The Mechanism Of Arginine Methyltransferase Inhibitor 1 And Its Synergistic Antitumor Effect With Compound Kushen Injection In Lung Cancer

Background:Globally,the lung cancer is the deadly leading cause of cancer and exhibits high incidence on human,especially occurs in male human.The mortality is continued to increase and the survival of late-stage lung cancer is short and cure uncommon which is much lower than other types of cancers.At present,the major management still is surgical resection and chemotherapy,which easily contribute to the trauma and toxic response which is underpowered.Therefore,it is necessary to explore a functional but less toxic drug.According to previous research,Protein arginine methyltransferases(PRMTs)could catalyze arginine methylation which encoded in mammalian genomes,it has been verified that the enzymes is implicated in several of cancers.PRMT5(Type ? PRMTs)plays an important role in cancer,which is overexpressed in many types malignant tissue.Studies have confirmed that arginine methyltransferase inhibitor 1(AMI-1)can inhibit the activity of type I PRMT(including PRMT1,PRMT3,PRMT4 and PRMT6)enzymes in vitro,and there is an evidence demonstrated that AMI-1 can regulate type I PRMTs and its related signal pathways to inhibit the proliferation of pancreatic cancer,colorectal cancer and other cells in vitro,but the inhibitory effect of AMI-1 combined with compound kushen injection(CKI)on type I PRMT5,lung cancer and its mechanism are still unclear.Objective:This study aims to investigate the effect and potential mechanism of AMI-1combined with CKI on lung cancer cells in vitro and in vivo,so as to provide a theoretical basis and therapeutic option for the diagnosis and treatment of patients with lung cancer.Methods:(1)The effects of AMI-1 on proliferation and colony of lung cancer cells were measured by CCK-8 and colony formation assay.(2)The anti-tumor activity of AMI-1 was evaluated by BALB/c nude mice xenograft models.(3)The effects of AMI-1 on cycle and apoptosis of lung cancer cells were analyed by flow cytometry.(4)The effects of AMI-1 on lung cancer cell migration were evaluated by Transwell and Scratch assay.(5)The effects of AMI-1 on PRMT5 and other PRMTs activity were detected using Chemiluminescent Assay Kit.(6)The effects of AMI-1 on PRMT5 and its related signal pathway protein in lung cancer cells and/or tissues were analyed by Western blot.(7)To verify the role and mechanism of PRMT5 in lung cancer by PRMT5-specific si RNA.(8)The effects of AMI-1 combined with CKI on growth of lung cancer cells were evaluated by MTT assay and mice xenograft models.Results:(1)The CCK8 and colony formation assay showed that AMI-1 treatment significantly inhibited proliferation and colony formation of lung cancer cells in a time-and dose-dependent manner.(2)The in vivo tumor models showed that AMI-1 treatment significantly inhibited the growth of nude mice xenograft models compared with control group.(3)Flow cytometry analysis showed that AMI-1 induced G0/G1 arrest and apoptosis of lung cancer cells compared with control group.(4)Transwell and scratch assays showed that treatment of lung cells with AMI-1resulted in marked reduction in migration activity compared with control group.(5)Chemiluminescent assay showed that AMI-1 significantly inhibited the activity of type ? PRMT5 enzyme.(6)Western blot analysis showed that AMI-1 reduced PRMT5/e IF4 E expression and H4R3me2s/H3R8me2 s accumulation while increased that of p53 in lung cancer cells and/or tissues.(7)PRMT5 silencing inhibited cell proliferation,reduced H4R3me2s/H3R8me2 s accumulation and e IF4 E expression in lung cancer cells.(8)The MTT and mice xenogratf models showed that the combination of AMI-1and CKI synergistically inhibited the proliferation and growth of lung cancer.Conclusion:AMI-1 significantly inhibited lung cancer growth in vitro and in vivo,and inhibited the activity of PRMT5 enzyme,reduced “oncoprotein” PRMT5 expression and H4R3me2 s and H3R8me2 s accumulation.In addition,AMI-1 down-regulated the expression of “oncoprotein” e IF4 E while up-regulated tumor suppressor p53 levels,and regulated the proliferation,cell cycle progression,apoptosis and migration of lung cancer cells.The combination of AMI-1 and CKI synergistically inhibited the growth of lung cancer cells in vitro and in vivo.