小檗碱对人肾细胞癌细胞增殖、凋亡、DNA断裂及损伤修复的影响

Berberine's effects on the proliferation,apoptosis and DNA damage and repair of human renal cell carcinoma cells

目的探讨小檗碱(BBR)对人肾细胞癌(RCC)细胞生长及DNA断裂的影响。方法采用CCK-8法测定BBR在0~240μmol/L不同浓度下对RCC细胞(A498、786-O)增殖的影响;根据CCK-8检测结果,将RCC细胞分为对照组(0μmol/L)、30μmol/L组和60μmol/L组,应用Annexin V-FITC/PI双染色法及流式细胞术检测BBR对RCC细胞凋亡的影响;Western blotting检测RCC细胞中凋亡执行因子前体和降解体(pro-caspase3、cleavedcaspase3)、组蛋白H2A变构体(γH2A.X)和KU70蛋白的表达。结果用BBR处理RCC细胞后,细胞增殖受到抑制,并呈时间及药物浓度依赖性,差异有统计学意义(A498:P<0.001;786-O:P=0.002);有效浓度的BBR可增加RCC细胞凋亡率;30、60μmol/L组较0μmol/L组cleaved-caspase3均表达增加(A498:P=0.018、P<0.001;786-O:P=0.038、P<0.001),γH2A.X均表达增加(A498:P<0.001、P<0.001;786-O:P<0.001、P<0.001),而KU70均表达下降(A498:P=0.002、P<0.001;786-O:P<0.001、P<0.001);60较30μmol/L组cleaved-caspase3表达增加(A498:P=0.020;786-O:P=0.010),γH2A.X表达增加(A498:P=0.002;786-O:P<0.001),而KU70下调(A498:P<0.001;786-O:P=0.005)。结论 BBR可抑制人RCC细胞的增殖,诱导RCC细胞凋亡,并促使DNA断裂、抑制DNA的损伤修复。

Objective To determine the effects of berberine（ BBR） on the growth and DNA damage of human renal cell carcinoma（ RCC） cells. Methods The effects of BBR on the proliferation of A498 and 786-O RCC cells at concentrations of 0-240 μmol/L were tested with CCK-8 testing kit. Based on the results of CCK-8 test,RCC cells were divided into control group（ 0 μmol/L）,30 μmol/L group and 60 μmol/L group. The cell apoptosis after BBR treatment was evaluated with Annexin V-FITC/PI Apoptosis Detection Kit. The expressions of proteins involved in the apoptotic pathway and DNA damage repair pathways,including pro-caspased3,cleaved-caspased3,γH2 A.X and KU70,were assessed with Western blotting. Results The proliferation of RCC cells was significantly suppressed by BBR in a timeand dose-dependent manner（ A498： P〈0.001; 786-O： P= 0.002）. Cell apoptosis was obviously increased after BBRtreatment at an optimal concentration and time point. In comparison with the control group,the 30 and 60 μmol/L groups showed increased expressions of cleaved-caspase3（ A498： P= 0.018,P〈0.001; 786-O： P= 0.038,P〈0.001）,and γH2 A.X（ A498： P〈0.001,P〈0.001; 786-O： P〈0.001,P〈0.001）,but decreased expression of KU70（ A498：P= 0.002,P〈0.001; 786-O： P〈0.001,P〈0.001）. Compared with the 30 μmol/L group,the 60 μmol/L group showed increased expressions of cleaved-caspase3（ A498： P = 0.020; 786-O： P = 0.010）,and γH2 A.X（ A498： P = 0.002;786-O： P〈0.001）,but decreased expression of KU70（ A498： P〈0.001; 786-O： P = 0.005）. Conclusion BBR can effectively suppress RCC cell proliferation,induce RCC cell apoptosis and DNA damage,and inhibit DNA damage and repair.