Knockdown Of NOX5-S Research Articles

Silencer-of-Death Domain Mediates Acid-Induced Decrease in Cell Apoptosis in Barrett's Associated Esophageal Adenocarcinoma Cells.

We have shown that NADPH oxidase (NOX)5-S may mediate the acid-induced decrease in cell apoptosis. However, mechanisms of NOX5-S-dependent decrease in cell apoptosis are not fully understood. In this study, we found that silencer-of-death domain (SODD) was significantly increased in esophageal adenocarcinoma (EA) tissues, EA cell lines FLO and OE33, and a dysplastic cell line CP-B. Strong SODD immunostaining was significantly higher in low-grade dysplasia (66.7%), high-grade dysplasia (81.2%), and EA (71.2%) than in Barrett's mucosa (10.5%). Acid treatment significantly increased SODD protein and mRNA expression and promoter activity in FLO cells, an increase that was significantly decreased by the knockdown of NOX5-S and nuclear factor κB (NF-κB)1 p50 with their small interfering RNAs. Similarly, acid-induced increase of SODD mRNA was blocked by knockdown of NOX5-S and p50 in a BE cell line CP-A. Overexpression of NOX5-S significantly increased SODD protein expression in FLO cells. Moreover, overexpression of NOX5-S or p50 significantly increased the SODD promoter activity and decreased the caspase 9 activity or apoptosis. NOX5-S overexpression-induced increase in SODD promoter activity was significantly decreased by knockdown of p50. In addition, acid treatment significantly decreased the caspase 9 activity, a decrease that was significantly inhibited by knockdown of SODD. Furthermore, chromatin immunoprecipitation assay showed that NF-κB1 p50 bound to SODD genomic DNA containing a NF-κB-binding element GGGGACACCCT. This binding element was further confirmed by a gel mobility shift assay. We conclude that acid-induced increase in SODD expression and decrease in cell apoptosis may depend on the activation of NOX5-S and NF-κB1 p50 in FLO cells.

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.

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.

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.

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.

Role of Rac1 in regulation of NOX5-S function in Barrett's esophageal adenocarcinoma cells

We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H(2)O(2) production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H(2)O(2) production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca(2+)-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.

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.

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.

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.

STAT5 mediates PAF-induced NADPH oxidase NOX5-S expression in Barrett's esophageal adenocarcinoma cells

We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368-20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H(2)O(2)) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H(2)O(2) production. PAF-dependent induction of NOX5-S expression and H(2)O(2) production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA(2), and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA(2), whereas PAF-induced cPLA(2) phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H(2)O(2) production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA(2), and STAT5 in these cells.

NADPH Oxidase NOX5-S Mediates Acid-induced Cyclooxygenase-2 Expression via Activation of NF-κBin Barrett’s Esophageal Adenocarcinoma Cells

We have shown that the NADPH oxidase NOX5-S may play an important role in the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) by increasing cell proliferation and decreasing apoptosis. However, the mechanism of the acid-induced NOX5-S-mediated increase in cell proliferation is not known. We found that, in SEG1 EA cells, the acid-induced increase in prostaglandin E2 (PGE2) production was mediated by activation of cyclooxygenase-2 (COX2) but not by COX1. Acid treatment increased intracellular Ca2+, and a blockade of intracellular Ca2+ increase inhibited the acid-induced increase in COX2 expression and PGE2 production. Knockdown of NOX5-S or NF-kappaB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE2 production in SEG1 cells. Acid treatment significantly decreased IkappaBalpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappaB in vivo activation reporter plasmid, pNF-kappaB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IkappaBalpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappaB-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H2O2 production, COX2 expression, PGE2 production, and thymidine incorporation. The increase in thymidine incorporation occurring in NOX5-S-overexpressing Barrett's cells or induced by acid treatment in SEG1 EA cells was significantly decreased by COX2 inhibitors or small interfering RNA. We conclude that acid-induced COX2 expression and PGE2 production depend on an increase in cytosolic Ca2+ and sequential activation of NOX5-S and NF-kappaB in SEG1 cells. COX2-derived PGE2 production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.