Barrett's Esophageal Adenocarcinoma Cells Research Articles

Cancer Stem Cells of Esophageal Adenocarcinoma are Suppressed by Inhibitors of TRPV2 and SLC12A2.

The potential of membrane transporters activated in cancer stem cells (CSCs) as new therapeutic targets for cancer is attracting increasing interest. Therefore, the present study examined the expression profiles of ion transport-related molecules in the CSCs of esophageal adenocarcinoma (EAC). Cells that highly expressed aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from OE33 cells, a human Barrett's EAC cell line, by fluorescence-activated cell sorting. CSCs were identified based on the formation of tumorspheres. Gene expression profiles in CSCs were examined by a microarray analysis. Among OE33 cells, ALDH1A1 messenger RNA levels were higher in CSCs than in non-CSCs. Furthermore, CSCs exhibited resistance to cisplatin and had the capacity to redifferentiate. The results of the microarray analysis of CSCs showed the up-regulated expression of several genes related to ion channels/transporters, such as transient receptor potential vanilloid 2 (TRPV2) and solute carrier family 12 member 2 (SLC12A2). The cytotoxicities of the TRPV2 inhibitor tranilast and the SLC12A2 inhibitor furosemide were higher at lower concentrations in CSCs than in non-CSCs, and both markedly reduced the number of tumorspheres. The cell population among OE33 cells that highly expressed ALDH1A1 also was significantly decreased by these inhibitors. Based on the present results, TRPV2 and SLC12A2 are involved in the maintenance of CSCs, and their specific inhibitors, tranilast and furosemide, respectively, have potential as targeted therapeutic agents for EAC.

Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

BackgroundChronic gastroesophageal reflux disease (GERD) is a major risk factor for the development of metaplastic Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Uncontrolled accumulation of reactive oxygen species (ROS) in response to acidic bile salts (ABS) in reflux conditions can be lethal to cells. In this study, we investigated the role of APE1/REF1 in regulating nuclear erythroid factor-like 2 (NRF2), the master antioxidant transcription factor, in response to reflux conditions. ResultsWe found that APE1 protein was critical for protecting against cellular ROS levels, oxidative DNA damage, double strand DNA breaks, and cell death in response to conditions that mimic reflux. Analysis of cell lines and de-identified tissues from patients with EAC demonstrated overexpression of both APE1 and NRF2 in EAC cells, as compared to non-neoplastic esophageal cells. Using reflux conditions, we detected concordant and prolonged increases of APE1 and NRF2 protein levels for several hours, following transient short exposure to ABS (20 min). NRF2 transcription activity, as measured by ARE luciferase reporter, and expression of its target genes (HO-1 and TRXND1) were similarly increased in response to ABS. Using genetic knockdown of APE1, we found that APE1 was required for the increase in NRF2 protein stability, nuclear localization, and transcription activation in EAC. Using knockdown of APE1 with reconstitution of wild-type and a redox-deficient mutant (C65A) of APE1, as well as pharmacologic APE1 redox inhibitor (E3330), we demonstrated that APE1 regulated NRF2 in a redox-dependent manner. Mechanistically, we found that APE1 is required for phosphorylation and inactivation of GSK-3β, an important player in the NRF2 degradation pathway. ConclusionAPE1 redox function was required for ABS-induced activation of NRF2 by regulating phosphorylation and inactivation of GSK-3β. The APE1-NRF2 network played a critical role in protecting esophageal cells against ROS and promoting cell survival under oxidative reflux conditions.

PS02.029: USEFULNESS OF TRIAMCINOLONE INJECTION TO PREVENT STRICTURE AFTER CIRCUMFERENTIAL ESOPHAGEAL ESD

Abstract Background ESD is a standard treatment for superficial esophageal cancer in Japan. Stricture is one of important complication of esophageal ESD, and it makes quality of life of the patient worse. Usefulness of Triamcinolone (TA) injection to prevent stricture after semi-circumferential ESD has been reported. However, usefulness of TA injection after circumferential esophageal ESD is still unclear. Methods The aim of this study is to clarify the usefulness of triamcinolone injection to prevent stricture after circumferential ESD. A total of forty-nine patients treated by circumferential esophageal ESD from 2004 to 2017 in Saku Central Hospital Advanced Care Center were enrolled to this retrospective study. Fifty mg TA was injected into submucosal layer just after ESD, and TA injection was repeated in two-weeks interval by the ESD ulcer healed. When the length of circumferential ESD was 50mm or longer, 100 mg Triamcinolone was injected just after ESD, and 50mg TA injection was repeated in two-weeks interval. Fifteen mm endoscopic balloon dilatation (EBD) was performed when the scope couldn’t pass the ESD ulcer. The primary endpoint was the number of balloon dilatation. The secondary endpoints were duration from ESD to ulcer healing, complications and the difference between Barrett's esophageal adenocarcinoma (EAC) and squamous cell carcinoma (SCC). Results 1. Median number of EBD in Non-TA and TA group were 15 (13–33) and 3 (0–23), respectively (P < 0.01) 2. Median duration from ESD to ulcer healing were 6 (3–23) and 4 (1–24) months. respectively. (P = 0.47) 3. Complications: Perforation rate due to EBD was 6.3% (1/16) and 3.0%(1/33). Both patients were treated by conservative therapy. 4. Difference between EAC and SCC: There were nine EAC and forty SCC patients in TA group. The number of EBD were 5.3 (5–11) and 5.4 (3 -11), respectively. Duration of ulcer healing were 10 (4–14) and 6.5 (1–24), respectively. There was no significant difference between two groups. Conclusion Triamcinolone injection is safe and effective treatment to prevent stricture after circumferential ESD, not only for SCC but also for EAC. Disclosure All authors have declared no conflicts of interest.

334 - APE1 Upregulates MMP14 Expression to Promote Invasion of Barrett's Esophagus Cells and Esophageal Adenocarcinoma Cells Through Novel Redox-Sensitive ARF6-Mediated Exocytosis

334 - APE1 Upregulates MMP14 Expression to Promote Invasion of Barrett's Esophagus Cells and Esophageal Adenocarcinoma Cells Through Novel Redox-Sensitive ARF6-Mediated Exocytosis

Review of the gut microbiome and esophageal cancer: Pathogenesis and potential clinical implications.

Esophageal cancer ranks among the most aggressive malignant diseases. The limited improvements in treatment outcomes provided by conventional therapies have prompted us to seek innovative strategies for treating this cancer. More than 100 trillion microorganisms inhabit the human intestinal tract and play a crucial role in health and disease conditions, including cancer. The human intestinal microbiome is thought to influence tumor development and progression in the gastrointestinal tract by various mechanisms. For example, Fusobacterium nucleatum, which primarily inhabits the oral cavity and causes periodontal disease, might contribute to aggressive tumor behavior through activation of chemokines such as CCL20 in esophageal cancer tissue. Composition of the intestinal microbiota is influenced by diet, lifestyle, antibiotics, and pro‐ and prebiotics. Therefore, by better understanding how the bacterial microbiota contributes to esophageal carcinogenesis, we might develop novel cancer prevention and treatment strategies through targeting the gastrointestinal microflora. This review discusses the current knowledge, available data and information on the relationship of microbiota with esophagitis, Barrett's esophagus, esophageal adenocarcinoma and squamous cell carcinoma.

Apigenin inhibits proliferation and migratory properties of Barrett's esophageal adenocarcinoma cells by targeting PI3K/Akt/mTOR pathway

Purpose: To investigate the effect of apigenin on Barrett's esophagus–associated esophageal adenocarcinoma (BEAC) cells OE33, and also to ascertain the mechanism by which it inhibits cellular proliferation and motility. Methods: Proliferation index of OE33 in the absence and presence of apigenin was determined by methyl-thiazolyl-tetrazolium (MTT) assay and apoptosis was determined by enzyme-linked immunosorbent assay (ELISA) method. Boyden Chamber’s assay was applied to determine the migration and invasion of control and apigenin-treated OE33 cells. Status of PI3K/Akt/mTor signaling was further determined by Western blotting in control and apigenin-treated cells. Results: Apigenin resulted in the inhibition of the proliferation of OE33 cells in a dose- and timedependent fashion, with an IC 50 of 75 μM, after 72 h of incubation, and also induced apoptosis, with modulation of pro- and anti-apoptotic genes. Furthermore, apigenin inhibited the motility of OE33 by targeting PI3K/Akt/mTOR signaling. Conclusion: Apigenin effectively inhibits the oncogenicity of OE33 cells by targeting PI3K/Akt/mTOR pathway. Keywords: Barrett's esophagus–associated esophageal adenocarcinoma (BEAC), Apoptosis, Migration, Anticancer

Su2063 Targeting HSP90 in Patient derived Barrett's Esophagus and Esophageal adenocarcinoma cells

Su2063 Targeting HSP90 in Patient derived Barrett's Esophagus and Esophageal adenocarcinoma cells

Tu1176 HOX Gene Expression In Barrett's Esophageal Dysplastic and Adenocarcinoma Cells

Tu1176 HOX Gene Expression In Barrett's Esophageal Dysplastic and Adenocarcinoma Cells

Gold nanorods and curcumin-loaded nanomicelles for efficient in vivo photothermal therapy of Barrett's esophagus.

Provide an enhanced local drug delivery, nanoparticle(s) to minimize systemic effects and achieve enhanced permeability and drug retention into abnormal cells and stroma. Here a simultaneous loading of lipophilic gold nanorods (GNRs) and curcumin into polymeric nanomicelles made of biocompatible PLGA-b-PEG copolymer through a double re-emulsification process has been developed. Initial results in vitro on Barrett's esophagus and esophageal adenocarcinoma cell lines demonstrated a significant reduction in cell viability with curcumin and GNRs exposure (p < 0.05). In vivo Barrett's-associated animal model confirmed these results with successful in vivo demonstrated eradication of all high-grade dysplastic premalignant cancer cells. The synthesis of this novel nanosystem containing GNRs and curcumin is safe and effective in treating and eradicating premalignant esophageal adenocarcinoma.

Tu1671 Calcium Signaling in Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Tu1674 Cannabinoid Agonist Interfere the Human Bone Marrow-Derived Mesenchymal Stem Cell-Induced Gastric Cancer Cell Migration

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Tu1672 Involvement of p21-Activated Kinase 4 (PAK4) in Gastrin-Induced Migration of Gastric Cancer Cells

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Mo1658 Acid-Induced Decrease in Cell Apoptosis Is Mediated by Activation of NADPH Oxidase NOX5-S, NF-κB and Sodd in Barrett's Esophageal Adenocarcinoma Cells

Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood. Acid reflux may play an important role in this progression. We have previously shown that acid exposure increases H2O2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-dependent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers. In this study we examined the role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD. SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T. Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells. Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immunoprecipitation (ChIP) assay showed that SODDDNAwas detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT. Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleotide significantly reduced binding. The addition of the mutant SODD oligonucleotide had less effect on binding. We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD. Supported by NIH NIDDK R01 DK080703.

Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca(2+) in FLO cells, an increase that is blocked by Ca(2+)-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca(2+) increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA.

Mo1855 Role of HOXD9 in Acid-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

G A A b st ra ct s investigated variables were not associated with progression to HGD or EAC. CONCLUSION: Older patients with BE and LGD with a repeat diagnosis of LGD during follow-up and followed-up in a university hospital, probably, probably reflecting selection bias, are at a higher risk of progression to HGD or EAC. These data can be used to tailor endoscopic surveillance intervals in selected groups of patients with BE and LGD.

Sa2015 Signaling in Bile Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

G A A b st ra ct s was recorded at termination. Blood was collected for determination of serum gastrin. The histological changes in the stomachs were examined. The presence of dysplasia and carcinoma on the anterior and posterior side of the stomachs were compared, and the oxyntic mucosal thickness and neuroendocrine (chromogranin A positive) cell density were calculated. Results: Ten of fifteen animals operated by anterior vagotomy and 11 of 16 sham-operated developed hypergastrinemia. Vagotomy did not affect intragastric pH or serum gastrin. There was no difference between innervated and denervated oxyntic mucosa in dysplasia and carcinoma development. However, vagotomy resulted in lower stomach weight in hypergastrinemic animals (2.8 ± 0.26 g vs. 3.7 ± 0.39 g, p=0.027) and reduced oxyntic mucosal thickness on the anterior side (0.83 ± 0.06 mm vs. 1.13 ± 0.1 mm, p ,0.01). Vagotomy also resulted in a reduction in volume density of chromogranin A positive cells in the oxyntic mucosa (12.1 ± 1.8 % vs. 18.8 ± 3.0 %, p=0.04). Conclusions: Unilateral truncal vagotomy reduced the trophic effects of hypergastrinemia on the ECL cell and oxyntic mucosa, but did not prevent gastric carcinogenesis in female Japanese cotton rats. The effects of vagotomy on gastric carcinogenesis in animal models are conflicting and further studies in patients should be done to clarify the clinically significant effects of vagotomy.

Targeting homologous recombination and telomerase in Barrett’s adenocarcinoma: impact on telomere maintenance, genomic instability and tumor growth

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in BAC. The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contribute to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase, makes telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy, targeting HR and telomerase, has potential to prevent both the tumor growth and genomic evolution in BAC.

Dendritic Cells in Barrett's Esophagus Carcinogenesis: An Inadequate Microenvironment for Antitumor Immunity?

Barrett's esophagus corresponds to the replacement of the normal esophageal squamous epithelium by a columnar epithelium through a metaplastic process. This tissue remodeling is associated with chronic gastroesophageal reflux and constitutes a premalignant lesion leading to a 30- to 60-fold increase in the risk to evolve into esophageal adenocarcinoma. The present study aimed to investigate a possible immune evasion in Barrett's esophagus favoring esophageal adenocarcinoma development. We demonstrated that myeloid and plasmacytoid dendritic cells are recruited during the esophageal metaplasia-dysplasia-carcinoma sequence, through the action of their chemoattractants, macrophage inflammatory protein 3α and chemerin. Next, we showed that, in contrast to plasmacytoid dendritic cells, myeloid dendritic cells, co-cultured with Barrett's esophagus and esophageal adenocarcinoma cell lines, display a tolerogenic phenotype. Accordingly, myeloid dendritic cells co-cultured with esophageal adenocarcinoma cell lines stimulated regulatory T cell differentiation from naïve CD4(+) T cells. In agreement with those results, we observed that both metaplastic areas and (pre)malignant lesions of the esophagus are infiltrated by regulatory T cells. In conclusion, soluble factors secreted by epithelial cells during the esophageal metaplasia-dysplasia-carcinoma sequence influence dendritic cell distribution and promote tumor progression by rendering them tolerogenic.

Abstract 2720: Newly established Barrett's adenocarcinoma cell line (TYAE-1).

Abstract Although 10 esophageal adenocarcinoma cell lines have been reported, only 5 were Barrett's adenocarcinoma cell lines. Furthermore, there was only one mouse-inoculated esophageal adenocarcinoma cell line. In this paper, we present a newly established Barrett's esophageal adenocarcinoma cell line (TYAE-1). Material and Method: The Barrett's esophageal adenocarcinoma cells were obtained from the resected tumor of a patient (74 years-old male). The resected tumor was confirmed as well-differentiated intraepithelial Barrett's adenocarcinoma without lymphnode metastasis. The tumor was too small (T1a) for a primary culture, so we transplanted the tumor in the back of a nude mouse. Results: One month later, the tumor grew rapidly and was able to be re-transplanted to other mice. Up to the writing of this report, the tumor (TYAE-1m) was able to be re-transplanted 10 times every month. The histology of the transplanted tumor was slightly different from the primary tumor. The majority of the tumor was cribriform type and there were a few tubular-trabecular type cells. The expression of beta-Catenin was cytoplasmic in the cribriform part and membranous in the tubular part. Both cell types did express VEGF slightly however they did not express HER-2. We also started to perform in vitro cultures from the mice transplanted tumor and established TYAE-1c cell. TYAE-1c cells were able to be transplanted in nude mice and transplanted tumor showed the same histological features as TYAE-1m. TYAE-1m and TYAE-1c cells were sensitive to Gemcitabine treatment. On the other hand, both tumors did not show sensitivity for Docetaxel treatment. In conclusion: Although the tumor was small and had no invasive characteristics by clinicopathological examination, this tumor has aggressive behavior. This in vitro and in vivo Barrett's adenocarcinoma model may useful for understanding the behavior of Barrett's adenocarcinoma. Citation Format: Yutaka Shimada, Makoto Moriyama, Tomoyuki Okumura, Shinichi Sekine, Shigeaki Sawada, Koshi Matsui, Shozo Hojo, Kazuto Shibuya, Isaku Yoshioka, Toru Yoshida, Takuya Nagata, Kazuhiro Tsukada. Newly established Barrett's adenocarcinoma cell line (TYAE-1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2720. doi:10.1158/1538-7445.AM2013-2720

Targeting the intrinsic inflammatory pathway: honokiol exerts proapoptotic effects through STAT3 inhibition in transformed Barrett's cells

One way to link chronic inflammation with cancer is through the intrinsic inflammatory pathway, in which genetic alterations that induce malignant transformation also produce a cancer-promoting, inflammatory microenvironment. Signal transducer and activator of transcription 3 (STAT3) contributes to the intrinsic inflammatory pathway in Barrett's esophagus. In human tumors, honokiol (a polyphenol in herbal teas) has growth-inhibitory and proapoptotic effects associated with suppressed activation of STAT3. We used human Barrett's epithelial and esophageal adenocarcinoma cell lines to determine effects of honokiol on cell number, necrosis, apoptosis, and anchorage-independent growth and to explore STAT3's role in those effects. We determined Ras activity and expression of phosphorylated ERK1/2, phosphorylated Akt, and phosphorylated STAT3 in the presence or absence of honokiol. Cells were infected with constitutively active Stat3-C to assess effects of honokiol-induced STAT3 inhibition on apoptosis. Honokiol decreased cell number and increased necrosis and apoptosis in transformed Barrett's cells, but not in nontransformed cells. In adenocarcinoma cells, honokiol also increased necrosis and apoptosis and decreased anchorage-independent growth. Within 30 min of honokiol treatment, transformed Barrett's cells decreased expression of phosphorylated STAT3; decreases in Ras activity and phosphorylated ERK1/2 expression were detected at 24 h. Infection with Stat3-C significantly reduced apoptosis after honokiol treatment. Honokiol causes necrosis and apoptosis in transformed Barrett's and esophageal adenocarcinoma cells, but not in nontransformed Barrett's cells, and the proapoptotic effects of honokiol are mediated by its inhibition of STAT3 signaling. These findings suggest a potential role for targeting the intrinsic inflammatory pathways as a therapeutic strategy to prevent Barrett's carcinogenesis.