苦参碱通过调控β-catenin信号通路抑制肝癌细胞干性  

Matrine suppresses stemness of hepatocellular carcinoma cells by regulating β-catenin signaling pathway

作　　者：戴美琴 蔡茁 陈娜娜[2] 李金州 温嘉泳 谭丽转 郭丹[1] DAI Meiqin;CAI Zhuo;CHEN Nana;LI Jinzhou;WEN Jiayong;TAN Lizhuan;GUO Dan(Department of Pharmacy,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;College of Pharmaceutical Science,Southern Medical University,Guangzhou 510515,China;Department of Pharmacy,Air Force Hospital of Southern Military Command,Guangzhou 510602,China)

机构地区：[1]南方医科大学南方医院药学部,广东广州510515 [2]南方医科大学药学院,广东广州510515 [3]南部战区空军医院药剂科,广东广州510602

出　　处：《南方医科大学学报》2019年第10期1239-1245,共7页Journal of Southern Medical University

基　　金：广东省自然科学基金(2016A030313595);广州市科技计划项目(201707010074)

摘　　要：目的探讨苦参碱对人肝癌HepG2和Huh7细胞增殖活性、细胞干性、β-catenin转录活性以及AKT/GSK3β/β-catenin信号通路的影响。方法应用MTT法检测细胞增殖活性,平板克隆形成实验检测0 g/mL、200 g/mL、400μg/mL、800μg/mL苦参碱对人肝癌HepG2和Huh7细胞的克隆形成能力,荧光定量PCR分析0 g/mL、200 g/mL、400μg/mL、800μg/mL苦参碱对人肝癌HepG2和Huh7细胞干性基因CD90、EpCAM(上皮细胞粘附分子)和CD133 mRNA的表达水平,双荧光素酶报告基因检测0 g/mL、200 g/mL、400μg/mL、800μg/mL苦参碱对人肝癌HepG2和Huh7细胞内β-catenin转录活性,Western blotting检测0 g/mL、400μg/mL、800μg/mL苦参碱对人肝癌HepG2和Huh7细胞内AKT(蛋白激酶B)、GSK-3β和β-catenin及其相应磷酸化蛋白的表达水平。结果MTT实验结果显示,苦参碱呈时间-浓度依赖性地抑制肝癌HepG2和Huh7细胞的增殖,处理24、48、72 h对HepG2细胞的半数抑制浓度依次为2369、1565、909.1μg/mL,对Huh7细胞的半数抑制浓度依次为1355、781.8、612.8μg/mL。苦参碱浓度依赖性地抑制肝癌细胞的克隆形成能力,400μg/mL、800μg/mL苦参碱能够显著抑制HepG2细胞的克隆形成能力(P<0.01,P<0.001),200 g/mL、400μg/mL、800μg/mL苦参碱能够显著抑制Huh7细胞的克隆形成能力(P<0.05,P<0.01,P<0.001)。苦参碱能够显著肝癌细胞内CD90、EpCAM和CD133等干细胞标志物mRNA的表达,其中400μg/mL、800μg/mL苦参碱能够显著抑制HepG2细胞中CD90(P<0.01,P<0.001)、EpCAM(P<0.05,P<0.01)和CD133(P<0.01,P<0.01)mRNA的表达,400μg/mL、800μg/mL苦参碱能够显著抑制Huh7细胞中CD90(P<0.01,P<0.01)、EpCAM(P<0.05,P<0.01)和CD133(P<0.01,P<0.01)mRNA的表达。同时400μg/mL、800μg/mL苦参碱显著抑制HepG2细胞(P<0.001,P<0.001)和Huh7细胞(P<0.01,P<0.001)内β-catenin的转录活性。研究结果显示400μg/mL、800μg/mL苦参碱能够显著降低HepG2和Huh7细胞内β-catenin和磷酸化的AKT和GSK-3β蛋白水平,增加β-catenin�Objective To explore the effects of matrine on the proliferation,tumor cell stemness,β-catenin transcriptional activity and AKT/GSK3β/β-catenin signaling pathway in human hepatocellular carcinoma(HCC)HepG2 and Huh7 cells.Methods The proliferation and colony formation ability of HepG2 and Huh7 cells treated with 200,400,and 800μg/mL matrine were evaluated with MTT assay and colony formation assay,respectively.Real-time quantitative PCR was performed to detect the mRNA expressions of CD90,epithelial cell adhesion molecule(EpCAM)and CD133,and dual-luciferase assay was used to detect the transcriptional activity ofβ-catenin in the treated cells.The effects of matrine on the expressions of protein kinase B(AKT),P-AKT,GSK-3β,P-GSK-3β,P-β-catenin andβ-catenin proteins in the Wnt/β-catenin signaling pathway were assessed using Western blotting.Results Matrine inhibited the proliferation of the two HCC cell lines in a time-and concentration-dependent manner.The half-inhibitory concentrations of matrine were 2369,1565 and 909.1μg/mL at 24,48 and 72 h in HepG2 cells,respectively,and were 1355,781.8,and 612.8μg/mL in Huh7 cells,respectively.Matrine concentrationdependently suppressed colony formation of the HCC cells,producing significant inhibitory effects at 400μg/mL(P<0.01)and 800μg/mL(P<0.001)in HepG2 cells and at 200μg/mL(P<0.05),400μg/mL(P<0.01),and 800μg/mL(P<0.001)in Huh7 cells.Matrine at 400 and 800μg/mL significantly inhibited the mRNA expression of CD90,EpCAM and CD133 and the transcriptional level ofβ-catenin in both HepG2 and Huh7 cells(P<0.05 or 0.01).Matrine at 400 and 800μg/mL also significantly decreased the protein levels ofβ-catenin,P-AKT and P-GSK-3βand increased the phosphorylation level ofβ-catenin in both of the cell lines.Conclusion Matrine inhibits the proliferation,colony formation,and the expressions of tumor stem cell markers CD90,EpCAM and CD133 in both HepG2 and Huh7 cells probably by inhibiting Wnt/β-catenin signaling pathway and the transcriptional activity ofβ-catenin

关 键 词：苦参碱 肝细胞癌 肿瘤干性 Β-CATENIN 

分 类 号：R73[医药卫生—肿瘤]