Human gastric carcinoma cell line, MKN-45, was obtained from American Type Culture Collection (Manassas, VA, USA). MKN-45 cells were maintained in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. On the day of experiment, cells were refed with fresh medium and co-cultured with H pylori.

Cag pathogenicity island-positive H pylori (ATCC 43504) strain was used in experiments described in this study. Stock cultures were maintained at -70 °C in brucella broth supplemented with 30% glycerol. The bacteria were grown at 37 °C in 5% horse blood agar plates and in a microaerobic condition. Cultures were routinely screened for urease activity. For co-infection studies, H pylori were harvested between 48 and 72 h after inoculation of agar plates, resuspended in sterile phosphate buffered saline (PBS), and enumerated by absorbance at 600 nm (1 optical density (A) at 600 nm = 2.4×10⁸ colony-forming units/mL). MKN-45 cells were seeded into 65 mm dishes, and H pylori at a multiplicity of infection 80 were added in the culture.

The effect of H pylori infection on cell migration was examined using the Matrigel Invasion chamber, as suggested by the manufacturer (BD Bioscience). The lower surface of the chamber contained a transwell filter (8-μm pores), coated with fibronectin, and vitronectin. MKN 45 cells (1×10⁵) and H pylori were added to the upper chamber, in the presence or absence of NS-398, and incubated overnight in a humidified tissue culture incubator, at 37 °C, 50 mL/L CO₂ atmosphere. The next day, a cotton tipped swab was inserted into chambers to remove non-invading cells by applying gentle but firm pressure while moving the tip around the membrane surface. The cells on the lower surface of insert chambers were stained with hematoxylin for 10 min, and the cell number was counted under a microscope (40 to 200×magnifications). The extent of cell invasion was expressed as fold increases of total number of cells on the lower surface of chambers in treated over untreated samples.

To obtain whole-cell extracts, cells were washed twice with ice-cold phosphate-buffer saline (PBS), and pelleted by centrifugation (200 r/min). Cell pellets were then lysed in a standard RIPA buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS), containing protease inhibitors. Protein concentrations were determined by Bio-Rad assays. Protein samples were dissolved in the loading buffer (60 mmol/L Tris-HCl, pH 6.8, 2% SDS, 100 mmol/L dithiothreitol, and 0.01% bromophenol blue), heated to 100 °C for 3 min, and loaded onto the gel in an electrophoresis buffer containing 25 mmol/L Tris-HCl, pH 8.3, 250 mmol/L glycine, and 0.1% SDS. At the completion of electrophoresis, protein was transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Life Science). The membrane was incubated in the blocking buffer (10 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20), containing 5% nonfat powdered milk for 2 h. The membrane was immunoblotted with COX-2, MMP-9, or VEGF antiserum (obtained from Santa Cruz Biotech, Santa Cruz, CA, USA). After incubation with the secondary antibody, the membrane was visualized with Enhanced Chemiluminescence kit from Amersham.

To examine transcriptional regulation of COX-2 promoters by NF-κB, MKN-45 cells were transiently transfected with pMT2-LacZ and COX-2-Luc or phPES2 (KBM)-Luc DNAs, in the presence of NF-κB p65, p50, or control pMT2 plasmid (kindly supplied by Dr. Gail Sonenshein, Boston Medical Center, Boston, MA, USA). The phPES2 (KBM) plasmid contains a full-length COX-2 promoter in which a putative NF-κB binding site is mutated, as described previously[16]. For luciferase assays, cells were washed twice with PBS and then lysed in 500 μL of lysis buffer following the manufacturer’s instructions (Analytical Luminescence, San Diego, CA, USA). To assay luciferase activity, cell lysate (100 μL) was mixed with 100 μL of luciferase substrate solution A (Analytical Luminescence). Using a luminometer with automatic injection, 100 μL of luciferase solution B was added (Analytical Luminescence) and luciferase activity was detected as the light emission over a 30-s period.

The β-galactosidase activity in 40 μL of the cell lysate was determined after a 5-30-min incubation at 37 °C with 2 mmol/L chlorophenol red β-galactopyranoside (Boehringer Mannheim) in 2 mmol/L MgCl₂, 0.1 mmol/L MnCl₂, 45 mmol/L 2-mercaptoethanol, and 100 mmol/L NaHPO₄, pH 8.0. The reactions were terminated by adding 500 μL of 0.5 mol/L EDTA, pH 8.0, and the absorbance at 570 nm was measured using a spectrophotometer. With each experiment, the luciferase activity was determined in duplicate and normalized to the β-galactosidase activity for each dish.

Nuclear extracts were prepared using Nuclear Extract kit (Active Motif, Carlsbad, CA, USA). A double-strand oligonucleotide (5’-AGTTGAGGGGACTTTCCCAGGC-3’), corresponding to the putative NF-κB binding domain on the COX-2 promoter was synthesized and was labeled with [γ-³²P] ATP (3000 Ci/mmol at 10 mCi/mL) using a T4 polynucleotide kinase. Nuclear protein (1 μg) was incubated in a buffer containing 20% glycerol, 5 mmol/L MgCl₂, 2.5 mmol/L EDTA, 2.5 mmol/L DTT, 250 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 0.25 mg/mL poly (dl-dC)·poly (dl-dC), for 10 min at room temperature, and ³²P-labeled NF-κB oligo was added to each reaction and incubated for additional 20 min. Samples were subjected to electrophoresis at room temperature on a 4% acrylamide gel at 25 mA using 0.5×TBE buffer. The gels were dried at 80 °C for 2 h and exposed to radiography film at -70 °C.

To further investigate the role of MMP-9 and VEGF in gastric cancer invasion, the expression of these two proteins were examined in H pylori-positive gastric cancer tissues. Tissue sections from gastric cancer were de-paraffinized, rehydrated, and immersed in 3% hydrogen peroxide–methanol solution for 10 min at room temperature to inhibit endogenous peroxidase activity. The sections were then incubated with unmasking solution (0.01 mol/L citrate buffer pH 6.0), heated for 10 min. The sections were allowed to cool down to room temperature and washed twice in PBS buffer for 5 min. The sections were pre-incubated with diluted normal serum for 10 min, and then incubated with 1:40 mouse anti-MMP-9 monoclonal antibody (Novocastra Lab. Ltd, Newcastle, UK), 1:100 rabbit anti-VEGF polyclonal antibody (Zymed Lab. Inc., South San Francisco, CA, USA), or 1:40 mouse anti-COX-2 monoclonal antibody (Cayman Chemical Co., Ann Arbor, MI, USA) for 1 h at room temperature. The sections were washed in PBS for 5 min twice, and incubated with appropriate biotinylated secondary antibody. After washing in PBS for 5 min twice, the slides were incubated with ABC reagents, followed with DAB or other suitable peroxidase substrates. The sections were counterstained with hematoxylin for 30 s, washed, dried, and mounted.

In vivo tumor invasion includes not only migration process, but also adhesion, proliferation and angiogenesis, etc., dissociation of in vitro assay for tumor cell invasion (migration) with in vivo tumor invasion might exist. However, similar migration assay to assess tumor cell invasion has been used by several articles published recently[17].

We used Matrigel invasion chamber to examine whether H pylori infection induces gastric cancer cells invasion and to determine the effect of COX-2 inhibitor on this process. MKN-45 cells were incubated with H pylori in the presence or absence of COX-2 inhibitor, NS-398 (100 ng/mL). As illustrated in Figure 1A co-infection with H pylori induced a 2.5 fold increase in MKN-45 cells migrated through Matrigel-coated filters, indicating that H pylori promoted gastric cancer cells invasion. The COX-2 inhibitor, NS398, significantly reduced cell invasion in H pylori stimulated, but not in untreated cells. This result suggested that H pylori induced gastric cancer cells invasion was, in part, mediated through a COX-2-dependent mechanism.

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Figure 1 Effects of H pylori infection, a cox-2 (NS398), or a NF-κB inhibitor (PDTC) on gastric cancer cell invasion. A: MKN-45 cells were treated with H pylori in the presence or absence of NS398. Cells on the lower surface of insert chamber were stained with hematoxylin for 10 min and counted under microscope with 200× magnifications. Data are presented as mean±SD of three separate experiments (P<0.05); B: MKN-45 cells were treated with H pylori in the presence or absence of PDTC (P<0.05); microscopic photos of stained migration cells: C: control; D: with H pylori; E: with H pylori and NS-398; F: control; G: with H pylori; H: with H pylori and PDTC.

To examine the effect of NF-κB on the migration of MKN-45 cells, cells were incubated with H pylori in the presence or absence of NF-κB inhibitor, (pyrrolidine dithiocarbamate (PDTC), 0.1 μmol/L, purchased from Sigma Chem, St. Louis, MO, USA). As illustrated in Figure 1B the effect of H pylori infection on gastric cancer cells migration was completely abolished by NF-κB inhibitor, suggesting a potential involvement NF-κB in this process. The representative microscopic photos of stained cells treated with H pylori in the presence or absence of COX-2 inhibitor were shown in Figures 1C-1E.

Several proteins, including MMP-9 and VEGF, have been reported to play an important role in tumor invasion. The effects of H pylori on the expression level of MMP-9 and VEGF were examined in MKN 45 cells after co-infection with H pylori for a different period of time. The induction of VEGF protein expression was noticeable within an hour after H pylori infection and reached the highest level in 24 h (Figure 2A). Although the increment of MMP-9 level after H pylori infection was smaller than that of VEGF, a significant increase was observed at 24 h (Figure 2A).

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Figure 2 H pylori infection increases MMP-9, VEGF, and COX-2 expressions in gastric epithelial cell. MKN-45 gastric cancer cells were incubated with H pylori for 0-24 h and total cellular protein was extracted for Western blot analysis for the expression of A: MMP-9 and VEGF and B: COX-2 proteins. The blots were stripped and probed with β-actin to document equal protein loading. The experiment was performed for thrice with similar results.

Recent studies showed that COX-2 inhibitor reduced the release of MMP and COX-2-induced MMP-9 expression[18,19]. Therefore, we examined whether the activation of MMP-9 and VEGF by H pylori infection was also dependent on COX-2 expression. The MKN-45 cells were co-cultured with H pylori for 24 h in the presence or absence of a COX-2 inhibitor, NS-398, and our results showed that co-culture with H pylori resulted in a time-dependent increase in COX-2 protein concentration in MKN-45 cells (Figure 2B). Moreover, NS398 significantly reduced the expression levels of COX-2, MMP-9, and VEGF, induced by H pylori in MKN-45 cells at 24 h (Figure 3A). These data suggest that the induction of MMP-9 and VEGF by H pylori is mediated through a COX-2-dependent mechanism.

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Figure 3 Effect of COX-2 or NF-κB inhibitor on COX-2, MMP-9, or VEGF protein level in MKN-45 cells. A: Cells were cultured in the presence or absence of H pylori and NS398 for 24 h, and cellular protein was extracted and subjected to Western blot analysis; B: MKN-45 cells were treated with or without PDTC and in the presence or absence of H pylori for 24 h. The experiments were repeated on at least three occasions, and the results were identical to these presented here. All blots were stripped and probed with β-actin to check for equal protein loading.

To examine whether activations of MMP-9 and VEGF by H pylori infection were also dependent on NF-κB expression, MKN-45 cells were co-cultured with H pylori for 24 h in the presence or absence of a NF-κB inhibitor, PDTC. As demonstrated in Figure 3B, the effects of H pylori on COX-2, MMP-9, and VEGF expression were significantly reduced by PDTC. These results suggest that the induction of COX-2, MMP-9, and VEGF by H pylori is also NF-κB-dependent.

A previous study has shown that NF-κB regulated COX-2 expression and affected cell proliferation in human gastric cancer cells[14]. We hypothesize that the induction of MMP-9 and VEGF by H pylori is associated with NF-κB mediated COX-2 expression. To investigate this hypothesis, we first examined the effect of NF-κB on COX-2 promoter activities in MKN45 cells. As illustrated in Figure 4A co-transfection with NF-κB p65 or p50 plasmid DNA significantly enhanced COX-2 promoter activity, and the induction was completely abolished in phPES2 (KBM) construct where a putative NF-κB binding domain was mutated. Furthermore, the interaction between NF-κB and COX-2 promoter was also enhanced by H pylori infection in MKN-45 cells (Figure 4B).

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Figure 4 A: Transactivation of COX-2 promoter by NF-κB. MKN-45 cells were transiently transfected with full-length COX-2 promoter (Black bar), or mutated COX-2 construct, phPES2 (KBM), where the putative NF-κB binding domain was mutated (open bar), in the presence of p65, p50, or control vector (pMT2) plasmid. Luciferase and β-galactosidase activities were performed 48 h after transfection. (n = 4, P<0.05); B: Binding of nuclear NF-κB to COX-2 promoter DNA in MKN-45 cells. Nuclear protein was extracted from cells cultured in the absence (lane 2), or presence (lane 3) of H pylori. The protein-DNA interaction bands were shown as (→). Lane 1 showed free probe only.

To investigate whether these observations were also present in vivo, we randomly selected six gastric cancer patients (three cases with H pylori infection, confirmed by Giemsa stain and CLO test; three cases without H pylori infection) and examined the expression of COX-2, MMP-9, or VEGF protein in the surgical specimens. The immunostain of COX-2 (Figures 5A and 5B), MMP-9 (Figures 5C and 5D), or VEGF (Figures 5E and 5F) was located predominantly on the surface epithelial cells, and the intensity was more abundant in H pylori-positive than H pylori-negative tissue samples (Figure 5).

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Figure 5 Immunohistochemical detection of COX-2, MMP-9, and VEGF in gastric cancer tissues. The serial sections of gastric cancer surgical specimens were stained with anti-COX-2 (A and B), anti-MMP-9 (C and D), and anti-VEGF (E and F) antibodies. Sections were counterstained with hematoxylin. The immunoreactivities of COX-2, MMP-9 and VEGF are predominantly detected in the cytoplasm of the tumor cell. Tissues in A, C, and E are from H pylori-positive patients, whereas, sections in B, D, and F are from H pylori-negative individuals. (Magnification: A-F: 400×).