Endostatin Adonia E. Papathanassiu, Shawn J. Green*, Davida K. Grella and B. Kim Lee Sim EntreMed, Inc., 9640 Medical Center Drive, Rockville, MD 20850, USA * corresponding author tel: 301 738 2494, fax: 301 217 9858, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.08009.
SUMMARY Similarly to angiostatin protein, endostatin protein is a proteolytic fragment of collagen XVIII that inhibits proliferation and migration of endothelial cell in vitro and tumor angiogenesis in vivo.
BACKGROUND
Discovery Endostatin protein, an inhibitor of angiogenesis, was isolated from the culture supernatants of a murine hemangioendothelioma cell line (EOMA). N-terminal sequence analysis revealed that endostatin protein is a fragment of collagen XVIII.
Structure Endostatin protein is a 20 kDa fragment of the noncollagenous (NC1) domain of the C-terminal region of collagen XVIII, expressed by endothelial cells and found in the basement membrane and surrounding perivascular spaces. Studies of the X-ray crystal structure of mouse endostatin protein suggest that the protein folds autonomously into a compact globular unit and this unit is structurally related to the carbohydrate-recognition domain of C-type lectins. Crystallization of human endostatin protein revealed a Zn2+-binding pocket.
factor-induced migration and proliferation of endothelial cells. Systemic treatment with endostatin protein results in suppression of tumor growth and regression of established tumors, and to the first demonstration of tumor dormancy therapy in murine models.
GENE AND GENE REGULATION Angiostatin protein and endostatin protein are proteolytic products of plasminogen and collagen XVIII, respectively. Transcripts of a gene encoding angiostatin protein or endostatin protein have not been detected in various organs or tumors. However, plasminogen transcripts are found primarily in the liver and collagen XVIII transcripts are found in a variety of organs and tissue types. This section is not applicable to 2ME2, since it is a small organic molecule. The accession numbers, chromosome location, and tissue expression of the parent molecules, human plasminogen and human collagen XVIII, are given below:
Accession numbers Human collagen XVIII: L22548
Chromosome location Human collagen XVIII: chromosome 21q22.3
Main activities and pathophysiological roles
Cells and tissues that express the gene
Endostatin protein inhibits angiogenesis and tumor growth by specifically inhibiting growth
Human collagen XVIII is expressed in a variety of tissues such as liver, lung, kidney, and, to a lesser
830 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim extent, heart, brain, skeletal muscles, and testis. It has also expressed in cells such as embryonic fibroblasts and vascular endothelial cells.
PROTEIN
Accession numbers Human collagen XVIII: P39060
Important homologies
Sequence See Figure 1.
Description of protein The parent molecule of endostatin protein, collagen XVIII, belongs to a family of collagen-like proteins referred to as multiplexins. Collagen XVIII consists of an N-terminal region with three splice variants, a series of collagen-like domains, and an NC1 domain of approximately 300 residues. Endostatin protein corresponds to the last 184 amino acid residues of the NC1 domain. Although very little is known about endostatin protein, studies have shown that it binds to microfibrillar fibulin-1 and -2 as well as heparin and sulfatides. The last two interactions appear to depend on different binding epitopes.
Discussion of crystal structure Human endostatin protein monomer consists of a seven-stranded sheet at the center of the core structure. One side of the sheet is occupied by an helix ( 1), while the other side is occupied by a sequence of elaborate loops, a short antiparallel sheet, and a second, shorter helix ( 2). The endostatin protein structure contains two disulfide bridges: one between Cys164 and Cys304 and a second one between Cys266 and Cys296. The molecule also possesses a zincbinding site located at the N-terminus of the protein immediately adjacent to the precursor cleavage site. Figure 1 protein. HSHRDFQFVL RLQDLYSIVR FDGKDVLRHP LGRLLGQSAA
Amino acid sequence for human endostatin HLVALNSPLS RADRAAVPIV TWPQKSVWHG SCCHHAYIVL
The site is tetrahedral, with three zinc ligands from the N-terminal region (His1, His3, and His11) and a fourth (Asp76) from the central sheet. Interestingly, the zinc-binding site of endostatin protein resembles the zinc site of matrix metalloproteases. Although the function of such a site in endostatin protein molecules is not clear, X-ray crystallography shows that endostatin protein crystals contain a number of symmetric dimers and that this dimerization may be zinc-dependent.
GGMRGIRGAD NLKDELLFPS SDPNGRRLTE CIEENSFMTA
FQQARAVGLS SGTFRAFLSS WEALFSGSEG PLKPGARIFS SYCETWRTEA PSATGQASSL FSK
As a fragment of collagen XVIII, endostatin protein exhibits a 58% homology with the C-terminus of collagen XV. It also possesses a high degree of structural similarity with the carbohydrate recognition domain (CRD) of mammalian C-type lectins, especially E-selectin and lithostathine, although the corresponding sequence identity is only 9%. Comparison of the endostatin protein and E-selectin structures revealed that the disulfide bridges and helices are in topologically equivalent positions in the two proteins, whereas the position of the central sheet varies. However, unlike E-selectin, endostatin protein does not possess calcium-binding sites. In addition to E-selectin, endostatin protein is structurally similar to human tetranectin, a trimeric plasminogen-binding protein and coagulation factors IX/X-binding protein from snake venom.
Posttranslational modifications As a product of a proteolytic cleavage of collagen XVIII, endostatin protein would not be subject to posttranslational modifications. Endostatin protein does not contain an N-linked glycosylation site.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce Studies have failed to detect endostatin protein in the conditioned media of 10 different human and mouse cell lines including fibroblasts, epithelial, and tumor cells, although low levels of protein (5±10 ng/mL) have been found in the medium of endothelial and teratocarcinoma cells. Increasing concentrations of endostatin protein have been detected in tissue extracts from mouse brain, skeletal muscle, heart,
Endostatin kidney, testis, and liver, indicating that proteolytic digest of collagen XVIII is the source of endostatin protein. This hypothesis is corroborated by studies showing that, in many tissues, NC1 domain of collagen is released from collagen XVIII and retained in the submolecular assembly of basement membranes through noncovalent interactions with other ligands. These studies also suggest that a two-step proteolysis may be required for the release of soluble endostatin protein (Sasaki et al., 1998).
RECEPTOR UTILIZATION The endostatin protein receptor is not known.
IN VITRO ACTIVITIES
In vitro findings
831
Bioassays used The following bioassays were used for the in vitro experiments. Proliferation assay. Subconfluent cultures of endothelial or tumor cells are cultured with growth factor(s) in the presence or absence of an angiogenic inhibitor. The assay is terminated 72±96 hours later. Cell growth is determined by cell counting or DNA synthesis via thymidine or uridine incorporation. Wound migration assay. Confluent endothelial cell monolayers are wounded with a razor blade. Cells from the wound area migrate in the presence of growth factors. After a short incubation in the presence or absence of an angiogenic inhibitor, the cells are fixed and stained and the number of migrating cells are counted under a light microscope.
The anti-angiogenic activity of endostatin protein is summarized in Table 1.
The following bioassays were used for the in vivo experiments:
Regulatory molecules: inhibitors and enhancers
Chorioallantoic membrane (CAM). Disks containing various concentrations of an angiogenic inhibitor are placed on the chorioallantoic membrane of 6-day-old chicken embryos. After 48 hours of incubation, CAMs are checked for the presence of avascular zones in the periphery of the disk using a stereomicroscope.
None known, although ability to bind to Zn2+ may be essential for anti-angiogenic activity (Boehm et al., 1998).
Table 1 Anti-angiogenic activity of endostatin protein Cell type Bovine capillary endothelial (BCE)a,c Human umbilical vein (HUVEC) Bovine aortic smooth muscle
b
a
Inhibition of cell proliferation
Inhibition of cell migration
+,+
+
+
+
ÿ
*ND
Bovine retinal pigment epitheliala
ÿ
ND
3T3 Fibroblasts
ÿ
ND
ÿ
ND
ÿ
ND
ÿ
ND
Mink lung epithelial
a
Hemangioendothelioma (EOMA)a Lewis lung carcinoma (LLC)
a
ND, not done. a
Described by O'Reilly et al. (1997). Reported IC50 value for inhibition of BCE proliferation was 600 ng/mL or 30 nM protein. No significant inhibition was observed for nonendothelial cells, when endostatin protein was tested at doses up to 1 log higher than those used for endothelial cells.
b
Described by Hohenester et al. (1998). Reported IC50 value for inhibition of HUVEC proliferation was 100 ng/mL or 5 nM of protein.
c
Described by Sim et al. (1997). Reported IC50 value for inhibition of BCE proliferation was 0.5±1 mg/mL.
832 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim Mouse cornea micropocket assay. A pellet containing sucrose octasulfate, hydron, and 80±100 ng bFGF is placed into the cornea micropocket of a mouse and the cornea angiogenesis is evaluated by slit-lamp microscopy. Primary tumor model. Mice or rats are implanted with a specific number of a certain tumor cell type. Systemic administration of an angiogenic inhibitor is initiated after the tumors have grown to a palpable size. Tumor size is assessed with a caliper and the tumor volume is determined. Metastatic tumor model. Mice are implanted with tumor cells that exhibit a metastatic potential. The tumors are allowed to grow to a certain size before they are surgically removed. After tumor resection, systemic treatment with an angiogenic inhibitor is initiated. At the end of the experiment, the number of pulmonary metastases and/or the lung weight of treated mice are compared to those of mice that received a control treatment or they did not undergo tumor resection.
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles Endostatin protein inhibits neovascularization via inhibition of growth factor-induced migration and proliferation of endothelial cells. Since detectable levels of circulating protein have been found in healthy subjects, endostatin protein may play a role in the homeostatic regulation of angiogenesis (Sasaki et al., 1998).
Species differences A similarity index of greater than 85% exists between endostatin protein amino acid sequences from various species. Posttranslational modification differences have not yet been studied.
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects A study involving a small group of healthy human subjects suggested that concentrations of
120±300 ng/mL are the physiological levels of circulating endostatin protein (Sasaki et al., 1998). Another study determined the levels of circulating endostatin protein in patients with chronic renal insufficiency to be in the range of 10ÿ10 M (2 ng/mL) or higher (Standker et al., 1997).
Role in experiments of nature and disease states Endostatin protein was shown to inhibit formation of new blood vessels in CAM assay (doses of 10±20 mg per embryo) and in the cornea micropocket assay (O'Reilly et al., 1997). It was also shown to inhibit tumor growth and metastasis in a variety of primary and experimental or spontaneous metastatic tumor models. The inhibitory activity of endostatin protein was attributed to its ability to increase the apoptotic index of the treated tumors while leaving their proliferative index unaltered. Systemic administration of endostatin protein led to complete regression of established tumors due to a potent inhibition of angiogenesis. Discontinuation of the treatment resulted in tumor growth, although repeated cycles of endostatin protein therapy produced permanent tumor dormancy (O'Reilly et al., 1997).
IN THERAPY
Preclinical ± How does it affect disease models in animals? The in vivo biological effects of endostatin protein were determined by the following bioassays: CAM, cornea micropocket assay, primary tumor model, and spontaneous metastatic tumor models. The effects of endostatin protein in numerous disease models are summarized in Table 2 and Table 3.
Toxicity Thus far, no drug-related toxicity has been associated with prolonged endostatin protein treatment of mice bearing Lewis lung carcinoma, T24, fibrosarcoma, or B16F10 melanoma cells. The mice develop normally and weight loss is not observed. In addition, endostatin protein treatment did not result in development of tumor resistance (Boehm et al., 1997).
Endostatin
833
Table 2 Effect of recombinant murine endostatin protein in various tumor models Tumor model
Biological effect
Lewis lung carcinoma (LLC-LM) spontaneous metastatica
Suppression of pulmonary metastases after daily administration of 0.3 mg/kg
Lewis lung carcinoma (LLC) primarya
1. 50% inhibition of tumor growth after daily administration of 2.5 mg/kg 2. Complete inhibition of tumor growth after daily administration of 10 mg/kg 3. Tumor regression after daily administration of 20 mg/kg
B16F10 primarya
Tumor regression
T241 fibrosarcoma primarya
Tumor regression
Hemangioendothelioma (EOMA) primary Gliosarcoma primary
a
Tumor regression
b
Tumor regression
Renal cell carcinoma (768-O RCC)
c
Tumor suppression at 10 mg/kg per day
a
Described by O'Reilly et al. (1997).
b
Described by Sasaki et al. (1998).
c
Described by Dhanabal et al. (1998).
Table 3 Combined effect of angiostatin protein and ionizing radiation in various tumor models Primary tumor model
Angiostatin protein regimen
Radiation regimen
Effect
Lewis lung carcinoma
25 mg/kg daily
20 Gy on days 0 and 1
Tumor regression
D54 human glioma
25 mg/kg once
5 Gy per day: 30 Gy total
Tumor regression
SQ-20B squamous cell carcinoma
25 mg/kg daily
5 Gy per day: 50 Gy total
Tumor regression
PC3 prostate adenocarcinoma
25 mg/kg daily
5 Gy per day: 40 Gy total
Tumor regression
References Boehm, T., Folkman, J., Browder, T., and O'Reilly, M. S. (1997). Antiangiogenic therapy of experimental cancer does not induce acquired resistance. Nature 390, 404±407. Boehm, T., O'Reilly, M. S., Keough, K., Shiloach, J., Shapiro, R., and Folkman, J. (1998). Zinc-binding of EndostatinTM protein is essential for its antiangiogenic activity. Biochem. Biophys. Res. Commun. 252, 190±194. Dhanabal, M., Ramchandran, R., Volk, R., Stillman, I. E., Lombardo, M., Iruela-Arispe, M. L., Simons, M., and Sukhatme, V. (1998). Endostatin: yeast production, mutants, and anti-tumor effect in renal cell carcinoma. Cancer Res. 59, 189±197. Hohenester, E., Sasaki, T., Olsen, B. R., and Timpl, R. (1998). Crystal structure of the angiogenesis inhibitor EndostatinTM protein at 1.5 AÊ resolution. EMBO J. 17, 1656±1664. O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997). Endostastin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 1±20.
Sasaki, T., Fukai, N., Mann, K., Gohring, W., Olsen, B. R., and Timpl, R. (1998). Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J. 17, 4249±4256. Sim, B. K. L., O'Reilly, M. S., Liang, H., Fortier, A. H., He, W., Madsen, J. W., Lapcevich, R., and Nacy, C. A. (1997). A recombinant human Angiostatin1 protein inhibits experimental primary and metastatic cancer. Cancer Res. 57, 1329±1334. Standker, L., Schrader, M., Kanse, S. M., Jurgens, M., Forssmann, W. G., and Preissner, K. T. (1997). Isolation and characterization of the circulating form of human endostatin. FEBS Lett. 420, 129±133.
LICENSED PRODUCTS EndostatinTM Protein is a trademark product of EntreMed, Inc. (Rockville, MD). An endostatin protein ELISA kit is sold by CytoImmune, Inc. (College Park, MD) and EntreMed, Inc.
