Cf-GP was purchased in 2006 in Korea. C. fulvescens powder (40 g) was diluted in 1 liter water and stirred for 3 h at 80°C using a heating mantle, followed by centrifugation at 1,500 × g for 15 min at 4°C.

Three volumes of 95% ethanol were added and precipitate was removed by vacuum filtration. To the supernatants, 80% ammonium sulfate was added, followed by stirring for 24 h. Next, salt was removed by membrane dialysis (Por Membrane MW 3,500 Da, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) for 1 day at 4°C. The concentrated solution was aliquoted into 1.5 ml tubes and stored at −70°C until use. These samples are hereafter termed Cf-GP.

Human gastric cancer AGS cells (American Type Culture Collection, Manassas, VA, USA) were maintained at 37°C in a 5% CO₂ humidified atmosphere. Cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/l penicillin and 100 mg/l streptomycin. Cells were cultured to 60–80% confluence in 100-mm diameter dishes. The medium was replaced every day.

AGS cell proliferation was measured using a CellTiter 96^® aqueous non-radioactivity cell proliferation assay (Promega Corporation, Madison, WI, USA). The assay is based on the cleavage of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) into a formazan product that is soluble in tissue culture media. Cells were seeded onto 96-well plates at 2×10⁴ cells/well in 100 μl medium. Cells were maintained for 24 h and the medium was then replaced with serum-free medium (SFM). Following 24 h, the medium was replaced with SFM containing Cf-GP (0, 5, 10 or 20 μg/ml) for 24 h. Cells were then incubated with MTS solution for 30 min at 37°C. Cell proliferation was measured by means of absorbance at 490 nm using the Benchmark enzyme-linked immunosorbent assay (ELISA) plate reader (Bio-Rad, Hercules, CA, USA).

Cell invasion was measured using an 8.0-μm pore size insert in Transwell^® plates (Corning Costar Inc., Corning, NY, USA). AGS cells were seeded into the upper chamber (Matrigel coated; Corning Costar Inc.) and maintained for 24 h. The medium was then replaced with SFM containing Cf-GP (0, 5, 10 or 20 μg/ml) for 24 h. The lower chamber was maintained with 10% FBS. Following 24 h, the cut on the bottom of the filter (Matrigel coated) was stained with hematoxylin. Stained cells were calculated by extrapolation from the number counted.

Using a voltohmmeter (EVOM Epithelial Tissue Voltohmmeter; World Precision Instruments, Sarasota, FL, USA), TEER was measured. AGS cells were seeded into the upper chamber (Matrigel coated) of the transwell plates and maintained for 24 h. Next, SFM was replaced with medium containing Cf-GP (0, 5, 10 or 20 μg/ml) for 24 h. The lower chamber was maintained with 10% FBS. Following 24 h, the upper chamber was separated and TEER values were determined using the voltohmmeter.

TEER was calculated using the following formula: TEER [(resistance (Ω cm²)] = (Ω - background Ω) × membrane area (cm²); background resistance was 14 and the membrane area was 1.54 cm². The change in TEER values for each insert was calculated using the following formula: change in TEER (%) = TEER (Ω cm²)/initial TEER (Ω cm²) - 100.

AGS cells were seeded into six-well plates at 2×10⁴ cells/well in 2 ml medium. Cells were incubated for 24 h and the medium was replaced with SFM. Following 24 h, the medium was replaced again with SFM containing Cf-GP (0, 5, 10 or 20 μg/ml) for 24 h. Cells were treated with the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and the RNA extracted was quantified using Oligo(dT) primers (Intron Biotechnology Co. Ltd., Seongnam, Korea); the corresponding cDNA was then synthesized. cDNA was subjected to amplification using a PCR kit (dNTP mix, 10X Ex Taq Buffer and Ex Taq; Takara Bio, Inc., Shiga, Japan) with primers (Table I) in 0.1% diethylpyrocarbonate water. PCR products were resolved on 1% agarose gels. Gels were stained with 10 mg/ml ethidium bromide to visualize amplification products.

AGS cells were cultured in 100-mm diameter dishes. Cells were cultured to 60–80% confluence and the medium was replaced with SFM for 4 h. Next, the medium was replaced with fresh SFM containing Cf-GP (0, 5, 10 or 20 μg/ml) for 24 h. Cells were washed with phosphate-buffered saline and added to lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% NP-40, 1 mM NaF, 1 mM Na₃VO₄, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 0.25% Na-deoxycholate and 1 mM PMSF]. Lysates were separated using 10–15% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 1% bovine serum albumin in TBS-T [10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% Tween 20] at room temperature and incubated with agitation with specific antibodies: anti-claudin-1, −2, −3 and −4 (1:1,000), anti-β-catenin (1:1,000), anti-E-cadherin (1:1,000), anti-MMP-2 and −9 (1:1,000) and anti-tissue inhibitors of metalloproteinases-1 (TIMP-1; 1:1,000; all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The secondary peroxidase-conjugated goat, mouse and rabbit antibodies (1:10,000) were purchased from GE Healthcare Bio-Sciences (Piscataway, NJ, USA). Bands were visualized using Super Signal West Pico Stable Peroxide solution and the Super Signal West Pico Luminol/Enhancer solution (Pierce Biotechnology, Inc., Rockford, IL, USA) and developed using Kodak X-ray film (Eastman Kodak Company, Rochester, NY, USA).

AGS cells were cultured in six-well plates to 60–80% confluence and the medium was replaced with SFM for 4 h. Next, the medium was replaced with fresh SFM containing Cf-GP (0, 5, 10 or 20 μg/ml) for 24 h. The obtained conditioned medium was loaded in 10% SDS-free acrylamide gels with 0.1% gelatin. The completed loading gel was treated with 2.5% Triton X-100 and incubated under agitation for 30 min, followed by incubation with developing buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂ and 1 μM ZnCl₂; pH 7.5) at 37°C for 2 days. The gel was fixed (7% acetic acid) and stained (0.5% Coomassie Brilliant Blue 250 in dilute fixing solution).

Data are presented as the mean ± SD and were calculated using SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA). Data were validated by analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference and was determined by Duncan's multiple range test for group comparisons.

MTS assays were used to investigate the effect of Cf-GP (0, 5, 10 or 20 μg/ml) on AGS cell proliferation. Cf-GP at 20 μg/ml resulted in a 50% decrease in proliferation (Fig. 1). In addition, Cf-GP inhibited AGS cell growth in a dose-dependent manner. We previously reported that Cf-GP induced proliferation of human intestinal epithelial IEC-6 cells (17).

In general, increased proliferation of cancer cells to other tissues is a result of accelerated invasion. Since Cf-GP inhibited AGS cell growth, the effect of Cf-GP on invasion was investigated using hematoxylin staining (Fig. 2A). As demonstrated in Fig. 2B, the Cf-GP treatment group exhibited decreased cell invasion compared with control cells. Treatment with 20 μg/ml Cf-GP led to a 50% reduction compared with the control group.

Increased cell membrane permeability is mediated by weakening of TEER. We thus evaluated the effect of Cf-GP on AGS cell invasion by measuring TEER. Decreases of TEER values allowed easier penetration of intracellular material. Normal cells indicate higher TEER values by the strong electrical resistance of the cell membrane. However, MMP activity induced degradation of cell membranes and permeability in cancer cells. Therefore, cancer cells have lower TEER values and this increases invasion. In the present study, TEER values in AGS cells were increased upon Cf-GP treatment in a dose-dependent manner (Fig. 3).

The increased membrane permeability in cancer cells is due to mutations in a variety of genes associated with TJ proteins. Typically, overexpression of TJ proteins (claudin, zo-1 and occludin) and TJ-related proteins (β-catenin) is observed in cancer cells. In addition, β-catenin levels are increased due to loss of E-cadherin. Therefore, β-catenin increases cancer cell adhesion. In the present study, protein and mRNA levels were determined by western blot analysis and RT-PCR, respectively. As demonstrated in Fig. 4A and B, in control cells, expression levels of the TJ proteins, claudin, zo-1 and occludin increased, but were reduced in a dose-dependent manner upon Cf-GP treatment. In addition, β-catenin levels were reduced while that of the inhibitor of β-catenin, E-cadherin, was increased by Cf-GP treatment at the protein and mRNA levels (Fig. 4C and D).

The interaction between cancer cells and the basement membrane is a major step in metastasis and invasion (18). The basement membrane is composed of substances, including collagen and lamin. Cell membrane degradation by MMP proteins and cancer cells is an important process for invasion and metastasis. In particular, MMP-2 and MMP-9 are important mediators of basement membrane degradation of gelatin and factors involved in angiogenesis and cancer cell invasion are known to induce MMP-2 and −9 (19). Thus, inhibition of MMP-2 and −9 is essential for preventing basement membrane destruction. MMP proteins were associated with TJ proteins and upregulation of TJ proteins induced MMP activation in cancer cells. MMP activity leads to cell invasion and metastasis. In general, normal cells have tissue inhibitors of metalloproteinases (TIMPs). Therefore MMP activation and degradation of cell basement membrane are inhibited. However, cancer cells have constant proliferation and invasion by upregulation of TJ proteins and do not have TIMPs (20). In our previous study, expression of TJ proteins was confirmed. Therefore, MMP and TIMP levels were evaluated in AGS cells following treatment with Cf-GP. MMP-2 and −9 levels were decreased due to increased TIMP-1 as determined by western blot analysis and RT-PCR (Fig. 5A and B). In addition, gelatin zymography assays indicated that MMP protein levels decreased with increasing Cf-GP concentrations (Fig. 5C).