Helicobacter pylori induces malignant transformation of

gastric epithelial cells in vitro

XIU-WEN YU,1,2 YING XU,1 YUE-HUA GONG,1 XU QIAN and YUAN YUAN1

1Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First AffiliatedHospital of China Medical University; the key Cancer Control Laboratory in Liaoning Province, Shenyang; and

2Department of Pathology, Qiqihar Medical College, Qiqihar, Heilongjiang Province, China

Yu X-W, Xu Y, Gong Y-H, Qian X, Yuan Y. Helicobacter pylori induces malignant transformation ofgastric epithelial cells in vitro. APMIS 2011; 119: 187–97.

Epidemiologic studies have demonstrated thatHelicobacter pylori infection is associated with increasedrisk for the development of gastric cancer. Animal studies have also shown thatH. pylori infection leadsto gastric carcinogenesis, especially intestinal phenotypes. However, no in vitro study has been carriedout for cell transformation induced by H. pylori. The present study aimed to investigate whether‘chronic’ H. pylori infection induces gastric epithelial cell transformation, and elucidate the underlyingmechanisms of transformation induced by H. pylori. The immortalized ‘normal’ gastric epithelial cellline, GES-1, was co-cultured for 45 days with H. pylori strains B975 and L301. The cell proliferationwas measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Ki-67 antigen,and colony formation assay. The cell transformation was determined by observing cell morphology andmeasuring the expression of E-cadherin, b-catenin, and transcription factor-4 (TCF-4) at both proteinand mRNA levels. H. pylori induced morphologic changes in GES-1 cells and significantly increasedthe proliferation of GES-1 cells. Moreover, H. pylori up-regulated the expression of b-catenin andTCF-4, and also induced the nuclear accumulation of b-catenin. In addition, the diffusive gastric can-cer-related gene, E-cadherin, was up-regulated at the protein level, but down-regulated at the mRNAlevel.H. pylori infection is capable of inducing GES-1 transformation to present with the characteristicsof intestinal-type gastric cancers in vitro, likely through the b-catenin ⁄TCF-4 signaling pathway.

Key words: Helicobacter pylori; GES-1 cell; cell transformation; intestinal phenotypes of gastriccancers.

Yuan Yuan, No. 115 Nanjing Northern Street, Heping District, Shengyang, Liaoning Province,110001 China. e-mail: yyuan3@hotmail.com

Chronic infection of the gastric mucosa by thehuman pathogen Helicobacter pylori is a leadingcause for the development of gastroduodenaldiseases such as chronic gastritis, peptic ulcers,and mucosa-associated lymphoid tissue lym-phoma (1). More importantly, chronic H. pyloriinfection has been classified as a definite carcin-ogen for the development of gastric carcinoma(2), which is the fifth most common malignancy

worldwide, and is still the second most commoncancer and first leading cause of cancer death inChina (3). In addition to H. pylori infection, theenvironmental and dietary factors and geneticbackground of the host all contribute to thedevelopment of gastric cancer. It has beenwidely accepted that the development of gastriccancer, especially the intestinal type, is a compli-cated multi-step process in vivo, in which themalignant transformation is involved. The pro-cess usually begins from the normal mucosa tochronic gastritis, gastric glandular atrophy,

Received 23 October 2010. Accepted 25 November2010

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APMIS � 2011 APMIS

DOI 10.1111/j.1600-0463.2010.02709.x

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intestinal metaplasia, then dysplasia and finallyto early adenocarcinoma (4). The previous stud-ies on H. pylori-infected Mongolian gerbils con-firmed that H. pylori infection could lead togastric cancer, especially the intestinal type(5–9). However, to our knowledge, thereremains no study that determines whether andhow (if any) H. pylori induces the transforma-tion of gastric epithelial cells in vitro.During the development of gastric cancer,

there are malfunctions or alterations of signalingpathways and the abnormal expression ofrelated genes and factors, such as oncogenes,tumor suppressor genes, cellular adhesion mole-cules (CAM), and telomeres (10, 11). Currentstudies indicate that the Wnt ⁄b-catenin signalingpathway plays a crucial role in the pathologicprocess of carcinogenesis and advance (11, 12),and is particularly related to the development ofgastrointestinal tumors (13, 14). The activationof the Wnt ⁄b-catenin signaling pathway hasbeen observed in about 30% of gastric cancerpatients, often as a result of N-terminal muta-tions in b-catenin injuring its proper degrada-tion (15). b-catenin and transcription factor-4(TCF-4), which are the proteins involved in theWnt ⁄b-catenin signaling pathway, are associatedwith intestinal phenotypic expression in humangastric cancer, and thus regarded as biomarkersfor intestinal-type gastric cancer (16–20).E-cadherin, a kind of calcium-dependent

CAM, normally binds with b-catenin to form thecomplex at the cell membrane and mediates thecellular adhesion between the same kinds of cells.E-cadherin is also involved in the carcinogenesisof diffusive gastric cancer. Recently, severalstudies have demonstrated that the expression ofE-cadherin is down-regulated in diffuse gastriccancer because of mutations of E-cadherin gene(CDH1) (21–24). In addition, alterations in theE-cadherin ⁄b-catenin cell adhesion complexfrequently occur in gastric cancer and are associ-ated with increased nuclear localization ofb-catenin (25). Thus, perturbation in the expres-sion or function of E-cadherin ⁄b-catenin genescould result in consequent malignant transfor-mation and tumor progression.GES-1 is a kind of immortal gastric epithelial

cell line, which is derived from fetal gastric epi-thelial cell after SV40 transfection (26). It hasbeen demonstrated that GES-1 is basically a nor-mal gastric epithelial cell line that can be used for

further investigation on the in vitro characteris-tics of normal gastric mucosa and the develop-ment mechanism of gastric cancer (26). UsingGES-1, the present study aimed to investigatewhether ‘chronic’H. pylori infection induces gas-tric epithelial cell transformation, and elucidatethe underlying mechanisms of transformationinduced byH. pylori bymonitoring themorphol-ogy of GES-1 cells, and measuring the expres-sion of E-cadherin, b-catenin, and TCF-4 inGES-1 cells after co-culture withH. pylori.

MATERIALS AND METHODS

Culture of human gastric epithelial cells

We used an immortalized human fetal gastric epithe-lial cell line, GES-1 (27) (kindly provided by theDepartment of Cell Genetics at Beijing Institute forCancer Research, Beijing, China), for the study.GES-1 cells were grown in RPMI 1640 medium (pH7.2–7.4; Gibco, Carlsbad, CA, USA) supplementedwith 10% fetal bovine serum (FBS; Beijing ZhongShan-Golden Bridge Biological Technology Co., Beij-ing, China) and penicillin ⁄ streptomycin (both100 U ⁄mL) at 37 �C in a humidified atmosphere of5% CO2. The cells were allowed to reach 80% conflu-ency before passage. The culture medium was replen-ished with the fresh medium every 2 or 3 days.

Culture ofHelicobacter pylori

We used two H. pylori strains, B975 and L301 (bothprovided by the Third Laboratory of Institute of Can-cer Research at China Medical University, Shenyang,China). B975 and L301 were isolated from a patientwith active chronic gastritis and a patient with gastriccancer, respectively. Both strains share the same viru-lence factors including cytotoxin-associated gene Aprotein (CagA), vacuolating cytotoxin A (VacA), andblood group antigen-binding adhesion (BabA).H. pylori was cultured on brain heart infusion agarplates with 10% sheep blood at 37 �C in a 97%humidified atmosphere of 85% N2, 5% O2, and 10%CO2 under microaerobic conditions. Single colonieswere subcultured on the agar plates for 3–4 days, andthen further subcultured for 48 h before the bacterialcells were harvested into RPMI 1640 medium with10% FBS for immediate use.

Co-culture of GES-1 cells withHelicobacter pylori

GES-1 cells were grown in RPMI 1640 medium sup-plemented with 10% FBS without antibiotics in cellculture flasks overnight. Then, bacteria were added in

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the ratio (GES-1 cells: bacterial cells) of 1:1 forco-culture. The cells were allowed to reach 80% con-fluency before passage. The culture was replenishedwith fresh medium every 2 or 3 days, after threewashes with aseptic phosphate-buffered saline (PBS).GES-1 cells cultured under the same conditions butwithout co-culture with H. pylori were used as con-trols. The culture continued for 45 days, and the cellswere used for experiments and analysis during thatperiod of time.

Cell morphology observation

The morphology of viable GES-1 cells cultured withor without H. pylori was observed under an invertedphase contrast microscope at 72 h and 45 days.In addition, GES-1 cells cultured with or withoutH. pylori for 45 days were placed on chamber slides.The slides were fixed with acetone at 4 �C and stainedwith hematoxylin and eosin (H&E) for morphologicobservation. Moreover, the GES-1 cells harvestedfrom culture flasks after 45 days of co-culture werefixed with 2.5% glutaraldehyde for observation undera transmission electron microscope (TEM, EM208S;Philips, Eindhoven, The Netherlands).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay for cell proliferation

GES-1 cells cultured with or without H. pylori for45 days (at a concentration of approximately 1 · 104

cells ⁄well) were seeded into wells containing 100 lLof the culture medium of a 96-well plate and incu-bated overnight at 37 �C. Then, the cells were cul-tured, replenishing with fresh medium at differenttime points (i.e., 12, 24, and 48 h). Then, 25 lL of5 lg ⁄mL MTT (Sigma Chemical Co., St. Louis,MO, USA) labeling reagent was added to the desig-nated wells and cells were further incubated at 37 �Cfor 4 h. The supernatant was removed, and then150 lL of dimethyl sulfoxide was added. After theplate was incubated at 37 �C for 10 min, the absor-bency was measured using a micro ELISA reader(Bio-Tek Instruments, Winooski, VT, USA) at570 nm. The experiments were repeated twice, andduplicated wells were used for cells cultured with orwithout H. pylori for 45 days at each time point foreach experiment.

Immunocytochemistry assay for Ki-67 protein

expression

Immunostaining was performed to determine Ki-67protein expression in GES-1 cells cultured with orwithout H. pylori for 45 days using the labeled strep-tavidin–biotin technique, according to the manufac-turer’s (Zhong Shan-Golden Bridge Biological

Technology Co.) instructions. The polyclonal anti-Ki-67 (Biotechnology, Inc., Santa Cruz, CA, USA)was used as the primary antibody. GES-1 cells wereplaced on the chamber slide in PBS, and then treatedwith normal goat serum for 30 min to block any non-specific binding, which was followed by incubationovernight at 4 �C with an optimum dilution of theprimary antibody (·100 dilution). The negative con-trol was prepared by processing the slides in the samemanner, but without the primary antibody. The aver-age optical density was obtained by measuring threerandomly selected fields per slide using a microELISA reader (Bio-Tek Instruments).

Colony formation assay for cell viability

Viable cells cultured with or without H. pylori for45 days (200 cells ⁄well) were seeded into wells of asix-well plate (in triplicate) and cultured in an incuba-tor with 5% CO2. The medium was replaced 1 weeklater and the colony count was performed after incu-bation for another week. Cells in the plate werewashed with PBS thrice after removal of the medium,and then fixed with 4% paraformaldehyde for 5 mintwice, followed by staining with 0.1% crystal violetfor 5 min. The stain was washed off under runningtap water, and the plate was allowed to dry. The num-ber of distinctly stained colonies containing at least50 cells per colony was counted under an invertedmicroscope. Colony-forming efficiency (CFE) wascalculated as the number of colonies generateddivided by the seeded cells (i.e., 200) · 100%.

Immunofluorescence assay for protein expression

of b-catenin, E-cadherin, and TCF-4

GES-1 cells cultured with or without H. pylori for45 days were placed on chamber slides, and were fixedwith acetone at 4 �C for 10 min. Then, the cells werewashed in 2-ethanesulfonic acid buffer five times(each for 5 min) before addition of 10% goat serumalbumin and incubation for 30 min at room tempera-ture. After further three washes (each for 5 min), thecells were permeabilized by 0.5% Triton X-100 for10 min, washed again for 3–5 min, and then incu-bated overnight at 4 �C with one of the following pri-mary antibodies: a concentrated murine monoclonalanti-human b-catenin (E5, ·100 dilution; SantaCruz), a concentrated rabbit polyclonal anti-humanE-cadherin (H-108, ·100 dilution; Santa Cruz), or aconcentrated rabbit monoclonal anti-human TCF-4(EP2033Y, ·100 dilution; Epitomics, CA, USA).Then, goat anti-mouse IgG ⁄FITC (·50 dilution;Santa Cruz) or goat anti-rabbit IgG ⁄RBITC (·50dilution; Santa Cruz) antibodies were added, respec-tively, and the cells were incubated at 37 �C in thedark for 30 min and washed with PBS. Finally, thecells were mounted on slides with 50% glycerin and

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examined under a fluorescence microscope (OlympusCX41-32RFL, Tokyo, Japan).

RNA extraction and real-time reverse transcription-

polymerase chain reaction (RT-PCR) for mRNA

expression of b-catenin, E-cadherin, and TCF-4

Total RNA was extracted from GES-1 cells culturedwith or without H. pylori for 45 days using a totalRNA kit (Tiangen Biotech, Beijing, China). RNA(1.5 lg) was reverse transcribed to cDNA usingImProm-II� Reverse Transcription System Kit (Pro-mega, Southampton, UK) according to the manufac-turer’s instructions. RT-PCR was carried out usingTaqMan primer sets specific for human E-cadherin,b-catenin, TCF-4, and b-actin, as described previously(Table 1) (28–31). The densitometry analysis was per-formed using the GDS-8000 System for gel documen-tation (UVPBioImaging Systems,Upland, CA,USA).

Statistical analysis

Statistical analysis was performed using SPSS� ver-sion 11.5 (SPSS, Chicago, IL, USA). Continuousvariables were expressed as mean ± standard devia-tion (SD) and their differences percentages were com-pared between groups by the Mann–Whitney test orchi-squared test, where appropriate. A p-value of lessthan 0.05 (two-sided) was considered statistically sig-nificant.

RESULTS

Effect ofHelicobacter pylori on the morphology

of GES-1 cells

As observed using the inverted phase contrastmicroscope, GES-1 control cells presented with

a polygonal or fusiform shape, a regular appear-ance, clear edge, and anchorage-dependentgrowth. Floating cells were rarely observed(Fig. 1, A1). After 72 h of co-culture withH. pylori, irregular appearance emerged, withunclear edges. There were numerous increasingfloating cells. After 45 days of co-culture, mostGES-1 cells exhibited significant morphologicchanges including enlarged cellular size, moreirregular appearance, and enlarged cellularnuclei with increased number of nucleoli. Occa-sionally, giant cells were observed. The numberof viable cells declined (Fig. 1, A2 and A3). Sim-ilar findings were observed with H&E stainingas shown in Fig. 1, B1–B3. The significantlyincreased pathologic karyokinesis was observedin nuclei. TEM revealed that the cells co-cul-tured with H. pylori for 45 days of co-culture appeared with an irregular shape,increased cellular microvilli, enlarged nucleiwith increased chromatin, and thickenednuclear membrane (Fig. 1, C1–C3). Occasion-ally, H. pylori was observed surrounding thecells (Fig. 1, C3).

Effect ofHelicobacter pylori on proliferation

of GES-1 cells

The MTT assay showed that H. pylori signifi-cantly inhibited the proliferation of GES-1 cellsfor up to 48 h after co-culture with the cells;however, after 72 h of co-culture, H. pylorisignificantly increased the proliferation of thecells (p < 0.001; Table 1). Moreover, L301 hadmore power than B975 on the proliferation ofGES-1 cells (p < 0.001; Table 2).

Table 1. TaqMan primer sets and reverse transcription-polymerase chain reaction procedures for mRNAexpression

mRNA Forward primer Reverse primer Procedures

E-cadherin (29) 5¢-TGA AGG TGA CAGAGC CTC TGG AT-3¢

5¢-TGG GTG AAT TCGGGC TTG TT-3¢

94 �C 1 min, 94 �C 39 s,55 �C 30 sec, 72 �C 30 s,

30 cycles, 72 �C 5 minb-catenin (30) 5¢-ACA AAC TGT TTT

GAA ATT CCA-3¢5¢-CGA GTC ATT GCA

TAC TGT CC-3¢95 �C 2 min, 95 �C 1 min,

58 �C 30 s, 72 �C 30 s,35 cycles, 72 �C 10 min

Transcriptionfactor-4 (31)

5¢-TCA CCA ACA GCGAAT GGC-3¢

5¢-AGG AAGGAT AGCCTG GCG-3¢

94 �C 4 min, 94 �C 45 s,60 �C 30 s, 72 �C 1 min,

33 cycles, 72 �C 5 minb-actin (32) 5¢-GCA TGG AGT CCT

GTG GCA T-3¢5¢-CTA GAA GCA TTT

GCGGTG G-3¢94 �C 2 min, 94 �C 45 s,

58 �C 45 s, 72 �C 45 s,30 cycles, 72 �C 7 min

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As shown in Fig. 2, the expression of Ki-67protein intensity was significantly increased inGES-1 cells co-cultured with the two H. pyloristrains, B975 (OD: 0.327 ± 0.009) or L301(OD: 0.358 ± 0.008), compared with that ofcontrol cells (OD: 0.308 ± 0.006, p = 0.040and p = 0.001, respectively). In addition, L301induced a more significant increase in Ki67

antigen expression than did B975 in GES-1 cells(p = 0.009).As shown in Fig. 3, H. pylori significantly

enhanced the colony formation of GES-1 cells.L301 significantly increased the CFE of GES-1cells, compared with that of the control cells(40.6% vs 27.2%, p = 0.042). There was aslight, but not statistically significant, increase

A1 A2 A3

B1 B2 B3

C1 C2 C3

Fig. 1. The effect of Helicobacter pylori on GES-1 cell morphology. (A) Inverted microscopy showingmorphology (arrows) of control GES-1 cells (A1, 200·), GES-1 cells co-cultured with H. pylori strain, B975,for 45 days (A2, 200·), and GES-1 cells co-cultured with H. pylori strain, L301, for 45 days (A3, 200·);(B) hematoxylin and eosin staining (200·) showing morphology (arrows) of control GES-1 cells (B1, 200·),and GES-1 cells co-cultured with H. pylori strain, B975, for 45 days (B2, 200·), and cellular changes andmitosis (arrow) of GES-1 cells co-cultured with H. pylori strain, L301 for 45 days (200·); (C) transmissionelectron microscopy showing the morphology (arrows) of control GES-1 cells (C1, 5000·), GES-1 co-culturedwith H. pylori strain, B975, for 45 days (C2, 5000·), and H. pylori cells (arrow) surrounding the GES-1 cells(C3, 8000·).

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in the CFE in GES-1 cells co-cultured withB975, compared with control cells (32.3% vs27.2%, p = 0.229).

Effect ofHelicobacter pylori on the protein and mRNA

expression of E-cadherin, b-catenin, and TCF-4 inGES-1 cells

Immunofluorescence assay showed that afterco-culture withH. pylori for 45 days, the expres-sion of E-cadherin, b-catenin, and TCF-4 wassignificantly up-regulated in GES-1 cells, com-pared with that in control cells (Fig. 4). More-over, b-catenin expression was mostly located inthe cytoplasm and nuclei of GES-1 cells co-cultured with H. pylori, but rarely in the cellmembrane (arrows; Fig. 4E,J) as were in theGES-1 control cells (arrows; Fig. 4B,G). Inaddition, fluorescent double-staining alsoshowed the expression of E-cadherin, b-catenin,and TCF-4 (Fig. 4C,F,I,L).As measured by real-time RT-PCR, the

expression of E-cadherin mRNA was signifi-cantly down-regulated, but the expression ofb-catenin and TCF-4 mRNA was up-regulated(Fig. 5 and Table 3).

DISCUSSION

Recently, H. pylori infection has been confirmedto be related to the development of gastric can-cer by Mongolian gerbil models (6–9). Althoughmechanisms of H. pylori-induced carcinogenesisare only beginning to be understood, inflamma-tion is the most commonly cited mechanismin the carcinogenic process. Inflammation isthought to induce cancer by increasing the pro-duction of free radicals, inducing apoptosis andnecrosis of epithelial cells and augmenting cellproliferation (32). An important mechanismposited other than inflammation is thatH. pylori directly interacts with epithelial cells,resulting in protein modulation, gene altera-tions, and consequently epithelial cell transfor-mation (33). In the present study, we used anon-tumorous gastric epithelial cell line, GES-1,to construct a cell transformation model with‘chronic’ H. pylori infection in vitro, to avoid orminimize the impact of inflammation presentin vivo. To our knowledge, this is the first timethat such a model was used to study the effectsofH. pylori on the transformation of gastric epi-thelial cells in vitro. We observed that H. pylori

Table 2. Effect ofHelicobacter pylori on proliferation of GES-1 cells

Cell group Proliferation (OD value) at different time points

24 h 48 h 72 h

Control GES-1 cells 0.687 ± 0.059 0.899 ± 0.174 1.224 ± 0.096GES-1 cells co-cultured with B975 0.300 ± 0.026*,** 0.702 ± 0.108* 1.506 ± 0.084*GES-1 cells co-cultured with L301 0.492 ± 0.505*,** 0.933 ± 0.131** 1.757 ± 0.101*,**

The experiment was performed twice with duplicate samples in each experiment.*p < 0.001 vs GES-1 control cells.**p < 0.001 vs GES-1 cells co-cultured with B975.

A B

Fig. 2. Effect of Helicobacter pylori on the expression of Ki-67 protein in GES-1 cells (200·). (A) GES-1 controlcells (arrow); and (B) GES-1 cells co-cultured withH. pylori for 45 days (arrow).

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induced GES-1 cells into morphologic changesand significantly increased the proliferation ofGES-1 cells. Moreover, H. pylori up-regulatedthe expression of b-catenin and TCF-4, knownas intestinal phenotypes of gastric cancer-related genes, and also induced the nuclearaccumulation of b-catenin. In addition, thediffusive gastric cancer-related gene, E-cadherin,was up-regulated at the protein level, but down-regulated at the mRNA level.Although the two H. pylori strains we used

were isolated from patients with different gastricdiseases, both possessed the virulence factor,CagA. It has been demonstrated that CagA acti-vates the anti-apoptotic pathways in gastric epi-thelial cells to overcome self-renewal of the hostcell and help sustain H. pylori infection (34).In the present study, after co-culture with theCagA+ H. pylori strains for 45 days, prolifera-tion and morphology of GES-1 cells presentedwith the features of transformation cells. More-over, the colony-forming assay also confirmedthat GES-1 proliferation ability was enhancedby H. pylori infection. These findings potentiallyindicate that H. pylori is able to induce GES-1to transform in vitro; however, further extensiveinvestigation based on our preliminary observa-tions is required. It is noted that the strain,

L301, isolated from a patient with gastric cancerwas more potent than the strain, B975, isolatedfrom a patient with active chronic gastritis, instimulating the cell proliferation and pathologickaryokinesis of GES-1. This observation indi-cates that there may be differences in the viru-lence and pathogenicity between strains isolatedfrom gastric cancer patients and those fromactive gastritis patients, which should be eluci-dated in further studies.A large body of evidence supports a causal

role of H. pylori in the majority of gastric malig-nancies. Great strides have been made in under-standing the pathogenesis of this bacterium.However, much remains to be studied. Geneticchanges can already be detected in intestinalmetaplasia, with p16 methylation being signifi-cantly associated with H. pylori infection in pre-cancerous lesions (35). Studies have also showndecreased E-cadherin expression in the gastricmucosa of H. pylori-infected individuals (36),and the interaction of CagA with E-cadherin,which causes cytoplasmic and nuclear accumu-lation of b-catenin, has been documented andimplicated in the development of intestinalmetaplasia (37). In the present study, H. pyloriled to the up-regulated protein and mRNAof intestinal-type gastric cancer-related genes,

A B

C D

Fig. 3. Colony formation of GES-1 cells observed with naked eyes and under a microscope (40·). (A, C) GES-1control cells; and (B, D) GES-1 cells co-cultured with Helicobacter pylori strain, B975. The number of colonies ofGES-1 was significantly increased for cells co-cultured withH. pylori, compared with control cells.

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that is, b-catenin and TCF-4, as shown in Figs 4and 5. In addition, b-catenin presented withcytoplasmic and nuclear translocation. Similarresults were reported in a study using MCF-7breast cancer cell line, which showed that a pre-dominant cytoplasmic localization of b-cateninafter prolonged H. pylori infection was associ-ated with deregulation of cell adhesion throughdisconnection of the E-cadherin–catenin com-plex from the cytoskeleton (38). Furthermore,we observed that the diffusive gastric cancer-related gene, E-cadherin, was up-regulatedat the protein level, but down-regulated atthe mRNA level, which further supports that

A B C

D E F

G H I

J K L

Control cells

Control cells

Co-cultured cells

Co-cultured cells

Fig. 4. The expression of E-cadherin, b-catenin, and transcription factor-4 (TCF-4) in GES-1 co-culture andcontrol groups (200·). (A–C and G–I) GES-1 control cells; (D–F and J–L) GES-1 cells exposed to Helicobacterpylori. (A) and (D) show the expression of E-cadherin; (B) and (G) show the expression of b-catenin on cell mem-brane, and (E) and (J) show the expression of b-catenin in the cytoplasm and nucleus; (H) and (K) show theexpression of TCF-4; (C) and (F) show the co-localization of E-cadherin and b-catenin; and (I) and (L) show theco-localization of b-catenin and TCF-4.

Fig. 5. Effects of Helicobacter pylori on the expres-sion of E-cadherin, b-catenin, and transcription fac-tor-4 mRNA in GES-1 cells. Lane 1, GES-1 controlcells; lane 2, GES-1 cells co-cultured with B975; andlane 3, GES-1 cells co-cultured with L301.

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H. pylori interacts with b-catenin ⁄TCF-4 signal-ing pathway to exert its role in the intestinal-type transformation.In the in vitro model we established that the

impact of host inflammation was attenuated (ifnot eliminated), indicating that the malignanttransformation should be mainly attributed tothe virulence factors of H. pylori. Phosphory-lated CagA is known to disrupt tight cell junc-tions, resulting in cell elongation and theformation of the so-called ‘hummingbird phe-notype’ (39). This process may result in thesloughing off of epithelial cells and compensa-tory cell proliferation. Suzuki et al. (40)reported that the non-phosphorylated CagAactivity was involved in interaction with acti-vated Met, a hepatocyte growth factor receptor,which in turn led to the activation of b-cateninand NF-jB signaling, and contributed to theepithelial proliferative and proinflammatoryresponses associated with the development ofchronic gastritis and gastric cancer. Sokolovaet al. (41) reported that, in Madin-Darby caninekidney cell line, H. pylori infection suppressedSer ⁄Thr phosphorylation and ubiquitin-depen-dent degradation of b-catenin, resulting inup-regulation of lymphoid enhancer-bindingfactor ⁄T-cell factor (LEF ⁄TCF)-dependenttranscription. Therefore, CagA may play apivotal role in the malignant transformation ofgastric epithelial cells by means of activation ofthe signaling pathways; however, further exten-sive investigation is required to reveal howCagA plays the role and whether and how othervirulence factors are involved in the process.In conclusion, H. pylori infection is capable of

inducing GES-1 transformation to present withthe characteristics of intestinal-type gastric can-cers in vitro, likely through b-catenin ⁄TCF-4signaling pathway. Further investigation onmolecular mechanisms with regard to howH. pylori induces malignant transformation isrequired.

The authors wish to acknowledge the kind offer ofthe GES-1 cell line by the Beijing Institute for Can-cer Research. They are also grateful to the CentralLaboratory of the First Hospital of China MedicalUniversity for its excellent technical assistance. Thiswork was supported by grant 2010CB529304 fromthe National Basic Research Development Programof China (973 Program) the Foundation of the KeyLaboratory of Cancer Intervention in LiaoningProvince (Ref No. 2008S231) and D200866 fromthe Natural Science Foundation of HeilongjiangProvince.

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Table 3. The expression of E-cadherin, b-catenin, and transcription factor-4 (TCF-4) mRNA in GES-1 cells withor without co-culture withHelicobacter pylori

Cell group E-cadherin ⁄ b-actin b-catenin ⁄ b-actin TCF-4 ⁄ b-actinControl GES-1 cells 0.568 ± 0.010 0.658 ± 0.027 0.200 ± 0.080GES-1 cells co-cultured with B975 0.461 ± 0.016* 1.413 ± 0.124* 0.925 ± 0.062*GES-1 cells co-cultured with L301 0.389 ± 0.013* 0.862 ± 0.016* 1.251 ± 0.069*

*p < 0.01 vs GES-1 control cells.

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� 2011 The 1st Affiliated Hospital, China Medical University � 2011 APMIS 195

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� 2011 The 1st Affiliated Hospital, China Medical University � 2011 APMIS 197

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