A Cell Binding Domain from the α3 Chain of Type IV Collagen Inhibits Proliferation of Melanoma Cells

Abstract

Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the ␣3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the ␣1, ␣2, ␣4, and ␣5(IV) chains did not exhibit this property. Because of the intimate relationship between metastasizing neoplastic cells and vascular as well as epithelial basement membranes, we measured the cell adhesionpromoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide ␣3(IV)185-203 (CNYYSNSYSFWLASLNPER) not only promotes adhesion of human melanoma cells but also inhibits their proliferation. Adhesion increased by 50 -60% over control. Melanoma cell proliferation was inhibited by 40% when cells were grown in a medium containing 5 g/ml peptide for 5 days. Studies showed that replacement of serine in position 189 or 191 by alanine resulted in significantly reduced adhesion. Similarly, serine replacement resulted in reduced ability to inhibit proliferation. Our data suggest that a region of the NC1 domain of the ␣3(IV) chain, contained within the sequence 185-203, not only specifically promotes adhesion but also inhibits proliferation of melanoma cells. These properties appear to be dependent on the presence of the triplet sequence -SNS-(residues 189 -191), which is unique to the ␣3 chain and may represent an important functional epitope.

VEGF165 requires extracellular matrix components to induce mitogenic effects and migratory response in breast cancer cells

“…Integrins might also play a role in inducing the inhibitory e ect of collagen type IV. Collagen type IV was shown to inhibit proliferation of melanoma cells (Han et al, 1997) and breast cancer cells (Shahan et al, 1999a) by up to 67%. This inhibition was attributed to the Serine-N-Serine sequence of the a3(IV) collagen chain.…”

αvβ3 and αvβ5 Integrins Bind Both the Proximal RGD Site and Non-RGD Motifs within Noncollagenous (NC1) Domain of the α3 Chain of Type IV Collagen

“…These sites correspond to residues 56 -75 (designated peptide T3) and 185-203 of ␣3NC1 domain (17,18). Biological activity of both peptides was shown to be dependent on ␣ v ␤ 3 integrin binding.…”

“…Instead, they identified a non-RGD region comprising residues 54 -132 of the ␣3NC1 domain that bound the ␣ v ␤ 3 integrin, which was later narrowed down to 25 residues using deletion mutagenesis and synthetic peptides (17). Another non-RGD region of the ␣3NC1 domain, residues 185-203, identified by Han et al (18) was demonstrated to inhibit proliferation of melanoma cells, and the receptor for this synthetic peptide was identified as ␣ v ␤ 3 integrin by affinity chromatography (19). Whether these two non-RGD motifs quantitatively account for the adhesive activity of the native RGD-␣3NC1 domain and its capacity to bind ␣ v ␤ 3 integrin has not been addressed.…”

Mammalian collagen IV

“…Recently, we directly identified α v β 3 and α v β 5 integrins as endothelial receptors for α3 NC1 domain by affinity chromatography and confirmed binding of purified integrins in vitro (Pedchenko et al, 2004). So far, specific integrin binding sites have been characterized only for α3 NC1 domain (Han et al, 1997;Maeshima et al, 2000a). One region was initially identified within the first half of α3 NC1 domain (residues 54-132), since corresponding recombinant protein inhibited proliferation of endothelial cells, induced apoptosis, and decreased tumor growth in nude mice (Maeshima et al, 2001a).…”

“…This results in inhibition of the Cap-dependent protein synthesis mediated by focal adhesion/ phosphatidylinositol-3/Akt/mTOR kinases (Maeshima et al, 2002;Sudhakar et al, 2003). A second cell-binding site was ascribed to amino acids 185-203 of α3 NC1 domain based on the inhibition of melanoma cells proliferation by the corresponding synthetic peptide (Han et al, 1997). This peptide binds α v β 3 integrin and CD47/IAP from the cell lysate through specific interaction of β 3 subunit with amino acid triplet SNS (Pasco et al, 2000;Shahan et al, 1999).…”