ATPR Induce Leukemia Cell Differentiation By Decreasing EIF6 And Its Possible Mechanisms

Chronic myeloid leukemia?CML?is an abnormal proliferative hematological malignancy of bone marrow hematopoietic stem cells caused by the sustained activation of tyrosine kinase due to the generation of abnormal chromosomes Ph.Maintaining the undifferentiated state and increasing the proliferation level of cells is a typical feature of CML.Therefore,inhibiting the proliferation of malignant cells and promoting their normal differentiation is a new hot spot strategy for the treatment of various hematological malignancies.All-trans retinoic acid?ATRA?is a mature clinical treatment which co?ld successf?lly attenuates acute promyelocytic leukemia?APL?,but due to its severe drug resistance and problems such as side effects have limited their clinical application.Our group designed and modified the carbon chain ends of ATRA andsynthesizedavarietyofretinoicacidderivatives.Invitro,4-amino-2-trifluoromethyl-phenyl retinate?ATPR?has been shown to successf?lly induce tumor cell differentiation and inhibit cell proliferation,but the specific molec?lar mechanisms of its role is still to be explored.Therefore,to explore the how ATPR induce leukemia cells differentiation,this study experimentally designed to investigate the specific role of the eukaryotic translation initiation factor eIF6.1.Effect of ATPR on eIF6 expression in different leukemia cell lines.K562 cells were treated with an ATPR concentration gradient?0,10-5,10-6,10-7,10-8,10-9 M?and different time points?0,24,48,72h?.The expression of eIF6 was detected by western blot.Then,NB4 cells and THP-1 cells were treated with ATPR under optimal conditions to detect the effect of ATPR on eIF6 expression in different types of leukemia.These res?lts indicated that ATPR decrease the protein level of eIF6 in different leukemia cell lines.2.The mechanism of eIF6 in ATPR-induced K562 cell differentiation.ATPR?10-6 M?was used to treat K562 cells after transfected with siRNA-eIF6,then the protein level of eIF6,cyclin D1,c-Myc and C/EBP?were measured by western blot.Wright-Giemsa staining was applied to detected the morphological changes.Flow cytometry was applied to examine the cell cycle distribution and the expression of CD11b.The res?lts showed that silencing eIF6 co?ld suppress the expression of cyclin D1 and c-Myc and promoted the expression of C/EBP?in K562 cells.Down-reg?lation of Eif6 also had a significant effect on the morphological changes of K562 cells and eIF6 siRNA co?ld accelerate the expression of CD11b and induced cell cycle arrest.To further confirm the role of eIF6,GV141-eIF6 was used to up-reg?late the expression of eIF6 in K562 cells.Then,the transfected K562 cells were stim?lated by ATPR?10-6M?,and the protein level of eIF6,cyclin D1,c-Myc and C/EBP?were measured by western blot.Wright-Giemsa staining was applied to detected the morphological changes.Flow cytometry was applied to examine the cell cycle distribution and the expression of CD11b.The res?lts showed that overexpression of eIF6 promoted the expression of cyclin D1 and c-Myc in K562 cells which inhibited by ATPR,while the expressions of C/EBP?changed in opposites.The characteristics of matured gran?locytes,cell cycle arrest and CD11b expression were prevented with overexpression of eIF6.3.The role of Notch1/CBF-1 signaling pathway in ATPR-induced K562 cell differentiation.In order to determine whether ATPR co?ld inhibit Notch1/CBF-1 signaling pathway to decrease eIF6 and induce leukemia cell differentiation,K562 cells were treated with ATPR(10^-6 M)for 72h after pretreatment with DAPT or Chrysin.the protein level of eIF6,Notch1,CBF-1?RBP-Jk?,cyclin D1,c-Myc and C/EBP?were measured by western blot.Wright-Giemsa staining was applied to detected the morphological changes.Flow cytometry was applied to examine the cell cycle distribution and the expression of CD11b.The res?lts shown that Notch1/CBF-1 signal activated by Chrysin co?ld increase expression of eIF6 and restrain the differentiation in ATPR-induced K562 cells,which co?ld be reversed by DAPT treatment,a Notch signaling pathway inhibitor.