Hyperplastic gastric tumors induced by activated macrophages in COX-2/mPGES-1 transgenic mice

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme for prostanoid biosynthesis, plays a key role in gastrointestinal carcinogenesis. Among various prostanoids, prostaglandin E2 (PGE2) appears to be most responsible for cancer development. To investigate the role of PGE2 in gastric tumorigenesis, we constructed transgenic mice simultaneously expressing COX-2 and microsomal prostaglandin E synthase (mPGES)-1 in the gastric epithelial cells. The transgenic mice developed metaplasia, hyperplasia and tumorous growths in the glandular stomach with heavy macrophage infiltrations. Although gastric bacterial counts in the transgenic mice were within the normal range, treatment with antibiotics significantly suppressed activation of the macrophages and tumorous hyperplasia. Importantly, the antibiotics treatment did not affect the macrophage accumulation. Notably, treatment of the transgenic mice with lipopolysaccharides induced proinflammatory cytokines through Toll-like receptor 4 in the gastric epithelial cells. These results indicate that an increased level of PGE2 enhances macrophage infiltration, and that they are activated through epithelial cells by the gastric flora, resulting in gastric metaplasia and tumorous growth. Furthermore, Helicobacter infection upregulated epithelial PGE2 production, suggesting that the COX-2/mPGES-1 pathway contributes to the Helicobacter-associated gastric tumorigenesis.     

Establishment of a gastric epithelial progenitor cell line from a transgenic mouse expressing the simian virus 40 large T antigen gene in the parietal cell lineage

Abstract.     Objective:  In this study the gastric mucosa of transgenic mice expressing the simian virus 40 large T antigen gene in the parietal cell lineage is used to establish and characterize a new epithelial progenitor cell line. In these mice, proliferation and amplification of preparietal cells preclude their maturation into acid-secreting parietal cells leading to achlorohydria, hyperplasia, dysplasia and eventually gastric adenocarcinoma. Materials and methods:  Enzymatically dispersed gastric epithelial cells were cultured, cloned and screened using immunohistochemical methods, for expression of a variety of biomarkers of differentiated pit, parietal, enteroendocrine and neck/zymogenic cells. Results:  A biomarker-deficient cell line whose ultrastructural features resembled those of mouse gastric epithelial progenitor cells was established. Treatment with either hydrocortisone or oestrogen significantly enhanced proliferation of these cells, whereas retinoic acid inhibited their growth. No change in differentiation was detected with any of these treatments; however, when these cells were injected subcutaneously into nude mice, they proliferated to form tumours and undergo partial differentiation towards parietal cell lineage. Conclusion:  This mouse gastric epithelial progenitor cell line could be useful as an in vitro model to study growth properties, proliferation and differentiation of a subpopulation of gastric epithelial progenitor cells and also to study gastric carcinogenesis.     

Immunohistochemical detection of carcinoembryonic antigen (CEA) in non-cancerous and cancerous gastric mucosa

Carcinoembryonic antigen (CEA) was stained by the PAP immunoperoxidase method in cancerous and non-cancerous gastric mucosa of 40 patients (25 non-cancerous dyspeptic patients and 15 patients with gastric carcinoma). The pattern of CEA localization was apical or membranous-cytoplasmic and immuno-reactivity was mild (+), moderate (++) or intensive ( ). No CEA immunoreactivity was detected in normal gastric mucosa whereas it was marked in gastric mucosa of non-cancerous dyspeptic patients with chronic atrophic gastritis and dysplasia (intense). In patients with superficial gastritis and epithelial hyperplasia it was mild or absent. The CEA localization pattern was also apical in non-cancerous dyspeptic patients with microscopic changes, e.g. superficial or chronic atrophic gastritis, epithelial hyperplasia and dysplasia, and in non-cancerous mucosa and cancerous tissue of patients with well (G1) and moderately (G2) differentiated adenocarcinoma.     

Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling

Chronic hypergastrinemia is associated with enterochromaffin-like (ECL) cell hyperplasia, which may progress to gastric carcinoid tumors. The latter consists of epithelial cells and stroma, and both compartments usually regress after normalization of hypergastrinemia. We previously showed that matrix metalloproteinase (MMP)-7 in gastric epithelial cells was upregulated by Helicobacter pylori and described MMP-7-dependent reciprocal signaling between the epithelium and a key stromal cell type, the myofibroblast. Here, we describe the regulation of gastric MMP-7 by gastrin and the potential significance for recruiting and maintaining myofibroblast populations. Biopsies of the gastric corpus and ECL cell carcinoid tumors were obtained from hypergastrinemic patients. Western blot analysis, ELISA, immunohistochemistry, and promoter-luciferase (luc) reporter assays were used to study MMP-7 expression. Gastric myofibroblasts were identified by alpha-smooth muscle actin (alpha-SMA) expression, and the effects of MMP-7 on myofibroblast proliferation were investigated. In hypergastrinemic patients, there was an increased abundance of MMP-7 and alpha-SMA in gastric corpus biopsies and ECL cell carcinoid tumors. In the latter, MMP-7 was localized to ECL cells but not stromal cells, which were nevertheless well represented. Gastrin stimulated MMP-7-luc expression in both AGS-G(R) and primary human gastric epithelial cells. Conditioned medium from gastrin-treated human gastric glands stimulated myofibroblast proliferation, which was inhibited by neutralizing antibodies to MMP-7. MMP-7 increased the proliferation of myofibroblasts via the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, stimulation of gastric MMP-7 by elevated plasma gastrin may activate epithelial-mesenchymal signaling pathways regulating myofibroblast function via MAPK and PI3K pathways and contribute to stromal deposition in ECL cell carcinoid tumors.     

Age dependent association of endometrial polyps with increased risk of cancer involvement

Background

Most of gastric adenocarcinoma can be simply diagnosed by microscopic examination of biopsy specimen. Rarely the structural and cellular atypia of tumor cells is too insignificant to discriminate from benign foveolar epithelium.

Case presentation

A 67-year-old male presented with a gastric mass incidentally found on the abdominal computed tomography (CT) for routine medical examination. Gastric endoscopic examination revealed a huge fungating mass at the cardia and mucosal biopsy was performed. Microscopically the biopsy specimen showed proliferation of bland looking foveolar epithelia in the inflammatory background and diagnosed as foveolar epithelial hyperplasia. Because the clinical and endoscopic features of this patient were strongly suggestive of malignancy, the patient underwent radical total gastrectomy. The resected stomach revealed a huge fungating tumor at the cardia. The cut surface of the tumor was whitish gelatinous. Microscopically the tumor was sharply demarcated from surrounding mucosa and composed of very well formed glandular structures without significant cellular atypia, which invaded into the whole layer of the gastric wall. Tumor glands were occasionally complicated or dilated, and glandular lumina were filled with abundant mucin. Immunohistochemically the tumor cells revealed no overexpression of p53 protein but high Ki-67 labeling index. The tumor cells and intraluminal mucin were diffusely expressed MUC1 and MUC5AC and only focally expressed MUC2. On abdominal CT taken after 12 months demonstrated peritoneal carcinomatosis and multiple metastatic foci in the lung.

Conclusion

The clinicopathologic profiles of gastric extremely well differentiated adenocarcinoma of gastric phenotype include cardiac location, fungating gross type, very similar histology to foveolar epithelial hyperplasia, foveolar mucin phenotype, lack of p53 overexpressoin and high proliferative index.     

Gastric Pit Cell Hyperplasia and Glandular Atrophy in Carbonic Anhydrase IX Knockout Mice: Studies on Two Strains C57/BL6 and BALB/C

Carbonic anhydrase (CA) isoenzyme IX is a hypoxia-inducible enzyme, which is expressed in the human and rodent gastrointestinal tract and overexpressed in several different tumors. Functionally, it has probably an effect on proliferation and differentiation of gastrointestinal epithelial cells. It may also participate in gastric morphogenesis, since a recent study has shown gastric pit cell hyperplasia and glandular atrophy in CA IX-knockout mice. However, it is not known whether CA IX produces morphological changes in the gastric mucosa, which can turn into a dysplasia or malignancy in the presence of some carcinogenic factors. High-salt diet is considered such a factor which has been shown to modulate Helicobacter pylori-associated carcinogenesis. We produced two strains of CA IX-knockout mice, C57/BL6 and BALB/c, and the mice ate either standard or high-salt feed for 20 weeks. Stomach samples were collected from 40 Car9^−/− knockout mice and 37 wildtype littermates, and the tissue sections were examined for histology. CA IX-deficiency caused gastric pit cell hyperplasia and glandular atrophy in both BALB/c and C57/BL6 strains. Excess dietary salt had no significant effect on the severity of pit cell hyperplasia. No dysplasia was found in any of the groups. In C57/BL6 mice, CA IX-deficiency was associated with gastric submucosal inflammation. The results indicate that CA IX-deficiency provides a useful model to study the mechanisms of gastric morphogenesis and epithelial integrity. Further studies are needed to see whether CA IX has a role in the regulation of immune response. The findings suggest that although CA IX-deficiency is not a tumor-promoting factor per se, it induces glandular atrophy in the body mucosa, a lesion which is considered to be a preneoplastic alteration in the stomach.     

Helicobacter pylori infection and CagA protein translocation in human primary gastric epithelial cell culture

BACKGROUND: Increasing evidence has shown that Helicobacter pylori CagA protein translocation into gastric epithelial cells plays an important role in the development of gastric inflammation and malignancy. Translocated CagA undergoes tyrosine phosphorylation in gastric adenocarcinoma cell line cells, and CagA involves disruption of cellular apical-junction complex in Madin-Darby canine kidney cells. METHODS: To elucidate whether these events take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. RESULTS: Our results demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeletal rearrangement and the disruption of tight junctions in primary gastric epithelial cells. CONCLUSIONS: This study provides direct evidence of the modulation of gastric epithelial cells by CagA protein translocation, and advances our understanding of the pathogenesis of H. pylori infection.     

Helicobacter pylori infection activates NF-kappaB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-kappaB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-kappaB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-kappaB signaling pathway via NF-kappaB-inducing kinase and IkappaB kinase-beta activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IkappaBalpha superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-kappaB. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 microM), decreased caspase-3 activity and apoptosis in NF-kappaB-suppressed cells infected with H. pylori. These results suggest that NF-kappaB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.     

Nutrients released by gastric epithelial cells enhance Helicobacter pylori growth

BACKGROUND: Helicobacter pylori survives and proliferates in the human gastric mucosa. In this niche, H. pylori adheres to the gastric epithelial cells near the tight junctions. In vitro, H. pylori proliferated well in tissue-culture medium near gastric epithelial cells. However, in the absence of epithelial cells, growth of H. pylori could only be established in tissue-culture medium when, prior to the experiment, it was preincubated near gastric epithelial cells. Therefore, we aimed to determine whether diffusion of nutrients derived from epithelial cells was required for H. pylori growth in Dulbecco's modified Eagle's minimal essential medium (DMEM) cell culture medium. MATERIALS AND METHODS: Cell culture conditions essential for H. pylori growth in vitro were determined with gastric epithelial HM02 cells. RESULTS: Deprivation of iron in cell-culture-conditioned DMEM resulted in a growth arrest of H. pylori. However, near gastric epithelial cells, growth of H. pylori was resistant to iron deprivation. Evidently, when residing close to epithelial cells, H. pylori was able to fulfil its iron requirements, even when the DMEM was deprived of iron. Nevertheless, supplementation with iron alone did not restore H. pylori growth in DMEM, hence other nutrients were deficient as well in the absence of epithelial cells. Growth of H. pylori in DMEM was restored when hypoxanthine, L-alanine and L-proline were added to the DMEM. CONCLUSIONS: Diffusion of (precursors of) these nutrients from the gastric epithelial cells is essential for H. pylori growth in vitro. We hypothesize that in vivo, H. pylori favors colonization near the tight junctions, to gain maximal access to the nutrient(s) released by gastric epithelial cells.     

Grb2 is a key mediator of helicobacter pylori CagA protein activities

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.     

Up-regulation of production of TGF-beta and IL-4 and down-regulation of IL-6 by apoptotic human bronchial epithelial cells

Human bronchial epithelial cells secrete cytokines that play a role in immune responses in the lung. However, the roles of these cytokines in regulating epithelial repair following acute lung injury are largely unknown. Responses to injury include hyperplasia of epithelial cells and squamous metaplasia. The resolution stage is characterized by discontinuation of hyperplasia. Apoptosis is considered to be the most efficient mechanism of removal of unwanted cells without causing inflammation. The presence of TGF-beta1 increases apoptosis, induces squamous metaplasia and inhibits proliferation of airway epithelial cells. Interleukin-4 increases the ability of macrophages to phagocytose epithelial cells and produce inflammatory cytokines. The purpose of this study was to investigate the hypothesis that apoptotic lung epithelial cells produce cytokines, which could act in an autocrine manner to control hyperplasia and induce squamous differentiation following acute lung injury. A bronchial epithelial cell line (16 HBE) was used as an in vitro model, to study the production of TGF-beta, IL-4 and IL-6 by lung epithelial cells undergoing apoptosis. Apoptotic and live cells were sorted on the basis of bright and negative staining with FITC-conjugated Annexin V, respectively. Intracellular IL-6, TGF-beta and IL-4 was measured using flow cytometric techniques. Electron microscopy, immunohistochemistry and ELISA were used as supportive techniques. Apoptotic cells produced significantly more TGF-beta and IL-4 (but less IL-6) than viable cells. Increased production of TGF-beta and IL-4 by epithelial cells undergoing apoptosis may contribute to the inhibition of proliferation, squamous metaplasia, and reduction of the inflammatory response in acute lung injury.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Epidermal growth factor receptor-dependent PI3K-activation promotes restitution of wounded human gastric epithelial monolayers

Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.     

树突状细胞对胃癌前病变的免疫保护分析

摘要 目的：探究树突状细胞(Dendritic cells,DC)对胃癌的免疫保护作用。方法：选择2016年1月至2018年1月于我院接受治疗的145例胃癌、39例慢性萎缩性胃炎、21例不典型增生、27例肠上皮化生以及20例正常对照组患者为研究对象,分别采集其胃粘膜标本进行染色,记录和比较其胃粘膜中S100⁺、CD4⁺和CD8⁺细胞的数量、平均面积以及平均吸光度,并将胃癌患者分为中分化腺癌(49例)、低分化腺癌(53例)和未分化癌(43例)进行对比。结果：(1)胃癌组、慢性萎缩性胃炎组、不典型增生、肠上皮化生组的胃粘膜S100⁺阳性细胞计数明显高于正常对照组（P<0.05）,胃癌组平均吸光度低于对照组,其他3组平均吸光度显著高于对照组,(P<0.05);胃癌组平均面积与正常对照组相比无差异(P>0.05),其他三组平均面积显著高于对照组（P<0.05）;(2)慢性萎缩性胃炎组、肠上皮化生组、不典型增生组患者CD4⁺细胞数均低于对照组(P<0.05);胃癌组、慢性萎缩性胃炎组、肠上皮化生组患者平均面积均低于对照组(P<0.05);胃癌组、慢性萎缩性胃炎组、不典型增生、肠上皮化生组平均吸光度均低于对照组(P<0.05);(3)慢性萎缩性胃炎组、肠上皮化生组、不典型增生组患者CD8⁺细胞数明显高于对照组(P<0.05),胃癌组稍低于对照组(P>0.05);胃癌组患者平均面积低于对照组(P<0.05);胃癌组患者平均吸光值低于对照组,慢性萎缩性胃炎组、肠上皮化生组患者高于对照组(P均<0.05);(4)随着胃癌分化程度的降低,胃癌患者DC细胞数有降低趋势。结论：胃癌前病变患者胃粘膜中DC数量会显著增多,免疫功能加强,DC细胞数量会随胃癌分化程度的降低而减少,分析其原因与DC细胞能够抑制癌前病变有关。     

The role of cellular migration in the repair process of gastric epithelial cells.

The epithelial cells of stomach are continuously exposed to various toxic agents that may cause mucosal injury. The epithelial lining is rapidly replaced by cells that migrate from the proliferative zone of the gastric gland, to maintain the integrity of the gastric mucosa. Thus, cell migration is an essential part of the early process of gastric mucosal repair. After various forms of gastric injury, mucosal integrity is reestablished by the rapid migration of epithelial cells. However, the cellular mechanisms of the restitution remain unclear to date. In this report, we will review the role of cellular migration in the repair process of gastric epithelial cells in culture. It has been reported that hepatocyte growth factor (HGF) has the potency of acceleration of cellular repair process. In this review, we also report that HGF plays a leading role in the mucosal repair after damage by using a novel cell culture model.