Epigenetic alterations in gastric carcinogenesis

Gastric cancer is believed to result in part from the accumulation of multiple genetic alterations leading to oncogeneoverexpression and tumor suppressor loss. Epigenetic alterations as a distinct and crucial mechanism to silence a varietyof methylated tissue-specific and imprinted genes, have been extensively studied in gastric carcinoma and play impor-tant roles in gastric carcinogenesis. This review will briefly discuss the basic aspects of DNA methylation and CpGisland methylation, in particular the epigenetic alterations of certain critical genes implicated in gastric carcinogenesisand its relevance of clinical implications.

CagA+ H pylori infection is associated with polarization of T helper cell immune responses in gastric carcinogenesis

AIM:To characterize the immune responses including local and systemic immunity induced by infection with H pylori,especially with CagA+ H pylori strains and the underlying immunopathogenesis. METHODS:A total of 711 patients with different gastric lesions were recruited to determine the presence of H pylori infection and cytotoxin associated protein A (CagA),the presence of T helper (Th) cells and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMCs),expression of plasma cytokines,and RNA and protein expression of IFN-γ and IL-4 in gastric biopsies and PBMCs were determined by rapid urease test,urea 14C breath test,immunoblotting test,flow cytometry ,real time RT-PCR and immunohistochemistry. RESULTS:Of the patients,629 (88.47%) were infected with H pylori ; 506 (71.16%) with CagA+ and 123 (17.30%) with CagA- strains. Among patients infected with CagA+ H pylori strains,Th1-mediated cellular immunity was associated with earlier stages of gastric carcinogenesis,while Th2-mediated humoral immunity dominated the advanced stages and was negatively associated with an abundance of Treg cells. However,there was no such tendency in Th1/Th2 polarization in patients infected with CagA- H pylori strains and those without H pylori infection. CONCLUSION:Polarization of Th cell immune responses occurs in patients with CagA+ H pyloriinfection,which is associated with the stage and severity of gastric pathology during the progression of gastric carcinogenesis. This finding provides further evidence for a causal role of CagA+ H pylori infection in the immunopathogenesis of gastric cancer.

Cyr61/CTGF/Nov family proteins in gastric carcinogenesis

Gastric cancer(GC)is the second leading cause of cancer-related death.The poor survival rate may reflect the relatively aggressive tumor biology of GC.Recently,the importance of the tumor microenvironment in carcinogenesis has emerged.In the tumor microenvironment,tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins capable of modulating carcinogenesis and regulating metastasis.The Cyr61/CTGF/Nov(CCN)proteins are a family of matricellular proteins with variable roles in many physiological and pathological processes.The evidence suggests that CCN family proteins contribute to GC carcinogenic processes.Here,we briefly review recent research on the effects of CCN family proteins in GC carcinogenesis and the development of new targeted agents in this field.

Is NEDD4-1 a negative regulator of phosphatase and tensin homolog in gastric carcinogenesis?

The expression of phosphatase and tensin homolog (PTEN ), a tumor suppressor gene, is frequently downregulated in gastric carcinomas due to mutation, loss of heterozygosity, and promoter hypermethylation. However, it is unknown if additional mechanisms may account for the down-regulation of PTEN expression. While neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) is believed to be a potential dual regulator of PTEN, there are conflicting reports regarding their interaction. To gain further insight into the role of NEDD4-1 and its association with PTEN in gastric carcinoma development, we measured the protein expression of NEDD4-1 and PTEN in gastric mucosae with various pathological lesions and found that NEDD4-1 increased from normal gastric mucosa to intestinal metaplasia and decreased from dysplasia to gastric carcinoma. These changes did not correlate with PTEN expression changes during gastric carcinogenesis. Moreover, we found similar results in protein levels in the primary tumors and adjacent non-tumorous tissues. These results differ from a previous report showing that expression of NEDD4-1 is up-regulated in gastric carcinomas, and show a more complex pattern of NEDD4-1 gene expression during gastric carcinogenesis.

Histological and ultrastructural changes induced by selenium in early experimental gastric carcinogenesis

AIM： To investigate the effect and significance of selenium in early experimental gastric carcinogenesis. METHODS： Weaning male Wistar rats were divided randomly into normal control group, experiment control group, low selenium （2 mg/L） group and high selenium （4 mg/L） group. Wistar rat gastric carcinogenesis was induced by N-methyl-N-nitro-N-nitroso guanidine （MNNG） （20 mg/kg） gavage dally for 10 d. Na2SeO3 was given by piped drinking 1 wk prior to MNNG gavage. The rats were killed at the 43^rd wk. The surface characteristics of gastric mucosa were observed with naked eyes. Histopathologic changes of rat gastric mucosa were observed by HE staining and AB-PAS methods. The changes of cellular ultrastructure were observed under transmission electron microscope. Statistical analysis was carried out by SPSS. RESULTS： The incidence rate of gastric mucosa erosion, hemorrhage and intestinal metaplasia was 0, 45.5%, 66.7%, and 92.9%, respectively （92.9% vs 45.5%, P〈0.05） in the normal control group, experiment control group, low selenium group, and high selenium group. Leiomyoma formed in the process of inducement of rat gastric carcinoma. Dietary Na2SeO3 （2 and 4 mg/L） slightly increased the incidence rate of leiomyoma （0.23%, 46.6%, and 46.6%）. gastric mucosa did not change in the course of rat gastric carcinogenesis. Dietary Na2SeO3 by pipe drinking could expand the intracellular secretory canaliculus of parietal cells and increase the number of endocrine cells and lysosomes. CONCLUSION： Dietary Na2SeO3 by pipe drinking aggravates gastric erosion, hemorrhage and promotes intestinal metaplasia of gastric mucosa. The mechanism may be related with the function of parietal cells.

Effect of 1, 25-Dihydroxyvitamin D_3 on Gastric Carcinogenesis Induced by N-Methyl-N'-nitro-N-nitrosoguanidine in Rats

The effect of 1, 25-dihydroxyvitamin D3 (1, 25 (OH)2D3) given in the post-initiation stage of gastric carcinogenesis induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was investigated in male Wistar rats. Gastric carcinogenesis in rats was induced by administration of MNNG (150 mg·L-1) in drinking water. Four weeks after MNNG exposure, the rats were switched to the diet containing 1, 25 (OH)2D3 (2. 5 μg·kg-1 and 5. 0 μg·kg-1) and maintained on the diet. Animals were killed at week 16 and week 32 for immunohistochemical and histopathological studies. At week 16, the proliferating cell nuclear antingen (PCNA) labeling index in epithelium from the glandular stomach of rats that received 1, 25 (OH)2D3 (5.0 μg·kg-1) in combination with MNNG (150 mg·L-1) were significantly higher when compared with the rats receiving MNNG alone. Supplementation of 1, 25 (OH)2D3 (5. 0 μg·kg-1) in the rats' diet caused a dramatic increase in carcinoma incidence, and the number of individual cancer foci in the glandular stomach of rats receiving MNNG at week 32. It was concluded that certain dose of 1, 25 (OH)2D3 enhanced gastric carcinogenesis induced by MNNG in rats.

Could microbiome analysis be a new diagnostic tool in gastric carcinogenesis for high risk, Helicobacter pylori negative patients?

Helicobacter pylori(H.pylori)has long been believed to be the major colonizer of the stomach,but recent advances in genetic sequencing have allowed for further differentiation of the gastric microbiome and revealed the true complexity of the gastric microbiome.One of the few studies specifically evaluated the microbiome in the H.pylori negative patient population.They concluded that various stages of gastric carcinogenesis are associated with distinct bacterial taxa that could service both a predictive and diagnostic purpose.While the study has some limitations,the conclusions they make are intriguing and should prompt a larger prospective study to be done that spans multiple geographic regions.

MYC and gastric adenocarcinoma carcinogenesis

MYC is an oncogene involved in cell cycle regulation, cell growth arrest, cell adhesion, metabolism, ribosome biogenesis, protein synthesis, and mitochondrial function. It has been described as a key element of several carcinogenesis processes in humans. Many studies have shown an association between MYC deregulation and gastric cancer. MYC deregulation is also seen in gastric preneoplastic lesions and thus it may have a role in early gastric carcinogenesis. Several studies have suggested that amplification is the main mechanism of MYC deregulation in gastric cancer. In the present review, we focus on the deregulation of the MYC oncogene in gastric adenocarcinoma carcinogenesis, including its association with Helicobacterpylori （Hpylon] and clinical applications.

Cellular stress and redox activity proteins are involved in gastric carcinogenesis associated with Helicobacter pylori infection expressing high levels of thioredoxin-1

Heficobacterpylori infection is related to the development of gastric diseases. Our previous studies showed that high thioredoxin-1 （Trxl） expression in H. pylori can promote gastric carcinogenesis. To explore the underlying molecular mechanisms, we performed an isobaric tags for relative and absolute quantitation （iTRAQ）-based quantitative proteomic analysis of stomach tissues from Mongolian gerbil infected with H. pylori expressing high and low Trxl Differences in the profiles of the expressed proteins were analyzed by bioinformatics and verified using Western blot analysis. We found three candidate proteins, 14-3-3α/β, glutathione-S-transferase （GST）, and heat shock protein 70 （HSP70）, in high Trxl tissues compared with low Trxl tissues and concluded that cellular stress and redox activity- related proteins were involved in the pathogenesis of gastric cancer associated with H. pylori Trxl.

O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group to a cysteine residue in its active site and became inactive.The chemical carcinogen N-nitroso compounds(NOCs)can directly bind to the DNA and induce the O_(6)-methylguanine adducts,which is an important cause of gene mutation and tumorigenesis.However,the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis,especially in the initiation phase,remains largely unclear.AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODS We established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)or N-methyl-N-nitroso-urea(MNU)treatment.Cell proliferation,colony formation,soft agar,cell migration,and xenograft assays were used to verify the malignant phenotype.By using quantitative real-time polymerase chain reaction(qPCR)and Western blot analysis,we detected the MGMT expression in malignant transformed cells.We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry.MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR.The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.RESULTS We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment.Moreover,we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells.Inhibition of the MGMT expression by O_(6)-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes.Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU.Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues,but downregulated in the gastric cancer tissues.CONCLUSION Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation.The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis,which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.

α-catenin expression is decreased in patients with gastric carcinoma

AIM: To assess the expression of α-catenin in gastric carcinoma and to determine the role of α-catenin expression in gastric carcinogenesis.METHODS: α-catenin expression was assessed by semi quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical staining in 49 gastric carcinomas,26 adjacent non-cancerous mucosae, and gastric biopsy specimens from 11 healthy controls.RESULTS: mRNA levels of α-catenin were reduced or absent in 34 of 49 (69%) gastric carcinoma tissues and in 5 of 26 (19%) tumor-free gastric mucosae of carcinomapatients, respectively. Of the carcinoma samples with altered α-catenin mRNA levels, α-catenin expression was negative in 20 and decreased in 14 cases. Up to 69% of tumors were stained abnormally for α-catenin. Of the 34 cases whose mRNA expression of α-catenin was reduced, 32 (94%) showed abnormal immunostaining patterns, while only 2 showed a normal α-catenin expression. The frequency of reduced expression of α-catenin mRNA was 14% in well-differentiated carcinomas, higher than that in poorlydifferentiated carcinomas (86%). A significant correlation was not shown between α-catenin expression and bothdepth of invasion and lymph node metastasis. Moreover, there was no statistical difference between loss or down-regulation of α-catenin mRNA and Helicobacter pylori ( H pylori) infection.CONCLUSION: Downregulation of α-catenin expressionis common in gastric carcinoma, and α-catenin expression may be used as a differentiation marker. Downregulation of α-catenin expression may be an early event in tumorigenesis. Reduced α-catenin expression is not correlated with H pylori infection.

Metastasis-associated lung adenocarcinoma transcript 1 molecular mechanisms in gastric cancer progression

Gastric cancer(GC)remains among the most common cancers worldwide with a high mortality-to-incidence ratio.Accumulated evidence suggests that long noncoding RNAs(lncRNAs)are involved in gastric carcinogenesis.These transcripts are longer than 200 nucleotides and modulate gene expression at multiple molecular levels,inducing or inhibiting biological processes and diseases.Metastasis-associated lung adenocarcinoma transcript 1(MALAT1)is one of the best-studied lncRNAs with comprehensive actions contributing to cancer progression.This lncRNA regulates gene expression at the transcriptional and posttranscriptional levels through interactions with microRNAs and proteins.In the present review,we discussed the molecular mechanism of MALAT1 and summarized the current knowledge of its expression in GC.Moreover,we highlighted the potential use of MALAT1 as a biomarker,including liquid biopsy.

INDUCTION OF GASTRIC INTRAEPITHELIAL NEOPLASIA OF GLANDULAR STOMACH OF MONGOLIAN GERBILS BY ELICOBACTER PYLORI

Objective： To setup an animal model of gastric carcinogenesis by Helicobacter pylori （Hp） for basic, prevention and therapeutic research of Hp-related diseases. Methods： 22 young male Mongolian gerbils were administrated with suspension of Hp strain TN2 by intragastric garage for 5 consecutive times （4×10^8 CFU/time, 1 time/4 days）. 10 male gerbils were used as negative control. Two infected gerbils were killed at 10, 20, and 30 weeks, respectively, after inoculation to monitor the development of gastric lesions. Other animals were killed at 40 experimental weeks. Pathological changes of glandular stomach were examined histologically. Results： Gastric intraepithelial neoplasias （GIN） and low-grade dysplasias were observed only in the pyloric antrum of Hp-treated gerbils （3 and 2 ones, respectively）, but not in control group （5/13 vs. 0/10, P〈0.04）. High incidence of chronic active gastritis and chronic atrophic gastritis were observed in Hp-treated animals （10/13, 76.9%）. Low incidence of chronic atrophic gastritis was also detected in negative control gerbils （3/10, 30%; P〈0.04）. Conclusion： Hp inoculation could induce chronic inflammation and malignant lesions of the glandular stomach of Mongolian gerbils conveniently.

Bioactive Fatty Acids Reduce Development of Gastric Cancer Following Duodenogastric Reflux in Rats

Background: Bioactive fatty acids such as the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the modified fatty acid analogue, tetradecylthioacetic acid (TTA), are known to influence inflammatory processes in the body. Our aim was to investigate if diets containing fish oil (FO) enriched with bioactive fatty acids could affect inflammation and development of glandular stomach carcinogenesis in a duodenogastric reflux (DGR) animal model. We also wanted to evaluate if a high-fat diet might increase the risk of developing gastric cancer compared to a low-fat diet. Methods: 185 rats operated on with a gastroenterostomy were randomly allocated to 5 different treatment groups given: low-fat, high-fat, high-fat + FO, high-fat + TTA or high-fat + FO + TTA. The stomachs were removed after 50 weeks and examined by light microscopy with hematoxylin and eosin staining (HE). Immunohistochemical staining against COX-2, PCNA and p53 was performed when adenocarcinomas were found. The plasma fatty acid profile was determined. Results: Adenocarcinomas developed in 21% of animals fed the low-fat diet, 35% in the high-fat group, 16% in the high-fat + TTA group, 21% in the high-fat + FO group and 8.6% in the high-fat + FO + TTA treatment group. COX-2 and PCNA were positive whereas p53 was negative in the majority of the samples. The anti-inflammatory fatty acid index increased after treatment with FO and in combination with FO and TTA. Conclusion: FO and TTA in combination with a high-fat diet significantly lower the risk of developing adenocarcinomas in rats subjected to duodenogastric reflux. This is most likely due to a selective modulation of inflammation.

Potential implications of Helicobacter pylori-related neutrophil-activating protein

Helicobacter pylori （H. pylori） virulence factors pro- mote the release of various chemoattractants/inflam- matory mediators, including mainly the neutrophil- attractant chemokine interleukin-8 and neutrophil- activating protein （NAP）, involved in H. pylor/-induced gastric pathologies. Co-administration of Chios mastic gum （CMG）, which inhibits H. pylor/NAP, with an H. pylori eradication regimen might add clinical benefits against H. pylori-related gastric pathologies, but pos- sibly not CMG as main therapy. Although H. pylori NAP and other H. pylori-related cytotoxins [i.e., vaculating cytotoxin （VacA）] appear to play a major role in gener- ating and maintaining the H. pylori-associated gastric inflammatory response and H. pylor/NAP is a promising vaccine candidate against H. pylori infection （H. pylori-1）, concerns regarding its potential drawbacks, particularly neurogenic ones, due to possible cross- mimicry, should be considered. Possible cross-mimicry between H. p, vlor/ NAP and/or bacterial aquaporin （AQP） and neural tissues may be associated with the anti-AQP-4 antibody-related neural damage in multiple sclerosis （MS）/neuromyelitis optica patients. Moreover, the sequence homology found between H. pylori VacA and human Na＋/K＋-ATPase A subunit suggests that antibodies to VacA involve ion channels in abaxonal Schwann cell plasmalemma resulting in demyelination in some patients. A series of factors have been im- plicated in inducing blood-brain barrier （BBB） disrup- tion, including inflammatory mediators （e.g., cytokines and chemokines induced by H. pylor/-I） and oxidative stress. BBB disruption permits access of AQP4-specific antibodies and T lymphocytes to the central nervous system, thereby playing a major role in multiple sclero- sis pathogenesis. Relative studies show a strong asso- ciation between H. pylori-I and MS. H. pylor/-I induces humoral and cellular immune responses that, owing to the sharing of homologous epitopes （molecular mim- icry）, cross-react with components of nerves, thereby contributing and perpetuating neural tissue damage. Finally, H. pylori NAP also plays a possible pathoge- netic role in both gastric and colon oncogenesis.