Cyclooxygenase-2 (COX-2) has been linked to neoplastic progression in Barrett's esophagus. Acid exposure has been shown both to activate the MAPK pathways and to increase COX-2 protein expression in Barrett's metaplasia, but it is not known whether these effects are interrelated. We hypothesized that acid-induced activation of the MAPK pathways mediates an increase in COX-2 expression in Barrett's esophagus, and we tested this hypothesis in a Barrett's-associated adenocarcinoma cell line (SEG-1). We exposed SEG-1 cells to acidic or neutral media in the presence and absence of two MAPK inhibitors: U-0126 (an ERK inhibitor) or SB-203580 (a p38 inhibitor). We quantitated COX-2 protein levels using an enzyme immunometric assay and COX-2 mRNA levels using real-time PCR. We also determined how acid affects the activity of the COX-2 promoter and mRNA stability. Compared with SEG-1 cells exposed to neutral media, acid-exposed cells exhibited a 2.8-fold increase in COX-2 mRNA levels within 30 min. Both U-0126 and SB-203580 attenuated the acid-induced increase in COX-2 mRNA. Acid significantly increased COX-2 protein expression and promoter activity, and both of these effects were abolished by treatment with U-0126 and SB-203580. Acid exposure also stabilized COX-2 mRNA levels, an effect that was abolished by U-0126 but not by SB-203580. We conclude that acid increases COX-2 expression through activation of the MAPK pathways. Acid-induced activation of both ERK and p38 causes a significant increase in COX-2 promoter activity, and acid-activated ERK stabilizes COX-2 mRNA. These findings suggest potential mechanisms whereby acid reflux might promote carcinogenesis in Barrett's esophagus.

BACKGROUND & AIMS:To explore mechanisms whereby acid reflux might contribute to carcinogenesis in Barrett's esophagus (BE) we studied: (1) the effects of acid on the mitogen-activated protein kinase (MAPK) pathways, cell proliferation, and apoptosis in a Barrett's adenocarcinoma cell line (SEG-1); and (2) the ability of acid to activate the MAPK pathways in vivo in patients with BE.METHODS:SEG-1 cells were exposed to acidic media for 3 minutes, and the activities of 3 MAPKs (ERK, p38, and JNK) were determined. Proliferation was assessed using flow cytometry; cell growth and apoptosis were assessed using cell counts and an apoptosis ELISA assay. MAPK activation was studied in biopsy specimens taken from patients with BE before and after esophageal perfusion for 3 minutes with 0.1N HCl.RESULTS:Acid-exposed SEG-1 cells exhibited a significant increase in proliferation and total cell numbers, and a significant decrease in apoptosis. These effects were preceded by a rapid increase in the activities of ERK and p38, and a delayed increase in JNK activity. PD 98059 abolished the acid-induced increase in G0/G1 and decrease in subG0 phases of the cell cycle. Both SB 203580 and DN-JNK 1/2 inhibited the acid-induced progression from G0/G1 to G2/M. The acid-induced decrease in apoptosis was abolished by inhibition of either ERK or p38. In the patients, acid exposure significantly increased the activity of p38 in the metaplastic epithelium.CONCLUSIONS:Acid increases proliferation and survival, and decreases apoptosis in SEG-1 cells by activating the MAPK pathways. Acid also activates the MAPK pathways in BE in vivo. These findings suggest that acid might contribute to carcinogenesis in BE through activation of MAPK pathways.

luciferase measurement.Suhconfluent cultures (70%) of HCA7 colon cancer cell lines were incubated in serum free medium for 24 hours to induce partial growth arrest.Proliferation was stimulated by 10% foetal calf serum for 24 hours.The effect of methylclofenapate (1-lO0/Lg), and other ligands on DNA synthesis were measured using [3H] thymidine incorporation assay whilst rates of apoptosis and necrosis were assessed by flow cytometric analysis of cells stained with Annexin V FITC and propidium iodine.Results Methylclopfenapate caused a significant dose-dependent increase in PPAR~PPARE activation (luciferase response enhanced 1.6 -+ 0.15 [SEM] fold at lp.M, 2.6 -+ 0.2 fold at IO/~M and 4.2 -+ 0.6 fold at lOOp.M, p = 0.001 ).Methylclofenapate caused no significant activation of PPAR/~ or PPAR'y at concentrations -

9%) 12 (9%) 82/109 (75%) ReJ GERD-65 2 (3%) 5 (7.7%/ 28/58 (48%) Con GERD.~ 196 9 (4.6%) 5 (2.6%) t00

Adenocarcinoma in Barrett’s esophagus has been increasing in incidence at a rapid rate for more than two decades. Cyclooxygenase (COX)-2 appears to play an important role in gastrointestinal carcinogenesis, and COX-2 overexpression has been demonstrated both in esophageal adenocarcinomas and in the metaplastic epithelium of Barrett’s esophagus. The aim of our study was to determine whether selective inhibition of COX-2 by NS-398 would alter the rates of cell growth and apoptosis in human Barrett’s-associated esophageal adenocarcinoma cell lines. COX-1 and COX-2 expression in adenocarcinoma cell lines was determined using reverse transcription-PCR and Western blotting for mRNA and protein, respectively. Esophageal adenocarcinoma cell lines were treated with various concentrations of NS-398 (selective for COX-2 inhibition) and flurbiprofen (selective for COX-1 inhibition). Cell growth was compared in flurbiprofen-treated and untreated tumor cell lines; cell growth and apoptosis were compared in NS-398-treated and untreated tumor cell lines. COX-2 mRNA and protein were detected in two of three cell lines (SEG-1 and FLO); the third cell line, BIC-1, did not express COX-2 mRNA or protein under basal conditions or after stimulation with phorbol 12-myristate 13-acetate. Treatment with COX-1-selective concentrations of flurbiprofen did not affect cell growth in any of the three tumor cell lines. In contrast, treatment with COX-2-selective concentrations of NS-398 significantly suppressed cell growth and increased apoptosis in the cell lines that expressed COX-2 (SEG-1 and FLO), but not in the cell line that did not express COX-2 (BIC-1). We conclude that the administration of a selective inhibitor of COX-2 significantly decreases cell growth and increases apoptosis in Barrett’s-associated adenocarcinoma tumor cells that express COX-2. These observations suggest a potential role for selective COX-2 inhibitors in the prevention and treatment of esophageal adenocarcinoma for patients with Barrett’s esophagus.

Aspirin causes peptic ulcers predominately by reducing gastric mucosal cyclooxygenase (COX) activity and prostaglandin synthesis. Because aspirin circulates for only a few hours, we hypothesized that aspirin's inhibitory effect on gastric COX activity must be prolonged. We performed a placebo-controlled experiment in healthy humans to determine the duration of inhibition of aspirin on gastric mucosal COX activity (PGE(2) and PGF(2 alpha) synthesis rates). Recovery of gastric COX activity after stopping aspirin was slow and linear. Seventy-two hours after 325-mg aspirin, gastric COX activity was still reduced by 57% (P< 0.001). Duration of inhibition of gastric COX activity was estimated to be 7-8 days after 325-mg aspirin and 5 days after 81-mg aspirin. Recovery of gastric prostaglandin synthesis after 325-mg but not after 81-mg aspirin occurred at slower rates in subjects with Helicobacter pylori-associated gastritis than in those with normal histology. In conclusion, aspirin inhibits gastric COX activity for much longer than predicted from its pharmacokinetic profile, explaining why aspirin at widely spaced intervals is ulcerogenic.