A potential role for Helicobacter pylori heat shock protein 60 in gastric tumorigenesis

doi:10.1016/j.bbrc.2010.01.010

Biochemical and Biophysical Research Communications

Volume 392, Issue 2, 5 February 2010, Pages 183-189

https://doi.org/10.1016/j.bbrc.2010.01.010 Get rights and content

Abstract

Helicobacter pylori has been found to promote the malignant process leading to gastric cancer. Heat shock protein 60 of H. pylori (HpHSP60) was previously been identified as a potent immunogene. This study investigates the role of HpHSP60 in gastric cancer carcinogenesis. The effect of HpHSP60 on cell proliferation, anti-death activity, angiogenesis and cell migration were explored. The results showed that HpHSP60 enhanced migration by gastric cancer cells and promoted tube formation by umbilical vein endothelial cells (HUVECs); however, HpHSP60 did not increase cell proliferation nor was this protein able to rescue gastric cancer cells from death. Moreover, the results also indicated HpHSP60 had different effects on AGS gastric cancer cells or THP-1 monocytic cells in terms of their expression of pro-inflammatory cytokines, which are known to be important to cancer development. We propose that HpHSP60 may trigger the initiation of carcinogenesis by inducing pro-inflammatory cytokine release and by promoting angiogenesis and metastasis. Thus, this extracellular pathogen-derived HSP60 is potentially a vigorous virulence factor that can act as a carcinogen during gastric tumorigenesis.

Introduction

Helicobacter pylori is an important human pathogen and infection with this bacterium may lead to various gastric diseases [1]; particularly, it has been proposed to act as a carcinogen causing gastric tumors. H. pylori has been demonstrated to promote tumorigenesis. Cancer initiate processes such as preventing cells from apoptosis [2], enhancing cell proliferation [3], increasing blood vessel density [4], and augmenting cell invasion/migration ability [5] were found to be associated with H. pylori infection.

Persistent infection by H. pylori is known to trigger chronic inflammation, which is an important risk factor for malignancy [6]. Chemokine productions during inflammation have multiple effects on tumor growth, invasion, and angiogenesis [7]. Recent studies have indicated that patients with H. pylori infection have higher CXC chemokine expression levels of various factors, including IL-8, GRO, and epithelial neutrophil activating protein-78 (ENA-78), in the gastric mucosa, gastric epithelium cells, and macrophages [8], [9].

Based on detection of auto-antibodies in patients, it has been suggested that overexpression of human heat shock protein 60 (HSP60) during the early stage of breast cancer development may be functionally correlated with tumor growth and/or progression [10]. Additionally, human HSP60 has been suggested as facilitating metastasis [11] and anti-apoptosis [12] in tumor cells. If we consider pathogen-derived HSP60s, however, there have been only a few reports suggesting an association between these proteins and tumorigenesis. HSP60 from the intracellular bacterium Chlamydia trachomatis has been proposed as a risk factor for ovarian cancer since it is able to inhibit apoptosis [13]; nonetheless, the effect of extracellular HSP60 from a bacterial pathogen on cancer development remains unclear. H. pylori HSP60 has been previously identified as an adhesion molecule that is able to interact with gastric epithelial cells and mucin [14]. It is also known to be involved in the induction of host inflammatory responses. As a potent immunogen, HpHSP60 can stimulate human monocytes/macrophages and/or human gastric epithelium cells to produce pro-inflammatory cytokines including IL-1, IL-6, IL-8, and GRO [15], [16], [17], [18]. Recently, by measuring the level of anti-HpHSP60 antibodies in the sera of patients with different gastric diseases, Tanaka et al. proposed that H. pylori HSP60 might be associated with gastric carcinogenesis [19].

In this study, we investigate how HpHSP60 may act during tumor progression and malignancy. By screening H. pylori patient sera and a general examination of the cancer growth properties of HpHSP60-treated cells, we found that HpHSP60 promote inflammation, cell migration, and angiogenesis but does not increase cell proliferation or anti-death activity. These results suggest that HpHSP60 may act as a potent carcinogen during H. pylori infection.

Section snippets

Materials and methods

Cell culture. AGS and SNU-1 cells (BCRC, Hsinchu, Taiwan) were cultured in RPMI 1640 medium (Invitrogen, MD, USA) supplemented with 2 g/L sodium bicarbonate (BIO BASIC Inc., Canada), 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, MD, USA) and 50 μg/mL penicillin/streptomycin (Biological industries, Beithaemek, Israel). Human umbilical vein endothelial cells (HUVECs) (BCRC, Hsinchu, Taiwan) were cultured in M199 medium (Invitrogen, MD, USA) supplemented with 10% heat-inactivated FBS, 50

Analysis of the anti-HpHSP60 antibody titers in H. pylori-positive patients with gastric cancer or other gastro-duodenal diseases

To determine whether HpHSP60 was associated with gastric diseases, the levels of anti-HpHSP60 antibodies in the sera of H. pylori-infected patients were measured as an indicator. According the clinical symptoms, samples were collected from patients suffering from gastric cancer (HC, n = 45), gastritis (HS, n = 26), duodenal ulcer (HD, n = 76), and gastric ulcer (HU, n = 16). The titers of anti-HpHSP60 antibody in sera of patients with gastric cancer were significant lower than in the sera of the other

Discussion

In this study, we demonstrated the role of HpHSP60 in gastric tumor progression. This seems to occur by promoting the migration ability of cancer cells (Fig. 3A), by increasing the angiogenic activity of endothelial cells (Fig. 3B), and by inducing inflammatory cytokines production by both gastric epithelial cells and monocytes (Fig. 4). These results suggest that HpHSP60 is able to accelerate tumorigenesis by multiple mechanisms.

Microbial HSP60s are known as immunodominant antigens because

Acknowledgments

This study was supported by Grants from the National Science Council, Taipei, Taiwan (NSC 98-2320-B-009-002) and in parts by the ATU-MOE project.

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However, many studies have shown that HpHSP60 acts as a potent immunogen, leading to the strong induction of proinflammatory cytokines, such as TNF-α, IL-8, and IL-6 [28,29]. These cytokines determine inflammation at the site of infection, and such HpHSP60-induced inflammation might have the potential to promote processes of malignant tumorigenesis, including angiogenesis and metastasis [30,31]. Taken together, the relationship between HpHSP60 and Treg cells is intriguing and deserves to be further investigated.
Local Treg responses are involved in Helicobacter pylori-related inflammation and clinical outcomes after infection, and H. pylori-derived HSP60 (HpHSP60) is an important virulence factor associated with gastric carcinogenesis. This study to investigate the role of HpHSP60 in immunosuppression, particularly with regard to whether it could induce the production of Treg cells. For this purpose, human peripheral blood mononuclear cells (PBMCs) were treated with or without HpHSP60 in the presence of an anti-CD3 mAb to determine the effect of HpHSP60 on cell proliferation. In this report, HpHSP60 decreased the expression of CDK4 to significantly arrest the proliferation of mitogen-stimulated T-cells, which correlated with the induction of Treg cells. Moreover, monocytic cells were essential for the induction of HpHSP60-induced Treg cells via the secretion of IL-10 and TGF-β after treatment with HpHSP60. Blockage of HpHSP60 with specific monoclonal antibodies significantly reduced the colonization of H. pylori and the expression of Treg cells in vivo. Overall, our results suggest that HpHSP60 could act on macrophages to trigger the expression of IL-10 and TGF-β, thereby leading to an increase in Treg cells and inhibition of T-cell proliferation.
Toll-like receptor 2: An important immunomodulatory molecule during Helicobacter pylori infection
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Toll like receptors (TLRs) are an essential subset of pathogen recognition receptors (PRRs) which identify the microbial components and contribute in the regulation of innate and adaptive immune responses against the infectious agents. The TLRs, especially TLR2, TLR4, TLR5 and TLR9, participate in the induction of immune response against H. pylori. TLR2 is expressed on a number of immune and non-immune cells and recognizes a vast broad of microbial components due to its potential to form heterodimers with other TLRs, including TLR1, TLR6 and TLR10. A number of H. pylori–related molecules may contribute to TLR2-dependent responses, including HP-LPS, HP-HSP60 and HP-NAP. TLR2 plays a pivotal role in regulation of immune response to H. pylori through activation of NF-κB and induction of cytokine expression in epithelial cells, monocytes/macrophages, dendritic cells, neutrophils and B cells. The TLR2-related immune response that is induced by H. pylori-derived components may play an important role regarding the outcome of the infection toward bacterial elimination, persistence or pathological reactions. The immunomodulatory and immunoregulatory roles of TLR2 during H. pylori infection were considered in this review. TLR2 could be considered as an interesting therapeutic target for treatment of H. pylori-related diseases.
Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis
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Helicobacter pylori pathogenesis and disease outcomes are mediated by a complex interplay between bacterial virulence factors, host, and environmental factors. After H. pylori enters the host stomach, four steps are critical for bacteria to establish successful colonization, persistent infection, and disease pathogenesis: (1) Survival in the acidic stomach; (2) movement toward epithelium cells by flagella-mediated motility; (3) attachment to host cells by adhesins/receptors interaction; (4) causing tissue damage by toxin release. Over the past 20 years, the understanding of H. pylori pathogenesis has been improved by studies focusing on the host and bacterial factors through epidemiology researches and molecular mechanism investigations. These include studies identifying the roles of novel virulence factors and their association with different disease outcomes, especially the bacterial adhesins, cag pathogenicity island, and vacuolating cytotoxin. Recently, the development of large-scale screening methods, including proteomic, and transcriptomic tools, has been used to determine the complex gene regulatory networks in H. pylori. In addition, a more available complete genomic database of H. pylori strains isolated from patients with different gastrointestinal diseases worldwide is helpful to characterize this bacterium. This review highlights the key findings of H. pylori virulence factors reported over the past 20 years.
Antibodies against Helicobacter pylori heat shock protein 60 aggravate HSP60-mediated proinflammatory responses
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An array of proinflammatory cytokines such as IL-1β, IL-8, and TNF-α [2] are stimulated by H. pylori to modulate host immune functions [3] or promote apoptosis of gastric epithelial cells [1], which have been suggested to play an important role in the pathogenesis of chronic gastritis or gastric cancer. H. pylori heat shock protein 60 (HpHSP60) has been identified as one of the potential immunogens of the bacterium that induces IL-6, IL-8, TNF-α, and GRO production from monocytes or gastric epithelial cells [4,5]. Several examples have indicated that HpHSP60 plays a role in the induction of chronic mucosal inflammation and gastric atrophy [6–8].
Anti-Helicobacter pylori heat shock protein 60 (HpHSP60) antibodies are usually found in H. pylori-infected patients and are known to be associated with the progression of gastric diseases. However, the effects of these antibodies on the functions of HpHSP60 have not been identified. This study aims to investigate the effects of the interaction between anti-HSP60 antibodies and HpHSP60 on inflammatory responses. Anti-HpHSP60 polyclonal sera and monoclonal antibodies (mAbs) were produced to evaluate their effects on HpHSP60-induced IL-8 and TNF-α activity. The results indicated that anti-HpHSP60 polyclonal sera collected from patients infected with H. pylori or from rabbit and mice immunized with HpHSP60 could significantly enhance HpHSP60-mediated IL-8 and TNF-α secretion from monocytic THP-1 cells. Similar effects were also found with anti-HpHSP60 mAbs. Further analysis revealed that this phenomenon was only carried out by anti-HpHSP60 antibody but not by other non-specific mAbs. Moreover, the non-specific mAbs decreased the synergism of HpHSP60 and anti-HpHSP60 mAbs in proinflammatory cytokine induction. Herein, we have examined the role of anti-HpHSP60 antibody in host immune responses for the first time. This study demonstrated that H. pylori HSP60/mAbs could modulate helicobacterial pathogenesis by increasing IL-8 and TNF-α production. The pathogen-specific antibodies may execute potential immune functions rather than recognize or neutralize microbes.
Helicobacter pylori-derived heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway
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Additionally, H. pylori may promote the expression of angiogenesis-promoting factors such as IL-8, IL-6, TNF-α, and growth related oncogene (GRO) in endothelial cells [16]. As previously mentioned, HpHSP60 induces expression of pro-inflammatory genes related to angiogenesis, and recent evidence suggests that HpHSP60 directly enhances blood vessel formation [11]. Chemokines comprise a family of approximately 50 low molecular weight chemotactic cytokines that were initially described as being important for leukocyte recruitment to sites of infection and inflammation [17].
Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCβ2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCβ2/Ca2+ signal transduction in endothelial cells.
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¹: These authors contributed equally to this study.

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A potential role for Helicobacter pylori heat shock protein 60 in gastric tumorigenesis

Abstract

Introduction

Section snippets

Materials and methods

Analysis of the anti-HpHSP60 antibody titers in H. pylori-positive patients with gastric cancer or other gastro-duodenal diseases

Discussion

Acknowledgments

Best Pract. Res. Clin. Gastroenterol.

Gastroenterology

Adv. Cancer Res.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Cell

Biochem. Pharmacol.

J. Biol. Chem.

Clin. Microbiol. Infect.

Vet. Immunol. Immunopathol.

Late reactivation of sonic hedgehog by Helicobacter pylori results in population of gastric epithelial cells that are resistant to apoptosis: implication for gastric carcinogenesis

Cancer Lett.

Overexpression of cyclin E in Mongolian gerbil with Helicobacter pylori-induced gastric precancerosis

World J. Gastroenterol.

Novel action of gastric proton pump inhibitor on suppression of Helicobacter pylori induced angiogenesis

Gut

P120 and Kaiso regulate Helicobacter pylori-induced expression of matrix metalloproteinase-7

Mol. Biol. Cell

CXC chemokines Gro(alpha)/IL-8 and IP-10/MIG in Helicobacter pylori gastritis

Clin. Exp. Immunol.

Gastric epithelial cell CXC chemokine secretion following Helicobacter pylori infection in vitro

J. Gastroenterol. Hepatol.

Proteomics-based identification of HSP60 as a tumor-associated antigen in early stage breast cancer and ductal carcinoma in situ

J. Proteome Res.

Interaction between HSP60 and beta-catenin promotes metastasis

Carcinogenesis