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W1874 Differential Expression and Activity of Toll-Like Receptor 2 and 4 in Barrett's Esophagus and Esophageal Adenocarcinoma
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prominent reaction during the acute phase of social isolation stress and leads to deterioration in quality of life. We investigated the effect of ghrelin on loss of appetite caused by social isolation. Methods:Male C57BL/6J mice (6 weeks old) were housed in groups (five mice per cage) and were then switched to individual housing (isolation). Food intake, serum corticosterone, and plasma ghrelin were measured over time. In addition, mice deprived of food for 24 h were administered rat ghrelin (i.v.) or ghrelin receptor antagonist (D-Lys3GHRP-6, i.v.) immediately after isolation and effects on food intake were examined. Similarly, rikkunshito (RKT, 250 or 500 mg/kg, p.o.), an endogenous ghrelin secretagogue (Gastroenterology 2009, Endocrinology 2010), was administered alone or in combination with a receptor antagonist, and food intake was measured. Results:After 30 minutes of isolation, serum corticosterone had increased significantly and plasma ghrelin had decreased. As shown in the figure, i.v. administration of rat ghrelin significantly alleviated the decrease in food intake caused by isolation (0.01 ± 0.01 g/h to 0.46 ± 0.13 g/h; p < 0.01). The administration of ghrelin receptor antagonist significantly decreased food intake in isolation group. RKT significantly increased the plasma ghrelin level in isolated mice (90.6 ± 7.4 fmol/mL to 120.2 ± 12.4 fmol/mL; p < 0.05) and significantly alleviated the decrease in food intake (0.46 ± 0.05 g/h to 0.98 ± 0.08 g/h;p < 0.001). In contrast, the administration of RKT in combination with ghrelin receptor antagonist blocked the increase in appetite caused by RKT (0.63 ± 0.12 g/h; p < 0.05 vs rikkunshito treated group). Conclusion:These results suggest that an abnormality in secretion of peripheral ghrelin may be in part responsible for the decrease in appetite caused by social isolation.
inhibition of telomerase leads to the induction of ALT in premalignant and malignant esophageal squamous epithelial and Barrett cells. Methods: We generated immortalized human esophageal epithelial cells overexpressing hTERT and squamous cancer cells overexpressing Cyclin D1, d.n.p53, EGFR and c-myc using retroviral transduction. Furthermore, we used genetically defined Barrett cells, which contain a p16 or p16 and p53 alterations and the esophageal adenocarcinoma cell line OE19. To genetically inhibit telomerase, cell types were transduced with mutated versions of hTER, anti-hTER siRNA or a combination of both using lentiviral transduction. Growth, telomerase activity (TRAP-assay), overall telomere length (TRF) as well as chromosome specific telomere length (Q-FISH) and ALT by indirect immunofluorescence (APB) were assayed. Moreover, we directly investigated telomere recombination with chromosome orientation CO-FISH. Results: MT-hTER, antihTER siRNA and the combination inhibit cell growth in immortalized squamous epithelial cells, malignant transformed squamous cancer cells and all Barrett cells. Nevertheless, growth inhibition is less pronounced in cancer cells as compared to premalignant cells. Furthermore, we could observe a reduction of telomerase activity in hTER-inhibited cell types, whereas TRF analysis revealed telomere maintenance or heterogeneous telomere length. This was observed in all premalignant and malignant cell types investigated regardless of the differentiation but dependent on the mode of inhibition. Q-FISH confirmed a heterogeneous telomere length on the chromosomal level, whereas controls displayed homogenous telomere length. Moreover, APBs appeared in a higher frequency in transduced immortalized cells compared to controls. Summary: We could demonstrate that genetic inhibition of telomerase could lead to ALT in different immortalized and malignant esophageal cells. This was independent of differentiation but correlated with their malignant status. Nevertheless, these findings suggest that telomerase positive cells especially cancer cells might find alternative ways to maintain their telomeres when treated with telomerase inhibitors. W1873 Honokiol Exerts Pronounced Anti-Proliferative and Pro-Apoptotic Effects in Transformed Barrett's Epithelial Cells That Have Active RAS and Inactive p53. Chunhua Yu, Hui Ying Zhang, Xi Zhang, Xiaofang Huo, Kathy K. Hormi-Carver, Jack Arbiser, Stuart J. Spechler, Rhonda F. Souza Introduction: Honokiol (HNK), a polyphenol found in Asian herbal teas, can inhibit the Ras pathway and cause anti-proliferative/pro-apoptotic effects in a number of human tumors, especially in those with p53 inactivation. Both Ras activation and p53 inactivation occur frequently during carcinogenesis in Barrett's metaplasia, suggesting a potential role for HNK in the treatment of Barrett's cancers. We have developed Barrett's epithelial cell lines that express oncogenic Ras, with and without p53 knockdown, and we have used those lines to study effects of HNK on proliferation and apoptosis. Methods: Telomerase-immortalized, non-neoplastic Barrett's (BAR-T) cells were stably infected with the retroviral vector pBabeoncogenic H-RasG12V to activate the Ras pathway, with or without co-infection by pSUPERsiRNA-p53 to knockdown p53. The resulting cell lines expressed active H-Ras (R), with and without p53 knockdown (p53kd). The cells were cultured in media containing 0.5% serum without additional growth factors, and were treated with HNK in doses of 10, 20, 40, and 80 μM for up to 48 hours. Proliferation was determined by cell count. Apoptosis and necrosis were determined by Cell Death Elisa and morphology. Results: HNK in doses of 40 μM and higher induced necrosis in all cell lines. HNK in a dose of 10 μM had no apparent effect on proliferation in BAR-T and R1 cells, which are not transformed. At a dose of 20 μM, however, HNK treatment caused a significant decrease in proliferation at 24 hours in the BAR-T and R1 cells. In contrast, HNK in a dose of only 10 μM caused a significant decrease in proliferation at 24 hours in R1p53kd1 cells, which are transformed. By 48 hours, cell numbers had returned to those of untreated control cells in the BAR-T and R1 cells treated with 10 and 20 μM HNK, and in the R1p53kd1 cells treated with 10 μM HNK. In contrast, cell numbers were significantly lower at 48 hours in R1p53kd1 cells treated with 20 μM HNK. This finding was confirmed in another transformed cell line (R1p53kd2). Treatment with 20 μM HNK also significantly increased apoptosis in both the R1p53kd1 and R1p53kd2 cells (R1p53kd1: 0.2 ± 0.0 SEM to .63 ± 0.04; R1p53kd2: 0.08 ± 0.01 to .61 ± 0.03), but not in the BAR-T or R1 cells. Conclusions: HNK exerts pronounced anti-proliferative and pro-apoptotic effects in transformed Barrett's epithelial cells that express oncogenic Ras and lack functional p53. The transformed cells are far more sensitive to those effects than the non-transformed Barrett's cells, including those that express Ras. These findings support a potential role for HNK as a chemotherapeutic agent for Barrett's cancers.
Figure Effect of ghrelin administering to appetite decrease by isolation W1865 Apigenin Inhibits NNK-Induced FAK Y397 Activation in Pancreatic Cells Hung Pham, Monica C. Chen, Jonathan C. King, Hiroki Takahashi, Stephen J. Pandol, Howard A. Reber, Oscar J. Hines, Guido Eibl Background: Apigenin (4′, 5, 7-trihydroxyflavone) is a non-toxic flavonoid purported to attenuate growth and proliferation of cancer cells. The carcinogen, NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), derived from tobacco products induces pancreatic cancer In Vivo. NNK binds to and activates the β-adrenergic rececptor signaling, with a subsequent activation of Src signaling. The receptor-associated Src family of kinases/focal adhesion kinases (FAK) modulates cancer cell survival, migration and invasion. Human pancreatic cell lines were employed to determine whether apigenin could inhibit NNK-induced metastatic potential of these cells involving FAK activation. Methods: Human pancreatic cancer ductal cell lines MIA PaCa-2 and BxPC-3 were cultured to confluency and serum-starved overnight prior to experiments. Proliferation studies were performed using MTT assay at 24, 48 or 72 hrs. Scratch assays were performed on confluent monolayers of cells using a 20 μL pipette tip and recorded at 0, 2, 6, and 24 hrs. Protein expression for FAK Y397 and total FAK were determined by Western blotting. Results: Addition of 100 μM of NNK enhanced growth of both MiaPaCa-2 and BxPC-3 cells by a factor of 1.6 ± 0.2 and 1.5 ± 0.3, respectively as determined by MTT. This NNK-enhanced proliferation was significantly suppressed by 50 μM of the specific β-adrenergic receptor inhibitor propranolol in MiaPaCa-2 at 26.6 ± 3.2 % and BxPC-3 at 26.7 ± 1.3 %. Similarly, pre-incubation with 50 μM of apigenin for 24 hr prior to NNK stimulation resulted in a 22.5 ± 1.7 % decrease in MiaPaCa-2 and a 38.0 ± 2.6 % decrease in BxPC-3 cellular proliferation. Scratch assays showed that addition of 100 μM of NNK to both cells types enhanced cellular migration and growth with complete closure by 24 hr that was blocked by 50 μM of propranolol. Antibody staining for activated FAK Y397 expression revealed a time-dependent increase in FAK activation at 10 min that was sustained at 30 min when stimulated with 100 μM of NNK for both cell types. FAK activation by 100 μM of NNK was suppressed with a pre-incubation with 50 μM of propranolol or, interestingly, with 50 μM of apigenin. Antibody staining for FAK expression showed no discernible difference when both cell types were treated with 100 μM of NNK, 50 μM of propranolol or 50 μM of apigenin compared to untreated cells. Conclusion: Overall, the findings demonstrate that FAK Y397 activation by NNK seemingly proceeds through βadrenergic receptor activation that could be inhibited by apigenin. These results suggest a therapeutic role for apigenin in attenuating NNK-mediated growth and migration of pancreatic cells.
W1874 Differential Expression and Activity of Toll-Like Receptor 2 and 4 in Barrett's Esophagus and Esophageal Adenocarcinoma Romy E. Verbeek, Fiebo J. ten Kate, Frank P. Vleggaar, Peter D. Siersema, Jantine W. van Baal BACKGROUND: In Barrett's Esophagus (BE), the normal squamous epithelium is replaced by intestinal columnar epithelium as a result of chronic gastro-esophageal reflux. BE is a premalignant condition predisposing to the development of esophageal adenocarcinoma (EAC). Inflammation is likely to be involved in the pathogenesis of BE and EAC. Toll-like receptors (TLRs) are innate immune receptors that have been shown to be involved in inflammation and carcinogenesis. TLRs act in the presence of micro-organisms and signals produced by tissue damage. AIM: To investigate the expression of TLR 2 and 4 in BE and EAC and its functional activity in the esophagus. METHODS: Esophageal biopsies were obtained during endoscopy from normal squamous epithelium (SQ), reflux esophagitis (RE), BE and EAC of 14 patients with BE and 8 with BE associated EAC. Paraffin and frozen sections were used to assess TLR 2 and 4 protein expression by immunohistochemistry. RNA was isolated from frozen esophageal biopsies and subsequent TLR 2 and 4 mRNA expression was quantified by Q-RT-PCR. Functional activity of TLR 2 and 4 was assessed in Het1a (normal esophageal squamous epithelium) cells by IκBα phosphorylation and degradation (Western Blot) and IL8 production (ELISA) following stimulation with Pam3CSK4 and LPS, TLR 2 and 4 agonists respectively. RESULTS: Immunohistochemistry showed expression of TLR 2 and 4, which was for a large part seen at the epithelial surface in BE and EAC and confined to the epithelial base in SQ and RE. Q-RT-PCR showed a 9.1
W1872 Regulation of Telomerase and ALT in Esophageal Squamous Cell Cancer and Barrett Cancer Angela Queisser, Michaela Thaler, Alexander von Werder, Heike Kunert, Hiroshi Nakagawa, Oliver G. Opitz Introduction: In a cellular model of squamous carcinogenesis epithelial cells are immortalized by an ALT mechanism and still recruit telomerase during tumor progression. Nevertheless, it is not known whether these two telomere maintenance mechanisms telomerase (containing an RNA-template (hTER) and a core protein (hTERT)) and the recombination based ALT are recruited in the same cell type and how they are regulated. Here we investigate if genetic
AGA Abstracts
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AGA Abstracts
fold (p=0.008) increase in TLR 4 expression in EAC (1.5± 0.6) and a 7.3 fold (p=0.002) increase in BE (1.2± 0.2) compared to SQ (0.2± 0.1). TLR 2 mRNA expression was increased in EAC (2.7± 0.6, p=0.04) compared to SQ (1.0± 0.3), with no significant difference found comparing BE (1.5± 0.6) with EAC or SQ. Pam3CSK4 incubation of Het1a cells resulted in increased IL8 production (1.5± 0.09 ng/ml vs. control 0.6± 0.07 ng/ml, p<0.001); LPS incubation showed an increased IL8 production of 2.2 ± 0.01 ng/ml (p<0.001), indicating TLR 2 and 4 pathway activation respectively. Furthermore, phosphorylation and degradation of IκBα was observed after TLR 2 and 4 stimulation in Het1a cells as a consequence of NFκB activation. CONCLUSION: Our results showed differential expression of TLR 2 and 4 comparing EAC, BE and SQ and functional activity of these receptors in an esophageal cell line. Binding of natural ligands to TLR 2 and 4 on the surface of patients with BE and EAC may result in activation of downstream targets, contributing to inflammation and carcinogenesis. W1875 Clinicopathological Features of Superficial Esophageal Carcinoma With Nodal or Distant Organ Recurrence After Complete Endoscopic Resection: MultiInstitutional Analysis of 24 Endoscopically Resected Cases Yukihiro Nakanishi, Tatsuyuki Kawano W1877
Background: Little is known about clinicopathological features of superficial esophageal carcinoma (SEC) which shows nodal or distant organ recurrence after complete endoscopic resection of primary tumor. This study aims to clarify the clinicopathological features of SEC which showed nodal or distant organ recurrence after endoscopic resection. Patients and Methods: We retrospectively investigated the clinicopathological features of 24 endoscopically resected SECs collected from multiple institutions in Japan, all of which showed either nodal or distant organ recurrence after complete endoscopic resection of primary lesion. Case selection was restricted to include tumors invading the lamina propria (M2), tumors in contact with or invading the muscularis mucosa (M3), or tumors invading less than 200 microns of the submucosa (SM1). Clinicopathological factors including recurrence mode, macroscopic type, histologic type, invasion depth, lymphovascular invasion, and growth pattern were assessed. Results: Of 24 SECs with recurrence, 23 (96%) cases showed nodal recurrence and one (4%) case showed both nodal and distant organ recurrences. Twenty one (88%) cases were superficial depressed type, 2 (8%) cases were combined superficial elevated and depressed type, and one (4%) case was completely flat type. Twenty two (92%) cases were squamous cell carcinomas and 2 (8%) cases were high grade neuroendocrine carcinomas. Two (8%) cases showed M2 invasion, 12 (50%) cases M3 invasion, and 2 (8%) cases SM1 invasion. Sixteen (67%) cases showed lymphovascular invasion. One (4%) case showed pushing growth pattern, 19 (79%) cases showed intermediate growth pattern, and 4 (17%) cases showed infiltrative growth pattern. Conclusions: High incidence of lymphovascular invasion was found in recurrent cases even though they were minimally invasive SECs. Lymphovascular invasion is considered to be the most important histopathologic factor for recurrence after complete endoscopic resection of the primary lesion.
The Effect of Diet on the Development of Oesophageal Adenocarcinoma: Comparison With Community and Reflux Oesophagitis Controls From MOSES (Midlands Oesophageal Adenocarcinoma Epidemiology Study) Sheldon C. Cooper, Sandra Prew, Laura Podmore, Nigel J. Trudgill Diet is a key modifiable risk factor in the aetiology of cancer. The UK has the highest incidence of oesophageal adenocarcinoma (OAC) in the world. We have undertaken the largest case-control study to date, to identify dietary risk factors associated with development of OAC. Incident cases of OAC from 12 hospitals in England and age-, gender-, ethnicallyand geographically-matched community control (CC) subjects (primary care) and reflux oesophagitis (RO) control patients (endoscopy units) were interviewed. Frequency of ingestion (days per week) and weekly portion consumption of vegetables, fruit, fruit juice, red and white meat, fish, tea and coffee were divided into quintiles for analysis. Logistic regression generated odds ratios (OR) for potential dietary aetiological factors 1 and 10 years prior to diagnosis. Results presented as highest versus lowest quintile. 207 OAC (177 men, median (IQR) age 68 (60-75) years), 283 CC subjects (236 men, age 67 (60-74) years), and 224 RO patients (178 men, age 66 (59-75) years) were recruited. Frequency of vegetable consumption 1 year prior to diagnosis was negatively associated with OAC (vs. CC) OR (95% CI) 0.55 (0.34-0.88) (p=0.004) and total consumption both 1 and 10 years prior 0.38 (0.21-0.69) (p=0.002) and 0.42 (0.23-0.75) (p=0.004) respectively. No association was seen with vegetable consumption vs RO. Fruit, but not fruit juice, consumption was negatively associated with OAC for both time points (p<0.0001 unless stated): frequency 0.35 (0.23-0.54) 1 year and 0.46 (0.31-0.69) 10 years prior; weekly consumption 0.32 (0.18-0.5) 1 year and 0.39 (0.2-0.77) (p=0.007) 10 years prior; and vs. RO for frequency 1 year prior 0.62 (0.4-0.97) (p=0.035). Red meat was associated with OAC vs. CC 1 year prior to diagnosis: frequency 2.1 (1.25-3.54) (p=0.005) and weekly consumption 2.44 (1.334.48) (p=0.004); and vs. RO weekly consumption 1 year prior 1.95 (1.03-3.68) (p=0.04). White meat, fish and coffee consumption showed no association with OAC. Tea consumption (cups/day) was associated with OAC (vs CC only): 2.07 (1.11-3.85) (p=0.021) 1 year and 2.38 (1.34-4.21) (p=0.003) 10 years prior to diagnosis. Use of salt in cooking and at the table was associated with OAC (vs CC) for both time periods and vs RO at the table 1-year prior to diagnosis. Eating five-a-day (fruit, juice, and vegetables) was negatively associated with OAC (vs CC): 0.53 (0.33-0.85) (p=0.008). Vegetable and more strikingly fruit consumption were negatively associated with OAC. Greater consumption of red meat, salt and tea were positively associated with OAC. Dietary modification may offer opportunities to impact upon the increasing incidence of OAC
W1876 Survival After Curative Resection for Adenocarcinoma at the GastroEsophageal Junction With Postoperative Chemoradio Therapy or Surgery Alone Steen C. Kofoed, Lene Baeksgaard, Lars Bo Svendsen We present the survival data of a 350 patients curatively resected for adenocarcinoma at the gastro-esophageal junction and postoperatively treated with chemoradio therapy according to the Intergroup 0116 protocol. The survival was compared with the survival after curative resection only. From 1992 through 2003, 137 patients underwent curative resection(group 1). In 2003, the Intergroup 0116 protocol was implemented in our institution. From 2003 through 2009, 96 patients underwent surgery only(group 2) and 117 patients received postoperative therapy(group 3). The operation was carried out as a two-phase abdominal and right chest approach for en-bloc subtotal esophagectomy. The postoperative treatment consisted of chemotherapy with fluorouracil plus leucovorin and loco-regional radiation. To calculate the disease-free survival, information regarding diagnosis for metastasis or relapse of disease was collected from the Danish Cancer Register. All deaths due to complications after surgery and patients with superficial tumours (T0N0/T1N0) were excluded from the analysis. The overall disease-free 3-year survival in group 1, 2 and 3 was 39%, 45% and 53% respectively (Log rank test:P=0.58). Figure 1 shows disease-free survival after curative resection where lymph node involvement was found in the resected specimen. The 3 year survival in the surgery only group (group 2) was 29%. Median time of survival was 16 month(CI:1220). In the postoperative therapy group, the survival was 48%. Median time of survival was 29 month(CI:11-47)(Log rank test:P=0.014). From 2003 through 2009, 64(30%) patients underwent a D1 resection (≥15 nodes) and 57(27%) patients underwent D2 resection (≥26 nodes). Our results support, that postoperative chemoradio therapy is a reasonable option in curatively resected patients with adenocarcinoma at the gastro-esophageal junction and positive node status in the resected specimen.
W1878 The Effect of Smoking and Alcohol Consumption on the Development of Oesophageal Adenocarcinoma: Comparison With Community and Reflux Oesophagitis Controls From MOSES (Midlands Oesophageal Adenocarcinoma Epidemiology Study) Sheldon C. Cooper, Sandra Prew, Laura Podmore, Nigel J. Trudgill The UK has the highest incidence of oesophageal adenocarcinoma (OAC) in the world. Smoking and alcohol consumption have typically been associated with oesophageal squamous cell carcinoma. We have undertaken the largest case-control study to date, to identify risk factors associated with the development of OAC. Incident cases of OAC from 12 hospitals in England and age-, gender-, ethnically- and geographically-matched community control subjects (CC) subjects (primary care) and reflux oesophagitis (RO) control patients (endoscopy units) were interviewed. Logistic regression generated odds ratios (OR) for smoking and alcohol consumption 1 and 10 years prior to diagnosis. 207 OAC (177 men, median (IQR) age 68 (60-75) years), 283 CC subjects (236 men, age 67 (60-74) years) and 224 RO patients (178 men, age 66 (59-75) years) were recruited. Being a current smoker (vs. never smoked) is associated with OAC: vs. CC 4.37 (2.46-7.76) (p<0.0001) and vs. RO 2.28 (1.29-4.03) (p=0.005). Previously smoking (vs. never) was also associated with OAC: vs. CC 2.32 (1.5-3.61) and vs. RO 1.9 (1.19-3.04) (p=0.007). Quantity smoked 1 year (CC 2.45 (1.45-4.15) (p=0.001) and RO 2.29 (1.22-4.31) (p=0.01)) and 10 years prior to diagnosis (CC 2.7 (1.6-4.52) (p<0.0001) and RO 1.37 (0.83-2.27) (p=0.02)) were associated with OAC. Being a drinker of alcohol was negatively associated with OAC (vs CC only) (0.63 (0.2-0.93) (p=0.033)), including increasing duration of consumption (0.51 (0.29-0.9) (p=0.019)), but not quantity. Smoking is strongly associated with the development of OAC, when compared with both community controls and reflux oesophagitis patients. There is also clear evidence of a dose-response relationship. In contrast to oesophageal squamous cell carcinoma, there was some evidence to suggest that alcohol intake is negatively associated with OAC.
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prominent reaction during the acute phase of social isolation stress and leads to deterioration in quality of life. We investigated the effect of ghrelin on loss of appetite caused by social isolation. Methods:Male C57BL/6J mice (6 weeks old) were housed in groups (five mice per cage) and were then switched to individual housing (isolation). Food intake, serum corticosterone, and plasma ghrelin were measured over time. In addition, mice deprived of food for 24 h were administered rat ghrelin (i.v.) or ghrelin receptor antagonist (D-Lys3GHRP-6, i.v.) immediately after isolation and effects on food intake were examined. Similarly, rikkunshito (RKT, 250 or 500 mg/kg, p.o.), an endogenous ghrelin secretagogue (Gastroenterology 2009, Endocrinology 2010), was administered alone or in combination with a receptor antagonist, and food intake was measured. Results:After 30 minutes of isolation, serum corticosterone had increased significantly and plasma ghrelin had decreased. As shown in the figure, i.v. administration of rat ghrelin significantly alleviated the decrease in food intake caused by isolation (0.01 ± 0.01 g/h to 0.46 ± 0.13 g/h; p < 0.01). The administration of ghrelin receptor antagonist significantly decreased food intake in isolation group. RKT significantly increased the plasma ghrelin level in isolated mice (90.6 ± 7.4 fmol/mL to 120.2 ± 12.4 fmol/mL; p < 0.05) and significantly alleviated the decrease in food intake (0.46 ± 0.05 g/h to 0.98 ± 0.08 g/h;p < 0.001). In contrast, the administration of RKT in combination with ghrelin receptor antagonist blocked the increase in appetite caused by RKT (0.63 ± 0.12 g/h; p < 0.05 vs rikkunshito treated group). Conclusion:These results suggest that an abnormality in secretion of peripheral ghrelin may be in part responsible for the decrease in appetite caused by social isolation.
inhibition of telomerase leads to the induction of ALT in premalignant and malignant esophageal squamous epithelial and Barrett cells. Methods: We generated immortalized human esophageal epithelial cells overexpressing hTERT and squamous cancer cells overexpressing Cyclin D1, d.n.p53, EGFR and c-myc using retroviral transduction. Furthermore, we used genetically defined Barrett cells, which contain a p16 or p16 and p53 alterations and the esophageal adenocarcinoma cell line OE19. To genetically inhibit telomerase, cell types were transduced with mutated versions of hTER, anti-hTER siRNA or a combination of both using lentiviral transduction. Growth, telomerase activity (TRAP-assay), overall telomere length (TRF) as well as chromosome specific telomere length (Q-FISH) and ALT by indirect immunofluorescence (APB) were assayed. Moreover, we directly investigated telomere recombination with chromosome orientation CO-FISH. Results: MT-hTER, antihTER siRNA and the combination inhibit cell growth in immortalized squamous epithelial cells, malignant transformed squamous cancer cells and all Barrett cells. Nevertheless, growth inhibition is less pronounced in cancer cells as compared to premalignant cells. Furthermore, we could observe a reduction of telomerase activity in hTER-inhibited cell types, whereas TRF analysis revealed telomere maintenance or heterogeneous telomere length. This was observed in all premalignant and malignant cell types investigated regardless of the differentiation but dependent on the mode of inhibition. Q-FISH confirmed a heterogeneous telomere length on the chromosomal level, whereas controls displayed homogenous telomere length. Moreover, APBs appeared in a higher frequency in transduced immortalized cells compared to controls. Summary: We could demonstrate that genetic inhibition of telomerase could lead to ALT in different immortalized and malignant esophageal cells. This was independent of differentiation but correlated with their malignant status. Nevertheless, these findings suggest that telomerase positive cells especially cancer cells might find alternative ways to maintain their telomeres when treated with telomerase inhibitors. W1873 Honokiol Exerts Pronounced Anti-Proliferative and Pro-Apoptotic Effects in Transformed Barrett's Epithelial Cells That Have Active RAS and Inactive p53. Chunhua Yu, Hui Ying Zhang, Xi Zhang, Xiaofang Huo, Kathy K. Hormi-Carver, Jack Arbiser, Stuart J. Spechler, Rhonda F. Souza Introduction: Honokiol (HNK), a polyphenol found in Asian herbal teas, can inhibit the Ras pathway and cause anti-proliferative/pro-apoptotic effects in a number of human tumors, especially in those with p53 inactivation. Both Ras activation and p53 inactivation occur frequently during carcinogenesis in Barrett's metaplasia, suggesting a potential role for HNK in the treatment of Barrett's cancers. We have developed Barrett's epithelial cell lines that express oncogenic Ras, with and without p53 knockdown, and we have used those lines to study effects of HNK on proliferation and apoptosis. Methods: Telomerase-immortalized, non-neoplastic Barrett's (BAR-T) cells were stably infected with the retroviral vector pBabeoncogenic H-RasG12V to activate the Ras pathway, with or without co-infection by pSUPERsiRNA-p53 to knockdown p53. The resulting cell lines expressed active H-Ras (R), with and without p53 knockdown (p53kd). The cells were cultured in media containing 0.5% serum without additional growth factors, and were treated with HNK in doses of 10, 20, 40, and 80 μM for up to 48 hours. Proliferation was determined by cell count. Apoptosis and necrosis were determined by Cell Death Elisa and morphology. Results: HNK in doses of 40 μM and higher induced necrosis in all cell lines. HNK in a dose of 10 μM had no apparent effect on proliferation in BAR-T and R1 cells, which are not transformed. At a dose of 20 μM, however, HNK treatment caused a significant decrease in proliferation at 24 hours in the BAR-T and R1 cells. In contrast, HNK in a dose of only 10 μM caused a significant decrease in proliferation at 24 hours in R1p53kd1 cells, which are transformed. By 48 hours, cell numbers had returned to those of untreated control cells in the BAR-T and R1 cells treated with 10 and 20 μM HNK, and in the R1p53kd1 cells treated with 10 μM HNK. In contrast, cell numbers were significantly lower at 48 hours in R1p53kd1 cells treated with 20 μM HNK. This finding was confirmed in another transformed cell line (R1p53kd2). Treatment with 20 μM HNK also significantly increased apoptosis in both the R1p53kd1 and R1p53kd2 cells (R1p53kd1: 0.2 ± 0.0 SEM to .63 ± 0.04; R1p53kd2: 0.08 ± 0.01 to .61 ± 0.03), but not in the BAR-T or R1 cells. Conclusions: HNK exerts pronounced anti-proliferative and pro-apoptotic effects in transformed Barrett's epithelial cells that express oncogenic Ras and lack functional p53. The transformed cells are far more sensitive to those effects than the non-transformed Barrett's cells, including those that express Ras. These findings support a potential role for HNK as a chemotherapeutic agent for Barrett's cancers.
Figure Effect of ghrelin administering to appetite decrease by isolation W1865 Apigenin Inhibits NNK-Induced FAK Y397 Activation in Pancreatic Cells Hung Pham, Monica C. Chen, Jonathan C. King, Hiroki Takahashi, Stephen J. Pandol, Howard A. Reber, Oscar J. Hines, Guido Eibl Background: Apigenin (4′, 5, 7-trihydroxyflavone) is a non-toxic flavonoid purported to attenuate growth and proliferation of cancer cells. The carcinogen, NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), derived from tobacco products induces pancreatic cancer In Vivo. NNK binds to and activates the β-adrenergic rececptor signaling, with a subsequent activation of Src signaling. The receptor-associated Src family of kinases/focal adhesion kinases (FAK) modulates cancer cell survival, migration and invasion. Human pancreatic cell lines were employed to determine whether apigenin could inhibit NNK-induced metastatic potential of these cells involving FAK activation. Methods: Human pancreatic cancer ductal cell lines MIA PaCa-2 and BxPC-3 were cultured to confluency and serum-starved overnight prior to experiments. Proliferation studies were performed using MTT assay at 24, 48 or 72 hrs. Scratch assays were performed on confluent monolayers of cells using a 20 μL pipette tip and recorded at 0, 2, 6, and 24 hrs. Protein expression for FAK Y397 and total FAK were determined by Western blotting. Results: Addition of 100 μM of NNK enhanced growth of both MiaPaCa-2 and BxPC-3 cells by a factor of 1.6 ± 0.2 and 1.5 ± 0.3, respectively as determined by MTT. This NNK-enhanced proliferation was significantly suppressed by 50 μM of the specific β-adrenergic receptor inhibitor propranolol in MiaPaCa-2 at 26.6 ± 3.2 % and BxPC-3 at 26.7 ± 1.3 %. Similarly, pre-incubation with 50 μM of apigenin for 24 hr prior to NNK stimulation resulted in a 22.5 ± 1.7 % decrease in MiaPaCa-2 and a 38.0 ± 2.6 % decrease in BxPC-3 cellular proliferation. Scratch assays showed that addition of 100 μM of NNK to both cells types enhanced cellular migration and growth with complete closure by 24 hr that was blocked by 50 μM of propranolol. Antibody staining for activated FAK Y397 expression revealed a time-dependent increase in FAK activation at 10 min that was sustained at 30 min when stimulated with 100 μM of NNK for both cell types. FAK activation by 100 μM of NNK was suppressed with a pre-incubation with 50 μM of propranolol or, interestingly, with 50 μM of apigenin. Antibody staining for FAK expression showed no discernible difference when both cell types were treated with 100 μM of NNK, 50 μM of propranolol or 50 μM of apigenin compared to untreated cells. Conclusion: Overall, the findings demonstrate that FAK Y397 activation by NNK seemingly proceeds through βadrenergic receptor activation that could be inhibited by apigenin. These results suggest a therapeutic role for apigenin in attenuating NNK-mediated growth and migration of pancreatic cells.
W1874 Differential Expression and Activity of Toll-Like Receptor 2 and 4 in Barrett's Esophagus and Esophageal Adenocarcinoma Romy E. Verbeek, Fiebo J. ten Kate, Frank P. Vleggaar, Peter D. Siersema, Jantine W. van Baal BACKGROUND: In Barrett's Esophagus (BE), the normal squamous epithelium is replaced by intestinal columnar epithelium as a result of chronic gastro-esophageal reflux. BE is a premalignant condition predisposing to the development of esophageal adenocarcinoma (EAC). Inflammation is likely to be involved in the pathogenesis of BE and EAC. Toll-like receptors (TLRs) are innate immune receptors that have been shown to be involved in inflammation and carcinogenesis. TLRs act in the presence of micro-organisms and signals produced by tissue damage. AIM: To investigate the expression of TLR 2 and 4 in BE and EAC and its functional activity in the esophagus. METHODS: Esophageal biopsies were obtained during endoscopy from normal squamous epithelium (SQ), reflux esophagitis (RE), BE and EAC of 14 patients with BE and 8 with BE associated EAC. Paraffin and frozen sections were used to assess TLR 2 and 4 protein expression by immunohistochemistry. RNA was isolated from frozen esophageal biopsies and subsequent TLR 2 and 4 mRNA expression was quantified by Q-RT-PCR. Functional activity of TLR 2 and 4 was assessed in Het1a (normal esophageal squamous epithelium) cells by IκBα phosphorylation and degradation (Western Blot) and IL8 production (ELISA) following stimulation with Pam3CSK4 and LPS, TLR 2 and 4 agonists respectively. RESULTS: Immunohistochemistry showed expression of TLR 2 and 4, which was for a large part seen at the epithelial surface in BE and EAC and confined to the epithelial base in SQ and RE. Q-RT-PCR showed a 9.1
W1872 Regulation of Telomerase and ALT in Esophageal Squamous Cell Cancer and Barrett Cancer Angela Queisser, Michaela Thaler, Alexander von Werder, Heike Kunert, Hiroshi Nakagawa, Oliver G. Opitz Introduction: In a cellular model of squamous carcinogenesis epithelial cells are immortalized by an ALT mechanism and still recruit telomerase during tumor progression. Nevertheless, it is not known whether these two telomere maintenance mechanisms telomerase (containing an RNA-template (hTER) and a core protein (hTERT)) and the recombination based ALT are recruited in the same cell type and how they are regulated. Here we investigate if genetic
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fold (p=0.008) increase in TLR 4 expression in EAC (1.5± 0.6) and a 7.3 fold (p=0.002) increase in BE (1.2± 0.2) compared to SQ (0.2± 0.1). TLR 2 mRNA expression was increased in EAC (2.7± 0.6, p=0.04) compared to SQ (1.0± 0.3), with no significant difference found comparing BE (1.5± 0.6) with EAC or SQ. Pam3CSK4 incubation of Het1a cells resulted in increased IL8 production (1.5± 0.09 ng/ml vs. control 0.6± 0.07 ng/ml, p<0.001); LPS incubation showed an increased IL8 production of 2.2 ± 0.01 ng/ml (p<0.001), indicating TLR 2 and 4 pathway activation respectively. Furthermore, phosphorylation and degradation of IκBα was observed after TLR 2 and 4 stimulation in Het1a cells as a consequence of NFκB activation. CONCLUSION: Our results showed differential expression of TLR 2 and 4 comparing EAC, BE and SQ and functional activity of these receptors in an esophageal cell line. Binding of natural ligands to TLR 2 and 4 on the surface of patients with BE and EAC may result in activation of downstream targets, contributing to inflammation and carcinogenesis. W1875 Clinicopathological Features of Superficial Esophageal Carcinoma With Nodal or Distant Organ Recurrence After Complete Endoscopic Resection: MultiInstitutional Analysis of 24 Endoscopically Resected Cases Yukihiro Nakanishi, Tatsuyuki Kawano W1877
Background: Little is known about clinicopathological features of superficial esophageal carcinoma (SEC) which shows nodal or distant organ recurrence after complete endoscopic resection of primary tumor. This study aims to clarify the clinicopathological features of SEC which showed nodal or distant organ recurrence after endoscopic resection. Patients and Methods: We retrospectively investigated the clinicopathological features of 24 endoscopically resected SECs collected from multiple institutions in Japan, all of which showed either nodal or distant organ recurrence after complete endoscopic resection of primary lesion. Case selection was restricted to include tumors invading the lamina propria (M2), tumors in contact with or invading the muscularis mucosa (M3), or tumors invading less than 200 microns of the submucosa (SM1). Clinicopathological factors including recurrence mode, macroscopic type, histologic type, invasion depth, lymphovascular invasion, and growth pattern were assessed. Results: Of 24 SECs with recurrence, 23 (96%) cases showed nodal recurrence and one (4%) case showed both nodal and distant organ recurrences. Twenty one (88%) cases were superficial depressed type, 2 (8%) cases were combined superficial elevated and depressed type, and one (4%) case was completely flat type. Twenty two (92%) cases were squamous cell carcinomas and 2 (8%) cases were high grade neuroendocrine carcinomas. Two (8%) cases showed M2 invasion, 12 (50%) cases M3 invasion, and 2 (8%) cases SM1 invasion. Sixteen (67%) cases showed lymphovascular invasion. One (4%) case showed pushing growth pattern, 19 (79%) cases showed intermediate growth pattern, and 4 (17%) cases showed infiltrative growth pattern. Conclusions: High incidence of lymphovascular invasion was found in recurrent cases even though they were minimally invasive SECs. Lymphovascular invasion is considered to be the most important histopathologic factor for recurrence after complete endoscopic resection of the primary lesion.
The Effect of Diet on the Development of Oesophageal Adenocarcinoma: Comparison With Community and Reflux Oesophagitis Controls From MOSES (Midlands Oesophageal Adenocarcinoma Epidemiology Study) Sheldon C. Cooper, Sandra Prew, Laura Podmore, Nigel J. Trudgill Diet is a key modifiable risk factor in the aetiology of cancer. The UK has the highest incidence of oesophageal adenocarcinoma (OAC) in the world. We have undertaken the largest case-control study to date, to identify dietary risk factors associated with development of OAC. Incident cases of OAC from 12 hospitals in England and age-, gender-, ethnicallyand geographically-matched community control (CC) subjects (primary care) and reflux oesophagitis (RO) control patients (endoscopy units) were interviewed. Frequency of ingestion (days per week) and weekly portion consumption of vegetables, fruit, fruit juice, red and white meat, fish, tea and coffee were divided into quintiles for analysis. Logistic regression generated odds ratios (OR) for potential dietary aetiological factors 1 and 10 years prior to diagnosis. Results presented as highest versus lowest quintile. 207 OAC (177 men, median (IQR) age 68 (60-75) years), 283 CC subjects (236 men, age 67 (60-74) years), and 224 RO patients (178 men, age 66 (59-75) years) were recruited. Frequency of vegetable consumption 1 year prior to diagnosis was negatively associated with OAC (vs. CC) OR (95% CI) 0.55 (0.34-0.88) (p=0.004) and total consumption both 1 and 10 years prior 0.38 (0.21-0.69) (p=0.002) and 0.42 (0.23-0.75) (p=0.004) respectively. No association was seen with vegetable consumption vs RO. Fruit, but not fruit juice, consumption was negatively associated with OAC for both time points (p<0.0001 unless stated): frequency 0.35 (0.23-0.54) 1 year and 0.46 (0.31-0.69) 10 years prior; weekly consumption 0.32 (0.18-0.5) 1 year and 0.39 (0.2-0.77) (p=0.007) 10 years prior; and vs. RO for frequency 1 year prior 0.62 (0.4-0.97) (p=0.035). Red meat was associated with OAC vs. CC 1 year prior to diagnosis: frequency 2.1 (1.25-3.54) (p=0.005) and weekly consumption 2.44 (1.334.48) (p=0.004); and vs. RO weekly consumption 1 year prior 1.95 (1.03-3.68) (p=0.04). White meat, fish and coffee consumption showed no association with OAC. Tea consumption (cups/day) was associated with OAC (vs CC only): 2.07 (1.11-3.85) (p=0.021) 1 year and 2.38 (1.34-4.21) (p=0.003) 10 years prior to diagnosis. Use of salt in cooking and at the table was associated with OAC (vs CC) for both time periods and vs RO at the table 1-year prior to diagnosis. Eating five-a-day (fruit, juice, and vegetables) was negatively associated with OAC (vs CC): 0.53 (0.33-0.85) (p=0.008). Vegetable and more strikingly fruit consumption were negatively associated with OAC. Greater consumption of red meat, salt and tea were positively associated with OAC. Dietary modification may offer opportunities to impact upon the increasing incidence of OAC
W1876 Survival After Curative Resection for Adenocarcinoma at the GastroEsophageal Junction With Postoperative Chemoradio Therapy or Surgery Alone Steen C. Kofoed, Lene Baeksgaard, Lars Bo Svendsen We present the survival data of a 350 patients curatively resected for adenocarcinoma at the gastro-esophageal junction and postoperatively treated with chemoradio therapy according to the Intergroup 0116 protocol. The survival was compared with the survival after curative resection only. From 1992 through 2003, 137 patients underwent curative resection(group 1). In 2003, the Intergroup 0116 protocol was implemented in our institution. From 2003 through 2009, 96 patients underwent surgery only(group 2) and 117 patients received postoperative therapy(group 3). The operation was carried out as a two-phase abdominal and right chest approach for en-bloc subtotal esophagectomy. The postoperative treatment consisted of chemotherapy with fluorouracil plus leucovorin and loco-regional radiation. To calculate the disease-free survival, information regarding diagnosis for metastasis or relapse of disease was collected from the Danish Cancer Register. All deaths due to complications after surgery and patients with superficial tumours (T0N0/T1N0) were excluded from the analysis. The overall disease-free 3-year survival in group 1, 2 and 3 was 39%, 45% and 53% respectively (Log rank test:P=0.58). Figure 1 shows disease-free survival after curative resection where lymph node involvement was found in the resected specimen. The 3 year survival in the surgery only group (group 2) was 29%. Median time of survival was 16 month(CI:1220). In the postoperative therapy group, the survival was 48%. Median time of survival was 29 month(CI:11-47)(Log rank test:P=0.014). From 2003 through 2009, 64(30%) patients underwent a D1 resection (≥15 nodes) and 57(27%) patients underwent D2 resection (≥26 nodes). Our results support, that postoperative chemoradio therapy is a reasonable option in curatively resected patients with adenocarcinoma at the gastro-esophageal junction and positive node status in the resected specimen.
W1878 The Effect of Smoking and Alcohol Consumption on the Development of Oesophageal Adenocarcinoma: Comparison With Community and Reflux Oesophagitis Controls From MOSES (Midlands Oesophageal Adenocarcinoma Epidemiology Study) Sheldon C. Cooper, Sandra Prew, Laura Podmore, Nigel J. Trudgill The UK has the highest incidence of oesophageal adenocarcinoma (OAC) in the world. Smoking and alcohol consumption have typically been associated with oesophageal squamous cell carcinoma. We have undertaken the largest case-control study to date, to identify risk factors associated with the development of OAC. Incident cases of OAC from 12 hospitals in England and age-, gender-, ethnically- and geographically-matched community control subjects (CC) subjects (primary care) and reflux oesophagitis (RO) control patients (endoscopy units) were interviewed. Logistic regression generated odds ratios (OR) for smoking and alcohol consumption 1 and 10 years prior to diagnosis. 207 OAC (177 men, median (IQR) age 68 (60-75) years), 283 CC subjects (236 men, age 67 (60-74) years) and 224 RO patients (178 men, age 66 (59-75) years) were recruited. Being a current smoker (vs. never smoked) is associated with OAC: vs. CC 4.37 (2.46-7.76) (p<0.0001) and vs. RO 2.28 (1.29-4.03) (p=0.005). Previously smoking (vs. never) was also associated with OAC: vs. CC 2.32 (1.5-3.61) and vs. RO 1.9 (1.19-3.04) (p=0.007). Quantity smoked 1 year (CC 2.45 (1.45-4.15) (p=0.001) and RO 2.29 (1.22-4.31) (p=0.01)) and 10 years prior to diagnosis (CC 2.7 (1.6-4.52) (p<0.0001) and RO 1.37 (0.83-2.27) (p=0.02)) were associated with OAC. Being a drinker of alcohol was negatively associated with OAC (vs CC only) (0.63 (0.2-0.93) (p=0.033)), including increasing duration of consumption (0.51 (0.29-0.9) (p=0.019)), but not quantity. Smoking is strongly associated with the development of OAC, when compared with both community controls and reflux oesophagitis patients. There is also clear evidence of a dose-response relationship. In contrast to oesophageal squamous cell carcinoma, there was some evidence to suggest that alcohol intake is negatively associated with OAC.
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