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 matrices such as fibronectin and vitronectin has been shown to be mediated through an arginine-glycine-aspartic acid (RGD) sequence within adhesive proteins. Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins. Disintegrins have been isolated from the venom of various vipers and have been shown to be potent inhibitors of platelet aggregation. In this study, we found that human hepatoma cell adhesion to immobilized matrix proteins (i.e, fibronectin, collagen, laminin, and vitronectin) was differentially affected by various anti-integrin monoclonal antibodies (mAbs) (i.e., α₃β₁, α₅β₁, α₆β₁, and α(v)β₃) as well as by the peptide GRGDS. Indirect flow cytometric analysis of hepatoma cells with anti-integrin mAbs demonstrated that α₆β₁ was uniformly expressed at a high density, while α₃β₁ and α₅β₁ were moderately expressed and α(v)β₃ was expressed in small amounts on hepatoma cells, consistent with the results obtained from immunofluorescence microscopic analysis. When immobilized on plastic wells, triflavin promoted hepatoma cell attachment; this cell attachment was inhibited by either GRGDS or mAbs against integrins (α₃β₁, α₅β₁, and α(v)β₃). In addition, the binding of FITC- conjugated triflavin to hepatoma cells was inhibited by GRGDS, anti-α₃β₁, anti-α₅β₁, and anti-α(v)β₃ mAbs. Among these mAbs, anti-α₅β₁ exerted the most pronounced inhibitory effect (>70%) on the binding of triflavin to hepatoma cells. Taken together, these results suggest that triflavin binds via its RGD sequence to multiple integrin receptors (i.e., α₅β₁, α₃β₁, and α(v)β₃) expressed on the surface of hepatoma cells, resulting in inhibition of hepatoma cell adhesion to extracellular matrices (i.e., fibronectin and vitronectin).