The Survival Strategies Of OE-19 Cells Under Acid Or Acid/bile Condition: The Molecular Mechanisms Behind TRAIL Resistance

Objective Despite the fact that tumor necrosis factor related apoptosis inducing ligand(TRAIL)kills cancer cells selectively,more and more cancer cells have been found resist to TRAIL treatment.Esophageal adenocarcinoma(EAC)is such an example.EAC is generally believed to develop from esophageal inflammation caused by chronic gastroesop Hageal reflux disease(GERD).During GERD episodes,the esop Hageal epithelium is frequently exposed to stomach acid,which often contains duodenal bile salts that could lead to carcinogenesis.In response to GERD episodes,a number of cytokines,such as TRAIL,are produced to the region to cause inflammation and to repair tissue damage.The aim of this study is to investigate how GERD episodes affect TRAIL signaling in EAC cells and to explore the mechanisms behind TRAIL resistance.Method To simulate GERD episodes in vitro,we exposed OE-19 cells to acidic medium(p H 4.5)or acid bile salts,including acute and chronic stimuli.Expressions of TRAIL and its receptors were evaluated by Western blot analyses.Apoptosis was assessed using an Apopxin-based fluorescence technique.To determine the roles of TRAIL and its receptors in apoptosis,cells were transfected with plasmid vectors carrying either the open reading frame or sh RNA sequence for the targeted gene.The empty vector or the vector carrying a negative sequence was used as control.Results(1)Under the regular growth condition,OE-19 cells expressed the death receptor DR4 and three decoy receptors including DcR1,DcR2 and OPG.In response to acid exposure,regardless of acute or chronic,both TRAIL ligand and its second death receptor DR5 were activated to express and DR4 was down significantly(P<0.05),while the decoy receptors remained unchanged.When bile salts came along with the acid attacks,in addition to the downregulation of DR4,all decoy receptors were gone almost completely(P<0.05).(2)Apoptosis assays demonstrated that acid alone had no effect on cell death,but in the presence of bile salts,especially the combination of acid/bile,apoptosis increased significantly(P<0.05).No necrosis was detected.(3)Western blotting showed that transfection of the oesophageal adenocarcinoma OE-19 cells with p RS plasmid containing a specific sh RNA sequence for either TRAIL or DR5 effectively inhibited the targeted gene.As a result,the apoptosis in response to acid/bile exposure came down significantly(P<0.05).(4)Cells transfected with empty plasmids screened from normal medium were used as the control group.Compared with the control group,cells transfected with p CMV6 plasmids loaded with target gene sequences induced high expression of receptors DcR1,DcR2 and OPG(P<0.05),while apoptosis was significantly inhibited in the transfection group,especially in the DcR2 transfection group(P<0.05).(5)Compared with the control group,namely the basal medium stimulation group,the PARP protein in the acid bile salt group was cleaved into two proteins,while the PARP protein in the other group was not cleaved significantly(P<0.05).Conclusions(1)Acid and bile both can induce massive production of TRAIL and DR5,but TRAIL triggers OE-19 cell apoptosis promptly only when acid and bile are present simultaneously.(2)Each of the decoy receptors contributes to TRAIL resistance in OE-19 cells,but DcR2 plays the major part.