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O R I G I N A L A R T I C L E

Role of cagA-Positive Helicobacter pylori on Cell Proliferation,Apoptosis, and Inflammation 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

Ó 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 inflammation 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 significant

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%, respec-

tively), 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 significant 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, caspase-

8 and -3 protein were activated. The H. pylori cagA? strain

had significantly 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: [email protected]

M.-J. Cho


W.-K. Lee


H.-L. Kange-mail: [email protected]

K.-H. Rhee


B. Sripa

Department of Pathology, and Liver Fluke and

Cholangiocarcinoma Research Center, Faculty of Medicine,

Khon Kaen University, Khon Kaen 40002, Thailand


Electronic supplementary material The online version of thisarticle (doi:10.1007/s10620-010-1512-y ) contains supplementarymaterial, which is available to authorized users.

W. Boonyanugomol Á C. Chomvarin (&) Á C. Hahnvajanawong

Department of Microbiology, and Liver Fluke and

Cholangiocarcinoma Research Center, Faculty of Medicine,

Khon Kaen University, Khon Kaen 40002, Thailand


W. Boonyanugomol


C. Hahnvajanawonge-mail: [email protected]

S.-C. Baik Á J.-Y. Song Á K.-M. Kim Á M.-J. Cho Á W.-K. Lee Á

H.-L. Kang Á 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


J.-Y. Song


123

Dig Dis Sci (2011) 56:1682–1692

DOI 10.1007/s10620-010-1512-y

http://dx.doi.org/10.1007/s10620-010-1512-y

http://dx.doi.org/10.1007/s10620-010-1512-y



of biliary cancer by disturbing cell proliferation, apoptosis,

and promoting cell inflammation in the CCA cell line.

Keywords Helicobacter pylori Á Cell proliferation Á

Apoptosis Á Inflammation Á Hepatobiliary diseases Á

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 fluke Opisthorchis viverrini is

intimately related to pathogenesis of CCA in this region

[2]. However, pathogenesis of CCA or other biliary cancers

in non-liver fluke 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 diseasesincluding 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 [5–7]. H. pylori was

detected more often in both CCA and hepatocellular car-

cinoma (HCC) patients than in patients with non-malignant

tumors and the control group [6, 8], suggesting a correla-

tion 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 dem-

onstrated that prolonged infection with H. pylori resulted in

the development of HCC [10]. Additionally, the cytotoxin-

associated 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 mito-

gen-activating protein kinases (MAPK) and NF- jB in both

CagA tyrosine phosphorylation-dependent and -indepen-

dent manners [15]. IL-8, a proinflammatory cytokine, can

induce neutrophil and monocyte infiltration leading to

inflammation [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 [17–19]. Recently, an in vitro study showed that a

putatively more virulent strain of H. pylori induced apop-

tosis 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 (especiallycagA? strain) in biliary cells including cell proliferation,

induction of apoptosis and inflammation have not been

extensively studied. In this study, therefore, we investi-

gated the molecular mechanisms of H. pylori cagA? and

cagA- strains in cell proliferation, induction of apoptosis,

and pro-inflammatory 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 37°C 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 37°C in a humidified 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 tetra-

zoliumbromide] assay. KKU-100 cells (8 9 104 cells/well)

were seeded in a 96-well microtiter plate and incubated at

37°C 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 dimethyl-

sulfoxide (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.

Dig Dis Sci (2011) 56:1682–1692 1683

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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-bromo-

20-deoxyuridine) incorporation was performed as previ-

ously described [20]. Chromogenic substrate tetra-meth-

ylbenzidine (TMB) was then added and optical density wasmeasured 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 per-

centage of DNA synthesis compared to the control.

Determination of Apoptotic Cells

Nuclear staining using 4,6-diamidino-2-phenylindole

(DAPI) was performed to determine chromatin condensa-

tion and fragmentation. KKU-100 cells (1 9 106 cells/

well) were grown in a cell culture dish at 37°C for 24 h andco-cultured with medium alone (control) or H. pylori

(1:200) for 12 and 24 h. The treated cells were washed with

PBS, fixed with methanol, stained with 1 lg/ml of DAPI at

37°C for 15 min and observed under a fluorescence

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 QIAampÒ DNA

Mini Kit (Qiagen, USA) according to the manufacturer’s

instructions. DNA was electrophoresed in a 1.5% agarose

gel and the DNA band was visualized using a UV-illumi-

nator (Bio-Rad, USA).

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 TRIzolÒ

reagent (Invitrogen, Grand Island, NY, USA) according tothe manufacturer’s 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 [23–25].

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 quantified 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,

Table 1 Primers used

for RT-PCRGenes Primer sequences Product

size (bp)

References

iNOS 50-CGGTGCTGTATTTCCTTACGAGGCGACGAAGG-3 0

50-GGTGCTGCT TGT TAGGAGGTCAAGTAAAGGGC-30

259 [24]

p53 50-TTCTTGCATTCTGGGACAGCC-3 0

50-GCCTCATTCAGCTCTCGGAAC-3 0

650 [24]

bax 50-TGGCAGCTGACATGTTTTCTGAC-3 0

50-CGTCCCAACCACCCTGGTCT-3 0

195 [24]

bcl-2 50-CTGTACGGCCCCAGCATGCG-3 0

50-GCTTTGTTTCATGGTACATC-3 0

231 [23]

caspase-3 50-TTCAGAGGGGATCGTTGTAGAAGTC-3 0

50-CAAGCTTGTCGGCATACTGTTTCAG-3 0

264 [25]

GAPDH 50-CGGAGT CAACGGATT TGGTCGTAT-30

50-AGCCTTCTCCATGGTGGTGAAGAC-3 0

306 [24]

1684 Dig Dis Sci (2011) 56:1682–1692

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NF- jB (Cell Signaling Technology, MA, USA) overnight

at 4°C. HRP-conjugated secondary antibody was added,

and protein was visualized using the enhanced chemilu-

minescence (ECL) system (Thermo Scientific, IL, USA).

The intensity of the protein bands was quantified 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 tocontrol (intensity of protein band of H. pylori-treated cells

divided by protein band of control).

Measurement of IL-8 Production for Cell Inflammation

KKU-100 cells were co-cultured with H. pylori at a cell-

bacteria 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 manufacturer’s 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 Student’s t test.

p\ 0.05 and p\ 0.01 were considered significant.

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 cellgrowth 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-bac-

teria ratio of 1:1 was significantly 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

(1:10–1:200) 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 significantly lower (49 ± 2.47%) than thattreated with the cagA- strain (60 ± 1.30%) (Fig. 1a).

For DNA synthesis of KKU-100 cells at a low cell-

bacteria 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 syn-

thesis in KKU-100 cells at cell-bacterial ratios starting

from 1:20 to 1:200 ( p\ 0.05) (Fig. 1b). At the high cell-

bacteria 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 cagA-

strains 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


strains

Dig Dis Sci (2011) 56:1682–1692 1685

123



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 prolifera-

tion was performed using RT-PCR and Western blotting to

determine bcl-2, bax, p53 gene expression, and phosphor-ylated MAPK and NF-jB protein expression. At low

inocula of the H. pylori cagA? and cagA- strains (cell-

bacteria ratio of 1:1), the expression of pro-apoptotic

genes, bax and p53, were not significantly changed com-

pared 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 significantly higher than in the cells treated with the

H. pylori cagA- strain (Fig. 2a).

Figure 2b illustrates the time dependent of phosphor-

ylated 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). Phosphor-

ylation 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 acti-

vation at 30 min.

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 usingDAPI 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 homoge-

neous chromatin distribution (Fig. 3a, left panel). At 24 h,

the number of apoptotic cells resulting from treatment with

the H. pylori cagA? was significantly 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 pro-

grammed cell death, the mRNA level was evaluated. Using

RT-PCR analysis, a significance 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

Fig. 2 Role of H. pylori on cellproliferation in KKU-100 cells,

co-cultured with the H. pylori

cagA? and cagA- strains at a

low inoculum (1:1); apoptosis-

related 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





strains

1686 Dig Dis Sci (2011) 56:1682–1692

123



(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 significantly higher than the H. pylori cagA-

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 significantly higher than that exposed to

the H. pylori cagA- strain (1.35-fold for the p53 and 1.32-

fold for the caspase-3) at 24 h of treatment (Fig. 4). Cells

treated with the H. pylori cagA? and cagA- strains showed

significant 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 significantly 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 significantly 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 significant

reduction of Bcl-xL while increasing the Fas in a time-

dependent 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. 3 Determination of

apoptotic cell death in KKU-

100 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





strains

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 cagA-

strains 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

Dig Dis Sci (2011) 56:1682–1692 1687

123



(6.41-fold) and activated caspase-3 (9.39-fold) was sig-

nificantly higher in the H. pylori cagA?-treated cells than

in the H. pylori cagA--treated cells (2.67-fold and 3.94-

fold, 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 cagA-

treated cells were similar (Fig. 5).

H. pylori Stimulates IL-8 Production in Biliary Cells

To evaluate H. pylori affect on biliary cell inflammation,

ELISA was used to measure IL-8 secretion in KKU-100

cells infected with H. pylori (Fig. 6). The concentration of

IL-8 was significantly 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 bac-

terial concentrations (Fig. 6).

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

significantly 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

inflammation 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

Fig. 5 Assessment of apoptosis-related protein expression in KKU-

100 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-actinintensity. Data (shown beloweach band)are presented as a fold-

change ofthe treated cells comparedto thecontrol 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

Table 2 Bax/Bcl-2 ratios of KKU-100 cell line infected with

H. pylori

H. pylori strains Bax/Bcl-2 ratio

0 h 6 h 12 h 24 h

H. pylori cagA? 0.24 0.58 1.45* 5.71*, #

H. pylori cagA- 0.24 0.53 1.75* 3.38*

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

Fig. 6 H. pylori stimulates IL-8 production. The KKU-100 cells wereco-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 experi-

ment (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

1688 Dig Dis Sci (2011) 56:1682–1692

123



depended on the diversity of H. pylori strains [10]. Addi-

tionally, H. pylori inoculated orally in the C57BL/6 mice

resulted in the development of severe gastric mucosal

inflammation, mild to moderate hepatitis, and gallbladder

mucosa thickening with mild submucosal lymphocytic

infiltration [28]. Goo and colleagues showed that H. pylori

promotes hepatic fibrosis and increases hepatocytes pro-

liferation 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. hepaticus—the causative agent of hepatocellular carci-

noma 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 proposethat 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 inflammation. The

inflammation can be caused by stimulation of many cyto-

kine productions [34]. H. pylori infection is well known as

a cause of gastric inflammation and to stimulate pro-

inflammatory cytokines production (i.e., TNF-alpha, IFN-

gamma, IL-1, IL-6, IL-8) [35]. In inflammatory conditions,

iNOS can be induced, in almost any cell type, through

stimulation by inflammatory 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 proinflammatory CXC chemokine (IL-8) have

been shown to be regulated by the NO [37–39].

IL-8 was shown to activate multiple intracellular sig-

naling pathways leading to an increase in proliferation and

survival of cancer cells [16]. In this study, using the KKU-

100 cell line, we found that at the low cell-bacteria ratio

(1:1), the H. pylori cagA? strain significantly 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 pro-

duction, 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 significant 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 specific H. pylori strains used in

each study.

Many studies failed to culture H. pylori from hepatob-

iliary 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 confirms 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 cell-

bacteria 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., anti-

apoptotic 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-pro-

moting genes and anti-apoptotic genes) which might con-

tribute to the malignant transformation [14].

In a previous report, high concentration of NO, pro-

ceeding 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 con-

centrations [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 significantly 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 cas-

pase-8 and -3 [44]. Our results showed a higher up-regu-

lation of activated caspase-8 and -3 protein expression in

Dig Dis Sci (2011) 56:1682–1692 1689

123



the KKU-100 cell line co-cultured with the H. pylori

cagA? strain than the H. pylori cagA- strain. Our findings

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 throughthe mitochondrial pathway [47]. Our study found a sig-

nificant increase in the pro-apoptotic protein, Bax, and a

significant 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 cyto-

chrome 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 caspase-

9 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 significantly 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

findings agree with other studies of gastroduodenal dis-

eases 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 vir-

ulence factors of H. pylori are also involved in apoptosis on

KKU-100 cells. In order to clarify whether CagA influ-

ences apoptosis in biliary cells, a purified CagA protein

should be used to examine its exact role in apoptosis.

Disturbance of the cell death process by apoptosis-

enhanced cell turnover will result in increased prolifera-

tion, 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 Helico-bacter 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 inflammation and proliferation in tissues of

CCA patients.

In conclusion, our study shows that H. pylori has mul-

tiple effects on biliary cells, including: (1) stimulation of

DNA synthesis and increased cell proliferation via activa-

tion 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 dosesof bacteria; and, (3) stimulation of IL-8 production. We

conclude that CagA play an important role in the carci-

nogenesis of the biliary cancer through the disturbance of

cellular homeostasis by stimulation of cell proliferation and

induction of apoptosis (which depends on bacterial con-

centration) and promotes inflammation by stimulating pro-

inflammatory 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 onHigher 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 Medi-

cine, 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 English-

language presentation of the manuscript.

References

1. Sripa B, Pairojkul C. Cholangiocarcinoma: lessons from Thai-

land. Curr Opin Gastroenterol. 2008;24:349–356.

2. Sripa B, Kaewkes S, Sithithaworn P et al. Liver fluke induces

cholangiocarcinoma. PLoS Med . 2007;4:e201.

3. Pellicano R, Menard A, Rizzetto M, Megraud F. Helicobacter

species and liver diseases: association or causation? Lancet Infect

Dis. 2008;8:254–260.

4. Fox JG, Dewhirst FE, Shen Z, et al. Hepatic Helicobacter species

identified in bile and gallbladder tissue from Chileans with

chronic cholecystitis. Gastroenterology . 1998;114:755–763.

1690 Dig Dis Sci (2011) 56:1682–1692

123



5. Avenaud P, Marais A, Monteiro L, et al. Detection of Helico-

bacter species in the liver of patients with and without primary

liver carcinoma. Cancer . 2000;89:1431–1439.

6. Nilsson HO, Mulchandani R, Tranberg KG, Stenram U, Wad-

strom T. Helicobacter species identified in liver from patients

with cholangiocarcinoma and hepatocellular carcinoma. Gastro-

enterology. 2001;120:323–324.

7. Pellicano R, Mazzaferro V, Grigioni WF, et al. Helicobacter

species sequences in liver samples from patients with and without

hepatocellular carcinoma. World J Gastroenterol. 2004;10:

598–601.

8. Huang Y, Fan XG, Wang ZM, et al. Identification of Helico-

bacter species in human liver samples from patients with primary

hepatocellular carcinoma. J Clin Pathol. 2004;57:1273–1277.

9. Abu Al-Soud W, Stenram U, Ljungh A, et al. DNA of Helico-

bacter spp. and common gut bacteria in primary liver carcinoma.

Dig Liver Dis. 2008;40:126–131.

10. Wang X, Willen R, Svensson M, Ljungh A, Wadstrom T. Two-

year follow-up of Helicobacter pylori infection in C57Bl/6 and

Balb/ca mice. Apmis. 2003;111:514–522.

11. Tiwari SK, Khan AA, Ibrahim M, Habeeb MA, Habibullah CM.

Helicobacter pylori and other Helicobacter species DNA in

human bile samples from patients with various hepato-biliary

diseases. World J Gastroenterol. 2006;12:2181–2186.

12. Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helico-

bacter pylori infection. Clin Microbiol Rev. 2006;19:449–490.

13. Hatakeyama M. The role of Helicobacter pylori CagA in gastric

carcinogenesis. Int J Hematol. 2006;84:301–308.

14. Hatakeyama M. SagA of CagA in Helicobacter pylori patho-

genesis. Curr Opin Microbiol. 2008;11:30–37.

15. Brandt S, Kwok T, Hartig R, Konig W, Backert S. NF-kappaB

activation and potentiation of proinflammatory responses by the

Helicobacter pylori CagA protein. Proc Natl Acad Sci USA.

2005;102:9300–9305.

16. Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin

Cancer Res. 2008;14:6735–6741.

17. Cabral MM, Mendes CM, Castro LP, et al. Apoptosis in Heli-

cobacter pylori gastritis is related to cagA status. Helicobacter .

2006;11:469–476.

18. Zhang ZW, Patchett SE, Farthing MJ. Role of Helicobacter

pylori and p53 in regulation of gastric epithelial cell cycle phase

progression. Dig Dis Sci. 2002;47:987–995.

19. Moss SF, Sordillo EM, Abdalla AM, et al. Increased gastric

epithelial cell apoptosis associated with colonization with cagA?

Helicobacter pylori strains. Cancer Res. 2001;61:1406–1411.

20. Ito K, Yamaoka Y, Yoffe B, Graham DY. Disturbance of apop-

tosis and DNA synthesis by Helicobacter pylori infection of

hepatocytes. Dig Dis Sci. 2008;53:2532–2540.

21. Kim KM, Lee SG, Park MG, et al. Gamma-glutamyl-

transpeptidase of Helicobacter pylori induces mitochondria-

mediated apoptosis in AGS cells. Biochem Biophys Res Commun.

2007;355:562–567.

22. Sripa B, Leungwattanawanit S, Nitta T, et al. Establishment and

characterization of an opisthorchiasis-associated cholangiocarci-noma cell line (KKU-100). World J Gastroenterol. 2005;11:

3392–3397.

23. Greenlund LJ, Korsmeyer SJ, Johnson EM Jr. Role of bcl-2 in the

survival and function of developing and mature sympathetic

neurons. Neuron. 1995;15:649–661.

24. Perfetto B, Buommino E, Canozo N, et al. Interferon-gamma

cooperates with Helicobacter pylori to induce iNOS-related

apoptosis in AGS gastric adenocarcinoma cells. Res Microbiol.

2004;155:259–266.

25. Winter RN, Kramer A, Borkowski A, Kyprianou N. Loss of

caspase-1 and caspase-3 protein expression in human prostate

cancer. Cancer Res. 2001;61:1227–1232.

26. Lin TT, Yeh CT, Wu CS, Liaw YF. Detection and partial

sequence analysis of Helicobacter pylori DNA in the bile sam-

ples. Dig Dis Sci. 1995;40:2214–2219.

27. Myung SJ, Kim MH, Shim KN, et al. Detection of Helicobacter

pylori DNA in human biliary tree and its association with

hepatolithiasis. Dig Dis Sci. 2000;45:1405–1412.

28. Huang Y, Tian XF, Fan XG, Fu CY, Zhu C. The pathological

effect of Helicobacter pylori infection on liver tissues in mice.

Clin Microbiol Infect . 2009;15:843–849.

29. Goo MJ, Ki MR, Lee HR, et al. Helicobacter pylori promotes

hepatic fibrosis in the animal model. Lab Invest . 2009;89:

1291–1303.

30. Ward JM, Fox JG, Anver MR, et al. Chronic active hepatitis and

associated liver tumors in mice caused by a persistent bacterial

infection with a novel Helicobacter species. J Natl Cancer Inst .

1994;86:1222–1227.

31. Boutin SR, Rogers AB, Shen Z, et al. Hepatic temporal gene

expression profiling in Helicobacter hepaticus-infected A/JCr

mice. Toxicol Pathol. 2004;32:678–693.

32. Taylor NS, Fox JG, Yan L. In vitro hepatotoxic factor in Heli-

cobacter hepaticus, H. pylori and other Helicobacter species.

J Med Microbiol. 1995;42:48–52.

33. Avenaud P, Castroviejo M, Claret S, et al. Expression and

activity of the cytolethal distending toxin of Helicobacter hepa-

ticus. Biochem Biophys Res Commun. 2004;318:739–745.

34. Balkwill F, Mantovani A. Inflammation and cancer: back to

virchow? Lancet . 2001;357:539–545.

35. Bodger K, Crabtree JE. Helicobacter pylori and gastric inflam-

mation. Br Med Bull. 1998;54:139–150.

36. Pfeilschifter J, Eberhardt W, Beck KF. Regulation of gene

expression by nitric oxide. Pflugers Arch. 2001;442:479–486.

37. Beck KF, Eberhardt W, Frank S, et al. Inducible NO synthase:

role in cellular signalling. J Exp Biol. 1999;202:645–653.

38. Brown Z, Robson RL, Westwick J. L-arginine/nitric oxide path-

way: a possible signal transduction mechanism for the regulation

of the chemokine IL-8 in human mesangial cells. Adv Exp Med

Biol. 1993;351:65–75.

39. Allen RG, Tresini M. Oxidative stress and gene regulation. Free

Radic Biol Med . 2000;28:463–499.

40. Ito K, Nakamura M, Toda G, et al. Potential role of Helicobacter

pylori in hepatocarcinogenesis. Int J Mol Med . 2004;13:221–227.

41. Lala PK, Chakraborty C. Role of nitric oxide in carcinogenesis

and tumour progression. Lancet Oncol. 2001;2:149–156.

42. Brune B, von Knethen A, Sandau KB. Nitric oxide and its role in

apoptosis. Eur J Pharmacol. 1998;351:261–272.

43. Obst B, Wagner S, Sewing KF, Beil W. Helicobacter pylori

causes DNA damage in gastric epithelial cells. Carcinogenesis.

2000;21:1111–1115.

44. Gupta S. Molecular signaling in death receptor and mitochondrial

pathways of apoptosis (review). Int J Oncol. 2003;22:15–20.

45. Rudi J, Kuck D, Strand S, et al. Involvement of the CD95 (APO-

1/Fas) receptor and ligand system in Helicobacter pylori-induced

gastric epithelial apoptosis. J Clin Invest . 1998;102:1506–1514.

46. Le’Negrate G, Ricci V, Hofman V, et al. Epithelial intestinal cellapoptosis induced by Helicobacter pylori depends on expression

of the cag pathogenicity island phenotype. Infect Immun.

2001;69:5001–5009.

47. Gogvadze V, Orrenius S. Mitochondrial regulation of apoptotic

cell death. Chem Biol Interact . 2006;163:4–14.

48. Perlman H, Zhang X, Chen MW, Walsh K, Buttyan R. An ele-

vated bax/bcl-2 ratio corresponds with the onset of prostate epi-

thelial cell apoptosis. Cell Death Differ . 1999;6:48–54.

49. Narita M, Shimizu S, Ito T, et al. Bax interacts with the perme-

ability transition pore to induce permeability transition and

cytochrome c release in isolated mitochondria. Proc Natl Acad

Sci USA. 1998;95:14681–14686.

Dig Dis Sci (2011) 56:1682–1692 1691

123



50. Elmore S. Apoptosis: a review of programmed cell death. Toxicol

Pathol. 2007;35:495–516.

51. Bras M, Queenan B, Susin SA. Programmed cell death via

mitochondria: different modes of dying. Biochemistry (Mosc).

2005;70:231–239.

52. Crabtree JE, Farmery SM, Lindley IJ, et al. CagA/cytotoxic

strains of Helicobacter pylori and interleukin-8 in gastric epi-

thelial cell lines. J Clin Pathol. 1994;47:945–950.

53. Kawahara T, Teshima S, Kuwano Y, et al. Helicobacter pylori

lipopolysaccharide induces apoptosis of cultured guinea pig

gastric mucosal cells. Am J Physiol Gastrointest Liver Physiol.

2001;281:G726–G734.

54. Kuck D, Kolmerer B, Iking-Konert C, et al. Vacuolating cyto-

toxin of Helicobacter pylori induces apoptosis in the human

gastric epithelial cell line AGS. Infect Immun. 2001;69:

5080–5087.

55. Schulte-Hermann R, Bursch W, Grasl-Kraupp B, et al. Role

of active cell death (apoptosis) in multi-stage carcinogenesis.

Toxicol Lett . 1995;82–83:143–148.

1692 Dig Dis Sci (2011) 56:1682–1692

123

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