氯喹联合顺铂对结肠癌HCT116细胞的增殖和凋亡的影响

目的探讨氯喹(CQ)联合顺铂(DDP)对人结肠癌HCT116细胞增殖和凋亡的影响及其可能机制。方法将HCT116细胞分组为16组:空白对照组(不加药物)、5个浓度(5,10,20,40,80μmol·L^-1)氯喹-1组至氯喹-5组,5个浓度(2,4,8,16,32μmol·L^-1)顺铂-1组至顺铂-5组和5个浓度(20μmol·L^-1氯喹+2,4,8,16,32μmol·L^-1顺铂)联合-1组至联合-5组,各组均培养24 h。用噻唑蓝法检测细胞的存活率,用PI单染流式细胞术检测细胞的凋亡情况,用蛋白质印迹法检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)等凋亡相关蛋白的表达水平(灰度值)。结果空白对照组、氯喹-1组至氯喹-5组、顺铂-1组至顺铂-5组和联合-1组至联合-5组于24 h的细胞存活率分别为(100.00±1.05)%,(99.65±1.98)%,(97.43±0.72)%,(83.88±0.51)%,(69.65±3.33)%,(51.87±0.49)%,(98.55±1.50)%,(92.33±0.50)%,(81.66±4.29)%,(70.41±0.50)%,(48.34±1.50)%,(78.61±4.37)%,(59.01±3.52)%,(37.23±0.57)%,(32.88±3.96)%和(21.09±2.57)%。空白对照组、氯喹-3组、顺铂-3组和联合-3组的细胞凋亡率分别为(1.63%±0.50%),(15.97%±1.40%),(24.83%±2.12%)和(47.23±4.29)%;这4组的Bcl-2相对表达量分别为1.11±0.01,0.92±0.03和0.73±0.08,0.46±0.07;这4组的Bax蛋白相对表达量分别为0.18±0.01,0.27±0.03,0.46±0.09和0.58±0.04。氯喹联合顺铂能显著减少抗凋亡Bcl-2蛋白的表达,并上调促凋亡Bax蛋白的表达。联合组与单用组比较,上述指标的差异均有统计学意义(均P<0.05);各给药组与空白对照组比较,上述指标的差异均有统计学意义(均P<0.05)。结论氯喹可以增强顺铂对HCT116细胞的增殖抑制作用,同时可以增强顺铂诱导结肠癌细胞的凋亡,其机制可能与下调Bcl-2和上调Bax有关联。

Effects of chloroquine combined with cisplatin on proliferation and apoptosis of colon cancer HCT116 cells

Objective To investigate the effects of chloroquine(CQ)combined with cisplatin(DDP)on proliferation and apoptosis of human colon cancer HCT116 cells,and its possible mechanism.Methods HCT116 cells were divided into 16 groups:blank control group(without drugs),five concentrations(5,10,20,40 and 80μmol·L^-1)of CQ-1,CQ-2,CQ-3,CQ-4,CQ-5 groups,five concentrations(2,4,8,16,32μmol·L^-1)of DDP-1,DDP-2,DDP-3,DDP-4,DDP-5 groups,and five concentrations(20μmol·L^-1 CQ combined with 2,4,8,16,32μmol·L^-1 DDP)of combined-1,c ombined-2,combined-3,combined-4,combined-5 groups,for 24 h.Cell viability was detected by methyl thiazolyl tetrazolium.The apoptosis of cells was assessed using flow cytometry with PI staining.The relative expressions(gray value)of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X(Bax)were analyzed using Western blotting.Results The cell survival rate at 24 h in blank control group,CQ-1,CQ-2,CQ-3,CQ-4,CQ-5 groups,DDP-1,DDP-2,DDP-3,DDP-4,DDP-5 groups,combined-1,combined-2,combined-3,combined-4,combined-5 groups were(100.00±1.05)%,(99.65±1.98)%,(97.43±0.72)%,(83.88±0.51)%,(69.65±3.33)%,(51.87±0.49)%,(98.55±1.50)%,(92.33±0.50)%,(81.66±4.29)%,(70.41±0.50)%,(48.34±1.50)%,(78.61±4.37)%,(59.01±3.52)%,(37.23±0.57)%,(32.88±3.96)%,and(21.09±2.57)%.The apoptosis rate in balnk control group,CQ-3 group,DDP-3 group and combined group were(1.63±0.50)%,(15.97±1.40)%,(24.83±2.12)%,(47.23±4.29)%;the relative expressions of Bcl-2 protein in the 4 groups were 1.11±0.01,0.92±0.03,0.73±0.08,0.46±0.07;the relative expressions of Bax protein in the 4 groups were 0.18±0.01,0.27±0.03,0.46±0.09,0.58±0.04.Both combined group could significantly down regulate the expression of anti apoptotic Bcl-2 protein and up regulate the expression of Pro apoptotic Bax protein.Comparison between combined group and CQ group,DDP group,the difference of the factors were significant(all P<0.05);compared between each treatment groups and blank control group,the difference of the factors were significant(all P<0.05).Conclusion CQ could enhance the proliferation inhibition of HCT116 cells by DDP,and enhance the DDP-induced apoptosis,the mechanism of which may involve the down-regulation of Bcl-2 and up-regulation of Bax expression.