盐酸小檗胺通过调控自噬和PI3K/Akt/mTOR信号通路改善索拉非尼耐药的机制  

Berbamine Hydrochloride Ameliorates Sorafenib Resistance by Regulating Autophagy and PI3K/Akt/mTOR Signaling Pathway

作　　者：吴泽明 黄欣慧 彭琴 肖灵 黄梓源 林翊雅 谭宇蕙[1] WU Zeming;HUANG Xinhui;PENG Qin;XIAO Ling;HUANG Ziyuan;LIN Yiya;TAN Yuhui(School of Basic Medical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;School of Life Sciences and Technology,Tongji University,Shanghai 200092,China;Bao'an Traditional Chinese Medicine Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518133,China)

机构地区：[1]广州中医药大学基础医学院,广州510006 [2]同济大学生命科学与技术学院,上海200092 [3]广州中医药大学附属宝安中医院,广东深圳518133

出　　处：《中国实验方剂学杂志》2024年第14期78-88,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：广东省自然科学基金面上项目(2023A1515012708);国家自然科学基金面上项目(81774028)。

摘　　要：目的:研究盐酸小檗胺改善肝癌细胞对索拉非尼耐药的作用及相关机制。方法:以1.25μmol·L^(-1)索拉非尼为起始浓度采用药物浓度递增法筛选索拉非尼耐药细胞株SMMC-7721/S,SMMC-7721和SMMC-7721/S分别设置0、2.5、5、10、15、20μmol·L^(-1)索拉菲尼给药组,采用细胞增殖与活性检测试剂盒(CCK-8)实验检测半抑制浓度(IC50)、计算耐药指数(RI);采用蛋白免疫印迹法(Western blot)比较SMMC-7721和SMMC-7721/S细胞自噬和磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路相关蛋白表达差异。设置5μmol·L^(-1)盐酸小檗胺与2.5、5、10μmol·L^(-1)索拉非尼单独和联合组,进行CCK-8实验检测各组对肝癌细胞SMMC-7721/S细胞生长的影响;分别设置5μmol·L^(-1)小檗胺与5μmol·L^(-1)索拉非尼单独和联合组,进行平板克隆形成实验检测各组对SMMC-7721和SMMC-7721/S细胞增殖的影响;分别设置5μmol·L^(-1)盐酸小檗胺、0.1μmol·L^(-1)自噬抑制剂巴氟洛霉素(Baf)组,进行免疫荧光实验检测溶酶体酸化标志物微管相关蛋白1轻链3(LC3)及LysoTracker探针检测SMMC-7721细胞溶酶体酸化情况;设置盐酸小檗胺5μmol·L^(-1)、Baf 0.1μmol·L^(-1)设置0、2.5、5、10μmol·L^(-1)盐酸小檗胺组及0.1μmol·L^(-1)Baf组,进行Western blot实验比较各组对SMMC-7721细胞自噬和PI3K/Akt/mTOR信号通路相关蛋白表达的影响。结果:CCK-8实验表明索拉非尼对SMMC-7721/S细胞24 h的IC50为9.56 mol·L^(-1)(P<0.01),对SMMC-7721细胞24 h的IC50为7.99 mol·L^(-1),耐药指数RI为1.20(P<0.01),为轻度耐药;Western blot实验表明,对比SMMC-7721细胞,SMMC-7721/S细胞内磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、磷酸化蛋白激酶B(p-Akt)、微管相关蛋白1轻链3-Ⅱ(LC3Ⅱ)表达增加,螯合体1(p62)蛋白表达显著减少(P<0.01),化蛋白激酶B(Akt)蛋白表达水平差异无统计学意义。CCK-8实验和平板克隆形成实验表明盐酸小檗胺�Objective:To investigate the effects of berbamine hydrochloride on sorafenib resistance in hepatocellular carcinoma cells and the underlying mechanisms.Method:The sorafenib-resistant cell line SMMC-7721/S was selected by the concentration increment method starting at 1.25μmol·L^(-1) sorafenib.Both SMMC-7721 and SMMC-7721/S cells were treated with 0,2.5,5,10,15,20μmol·L^(-1) sorafenib,and the cell counting kit-8(CCK-8)assay was employed to determine the half maximal inhibitory concentration(IC50)and calculate the resistance index(RI).Western blot was conducted to compare the expression of proteins involved in autophagy and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway between SMMC-7721 and SMMC-7721/S cells.Furthermore,SMMC-7721/S cells were treated with 5μmol·L^(-1) berbamine hydrochloride alone or in combination with 2.5,5,10μmol·L^(-1) sorafenib,and the cell growth was assessed by the CCK-8 assay.In addition,SMMC-7721 and SMMC-7721/S cells were treated with 5μmol·L^(-1) berbamine hydrochloride alone or in combination with 5μmol·L^(-1) sorafenib,and the cell proliferation was examined by the colony formation assay.The immunofluorescence assays with Microtubule-associated protein 1 light chain 3(LC3)and LysoTracker as probes were employed to assess the lysosomal acidification in SMMC-7721 cells treated with 5μmol·L^(-1) berbamine hydrochloride or 0.1μmol·L^(-1) autophagy inhibitor bafilomycin A1(Baf).Further,the expression of proteins involved in autophagy and PI3K/Akt/mTOR signaling pathway was determined by Western blot and compared between groups.Result:Sorafenib showed the IC50 of 9.56 mol·L^(-1)(P<0.01)and 7.99 mol·L^(-1) for SMMC-7721/S and SMMC-7721 cells,respectively,at 24 h.The resistance index(RI)of SMMC-7721/S for sorafenib was 1.20(P<0.01),which indicated mild resistance.Compared with SMMC-7721 cells,SMMC-7721/S cells exhibited up-regulated expression of p-mTOR,p-Akt,and LC3Ⅱ,down-regulated expression of p62 protein(P<0

关 键 词：盐酸小檗胺 索拉非尼耐药 自噬 磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR) 肝癌 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33