Phorbol-12-myristate 13-acetate, PD-98059, SB-203580, SP-600125, TAPI-0, ionomycin, thrombin, and recombinant hirudin were from Calbiochem (Schwalbach, Germany). Human plasma-derived PC and aPC were from Hematological Technologies Inc. (Cell Systems, Biotechnologie Vertrieb GmbH, St. Katharinen, Germany). Calcein AM, bovine serum albumin (BSA), 4-aminophenylmercuric acetate, and methyl-β-cyclodextrin were purchased from Sigma-Aldrich (Deisenhofen, Germany). Recombinant human IL-1β, TNF-α, IFN-γ, and IL-6 were from Roche Diagnostics GmbH (Mannheim, Germany), chromogenic substrate S-2366 from Haemochrom Diagnostica GmbH (Essen, Germany). Calcein AM, PMA, PD-98059, SB-203580, SP-600125, TAPI-0, ionomycin, and APMA were dissolved in dimethyl sulfoxide (DMSO). The final concentrations of DMSO were 0.3% or less, and controls using DMSO alone were run in all cases. Other agents were used as aqueous solutions. Monoclonal anti-EPCR antibody produced in rat, RCR-252, and anti-rat IgG-FITC conjugated antibody produced in goat were from Sigma-Aldrich (Deisenhofen, Germany).
Cell culture and incubation
Normal human prostate epithelial cells (PrEC; Cambrex Bio Science, Walkersville, MD, USA) were maintained up to a maximum of six passages in prostate epithelial growth medium supplemented with bovine pituitary extract, epidermal growth factor, insulin, transferrin, hydrocortisone, retinoic acid, epinephrine, triiodothyronine and gentamicin-amphotericin solution, according to the manufacturer's instruction. Every two to three days the medium was changed and before reaching confluence, cells were passaged using trypsin/EDTA. Human prostate malignant cell lines (PC-3, DU-145, and LNCaP cells) were purchased from German Collection of Microorganisms and Cell Cultures (Berlin, Germany). They were cultured in standard cell culture medium RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2.
RNA Extraction and RT-PCR Analysis
RNA was isolated after lysis of cells in TRI Reagent according to the manufacturer`s instructions. Isolated RNA was converted to cDNA using the GeneAmp RNA-PCR Kit (PerkinElmer LAS GmbH, Jügesheim, Germany). A portion of the RT reaction products was then amplified for identification of EPCR- and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific mRNA as a reference gene using PCR. Early described primers were used to amplify the coding sequences of human EPCR: 5'-TGG CCT TTC CTC TGA CCA TCC-3` (sense) and 5'-GGA GCT CCC ATT CAC AGC CAC-3` (antisense) giving PCR products with a length of 100 bp . The further applied primer pair was 5`-CGG AGT CAA CGG ATT TGG TCG TAT TG-3` and 5`-GCA GGA GGC ATT GCT GAT GAT CTT G-3` for GAPDH amplifying products with 439 bp length. Primer pairs were applied in a final concentration of 0.8 μM. The buffers and reagents used were from GeneAmp Kit (PerkinElmer LAS GmbH). After amplification, the PCR products were subjected to agarose gel electroforesis and photographed using a G:BOX Chemi, GelVue UV Transilluminator devise (SynGene, USA). Images were analysed using GeneTools software from SynGene.
After incubation cells were scraped off the culture dishes, washed in phosphate buffered saline (PBS), pH 7.4, and resuspended at 1 × 106 cells/ml in FACS buffer (PBS supplemented with 1% BSA and 0.1% sodium azide). Cells were then incubated with anti-EPCR rat monoclonal antibody RCR-252 added to a final concentration of 2.5 μg/ml for 30 min at 4°C. Subsequently, cells were washed twice with FACS-buffer and incubated under light protected conditions for 30 min at 4°C with FITC-conjugated anti-rat secondary antibodies, which were added to a final dilution of 1:100 of the commercially supplied stock solution. Finally, cells were again washed twice, fixed in 4% paraformaldehyde in PBS, and analyzed on EPICS XL flow cytometer (Beckman Coulter GmbH, Krefeld, Germany). Isotype rat IgG was used instead of primary antibodies as controls for EPCR determination.
ELISA based quantitative determination of sEPCR
Amounts of sEPCR released by prostate cells were determined using Asserachrom sEPCR ELISA kits (Diagnostica Stago, Asnieres, France) according to the manufacturer`s instructions. For this purpose, cells were grown to confluence in 96-well microplate in complete medium. After this, the medium was refreshed and cells were further incubated with inducers or inhibitors of EPCR shedding. At the end of incubations, medium was removed, centrifuged at 800 g for 10 min to remove the cell debris and used for analysis without further dilution to determine sEPCR levels released by cells. Total cell protein was determined using a Bicinchoninic Acid assay kit (Sigma-Aldrich, Deisenhofen, Germany) with bovine serum albumin as internal standard.
Determination of ERK 1/2 phosphorylation with cell-based ELISA
Prostate cancer cells were cultured in 96-well microplates for quantitative determination of ERK 1/2 phosphorylation. On the day of experiments, culture medium was replaced by serum-free growth medium. After a 30-minute pre-incubation period with or without 50 μM PD-98059 as a selective inhibitor of the MEK/ERK pathway, either 25 ng/ml IL-1β or 25 ng/ml TNF-α was added directly into wells and cells were further incubated for set periods of time. Levels of total ERK and phosphorylated ERK (P-ERK) were quantified in fixed cells using a RayBio Cell-based P-ERK 1/2 (Thr202/Tyr204) ELISA kit (BioCat GmbH, Heidelberg, Germany) according to the manufacturer's instructions.
Prostate cancer cell 3D invasion assay
Cell invasion was measured in vitro using Oris™ Cell Invasion & Detection Assay (AMS Biotechnology Ltd, Abingdon OX14 4SE, UK) according to the manufacturer`s instructions. Briefly, after serum starvation for 18 hr cells (50,000 cells/well) were seeded on the Oris™ BME (Basement Membrane Extract) coated microplate and allowed to adhere overnight. Stoppers were removed and cells were overlaid with BME in the presence of 10% FBS. After a 48-hr incubation, cells were stained with Calcein AM reagent. The detection mask was applied to the bottom of the microplate and fluorescence from cells in the detection zone was quantified using a Victor3 1420 Multilabel Counter reader (PerkinElmer LAS GmbH, Rodgau Jügesheim, Germany) at excitation/emission wavelengths of 485/520 nm.
Protein C activation assay
Cells were cultured in 24-well plates, treated as indicated and subsequently washed three times in buffer A containing 50 mM Tris-HCI (pH 7.5), 2 mM CaCl2, 100 mM NaCl, and 0.1% BSA. Washed cells were incubated for 2 h at 37°C in the presence of human protein C (4 μg/ml), thrombin (0.12 NIH U pro well), and buffer A in a final volume of 200 μl/well. Thereafter, 150 μl of supernatants were transferred into 96-well plates and assayed for the generation of aPC using 0.8 mM chromogenic substrate S-2366. To prevent nonspecific cleavages of S-2366 by thrombin, hirudin (10 antithrombin units pro well) was added to each probe. Extinction of reaction product was measured at 405 nm on Victor3 1420 Multilabel Counter reader. Amounts of generated aPC were calculated using aPC standards and normalized to cell protein content.
Data were analyzed by one-way analysis of variance coupled with Dunnett's post hoc test to compare each experimental group with a nominated control group using SPSS 14.0 software. Differences were considered significant at P < 0.05.