NADPH Oxidase NOX5-S Research Articles

Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett's Esophageal Adenocarcinoma Cells.

The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA.

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

Tu1906 Direct Activation of MEK1 Kinase by Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Barrett's Associated Esophageal Adenocarcinoma Cells

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.

Tu1671 Calcium Signaling in Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

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. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Tu1674 Cannabinoid Agonist Interfere the Human Bone Marrow-Derived Mesenchymal Stem Cell-Induced Gastric Cancer Cell Migration

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. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Tu1672 Involvement of p21-Activated Kinase 4 (PAK4) in Gastrin-Induced Migration of Gastric Cancer Cells

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. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Mo1660 FOXP3 Inhibits Cell Proliferation of Gastric Cancer Cells Through Modulation of the TGF-β/SMAD Signaling Pathway

Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood. Acid reflux may play an important role in this progression. We have previously shown that acid exposure increases H2O2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-dependent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers. In this study we examined the role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD. SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T. Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells. Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immunoprecipitation (ChIP) assay showed that SODDDNAwas detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT. Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleotide significantly reduced binding. The addition of the mutant SODD oligonucleotide had less effect on binding. We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD. Supported by NIH NIDDK R01 DK080703.

Mo1658 Acid-Induced Decrease in Cell Apoptosis Is Mediated by Activation of NADPH Oxidase NOX5-S, NF-κB and Sodd in Barrett's Esophageal Adenocarcinoma Cells

Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood. Acid reflux may play an important role in this progression. We have previously shown that acid exposure increases H2O2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-dependent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers. In this study we examined the role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD. SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T. Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells. Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immunoprecipitation (ChIP) assay showed that SODDDNAwas detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT. Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleotide significantly reduced binding. The addition of the mutant SODD oligonucleotide had less effect on binding. We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD. Supported by NIH NIDDK R01 DK080703.

Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca(2+) in FLO cells, an increase that is blocked by Ca(2+)-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca(2+) increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA.

Role of NADPH oxidase NOX5-S, NF-κB, and DNMT1 in acid-induced p16 hypermethylation in Barrett's cells

Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.

Signaling in H2O2-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have previously shown that NADPH oxidase NOX5-S generates reactive oxygen species (ROS) when Barrett's metaplastic cells are exposed to acid. Besides metaplastic cells, other H(2)O(2)-producing cells (e.g., inflammatory cells) present in BE mucosa may produce additional ROS, which may also affect metaplastic cells contributing to esophageal tumorigenesis. In this study, we investigate whether exogenous H(2)O(2) stimulates cell proliferation by increasing NOX5-S expression. Low dose (10(-13) M) of H(2)O(2) significantly increased thymidine incorporation, NOX5-S mRNA, and protein expression in a Barrett's EA cell line FLO. H(2)O(2)-induced increase in NOX5-S expression was significantly inhibited by knockdown of nuclear factor (NF)-κB1 p50 with p50 small interfering RNA (siRNA) in EA cell lines FLO and OE33. H(2)O(2) significantly increased p65 phosphorylation and the luciferase activity in FLO cells transfected with a NF-κB activation reporter plasmid pNF-κB-Luc. H(2)O(2)-induced increase in luciferase activity in FLO cells was significantly decreased by knockdown of extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase (MAPK). Overexpression of p50 and p65 remarkably increased the luciferase activity in FLO cells transfected with a NOX5-S reporter plasmid NOX5-LP. In addition, H(2)O(2)-induced thymidine incorporation in FLO cells was significantly decreased by the MAPK kinase 1/2 inhibitor 2'-amino-3'methoxyflavone (PD98059) and ERK2 siRNA but not by ERK1 siRNA. Likewise, H(2)O(2)-induced increase in NOX5-S expression was significantly decreased by ERK2 siRNA in FLO and OE33 cells. We conclude that a low dose of H(2)O(2) increases cell proliferation. H(2)O(2)-induced increase in cell proliferation may depend on sequential activation of ERK2 MAPK, NF-κB1 p50, and NOX5-S.

NADPH Oxidase NOX5-S Mediates Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

G A A b st ra ct s study we examine the role of mPGES1 in the acid-induced increase in PGE2 production and cell proliferation, and studied the role of NADPH oxidase NOX5-S in acid-induced upregulation of mPGES1 in an EA cell line FLO. RT-PCR shows that mPGES1, mPGES2 and cytosolic PGES (cPGES) are present in FLO cells. Pulsed acid treatment increased mPGES1 mRNA by 110% and mPGES2 by 10%, but did not have any effect on cPGES mRNA. Acid treatment significantly increased mPGES1 protein expression. Knockdown of mPGES1 by mPGES1 siRNA blocked acid-induced increase in PGE2 production and thymidine incorporation (an indicator of cell proliferation rate). Knockdown of NOX5-S by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a NF-κB In Vivo activation reporter plasmid pNF-κBLuc. Knockdown of NF-κB1 p50 by p50 siRNA almost abolished acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. In a chromatin immunoprecipitation assay, the promoter region of mPGES1 DNA was detectable in the immunoprecipitated chromatin sample of FLO cell lysate with a p50 antibody, indicating that p50 binds to mPGES1 promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. Supported by NIH NIDDK R01 DK080703.

NADPH Oxidase NOX5-S Mediates Acid-Induced mPGES1 Expression in Barrett's Esophageal Adenocarcinoma Cells

NADPH Oxidase NOX5-S Mediates Acid-Induced mPGES1 Expression in Barrett's Esophageal Adenocarcinoma Cells

Acid-induced p16 hypermethylation contributes to development of esophageal adenocarcinoma via activation of NADPH oxidase NOX5-S

Inactivation of tumor suppressor gene p16 may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Hypermethylation of p16 gene promoter is an important mechanism inactivating p16. However, the mechanisms of p16 hypermethylation in EA are not known. Therefore, we examined whether acid increases methylation of p16 gene promoter and whether NADPH oxidase NOX5-S mediates acid-induced p16 hypermethylation in a Barrett's cell line BAR-T and an EA cell line OE33. We found that NOX5-S was present in BAR-T and OE33 cells. Acid-induced increase in H(2)O(2) production and cell proliferation was significantly reduced by knockdown of NOX5-S. Exogenous H(2)O(2) remarkably increased p16 promoter methylation and cell proliferation. In addition, acid treatment significantly increased p16 promoter methylation and decreased p16 mRNA level. Knockdown of NOX5-S significantly increased p16 mRNA, inhibited acid-induced downregulation of p16 mRNA, and blocked acid-induced increase in p16 methylation and cell proliferation. Conversely, overexpression of NOX5-S significantly decreased p16 mRNA and increased p16 methylation and cell proliferation. In conclusion, NOX5-S is present in BAR-T cells and OE33 cells and mediates acid-induced H(2)O(2) production and cell proliferation. NOX5-S is also involved in acid-induced hypermethylation of p16 gene promoter and downregulation of p16 mRNA. It is possible that acid reflux present in BE patients may activate NOX5-S and increase production of reactive oxygen species, which in turn increase p16 promoter methylation, downregulate p16 expression, and increase cell proliferation, thereby contributing to the progression from BE to EA.

S1063 The Bile Acid Receptor TGR5 and NADPH Oxidase NOX5-S Mediate Bile Acid-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

S1063 The Bile Acid Receptor TGR5 and NADPH Oxidase NOX5-S Mediate Bile Acid-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

S1062 Acid-Induced p16 Hypermethylation Contributes to Development of Esophageal Adenocarcinoma via Activation of NADPH Oxidase NOX5-S

S1062 Acid-Induced p16 Hypermethylation Contributes to Development of Esophageal Adenocarcinoma via Activation of NADPH Oxidase NOX5-S

Bile Acid Reflux Contributes to Development of Esophageal Adenocarcinoma via Activation of Phosphatidylinositol-Specific Phospholipase Cγ2 and NADPH Oxidase NOX5-S

Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

Role of a novel bile acid receptor TGR5 in the development of oesophageal adenocarcinoma

Background and aimsMechanisms of the progression from Barrett's oesophagus to oesophageal adenocarcinoma (OA) are not fully understood. Bile acids may have an important role in this progression. This study aimed...

578 MEK1 Kinase and Rho Kinase ROCK2 Mediate Acid-Induced NOX5-S Expression and H 2O 2 Production in Barrett's Esophageal Adenocarcinoma Cells

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 therefore investigated the role of NADPH oxidases in acid-induced changes in Barrett's EA cell line FLO. RT-PCR and 5'-RACE showed that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression was significantly greater in EA cell lines (FLO, and OE33) than in esophageal squamous epithelial cell line HET-1A. The levels of NOX5 mRNA were also markedly increased in human Barrett's mucosal biopsies with moderate dysplasia and in EA tissues, when compared with normal esophageal mucosa or BE mucosa. Immunohistochemical studies showed that NOX5-S was present in the cytosol of FLO cells. This result was further confirmed by transfection of FLO cells with GFP-tagged NOX5-S plasmid. In FLOEA cells, knockdown of NOX5-S by NOX5 siRNA significantly decreased cell proliferation at basal condition and inhibited acid-induced increase in cell proliferation, suggesting that NOX5-S may play a role in growth and proliferation of EA cells. Acid treatment significantly increased H2O2 production in both FLO cells and BE mucosa. Acid treatment significantly increased NOX5-S mRNA expression and H2O2 production, and knockdown of NOX5-S with NOX5 siRNA abolished acid-induced H2O2 production in FLO cells, indicating that NOX5-S mediates acid-induced H2O2 production. Acid-induced NOX5-S expression and H2O2 production were significantly decreased by knockdown of Rho kinase ROCK2 and MEK1 kinase with their siRNAs. Overexpression of constitutively active ROCK2 Rho kinase and constitutively active MEK1 kinase significantly increased NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased Rho kinase activity, an increase which was not affected by knockdown of MEK1 kinase. Overexpression of constitutively active ROCK2 significantly increased MEK1 phosphorylation. These data suggest that acidinduced NOX5-S expression depends on sequential activation of Rho kinase ROCK2 and MEK1 kinase. In conclusion, in EA cells acid induces H2O2 production by activation of NADPH oxidase NOX5-S and causes upregulation of NOX5-S expression through sequential activation of Rho kinase ROCK2 and MEK1 kinase. In these cells NOX5-S contributes to increased cell proliferation. Supported by NIH NIDDK R21 DK073327-01.

582 Manometry and Impedance Characteristics of the Esophagus in Patients with Early Stage of Idiopathic Parkinson's Disease

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 therefore investigated the role of NADPH oxidases in acid-induced changes in Barrett's EA cell line FLO. RT-PCR and 5'-RACE showed that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression was significantly greater in EA cell lines (FLO, and OE33) than in esophageal squamous epithelial cell line HET-1A. The levels of NOX5 mRNA were also markedly increased in human Barrett's mucosal biopsies with moderate dysplasia and in EA tissues, when compared with normal esophageal mucosa or BE mucosa. Immunohistochemical studies showed that NOX5-S was present in the cytosol of FLO cells. This result was further confirmed by transfection of FLO cells with GFP-tagged NOX5-S plasmid. In FLOEA cells, knockdown of NOX5-S by NOX5 siRNA significantly decreased cell proliferation at basal condition and inhibited acid-induced increase in cell proliferation, suggesting that NOX5-S may play a role in growth and proliferation of EA cells. Acid treatment significantly increased H2O2 production in both FLO cells and BE mucosa. Acid treatment significantly increased NOX5-S mRNA expression and H2O2 production, and knockdown of NOX5-S with NOX5 siRNA abolished acid-induced H2O2 production in FLO cells, indicating that NOX5-S mediates acid-induced H2O2 production. Acid-induced NOX5-S expression and H2O2 production were significantly decreased by knockdown of Rho kinase ROCK2 and MEK1 kinase with their siRNAs. Overexpression of constitutively active ROCK2 Rho kinase and constitutively active MEK1 kinase significantly increased NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased Rho kinase activity, an increase which was not affected by knockdown of MEK1 kinase. Overexpression of constitutively active ROCK2 significantly increased MEK1 phosphorylation. These data suggest that acidinduced NOX5-S expression depends on sequential activation of Rho kinase ROCK2 and MEK1 kinase. In conclusion, in EA cells acid induces H2O2 production by activation of NADPH oxidase NOX5-S and causes upregulation of NOX5-S expression through sequential activation of Rho kinase ROCK2 and MEK1 kinase. In these cells NOX5-S contributes to increased cell proliferation. Supported by NIH NIDDK R21 DK073327-01.