Diallyl Disulfide Downregulates Calreticulin And Promotes C/EBP? Expression In Differentiation Of HL-60 Cells

BACKGROUND: Leukemia seriously harms human health and it has practical significance to induce leukemia differentiation and treatment.DADS is a fat-soluble active ingredient of garlic.It is an anti-tumor drug with great development potential.Our previous work showed that low-dose DADS can induce differentiation of HL-60 cells in vitro and in vivo and increase the production of reactive oxygen species?ROS?.We used proteomics to screen out 18 differentially expressed proteins that induce DADS-induced leukemic cell differentiation.The expression of the Ca²⁺ binding chaperone protein,calreticulin?CRT?,in the endoplasmic reticulum was significantly down-regulated.It has been reported that CRT plays an important regulatory role in the differentiation of tumor cells,C/EBP? is an important regulator of granulocyte differentiation,inhibition of CRT can promote the expression of C/EBP?.OBJECTIVE: On the basis of previous work,to explore the mechanism of DADS down-regulates the expression of C/EBP? and induces differentiation of HL-60 cells.METHODS: Peripheral blood samples were collected and flow cytometry?FCM?was used to detect the expression of CRT and C/EBP? in peripheral blood leukocytes,and the correlation between them was studied.Hematoxylin-eosin?HE?staining was used to observe the morphological changes after differentiation of HL-60 cells induced by DADS.RT-PCR and western blot were used to detect the expression of CRT and C/EBP? after DADS treatment in HL-60 cells.The tumorigenesis model of SCID mice was constructed to detect the biological behaviors of DADS on tumorigenesis and the effects on the expression of CRT and C/EBP? in SCID mice.The reactive oxygen species?ROS?levels in DADS-treated HL-60 cells were detected by FCM,and the expression of AKT,p-AKT,PERK,p-PERK,CRT,and C/EBP? were detected by western blot.RNA immunoprecipitation?RIP?was used to study the binding of CRT with C/EBP? mRNA.RESULT: 1.The expression of CRT and C/EBP? in peripheral blood leukocytes of leukemia patients and their correlation The results of FCM demonstrated that the expression of CRT was higher in leukemia patients than in controls?p<0.05?.The expression of C/EBP? in leukemia patients was lower than normal human?p<0.05?.There was a correlation between the expression of both CRT and C/EBP? in each group.2.Differential expression of CRT and C/EBP? in DADS-differentiated HL-60 cells The results of HE staining showed that compared with the control group,the cells showed obvious differentiation after DADS treated HL-60 cells for 48 h.After DADS treated with HL-60 cells for 0 h,12 h,24 h,and 48 h,the RT-PCR and western blot data showed that the expression of CRT at the mRNA and protein levels gradually decreased with the prolonged DADS treatment time.The characteristics?p<0.05?of C/EBP? at mRNA and protein levels were gradually increased?p<0.05?.3.The effect of DADS on the biological behavior and the expression of CRT and C/EBP? in tumorigenesis of SCID mice HE staining was performed on SCID sections.The staining results showed that the cells treated with drugs showed a clear differentiation and the canceration was reduced.Compared with the control group,the drug significantly suppressed the tumor volume.Tumor growth in the 21mg/kg DADS?LD DADS?,42mg/kg DADS?HD DADS?and 11mg/kg ATRA groups was inhibited by 50.34%,76.34% and 80.35%,respectively.Western blot results showed that compared with the control group,the expression of CRT in the drug-treated group was decreased?p<0.05?,and the expression of C/EBP? was increased?p<0.05?.4.DADS induces CRT downregulation and translocation through ROS signaling pathway The results of cell immunofluorescence showed that after 48 h of DADS and HL-60 cells,CRT was transferred from the cell to the cell membrane.FCM results showed that ROS levels in cells were increased after DADS treatment for 0 h,12 h,24 h,48 h,and 72 h in HL-60 cells?p<0.05?.After DAHS pretreated with 10 m M NAC for 1 h,DADS were separated.After HL-60 cells were treated for 0 h,12 h,24 h,48 h,the increase of ROS content in HL-60 cells was decreased?p<0.05?.Western blot results showed that compared with the control group,the expression of CRT and C/EBP? in DADS-treated and NAC+DADS treated cells changed?p<0.05?.Compared with the control group,the expression of intracellular PERK,p-PERK,AKT,p-AKT,CRT,C/EBP? and other proteins changed in DADS-treated cells?p<0.05?.5.DADS down-regulates CRT to promote C/EBP? mRNA translation and induce differentiation of HL-60 cells Western blot results showed that after DADS treated HL-60 cells for 48 hours,the CRT content in the treated group was significantly lower than that in the control group?p<0.05?.The result of RNA immunoprecipitation experiments showed that the interaction of CRT and C/EBP? mRNA in HL-60 cells resulted in the obstruction of C/EBP? expression.CONCLUSION: 1.Compared with control group,CRT is highly expressed in clinical leukemia patients,C/EBP? is lowly expressed in clinical leukemia patients,and there is a correlation between the two expressions.2.DADS can down-regulate CRT expression in vivo and in vitro,up-regulate C/EBP? expression and induce differentiation of HL-60 cells.3.DADS can increase the production of ROS in HL-60 cells and induce differentiation of HL-60 cells through the ROS signaling pathway.4.DADS down-regulates CRT to promote C/EBP? mRNA expression and induce differentiation of HL-60 cells...