LBH589或联合硼替佐米逆转髓系白血病耐药及其分子机制研究

Reversal effect of LBH589 alone or in combination with bortezomib on drug-resistance in myeloid leu- kemia and its mechanism

目的探讨新一代组蛋白去乙酰化酶抑制剂LBH589联合蛋白酶体抑制剂硼替佐米逆转急性髓系白血病（AML）细胞耐药效应及其分子机制。方法耐药AML细胞株HL-60／ADM和难治性AML原代细胞与不同浓度LBH589、硼替佐米或两者联合进行体外培养，采用MTT法检测细胞增殖活力，Hoechst33342染色和Annexin V—FITC／PI双染流式细胞术检测细胞凋亡，用阿霉素摄取率并结合细胞增殖抑制率评估逆转耐药效应。进一步检测处理前后细胞MRP1及信号通路蛋白变化。结果LBH589、硼替佐米均能够抑制HL-60／ADM和难治性AML原代细胞增殖，诱导凋亡，其中21nmol/L LBH589和12nmol/L硼替佐米联合时作用最强，经Calcusyn软件分析证明两者联合具有明显的协同作用（叫值分别为0．531和0．498）。联合组MRP1阳性率为（57．78±3．34）％，显著低于LBH589、硼替佐米单药组[（76．06±5．17）％和（71．83±4．53）％，P值均〈0．05]，而单药组与对照组之间差异也有统计学意义（P值均〈0．01）。联合组细胞内阿霉素摄取率为（64．81±3．69）％，明显高于单药组[（28．96±2．52）％和（37．29±3．71）％]（P值均〈0．05）。LBH589联合硼替佐米可以明显抑制磷酸化Akt和磷酸化IKKα／β蛋白的表达，降低P13K／Akt／NF-κB通路的活性，明显上调P53蛋白的表达，抑制NF-κBP65、Bcl-2、XIAP和MRP1蛋白活性，促进Caspase-8、Caspase-3和PARP的裂解激活。结论LBH589、硼替佐米能够抑制耐药AML细胞增殖和促进凋亡，并逆转耐药，两者联合具有明显的协同作用。P13K／Akt／NF-κB信号传导通路受抑或阻断可能是其主要的作用机制。

Objective To investigate reversal effect of histone deacetylase inhibitor LBH589 alone or in combination with proteasome inhibitor bortezomib on drug resistance in acute myeloid leukemia（AML） and its mechanism. Methods Ex vivo cultures of HL-60/ADM cells and fresh refractory AML cells were treated with LBH589, bortezomib or their combination at varying concentrations. Proliferation capacity, apoptosis rate and reversal of drug resistance were evaluated by M3T assay, dual staining of Hoechst33342 and AnnexinV- FITC/PI by flow cytometry, and adriamycin uptake rate with proliferation inhibition, respectively. The change of signal pathway at protein level was analyzed by Western blot. Results Synergistic cytotoxicity was observed in the combination treatment with LBH589 and bortezomib against HL-60/ADM cells, as well as the fresh AML cells, the most powerful synergy being observed at 21nmol/L LBH589 plus 12 nmol/L bortezomib, with CI values of 0. 531 and 0. 498, respectively by Calcusyn software analysis. Moreover, the accumulation of adriamycin in HL-60/ADM cells was increased more in combinalion treatment [ （64.81 ± 3.69）% ] than in either LBH589 [ （28.96 ± 2.52） % ] or bortezomib [ （ 37.29 ± 3.71 ） % ] alone （ P 〈 0.05 ）, and so did the uptake rate of adriamycin being （64.81 ± 3.69 ） % , （28.96 ± 2.52 ） % and （ 37.29 ± 3.71 ） % respectively （ P 〈 0.05 ）. The combination treatment induced multiple apoptotic molecules co-action and intraeellular drug accumulation contributed to the synergistic eytotoxity, including caspase activation, PARP cleavage, XIAP downregulation, p53-dependent suppression of Bcl-2 and MRP1 expression via the inhibition of phosphoinositide 3-kinase （ P13K ）/Akt/nuelear factor-κB （ NF-κB ） signaling pathway. Conclusions Combination treatment of drug resistant AML cells with LBH589 and bortezomib produces a synergistic effect of in creasing sensitivity to chemotherapy. The mechanism may be mainly resulted from inhibition of PI3K/ Akt/NF-κB signaling pathway.