Bicalutamide

(S)-niphatenone B or (R)-niphatenone B were synthesized with greater than 98% enantiometrically purity as previously reported [6] (link) and their structures are shown on Figure. 1A. EPI-002 was made in house. The synthetic androgen, R1881 was purchased from Perkin–Elmer (Woodbridge, ON). Enzalutamide (MDV-3100) was purchased from Omega Chem, St-Romuald, Quebec. Bicalutamide was a gift from Dr Marc Zarenda, AstraZeneca Pharmaceuticals (Mississauga, ON). Interleukin-6 was purchased from R&D Systems (Minneapolis, MN). Progesterone (4-pregnene-3,20-dione), dexamethasone, HEPES, TCEP and glutathione were obtained from Sigma-Aldrich (Oakville, ON). AR PG21 antibody was purchased from Millipore (Temecula, CA). β-actin antibody was purchased from Abcam (Cambridge, MA).
LNCaP, PC3, Cos-1 and CV-1 cell lines as well as PSA(6.1 kb)-luciferase, probasin (PB)-luciferase, PRE-luciferase, GRE-Luciferase, 5xGal4UAS-TATA-luciferase and AR-(1-558)-Gal4 DBD, AR-YFP and AR^var567es have been described previously [3] (link), [7] (link).
All experiments were carried out at concentration of 7.8 µM niphatenone, 25 µM EPI-002, 1 nM R1881, and 10 µM antiandrogens (Bicalutamide and enzalutamide) except where specifically indicated.

All subjects received standard androgen-blocking endocrine therapy: Bicalutamide (AstraZeneca), 5 mg once daily, Goserelin acetate (AstraZeneca) 3.6 mg subcutaneously, once/28 days.

Testosterone was purchased from Fluka, mibolerone and [³H]mibolerone from Perkin Elmer, and DHT from Sigma. Bicalutamide was extracted from pellets (AstraZeneca), and ODM-201 and ORM-15341 were synthetized by Orion Pharma.

Human prostate cancer cell lines LNCaP‐FGC, PC‐3 and DU145 were purchased from ATCC (Rockville, MD, USA). LNCaP cells had been passaged directly from original low‐passage stocks (2009), and we confirmed PC‐3, LNCaP and DU145 cell identity by STR profiling in 2010 and 2014 (Cellbank, Sydney). Cells were cultured in RPMI 1640 medium containing 10% v/v fetal bovine serum (FBS), penicillin–streptomycin solution and 1 mm sodium pyruvate. Cells were maintained at 37 °C in an atmosphere containing 5% CO₂. Chemicals were diluted as follows, with control wells treated with the appropriate vehicle controls: H‐Ser(Bzl)‐OH (BenSer; Bachem; diluted in H₂O); l‐γ‐glutamyl‐p‐nitroanilide (GPNA; MP Biochemicals; diluted in H₂O); bicalutamide (Astra Zeneca; diluted in DMSO). The [³H]‐l‐leucine and [³H]‐l‐glutamine uptake assays were performed as detailed previously 18, 32.

HeLa cells and the LNCaP prostate cancer cell line were obtained from ATCC and cultured at 37 °C, 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute (RPMI) medium 1640 (Invitrogen, Strathclyde, UK) respectively, both supplemented with 2 mml-Glutamine, 100units/ml penicillin, 100 mg/ml streptomycin (Sigma Aldrich, St Louis, MO) and 10% fetal bovine serum. The LNCaP-PHB cell line has been previously described (16 (link)) and was grown in media supplemented as above with the exception that 10% tetracycline-free fetal bovine serum was used and the cells additionally supplemented with 12 μg/ml blasticidin (Invitrogen, Carlsbad, CA), 500 μg/ml G418 (Sigma-Aldrich), and 0.3 mg/ml zeocin (Invitrogen).
Mibolerone (Perkin-Elmer, Beaconsfield, UK), cyproterone acetate (Sigma-Aldrich, Dorsett, UK), bicalutamide (Astra-Zeneca, Cheshire, UK), and hydroxyflutamide (Schering-Plough, Hertfordshire, UK) were resuspended in ethanol and stored at −20 °C until use, final concentrations were 10 nm for Mibolerone and 1 μm for antiandrogens.

Bicalutamide was obtained from AstraZeneca (North Ryde, NSW, Australia) and dissolved in ethanol. Enzalutamide (MDV3100) was obtained from SelleckChem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO).