摘要
目的:探讨左金丸(ZJW)对KRAS突变的人结肠癌细胞西妥昔单抗(CET)抵抗的效果及机制。方法:采用Sanger测序法检测SW620、Lovo、HCT116、HT29和Caco2细胞KRAS基因突变状态;采用CCK-8法检测ZJW、CET、ZJW联合CET以及联合不同细胞死亡抑制剂对上述细胞存活率的影响,并观察ZJW联合CET对KRAS突变型细胞(SW620、Lovo及HCT116)耐药性的影响;流式细胞术、比色法和FerroOrange荧光探针检测和观察ZJW、CET以及它们联合作用对KRAS突变型细胞内ROS、GSH及Fe^(2+)水平的影响;Western blot和Real-time PCR法检测ZJW联合CET对铁死亡相关通路蛋白和mRNA的调控作用。结果:KRAS突变型细胞为HCT116(KRAS^(G13D)),Lovo(KRAS^(G13D)),SW620(KRAS^(G12V)),KRAS野生型细胞为HT29和Caco2;与125μg/mL CET处理组比较,相同浓度CET明显降低KRAS野生型细胞Caco2和HT29的存活率,分别为34.03%和40.68%(P<0.01);与空白对照组比较,ZJW(50μg/mL)联合CET(125μg/mL)后,HCT116、Lovo和SW620细胞存活率均有显著下降,分别为59.97%,59.47%,52.58%(P<0.05);ZJW能够增强KRAS突变细胞内ROS和Fe^(2+)水平,降低GSH水平;当ZJW联合CET后,效果更加显著(P<0.05,P<0.01)。ZJW联合CET抑制SW620细胞SLC7A11、GPX4、Nrf2、MDM2蛋白和mRNA的表达,增强p53蛋白的表达,但不影响p53 mRNA的水平。结论:ZJW能够调控KRAS突变细胞Nrf2和p53途径从而抑制SLC7A11和GPX4促进铁死亡,可能是逆转CET耐药的机制。
AIM:To investigate the mechanism and reversal effect of Zuo Jin Wan(ZJW)on cetuximab(CET)resistance in KRAS mutant colorectal cancer cell.METHODS:The mutation status of KRAS gene in SW620,Lovo,HCT116,HT29 and Caco2 cells were detected by Sanger sequencing.CCK-8 assay was used to detect the effects of ZJW,CET,ZJW combined with CET and CET,ZJW in combination with other cell death inhibitors on the survival rate of the above cells,and to observe the reversal effects of ZJW on CET-treated KRAS mutant cells(SW620,Lovo and HCT116).Flow cytometry,colorimetric method,and Fe^(2+)ions fluorescent probe FerroOrange were used to detect the effects of ZJW,CET and their combination on ROS,GSH and Fe^(2+)levels in KRAS mutant cells.Western blot and real-time PCR were used to detect the regulatory effects of ZJW combined with CET on proteins and mRNA of ferroptosis-related pathway.RESULTS:KRAS mutant cells include HCT116(KRAS^(G13D)),Lovo(KRAS^(G13D))and SW620(KRAS^(G12V)),while HT29 and Caco2 were KRAS wild-type cells.Compared with 125μg/mL CET-treated KRAS mutant cells,CET at same concentration significantly reduced cell viabilities of KRAS wild-type cells(Caco2 and HT29 cells)by 34.03%and 40.68%,respectively(P<0.01).ZJW(50μg/mL)combined with CET(125μg/mL)markedly reduced cell viability of HCT116,Lovo and SW620 cells,which were 59.97%,59.47%and 52.58%,respectively(P<0.05),compared with the controls.ZJW could enhance ROS and Fe^(2+)levels,and decrease GSH levels in KRAS mutant cells.When ZJW combined with CET,the effects were more significant(P<0.05,P<0.01).ZJW combined with CET inhibited protein and mRNA expressions of SLC7A11,GPX4,Nrf2 and MDM2 in SW620 cells,and increased the expression of p53 protein.However,the combined treatment did not affect the level of p53 mRNA.CONCLUSION:ZJW could regulate Nrf2 and p53 pathways in KRAS mutant cells,thereby inhibiting SLC7A11 and GPX4 expressions and promoting ferroptosis,which may be the mechanism of reversing CET resisgtance.
作者
卫真真
隋华
姜宇朗
孙明瑜
闫化茹
王子元
WEI Zhenzhen;SUI Hua;JIANG Yulang;SUN Mingyu;YAN Huaru;WANG Ziyuan(Department of Pathology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Medical Experiment Center,Jiading Branch of Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 201803,China;Department of Medical Oncology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Key Laboratory of Liver and Kidney Diseases,Institute of Liver Diseases,Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,528 Zhangheng Road,Shanghai 201203,China)
出处
《中国临床药理学与治疗学》
CAS
CSCD
2023年第2期130-137,共8页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
国家自然科学基金资助项目(81703885,81874399)
上海中医药大学附属曙光医院“四明临床研究专项”(SGKJLC-202030)
上海市临床重点专科建设项目(shslczdzk01201)
国家中医药管理局中医肝胆病重点学科、慢性肝病虚损重点研究室和上海市中医临床重点实验室(20DZ2272200)
国家中医药管理局第四批中医优才(2017-124)
上海市科委专项(19401972300)
上海中医药大学研究生创新课程建设(2017)
山东省重点研发计划(重大科技创新工程,2021CXGC010509)。
