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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2014  |  Volume : 57  |  Issue : 1  |  Page : 13-18
CagA+ H. pylori filtrate induces cytokine IL-8 secretion by esophageal squamous carcinoma EC 109 cells via a p38 pathway


1 Department of Pathology, Shaanxi Provincial People's Hospital, Third Affiliated Hospital of the School of Medicine, Xi'an Jiaotong University, Xi'an, 710068, China
2 Department of Clinical Laboratory, Shaanxi Provincial People's Hospital, Third Affiliated Hospital of the School of Medicine, Xi'an Jiaotong University, Xi'an, 710068, China
3 Department of Thoracic Surgery, Shaanxi Provincial People's Hospital, Third Affiliated Hospital of the School of Medicine, Xi'an Jiaotong University, Xi'an, 710068, China

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Date of Web Publication17-Apr-2014
 

   Abstract 

Background: The relationship between Helicobacter pylori (Hp) infection and the risk of esophageal squamous cell carcinoma is unclear. Aim: The purpose was to investigate the effects of CagA+ Hp on esophageal squamous carcinoma Ec 109 cells in vitro and explore the molecular mechanisms underlying these effects. Materials and Methods: Ec 109 cells were treated with CagA+ Hp filtrate at a concentration of 1.0 mg/mL or 50 μg/mL in vitro, proliferation and apoptosis of Ec 109 cells were assayed, the secretion of IL-8 was measured by ELISA, and the levels of Src homology-2 domain-containing phosphatase (SHP-2) mRNAs was assayed by RT-PCR.. Furthermore, after pretreatment of Ec109 cells with the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the p38 pathway was detected. Results: CagA+ Hp filtrate enhanced both the proliferation and apoptosis of Ec 109 cells. In addition, cytokine IL-8 release was significantly increased, and the expression of SHP-2 mRNA declined sharply in the CagA+ Hp group. Furthermore, after pretreatment of Ec109 cells with the specific p38 MAPK inhibitor SB203580, Ec109 cells proliferation and IL-8 secretion were inhibited. Conclusions: Our results suggest that CagA+ Hp filtrates could induce proliferation and the secretion of IL-8 by Ec109 cells in vitro. IL-8 secretion was induced through the activation of the p38 MAPK signal pathway.

Keywords: CagA, Helicobacter pylori, Ec109 cells, IL-8, P38 MAPK

How to cite this article:
Ying GX, Wen Sheng L I, Xia ZL, Tao WH. CagA+ H. pylori filtrate induces cytokine IL-8 secretion by esophageal squamous carcinoma EC 109 cells via a p38 pathway. Indian J Pathol Microbiol 2014;57:13-8

How to cite this URL:
Ying GX, Wen Sheng L I, Xia ZL, Tao WH. CagA+ H. pylori filtrate induces cytokine IL-8 secretion by esophageal squamous carcinoma EC 109 cells via a p38 pathway. Indian J Pathol Microbiol [serial online] 2014 [cited 2020 Sep 20];57:13-8. Available from: http://www.ijpmonline.org/text.asp?2014/57/1/13/130866



   Introduction Top


The chronic infection of the human stomach by  Helicobacter pylori Scientific Name Search  Gram-negative bacterium, is a major cause of chronic gastritis, peptic ulcers, and gastric malignancies, including gastric non-cardia adenocarcinoma and mucosal-associated lymphoid tissue (MALT) lymphoma. [1] An inverse association between Hp infection and esophageal adenocarcinoma has been reported; this relationship may be attributed to a reduction in acidity because Hp induces atrophic gastritis and produces ammonia. However, compared with adenocarcinomas of the esophagus, few studies have examined the association between Hp infection and esophageal squamous cell carcinoma, and the relationship between Hp infection and esophageal squamous cell carcinoma risk is still controversial. [2] Epidemiological data suggest that Hp infection is more prevalent in esophageal squamous cell carcinoma samples than in normal esophageal mucosa and adjacent tissues. [3] Furthermore, one study used an IgG enzyme-linked immunosorbent assay to demonstrate that infection with cytotoxin-associated gene A (CagA)-positive strains of Hp may increase the risk of esophageal squamous cell carcinoma. [4] Wu et al. found an inverse association between Hp infection and esophageal squamous cell carcinoma and found that Hp induces apoptosis in esophageal squamous cell carcinoma cells in vitro. [5],[6] Our previous study found that the filtrate from a CagA+ Hp could enhance the proliferation of human immortalized esophageal epithelial (NE3) cells, concomitant with a decrease in SHP-2 mRNA expression and an increase in cytokine IL-8 release in vitro, but the mechanisms underlying these effects are still unknown.

Thus, we conducted further in vitro studies to clarify this issue. In the present study, we provide direct evidence of an association between esophageal squamous cell carcinoma and Hp infection. We treated Ec109 cells with CagA + Hp filtrate to investigate the effects of CagA + Hp on Ec109 cells in vitro and subsequently explored the potential molecular mechanisms underlying these effects.


   Materials and Methods Top


Bacterial strains and filtrate preparation

CagA-positive and CagA-negative culture filtrates were provided by Prof. Quanming Zou from Third Military Medical University, China. Hp was grown in Brinell broth, supplemented with 5% fetal calf serum (Gibco, Grand Island, NY, USA), for 24-36 h at 37°C in a thermostatic shaker under microaerophilic conditions. As described by Sommi et al., [7] when bacterial suspensions reached A450 1.2 (corresponding to a bacterial concentration of 5 × 10 8 CFU/mL), Hp culture filtrate was obtained by removing the bacterial by centrifugation (1200 × g for 10 min) and sterilization of the supernatants by passage through a 0.22 μm cellulose acetate filter (Nalge, Rochester, NY, USA). The resulting supernatant was filtered through a 0.22 μm cellulose acetate filter (Nalge, Rochester, NY, USA). A non-inoculated broth filtrate was used as a blank control. The presence of CagA in the filtrate was confirmed with Western blots. The culture filtrate was stored at -70°C until use.

Ec 109 cell culture

The human esophageal carcinoma cell line Ec109 was kindly provided by the Central Laboratory of Shaanxi Provincial People's Hospital. Ec109 cells were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (SIJIQING), 100 U/Ml penicillin, 100 U/mL streptomycin and 2 mM l-glutamine at 37°C in a humidified atmosphere containing 5% CO 2 . The culture medium was changed every 2-3 days to maintain exponential growth.

MTT assay for Ec 109 cell proliferation

The Ec109 cells were seeded at a density of 5 × 10 5 cells per well in 96-well flat-bottom microtiter plates allowed to adhere overnight, then treated with CagA+ Hp filtrate at a concentration of 1.0 mg/mL or 50 μg/mL for 24, 48, 72 or 96 hours. CagA-Hp filtrate and Brinell broth were used as negative control. MTT solution (5 mg/mL) was added 4 h before the end of the incubation, and the reaction was terminated by adding 10% acidified SDS. The absorbance value per well at 570 nm was read using an ELISA microplate reader (Thermo, USA). All MTT assays were performed in triplicate.

Flow cytometry analysis of apoptosis in Ec109 cells

Annexin V-FITC/PI double staining was performed to detect cell apoptosis. Ec109 cells were harvested in Annexin V binding buffer after treatment with 1.0 mg/mL of CagA+ Hp filtrate for 48 hours. 1 × 10 6 cells of Ec109 were used for flow cytometric evaluation. The suspension was incubated with 5 μL Annexin V-FITC (Beijing 4A Biotech Co., Ltd) and 10 μL PI for 10 min at room temperature in the dark, followed by cytometric analysis (BD, USA) within 30 min of staining. All experiments were performed in triplicate.

Induction of IL-8

Ec109 cells were treated with 1.0 mg/mL CagA+ Hp filtrate for 48 hours. The level of IL-8 was measured by the enzyme-linked immunosorbent assay (ELISA) using a Quantikine Immunoassay Kit (Mega, USA) as recommended by the manufacturer. After the development of the colorimetric reaction, the optical density (OD) at 450 nm was quantified by the ELISA microplate reader (Thermo, USA), and OD readings were converted to pg/mL based on standard curves obtained with known concentrations of the cytokine in each assay. Each sample was tested in duplicate. The cytokine concentrations varied by less than 10% between the replicates and were expressed as the mean values.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SHP-2

Ec109 cells were treated with 1.0 mg/mL CagA+ Hp filtrate for 48 hours. The levels of SHP-2 and β-actin mRNAs were assayed by RT-PCR. Total cellular RNA was extracted from the Ec109 cells using an RNAzol kit (Tiangen Biotech Co., Ltd) according to the manufacturer's instructions (Biotec Laboratories Inc.) The RNA (2 μg) was reverse transcribed, and the complementary DNA was amplified in a 50 μL reaction in a thermocycler (MJ Research, USA) for 30 cycles with commercially available PCR primers for SHP-2. The PCR reaction was carried out in a volume of 50 μL containing 2 μL of cDNA template, 2.5 mM/L MgCl2, 200 μM/L of each deoxynucleotide triphosphate, 200 nM/L of each primer, and 0.05 U TaqDNA polymerase (Fermentas, USA). The primer sequences used for SHP-2 were: Forward, 5΄-CAA CAC CTT TCT CTC TGA TGATGT C-3΄; reverse, 5΄-GTG GAG AGA GGA AAGAGT AAA TGT G-3΄. The primer sequences used for β-actin were: Forward, 5΄-GAG ACC TTC AAC ACC CCA GCC-3΄; reverse, 5΄-GGA CTA CAG GTC TTT GCG GAT G-3΄. Negative and positive controls were included in each experiment. β-actin (200 bp) was used as positive control. The PCR protocol used began with an initial denaturation at 95°C for 5 min, followed by 38 amplification cycles (94°C for 50 s, 58°C for 45 s, and 72°C for 50 s), followed by a 10 min final amplification at 72°C. The PCR products were subjected to electrophoresis on 1.5% agarose gels and stained with ethidium bromide.

Western blot analysis of phosphospecific p38 MAPK activation

Monolayers of Ec109 cells were treated with Hp filtrate for 48 h, washed three times with PBS and then lysed with sodium dodecyl sulfate (SDS) buffer. The cells were subsequently harvested, heated to 100°C for 5 min, and loaded onto a 10% SDS-polyacrylamide gel and separated by routine SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Huamei Co., Ltd). The membranes were blocked for 1 h at room temperature with a 5% solution of non-fat dry milk in Tris-buffered saline (pH 7.4) and were subsequently incubated with p38 MAPK antibodies or phospho-p38 MAPK antibodies diluted 1/1000 in a dilution buffer at room temperature for 1 h. The membranes were then incubated at room temperature for 1 h with peroxidase-conjugated goat anti-rabbit antibody (1/1000 dilution; BOAOSEN Co., Ltd). Each sample was probed with nonphosphospecific p38 MAPK antibodies to confirm that equivalent amounts of protein were loaded in each lane. A phosphospecific p38 MAPK antibody (Santa Cruz Biotechnology, USA) was used to detect phosphorylated p38 MAPK. Nonphosphospecific p38 MAPK antibody was also obtained from Santa Cruz Biotechnology (Santa Cruz, USA).

Inhibition of p38 signaling in Ec109 cells

The stock solution of p38 MAPK inhibitor SB203580 (Alexis, USA) was prepared in dimethyl sulfoxide. Ec109 cells were treated with SB203580 at the indicated final concentrations for 1 h before exposure to Hp filtrate. Control cells without inhibitor were treated with dimethyl sulfoxide alone.

Detection of IL-8 following the inhibition of p38

After 1 h of treatment with SB203580, Ec109 cells were washed twice with PBS, then Ec109 cells were treated with Hp filtrate for 48 h at a concentration of 1.0 mg/mL. The level of IL-8 was assayed by the ELISA as described above.

Analysis of SHP-2 mRNA following the

inhibition of p38


After 1 h of treatment with SB203580, Ec109 cells were washed twice with PBS, then Ec109 cells were treated with CagA+ Hp filtrate for 48 h at a concentration of 1.0 mg/mL. SHP-2 mRNA was detected as described above.

Statistical analysis

All data were expressed as the mean ± SE. SPSS 10.0 software was used for one-way analysis of variance and q test. P < 0.05 was considered statistically significant.


   Results Top


CagA+ Hp filtrate promoted Ec109 cell proliferation

The effects of CagA+ Hp filtrate on Ec109 cell proliferation was evaluated by the MTT assay. After Ec109 cells were treated with CagA+ Hp filtrate, in the first 2 days, Ec109 cell showed slight growth trend, following sharply proliferation, and arriving peak(OD 0.87) in the third day. Compared with CagA+ Hp, Ec109 cells were treated with CagA- Hp or control group only showed a slight proliferation trend, there was difference on Ec109 cell proliferation between the CagA+ Hp group and other two groups (P < 0.05). These results showed that CagA+ Hp filtrate enhanced the proliferation of Ec109 cells relative to treatment with CagA- or control broth at concentrations of either 1.0 mg/mL or 50 μg/mL [Figure 1].
Figure 1: Growth curve of Ec109 cells treated with Hp fi ltrates. (a) Growth curve of Ec109 cells treated with Hp fi ltrates at a concentration of 1.0 mg/mL. (a) Growth curve of Ec109 cells treated with Hp fi ltrates at a concentration of 50 μg/mL. Both (a) and (b) showed that CagA+ Hp filtrates stimulate Ec109 cell proliferation. Error bars indicate the mean ± S.D. *, P < 0.05, statistically signifi cant

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CagA+ Hp filtrate enhanced apoptosis in Ec109 cells

When Ec109 cells treated with CagA+ Hp filtrate at 1.0 mg/mL concentration, the percentages of apoptotic cells were 35.45%, 25.14%, and 26.11% for Ec109 cells treated with CagA+ and CagA- Hp filtrate and Brinell broth, respectively. Flow cytometry showed that CagA+ Hp filtrate increased the apoptosis of Ec109 cells. The percentage of apoptotic cells was clearly enhanced in Ec109 cells treated with CagA+ Hp filtrate compared with the CagA- Hp group and the control group (P < 0.05). The percentages of apoptotic cells were 31.83%, 29.38%, and 26.53% for Ec109 cells treated with CagA+ and CagA- Hp filtrate and Brinell broth, respectively. There were no significant differences between the three groups when 50 μg/mL filtrate was used [Figure 2], [Figure 3].
Figure 2: Flow cytometry Dot plots showing Annexin/PI Ec 109 cell apoptosis following treatment with Hp filtrates at a concentration of 1.0 mg/mL. (a) Flow cytometry Dot plots for the CagA+ Hp group, (b) flow cytometry Dot plots for the CagA- Hp group, (c) flow cytometry Dot plots for the control group


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Figure 3: Ec 109 cell apoptosis analysis following treatment with Hp filtrates. (a) Ec 109 cell apoptosis analysis following treatment with Hp filtrates at a concentration of 1.0 mg/mL. (b) Ec109 cells apoptosis analysis following treatment with Hp filtrates at a concentration of 50 mg/mL. These results showed that CagA+ Hp filtrates induced apoptosis in Ec109 cells at a concentration of 1.0 mg/mL. Error bars indicate the mean ± S.D. *P < 0.05 vs. control, statistically significant


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CagA+ Hp induced p38 MAPK activation in Ec109 cells

After applying Hp filtrate to Ec109 cells for 48 h, the total of p38 MAPK and the phosphorylation of p38 MAPK were detected by western blotting analysis. The ratio of phospho-p38 MAPK/ p38 MAPK ratio was significantly increased in the CagA+ Hp group compared with the CagA- Hp group and control group (P < 0.05). After pretreatment of Ec109 cells with the p38 MAPK inhibitor SB203580 for 1 h, cells were cultured in Hp filtrate for 48 h, the phospho-p38 MAPK / p38 MAPK ratio decreased significantly compared to the untreated phospho-p38 MAPK/p38 MAPK ratio (P < 0.05) in all four experiment groups (10 μmol/L SB203580+ CagA+, 20 μmol/L SB203580+ CagA+, 10 μmol/L SB203580+ CagA-, and 20 μmol/L SB203580+ CagA-). These results demonstrated that CagA+ Hp filtrate induced p38 MAPK activation [Figure 4].
Figure 4: (a) Western blot analysis of phospho-p38 MAPK, total p38 MAPK in Ec109 cells treated with CagA+ Hp for 48 h. (b) Western blot of phospho-p38 MAPK, total p38 MAPK in Ec109 cells treated with CagA+ Hp for 48 h following pretreatment with a p38 inhibitor. (c) The ratio of p-p38 MAPK/p38 MAPK was calculated. The results suggested that CagA+ Hp filtrates could stimulate the phosphorylation of p38 MAPK; (d) After pretreatment with a p38 MAPK inhibitor, the phosphorylation of p38 MAPK stimulated by CagA+ Hp filtrates was decreased. Error bars indicate the mean ± S.D. *P < 0.05, statistically significant


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CagA+ Hp filtrate induced IL-8 secretion in a p38-dependent manner

Secreted IL-8 was detected in all three treated groups, at concentrations of 863.3, 582 and 569.80, in the CagA+ Hp, CagA- Hp and control groups, respectively, upon treatment with 1.0 mg/mL filtrate. The concentration of IL-8 was obviously increased in the CagA+ group compared with the CagA- and control groups (P < 0.05). When Ec109 cells were pretreated with the specific p38 MAPK inhibitor SB203580 for 1 h before Hp filtrate was applied for 48 h (10 μmol/L SB203580+ CagA+, 20 μmol/L SB203580+ CagA+) and CagA- Hp (10 μmol/L SB203580+ CagA-, 20 μmol/L SB203580+ CagA-), the level of IL-8 secreted by cells treated with CagA+ Hp decreased compared with that of the non-pretreated CagA+ Hp filtrate group (P < 0.05). These results demonstrated that CagA+ Hp filtrates significantly increased the level of IL-8 via the p38 MAPK pathway [Figure 5].
Figure 5: CagA+ Hp filtrate induced IL-8 secretion in a p38-dependent manner. (a) IL-8 secretion was analyzed in Ec 109 cells stimulated by Hp filtrates for 48 h in vitro; CagA+ Hp obviously stimulated IL-8 secretion. (b) Pretreatment with a p38 MAPK inhibitor for 1 h prior to treatment with Hp filtrates for 48 h inhibited IL-8 secretion. Error bars indicate the mean ± S.D. *P < 0.05 vs control, statistically significant


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The reduction of SHP-2 expression by CagA+ Hp filtrate was enhanced by p38 inhibition

The expression of SHP-2 mRNA was assayed by RT-PCR. Agarose gel electrophoresis analysis of SHP-2 PCR products showed a specific band at 376 bp in both the CagA+ Hp and CagA- Hp groups. A comparison with the sequences in GenBank showed that these PCR products represented SHP-2. No specific band was detected in the broth control group. These results indicated that the expression of SHP-2 was significantly lower in the CagA+ Hp group than in the CagA- Hp group and broth control group (P < 0.05). After pretreatment with the specific p38 MAPK inhibitor SB203580, the Ec109 cells were treated with Hp filtrate for 48 h, and the level of SHP-2 in the CagA+ Hp group (10 μmol/L SB203580+ CagA+, 20 μmol/L SB203580+ CagA+) and CagA- Hp groups (10 μmol/L SB203580+ CagA-, 20 μmol/L SB203580+ CagA-) decreased compared with that in the non-pretreated CagA+ Hp group (P < 0.05). These results showed that the expression of SHP-2 was downregulated in Ec109 cells following treatment with Hp filtrate. Pretreatment with a specific p38 MAPK inhibitor not only failed to prevent the decrease in SHP-2 expression but actually decreased SHP-2 expression even further (P < 0.05). These data suggest that the inhibition of SHP-2 expression by CagA+ Hp filtrate is not mediated by the p38 MAPK pathway [Figure 6].
Figure 6: SHP-2 mRNA expression analysis in Ec109 cells. (a) SHP-2 mRNA expression analysis in Ec109 cells treated with CagA+ Hp filtrates for 48 h. CagA+ Hp filtrates reduced the expression of SHP-2. (b) Pretreatment with a p38 MAPK inhibitor for 1 h prior to treatment with Hp filtrates for 48 h did not prevent the decrease in SHP-2 mRNA expression. Error bars indicate the mean ± S.D. *P < 0.05 vs. control, statistically significant


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   Discussion Top


Our previous study showed that CagA+ Hp filtrates could increase the proliferation of human immortalized esophageal epithelial (NE3) cells, accompanied by a decrease in SHP-2 mRNA expression and an increase in cytokine IL-8 secretion in vitro, but the detailed mechanisms underlying these effects remained unknown. Thus, we conducted in vitro studies to clarify this issue.

The association between Hp infection and esophageal squamous cell carcinoma remains uncertain. Our data showed that CagA+ Hp filtrate could promote the proliferation of Ec109 cells at both high (1.0 mg/mL) and low (50 μg/mL) concentrations. This result was consistent with our previous observations in NE3 cells treated with CagA+ Hp. Flow cytometry showed that treatment with 1.0 mg/mL CagA+ Hp enhanced apoptosis in Ec109 cells. Wu et al. also observed a pro-apoptotic effect of Hp on esophageal squamous cell carcinoma cells in vitro, but they considered this to be indirect evidence of the inverse association between Hp infection and ESCC risk. [5],[6] However, in this study, though we observed increased apoptosis in Ec109 cells, this was not correlated with growth inhibition; on the contrary, increased cell proliferation appeared to compensate for the increased apoptosis. These results are supported by data published by Kacar et al, who used immunohistochemical methods to show that Hp infection increases the proliferative rate of gastric foveolar cells in conjunction with increased apoptosis.

Previous investigations of the correlation between Hp and gastric disease have shown that SHP-2 is a critical intracellular target of the Hp CagA protein. [8] CagA is delivered into gastric epithelial cells via the bacterial type IV secretion system, where it undergoes tyrosine phosphorylation by Src and Abl kinases. Tyrosine-phosphorylated CagA then acquires the ability to interact with and deregulate SHP-2 phosphatase, a bona-fide oncoprotein, and the deregulation of SHP-2 is involved in a variety of human malignancies. [9],[10],[11],[12] The present study showed that CagA+ Hp inhibited the expression of SHP-2 mRNA in Ec109 cells, which suggested that CagA binds and deregulates the SHP-2 phosphatase. Our results further indicated that the deregulation of SHP-2 by CagA may induce abnormal proliferation and apoptosis in Ec109 cells. We speculated that CagA entered Ec109 cells, leading to the activation of the SHP-2 phosphatase and the formation of a Cag/SHP-2 complex to initiate a cascade that blocks the related signaling pathway, thereby affecting Ec109 cell growth, proliferation, and apoptosis.

Interleukin-8 (IL-8) is an important chemokine in tumor angiogenesis and cancer progression. The over secretion of IL-8 has been detected in a variety of human tumors, including gastric cancers, and IL-8 is associated with adhesion, migration and invasion in gastric cancer. [13] IL-8 plays a central role in the pathogenesis of Hp infection. Cag PAI induces IL-8 secretion, increases epithelial cell proliferation and may be important in carcinogenesis. [14] Here, we found that CagA+ Hp filtrate increased IL-8 secretion in Ec109 cells. These results not only demonstrated that CagA+ Hp is able to induce the secretion of IL-8 but also supported the hypothesis that IL-8 release is a universal feature of Hp-infected cells, and this feature may participate in a CagA+ Hp-associated inflammatory reaction.

What is the molecular mechanism by which CagA+ Hp induces IL-8 secretion in Ec109 cells? CagA+ Hp, when translocated into gastric epithelial cells induces IL-8 secretion through multiple signaling pathways, including extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB). [15] An analysis of intracellular signaling pathways indicates that clinical isolates of Hp induce IL-8 gene transcription through NF-κB p65 but also by a MOI-dependent differential activation of MAPK pathways. [16] Some researchers have reported that the IL-8 release induced by CagA occurs via a Ras®Raf®Mek®Erk®NF-κB signaling pathway in a SHP-2- and c-Met-independent manner and that the secretion of IL-8 by gastric epithelial cell upon H. pylori infection is dependent on the activation of NF-kappaB. [17] Our data revealed that CagA+ Hp induces the strong secretion of IL-8 in Ec109 cells in a p38 MAPK-dependent and possibly SHP-2-independent manner; our experiments showed that the presence of a p38 MAPK inhibitor did not prevent the CagA-induced decrease in SHP-2. The pharmacological inhibition of p38 and ERK activity significantly reduced IL-8 production in response to Hp, further emphasizing the importance of MAPKs in the innate immune response to this pathogen.

In conclusion, CagA+ Hp filtrates induced both proliferation and apoptosis, enhanced IL-8 secretion, and downregulated SHP-2 mRNA expression in Ec109 cells. The secretion of the cytokine IL-8 may participate in the CagA+ Hp-associated inflammatory reaction. IL-8 secretion was induced through the activation of the p38 mitogen-activated protein kinase pathway and was not associated with the decrease in SHP-2 mRNA.

However, in the study, our research object is Ec109 cells, which is esophageal squamous carcinoma cells, it is not a very suitable cell line for investigating the relation between Hp infection and cause of esophageal cancer occurs, it is a possible limitation of the study. After all, as Ec109 cells have biological characteristics of cancer cells, we pay attention to molecular mechanism and pathway research, relatively. Esophageal epithelial cell is a more appropriate cell line, which could be use for observation possible carcinogenesis from CagA+ Hp. Furthermore, p38 MAPK was selected to research the signal pathway, more from gastric cancer research thinking. May be there are other more critical pathways that play a role.

For future studies, we need to deeply study the relationship between esophageal squamous cell DNA damage as well as chromosome instability and Hp in vitro; furthermore, we should explore whether Hp has malignant transformation on esophageal squamous cell.

 
   References Top

1.Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347:1175-86.   Back to cited text no. 1
    
2.Simán JH, Engstrand L, Berglund G, Forsgren A, Florén CH. Helicobacter pylori and CagA seropositivity and its association with gastric and oesophageal carcinoma. Scand J Gastroenterol 2007;42:933-40.  Back to cited text no. 2
    
3.Yu DH, Cheng ZN, Jia JH, Tang SL, Wu Y, Wang QZ, et al. Relation between helicobacter pylori L-form infection and tumor angiogenesis in human esophageal carcinoma. Zhonghua Zhong Liu Za Zhi 2003;25:51-4.   Back to cited text no. 3
    
4.Ye W, Held M, Lagergren J, Engstrand L, Blot WJ, McLaughlin JK, et al. Helicobacter pylori infection and gastric atrophy: Risk of adenocarcinoma and squamous-cell carcinoma of the esophagus and adenocarcinoma of the gastric cardia. J Natl Cancer Inst 2004;96:388-96.  Back to cited text no. 4
    
5.Wu IC, Wu DC, Yu FJ, Wang JY, Kuo CH, Yang SF, et al. Association between Helicobacter pylori seropositivity and digestive tract cancers. World J Gastroenterol 2009;43:5465-71.  Back to cited text no. 5
    
6.Wu IC, Wu MT, Chen YK, Hsu MC, Chou SH, Lee CH, et al. Apoptotic effect of Helicobacter pylori on oesophageal squamous-cell carcinoma cells in vitro. Eur J Clin Invest 2008;38:760-5.  Back to cited text no. 6
    
7.Sommi P, Ricci V, Fiocca R, Romano M, Ivey KJ, Cova E, et al. Significance of ammonia in the genesis of gastric epithelial lesions induced by Helicobacter pylori: An in vitro study with different bacterial strains and urea concentrations. Digestion 1996;57:299-304.  Back to cited text no. 7
    
8.Hatakeyama M. Deregulation of SHP-2 tyrosinephosphatase by the Helicobacter pylori virulencefactor CagA. Keio J Med 2002;51: 26-32.  Back to cited text no. 8
[PUBMED]    
9.Tanaka H, Yoshida M, Azuma T. The role of CagA in H. pylori infection. Nippon Rinsho 2009;67:2245-9.  Back to cited text no. 9
    
10.Lu H, Murata-Kamiya N, Saito Y, Hatakeyama M. Role of partitioning-defective 1/microtubule affinity-regulating kinases in the morphogenetic activity of Helicobacter pylori CagA. J Biol Chem 2009;284: 23024-36.  Back to cited text no. 10
    
11.Hatakeyama M. Helicobacter pylori and gastric carcinogenesis. J Gastroenterol 2009;44:239-48.   Back to cited text no. 11
[PUBMED]    
12.Botham CM, Wandler AM, Guillemin K. A transgenic Drosophila model demonstrates that the Helicobacter pylori CagA protein functions as a eukaryotic gab adaptor. PLoS Pathog 2008;4:e1000064.  Back to cited text no. 12
    
13.Ju D, Sun D, Xiu L, Meng X, Zhang C, Wei P. Interleukin-8 is associated with adhesion, migration and invasion in human gastric cancer SCG-7901 cells. Med Oncol 2010;43:419-34.  Back to cited text no. 13
    
14.Ritter B, Kilian P, Reboll MR, Resch K, Distefano JK, Frank R, et al. Differential Effects of Multiplicity of Infection on Helicobacter pylori-Induced Signaling Pathways and Interleukin-8 Gene Transcription. J Clin Immunol 2011;31:60-8.  Back to cited text no. 14
    
15.Lim JW, Kim KH, Kim H. alphaPix interacts with Helicobacter pylori CagA to induce IL-8 expression in gastric epithelial cells. Scand J Gastroenterol 2009;44:1166-72.  Back to cited text no. 15
    
16.Brandt S, Kwok T, Hartig R, König W, Backert S. NF-κB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc Natl Acad Sci U S A 2005;102:9300-5.  Back to cited text no. 16
    
17.Allison CC, Kufer TA, Kremmer E, Kaparakis M, Ferrero RL. Helicobacter pylori Induces MAPK Phosphorylation and AP-1 Activation via a NOD1-Dependent Mechanism. J Immunol 2009;183:8099-109.  Back to cited text no. 17
    

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Correspondence Address:
L I Wen Sheng
Shaanxi Provincial People's Hospital, Third Affiliated Hospital of the School of Medicine, Xi'an Jiaotong University, Xi'an, 710068
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.130866

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Molecular Medicine Reports. 2015; 12(1): 945
[Pubmed] | [DOI]



 

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    Abstract
   Introduction
    Materials and Me...
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