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-G₁ 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_L 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_L ratio.

Biochanin A是红三叶草中的主要异黄酮，是一种有效的抗癌化学药物。但是，尚未阐明生物chanin A对人骨肉瘤细胞的作用。这项研究调查了生物chanin A在骨肉瘤细胞中的抗增殖潜力。结果表明，生物chanin A以剂量依赖的方式抑制细胞生长和集落形成，对正常细胞的毒性最小。阿霉素和生物chanin A的组合可以协同抑制骨肉瘤细胞的生长。凋亡素的形成，磷脂酰丝氨酸的外在化，sub-G _1的积累证明了生物chanin A通过诱导细胞凋亡的细胞毒性作用。相细胞，caspase 3激活和PARP裂解。细胞凋亡与线粒体膜电位的损失相关，细胞色素的释放Ç，胱天蛋白酶9活化，增加的Bax的表达，并减少的Bcl-2和Bcl-X_大号表达。用caspase-9特异性抑制剂（Z-LEHD-FMK）进行的预处理可以部分减轻细胞死亡，提示内在的线粒体凋亡级联反应。但是，用JNK抑制剂SP600125，MEK抑制剂PD-98059和p38 MAPK抑制剂SB203580或抗氧化剂维生素E，N-乙酰半胱氨酸和谷胱甘肽进行的预处理无法阻止生物素A诱导的细胞死亡。我们的结果表明，生物chanin A通过触发内在的线粒体途径和caspase-9和-3的激活并增加Bax：Bcl-2 / Bcl- _XL比率来抑制骨肉瘤细胞的生长并诱导其凋亡。