Effects of resveratrol on LNCaP cell growth in culture. (A) Concentration-dependent effects of resveratrol on LNCaP cells cultured in 10% FBS. LNCaP cells were plated in 24-well plates; 24 h after plating, cells were treated with 0, 1, 5 or 25 μM resveratrol for 96 h and cell growth determined as described in Materials and Methods. Results are expressed as mean ± SD (n = 4). Bars with *are significantly different from vehicle control (0) at P < 0.05. (B) Effects of resveratrol on R1881-induced cell growth. LNCaP cells were plated in 24-well plates; 24 h after plating, the medium was switched to Media B, which contains 10% CDS, for an additional 24 h. Cells were then treated with and without R1881 (1 nM) in the presence of 0, 1, 5 or 25 μM resveratrol for 96 h and cell growth determined as described in Materials and Methods. Results are expressed as percent inhibition (mean ± SD, n = 4). Bars with different letters are significantly different from each other at P < 0.05. (C) Effects of resveratrol on 17β-estradiol-induced growth. LNCaP cells were seeded in 24-well plates; 24 h after plating, medium was switched to Media B, which contains 10% CDS, for an additional 24 h. Cells were then treated with and without 17β-estradiol (1 nM) in the presence of 0, 1, 5 and 25 μM resveratrol for 96 h, and cell growth was determined as described in Materials and Methods. Results are expressed as percent inhibition (mean ± SD, n = 4). Bars with different letters are significantly different from each other at P < 0.05.

Effects of resveratrol on R1881- and 17β-estradiol-induced ARG mRNA and protein levels. (A) Effects of resveratrol on the R1881-induced increase in ARG mRNA levels. LNCaP cells were plated in six-well plates; 24 h after plating, the medium was switched to Media B, which contains 10% CDS, for an additional 24 h. Cells were then treated with or without R1881 (1 nM) in the presence or absence of resveratrol (25 μM) for 48 h, total RNA was isolated and mRNA levels of selected ARGs (B2M, PSA, SEPP1 and STK39) were determined as described in Materials and Methods. Results are expressed as mean ± SD (n = 3). Bars with different letters are significantly different from each other at P < 0.05. (B) Effects of resveratrol on the 17β-estradiol-induced increase in ARG mRNA levels. LNCaP cells were plated in six-well plates; 24 h after plating, the medium was switched to Media B, which contains 10% CDS, for an additional 24 h. Cells were then treated with or without 17β-estradiol (1 nM) in the presence or absence of resveratrol (25 μM) for 48 h, total RNA was isolated and mRNA levels of selected ARGs (PSA and STK39) were determined as described in Materials and Methods. Results are expressed as mean ± SD (n = 3). Bars with different letters are significantly different from each other at P < 0.05. (C) Effects of resveratrol on PSA protein levels. LNCaP cells were plated in 100 mm plates; 24 h after plating, the medium was switched to Media B, which contains 10% CDS, for an additional 24 h. Cells were then treated with or without R1881 or 17β-estradiol (1 nM) in the presence or absence of resveratrol (25 μM) for 48 h, protein was isolated and immunodetection of PSA was performed as described in Materials and Methods.

IHC analysis of markers for the steroid hormone-responsive pathway, proliferation, apoptosis and angiogenesis. IHC analyses for the steroid hormone-responsive gene PSA, the proliferation marker PCNA, apoptosis and the angiogenesis marker PECAM-1 were performed on paraffin-embedded tumor samples as described in Materials and Methods. (A) PSA. PSA expression index = average PSA-positive cells/mm² (B) PCNA. Proliferation index = average PCNA-positive cells/mm² (C) ApopTag. Apoptosis index = average apoptotic cells per field (D) PECAM-1. Angiogenesis index = average vessel counts per field *indicates significantly different from control at P < 0.05.

Effects of resveratrol on ARG expression and affinity of resveratrol for AR. (A) Microarray analysis of ARG gene expression. LNCaP cells cultured in 10% FBS were treated with 0, 1, 5 or 25 μM resveratrol for 48 h, and total RNA isolation and microarray analysis for ARG expression were performed as described in Materials and Methods. Triplicate treatments and analyses were conducted for each concentration. Results are expressed as a heat map. (Red color: upregulated gene and green color: downregulated gene.) Intensity of the color is proportional to the magnitude of effects on a log 2 scale, −3 to +3. (B) RT–PCR analysis of effects of resveratrol on ARG expression. LNCaP cells cultured in 10% FBS were treated with or without resveratrol (25 μM) for 48 h, total RNA was isolated and mRNA levels of selected ARGs (B2M, PSA, SEPP1 and STK39) were determined as described in Materials and Methods. Results are expressed as mean ± SD (n = 3). Bar with * is significantly different from vehicle control at P < 0.05. (C) RT–PCR analysis of effects of resveratrol on IGF-1R expression. LNCaP cells cultured in 10% FBS were treated with 0, 1, 5 or 25 μM resveratrol for 48 h, and total RNA was isolated and mRNA levels of IGF-1R determined as described in Materials and Methods. Results are expressed as mean ± SD (n = 3). Bars with *are significantly different from vehicle control (0) at P < 0.05. (D) RT–PCR analysis of effects of resveratrol on CDKN1A expression. LNCaP cells cultured in 10% FBS were treated with 0, 1, 5 or 25 μM resveratrol for 48 h, and total RNA was isolated and mRNA levels of CDKN1A determined as described in Materials and Methods. Results are expressed as mean ± SD (n = 3). Bar with *is significantly different from vehicle control (0) at P < 0.05. (E) Affinity of resveratrol for AR. AR binding assays were performed as described in Materials and Methods. Dihydrotestosterone (DHT) was used as positive control. Results are expressed as percent control ± SD (n = 3). Points with *are significantly different from vehicle control (0) at P < 0.05.

Effects of resveratrol on LNCaP cell-derived tumor growth in athymic nude mice. LNCaP cell tumor xenografts were established in athymic nude mice (n = 22 per diet group) as described in Materials and Methods. Tumor volumes were measured twice a week and calculated as described in Materials and Methods. The linear coefficients and standard errors from the random effects model are shown in the graph. (A) Body weights of control, RES50 and RES100 groups during the study. (B) Temporal effects on tumor growth in animals fed control (filled circles), RES50 (open circles) or RES100 (filled triangles) diets. (C) Comparison of tumor volumes between animals fed control, RES50 or RES100 diet in week 3. Week 3 results from Panel B were regraphed, and *indicates significantly different from control at P < 0.05. (D) Comparison of tumor volumes between animals fed control, RES50 or RES100 diets in week 4. Week 4 results from Panel B were regraphed, and *indicates significantly different from control at P < 0.05.