Levonorgestrel

Simian Virus 40 large T-antigen p53-inactivated human FTE cell lines FT190 and FT194 were obtained from Dr. Ronny Drapkin [15 (link)]. The FT190 and FT194 cell lines were cultured according to Karst et al [16 (link)] and treated with different concentrations of medroxyprogesterone acetate (Sigma; M1629), progesterone (Sigma; P8783), levonorgestrel (Sigma; N2260) and norethindrone (Sigma; N4128) for 72hrs on 6cm cell culture plates. At the end of treatment, 20–40μg of protein was used for standard western blotting analysis for cleaved caspase-3. To measure protein fold changes, densitometry values of cleaved caspase-3 normalized to actin protein bands were quantified using LabWorks software for both untreated and progestin-treated FTE cells. FTE cell viability, cytotoxicity and caspase activity were examined using ApoTox-Glo™ Triplex Assay (#G6321 Promega) according to the manufacturer’s instructions using 5000 cells/well on a 96-well white plate in phenol-free medium with different concentrations of MPA.

Androstanedione, bicalutamide, dexamethasone, dibutylphthalate, dihydrotestosterone (DHT), flutamide, hydroxyflutamide, levonorgestrel, lidocaine hydrochloride, 1-ethyl-3-(3-methylaminopropyl)carbodiimide (EDC), gluteraldehyde, hydroxylamine hydrochloride, N-hydroxysulfosuccinimide (Sulfo-NHS), potassium phosphate dibasic, pyridine (99.8%), sodium azide, sodium cyanoborohydride, sodium chloride, trizma base, calcium chloride, glycerol and sodium phosphate monobasic were purchased from Sigma–Aldrich (Stainhaim, Germany). HPLC grade Acetonitrile (ACN) and methanol (MeOH) were from POCh (Gli-wice, Poland). Human androgen receptor (AR) full length protein was from Abcam (Cambridge, UK).
Commercially available amine terminated magnetic beads (BcMag) (50 mg/mL, 1 μm diameter) of “sophistically coated iron oxide particles to provide primary amino groups”, were purchased from Bioclon Inc. (San Diego, CA, USA). A manual magnetic separator Dynal MPC-S was purchased from Invitrogen (Carlsbad, CA, USA). Solutions were prepared using purified water used in the study using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA).

P₄, MPA, NET-A, levonorgestrel (LNG), gestodene (GES), nestorone (NES), nomegestrol acetate (NoMAC), drospirenone (DRSP), dihydrotestosterone (DHT), hydroxyflutamide (OHF), estradiol (E₂), fulvestrant (ICI 182,780) and phorbol 12-myristate13-acetate (PMA) were obtained from Sigma-Aldrich, RSA. Human tumor necrosis factor-alpha (TNFα) was obtained from Celtic Diagnostics, RSA. Unlabeled mibolerone (MIB) and [³H]-MIB (84.3 Ci/mmol) were purchased from PerkinElmer Life and Analytical Science, RSA, while [³H]-E₂ (100 Ci/mmol) was obtained from AEC-Amersham, RSA.

The human MDA-MB-231 breast cancer cell line was gifted by Prof. Adrienne Edkins (Rhodes University, RSA) and was maintained as previously described in [12 (link)], with only mycoplasma negative cells used in experiments. R5020 was purchased from Perkin Elmer Life and Analytical Science, while P₄, medroxyprogesterone acetate (MPA), norethisterone (NET), levonorgestrel (LNG), gestodene (GES), nestorone (NES), nomegestrol acetate (NOMAC), drospirenone (DRSP), and phorbol myristate acetate (PMA) were purchased from Sigma-Aldrich. The human PR-A expression vector (pSG5-hPR-A) [13 (link)] and the pSG5-empty expression vector containing no eukaryotic promoter or enhancer sequences [14 (link)] were received from Dr. Eric Kalkhoven (University Medical Centre, Utrecht, Netherlands) and Prof. Gunnar Mellgren (University of Bergen, Norway), respectively. The pTAT-2x-progesterone response element (PRE)-E1b-luciferase promoter-reporter construct [15 (link)] was received from Prof. Guido Jenster (Erasmus University of Rotterdam, Netherlands), while the 5x-nuclear factor kappa beta (NFκB)-luciferase promoter-reporter construct was purchased from Stratagene (Houston, Texas).