TRIFLAVIN, AN ARG-GLY-ASP-CONTAINING PEPTIDE, INHIBITS THE ADHESION OF TUMOR-CELLS TO MATRIX PROTEINS VIA BINDING TO MULTIPLE INTEGRIN RECEPTORS EXPRESSED ON HUMAN HEPATOMA-CELLS

Integrins are a superfamily of cell surface glycoproteins that promote cellular adhesion,.The interaction of integrins with extracellular ma trices such as fibronectin and vitronectin has been shown to be mediat ed through an arginine-glycine-aspartic acid (RGD) sequence within adh esive proteins. Triflavin, a 7.5-kDa cysteine-rich polypeptide purifie d from Trimeresurus flavoviridis snake venom, belongs to a family of R GD-containing peptides, termed disintegrins. Disintegrins have been is olated from the venom of various vipers and have been shown to be pote nt inhibitors of platelet aggregation. In this study, we found that hu man hepatoma cell adhesion to immobilized matrix proteins (i.e, fibron ectin, collagen, laminin, and vitronectin) was differentially affected by various anti-integrin monoclonal antibodies (mAbs) (i.e., alpha(3) beta(1), alpha(5) beta(1), alpha(6) beta(1), and alpha(v) beta(3)) as well as by the peptide GRGDS. Indirect flow cytometric analysis of he patoma cells with anti-integrin mAbs demonstrated that alpha(6) beta(1 ) was uniformly expressed at a high density, while alpha(3) beta(1) an d alpha(5) beta(1) were moderately expressed and alpha(v) beta(3) was expressed in small amounts on hepatoma cells, consistent with the resu lts obtained from immunofluorescence microscopic analysis. When immobi lized on plastic wells, triflavin promoted hepatoma cell attachment; t his cell attachment was inhibited by either GRGDS or mAbs against inte grins (alpha(3) beta(1), alpha(5) beta(1), and alpha(v) beta(3)). In a ddition, the binding of FITC-conjugated triflavin to hepatoma cells wa s inhibited by GRGDS, anti-alpha(3) beta(1), anti-alpha(5) beta(1), an d anti-alpha(v) beta(3) mAbs. Among these mAbs, anti-alpha(5) beta(1) exerted the most pronounced inhibitory effect (>70%) on the binding of triflavin to hepatoma cells, Taken together, these results suggest th at triflavin binds via its RGD sequence to multiple integrin receptors (i.e., alpha(5) beta(1), alpha(3) beta(1), and alpha(v) beta(3)) expr essed on the surface of hepatoma cells, resulting in inhibition of hep atoma cell adhesion to extracellular matrices (i.e., fibronectin and v itronectin).