全反式维甲酸调控内质网应激诱导FLT3-ITD蛋白自噬降解促进白血病细胞凋亡

Effect of endoplasmic reticulum stress induced by all-trans retinoic acid on apoptosis of FLT3-ITD mutated leukemia cells by activating autophagy in FLT3-ITD mutated protein

目的:以内质网应激(ERS)为切入点,深入研究全反式维甲酸(ATRA)调控FLT3-ITD突变蛋白表达下降,导致FLT3-ITD突变阳性白血病细胞凋亡的分子机制。方法:ATRA处理FLT3-ITD突变阳性白血病细胞株(MV4-11和MOLM13),流式细胞术检测细胞凋亡,实时荧光定量PCR和Western blot法分别检测细胞ERS相关和(或)自噬相关基因及蛋白的表达。结果:低剂量ATRA可提高FLT3-ITD突变阳性白血病细胞的ERS水平;ATRA作用于ERS相关的PERK/eif2ɑ信号通路,使FLT3-ITD突变阳性细胞的ERS水平持续升高,CHOP基因表达上调;FLT3-ITD突变阳性细胞经ATRA处理后,FLT3-ITD蛋白表达水平降低;细胞内与ERS有关的并且主要的两个蛋白降解途径中,与内质网关联降解(ERAD)相关的蛋白ATF6表达无明显变化,而与内质网激活自噬(ERAA)相关的自噬相关蛋白Atg5和Atg7表达明显升高。结论:ATRA通过激活PERK/eif2ɑ信号通路持续上调FLT3-ITD突变阳性细胞的ERS水平,通过ERAA途径自噬降解FLT3-ITD蛋白,促进白血病细胞凋亡。研究结果为临床应用ATRA治疗难治性FLT3-ITD突变阳性白血病提供了初步实验依据。

Objective Endoplasmic reticulum stress(ERS)was used as the research emphasis to further investigate the mechanisms of apoptosis of FLT3-ITD-mutated leukemia cells and decreased expression of FLT3-ITD mutated protein induced by all-trans retinoic acid(ATRA).Methods FLT3-ITD-mutated leukemia cell lines(MV4-11 and MOLM13)were treated with ATRA.Flow cytometry was conducted to assess cell apoptosis.Real-time fluorescent quantitative PCR(RT-qPCR)and Western blot were used to detect the expression of ERS-related and autophagy-related genes and protein,respectively.Results A low-dose ATRA further increased FLT3-ITD cells and ERS levels.ATRA acted on the ERS-related PERK/eif2ɑsignaling pathway and continued to increase the ERS of FLT3-ITD cells,resulting in an upregulation of apoptotic gene CHOP expression.After the treatment with ATRA,FLT3-ITD protein in FLT3-ITD cells was decreased.Of the two main ERS-related protein degradation pathways,ER-associated degradation(ERAD)and ER-activated autophagy(ERAA),the expression of ERAD-related protein ATF6 in FLT3-ITD cells was not significantly changed on ATRA,whereas the expression of ERAA-related proteins Atg7 and Atg5 were significantly increased.Conclusions ATRA further raises the ERS level of FLT3-ITD cells continuously by activating the ERS-related PERK/eif2ɑsignal pathway and induces FLT3-ITD protein autophagy degradation through ERAA pathway,which induces apoptosis of FLT3-ITD-mutated leukemia cells.These results provide preliminary evidence on the use of ATRA in the treatment of refractory leukemia with FLT3-ITD.