Wan X, Li J, Xie X, Lu W
Gynecology Oncology Department, Women's Hospital, Zhejiang University, Hangzhou, China.
Int J Gynecol Cancer. 2007 Jul-Aug;17(4):808-12. doi: 10.1111/j.1525-1438.2007.00890.x. Epub 2007 Mar 13.
To investigate whether PTEN can augment doxorubicin-induced apoptosis in PTEN-null Ishikawa cells. We previously demonstrated that Ishikawa cells do not possess functional PTEN protein because of protein truncations. Clones expressing the steady-state level of the PTEN protein from PTEN-null Ishikawa cells have been established and were used in this study. Doxorubicin is a commonly used anticancer drug in endometrial carcinoma. The cytotoxic effect of doxorubicin was evaluated using the methyl thiazoleterazolium (MTT) assay. We used the Hoechst 33258 staining to confirm the induction of apoptosis. Immunoprecipitation and Western blot analysis were performed to evaluate the effects of doxorubicin on phosphorylation of Bcl-2 antagonist of cell death (Bad) and protein kinase B (Akt/PKB). Doxorubicin induced death of all cell lines in a dose-dependent manner, but the death was more significant in PTEN-expressing clones than in parent Ishikawa cells. A low concentration of doxorubicin (0.1 muM) did not affect apoptosis in PTEN-null Ishikawa cells, but it induced apoptosis in PTEN-expressing clones. A high concentration (1 microM) induced apoptosis in all cell lines, but the percentages of apoptotic cells were higher in PTEN-expressing clones than in parent Ishikawa cells. In the clones, phospho-Akt/PKB and phospho-Bad (Ser-136) were downregulated. Doxorubicin reduced the levels of phospho-Akt/PKB and phospho-Bad (Ser-136) in all the cell lines, but the reduction was most significant in the PTEN-expressing clones. Our present results indicate that PTEN transfection significantly enhances doxorubicin chemosensitivity through effective induction of apoptosis by downregulation of the PI3K/Akt/PKB signaling pathway in Ishikawa cells.
为了研究PTEN是否能增强阿霉素诱导的PTEN缺失的Ishikawa细胞凋亡。我们之前证明，由于蛋白质截短，Ishikawa细胞不具有功能性PTEN蛋白。已建立了从PTEN缺失的Ishikawa细胞中表达稳定水平PTEN蛋白的克隆，并用于本研究。阿霉素是子宫内膜癌中常用的抗癌药物。使用噻唑蓝（MTT）法评估阿霉素的细胞毒性作用。我们使用Hoechst 33258染色来确认凋亡的诱导。进行免疫沉淀和蛋白质印迹分析以评估阿霉素对细胞死亡拮抗剂Bcl-2（Bad）和蛋白激酶B（Akt/PKB）磷酸化的影响。阿霉素以剂量依赖性方式诱导所有细胞系死亡，但在表达PTEN的克隆中死亡比在亲代Ishikawa细胞中更显著。低浓度阿霉素（0.1μM）不影响PTEN缺失的Ishikawa细胞凋亡，但可诱导表达PTEN的克隆凋亡。高浓度（1μM）诱导所有细胞系凋亡，但表达PTEN的克隆中凋亡细胞百分比高于亲代Ishikawa细胞。在这些克隆中，磷酸化Akt/PKB和磷酸化Bad（Ser-136）下调。阿霉素降低了所有细胞系中磷酸化Akt/PKB和磷酸化Bad（Ser-136）的水平，但在表达PTEN的克隆中降低最为显著。我们目前的结果表明PTEN转染通过下调Ishikawa细胞中PI3K/Akt/PKB信号通路有效诱导凋亡，从而显著增强阿霉素的化疗敏感性。
