辛伐他汀逆转KRAS基因突变结肠癌细胞西妥昔单抗耐药研究  

Simvastatin reverses cetuximab-resistant KRAS-mutant colorectal cancer cells to cetuximab

作　　者：陈雪岩 虞凯杰 郭翠翠 张一山 薛佳辰[1] 陈雅敏[2] CHEN Xue-yan;YU Kai-jie;GUO Cui cui;ZHANG Yi-shan;XUE Jia-chen;CHEN Ya -min(Oncology Teaching and Research Department ,First Clinical College ,Dalian Medical University, Dalian 116011 ,P. R. China;Department of Oncology ,First Affiliated Hospital of Dalian Medical University, Dalian 116011 ,P. R. China)

机构地区：[1]大连医科大学第一临床学院肿瘤教研室,辽宁大连116011 [2]大连医科大学附属第一医院肿瘤一科,辽宁大连116011

出　　处：《中华肿瘤防治杂志》2017年第23期1629-1634,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：CSCO-默克雪兰诺肿瘤研究基金(Y-MX2016-057)

摘　　要：目的鼠类肉瘤病毒癌基因(kirsten ratsarcoma viral oncogene,KRAS)突变型结直肠癌患者对西妥昔单抗治疗耐药,为了使更多患者获益,本实验旨在研究辛伐他汀能否逆转KRAS基因突变所致结肠癌细胞对西妥昔单抗的耐药。方法将KRAS突变型结肠癌DLD-1随机分为正常组、西妥昔单抗(150μg/mL)组、辛伐他汀(0.2μg/mL)组和两药联合组4组,采用MTT法检测细胞增殖能力,流式细胞法检测细胞凋亡率,平板克隆形成法检测细胞克隆能力,Transwell小室法检测细胞侵袭能力,蛋白质印迹法检测BRAF、p-ERK1/2、Bcl-xl、Bad和Caspase-3的表达。结果 MTT结果显示,正常组、西妥昔单抗组、辛伐他汀组和两药联合组细胞增殖率分别为(100.00±0.00)%、(90.70±1.30)%、(88.00±0.60)%和(72.00±1.20)%,两药联合处理组的细胞增殖率明显下降,两者存在明显的交互作用,F=22.39,P<0.001。克隆形成结果显示,两药联合组的克隆形成率相对于西妥昔单抗组由(87.05±2.47)%下降至(52.28±1.51)%,克隆形成受到抑制,F=197.86,P<0.001。流式结果显示,两药联合组相对于西妥昔单抗单药组的细胞凋亡率由(9.36±0.33)%上升至(26.16±2.91)%,诱导凋亡,F=63.97,P<0.001。Transwell小室结果显示,两药联合组的细胞侵袭率相对于单用西妥昔单抗组由(127.45±17.06)%下降至(42.22±4.78)%,F=37.59,P<0.001。蛋白质印迹结果显示,BRAF及p-ERK1/2表达降低,MAPK信号通路受到抑制、抗凋亡蛋白Bcl-xl表达下降,凋亡前蛋白Bad表达升高,凋亡相关蛋白Procaspase-3表达下降,Cleaved caspase-3表达升高。结论辛伐他汀与西妥昔单抗联合应用可以抑制DLD-1增殖、克隆、侵袭,可能是通过抑制MAPK通路,活化凋亡相关蛋白,诱导细胞凋亡,从而有效逆转其耐药。OBJECTIVE KRAS-mutant CRC patients are resistant to cetuximab treatment.In order to benefit more patients from cetuximab targeted therapy,this study aims to investigate the effect of simvastatin on resensitizing cetuximab-resistant human CRC cells invitro.METHODS The KRAS mutant DLD-1 cells were randomly divided into four groups：normal,cetuximab（150μg/mL）,simvastatin（0.2μg/mL）and cetuximab plus simvastatin.Then cell proliferation was detected by MTT assay;cell apoptosis was dectected by flow cytometry;cell cloning ability was determined by colony formation assay;cell invasion was detected by Transwell assay;the expressions of BRAF,p-ERK1/2,Bcl-xl,Bad,caspase-3 were determined by Western blot.RESULTS MTT assay showed that the proliferation rate of DLD-1 cells stimulated by four methods respectively were（100.00±0.00）% ,（90.70±1.30）% ,（88.00±0.60）% and（72.00±1.20）% .Simvastatin plus cetuximab contrasting to cetuximab alone could significantly reduce the cell proliferation,and there was great interactional effect between these two drugs,F=22.39,P〈0.001.Colony formation showed that the cloning rate of cetuximab group and simvastatin plus cetuxiamb gruop were respectively（87.05±2.47）% and（52.28±1.51）% ,F=197.86,P〈0.001.Flow cytometry showed that simvastatin combined with cetuximab could significantly induce the cell apoptosis rate,which was increased from（9.36±0.33）% to（26.16±2.91）% ,F=63.97,P〈0.001.Transwell assay showed the invasion rate was significantly reduced when simvastatin combined with cetuximab,which was decreased from（127.45±17.06）% to（42.22±4.78）% ,F=37.59,P〈0.001.Western blot results showed that BRAF and p-ERK1/2 expressions were decreased,which inhibited the MAPK signaling pathway.The expressions of apoptosis protein such as Bcl-xl,procaspase-3 were down regulated,while Bad and Cleaved caspase-3 were up regulated.CONCLUSIONS Simvastatin combines with cetuximab may siginificantly inhibit the DLD-1 cell proliferation,colony formation and

关 键 词：结直肠癌 辛伐他汀 西妥昔单抗 鼠类肉瘤病毒癌基因 

分 类 号：R735.34[医药卫生—肿瘤]