Optimization of estrogenic activity detection method using human breast cancer MCF-7 cells

We tried to optimize the assay system using human breast cancer MCF-7 cells for detection of estrogenic activity of environmental estrogens. Since fetal bovine serum (FBS) contains various growth factors including estrogen, 100 ml of FBS was treated with 5 g of activated charcoal to remove estrogen. MCF-7 cells were cultured with RPMI 1640 medium supplemented with FBS or charcoal-treated FBS (cFBS), in the presence of 17β-estradiol or flavonoids. Then, the cells were trypsinized and cell number was counted using a Coulter Counter. When the cells were cultured in the medium containing 0 to 5% FBS, proliferation-stimulating activity of daidzein and genistein was detected most strongly in the medium containing 1% FBS. In the case cFBS-supplemented medium, the effect of flavonoids was detectable, in all cFBS concentrations. The proliferation-stimulating effect of daidzein and genistein was detectable after a 72-hr lag period. In the presence of 1% FBS or 1% cFBS, 17 β -estradiol enhanced proliferation of the cells at the concentrations between 10^-11 and 10^-8M, while daidzein and genistein enhanced it only at 10^-8M. On the other hand, quercetin and luteolin exerted a proliferation-inhibiting activity against MCF-7 cells at the concentrations over 10^-11M. These results indicate that estrogenic activity of flavonoids against MCF-7 cells was detectable in the medium supplemented with 1% FBS or 1 to 5% cFBS. Proliferation-inhibitory activity of quercetin and luteolin suggests that these compounds exert anti-estrogenic and anti-cancer activities.