Example 33
This example details the use of immunocytochemistry to visualize AR translocation.
In order to determine the extent to which the compounds of the present invention can inhibit AR translocation from the cytoplasm to the nucleus after addition of the synthetic androgen R1881, LNCaP cells were treated with either DMSO, 1 nM R1881, 10 μM bicalutamide, followed by 1 nM R1881, or 10 μM Compound 11, followed by 1 nM R1881. LNCaP cells were incubated on cover slips in phenol red-free RPMI 1640, with 10% charcoal-stripped FBS, and 1% antimycotic-antibiotic solution, for three days at 37° C. Cells were then treated with DMSO, bicalutamide or Compound 11 for three hours, followed by treatment with R1881 for three hours. After fixation and permeabilization, cells were stained, fluorescence images were obtained using a Leica® fluorescence microscope, and nuclear to cytoplasmic ratios of AR staining intensity were obtained using OpenLab® image analysis software (Perkin Elmer, Waltham, Mass.). Compound 11 was markedly more active than bicalutamide in inhibiting R1881-induced AR translocation to the nucleus (