Anti-proliferative activity of biochanin A in human osteosarcoma cells via mitochondrial-involved apoptosis.

Biochanin A is a major isoflavone in red clover and a potent chemopreventive agent against cancer. However, the effects of biochanin A on human osteosarcoma cells have never been clarified. This study investigated the anti-proliferative potential of biochanin A in osteosarcoma cells. The results indicate that biochanin A inhibited cell growth and colony formation in a dose-dependent manner with a minimal toxicity to normal cells. The combination of doxorubicin and biochanin A could synergistically inhibit osteosarcoma cell growth. The cytotoxic effect of biochanin A via the induction of apoptosis as evidenced by formation of apoptotic bodies, externalization of phosphatidylserine, accumulation of sub-G phase cells, caspase 3 activation, and cleavage of PARP. Apoptosis was associated with loss of the mitochondrial membrane potential, release of cytochrome c, caspase 9 activation, increased Bax expression, and reduced Bcl-2 and Bcl-X expression. Pre-treatment with a caspase-9 specific inhibitor (Z-LEHD-FMK) partially attenuated cell death, suggesting involvement of the intrinsic mitochondrial apoptotic cascade. However, pre-treatment with the JNK inhibitor SP600125, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB203580 or the antioxidants vitamin E, N-acetylcysteine, and glutathione failed to prevent biochanin A-induced cell death. Our results suggest that biochanin A inhibits cell growth and induces apoptosis in osteosarcoma cells by triggering activation of the intrinsic mitochondrial pathway and caspase-9 and -3 and increasing the Bax: Bcl-2/Bcl-X ratio.