吴茱萸碱逆转人肺癌细胞株A549/DDP耐药机理的实验研究

Mechanism of reversing MDR of human lung adenocarcinoma cell line A549/DDP by evodiamine

目的观察吴茱萸碱逆转人肺腺癌耐药株A549/DDP细胞耐药性的效果并探讨其与阻断NF-κB信号传导通路的相关性。方法采用MTT法检测单用吴茱萸碱、顺铂(DDP)以及两药联用在不同时间对A549/DDP细胞的增殖影响,计算IC50及耐药逆转倍数。流式细胞仪检测各组细胞的凋亡情况。RT-PCR检测各组MDR1、NF-κB、Bcl-2、MMP-2和VEGF的mRNA表达。Westernblot法检测各组细胞的pIκB-α、pIKKα蛋白表达水平。结果吴茱萸碱0.125mg/L、0.25mg/L针对A549/DDP细胞对DDP的耐药逆转倍数分别为3.668和11.48。RT-PCR显示在吴茱萸碱作用下检测基因的mRNA表达随着吴茱萸碱浓度的增加和时间的延长其表达逐渐下降。当吴茱萸碱与DDP联用时,可明显提高A549/DDP细胞对化疗药的敏感性,凋亡细胞显著增加(P<0.05)。Westernblot法结果提示A549/DDP细胞中pIκB-α的表达水平随着吴茱萸碱作用时间的延长逐渐下降,pIKKα表达则无显著变化。结论吴茱萸碱可以通过抑制IκB-α的磷酸化阻断NF-κB信号通路,促进细胞凋亡,抑制细胞增殖,增加耐药细胞对DDP的敏感性。

Objective This study was to evaluate the reversal effect of evodiamine（EV）on human lung adenocarcinoma cell line A549/DDP and its possible mechanisms through inhibiting NF-κB signaling pathway.Methods Cytotoxicity and the reversal effect of EV and cisplatin（DDP）alone and combination of these two drugs were detected by MTT assay,and then the IC50 and the drug resistance reversal folds were calculated.Apoptosis was analyzed by flow cytometry.The mRNA expression of MDR1,NF-κB,Bcl-2,MMP-2,VEGF was measured by reverse transcriptase polymerase chain reaction（RT-PCR）.Expressions of pIκB-α,pIKKα were detected by Western blot.Results When added 0.125mg/L,0.25mg/L EV,the drug resistance reversal of A549/DDP cells to DDP was increased to 3.668 and 11.48 folds.mRNA levels of MDR1,NF-κB,Bcl-2,MMP-2,VEGF were in a concentration-dependent and time-dependant manner.Evodiamine could increase the chemo sensitivity of A549/DDP cells to DDP.In A549/DDP cells,evodiamine inhibited IκBα phosphorylation also in a concentration-dependent and time-dependant manner,but pIKKα was not in the same situation.Conclusion Evodiamine can lead to suppression of proliferation,induction of apoptosis and an increase of the sensitivity of lung adenocarcinoma cell line A549/DDP to DDP by inhibiting the phosphorylation of IκBα to block NF-κB signaling pathway.