OVCAR-5 is a human epithelial carcinoma cell line of the ovary, established from the ascitic fluid of a patient with progressive ovarian adenocarcinoma without prior cytotoxic treatment. The unique growth pattern of ovarian carcinoma makes it an ideal model for examining the anticancer activity of contortrostatin (CN), a homodimeric disintegrin from southern copperhead venom. FACS analysis revealed that OVCAR-5 is integrin ·vß3 negative, but ·vß5 positive. CN effectively blocks the adhesion of OVCAR-5 cells to several extracellular matrix proteins and inhibits tumor cell invasion through an artificial basement membrane. In a xenograft nude mouse model with intraperitoneal introduction of OVCAR-5 cells, intraperitoneal injection of CN was used for therapy. Tumor dissemination in CN-treated versus control groups was studied by gross examination, and antiangiogenic potential was examined by factor VIII immunohistochemistry and image analysis. CN not only significantly inhibited ovarian cancer dissemination in the nude mouse model, but it also dramatically prevented the recruitment of blood vessels to tumors at secondary sites.

Contortrostatin, a 13.5 kDa disulfide-linked homodimeric polypeptide possessing an Arg-Gly-Asp sequence, was isolated from venom of the southern copperhead snake. Daily injection of contortrostatin into the primary tumor of human breast cancer MDA-MB-435 carried in nude mice significantly inhibited tumor growth and neovascularization of the tumor tissue. On the chick embryo chorioallantoic membrane, contortrostatin inhibited angiogenesis induced by MDA-MB-435 cells, basic fibroblast growth factor, and vascular endothelial growth factor. In addition, contortrostatin effectively blocked adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and significantly inhibited invasion of HUVEC through a Matrigel barrier. Competitive binding assays and adhesion assays with different integrin antibodies suggested that integrin alpha(v)beta3 is a binding site for contortrostatin on vascular endothelial cells. Detachment of HUVEC from vitronectin by contortrostatin induced apoptosis. HUVEC adhered and spread well on immobilized contortrostatin without undergoing apoptosis, suggesting that it is the inhibition of adhesion and spreading of HUVEC on extracellular matrix proteins, rather than binding of contortrostatin to integrins per se, that triggers apoptosis. We conclude that contortrostatin binds to alpha(v)beta3, and interferes with the anchorage-dependent survival mechanism of the vascular endothelial cells, and the mobility of the cells. The consequent suppression of angiogenesis is an important component of the antineoplastic activity of contortrostatin.

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