Yu Fu, Watts Ralph N, Zhang Xu Dong, Borrow Jodie M, Hersey Peter
Oncology and Immunology Unit, Newcastle Mater Hospital, New South Wales, Australia.
Anticancer Drugs. 2006 Nov;17(10):1151-61. doi: 10.1097/01.cad.0000231484.17063.9a.
Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.
苯氧二醇是一种具有抗肿瘤活性的植物激素异黄酮的化学修饰类似物。在本研究中，我们检测了其诱导人黑色素瘤细胞凋亡的能力及相关机制。通过使用膜联蛋白V/碘化丙啶染色并测定线粒体膜电位，观察了苯氧二醇处理细胞中的凋亡情况。为了确定哪些半胱天冬酶途径参与了苯氧二醇诱导的凋亡，使用特异性半胱天冬酶抑制剂进行了研究。进行蛋白质免疫印迹研究以确定凋亡级联反应中的哪些蛋白质受到影响从而导致苯氧二醇诱导的凋亡。我们发现，苯氧二醇诱导的凋亡在48小时时达到最大值，不同黑色素瘤细胞系中的凋亡率在12±4%至48±5%之间。这种凋亡主要依赖于半胱天冬酶-3和半胱天冬酶-9的激活。凋亡与线粒体膜电位变化的诱导相关，并被Bcl-2的过表达所抑制。对苯氧二醇敏感性的差异似乎与线粒体上游事件以及Bax构象变化的程度有关。p53调节的仅含BH3结构域的蛋白质（Bad、PUMA和Noxa）在敏感细胞系中增加，而在耐药细胞系中未增加，而Bim在所有测试细胞系中均增加。然而，Bim似乎仅部分参与其中，因为通过RNA干扰降低Bim会导致凋亡水平降低。总之，这些研究表明，苯氧二醇通过诱导p53依赖性的含BH3结构域蛋白质（Bad、PUMA和Noxa）以及p53非依赖性的Bim蛋白，导致Bax激活及其下游事件，从而诱导黑色素瘤细胞凋亡。
