Tian Xin, Luo Ying, Yan Yong-bo, Sui Cheng-guang, Meng Fan-dong, Liu Yun-peng
China Medical University, Shenyang, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2012 Dec;34(6):556-62. doi: 10.3881/j.issn.1000-503X.2012.06.004.
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.
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 (IC(50)) 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 immunofluorescence labling combined with laser confocal scanning microscopy.
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 G(1) phase and increased in S and G(2)/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).
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 G(2)/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-induced apoptosis.
通过探讨蟾毒灵对人食管鳞状癌细胞EC9706增殖和凋亡的影响，研究其对人端粒酶逆转录酶（hTERT）核-线粒体定位的作用。
用不同浓度的蟾毒灵处理EC9706细胞，采用CCK-8法检测细胞生长抑制情况并计算50%抑制浓度（IC50）。用PI染色通过流式细胞术进行细胞周期分析，用Hoechst 33342染色通过荧光显微镜观察凋亡细胞核形态。用Annexin V-FITC/PI双染通过流式细胞术检测凋亡指数。通过蛋白质免疫印迹法以及多重免疫荧光标记结合激光共聚焦扫描显微镜确定hTERT亚细胞定位和蛋白表达。
蟾毒灵可显著抑制EC9706细胞的增殖，且随着处理时间和浓度的增加抑制作用增强（p<0.01）。EC9706细胞暴露于100 nmol/L蟾毒灵后，G1期细胞数量逐渐减少，S期和G2/M期细胞数量增加（p<0.05）。观察到典型的凋亡细胞核形态变化，凋亡指数升高（p<0.01）。hTERT在细胞核中的表达降低，但在线粒体中的表达增加（p<0.05）。
蟾毒灵可时间和剂量依赖性地抑制人食管鳞状癌细胞EC9706的增殖。它可使细胞周期阻滞于S期和G2/M期，并诱导EC9706细胞凋亡。hTERT定位于细胞核和线粒体，在蟾毒灵诱导的凋亡过程中可部分从细胞核转位至线粒体。
