Effects Of Autophagy On ATPR-induced Differentiation Of Human Acute Promyelocytic Leukemia NB4 Cells And Its Possible Mechanisms

Acute promyelocytic leukemia(APL)is a malignant disease of hematopoietic tissue,accounting for about 10%-15% of acute myeloid leukemia(AML).PML-RARα fusion protein is considered to be the leading cause contributing to the pathogenesis of APL.As a classical differentiation agent,all-trans retinoic acid(ATRA)has been widely used in the clinical treatment of APL.ATRA and arsenic trioxide(As2O3)are recognized as the most successful example of tumor-targeted therapy.Both ATRA and As2O3 could interact directly with PML-RARα to stimulate proteasome and autophagy-dependent proteolysis of this oncoprotein.Moreover,both drugs could stimulate PML-RARα degradation by activation of autophagy through a mechanism that involves inhibition of the m TOR kinase.Finally,increased levels of autophagy promote diffrentiation of APL cells.However,the clinical application of ATRA has limitations of retinoic acid syndrome and drug resistance.4-Amino-2-Trifluoromethyl-Phenyl Retinate(ATPR),a novel all-trans retinoic acid(ATRA)derivative designed and synthesized by our team,could induce differentiation of APL cells in vivo and in vitro,but the molecular mechanism remains unrevealed.To further explore the underlying mechanism of ATPR,the APL cell line NB4 cells were choosed as the object to detect the autophagy effect of ATPR and the role of autophagy in ATPR-induced differentiation in NB4 cells in vitro.This study mainly contains three sections,as belows:1.Expression of autophagy in ATPR-induced differentiation of NB4 cells.NB4 cells were treated with an ATPR concentration gradient(10-5-10-9M,48h)and different time points(0-72 h,10-6M).The protein expression of LC3B-II and Beclin1 were assessed by Western blot and the m RNA expression of ATG5 and Beclin1 were analyzed by q RT-PCR.The state of autophagosome and autolysosome were detected via MDC staining,the m Cherry-EGFP-LC3 B plasmid transfection,and transmission electron microscopy(TEM).Experimental results showed that ATPR could induce autophagy of NB4 cells in vitro.The protein expression of LC3B-II and Beclin1 as well as the m RNA expression of ATG5 and Beclin1 were up-regulated.ATPR(10-6M)could also dramatically increased the autophagic vacuole in vitro.2.Effect of autophagy on ATPR-induced differentiation in NB4 cellsNB4 cells were treated with ATPR(10-6M)for 48 h in the presence or absence of 3-methyladenine(3-MA,5m M)for 1h.The levels of Beclin1,LC3B-II,and PML-RARα protein expression were determined by Western blot.The cell morphological features were observed by Wright–Giemsa dye under a microscopy.And the cell surface differentiation marker CD11 b expression and cell cycle proportion were analyzed by flow cytometer.Experimental results showed that LC3B-II,Beclin1 and the degradation of PML-RARα were reduced after pretreatment with 3-MA.The morphologically granulocytic differentiation appeared stunted in 3-MA pretreated cells,and the CD11 b expression,the change of cell cycle distribution were suppressed after pretreatment with 3-MA.To further explore the relationship between autophagy and ATPR-induced differentiation.NB4 cells were transfected with control siRNA or ATG5 siRNA for 6h,and then incubated with 10-6 M ATPR for 48 h.The protein expression of ATG5,LC3B-II and PML-RARα were measured by Western blot.Cell morphological features and CD11 b expression were analyzed by Wright–Giemsa staining and flow cytometer.Experimental results showed that ATG5,LC3B-II protein expression and PML-RARα degradation were decreased pretreatment with ATG5 siRNA.The characteristics of matured granulocytes and CD11 b expression were prevented pretreatment with ATG5 siRNA.3.Effect of Notch1/Hes1 signaling pathway on ATPR-induced autophagy and differentiation in NB4 cellsNB4 cells were exposed to 10-6M ATRA or 10-6M ATPR for 48 h,the protein levels of Notch1 and Hes1 were assessed by Western blot.Experimental results showed that the protein levels of Notch1 and Hes1 were remarkably increased after ATPR treatment.To explore whether Notch1 signaling was involved in ATPR-induced cell autophagy and differentiation,cells were incubated with a γ-secretase inhibitor DAPT(10μM)for 1h,and then treated with ATPR(10-6M)for 48 h.The protein levels of Notch1,LC3B-II and PML-RARα were assessed by Western blot.MDC staining for acidic vacuoles was analyzed by fluorescence microscopy and cell morphological features were assayed by Wright–Giemsa staining.Surface CD11 b expression and the distribution of cell cycle were analyzed by flow cytometer.Experimental results showed that ATPR-induced Notch1,LC3B-II and the degradation of PML-RARα were reduced by DAPT.Moreover,NB4 cells failed to successfully differentiate when pretreated with DAPT.In addition,pretreatment with DAPT prevented cell differentiation by down-regulating the expression of CD11 b on the surface of NB4 cells and decreased the G0/G1 phase of cell cycle distribution.