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

Yohei Maeshima,Mark Manfredi,Corinne Reimer,Kathryn A Holthaus,Helmut Hopfer,Babi R Chandamuri,Surender Kharbanda,Raghu Kalluri

doi:10.1074/jbc.m007764200

Abstract

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 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 alpha(v)beta(3) 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, alpha(v)beta(3) 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.

Highlights

Components of vascular basement membrane are involved in regulating angiogenesis
Tumstatin consists of 244 amino acids including 12 amino acids from the triple helical portion located in the N-terminal portion, and 232 amino acids derived from the NC1 domain
The switch to an angiogenic phenotype requires both up-regulation of angiogenic stimulators and down-regulation of angiogenesis inhibitors [1]

Summary

Introduction

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of ␣3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. 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 show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. These studies for the first time suggest that through the action of endogenous inhibitors, ␣v␤3 integrin may 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. Recent studies from several different laboratories have shown that endogenous inhibitors of angiogenesis are produced de novo to orchestrate a systematic regulation of tumor uptake and growth [3,4,5,6,7]

Methods

Results

Conclusion