br Materials and methods br Chemistry procedures for synthes
2. Materials and methods
2.1. Chemistry procedures for synthesis of 11β-aryloxy-estradiol derivatives
Reagents: Tetrahydrofuran (THF) was distilled from benzoquinone ketyl radical under an argon atmosphere. Dichloromethane, toluene, benzene, and pyridine were distilled from calcium hydride under an argon atmosphere. Anhydrous N, N-dimethylformamide (DMF) was purchased from Sigma-Aldrich. All other solvents or reagents were purified according to standard procedures. (8S, 9S, 13S, 14S, 17S)-3,17-bis(Benzyloxy)-13-methyl-6, 7, 8, 9, 12, 13, 14, 15, 16, 17-decahydro-11H-cyclopenta[a]phenanthren-11-one (11-ketone) was prepared using established procedures [31–34].
Instrumentation: 1H NMR, 13C NMR, and 19F NMR spectra were obtained at 300 MHz, 400 MHz, or 500 MHz for proton, 75 MHz, 100 MHz, or 125 MHz for carbon, and 282 MHz, or 376 MHz for fluorine are so indicated. The chemical shifts are reported in parts per million (ppm, δ). The coupling constants are reported in Hertz (Hz) and the resonance patterns are reported with notations as the following: br (broad), s (singlet), d (double), t (triplet), q (quartet) and m (multiplet). High-resolution mass spectra were measured on a time-of-flight LCeMS. Thin-layer chromatography (TLC) was carried out using pre-coated silica gel sheets. Visual detection was performed with ultraviolet light, p-anisaldehyde stain, potassium permanganate stain or iodine. Flash chromatography was done using silica gel P60 (60 A, 40–63 μm)
with compressed air.
General chemistry procedures to prepare the several antiestrogen compounds described in this report are presented in detail in Supplementary Methods.
Cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. Briefly, ERα-positive human BC LY500307 MCF-7, T47D and ZR-75 were cultured in DMEM or RPMI-1640 media as before [35,36], and MCF-7 cells with HER-2 overexpression  and MCF-7 cells with acquired tamoxifen resistance were established and cultivated as reported previously [38,39]. Mouse triple-negative (ERα-/PR-/HER2-) 4T1 breast tumor cells were cultured in RPMI-1640 medium. Media were supplemented with 10% fetal bovine serum (FBS; Gemini Bio-Products), 100 units/ml penicillin, 100 μg/ml streptomycin sulfate and 2.5 μg/ml amphotericin B (Gemini Bio-Products). Cultures were maintained at 37 °C in a 5% CO2 incubator. For steroid-free conditions, medium was changed 48 h before studies to phenol red-free DMEM or phenol red-free RPMI-1640 with 5% dextran-coated, charcoal-treated FBS (DCC-FBS) as before .
2.3. Cell proliferation assays
MCF-7 and other selected BC cells were seeded in 96-well plates at 3–5 × 105 cells/well in complete medium. After 24 h, medium was switched to estrogen-free conditions as described above. After 48 h, cells were treated with indicated concentrations of antiestrogens for 72 h with or without estradiol-17β (E2). Cell number and viability were determined by either cell counts or by colorimetric assays using the CellTiter 96 Aqueous (Promega) assay or the cell proliferation ELISA BrdU assay (Roche) as per manufacturer’s instructions. Treatments were done in quadruplicate, and experiments were repeated at least three times. In selected experiments using the Incucyte™ Live Cell System (Essen Bioscience) as per the manufacturer’s instructions, the proliferation of 4T1 cells maintained in a tissue culture incubator was monitored by using the NucLight Rapid Red Reagent for cell labeling in 6-well plates. Images for cell confluence were obtained every 4–6 h; as cells proliferate, the confluence increases, and confluence is therefore a surrogate for proliferation. Images were analyzed using the Live-Cell Analysis System (Essen Bioscience).
2.4. Polyacrylamide gel electrophoresis and Western immunoblotting
MCF7 cells were plated in regular medium. After 24 h, cells were incubated in the presence of antiestrogens or fulvestrant for 4 h in phenol-red free medium without FBS. Cell lysates were prepared using RIPA buﬀer, and protein concentration was determined using the BCA Protein Assay Kit (PIERCE/ThermoFisher Scientific). Forty micrograms of total cell protein was resolved by 4–15% SDS-PAGE, transferred to a PVDF membrane and probed with antibody directed against ERα (1D5, 1:100, ThermoFisher cat# MA5-13191). Ribosomal protein L13A (RPL13A), an established housekeeping gene that is not regulated by estradiol-17β or tamoxifen was used as loading control (dilution 1:1000, Invitrogen/ThermoFisher cat# PA5-58528) .
2.5. Competition binding assays in ER-positive human breast tumor cells
Specific estradiol-17β (E2) binding and competition for binding by antiestrogen JD128 or fulvestrant was assessed in human MCF-7 breast cancer cells using methods as described before [36,41]. In brief, MCF-7 cells were suspended in phenol red-free RPMI medium to a con-centration of 1 × 107 cells/ml, and incubations for 60 min were begun with the addition of [2,4,6,7-3H (N)]-estradiol-17β (99 Ci/mmol; New England Nulcear/Perkin Elmer, Waltham, MA) at 37 °C with shaking. A