cDNA microarray analysis of Helicobacter pylori-mediated alteration of gene expression in gastric cancer cells

Helicobacter pylori infection stimulates several intracellular signaling pathways and is accompanied by increased gene expression in gastric epithelial cells. High-density cDNA microarray was used to characterize the mRNA expression profile of genes in human gastric cancer cells (MKN45, AGS) cocultured with H. pylori. Coculture with cag pathogenicity island (PAI)-positive H. pylori (wild-type) significantly up-regulated mRNA expression in 8 of 2304 genes tested. In 6 (interleukin-8, I(kappaB)alpha, A20, ERF-1, keratin K7, glutathione peroxidase) of the 8 genes, up-regulation was confirmed by RT-PCR. In coculture with isogenic cagE-negative mutant ((Delta)cagE), which encodes a type IV secretion system with other genes in the cag PAI, no significant up-regulation was found. We further analyzed the role of A20. Transfection of expression vector encoding A20 resulted in an inhibition of H. pylori-mediated NF-kappaB activation, indicating that H. pylori-mediated A20 expression could be a negative regulator of NF-kappaB activation. Taken together, these results indicate the importance of microarray technology as a tool for analyzing the complex interplay between H. pylori and the host.     

Helicobacter pylori-induced epithelial cell signalling in gastric carcinogenesis

Helicobacter pylori represents a highly successful human microbial pathogen that infects the stomach of more than half of the world's population. H. pylori induces gastric inflammation, and the diseases that can follow such infection include chronic gastritis, peptic ulcers and, more rarely, gastric cancer. The reasons why a minority of patients with H. pylori develops gastric cancer could be related to differences in host susceptibility, environmental factors and the genetic diversity of the organism. This review examines the features of H. pylori-induced epithelial cell signalling in gastric diseases. Clinical studies and animal models, and also evidence for H. pylori strain-related differences in gastric epithelial cell proliferation in vivo are discussed. In addition, the mechanisms by which H. pylori triggers hyperproliferative processes and takes direct command of epithelial cell signalling, including activation of tyrosine kinase receptors, cell-cell interactions and cell motility are reviewed.     

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21^Cip1 and p27^Kip1 expression levels were examined by bromodeoxyuridine assay, flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as their location. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G₁ arrest, up-regulation of cell cycle-regulatory proteins p21^Cip1 and p27^Kip1 was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins. [BMB Reports 2013; 46(1): 25-30]     

Distinct mechanism of Helicobacter pylori-mediated NF-kappa B activation between gastric cancer cells and monocytic cells

NF-kappaB is a critical regulator of genes involved in inflammation. Gastric epithelial cells and macrophages are considered the main sources of pro-inflammatory cytokines. We investigated NF-kappaB activation by Helicobacter pylori in MKN45 gastric epithelial cells and THP-1 monocytic cells. Although, cag pathogenicity island (PAI)-positive H. pylori (wild type) activated NF-kappaB in both cells, isogenic mutant of cagE (DeltacagE) activated it only in THP-1 cells. Supernatant from the wild type culture could activate NF-kappaB in THP-1 cells but not in MKN45 cells. High density cDNA array analysis revealed that mRNA expression of NF-kappaB-regulated genes such as interleukin (IL)-8, tumor necrosis factor-alpha (TNFalpha), and IL-1beta was significantly up-regulated by the wild type in both cells, whereas it was up-regulated by DeltacagE only in THP-1 cells. Experiments using CD14-neutralizing antibody and IL-1 receptor-associated kinase (IRAK) assay showed that both wild type and DeltacagE H. pylori activated NF-kappaB through CD14 and IRAK in THP-1 cells but not in MKN45 cells. Macrophages from C3H/HeJ mice carrying point mutation in the Toll-like receptor 4 (TLR4) gene showed decreased NF-kappaB activation and TNFalpha secretion compared with C3H/HeN mouse macrophage when treated with H. pylori. In conclusion, H. pylori-induced NF-kappaB activation in epithelial cells is dependent on cag PAI and contact but does not involve CD14 and IRAK, whereas in macrophage/monocytic cells it is independent of cag PAI or contact but involves CD14 and TLR4.     

Cellular DNA, ras p21 and p53 expression in the carcinogenesis of adenomatous colorectal polyps

OBJECTIVE: To explore the possible roles of cellular DNA, oncogene ras and tumor suppressor gene p53 in the carcinogenesis of colorectal adenomatous polyps (CAP). STUDY DESIGN: Cellular DNA content, oncogene ras and tumor suppressor gene p53 expression at the protein level were quantitatively studied with flow cytometry (FCM) in 16 cases of CAP with mild epithelial dysplasia (CAP-MD), 16 cases of CAP with moderate/severe epithelial dysplasia (CAP-M/SD) and 11 cases of cancer in adenomatous polyps (CIAP). RESULTS: Nuclear DNA contents of CAP-M/SD (DNA [DI] = 1.11 +/- 0.06) and CIAP (DI = 1.29 +/- 0.03) were significantly higher than those of CAP-MD (DI = 1.06 +/- 0.06) and normal controls (DI = 1.00, P < .005) and were in the FCM DNA aneuploidy range. The rates and amount (as determined by the fluoresence index) of mutant p53 protein expression in CAP-M/SD and CIAP were significantly higher than those in the control and CAP-MD groups. Positive rates of ras p21 expression were all high in CAP-MD, CAP-M/SD and CIAP (80%, 75% and 100%, respectively), yet the intensity of expression in the last was significantly stronger than those in the former two groups. DNA aneuploid, ras p21 and p53 coexpression were found in 10 of 11 cases of CIAP. CONCLUSION: The results suggest that cellular DNA, ras p21 and p53 are all involved in the carcinogenesis of CAP. Clinically, the appearance of DNA aneuploidy, ras p21 or p53 overexpression should be considered markers of malignant conversion in CAP.     

Redox biology and gastric carcinogenesis: the role of Helicobacter pylori

Almost half the world's population is infected by Helicobacter pylori (H. pylori). This bacterium increases the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in human stomach, and this has been reported to impact upon gastric inflammation and carcinogenesis. However, the precise mechanism by which H. pylori induces gastric carcinogenesis is presently unclear. Although the main source of ROS/RNS production is possibly the host neutrophil, H. pylori itself produces O???. Furthermore, its cytotoxin induces ROS production by gastric epithelial cells, which might affect intracellular signal transduction, resulting in gastric carcinogenesis. Excessive ROS production in gastric epithelial cells can cause DNA damage and thus might be involved in gastric carcinogenesis. Understanding the molecular mechanism of H. pylori-induced carcinogenesis is important for developing new strategies against gastric cancer.     

Several GTP-binding proteins, including p21c-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzyme S

Pseudomonas aeruginosa exoenzyme S has appeared to be relatively indiscriminate in its choice of substrates, but in fact it ADP-ribosylates only a small subset of cellular proteins and exhibits a marked preference for several different membrane-associated proteins of apparent Mr = 23,000-25,000, at least some of which appear to bind GTP. One of these is the p21 product of the proto-oncogene c-H-ras, which can be labeled to completion. ADP-ribosylation does not alter the interaction of p21c-H-ras with guanyl nucleotides, but does cause a shift in electrophoretic mobility that implies a large conformational change. Exoenzyme S modifies all of its substrates at arginine residues.     

Multifaceted p21 in carcinogenesis,stemness of tumor and tumor therapy

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21^Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.     

Effect of Helicobacter pylori-induced cyclooxygenase-2 on gastric epithelial cell kinetics: implication for gastric carcinogenesis

Background. Cyclooxygenase (COX)‐2 induced by Helicobacter pylori is thought to enhance gastric carcinogenesis by affecting the maintenance of epithelial homeostasis. Materials and Methods. Gastric biopsies from 160 subjects, 97 with nonulcer dyspepsia (47 H. pylori negative, 50 H. pylori positive) and 63 with gastric cancer were examined immunohistochemically for COX‐2 expression, cell proliferation and apoptotic indices. Results. COX‐2 expression in corpus was significantly higher in H. pylori positive than in negative non‐ulcer dyspepsia (NUD) (p < .05). Regardless of site, gastric cancer subjects had higher COX‐2 expression in both antrum and corpus compared with H. pylori negative and positive NUD (p < .005). Proliferation was higher in cancer and H. pylori positive than in negative NUD (p < .0001). Moreover, cancer had enhanced proliferation than H. pylori positive NUD in corpus greater (p = .0454) and antrum lesser (p = .0215) curvatures. Apoptosis was higher in H. pylori positive than in negative NUD (p < .05). However, both had a higher index than the cancer subjects (p < .0001). Apoptosis : proliferation ratio was higher in corpus of H. pylori negative than in positive NUD in greater (p = .0122) and lesser (p = .0009) curvatures. However, both had a higher A:P ratio than cancer cases (p = .0001). A negative correlation between COX‐2 expression and A:P ratio was found in corpus greater (r = –.176, p= .0437) and lesser (r = –.188, p= .0312) curvatures. Conclusion. The expression of COX‐2 is associated with disruption in gastric epithelial kinetics and hence may play a role in gastric carcinogenesis.     

p16 hypermethylation during gastric carcinogenesis of Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine

Inactivation of the tumor suppressor gene, p16 by CpG hypermethylation is a common event in various tumors including gastric carcinoma. The aim of this study is to investigate if p16 hypermethylation is an early and frequent event in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The frequency and timing of p16 hypermethylation during the multistep gastric carcinogenesis in Wistar rats were analyzed in various microdissected gastric lesions. The p16 methylation status and the presence of p16 protein were analyzed by methylation-specific PCR and immunohistochemistry, respectively. Results showed that p16 methylation frequency was correlated with the severity of gastric pathologic lesions, positively. For instance, p16 methylation was found in 2.7% of normal gastric epithelium (n = 36), 16.7% of chronic atrophy gastritis (n = 24), 37.5% of dysplasia (n = 24), 67.4% of gastric adenoma (n = 43), and 85.2% of gastric carcinoma (n = 27). The p16 methylation in the distal stomach epithelium was higher than that in the proximal stomach. p16 protein was expressed in all of 15 p16 unmethylated gastric epithelial samples, but not expressed in all of 12 p16 methylated samples. These results suggest that CpG island hypermethylation may account for the silencing of p16 in rat stomach and is an early event whose accumulation will finally lead to gastric carcinogenesis.     

Critical role of an endogenous gastric peroxidase in controlling oxidative damage in H. pylori-mediated and nonmediated gastric ulcer

The objective of the present study is to delineate the mechanism of oxidative damage in human gastric ulcerated mucosa despite the presence of some antioxidant enzymes. We report for the first time the critical role of an endogenous peroxidase, a major H(2)O(2) metabolizing enzyme, in controlling oxidative damage in gastric mucosa. Human gastric mucosa contains a highly active peroxidase in addition to the myeloperoxidase contributed by neutrophil. In both non-Helicobacter pylori (H. pylori)- and H. pylori-mediated gastric ulcer, when myeloperoxidase level increases due to neutrophil accumulation, gastric peroxidase (GPO) level decreases significantly. Moreover, gastric ulcer is associated with oxidative damage of the mucosa as evidenced by significant increase in lipid peroxidation, protein oxidation, and thiol depletion indicating accumulation of reactive oxygen metabolites (ROM). Mucosal total superoxide dismutase (Mn and Cu-Zn SOD) level also decreases significantly leading to increased accumulation of O(2)(*-). To investigate the plausible ROM-mediated inactivation of the GPO during ulceration, the enzyme was partially purified from the mucosa. When exposed to an in vitro ROM generating system, using Cu(2+), ascorbate, and H(2)O(2,) the enzyme gets inactivated, which is dependent on Cu(2+), ascorbate, or H(2)O(2). Insensitivity to SOD excludes inactivation by O(2)(*-). However, complete protection by catalase indicates that H(2)O(2) is essential for inactivation. Sensitivity to EDTA and hydroxyl radical *OH) scavengers indicates that GPO is inactivated most probably by *OH generated from H(2)O(2). We propose that GPO is inactivated in vivo by ROM generated by activated neutrophil. This leads to further accumulation of endogenous H(2)O(2) to cause more oxidative damage to aggravate the ulcer.     

3p21, 5q21, 9p21 and 17p13 allelic deletions accumulate in the dysplastic spectrum of laryngeal carcinogenesis and precede malignant transformation

A tissue field of somatic genetic alterations precede the histopathological phenotypic changes of carcinoma. Loss of Heterozygosity (LOH) at the sites of known or putative tumor suppressor genes is a common genetic abnormality detected in precancerous conditions. These genomic changes could be of potential use in the diagnosis and prognosis of pre-malignant laryngeal lesions. Recently the concept of laryngeal intraepithelial neoplasia (LIN) was introduced. To evaluate patients with an increased risk of developing invasive laryngeal carcinoma via a dysplasia-carcinoma progression we investigated 102 microdissected cell populations. Cell populations were procured from 15 laryngectomy specimens with different peritumoral histological changes adjacent to the squamous cell carcinoma cells and 15 laryngeal endoscopic biopsies with no evidence of malignant transformation in a 6-10-year follow-up period. Histological diagnoses were subdivided into keratosis without dysplasia (KWD), with mild dysplasia (LIN 1), with moderate dysplasia (LIN 2), and with severe dysplasia or carcinoma in situ (LIN 3). Microsatellite analysis was performed with the aim of studying LOH of 5q21 (APC), 9p21 (p16), 3p21 and 17p13 (p53) chromosomal regions. Frequent allelic losses were found in carcinoma cells at p53 (54%), p16 (66%), 3p21(87%) and 5q21(58%). Identical LOH patterns were determined in 100% of the LIN3 peritumoral cells, 60% of LIN2, 50% of LIN 1 and 25% of KWD. In contrast, histologically normal mucosae, KWD and LIN1 lesions without malignant progression showed no allelic loss. These results show that dysplasia correlates with LOH at 3p21, 5q21, 9p21 and 17p13 in early laryngeal carcinogenesis. These genomic changes in pre-malignant laryngeal lesions could be of potential use as markers for cancer risk assessment.