Impact of Helicobacter pylori on the growth of hepatic orthotopic graft tumors in mice

  • Authors:
    • Junwei Wang
    • Xiaoqian Wang
    • Nanhong Tang
    • Yanling Chen
    • Feifei She
  • View Affiliations

  • Published online on: July 28, 2015     https://doi.org/10.3892/ijo.2015.3107
  • Pages: 1416-1428
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Helicobacter pylori is a well-known causative organism of chronic gastric diseases and has been found in many hepatic carcinoma samples. To explore the expression of apoptosis-related proteins and carcinoma development in H. pylori-infected livers, we utilized BALB/cAnSlac mice to establish an H. pylori-infected model by oral inoculation and orthotopic grafts of hepatic tumors by H22 cells, respectively. We found that H. pylori colonies could not be cultured from all liver and tumor samples. However, its 16S rRNA was detectable in 85.3% of livers and 66.7% of tumors in the infected mice. Inflammatory cells were observed and thinly distributed in the lobule portions of the liver, and H. pylori mainly existed in the infected hepatic sinusoids and the necrotic areas of the infected tumors. No significant difference was found in liver to body weight ratio between the infected and uninfected. Moreover, the pathological tumor difference was unremarkable between the two. The proliferating cell nuclear antigen (PCNA) and Bcl-2-associated X protein (Bax) expression in the infected tumors was significantly higher and lower, respectively, than those of the uninfected tumors. However, no significant difference in Bcl-2 (B-cell lymphoma 2) expression existed. The results indicate that H. pylori found in the livers which were infected by H. pylori oral inoculation could contribute to the infiltration of inflammatory cells in livers. Although H. pylori has no significant impact on the liver to body weight ratio or tumor Bcl-2 expression, it may upregulate PCNA expression and downregulate Bax expression, respectively. All our findings show that H. pylori may promote proliferation and inhibit apoptosis of tumor cells.

Introduction

As one of the most common pathogens in human bacteria-related chronic infection and carcinomatous diseases, Helicobacter pylori infects almost 50% of the world population (1). In 1994, the World Health Organization (WHO) and International Agency for Research on Cancer (IARC) classified it as a type I carcinogen (2). It has been reported that H. pylori plays a crucial role in the occurrence and development of chronic active gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma (MALT) and even gastric adenocarcinoma (3). Moreover, H. pylori is also closely related to some hematological systemic disorders, for example, hypoferric anemia and idiopathic thrombocytopenic purpura. Recently, it was demonstrated that it is involved in extragas-tric diseases such as atherosclerosis, ischemic heart disease, immunologic dysfunction, migraines and pediatric growth retardation (47). According to abundant clinical studies which had demonstrated morphological and molecular biological evidence, the infection rate of H. pylori is higher in patients with chronic hepatic diseases such as chronic hepatitis, liver cirrhosis and liver cancer than the rate of healthy persons (814). Xuan et al (14) even positively cultured H. pylori in 3 (10.7%) of their 28 primary hepatic carcinoma patients.

Primary hepatic cancer is an epidemic malignant tumor worldwide, and ~665,000 new cases are diagnosed every year (15). With an extremely high mortality rate, it ranks second as a cancer-related cause of death and is responsible for 746,000 deaths worldwide in 2012 (16). Its 5-year survival rate in developed countries, such as the United States, is 9% (15), whereas <5% worldwide (17), due to its high postoperative recurrence rate and poor prognosis (18,19). Thus, interference from the source is the most effective measure that might etiologically prevent the occurrence and development of primary hepatic cancer. Hepatitis B virus, hepatitis C virus, aflatoxin, chemical carcinogens and parasitic infections have been identified as pathogenic factors in primary hepatic cancer (2022). However, basing on the discovery that H. pylori was found in the liver of patients who suffered from chronic hepatic diseases, researchers have proposed that it plays a role in hepatitis and hepatic cancer. This novel hypothesis prompted studies concerning the correlation between H. pylori and hepatic cancer (23,24). The findings may also be helpful in prophylaxis and treatment of hepatic cancer.

Ito et al (25), Zhang et al (26) and Chen et al (27) co-cultured human hepatoma cells with H. pylori in vitro and determined the interaction of H. pylori with hepatocyte surface molecular receptors. H. pylori adhered to and invaded hepatoma cells. As a result, it caused a cytotoxic effect that upregulated tumor-related cyclin D1 and PCNA (proliferating cell nuclear antigen). However, they mainly performed studies in vitro that could hardly simulate the microenvironment of liver in vivo. Thus, the function of H. pylori in liver cancer remains unclear. In the present study, BALB/cAnSlac mice were orally inoculated with an infective dose of H. pylori. Then, we developed a tumor-bearing mouse model. Finally, we evaluated the infection status and explored the role of H. pylori in the development of hepatic cancer.

Materials and methods

Animals

Six to eight weeks old male and female specific-pathogen-free (SPF) BALB/cAnSlac mice whose fecal DNA were determined by C97/C98 Helicobacter spp. primers were obtained from Slaccas Laboratory Animal Co., Ltd., (Shanghai, China; License no. SCXK 2002–0003, Certificate no. 2007000551273) and housed under SPF conditions in Fujian Medical University Laboratory Animal Center (Fuzhou, China; License no. SYXK 2012–0001). All the following pathogens were excluded from SPF BALB/cAnSlac mice [SPF Laboratory Animal Standard of the People's Republic of China (GB 14922.2–2011)]. Salmonella spp.; Yersinia pseudotuberculosis; Yersinia enterocolitica; Pathogenic dermal fungi; Streptobacillus moniliformis; Bordetella bronchiseptica; Mycoplasma spp.; Corynebacterium kutscheri; Tyzzer's organism; Escherichia coli O115 a, C, K (B); Pasteurella pneumotropica; Klebsiella pneumonia; Staphylococcus aureus; Streptococcus pnemoniae; β-hemolytic streptococcus; Pseudomonas aeruginosa; lymphocytic choriomenigitis virus (LCMV); hantavirus (HV); ectromelia virus (Ect.); mouse hepatitis virus (MHV); sendai virus (SV); pneumonia virus of mice (PVM); reovirus type III (Reo-3); minute virus of mice (MVM); Theiler's mouse encephalomyelitis virus (TMEV); mouse adenovirus (Mad); polyomavirus (POLY); rat parvovirus (KRV); rat parvovirus (H-1); rat coronavirus (RCV)/sialodacryoadenitis virus (SDAV). All mice were fed a sterilized commercial diet, given water ad libitum, and allowed to acclimatize for at least 1 week before the experiments. All animal protocols met the approval of the Institutional Animal Care Committee.

Bacterial strains and culture conditions

H. pylori type strain NCTC 11637 (National Institute For Communicable Disease Control And Prevention, Chinese Center For Disease Control And Prevention, Beijing, China) was used in the present study. Columbia agar base was used (Oxoid Ltd., Hampshire, UK) that was supplemented with 8% sheep blood (Bio-Kont Technology Co., Ltd., Xiamen, China) and H. pylori selective supplements (Oxoid) containing vancomycin (0.01 mg/ml), cefsulodin (0.5 mg/ml), trimethoprim (0.5 mg/ml) and amphotericin B (0.5 mg/ml). The bacteria were incubated in microaerophilic conditions (5% O2, 10% CO2, 85% N2, 37°C, humidity >90%; LEEC Touch 190S; LEEC Ltd., Nottingham, UK) and harvested in sterile phosphate-buffered saline (PBS) after 48 h of growth, centrifuged at 4000 × g for 2 min, and adjusted in sterile PBS to a final concentration of 5×109 colony-forming units per milliliter (CFU/ml). Additionally, H. pylori was not only assessed by Gram staining and phase microscopy for purity, morphology and motility, but also tested for urease, catalase and oxidase activity before any animal experiments.

Cell line and cultivation

H22 murine hepatic hepatoma cells were obtained from China Center for Type Culture Collection (Wuhan, China) and syngeneic to BALB/cAnSlac mice. The cells were initially grown in a complete RPMI-1640 medium (HyClone Laboratories Inc., Logan, UT, USA) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 in vitro for 2–3 days. Subsequently, the cell suspension was tested for Helicobacter and other microorganisms prior to mouse injection or implantation. The cell suspension was spread evenly over Columbia agar base and kept in microaerophilic condition for microaerophilic bacteria detection. So did the Luria-Bertani base in usual condition (37°C) for aerobic bacteria detection. Additionally, after centrifugation the DNA was extracted by Genomic DNA Mini extraction kit (Beyotime Institute of Biotechnology, Haimen, China) for Helicobacter genus-specific 16S rRNA (C97/C98) test. Under the circumstance that all detection methods above were negative for microorganisms, we conducted the further protocols. The cell suspension was collected and centrifuged at 2000 × g for 5 min and adjusted with a serum-free RPMI-1640 medium to a final concentration of 1×107/ml. Every BALB/cAnSlac mouse was intraperitoneally injected with a 0.2-ml cell suspension for the first in vivo subcultivation for a 7-day period. Finally, the mouse was euthanized by cervical dislocation, and its ascites was collected and centrifuged. Ascitic precipitate cells were washed and adjusted to the same concentration. The cell suspension was intraperitoneally injected into another BALB/cAnSlac mouse for the second in vivo subcultivation. Following similar, previously discussed protocols, a third in vivo subcultivation was performed. Then, the centrifuged cells were adjusted to 4×107/ml and were used in an orthotopic implantation.

Drug pretreatment

Lactobacilli which inhabit the stomachs of the mice would interfere with the growth of H. pylori even eradicate it (28). All 70 experimental mice were treated on the 1st to 3rd day with the drugs listed in Table I, according to the methods described by Thalmaier et al (29). A mixed solution of ciprofloxacin (Zhejiang Jingxin Pharmaceutical, Co., Ltd., Zhejiang, China), amikacin (Shanghai Harvest Pharmaceutical Co., Ltd., Shanghai, China), imipenem (Merck Sharp & Dohme Corp., Kenilworth, NJ, USA), vancomycin (Zhejiang Hisun Pharmaceutical Co., Ltd., Zhejiang, China), and fluconazole (Yangtze River Pharmaceutical Group, Taizhou, China) were administered once daily.

Table I

Drugs and dose scheme (μg/mouse/day).

Table I

Drugs and dose scheme (μg/mouse/day).

AntibioticsOral doseIntraperitoneal dose
Ciprofloxacin5000
Amikacin375375
Imipenem12501250
Vancomycin10001000
Fluconazole1500

[i] μg, microgram.

Infection protocols

On day 3 (after the last drug treatment), 70 mice were randomly divided into group A (n=40, H. pylori infected mice) and group B (n=30, uninfected mice). From 3rd to 9th day, all mice were fasted for 15–18 h overnight before the treatment on the following day. The next day, every mouse was orally inoculated with a 0.2-ml sterilized alkalescent buffer (300 mM NaHCO3) to neutralize gastric acidity. Then, 15–30 min later, each mouse in group A was administered an oral H. pylori suspension (1×109 CFU; in 0.2 ml of sterilized PBS) once daily for a continuous 7-day period (4th to 10th day; defined as the first week); group B was administered a 0.2-ml sham dose of sterilized PBS in a same manner and schedule (3032). All mice underwent hepatic surgery during the 9th week.

Orthotopic hepatic carcinoma model

At the beginning of the 9th week, the mice in group A were randomly divided into group A1 (n=20; H. pylori infected and tumor positive mice) and group A2 (n=20; H. pylori infected and tumor negative mice), and group B was divided into group B1 (n=20, uninfected and tumor positive mice) and group B2 (n=10, uninfected and tumor negative mice). The left liver lobe of the mice was implanted with 50 μl of H22 cell suspension (2×106 cells; mice in group A1 and B1) or just 50 μl serum-free RPMI-1640 medium (mice in group A2 and B2) with a subcapsular intrahepatic injection according to Yao et al (33) and Aprahamian et al (34). Each mouse was anesthetized with 80 mg/kg ketamine (Sunkind Co., Ltd., Shanxi, China) by intraperitoneal injection. The mouse was placed in a supine position on the operating table when it was fully anesthetized. A small median longitudinal incision was made below the xiphoid to expose the left lobe of the liver, and sterile gauze was placed under it to prevent peritoneal sowing. H22 cells in a serum-free RPMI-1640 medium were slowly injected into the parenchyma of the left hepatic lobe at a 30-degree angle with a 100-μl microinjector (Shanghai Bolige Industry & Trade Co., Ltd., Shanghai, China), thus, a transparent bleb of medium could be seen within the hepatic capsule. A sterile cotton swab was gently compressed on the injection site for hemostasis, and the abdomen was closed separately in a two layer method (peritoneum and skin) with a 4-0 silk suture. The mice were kept in SPF warm incubator for observation and finally returned to the SPF animal room when they had fully recovered from the anesthesia.

Tissue collection and bacterial isolation

The mice were fasted for 24 h overnight before necropsy. At the end of the 13th week, all mice were euthanized by cervical dislocation and necropsied for bacterial isolation and histopathology. The hemorrhagic ascites was firstly collected from bulge abdomen of tumor positive mouse by a 2-ml aseptically injector. The ascites was fully mixed and piped (200 μl) onto the Columbia agar base for H. pylori culture in microaerophilic environment. The remainder was centrifuged at 4000 × g for 5 min. The supernatant was tested for AFP (α-fetoprotein) by ELISA kit (Wuhan Boster Biological Technology, Co., Ltd., Wuhan, China) and compared with that in serum of both tumor positive and negative mouse. The DNA of cellular precipitate was extracted by Genomic DNA Mini extraction kit (Beyotime Institute of Biotechnology) and tested for Helicobacter genus-specific 16S rRNA (C97/C98). Samples of blood, stomach and liver were aseptically collected and cultured during necropsy. A total of 150 μl of blood samples were collected from inferior vena cava puncture. One hundend microliters were spread evenly over a Columbia agar plate for H. pylori cultivation and 50 μl were stored at −80°C until for DNA and AFP analysis. The liver, as a whole, was removed and weighed. Then, the left liver lobe (group A2 and B2) was directly divided into three sections. The first section (50–100 mg) was ground in sterile grinders with 100 μl of sterile PBS and spread evenly over a Columbia agar plate for H. pylori cultivation. The second section (15×15×3 mm) used for histological analysis was placed into 10% normal buffered formalin for 24 h, and embedded in paraffin. Sections (3-μm) were cut. The slides were stained with hematoxylin and eosin (H&E; conducted by the Pathology Department of Affiliated Union Hospital of Fujian Medical University, Fuzhou, China) for assessment of histopathological changes and immunohistochemistry (IHC) staining for H. pylori, PCNA, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). The third section was stored at −80°C for DNA and protein analysis. Equally, the tumor in the left liver lobe (A1 and B1 group) was also divided, processed and analyzed as the protocols described above. Finally, the stomach was removed, opened along its greater curvature, and rinsed gently in sterile cold PBS. When the contents had been totally removed, the stomach was placed (mucosal side up) on sterile gauze and longitudinally dissected along the greater curvature into three fragments, so that each one contained the gastric cardia, body and antrum. The three gastric fragments were processed and analyzed. All Columbia agar plates were incubated in a microaerophilic condition (5% O2, 10% CO2, 85% N2, humidity >90%) at 37°C. The plates were evaluated for H. pylori growth from 3rd to 10th day after necropsy. The presence of colonies were confirmed using morphology, Gram staining, biochemical tests (urease, catalase and oxidase reactions) and polymerase chain reaction (PCR). The plates were discarded and defined as negative if no positive colony was detected until the 10th day.

DNA extraction and PCR amplification

DNA of samples (blood, stomachs, livers and tumors) were isolated with Genomic DNA Mini extraction kit (Beyotime Institute of Biotechnology). According to the manufacturer's instruction, ~25 mg of tissue was completely triturated with an electric grinder (Tiangen Biotech, Co., Ltd., Beijing, China), and then, the homogenate (or 50 μl of blood) was mixed in a vortex with 180 μl of sample lysis buffer A and 20 μl of Proteinase K. The mixture was incubated overnight at 55°C, thus, the tissues could be fully cleaved. The next day, 200 μl of sample lysis buffer B was added to and incubated with them for 10 min at 70°C. The sample was fully mixed with 200 μl of dehydrated ethanol and then added to a clean spun column. The mixture was washed and centrifuged with a series of washing buffer. Finally, 50 μl of elution buffer was pipetted into the spun column. The DNA was collected with the last high-speed centrifugation, and its concentration was adjusted to 80 ng/μl for the following analysis. The extracted DNA was amplified with Helicobacter genus-specific 16S rRNA primers (35): sense primer, 5′-GCT ATG ACG GGT ATC C-3′ (C97F) and antisense primer, 5′-GAT TTT ACC CCT ACA CCA-3′ (C98R). The sense and antisense primers amplified a 400-bp product. We mixed 2 μl of 80 ng/μl extracted DNA, 1 μl of 20 pmol from each primer, 12.5 μl of 2X Power Taq PCR MasterMix (Bioteke Corp., Beijing, China), and 8.5 μl of sterilized ultrapure water. The mixture was amplified after a PCR cycle: initial denaturation with a Taq polymerase at 95°C for 10 min, denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 75°C for 30 sec (35 cycles), and a final extension step at 75°C for 5 min. The amplified products were loaded onto 1.5% (weight/volume) agarose (Solarbio Technology Co., Ltd., Beijing, China) gels containing 0.04% (volume/volume) GoodView (Beijing SBS Genetech Co., Ltd., Beijing, China), and the production size was compared with a 100-bp DNA marker ladder (Pregene Biotechnology Co., Ltd., Beijing, China). H. pylori type strain NCTC 11637 was used as a positive control, while sterilized ultrapure water was used as a negative control for each PCR cycle. Bands were visualized and photographed under UV light. The 16S rRNA positive samples were sequenced by Biosune Biotechnology Co., Ltd., (Beijing, China) and blasted with H. pylori type strain NCTC 11637.

Immunohistochemical staining and quantitative analysis

Tissue sections were deparaffinized in xylene and rehydrated through graded alcohols to water. Slides were antigen unmasked by maintaining in a citrate buffer (10 mmol/l; pH 6.0) at a sub-boiling temperature for 20 min. The sections were cooled on a bench top for 30 min and washed with PBS (pH 7.4) three times for 5 min each. Hydrogen peroxide (3%) was incubated with the slides for 10 min and washed away, so endogenous peroxidase activity would be ceased. Sections were then incubated with polyclonal rabbit anti-H. pylori antibody (1:50 diluted in PBS, Product no. B047101; Dako, Glostrup, Denmark), monoclonal mouse anti-PCNA antibody (1:200 diluted in PBS, Product no. sc-56; Santa Cruz Biotechnology, Dallas, TX, USA), mono-clonal mouse anti-Bcl-2 antibody (1:50 diluted in PBS, Product no. sc-7382; Santa Cruz Biotechnology), or polyclonal rabbit anti-Bax antibody (1:50 diluted in PBS, Product no. sc-526; Santa Cruz Biotechnology) overnight at 4°C. The next day, we removed the primary antibody and washed the sections. A prepared rabbit or mouse SP kit (ZSGB-BIO, Beijing, China) was used according to the manufacturer's instructions, and we incubated the sections with solution A for 30 min and then with solution B for 45 min at 37°C. We removed the reagents and washed them. DAB (ZSGB-BIO) was added to each section and the staining closely monitored. As soon as the sections had developed, they were immersed in PBS and counterstained in hematoxylin. Finally, the sections were dehydrated with graded alcohols to form xylene before mounting. The tissue sections must not dry out during the whole immunocytochemical staining procedure. Brown granules which were observed in the gastric lumens, hepatic sinusoid, cytoplasm, or nucleus were regarded as positive staining. The slides were observed under a light microscope (Olympus Corp., Tokyo, Japan), and five photographs of each section were randomly obtained at ×40 magnification using the same conditions (light source, color saturation, brightness, gain and contrast). Photograph quantification was performed using Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA). In each field, the integrated optical density (IOD) for all positive staining was measured.

Protein extraction and western blotting

Twenty milligrams of the liver or tumor was completely triturated with an electric grinder, and the homogenate was mixed with 250 μl of cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1 mM of phenylmethylsulfonyl fluoride (PMSF). The mixture was centrifuged (14,000 × g, 4°C for 5 min), and the supernatant was collected. The protein concentrations were detected using a BCA kit (Beyotime Institute of Biotechnology). The proteins (50–100 μg/lane) were electrophoresed in a 12% SDS-polyacrylamide gel and then transferred onto a PVDF membrane (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5% skim milk in TBST at room temperature for 1 h and subsequently incubated overnight at 4°C with a primary antibody (PCNA at 1:500, Product no. sc-56; Bax at 1:500, Product no. sc-526; and Bcl-2 at 1:500, Product no. sc-7382). The next day, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at 1:5,000. Finally, the protein bands on the membranes were detected using an ECL kit (Beyotime Institute of Biotechnology) and scanned with the ImageQuant LAS 4000 digital imaging system (GE Healthcare, Buckinghamshire, UK) or exposed to a medical radiography film (Kodak, Tokyo, Japan). The bands were quantified by Image-Pro Plus software. The IOD of each band was measured and expressed as mean ± standard deviation (mean ± SD).

Statistical analysis

Measurement data are presented as mean ± SD for normal data and median with ranges for non-normal data, while enumeration data are presented as ratios. The data were analyzed using one-way ANOVA if they corresponded with both a normal distribution and homoscedasticity. The SPSS program package (version 11.0; SPSS, Inc., Chicago, IL, USA) was used for our statistical analysis. Differences were considered significant at P-value <0.05.

Results

Morphological, serologic and ascitic features of mice

The skin ulceration (Fig. 1) was observed in 27.8% (5/18) and 30% (6/20) of mice in group A1 and B1, respectively. However, there was no significant (P=0.583) difference between the two groups. We were unable to collect the skin ulceration for morphological detection. But we analyzed the ‘link tissue’ which connected hepatic tumor to abdominal wall instead of ulceration by H&E stain. Consequently, we were not surprised at finding that it was full of tumor cells which were characteristic of cellular pleomorphism. Additionally, 50% (9/18) and 45% (9/20) of mice in group A1 and B1 had developed ascites before they were necropsied. Moreover, H. pylori could not be cultured from any blood and ascites samples. AFP level (mean ± SD, ng/ml) of each group was 11.11±0.69 (ascites of A1 group), 10.89±0.70 (serum of A1 group), 10.99±0.67 (serum of A2 group), 10.98±0.72 (ascites of B1 group), 10.88±0.54 (serum of B1 group) and 11.08±0.71 (serum of B2 group), respectively. However, no significant difference (P=0.962) could be found between any of the two groups above. Moreover, Helicobacter genus-specific 16S rRNA was not detected in blood or ascites samples.

Gastric H. pylori determination and histopathological assessment

H. pylori was cultured from gastric homogenate from 90% (18 of 20) of the mice in group A1 (H. pylori infected and tumor positive mice) and 80% (16 of 20) of the mice in group A2 (H. pylori infected and tumor negative mice). Helicobacter genus-specific 16S rRNA (Fig. 2) was detected using DNA extracted from the stomach, and a 95% (19 of 20) positive rate was found in both group A1 and A2. Three to five positive PCR products in each group were randomly selected and sequenced. All the sequenced samples were completely homologous to H. pylori type strain NCTC 11637. IHC staining for H. pylori in Helicobacter genus-specific 16S rRNA positive gastric slides showed that it mainly colonized the gastric glands (Fig. 3). Although there was no apparent ulceration in any H. pylori-infected stomach (group A1 and A2), the histopathological changes (Fig. 4) indicated that chronic inflammatory cells infiltrated the pyloric antrum area, and the inflammatory changes were mild to moderate. All the pathogen detections above were negative for H. pylori in the uninfected stomachs (group B1 and B2), and chronic inflammatory cells were not apparent.

Detection of H. pylori in livers

The mice in group A1 (n=18) and A2 (n=16) whose gastric homogenates were positive for H. pylori by culture method as well as all individuals in group B1 (n=20) and B2 (n=10) were taken into the following detection and analysis procedures. H. pylori could not be cultured in any liver or tumor sample. The positive rate of Helicobacter genus-specific 16S rRNA (Fig. 2) for the liver and tumor samples in group A1 was 83.3% (15 of 18) and 66.7% (12 of 18), respectively. The rate for the liver samples in group A2 was 87.5% (14 of 16). Three to five positive PCR products were also sequenced as previously described. All sequenced products were also completely homologous to H. pylori type strain NCTC 11637. H. pylori was mainly observed in the hepatic sinusoid and necrotic area of the tumors in the infected mice using IHC staining detection (Fig. 3), while none could be found in the tumor parenchyma. All the H. pylori detection methods were negative for the uninfected livers and tumors (group B1 and B2).

Morphological changes in the livers

We determined liver to body weight ratio for each group and show the average ratio (mean ± SD) of the mice in groups A1, A2, B1 and B2 was 0.072261±0.024696,0.045331±0.003885,0.062900±0.020324, and 0.042018±0.004516, respectively. The ratio of tumor positive mice (group A1 and B1) was significantly higher (P<0.001) than that of tumor negative mice (group A2 and B2), while there was no significant difference between H. pylori infected and uninfected mice, namely group A1 and B1 (P=0.178) or group A2 and B2 (P=0.635). Cytoplasmic vacuolation (ballooning degeneration) and hepatic binucleate cells could be observed in the H. pylori infected livers, and the sinusoids were compressed by swollen hepatic cells. In addition, the infiltrated inflammatory cells were thinly distributed in the hepatic lobules and also observed in the H. pylori infected livers. However, no visible microscopic difference existed between group A1 and B1 tumors. Both had characterized structural disorders and cellular pleomorphism such as atypia, increased nuclear-cytoplasmic ratio, hyperchromatic nuclei and pathological mitosis. Additionally, inflammatory cells and vasodilation could be observed among the tumor cells. The necrotic areas, which were homogeneous and lightly stained, were distributed around the tumor parenchyma (Figs. 1 and 5).

Protein expression in livers and tumors

We detected the expression of specific proteins in the livers and tumors by performing IHC staining and western blot analysis. Similar trends with IHC staining and western blot analysis were noted regarding PCNA and Bcl-2 expression. The PCNA expression (Figs. 6 and 7 and Table II) was significantly (P≤0.01) increased in the H. pylori infected tumors (group A1) as being compared with the uninfected tumors (group B1). Similarly, it was also significantly (P<0.05) increased in the H. pylori infected livers (group A1 and A2) as being compared with the uninfected livers (group B1 and B2). Additionally, the tumor PCNA expression (group A1 and B1) was dramatically increased (P<0.001) as compared with the liver (group A1, A2, B1 and B2). There was no difference (P>0.1) in the Bcl-2 expression (Figs. 6 and 7 and Table II) in any tumor or liver. With western blot analysis, a significant reduction (P=0.035) in the Bax expression (Fig. 7 and Table II) of the H. pylori-infected tumors was demonstrated (group A1) as compared with the uninfected tumors (group B1); there was no difference (P=0.176) in Bax expression (Fig. 6 and Table II) between these two groups with IHC staining. However, both western blot analysis and IHC staining demonstrated a significant upregulation (P<0.05) of Bax expression in the H. pylori-infected livers (group A1 and A2), as compared with the uninfected livers (group B1 and B2). Moreover, the Bax expression in the tumors (group A1 and B1) was significantly decreased (P<0.001), as compared with the livers (group A1, A2, B1 and B2).

Table II

Integrated optical density of the proteins expressed in representative liver or tumor samples detected by immunohisto-chemical stain and western blot analysis (mean ± SD).

Table II

Integrated optical density of the proteins expressed in representative liver or tumor samples detected by immunohisto-chemical stain and western blot analysis (mean ± SD).

PCNABcl-2Bax



GroupSampleIHCWBIHCWBIHCWB
A1Liver 660.384±24.196a,b 804.811±21.937a,b257.009±25.241426.883±31.775 568.872±35.024a,b 1473.967±25.356a,b
A1Tumor 769.849±16.381c 1406.576±100.647c284.988±12.188458.303±43.060309.306±20.036 822.853±38.193c
A2Liver 657.051±13.166a,b 812.288±15.339a,b243.345±25.150441.563±20.182 533.919±15.386a,b 1395.167±85.053a,b
B1Liver452.964±15.665677.803±17.419283.233±38.032440.803±36.415449.545±30.011 1145.367±50.639
B1Tumor725.417±17.978 1226.722±124.089275.016±21.806419.970±26.492336.763±15.308961.160±18.509
B2Liver442.964±18.235672.141±18.418262.746±40.646405.830±41.563481.272±16.762 1248.333±134.814

{ label (or @symbol) needed for fn[@id='tfn2-ijo-47-04-1416'] } IHC, immunohistochemical stain. WB, western blotting. Results are shown with the mean ± SD.

a P<0.05 (vs. B1 liver);

b P<0.05 (vs. B2 liver);

c P<0.05 (vs. B1 tumor).

Discussion

H. pylori has been classified as a type I carcinogen (2), that is a major causative factor in many gastric diseases. Furthermore, some extragastric diseases are also correlated with its pre-infection during childhood (47). Recent studies on the correlation between H. pylori infection and chronic hepatic disease have found that the anti-H. pylori antibody level in the serum of patients with chronic liver disease is significantly higher than that of healthy patients (812,3640). In addition, H. pylori was also detected and confirmed in many liver samples from chronic hepatic disease patients using morphological and genetic detection (8,13,14,24,4143). Moreover, researchers have successfully cultured H. pylori from the liver samples of some clinical hepatic disease cases (14,44). All the above mentioned details suggest that H. pylori could infect the human liver and may play a role in the development or progression of hepatic disease.

However, when talking about the role of H. pylori in the progression of hepatic diseases, there is no consistent opinion among researchers. Some investigator, such as Fox et al (35), Matsukura et al (45) and García et al (46) claimed that it could not colonize livers and was barely able to promote hepatic diseases. They rather regard it as contaminant instead of infection. However, others, such as Goo et al (23), Ito et al (24) and Rocha et al (47), reported that H. pylori not only colonize the livers, but also contribute to the hepatic disease evolution. Nowadays, investigators are gradually discovering a growing kinds of Helicobacter spp., such as H. bilis, H. pullorum and H. pylori, which has been found in the liver of hepatic disease patients. However, attention is mainly focused on H. bilis or H. pullorum (35,45,46) rather than H. pylori for the following reasons. It has been confirmed by in vitro study that H. pylori was unable to survive in bile products (48). Moreover, cholecystectomy would increase the gastric Helicobacter infection risk (49). Relying on the crucial findings that H. pylori was unable to survive in hepatobiliary condition, the presence of H. pylori was considered a result of contamination rather than colonization, even though it had ever been found in gallbladder and bile of patients (50). In humans, bile is produced continuously by the hepatocytes (liver bile), but stored and concentrated in the gallbladder (gallbladder bile) (51). Ito et al (25), Zhang et al (26) and Chen et al (27) found H. pylori exhibited hepatotoxicity while it was co-cultured with hepatocytes which were capable of producing bile in vitro. Hence, they insisted H. pylori might play a potential role in hepatocarcinogenesis. Additionally, Goo et al (23), Ito et al (24) and Rocha et al (47) found all liver samples were negative for other gut organisms, such as E. coli, which mainly inhabited gut and more likely present in liver than H. pylori as a contaminant. Moreover, the successful H. pylori-cultivation from livers of patients (14,44) also suggest its colonization rather than contamination. Finally, basing on the finding that H. pylori could be recovered from feces, Kelly et al (52) and Thomas et al (53) agreed with the opinion that it would survived even after exposure to bile. We came to the same conclusion as that summarized by Ito et al (25), Zhang et al (26) and Chen et al (27). However, bile products might transform H. pylori from virulent helical form to insufficient coccoid form (54). The colonization and pathology ability of H. pylori in liver may depend on its virulence factors, such as CagA or VacA (25,5557) and forms. Therefore, we thought the reasons for low H. pylori frequency in the study of Fox et al (35) may due to the insufficient strain or form of H. pylori which the researchers had adopted.

By using morphological (IHC staining) and genetic detection (PCR), the present study confirmed that H. pylori or only its structural components were both detected in stomachs and livers. It was mainly detected in the hepatic sinusoids and necrotic areas of the tumors. Currently, the following possible perspectives or hypotheses are widely accepted regarding the presence of H. pylori in the liver. Firstly, because its genetic components had been detected in cholecystic samples or the bile of gallbladder disease patients, some researchers consider the findings related to duodenal reflux (5862). Gastrointestinal motility will transport H. pylori from the stomach to the descending duodenum. It would be gradually transmitted in a retrograde manner through the major duodenal papilla, sphincter of Oddi, common bile duct, common hepatic duct, left and right hepatic ducts, and cholangiole to its final destination in the liver or gallbladder. Secondly, the genetic components of H. pylori were detectable in the endarterium samples of atherosclerotic patients using PCR detection by Danesh et al (7) and others who indicated that H. pylori might enter into the circulatory system (5,6,63). A long-term chronic H. pylori infection would result in chronic gastritis and a chronic gastric ulcer. Consequently, a small amount of H. pylori would constantly leak into the blood through impaired vessels that are in or around gastric ulcers and then wander aimlessly along the portal or lymph circulation. Tian et al (66) found that inflammatory cells were mostly thinly distributed along the central veins, hepatic sinusoids, arteriolar, and venules in the periportal area of H. pylori-infected livers. Although similar morphological changes were found in H. pylori-infected mouse livers of the present study, we could not detect H. pylori genus-specific 16S rRNA in any of the blood samples. The low bacteria concentration (<103 CFU/μl) in the blood may be responsible for the negative results (60). Thus, even though it was undetectable in the blood of hepatic positive individuals, we cannot deny the abovementioned secondary hypothesis. Thirdly, inflammatory cells would phagocytize H. pylori and arrive at liver when the hepatic homeostatic equilibrium had been broken, for example, an increasing of bile pH value.

With a growing concern regarding the correlativity of H. pylori infection and hepatic disease, the positivity rate of H. pylori genetic components detected in hepatic samples is increasing. Although it is not uncommon for H. pylori to be cultured from the gastric samples of infected patients and animals, a positive culture from hepatic samples is still rarely reported. It has been reported that H. pylori could be cultured from clinical hepatic samples (14,44). However, no positive culture result could be found in hepatic or blood samples of H. pylori-infected animal models which were developed by various researchers, including ourselves. By referring to the findings of researchers in the field, we analyzed the results and summarized the possible reasons as follows. First, by analyzing the results of H. pylori-specific IHC staining, we found that the densities of a positive reaction in H. pylori-infected livers were significantly lower than those of the stomachs. Silva et al (60) indicated the reasons why they were unable to visualize the H. pylori colonies stating that the bacteria were cultured in the conventional conditions and methods. Hence, a low detection sensitivity of bacterial culture may produce a false-negative result. H. pylori prefers gaseous conditions (2–5% of oxygen and 5–10% of carbon dioxide), certain temperatures (34 to 40°C; 37°C is its optimum), humidity (>90%), and acidity (pH 5.5 to 8.0; neutral is the optimum). H. pylori transforms from its normal form to a coccoid form if its growing environment changes (54), such as cholestasis in which hepatic cells excrete abnormal acidic bile. The coccoid form of H. pylori is widely known as a degenerative dead form or a nonculturable form (64,65). In addition to its alkalinity, bile may also contain some other unfavorable ingredients that are unsuitable for the growth of H. pylori and would play a crucial role in restraining or killing it. Because the genetic components or structural proteins would not be eliminated completely by inflammatory or hepatic cells. Thus, we were able to realize the existence of H. pylori in samples by a more sensitive method, such as morphological IHC stain and PCR. Narikawa et al (54) also hypothesized that the coccoid transformation of H. pylori would contribute to the inability of H. pylori culture in hepatic samples despite a positive genetic detection.

Although, the liver to body weight ratio in H. pylori-infected livers was not significant as compared with uninfected livers, cytoplasmic vacuolation and hepatic binucleate cells were observed by Goo et al (23), and the present study. In addition, infiltrated inflammatory cells that were thinly distributed in the H. pylori-infected hepatic lobules were also found by Tian et al (66). No macroscopic or microscopic differences existed in the H. pylori-infected and uninfected tumors, except for the pathogenic detection of H. pylori. In the present study, the skin ulceration bearing mouse usually bore an extremely gross exophytic growing hepatic tumor which stuck to the abdominal wall. We did not have the resources for skin ulceration detection, we analyzed the ‘link tissue’ instead and found that it was full of pleomorphic tumor cells. On that account, we insist that the skin ulceration was a part of the necrotic tumor, which had invaded into the abdominal wall. In order to explore the impact of H. pylori in the development and progression of liver and hepatic orthotopic graft tumors, we introduced three tumor markers, namely PCNA, Bax and Bcl-2, which are cellular growth, apoptosis-promotion and apoptosis-inhibition proteins, respectively. In the present study, H. pylori may have upregulated the expression of PCNA both in tumor and liver, whereas may have downregulated the Bax expression in the tumor and upregulated it in the liver. No significant impact of H. pylori on the Bcl-2 expression could be seen in the tumor or the liver.

PCNA was a 36-kDa antigen which was associated with cellular growth activity (6770). It plays an important role in cellular proliferation and DNA synthesis, and its expression is usually proportional to DNA synthesis. Hence, it is widely thought to be an indicator and is used to estimate the occurrence, development, metastasis, progression, classification, neoplasm staging, treatment evaluation and prognosis of many tumors (7174). In actively proliferative cells, such as tumor cells, the expression of PCNA is significantly upregulated (7580). In the present study, besides the significantly unregulated expression in tumors, PCNA in H. pylori-infected livers and tumors was also markedly higher than that in the uninfected ones. Our results corresponded to the findings of Goo et al (23) and Tian et al (81). We noted that the swollen cells in the H. pylori-infected livers compressed the hepatic sinusoids, and subsequently, might lead to the blood and oxygen deficits. The impaired hepatic cells might induce the expression of PCNA indirectly as the deficits in nutrition, oxygen and blood supply. Moreover, inflammatory cells and their infection factors might contribute to PCNA upregulation in hepatic cells. This is our hypothesis for the reasons for PCNA upregulation that was detected in H. pylori-infected livers. Moreover, Tian et al (81) performed an in vitro experiment involving the human liver HepG2 cells and H. pylori co-culture indicating an increasing PCNA expression. However, the liver cells in their study were in an abundant nutritious, oxygen and blood condition (82). Thus, we infer from the results that H. pylori may promote cellular proliferation in some direct but unknown pathways.

The Bcl-2 family is divided into two categories. One is a type of regulator protein that induces cell apoptosis, such as Bax. The other, especially Bcl-2, is considered an important anti-apoptosis protein. The Bcl-2 family antigen would combine with each other and become a protein dimer which act as a molecular switch in the progression of cell death (8388). The occurrence of carcinoma is an imbalance between cellular growth or proliferation and cellular death or apoptosis. The upregulation of proto-oncogenes or downregulation of anti-oncogenes would finally promote tumor progression. When the Bax expression were higher than that of Bcl-2, they would combine as Bax/Bax homologous dimers and promote cellular apoptosis. Alternatively, the Bcl-2 and Bax antigens would combine as Bcl-2/Bax heterodimers and Bcl-2/Bcl-2 homodimers and play a role in inhibiting cellular apoptosis (86,8992). Deficits in nutrients, oxygen, and blood supply might be a crucial reason for cellular apoptosis in the present study (93). Additionally, inflammatory factors or mediators excreted by infiltrating inflammatory cells were also responsible for cellular apoptosis. All the above mentioned reasons enabled the cellular apoptosis in H. pylori-infected livers and led them to have a higher Bax expression. Hemangiectasis was noted in the tumors, thus, their blood and oxygen supply were abundant. Moreover, the percentage of death and apoptosis was significantly lower in the tumor than the normal liver tissue. Thus, we were able to explain the downregulation of the Bax expression demonstrated in the present study. We conclude from the results that inflammation might be the primary cause of apoptosis in the H. pylori-infected livers, but H. pylori might act as a crucial factor to inhibit or reduce tumorous apoptosis.

The present study has some shortcomings. We could not establish the H. pylori forms and the exact reasons for hepatic inflammation. Additionally, it is unknown whether the difference among groups will be more significantly visible when the model-build period is prolonged. Therefore, further research is essential for the worldwide unsolved issues that were found in exploring the relationship between H. pylori and hepatic diseases.

Acknowledgements

Our research was supported by the Provincial Natural Science Foundation of Fujian (code: 2012J01358) and by the Construction Project of National Key Clinical Subject of General Surgery.

Abbreviations:

H. pylori

Helicobacter pylori

PCNA

proliferating cell nuclear antigen

Bcl-2

B-cell lymphoma 2

Bax

Bcl-2-associated X protein

References

1 

Rybicka M, Nakonieczna J, Stalke P and Bielawski KP: Host response to the presence of Helicobacter spp. DNA in the liver of patients with chronic liver diseases. Pol J Microbiol. 60:175–178. 2011.PubMed/NCBI

2 

Schistosomes, liver flukes and Helicobacter pylori. In: IARC Working Group on the Evaluation of Carcinogenic Risks to Humans; Lyon. 7–14 June 1994; IARC monographs on the evaluation of carcinogenic risks to humans. World Health Organization, International Agency for Research on Cancer; 61. pp. 1–241. 1994

3 

Wedi B and Kapp A: Helicobacter pylori infection in skin diseases: a critical appraisal. Am J Clin Dermatol. 3:273–282. 2002. View Article : Google Scholar : PubMed/NCBI

4 

Pellicano R, Menard A, Rizzetto M and Megraud F: Helicobacter species and liver diseases: association or causation? Lancet Infect Dis. 8:254–260. 2008. View Article : Google Scholar : PubMed/NCBI

5 

Farsak B, Yildirir A, Akyön Y, Pinar A, Oç M, Böke E, Kes S and Tokgözoğlu L: Detection of Chlamydia pneumoniae and Helicobacter pylori DNA in human atherosclerotic plaques by PCR. J Clin Microbiol. 38:4408–4411. 2000.PubMed/NCBI

6 

Ameriso SF, Fridman EA, Leiguarda RC and Sevlever GE: Detection of Helicobacter pylori in human carotid atheroscle-rotic plaques. Stroke. 32:385–391. 2001. View Article : Google Scholar : PubMed/NCBI

7 

Danesh J, Koreth J, Youngman L, Collins R, Arnold JR, Balarajan Y, McGee J and Roskell D: Is Helicobacter pylori a factor in coronary atherosclerosis? J Clin Microbiol. 37:16511999.PubMed/NCBI

8 

Nilsson I, Lindgren S, Eriksson S and Wadstrom T: Serum antibodies to Helicobacter hepaticus and Helicobacter pylori in patients with chronic liver disease. Gut. 46:410–414. 2000. View Article : Google Scholar : PubMed/NCBI

9 

Pellicano R, Leone N, Berrutti M, Cutufia MA, Fiorentino M, Rizzetto M and Ponzetto A: Helicobacter pylori seroprevalence in hepatitis C virus positive patients with cirrhosis. J Hepatol. 33:648–650. 2000. View Article : Google Scholar : PubMed/NCBI

10 

Ponzetto A, Pellicano R, Leone N, Berrutti M, Turrini F and Rizzetto M: Helicobacter pylori seroprevalence in cirrhotic patients with hepatitis B virus infection. Neth J Med. 56:206–210. 2000. View Article : Google Scholar : PubMed/NCBI

11 

Ponzetto A, Pellicano R, Leone N, Cutufia MA, Turrini F, Grigioni WF, D'Errico A, Mortimer P, Rizzetto M and Silengo L: Helicobacter infection and cirrhosis in hepatitis C virus carriage: is it an innocent bystander or a troublemaker? Med Hypotheses. 54:275–277. 2000. View Article : Google Scholar : PubMed/NCBI

12 

Queiroz DM, Rocha AM, Rocha GA, Cinque SM, Oliveira AG, Godoy A and Tanno H: Association between Helicobacter pylori infection and cirrhosis in patients with chronic hepatitis C virus. Dig Dis Sci. 51:370–373. 2006. View Article : Google Scholar : PubMed/NCBI

13 

Avenaud P, Marais A, Monteiro L, Le Bail B, Bioulac Sage P, Balabaud C and Mégraud F: Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma. Cancer. 89:1431–1439. 2000. View Article : Google Scholar : PubMed/NCBI

14 

Xuan SY, Li N, Qiang X, Zhou RR, Shi YX and Jiang WJ: Helicobacter infection in hepatocellular carcinoma tissue. World J Gastroenterol. 12:2335–2340. 2006.PubMed/NCBI

15 

Farazi PA and DePinho RA: Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer. 6:674–687. 2006. View Article : Google Scholar : PubMed/NCBI

16 

Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar

17 

Parkin DM, Bray F, Ferlay J and Pisani P: Global cancer statistics, 2002. CA Cancer J Clin. 55:74–108. 2005. View Article : Google Scholar : PubMed/NCBI

18 

Iwatsuki S, Starzl TE, Sheahan DG, Yokoyama I, Demetris AJ, Todo S, Tzakis AG, Van Thiel DH, Carr B, Selby R, et al: Hepatic resection versus transplantation for hepatocellular carcinoma. Ann Surg. 214:221–228; discussion 228–229. 1991. View Article : Google Scholar : PubMed/NCBI

19 

McPeake JR, O'Grady JG, Zaman S, Portmann B, Wight DG, Tan KC, Calne RY and Williams R: Liver transplantation for primary hepatocellular carcinoma: tumor size and number determine outcome. J Hepatol. 18:226–234. 1993. View Article : Google Scholar : PubMed/NCBI

20 

Kensler TW, Roebuck BD, Wogan GN and Groopman JD: Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicol Sci. 120(Suppl 1): S28–S48. 2011. View Article : Google Scholar :

21 

Chuang SC, La Vecchia C and Boffetta P: Liver cancer: Descriptive epidemiology and risk factors other than HBV and HCV infection. Cancer Lett. 286:9–14. 2009. View Article : Google Scholar

22 

Arzumanyan A, Reis HM and Feitelson MA: Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma. Nat Rev Cancer. 13:123–135. 2013. View Article : Google Scholar : PubMed/NCBI

23 

Goo MJ, Ki MR, Lee HR, Yang HJ, Yuan DW, Hong IH, Park JK, Hong KS, Han JY, Hwang OK, et al: Helicobacter pylori promotes hepatic fibrosis in the animal model. Lab Invest. 89:1291–1303. 2009. View Article : Google Scholar : PubMed/NCBI

24 

Ito K, Nakamura M, Toda G, Negishi M, Torii A and Ohno T: Potential role of Helicobacter pylori in hepatocarcinogenesis. Int J Mol Med. 13:221–227. 2004.PubMed/NCBI

25 

Ito K, Yamaoka Y, Ota H, El-Zimaity H and Graham DY: Adherence, internalization, and persistence of Helicobacter pylori in hepatocytes. Dig Dis Sci. 53:2541–2549. 2008. View Article : Google Scholar : PubMed/NCBI

26 

Zhang Y, Fan XG, Huang YK, Chen R and Dai H: Helicobacter pylori enhances cyclin D1, PCNA expression in HepG2 cell line. Zhonghua Gan Zang Bing Za Zhi. 12:695–696. 2004.(In Chinese). PubMed/NCBI

27 

Chen R, Fan XG, Huang Y, Li N and Chen CH: In vitro cytotoxicity of Helicobacter pylori on hepatocarcinoma HepG2 cells. Ai Zheng. 23:44–49. 2004.(In Chinese). PubMed/NCBI

28 

Karita M, Li Q, Cantero D and Okita K: Establishment of a small animal model for human Helicobacter pylori infection using germ-free mouse. Am J Gastroenterol. 89:208–213. 1994.PubMed/NCBI

29 

Thalmaier U, Lehn N, Pfeffer K, Stolte M, Vieth M and Schneider-Brachert W: Role of tumor necrosis factor alpha in Helicobacter pylori gastritis in tumor necrosis factor receptor 1-deficient mice. Infect Immun. 70:3149–3155. 2002. View Article : Google Scholar : PubMed/NCBI

30 

Algood HM, Allen SS, Washington MK, Peek RM Jr, Miller GG and Cover TL: Regulation of gastric B cell recruitment is dependent on IL-17 receptor A signaling in a model of chronic bacterial infection. J Immunol. 183:5837–5846. 2009. View Article : Google Scholar : PubMed/NCBI

31 

Maurer KJ, Ihrig MM, Rogers AB, Ng V, Bouchard G, Leonard MR, Carey MC and Fox JG: Identification of cholelithogenic enterohepatic Helicobacter species and their role in murine cholesterol gallstone formation. Gastroenterology. 128:1023–1033. 2005. View Article : Google Scholar : PubMed/NCBI

32 

Lee CW, Rickman B, Rogers AB, Muthupalani S, Takaishi S, Yang P, Wang TC and Fox JG: Combination of sulindac and antimicrobial eradication of Helicobacter pylori prevents progression of gastric cancer in hypergastrinemic INS-GAS mice. Cancer Res. 69:8166–8174. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Yao X, Hu JF, Daniels M, Yien H, Lu H, Sharan H, Zhou X, Zeng Z, Li T, Yang Y, et al: A novel orthotopic tumor model to study growth factors and oncogenes in hepatocarcinogenesis. Clin Cancer Res. 9:2719–2726. 2003.PubMed/NCBI

34 

Aprahamian M, Bour G, Akladios CY, Fylaktakidou K, Greferath R, Soler L, Marescaux J, Egly JM, Lehn JM and Nicolau C: Myo-InositolTrisPyroPhosphate treatment leads to HIF-1α suppression and eradication of early hepatoma tumors in rats. Chem Biochem. 12:777–783. 2011.

35 

Fox JG, Dewhirst FE, Shen Z, Feng Y, Taylor NS, Paster BJ, Ericson RL, Lau CN, Correa P, Araya JC, et al: Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology. 114:755–763. 1998. View Article : Google Scholar : PubMed/NCBI

36 

Leone N, Pellicano R, Brunello F, Cutufia MA, Berrutti M, Fagoonee S, Rizzetto M and Ponzetto A: Helicobacter pylori seroprevalence in patients with cirrhosis of the liver and hepatocellular carcinoma. Cancer Detect Prev. 27:494–497. 2003. View Article : Google Scholar : PubMed/NCBI

37 

Ponzetto A, Pellicano R, Redaelli A, Rizzetto M and Roffi L: Helicobacter pylori infection in patients with hepatitis C Virus positive chronic liver diseases. New Microbiol. 26:321–328. 2003.PubMed/NCBI

38 

Spinzi G, Pellicano R, Minoli G, Terreni N, Cutufia MA, Fagoonee S, Rizzetto M and Ponzetto A: Helicobacter pylori seroprevalence in hepatitis C virus positive patients with cirrhosis. The Como cross-sectional study. Panminerva Med. 43:85–87. 2001.PubMed/NCBI

39 

Fan XG, Zou YY, Wu AH, Li TG, Hu GL and Zhang Z: Seroprevalence of Helicobacter pylori infection in patients with hepatitis B. Br J Biomed Sci. 55:176–178. 1998.

40 

Zhang S, Bao Y and Zu MH: Research of relationship between Hp infection and HCC. Chin J Clin Oncol. 45–48. 2004.

41 

Huang Y, Fan XG, Wang ZM, Zhou JH, Tian XF and Li N: Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma. J Clin Pathol. 57:1273–1277. 2004. View Article : Google Scholar : PubMed/NCBI

42 

Pellicano R, Mazzaferro V, Grigioni WF, Cutufia MA, Fagoonee S, Silengo L, Rizzetto M and Ponzetto A: Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma. World J Gastroenterol. 10:598–601. 2004. View Article : Google Scholar : PubMed/NCBI

43 

Leelawat K, Suksumek N, Leelawat S and Lek-Uthai U: Detection of VacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients. Southeast Asian J Trop Med Public Health. 38:881–885. 2007.PubMed/NCBI

44 

de Magalhaes Queiroz DM and Santos A: Isolation of a Helicobacter strain from the human liver. Gastroenterology. 121:1023–1024. 2001. View Article : Google Scholar : PubMed/NCBI

45 

Matsukura N, Yokomuro S, Yamada S, Tajiri T, Sundo T, Hadama T, Kamiya S, Naito Z and Fox JG: Association between Helicobacter bilis in bile and biliary tract malignancies: H. bilis in bile from Japanese and Thai patients with benign and malignant diseases in the biliary tract. Jpn J Cancer Res. 93:842–847. 2002. View Article : Google Scholar : PubMed/NCBI

46 

García A, Feng Y, Parry NM, McCabe A, Mobley MW, Lertpiriyapong K, Whary MT and Fox JG: Helicobacter pylori infection does not promote hepatocellular cancer in a transgenic mouse model of hepatitis C virus pathogenesis. Gut Microbes. 4:577–590. 2013. View Article : Google Scholar : PubMed/NCBI

47 

Rocha M, Avenaud P, Menard A, Le Bail B, Balabaud C, Bioulac-Sage P, de Magalhães Queiroz DM and Mégraud F: Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma. Gut. 54:396–401. 2005. View Article : Google Scholar : PubMed/NCBI

48 

Hanninen ML: Sensitivity of Helicobacter pylori to different bile salts. Eur J Clin Microbiol Infect Disy. 10:515–518. 1991. View Article : Google Scholar

49 

Caldwell MT, McDermott M, Jazrawi S, O'Dowd G, Byrne PJ, Walsh TN, Hourihane DO and Hennessy TP: Helicobacter pylori infection increases following cholecystectomy. Ir J Med Sci. 164:52–55. 1995. View Article : Google Scholar : PubMed/NCBI

50 

Kawaguchi M, Saito T, Ohno H, Midorikawa S, Sanji T, Handa Y, Morita S, Yoshida H, Tsurui M, Misaka R, et al: Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa. J Gastroenterol. 31:294–298. 1996. View Article : Google Scholar : PubMed/NCBI

51 

Guyton JE and Hall AC: Textbook of Medical Physiology. Saunders Elsevier; Philadelphia, PA: 2011

52 

Kelly SM, Pitcher MC, Farmery SM and Gibson GR: Isolation of Helicobacter pylori from feces of patients with dyspepsia in the United Kingdom. Gastroenterology. 107:1671–1674. 1994.PubMed/NCBI

53 

Thomas JE, Gibson GR, Darboe MK, Dale A and Weaver LT: Isolation of Helicobacter pylori from human faeces. Lancet. 340:1194–1195. 1992. View Article : Google Scholar : PubMed/NCBI

54 

Narikawa S, Kawai S, Aoshima H, Kawamata O, Kawaguchi R, Hikiji K, Kato M, Iino S and Mizushima Y: Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori. Clin Diagn Lab Immunol. 4:285–290. 1997.PubMed/NCBI

55 

Dore MP, Realdi G, Mura D, Graham DY and Sepulveda AR: Helicobacter infection in patients with HCV-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Dig Dis Sci. 47:1638–1643. 2002. View Article : Google Scholar : PubMed/NCBI

56 

Silva LD, Rocha AM, Rocha GA, de Moura SB, Rocha MM, Dani R, de Melo FF, Guerra JB, de Castro LP, Mendes GS, et al: The presence of Helicobacter pylori in the liver depends on the Th1, Th17 and Treg cytokine profile of the patient. Mem Inst Oswaldo Cruz. 106:748–754. 2011. View Article : Google Scholar : PubMed/NCBI

57 

Boonyanugomol W, Chomvarin C, Sripa B, Bhudhisawasdi V, Khuntikeo N, Hahnvajanawong C and Chamsuwan A: Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation. HPB (Oxford). 14:177–184. 2012. View Article : Google Scholar

58 

Myung SJ, Kim MH, Shim KN, Kim YS, Kim EO, Kim HJ, Park ET, Yoo KS, Lim BC, Seo DW, et al: Detection of Helicobacter pylori DNA in human biliary tree and its association with hepatolithiasis. Dig Dis Sci. 45:1405–1412. 2000. View Article : Google Scholar : PubMed/NCBI

59 

Chen W, Li D, Cannan RJ and Stubbs RS: Common presence of Helicobacter DNA in the gallbladder of patients with gallstone diseases and controls. Dig Liver Dis. 35:237–243. 2003. View Article : Google Scholar : PubMed/NCBI

60 

Silva CP, Pereira-Lima JC, Oliveira AG, Guerra JB, Marques DL, Sarmanho L, Cabral MM and Queiroz DM: Association of the presence of Helicobacter in gallbladder tissue with cholelithiasis and cholecystitis. J Clin Microbiol. 41:5615–5618. 2003. View Article : Google Scholar : PubMed/NCBI

61 

Maurer KJ, Rogers AB, Ge Z, Wiese AJ, Carey MC and Fox JG: Helicobacter pylori and cholesterol gallstone formation in C57L/J mice: A prospective study. Am J Physiol Gastrointest Liver Physiol. 290:G175–G182. 2006. View Article : Google Scholar

62 

Abayli B, Colakoglu S, Serin M, Erdogan S, Isiksal YF, Tuncer I, Koksal F and Demiryurek H: Helicobacter pylori in the etiology of cholesterol gallstones. J Clin Gastroenterol. 39:134–137. 2005.PubMed/NCBI

63 

Kowalski M, Rees W, Konturek PC, Grove R, Scheffold T, Meixner H, Brunec M, Franz N, Konturek JW, Pieniazek P, et al: Detection of Helicobacter pylori specific DNA in human atheromatous coronary arteries and its association to prior myocardial infarction and unstable angina. Dig Liver Dis. 34:398–402. 2002. View Article : Google Scholar : PubMed/NCBI

64 

Kusters JG, Gerrits MM, Van Strijp JA and Vandenbroucke-Grauls CM: Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun. 65:3672–3679. 1997.PubMed/NCBI

65 

Andersen LP, Dorland A, Karacan H, Colding H, Nilsson HO, Wadström T and Blom J: Possible clinical importance of the transformation of Helicobacter pylori into coccoid forms. Scand J Gastroenterol. 35:897–903. 2000. View Article : Google Scholar : PubMed/NCBI

66 

Tian XF, Fan XG, Fu CY, Huang Y and Zhu C: Experimental study on the pathological effect of Helicobacter pylori on liver tissues. Zhonghua Gan Zang Bing Za Zhi. 13:780–783. 2005.(In Chinese). PubMed/NCBI

67 

Egelkrout EM, Mariconti L, Settlage SB, Cella R, Robertson D and Hanley-Bowdoin L: Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell. 14:3225–3236. 2002. View Article : Google Scholar : PubMed/NCBI

68 

Essers J, Theil AF, Baldeyron C, van Cappellen WA, Houtsmuller AB, Kanaar R and Vermeulen W: Nuclear dynamics of PCNA in DNA replication and repair. Mol Cell Biol. 25:9350–9359. 2005. View Article : Google Scholar : PubMed/NCBI

69 

Shivji KK, Kenny MK and Wood RD: Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 69:367–374. 1992. View Article : Google Scholar : PubMed/NCBI

70 

Leonardi E, Girlando S, Serio G, Mauri FA, Perrone G, Scampini S, Dalla Palma P and Barbareschi M: PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J Clin Pathol. 45:416–419. 1992. View Article : Google Scholar : PubMed/NCBI

71 

Kobayashi I, Matsuo K, Ishibashi Y, Kanda S and Sakai H: The proliferative activity in dysplasia and carcinoma in situ of the uterine cervix analyzed by proliferating cell nuclear antigen immunostaining and silver-binding argyrophilic nucleolar organizer region staining. Hum Pathol. 25:198–202. 1994. View Article : Google Scholar : PubMed/NCBI

72 

Allegranza A, Girlando S, Arrigoni GL, Veronese S, Mauri FA, Gambacorta M, Pollo B, Dalla Palma P and Barbareschi M: Proliferating cell nuclear antigen expression in central nervous system neoplasms. Virchows Arch A Pathol Anat Histopathol. 419:417–423. 1991. View Article : Google Scholar : PubMed/NCBI

73 

Tsai ST and Jin YT: Proliferating cell nuclear antigen (PCNA) expression in oral squamous cell carcinomas. J Oral Pathol Med. 24:313–315. 1995. View Article : Google Scholar : PubMed/NCBI

74 

Willett CG1, Warland G, Hagan MP, Daly WJ, Coen J, Shellito PC and Compton CC: Tumor proliferation in rectal cancer following preoperative irradiation. J Clin Oncol. 13:1417–1424. 1995.PubMed/NCBI

75 

Prosperi E: Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. Prog Cell Cycle Res. 3:193–210. 1997. View Article : Google Scholar : PubMed/NCBI

76 

Moldovan GL, Pfander B and Jentsch S: PCNA, the maestro of the replication fork. Cell. 129:665–679. 2007. View Article : Google Scholar : PubMed/NCBI

77 

Wang SC, Nakajima Y, Yu YL, Xia W, Chen CT, Yang CC, McIntush EW, Li LY, Hawke DH, Kobayashi R and Hung MC: Tyrosine phosphorylation controls PCNA function through protein stability. Nat Cell Biol. 8:1359–1368. 2006. View Article : Google Scholar : PubMed/NCBI

78 

Pal HC1, Sharma S, Strickland LR, Agarwal J, Athar M, Elmets CA and Afaq F: Delphinidin reduces cell proliferation and induces apoptosis of non-small-cell lung cancer cells by targeting EGFR/VEGFR2 signaling pathways. PLoS One. 8:e772702013. View Article : Google Scholar : PubMed/NCBI

79 

Aziz MH, Wheeler DL, Bhamb B and Verma AK: Protein kinase C delta overexpressing transgenic mice are resistant to chemically but not to UV radiation-induced development of squamous cell carcinomas: A possible link to specific cytokines and cyclo-oxygenase-2. Cancer Res. 66:713–722. 2006. View Article : Google Scholar : PubMed/NCBI

80 

Ojeh N, Hiilesvuo K, Warri A, Salmivirta M, Henttinen T and Maatta A: Ectopic expression of syndecan-1 in basal epidermis affects keratinocyte proliferation and wound re-epithelialization. J Invest Dermatol. 128:26–34. 2008. View Article : Google Scholar

81 

Tian XF, Fan XG, Huang Y, Zhang Y and Zhu C: Expression of cyclin D1 proliferating cell nuclear antigen in liver of C57BL/6 mice infected with Helicobacter pylori. World Chin J Digestol. 14:1341–1345. 2006.

82 

Zhang Y, Fan XG, Chen R, Xiao ZQ, Feng XP, Tian XF and Chen ZH: Comparative proteome analysis of untreated and Helicobacter pylori-treated HepG2. World J Gastroenterol. 11:3485–3489. 2005. View Article : Google Scholar : PubMed/NCBI

83 

Komatsu K1, Miyashita T, Hang H, Hopkins KM, Zheng W, Cuddeback S, Yamada M, Lieberman HB and Wang HG: Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis. Nat Cell Biol. 2:1–6. 2000.PubMed/NCBI

84 

Lin B, Kolluri SK, Lin F, Liu W, Han YH, Cao X, Dawson MI, Reed JC and Zhang XK: Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell. 116:527–540. 2004. View Article : Google Scholar : PubMed/NCBI

85 

Hoetelmans RW: Nuclear partners of Bcl-2: Bax and PML. DNA Cell Biol. 23:351–354. 2004. View Article : Google Scholar : PubMed/NCBI

86 

Oltvai ZN, Milliman CL and Korsmeyer SJ: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 74:609–619. 1993. View Article : Google Scholar : PubMed/NCBI

87 

Tsujimoto Y, Finger LR, Yunis J, Nowell PC and Croce CM: Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science. 226:1097–1099. 1984. View Article : Google Scholar : PubMed/NCBI

88 

Cleary ML, Smith SD and Sklar J: Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 47:19–28. 1986. View Article : Google Scholar : PubMed/NCBI

89 

Yang LJ, Cao XT and Yu LZ: Bcl-2 and Bax in apoptosis of tumor cells. Chin J Cancer Biother. 10:232–234. 2003.

90 

Yin XM, Oltvai ZN and Korsmeyer SJ: BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature. 369:321–323. 1994. View Article : Google Scholar : PubMed/NCBI

91 

Yang E and Korsmeyer SJ: Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood. 88:386–401. 1996.PubMed/NCBI

92 

Yang E, Zha J, Jockel J, Boise LH, Thompson CB and Korsmeyer SJ: Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 80:285–291. 1995. View Article : Google Scholar : PubMed/NCBI

93 

Cotran RS, Kumar V and Collins T: Robbins Pathologic Basis of Disease. 6th edition. W.B Saunders Company; Philadelphia, PA: 1998

Related Articles

Journal Cover

October-2015
Volume 47 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Wang J, Wang X, Tang N, Chen Y and She F: Impact of Helicobacter pylori on the growth of hepatic orthotopic graft tumors in mice. Int J Oncol 47: 1416-1428, 2015
APA
Wang, J., Wang, X., Tang, N., Chen, Y., & She, F. (2015). Impact of Helicobacter pylori on the growth of hepatic orthotopic graft tumors in mice. International Journal of Oncology, 47, 1416-1428. https://doi.org/10.3892/ijo.2015.3107
MLA
Wang, J., Wang, X., Tang, N., Chen, Y., She, F."Impact of Helicobacter pylori on the growth of hepatic orthotopic graft tumors in mice". International Journal of Oncology 47.4 (2015): 1416-1428.
Chicago
Wang, J., Wang, X., Tang, N., Chen, Y., She, F."Impact of Helicobacter pylori on the growth of hepatic orthotopic graft tumors in mice". International Journal of Oncology 47, no. 4 (2015): 1416-1428. https://doi.org/10.3892/ijo.2015.3107