The human pancreatic cancer cell lines, BxPC-3, Capan-2, and SW1990, were from the American Type Culture Collection (Rockville, MD). The BxPC-3 cells were maintained in RPMI 1640 (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS). SW1990 and Capan-2 cells were maintained in Dulbecco modified Eagle medium (Gibco BRL) with high glucose and 10% FCS. All cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Human pancreatic tissues were obtained in Department of Gastroenterological Surgery, Nagoya City University Hospital with patients' or their relatives' informed consent. Tissue samples were fixed in 10% formalin and then embedded in paraffin. Immunohistochemical studies on tumor-free pancreatic tissue were performed using non-cancerous regions of tumor-containing pancreas.
Recombinant human IL-1α (rIL-1α) was provided by Gibco BRL. The monoclonal antibodies (mAbs) used included anti-β1 (P5D2), anti-α6 (GoH3), anti-αv (AV1), and anti-β4 (439-9B) from Chemicon International, Inc. (Temecula, CA, USA); anti-IL-1RI (35730) from Genzyme/Techne; anti-uPA-specific antibody (#3471) and uPAR specific antibody (#3936) from American Diagnostica (Temecula, CA, USA); anti-phospho-ERK 1/2 (Thr 202/Tyr 204), anti-ERK 1 (C-16), and anti-ERK 2 (C-14) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Western blot analysis
The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100, 0.1% SDS, 0.1% Nonidet P-40, 2 mM PMSF, 1 mM vanadate, 5 μg/ml Trasylol, 10 μM Pepstatin A and 10 μM leupeptin). Following a low-speed spin (500 rpm, 5 min) to pellet nuclei and cell debris, the supernatant fraction was further centrifuged (100,000 g, 30 min), and the crude plasma membranes obtained in the pellet were re-suspended in 20 mM Tris-HCl (pH 7.4). Protein concentrations were determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). The amounts of samples were 50 μg per each lane. Western blot analyses were performed following SDS-PAGE. The lysates were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Immobilon PVDF; Nihon Millipore Ltd., Tokyo, Japan) and immunoblotted with each antibody.
Flow cytometric analyses
Flow-cytometric analysis was performed using FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA). The indirect immunofluorescence method was applied to stain the cancer cells with various monoclonal antibodies as the primary antibody (stained for 30 min at room temperature), followed by the addition of the secondary antibody conjugated fluorescein isothiocyanate (Dako, Glostrup, Denmark). Results are expressed as mean fluorescence intensity for triplicate determinations.
Cell proliferation assay
Pancreatic cancer cell proliferation was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction method] assay and cell count. In MTT assay, pancreatic cancer cells were seeded at a density of 2 × 103 cells/100 μl into 96-well plates and allowed to adhere overnight. Culture media were replaced, and the cells then cultured in medium alone (control) or in medium with/without 10 ng/ml of rIL-1α. After 24 h of incubation, cells were cultured for 4 h with the metabolic substrate tetrazolium salt MTT at a final concentration of 0.5 mg/ml. Formazan was detected spectorphotometically at 540 nm with a multiwell spectrophotometer (ELISA Reader; Biotek Instruments, Burlington, VT, USA).
In cell count, pancreatic cancer cells were seeded at a density of 2 × 105 cells on 35 mm well in media containing 10% FCS. After 24 h, cells were starved with 0.5% FCS for another 24 hours. Culture media was replaced to the fresh serum free media, and added rIL-1α at concentration of 10 ng/ml. After 24 h incubation, cells were washed once with phosphate-buffered saline (PBS), trypsinized, and centrifuged for 3 min at 1,500 rpm. The cell pellet was re-suspended in 2 ml of PBS and cells were counted using a light microscope.
Before the stimulating experiments with IL-1α were attempted, the lowest effective concentration was determined using rIL-1α at concentrations of 0.1 ng/ml, 0.5 ng/ml, 1.0 ng/ml, 10 ng/ml, and 100 ng/ml. A concentration of 10 ng/ml was determined to be the lowest effective concentration for stimulating experiments (data not shown). In some experiments, 0.5 μg/ml anti-α6 integrin or anti-β1 integrin mAbs was added to the cancer cells for 24 h. Before the blocking experiments were attempted, the lowest effective antibody concentration was determined using antibodies at concentrations of 0.1 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 0.75 μg/ml, and 1.0 μg/ml. A concentration of 0.5 μg/ml was determined to be the lowest effective concentration for blocking experiments (data not shown). Experiments were performed in triplicate and repeated three times.
Adhesion assay was performed as described previously with some modifications . 24-well plates coated with laminin, the putative ligand of the α6β1-integrin, were obtained from Becton-Dickinson Labware (Franklin Lakes, NJ, USA). Briefly, cancer cells were incubated for 24 hours with/without rIL-1α (10 ng/ml) and then added (2 × 105 cells/well) to each well and incubated at 37°C for 30 min. The wells were then washed three times with PBS to remove unattached cells. In some experiments, 0.5 μg/ml anti-α6 or anti-β1 integrin antibodies were added to the cancer cells for >30 min prior to addition of rIL-1α.
The migration response of pancreatic cancer cells in response to IL-1α was determined by using Matrigel-coated invasion chambers (Becton and Dickinson, USA). Cancer cells were added (1 × 105 cells/well) to the inner chamber of a cell culture insert and incubated at 37°C for 24 h, either with culture media containing 10 ng/ml rIL-1α or with culture media containing 10 ng/ml rIL-1α and 0.5 μg/ml anti-α6 integrin, anti-β1 integrin, or anti-uPAR antibodies. Complete medium containing 20% fetal bovine serum served as a chemo-attractant in the lower chamber. To quantitate migration, the filters were fixed in 70 % ethanol for 30 min and stained with Giemsa. Cells were removed from the upper surface of the filters by rubbing gently with a cotton-tipped applicator. Cells that had migrated through the membrane were counted in five random microscope fields of the lower filter surface.
Ras activation assay
The activation state of Ras was determined using the Ras Activation Assay Kit provided by Upstate (Lake Placid, NY, USA). Briefly, pancreatic cancer cells were serum starved for 24 h, and then incubated in serum-free medium with/without rIL-1α (10 ng/ml) for 30 min. Cells were harvested and lysed in lysis buffer (100 mM HEPES, pH 7.5, 200 mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 5 mM EDTA and 10% glycerol), and supernatant prepared by centrifugation for 5 min at 4°C at 14,000 g. Ras-GTP from various treated lysates was "pulled down" using the GST fusion protein corresponding to human Ras binding domain of Raf-1 bound to agarose. The presence of Ras-GTP was detected by Western blotting using anti-Ras antibody (Upstate).
Pancreatic tissues were studied using the labeled streptavidin biotin method [40, 41]. Specimens were sectioned at 3.5-μm thick and deparaffinized. After rinsing in phosphate-buffered saline (pH 7.2), 10% bovine serum (Wako, Osaka, Japan) was applied for 10 min to block nonspecific binding. Sections were then incubated with anti-α6 integrin (overnight at 4°C), anti-β1 integrin (over night at 4°C), or anti-uPAR (60 min at 37°C) mAbs as primary antibodies. After rinsing in phosphate-buffered saline, sections were treated with biotinylated anti-mouse immunoglobulin (Ig) (Dako, Copenhagen, Denmark) for 10 min. After rinsing in phosphate-buffered saline, sections were treated with horseradish peroxidase-labeled streptavidin (Dako, Copenhagen, Denmark) for 10 minutes. The peroxidase reaction was visualized by incubating the sections with 0.02 % 3,3'-diaminobenzidine tetrahydrochloride in 0.05 M Tris buffer (pH 7.6) with 0.01 % hydrogen peroxide, followed by hematoxylin counterstaining. Negative control sections were prepared using normal mouse IgG instead of primary antibody.
Two observers (H.S. and H.F.) independently evaluated the immunostaining results. The concordance ratio was > 90%. Differences of opinion were resolved by reaching a consensus with the assistance of a third evaluator (Y.M.). The intensity of tissue staining was graded semiquantitatively on a 4-point scale (-, +, ++, and +++). Likewise, the proportion of cells stained was assessed on a 4-point scale (1, 0–15%; 2, 15–50%; 3, 50–85%; 4, 85–100% cells stained). To evaluate immunohistochemical findings from pancreatic cancer tissues, cases were classed in strongly staining (Group S) and weakly staining groups (Group W) by intensity and proportion of immunostaining. Immunostaining of intensity more than +++ or a staining area was more than 3 for α6 integrin subunit, β1 integrin subunit, or uPAR was defined as Group S.
Statistical comparisons were made using the Student's t test for paired observations or by one-way ANOVA for multiple comparisons. The Mann-Whitney U test was used to compare the immunohistochemical characteristics. Differences between Kaplan-Meier survival curves based on Immunohistochemical analysis were tested with the Wilcoxon test. Multiple survival analysis was calculated according to Cox's proportional hazards model. Statistical significance was indicated by p < 0.05. Data are presented as mean ± standard deviations (s.d.). Each experiment was repeated three times and was carried out in triplicate.