The Mechanism Of Peimine Inducing Apoptosis Of Gastric Cancer MKN-45 Cells By Regulating ROS Mediated Signaling Pathways

As the main treatment for cancer patients after surgery,chemotherapy often destroys healthy normal cells,leads to the disorder of human immune metabolism,and has great side effects on the body.Therefore,it is urgent to find a highly effective drug with low toxic side effects.Many natural extracts have been found to have potential therapeutic effects on cancer cells while having few side effects on normal cells.Fritillaria thunbergii is often used as an anticancer Chinese medicine in various prescriptions,which has been proved to have the effect of clearing heat and detoxifying,removing carbuncle.Peimine(PM)is a natural product extracted from Fritillaria thunbergii,which has anti-cancer,anti-inflammatory and analgesic effects,but its specific anti-cancer mechanism is very limited.In this study,gastric cancer MKN-45 cells were used as an in vitro experimental model to explore the killing effect of PM on gastric cancer cells,and further study its apoptosation-inducing effect,cycle-arresting effect,migration-inhibiting effect and potential signal transduction pathway on gastric cancer MKN-45 cells.In this study,CCK-8 assay was used to detect the killing effect of PM on 12 types of gastric cancer cells from different sites and the toxic effects of PM on 4 types of normal cells from different tissues.Annexin V-FITC/PI staining and flow cytometry were used to detect the apoptosis,mitochondrial membrane potential and reactive oxygen species(ROS)levels of MKN-45 cells treated with PM.Flow cytometry was used to detect the MKN-45 cell cycle arrest induced by PM.Western blotting was used to detect the expression of apoptotic proteins,signaling pathway proteins and the relationship between apoptosis and ROS accumulation in MKN-45 cells treated with PM.Wound healing assay,Transwell assay and Western blotting were used to evaluate the migration inhibition effect of PM on MKN-45 cells.The results of CCK-8 assay showed that PM significantly decreased the activity of gastric cancer cells,and the effect on MKN-45 cells was the most obvious.Compared with the common chemotherapy drugs 5-fluorouracil(5-FU),PM showed lower toxic and side effects on normal cells.Annexin V-FITC/PI staining and flow cytometry showed that PM induced apoptosis of MKN-45 cells by decreasing mitochondrial membrane potential.Flow cytometry and Western blotting showed that PM regulated mitogen activated protein kinase(MAPK),signal transduction and transcriptional activator protein 3(STAT3)and nuclear factor κB(NF-κB)signaling pathways dependent on ROS accumulation.PM also down-regulated the expression levels of p-AKT,Cyclin B1 and CDK1/2,and up-regulated the expression levels of p21 and p27 by increasing ROS accumulation,leading to cell cycle arrest in G2/M phase.In addition,PM regulated the Wnt/β-catenin pathway by regulating ROS,down-regulated the expression level of N-cadherin protein,up-regulated the expression level of E-cadherin protein,and inhibited the migration of MKN-45 cells.In conclusion,this study is the first to explore the anticancer effect of PM through ROS mediated endogenous apoptosis,G2/M phase cycle arrest and inhibition of cell migration,which may become an effective treatment for gastric cancer.