Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells
Huiqing Yuan1,2,
Aiyu Gong1 and
Charles Y.F. Young1,*
1 Departments of Urology and Biochemistry/Molecular Biology, Mayo Graduate School, Mayo Clinic, Rochester, MN 55905, USA and 2 Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, P. R. China
* To whom correspondence should be addressed at: Departments of Urology and Biochemistry/Molecular Biology, Mayo Clinic/Foundation, Guggenheim Building 502, 200 First Street SW, Rochester, MN 55905, USA. Tel: +1 507 284 8336; Fax: +1 507 284 2384; Email: young.charles{at}mayo.edu
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Abstract
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The transactivation function of the human androgen receptor (AR) can be regulated by several coregulators that may be either positive or negative. Ubiquitous transcription factor Sp1 not only regulates the basal expression of the AR but also acts as its coregulator. Our previous study has shown that quercetin, one of the main polyphenols, can effectively inhibit the expression and function of the AR. The present study is to address if quercetin may affect Sp1's action on AR transactivation activity in human prostate adenocarcinoma cell lines, LNCaP and PC-3. First, we showed that indeed in transient transfections Sp1 could enhance transcriptional activity of the AR promoter and of androgen upregulated gene promoters, i.e. the prostate-specific antigen and the hK2 genes. Interestingly, the enhancing activity of Sp1 could be repressed by quercetin. The gel shift and western blot analyses indicated that the specific DNA motif binding activity of Sp1 and its protein levels were not altered by quercetin. However, the state of interaction of Sp1 with the AR treated by quercetin plus androgen was different from that by androgen treatment or none as demonstrated by coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down assays. Moreover, we showed that quercetin caused changes in post-translational modification of AR protein. The above findings strongly suggest that changes induced by quercetin in post-translational modification of the AR and in states of physical interaction of Sp1 with the AR may be critical for the attenuation of AR's function.
Abbreviations: AR, androgen receptor; AP-1, activator protein-1; ARE, androgen responsive element; ß-gal, ß-galactosidase; DTT, dithiothreitol; GST, glutathione S-transferase; hK2, human glandular kallikrein; Mib, mibolerone; PBS, phosphate buffered saline; PMSF, phenylmethanesulfonyl fluoride; PSA, prostate-specific antigen; Sp1, promoter specificity protein 1; CREB, cAMP response element binding protein; CBP, CREB-binding protein
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Introduction
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The androgen receptor (AR) is a member of nuclear receptor superfamily of transcription factors (1,2), and plays an important role in proliferation, differentiation, maintenance and function of the prostate (3,4). The aberrant regulation of the AR is implicated in the formation and progression of prostate cancer. Evidence has shown that the AR is expressed in most of primary, metastatic and hormone-refractory malignant stages of prostate cancer (5,6). An increase in heterogeneity of AR expression may be related with higher grade or poorer prognosis of the tumor (7,8). Transactivation of the AR can be regulated by interacting with coregulators (911) including cAMP response element binding protein (CREB)-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). Also, it has been found that the AR interacts with transcription factors such as Smad3 (12), AP1/c-Jun (13) and Ets family of transcription factors (14,15).
Sp1 is an ubiquitously expressed transcription factor that belongs to a zinc finger family (1619). It is believed that Sp1 regulates gene transcription by binding to a GC rich element (or GC box) in the promoter of target genes. Sp1 has been recently shown not only to interact directly with basal transcription factors including TFIID (20), and TBP (21), but also to physically associate and functionally cooperate with a number of sequence-specific transcription factors such as NF-
B (22,23), Oct-1 (24) and GATA-1 protein (25). On the other hand, some of the transcription factors can impair Sp1-mediated transcriptional activity. For example, the promyelocytic leukemia protein represses Sp1-mediated transcription by preventing it from binding to DNA (26). Tumor suppressor protein von Hipple-Lindau (VHL) interacts with Sp1 and inhibits vascular endothelial growth factor (VEGF) promoter activity (27).
Steroid hormone receptors have also been found to interact with Sp1 in the regulation of target genes. The estrogen receptor and Sp1 can form a complex and result in transactivation of heat shock protein p27 gene promoter in an estrogen responsive element-independent manner (28). Similarly, progesterone activates p21WAF1 gene expression through a mechanism that involves interaction between the progesterone receptor and Sp1 (29). More recently, it was reported that the AR could interact with Sp1 for either transactivation or suppression of target gene expression. Studies of Curtin et al. (30) demonstrated that androgens suppress GnRH-stimulated rat LHß gene expression, which occurs through proteinprotein interaction of AR and Sp1 that reduces Sp1 DNA binding activity. However, the association of AR and Sp1 results in synergistic activation of p21WAF1 gene promoter (31). Therefore, whether the interaction of Sp1 and the AR can result in transactivation or suppression may depend on a particular target gene, a cell type or both.
Quercetin, an abundant polyphenol compound in edible fruits and vegetables, has shown its inhibitory effect on tumor development via various mechanisms (3234). Our previous studies demonstrated the inhibitory effect of quercetin on the expression and function of the AR in LNCaP and/or LAPC-4 human prostate cancer cell lines, and consequently on the androgen-induced synthesis and the secretion of both prostate-specific antigen (PSA) and human glandular kallikrein (hK2) as well as other androgen regulated genes (35). We propose that the AR inhibitory property of quercetin may potentially have a role in reducing the risk of developing prostate cancer. Although, as mentioned above, quercetin can be an effective inhibitor to the AR, it is not completely clear how it actually affects AR's functions. The aim of this study is to gain a better understanding of the potential role of Sp1 in the quercetin-mediated AR inhibitory effect.
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Materials and methods
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Cell culture and chemicals
Human prostate cancer cell lines, LNCaP and PC-3 (obtained from The American Type Culture Collection, Rockville, MD) were routinely grown in 100 mm, 60 mm or 12-well plate culture dishes in RPMI 1640 medium (Celox, St Paul, MN) supplemented with 5% fetal bovine serum (FBS) (Biofluids, Rockville, MD) and 5% CO2 at 37°C until reaching
5070% confluence. Cells were maintained in serum-free RPIM 1640 medium for a further 24 h to deplete endogenous steroid hormones before experiments, and then treated with mibolerone (Mib, 1 nM) with or without quercetin (100 µM) in the medium containing 5% charcoal stripped serum. Mibolerone (Mib) (New England Nuclear) is a synthetic androgen that is not metabolized in cell culture. The dose 1 nM of androgen is at the physiologic concentration range and used throughout the study. Quercetin (LKT, Inc., St Paul, MN) was dissolved in dimethyl sulfoxide (DMSO). DMSO was also used as a control vehicle.
Transient transfection and luciferase reporter gene assay
Cells were seeded in 60 mm dishes or 12-well plates at a density of 2.5 x 105 cells/dish or 6 x 104 cells/well and grown under the conditions described above for 48 h before transfection. For transfection into LNCaP cells, pGL-3 basic vector (Promega, Madison, WI) containing the AR promoter (1380/+577) (AR promoter, 2.5 µg/dish), pGL3 basic vector with 6 kb PSA promoter (5824/+12) (PSA6 kb, 2.5 µg/dish) or pGL3-SV-40 with three copies of Sp1 binding motif of the AR promoter (AR-3Sp1, 1 µg/well) and pcDNA-Sp1 expressing human Sp1 (0.5, 1.0 or 2.0 µg/dish or 0.2 µg/well) were cotransfected by using GeneFECTORTM Transfection Reagent (Venn Nova, LLC). For transfection into PC-3 cells, a human AR expression vector pSG5-AR (pSG5-hAR, 1 µg/well) was cotransfected with pGL3-SV-40-hK2-3ARE (1 µg/well) and pcDNA-Sp1 expressing human Sp1 (0.2 µg/well) by using Lipofectamine (Life Technologies, Inc.). The parental vectors pGL3 basic (1 µg/well) and pGL3-SV40 (1 µg/well) were used as controls. A plasmid containing a CMV promoter/ß galactosidase (ß-gal expression vector, 0.4 µg/dish or 0.2 µg/well) was cotransfected to normalize transfection efficiency. After 24 h of transfection, cells were either treated with quercetin (100 µM) or remained untreated in the presence or absence of 1 nM Mib for an additional 24 h in the medium with 1% charcoal stripped serum. Cell extracts were prepared and used for ß-gal assay and luciferase assays (Promega, Madison, WI). At least three independent transfections were performed.
Western blot-analysis
Cells were grown in 100 mm dishes under the same conditions described above and treated with or not with quercetin (100 M) in the presence or absence of 1 nM Mib for 24 h. Removal of the media was followed by a brief rinse with 6 ml cold phosphate buffered saline (PBS), and cell pellets were obtained by centrifugation. Whole cell lysates were prepared with slight modification according to the method described previously (36). Briefly, the cell pellets were resuspended with three packed cell volumes of extraction buffer [20 mM HEPES, pH 7.9, 20% glycerol, 0.5 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT)] containing freshly prepared protease inhibitors [50 µg/ml aprotinin, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM ß-glycerolphosphate] and subjected to one cycle of freezethawing. The samples were then centrifuged at 14 000 g for 30 s and supernatant was diluted with 2.5 volumes of dilution buffer (extraction buffer containing 150 mM of KCl) to reduce the salt concentration. The Bradford protein assay (Bio-Rad, Hercules, CA, USA) was employed for quantifying protein content and the samples were stored at 80°C in small aliquots. Proteins were run with a reducing SDS polyacrylamide gel (412%) and electro-transferred onto nitrocellulose membrane (Bio-Rad). The nitrocellulose membranes were immediately stained by Ponceau S (0.1% Ponceau S, 5% acetic acid) to show loading and transfer efficiency of proteins and photographed. The blots were then blocked with 5% non-fat milk in TBST buffer (20 mM TrisHCl, 137 mM NaCl and 0.1% Tween-20, pH 8.0) prior to incubation with specific antibody to Sp1 (Santa Cruze Biotechnology Inc., Santa Cruz, CA, sc-59) or AR (BD Biosciences, 554225) for 1 h at room temperature. Membranes were incubated with an anti-rabbit or anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (1:10000 dilution; Amersham Pharmacia Biotech UK limited, Buckinghamshire, UK) at room temperature for 1 h and visualized by enhanced chemiluminescence substrate (ECL, Amersham Corporation, Arlington Heights, IL).
Nuclear extracts
LNCaP cells were grown in 100 mm dishes and treated with 100 µM quercetin in the presence or absence of 1 nM Mib for 24 h. Nuclear extracts were prepared according to the user manual of TransFactor Extraction kit (Clontech Lab, Inc.). Briefly, cells were harvested with cold Ca2+, Mg2+ free PBS and centrifuged at 1000 r.p.m., 4°C for 10 min. Cell pellets were then washed with ice-cold PBS and resuspended in a lysis buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT and Boehringer complete protease inhibitor cocktail. The suspension was then passed through a 27-gauge needle and centrifuged for 20 min at 10 000 g. After removing the supernatant, the crude nuclear pellet was resuspended in nuclear extraction buffer (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT and protease inhibitor cocktail). The nuclear pellet was disrupted by passing through a 27-gauge needle and shaken gently at 4°C for 30 min. The soluble supernatant was collected by centrifugation at 20 000 g for 5 min and stored at 80°C in small aliquots. The protein concentrations of nuclear extracts were measured using Bio-Rad protein assay kit (Bradford assay).
Electrophoretic mobility shift assay (EMSA)
Double stranded oligonucleotides corresponding to the specific binding sequence of Sp1 in the AR promoter as described before (37) were end-labeled with [
-32P]ATP by T4 polynucleotide kinase to a specific activity of 2.4 x 1072.4 x 108 cpm/µg. The EMSA was performed according to the user instructions of Promega. Briefly, the 32P-labeled Sp1 probe (1 x 105 cpm) was mixed with or not with nuclear extracts (4 µg/reaction) in the presence or absence of 100-fold molar excess of unlabeled Sp1 oligonucleotides as a competitor for 20 min at room temperature. The final reaction contained 10 mM TrisHCl, pH 7.5, 1 mM MgCl2, 50 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT and 2 µg poly(dIdC). Finally, the reaction mixtures were eletrophoresed in a prerun 6% polyacrylamide with 0.5 x TBE buffer at 150 V for 11.5 h. The gel was vacuum dried for autoradiography.
Coimmunoprecipitation
LNCaP cells were plated in 100 mm culture dishes and kept in serum-free RPMI 1640 medium for 24 h before treatment. The cells were treated with mibolerone (1 nM) or quercetin (100 µM) for an additional 24 h in a medium containing 5% charcoal stripped serum. Whole cell lysates were prepared as described above and precleared with anti-rabbit or anti-mouse IgG and protein A-agarose or protein G-agarose (Santa Cruz Biotechnology, Inc.). Aliquots of 500 µg of proteins were incubated in binding buffer (20 mM HEPES, pH 7.9, 20% glycerol, 150 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM ß-glycerolphosphate) with 2 µg of antibody directed against AR (BD Biosciences), or Sp1 (Santa Cruz Biotechnology, Inc.) at 4°C overnight. The protein A- or protein G-agarose beads were added and incubated for 2 h at 4°C. The immunoprecipitates were washed four times with buffer containing 50 mM TrisHCl, pH 7.5, 0.5% IGEPAL CA-630, 150 mM NaCl, 0.2 mM EDTA, 0.5 mM PMSF, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM ß-glycerolphosphate. Immunocomplexes were recovered by heating at 75°C for 10 min in SDS sample buffer and analyzed by electrophoresis using 7% NuPAGE Trisacetate gel (Invitrogen). Western blot detection was performed as described above.
Purification of GST fusion protein and GST pull-down assay
Full-length and truncated GST-Sp1 fusion protein expression constructs pGEX-2TK-MCS-Sp1-full, pGEX-2TK-MCS-Sp1 (1611) and pGEX-2TK-MCS-Sp1 (612778) were kindly provided by Dr Erhard Wintersberger (Universität Wien, Vienna, Austria) (38). BL21 bacterial cells (Escherichia coli) harboring the various GST-Sp1 or GST (pGEX-4x) plasmids mentioned above, were grown at 37°C until the absorbance at 600 nm reached 0.60.8. Cells were induced with 0.2 mM isopropylthiogalactopyranoside (IPTG) and grown for an additional 3 h. Cell pellets were obtained by centrifugation and resuspended in PBS buffer with 0.5 mM PMSF, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM ß-glycerolphosphate and 1 mg/ml lysozyme. The bacterial cells were disrupted by three freezethaw cycles and the suspensions were centrifuged at 13 000 r.p.m. for 30 min at 4°C. The supernatant was aliquoted and stored at 80°C until use.
The above GST-fusion or GST protein extracts were incubated with glutathione Sepharose beads (Amersham Pharmacia Biotech) in PBS buffer with protease inhibitors for 4 h at 4°C. Pelleted beads were washed three times with PBS buffer, twice with wash buffer (50 mM TrisHCl, pH 7.5, 0.5% IGEPAL CA-630, 150 mM NaCl, 0.2 mM EDTA, 0.5 mM PMSF, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM ß-glycerolphosphate) with the addition of 0.5% dry milk and twice with binding buffer (20 mM HEPES, pH 7.9, 20% glycerol, 150 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 50 µg/ml aprotinin, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM ß-glycerolphosphate). The beads were incubated with equal amounts of whole LNCaP cell lysates (500 µg) prepared in the last section at 4°C overnight. The beads were washed four times with wash buffer, resuspended in 40 µl of SDS sample loading buffer and heated at 75°C for 10 min and 15 µl of the supernatant was loaded onto 7% SDSPAGE gel for western blot-analysis.
Statistics
The data were analyzed by 2-tailed Student's t-test. P < 0.05 was accepted as the level of significance.
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Results
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Sp1 regulated AR promoter activity is repressed by quercetin
As shown previously, quercetin can suppress AR promoter activity (35). Since the Sp1 binding sequence is the major positive regulatory element in the AR promoter (3941) and is the target for downregulation by green tea polyphenols (37), it prompted us to examine the potential role of Sp1 on quercetin-mediated inhibition effect in prostate cancer cell lines by performing transient transfection experiment. The data in Figure 1A showed that cotransfection of LNCaP cells with a construct encoding full-length Sp1 along with an AR promoter-luciferase vector resulted in a significant increase in AR promoter-mediated luciferase expression in a dose-dependent manner. However, the luciferase activities of the AR promoter alone or enhanced by Sp1 were drastically inhibited in the presence of quercetin. To further demonstrate the role of the Sp1 binding site, a construct containing three copies of the Sp1 binding site in the AR promoter (pGL3-SV40-AR-3Sp1) was used in transfections as shown in Figure 1B. Sp1-mediated stimulation was seen in reporter activity, and repression of the reporter activity was achieved with quercetin treatment. Together, the data suggest that stimulatory function of Sp1 on the AR promoter can be suppressed by quercetin.

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Fig. 1. Inhibition of Sp1-mediated transactivation on the AR promoter by quercetin in LNCaP cells. (A) LNCaP cells were cotransfected with pGL3-AR spromoter-luciferase reporter plasmid and increasing amounts of pcDNA-Sp1 expression plasmid as indicated. The cells were treated with 1 nM Mib or 1 nM Mib + 100 µM quercetin for an additional 24 h after transfection. (B) LNCaP cells were cotransfected with luciferase reporter plasmid containing three copies of the Sp1 binding motif of the AR promoter (pGL3-SV40-AR-3Sp1) and pcDNA-Sp1 expression plasmid, or the parental vector (pGL3-SV40) as a control. In each transfection, the pCMV-ß-galactosidase was cotransfected to monitor transfection efficiency. Cell extracts were prepared from above transfections for luciferase and ß-gal assays. The resulting luciferase activities were further normalized to ß-gal and expressed as relative activity ± standard error (SE). At least three separate experiments were performed. In panel (A), **P < 0.01 and ***P < 0.001 when compared with group AR promoter + Mib only. In panel (B), *P < 0.05, when compared with group AR-3Sp1 alone; **P < 0.01, when compared with group AR3Sp1 +Sp1.
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Sp1-mediated stimulation on transactivation function of AR is inhibited by quercetin
We wanted to investigate further whether Sp1 has a general role as a coactivator in transactivation function of the AR. PSA and hk2 are androgen-inducible genes that contain androgen responsive elements (AREs) to which the AR binds. The expressions of these genes are highly dependent on the regulation of androgens through the action of the AR. As seen in Figure 2A, Sp1-enhanced androgen induction of reporter gene activity was observed by cotransfection of LNCaP cells with a PSA 6 kb promoter-luciferase reporter construct and different amounts of the Sp1 expression vector. Sp1 alone had no effect on the PSA 6 kb-luciferase activity. A strong suppressive effect of quercetin was clearly seen on androgen induction of the PSA 6 kb promoter even with cotransfection of Sp1. We then used luciferase reporter plasmid containing three copies of ARE in hk2 gene to test whether Sp1 truly acts as coactivator of the AR and whether Sp1 coactivating function could be affected in the presence of quercetin in a human prostate cancer cell line, PC-3, which expresses very little of the AR. As expected in Figure 2B, the androgen induced activity of hk2-3ARE plasmid was further increased by Sp1, However, the activity stimulated by Sp1 was blunted with quercetin treatment.

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Fig. 2. Functional synergy of Sp1 on the AR transcription activity of androgen-response genes was repressed by quercetin. (A) LNCaP cells were cotransfected with pGL3-PSA6 kb promoter luciferase reporter, the indicated amounts of pcDNA-Sp1 expression plasmid and the pCMV-ß-galactosidase. The pGL3 basic vector was included as a control. The cells were incubated with none (control), 1 nM Mib or 1 nM + 100 µM quercetin for 24 h after transfection. *P < 0.05, a comparison between group PSA6 kb + Mib and group PSA6 kb + 0.5 µg Sp1+ Mib; **P < 0.01, a comparison between group PSA6 kb + Mib and group PSA6 kb + Mib + 1 µg Sp1; ***P < 0.001, a comparison between group PSA6 kb + Mib and group PSA6 kb + Mib + quercetin, group PSA6 kb + Mib + 0.5 µg Sp1 and group PSA6 kb + Mib + 0.5 µg Sp1 + quercetin or group PSA6 kb + Mib + 1 µg Sp1 and group PSA6 kb + Mib + 1 µg Sp1 + quercetin. (B) PC3 cells were cotransfected with luciferase reporter plasmid pGL3-SV-40 with three copies of ARE of the hk2 gene (pGL3-SV40-hk2-3ARE), human AR expression construct or pcDNASp1 expression vector as indicated. A parental vector (pGL3-SV40) was also included as a control. The normalized, relative luciferase activities (mean ± SE) of at least three independent experiments were shown. *P < 0.05, a comparison between group hAR+hK2-3ARE and group hAR+hK2-3ARE + Mib; **P < 0.01, a comparison between group hAR+hK2-3ARE + Mib and group hAR+hK2-3ARE +Mib + quercetin or group hAR + hK2-3ARE + Mib + Sp1; ***P < 0.001, a comparison between groups hAR + hK2-3ARE + Mib + Sp1 with and without quercetin.
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Sp1 DNA binding activity is not affected by quercetin
The above results led us to investigate whether the function and expression of Sp1 could be affected by quercetin. As shown in Figure 3, specific DNAprotein complexes were detected with a double stranded DNA probe containing a putative Sp1-binding site of the AR promoter (nt 60 to 40). Nuclear extracts from LNCaP cells treated with quercetin showed almost the same Sp1 binding activity to the Sp1 binding element when compared with the no treatment or Mib treatment alone (lanes 2, 3 and 4), and these binding activities were androgen-independent. All detected DNAprotein complexes were specific for the Sp1 binding element, as they were fully competed out by a 100-fold molar excess of cold Sp1 oligonucleotide (lanes 5, 6 and 7) but not by the AP1 oligonucleotide (lanes 8, 9 and 10). However, the intensities of the retarded bands (lanes 8, 9 and 10) were slightly decreased in competition with the 100-fold excess of the AP1 oliognucleotide. Similar results were also obtained with a Sp1 DNA probe containing a consensus Sp1 binding site (data not shown).

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Fig. 3. Specific DNA binding activity of Sp1 to the Sp1 binding site in the AR promoter was not affected by quercetin. Nuclear extracts (4 µg/reaction), prepared from LNCaP cells treated with none, Mib alone or Mib + quercetin for 24 h, were incubated with 32P-labeled double-stranded Sp1 oligonucleotides ± 100 x unlabeled Sp1 or AP1 oligonucleotides for 20 min at room temperature. The Sp1DNA complexes were resolved on a 5% non-denaturing polyacrylamide gel. The retarded Sp1 bands (see arrow) were visualized by autoradiography. NS, non-specific competitor. These experiments were repeated several times with essentially identical results.
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Sp1 protein level is not decreased in the presence of quercetin
Given the fact that quercetin did not affect Sp1 function such as its in vitro DNA binding activity it might also indicate that quercetin did not affect Sp1 protein expression. Further, we used western blot-analysis to show Sp1 protein levels in whole cell lysates, cytosolic extracts and nuclear extracts, respectively. First, Figure 4A showed that there was no significant difference in Sp1 protein expression between whole cell lysates treated with quercetin or untreated cell lysates in the presence of Mib (lanes 2 and 3). Since Sp1 is a nuclear protein and functions in the nucleus, the presence of Sp1 in the cytosolic and nuclear extracts was further examined. As seen in Figure 4B, Sp1 protein was mainly present in the nucleus but not in the cytosolic fraction (compare lanes 1, 2 and 3 with lanes 4, 5 and 6) regardless of the treatment cells received. Thus, quercetin had little effect on nuclear localization of Sp1 protein in LNCaP cells (lanes 5 and 6). In addition, we showed in Figure 4C that the androgen stimulated the nuclear localization of the AR. Interestingly, the results in Figure 4C suggested that although quercetin could reduce AR protein, it might somewhat facilitate AR nuclear localization.

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Fig. 4. Western blot analysis of Sp1 expression level in LNCaP cells. (A) Whole cell lysates from LNCaP cells treated with none, Mib or Mib + quercetin were resolved on a 412% SDSPAGE gel for western blot-analysis of anti-Sp1 antibody. (B) Cytosolic and nuclear extracts prepared from LNCaP cells treated with none, Mib or Mib with quercetin were subjected to gel electrophoresis for western blot-analysis of anti-Sp1 antibody. (C) Western blot-analysis of AR protein expression in whole cell lysates, cytosolic and nuclear extracts from LNCaP cells treated with or without the above chemical was performed. Ponceau S-stained protein bands were served to monitor protein loading and transferring efficiency.
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Quercetin affects the interaction state of Sp1 and the AR: coimmunoprecipitation and GST pull-down
The studies described above demonstrated that the quercetin might have little effect on certain Sp1 functions including DNA binding and nuclear localization as well as protein expression levels. Therefore we speculated that the inhibitory effect of quercetin on Sp1's coactivator activity might be due to the alteration of proteinprotein interaction between Sp1 and the AR. In an attempt to obtain direct evidence for such an interaction between Sp1 and the AR in cells, we performed coimmunoprecipitation followed by western blot-analysis to test this possibility. Proteins potentially associated with Sp1 were first precipitated with anti-Sp1 antibody and subsequently probed with AR-specific antibody and Sp1 antibody by western blot assay. As indicated in Figure 5A, AR protein was clearly detectable in the immunoprecipitates from cells treated with quercetin in the presence of Mib (lane 6), while less AR protein was precipitated from the cell extracts treated with Mib alone (lane 5) and no AR protein was detected in the untreated cells (lane 4). When using normal IgG very little signal was produced in all above experiments (lanes 7, 8 and 9). Reciprocally, we extended our coimmunoprecipitation analyses by using an anti-AR antibody. Endogenous Sp1 protein was coimmunoprecipitated with the AR from cells treated with quercetin in Figure 5B (lane 6). Somewhat surprisingly, almost no detectable Sp1 protein could be seen in cells treated with Mib alone (lane 5). Note that there is a band above Sp1 protein found across all the three groups (lanes 4, 5 and 6) that is considered as a non-specific band.

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Fig. 5. Coimmunoprecipitation analysis of the association of Sp1 and the AR. Proteins in whole cell extracts from LNCaP cells treated with none, Mib or Mib + quercetin were prepared. Immunoprecipitation experiments were performed with either anti-Sp1 antibody (A), or anti-AR antibody (B). Protein complexes were fractioned on polyacrylamide gels and the coprecipitated Sp1 and AR proteins were detected by using either anti-Sp1 antibody or anti-AR antibody. Normal IgG was used as the negative control. Input represents 10% of the whole cell extracts used in the above experiments.
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To further verify the interaction between Sp1 and the AR, glutathione S-transferase (GST) pulldown experiments were employed to determine the region(s) of Sp1 that might be involved in proteinprotein interaction with the AR. Fusion proteins containing full-length of human Sp1 (GST-Sp1-full), two truncated forms of Sp1 fused at the C-terminus of GST or GST alone were used for this purpose (Figure 6A). For detecting Sp1 binding proteins, precleared cell extracts from LNCaP cells, untreated, or treated with Mib or quercetin plus Mib were incubated with the above GST fusion proteins or GST bound to glutathioneagarose beads. The proteinprotein complexes were analyzed by anti-AR antibody. As shown in Figure 6B, AR was not retained by GST, whereas it bound itself to the GST-Sp1-full in accordance with the immunoprecipitation results shown in Figure 5, suggesting that the interaction between the two factors was specific. Again, Sp1 did not seem to be able to pull down the AR in the absence of androgen treatment. Moreover, pull-downs with two truncated Sp1 forms (Figure 6A) showed that the liganded AR under influence of quercetin could provide further interaction toward the C-terminal region of Sp1 protein.

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Fig. 6. Mapping region(s) of Sp1 involved in its association with the AR in the presence of quercetin. (A) Schematic representation of the full-length or truncated Sp1 forms used in the GST pull-down assay. The numbers in parentheses indicate the amino acids of Sp1 protein linked to the GST. The letters AD indicate different domains of Sp1 protein, the letter ZF indicates three zinc finger DNA binding motifs of Sp1. (B) Whole cell lysates from LNCaP cells free of treatment, or treated with Mib alone or combined with quercetin were incubated with GST or the indicated GST-Sp1 fusion proteins coupled sepharose beads as described under Materials and methods. Bound proteins were recovered and analyzed by 7% of SDSPAGE gel and immunoblotting with an anti-AR antibody. Input represents 10% of the whole cell extracts used in the binding experiments. The images were obtained by 15 min exposure of the blots.
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Androgen dependent phosphorylation of the AR is reduced in the presence of quercetin
AR is a phosphoprotein and phosphorylation of the AR is important for AR function. Blok's studies (42,43) have indicated that androgen-induced hyperphosphorylation states or hyperphosphorylated isoforms of the AR can be differentiated from hypophosphorylation states or hypophosphorylated isoforms of the unliganded AR by comparison of the migration rates of the AR isoforms with and without androgen treatment in western blots. The results in Figure 7 indicate that Mib induced slow migrated AR isoforms (lane 2 or 4) whereas fast migrated isoforms without Mib treatment were seen (lane 1), indicating occurrence of hyperphosphorylation in the AR by Mib. Quercetin seemed to repress AR hyperphosphorylation by Mib (lane 3 or 5). Since the AR protein level was reduced by quercetin, we also increased the protein amount (30 µg/lane in lane 5 of Figure 7) in quercetin-treated sample to get better observation. The result remained the same as in the migration rates of the AR isoforms.

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Fig. 7. Effect of quercetin on the AR phosphorylation status. Indicated amounts of proteins in whole cell extracts from LNCaP cells treated with none, Mib or Mib with quercetin were subjected to western blot-analysis. The AR migrated differentially due to its phosphorylation states around the area of 110 kDa.
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Discussion
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We have previously reported that quercetin, one of the major plant polyphenols, showed its inhibitory effect on the expression and function of the AR (35). In the same study we showed that quercetin at concentrations as low as 10 µM could have effects on inhibiting AR-mediated gene expression in LNCaP cells. Quercetin at concentrations of 50100 µM demonstrated optimal inhibitory effects on AR's function. Therefore, a relatively high concentration, i.e. 100 µM, of quercetin was used in this study. Currently, whether this concentration can be achieved in human local tissues is unknown. A very recent study (44) suggested that >50 µM of quercetin might be obtainable in rat circulation when quercetin at 150 mg/kg body weight was given orally. Moreover, quercetin administered at dose levels of 401900 mg/kg/day in male or female rats showed no signs of treatment-related clinical signs of toxicity or death (45). This seems to suggest that quercetin is a safe chemical for potential clinical utility.
Previously, we showed (37) that EGCG, a green tea polyphenol, could downregulate Sp1 protein levels and therefore affected AR's expression and function. Note that the AR gene promoter harbors a Sp1-binding GC box site, which is the major factor for regulating the basal expression of the AR (39). We confirmed that the Sp1 binding site in the AR promoter could be regulated by ectopic coexpression of Sp1 in prostate cancer cells. We now demonstrated that quercetin could inhibit the Sp1-mediated transcription activity of the AR gene promoter. The results of this study seem to suggest that the mechanism by which quercetin inhibits AR function may be different from that of the tea polyphenol, EGCG.
The present study revealed that transcription factor Sp1 as a coactivator for the AR was subjected to the influence of quercetin. Our previous study (35) demonstrated that quercetin could repress AR's function without affecting endogenous levels of Sp1 protein in our model cell system, LNCaP. Nevertheless, Figures 1 and 2 helped establish the inhibitory effect of quercetin on the role of Sp1 in AR-mediated gene expression. However, the results of our gel shift analysis and western blot assay seemed to rule out the possibility that the inhibition of quercetin on the AR was achieved via inhibiting Sp1's functions such as DNA binding ability and nuclear localization or by reducing its expression levels.
We then examined whether the suppressive effect of the quercetin might, at least in part, occur at the interaction of Sp1 and the AR. This possibility was confirmed by coimmunoprecipitations and GST pull-downs as in Figures 5 and 6, respectively. Using anti-Sp1 antibody for coimmunoprecipitation, showed more AR molecules being retained in the precipitates from cells treated with quercetin plus androgen than that from androgen alone (Figure 5A), suggesting quercetin may increase the binding affinity between AR and Sp1. Further, with anti-AR antibody, endogenous Sp1 protein was coimmunoprecipitated with the AR from cells treated with Mib plus quercetin (lane 6 in Figure 5B) but not in cells treated with Mib alone or untreated cells. Taken together from Figure 5A and B, although we cannot pinpoint how exactly the AR and Sp1 interact, the evidence is compelling to infer that quercetin induces much stronger interaction of the AR and Sp1 than that in the treatment of Mib alone. Potentially, a relatively weak interaction of the AR and Sp1 in the treatment of Mib might cause the inability of pull-down of Sp1 by anti-AR antibody. It is possible that the AR antibody could also weaken the interaction of the AR and Sp1 owing to conformation change or steric interference induced by the antibody. Regardless, the states of interaction of the AR and Sp1 must be somewhat different under the above conditions.
The GST-pull-down seemed to confirm the above notion. These pull-down experiments demonstrated that there was an interaction of the ligand-activated AR and Sp1. Moreover, the same experiments (Figures 5A and 6) seemed to indicate that there was no interaction between the AR and Sp1 when the AR was not activated by androgens. Importantly, for the first time we showed that there were some subtle differences in the states of the interaction of the AR and Sp1 depending on the treatments received. These different binding states of Sp1 to the AR may affect transactivation activity of the AR. We postulate that quercetin may increase the binding affinity of Sp1 to the AR that may reduce AR's function in prostate cancer cells.
Consistent with previous reports (30,31), we showed that the AR forms a complex with Sp1 in a ligand-dependent manner. Sp1 seems to act as a coactivator for the ligand-activated AR in gene activation in LNCaP cells. One of the previous studies suggested that the DNA-binding domain of the AR is the site for binding Sp1 (30). Porter's studies showed that the association of the estrogen receptor with the C-terminal region of Sp1 resulted in synergistically functional induction of heat shock protein 27 (28). On the contrary, the interaction of the ligand-activated AR and Sp1 resulted in reducing Sp1 binding to the rat LHß gene and suppressing the gene expression (30). Recently, it was reported that Sp1 protein bound to GC-rich elements in the promoter of a housekeeping gene MAZ and repressed the expression of the gene. A novel repressive domain was identified in the C-terminal region of Sp1 (amino acids 622788) (46). Our study seems to suggest that quercetin may induce an additional interaction of the AR with the C-terminal area of Sp1, resulting in repression of AR-mediated gene activation. At any rate, Sp1 seems to act as a coregulator, either positive or negative, for AR.
Currently, it is not clear how quercetin can induce such an interaction of AR and Sp1 as presented in this study. One clue is that the change in phosphorylation states of the AR by quercetin may have a role in regulating the state of interaction of these two factors. However, whether the AR phosphorylation states can affect interaction of AR with its transcriptional coregulators has not been established yet in the literature or in this study. In addition, quercetin might cause other coregulators to be involved in the formation of the repressive ARSp1 complex. Further investigations will be needed to confirm the possibilities and provide better understanding of the mechanism by which quercetin exerts inhibitory effect on the AR in prostate cancer cells.
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Acknowledgments
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This work is supported in part by NIH grants NCI/DK89000 and NCI 70892.
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Received September 21, 2004;
revised December 14, 2004;
accepted January 8, 2005.