组蛋白去乙酰化酶抑制剂LBH589对急性髓系白血病细胞株HL60/ADM增殖、凋亡及耐药的影响

Effect of histone deacetylase inhibitor LBH589 on proliferation, apoptosis and drug resistance of acute myeloid leukemia cell line HL60/ADM

目的 探讨组蛋白去乙酰化酶抑制剂LBH589对急性髓系白血病细胞株HL660/ADM的增殖、凋亡和耐药的影响.方法 采用不同浓度LBH589处理耐药的HL60/ADM细胞,四甲基偶氮唑蓝（MTT）法检测细胞增殖和多柔比星作用24h IC50值,AnnexinV-FITC/PI荧光染色流式细胞术检测细胞凋亡、多柔比星摄取率和多药耐药蛋白1（MRP1）表达,评估LBH589逆转耐药效应.Westemblot检测p53、Akt、p-Akt、组蛋白-3、乙酰化组蛋白-3、β-actin蛋白表达.结果 10～80 nmol/L LBH589能够抑制HL60/ADM细胞增殖和诱导凋亡,70 nmol/LLBH589作用60 h抑制效果最佳.20 nmol/LLBH589显著下调HL60/ADM细胞表面MRP1的表达[（93.90±4.20）％比（76.19±6.53）％,P＜0.05]、提高HL60/ADM细胞多柔比星摄取率[（8.53±0.68）％比（25.67±1.34）％,P＜ 0.01]、降低多柔比星24 hIG0值[（6.833±0.319） μg/ml比（1.382±0.104）μg/ml,P＜ 0.01],其逆转耐药倍数为4.9倍.LBH589处理HL60/ADM细胞24、48 h,乙酰化组蛋白-3相对表达水平均高于LBH589处理前（P＜0.01）,处理后24h和48 h p-Akt相对表达水平分别为1.07±0.09和0.59±0.01,低于处理前表达水平（2.03±0.12）（P＜0.01）,p53蛋白相对表达水平分别为0.57±0.04和1.31±0.09,明显高于处理前的表达（0.21±0.02）（P＜ 0.01）.结论 LBH589通过阻断PI3K-Akt通路、下调其MRP1的表达及提高多柔比星摄取率有效地抑制HL60/ADM细胞的增殖和诱导其凋亡,并逆转其耐药.

Objective To investigate the effect of histone deacetylase inhibitor LBH589 on proliferation,apoptosis and drug resistance of chemoresistant acute myeloid leukemia cells HL60/ADM.Methods HL60/ADM cells were treated with LBH589.Proliferation,apoptosis and adriamycin IC50 were evaluated by MTT assay and AnnexinV-FITC/PI stain.The change in MRP1 expression and intercellular adriamycin accumulatiom were analyzed by flow cytometry.Results Effective proliferative inhibition and apoptotic induction in HL60/ADM cells were observed after treatment with 10-80 nmol/L LBH589 with maximal effect detected after treatment with 70 nmol/L LBH589 for 60 hours.However,inhibition ratio remain unchanged with the further increase of drug dose and incubation time （P ＞ 0.05）.Downregulation of MRP1 [（93.90±4.20） ％ vs （76.19±6.53） ％],upregulation of adriamycin accumulation [（8.53±0.68） ％ vs （25.67±1.34） ％] and decrease in adriamycin IC50 [（6.833±0.319） μg/ml vs （1.382±0.104） μg/ml] were induced by the treatment with 20 nmol/L LBH589 （P ＜ 0.01）,whose reversal fold was 4.9.The expression of acetylated histone 3 after treatment with LBH589 was higher than that before treatment （P ＜ 0.01）.However,relative p-Akt levels after treatment for 24 h and 48 h were 1.07±0.09 and 0.59±0.01,respectively,which were lower than that before treatment （2.03±0.12） （P ＜ 0.01）.Meanwhile,expression levels of p53 were 0.57±0.04 and 1.31±0.09,respectively,which were higher than that before treatment （0.21 ±0.02） （P ＜ 0.01）.Conclusion Treatment with LBH589 has the capability of inhibiting proliferation and inducing apoptosis,as well as increasing intercellular adriamycin accumulation and sensitivity through downregulation of MRP1 expression and inhibition of PI3K-Akt signaling pathway in HL60/ADM cells.