Acetylsalicylic Acid Exhibits Antitumor Effects in Esophageal Adenocarcinoma Cells In Vitro and In Vivo.

BACKGROUND AND AIM: 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). METHODS: 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. RESULTS: In vitro cell proliferation and migration were significantly inhibited while apoptosis increased (p < 0.05) by ASA. Although ASA did not induce tumor remission, tumor progression was significantly lower in ASA-treated mice when compared to non-treated animals (478 % in mice treated with 5 mg/kg/day ASA vs. 2696 % control; 748 % in mice treated with 50 mg/kg/day ASA vs. 2670 % control). Maximum tumor inhibition was 92 and 85 %, respectively. This effect was associated with a significant decrease of proliferation index in tumors. ASA 5 mg/kg/day did not modify tumor PGE2 levels. Whereas tumor PGE2 content in mice treated with ASA 50 mg/kg was lower than in control mice, the difference was not significant. CONCLUSION: Although these results need to be confirmed in other EAC cells, our data suggest a role for ASA in the treatment of this tumor.

Association of PSCA rs2294008 gene variants with poor prognosis and increased susceptibility to gastric cancer and decreased risk of duodenal ulcer disease.

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.

NSAIDS and gastrointestinal cancer.

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.

Optimizing aspirin dose for colorectal cancer patients through deep phenotyping using novel biomarkers of drug action.

Background: Low-dose aspirin's mechanism of action for preventing colorectal cancer (CRC) is still debated, and the optimal dose remains uncertain. We aimed to optimize the aspirin dose for cancer prevention in CRC patients through deep phenotyping using innovative biomarkers for aspirin's action. Methods: We conducted a Phase II, open-label clinical trial in 34 CRC patients of both sexes randomized to receive enteric-coated aspirin 100 mg/d, 100 mg/BID, or 300 mg/d for 3 ± 1 weeks. Biomarkers were evaluated in blood, urine, and colorectal biopsies at baseline and after dosing with aspirin. Novel biomarkers of aspirin action were assessed in platelets and colorectal tissues using liquid chromatography-mass spectrometry to quantify the extent of cyclooxygenase (COX)-1 and COX-2 acetylation at Serine 529 and Serine 516, respectively. Results: All aspirin doses caused comparable % acetylation of platelet COX-1 at Serine 529 associated with similar profound inhibition of platelet-dependent thromboxane (TX)A2 generation ex vivo (serum TXB2) and in vivo (urinary TXM). TXB2 was significantly reduced in CRC tissue by aspirin 300 mg/d and 100 mg/BID, associated with comparable % acetylation of COX-1. Differently, 100 mg/day showed a lower % acetylation of COX-1 in CRC tissue and no significant reduction of TXB2. Prostaglandin (PG)E2 biosynthesis in colorectal tumors and in vivo (urinary PGEM) remained unaffected by any dose of aspirin associated with the variable and low extent of COX-2 acetylation at Serine 516 in tumor tissue. Increased expression of tumor-promoting genes like VIM (vimentin) and TWIST1 (Twist Family BHLH Transcription Factor 1) vs. baseline was detected with 100 mg/d of aspirin but not with the other two higher doses. Conclusion: In CRC patients, aspirin 300 mg/d or 100 mg/BID had comparable antiplatelet effects to aspirin 100 mg/d, indicating similar inhibition of the platelet's contribution to cancer. However, aspirin 300 mg/d and 100 mg/BID can have additional anticancer effects by inhibiting cancerous tissue's TXA2 biosynthesis associated with a restraining impact on tumor-promoting gene expression. EUDRACT number: 2018-002101-65. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03957902.

Antitumor effects of lactate transport inhibition on esophageal adenocarcinoma cells.

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.

Serum lipid mediator profiles in COVID-19 patients and lung disease severity: a pilot study.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is highly heterogeneous, ranging from asymptomatic to severe and fatal cases. COVID-19 has been characterized by an increase of serum pro-inflammatory cytokine levels which seems to be associated with fatal cases. By contrast, the role of pro-resolving lipid mediators (SPMs), involved in the attenuation of inflammatory responses, has been scarcely investigated, so further studies are needed to understand SPMs metabolism in COVID-19 and other infectious diseases. Our aim was to analyse the lipid mediator metabolome, quantifying pro- and anti-inflammatory serum bioactive lipids by LC-MS/MS in 7 non-infected subjects and 24 COVID-19 patients divided into mild, moderate, and severe groups according to the pulmonary involvement, to better understand the disease outcome and the severity of the pulmonary manifestations. Statistical analysis was performed with the R programming language (R Foundation for Statistical Computing, Vienna, Austria). All COVID-19 patients had increased levels of Prostaglandin E2. Severe patients showed a significant increase versus controls, mild- and moderate-affected patients, expressed as median (interquartile range), in resolvin E1 [112.6 (502.7) vs 0.0 (0.0) pg/ml in the other groups], as well as in maresin 2 [14.5 (7.0) vs 8.1 (4.2), 5.5 (4.3), and 3.0 (4.0) pg/ml, respectively]. Moreover, 14-hydroxy docosahexaenoic acid (14-HDHA) levels were also increased in severe vs control and mild-affected patients [24.7 (38.2) vs 2.4 (2.2) and 3.7 (6.4) ng/mL, respectively]. Resolvin D5 was also significantly elevated in both moderate [15.0 (22.4) pg/ml] and severe patients [24.0 (24.1) pg/ml] versus controls [0.0 (0.0) pg/ml]. These results were confirmed by sparse partial least squares discriminant analysis which highlighted the contribution of these mediators to the separation between each of the groups. In conclusion, the potent inflammatory response to SARS-CoV-2 infection involves not only pro- but also anti-inflammatory lipid mediators that can be quantified in easily accessible serum samples, suggesting the need to perform future research on their generation pathways that will help us to discover new therapeutic targets.

Biomarkers of Response to Low-Dose Aspirin in Familial Adenomatous Polyposis Patients.

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.

Relevance of GSTM1, GSTT1, and GSTP1 gene polymorphisms to gastric cancer susceptibility and phenotype.

Human glutathione S-transferases (GSTs) are phase II metabolizing enzymes that play a key role in protecting against cancer by detoxifying numerous potentially cytotoxic/genotoxic compounds. The genes encoding the human GST isoenzymes GSTM(mu)1, GSTT(theta)1 and GSTP(pi)1 harbour polymorphisms, which have been considered important modifiers of the individual risk for environmentally induced cancers such as gastric cancer (GC). However, results are inconsistent among studies from different geographic areas and ethnic groups. Our goal was to perform a nationwide, case-control study in Spain to evaluate the relevance of several functional GST gene polymorphisms and environmental factors to GC risk and phenotype. DNA from 557 GC patients and 557 sex- and age-matched healthy controls (HC) was typed for two deletions in the GSTM1 and GSTT1 genes and two SNPs in the GSTP1 gene (rs1695 and rs1138272) using polymerase chain reaction-restriction fragment length polymorphism methods. Logistic regression analysis identified Helicobacter pylori infection with CagA strains [odds ratio (OR): 2.36; 95% confidence interval (CI): 1.78-3.15], smoking habit (OR: 2.10; 95% CI: 1.48-2.97) and family history of GC (OR: 3.2; 95% CI: 2.02-5.16) as independent risk factors for GC. No differences in the frequencies of GSTM1 or GSTT1 null genotypes were observed between cases and controls (GSTM1: 50.8% vs. 48%; GSTT1: 21.5% vs. 21%). Moreover, simultaneous carriage of both, the GSTM1 and the GSTT1 null genotypes, was almost identical in both groups (10.7% in GC vs. 10.6% in HC). In addition, no significant differences in GSTP1 Ile105Val (rs1695) and GSTP1 Val114Ala (rs1138272) genotype distribution were observed between GC patients and controls. Subgroup analysis for age, gender, Helicobacter pylori status, smoking habits, family history of GC, anatomic location and histological subtype revealed no significant association between GST variants and GC risk. Our results show that the GST polymorphisms evaluated in this study are not relevant when determining the individual susceptibility to GC or phenotype in a South-European population.

Cardiac function and aminoterminal pro-brain natriuretic peptide levels in liver-transplanted cirrhotic patients.

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/m(2) ; p < 0.05), diastolic cardiac function deteriorated, expressed as a reduction in E/A wave ratio (1.105 ± 0.295 vs. 0.798 ± 0.248; p < 0.001), and NT-proBNP levels decreased significantly in patients compared to pre-transplantation values (1759 ± 1154 vs. 1117 ± 600 pg/mL; p < 0.001), although they were still above levels found in controls (1117 ± 600 vs. 856 ± 123 pg/mL; p < 0.05). NT-proBNP levels above 2000 pg/mL before transplantation were significantly associated with risk for cardiovascular events after procedure (37% vs. 9%, p = 0.008). CONCLUSIONS: In cirrhotic patients, diastolic function and cardiac structure deteriorate after LT. Compared to controls, NT-proBNP levels tend to be higher before and after transplantation. The mechanisms and consequences of these results require further study.

Blood-Cell-Based Inflammatory Markers as a Useful Tool for Early Diagnosis in Colorectal Cancer.

Background: Systemic inflammation seems to be involved in the pathogenetic pathways of colorectal cancer (CRC). Analytical markers that reflect the inflammatory status, such as neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR) or systemic immune-inflammation index (SII), have been proposed as tools for the prognosis of CRC. Nevertheless, their use for diagnosis has been scarcely investigated. Aims: To analyze the ability of these markers and of a new marker combining SII and hemoglobin concentration, named NP/LHb = [neutrophils x platelets]/[lymphocytes x hemoglobin], as tools for CRC diagnosis. Furthermore, we studied their association with CRC-related variables. Methods: Case-control study including 214 CRC patients and 214 controls without CRC, matched by age (±5 years) and sex. We collected demographic, CRC-related and laboratory variables to calculate NLR, PLR, SII, and NP/LHb. In the case group, the laboratory variables were collected at two different period times, 6 months (IQR 4-8) before the CRC diagnosis and at the time of the diagnosis. ROC analysis was performed to evaluate the discriminatory accuracy of each index and we calculated Se, Sp, PPV, NPV, and OR to identify the diagnostic performance of each positive marker. Results: NP/LHb showed high Sp (92.06%) and PPV (87.50%) to diagnose patients with CRC. This index exhibited an OR of 14.52 (8.26-25.52) and the best area under the curve (AUC: 0.78) for a positive CRC diagnosis. We found significant differences in all indices according to the presence of CRC, observing the highest values in CRC patients at time of diagnosis, in comparison with the analysis performed in the previous months to diagnosis or with control patients. There were significant differences in all ratios according to TNM stages (p < 0.05). PLR, SII and NP/LHb (but not NLR) showed significant differences according to tumor location (p < 0.05). Right-sided colon cancers presented the highest values, in comparison with left-sided and rectal cancers. Conclusions: Systemic inflammatory cell ratios (especially NP/LHb) change over time with the development of CRC, so they could be useful in its early diagnosis. We suggest that they could be routinely measured in patients with suspicion of CRC, to identify those ones with a higher risk of cancer, considering the high positive predictive value they have shown in our study.

1,4-Dihydropyridine as a Promising Scaffold for Novel Antimicrobials Against Helicobacter pylori.

The increasing occurrence of multidrug-resistant strains of the gastric carcinogenic bacterium Helicobacter pylori threatens the efficacy of current eradication therapies. In a previous work, we found that several 1,4-dihydropyridine (DHP)-based antihypertensive drugs exhibited strong bactericidal activities against H. pylori by targeting the essential response regulator HsrA. To further evaluate the potential of 1,4-DHP as a scaffold for novel antimicrobials against H. pylori, we determined the antibacterial effects of 12 novel DHP derivatives that have previously failed to effectively block L- and T-type calcium channels. Six of these molecules exhibited potent antimicrobial activities (MIC ≤ 8 mg/L) against three different antibiotic-resistant strains of H. pylori, while at least one compound resulted as effective as metronidazole. Such antimicrobial actions appeared to be specific against Epsilonproteobacteria, since no deleterious effects were appreciated on Escherichia coli and Staphylococcus epidermidis. The new bactericidal DHP derivatives targeted the H. pylori regulator HsrA and inhibited its DNA binding activity according to both in vitro and in vivo analyses. Molecular docking predicted a potential druggable binding pocket in HsrA, which could open the door to structure-based design of novel anti-H. pylori drugs.

Low-dose aspirin affects the small bowel mucosa: results of a pilot study with a multidimensional assessment.

BACKGROUND & AIMS: Whether low-dose aspirin (acetylsalicylic acid [ASA]) produces intestinal damage is controversial. Our aim was to determine whether the small bowel is damaged by low-dose ASA on a short-term basis. METHODS: Twenty healthy volunteers (age range, 19-64 years) underwent video capsule endoscopy (VCE), fecal calprotectin, and permeability tests (sucrose and lactulose/mannitol [lac/man] ratio) before and after ingestion of 100 mg of enteric-coated ASA daily for 14 days. Video capsule images were assessed by 2 independent expert endoscopists, fully blinded to the treatment group, by using an endoscopic scale. RESULTS: Post-ASA VCE detected 10 cases (50%) with mucosal damage not apparent in baseline studies (6 cases had petechiae, 3 had erosions, and 1 had bleeding stigmata in 2 ulcers). The median baseline lac/man ratio (0.021; range, 0.011-0.045) increased after ASA use (0.036; range, 0.007-0.258; P = .08), and the post-ASA lac/man ratio was above the upper end of normal (>0.025) in 10 of 20 volunteers (vs baseline, P < .02). The median baseline fecal calprotectin concentration (6.05 microg/g; range, 1.9-79.2) also increased significantly after ASA use (23.9 microg/g; range, 3.1-75.3; P < .0005), with 3 patients having values above the cutoff (>50 microg/g). Five of 10 subjects with abnormal findings at VCE also had lac/man ratios above the cutoff. Median baseline sucrose urinary excretion (70.0 mg; range, 11.8-151.3) increased significantly after ASA administration (107.0 mg; range, 22.9-411.3; P < .05). CONCLUSIONS: The short-term administration of low-dose ASA is associated with mucosal abnormalities of the small bowel mucosa, which might have implications in clinical practice.

Omega-3 Polyunsaturated Fatty Acids and Their Bioactive Metabolites in Gastrointestinal Malignancies Related to Unresolved Inflammation. A Review.

Chronic inflammation takes part in the pathogenesis of some malignancies of the gastrointestinal tract including colorectal (CRC), gastric, and esophageal cancers. The use of ω3 polyunsaturated fatty acid (ω3-PUFA) supplements for chemoprevention or adjuvant therapy of gastrointestinal cancers is being investigated in recent years. Most evidence has been reported in CRC, although their protective role has also been reported for Helicobacter pylori-induced gastric cancer or Barrett's esophagus-derived adenocarcinoma. Studies based on ω3-PUFA supplementation in animal models of familial adenomatous polyposis (FAP) and CRC revealed positive effects on cancer prevention, reducing the number and size of tumors, down-regulating arachidonic acid-derived eicosanoids, upregulating anti-oxidant enzymes, and reducing lipid peroxidation, whereas contradictory results have been found in induced colitis and colitis-associated cancer. Beneficial effects have also been found in FAP and ulcerative colitis patients. Of special interest is their positive effect as adjuvants on radio- and chemo-sensitivity, specificity, and prevention of treatment complications. Some controversial results obtained in CRC might be justified by different dietary sources, extraction and preparation procedures of ω3-PUFAs, difficulties on filling out food questionnaires, daily dose and type of PUFAs, adenoma subtype, location of CRC, sex differences, and genetic factors. Studies using animal models of inflammatory bowel disease have confirmed that exogenous administration of active metabolites derived from PUFAs called pro-resolving mediators like lipoxin A4, arachidonic acid-derived, resolvins derived from eicosapentaenoic (EPA), docosahexaenoic (DHA), and docosapentaenoic (DPA) acids as well as maresin 1 and protectins DHA- and DPA-derived improve disease and inflammatory outcomes without causing immunosuppression or other side effects.

Relevance of DNA repair gene polymorphisms to gastric cancer risk and phenotype.

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.

Effects of selective PGE2 receptor antagonists in esophageal adenocarcinoma cells derived from Barrett's esophagus.

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.

Proton Pump Inhibitors Display Antitumor Effects in Barrett's Adenocarcinoma Cells.

Recent evidence has reported that proton pump inhibitors (PPIs) can exert antineoplastic effects through the disruption of pH homeostasis by inhibiting vacuolar ATPase (H+-VATPase), a proton pump overexpressed in several tumor cells, but this aspect has not been deeply investigated in EAC yet. In the present study, the expression of H+-VATPase was assessed through the metaplasia-dysplasia-adenocarcinoma sequence in Barrett's esophagus (BE) and the antineoplastic effects of PPIs and cellular mechanisms involved were evaluated in vitro. H+-VATPase expression was assessed by immunohistochemistry in paraffined-embedded samples or by immunofluorescence in cultured BE and EAC cell lines. Cells were treated with different concentrations of PPIs and parameters of citotoxicity, oxidative stress, and autophagy were evaluated. H+-VATPase expression was found in all biopsies and cell lines evaluated, showing differences in the location of the pump between the cell lines. Esomeprazole inhibited proliferation and cell invasion and induced apoptosis of EAC cells. Production of reactive oxygen species (ROS) seemed to be involved in the cytotoxic effects observed since the addition of N-acetylcysteine significantly reduced esomeprazole-induced apoptosis in EAC cells. Esomeprazole also reduced intracellular pH of tumor cells, whereas only disturbed the mitochondrial membrane potential in OE33 cells. Esomeprazole induced autophagy in both EAC cells, but also triggered a blockade in autophagic flux in the metastatic cell line. These data provide in vitro evidence supporting the potential use of PPIs as novel antineoplastic drugs for EAC and also shed some light on the mechanisms that trigger PPIs cytotoxic effects, which differ upon the cell line evaluated.

Genomic rearrangements in MSH2 and MLH1 are rare mutational events in Spanish patients with hereditary nonpolyposis colorectal cancer.

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.

Superoxide dismutase prevents development of adenocarcinoma in a rat model of Barrett's esophagus.

AIM: To test whether antioxidant treatment could prevent the progression of Barrett's esophagus to adenocarcinoma. METHODS: In a rat model of gastroduodenoesophageal reflux by esophagojejunal anastomosis with gastric preservation, groups of 6-10 rats were randomized to receive treatment with superoxide dismutase (SOD) or vehicle and followed up for 4 mo. Rat's esophagus was assessed by histological analysis, superoxide anion and peroxinitrite generation, SOD levels and DNA oxidative damage. RESULTS: All rats undergoing esophagojejunostomy developed extensive esophageal mucosal ulceration and inflammation by mo 4. The process was associated with a progressive presence of intestinal metaplasia beyond the anastomotic area (9% 1st mo and 50% 4th mo) (94% at the anastomotic level) and adenocarcinoma (11% 1st mo and 60% 4th mo). These changes were associated with superoxide anion and peroxinitrite mucosal generation, an early and significant increase of DNA oxidative damage and a significant decrease in SOD levels (P<0.05). Exogenous administration of SOD decreased mucosal superoxide levels, increased mucosal SOD levels and reduced the risk of developing intestinal metaplasia beyond the anastomotic area (odds ratio = 0.326; 95%CI: 0.108-0.981; P = 0.046), and esophageal adenocarcinoma (odds ratio = 0.243; 95%CI: 0.073-0.804; P = 0.021). CONCLUSION: Superoxide dismutase prevents the progression of esophagitis to Barrett's esophagus and adenocarcinoma in this rat model of gastrointestinal reflux, supporting a role of antioxidants in the chemoprevention of esophageal adenocarcinoma.

Free radicals and antioxidant systems in reflux esophagitis and Barrett's esophagus.

AIM: Experimental studies suggest that free radicals are involved in acid and pepsin-induced damage of esophageal mucosa. The profile and balance between free radicals and antioxidant systems in human esophagitis are unknown. METHODS: Superoxide anion and its powerful oxidant reaction with nitric oxide (peroxynitrite) generation were determined in esophageal mucosal biopsies from 101 patients with different gastro-esophageal reflux diseases and 28 controls. Activity of both superoxide dismutase (SOD) and catalase, and reduced glutathione (GSH) levels, were also assessed. Expression of Cu, ZnSOD, MnSOD and tyrosine-nitrated MnSOD were analyzed by Western blot and/or immunohistochemistry. RESULTS: The highest levels of superoxide anion generation were found in patients with severe lesions of esophagitis. Peroxynitrite generation was intense in Barrett's biopsies, weaker in esophagitis and absent/weak in normal mucosa. Expression of Cu, ZnSOD and MnSOD isoforms were present in normal mucosa and increased according to the severity of the lesion, reaching the highest level in Barrett's esophagus. However, SOD mucosal activity significantly decreased in patients with esophagitis and Barrett's esophagus, which was, at least in part, due to nitration of its tyrosine residues. Catalase activity and GSH levels were significantly increased in mucosal specimens from patients with esophagitis and/or Barrett's esophagus. CONCLUSION: A decrease in SOD antioxidant activity leading to increased mucosal levels of superoxide anion and peroxynitrite radicals may contribute to the development of esophageal damage and Barrett's esophagus in patients with gastroesophageal reflux. Administration of SOD may be a therapeutic target in the treatment of patients with esophagitis and Barrett's esophagus.

Angiogenesis and proliferation markers in adjacent cirrhotic tissue could predict hepatocellular carcinoma outcome after liver transplantation.

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.