| Androgens are produced primarily in the form of testosterone by Leydig cells in the testes and are generally found circulating throughout the body. Once testosterone has entered the cells, it is usually converted to dihydrotestosterone (DHT) by 5 α -reductase. Androgens play an important role in the proliferation, differentiation, maintenance and function of the prostate. Intriguingly, they may also be involved in the development and progression of prostate cancer. The androgen receptor (AR) is a phosphoprotein that mediates the actions of testosterone and DHT by acting as a transcription factor. The AR is capable of binding to both testosterone and DHT, although DHT has a higher affinity for the AR (approximately 2-fold to 10-fold) and is consequently the primary androgen bound by the AR. The AR is a member of the steroid receptor superfamily and is composed of three major domains: an N-terminal transcriptional activation domain, a central DNA-binding domain, and a C-terminal steroid-binding domain. Before binding its ligand, the AR is thought to be in an inactive state, in which it is bound to at least three heat shock proteins. Once the AR has bound DHT, some of these proteins dissociate, and there is a conformational change in the AR. The AR then translocates into nucleus and binds as a homodimer to a specific DNA site, called androgen responsive element (ARE), in the promoter of androgen-responsive genes such as prostate-specific antigen(PSA), human glandular kallikrein(hk2) etc, to activate transcription of these genes. Evidence suggests that the AR is still expressed and functional in many advanced or hormone refractory prostate tumors. Minimization orelimination of the function of AR might provide a very effective strategy for preventing the development and progression of the cancer.Quercetin, abundant polyphenols in fruits, vegetables and red wine, has shown its inhibitory effects on tumor development via various mechanisms. The low incidence of prostate cancer in oriental countries compared to that in western countries, in part, may be attributable to the consumption of a large amount of vegetables, soya and fruits containing plant polyphenols. Our previous studies demonstrated that quercetin exhibited inhibitory effect on the expression and function of the AR in two androgen responsive human prostate cancer cell lines, LNCaP and/or LAPC-4. However, it was not clear how the quercetin actually affects AR's activities.The first question we addressed was whether quercetin had any effect on the growth properties of LNCaP cells. Cells proliferation was measured by MTS analysis. Cells treated with the androgen DHT alone showed growth enhancement. In the presence of DHT, quercetin had inhibitory effect on the cell growth in a dose-dependent fashion, and significant inhibition on cell proliferation was achieved with higher concentrations of quercetin (50 |imol/L and 100 |imol/L respectively, p< 0.01). Since the AR is a nuclear protein and functions in nucleus. We prepared whole cell lysates, cytosolic extracts and nuclear extracts for western blot after quercetin treatment to get more detailed information. The AR protein level was stimulated significantly by androgen, and was decreased by quercetin treatment. Further more, quercetin, although it could reduce AR protein, might somewhat facilitate AR nuclear localization in the presence of androgen.The transactivation function of the AR can be regulated by several co-regulators that may be either positive or negative. c-Jun, a member of the basic leucine zipper family, is a main component of AP-1 protein complex which can directly activate or induce transcription of many genes containing AP-1 DNA binding sequences. It has been shown that the DNA binding domain of nuclear receptors may interact with the leucine zipper of c-Jun and result in mutualrepression. Our studies showed that qucrcctin caused an increase in expression of c-Jun as well as its phosphorylatcd form in a dose-dependent manner in prostate cell lines. Interestingly, the c-Jun and phosphorylatcd c-Jun protein levels were increased after 15 h and 24 h treatments with qucrcctin, and more strong band was observed in cells treated with qucrcctin for 24 h than that of 15 h treatment. Although c-Jun is an early response gene and usually has a short half-life, in this case, the protein levels of c-Jun remained obviously detectable for a long period after treatment. This suggests that c-Jun's transactivation ability may be separated from c-Jun's ability to the AR transactivation in the presence of quercetin. Gel shift assay demonstrated that the c-Jun induced by quercetin was functional because c-Jun has more strong affinity to a specific DNA sequence in quercetin treatment when compared to androgen treatment. Transient transfections showed that c-Jun repressed prostate-specific antigen(PSA) promoter activity and transcriptional activity of the AR promoter. Further, a luciferase reporter plasmid containing three copies of ARE in hk2 gene(hk2-3AR￡) was cotransfected with c-Jun in LNCaP cells, the androgen induced activity of hk2-3ARE plasmid was dramatically blunted with cotransfection of c-Jun expression vector in a dose-dependent manner. A similar result was also obtained in PC3 cells, which is androgen-independent and expresses very little of the AR, both PSA promoter and hk2-3ARE activities were repressed by cotransfection of c-Jun and human AR expression vectors. Since the hk2-3AR￡ plasmid only contains ARE sequence to which the AR binds specifically, the results support a mechanism in which overexpressed c-Jun mediates inhibitory effect on expression and function of the AR in a DNA-independent manner. One of the putative co-activator for the AR, CREB (cAMP response element-binding protein) binding protein (CBP) has intrinsic histone acetyltransferase activity. The CBP can increase the transcriptional activity of endogenous AR in LNCaP cells. In addition, CBP was also shown to act as a coactivator for c-Jun. It has been reported that the transcriptional interference between AR and API could be mediated through competition forlimiting amounts of CBP in cells [102,103). The expression of CBP in cells treated with quercetin and no treatment showed no visible difference. Transfection result demonstrated that CBP could stimulate androgcn-dcpcndcnt reporter activity, however, the CBP-mediated stimulation on the AR function was impaired by quercetin. These data showed that ectopic expression of CBP could not reverse quercetin-induced inhibition of AR transactivity. The results described above suggest that quercetin-mediated repression on PSA expression may be via, in part, c-Jun-AR interaction.Spl is a ubiquitously expressed transcription factor that belongs to a zinc finger family. It is believed that Spl regulates gene transcription by binding to a GC rich element (or GC box) in the promoter of target genes. Since the Spl binding sequence is the major, positive regulatory element in the AR promoter [88,89], this prompted us to examine the potential role of Spl on quercetin-mediated inhibition effect in prostate cancer cell lines. Cotransfection of LNCaP cells with a construct encoding full length Spl along with an AR promoter-luciferase vector resulted in a significant increase in AR promoter expression in a dose-dependent manner. However, the activities of the AR promoter alone or enhanced by Spl were drastically inhibited in the presence of quercetin. This result revealed that transcription factor Spl for the AR gene was subjected to the influence of quercetin.We next wanted to investigate whether Spl had a general role as a co-activator in transactivation function of the AR. Spl-enhanced androgen induction of reporter gene activity was observed by cotransfection of LNCaP cells with PSA promoter and different amounts of the Spl expression vector, a strong suppressive effect of quercetin was clearly seen on androgen induction of the PSA 6kb promoter even with cotransfection of Spl. Similarly, the androgen induced activity of hk2-3ARE plasmid which doesn't harbor any Spl binding site was further increased by Spl, while the activity stimulated by Spl was blunted with quercetin treatment. However, the results of our gel shift analysis showed that Spl binding activity to the specific DNA sequence was not affectedby quercetin, and western blot assay provided an evidence that Spl protein level was not reduced by quercetin neither. This seemed to rule out the possibility that the inhibition of quercetin on the AR was achieved via inhibiting Spl's functions such as DNA binding ability and nuclear localization or by reducing its expression levels. Together, the results implicate that Spl can be a positive transcription factor for the AR gene, and acts as a co-activator for the AR. A potential association of Spl and AR can be affected by quercetin.The above findings prompt us to investigate whether the suppressive effect of the quercetin might, at least in part, occur at the interactions of c-Jun, Spl and the AR. Indeed, cotransfection of increasing amounts of c-Jun caused a dramatic decrease in PSA promoter and hk2-3AR￡ activities in a dose response manner, while inhibition of c-Jun was reversed when Spl was cotransfected together. The profile of the inhibitory effect of c-Jun and induction of Spl on reporter activation in LNCaP cells was similar to that in PC-3 cells. Taken together, the results showed (i) overexpression of c-Jun was critical for inhibition of androgen-regulated reporter activity, (ii) functional interaction of c-Jun and Spl suppressed Spl-induced transactivation on androgen-response reporter activity. Since induction of PSA promoter and hk2-3ARE luciferase activities is dependent on AR, the transcriptional activity of AR could be regulated by ectopic expression of c-Jun and Spl without any apparent DNA binding. As proposed, there were functional interactions of c-Jun, Spl and AR in prostate cancer cells.This observation seems to suggest that there might be some physical associations among these three factors. We performed coimmunoprecipitation followed by western blotting analysis to test this possibility. Proteins associated with Spl were first precipitated with anti-Spl antibody and subsequently analyzed by western blotting with anti-c-Jun antibody or anti-AR antibody. Both c-Jun and the AR proteins were predominantly detected by enhanced bands in immunoprecipitates from cells treated with quercetin in the presence of Mib, precipitate from no treatment cells resulted in no detectable c-Jun or AR in it.Reciprocally, we extended our coimmunoprccipitaion analyses by using anti-c-Jun antibody. Both endogenous Spl and AR proteins were co-immunoprccipitatcd with the c-Jun from cells treated with quercctin but could not be precipitated from control. Finally, we performed coimmunoprecipitation by using three anti-AR antibodies against to N-terminal region, DNA-binding domain and C-terminal region of the AR, respectively. The immunoprecipitated c-Jun by anti-DNA binding domain of the AR was found significantly in cells treated with quercetin, and Spl bound to AR was observed in precipitates by antibodies against-DNA binding domain and C-terminal region of the AR respectively. Collectively, these results supported the presumption that c-Jun, Spl and the AR protein did physically interact in vivo and form a protein-protein complex in quercetin treatment cells. Associations of N-terminal of the AR and Spl, N-terminal and/or C-terminal of the AR with c-Jun resulted in down regulation of AR transcriptional activity on androgen-response genes in prostate cancer cells.To further obtain the direct evidence of interaction between Spl and the AR in vitro, glutathione S-transferase (GST) pulldown experiments were employed to determine the region(s) of Spl that might be involved in protein-protein interaction with the AR. Fusion proteins containing full-length of human Spl (GST-Spl-full), three truncated forms of Spl fused at the C terminus of GST or GST alone were used for this purpose. These pull-down experiments demonstrated that the AR was not retained by GST, whereas it bound to the GST-Spl-full in accordance with the immunoprecipitation results, suggesting that the interaction between the two factors was specific. Moreover, pull-downs with three truncated Spl forms showed that the liganded-AR under influence of quercetin could provide further interaction towards the C-terminal region of Spl protein.Currently it is not clear how quercetin can induce such interactions of the AR, c-Jun and Spl as presented in this study. One clue is that the change in phosphorylation states of proteins. Phosphorylation/dephosphorylation ofproteins is a common principle to regulate their biological activities including DNA binding affinity, interactions with other transcription factors, protein stability etc. The transactivation activity of c-Jun can be regulated through phosphorylation of clusters of serinc/threonine residues in N-terminus. The higher induction of phosphorylated c-Jun by quercetin might play an important role in repression of the AR function through protein-protein interactions with Spl and AR. In case of the AR, decreasing in phosphorylation states of the AR by quercetin was observed, the change in phosphorylation of the AR may have a role in regulating the states of interaction of these three factors.Collectively, for the first time we showed: (1) quercetin decreases phosphorylation states of the AR in the presence of androgen, whether the AR phosphorylation states can affect interaction of AR with its transcriptional co-regulators has not been established yet in the literature or this study. (2) Overexpression of c-Jun and phospho-c-Jun in quercetin-treated cells repressed the reporter activities of AR promoter, PSA promoter and hk2-3ARE, the inhibition of c-Jun on the AR was specific, because the reporter activity of hk2-3AR￡ which only contains three copies of ARE elements in plasmid depends on the AR transcriptional function. (3) The quercetin-mediated response was not attributable to the decrease in Spl biosynthesis and DNA binding activity, because no changes in Spl expression and binding activity to the specific sequence were observed in cells treated with quercetin. (4) Our reporter assays demonstrated that ectopic transfection of Spl specifically enhanced the AR transactivation, while cotransfection of c-Jun together with Spl inhibited the Spl-mediated transcriptional activity on the AR. This finding supported the notion that functional interactions of c-Jun, Spl and the AR were responsible for down-regulation of the AR transcriptional activity, and overexpression of c-Jun induced by quercetin might be a crucial role in this regulation. (5) Direct associations of these three factors were confirmed reciprocally by coimmunoprccipitation and GST pulldown. A multiple protein complex was formed in cells by quercetin treatment, and inhibited the AR transcriptionactivity in prostate-specific manner. In addition, quercetin might cause other co-regulators to be involved in the formation of the repressive AR-Spl complex. Further investigations will be needed to confirm the possibilities and provide with better understanding of the mechanism by which quercetin exerts inhibitory effect on the AR in prostate cancer cells. |
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