奥利司他逆转肺腺癌A549/DDP细胞株顺铂耐药及机制研究

本研究的目的是观察奥利司他(Orlistat)逆转肺腺癌耐顺铂细胞株A549/DDP的耐药作用并考察其可能的分子机制。应用CCK-8法检测并计算肺腺癌耐药细胞株A549/DDP对顺铂(cisplatin,DDP)的耐药指数(resistance index,RI),筛选奥利司他的最佳实验浓度并观察其对A549/DDP细胞的耐药逆转效果;倒置荧光显微镜下观察各实验组细胞的形态学变化及Hoechst 33258荧光染色后细胞凋亡形态学改变;采用流式细胞术检测不同药物处理对细胞凋亡率的影响;Western blotting检测各实验组细胞P-gp、FASN、PI3K、Akt、p-Akt、NF-κB、Bcl-2和cleved-caspase-3的表达情况。结果显示,奥利司他抑制了A549/DDP耐药细胞株的增殖,且具有浓度依赖性。奥利司他与DDP联用增加了耐药株A549/DDP细胞对DDP的敏感性,耐药逆转倍数为5.02。倒置荧光显微镜观察到联合用药组细胞出现了明显的凋亡形态学改变。流式细胞术结果表明,与对照组和单药组比较,两种药物联用后细胞的凋亡率显著提高(p<0.05)。Western blotting检测结果显示,相较于其它3组,联合用药组中耐药相关蛋白P-gp表达下调(p<0.01),PI3K、p-Akt、NF-κB表达水平均显著降低(p<0.01),抗凋亡蛋白Bcl-2表达下降(p<0.01),凋亡相关蛋白cleved-caspase-3表达上调(p<0.01);虽然FASN的表达在单用奥利司他组及联合用药组中均降低,但这两组间的FASN表达水平并无统计学差异。以上结果表明,奥利司他能够提高A549/DDP耐药细胞株对化疗药物DDP的敏感性,具有逆转肺腺癌细胞耐药性的作用,其机制与降低耐药蛋白P-gp的表达、抑制PI3K/Akt/NF-κB信号通路以及其下游凋亡相关蛋白有关。

Reversal Effect and Its Mechanism of Orlistat on Cisplatin Resistance in Lung Adenocarcinoma Cell Lines A549/DDP

We aim to observe the reversal effect of drug resistance on the cisplatin-resistant lung adenocarcinoma cell line A549/DDP via orlistat and to explore the possible molecular mechanisms.The resistance index of A549/DDP against cisplatin was detected and computed by CCK-8.Besides,CCK-8 was used to screen the best experimental concentration of orlistat and to observe the reversal effect.Observation of the morphological changes and apoptosis effect after staining of Hoechst 33258 in each experimental group was accomplished by inverted fluorescence microscope.The impacts of different drugs on apoptosis rate were detected by flow cytometry.Western blotting was used to detect the expression value of P-gp,FASN,PI3K,Akt,p-Akt,NF-κB,Bcl-2 and cleved-caspase-3.The results showed that orlistat inhibits the proliferation of A549/DDP with a concentration-dependent manner.The combination of orlistat and DDP increased the sensitivity of DDP on A549/DDP cell line,and the reversal fold was 5.02.Inverted fluorescence microscope showed that the combination group had significant morphological changes of apoptosis.Flow cytometry revealed that comparing with the control group and the single-drug group,the combination group had a sharp increase in apoptosis rate(p<0.05).Western blotting showed that comparing with other three groups,the combination group had a lower expression level of P-gp(p<0.01)and lower expressions level of PI3K,p-Akt and NF-κB(p<0.01).The expression of anti-apoptosis protein Bcl-2 was decreased(p<0.01),while the expression of pro-apoptosis protein cleved-caspase-3 was up-regulated(p<0.01).And there was no sign­ificant difference of decreased FASN expression between the orlistat group and combination group.These results suggested that orlistat could increase the sensitivity of DDP on A549/DDP cell line and reverse the drug-resistant effect.The mechanism was associated with down-regulation of drug-resistant protein P-gp,inhibition of PI3K/Akt/NF-κB signaling pathway and regulation its downstream apoptosis-related proteins.