【摘要】 目的探讨c-Met抑制剂SU11274逆转肝细胞生长因子(HGF)诱导非小细胞肺癌(NSCLC)对厄洛替尼耐药的作用机制。方法选择人NSCLC细胞PC-9(EGFR突变型,敏感株)及人胚肺成纤维细胞MRC-5,常规规养48 h,采用ELISA法检测两种细胞培养上清液HGF水平,采用MTT法检测加入50%MRC-5细胞培养上清液后IC50浓度厄洛替尼下PC-9细胞的增殖率,采用Western blotting法检测PC-9细胞c-Met及其下游通路蛋白(Akt、Stat3、Erk1/2及p-Akt、p-Stat3、p-Erk1/2)表达。选择雌性BALB/c裸鼠49只,随机分为对照组、HGF组、HGF+DMSO组、HGF+SU11274组、厄洛替尼组、HGF+厄洛替尼+SU11274组、HGF+厄洛替尼组,每组7只。对照组和厄洛替尼组于右肋皮下接种密度为5×106个/m L PC-9细胞悬液100μL,其他组均于右肋皮下接种100μL密度相同的PC-9细胞与MRC-5细胞培养上清液的混合液。当肿瘤直径生长至约4 mm时,对照组、HGF组以1%Tween-80灌胃,厄洛替尼组、HGF+厄洛替尼组、HGF+厄洛替尼+SU11274组以厄洛替尼25 mg/(kg·d)灌胃,每周灌胃5天,连续4周。HGF+SU11274组、HGF+厄洛替尼+SU11274组灌胃后腹腔注射SU11274 0. 18 mg/(kg·d),每周5次,连续4周。分别于灌胃4、7、11、14、18、21、25天计算肿瘤体积,末次灌胃结束,断颈处死,完整剥离移植瘤组织,称取肿瘤质量,计算抑瘤率。取部分移植瘤组织,采用免疫组化法检测c-Met及其下游通路蛋白(Akt、Stat3、Erk1/2及p-Akt、p-Stat3、p-Erk1/2)表达。结果 PC-9细胞培养上清液HGF水平<0. 1 pg/m L,MRC-5细胞培养上清液HGF水平为(1 262. 07±89. 78) pg/m L。在加入50%MRC-5细胞培养上清液后IC50浓度厄洛替尼下PC-9细胞增殖率为78. 24%。与未加入MRC-5细胞培养上清液的PC-9细胞比较,加入MRC-5细胞培养上清液的PC-9细胞p-c-Met、p-Akt、p-Stat3、p-Erk1/2蛋白相对表达量明显升高(P均<0. 05),而c-Met、Akt、Stat3、Erk1/2蛋白相对表达量变化不明显(P均> 0. 05)。厄洛替尼组、HGF+厄洛替尼组及HGF+厄洛替尼+SU11274组均能明显抑制移植瘤生长,其他各组抑制肿瘤生长效果不明显。与HGF+厄洛替尼组比较,厄洛替尼组与HGF+厄洛替尼+SU11274组抑瘤率明显升高(P均<0. 05),而厄洛替尼组与HGF+厄洛替尼+SU11274组比较P> 0. 05。与HGF组、HGF+DMSO组和HGF+厄洛替尼组比较,厄洛替尼组、HGF+SU11274组和HGF+厄洛替尼+SU11274组移植瘤组织pMet、p-Stat3、p-Akt、p-Erk1/2蛋白相对表达量均明显降低(P均<0. 05)。结论 HGF可诱导NSCLC对厄洛替尼的耐药,c-Met及其下游信号通路持续激活是NSCLC对厄洛替尼耐药的重要机制; c-Met抑制剂SU11274和厄洛替尼联合应用可逆转HGF诱导的NSCLC对厄洛替尼的耐药,其机制可能与抑制c-Met及其下游信号通路激活有关。更多还原

【Abstract】 Objective To investigate the mechanism of c-Met inhibitor SU11274 reversing the effect of hepatocyte growth factor( HGF) on erlotinib resistance in non-small-cell lung cancer( NSCLC). Methods Human NSCLC cell line PC-9( EGFR mutant,sensitive strain) and human embryonic lung fibroblast MRC-5 were selected,and were cultured for48 h. The HGF levels in the cell culture supernatants were determined by using ELISA. MTT assay was used to detect the proliferation of PC-9 cells under erlotinib IC50 after adding 50% MRC-5 cell culture medium. Western blotting was used to detect the effect of HGF on the expression of c-Met and downstream pathway proteins( Akt,Stat3,Erk1/2 and p-Akt,pStat3,p-Erk1/2) in PC-9 cells. Forty-nine female BALB/c nude mice were randomly divided into the control group,HGF group,HGF + DMSO group,HGF + SU11274 group,erlotinib group,HGF + erlotinib + SU11274 group,and HGF + erlotinib group,with 7 in each group. The mice in the control group and the erlotinib group were inoculated subcutaneously with a density of 5 × 105 PC-9 cells into the right flank; in the other groups,the mice were inoculated subcutaneously with the same density of PC-9 + MRC-5 cells into the right flank. When the diameter of the tumor grew to about 4 mm,the control group and the HGF group were intragastrically administered 1% Tween-80,the erlotinib group,HGF + erlotinib group,erlotinib group,and HGF + erlotinib + SU11274 group were intragastrically administered erlotinib 25 mg/( kg·d)for 5 days per week for 4 weeks. After intragastric administration,the mice in the HGF + SU11274 group and HGF + erlotinib + SU11274 group were intraperitoneally injected with SU11274 0. 18 mg/( kg·d) 5 times a week for 4 weeks. The tumor volume was calculated on the 4 th,7 th,11 th,14 th,18 th,21 st and 25 th day of the lavage. At the end of the last lavage,the nude mice were sacrificed by cervical dislocation,the tumor tissue was removed,the tumor mass was weighed,and the tumor inhibition rate was calculated. Immunohistochemistry was used to detect the expression of c-Met and downstream pathway proteins( Akt,Stat3,Erk1/2 and p-Akt,p-Stat3,p-Erk1/2) in the tumor tissues. Results The HGF level of PC-9 cell culture supernatant was < 0. 1 pg/m L,while MRC-5 cell culture supernatant was( 1 262. 07 ± 89. 78)pg/m L. After we added HGF,the PC-9 cell proliferation rate at erlotinib IC50 was 78. 24%. Compared with the control group,the expression of c-Met,p-Akt,p-Stat3,and p-Erk1/2 protein in the PC-9 cells supplemented with MRC-5 cell culture supernatant significantly increased( all P < 0. 05),and the expression of c-Met,Akt,Stat3 and Erk1/2 protein did not significantly change( all P > 0. 05). The erlotinib group,HGF + erlotinib group and HGF + erlotinib + SU11274 group significantly inhibited the growth of transplanted tumors,while other groups inhibited tumor growth without obvious significant. Compared with the HGF + erlotinib group,the inhibition rate of erlotinib group and HGF + erlotinib + SU11274 group significantly increased( P < 0. 05),but there was no significant difference as compared with that in the HGF + erlotinib +SU11274 group( P > 0. 05). Compared with the HGF group,HGF + DMSO group,and HGF + erlotinib group,the expression of p-Met,p-Stat3,p-Akt and p-Erk1/2 in the tumor tissues significantly decreased in the erlotinib group,HGF +SU11274 group and HGF + erlotinib + SU11274 group( all P < 0. 05). Conclusions HGF can induce the resistance of NSCLC cells to erlotinib,and the continuous activation of c-Met and downstream signaling pathway is an important mechanism for its resistance to erlotinib. The c-Met inhibitor SU11274 combined erlotinib reverses HGF-induced erlotinib resistance in NSCLC cells,which may be related to the inhibition of c-Met and its downstream pathway activation.更多还原