Abstract
We report the results of a multidisciplinary study on the inhibitory effect of a snake venom disintegrin, contortrostatin, a 13.5 kDa homodimeric protein isolated from Agkistrodon contortrix contortrix (southern copperhead) venom, on breast cancer progression. We demonstrate that contortrostatin binds to integrins and blocks the adhesion of human breast cancer cells (MDA-MB-435) to extracellular matrix (ECM) proteins including fibronectin and vitronectin, but it has no effect on adhesion of the cells to laminin and Matrigel. Contortrostatin also prevents invasion of MDA-MB-435 cells through an artificial Matrigel basement membrane. Daily local injection of contortrostatin (5 μg per mouse per day) into MDA-MB-435 tumor masses in an orthotopic xenograft nude mouse model inhibits growth of the tumor by 74% (p = 0.0164). More importantly, it reduces the number of pulmonary macro-metastasis of the breast cancer by 68% (p < 0.001), and micro-metastasis by 62.4% (p < 0.001). Contortrostatin is not cytotoxic to cancer cells, and does not inhibit proliferation of the breast cancer cells in vitro. However, contortrostatin inhibits angiogenesis induced by the breast cancer, as shown by immunohistochemical quantitation of the vascular endothelial cells in tumor tissue removed from the nude mice. We have identified αvβ3, an important integrin mediating cell motility and tumor invasion, as one of the binding sites of contortrostatin on MDA-MB-435 cells. We conclude that contortrostatin blocks αvβ3, and perhaps other integrins, and thus inhibits in vivo progression.
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Hynes RO: Integrins: Versatility modulation, and signaling in cell adhesion. Cell 69: 11–25, 1992
Cheresh DA: Structural and biologic properties of integrinmediated cell adhesion. Clin Lab Med 12: 217–236, 1992
Giancotti FG, Mainiero F: Integrin-mediated adhesion and signaling in tumorgenesis. Biochim Biophys Acta 1198: 47–64, 1994
Juliano RL, Varner JA: Adhesion molecules in cancer: the role of integrins. Curr Opin Cell Biol 5: 812–818, 1993
Juliano R: Signal transduction by integrins and its role in the regulation of tumor growth. Cancer Metastasis Rev 13: 25–30, 1994
Jacob K, Bosserhoff AK, Wach F, Knuchel R, Klein EC, Hein R, Buettner R: Characterization of selected strongly and weakly invasive sublines of a primary human melanoma cell line and isolation of subtractive cDNA clones. Int J Cancer 60: 668–675, 1995
Kawahara E, Imai K, Kumagai S, Yamamoto E, Nakanishi I: Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of av integrins. J Cancer Res Clin Oncol 121: 133–140, 1995
Liapis H, Adler LM, Wick MR, Rader JS: Expression of αvβ3 integrin is less frequent in ovarian epithelial tumors of low malignant potential in contrast to ovarian carcinomas. Hum Pathol 28: 443–449, 1997
Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, Damsky C: Cooperative signaling by α5β1 and α4β1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol 129: 867–879, 1995
Seftor REB, Seftor EA, Gehlsen KR, Stetler-Stevenson WG, Brown PD, Ruoslahti E, Hendrix MJC: Role of the αvβ3 integrin in human melanoma cell invasion. Proc Natl Acad Sci 89: 1557–1561, 1992
Seftor REB, Seftor EA, Stetler-Stevenson WG, Hendrix MJC: The 72 kDa Type IV collagenase is modulated via differential expression of αvβ3 and α5β1 integrins during human melanoma cell invasion. Cancer Res 53: 3411–3415, 1993
Brooks PC, Stromblad S, Klemke R, Visscher D, Sarkar FH, Cheresh DA: Antiintegrin αvβ3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest 96: 1815–1822, 1995
Brooks PC, Clark RA, Cheresh DA: Requirement of vascular integrin αvβ3 for angiogenesis. Science 264: 569–571, 1994
Brooks PC, Montgomery AMP, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA: Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79: 1157–1164, 1994
Pierschbacher MD, Rouslahti E: The cell attachment activity of fibronectin can be duplicated by small fragments of the molecule. Nature 309: 30–35, 1984
Knudsen KA, Tuszynski GP, Huang TF, Niewiarowski S: Trigramin, an RGD-containing peptide from snake venom, inhibits cell-substratum adhesion of human melanoma cells. Exp Cell Res 179: 42–49, 1988
Trikha M, De Clerk YA, Markland FS: Contortrostatin, a snake venom disintegrin, inhibits α1 integrin-mediated human metastatic melanoma cell adhesion, and blocks experimental metastasis. Cancer Res 54: 4993–4998, 1994
Savage B, Marzec UM, Chao BH, Harker LA, Maraganore JM, Ruggeri ZM: Binding of the snake venom-derived proteins applaggin and echistatin to the arginine- glycine- aspartic acid and recognition site(s) on platelet glycoprotein IIb/IIIa. J Biol Chem 268: 1066–1073, 1990
Juliano D, Wang Y, Marcinkiewicz C, Rosenthal LA, Stewart GJ, Niewiarowski S: Disintegrin interaction with αvβ3 integrin on human umbilical vein endothelial cells: Expression of ligand-induced binding site on α3 subunit. Exp Cell Res 225: 132–142, 1996
Dennis MS, Henzel WJ, Pitti RM, Lipari MT, Napier MA, Deisher TA, Bunting S, Lazarus RA: Platelet glycoprotein IIb/IIIa protein antagonists from snake venoms: Evidence for a family of platelet-aggregation inhibitors. Proc Natl Acad Sci 87: 2471–2475, 1990
Gould RJ, Polokoff MA, Friedman PA, Huang T-F, Holt JC, Cook JJ, Niewiarowski S: Disintegrins: A family of integrin inhibitory proteins from viper venoms. Proc Soc Exper Biol Med 195: 168–171, 1990
Niewiarowski S, McLane MA, Kloczewiak M, Stewart GJ: Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors. Semin Hematol 31: 289–300, 1994
McLane M, Marcinkiewica C, Vijay-Kumar S, Wierzbicka-Patynowski I, Niewiarowski S: Viper venom disintegrins and related molecules [Review]. Proc Soc Exp Biol Med 219: 109–119, 1998
Trikha M, Rote WE, Manley PJ, Lucchesi BR, Markland FS: Purification and characterization of platelet aggregation inhibitors from snake venoms. Thromb Res 73: 39–52, 1994
Sheu J-R, Teng C-M, Huang T-F: Triflavin, an RGDcontaining antiplatelet peptide, binds to GPIIIa of ADPstimulated platelets. Biochem Biophys Res Commun 189: 1236–1242, 1992
Kang I-C, Lee Y-D, Kim D-S: A Novel disintegrin salmosin inhibits tumor angiogenesis. Cancer Res 59: 3754–3760, 1999
Price JE, Polyzos A, Zhang RD, Daniels LM: Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 50: 717–721, 1990
Repesh LA: A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9: 192–208, 1989
Osborne CK, Hobbs K, Clark GM: Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice. Cancer Res 45: 584–590, 1985
Gasparini G, Harris AL: Prognostic significance of tumor vascularity. In: Teicher BA (ed.) Antiangiogenic Agents in Cancer Therapy, Totowa, Humana Press Inc., 1999, pp. 317–339
Long JS: Hypothesis testing and goodness of fit. In: Long JS (ed.) Regression Models for Categorical and Limited Dependent Variables, Thousand Oaks, SAGE, 1997, pp. 85–93
Coller BS, Peerschke EI, Scudder LE, Sullivan CA: A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest 72: 325–338, 1983
Charo IF, Bekeart LS, Phillips DR: Platelet glycoprotein IIb-IIIa like proteins mediate endothelial cell attachment to adhesive proteins and the extracellular matrix. J Biol Chem 262: 9935–9938, 1987
Phillips DR, Charo IF, Scarborough RM: GP IIb-IIIa: The responsive integrin. Cell 65: 359–362, 1991
Chang YS, Chen YQ, Timar J, Nelson KK, Grossi IM, Fitzgerald LA, Diglio CA, Honn KV: Increased expression of aIIbb3 integrin in subpopulations of murine melanoma cells with high lung-colonizing ability. Int J Cancer 51: 445–451, 1992
Chen YQ, Gao X, Timar J, Tang D, Grossi IM, Chelladurai M, Kunicki TJ, Fligiel SEG, Taylor JD, Honn KV: Identification of the αIIbβ3 integrin in murine tumor cells. J Biol Chem 267: 17314–17320, 1992
Peerschke EI, Coller BS: A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin-treated platelets. Blood 64: 59–63, 1984
Liotta LA: Tumor invasion and metastasis - Role of the extracellular matrix: Rhoads Memorial Award Lecture. Cancer Res 46: 1–7, 1986
Felding-Habermann B, Cheresh DA: Vitronectin and its receptors. Curr Opin Cell Biol 5: 864–868, 1993
Wayner EA, Orlando RA, Cheresh DA: Integrins αvβ3 and αvβ5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J Cell Biol 113: 919–929, 1991
Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR: Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21: 6188–6193, 1982
Wong N, Mueller B, Barbas C, Ruminski P, Quaranta V, Lin E, Smith J: αv Integrins mediate adhesion and migration of breast carcinoma cell lines. Clin Exp Metastasis 16: 50–61, 1998
Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA: Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 137: 481–492, 1997
Yeh C, Peng H, Huang T: Accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as integrin αvβ3 antagonist and inducing apoptosis. Blood 92: 3268–3276, 1998
Folkman J: Editorial: Angiogenesis and breast cancer. J Clin Oncol 12: 441–443, 1994
O'Reilly MS, Holmgren L, Chen C, Folkman J: Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 2: 689–692, 1996
O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn ERBJ, Olsen BR, Folkman J: Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277–285, 1997
Boehm T, Folkman J, Browder T, O'Reilly MS: Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390: 404–407, 1997
Honn KV, Tang DG, Chen YQ: Platelets and cancer metastasis: more than an epiphenomenon. Semin Thrombos Hemostas 18: 392–415, 1992
Boukerche H, Berthier-Vergnes O, Tabone E, Dore JF, Laung CL, McGregor JL: Platelet- melanoma cell interaction is mediated by the glycoprotein IIb-IIIa complex. Blood 74: 658–663, 1989
Karpatkin S, Pearlstein E, Ambrogio C, Coller BS: Role of adhesive proteins in platelet tumor interaction In Vitro and metastasis formation In Vivo. J Clin Invest 81: 1012–1019, 1988
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Zhou, Q., Sherwin, R.P., Parrish, C. et al. Contortrostatin, a dimeric disintegrin from contortrix contortrix, inhibits breast cancer progression. Breast Cancer Res Treat 61, 249–259 (2000). https://doi.org/10.1023/A:1006457903545
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DOI: https://doi.org/10.1023/A:1006457903545