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Archived Hematoxylin and Eosin (H&E) stained pathology slides are routinely stored to index formalin-fixed paraffin-embedded (FFPE) sample tissue blocks. FFPE blocks are clinically annotated human tumor specimens that can be valuable in studies decades after the tissue is collected. If stored properly, they have the potential to yield a valuable number of serial sectioned slides for diagnostic or research purposes. However, some retrospective studies are limited in scope because the tissue samples have been depleted or not enough material is available in stored blocks for serial sections. The goal of these studies was to determine if archived H&E-stained slides can be directly reutilized by optimizing methods to de-stain and then re-stain the H&E stained slides to allow the detection of several biomarkers of interest using a conjugated antibody with chromogen multiplex immunohistochemistry procedure. This simple but innovative procedure, combined with image analysis techniques, demonstrates the ability to perform precise detection of relevant markers correlated to disease progression in initially identified tumor regions in tissue. This may add clinical value in retaining H&E slides for further use.
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The major aim of this study was to evaluate the performance of anti-BRAF V600E (VE1) antibody in colorectal tumors with and without KRAS mutation. KRAS and BRAF are two major oncogenic drivers of colorectal cancer (CRC) that have been frequently described as mutually exclusive, thus the BRAF V600E mutation is not expected to be present in the cases with KRAS mutation. In addition, a review of 25 studies comparing immunohistochemistry (IHC) using the anti-BRAF V600E (VE1) antibody with BRAF V600E molecular testing in 4041 patient samples was included. One-hundred and twenty cases with/without KRAS or BRAF mutations were acquired. The tissue were immunostained with anti-BRAF V600E (VE1) antibody with OptiView DAB IHC detection kit. The KRAS mutated cases with equivocal immunostaining were further evaluated by Sanger sequencing for BRAF V600E mutation. Thirty cases with BRAF V600E mutation showed unequivocal, diffuse, uniform, positive cytoplasmic staining and 30 cases with wild-type KRAS and BRAF showed negative staining with anti-BRAF V600E (VE1) antibody. Out of 60 cases with KRAS mutation, 56 cases (93.3%) were negative for BRAF V600E mutation by IHC. Four cases showed weak, equivocal, heterogeneous, cytoplasmic staining along with nuclear staining in 25-90% of tumor cells. These cases were confirmed to be negative for BRAF V600E mutation by Sanger sequencing. Overall, IHC with anti-BRAF V600E (VE1) antibody using recommended protocol with OptiView detection is optimal for detection of BRAF V600E mutation in CRC. Our data are consistent with previous reports indicating that KRAS and BRAF V600E mutation are mutually exclusive.
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Anticuerpos Monoclonales/inmunología , Neoplasias Colorrectales/diagnóstico , Inmunohistoquímica/métodos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/inmunología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Humanos , Ratones , Pronóstico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
BACKGROUND: Although normally linked to bone and cartilage development, the Runt-related transcription factor, RUNX2, was reported in the mouse heart during development of the valves. We examined RUNX2 expression and function in the developing avian heart as it related to the epithelial-mesenchymal transition (EMT) in the atrioventricular canal. EMT can be separated into an activation stage involving hypertrophy and cell separation and an invasion stage where cells invade the extracellular matrix. The localization and activity of RUNX2 was explored in relation to these steps in the heart. As RUNX2 was also reported in cancer tissues, we examined its expression in the progression of esophageal cancer in staged tissues. RESULTS: A specific isoform, RUNX2-I, is present and required for EMT by endothelia of the atrioventricular canal. Knockdown of RUNX2-I inhibits the cell-cell separation that is characteristic of initial activation of EMT. Loss of RUNX2-I altered expression of EMT markers to a greater extent during activation than during subsequent cell invasion. Transforming growth factor beta 2 (TGFß2) mediates activation during cardiac endothelial EMT. Consistent with a role in activation, RUNX2-I is regulated by TGFß2 and its activity is independent of similarly expressed Snai2 in regulation of EMT. Examination of RUNX2 expression in esophageal cancer showed its upregulation concomitant with the development of dysplasia and continued expression in adenocarcinoma. CONCLUSIONS: These data introduce the RUNX2-I isoform as a critical early transcription factor mediating EMT in the developing heart after induction by TGFß2. Its expression in tumor tissue suggests a similar role for RUNX2 in the EMT of metastasis. Developmental Dynamics 247:542-554, 2018. © 2017 Wiley Periodicals, Inc.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Transición Epitelial-Mesenquimal , Animales , Embrión de Pollo , Pollos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Neoplasias/metabolismo , Isoformas de Proteínas , Activación Transcripcional , Factor de Crecimiento Transformador betaRESUMEN
The most common of all activating BRAF mutations (T1799A) leads to a substitution of valine (V) to glutamic acid (E) at the position 600 of the amino acid sequence. The major goal of this study was to compare detection of the BRAF V600E mutation by DNA sequencing with immunohistochemistry (IHC) using the anti-BRAF V600E (VE1) antibody. Archival formalin fixed, paraffin embedded tissues from 352 patients with colon adenocarcinoma (nâ=â279) and papillary thyroid carcinoma (nâ=â73) were evaluated for the BRAF V600E mutation by sequencing and IHC. The discordant cases were re-evaluated by repeat IHC, SNaPshot and next-generation sequencing (NGS). Furthermore, the effect of pre-analytical variables on the utility of this antibody was evaluated in two xenograft mouse models.After resolving 15 initially discordant cases, 212 cases were negative for the BRAF V600E mutation by IHC. Of these, 210 cases (99.1%) were also negative by sequencing and two cases (0.9%) remained discordant. Of the 140 cases that were IHC positive for BRAF V600E, 138 cases were confirmed by sequencing (98.6%) and two cases remained discordant (1.4%). Overall, the negative predictive value was 99.1%, positive predictive value 98.6%, sensitivity 98.6%, specificity 99.1% and overall percentage agreement 98.9% (348/352 cases). Tissue fixation studies indicated that tissues should be fixed for 12-24âh within 2âh of tissue collection with 10% neutral buffered formalin.
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Adenocarcinoma/patología , Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/metabolismo , Carcinoma/patología , Neoplasias Colorrectales/patología , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/patología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma Papilar , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Masculino , Ratones , Mutación , Proteínas Proto-Oncogénicas B-raf/inmunología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismoRESUMEN
This paper presents commentaries on animal models used for Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) research; acid- and bile-induced chromosomal instability and clonal selection during the progression of BE to EAC; how the components of gastric refluxate, especially acid and bile salts, promote carcinogenesis in metaplastic BE; genome-wide changes in DNA methylation and transcription involved in BE carcinogenesis; the potential role of miRNA in the development of BE and EAC; the effect of inflammatory cytokines linked to obesity on the activation of cell-death pathways and cell survival in BE and esophageal cancer; and the role of autophagy in esophageal cancer development.
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Adenocarcinoma/patología , Esófago de Barrett/patología , Transformación Celular Neoplásica/patología , Neoplasias Esofágicas/patología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Metilación de ADN , Modelos Animales de Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , HumanosRESUMEN
The following paper on the molecular biology of Barrett's esophagus (BE) includes commentaries on signaling pathways central to the development of BE including Hh, NF-κB, and IL-6/STAT3; surgical approaches for esophagectomy and classification of lesions by appropriate therapy; the debate over the merits of minimally invasive esophagectomy versus open surgery; outcomes for patients with pharyngolaryngoesophagectomy; the applications of neoadjuvant chemotherapy and chemoradiotherapy; animal models examining the surgical models of BE and esophageal adenocarcinoma; the roles of various morphogens and Cdx2 in BE; and the use of in vitro BE models for chemoprevention studies.
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Adenocarcinoma/patología , Esófago de Barrett/patología , Transformación Celular Neoplásica/patología , Neoplasias Esofágicas/patología , Esófago/patología , Adenocarcinoma/genética , Adenocarcinoma/cirugía , Esófago de Barrett/genética , Esófago de Barrett/cirugía , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/cirugía , Esofagectomía , Esófago/metabolismo , Esófago/cirugía , Humanos , Interleucina-6/genética , FN-kappa B/genéticaRESUMEN
Enterohepatic helicobacter species (EHS) infect the intestinal tract and biliary tree, triggering intestinal and hepatic disorders. Helicobacter hepaticus, the prototypic murine EHS, is also associated with inflammation. Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. The cause of NEC is not fully understood, but anomalies of bacterial colonization (dysbiosis) are thought to play an important role in disease onset. To evaluate the effect of H. hepaticus infection on the development of NEC, premature formula-fed rats were kept either in H. hepaticus-free conditions or colonized with H. hepaticus; both groups were exposed to asphyxia and cold stress. The incidence of NEC, expression of Toll-like receptors (TLRs), production of cytokines and mucins, and presence of autophagy regulators were evaluated at the site of injury. H. hepaticus infection increased the incidence of NEC from 39 to 71% and significantly increased levels of TLR4 receptor, expression of proinflammatory cytokines CXCL1, IL-1ß, IL-12, and IL-23, and altered activation of autophagy. H. hepaticus induces inflammation and increases the incidence and severity of experimental NEC; this is consistent with observations in neonates of blooms of proinflammatory microbes just before the onset of NEC. Future studies using rodent NEC models should include testing for H. hepaticus infection. Further studies in neonates of early identification and/or diminution of proinflammatory microbes may be beneficial in decreasing the incidence of NEC.
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Enterocolitis Necrotizante/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter hepaticus/fisiología , Animales , Autofagia , Citocinas/genética , Citocinas/metabolismo , Enterocolitis Necrotizante/patología , Femenino , Regulación de la Expresión Génica/fisiología , Infecciones por Helicobacter/patología , Íleon/metabolismo , Íleon/microbiología , Íleon/patología , Inflamación/metabolismo , Inflamación/patología , Embarazo , ARN Bacteriano/aislamiento & purificación , Ratas , Ratas Sprague-DawleyRESUMEN
Barrett's esophagus (BE) is a metaplastic lesion of the distal esophagus arising as a consequence of chronic gastroesophageal reflux disease. Multiple studies show that BE is associated with increased risk of esophageal adenocarcinoma (EAC). Epidemiological studies and animal models demonstrate that chronic inflammation triggered by repeated exposure to refluxate predisposes to the development of BE and EAC. The chronic inflammation is associated with cytokine alterations. Interleukin 6 (IL-6) is a cytokine that stimulates cell proliferation and apoptosis resistance is frequently increased in different cancers. Importantly, IL-6 and transcriptional factor signal transducer and activator of transcription 3 (STAT3) that is activated by IL-6 are also increased in BE and EAC. This review critically appraises the role of IL-6/STAT3 pathway in progression of BE to EAC from the published evidence currently available.
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Esófago de Barrett/metabolismo , Regulación de la Expresión Génica , Interleucina-6/fisiología , Animales , Apoptosis , Ácidos y Sales Biliares/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias Esofágicas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación , Masculino , Ratones , Factor de Transcripción STAT3/metabolismo , Factores SexualesRESUMEN
Chronic inflammation increases lymphoma risk. Chronic inflammation exposes cells to increased reactive oxygen species (ROS). Constant exposure to ROS selects for oxidative stress-resistant cells with upregulated anti-oxidant defense enzymes. The impact of oxidative stress resistance on the redox biology and chemotherapy response in lymphoma has not been rigorously tested. To measure the effect of antioxidant defense enzyme upregulation in lymphoid cells, we created oxidative stress-resistant WEHI7.2 thymic lymphoma cell variants. We selected a population of WEHI7.2 cells for resistance to hydrogen peroxide and constructed catalase-overexpressing WEHI7.2 transfectants. The WEHI7.2 variants had: i) increased catalase and total superoxide dismutase activities; ii) an altered GSSG/2GSH redox potential; iii) a more oxidized NADP(+)/NADPH pool; and iv) increased phase 2 enzymes, NAD(P)H:quinone oxidoreductase and glutathione S-transferases µ and π. Regression analysis showed a correlation between the GSSG/2GSH redox potential and the increased phase 2 enzyme activities. As predicted from the anti-oxidant defense enzyme profile, the variants were more resistant to the oxidants hydrogen peroxide and paraquat. The variants exhibited resistance to the common lymphoma chemotherapeutics, cyclophosphamide, doxorubicin, vincristine and glucocorticoids. These data indicate that chronic ROS exposure results in lymphoid cells with multiple changes in their redox biology and a chemoresistance phenotype. These data further suggest that lymphomas that arise at the site of chronic inflammation develop chemoresistance due to a combination of drug detoxification and removal of ROS.
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Beclin-1 has a central role in the regulation of autophagy. Barrett's esophagus (BE) is associated with a significantly increased risk for the development of esophageal adenocarcinoma (EAC). In the current study, we evaluated the role of Beclin-1 and autophagy in the EAC. Biopsies obtained from patients with BE and EAC, tissues from a rat model of BE and EAC, and esophageal cell lines were evaluated for the expression of Beclin-1 by immunohistochemistry, immunoblotting, or RT-PCR. Since reflux of bile acids is important in EAC, we also evaluated the effect of exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression. Beclin-1 expression was high in squamous epithelium and nondysplastic BE, whereas its expression was low in dysplastic BE and EAC. The same pattern of expression was observed in rat tissues and in esophageal cell lines. Normal esophageal epithelium and HET-1A cells (derived from normal squamous epithelium) show high levels of Beclin-1, but lower levels of Beclin-1 were found in BE and EAC cell lines (CP-A, CP-C, and OE33). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy as evaluated by electron microscopy and counting percentage of GFP-LC3-positive BE cells with punctate pattern. In contrast, chronic exposure to DCA did not result in the alteration of Beclin-1 levels or autophagy. In summary, these data suggest that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance.
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Adenocarcinoma/patología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/fisiología , Ácido Desoxicólico/fisiología , Neoplasias Esofágicas/patología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Aminoácidos/deficiencia , Animales , Autofagia , Esófago de Barrett/patología , Beclina-1 , Ácidos y Sales Biliares/metabolismo , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Ácido Desoxicólico/farmacología , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Análisis por Micromatrices , Microscopía Confocal , Microscopía Electrónica de Transmisión , ARN/biosíntesis , ARN/genética , ARN Interferente Pequeño/farmacología , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.
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Apoptosis/efectos de los fármacos , Cationes/metabolismo , Ácido Desoxicólico/farmacología , Esófago/patología , Neoplasias Gastrointestinales/patología , Intercambiadores de Sodio-Hidrógeno/fisiología , Ácidos y Sales Biliares/farmacología , Células Cultivadas , Neoplasias Gastrointestinales/etiología , Humanos , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Lisosomas , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidoresRESUMEN
Although the prevalence of Barrett's esophagus (BE) is rising no data exist for racial minorities on prevalence in the general population. Minorities have a lower prevalence than Caucasians, and yet age, smoking, abdominal obesity, and Helicobacter pylori are all risk factors. Metabolic changes induced by adipocytokines and the apparently strong association between obesity, central adiposity, and BE may lead to reconsideration of some aspects of the natural history of BE. There is lack of experimental evidence on acid sensitivity and BE, which is hyposensitive compared to esophageal reflux disease. Reactive nitrogen and oxygen species lead to impaired expression of tumor suppressor genes, which can lead to cancer development; thus, antioxidants may be protective. Gastroesophageal reflux disease may be considered an immune-mediated disease starting at the submucosal layer; the cytokine profile of the mucosal immune response may explain the different outcome of gastroesophageal reflux.
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Esófago de Barrett/epidemiología , Esófago de Barrett/etiología , Humanos , Incidencia , PrevalenciaRESUMEN
The following on proton pump inhibitors and chemoprevention in Barrett's esophagus includes commentaries on normalization of esophageal refluxate; the effects of 5-HT(4) agonists on EGF secretion and of lubripristone on chloride channels agents; the role of Campylobacter toxin production; the deleterious effects of unconjugated bile acids; the role of baclofen in nonacid reflux; the threshold for adequate esophageal acid exposure; the effects of proton pump inhibitor (PPI) therapy on normalization of esophageal pH and on cell proliferation; the role of the phenotype of cellular proliferation on the effects of PPI therapy; and the value of Symptom Index and Symptom Association Probability in the evaluation of potential response to treatment.
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Esófago de Barrett/tratamiento farmacológico , Inhibidores de la Bomba de Protones/uso terapéutico , Esófago de Barrett/patología , Esófago de Barrett/prevención & control , Quimioprevención , HumanosRESUMEN
The following on proton pump inhibitors (PPIs) and chemoprevention in relation to Barrett's esophagus includes commentaries on 48-h pH monitoring, pH-impedence, bile acid testing, dyspepsia, long/short segment Barrett's esophagus, nonerosive reflux disease (NERD), functional heartburn, dual-release delivery PPIs, immediate-release PPIs, long-term PPI use, prokinetic agents, obesity, baclofen, nocturnal acid breakthrough, nonsteroidal anti-inflammatory drugs (NSAIDs), and new PPIs.
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Esófago de Barrett/tratamiento farmacológico , Quimioprevención , Inhibidores de la Bomba de Protones/uso terapéutico , Humanos , Monitoreo Fisiológico , Resultado del TratamientoRESUMEN
The following on endoscopic treatments of Barrett's esophagus includes commentaries on indications for endoscopic treatments; endo-luminal plication procedures; the cellular modifications induced by the endoscopic ablation therapies; eradication by banding without resection; the evaluation of complete ablation; recurrence after ablation; association of antireflux surgery; radiofrequency ablation; and nondysplastic Barrett's esophagus.
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Esófago de Barrett/terapia , Endoscopía/métodos , HumanosRESUMEN
The following on surveillance and reversal of Barrett's esophagus (BE) includes commentaries on criteria for surveillance even when squamous epithelium stains normally with a variety of biomarkers; the long-term follow-up of surgery versus endoscopic ablation of BE; the recommended surveillance intervals in patients without dysplasia; the sampling problems related to anatomic changes following fundoplication; the value of tissue spectroscopy and optical coherence tomography; the cost-effectiveness of biopsy protocols for surveillance; the quality of life of Barrett's patients; and risk stratification and surveillance strategies.
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Esófago de Barrett/epidemiología , Vigilancia de la Población , Esófago de Barrett/terapia , Humanos , Tomografía de Coherencia ÓpticaRESUMEN
The following on prevalence and incidence of adenocarcinomas in Barrett's esphophagus (BE) includes commentaries on the mechanisms of a potential protective effect of proton pump inhibitors (PPIs) on progression of BE to high-grade dysplasia; evaluation of the role of PPIs in decreasing the risk of degeneration; the geographical variations of incidence of BE; the role of the nonmorphologic biomarkers; the relationship between length of BE and development of cancer; the confounding factors in incidence rates of BE; the role of the increase of cell differentiation and apoptosis induced by PPIs in the diminution of cancer risk; the frequency of occult neoplastic foci and unsuspected invasive cancer in surgical specimens; the influence on the indications of endoscopic therapy; the overestimation of regression in surgical series; attempts to evaluate the reasons for variations of cancer incidence in the literature; and progress in screening and surveillance for BE.
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Adenocarcinoma/epidemiología , Esófago de Barrett/epidemiología , Neoplasias Esofágicas/epidemiología , Progresión de la Enfermedad , Humanos , Incidencia , Prevalencia , Inhibidores de la Bomba de Protones/uso terapéuticoRESUMEN
The following on molecular mechanisms of Barrett's esophagus and adenocarcinoma contains commentaries on the mechanism of bile and gastric acid induced damage; the roles of BMP-4 and CDX-2 in the development of intestinal metaplasia; the transcription factors driving intestinalization in Barrett's esophagus; the contribution of bone marrow to metaplasia and adenocarcinoma; activation and inactivation of transcription factors; and a novel study design targeting molecular pathways in Barrett's esophagus.
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Adenocarcinoma/genética , Esófago de Barrett/genética , Neoplasias Esofágicas/genética , Adenocarcinoma/fisiopatología , Esófago de Barrett/fisiopatología , Ácidos y Sales Biliares/fisiología , Proteína Morfogenética Ósea 4/fisiología , Factor de Transcripción CDX2 , Daño del ADN , Neoplasias Esofágicas/fisiopatología , Proteínas de Homeodominio/fisiología , Humanos , Transactivadores/fisiologíaRESUMEN
Barrett's esophagus (BE) is a premalignant condition, where normal squamous epithelium is replaced by intestinal epithelium. BE is associated with an increased risk of developing esophageal adenocarcinoma (EAC). However, the BE cell of origin is not clear. We hypothesize that BE tissue originates from esophageal squamous cells, which can differentiate to columnar cells as a result of repeated exposure to gastric acid and bile acids, two components of refluxate implicated in BE pathology. To test this hypothesis, we repeatedly exposed squamous esophageal HET1A cells to 0.2 mM bile acid (BA) cocktail at pH 5.5 and developed an HET1AR-resistant cell line. These cells are able to survive and proliferate after repeated 2-h treatments with BA at pH 5.5. HET1AR cells are resistant to acidification and express markers of columnar differentiation, villin, CDX2, and cytokeratin 8/18. HET1AR cells have increased amounts of reactive oxygen species, concomitant with a decreased level and activity of manganese superoxide dismutase compared with parental cells. Furthermore, HET1AR cells express proteins and activate signaling pathways associated with inflammation, cell survival, and tumorigenesis that are thought to contribute to BE and EAC development. These include STAT3, NF-κB, epidermal growth factor receptor (EGFR), cyclooxygenase-2, interleukin-6, phosphorylated mammalian target of rapamycin (p-mTOR), and Mcl-1. The expression of prosurvival and inflammatory proteins and resistance to cell death could be partially modified by inhibition of STAT3 signaling. In summary, our study shows that long-term exposure of squamous cells to BA at acidic pH causes the cells to display the same characteristics and markers as BE.
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Esófago de Barrett/etiología , Ácidos y Sales Biliares/farmacología , Esófago/efectos de los fármacos , Ácidos/farmacología , Adenocarcinoma/metabolismo , Biomarcadores/metabolismo , Línea Celular Transformada , Regulación hacia Abajo , Resistencia a Medicamentos , Epitelio/metabolismo , Neoplasias Esofágicas/metabolismo , Esófago/citología , Esófago/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Interleucina-6/metabolismo , Metaplasia/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Superóxido Dismutasa/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Barrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pH(i)) and its effect on DNA damage in cells derived from normal oesophagus (HET1A) or Barrett's oesophagus (CP-A). DESIGN: Cells were exposed to bile acid cocktail (BA) and/or acid in the presence/absence of inhibitors of nitric oxide synthase (NOS) or sodium-hydrogen exchanger (NHE). Nitric oxide (NO), pH(i) and DNA damage were measured by fluorescent imaging and comet assay. Expression of NHE1 and NOS in cultured cells and biopsies from Barrett's oesophagus or normal squamous epithelium was determined by RT-PCR, immunoblotting or immunohistochemistry. RESULTS: A dose dependent decrease in pH(i) was observed in CP-A cells exposed to BA. This effect of BA is the consequence of NOS activation and increased NO production, which leads to NHE inhibition. Exposure of oesophageal cells to acid in combination with BA synergistically decreased pH(i). The decrease was more pronounced in CP-A cells and resulted in >2-fold increase in DNA damage compared to acid treatment alone. Examination of biopsies and cell lines revealed robust expression of NHE1 in Barrett's oesophagus but an absence of NHE1 in normal epithelium. CONCLUSIONS: The results of this study identify a new mechanism of bile acid-induced DNA damage. We found that bile acids present in the refluxate activate immediately all three isoforms of NOS, which leads to an increased NO production and NHE inhibition. This consequently results in increased intracellular acidification and DNA damage, which may lead to mutations and cancer progression. Therefore, we propose that in addition to gastric reflux, bile reflux should be controlled in patients with Barrett's oesophagus.