厄洛替尼联合二甲双胍对人肺癌细胞的抑制作用及其凋亡机制研究

Inhibitory Effects of Erlotinib Combined with Metformin on Human Lung Cancer Cells and Its Mechanism of Inducing Apoptosis

目的探讨厄洛替尼联合二甲双胍对人肺癌H1975细胞增殖、迁移、侵袭及凋亡的影响及可能凋亡机制。方法将人肺癌H1975细胞分为对照组、厄洛替尼组(25μmol·L-1)、二甲双胍组(5 mmol·L-1)及联合用药组(25μmol·L-1厄洛替尼+5 mmol·L-1二甲双胍)。MTT检测各组细胞的增殖能力;划痕实验观察各组细胞的迁移能力;Transwell小室实验观察各组细胞的侵袭能力;TUNEL检测各组细胞的凋亡情况;Western blotting检测各组细胞Mcl-1、Bax、Bcl-xl蛋白及EMT标志分子的表达水平。结果联合用药组H1975细胞增殖、迁移、侵袭能力均低于对照组和单药组(P<0.05);TUNEL实验结果显示,各组细胞凋亡率为联合用药组>单药组>对照组(P<0.05);Western blotting结果显示,联合用药组抗凋亡蛋白Mcl-1、Bclxl及细胞间质标志Vimentin蛋白的表达水平显著低于对照组和单药组(P<0.05),促凋亡蛋白Bax及细胞上皮标志E-cadherin蛋白的表达水平显著高于对照组和单药组(P<0.05)。结论厄洛替尼联合二甲双胍具有协同增效作用,可显著抑制人肺癌H1975细胞的增殖、迁移和侵袭能力,促进细胞凋亡,其机制可能与逆转EMT发生及激活线粒体凋亡途径有关。

Objective To investigate the effects of erlotinib combined with metformin on proliferation,migration,invasion and apoptosis of human lung cancer cell line H1975 and its possible apoptosis mechanism.Methods Human lung cancer H1975 cells were divided into control group,erlotinib group(25μmol·L-1),metformin group(5 mmol·L-1)and combination group(25μmol·L-1 erlotinib+5 mmol·L-1 metformin).The cell proliferation ability was detected by MTT.The migration ability was observed by scratch test.The invasion ability was observed by Transwell.The apoptosis was detected by TUNEL method.The expression levels of Mcl-1,Bax and Bcl-xl proteins,and EMTrelated molecular markers were detected by Western blotting.Results In this experiment,the proliferation,migration and invasion ability of H1975 cells in combination group were weaker than that of the control group and the single drug group(P<0.05).The TUNEL experiment showed that the apoptosis rate was the highest in combination group,and second highest in single drug group,and the lowest in control group(P<0.05).The results of Western blotting showed that the combination group significantly reduced the expression levels of anti-apoptotic proteins Mcl-1 and Bcl-xl,and interstitial marker Vimentin protein,as compared with the control group or the single drug group(P<0.05).The combination group also had higher expression levels of Bax and E-cadherin than the control group or the single drug group(P<0.05).Conclusion Erlotinib and metformin synergistically and remarkably inhibited the growth,invasion and migration and induced the apoptosis of H1975 cells.The potential mechanism for antitumor might be related with their activation on EMT reversal and mitochondrial apoptosis pathway.