Minnie Y.Y. Go

Cyclooxygenase-2 Expression in Helicobacter pylori-Associated Premalignant and Malignant Gastric Lesions

Expression of cyclooxygenase-2 (COX-2) in various stages of the Helicobacter pylori-associated gastric carcinogenesis pathway has not been elucidated. We investigated the distribution and intensity of COX-2 expression in premalignant and malignant gastric lesions, and monitored the changes after H. pylori eradication. Gastric biopsies from H. pylori-infected patients with chronic active gastritis, gastric atrophy, intestinal metaplasia (IM), gastric adenocarcinoma, and noninfected controls were studied. Expression of COX-2 was evaluated by immunohistochemistry and in situ hybridization. Endoscopic biopsies were repeated 1 year after successful eradication of H. pylori in a group of IM patients for comparing COX-2 expression and progression of IM. In all H. pylori-infected patients, COX-2 expression was predominantly found in the foveolar and glandular epithelium and, to a lesser extent, in the lamina propria. In the noninfected group, only 35% of cases demonstrated weak COX-2 expression. Intensity of COX-2 was not significantly different between the chronic active gastritis, gastric atrophy, IM, and gastric adenocarcinoma groups. In 17 patients with IM, COX-2 expressions in the epithelial cells and stromal cells were reduced 1 year after H. pylori eradication. However, the changes in COX-2 expression did not correlate with progression/regression of IM. Both premalignant and malignant gastric lesions demonstrate strong COX-2 expression. Successful eradication of H. pylori leads to down-regulation of COX-2 expression but failed to reverse IM at 1 year.

Concurrent hypermethylation of multiple tumor-related genes in gastric carcinoma and adjacent normal tissues

Transcriptional silencing by CpG‐island hypermethylation now is believed to be an important mechanism of tumorigenesis. To date, studies on CpG‐island hypermethylation in gastric carcinoma and adjacent normal tissues are few.

Effects of Helicobacter pylori Eradication on Methylation Status of E-Cadherin Gene in Noncancerous Stomach

Purpose: Promoter hypermethylation of E-cadherin plays an important role on gastric cancer development. Whereas E-cadherin methylation was frequently detected in the stomach of Helicobacter pylori–infected individuals, we tested whether eradication of H. pylori alters the methylation status of the noncancerous gastric epithelium. Experimental Design: Endoscopic biopsies were taken from the antrum and corpus of H. pylori–infected subjects without gastric cancer. Presence of methylated E-cadherin sequences in the gastric specimens was detected by methylation-specific PCR. Bisulfite DNA sequencing was done to determine the topographical distribution and changes in methylation profiles with H. pylori eradication. Results: Among the 28 H. pylori–infected subjects (median age, 44.5 years), 15 (53.6%) had E-cadherin methylation detected in stomach at baseline. Discordant methylation patterns between the antrum and corpus were noted in six patients. One year after successful H. pylori eradication, there was a significant reduction in the methylation density of the promoter region and exon 1 of the E-cadherin gene as detected by bisulfite DNA sequencing (P < 0.001). Conclusion: Promoter methylation in E-cadherin was frequently detected in the stomach of H. pylori–infected individuals. Eradication of H. pylori might possibly reduce the methylation density in E-cadherin gene and the chance of subsequent neoplastic transformation.

Prevalence and distribution of Helicobacter pylori in gastroesophageal reflux disease: a study from the East

OBJECTIVES:The relationship between Helicobacter pylori infection and gastroesophageal reflux (H. pylori) disease (GERD) is controversial. In Asian populations, the prevalence of H. pylori infection is high and GERD is relatively uncommon. The aim of this study was 1) to test the hypothesis that H. pylori protects the esophagus against GERD, and 2) to study the pattern of H. pylori colonization and gastritis in GERD.METHODS:We conducted a prospective case-control study in which patients with GERD and asymptomatic controls were compared for the prevalence of H. pylori infection. Diagnosis of GERD was based on symptoms of heartburn that improved with acid-suppressive therapy and/or endoscopic evidence of erosive esophagitis. H. pylori status was determined by serology and, when endoscopy was indicated, was confirmed by rapid urease test and histology. Gastric biopsies were examined under hematoxylin and eosin and Giemsa stains. Density of H. pylori colonization and activity of gastritis at different parts of stomach were graded and compared according to Updated Sydney system.RESULTS:A total of 106 patients with GERD and 120 age- and sex-matched, asymptomatic controls were enrolled. The prevalence of H. pylori infection was significantly lower in GERD patients (31%) compared with controls (61%, p < 0.001, odds ratio 0.229, 95% confidence interval 0.13–0.41). H. pylori-infected GERD patients showed significantly more severe gastritis in the antrum than in other parts of stomach (mean inflammatory scores: antrum; 3.3 ± 1.63*, body; 1.85 ± 1.31; fundus; 1.65 ± 0.58; cardia, 1.65 ± 1.39; *p < 0.005). H. pylori colonization was found less commonly and at lower density at the cardia compared with other parts of the stomach.CONCLUSIONS:H. pylori infection protects against the development of GERD, and carditis is unlikely to play an important role.

Expression of HBx and COX-2 in chronic hepatitis B, cirrhosis and hepatocellular carcinoma: implication of HBx in upregulation of COX-2

Hepatitis B virus is a major etiological factor of hepatocellular carcinoma, but the underlying mechanisms remain unclear. We have previously demonstrated that upregulation of cyclooxygenase (COX)-2 in chronic hepatitis B persisted despite successful antiviral therapy. In this study, we investigated the relationship between the transactivator HBx and COX-2 in hepatitis B virus-associated chronic liver diseases. Expressions of HBx and COX-2 in tissue specimens were determined by single and double immunohistochemistry. The effects of HBx on COX-2 and prostaglandin E2 production were studied by transfection. HBx was expressed in 11/11 (100%) of chronic hepatitis B, 23/23 (100%) of cirrhosis, and 18/23 (78%) of hepatocellular carcinoma, whereas no immunoreactivity was found in four nonalcoholic steato-hepatitis controls. COX-2 expression was also detected in all specimens of liver lesions except in only 29% of poorly differentiated hepatocellular carcinoma. Significant correlation between HBx and COX-2 immunoreactivity scores was found in different types of chronic liver diseases (chronic hepatitis B, rs=0.68; cirrhosis, rs=0.57; hepatocellular carcinoma, rs=0.45). Double immunohistochemistry showed colocalization of HBx and COX-2 in hepatic parenchymal cells. Similar to COX-2, there was no significant change in HBx expression in patients with chronic hepatitis B after interferon and lamivudine therapy when hepatitis B virus DNA became undetectable and inflammation subsided. Transfection of Hep3B hepatocellular carcinoma cells with HBx increased COX-2 expression and prostaglandin E2 production. HBx was localized mainly in the cytoplasm and less in nucleus, as found in the liver lesions. In conclusion, our results strongly suggested that there was a close relationship between HBx and COX-2. COX-2 might represent an important cellular effector of HBx that contributes to hepatitis B virus-associated hepatocarcinogenesis.

T1ρ MR Imaging Is Sensitive to Evaluate Liver Fibrosis: An Experimental Study in a Rat Biliary Duct Ligation Model

PURPOSE To correlate spin-lattice relaxation time in the rotating frame (T1ρ) measurements with degree of liver fibrosis in a rat model. MATERIALS AND METHODS The protocols and procedures were approved by the local Animal Experimentation Ethics Committee. Liver fibrosis was induced with biliary duct ligation (BDL). Two studies, 1 month apart, were performed with a 3-T clinical imager. The first study involved longitudinal magnetic resonance (MR) imaging follow-up of BDL rats (n = 8) and control rats (n = 4) on days 8, 15, 21, and 29 after BDL. The second study involved MR imaging of another group of BDL and control rats (n = 5 for each) on days 24 and 38 after BDL. Hematoxylin-eosin and picrosirius red staining were performed in liver specimens from days 8, 15, 24, and 38 after BDL. Repeated-measures analysis of variance was used, and treatment groups were compared (Bonferroni adjustment). RESULTS On day 8, there were proliferation of bile duct and inflammatory cell infiltration around portal triads. While there was overlap, BDL rats (n = 8) demonstrated higher mean liver T1ρ values than did control rats (n = 4) on day 8 (46.7 msec ± 2.9 [standard deviation] vs 44.7 msec ± 1.2, P = .4). On day 15, BDL rats demonstrated liver fibrosis with a background of inflammatory infiltration. On day 15, mean T1ρ values in BDL rats could be largely separated from those in control rats (52.6 msec ± 6.0 vs 43.8 msec ± 1.5, P = .02). On day 24, BDL rats had liver T1ρ values 23.5% higher than in control rats (n = 5 for each group, P = .0007). Histomorphometric analysis showed that collagen content increased after surgery from days 8 to 24 (n = 6 for each group, P < .0001), with no further increase between days 24 and 38 (n = 6 for each group, P >.99). CONCLUSION In this model, liver fibrosis was detected with T1ρ MR imaging; the degree of fibrosis was correlated with degree of increase in T1ρ measurements. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11101638/-/DC1.

Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro

Nonalcoholic steatohepatitis with fibrosis is a more severe form of nonalcoholic fatty liver disease, one of the most common liver diseases. We have previously shown that peroxisome proliferator-activated receptors gamma (PPARgamma) ligand, rosiglitazone, prevented the development of the methionine choline deficient (MCD) diet-induced fibrosing steatohepatitis. We have now tested whether overexpression of PPARgamma ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice fed with MCD diet for 8 weeks developed hepatic fibrosis with increased hepatic expression of collagen1alpha(I), inhibitors of fibrosis reversal-1, regulator involved in matrix degradation-9 and connective tissue growth factor. After 2 weeks of transduction of PPARgamma through an adenovirus-expressing PPARgamma (Ad-PPARgamma), expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells (HSCs) and resolution of liver fibrosis. On the in vitro study, PPARgamma is expressed in primary quiescent HSC, but depleted in culture activated HSC. Conversely, ectopic expression of PPARgamma in activated HSC achieved the phenotypic reversal to the quiescent cell. Such induction markedly suppressed cell viability and cell proliferation, downregulated proliferating cell nuclear antigen, and caused cell cycle arrest at G0/G1 phase. Further, introduction of PPARgamma in HSC increased cell apoptosis, this was confirmed by enhanced expression of FasL, cleaved caspase-3, cleaved caspase-7 and poly ADP-ribose polymerase, indicating an extrinsic apoptosis pathway. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by overexpression of PPARgamma. It is likely that PPARgamma reverses fibrosis by reducing HSCs proliferation, inducing cell cycle arrest and apoptosis.

Epigenetic inactivation of BCL6B, a novel functional tumour suppressor for gastric cancer, is associated with poor survival

Objective Using genome-wide promoter methylation assay, B cell CLL/lymphoma 6 member B (BCL6B) was found to be preferentially methylated in cancer. A study was undertaken to examine the epigenetic regulation, biological function and clinical significance of BCL6B in gastric cancer (GC). Methods BCL6B promoter methylation was evaluated by combined bisulfite restriction analysis and sequencing. The biological functions of BCL6B were determined by cell viability, colony formation, flow cytometry and in vivo tumorigenicity assays. The molecular targets of BCL6B were identified by cDNA expression array. Results BCL6B was silenced or downregulated in all nine GC cell lines and readily expressed in normal gastric tissues. Loss of BCL6B expression was regulated by promoter hypermethylation. Re-expression of BCL6B in GC cell lines inhibited colony formation, suppressed cell viability, induced apoptosis and restrained the tumorigenecity in nude mice. These effects were associated with upregulation of the pro-apoptosis genes tumour necrosis factor receptor superfamily member 1A, caspase-8, caspase-9, caspase-3 and caspase-7 and nuclear enzyme poly (ADP-ribose) polymerase, downregulation of the pro-proliferation genes S100 calcium binding protein A4 and vascular endothelial growth factor A, and induction of the tumour suppressor genes ataxia telangiectasia mutated homologue and p53. BCL6B hypermethylation was detected in 49.0% (102/208) and 66.3% (67/101) of two independent cohorts of patients with GC, respectively. BCL6B methylation was an independent factor for the survival of patients with GC (p=0.001 for cohort I, p=0.02 for cohort II). Conclusions BCL6B plays a pivotal role as a potential tumour suppressor in GC. Detection of methylated BCL6B may serve as an independent biomarker for the prognosis of GC.

CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis

BACKGROUND & AIMS Perpetuate liver inflammation is crucial in the pathogenesis of non-alcoholic steatohepatitis (NASH). Expression of CXCL10, a pro-inflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 plays a role in NASH was unknown. We aimed to investigate the functional and clinical impact of CXCL10 in NASH. METHODS Cxcl10 gene-deleted (Cxcl10(-/-)) and C57BL/6 wild type (WT) mice were fed a methionine- and choline-deficient (MCD) diet for 4 or 8 weeks. In other experiments, we injected neutralizing anti-CXCL10 mAb into MCD-fed WT mice. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 control subjects. RESULTS WT mice, fed the MCD diet, developed steatohepatitis with higher hepatic CXCL10 expression. Cxcl10(-/-) mice were refractory to MCD-induced steatohepatitis. We further revealed that CXCL10 was associated with the induction of important pro-inflammatory cytokines (TNF-α, IL-1β, and MCP-1) and activation of the NF-κB pathway. CXCL10 was linked to steatosis through upregulation of the lipogenic factors SREBP-1c and LXR, and also to oxidative stress (upregulation of CYP2E1 and C/EBPβ). Blockade of CXCL10 protected against hepatocyte injury in vitro and against steatohepatitis development in mice. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, the circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. CONCLUSIONS We demonstrate for the first time that CXCL10 plays a pivotal role in the pathogenesis of experimental steatohepatitis. CXCL10 maybe a potential non-invasive biomarker for NASH patients.

Paired box gene 5 is a novel tumor suppressor in hepatocellular carcinoma through interaction with p53 signaling pathway

The paired box 5 (PAX5) is a member of PAX transcription factors family involved in the regulation of embryonic development. However, the role of PAX5 in carcinogenesis is largely unclear. We identified that PAX5 is involved in human cancer by methylation‐sensitive representational difference analysis. We examined the biological functions and related molecular mechanisms of PAX5 in hepatocellular carcinoma (HCC). Promoter methylation of PAX5 was evaluated by methylation‐specific polymerase chain reaction (PCR) and bisulfite genomic sequencing (BGS). The functions of ectopic PAX5 expression were determined by viability assay, colony formation, and cell cycle analyses, along with in vivo tumorigenicity assays. The PAX5 target signal pathway was identified by promoter luciferase assay, chromosome immunoprecipitation (ChIP), and pathway PCR array. PAX5 is expressed in normal human liver tissue, but silenced or down‐regulated in 83% (10/12) of HCC cell lines. The mean expression level of PAX5 was significantly lower in primary HCCs as compared to their adjacent normal tissues (P < 0.0001). The promoter methylation contributes to the inactivation of PAX5. Restoring PAX5 expression in silenced HCC cell lines suppressed cell proliferation, induced apoptosis in vitro, and inhibited tumor growth in nude mice (P < 0.0001). The pathway luciferase reporter assay indicated that PAX5 activated p53 and p21 signaling. ChIP analysis demonstrated that PAX5 directly bound to the p53 promoter. The antitumorigenic function of PAX5 was at least up‐regulated by p53 and its downstream targets including tumor necrosis factor, Fas ligand, leucine‐rich repeats, and death domain‐containing, poly(rC) binding protein 4, p21, and growth arrest and DNA‐damage‐inducible alpha. Conclusion: PAX5 is frequently inactivated by promoter methylation in HCC. PAX5 appears to be a functional tumor suppressor involved in liver carcinogenesis through direct regulation of the p53 signaling pathway. (HEPATOLOGY 2011)