Mechanism Study Of Dihydroartemisinin Inhibiting The Progression Of Melanoma By IL-10-mediated Anti-tumor Immunity In Mice

Background:In recent years,the incidence and death of melanoma have been increasing year by year.Because melanoma is accompanied by a variety of gene locus mutations and high degree of malignancy,the general clinical treatment of melanoma often fails to achieve the desired effect.Tumor immunotherapy shows a good prospect in the treatment of melanoma,but it still has some defects.Dihydroartemisinin(DHA)has shown significant inhibitory effects on a variety of tumor cells,and several studies have shown that DHA affects T cell differentiation and function.However,studies on the treatment of melanoma by DHA are rare,and the specific molecular mechanism is still unclear.Objectives:To investigate the effects of DHA on the proliferation and migration of melanoma and to explore its possible mechanism.Methods:The inhibitory effect of DHA at different concentrations on the proliferation of B16F10 cells was detected by CCK8.The wound-healing experiment and the clone formation experiment were used to examine the effects of DHA on the lateral migration and community formation of B16F10 cells,respectively.Hoechst 33342 staining was used to observe the apoptosis of B16F10 cells.B16F10 cells were inoculated subcutaneously in BALB/c mice to construct a mouse melanoma model for 14 days.All mice were randomly divided into three groups,named model group,DHA-L group and DHA-H group.They were given 0.2 ml of carrier or 25 mg/kg concentration of DHA or 50 mg/kg concentration of DHA per day.During the modeling period,melanoma-bearing mice were weighed and tumor growth was observed,daily.The expression of MMP2,apoptosis-related proteins,STAT1,STAT3,NF-κB and Akt signaling pathways related proteins,PD-L1 and IFN-γwere detected by western blot The levels of ALT,AST,Cr,β2-MG,IL-6 and IL-10 in the serum and the level of IL-10 in cell culture supernatant were determined by ELISA.H&E staining was used to observe the histological changes in melanoma tissues of mice among the three groups.Immunohistochemical staining was used to detect the expression of Ki-67,CD8 and Foxp3 in mice melanoma.The mRNA expression levels of T-bet,GATA3,ROR-γt and Foxp3 in melanoma were detected by RT-PCR.The percentages of CD4+D25+Foxp3+regulatory T cells,CD8+T cells,CD4+IFN-y+T cells and CD8+IFN-γ+T cells in the spleen of the mice in each group were detected by flow cytometry.Results:1.CCK8 showed that DHA inhibited the proliferation of B16F10 cells in a time-and dose-dependent manner(P<0.05).2.The result of wound healing experiment showed that DHA inhibited the lateral migration of B16F10 cells in a dose-dependent manner.3.The result of clone formation experiment showed that DHA inhibited the community formation of B16F10 cells in a dose-dependent manner.4.Hoechst 33342 staining showed that the bright blue apoptotic nuclei were significantly more in the DHA groups than those in the model group.5.The results of western blot showed that DHA inhibited the expression of MMP2,Bcl-2,p-STAT3 and p-p65(P<0.05),increased the expression of Bax,cleaved caspase 9,cleaved caspase 3,cleaved PARP,p-STAT1 and IFN-γ(P<0.05).However,DHA did not alter the expression of PD-L1 and p-Akt(P>0.05).6.ELISA showed that there were no differences in ALT,AST,Cr and β2-MG levels among the three groups(P>0.05),and DHA treatment reduced the expression of IL-6 and IL-10(P<0.05).7.The melanoma bearing mouse model showed that the treatment with DHA significantly alleviated melanoma and significantly reduced tumor volume,compared with model group(P<0.05).8.H&E staining showed that a large number of melanoma cells were nested,with abundant cytoplasm and common pathological mitosis in model group.In the DHA groups,melanoma cells were scattered,and tumor cell nests were occasionally observed.The cytoplasm of melanoma cells was swollen,and the nuclei were solid,fragmented and dissolved.9.Immunohistochemical staining showed that DHA reduced the expression of the proliferation marker Ki-67,increased the number of CD8+ cells,and decreased the number of Foxp3+cells.10.The results of RT-PCR showed that DHA reduced the expression of GATA3,ROR-yt and Foxp3 in melanoma(P<0.05),but DHA had no significant effect on the expression of T-bet(P>0.05).11.The results of flow cytometry showed that DHA significantly inhibited CD4+CD25+Foxp3+regulatory T cells,promoted CD8+T cells and CD8+IFN-γ+ T cells(P<0.05),but had no significant effect on CD4+IFN-γ+ T cells(P>0.05).Conclusion:DHA induced mitochondrial apoptosis and altered cytokines expression,such as IL-10,in tumor microenvironment by inhibiting the phosphorylation of STAT3.Changes of cytokines in tumor microenvironment weakened the immunosuppressive effect of Treg cells and enhanced the killing effect of CD8+T cells in melanoma bearing mice.