Genistein converts Bcl-2 into a proapoptotic protein in Bcl-2 overexpressing MCF-7 breast cancer cells.

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Genistein is a soy isoflavone with multiple anti-tumor properties. Downregulation of Bcl-2, a critical anti-apoptotic protein, is associated with genistein-induced apoptosis. More interestingly, Bcl-2 overexpression, which is frequently detected in breast cancers, is associated with a better prognosis. The goal of this project was to study the role of Bcl-2 in genistein mediated cell death in MCF-7 breast cancer cells. We first examined the effect of genistein on endogenous Bcl-2 protein and mRNA levels using Western blot and RT-PCR. Genistein induced Bcl-2 expression in MCF-7 cells at lower concentration but downregulated Bcl-2 expression at higher concentration, suggesting a bi-phasic effect. With little changes in Bax expression, Bax/Bcl-2 ratio was modified in these cells upon genistein treatment. To study the effect of Bcl-2 overexpression on genistein mediated growth inhibition, we overexpressed Bcl-2 in MCF-7 cells and examined their responses to genistein using MTT and clonogenic assays. Surprisingly, overexpression of Bcl-2 rendered MCF-7 cells more sensitive to genistein. To study the mechanism underlying sensitization of MCF-7 cells; cyochrome c release and JC-1 staining was assessed as an apoptotic indicator. Cytochrome c increased significantly in the cytosol of genistein treated MCF-7/Bcl-2 cells, as compared to MCF-7 control cells. Modified cytochrome c release was accompanied by increased depolarization of mitochondrial membrane and the enrichment of Bcl-2 in mitochondrial fraction, suggesting regulation of mitochondrial permeability by Bcl-2 contributing to the sensitization. To test whether Bcl-2 overexpression modulates genistein-induced cell cycle arrest, we examined the cell cycle distribution of genistein treated cells. In contrast to the G1/G0 arrest in genistein treated MCF-7/control cells, G2/M arrest was the prominent in genistein treated MCF-7/Bcl-2 cells accompanied by increased induction of p21, but not p53 and p27. Our results indicate that genistein-Bcl-2 interaction is closely associated with genistein sensitivity, suggesting that genistein may convert Bcl-2 into a proapoptotic molecule in Bcl-2 overexpressing breast cancers. Therefore, Bcl-2 breast cancers overexpressing high levels of Bcl-2 might be benefited from soy/genistein mediated prevention or treatment.