Mabuchi Seiji, Ohmichi Masahide, Kimura Akiko, Hisamoto Koji, Hayakawa Jun, Nishio Yukihiro, Adachi Kazushige, Takahashi Kazuhiro, Arimoto-Ishida Emi, Nakatsuji Yuki, Tasaka Keiichi, Murata Yuji
Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2002 Sep 6;277(36):33490-500. doi: 10.1074/jbc.M204042200. Epub 2002 Jun 26.
We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.
我们研究了磷脂酰肌醇3激酶（PI-3K）-Akt-BAD级联、ERK-BAD级联和Akt-Raf-1级联在缺乏功能性p53的耐紫杉醇SW626人卵巢癌细胞系中的作用。用紫杉醇处理SW626细胞会在不同时间激活Akt和ERK。通过用PI-3K抑制剂（渥曼青霉素或LY294002）处理或在SW626细胞中外源表达显性负性Akt来干扰Akt级联，会导致用紫杉醇处理后细胞活力下降。在SW626细胞中用MEK抑制剂PD98059处理来干扰ERK级联，也会导致用紫杉醇处理后细胞活力下降。用紫杉醇处理细胞还会刺激BAD在Ser-112和Ser-136位点的磷酸化。渥曼青霉素处理或与显性负性Akt共转染可阻断BAD在Ser-136的磷酸化。另一方面，PD98059可阻断BAD在Ser-112的磷酸化。我们使用BAD突变体进一步研究了BAD在紫杉醇处理后细胞活力中的作用。在SW626细胞中外源表达双取代的BAD2SA会导致用紫杉醇处理后细胞活力下降。此外，由于紫杉醇诱导的凋亡由活化的Raf-1介导，且Raf-1中Ser-259周围区域符合Akt磷酸化的共有序列，因此研究了Akt对Raf-1的调节作用。我们证明了Akt与Raf-1之间的关联，并表明紫杉醇诱导的Raf-1在Ser-259的磷酸化被渥曼青霉素或LY294002处理所阻断。此外，干扰紫杉醇诱导的Akt级联会上调Raf-1活性，而组成型活性Akt的表达会抑制Raf-1活性，这表明Akt负向调节Raf-1。我们的研究结果表明，紫杉醇通过ERK级联诱导BAD Ser-112的磷酸化，通过PI-3K-Akt级联诱导BAD Ser-136和Raf-1 Ser-25 的磷酸化，并且抑制这些级联中的任何一个都会使卵巢癌细胞对紫杉醇敏感。
