双氯芬酸联合顺铂通过热疗抑制肺腺癌A549细胞株的协同作用

Diclofenac combined with cisplatin synergisticallyexhibits inhibitoryeffect on lung adenocarcinoma cell line A549 in hyperthermia

目的:探讨双氯芬酸能否增强顺铂对肺腺癌A549细胞株的热化疗作用。方法:应用MTS法测定双氯酚酸和顺铂在37℃、43℃下对A549细胞的半数致死率(IC_(50)),以此确定两药物的后续实验浓度。进一步实验分组分为对照组、顺铂组、双氯酚酸+顺铂组。用流式细胞技术检测各组细胞凋亡率;RT-PCR技术检测Bax、Bcl-2 mRNA的表达情况;蛋白免疫印迹法检测凋亡相关基因Bax、Bcl-2蛋白的表达。结果:MTS实验结果为双氯酚酸37℃、43℃的IC_(50)分别为0.138μmol/L、0.144μmol/L,顺铂37℃、43℃的IC_(50)分别为4.702 ug/mL、3.234 ug/mL。流式细胞技术检测结果显示,对照组、顺铂组、双氯酚酸+顺铂组的细胞总凋亡率分别为(12.53±0.51)%、(24.82±3.93)%、(56.07±1.97)%,组间差异具有统计学意义(P<0.05)。RT-PCR检测结果为,与对照组相比,顺铂组和双氯酚酸+顺铂组的Bax mRNA表达均上调,而Bcl-2表达均下调,组间差异具有统计学意义(P<0.05);与顺铂组相比,双氯酚酸+顺铂组的Bcl-2/Bax比值下降,差异具有统计学意义(P<0.01)。蛋白免疫印迹实验结果显示,与对照组相比,顺铂组和双氯酚酸+顺铂组Bax蛋白表达均上调,且双氯酚酸+顺铂组上调更明显;Bcl-2蛋白表达均下调,且双氯酚酸+顺铂组下调更明显。结论:双氯酚酸能增强顺铂对A549细胞的热化疗作用,促进A549细胞的凋亡。

Objective:To investigate whether diclofenac can enhance the thermochemotherapeuticeffect of cisplatin on lung adenocarcinoma A549 cell line.Methods:MTS method was used to determine the half maximum inhibitory concentrations(IC_(50))of diclofenac and cisplatin on A549 cells at 37℃and 43℃,so as to determine the concentrations of the two drugs in subsequent parts of experiment.Further,the experiment were done with A549 cells divided into control group,cisplatin group,and diclofenac+cisplatin group.Flow cytometry was used to detect the apoptosis of each group;rT-PCR was used to detect the mRNA expression of apoptosis-related Bax and Bcl-2 genes;western blotting was used to detect the protein expression of Bax and Bcl-2.Results:MTS experiment showed that the Ics50 of diclofenac at 37℃and 43℃were 0.138μmol/L and 0.144μmol/L respectively,and those of cisplatin at 37℃and 43℃were 4.702 ug/mL and 3.234 ug/mL,respectively.Flow cytometry showed that the total apoptosis rates in the control group,cisplatin group,and diclofenac+cisplatin group were(12.53±0.51)%,(24.82±3.93)%,and(56.07±1.97)%,respectively,with statistical difference between groups(P<0.05).RT-PCR showed that,compared with the control group,the Bax mRNA expression in the cisplatin group and the diclofenac+cisplatin group was both up-regulated,while the Bcl-2 mRNA expression was down-regulated,with statistical differences between groups(P<0.05).Compared with the cisplatin group,the Bcl-2/Bax ratio was lower in the diclofenac+cisplatin group,with statistically significant difference(P<0.01).Western blotting showed that,compared with the control group,the Bax protein expression was up-regulated in the cisplatin group and the diclofenac+cisplatin group,and the up-regulation was more obvious in the diclofenac+cisplatin group.The expression of Bcl-2 protein was down-regulated in both treatment groups,and the down-regulation was more obvious in the diclofenac+cisplatin group.Conclusion:Diclofenac can enhance the thermochemotherapeutic effect of cisplatin on A549 cells and promote apoptosis of A549 cells.