Differential sensitivity of normal and malignant breast epithelial cells to genistein is partly mediated by p21(WAF11)

Genistein, a soy metabolite, is a potential chemopreventive agent against v arious types of cancer, There are several studies documenting molecular alt erations leading to cell cycle arrest and induction of apoptosis in a varie ty of cancer cells; however, no studies, to date, have shown the effect of genistein in isogenic normal and malignant breast epithelial cells, In this study, we investigated whether genistein shows any differential sensitivit y to normal (MCF10A and MCF12A) and malignant (MCF10CA1a and MDA-MB-231) br east epithelial cells, We found that genistein causes a greater degree of G (2)-M arrest and induces apoptosis in malignant cell lines compared with no rmal breast epithelial cells. After genistein treatment, flow cytometric an alysis revealed a hyperdiploid population in malignant cells that was not o bserved in normal cells. Cell cycle regulator p21(WAF1), which is known to be up-regulated by genistein treatment, was greatly induced at RNA and prot ein levels in normal cells, whereas its level was only slightly induced in malignant MDA-MB-231 cells and not detectable in malignant MCF10CA1a cells, Therefore, we investigated the causal role of p21(WAF1), the differential sensitivity of genistein among these cell lines. We examined the effects of genistein on p21(WAF1) -/- and p21(WAF1) +/+ HCT 116 cells, which were used as controls prior to studies on breast cancer ce lls. We found that there was a greater degree of cell cycle arrest and apop tosis in p21(WAF1) -/- cells compared with p21(WAF1) +/+ HCT116 cells after genistein treatment, Flow cytometric analysis after genistein treatment sh owed a significant number of p21(WAF1) -/- cells in the hyperdiploid popula tion, which are probably programmed to die through apoptotic processes, To further confirm the causal role of p21(WAF1) in genistein-mediated cell cyc le arrest and apoptosis, we down-regulated p21(WAF1) by antisense p21(WAF1) cDNA transfection experiments. We found that both normal and malignant p21 (WAF1) antisense (AS)-expressing clones became more sensitive to G(2)-M arr est after genistein treatment. Flow cytometric analysis showed an increase in the hyperdiploid population in the AS clones. Further evaluation showed an increase in apoptosis in malignant AS clones but not in normal breast ep ithelial AS clones. These results suggest that p21(WAF1) may play an import ant role in determining the sensitivity of normal and malignant breast epit helial cells to genistein.