Shingo Tsuji

Helicobacter pylori eradication to prevent gastric cancer: Underlying molecular and cellular mechanisms

Bone marrow is reported to contain hematopoietic stem cells and other adult somatic stem cells that have phenotypes of cells composing tissues other than bone marrow. To explore the implication of bone marrow‐derived cells in the treatment of inflammatory bowel diseases, experimental colitis was induced in wild‐type rats after transplantation of bone marrow from transgenic rats expressing green fluorescence protein (GFP). Chronic colitis was induced 21 days later using 30 mg 2,4,6‐trinitrobenzenesulfonic acid (TNBS). Control rats received saline. At 28, 56, and 224 days after TNBS administration, rats were euthanized, and tissues were removed and processed for paraffin‐embedded sections. Cells derived from bone marrow were identified by immunohistochemistry using anti‐GFP antibody. To identify the phenotypes of the cells expressing GFP, we conducted serial‐section analysis and double‐staining analysis using antibodies against cytokeratin (epithelial cells) or vimentin (interstitial cells). In the present study, GFP‐positive, bone marrow‐derived cells occupied 37.6% and 4.25% of the colonic epithelium at 28 days and 56 days after the induction of TNBS‐colitis, respectively. Also, significant amounts of mucosal and submucosal interstitial cells were derived from the bone marrow. These findings showed that a large amount of bone marrow‐derived cells were involved in regeneration of the colon after experimental colitis in rats.

Summary: Chronic inflammation has been reported to accelerate neoplasmas in gastrointestinal tract. Certain bacteria including Helicobacter pylori directly interact with host cells, induce proinflammatory cytokines and stimulate production of free radicals. Free radicals cause mutations in target cells so that neoplastic clones are established. Accumulation of such genetic alterations may cause malignant transformation of some established clones. In addition, inflammatory alterations may promote growth, expansion and invasion of gastrointestinal epithelial cells. The latter changes caused by inflammation may occur even without further genetic mutations or epigenetic alterations, and therefore may be categorized as ‘perigenetic alterations’ of neoplastic cells. For an example, tumour necrosis factor α (TNF‐α) plays pivotal roles not only in the reduction but also in the growth, invasion and metastases of certain neoplasmas. Our studies show that TNF‐α increases intracellular radical production, degradates E‐cadherin / β‐catenin complex and promotes dispersion and migration in epithelial cells transformed with an activated src oncogene (v‐src). These data indicate that an inflammatory cytokine induces the malignant potential of src‐activated neoplastic cells. Interestingly, TNF‐α also induced these phenotypic changes in nonmutated cells whose c‐Src was activated by TGF‐α, suggesting that the invasive properties of the cell were not necessarily related to gene mutation. Furthermore, certain radical scavengers suppressed the invasive phenotype of the cells. These results indicate that perigenetic alterations are an important target of pharmacological intervention of carcinogenesis.

Gastrin, PGs, and growth factors have important roles in maintaining gastrointestinal mucosal integrity. Cyclooxygenases (COX-1 and COX-2) are the key enzymes involved in PG synthesis. This study aimed to clarify the mechanisms of gastric mucosal protection by gastrin. Fasted rats were administered subcutaneous gastrin 17 with or without gastrin receptor antagonist YM022 pretreatment. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and COX-2 expression were examined using Western blot analysis. Another series of experiments investigated 1) PGE₂ levels in gastric mucosa, 2) the protective action of gastrin against gastric damage by acidified ethanol,3) the effects of a specific HB-EGF-neutralizing antibody on gastrin-induced COX-2 expression, and 4) the effects of a specific COX-2 inhibitor NS-398 on PGE₂ synthesis and the mucosal protection afforded by gastrin. Gastrin dose-dependently increased HB-EGF, COX-2 expression, and PGE₂ levels and reduced gastric damage. However, pretreatment with YM022 dose-dependently abolished such effects of gastrin. A specific HB-EGF- neutralizing antibody and an EGF receptor inhibitor decreased gastrin-induced COX-2 expression. NS-398 blocked gastrin-induced PGE₂ synthesis and mucosal protection. In conclusion, this study demonstrates that gastrin enhances gastric mucosal integrity through COX-2, which is partially mediated by HB-EGF, and PGE₂ upregulation in rats.

Proton pump inhibitors (PPIs) are antiulcer agents that have both gastric antisecretory and mucosal protective actions. The mechanisms of PPI-induced gastric mucosal protection are not known. The present study was designed to examine the mechanism for lansoprazole-induced gastric mucosal protection in rats. Rats were given 0.5, 5, and 50 mg/kg/day lansoprazole alone or both lansoprazole (50 mg/kg/day) and a specific gastrin receptor antagonist 3R-1-(2,2-diethoxyethyl)-((4-methylphenyl)amino-carbonyl methyl)-3-((4-methylphenyl)ureidoindoline-2- one) (AG-041R) (3, 10, and 30 mg/kg/day) for 14 days. Serum gastrin concentrations were measured. The expression of cyclooxygenases (COX-1 and COX-2) in the gastric mucosa was analyzed using Western blotting and immunohistochemical staining. Another series of rats was used to examine the 1) levels of prostaglandin (PG) E₂ in gastric mucosa, 2) influences of the drugs on gastric damage caused by absolute ethanol, and 3) effects of a COX-2-specific inhibitor on PGE₂ in the gastric mucosa and the mucosal protection afforded by lansoprazole. Lansoprazole dose dependently increased the serum gastrin concentration and enhanced the mucosal expression of COX-2 but not that of COX-1. Lansoprazole increased gastric mucosal PGE₂ and reduced gastric damage caused by ethanol. Concomitant administration of AG-041R abolished the lansoprazole-induced COX-2 expression, and increased mucosal PGE₂ and mucosal protection. A specific COX-2 inhibitor blocked the lansoprazole-induced increase in mucosal PGE₂and mucosal protection. Activation of gastrin receptors by endogenous gastrin has a pivotal role in the effects of lansoprazole on COX-2 up-regulation and mucosal protection in the rat stomach.

The present paper reviews current concepts on the role of cyclooxygenase (COX) in the development of malignant tumors. An inducible isoform of cyclooxygenase is expressed in neoplastic, pre-neoplastic, and peri-neoplastic cells by mutation of oncogenes (such as ras), tumor promoters, mitogens, cytokines, their receptors, and pathogenic factors such as Helicobacter. Cells overexpressing cox-2 escape apoptosis, have abnormal cell-to-cell interactions, and acquire invasive phenotypes. On the other hand, angiogenesis plays a key role in the development of malignant tumors. Both in vitro and in vivo studies indicate that cox-2 overexpression upregulates angiogenic factors in neoplastic cells and promotes tumor angiogenesis. It is also possible that cox-2 expression upregulates angiogenic factors in peri-neoplastic cells that express the isozyme. Interestingly, cox-1, the other isozyme that is expressed in tumor vascular endothelia, participates in tumor angiogenesis, because an anti-sense oligonucleotide of cox-1 suppresses in vitro angiogenesis induced by cox-2-overexpressing cells. A non-specific COX inhibitor, not a specific COX-2 inhibitor, reduced growth and angiogenesis in cancer xenografts by inhibition of COX-1 in vascular endothelial cells, even when the tumor did not express COX-2. These results demonstrate that COX inhibitors suppress angiogenesis and tumor growth by inhibiting expression of angiogenic factors and vascular endothelial cell migration. Furthermore, another concept is emerging to indicate that prostaglandins (COX-2 products and mediators of classic inflammation) suppress host immunity against tumors. This evidence supports the hypothesis that COX is an important perigenetic factor in the development of cancer growth, and offers a new strategy against cancer using COX inhibitors (nonsteroidal anti-inflammatory drugs).

T-593 (osutidine, CAS 140695-21-2) is a new anti-ulcer agent that consists of two enantiomers, (+) (R) T-593 and (-)-(S)-T-593. The present study was designed to investigate the effects of T-593, (+)-(R)-T-593 and (-) (S) T-593 on ethanol-induced gastric damage in rats. Rats were given intraperitoneal administration of vehicle, racemic T-593, (+) (R)-T-593 or (-) (S)-T-593. 30 min later, the rats intragastrically received a 1 ml solution of 40 % ethanol, and 30 min thereafter, they were sacrificed for assessment of gastric damage. Gastric hemodynamics were assessed by reflectance spectrophotometry and laser Doppler flowmetry during the experiment. Gastric damage was significantly suppressed by racemic T-593 in a dose-depenent manner. While 60 mg/kg of (+)-(R) T-593 and (-)-(S) T-593 also suppressed ethanol-induced gastric injury, the same dose of racemic T-593 exerted the most potent anti-ulcerative activity. Both (+) (R) T-593 and racemic T-593 in creased gastric mucosal blood flow and abolished gastric mucosal blood flow stasis induced by 40 % ethanol. Although (-) (S) T-593, which antagonizeshist amine H₂-receptors, exerts an anti-ulcerative effect, (+) (R) T-593 also protects the gastric mucosa from ethanol induced injury, possibly by influencing gastric mucosal hemodynamics. Thus racemic T-593 may protect the gastric mucosa by antagonizing H₂receptors and by enhancing gastric circulation in rats. Wirkungen von Osutidin (T-593) und seiner Enantiomere auf die Hämodynamik der Magenschleimhaut und die Schleim hautintegrität narkotisierter Ratten T-593 (Osutidin, CAS 140695-21-2) ist eine neue Substanz gegen Magenge schwüre, die aus den beiden Enantiome ren (+) (R)-T-593 und (-)-(S)-T-593 be steht. Die vorliegende Studie untersucht die Wirkungen von T-593, (+)-(R)-T-593 und (-)-(S)-T-593 auf durch Ethanol induzierte Magenschäden bei Ratten. Rattenwurde intraperitoneal ein Vehikel, razemisches T-593, (+) (R)-T-593 oder (-)-(S)-T-593 verabreicht. 30 min später erhielten die Ratten intragastrisch 1 ml einer 40 %igen Ethanol-Lösung und wurden nach weiteren 30 min zur Untersuchung auf Magenschäden getötet. Während des Experiments erfolgte die Beurteilung der gastrischen Hämodynamik mit Hilfe von Reflexions Spektrophotometrie und Doppler Laser Flowmeter. Razemisches T-593 führte abhängig von der verabreichten Dosis zu einer signifikanten Unterdrükkung von Magenschäden. Während eine Dosis von 60 mg/kg (+)-(R)-T-593 und (-)-(S)-T-593 auch eine Unterdrückung Ethanol-induzierter gastrischer Verletzungen bewirkte, entwickelte dieselbe Dosis von razemischem T-593 eine außerordentlich starke Aktivität gegen Magengeschwüre. Sowohl (+)-(R)-T-593 als auch razemisches T-593 steigerte die Durchblutung der Magenschleimhaut und beseitigte die durch 40 %iges Ethanol verursachte Stauung des Blutflusses in der Magenschleimhaut. Das Histamin-H₂-Rezeptoren entgegenwirkende (-)-(S)-T-593 hat zwar einen Effekt gegen Magengeschwüre, aber (+)-(R)-T-593 schützt darüber hinaus auch die Magenschleimhaut vor Ethanol-induzierten Verletzungen, wahrscheinlich durch Beeinflussung der Hämodynamik der Magenschleimhaut. Dies legt den Schlußnahe, daß bei Ratten die Schutzwirkung von T-593 für die Magenschleimhaut auf einem Antagonismus von H₂-Rezeptoren und der Verbesserung der Magendurchblutung beruht.

Background and Aims : The present study examined the effects of NS‐398, a specific cyclo‐oxygenase‐2 inhibitor, on gastric mucosal cell kinetics and gastric wound healing following acid‐induced injury. Methods : Male Sprague‐Dawley rats were fasted for 24 h and then 0.6 mol/L hydrochloric acid (HCl; 1 mL) was administered into the stomach; NS‐398 or indomethacin was administered to the animals 10 min after the acid. Levels of constitutive cyclo‐oxygenase (COX‐1) and mitogen‐inducible cyclo‐oxygenase (COX‐2) in the gastric mucosa were analysed using western blotting and immunohistochemical staining. The grade of the lesion was assessed using planimetry and histological examination, including immunohistochemistry for proliferating cell nuclear antigen (PCNA). Results : Although there was strong expression of COX‐1, there was minimal expression of COX‐2 in the gastric mucosa. Expression of COX‐2 was enhanced mainly in surface epithelial cells and neck cells following HCl administration. Gastric mucosal ulcers and erosions healed within 48 h, during which time the proliferative zone expanded in the control animals. Indomethacin and NS‐398 suppressed the expansion of the proliferative zone and delayed the healing of the gastric injury. Conclusion : The present study demonstrated that cyclo‐oxygenase‐2 inhibitors delay gastric wound healing by suppressing expansion of the mucosal proliferative zone. These results provide evidence that cyclo‐oxygenase‐2 has an important role in gastric mucosal regeneration.

Background and Study Aims: The aim of the present study was to evaluate a new endoscope disinfector (WM-1) that uses acidic electrolytic water (AEW). Materials and Methods: AEW was produced by electrolysis of a 0.05 % NaCl-water mixture, with a redox potential greater than 1000 mV and a pH lower than 2.7. In the first study, an endoscope artificially contaminated with 15 species of bacteria and four strains of viruses was treated using the WM-1. In the second study, endoscopic contamination after clinical use was examined by culture for Helicobacter pylori and other bacteria, and by polymerase chain reaction for the H. pylori urease gene and hepatitis C virus. The extent of contamination was then examined after exposing the WM-1 to AEW. The safety of AEW was examined using both in vivo and in vitro studies. Results: All of the bacteria and viruses were destroyed or inactivated after the instrument had been exposed to AEW. Clinical contamination was detected from the instrument in 19 of 30 endoscopic procedures, whereas no bacteria or viruses were detected after five minutes' exposure to AEW. AEW was found to be nonirritant, nontoxic to cells, and nonmutagenic. Conclusion: The WM-1 successfully and safely disinfected the endoscopes. With running costs of ¥24 per day ($0.21 per day), the WM-1 provides an effective and inexpensive alternative to conventional disinfection equipment.