- Email: [email protected]
Functional profile of gastric epithelial cells infected with Helicobacter pylori strains
Accepted Manuscript Functional profile of gastric epithelial cells infected with Helicobacter pylori strains Ying Zhang, Hui Sun, Xingxing Chen, Jiaojiao Li, Huilin Zhao, Li Geng, Boqing Li PII:
S0882-4010(16)30040-7
DOI:
10.1016/j.micpath.2016.03.007
Reference:
YMPAT 1801
To appear in:
Microbial Pathogenesis
Received Date: 19 January 2016 Revised Date:
21 March 2016
Accepted Date: 22 March 2016
Please cite this article as: Zhang Y, Sun H, Chen X, Li J, Zhao H, Geng L, Li B, Functional profile of gastric epithelial cells infected with Helicobacter pylori strains, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Functional profile of gastric epithelial cells infected with Helicobacter pylori strains Ying Zhang#, Hui Sun#, Xingxing Chen, Jiaojiao Li, Huilin Zhao, Li Geng, Boqing Li*
#: these authors contributed equally to the paper Abstract
RI PT
School of Basic Medical Sciences, Binzhou Medical University
H. pylori infection represents a key factor in the etiology of various
SC
gastro-duodenal diseases, ranging from chronic gastritis to the development of peptic
ulcer disease and end-stage gastric cancer. In the present study, the 26695 and SS1
M AN U
strains of H. pylori were used to study the differential functional profiles of gastric epithelial cells infected with H. pylori. The apoptosis rates in GES-1 cells were significantly increased 3, 12 and 24 h after H. pylori 26695 and SS1 infection. Moreover, apoptosis by cells infected with the H. pylori 26695 strain was significantly higher than cells infected with the SS1 strain of H. pylori. No significant changes in the proliferation rates of GES-1 cells were observed after H. pylori 26695
TE D
or SS1 infection at any time during the experimental period. Exposure to H. pylori 26695 and SS1 induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner. Furthermore, H. pylori 26695 infection increased migration
EP
of GES-1 cells every hour during the whole experimental period compared withcontrol cells. However, GES-1 cells infected with the H. pylori SS1 strain
AC C
exhibited migration rates almost stable and comparable to those of control cells. These results indicate that the gastric epithelial cells respond differently depending on the H. pylori strain. This study indicates that the development of different gastric-related diseases may be a H. pylori strain-specific response. Keywords: Helicobacter pylori; apoptosis; proliferation; adhesion; migration
1. Introduction Helicobacter pylori is one of the most prevalent pathogens that contribute to human diseases [1, 2]. According to epidemiological studies, approximately 50% to 80% of the world population is infected with H. pylori, and areas of high incidence
ACCEPTED MANUSCRIPT
are concentrated in East Asia, including China, Japan and South Korea [3，4]. Chronic infection with H. pylori can result in DNA damage and genetic instability of the gastric mucosal cells, causing chronic gastritis and atrophy. The International Agency
RI PT
for Cancer Research (IACR) classified H. pylori as a class I carcinogen in 1994 [5], as numerous studies evidenced the relationship of H. pylori and gastric cancer from a combination of environmental and host-dependent factors.
The infection strategy of H. pylori and the defense capacity of the gastric
SC
epithelial cells in the gastric mucosa collectively influence colonization, survival, and
development of H. pylori infection-derived diseases. Mucosal epithelial cells are not only responsible for digestive processes, but also exert the crucial function of
M AN U
protecting the underlying tissue from pathogenic microorganisms that otherwise would invade the lumen [6]. Gastric epithelial cells form the first line of defense, thus contributing an innate defense such as cell barrier integrity, cell turnover, autophagy, and innate immune responses [6]. However, H. pylori can break through the gastric mucosal barrier and remain as a persistent infection via highly specialized
TE D
mechanisms, which facilitate H. pylori to avoid host defense mechanisms [7]. The failure of the host response in clearing the H. pylori infection promotes the development of chronic infection such as chronic gastritis. H. pylori infection represents a key factor in the etiology of various
EP
gastro-duodenal diseases, ranging from chronic gastritis to the development of peptic ulcer disease and gastric cancer (GC) [8]. The significant variation of the different
AC C
strains of H. pylori might be one of the important inducing factors that determines prevalence and incidence of H. pylori-associated diseases [9, 10]. Determining the
effects of the different H. pylori strains on the biological function of gastric epithelial cells will allow for better understanding of their cytotoxic effect and their regulatory mechanisms including the integration and rehabilitation of the mucosal epithelial barrier. In this context, the present study was performed with the following objectives (1) to investigate the time-response in cell apoptosis and proliferation in gastric epithelial cells (GES-1) after H. pylori infection, (2) to characterize cell adhesion and migration rates in GES-1 cells after H. pylori infection, (3) to further explore the
ACCEPTED MANUSCRIPT
cytotoxic diversity of H. pylori strains. 2. Materials and Methods 2.1. H. pylori
RI PT
Human-adapted strains of H. pylori 26695 and SS1, were provided by the H. pylori Research Laboratory of the Chinese Center for Disease Control and Prevention
(Beijing, China). H. pylori 26695 strain was originally isolated from a patient in the
United Kingdom with gastritis and Jcan-F Tomb et al. obtained the complete genome
SC
sequence of the gastric pathogen H. pylori 26695 in 1997 [11]. H. Pylori SS1 came
from a 42-year-old Greek-born woman in 1997, who had been diagnosed with a peptic ulcer [12]. Introduced H. Pylori SS1 bacteria were grown on chocolate agar
M AN U
plates supplemented with 10% sheep’s blood at 37˚C under microaerophilic conditions (5% O2, 10% CO2 and 85% N2) and subcultured every 3 days. For infection, H. pylori was harvested in chocolate agar broth and quantified by a spectrophotometer reaching 1×108 CFU/ml. 2.2. Human gastric epithelial cells
TE D
The human gastric epithelial cell line GES-1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco Modified Essential Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere of 5% CO2 at 37°C. When cells reached approximately 80%
passaged.
EP
confluence in 25 cm2 culture flasks, cells were treated with 0.25% trypsin and
AC C
2.3. Co-culture of GES-1 cells with H. pylori GES-1 cells were plated onto 6-well plates in DMEM containing 10% FBS (1×105
cells/2ml/well) overnight. The 26695 and SS1 strains of H. pylori were harvested and suspended in DMEM (including 10% FBS but no antimicrobial agents). Bacteria were added into the cells at a multiplicity of infection (MOI) of 200:1 and co-cultured for 0, 3, 12 and 24h. GES-1 cells were immediately used to detect cell apoptosis, proliferation, adhesion and motility rates [13]. 2.4. Cell apoptosis assays GES-1 cell apoptosis assay was performed using the Annexin V-FITC Apoptosis
ACCEPTED MANUSCRIPT
Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the instructions provided by the manufacturer. Briefly, 1×105 GES-1 cells were mixed together with 500µl of Annexin binding buffer, 5µl of FITC-conjugated Annexin V antibody and 5µl
RI PT
of propidium iodide (PI). The mixture was incubated in the dark for 15 min at room temperature. The relative number of apoptotic cells was determined using flow cytometry [14]. 2.5. EdU cell proliferation assay
SC
Cell proliferation was measured using a key fluor 488-EdU Proliferation Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s
instructions. GES-1 cells were incubated with H. pylori at 200:1 of bacterium to cell
M AN U
ratio in DMEM containing 10% FBS for 0, 3, 12 and 24h. All cells were treated with 50 µmol/L of EdU for 3 h at 37°C. After being fixed, the treated cells on coverslips were permeabilized with 0.5% TritonX-100 for 10 min. Cells were stained with Click-iT reaction mixture and incubated with Hoechst 33342 to stain the cell nuclei. Images were captured using inverted fluorescence microscope (Olympus, Tokyo,
TE D
Japan) [15]. The numbers of EdU- and DAPI-positive cells were quantified by IMAGEJ software. The percentages of immunopositive cells [(the number of immunopositive cells/total cells) × 100] were expressed. Five random fields of each well were chosen to perform the calculation.
EP
2.6. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell adhesion
AC C
A 96-well plate was treated with matrigel (0.04mg/mL, 50µl) (BD Biosciences, San Jose, CA) in DMEM overnight to facilitate cell attachment. GES-1 cells were cultured with the 26695 and SS1 strains of H. pylori for 0, 3, 12 and 24h, and then trypsinized and seeded into 100 mL of culture medium. MTT solution (5mg/mL, 20µL) was
added to the cells, and plates were further incubated at 37°C for 4h. The supernatant was carefully removed, and dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) was added to each well to dissolve formazan crystals. After 10 min shaking, the optical density was measured using ELISA at 490 nm wavelength. Cell adhesion rates
ACCEPTED MANUSCRIPT
= (OD of the infection group cells/ OD of the control group cells) x 100% [16]. 2.7. Scratch motility (wound-healing) assay GES-1 cells (1 x 105cells/well) were cultured on glass bottom dishes and allowed to
RI PT
form a confluent monolayer for 24h. Cells were then scratched using a sterile 10 µL pipette tip and the edge of each gap was straight and smooth. Single Cell Transferred Speed (SCTS) of treated cells were observed under the Living Cell Working Station.
Six random fields were selected and imaged continuously taken every 10 minutes for
SC
a period of 24h. Afterwards, the migrated distances were measured and the single cell migration velocity was calculated by manual counting. The analysis of image data
2.8. Statistical analysis
M AN U
was obtained by the Image Pro Plus 6.2.
The SPSS version 16.0 was used for statistical analysis. Continuous variables were expressed as mean ± standard deviation (SD) and the differences between the two data sets were determined by the Student’s t-test. A P < 0.05 (two-tailed) was considered statistically significant.
TE D
3. Results
3.1. Apoptosis in H. pylori-infected GES-1 cells The apoptosis rates in GES-1 cells were significantly increased 3, 12 and 24h after infection with the 26695 and SS1 strains of H. pylori (Fig. 1A and Fig. 1B). A cell
EP
apoptosis rate of 31.83±4.39% was observed in GES-1 cells 3 h after H. pylori infection with the 26695 strain, whereas the apoptosis rate was 5.00±3.15% in control
AC C
GES cells. At the 12 and 24h time-point after H. pylori 26695 infection, cell apoptosis increased to 50.80±7.54% and 54.00±6.41%, respectively, in other words, a 10-fold (P<0.01) and a 11-fold (P<0.01) increase compared withthe control group. GES-1
cells infected with the strain SS1 of H. pylori displayed apoptotic rates of 12.80±1.85%, 15.23±3.18% and 33.30±5.89% at 3, 12, and 24h after infection, respectively. This represented a significant increase (P < 0.01) compared withthe
control group. Altogether, the cell apoptosis rates in the 26695 strain of H. pylori-treated GES cells were significantly higher than the ones treated with the SS1
ACCEPTED MANUSCRIPT
strain (P < 0.05). 3.2. Proliferation of GES-1 after H. pylori infection BrdU cell proliferation assay was employed to examine the proliferation of GES-1
RI PT
after infection with the 26695 and the SS1 strains of H. pylori. No significant change in proliferation of GES-1 cells was observed after infection with the 26695 strain or with the SS1 strain throughout the course of the experiment. Additionally, no
significant difference in proliferation of GES-1 cells between the 26695 and SS1
SC
strains of H. pylori was detected (Fig. 2A and Fig. 2B). 3.3. Cell adhesion rates in H. pylori-infected GES-1 cells
The MTT cell adhesion assay was performed to determine the adhesion rate of
M AN U
GES-1 cells after infection with the 26695 and the SS1 strains of H. pylori. Treatment with both the H. pylori 26695 and the SS1 strain induced a significant decline in the cell adhesion rate of GES-1 cells in a time-dependent manner (Fig. 3). The cell adhesion rate of GES-1 cells treated with the H. pylori 26695 strain reached approximately a 4/5-fold, 3/5-fold and 1/2-fold-decrease at 3, 12 and 24 h after
TE D
bacterial challenge compared withthe control cells. The cell adhesion rate of GES-1 was down-regulated at 3, 12 and 24 h in SS1-infected GES-1 cells. Finally, no significant differences were found in the adhesion rates of GES-1 cells infected with the strains 26695 and SS1 of H. pylori.
EP
3.4. Motility of GES-1 cells after H. pylori infection The scratch motility (wound-healing) assay was performed to determine the
AC C
migration and invasion capacities of GES-1 cells after infection with the 26695 and the SS1 strains of H. pylori. After the simulation of H. pylori, Migration of
26695-infected GES-1 cells displayed an hourly two-fold increase (P < 0.05) throughout the experimental period compared withthe control cells. SS1 strain-infected GES-1 cells showed the same stability as control cells in this experiment (Fig. 4). 4. Discussion The gastric epithelium is a dynamic tissue with a high cellular turnover rate thus epithelial renewal occurs every three to five days [17]. This unique system is
ACCEPTED MANUSCRIPT
maintained by the balance between proliferation and apoptosis in gastric epithelial cells, which is essential to maintain normal homeostasis and gastric epithelial morphology and physiology [18, 19]. H. pylori infection contributes to the disruption
RI PT
of cellular integrity and turnover rate in the gastric epithelium, thus favoring the development of H. pylori infection-associated diseases and end-stage gastric cancer
[20, 21]. H. pylori 26695 strain was originally isolated from a patient with gastritis [11]. H. Pylori SS1 strain came from a 42-year-old woman diagnosed with a peptic
SC
ulcer, and it could significantly promoted gastric cancers [12, 22]. H. pylori is a genetically diverse species due to high mutation and recombination frequence.
Therefore, the functional analysis of H. pylori 26695 and SS1 strains on GES-1, a
M AN U
gastric epithelial cell line might provide a better understanding on the regulatory mechanism of bacteria integration in the gastric epithelium.
An important feature in the pathogenesis of H. pylori infection-associated diseases is increased gastric epithelial apoptosis which leads to destruction and loss of integrity in the epithelial tissue [23-25]. In the present study, the rates of apoptosis in GES-1
TE D
cells were significantly increased 3, 12 and 24 h after the 26695 and SS1 strains of H. pylori infection. However, no significant changes in proliferation were observed in the 26695 and SS1 strain-infected GES-1 throughout the experimental period. Collectively, these results suggest that H. pylori infection elicits an acute-phase
EP
response activating the immune host response, and causing an imbalance between cell proliferation and cell apoptosis, thus facilitating H. pylori colonization and increased
AC C
host susceptibility to bacterial infection. Unexpectedly, our data indicate that neither of the strains exerted an effect on cell proliferation. The colonization of H. pylori
relies on multiple virulence factors such as vacuolating cytotoxin (VacA), cytotoxin-associated gene (CagA) and gamma-glutamyl-transpeptidase enzyme (GGT) to persist in the human stomach for long periods of time [26-29]. A previous report showed in vitro and in vivo that virulence factors can cause death of gastric epithelial cells through both apoptosis and necrosis, mechanisms not only involved in the pathogenesis of H. pylori infection, but also important in the early stages of carcinogenesis [30]. Furthermore, increased levels of inflammatory cytokines such as
ACCEPTED MANUSCRIPT
TNF and IFN-γ can also induce apoptosis and contribute significantly to the H. pylori infection-associated disease pathology [31, 32]. Cell adhesion and migration are of pivotal importance in many biological processes,
RI PT
especially for mechanisms of repair in gastric epithelial cells infected with H. pylori [33, 34]. In the present study, infection with the strains 26695 and SS1 of H. pylori
induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner, suggesting that H. pylori infection promotes the disintegration of
SC
proliferative gastric epithelial cells. The scratch motility (wound-healing) assay was employed to evaluate the migration and invasion capacity of GES-1 cells after
bacterial infection. After infection with the 26695 strain GES-1 cells migration rates
M AN U
increased hourly during the experiment compared withthe controls. Altogether, these results indicate that the 26695 strain of H. pylori infection induced migration of proliferative cells and promoted regeneration of the gastric epithelium. However, migration of GES-1 cells was practically stable when these cells were infected with the SS1 strain, suggesting that different H. pylori strains elicits different responses in
TE D
gastric epithelia cells. It is well known that mutations and recombination frequently occur during mixed strain H. pylori infection, which may result in extensive genetic diversity and occurrence of gastrointestinal-related diseases [35, 36]. Although approximately 50% to 80% of the world population is infected with H. pylori, only a
EP
fraction of infected individuals develop gastric-related disorders [37]. The significant variation of the different strains of H. pylori might determine the prevalence and
AC C
incidence of several H. pylori-associated diseases. 5. Conclusions
H. pylori infection elicits an acute-phase response, activating the immune host
response, and causing imbalance between cell proliferation and cell apoptosis. Infection with H. pylori induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner. Each H. pylori strain expresses differential migration rates in gastric epithelial cells. These results together indicate that H. pylori infection facilitates it colonization and increases host susceptibility to bacterial infection, and the different development of gastric-related diseases might be a H.
ACCEPTED MANUSCRIPT
pylori strain-specific response. Acknowledgements: This research was supported by a grant (No. 81471561) from National Natural
RI PT
Science Foundation of China, Scientific research project of Binzhou Medical University (No. BY2014KYQD06) and Science and technology program of Shandong Province (No. J15LK02). References:
Gastroenterol Hepatol 9(2012) 17-20.
SC
[1] J.J. Misiewicz, Management of Helicobacter pylori related disorders. Eur J
Cancer 10 (2010) 403-414.
M AN U
[2] D.B. Polk, R.M. Peek , Helicobacter pylori: gastric cancer and beyond. Nat Rev
[3] C. Chomvarin, K. Phusri, K. Sawadpanich, P. Mairiang, W. Namwat, C. Wongkham and C. Hahnvajanawong, Prevalence of CagA EPIYA motifs in Helicobacter pylori among dyspeptic patients in Northeast Thailand. Southeast Asian J Trop Med Public Health 43 (2012) 105-115.
TE D
[4] L. Shokrzadeh, K. Baghaei, Y. Yamaoka, H. Dabiri, F. Jafari, N. Sahebekhtiari, A. Tahami, M. Sugimoto, H. Zojaji and M. R. Zali, Analysis of 3ʹ-end variable region of the CagA gene in Helicobacter pylori isolated from Iranian Population. J Gastroen Hepatol 25 (2010) 172-177.
EP
[5] IARC Working Group, Monographs on the evaluation of the carcinogenic risk to humans: schistosomes, liver flukes and Helicobacter pylori. Geneva: IARC
AC C
Publications (1994) 177-241. [6] L.E. Wroblewski, R.M. Peek, Targeted disruption of the epithelial-barrier by Helicobacter pylori. Cell Commun Signal 9 (2011) 29.
[7] I. Saadat, H. Higashi, C. Obuse, M. Umeda, N. Murata-Kamiya, Y. Saito, H. Lu, N. Ohnishi, T. Azuma, A. Suzuki, S. Ohno and M. Hatakeyama, Helicobacter pylori
CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature 447 (2007) 330-333. [8] R.M. Peek, J.E. Crabtree, Helicobacter infection and gastric neoplasia. J Pathol 208 (2006) 233-248
ACCEPTED MANUSCRIPT
[9] N. Salama, K. Guillemin, T.K. McDaniel, G. Sherlock, L. Tompkins and S. Falkow, A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains. Proc Natl Acad Sci U S A 97 (2000) 14668-14673.
RI PT
[10] D.A. Israel, N. Salama, C.N. Arnold, S.F. Moss, T. Ando, H.P. Wirth, K.T. Tham, M. Camorlinga, M.J. Blaser, S. Falkow and R.M. Peek, Jr, Helicobacter pylori
strain-specific differences in genetic content identified by microarray influence host inflammatory responses. J Clin Invest 107 (2001) 611-620.
SC
[11] A. Lee, J. O'Rourke, M. C. De Ungria, B. Robertson, G. Daskalopoulos and M. F.
Dixon, A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112 (1997) 1386-1397.
M AN U
[12] J. F. Tomb, O. White, A. R. Keriavage, R. A. Clayton, G. G. Sutton and et al., The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388 (1997) 539-547.
[13] J. Feng, A. Liu, Y. Yuan and L. Ge, Effects of different Helicobacter pylori loads on proliferation, apoptosis and FAF1 mRNA expression in gastric cancer cell.
TE D
Chin J of Oncol Prev and Treat 6 (2014) 371-375.
[14] J.Q. Zhang, Y. M. Li, T. Liu, W. T. He, Y. T. Chen, X. H. Chen, X. Li, W. C. Zhou, J. F. Yi and Z. J. Ren. Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy. World J Gastroenterd 16 (2010)
EP
4281-4290.
[15] Y. Cai, X. Cong, R. Fei and J. Wang. Effects of Helicobacter pylori omeprazole
AC C
and gastin on the proliferation and apoptosis of gastric epithelial cell. Natl Med J China 90 (2010) 2558-2563.
[16] I. Miki, N. Ishihara, M. Otoshi and H. Kase. Simple colorimetric cell-cell adhesion assay using MTT-stained leukemia cells. J Immunol Methods 164 (1993) 255-261.
[17] P.A. Hall, P.J. Coates, B. Ansari and D. Hopwood, Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107 (1994) 3569-3577. [18] M.R. Amieva and E.M. El-Omar, Host-bacterial interactions in Helicobacter
ACCEPTED MANUSCRIPT
pylori infection. Gastroenterology 134 (2008) 306-323 [19] H. Schmitz, C. Barmeyer, M. Fromm, N. Runkel, H. Foss, C. J. Bentzel, E. Riecken and J. Schulzke, Altered tight junction structure contributes to the
RI PT
impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116 (1999) 301-309.
[20] S. Maeda, A.F. Mentis, Pathogenesis of Helicobacter pylori infection. Helicobacter 12 (2007) 10-14.
SC
[21] X.W. Yu, Y. Xu, Y.H. Gong, X. Qian and Y. Yuan, Helicobacter pylori induces malignant transformation of gastric epithelial cells in vitro. APMIS 119 (2011) 187-197.
M AN U
[22] T. Kawazoe, T. Sakagami, K. Nakajima, K. Hori, Y. Fukuda, T. Matsumoto and H. Miwa, Role of bacterial strain diversity of Helicobacter pylori in gastric carcinogenesis induced by N-methyl-N-nitrosourea in Mongolian gerbils. Helicobacter 12 (2007) 213-223.
[23] A.L. Werneck-Silva, E.P. Alvares, P. Gama, A.O. Mourão, C. Damião, L.H. Osaki,
TE D
D. Ogias and A.M Sipahi, Intestinal damage in strongyloidiasis: the imbalance between cell death and proliferation. Dig Dis Sci51 (2006) 1063-1069. [24] S. Maeda, H. Yoshida, Y. Mitsuno, Y. Hirata, K. Ogura, Y. Shiratori and M. Omata, Analysis of apoptotic and antiapoptotic signalling pathways induced by
EP
Helicobacte pylori. Gut 50 (2002) 771-778. [25] D. Olivares, J.P. Gisbert and J.M. Pajares, Helicobacter pylori infection and
AC C
gastric mucosal epithelial cell apoptosis. Rev Esp Enferm Dig 97 (2005) 505-520.
[26] J.N. Radin, C. Gonzalez-Rivera, S.E. Ivie, M.S. McClain and T.L. Cover, Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells.
Infect Immun 79 (2011) 2535–2543.
[27] J.N. Radin, C. Gonzalez-Rivera, A.E. Frick-Cheng, J. Sheng, J.A. Gaddy, D.H. Rubin, H.M. Algood, M.S. McClain and T.L. Cover, Role of connexin 43 in Helicobacter pylori VacA-induced cell death. Infect Immun 82 (2014) 423-432. [28] X. Song, H.X. Chen, X.Y. Wang, X. Y. Deng, Y. X. Xi, Q. He, T.L. Peng, J. Chen,
ACCEPTED MANUSCRIPT
W. Chen, B.C.Y. Wong and M.H. Chen, H. pylori-encoded CagA disrupts tight junctions and induces invasiveness of AGS gastric carcinoma cells via Cdx2-dependent targeting of Claudin-2. Cell Immunol 286 (2013) 22-30.
RI PT
[29] M. Gong, S.S. Ling, S.Y. Lui, K.G. Yeoh and B. Ho, Helicobacter pylori gamma-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gastroenterology 139(2010) 564-573.
[30] M.E. Martin, S. Bhatnagar, M.D. George, B.J. Paster, D.R. Canfield, J.A. Eisen
SC
and J.V. Solnick, The impact of Helicobacter pylori infection on the gastric microbiota of the rhesus macaque. PLoS ONE 8 (2013) e76375.
[31] G. Bouma, W. Strober, The immunological and genetic basis of inflammatory
M AN U
bowel disease. Nat Rev Immunol 3 (2003) 521-533.
[32] M. Koshiji, Y. Adachi, S. Sogo, S. Taketani, N. Oyaizu, S. Than, M. Inaba, S. Phawa, K. Hioki and S. Ikehara, Apoptosis of colorectal adenocarcinoma (COLO
201)
by
tumour
necrosis
factor-alpha
(TNF-alpha)
and/or
interferon-gamma (IFN-gamma), resulting from down-modulation of Bcl-2
TE D
expression. Clin Exp Immunol 111 (1998) 211-218.
[33]A.L. Herard, D. Pierrot, J. Hinnrasky, H. Kaplan, D. Sheppard, E. Puchelle and J.M. Zahm, Fibronectin and its α5β1-integrin receptor are involved in the wound-repair progress of airway epitheliem. Am J Physiol 271 (1996) 726-733.
EP
[34] J.M. Zahm, H. Kaplan, A.L. Hérard, F. Doriot, D. Pierrot, P. Somelette and E. Puchelle, Cell migration and proliferation during the in vitro wound repair of the
AC C
respiratory epithelium. Cell Motil Cytoskele 37 (1997) 33-43. [35] L.E. Wroblewski, R.M. Peek and K.T. Wilson, Helicobacter pylori and gastric cancer: Factors that modulate disease risk. Clin Microbiol Rev 23 (2010) 713-739.
[36] I.M. Toller, K.J. Neelsen, M. Steger, M.L. Hartung, M.O. Hottiger, M. Stucki, B. Kalali, M. Gerhard, A.A. Sartori, M. Lopes and A. Müller, Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA doublestrand breaks and a DNA damage response in its host cells. Proc Natl Acad Sci USA 108 (2011) 14944-14949.
ACCEPTED MANUSCRIPT
[37] P. C. Konturek, S. J. Konturek and T. Brzozowski, Helicobacter pylori infection in gastric cancerogenesis. J Physiol Pharmacol 60 (2009) 3-21. Figures:
RI PT
Fig.1. Flow cytometry analysis of differentiated GES-1 cells infected with the 26695 and SS1 strains of H. pylori at different time points was performed to study
apoptosis. GES-1 cells were treated with the 26695 and SS1 strains of H. pylori at 200:1 of bacterium to cell ratio from 0 to 24 h and apoptosis was detected
SC
using AnnexinV-FITC/PI double staining. Representative flowcharts (A) and the rate of apoptotic cells (B,) are shown (mean±SD; n=3,*P < 0.05).
Fig.2. EdU staining assay to assess proliferation in differentiated GES-1 cells infected
M AN U
with the 26695 and SS1 strains of H. pylori at different time points. GES-1 cells were treated with the 26695 and SS1 strains of H. pylori at 200:1 of bacterium to cell ratio for the indicated time and proliferation was detected using BrdU incorporation into the cellular DNA and measured by fluorescence microscopy. Representative micrographs (A) and the rate of cell proliferation (B) are shown.
TE D
No significance was observed ( mean±SD, n=3).
Fig.3.Cell adhesion of GES-1 infected with the strains SS1 or 26695 of H. pylori at different time points. GES-1 cells were treated with the H. pylori SS1 or 26695 strains at 200:1 of bacterium to cell ratio at the indicated time-points, and the
EP
adhesive capacity was detected using the cell adhesion MTT assay. The cell adhesion rate is shown (mean±SD, n=3; *P<0.05 vs. 0 h group).
AC C
Fig.4. Scratch motility assay for the study of migration of differentiated GES-1 cells infected with the strains SS1 or 26695 of H. pylori. GES-1 cells were treated with the H. pylori SS1 or 26695 strains at 200:1 of bacterium to cell ratio and
observed under the Living Cell Working Station for 24h. Representative micrographs (A) and the cell migration rate (B) are shown (mean±SD, n=3).
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
1. H. pylori infection causes imbalance between cell proliferation and cell apoptosis. 2. H. pylori infection promotes the disintegration of proliferative gastric epithelial cells. 3. Each H. pylori strain elicits differential responses in gastric epithelial cells.
S0882-4010(16)30040-7
DOI:
10.1016/j.micpath.2016.03.007
Reference:
YMPAT 1801
To appear in:
Microbial Pathogenesis
Received Date: 19 January 2016 Revised Date:
21 March 2016
Accepted Date: 22 March 2016
Please cite this article as: Zhang Y, Sun H, Chen X, Li J, Zhao H, Geng L, Li B, Functional profile of gastric epithelial cells infected with Helicobacter pylori strains, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Functional profile of gastric epithelial cells infected with Helicobacter pylori strains Ying Zhang#, Hui Sun#, Xingxing Chen, Jiaojiao Li, Huilin Zhao, Li Geng, Boqing Li*
#: these authors contributed equally to the paper Abstract
RI PT
School of Basic Medical Sciences, Binzhou Medical University
H. pylori infection represents a key factor in the etiology of various
SC
gastro-duodenal diseases, ranging from chronic gastritis to the development of peptic
ulcer disease and end-stage gastric cancer. In the present study, the 26695 and SS1
M AN U
strains of H. pylori were used to study the differential functional profiles of gastric epithelial cells infected with H. pylori. The apoptosis rates in GES-1 cells were significantly increased 3, 12 and 24 h after H. pylori 26695 and SS1 infection. Moreover, apoptosis by cells infected with the H. pylori 26695 strain was significantly higher than cells infected with the SS1 strain of H. pylori. No significant changes in the proliferation rates of GES-1 cells were observed after H. pylori 26695
TE D
or SS1 infection at any time during the experimental period. Exposure to H. pylori 26695 and SS1 induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner. Furthermore, H. pylori 26695 infection increased migration
EP
of GES-1 cells every hour during the whole experimental period compared withcontrol cells. However, GES-1 cells infected with the H. pylori SS1 strain
AC C
exhibited migration rates almost stable and comparable to those of control cells. These results indicate that the gastric epithelial cells respond differently depending on the H. pylori strain. This study indicates that the development of different gastric-related diseases may be a H. pylori strain-specific response. Keywords: Helicobacter pylori; apoptosis; proliferation; adhesion; migration
1. Introduction Helicobacter pylori is one of the most prevalent pathogens that contribute to human diseases [1, 2]. According to epidemiological studies, approximately 50% to 80% of the world population is infected with H. pylori, and areas of high incidence
ACCEPTED MANUSCRIPT
are concentrated in East Asia, including China, Japan and South Korea [3，4]. Chronic infection with H. pylori can result in DNA damage and genetic instability of the gastric mucosal cells, causing chronic gastritis and atrophy. The International Agency
RI PT
for Cancer Research (IACR) classified H. pylori as a class I carcinogen in 1994 [5], as numerous studies evidenced the relationship of H. pylori and gastric cancer from a combination of environmental and host-dependent factors.
The infection strategy of H. pylori and the defense capacity of the gastric
SC
epithelial cells in the gastric mucosa collectively influence colonization, survival, and
development of H. pylori infection-derived diseases. Mucosal epithelial cells are not only responsible for digestive processes, but also exert the crucial function of
M AN U
protecting the underlying tissue from pathogenic microorganisms that otherwise would invade the lumen [6]. Gastric epithelial cells form the first line of defense, thus contributing an innate defense such as cell barrier integrity, cell turnover, autophagy, and innate immune responses [6]. However, H. pylori can break through the gastric mucosal barrier and remain as a persistent infection via highly specialized
TE D
mechanisms, which facilitate H. pylori to avoid host defense mechanisms [7]. The failure of the host response in clearing the H. pylori infection promotes the development of chronic infection such as chronic gastritis. H. pylori infection represents a key factor in the etiology of various
EP
gastro-duodenal diseases, ranging from chronic gastritis to the development of peptic ulcer disease and gastric cancer (GC) [8]. The significant variation of the different
AC C
strains of H. pylori might be one of the important inducing factors that determines prevalence and incidence of H. pylori-associated diseases [9, 10]. Determining the
effects of the different H. pylori strains on the biological function of gastric epithelial cells will allow for better understanding of their cytotoxic effect and their regulatory mechanisms including the integration and rehabilitation of the mucosal epithelial barrier. In this context, the present study was performed with the following objectives (1) to investigate the time-response in cell apoptosis and proliferation in gastric epithelial cells (GES-1) after H. pylori infection, (2) to characterize cell adhesion and migration rates in GES-1 cells after H. pylori infection, (3) to further explore the
ACCEPTED MANUSCRIPT
cytotoxic diversity of H. pylori strains. 2. Materials and Methods 2.1. H. pylori
RI PT
Human-adapted strains of H. pylori 26695 and SS1, were provided by the H. pylori Research Laboratory of the Chinese Center for Disease Control and Prevention
(Beijing, China). H. pylori 26695 strain was originally isolated from a patient in the
United Kingdom with gastritis and Jcan-F Tomb et al. obtained the complete genome
SC
sequence of the gastric pathogen H. pylori 26695 in 1997 [11]. H. Pylori SS1 came
from a 42-year-old Greek-born woman in 1997, who had been diagnosed with a peptic ulcer [12]. Introduced H. Pylori SS1 bacteria were grown on chocolate agar
M AN U
plates supplemented with 10% sheep’s blood at 37˚C under microaerophilic conditions (5% O2, 10% CO2 and 85% N2) and subcultured every 3 days. For infection, H. pylori was harvested in chocolate agar broth and quantified by a spectrophotometer reaching 1×108 CFU/ml. 2.2. Human gastric epithelial cells
TE D
The human gastric epithelial cell line GES-1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco Modified Essential Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere of 5% CO2 at 37°C. When cells reached approximately 80%
passaged.
EP
confluence in 25 cm2 culture flasks, cells were treated with 0.25% trypsin and
AC C
2.3. Co-culture of GES-1 cells with H. pylori GES-1 cells were plated onto 6-well plates in DMEM containing 10% FBS (1×105
cells/2ml/well) overnight. The 26695 and SS1 strains of H. pylori were harvested and suspended in DMEM (including 10% FBS but no antimicrobial agents). Bacteria were added into the cells at a multiplicity of infection (MOI) of 200:1 and co-cultured for 0, 3, 12 and 24h. GES-1 cells were immediately used to detect cell apoptosis, proliferation, adhesion and motility rates [13]. 2.4. Cell apoptosis assays GES-1 cell apoptosis assay was performed using the Annexin V-FITC Apoptosis
ACCEPTED MANUSCRIPT
Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the instructions provided by the manufacturer. Briefly, 1×105 GES-1 cells were mixed together with 500µl of Annexin binding buffer, 5µl of FITC-conjugated Annexin V antibody and 5µl
RI PT
of propidium iodide (PI). The mixture was incubated in the dark for 15 min at room temperature. The relative number of apoptotic cells was determined using flow cytometry [14]. 2.5. EdU cell proliferation assay
SC
Cell proliferation was measured using a key fluor 488-EdU Proliferation Detection Kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s
instructions. GES-1 cells were incubated with H. pylori at 200:1 of bacterium to cell
M AN U
ratio in DMEM containing 10% FBS for 0, 3, 12 and 24h. All cells were treated with 50 µmol/L of EdU for 3 h at 37°C. After being fixed, the treated cells on coverslips were permeabilized with 0.5% TritonX-100 for 10 min. Cells were stained with Click-iT reaction mixture and incubated with Hoechst 33342 to stain the cell nuclei. Images were captured using inverted fluorescence microscope (Olympus, Tokyo,
TE D
Japan) [15]. The numbers of EdU- and DAPI-positive cells were quantified by IMAGEJ software. The percentages of immunopositive cells [(the number of immunopositive cells/total cells) × 100] were expressed. Five random fields of each well were chosen to perform the calculation.
EP
2.6. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell adhesion
AC C
A 96-well plate was treated with matrigel (0.04mg/mL, 50µl) (BD Biosciences, San Jose, CA) in DMEM overnight to facilitate cell attachment. GES-1 cells were cultured with the 26695 and SS1 strains of H. pylori for 0, 3, 12 and 24h, and then trypsinized and seeded into 100 mL of culture medium. MTT solution (5mg/mL, 20µL) was
added to the cells, and plates were further incubated at 37°C for 4h. The supernatant was carefully removed, and dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) was added to each well to dissolve formazan crystals. After 10 min shaking, the optical density was measured using ELISA at 490 nm wavelength. Cell adhesion rates
ACCEPTED MANUSCRIPT
= (OD of the infection group cells/ OD of the control group cells) x 100% [16]. 2.7. Scratch motility (wound-healing) assay GES-1 cells (1 x 105cells/well) were cultured on glass bottom dishes and allowed to
RI PT
form a confluent monolayer for 24h. Cells were then scratched using a sterile 10 µL pipette tip and the edge of each gap was straight and smooth. Single Cell Transferred Speed (SCTS) of treated cells were observed under the Living Cell Working Station.
Six random fields were selected and imaged continuously taken every 10 minutes for
SC
a period of 24h. Afterwards, the migrated distances were measured and the single cell migration velocity was calculated by manual counting. The analysis of image data
2.8. Statistical analysis
M AN U
was obtained by the Image Pro Plus 6.2.
The SPSS version 16.0 was used for statistical analysis. Continuous variables were expressed as mean ± standard deviation (SD) and the differences between the two data sets were determined by the Student’s t-test. A P < 0.05 (two-tailed) was considered statistically significant.
TE D
3. Results
3.1. Apoptosis in H. pylori-infected GES-1 cells The apoptosis rates in GES-1 cells were significantly increased 3, 12 and 24h after infection with the 26695 and SS1 strains of H. pylori (Fig. 1A and Fig. 1B). A cell
EP
apoptosis rate of 31.83±4.39% was observed in GES-1 cells 3 h after H. pylori infection with the 26695 strain, whereas the apoptosis rate was 5.00±3.15% in control
AC C
GES cells. At the 12 and 24h time-point after H. pylori 26695 infection, cell apoptosis increased to 50.80±7.54% and 54.00±6.41%, respectively, in other words, a 10-fold (P<0.01) and a 11-fold (P<0.01) increase compared withthe control group. GES-1
cells infected with the strain SS1 of H. pylori displayed apoptotic rates of 12.80±1.85%, 15.23±3.18% and 33.30±5.89% at 3, 12, and 24h after infection, respectively. This represented a significant increase (P < 0.01) compared withthe
control group. Altogether, the cell apoptosis rates in the 26695 strain of H. pylori-treated GES cells were significantly higher than the ones treated with the SS1
ACCEPTED MANUSCRIPT
strain (P < 0.05). 3.2. Proliferation of GES-1 after H. pylori infection BrdU cell proliferation assay was employed to examine the proliferation of GES-1
RI PT
after infection with the 26695 and the SS1 strains of H. pylori. No significant change in proliferation of GES-1 cells was observed after infection with the 26695 strain or with the SS1 strain throughout the course of the experiment. Additionally, no
significant difference in proliferation of GES-1 cells between the 26695 and SS1
SC
strains of H. pylori was detected (Fig. 2A and Fig. 2B). 3.3. Cell adhesion rates in H. pylori-infected GES-1 cells
The MTT cell adhesion assay was performed to determine the adhesion rate of
M AN U
GES-1 cells after infection with the 26695 and the SS1 strains of H. pylori. Treatment with both the H. pylori 26695 and the SS1 strain induced a significant decline in the cell adhesion rate of GES-1 cells in a time-dependent manner (Fig. 3). The cell adhesion rate of GES-1 cells treated with the H. pylori 26695 strain reached approximately a 4/5-fold, 3/5-fold and 1/2-fold-decrease at 3, 12 and 24 h after
TE D
bacterial challenge compared withthe control cells. The cell adhesion rate of GES-1 was down-regulated at 3, 12 and 24 h in SS1-infected GES-1 cells. Finally, no significant differences were found in the adhesion rates of GES-1 cells infected with the strains 26695 and SS1 of H. pylori.
EP
3.4. Motility of GES-1 cells after H. pylori infection The scratch motility (wound-healing) assay was performed to determine the
AC C
migration and invasion capacities of GES-1 cells after infection with the 26695 and the SS1 strains of H. pylori. After the simulation of H. pylori, Migration of
26695-infected GES-1 cells displayed an hourly two-fold increase (P < 0.05) throughout the experimental period compared withthe control cells. SS1 strain-infected GES-1 cells showed the same stability as control cells in this experiment (Fig. 4). 4. Discussion The gastric epithelium is a dynamic tissue with a high cellular turnover rate thus epithelial renewal occurs every three to five days [17]. This unique system is
ACCEPTED MANUSCRIPT
maintained by the balance between proliferation and apoptosis in gastric epithelial cells, which is essential to maintain normal homeostasis and gastric epithelial morphology and physiology [18, 19]. H. pylori infection contributes to the disruption
RI PT
of cellular integrity and turnover rate in the gastric epithelium, thus favoring the development of H. pylori infection-associated diseases and end-stage gastric cancer
[20, 21]. H. pylori 26695 strain was originally isolated from a patient with gastritis [11]. H. Pylori SS1 strain came from a 42-year-old woman diagnosed with a peptic
SC
ulcer, and it could significantly promoted gastric cancers [12, 22]. H. pylori is a genetically diverse species due to high mutation and recombination frequence.
Therefore, the functional analysis of H. pylori 26695 and SS1 strains on GES-1, a
M AN U
gastric epithelial cell line might provide a better understanding on the regulatory mechanism of bacteria integration in the gastric epithelium.
An important feature in the pathogenesis of H. pylori infection-associated diseases is increased gastric epithelial apoptosis which leads to destruction and loss of integrity in the epithelial tissue [23-25]. In the present study, the rates of apoptosis in GES-1
TE D
cells were significantly increased 3, 12 and 24 h after the 26695 and SS1 strains of H. pylori infection. However, no significant changes in proliferation were observed in the 26695 and SS1 strain-infected GES-1 throughout the experimental period. Collectively, these results suggest that H. pylori infection elicits an acute-phase
EP
response activating the immune host response, and causing an imbalance between cell proliferation and cell apoptosis, thus facilitating H. pylori colonization and increased
AC C
host susceptibility to bacterial infection. Unexpectedly, our data indicate that neither of the strains exerted an effect on cell proliferation. The colonization of H. pylori
relies on multiple virulence factors such as vacuolating cytotoxin (VacA), cytotoxin-associated gene (CagA) and gamma-glutamyl-transpeptidase enzyme (GGT) to persist in the human stomach for long periods of time [26-29]. A previous report showed in vitro and in vivo that virulence factors can cause death of gastric epithelial cells through both apoptosis and necrosis, mechanisms not only involved in the pathogenesis of H. pylori infection, but also important in the early stages of carcinogenesis [30]. Furthermore, increased levels of inflammatory cytokines such as
ACCEPTED MANUSCRIPT
TNF and IFN-γ can also induce apoptosis and contribute significantly to the H. pylori infection-associated disease pathology [31, 32]. Cell adhesion and migration are of pivotal importance in many biological processes,
RI PT
especially for mechanisms of repair in gastric epithelial cells infected with H. pylori [33, 34]. In the present study, infection with the strains 26695 and SS1 of H. pylori
induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner, suggesting that H. pylori infection promotes the disintegration of
SC
proliferative gastric epithelial cells. The scratch motility (wound-healing) assay was employed to evaluate the migration and invasion capacity of GES-1 cells after
bacterial infection. After infection with the 26695 strain GES-1 cells migration rates
M AN U
increased hourly during the experiment compared withthe controls. Altogether, these results indicate that the 26695 strain of H. pylori infection induced migration of proliferative cells and promoted regeneration of the gastric epithelium. However, migration of GES-1 cells was practically stable when these cells were infected with the SS1 strain, suggesting that different H. pylori strains elicits different responses in
TE D
gastric epithelia cells. It is well known that mutations and recombination frequently occur during mixed strain H. pylori infection, which may result in extensive genetic diversity and occurrence of gastrointestinal-related diseases [35, 36]. Although approximately 50% to 80% of the world population is infected with H. pylori, only a
EP
fraction of infected individuals develop gastric-related disorders [37]. The significant variation of the different strains of H. pylori might determine the prevalence and
AC C
incidence of several H. pylori-associated diseases. 5. Conclusions
H. pylori infection elicits an acute-phase response, activating the immune host
response, and causing imbalance between cell proliferation and cell apoptosis. Infection with H. pylori induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner. Each H. pylori strain expresses differential migration rates in gastric epithelial cells. These results together indicate that H. pylori infection facilitates it colonization and increases host susceptibility to bacterial infection, and the different development of gastric-related diseases might be a H.
ACCEPTED MANUSCRIPT
pylori strain-specific response. Acknowledgements: This research was supported by a grant (No. 81471561) from National Natural
RI PT
Science Foundation of China, Scientific research project of Binzhou Medical University (No. BY2014KYQD06) and Science and technology program of Shandong Province (No. J15LK02). References:
Gastroenterol Hepatol 9(2012) 17-20.
SC
[1] J.J. Misiewicz, Management of Helicobacter pylori related disorders. Eur J
Cancer 10 (2010) 403-414.
M AN U
[2] D.B. Polk, R.M. Peek , Helicobacter pylori: gastric cancer and beyond. Nat Rev
[3] C. Chomvarin, K. Phusri, K. Sawadpanich, P. Mairiang, W. Namwat, C. Wongkham and C. Hahnvajanawong, Prevalence of CagA EPIYA motifs in Helicobacter pylori among dyspeptic patients in Northeast Thailand. Southeast Asian J Trop Med Public Health 43 (2012) 105-115.
TE D
[4] L. Shokrzadeh, K. Baghaei, Y. Yamaoka, H. Dabiri, F. Jafari, N. Sahebekhtiari, A. Tahami, M. Sugimoto, H. Zojaji and M. R. Zali, Analysis of 3ʹ-end variable region of the CagA gene in Helicobacter pylori isolated from Iranian Population. J Gastroen Hepatol 25 (2010) 172-177.
EP
[5] IARC Working Group, Monographs on the evaluation of the carcinogenic risk to humans: schistosomes, liver flukes and Helicobacter pylori. Geneva: IARC
AC C
Publications (1994) 177-241. [6] L.E. Wroblewski, R.M. Peek, Targeted disruption of the epithelial-barrier by Helicobacter pylori. Cell Commun Signal 9 (2011) 29.
[7] I. Saadat, H. Higashi, C. Obuse, M. Umeda, N. Murata-Kamiya, Y. Saito, H. Lu, N. Ohnishi, T. Azuma, A. Suzuki, S. Ohno and M. Hatakeyama, Helicobacter pylori
CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature 447 (2007) 330-333. [8] R.M. Peek, J.E. Crabtree, Helicobacter infection and gastric neoplasia. J Pathol 208 (2006) 233-248
ACCEPTED MANUSCRIPT
[9] N. Salama, K. Guillemin, T.K. McDaniel, G. Sherlock, L. Tompkins and S. Falkow, A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains. Proc Natl Acad Sci U S A 97 (2000) 14668-14673.
RI PT
[10] D.A. Israel, N. Salama, C.N. Arnold, S.F. Moss, T. Ando, H.P. Wirth, K.T. Tham, M. Camorlinga, M.J. Blaser, S. Falkow and R.M. Peek, Jr, Helicobacter pylori
strain-specific differences in genetic content identified by microarray influence host inflammatory responses. J Clin Invest 107 (2001) 611-620.
SC
[11] A. Lee, J. O'Rourke, M. C. De Ungria, B. Robertson, G. Daskalopoulos and M. F.
Dixon, A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112 (1997) 1386-1397.
M AN U
[12] J. F. Tomb, O. White, A. R. Keriavage, R. A. Clayton, G. G. Sutton and et al., The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388 (1997) 539-547.
[13] J. Feng, A. Liu, Y. Yuan and L. Ge, Effects of different Helicobacter pylori loads on proliferation, apoptosis and FAF1 mRNA expression in gastric cancer cell.
TE D
Chin J of Oncol Prev and Treat 6 (2014) 371-375.
[14] J.Q. Zhang, Y. M. Li, T. Liu, W. T. He, Y. T. Chen, X. H. Chen, X. Li, W. C. Zhou, J. F. Yi and Z. J. Ren. Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy. World J Gastroenterd 16 (2010)
EP
4281-4290.
[15] Y. Cai, X. Cong, R. Fei and J. Wang. Effects of Helicobacter pylori omeprazole
AC C
and gastin on the proliferation and apoptosis of gastric epithelial cell. Natl Med J China 90 (2010) 2558-2563.
[16] I. Miki, N. Ishihara, M. Otoshi and H. Kase. Simple colorimetric cell-cell adhesion assay using MTT-stained leukemia cells. J Immunol Methods 164 (1993) 255-261.
[17] P.A. Hall, P.J. Coates, B. Ansari and D. Hopwood, Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107 (1994) 3569-3577. [18] M.R. Amieva and E.M. El-Omar, Host-bacterial interactions in Helicobacter
ACCEPTED MANUSCRIPT
pylori infection. Gastroenterology 134 (2008) 306-323 [19] H. Schmitz, C. Barmeyer, M. Fromm, N. Runkel, H. Foss, C. J. Bentzel, E. Riecken and J. Schulzke, Altered tight junction structure contributes to the
RI PT
impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116 (1999) 301-309.
[20] S. Maeda, A.F. Mentis, Pathogenesis of Helicobacter pylori infection. Helicobacter 12 (2007) 10-14.
SC
[21] X.W. Yu, Y. Xu, Y.H. Gong, X. Qian and Y. Yuan, Helicobacter pylori induces malignant transformation of gastric epithelial cells in vitro. APMIS 119 (2011) 187-197.
M AN U
[22] T. Kawazoe, T. Sakagami, K. Nakajima, K. Hori, Y. Fukuda, T. Matsumoto and H. Miwa, Role of bacterial strain diversity of Helicobacter pylori in gastric carcinogenesis induced by N-methyl-N-nitrosourea in Mongolian gerbils. Helicobacter 12 (2007) 213-223.
[23] A.L. Werneck-Silva, E.P. Alvares, P. Gama, A.O. Mourão, C. Damião, L.H. Osaki,
TE D
D. Ogias and A.M Sipahi, Intestinal damage in strongyloidiasis: the imbalance between cell death and proliferation. Dig Dis Sci51 (2006) 1063-1069. [24] S. Maeda, H. Yoshida, Y. Mitsuno, Y. Hirata, K. Ogura, Y. Shiratori and M. Omata, Analysis of apoptotic and antiapoptotic signalling pathways induced by
EP
Helicobacte pylori. Gut 50 (2002) 771-778. [25] D. Olivares, J.P. Gisbert and J.M. Pajares, Helicobacter pylori infection and
AC C
gastric mucosal epithelial cell apoptosis. Rev Esp Enferm Dig 97 (2005) 505-520.
[26] J.N. Radin, C. Gonzalez-Rivera, S.E. Ivie, M.S. McClain and T.L. Cover, Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells.
Infect Immun 79 (2011) 2535–2543.
[27] J.N. Radin, C. Gonzalez-Rivera, A.E. Frick-Cheng, J. Sheng, J.A. Gaddy, D.H. Rubin, H.M. Algood, M.S. McClain and T.L. Cover, Role of connexin 43 in Helicobacter pylori VacA-induced cell death. Infect Immun 82 (2014) 423-432. [28] X. Song, H.X. Chen, X.Y. Wang, X. Y. Deng, Y. X. Xi, Q. He, T.L. Peng, J. Chen,
ACCEPTED MANUSCRIPT
W. Chen, B.C.Y. Wong and M.H. Chen, H. pylori-encoded CagA disrupts tight junctions and induces invasiveness of AGS gastric carcinoma cells via Cdx2-dependent targeting of Claudin-2. Cell Immunol 286 (2013) 22-30.
RI PT
[29] M. Gong, S.S. Ling, S.Y. Lui, K.G. Yeoh and B. Ho, Helicobacter pylori gamma-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gastroenterology 139(2010) 564-573.
[30] M.E. Martin, S. Bhatnagar, M.D. George, B.J. Paster, D.R. Canfield, J.A. Eisen
SC
and J.V. Solnick, The impact of Helicobacter pylori infection on the gastric microbiota of the rhesus macaque. PLoS ONE 8 (2013) e76375.
[31] G. Bouma, W. Strober, The immunological and genetic basis of inflammatory
M AN U
bowel disease. Nat Rev Immunol 3 (2003) 521-533.
[32] M. Koshiji, Y. Adachi, S. Sogo, S. Taketani, N. Oyaizu, S. Than, M. Inaba, S. Phawa, K. Hioki and S. Ikehara, Apoptosis of colorectal adenocarcinoma (COLO
201)
by
tumour
necrosis
factor-alpha
(TNF-alpha)
and/or
interferon-gamma (IFN-gamma), resulting from down-modulation of Bcl-2
TE D
expression. Clin Exp Immunol 111 (1998) 211-218.
[33]A.L. Herard, D. Pierrot, J. Hinnrasky, H. Kaplan, D. Sheppard, E. Puchelle and J.M. Zahm, Fibronectin and its α5β1-integrin receptor are involved in the wound-repair progress of airway epitheliem. Am J Physiol 271 (1996) 726-733.
EP
[34] J.M. Zahm, H. Kaplan, A.L. Hérard, F. Doriot, D. Pierrot, P. Somelette and E. Puchelle, Cell migration and proliferation during the in vitro wound repair of the
AC C
respiratory epithelium. Cell Motil Cytoskele 37 (1997) 33-43. [35] L.E. Wroblewski, R.M. Peek and K.T. Wilson, Helicobacter pylori and gastric cancer: Factors that modulate disease risk. Clin Microbiol Rev 23 (2010) 713-739.
[36] I.M. Toller, K.J. Neelsen, M. Steger, M.L. Hartung, M.O. Hottiger, M. Stucki, B. Kalali, M. Gerhard, A.A. Sartori, M. Lopes and A. Müller, Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA doublestrand breaks and a DNA damage response in its host cells. Proc Natl Acad Sci USA 108 (2011) 14944-14949.
ACCEPTED MANUSCRIPT
[37] P. C. Konturek, S. J. Konturek and T. Brzozowski, Helicobacter pylori infection in gastric cancerogenesis. J Physiol Pharmacol 60 (2009) 3-21. Figures:
RI PT
Fig.1. Flow cytometry analysis of differentiated GES-1 cells infected with the 26695 and SS1 strains of H. pylori at different time points was performed to study
apoptosis. GES-1 cells were treated with the 26695 and SS1 strains of H. pylori at 200:1 of bacterium to cell ratio from 0 to 24 h and apoptosis was detected
SC
using AnnexinV-FITC/PI double staining. Representative flowcharts (A) and the rate of apoptotic cells (B,) are shown (mean±SD; n=3,*P < 0.05).
Fig.2. EdU staining assay to assess proliferation in differentiated GES-1 cells infected
M AN U
with the 26695 and SS1 strains of H. pylori at different time points. GES-1 cells were treated with the 26695 and SS1 strains of H. pylori at 200:1 of bacterium to cell ratio for the indicated time and proliferation was detected using BrdU incorporation into the cellular DNA and measured by fluorescence microscopy. Representative micrographs (A) and the rate of cell proliferation (B) are shown.
TE D
No significance was observed ( mean±SD, n=3).
Fig.3.Cell adhesion of GES-1 infected with the strains SS1 or 26695 of H. pylori at different time points. GES-1 cells were treated with the H. pylori SS1 or 26695 strains at 200:1 of bacterium to cell ratio at the indicated time-points, and the
EP
adhesive capacity was detected using the cell adhesion MTT assay. The cell adhesion rate is shown (mean±SD, n=3; *P<0.05 vs. 0 h group).
AC C
Fig.4. Scratch motility assay for the study of migration of differentiated GES-1 cells infected with the strains SS1 or 26695 of H. pylori. GES-1 cells were treated with the H. pylori SS1 or 26695 strains at 200:1 of bacterium to cell ratio and
observed under the Living Cell Working Station for 24h. Representative micrographs (A) and the cell migration rate (B) are shown (mean±SD, n=3).
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
1. H. pylori infection causes imbalance between cell proliferation and cell apoptosis. 2. H. pylori infection promotes the disintegration of proliferative gastric epithelial cells. 3. Each H. pylori strain elicits differential responses in gastric epithelial cells.