Diallyl Disulfide Downregulates Calreticulin And Promotes C/EBPα 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/EBPα is an important regulator of granulocyte differentiation,inhibition of CRT can promote the expression of C/EBPα.OBJECTIVE: On the basis of previous work,to explore the mechanism of DADS down-regulates the expression of C/EBPα 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/EBPα 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/EBPα 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/EBPα 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/EBPα were detected by western blot.RNA immunoprecipitation（RIP）was used to study the binding of CRT with C/EBPα mRNA.RESULT: 1.The expression of CRT and C/EBPα 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/EBPα in leukemia patients was lower than normal human（p<0.05）.There was a correlation between the expression of both CRT and C/EBPα in each group.2.Differential expression of CRT and C/EBPα 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/EBPα 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/EBPα 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/EBPα 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/EBPα 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/EBPα and other proteins changed in DADS-treated cells（p<0.05）.5.DADS down-regulates CRT to promote C/EBPα 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/EBPα mRNA in HL-60 cells resulted in the obstruction of C/EBPα expression.CONCLUSION: 1.Compared with control group,CRT is highly expressed in clinical leukemia patients,C/EBPα 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/EBPα 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/EBPα mRNA expression and induce differentiation of HL-60 cells...