A novel dietary flavonoid fisetin inhibits androgen receptor signaling and tumor growth in athymic nude mice

Abstract

Androgen receptor (AR)-mediated signaling plays an important role in the development and progression of prostate cancer (PCa). Hormonal therapies, mainly with combinations of antiandrogens and androgen deprivation, are the mainstay treatment for advanced disease. However, emergence of androgen resistance largely due to inefficient antihormone action limits their therapeutic usefulness. Here, we report that fisetin, a novel dietary flavonoid, acts as a novel AR ligand by competing with the high-affinity androgen to interact with the ligand binding domain of AR. We show that this physical interaction results in substantial decrease in AR stability and decrease in amino-terminal/carboxyl-terminal (N-C) interaction of AR. This results in blunting of AR-mediated transactivation of target genes including prostate-specific antigen (PSA). In addition, treatment of LNCaP cells with fisetin decreased AR protein levels, in part, by decreasing its promoter activity and by accelerating its degradation. Fisetin also synergized with Casodex in inducing apoptosis in LNCaP cells. Treatment with fisetin in athymic nude mice implanted with AR-positive CWR22Rupsilon1 human PCa cells resulted in inhibition of tumor growth and reduction in serum PSA levels. These data identify fisetin as an inhibitor of AR signaling axis and suggest that it could be a useful chemopreventive and chemotherapeutic agent to delay progression of PCa.

Figure 1

Effect of fisetin on cell viability and its interaction with the LBD of AR. A, chemical structure of fisetin. B, effect of fisetin on cell growth. As detailed in Materials and Methods, LNCaP, CWR22Rυ1, and prostate epithelial cells (PrEC) were treated with fisetin for 48 h and the viability of cells was determined by the MTT assay. Points, mean percentage of viable cells from three experiments, with each treatment done in multiple wells; bars, SE. *, P < 0.001, compared with the control (0 μmol/L) group. C, effect of fisetin on R1881-induced cell growth. As detailed in Materials and Methods, LNCaP cells were treated with R1881 (1 nmol/L), Casodex (10–7 mol/L), and the combination of R1881 (1 nmol/L) and fisetin (10–60 μmol/L) for 48 h, and cell viability was determined by the MTT assay. Columns, mean percentage of cell viability from thee experiments, with each treatment done in multiple wells; bars, SE. *, P < 0.001, compared with control (0 μmol/L); ^§, P < 0.001, compared with R1881. D, fisetin competes with DHT and physically interacts with the LBD of AR. Points, average of two sample wells; bars, SE. The curve was fit using a sigmoidal dose-response equation with varying slope using Prism software from GraphPad Software, Inc.

Figure 2

Effect of fisetin on protein and mRNA expression of AR and its interference with N-C interaction of AR. A, effect of fisetin on the protein expression (top left) and mRNA expression (bottom left) of AR as determined by RT-PCR in LNCaP cells. Right, LNCaP cells were transiently transfected with AR promoter reporter (pLARS) along with Renilla luciferase and treated with solvent (DMSO) and fisetin (10–60 μmol/L) for 48 h with or without R1881 (1 nmol/L). Columns, average relative luciferase values (normalized to Renilla luciferase) from triplicate experiments; bars, SE. B, effect of fisetin on DHT-stimulated protein expression of AR in LNCaP cells. The cells were treated with 40 μmol/L fisetin for 48 h and then harvested. Cells were grown in 10% FBS, charcoal-stripped serum with 10, 50, and 100 nmol/L of DHT with or without fisetin (40 μmol/L) for 48 h. The details are described in Materials and Methods. Total cell lysates were prepared and 40 μg of protein were subjected to SDS-PAGE followed by Western blot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The Western blots shown are representative of three independent experiments with similar results. The values above the figures represent relative density of the bands normalized to β-actin. C, effect of fisetin on AR protein turnover in LNCaP cells. The cells were treated with 40 μmol/L fisetin and 50 μg/mL cycloheximide for the indicated time periods, followed by the preparation of whole cell lysates. Cycloheximide was added to the media 30 min before the addition of fisetin. AR protein levels were determined by Western blot analysis with antibody specifically against AR and normalized to β-actin control. Western blots were quantified by the relative denisties of the bands and the relative percentage of AR protein was plotted against hours after cycloheximide treatment; bars, SD. D, AR N-C interaction assay was done on CV1 cells as described in Materials and Methods. Eight hours after transfection, cells were washed, given fresh media, and treated with R1881 (1 nmol/L) and/or Casodex (10–7 mol/L) along with 10 and 20 μmol/L of fisetin for 48 h; after which cells were lysed and luciferase activity was measured. The graphs represent the fold hormone induction compared with non–hormone-treated group, which was set as 1.

Figure 3

Effects of fisetin on protein and mRNA expression of PSA and AR-regulated reporter genes. A, effect of fisetin on the protein expression (top) and mRNA expression (bottom) of PSA as determined by RT-PCR in LNCaP cells. B, effect of fisetin on DHT-stimulated protein expression of PSA in LNCaP cells. The cells were treated with 40 μmol/L fisetin for 48 h and then harvested. Cells were grown in 10% FBS, charcoal-stripped serum with 10, 50, and 100 nmol/L of DHT with or without fisetin (40 μmol/L) for 48 h. The details are described in Materials and Methods. Total cell lysates were prepared and 40 μg protein was subjected to SDS-PAGE followed by Western blot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the Western blots and reprobing them for β-actin. Representative Western blots of three independent experiments with similar results. The values above the figures represent relative density of the bands normalized to β-actin. C, effect of fisetin on secreted levels of PSA in LNCaP cells. The cells were treated with fisetin (10–60 μmol/L) for 48 h and then harvested. The PSA levels were determined by ELISA. The details are described in Materials and Methods; the figures represent the data of three experiments, each conducted in duplicate. *, P < 0.001, compared with the control (0 μmol/L) group. D, LNCaP cells transiently transfected with either PSA-Luc (left) or MMTV-Luc (right) reporters were treated 48 h posttransfection with or without 1 nmol/L R1881 and with fisetin (10–60 μmol/L). The graphs represent the fold hormone induction compared with non–hormone-treated group, which was set as 1.

Figure 4

Effect of fisetin on induction of apoptosis. LNCaP cells were treated with fisetin (10 and 20 μmol/L) and/or Casodex (10–7 mol/L) for 48 h. The fluorescence was measured with a Zeiss 410 confocal microscope. The details are described in Materials and Methods. Data are from one representative experiment repeated twice with similar results; magnification, ×400.

Figure 5

Effect of fisetin on CWR22Rυ1 tumor growth and PSA secretion in athymic nude mice. Twenty-four animals were then randomly divided into 8 animals in group 1 and 16 animals in group 2. Approximately 1 million CWR22Rυ1 cells were s.c. injected in each flank of mouse to initiate tumor growth. Twenty-four hours after cell implantation, the eight animals of the first group of animals received i.p. injection of DMSO (30 μL) and served as control. The 16 animals of group 2 received i.p. injection of fisetin (1 mg/animal) in 30 μL of DMSO twice weekly. Once tumors started to grow, their sizes were measured twice weekly and the tumor volume was calculated. A, top, photographs of representative mice with tumors from each group. Bottom, photographs of excised tumors from each group. B, average tumor volume of control and fisetin-treated mice plotted over days after tumor cell inoculation. Points, mean of 16 tumors in eight animals; bars, SD. *, P < 0.001, versus the control group. C, number of mice remaining with tumor volumes of 1,200 mm³ after they received treatment with fisetin for the indicated days. D, serum PSA levels were analyzed by ELISA. Points, mean of eight animals; bars, SE. *, P < 0.001, versus the control group. Details are described in Materials and Methods.

Figure 6

Summary of the mechanism of fisetin action in prostate cancer cells. Fisetin decreases AR promoter activity leading to decreased AR expression. In addition, fisetin competes with natural AR agonist DHT to physically interact with the expressed AR protein. Once bound, it decreases the interdomain N-C interaction of AR leading to (a) a decrease in AR stabilization and (b) a decrease in AR transactivation function. This results in decreased expression of AR target genes and, hence, negatively influences the growth of PCa cells in vitro and in vivo.