THE LIPOXYGENASE METABOLITE 12(S)-HETE PROMOTES-ALPHA-IIB-BETA-3 INTEGRIN-MEDIATED TUMOR-CELL SPREADING ON FIBRONECTIN

Tumor-cell interaction with the vessel wall during metastasis involves adhesion, induction of endothelial-cell retraction and spreading on the exposed sub-endothelial matrix. The signals for initiation of tumor-cell spreading and the receptors involved are unknown. A protocol was developed to distinguish between initial tumor-cell (B16 amelanotic melanoma; B16a) adhesion to and spreading on fibronectin. The time for maximum spreading was 50 min. Treatment with a lipoxygenase metabolite of arachidonic: acid [12(S)-HETE] resulted in maximum spreading in 15 min (max. effect approx. 0.1 muM). Other lipoxygenase metabolites were ineffective. 12(S)-HETE treatment induced a rearrangement of F-actin, vinculin, vimentin intermediate filaments and integrin alpha(IIb)beta3, but: not integrin alpha5beta1. Antibodies to alpha(IIb)beta3 but not alpha5beta1 blocked the 12(S)-HETE effect on B16a spreading. B16a-cell attachment to fibronectin resulted in increased metabolism of arachidonic acid to 12(S)-HETE, which was inhibited by lipoxygenase but not by cyclo-oxygenase inhibitors. Accordingly, lipoxygenase inhibitors but not cyclo-oxygenase inhibitors blocked spontaneous B16a-cell spreading. The protein-kinase-C inhibitors calphostin C, H7 and staurosporine also inhibited spreading, while the protein-kinase-A inhibitor H8 was ineffective. These data suggest that B16a-cell spreading on fibronectin is initiated by a lipoxygenase metabolite [12(S)-HETE] of arachidonic acid and is mediated by protein kinase C.