours. Controls were made by adding 20 µl DMSO (1% v/v) to the culture milieu. The medium was then removed and stored at –20 °C until analysis. PGE2 was assayed by using the PGE2 enzyme immunoassay (ELISA) kit according to the procedure described by the manufacturer (Cat No. 514010, Cayman Chemical Company, Ann Arbor-USA).

2.6. Measuring of the Equine MPO by ELISA The total MPO concentration release by activated PMNs was measured by an ELISA method, developed by Franck et al. (Franck et al., 2005) and distributed by Biocode Hycel (Liège, Belgium). Anti-equine MPO polyclonal antibodies obtained from rabbit were coated on 96-wells). Samples (100 µl) or dilutions of the standard equine MPO were added into the wells and incubated overnight at 4°C. After washing, an anti MPO polyclonal antibody obtained in guinea pig, coupled to alkaline phosphatase, was added and incubated 2 h at 37°C. After washing, the phosphatase activity was detected by incubation (30 min, 25°C, in the darkness) with a paranitrophenyl phosphate solution. The reaction was stopped and the absorbance (405 nm) was read with the Multiscan Ascent (Labsystem).

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The samples used for MPO measurement were obtained as follows: equine neutrophil suspensions (106 cells/ml) were incubated for 10 min at 37 °C with the drug at the final concentration of 10-4, 10-5 or 10-6 M, and then activated with 8 x 10-7 M PMA for 30 min at 37 °C. After the activation, the cell suspension was centrifuged (450 x g, for 10 min), the supernatant was collected and used for ELISA measurement.

2.7. Cell-Free Systems 2.7.1. Evaluation of the Antioxidant Capacity of COX-2 Inhibitors Versus Traditional Nsaids The enzymatic system 100 µg/ml HRP/0.5 mM H2O2/450 µM ABTS was used. The compound I resulting from the reaction of HRP with H2O2 induced the formation of the ABTS radical cation (ABTS·+) with a maximal absorbance at 690 nm. The antioxidant capacity of the drug under investigation was evaluated by following the absorbance intensity of the ABTS oxidation. The tested compounds were used at the final concentrations of 2.10-4 and 2.10-5 M and added just after the reaction has been started by 5 µl H2O2 addition (500 µM final concentration). All measurements were carried out at room temperature with the Fluoroskan Ascent device (Thermolab Systems, Belgium). 2.7.2. Measurement of the MPO Activity by SIEFED The measurement of equine MPO activity in the presence or absence of the drug was conducted by an original method, the SIEFED (“Specific Immunological Extraction Followed by Enzymatic Detection”), recently developed by Franck et al. (Franck et al., 2005). Briefly, the method consists in the capture of MPO from biochemical or biological samples by specific immobilized antibodies, followed by a washing (elimination of proteins or interfering molecules) and a direct detection of the MPO activity. Black microtitration wells (Cliniplate EB, Thermo Labsystems) were coated (overnight, at 4 °C) with 600 ng/well of rabbit-anti MPO IgG. Equine MPO standard (ranging from 0.25 mU/ml to 6.4 mU/ml) and samples containing MPO were added (200 µl) to the microplate and incubated 2 h at 37°C. After 3 washings, the peroxidase activity of MPO was detected by adding 100 µl of a 40 µM Amplex red, freshly prepared in phosphate buffer (50 mM) at pH 7.5 containing 10 µM H2O2 and 10 mM nitrite. After incubation in the darkness (30 min, 37°C), the fluorescence was measured with a Fluoroscan Ascent (Thermo Labsystems) at the excitation and emission wavelengths of 544 and 590 nm respectively. The fluorescence value was directly proportional to the quantity of active MPO in the sample. COX-2 inhibitors and traditional NSAIDs stock solutions in DMSO or ethanol were diluted with the SIEFED diluting buffer and used at the final concentrations of 10-4, 10-5 or 10-6 M. Drugs were incubated with 20 ng/ml equine MPO for 10 min at room temperature. Controls were made with MPO alone. Statistical analysis. Data were expressed as means ± SD and performed by Student’s t test to determine the statistical significance between drugs or control (cell + vehicle). A value of p < 0.05 was considered statistically significant.

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3. Results 3.1. Cellular Systems 3.1.1. Effect of COX-2 Inhibitors on the ROS Production by the Undifferentiated THP-1 Cells Figure 4 shows the effect of the COX-2 inhibitors on the production of ROS by 1.106 THP-1 cells, activated with 5.10-7 M PMA. We observed that the fluorescence intensity was very weak in the absence of HRP and moderate in the absence of PMA. When the cells were treated with the drug (nimesulide) at 10-4 or 10-5 M, a decrease of the fluorescence intensity was observed (20% and 10% inhibition respectively), compared to the control with ethanol (drug vehicle) (Fig. 4A). The nimesulide analogue NS-398 had a very weak inhibiting effect at 10-4 M but an increasing effect at 10-5 M. For the coxib’s family, Vioxx® at both concentrations induced a concentration-dependent inhibiting effect (20% and 15% inhibition respectively) (Fig. 4B). Celebrex® at 10-4 M reduced the fluorescence amplitude by 50%, compared with the DMSO control (Fig. 4B). In contrast, the traditional NSAID, ibuprofen, slightly increased the fluorescence.

Figure 4. Effects of COX-2 inhibitors on ROS produced by 1x106 undifferentiated THP-1 cells stimulated with 5x10-7 M PMA. Oxidant (H2O2)-induced DCFH oxidation was monitored by fluorescence technique at 37°C for 30 min, in the presence of 15 µl HRP (45 µg/ml). Drugs were used at 10-4 M (white column) and 10-5 M (grey column). Results are means ± SD (n = 4). C: control (no drugs); EtOH: ethanol (drug vehicle); NIM: nimesulide. *p<0.01 and **p<0.001 by Student’s t test. Comparison of NIM with NS-398 or Celebrex with Vioxx and ibuprofen respectively

3.1.2. Effect of COX-2 Inhibitors on the ROS Production by THP-1 Cells Differentiated by PMA Figure 5 shows the effects of the drugs on THP-1 cells differentiated into macrophages by treatment with low dose of PMA (10-8 M). The effects of COX-2 inhibitors and two NSAIDs at 10-5 M on the ROS produced by the macrophages after an instantaneous stimulation with either 5.10-7 M PMA (Fig. 5A) or 3 µg/ml LPS (Fig. 5B) were investigated. In both models, neither the COX-2 inhibitors nor the two traditional NSAIDs ibuprofen and ASA had a significant effect, except for Celebrex® that exerted a weak inhibition on PMA-

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activated differentiated cells. We observed that most of the tested compounds rather enhanced the fluorescence intensity.

Figure 5. Effects of COX-2 inhibitors on ROS produced by PMA-induced differentiation of THP-1 cells, after activation with either 5x10-7 M PMA (part A) or 3 µg/ml LPS (part B). The modulating effects of the COX-2 inhibitors were compared to traditional NSAIDs. The fluorescence was monitored at 37°C for 30 min, in the presence of 15 µl HRP (45 µg/ml) in PBS (pH 7.4). 40 µM DCFH were preincubated for 1 h and removed before the stimulation with the activator agent. Results are means ± SD (n = 3)

3.1.3. Effect of COX-2 Inhibitors in THP-1 Cells Differentiated by Chlamydia Pneumoniae Figure 6 compares these effects on macrophages that were further stimulated with either 5.10-7 M PMA (Fig. 6A) or 3 µg/ml LPS (Fig. 6B), in the presence or absence of the drug. On the PMA-stimulated differentiated THP-1 cells, a decrease of the fluorescence intensity was observed with 10-5 M NS-398 (20 % inhibition), nimesulide (40 % inhibition), resveratrol (15%), Celebrex® (12%) and Vioxx® (18%). In this model, ASA rather increased the fluorescence intensity (Fig. 6A). The combination of the COX-2 inhibitor with resveratrol did not significantly change the effect obtained with the drug alone. In contrast to the PMA-stimulated macrophages, the stimulation of the differentiated THP-1 cells with 3 µg/ml LPS led to different results. All the drugs tested in this model showed an inhibiting effect on the ROS production (Fig. 6B). The combination of the drug with resveratrol led to a little or no effect or even a suppression of the drug inhibiting effect for Celebrex® and Vioxx®. In this model, 10-5 M ASA (P10) strongly decreased the fluorescence intensity.

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Figure 6. Effects of COX-2 inhibitors on the ROS production by Cp primed THP-1 cells 1x106 and comparison of the cellular activation with either 5x10-7 M PMA (part A) or 3 µg/ml LPS (part B). The potential inhibiting or triggering effect of COX-2 inhibitors was compared to that of traditional NSAIDs. The fluorescence was monitored at 37°C for 30 min, in the presence of 15 µl HRP (45 µg/ml) in PBS (pH 7.4). 40 µM DCFH were pre-incubated for 1 h and removed before the stimulation with the activator agent. Results are means ± SD (n = 3). C: control (no drug)

Figure 6C. Light microscopic observations of THP-1 cells (1), THP-1 cells transformed into macrophages by pre-incubation with 10-8 M PMA (2) or Chlamydia pneumoniae (3) 1. flotting cell, round-shaped cell 2. transformed cell (apoptotic process) 3. adherent macrophage with a characteristic spiked-aspect

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On the figure 6C is shown the effects of pre-incubation of THP-1 cells with Cp or PMA. The cells pre-treated with PMA underwent transformation characteristic of apoptosis or necrosis, while the cells pre-treated with Cp were transformed into well-defined macrophages. 3.1.4. Modulation of PGE2 Production in Cp Infected A549 Cells by COX-2 Inhibitors The figure 7 shows that the pre-incubation of A549 cells with Cp for 19 h resulted in a strong production of PGE2 compared to the control (A549 without Cp). The PGE2 release when the cells were activated was about 2 times higher than that of non-activated cells. When the cells were pre-incubated together with the drug, at the final concentration of 10-5 M, a dramatic decrease of the PGE2 release was observed compared to vehicle (10 µl DMSO, 1% v/v). It should be noted that DMSO induced an inhibiting effect but less pronounced. Similar decrease was observed with NS-398 and resveratrol. We then investigated the effect of the pre-incubation of A549 cells together with a combination of 10-5 M nimesulide with 10-5 M resveratrol (or NS-398 + resveratrol) on the production of PGE2. The combination of NS-398 with resveratrol had an additional reducing effect on the PGE2 release of PMA-activated cells, while the other combination (nimesulide + resveratrol) was without significant additional effect (Fig. 7).

Figure 7. Production of PGE2 by Cp infected alveolar cells (A549). (A): PGE2 production by 5x105 infected cells and modulating effect of COX-2 inhibitors. (B): PGE2 production by 5x105 infected cells, after activation by PMA and effects of COX-2 inhibitors. The combined effect of the drug with resveratrol was also studied. Experiments were made in duplicate each (n = 2)

3.1.5. Modulator Effect of COX-2 Inhibitors on the CL of the Activated Neutrophils Figure 8 summarizes the effect of COX-2 inhibitors on the CL produced by activated neutrophils. The effect of the drug was studied on the stimulated and non-stimulated neutrophils. Controls were made in the presence of ethanol (Fig. 8A) or DMSO (Fig. 8B). Ethanol had no effect on the CL response while DMSO increased the CL of PMA-stimulated

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neutrophils. The CL produced by the non-stimulated PMNs was increased by ibuprofen, aceclofenac and diclofenac, but not by nimesulide and its analogue NS-398 (Fig. 8A). The CL produced by the PMA-stimulated PMN was inhibited by NS-398 or nimesulide, at the concentration of 10-4 and 10-5 M for NS-398 and at 10-4 M for nimesulide. At both concentrations, the traditional NSAIDs (ibuprofen, aceclofenac and diclofenac) enhanced the CL response. On figure 8B, the two coxib’s derivatives are compared to Tiron (P8) and resveratrol (P9). At 10-4 M celecoxib seemed strongly inhibitor, but this effect was due to cell death as assessed by the cell viability test. Nevertheless, at 10-5 M celecoxib induced a weak decrease of the CL response, but this effect seemed not to be in connected to its toxic effect. Rofecoxib (Vioxx®) caused 50% of inhibition at 10-4 M but only 10% inhibition at 10-5M (Fig. 8B).

Figure 8. Lucigenin-enhanced CL of stimulated equine neutrophils and effects of Cox-2 inhibitors compared to traditional NSAIDs and two antioxidant compounds (Tiron and resveratrol). The CL response was monitored for 30 min. Each assay was perfomed in triplicate (n ≥ 3). *p<0.01 and **p<0.001 versus control (cell + vehicle)

3.1.6. Effect of COX-2 Inhibitors on the Release of MPO by Neutrophils Figure 9 shows the effect of the COX-2 inhibitors on the MPO release by PMA-activated PMNs, in comparison with traditional NSAIDs. The drugs had no effect on the MPO release by non-activated PMNs (Fig. 9 A and B, left part). On activated neutrophils, ethanol in the milieu had a slight reducing effect on the MPO release, and the drugs only had slightly inhibiting effects, except for ibuprofen which was strongly inhibitor at 10-4 M (48%) but not at 10-5 M (Fig. 9A, right part).

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Figure 9. Measurement of MPO released in the medium by non-activated or PMA-activated equine neutrophils and effects of COX-2 inhibitors and NSAIDs. 1x106 PMNs were stimulated with 5x10-7 M PMA in PBS (pH 7.4). Each assay was performed in triplicate (n = 3)

Regarding the effects of the coxib’s derivatives on the MPO release (Fig. 9B), only Celecoxib presented a reducing activity at 10-4 M, compared to the control with DMSO vehicle. The addition of 10µl DMSO (1%, v/v) resulted in a little increase of the MPO release. For the two antioxidant molecules, only tiron at 10-4 M reduced the MPO release by 15%.

3.2. Cell-Free Systems 3.2.1. Evaluation of the Radical-Scavenging Activity of COX-2 Inhibitors on the ABTS·+ Radical Formation COX-2 inhibitors were compared to traditional NSAIDs and a phenolic antioxidant resveratrol. The use of H2O2 (0.5 mM) and HRP (100 µg/ml) allowed the formation of oxoferryl species [P-Fe(IV)=O], with subsequent electron transfer from ABTS substrate, resulting in the formation of the ABTS·+ radical cation. The ABTS·+ control was obtained in the absence of COX-2 inhibitors or NSAIDs (Fig. 10). At the concentrations of 10-4 and 10-5 M, resveratrol (P8) was inhibitor (90% and 20% inhibition, respectively). Aceclofenac (P4) and diclofenac (P5) also exhibited a good radical-scavenging capacity at 10-4 M (40% and 39%, inhibition respectively). In contrast, NS-398 (P1) and Celebrex® (P6) only had displayed little effect while nimesulide (P2), ibuprofen (P3) and Vioxx® (P7) were without any radical-scavenging effect even at 10-4 M. None of the studied drugs increased the formation of ABTS·+ radical cation. The efficacy of the compounds tested in this model can

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be summarized as follows: resveratrol > diclofenac > aceclofenac > celebrex > NS-398 > nimesulide = vioxx.

Figure 10. Evaluation of the radical-scavenging capacity of COX-2 inhibitors on an enzymatic model (100 µg/ml HRP/ 0.5 mM H2O2/ 450 µM ABTS) in phosphate buffer (PBS) pH 7.4. The ABTS·+ radical was followed at 690 nm in the presence of the drug at two final concentrations. An antioxidant drug, the polyphenolic molecule resveratrol was included in this study for comparison. Results are means ± SD (n = 3)

3.2.2. Modulation of the MPO Activity by COX-2-Inhibitors and Traditional NSAIDs The stock solution of the drug was made either in ethanol or DMSO. Both vehicles were tested at the two different concentrations. For comparison, two well-known antioxidant compounds (resveratrol and tiron) were also studied. The addition of ethanol to the reaction mixture led to a strong inhibiting effect on the MPO activity, while DMSO had only little effect. In this context, the potential modulating capacity of the drug was compared to its appropriate control (Fig. 11). Nimesulide and ibuprofen at 10-4 M showed an inhibiting effect of 30% and 50% respectively, but increased the MPO activity at 10-5 M. NS-398, aceclofenac and diclofenac had no effect at 10-4 M but increased the MPO activity at 10-5 M. For the experiments carried out with DMSO as vehicle solution, a strong inhibiting effect on the MPO activity was observed for resveratrol at both tested concentrations (80% and 60% inhibition). Tiron at 10-4 M inhibited the activity of MPO by 50% but was without effect at 10-5 M. Vioxx® exhibited a concentration-dependent inhibiting effect on the MPO, but Celebrex® at 10-4 M only had a little inhibiting effect (5%) and even increased the MPO activity at 10-5 M (Fig. 11).

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Figure 11. Effect of COX-2 inhibitors, NSAIDs and two antioxidant drugs on the equine MPO activity measured by a specific enzymatic assay (SIEFED). The enzymatic assay was performed with MPO, in the presence of 10 µM H2O2 and nitrite. Controls were made with either ethanol or DMSO at two final concentrations. Each assay was performed in triplicate (n = 3)

4. Discussion Since the withdrawal of one of the most popular coxib’s derivative, Vioxx®, followed by the two others, Celebrex® and Bextra®, concerns regarding the safety of the COX-2 inhibitors due to adverse cardiovascular events have been raised (FitzGerald, 2004; Mukherjee et al., 2001). However, despite these concerns, the use of COX-2 inhibitors still remains a subject of intense debate (Wang, 2005), and the clinicians working in the cancer area think that COX-2 inhibitors still present a huge benefit for the treatment of cancer and even for other chronic diseases. They also think that COX-2 inhibitors might be used, but only in patients with low cardiovascular risks. On the other hand, the new insight is that COX-2 inhibitors may act on various molecular targets, including the signalling pathways, cytokines and reactive oxygen species. This hypothesis is sustained by several studies which have shown that the COX-2 inhibitor Celebrex® exerted its anticancer effect both on COX-2-dependent and independent pathways (Buecher et al., 2005; Kashfi and Rigas, 2005). Our results in cellular models (undifferentiated THP-1 cells) indicated that COX-2 inhibitors only had a weak inhibiting effect on the ROS production by the stimulated cells, except for Celebrex®, which seemed to exhibit a strong inhibitor effect, but due to a toxic effect, showing massive cell death as evidenced by the Trypan blue assay. We also showed that in the PMA-differentiated cell model, the drugs tested had no real effect, independently of the stimulating agent, but the priming of THP-1 cells by PMA induced the cell apoptosis or necrosis (Fig. 6B), and in this model, the infection of THP-1 cells by Cp did not cause necrosis but favoured the conversion of monocytes into macrophages. We have already shown that the infection of monocyte by Cp was accompanied by an increased oxidative activity, through the NADPH oxidase pathway (Mouithys-Mickalad et al., 2001; Mouithys-Mickalad et al., 2004). Yamaguchi et al. also observed an increased

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enzyme activity when human monocytic cells were infected by Cp (Yamaguchi et al., 2002). These results were in agreement with the hypothesis of a cellular oxidation of the LDL by cytokines or oxidant species produced during chronic inflammation (Cathcart, 2004; Boivert, 2001; Nguyen-Khoa et al., 1999). Our study also demonstrated that the use of LPS, to stimulate the Cp-differentiated THP1 cells, led to a good inhibiting profile of the ROS production, for the overall tested drugs, including resveratrol; but in the model using PMA as stimulator the inhibiting profile was less pronounced. In addition, we showed that in the presence of LPS, the combination of the COX-2 inhibitor, NS-398, with resveratrol led to an additional inhibiting effect, what we explained by difference in the action of the two stimulators, since LPS acted through the receptor pathway and PMA directly on the protein kinase C (PKC). These results might have a biological relevance in the new insight that NSAIDs modulate various molecular targets, including cytokines and oxidant species production during chronic inflammatory diseases. Moreover, there is a strong link between the chronic inflammation and some diseases such as atherosclerosis, prostate and colorectal cancers or acute lung injury (Wang et al., 2005; Hernandez-Presa et al., 2002; Lusis, 2000; Becker et al., 1999; Speir et al., 1998). In this context, the role of infectious agent in chronic inflammatory diseases appears crucial and Chlamydia pneumoniae has been involved in atherosclerosis, lung pulmonary infection and in asthma (Webley et al., 2005; Branden et al., 2005; Danesh et al., 1997). Despite the efficacy of antibiotics in certain situations, their use to eradicate the pathogens in the chronic inflammation still remains a huge health public problem, and therefore other therapeutic approaches are needed. More recently, Lee and co-workers demonstrated that the expression of COX-2 enzyme by human A549 non-small lung cancer cells, activated by IFN-α, was inhibited in the presence of curcumin, a phenol antioxidant (Lee et al., 2005). But the production of PGE2 by Cp-infected A549 cells has never been studied in the presence of the COX-2 inhibitors. We first investigated whether Cp infection of A549 cells induced the PGE2 production and if the use of COX-2 inhibitors could inhibit this production. We found, a strong PGE2 production by Cp infected of A549 cells, and this production was more important when the infected cells were further stimulated with PMA. Interestingly, the two COX-2 inhibitors, tested at the therapeutic plasmatic concentration of 10 µM, reduced the production of PGE2. We observed a similar decreasing effect on PGE2 production when resveratrol was used at low concentration, and that the combination of NS-398 with resveratrol led to an additional inhibiting effect. This would mean an implication of the protein kinase (PK)-C pathway in the production of prostaglandins. These preliminary results demonstrated that the use of the preferential COX-2 inhibitor, NS-398, in association with an antioxidant resveratrol might be of therapeutical interest, even if more investigations are needed to prove their potential efficacy. Our findings are in agreement with the recent study by Yang et al. who reported the induction of pro-inflammatory cytokines in human lung epithelial cells during Cp infection, and showed that the cyto-adherence of Cp to cells was important in the induction of the host cytokine responses (Yang et al., 2003). In our cellular model, the infection was carried out by a simple incubation of the A549 cells with Cp without centrifugation. Taking into account that NSAIDs are also known to inhibit the neutrophil activation by different ways (Bevilacqua et al., 1994; Abramson et al., 1990; Umeki, 1990), and that during inflammation

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there is an increased MPO release by activated neutrophils, we studied the interactions of the COX-2 inhibitors and the traditional NSAIDs with MPO release and MPO activity. Myeloperoxidase (MPO) contributes to the host defence by killing the exogenous pathogens (Deby-Dupont et al., 1999). There are a growing number of studies dealing with the role of MPO in the inflammatory diseases, including atherosclerosis (Podrez et al., 2000; Nambi, 2005). Since COX-2 enzyme is increased in the atherosclerotic lesions and MPO is also found within these lesions, we investigated the potential inhibiting effect of COX-2 inhibitors on the enzyme activity and compared COX-2 inhibitors with traditional NSAIDs and two antioxidant compounds (resveratrol and tiron). We first showed that, in the model of activated neutrophils, COX-2 inhibitors decreased the lucigenin-enhanced CL response while the traditional NSAIDs rather increased it. These results are in disagreement with other literature data (Parij et al. 1998), but can be explained by the nature of the enhancer used to investigate the CL assay. Indeed, if luminol or DCF can readily react with reactive species produced within the cells (Rota et al., 1999; Li et al., 1999), conversely, lucigenin, at low concentration does not undergo redox cycling. To control the absence of redox cycling artefacts, we tested the COX-2 inhibitors or NSAIDs reaction with the chemiluminescent probe (lucigenin) alone and with non-stimulated neutrophils. Surprisingly, we found that traditional NSAIDs increased the CL response of the non-stimulated PMNs. These results suggest a direct reaction of NSAIDs with lucigenin, increasing so the CL response, or the drugs can stimulate the ROS production through the NADPH oxidase pathway. MaffeiFacino and co-workers and our group had already observed that the COX-2 inhibitor nimesulide and its main analogue, 4’hydroxy-nimesulide, inhibited the ROS production both in vitro and in the cellular models (Maffei-Facino et al., 1995; Mouithys-Mickalad et al., 2000). In this model, resveratrol, but not Tiron at 10-4 M, strongly decreased the CL response. But, in contrast to the CL results, most of the COX-2 inhibitors remained without effect on the enzyme (MPO) release, except for Vioxx® which had a decreasing effect. Our results clearly indicate that COX-2 inhibitors were inhibitor of the ROS production, but were without effect on the MPO release. In order to better understand the mechanisms of action of the COX-2 inhibitors towards the ROS production during the oxidative processes, two enzymatic systems (cell-free systems) were investigated. We observed that the formation of the ABTS* radical, from the HRP/ABTS/H2O2 system, was strongly inhibited by resveratrol. The two traditional NSAIDs, aceclofenac and diclifenac also showed a good radical scavenging capacity, but the COX-2 inhibitors showed almost no effect, except NS-398 and Celebrex® which were weakly inhibitors. Similarly, in the second enzymatic (MPO) system, the COX-2 inhibitors were weakly inhibitor of the activity of the enzyme, but rather increased the enzyme activity at the low concentration, except for Vioxx®. Once again, resveratrol and Tiron to a lesser degree, showed a good inhibiting profile on the MPO activity.

5. Conclusion Overall, the cellular results presented in this report indicate that the COX-2 inhibitors are moderately active on the intracellular oxidant processes, but modulator effects on the

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prostaglandins (PGE2) and ROS production by infected cells. Our cellular observations also demonstrated that the combination of a COX-2 inhibitor with resveratrol might be of therapeutic interest, especially for the treatment of chronic inflammatory diseases, and particularly when infectious agent is involved. We also observed, by using cell-free systems, that the tested drugs were only weakly inhibitor on the enzymatic activity of myeloperoxidase and had no scavenging capacity on radicals derived from horseradish peroxidase activity. Despite concerns raised by the observations of increased adverse cardiovascular events when the drugs of coxib’s family are used in humans, the research in the area of the COX-2 inhibitors will continue. We think that the use of selective preferential COX-2 inhibitors such as nimesulide and its related derivatives, which already keep some activity on the COX-1 enzyme, will represent an alternative to avoid the cardiovascular risks described for the other coxibs. Anyway, the coxib’s molecules would continue to be given to patients with low cardiovascular risks, and their role in the chemoprevention of the colorectal and prostate cancers is more and more growing.

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In: COX-2 Inhibitor Research Editor: Maynard J. Howardell, pp. 209-235

ISBN 1-59454-994-X © 2006 Nova Science Publishers, Inc.

Chapter VI

Theoretical Mechanism Studies on Dual Inhibition of Human Cyclooxygenase-2 and 5-Lipoxygenase by DiarylPyrrolizine Derivatives R. Pouplana∗, C. Pérez and J. Ruiz Departament de Fisicoquímica. Facultat de Farmàcia, Universitat de Barcelona, Spain

Abstract For this paper we are modelled the active site of the human 5-LOX on the basis of the X-ray coordinates obtained for the rabbit 15-lipoxygenase and introduced in a dynamic approach the diaryl-pyrrolizine inhibitor compounds. Also we are modelled the binding mode for these compounds in the active site of the human COX-2. The binding mode on the COX-2 proposed for 6-7-diaryl-2,3— dihydropyrrolizine derivatives compounds have been shown a major anchor point defined by residues Tyr-355, Val-523, His-90, Gln-192, and Arg-513. Another mode of interaction for Licofelone inside the COX-2 active site was the polar moiety carboxylic group lying in the proximity of Tyr-385 and Ser-530. The binding mode on 5-LOX proposed for these compounds inserts the “COX fragment” deep in the cavity with the methylsulfonyl moiety at the bottom, interacting with Gln-413, Lys-423 and Asn-425. The “5-LOX part” fills the entrance of the active site interacting with Phe-421, Leu-414 and Gln-363 and also forms a salt bridge with the carboxylic oxygen (licofelone) and Lys-423 and Gln-413. All of these drugs do not present a selective COX-2 inhibition and the future clinical data of compounds, such as licofelone and 6-7-diaryl-2,3-dihydropyrrolizine derivatives, could point out the interest of a balanced inhibition of the two COX isoforms, associated with the blockade of the 5-LOX pathway. ∗

Departament de Fisicoquímica. Facultat de Farmàcia. Universitat de Barcelona, Av. Diagonal 643, Barcelona 08028, Spain. Email: [email protected]

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It appears that selective COX-2 inhibitors do not fully satisfy the search for new safer antiinflammatory agents. The 5-LOX pathway, which generates products particularly important in inflammation, is up-regulated during COX blockade and is thus potentially responsible for undesirable adverse effects, such as asthma. Both are key enzymes involved in the arachidonic acid (AA) cascade, leading to important bioactive fatty acids known as eicosanoids and leukotrienes (LTs), respectively. Dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) is, therefore, an interesting alternative to provide safer NSAIDs. In addition, both COX-2 and 5-LOX enzymes have been involved in the development and progression of numerous types of cancer such as pancreatic, lung, colorectal, prostate. So, the use of dual inhibitors opens up new perspectives in the prophylactic treatment of this dreadful disease. In regard to its characteristics and mechanism of action, several new strategies have been considered, notably the dual inhibition of COX-2 and 5-LOX. Several pyrrolizine derivatives possess a dual inhibitory activity. Licofelone inhibits 5LOX and preferentially COX-1, it does not cause any GI damage and compound 6-7-diaryl2,3--dihydropyrrolizine, combine the structural requirements for COX-1/COX-2 and 5-LOX inhibition, with a higher selectivity towards COX-2. Unlike most of the described compounds, they are non-redox competitive inhibitors, which compete with AA to bind the enzyme active site. The emphasis will be focused on structure-function relationships with a view to delineating the influence of key COX/LOX binding site groups on cell proliferation inhibition and/or apoptosis induction.

Introduction New studies on relationships between polyunsaturated fatty acid metabolism and carcinogenesis have led to the identification of new molecular targets in cancer chemoprevention research. These targets include arachidonic acid (AA) metabolizing enzymes such as cyclooxygenases (COXs) and lipoxygenases (LOXs), which lead to the formation of various eicosanoids involved in a variety of human diseases, such as inflammation, fever, arthritis, and, more recently discovered, cancer.[1-4] The implications of COXs and LOXs have been discussed in numerous types of cancers, including colon, pancreas, breast, lung, skin, urinary bladder, or liver cancers, but inhibiting these enzymes seems to be even more promising to halt or reverse the progression of prostate cancer. In fact, because of the unavailability of effective systemic therapies, this cancer is usually fatal once the tumour cells invade the outer area of the gland. Recent data has demonstrated the involvement of COX-2 in both in vitro proliferation and in vivo tumour growth rate. [5-9] Other works have highlighted the role played by COX2 in disturbing the balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) in prostate cancer cells, indicating the potential use of COX inhibitors in the prevention and therapy of prostate cancer invasion.

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Moreover, dynamically evolving research shows the different roles of LOXs and their metabolic products in carcinogenesis and chemoprevention. Many compounds widely used in the treatment of pain and inflammation, such as celecoxib, rofecoxib, zileuton, and indomethacin, have been tested in vitro and in vivo on cell growth and non-necrotic cell death. However, the signalling mechanism used by COX2/LOXs inhibitors to mediate apoptotic death in cancer cells remains the focus of many investigations, and there is increasing evidence to suggest that COX-2 inhibition may have no role in NSAID-mediated apoptotic cell death.

Cycloxygenase Despite the wide use of non-steroidal anti-inflammatory drugs (NSAIDs) over the last century, their mechanism of action was not fully appreciated until 1971 when Vane [10] identified their molecular target, the COX enzyme. In the early 1990s, a second isoform (COX-2) was discovered, distinct from the first one, and then renamed COX-1 [11, 12]. While both enzymes carry out essentially the same catalytic reaction, they differ in expression [13], function and structure [14]. The prostaglandins (PGs) produced by COX-2 play a major role in inflammatory reactions and are responsible for the characteristic inflammatory symptoms (redness, pain, edema, fever and loss of function) [15]. The inducible isozyme has also been implicated in pathological processes such as various cancer types (colorectal [16, 17], breast [18]), and Alzheimer and Parkinson’s diseases [19].

Figure 1. Ribbon representation of the modelled human COX-2 homonomer. The binding inhibitors site with highlighted blue residues

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However, recent studies have shown that the relation between the two isoforms is not so straightforward. The three-dimensional (3D) structure of these two enzymes was determined by X-ray diffraction [20, 21]. The COX active site, quite similar in both isozymes [22], consists of a long narrow hydrophobic channel extending from the membrane-binding domain (the lobby) to the core of the catalytic one [23].

Figure 2. Model of the cyclooxygenase-binding site (green) of human COX-2. Only residues relevant to the discussion are displayed. The isopotential surfaces (blue) at -7,0 kcal/mol using hydrophilic probe and -3,0 kcal/mol using methyl probe (yellow) determined from GRID calculations are shown

Despite their similarity, the COX-2 active site is about 20% larger and has a slightly different form than that of COX-1 (Fig.1-2). These size and shape differences are caused mainly by two changes in the amino acid sequence [24]. Ile-523 in COX-1 is replaced by a valine in COX-2. This difference opens up a small hydrophilic side pocket off the main channel, appreciably increasing the volume of the COX-2 active site. Access to this nook is sterically denied in COX-1 by the longer side chain of Ile-523. In addition, the exchange of Ile-434 for a valine in COX-2 allows a neighbouring residue Phe-518 to swing out of the way, increasing further access to the side cavity. Another essential amino acid difference between the two isoforms exists, which does not alter the shape of the drug-binding site but rather changes its chemical environment. Within the side pocket of COX-2 is an arginine in place of His-513 in COX-1, which can interact with polar moieties [25].

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These differences between the COX active sites have major implications for the selectivity profile of inhibitors to compete with araquidonic acid (AA) for binding to the COX active site [26]. These drugs can be subdivided into two classes: isozyme non-specific NSAIDs and selective COX-2 inhibitors. Despite an extensive chemical diversity, they all NSAIDs possess a carboxylate function that, like one of the AA, forms an ion pair with Arg-120 at the bottom of the COX active site [27]. They share the same therapeutic properties but are also responsible for GI lesions and renal toxicity, leading at high doses to erosions, ulcerations, bleedings, and even to death [28]. Indeed, because of their non-specific inhibition of both COX isoforms, classical NSAIDs reduce the production of pro-inflammatory PGs at sites of injury (via COX-2 inhibition) but also the formation of physiological PGs in the stomach and the kidney (via COX-1 inhibition). Currently, more than 500 COX-2-specific inhibitors have been designed. The main structural features of these compounds are the absence of the carboxylate group, characteristic of classical NSAIDs, and generally, the presence of a sulfone (SO2) or sulfonamide (SO2NH2) moiety, which can interact with Arg-513 in the hydrophilic side pocket of the COX-2 active site, defined by residues Tyr-355, Val-523, His-90, Gln-192 and Arg-513 [29]. Although the majority of these compounds were discovered before the structure of COX-2 was solved, crystallographic data can now be used to rationally design selective inhibitors [30]. So far, two compounds, celecoxib and rofecoxib, have been launched for agents accompanied by a reduced risk of GI toxicity compared with classical NSAIDs [31]. These compounds also open new therapeutic insights in the treatment of several diseases where COX-2 implication has been shown, notably in various cancer types [18] and in Alzheimer’s disease [19].

Controversy Concerning Selective COX-2 Inhibitors NSAIDs are widely used for the treatment of inflammatory diseases, such as arthritis. However, their chronic use has often been impaired by the adverse effects they cause, especially in the GI tract and the kidney. Selective COX-2 inhibitors have been developed and marketed in order to reduce NSAIDs-induced side effects associated with COX-1 inhibition. The isoform COX-2 is constitutively expressed in the kidney and the reproductive tract. In addition to its implication in the kidney development, this isoform plays an important part in the regulation of renal function (perfusion, water handling, and renin release) in both normal and paraphysiological conditions (i.e., in patients with liver cirrhosis, renal insufficiency or congestive heart failure). These patients are, therefore, at risk of renal ischemia when NSAIDs and/or selective COX-2 inhibitors reduce vasodilator PG synthesis [32, 33]. Moreover, cyclic hormonal induction of COX-2 plays an important role in ovulation.

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In addition, COX-2 may be involved in the ‘‘adaptative cytoprotection’’ response in GI mucosa. When the latter is inflamed or ulcerated, COX-2 is rapidly induced at sites of injury where it produces large amounts of PGs involved in the healing process. So, selective COX-2 inhibitors should be avoided in patients with gastric susceptibility [34]. The incidence of the use of these compounds on cardiovascular diseases still requires vigilance. Indeed, COX-2 has been shown to generate PGI2 in endothelial cells [35]. Therefore, by decreasing vasodilator and antiaggregatory PGI2 production, selective COX-2 inhibitors may tip the natural balance PGI2/TXA2 in favour of prothrombotic TXA2 and may lead to increased cardiovascular thrombotic events [36]. However, recent findings have highlighted an important role for COX-2 in several physiological processes, as well as a key role in inflammation and pain perception for COX1, therefore, raising questions about the ‘‘selective COX-2 inhibitor’’ theory. Moreover, COX inhibition leads to an up-regulation of the 5-LOX pathway, yielding various adverse effects. Finally, PGs produced by COX-1 have also been shown to contribute to inflammatory responses and hyperalgesia. In these cases, the anti-inflammatory efficacy of selective COX-2 inhibitors was only observed at doses that inhibited COX-1 [37]. In conclusion, it appears that selective COX-2 inhibitors do not fully satisfy the search for new safer antiinflammatory agents.

Lipoxygenases Until now, three major isozymes have been observed in human beings, classified according to their positional specificity of AA oxygenation: the 5-, 12- and 15-LOX insert O2 at the C-5, -12 and -15 positions of AA, respectively and produce the 5-, 12- and 15-HPETE (hydroperoxy-eicosatetraenoic acid) [38]. The knowledge about the biological roles of 12- and 15-LOX is limited [39-40] and needs to be investigated further. 5-LOX, in contrast, has been widely studied and it might be biologically the most important LOX. 5-LOX belongs to a family of lipid peroxidising enzymes, which are expressed in both the vegetal and animal kingdoms [41, 42]. These dioxygenases catalyse the oxygenation of polyunsaturated fatty acids containing a 1,4-cis,cis-pentadiene moiety to produce hydroperoxy derivatives. They require one non-heme iron atom per molecule, which oscillates between Fe2+ (inactive enzyme) and Fe3+ (active form) during the catalytic cycle [43]. The detailed mechanism of the LOX reaction consists of three consecutive steps: (a) stereo-selective hydrogen abstraction from a doubly allelic methylene group, (b) radical rearrangement, and (c) stereo-specific insertion of molecular oxygen and reduction of hydroperoxy-radical intermediate to the corresponding anion [44]. In addition, the 5-LOX pathway, which generates products particularly important in inflammation, leukotriene (LTs), is up-regulated during COX blockade and is thus potentially responsible for undesirable adverse effects, such as asthma. The term leukotriene reflects the cells of origin of these compounds, the leukocytes, and their characteristic structure of conjugated trienes [45].

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5-LOX has been characterised from various mammalian species. It is a monomeric protein of 75-80 kDa, containing about 673 amino acids [46]. While its 3D structure has not been determined yet, several structures of LOXs (two isoforms from soybeans, LOX-1 and LOX-3 [47] and one from rabbit, 15-LOX [48]) have been reported. Based on these crystallographic data, the different LOX enzymes are known to share an overall folding pattern (Fig. 3-4) comprised of two distinct units: a small N-terminal, β-barrel domain, suggested to interact with lipids according to its similarity with the Cterminal domain of human pancreatic lipase [49], and a larger C-terminal catalytic domain, mainly composed of α-helices and containing the active site with the catalytic non-heme iron atom [50].

Figure 3. Ribbon representation of the modelled human 5-LOX. The binding inhibitors site cavity in red.

Unlike other isoforms, 5-LOX is activated by calcium. Indeed, it binds, through its Nterminal domain, two calcium ions in a reversible manner [51]. This enzyme is also stimulated by ATP in the presence of Ca2+ (costimulatory effect), by lipid hydroperoxides and phosphatidylcholine [52]. Then, it interacts with a small membrane protein of 18 kDa designated 5-LOX activating protein or FLAP [53]. Although the mechanism of action still remains obscure, it seems that this protein transfers the substrate AA to 5-LOX, this interaction between the enzyme and FLAP being crucial for cellular LT biosynthesis [54]. In regard to its characteristics and mechanism of action, different strategies have been developed to inhibit the 5-LOX pathway [55-56]. Direct approaches, on the one hand, involve (a) redox inhibitors or antioxidants, which interfere with the redox cycle of 5- LOX, (b) iron-chelator agents, and (c) non-redox competitive inhibitors, which compete with AA to bind the enzyme active site. On the other hand, indirect inhibitors, namely FLAP inhibitors, can prevent 5-LOX from interacting with FLAP and, therefore, inhibit the LT biosynthesis.

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Figure 4. Model of the lipoxygenase-binding site (green) of human 5-LOX. Only residues relevant to the discussion are displayed. The isopotential surfaces (blue) at -7,0 kcal/mol using hydrophilic probe and -3,0 kcal/mol using methyl probe (yellow) determined from GRID calculations are shown

Antioxidants are generally small lipophilic aromatic molecules, such as phenols and quinones [57]. The prototypes of this class are the pyrazoline derivatives and phenidone [58]. Despite diffuse structure-activity relationships (SAR), lipophilicity is an essential feature for the activity of these compounds [59].

Figure 5. FLAP inhibitors

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Whereas most are potent inhibitors, they reveal many side effects due to their poor selectivity against 5-LOX. They readily interfere with other biological redox systems, yielding mainly methaemoglobin formation and genotoxicity [60]. Considering the toxicities and multiple difficulties encountered in the design of redox and iron-chelator inhibitors, the search for active-site directed 5-LOX inhibitors was considered as a new strategy [61]. Several compounds, which form enantio-specific interactions with the enzyme, were discovered, such as ZD-2138 [62]. Several compounds able to interact with FLAP, such as MK-866 and Bay-X-1005, (Figure 5) have been evaluated in clinical trials for asthma [63]. While intensive efforts to develop drugs from LT biosynthesis inhibitors have been rewarded by only one marketed compound, the design of LT receptor antagonists has been more fruitful, leading to three marketed drugs. All these compounds, though effective in the treatment of asthma, appear to be an insufficient single therapeutic modality in other inflammatory diseases. Therefore, a promising approach consists of dual inhibition of both 5-LOX and COX-2.

Dual COX/5-LOX Inhibitors Considering the pro-inflammatory properties of LTs and prostanoids, drugs able to block equally the synthesis of both eicosanoids (dual inhibitors), should not only present a superior anti-inflammatory profile but also fewer side effects than NSAIDs and selective COX-2 inhibitors [64]. Dual inhibition of COX-2 and 5-LOX is, therefore, an interesting alternative to provide safer NSAIDs [65]. Indeed, it has been shown that COX inhibition by NSAIDs, besides causing a reduction in the synthesis of vasodilator and gastroprotective PGs, leads to an up-regulation of AA metabolism by the 5-LOX pathway [66], increasing the formation of LTs and contributing to inflammation and NSAIDs-induced adverse effects. Furthermore, LTs promote the development of GI damage, the most troublesome side effect of NSAIDs. Therefore, dual inhibitors can be expected to present an enhanced anti-inflammatory potency as well as not to cause GI injury and/or allergic adverse reactions. In addition, both COX-2 and 5-LOX enzymes have been involved in the development and progression of numerous types of cancer such as pancreatic, lung, colorectal, prostate, [67-68]. So, the use of dual inhibitors opens up new perspectives in the prophylactic treatment of this dreadful disease. As seen previously, inhibition of both COX isoenzymes, in spite of producing a high anti-inflammatory efficacy, can lead to GI and renal toxicity through COX-1 inhibition. Inhibition of 5-LOX, in contrast, prevents pro-inflammatory and GI damaging effects of LTs. Future clinical data on dual inhibitors (COX-2/5- LOX as well as COX/5-LOX) should, therefore, inform us about the interest of a balanced inhibition of the two COX isoforms, associated with the blockade of the 5- LOX pathway.

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While inhibitors of COXs and of 5-LOX in combination have been proved to be more effective than either class of drugs used alone [69], a single agent inhibiting both enzymes should avoid potential dosing and drug interaction complications of such polypharmacy [70]. Table 1. IC50 values of compounds tested from H. Ulbrich et al., (2002) [39] Compound Licofelone Methyl-sulfonyl H-sulfonyl

IC50 COX-1 (µM) 0.16 0.7 (25%)*

IC50 COX-2 (µM) 0.37 0.005 0.03

IC50 5-LOX (µM) 0.21 10 10

∆Ubin(COX-2) (kcal/mol) -34.48 -32.51 -20.52

∆Ubin(5-LOX) (kcal/mol) -22.68 -19.17 -18.60

* percentage inhibition at a concentration of 10 µM. Changes in energy from MD simulations using bond and unbound inhibitors (∆Ubinding = [∆Uvdw + ∆Uelec]).

Figure 6. Non-redox competitive inhibitors

Several pyrrolizine derivatives possess a dual inhibitory activity. Licofelone (71) inhibits 5-LOX and preferentially COX-1, it does not cause any GI damage and 6,7-diaryl-pirrolyzine derivative compounds, combine the structural requirements for COX-1/COX-2 and 5-LOX inhibition, with a higher selectivity towards COX-2 (Table 1). Unlike most of the described

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compounds, they are non-redox competitive inhibitors, which compete with AA to bind the enzyme active site. All of these drugs do not present a selective COX-2 inhibition and the future clinical data of compounds, such as [6-(4-chlorophenyl)-1,1-dimethyl-7-phenyl-2,3-dihydro-1Hpyrrolizin-5-yl]acetic acid (licofelone) and 6,7-diaryl-2,3-dihydropyrrolizine derivatives (Figure 6), could point out the interest of a balanced inhibition of the two COX isoforms, associated with the blockade of the 5-LOX pathway, it seems in any case interesting to design dual COX-2/LOX inhibitors, first to prevent a drift of AA metabolism toward the other pathway, which would lead to potential side effects, and second to force cell death, that is, to kill specific cells possessing a high flux of arachidonic acid and its metabolites in prostate and colon cancer cells. For this paper we are modelled the active site of the human 5-lipoxygenase on the basis of the X-ray coordinates obtained for the rabbit 15-lipoxygenase and introduced in a dynamic approach a Licofelone acid, methylsulfonyl, methyl-6,7-diaryl-2,3-dihydropyrrolizine (Methyl-sulfonyl) or methylsulfonyl-6,7-diaryl-2,3-dihydro-pyrrolizine (H-sulfonyl) molecules in a straight orientation. Also molecular models of the complexes between these compounds with the cyclooxygenase active site of human COX-2 have been built by combining conformational searching and automated docking techniques. The stability of the resulting complexes has been assessed by molecular dynamics simulations and the binding mode was determined.

Materials and Methods Defining the Binding Cavity of Human 5-Lipoxygenase Model Crystallographic data for several LOX/substrate complexes would be very useful in deciding between the space-determined and the orientation-determined models for LOX positional specificity. This is, however, a rather difficult task for several reasons, an high quality crystals are currently not available for any 5-LOX family member and crystallisation of complexes of substrate with native LOXs must be carried out under strictly anaerobic conditions to avoid formation of hydroperoxy fatty acids, which are potent inactivates of LOXs and may cause structural alterations. The high similarity between the catalytic domains of human 5-LOX and rabbit 15-LOX might be a good template for modelling the catalytic domain of 5-LOX by homology. Human (h) 5-LOX was built by using Amino acid sequences obtained from Swiss-Prot from the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics. Primary accession numbers for proteins are for 5-LOXs, human P09917, mouse P48999, rat P12527, hamster P51399; for 15-LOX, rabbit P12530, human P16050; for soybeans, LOX1 P08170, LOX3 P09186. Modelling by homology was performed by Swiss-Model, 3.5 (Automated Protein Modelling Server) (http://www.expasy.ch/sw/issmod/SWISS-MODEL.html) based upon sequence alignment of residues 42-673 of h-5LOX protein sequence and the crystal structures of rabbit reticulocyte 15-LOX (1LOX.pdb) complexed with RS7, soybean 3-LOX (1IK3.pdb)

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complexed with 13(S)-Hydroperoxy-9, 11(E)-octadecadienoic acid, soybean 3-LOX (1LNH.pdb), soybean 3-LOX (1BYT.pdb) complexed with 4-nitrocatechol, and oxidoreductase (2SBL.pdb). All ionizable residues were considered in the standard Ionisation State at neutral pH. An all atom model was initially built up with the ProModII program (72). Hydrogens were added using standard geometries and a cap of 220 TIP3P water molecules centred in the catalytic iron were included. Then was further gradually energy-minimised using the molecular mechanics program AMBER (73). The resulting structure was used as the starting coordinate file in docking studies. A solvent-accessible cavity within the catalytic domain is proposed to be the substratebinding site (74). Since the structure was solved in the absence of any bound substrate or inhibitor, it is difficult to assign functional relevance to particular structural features. The structural model of the human 5-LOX does, in fact, suggest that the substratebinding cavity is some 20% larger than that of the rabbit 15-LOX, supporting the spacedetermined model. The walls of the cavity of the substrate-binding pocket are lined by hydrophobic residues, but in the proximity of the iron center there are several polar amino acids. Our initial attempts to dock licofelone into the inner cavity of human lipoxygenase-5 indicated that this compound could be placed in the cavity in several different conformations, as well as in several orientations. In an effort to clarify which possible binding mode might be more realistic, assumptions concerning the interaction between the enzyme and inhibitor acid had to be made. The GRID was used to explore the most relevant regions of the target in the active site of h-5LOX for selective interactions. The program GRID (75-77) is a computational procedure energetically favorable binding sites on molecules of known 3D structure. The energies were calculated as Lennard-Jones, electrostatic, and hydrogen bond interactions between a small chemical group (probe) and the 3D structure (target), using a position-dependent dielectric function to modulate the strong electrostatic interaction between charged centres. The GRID origin (Fe) and axes were chosen in such a way that all the atoms around 20 Å of the active centre of the protein were included in the calculations. The probe selected for this study includes the methyl group (C3), the oxygen of sulfone (OS), the aliphatic anionic carboxylate group (COO-), hydrophilic group (OH2) and the hydrophobic group (DRY). The energy calculations were performed using 0.3Å spacing between the grid points in a rectangular box measuring 25x35x28Å. The resulting grids were contoured at appropriate energy levels and graphically displayed to aid in the visualisation of complementarily regions with the selected conformer They give precise spatial information, and this specificity and sensitivity are an advantage since the probes may then be representative of the important chemical groups present in diaryl-2,3-dihydropyrrolizine derivatives, provided that the analysis can distinguish between different types of interaction. Several amino acids have been implicated in positioning the inhibitor at the active site in addition to those described above as determinants of positional specificity. Analysis of the residues in the binding cavity shows that a histidine is located at position 600 and that an asparagine occupies position 425. In the model of h5-LOX, the histidine and asparagine side chains are oriented towards the iron atom.

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A potential role of this 5-LOX histidine in positional specificity has been speculated upon before on the basis of sequence alignment and modelling studies. In addition, the conserved tryptophan just before the histidine in the 5-LOX primary structures has a related character to Tyr603 of the 8-LOX. In developing a possible docking mode for licofelone acid, we postulated that there may be a positively charged residue in the vicinity of the binding site to interact with the carboxylate group. Three positively charged amino acid residues, Lys409, Lys423, Asn425, and His600 are possible candidates for this interaction based on the human 5-lipoxygenase structure. Another possible interaction between the enzyme and the inhibitor could be that of an aromatic residue with the diaryl group of the compound. In considering various docking orientations of inhibitor acid, the existence of possible π-electron interactions (Phe359, Phe421) was noted.

Molecular Modelling of Ligands Molecular modelling of inhibitors was carried out on an O2 Silicon Graphics computer using the Biosym/MSI molecular modelling software [78]. Molecular geometry (Figure 6) of licofelone, methylsulfonyl, methyl-6,7-diaryl-2,3dihydropyrrolizine and methylsulfonyl-6,7-diaryl-2,3-dihydro-pyrrolizine were obtained by combining X-ray crystallography data in the Cambridge Structural Database and conformational analysis. The conformational analysis was performed combining quenched molecular dynamics and energy minimisation techniques using the AMBER program [73]. The X-ray conformation was heated from 6 K to 2000 K in 100 ps using classical molecular dynamics and a time step of 1.0 fs, with coordinates saved every 0.05 ps, resulting in 2000 conformations. Finally, 10% of all conformers were randomly selected and saved in a database, ultimately containing 200 conformers. All conformers in the database were subjected to a two-step energy minimization using the same forcefield as for the MD calculations. A Steepest-Descent algorithm was first used, with convergence obtained at 0.05 RMSG, followed by a Newton-Raphson algorithm with convergence obtained when the gradient was below 0.001 RMSG. After a further 25 ps of equilibration, it was slowly cooled down back to the initial temperature and energy minimized by using the Conjugate-Gradient method until the root-mean-square gradient was less than 0.01 kcal mol−1 Å−1. The resulting structures were subjected to the same simulated annealing protocol, and the whole procedure was repeated 30 times. All conformers of the inhibitors having an rmsd greater 0.25Å and energies within 3 kcal/mol of the global minimum were used as the starting co-ordinate file in the docking study. For each ligand, various starting geometries were used to ensure a proper exploration of conformational space. All minimization and dynamic calculations were performed in the absence of water, assuming a dielectric constant equal to 4r. Finally, two minimum energy conformations with significant geometrical differences were selected for submission to the docking procedure.

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The inhibitors were considered in the standard ionisation state at neutral pH, and their geometry was fully optimised at the ab initio HF/6-31G(d) level using the program Gaussian 98 [79].

Ligand Docking in Human COX-2 and 5-LOX Models The COX-2 protein exists in situ as a dimmer; however, the reason for dimmerization is not known [80]; the monomer structure alone has always been considered in molecular modelling studies assuming the interactions governing the COX-2 inhibitor binding to be reproducible using one monomer. A model of human COX-2 (Figure 2), recently built in our group, was used to examine the binding of inhibitors to this enzyme [81]. The structural model of the human 5-LOX proposed by our group (Figure 4) was used to examine the dual inhibitors binding to the lipoxygenase. The AutoDock 3.0 program [82] was used to explore the docking for different conformations of the 3 inhibitors in the active site of each enzyme. The AutoDock exploration was carried out within a 30Å cube by using 0.25Å grid spacing. Six affinity grids were calculated (C, N, O, S, H, Cl). The simulated annealing protocol consisted of 100 runs of 50 cycles; each cycle including 25000 accepted or 25000 rejected relative positions. A distance dependent dielectric constant equal to 4r was used to simulate a partially solvated state. The annealing temperature was set to 310 K during the first cycle and then linearly reduced at the end of each cycle. Ligands were considered conformationally flexible by defining the torsion angles about which rotation was allowed. AutoDock was used to generate conformers within the binding site by randomly changing torsion angles and overall orientation of each molecule. From the 100 simulations with each compound, the binding mode with the lowest docked energy structures in the top ranked cluster was selected. To take into account protein flexibility, the stability and behaviour of all complexes was studied in a dynamic context and the van der Waals and electrostatic components of the interaction energy monitored.

Molecular Dynamics Simulations The resulting complexes were energy minimised by molecular mechanics (MM) method in AMBER. Restricted electrostatic potential fitted charges determined at the HF/6-31G(d) level, and van der Waals parameters taken for related atom types in the AMBER-98 force field were used for each inhibitor. SHAKE was used to maintain all the bonds at their equilibrium distances and a nonbonded 11Å cutoff and a distance-dependent dielectric constant were used throughout. In each case, 100 steps of steepest descent were followed by conjugate gradient until the root-mean-square value of the potential energy gradient was below 0.01 kcal mol−1 Å−1. Ligand and a cap of 220 TIP3P water molecules centred at the inhibitor, together with the enzyme, were used as input for the subsequent molecular dynamics simulation. The

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resulting systems were subjected to full conjugate gradient minimisation until the rms value of the potential energy gradient was below 0.01 kcal mol−1 Å−1, using the AMBER force field for all parameters except for the charges for the ligand atoms, which were obtained quantum mechanically as described above. First, for each protein a short optimisation run restraining the backbone to their initial coordinates was conducted. This allowed readjustment of covalent bonds and van der Waals contacts without changing the overall conformation of the protein. Then, only the ligand atoms were allowed to move and finally the whole complex was energy minimised although the protein backbone atoms were restrained to their initial positions by a harmonic potential with a force constant of 32 kcal mol−1 Å−1, while the hydrogen atoms, the inhibitor and water molecules were unrestrained. In each case, 100 steps of steepest descent were followed by conjugate gradient until the root-mean square value of the potential energy gradient was below 0.01 kcal mol-1 Å-1. The final coordinate set was used as input for the subsequent molecular dynamic (MD) simulations under the same conditions. In 100 ps heating phase, the temperature was raised from 0.2 to 298 K, equilibrated for 110 ps at 298 K. A 2 ns trajectory was then simulated at 298K employing a time step of 2 fs. The SHAKE was used to maintain all the bonds at their equilibrium values. The non-bonded pair-list was updated every 20 cycles and coordinates were saved every 10 ps for from the last 1000 ps and energy-minimised for further analysis of complexes. The reported flexible nature of the hCOX-2 and 5-LOX binding sites was taken into account in our MD simulations, which were carried out for the two orientations of inhibitors suggested by the automated docking program. For each inhibitor one MD simulation was performed for the unbound inhibitor in solvent, using a cap of 220 TIP3P water molecules centred at the inhibitor. The starting conformation for the unbound inhibitors was obtained from the refined complexes. The MD simulations consisted of an initial solvent equilibration for 30 ps equilibrated for 110 ps at 298 K and then a 300 ps molecular dynamics was performed. Integration was carried out with time-step of 2 fs, with scaling factor 2.0 for 1–4 interaction. Energy averages were then accumulated over final 100 configurations during which atomic co-ordinates were saved after each 1ps. Separate running averages were determined for the Lennard-Jones and coulombic components of the nonbonded interaction energies. The binding affinity can be expressed as the Uvdw and Uelec refer to the Lennard-Jones and electrostatic average interaction energies for the bound and unbound states of the inhibitor.

Results and Discussion Docking in Human COX-2 and 5-LOX Models High-resolution structural information on COX-inhibitor complexes led to a detailed description of the COX active site. The change of two isoleucines (Ile-434 and Ile-523) in COX-1 by two valines in COX-2 opens up an extra hydrophilic nook off the main channel, appreciably increasing the volume of the COX-2 active site. Another essential amino acid

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difference consists of Arg-513 inside this side pocket, in place of a histidine in COX-1. It generates a specific interaction site for inhibitors in COX-2. Docking of each of the three representative ligands into the COX-2 active site generated a number of possible structures with different orientations (and energies) of ligand inside the active site. The most energetically favorable conformation for each ligand in the COX-2 complex was chosen for further analysis. Two different binding modes can be considered among the inhibitors studied. The first (Figures 7-8), “SC558-like”, is close to that adopted by SC-558 [27]. However, despite these interactions, the lesser stability of these sulfonyl complexes, in comparison to that of licofelone complex in COX-2, can be assessed by the smaller electrostatic interaction energy (∆E ranging from 6.3 to 7.9 kcal mol-1).

Figure 7. Docking of the molecule Licofelone-COX-2 complex: a) “diclofen-like” orientation in the active site (the ligand is shown in white sticks), b) “SC558-like” orientation in the active site (the ligand is shown in green ball and sticks). Only residues relevant to the discussion are displayed. The hydrogenbonding interactions are shown as broken lines. All protein hydrogens are removed for clarity

Another mode of interaction “diclofen-like” (Figure 7) for licofelone inside the COX-2 active site was also proposed by AutoDock, i.e., the polar moiety carboxylic group lying in the proximity of Tyr385 and Ser530. This was similar to a new inverted orientation, recently revealed by the crystallographic structure of diclofenac bound to COX-2, [83]. However, the resulting licofelone-COX-2 complex was much less stable than the “SC558-like” one ( E 7.6 kcal mol-1). In contrast to COX enzymes, structural knowledge about the 5-LOX active site is much more limited. It was therefore first explored with different GRID probes. The program GRID was applied to the active site of the structure h-5LOX model. A methyl group (C3) and the hydrophilic group (OH2) probe were used to map key electrostatic and van der Waals interaction sites. A detailed examination of the 3D regions in the active site (Fig. 4) at which the GRID probes would interact most selectively with the enzyme shows a completely filed van der Waals contact channel (contour yellow at –3,0 kcal/mol) from Phe359 to Trp599. The OH2 probe contoured at –7,0 kcal/mol (blue) mapped polar

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interaction sites, including a large area bordering Gln413, Lys423, Asn425 and His600, and other area including Lys409 and Tyr181 residues.

Figure 8. Docking of the molecule Methyl-sulfonyl-COX2 and H-sulfonyl-COX-2 complexes: a) “Methyl-sulfonyl” orientation in the active site (the ligand is shown in ball and sticks), b) “H-sulfonyl” orientation in the active site (the ligand is shown in sticks). Only residues relevant to the discussion are displayed. The hydrogen-bonding interactions are shown as broken lines. All protein hydrogens are removed for clarity

Our working models (Fig.9) docked the chloride end of Licofelone acid near the narrow neck of the inner cavity defined by Phe359, Ala424, Leu414, and Phe421. The carboxylate group was docked near Lys423 and Gln413, as this orientation of the ligand allowed the 2,3-dihydropyrrolizine moiety to be very close to the catalytic iron. The quality of the binding modes was visually assessed to determine which amino acid residues could be critical for proposed ligand binding modes.

Figure 9. Docking of the molecule Licofelone-5-LOX complex (the ligand is shown in ball and sticks). Only residues relevant to the discussion are displayed. The hydrogen-bonding interactions are shown as broken lines. All protein hydrogens are removed for clarity

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The binding mode on the 5-LOX proposed for the other compounds have been shown (Fig. 10). It inserts the “COX fragment” deep in the cavity with the methylsulfonyl moiety at the bottom, interacting with Asn425, Lys423, Phe421 and Ala424. While, the “LOX fragment” deep in the cavity, interacting with Phe359, Gln413, and Leu414.

Figure 10. Docking of the molecules Methyl-sulfonyl and H-sulfonyl-5-LOX complexes: a) “Methylsulfonyl” orientation in the active site (the ligand is shown in ball and sticks), b) “H-sulfonyl” orientation in the active site (the ligand is shown in sticks). Only residues relevant to the discussion are displayed. The hydrogen-bonding interactions are shown as broken lines. All protein hydrogens are removed for clarity

COX-2 Binding Mode The orientations and binding interactions of ligand licofelone within the COX-2 active site are shown in Figure 7. Molecule licofelone is involved in a different pattern of interactions throughout the COX-2 active site, as might be expected. These orientations are similar to that of SC-558 (6COX) and diclofenac. Molecule licofelone “like-SC558” showed favorable van der Waals and electrostatic interactions with Val523 Val349, Leu352, Ser353, Arg120, and Tyr355. It also showed some interactions with Tyr385, Arg513, Phe518, Met522, Ala527 and Ser530. Different sets of hydrogen bonding interactions with residues Arg120 (C=O…H-N 2.65Å, all distances are for dH-X), and Tyr355 (C=O…H-O 1.88Å) are observed. Molecule licofelone “like-diclofen” showed favorable van der Waals and electrostatic interactions with Val349, Leu352, Ser353, Arg120, Tyr355, Ala527, Leu531, and Ser530. It also showed some interactions with Tyr385, Phe518, Met522, Ala527 and Ser530, but showed not interactions with Arg513 and Ala516.

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Its carboxylic acid moiety, in contrast to other NSAIDs, is situated in the upper part of the channel, interacting through H-bonds with Ser530 (C=O…H-O 3.05Å), and Tyr355 (C=O…H-O 2.88Å). In the case of molecules methyl and H-sulfonyl, the preferred orientations indicate that while the position of pyrrolizine ring is conserved, the positions of phenyl rings are interchanged. This is easier to understand because the electronegative –CH2COO group in licofelone has been replaced with an electropositive H atom. Because this electronegative group interacts strongly with Arg120, the change in functional group results in an orientation change bringing the nitrogen of pyrrolizine ring in methyl-sulfonyl close to Arg120. The number of observed hydrogen-bonding interactions between the methanesulfonyl moiety and the various amino acid residues at the entrance of the COX-2 active site enables the compound H-sulfonyl to be a strongly binding and selective COX-2 inhibitor. The substituents (4-CH3) on the ring A also induce favorable electrostatic interactions between various amino acid residues at the bottom of the active site (hydrophobic cleft) and ring A (Figure 8). The orientation of the moderately active molecule H-sulfonyl is similar to that of the highly active molecule Methyl-sulfonyl. The steric interaction energies of diarylpyrrolizine inhibitors with various amino acid residues in the active site are plotted in Figure 11a while the electrostatic contributions are given in Figure 11b. In general, molecules methyl and H-sulfonyl show similar types of interactions with various amino acid residues in the active site. Molecule Methyl-sulfonyl, which is a more active COX-2 inhibitor, is bound in a totally different orientation from that of molecule licofelone at the COX-2 active site and is shown in Figure 8. The CH3-substituted ring of the molecule Methyl-sulfonyl moved toward the hydrophobic cleft of the active site. Such an orientation preference may be due to electrostatic interaction between the Arg120 and the pyrrolizine moiety of the ring. This also resulted in some favorable interactions throughout the active site with residues such as Arg120, Val349, Ser353, and Val523 (important for selectivity) [84-85]. Hydrogen-bonding interactions of Methyl-sulfonyl with Arg513 (N-H…O=S 3.15Å), and Ser353 (O-H…O=S 2.97Å) are observed. The replacement of the methyl group in molecule Methyl-sulfonyl with the H is the main reason for these weaker interactions and hence lower activity. Interactions with various residues at the entrance of the active site, Val116, Leu359, and Met522, are less favorable, and interactions with other residues, Val344 and His351, are very weak. Substituent effects such as these have been reported recently. The ∆Uvdw values (Table 1) are attractive and similar in magnitude to those for the neutral inhibitors. More favourable inhibitor-protein van der Waals interactions indicate a good steric fit and enhance binding. As might be expected from the favourable salvation of a charged species, the ∆Uelec values are generally positive. This is reflected in the free energies of solvation in aqueous solution of the ligands. In fact, it has been estimated that the loss of hydrogen bonds upon binding of the ligand, relative to the free hydrated state, costs around 2 kcal/mol to the free energy of binding [40]. There still remains, however, a general trend that more active inhibitors have larger ∆Ubinding (∆Uvdw + ∆Uelec) values (Table 1).

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a) electrostatic interaction energy (kcal/mol) 2.00 0.00 -2.00 -4.00 -6.00 L531

S530

A527

E524

V523

F518

M522

A516

R513

W387

L359

Y385

Y355

L352

S353

V349

R120

Q192

V116

H89

-8.00

residues number H-sulfonyl

Methyl-sulfonyl

Licofelone-SC558

Licofelone-diclofen

b) vdW interaction energy (kcal/mol)

0.00 -2.00 -4.00 -6.00

L531

S530

A527

E524

V523

M522

F518

A516

R513

W387

Y385

L359

Y355

L352

S353

V349

Q192

V116

R120

H89

-8.00

residues number H-sulfonyl

Methyl-sulfonyl

Licofelone-SC558

Licofelone-diclofen

Figure 11.- Steric (a) and electrostatic (b) contributions to the nonbonded interaction energies between molecules H-sulfonyl, Methyl-sulfonyl, and Licofelone (like-SC558 and like-diclofen orientations) with various residues of the active site of COX-2

5-LOX Binding Mode The observation that the volume of this active site of mammalian 5-LOXs is bigger than that of other isoforms may be useful for the development of specific 5-LOX binding site for interactions with substrates and inhibitors. The amino acids Phe359, Ala424, Asn425 and Ala603, have been identified previously as sequence determinants for the positional specificity of other LOX isoforms. The binding mode on the 5-LOX proposed for the licofelone compound has been shown (Fig. 9). The “COX part” fills the entrance of the active site and also forms an H-bond with the carboxylic oxygen and Lys423/Gln413. The “5-LOX part” fills the entrance of the active site and also forms an H-bond with the pyrrolizine nitrogen and Gln363. The binding modes proposed for the methylsulfonyl compounds are shown in Figure 10. It inserts the “COX fragment” deep in the cavity with the methanesulfonyl moiety at the bottom, interacting with Phe421, Asn425, Lys423 and Ala424. The “5-LOX part” fills the entrance of the active site and also forms an H-bond with the pyrrolizine nitrogen Gln363 and Lys409.

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a) electrostatic interaction energy (kcal/mol) 0,00 -2,00 -4,00 -6,00

H600

H432

G431

N425

A424

K423

F421

C418

L414

A410

Q413

K409

L368

Q363

F359

H360

I292

F177

-8,00

residues number H-sulfonyl

Methyl-sulfonyl

Licofelone

b) vdW interaction energy (kcal/mol) 0,00 -2,00 -4,00 -6,00 -8,00 H600

H432

G431

N425

A424

K423

F421

C418

L414

Q413

A410

K409

L368

Q363

F359

H360

I292

F177

-10,00

residues number H-sulfonyl

Methyl-sulfonyl

Licofelone

Figure 12.- Steric (a) and electrostatic (b) contributions to the nonbonded interaction energies between molecules H-sulfonyl, Methyl-sulfonyl, and Licofelone with various residues of the active site of 5LOX.

In order to evaluate the relative contributions of the different residues to complex, stabilization the 100 structures collected from the last 1000 ps of the simulations were averaged and energy minimised, and the interaction energy between substrate and the binding site was discomposed on a residue basis using the ANAL module of AMBER (Fig. 12). Our working model suggest that Phe359, Gln363, Gln413, Leu414, Phe421, Ala424, Asn425, and Ala603 are involved in positioning inhibitor at the active site and, thus, may be considered as sequence determinants for the positional capacity. These data indicate that there are electrostatic (Gln413/Lys423/Asn425) preference for either inhibitor orientation at the active site of the human 5-lipoxygenase (Licofelone to be more effective). M. Arockia Babu et al., [86] based on yours 3D-QSAR biophore models for 5lipoxygenase antagonists chalcones were suggested that there are three similar biophoric sites, one is involved in hydrogen bonding, second site is for electrostatic and ionic interactions and the third is involved in π−π interactions. Surprisingly, when K. Schwartz et al, [87] mutated the Lys-409 (K409L and K409R), they did not observe major differences when the positional specificity and the specific activities of the wild type and mutant enzyme species were compared. These data suggest that Lys-409 of the h5-LOX may not be of major importance for enzyme/inhibitor interaction.

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Conclusion Selective Cyclooxygenase Inhibitors NSAIDs differ dramatically with regard to how quickly they productively bind in the COX active site and how quickly they come out of the COX channel. Some NSAIDs have very rapid on and off rates, such as ibuprofen [80]. Such drugs do not show time dependence. They inhibit COX activity essentially instantaneously after addition of the NSAID, and they readily wash out of the COX active site when the NSAID is removed from the environment of the enzyme. In contrast, many NSAIDs such as indomethacin and diclofenac are timedependent. They require typically seconds to minutes to bind the COX active site. Once bound, however, these drugs typically have low off-rates that may require hours for the NSAID to wash out of the active site. Time-dependent NSAIDs compete very poorly with AA in instantaneous assays of COX activity site. Time-dependent NSAIDs bind the COX active site first in a loose interaction and then in a productive tight complex. The rate-limiting step in drug binding is the COX-2. Like timedependent carboxyl-containing NSAIDs, time dependence for Methyl-sulfonyl and Hsulfonyl formation of the tight binding conformation of the NSAID within the COX channel. Of particular importance to this second step in NSAID binding is the constriction point created by the hydrogen bonding network of Arg120, Tyr355, and Glu524 and the proposed difficulty for some NSAIDs to traverse it [84]. One open state of the COX-2 enzyme has been identified crystallographically. An open state of the COX-1 enzyme that allows NSAIDs to pass the constriction point is likely to be transient since crystallographic studies show no difference in COX-1 conformation bound to time-dependent or nondependent NSAIDs [80]. Once having passed through the constriction site into the catalytic pocket, carboxylcontaining NSAIDs (Licofelone) form a salt bridge between the carboxylate group and the guanidinium moiety of Arg120 and hydrogen bonding with Tyr355 in COX-2. Hydrophobic interactions between the aromatic rings of compounds and the hydrophobic amino acids lining the channel further stabilize binding. The sum of these interactions results in tight binding of Licofelone at the constriction point of the channel, where they totally block entry of AA. Methyl-sulfonyl and H-sulfonyl are diaryl compounds containing a methylsulfone. Each of these compounds is a weak time-independent inhibitor of COX-1, but a potent time-dependent inhibitor of COX-2. Like time-dependent carboxyl-containing NSAIDs, time dependence for Methyl-sulfonyl and H-sulfonyl requires these compounds to enter and be stabilized in the catalytic pocket. However, because these drugs lack a carboxyl group, stabilization of binding for both of these drugs does not require Arg120. Instead, a sum of hydrophobic and hydrogen bonding interactions stabilizes binding. Of particular importance is penetration of the sulfur-containing phenyl ring into the hydrophobic out pocketing (Val349, Ser353, Trp359, and Val523) in the COX-2 catalytic pocket shown in Figure 8 [27], and the better electrostatic interactions with Arg513. The binding of methyl-sulfonyl should involve a direct hydrogen bond between Arg513 and the sulfonyl group of the drug. There is a loss of favorable van der Waals interactions in the region Val116, Arg120, Tyr385, and Leu531, since the loss of favorable electrostatic

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interactions with Tyr355 is mostly compensated by better electrostatic interactions with His89 and Asn192

Selective 5-Lypoxygenase Inhibitors In, summary, we have developed a theoretical model for the structure of the catalytic domain in the human 5-LOX. This model allows inspection of specific residues and domains in the context of the three dimensional molecule. To take into account 5-LOX protein flexibility, which may be a functional component of ligand binding, as well as the possibility conformational changes, the behaviour of all complexes was studied in a dynamic context and the van der Waals and electrostatic components of the interaction energy were determined. These complexes remained stable during the whole trajectory and yielded almost equivalent overall interaction energies. These results also demonstrate the importance of both a positively charged amino acid residue and an aromatic amino acid residue for inhibitor binding. Our working model suggest that Phe359, Gln363, Gln413, Leu414, Phe421, Ala424, Asn425, and 603 Ala are involved in positioning inhibitor at the active site and, thus, may be considered as sequence determinants for the positional capacity. These data indicate that there are electrostatic (Gln413/Lys423/Asn425) preference for either inhibitor orientation at the active site of the human 5-lipoxygenase.

Dual COX/5-LOX Inhibitors A new strategy has been considered: the dual inhibition of 5-LOX and COX enzymes. Various structural families of dual inhibitors have been designed, and several compounds are currently undergoing preclinical or clinical development. By preventing the biosynthesis of both prostanoids and LTs, they are potent anti-inflammatory agents. Though none of these compounds have reached the market yet, they might represent a valuable therapeutic alternative to classical NSAIDs and to some extent, to selective COX-2 inhibitors, notably because of their almost complete lack of GI toxicity. The results of this study may provide valuable information to researchers who are working on the development of safer anti-inflammatory agents. All of these drugs do not present a selective COX-2 inhibition and the future clinical data of compounds, such as the diaryl-pyrrolizine derivatives, could point out the interest of a balanced inhibition of the two COX isoforms, associated with the blockade of the 5-LOX pathway. The steric interaction energies of diarylpyrrolizine inhibitors with various amino acid residues in the active site of COX-2 are favorable at the inhibition, while the electrostatic contributions are favourable in the active site of 5-LOX. Finally, as COX-2 and 5-LOX are up-regulated in various cancers, development of drugs targeting both enzymes would be a useful future direction for chemoprevention.

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Acknowledgments We thank Drs. M. G. Morris and P.J. Goodford for provision of the software AutoDock and GRID. This work has been supported by the Spanish Ministry of Science and Technology (Grant SAF2002-0482-C02-01).

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Index A access, 212 accounting, 44, 86 accumulation, 2, 8, 18, 21, 44, 45, 71, 110, 123, 127, 128, 157 acetaminophen, 66, 131, 203 acetic acid, 188, 219 acid, vii, 1, 3, 4, 5, 6, 7, 9, 17, 24, 33, 43, 45, 46, 51, 56, 63, 74, 75, 77, 93, 96, 98, 111, 116, 120, 122, 127, 129, 134, 136, 147, 148, 154, 156, 175, 182, 183, 184, 188, 189, 210, 212, 213, 214, 219, 220, 221, 223, 225, 227, 231 acidity, 97 acidosis, ix, 146, 171 activation, 15, 17, 18, 19, 21, 32, 33, 36, 37, 38, 40, 43, 44, 45, 46, 66, 69, 70, 71, 82, 95, 113, 121, 122, 123, 124, 125, 126, 129, 134, 139, 140, 141, 147, 186, 191, 193, 194, 195, 200, 204 active site, x, 209, 210, 212, 213, 215, 219, 220, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 acute interstitial nephritis, ix, 146, 152, 179, 180 acute lung injury, 200 acute renal failure, ix, 145, 146, 151, 152, 159, 160, 163, 164, 173, 174, 175, 177 acute tubular necrosis, 172 adaptation, 158, 176 adenoma, 9, 25, 27, 37, 57, 59, 131 adenosine, 118 ADH, 150, 154, 155 adhesion, 7, 33, 38, 39, 80, 138 adhesive interaction, 38 adipocyte, 134 adipose, 138 adults, 49, 91

adverse event, 163, 165, 168, 169 affect, 23, 33, 41, 95, 96, 116, 121, 160, 162, 175 Africa, 87 African Americans, 73 agar, 36, 42 age, viii, 3, 9, 14, 30, 86, 87, 91, 96, 103, 159, 168, 178 agent, viii, 26, 40, 48, 54, 57, 60, 61, 64, 70, 85, 103, 142, 157, 171, 193, 194, 199, 200, 202, 206, 218 aggressive behavior, 3, 13 agonist, 135, 136 AIDS, 53 airways, 207 alcohol, 116 aldosterone, ix, 146, 150, 156, 159, 171 algorithm, 221 alternative(s), viii, 34, 56, 62, 85, 97, 103, 125, 138, 156, 202, 210, 217, 231 alveolar macrophage, 129 Alzheimer's disease, 24, 62 amines, 95, 110 amino acids, 215, 220, 228, 230 ammonia, 33 amplitude, 192 analgesic, 23, 24, 53 androgen, 142 angina, 171, 179 angiogenesis, vii, 1, 3, 7, 9, 10, 18, 19, 21, 25, 28, 34, 35, 36, 37, 38, 39, 40, 42, 43, 45, 55, 56, 57, 58, 60, 65, 67, 68, 69, 71, 72, 73, 78, 80, 101, 112, 113, 114, 116, 122, 124, 127, 135, 136, 137, 141 angiotensin II, 46, 82, 150, 151, 157, 176, 177 anhydrase, 171 animals, 7, 27, 45, 87, 96, 119, 121, 157, 166 annealing, 221, 222

238

Index

anti-angiogenic agents, 61 antibody, 40, 109, 190 antidiuretic hormone, 150 antigen-presenting cell, 21 antihypertensive agents, 168, 169 antihypertensive drugs, 155 anti-inflammatory agents, 23, 65, 139 anti-inflammatory drugs, vii, viii, 2, 3, 9, 22, 24, 50, 62, 65, 66, 67, 68, 71, 72, 74, 76, 77, 83, 85, 98, 112, 114, 131, 135, 137, 139, 141, 142, 146, 149, 152, 173, 174, 175, 176, 177, 178, 183, 205, 206 anti-inflammatory medications, 48 antimicrobial therapy, 96 antioxidant, x, 92, 93, 95, 98, 108, 111, 181, 184, 187, 191, 196, 197, 198, 199, 200, 205 antipyretic, 66, 131, 203 antisense oligonucleotides, 124 antitumor, viii, 21, 35, 40, 42, 43, 60, 61, 74, 78, 80, 82, 115, 116, 126, 127, 128, 136, 141, 205 antrum, 14, 33 APC, 15, 18, 26, 44, 70, 120, 142 apoptosis, vii, viii, 1, 2, 3, 9, 17, 20, 21, 26, 28, 29, 31, 32, 33, 34, 35, 40, 42, 43, 44, 45, 47, 52, 56, 57, 58, 60, 62, 65, 66, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79, 80, 81, 85, 93, 98, 99, 101, 103, 112, 113, 114, 116, 122, 123, 126, 127, 133, 135, 136, 139, 140, 141, 158, 186, 195, 199, 204, 210 appetite, 127 arginine, 212 aromatic hydrocarbons, 95 aromatic rings, 230 arrest, 55, 69, 73, 112 arterioles, 149, 150, 159 artery(ies), 130, 149, 158, 159, 164, 202 arthritis, vii, 22, 30, 185 ascorbic acid, 98 Asia, 96 assessment, 91 assignment, 111, 129 association, 11, 19, 20, 32, 35, 43, 56, 58, 60, 66, 72, 80, 86, 89, 90, 91, 92, 93, 96, 100, 109, 124, 156, 165, 179, 187, 200, 206 assumptions, 220 asthma, 187, 200, 205, 207, 210, 214, 217 atherogenesis, 205 atherosclerosis, 186, 187, 201, 204 atherosclerotic plaque, 187 atoms, 220, 223 ATP, 215 atrophy, viii, 13, 33, 86, 97, 104

attention, viii, 85, 98, 116 availability, 61, 119

B bacteria, 33 basic research, 76 behavior, 13 beneficial effect, ix, 36, 58, 63, 103, 104, 120, 181 benzene, 188 beta-carotene, 91, 93, 108 beverages, 92, 106 bias, 91 bicarbonate, 7, 122, 164 binding, x, 95, 116, 118, 120, 121, 130, 152, 183, 209, 210, 211, 212, 213, 215, 216, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 234 bioassay, 109 bioavailability, 120 biopsy, 52 biosynthesis, 51, 80, 125, 137, 174, 215, 217, 231 birth, viii, 86, 103 black tea, 88, 106 bladder, 74, 76, 121 bladder carcinogenesis, 74 bleeding, ix, 3, 23, 26, 47, 49, 181, 183 blocks, 140 blood, ix, 4, 9, 14, 19, 23, 37, 39, 43, 51, 82, 96, 121, 129, 145, 146, 151, 154, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 175, 177, 178, 179, 183, 189, 202 blood flow, ix, 4, 51, 121, 145, 151, 154, 158, 159, 163, 171, 175, 183 blood group, 9 blood monocytes, 129 blood plasma, 43 blood pressure, ix, 146, 151, 154, 166, 167, 168, 169, 170, 171, 177, 178, 179 blood supply, 39 blood urea nitrogen, 164 blood vessels, 37 body, 30, 33, 52, 63, 103, 104, 105, 109, 147, 155, 184 body mass index, 104 body size, 105 body weight, 104 bonding, 224, 225, 226, 227, 229, 230 bonds, 222, 223, 227 bowel, 7, 24, 36, 54, 67, 78, 114

Index brain, 118, 127, 130, 142, 184 Brazil, 88, 105 breakfast, 90 breast cancer, 20, 36, 59, 70, 77, 125, 132, 134, 137, 138, 141 breast carcinoma, 138

C C. pneumoniae, 189, 190 cabbage, 95 calcium, 45, 71, 96, 155, 168, 169, 215 calcium channel blocker, 155, 168, 169 caloric restriction, 87 Canada, 88 cancer, vii, viii, 1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 20, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 120, 121, 123, 125, 127, 128, 130, 131, 132, 133, 134, 135, 137, 139, 141, 186, 199, 204, 206, 207, 210, 211, 213, 217 cancer cells, 3, 10, 26, 29, 36, 38, 40, 42, 44, 45, 46, 58, 61, 73, 76, 81, 112, 123, 125, 127, 128, 135, 211 cancer progression, 2 candidates, 221 cannabinoids, 142 capillary, 124 carbon, 116 carcinogen, 13, 24, 27, 30, 36, 58, 67, 92, 95, 96, 99, 121 carcinogenesis, vii, viii, 1, 2, 3, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 20, 25, 30, 31, 34, 35, 36, 37, 42, 47, 55, 56, 57, 59, 64, 71, 75, 76, 77, 78, 79, 80, 86, 87, 95, 96, 97, 98, 99, 104, 109, 110, 111, 115, 116, 119, 122, 126, 127, 128, 131, 132, 133, 134, 135, 139, 186, 210, 211 carcinoma, 2, 3, 4, 8, 9, 12, 13, 14, 16, 21, 23, 25, 27, 28, 29, 30, 35, 37, 39, 59, 64, 65, 68, 70, 71, 72, 73, 74, 75, 76, 81, 82, 83, 105, 106, 109, 111, 112, 113, 123, 124, 135, 136, 137, 138, 140, 141, 203 cardiovascular disease, 24, 54, 68, 121, 187, 204, 214 cardiovascular risk, 54

239

carotene, 88, 90, 93, 94, 108 carotenoids, 91, 93 catalytic activity, 98, 121 catecholamines, 151 cation, 156, 187, 191, 197 causation, 111 cDNA, 5, 99, 111, 129 cell, vii, x, 1, 4, 5, 9, 10, 14, 16, 17, 19, 20, 21, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 53, 55, 56, 57, 62, 65, 66, 67, 68, 69, 70, 72, 73, 75, 77, 79, 80, 81, 92, 93, 95, 96, 98, 99, 101, 111, 112, 113, 114, 116, 117, 118, 122, 123, 124, 125, 126, 127, 129, 130, 131, 133, 135, 136, 138, 139, 140, 141, 142, 147, 148, 149, 158, 176, 182, 187, 189, 190, 191, 194, 196, 199, 201, 202, 203, 204, 205, 206, 210, 211, 219 cell culture, 55, 57, 93, 96 cell cycle, 44, 69, 73, 112 cell death, 21, 27, 31, 33, 45, 68, 77, 113, 124, 158, 196, 199, 211, 219 cell line, x, 5, 16, 21, 26, 28, 30, 40, 41, 44, 45, 46, 65, 68, 70, 73, 80, 81, 98, 99, 101, 112, 113, 126, 130, 135, 136, 139, 141, 176, 182, 187, 189, 203, 204 cell surface, 39, 125 cerebral cortex, 118 cervical cancer, 124, 137 channels, 150, 151, 155 chemiluminescence, 190, 205 chemokines, 33 chemoprevention, vii, viii, 2, 15, 25, 27, 30, 33, 34, 46, 47, 50, 53, 54, 57, 58, 59, 60, 61, 62, 64, 66, 74, 75, 76, 79, 85, 86, 96, 98, 103, 104, 114, 202, 204, 207, 210, 211, 231 chemotaxis, 16 chemotherapy, 36, 58, 60, 61, 63, 64, 80, 86, 125, 127, 128, 186 childhood, 96 China, 1, 8, 72, 80, 88, 93, 94, 97, 108, 109 cholangiocarcinoma, 81, 140 cholesterol, 87, 88, 187 chromosome, 117, 148 chronic inflammatory cells, 21, 41 chronic renal failure, 163 circulation, 151, 152 cirrhosis, ix, 23, 146, 151, 154, 159, 160, 163, 173, 177 classes, x, 23, 168, 181, 185, 213 classification, 9 cleavage, 31

240

Index

clinical presentation, 172 clinical syndrome, 151 clinical trials, 24, 26, 27, 38, 48, 49, 50, 55, 59, 60, 61, 62, 92, 128, 165, 167, 171, 177, 217 cloning, 66, 111, 129, 131, 203 CO2, 189 coding, 118 cohort, 29, 59, 67, 89, 90, 91, 92, 96, 103, 106, 107, 114, 154 colitis, 7 collateral, 55 colon, 2, 18, 20, 21, 22, 23, 25, 30, 35, 36, 37, 39, 42, 43, 44, 45, 46, 48, 53, 54, 56, 58, 59, 60, 61, 62, 65, 66, 67, 69, 70, 71, 72, 73, 75, 77, 80, 82, 83, 121, 124, 126, 127, 130, 131, 133, 135, 136, 138, 139, 140, 141, 186, 204, 206, 210, 219 colon cancer, 20, 25, 36, 37, 44, 45, 46, 48, 58, 65, 66, 67, 69, 70, 73, 75, 77, 80, 83, 124, 126, 127, 130, 133, 136, 138, 139, 140, 141, 186, 219 colonization, 97 colorectal adenocarcinoma, 56 colorectal cancer, 8, 25, 36, 38, 58, 63, 65, 68, 78, 80, 98, 119, 133, 140, 142 combination therapy, 46, 49, 128 combined effect, 187, 195 community, 54, 182 compensation, 121 competition, 154 complementary DNA, 129 complexity, 57 compliance, 49 complications, ix, 47, 48, 53, 65, 66, 78, 146, 173, 204, 218 components, 87, 95, 222, 223, 231 compounds, x, 9, 22, 24, 43, 48, 49, 62, 95, 96, 97, 127, 128, 181, 182, 185, 191, 193, 196, 197, 198, 201, 209, 210, 211, 213, 214, 216, 217, 218, 219, 226, 228, 230, 231 concentration, 32, 38, 42, 45, 49, 95, 151, 156, 162, 171, 172, 189, 190, 191, 192, 195, 196, 198, 200, 201, 218 conduct, 104 confidence, 29 confidence interval, 29 conformational analysis, 221 congestive heart failure, 151, 158, 159, 160, 164, 165, 171, 173, 175, 213 consumers, 92, 97 consumption, 87, 91, 92, 93, 97, 107, 108, 110, 172 consumption frequency, 87

context, 179, 186, 198, 200, 222, 231 control, viii, 4, 10, 14, 22, 28, 29, 49, 51, 65, 72, 82, 85, 88, 91, 95, 96, 98, 100, 102, 103, 105, 106, 109, 114, 118, 121, 125, 136, 142, 154, 161, 165, 166, 168, 170, 171, 191, 192, 194, 195, 196, 197, 198, 201 control group, 14, 15, 28 controlled studies, 81 controlled trials, 48, 169 convergence, 221 conversion, vii, 1, 4, 17, 56, 96, 98, 123, 184, 199 cooking, 90 COPD, 203 coronary arteries, 202 coronary heart disease, 179, 203, 206 correlation, 9, 11, 12, 13, 29, 69, 70, 81, 96, 98, 114, 133 cortex, 149, 158 corticosteroids, 3 costs, 227 covalent bond, 223 COX-2 enzyme, vii, ix, x, 121, 124, 127, 145, 147, 157, 158, 159, 179, 181, 184, 187, 200, 201, 230 cox-2 inhibitor, 23 creatinine, 156, 160, 161, 164, 172 crime, 206 crystallisation, 219 crystals, 219 culture, 127, 188, 189, 190 cycles, 222, 223 cycling, 201 cyclooxygenase, vii, ix, 1, 2, 4, 5, 6, 22, 24, 32, 34, 43, 44, 56, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 111, 112, 113, 114, 115, 116, 119, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 156, 160, 173, 174, 176, 177, 178, 179, 181, 182, 183, 203, 204, 205, 206, 207, 210, 212, 219, 233 cyclophosphamide, 61 cytochrome, 4, 20, 23, 43, 73, 125, 138, 183 cytokines, 5, 7, 17, 22, 33, 47, 51, 98, 118, 186, 199, 200, 207 cytomegalovirus, 206 cytometry, 12, 31 cytotoxicity, 52, 53, 61, 204

Index

D damage, 24, 32, 33, 47, 51, 68, 78, 92, 95, 110, 120, 207, 210, 217, 218 database, 72, 103, 114, 168, 169, 170, 221 death, viii, 2, 18, 20, 21, 42, 43, 45, 48, 85, 92, 107, 158, 211, 213 defense, 33 defense mechanisms, 33 deficiency, 119, 155 definition, 165 degradation, 112, 158 dehydration, 121 delivery, 156 dendritic cell, 21, 77, 79, 82 density, 11, 13, 19, 28, 105, 124, 189, 206 deprivation, 166 derivatives, x, 42, 140, 196, 197, 202, 209, 210, 214, 216, 218, 219, 220, 231 detection, 2, 12, 37, 191 developed countries, 187 diabetes, ix, 146, 160, 164, 172 diabetic patients, 168 dialysis, 164 diarrhea, ix, 145, 151 diastolic blood pressure, 168, 169 dielectric constant, 221, 222 diet, 8, 28, 30, 87, 91, 92, 97, 98, 104, 105, 106, 107, 110, 158, 160, 161, 162, 163, 166, 167, 176, 177, 178 dietary fat, 70 dietary habits, viii, 85, 97 dietary supplementation, 98 differentiation, 4, 9, 21, 27, 95, 131, 134, 141, 187, 189, 190, 193, 205, 207 diffusion, 147, 156 disorder, 156 distribution, 7, 44, 45, 176 diuretic, ix, 145, 151, 154, 161, 165, 166, 171 diversity, 71 division, 3, 36, 45, 58 DNA, 2, 26, 36, 93, 95, 97, 109, 110, 113, 123, 135, 148 DNA repair, 2 dogs, 163 domain, 18, 212, 215, 219, 220, 231 dominance, 60 dosage, 62, 64, 154 dosing, 60, 218 double blind study, 25, 48

241

double-blind trial, 167 down-regulation, 22, 44, 46, 99 drug efflux, 139 drug half-life, 165 drug interaction, 218 drug therapy, 21, 41, 161, 163, 175 drug treatment, 28, 172 drugs, vii, ix, x, 3, 23, 24, 28, 29, 32, 34, 44, 46, 48, 50, 54, 55, 62, 63, 66, 70, 75, 77, 119, 120, 128, 131, 134, 146, 154, 156, 160, 163, 164, 165, 166, 167, 171, 173, 182, 183, 184, 187, 189, 191, 192, 193, 196, 197, 199, 200, 201, 202, 203, 206, 209, 213, 217, 218, 219, 230, 231 duodenal ulcer, 65 duration, 30, 57, 62, 153 dysplasia, 9, 13, 76, 93, 108, 112

E eating, 87, 91, 93 ECM, 38 economic status, 87 edema, ix, 22, 146, 151, 152, 154, 160, 164, 165, 166, 169, 173, 177, 178, 211 effusion, 164 elderly, 54, 153, 154, 155, 159, 160, 161, 169, 173, 175, 176, 177, 178 elders, 167 electrolyte, ix, 146, 154, 159, 163 ELISA, 188, 190, 191 ELISA method, 190 embryo, 83 emission, 190, 191 encoding, 41, 117 endothelial cells, 19, 20, 39, 42, 57, 72, 99, 118, 124, 130, 136, 159, 214 endothelium, 80, 155 enthusiasm, viii, 85, 103 environment, 96, 212, 230 environmental factors, 8, 86, 87, 111 enzymatic activity, vii, 1, 48, 56, 98, 118, 187, 202 enzyme immunoassay, 32, 190 enzymes, viii, 4, 7, 8, 22, 23, 34, 36, 43, 58, 59, 109, 115, 116, 118, 121, 123, 147, 148, 158, 184, 186, 210, 211, 212, 214, 215, 217, 218, 224, 231 eosinophilia, 156 eosinophils, 50 epidemiology, 64, 78, 87, 105 epithelia, 118

Index

242

epithelial cells, 5, 7, 10, 13, 28, 32, 38, 42, 58, 64, 71, 77, 78, 92, 97, 119, 129, 130, 186, 200, 207 epithelium, 7, 9, 12, 18, 35, 92, 97, 113 equilibrium, 222, 223 esophagus, 59, 106, 108, 113 ESR, 205 ester, 5, 78, 99, 102, 113, 188, 206 estrogen, 25, 125 ethanol, 7, 188, 191, 192, 195, 196, 198, 199 ethnicity, 87 evidence, 14, 21, 24, 25, 30, 34, 37, 52, 57, 59, 61, 86, 87, 91, 96, 103, 108, 109, 113, 115, 116, 119, 120, 123, 124, 125, 126, 127, 128, 129, 135, 140, 160, 171, 203, 206, 211 evolution, 187 excitation, 190, 191 exclusion, 160 excretion, ix, 91, 120, 145, 146, 149, 150, 151, 154, 155, 156, 160, 161, 162, 164, 166, 167, 171, 174 exons, 117, 138 experimental condition, 9 exposure, 29, 40, 45, 107, 156, 157 expression, viii, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 46, 47, 49, 51, 56, 58, 62, 63, 64, 65, 66, 69, 71, 72, 73, 76, 77, 79, 80, 81, 82, 83, 95, 98, 99, 111, 112, 113, 115, 116, 118, 119, 123, 124, 125, 126, 127, 129, 130, 133, 135, 136, 137, 138, 139, 140, 147, 149, 157, 158, 159, 175, 176, 186, 200, 204, 206, 207, 211

F failure, ix, 146, 158, 163, 176 family, x, 5, 21, 36, 38, 121, 127, 181, 185, 192, 202, 214, 219 fat, 87, 88 fatty acids, 116, 134, 210, 219 FDA, 23, 24, 25, 48, 49, 54, 169, 170, 177, 178, 185 feedback, 39, 150, 158, 176 females, 54, 87, 91, 92, 119 fever, 24, 146, 149, 156, 183, 210, 211 fibers, 90 fibroblast growth factor, 7, 17, 37, 38, 124, 136 fibroblasts, 5, 7, 20, 37, 42, 65, 83, 138 filtration, ix, 145, 150, 151, 161, 162, 163, 164 first generation, 87 fish, 87, 88, 89, 90, 91, 100 flavonoids, 91

flexibility, 222, 231 fluid, 93, 122, 154, 165 fluorescence, 129, 189, 190, 191, 192, 193, 194 focusing, 24, 87, 91, 97, 98 folate, 88 folic acid, 93, 108 food, 87, 88, 90, 91, 92, 95, 107, 108 food intake, 107, 108 free energy, 227 fruits, 87, 96, 106, 108

G gastrectomy, 8 gastric mucosa, 4, 6, 7, 9, 10, 12, 13, 16, 32, 33, 34, 36, 38, 49, 51, 52, 70, 72, 76, 79, 82, 93, 97, 98, 110, 112, 183, 207 gastric ulcer, 7, 79, 111 gastrin, 30, 47, 72 gastritis, 7, 13, 32, 47, 76, 79, 82, 92, 93, 108, 109, 110, 111 gastroesophageal reflux, 86 gastrointestinal bleeding, 119 gastrointestinal tract, viii, 3, 5, 23, 31, 47, 49, 51, 74, 76, 80, 85, 103, 121, 207 gene, x, 5, 6, 7, 8, 9, 18, 20, 21, 23, 26, 27, 36, 37, 40, 41, 43, 44, 47, 58, 63, 71, 78, 79, 80, 83, 95, 99, 111, 116, 117, 118, 119, 125, 129, 130, 132, 136, 137, 138, 140, 141, 148, 149, 158, 173, 181, 186, 203, 206 gene expression, 7, 20, 36, 47, 58, 63, 79, 80, 95, 118, 125, 130, 136, 137, 138, 141, 158, 186, 203, 206 gene promoter, 118 gene transfer, 41 generalization, 24 generation, 4, 52, 92, 97, 99, 116, 118, 158 genes, 3, 17, 18, 37, 38, 39, 43, 45, 56, 83, 117, 118, 121, 126 genetic factors, 8, 104 genetic mutations, 21 genotype, 34 gland, 8, 58, 119, 129 glioma, 127, 142 glucose, 189 glutathione, 61, 95, 109 glycerol, 127 glycosylation, 129 grains, 89, 91 grids, 220, 222

Index GRIN, 234 grouping, 10 groups, x, 25, 27, 28, 30, 31, 32, 33, 51, 87, 161, 166, 167, 168, 181, 210, 220 growth, vii, 1, 5, 7, 10, 12, 17, 19, 20, 21, 26, 28, 29, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 46, 47, 53, 56, 57, 58, 60, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 80, 81, 82, 93, 95, 99, 102, 105, 109, 112, 114, 116, 118, 123, 125, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 148, 184, 189, 211 growth factor, vii, 1, 5, 7, 10, 17, 20, 36, 37, 46, 56, 57, 65, 66, 67, 68, 70, 71, 72, 73, 74, 80, 82, 112, 118, 130, 132, 136, 137, 141, 148, 184 growth hormone, 47 gut, 23, 25, 48, 51, 54, 69, 81, 133

H half-life, 118, 165, 167 HE, 83 head and neck cancer, 19, 40, 124, 137 healing, 7, 28, 34, 37, 51, 53, 66, 71, 82, 137, 206, 214 health, 53, 81, 92, 93, 103, 104, 200, 203 heart attack, 179 heart disease, ix, 146, 154 heart failure, 153 heat, 109, 189 heating, 223 helicobacter pylori, 70 hematochezia, 28 hematuria, 172 heme, 214, 215 hemodialysis, 163 hepatocellular carcinoma, 24, 26, 125, 134, 136, 138, 139, 140, 141 hepatocytes, 123, 135 hepatoma, 127, 141 high fat, 96 histidine, 220, 221, 224 histology, 10, 27, 34, 51 homeostasis, 6, 151, 152, 156, 159 homogeneity, 4, 87 hormone, 121, 150 hospitalization, 153, 154, 169, 174 host, 8, 21, 40, 41, 87, 200 human neutrophils, 130, 205 human subjects, 160 humoral immunity, 21

243

hydrogen, 116, 206, 214, 220, 223, 224, 225, 226, 227, 229, 230 hydrogen abstraction, 214 hydrogen atoms, 223 hydrogen bonds, 227 hydrogen peroxide, 206 hydroperoxides, 215 hydroxyl, 205 hypercholesterolemia, 206 hyperkalemia, ix, 146, 155, 163, 164, 171, 175, 177 hyperplasia, 9, 58, 207 hypertension, ix, 78, 146, 151, 152, 153, 154, 158, 160, 164, 165, 166, 167, 168, 169, 170, 171, 173, 176, 178, 179 hypertrophy, 36 hyponatremia, 155, 160, 171, 173 hypothesis, 63, 70, 126, 157, 171, 187, 199, 200, 203 hypoxia, 124, 136, 141 hypoxia-inducible factor, 136

I ibuprofen, x, 27, 49, 99, 102, 120, 142, 154, 158, 165, 174, 182, 187, 188, 189, 192, 196, 197, 198, 230 identification, ix, 2, 22, 129, 146, 210 identity, 98 IFN, 5, 22, 200 IL-13, 118, 130 IL-6, 22 IL-8, 30, 47, 129 immigrants, 87 immune function, vii, 1, 17, 35, 56, 157 immune response, 21, 42, 121, 127 immune system, 22 immunity, 21, 41 immunodeficiency, 82 immunogenicity, 157 immunohistochemistry, 12, 13, 27, 31 immunoreactivity, 7, 12, 13, 159 immunosuppression, 9 in situ hybridization, 27, 129 in vitro, x, 10, 19, 21, 23, 26, 31, 37, 38, 40, 42, 43, 46, 49, 52, 57, 62, 64, 76, 81, 93, 99, 109, 112, 126, 130, 133, 141, 143, 158, 182, 185, 187, 201, 206, 210, 211 incidence, viii, 8, 29, 31, 42, 48, 49, 53, 57, 59, 77, 78, 85, 86, 92, 93, 94, 97, 99, 102, 104, 105, 106, 107, 108, 115, 119, 131, 156, 160, 165, 167, 169, 174, 179, 214

244

Index

incubation period, 189 independence, 45, 68 indication, 25 indices, 33 individual differences, 56 inducer, 43 induction, 2, 9, 12, 21, 27, 31, 32, 34, 38, 40, 42, 43, 44, 45, 52, 57, 65, 69, 70, 71, 73, 77, 79, 81, 82, 95, 98, 99, 112, 114, 118, 124, 126, 131, 133, 138, 139, 140, 187, 200, 206, 210, 213 industrialized countries, 86 industry, 22 infection, viii, 7, 8, 11, 12, 13, 14, 15, 16, 30, 31, 32, 33, 34, 65, 66, 69, 71, 80, 82, 86, 96, 97, 100, 104, 109, 110, 111, 187, 188, 199, 200, 202, 203, 207 inflammation, vii, x, 1, 3, 4, 5, 15, 17, 22, 24, 27, 32, 33, 35, 49, 51, 54, 56, 63, 69, 93, 97, 98, 118, 139, 146, 147, 149, 156, 182, 183, 184, 186, 187, 200, 202, 206, 210, 211, 214, 217 inflammatory cells, 137, 202 inflammatory disease, ix, 22, 81, 121, 181, 187, 200, 201, 202, 206, 213, 217 inflammatory mediators, 33, 149, 186 inflammatory responses, 147, 214 influence, 12, 30, 47, 81, 95, 96, 122, 167, 210 ingest, 146, 152 ingestion, 96 inhibition, vii, ix, x, 1, 3, 9, 12, 14, 16, 21, 22, 24, 25, 27, 28, 30, 33, 34, 35, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 65, 66, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 81, 92, 98, 99, 112, 116, 120, 121, 123, 124, 125, 126, 127, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 145, 151, 154, 155, 157, 158, 160, 161, 164, 166, 171, 175, 176, 177, 178, 179, 184, 185, 186, 192, 193, 196, 197, 198, 203, 205, 206, 209, 210, 211, 213, 214, 217, 218, 219, 231, 233 inhibitor, ix, x, 2, 3, 21, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 57, 59, 60, 61, 62, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 78, 80, 81, 82, 83, 99, 104, 112, 113, 114, 121, 128, 132, 133, 134, 140, 141, 142, 146, 151, 158, 161, 163, 165, 166, 167, 168, 169, 171, 172, 177, 178, 179, 180, 185, 193, 196, 197, 199, 200, 201, 202, 204, 207, 209, 220, 221, 222, 223, 227, 229, 230, 231 initiation, 2, 35, 60, 155, 166, 169, 186

injury, viii, 7, 33, 34, 47, 48, 51, 67, 76, 79, 81, 85, 103, 157, 204, 207, 213, 214, 217 input, 222, 223 insertion, 214 insight, 71, 137, 156, 160, 166, 186, 199, 200 instability, 11, 72, 82 integrin, 21, 37, 38, 40, 67 integrity, 28, 121 intensity, 7, 28, 31, 191, 192, 193 interaction, x, 14, 86, 97, 98, 110, 121, 175, 209, 215, 220, 221, 222, 223, 224, 227, 228, 229, 230, 231 interactions, x, 32, 50, 182, 201, 217, 220, 221, 222, 224, 225, 226, 227, 228, 229, 230 interest, vii, ix, x, 2, 3, 40, 59, 95, 128, 165, 181, 186, 200, 202, 209, 217, 219, 231 interference, 33 interferon, 33, 205 interferon-γ, 33 interleukins, 17, 18 interstitial nephritis, 156, 179 intervention, 35, 58, 93, 94, 104, 108, 154 invasive cancer, 34 inversion, 46, 47, 63 ion transport, 43 ionizing radiation, 61 ions, 215 iron, 214, 215, 217, 220, 225 ischemia, 7, 213 isolation, 190 isomers, 46, 63, 148 isozyme(s), 6, 78, 159, 211, 212, 213, 214

K kidney, ix, 118, 130, 145, 146, 147, 149, 151, 156, 157, 158, 159, 160, 161, 162, 163, 164, 173, 174, 175, 176, 179, 184, 213 kidneys, 158, 159 kinase activity, 44 knowledge, 60, 62, 104, 214

L labeling, 32 language, 24, 55 LDL, 187, 200, 203 lead, 13, 15, 16, 22, 30, 49, 155, 158, 165, 166, 171, 185, 210, 214, 217, 219

Index lesions, x, 3, 7, 8, 9, 13, 14, 34, 51, 52, 53, 57, 92, 93, 97, 98, 108, 181, 187, 201, 202, 213 leucocyte, 7 leukotrienes, 49, 50, 156, 210 liberation, 4, 147 life span, 105 lifestyle, 58, 187 ligands, 121, 134, 224, 227 likelihood, 63, 97 linkage, 103 links, 97, 148 lipids, 116, 127, 215 lipooxygenase, 96 liver, ix, 18, 20, 60, 66, 68, 70, 72, 82, 109, 121, 124, 125, 146, 210, 213 liver cancer, 70, 124, 125 liver cirrhosis, 213 liver disease, ix, 146 liver metastases, 20 localization, 121, 136, 157, 158, 159, 184, 207 location, 9, 14, 159 low risk, 62, 161 low-density lipoprotein, 187, 204, 205 LTB4, 56 luminescence, 190 lung cancer, 67, 138, 141, 186, 200, 204, 205 lupus, 159, 173, 174 lycopene, 90 lying, x, 209, 224 lymph, 3, 9, 12, 13, 14, 59, 124, 137 lymph node, 3, 9, 13, 14, 137 lymphocytes, 21, 41 lymphoid, 10, 13, 14 lymphoid tissue, 13 lymphoma, 13, 61, 75

M machinery, 117 macromolecules, 234 macrophages, 5, 7, 20, 21, 41, 57, 130, 131, 149, 189, 192, 193, 194, 195, 199, 202, 203, 205, 207 males, 87, 91, 92 malignancy, 58, 110 malignant tumors, 2 management, 24, 67, 68, 69, 177, 180, 183 market, 23, 104, 128, 171, 231 marketing, 23, 165 mast cells, 118, 131 matrix, 33, 138, 210

245

matrix metalloproteinase, 138, 210 maturation, 79 mean arterial pressure, 155 measurement, 52, 165, 189, 190, 191, 204 measures, 92, 167 meat, 87, 88, 89, 90, 91, 104, 107 media, 99 median, 169 Medicaid, 155 Medicare, 70 medulla, 158 MEK, 140 membranous glomerulonephritis, 172, 173 memory, 127 men, 8, 89, 90, 91, 104, 107 mesangial cells, 157 messenger RNA, 138 meta analysis, 48 metabolism, 4, 50, 74, 92, 95, 116, 120, 127, 143, 147, 159, 171, 175, 183, 210, 217, 219 metabolites, 35, 38, 45, 121, 128, 147, 157, 177, 202, 219 metabolizing, 9, 23, 210 metalloproteinase, 12, 125 metastasis, 2, 9, 11, 12, 13, 14, 29, 36, 37, 46, 60, 66, 82, 124, 137 methylation, 64, 95, 109 mice, 3, 7, 8, 9, 16, 27, 28, 30, 32, 33, 35, 36, 37, 38, 42, 44, 46, 54, 56, 58, 60, 61, 63, 66, 72, 73, 76, 78, 82, 99, 102, 113, 114, 119, 120, 121, 131, 132, 133, 134, 135, 158, 203 microcirculation, 7, 174 micronutrients, 91, 92, 93, 98 migrants, 87, 105 migration, 19, 20, 35, 38, 39, 40, 66, 67, 99, 124, 138 milk, 88, 89 minimal change disease, 156, 172 mitogen, 18, 116, 123, 129, 130, 139, 140 MMP, 125, 127 MMP-2, 125 MMP-9, 127 MMPs, 210 mode, x, 209, 219, 220, 221, 222, 224, 226, 228 models, x, 2, 16, 23, 24, 28, 29, 30, 34, 53, 54, 55, 58, 60, 61, 62, 76, 81, 99, 103, 120, 124, 127, 133, 159, 163, 182, 187, 192, 199, 201, 219, 225, 229 molecular dynamics, 219, 221, 222, 223 molecular oxygen, 214

Index

246

molecular pathology, 68 molecular weight, 118 molecules, 4, 128, 185, 191, 197, 202, 216, 219, 220, 222, 223, 226, 227, 228, 229 molybdenum, 94 monitoring, ix, 146, 168, 173 monocyte chemoattractant protein, 207 morbidity, 4, 62, 93, 184, 187 mortality, viii, 4, 8, 29, 62, 85, 86, 93, 94, 106, 107, 115, 119, 187 mortality rate, 86 mortality risk, 8 mRNA, 4, 5, 7, 9, 12, 14, 16, 18, 26, 27, 51, 52, 63, 71, 76, 79, 99, 101, 117, 118, 129, 130, 136, 138, 157, 175 mucin, 92 mucosa, viii, 5, 6, 7, 9, 12, 13, 21, 32, 33, 36, 51, 52, 53, 62, 72, 81, 85, 103, 110, 149, 186, 214 mucus, 7, 9, 122 mutant, 25, 133, 229 mutation, 15 myocardial infarction, 179, 187

N NaCl, 150, 151, 156, 158, 188 natural killer cell, 21, 36 nausea, ix, 127, 145 necrosis, 17, 52, 129, 157, 160, 199 needs, 6, 165, 214 neoangiogenesis, 98, 99 neovascularization, 124, 137 nephrectomy, 159 nephritis, 156, 179 nephron, 150, 154, 155, 158 nephropathy, 156, 160, 173 nephrosis, 151, 159, 160 nephrotic syndrome, ix, 146, 151 nervous system, 127 network, 37, 230 neurodegeneration, 50 neurons, 130 neutrophils, 50, 57, 188, 189, 195, 196, 197, 201, 202 niacin, 94 nicotinamide, 206 nitric oxide, 16, 18, 49, 62, 75, 81, 97, 202, 206 nitric oxide synthase, 18, 75, 81, 202 nitrogen, 188, 227, 228 nitroso compounds, 95, 97, 107

N-N, 99 non-steroidal anti-inflammatory drugs (NSAIDs), vii, viii, ix, 1, 2, 3, 5, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 70, 72, 74, 75, 76, 77, 78, 79, 80, 83, 85, 98, 99, 102, 103, 112, 113, 115, 119, 120, 123, 124, 125, 126, 127, 128, 140, 141, 145, 146, 149, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 178, 181, 182, 183, 184, 186, 187, 188, 191, 192, 193, 194, 196, 197, 198, 199, 200, 201, 202, 205, 206, 207, 210, 211, 213, 217, 227, 230, 231 norepinephrine, 177 nutrients, 38, 93

O observations, 3, 4, 42, 76, 87, 93, 147, 159, 166, 194, 202 offenders, 175 oils, 90 old age, ix, 146, 160 oncogenes, 8, 17, 18, 37, 38, 58, 71 optimism, 62 organization, 38 orientation, 219, 222, 224, 225, 226, 227, 229, 231 osmolality, 151 osmotic pressure, 92 osteoarthritis, ix, 23, 24, 48, 49, 65, 69, 165, 166, 168, 173, 178, 181 outline, 4 output, 7, 161 ovarian cancer, 119, 124 overload, 151, 154 ovulation, 4, 213 oxidation, 22, 43, 192, 200, 204, 206 oxidative stress, 33, 204, 206 oxygen, x, 38, 116, 182, 209, 220, 228

P p53, 11, 12, 18, 71, 72, 75, 79, 99 pain, vii, ix, 3, 7, 22, 23, 24, 30, 48, 49, 51, 127, 146, 149, 181, 183, 203, 206, 211, 214 palliative, 86 pancreas, 2, 22, 58, 210 pancreatic cancer, 57, 68, 125, 138, 139

Index pancreatitis, 24 Parkinson’s disease, 211 pathogenesis, viii, 60, 78, 86 pathogens, 200, 201 pathology, 42, 173 pathways, 2, 4, 17, 18, 20, 34, 45, 56, 60, 61, 65, 71, 74, 95, 113, 125, 127, 128, 139, 157, 183, 186, 199 PCR, 9, 11, 12, 41, 52 penetrance, 27 penicillin, 189 peptic ulcer, viii, 3, 24, 32, 85, 103 peptides, 5 perforation, 26, 47 perfusion, 149, 151 peripheral blood, 22 permeability, 38, 43, 49, 51, 67, 77, 122 perspective, viii, 2, 62, 108, 178, 206 PGE, 4, 36, 43, 44, 46, 79, 122, 129, 136 pH, 97, 188, 189, 190, 191, 193, 194, 197, 198, 220, 222 pharmacokinetics, 49, 167 pharmacological treatment, 125 pharmacology, 66, 142, 143, 204 phenol, 200 phenotype, vii, 1, 2, 17, 35, 56, 125, 139 phosphatidylcholine, 215 phospholipids, 4, 43, 116, 147, 183 phosphorylation, 36, 46, 127, 140 physiology, 51, 149, 157, 203 pilot study, 177 placebo, 25, 48, 54, 55, 96, 161, 162, 163, 165, 167, 168, 169, 178, 180 plasma, 23, 26, 47, 52, 151, 155, 156, 166, 171, 187 plasma membrane, 155 platelet aggregation, 4, 54, 122, 183 platelets, 49 pleurisy, 6 PM, 73, 75, 78, 177 Poland, 88 polymerase, 9, 12, 138, 159 polymerase chain reaction, 9, 12, 138, 159 polyp(s), 9, 25, 36, 38, 48, 54, 55, 61, 119, 121, 122 polypectomy, 59 polyunsaturated fatty acids, 4, 214 poor, 8, 10, 36, 217 population, viii, 25, 65, 85, 86, 88, 91, 92, 93, 96, 98, 103, 104, 106, 114, 131, 146, 153, 160, 165, 171, 174 positive correlation, 10, 38, 124, 125

247

potassium, ix, 145, 146, 150, 152, 156, 159, 161, 162, 164, 171 potatoes, 90 PPAR ligands, 121 preference, 227, 229, 231 pressure, 154, 166, 167, 168, 169, 170, 171, 175, 178, 179 prevention, vii, viii, 1, 2, 3, 8, 9, 13, 24, 25, 30, 34, 35, 39, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 67, 69, 70, 72, 73, 74, 75, 76, 78, 79, 82, 85, 86, 93, 94, 98, 102, 103, 104, 105, 108, 109, 111, 113, 120, 123, 128, 132, 179, 186, 210 primary tumor, 20, 59 priming, 187, 199 principle, 53, 60 probability, viii, 86, 103 probe, 189, 201, 204, 212, 216, 220, 224 production, vii, x, 1, 3, 4, 5, 16, 17, 19, 21, 22, 23, 32, 33, 35, 36, 37, 39, 45, 49, 51, 54, 56, 58, 110, 122, 123, 124, 126, 127, 132, 137, 138, 147, 148, 151, 156, 157, 158, 159, 166, 181, 186, 192, 193, 194, 195, 199, 200, 201, 202, 203, 205, 213, 214 prognosis, 8, 9, 11, 12, 36, 113, 115, 137 program, 220, 221, 222, 223, 224 proliferation, vii, viii, 1, 10, 19, 20, 26, 28, 29, 32, 33, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 57, 65, 70, 72, 73, 76, 81, 85, 92, 95, 96, 98, 99, 101, 103, 112, 122, 123, 124, 126, 127, 135, 136, 139, 141, 142, 210 promoter, 8, 13, 18, 125 prophylactic, 210, 217 prophylaxis, 24, 70 prostaglandins, vii, ix, 1, 3, 4, 5, 6, 15, 17, 19, 20, 32, 35, 36, 40, 44, 49, 50, 51, 56, 75, 80, 98, 116, 121, 123, 124, 135, 141, 142, 145, 146, 147, 148, 149, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162, 166, 174, 175, 183, 186, 200, 202, 211 prostate, 22, 42, 44, 45, 46, 53, 58, 59, 61, 70, 119, 124, 127, 132, 136, 140, 141, 142, 186, 200, 202, 204, 210, 217, 219 prostate cancer, 44, 45, 46, 59, 70, 127, 136, 140, 141, 142, 186, 204, 210 protective role, 91, 92, 149 protein kinase C, 200 protein kinases, 18, 129, 140 protein sequence, 219 protein synthesis, 175 proteins, 7, 19, 21, 38, 44, 47, 72, 95, 118, 124, 130, 191, 202, 219 proteomics, 219

Index

248 protocol, 221, 222 proto-oncogene, 15 public health, 171, 187 pylorus, 27 pyuria, 172

Q quartile, 87

R radiation, 40, 61, 62, 64 radiation therapy, 40, 61 radical formation, 206 radio, 86 radiotherapy, 61, 63, 128 randomized controlled clinical trials, 24 range, 5, 53, 54, 91, 115, 120, 121, 156, 157, 160, 167 rash, 156 reactive oxygen, 97, 110, 186, 199, 204, 205 reasoning, 61 recall, 91, 162 receptors, 9, 20, 36, 38, 79, 116, 121, 125, 127, 128, 132, 134, 141, 142, 147 recurrence, 34, 55 reduction, ix, 21, 23, 25, 29, 30, 32, 40, 42, 47, 48, 50, 55, 58, 59, 91, 93, 99, 103, 119, 120, 122, 145, 147, 156, 158, 160, 162, 214, 217 redundancy, 60 registry, 92, 103 regression, 25, 27, 34, 48, 97, 98, 121 regulation, ix, 4, 18, 20, 36, 38, 44, 45, 47, 70, 75, 77, 79, 99, 112, 118, 119, 124, 127, 130, 132, 137, 139, 147, 148, 151, 154, 158, 159, 177, 181, 183, 186, 203, 213, 214, 217 regulators, 79, 149 relationship(s), vii, 2, 14, 15, 28, 47, 53, 58, 70, 77, 87, 96, 106, 109, 210, 216 relatives, viii, 77, 86, 104 relevance, 60, 76, 78, 200, 220 reliability, 165 renal artery stenosis, ix, 146 renal dysfunction, 174 renal failure, ix, 146, 153, 160, 172, 177 renal medulla, 149, 158, 166, 176 renin, ix, 146, 147, 150, 155, 156, 158, 159, 165, 176, 213

repair, 93 replacement, 227 replication, 29, 77 repression, 21, 41, 95 residues, x, 184, 209, 211, 212, 213, 216, 219, 220, 221, 224, 225, 226, 227, 228, 229, 231 resistance, 21, 33, 35, 58, 125, 128, 139, 154, 155, 165, 166 resolution, 5, 223 resources, viii, 86, 103 respiratory, 187, 205 restitution, 7 retention, 150, 151, 154, 158, 161, 163, 164, 165, 166, 167, 171 reticulum, 71, 116, 129 retinol, 88 reverse transcriptase, 12 rheumatic diseases, 47, 50, 182 rheumatoid arthritis, ix, 23, 24, 29, 48, 68, 103, 143, 165, 173, 174, 178, 180, 181, 185 rhodopsin, 121 riboflavin, 94 rice, 88, 91, 98, 100 RIE, 21 risk, viii, ix, 2, 3, 7, 13, 16, 22, 23, 24, 25, 29, 30, 32, 33, 48, 53, 54, 55, 57, 59, 65, 66, 67, 68, 72, 73, 75, 78, 79, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 100, 102, 103, 104, 105, 106, 107, 108, 110, 114, 119, 131, 145, 146, 152, 153, 154, 155, 160, 161, 163, 165, 166, 168, 171, 173, 174, 175, 187, 203, 204, 205, 213 risk factors, 3, 65, 105, 106, 153, 160, 163, 171, 187 risk profile, ix, 146, 160 RNA, 118, 130, 159 rodents, 28, 36, 87, 99 room temperature, 189, 191 root-mean-square, 221, 222

S sacrifice, 31, 32 safety, vii, viii, x, 2, 3, 22, 23, 48, 49, 53, 55, 60, 61, 64, 69, 76, 81, 86, 104, 128, 147, 162, 177, 178, 181, 199 salts, 92, 188 sample, viii, 85, 103, 191 saturated fat, 87, 88, 91 scaling, 223 scores, 32 search, 24, 55, 62, 210, 214, 217

Index searching, 219 secretion, 7, 36, 121, 122, 123, 150, 156, 159 sediment, 172 selectivity, 23, 49, 120, 173, 184, 185, 203, 210, 213, 217, 218, 227 selenium, 91, 93, 94, 108, 109 self, 20, 108 seminal vesicle, 118, 129 sensitivity, 220 series, 7, 8, 16, 28, 87, 141, 156, 163 serology, 96 serum, 14, 30, 36, 47, 70, 72, 109, 151, 156, 162, 171, 172, 188, 189 shape, 212 shares, 120 sharing, 98 sheep, 129 side effects, vii, 2, 22, 23, 24, 26, 48, 49, 53, 54, 61, 183, 213, 217 signaling pathways, 4, 20, 127, 136 signalling, 18, 37, 45, 77, 142, 186, 199, 211 signalling pathways, 186, 199 signals, 121 similarity, 212, 215, 219 simulation, 222, 223 sites, 18, 49, 51, 67, 114, 118, 124, 125, 147, 148, 149, 160, 186, 213, 214, 220, 223, 224, 229, 234 skeletal muscle, 206 skin, 2, 22, 53, 58, 59, 119, 121, 131, 134, 135, 138, 210 skin cancer, 134 smokers, 93, 108 smooth muscle cells, 57, 129, 136, 157, 159, 176, 204, 206 sodium, ix, 7, 22, 69, 88, 91, 139, 145, 146, 149, 150, 151, 152, 154, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 169, 171, 176, 188, 189 software, 28, 221, 232 solid tumors, 40 solvation, 227 soy bean(s), 98, 100, 215, 219 species, 47, 63, 97, 182, 186, 189, 197, 199, 200, 201, 204, 205, 215, 227, 229 specificity, 48, 54, 184, 214, 219, 220, 221, 228, 229 spectrophotometry, 33 spectrum, vii, 2, 8, 13, 59, 64, 118 spin, 205 spinal cord, 184 sprouting, 124 squamous cell, 66

249

squamous cell carcinoma, 66 stability, 118, 130, 219, 222, 224 stabilization, 71, 124, 136, 229, 230 stages, 9, 34, 58, 75, 86, 95, 133, 137, 163 starch, 104 starch polysaccharides, 104 stasis, 7 statistics, 69, 104 stenosis, 159 steroids, 183 stimulus, 60 stock, 189, 191, 198 stomach, 3, 6, 7, 8, 9, 13, 15, 28, 29, 30, 31, 42, 51, 52, 53, 58, 59, 62, 70, 76, 78, 80, 86, 92, 93, 94, 97, 105, 106, 107, 108, 109, 111, 112, 113, 114, 118, 149, 213 storage, 95 strain, 188 strategies, 2, 49, 53, 59, 60, 61, 62, 86, 210, 215 stress, ix, 140, 145, 157, 162 stroke, 171 stroma, 35, 37 stromal cells, 13, 28, 40, 58, 138 structural knowledge, 224 structural modifications, 43, 60 submucosa, 10 substrates, 22, 228 sucrose, 188 sugar, 88 sulfonamide, 133, 213 sulfur, 230 Sun, 65, 81, 108, 129, 130, 137, 140, 176 supply, 38, 58 suppression, 35, 36, 37, 42, 43, 45, 49, 52, 55, 77, 99, 127, 135, 155, 193 surface area, 63 survival, 8, 19, 21, 26, 30, 37, 39, 40, 44, 45, 46, 58, 63, 73, 79, 86, 115, 127, 174, 176 survival rate, 8, 86 susceptibility, 33, 56, 69, 86, 87, 109, 173, 214 suspensions, 191 swelling, vii switching, 125 symptoms, 22, 50, 91, 211 syndrome, ix, 8, 26, 146, 176, 179 synthesis, vii, 1, 3, 4, 15, 18, 19, 27, 32, 33, 34, 36, 39, 40, 42, 43, 44, 47, 49, 56, 76, 78, 79, 93, 116, 123, 124, 126, 130, 135, 147, 148, 149, 150, 151, 152, 155, 156, 157, 158, 164, 166, 175, 177, 206, 213, 217

Index

250

systems, x, 23, 36, 57, 182, 201, 202, 206, 217, 223 systolic blood pressure, 166, 167, 168, 169, 170 systolic pressure, 171

T T cell, 137 T lymphocytes, 9 targets, 33, 57, 60, 61, 62, 74, 108, 116, 118, 126, 128, 141, 186, 187, 199, 200, 204, 210 TCC, 189 temperature, 88, 190, 221, 222, 223 TGF, 77, 118, 123, 129, 135, 158 theory, 97 therapeutic agents, 24, 156 therapeutic approaches, 200 therapeutic targets, 134 therapeutics, 3, 67, 71 therapy, vii, viii, ix, 2, 3, 21, 38, 58, 59, 61, 63, 64, 67, 75, 85, 86, 104, 110, 111, 145, 146, 151, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 169, 171, 172, 173, 175, 178, 179, 204, 210 threshold, 40 thromboxanes, 4, 117 time, viii, 6, 9, 26, 34, 44, 45, 57, 64, 86, 87, 99, 104, 120, 128, 167, 168, 169, 171, 173, 221, 223, 230 tissue, 6, 7, 10, 14, 51, 52, 53, 58, 61, 62, 63, 109, 113, 119, 125, 138, 157, 159, 183, 210 tissue homeostasis, 183 TNF, 22, 30, 47, 49, 118, 174, 184 TNF-alpha, 30, 47, 49, 184 TNF-α, 22 tobacco, 27, 111 toxic effect, viii, 43, 85, 103, 196, 199 toxicity, 23, 24, 25, 26, 43, 47, 49, 59, 61, 62, 81, 119, 128, 131, 133, 143, 146, 161, 173, 175, 189, 213, 217, 231 toxin, 16 trace elements, 14 trajectory, 223, 231 transcription, x, 9, 18, 20, 39, 45, 118, 121, 126, 127, 138, 139, 148, 158, 159, 181 transcription factors, 121, 127 transduction, 121, 125 transfection, 35 transformation, 8, 58, 83, 97, 119, 195 transforming growth factor, 12, 118, 123 transition, 57, 77

translation, 118 translocation, 45, 66, 112 transmission, 7 transport, 150, 156 trend, 87, 91, 92, 169, 227 trial, 27, 48, 54, 59, 65, 69, 93, 108, 110, 111, 131, 160, 169, 170, 173, 177, 178, 179, 180 triggers, 71, 148, 187, 205 tryptophan, 221 tumor, vii, 1, 2, 3, 5, 6, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 25, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 51, 53, 56, 57, 58, 59, 60, 62, 63, 64, 66, 67, 69, 71, 72, 74, 75, 76, 77, 81, 93, 98, 99, 102, 103, 108, 110, 113, 115, 116, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 131, 132, 133, 134, 135, 137, 138, 140, 141, 157, 204 tumor cells, vii, 1, 14, 17, 21, 28, 29, 35, 36, 40, 43, 56, 72, 93, 108, 116, 125 tumor growth, vii, 1, 2, 10, 19, 34, 35, 37, 38, 39, 40, 42, 46, 47, 57, 58, 81, 93, 121, 122, 128, 133, 135, 137 tumor invasion, 36 tumor necrosis factor, 5, 33, 51, 77, 118, 140, 157 tumor progression, 3, 21, 37 tumour growth, 17, 20, 210 turnover, 32 twins, 106 type 2 diabetes, 179 tyrosine, 36, 38, 61, 71, 80, 175

U ulcer, 7, 13, 23, 33, 34, 37, 51, 71, 76, 137 uncertainty, 33 urinary bladder, 2, 22, 134, 210 urinary bladder cancer, 134 urine, 91, 161, 167, 172

V valine, 212 values, 42, 55, 160, 218, 223, 227 variability, 23, 91 variables, 11 variation, 87, 91, 168 vascular endothelial growth factor (VEGF), 17, 122 vasculature, 37, 38, 124, 157 vasoconstriction, 54, 151, 152, 153, 155

Index vasodilator, 166, 213, 214, 217 vasopressin, 151 vector, 35, 41, 99 vegetables, 87, 88, 89, 90, 91, 92, 95, 104, 106, 108 VEGF expression, 10, 37, 99, 124, 136, 137 vehicles, 198 vein, 99, 189 vessels, 155 vitamin C, 106 vitamin supplementation, 93

W warrants, 61 water, ix, 95, 145, 146, 147, 149, 150, 151, 152, 154, 155, 156, 158, 159, 160, 166, 171, 213, 220, 221, 222, 223 water absorption, 150 wavelengths, 191 weight gain, 46, 165, 166 well-being, 104

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wild type, 35, 229 withdrawal, x, 54, 71, 181, 199 women, 8, 89, 90, 91, 104, 107 work, 1, 64, 128, 182, 232 workers, 107, 200, 201 World Health Organization, 87, 107 writing, 64

X xenografts, 28, 29, 44, 114, 121, 133 X-ray diffraction, 212

Y yield, 38, 60, 116

Z zinc, 94