Dig Dis Sci (2011) 56:16821692 DOI 10.

1007/s10620-010-1512-y

ORIGINAL ARTICLE

Role of cagA-Positive Helicobacter pylori on Cell Proliferation, Apoptosis, and Inammation in Biliary Cells
Wongwarut Boonyanugomol Chariya Chomvarin Seung-Chul Baik Jea-Young Song Chariya Hahnvajanawong Kyung-Mi Kim Myung-Je Cho Woo-Kon Lee Hyung-Lyun Kang Kwang-Ho Rhee Banchob Sripa

Received: 28 September 2010 / Accepted: 19 November 2010 / Published online: 22 December 2010 Springer Science+Business Media, LLC 2010

Abstract Background and Aims The pathogenesis of Helicobacter pylori in the human hepatobiliary system has not been clearly elucidated. We compared the effects of H. pylori cagA? and cagA- mutant strains on cell proliferation, apoptosis, and inammation in a cholangiocarcinoma (CCA) cell line (KKU-100). Methods MTT and BrdU were used to determine cell viability and DNA synthesis, respectively. The results were further investigated by RT-PCR and Western-blot analysis. The production of interleukin-8 (IL-8) was measured by ELISA assay. Results At low H. pylori inocula (cell-bacteria ratio of 1:1), the H. pylori cagA? strain showed a signicant
Electronic supplementary material The online version of this article (doi:10.1007/s10620-010-1512-y) contains supplementary material, which is available to authorized users.
W. Boonyanugomol C. Chomvarin (&) C. Hahnvajanawong Department of Microbiology, and Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: chariya@kku.ac.th W. Boonyanugomol e-mail: kong_55@hotmail.com C. Hahnvajanawong e-mail: hchari@kku.ac.th S.-C. Baik J.-Y. Song K.-M. Kim M.-J. Cho W.-K. Lee H.-L. Kang K.-H. Rhee Department of Microbiology, and Research Institute of Life Science, Gyeongsang National University College of Medicine, Chiram-dong 90, Jinju, Gyeongsangnam-do 660-751, Republic of Korea e-mail: scbaik@gaechuk.gsnu.ac.kr J.-Y. Song e-mail: songjy7183@hanmail.net

stimulation in KKU-100 cell growth (109 1.79%) and DNA synthesis (131 3.39%) than did the H. pylori cagA- strain (95 3.06% and 120 2.32%, respectively), through activation of the anti-apoptotic bcl-2 gene, MAP kinase and NF- jB cascade. By contrast, at high H. pylori inocula (cell-bacteria ratio of 1:200), the H. pylori cagA? strain showed a signicant reduction in KKU-100 cell survival (49 2.47%) and DNA synthesis (49 1.14%) than did the H. pylori cagA- strain (60 1.30% and 75 4.00%, respectively), by increased iNOS, p53 and bax, while decreased bcl-2. Additionally, caspase8 and -3 protein were activated. The H. pylori cagA? strain had signicantly stronger effect on IL-8 production than did the cagA- strain. Conclusions These results suggest that the H. pylori cagA? strain may play an important role in the development

K.-M. Kim e-mail: zxc103@hanmail.net M.-J. Cho e-mail: mjecho@gshp.gsnu.ac.kr W.-K. Lee e-mail: wklee@gshp.gsnu.ac.kr H.-L. Kang e-mail: kangssi@nongae.gsnu.ac.kr K.-H. Rhee e-mail: klee@gshp.gsnu.ac.kr B. Sripa Department of Pathology, and Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: banchob@kku.ac.th

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of biliary cancer by disturbing cell proliferation, apoptosis, and promoting cell inammation in the CCA cell line. Keywords Helicobacter pylori Cell proliferation Apoptosis Inammation Hepatobiliary diseases Cholangiocarcinoma

Introduction Cholangiocarcinoma (CCA) is a fatal bile duct epithelial cancer. The highest incidence of this primary liver cancer in the world has been reported in northeast Thailand [1]. The infection with the liver uke Opisthorchis viverrini is intimately related to pathogenesis of CCA in this region [2]. However, pathogenesis of CCA or other biliary cancers in non-liver uke endemic areas is still poorly understood. Recently, a new synergistic role of bacterial infection including Helicobacter spp. in the hepatobiliary tract has been proposed to be involved in severe liver diseases including cancer [3]. Therefore, it is useful to explore roles of Helicobacter in biliary cancer development. The entero-hepatic Helicobacter spp. was detected in the hepatobiliary tract of humans [4]. Among this genus, H. pylori was the most common species detected in patients with hepatobiliary diseases [57]. H. pylori was detected more often in both CCA and hepatocellular carcinoma (HCC) patients than in patients with non-malignant tumors and the control group [6, 8], suggesting a correlation between H. pylori and liver cancer development. Partial DNA sequences of H. pylori from liver samples (HCC, CCA) clustered separately from gastric H. pylori strains [9]. The animal model with C57BL/6 mice demonstrated that prolonged infection with H. pylori resulted in the development of HCC [10]. Additionally, the cytotoxinassociated gene A (cagA) was reported to be associated with liver tract diseases [11]. Therefore, we hypothesize that the H. pylori cagA? strain could be a co-factor in the development of liver diseases, especially CCA. The cagA is an important virulence gene of H. pylori associated with the pathogenesis of H. pylori infection [12]. CagA, a product of cagA, has an effect on biological activities such as cell motility, cytoskeleton rearrangement, cell proliferation, and apoptosis [13, 14]. Moreover, cagA can induce interleukin-8 (IL-8) gene expression via mitogen-activating protein kinases (MAPK) and NF- jB in both CagA tyrosine phosphorylation-dependent and -independent manners [15]. IL-8, a proinammatory cytokine, can induce neutrophil and monocyte inltration leading to inammation [16]. Additionally, IL-8 has been shown to activate multiple intracellular signaling pathways, leading to an increase in proliferation and survival of cancer cells [16].

H. pylori has been reported to induce apoptosis as well as to increase cellular proliferation in gastric epithelial cells [1719]. Recently, an in vitro study showed that a putatively more virulent strain of H. pylori induced apoptosis in a hepatocyte cell line by inhibiting DNA synthesis and activating caspase-3 at high doses of bacteria, while low doses of H. pylori increase DNA synthesis [20]. However, the mechanisms by which H. pylori (especially cagA? strain) in biliary cells including cell proliferation, induction of apoptosis and inammation have not been extensively studied. In this study, therefore, we investigated the molecular mechanisms of H. pylori cagA? and cagA- strains in cell proliferation, induction of apoptosis, and pro-inammatory cytokine production (IL-8) in biliary cells.

Materials and Methods Bacterial Strains H. pylori (Korean strain 51) cagA wild-type (cagA?) and cagA isogenic mutant (cagA-) strains were obtained from the H. pylori Korean-type culture collection (Gyeongsang National University, School of Medicine). The two strains were grown on Brucella agar supplemented with 10% bovine serum for 24 h at 37C under microaerophilic conditions [21]. Cell Culture The human CCA cell line (KKU-100) was cultured in Ham F12 medium containing 10% heat-inactivated FBS, 100 units/ml of penicillin, and 100 lg/ml of streptomycin at 37C in a humidied incubator containing 5% CO2 [22]. Cytotoxicity by MTT Assay The effect of H. pylori on cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] assay. KKU-100 cells (8 9 104 cells/well) were seeded in a 96-well microtiter plate and incubated at 37C for 24 h. After co-culturing with H. pylori at low inocula (cell-bacteria ratio of 1:1) to a high inocula (1:200) for 24 h, MTT assay was performed according to the procedure of Kim et al. (2007). After removing the supernatant, formazan crystal was dissolved in dimethylsulfoxide (DMSO), and the optical density of each well was measured by a microplate reader at a wavelength of 540 nm. Culture medium without H. pylori was used as the control. The absorbance value was calculated as a percentage of cell viability compared to the control.

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Measurement of DNA Synthesis by BrdU Assay After co-culturing KKU-100 cells with H. pylori at low inocula (cell-bacteria ratio of 1:1) to a high inocula (1:200) in 96-well microtiter plates for 24 h, BrdU (5-bromo20 -deoxyuridine) incorporation was performed as previously described [20]. Chromogenic substrate tetra-methylbenzidine (TMB) was then added and optical density was measured using a microplate reader at a wavelength of 520 nm. Culture medium without H. pylori was used as the control. The absorbance value was calculated as a percentage of DNA synthesis compared to the control.

RT-PCR for iNOS and Apoptosis-Related Gene Expression KKU-100 cells (1 9 106 cells) were cultured in a cell culture dish for 24 h. After co-culturing with medium alone (control) or H. pylori (1:1 or 1:200), total RNA was extracted from the control and treated cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturers instructions. The quantity of RNA and its purity were measured with a spectrophotometer. RT-PCR of iNOS, p53, bax, bcl-2, caspase-3, and GAPDH were performed as previously described [2325]. The PCR primers are presented in Table 1. The PCR product was electrophoresed in 1.5% agarose, stained with ethidium bromide, and visualized under UV light. The intensity of the PCR product bands was quantied using Quantities One software version 4.6.2 (Bio-Rad, USA). The intensity of PCR product band was normalized to GAPDH intensity and data were expressed as fold changed compared to control (intensity of PCR product band of H. pylori-treated cells divided by PCR product band of control). Western-Blot Analysis for Protein-Involved Apoptosis and Cell Proliferation After co-culturing KKU-100 cells (1 9 106) with medium alone (control) or H. pylori (1:1 or 1:200) for 0, 6, 12, and 24 h, the protein extraction and Western blotting were performed as previously described [21]. After blocking, the membranes were incubated with the following antibodies: Bax, Bcl-2, Bcl-xL, Fas, activated caspase-8, cytochrome c, b-actin (Santa Cruz Biotechnology, CA, USA), activated caspase-9, activated and -3, cleaved PARP, pMAPK,

Determination of Apoptotic Cells Nuclear staining using 4,6-diamidino-2-phenylindole (DAPI) was performed to determine chromatin condensation and fragmentation. KKU-100 cells (1 9 106 cells/ well) were grown in a cell culture dish at 37C for 24 h and co-cultured with medium alone (control) or H. pylori (1:200) for 12 and 24 h. The treated cells were washed with PBS, xed with methanol, stained with 1 lg/ml of DAPI at 37C for 15 min and observed under a uorescence microscope (Olympus, Japan). Apoptotic cell death was enumerated by counting a total of 500 cells, and the result was expressed as a percentage of apoptotic cells. DNA fragmentation was assessed using agarose gel electrophoresis. Cells treated with H. pylori (1:200) were washed with PBS, and DNA extracted by QIAamp DNA Mini Kit (Qiagen, USA) according to the manufacturers instructions. DNA was electrophoresed in a 1.5% agarose gel and the DNA band was visualized using a UV-illuminator (Bio-Rad, USA).

Table 1 Primers used for RT-PCR

Genes iNOS p53 bax bcl-2 caspase-3 GAPDH

Primer sequences 50 -CGGTGCTGTATTTCCTTACGAGGCGACGAAGG-30 50 -GGTGCTGCT TGT TAGGAGGTCAAGTAAAGGGC-30 50 -TTCTTGCATTCTGGGACAGCC-30 50 -GCCTCATTCAGCTCTCGGAAC-30 50 -TGGCAGCTGACATGTTTTCTGAC-30 50 -CGTCCCAACCACCCTGGTCT-30 50 -CTGTACGGCCCCAGCATGCG-30 50 -GCTTTGTTTCATGGTACATC-30 50 -TTCAGAGGGGATCGTTGTAGAAGTC-30 50 -CAAGCTTGTCGGCATACTGTTTCAG-30 50 -CGGAGT CAACGGATT TGGTCGTAT-30 50 -AGCCTTCTCCATGGTGGTGAAGAC-30

Product size (bp) 259 650 195 231 264 306

References [24] [24] [24] [23] [25] [24]

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NF- jB (Cell Signaling Technology, MA, USA) overnight at 4C. HRP-conjugated secondary antibody was added, and protein was visualized using the enhanced chemiluminescence (ECL) system (Thermo Scientic, IL, USA). The intensity of the protein bands was quantied using Quantities One software version 4.6.2 (Bio-Rad, USA). The intensity of protein band was normalized to b-actin intensity and data expressed as fold changed compared to control (intensity of protein band of H. pylori-treated cells divided by protein band of control). Measurement of IL-8 Production for Cell Inammation KKU-100 cells were co-cultured with H. pylori at a cellbacteria ratio of 1:1 or 1:200 then the supernatant was collected and centrifuged at 7,000 rpm for 10 min to eliminate unattached bacteria. The concentration of IL-8 was measured using IL-8 enzyme-link immunosorbent assay (ELISA) (BioSource International) according to the manufacturers instructions. Culture medium without H. pylori was used as the control. The absorbance value was calculated as a concentration of IL-8 compared to standard IL-8. Statistical Analysis All of the data were reported as means SE. Differences between the untreated control, and the H. pylori cagA? and cagA- strains were analyzed using Students t test. p \ 0.05 and p \ 0.01 were considered signicant.

Results Effect of H. pylori on Biliary Cell Growth and DNA Synthesis In order to determine the cytotoxic and genotoxic potential of H. pylori, the biliary cells were treated with both the H. pylori cagA? and cagA- strains. The KKU-100 cell growth and DNA synthesis were performed using MTT and BrdU assay, respectively. Cell viability of the KKU-100 cells treated with the H. pylori cagA? strain at a cell-bacteria ratio of 1:1 was signicantly higher (109 1.79%) than that treated with the cagA- strain (95 3.06%). However, treatment of the KKU-100 cells with the H. pylori cagA? and cagA- strains at high cell-bacteria ratio (1:101:200) inhibited cell growth in a dose-dependent manner at 24 h, compared to the control (p \ 0.05) (Fig. 1a). Interestingly, at a high cell-bacteria ratio of 1:200, the KKU-100 cell growth treated with the H. pylori cagA? strain was signicantly lower (49 2.47%) than that treated with the cagA- strain (60 1.30%) (Fig. 1a). For DNA synthesis of KKU-100 cells at a low cellbacteria ratio of 1:1, the H. pylori cagA? strain was associated with a markedly increased DNA synthesis (131 3.39%), compared to the H. pylori cagA- strain (120 2.32%) (p \ 0.05) (Fig. 1b). Indeed, both the H. pylori cagA? and cagA- strains inhibited DNA synthesis in KKU-100 cells at cell-bacterial ratios starting from 1:20 to 1:200 (p \ 0.05) (Fig. 1b). At the high cellbacteria ratio of 1:200, the H. pylori cagA? strain resulted in a reduction of DNA synthesis of 49 1.14% versus

Fig. 1 Effects of H. pylori on the KKU-100 cell line in cell growth and DNA synthesis at various bacterial concentrations at 24 h. Cytotoxic effect of the H. pylori cagA? and cagAstrains determined cell viability using the MTT assay (a), genotoxic effect of the H. pylori cagA? and cagA- strains determined DNA synthesis using the BrdU assay (b). Values are the mean SE for the independent experiment (n = 3). *p value \ 0.05 was compared between treated cells and control, #p value \ 0.05 was compared between the H. pylori cagA? and cagAstrains

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75 4.00% in the H. pylori cagA- strain (p \ 0.05) (Fig. 1b). H. pylori Induced Biliary Cell Proliferation Determination of H. pylori-induced biliary cell proliferation was performed using RT-PCR and Western blotting to determine bcl-2, bax, p53 gene expression, and phosphorylated MAPK and NF-jB protein expression. At low inocula of the H. pylori cagA? and cagA- strains (cellbacteria ratio of 1:1), the expression of pro-apoptotic genes, bax and p53, were not signicantly changed compared to control (Fig. 2a). Interestingly, at 6, 12, and 24 h of treatment, the increase in the anti-apoptotic gene bcl-2 in the KKU-100 cells exposed to the H. pylori cagA? strain was signicantly higher than in the cells treated with the H. pylori cagA- strain (Fig. 2a). Figure 2b illustrates the time dependent of phosphorylated MAP kinase and NF- jB activation following exposure of the KKU-100 cells to the H. pylori cagA? and cagA- strains at a low inoculation (1:1). Phosphorylation of the MAP kinase was markedly activated in the KKU-100 cells treated with the H. pylori cagA? strain, while in the H. pylori cagA--treated cells showed slightly activated. The activation of the NF- jB was clearly evident within 15 min of the KKU-100 cells treated with the H. pylori cagA? strain, but cells treated with the H. pylori cagA- strain exhibited marked activation at 30 min.
Fig. 2 Role of H. pylori on cell proliferation in KKU-100 cells, co-cultured with the H. pylori cagA? and cagA- strains at a low inoculum (1:1); apoptosisrelated gene expression (a), and activation of MAP kinase and NF- jB protein expression (b). Density of each PCR and protein band was measured and normalized with GAPDH and b-actin intensity, respectively. Data (shown below each band) are presented as a fold-change of the treated cells compared to the control cells (mean SE) in the independent experiment (n = 3). *p value \ 0.05 was compared between treated cells and control, #p value \ 0.05 was compared between the H. pylori cagA? and cagAstrains

H. pylori Induces Biliary Cell Apoptosis Determination of Apoptotic Morphology Inhibition of cell growth and DNA synthesis was found in the KKU-100 cells exposed to high doses of bacteria; a cell-bacteria ratio of 1:200 was used to determine biliary cell apoptosis. Apoptotic cell death was determined using DAPI staining and DNA fragmentation assay. Using DAPI staining, cells with chromatin condensation and nuclear fragmentation, the typical characteristics of apoptosis, were observed in KKU-100 cells treated both with the H. pylori cagA? and cagA- (Fig. 3a, middle and right panels). However, the control cells showed nuclei with homogeneous chromatin distribution (Fig. 3a, left panel). At 24 h, the number of apoptotic cells resulting from treatment with the H. pylori cagA? was signicantly higher than in cells treated with the H. pylori cagA- strain (Fig. 3b). Typical DNA fragmentation, using agarose gel electrophoresis, was observed in the cells exposed to both the H. pylori cagA? and cagA- strains (Fig. 3c). Expression of iNOS Gene To investigate the role of the iNOS gene involved in programmed cell death, the mRNA level was evaluated. Using RT-PCR analysis, a signicance of the fold increase of iNOS mRNA in cells treated with the H. pylori cagA? and cagA- was observed at a cell-bacteria ratio of 1:200

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Dig Dis Sci (2011) 56:16821692 Fig. 3 Determination of apoptotic cell death in KKU100 cells treated with H. pylori at a high cell-bacteria ratio (1:200). Apoptotic cell death was assessed by DAPI staining (a), counting of apoptotic cells (b), and DNA fragmentation assay (c). Lane M molecular mass marker; lane 1 control cell; lane 2 H. pylori cagA?; lane 3 H. pylori cagA-. Values are mean SE of the independent experiment (n = 3). *p value \ 0.05 was compared between treated cells and control, #p value \ 0.05 was compared between the H. pylori cagA? and cagAstrains

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treated cells (1.45-fold), especially at 24 h of treatment (Fig. 4). Expression of Apoptosis-Related Genes and Proteins The apoptosis-related genes and proteins expression were determined by RT-PCR (Fig. 4) and Western blotting (Fig. 5). At a high dose of bacteria (1:200), the fold increase of p53 (4.16-fold) and caspase-3 gene expression (1.62-fold) in the KKU-100 cells exposed to the H. pylori cagA? strain was signicantly higher than that exposed to the H. pylori cagA- strain (1.35-fold for the p53 and 1.32fold for the caspase-3) at 24 h of treatment (Fig. 4). Cells treated with the H. pylori cagA? and cagA- strains showed signicant time-dependent, up-regulation of pro-apoptotic Bax at both the mRNA (Fig. 4) and protein (Fig. 5) levels, whereas the mRNA (Fig. 4) and protein levels of Bcl-2 (Fig. 5) were signicantly down-regulated, leading to an increased Bax/Bcl-2 ratio (Table 2). At 24 h of treatment, the Bax/Bcl-2 ratio of the H. pylori cagA?-treated cells (5.71) was signicantly higher than in the H. pylori cagA-treated cells (3.38) (p \ 0.05) (Table 2). Western-blot analysis revealed that exposure of cells to the H. pylori cagA? and cagA- resulted in a signicant reduction of Bcl-xL while increasing the Fas in a timedependent manner activated caspase-8, -9, -3, cytochrome c, and cleaved PARP (Fig. 5). When the relative intensities of the apoptosis-related proteins to b-actin of cells exposed to the H. pylori cagA? and cagA- were compared to control cells (0 h), the fold increase of activated caspase-8

Fig. 4 RT-PCR of inducible nitric oxide synthase (iNOS), p53 and apoptosis-related gene expression in KKU-100 cells co-cultured with a high cell-bacteria ratio (1:200) of the H. pylori cagA? and cagAstrains at 6, 12, and 24 h. Density of each PCR band was measured and normalized with GAPDH intensity. Data (shown below each band) are presented as a fold-change of the treated cells compared to the control cells (mean SE) in the independent experiment (n = 3). * p value \ 0.05 and **p value \ 0.01 were compared between treated cells and control, #p value \ 0.05 was compared between the H. pylori cagA? and cagA- strains

(Fig. 4). The fold increase of the iNOS gene in the cells treated with the H. pylori cagA? strain was 3.17-fold, which is signicantly higher than the H. pylori cagA-

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Fig. 5 Assessment of apoptosis-related protein expression in KKU100 cells co-cultured with the H. pylori cagA? and cagA- strains (1:200) at 6, 12, and 24 h was determined by Western-blot analysis. Density of each protein band was measured and normalized with b-actin intensity. Data (shown below each band) are presented as a foldchange of the treated cells compared to the control cells (mean SE) in the independent experiment (n = 3). *p value \ 0.05 and **p value \ 0.01 were compared between treated cells and control. #p value \ 0.05 was compared between the H. pylori cagA? and cagA- strains

Fig. 6 H. pylori stimulates IL-8 production. The KKU-100 cells were co-cultured with the H. pylori cagA? and cagA- strains at 1, 3, 6, 12, and 24 h with the inoculation of H. pylori of 1:1 and 1:200. Culture supernatant was collected and measured IL-8 using ELISA. Data (shown on each bar) are presented as a fold-increase of treated cells compared to control cells (mean SE) in the independent experiment (n = 3). *p value \ 0.05 was compared between treated cells and control. #p value \ 0.05 was compared between the H. pylori cagA? and cagA- strains

Table 2 Bax/Bcl-2 ratios of KKU-100 cell line infected with H. pylori H. pylori strains Bax/Bcl-2 ratio 0h H. pylori cagA
?

6h 0.58 0.53

12 h 1.45
*

24 h 5.71*, 3.38*
#

0.24 0.24

H. pylori cagA-

1.75*

IL-8 was signicantly increased in the KKU-100 cells exposed to the H. pylori cagA? and cagA- strains in a time-dependent manner at both low (1:1) and high (1:200) bacterial concentrations. The H. pylori cagA? strain showed a higher level of IL-8 production than the H. pylori cagA--treated cells (p \ 0.05) at both low and high bacterial concentrations (Fig. 6).

Data are shown as a ratio of mean of protein expression level of Bax to Bcl-2 in control and H. pylori-treated cells. * p value \ 0.05 was compared between treated cells and control, #p value \ 0.05 was compared between the H. pylori cagA? and cagA- strain

Discussion Helicobacter spp. (especially H. pylori) has been detected in human beings [26, 27]. Nilson and colleagues detected H. pylori in 73% of cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) patients, while the control group (liver metastases) showed negative PCR results [6]. In agreement with our previous study, H. pylori was found signicantly more often in CCA patients than in patients with benign or the control group. Moreover, we found that the presence of H. pylori was associated with biliary inammation and proliferation in tissues of CCA patients (manuscript in preparation). Study of an animal model (with C57BL/6 mice) revealed that prolonged infection with H. pylori resulted in the development of HCC after 23 months; albeit outcomes

(6.41-fold) and activated caspase-3 (9.39-fold) was signicantly higher in the H. pylori cagA?-treated cells than in the H. pylori cagA--treated cells (2.67-fold and 3.94fold, respectively) (Fig. 5). By comparison, the increase of Fas, cytochrome c, and activated caspase-9 and the fold decrease of Bcl-xL in both the H. pylori cagA? and cagAtreated cells were similar (Fig. 5). H. pylori Stimulates IL-8 Production in Biliary Cells To evaluate H. pylori affect on biliary cell inammation, ELISA was used to measure IL-8 secretion in KKU-100 cells infected with H. pylori (Fig. 6). The concentration of

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depended on the diversity of H. pylori strains [10]. Additionally, H. pylori inoculated orally in the C57BL/6 mice resulted in the development of severe gastric mucosal inammation, mild to moderate hepatitis, and gallbladder mucosa thickening with mild submucosal lymphocytic inltration [28]. Goo and colleagues showed that H. pylori promotes hepatic brosis and increases hepatocytes proliferation by stimulation of a-smooth muscle actin (a-SMA) and transforming growth factor-b1 (TGF-b1), suggesting that H. pylori induces the development of liver cirrhosis [29]. Studies of other Helicobacter spp. infections such as H. hepaticusthe causative agent of hepatocellular carcinoma in mice [30]revealed that inoculated A/JCr mice developed chronic hepatitis and hepatocellular neoplasms, which led to hepatocellular carcinoma [30, 31]. While H. hepaticus produces toxins that cause cytotoxic activity in a mouse liver cell line [32] and a cytolethal distending toxin (Cdt), which has DNase activity [33], H. pylori produces many toxins such as CagA and VacA. We therefore propose that the enteric Helicobacter spp., including H. pylori, may be the infectious factor involved in the development of hepatobiliary diseases, including liver carcinoma. A trigger in the development of cancer is chronic infection and the resultant long-term inammation. The inammation can be caused by stimulation of many cytokine productions [34]. H. pylori infection is well known as a cause of gastric inammation and to stimulate proinammatory cytokines production (i.e., TNF-alpha, IFNgamma, IL-1, IL-6, IL-8) [35]. In inammatory conditions, iNOS can be induced, in almost any cell type, through stimulation by inammatory cytokines and/or bacterial products (i.e., lipopolysaccharide) leading to nitric oxide (NO) production [36]. The transcription factors, including nuclear factor kappa-B (NF-jB), tumor suppressor protein p53, and proinammatory CXC chemokine (IL-8) have been shown to be regulated by the NO [3739]. IL-8 was shown to activate multiple intracellular signaling pathways leading to an increase in proliferation and survival of cancer cells [16]. In this study, using the KKU100 cell line, we found that at the low cell-bacteria ratio (1:1), the H. pylori cagA? strain signicantly enhanced IL-8 production, DNA synthesis and cell proliferation compared to the H. pylori cagA- strain. These results imply that the H. pylori cagA? can stimulate IL-8 production, which in turn increased DNA synthesis and cell proliferation of the KKU-100 cells. Our results are similar to a report of a virulent H. pylori strain (cagA?/vacA?/ babA?/oipA?) that at low concentrations induced higher DNA synthesis on a hepatocyte cell line (Huh7) than a less virulent strain [20]. However, no signicant difference between gastric epithelial cells treated with the type I cytotoxic strains (cagA?/vacA?) and non-cytotoxic strains

(cagA-/vacA-) in the induction of apoptosis and cell proliferation was reported [18]. The discrepancies of results may depend on the specic H. pylori strains used in each study. Many studies failed to culture H. pylori from hepatobiliary samples [8, 11, 28]. It could be that the number of bacteria was too small (since the bacteria were not in their usual niche) [8, 9], indicating a low concentration of H. pylori infection. However, the detection of H. pylori using immunohistochemistry conrms the true colonization of H. pylori in the liver [8, 40]. We propose that there is a low density of H. pylori infection in the biliary cells and that persistent H. pylori infection could lead to subtle modulations in cell replication as a previous report in hepatocytes [20]. An increasing rate of DNA synthesis at low dose of H. pylori may be a mechanism of the cell to compensate the high rate of cell death [20]. Taken together, our results suggest that at a low concentration of H. pylori, CagA may play an important role in DNA synthesis and proliferation of biliary cells. To understand the molecular mechanism in KKU-100 cell proliferation, several gene and protein expressions were examined. Our results indicate that at the low cellbacteria ratio of 1:1, the KKU-100 cells treated with the H. pylori cagA? strain had a marked, time-dependent, increase in intracellular signaling molecules; viz., antiapoptotic bcl-2 gene expression, phosphorylated MAP kinase and NF- jB protein expression, while the gene expression levels of bax and p53 were not changed. These effects may be due to an interaction of CagA with several intracellular signaling molecules, MAPK [13] and NF- jB, leading to the activation of many genes (i.e., growth-promoting genes and anti-apoptotic genes) which might contribute to the malignant transformation [14]. In a previous report, high concentration of NO, proceeding mainly by the iNOS pathway cause DNA damage [41] and induce apoptosis [36, 42]. The tumor suppressor protein (p53) has a dual role in NO-mediated apoptosis because p53 protects the cell from apoptosis at low NO concentrations and stimulates apoptosis at high NO concentrations [41]. H. pylori was reported as a cause of gastric epithelial AGS cell line damage by inducing iNOS and p53-dependent apoptosis [24]. In our study, at a high cell-bacteria ratio (1:200), the H. pylori cagA? strain showed signicantly stronger growth inhibition, decreased DNA synthesis, induced apoptosis, increased iNOS, and p53 gene expression in the KKU-100 cell line than the H. pylori cagA- strain. These results agree with previous reports on various cell lines [20, 24, 43]. Apoptosis induced through the extrinsic pathway involves death receptor stimulation and activation of caspase-8 and -3 [44]. Our results showed a higher up-regulation of activated caspase-8 and -3 protein expression in

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the KKU-100 cell line co-cultured with the H. pylori cagA? strain than the H. pylori cagA- strain. Our ndings agree with the previous studies in epithelial intestinal cells and in gastric cell lines co-cultured with the toxigenic H. pylori strains (vacA?/cagA?) than the non-toxigenic H. pylori strains (vacA-/cagA-) [45, 46]. In the intrinsic signaling pathway, the Bcl-2 family of proteins plays major roles in apoptotic regulation through the mitochondrial pathway [47]. Our study found a signicant increase in the pro-apoptotic protein, Bax, and a signicant decrease in the anti-apoptotic proteins Bcl-2 and Bcl-xL, in KKU-100 cells treated with the H. pylori cagA? strain leading to an increased Bax/Bcl-2 ratio induced apoptosis as a previous report [48]. The Bax protein interacts with the Bcl-2 [47] thereby the release of cytochrome c via pore-forming activity at the mitochondrial outer membrane [49]. Cytochrome c forms a complex with Apaf-1 and pro-caspase-9, leading to activation of caspase9 and -3 and DNA fragmentation [50, 51]. Our results showed a higher increase in the level of cytochrome c, activated caspase-9 and -3 and cleaved PARP in KKU-100 cells treated with both H. pylori strains compared to the control. These results suggest that H. pylori (especially the H. pylori cagA? strain) induces apoptosis in the KKU-100 cell line through both a receptor/ligand interaction and mitochondria-mediated apoptosis. At the high cell-bacteria ratio of 1:200, the H. pylori cagA? strain enhanced iNOS gene expression and IL-8 production in KKU-100 cells at a signicantly higher level than the H. pylori cagA- strain, and this agrees with a previous study [52]. Since iNOS can cause NO production, leading to DNA damage and up-regulation of IL-8, it may increase proliferation and survival of cancer cells [16]. It is, therefore, possible that in an environment with high NO and IL-8 level, cells with damaged DNA are stimulated to proliferate, which leads to the proliferation of unrepaired DNA-containing cells that may be involved in tumor development. Our data indicate that CagA play an important role in apoptosis-induced by H. pylori in KKU-100 cells. These ndings agree with other studies of gastroduodenal diseases that show that infection with H. pylori cagA? strains may have a role in atrophy development [17] and induction of apoptosis [46]. Recently, the apoptosis-inducing factors of H. pylori have been reported to play an important role in the pathogenesis [21, 53, 54]. We propose that other virulence factors of H. pylori are also involved in apoptosis on KKU-100 cells. In order to clarify whether CagA inuences apoptosis in biliary cells, a puried CagA protein should be used to examine its exact role in apoptosis. Disturbance of the cell death process by apoptosisenhanced cell turnover will result in increased proliferation, which will increase the probability of accumulating

genetic alterations and may ultimately lead to tumor development [55]. In this study, we proposed the role of H. pylori (especially cagA? strain) on biliary cells in a different density of bacteria. Our results implicate that both low and high doses of H. pylori infection are involved in cancer development of hepatobiliary cells by stimulation of cell proliferation and induction of apoptosis pathway. However, based on evidence that the density of Helicobacter in hepatobiliary tract is too low, we thus speculate that development of biliary cancer may relate in a low number of H. pylori infections. This result agrees with our previous study in the presence of H. pylori associated with biliary cell inammation and proliferation in tissues of CCA patients. In conclusion, our study shows that H. pylori has multiple effects on biliary cells, including: (1) stimulation of DNA synthesis and increased cell proliferation via activation of MAP kinase and NF- KB at low doses of bacteria; (2) induction of apoptosis through both the extrinsic and mitochondria-dependent apoptosis pathways at high doses of bacteria; and, (3) stimulation of IL-8 production. We conclude that CagA play an important role in the carcinogenesis of the biliary cancer through the disturbance of cellular homeostasis by stimulation of cell proliferation and induction of apoptosis (which depends on bacterial concentration) and promotes inammation by stimulating proinammatory cytokine IL-8 production. Further studies regarding the other mechanisms of H. pylori in the CCA cell line are warranted. Additionally, the in vivo roles of H. pylori on the hepatobiliary tract need to be elucidated.
Acknowledgments I would like to thank the Commission on Higher Education, Thailand, for supporting with grant funds under the program Strategic Scholarships for Frontier Research Network for the Join Ph.D. Program Thai Doctoral degree for this research and also thank Khon Kaen University for supporting some parts of this work. I am grateful to the Helicobacter pylori Research Center, Department of Microbiology, Gyeongsang National University School of Medicine, for providing H. pylori strains and materials used in my experiments. I also thank the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University for providing the cell line. I thank Mr. Bryan Roderick Hamman for assistance with the Englishlanguage presentation of the manuscript.

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