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Bile Acid-Induced APE-1 Mediates Stat3 Activation in Barrett's and Esophageal Adenocarcinoma Cells
Mo1029
INCREASED FREQUENCY OF AIR SWALLOWS DURING 24 HOURS IN NERD PATIENTS NON RESPONDER TO PROTON PUMP INHIBITORS Mentore Ribolsi, Dario Biasutto, Paola Balestrieri, Michele Cicala
BILE ACID-INDUCED APE-1 MEDIATES STAT3 ACTIVATION IN BARRETT'S AND ESOPHAGEAL ADENOCARCINOMA CELLS Ajaz A. Bhat, Heng Lu, Mohammed Soutto, Zheng Chen, Dunfa Peng, Jun Hong, Abbes Belkhiri, Wael M. El-Rifai
Background: An increased overall number of refluxes and enhanced sensitivity to mixed and proximal reflux episodes has been claimed to play a role in the symptoms resistance to PPIs NERD patients. Moreover, it has been shown that non-responder patients swallow more air at mealtime than responder patients. Aim: To explore the relationship between swallowing, reflux pattern and PPI response in NERD patients. Methods: 24-hour MII-pH was performed, following a 2-week washout from PPIs, in 85 NERD non-responders to double dose of PPIs and in 102 NERD responders, presenting with typical symptoms, following esophageal manometry. All patients filled out a questionnaire with symptom score. Acid exposure time (AET) and Symptom Association Probability (SAP) index were calculated. Patients, both responders and non-responders, were classified according to AET and SAP. For each patient, the number of swallows and of swallows containing air was calculated during the entire length of MII-pH study, excluding meal periods. Results: Non-responder patients AET+ and/or SAP+ were characterized, compared to non-responders AET- and SAPand to responder patients, by a higher overall number of reflux episodes. The proportion of weakly and mixed reflux episodes did not differ between groups. During MII-pH, both non-responders AET+ and/or SAP+ and AET- and SAP- patients showed a significantly higher proportion of swallows than responder patients. Moreover, both non-responders AET+ and/or SAP+ and AET- and SAP- patients showed a significantly higher proportion of air swallows than that of responder patients. Conclusions: Non-responder NERD patients are characterized by an increased frequency of air swallows, during 24 hours, compared to NERD responders patients. The increased air volume swallowed, together with a higher mucosal sensitivity to esophageal distention, may lead to symptom persistence despite acid suppression in these patients. Table 1
Background: Barrett's esophagus (BE) develops as a consequence of chronic gastroesophageal reflux disease (GERD), where gastric juice containing acid and bile salts abnormally refluxate into the distal region of esophagus generating high levels of oxidative stress. BE is a premalignant metaplastic columnar epithelial lesion that can progress to EAC. APE-1/Ref-1 is a dual function protein with DNA repair and redox capabilities. STAT3 signaling has been implicated in regulating cell survival, angiogenesis, and metastasis. In this study we investigated the role of APE-1 in regulating STAT3. Methodology: Non-neoplastic Barrett's (CPA, BAR10T and CPB) and esophageal adenocarcinoma (OE33, FLO-1) cells were exposed to acidic bile salts (pH4.0 and 5.5, 100 and 200uM) for 20 minutes. The mRNA and protein expression levels of APE-1, STAT3 and target genes were evaluated by real time PCR and immunoblotting. STAT3 transcription activity was studied by luciferase reporter assay. Immunofluorescence and cytoplasmic-nuclear fractionation were used to examine localization of APE-1 and STAT3 after exposure to acidic bile salts. Results: We detected low to absent levels of APE-1 in non-neoplastic BE cells, whereas EAC expressed constitutively high levels of APE-1. Exposure of non-neoplastic Barrett's cells to acidic bile salts led to transient non-transcriptional induction of protein expression of APE-1. The increase in APE-1 protein expression correlated with an increase in STAT3 phosphorylation and nuclear localization. Using a STAT3 luciferase reporter assay, we also found that exposure to acidic bile salts increases the STAT3 transcription activity. Using APE-1 knockdown models, we found a significant decrease in activation of STAT3 at protein and transcription activity levels, following exposure to acidic bile salts, as compared to control cells. Using qPCR, we also detected downregulation of survivin, a STAT3 target gene, in APE-1 knockdown cells. Conversely, transient overexpression of APE1 by adenoviral infection induced a marked increase in the phosphorylation of STAT3 (Y705), nuclear localization, transcription activity, and expression of survivin. Of note, immunofluorescence and cytoplasmic-nuclear fractionation revealed that exposure to acidic bile salts increases the nuclear co-localization of both APE-1 and STAT3. Pharmacological inhibition of redox activity of APE-1 using E3330 resulted in a decrease in STAT3 activation. Conclusion: The redox activity of APE-1 mediates activation of STAT3 as a survival mechanism in response to acidic bile salts and oxidative stress. Constitutive activation of APE-1 in EAC may present a therapeutic opportunity to targeting redox vulnerabilities in cancer cells. Further studies are underway to delineate the signaling axis by which APE-1 mediates STAT3 activation under acidic bile salt exposure.
Mo1030 IN NON-NEOPLASTIC BARRETT'S EPITHELIAL CELLS, ACID AND BILE SALTS INDUCE EPITHELIAL MESENCHYMAL TRANSITION (EMT) THROUGH A HIF-MEDIATED REDUCTION IN MICRORNA-200A AND -200B EXPRESSION Tianhong Su, Xi Zhang, Chunhua Yu, Xiaofang Huo, Thai Pham, Kerry B. Dunbar, Edaire Cheng, Manisha Bajpai, Kiron M. Das, David H. Wang, Stuart J. Spechler, Rhonda F. Souza, Qiuyang D. Zhang
Mo1028 GERDQ IS HIGHLY PREDICTIVE OF EROSIVE ESOPHAGITIS IN OBESE PATIENTS EVALUATED FOR BARIATRIC SURGERY Ala I. Sharara, Hussein H. Rimmani, Rani Shayto, Carla Abou Fadel, Hanaa Aridi, Amr I. Al Abbas, Chadi Mamlouk, Yasser H. Shaib, Bassem Safadi, Ramzi Alami
Background: Epithelial-mesenchymal transition (EMT), which might be involved in Barrett's pathogenesis, is induced by transcription factors like ZEB1 that repress epithelial genes (e.g. E-cadherin) and activate mesenchymal genes (e.g. vimentin, MMPs). Members of the microRNA (miR)-200 family can prevent EMT by inhibiting ZEB1 expression in epithelial cells. In earlier studies, we reported that acidic bile salts increased the activity of hypoxia inducible factors (HIFs, which might mediate EMT) in esophageal squamous cells, and induced features of EMT in Barrett's cells. In this study, we explored whether acidic bile salts induce EMT in Barrett's cells by activating HIFs and suppressing miR-200 a&b. Methods: Non-neoplastic Barrett's epithelial cell lines (BAR-T and BAR-10T) were treated with: 1) acidic media (pH 5.5) containing conjugated bile acids (400 µM) for 15 or 30 minutes, 2) miR-200 a&b inhibitors or 3) transfections with HIF-1α and HIF-2α cDNAs or shRNAs, or a miR-200 promoter construct with or without a point mutation in the HIF binding site. We evaluated: 1) miR-200 a&b expression, 2) miR-200 promoter activity, and 3) ZEB1, Ecadherin, MMP2, and vimentin expression. miR-200 expression also was evaluated in BART cells treated with acidic bile salts for 4 weeks (BEC-4W) and 20 weeks (BEC-20W), and in a rat model of Barrett's metaplasia induced by esophago-duodenostomy. Results: In BART and BAR-10T, a 30-minute exposure to acidic bile salts significantly decreased miR-200 a&b expression and miR-200 promoter activity. In BAR-T, treatment with miR-200 inhibitors decreased E-cadherin and increased ZEB1, MMP2, and vimentin expression. In BAR-T, a 15-minute exposure to acidic bile salts increased nuclear protein levels of HIF-1α and HIF2α. In BAR-T and BAR-10T, HIF cDNA transfection significantly decreased miR-200 promoter activity, and HIF knockdown by shRNA prevented the decrease in miR-200 promoter activity induced by acidic bile salts. However, a point mutation in the HIF binding site did not prevent the decrease in miR-200 promoter activity induced by acidic bile salts in either cell line. Compared to BAR-T cells, BEC-4W and BEC-20W cells had significantly reduced baseline expression of miR-200 a&b. In esophageal columnar metaplasia that developed in rats, miR-200 a&b expression was significantly lower at 12 and 16 weeks than at 6 weeks after esophago-duodenostomy. Conclusions: In Barrett's epithelial cells, acidic bile salts decrease expression of miR-200 a&b and increase expression of EMT markers. Acidic bile salts also increase nuclear levels of HIFs, leading to suppression of miR-200 promoter activity, an effect that is not mediated by direct binding of HIFs to this promoter. These findings elucidate molecular mechanisms whereby refluxed acid and bile can induce EMT via HIF-mediated suppression of miR-200 a&b in Barrett's esophagus.
Background: Gastroesophageal reflux disease (GERD) is common in obese individuals. Studies investigating validated GERD questionnaires and other clinical factors at identifying erosive esophagitis (EE) in this population are limited. Aims: To prospectively evaluate the prevalence of GERD in obese patients considered for bariatric surgery and to identify risk and predictive factors for erosive esophagitis (EE). Methods: Consecutive patients undergoing routine esophagogastroduodenoscopy (EGD) prior to bariatric surgery were enrolled after informed consent. Exclusion criteria included age<18 and prior esophageal or gastric surgery. Patients completed 2 validated questionnaires, the GERDQ and the Nocturnal GERD Symptom Severity and Impact Questionnaire (N-GSSIQ) prior to endoscopy. Demographic data including gender, age, height, weight, BMI, waist circumference (WC), and use of PPI or H2 antagonists were collected. Endoscopic data included presence or absence of EE, grade of EE, presence and size of hiatal hernia (HH), and gastroesophageal flap valve endoscopic grading (Hill grade). Data pertaining to type of bariatric surgery planned before and after EGD findings was retained from the surgical clinic visit records. Results: 130 patients have been recruited to date (61M:69F). The mean age was 38.5±12.2 and mean BMI was 40.4±5.6, nearly equally divided between BMI ≤ or >40. HH was present in 15.4% of patients. Twelve patients on PPI were excluded from the EE analysis because of absence of erosive disease. EE was documented in 40 of 97 patients on no PPI (41.2%). On multivariate analysis, presence of HH and a high GERD score were significantly associated with EE (p=0.029 and 0.001 respectively). Patients with high probability GERDQ scores had a higher prevalence of EE vs. low probability GERDQ scorers (52% vs. 17%, p=0.001). EE was more common in males vs. females (70% vs. 30%, p=0.042) and in patients with HH vs. those without (53.3% vs. 42.7%, p=0.043). BMI (≤40 vs. >40) and WC were not associated with EE. Logistic regression showed that GERDQ scores of ≥8 was the only independent risk factor for EE (OR 3.52, 95% CI 1.67-7.458). A GERDQ score of ≥8 had a PPV of 62.5% at identifying EE while a low score of <8 had a NPV of 80.5% to exclude EE. Total nocturnal and morning-impact-of-nocturnal scores on the N-GSSIQ questionnaire were not significantly different between EE and non-EE patients. Six patients (4.6%) had the initial choice of surgery altered by the referring surgeon because of endoscopic findings (HH and EE). Conclusion: Erosive GERD is highly prevalent in this cohort of obese patients considered for bariatric surgery. A high GERDQ score and presence of HH correlated with the presence of EE. Waist circumference and BMI were not associated with EE. A low GERDQ score was strongly predictive of the absence of EE in this patient population.
S-661
AGA Abstracts
AGA Abstracts
Mo1027
INCREASED FREQUENCY OF AIR SWALLOWS DURING 24 HOURS IN NERD PATIENTS NON RESPONDER TO PROTON PUMP INHIBITORS Mentore Ribolsi, Dario Biasutto, Paola Balestrieri, Michele Cicala
BILE ACID-INDUCED APE-1 MEDIATES STAT3 ACTIVATION IN BARRETT'S AND ESOPHAGEAL ADENOCARCINOMA CELLS Ajaz A. Bhat, Heng Lu, Mohammed Soutto, Zheng Chen, Dunfa Peng, Jun Hong, Abbes Belkhiri, Wael M. El-Rifai
Background: An increased overall number of refluxes and enhanced sensitivity to mixed and proximal reflux episodes has been claimed to play a role in the symptoms resistance to PPIs NERD patients. Moreover, it has been shown that non-responder patients swallow more air at mealtime than responder patients. Aim: To explore the relationship between swallowing, reflux pattern and PPI response in NERD patients. Methods: 24-hour MII-pH was performed, following a 2-week washout from PPIs, in 85 NERD non-responders to double dose of PPIs and in 102 NERD responders, presenting with typical symptoms, following esophageal manometry. All patients filled out a questionnaire with symptom score. Acid exposure time (AET) and Symptom Association Probability (SAP) index were calculated. Patients, both responders and non-responders, were classified according to AET and SAP. For each patient, the number of swallows and of swallows containing air was calculated during the entire length of MII-pH study, excluding meal periods. Results: Non-responder patients AET+ and/or SAP+ were characterized, compared to non-responders AET- and SAPand to responder patients, by a higher overall number of reflux episodes. The proportion of weakly and mixed reflux episodes did not differ between groups. During MII-pH, both non-responders AET+ and/or SAP+ and AET- and SAP- patients showed a significantly higher proportion of swallows than responder patients. Moreover, both non-responders AET+ and/or SAP+ and AET- and SAP- patients showed a significantly higher proportion of air swallows than that of responder patients. Conclusions: Non-responder NERD patients are characterized by an increased frequency of air swallows, during 24 hours, compared to NERD responders patients. The increased air volume swallowed, together with a higher mucosal sensitivity to esophageal distention, may lead to symptom persistence despite acid suppression in these patients. Table 1
Background: Barrett's esophagus (BE) develops as a consequence of chronic gastroesophageal reflux disease (GERD), where gastric juice containing acid and bile salts abnormally refluxate into the distal region of esophagus generating high levels of oxidative stress. BE is a premalignant metaplastic columnar epithelial lesion that can progress to EAC. APE-1/Ref-1 is a dual function protein with DNA repair and redox capabilities. STAT3 signaling has been implicated in regulating cell survival, angiogenesis, and metastasis. In this study we investigated the role of APE-1 in regulating STAT3. Methodology: Non-neoplastic Barrett's (CPA, BAR10T and CPB) and esophageal adenocarcinoma (OE33, FLO-1) cells were exposed to acidic bile salts (pH4.0 and 5.5, 100 and 200uM) for 20 minutes. The mRNA and protein expression levels of APE-1, STAT3 and target genes were evaluated by real time PCR and immunoblotting. STAT3 transcription activity was studied by luciferase reporter assay. Immunofluorescence and cytoplasmic-nuclear fractionation were used to examine localization of APE-1 and STAT3 after exposure to acidic bile salts. Results: We detected low to absent levels of APE-1 in non-neoplastic BE cells, whereas EAC expressed constitutively high levels of APE-1. Exposure of non-neoplastic Barrett's cells to acidic bile salts led to transient non-transcriptional induction of protein expression of APE-1. The increase in APE-1 protein expression correlated with an increase in STAT3 phosphorylation and nuclear localization. Using a STAT3 luciferase reporter assay, we also found that exposure to acidic bile salts increases the STAT3 transcription activity. Using APE-1 knockdown models, we found a significant decrease in activation of STAT3 at protein and transcription activity levels, following exposure to acidic bile salts, as compared to control cells. Using qPCR, we also detected downregulation of survivin, a STAT3 target gene, in APE-1 knockdown cells. Conversely, transient overexpression of APE1 by adenoviral infection induced a marked increase in the phosphorylation of STAT3 (Y705), nuclear localization, transcription activity, and expression of survivin. Of note, immunofluorescence and cytoplasmic-nuclear fractionation revealed that exposure to acidic bile salts increases the nuclear co-localization of both APE-1 and STAT3. Pharmacological inhibition of redox activity of APE-1 using E3330 resulted in a decrease in STAT3 activation. Conclusion: The redox activity of APE-1 mediates activation of STAT3 as a survival mechanism in response to acidic bile salts and oxidative stress. Constitutive activation of APE-1 in EAC may present a therapeutic opportunity to targeting redox vulnerabilities in cancer cells. Further studies are underway to delineate the signaling axis by which APE-1 mediates STAT3 activation under acidic bile salt exposure.
Mo1030 IN NON-NEOPLASTIC BARRETT'S EPITHELIAL CELLS, ACID AND BILE SALTS INDUCE EPITHELIAL MESENCHYMAL TRANSITION (EMT) THROUGH A HIF-MEDIATED REDUCTION IN MICRORNA-200A AND -200B EXPRESSION Tianhong Su, Xi Zhang, Chunhua Yu, Xiaofang Huo, Thai Pham, Kerry B. Dunbar, Edaire Cheng, Manisha Bajpai, Kiron M. Das, David H. Wang, Stuart J. Spechler, Rhonda F. Souza, Qiuyang D. Zhang
Mo1028 GERDQ IS HIGHLY PREDICTIVE OF EROSIVE ESOPHAGITIS IN OBESE PATIENTS EVALUATED FOR BARIATRIC SURGERY Ala I. Sharara, Hussein H. Rimmani, Rani Shayto, Carla Abou Fadel, Hanaa Aridi, Amr I. Al Abbas, Chadi Mamlouk, Yasser H. Shaib, Bassem Safadi, Ramzi Alami
Background: Epithelial-mesenchymal transition (EMT), which might be involved in Barrett's pathogenesis, is induced by transcription factors like ZEB1 that repress epithelial genes (e.g. E-cadherin) and activate mesenchymal genes (e.g. vimentin, MMPs). Members of the microRNA (miR)-200 family can prevent EMT by inhibiting ZEB1 expression in epithelial cells. In earlier studies, we reported that acidic bile salts increased the activity of hypoxia inducible factors (HIFs, which might mediate EMT) in esophageal squamous cells, and induced features of EMT in Barrett's cells. In this study, we explored whether acidic bile salts induce EMT in Barrett's cells by activating HIFs and suppressing miR-200 a&b. Methods: Non-neoplastic Barrett's epithelial cell lines (BAR-T and BAR-10T) were treated with: 1) acidic media (pH 5.5) containing conjugated bile acids (400 µM) for 15 or 30 minutes, 2) miR-200 a&b inhibitors or 3) transfections with HIF-1α and HIF-2α cDNAs or shRNAs, or a miR-200 promoter construct with or without a point mutation in the HIF binding site. We evaluated: 1) miR-200 a&b expression, 2) miR-200 promoter activity, and 3) ZEB1, Ecadherin, MMP2, and vimentin expression. miR-200 expression also was evaluated in BART cells treated with acidic bile salts for 4 weeks (BEC-4W) and 20 weeks (BEC-20W), and in a rat model of Barrett's metaplasia induced by esophago-duodenostomy. Results: In BART and BAR-10T, a 30-minute exposure to acidic bile salts significantly decreased miR-200 a&b expression and miR-200 promoter activity. In BAR-T, treatment with miR-200 inhibitors decreased E-cadherin and increased ZEB1, MMP2, and vimentin expression. In BAR-T, a 15-minute exposure to acidic bile salts increased nuclear protein levels of HIF-1α and HIF2α. In BAR-T and BAR-10T, HIF cDNA transfection significantly decreased miR-200 promoter activity, and HIF knockdown by shRNA prevented the decrease in miR-200 promoter activity induced by acidic bile salts. However, a point mutation in the HIF binding site did not prevent the decrease in miR-200 promoter activity induced by acidic bile salts in either cell line. Compared to BAR-T cells, BEC-4W and BEC-20W cells had significantly reduced baseline expression of miR-200 a&b. In esophageal columnar metaplasia that developed in rats, miR-200 a&b expression was significantly lower at 12 and 16 weeks than at 6 weeks after esophago-duodenostomy. Conclusions: In Barrett's epithelial cells, acidic bile salts decrease expression of miR-200 a&b and increase expression of EMT markers. Acidic bile salts also increase nuclear levels of HIFs, leading to suppression of miR-200 promoter activity, an effect that is not mediated by direct binding of HIFs to this promoter. These findings elucidate molecular mechanisms whereby refluxed acid and bile can induce EMT via HIF-mediated suppression of miR-200 a&b in Barrett's esophagus.
Background: Gastroesophageal reflux disease (GERD) is common in obese individuals. Studies investigating validated GERD questionnaires and other clinical factors at identifying erosive esophagitis (EE) in this population are limited. Aims: To prospectively evaluate the prevalence of GERD in obese patients considered for bariatric surgery and to identify risk and predictive factors for erosive esophagitis (EE). Methods: Consecutive patients undergoing routine esophagogastroduodenoscopy (EGD) prior to bariatric surgery were enrolled after informed consent. Exclusion criteria included age<18 and prior esophageal or gastric surgery. Patients completed 2 validated questionnaires, the GERDQ and the Nocturnal GERD Symptom Severity and Impact Questionnaire (N-GSSIQ) prior to endoscopy. Demographic data including gender, age, height, weight, BMI, waist circumference (WC), and use of PPI or H2 antagonists were collected. Endoscopic data included presence or absence of EE, grade of EE, presence and size of hiatal hernia (HH), and gastroesophageal flap valve endoscopic grading (Hill grade). Data pertaining to type of bariatric surgery planned before and after EGD findings was retained from the surgical clinic visit records. Results: 130 patients have been recruited to date (61M:69F). The mean age was 38.5±12.2 and mean BMI was 40.4±5.6, nearly equally divided between BMI ≤ or >40. HH was present in 15.4% of patients. Twelve patients on PPI were excluded from the EE analysis because of absence of erosive disease. EE was documented in 40 of 97 patients on no PPI (41.2%). On multivariate analysis, presence of HH and a high GERD score were significantly associated with EE (p=0.029 and 0.001 respectively). Patients with high probability GERDQ scores had a higher prevalence of EE vs. low probability GERDQ scorers (52% vs. 17%, p=0.001). EE was more common in males vs. females (70% vs. 30%, p=0.042) and in patients with HH vs. those without (53.3% vs. 42.7%, p=0.043). BMI (≤40 vs. >40) and WC were not associated with EE. Logistic regression showed that GERDQ scores of ≥8 was the only independent risk factor for EE (OR 3.52, 95% CI 1.67-7.458). A GERDQ score of ≥8 had a PPV of 62.5% at identifying EE while a low score of <8 had a NPV of 80.5% to exclude EE. Total nocturnal and morning-impact-of-nocturnal scores on the N-GSSIQ questionnaire were not significantly different between EE and non-EE patients. Six patients (4.6%) had the initial choice of surgery altered by the referring surgeon because of endoscopic findings (HH and EE). Conclusion: Erosive GERD is highly prevalent in this cohort of obese patients considered for bariatric surgery. A high GERDQ score and presence of HH correlated with the presence of EE. Waist circumference and BMI were not associated with EE. A low GERDQ score was strongly predictive of the absence of EE in this patient population.
S-661
AGA Abstracts
AGA Abstracts
Mo1027