水飞蓟宾联合5-FU抑制胃癌MGC-803细胞增殖及机制

摘要

目的：研究水飞蓟宾联合5-氟尿嘧啶（5-FU ）抑制胃癌MGC-803细胞增殖，探讨其协同增效作用及机制．方法：采用噻唑蓝（MTT）法检测不同浓度的水飞蓟宾对人胃癌MGC-803细胞，人正常肝L02细胞的增殖抑制作用，并计算IC20，IC50；采用IC20浓度的水飞蓟宾联合不同浓度的5-氟尿嘧啶，观察对人胃癌MGC-803细胞增殖抑制作用；水飞蓟宾单独或联合5-FU作用于胃癌MGC-803细胞，碘化丙锭（PI）单染色检测细胞周期改变，Annexin V-FITC/PI双染流式细胞术检测细胞凋亡水平，Western blotting检测细胞周期和凋亡相关蛋白的表达．结果：MTT法检测显示，水飞蓟宾对胃癌MGC-803细胞有增殖抑制作用，且呈剂量-效应关系，对人正常肝L02细胞增殖抑制作用较弱．水飞蓟宾作用于胃癌MGC-803细胞48 h 的IC20、IC50浓度分别为144.6、214.7μmol /L；水飞蓟宾与不同浓度的5-氟尿嘧啶联合作用于MGC-803细胞48 h，可提高MGC-803细胞对5-氟尿嘧啶的敏感性，增敏5.73倍．流式结果显示，无论是单独使用水飞蓟宾、5-氟尿嘧啶还是水飞蓟宾与5-氟尿嘧啶联合使用都能使细胞抑制在G0/G1期和出现凋亡细胞群；Western blotting结果显示，水飞蓟宾与5-氟尿嘧啶联合使用，能使细胞的细胞周期相关蛋白P15表达升高，CDK6表达下降，Bcl-2蛋白家族中抗凋亡蛋白Bcl-2，Bcl-xL表达明显降低，促凋亡蛋白Bax表达基本不变，Casepase-9，Caspase-3活化降解．结论：水飞蓟宾提高胃癌MGC-803细胞对5-FU的敏感性，水飞蓟宾在联合5-FU之后能够使MGC-803细胞抑制在G0/G1期，并通过线粒体凋亡途径使细胞发生凋亡．%Aim:To investigate the synergistic effect of Silibinin combined with 5-fluorouracil (5-FU) on gastric cancer MGC-803 cells and the possible mechanism.Methods:MTT assay was used to deter-mine the inhibitory effect of Silibinin on gastric cancer MGC-803 cells and hepatocyte L02 cells.Changes in cell cycle and the apoptotic cell percentage were determined by Flow Cytometry.The expression of cell cycle related proteins and apoptosis associated proteins were analyzed by Western blotting.Results:MTT method was used to determine the IC20 value and IC50 value for hepatocyte L02 cells.IC20 concentra-tion of Silibinin combined with 5-FU at different concentrations were incubated with MGC-803 cells for 48 hours,MTT assay was used to measure the proliferation inhibition effects.After MGC-803 cells were in-cubated with Silibinin combined with 5-FU for 48 hours,the cell cycle of MGC-803 was inhibited by Sili-binin at different concentrations,in a dose-dependent manner(P<0.05 ),but no obvious effects were found on the proliferation of hepatocyte L02 cells.The IC20 and IC50 for Silibinin against gastric cancer MGC-803 cells after incubation for 48 hours were found to be 144.6 and 214.7 μmol /L,respectively. The chemo-therapeutic effects of 5-fluorouracil on MGC-803 cells were enhanced when combined with Sil-ibinin,and the drug sensitivity was increased by about 5 .73 times.PI staining analysis showed that cell cycle was arrested in G0/G1 phase.Annexin V-FITC /PI staining analysis also showed a dose-dependent effect on the apoptotic cells.Western blotting showed that the expression of cell cycle related protein CDK6 was decreased while that of P15 was increased.The expressions of apoptosis associated proteins, pro-Caspase-9,pro-Caspase-3,Bcl-2,Bcl-xL,were found to be decreased significantly,but expression of Bax remained unchanged.Conclusion:Silibinin has synergistic inhibitory effect with 5-fluorouracil on MGC-803 cells.Silibinin combined with 5-fluorouracil might inhibit cell cycle in G0/G1 phase and in-duce cell apoptosis through the mitochondrial pathway.