Effects of 23-hydroxybetulinic acid on the growth of mouse colon cancer by inhibiting myeloid-derived suppressor cells
AIM To investigate the mechanism of 23-hydroxybetulinic acid(23-HBA)in inhibiting the growth of mouse colon cancer via myeloid-derived suppressor cells(MDSCs).METHODS The in vivo experiment of mouse colon cancer models establishment by subcutaneous CT-26 transplantation was followed by 18 days intraperitoneal injection of 5-FU at a frequency of twice every 3 days or daily injection of 23-HBA at the tail vein.After the last dose,the killed mice had their changes of body weight,tumor weight and volume,levels of organ index and blood routine test observed;their proportions of MDSCs and CD8+T cells in tumor tissues detected by flow cytometry;and their expressions of arginase 1(Arg1),nitric oxide synthase(iNOS)and cytokine granzyme B(GZMB),perforin 1(PRF1)and interferon-γ(IFN-γ)mRNA related to the immunosuppression function of MDSCs cells in tumor tissues detected by RT-qPCR method.In the in vitro experiment,the survival rates of bone marrow(BM)cells and MDSCs cells were detected by CCK8 method to determine the safe concentration of 23-HBA.MDSCs derived from BM mediated by 40 ng/mL granulocyte-macrophage colony stimulating factor(GM-CSF)and 40 ng/mL interleukin-6(IL-6)were dosed with,different concentrations of 23-HBA(0,10.5,21,42 μmol/L)for 4 days simultaneously,and had their ratio detected by flow cytometry afterwards.MDSCs inducted from BM in vitro were further divided into the control group and the low,medium and high dose 23-HBA groups(10.5,21 and 42 μmoL/L)for 24 hrs corresponding intervention,and had their expressions of Arg1 and iNOS mRNA detected by RT-qPCR,and their expressions of Arg1 and iNOS proteins detected by Western blot.RESULTS The result of the in vivo experiments revealed that in contrast to the model group,the 23-HBA groups displayed decreased volume and weight of transplanted mouse tumors(P<0.05,P<0.01);decreased proportion of MDSCs in tumor tissues(P<0.05);increased proportion of CD8+T cells(P<0.05,P<0.01);decreased Arg1 mRNA expression(P<0.05);and increased expressions of GZMB and IFN-γ mRNA(P<0.05,P<0.01).It turned out that in the in vitro experiments,23-HBA at concentrations of 10.5,21,42 μmoL/L had no significant effect on the survival rate of BM and MDSCs(P>0.05);23-HBA at high dose inhibited the differentiation of BM into MDSCs(P<0.01);and 23-HBA dose-dependently decreased the expressions of Arg1 and iNOS mRNA(P<0.05,P<0.01),and downregulated the protein expressions of Arg1 and iNOS in MDSCs(P<0.05,P<0.01).CONCLUSION 23-HBA can regulate the proportion of MDSCs in tumor by intervening their differentiation,weaken their immunosuppressive function and thus restore the anti-tumor activity of CD8+T cells in their role in anti-colon cancer action.
23-hydroxybetulinic acidcolon cancermyeloid-derived suppressor cellsdifferentiationimmunosuppressive function
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