The Effect Of Diallyl Disulfide On Differentiation In HL-60Cells Of Overexpression CRT

Objective: To study CRT over-expression on human leukemia HL-60cellsdifferentiation induced by diallyl disulfide and related molecular mechanisms ofinduction of differentiation.Methods: The effects of DADS on expression of C/EBPα and HL-60cellsdifferentiation was detected by RT-PCR, western blotting. By building the CRT highexpression plasmid, the CRT over-expression plasmid pcDNA3.1-12GS0643-IG-3was transfected into HL-60cells and detected the HL-60cells differentiation withCRT over-expression and the effects of DADS. The influence of the ability ofmigration and invasion in HL-60cell after the effect of DADS was tested withtranswell experiment. MTT assay and NBT assay detected the ability of proliferationand reducing ability of the HL-60with CRT over-expression after the effects ofDADS.Results:1. DADS influence CRT expression of human HL-60leukemia cells.Western blot, RT-PCR results show, human leukemia HL-60cells was treatedwith1.25mg·L-1DADS for12,24,48h, comparing with the untreated group, theexpression of CRT decreased with time-dependent characteristics (P<0.05).2. DADS influence C/EBPα expression of human HL-60leukemia cellsWestern blot, RT-PCR results show, human leukemia HL-60cells was treatedwith1.25mg·L-1DADS for12,24,48h, comparing with the untreated group, theexpression of C/EBPα increased with time-dependent characteristics (P<0.05). 3. DADS influence migration of HL-60cellsMigration experiments showed that the human leukemia HL-60cells was treatedwith1.25mg·L-1for12,24h, HL-60cells migration decreased in a time-dependentways comparing with the untreated group (P<0.05).4. CRT over-expression on the effect of CRT expression of HL-60cells inducedwith DADS.After the CRT over-expression plasmids was transiently transfected into HL-60cells and were cultured in a cell incubator for48h. The transfected cells show greenfluorescence under an inverted fluorescence microscope, indicating a successfultransfection.RT-PCR results showed that CRT mRNA expression increased comparing withnegative transfected group, and CRT mRNA of each experimental group was lowerthan the untreated group after treated with DADS.Western blot results showed that CRT protein increased comparing with negativetransfected group, and CRT of each experimental group was lower than the untreatedgroup after treated with DADS.RT-PCR and Western blot results showed that, the negative plasmid transfectiongroup, non-transfected group and high expression group were treated with1.25mg·L-1DADS for48h, levels of CRT mRNA and protein were significantly lower thanthe untreated groups (P<0.05).It shows that mRNA and protein expression of CRTover-expression group were higher than the negative transfected and non-transfectedgroup, and DADS could down-regulate CRT expression in HL-60cells.5. CRT over-expression on the effect of C/EBPα expression of HL-60cellsinduced by DADS.After the CRT over-expression plasmids was transiently transfected into HL-60cells and were cultured in a cell incubator for48h. The transfected cells show greenfluorescence under an inverted fluorescence microscope, indicating a successfultransfection.RT-PCR results showed that C/EBPα mRNA expression increased comparingwith negative transfected group, and C/EBPα mRNA of each experimental group was higher than the untreated group after treated with DADS.Western blot results showed that C/EBPα gene expression increased comparingwith negative transfected group, and C/EBPα gene level of each experimental groupwere higher than the untreated group after the DADS affect the group.RT-PCR and Western blot results showed that, the negative plasmid transfectiongroup, non-transfected group and high CRT expression group were treated with1.25mg·L-1DADS for48h, levels of C/EBPα mRNA and protein were significantly higherthan the untreated groups (P<0.05).It shows that mRNA and protein expression ofC/EBPα with CRT over-expression group were lower than the negative transfectedand non-transfected group, and DADS could up-regulate C/EBPα expression inHL-60cells.6. CRT over-expression on the differentiation of HL-60cells induced by DADS.NBT results show that the abilities of differentiation with transfected CRTover-expression higher than negative group, and lower than untreated group aftertreated with DADS or ATRA(P<0.05). No obvious difference was found between theARTA and1.25mg·L-1DADS group (P>0.05).MTT experiments showed that the proliferation of transfected CRTover-expression group, the negative group and the non-transfected group werereduced compared with untreated group. We can conclude that DADS can inhibit theability of proliferation of HL-60cells.Transwell invasion assays showed that the number of HL-60cells whichpenetrated through the Matrigel in the CRT over-expression group, the negative groupand the non-transfected group treated with DADS were lesser than before(P<0.05).No obvious difference was detected between non-transfected group and the negativetransfected group (P>0.05).It indicate that DADS can inhibit the invasion of HL-60cells.Conclusion:1. DADS can inhibit the proliferation, migration, invasion and induct thedifferentiation of HL-60cells by down-regulating the expression of CRT. 2. DADS could increase the expression of C/EBPα in HL-60cells, increasedexpression of CD11b and decrease expression of CD33.