Ribavirin Inhibits Growth In Soft Tissue Sarcomas By Regulating PRMT1 And PRMT5

Background:Soft tissue sarcomas(STS)are malignant tumors of mesenchymal tissues.Although a variety of treatments have emerged in recent years,these are still insufficient for sarcoma patients with advanced tumors.Thus,developing new therapeutics is necessary to improve the treatment of deadly sarcomas.The methylation of arginine residues is a post-translational modification that occurs on many proteins,such as histone and non-histone proteins.It is carried out by the protein arginine methyltransferases(PRMTs),which classified as typeⅠ,type Ⅱ and type Ⅲ enzymes according to their catalytic activity.Type Ⅰ protein arginine methyltransferase 1(PRMT1)and TypeⅡ PRMT5 catalyze asymmetric and symmetric dimethylation of arginine(ADMA and SDMA)on proteins,respectively.PRMT1 and PRMT5 are frequently overexpression in cancer cells,and their elevated expression correlates with poor prognosis,thus making them excellent therapeutic targets.Ribavirin,an anti-viral molecule used in hepatitis C,is emerging anti-tumor agents.However,little is known about their effect in soft tissue sarcomas.Objective:This study intends to investigate the antitumor activity of ribavirin on soft tissue sarcomas cells in vivo and in vitro,and explore the underlying relationship between ribavirin and PRMT1/PRMT5.We aim to elucidate the molecular mechanism of ribavirin.Methods:1.To determine the levels of PRMT1 and PRMT5 in mouse normal fibroblast cell L929 and sarcoma cells including S180,HT-1080,A-673,U2OS and MG-63,we carried out western blot analysis using the total protein isolated from each cell lines.The role of ribavirin in sarcoma cells was investigated by MTT assay and the correlation of ribavirin with the expression of PRMT1/PRMT5 was explored.2.The colony formation,cell cycle and apoptosis were tested by clonogenic assay and flow cytometry.3.The effect of ribavirin on soft tissue sarcomas in vivo was examined by S180xenograft model.The role of ribavirin in lung colonization was explored by inoculating S180 cells directly into the tail veins of KM mice.Survival outcome was evaluated using S180-induced ascites tumor model.The peritoneal permeability assay was performed to determine vessel permeability in animal models.The effect of ribavirin on the immune functions was evaluated by immune organ indexes of mice.4.The levels of PRMT1 and PRMT5 after treatment with ribavirin were detected by q RT-PCR and western blot.Subsequently we examined changes in H4R3me2a and H4R3me2s/H3R8me2s after treatment with ribavirin.The concentration of VEGF in the ascites was determined using ELISA kit.Ch IP assay was performed to detect the enrichment of PRMT1 and PRMT5 at the promoter region of VEGF gene.Results:1.The analysis showed that PRMT1 level was lowest in HT-1080 and highest in A-673 cells,while PRMT5 level was lowest in U2OS cells and highest in S180 cells.Interestingly,the MTT assays revealed a significantly slower proliferation rate in A-673 and S180 cells when compared with control cells,whereas ribavirin was less effective in treating HT-1080 and U2OS cells.Collectively,our data indicated that the antitumor activity of ribavirin was positively correlated with the protein levels of PRMT1 and PRMT5 in sarcoma cells.2.Ribavirin inhibited soft tissue sarcoma cells growth and induced G0/G1 phase cell-cycle arrest and cell death.Moreover,we observed that the expression of Cyclin D1 and PCNA protein decreased after ribavirin administration to soft tissue sarcoma cells compared with that of vehicle control cells.3.Ribavirin or doxorubicin as single agent significantly inhibited tumor growth compared with vehicle-treated mice,but more importantly,we noticed that ribavirin treatment significantly potentiated the effect of doxorubicin increasing the IR from44.1%to 78.0%.In addition,ribavirin reduced lung metastasis and prolonged survival of mice implanted with S180 cells.Furthermore,the results showed that the indices of thymus and spleen in ribavirin group were much higher than those of control group.The total number of CD3~+,CD4~+,CD8~+and CD19~+cells in ribavirin group were significantly more than those in control group.4.We observed that ribavirin significantly reduced accumulation of H4R3me2a and H4R3me2s/H3R8me2s that followed from the PRMT1-and PRMT5-specific activity,respectively,which was coincident with PRMT1 and PRMT5 reduction.Ribavirin lowered the permeability of the peritoneum via decreasing the level of vascular endothelial growth factor(VEGF).Strikingly,ribavirin decreased the enrichment of PRMT1 while increased that of PRMT5 at VEGF promoter region.Conclusions:Our results demonstrate that ribavirin inhibites growth and metastasis,and prolongs survival in soft tissue sarcomas by downregulating PRMT1and PRMT5.Ribavirin decrease the expression levels of VEGF by repressing transcription of VEGF through PRMT1 and PRMT5.Collectively,the therapeutic activity against soft tissue sarcomas supports the potential of ribavirin in the treatment of this neoplasm and forms the basis for future investigations aimed at optimizing the therapeutic activity of ribavirin given alone or in combination with other drugs.