摘要
目的:研究辛伐他汀(simvastatin,Sim)联合贝伐珠单抗(bevacizumab,Beva)对人肺腺癌A549细胞增殖、迁移、侵袭及凋亡的影响,及可能的作用机制。方法:采用CCK-8法和平板克隆形成实验检测不同浓度的Sim(4、8、12、16和20μmol/L)和Beva(1、2、4、5和6mg/mL)单药或2药联合对A549细胞增殖的影响,并计算Beva的半数抑制浓度(half maximal inhibitory concentration,IC_(50))以及联合用药指数(combination index,CI)。分别采用划痕愈合实验、Transwell小室实验和FCM法检测Sim单药、Beva单药以及2药联合处理对A549细胞迁移、侵袭及凋亡的影响,应用实时荧光定量PCR法检测不同处理对A549细胞内缺氧诱导因子1α(hypoxia-induciblefactor-1α,HIF-1α)m RNA表达水平的影响,用蛋白质印迹法检测不同处理对A549细胞内增殖相关蛋白增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、迁移和侵袭相关蛋白[基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)和波形蛋白(vimentin)]、凋亡相关蛋白(Bax和Bcl-2)以及HIF-1α和β-连环蛋白(β-catenin)表达水平的影响。结果:不同浓度的Sim单药、Beva单药及2药联合处理后,均可抑制A549细胞的增殖(P<0.05),Beva在24和48 h时的IC_(50)值分别为(5.272±0.345)mg/m L和(3.434±0.312)mg/mL,2药联合的CI值均<1,表现为协同效应。Sim联合Beva组A549细胞的克隆形成数、迁移及侵袭能力均明显低于对照组和各单药组(P<0.05),且2药联合处理组细胞中PCNA、MMP-9和vimentin蛋白的表达水平明显低于对照组和各单药组(P<0.05)。Sim单药浓度为8μmol/L时对A549细胞无明显的促凋亡作用(P>0.05),但联合Beva可增加Beva的促凋亡作用,且可上调促凋亡蛋白Bax的表达水平,下调抗凋亡蛋白Bcl-2的表达水平(P<0.05)。与对照组相比,Beva作用于A549细胞后HIF-1αm RNA和蛋白以及β-catenin蛋白的表达水平均明显增高,而Sim作用后其表达水平均明显降低(P<0.05);与Beva单药组相比,2药联合处理后A549细胞中的HIF-1αm RNA和蛋白以及β-catenin蛋白表达水平明显下调(P<0.05)。结论:Sim联合Beva可协同抑制肺腺癌A549细胞的增殖、迁移和侵袭能力,并促进其凋亡;其机制可能与Sim可削弱Beva诱导的HIF-1α表达升高,进而负向调控HIF-1α-Wnt/β-catenin信号通路相关。
Objective:To investigate the effect of Sim(simvastatin)combined with Beva(bevacizumab)on the proliferation,migration,invasion and apoptosis of pulmanory adenocarcinoma A549 cells,and its underlying mechanism.Methods:The effect of different concentrations of Sim(4,8,12,16 and 20μmol/L)and Beva(1,2,4,5 and 6 mg/mL),alone or in combination,on the proliferation of A549 cells was evaluated by CCK-8 assay and colony formation assay.The IC_(50)(half inhibitory concentration)of Beva and the CI(combination index)values of Sim in combination with Beva were calculated.The effects of Sim and Beva,alone or in combination,on the migration,invasion and apoptosis of A549 cells were assessed by wound healing assay,Transwell assay and FCM assay,respectively.The expression levels of HIF-1α(hypoxia-inducible factor-1α)mRNA in A549 cells after different treatment was examined by real-time fluorescence quantitative PCR.The expression levels of proliferation associated protein PCNA(proliferating cell nuclear antigen),migration and invasion associated proteins[MMP-9(matrix metalloproteinase-9)and vimentin],apoptosis associated proteins[Bax and Bcl-2],as well as HIF-1αandβ-catenin in A549 cells after different treatment were detected by Western blotting.Results:Different concentrations of Sim and Beva,alone or in combination,could inhibit the proliferation of A549 cells(P<0.05).The IC_(50)values of Beva were(5.272±0.3448)mg/mL at 24 h and(3.434±0.3115)mg/mL at 48 h of treatment,and the 2 drugs had synergistic effect with their CI<1.The number of colonies formed and the activities of migration and invasion of A549 cells in the Sim+Beva treatment group were significantly lower than those in the control and single-drug treatment groups(P<0.05),and the expression levels of PCNA,MMP-9 and vimentin in A549 cells treated with Sim+Beva were significantly lower than those in the control and single-drug treatment groups(P<0.05).Sim(8μmol/L)alone had little pro-apoptotic effect on A549 cells(P>0.05),but it could increase the pro-apoptotic effect of Beva when used in combination with Beva.Meanwhile,the expression level of the pro-apoptotic protein Bax was up-regulated while that of the anti-apoptotic protein Bcl-2 was down-regulated in the Sim+Beva treatment group(P<0.05).Compared with the control group,the expression levels of HIF-1αmRNA,HIF-1αprotein andβ-catenin protein were significantly increased in A549 cells after Beva treatment,whereas their expression levels were significantly decreased after Sim treatment(P<0.05).Compared with Beva treatment alone,the expression levels of HIF-1αmRNA,HIF-1αprotein andβ-catenin protein in the Sim+Beva treatment group were significantly down-regulated(P<0.05).Conclusion:The combination of Sim and Beva can synergistically inhibit the proliferation,migration and invasion but promote the apoptosis of pulmonary adenocarcinoma A549 cells.The underlying mechanism may be related to the ability of Sim in attenuating the elevated HIF-1αexpression induced by Beva,which in turn negatively regulates the HIF-1α-Wnt/β-catenin signaling pathway.
作者
涂昕
刘单
邓述恺
TU Xin;LIU Dan;DENG Shukai(Department of Respiratory and Critical Care Medicine,The Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2022年第4期229-240,共12页
Tumor
关键词
肺腺癌
辛伐他汀
贝伐珠单抗
细胞增殖
细胞凋亡
Pulmonary adenocarcinoma
Simvastatin
Bevacizumab
Cell proliferation
Apoptosis
作者简介
Correspondence to:邓述恺,E-mail:2900880171@qq.com
