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. Immunofluores cence microscopy and flow cytometric analysis using anti-integrin mAbs as the primary binding ligands demonstrated that the platelet integri n receptor alpha(IIb)beta(3), as well as alpha(v) beta(3), alpha(5) be ta(1) and alpha(6) beta(1), are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against alph a(IIb)beta(3) and alpha(5) beta(1), inhibited unstimulated basal adhes ion to fibronectin by approximately 30% and 40%, respectively. The sur face immunoreactivity of tumor cells for alpha(IIb)beta(3) Was enhance d by pretreatment (5 min) with a phorbol ester (12-0-tetradecanoylphor bol-13-acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid , 12-hydroxyeicosatetraenoic acid (12-HETE) in a dose- and time-depend ent manner. SW-480 cells possess a large intracellular poor of alpha(I Ib)beta(3) from which the receptor complex translocates to the cell su rface following pretreatment with TPA or 12(S)-HETE. This pretreatment enhances adhesion to fibronectin, which is mediated exclusively by al pha(IIb)beta(3) integrins. Staurosporine was found to block alpha(IIb) beta(3) up-regulation and enhanced-adhesion. TPA and 12(S)-HETE also f acilitated the redistribution of alpha(IIb)beta(3) during the enhanced -spreading process. Rhodostomin, an Arg-Gly-Asp- (RGD) containing anti platelet snake venom peptide, was about 400-times more potent than RGD S at inhibiting control, TPA- or 12(S)-HETE-enhanced adhesion of SW-48 0 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 w ith rhodostomin, suggesting that rhodostomin binds via its RGD sequenc e to multiple integrin receptors (i.e., alpha(IIb)beta(3), alpha(v) be ta(3), alpha(5) beta(1)) expressed on the SW-480 cell surface, inhibit ing cell adhesion to ECM.