没食子儿茶素没食子酸酯诱导人肝癌细胞凋亡

目的 探讨没食子儿茶素没食子酸酯(EGCG)诱导人肝癌细胞株凋亡的作用和机制.方法 以不同浓度EGCG处理人肝癌细胞株HepG2和SMMC-7721细胞24 h和48 h.四甲基偶氮唑蓝比色法和锥虫蓝染色细胞计数评价细胞生长情况;流式细胞术检测细胞凋亡和环氧合酶-2(COX-2)、Bcl-2蛋白;比色法测定天冬氨酸蛋白酶-9和caspase-3活性;RT-PCR检测COX-2和Bcl-2家族mRNA的表达.结果 EGCG(50、100、200、400μg/ml)处理48 h后,HepG2细胞活性下降至93.8%±2.8%,62.3%±5.4%,33.9%±2.5%和17.6%±3.2%,与对照组(100.0%±2.8%)比较差异均有统计学意义(均P＜0.05);SMMC-7721细胞活性下降至49.6%±3.5%,30.3%±3.8%,17.7%±2.2%和13.0%±2.5%,与对照组(100.0%±0.8%)比较差异均有统计学意义(均P＜0.05);100 μg/ml EGCG处理细胞24、48、72和96 h后,HepG2活细胞计数(×104)分别是8.0±1.5,22.0±3.1,37.0±5.4和61.0±8.7,与对照组(15.0±2.5,45.0±5.3,86.0±11.0和210.0±23.0)相比明显减少,差异均有统计学意义(均P＜0.05);SMMC-7721活细胞计数(×104)分别是7.0±2.2,13.0±2.5,20.0±3.7和31.0±4.0,与对照组(14.0±2.2,40.0±4.3,75.0±8.8和182.0±28.0)相比明显减少,差异均有统计学意义(均P＜0.05).EGGG(50、100、200μg/ml)处理细胞12 h后,HepG2细胞凋亡率分别是8.7%±0.4%,18.1%±1.1%和22.1%±1.8%;SMMC-7721细胞凋亡率分别是5.9%±0.3%,7.8%±0.6%和12.2%±0.8%,与对照组(3.3%±0.3%)和(3.7%±0.4%)比较,差异均有统计学意义(P＜0.05);EGCG(100、200μg/ml)处理细胞12 h后,HepG2细胞caspase-9活性为1.8±0.4和2.5±0.4;caspase-3活性为2.0±0.4和2.8±0.5,两者与对照组(1.0±0.1和1.0±0.2)比较差异均有统计学意义(均P＜0.05);SMMC-7721细胞caspase-9活性为1.7±0.4和2.5±0.4,caspase-3活性为1.9±0.4和2.6±0.3,均显著高于对照组(1.0±0.1和1.0±0.2,P＜0.05).EGCG(200μg/ml)下调COX-2和Bcl-2的表达,而对Bcl-2家族其他成员表达无明显影响.结论 EGCG可能通过下调COX-2和Bcl-2的表达,激活caspase-9和caspase-3诱导肝癌细胞凋亡.

Epigallocatechin-3-ganate induces apoptosis in human hepatocellular carcinoma cells

Objective To investigate the effects of epigalloeateehin-3-gallate(EGCG)on human hepatocellular carcinoma(HCC)cells and mechanism thereof.Method Human HCC cells of the lines HepG2 and SMMC-7721 were cultured and treated with of EGCG of the concentrations of 6.25,12.5,25,50,100,200,and 400 μg/ml respectively for 24 h and 48 h.The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.Trypan blue staining was used to count the cells.Flow cytometry was conducted to detect the cell apoptosis.The protein levels of Bcl-2,an anti-apoptosis factor,and cyclooxygenase-2(COX-2),an up-regulator of Bcl-2.The activities of caspase-9 and caspase-3 hat promote the apoptosis of HCC cells,were measured using colorimetric method.RT-PCR was used to dmect the mRNA expression of COX-2 and Bcl-2 family.Results The viabilities of the HepG2 and SMMC-7721 cells treated with EGGG of the concentrations of 50-400 μg/ml for 48 h reduced to 93.8%±2.8%.62.3%±5.4%,33.9%±2.5%,and 17.6%±3.2% respectively,all significantly lower than that of the control group[(100.0%±2.8%),all P＜0.05];and the viabilities of the SMMC-772 cells treated with EGCG of the concentrations of 50-400 μg/ml for 48 h reduced to 49.6%±3.5%,30.3%±3.8%,17.7%±2.2%,and 13.0%±2.5%respectively,all significantly lower than that of the control group[(100.0%±0.8%),all P＜0.05].After treatment with 100 μg/ml EGCG for 24 h,48 h,72 h,and 96 h,the live HepG2 cell numbers were(8.0±1.5),(22.0±3.1),(37.0±5.4),and (61.0±8.7)104 respectively,all significantly lower than those of the control cells[(15.0±2.5),(45.0±5.3),(86.0±11.0),and(210.0±23.0)104 respectively,all P＜0.05];and the live SMMC-7721 cell numbers were(7.0±2.2),(13.0±2.5),(20.0±3.7),and(31.0±4.0)104 respectively,all significantly lower than those of the control cells[(15.0±2.5),(45.0±5.3),(86.0±11.0),and(210.0±23.0)104 respectively,all P＜0.05].The apoptotic rates of HepG2 cells treated with EGCG of the concentrations of 50,100,and 200 μg/ml for 12 h were 8.7%±0.4%,18.1%±1.1%,and 22.1%±1.8% respectively,all significantly higher than that of the control group(3.3%±0.3%,P＜0.05);and the apoptotic rates of SMMC-7721 ceHs were 5.9%±0.3%,7.8%±0.6%,and 12.2%±0.8% respectively,all significantly higher than that of the contrel group(3.7%±0.4%,P＜0.05).After treatment with EGCG of the concentrations of 100 and 200 μg/ml for 12 h,the caspaae-9 activities of the HepG2 cells increased to(1.8±0.4)and(2.5±0.4)respectively,both significantly higher than that of the control group(1.0±0.1.both P＜0.05);and the caspase-3 activities of the HepG2 cells increased to(2.0±0.4)and(2.8±0.5)respectively,both significantly higher than that of the control group(1.0±0.2.P＜0.05);and the caspase-9 activities of the SMMC-7721 cells increased to(1.7±0.4)and(2.5±0.4),both significantly higher than that of the control group(1.0±0.1,both P＜0.05),and the caspase-3 activities of the SMMC-7721 cells increased to(1.9±0.4)and(2.6±0.3)respectively,both significantly higher than that of the control group[(1.0±0.2),both P＜0.05].When the concentration of EGCG was over 200μg/ml,it down-regulated the expression of COX-2 and Bcl-2 in both cell lines,however,ECCG resulted in no significant changes of Bcl-xl,Bax,Bad,and Bid.Conclusion EGCG induces apoptosis in HCC cells through down-regulation of COX-2 and Bcl-2 and consequently activating caspase-9 and caspase-3.