Study On The Anti-melanoma Effect And Mechanism Of Pimozide Enhanced By Attenuated Salmonella PD-1-siRNA

BackgroundMelanoma,as a malignant skin tumor,is a serious threat to human health.Pimozide as a drug that has been used for the treatment of metastatic melanoma patients for a long time,has not achieved satisfactory results.With the development of tumor immuno-therapy,immunotherapy combined with chemotherapy has attracted more and more attention.Programmed death receptor 1(Programmed cell death protein 1,PD-1),is a kind of important immunosuppressive molecules,blocking the combination of PD-1 and PD-L1 to treat tumor,achieved good effect in clinic.Three interfering PD-1-siRNA sequences were designed in the early stage.This study will explore whether pd-1-sirna combined with Pimozide can synergistically enhance the therapeutic effect of anti-melanoma mice,and initially explore its mechanism.ObjectiveTo investigate the effect and mechanism of Pimozide anti-melanoma therapy enhanced by attenuated salmonella PD-1-siRNA.Methods1.Cell experimentEl-4(mouse lymphoma cells)was used to prepare cell plates to verify the function of PD-1-siRNA.Well plates of B16 cells(Mouse melanoma cells)were prepared,and Pimozide drugs of different concentrations were added after 14h-16h.The apoptosis was detected by flow analysis at 24h and 48h,respectively.In addition,at 72h,transwell assay was used to detect the invasion ability of B16 cells under the action of Pimozide at different concentrations.2.Animal experimentMale C57BL/6 mice purchased at about 6 weeks of age were randomLy divided into5 groups,namely PBS group,Scramble group,Pimozide group,pSi-PD-1 group and Pimozide combined with pSi-PD-1 group,to construct a melanoma bearing mouse model,and each group was treated one week later.To observe the incidence of tumor in C57BL/6 mice,measure the tumor weight and record the survival period of melanoma mice;The expressions of CD4,CD8,Caspase-3,PD-1 and other related proteins in tumor tissues were detected by western blot.Immunohistochemistry was used to detect the infiltration of immune cells in the tumor tissue of mice,and TUNEL apoptosis fluorescence was used to detect the expression level of apoptotic cells in the tumor tissue of melanoma mice.The proportions of CD4~+T cells and CD8~+T cells,PD-1~+immune cells,and NK1.1~+immune cells,CD25~+Foxp3~+regulatory T cells(Treg)in the mouse spleen were measured using FACS.Results1.Pimozide inhibited the invasion of B16 cells,increased apoptosis of B16 cells.2.Our self-designed PD-1-siRNA can effectively interfere with the expression of PD-1 in EL-4 cells.3.In vivo experiments on melanoma bearing mice,PD-1-siRNA delivered by attenuated salmonella can be combined with Pimozide treatment to extend the survival period of mice and inhibit tumor growth,showing synergistic anti-tumor treatment effect.4.In vivo experiments on melanoma tumor-bearing mice,attenuated salmonella carrier PD-1-siRNA combined with Pimozide treatment can significantly inhibit the expression of PD-1 on the surface of T cells in tumor microenvironment and induce tumor cell apoptosis.In addition,T lymphocyte infiltration in tumor tissues of melanoma bearing mice was increased and the expression of PD-1 was inhibited.5.Treatment of melanoma bearing mice by attenuated salmonella carrier PD-1-siRNA combined with Pimozide,increased the proportion of CD4~+and CD8~+T cells and NK1.1~+immune cells in the spleen of melanoma bearing mice,and decreased the proportion of PD-1~+immune cells and CD25~+Foxp3~+Treg.ConclusionsPimozide can effectively inhibit the invasion ability of B16 cells,and attenuated salmonella PD-1-siRNA combined with Pimozide can effectively inhibit the tumor growth and prolong the survival period of melanoma mice,both of which have synergistic anti-melanoma effect in mice,and without obvious toxic and side effects.Therefore,the way of attenuated salmonella carriage PD-1-siRNA combined with Pimozide treatment may become a potential new treatment strategy for melanoma.