三氧化二砷对肝癌细胞系SMMC-7721凋亡及Smac caspase-9、caspase-3蛋白表达的影响

Effect of Arsenic Trioxide on Apoptosis and Expression of Smac,Caspase-9,Caspase-3 in Human Hepatocellular Carcinoma SMMC-7721 Cells

目的:研究三氧化二砷(As2O3)对人肝癌细胞SMMC-7721的促凋亡作用及对Smac、caspase-9、caspase-3表达的影响。方法:人肝癌细胞SMMC-7721经As2O3处理,共分为四组,分别为空白对照组、低剂量组、中等剂量组、高剂量组。分别采用MTT、Hoechst 33258染色法、Annexin V-FITC/PI双染法观察其对SMMC-7721细胞增殖的抑制,凋亡细胞核的形态学变化,以及诱导凋亡作用;采用Western blot法检测凋亡相关蛋白Smac、caspase-9、caspase-3表达的变化。结果:MTT显示:As2O3在体外能明显抑制SMMC-7721的生长,具有时间剂量依赖关系,与空白对照组相比,其余三组细胞生存率明显下降,差异均有统计学意义(P<0.05);Hoechst 33258显示细胞呈明显的凋亡细胞形态学特征,具有剂量依赖性;Annexin V-FITC/PI双染法显示:As2O3作用24小时可诱导SMMC-7721细胞凋亡,且呈剂量依赖性,与空白对照组相比(2.69±0.58),其余三组(4.01±0.58)、(5.99±1.69)、(9.26±2.34)差异均有统计学意义(P<0.05);Western blot显示:As2O3作用SMMC-7721细胞24小时,Smac、caspase-9、caspase-3表达上升,呈剂量依赖性,与空白对照组相比,其余三组蛋白表达量明显增加,差异均有统计学意义(P<0.05)。结论:一定量的As2O3能抑制SMMC-7721细胞增殖,促进其凋亡,其机制可能与调控Smac、caspase-9、caspase-3表达有关。

Objective： To investigate the effect of As203 on apoptosis and suppress the proliferation of the hepatocellular carcinoma cells and the effect on the expression of Smac, caspase-9, caspase-3 in hepatocellular carcinoma cells. Methods： SMMC-7721 cells were treated with AS203, which were divided into four groups, including blank control group, low dose group, medium dose group and high dose group. Cell growth inhibitory rate was detected by MTT colorimetric assay every 24 hours. The nuclear morphology of apoptotic cells was observed by Hoechst 33258 staining, and the apoptotic effect of As203 in cell nucleus in the way of fluorescence was obtained. Apoptosis rate was analyzed by Annexin V-FITC/PI fluorescence staining. The expression of Smac and caspase-9, caspase-3 was measured by Western blot. This expression value ofapoptotic protein can illustrate the changes of the gene with the As2O3. Results： MTT tests suggested SMMC-7721 cells proliferation were inhibited by As2O3 in time and dose-dependent in vitro. Compared with that in the blank control group, the rest of the three groups of cell survival rate decreased obvious/y, and difference have statistical significance （P〈0.05）. Nuclear morphology changes characterized by apoptotic cells were observed clearly by Hoechst 33258 staining in dose-dependent manner. Annexin V-FITC/PI showed that AS203 could induce SMMC-7721 cells apoptosis after 24 hours in dose-dependent manner. Compared with that in the blank control group （2.69± 0.58）, the rest of the three groups were statistically significant differences （4.01 ± 0.58）, （5.99± 1.69）, （9.26± 2.34） （P〈0.05）. Western blot indicated that the expression of Smac and caspase-9, caspase-3 increased in SMMC-7721 cells induced by As203 after 24 hours. Compared with that in the blank control group, the rest of the three groups of protein expression quantity increased significantly, and difference have statistical significance （P〈0.05）. Conclusions： The mechanistic study showed that As2O3 acted through inhibit of SMMC-7721 cells proliferation and induce apoptosis in which Smac and caspase-9, caspase-3 were activated.