Relationship between peroxisome proliferator-activated receptor-gamma expression and differentiation of human esophageal squamous cell carcinoma.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, is involved in suppressing the growth of several tumors. We showed that PPAR-gamma is expressed in Barrett's adenocarcinoma cell lines and inhibited the growth of these lines through the induction of G1 cell cycle arrest and apoptosis. We examined PPAR-gamma expression in human esophageal squamous cell carcinoma (SCC) in vitro and in vivo and investigated whether PPAR-gamma ligands affect the proliferation and apoptosis of human SCC cell lines. Biopsy specimens (n=46) obtained from human SCC of the esophagus were stained using a monoclonal antibody against human PPAR-gamma. We assessed the effects of PPAR-gamma ligands on the growth of SCC cells by adding 15-deoxy prostaglandin J2 (15d-PGJ2), or troglitazone to six human esophageal SCC cell lines (TE-1, TE-2, TE-3, TE-5, TE-8, and TE-9). Immunohistochemical staining showed that 34 of 46 (73.9%) SCC of the esophagus expressed PPAR-gamma. All SCC cell lines expressed PPAR-gamma mRNA and protein, especially when poorly differentiated (TE-2, TE-5, and TE-9). The PPAR-gamma ligands significantly and dose-dependently inhibited the proliferation of SCC lines, except for well-differentiated TE-1 and TE-3. Apoptosis was induced by 15d-PGJ2 (10 microM) in all tested SCC lines except TE-1, whereas troglitazone (50 microM) was marginally effective in only the TE-2 and TE-3 cell lines. The present findings suggest that PPAR-gamma could be a therapeutic target for treating squamous cell carcinoma of the esophagus, possibly through the induction of apoptosis.

Roles of epidermal growth factor and Na+/H+ exchanger-1 in esophageal epithelial defense against acid-induced injury.

Epidermal growth factor (EGF) is predominantly secreted by salivary glands and activates Na(+)/H(+) exchanger-1 (NHE-1), which regulates intracellular pH (pH(i)). We investigated the roles of EGF and NHE-1 in esophageal epithelial defense against acid using human esophageal epithelial cell lines and a rat chronic esophagitis model. Esophageal epithelial cells were incubated with acidified medium in the absence or presence of EGF. Cell viability and changes in pH(i) were measured. Chronic acid reflux esophagitis was induced in rats with and without sialoadenectomy. Esophageal lesion index, epithelial proliferation, and expression of EGF receptors and NHE-1 were examined. EGF protected esophageal epithelial cells against acid in a dose-dependent manner, and the cytoprotective effect of EGF was completely blocked by treatment with NHE-1 inhibitors. Tyrosine kinase, calmodulin, and PKC inhibitors significantly inhibited cytoprotection by EGF, whereas MEK, phosphatidylinositol 3-kinase, and PKA inhibitors had no effect. EGF significantly increased pH(i) recovery after NH(4)Cl pulse acidification, and this increase in pH(i) recovery was significantly blocked by inhibitors of calmodulin and PKC. Sialoadenectomy led to an increase in the severity of chronic esophagitis but affected neither epithelial proliferation nor expression of EGF receptors. Expression of NHE-1 mRNA was increased in esophagitis and upregulated in rats with sialoadenectomy. The increasing severity of esophagitis in rats with sialoadenectomy was prevented by exogenous administration of EGF. In conclusion, EGF protects esophageal epithelial cells against acid through NHE activation via Ca(2+)/calmodulin and the PKC pathway. Deficiency in endogenous EGF is associated with increased severity of esophagitis. EGF and NHE-1 play crucial roles in esophageal epithelial defense against acid.

Rebamipide prevents delay of acetic acid-induced gastric ulcer healing caused by Helicobacter pylori infection in Mongolian gerbils.

In this study, we examined the effect of rebamipide, a mucoprotective drug, on gastric ulcer healing in Mongolian gerbils infected with H. pylori. Male Mongolian gerbils were inoculated with H. pylori or vehicle alone 12 hr after the production of an acetic acid-induced gastric ulcer. On day 5, the gerbils inoculated with H. pylori were divided into three groups and fed rebamipide-containing diet (0.038%, 60 mg/kg, or 0.0038%, 6 mg/kg), or standard laboratory chow. The gerbils inoculated with the vehicle were fed standard laboratory chow throughout the experiment. The gerbils were killed on day 5, 15, or 30 after ulcer production, and removed stomachs were subjected to calculation of ulcer size, culture for H. pylori, and measurement of myeloperoxidase activity, a marker for neutrophil infiltration, in ulcerated tissue. Apoptotic and proliferating cells of gastric epithelium in ulcer margins were detected by the in situ DNA nick end-labeling method and immunohistochemical staining for 5-bromo-2'-deoxyuridine (BrdU), respectively. Rebamipide did not affect colonization levels of H. pylori. Infection with H. pylori did not affect ulcer size by day 5 but significantly delayed ulcer healing by days 15 and 30, accompanied by an increase in the number of apoptotic cells, a decrease in the number of BrdU-positive cells, and an increase in myeloperoxydase activity. Rebamipide prevented delay of ulcer healing and abolished these effects of H. pylori on cell kinetics and neutrophil infiltration. In conclusion, rebamipide may prevent the delay of acetic acid-induced gastric ulcer healing caused by H. pylori through modulating cell kinetics and inhibiting neutrophil infiltration.

Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha.

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.

Increased expression of transforming growth factor-alpha and epidermal growth factor receptors in rat chronic reflux esophagitis.

BACKGROUND AND AIM: Transforming growth factor-alpha (TGF-alpha), which binds to epidermal growth factor receptors (EGF-R), stimulates esophageal epithelial cell proliferation, enabling rapid repair after mucosal injury. The aim of the present study was to examine epithelial proliferation and dynamics of TGF-alpha and EGF-R gene and protein expression in rat chronic acid reflux esophagitis. METHODS: Gastric acid reflux esophagitis was induced in Wistar rats by ligating the transitional region between the forestomach and the glandular portion, and by covering the duodenum near the pyloric ring with a small piece of an 18Fr Nélaton catheter. Epithelial cell proliferation was assessed by bromodeoxyuridine (BrdU) uptake. Expression of TGF-alpha and EGF-R mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Esophageal lesions were observed in the lower and middle esophagus. Histologically, a significant increase in mucosal thickening with elongation of lamina propria papillae and basal cell hyperplasia was observed. The BrdU labeling index was significantly increased from 2.7 +/- 1.0 in normal mucosa and 2.8 +/- 1.2 in background mucosa adjacent to the esophageal lesion, to 60.3 +/- 32.7 in the lesions of chronic esophagitis. Expression of TGF-alpha and EGF-R mRNA in the esophageal lesion significantly increased compared to those in the control and background tissue, whereas treatment with rabeprazole significantly inhibited increases in TGF-alpha and EGF-R mRNA expression. According to immunohistochemical study, TGF-alpha and EGF-R revealed strong expression in esophageal lesions compared with control and background mucosa. The superficial layer of the esophagus was strongly positive for TGF-alpha and most cells in regions of basal hyperplasia had a positive reaction for EGF-R in the esophagitis lesion. CONCLUSION: Epithelial proliferation and expression of TGF-alpha and EGF-R were significantly increased in rat chronic reflux esophagitis. Activation of TGF-alpha and EGF-R genes in response to acid reflux may facilitate rapid mucosal healing by stimulating epithelial proliferation. These results suggest that TGF-alpha and EGF-R play crucial roles in rat chronic reflux esophagitis.

Increased expression of cytokines and adhesion molecules in rat chronic esophagitis.

BACKGROUND/AIMS: Cytokines and adhesion molecules regulate many inflammatory processes in several gastrointestinal diseases. The dynamics of cytokines and adhesion molecules in reflux esophagitis are unknown in detail. We examined the expression and dynamics of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2, GRO/cytokine-induced neutrophil chemoattractant-2alpha (CINC-2alpha), intercellular adhesion molecule-1 (ICAM-1), leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18), and Mac-1 (CD11b/CD18) in rat chronic reflux esophagitis. METHODS: Chronic acid reflux esophagitis was induced in Wistar rats by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus with a small piece of an 18-Fr Nélaton catheter. Rats were killed 3 or 21 days after operation. The levels of mRNA expression of cytokines and ICAM-1 were determined by real-time quantitative RT-PCR. Localization of adhesion molecules and cytokines was investigated by immunohistochemical staining, and numbers of LFA-1- or Mac-1-positive cells were quantified. RESULTS: IL-1beta, TNF-alpha, MCP-1, MIP-1alpha, MIP-2, CINC-2alpha, and ICAM-1 mRNA expression was significantly increased in esophageal lesions compared with normal esophagus. There were few these cytokines- or adhesion molecule-positive cells in normal esophagus. In regions of esophagitis, numerous inflammatory leukocytes in lamina propria and the submucosal layer exhibited positive reactions for these cytokines and endothelial cells were intensely stained for ICAM-1. Numbers of LFA-1- and Mac-1-positive cells were significantly increased in rat chronic esophagitis. Treatment with rabeprazole almost completely inhibited development of chronic acid reflux esophagitis and significantly decreased expression of cytokines and ICAM-1 mRNA in esophageal tissue compared with control. CONCLUSION: Cytokines and adhesion molecules play important roles in the pathogenesis of chronic reflux esophagitis in this rat model.

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2.

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.

In vivo action of novel alkyl methyl quinolone alkaloids against Helicobacter pylori.

Previously purified and isolated compounds of novel alkyl methyl quinolone alkaloids (AM quinolones) from Gosyuyu (Wu-Chu-Yu), a Chinese herbal medicine, have a strong and highly selective antibacterial activity against Helicobacter pylori in vitro. To clarify the antibacterial mechanism(s) of AM quinolones, we examined the effects of AM quinolones on respiration of H. pylori in vitro. One week after treatment with AM quinolones alone (2, 10 or 20 mg/kg/day, orally) or combinations of AM quinolones and omeprazole (30 mg/kg/day) for H. pylori (1 x 10(8) cfu)-infected Mongolian gerbils, we checked viable H. pylori and myeloperoxidase (MPO) activity in the gastric tissues. AM quinolones decreased the number of H. pylori and inhibited H. pylori respiration in a dose-dependent manner. AM quinolones decreased the number of viable H. pylori (AM quinolones alone: 46.0 +/- 22.6 x 10(4), 17.3 +/- 4.9 x 10(4) and 8.1 +/- 6.6 x 10(4) cfu/stomach, respectively; and combinations of AM quinolones and omeprazole: 8.0 +/- 5.6 x 10(4), 4.2 +/- 2.5 x 10(4) and 5.5 +/- 2.7 x 10(4) cfu/stomach) compared with the vehicle-treated group (control: 359.9 +/- 180.3 x 10(4) cfu/stomach). AM quinolones significantly decreased MPO activity of H. pylori-inoculated gastric tissues. These findings suggest that AM quinolones have a potent antibacterial effect against H. pylori through respiratory inhibition, and reduced bacterial growth in vivo. AM quinolones might be novel therapeutic agents for H. pylori eradication.

Safety and efficacy of S-1, a novel oral fluorouracil antitumor drug, for a chronic renal failure patient maintained on hemodialysis.

OBJECTIVE: S-1 is a novel oral fluorouracil antitumor drug that combines three pharmacological agents: tegafur (FT), a prodrug of 5-fluorouracil (5-FU); 5-chloro-2,4-dihydroxypyridine (CDHP), an inhibitor of dihydropyrimidine dehydrogenase, and potassium oxonate (Oxo), a reducer of gastrointestinal toxicity. S-1 has safe and potent antitumor effects in patients with gastric cancer via these respective functions. However, the plasma 5-FU concentration is suspected to accumulate in patients with renal dysfunction, because 50% of the CDHP is excreted into the urine. There are no useful data on safety and efficacy of S-1 in chronic renal failure patients maintained on hemodialysis (HD). We examined the influence of HD on the pharmacokinetics (PK) of S-1 and its therapeutic efficacy in liver metastases from gastric cancer. METHODS: For the HD patient, the dose of S-1 in a single-administration study was set at 50 mg/body/day (41.7% of the recommended dose of 80 mg/m2/day). S-1 was given to the patient 24 h after HD. Blood samples were obtained before administration and 2, 4, 6, 8, and 24 h thereafter and 1, 2, 4, and 72 h after the following HD session. The PK parameters (5-FU, CDHP, Oxo, and FT) were measured, and Cmax, Tmax, AUC0-24, and T1/2 were calculated. The dose of consecutive or maintained administrations was determined. RESULTS: Both an increase in Cmax and an elongation of T1/2 for 5-FU, CDHP, and Oxo, but not for FT, occurred in this case as compared with controls. The AUC0-24 of 5-FU in this case was similar to that of controls at the standard dose. After HD, 87.8, 54.5, 77.4, and 66.2% of 5-FU, CDHP, Oxo, and FT, respectively, were eliminated. A slight accumulation of CDHP did not alter the 5-FU PK. Consecutive or maintained S-1 oral administration at the same dose showed similar effects on all PK parameters of a single-administration test. Liver metastases almost totally regressed with no adverse events 4 weeks after S-1 treatment (50 mg/body/day three times a week). CONCLUSION: Adjusted doses of S-1 according to the results of PK studies may provide therapeutic safety and high efficacy in liver metastases from gastric cancer, even in chronic renal failure patients maintained on HD.