Preferential induction of G1 arrest in androgen-responsive human prostate cancer cells by androgen receptor signaling antagonists DL3 and antiandrogen bicalutamide

Abstract

The purpose of this study was to further characterize cell growth-inhibitory effects of a recently identified androgen receptor (AR) signaling inhibitor 6-amino-2-[2-(4-tert-butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one (DL3)(5) and antiandrogen bicalutamide (Bic). DL3 was more potent than Bic in induction of G1 arrest and reduction of G1-related cell cycle protein expression in AR-positive LNCaP cells. DL3, but not Bic, moderately inhibited growth of AR-negative PC-3 cells independent of G1 arrest. The data indicated that DL3 inhibit cell growth in both AR-dependent and -independent manners and is potentially a potent therapeutic agent for the management of advanced human prostate cancer.

Fig. 1. Effects of DL3 and Bic on growth of LNCaP and PC-3 cells

LNCaP (A) and PC-3 (B) cells (2000/well) were incubated for 4 days with various concentrations DL3 or Bic. Viable cells were stained with MTT and effects of treatments on cell growth were calculated. Data shown are mean ± SD of one representative experiment of three to five. *, **, and *** are p<0.05, p<0.01, and p<0.001, respectively, in comparison with the cultures in the absence of the drugs.

Fig. 2. DL3 and Bic did not induce apoptosis in LNCaP cells

A. LNCaP-ARR₂PB-GFP cells were incubated for 72 hr in phenol-free medium supplemented with 5% charcoal-dextran stripped FBS, followed by treatment with 20 uM of DL3 or Bic in the absence or presence of 1 nM of DHT for 96 hr. The cultures were observed under a fluorescent microscope and recorded. B. LNCaP cells were incubated for 72 hr in phenol-free medium supplemented with 5% FBS, followed by treatment with various concentrations of DL3 in the absence or presence of 1 nM of DHT for 48 hr. Cellular caspase 3 level was revealed by immunoblotting with β-actin as loading control. Data shown were one representative experiment of 3.

Fig. 3. DL3 and Bic induced G1 phase arrest in LNCaP cells

LNCaP and PC-3 cells in 6-well plates (5 × 10⁵ and 2.5 × 10⁵/well, respectively) were starved in serum-free medium for 24 hr, followed by stimulation with 10% FBS plus 1 nM DHT in the absence or presence of 10 or 20 uM of DL3 or Bic. The cells were sampled every 24 hr up to 72 hr for cell cycle analysis by FACS as detailed in Materials and Methods. Data shown were the cell cycle distribution pattern of cells treated for 48 hr. This is one representative experiment of 5.

Fig. 4. Effects of DL3 and Bic on the expression of G1 phase-related cell cycle-regulating proteins

A. LNCaP and PC-3 cells were treated for 48 hr with 20 uM of DL3 or Bic. Cell lysates were prepared and analyzed by immunoblotting with β-actin as loading control. B and C. LNCaP and PC-3 cells were starved in serum-free medium for 24 hr and then treated for 24 hr with 10% FBS in the absence or presence of 20 uM of DL3 or Bic. Cell lystates were analyzed by western blotting with β-actin as loading control. D. LNCaP and PC-3 cells were starved in serum-free medium for 24 hr and then treated for 24 hr with 10% FBS in the absence or presence of 20 uM of DL3 or Bic. Cell lystates were immunoprecipitated with an antibody to Rb and analyzed by western blotting with antibodies against CDK substrates and pRb (ser807/811). Data shown is from one representative experiment of 3.