Effects Of Luteolin On DNA Double-strand Breaks And Homologous Recombination Repair In Human Gastric Cancer Cell Lines

Objectivesluteolin(3,4,5,7-tetrahydroxyflavone)is a class of flavonoids widely found in the rhizome of many plants,It has a variety of biological activities,such as anti-tumor,anti-inflammatory,antioxidant and neuroprotective,and it can reverse the drug resistance of a variety of cancer cells.Related studies have shown that flavonoid antioxidants can trigger apoptosis by inducing DNA double-strand breaks and blocking the initiation process of homologous recombination repair.This is related to the weakening of Ataxia-telangiectasia mutated(ATM)activation and inhibition of phosphorylated kinase ATM(pATM)is associated with the binding of the MRE11-RAD50-NBS1(MRN)complex.However,there are few studies on the effects and mechanisms of luteolin on DNA damage repair in gastric cancer cells.In this study,luteolin was used to act on gastric epithelial cell GES-1?gastric cancer cells AGS?MKN-45?NCI-N87 and HEK-293(DR-GFP)cells in vitro.To explore the mechanism of inhibiting tumor cells and provide experimental basis for clinical treatment application in the future.Materials and Methods1.Luteolin and GES-1,AGS,MKN-45,NCI-N87 cell were cultured together.Western Blot was used to detect the expression of DNA damage marker protein ?H2AX,and HR repair key protein(Rad51?pCtIP?pMRE11)to evaluate the effect of luteolin on the damage and repair DNA normal gastric epithelial cells and multiple gastric cancer cells.2.AGS cells were co-cultured with luteolin,using neutral comet assay(single-cell gel electrophoresis)to further detect DNA damage(double-strand break).3.AGS cells were co-cultured with luteolin.immunofluorescence was used to detect the recruitment of DNA damage marker protein ?H2AX,and HR repair protein Rad51.4.To evaluate the efficiency of DNA damage repair,HEK-293(DR-GFP)cells were cultured with luteolin and the number of positive cells in total cells was measured by flow cytometry.Results1.After drug treatment of GES-1 cells,the expression of marker protein ?H2AX and repair protein Rad51were significantly higher than that of blank control group(P<0.05).2.After drug treatment of AGS cells,the expression of ?H2AX was up-regulated compared with the blank control group(P<0.05);While the expression of repair protein Rad51 and pCtIP was down-regulated in the blank group,the difference was statistically significant.3.After drug treatment of NCI-N87 cells,the expression of ?H2AX was up-regulated compared with the blank control group(P<0.05);The expression of Rad51,pCtIP,pMRE11was down-regulated compared with the blank control group at drug concentration of 40 mmol/L(P<0.05).4.After drug treatment of MKN-45 cells,the expression of ?H2AX and pCtIP were up-regulated compared with the blank control group(P<0.05);The expression Rad51 was down-regulated at 10 mmol/L compared with that of blank control group,the decrease is more pronounced at 20 mmol/L(P<0.05).The expression of pMRE11 was down-regulated at 20 mmol/L and 40 mmol/L compared with the control group(P<0.05),there was no significant difference between the blank group and at the concentration of 10 mmol/L(P=0.206),we need to do further analysis and validation.5.After drug treatment of AGS cells,the expression and recruitment of DNA damage marker protein ?H2AX was up-regulated compared with the control group,and the drug concentrations at 20 mmol/L was up-regulated compared with 10 mmol/L(P<0.01).The expression and recruitment of HR repair key protein(Rad51)was up-regulated in the dosing group compared with the control group,and up-regulated in the drug concentration at 10 mmol/L compared with 20 mmol/L(P<0.05).6.Comet experiments show that the tailing phenomenon increases when the drug concentration is 20 mmol/L.7.The number of positive cells in the I-Scel plasmid-transfected group was lower than that in the non-dosing GFP group(P<0.05).Reduction in the number of GFP-positive cells in the dosing group(20 mmol/L)after transfection of I-Scel plasmid compared with 10 mmol/L(P<0.01).ConciusionsLuteolin acts on different human gastric cancer cells and induces DNA double-strand breaks in DNA damage repair,which reduces HR repair to a certain extent and promotes gastric cancer cell apoptosis.