Volume 2013, Article ID 682418, 12 pages http://dx.doi.org/10.1155/2013/682418
Research Article
Inhibition of
Helicobacter pylori CagA-Induced Pathogenesis by
Methylantcinate B from
Antrodia camphorata
Chun-Jung Lin,
1Yerra Koteswara Rao,
2Chiu-Lien Hung,
3Chun-Lung Feng,
4Hsien-Yuan Lane,
1David T. W. Tzeng,
5Ping-Ning Hsu,
6Chih-Ho Lai,
1, 7and Yew-Min Tzeng
21Graduate Institute of Clinical Medical Science, China Medical University, Taichung 40402, Taiwan
2Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Taichung 41349, Taiwan 3Department of Biochemistry and Molecular Medicine, University of California Davis Comprehensive Cancer Center,
Sacramento, CA 95817, USA
4Department of Internal Medicine, China Medical University Hospital, Taichung 40402, Taiwan 5Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung 40227, Taiwan 6Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan
7Department of Microbiology and Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan
Correspondence should be addressed to Yew-Min Tzeng; [email protected], and Chih-Ho Lai; [email protected] Received 11 October 2012; Accepted 23 December 2012
Academic Editor: Mohd Roslan Sulaiman
Copyright © 2013 Chun-Jung Lin et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e bacterial pathogen Helicobacter pylori (Hp) is the leading risk factor for the development of gastric cancer. Hp virulence factor, cytotoxin-associated gene A (CagA) interacted with cholesterol-enriched microdomains and leads to induction of in�ammation in gastric epithelial cells (AGS). In this study, we identi�ed a triterpenoid methylantcinate B (MAB) from the medicinal mushroom Antrodia camphorata which inhibited the translocation and phosphorylation of CagA and caused a reduction in hummingbird phenotype in HP-infected AGS cells. Additionally, MAB suppressed the Hp-induced in�ammatory response by attenuation of NF-𝜅𝜅B activation, translocation of p65 NF-NF-𝜅𝜅B, and phosphorylation of INF-𝜅𝜅B-𝛼𝛼, indicating that MAB modulates CagA-mediated signaling pathway. Additionally, MAB also suppressed the IL-8 luciferase activity and its secretion in HP-infected AGS cells. On the other hand, molecular structure simulations revealed that MAB interacts with CagA similarly to that of cholesterol. Moreover, binding of cholesterol to the immobilized CagA was inhibited by increased levels of MAB. Our results demonstrate that MAB is the �rst natural triterpenoid which competes with cholesterol bound to CagA leading to attenuation of Hp-induced pathogenesis of epithelial cells. us, this study indicates that MAB may have a scope to develop as a therapeutic candidate against Hp CagA-induced in�ammation.
1. Introduction
Chronic infection with the human bacterial pathogen Heli-cobacter pylori (Hp) causes gastritis and peptic ulceration and increases the carrier’s risk of developing gastric cancer [1]. Hp possesses various virulence factors known to be important for the induction of disease during infection. One of the best described effectors molecule is cytotoxin-associated gene A (CagA) which translocated into host gastric epithelial cells by type IV secretion system (T4SS) [2, 3]. is T4SS represents a needle-like structure (also called T4SS pilus) protruding from
the Hp surface and is induced by host cell contact to inject virulence factors including CagA [3]. CagA and the T4SS played a crucial role in gastric cancer development of gerbils [4]. Once intracellular, CagA localizes on the inner surface of the plasma membrane and becomes phosphorylated on tyro-sine residues by Src family kinases [5]. e translocation and phosphorylation of CagA are triggered by direct interaction of the Hp T4SS with the 𝛼𝛼5𝛽𝛽1integrin, which is found on the
surfaces of epithelial cells [5, 6]. is interaction triggers the delivery of CagA into the host as well as subsequent Src kinase activation. e phosphorylated CagA (pCagA) subsequently
induces a signaling cascade, resulting in the induction of proin�ammatory responses in epithelial cells [5]. Previous reports described that pCagA leads to the activation of interleukin (IL)-8 transcriptions through the nuclear factor (NF)-𝜅𝜅B signaling pathway [3, 5–7]. Recently, CagA has been recognized as an oncoprotein that acts in mammals which were demonstrated by a transgenic mouse model [8].
Several reports demonstrated that translocation and phosphorylation of CagA are mediated by cholesterol-rich microdomains of the plasma membrane, commonly termed lipid ras [9–11]. ese domains not only are enriched in cholesterol but also contain phospholipids and glycosylphosphatidylinositol-anchored proteins and have been reported to be sites utilized by Hp to interact with host cells [12, 13]. It is known that Hp migrates toward and acquires exogenous cholesterol from the plasma membranes of host epithelial cells [9–11]. Cholesterol depletion can lead to reduction of CagA-induced pathogenesis in host cells [9]. erefore, development of a new strategy to target bacterial virulence factor mediated by competing cholesterol-enriched binding sites was thought as a potential therapeutic approach.
Current therapies for Hp infection are antibiotics based and oen compromised by antimicrobial resistance. e search for new chemical compounds against Hp is warranted. Natural products have great potential to serve as sources of bioactive molecules and have been shown to in�uence the outcome of Hp infection [14]. Traditional medicines have been used to treat a wide range of ailments, including gastrointestinal disorders, such as dyspepsia, gastritis, and peptic ulcer disease [14]. Antrodia camphorata (AC), named “Niu-chang-chih” in Chinese, is a medicinal mushroom being widely used as a food dietary supplement for can-cer prevention in several Asian and European countries [15]. Although, more than hundred secondary metabolites have been identi�ed from Ac, particular attention has been directed to a triterpenoid, methylantcinate B (MAB). MAB has been reported to display tumor-speci�c cytotoxicity against various cancer cell lines [16]. Additionally, our recent study also indicates that MAB attenuates the Hp-associated in�ammation in gastric epithelial cell line (AGS cells) [17]. However, the molecular mechanism underlying the inhibition of Hp-induced in�ammatory responses is not yet completely dissolved.
In this paper, we investigated the effects of MAB on CagA translocation and phosphorylation in Hp-infected AGS cells. In addition, we also examined the effects of MAB on CagA-induced in�ammatory responses by the determina-tion of NF-𝜅𝜅B activadetermina-tion, translocadetermina-tion of p65 NF-𝜅𝜅B, and phosphorylation of I𝜅𝜅B𝛼𝛼, and IL-8 secretion. It is known that CagA interacts with membrane cholesterol leading to induction of in�ammation in Hp-infected AGS cells. Since the chemical structure of MAB is similar to that of cholesterol, determining the binding mode of MAB to CagA should enhance our understanding of the pharmacophoric features of MAB responsible for its inhibitory activity against Hp CagA-induced in�ammation. is led us to investigate the mechanism of MAB interaction with CagA by the competi-tion of its binding to cholesterol.
2. Materials and Methods
2.1. Antibodies and Chemicals. Antibodies against CagA, ubiquitin, and 𝛽𝛽-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For detection of CagA phosphorylation, 4G10 platinum antiphosphotyrosine was purchased from Millipore (Temecula, CA). Antiphospho-NF-𝜅𝜅B p65 (Ser536), anti-I𝜅𝜅B-𝛼𝛼, PCNA antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA). MG132 was purchased from Sigma-Aldrich (St. Louis, MO). Lipofectamine 2000 and 4′ ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carls-bad, CA). NF-𝜅𝜅B-Luc plasmid was purchased from Strata-gene (La Jolla, CA), and the IL-8 promoter construct (IL-8/wild-type, nucleotides −162 to +44) was kindly provided by Dr. Chih-Hsin Tang of the Department of Pharmacology, China Medical University [18]. Luciferase substrate was purchased from Promega (Madison, WI). NE-PER nuclear and cytoplasmic extraction reagent kit was purchased from ermo Scienti�c Inc. (Rockford, IL).
2.2. Cell Culture and Cytotoxicity Assay. Human gastric epithelial cell line, AGS cells, was cultured in Ham’s F12 medium (Hyclone, Logan, UT) with 10% of decomplement fetal bovine serum (Hyclone, Logan, UT) and incubated in 5% CO2 at 37∘C. All experiments related to bacterial
infec-tion were not supplemented with antimicrobial agents. e 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide (MTT) assay was employed to assess the cytotoxicity of MAB in AGS cells as described previously [17]. Treatment of AGS cells with maximum concentration of MAB (50 𝜇𝜇M) barely in�uenced the viability of AGS cells.
2.3. Helicobacter pylori (Hp) Strains. Hp 26695 (ATCC 700392) wild-type strain containing CagA gene was used, and its isogenic mutant strain ΔCagA was generated as previously described [9]. Hp strain with CagA-EGFP was kindly provided by Dr. Ping-Ning Hsu of the Graduate Insti-tute of Immunology, College of Medicine, National Taiwan University [19]. All Hp strains were cultured and maintained on Brucella agar plates (Becton Dickinson, Franklin Lakes, NJ) containing 10% sheep blood. Bacteria were cultured for 36–48 h and then added to epithelial cells at a multiplicity of infection (MOI) of 100.
2.�. �uri�cation o� �ethylantcinate B (�AB). e compound MAB used in this study was isolated from chloroform extracts of the fruiting bodies of A. camphorata, following the extraction and isolation procedures as we described previously [16, 17]. e purity of the MAB was >98% by HPLC. Its structure was con�rmed by comparison of its mass and nuclear magnetic resonance spectral data with those in the literature [16, 17].
2.5. Western Blot Analysis. AGS cells (1 × 106) were seeded in 6-well plates and performed assigned experiments. Cells were lysed with 100 𝜇𝜇L RIPA reagent (150 mM NaCl, 50 mM
Tris Base pH 7.4, 1 mM EDTA, 1% NP-40, and 0.25% deoxy-cholate), and the supernatants were utilized to performed western blot assay. e relative of bands were quanti�ed using ImageJ and UN-SCAN-IT soware (Silk Scienti�c Corporation, Orem, UT) [20].
2.6. Hummingbird Phenotype Analysis. AGS cells (1 × 106)
were cultured in 12-well plates at 37∘C for 24 h followed by infection with wild-type Hp strain 26695 at an MOI of 100 for 6 h. Elongated cells (hummingbird phenotype) were de�ned as thin needle-like protrusions that were longer than 20 𝜇𝜇m the detail of a typical elongated cell phenotype was described by pervious report [10]. All samples were performed in duplicate from three independent experiments. e proportion of elongated cells was calculated from the numbers of cells having the hummingbird phenotypes. 2.7. Luciferase Activity Assay. AGS cells (3 × 105) were seeded in 12-well plates and transfect cells with either IL-8-Luc or NF-𝜅𝜅B-Luc plasmid by using Lipofectamine 2000. Aer 24 h incubation, cells were then cocultured with wild-type or ΔCagA strain of Hp for 6 h. To prepare total cell lysates, cells were washed once with PBS and 100 𝜇𝜇L of reporter lysis buffer (Promega) was added, and cells were scraped from dishes. Samples were centrifuge with 13,000 rpm for 5 min, and 80 𝜇𝜇L of luciferase substrate was added to 20 𝜇𝜇L samples. Luminescence was measured by using a 96-well microplate luminometer (ermoLabsystems, Helsinki, Finland). 2.8. Analysis of IL-8 Secretion. AGS cells were grown to 90% con�uence in 12-well plates and then experimental design was performed as described previously [21]. Aer 6 h incubation, cultured supernatants were collected, and the IL-8 concentration was determined by enzyme-linked immunosorbent assay (ELISA) by using a sandwich ELISA kit (R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.
2.�. Immuno�uorescence and Confocal Microscopy Analysis. To visualize localization of Hp and the CagA-EGFP transloca-tion, AGS cells (2 × 105) were seeded on coverslips in six-well
plates and incubated for 16 h. Cells were infected with Hp for 6 h and then washed three times with PBS and �xed with 3.7% paraformaldehyde (Sigma-Aldrich) for at least 1 h. e cells were then permeabilized with 0.1% Triton X-100 for 30 min and stained with DAPI (5 𝜇𝜇g/mL). e stained cells were then analyzed by confocal laser scanning microscope (LSM 510, Carl Zeiss, Göttingen, Germany) with a 100x objective (oil immersion, aperture 1.3). e quanti�cation of �uorescence intensity was analyzed by Image J (US NIH) as described previously [18].
2.10. Structural Comparisons and Modeling. e model of CagA C-terminal was compared with all protein structures available in the DALI server (http://www.ebi.ac.uk/dali/). Structural comparisons with the cholesterol binding protein (Protein Data Bank code: 3IW1) [22], were carried out
using the program Discovery Studio to superimpose C𝛼𝛼 atoms. Combined sequence and secondary structure align-ments, the �gure preparation was done with the program ESPript [23]. Structural �gures were prepared with the program PyMol (http://www.pymol.org). To build the CagA C-terminal region with cholesterol and MAB model, the sub-strate cholesterol and MAB were docked using the program iGEMDOCK, version 2.1 [24]. First, the 3IW1 was chosen as the template for CagA C-terminal modeling. Residues from 645 to 1188 for CagA C-terminal region were modeled by Discovery Studio (http://accelrys.com/products/discovery-studio/). e binding site for docking was determined by con-sidering the protein atoms located ≤10 Å from the binding residues of CagA C-terminal model. e default parameters of iGEMDOCK were used.
2.11. Dot Blot Analysis. Polyvinylidene �uoride (PVDF) membranes (Millipore, Billerica, MA) were prepared and immersed into PBS to eliminate methanol. A series con-centrations of MAB (0, 10, 20, and 50 𝜇𝜇M) and cholesterol (0, 0.5, 1.0, and 2.0 mg/mL) were added onto membranes at the center of grid with vacuums. e membranes were blocked by 3% BSA in PBS for 1 h. RIPA-lysed-Hp (2 𝜇𝜇g/mL) were applied onto the membranes and incubated at 4∘C for
16 h without shaking. e membranes were washed with PBS twice and incubated with goat-anti-CagA antibody (Santa Cruz Biotechnology) at 4∘C for 16 h. e membranes were washed with PBS and then incubated with anti-goat-HRP antibody (Santa Cruz). Aer 1 h incubation, the membranes were washed, and the images were visualized by using Image �uant LAS-4000 (Fuji�lm, Tokyo, Japan). e relative density of images was quanti�ed by using UN-SCAN-IT soware (Silk Scienti�c Corporation, Orem, UT).
2.12. Binding Inhibition Assay. Cholesterol and CagA bind-ing inhibition by MAB was determined by dot blot analysis. RIPA-lysed-Hp (2 𝜇𝜇g/mL) were applied onto the PVDF mem-branes. DMSO, MAB (5 𝜇𝜇M), cholesterol (4 mg/mL), and various ratios of cholesterol : MAB (1 : 1–1 : 8) were added onto the membranes. e membranes were blocked in 3% BSA for 1 h, washed with PBS, and then incubated with 0.1 mg/mL recombinant rat Apo-A1 (Biovision, CA) at 4∘C for 16 h. e membranes were incubated with rabbit Apo-A1 antibody (Biovision), followed by probed with anti-rabbit-HRP antibody (Santa Cruz). Bound cholesterol was visualized by developing the blot and quanti�ed as done for the dot blot.
2.13. Statistical Analysis. e Student’s 𝑡𝑡-test was performed to calculate the statistical signi�cance of experimental results between two groups. 𝑃𝑃 𝑃 𝑃𝑃𝑃5 was considered signi�cant.
3. Results
3.1. MAB Attenuates Hp CagA Translocation and Phosphory-lation. CagA translocation is required in order to exert its effects on host AGS cells, and once within the host cytoplasm CagA can be phosphorylated by tyrosine kinases. erefore,
we �rst sought to determine if MAB treatment could affect the expression and function of Hp CagA phosphorylation and translocation in AGS cells. We utilized wild-type Hp (WT Hp) in this set of experiments. e levels of CagA translocation and tyrosine phosphorylation were analyzed by immunoprecipitation followed by western blot assays. MAB treatment for 6 h dose dependently (10, 20, and 50 𝜇𝜇M) reduced the levels of CagA protein expression compared to untreated cells (Figure 1(a)). Additionally, upon Hp infection the translocation and phosphorylation of CagA (pCagA) were increased to levels higher than those in noninfected cells (Figures 1(b) and 1(c)). However, with the MAB con-centrations of 10, 20, and 50 𝜇𝜇M, the levels of translocated and phosphorylated CagA were dose dependently decreased (Figure 1(a)). In particular, when Hp-infected AGS cells was treated with 50 𝜇𝜇M of MAB, the levels of translocated CagA (Figure 1(b)) and phosphorylated CagA (Figure 1(c)) were reduced to 54.6% and 61.2%, respectively, as compared with nontreated cells. Next, we examined the effect of MAB on Hp-infected AGS cells polarity defect, known as the “hummingbird phenotype.” Induction of this characteristic loss of cell polarity (indicated by pronounced cell elongation) was clearly acquired in Hp-infected AGS cells (Figures 1(d) and 1(e)). In particular, in Hp-infected AGS cells, nearly 34.6% of AGS cells, exhibited the hummingbird phenotype as compared with the nontreated cells (Figures 1(d) and 1(e)). ese data are consistent with the results obtained by western blot, suggesting that p-CagA molecule induces signaling that leads to profound AGS cell elongation. However, before treat-ment of Hp-infected AGS cells with MAB, the proportions of elongated cells were reduced in a dose dependent manner (Figure 1(e)). Speci�cally, the elongated Hp-infected AGS cells were reduced from 34.6% to 13.5% when pretreated with 50 𝜇𝜇M of MAB.
Given the above results, we then directly examined the inhibition of CagA translocation into cytoplasm by MAB using confocal microscopy. As shown in Figure 2, in cultured CagA-EGFP Hp with AGS cells, most of the Hp-associated CagA was delivered into the cells and localized in the cyto-plasm. In contrast, when cells were treated with 50 𝜇𝜇M MAB, it was observed that major portion of CagA accumulated around the cells at sites of Hp infection. e distributions of �uorescence intensity for translocated CagA-EGFP signals in cytoplasm were determined and presented as intensity histograms (Figure 2, right panel). Our data clearly indicate that MAB has the activity to inhibit Hp CagA translocation in AGS cells.
3.2. MAB Inhibits Hp CagA Functions. Furthermore, we sought to identify the signal transduction pathway that might mediate CagA-dependent in�ammation. erefore, we next carried out transient transfection of AGS cells with NF-𝜅𝜅B-luc construct. e transfected AGS cells were treated with MAB and infected with WT Hp or the isogenic CagA-deleted mutant Hp (ΔCagA Hp). Treatment of AGS cells without WT Hp or with MAB alone, the expressions on the induction of NF-𝜅𝜅B activity, were at the basal levels (Figure 3(a)). Infection of AGS cells with WT Hp or ΔCagA Hp showed a notice-able increment in the NF-𝜅𝜅B luciferase activity. However,
pretreatment of AGS cells with 50 𝜇𝜇M MAB reduced the levels of luciferase activity in samples exposed to WT Hp (Figure 3(a)). is phenomenon was minimal in ΔCagA Hp-induced luciferase activity as compared with WT Hp (Figure 3(a)). In line with this, we next examined the effect of MAB in p65 nuclear translocation, which plays an essential role in the regulation of NF-𝜅𝜅B activation [4]. We assessed the nuclear content of p65 NF-𝜅𝜅B in response to WT Hp or ΔCagA mutant Hp alone or preincubation with MAB. e results of immunoblots analysis revealed that incubation of the AGS cells with the WT Hp leads to an increase of p65 NF-𝜅𝜅B in the nuclear extracts as compared with mock treated or MAB alone or ΔCagA Hp treated cells (Figure 3(b)). However, in the presence of preincubation with 50 𝜇𝜇M MAB, the WT Hp-induced nuclear translocation of p65 NF-𝜅𝜅B was signi�cantly diminished (Figure 3(b)). e quantitative data showed that 50 𝜇𝜇M MAB noticeably reduced the WT Hp-induced p65 translocation into the nucleus (Figure 3(b)). Together, these data indicate that MAB suppressed the Hp-induced translocation of p65 NF-𝜅𝜅B into the nucleus followed by attenuation of NF-𝜅𝜅B activation.
3.3. MAB Inhibits Hp CagA-Induced Phosphorylation of I𝜅𝜅B𝛼𝛼 and IL-8 Secretion. As NF-𝜅𝜅B activation requires phospho-rylation and degradation of the inhibitory protein I𝜅𝜅B-𝛼𝛼 [5, 25], we exposed the AGS cells to WT Hp in the absence or presence of MAB, and the cell lysates were analyzed for I𝜅𝜅B𝛼𝛼 phosphorylation. Infection of AGS cells with WT Hp results in the increased level of proteasome target protein phospho-rylation of I𝜅𝜅B𝛼𝛼. As shown in Figure 4(a), the compound MAB and NF-𝜅𝜅B inhibitor (MG132) abolished the increased level of phosphorylated I𝜅𝜅B𝛼𝛼. Additionally, MAB at 50 𝜇𝜇M also attenuated the accumulation of ubiquitinated proteins by Hp CagA in Hp-infected AGS cells (Figure 4(b)). Next, we examined MAB inhibitory effect on Hp-induced AGS cell IL-8 luciferase activity and secretion. As shown in Figure 5(a), parallel to the results obtained from NF-𝜅𝜅B measurements (Figure 3(a)), we observed a similar inhibitory effect on cytokine IL-8 luciferase activity. We also measured IL-8 protein levels in supernatants of AGS cells cocultured with WT Hp at an MOI of 100 for 6 h (Figure 5(b)). Hp-infected AGS cells grown without MAB produced 5316 pg/mL IL-8, a 11.4-fold increase over control cells. When Hp cultured in the presence of 50 𝜇𝜇M MAB were used, AGS cells generated only 3432 pg/mL IL-8, a 46.8% inhibition.
3.4. Structure-Based Virtual Docking of Cholesterol and MAB for CagA Model. Once Hp’s intimate contact with AGS cells is established, the T4SS of Hp further interacts with speci�c host cell surface molecules including cholesterol in lipid ras to facilitate injection of CagA translocation and proin�amma-tory signaling. As the chemical structure of MAB resembles with cholesterol (Figure 6(a)), we hypothesized that MAB may bind with CagA C-terminal domain similar to that of cholesterol. is led us to investigate molecular modeling simulations to examine the interaction of cholesterol and MAB with CagA C-terminal domain. Structural comparisons with CagA and the cholesterol binding protein (Protein Data Bank code number: 3IW1) were then chosen as the
0 10 20 50 CagA Actin p-CagA AGS only Hp H. pylori MAB (µM) (a) C agA t ranslo cat io n (%) 0 20 40 60 80 100 120 0 10 20 50 H. pylori MAB (µM) AGS only ∗∗ ∗∗ (b) 0 20 40 60 80 100 120 0 10 20 50 H. pylori MAB (µM) AGS only ∗ ∗∗ C agA p hosp ho ry lat io n (%) (c) (d) 0 10 20 30 40 50 0 10 20 50 H. pylori MAB (µM) AGS only ∗ ∗∗ ∗∗ H ummingb ir d c el ls (%) (e)
F 1: Methylantcinate B (MAB) in�uences H. pylori (Hp) CagA expression, translocation, and phosphorylation. AGS cells were treated with indicated concentrations of MAB prior infection by Hp at MOI 100 for 6 h. (a) e samples were immunoprecipitated for CagA and subjected to western blot analysis. Actin was represented as an internal control for equal loading. e levels of CagA translocation (b) and CagA phosphorylation (c) were determined by densitometric analysis. (d) e proportions of elongated cells were evaluated aer cells cocultured with Hp and MAB for 6 h. (e) e hummingbird phenotype cells were determined. Results were shown as mean values ± standard
deviations from 3 independent experiments.∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃 and∗∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃 were considered as statistically signi�cant. Arrows in (d) represented
DAPI GFP Merge A GS o nl y C agA -EGFP MAB C agA -EGFP/MAB (a) T ranslo cat ed EGFP/D AP I (%) CagA-EGFP CagA-EGFP + MAB 0 20 40 60 80 100 120 ∗∗ (b)
F 2: Translocation of CagA was attenuated by MAB. AGS cells were treated or untreated with MAB and infected with CagA-EGFP H. pylori at 37∘C for 6 h. Cells were �xed and stained with DAPI (blue) to visualize bacteria and the cell nucleus. Samples were analyzed by
confocal microscopy. e colocalization of GFP with DAPI appears cyan in the overlay to show bacteria (arrows). e translocated CagA-EGFP of green �uorescence signals (arrow heads) were �uanti�ed and normalized with DAPI signals. e data were analyzed using the
Pearson correlation coe�cient and presented in the right panel. Statistical signi�cance was determined for 3 independent experiments.∗∗𝑃𝑃 𝑃
0.01; Bar, 10 𝜇𝜇m.
template for CagA C-terminal region modeling (Figure 6(b)). By using iGEMDOCK, the structural modeling showed that cholesterol binding site of CagA model took the shape of a hydrophobic pocket (Figure 6(c)). Accordingly, as shown in Figure 6(d), a MAB molecule was inserted into the CagA C-terminal domain. Comparing their contact residues between the interaction of cholesterol and MAB with CagA C-terminal domain, the data showed 12 amino acids in the same interaction sites of CagA. e putative binding residues shown in CagA C-terminal region were D761, F762, S763, K764, N889, N890, G891, K932, T1160, G1161, Y1162, and Y1163.
3.5. Interaction of CagA with Cholesterol Was Inhibited by MAB. e structure-based simulations raised the possibility that CagA directly binds to MAB which may compete the interaction of cholesterol with CagA upon Hp infection. To explore this idea, series concentrations of cholesterol and
MAB were prepared and overlaid onto PVDF membranes. As shown in Figure 7, CagA binds to various concentrations of immobilized cholesterol (0.5–2 mg/mL). Similarly, binding of CagA to the immobilized MAB was also shown in a dose-dependent manner (10–50 𝜇𝜇M). e results from the dot blot analysis indicate that both of cholesterol and MAB are able to bind CagA, consistent with the observation in structural simulations.
To further investigate the competitive effect of cholesterol and MAB, the binding inhibition assay was performed. CagA was dotted onto PVDF membranes, and a series ratio of cholesterol and MAB were added (cholesterol : MAB, 1 : 1, 1 : 2, 1 : 4, and 1 : 8). As shown in Figure 8(a), as MAB pro-portion raised, the amount of cholesterol bound to CagA was decreased as expected. e cholesterol signal was �uanti�ed, and the competitive effect dropped signi�cantly from 82.9% to 18.5% (Figure 8(b)). To explore whether MAB has an exclusive inhibitory effect on cholesterol binding to CagA,
0 1 2 3 4 WT MAB + + + + N F-κB l ucif er ase a ct iv it y (f old) − − − − H. pylori ∆CagA ∗∗ (a) WT MAB p65 PCNA + + + + + + + − − − − − − − − − − − ∆CagA WT MAB + + + + + − − − − − − − − − + + − − ∆CagA p65/PCN A (f old) 0 0.5 1 1.5 2 ∗∗ (b)
F 3: Effects of MAB on H. pylori (Hp) CagA-mediated NF-𝜅𝜅B activation and p65 nuclear translocation. e levels of NF-𝜅𝜅B luciferase activity (a) and nuclear p65 (b) were determined in the cell lysates as described in Section 2. Proliferating cell nuclear antigen (PCNA) was used as a loading control for the nuclear fraction of cell lysates. Results are shown as mean values ± standard deviations from 3 independent
e�periments. Statistical signi�cance was determined using the Student�s 𝑡𝑡-test when compared to the mock control.∗∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃 was considered
as statistically signi�cant. �T, wild-type Hp� ΔCagA, cagA mutant Hp.
a series ratio of cholesterol, and antrocin, a sesquiterpene lactone from Ac were added, and the binding inhibition assay was performed. It was found that antrocin does not affect the cholesterol binding to CagA (Figures 8(a) and 8(b)). ese results reveal that MAB may interfere with the interaction between cholesterol and CagA by competitive with the binding site of CagA.
4. Discussion
Although Hp is not an intracellular pathogen, its interac-tions with host cells may provide certain survival bene�ts for Hp and contribute to the establishment of a chronic infection. Indeed, Hp uses host plasma membranes as a site for replication and formation of microcolonies [4]. Additionally, colonization of the epithelial cell membranes by Hp is enhanced when the apoptotic response of AGS cells is impaired. Epidemiological studies reveal that Hp physically interacts with AGS cells and introduces CagA protein into the host cells [4]. CagA is known for its role as inducer of proin�ammatory responses in AGS cells, and as such, it enhances the activation of IL-8 through NF-𝜅𝜅B signaling pathway [3, 5]. e triterpenoid compound MAB has recently reported for its anti-in�ammatory activities in
Hp-infected AGS cells [17]. However, there is no study addressing the detailed protective mechanism of MAB in Hp CagA-induced pathogenesis. For the �rst time, we now provide the mechanism responsible for MAB inhibited Hp-induced in�ammation by directly targeting CagA and this feature is likely by competing the cholesterol interaction with CagA and, thereby, attenuates CagA function in AGS cells.
Current literature indicates that CagA molecules are directly translocated into AGS cells via a Hp T4SS [3, 8]. It is also reported that CagA is rapidly tyrosine phosphorylated aer its injection into host AGS cells and mimics a host cell factor for the activation or inactivation of some speci�c intra-cellular signaling pathways [3]. Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in the C-terminal region of CagA protein have been identi�ed as phosphorylation sites using site-directed muta-genesis of injected and/or transfected CagA [3]. Increased CagA signal intensity could result from increased amounts of injected CagA molecules undergoing phosphorylation at a speci�c site and/or by increased phosphorylation of multiple sites per CagA molecule. Accordingly, in this study we found that the compound MAB dose-dependently reduced the CagA translocation and phosphorylation (Figures 1(a)– 1(c)). e most striking Hp-induced morphological change to host AGS cells is the induction of an elongated cell
0 0.5 1 1.5 2 MAB p-I κB-α/a ct in (f old) ∗∗ − + + − − − − + − + + − − + + − − − MG132 H. pylori MAB MG132 Actin − − + + − − + − − − + + + − − − + − H. pylori p-IκB-α (a) Ubn-Prs Actin 100 48 (KDa) MAB − + + − − − − + − + + − − + + − − − MG132 H. pylori (b)
F 4: MAB prevents H. pylori (Hp) CagA-induced I𝜅𝜅B-𝛼𝛼 phosphorylation and protein ubiquitination. AGS cells were treated with MAB prior infection by Hp at MOI 100 for 24 h. (a) e cell lysates were subjected to western blot analysis. e levels of I𝜅𝜅B-𝛼𝛼 phosphorylation were determined by densitometric analysis. (b) e levels of protein ubiquitination were detected by western blot using a speci�c antibody
to ubiquitin. Molecular mass markers (KDa) are shown on the le. Actin was represented as an internal control for equal loading.∗∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃
was considered as statistically signi�cant. p-I𝜅𝜅B-𝛼𝛼, phosphorylated I𝜅𝜅B-𝛼𝛼; Ubn-Prs, ubiquitinated proteins.
0 1 2 3 4 IL -8 l ucif er ase a ct iv it y (f old) + − ∗∗ H. pylori MAB− MAB + (a) + − ∗∗ H. pylori MAB− MAB + 0 1000 2000 3000 4000 5000 6000 IL -8 se cr et io n (p g/mL) (b)
F 5: MAB attenuates H. pylori-induced IL-8 activity. AGS cells were infected with H. pylori in the absence or presence of MAB for
6 h. Cell lysates and culture supernatants were prepared for IL-8 luciferase activities (a) and IL-8 secretions (b), respectively.∗∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃 was
Cholesterol MAB CH3 CH 3 CH3 CH3 COOCH3 O O O HO H3C (a) (b) (c) (d)
F 6: Structural comparisons and modeling simulations for cholesterol and MAB binding to CagA. (a) Chemical structures of cholesterol and triterpenoid MAB. (b) e overall structure of CagA C-terminal domain colored in green and cholesterol binding protein (PDB code: 3IW1) colored in cyan. (c) e model of CagA C-terminal region with cholesterol (stick, colored in yellow). (d) e model of CagA C-terminal domain with MAB (stick, colored in magenta). e numbers of amino acids shown in CagA were directly contacted with the cholesterol (c) or MAB (d) binding sites.
phenotype termed hummingbird [1]. is phenotype results from two successive events: the induction of cell scattering and cell elongation. e induction of early cell motility mainly depends on a CagA-dependent T4SS factor and cell elongation is clearly triggered by phosphorylated CagA. is phenotype may impact pathogenesis by in�uencing immune responses, wound healing, metastasis, or invasive growth of cancer cells in vivo [26, 27]. In this study, MAB dose-dependently suppressed the number of elongated cells (Fig-ure 1(d)). Taken together, this data indicate that expos(Fig-ure to MAB affects the CagA expression, translocation, and leading to a reduction in the amount of CagA phosphorylation followed by reducing the hummingbird phenotype of AGS cells.
Next, a hallmark of Hp infection is increased chronic in�ammation. Previous reports have been demonstrated that
CagA is an important determinant for the induction of tran-scription factor NF-𝜅𝜅B and this induced NF-𝜅𝜅B has a dom-inant role in IL-8 production in AGS cells [5]. e induced NF-𝜅𝜅B can be activated by phosphorylation via different signaling pathways leading to subsequent proteolytic degra-dation of I𝜅𝜅B [3]. Additionally, activated NF-𝜅𝜅B translocates to the nucleus where it upregulates IL-8 gene transcription [4, 5]. erefore, the inhibition of NF-𝜅𝜅B signaling is an ideal strategy for eradication of Hp infection. In this study, MAB suppressed the 𝜅𝜅B activation, translocation of p65 NF-𝜅𝜅B into the nucleus, and phosphorylation of INF-𝜅𝜅B-𝛼𝛼 (Figures 2–4). us, these results indicate that MAB attenuated Hp CagA-induced in�ammation through the NF-𝜅𝜅B-mediated signaling pathway. On the other hand, they are consistent with previous report that constitutive activation of NF-𝜅𝜅B was thought to be associated with progression of several
MAB Cholesterol 0 0.5 1 2 0 10 20 50 (mg/mL) (µM) (a) B ound C agA (f old) 0 0.2 0.4 0.6 0.8 1 1.2 2 1 0.5 0 Cholesterol (mg/mL) ∗∗ ∗∗ ∗∗ (b) 50 20 10 0 B ound C agA (f old) 0 0.2 0.4 0.6 0.8 1 1.2 ∗∗ ∗∗ ∗ MAB (µM) (c)
F 7: Binding of CagA to the immobilized cholesterol and MAB. (a) Representative dot blot that shows binding of CagA to various concentrations of cholesterol and MAB. e binding activities of CagA to the immobilized cholesterol (b) and MAB (c) were quanti�ed by
densitometric analysis with 3 independent experiments.∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃;∗∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃 compared to each highest concentration group, as determined
by Student’s 𝑡𝑡-test. Chol, cholesterol.
tumor-cell types [1]. Here, we showed that MAB not only suppresses Hp CagA-induced NF-𝜅𝜅B activation but also blocks p65 nuclear translocation (Figure 3), implicating that MAB harbors anticancer activity. ese results may provide molecular evidence for our previous report that MAB displayed cytotoxicity against various cancer cell types [16]. On the other hand, it is known that the expression of IL-8 in response to Hp infection is a key cause of Hp-induced in�ammation and IL-8 is regulated by NF-𝜅𝜅B [5, 25]. In this stydy, MAB suppressed the IL-8 luciferase activity as well as its secretion (Figure 5), indicating that the immunos-timulatory effect of the Hp was lessened by exposure to MAB. Collectively, these data indicate that pretreatment of AGS cells with MAB inhibited the Hp CagA-induced in�ammatory response.
Previously it is reported that the natural triterpenoids possess potent serum cholesterol level reduced activities [15, 28, 29], indicating that triterpenoids have the potential in
antihyperlipidaemia through modulation of cholesterol syn-thesis. Additionally, a previous study demonstrated that Ac can downregulate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a key enzyme for cholesterol synthesis [30]. However, in this study, our preliminary data showed that MAB which was isolated from Ac did not alter the level of cellular cholesterol (data not shown). is evidence indicated that MAB-attenuated CagA function was not through the depletion of cholesterol in membrane ras.
Several studies have been found that CagA C-terminal domain containing EPIYA motif in response to CagA targeted membrane ras [10, 31]. Our recent report showed that tethering of CagA to cholesterol-enriched membrane microdomains is important for IL-8 induction in AGS cells [31]. Modulation of cellular cholesterol levels alter the partitioning of CagA into membrane ras [9, 11, 13], indicating that CagA may interact with membrane cholesterol. Analysis of amino acid sequence of CagA
MAB Antrocin Mock Chol Chol : antrocin Chol : MAB 1 : 1 1 : 2 1 : 4 1 : 8 1 : 1 1 : 2 1 : 4 1 : 8 (a) Sig nal o f c holest er ol (%) 0 20 40 60 80 100 120 Chol : antrocin Chol : MAB Mix proportion 1 : 1 1 : 2 1 : 4 1 : 8 ∗∗ ∗∗ ∗∗ (b)
F 8: Binding of cholesterol to CagA was inhibited with increased levels of MAB. (a) A series of dot blots with various ratios of cholesterol : antrocin and cholesterol : MAB (1 : 1 to 1 : 8) were overlaid to the immobilized CagA and detected by antibody as described in
Section 2. (b) e binding inhibition analyses were �uanti�ed and the results were representative as 3 independent experiments.∗∗𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃
was considered as statistically signi�cant. Chol, cholesterol. revealed that some cholesterol-recognition/interaction-amino-acid-consensus-pattern- (CRAC-) like regions were represented in C-terminal domain (GenBank accession number: AE000511.1). e CRAC region contains the conserved motif L/V(X)1-5Y(X)1-5R/K which may contribute to the association of protein with cholesterol [32]. We then carried out the docking simulations using iGEMDOCK (version 2.1) [24]. Our results indicated that the cholesterol and MAB similarly interacted with CagA (Figure 6). Moreover, the same contact regions of 12 amino acids were localized in the C-terminal domain of CagA (Figures 6(c) and 6(d)). ese results support our hypothesis that the direct binding of MAB to CagA lead to preventing cholesterol from interaction with CagA.
In conclusion, we have shown that a triterpenoid MAB attenuated the Hp-induced in�ammatory responses by com-peting the binding of cholesterol with CagA. We also demon-strated that CagA-induced pathogenesis was inhibited by treatment of cells with MAB. us, MAB may have a scope to develop as potential candidate against Hp CagA-mediated pathogenesis. However, further studies are necessary to investigate the importance of these �ndings in vivo.
�on��ct of �nterests
e authors declare that there is no potential con�ict of interests.
Acknowledgments
is work was supported by the National Science Coun-cil of Taiwan (NSC 100-2113-M-324-001-MY3, 101-2811-M-324-001, and 101-2313-B-039-004-MY3), China Medi-cal University (CMU101-S-13), CliniMedi-cal Trial and Research
Center of Excellence Funds (DOH101-TD-B-111-004) from the Taiwanese Department of Health, and the Tomorrow Medicine Foundation. e authors thank Shu-Chen Shen (Scienti�c Instrument Center, Academia Sinica) for confocal microscopy analysis.
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