Human Esophageal Adenocarcinoma Cell Lines Research Articles

A possible molecular mechanism of two flavones and two flavonols on the induction of differentiation in a human oesophageal adenocarcinoma cell line (OE33)

The influences of two flavones (baicalein, chrysin) and two flavonols (galangin, kaempferol) selected on induction of differentiation in a human esophageal adenocarcinoma (OE33) cell line were studied in the present work. All compounds selected could be able to inhibit proliferation of OE33 cells in a dose- and time-dependent manner. The inhibitory potency of these compounds on OE33 cells was in the order of: galangin > chrysin > baicalein >kaempferol. OE33 cells treated with these compounds for 24 h showed differentiation characteristics including the differentiation-specific morphological changes, the augmentation of differentiation markers and the inhibition of human telomerase reverse transcriptase. The potency of these compounds on induction of differentiation of OE33 cells was similar to that of them on growth inhibition. To reveal the possible molecular mechanisms by which these compounds modulate proliferation and differentiation in OE33 cells, the alterations of three genes, 14-3-3s, cyclin B1 and cyclin D1, which are related to proliferation and differentiation of cells, were investigated by real-time polymerase chain reaction (RT-PCR) and Western blot. The results showed that up-regulation of 14-3-3s and down-regulation of cyclin B1 and cyclin D1 at the mRNA and protein levels were observed in OE33 cells treated with these compounds, indicating that 14-3-3s, cyclin B1 and cyclin D1 might be target genes of these flavonoids in inducing differentiation of OE33 cells. Key words: Flavones, flavonols, differentiation, esophageal cancer, molecular mechanism.

Cytogenetic studies of human esophageal squamous cell carcinoma and gastric adenocarcinoma cell lines

Esophagus and gastric cancer are one of the most common in Asian countries, but molecular event in the carcinogenesis remains as largely unidentified associated oncogenes. Detailed karyotypic analyses were performed on 4 cell lines derived from malignant cells of a patient with esophagus carcinoma of both diagnosis and metastasis stages and 2 cell lines from 2 independent patients with gastric cancer of metastasis stages. Most of these cells examined were near triploid with modal chromosome number of 59-73. These cells had a complex karyotype with more than 10 rearrangements. The karyotypic pattern was relatively stable; consistent alterations involved long arm of chromosomes 2, 9 and 12 and short arm of chromosomes 3 in both diagnosis and metastasis stages in esophagus cell lines. Those alterations of gastric cancer involved chromosomes 7 and 11. Higher rearrangements associated with translocations and deletions were observed at 2q23 of chromosome2, 3p14 of chromosome 3, 4q11 of chromosome 4 and 11q25 of chromosome 11 in esophagus cancer; while 7p13 of chromosome 7 and 11p15 of chromosome 11 in gastric cancer. The overall chromosome pattern was not similar between these cancers, although these tumors arise from anatomically or biologically related site. Unknown oncogenes localizing on these chromosome breakpoints for translocation or deletion region might be associated with the pathogenesis of esophagus or gastric cancer. These newly established cell lines will be useful tools in the study of the molecular pathogenesis and for testing new therapeutic reagents for these cancers in the future.

Abstract 1098: Interactions between P53 and IGF2 in human esophageal adenocarcinoma tissues and cell lines

Abstract To define the role of the insulin-like growth factor (IGF) axis in esophageal adenocarcinoma (EADC), the objective of this study was to evaluate interactions between IGF2 and P53 in a well characterized series of surgically-resected esophageal tissues and the following cell lines: Het1A (derived from immortalized normal esophageal epithelium), OE33 and JH-EsoAd1 (each derived from a primary EADC). Nucleic acids were extracted from 68 primary EADC (and matched histologically normal) tissues. PCR was performed on gDNA and RT-PCR on RNA followed by ApaI digestion to identify informative (heterozygous) cases and imprinting status, respectively. Expression of IGF2 mRNA was determined by quantitative PCR, and of IGF2 and P53 protein by Western analysis and immunohistochemistry. Molecular findings in tissues were correlated with pathologic and clinical characteristics including patient survival. Functional studies utilizing cell lines evaluated IGF2/P53 interactions. Of 44 informative cases, P53 protein overexpression was significantly higher in normally imprinted tumors (12/30) vs. tumors with loss of IGF2 imprinting (1/14; p<0.05). Multivariable logistic regression confirmed IGF2 imprinting status to be independently associated with P53 expression (OR 0.12, 95% CI 0.01-1.00; p=0.05). Tumors overexpressing both IGF2 and P53 were found to be of advanced stage with poor survival. In patients below age 65 years, IGF2 mRNA expression was significantly higher in tumors with wild type P53, and multivariate analysis showed IGF2 mRNA expression to be independently associated with p53 mutation (OR 0.74, 95% CI 0.58-0.94; p<0.05) and P53 protein expression (OR 0.83, 95% CI 0.69-1.00; p<0.05). Whereas treatment of Het1A cells with IGF2 significantly increased P53 expression (1.34+/-0.06 vs. 1.00+/-0.00 untreated cells; p=0.003), IGF2 did not modulate P53 expression in either of the two tumor cell lines (OE33 and JH-EsoAd1). IGF1 Receptor (IGF1R) inhibition with AG1024 reduced P53 expression only in OE33 (0.48+/-0.22 vs. 1.00+/-0.00 untreated cells; p=0.05) and JH-EsoAd1 (0.71 +/- 0.23 vs. 1.00+/- 0.00 untreated; p = 0.15) cells, an effect not reversed by IGF2 treatment. In conclusion: 1) these studies identify novel molecular regulatory interactions between P53 and IGF2 in esophageal malignancy, which are dependent on IGF2 imprinting status and tissue/cell type. 2) Overexpression of both IGF2 and P53 in esophageal tumors is associated with aggressive disease in younger patients (below age 65 years), and may represent clinically useful biomarkers to define a biologically distinct subset of EADC. 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 1098. doi:10.1158/1538-7445.AM2011-1098

Effect of RNA interference targeting HIF-1α gene on biological behavior of human esophageal squamous cell carcinoma and gastric adenocarcinoma cells in vitro

To investigate the effect of hypoxia inducible factor-1α (HIF-1α) on the proliferation, migration and vasculogenic mimicry(VM) in human esophageal squamous cell carcinoma cell line Eca-109 and gastric adenocarcinoma cell line SGC-7901 in vitro. The recombinant plasmid pGCsi-shHIF-1α was transfected into Eca-109 and SGC-7901 cells by Lipofectamine(TM) 2000. The inhibitory effect of HIF-1α was measured at protein level by Western blot under normoxia and hypoxia. The cell proliferation was detected by colony formation and MTT assays. The migration of transfected cells was assayed using Transwell chambers. Whether Eca-109 and SGC-7901 cells could form the capillary tube-like structures (TLSs) was observed by 3-dimensional culture, and the tube formation of transfected cells was detected by tube-like structure formation assay. The expression of HIF-1α protein in each group of transfected cells was significantly suppressed under normoxia and hypoxia (Eca-109: 0.00, 0.74 ± 0.05; 0.00, 1.11 ± 0.06; SGC-7901: 0.00, 0.60 ± 0.05; 0.00, 0.96 ± 0.07, P < 0.01). Colony formation and MTT assays showed that the cell proliferation of the pGCsi-shHIF-1α transfected cells was slower than that of the control groups (104.7 ± 9.6, 151.7 ± 4.5; 88.3 ± 5.1, 128.3 ± 6.7, P < 0.05). The migration of the recombinant plasmid-transfected cells was significantly suppressed compared with that of cells transfected with empty vector (55.5 ± 11.2, 121.9 ± 17.3; 64.7 ± 10.8, 132.3 ± 16.0, P < 0.01). Both the Eca-109 and SGC-7901 cells could form TLSs when cultured on matrigel, and the number of tubules was significantly increased under hypoxia (30.8 ± 3.9, 34.3 ± 3.4; 26.2 ± 3.4, 30.1 ± 4.1, P < 0.05), the tubule-forming ability of transfected groups was significantly inhibited under normoxia and hypoxia (Eca-109: 3.7 ± 2.8, 30.8 ± 3.9; 3.9 ± 2.7, 34.3 ± 3.4; SGC-7901: 4.9 ± 3.5, 26.2 ± 3.4; 5.3 ± 3.6, 30.1 ± 4.1, P < 0.01). Both the esophageal squamous cell carcinoma cell line Eca-109 and gastric adenocarcinoma cell line SGC-7901 are capable of forming vasculogenic mimicry structures in vitro. The recombinant plasmid pGCsi-shHIF-1α can efficiently suppress their proliferation, migration and vasculogenic mimicry formation.

Abstract B67: In vitro and in vivo inhibitory effects of cranberry proanthocyanidins against esophageal adenocarcinoma

Abstract This study investigated the cancer inhibitory potential of a proanthocyanidin rich cranberry extract utilizing a panel of authenticated human esophageal adenocarcinoma cell lines (EAC), a nude mouse xenograft model, and the rat esophagogastrodoudenal anastomosis (EGDA) model of EAC. PACs effects on cell morphology, global gene expression, phase of cell cycle, and cell death induction via apoptosis, autophagy, and necrosis were evaluated. Methods included RapidDiff and MDC staining, Annexin/PI and BrdU staining with flow cytometric evaluation, transmission electron microscopy (TEM), protein evaluation utilizing standard Western blot techniques, and validation of global gene expression changes by real-time PCR. PAC treatment [50-100µg/ml] of EAC cancer cell lines resulted in significant increases in apoptosis, autophagy and necrosis. Differential cell death induction was noted based upon the cell lines resistance to a bile/acid cocktail mimicking acid exposure in humans as well as the status of the apoptotic and autophagic machinery of the select cell line. PACs cell death inducing capacity correlated with altered MAPKinase signaling as evidenced by activation of P-p38 and P-JNK and inhibition of P-ERK1/2. PAC treatment induced other proapoptotic markers including cleaved PARP, BAK1, BAX, and cytochrome C, but in a cell line dependent manner. Mild increases in Beclin-1 were noted following PAC treatment, as were significant increases in LC3II, a marker of autophagasome formation which occurs during autophagy induction. Results were confirmed by TEM and MDC staining. PAC induced cell cycle arrest at the G2/M transition, increased the percent of cells in G1, caused an S-phase delay and increased P21 levels in all three cell lines. PAC administration significantly inhibited the growth of OE19 xenografts via modulation of cell cycle and MAPK signaling pathways. Oral gavage of PAC [250µg/mouse] six times a week to nude mice resulted in a 67% decrease in OE19 tumor volume, increased cytochrome C protein levels and decreased levels of P-ERK1/2, cyclin A, PCNA, and CD31 in PAC treated tumors compared to vehicle treated tumors. Lastly, a four week pilot study was performed to evaluate the safety of daily administration of PAC in the rodent EGDA model. PAC appeared well tolerated given the average daily dose of 450µg/rat/day as evidenced by normal body weight and food consumption, serology profiles and histopathology of the major organs. Further long-term studies are planned to more fully access the chemopreventive potential of PAC against esophageal adenocarcinoma and premalignancy utilizing the clinically relevant rat EGDA surgical model. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B67.

Associations between leptin and adiponectin receptor upregulation, visceral obesity and tumour stage in oesophageal and junctional adenocarcinoma

Obesity is associated with oesophageal adenocarcinoma, but mechanisms linking fat and carcinogenesis remain poorly understood. Altered circulating adipocytokines may be important. This study aimed to identify pathways through which visceral fat impacts on tumour biology. Seventy-five patients with oesophageal adenocarcinoma underwent anthropometric and radiological assessment of obesity. Expression of leptin receptor (ObR) and adiponectin receptors 1 and 2 (AdipR1, AdipR2) was quantified by real-time reverse transcriptase-polymerase chain reaction. The human oesophageal adenocarcinoma cell line OE33 was used as the calibrator sample. Ninety-one per cent of tumours expressed ObR, 95 per cent expressed AdipR1 and 100 per cent expressed AdipR2. Relative expression of ObR was upregulated in 67 per cent, and AdipR1 and AdipR2 were downregulated in 55 and 68 per cent respectively, relative to the calibrator sample. Upregulated ObR and AdipR2 expression was significantly associated with anthropometric and radiological measures of obesity. Upregulated ObR was associated with advanced tumour and node category (P = 0.036 and P = 0.025 respectively), and upregulated AdipR2 with nodal involvement (P = 0.037). Obesity is associated with upregulated ObR and AdipR2 expression in oesophageal adenocarcinoma. The association of ObR and AdipR2 with tumour stage suggest that pathways involving adipocytokines affect tumour biology.

S1634 Bone Morphogenetic Protein-4 Has an Important Role in the Transformation of Normal Esophageal Cells to an Intestinal Phenotype in Barrett's Esophagus via CDx2 Pathway

Background & Aims: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. A recent study has shown that bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor-β superfamily, is uniquely overexpressed in BE. The mechanisms of the BMP signaling pathway in the metaplastic transformation in BE, however, remain unclear. The aim of this study was to examine the role of the BMP signaling pathway in the metaplastic transformation of normal squamous cells into columnar cells. Methods: The expression of BMP signaling related molecules, including BMP-4, ID2, SMAD4 in biopsy samples was assessed. Samples were taken fromBE (16 cases), with or without reflux esophagitis, and fromnormal esophagus (12 cases) and assayed by immunohistochemistry and real-time PCR. In addition, the In Vitro effects of acid and/or bile acid on BMP-4 expression were analyzed in HET1A, an immortalized human esophageal squamous cell line, and OE33, a human esophageal adenocarcinoma cell line. These cells were treated with recombinant human BMP-4 (10-100 ng/ mL), and cell biological changes were examined by immunoblot analysis and real-time PCR. Cell proliferation was assessed by MTS assay. Results: Immunohistochemistry of biopsy specimens demonstrated that the expression of BMP-4 protein was observed in all the cases of BE, but not in normal squamous epithelium. Consistent with the immunohistochemistry results, BMP-4 mRNA expression was 13 fold greater in BE as compared with normal squamous epithelium. Up-regulation of BMP-4 expression was more evident in BE with reflux esophagitis. In Vitro, acid (pH4) and bile acid exposure synergistically induced BMP4 expression in both HET1A and OE33 cells. Treatment of these cells with recombinant BMP-4 resulted in increased expression of the intestine-specific transcription factor, CDX2. Blocking BMP signalling by preincubating cells with the BMP antagonist Noggin before treatment with BMP-4, resulted in decreased expression of CDX2. In addition, Noggin also blocked the proliferation of OE33 cells. Conclusion: These results suggest that the BMP signaling pathway is activated in BE, and that BMP-4 has an important role in the process of transformation of squamous epithelium toward a mucosa that resembles columnar metaplasia. The results suggest that transformation occurs via a CDX2-dependent pathway, and that this process might be promoted in the inflamed condition. We propose that the premalignant metaplastic development of BE is mediated by BMP-4, and that inhibition of BMP-4 signaling offers a novel therapeutic approach for this disease.

Abstract 3363: Identification and characterization of stem-like cells in human esophageal epithelial and adenocarcinoma cells

Abstract The aim of this study was to identify and characterize stem-like cells in human normal esophageal epithelial and adenocarcinoma cell lines. The immortalized esophageal epithelial cell line, Het-1A, and the primary esophageal adenocarcinoma (EADC) cell line, OE33, were first studied by flow cytometry and immunofluorescence using a panel of antibodies to putative stem cell biomarkers which included CD34, CD44, CD44/CD24, CD117/integrin α6, Musashi I, EpiCam, and Oct 4. Fluorescence activated cell sorting (FACS) was then used to isolate 15% of cells with the highest CD44 staining (CD44high) or reactive oxygen species (ROS) (ROShigh) and 15% of cells with the lowest (including negative) CD44 staining (CD44low) or ROS (ROSlow). Each cell population was further characterized using sphere-forming assays, soft agar clonogenic assays, flow cytometry for ROS, and synchrotron mid-infrared (IR) spectromicroscopy. CD44 positivity was found in 90% of Het-1A and 75% of OE33 cells overall. For both Het-1A and OE33 cell lines, spheroid formation (mean±SD/5000 cells) was significantly higher in cells expressing high levels of CD44 (Het-1A:138.33±2.08 vs. 97.67±1.53, p&amp;lt;0.01; OE33: 150.00±31.19 vs. 100.67±7.02, p=0.056) and low levels of ROS (Het-1A: 149.33±20.43 vs. 92.00±8.89, p=0.011; OE33: 199.67±31.63 vs. 119.33±14.47, p=0.016). Colony formation (mean±SD/1000 cells) was also found to be significantly higher in CD44high (Het-1A: 15.33±4.51 vs. 8.00±1.00, p=0.051; OE33: 92.67±11.02 vs. 18.00±1.73, p&amp;lt;0.01) and ROSlow (Het-1A: 45.00±1.00 vs. 31.67±3.79, p&amp;lt;0.01; OE33: 33.00±5.29 vs. 14.67±5.03, p=0.012) cell populations. Whereas OE33 CD44high cells were found to express consistently lower levels of ROS compared with OE33 CD44low cells (p&amp;lt;0.01), no differences in ROS expression were found in Het-1A cells sorted by CD44 expression. Spectromicroscopic analysis revealed only small non-significant differences between cell populations sorted by CD44 and ROS expression. We conclude that the higher rates of spheroid formation and clonogenicity of Het-1A and OE33 cells expressing high levels of CD44 (CD44high) and low levels of ROS (ROSlow) reflect an enrichment of stem-like cell populations in esophageal cell lines. However, the significant differences in ROS levels between CD44high OE33 cells and CD44high Het-1A cells suggests unique and biologically distinct pathways in stem-like cells derived from normal and malignant esophageal epithelia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3363.

Complementary use of fluorescence and magnetic resonance imaging of metastatic esophageal cancer in a novel orthotopic mouse model

We describe the development of an aggressive orthotopic metastatic model of esophageal cancer, which is visualized in real time with combined magnetic resonance imaging (MRI) and fluorescence imaging. The aim of the study was to describe the development of a novel model of metastatic tumor disease of esophageal carcinoma and use this model to evaluate fluorescence and MRI in early detection of local and metastatic disease. The human esophageal adenocarcinoma cell line PT1590 was stably transfected with green fluorescent protein (GFP). Nude mice were orthotopically implanted with PT1590-GFP cells. Orthotopic tumor growth as well as metastatic spread was examined by fluorescence imaging and high-resolution MRI at defined intervals after orthotopic implantation. Highly aggressive novel fluorescent cell lines were isolated from metastatic tissues and put into culture. After implantation of these cells, 100% of the animals developed orthotopic primary tumors. In 83% of animals, metastatic spread to liver, lung and lymph nodes was observed. Primary tumor growth could be visualized with fluorescence imaging and with MRI with high correlation between the 2 methods. Fluorescence imaging allows fast, sensitive, and economical imaging of the primary and metastatic tumor without anesthesia. With MRI, anatomical structures are visualized more precisely and tumors can be more accurately localized to specific organs. This model should prove highly useful to understand esophageal carcinoma and to identify novel therapeutics for this treatment-resistant disease.

Verification and Unmasking of Widely Used Human Esophageal Adenocarcinoma Cell Lines

For decades, hundreds of different human tumor type-specific cell lines have been used in experimental cancer research as models for their respective tumors. The veracity of experimental results for a specific tumor type relies on the correct derivation of the cell line. In a worldwide effort, we verified the authenticity of all available esophageal adenocarcinoma (EAC) cell lines. We proved that the frequently used cell lines SEG-1 and BIC-1 and the SK-GT-5 cell line are in fact cell lines from other tumor types. Experimental results based on these contaminated cell lines have led to ongoing clinical trials recruiting EAC patients, to more than 100 scientific publications, and to at least three National Institutes of Health cancer research grants and 11 US patents, which emphasizes the importance of our findings. Widespread use of contaminated cell lines threatens the development of treatment strategies for EAC.

Fourier transform infrared (FTIR) spectromicroscopic characterization of stem-like cell populations in human esophageal normal and adenocarcinoma cell lines

We have tested an approach to identify putative cancer stem cells that involves measurement of the infrared absorption spectrum of individual cells in an aqueous environment, and their subsequent classification using multivariate data analysis techniques. Two primary esophageal cell lines were characterized: the immortalized normal esophageal epithelial cell line, Het-1A, and the esophageal adenocarcinoma cell line, OE33. In addition, we also evaluated spheroids, reflecting stem-like cell populations, which were derived from each parent cell line when grown in serum-free media. As differences in cell size appeared to be a strong discriminating factor, a correction needs to be performed to allow a reliable classification based on infrared absorption spectra. We demonstrated that stem-like cells derived from Het-1A could easily be discriminated on the basis of absorbance differences in the 1000-1200 cm(-1) spectral interval, whereas this was not possible for OE33. Furthermore, we found that changes due to aging of OE33 cells in culture dominated the infrared absorption spectra and somewhat limited the potential of this approach to identify stem-like cell populations using this in vitro model system.

Flavones and flavonols exert cytotoxic effects on a human oesophageal adenocarcinoma cell line (OE33) by causing G2/M arrest and inducing apoptosis

Dietary flavonoids have been shown to exert specific cytotoxicity towards some cancer cells, but the precise molecular mechanisms are still not completely understood. In our study, cytotoxic effects of structurally related flavones and flavonols on a human oesophageal adenocarcinoma cell line (OE33) were compared, and the molecular mechanisms responsible for their cytotoxic effects were explored. The results of MTT assay showed that flavones (luteolin, apigenin, chrysin) and flavonols (quercetin, kaempferol, myricetin) were all able to induce cytotoxicity in OE33 cells in a dose- and time-dependent manner, and the cytotoxic potency of these compounds was in the order of quercetin>luteolin>chrysin>kaempferol>apigenin>myricetin. Flow cytometry and DNA fragmentation analysis indicated that the cytotoxicity induced by flavones and flavonols was mediated by G2/M cell cycle arrest and apoptosis. Furthermore, the expression of genes related to cell cycle arrest and apoptosis was assessed by oligonucleotide microarray, real-time RT-PCR and Western blot. It was found that the treatment of OE33 cells with flavones and flavonols caused G2/M arrest through up-regulation of GADD45β and 14-3-3σ and down-regulation of cyclin B1 at the mRNA and protein levels, and induced p53-independent mitochondrial-mediated apoptosis through up-regulation of PIG3 and cleavage of caspase-9 and caspase-3. The results of western blot analysis further showed that increases of p63 and p73 protein translation or stability might be contribute to the regulation of GADD45β, 14-3-3σ, cyclin B1 and PIG3.

Activation of MUC1 mucin expression by bile acids in human esophageal adenocarcinomatous cells and tissues is mediated by the phosphatidylinositol 3-kinase

In esophageal adenocarcinoma, MUC1 mucin expression increases in early stages of the carcinogenetic sequence, during which bile reflux has been identified as a major carcinogen. However, no link between MUC1 overexpression and the presence of bile acids in the reflux has been established so far, and molecular mechanisms regulating MUC1 expression during esophageal carcinogenetic sequence are unknown. Our aim was to identify (1) the bile acids able to upregulate MUC1 expression in esophageal cancer cells and mucosal samples, (2) the regulatory regions in MUC1 promoter responsive to bile acids, and (3) the signaling pathway(s) involved in this regulation. MUC1 mRNA and mucin expression were studied by the means of real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, both in the human esophageal OE33 adenocarcinoma cell line and in an ex vivo explant model. MUC1 promoter was cloned and transcription regulation was studied by transient cell transfection to identify the bile acid-responsive regions. Signaling pathways involved were identified using specific pharmacologic inhibitors and siRNA approach. Taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic, sodium glycocholate, and deoxycholic bile acids upregulated MUC1 mRNA and protein expression. The highest induction was obtained with deoxycholic and taurocholic acids in both cellular and explant models. The bile acid-mediated upregulation of MUC1 transcription occurs at the promoter level, with responsive elements located in the -1472/-234 region of the promoter, and involves the phosphatidylinositol 3-kinase signaling pathway. Bile acids induce MUC1 mucin overexpression in human esophageal adenocarcinoma cells and tissues by activating its transcription through a process involving phosphatidylinositol 3-kinase.

Androgen receptors may act in a paracrine manner to regulate oesophageal adenocarcinoma growth

Background The role of androgen receptors (ARs) in tumorigenesis, including transcription of fibroblast growth factors (FGFs), is established in prostate cancer. This study examined the role of ARs and FGFs in oesophageal adenocarcinoma (EAC), where tumour incidence in males is higher. Methods AR gene expression was analysed using quantitative RT-PCR; AR, fibroblast growth factor receptor-1 (FGFR-1) and fibroblast growth factor-8 isoform b (FGF-8b) protein by immunohistochemistry; and serum steroid levels (testosterone, progesterone, luteinising hormone and follicle stimulating hormone (FSH)) by immunoassay. A human oesophageal adenocarcinoma cell line was grown subcutaneously in nude mice. Results AR gene expression was of significantly higher levels than oesophageal adenocarcinomas ( n = 21, p = 0.002) and in the squamous carcinoma line (OE21) compared with the adenocarcinoma lines (OE33 and OE19). Median serum testosterone levels in oesophageal carcinoma patients were higher than in age-matched controls ( p = 0.01) and reduced postoperatively, in patients undergoing curative resection ( p = 0.006). No significant differences were observed in hormones except FSH, where preoperative levels were significantly higher in the EAC group. AR protein was expressed in normal oesophageal squamous epithelial cells and also in the stroma of 18/23 EAC samples. FGFR-1 protein was expressed in malignant epithelium of 23/23 tumour samples. OE19 xenografts grew faster in male versus female mice (tumour weight at day 21, 1.14 g and 0.28 g, respectively, p = 0.005) and had elevated FGF receptor expression. Conclusions AR expressed in the stroma of oesophageal adenocarcinomas may induce paracrine effects following stimulation by androgens (including tumour-derived), possibly via FGFs, including FGF-8b.

Bile acids induce overexpression of homeobox gene CDX-2 and vascular endothelial growth factor (VEGF) in human Barrett's esophageal mucosa and adenocarcinoma cell line

Objective. Barrett's esophagus (BE) is an acquired precancerous condition that develops from mucosal injury incurred after chronic gastroesophageal acid and bile reflux. The mechanism of progression of carcinogenesis in BE is still not fully understood. Recently, the role of bile acids and the homeobox gene transcription factor CDX-2 has been suggested in the pathogenesis of BE. The aims of the present study were 1) to compare the mRNA and protein expression of CDX-2 in biopsies obtained from patients with BE and normal squamous epithelium and 2) to study the effect of two different bile salts, ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA), on the mRNA expression of CDX-2 and vascular endothelial growth factor (VEGF) in Barrett's the adenocarcinoma cell line (OE-33). Material and methods. CDX-2 expression was measured in Barrett's mucosa and normal esophageal mucosa obtained from 15 patients with BE histologically diagnosed by immunohistochemistry, Western blot, and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In in vitro experiments, OE-33 cells were incubated with DCA (100 µM) and UDCA (100 µM) in neutral and shortly acidified media (pulse acidification). The expression of CDX-2 and VEGF was assessed by quantitative RT-PCR. Results. Both mRNA and protein expression of CDX-2 were significantly up-regulated in Barrett's mucosa as compared to normal esophageal mucosa. In neutral medium, OE-33 cells showed an increase in CDX-2 expression after incubation with DCA or UDCA. After short acidification of the medium, expression of CDX-2 in OE-33 cells was significantly higher than that in cells incubated in neutral pH. The addition of DCA and UDCA did not cause any further alteration in CDX-2 expression. In neutral and acidified medium, VEGF mRNA expression was only significantly up-regulated by DCA, but not by UDCA. Conclusions. Bile acids, especially in acidic medium, increase expression of CDX-2. DCA appears to be a stronger stimulant of the expression of VEGF than UDCA in the Barrett's carcinoma cell line, indicating a stronger carcinogenic potential of this bile salt.

Regulation of Cdx2 expression by promoter methylation, and effects of Cdx2 transfection on morphology and gene expression of human esophageal epithelial cells

Caudal-related homeobox 2 (Cdx2) has been suggested as an early marker of Barrett's esophagus (BE), which is the premalignant lesion of esophageal adenocarcinoma (EAC). However, the mechanism of ectopic Cdx2 expression in the esophageal epithelial cells and its role in the development of BE remained unclear. RT-PCR, pyrosequencing and methylation-specific PCR were used to determine expression and promoter methylation of Cdx2 in human esophageal epithelial cells (HET1A and SEG1) after treatment with 5-aza-2'-deoxycytidine (DAC), acid, bile acids and their combination. HET1A cells with stable transfection of Cdx2 were characterized for morphology and gene expression profiles with Affymetrix array. We found Cdx2 was expressed in most human EAC cell lines, but not in squamous epithelial cell lines. DAC-induced demethylation and expression of Cdx2 in HET1A and SEG1 cells, and treatment with a DNA methylating agent counteracted the effect of DAC. Treatment of HET1A and SEG1 cells with acid, bile acids or both also resulted in promoter demethylation and expression of Cdx2. HET1A cells with stable transfection of human Cdx2 formed crypt-like structures in vitro. Microarray analysis and quantitative real-time PCR showed that stable transfection of Cdx2 up-regulated differentiation markers of intestinal columnar epithelial cells and goblet cells in HET1A cells. This may be partially due to modulation of Notch signaling pathway, as western blotting confirmed down-regulation of Hes1 and up-regulation of Atoh1 and Muc2. Our data suggest that exposure to acid and/or bile acids may activate Cdx2 expression in human esophageal epithelial cells through promoter demethylation, and ectopic Cdx2 expression in esophageal squamous epithelial cells may contribute to intestinal metaplasia of the esophagus.

Genotoxic effects of myosmine in a human esophageal adenocarcinoma cell line

The incidence of esophageal adenocarcinoma is rapidly rising in Western populations. Gastroesophageal reflux disease (GERD) is thought to be one of the most important risk factors. However, the mechanisms by which GERD enhances tumor formation at the gastroesophageal junction are not well understood. Myosmine is a tobacco alkaloid which has also a wide spread occurrence in human diet. It is readily activated by nitrosation and peroxidation giving rise to the same hydroxypyridylbutanone-releasing DNA adducts as the esophageal carcinogen N′-nitrosonornicotine. Therefore, the genotoxicity of myosmine was tested in a human esophageal adenocarcinoma cell line (OE33). DNA damage was assessed by single-cell gel electrophoresis (Comet assay). DNA strand breaks, alkali labile sites and incomplete excision repair were expressed using the Olive tail moment (OTM). The Fapy glycosylase (Fpg) enzyme was incorporated into the assay to reveal additional oxidative DNA damage. DNA migration was determined after incubation of the cells for 1–24 h. Under neutral conditions high myosmine concentrations of 25–50 mM were necessary to elicit a weak genotoxic effect. At pH 6 genotoxicity was clearly enhanced giving a significant increase of OTM values at 5 mM myosmine. Lower pH values could not be tested because of massive cytotoxicity even in the absence of myosmine. Co-incubation of 25 mM myosmine with 1 mM H 2O 2 for 1 h significantly enhanced the genotoxicity of H 2O 2 but not the oxidative lesions additionally detected with the Fpg enzyme. In the presence of the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) a dose-dependent significant genotoxic effect was obtained with 1–10 mM myosmine after 4 h incubation. NS-398, a selective inhibitor of cyclooxygenase 2, did not affect the SIN-1 stimulated genotoxicity of myosmine. Finally, the 23 h repair of N-methyl- N′-nitro- N-nitrosoguanidine-induced DNA lesions was significantly inhibited in the presence of 10 mM myosmine. In conclusion, myosmine exerts significant genotoxic effects in esophageal cells under conditions which may prevail in GERD such as increased oxidative and nitrosative stress resulting from chronic inflammation.

Effect of Photofrin II as a radio-sensitizing agent in two different oesophageal carcinoma cell lines

Background and Purpose: In spite of major advances in cancer treatment, the prognosis of patients with oesophageal carcinoma remains poor. Squamous cell carcinoma and adenocarcinoma account for 95% of all oesophageal tumors, although other histological subtypes are occasionally seen. We aimed to evaluate whether Photofrin II can enhance the effect of ionizing radiation on oesophageal cancer in an in vitro tumor model. Material and Methods: A human oesophageal squamous cancer cell line (OE-21) and a human oesophageal adenocarcinoma cell line (OE-33) were evaluated with and without incubation with Photofrin II. Cells were irradiated using doses ranging from 0 to 8 Gy. The response rate of the cells to irradiation was evaluated by a tetrazolium-based colorimetric assay, similar to the MTT test, with the aim to determine the efficiency of Photofrin II as a radiation sensitizer in comparison to irradiation alone. Results: The OE-21 cell line demonstrated a significantly reduced cellular survival rate, when irradiated in the presence of Photofrin, as compared to a control group irradiated in the absence of Photofrin II. For the OE-33 cell line, no significant differences were found between the group treated with Photofrin II and the control group. Conclusion: Our results demonstrate in an in vitro model that Photofrin II may act as a radio-sensitizer in squamous cell oesophageal cancer, but not in oesophageal adenocarcinoma.

Synthetic and naturally occurring COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest.

Epidemiological studies suggest that the use of NSAIDs and/or a high intake of fruit and vegetables reduce the risk of oesophageal adenocarcinoma. Since COX-2 is up-regulated in Barrett's oesophageal carcinogenesis, the protective effect of NSAIDs and natural food components might reflect COX-2 inhibition. We explored the effects of quercetin, a natural flavonoid with a potent COX-2 inhibitory activity, and two commercially available selective COX-2 inhibitors (NS-398 and nimesulide) on cell proliferation, apoptosis, PGE2 production and COX-2 mRNA expression in a human oesophageal adenocarcinoma cell line (OE33). Changes in the relative numbers of adherent and floating cells were quantified and apoptotic cells were identified using ethidium bromide and acridine orange staining under fluorescence microscopy. Flow cytometric analysis of adherent and floating cells was used to quantify apoptosis and to examine the effects of the agents on the cell cycle. After 48 h exposure at concentrations of > or =1 microM both COX-2 inhibitors and quercetin suppressed cell proliferation (P < 0.01) and increased the fraction of floating apoptotic cells. At higher concentrations (50 microM) and longer exposure (48 h) the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors (P < 0.01). Cell cycle analyses showed that quercetin blocked cells in S phase, while the selective COX-2 inhibitors blocked cells in G1/S interphase. COX-2 mRNA expression was suppressed by quercetin and the synthetic COX-2 inhibitors in a time- and dose-dependent manner. Quercetin and the synthetic COX-2 inhibitors (10 microM) suppressed PGE2 production by approximately 70% after 24 h exposure (P < 0.001). We conclude that OE33 is a useful model for the study of COX-2 expression and associated phenomena in human adenocarcinoma cells. Synthetic COX-2 inhibitors and the food-borne flavonoid quercetin suppress proliferation, induce apoptosis and cell cycle block in human oesophageal adenocarcinoma cells in vitro, and future studies should assess their effects in vivo.

Gastrin stimulates receptor-mediated proliferation of human esophageal adenocarcinoma cells

The prevalence of esophageal adenocarcinoma in the setting of Barrett's metaplasia continues to increase in Western nations at a rate greater than any other cancer. The trophic properties of gastrin have been documented in gastric, pancreatic and colon cancer cell lines, suggesting a potential role for this regulatory peptide in the growth of these malignancies. The aims of these studies were to identify and characterize the presence of functional cholecystokinin type-2 (gastrin) receptors on the membranes of human esophageal adenocarcinoma cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of cholecystokinin type-2 receptor transcripts in human esophageal adenocarcinoma cell lines. Competitive binding assays revealed specific binding of gastrin in SEG-1 cells (IC 50 of 2.4×10 −8 M). This finding was confirmed by laser scanning confocal microscopy through internalization of rhodamine green labeled gastrin heptapeptide in SEG-1 cells. Gastrin caused a dose-dependent increase in proliferation of SEG-1 cells when compared to controls. This effect was abolished by co-incubation with L365,260, a CCK-2-specific receptor antagonist. Gastrin-induced phosphorylation of the p44 and p42 mitogen-activated protein kinases was demonstrated by Western blot analysis. In conclusion, the studied human esophageal adenocarcinoma cell lines possess cholecystokinin type-2 (gastrin) receptors. Receptors bind gastrin, resulting in increased proliferation in SEG-1 cells.