Rat Gastric Epithelial Cells Research Articles

A Novel Cancer Therapy Using Proton Beam: An Integrated Proton-Dynamic Therapy, iPDT

[Background and Aims]A super-aged society, the prime example of which is Japan, is being flooded with increasing in numerous numbers of aged cancer patients, who often take anti-coagulant medicines to prevent the attacks of ischemic heart diseases and/ or strokes. Since they show bleeding tendency induced by the medicines, it’s hard for them to receive surgical operations. Thus, low invasive cancer treatments without bleedings are abundantly desired. In this study, the applicant will establish a new proton therapy, in which a resonance nuclear reaction (RNR) induced by collision of a proton with a stable nitrogen isotope (15N), can be a clue to injure cancer-cells specifically. The LET of the product ions (12C6+, 4He2+) of the 15N RNR is above 50 times in comparison with the LET of proton. To establish the cancer-specific 15N delivery, 15N-aminolevulinic acid (15N-ALA) will be used. This reagent has been already used and obtained regulatory approvals as a diagnostic medicine for the location of the glioblastoma in Japan.[Methods]In this study, a pair of rat gastric epithelial cells, one is normal cell RGM1, and another is cancerous characteristics cells RGK1 were used. The authors treated these cells with both 1mM 15N-ALA and 14N-ALA. Thereafter, we measured and compared porphyrin fluorescent intensity of these cells. Moreover, these cells will be irradiated with 4 nc proton beam generated by an electrostatic accelerator, then 4.43 MeV gamma-rays will be measured to demonstrate the amount of 15N collected specifically in the tumour cells. After these proton irradiations, cells were stained with hoechest33258 and dapi to observe cellular apoptosis.[Results] Cancer cells accumulated 9 folds of 15N in comparison with normal epithelial cells. After 3hours of proton irradiation, both cancer cells and normal cells were not injured. However, almost all cancer cells were died with apoptosis while normal cells showed few cellular injuries after 24 hours.[Conclusions] Cancer cells were completely killed by RNR involved with imM 15N-ALA and 4nc proton irradiation whereas this dose of proton showed few effects of ionizing radiation on normal cells. Thus, iPDT should be an ideal treatment for cancer. Moreover, the quantitative analysis of 15N in the tumour cells can be a new pre-diagnosis for iPDT.

Polphylipoprotein-induced autophagy mechanism with high performance in photodynamic therapy

Polphylipoprotein (PLP) is a recently developed nanoparticle with high biocompatibility and tumor selectivity, and which has demonstrated unprecedentedly high performance photosensitizer in photodynamic therapy (PDT) and photodynamic diagnosis. On the basis of these discoveries, PLP is anticipated to have a very high potential for PDT. However, the mechanism by which PLP kills cancer cells effectively has not been sufficiently clarified. To comprehensively understand the PLP-induced PDT processes, we conduct multifaceted experiments using both normal cells and cancer cells originating from the same sources, namely, RGM1, a rat gastric epithelial cell line, and RGK1, a rat gastric mucosa-derived cancer-like mutant. We reveal that PLP enables highly effective cancer treatment through PDT by employing a unique mechanism that utilizes the process of autophagy. The dynamics of PLP-accumulated phagosomes immediately after light irradiation are found to be completely different between normal cells and cancer cells, and it becomes clear that this difference results in the manifestation of the characteristic effect of PDT when using PLP. Since PLP is originally developed as a drug delivery agent, this study also suggests the potential for intracellular drug delivery processes through PLP-induced autophagy.

A New Cancer Therapy with a Resonance Nuclear Reaction between 15n-5-ALA and Proton Beam: iPDT

A super-aged society, the prime example of which is Japan, is being flooded with increasing in numerous numbers of aged cancer patients, who often take anti-coagulant medicines to prevent the attacks of ischemic heart diseases and/ or strokes. Since they have diseases with bleeding tendency induced by the medicines, it’s hard for them to receive surgical operations. Thus, low invasive cancer treatments without bleedings are abundantly desired. In this study, the applicant will establish a new proton therapy, in which a resonance nuclear reaction (RNR) induced by collision of a proton with a stable nitrogen isotope (15N), can be a clue to injure cancer-cells specifically. The LET of the product ions (12C6+, 4He2+) of the 15N RNR is above 50 times in comparison with the LET of proton. To establish the cancer-specific 15N delivery, 15N-aminolevulinic acid (15N-ALA) will be used. This reagent has been already used and obtained regulatory approvals as a diagnostic medicine for the location of the glioblastoma in Japan.In this study, a pair of rat gastric epithelial cells, one is normal cell, and another is cancerous characteristics cells were used, 15N-ALA treated porphyrin fluorescent intensity of these cells will be compared with 14N-ALA treatment. The cells will be irradiated with a proton beam generated by an electrostatic accelerator and 4.43 MeV gamma-rays will be measured to demonstrate the amount of 15N collected specifically in the tumour cells. Such quantitative analysis of 15N in the tumour cells can be a new pre-diagnosis of iPDT.

Abstract 1106: A novel cancer therapy using proton beam; an integrated proton dynamic therapy, iPDT

Abstract A super-aged society, the prime example of which is Japan, is being flooded with increasing in numerous numbers of aged cancer patients, who often take anti-coagulant medicines to prevent the attacks of ischemic heart diseases and/or strokes. Since they show bleeding tendency induced by the medicines, it’s hard for them to receive surgical operations. Thus, low invasive cancer treatments without bleedings are abundantly desired. In this study, the applicant will establish a new proton therapy, in which a resonance nuclear reaction (RNR) induced by collision of a proton with a stable nitrogen isotope (15N), can be a clue to injure cancer-cells specifically. The LET of the product ions (12C6+, 4He2+) of the 15N RNR is above 50 times in comparison with the LET of proton. To establish the cancer-specific 15N delivery, 15N-aminolevulinic acid (15N-ALA) will be used. This reagent has been already used and obtained regulatory approvals as a diagnostic medicine for the location of the glioblastoma in Japan. In this study, a pair of rat gastric epithelial cells, one is normal cell, and another is cancerous characteristics cells were used. 15N-ALA treated porphyrin fluorescent intensity of these cells will be compared with 14N-ALA treatment. The cells will be irradiated with a proton beam generated by an electrostatic accelerator and 4.43 MeV gamma-rays will be measured to demonstrate the amount of 15N collected specifically in the tumor cells. Such quantitative analysis of 15N in the tumor cells can be a new pre-diagnosis of iPDT. Citation Format: HIrofumi Matsui. A novel cancer therapy using proton beam; an integrated proton dynamic therapy, iPDT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1106.

ZINC40099027 Promotes Gastric Mucosal Repair in Ongoing Aspirin-Associated Gastric Injury by Activating Focal Adhesion Kinase.

Nonsteroidal anti-inflammatory drugs cause gastric ulcers and gastritis. No drug that treats GI injury directly stimulates mucosal healing. ZINC40099027 (ZN27) activates focal adhesion kinase (FAK) and heals acute indomethacin-induced small bowel injury. We investigated the efficacy of ZN27 in rat and human gastric epithelial cells and ongoing aspirin-associated gastric injury. ZN27 (10 nM) stimulated FAK activation and wound closure in rat and human gastric cell lines. C57BL/6J mice were treated with 300 mg/kg/day aspirin for five days to induce ongoing gastric injury. One day after the initial injury, mice received 900 µg/kg/6 h ZN27, 10 mg/kg/day omeprazole, or 900 µg/kg/6 h ZN27 plus 10 mg/kg/day omeprazole. Like omeprazole, ZN27 reduced gastric injury vs. vehicle controls. ZN27-treated mice displayed better gastric architecture, with thicker mucosa and less hyperemia, inflammation, and submucosal edema, and lost less weight than vehicle controls. Gastric pH, serum creatinine, serum alanine aminotransferase (ALT), and renal and hepatic histology were unaffected by ZN27. Blinded scoring of pFAK-Y-397 immunoreactivity at the edge of ZN27-treated lesions demonstrated increased FAK activation, compared to vehicle-treated lesions, confirming target activation in vivo. These results suggest that ZN27 ameliorates ongoing aspirin-associated gastric mucosal injury by a pathway involving FAK activation. ZN27-derivatives may be useful to promote gastric mucosal repair.

The gastroprotective effect of alpinia officinarum extract on indomethacin-induced topical injuries in RGM-1 Cells: Involvement of H+/K+-ATPase- and mitochondrial-mediated apoptosis

Background: Topical effects are essential mechanisms of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced gastric damage. Our previous study showed that the extract of Alpinia officinarum (AOE) may have some protective effects against the topical effects of indomethacin (INDO). The aim of this study was to elucidate the protective effects and mechanisms of A. officinarum against INDO-induced topical injuries to gastric mucosa. Materials and Methods: 0.5 mM INDO was used to cause topical effects to rat gastric epithelial cells (RGM-1). Meanwhile, AOE (2.5 μg/mL) and galangin (GAL) (0.05 mM) were added, respectively, to explore their protective effects. The cell proliferation, mitochondrial viability, and mitochondrial-mediated apoptosis were assessed by flow cytometry, inverted fluorescence microscope, or microplate reader. Pro- and cleaved-caspase-3 were detected by Western blot method. Results: AOE and GAL could significantly protect INDO-damaged RGM-1 cells by promoting cell proliferation, upregulating mitochondrial viability, inhibiting mitochondrial cytochrome c release into cytoplasm, inhibiting lipid peroxidation and caspase-3 activity, and suppressing H+/K+-ATPase activity. Conclusion: The gastroprotective effects of AOE and GAL were closely associated with suppressing the gastric acid secretion and restraining mitochondrial-mediated apoptosis. These data provided new perceptions into interpreting the underlying mechanisms of gastroprotective effects of A. officinarum and showed a promising clinical use in treating gastric mucosal injury induced by NSAIDs.

NSAID-induced injury of gastric epithelial cells is reversible: roles of mitochondria, AMP kinase, NGF, and PGE2.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1-4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR.NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.

Mitochondria in gastric epithelial cells are the key targets for NSAIDs-induced injury and NGF cytoprotection.

Gastric epithelial cells are important components of mucosal protection and targets of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced injury. Diclofenac (DFN) is one of the most widely used NSAIDs; however, even its short-term use can induce gastric erosions and ulcers. Nerve growth factor (NGF) has been reported to act not only on neuronal cells but also on endothelial cells; however, its action on gastric epithelial cells is unknown. This study was aimed to determine, whether NGF can protect gastric epithelial cells against DFN-induced injury, and to determine the underlying molecular mechanisms with a focus on mitochondria, survivin, and insulin-like growth factor 1 (IGF-1). Cultured normal rat gastric mucosal epithelial cells 1 (RGM1) were treated with phosphate-buffered saline (PBS; control), NGF (100 ng/mL) and/or DFN (0.25-1.00 mM) for 4 hours. We examined: (1) cell injury by confocal microscopy; (2) cell death/survival using Calcein AM live cell tracking dye; (3) mitochondrial structure and membrane potential function using MitoTracker in live cells; and (4) expression of NGF, its receptor - tropomyosin receptor kinase A (TrkA), survivin and IGF-1 by immunostaining. DFN treatment of RGM1 cells for 4 hours caused extensive cell injury, mitochondrial disintegration, reduced cell viability (from 94 ± 3% in controls to 14 ± 4% in 0.5 mM DFN-treated cells; P < 0.001), and expression of survivin and IGF-1. NGF treatment significantly increased survivin and IGF-1 expression by 41% and 75%, respectively versus PBS controls. Pretreatment with NGF before DFN treatment reduced mitochondrial damage and cell death by 73% and 82%, respectively versus treatment with DFN alone (all P < 0.001). This study also showed the presence of high-affinity TrkA receptors in the plasma membrane and mitochondria of RGM1 cells indicating novel actions of NGF.

Role of Helicobacter pylori infection in cancer-associated fibroblast-induced epithelial-mesenchymal transition in vitro.

BackgroundMajor human gastrointestinal pathogen Helicobacter pylori (H. pylori) colonizes the gastric mucosa causing inflammation and severe complications including cancer, but the involvement of fibroblasts in the pathogenesis of these disorders in H. pylori‐infected stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts. Their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue; however, the interaction between H. pylori and fibroblasts remains unknown. We determined the effect of coincubation of normal rat gastric fibroblasts with alive H. pylori (cagA+vacA+) and H. pylori (cagA−vacA−) strains on the differentiation of these fibroblasts into cells possessing characteristics of cancer‐associated fibroblasts (CAFs) able to induce epithelial‐mesenchymal transition (EMT) of normal rat gastric epithelial cells (RGM‐1).Materials and MethodsThe panel of CAFs markers mRNA was analyzed in H. pylori (cagA+vacA+)‐infected fibroblasts by RT‐PCR. After insert coculture of differentiated fibroblasts with RGM‐1 cells from 24 up to 48, 72, and 96 hours, the mRNA expression for EMT‐associated genes was analyzed by RT‐PCR.ResultsThe mRNA expression for CAFs markers was significantly increased after 72 hours of infection with H. pylori (cagA+vacA+) but not H. pylori (cagA−vacA−) strain. Following coculture with CAFs, RGM‐1 cells showed significant decrease in E‐cadherin mRNA, and the parallel increase in the expression of Twist and Snail transcription factors mRNA was observed along with the overexpression of mRNAs for TGFβR, HGFR, FGFR, N‐cadherin, vimentin, α‐SMA, VEGF, and integrin‐β1.Conclusion Helicobacter pylori (cagA+vacA+) strain induces differentiation of normal fibroblasts into CAFs, likely to initiate the EMT process in RGM‐1 epithelial cell line.

Autophagy impairment byHelicobacter pylori-induced methylation silencing ofMAP1LC3Av1promotes gastric carcinogenesis

Helicobacter pylori (H. pylori) infection induces methylation silencing of tumor suppressor genes causing gastric carcinogenesis. Impairment of autophagy induces DNA damage leading to genetic instability and carcinogenesis. We aimed to identify whether H. pylori infection induced methylation silencing of host autophagy-related (Atg) genes, impairing autophagy and enhancing gastric carcinogenesis. Gastric mucosae were obtained from 41 gastric cancer patients and 11 healthy volunteers (8 H. pylori-uninfected and 3 H. pylori-infected). Methylation status of Atg genes was analyzed by a methylation microarray and quantitative methylation-specific PCR (qMSP); mRNA expression was assessed by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation, migration and invasion were assessed in normal rat gastric epithelial cells. Gene knock-down was performed by siRNA. Autophagy was assessed by western blotting. Of 34 Atg genes, MAP1LC3A variant 1 (MAP1LC3Av1) and ULK2 were identified by methylation microarray analysis as exhibiting specific methylation in H. pylori-infected mucosae and gastric cancer tissues. Methylation silencing of MAP1LC3Av1 was confirmed by qMSP, qRT-PCR and de-methylation treatment in two gastric cancer cell lines. Knock-down of map1lc3a, the rat homolog of the human MAP1LC3Av1, inhibited autophagy response and increased cell proliferation, migration and invasion in normal rat gastric epithelial cells, despite the presence of map1lc3b, the rat homolog of the human MAP1LC3B gene important for autophagy. Furthermore, MAP1LC3Av1 was methylation-silenced in 23.3% of gastric cancerous mucosae and 40% of non-cancerous mucosae with H. pylori infection. MAP1LC3Av1 is essential for autophagy and H. pylori-induced methylation silencing of MAP1LC3Av1 may impair autophagy, facilitating gastric carcinogenesis.

421 Development and Validation of a NBI Classification System for the Prediction of Dysplasia in Barrett's Esophagus (BE): Consensus Results From an International Working Group

Background/Purpose: Visceral hypersensitivity for acid, extension and temperature stimuli in stomach and duodenum are involved in functional dyspepsia (FD). While the involvement of TRPV1 and TRPA1 have been reported in a number of animal experiments, data remains conflicting. On the other hand, ATP-gated ion channels, especially P2X3 receptor have recently been worthy of attention for its role in visceral hypersensitivity. We have reported that TRPV4 is expressed in esophageal, gastric and intestinal epithelia (J Physiol 2011, DDW2013, Am J Physiol 2013) and that ATP exocytosis is induced by TRPV4 activation in esophageal keratinocytes. We hypothesized that various physiological stimuli induced ATP release from gastric and duodenal (intestinal) epithelia through each different receptor and that excessive ATP release was involved in the pathophysiology of visceral hypersensitivity. METHODS: TRPV4 expression was examined in normal rat gastric epithelial cell line (RGE), human gastric cancer cell line (AGS) and rat intestinal cell line (IEC-6) by RT-PCR, immunostaining or Western blotting. ATP releases responding to various physiological stimuli (chemicals, low osmotic pressure, acid, extension, temperature) from each cell line were examined using luciferin-luciferase reaction. Acidic (pH 4.0), warm (40 °C) and standard (25 °C, pH7.4) solutions were used as acidic, warm stimuli or a control, respectively. Stretch stimuli was applied to cells cultured on a silicon chamber using a stretching apparatus (STREX Inc, Osaka, Japan). RESULTS: TRPV4 was expressed in RGE, IEC-6 but not in AGS. Specific TRPV4 agonist (GSK1016790A) or endogenous TRPV4 activator (5,6-EET) significantly enhanced ATP release from RGE cells but not AGS. Low osmotic, acidic, stretch or temperature stimuli significantly enhanced ATP release in RGE or IEC-6 cells. Stretchinduced ATP release from RGE cells was inhibited by pretreatment with a specific TRPV4 inhibitor, HC067047. Cell viability after each stimulus was confirmed by Trypan blue staining. Conclusion: Gastric and duodenal (intestinal) epithelial cells release ATP responding various physiological stimuli partially via TRPV4 activation . Excessive ATP release from epithelial cells might be involved in the pathophysiology of visceral hypersensitivity.

420 Quantitative Analysis of Volumetric Laser Endomicroscopy of Histologically Correlated Images Potentially Identifies Early Neoplasia in Barrett's Esophagus

Background/Purpose: Visceral hypersensitivity for acid, extension and temperature stimuli in stomach and duodenum are involved in functional dyspepsia (FD). While the involvement of TRPV1 and TRPA1 have been reported in a number of animal experiments, data remains conflicting. On the other hand, ATP-gated ion channels, especially P2X3 receptor have recently been worthy of attention for its role in visceral hypersensitivity. We have reported that TRPV4 is expressed in esophageal, gastric and intestinal epithelia (J Physiol 2011, DDW2013, Am J Physiol 2013) and that ATP exocytosis is induced by TRPV4 activation in esophageal keratinocytes. We hypothesized that various physiological stimuli induced ATP release from gastric and duodenal (intestinal) epithelia through each different receptor and that excessive ATP release was involved in the pathophysiology of visceral hypersensitivity. METHODS: TRPV4 expression was examined in normal rat gastric epithelial cell line (RGE), human gastric cancer cell line (AGS) and rat intestinal cell line (IEC-6) by RT-PCR, immunostaining or Western blotting. ATP releases responding to various physiological stimuli (chemicals, low osmotic pressure, acid, extension, temperature) from each cell line were examined using luciferin-luciferase reaction. Acidic (pH 4.0), warm (40 °C) and standard (25 °C, pH7.4) solutions were used as acidic, warm stimuli or a control, respectively. Stretch stimuli was applied to cells cultured on a silicon chamber using a stretching apparatus (STREX Inc, Osaka, Japan). RESULTS: TRPV4 was expressed in RGE, IEC-6 but not in AGS. Specific TRPV4 agonist (GSK1016790A) or endogenous TRPV4 activator (5,6-EET) significantly enhanced ATP release from RGE cells but not AGS. Low osmotic, acidic, stretch or temperature stimuli significantly enhanced ATP release in RGE or IEC-6 cells. Stretchinduced ATP release from RGE cells was inhibited by pretreatment with a specific TRPV4 inhibitor, HC067047. Cell viability after each stimulus was confirmed by Trypan blue staining. Conclusion: Gastric and duodenal (intestinal) epithelial cells release ATP responding various physiological stimuli partially via TRPV4 activation . Excessive ATP release from epithelial cells might be involved in the pathophysiology of visceral hypersensitivity.

Acetic acid induces cell death: an in vitro study using normal rat gastric mucosal cell line and rat and human gastric cancer and mesothelioma cell lines.

We recently reported that topical application of acetic acid promptly caused tumor necrosis in a mouse model of gastric cancer. The aim of the present study was to examine whether acetic acid can directly induce cancer cell death. Rat gastric epithelial cell line (RGM-1), rat gastric carcinoma cell line (RGK-1), human gastric cancer cell line (KATO III), and human mesothelioma cell lines (ACC-MESO1 and MSTO-211H) were used. Acetic acid was added into the cell culture at different concentrations for different time periods. Cell death was analyzed by MTT assay, flow cytometry, and trypan blue exclusion test. Acetic acid promptly induced the cell death of RGM-1, RGK-1 cells, and KATO III cells in a concentration-dependent manner from 0.01% to 0.5%. Acetic acid at 0.5% for 1 min induced the cell death by 80%. RGK-1 cells were more sensitive to acetic acid than RGM-l cells. KATO III cells were more sensitive to acetic acid than RGK-1 cells. Acetic acid at 0.5% for 10 min induced almost complete cell death of ACC-MESO1 and MSTO-211H. Acetic acid is a powerful anticancer agent. Topical application of acetic acid may be a feasible approach for the treatments of gastric cancer and possibly other malignancies.

Acetaldehyde is an oxidative stressor for gastric epithelial cells.

Alcohol drinking and smoking contain the risk of a carcinogenesis. Acetaldehyde is content in cigarette smoke and an ethanol metabolite. However the clear evidence for reactive oxygen species (ROS) generation by acetaldehyde in gastric cells in vitro is none. In this study, we elucidated acetaldehyde is an oxidative stress inducer on rat gastric epithelial cells by electron paramagnetic resonance measurement in living cells. We also confirmed whether acetaldehyde-induced cellular ROS was derived from mitochondria or not. The results of cellular ROS determination showed that an increment of cellular ROS was shown for 15 min in living cells from exposing 0.1% (v/v) acetaldehyde. Lipid peroxidation in cellular membrane also induced by 0.1% ethanol and the tendency is same in the results of cellular ROS determination. JC-1 stained showed the decrement of mitochondrial membrane potential. These results indicated that acetaldehyde is not merely a necrotizing factor for gastric epithelial cells, but also an oxidative stress inducer via injured mitochondria.

Alcohol is an oxidative stressor for gastric epithelial cells: detection of superoxide in living cells

Alcohol/ethanol has been reported to derived necrosis and apoptosis with an oxidative stress in gastric mucosal cells. However the clear evidence for reactive oxygen species (ROS) generation by alcohol in gastric cells in vitro is none. In this study, we elucidated ethanol is an oxidative stress inducer on rat gastric epithelial cells by electron paramagnetic resonance measurement in living cells. We also confirmed whether ethanol-induced cellular ROS was derived from mitochondria or not. The results of cellular ROS determination showed that an increment of cellular ROS was shown for 15 min from exposing 1% (v/v) ethanol. Lipid peroxidation in cellular membrane also induced by 1% ethanol and the tendency is same in the results of cellular ROS determination. JC-1 stained showed the decrement of mitochondrial membrane potential. Additionally the localization of cellular ROS coincided with mitochondria. These results indicated that ethanol is not merely a necrotizing factor for gastric epithelial cells, but also an oxidative stress inducer via injured mitochondria.

TFF1 is Differentially Expressed in Stationary and Migratory Rat Gastric Epithelial Cells (RGM-1) after in Vitro Wounding: Influence of TFF1 RNA Interference on Cell Migration

Background/Aims: The trefoil factor family (TFF) peptide TFF1 is typically secreted by gastric surface mucous cells. These cells are also the major players in gastric restitution, i.e., repair of the stomach mucosa by cell migration after injury. Methods: An established in vitro model of gastric restitution, i.e., migration of the non-transformed rat gastric cell line RGM-1 after scratch wounding, was investigated by expression profiling of selected genes from separated stationary and migratory cells. Also semi-quantitative immunocytochemistry was performed. Furthermore, RGM-1 cells were transfected with stealth RNAi™ duplexes targeting Tff1 and relative cell migration rates were analyzed. Results: Surprisingly, Tff1 expression was up-regulated in migratory cells. No unequivocal signs of the epithelial-mesenchymal transition were detectable in migratory cells. Transfection of RGM-1 cells with Tff1-siRNAi duplexes negatively influenced migration of these cells. Conclusion: This clearly points to a function of Tff1 as a motogen. A possible up-regulation of TFF1 synthesis in migratory surface mucous cells also in vivo would be an ideal mechanism to specifically enhance gastric restitution only where topologically needed and to minimize eventual negative side effects of TFF1 such as cell scattering and invasiveness.

Clopidogrel delays gastric ulcer healing in rats

Clopidogrel is not safe enough for the gastric mucosa in patients with high risk of peptic ulcer. This study aimed to explore if clopidogrel delays gastric ulcer healing and elucidate the involved mechanisms. Gastric ulcer was induced in rats and the ulcer size, mucosal epithelial cell proliferation of the ulcer margin, expression of growth factors [epidermal growth factor (EGF), basic fibroblast growth factor] and their receptors, and signal transduction pathways for cell proliferation were measured and compared between the clopidogrel-treated group and untreated controls. For the in vitro part, rat gastric mucosal epithelial cell line (RGM-1 cells) was used to establish EGF receptor over-expressed cells. Cell proliferation and molecular change under EGF treatment (10ng/ml) with and without clopidogrel (10−6M) were demonstrated. Ulcer size was significantly larger in the clopidogrel-treated group compared to the control and mucosal epithelial cell proliferation of the ulcer margin was significantly decreased in the clopidogrel-treated group (P<0.05). Clopidogrel (2mg and 10mg/kg/day) significantly decreased ulcer-induced gastric epithelial cell proliferation and ulcer-stimulated expressions of EGF receptor and phosphorylated extracellular signal-regulated kinase (PERK) at the ulcer margin (P<0.05). Clopidogrel (10−6M) also inhibited EGF-stimulated EGF receptor, PERK expression, and cell proliferation in RGM-1 cells (P<0.05), and caused much less inhibition of EGF-stimulated cell proliferation in EGF receptor over-expressed RGM-1 cells than in RGM-1 cells (22% vs. 32% reduction). In conclusion, clopidogrel delays gastric ulcer healing in rats via inhibiting gastric epithelial cell proliferation, at least by inhibition of the EGF receptor-ERK signal transduction pathway.

Transmural Pressure Loading Enhances Gastric Mucosal Cell Proliferation

Although increased intraluminal pressure in the stomach due to gastric outlet obstruction or functional gastric motor dysfunction, including gastroparesis, may affect gastric mucosal integrity, the direct effect of mechanical pressure on gastric mucosal cells has not yet been fully investigated. The aims of this study were to determine whether exposure to transmural pressure would affect the proliferation of gastric mucosal cells and to elucidate the intracellular signaling pathways involved. Cellular proliferation and DNA synthesis were evaluated in rat gastric epithelial cells exposed to high transmural pressures. The levels of activation of 3 MAP kinases, ERK, JNK, and p38, were assessed, and the induction of immediate early gene expression was examined. The activation of nuclear factor activator protein-1 (AP-1) was evaluated by an electrophoretic mobility shift assay. Exposure to high transmural pressure significantly increased DNA synthesis within 24 h, with the most marked increase observed after exposure to a pressure of 80 mmHg, and this increase was inhibited by the MEK1 inhibitor PD98059. Early activation of ERK kinase, but not of JNK or p38 kinase, was detected after pressure loading. Early induction of the c-fos and c-myc genes and activation of the AP-1 transcription factor were also demonstrated within 3 h of exposure to 80 mmHg of pressure. Gastric mucosal cell proliferation induced by exposure to high transmural pressure may be related to early activation of ERK, the induction of c-fos and c-myc, and the activation of AP-1.

Sulindac sulfide induces autophagic death in gastric epithelial cells via Survivin down-regulation: A mechanism of NSAIDs-induced gastric injury

Sulindac sulfide, a nonsteroidal anti-inflammatory drug (NSAID), has anti-tumorigenic and anti-inflammatory activities, but causes gastric mucosal damage. NSAIDs cause gastric injury in part by down-regulation of Survivin, an apoptosis inhibitor, resulting in apoptosis induction. Autophagy is a process that promotes cellular health by destroying unwanted cellular materials. Excessive autophagy induction could lead to a non-apoptotic cell death (autophagic cell death). The present study showed that sulindac sulfide at a physiological concentration also induces autophagic death in human gastric epithelial AGS and rat gastric epithelial RGM-1 cells, and that Survivin down-regulation is a mechanism involved: Sulindac sulfide treatment increased LC3b-II and APG7 levels and cytosolic vacuole formation, indications of autophagy induction, in AGS and RGM-1 cells. Sulindac sulfide treatment induced AGS and RGM-1 cell death, which was significantly reduced by pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine, indicating that sulindac sulfide induced autophagic cell death. Stable overexpression of Survivin in RGM-1 cells did not inhibit the induction of LC3b-II levels or vacuole formation by sulindac sulfide, but significantly reduced the resulting cell death, suggesting that Survivin may inhibit autophagic cell death downstream of LC3b-II induction and vacuole formation. Indeed, siRNA depletion of LC3b in AGS cells inhibited the down-regulation of Survivin levels and the induction of cell death by sulindac sulfide, confirming that down-regulation of Survivin occurs in the autophagy pathway downstream of LC3b-II induction by sulindac sulfide. Induction of Survivin-dependent autophagic cell death is a novel mechanism by which sulindac sulfide induces gastric mucosal injury.

Delayed production of arachidonic acid contributes to the delay of proteinase‐activated receptor‐1 (PAR1)‐triggered prostaglandin E2 release in rat gastric epithelial RGM1 cells

Proteinase-activated receptor-1 (PAR1), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E(2) (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR1-triggered PGE(2) release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE(2) release after PAR1 activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH(2), alone caused PGE(2) release at 18 h, but not 6 h, TFLLR-NH(2) in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH(2) plus linoleic acid caused a similar rapid response. CP-24879, a Δ(5)/Δ(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH(2) plus linoleic acid, but not by TFLLR-NH(2) alone. The TFLLR-NH(2)-induced PGE(2) release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca(2+)-independent PLA(2) (cPLA(2)) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA(2) isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PGE(2) release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA(2) including sPLA(2)-IIA may complementarily contribute to the liberation of free arachidonic acid.