PHOSPHOTYROSINE INHIBITION AND CONTROL OF VASCULAR ENDOTHELIAL-CELL PROLIFERATION BY GENISTEIN

Genistein (4',5,7-trihydroxyisoflavone) is a potent anti-angiogenic co mpound. We investigated the inhibition of phosphotyrosine as a putativ e signaling mechanism utilized by the drug in modulating basic fibrobl ast growth factor (bFGF)-mediated vascular endothelial cell proliferat ion. The studies included the effect of genistein on DNA synthesis, ce ll viability, phosphotyrosine induction and characterization of the FG F receptor (FGFR). DNA synthesis was attenuated significantly by genis tein in a concentration- and time course-dependent manner with relativ ely low cytotoxicity during a 16-24 hr exposure (IC50 = 12.5 mu M; LC( 50) = 300 mu M). Ligand-stimulated cells exhibited significant increas es in phosphotyrosine, affecting FGFR and several tyrosine kinase subs trates, ranging in size from M, 28 to 200 kDa. Inhibition of phosphoty rosine induction as shown by western blots occurred only at high conce ntrations of the drug (>500 mu M). These results were supported by res ults obtained using fluorescence immunocytochemistry. FGFR was shown t o be FGF-R1 beta 2, a dimer of approximately 85 and 62 kDa, which was prevented from being autophosphorylated when relatively high concentra tions of the drug were applied. Low dose (<20 mu M) inhibition of DNA synthesis by genistein did not correlate with the high concentration r equired for phosphotyrosine inhibition. The data suggest that although cell stimulation results in phosphotyrosine induction, inhibition of phosphotyrosine is not required for inhibition of DNA synthesis. Furth ermore, in endothelial cells, inhibition of DNA synthesis by genistein is not mediated primarily by the inhibition of protein tyrosine kinas e activity.