Anti-HB-EGF-C antibody was used to recognize HB-EGF-C, as previously described , and anti-HB-EGF antibody (R&D Systems, Minneapolis, MN) was used to recognize the proHB-EGF ectodomain. Cetuximab ((Merk, Darmstadt, Germany)), a monoclonal antibody to EGFR, was used to inhibit EGFR phosphorylation.
Immunohistochemical staining was performed using anti-HB-EGF antibody and anti-HB-EGF-C antibody. Consecutive 4-μm-thick sections were deparaffinized and hydrated through a graded series of alcohols. After inhibiting endogenous peroxidase activity by immersion in a 3 % H2O2/methanol solution, antigen retrieval was achieved by heating the samples in 10 mM citrate buffer (pH 6.0) in a microwave oven for 10 min at 98 °C. Next, sections were incubated overnight with primary antibodies. After thorough washing in PBS (−), the samples were incubated with biotinylated secondary antibodies and then with avidin-biotin-horseradish peroxidase complexes (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Finally, immune complexes were visualized by incubation in 0.01 % H2O2 and 0.05 % 3,3′-diaminobenzidine tetrachloride. Nuclear counterstaining was accomplished with Mayer’s hematoxylin.
Clinical patients and assessment
Using information from a computerized database, we obtained gastric cancer specimens from 96 patients who had undergone surgical operations for gastric adenocarcinoma between January 2002 and December 2006 at the Nagoya City University Hospital. Written general consent that included research uses of clinical data had been obtained from all patients. The study was performed in accordance with the Declaration of Helsinki and Japanese ethical guidelines for epidemiological research. We obtained an institutional review board (IRB) waiver to conduct this study from the chairperson of the IRB. All 96 patients were histologically diagnosed with gastric cancer. Clinical stage was determined according to the Seventh edition of the UICC-TNM classification . All immunostained specimens were assessed by 2 investigators who were blinded to all clinical information. When more than 10 % of the cancer cells in each section were stained for both anti-HB-EGF antibody and anti-HB-EGF-C antibody, immunostaining was defined as positive.
We used KATO III and MKN45 gastric cancer cell line (Japan Health Science Research Resources Bank, Tokyo, Japan) in our investigation. KATO III and MKN45 cells were maintained in RPMI1640 (Sigma-Aldrich Co., St. Louis, MO) medium that was supplemented with 10 % fetal bovine serum (FBS). Cells were cultured at 37 °C in 5 % CO2 humidified air.
cDNAs encoding wild-type HB-EGF (wt-HB-EGF) and the mutant that carry a point mutation at K201A of the C-terminus (HB-EGF-mC)  were subcloned into pME18SIII with hygromycin-resistance gene. As previously shown, HB-EGF-mC does not translocate from the plasma membrane to the nucleus after TPA stimulation. All cDNA constructs were verified by DNA sequencing using a CEQ 8000 DNA Analysis System (Beckman Coulter, Brea, CA).
The KATO III human gastric cancer cell line was transfected with wt-HB-EGF (KATO III/wt-HB-EGF) and HB-EGF-mC (KATOIII/HB-EGF-mC) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions, and stably transfected clones were isolated using hygromycin B (Invitrogen). As a control, KATO III was transfected with an empty plasmid (KATO III/mock). As well as KATO III cells, MKN45/wt-HB-EGF, MKN45/HB-EGF-mC and MKN45/mock cells were established. These cells were maintained in RPMI1640 medium that was supplemented with 10 % FBS and 800 μg/mL hygromycin B.
Samples were fixed with ethanol and acetone and incubated with primary antibodies against HB-EGF-C. Secondary antibodies was Alexa Fluor® 594 goat anti-rabbit IgG (H + L) (Invitrogen). All sections were counterstained with DAPI (KPL, Inc., Gaithersburg, MD). Images were obtained using an Eclipse 80i fluorescent microscope (Nikon, Tokyo, Japan).
Each gastric cancer cell in a subconfluent state was placed in serum-free medium for 24 h and stimulated with 200 nM TPA (Cell Signaling Technology, Danvers, MA) for 60 min. Cells were stimulated by TPA in order to investigate the localization of HB-EGF-C by shedding proHB-EGF , and the intracellular localization of HB-EGF-C was then analyzed by immunofluorescence.
Cells were washed with PBS and subsequently dissolved in 1× cell lysis buffer (Cell Signaling Technology) containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1 % Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin. After disruption in an ice bath by using a Bio-ruptor sonicator (Cosmo Bio, Tokyo, Japan) for 15 s, lysates were centrifuged at 15,000 rpm for 10 min at 4°C. Each sample was normalized against an equal protein concentration by using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). A equal quantity of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.5 mol/L Tris–HCl, pH 7.2, 1 % SDS, 100 mmol/L β-mercaptoethanol, and 0.01 % bromophenol blue) was added to each sample and boiled for 5 min at 100°C. Aliquots of sample were fractionated on 10 or 15 % SDS-PAGE and then electroblotted onto a nitrocellulose membrane. The membrane was blocked with 5 % skimmed milk in PBS for 1 h at room temperature. The membrane was incubated with the primary antibodies for HB-EGF-C, HB-EGF, EGFR (MILLIPORE, Temecula, CA) or phospho-EGFR (MILLIPORE) overnight at 4°C and then washed with 0.05 % Tween 20 in PBS 3 times at 5-min intervals. The membrane was incubated with secondary antibody for 1 h at room temperature, which was followed by 3 washes with 0.05 % Tween 20 in PBS 3 times at 5-min intervals. The membrane was treated with enhanced chemiluminescence detection reagents (ECL; Amersham, Arlington Heights, IL) for 1 min at room temperature and then exposed to scientific imaging films (Eastman Kodak, Rochester, NY). Proteins were visualized as bands on the images. Filters were stripped and reprobed with monoclonal β-actin antibody (Abcam plc, Tokyo, Japan) as an internal control.
Cell proliferation assay
Proliferation assays were performed as follows. Cells were seeded at 2.0 × 104 cells in the medium with 10 % FBS on 6-cm diameter dishes. Cells were counted on days 3, 5, and 8 by using an Automatic Cell Counter (Millipore, Billerica, MA). Each experiment was conducted independently in cell lines isolated from 3 independent clones.
Wound healing assay
A wound healing assay was conducted in order to measure cell motility. Cells were grown to confluence in 6-well plates and serum-starved for 24 h, and then a cross-shaped wound was made on the monolayers by using a sterile 200-μL pipette tip. Cells were washed with PBS, placed in the same media with 200nM TPA, and the cross-shaped wound was photographed under microscope at 0 h and 24 h.
Transwell invasion assay
Cell migration was assessed using the Cell Invasion Assay (CULTREX, Gaithersburg, MD), which consists of a 96-well transwell tissue culture plate with an 8-μm pore size membranes coated with matrigel (top chamber) and a black receiver plate compatible with a 96-well fluorescent plate reader (bottom chamber). After 24 h of serum starvation, cells (5000 cells/well) in serum-free medium with 200nM TPA were placed in the top chamber, and medium containing 10 % FBS was added to the bottom chamber. After 48 h of incubation in CO2 at 37°C, the cells that had invaded the bottom chamber were measured according to the manufacturer’s instructions. Each experiment was conducted in cell lines which was isolated from 3 independent clones.
Values are expressed as the mean ± SD. Data were analyzed using χ2 test as appropriate. Multiple comparison was done using Games-Howell’s method. P-values < 0.05 were considered statistically significant. Data analyses were performed using Dr. SPSS II for Windows release 11.0.1 J software (SPSS Japan, Tokyo, Japan).