Figure 4. Reversal of bicalutamide-mediated transcriptional and growth suppression of cells by SIRT1 depletion.

A) SIRT1 knockdown impairs bicalutamide-mediated PSA transcription repression. A SIRT1 knockdown cell line (RNAi) and control-transfected LNCaP cells (Ctrl) were cultured in charcoal-stripped serum, transfected with the PSA-LUC reporter vector, and then treated with DHT (1 nM) (D), or DHT plus bicalutamide (10 μM) (D+CDX), or vehicle (S). Cells were harvested after 24 hr and lysates were assayed for luciferase activity. The data are presented as a percent of the activity obtained in DHT alone (assigned the value of 100). B) Immunoblot analysis of SIRT1, AR and β-actin levels in control-transfected LNCaP cells (Ctrl) and a LNCaP line in which SIRT1 had been knocked down by stable expression of siRNA (RNAi). C) SIRT1 over-expression can partially rescue the defect of bicalutamide-mediated PSA transcription suppression induced by SIRT1 depletion. SIRT1 knockdown cells were cultured in charcoal-stripped serum, co-transfected with PSA-LUC and empty-vector (RNAi) or with PSA-LUC and SIRT1 expression vector (RNAi+SIRT1wt), or with PSA-LUC and SIRT1 catalytic inactive mutant (RNAi+SIRT1H363Y) and then treated with DHT (1 nM) (D), or DHT plus bicalutamide (10 μM) (D+CDX), or vehicle (S). Cells were harvested after 24 hr and lysates were assayed for luciferase activity. The data are presented as a percent of the activity obtained with exposure to DHT alone (assigned the value of 100). D) Immunoblot analysis of SIRT1 and AR protein levels in SIRT1 knockdown cell lines transfected with empty vector (RNAi), or wt-SIRT (SIRT1wt), or catalytic inactive mutant (SIRT1H363Y). E) SIRT1 is required for bicalutamide-mediated cell growth suppression. Parental empty-vector transfected LNCaP cells (Ctrl) or LNCaP cells in which SIRT1 had been stably knocked down by siRNA expression (RNAi) were cultured in charcoal-stripped serum three days, and exposed to DHT (1 nM) for another three days, then treated with addition of bicalutamide (2.5 μM) (D+CDX) or vehicle (D) for 48 hr. Viable cells were quantitated by MTS assay, and the results expressed relative to the values obtained from wells cultured without added bicalutamide (assigned as value of 100). Asterisks (*) indicates significant differences between two groups (p<0.05).