Experimental Studies For The Effect Of Dual Inhibition Of COX-2 And 5-LOX On Human AGS Cell Line And For Its Possible Mechanisms

Objective: We tested the efficacy of the selective COX-2 inhibitor nimesulide,5-LOX inhibitor AA861 or their combination on human AGS gastric carcinoma cell line,then investigated whether the dual inhibition of COX-2 and 5-LOX has a more power anticancer effect than single inhibition of either COX-2 or 5-LOX alone on human AGS cell line,and studied its possible mechanisms.Methods: AGS cell were passaged into a RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 U/mL penicillin G and 100 U/mL streptomycin in an incubator at 37°C, with 100% humidity and 5% CO2。AGS was used as a model,and nimesulide(50μmol/L,100μmol/L,200μmol/L)and the combination of nimesulide and AA861, were used in vitro .Morphological changes of AGS cells was observed and photographed with inverted microscope;The proliferation was detected by thiazolyl blue tetrazolium bromide(MTT)assays;and AO-EB fluorescence staining,TUNEL staining and DNA-fragment assay were used to evaluate the apoptotic index of AGS cells .Wherease reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of 5-LOX mRNA. And western blot was conducted to evaluate the expression of 5-LOX and p-Akt proteins .Results: Morphological changes of AGS cells which were treated with Nimesulide,AA861 or their combination were obvious compared with control under inverted microscope;MTT assay revealed that Nimesulide inhibited the proliferation of AGS cells in a dose-and time-dependent manner, except Nimesulide 200μmol/L group at 24h and 48h group;The apoptosistic rates of AGS cells tested by TUNEL staining showed that Nimesulide induces apoptosis in a dose-dependent fashion on AGS cells. Meanwhile,MTT(AA861 vs combination, p<0.01; nimesulide vs combination, p<0.05),AO-EB staining(single vs combination, p<0.01),TUNEL staining (single vs combination, p<0.01) and DNA-fragment assay showed that combined treatment with both COX-2 and 5-LOX inhibitors further inhibited the proliferation and induced the apoptosis of AGS cells over treatment with either COX-2-inhibitor or 5-LOX-inhibitor alone.Further more, RT-PCR and Western blot revealed that the expression of 5-LOX mRNA and protein in the cells which were treated with Nimesulide or the combination of Nimesulide and AA861 was higher than that of control(p<0.01),meanwhile,AA861 had not effect on the expression of 5-LOX (p>0.05). Western blot also showed that the expression of p-AKt protein in the cells which were treated with AA861 or the combination of Nimesulide and AA861 was lower than that of control(p<0.05),meanwhile,the cell with Nimesulide had higher expression of p-AKt protein than that of control(p<0.01).Conclusion:Both COX-2 inhibitor Nimesulide and 5-LOX inhibitor AA861 inhibits proliferation and induces apoptosis in human AGS cells.The dual inhibition of COX-2 and 5-LOX may present a superior anticancer profile than single inhibition of either COX-2 or 5-LOX alone. Further more, Nimesulide upset the expression of 5-LOX and p-AKt,and AA861 reduce the expression of p-AKt,but has not effect on the expression of 5-LOX, meanwhile, the combination of Nimesulide and AA861 increase the expression of 5-LOX,and reduce the expression of p-AKt powerly.