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.
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.
Chiang
H S
HS
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
Peng
H C
HC
Huang
T F
TF
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001666:Binding Sites, Antibody; D002448:Cell Adhesion / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D016023:Integrins / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D006893:Hydroxyeicosatetraenoic Acids; D016023:Integrins; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; C072026:rhodostomin; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D013755:Tetradecanoylphorbol Acetate