Effect of Proton Pump Inhibitor Therapy on NOX5, mPGES1 and iNOS expression in Barrett’s Esophagus

Acid reflux may contribute to the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA). However, it is not clear whether the molecular changes present in BE patients are reversible after proton pump inhibitor (PPI) treatment. In this study we examined whether PPI treatment affects NOX5, microsomal prostaglandin E synthase (mPGES)-1 and inducible nitric oxide synthase (iNOS) expression. We found that NADPH oxidase 5 (NOX5), mPGES-1 and iNOS were significantly increased in BE mucosa. One-month PPI treatment significantly decreased NOX5, mPGES1 and iNOS. In BAR-T cells, NOX5 mRNA and p16 promoter methylation increased after pulsed acid treatment in a time-dependent manner. Four or eight-week-acid induced increase in NOX5 mRNA, NOX5 protein and p16 methylation may be reversible. Twelve-week acid treatment also significantly increased NOX5, mPGES1 and iNOS mRNA expression. However, twelve-week-acid-induced changes only partially restored or did not recover at all after the cells were cultured at pH 7.2 for 8 weeks. We conclude that NOX5, mPGES1 and iNOS may be reversible after PPI treatment. Short-term acid-induced increase in NOX5 expression and p16 methylation might be reversible, whereas long-term acid-induced changes only partially recovered 8 weeks after removal of acid treatment.

isoforms: a cytosolic (cPGES) and two microsomal (mPGES) isomerases [12][13][14] . Microsomal PGES1 has been reported to be increased in an animal model of BE 15 and in human EA 16 . We have also reported that mPGES1 is identifiable in FLO-1 EA cells and that mPGES1 mRNA and protein levels are significantly enhanced in response to pulsed acid treatment in FLO-1 cells 7 . Therefore, mPGES1 may also contribute to the development of EA. It is not known whether PPI treatment may reverse mPGES1.
It is known that nitric oxide (NO) is involved in angiogenesis, apoptosis, gene expression, and DNA damage and may be important in carcinogenesis and tumor progression in the gastrointestinal tract 17,18 . NO may produce peroxynitrite and N 2 O 3 when it reacts with the superoxide radical and oxygen, respectively 19 . Three isoforms of NO synthase have been identified: endothelial NO synthase (NOS), neuronal NOS and inducible NOS. Large amount of NO may be generated by inducible NOS during inflammation and may in part mediate the progression from BE to EA 20,21 .
Our aim in this study is to examine NOX5, mPGES1 and iNOS in BE and to investigate whether PPI treatment reverses these genes. We found that NOX5, mPGES1 and iNOS were significantly increased in BE mucosa. PPI treatment for one month significantly decreased all three genes.

Results
NADPH oxidases in BE mucosa. Eight male BE patients aged from 58 to 75 (average 67.5 ± 2.3) were enrolled in this study. The duration of the disease was 3-17 years (11.2 ± 2.1 years). The length of the BE was 2-11 cm (4.6 ± 1 cm). BE patients were asked to discontinue PPI for one month and then the first biopsy was obtained. After the first biopsy, the treatment with proton pump inhibitor (PPI) was started twice a day for one month. At the end of this one-month period of PPI treatment, biopsies were repeated. Figure 1 showed that NOX5 mRNA was significantly increased in BE mucosa, when compared with normal esophageal mucosa. Dual oxidase 1 (DUOX1) and DUOX2 mRNAs did not have significant changes between BE mucosa and normal esophageal mucosa. PPI treatment for a month significantly decreased NOX5 mRNA in BE mucosa. However, PPI did not have any effect on NOX5 mRNA in normal esophageal mucosa ( Fig. 2A). The data suggest that the increased NOX5 mRNA may be reversible in BE mucosa after PPI treatment. Effect of PPI treatment on the expression of NADPH oxidases. NOX5 mRNA was significantly increased, when compared with normal esophageal mucosa. DUOX1 and DUOX2 mRNAs did not have significant changes between BE mucosa and normal esophageal mucosa. PPI treatment for a month significantly decreased NOX5 mRNA. The data suggest that the increased NOX5 mRNA may be reversible in BE mucosa after PPI treatment. N = 5; ANOVA *P < 0.05, compared normal, **P < 0.05, compared with BE without PPI www.nature.com/scientificreports www.nature.com/scientificreports/ Microsomal PGE synthase 1 in BE mucosa. We have previously reported that mPGES1 is identifiable in FLO-1 EA cells and is increased in response to pulsed acid treatment 7 . Figure 3A showed that mPGES1 mRNA was significantly increased in BE mucosa, an increase which was significantly attenuated by PPI treatment, suggesting that mPGES1 may be reversible in BE mucosa after PPI treatment. PPI did not have any effect on mPGES1 in normal esophageal mucosa (Fig. 2B).
Inducible nitric oxide synthase in BE mucosa. Inducible NOS expression has been shown to be gradually upregulated in the progression from BE to EA, but not in normal esophageal/gastric mucosa 20,21 . We found that inducible NOS was significantly increased in the BE mucosa, an increase which was significantly reduced by PPI treatment (Fig. 3B), suggesting that the overexpression of iNOS might be reversible in BE mucosa after PPI treatment. PPI did not have any effect on iNOS in normal esophageal mucosa (Fig. 2C).

Effect of omeprazole on NOX5, mPGES1 and iNOS expression in Barrett's EA cells. Barrett's
EA cell line FLO-1 was treated with omeprazole 1μM for 24 hours and then mRNAs were measured by real-time PCR. Figure 4 showed that omeprazole had no effect on NOX5, mPGES1 and iNOS mRNA expression.
Effect of long-term acid treatment on NOX5-S expression, p16 promoter methylation, mPGES1 and iNOS expression. We have previously shown that NADPH oxidase NOX5-S exists in FLO-1 EA cells and that it is upregulated in Barrett's mucosa with high-grade dysplasia and responsible for the acid-induced ROS production 4 .
To examine whether the overexpression of NOX5-S is reversible, BAR-T cells were first treated with acidic culture medium (pH 4.0) for 5 min, three times a day for 2, 4, 8 and 12 weeks. Then BAR-T cells were cultured at normal culture medium (pH 7.2) for additional 2, 4 and 8 weeks after acid treatment for 2, 4, 8 and 12 weeks, respectively. Figure 5A showed that NOX5 mRNA increased after pulsed acid treatment in a time-dependent manner. Figure 5B and 5c showed that the increase in NOX5 mRNA induced by pulsed acid treatment for 4 and 8 weeks almost recovered after cells were cultured in normal culture medium for 8 weeks. However, 12-week-acid-induced increase in NOX5 mRNA only partially restored after the cells were cultured in normal culture medium for 8 weeks (Fig. 5D). Similarly, pulsed acid treatment for 4 and 12 weeks significantly increased NOX5-S protein levels. The increase in NOX5-S protein induced by pulsed acid treatment for 4 weeks almost recovered after the cells were cultured in normal culture medium for 8 weeks. However, 12-week-acid-induced increase in NOX5 protein only partially restored after the cells were cultured in normal culture medium for 8 weeks (Fig. 6). These data suggest that, if BAR-T cells exposed to pulsed acid treatment for a longer period, the cells would take a much longer time to recover.
Similarly, p16 promoter methylation increased after pulsed acid treatment in a time-dependent manner (Fig. 7A). This increase in p16 promoter methylation induced by pulsed acid treatment for 4 and 8 weeks almost recovered after the cells were cultured in normal culture medium for 8 weeks (Fig. 7B,C). However, 12-week-acid-induced increase in p16 promoter methylation did not restore after the cells were cultured in normal culture medium for 8 weeks (Fig. 7D). In addition, twelve-week acid treatment significantly increased mPGES1 and iNOS mRNA expressions (Fig. 8). These increases did not restore after the cells were cultured in normal culture medium for 8 weeks. These data suggest that long-term acid treatment may cause irreversible gene changes. Figure 3. Expression of mPGES1 and iNOS. (A) mPGES1 mRNA was significantly increased in BE mucosa, an increase which was significantly decreased by PPI treatment, suggesting that mPGES1 may be reversible in BE mucosa by PPI treatment. (B) Inducible NOS was significantly increased in the BE mucosa, an increase which was significantly reduced by PPI treatment, suggesting that the overexpression of iNOS might be reversible. N = 5; ANOVA # P < 0.02, compared normal, **P < 0.05, compared with BE without PPI. Omeprazole had no effect on NOX5 mRNA. (B) Omeprazole had no effect on mPGES1 mRNA. (C) Omeprazole had no effect on iNOS mRNA. These data suggest that, if BAR-T cells exposed to pulsed acid treatment for a longer period of time, the cells would take a much longer time to recover. N = 3, ANOVA. *P < 0.01, **P < 0.001, compared with control, # P < 0.05, ## P < 0.01 compared with acid group.

Discussion
We have previously reported that NOX5-S is responsible for the increase in cell proliferation in response to acid treatment in Barrett's cells BAR-T and EA cells (OE33 and FLO-1) 1,7 , and is involved in acid-induced DNA damage 9 . In this study we examined whether PPI reverses oxidative stress genes.
We found that NOX5 and iNOS were markedly elevated in BE mucosa. We have previously reported that NOX5-S is the major isoform of NADPH oxidases in FLO-1 EA cells. The increase of NOX5 and iNOS may increase the production of ROS and thereby cause gene damage. Reactive oxygen species may increase gene mutation and modify the functions of enzyme and proteins (e.g. activation of oncogene products and/or inhibition of tumor suppressor proteins) via the damage of DNA, RNA, lipids and proteins 22,23 . Microsomal PGES1 mRNA was also significantly increased in BE mucosa. Similarly, pulsed acid treatment significantly enhanced the expressions of NOX5, iNOS, and mPGES1 mRNAs in BAR-T cells.
We also found that NOX5, mPGES1 and iNOS may be reversible in BE mucosa after PPI treatment since PPI treatment significantly downregulated the NOX5 mRNA, mPGES1 mRNA and iNOS mRNA expression. However, PPI did not affect these gene expression in normal esophageal mucosa.
To further confirm these in vivo data, we examined whether the overexpression of NOX5-S and hypermethylation of p16 may be reversible in vitro. We found that short-term (such as 4 weeks) acid-induced increase in NOX5 mRNA and p16 promoter methylation may be reversible since the increase in NOX5 mRNA and p16 methylation induced by pulsed acid treatment for 4 and 8 weeks almost completely recovered after the cells were cultured in normal culture medium for 8 weeks. However, long-term (such as 12 weeks) acid treatment-induced increase in NOX5 mRNA, iNOS, mPGES1, and p16 methylation may take more than 8 weeks to recover since these genes were not restored after the cells were cultured in normal culture medium for 8 weeks.
PPI has both anti-acid secretion and anti-inflammatory effects 24 . Our in vitro data suggest that inhibition of acid reflux by PPI may be involved in the reversal of NOX5, mPGES1 and iNOS in vivo since 1) short-term (such as 4 weeks) acid-induced increase in NOX5 mRNA and p16 promoter methylation was recovered after removal of acid treatment; 2) Omeprazole did not have any effect on the expression of NOX5, mPGES1 and iNOS in cultured FLO-1 cells.
In conclusion, NOX5, mPGES1 and iNOS were significantly increased in BE mucosa. Proton pump inhibitor treatment for one month significantly decreased these three gene expression. In BAR-T cells, NOX5 mRNA, iNOS mRNA, mPGES1 mRNA and p16 promoter methylation increased after pulsed acid treatment. Four or (C) Typical image of three Western blot analysis and (D) summarized data showed that the increase in NOX5 protein induced by pulsed acid treatment for 12 weeks only partially restored after the cells were cultured in normal culture medium for 8 weeks. These data suggest that, if BAR-T cells exposed to pulsed acid treatment for a longer period, the cells would take a much longer time to recover. N = 3, ANOVA. *P < 0.01, compared with control, **P < 0.01 compared with acid group. Original Western blot images are available in the supplemental file at https://doi.org/10.1038/s41598-019-52800-7. (2019) 9:16242 | https://doi.org/10.1038/s41598-019-52800-7 www.nature.com/scientificreports www.nature.com/scientificreports/ 12-week-acid induced increase in p16 promoter methylation did not restore after the cells were cultured in normal culture medium for 8 weeks. N = 3, ANOVA, *P < 0.05, **P < 0.01, compared with control; # P < 0.05, ## P < 0.01, compared with acid group. Figure 8. Effect of long-term acid treatment on mPGES1 and iNOS mRNA expression. BAR-T cells were first treated with acidic culture medium (pH 4.0) for 5 min, three times a day for 12 weeks. Then BAR-T cells were cultured at normal culture medium (pH 7.2) for additional 8 weeks after acid treatment for 12 weeks. (A) 12-week acid treatment significantly increased mPGES1 mRNA. The increase did not restore after the cells were cultured in normal culture medium for 8 weeks. (B) 12-week acid treatment significantly increased iNOS mRNA. The increase did not restore after the cells were cultured in normal culture medium for 8 weeks. ANOVA. *P < 0.05, compared with control.