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Tu1910 The BARHL2 Gene Is Silenced by Promoter Hypermethylation in Early Stage of Gastric Cancer
Tu1906
important mechanism for the differential expression of NTR1 and NTR2 in NET cells. Furthermore, we find that NTR1 contributes to NET cell tumorigenesis and may be utilized as a potential therapeutic target in NETs.
Direct Activation of MEK1 Kinase by Rho Kinase ROCK2 Mediates AcidInduced NADPH Oxidase NOX5-S Expression in Barrett's Associated Esophageal Adenocarcinoma Cells Jie Hong, Dan Li, Weibiao Cao
Regulation of Glucose Metabolism via the Portal Neural System Hiroshi Yamamoto, Tsuyoshi Yamaguchi, Sachiko Kaida, Satoshi Murata, Masaji Tani It is well known that bariatric surgery not only reduces body weight but also dramatically improves obesity related diseases including diabetes. The underlying mechanisms of improved glycemic control after surgery has not been undetermined yet. Gastrointestinal hormones including GLP-1 have been thought to play an important role of improving diabetes. Recent studies have shown that a gut-brain-liver neuronal network is a key signal of glucose metabolism. In addition, GLP-1 receptors have been shown to be within the nerve terminals of vagal afferent nerve around the portal vein. The aim of this study was to clarify the role of the portal neural system on the regulation of glucose homeostasis and food intake in the physiological condition. (Materials & Methods) Twenty male 12-weekold Wistar rats were randomly divided into two groups of 10 animals each: a denervation of whole portal vein (DV) group and a sham operation (SO) group. Body weight and food intake per day were monitored before and one week after surgery. A week after surgery, glucose tolerance tests were performed in conscious rats and glucose,GLP-17-36 and insulin levels during OGTT were assayed at 0, 10, 30, 60, 120 min The insulinogenic index and HOMA-IR and Matsuda index were calculated. (Results) There was no significant difference in food intake per day and body weight between DV and SO rats before and one week after surgery. Portal denervation significantly increased insulin release during OGTT. Insulinogenic indexes were significantly augmented in DV rats compared with those in SO rats HOMAIR was significantly increased and the Matsuda index significantly decreased in DV rats compared with those in SO rats GLP-1 levels at 0, 10, 30, 60, and 120 min during OGTT were significantly higher in DV rats compared with those in SO rats. (Conclusion) In the physiological state, loss of the portal neural system leads to decreased insulin sensitivity and increased blood glucose levels but does not affect food intake. These data indicate that an intact portal neural system is important for maintaining normal glucose metabolism.
Tu1907 Curcumin Reduces the Inflammatory Factors of Reflux Esophagitis Through Non PKC Pathway in Part in Rat Model Xiue Yan, Yingchun Wang, Kuo Zhang, Yong Liu, Jun Li, Liya Zhou
Tu1910
Background & Aims: Inflammatory factors are released in esophageal mucosa with gastroesophageal reflux disease (GERD) and may be involved in the pathogenesis of GERD. Curcumin is known to be an anti-inflammatory, antioxidant and anticancer agent. Thus the aim of this study is to investigate whether curcumin affects IL-6,TNF-α,GM-CSF and COX2 expression of reflux esophagitis in rat by WB and real-time PCR and the possible signaling pathways. Methods:(1) The first part: Thirty-two Eight-week old Sprague-Dawley rats were separated into four groups according to different doses of curcumin (10mg/kg, 20mg/kg, 50mg/kg and control). After introperitoneal administration for 3 days, operation was performed to establish the reflux esophagitis model. Ten days later, esophageal tissues were obtained by sacrificing rats. Inflammatory factors including IL-6,TNF-α,GM-CSF and COX2 and PKC subtype including Pkcαβγθεηδ were assessed by using WB and real-time PCR. (2)The second part: Experimental rats were divided into five intraperitoneal injection groups(n=36). GroupA(n=8): BIM 0.2mg/kg/d(PKC inhibitor), Group B(n=8): Curcumin 10mg/kg/d, Group C(n=8): BIM 0.2mg/kg/+ Curcumin 10mg/kg/d , Group D(n=8): Solvent control, Group E(n=4): Blank control. The process and the content to be assessed were the same as above. Results: The first part: All doses of curcumin reduced the IL-6,TNF-α,GMCSF and COX-2 levels significantly(P<0.05). All PKC subtype levels were reduced by different doses of curcumin except Pkcαθ.The second part: The inflammatory factors levels and PKC subtypes of Group C (BIM 0.2mg/kg/d+ Curcumin 10mg/kg/d ) were higher than other groups significantly except IL-6 and Pkcθ(P<0.05). Conclusions: Because of its anti-inflammatory effects, curcumin treatment may have preventive effects on reflux esophagitis in rat model through non PKC pathway in part.
The BARHL2 Gene Is Silenced by Promoter Hypermethylation in Early Stage of Gastric Cancer Yoshiyuki Watanabe, Ritsuko Oikawa, Hiroyuki Yamamoto, Fumio Itoh Aim: Although minimal invasive treatment is accepted for gastric cancer, we still do not have any appropriate risk markers to detect residual neoplasia and recurrence. We previously reported that aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of neoplasia. Our goal is to find some candidate genes as a treatment marker using genome wide DNA methylation analysis for early gastric cancer (EGC). Methods and Results: We studied Methylated CpG Island Amplification Microarray (MCAM) analysis using 12 gastric washes (each 6 before (Pre) and after (Post) endoscopic treatment in same patients). One of them is BARHL2. We examined the DNA methylation status of BARHL2 in a validation set consisting of 128 washes samples (Pre, 64: Post, 64) at EGC. We next identified functional studies about BARHL2 as a putative tumor suppressor gene. Treating GCa cells that lacked BARHL2 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Introduction of exogenous BARHL2 into silenced cells suppressed colony formation. Moreover, BARHL2 showed significantly differential methylation between before and after treatment in EGC patient (BARHL2, p<0.0001). Discussion: Our data suggest that silencing of BARHL2 occurs frequently in EGC and may play a key role in development and progression of the disease. Tu1911
Tu1908
A Novel Mechanism for Radiocontrast-Induced Pancreatic NF-KB Translocation and Injury Through Triggering Calcium and Calcineurin Activation Shunqian Jin, Abrahim I. Orabi, Tianming Le, Swati Sah, Rita Bottino, Sohail Z. Husain
Differential Expression and Tumorigenic Function of Neurotensin Receptor 1 in Neuroendocrine Tumor Cells Ji Tae Kim, Jing Li, Heidi L. Weiss, Courtney M. Townsend, B. Mark Evers
Radiocontrast (RC) is essential to many modern radiographic procedures, but RC-induced injury is a major iatrogenic problem whose mechanisms are poorly understood. We show here for the first time that exposure of the two common RCs iohexol and isovue to mouse and human pancreatic acinar cells, a scenario mimicking aspects of post-ERCP pancreatitis in vitro, triggers phospholipase C activation, the opening of inositol 1,4,5-trisphosphate receptors, the release of intracellular Ca2+, and subsequent extracellular Ca2+ entry. An aberrant peak-plateau Ca2+ signal ensues, which is independent of osmolality and is not seen in a non-specific cell type such as HEK293 cells. RC is cell impermeant, and the finding would suggest ligand binding to a G-protein-coupled receptor, but muscarinic and CCK receptors do not appear to be playing a role. Importantly, the Ca2+/calmodulin phosphatase is activated, which transduces to NF-κB nuclear translocation and cell injury. In vivo in mice, pre- or post-treatment with calcineurin inhibitors or calcineurin deficiency protects against post-ERCP pancreatitis. Using the context of the pancreas, the results establish a novel mechanism by which RC can provoke inflammatory signals and induce organ injury by triggering Ca2+ and activating calcineurin.
The incidence rates of neuroendocrine tumors (NETs), relatively rare tumors that produce and secrete a variety of active peptides and amines, have been increasing over the last decade. Neurotensin (NT), a tridecapeptide localized predominantly in the small bowel and present in some NETs, stimulates the growth of many cancers including NETs, acting mainly through the interaction with the high-affinity NT receptor 1 (NTR1) although binding to either NTR2 or NTR3 may also contribute to this effect. The purpose of our present study was to delineate the expression and function of NTRs in NETs. Here, we found that the expression of NTR1 and NTR2 was highly variable in human NET cell lines including BON (pancreatic carcinoid), QGP-1 (pancreatic somatostatinoma), NCI-H727 (bronchial carcinoid) and UMC-11 (bronchial carcinoid), whereas NTR3 and NT were expressed in all NET cells. DNA methylation contributed to the variable expression of NTR1 and NTR2 as noted by increased mRNA and protein levels after treatment with 5-aza-2'-deoxycytidine. Methylation analyses (i.e., methylation-specific PCR and bisulfite sequencing) demonstrated that the NTR1 and NTR2 promoters were methylated in NET cells. In addition, we found that knockdown of NTR1 decreased proliferation, expression levels of growth-related proteins, c-Myc and Cyclin D1, and anchorage-independent growth of BON cells. Moreover, knockdown of NTR1 suppressed BON cell adhesion and migration. Our results indicate that promoter methylation is an
S-933
AGA Abstracts
AGA Abstracts
Tu1909
Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.
important mechanism for the differential expression of NTR1 and NTR2 in NET cells. Furthermore, we find that NTR1 contributes to NET cell tumorigenesis and may be utilized as a potential therapeutic target in NETs.
Direct Activation of MEK1 Kinase by Rho Kinase ROCK2 Mediates AcidInduced NADPH Oxidase NOX5-S Expression in Barrett's Associated Esophageal Adenocarcinoma Cells Jie Hong, Dan Li, Weibiao Cao
Regulation of Glucose Metabolism via the Portal Neural System Hiroshi Yamamoto, Tsuyoshi Yamaguchi, Sachiko Kaida, Satoshi Murata, Masaji Tani It is well known that bariatric surgery not only reduces body weight but also dramatically improves obesity related diseases including diabetes. The underlying mechanisms of improved glycemic control after surgery has not been undetermined yet. Gastrointestinal hormones including GLP-1 have been thought to play an important role of improving diabetes. Recent studies have shown that a gut-brain-liver neuronal network is a key signal of glucose metabolism. In addition, GLP-1 receptors have been shown to be within the nerve terminals of vagal afferent nerve around the portal vein. The aim of this study was to clarify the role of the portal neural system on the regulation of glucose homeostasis and food intake in the physiological condition. (Materials & Methods) Twenty male 12-weekold Wistar rats were randomly divided into two groups of 10 animals each: a denervation of whole portal vein (DV) group and a sham operation (SO) group. Body weight and food intake per day were monitored before and one week after surgery. A week after surgery, glucose tolerance tests were performed in conscious rats and glucose,GLP-17-36 and insulin levels during OGTT were assayed at 0, 10, 30, 60, 120 min The insulinogenic index and HOMA-IR and Matsuda index were calculated. (Results) There was no significant difference in food intake per day and body weight between DV and SO rats before and one week after surgery. Portal denervation significantly increased insulin release during OGTT. Insulinogenic indexes were significantly augmented in DV rats compared with those in SO rats HOMAIR was significantly increased and the Matsuda index significantly decreased in DV rats compared with those in SO rats GLP-1 levels at 0, 10, 30, 60, and 120 min during OGTT were significantly higher in DV rats compared with those in SO rats. (Conclusion) In the physiological state, loss of the portal neural system leads to decreased insulin sensitivity and increased blood glucose levels but does not affect food intake. These data indicate that an intact portal neural system is important for maintaining normal glucose metabolism.
Tu1907 Curcumin Reduces the Inflammatory Factors of Reflux Esophagitis Through Non PKC Pathway in Part in Rat Model Xiue Yan, Yingchun Wang, Kuo Zhang, Yong Liu, Jun Li, Liya Zhou
Tu1910
Background & Aims: Inflammatory factors are released in esophageal mucosa with gastroesophageal reflux disease (GERD) and may be involved in the pathogenesis of GERD. Curcumin is known to be an anti-inflammatory, antioxidant and anticancer agent. Thus the aim of this study is to investigate whether curcumin affects IL-6,TNF-α,GM-CSF and COX2 expression of reflux esophagitis in rat by WB and real-time PCR and the possible signaling pathways. Methods:(1) The first part: Thirty-two Eight-week old Sprague-Dawley rats were separated into four groups according to different doses of curcumin (10mg/kg, 20mg/kg, 50mg/kg and control). After introperitoneal administration for 3 days, operation was performed to establish the reflux esophagitis model. Ten days later, esophageal tissues were obtained by sacrificing rats. Inflammatory factors including IL-6,TNF-α,GM-CSF and COX2 and PKC subtype including Pkcαβγθεηδ were assessed by using WB and real-time PCR. (2)The second part: Experimental rats were divided into five intraperitoneal injection groups(n=36). GroupA(n=8): BIM 0.2mg/kg/d(PKC inhibitor), Group B(n=8): Curcumin 10mg/kg/d, Group C(n=8): BIM 0.2mg/kg/+ Curcumin 10mg/kg/d , Group D(n=8): Solvent control, Group E(n=4): Blank control. The process and the content to be assessed were the same as above. Results: The first part: All doses of curcumin reduced the IL-6,TNF-α,GMCSF and COX-2 levels significantly(P<0.05). All PKC subtype levels were reduced by different doses of curcumin except Pkcαθ.The second part: The inflammatory factors levels and PKC subtypes of Group C (BIM 0.2mg/kg/d+ Curcumin 10mg/kg/d ) were higher than other groups significantly except IL-6 and Pkcθ(P<0.05). Conclusions: Because of its anti-inflammatory effects, curcumin treatment may have preventive effects on reflux esophagitis in rat model through non PKC pathway in part.
The BARHL2 Gene Is Silenced by Promoter Hypermethylation in Early Stage of Gastric Cancer Yoshiyuki Watanabe, Ritsuko Oikawa, Hiroyuki Yamamoto, Fumio Itoh Aim: Although minimal invasive treatment is accepted for gastric cancer, we still do not have any appropriate risk markers to detect residual neoplasia and recurrence. We previously reported that aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of neoplasia. Our goal is to find some candidate genes as a treatment marker using genome wide DNA methylation analysis for early gastric cancer (EGC). Methods and Results: We studied Methylated CpG Island Amplification Microarray (MCAM) analysis using 12 gastric washes (each 6 before (Pre) and after (Post) endoscopic treatment in same patients). One of them is BARHL2. We examined the DNA methylation status of BARHL2 in a validation set consisting of 128 washes samples (Pre, 64: Post, 64) at EGC. We next identified functional studies about BARHL2 as a putative tumor suppressor gene. Treating GCa cells that lacked BARHL2 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Introduction of exogenous BARHL2 into silenced cells suppressed colony formation. Moreover, BARHL2 showed significantly differential methylation between before and after treatment in EGC patient (BARHL2, p<0.0001). Discussion: Our data suggest that silencing of BARHL2 occurs frequently in EGC and may play a key role in development and progression of the disease. Tu1911
Tu1908
A Novel Mechanism for Radiocontrast-Induced Pancreatic NF-KB Translocation and Injury Through Triggering Calcium and Calcineurin Activation Shunqian Jin, Abrahim I. Orabi, Tianming Le, Swati Sah, Rita Bottino, Sohail Z. Husain
Differential Expression and Tumorigenic Function of Neurotensin Receptor 1 in Neuroendocrine Tumor Cells Ji Tae Kim, Jing Li, Heidi L. Weiss, Courtney M. Townsend, B. Mark Evers
Radiocontrast (RC) is essential to many modern radiographic procedures, but RC-induced injury is a major iatrogenic problem whose mechanisms are poorly understood. We show here for the first time that exposure of the two common RCs iohexol and isovue to mouse and human pancreatic acinar cells, a scenario mimicking aspects of post-ERCP pancreatitis in vitro, triggers phospholipase C activation, the opening of inositol 1,4,5-trisphosphate receptors, the release of intracellular Ca2+, and subsequent extracellular Ca2+ entry. An aberrant peak-plateau Ca2+ signal ensues, which is independent of osmolality and is not seen in a non-specific cell type such as HEK293 cells. RC is cell impermeant, and the finding would suggest ligand binding to a G-protein-coupled receptor, but muscarinic and CCK receptors do not appear to be playing a role. Importantly, the Ca2+/calmodulin phosphatase is activated, which transduces to NF-κB nuclear translocation and cell injury. In vivo in mice, pre- or post-treatment with calcineurin inhibitors or calcineurin deficiency protects against post-ERCP pancreatitis. Using the context of the pancreas, the results establish a novel mechanism by which RC can provoke inflammatory signals and induce organ injury by triggering Ca2+ and activating calcineurin.
The incidence rates of neuroendocrine tumors (NETs), relatively rare tumors that produce and secrete a variety of active peptides and amines, have been increasing over the last decade. Neurotensin (NT), a tridecapeptide localized predominantly in the small bowel and present in some NETs, stimulates the growth of many cancers including NETs, acting mainly through the interaction with the high-affinity NT receptor 1 (NTR1) although binding to either NTR2 or NTR3 may also contribute to this effect. The purpose of our present study was to delineate the expression and function of NTRs in NETs. Here, we found that the expression of NTR1 and NTR2 was highly variable in human NET cell lines including BON (pancreatic carcinoid), QGP-1 (pancreatic somatostatinoma), NCI-H727 (bronchial carcinoid) and UMC-11 (bronchial carcinoid), whereas NTR3 and NT were expressed in all NET cells. DNA methylation contributed to the variable expression of NTR1 and NTR2 as noted by increased mRNA and protein levels after treatment with 5-aza-2'-deoxycytidine. Methylation analyses (i.e., methylation-specific PCR and bisulfite sequencing) demonstrated that the NTR1 and NTR2 promoters were methylated in NET cells. In addition, we found that knockdown of NTR1 decreased proliferation, expression levels of growth-related proteins, c-Myc and Cyclin D1, and anchorage-independent growth of BON cells. Moreover, knockdown of NTR1 suppressed BON cell adhesion and migration. Our results indicate that promoter methylation is an
S-933
AGA Abstracts
AGA Abstracts
Tu1909
Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.