Sunters Andrew, Fernández de Mattos Silvia, Stahl Marie, Brosens Jan J, Zoumpoulidou Georgia, Saunders Catherine A, Coffer Paul J, Medema René H, Coombes R Charles, Lam Eric W-F
Cancer Research-UK Laboratories, Department of Cancer Medicine, Medical Research Council Cyclotron Building, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom.
J Biol Chem. 2003 Dec 12;278(50):49795-805. doi: 10.1074/jbc.M309523200. Epub 2003 Oct 3.
Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Consequently, we have studied the role of the FoxO transcription factors in determining cellular response to paclitaxel. Western blotting revealed that in a panel of nine breast cancer cell lines expression of FoxO1a and FoxO3a correlated with the expression of the pro-apoptotic FoxO target Bim, which was associated with paclitaxel-induced apoptosis. In MCF-7 cells, which were paclitaxel-sensitive, the already high basal levels of FoxO3a and Bim protein increased dramatically after drug treatment, as did Bim mRNA, which correlated with apoptosis induction. This was not observed in MDA-231 cells, which expressed low levels of FoxOs and Bim. Gene reporter experiments demonstrated that in MCF-7 cells maximal induction of Bim promoter was dependent on a FoxO binding site, suggesting that FoxO3a is responsible for the transcriptional up-regulation of Bim. Gene silencing experiments showed that small interference RNA (siRNA) specific for FoxO3a reduced the levels of FoxO3a and Bim protein as well as inhibited apoptosis in paclitaxel-treated MCF-7 cells. Furthermore, siRNA specific for Bim reduced the levels of Bim protein and inhibited apoptosis in paclitaxel-treated MCF-7 cells. This is the first demonstration that up-regulation of FoxO3a by paclitaxel can result in increased levels of Bim mRNA and protein, which can be a direct cause of apoptosis in breast cancer cells.
紫杉醇用于治疗乳腺癌，但其诱导细胞凋亡的机制尚不清楚。因此，我们研究了FoxO转录因子在决定细胞对紫杉醇反应中的作用。蛋白质免疫印迹分析显示，在一组9种乳腺癌细胞系中，FoxO1a和FoxO3a的表达与促凋亡的FoxO靶标Bim的表达相关，而Bim与紫杉醇诱导的细胞凋亡有关。在对紫杉醇敏感的MCF-7细胞中，药物处理后，原本就较高的FoxO3a和Bim蛋白基础水平显著升高，Bim mRNA也同样升高，这与细胞凋亡的诱导相关。在表达低水平FoxO和Bim的MDA-231细胞中未观察到这种现象。基因报告实验表明，在MCF-7细胞中，Bim启动子的最大诱导依赖于一个FoxO结合位点，这表明FoxO3a负责Bim的转录上调。基因沉默实验表明，针对FoxO3a的小干扰RNA（siRNA）降低了FoxO3a和Bim蛋白的水平，并抑制了紫杉醇处理的MCF-7细胞的凋亡。此外，针对Bim的siRNA降低了Bim蛋白的水平，并抑制了紫杉醇处理的MCF-7细胞的凋亡。这首次证明了紫杉醇上调FoxO3a可导致Bim mRNA和蛋白水平升高，这可能是乳腺癌细胞凋亡的直接原因。
