Study On The Mechanism Of Dihydroartemisinin Against Diffuse Large B Cell Lymphoma Through Inhibition Of STAT3 Signaling Pathway

Objective: Constitutively activated STAT3 is detected in a wide variety of human cancer cells,and aberrant activation of STAT3 protein is often associated with cellular transformation by various oncoproteins.In particular,the multiple pathogenic effects of STAT3 activation have been demonstrated in hematologic tumors.Although rituximab has significantly improved the survival of patients with diffuse large B-cell lymphoma(DLBCL),there are still patients with relapses and drug resistance whose prognosis is poor.In recent years,the anticancer activity of dihydroartemisinin(DHA)has received attention.To this end,the aim of this study was to investigate the effects of DHA on the proliferation and apoptotic phenotype of human DLBCL cell line SU-DHL4 and STAT3 signaling pathway,and to preliminarily explore its molecular mechanisms.Methods: 1.SU-DHL4 cells were incubated for 24 h,48h and 72 h in the surrounding environment with different DHA concentrations(2.5μM,5μM,10μM,20μM,40μM and80μM),respectively,and the cell proliferation rate was analyzed by CCK-8 method;2.SU-DHL4 cells were incubated for 24 h in the surrounding environment with different DHA concentrations(10μM,20μM,and 40μM),and the cells were double-stained with Annexin V-FITC/PI,and the percentage of apoptotic cells was detected on flow cytometry;3.Western Blot was performed on SU-DHL4 cells treated with different concentrations of DHA(10μM,20μM,40μM)for 6h and 24 h,respectively,using β-actin as an internal reference to detect the changes in the relative expression of STAT3,p-STAT3(Tyr705)and c-Myc protein before and after DHA treatment,and RT-PCR was performed using GAPDH as an internal reference to detect the changes in the relative expression of c-Myc m RNA;4.Western Blot was performed on SU-DHL4 cells treated with different concentrations of DHA(10μM,20μM,40μM)for 24 h,and β-actin was used as an internal reference to detect the changes in the relative expression of apoptosis-related proteins Bcl-2,Bax,caspase-9,caspase-3,cleaved caspase-3 and cleaved-PARP proteins before and after DHA treatment.Results: 1.DHA significantly inhibited the proliferation of SU-DHL4 cells,and the inhibitory effect of 2.5μM DHA and its subsequent DHA action concentrations increased sequentially at the same assay time points;the inhibitory effect of 24 h and its subsequent assay time points also increased sequentially at the same DHA action concentrations(P<0.05).The half-inhibitory concentration(IC50)of DHA acting on SU-DHL4 cells for24 h was 33.14μM;2.DHA was able to significantly induce apoptosis in SU-DHL4 cells,and the apoptosis rate increased from(23.01±2.98)% in the control group to(52.36±4.54)%,(60.03±0.67)%,(74.3±2.6)%,respectively,after 10μM,20μM and 40μM DHA acting on SU-DHL4 cells for 24 h(P<0.05);3.Western Blot results showed that DHA had the function of reducing the expression level of p-STAT3(Try705)protein.20μM DHA acting on SU-DHL4 cells for 6 h caused a 32.38% downregulation of p-STAT3(Try705)protein expression,and 40μM DHA acting on SU-DHL4 cells for 6 h caused a46.34% downregulation of p-STAT3(Try705)protein expression(P<0.05),while all concentrations of DHA had no effect on the expression level of total STAT3 protein(P<0.05).According to the results of Western Blot and RT-PCR,DHA could significantly inhibit the expression of STAT3 target gene c-Myc.After 10μM,20μM and 40μM of DHA acted on SU-DHL4 cells for 24 h,the expression of c-Myc m RNA decreased by44.95%,54.66%,61.42%,respectively,compared with the control group;the expression of c-Myc protein decreased by 82.99%,93.35% and 96.7%,respectively,compared with the control group(P<0.05);4.Western Blot results showed that the expression of apoptosis-related proteins caspase-9 and caspase-3 decreased and the expression of cleaved caspase-3 and cleaved-PARP increased after the effect of 10μM,20μM and 40μM DHA on SU-DHL4 cells for 24 h,while the expression levels of Bcl-2 and Bax did not change significantly.Conclusion: DHA inhibits cell proliferation and induces apoptosis in human DLBCL cells SU-DHL4.The molecular mechanism may be that DHA inhibits STAT3 activity thereby reducing c-Myc expression at the transcriptional and translational levels as well as inducing apoptosis by the endogenous apoptotic pathway,independent of Bcl-2 and Bax proteins.