Apigenin blocks induction of vascular endothelial growth factor mRNA and protein in progestin-treated human breast cancer cells

Objective: The results of recent clinical trials indicate that hormone therapy with estrogen and progestin is associated with higher risk of breast cancer in postmenopausal women than treatment with estrogen alone or placebo. This observation is consistent with studies showing that progestins stimulate the expression of vascular endothelial growth factor (VEGF), which in turn stimulates angiogenesis. The objective of this study was to examine whether apigenin, a natural flavone, inhibits the progestin-dependent induction of VEGF in human breast cancer cells. Methods: T47-D human breast cancer cells were treated with medroxyprogesterone acetate (MPA; 10 nM) or other synthetic progestins in the presence or absence of antiprogestin RU-486 and variable doses of apigenin (1-100 mu M). BT-474 cells were also treated with MPA +/- 100 mu M apigenin. Secreted VEGF was quantified by enzyme-linked immunosorbent assay, and total cellular VEGF mRNA was quantified by reverse transcriptase polymerase chain reaction. The expression of VEGF receptor-1 (flt), VEGF receptor-2 (flk), progesterone receptor, and estrogen receptor-alpha was also quantified by Western blot analysis and/or reverse transcriptase polymerase chain reaction. Results: Apigenin (50 or 100 mu M) prevented progestin-dependent induction of both VEGF mRNA and protein and reduced progesterone receptor levels in T47-D cells. Apigenin also blocked MPA-dependent secretion of VEGF from BT-474 cells. mRNA levels of progesterone and estrogen receptor-alpha were unaffected by apigenin, which did exert somewhat suppressive, although complex, effects on VEGF receptor expression in MPA-treated T47-D cells. Conclusions: Apigenin blocks progestin-dependent induction of VEGF mRNA and protein and broadly inhibits the ability of progestins to alter the expression of other components of the angiogenesis pathway, including progesterone receptor and VEGF receptors, in human breast cancer cells. Further studies are warranted to explore the potential of apigenin as a chemopreventive agent in postmenopausal women exposed to oral progestins.