Identification of the Anti-angiogenic Site within Vascular Basement Membrane-derived Tumstatin

Abstract

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of a3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by α vβ3 integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, α vβ3 integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.