CHARACTERIZATION OF INTEGRIN EXPRESSION AND REGULATION ON SW-480 HUMAN COLON ADENOCARCINOMA CELLS AND THE EFFECT OF RHODOSTOMIN ON BASAL AND UP-REGULATED TUMOR-CELL ADHESION

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-integrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor alpha(IIb)beta(3), as well as alpha(v) beta(3), alpha(5) beta(1) and alpha(6) beta(1), are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against alpha(IIb)beta(3) and alpha(5) beta(1), inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for alpha(IIb)beta(3) Was enhanced by pretreatment (5 min) with a phorbol ester (12-0-tetradecanoylphorbol-13-acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular poor of alpha(IIb)beta(3) from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)-HETE. This pretreatment enhances adhesion to fibronectin, which is mediated exclusively by alpha(IIb)beta(3) integrins. Staurosporine was found to block alpha(IIb)beta(3) up-regulation and enhanced-adhesion. TPA and 12(S)-HETE also facilitated the redistribution of alpha(IIb)beta(3) during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp- (RGD) containing antiplatelet snake venom peptide, was about 400-times more potent than RGDS at inhibiting control, TPA- or 12(S)-HETE-enhanced adhesion of SW-480 cells to fibronectin. The binding of mAbs against alpha(IIb)beta(3), alpha(v) beta(3) and alpha(5) beta(1) was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrin receptors (i.e., alpha(IIb)beta(3), alpha(v) beta(3), alpha(5) beta(1)) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.