HDAC抑制剂Quisinostat对肝癌Huh7细胞增殖和凋亡的影响及作用机制

Effect of HDAC inhibitor Quisinostat on hepatocellular carcinoma Huh7 cells and its mechanism

目的研究组蛋白去乙酰化酶抑制剂Quisinostat对肝癌Huh7细胞增殖和凋亡的影响,并分析其作用机制。方法以空白药物处理组为对照组,不同浓度Quisinostat处理组作为实验组,采用CCK-8法检测各组细胞增殖抑制率,流式细胞术检测各组细胞周期占比,Annexin-V FITC/PI双染法检测各组细胞凋亡率。以空白药物处理组为对照组,50 nM Quisinostat组、50 nM Quisinostat+50μMZ-VAD-FMK组(作为实验组),采用Annexin-V FITC/PI双染法测定各组细胞凋亡率。采用Western blot法检测不同浓度Quisinostat处理组细胞周期和凋亡相关蛋白表达水平。结果随着Quisinostat浓度增加,Huh7细胞增殖抑制率逐渐增加(P<0.05);同一浓度Quisinostat下Huh7细胞增殖抑制率随着时间延长也逐渐增强(P<0.05)。与0 nM组相比,25、50 nM组G0/G1期细胞明显增加,S期细胞明显减少,S+G2/M期细胞明显下降(均P<0.05),而3组比较G2/M期差异无统计学意义(P>0.05)。与0 nM比较,25、50、100 nM组Huh7细胞晚期、早期凋亡率均更高(均P<0.05)。3组间Huh7细胞晚期、早期凋亡率比较差异均有统计学意义(均P<0.05);与0 nMQ组比较,50 nMQ组、50 nMQ+50μm Z组Huh7细胞晚期、早期凋亡率均更高(均P<0.05);与50 nM Q组比较,50 nMQ+50μm Z组Huh7细胞晚期、早期凋亡率均更低(均P<0.05)。Western blot结果显示,随着Quisinostat浓度升高,B淋巴细胞瘤-2蛋白相关X蛋白、剪切型半胱胺酸天冬氨酸蛋白酶-3、剪切型半胱胺酸天冬氨酸蛋白酶-9、p53、p21、p27蛋白表达水平均逐渐增高,B淋巴细胞瘤-2蛋白、细胞周期蛋白D1表达水平均逐渐降低(均P<0.05)。结论高浓度Quisinostat能有效将肝癌Huh7细胞周期阻滞在G0/G1期,抑制细胞增殖,并且诱导肝癌细胞发生凋亡,从而导致肝癌细胞的生长抑制。

Objective To examine the effect of HDAC inhibitor Quisinostat for the hepatocellular carcinoma(HCC)Huh7 cells,and its mechanism.Methods Human hepatocellular carcinoma(HCC)Huh7 cells were treated with different concentrations of Quisinostat(0 nM,25 nM,50 nM,100 nM Quisinostator)and 50 nM Quisinostat+50μM Z-VAD-FMK,respectively.CCK-8 method was used to determine the cell viability;flow cytometry with PI staining was applied to determine cell cycle,annexin-V FITC/PI was used to determine the cell apoptosis;Western blot was used to determine the expression of apoptosis and cell cycle-related proteins.Results Quisinostat inhibited proliferation of Huh7 cells in a dose/time dependent manner(P<0.05).Compared with the control group,the proportions of cells in the G0/G1 phase were significantly increased in 25 nM and 50 nM treatment groups,while the proportions of cells in the S+G2/M phase were decreased(P<0.05).There were no significant difference for the proportion of cells in the G2/M phase among 3 experiment groups(P>0.05).Compared with the control group,the apoptosis rates were increased in 25 nM,50 nM,100 nM treatment groups(P<0.05).There were obvious difference for the apoptosis rate among 3 treatment groups(P<0.05).Compared with the control group,the apoptosis rates were higer in 50 nM Quisinostat and 50 nMQuisinostat+50μM Z-VAD-FMK group(P<0.05).Compared with 50 nMQuisinostat group,the apoptosis rate was decreased in 50 nM Q+50μM Z group(P<0.05).With the dose of Quisinostat increasing,the expression of Bcl-2 and Cyclin D1 decreased;and the expression of BAX,p21,p27,cleaved Caspase-9,cleaved Caspase-3 and p53 increased(P<0.05).Conclusion High concentration of Quisinostat can inhibit the growth of Huh7cells by blocking the cell cycle at G0/G1 stage and prompting the apoptosis.The cell cycle arrest was induced by the elevation of p21 and p27 and reduction of Cyclin D1.Apoptosis was induced by the activation of p53 signaling,increasing expression of BAX,Cleaved Capase-3 and Cleaved Caspase-9,and decreasing expression of Bcl-2.