南方红豆杉水提物抑制T790M突变及c-MET扩增导致的吉非替尼耐药体外研究

Aqueous Extract ofTaxus Chinensis var.Mairei Inhibits Gefitinib Resistance Induced by T790M Mutation and c-MET AmplificationIn Vitro

目的研究南方红豆杉水提物抑制T790M突变及c-MET扩增所致吉非替尼耐药机制。方法应用MTT法检测不同浓度南方红豆杉水提物(0.25、0.5、1、2、4 mg/mL)和吉非替尼(3、6、12、18、30μmol/L)对H1975、H820细胞株增殖的影响,根据结果筛选最佳后续实验药物浓度,计算各组半数抑制浓度(IC50)和两药联合指数(CI);将对照组(1640培养液)、南方红豆杉组(0.25μmol/L)、吉非替尼组(3μmol/L)及联合组(南方红豆杉0.25μmol/L+吉非替尼3μmol/L)H1975和H820细胞药物处理72 h后,采用Western Blot检测EGFR、p-EGFR、AKT、p-AKT及PI3K蛋白表达,采用qRT-PCR检测EGFR、PI3K及AKT mRNA表达。结果在H1975和H820细胞中联合组达到IC50所需的吉非替尼浓度明显小于吉非替尼组,且CI<1。在H1975细胞中,与对照组比较,南方红豆杉组EGFR、p-EGFR、PI3K及p-AKT蛋白表达降低(P<0.01,P<0.05),吉非替尼组AKT蛋白表达降低(P<0.05),联合组EGFR、p-EGFR、AKT、p-AKT及PI3K蛋白表达降低(P<0.01,P<0.05)。与吉非替尼组比较,南方红豆杉组EGFR、p-EGFR、p-AKT蛋白表达降低(P<0.01),联合组EGFR、p-EGFR、PI3K及p-AKT蛋白表达降低(P<0.01)。在H820细胞中,与对照组比较,南方红豆杉组EGFR、PI3K、p-AKT蛋白表达下调(P<0.01),联合组p-EGFR、PI3K、p-AKT蛋白表达降低(P<0.01)。与吉非替尼组比较,南方红豆杉组EGFR、p-AKT蛋白表达降低(P<0.01,P<0.05),联合组p-EG-FR、PI3K、p-AKT蛋白表达降低(P<0.01)。在H1975及H820细胞中,与对照组比较,吉非替尼组AKT及PI3K mRNA表达降低(P<0.01,P<0.05),南方红豆杉组及联合组EGFR、AKT及PI3K mRNA表达降低(P<0.01)。与吉非替尼组比较,联合组EGFR、AKT及PI3K mRNA表达降低(P<0.01)。结论南方红豆杉水提物能增强吉非替尼对H1975、H820细胞增殖抑制作用,可抑制T790M突变和c-MET扩增所致的吉非替尼耐药,其机制与抑制EGFR/PI3K/AKT通路有关。

Objective To explore the mechanism that aqueous extract Taxus chinensis var.mairei(AETC)inhibits gefitinib resistance which was caused by T790M gene mutation and c-MET gene amplification.Methods The inhibitory effect of AETC(0.25,0.5,1,2,4 mg/mL)and gefitinib(3,6,12,18,30μmol/L)against H1975 and H820 cell lines was detected by MTT assay.On the basis of MTT results,the best concentration of follow-up experimental drugs was screened,the 50%inhibitory concentration(IC50)and the combined index(CI)were calculated.H1975 and H820 cell were divided to four groups,i.e.control group(H1640 Culture medium),AECT group(0.25μmol/L),gefitinib group(3μmol/L)and combination group(AECT 0.25μmol/L+gefitinib 3μmol/L).After 72 hours of intervention,Western Blot and qRT-PCR were used to determine the protein and mRNA expression of EGFR and its downstream pathways of each group.Results In H1975 and H820 cells,the concentration of gefitinib required for IC50 in the combination group was significantly lower than that in the gefitinib group,and the CI value was less than 1.In H1975 cells,compared with the control group,the expression of EGFR,p-EGFR,PI3K and p-AKT in AETC group decreased(P<0.01,P<0.05),the expression of AKT in gefitinib group decreased(P<0.05),and the expression of EGFR,p-EGFR,AKT,p-AKT and PI3K in combination group decreased(P<0.01,P<0.05).Compared with gefitinib group,the expressions of EGFR,p-EGFR and p-AKT in AETC group decreased(P<0.01),while the expression of EGFR,p-EGFR,PI3K and p-AKT in combination group decreased(P<0.01).In H820 cells,compared with the control group,the expression of EGFR,PI3K and p-AKT protein was down-regulated in AETC group(P<0.01),while the expression of p-EGFR,PI3K and p-AKT protein was down-regulated in combination group(P<0.01).Compared with gefitinib group,the expression of EGFR and p-AKT protein in AETC group decreased(P<0.01,P<0.05),while the expression of p-EGFR,PI3K and p-AKT protein in combination group decreased(P<0.01).In H1975 and H820 cells,the expression of AKT and PI3K mRNA in gefitinib group was lower than that in control group(P<0.01,P<0.05),while the expression of EGFR,AKT and PI3K mRNA in AETC group and combination group was lower than that in control group(P<0.01).Compared with gefitinib group,the expression of EGFR,AKT and PI3K mRNA in combined group was decreased(P<0.01).Conclusion AETC can enhance the inhibitory effect of gefitinib on the proliferation of H1975 and H820 cells and inhibit the gefitinib resistance induced by T790M mutation and c-MET amplification through controlling EGFR/PI3K/AKT pathway.