Rapamycin Enhances DADS-induced Autophagy And Apoptosis In Human Leukemia HL-60 Cells

Objective : To study the effects of diallyl disulfide(DADS),autophagy inducer rapamycin(RAPA)and their combination on proliferation inhibition and apoptosis of human leukemia HL-60 cells.To explore the role of autophagy in human leukemia HL-60 cells,and provide a theoretical basis for the optimal treatment of leukemia by DADS.Methods:1.The effects of RAPA,DADS,DADS and RAPA on the proliferation inhibition of HL-60 cells were detected by CCK-8method.2.Immunofluorescence was used to detect the expression of autophagy marker protein LC3 in HL-60 cells treated with DADS and RAPA.3.The expression of autophagy proteins LC3 B and P62 in HL-60 cells treated with DADS and RAPA was detected by Western Blot.4.Annexin V-PI double staining combined with flow cytometry was used to detect the apoptosis rate of HL-60 cells by RAPA,DADS,DADS and RAPA.Results : 1.Evaluation of the effects of RAPA,DADS andDADS combined with RAPA on the proliferation inhibition of human leukemia HL-60 cells by CCK-8 assay.Treatment of HL-60 cells with different concentrations of RAPA at 0,20,40,80,160,320,640 nM for48h,the inhibition rates of each group were 0,25.68±2.94%,26.33±3.10%,26.95±1.33%,27.68±2.78%,30.93±3.25%,32.68±2.98%,the inhibition rate of each experimental group was higher than that of the blank group(P<0.05),and increased slightly with the increase of RAPA concentration.After treatment with HL-60 cells at different concentrations of DADS at 5,10,20,40,60?M for 48 h,the inhibition rates of the groups were 2.51±3.24%,18.96±5.68%,29.52±0.65%,45.25±3.23% and 51.93±3.51%,compared with the vehicle group(0.07±3.69%),the concentrations of 5?M DADS could not inhibit the proliferation of HL-60cells(P>0.05),However the 10,20,40,60?M concentrations of DADS could inhibit the proliferation of HL-60 cells(P<0.05),and in a concentration-dependent manner.After treated with RAPA,DADS,DADS and RAPA for 48 h,the inhibition rate of each group was 25.19±1.89%,51.98±2.70% and 61.61±1.08%,compared with the blank group(P<0.05),the inhibition rate of each experimental group increased,and DADS combined with RAPA group had the highest inhibition rate of HL-60 cells.The results suggest that RAPA can enhance the inhibition rate of DADS on HL-60 cells.2.Immunofluorescence detection of DADS and RAPA treatment of HL-60 cells for 24 h,to find the expression ofautophagy protein LC3,the results showed that compared with the control group,red highlight vesicular LC3 appeared in the experimental group.It is suggested that DADS combined with RAPA treatment of HL-60 cells can induce autophagy in cells.Western blot was used to detect the expression of LC3B-II and LC3B-I protein in HL-60 cells treated with DADS,RAPA,DADS and RAPA group for 4 hours.The results showed that compared with the control group(P<0.05),the ratios of LC3B-II and LC3B-I in each experimental group increased significantly,and the ratio of LC3B-II and LC3B-I in DADS and RAPA group increased most significantly.Moreover,in the experimental group of HL-60 cells treated with DADS and RAPA for 2h and 6h,compared with the control group(P<0.05),the ratio of LC3 BII and LC3B-I increased significantly after 6h treatment.At the same time,Western blot was used to detect the expression of RAPA,DADS,DADS and RAPA on P62 protein in HL-60 cells for 24 hours.The results showed that compared with the untreated group(P<0.05),the expression level of p62 protein in each experimental group was decreased,in which DADS and RAPA group the p62 protein expression was the lowest.The results suggest that RAPA can enhance DADS-induced autophagy in human leukemia HL-60 cells,and the autophagy process is unobstructed.3.Flow cytometry was used to detect the apoptosis rate of HL-60 cells treated with different concentrations of RAPA for 48 h.The results showed that the apoptosis rate of eachexperimental group had no significance obviously(P>0.05).Moreover,After treatment with HL-60 cells with DADS,DADS and RAPA for48 h,the apoptosis rate of the two groups was significantly higher than that of the RAPA-treated group(P<0.05).The apoptosis rate of DADS and RAPA group was the most significant.It is suggested that rapamycin can increase the apoptosis of human promyelocytic leukemia HL-60 cells by DADS,which may improve the effect of DADS on leukemia.Conclusion : Rapamycin enhances the inhibitory effect of DADS on the proliferation of human leukemia HL-60 cells,and its mechanism is related to the induction of autophagic cell death and apoptosis.