蟾蜍灵对人食管鳞癌EC9706细胞增殖及凋亡的影响

摘要

目的 观察蟾蜍灵对人食管鳞癌EC9706细胞增殖及凋亡的影响,探讨其对人端粒酶逆转录酶(hTERT)核-线粒体定位的影响.方法 采用不同浓度蟾蜍灵作用EC9706细胞,CCK-8法测定细胞增殖抑制并计算半数抑制浓度(IC50),PI染色流式细胞仪分析细胞周期,Hoechst 33342染色荧光显微镜观察凋亡核形态改变,Annexin V-FITC/PI双染流式细胞仪检测细胞凋亡率,Western blot法及多重免疫荧光标记激光共聚焦显微镜检测hTERT亚细胞定位及蛋白表达.结果 随作用时间及浓度的增加,蟾蜍灵可显著抑制人食管鳞癌EC9706细胞的增殖(P均＜0.01).100 nmol/L蟾蜍灵作用EC9706细胞时,随着作用时间的延长,G1期细胞逐渐减少,S及G2/M期细胞显著增加(P均＜0.05);细胞核发生典型的凋亡形态学改变,凋亡率逐渐增加(P＜0.01).hTERT可表达于EC9706细胞线粒体中,在蟾蜍灵诱导的凋亡过程中,细胞核hTERT的表达减少,而线粒体hTERT的表达则有所增加(P＜0.05).结论 蟾蜍灵可抑制EC9706细胞增殖,诱导其凋亡,细胞周期阻滞于S及G2/M期.hTERT除细胞核外还定位于线粒体中,并在蟾蜍灵诱导的凋亡过程中由细胞核向线粒体转位.%Objective To investigate the effect of bufalin on nucleus-mitochondria localization of human telomerase reverse transcriptase (hTERT) by exploring its effect on proliferation and apoptosis in human esophageal squamous carcinoma EC9706 cells. Methods EC9706 cells were treated with bufalin at various concentrations, and then the cell growth inhibition of EC9706 cells was examined by CCK-8 assay and the 50% inhibitory concentration (IC50) was calculated. Cell cycle analysis was performed by flow cytometry with PI staining, and nucleus morphology of apoptosis were observed by fluorescence microscopy with Hoechst 33342 staining. The apoptotic index was measured by flow cytometry with Annexin V-FITC/PI double staining. hTERT subcellular localization and protein expression were determined by Western blotting and multiple immu-nofluorescence labling combined with laser confocal scanning microscopy. Results The proliferation of EC 9706 cells was significantly inhibited by bufalin along with the increase of processing time and concentrations (P< 0. 01). After the EC9706 cells were exposed to 100 nmol/L bufalin, the number of cells gradually decreased in G1 phase and increased in S and G2/M phases (P <0.05). The typical nucleus morphological changes of apoptosis were observed and the apoptotic index was increased (P<0.01). The expression of hTERT decreased in nucleus but increased in mitochondria (P<0.05). Conclusions Bufalin can inhibit the proliferation of human esophageal squamous carcinoma EC9706 cells in a time- and dose-dependent manner. It can arrest cell cycle in S and G2/M phases and induce the apoptosis of EC 9706 cells. hTERT is localized in both nucleus and mitochondria, and can be partially translocated from nucleus to mitochondria during the bufalin-in-duced apoptosis.