吉非替尼联合二甲双胍对EGFR-TKIs原发性耐药非小细胞肺癌H1975细胞的抑制作用及其机制研究

目的 探讨吉非替尼联合二甲双胍对肺癌H1975细胞的抑制增效作用及其机制.方法 采用MTT、克隆形成实验观察吉非替尼与二甲双胍单用及联用对原发性耐药非小细胞肺癌H1975细胞增殖活性的影响;Matrigel包被的Transwell小室检测细胞侵袭能力;划痕愈合实验检测细胞迁移能力;TUNEL检测细胞凋亡率;Western blot、免疫荧光法检测凋亡和上皮细胞-间充质转化(epithelial tomesenchymal transition,EMT)相关蛋白的变化.结果 吉非替尼联合二甲双胍组对H1975细胞增殖、侵袭及迁移抑制效应优于吉非替尼组,协同增效作用明显(P＜0.05);吉非替尼和二甲双胍单用时较对照组的细胞凋亡率增加,当两者联合作用时细胞凋亡率增加更显著(P＜0.05),且明显下调抗凋亡相关蛋白(Bcl-xl、Mcl-1)和上调促凋亡蛋白Bax;两药联用明显逆转H1975细胞EMT的发生;间质细胞相关蛋白(Vimentin、N-cadherin、Slug、Snail)表达水平降低,上皮细胞相关蛋白E-cadhenn表达水平升高.结论 吉非替尼联合二甲双胍可显著抑制EGFR-TKIs原发性耐药非小细胞肺癌H1975细胞的增殖、侵袭和迁移,促使肿瘤细胞凋亡;其机制可能通过激活线粒体凋亡途径及逆转EMT的发生来发挥抗肿瘤作用.

Inhibitory effect and mechanism of gefitinib combined with metformin on EGFR-TKI intrinsic resistant NSCLC H1975 cells

Objective To investigate the synergistic inhibitory effect of gefitinib combined with metformin on non-small cell lung cancer cell line H1975 with intrinsic resistance to EGFR-tyrosine kinase inhibitors (TKIs),and explore the underlying mechanism.Methods After H1975 cells were treated by gefitinib or/and metformin,MTT assay and colony formation assay were used to observe the proliferation of the cells.Cell invasion was tested by matrigel-coated Transwell chamber assay,and migration ability was measured by wound healing assay.TUNEL assay was employed to observe apoptotic rate.The protein levels of apoptosis and epithelial to mesenchymal transition (EMT) related proteins were assayed by immunofluorescence assay and Western blotting.Results Gefitinib combined with metformin showed more obviously synergistic inhibitory effect on the proliferation,invasion and migration in H1975 cells than gefitinib alone (P ＜ 0.05).As compared to the untreated control cells,gefitinib and metformin alone promoted the cell apoptosis.Their combination induced an even higher apoptotic rate in H1975 cells (P ＜ 0.05),downregulated anti-apoptotic proteins Bcl-xl and Mcl-1,up-regulated pro-apoptotic protein Bax,enhanced the EMT reversion,decreased the expression levels of mesenchymal markers,Vimentin,N-cadherin,Slug,Snail while increased the epithelial marker E-cadherin.Conclusion Combination of gefitinib and metformin remarkably inhibits the growth,invasion and migration and induces the apoptosis in inherent EGFR-TKIs resistant H1975 cells.The potential mechanism for antitumor might be due to their activation on mitochondrial apoptosis pathway and EMT reversal.