DADS Induced HL-60 Cells To Autophagy And Apoptosis Via ROS-dependent Manner

[Objective] To study the effect of diallyl disulfide(DAD S)on the regulation of autophagy to apoptosis in human promyeloc ytic leukemia HL-60 cells by ROS,and to prove that ROS concentr ation changes are the starting point of DADS-induced apoptosis of human HL-60 cells,found that the working point of anti-tumor drug DADS to improve the efficiency of DADS,reduce its resistance to leukemia,conducive to provide faster and better treatment.[Method] Western blot was used to detect the expression of apoptotic marker protein Cleaved PARP and the expression of autophagy marker LC-3B ?.Morphology was used to detect the formation of autophagic bubbles in the cells by AO staining and MDC staining.The formation of autophagic bubbles and the occurrence of apoptotic bodies were observed by electron microscopy.The apoptotic rate was measured by Annexin V-PI combined with flow cytometry.The autophagy rate was measured by AO staining combined with flow cytometry.The levels of reactive oxygen species(ROS)were measured by DCFDA staining combined with flow cytometry.[Results](1)The results of AO staining showed that red dot aggregates appeared in HL-60 cells treated with 2.5 and 5mg/L DADS,compared with the control group.The results of MDC staining showed that the number of autophagic vacuole in HL-60 cells treated with 2.5 and 5mg/L DADS increased significantly.The results of electron microscopy showed that autophagic vacuole appeared in the cells under the action of 2.5mg/L DADS,and apoptotic bodies appeared at 5mg/L.Western blot results showed that:different concentrations of DADS treatment of human leukemia HL-60 cells on 24 hours,compared with the untreated group,the expression of Cleaved PARP protein in the 2.5,5,10mg/L group was significantly higher than that in the control group(P<0.05),and at 5mg/L is the highest.When the concentration of DADS was1.25mg/L,the expression of Cleaved PARP protein decreased(P<0.05).Compared with untreated group,the expression level of autophagy marker LC-3B ? was significantly increased in 1.25,2.5,5,10mg/L DADS treatment group(P<0.05),and reached the highest peak at the concentration of 1.25mg/L,and then decreased at the apoptotic peak(5mg/L).Suggesting that autophagy in HL-60 cells with different concentrations of DADS-induced may have different functions:protection or death.Flow cytometry showed that the autophagy rate of the0,2.5,5mg/L DADS group was 88.87%,98.98% and 93.13%.which indicated that the proportion of autophagy cells in HL-60 cells wasincreased by DADS,and the peak level was 2.5mg/L.The apoptotic rates of the 0,2.5,5mg/L DADS group was 12.3%,19.75%,26.44%,and which indicated that the proportion of apoptotic cells in HL-60 cells was increased by DADS.At low concentrations(2.5mg/L)of DADS,the expression of autophagy increased,while at high concentrations(5mg/L)of DADS,the expression of apoptosis increased.(2)Western blot results showed that autophagy inhibitors 3-MA could significantly inhibit the expression of LC-3B II in HL-60 cells induced by DADS,and decreased the expression of Cleaved PARP protein(P<0.05).The results of flow cytometry showed that the apoptotic rate of 2.5mg/L DADS treated group was 18.75%,and the apoptosis rate was reduced to 12.41% under the combined action with 3-MA.The apoptotic rate of 5mg/L DADS treated group was 21.54%,and the apoptosis rate was reduced to 15.55% under the combined action with 3-MA.It was confirmed that autophagy inhibitor 3-MA could inhibit DADS-induced apoptosis(p<0.05),indicating that part of the apoptosis is caused by autophagy.(3)Flow cytometry showed that the mean fluorescence intensity(Geo Mean)of the 0,2.5,5mg/L DADS treated group was 338.55,573.15,607.02,suggesting that DADS could cause elevated levels of reactive oxygen species in HL-60cells(p<0.05).(4)Western blot analysis showed that apoptosis of HL-60 cells induced by 2.5mg/L DADS was significantly inhibited when NAC was 2mM,and the induction of apoptosis was induced by 5mg/L DADS(P<0.05).Apoptosis was significantly increased by 2.5mg/L DADS interaction with 4mM NAC,whereas apoptosis was significantly inhibited by 4mM NAC interaction with 5mg/L DADS(P<0.05).The autophagy of HL-60 cells induced by 2.5mg/L DADS was significantly inhibited when NAC was 2mM,and 5mg/L DADS induced autophagy was significantly increased when the cells were treated with 4mM NAC(P<0.05).The flow cytometry showed that the apoptotic rate was 9.78% when DADS was 2.5mg/L,and the apoptosis rate was 6.33% when NAC was 2mmol/L,and NAC inhibited DADS induced apoptosis(P<0.05).The apoptotic rate was10.79% when DADS was 5mg/L,and the apoptosis rate was 7.4% when NAC was 4mmol/L,and NAC inhibited DADS induced apoptosis(P<0.05).The average fluorescence intensity is 567.13 When DADS is 2.5mg/L,and combined with 2mmol/L and 4mmol/L NAC the average fluorescence intensity were 562.08,520.55,the above shows that NAC can inhibit DADS-induced ROS production(P<0.05).Combined with the previous results of apoptosis,2mmol/L NAC and 2.5mg/L DADS combined inhibition of apoptosis was the most significant,Suggesting that the ROS level at this time may be the "threshold" concentration(Geo Mean 556.57)of apoptotic transformation of HL-60 cells induced by DADS.It is speculated that when DADS induced ROS level is greater than ROS "threshold" concentration can cause HL-60 cell apoptosis or autophagic death increased,while DADS-induced ROS levels less thanthis "threshold" concentration can cause HL-60 cells to develop cytoplasmic autophagy.[Conclusion]1.DADS can induce atuophagy to apoptosis in HL-60 cells.2.DADS-induced HL-60 cell autophagic protection,apoptosis or autophagic cell death,is associated with the threshold of ROS.