二氯乙酸钠联合顺铂诱导人肺腺癌A549细胞株凋亡的实验研究

摘要

目的 研究二氯乙酸钠(Sodium dichloroacetate,DCA-Na)、顺铂(DDP)联合对人肺腺癌A549细胞株增殖和凋亡的影响及其作用机制.方法 用MTT法检测DCA-Na、DDP应用对A549细胞增殖抑制作用的影响;流式细胞仪(Annexin V-FITC/PI法)检测DCA-Na、DDP单药及两药联合作用于A549细胞凋亡率的变化;用分光光度法检测DCA-Na作用于A549细胞后半胱氨酸天门冬氨酸蛋白酶(Caspase)-3、-8、-9蛋白的活性.结果 DCA-Na、DDP单药组均对A549细胞增殖有抑制作用,且呈明显的剂量-时间依赖性.0.4、2 μg/mL的DDP与37.5、75、150 μg/mL的DCA-Na联合作用A549细胞24、48、72 h后的抑制率显著高于同浓度DDP单药组(P<0.05),且2 μg/mL的DDP与75 μg/mL的DCA联合在48 h表现为协同作用.流式细胞仪检测显示,A549细胞联合组凋亡率显著高于各单药组(P<0.05).DCA-Na作用于A549细胞后,分别在12、24、48、72 h测得Caspase-3、-8、-9蛋白活性显著高于对照组(P<0.05).结论 DCA-Na对人肺腺癌A549细胞的增殖有抑制作用,并且随着药物浓度增加、作用时间延长,其抑制作用也增加(呈剂量和时间依赖性).一定浓度范围的DCA-Na和DDP联合作用于人肺腺癌A549细胞能够产生协同作用.DCA-Na可诱导人肺腺癌A549细胞凋亡,且与DDP联合后其诱导凋亡作用更为显著.%Objective To explore the proliferation and apoptosis effects of dichloroacetate( DCA-Na ) combined with DDP on human lung adenocarcinoma cell line-A549 in vitro and to investigate the possible underlying mechanisms. Methods The inhibitory effects of DCA-Na combined with DDP on proliferation of A549 cells were determined by MTT assay. A549 cells apoptosis was evaluated by FCM with Annexin V-FITC and PI. The activity of Caspase-3 , Caspase-8, Caspase-9 of A549 cells, which were affected by DCA-Na at different time points, was evaluated by spectrophotometry. Results DCA-Na and DDP used alone could inhibit the proliferation of A549 cells, and the inhibition effect showed dose-time dependent relationship. The combination of DDP( 0. 4 μg/mL,2 μg/mL )with DCA-Na( 37. 5 μg/mL,75 μg/mL, 150 μg/mL )significantly increased the inhibition effect of A549 cells, compared with DDP group( P <0. 05 ). The combination of DCA-Na( 75 μg/mL )and DDP( 2 μg/mL ) showed synergy at 48 h. The number of A549 cells which were treated by DCA-Na( 75 μg/mL )and/or DDP( 2 μg/mL )was obviously fewer than that of control group. FCM showed that the rate of apoptosis of A549 cells combination group was significantly higher than that in DCA-Na and ( DDP group )( P <0. 05 ). When A549 cells had been treated with DCA( 37. 5 μg/mL,75 μg/mL,150 μg/mL )for different time, the activity of Caspase-3, Caspase-8, Caspase-9 was significantly higher than those of control group( P < 0. 05 ). Conclusion DCA-Na could inhibit the proliferation of A549 cells in a dose-time dependent manner. DCA-Na and DDP in certain concentration has synergy effects. DCA-Na could induce the apoptosis of A549 cells, and the induction effect of DCA-Na combined with DDP is more significant.