Normal Esophageal Cell Line Research Articles

Abstract 3023: RNA-Seq identifies neuropilin-2 as a novel oncogene in esophageal squamous cell carcinoma.

Abstract Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is one of the most common and deadliest cancers in the world, with survival rates of less than 15%. Like many other solid tumors, the pathogenesis of ESCC is believed to be a multistep process with accumulation of numerous genetic alterations involving de-regulation of both tumor-suppressor genes and oncogenes. An understanding of the molecular events that result in the development and progression of the disease may lead to treatments that will increase survival rates of ESCC patients. Recent advances in sequencing technologies offer the opportunity to characterize the cancer genome at unprecedented depth and sensitivity. RNA-Seq has proved an effective means of precisely quantifying the changing expression levels of each transcript. In this study, we performed integrative RNA-Seq analysis on 3 paired patient-derived non-tumor and ESCC clinical specimens and discovered a number of commonly differentially expressed genes, of which included a significantly de-regulated neuropilin (NRP) family including neuropilin-1 (NRP1) and neuropilin-2 (NRP2). NRP is a single-spanned membrane glycoprotein. Members of the family have previously been shown to play an important role in various cancer types and to be a co-receptor of vascular endothelial growth factors. Yet to date, its role in ESCC has not been explored. Up-regulation of both NRP1 and NRP2 in ESCC was subsequently validated in the original 3 pairs of clinical samples used for RNA-Seq as well as in a larger cohort of samples (n = 40) by qPCR. Given that NRP2 de-regulation in ESCC is more significant, we chose to first focus our study on this member of the family. Immunohistochemical studies likewise found NRP2 to be up-regulated in ESCC as compared with its non-tumor counterparts in a tissue microarray. Consistently, expression studies in a panel of esophageal cell lines found NRP2 to be expressed at a significantly higher level in 7 of the 8 ESCC cell lines examined compared with immortalized normal esophageal cell lines, NE1 and NE3. Functional studies in ESCC cells, KYSE180 and KYSE140, with NRP2 stably repressed by lentiviral-based shRNA approach impaired proliferation and metastatic abilities in vitro, as demonstrated by foci formation and chemotaxis migration assays, respectively. Moreover, conditioned medium collected from NRP2 repressed cells displayed a reduced ability to induce capillary tube formation in HUVEC cells. These results suggest that NRP2 plays an important role in regulating cell proliferation, migration and angiogenesis in ESCC. Additional work on the mechanism by which NRP2 drives ESCC and the effectiveness of the use of a NRP2 neutralizing antibody against ESCC is currently being studied. We believe with further study, NRP2 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target for ESCC treatment. Citation Format: Tsun Ming Fung, Man Tong, Pak Shing Kwan, Yuen Piu Chan, Xin-Yuan Guan, Stephanie Ma. RNA-Seq identifies neuropilin-2 as a novel oncogene in esophageal squamous cell carcinoma. [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 3023. doi:10.1158/1538-7445.AM2013-3023

Glutathione peroxidase 7 has potential tumour suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma

ObjectiveTo investigate the potential tumour suppressor functions of glutathione peroxidase 7 (GPX7) and examine the interplay between epigenetic and genetic events in regulating its expression in oesophageal adenocarcinomas (OAC).DesignIn vitro...

Nano-Curcumin Inhibits Proliferation of Esophageal Adenocarcinoma Cells and Enhances the T Cell Mediated Immune Response

In Western countries the incidence of the esophageal adenocarcinoma (EAC) has risen at a more rapid rate than that of any other malignancy. Despite intensive therapies this cancer is associated with extreme high morbidity and mortality. For this reason, novel effective therapeutic strategies are urgently required. Dendritic Cell (DC)-based immunotherapy is a promising novel treatment strategy, which combined with other anti-cancer strategies has been proven to be beneficial for cancer patients. Curcumin (diferuloylmethane), is a natural polyphenol that is known for its anti-cancer effects however, in it’s free form, curcumin has poor bioavailability. The aim of this study was to investigate whether using a highly absorptive form of curcumin, dispersed with colloidal nano-particles, named Theracurmin would be more effective against EAC cells and to analyze if this new compound affects DC-induced T cell response. As a result, we show efficient uptake of nano-curcumin by the EAC cell lines, OE33, and OE19. Moreover, nano-curcumin significantly decreased the proliferation of the EAC cells, while did not affect the normal esophageal cell line HET-1A. We also found that nano-curcumin significantly up-regulated the expression of the co-stimulatory molecule CD86 in DCs and significantly decreased the secretion of pro-inflammatory cytokines from in vitro activated T cells. When we combined T cells with nano-curcumin treatment in OE19 and OE33, we found that the basic levels of T cell induced cytotoxicity of 6.4 and 4.1%, increased to 15 and 13%, respectively. In conclusion, we found that nano-curcumin is effective against EAC, sensitizes EAC cells to T cell induced cytotoxicity and decreases the pro-inflammatory signals from T cells. Combining DC immunotherapy with nano-curcumin is potentially a promising approach for future treatment of EAC.

Identification of PTK6, via RNA Sequencing Analysis, as a Suppressor of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is the most commonly observed histologic subtype of esophageal cancer. ESCC is believed to develop via accumulation of numerous genetic alterations, including inactivation of tumor suppressor genes and activation of oncogenes. We searched for transcripts that were altered in human ESCC samples compared with nontumor tissues. We performed integrative transcriptome sequencing (RNA-Seq) analysis using ESCC samples from 3 patients and adjacent nontumor tissues to identify transcripts that were altered in ESCC tissue. We performed molecular and functional studies of the transcripts identified and investigated the mechanisms of alteration. We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, leading to activation of β-catenin. PTK6 was identified as a transcript that is down-regulated in human ESCC tissues via epigenetic modification at the PTK6 locus. Its product appears to regulate cell proliferation by reducing phosphorylation of Akt and GSK3β, leading to activation of β-catenin. Reduced levels of PTK6 promote growth of xenograft tumors in mice; it might be developed as a marker of ESCC.

Abstract 5042: A-AS1, a long non-protein coding RNA overexpressed in esophageal adenocarcinoma, influences cancer cell proliferation and apoptosis

Abstract Esophageal adenocarcinoma (EAC) is one of the fastest-growing cancers in the Western world. 95% of EACs occur without any antecedent diagnosis of Barrett's esophagus (BE). In these 95% of EAC patients, 5-year survival is only 15%. The rising incidence and poor prognosis of EAC have intensified research efforts into earlier methods to detect this disease. The majority of the human genome is transcribed and generates mostly non-coding (nc)RNAs that do not encode protein information. Long non-coding RNAs (lncRNAs) are emerging as a novel class of ncRNAs, but knowledge about these ncRNAs is limited. Recently, dysregulation of lncRNAs has been shown in various types of cancer, suggesting that lncRNAs may be involved in tumorigenesis or tumor progression. In the present study, we report that a number of lncRNAs are differentially expressed in EAC tissues and cell lines relative to normal esophageal tissues and cell lines. One of these lncRNAs, A-AS1 is derived from the antisense strand of the A gene locus and is overexpressed in EACs relative to matched normal esophagus (NE). Cell proliferation and colony formation assays revealed that A-AS1 siRNA-mediated knockdown reduces cell proliferation and colony-forming ability. In vitro scratch assays, migration and invasion assays in vitro also demonstrated decreased migration and invasion induced by A-AS1 siRNA knockdown. Moreover, we found that inhibition of A-AS1 expression results in increased apoptosis in EAC cell lines. Taken together, these results suggest that the elevated expression of A-AS1 observed in EACs may have contributed to their development or progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5042. doi:1538-7445.AM2012-5042

Malignant Progression in O6-Methylguanine-DNA Methyltransferase-Deficient Esophageal Cancer Cells Is Associated with Ezrin Protein

The abnormal function of O(6)-methylguanine-DNA methyltransferase (MGMT) is reported to be associated with the occurrence of various tumors and malignant tumor progression. However, little evidence is available to describe its role in esophageal carcinogenesis. To address this issue, we constructed a stable MGMT-silenced esophageal cancer cell line by RNA interference, and exposed the cells to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) to investigate the role that MGMT plays in toxicity. During this time, we also observed the malignant behavior of cells in vitro and in vivo. In addition, two-dimensional electrophoresis and mass spectrometry were used to detect and confirm the proteins that were differentially expressed in the MGMT-deficient and MGMT-proficient cells, which might be responsible for the malignant alteration of cells. Results showed that the IC(50) of MGMT-deficient and MGMT-proficient cells exposed to MNNG was 30 μM and 65 μM, respectively, and MGMT-deficient cells had more aggressive motility and invasive abilities compared with MGMT-proficient cells. Nineteen differentially expressed proteins were detected between the MGMT-deficient and MGMT-proficient cells, 14 of which were identified, including the membrane-cytoskeleton linker protein, Ezrin, which was confirmed by both mass spectrometry and western blot analysis. The correlation between MGMT, Ezrin expression, and the malignant behavior of one normal epithelial esophageal cell line and seven esophageal cancer lines is discussed. In conclusion, loss of MGMT expression leads EC109 esophageal cancer cells to have increased malignant behavior, which may correlate with its high Ezrin protein expression.

An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition

BackgroundThe evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barrett's esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents.MethodsOne fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT) was grown in competition with one of four Barrett's esophagus cell lines (CP-A, CP-B, CP-C, CP-D) under varying conditions and the outcome of competition measured over 14 days by flow cytometry.ResultsWe demonstrate that ascorbic acid (vitamin C) can help squamous cells outcompete Barrett's cells in this system. We are also able to show that ascorbic acid's boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus.ConclusionsThis model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.

Abstract 2105: RNA-seq identifies protein tyrosine kinase 6 (PTK6) as a candidate tumor-suppressor gene in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is one of the most common and deadliest cancers in the world, with survival rates of less than 15%. Like many other solid tumors, the pathogenesis of ESCC is also believed to be a multistep process with accumulation of numerous genetic alterations involving inactivation of tumor-suppressor genes and activation of oncogenes. An understanding of the molecular events that result in the development and progression of the disease may lead to treatments that will increase survival rates of ESCC patients. Recent advances in sequencing technologies offer the opportunity to characterize the cancer genome at unprecedented depth and sensitivity. Large-scale transcriptome sequencing (RNA-Seq) has proved an effective means of precisely quantifying the changing expression levels of each transcript. In this study, we performed integrative RNA-Seq analysis on 14 patient-derived ESCC clinical specimens (4 paired ESCC and their adjacent non-tumorous (NT) counterparts, 1 unpaired NT and 5 unpaired ESCC) and discovered a number of commonly differentially expressed genes in ESCC compared with NT tissues. A total of 526 genes were found to be significantly aberrantly expressed, including 452 genes that were up-regulated and 74 genes that were down-regulated. One candidate tumor suppressor gene, protein tyrosine kinase 6 (PTK6), was chosen for further characterization. By both quantitative real-time PCR and immunohistochemistry analysis, PTK6 was found to be significantly down-regulated in a larger cohort of ESCC tumors compared with NT counterparts. Consistently, expression studies in a panel of esophageal cell lines found PTK6 to be either absent or expressed in low amounts in all eight ESCC cell lines examined compared with two immortalized normal esophageal cell lines, NE1 and NE3. Down-regulation of PTK6 in both ESCC cell lines and clinical samples was found to be significantly associated with promoter hypermethylation, as evident by results obtained from studies involving demethylation treatment with 5-aza-dC, methylation specific PCR as well as bisulfite genomic sequencing. Functional studies in ESCC cell lines, EC109 and KYSE30, with PTK6 stably repressed by lentiviral-based approach stimulated both in vitro and in vivo tumorigenicity ability of the cells including foci formation, colony formation in soft agar as well as tumor formation in nude mice. Taken together, our findings define a function for PTK6 as an important tumor suppressor gene in ESCC development. Additional work on the mechanism by which PTK6 drives ESCC is currently being studied in our laboratory. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2011-2105

HOXB cluster genes are novel potential regulators of Barrett's oesophagus development

<h3>Introduction</h3> Barrett9s oesophagus (BO) is a preneoplastic condition to oesophageal adenocarcinoma (OAC) where the squamous epithelium is replaced by a columnar epithelium as a consequence of chronic gastro-oesophageal reflux disease. The molecular events driving this metaplastic change are still poorly understood. Analysis of an in-house microarray screening showed activation in BO of three HOXB cluster genes (HOXB5, HOXB6 and HOXB7). Thus, we set out to validate whether these genes are causally linked to the development of BO <h3>Methods</h3> To externally validate microarray data, RNA was extracted from endoscopic biopsies of colon (n = 14), duodenum (n = 13), gastric body (n = 11), normal oesophagus (n = 14), BO without dysplasia (n = 13), BO with low grade dysplasia (n = 9) and high grade dysplasia (n = 6), as well as surgical specimens of OAC (n = 12). Expression of the mid cluster HOXB genes (HOXB5, HOXB6 and HOXB7), as well as neighbouring genes (HOXB4 and HOXB8), were assessed by qPCR. A normal oesophageal cell line (NES) was transiently transfected with vectors coding for HOXB5, HOXB6 and HOXB7 and the intestinal markers villin, cytokeratin 20 (KRT20), sucrase isomaltase (SI) and MUC2 were assessed by qPCR. Chromatin was isolated from surgical specimens of normal oesophagus (n = 5) and OAC (n = 5) and immunoprecipitated with antibodies against methylated histone H3 at Lysine 27 (H3K27me3) and Lysine 4 (H3K4me3) and acetylated histone H3 (AcH3). <h3>Results</h3> HOXB5, HOXB6 and HOXB7 genes were consistently up-regulated in all pathological stages of Barrett9s disease, including OAC, as compared to normal oesophagus (p &lt; 0.0001 for all three genes). HOXB4 and HOXB8 did not show any transcriptional activation in BO and OAC. When compared with other GI segments, expression levels of these five HOXB genes in BO resembled that of the colon rather than other columnar epithelia (stomach and duodenum). Over-expression of mid cluster HOXB genes in NES cells was associated with activation of villin, KRT20 and MUC2 but not SI. In particular HOXB5 induced villin and MUC2 (2- and 4-fold, respectively); HOXB6 activated villin, KRT20 and MUC2 (4-, 3- and 4-fold, respectively) and HOXB7 induced KRT20 and MUC2 (14-fold for both). Chromatin immunoprecipitation showed significant enrichment of the active mark AcH3 and significant loss of the repressive mark H3K27me3 in OAC compared to normal oesophagus for HOXB5, HOXB6 and HOXB7, but not HOXB4 and HOXB8. No change was detected for the active mark H3K4me3. <h3>Conclusion</h3> Mid cluster HOXB genes are novel potential mediators of the preneoplastic intestinal phenotype in the oesophagus and they are regulated through an epigenetic mechanism acting on the histone code. The HOXB gene signature of oesophageal intestinal metaplasia is similar to that of large bowel, rather than small bowel and gastric epithelia.

A comparison of primary oesophageal squamous epithelial cells with HET‐1A in organotypic culture

Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5-year survival of less than 15%. Recent evidence suggests that stromal-epithelial interactions are fundamental in carcinogenesis. The advent of co-culture techniques permits the investigation of cross-talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET-1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET-1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET-1A cells were highly proliferative and did not express the epithelial markers E-cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N-cadherin. Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET-1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.

Physiological and molecular analysis of acid loading mechanisms in squamous and columnar-lined esophagus

Barrett's esophagus (BE) may be an adaptive cellular response to repeated acid exposure. The aims of this study were to compare intracellular acid loading in BE cells with normal squamous esophageal cells. Primary squamous and BE cells were obtained endoscopically and cultured for up to 24 h. Barrett's adenocarcinoma cell lines TE7 and OE-33 were compared with a normal esophageal (NE) cell line OE-21. Extracellular pH was lowered to 6.0 using HCl; specific ion exchangers were blocked pharmacologically and pH microfluorimetry was performed using 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The effect of prolonged acid preincubations and repeated acid exposure on acid loading and recovery were examined. Acid loading was greater in primary BE than NE cells (DeltapHi -0.22 +/- 0.08 vs.-0.13 +/- 0.01) and maximal in the BE carcinoma cell line TE7 (DeltapHi -0.30 +/- 0.01). Whereas TE7 cells were able to recover fully from repeated acid exposure, OE-21 cells remained profoundly acidic. BE primary and transformed cells utilize DIDS inhibitable sodium-independent chloride/bicarbonate exchange as well as sodium/hydrogen ion exchange for acid loading. In contrast, SE only requires sodium-independent chloride/bicarbonate exchange for acidification. The degree of acid loading is greater in BE than NE cells and it occurs via dual ion exchangers similar to gastric mucosa. Only Barrett's epithelial cells can maintain a physiological pHi following prolonged and repeated reflux exposure, which may confer a teleological advantage.

The overexpression of caveolin-1 and caveolin-2 correlates with a poor prognosis and tumor progression in esophageal squamous cell carcinoma

Caveolin-1 (CAV1) and caveolin-2 (CAV2) are the major structural proteins of caveolae. We investigated the relationship between the clinicopathological factors of esophageal squamous cell carcinoma (ESCC) and the expression of CAV1 and CAV2. CAV1 and CAV2 expression were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 15 esophageal cancer cell lines (TE1-15) and a normal esophageal epithelium cell line (Het-1A). CAV1 and CAV2 expression was examined by RT-PCR and immunohistochemical analysis in 47 ESCC specimens. High levels of CAV1 and CAV2 mRNA were detected in TE1-15, but neither CAV1 nor CAV2 mRNA were detected in Het-1A. In the ESCC samples CAV1 and CAV2 mRNA expression in the ESCC samples were significantly higher than in the corresponding normal esophageal mucosa (CAV1, P=0.0024; CAV2, P=0.0136). However, we could not find any significant relationship between CAV1 or CAV2 mRNA expression and clinicopathological factors. Immunostaining for CAV1 was positive in 13 of 47 patients (27.7%), whereas CAV2 was positive in 22 of 47 patients (46.8%). A significant correlation was observed between CAV1 and CAV2 immunostaining and T factor, lymphatic invasion, vein invasion and differentiation. The patients with positive staining for CAV1 or CAV2 had a significantly shorter survival than those with negative staining (P=0.0105 and 0.0424 for CAV1 and CAV2, respectively). These results suggest that positive staining for CAV1 and CAV2 could be a potentially useful prognostic marker of ESCC.

The tumor suppressor Wnt inhibitory factor 1 is frequently methylated in nasopharyngeal and esophageal carcinomas

Aberrant activation of the wingless-type- (Wnt)-signaling pathway is common in many cancers including nasopharyngeal (NPC) and esophageal squamous cell (ESCC) carcinomas, both prevalent in Southern China and Southeast Asia. However, the molecular mechanism leading to this abnormality is still obscure. Wnt inhibitory factor-1 (WIF1) is a secreted antagonist of the Wnt pathway, and is recently shown to be inactivated by epigenetic mechanism in some tumors. Here, we examined whether WIF1 is also inactivated epigenetically in NPC and ESCC. With semiquantitative reverse transcription-PCR and methylation-specific PCR, we detected WIF1 downregulation or silencing in 6/6 of NPC and 12/19 of ESCC cell lines, which is well correlated with its methylation status. Methylation was further confirmed by high-resolution bisulfite genomic sequencing. Methylation was also frequently observed in a large collection of primary tumors of NPC (85%, 55/65) and ESCC (27%, 25/92), with WIF1 expressed and unmethylated in normal NPC and esophageal cell lines and normal tissues. Treatment of 5-aza-2′-deoxycytidine demethylated WIF1 and induced its expression in NPC and ESCC cell lines, highlighting a direct role of epigenetic inactivation. Ectopic expression of WIF1 in NPC and ESCC tumor cells resulted in significant inhibition of tumor cell colony formation, similar to TP53, and also significant downregulation of β-catenin protein level in NPC cells. Thus, WIF1 functions as a tumor suppressor for both NPC and ESCC through suppressing the Wnt-signaling pathway, but is frequently silenced by epigenetic mechanism in a tumor-specific way. Our study indicates that epigenetic inactivation of WIF1 contributes to the aberrant activation of Wnt pathway and is involved in the pathogenesis of both tumors. WIF1 methylation could also serve as a specific biomarker for these tumors.

Pathogenesis of Barrett Esophagus

Bile acid exposure can induce caudal-related homeobox 2 (CDX2) messenger RNA (mRNA) expression, a transcription factor that plays a crucial role in the development of Barrett esophagus. We investigated mucin 2 (MUC2) and CDX2 mRNA expression before and after treatment with deoxycholic acid in 4 human esophageal cell lines. Four human esophageal cell lines-(1) normal squamous cells immortalized by SV40 (Het-1A), (2) adenocarcinoma (SEG-1), and (3 and 4) squamous cell carcinoma (HKESC-1 and HKESC-2)-were exposed in culture for 1 to 24 hours to 100 microM to 1000 microM deoxycholic acid. Total RNA was extracted before and after bile acid treatment and reverse transcribed to complementary DNA. MUC2 and CDX2 mRNA expression as determined by semiquantitative reverse transcription-polymerase chain reaction. MUC2 mRNA expression was absent before deoxycholic acid exposure in all 4 cell lines. MUC2 expression increased in a dose- and time-dependent manner with deoxycholic acid in all cell lines. Deoxycholic acid activated MUC2 up-regulation, which correlated directly with CDX2 up-regulation in all 4 cell lines. Bile acids up-regulate both intestinal differentiation factor CDX2 and goblet cell-specific gene MUC2 in normal esophageal and cancer cell lines. Further, bile acid-stimulated MUC2 up-regulation correlates directly with CDX2 up-regulation. The simultaneous up-regulation of both CDX2 and MUC2 after bile acid exposure provides molecular evidence of the role of bile acid in the pathogenesis of Barrett esophagus.

Induction of prostaglandin E synthase by gastroesophageal reflux contents in normal esophageal epithelial cells and esophageal cancer cells

The synthesis of prostaglandin E2 (PGE2) requires cyclooxygenase (COX) and prostaglandin E synthase (PGES). There are two forms of PGES: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1. In this study, we investigated the effects of gastroesophageal reflux (GER) contents on PGES and COX-2 in esophageal cells. We incubated a human normal esophageal cell line, two esophageal squamous cell carcinoma (SCC) cell lines, and two esophageal adenocarcinoma (ADC) cell lines with GER contents. The production of PGE2 by these cells was assayed with an enzyme immunoassay kit. The protein expression of COX-2, cPGES, and mPGES-1 was confirmed by immunoblot analysis. The following results were obtained: GER contents induced the expression of COX-2 in all five cell lines. In normal esophageal cells, cPGES, but not mPGES-1, was detected in the cytosolic fraction. GER contents induced the expression of cPGES in the microsomal fraction. In SCC cells, cPGES was expressed in the cytosolic fraction, and mPGES-1 was expressed in the microsomal fraction. GER contents induced the expression of mPGES-1 in the microsomal fraction. In ADC cells, cPGES was expressed in both the cytosolic and microsomal fractions. GER contents induced the expression of both cPGES and mPGES-1 in the microsomal fraction. In conclusion, our results suggest that GER contents induce PGE2 production in esophageal cells. However, there are different isoforms of PGES in normal cells, SCC cells, and ADC cells.

Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis

Background: The antiapoptotic and epithelial–mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of...