Study The Synergistic Antitumor Effect And Mechanism Of Compound Kushen Injection And Arginine Methyltransferase Inhibitor 1 On Pancreatic Cancer

Objective:1.To study the effects of Arginine methyltransferase inhibitor 1(AMI-1)on pancreatic cancer,and explore the relationship between AMI-1 and protein arginine methyltransferase 5(PRMT5)in pancreatic cancer.2.To study the potential regulatory relationship between Compound Kushen Injection(CKI)and PRMTs in pancreatic cancer.3.To study whether the combination of AMI-1 and CKI have synergistic antitumor effects on pancreatic cancer in vivo and in vitro,then explore the mechanism of synergistic antitumor effects.Method:1.The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)database were used to analyze the differential expression of PRMT1 and PRMT5 in pancreatic cancer tissues and normal pancreatic tissues,and the correlation between PRMT1 and PRMT5 in pancreatic cancer.2.The effects of AMI-1 and CKI alone and combined on the proliferation of pancreatic cancer cells in vitro were measured by MTT assay.The effect of the combination of AMI-1and CKI on clone formation,migration ability and apoptosis of pancreatic cancer cells were measured by colony formation,scratching,Transwell,flow cytometry assay respectively.The effects of AMI-1 on the expression of PRMT5 and H4R3me2 s,the effects of CKI on the expression of PRMTs and the effects of the combination of AMI-1 and CKI on the expression of PRMT5,H4R3me2 s,c-Myc,β-catenin,and PCNA were analyzed in pancreatic cancer by Western blot.3.The effect of AMI-1 and CKI alone and in combination on tumor growth in vivo were examined by BALB/c nude mice xenograft models.Nude mice were divided into model group,AMI-1 group,CKI group,and AMI-1+CKI group.The effects of the combination of AMI-1 and CKI on the expression of PRMT5,H4R3me2 s in pancreatic cancer tissues were analyzed by Western blot.Result:1.Bioinformatics analysis showed that the expressions of PRMT1 and PRMT5 in pancreatic cancer tissues were higher than in normal pancreas tissues.In pancreatic cancer,PRMT1 has a strong positive correlation with PRMT5.2.(1)MTT assay showed that compared with negative control group,after single agent AMI-1 and CKI treatment,the activity of pancreatic cancer cells significantly decreased in a time-and dose-dependent manner(P<0.001).(2)Colony formation,scratch,Transwell and flow cytometry assay showed that compared with negative control,AMI-1 and CKI groups,the combination of AMI-1 and CKI synergistically inhibited the colony formation in pancreatic cancer cells(P < 0.001),inhibited the cell migration ability(P < 0.001),and induced apoptosis(P < 0.05).(3)Western blot analysis showed that compared with negative control group,AMI-1 reduced PRMT5 expression and H4R3me2 s accumulation in pancreatic cancer(P<0.001),CKI significantly down-regulated the protein levels of PRMT1(P<0.001),and up-regulated the protein levels of PRMT2,PRMT3,PRMT4,PRMT6 and PRMT8 in pancreatic cancer(P<0.001);compared with negative control,AMI-1 and CKI groups,the combination of AMI-1 and CKI significantly reduced PRMT5,H4R3me2 s,c-Myc,β-catenin and PCNA expression in pancreatic cancer cells(P<0.001).3.The results of the nude mice xenograft models:(1)After 31 days of pharmacological intervention,the tumor weight and tumor volume of nude mice in each treatment group was significantly lower than that in the model group(P<0.001),and compared with AMI-1 group and CKI group,the tumor inhibition rate of AMI-1+CKI group had an increasing trend but there was no statistical significance(P>0.05).(2)Compared with model group,the body weight and organ index of nude mice in each treatment group were not significantly different(P>0.05),there were different degrees of pathological necrosis in each treatment group.(3)Western blot analysis showed that compared with the model,AMI-1 and CKI groups,AMI-1+CKI group significantly reduced PRMT5,H4R3me2 s expression in pancreatic cancer tissues(P<0.001).Conclusion:1.AMI-1 significantly inhibited pancreatic cancer growth,and reduced PRMT5 expression and H4R3me2 s accumulation in vitro.2.The process of CKI inhibited the proliferation of pancreatic cancer may be by downregulating the expression of PRMT1 and up-regulating the expression of PRMT2,PRMT3,PRMT4,PRMT6,and PRMT8 in vitro.3.The combination of AMI-1 and CKI synergistically inhibited the growth of pancreatic cancer cells in vitro.In vivo,AMI-1 and CKI alone significantly inhibited the growth of pancreatic cancer,the combination of AMI-1 and CKI had a tendency to increase the tumor inhibition rate of single agent.The mechanism of synergistic antitumor effects may be as follows.(1)In vitro,AMI-1 and CKI alone and in combination inhibited the migration ability and induced apoptosis of pancreatic cancer cells.(2)In vitro,AMI-1 and CKI alone and in combination reduced PRMT5,H4R3me2 s,c-Myc,β-catenin and PCNA protein levels.(3)In vivo,AMI-1 and CKI alone and in combination reduced PRMT5 and H4R3me2 s protein levels.