Abstract
Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.
Highlights
Until the peptide region is exposed by truncation of the ␣3(IV)noncollagenous 1 (NC1) domain, a requirement not essential for the anti-angiogenic activity of this domain
Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen ␣2 chain with specific anti-angiogenic properties
We demonstrate the pivotal role of the NC1 domain of the ␣3 chain of human type IV collagen [13, 26] produced as a recombinant protein, in inhibiting the proliferation of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angiogenesis and tumor growth and in inducing endothelial cell-specific apoptosis
Summary
Until the peptide region is exposed by truncation of the ␣3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Type IV collagen is expressed as six distinct ␣-chains, namely, ␣1-␣6 [12], assembles into triple helices, and further forms a network to provide a scaffold for other macromolecules in basement membranes. These ␣-chains are composed of three domains, the N-terminal 7 S domain, the middle triple helical domain, and the C-terminal globular noncollagenous domain (NC1)1 [13]. They found that the specific sequence, -SNS-, located within amino acids 189 –191, was required for both the melanoma cell adhesion and inhibition of proliferation [19] These investigators did not use the isolated human ␣3(IV) NC1 domain in these studies [19]. The 185–203 ␣3(IV) NC1 synthetic peptide was not tested on other cell types, including endothelial cells
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