DHODH Inhibition Induced Cell Cycle Arrest And Autophagy In Melanoma Cells In Vitro And In Vivo

Objective and backgrond:Malignant melanoma is a highly malignant tumorwith a propensity for distant metastasis resulting in poor prognosis,which originated in the neural crest cell.In recent years,the incidence of malignant melanoma increases rapidly in China.The majority of patients with early~stage melanoma can be cured by surgery,but for the advanced tumor,the standard chemotherapeutic approaches used in the treatment have been disappointing.So to explore new chemotherapeutic regimens with high efficacy and low toxicity is particularly needed.Dihydroorotate dehydrogenase(DHODH)is the fourth enzyme of pyrimidine nucleotide de novo synthesis[1].Leflunomide is a kind of 5~methylisopropyl oxazole~4~formamide derivatives,which is the first approved for use in clinical treatment as a DHODH inhibitor.In recent years,more and more studies confirmed that DHODH expression was associated with the development of a variety of tumors.Therefore,DHODH has some research prospects to be a new anticancer targets.From this study observed by inhibiting DHODH or interference DHODH for malignant melanoma A375 and MV3 cell proliferation,cycle,the influence of the death,explore DHODH inhibition on soft agar cloning formation and a tumor~burdened inhibition of tumor growth in mice,further to explore the inhibition mechanism DHODH cause tumor cell proliferation and death.Methods:1 By cell counting method,determined by MTT(methyl thiazolyl tetrazolium)colorimetry to detect the effect of different concentrations of leflunomide(0μM,50μM,100μM,200μM),at four time points(24 h,48 h,72 h,96 h)on human melanoma cell proliferation inhibition,at the same time,the BrdU immunofluorescence assay was applicated to analyze the effect of leflunomide or shDHODH on A375 and MV3 cell proliferation;2 In order to explore the mechanism of the apoptosis and cycle arrest induced by leflunomide or shDHODH,The effect of on cell cycle was analyzed by PI staining and flow cytometry.3 The effect of on apoptosis was detected by Annexin V~FITC/PI staining and Hoechst 33342 staining;4 Western Blot,immunofluorescence and GFP~Lc3B instantaneous transfer experiments were applied to test thar whether autophagy took plase on A375 and MV3 cells;5 Through the cell cycle analysis and Western Blot analysis evaluated whether supplementary exogenous uracil or overexpression of DHODH could save the cell cycle arrest and autophagy;6 Soft agar analysis and and the tumor formation in vivo of mice were used to measure the effect of tumorigenic capacity of leflunomide on A375 and MV3 melanoma cells.Results:1 We confirmed cell growth and proliferation,by MTT and Brdu assays in human melanoma cells A375 and MV3.Cells delt with different concentrations of leflunomide for 72 h,resulting in cell proliferation inhibition in a dosage~dependent manner in human melanoma.BrdU immunofluorescence staining further confirmed that compared to the control group the percentage of BrdU~positive cells of the group which was treated with leflunomide decreased significantly;2 The flow cytometry show a significant increase in the proportionn of cells in the S phase,and a significant decrease in the G0/G1 phases.Then,by western blot assay we confirmed it.And supplementary exogenous uracil could save the cell cycle arrest;3 Through the cell morphological observation and Hoechst 33342 staining we found that there was typical change of apoptosis in Annexin V~FITC/PI dye flow cytometry analysis showed that there was obvious apoptosis peak in A375 cells after treated with leflunomide but not in MV3;4 Western Blot results showed that compared to delt whith DMSO LC3B~Ⅰwas degradated in to LC3B~Ⅱafter treated with leflunomide.This indicated autophagy was occured.Immunofluorescence and GFP~Lc3B result also showed that there were significant differences between the experimental group and control group,In contrast with control group,there was obviously LC3B dot gathered;5 By Western Blot and MTT assay,we confirmed that shDHODH could inhibit the A375 and MV3 cell proliferation;Brd U immunofluorescence staining aslo confirmed it;6 The flow cytometry show a significant increase in the proportionn of cells in the S phase,and a significant decrease in the G0/G1 phases in the shDHODH cells.Then,by western blot assay we confirmed it.And supplementary exogenous uracil or overexpression DHODH could save the cell cycle arrest;7 The cell morphological observation and Hoechst 33342 staining showed that there was no significant difference between sh DHODH cells and scramble cells;8 Western Blot results showed that compared to scramble cells LC3B~Ⅰwas degradated in to LC3B~Ⅱafter knockdown of DHODH.This indicated autophagy was occured.Immunofluorescence and GFP~Lc3B result also showed that there were significant differences between the experimental group and control group,In contrast with control group,there was obviously LC3B dot gathered;9 Soft agar analysis showed that leflunomide or knockdown DHODH could inhibit the effect of tumorigenic capacity of A375 and MV3 melanoma cells.Melanoma cells in vivo xenotransplantation NOD/SCID mouse model showed that the tumor volume and tumor weight of leflunomide treatment group were significantly lower than control group.Conclusion:1 This study shows that two methods of leflunomide inhibits DHODH,interference DHODH can inhibit melanoma cell proliferation and clone formation,leflunomide in vivo experiments show DHODH inhibitors or interference DHODH can inhibit the ability into tumor cells in vitro and mice.Thus,DHODH may be necessary to melanoma cell proliferation and survival genes,DHODH can serve as a potential target of cancer therapy,leflunomide is a new promising targeted candidate drugs for the treatment of melanoma2 Leflunomide DHODH inhibitors or interference DHODH induced melanoma cell cycle arrest in S phase.Leflunomide DHODH inhibitors induced A375 apoptosis and autophagy,MV3 cells induced autophagy.Interference DHODH melanoma cells induced autophagy.That limited inhibitory DHODH melanoma cell viability and induction of cycle arrest and apoptosis associated with autophagy.