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NF-κB, inducible nitric oxide synthase and apoptosis by Helicobacter pylori infection
Free Radical Biology & Medicine, Vol. 31, No. 3, pp. 355–366, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(01)00592-5
Original Contribution NF-B, INDUCIBLE NITRIC OXIDE SYNTHASE AND APOPTOSIS BY HELICOBACTER PYLORI INFECTION JOO WEON LIM, HYEYOUNG KIM,
and
KYUNG HWAN KIM
Department of Pharmacology and Institute of Gastroenterology, Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Received 18 September 2000; Accepted 3 May 2001)
Abstract—Oxygen radicals are considered as an important regulator in the pathogenesis of Helicobacter pylori (H. pylori)-induced gastric ulceration and carcinogenesis. Inflammatory genes including inducible nitric oxide synthase (iNOS) may be regulated by oxidant-sensitive transcription factor, nuclear factor-B (NF-B). iNOS induction has been related to gastric apoptosis. We studied the role of NF-B on iNOS expression and apoptosis in H. pylori-stimulated gastric epithelial AGS cells. AGS cells were treated with antisense oligonucleotide (AS ODN) for NF-B subunit p50, an antioxidant enzyme catalase, an inhibitor of NF-B activation pyrrolidine dithiocarbamate (PDTC), iNOS inhibitors NG-nitro-L-arginine-methyl ester (L-NAME) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a peroxynitrite donor SIN-1, and a nitric oxide donor NOC-18 in the presence or absence of H. pylori. H. pylori induced cytotocixity time- and dose-dependently, which occurred with induction in iNOS expression and nitrite production. SIN-1 and NOC-18 induced dose-dependent cytotoxicity in AGS cells. Catalase, PDTC, L-NAME, and AMT prevented H. pylori-induced cytotoxicity and apoptosis. It was related to their inhibition on iNOS expression and nitrite production. The cells treated with AS ODN had low levels of p50 and NF-B and inhibited H. pylori-induced cytotoxicity, apoptosis, iNOS expression, and nitrite production. In conclusion, NF-B plays a novel role in iNOS expression and apoptosis in H. pylori-infected gastric epithelial cells. © 2001 Elsevier Science Inc. Keywords—Helicobacter pylori, NF-B, Inducible nitric oxide synthase, Apoptosis, Free radicals
INTRODUCTION
during H. pylori infection could result in the recruitment of leukocytes to infected tissues and therefore may be important in the regulation of inflammatory and immune processes in response to H. pylori. Nitric oxide (NO) produced by the inducible NO synthase (iNOS) is a critical component of host defenses against bacteria, viruses, and parasites [6,7]. In contrast to the other constitutive NOS isoforms that are active only when intracellular calcium concentrations are elevated, iNOS is always active. The regulation of iNOS is thus primarily at the level of transcription. Transcription of iNOS is induced by a variety of stimuli, including lipopolysaccharide, cytokines, and bacterial wall products. Large amounts of NO produced by iNOS are harmful to the tissue by producing peroxynitrite, which is a reaction product between NO and superoxide. Therefore, large production of NO may contribute to gastric cell injury. iNOS activity was enhanced in gastric mucosa of patients with H. pylori-positive duodenal ulcers [8,9]. Apoptosis, programmed cell death, has been charac-
Helicobacter pylori (H. pylori) has been shown to be an important pathogen of gastroduodenal inflammation and gastric carcinogenesis [1,2]. However, the pathogenic mechanisms are not well defined. One of the potential toxic factors involving H. pylori-induced gastric injury is oxygen radicals which are released from activated neutrophils, because H. pylori exhibits chemotactic activity for neutrophils [3]. Thus, neutrophil infiltration of the gastric epithelium was the initial pathological abnormality described in H. pylori gastritis and remains a hallmark of active infection. However, H. pylori itself induced the production of oxygen radicals in gastric epithelial cells, and enhances membrane damage [4,5]. Thus, prolonged production of oxygen radicals by gastric epithelial cells Address correspondence to: Hyeyoung Kim, Ph.D., Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea; Tel: ⫹82 (2) 361-5232; Fax: ⫹82 (2) 313-1894; E-Mail: [email protected]. 355
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terized morphologically by cell shrinkage and chromatin condensation, and biochemically by DNA laddering [10, 11]. NO induced apoptotic cell death in several cell systems [12–15] while H. pylori infection resulted in apoptosis of gastric epithelial cells [16,17]. Recently, the relation between iNOS expression and gastric epithelial cell apoptosis was reported in H. pylori-infected gastric epithelial cells [18,19]. The evidence that increased levels of oxidative DNA damage was related to increased occurrences of apoptosis and increased expression of iNOS, demonstrates the mechanistic links between H. pylori infection and gastric carcinogenesis [20,21]. Therefore, chronic expression of iNOS may play an important role in H. pylori-associated gastric apoptosis and carcinogenesis, in addition to propagation of gastric inflammation. iNOS gene has nuclear factor-B (NF-B)-binding sites in their promoter regions, and H. pylori is known to stimulate activation of NF-B [22,23]. NF-B is an inducible transcription factor that mediates signal transduction between cytoplasm and nucleus in many cell types [24]. NF-B is a member of the Rel family including p50 (NF-B1), p52(NF-B2), Rel A (p65), c-Rel, Rel B, and Drosophila morphogen dorsal gene product [25]. In resting cells, NF-B is localized in the cytoplasm as a hetero- or homodimer, which are noncovalently associated with cytoplasmic inhibitory proteins, including I B-␣. Upon stimulation by a variety of pathogenic inducers such as viruses, mitogens, bacteria, agents providing oxygen radicals, and inflammatory cytokines, the NF-B complex migrates into the nucleus and binds DNA recognition sites in the regulatory regions of the target genes [26]. However, it remains to be investigated whether H. pylori induce iNOS expression through a specific transcription regulation via NF-B. NF-B activation is induced by oxygen radicals such as hydrogen peroxide [27] and repressed by antioxidants [28]. Previously we found that H. pylori increased hydrogen peroxide production and induced NF-B activation in gastric epithelial cells [29,30]. Pyrrolidine dithiocarbamate (PDTC), a proven free radical scavenger and an inhibitor of NF-B activation, potentially inhibits NF-B interaction with its upstream regulatory binding site, thereby preventing NFB-mediated transcriptional activation [31]. N-acetylcysteine, an antioxidant as a precursor of glutathione (GSH), inhibited NF-B activation and protected against alloxan-induced diabetes in mice [32]. The results suggest the hypothesis that antioxidants and NF-B inhibitors might inhibit H. pylori-induced expression of iNOS and apoptosis by inhibiting oxidant-mediated activation of a transcription factor, NF-B. Antisense oligonucleotides (AS ODN) are small, synthetic molecules, 15–25 base pairs in length, and are
usually single-stranded DNA complementary to the mRNA transcribed from the target gene. Formation of a duplex structure occurs through base-pairing between the antisense DNA and its target mRNA, inhibiting gene expression [33]. Postulated mechanism of AS ODN include steric hindrance of gene transcription or gene translation, blockage of mRNA processing or splicing, and degradation of mRNA through the action of RNase H activity [34]. An additional benefit is that ODN can be chemically manipulated by replacement of a nonbridging oxygen with sulfur in a phosphate linkage to form phosphorothioate-modified ODN, making ODN more resistant to degradation by nucleases [35]. Thus, AS ODN can be made to bind to a single gene or its transcription product and act within the constraints of that gene’s expression, minimizing side effects. H. pylori induced the activation of two species of NF-B dimers (a p50/ p65 heterodimer and a p50 homodimer) in gastric epithelial cells [35,36]. As a choice for targeting by AS ODN, p50 offers the best potential for effects upon NF-B expression and the genes that NF-B regulates. We conducted the present study to evaluate the role of NF-B on H. pylori-induced expression of iNOS and apoptosis in gastric epithelial AGS cells by using an antioxidant enzyme catalase, and an inhibitor of NF-B activation PDTC to inhibit oxidant-mediated activation of NF-B and by using phosphorothioate-modified AS ODN for p50 to inhibit NF-B expression. The cells were also treated with iNOS inhibitors such as NG-nitroL-arginine-methyl ester (L-NAME) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), or a peroxynitrite donor 3-morpholinosyldononimine (SIN-1) or an NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-l-ium-1,2-diolate] (NOC-18) in the presence or absence of H. pylori. Cytotoxicity index (viable cell number), iNOS expression, nitrite production, and apoptosis indices (Hoechst staining, quantitation of DNA fragmentation) were determined. MATERIALS AND METHODS
Bacterial strain and culture condition H. pylori, strain NCTC 11637, was obtained from the American Type Culture Collection (Rockville, MD, USA). The bacteria was inoculated in a chocolate agar plate (Becton Dickinson Microbiology, Cockeysville, MD, USA) and incubated for 24 h under micro-aerobic conditions using an anaerobic chamber (BBL Campy Pouch System, Becton Dickinson Microbiology Systems) at 37°C. Whole H. pylori was harvested from a culture plate, suspended in antibiotic-free RPMI-1640 medium supplemented with 10% fetal bovine serum, and treated to AGS cells.
NF-B, iNOS and apoptosis
Gastric epithelial cell culture A human gastric epithelial cell line AGS (gastric adenocarcinoma, ATCC CRL 1739) was purchased from the American Type Culture Collection. The cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin). The cells were seeded onto a 24 well culture plate at a density of 4 ⫻ 105 cells per well in a volume of 1 ml and cultured overnight to reach 80% confluency. Prior to stimulation, each well was washed three times with 1 ml of fresh culture medium containing no antibiotics. Experimental protocol For cytotoxicity experiment, AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, 300:1, 400:1, and 500:1 for 36 h. As a cytotoxicity index, cell number was assessed by trypan blue exclusion test. After treatment of either AS ODN or S ODN (0.2 M, 0.5 M), the cells were cultured in the presence of H. pylori (at a bacterium/cell, 300:1) for 4 h and levels of p50 and NF-B were determined in nuclear extract by Western blot analysis and electrophoretic mobility shift assay (EMSA). For iNOS expression and nitrite production by H. pylori, AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, and 300:1 for 12 h. mRNA expression of iNOS was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Nitrite production was determined in the medium from the cells cultured in the presence of H. pylori at a bacterium/cell ratio of 200:1 and 300:1 for 36 h. Nitrite content in the medium was standardized to the cell number at each time point of the experiments. To determine the relation between NO and cytotoxicity, AGS cells (4 ⫻ 105 cells/well) were treated with 0.2 mM, 0.5 mM, and 1 mM of SIN-1 (Sigma Biochemical Co., St. Louis, MO, USA) or NOC-18 (Alexis Biochemicals, San Diego, CA, USA) in the absence of H. pylori for 24 h and cytotoxicity was determined. For treatment experiment, AGS cells (4 ⫻ 105cells/well) were treated with or without an antioxidant enzyme catalase (200 units/ml), an inhibitor of NF-B activation PDTC (30 M), and iNOS inhibitors such as NAME (1 mM), and AMT (5 M) or 0.5 M of AS ODN or S ODN in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 12 h (iNOS expression) and 24 h (cytotoxicity index, nitrite production, and apoptosis indices). As apoptosis indices, Hoechst staining and quantification of DNA fragmentation were used in present study. In addition, H. pylori (1.2 ⫻ 108/ml) was cultured in fresh culture medium containing no
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antibiotics in the absence of AGS cells. After 24 h, nitrite content in the medium released from H. pylori alone was determined. Catalase, PDTC, NAME, and AMT were obtained from Sigma.
ODNs preparation Single-stranded ODNs were produced commercially (GIBCO BRL, Rockville, MD, USA). ODNs were phosphorothioate-modified to reduce intracellular nuclease digestion. Antisense (AS) and sense (S) ODNs targeted the ATG start codon of the p50 mRNA. The sequence of the p50 AS ODN was 5⬘ GGA TCA TCT TCT GCC ATT CTG 3⬘. The sequence of p50 S ODN was 5⬘ CAG AAT GGC AGA AGA TGA TCC 3⬘.
Treatment with ODNs using cationic liposome AGS cells were treated with ODNs using a cationic liposome, a commercially available transfection-reagent DOTAP (N-[1-(2,3-dioleoyloxy) propyl]-N,N,Ntrimethyl ammonium methylsulfate) (Boehringer-Mannheim, Pentz berg, Germany) to improve stability and intracellular delivery of ODNs [37]. When DOTAP was employed, the appropriate amount of ODNs were incubated with DOTAP (15 l/ml) to achieve the respective final concentration of the ODNs to either 0.2 or 0.5 M at 37°C for 15 min. Then the mixture was added directly to the cells, plated at 1 ⫻ 105 cells/ml in 12 well plates, and incubated for 55 h. Cells were trypsinized and plated again at the density of 1 ⫻ 106 cells/ml in 12 well plates. 0.2 or 0.5 M (final concentration) of ODN were added to the cells and incubated for another 17 h. After medium was changed with antibioticfree medium, ODN-transfected cells were cultured in the presence of H. pylori. Time point for the determination of NF-B and p50 was 4 h, which was adopted from our previous studies [29,30]. Western blot analysis for NF-B subunit p50 Nuclear extract was prepared from the cells treated with or without ODNs described below and nuclear protein was determined by the method of Bradford [38]. One hundred g of protein was loaded per lane, separated by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions, and transferred onto Hybond-PVDF membranes (Amersham Inc., Arlington Heights, IL, USA) by electroblotting. The transfer of protein and equality of loading in all lanes was verified using reversible staining with Ponceau S. Membranes were blocked using 5% nonfat dry milk. NF-B subunit p50 protein was detected by incubation of blots with a
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specific monoclonal antibody to synthetic peptides derived from human p50 amino acid sequences (Cayman Chemical, Ann Arbor, MI, USA) at a dilution of 1:1000 overnight at 4°C, followed by sheep anti-mouse secondary antibody conjugated to horseradish peroxidase and determination of enhanced chemiluminescence (Amersham) using exposure to BioMax MR film (Kodak). Extraction of nuclei AGS cells treated with or without AS ODN or S ODN were cultured in the absence or the presence of H. pylori. The culture medium was aspirated and the cells were rinsed with ice-cold phosphate-buffered saline (PBS), harvested by scraping into PBS, and pelleted by centrifugation at 1500 ⫻ g for 5 min. The cells were lysed in buffer containing 10 mM Hepes, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM MgCl2, 0.2% Nonidet P-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonylfluoride (PMSF). The nuclear pellet was resuspended on ice in nuclear extraction buffer containing 20 mM Hepes, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF [39], and the nuclear protein concentration was determined by the method of Bradford [38]. NF-B activation by EMSA NF-B gel shift oligonucleotide, 5⬘AGT TGA GGG GAC TTT CCC AGG C-3⬘ (Promega Corp., Madison, WI, USA) was labeled with [32P] dATP (Amersham) using T4 polynucleotide kinase (GIBCO-BRL, Grand Island, NY, USA). End-labeled probe was purified from unincorporated [32P] dATP using a Bio-Rad purification column (Bio-Rad Laboratories, Hercules, CA, USA) and recovered in tris-EDTA buffer (TE). Nuclear extracts (5 g) were preincubated in buffer containing 12% glycerol; 12 mM Hepes, pH 7.9; 4 mM Tris-HCl, pH 7.9; 1 mM EDTA; 1 mM DTT; 25 mM KCl; 5 mM MgCl2; 0.04 g/ml poly[d(I-C)] (Boehringer Mannheim, Indianapolis, IN, USA); 0.4 mM PMSF; and TE. The labeled probe was added and samples were incubated on ice for 10 min. Samples were subjected to electrophoretic separation at room temperature on a nondenaturing 5% acrylamide gel at 30 mA using 0.5 X Tris borate EDTA buffer. The gels were dried at 80° for 1 h and exposed to the radiography film for 6 –18 h at ⫺70° with intensifying screens [40]. mRNA expression of iNOS by RT-PCR Total RNA was isolated from cells by guanidine thiocyanate extraction method [41]. The internal standard
(-actin) was coamplified with iNOS. RNA was reverse transcribed into cDNA and used for PCR with human iNOS and -actin specific primers. For iNOS, primers were 5⬘-CTGCATGGATAAGTACAGGCTGAGC-3⬘ (a 25-mer forward primer at position 1593) and 5⬘-AGCTTCTGATCAATGTCATGAGCAA-3⬘ (a 25-mer reverse primer at position 1817), giving rise to a 225 bp PCR product [22]. For -actin, primers were 5⬘-ACCAACTGGGACGACATGGAG-3⬘ (a 21-mer forward primer at position 270) and 5⬘-GTGAGGATCTTCATGAGGTAGTC-3⬘ (a 23-mer reverse primer at position 624), giving rise to a 353 bp PCR product [42]. Briefly, the PCR was amplified by 30 repeat denaturation cycles at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s. During the first cycle, the 95°C step extended to 2 min, and on the final cycle the 72°C step extended to 5 min. PCR products were separated on 1.5% agarose gels containing 0.5 g/ml ethidium bromide and visualized by UV transillumination.
Fluorometric determination of nitrite 2,3-Diaminonaphthalene (DAN) is reacted with nitrite under acidic conditions to form 1-(1)-naphthotriazole, a fluorescent product. Culture medium (500 l) was first brought to volume (1 ml) with deionized water. To this, 100 l of freshly prepared DAN (0.05 mg/ml in 0.62 M HCl) was added and mixed immediately. After 10 min incubation at 20°C, the reaction was terminated with 50 l of 2.8 N NaOH [43]. Formation of the 2,3-diaminonaphthtriazol was measured using a SPF-500C spectrofluorometer (SLM Instruments, Inc., Urbana, IL, USA). Nitrite content was standardized to the cell number at the end of the experiment and expressed as nmol/ 106 cells.
Morphological characterization of cell death by Hoechst staining AGS cells (4 ⫻ 105/well), plated onto glass coverslips in 24 well plates, were treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. The cells were washed twice with PBS, fixed with 4% paraformadehyde for 30 min at room temperature, and stained with 1 mM bisbenzimide (Hoechst 33258; Sigma) for 10 min at 37°C. After washing with tap water for 5 min, cover-slips were mounted onto microscope slide and nuclear morphology was observed under a fluorescence microscope at 400⫻ [44].
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Apoptotic cell count by Hoechst staining Assessment of apoptotic cell death was performed by staining with DNA-specific dye Hoechst 33258. AGS cells (4 ⫻ 105cells/well) were treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. The cells were washed with PBS (pH 7.4), fixed with 4% paraformaldehyde for 30 min, and then stained with 1 mM Hoechst 33258 for 10 min at 37°C. Cells were washed again with PBS prior to viewing stained nuclei under an inverted microscope [45]. Percentage of apoptotic cells, assessed by staining with Hoechst 33258, was calculated based on total numbers of the cells at the end of the experiment. Quantitation of DNA fragmentation Nucleosomes were quantified by means of a sandwich enzyme-linked immunosorbent assay (ELISA) (Cell Death Detection ELISAplus kit; Boehringer Mannheim GmbH, Germany). AGS cells (4 ⫻ 105cells/well) were treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. The cells were detached and 1 ⫻ 105 of cells were lysed. After cell lysis, oligonucleasome-bound DNA was quantitated by using biotin-coupled mouse monoclonal anti-histone antibody as a capturing antibody, peroxidase-conjugated mouse monoclonal anti-DNA antibody as a detecting antibody, and 2,2⬘-azio-di[3ethylbenhioazolin-sulfonate (ABTC) as a developing reagent. The relative increase in nucleosomes in the cell lysate, determined at 405 nm, was expressed as an enrichment factor. Enrichment factor of the cells received no treatment and cultured in the absence of H. pylori (none) was considered as 1. Statistical analysis Results are expressed as means ⫾ SE of four different experiments. Analysis of variance (ANOVA) followed by Newman-Keul’s test was used for statistical analysis [46]. p ⬍ .05 was considered statistically significant. RESULTS
Cytotoxicity by H. pylori in AGS cells, depending on the number of bacterium treated to the cells To determine whether H. pylori infection induces cytotoxicity of AGS cells, the cells were cultured in
Fig. 1. Cytotoxicity by H. pylori in AGS cells, depending on the number of bacterium treated to the cells. (A) AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/ cell ratio of 200:1 and 300:1 for 36 h. At an indicated time point, cell number was determined by trypan blue exclusion test. (B) AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, 300:1, 400:1, and 500:1 for 24 h. Cell number was determined by trypan blue exclusion test. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the corresponding cells at the start of experiment (0 h) (A) or none (B). None ⫽ the cells cultured in the absence of H. pylori.
the presence of H. pylori and the viable cells were counted at an indicated time point (Fig. 1A). Cytotoxicity of AGS cells increased with the number of bacterium treated to the cells. It started within 12 h of culture at a bacterium/cell ratio of 300:1 and 24 h at a bacterium/cell ratio of 200:1. At 36 h, the number of viable cells, cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1, decreased to 26% of that of the cells cultured in the absence of H. pylori (none). H. pylori-induced cytotoxicity was determined by the experiment using more defined ratios of bacterium/cell from 100:1 to 500:1 (Fig. 1B). At 24 h, reduction in viable cell number was shown at a bacterium/cell ratio, 200:1. The cell number was lower than that at the start of the experiment with the number of bacte-
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Fig. 2. iNOS expression and nitrite production by H. pylori in AGS cells, depending on the number of bacterium treated to the cells. (A) AGS cells (4 ⫻ 105cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, and 300:1 for 12 h. mRNA expression of iNOS was determined by RT-PCR analysis. The internal standard (-actin) was coamplified with iNOS. (B) AGS cells (4 ⫻ 105cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 200:1 and 300:1 for 36 h. Nitrite content in medium was determined at an indicated time point and expressed as nmol/106 cells. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to nitrite production from the corresponding cells at the start of experiment (0 h). None ⫽ the cells cultured in the absence of H. pylori.
rium treated to AGS cells up to the ratio of bacterium/ cell, 500:1. Therefore, in further experiments on NFB, iNOS, and apoptosis, we used a bacterium/cell ratio of 300:1 and a 24 h culture period. iNOS expression and nitrite production by H. pylori in AGS cells, depending on the number of bacterium treated to the cells RT-PCR analysis shows that H. pylori induced mRNA expression of iNOS with the number of bacterium treated to the cells at 12 h (Fig. 2A). At a bacterium/ cell ratio, 200:1, iNOS expression was shown in the cells and more increased at a bacterium/cell ratio, 300:1. Nitrite production from the cells, cultured in the presence of H. pylori at bacterium/cell ratios of 200:1 and 300:1, was significantly higher at 24 h and even increased at 36 h (Fig. 2B). Nitrite released from the cells (nmol/106 cells), standardized to the cell number at each time point of the experiment, were 3.9 ⫾ 2.1, 4.5 ⫾ 1.9, 5.0 ⫾ 1.7, and 5.4 ⫾ 1.5 at 0, 12, 24, and 36 h in the absence of H. pylori (none). At a bacterium/cell ratio of 300:1, nitrite
Fig. 3. Cytotoxicity by SIN-1 and NOC-18 in AGS cells. AGS cells (4 ⫻ 105 cells/well) were treated with SIN-1 (0.2 mM, 0.5 mM, 1.0 mM) or NOC-18 (0.2 mM, 0.5 mM, 1.0 mM) and cultured in the absence of H. pylori for 24 h. Cell number was determined by trypan blue exclusion test. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment (none).
contents released into the medium, which was standardized to the cell number at each time point (nmol/106 cells), were 4.1 ⫾ 3.0, 32.6 ⫾ 3.3, 57.0 ⫾ 6.1, and 77.5 ⫾ 5.9 at 0, 12, 24, and 36 h, respectively. H. pylori alone, plated at 1.2 ⫻ 108/ml, produced a relatively small amount of nitrite (6.9 ⫾ 0.7 nmol/ml) at 24 h in the absence of AGS cells. Cytotoxicity by SIN-1 and NOC-18 in AGS cells To determine the relation between iNOS expression and cytotoxicity, AGS cells were treated with a peroxynitrite donor, SIN-1 (0.2 mM, 0.5 mM, 1 mM) and an NO donor NOC-18 (0.2 mM, 0.5 mM, 1 mM) in the absence of H. pylori (Fig. 3). At 24 h, dose-dependent reduction in viable cell number was shown in the cells treated with SIN-1 and NOC-18. These results demonstrate the possible involvement of high amounts of NO in apoptosis of AGS cells.
NF-B, iNOS and apoptosis
Fig. 4. Western blot analysis of p50 and NF-B activation by H. pylori in AGS cells treated with ODNs. Either AS ODN or S ODN (0.2 M, 0.5 M) was treated to the cells and cultured in the presence of H. pylori for 4 h. Nuclear protein was analyzed for p50 protein by Western blot analysis (A) and NF-B activation by EMSA (B). None ⫽ the cells received no ODNs and cultured in the absence of H. pylori. H. pylori alone ⫽ the cells received ODNs, but cultured in the presence of H. pylori.
Western blot analysis of p50 and NF-B activation by H. pylori in AGS cells treated with ODNs Either AS ODN or S ODN (0.2 M, 0.5 M) was treated directly to the cells and cultured in the presence of H. pylori for 4 h. Transfection efficiency of ODNs was assessed by determining the level of p50 protein in the nucleus (Fig. 4A). Western blot analysis showed that H. pylori induced increase in p50 in the nucleus. The increase in p50 by H. pylori was inhibited by the treatment of AS ODN, but not by S ODN. H. pylori induced the activation of two species of NF-B dimers (a p50/p65 heterodimer and a p50 homodimer) in gastric epithelial cells [35]. Thus, reduction in p50 protein may cause inhibition of NF-B activation. As shown in Fig. 4B, H. pylori induced NF-B activation, and treatment of AS ODN inhibited NF-B
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Fig. 5. Cytotoxicity by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation and iNOS inhibitors or ODNs. AGS cells (4 ⫻ 105cells/well), treated with or without catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A) or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), were cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Cell number was determined by trypan blue exclusion test. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none control). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori control). None ⫽ the cells cultured in the absence of H. pylori.
activation dose-dependently. The cells treated with S ODN had significant NF-B activation in the nucleus by H. pylori infection. These results demonstrate that H. pylori-induced NF-B activation was inhibited by treatment of p50 AS ODN to the cells. Cytotoxicity by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs Viable cell number was determined in the cells, treated with or without catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M), or 0.5 M of AS ODN or S ODN in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h (Fig. 5). H. pylori
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caused 38% reduction in viable cell number as compared to those of the cells cultured in the absence of H. pylori (none control). Treatment of an antioxidant enzyme catalase and an inhibitor of NF-B activation PDTC inhibited the H. pylori-induced cytotoxicity of the cells, resulting in 10 –14% increase in viable cell number as compared to the cells receiving no treatment and cultured in the presence of H. pylori (H. pylori control). iNOS inhibitors, NAME and AMT, significantly inhibited H. pylori-mediated reduction in viable cell number. Treatment of AS ODN showed similar inhibition on H. pyloriinduced cytotoxicity while S ODN had no inhibitory effect on the reduction in viable cells caused by H. pylori (Fig. 5). Treatment of an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs did not itself induce cytotoxicity of the cells in the absence of H. pylori, determined by viable cell number. iNOS expression and nitrite production by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation and ODNs AGS cells were treated with or without catalase (200 units/ml), PDTC (30 M), or 0.5 M AS ODN or S ODN and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 12 h. H. pylori-induced iNOS expression was inhibited by catalase, PDTC, and AS ODN, but not by S ODN (Fig. 6A). Nitrite production from the cells was increased by H. pylori infection; 10 –12-fold increase compared to the cells in the absence of H. pylori (Fig. 6B). Increase in nitrite production by H. pylori was inhibited by catalase, PDTC, and AS ODN, but treatment of S ODN showed no effect on nitrite production. In the absence of H. pylori, catalase, PDTC, and ODNs affected neither iNOS expression nor nitrite production from the cells. Apoptosis by H. pylori in AGS cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs Staining of DNA-specific dye Hoechst 33258 demonstrated that H. pylori caused severely fragmented nuclei and contained highly condensed chromatin (Fig. 7). This apoptotic phenomenon was inhibited by catalase (200 units/ml), PDTC (30 M), NAME (1 mM), AMT (5 M), and AS ODN (0.5 M). Treatment of S ODN showed similar apoptotic nuclear morphology (nuclear condensation and fragmentation) as that shown in the cells receiving no treatment, but cultured in the presence of H. pylori (H. pylori alone). Percentage of apopotic cells determined by staining with Hoechst 33258, was calculated based on total numbers of the cells (Fig. 8). H.
Fig. 6. iNOS expression and nitrite production by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs. AGS cells (4 ⫻ 105cells/well), were treated with or without catalase (200 units/ml), PDTC (30 M), NAME (1 mM), AMT (5 M), or 0.5 M of antisense (AS) ODN or sense (S) ODN, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 12 h. (A) mRNA expression of iNOS was determined by RT-PCR analysis. The internal standard (-actin) was coamplified with iNOS. (B) Nitrite content in culture medium was determined and expressed as nmol/106 cells. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none control). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori control). None ⫽ the cells cultured in the absence of H. pylori.
pylori induced 7-fold increase in the number of apoptotic cells, which was inhibited by catalase, PDTC, NAME, AMT, and AS ODN. S ODN had no effect on H. pyloriinduced increase in apoptotic cells. H. pylori-induced DNA fragmentation was determined by the amount of oligonucleasome-bound DNA in the cell lysate (Fig. 9). Apoptosis-associated increase in nucleosome-associated
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Fig. 8. Assessment of apoptotic cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, determined by Hoechst staining. AGS cells (4 ⫻ 105cells/well), were treated with catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A), or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Monolayers of cells were fixed in 4% paraformaldehyde and were stained with DNA-specific dye Hoechst 33258. % of apoptotic cells were assessed based on total numbers of the cells. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori alone). Fig. 7. Hoechst staining of AGS cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs. AGS cells (4 ⫻ 105cells/well), were treated either with catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A), or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Monolayers of cells onto coverslips were fixed in 4% paraformaldehyde and were stained with DNA-specific dye Hoechst 33258. Nuclear morphology was observed under a fluorescence microscope at 400⫻. None ⫽ the cells received no treatment and cultured in the absence of H. pylori. H. pylori alone ⫽ the cells received no treatment, but cultured in the presence of H. pylori.
low-molecular-weight DNA was inhibited by catalase, PDTC, iNOS inhibitors, and AS ODN, but not by S ODN. DISCUSSION
Helicobacter pylori (H. pylori) may affect normal balance between cell proliferation and cell death in gas-
tric mucosa. Recently, several investigators reported that H. pylori activated NF-B in human gastric mucosa in vivo and cultured gastric epithelial cells in vitro [47– 49]. This bacterium directly induced apoptosis of human gastric epithelial cells [16,17]. In the present study, we showed that H. pylori activated NF-B and induced apoptosis of gastric epithelial AGS cells. To inhibit NF-B activation, we used antioxidants, catalase and PDTC, because these antioxidants, which scavenge oxygen radicals, are known to inhibit the activation of NF-B [50,51]. Additionally, we used AS ODN to p50 of NF-B, which specifically inhibited NF-B expression. Inhibition of NF-B activation suppressed H. pylori-induced apoptosis in AGS cells. These results demonstrate that NF-B may play an important role in H. pylori-induced apoptosis of gastric epithelial cells. Because the promoter of human iNOS gene contains a regulatory DNA sequence to which NF-B binds [22]
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Fig. 9. Quantification of DNA fragmentation of AGS cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs. AGS cells (4 ⫻ 105cells/well), were treated either with catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A), or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Cell lysates were isolated from 1 ⫻ 105 cells. DNA fragmentation was determined by the amount of oligonucleasomebound DNA in the cell lysate. The relative increase in nucleosomes in the cell lysate, determined at 405 nm, was expressed as an enrichment factor. Enrichment factor of none was considered as 1. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori alone).
and the expression of iNOS have been regulated by NF-B in several cell lines [22,23], we next investigated whether NF-B would regulate iNOS expression and nitrite production in H. pylori-stimulated AGS cells. Furthermore, the relation between iNOS and apoptosis was reported in several cells [12–15]. iNOS expression was induced in gastric mucosa of patients with H. pyloripositive duodenal ulcers in vivo [8] and in macrophages treated with H. pylori in vitro [52,53]. Furthermore, Mannick et al. reported that dietary supplement with antioxidant -carotene reduced nitrotyrosine staining, a marker for peroxynitrite production, in gastric mucosa of patients with H. pylori infection [54]. In the present study, we showed that H. pylori infection increased iNOS expression and nitrite production with the number of bacterium treated to the cells, and inhibition of NF-B activation blocked iNOS expression and nitrite produc-
tion in H. pylori-stimulated AGS cells. These results indicate that H. pylori may cause NF-B activation and iNOS expression, which induces high NO production and apoptosis in gastric epithelial cells. We showed that iNOS inhibitors suppressed H. pylori-mediated cytotoxicity and apoptosis, determined by DNA fragmentation and chromatin condensation in gastric epithelial cells. Furthermore, a peroxynitrite donor SIN-1 and an NO donor NOC-18 dose-dependently induced reduction in viable cells. Together with inhibition on cytotoxicity and apoptosis by NF-B inhibition, effects of iNOS inhibitors confirms the above hypothesis that iNOS expression mediated by NF-B activation is associated with H. pylori-induced apoptosis in gastric epithelial cells. The exact mechanism how NO leads to DNA damage in the cells undergoing apoptosis has not been clarified. One possible explanation is that the reaction of NO with oxygen radicals results in production of highly toxic nitrous radical peroxynitrite. Peroxynitrite may attack aromatic amines such as pyridine and purine, finally leading to DNA strand breaks [55]. NO effect on nucleic acids has been determined for naked DNA in normal cells [56,57]. In addition, NO produced by iNOS may react with ferrous-sulfate-containing proteins and form nitrosyl complexes, which lead to cellular damage [58]. Finally, NO derivatives such as nitrosamine might be one toxic material produced by H. pylori in gastric mucosa. Recent studies show that NF-B activation may be associated with cell apoptosis [59,60]. In particular, NF-B stimulates the tumor suppressor gene p53, and NF-B subunit p65 interacts with cell cycle inhibitor p21WAF1 [61,62]. Grilli et al. reported that blocking NF-B could protect neurons against neurotoxicity [63]. Additionally, several investigators reported that NF-B is concomitantly activated in TNF-␣-induced apoptosis [64,65] and inhibition of NF-B activation by certain antioxidants prevent apoptosis [66]. Apoptosis in gastric mucosa was reduced by supplementation with either antioxidant ascorbic acid or -carotene in H. pyloripositive patients [54]. These evidences suggest that NF-B may promote apoptosis in gastric epithelial cells. While NF-B activation is detrimental in the present study, several studies have shown that NF-B activation blocks apoptosis to a variety of insults [67–70]. Thus, transcription factor NF-B can be both detrimental and protective, depending on cell type in which it is expressed and the nature of the insult. Present study demonstrates the link between H. pylori-induced NF-B activation and apoptosis, which is mediated by iNOS expression induced by H. pylori in gastric epithelial cells. Further studies on the nature and the time point of gastric epithelial cell death induced by H. pylori infection, such as early apoptosis, late apoptosis, and possible
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necrosis, are necessary to provide a better understanding and therapeutic approach towards controlling H. pyloriinduced gastric injury. Acknowledgement — This study was supported by a Science Research Center grant from Korea Science and Engineering Foundation (KOSEF) to the Nitric Oxide Radical Toxicity Research Center (NORTReC) made in the program year of 2000 (H.K.).
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ABBREVIATIONS
H. pylori—Helicobacter pylori NF-B—nuclear factor-B INOS—inducible nitric oxide synthase PDTC—pyrrolidine dithiocarbamate L-NAME—NG-nitro-L-arginine-methyl ester AMT—2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine AS ODN—antisense oligonucleotide S ODN—sense oligonucleotide RT-PCR—reverse transcription-polymerase chain reaction EMSA— electrophoretic mobility shift assay
PII S0891-5849(01)00592-5
Original Contribution NF-B, INDUCIBLE NITRIC OXIDE SYNTHASE AND APOPTOSIS BY HELICOBACTER PYLORI INFECTION JOO WEON LIM, HYEYOUNG KIM,
and
KYUNG HWAN KIM
Department of Pharmacology and Institute of Gastroenterology, Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea (Received 18 September 2000; Accepted 3 May 2001)
Abstract—Oxygen radicals are considered as an important regulator in the pathogenesis of Helicobacter pylori (H. pylori)-induced gastric ulceration and carcinogenesis. Inflammatory genes including inducible nitric oxide synthase (iNOS) may be regulated by oxidant-sensitive transcription factor, nuclear factor-B (NF-B). iNOS induction has been related to gastric apoptosis. We studied the role of NF-B on iNOS expression and apoptosis in H. pylori-stimulated gastric epithelial AGS cells. AGS cells were treated with antisense oligonucleotide (AS ODN) for NF-B subunit p50, an antioxidant enzyme catalase, an inhibitor of NF-B activation pyrrolidine dithiocarbamate (PDTC), iNOS inhibitors NG-nitro-L-arginine-methyl ester (L-NAME) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a peroxynitrite donor SIN-1, and a nitric oxide donor NOC-18 in the presence or absence of H. pylori. H. pylori induced cytotocixity time- and dose-dependently, which occurred with induction in iNOS expression and nitrite production. SIN-1 and NOC-18 induced dose-dependent cytotoxicity in AGS cells. Catalase, PDTC, L-NAME, and AMT prevented H. pylori-induced cytotoxicity and apoptosis. It was related to their inhibition on iNOS expression and nitrite production. The cells treated with AS ODN had low levels of p50 and NF-B and inhibited H. pylori-induced cytotoxicity, apoptosis, iNOS expression, and nitrite production. In conclusion, NF-B plays a novel role in iNOS expression and apoptosis in H. pylori-infected gastric epithelial cells. © 2001 Elsevier Science Inc. Keywords—Helicobacter pylori, NF-B, Inducible nitric oxide synthase, Apoptosis, Free radicals
INTRODUCTION
during H. pylori infection could result in the recruitment of leukocytes to infected tissues and therefore may be important in the regulation of inflammatory and immune processes in response to H. pylori. Nitric oxide (NO) produced by the inducible NO synthase (iNOS) is a critical component of host defenses against bacteria, viruses, and parasites [6,7]. In contrast to the other constitutive NOS isoforms that are active only when intracellular calcium concentrations are elevated, iNOS is always active. The regulation of iNOS is thus primarily at the level of transcription. Transcription of iNOS is induced by a variety of stimuli, including lipopolysaccharide, cytokines, and bacterial wall products. Large amounts of NO produced by iNOS are harmful to the tissue by producing peroxynitrite, which is a reaction product between NO and superoxide. Therefore, large production of NO may contribute to gastric cell injury. iNOS activity was enhanced in gastric mucosa of patients with H. pylori-positive duodenal ulcers [8,9]. Apoptosis, programmed cell death, has been charac-
Helicobacter pylori (H. pylori) has been shown to be an important pathogen of gastroduodenal inflammation and gastric carcinogenesis [1,2]. However, the pathogenic mechanisms are not well defined. One of the potential toxic factors involving H. pylori-induced gastric injury is oxygen radicals which are released from activated neutrophils, because H. pylori exhibits chemotactic activity for neutrophils [3]. Thus, neutrophil infiltration of the gastric epithelium was the initial pathological abnormality described in H. pylori gastritis and remains a hallmark of active infection. However, H. pylori itself induced the production of oxygen radicals in gastric epithelial cells, and enhances membrane damage [4,5]. Thus, prolonged production of oxygen radicals by gastric epithelial cells Address correspondence to: Hyeyoung Kim, Ph.D., Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea; Tel: ⫹82 (2) 361-5232; Fax: ⫹82 (2) 313-1894; E-Mail: [email protected]. 355
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terized morphologically by cell shrinkage and chromatin condensation, and biochemically by DNA laddering [10, 11]. NO induced apoptotic cell death in several cell systems [12–15] while H. pylori infection resulted in apoptosis of gastric epithelial cells [16,17]. Recently, the relation between iNOS expression and gastric epithelial cell apoptosis was reported in H. pylori-infected gastric epithelial cells [18,19]. The evidence that increased levels of oxidative DNA damage was related to increased occurrences of apoptosis and increased expression of iNOS, demonstrates the mechanistic links between H. pylori infection and gastric carcinogenesis [20,21]. Therefore, chronic expression of iNOS may play an important role in H. pylori-associated gastric apoptosis and carcinogenesis, in addition to propagation of gastric inflammation. iNOS gene has nuclear factor-B (NF-B)-binding sites in their promoter regions, and H. pylori is known to stimulate activation of NF-B [22,23]. NF-B is an inducible transcription factor that mediates signal transduction between cytoplasm and nucleus in many cell types [24]. NF-B is a member of the Rel family including p50 (NF-B1), p52(NF-B2), Rel A (p65), c-Rel, Rel B, and Drosophila morphogen dorsal gene product [25]. In resting cells, NF-B is localized in the cytoplasm as a hetero- or homodimer, which are noncovalently associated with cytoplasmic inhibitory proteins, including I B-␣. Upon stimulation by a variety of pathogenic inducers such as viruses, mitogens, bacteria, agents providing oxygen radicals, and inflammatory cytokines, the NF-B complex migrates into the nucleus and binds DNA recognition sites in the regulatory regions of the target genes [26]. However, it remains to be investigated whether H. pylori induce iNOS expression through a specific transcription regulation via NF-B. NF-B activation is induced by oxygen radicals such as hydrogen peroxide [27] and repressed by antioxidants [28]. Previously we found that H. pylori increased hydrogen peroxide production and induced NF-B activation in gastric epithelial cells [29,30]. Pyrrolidine dithiocarbamate (PDTC), a proven free radical scavenger and an inhibitor of NF-B activation, potentially inhibits NF-B interaction with its upstream regulatory binding site, thereby preventing NFB-mediated transcriptional activation [31]. N-acetylcysteine, an antioxidant as a precursor of glutathione (GSH), inhibited NF-B activation and protected against alloxan-induced diabetes in mice [32]. The results suggest the hypothesis that antioxidants and NF-B inhibitors might inhibit H. pylori-induced expression of iNOS and apoptosis by inhibiting oxidant-mediated activation of a transcription factor, NF-B. Antisense oligonucleotides (AS ODN) are small, synthetic molecules, 15–25 base pairs in length, and are
usually single-stranded DNA complementary to the mRNA transcribed from the target gene. Formation of a duplex structure occurs through base-pairing between the antisense DNA and its target mRNA, inhibiting gene expression [33]. Postulated mechanism of AS ODN include steric hindrance of gene transcription or gene translation, blockage of mRNA processing or splicing, and degradation of mRNA through the action of RNase H activity [34]. An additional benefit is that ODN can be chemically manipulated by replacement of a nonbridging oxygen with sulfur in a phosphate linkage to form phosphorothioate-modified ODN, making ODN more resistant to degradation by nucleases [35]. Thus, AS ODN can be made to bind to a single gene or its transcription product and act within the constraints of that gene’s expression, minimizing side effects. H. pylori induced the activation of two species of NF-B dimers (a p50/ p65 heterodimer and a p50 homodimer) in gastric epithelial cells [35,36]. As a choice for targeting by AS ODN, p50 offers the best potential for effects upon NF-B expression and the genes that NF-B regulates. We conducted the present study to evaluate the role of NF-B on H. pylori-induced expression of iNOS and apoptosis in gastric epithelial AGS cells by using an antioxidant enzyme catalase, and an inhibitor of NF-B activation PDTC to inhibit oxidant-mediated activation of NF-B and by using phosphorothioate-modified AS ODN for p50 to inhibit NF-B expression. The cells were also treated with iNOS inhibitors such as NG-nitroL-arginine-methyl ester (L-NAME) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), or a peroxynitrite donor 3-morpholinosyldononimine (SIN-1) or an NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-l-ium-1,2-diolate] (NOC-18) in the presence or absence of H. pylori. Cytotoxicity index (viable cell number), iNOS expression, nitrite production, and apoptosis indices (Hoechst staining, quantitation of DNA fragmentation) were determined. MATERIALS AND METHODS
Bacterial strain and culture condition H. pylori, strain NCTC 11637, was obtained from the American Type Culture Collection (Rockville, MD, USA). The bacteria was inoculated in a chocolate agar plate (Becton Dickinson Microbiology, Cockeysville, MD, USA) and incubated for 24 h under micro-aerobic conditions using an anaerobic chamber (BBL Campy Pouch System, Becton Dickinson Microbiology Systems) at 37°C. Whole H. pylori was harvested from a culture plate, suspended in antibiotic-free RPMI-1640 medium supplemented with 10% fetal bovine serum, and treated to AGS cells.
NF-B, iNOS and apoptosis
Gastric epithelial cell culture A human gastric epithelial cell line AGS (gastric adenocarcinoma, ATCC CRL 1739) was purchased from the American Type Culture Collection. The cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin). The cells were seeded onto a 24 well culture plate at a density of 4 ⫻ 105 cells per well in a volume of 1 ml and cultured overnight to reach 80% confluency. Prior to stimulation, each well was washed three times with 1 ml of fresh culture medium containing no antibiotics. Experimental protocol For cytotoxicity experiment, AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, 300:1, 400:1, and 500:1 for 36 h. As a cytotoxicity index, cell number was assessed by trypan blue exclusion test. After treatment of either AS ODN or S ODN (0.2 M, 0.5 M), the cells were cultured in the presence of H. pylori (at a bacterium/cell, 300:1) for 4 h and levels of p50 and NF-B were determined in nuclear extract by Western blot analysis and electrophoretic mobility shift assay (EMSA). For iNOS expression and nitrite production by H. pylori, AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, and 300:1 for 12 h. mRNA expression of iNOS was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Nitrite production was determined in the medium from the cells cultured in the presence of H. pylori at a bacterium/cell ratio of 200:1 and 300:1 for 36 h. Nitrite content in the medium was standardized to the cell number at each time point of the experiments. To determine the relation between NO and cytotoxicity, AGS cells (4 ⫻ 105 cells/well) were treated with 0.2 mM, 0.5 mM, and 1 mM of SIN-1 (Sigma Biochemical Co., St. Louis, MO, USA) or NOC-18 (Alexis Biochemicals, San Diego, CA, USA) in the absence of H. pylori for 24 h and cytotoxicity was determined. For treatment experiment, AGS cells (4 ⫻ 105cells/well) were treated with or without an antioxidant enzyme catalase (200 units/ml), an inhibitor of NF-B activation PDTC (30 M), and iNOS inhibitors such as NAME (1 mM), and AMT (5 M) or 0.5 M of AS ODN or S ODN in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 12 h (iNOS expression) and 24 h (cytotoxicity index, nitrite production, and apoptosis indices). As apoptosis indices, Hoechst staining and quantification of DNA fragmentation were used in present study. In addition, H. pylori (1.2 ⫻ 108/ml) was cultured in fresh culture medium containing no
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antibiotics in the absence of AGS cells. After 24 h, nitrite content in the medium released from H. pylori alone was determined. Catalase, PDTC, NAME, and AMT were obtained from Sigma.
ODNs preparation Single-stranded ODNs were produced commercially (GIBCO BRL, Rockville, MD, USA). ODNs were phosphorothioate-modified to reduce intracellular nuclease digestion. Antisense (AS) and sense (S) ODNs targeted the ATG start codon of the p50 mRNA. The sequence of the p50 AS ODN was 5⬘ GGA TCA TCT TCT GCC ATT CTG 3⬘. The sequence of p50 S ODN was 5⬘ CAG AAT GGC AGA AGA TGA TCC 3⬘.
Treatment with ODNs using cationic liposome AGS cells were treated with ODNs using a cationic liposome, a commercially available transfection-reagent DOTAP (N-[1-(2,3-dioleoyloxy) propyl]-N,N,Ntrimethyl ammonium methylsulfate) (Boehringer-Mannheim, Pentz berg, Germany) to improve stability and intracellular delivery of ODNs [37]. When DOTAP was employed, the appropriate amount of ODNs were incubated with DOTAP (15 l/ml) to achieve the respective final concentration of the ODNs to either 0.2 or 0.5 M at 37°C for 15 min. Then the mixture was added directly to the cells, plated at 1 ⫻ 105 cells/ml in 12 well plates, and incubated for 55 h. Cells were trypsinized and plated again at the density of 1 ⫻ 106 cells/ml in 12 well plates. 0.2 or 0.5 M (final concentration) of ODN were added to the cells and incubated for another 17 h. After medium was changed with antibioticfree medium, ODN-transfected cells were cultured in the presence of H. pylori. Time point for the determination of NF-B and p50 was 4 h, which was adopted from our previous studies [29,30]. Western blot analysis for NF-B subunit p50 Nuclear extract was prepared from the cells treated with or without ODNs described below and nuclear protein was determined by the method of Bradford [38]. One hundred g of protein was loaded per lane, separated by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions, and transferred onto Hybond-PVDF membranes (Amersham Inc., Arlington Heights, IL, USA) by electroblotting. The transfer of protein and equality of loading in all lanes was verified using reversible staining with Ponceau S. Membranes were blocked using 5% nonfat dry milk. NF-B subunit p50 protein was detected by incubation of blots with a
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specific monoclonal antibody to synthetic peptides derived from human p50 amino acid sequences (Cayman Chemical, Ann Arbor, MI, USA) at a dilution of 1:1000 overnight at 4°C, followed by sheep anti-mouse secondary antibody conjugated to horseradish peroxidase and determination of enhanced chemiluminescence (Amersham) using exposure to BioMax MR film (Kodak). Extraction of nuclei AGS cells treated with or without AS ODN or S ODN were cultured in the absence or the presence of H. pylori. The culture medium was aspirated and the cells were rinsed with ice-cold phosphate-buffered saline (PBS), harvested by scraping into PBS, and pelleted by centrifugation at 1500 ⫻ g for 5 min. The cells were lysed in buffer containing 10 mM Hepes, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM MgCl2, 0.2% Nonidet P-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonylfluoride (PMSF). The nuclear pellet was resuspended on ice in nuclear extraction buffer containing 20 mM Hepes, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF [39], and the nuclear protein concentration was determined by the method of Bradford [38]. NF-B activation by EMSA NF-B gel shift oligonucleotide, 5⬘AGT TGA GGG GAC TTT CCC AGG C-3⬘ (Promega Corp., Madison, WI, USA) was labeled with [32P] dATP (Amersham) using T4 polynucleotide kinase (GIBCO-BRL, Grand Island, NY, USA). End-labeled probe was purified from unincorporated [32P] dATP using a Bio-Rad purification column (Bio-Rad Laboratories, Hercules, CA, USA) and recovered in tris-EDTA buffer (TE). Nuclear extracts (5 g) were preincubated in buffer containing 12% glycerol; 12 mM Hepes, pH 7.9; 4 mM Tris-HCl, pH 7.9; 1 mM EDTA; 1 mM DTT; 25 mM KCl; 5 mM MgCl2; 0.04 g/ml poly[d(I-C)] (Boehringer Mannheim, Indianapolis, IN, USA); 0.4 mM PMSF; and TE. The labeled probe was added and samples were incubated on ice for 10 min. Samples were subjected to electrophoretic separation at room temperature on a nondenaturing 5% acrylamide gel at 30 mA using 0.5 X Tris borate EDTA buffer. The gels were dried at 80° for 1 h and exposed to the radiography film for 6 –18 h at ⫺70° with intensifying screens [40]. mRNA expression of iNOS by RT-PCR Total RNA was isolated from cells by guanidine thiocyanate extraction method [41]. The internal standard
(-actin) was coamplified with iNOS. RNA was reverse transcribed into cDNA and used for PCR with human iNOS and -actin specific primers. For iNOS, primers were 5⬘-CTGCATGGATAAGTACAGGCTGAGC-3⬘ (a 25-mer forward primer at position 1593) and 5⬘-AGCTTCTGATCAATGTCATGAGCAA-3⬘ (a 25-mer reverse primer at position 1817), giving rise to a 225 bp PCR product [22]. For -actin, primers were 5⬘-ACCAACTGGGACGACATGGAG-3⬘ (a 21-mer forward primer at position 270) and 5⬘-GTGAGGATCTTCATGAGGTAGTC-3⬘ (a 23-mer reverse primer at position 624), giving rise to a 353 bp PCR product [42]. Briefly, the PCR was amplified by 30 repeat denaturation cycles at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s. During the first cycle, the 95°C step extended to 2 min, and on the final cycle the 72°C step extended to 5 min. PCR products were separated on 1.5% agarose gels containing 0.5 g/ml ethidium bromide and visualized by UV transillumination.
Fluorometric determination of nitrite 2,3-Diaminonaphthalene (DAN) is reacted with nitrite under acidic conditions to form 1-(1)-naphthotriazole, a fluorescent product. Culture medium (500 l) was first brought to volume (1 ml) with deionized water. To this, 100 l of freshly prepared DAN (0.05 mg/ml in 0.62 M HCl) was added and mixed immediately. After 10 min incubation at 20°C, the reaction was terminated with 50 l of 2.8 N NaOH [43]. Formation of the 2,3-diaminonaphthtriazol was measured using a SPF-500C spectrofluorometer (SLM Instruments, Inc., Urbana, IL, USA). Nitrite content was standardized to the cell number at the end of the experiment and expressed as nmol/ 106 cells.
Morphological characterization of cell death by Hoechst staining AGS cells (4 ⫻ 105/well), plated onto glass coverslips in 24 well plates, were treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. The cells were washed twice with PBS, fixed with 4% paraformadehyde for 30 min at room temperature, and stained with 1 mM bisbenzimide (Hoechst 33258; Sigma) for 10 min at 37°C. After washing with tap water for 5 min, cover-slips were mounted onto microscope slide and nuclear morphology was observed under a fluorescence microscope at 400⫻ [44].
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Apoptotic cell count by Hoechst staining Assessment of apoptotic cell death was performed by staining with DNA-specific dye Hoechst 33258. AGS cells (4 ⫻ 105cells/well) were treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. The cells were washed with PBS (pH 7.4), fixed with 4% paraformaldehyde for 30 min, and then stained with 1 mM Hoechst 33258 for 10 min at 37°C. Cells were washed again with PBS prior to viewing stained nuclei under an inverted microscope [45]. Percentage of apoptotic cells, assessed by staining with Hoechst 33258, was calculated based on total numbers of the cells at the end of the experiment. Quantitation of DNA fragmentation Nucleosomes were quantified by means of a sandwich enzyme-linked immunosorbent assay (ELISA) (Cell Death Detection ELISAplus kit; Boehringer Mannheim GmbH, Germany). AGS cells (4 ⫻ 105cells/well) were treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. The cells were detached and 1 ⫻ 105 of cells were lysed. After cell lysis, oligonucleasome-bound DNA was quantitated by using biotin-coupled mouse monoclonal anti-histone antibody as a capturing antibody, peroxidase-conjugated mouse monoclonal anti-DNA antibody as a detecting antibody, and 2,2⬘-azio-di[3ethylbenhioazolin-sulfonate (ABTC) as a developing reagent. The relative increase in nucleosomes in the cell lysate, determined at 405 nm, was expressed as an enrichment factor. Enrichment factor of the cells received no treatment and cultured in the absence of H. pylori (none) was considered as 1. Statistical analysis Results are expressed as means ⫾ SE of four different experiments. Analysis of variance (ANOVA) followed by Newman-Keul’s test was used for statistical analysis [46]. p ⬍ .05 was considered statistically significant. RESULTS
Cytotoxicity by H. pylori in AGS cells, depending on the number of bacterium treated to the cells To determine whether H. pylori infection induces cytotoxicity of AGS cells, the cells were cultured in
Fig. 1. Cytotoxicity by H. pylori in AGS cells, depending on the number of bacterium treated to the cells. (A) AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/ cell ratio of 200:1 and 300:1 for 36 h. At an indicated time point, cell number was determined by trypan blue exclusion test. (B) AGS cells (4 ⫻ 105 cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, 300:1, 400:1, and 500:1 for 24 h. Cell number was determined by trypan blue exclusion test. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the corresponding cells at the start of experiment (0 h) (A) or none (B). None ⫽ the cells cultured in the absence of H. pylori.
the presence of H. pylori and the viable cells were counted at an indicated time point (Fig. 1A). Cytotoxicity of AGS cells increased with the number of bacterium treated to the cells. It started within 12 h of culture at a bacterium/cell ratio of 300:1 and 24 h at a bacterium/cell ratio of 200:1. At 36 h, the number of viable cells, cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1, decreased to 26% of that of the cells cultured in the absence of H. pylori (none). H. pylori-induced cytotoxicity was determined by the experiment using more defined ratios of bacterium/cell from 100:1 to 500:1 (Fig. 1B). At 24 h, reduction in viable cell number was shown at a bacterium/cell ratio, 200:1. The cell number was lower than that at the start of the experiment with the number of bacte-
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Fig. 2. iNOS expression and nitrite production by H. pylori in AGS cells, depending on the number of bacterium treated to the cells. (A) AGS cells (4 ⫻ 105cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 100:1, 200:1, and 300:1 for 12 h. mRNA expression of iNOS was determined by RT-PCR analysis. The internal standard (-actin) was coamplified with iNOS. (B) AGS cells (4 ⫻ 105cells/well) were cultured in the presence of H. pylori at a bacterium/cell ratio of 200:1 and 300:1 for 36 h. Nitrite content in medium was determined at an indicated time point and expressed as nmol/106 cells. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to nitrite production from the corresponding cells at the start of experiment (0 h). None ⫽ the cells cultured in the absence of H. pylori.
rium treated to AGS cells up to the ratio of bacterium/ cell, 500:1. Therefore, in further experiments on NFB, iNOS, and apoptosis, we used a bacterium/cell ratio of 300:1 and a 24 h culture period. iNOS expression and nitrite production by H. pylori in AGS cells, depending on the number of bacterium treated to the cells RT-PCR analysis shows that H. pylori induced mRNA expression of iNOS with the number of bacterium treated to the cells at 12 h (Fig. 2A). At a bacterium/ cell ratio, 200:1, iNOS expression was shown in the cells and more increased at a bacterium/cell ratio, 300:1. Nitrite production from the cells, cultured in the presence of H. pylori at bacterium/cell ratios of 200:1 and 300:1, was significantly higher at 24 h and even increased at 36 h (Fig. 2B). Nitrite released from the cells (nmol/106 cells), standardized to the cell number at each time point of the experiment, were 3.9 ⫾ 2.1, 4.5 ⫾ 1.9, 5.0 ⫾ 1.7, and 5.4 ⫾ 1.5 at 0, 12, 24, and 36 h in the absence of H. pylori (none). At a bacterium/cell ratio of 300:1, nitrite
Fig. 3. Cytotoxicity by SIN-1 and NOC-18 in AGS cells. AGS cells (4 ⫻ 105 cells/well) were treated with SIN-1 (0.2 mM, 0.5 mM, 1.0 mM) or NOC-18 (0.2 mM, 0.5 mM, 1.0 mM) and cultured in the absence of H. pylori for 24 h. Cell number was determined by trypan blue exclusion test. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment (none).
contents released into the medium, which was standardized to the cell number at each time point (nmol/106 cells), were 4.1 ⫾ 3.0, 32.6 ⫾ 3.3, 57.0 ⫾ 6.1, and 77.5 ⫾ 5.9 at 0, 12, 24, and 36 h, respectively. H. pylori alone, plated at 1.2 ⫻ 108/ml, produced a relatively small amount of nitrite (6.9 ⫾ 0.7 nmol/ml) at 24 h in the absence of AGS cells. Cytotoxicity by SIN-1 and NOC-18 in AGS cells To determine the relation between iNOS expression and cytotoxicity, AGS cells were treated with a peroxynitrite donor, SIN-1 (0.2 mM, 0.5 mM, 1 mM) and an NO donor NOC-18 (0.2 mM, 0.5 mM, 1 mM) in the absence of H. pylori (Fig. 3). At 24 h, dose-dependent reduction in viable cell number was shown in the cells treated with SIN-1 and NOC-18. These results demonstrate the possible involvement of high amounts of NO in apoptosis of AGS cells.
NF-B, iNOS and apoptosis
Fig. 4. Western blot analysis of p50 and NF-B activation by H. pylori in AGS cells treated with ODNs. Either AS ODN or S ODN (0.2 M, 0.5 M) was treated to the cells and cultured in the presence of H. pylori for 4 h. Nuclear protein was analyzed for p50 protein by Western blot analysis (A) and NF-B activation by EMSA (B). None ⫽ the cells received no ODNs and cultured in the absence of H. pylori. H. pylori alone ⫽ the cells received ODNs, but cultured in the presence of H. pylori.
Western blot analysis of p50 and NF-B activation by H. pylori in AGS cells treated with ODNs Either AS ODN or S ODN (0.2 M, 0.5 M) was treated directly to the cells and cultured in the presence of H. pylori for 4 h. Transfection efficiency of ODNs was assessed by determining the level of p50 protein in the nucleus (Fig. 4A). Western blot analysis showed that H. pylori induced increase in p50 in the nucleus. The increase in p50 by H. pylori was inhibited by the treatment of AS ODN, but not by S ODN. H. pylori induced the activation of two species of NF-B dimers (a p50/p65 heterodimer and a p50 homodimer) in gastric epithelial cells [35]. Thus, reduction in p50 protein may cause inhibition of NF-B activation. As shown in Fig. 4B, H. pylori induced NF-B activation, and treatment of AS ODN inhibited NF-B
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Fig. 5. Cytotoxicity by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation and iNOS inhibitors or ODNs. AGS cells (4 ⫻ 105cells/well), treated with or without catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A) or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), were cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Cell number was determined by trypan blue exclusion test. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none control). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori control). None ⫽ the cells cultured in the absence of H. pylori.
activation dose-dependently. The cells treated with S ODN had significant NF-B activation in the nucleus by H. pylori infection. These results demonstrate that H. pylori-induced NF-B activation was inhibited by treatment of p50 AS ODN to the cells. Cytotoxicity by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs Viable cell number was determined in the cells, treated with or without catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M), or 0.5 M of AS ODN or S ODN in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h (Fig. 5). H. pylori
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caused 38% reduction in viable cell number as compared to those of the cells cultured in the absence of H. pylori (none control). Treatment of an antioxidant enzyme catalase and an inhibitor of NF-B activation PDTC inhibited the H. pylori-induced cytotoxicity of the cells, resulting in 10 –14% increase in viable cell number as compared to the cells receiving no treatment and cultured in the presence of H. pylori (H. pylori control). iNOS inhibitors, NAME and AMT, significantly inhibited H. pylori-mediated reduction in viable cell number. Treatment of AS ODN showed similar inhibition on H. pyloriinduced cytotoxicity while S ODN had no inhibitory effect on the reduction in viable cells caused by H. pylori (Fig. 5). Treatment of an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs did not itself induce cytotoxicity of the cells in the absence of H. pylori, determined by viable cell number. iNOS expression and nitrite production by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation and ODNs AGS cells were treated with or without catalase (200 units/ml), PDTC (30 M), or 0.5 M AS ODN or S ODN and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 12 h. H. pylori-induced iNOS expression was inhibited by catalase, PDTC, and AS ODN, but not by S ODN (Fig. 6A). Nitrite production from the cells was increased by H. pylori infection; 10 –12-fold increase compared to the cells in the absence of H. pylori (Fig. 6B). Increase in nitrite production by H. pylori was inhibited by catalase, PDTC, and AS ODN, but treatment of S ODN showed no effect on nitrite production. In the absence of H. pylori, catalase, PDTC, and ODNs affected neither iNOS expression nor nitrite production from the cells. Apoptosis by H. pylori in AGS cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs Staining of DNA-specific dye Hoechst 33258 demonstrated that H. pylori caused severely fragmented nuclei and contained highly condensed chromatin (Fig. 7). This apoptotic phenomenon was inhibited by catalase (200 units/ml), PDTC (30 M), NAME (1 mM), AMT (5 M), and AS ODN (0.5 M). Treatment of S ODN showed similar apoptotic nuclear morphology (nuclear condensation and fragmentation) as that shown in the cells receiving no treatment, but cultured in the presence of H. pylori (H. pylori alone). Percentage of apopotic cells determined by staining with Hoechst 33258, was calculated based on total numbers of the cells (Fig. 8). H.
Fig. 6. iNOS expression and nitrite production by H. pylori in AGS cells treated with or without an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs. AGS cells (4 ⫻ 105cells/well), were treated with or without catalase (200 units/ml), PDTC (30 M), NAME (1 mM), AMT (5 M), or 0.5 M of antisense (AS) ODN or sense (S) ODN, and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 12 h. (A) mRNA expression of iNOS was determined by RT-PCR analysis. The internal standard (-actin) was coamplified with iNOS. (B) Nitrite content in culture medium was determined and expressed as nmol/106 cells. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none control). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori control). None ⫽ the cells cultured in the absence of H. pylori.
pylori induced 7-fold increase in the number of apoptotic cells, which was inhibited by catalase, PDTC, NAME, AMT, and AS ODN. S ODN had no effect on H. pyloriinduced increase in apoptotic cells. H. pylori-induced DNA fragmentation was determined by the amount of oligonucleasome-bound DNA in the cell lysate (Fig. 9). Apoptosis-associated increase in nucleosome-associated
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Fig. 8. Assessment of apoptotic cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs, determined by Hoechst staining. AGS cells (4 ⫻ 105cells/well), were treated with catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A), or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Monolayers of cells were fixed in 4% paraformaldehyde and were stained with DNA-specific dye Hoechst 33258. % of apoptotic cells were assessed based on total numbers of the cells. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori alone). Fig. 7. Hoechst staining of AGS cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs. AGS cells (4 ⫻ 105cells/well), were treated either with catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A), or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Monolayers of cells onto coverslips were fixed in 4% paraformaldehyde and were stained with DNA-specific dye Hoechst 33258. Nuclear morphology was observed under a fluorescence microscope at 400⫻. None ⫽ the cells received no treatment and cultured in the absence of H. pylori. H. pylori alone ⫽ the cells received no treatment, but cultured in the presence of H. pylori.
low-molecular-weight DNA was inhibited by catalase, PDTC, iNOS inhibitors, and AS ODN, but not by S ODN. DISCUSSION
Helicobacter pylori (H. pylori) may affect normal balance between cell proliferation and cell death in gas-
tric mucosa. Recently, several investigators reported that H. pylori activated NF-B in human gastric mucosa in vivo and cultured gastric epithelial cells in vitro [47– 49]. This bacterium directly induced apoptosis of human gastric epithelial cells [16,17]. In the present study, we showed that H. pylori activated NF-B and induced apoptosis of gastric epithelial AGS cells. To inhibit NF-B activation, we used antioxidants, catalase and PDTC, because these antioxidants, which scavenge oxygen radicals, are known to inhibit the activation of NF-B [50,51]. Additionally, we used AS ODN to p50 of NF-B, which specifically inhibited NF-B expression. Inhibition of NF-B activation suppressed H. pylori-induced apoptosis in AGS cells. These results demonstrate that NF-B may play an important role in H. pylori-induced apoptosis of gastric epithelial cells. Because the promoter of human iNOS gene contains a regulatory DNA sequence to which NF-B binds [22]
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Fig. 9. Quantification of DNA fragmentation of AGS cells treated with an antioxidant enzyme, an inhibitor of NF-B activation, iNOS inhibitors, and ODNs. AGS cells (4 ⫻ 105cells/well), were treated either with catalase (200 units/ml), PDTC (30 M), NAME (1 mM), and AMT (5 M) (A), or 0.5 M of antisense (AS) ODN or sense (S) ODN (B), and cultured in the presence of H. pylori (at a bacterium/cell ratio, 300:1) for 24 h. Cell lysates were isolated from 1 ⫻ 105 cells. DNA fragmentation was determined by the amount of oligonucleasomebound DNA in the cell lysate. The relative increase in nucleosomes in the cell lysate, determined at 405 nm, was expressed as an enrichment factor. Enrichment factor of none was considered as 1. Data represent means ⫾ SE of four different experiments. *p ⬍ .05 compared to the cells received no treatment in the absence of H. pylori (none). ⫹p ⬍ .05 compared to the cells received no treatment, but cultured in the presence of H. pylori (H. pylori alone).
and the expression of iNOS have been regulated by NF-B in several cell lines [22,23], we next investigated whether NF-B would regulate iNOS expression and nitrite production in H. pylori-stimulated AGS cells. Furthermore, the relation between iNOS and apoptosis was reported in several cells [12–15]. iNOS expression was induced in gastric mucosa of patients with H. pyloripositive duodenal ulcers in vivo [8] and in macrophages treated with H. pylori in vitro [52,53]. Furthermore, Mannick et al. reported that dietary supplement with antioxidant -carotene reduced nitrotyrosine staining, a marker for peroxynitrite production, in gastric mucosa of patients with H. pylori infection [54]. In the present study, we showed that H. pylori infection increased iNOS expression and nitrite production with the number of bacterium treated to the cells, and inhibition of NF-B activation blocked iNOS expression and nitrite produc-
tion in H. pylori-stimulated AGS cells. These results indicate that H. pylori may cause NF-B activation and iNOS expression, which induces high NO production and apoptosis in gastric epithelial cells. We showed that iNOS inhibitors suppressed H. pylori-mediated cytotoxicity and apoptosis, determined by DNA fragmentation and chromatin condensation in gastric epithelial cells. Furthermore, a peroxynitrite donor SIN-1 and an NO donor NOC-18 dose-dependently induced reduction in viable cells. Together with inhibition on cytotoxicity and apoptosis by NF-B inhibition, effects of iNOS inhibitors confirms the above hypothesis that iNOS expression mediated by NF-B activation is associated with H. pylori-induced apoptosis in gastric epithelial cells. The exact mechanism how NO leads to DNA damage in the cells undergoing apoptosis has not been clarified. One possible explanation is that the reaction of NO with oxygen radicals results in production of highly toxic nitrous radical peroxynitrite. Peroxynitrite may attack aromatic amines such as pyridine and purine, finally leading to DNA strand breaks [55]. NO effect on nucleic acids has been determined for naked DNA in normal cells [56,57]. In addition, NO produced by iNOS may react with ferrous-sulfate-containing proteins and form nitrosyl complexes, which lead to cellular damage [58]. Finally, NO derivatives such as nitrosamine might be one toxic material produced by H. pylori in gastric mucosa. Recent studies show that NF-B activation may be associated with cell apoptosis [59,60]. In particular, NF-B stimulates the tumor suppressor gene p53, and NF-B subunit p65 interacts with cell cycle inhibitor p21WAF1 [61,62]. Grilli et al. reported that blocking NF-B could protect neurons against neurotoxicity [63]. Additionally, several investigators reported that NF-B is concomitantly activated in TNF-␣-induced apoptosis [64,65] and inhibition of NF-B activation by certain antioxidants prevent apoptosis [66]. Apoptosis in gastric mucosa was reduced by supplementation with either antioxidant ascorbic acid or -carotene in H. pyloripositive patients [54]. These evidences suggest that NF-B may promote apoptosis in gastric epithelial cells. While NF-B activation is detrimental in the present study, several studies have shown that NF-B activation blocks apoptosis to a variety of insults [67–70]. Thus, transcription factor NF-B can be both detrimental and protective, depending on cell type in which it is expressed and the nature of the insult. Present study demonstrates the link between H. pylori-induced NF-B activation and apoptosis, which is mediated by iNOS expression induced by H. pylori in gastric epithelial cells. Further studies on the nature and the time point of gastric epithelial cell death induced by H. pylori infection, such as early apoptosis, late apoptosis, and possible
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necrosis, are necessary to provide a better understanding and therapeutic approach towards controlling H. pyloriinduced gastric injury. Acknowledgement — This study was supported by a Science Research Center grant from Korea Science and Engineering Foundation (KOSEF) to the Nitric Oxide Radical Toxicity Research Center (NORTReC) made in the program year of 2000 (H.K.).
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ABBREVIATIONS
H. pylori—Helicobacter pylori NF-B—nuclear factor-B INOS—inducible nitric oxide synthase PDTC—pyrrolidine dithiocarbamate L-NAME—NG-nitro-L-arginine-methyl ester AMT—2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine AS ODN—antisense oligonucleotide S ODN—sense oligonucleotide RT-PCR—reverse transcription-polymerase chain reaction EMSA— electrophoretic mobility shift assay