索拉非尼联合5-氟尿嘧啶对人肝癌细胞株MHCCLM3增殖的抑制作用及其机制  

Anti-proliferation effect of sorafenib in combination with 5-FU for hepatocellular carcinoma in vitro： antagonistic performance and mechanism

作　　者：邓丽芬[1] 王艳红[1] 贾庆安[1] 任正刚[1] 沈沪佳 孙晓静[1] 李璟寰[1] 

机构地区：[1]复旦大学附属中山医院肝癌研究所,上海200032

出　　处：《中华肝脏病杂志》2013年第11期845-849,共5页Chinese Journal of Hepatology

基　　金：上海市卫生局项目（2010114）

摘　　要：目的观察分子靶向药物索拉非尼与常用化学治疗药物5-氟尿嘧啶（5-FU）合用模式对人肝癌细胞株MHCCLM3增殖的抑制作用，并初步探讨其机制。方法以人肝癌细胞系MHCCLM3细胞为靶细胞，用索拉非尼和（或）5-FU干预细胞。将细胞分4组，即索拉非尼组、5-FU组、两药联用组、空白对照组。使用CCK-8的方法检测药物对细胞的增殖抑制作用，参考中效原理的方法计算两药合用时的联合指数（凹）；应用流式细胞技术检测药物对细胞周期的影响；用Westernb10t方法检测增殖相关信号通路RAF／MEK／ERK和STAT3的相关蛋白，以及细胞周期相关蛋白cyclinDl的表达。两两比较使用最小显著差异法检验。结果（1）与空白对照组比较，索拉非尼组、5-FU组、两药联用组对MHCCLM3细胞的抑制率分别为46．16％±2．52％、28．67％±6．16％、22．59％±6．89％；索拉非尼、5-FU分别单用或合用均能明显抑制MHCCLM3细胞的增殖。（2）索拉非尼与5-FU按-定的药物浓度比例（索拉非尼：5-FU=2μmol：1mg／L）合用时，CI〉1，提示两药合用产生拮抗作用。（3）5-FU单药对细胞的半数抑制浓度（Ic50）为（102．86±27．84）mg／L，当与-定浓度（8μm01）的索拉非尼合用后，5-FU对细胞的Ic50增加到（178．61土20．73）rng／L。提示与索拉非尼合用时，MHCeLM3细胞对5-FU的敏感性下降。（4）细胞周期检测结果显示，G，期细胞比例，空白对照组为44．73％土1．63％，索拉非尼组为65．80％±0．56％。S期细胞的比例，空白对照组为46．63％±0．65％，索拉非尼组为22．83％土1．75％。提示索拉非尼作用后，能把MHCCLM3细胞阻滞在Gl期。（5）索拉非尼能明显阻断RAF／MEK／ERK和STAT3信号通路，下调cyclinDl蛋白的表达量，索拉非尼作用后，磷酸化C—RAF、磷酸化ERKl／2、磷酸化sTAT3（Y705）和cyclinDl4种蛋白的表达量分别下降至对照�Objective To investigate the anti-cancer efficacy and mechanism of sorafenib and5-fluorouracil （5-FU） therapy in vitro using the HCC cell line MHCCLM3. Methods The effects of sorafenib and 5-FU, alone or in combination, on the proliferation of MHCCLM3 cells were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay. Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting. Results Sorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells, with the following inhibition rates： somfenib： 46.16% ± 2.52%, 5-FU： 28.67% ± 6.16%, and sorafenib ＋ 5-FU： 22.59% ±6.89%. The sorafenib ＋ 5-FU combination did not provide better results than treatment with either drug alone. The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1, indicating that the two agents induced antagonistic, instead of synergistic, effects on the MHCCLM3 cells. In addition, the MHCCLM3 cells were less sensitive to 5-FU when administrated in combination with sorafenib, as evidenced by the half inhibitory concentration （IC50） significantly increasing from （102.86± 27.84） mg/L to （178.61 ± 20.73） mg/L （P = 0.003）. Somfenib alone induced G1 phase arrest （increasing from 44.73% ± 1.63% to 65.80%± 0.56%; P 〈 0.001） and significantly decreased the proportion of cells in S phase （decreasing from 46.63%± 0.65% to 22.83% ±1.75%; P 〈 0.01）, as well as down-regulated cyclinD1 expression （0.57 ± 0.03） fold change vs. untreated control group; P 〈 0.01）. 5-FU alone up-regulated cyclinD 1 expression （1.45 ±0.12） fold change vs. untreated control group; P 〈 0.01）. Moreover, somfenib alone significantly inhibited the RAF/ MEK/ERK and STAT3 pathways, with the fold-changes of

关 键 词：癌 肝细胞 拮抗剂 索拉非尼 

分 类 号：R735.7[医药卫生—肿瘤]