摘要
目的探讨PTEN基因是否增强子宫内膜癌细胞系Ishikawa细胞对阿霉素的敏感性。方法将PTEN基因转染前、后的Ishikawa细胞分别暴露于系列浓度的阿霉素,以MTT法测定这些细胞对阿霉素的敏感性,并通过Hoechst33258染色荧光显微镜观察细胞凋亡情况。同时,以免疫沉淀Westernblot法观察阿霉素对Bad和Akt/PKB蛋白磷酸化的影响。结果阿霉素以剂量依赖方式诱导全部细胞系的细胞凋亡,但对表达PTEN的克隆细胞所诱导的凋亡比对亲本株Ishikawa细胞更明显。低浓度阿霉素(0.1μmol/L)不引起PTEN蛋白缺失的Ishikawa细胞凋亡,但却能诱导表达PTEN蛋白克隆细胞凋亡;高浓度阿霉素(1μmol/L)诱导全部细胞系的细胞凋亡,但凋亡的发生率在表达PTEN的克隆细胞中明显高于亲本株Ishikawa细胞。在表达PTEN的克隆细胞中,磷酸化Akt/PKB和磷酸化Bad(Ser-136)的水平均下降。阿霉素降低了全部细胞系的磷酸化Akt/PKB和磷酸化Bad(Ser-136)水平,但在表达PTEN的克隆细胞中降低最明显。结论PTEN基因转染明显地增强了Ishikawa细胞对阿霉素的化学敏感性,阿霉素可能是通过下调PI3k/Akt/PKB信号途径而与PTEN蛋白共同发挥凋亡诱导作用。
Objective To investigate whether PTEN can increase sensitivity of lshikawa cells, an endometrial carcinoma cell line, to doxorubicin. Methods Ishikawa cells transfected by PTEN gene or not were separately treated with serial concentrations of doxorubicin. The sensitivity of cells to doxorubicin was determined by MTY assay. The cells were stained with Hoechst 33258 and examined under fluorescence microscope to determine cell apoptosis, lmmunoprecipitation and Western blotting analysis were performed to evaluate the effects of doxorubicin on phosphorylation of Bad and Akt/PKB. Results Doxorubicin induced cell death of the PTEN-transfected and non-transfected Ishikawa cells in a dose-dependent manner, but the cell death was more significant in PTEN-expressing clones than in parental Ishikawa cells. A low concentration of doxorubicin （0.1 μmol/L） did not affect cell apoptosis in PTEN-null Ishikawa cells, but it induced cell apoptosis in PTEN-expressing clones. A high concentration of doxorubicin （ 1 μmol/L） induced cell apoptosis in both cell lines. However, the percentage of apoptotic cells was higher in PTEN-expressing clones than that in parental Ishikawa cells. In the PTEN-expressing clones, expression of phospho-Akt/PKB and phospho-Bad（Ser-136）was down regulated. Doxorubicin reduced the levels of phospho-Akt/PKB and phospho-Bad （Ser-136） in both cell lines, but the most significant reduction occurred in the PTEN- expressing clones. Conclusion PTEN significantly enhances chemosensitivity of Ishikawa cells to doxorubicin. With PTEN expression, doxorubicin may exert apoptosis-induction activity by downregulation of the P13k/Akt/PKB signaling pathway in lshikawa cells.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2005年第9期513-515,共3页
Chinese Journal of Oncology
