Casodex

DHT (dihydrotestosterone) and Casodex (Bicalutamide) (Sigma-Aldrich, USA) were applied in vitro to activate or inhibit AR signaling at final concentrations of 10 nM and 1 μM, respectively. siRNA was used to knock-down CXCL5, P65 and P110 in RCC samples. Anti-GAPDH (6c5), -β-actin (I-19), -AR (N-20), and - tetramethylrhodamine isothiocyanate (TRITC) IgG antibodies were purchased from Santa Cruz Biotechnology. Anti-CD31 and -P65 antibodies were obtained from Millipore. 5-Bromo-2-deoxyuridine (BrdU) and crystal violet were obtained from Fisher Scientific. Anti-mouse/rabbit secondary antibodies for western blotting were obtained from Invitrogen.
To functionally inhibit/activate potential signaling pathways, we utilized LY29400/IGF-1 (a specific inhibitor/activator, respectively, of the PI3K/Akt pathway) and PDTC/TNF-α (a specific inhibitor/activator, respectively, of NF-κB signaling). To determine the role of CXCL5 in EC recruitment, before analysis, we applied a CXCL5 neutralizing antibody for 1 hr at room temperature at a final dilution of 1:300.

Cell cultures were maintained following ATCC recommendations. LNCaP (ATCC) and LNCaP stable derivatives were grown in RPMI 1640 (Gibco, Life Technologies) culture supplemented with 100 units/ml of penicillin, 1 mg/ml of streptomycin, and 10% FBS. For androgen treatment experiments cells were cultured in the same medium but replacing 10% FBS with 5% charcoal-filtered FBS (One Shot, Life Technologies) for 48 hours prior to addition of 5nM R1881 (metribolone, RM10, TSZCHEM), and after that were incubated for an additional 48 hours. Cells were collected by scraping and protein was isolated (Nuclear Extract kit, Active Motif) for Western blot analysis. Doxorubicin and casodex were purchased from Sigma-Aldrich Inc.