Study The Effects And Mechanism Of Combination Application Pimozide And IDO Inhibitor L-MT In Melanoma

BackgroundMelanoma is one of the deadliest and therapy resistant malignant.Finding new candidates to improve patient survival is a continuous effect.Previous studies showed that the effect of pimozide was not satisfied when treated with melanoma and find the way to enhance the treatment is required.Indoleamine-2,3-dioxygenase(IDO)is an immunosuppressiveintracellularrate-limitingenzyme,whichissignificantly overexpressed in melanoma and can promote immune tolerance in patients with various tumors,including melanoma.Therefore,we propose the combination of ido inhibitor L-1-methyl tryptophan(L-MT)and pimozide as a potential new treatment strategy for melanoma therapy.ObjectiveTo estimate the effect and mechanism of combination application pimozide and IDO inhibitor L-MT in melanoma..Methods1.Cell experimentThe best state of B16 cells were seeded into l plates and incubated for 24 hours.Then the control group was added with PBS and the experimental group was added with different concentrations of pimozide.The morphology of the cells was recorded by microscope at 24 h and 48 h,respectively.The viability of B16 cells was detected by cck-8 assay.The cell migration ability was detected by wound healing assay.The expression of cell proliferation,apoptosis and migration related proteins were detected by western blot.2.Animal experimentBuild melanoma bearing mice model,on the 7th day after B16 cells were seeded,the melanoma bearing mice were randomly divided into four groups:PBS group,pimozide group,L-MT group and pimozide plus L-MT group.Each day was treated with PBS(100ul)、pimozide(200μg/mouse)、L-MT(2 mg/mouse)or pimozide(200μg/mouse)combined with L-MT(2 mg/mouse)for one week,respectively.The incidence and weight of the tumor were observed daily.Some related proteins were detected by western blot method,including Cleaved Caspase 3,Cleaved Caspase 7,MMP2,CD4,CD8,and other related proteins.The changes of tumor tissue morphology were observed by hematoxylin-eosin staining.The expression of immunological cells and apoptotic cells were detected by immunohistochemical method and TUNEL assay.The proportion of CD4~+T cells and CD8~+T cells、NK1.1 cells and CD25~+Foxp3~+Treg in the spleen were detected by FACS.The trial was completed on the 30th day.Results1.Pimozide inhibited the growth of B16 cells in vitro without toxicity when the concentration less than 10μg/ml.2.Pimozide inhibited the expression of proliferation-associated protein and up-regulated the expression of apoptosis-related protein in B16 cells.3.Pimozide inhibited the migration of B16 cells in a dose-dependent and time-dependent manner.4.In melanoma bearing mice experiment,the combination of pimozide and L-MT can effectively inhibit the growth of melanoma,delay the incidence of tumor,and prolong the survival rate of melanoma bearing mice.5.Pimozide plus L-MT treatment significantly inhibited melanoma cells proliferation and induced apoptosis in melanoma bearing mice.6.In melanoma bearing mice experiment,the combination of pimozide and L-MT increased the infiltration of CD4~+T cells and CD8~+T cells in tumor tissue,especially CD8~+T cells.And the expression of CD8 protein in the combined treatment group was also significantly increased.The results were consistent.7.In melanoma bearing mice experiment,the combination of pimozide and L-MT increased the proportion of CD4~+T cells,CD8~+T cells and NK cells,but reduced the proportion of CD25~+Foxp3~+Treg in the spleen.ConclusionsPimozide can effectively promote cell apoptosis and inhibit cell proliferation and migration.The combination of pimozide and IDO inhibitor L-MT can effectively inhibit the tumor growth and improve the survival rate of melanoma in mice.Therefore,the use of pimozide in combination with IDO inhibitor may be a potential new treatment strategy for melanoma.