Study On The Effect Of NEDDylation Inhibitor MQS-2-46 On Gastric Carcinoma And Its Molecular Mechanism

Purpose:The morbidity and mortality of gastric cancer rank among the top five in the world.The treatment of gastric cancer continues to develop,but the treatment method with both high efficiency and safety is still in the process of exploring.This subject has carried out experimental design around the small molecule inhibitor MQS-2-46,indepth exploration of its anti-gastric cancer proliferation mechanism from both autophagy and DNA damage,providing new ideas and new ideas for the design of safer and more effective gastric cancer anti-tumor drugs.Methods:1.Screen the MQS series of compounds in the MTT experiment,and calculate their 24 h IC50 in different cell lines;2.Clone formation experiment and EdU experiment were used to detect the proliferation ability of gastric cancer cells MGC-803 and HGC-27 after 24 hours of compound treatment;flow cytometry was used to detect the changes of gastric cancer cell apoptosis and mitochondrial membrane potential levels after compound treatment;by Western Blotting detects the expression of apoptotic protein in gastric cancer cells after the compound’s action;3.Western blotting and mRFP-GFP-LC3 double-labeled plasmid transfection experiments,to detect the expression of LC3B and p62 in gastric cancer cells after the compound’s action,and the changes in autophagy flow in the cells;4.Immunofluorescence experiment and Western blotting after nucleoprotein extraction were used to detect the location and content of p62 in gastric cancer cells after the compound was treated;LC3B was knocked down by si-RNA transfection experiment,and flow cytometry was used to detect the apoptosis of gastric cancer cells after the compound was treated.Changes in death levels5.The comet assay detects the occurrence of DNA damage in gastric cancer cells after the action of lower concentrations of the compound;Western blotting detects the protein expression in the DNA damage response pathway in gastric cancer cells after the compound action.Results:1.MQS-2-46 was screened as the lead compound,and gastric cancer cells MGC-803 and HGC-27 were selected as the experimental objects.The 24 h IC50 of the compounds in both cells was 5 μM.2.The compound has good anti-proliferation and pro-apoptosis abilities on gastric cancer cells MGC-803 and HGC-27.3.The compound increases the production of autophagosomes in the cell and partially blocks the autophagy flow.4.The compound causes nuclear translocation of p62 in cells,and the LC3B knockdown experiment shows that autophagy is a protective autophagy in this process.5.Gastric cancer cells have obvious DNA damage after the compound is used,and the content of DNA damage protein γ-H2AX is significantly increased.Conclusion:The compound MQS-2-46 has good anti-proliferation ability of gastric cancer.It also promotes the production of autophagosomes in gastric cancer cells and partially blocks the autophagic flow,and obviously induces the DNA damage process.