Cells and reagents
LNCaP human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA), maintained in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS) and used within a range of 20–40 passages. VCaP human prostate cancer cells were purchased from the ATCC, maintained in DMEM supplemented with sodium pyruvate, non-essential amino acids and 10 % FBS, and used within 60–70 passages. Vorinostat was obtained from Merck (New Jersey, USA) and dissolved in DMSO. Bicalutamide was obtained from Astra Zeneca (London, UK) and dissolved in ethanol. Cycloheximide was obtained from Sigma (St. Louis, MO, USA) and dissolved in DMSO. Anti-AR (N-20), anti-prostate specific antigen (PSA; C-19) and anti-heat shock protein 90 (HSP90; H-114) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-IκBα antibody was obtained from Cell Signaling Technology Inc (Danvers, MA, USA). Anti-αtubulin antibody was obtained from Merck Millipore (Billerica, MA, USA). Horseradish peroxidase conjugated anti-rabbit, anti-mouse, and anti-sheep/goat secondary antibodies were obtained from DAKO (Botany, NSW, Australia). Non-specific, scrambled siRNA and ON-TARGETplus siRNAs targeting NFKBIA were purchased from Dharmacon (Lafayette, CO, USA) and the NFKBIA-IRES-eGFP lentiviral ORF plasmid was purchased from GeneCopoeia (Rockville, MD, USA). The pLV410 eGFP lentiviral ORF plasmid was kindly provided by Dr. Philip Gregory (University of Adelaide, Adelaide, Australia).
Cell viability assays
LNCaP or VCaP cells were seeded in triplicate in 24-well plates, and allowed to attach overnight before the growth medium was replaced with medium containing vehicle control or the indicated concentrations of vorinostat, bicalutamide, or the two agents in combination. Doses were calculated based on individual dose-response curves for each agent and cell line to ensure consistency in the antiproliferative response between different cell lines (, Additional file 1: Figure S1). Cells were counted every 2 days using a hemocytometer and cell viability was assessed using Trypan blue dye exclusion. For sequential treatments, cells were treated with drug one for 24 h, at which point the treatment medium was removed and replaced with drug two for 48 h. To assess the effect of cycloheximide, cells were pre-treated with 10 μM cycloheximide for 1 h, which was then removed and replaced with treatment medium. Wash out experiments were performed by allowing the treatment medium to remain on the cells for 1, 2, 4, 6, 8, 16, or 24 h, at which point it was removed and replaced with drug-free medium. At each end-point cells were counted using a hemocytometer and viability assessed as above.
LNCaP cells were cultured with vehicle control, 1 μM vorinostat, 5 μM bicalutamide, or the two agents in combination for 6 h. Adjustment of the dose of bicalutamide compared to the initial cell viability assays was necessary to ensure consistency in terms of cell death between the two experiments, due to variation in the sensitivity of LNCaP cells to this agent over time. Total RNA was extracted from the cells using Trizol reagent (Life Technologies, Carlsbad, CA, USA), and RNA integrity was analyzed on an Agilent Systems Bioanalyzer. RNA from cells treated with the combination of vorinostat and bicalutamide was compared with RNA from cells treated with either vehicle control or either of the agents individually using Affymetrix Human GeneChip ST 1.0 arrays at the Adelaide Microarray Centre, as described previously . Differential gene expression was assessed by ANOVA with the p-value adjusted using a step-up multiple test correction to control the false discovery rate (FDR) . Adjusted p-values < 0.05 were considered to be significant.
Quantitative real-time PCR
Independent RNA samples used to validate the microarray data were generated by culturing LNCaP cells with vehicle control, 1 μM vorinostat, 2.5 μM bicalutamide or the two agents in combination for 3, 6, 9 and 12 h. Total RNA (1 μg) was DNAse treated with Turbo DNA Free (Ambion, Austin, TX, USA), and then reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed with a 1:10 dilution of the cDNA using SYBR green (Bio-Rad) on a CFX Real-Time System (Bio-Rad). geNORM analysis was used to determine appropriate housekeeper genes for each sample set. Microarray RNA was normalized to HPRT1 and RPL19, and the independent sample set was normalized to GUSB and HPRT1. Primer sequences are listed in Additional file 2: Table S1.
LNCaP cells cultured with 2.5 μM bicalutamide or 1 μM vorinostat, individually and in combination, were lysed in radioimmunoprecipitation assay lysis buffer (10 mM Tris–HCl, 125 mM NaCl, 1 mM EDTA, 1 % Triton X-100) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates (20 μg) were electrophoresed through 7.5–15 % SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked overnight (4 °C) in 3 % non-fat milk powder in Tris-buffered saline containing 0.05 % Tween-20 (TBST). Immunodetection was performed overnight at 4 °C in 3 % non-fat milk powder in TBST using an anti-AR (1:1000) rabbit polyclonal antibody, anti-IκBα (1:1000) mouse monoclonal antibody, or anti-PSA (1:500) goat polyclonal antibody. Antibodies against HSP90 (1:1000, rabbit polyclonal) and α-tubulin (1:1000, mouse monoclonal) were used to assess loading. Proteins were detected with horseradish peroxidase-conjugated secondary antibodies and visualized on autoradiography film using enhanced chemiluminescence detection (GE Healthcare).
Enriched gene pathways were identified using Ingenuity Pathway Analysis (IPA), the Database for Annotation, Visualization and Integrated Discovery (DAVID), and Gene Set Enrichment Analysis. Significantly regulated genes (p < 0.05) were uploaded into IPA software v9.0 (Ingenuity Systems, CA, USA) in separate lists for the combination vs. bicalutamide alone and the combination vs. vorinostat alone. Each gene was mapped to its corresponding molecule in the Ingenuity pathways knowledge base, and core analysis identified enriched pathways and networks in the dataset against a background of the Affymetrix Human GeneChip ST 1.0 array. Lists of genes significantly regulated by the combination compared to either vehicle control or either of the individual agents were uploaded to the Functional Annotation Tool available through DAVID (https://david.ncifcrf.gov/; [29, 30]), converted to DAVID default IDs, and analyzed against a background of the microarray platform. Genes significantly regulated by the combination, either uniquely or when compared with the individual agents, were analyzed against a background of all genes significantly regulated by the combination over vehicle control. Enriched Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified using DAVID. Gene Set Enrichment Analysis  was implemented using the Broad Institute’s public GenePattern server (http://genepattern.broadinstitute.org/gp/pages/index.jsf), with default parameters.
Transfection of siRNA and expression constructs
For siRNA transfection, LNCaP or VCaP cells were seeded directly into transfection medium containing phenol red free (PRF) RPMI 1640, lipofectamine 2000 (Life Technologies), and reconstituted scrambled siRNA control or siRNA targeting NFKBIA at a concentration of 10 nM. Four siRNAs were tested, and #1 and #4 were found to be the most effective at achieving knockdown. After 4 h of culture, additional PRF-RPMI medium containing FBS and L-glutamine was added to the LNCaP transfection mixture, while DMEM containing FBS, L-glutamine and non-essential amino acids was added to the VCaP transfection mixture. Cells were harvested for counting and assessment of cell death using Trypan blue dye exclusion, and then lysed in RIPA buffer for immunoblot analysis three days post-treatment (LNCaP) or six days post-treatment (VCaP). The two timepoints used reflect the different growth kinetics between the two cell lines.
For transient transfection of lentiviral constructs expressing green fluorescent protein (GFP) or co-expressing NFKBIA and GFP, LNCaP cells were seeded at ~40 % confluency and allowed to attach overnight. Growth medium was removed and replaced with transfection medium containing PRF-RPMI, lipofectamine 2000, and 1.5 μg plasmid DNA. As for siRNA transfection, additional medium containing FBS, L-glutamine and either vehicle control or combination therapy was added to the transfection mix after 4 h of culture. At three days post-treatment, fluorescent cells were visualized using a fluorescent microscope, and transfection efficiency was estimated at between 40 and 50 %. Cells were then harvested and assessed for death using Trypan blue dye exclusion, after which they were lysed in RIPA buffer for subsequent immunoblot analysis.