The Molecular Mechanisms Of Apoptosis And Apoptosis Ligand Tolerance Induced By Tumor Necrosis Factors Related Apoptosis (TRAIL) In Esophageal Adenocarcinoma Cells

Part Ⅰ Acid/bile exposure activates expression of TRAIL and its death receptor TRAILR2 in various esophageal adenocarcinoma cell linesObjective:To investigate The effects of acid/bile exposure(simulating GERD episodes)on the expression of TRAIL and its receptors in esophageal adenocarcinoma cells,and to determine their relationship with apoptosis.Method:For chronic GERD simulation,four oesophageal adenocarcinoma cell lines(OE19,OE33,FLO-1 and SK-GT-4)were exposed to pH 4.2-4.5 medium in the presence or absence of bile salts four times/day for a week.For acute GERD simulation,on the other hand,cells were exposed to the conditioned medium for 30 min.Regular growth medium was used as control.Protein was extracted and examined for the expression of TRAIL and its five receptors,including TRAILR1,TRAILR2,TRAILR3,TRAILR4,and OPG,using Western-blotting technique.At the same time,apoptosis was measured by apopxin staining.Result:① Acid/bile exposure significantly upregulated TRAIL and TRAILR2 expression in all four cell lines.②Acid/bile exposure lowered expression of decoy receptors(TRAILR3,TRAILR4 and OPG)in OE19 and FLO-1 cells,whereas did not change them in OE33 and SK-GT-4 cells significantly.③Acid/bile exposure induced apoptosis in OE19 and FLO-1 cells,but not in OE33 and SK-GT-4 cellsConclusions:Different esophageal adenocarcinoma cells respond to acid/bile condition differently.While acid/bile salt exposure induces the expression of TRAIL and TRAILR2 in all the esophageal adenocarcinoma cells,apoptosis only takes place when the decoy receptors are suppressed.Part Ⅱ The molecular mechanisms of tumor necrosis factor related apoptotic ligand(TRAIL)mediated apoptosis in esophageal adenocarcinomaObjective:To investigate the molecular mechanisms acid/bile-induced apoptosis in OE19 and FLO-1 cells.Method:①Cells were transfected with shRNA pRS plasmids against TRAIL or TRAILR2 to knock down their expression.The empty vector pRS was used as control.The transfected cells were given chronic acid/bile treatment as before.Apoptosis was detected by apopxin staining.Gene expression was analyzed by Western blotting.②To create cell lines with overexpression of TRAILR3,TRAILR4 or OPG,cells were transfected with pCMV vector carrying the open reading frame for the targeted gene.The empty vector was used as control.The transfected cells were given chronic acid/bile treatment as before.Apoptosis was detected by apopxin staining.Gene expression was analyzed by Western blotting.③ To analyze the formation of death induced signaling complex(DISC)after acid/bile treatment,immunoprecipitation(IP)and Western blotting were performed to detect the possible components,including FADD,CASP8,c-FLIP,TRAILR2,and RIPK1.④To assess the possible effect of PKC α on c-FLIP stability,kinase assay was performed.In brief,c-FLIPR was first precipitated using a specific antibody,and phosphorylated serine was detected by Western blotting.Meanwhile,the expression of PKCa in OE19 cells was detected by Western blotting under the same conditions.PKC a agonist and PKC α inhibitor were added to the oe19 cell culture medium of the two groups respectively.After 6 hours,the protein was extracted and the expression of c-FLIP was detected by Western blotting.Two groups of OE19 cells were cultured at the same time for acid/bile salt stimulation.In one group,10 μPKC α activator sc-10 was added at the same time for stimulation.The protein was extracted from the two groups at the same time.The expression of PARP was analyzed by Western blotting,and the apoptosis of OE19 cells was detected under the same conditions.Result:①Knockdown of TRAIL or TRAILR2 prevented acid/bileinduced apoptosis,indicating the apoptosis under such condition was mediated by TRAIL and TRAILR2.②Overexpression of TRAILR3,TRAILR4 or OPG in OE19 cells effectively reduced acid/bile-induced apoptosis.③ Acid alone promoted c-FLIPR expression,while acid/bile combination inhibited it.As a result,Caspase-8 got activated to induce apoptosis.④Acid alone increased PKCaactivity,while acid/bile combination inhibited.PKCα agonist promoted the phosphorylation of c-FLIPR and its stability,while PKCainhibitor reduced cFLIPR phosphorylation and its stability.Conclusions:Acid/bile salts stimulate OE19 cells,on the premise of causing increased TRAIL expression,promote increased expression of the lethal receptor TRAILR2,suppress expression of all decoy receptors,inhibit PKCa activity,destabilize c-FLIP expression,promote FADD binding to pro-caspase-8 in the DISC and caspase-8 activation,leading to increased apoptosis.Part Ⅲ Molecular mechanisms behind acid/bile-induced tumor necrosis factor apoptosis related ligand(TRAIL)resistance in esophageal adenocarcinoma cellsObjective:To investigate the molecular mechanisms behind TRAIL resistance in OE33 and SK-GT-4 cells under acid/bile stimulation.Method:① OE33 cells were transfected with shRNA pRS plasmids against TRAILR3,TRAILR4 and OPG to obtain cell line with underexpression of decoy receptors,and we called it OE33DCR-.The OE33DCR-cells were given chronic acid/bile treatment.TRAIL recombinant protein was used as a reference.Apoptosis was measured by apopxin staining.② OE33DCR-cells were given chronic acid/bile treatment.RNA was extracted and hybridized with gene chips containing all the members of tumor necrosis factor,tumor necrosis factor receptor family as well as the related signaling molecules by reverse transcription polymerase chain reaction(RT-PCR).The correlation between gene expression and apoptosis was analyzed.② OE33DCR-Cells were transfected with the shRNA plasmid against c-FLIP to obtain OE33DCR-/c-FLIP-cells with effectively knockdown both the decoy receptor and c-FLIP.Acid/bile chronic experiment was repeated using the transfected cells(OE33DCR-/c-FLIP-)and apoptosis was detected by apopxin staining.By the same method,OE33DCR-cells were transfected with the shRNA plasmid against TRADD or TNFAIP3 to obtain OE33DCR-/TRADDand OE33DCR-/-TNFAIP3-Cells with TRADD or TNFAIP3 defficiency.Chronic acid/bile treatment and apoptosis assay were repeated for TNFAIP3DCR-/TRADD-And OE33DCR-/TNFAIP3cells.④OE33DCR-cells were given chronic acid/bile treatment.TRAILR2 was precipitated from the total protein using a specific antibody.The expression of FADD,TRADD,c-FLIP,TNFAIP3 and caspase-8 were detected by Western blotting to analyze the constituent proteins of the death inducing signalling complex(DISC)and association of FADD with TRADD and c-FLIP with caspase-8.⑤OE33DCR-cells were transfected with shRNA plasmids against c-FLIP and TRADD to obtain the OE33 DCR-/TRADD-/c-FLIP-cells with nockdown of all the decoy receptors,c-FLIP and TRADD simultaneously.Chronic acid/bile experiment and apoptosis assay were performed in OE33 DCR-/TRADD-/c-FLIP-cells.OE33 with simultaneous knockdown of the decoy receptor,TRADD,c-FLIP,and TNFAIP3 was generated by the same method.Chronic acid/bile experiment and apoptosis assay were performed in OE33DCR-/TRADD-/c-FLIP-/TNFAIP3 Cells.⑥ BOT-64(an NF κB inhibitor)was added to the experiment to assess the role of NFκB activity in association with TNFAIP3 by Western blotting in OE33DCR-、OE33DCR/TNFAlP3-And OE33DR/TRADD-Cells,which were given chronic acid/bile treatment.NFκB expression was also analyzed by FITC immunofluorescence stainingResults:(1)Knockdown of decoy receptors(TRAILR3,TRAILR4 and OPG)did not trigger OE33 apoptosis under acid/bile condition.Chronic acid/bile exposure upregulated almost all pro survival genes except TRAIL and TRAILR2.Knockdown of c-FLIP increased acid/bile-induced OE33 apoptosis,but knockdown of either TRADD or TNFAIP3 did not.(2)Chronic acid/bile exposure induced a DISC formation containing TRAILR2,TRADD,FADD and c-FLIP in OE33 cells.Caspase-8 was not involved.(3)Only when TRADD and c-FLIP expression were simultaneously inhibited,acid/bile exposure could induce apoptosis in OE33 cells.(4)The upregulation of TNFAIP3 during acid/bile exposure was to keep NF κB activity down,rather than to block apoptosis;activation of NF κB is induced by TRADD overexpressionConclusions:Acid/bile exposure promotes TRADD expression to block FADD from binding to TRAILR2,and promotes c-FLIP expression to keep procaspase-8 out of DISC.As a result,acid/bile exposure fails to induce apoptosis in OE33 cells.Overexpression of TRADD activates NFκ B,which promotes expression of TNFAIP3,forming a negative feedback loop.