Characterization of integrin expression and regulation on SW-480 human colon adenocarcinoma cells and the effect of rhodostomin on basal and upregulated tumor cell adhesion

Abstract

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-antigrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor α IIb β 3 , as well as α v β 3,α 5β 1 and α 6β 1 , are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against α IIb β 3 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for α IIb β 3 was enhanced by pretreatment (5 min) with a phorbol ester ( 12-O- tetradecanoylphorbol-13- acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hdyroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of α IIb β 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 exclusively by α IIb β 3 integrins. Staurosporine was found to block α IIb β 3 up-regulation and enhanced-adhesion. TPA and 12-(S)_- HETE also facilitated the redisitribution of α IIbβ 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp-(RGD) containing antiplatelet snake venom peptide, was about 400-times more patent than RGDS at inhibiting control, TPA- or 12-(S)- HETE -enhanced adhesion of SW-480 cells tp fibronectin. The binding of mAbs against α IIb β 3, α v β 3 and α 5 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrain receptors (i.e., α IIb β 3 , α v β 3 , α 5β 1 ) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.