奥希替尼联合贝伐珠单抗对EGFR-TKIs耐药H1975细胞增殖的抑制作用及机制研究

Inhibitory effect of osimertinib combined with bevacizumabon the proliferation of EGFRTKIS resistant H1975 cells and its mechanism

目的:探讨奥希替尼(AZD)联合贝伐珠单抗(BEV)对表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-TKIs)耐药H1975细胞增殖的作用及可能的机制。方法:体外培养人肺腺癌细胞株H1975(E21 L858R/E20 T790M),并通过低浓度加量递增方法诱导建立EGFR-TKIs奥希替尼耐药株H1975/OR。将H1975和H1975/OR细胞分为空白组(不做任何药物处理)、AZD组(不同浓度AZD处理)、BEV组(不同浓度BEV处理)和AZD+BEV组(先加入不同浓度BEV,6 h后加入不同浓度AZD)。分别利用MTT法和流式细胞术检测各组细胞增殖和凋亡活性,并计算半数抑制浓度(IC50)和联合应用指数(CI)。Western blotting检测各组细胞EGFR、蛋白激酶B(Akt)、细胞外调节蛋白激酶(ERK)、哺乳动物雷帕霉素靶蛋白(mTOR)、核糖体S6蛋白激酶(p70s6k)、5-磷酸腺苷激酶(AMPK)蛋白及相应磷酸化蛋白表达量。结果:成功建立AZD耐药细胞株H1975/OR细胞,对AZD的耐药指数为90.57。AZD+BEV抑制H1975细胞增殖的CI分别为0.749、0.790、0.409、0.508、0.298、0.009,CI均<1;抑制H1975/OR细胞增殖的CI分别为0.375、0.111、0.068、0.229、0.592、0.105,CI均<1。AZD+BEV组H1975细胞和H1975/OR细胞凋亡率均高于空白组、AZD组和BEV组(P<0.05)。与空白组、AZD组和BEV组比较,AZD+BEV组H1975细胞和H1975/OR细胞p-EGFR、p-Akt、p-ERK、p-mTOR、p-p70s6k蛋白表达量降低,同时p-AMPK蛋白表达量升高。结论:AZD联合BEV可协同抑制H1975细胞和H1975/OR细胞的增殖活力,其作用机制可能是通过下调VEGF表达进而抑制EGFR及其下游PI3K/AKT/mTOR通路和AMPK/ERK/mTOR通路的磷酸化激活。

Objective:To explore the synergistic effect of osimertinib(AZD)combined with bevacizumab(BEV)on the proliferation ofthe acquired resistance of H1975 cells to epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)and possible mechanism.Methods:Thelung adenocarcinomacell line H1975 was cultured in vitro and induced by a low-concentration incremental dose of AZD method to obtain AZD resistant cells H1975/OR.H1975 cells or H1975/OR cells were divided into control group(without any drug treatment),AZD group(with different concentrations of AZD treatment),BEV group(with different concentrations of BEV treatment)and AZD+BEV group(with different concentrations of BEV for 6 h,and then adding different concentrations of AZD).MTT assay and FCM were used to detected the proliferation and apoptosis activities of H1975 cells or H1975/OR cells and IC50 and combination index(CI)were calculated.And western blot was used to detected the expression levels of EGFR,protein kinase B(Akt),extracellular regulated protein kinase(ERK),mammalian target of rapamycin(mTOR),Ribosome S6 protein kinase(p70 s6 k),Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)and corresponding phosphorylated proteins.Results:AZD-resistant cell line H1975/OR was successfully estab-lished.And the resistance index(RI)of Azd was 90.57.MTTmethods results showed that CIs of AZD+BEV on inhibiting H1975 cells proliferation were 0.749,0.790,0.409,0.508,0.298,0.009,which all were less than 1;while CIs of AZD+BEV on inhibiting H1975/OR cells proliferation were 0.375,0.111,0.068,0.229,0.592,0.105,which all were less than 1.Flow cytometry results showed that the apoptosis rates of H1975 cells and H1975/OR cells in AZD+BEV group were both higher than that in control group,AZD group and BEV group(P<0.05).And then western blot results showed that the expression levels of p-EGFR,p-Akt,p-ERK,p-mTOR,p-p70 s6 k proteins in AZD+BEV group decreased,while AMPK protein levels increased,when compared with control group,AZD group and BEV group.Conclusion:AZD in combination with BEV can significantlysynergistically inhibit the proliferation of H1975 cells and H1975/OR cells,which would be related with the inhibition of EGFR and PI3 K/AKT/mTOR pathway or AMPK/ERK/mTOR pathway activation by decreasing VEGF expression.