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Journal of ZheJiang University(Medical Science)  2015, Vol. 44 Issue (5): 525-531    DOI: 10.3785/j.issn.1008-9292.2015.09.09
Everolimus combined with all-trans retinoid acid reverses drug resistance in acute promyelocytic leukemia NB4-R1 cells
LIAO Wei-chao, HE Ying, WANG Bin-sheng, HUANG He
Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
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Objective:To investigate the effect of everolimus(RAD001)combined with all-trans retinoid acid(ATRA) on drug resistance of ATRA-resistance acute promyelocytic leukemia(APL) cell line NB4-R1 and its molecular mechanism. Methods:APL NB4-R1 cells were treated with different concentrations of RAD001(1 nmol/L, 10 nmol/L and 100 nmol/L) with ATRA(1μmol/L) for 24, 48 and 72 h, respectively. The differentiation of NB4-R1 cells was analyzed by flow cytometry with CD11b staining and nitro blue tetrozolium(NBT) reduction test. Cell cycle was detected by cell cycle staining kit and apoptosis was detected by flow cytometry with Annexin V/PI staining. Protein expressions of LC-3II, PML-RARα, P-P70S6K and P-4E-BP1 were determined by Western blotting. Results:RAD001 combined with ATRA significantly induced NB4-R1 cells differentiation, but RAD001 or ATRA alone did not enhance NB4-R1 differentiation. The co-treatment induced accumulation of cells in G1 phase and decreased the proportion of cells in S phase. The combined treatment had no effect on cell apoptosis. The differentiation rate of NB4-R1 cells in 100 nmol/L RAD001, 1μmol/L ATRA, RAD001 combined with ATRA and control groups was(2.29±0.57)%,(17.06±2.65)%,(54.47±4.91)% and(2.54±0.53)%, respectively; the proportion of cells in G1 phase was(35.20±11.97)%,(33.54±6.25)%,(53.70±8.73)% and(27.40±6.01)%, respectively; cells apoptosis rate was(2.30±0.14)%,(2.25±0.21)%,(2.40±0.28)% and(1.95±0.07)%, respectively. The combination of RAD001 with ATRA significantly inhibited mTOR signaling downstream proteins P-P70S6K, P-4E-BP1 and enhanced autophagy-related protein LC3-II and Beclin 1. The co-treatment also induced degradation of fusion protein PML-RARα. Conclusion:RAD001 combined with ATRA can induce cell differentiation, inhibit cell cycle, resulting the reverse of drug resistance in NB4-R1 cells, which is associated with increase of autophagy level and degradation of PML-RARα.

Key wordsLeukemia, promyelocytic, acute/pathology      Everolimus      Cell differentiation/drug effects      Suppressor factors, immunologic      Drug resistance, neoplasm     
Received: 11 March 2015      Published: 12 November 2015
CLC:  R733.7  
Cite this article:

LIAO Wei-chao, HE Ying, WANG Bin-sheng, HUANG He. Everolimus combined with all-trans retinoid acid reverses drug resistance in acute promyelocytic leukemia NB4-R1 cells. Journal of ZheJiang University(Medical Science), 2015, 44(5): 525-531.

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目的:探讨依维莫司联合全反式维甲酸(简称维甲酸)逆转急性早幼粒细胞白血病(APL)细胞株NB4-R1耐药的作用。方法:应用CD11b染色流式细胞术及硝基四唑氮蓝(NBT)还原实验检测两药联合应用对细胞分化的影响,流式细胞术检测细胞周期情况,Annexin V/PI双染色检测细胞凋亡情况,蛋白质印迹法检测自噬相关蛋白微管结合蛋白轻链3(LC3)、Beclin 1及早幼粒白血病-维甲酸受体融合蛋白(PML-RARα)、磷酸化核糖体S6激酶(P-P70S6K)、磷酸化4E结合蛋白1(P-4E-BP1)等表达水平。结果:与维甲酸组比较,联用组能诱导耐药细胞株NB4-R1细胞的分化,并将细胞增殖阻止在G1期而对细胞凋亡无明显影响。100 nmol/L依维莫司组、1μmol/L维甲酸组、联用组、对照组NB4-R1细胞培养48 h后分化百分率分别为(2.29±0.57)%、(17.06±2.65)%、(54.47±4.91)%、(2.54±0.53)%;处于G1期的细胞百分率分别为(35.20±11.97)%、(33.54±6.25)%、(53.70±8.73)%、(27.40±6.01)%;四组细胞凋亡细胞百分率分别为(2.30±0.14)%、(2.25±0.21)%、(2.40±0.28)%、(1.95±0.07)%。与维甲酸组比较,联用组mTOR信号通路下游的P70S6K、4E-BP1分子磷酸化水平下降, LC3-II和Beclin 1的表达上调,且能部分降解融合蛋白PML-RARα。结论:依维莫司联合维甲酸能诱导NB4-R1细胞分化,且能阻滞细胞周期而不致细胞凋亡,其机制可能与依维莫司联合维甲酸抑制mTOR信号通路激活自噬作用从而降解PML-RARα蛋白有关。

关键词: 白血病,早幼粒细胞,急性/病理学,  依维莫司,  维甲酸/药理学,  细胞分化/药物作用,  抑制因子,免疫,  抗药性,肿瘤 
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