西达本胺增加慢性髓系白血病耐药株K562/ADM细胞对柔红霉素的敏感性

目的 观察西达本胺（CDM）能否影响人慢性髓系白血病耐药株K562/ADM细胞对柔红霉素（DNR）的敏感性，并探讨其可能的分子机制。方法 体外常规培养K562细胞和K562/ADM细胞，给予不同剂量CDM和（或）DNR处理48 h后，采用细胞计数试剂盒8（CCK-8）法检测CDM与DNR对K562和K562/ADM细胞的毒性作用，采用Chou-Talalay中效分析法对两药的联合效应进行评价，采用流式细胞术检测细胞增殖、细胞周期和凋亡，采用Western blotting方法检测组蛋白2AX（H2AX）、γH2AX（Ser139）、共济失调毛细血管扩张征突变基因（ATM）、p-ATM（Ser1981）、乳腺癌易感蛋白l（BRCA1）和p-BRCA1（Ser1524）的蛋白表达水平。 结果 DNR可剂量依赖性地抑制K562/ADM细胞活力（P<0.05），半数抑制浓度（IC50）为11.76 μmol/L，耐药倍数为18.09；CDM可协同增强DNR对K562/ADM细胞的抑制作用置信区间（CI）（CI<1），反转倍数为8.11；与对照组相比，DNR组细胞增殖率显著降低（P<0.05），G2/M期细胞比例和凋亡率明显升高（P<0.05），而无毒剂量的CDM可协同增强DNR引起的细胞增殖抑制、G2/M期阻滞和细胞凋亡（P<0.05）；耐药株K562/ADM细胞中ATM和BRCA1蛋白表达水平显著高于其亲代K562细胞（P<0.05）；DNR可上调K562/ADM细胞中H2AX、ATM和BRCA1蛋白的磷酸化水平（P<0.05）；CDM与DNR联用可使γH2AX蛋白水平进一步升高，但p-ATM和p BRCA1蛋白水平的变化则相反（P<0.05）。 结论 CMD可反转K562/ADM细胞对DNR的耐药性，这可能与上调H2AX蛋白的磷酸化水平以及下调ATM和BRCA1蛋白的磷酸化水平有关。

Enhancing effect of chidamide on the sensibility of human chronic myeloid leukemia K562/ADM cells to daunorubicin

Objective To investigate whether chidamide (CDM) could influence the sensibility of human chronic myeloid leukemia K562/ADM cells to daunorubicin (DNR) and its possible mechanism. Methods The K562 and K562/ADM cells were cultured in vitro and treated with CDM and(or) DNR for 48 hous, and then the cell viability was measured by cell counting kit-8(CCK-8) assay. The proliferation, cell cycle and apoptosis were analyzed by flow cytometry. Western blotting was performed to measure the protein levels of histon 2AX(H2AX), γH2AX (Ser139), ataxia telangiectasia mutated gene (ATM), p-ATM (Ser1981), breast cancer susceptibility protein 1(BRCA1), and p-BRCA1 (Ser1524). Results DNR remarkably inhibited the cell activity of K562/ADM cells in dose-dependent manner with a half maximal inhibitory concentration(IC50) value of 11.76 μmol/L, and the resistant factor was 18.09. Co-treatment with CMD and DNR produced a synergistic effect confidence interval(Cl)(CI<1) with a reversal fold of 8.11. DNR remarkably inhibited proliferation (P<0.05), induced G2/M phase arrest and apoptosis (P<0.05), these effects were enhanced under non-toxic concentration of CMD (P<0.05). K562/ADM cells had a significantly higher protein levels of ATM and BRCA1 than K562 cells (P<0.05). DNR significantly up-regulated the protein levels of γH2AX, p-ATM and p-BRCA1 (P<0.05), and the protein level of γH2AX appeared higher in the combination group compared to DNR alone (P<0.05); however, the co-treatment with CMD and DNR induced a decreased expression of p-ATM and p BRCA1 than the DNR alone (P<0.05). Conclusion CDM may enhance the sensibility of K562/ADM cells to DNR by up-regulating the protein level of γH2AX, and down-regulating the protein levels of p-ATM and p-BRCA1.