Patients and Specimen Processing
Between January 2001 and November 2004, 57 patients with high-risk localized prostate cancer (defined a cT2b or T3a or PSA ≥ 15 ng/ml or Gleason grade ≥ 4+3) were recruited for a phase II trial of neoadjuvant chemotherapy (using Docetaxel and Mitoxantrone). The design of the clinical trial has been previously described (18
). The study was approved by the Institutional Review Boards of the Oregon Health & Science University, Portland VA Medical Center, Kaiser Permanente Northwest Region, Legacy Health System, and the University of Washington and all patients provided signed informed consent. From each patient, ten standard prostate biopsies (bilateral at the apex, bilateral medial and lateral at mid-gland, bilateral medial and lateral at the base of the gland) were obtained under ultrasound guidance and snap-frozen in liquid nitrogen.
Tissue Microarray Construction
A tissue microarray (TMA) was constructed from formalin fixed representative tissues collected at prostatectomy from the first 50 patients enrolled on the neoadjuvant chemotherapy study. Tissue cores (0.6 mm diameter placed 0.2 mm apart) were removed from the paraffin-embedded prostate tissue blocks (donor blocks) and placed in a recipient paraffin block (30 × 25 mm) using a precision Tissue Arrayer (Beecher Instruments, Sun Prairie, WI). H&E slides of each donor block were examined microscopically and reviewed by a pathologist to determine the appropriate location to sample. From every study patient, three cores each of prostate cancer, normal prostate, and, where applicable, lymph nodes with metastatic cancer were placed in each block in a pseudo-randomized fashion. Dispersed amongst the study cores were control tissues from non-study patients (liver, prostate, lymph node, salivary gland, kidney, testis), untreated cell lines (DU-145, PC-3, LNCaP), and the same cell lines treated with mitoxantrone and docetaxel (singly or in combination). After completion, the block was heated at 37°C for 30 mins to ensure incorporation of the cores into the block. The block was then cut into 5μm thick sections and unstained slides were stored at 4°C until needed.
Microscopic evaluation of frozen sections of tissue samples identified the presence of adequate number of cancer cells in both pre-treatment and post-treatment samples for 31 subjects. Frozen sections (7μM) were cut from tissue frozen in ornithine carbamyl transferase blocks, stained with Mayer’s hematoxylin (Sigma, St. Louis, MO), dehydrated in 100% (v/v) ethanol and xylene, and used for laser capture microdissection using an Arcturus PixCell IIe microscope (Arcturus, Inc.). To evaluate gene expression alterations after chemotherapy, malignant epithelium from pretreated biopsy and post-treated prostatectomy specimens were captured separately (3,000 cells per sample). The histology of captured cells was verified both by review of an H&E-stained frozen section from each sample and by review of the pre/post–laser capture micro-dissection images.
Cell Lines and Antibodies
The androgen-sensitive and TGF-β responsive human prostate cancer cells LNCaP TβRII cells (19
), LNCaP, CWR22 and PC-3 prostate cancer cell lines were used in this study. To determine the effects of DHT and TGF-β, cells were grown in DMEM or RPMI1640 with 10% FBS (without phenol red). The antibodies against E-cadherin, β-catenin, and PARP were purchased from Cell Signaling Technology (Danvers, MA); The antibodies against the AR, tubulin, and N-cadherin proteins were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); The cofilin and actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO); The antibody against the human talin protein was purchased from Upstate Biotechnology (Billerica, MA); The GAPDH antibody was obtained from Novus Biologicals (Littleton, CO).
Cell lines: The LNCaP, CWR22 and PC-3 cell lines were obtained from ATCC and used within 6-12 months. The androgen sensitive and TGF-beta responsive human prostate cancer LNCaP TRII cells were generated and characterized in our laboratory (19
Western Blot Analysis
Total cellular protein was extracted from the cell pellets by homogenization in RIPA buffer (50 mM Tris HCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS). Protein samples (20-60μg) were loaded on 4%-12% SDS-polyacrylamide gels and subjected to electrophoretic analysis and subsequent blotting. Membranes were incubated with the primary antibody, overnight at 4°C and the relevant secondary antibodies (1hr at room temperature). Membranes were subsequently incubated with the enhanced chemiluminescence system (Amersham BioSciences, Piscataway, NJ) and autoradiographed using X-ray film (Amersham BioSciences). Densitometric analysis was performed using the Scion Image program (NIH, USA, http://rsb.info.nih.gov/scoin-image/
). All bands were normalized to actin and shown as fold-change compared to controls.
Cells were plated (1×105 cells/well) in chamber slides and after 24 hrs, cells were exposed to medium RPMI 1640 supplemented with 10% charcoal-stripped-serum (CSS) in the presence of DHT (1nM), TGF-β (5ng/ml), or combination of DHT and TGF-β as indicated. Following treatment, cells were fixed in 2% (v/v) paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 in PBS. Cells were incubated with the primary antibody overnight at 4°C and secondary antibody for 1hr at room temperature). Slides were mounted by Vectashield mounting medium (Vector laboratories Inc, Burlingame, CA) and were visualized and counted under fluorescence microscope (Olympus IX70 Inverted Microscope, Olympus America Inc. Center Valley, PA).
Prostate TMAs were subjected to immunohistochemical analysis using the following antibodies were used: the mouse monoclonal antibody against PSA and the rabbit polyclonal antibody against E-cadherin from Cell Signaling Technology Inc. (Danvers, MA); N-cadherin, and AR (# sc-7939, and sc815 respectively, Santa Cruz Biotechnology, Santa Cruz, CA). After blocking nonspecific binding with goat serum (1.5% in TBS-T) for 30min at room temperature, serial sections were exposed to the specific antibodies overnight at 4°C (Negative controls consisted of incubation with IgG). Sections were subsequently exposed to biotinylated goat anti-rabbit IgG (1hr, room temperature) and horseradish peroxidase-streptavidin conjugate (Chemicon, Billerica, MA). Color development was accomplished using a standard immunoperoxidase method (Dako cytomation LSAB2 system-HPR, Carpinteria, CA) and counterstaining with hematoxylin. Images were captured using an Olympus BX51 microscope system (Olympus America, Lake Success, NY). Protein expression pattern, intensity and localization were assessed in formalin-fixed, paraffin-embedded prostate cancer TMAs via light microscopy, was performed by two independent observers (NK and CB), blinded to treatment modality. Three different cores were measured for each patient. The overall pattern of staining in human prostate tumor cells in the TMAs was determined as the average number of positive epithelial cells in three different fields for each tissue core.
Cells were harvested in lysis buffer [10mM Tris pH 7.5, 1mM EDTA, 400mM NaCl, 10% (v/v) glycerol, 5 mM NaF, 0.5mM sodium orthovanadate, 1 mM dithiothreitol (DTT)], and Complete Mini Protease Inhibitor Cocktail (Roche Biochemicals, Indianapolis, IN). Cell extracts were homogenized and protein content was quantitated using the BioRad Protein Assay (BioRad, Hercules, CA). Cell lysates (400μg) were pre-cleared with protein A/G beads (Oncogene Research Products, #IP05, Boston, MA) and were subsequently incubated with the AR or the a-tubulin antibody (overnight at 4°C). Protein A/G beads were then added to the cell lysate/antibody mixture. Following incubation (1hr at 4°C), the lysate/antibody/bead mixture was centrifuged at 14,000g (30sec). Beads were subjected to elution with 100mM glycine pH 3.0 and eluate-fractions were centrifuged at 14,000g in 1M phosphate buffer pH8.0. Samples were lysed in SDS-PAGE buffer and analyzed by Western blotting as described above.
RNA extraction and Real-Time RT-PCR
RNA samples extracted with Trizol Reagent were treated with RNase-free DNase I and reverse transcript to cDNA (BioRad) Taqman realtime RT-PCR analysis of the cDNA samples was conducted in an ABI 7700 Sequence Detection System (Applied Biosystems Inc, Foster City, CA) using specific primers for PSA (Applied Biosystems Inc, CA).
Transient Transfections and Luciferase Activity Assays
Cells were plated (105 cells/well) in 6-well plates and treated as described above. After 48 hrs, cells were transfected with the ARE luciferase construct (1 mg/well) (from Dr. Zoran Culig, Innsbruck Medical University, Austria) in the presence of the control Renilla luciferase construct (Promega, Madison, WI) using Tfx-50 transfection reagent (Promega, # E1811). Following a 2hr-incubation with the DNA/Tfx50 mixture, serum-containing medium was added to the cells and incubation was continued for 22 hrs. After treatment, cells were harvested and luciferase activity was determined according to the manufacturer’s protocol (Promega, Dual Luciferase Assay, # E1920). Data are representative of three independent experiments in duplicate.
Student t-test and one-way analysis of variance (ANOVA) was performed to determine the statistical significance between values for the in vitro experiments. The data derived from the immunostaining analysis of human prostate tissue specimens were analyzed for statistical significance using the unpaired t-test. All numerical data are presented as the mean values ± SEM (standard error of the mean). Statistical significance was reached at a p value of less than 0.05.