扁蒴藤素对人结直肠癌细胞HCT116和HT29增殖、迁移和细胞周期的影响及作用机制研究  

Effects of pristimerin on proliferation,migration and cell cycle of colorectal cancer HCT116 and HT29 cells and its action mechanism

作　　者：吕蒙莹 罗照勇 王维民 梁巧玲 王杨 钱亚云 刘延庆 LYU Mengying;LUO Zhaoyong;WANG Weimin;LIANG Qiaoling;WANG Yang;QIAN Yayun;LIU Yanqing(Medical College of Yangzhou University,Institute of Translational Medicine,Yangzhou,Jiangsu,225001;The Key Laboratory of Syndrome Differentiation and Treatment of Toxic Pathogen-based Gastric Cancer of the State Administration of Traditional Chinese Medicine,Yangzhou,Jiangsu,225001)

机构地区：[1]扬州大学医学院/转化医学研究院,江苏扬州225001 [2]国家中医药管理局胃癌毒邪论治重点研究室,江苏扬州225001

出　　处：《实用临床医药杂志》2022年第13期60-67,共8页Journal of Clinical Medicine in Practice

基　　金：国家自然科学基金青年项目(81903906);江苏省高等学校自然科学研究重大项目(19KJA480003);江苏省自然科学基金青年项目(BK20180927);江苏省中医药局项目(YB201850);中国博士后科学基金面上项目(2018M642346);扬州大学创新培育基金项目(2019CXJ175);扬州大学高层次人才科研启动基金项目(2021)。

摘　　要：目的探讨扁蒴藤素对人结直肠癌HCT116细胞和HT29细胞增殖、迁移和细胞周期的影响,并初步探索潜在的分子机制。方法采用噻唑蓝(MTT)实验于不同时点分别检测不同浓度扁蒴藤素对HCT116细胞和HT29细胞存活率的影响;采用5-乙炔基-2'-脱氧尿苷(EdU)实验分别检测不同浓度扁蒴藤素对HCT116细胞和HT29细胞增殖能力的影响;采用Transwell实验观察不同浓度扁蒴藤素对HCT116细胞和HT29细胞迁移能力的影响;采用流式细胞术检测扁蒴藤素对HCT116细胞和HT29细胞周期的影响;采用蛋白质印迹法(Western blot)检测扁蒴藤素对HCT116细胞与HT29细胞周期相关蛋白cyclin E1、CDK2、p53、p21、p27表达和PI3K/Akt/mTOR信号转导通路相关蛋白表达的影响。结果MTT实验结果显示,经不同浓度扁蒴藤素分别处理12、24、36、48、60 h后,HCT116细胞和HT29细胞的存活率均较对照细胞降低,且呈时间和浓度依赖性。EdU实验结果显示,经0.5、1、2μmol/L扁蒴藤素处理24 h后,HCT116细胞中阳性细胞率低于对照组,差异有统计学意义(P<0.001);经1、2、4μmol/L扁蒴藤素处理24 h后,HT29细胞中阳性细胞率低于对照组,差异有统计学意义(P<0.001)。Transwell实验结果显示,加入0.5、1、2μmol/L扁蒴藤素作用24 h后,HCT116细胞穿透小室底膜的数量变少,且细胞迁移率低于对照细胞,差异有统计学意义(P<0.001);加入1、2、4μmol/L扁蒴藤素作用24 h后,HT29细胞穿透小室底膜的数量变少,且细胞迁移率低于对照细胞,差异有统计学意义(P<0.001)。流式细胞术结果显示,扁蒴藤素可诱导HCT116细胞和HT29细胞发生G_(0)/G_(1)期阻滞。Western blot结果显示,与对照细胞比较,HCT116细胞和HT29细胞经扁蒴藤素处理后p53、p21、p27蛋白表达量增加,cyclin E1、CDK2蛋白表达量降低,且p-PI3K、p-Akt、mTOR和p-mTOR蛋白表达量降低,差异有统计学意义(P<0.05或P<0.01或P<0.001)。结论扁蒴藤素可抑制HCT116细胞和HT2Objective To investigate impacts of pristimerin on proliferation,migration and cell cycle of colorectal cancer HCT116 and HT29 cells and uncover the potential mechanism.Methods Cell viability of HCT116 and HT29 cells treated with pristimerin at various concentrations was measured by Methyl Thiazolyl Tetrazolium(MTT)assay at different time points.The proliferation of HCT116 cells and HT29 cells treated with pristimerin at various concentrations was detected by 5-acetyney-2'-deoxyuridine(EdU)assay.Transwell experiment was performed to observe the effects of different concentrations of pristimerin on migration of HCT116 cells and HT29 cells.The influence of pristimerin on cell cycle of HCT116 cells and HT29 cells was tested by flow cytometry.Western blot was used to determine its impact on cell cycle-related proteins including cyclin E1,CDK2,p53,p27 and p21 as well as PI3K/Akt/mTOR signaling pathways.Results MTT tests revealed that the viability of HCT116 and HT29 cells after 12,24,36,48 and 60 h treated by different concentrations of pristimerin decreased significantly,and changed in a dose and time-dependent manner.EdU tests showed that the positive cell rates of HCT116 cells treated with 0.5,1,2μmol/L pristimerin for 24 h were lower than those of the control group,the difference was statistically significant(P<0.001).After treatment with 1,2 and 4μmol/L pristimerin for 24 h,the positive cell rates of HT29 cells were lower than those of the control group,the difference was statistically significant(P<0.001).Transwell experiment results showed that the number of HCT116 cells penetrating the subventricular membrane decreased,and the cell migration rates were lower than that of control cells after treatment with 0.5,1,2μmol/L pristimerin for 24 h(P<0.001).Flow cytometry results showed that pristimerin could induce cell cycle arrest of HCT116 and HT29 cells at G 0/G 1 stages.Western blot results displayed that p53,p21 and p27 protein expression were up-regulated and the expression of cyclin E1,CDK2,p-PI3K,p-Akt,mTO

关 键 词：扁蒴藤素 结直肠癌 增殖 迁移 细胞周期 信号通路 

分 类 号：R735.3[医药卫生—肿瘤] R285.5[医药卫生—临床医学]