The Mechanism Of Autophagy And Differentiation Of Human Acute Promyelocytic Leukemia NB4 Cells Induced By Low-dose PRF

ObjectiveAcute promyelocytic leukemia(APL)is characterized by t(15;17)(q22;Q21),the expression of PML-RARa fusion gene and protein,leading to the normal development and differentiation of myeloid cells blocked,is a key factor in the occurrence of APL.As a classic differentiation inducing agent,all-trans retinoic acid(ATRA)has been widely used in the clinical treatment of APL,and has synergistic effect with arsenic trioxide(ATO),which has been recognized as the most successful example of tumor targeted therapy.They can directly or synergically act on PML-RARa fusion protein,degrade pathogenic target protein through ubiquitin-protease or autophagy pathway,or inhibit mTOR kinase to activate autophagy degradation of fusion protein,and then promote APL cell differentiation.The total flavonoids of Puerariae radix flavone(PRF)is one of the main active components of Puerariae Radix.Our previous study found that PRF induced apoptosis of various acute leukemia cell lines,and NB4 cells were the most significant.In recent years,the research group has carried out original and progressive exploration on the molecular mechanism of the above research results,and achieved good results.In recent years,we found that low concentration PRF(10μg/ml)can effectively inhibit the proliferation of NB4 cells,but no apoptosis was observed.However,the levels of Bcl-2,ERK phosphorylation and Beclin1 protein expression were significantly increased,which was consistent with the trend of changes in the 30-50 μg/ml PRF treatment group.So,what is the relationship between autophagy and apoptosis of NB4 cells induced by PRF at a lower dose(10μg/ml)?Is there any necessary connection with cell differentiation?What is the possible mechanism of autophagy?Such problems arouse our interest in further research.Therefore,combined with relevant literature,this study aims to explore the role of low-dose PRF-induced autophagy in the survival and outcome of NB4 cells,and reveal the possible molecular mechanisms by using autophagy and changes in MEK/ERK related signaling molecules.Methods1.Effect of low-dose PRF on autophagy expression in NB4 cells NB4 cells were treated with PRF at gradient concentrations(0、5、10、15μg/ml)for 48h.The expression of autophagy related proteins LC3-Ⅱ and Beclin1 were detected by Western Blot(WB);qRT-PCR(quantitative real-time PCR)was used to detect the expression of autophagy-related genes Atg5 and Beclin1;After treated with 10μg/ml PRF for 48h,the changes of autophagosomes in NB4 cells were detected by MDC staining and transmission electron microscopy.2.Role of autophagy in differentiation of NB4 cells induced by low dose PRF NB4 cells were pretreated with 3MA(5mM)for 1h,and then treated with PRF(10μg/ml)for 48h.The expression of autophagy related proteins LC3-,Beclin1 and PML-RARa fusion proteins were detected by WB;Giemsa-Wright’s staining was used to detect the morphological changes of NB4 cells;Flow cytometry(FCM)was used to detect the positive expression rate of NB4 cell surface differentiation antigen CD 11b and cell changes.3.Possible molecular mechanisms of autophagy and differentiation of NB4 cells The expression levels of MEK and ERK1 proteins were detected by WB after the cells were treated by PRF(10μg/ml)for 48h.To further explore the role of MEK/ERK signaling pathway,we pretreated NB4 cells with SCH772984(10μM)for 1h,and then treated with PRF(10μg/ml)for 48h.WB technique was used to detect the changes of ERK1,LC3-Ⅱ and PML-RARa proteins;MDC staining and Giemsa-Wright’s staining were used to observe the changes of acidic autophagy lysosomes and cell morphology in NB4 cells;FCM was used to detect the expression of NB4 cell surface differentiation antigen CD11b and cell cycle changes;The expression levels of ERK1,LC3-Ⅱ and PML-RARa in NB4 cells treated with PRF at different concentration gradients(0,5,10,15μg/ml)for 48h and the inhibitor SCH772984(10μM)pretreated were analyzed by qRT-PCR.Results1.PRF can induce autophagy in NB4 cells in vitro.PRF(10μg/ml)after 48 h autophagy level ascending,autophagy related proteins LC3-Ⅱ and the expression of Beclin1 rise;The expression of Atg5 and Beclin1 increased at mRNA level,the expression of PML-RARa fusion protein decreased at the mRNA level.The number of autophagosomes in cells is increased.2.The autophagy inhibitor 3MA(5mM)was pretreated for 1h,and then treated with PRF(10μg/ml)for 48h,the expression of autophagy related proteins LC3-Ⅱ and Beclin1 were down-regulated,and the expression of fusion protein PML-RARa was down-regulated.The expressions of Beclin1,LC3-Ⅱ,MEK and ERK1 decreased at the mRNA level,while the expression of PML-RARα fusion protein increased at the mRNA level.Morphological changes that inhibited NB4 cells from differentiating into normal granulocytes were also observed.Occluding autophagy also reduced PRF-induced decrease in CD11b expression and inhibited the phenomenon of cell cycle arrest in G0/G1 phase.3.The PRF(10μg/ml)treated NB4 cells after 48 h can significantly increase the MEK and ERK1 protein expression level.After pretreatment with the pathway inhibitor SCH772984(10μM)for 1h,the protein expressions of ERK1 and LC3-Ⅱ and the degradation degree of PML-RARa induced by 10μg/mL PRF were inhibited,and the mRNA expressions of Beclinl,LC3-Ⅱ,MEK and ERK1 were decreased.The expression of PML-RARa fusion protein increased at the mRNA level.The decrease of the proportion of acidic autophagy lysosome in NB4 cells inhibited the cell morphological changes,and the expression of surface differentiation antigen CD11b was down-regulated,indicating that the differentiation of NB4 cells was blocked.ConclusionLow dose PRF(10μg/ml)can promote autophagy of NB4 cells by activating MEK/ERK signaling pathway,and induce the degradation of oncogenic protein PML-RARα,and finally induce NB4 cells to differentiate into normal granulocytes.The relationship between autophagy and differentiation of NB4 cells induced by PRF(10μg/ml)needs to be further verified by in vivo experiments.