羽扇豆醇Lupeol通过Wnt／β-连环蛋白信号通路抑制肝癌细胞增殖的机制  

The anti -proliferative effects of Lupeol on HepG2 cells via inhibition of Wnt/β-catenin signaling pathway

作　　者：张玲莉[1] 邱振鹏[2] 涂毅[1] 彭燕[1] 

机构地区：[1]武汉大学人民医院药学部,430060 [2]武汉大学基础医学院

出　　处：《中华实验外科杂志》2015年第8期1872-1875,共4页Chinese Journal of Experimental Surgery

基　　金：湖北省自然科学基金资助项目（2013CFB239）

摘　　要：目的探讨羽扇豆醇Lupeol通过Wnt／β-连环蛋白（母一catenin）信号通路抑制肝癌细胞增殖的作用机制。方法采用细胞计数试剂盒（CCK-8）法及碘化丙锭（PI）单染法检测Lupeol对肝癌细胞的生长抑制作用及细胞周期的影响；以荧光素酶TOPflash／FOPflash报告系统检测Lupeol对HepG2细胞中Wnt信号通路的影响；Westernblot法检测Lupeol对HepG2细胞中B—catenin蛋白表达的影响；荧光定量聚合酶链反应（Real—timePCR）检测Lupeol处理后c-Myc及细胞周期素D1（CyclinD1）的mRNA表达水平；运用pGL3-Basic荧光素酶报告系统研究Lupeol对CyclinD1启动子活性的影响。结果Lupeol作用48h后，对HepG2细胞具有显著的抑制作用[细胞半数致死量半数抑制剂量（IC50）为（57．2±5．0）μmoL／L]。Lupeol处理HepG2细胞24、48h使S期的细胞分布百分比上升至27％及42％；Top／Fopflash荧光素酶实验显示Lupeol使HepG2细胞中T细胞因子／淋巴增强因子（TCF／LEF）经典Wnt信号通路关键蛋白β-catenin介导的转录活性降低80％，并使细胞内累积的β-catenin的蛋白表达下调约30％，并减少了c—Myc及CyclinD1的mRNA表达；同时其作用于CyclinD1启动子使CyclinD1的转录激活降低了3倍。结论Lupeol可作用于Wnt／β-catenin信号通路进而抑制HepG2细胞增殖并呈现S期阻滞效应，其机制可能与减少β-catenin在细胞内累积及降低c-Myc与CyclinD1的表达有关。Objective To study the antiproliferative effects and the regulatingmechanism of Lupeol on hepatocellular carcinoma cells in vitro. Methods Three kinds of liver cancer cells were treated with different concentration from 10 - 100 μmol/L and cell counting kit - 8 （ CCK - 8 ） assay was employed to evaluate the antiproliferative effects. Propidium iodide （PI） staining assay was perferm to observe the mi- totic cycle in Lupeol- treated HepG2 cells. T- cell factor/lymphocyte enhancer factor （TCF/LEF） tran- scriptional activity was assessed by TOPflash/FOPflash lueiferase reporter plasmid kit. The β - eatenin pro- tein expression, mRNA expression of c - Myc and Cyclin D1 were determined by using Western blotting and reverse transcriptase - polymerase chain reaction （ RT - PCR ）. pGL3 - Basic vector loading with Cyclin D1 promoter region was transfected into HepG2 cells to clarity the effects of Lupeol on Cyclin D1 transcriptional activity. Results Lupeol dose- dependently suppressed the proliferation HepG2 celts and the 50% inhibitory dose （ IC50 ） is （57.2 ± 5.0） μmol/L. The cell percentage of S phase was significantly increased to 42% ＇after 48 h treatment of Lupeol. Top/Fop flash luciferase assay showed that Lupeol could supress the strongly activating status in HepG2. The β-catenin protein expression was decreased to ap- proximately 70% accompanied with the down - regulation of c - Myc and Cyelin D1 mRNA expression. The transcriptional activity of Cyelin D1 was also decreased to 25 % when cells were administrated with Lupeol, compared with control group. Conclusion Lupeol could inhibit the HepG2 cell proliferation and regulate the cell cycle in vitro via suppressing the Wnt/β-catenin signaling pathway, which was involved in sup- pressing expression of β- catenin, e - Mye and Cyelin D1.

关 键 词：羽扇豆醇 HEPG2细胞株 WNT信号通路 抗增殖 

分 类 号：R730[医药卫生—肿瘤]