组蛋白去乙酰化酶抑制剂LBH589诱导多发性骨髓瘤细胞凋亡及其逆转耐药的机制研究

Study on histone deacetylase inhibitor LBH589 induces apoptosis of multiple myeloma cells and its reversal of drug resistance mechanism

目的探讨新一代组蛋白去乙酰化酶抑制剂LBH589单药或联合蛋白酶体抑制剂硼替佐米对多发性骨髓瘤（MM）细胞增殖的影响及其机制。方法以MM细胞系U266细胞及地塞米松耐药细胞系MM1R细胞为对象，不同浓度LBH589或LBH589与硼替佐米联合作用上述细胞后，采用MTF法检测对细胞增殖的抑制作用，流式细胞术检测细胞周期和细胞凋亡率的变化，Westernblot分析组蛋白H4乙酰化程度及聚腺苷二磷酸核糖聚合酶（PARP）、Bcl—X蛋白表达水平的变化；实时荧光定量PCR分析caspase-3、APAF-1以及TOSO基因表达水平的变化。结果不同浓度LBH589（0、10、20、50nmol／L）单药及其50nmol／LLBH589与硼替佐米（10、20nmol／L）联合均能够抑制U266和MM1R细胞增殖，呈剂量和时间依赖性，且LBH589与硼替佐米联合组抑制作用均较单药组明显（P值均〈0．05）；MM1R细胞G0／G1期比例分别为36．60％、46．50％、51．40％、57．10％、75．48％、79．73％，凋亡率分别为5．27％、31．41％、39．78％、44．07％、73．60％、83．27％，作用呈剂量依赖性，且LBH589与硼替佐米联合组作用均较两者单药组明显（P值均〈0．05）；MM1R细胞组蛋白H4乙酰化程度和PARP蛋白表达水平逐渐上调，而Bcl-X蛋白表达水平则逐渐下调，变化呈剂量依赖性（P值均〈0．05）；MMlR细胞caspase-3、APAF-1基因表达水平逐渐上调，而TOSO基因表达水平则逐渐下调，变化呈剂量和时间依赖性（P值均〈0．05）。结论LBH589能够抑制MM细胞增殖，阻滞细胞周期，诱导细胞凋亡，对耐药细胞具有抗耐药作用，同时与硼替佐米联合应用对MM细胞有协同作用，其作用机制及逆转耐药机制涉及多个基因的表达变化。

Objective To explore the impact of a new generation of histone deacetylase inhibitor LBH589 alone or in combination with proteasome inhibitor bortezomib on multiple myeloma （ MM ） ceils pro-liferation and its mechanism. Methods MM cell line U266 and dexamethasone resistant cell line MM1R cells were treated with different concentrations of LBH589 alone or in combination with bortezomib, the inhibition of cells proliferation was detected by MTT, the cell cycle and apoptosis by flow cytometry. The expression level of histone H4 acetylation and PARP, BcI-X protein was analyzed by western blot, expression level of caspase-3, APAF-1 and TOSO gene by real-time fluorescence quantitative PCR. Results U266 and MM1R cell proliferation were inhibited by different concentrations of LBH589 （0, 10, 20, 50 nmol/L）alone or 50 nmol/L of LBH589 in combination with bortezomib （10, 20 nmol/L） in a dose - and time-dependent man- ner. Inhibition effect was significantly higher in all combinative groups than in single agent groups （ all P 〈 0.05）. The percentage of G0/Gl phase in MM1R cells were 36.60%, 46.50% , 51.40% , 57. 10% , 75.48%, 79.73%, respectively, and the apoptosis rate were 5.27%, 31.41%, 39.78%, 44.07%, 73.60%, 83.27%, respectively. The effects appeared to occur in a dose-dependent manner, and being sig- nificantly higher in all combinative groups than in single agent groups （ all P 〈 0.05 ）. The expression of the caspase-3 and APAF-1 gene up-regulated gradually, while TOSO gene expression in MM1R cells down-regula- ted gradually in a dose- and time-dependent manner （all P 〈 0.05）. Conclusions LBH589 can inhibit the growth of MM cells, block the cell cycle and induce cell apoptosis, which has anti-resistant effect on multi- drug resistant cell. At the same time LBH589 in combination with bortezomib on myeloma cell has a synergistic effect, its mechanism and reversal of drug resistance mechanism involves in multiple changes in gene expression.