DADS Induces Autophagic Cell Death In Human Leukemia HL-60 Cells

[Objective]To detect the autophagic death effect of DADS-induced leukemia HL-60 cells,provide experimental theoretical basis for optimizing DADS anti-leukemia effect and clinical application of DADS.[Methods]The effect of DADS on the proliferation of HL-60 cells was detected by CCK-8 method.The expression of autophagy marker LC3B-II in HL-60 cells treated with DADS was detected by Western blot.Autophagy inhibitors 3-MA combined with DADS treatment of HL-60 cells detected cell proliferation and induced autophagy.The expression of LC3B-II,p62 and Clevead PARP in cells was detected by Western blot.[Results]After CCK-8 assay,HL-60 cells were treated with different DADS concentrations of 0(solvent),25-,50-,75-,100-and 125 uM for 72 h,the cell proliferation rate was(99.44±4.32)%and(56.39±6.53)%,(47.53±3.21)%,(36.61±2.86)%,(32.22±5.92)%,(30.41±6.35)%,DADS significantly inhibited the proliferation of HL-60 cells.The expression of autophagy marker protein LC3B-II was positively correlated with increasing DADS concentration after Western blot detected the concentration of DADS at 25-,50-,75-and 100 uM for 72 h.Within the DADS treatment concentration range,the autophagic level of HL-60 cells was positively correlated with the DADS concentration.Autophagy inhibitors combined with DADS treatment of HL-60 cells,Western blot analysis showed that with the increase of 3-MA treatment concentration,the expression of autophagic marker protein LC3B-II gradually weakened,indicating that 3-MA can effectively weaken the DADS induced Autophagy.CCK-8 was used to detect the proliferation of HL-60 cells after 3-MA treatment in the vehicle group(DMSO),0uM group,25 uM group,100 uM group and blank control group.The results showed that HL-60 cells were treated with 25 and50 uM treatment conditions.The proliferation rate was(98.57±2.35)%and(97.87±2.71)%,which was not statistically significant compared with the vehicle group,whereas the proliferation rate of HL-60 cells was(90.15±3.55)%at 100μM treatment compared with the vehicle group.The difference was statistically significant.It was found that 50 uM was the optimal concentration of 3-MA in this experiment.Setup of DADS~-/3-MA~-(vehicle),DADS~+/3-MA~-,DADS~+/3-MA~+,and DADS~-/3-MA~+groups of experimental groups with 50 uM DADS and50 uM 3-MA,and blank control group treatment HL-60 cells.The CCK-8assay showed that the proliferation rates of HL-60 cells in the four groups were:(98.58±1.94)%,(53.49±3.63)%,(68.61±6.15)%,(98.86±5.99)%.The results show that 3-MA can effectively attenuate the proliferation inhibitory effect of DADS on HL-60 cells.Western blot was used to detect the expression of apoptotic marker Clevead PARP and autophagy marker proteins LC3B-II and p62 in HL-60 cells treated with 3-MA combined with DADS,and compared with vehicle group(DADS~-/3-MA~-).In contrast,the expression of LC3B-II and Cleaved PARP in the DADS~+/3-MA~-treated group was significantly increased,while the expression of p62 protein was significantly decreased,indicating that DADS can induce autophagy and apoptosis in HL-60 cells at the same time.DADS and 3-MA combined treatment group(DADS~+/3-MA~+)compared with single DADS treatment group(DADS~+/3-MA~-),LC3B-II protein expression was weakened,p62 protein expression was enhanced,but the expression of Cleaved PARP protein was not changed.Significant,it can be learned that autophagy inhibitor 3-MA can reduce DADS-induced autophagy,but has no effect on DADS-induced cell apoptosis.[Conclusion]1.Autophagy inhibitor 3-MA inhibited autophagy and attenuated the inhibitory effect of DADS on HL-60 cell proliferation;2.DADS could induce autophagy-related death of HL-60 cells,and may not be dependent on caspase apoptosis pathway.