Dihydroartemisinin Induces Apoptosis In Human Acute T Lymphocytic Leukemia Cells By Activating Oxidative Stress

Objective By observing the influnce of dihydroartemisinin(DHA)on the growth and apoptosis of human T-cell acute lymphoblastic leukemia(T-ALL)cells,we verified whether DHA induces T-ALL cell apoptosis by activating oxidative stress,and explored the molecular mechanism.In our research,we have explored a possibility of combination of DHA and ATO treatments to induce apoptosis of T-ALL cell and studied the molecular mechanism.Methods(1)Cultivated Jurkat cells and MOLT-4 cells of human T-ALL cell line,and took experiments in logarithmic growth phase;(2)Treated T-ALL cells with different concentrations of DHA and set a control group to observe different groups Changes in cell viability and proliferation ability(through CCK8 method,PI staining,clone formation,Ed U staining,immunofluorescence and other experiments);(3)T-ALL cells were treated with DHA at different concentrations and a control group was set.The reactive oxygen species,apoptosis rate,and apoptotic protein C-Caspase-3 activity level were observed in each groug(DCFH-DA probe flow cytometry to measure DCF fluorescence intensity,Annexin V-FITC / PI staining flow cytometry And immunofluorescence C-Caspase-3 activity measurement,etc.);(4)Western blot assay showed the cell DNA damage protein and apoptosis-related protein expression levels trested by DHA;(5)The cells were pretreated by ROS scavenger N-Acetyl-cysteine(NAC),then were treated with DHA,and set a control group to detect changes in ROS level,viability,proliferation ability,and apoptosis level,and observed whether ROS clearance can inhibitted the cells apotosis induced by DHA,restored cell viability(using CCK8 method,flow detection of ROS levels,immunofluorescence Ed U,C-Caspase-3 staining and other experiments);(6)We treated T-ALL cells with DHA and ATO single or combined,observed the changes in cell viability and proliferation ability of each group,and compared the differences in the ability to inhibit cell growth of combined drugs and single drugs(using CCK8 method,Ed U staining immunofluorescence and other experiments);(7)The cells were treated with DHA and ATO single or combined and NAC T-ALL cells were pretreated,and compared the amount of ROS produced in different groups;(8)Compared the expression levels of ?-H2 AX protein among the groups,and evaluated cells apoptosis rate changestreated by the different expression of the DNA damage protein;(9)Observed the DHA and ATO single or combined treatment T-ALL cell apoptosis rate changes in each group,evaluated whether the rate of apoptosis induced by the combination of the two drugs is higher than the sum of the rates of apoptosis induced by the two single drugs(using Annexin V-FITC / PI staining flow Measure the apoptosis rate,immunofluorescence C-Caspase-3 activity test,etc;(10)After pretreatment with NAC,observed the viability and proliferation ability of T-ALL cells treated with the combination of DHA and ATO.Results:(1)DHA inhibitted the activity and proliferation of T-ALL cell line Jurkat and MOLT-4 cells significantly in a dose-dependent manner;(2)DHA induced ROS production and apoptosis in a dose-dependent manner;(3)Western blot assay showed that the expression of DNA damage-related gene ?-H2 AX and apoptosis-related genes p53,c-Caspase3,Bax and c PARP were significantly increased,and Bcl-2 protein expression was decreased.(4)NAC saved cell apoptosis induced by DHA partly for eliminating ROS,and partly restored the inhibition of DHA on cell growth;(5)the inhibition rate of DHA and ATO on the activity and proliferation of Jurkat cells and MOLT-4 cells was much higher than the total effects of the two drugs,which proved that DHA and ATO can synergistically inhibit T-ALL cells;(6)The increased ROS level induced by the combination of DHA and ATO in Jurkat cells was significantly higher than the sum of single drug effects,which confirmed that ATO and DHA could synergistically enhance ROS production,while the pretreatment of ROC scavenger NAC could partially reduce ROS production level;(7)Western blot assay showed that the expression level of DNA damage protein ?-H2 AX in Jurkat cells was much higher than the sum of single drug effects after combined action of DHA and ATO(8)The results of annexin V-FITC/PI flow cytometry and immunofluorescence c-caspase-3 activity showed that compared with the control group,the apoptosis rate of cells treated with ATO or DHA was significantly increased,while the increase of apoptosis rate of cells caused by combination of two drugs was greater than the sum of apoptosis rate of cells caused by single drug.(9)After NAC pretreatment,the activity and proliferation of Jurkat cells and MOLT-4 cells treated with DHA and ATO were restored.Conclusion DHA has a significant effect in acute T-lymphocytic leukemia.It may be involved in the anti-tumor mechanism of DHA by activating DHA induced ROS production in Jurkat and MOLT-4 cells,causing DNA damage,activating the P53 apoptotic pathway,and promoting apoptosis.DHA and ATO can induce DNA damage and apoptosis of all cells by activating oxidative stress.