Prevention of DNA damage in Barrett’s esophageal cells exposed to acidic bile salts

Carcinogenesis, Dec 2016

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett’s metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

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Prevention of DNA damage in Barrett’s esophageal cells exposed to acidic bile salts

Carcinogenesis Prevention of DNA damage in Barrett's esophageal cells exposed to acidic bile salts Vikas Bhardwaj 1 2 Andela Horvat 1 Olga Korolkov 1 Mary K.Washington 0 Wael El-Rifa 1 Sergey I.Dikalov 3 Alexander I.Zaika 0 1 2 0 Department of Cancer Biology 1 SDAe,partment of Surgery 2 Department of Veterans Affairs, Tennessee Valley Healthcare System , Nashville, TN 37212, U , USA 3 Division of Clinical Pharmacology, Vanderbilt University Medical Center and Vanderbilt-Ingram Cancer Center , Nashville, TN 37232 , USA Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERDassociated tumorigenesis. - Introduction Esophageal adenocarcinoma (EA) has overtaken other histolo-gi Since Barrett’s esophagus is a precancerous lesion that may cal types of esophageal cancer in the USA with an incidence rate carry an increased risk of EA, preventive strategies have been that is more rapid than other types of cance1r,(2).The major risk proposed, including endoscopic and surgical ablative therapies. factor for EA is gastroesophageal reflux disease (GERD). Because Current analyses suggest that BE patients with high-grade-dys of the disease, esophageal cells are exposed to a gastric refl-ux plasia may benefit from these procedures 7(). However, ablative ate that is frequently mixed with duodenal bile. This causes -sig therapies do not warrant the risk and excessive cost for the nificant cellular and DNA damage and induces inflammation, vast majority of BE patients. The existing medical treatment which in turn, exacerbates the mucosal injury. If the damage for GERD almost exclusively uses drugs intended to suppress persists, it can cause Barrett’s esophagus (BE), a condition in production of acid, such as proton pump inhibitors. There is no which the normal squamous epithelial lining is replaced by a doubt that proton pump inhibitors provide symptomatic relief, metaplastic intestinal type of epithelium. Further damage of but this treatment has a limited effect on EA8).( Proton pump Barrett’s epithelium induced by reflux may cause tumorigenic inhibitor treatment does not correct the underlying reflux -dam alterations leading to EA3–(6). age in the Barrett’s esophagus, and gastroesophageal reflux of Abbreviations We developed a novel assay for analyses of DNA damage repair in esophageal cells exposed to acidic bile salts based on approaches p-re is to halt or reverse progression from pre-malignant lesions, viously described by Iliakiset  al. (23). Genomic DNA was collected from such as BE and low-grade dysplasia to high-grade dysplasia untreated and BA/A-treated cells using chloroform/isoamyl alcohol and or cancer 1(2). Natural compounds are of particular interest then purified using the High Pure PCR Product purification kit from Roche because of their safety, low toxicity, general acceptance as-die (Branchburg, NJ). Nuclear proteins were extracted from control (Cntr) or tary supplements and lack of deleterious side effects. apocynin-treated (Apo) cells. Briefly, control and apocynin-treated cells Inactivation of p53 is a strong indicator of neoplastic tr-ans were collected and resuspended in the cytoplasmic buffer [HEPES 1m0M formation of BE (13,14). p53 protein is a regulator of important pH 7.9, MgCl2 1.5 mM, KCl 10 mM, TritonX-100 0.01%, DTT 0.5mM, 1% pro cellular processes such as DNA damage response, cell cycle c-on tease inhibitors cocktail (Sigma-Aldrich)] at 4°C. The cells were lysed on trol and DNA damage repair 1(5). Prolonged exposure to acidic ice for 20min with gentle vortexing. The lysates were centrifuged at g400 bile salts has been shown to cause inactivation of p53 and tra-ns fTohre1p0emlilnetast w4°eCreantdhesnupweransahteadnotnscweawscitohllceycttoepdlaassmciyctobpulfafesrmiacnfdraccetni-otnris. formation of Barrett’s cells16() as well as p53 protein degrad-a fuged for 5min. The pellets were then resuspended in the nuclei buffer tion (17). Other members of the p53 protein family, p73 and p63, (HEPES 10 mM pH 7.9, MgCl 2 1.5 mM, KCl 400 mM, EDTA 0.2 mM, glycerol have also been shown to play significant roles in regulation of 25%, DTT 0.5 mM, 1% protease inhibitors cocktail) and kept on ice for DNA damage response and repair (18,19). Although much less is 20 min. During incubation, samples were frozen and thawed four times to known about their roles in EA, recent studies have shown that assist disruption of nuclear membrane and collection of nuclear proteins. levels and activities of p73 and p63 are altered by acidic bile salts The samples were then centrifuged at 21 00g0 for 40min and supernatant (18,20). was collected as nuclei extract. Protein concentration was determined In this study, we aim to understand the mechanisms of bile with the Bradford assay. For repair reaction,µ5g of nuclear protein extract acid-induced DNA damage and determine interventions to pr-e was incubated with 1µ g of DNA purified from BA/A-treated cells in the presence of reaction buffer (4mM HEPES-KOH, pH 7.5 at 37°C, KCl 2mM, vent damage induced by gastroesophageal reflux. MgCl2 0.3 mM, dNTPs 10  μM, ATP 1.5 mM and 1 mM β-mercaptoethanol) for 24 h. The reaction mixture was then separated on agarose gel to vis-ual Materials and methods ize high molecular weight and fragmented DNA. DNA damage repair was measured as a ratio of fragmented DNA in test and control (BA/A-treated Cell cultures only) samples using the ImageJ image analysis software (NIH). Human telomerase-immortalized esophageal cell lines—CP-A and CP-B (both from American Type Culture Collection, Manassas, VA) and BAR-T Comet assay (hTERT immortalized human Barrett’s metaplasia (BE) cells, a kind gift Alkaline comet assay was performed to determine DNA damage induced by from Dr. Souza (21)) were cultured in keratinocyte SFM (KSFM) medium BA/A (24). In brief, the cells were collected after appropriate treatment, mixed supplemented with 40 µg/ml bovine pituitary extract, 1.n0g/ml epidermal with LM Agarose (Trevigen, Gaithersburg, MD) and allowed to solidify on flare growth factor (Life Technologies, Carlsbad, CA) and 5% fetal bovine serum comet slides (Trevigen) at 4°C. The slides were then immersed in lysis buffer (Atlanta Biologicals, Lawrenceville, GA). Human SV40 T antigen imm-or (10mM Tris, pH 10, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10% DMSO) for talized non-tumorous esophageal epithelial cells HET-1A (ATCC) were 2h at 4°C. Electrophoresis was then performed on slides in alkaline con-di cultured in Dulbecco's modified Eagle's medium (Life Technologies) su-p tions (300mM NaOH, 1 mM EDTA, pH > 13). The slides were then washed and plemented with 10% fetal bovine serum. Cell lines were authenticated and fixed with 100% cold ethanol, and visualized with Olympus BX41 fluorescent characterized by the suppliers2(1). ATCC uses morphology, karyotyping microscope (Olympus, Pittsburg, PA).Tail DNA content was measure in a minand PCR-based approaches to confirm the identity of cell lines. imum of 50 cells per treatment using the Open Comet software. siRNA, antibodies and chemicals Analysis of DNA damage using p-H2AX antibody p73 was inhibited either by lentiviral transduction with shRNA or tr-ans Briefly, CP-A cells treated with acidic bile salts (100 uM fomri3n0) were colfection with siRNA, as described previously2(2). Both approaches targeted lected 12h after BA/A treatment using RIPA buffer. The lysates were se-pa the same p73 sequence (5′-GCAATAATCTCTCGCAGTA-3′) found in all p73 rated on SDS-PAGE gel, incubated with primary p-H2AX antibody (Millipore, isoforms (22). Billerica, MA; 1:100) overnight at 4°C and analyzed by Western blotting. Cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturers’ protocols. Reactive oxygen species (ROS) assay The following antibodies were used: p53 and p21 were from Calbiochem Cellular ROS levels were determined using′, 27′-dichlorofluorescin dia-c (Billerica, MA); p73 from Bethyl (Montgomery, TX); p63(A4A) and p73(H- etate (DCFDA) (Sigma-Aldrich) dye according to manufacturers’ protocol. 79) from Santa Cruz Biotechnologies (Dallas, TX); Rad51 from Abcam Briefly, 1× 106 cells were collected after appropriate treatment and stained (Cambridge, MA); BRCA1 and the double strand breaks Repair Antibody with DCFDA dye (20 µM) for 30min at 37°C. ROS levels were analyzed using Sampler Kit from Cell Signaling Technologies (Danvers, MA). The following flow cytometry. Dead cells were detected using propidium iodide staining. chemicals were used: resveratrol (3′,45-Trihydroxy-trans-stilbene, 5-((1E)-2(4-Hydroxyphenyl)ethenyl)-1,3-benzenediol), curcumin (1,7-bis(4-Hydroxy- Measurements of intracellular superoxide using 3-methoxyphenyl)-1,6-heptadiene-3,5-dione, Diferuloylmethane), apigenin TMH spin probe and electron paramagnetic (4′,5,7-Trihydroxyflavone,5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-benzopyrone), epigallocatechingallate(2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran- resonance (EPR) 3,5,7-triol 3-gallate) and apocynin (4-hydroxy-3-methoxyacetophenone), all Production of cellular superoxide was quantitatively measured by TMH from Sigma-Aldrich (St. Louis, MO). spin probe and EPR (25). Spin probe stock solutions was prepared in ice cold 0.9% NaCl in the presence of the chelating agent [diethylene- tri 3 h, following which the levels returned to those observed in amine penta-acetic acid, 0.2 mM]. Production of O2· in cells was measured untreated cells. Since ROS constitutes a variety of reactive- spe in Krebs HEPES buffer containing 5.8g/l NaCl, 0.35g/l KCl, 0.37g/l CaCl2, cies, we then sought to determine how BA/A treatment affect the 0.29g/l MgSO4, 2.1g/l NaHCO3, 0.14g/l K2HPO4, 5.21g/l Na-HEPES, 2g/ld-glu- induction of the specific ROS. Amplex Red assay and EPR speccaocisde., CpHell7s.3w,ienrethinecuprbeasteendcefoorf3m00.i1nmaMt d37ie°tChwylitehne0.t5rmiaMmTinMeHp.eTnhteaacceelltsic troscopy were used to determine the levels of hydrogen per-ox were then collected in 0.m6l of fresh Krebs HEPES buffer, snap frozen in ide and superoxide radicals, respectively. Our analyses revealed liquid nitrogen and analyzed for EPR spectra. The signal was quantified a rapid increase in the levels of hydrogen peroxide and supe-r using calibration with standard concentrations of the 3-carboxy-proxyl oxide radical in cells treated with BA/A compared to untreated nitroxide. The portion of the signal due to2·Owas determined by pre- cells F(igure  1C). The levels of hydrogen peroxide (left panel) incubation of duplicate samples with PEG-SOD (100 U/ml) for 3h which and superoxide radicals (right panel) returned to near baseline inhibited more than 75% of nitroxide formation2(6). between 3 and 6h after BA/A treatment which is similar to total ROS levels profile determined with DCFDA dyeF(igure 1B). Thus, Detection of cellular hydrogen peroxide production our data show that exposure to acidic bile salts results in a rapid by Amplex Red assay upregulation of ROS, such as hydrogen peroxide and superoxide radicals in BE cells. Natural compounds inhibit BA/A induced ROS production and DNA damage Cellular H2O2 was measured by Amplex Red assay (Thermo Fisher Scientific) which is based on oxidation of the non-fluorescent molecule N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red) to resorufin (excit-a tion at 530nm and emission at 590 nm) ( 27). Amplex Red provides specific and sensitive H2O2 detection of extracellular2OH2. Since H2O2 is diffusible, Amplex Red assay provides an index of total cellular2OH2 production (28). To further investigate the mechanism of BA/A induced DNA damage and develop a strategy to prevent it, we tested a panel Immunohistochemistry of natural compounds that are known to have antioxidant pr-op After institutional review board approval, 11 BE patients and 4 normal erties. We selected natural compounds because of their safety, esophageal samples resected at Vanderbilt University Medical Center availability and low toxicity, making them ideal for drug deve-lop were histologically verified, and representative regions were selected for ment. CP-A cells were treated with 10 uM of either compound for immunohistochemical analyses. The use of all human pathology spec-i 3 h followed by washing to remove drug and short exposure to mens for research was approved by the Institutional Review Board (IRB) of BA/A, as described above. The cells were then collectedh 1post Vanderbilt University Medical Center. Since only de-identified tissues were BA/A treatment, stained with DCFDA dye and analyzed for ce-llu included in this retrospective study, the IRB has waived requirements for lar ROS levels using flow cytometry. We found that pre-treatment informed consent. Immunohistochemical staining was done using the fo-l with either apigenin, apocynin or EGCG significantly reduced the laonwdinp5g3a(DnOti-b1o)dfireosm:p7C3a(Hlb-i7o9c)haenmd. pS6p3e(c4iAfi4c)itfryomofSsatnatinaiCnrguzwBaisotveecrhifnieodlobgyy, ROS levels F(igure 2A). Next, we investigated the effect of natural omitting a primary antibody step in the protocol. Immunohistochemical compounds on BA/A induced DNA damage using comet assay. results were evaluated for staining intensity. The intensity of staining was Cells were collected 1h8after BA/A treatment and analyzed for graded as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). DNA damage. Compared to BA/A treated CP-A cells, we found significantly less damage in cell pre-treated with either resv-era Statistics analyses trol, apigenin or apocyninF(igure 2B). We confirmed our observaStatistical analyses were performed using the Studentt-’tsest. Results are tions using BAR-T (another human Barrett’s esophageal cell line) expressed as averages ± SE, if not specifically indicated. Results were c-on and found that apigenin and apocynin significantly reduce DNA sidered significant at values oPf < 0.05. damage induced by BA/A in these cellsF(igure 2C, top left panel). Our results suggest that inhibition of ROS protects esophageal Results cells from DNA damage induced by acidic bile salts. However, the protective effect varies between cell lines. Acidic bile salts induce DNA damage and ROS Further, we analyzed the protective effect of natural c-om production in esophageal cells pounds on other esophageal cell lines (CP-B and Het-1A). Het-1A Since DNA damage in BE is an important contributing factor to cells are immortalized normal squamous epithelial esophageal tumorigenesis, we sought to understand the regulation of DNA cells and CP-B cells were derived from Barrett’s patient with damage by acidic bile salts (BA/A) in Barrett’s metaplastic cells. high grade dysplasia. Thus, tested cell lines represent dif-fer For our analyses, human BE cells, CP-A, were exposed to acidic ent stages of progression to BE-associated EA. Interestingly, all growth medium (pH 4.0) supplemented with 100 uM bile salts natural compounds protected CP-B cells from BA/A induced cocktail for 30min. The composition, total bile salts concent-ra DNA damage (Figure 2C, top right), while only apocynin signi-fi tion and pH were selected based on previous measurements that cantly reduced DNA damage induced by BA/A in Het-1A cells allowed us to mimic a typical episode of reflux in GERD patients (Figure  2C, bottom left). Taken together, our results show that (29–31). DNA damage in BA/A treated cells was analyzed using although a number of antioxidants confer protection, apocynin alkaline comet assay and phosphorylation of H2AX. We found is the most effective in reducing DNA damage induced by BA/A significant increase in comet tail DNA content and phosphory-la in esophageal cells (see summary table inFigure 2). tion of H2AX histone (p-H2AX) in BA/A treated cells compared to untreated controlsFi(gure 1A). Apocynin reduces BA/A induced damage by To understand the mechanism of DNA damage induced by promoting DNA repair acidic bile salts, we analyzed ROS in Barrett’s esophageal cells as To investigate whether the protective effect of apocynin was solely previous reports have shown an induction of ROS in reflux co-n due to inhibition of ROS production, CP-A cells treated with BA/A ditions (32,33). CP-A cells were treated with BA/A and collected were allowed to recover for 1h2and then treated with 1 uM apo-c at the indicated time F(igure  1B). The cells were then stained ynin for 3h. The 12-h time point represents the time by which the with the DCFDA fluorogenic dye and analyzed for ROS using flow ROS levels in BA/A treated cells returned to near baseline, thus cytometry. We found that BA/A treatment significantly elevated negating the protective effect of apocynin associated with inh-ibi ROS levels in CP-A cells. The ROS levels remained elevated for tion of ROS. Using comet assay, we investigated the dynamics of DNA damage and found that cells treated with apocynin recover p-H2AX proteins suggesting that primarily NHEJ is activated faster from DNA damage than control BA/A treated cells (18 and by esophageal cells in response to acidic bile salts treatment 24h, respectively) suggesting that apocynin enhances/accelerates (Supplementary Figure 1A, available atCarcinogenesis Online). the process of DNA damage repair (Figure 3A). To test this hypothesis, we developed a novelin vitro repair Apocynin affects expression of BRCA1 protein assay that allows us to investigate the effect of apocynin on To further characterize the role of apocynin in DNA repair, its repair of damaged DNA in BA/A-treated cellisn vitro. Its sche- effect on DNA repair proteins was analyzed using the double matic representation is shown inFigure  3B. Genomic DNA strand breaks Repair Antibody kit. CP-A and BAR-T cells were from BA/A treated CP-A cells were purified as described in the treated with apocynin (1µ M, 3 h) in the presence or absence Materials and Methods section and incubated with nuclear of BA/A treatment. We found that among tested DNA damage extracts (NE) from apocynin treated (Apo) or untreated control repair proteins, apocynin increases the levels of BRCA1 in both (Cntr) cells. Although both extracts were able to repair BA/A-CP-A and BAR-T cells F(igure 4A). The levels of other DNA repair treated DNA, nuclear extract from apocynin-treated cells was enzymes such as Rad50, Rad51 and KU80 were not increased by significantly more effective in reducing the amount of fr-ag BA/A or apocynin treatment. mented DNA than the corresponding extract from control untreated cellsFi(gure 3C). These results show that apocynin is Acidic bile salts and apocynin induce p73 protein able to enhance DNA damage repair of esophageal cells exposed p53 and other members of the p53 family, p63 and p73, are to acidic bile salts independently of ROS inhibition. involved in the regulation of DNA damage response. In addition, To explore the mechanism of DNA damage repair in BA/A we and others have previously shown that expression of these treated cells, we performed co-immunofluorescence analyses proteins are altered in esophageal carcinoma22(). Hence, we and assessed co-localization of DNA repair proteins in CP-A sought to investigate the modulation of p53, p73 and p63 pr-o cells challenged with BA/A. We found that 53BP1 and p-H2AX teins by apocynin. We found strong induction of p73 protein in proteins are co-localized in BA/A damaged cells, while no s-ig apocynin-treated CP-A and BAR-T cellsFi(gure 5Atop panel and nificant co-localization was observed between Rad51 and Supplementary Figure 1C, available atCarcinogenesis Online). In contrast to p73, no statistically significant changes of p53-pro with healthy individualsn(  =  4). Eight out of 11 GERD patients tein levels were observed in CP-A cells, and only a weak increase showed strong to moderate p73 staining (3–2), while 3 out 11 in BAR-T cells n( = 3). Levels of p63 were low in both tested cell patients showed a weaker p73 immunoreactivity between 1 and 2 lines. Furthermore, our data show that treatment with BA/A-fur (Figure 5B, top panels). Notably, strong increases in the p73 protein ther enhance the induction of p73 in esophageal cellFsig(ure 5A, were observed in both squamous and metaplastic Barrett’s epit-he compare lanes 2 with 4 andSupplementary Figure 1B, available lia (Figure 5B). In contrast to p73, expression of the p53 protein was at Carcinogenesis Online), suggesting that p73 may mediate the slightly elevated or undetectable in squamous and metaplastic protective role of apocynin. Barrett’s epitheliaF(igure 5B, middle panels). As a positive control To further investigate these proteinins vivo, we assessed their for p53 staining, esophageal tumors with elevated levels of the p53 expression in esophageal epithelium of control subjects and GERD protein due to p53 gene mutations were usedF(igure 5B; middle patients using immunohistochemistry. All GERD patients an-a panel). The p63 protein was expressed in the basal layer of esop-h lyzed (n = 11) had elevated protein levels of nuclear p73 compared ageal squamous epithelium in all patientsF(igure  5B, bottom panels). No p63 staining was found in all cases of BEF(igure 5B), confirming previous studies on low expression of p63 protein in BE (34–36). Thus, our data show that induction of p73 occurs both in vitro and in vivo. However, it is noteworthy that additional -fac tors such as inflammation may contribute to upregulation of p73 protein in Barrett’s patients. GERD and Barrett’s esophagus are the most prominent risk fa-c To elucidate the role of p73 in apocynin-treated cells, we do-wn tors for the development of EA. To better understand the tu-mo regulated p73 in CP-A cells using shRNA (shp73). Then, p73 rigenic alterations induced by GERD, we investigated cellular deficient and control cells transfected with scrambled shRNA damage induced by gastroesophageal reflux in metaplastic (scr shRNA) were treated with BA/A as described above and Barrett’s cells. Our studies showed that exposure of BE cells analyzed for DNA damage using comet assay. We found that to acidic bile salts at physiologically relevant concentrations inhibition of p73 significantly enhances DNA damage induced and pH leads to significant DNA damage. These data are co-n by acidic bile saltsF(igure 6A). Then, we investigated the effect sistent with the previous reports on damage induced by acidic of apocynin in p73-deficient cells. We found that apocynin reflux (18,37). Our studies also strongly emphasize the role ROS reduces DNA damage in scr shRNA control cells treated with in DNA damage as acidic bile salts were found to be potent BA/A. However, its protective effect was significantly dim-in inducers of ROS. Analyzing the specific ROS species, our stu-d ished in p73-deficient cells showing that p73 mediates act-iv ies reveal a rapid increase in hydrogen peroxide and supe-r ity of apocynin (Figure  6B; DNA damage in both scr shRNA oxide radicals that are known for their ability to cause DNA and shp73 treated with BA/A was arbitrarily set at 100%). To damage (38,39). investigate the underlying mechanisms, we downregulated Based on these findings, we screened a set of natural co-m p73 using specific siRNA and analyzed the levels of DNA repair pounds, which are known to inhibit ROS. Experimental inhib-i proteins. We found that inhibition of p73 reduced the levels of tion of ROS by these chemical compounds significantly reduced total and phosphorylated form of BRCA1F(igure  6C). Notably, the levels of DNA damage in esophageal cells exposed to acidic mRNA levels of BRCA1 were not altered by downregulation of bile salts. Interestingly, the preventive effect was varied with p73 suggesting that p73 regulate BRCA1 by non-transcriptional different antioxidants and cell lines, and did not correlate with mechanisms (data not shown). Thus, our results show that the their ability to scavenge ROS. Among tested antioxidants, ap-oc protective effect of apocynin is dependent on cellular p73 l-ev ynin, which is considered to be a weak ROS scavenger4(0), most els and inhibition of p73 sensitizes cells to BA/A induced DNA consistently reduced DNA damage in all analyzed esophageal damage by affecting BRCA1 protein. cell lines. Apocynin is a naturally occurring methoxy-substituted -cat in GERD patients. No p63 staining was found in all cases of BE. echol that was originally isolated from the Canadian hemp BE cell lines also showed a low expression of p63 protein. Thus, (Apocynum cannabinum). This compound presents a promis - among the p53 protein family, p73 is induced by reflux in esophing potential treatment for some inflammatory, cardiovascular ageal tissues and BE cell lines after treatment with BA/A. and neurodegenerative diseases and is known for its ability to Our data show that p73 helps to maintain the DNA integrity inhibit ROS production 4(1–43). However, our studies revealed as its experimental downregulation enhances DNA damage in that apocynin is able to reduce BA/A-induced DNA damage even cells exposed to acidic bile salts. p73 also mediates, at least in in post-BA/A treatment conditions when ROS levels were sign-ifi part, the protective effect of apocynin. When p73 protein was cantly decreased, suggesting that its protective effect cannot bedownregulated, apocynin became less effective in suppression of explained solely by its ability to suppress ROS. To further inv-es DNA damage induced by acidic bile salts. Downregulation of p73 tigate this phenomenon, we developed a novel assay that mea-s also decreased protein levels of BRCA1 and pBRCA1. Interestingly, ures the repair of genomic DNA that was damaged by acidic bile BRCA1 mRNA levels were not significantly changed, suggesting salts. Using this approach, we found that treatment with ap-oc that p73 affects BRCA1 at the post-transcriptional level. Further ynin not only suppresses ROS but also enhances the DNA repair studies are needed to investigate this regulatory mechanism. capacity of esophageal cells. Apocynin significantly accelerated In summary, a prolonged exposure to gastric acid and du-o the repair of damaged DNA. Among DNA repair proteins, we denal bile salts leads to excessive DNA damage, which causes were able to identify BRCA1 as a target of apocynin. Apocynin accumulation of tumorigenic alterations and progression to EA. increases protein levels of BRCA1 4(4). However, we cannot Mechanistically, acidic bile salts induce ROS such as hydrogen exclude that additional DDR proteins may also be involved. peroxide and superoxide in esophageal cells, which in turn, ra-p Given the important role of the p53 protein family in DNA idly induce DNA damage. We found that DNA damage can be si-g damage response and repair, we assessed the expression of the nificantly reduced by suppressing ROS with natural compounds. entire p53 family in condition of cellular damage induced by Apocynin was found to be the most effective compound as it has a acidic bile salts. We found that the p73 protein is strongly up- dual effect: reduction of ROS and facilitation of DNA damage repair. regulated by acidic bile salts in all tested cell lines. UpregulationThis process is mediated by p73 protein, which protects cells against of p73 was also observed in the esophagi of GERD patients both DNA damage induced by acidic bile salts. Collectively, our studies in squamous and Barrett’s epithelia. In striking contrast to support the concept that natural compounds can be used to prevent p73, expression of p53 was slightly increased or undetectable DNA damage and tumorigenic transformation induced by GERD. Supplementary material 21. Jaiswal, K.R. et al. (2007) Characterization of telomerase-immortalized, non-neoplastic, human Barrett’s cell line (BAR-T). Dis. Esophagus, 20, Supplementary Figure  1 can be found at http://carcin.oxford- 256–264. 22. Vilgelm, A.E. et al. (2010) Interactions of the p53 protein family in c-el lular stress response in gastrointestinal tumors. Mol. Cancer Ther., 9, Funding 693–705. 23. Iliakis, G. et  al. (2012)In vitro rejoining of double strand breaks in National Cancer Institute RO1 CA206564, the Department of genomic DNA. Methods Mol. Biol., 920, 471–484. Veteran Affairs BX002115, National Cancer Institute RO1 138833, 24. Bajpayee, M. et al. 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Bhardwaj, Vikas, Horvat, Andela, Korolkova, Olga, Washington, Mary K., El-Rifai, Wael, Dikalov, Sergey I., Zaika, Alexander I.. Prevention of DNA damage in Barrett’s esophageal cells exposed to acidic bile salts, Carcinogenesis, 2016, 1161-1169, DOI: 10.1093/carcin/bgw100