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LNCaP cells were plated on 100 mm Falcon tissue culture plates (2 × 106 cells per plate) in Media A. After 24 h, the medium was changed to Media B with 10% CDS, and the cells were incubated for an additional 24 h. After this incubation, the cells were then treated with either 1 nM R1881 or 1 nM 17β-estradiol in the presence or absence of 25 μM resveratrol. Medium containing the test compounds was replaced every 24 h. Cells were treated with the test compounds for a total of 96 h. Immunodetection of PSA was performed following the protocol described previously (27).
To assess the affinity of resveratrol for AR, the Androgen Receptor Competitor Assay Kit, Green (Invitrogen), a fluorescence polarization AR binding assay, was used according to the manufacturer’s protocol. Dihydrotestosterone (0–1.5 μM) was used as a positive control. The resveratrol concentration range used was 0–50 μM. Fluorescence polarization was detected using a TECAN ULTRA fluorescence plate reader (Tecan Systems, San Jose, CA) set at 485 nm excitation and 535 nm emission wavelengths. Results were calculated as percent control = [(no compound control fluorescence units (FU) − with compound FU)/(no compound control FU − with 1.5 μM dihydrotestosterone FU)] × 100%. Triplicate assays were performed and results expressed as percent control ± SD.
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Chuck Reynolds
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