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 H2O2 production and cell proliferation was significantly reduced by knockdown of NOX5-S. Exogenous H2O2 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 H2O2 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.
- Barrett's esophagus
- reactive oxygen species
- hydrogen peroxide
- cell proliferation
gastroesophageal reflux disease (GERD) complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA) (26). Approximately 10% of GERD patients develop BE where esophageal squamous epithelium damaged by acid reflux is replaced by a metaplastic, intestinal type epithelium. The specialized intestinal metaplasia of BE is associated with a 30- to 125-fold increased risk for the development of EA, with the best estimates of cancer incidence of ∼0.5–1.0% per year, i.e., one cancer per 100–200 patients for each year of observation (18, 39). A middle-aged individual with BE for 20 years or more has an estimated 10–20% lifetime risk of developing EA, which is similar to the risk of lung cancer among heavy smokers or of liver cancer among chronic hepatitis-B virus carriers (39). However, the mechanisms of the progression from BE (intestinal metaplasia) to EA are not fully understood. Many genetic and epigenetic alterations, chromosomal gains and losses, and hypermethylation of gene promoters may be involved in this progression (32, 39).
Increasing evidence suggests that inactivation of tumor suppressor gene p16 (INK4A/CDKN2A) may play an important role in the development of EA (32). Mechanisms of inactivation of p16 are multiple, including loss of heterozygosity, point mutation, and/or methylation (an addition of a methyl group to DNA) of p16 gene promoter. In EA, homozygous deletion of the p16 gene and point mutation of the remaining allele of p16 are infrequent (34, 40, 45). Hypermethylation (an increase in the methylation) of p16 gene promoter is present at a much higher frequency in BE with dysplasia and EA than in Barrett's intestinal metaplasia (25, 38, 40–41). Detection of p16 hypermethylation may be used to predict the neoplastic progression (24, 38). Therefore, hypermethylation of p16 gene promoter is an important mechanism inactivating p16. However, the mechanisms of p16 hypermethylation in BE with dysplasia or EA are not fully understood.
Role of reactive oxygen species (ROS) in carcinogenesis has been indicated in an animal model of hepatocellular carcinoma (17) and in other types of cancer such as colon cancer (1). ROS may damage DNA, RNA, lipids, and proteins, leading to increased mutation and altered functions of enzymes and proteins (e.g., activation of oncogene products and/or inhibition of tumor suppressor proteins) in tumor progression (13, 28). ROS have also been reported to induce DNA hypermethylation in tumor cells (27, 30). Whether ROS are involved in p16 hypermethylation in BE is not known.
We have previously shown that acid, a major refluxate in patients with BE, increases ROS production in Barrett's mucosal biopsies (16). This increase is blocked by the NADPH oxidase inhibitor apocynin, suggesting that NAPDH oxidases mediate the acid-induced increase in H2O2 production (16). Whether acid increases methylation of the p16 gene promoter and whether or not NADPH oxidases are involved in hypermethylation of p16 gene promoter are not known. In this study, we show that acid increases p16 DNA methylation and decreases p16 mRNA levels in Barrett's cell line BAR-T and in OE33 EA cells. To our knowledge we are the first to report that acid-induced increase in methylation of p16 gene promoter and reduction in p16 mRNA levels may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells.
MATERIALS AND METHODS
Cell culture and acid/H2O2 treatment.
Human esophageal squamous HET-1A cells (ATCC, Manassas, VA) were cultured in the bronchial epithelial cell medium (BEGM BulletKit, Cambrex, East Rutherford, NJ) containing a basal medium plus the additives (BEGM SingleQuots) in wells precoated with a mixture of 0.01 mg/ml fibronectin, 0.03 mg/ml vitrogen 100 (a type I collagen), and fetal bovine serum.
Human Barrett's cell line BAR-T was derived from esophageal mucosal biopsies of patients with BE (intestinal metaplasia) and immortalized with telomerase as described previously (23). Cells were cultured in wells precoated with collagen IV (1 μg/cm2; BD Bioscience, Bedford, MA) and in Keratinocyte Medium-2 (Ca2+-free solution, Cambrex, Rockland, ME) supplemented with 1.8 mM CaCl2, 5% fetal bovine serum, 400 ng/ml hydrocortisone, 20 ng/ml epidermal growth factor, 0.1 nM cholera toxin, 20 μg/ml adenine, 5 μg/ml insulin, 70 μg/ml bovine pituitary extract, and antibiotics.
Human Barrett's adenocarcinoma cell line OE33 was cultured in DMEM containing 10% fetal bovine serum and antibiotics. All the cell lines were cultured at 37°C in a 5% CO2 humidified atmosphere.
For acid treatment, OE33 cells were exposed to acidic DMEM (pH 4.0) or normal DMEM (control) for 1 h, washed, and cultured in fresh medium (pH 7.2, without phenol red) for an additional 24 h. Finally, the culture medium and cells were collected for measurements. Acidic DMEM (pH 4.0, 250 μl) was added to each well in a 12-well plate, and the final pH was ∼4.9 after 1-h incubation. BAR-T cells were exposed to acidic Keratinocyte Medium-2 (pH 6.0) or normal Keratinocyte Medium-2 (control) for 24 h, and then the culture medium and cells were collected for measurements.
For H2O2 treatment, OE33 cells or BAR-T cells were treated with H2O2 (10−11 M) for 24-h and then collected for measurements.
siRNA and plasmid transfection.
At 24 h before transfection at 70–80% confluence, cells were trypsinized and diluted 1:5 with fresh medium without antibiotics (1–3 ×105 cells/ml) and transferred to 12-well plates (1 ml/well). Transfection of siRNAs was carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer instructions. Per well, 75 pmol of small interfering RNA (siRNA) duplex of NOX5-S or control siRNA (a nontargeting siRNA) formulated into liposomes were applied; the final volume was 1.2 ml/well. Transfection efficiencies were determined by fluorescence microscopy after transfection of Block-it fluorescent oligonucleotide (Invitrogen) and were ∼70% at 48 h.
The pCMV-tag5a-NOX5-S plasmid was generously provided to us by Dr. David Lambeth (Emory University School of Medicine, Atlanta, GA). For transfection of pCMV-tag5a-NOX5-S plasmid, cells (70% confluence, ∼5×106 cells) were transfected with 2 μg of pCMV-tag5a-NOX5-S or control plasmid (pCMV-tag5a) using Amaxa-Nucleofector-System (Lonza) according to the manufacturer instructions. Transfection efficiencies were determined by fluorescence microscopy after transfection of pmax-GFP (Lonza) and were ∼80% at 48 h.
Bisulfite conversion of DNA sample.
Bisulfite conversion of cell samples were carried out as described previously (21). Genomic DNA from 104 cells of each different cell lines were extracted and modified using a CpG modification Kit (EZ DNA methylation-direct kit, Zymo Research) according to the manufacturer's protocol. Briefly, by bisulfite treatment, unmethylated cytosine bases were converted to uracil, whereas methylated cytosines remained unchanged. After treatment, methylated DNA sequence differed from unmethylated DNA, which was used to design methylation specific primers and probes.
Conventional methylation-specific PCR (MSP) was carried out as described previously (21, 42). The primers (21) for the bisulfite-converted methylated sequence were p16MF: 5′-TTATTAGAGGGTGGGGCGGATCGC-3′ and p16MR: 5′-GACCCCGAACCG CGACCGTAA-3′. p16MF and p16MR were designed to specifically amplify bisulfite-converted DNA in the promoter region of the methylated p16 gene. The primers (21) for the bisulfite-converted unmethylated sequence were p16UF: 5′-TTATTAGAGGGTGGGGTGGATTGT-3′and p16UR: 5′-CAACCCCAAACCACAACCATAA-3′. Forty cycles of PCR were carried out. PCR products were analyzed by using 8% nondenaturing polyacrylamide gels and ethidium bromide staining.
Real-time quantitative MSP.
Real-time quantitative MSP is based on the continuous optical monitoring of the progress of a fluorogenic PCR (7, 20). Primers (p16MF and p16MR) were used to detect methylated p16 gene. β-Actin was used as a reference control. Primers for β-actin were β-actin-forward (F): 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and β-actin-reverse (R): 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. The promoter region of β-actin covered by β-actin-F and β-actin-R does not have CpG sites. The cytosines at this region are always unmethylated and converted to uracil after bisulfite treatment (9). Thus this promoter region of β-actin is suitable to be used as an internal control to normalize methylated p16 DNA (10–11). The ratio of methylated p16 promoter DNA to β-actin DNA represented the relative p16 methylation level. Hot-start PCR was initiated at 95°C for 10 min and followed by 40 cycles of denaturation at 95°C 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. To overcome the primer dimer artifacts, an extra incubation step of 77°C for 30 s was added after the 72°C extension step, as previously described (7).
Total RNA was extracted by TRIzol reagent (Invitrogen, Grand Island, NY) for the cultured cells and purified by the total RNA purification system (Invitrogen). According to the protocols of the manufacturer, 1.5 μg of total RNAs from cultured cells was reversely transcribed by using a SUPERSCRIPT kit first strand synthesis system for reverse transcription (Invitrogen).
Detection of NOX5.
The primers used to detect NOX5 in FLO EA cells were 5-ATGGGCTACGTGGTAGTGGGGC-3 (2F), 5-ATGGAGAACCTGACCATCAGC-3 (3F), 5-TTGGGCCCATGAAAGATGAGCA-3 (2R), 5-GTGTGAGCCACAGTGTGCACG-3 (3R), 5-AGCCCCACTACCACGTAGCCC-3 (4R), 5-AGTGGGCAGCGCTGATGGTC-3 (5R), and 5-CTAGAAATTCTCTTGGAAAAATCTG-3 (6R). Three primers (3R for RT, 4R and 5R for nested PCR) were used to amplify the 5′-end of NOX5 by using a 5′-RACE kit (Invitrogen, Grand Island, NY). PCR products were gel extracted and sequenced by GENEWIZ (South Plainfield, NJ).
Real-time quantitative PCR.
Both the quantitative real-time MSP and quantitative real-time PCR were carried out on a Stratagene Mx4000 multiplex quantitative PCR system (Stratagene, La Jolla, CA). The primers used were p16F:5′-AAGGTCCCTCAGACATCCC-3′, p16R:5′-TGGACATTTACGGTAGTGGG-3′, 18sF:5′-CGGACAGGATTGACAGATTGATAGC-3′, 18sR: 5′-TGCCAGAGTCTCGTTCGTTATCG-3′. All reactions were performed in triplicate in a 25-μl total volume containing a 1× concentration of Brilliant SYBR Green QPCR Master Mix (Stratagene), the concentration of each sense and antisense primer were 100 nM, 1 μl cDNA, and 30 nM reference dyes. Reactions were carried out in a Stratagene Mx4000 multiplex quantitative PCR system for 1 cycle at 94°C for 5 min; 40 cycles at 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s; 1 cycle at 94°C for 1 min; and 1 cycle at 55°C for 30 s. Fluorescence values of SYBR Green I dye, representing the amount of product amplified at that point in the reaction, were recorded in real time at both the annealing step and the extension step of each cycle. The cycle threshold, defined as the point at which the fluorescence signal was statistically significant above background, was calculated for each amplicon in each experimental sample by use of Stratagene Mx4000 software. This value was then used to determine the relative amount of amplification in each sample by interpolating from the standard curve. The transcript level of each specific gene was normalized to 18s amplification.
Western blot analysis.
Cells were lysed in Triton X lysis buffer. The suspension was centrifuged at 15,000 g for 5 min, and the protein concentration in the supernatant was determined. Western blot was done as described previously (6). The NOX5 antibody prepared against a mixture of unique NOX5 peptides (NH2-YESFKASDPLGRGSKRC-COOH and NH2-YRHQKRKHTCPS-COOH) was generously provided by Dr. David Lambeth (5) and used at a dilution of 1:1,000. The GAPDH antibody was used at a dilution of 1:1,000.
For acid treatment, OE33 cells were cultured as described above and treated with acidic medium, pH 4.0, for 1 h at the confluence of 40–50%. After being washed three times, cells were cultured at pH 7.2 for additional 24 h. BAR-T cells were exposed to acidic Keratinocyte Medium-2 (pH 6.0) or normal Keratinocyte Medium-2 (control) for 24 h. For the H2O2 treatment, BAR-T cells and OE33 cells were treated with H2O2 (10−11 M) 24 h. For siRNA transfection, after siRNAs of NOX5 or control were introduced, cells were treated without or with acid for 1 h, cultured in regular medium for an additional 24 h, and then incubated with methyl-[3H]thymidine (0.05 μCi/ml) for 4 h. After being washed three times with PBS to remove unincorporated radioactivity, cells were collected and homogenized with a lysis buffer containing (pH 7.4): 50 mM HEPES, 50 mM NaCl, 1% Triton X-100, 1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol. Methyl-[3H]thymidine uptake was measured in a scintillation counter. The level of protein in the homogenates was also determined and the level of methyl-[3H]thymidine incorporation was normalized to protein content.
Amplex red hydrogen peroxide fluorescent assay.
Levels of H2O2 in culture medium were determined by using the Amplex Red H2O2 assay kit (Invitrogen). This assay uses the Amplex Red reagent (10-acetyl-3, 7-dihydroxyphenoxazine) to detect H2O2. In the presence of peroxidase, the Amplex Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce the red fluorescent oxidation product resorufin. Fluorescence is then measured with a fluorescence microplate reader by using excitation at 550 nm and emission detection at 590 nm.
The human NOX5 siRNA was from Ambion; p16 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); TRIzol reagent, total RNA purification system, and RNA reverse transcription kit were purchased from Invitrogen; and the Bisulfite Conversion CpG modification kit was from Zymo Research. Human Barrett's adenocarcinoma cell line OE33 and other reagents were purchased from Sigma.
Data were expressed as means ± SE. When data were normalized to the percentage of controls, standard errors of control groups were also normalized and calculated as follows: standard error of the raw data × 100/mean value of the raw data in control group. Statistical differences between two groups were determined by Student's t-test. Differences between multiple groups were tested by ANOVA and checked for significance by Fisher's protected least significant difference test.
Methylation of p16 gene promoter in HET-1A, BE, and EA OE33 cells.
It has been shown that aberrant p16 gene promoter methylation is an important mechanism to silence this gene (19, 40). Therefore, we examined methylation of the p16 gene promoter in human normal esophageal squamous cell line HET-1A, Barrett's cell line BAR-T, and EA cell line OE33 by MSP (21). All reactions were normalized to β-actin as previously described in the literature (9, 15). The methylation levels were significantly increased ∼2 times in BAR-T cells and 11 times in OE33 cells, compared with those in HET-1A cells (Fig. 1A). Furthermore, the p16 methylation level in OE33 cells was more than fourfold as high as in BAR-T cells. The data suggest that hypermethylation of the p16 gene promoter is present in BAR-T and OE33 cells.
Acid is involved in p16 epigenetic change in BE and OE33 cells.
Since acid reflux may play an important role in the progression from BE to dysplasia and to adenocarcinoma (14, 29), we examined whether acid treatment affects levels of p16 mRNA and promoter methylation. Acid treatment significantly decreased p16 mRNA level by 78% in BAR-T cells and by 43% in OE33 cells (Fig. 1B). Conversely, acid treatment significantly increased the methylation levels of p16 gene promoter (Fig. 1C) and cell proliferation level (Fig. 1D) in BAR-T and OE33 cells. The data suggest that acid treatment may reduce p16 mRNA expression and increase p16 methylation and cell proliferation in BAR-T cells and OE33 cells.
Role of ROS in acid-induced p16 hypermethylation.
We have shown that acid treatment significantly increases H2O2 production in Barrett's mucosal biopsies (16). In addition, ROS have been reported to induce hypermethylation of the E-cadherin promoter in hepatocellular carcinoma cell line (27). Thus we examined the role of ROS in acid-induced p16 hypermethylation. We found that acid treatment significantly increased H2O2 production both in BAR-T cells and OE33 cells (Fig. 2A). Exogenous H2O2 significantly increased p16 promoter methylation in BAR-T cells and OE33 cells (Fig. 2B). Exogenous H2O2 significantly increased cell proliferation in BAR-T cells and OE33 cells (Fig. 2C). The data suggest that H2O2 may be involved in acid-induced p16 promoter methylation and may increase cell proliferation in BAR-T cells and OE33 cells.
NOX5-S may contribute to acid-induced p16 hypermethylation in BAR-T and OE33 cells.
We have shown that NADPH oxidases may mediate acid-induced H2O2 production in Barrett's mucosal biopsies (16) and that NADPH oxidase NOX5-S is the major isoform of NADPH oxidases in FLO EA cells (22). Therefore, we examined whether NOX5-S is present in BAR-T and OE33 cells. NOX5 was detectable in BAR-T and OE33 cells by RT-PCR (data not shown). NOX5 has isoforms α, β, δ, and γ or NOX5-S (3). Figure 3A showed that only one band (∼200 bp) was detected by 5′-RACE using the primers abridged universal amplification primer and 5R (+11 to +31 from ATG) in BAR-T and OE33 cells. The size of the PCR products was consistent with NOX5-S but not with the other NOX5 isoforms. The PCR products were also sequenced and consistent with NOX5-S. Acid treatment significantly increased NOX5-S expression in BAR-T cells and OE33 cells (Fig. 3B).
Next we examined whether NOX5-S mediates acid-induced H2O2 production and cell proliferation in BAR-T cells and OE33 cells. We used NOX5 siRNA to knock down NOX5-S. NOX5 siRNA has been shown by us to effectively knock down NOX5 (16). We found that knockdown of NOX5-S remarkably decreased acid-induced increase in H2O2 production in BAR-T cells (Fig. 3C) and in OE33 cells (Fig. 3D), suggesting that acid-induced H2O2 production may be mediated by activation of NOX5-S. We also found that knockdown of NOX5-S remarkably decreased acid-induced increase in thymidine incorporation in BAR-T cells (Fig. 4A) and in OE33 cells (Fig. 4B), suggesting that acid-induced increase in cell proliferation may be mediated by activation of NOX5-S.
We further examined whether NOX5-S is involved in the regulation of the p16 gene expression. We found that knockdown of NOX5-S significantly increased mRNA level of p16 in BAR-T cells (Fig. 5A) and OE33 cells (Fig. 5B). In addition, knockdown of NOX5-S inhibited acid-induced downregulation of p16 mRNA expression in BAR-T (Fig. 5A) and OE33 cells (Fig. 5B). In contrast, knockdown of NOX5-S decreased acid-induced increase in p16 promoter methylation (Fig. 5, C and D). The data suggest that NOX5-S may play an important role in acid-induced increase in p16 promoter methylation and decrease in p16 mRNA expression.
To further confirm this result, NOX5-S was overexpressed in BAR-T cells and OE33 cells by transfection with the recombinant plasmid pCMV-tag5a-NOX5-S. Overexpression of NOX5-S significantly decreased p16 mRNA levels in BAR-T cells and OE33 cells (Fig. 6A). Conversely, overexpression of NOX5-S significantly increased p16 promoter methylation in these cells (Fig. 6B). In addition, overexpression of NOX5-S significantly increased thymidine incorporation in these cells (Fig. 6C). The data indicate that NOX5-S-derived ROS may increase cell proliferation in BAR-T cells and OE33 cells and that NOX5-S-mediated downregulation of p16 expression may depend on hypermethylation of p16 gene promoter.
The CDKN2/p16INK4A tumor-suppressor gene plays an important role in cell cycle regulation, deregulation of cell proliferation and phosphorylation of Rb (43–44). Inactivation of p16 due to homozygous deletion, point mutation, and/or epigenetic changes such as hypermethylation in its promoter region have been found in different tumor types (31, 34, 36). To date, hypermethylation of the p16 gene promoter region has been regarded as one of the most frequent genetic alterations in the progression from BE to dysplasia and to EA (4, 19, 40). However, the mechanisms of initiation and regulation of p16 hypermethylation in BE or EA are not clear.
Acid reflux may play an important role in the progression from metaplasia to dysplasia and to adenocarcinoma in patients with BE because of the following: 1) cultured biopsy specimens of intestinal metaplastic cells demonstrate a significant increase in cell proliferation as determined by tritiated thymidine uptake when explants are briefly exposed to acid (14); 2) long-term inhibition of esophageal acid exposure by administration of proton pump inhibitors to patients with BE has been shown to decrease proliferation of metaplastic cells (29); 3) a prospective study has shown that treatment with proton pump inhibitors significantly reduces the incidence of dysplasia in BE patients when compared with no therapy or treatment with an H2 receptor antagonist (12). However, whether acid promotes progression of intestinal metaplasia by inducing hypermethylation of p16 gene promoter in BE or EA is not known.
In this study, we found that acid treatment decreased p16 mRNA level in BAR-T cells as well as in OE33 cells (Fig. 1B). This decrease may be due to an increase in the methylation of p16 gene promoter as evidenced by our findings that acid treatment significantly increased p16 gene promoter methylation both in BAR-T cells and OE33 cells (Fig. 1C). OE33 cells were used in our study because OE33 cells have higher levels of p16 promoter methylation and the signal transduction pathways inducing p16 methylation may be easier to be detected in these cells.
The mechanisms whereby acid increases p16 gene promoter methylation are not clear. It has been shown that acid increases ROS production in Barrett's mucosal biopsies (16) and in esophageal squamous epithelial cell line HET-1A (8). We also found that acid significantly increased H2O2 production in BAR-T cells and OE33 cells (Fig. 2). We have previously shown that low doses of H2O2 increase cell proliferation and high doses cause cell apoptosis (16). In this study we found that exogenous H2O2 (10−11 M) significantly increased promoter methylation of the p16 gene and cell proliferation in BAR-T and OE33 cells (Fig. 2). This result is consistent with previous reports showing that ROS induce hypermethylation of the E-cadherin promoter in hepatocellular carcinoma cell line (27) and that oxidative stress-induced carcinogenesis may depend on hypermethylation of p15 and p16 genes in rat renal tumor cells (35). Therefore, our data suggest that ROS may mediate acid-induced p16 hypermethylation and increase cell proliferation.
The source of ROS in Barrett's cells is not known. Low levels of ROS, seen in nonphagocytic cells, were thought to be by-products of aerobic metabolism. More recently, however, superoxide-generating homologues of phagocytic NADPH oxidase catalytic subunit gp91phox (NOX1, NOX3-NOX5, DUOX1, DUOX2) and homologues of other subunits (p41phox or NOXO1, p51phox or NOXA1) have been found in several cell types (2, 16, 33), suggesting that ROS generated in these cells may have distinctive cellular functions related to immunity, signal transduction, and modification of the extracellular matrix. NOX5 has five isoforms: α, β, δ, and γ, and NOX5-S (3, 37). NOX5 α, β, δ, and γ have EF-hand (a helix-loop-helix structural domain) motifs at its NH2 terminal (3), whereas NOX5-S does not (15). In the present study we found that NOX5-S is present in BAR-T cells and OE33 EA cells (Fig. 3). Whether NOX5-S plays a role in acid-induced H2O2 production and cell proliferation in BAR-T and OE33 cells has not been established. We found that acid treatment significantly increased ROS production and cell proliferation in BAR-T cells and in OE33 EA cells (Fig. 2, A and C). Acid-induced ROS production and cell proliferation were dramatically decreased by knockdown of NOX5-S (Fig. 3, C and D; 4, A and B). The data therefore suggest that acid-induced H2O2 production and cell proliferation may be mediated by activation of the NADPH oxidase NOX5-S.
We also examined whether NOX5-S plays a role in p16 hypermethylation in BAR-T cells and OE33 EA cells. We found that NOX5-S is responsible for acid-induced decrease of p16 mRNA level and increase of p16 promoter methylation since 1) knockdown of NOX5-S inhibited acid-induced downregulation of p16 mRNA expression in BAR-T (Fig. 5A) and OE33 cells (Fig. 5B); 2) knockdown of NOX5-S decreased acid-induced increase in p16 promoter methylation (Fig. 5, C and D). The role of NOX5-S in p16 promoter hypermethylation was further supported by the following results: 1) knockdown of NOX5-S with NOX5-S siRNA significantly increased p16 mRNA expression level both in BAR-T cells and OE33 EA cells (Fig. 5, A and B) in basal conditions; 2) overexpression of NOX5-S decreased p16 mRNA level in BAR-T cells and OE33 cells (Fig. 6A); 3) the methylation level of p16 gene promoter was increased in BAR-T cells as well as in OE33 cells (Fig. 6B) after NOX5-S overexpression; 4) overexpression of NOX5-S significantly increased cell proliferation in BAR-T cells and OE33 cells (Fig. 6C).
The similar results obtained in BAR-T and OE33 cells suggest that acid-induced molecular changes may be general phenomena in different cell lines associated with BE.
In conclusion, the NADPH oxidase NOX5-S is present in BAR-T cells and OE33 EA cells and mediates acid-induced increase in H2O2 production and cell proliferation in these cells. NOX5-S is also involved in acid-induced hypermethylation of the p16 gene promoter and downregulation of p16 mRNA (Fig. 7). It is thus possible that acid reflux present in patients with BE may activate NADPH oxidase NOX5-S and increase ROS production. NOX5-S-derived ROS in turn increase p16 gene promoter methylation, downregulate p16 expression, and increase cell proliferation, thereby contributing to the progression from BE to EA.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK080703.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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