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Supplementary Figure 2 from Ectopic Expression of P-Cadherin Correlates with Promoter Hypomethylation Early in Colorectal Carcinogenesis and Enhanced Intestinal Crypt Fission In vivo

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential “field effect” of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia. [Cancer Res 2008;68(19):7760–8]

Supplementary Figure 1 from Ectopic Expression of P-Cadherin Correlates with Promoter Hypomethylation Early in Colorectal Carcinogenesis and Enhanced Intestinal Crypt Fission In vivo

Supplementary Materials from Ectopic Expression of P-Cadherin Correlates with Promoter Hypomethylation Early in Colorectal Carcinogenesis and Enhanced Intestinal Crypt Fission In vivo

Background: The results of Aspirin prevention of colorectal adenomas in patients with familial adenomatous polyposis (FAP) are controversial. Methods: We conducted a biomarker-based clinical study in eight FAP patients treated with enteric-coated low-dose Aspirin (100 mg daily for three months) to explore whether the drug targets mainly platelet cyclooxygenase (COX)-1 or affects extraplatelet cellular sources expressing COX-isozymes and/or off-target effects in colorectal adenomas. Results: In FAP patients, low-dose Aspirin-acetylated platelet COX-1 at Serine529 (>70%) was associated with an almost complete inhibition of platelet thromboxane (TX) B2 generation ex vivo (serum TXB2). However, enhanced residual urinary 11-dehydro-TXB2 and urinary PGEM, primary metabolites of TXA2 and prostaglandin (PG)E2, respectively, were detected in association with incomplete acetylation of COX-1 in normal colorectal biopsies and adenomas. Proteomics of adenomas showed that Aspirin significantly modulated only eight proteins. The upregulation of vimentin and downregulation of HBB (hemoglobin subunit beta) distinguished two groups with high vs. low residual 11-dehydro-TXB2 levels, possibly identifying the nonresponders and responders to Aspirin. Conclusions: Although low-dose Aspirin appropriately inhibited the platelet, persistently high systemic TXA2 and PGE2 biosynthesis were found, plausibly for a marginal inhibitory effect on prostanoid biosynthesis in the colorectum. Novel chemotherapeutic strategies in FAP can involve blocking the effects of TXA2 and PGE2 signaling with receptor antagonists.

As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition.

Background: As a consequence of altered glucose metabolism, cancer cells intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs).MCTs are frequently overexpressed in tumors, MCT1 in the well oxygenated areas and MCT4 in hypoxic regions, and recent evidence has reported that MCT inhibition can exert antineoplastic effects, but this aspect has not been investigated in esophageal adenocarcinoma (EAC) yet. In the present study, MCT1 and MCT4 levels were assessed in EAC cells and the effects of MCT-1 selective inhibitor AZD3965, hypoxia and a glucose overload were evaluated in vitro .Methods: Two EAC cell lines (OE33 and OACM5.1C) were treated with different concentrations of AZD3965 (10-100nM) in the presence or absence of a glucose overload under normoxic or hypoxic conditions and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi) and lactate levels were evaluated.Results: MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small population of the metastatic cells.Glucose addition did not have any effect on apoptosis nor cell proliferation.AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia.MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress.AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4, and also increased NHE-1 expression on these cells. Conclusion: These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition.

p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative (methylated/unmethylated) instead of accurate quantitative (percentage of CpG methylation) data. We aimed to measure the degree of p16 methylation in pure samples representing all the steps of Barrett's tumorogenesis and to evaluate the influence of sample heterogeneity in methylation analysis.77 paraffin-embedded human esophageal samples were analyzed. Histological grading was established by two pathologists in: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Areas of interest were selected by laser-capture microdissection. p16 methylation was quantified by pyrosequencing. An adjacent section of the whole sample was also analyzed to compare methylation data.After microdissection, we obtained 15 samples of squamous epithelium, 36 non-dysplastic Barrett's esophagus, 3 indefinite for dysplasia, 24 low-grade dysplasia, 4 high-grade dysplasia and 12 adenocarcinoma. Squamous epithelium showed the lowest methylation rates: 6% (IQR 5-11) vs. 11%(7-39.50) in negative/indefinite for dysplasia, p0.01; 10.60%(6-24) in low-grade dysplasia, p0.05; and 44.50%(9-66.75) in high-grade dysplasia/adenocarcinoma, p0.01. This latter group also exhibited higher methylation rates than Barrett's epithelium with and without low-grade dysplasia (p0.05). p16 methylation rates of microdissected and non-microdissected samples did not correlate unless the considered histological alteration comprised71% of the sample.p16 methylation is an early event in Barrett's carcinogenesis which increases with the severity of histological alteration. p16 methylation rates are profoundly influenced by sample heterogeneity, so selection of samples is crucial in order to detect differences.

Recent observational studies have shown therapeutic benefits of acetylsalicylic acid (ASA) in several types of cancer. We examined whether ASA exerts antitumor activity in esophageal adenocarcinoma (EAC). Human EAC cells (OE33) were treated with ASA (0–5 mM) to evaluate proliferation, apoptosis, and migration. In vivo model: OE33-derived tumors were subcutaneously implanted into athymic mice which were allocated to ASA (5 or 50 mg/kg/day)/vehicle (5–6 animals/group). Tumor growth was assessed every 2–3 days, and after 40 days, mice were euthanized. Plasma drug levels were determined by high-performance liquid chromatography. Histological and immunohistochemical (Ki67, activated caspase-3) analysis of tumors were performed. The effect of ASA on tumor prostaglandin E2 (PGE2) levels was also evaluated. In vitro cell proliferation and migration were significantly inhibited while apoptosis increased (p

A large body of evidence from epidemiological and preclinical studies have shown that nonsteroideal anti-inflammatory drugs (NSAIDs) have a chemopreventive effect on gastrointestinal cancers and, more specifically, in colorectal cancer. This review highlights recent advances in our understanding of the role of NSAIDs in colorectal cancer prevention and adjuvant treatment. Moreover, we have focused on randomized controlled studies assessing their efficacy to prevent adenoma recurrence and reduction of colorectal cancer incidence and mortality but also their gastrointestinal and cardiovascular side effects. Among NSAIDs, almost the unique agent with potential use as chemopreventive agent is aspirin at low dose since it has both no cardiovascular and low gastrointestinal risk. Furthermore, since aspirin has shown efficacy in secondary prevention of cardiovascular diseases, this drug carries a particular attractive intervention for selected populations. Nevertheless, before it can be prescribed, further studies are necessary to define some important questions, specially the most appropriate dose and time of aspirin use and the population who may benefit from it.

Two recent genome-wide association studies in Asians have reported the association between the PSCA (prostate stem cell antigen) rs2294008C>T gene polymorphism and two Helicobacter pylori infection-related diseases such as gastric cancer (GC) and duodenal ulcer (DU). Since rs2294008 allele frequencies differ notably among ethnicities, we aimed to assess the role of rs2294008 on the susceptibility to GC and DU in a Caucasian population in Spain. Moreover, the relevance of rs2294008 on GC prognosis was evaluated. Genomic DNA from 603 Spanish patients with primary GC, 139 with DU and 675 healthy controls was typed for the PSCA rs2294008C>T polymorphism by PCR-TaqMan assays. H. pylori infection [odds ratio (OR): 8.27; 95% confidence interval (CI): 3.45-15.33] and nonsteroidal anti-inflammatory drugs (OR: 6.54; 95% CI: 3.19-12.43) were identified as independent risk factors for DU whereas the rs2294008T allele was associated with reduced risk of developing the disease (OR: 0.52; 95% CI: 0.33-0.82). Infection with CagA strains (OR: 2.10; 95% CI: 1.63-2.34), smoking (OR: 1.93; 95% CI: 1.54-2.61), family history of GC (OR: 2.83; 95% CI: 2.01-3.83), and the rs2294008T allele (OR: 1.46; 95% CI: 1.07-1.99) were associated with increased risk of GC. Interestingly, the association with the rs2294008T allele was restricted to noncardia GC (OR: 1.43; 95% CI: 1.12-1.82), particularly of the diffuse histotype (OR: 1.59; 95% CI: 1.16-1.92). Finally, Cox regression analysis identified the rs2294008T variant as a prognosis factor associated with worse overall survival in patients with diffuse-type GC (hazard ratio: 1.85; 95% CI: 1.12-3.06). From these results we conclude that the PSCA rs2294008 polymorphism is involved in the susceptibility to GC and DU, as well as in the prognosis of the diffuse-type of GC in Caucasians.

The mechanism of action of low-dose aspirin in the prevention of colorectal cancer (CRC) remains largely hypothetical. We aimed to compare the effects of low-dose aspirin (100 mg/day for 7 days) given to 40 individuals undergoing CRC screening on the extent of cyclooxygenase (COX)-1 acetylation at serine-529 (AceCOX-1), in blood platelets vs. colorectal mucosa, at 7 (group 1) and 24 h (group 2) after dosing. A significantly (P < 0.01) lower %AceCOX-1 was detected in colonic and rectal mucosa (average 64%) vs. platelets (average 75%) in both groups. This effect was associated with an average 46% (P < 0.01) and 35% (P < 0.05) reduction in prostaglandin (PG) E2 levels and phosphorylated S6 (p-S6) levels, respectively. Rectal mucosal levels of p-S6/S6 significantly (P < 0.01) correlated with PGE2 . These findings demonstrate that low-dose aspirin produces long-lasting acetylation of COX-1 and downregulation of p-S6 in human colorectal mucosa, an effect that may interfere with early colorectal carcinogenesis.

Introduction The current staging systems for hepatocellular carcinoma (HCC) do not sufficiently predict outcomes after liver transplantation (LT). The present study assessed whether some tissue markers related to proliferation and angiogenesis have prognostic value. Patients and methods The expression of CD34, vascular endothelial growth factor (VEGF), VEGFR2, VEGFR1, angiopoietin-1, angiopoietin-2, TIE2, COX-2, and proliferating cell nuclear antigen (PCNA) in tumor and adjacent cirrhotic tissue samples from 36 patients with HCC (n=10 with tumor recurrence after LT) was determined by immunochemistry. Microvessel density was assessed by CD34 staining and the PCNA labeling index calculated as the percentage of positive cells among at least 1000 hepatocyte nuclei studied in each sample using the computer program ContimUZ. VEGF, VEGFR2, VEGFR-1, angiopoietin-1, angiopoietin-2, TIE2, and COX-2 staining were evaluated by two blinded pathologists. The tumor recurrence rate was analyzed after a minimum follow-up of 36 months. Results A higher proliferation index in both tumor and adjacent cirrhotic tissue was related to HCC recurrence. The proliferation index in tumor tissue was also related to microvascular invasion. High expression (staining in ≥50% of hepatocytes) of COX2 [P=0.025, odds ratio (OR)=7.5, 95% confidence interval (CI) 1.3-43.4], VEGF (P=0.01, OR=12, 95% CI 1.8-80.4), and its receptor VEGFR-2 (P=0.02, OR=8.5, 95% CI 1.4-49.5) in cirrhotic liver tissue, but not tumor tissue, was related to HCC recurrence after LT. Conclusion A high proliferation index in tumor and cirrhotic tissue and high expression levels of some angiogenic markers in adjacent cirrhotic tissue could be predictive of tumor recurrence after LT.

Purpose. To evaluate the dose effect of vitamin K3on wound healing mechanisms.Methods. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [3H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K31 mg/L, group 4/vitamin K32 mg/L, group 5/vitamin K34 mg/L, and group 6/vitamin K36 mg/L. Each experiment was carried out in triplicate and 4 times.Results. There were no differences among groups at the initial time.In vitrowound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K31 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K31 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented.Conclusions. Vitamin K3is able to inhibit fibroblast proliferation. Vitamin K32 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

Variations in DNA repair genes have been reported as key factors in gastric cancer (GC) susceptibility but results among studies are inconsistent. We aimed to assess the relevance of DNA repair gene polymorphisms and environmental factors to GC risk and phenotype in a Caucasian population in Spain. Genomic DNA from 603 patients with primary GC and 603 healthy controls was typed for 123 single nucleotide polymorphisms in DNA repair genes using the Illumina platform. Helicobacter pylori infection with CagA strains (odds ratio (OR): 1.99; 95% confidence interval (CI): 1.55-2.54), tobacco smoking (OR: 1.77; 95% CI: 1.22-2.57), and family history of GC (OR: 2.87; 95% CI: 1.85-4.45) were identified as independent risk factors for GC. By contrast, the TP53 rs9894946A (OR: 0.73; 95% CI: 0.56-0.96), TP53 rs1042522C (OR: 0.76; 95% CI: 0.56-0.96), and BRIP1 rs4986764T (OR: 0.55; 95% CI: 0.38-0.78) variants were associated with lower GC risk. Significant associations with specific anatomopathological GC subtypes were also observed, most notably in the ERCC4 gene with the rs1799801C, rs2238463G, and rs3136038T variants being inversely associated with cardia GC risk. Moreover, the XRCC3 rs861528 allele A was significantly increased in the patient subgroup with diffuse GC (OR: 1.75; 95% CI: 1.30-2.37). Our data show that specific TP53, BRIP1, ERCC4, and XRCC3 polymorphisms are relevant in susceptibility to GC risk and specific subtypes in Caucasians.

A large body of evidence from epidemiological, experimental and clinical studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors (COXIBs) have a chemopreventive effect on gastrointestinal cancers and, more specifically, in colorectal cancer (CRC). This chapter summarises scientific evidence derived from clinical studies assessing the role of NSAIDs and COXIBs in the prevention of both sporadic and hereditary CRC. Unfortunately, adverse side effects associated to both of them, mainly at gastrointestinal and cardiovascular level, make difficult their use for primary chemoprevention. Among NSAIDs, almost the unique agent with potential use as chemopreventive agent is aspirin at low dose since it has both no cardiovascular and low gastrointestinal risk. Therefore, currently, the use of nonaspirin NSAIDs (NA-NSAIDs) or COXIBs is restricted to hereditary colorectal cancer patients, to delay the progression of polyposis. In addition, the potential use of these drugs in established colorectal cancer is being tested in ongoing clinical trials. In the last years, different chemical modifications have been introduced in some conventional NSAIDs in an attempt to improve their potency and safety. Although some of these compounds have been evaluated in experimental studies with promising results, further studies are warranted before they can be prescribed in clinical setting to prevent CRC.

A large body of evidence from basic science, epidemiologic observations and population-based studies demonstrates that aspirin, as well as other non-steroidal anti-inflammatory drugs, has a chemopreventive effect on several cancer types and, more specifically, in CRC. This protective effect includes prevention of adenoma recurrence and reduction of CRC incidence and mortality. Although the protective effect appears to depend on the dose and the drug, the most important factor is the duration of exposure. However, the lowest effective dose, treatment duration, specific target populations, and effects on survival have not been defined yet. More important, data on the risk–benefit profile for cancer prevention are insufficient and, accordingly, no definitive recommendation can be made at present. In this article, besides reviewing current knowledge of the mechanisms involved in aspirin-based CRC chemoprevention, we will be focused on randomized controlled studies assessing its efficacy in high-, moderate- and average-risk populations.

Bernal V, Pascual I, Lanas A, Esquivias P, Piazuelo E, Garcia-Gil FA, Lacambra I, Simon MA. Cardiac function and aminoterminal pro-brain natriuretic peptide levels in liver-transplanted cirrhotic patients. Clin Transplant 2012: 26: 111–116. © 2011 John Wiley & Sons A/S. Abstract: Background: Cirrhosis is associated with structural and functional abnormalities of the heart. We examined the evolution of these abnormalities after liver transplantation (LT). Methods: Sixty cirrhotic patients, without cardiovascular disease, were included. Clinical data, echocardiography, and aminoterminal pro-brain natriuretic peptide (NT-proBNP) levels were analyzed before and after transplantation. Healthy controls (n = 25) were included for reference. Results: Before transplantation, cirrhotic patients had higher left atrium diameter, left ventricular (LV) mass index, and ejection fraction than controls. After transplantation, LV mass index increased (105 ± 31 vs. 119 ± 35 g/m2; p

Aliment Pharmacol Ther 31, 440–451 Summary Background Accumulating evidence suggests that cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is involved in oesophageal adenocarcinogenesis. PGE2 exerts its biological action by binding to specific receptors (EP1, EP2, EP3 and EP4). Aim To investigate which PGE2 receptor subtypes regulate PGE2 signals in the oesophageal adenocarcinoma sequence. Methods Expression was determined in oesophageal biopsies from 85 patients with oesophagitis, Barrett’s metaplasia, intraepithelial neoplasia, oesophageal adenocarcinoma and normal oesophagus. Levels of mRNA and protein expression were determined by quantitative PCR, immunohistochemistry and western-blot. Expression of EP receptors was also determined in response to acid and bile exposure in the Barrett’s adenocarcinoma cell line OE33. Results All four EP receptors subtypes were expressed in human oesophageal tissues. COX-2 and, especially, EP2 were increased in the Barrett’s metaplasia-intraepithelial neoplasia-adenocarcinoma sequence. Expression of the EP4 receptor protein was increased in oesophageal adenocarcinoma. In contrast, expression levels of COX-1 and EP3 receptor were decreased along the sequence. No differences in EP1 expression were found. Treatment with the bile acid deoxycholate increased COX-2, EP1, EP2 and EP4 expression in OE33 cells. Conclusions Our data suggest that in addition to COX-2, EP2 and EP4 receptors could be a selective target in the prevention and/or treatment of the Barrett’s-associated adenocarcinoma.

We aimed to evaluate the influence of Helicobacter pylori infection and IL-1/TNF gene polymorphisms on interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha gastric mucosal production. IL-1beta and TNF-alpha levels in homogenized biopsy specimens taken from the antrum and corpus of 81 patients were measured by enzyme-linked immunosorbent assay. Genomic DNA was typed for the IL1B-511, IL1B+3954, variable number of tandem repeat (VNTR) IL1RN, TNFA-308, TNFA-238, LTA NcoI, and LTA Bsi gene polymorphisms by polymerase chain reaction, restriction fragment length polymorphism, and TaqMan assays. H. pylori infection and CagA/VacA antibody status were determined by Western blot. IL-1beta and TNF-alpha protein levels were significantly higher in the gastric antrum of patients infected with H. pylori compared with uninfected patients [9.54 (5.07-16.28) vs. 4.55 (3.69-8.28) pg IL-1beta/mg protein, p = 0.004, and 1.5 (0.7-2.71) vs. 0.63 (0.3-1.26) pg TNF-alpha/mg protein, p = 0.001]. Among H. pylori-infected individuals, carriers of the IL1RN*2 allele had significantly higher antrum mucosal IL-1beta levels than noncarriers [15.97 (9.59-26.6) vs. 10.08 (7.72-13.33), p = 0.008]. No association between gastric mucosal TNF-alpha levels and genotypes of the TNFA and LTA gene polymorphisms was reported. Our results indicate that the VNTR polymorphism of the IL1RN gene influences IL-1beta gastric mucosal production in patients infected with H. pylori.

Background: Previous studies have reported a potential genetic predisposition to NSAID-related upper gastrointestinal (GI) bleeding. Objective: This study evaluated whether there was an association between 2 polymorphisms-the platelet glycoprotein (GP) IIIa PlA1/A2 polymorphism and the 27-bp VNTR (variable number of tandem repeats) polymorphism in intron 4 of the endothelial nitric oxide synthase ( e NOS) gene-and a risk for nonvariceal upper GI bleeding in Spanish patients taking low-dose aspirin for secondary prophylaxis of vascular occlusive diseases. Methods: Genotyping for the 2 polymorphisms was performed in patients hospitalized for upper GI bleeding associated with the use of low-dose aspirin between September 1998 and October 2000, and race-, age-, and sex-matched controls who were taking low-dose aspirin but had no history of upper GI bleeding. To ascertain allele frequencies in a healthy population, genotyping was also performed in an unmatched group of blood donors. Results: The study included 88 white patients (65 men, 23 women; mean age, 67.5 years) with an episode of upper GI bleeding, 108 matched controls with no history of upper GI bleeding (79 men, 29 women; mean age, 65.9 years), and 158 blood-donor controls (109 men, 49 women; mean age, 53.4 years). No significant differences were found between cases and controls in terms of genotype, carriage, or allele frequency of the GPIIIa PlA1/A2 polymorphism. However, after adjustment for confounding variables, logistic regression analysis indicated an association between carriage of the eNOS "a" allele and a reduced risk of upper GI bleeding (odds ratio [OR] ＝ 0.39; 95％ CI, 0.18-0.85; P ＝ 0.018). In this model, treatment with ni-trovasodilators (OR ＝ 0.28; 95％ CI, 0.12-0.66; P ＝ 0.004) and use of antisecretory drugs (OR ＝ 0.15; 95％ CI, 0.05-0.47; P ＝ 0.001) were also identified as protective factors. Helicobacter pylori infection (OR ＝ 3.07; 95％ CI, 1.23-7.70; P ＝ 0.017), alcohol consumption (OR ＝ 5.04; 95％ CI, 1.86-13.70; P ＝ 0.001), and a history of peptic ulcer (OR ＝ 13.41; 95％ CI, 3.78-47.64; P ＜ 0.001) were identified as risk factors for upper GI bleeding. Conclusion: In this small, selected population of individuals taking low-dose aspirin for secondary prevention, carriage of the "a" allele of the eNOS gene was associated with a decreased risk for upper GI bleeding.

Recent studies have reported an association between cytokine gene polymorphisms and GC risk. However, results are inconsistent among studies from different geographic regions and ethnic groups. Our goal was to evaluate the influence of Helicobacter pylori (H. pylori) infection and host genetic factors on GC susceptibility in a population of Spanish white GC patients.DNA from 404 unrelated patients with GC and 404 sex- and age-matched healthy controls was typed for several functional polymorphisms in pro- (IL-1B, TNFA, LTA, IL-12p40) and anti-inflammatory (IL-4, IL-1RN, IL-10, TGFB1) genes by PCR, RFLP, and TaqMan assays. H. pylori infection and CagA/VacA antibody status were also determined by western blot serology.Logistic regression analysis identified H. pylori infection with cagA strains (OR 2.54, 95% CI 1.77-3.66), smoking habit (OR 1.91, 95% CI 1.25-2.93), and positive family history of GC (OR 3.67, 95% CI 2.01-6.71) as independent risk factors for GC. None of the cytokine gene polymorphisms analyzed in this study were associated with susceptibility to GC development, whether GC patients were analyzed as a group or categorized according to anatomic location or histological subtype. Some simultaneous combinations of proinflammatory genotypes reportedly associated with greater GC risk yielded no significant differences between patients and controls.Our results show that, at least in some white populations, the contribution of the cytokine gene polymorphisms evaluated in this study (IL-1B, IL-1RN, IL-12p40, LTA, IL-10, IL-4, and TGF-B1) to GC susceptibility may be less relevant than previously reported.

Summary Background There is an overexpression of cyclo-oxygenase 2 (COX-2) in Barrett’s oesophagus (BO). Aim To determine the long-term effect of a COX-2 inhibitor on cellular mechanisms involved in BO. Methods A randomized controlled trial was conducted in BO patients allocated to continue the usual proton pump inhibitor (PPI) alone treatment, or PPI combined with rofecoxib (25 mg/day) for 6 months. Cell proliferation index and COX-2 expression in BO glands was determined in biopsy specimens at baseline and after treatment. Cell apoptosis, cyclin D1, p53 and vascular endothelial growth factor (VEGF) expression was also explored in a subset of patients. Student-t test and the U-Mann–Whitney test were used for quantitative and ordinal variables. Results Of 62 patients, 58 completed the study. A higher proportion of patients on rofecoxib + PPI exhibited a decrease in COX-2 expression compared to those treated with PPI alone, but cell proliferation index was not affected. Unlike PPI alone, rofecoxib + PPI was associated with an increase in the apoptotic cell index, a decrease in p53 cell staining and VEGF expression in mucosal vessels. No effect on low-grade dysplasia or cyclin D1 was observed. Conclusions The addition of rofecoxib to PPI therapy does not affect cell proliferation index in BO cells after 6 months of therapy, but does reduce COX-2 and VEGF expression and increases cell apoptosis.

Accumulating evidence suggests that COX-2-derived prostaglandin E 2 (PGE 2 ) plays an important role in esophageal adenocarcinogenesis. Recently, PGE 2 receptors (EP) have been shown to be involved in colon cancer development. Since it is not known which receptors regulate PGE 2 signals in esophageal adenocarcinoma, we investigated the role of EP receptors using a human Barrett's-derived esophageal adenocarcinoma cell line (OE33). OE33 cells expressed COX-1, COX-2, EP 1 , EP 2 and EP 4 but not EP 3 receptors as determined by real time RT-PCR and Western-blot. Treatment with 5-aza-dC restored expression, suggesting that hypermethylation is involved in EP 3 downregulation. Endogenous PGE 2 production was mainly due to COX-2, since this was significantly suppressed with COX-2 inhibitors (NS-398 and SC-58125), but not COX-1 inhibitors (SC-560). Cell proliferation ( 3 H-thymidine uptake) was significantly inhibited by NS-398 and SC-58125, the EP 1 antagonist SC-51322, AH6809 (EP 1 /EP 2 antagonist), and the EP 4 antagonist AH23848B, but was not affected by exogenous PGE 2 . However, treatment with the selective EP 2 agonist Butaprost or 16,16-dimethylPGE 2 significantly inhibited butyrate-induced apoptosis and stimulated OE33 cell migration. The effect of exogenous PGE 2 on migration was attenuated when cells were first treated with EP 1 and EP 4 antagonists. These findings suggest a potential role for EP selective antagonists in the treatment of esophageal adenocarcinoma.

Colorectal cancer (CRC) is one of the most common neoplasms and a leading cause of death related to cancer worldwide. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent autosomal dominant predisposition to the development of CRC, accounting for approximately 2.5% of the total CRC burden in Spain. Genomic rearrangements in the MSH2 and MLH1 genes have been reported to account for an important proportion of the mutation spectrum in HNPCC, and DNA dosage techniques have been developed facilitating molecular screening of such deletions/duplications. We screened for MSH2 and MLH1 genomic rearrangements by multiplex ligation-dependent probe amplification (MLPA) in 142 Spanish patients at risk for HNPCC prior to the exon-by-exon mutation scanning and found a deletion encompassing exons 9-16 of MSH2 and a duplication encompassing exons 11-16 of MSH2, both only in one case. These results showed that MSH2/MLH1 rearrangements in Spanish patients at risk for HNPCC seem to be a less frequent mutational event than in other populations.

There is extensive evidence that cyclooxygenase-2 (COX-2) plays a significant role in the process of carcinogenesis in different tumors. Although most of these evidences derive from studies in colorectal cancer, data obtained from recent studies strongly suggest that COX-2 might play an important role in the neoplastic transformation of esophageal epithelium. NSAIDs use is associated with a reduction of the risk of developing esophageal cancer, including adenocarcinoma. Up-regulation of COX-2 has been reported in different stages of the carcinogenic sequence leading to esophageal adenocarcinoma. Treatment with selective COX-2 inhibitors has been shown to reduce the damage induced by acid and pepsin in the esophageal mucosa of rabbits, the incidence of tumors in an animal model of esophageal adenocarcinoma and to decrease proliferation and induce apoptosis in both Barrett's epithelial and adenocarcinoma cells. The first clinical study has shown that selective inhibition of COX-2 is followed by a significant decrease of cell proliferation in human Barrett's metaplasia. Clinical trials have begun in order to assess the efficacy of selective COX-2 inhibitors to prevent the progression of Barrett's esophagus to adenocarcinoma. Bile salts and acid are likely to early induce COX-2 in this sequence, although other factors, such as proinflammatory cytokines, inducible nitric oxide synthase and growth factors such as TGF-beta, are potential COX-2 inducers in the esophagus. Further studies are necessary in order to better understand factors involved in COX-2 up-regulation and mechanisms of COX-2 associated tumorigenesis in the esophagus.

Nitric oxide (NO) exerts both protective and proinflammatory actions in the gastrointestinal tract. Enhanced gastric NO synthase (NOS) activity has been shown in duodenal ulcer patients. Recently, intron-4 polymorphism of the endothelial constitutive (ec) NOS gene has been associated with some pathological conditions. Our aim was to determine the genotype and allele frequencies of the ecNOS4 polymorphism in peptic ulcer patients. The distribution of the polymorphism ecNOS4a/b was studied in 188 ulcer patients and 120 healthy controls, from genomic DNA. Genotypes ab, bb, and aa and allele frequency were similar in both peptic ulcer patients and controls, and no differences were found when patients and controls were analyzed according to the presence of several etiological factors. However, alelle “a” carrier status was associated with decreased risk of bleeding in duodenal ulcer patients (OR = 0.49; 95% CI = 0.25–0.95; P = 0.03). In conclusion, this ecNOS4 polymorphism gene could be related to susceptibility of duodenal ulcer patients to bleeding.

Two recent genome-wide association studies in Asians have reported the association between the PSCA (prostate stem cell antigen) rs2294008CT gene polymorphism and two Helicobacter pylori infection-related diseases such as gastric cancer (GC) and duodenal ulcer (DU). Since rs2294008 allele frequencies differ notably among ethnicities, we aimed to assess the role of rs2294008 on the susceptibility to GC and DU in a Caucasian population in Spain. Moreover, the relevance of rs2294008 on GC prognosis was evaluated. Genomic DNA from 603 Spanish patients with primary GC, 139 with DU and 675 healthy controls was typed for the PSCA rs2294008CT polymorphism by PCR-TaqMan assays. H. pylori infection [odds ratio (OR): 8.27; 95% confidence interval (CI): 3.45-15.33] and nonsteroidal anti-inflammatory drugs (OR: 6.54; 95% CI: 3.19-12.43) were identified as independent risk factors for DU whereas the rs2294008T allele was associated with reduced risk of developing the disease (OR: 0.52; 95% CI: 0.33-0.82). Infection with CagA strains (OR: 2.10; 95% CI: 1.63-2.34), smoking (OR: 1.93; 95% CI: 1.54-2.61), family history of GC (OR: 2.83; 95% CI: 2.01-3.83), and the rs2294008T allele (OR: 1.46; 95% CI: 1.07-1.99) were associated with increased risk of GC. Interestingly, the association with the rs2294008T allele was restricted to noncardia GC (OR: 1.43; 95% CI: 1.12-1.82), particularly of the diffuse histotype (OR: 1.59; 95% CI: 1.16-1.92). Finally, Cox regression analysis identified the rs2294008T variant as a prognosis factor associated with worse overall survival in patients with diffuse-type GC (hazard ratio: 1.85; 95% CI: 1.12-3.06). From these results we conclude that the PSCA rs2294008 polymorphism is involved in the susceptibility to GC and DU, as well as in the prognosis of the diffuse-type of GC in Caucasians.

Summary Background Even though the acetylation of platelet cyclooxygenase (COX)-1 at serine-529 is the direct mechanism of action of low-dose aspirin, its antiplatelet effect has been characterized using indirect indexes of COX-1 activity. Objectives We performed a clinical study with enteric-coated low-dose aspirin (EC-aspirin), in healthy subjects, to evaluate the effects on the extent and duration of platelet COX-1 acetylation, using a novel proteomic strategy for absolute protein quantification (termed AQUA), as compared with traditional pharmacokinetic and pharmacodynamic parameters. Subjects and methods In a phase I, single-arm, open-label study of EC aspirin (100 mg day−1) administered to 24 healthy subjects, we compared, over a 24 h-period on day 1 and 7, % platelet acetylated COX-1 (AceCOX-1) with traditional pharmacokinetic and pharmacodynamics [i.e. serum thromboxane (TX) B2, platelet function by monitoring CEPI(collagen/epinephrine) closure time (CT) using whole-blood PFA-100 and urinary excretion of 11-dehydro-TXB2] parameters. Results Acetylation of platelet COX-1 was measurable before detection of aspirin levels in the systemic circulation and increased in a cumulative fashion upon repeated dosing. After the last dose of EC-aspirin, %AceCOX-1, serum TXB2 and CEPI-CT values were maximally and persistently modified throughout 24 h; they averaged 76 ± 2%, 99.0 ± 0.4% and 271 ± 5 s, respectively. EC-aspirin caused 75% reduction in urinary 11-dehydro-TXB2 excretion. After chronic dosing with aspirin, the pharmacokinetics of acetylsalicylic acid was completely dissociated from pharmacodynamics. Conclusions The demonstrated feasibility of quantifying the extent and duration of platelet COX-1 acetylation will allow characterizing the genetic, pharmacokinetic and pharmacodynamic determinants of the inter-individual variability in the antiplatelet response to low-dose aspirin as well as identifying extra-platelet sites of drug action.

Aspirin has been proposed in recent years as a candidate for chemoprevention of adenocarcinoma in patients with Barrett's esophagus. The aim of the present study was to evaluate the effect of acetylsalicylic acid (ASA) in an experimental model of esophageal adenocarcinoma. An animal model of gastroenteroesophageal reflux was established using Wistar rats undergoing esophagojejunostomy with gastric preservation. Following surgery, rats were divided into three groups: i) control (vehicle); ii) ASA 50 mg/kg/day; and iii) ASA 5 mg/kg/day. Four months after surgery, the surviving animals were sacrificed and the rat esophagi were assessed for histological and biochemical [prostaglandin E2 (PGE2) and lipoxin A4 (LXA4 ) levels] analysis. As in the control rats, those receiving aspirin treatment showed no decrease in inflammation grade, extent of ulcerated esophageal mucosa, length of intestinal metaplasia in continuity with anastomosis, presence of intestinal metaplasia beyond anastomosis, severity of dysplasia or incidence of adenocarcinoma. In contrast, aspirin-treated rats showed decreased esophageal tissue levels of PGE2 and increased LXA4, significantly in the high-dose aspirin group (p=0.008 and p=0.01, respectively). In this rat model of gastroesophageal reflux, the administration of aspirin modified esophageal tissue levels of PGE2 and LXA4, but was not effective in preventing the development of esophageal adenocarcinoma.

Objetivo Investigar la potencial contribucion de los alelos *0102 y *0301 del gen HLADQA1 en la infeccion por H. pylori y la enfermedad ulcerosa peptica en una poblacion caucasica espanola. Pacientes y Metodos Se incluyeron un total de 163 pacientes con ulcera peptica (117 ulcera duodenal [UD] y 46 ulcera gastrica [UG]; 111 con hemorragia digestiva alta reciente) y 90 controles. Los alelos *0102 y *0301 del gen HLADQA1 se tipificaron mediante PCR a partir de ADN genomico. La infeccion por H. pylori se determino en los pacientes mediante test de aliento y/o histologia y en los controles por test de aliento y/o serologia. En 98 pacientes y en 48 controles con infeccion por H. pylori se investigaron las citotoxinas CagA y VacA mediante serologia (Western-blot). Resultados La infeccion por H. pylori estaba presente en el 94,6% de UD, el 84,4% de UG y el 67,4% de los controles (p Conclusiones Ser portador de los alelos *0102 y *0301 del gen HLADQA1 no modifica ni la susceptibilidad a la infeccion por H. pylori ni su evolucion a ulcera peptica en una poblacion caucasica del sur de Europa.

It has been proposed that free radicals are involved in the pathogenesis of esophageal mucosal damage induced by acid and pepsin. Recent data have suggested that nitric oxide (NO) is involved in the mucosal defense of the esophagus and that superoxide anion plays a minor role in low-grade esophagitis. To study the role and potential interaction of NO and superoxide anion in an experimental model of high-grade esophagitis, acidified pepsin was perfused (45 min/12 hr) for five days in rabbits with different agents to modulate the generation of these radicals. Measurements included both macroscopic and microscopic mucosal damage, superoxide anion generation, NO synthase mucosal activity, and peroxynitrite formation. High-grade esophagitis was associated with mucosal superoxide anion generation. Treatment with exogenous superoxide dismutase completely prevented mucosal damage. The perfusion of acidified pepsin in the lumen of the esophagus was initially associated with increased NO synthase mucosal activity but decreased with the progression of damage. Generation of peroxynitrites was present in those cases with severe damage. Treatment with NO-modifying agents did not induce consistent modification of mucosal damage. It is concluded that superoxide anion is involved in the induction of high-grade esophagitis and that it interacts with nitric oxide to generate peroxynitrite radicals in this model. Superoxide dismutase but not NO-donor-modifying agents might have a therapeutic role in preventing severe esophageal mucosal damage induced by acid and pepsin.

Fundamento La presencia de anticuerpos frente a la citotoxina CagA constituye un marcador serologico de infeccion por cepas de Helicobacter pylori mas virulentas. El objetivo de este trabajo fue evaluar el riesgo para la aparicion de ulcera peptica en pacientes con infeccion por H. pylori en relacion con la presencia de anticuerpos frente a las citotoxinas CagA y VacA. Pacientes y metodo Estudio prospectivo de casos y controles en el que se incluyeron 104 pacientes con ulcera peptica con infeccion activa por H. pylori (test de ureasa y/o histologia positiva, o test del aliento positivo), apareados por edad y sexo con 104 individuos, sin historia previa ulcerosa, con infeccion activa por H. pylori (test del aliento positivo). La presencia de anticuerpos frente a las citotoxinas CagA y VacA se determino mediante Western blot. El consumo de antiinflamatorios no esteroides (AINE) se establecio mediante encuesta dirigida. Se realizo un analisis multivariable (regresion logistica) para estimacion de las odds ratio (OR). Resultados La presencia de anticuerpos anti-CagA fue mas frecuente en los pacientes con ulcera peptica (74%) que en los controles (46,2%) (OR, 5,7; IC del 95%, 2,1-15,6), no asi la presencia de anticuerpos anti-VacA (46,2 frente a 36,5%). El consumo de AINE tambien fue mas frecuente en los pacientes (51,9%) que en los controles (21,2%) (OR, 6,5; IC del 95%, 2, 2-19,5). Conclusiones La presencia de anticuerpos frente a la citotoxina CagA y el consumo de AINE son los dos factores de riesgo mas importantes para el desarrollo de ulcera peptica.

Background and Aims : Exposure to luminal acid induces mucosal damage in the oesophagus. The extent and degree of interaction of acid with wound repair mechanisms of the oesophagus have not been explored. Methods : Rabbit oesophageal cells were isolated, cultured and exposed to different extracellular pHs for several periods of time. Cell mortality was studied in both isolated and primary cultures of these cells by cell flow cytometry using fluorochrome propidium iodide and LDH release. Subconfluent cell cultures were used to study proliferative responses determined by [3H]-thymidine incorporation into DNA. Restitution was studied in confluent monolayers wounded by mechanical denudation. Results : Isolated cells were resistant to acid when exposed to pH > 2 for 15 min. Cell proliferation increased with small pH decreases (7–6.5) if the period of exposure was less than 6 h, but was inhibited with lower pHs and/or longer periods of acid exposure. However, restitution was gradually inhibited with further pH decreases (6.5–3) and time of acid exposure (0.5–24 h). Conclusions : The wound repair activities of oesophageal epithelial cells are deeply affected by even small decreases in pH. These findings may help to explain the need for profound acid inhibition in acid-related reflux oesophagitis.

Factors and mechanisms involved in esophagealmucosal injury and repair are not well known. The aim ofthis study was to assess the effects of growth factorsand prostaglandins on esophageal mucosal cell repair activities. Rabbit esophageal cells wereisolated, cultured, and exposed to different growthfactors and prostaglandin E2. Subconfluentcell cultures were used to study proliferative responsesdetermined by [3H]thymidine incorporation intoDNA. Restitution was studied in confluent monolayerswounded by mechanical denudation. Restitution was themain mechanism involved in wound repair within the first24 hr. HGF, IGF-I, and EGF dose-dependentlystimulated cell proliferation but did not affectrestitution. TGF-β1 inhibited bothproliferation and restitution while PDGF-BB andprostaglandin E2 had no effect. Esophageal epithelial cell restitution andproliferation are affected by growth factors. HGF,IGF-I, EGF (stimulation), and TGF-β1(inhibition) are major growth factors affecting in vitroesophageal wound repair activities, which, unlike those ofother areas of the digestive tract, are not affected byprostaglandins.

Fibroblasts (FIB) play an important role in the wound-healing process. It is not known whether human skin and gastric FIB show different responses to regulatory compounds. In this study, we have examined the collagen production by these FIB after different stimuli. In vitro release of collagen into the medium by steady-state confluent human FIB cultures was assessed over a 24-hour period by 3H-proline incorporation into collageneous protein. Serum and epidermal growth factor increased collagen secretion in both types of FIB, but gastric FIB produced less collagen than skin FIB. Prostaglandin E1 inhibited collagen production in both types of FIB, but nonsteroidal anti-inflammatory drugs and interleukin-1β, a cytokine involved in the wound-healing process, had opposite effects on gastric and skin FIB. The effects of lipoxygenase metabolites on collagen secretion was small, but different in both types of FIB. We conclude that, when compared to skin FIB, human gastric FIB produce less collagen and show pronounced different responses to different agents, which might be relevant to explain (in part) their clinical effects on ulcer healing. These data provide new insights into the wound-healing process.

Fibroblasts modulate epithelial biologicalactivities and play a key role in the ulcer healingprocess. There is no information regarding thebiological response of human gastric fibroblasts toregulatory compounds. The aim of this study was to assessthe effects of growth factors and prostaglandins on anin vitro model of human gastric fibroblast wound repair.Subconfluent fibroblast cultures were used to study proliferative responses, determined by[3H]thymidine incorporation into DNA. Invitro wound repair was determined in confluentfibroblast monolayers after mechanical denudation. Thepresence of putative growth factors secreted by fibroblastswas studied in conditioned medium by heparin-affinitychromatography and immunodetection with specificantibodies. Serum and platelet-derived growth factor (PDGF)-BB induced a dramatic increase in bothgastric fibroblast proliferation and closure of woundedcell monolayers, whereas these activities were inhibitedby both transforming growth factor(TGF)-β1 and prostaglandin E1. Basalactivities in unstimulated gastric fibroblasts werelower than those obtained in skin fibroblasts.Conditioned medium stimulated fibroblast proliferationand wound repair activity, which was inhibited by the addition of suramin,and was partially dependent on the presence of PDGF-likefactor. PDGF is a major, autocrine promotor of humangastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins andTGF-β. These findings might be important for futuretherapeutic ulcer healing approaches.

Unlike gastric mucosa, it has been considered that lipoxygenase metabolites protect the esophageal mucosa and that prostaglandins are only secreted in the presence of esophageal inflammation. The aim of this study was to determine the profile of arachidonic acid metabolites and their response to regulatory compounds in rabbit esophageal mucosal cells in culture. Eicosanoids secreted into the medium were extracted and identified by HPLC and RIA. Esophageal mucosal cells in culture metabolized arachidonic acid mainly through the cycloxygenase pathway and PGE2 was the major arachidonic acid metabolite secreted. The addition of IL-1β and A23187 (calcium ionophore) stimulated PGE2 synthesis. In basal conditions neither leukotrienes nor HETEs were detected. However, the addition of the NDGA induced the secretion of lipoxygenase metabolites identified as 12–15 HETEs. In conclusion, rabbit esophageal epithelial cells in culture metabolize arachidonic acid via both cycloxygenase and lipoxygenase pathways. In our system, PGE2 was the main arachidonic acid metabolite.

Gastrin is the main hormone responsible for the stimulation of gastric acid secretion; in addition, gastrin and its derivatives exert proliferative and antiapoptotic effects on several cell types. Gastrin synthesis and secretion are increased in certain situations, for example, when proton pump inhibitors are used. The impact of sustained hypergastrinemia is currently being investigated. In vitro experiments and animal models have shown that prolonged hypergastrinemia may be related with higher cancer rates; although, this relationship is less clear in human beings. Higher gastrin levels have been shown to cause hyperplasia of several cell types; yet, the risk for developing cancer seems to be the same in normo- and hypergastrinemic patients. Some tumors also produce their own gastrin, which can act in an autocrine manner promoting tumor growth. Certain cancers are extremely dependent on gastrin to proliferate. Initial research focused only on the effects of amidated gastrins, but there has been an interest in intermediates of gastrin in the last few decades. These intermediates aren't biologically inactive; in fact, they may exert greater effects on proliferation and apoptosis than the completely processed forms. In certain gastrin overproduction states, they are the most abundant gastrin peptides secreted. The purpose of this review is to examine the gastrin biosynthesis process and to summarize the results from different studies evaluating the production, levels, and effects of the main forms of gastrin in different overexpression states and their possible relationship with Barrett's and colorectal carcinogenesis.