CAJ | 학술논문

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21 nmol/LLBH589和12 nmol/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(CI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PI3K/Akt/NF-κB通路的活性,明显上调P53蛋白的表达,抑制NF-κB P65、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.PI3K/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.
목적 탐토신일대조단백거을선화매억제제LBH589연합단백매체억제제붕체좌미역전급성수계백혈병(AML)세포내약효응급기분자궤제.방법 내약AML세포주HL-60/ADM화난치성AML원대세포여불동농도LBH589、붕체좌미혹량자연합진행체외배양,채용MTT법검측세포증식활력,Hoechst33342염색화Annexin V-FITC/PI쌍염류식세포술검측세포조망,용아매소섭취솔병결합세포증식억제솔평고역전내약효응.진일보검측처리전후세포MRP1급신호통로단백변화.결과 LBH589、붕체좌미균능구억제HL-60/ADM화난치성AML원대세포증식,유도조망,기중21 nmol/LLBH589화12 nmol/L붕체좌미연합시작용최강,경Calcusyn연건분석증명량자연합구유명현적협동작용(CI치분별위0.531화0.498).연합조MRP1양성솔위(57.78±3.34)%,현저저우LBH589、붕체좌미단약조[(76.06±5.17)%화(71.83±4.53)%,P치균＜0.05],이단약조여대조조지간차이야유통계학의의(P치균＜0.01).연합조세포내아매소섭취솔위(64.81±3.69)%,명현고우단약조[(28.96±2.52)%화(37.29±3.71)%](P치균＜0.05).LBH589연합붕체좌미가이명현억제린산화Akt화린산화IKKα/β단백적표체,강저PI3K/Akt/NF-κB통로적활성,명현상조P53단백적표체,억제NF-κB P65、Bcl-2、XIAP화MRP1단백활성,촉진Caspase-8、Caspase-3화PARP적렬해격활.결론 LBH589、붕체좌미능구억제내약AML세포증식화촉진조망,병역전내약,량자연합구유명현적협동작용.PI3K/Akt/NF-κB신호전도통로수억혹조단가능시기주요적작용궤제.
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 MTT assay, dual staining of Hoechst33342 and AnnexinVFITC/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 21 nmol/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 combination 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 intracellular drug accumulation contributed to the synergistic cytotoxity, including caspase activation, PARP cleavage, XIAP downregulation, p53-dependent suppression of Bcl-2 and MRP1 expression via the inhibition of phosphoinositide 3-kinase ( PI3K )/Akt/nuclear 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.