Studies On The Mechanism Of Proliferation Inhibition And Apoptosis Induction By 15-LOX-1 Gene On Gastric Cancer Cell AGS

Objective: The study was to investigate the mechanism of proliferation inhibition and apoptosis induction by 15-lipoxygenase-1(15-LOX-1) gene on gastric cancer cell AGS.Methods: AGS cells were divided into three groups: AGS/15-LOX-1 group transfected with PCDNA3.1/15-LOX-1,vector control group transfected with PCDNA3.1 and blank control group without transfection.Plasmids of PCDNA3.1/15-LOX-1 encoding 15-LOX-1 gene were transiently transfected into AGS cells.The expression of 15-LOX-1 and PPARγat mRNA level were measured by reverse transcription polymerase chain reaction(RT-PCR).The expression of 15-LOX-1,PPARγ,Skp2/P27,Bax/Bcl-2 at protein level were measured by western blot.The proliferation of four groups cells(AGS/15-LOX-1,AGS,AGS/15-LOX-1 add with GW9662,AGS add with GW9662) were assessed by thiazolyl blue tetrazolium bromide(MTT) assay respectively.Results: (1) 15-LOX-1 mRNA and protein were expressed in AGS/15-LOX-1 cells compared with control groups detected by RT-PCR and western blot.(2) P27 which was expressed from AGS cells transfected with PCDNA3.1/15-LOX-1 is up-regulated, meanwhile, S-phase Kinase-associated Protein 2 (Skp2) is down-regulated compared with control groups(P<0.05).(3)There was a significant decline in cell proliferation ability of AGS/15-LOX-1 in comparision with control groups revealed by MTT assay(P<0.05).Cell survival rates were 58.5%,48h after transfection.After added with 10uM GW9662, cell proliferation ability of AGS/15-LOX-1 were 76.2%,48h after intervention.(4)PPARγmRNA and protein were expressed in AGS/15-LOX-1 cells,AGS and AGS/pcDNA3.1 cells detected by RT-PCR and western blot.Also,in AGS/15-LOX-1 cells,the expression of PPARγ was significantly higher than that in AGS and AGS/pcDNA3.1 cells(P<0.05).(5)Bax which was expressed from AGS cells transfected with 15-LOX-1 is up-regulated, meanwhile, Bcl-2 is down-regulated compared with control groups(P<0.05).But 48h after intervent with GW9662, western blot confirmed that there was no statistically significant differences in comparision with control groups(p>0.05).Conclusion:The expression of 15-LOX-1 could effectively regulate the expression of Skp2 and P27 to resort gastric carcinoma cell in period G0/G1 and inhibit carcinoma cell proliferation.The expression of 15-LOX-1 could up-regulated expression of bax and low-regulated expression of Bcl-2 through PPARγ,which may be the mechanism of 15-LOX-1 gene induce the apoptosis of human gastric cancer AGS cells.