Dihydroartemisinin Triggers C-Myc Proteolysis And Inhibits AKT Signaling Pathway In T-cell Lymphoma

Objective To study the effects of dihydroartemisinin(DHA)on phosphorylation of c-Myc protein in Jurkat and HuT 78 T-cell lymphoma cells,and to explore the potential therapeutic role and mechanism of DHA in T-cell lymphoma cells.Methods Jurkat and HuT 78 cell lines were selected as T-cell lymphoma cell models,and logarithmic growth phase cells were used for the following experiments.After Jurkat and HuT 78 cells treated with DHA,cell proliferation was detected by CCK-8 method and cell apoptosis was measured by flow cytometry.To determine the effect of DHA,the c-Myc,AKT and GSK3βmRNA was detected by RT-PCR,and GAPDH m RNA was used as an endogenous internal control.2~-??Ct??Ct method was applied to calculate the relative expression of c-Myc,AKT and GSK3βmRNA.We evaluated c-Myc,c-Myc phosphorylated at threonine 58,AKT,AKT phosphorylated at serine 473,GSK3βand GSK3βphosphorylated at serine 9 protein levels by Western blots analysis,with GAPDH as an internal control.Image J software was used to analyze the relative expression levels of c-Myc,p-c-Myc,AKT,p-AKT,GSK3βand p-GSK3βproteins.Then the effects of DHA on c-Myc proteolysisand and AKT/GSK3βsignaling pathway in T cell lymphoma cells were analyzed.Results DHA could damage the integrity of the cell membrane structure and induced a marked reduction of cell density.DHA inhibited the proliferation of Jurkat and HuT 78cells in a time-and concentration-dependent manner.There was a statistically significant difference compared with the control group(P<0.05).The IC50 values of DHA at 48h for Jurkat and HuT 78 cells were 16.63 and 33.35μM,respectively.Flow cytometry analyses showed that 15μM DHA significantly induced Jurkat cell apoptosis(P<0.05).Compared with the control group,which showed an apoptosis rate of 1.53%,the apoptosis rate of the DHA treatment group was increased to 32.44%.The apoptosis rate of HuT 78 cells also significantly increased to 22.81%upon 30μM DHA treatment compared with 1.65%in control cells(P<0.05).The results of RT-PCR showed that DHA could decrease the expression of Bcl-2 mRNA.Compared with the control group,the expression of Jurkat and HuT 78 cells Bcl-2 mRNA was decreased by 57%and 64%,respectively;there was a statistically significant difference(P<0.05).DHA could decrease the expression of c-Myc mRNA.Compared with the control group,the expression of Jurkat cells c-Myc mRNA was decreased by 43%,and the expression of HuT 78 cells c-Myc mRNA was decreased by 46%,there was a statistically significant difference(P<0.05).DHA had little effect on the expression of AKT and GSK3βmRNA(P>0.05).Western blot results showed that DHA could down-regulate the expression of Bcl-2,c-Myc,p-AKT and p-GSK3βin Jurkat by 42%,31%,20%and 16%,respectively(P<0.05).And the expression of Bcl-2,c-Myc,p-AKT and p-GSK3βin HuT 78 cells were decreased by 63%,30%,23%and 15%,respectively(P<0.05).The expression of Bax protein in Jurkat and HuT 78 cells was increased by 41%and 51%after DHA treatment,respectively.The expression of p-c-Myc protein in Jurkat and HuT 78 cells was increased by 34%and 25%after DHA treatment,respectively.There was a significant difference compared with the control group(P<0.01).Conclusion DHA can inhibit Jurkat and HuT 78 cells proliferation and promote cells apoptosis.The mechanism may be achieved through the following ways:DHA can down-regulate c-Myc mRNA expression,and then reduce the expression of c-Myc protein;DHA can enhance the phosphorylation of c-Myc protein,and accelerate c-Myc proteolysis;DHA can inhibit AKT/GSK3βsignaling pathway by decreasing the expression of p-AKT,p-GSK3βprotein.This study provides experimental basis for DHA treatment of T-cell lymphoma and gives a discussion of the specific mechanism of DHA.