Objective To investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3.Methods The effects of somfenib 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 somfenib + 5-FU combination did not provide better results than treatment with either drag 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 semitive 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).Sorafenib 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 cyclinD1 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 p-C-RAF,p-ERK1/2 and p-STAT3 being 0.56 ±0.05,0.54 ± 0.02 and 0.36 ± 0.02,respectively (allP ＜ 0.01); 5-FU alone produced no significant effects on these pathways.Conclusion Administered alone,both somfenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells.The somfenib + 5-FU combination treatment produces antagonistic,rather than synergistic,effects.Somfenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G lphase arrest and S phase reduction,thereby reducing the cells' sensitivity to 5-FU.%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(CI);应用流式细胞技术检测药物对细胞周期的影响；用Westem blot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cyclinD1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按一定的药物浓度比例(索拉非尼∶5-FU=2 μ mol∶1 mg/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;当与一定浓度(8μmol)的索拉非尼合用后,5-FU对细胞的IC50增加到(178.61±20.73) mg/L.提示与索拉非尼合用时,MHCCLM3细胞对5-FU的敏感性下降.(4)细胞周期检测结果显示,G1期细胞比例,空白对照组为44.73％±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％±1.75％.提示索拉非尼作用后,能把MHCCLM3细胞阻滞在G1期.(5)索拉非尼能明显阻断RAF/MEK/ERK和STAT3信号通路,下调cyclinD1蛋白的表达量,索拉非尼作用后,磷酸化C-RAF、磷酸化ERK1/2、磷酸化STAT3(Y705)和cyclinD 14种蛋白的表达量分别下降至对照组的(0.56±0.05)倍、(0.54±0.02)倍、(0.36±0.02)倍和(0.57±0.03)倍；而5-FU对这两条信号通路基本不起作用,但能上调cyclinD1的表达,5-FU作用后cyclinD1的表达量上调至对照组的(1.45±0.12)倍.结论 索拉非尼和5-FU对肝癌细胞均有显著的抗肿瘤作用,但两药联合应用并没有产生协同作用.其可能的机制为,索拉非尼通过阻断RAF/MEK/ERK信号通路以及STAT3相关通路,抑制了细胞周期相关蛋白cyclinD1的表达,使肿瘤细胞阻滞在G1期,而S期细胞数目减少,从而使肿瘤细胞对5-FU的敏感性降低.
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