曲古抑菌素A增加人肝癌Bel7402细胞对TRAIL敏感度的实验

doi:10.3971/j.issn.1000-8578.2015.06.002

曲古抑菌素A增加人肝癌Bel7402细胞对TRAIL敏感度的实验

Trichostatin A Increases Sensitivity of Hepatocellular Carcinoma Cells Bel7402 to TRAIL

摘要

摘要: 目的探讨曲古抑菌素 A（Trichostatin A, TSA）联合肿瘤坏死因子（tumor necrosis factor, TNF）相关凋亡诱导配体（TNF related apoptosis inducing ligand, TRAIL）对肝癌细胞Bel7402增殖凋亡的影响及其机制。方法采用四甲基偶氮唑盐（MTT）染色法分别检测TSA、TRAIL及低浓度TSA联合TRAIL处理Bel7402细胞的生长抑制率；4,6-二脒基-2-苯基吲哚二盐酸盐（DAPI）染色法对药物联合处理后的细胞进行凋亡形态学观察；免疫细胞化学和Western blot技术观察药物联合作用后p65蛋白在细胞中表达和定位的变化。结果不同浓度TSA作用6、12和24 h对人肝癌Bel7402细胞的增殖没有明显抑制作用，而作用48 h后的细胞增殖抑制率明显升高，和对照组比较差异有统计学意义（P<0.05）；不同浓度TRAIL处理Bel7402细胞存活率没有明显改变；低浓度TSA（20 ng/ml）预处理能够增加Bel7402细胞对TRAIL治疗的敏感度，TSA预处理联合TRAIL（100ng/ml)作用细胞24 h后，细胞生存率为（57.1±5.4）%，和单独药物处理组及对照组比较差异有统计学意义（P<0.05）；DAPI染色显示TSA和TRAIL联合作用后Bel7402细胞有核凋亡出现。荧光显微镜观察证明单独应用TSA（200 ng/ml）或TRAIL（100 ng/ml）处理的细胞p65蛋白有部分核内移位，而两种药物联合应用导致p65蛋白表达量降低，并且发生明显的核内转移和集聚。结论低浓度TSA能够增加肝癌Bel7402细胞对TRAIL的敏感度；其机制可能是两种药物联合应用降低p65的表达和活性，从而诱导Bel7402细胞凋亡。

Abstract: Objective To investigate the effect of Trichostatin A(TSA) combined with tumor necrosis factor(TNF) related apoptosis inducing ligand(TRAIL) on the proliferation and apoptosis of human hepatocellular carcinoma cell line Bel7402 in vitro, and to explore the possible mechanism. Methods The inhibitory effect of TSA, TRAIL, low concentration TSA combined with TRAIL on the proliferation of Bel7402 cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) staining method; 4,6-diamidino-2-phenylindole dihydrochlorid(DAPI) staining method was applied to display the apoptosome morphology of hepatocellular carcinoma cells; the expression and subcellular location of p65 were investigated by immunocytochemistry and Western blot. Results There was no influence of different concentrations of TSA on the proliferation of Bel7402 cells for 6, 12 and 24 h, but the proliferation of Bel7402 cells were inhibited significantly after 48 h, compared with control group(P<0.05). The proliferation of Bel7402 cells also had little change while treated with TRAIL; Pretreatment of TSA(20 ng/ml) could increase the sensitivity of Bel7402 cells to TRAIL. The survival rate of Bel7402 cells were (57.1±5.4)% after pretreated with TSA(20ng/ml) combined with TRAIL (100ng/ml) for 24h, compared with control group(P<0.05). DAPI staining method indicated that Bel7402 cells were trended to characteristic morphological changes of apoptosis when treated with TSA combined with TRAIL. Fluorescence microscope confirmed that the expression and location of p65 presented dominantly in ytoplasm, however, the translocation of p65 from the cytoplasm into the nucleus were repressed when treated with TSA (200ng/ml) or TRAIL(100ng/ml) for 24h. While the translocation and accumulation of p65 from cytoplasm to nucleus were increased and the expression of p65 were repressed when treated with low concentration of TSA combined with TRAIL. Conclusion TSA has a capacity to enhance TRAIL-induced apoptosis of hepatocellular carcinoma cells Bel7402; TSA combined with TRAIL inducing the apoptosis of Bel7402 cells may be involved in decreasing the expression and activity of p65．

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