Phosphotyrosine inhibition and control of vascular endothelial cell proliferation by genistein.
Genistein (4',5,7-trihydroxyisoflavone) is a potent anti-angiogenic compound. We investigated the inhibition of phosphotyrosine as a putative signaling mechanism utilized by the drug in modulating basic fibroblast growth factor (bFGF)-mediated vascular endothelial cell proliferation. The studies included the effect of genistein on DNA synthesis, cell viability, phosphotyrosine induction and characterization of the FGF receptor (FGFR). DNA synthesis was attenuated significantly by genistein in a concentration- and time course-dependent manner with relatively low cytotoxicity during a 16-24 hr exposure (IC50 = 12.5 microM; LC50 = 300 microM). Ligand-stimulated cells exhibited significant increases in phosphotyrosine, affecting FGFR and several tyrosine kinase substrates, ranging in size from M(r) 28 to 200 kDa. Inhibition of phosphotyrosine induction as shown by western blots occurred only at high concentrations of the drug (> 500 microM). These results were supported by results obtained using fluorescence immunocytochemistry. FGFR was shown to be FGF-R1 beta 2, a dimer of approximately 85 and 62 kDa, which was prevented from being autophosphorylated when relatively high concentrations of the drug were applied. Low dose (< 20 microM) inhibition of DNA synthesis by genistein did not correlate with the high concentration required 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. Furthermore, in endothelial cells, inhibition of DNA synthesis by genistein is not mediated primarily by the inhibition of protein tyrosine kinase activity.
Koroma
B M
BM
Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
de Juan
E
E
D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D007529:Isoflavones; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D019833:Genistein; D011505:Protein-Tyrosine Kinases