Figure 1. SIRT1 represses AR-dependent gene transcription.

A) Cell-based NAD-dependent HDAC activity assay of cells treated with 50 μM resveratrol or 10 mM NAM for 24 hr. B) Upper panel: The effects of resveratrol and nicotinamide on PSA-LUC transcription. LNCaP cells cultured in media containing charcoal-stripped serum were transfected with the PSA-LUC reporter vector plasmid. Transfected cells were then exposed to resveratrol (RES) at 50 μM or nicotinamide (NAM) at 10 mM for 24 hr, and treated with 10 nM DHT or vehicle-treated (Veh) for 24 hr before assay of luciferase activity, expressed here in arbitrary units. Insert shows the results from vehicle treatment, with an expanded y-axis. Lower panel: Immunoblot analysis of AR protein levels in cells treated with nicotinamide or resveratrol. C) Upper panel: Effects of SIRT1 over-expression or SIRT1 knockdown on PSA-LUC transcription. LNCaP cells were co-transfected with PSA-LUC and an empty control vector (pcDNA3.1), or a wt-SIRT1 expression vector (pcDNA3.1-SIRT1), or a vector expressing SIRT1 siRNA (pSUPER-SIRT1), or the empty siRNA expression vector (pSUPER), then treated with 10 nM DHT or vehicle-treated for 24 hr, and cells were harvested for luciferase assay. Insert shows the results from vehicle treatment, with an expanded y-axis. Lower panel: Immunoblot analysis of SIRT1 protein levels in cells transfected with wt-SIRT (pCDNA3.1-SIRT1), or DN-SIRT1 (pCDNA3.1-H363Y), or empty vector (pCDNA3.1), or empty siRNA vector (pSUPER), or SIRT1 siRNA expression vector (pSUPER-SIRT1). Cell extracts were normalized for protein content, separated by PAGE, transferred, probed with an anti-SIRT1 antibody or anti-AR antibody or an anti-β-actin antibody, and developed with a chemoluminescence kit. D) The effect of resveratrol or nicotinamide on endogenous AR-dependent and –independent PSA gene transcription. LNCaP cells cultured in media containing charcoal-stripped serum were exposed to 10 mM nicotinamide (NAM), 50 uM resveratrol (RES) or vehicle (control) for 2 hr, and treated with 10 nM DHT or vehicle-treated for 48 hr. Transcript levels of PSA was measured by quantitative RT-PCR analysis of RNA extracted from the cells. Transcript levels are expressed relative to β-actin transcripts. E) The effect of resveratrol or nicotinamide on endogenous AR-dependent and –independent KLK2 gene transcription. LNCaP cells were cultured in media containing charcoal-stripped serum were exposed to 10 mM nicotinamide (NAM), 50 uM resveratrol (RES) or vehicle (control) for 2 hr, and treated with 10 nM DHT or vehicle-treated for 48 hr. Transcript levels of KLK2 were measured by quantitative RT-PCR analysis of RNA extracted from the cells. Transcript levels are expressed relative to β-actin transcripts. F) Upper panel: SIRT1 knockdown increases endogenous AR-dependent PSA genes transcription. Transcript levels of PSA were measured by quantitative RT-PCR analysis of RNA extracted from empty vector-transfected LNCaP cells (Ctrl) and LNCaP cell lines in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). The cells were cultured under androgen-deprivation conditions for 3 days, followed by treatment with DHT or vehicle for 48 hr. Lower panel: Immunoblot analysis of SIRT1 and AR protein levels in an empty vector-transfected LNCaP cell line (Ctrl) and LNCaP cell lines in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). G) SIRT1 knockdown increases endogenous AR-dependent KLK2 gene transcription. Transcript levels of KLK2 were measured by quantitative RT-PCR analysis of RNA extracted from empty vector-transfected LNCaP cells (Ctrl) and a LNCaP cell line in which SIRT1 expression levels had been knocked down by stable expression of siRNA (RNAi). The cells were cultured under androgen-deprivation conditions for 3 days, followed by treatment with DHT or vehicle for 48 hr. H) SIRT1 deacetylase activity is required for SIRT1 effects on AR-dependent gene transcription. LNCaP cells cultured in media containing charcoal-stripped serum were co-transfected with the PSA-LUC vector plus an empty vector (Vector), or a SIRT1 expression vector (SIRT1), or a dominant-negative SIRT1 vector (H363Y), or SIRT1 expression vector plus DN-SIRT1 vectors (SIRT1+H363Y), then treated with 10 nM DHT or vehicle, and harvested for assay of luciferase activity. Insert shows the results from vehicle treatment, with an expanded y-axis. In all relevant figures, relative luciferase activities were normalized to β-gal activity to control for transfection efficiency. The error bars represent the SEM. Asterisks indicate significant differences between two groups (** p<0.01, *p<0.05).