Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin

Components of vascular basement membrane are involved in regulating angioge nesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV colla gen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putat ive 54-132-amino acid Tum-5 domain, and the activity mediated by alpha (v)b eta (3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coil and Pichia Pastoris specifically inhibit ed proliferation and caused apoptosis of endothelial cells with no signific ant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G(1) endothelial cell cycle arres t. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo usin g 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 hu man endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha (v)beta (3) integrin may also function as a negative regulator of angiogenesis. Taken together, the se findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candid ate for the treatment of cancer.