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., alpha3beta1, alpha5beta1, alpha6beta1, and alpha v beta3) as well as by the
peptide GRGDS. Indirect flow cytometric analysis of
hepatoma cells with anti-
integrin mAbs demonstrated that alpha6beta1 was uniformly expressed at a high density, while alpha3beta1, and alpha5beta1 were moderately expressed and alpha v beta3 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 alpha3beta1, alpha5beta1 and alpha v beta3). In addition, the binding of
FITC-conjugated
triflavin to
hepatoma cells was inhibited by
GRGDS, anti-alpha3beta1, antialpha5beta1, and anti-alpha v beta3 mAbs. Among these mAbs, anti-alpha5beta1 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., alpha5beta1, alpha3beta1, and alpha v beta3) expressed on the surface of
hepatoma cells, resulting in inhibition of
hepatoma cell adhesion to extracellular matrices (i.e.,
fibronectin and
vitronectin).