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-O-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 pool 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.