Androgen Receptor Nuclear Translocation Research Articles

Establishment and characterization of an androgen receptor‐dependent, androgen‐independent human prostate cancer cell line, LNCaP‐CS10

Hormone refractoriness is a lethal event for advanced prostate cancer patients, but the mechanisms of the disease are not well elucidated, especially for the so-called "outlaw" pathways of androgen receptor (AR)-dependent, androgen-independent hormone-refractory prostate cancer. Androgen-dependent prostate cancer LNCaP cells were treated with bicalutamide under an androgen-depleted condition to obtain refractory cells. In the obtained cell line, LNCaP-CS10, we analyzed the effects of androgen and bicalutamide on cell growth and prostate-specific antigen (PSA) production. In addition, AR gene mutation, AR expression levels, and AR subcellular localizations were analyzed. In LNCaP-CS10, cell growth and PSA production were found under an androgen-depleted condition and were induced by both R1881 and bicalutamide. Knocking down AR by siRNAs did suppress the growth and PSA production of LNCaP-CS10 cells in the androgen-depleted condition. In comparison to LNCaP, amplification or additional new mutations were not found in the AR genes, but AR nuclear translocation induced by bicalutamide was identified in the LNCaP-CS10 cells. The growth and PSA production of xenografted LNCaP-CS10 tumors, which secrete PSA not only in non-castrated SCID mice but also in castrated SCID mice, were not inhibited by bicalutamide. We have generated a bicalutamide-resistant and androgen-independent prostate cancer cell line, LNCaP-CS10, with outlaw activation both in vitro and in vivo. The LNCaP-CS10 cell line is beneficial for elucidating outlaw pathway mechanisms and evaluating the efficacy of new therapeutics for hormone-refractory prostate cancer.

Repressive Effects of Resveratrol on Androgen Receptor Transcriptional Activity

BackgroundThe chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.MethodologyThe AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+) cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(-) cells serving as controls. AR(+) cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP) assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE).ResultsAR in the AR (+) stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment.ConclusionWe demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

Decorin Suppresses Prostate Tumor Growth through Inhibition of Epidermal Growth Factor and Androgen Receptor Pathways

Epidermal growth factor receptor (EGFR) and androgen receptor (AR) pathways play pivotal roles in prostate cancer progression. Therefore, agents with dual-targeting ability may have important therapeutic potential. Decorin, a proteoglycan present in the tumor microenvironment, is known to regulate matrix assembly, growth factor binding, and receptor tyrosine kinase activity. Here, we show that in prostate-specific PtenP-/- mice, a genetically defined, immune-competent mouse model of prostate cancer, systemic delivery of decorin inhibits tumor progression by targeting cell proliferation and survival pathways. Moreover, in human prostate cancer cells, we show that decorin specifically inhibits EGFR and AR phosphorylation and cross talk between these pathways. This prevents AR nuclear translocation and inhibits the production of prostate specific antigen. Further, the phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathway is suppressed leading to tumor cell apoptosis. Those findings highlight the effectiveness of decorin in the presence of a powerful genetic cancer risk and implicate decorin as a potential new agent for prostate cancer therapy by targeting EGFR/AR-PI3K-Akt pathways.

NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth

Extracts from Pygeum africanum are used in the treatment of prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The ligand-activated human androgen receptor (AR) is known to control the growth of the prostate gland. Inhibition of human AR is therefore a major goal in treatment of patients. Here, we characterize the compound N-butylbenzene-sulfonamide (NBBS) isolated from P. africanum as a specific AR antagonist. This antihormonal activity inhibits AR- and progesterone receptor- (PR) mediated transactivation, but not the related human glucocorticoid receptor (GR) or the estrogen receptors (ERα or ERβ). Importantly, NBBS inhibits both endogenous PSA expression and growth of human PCa cells. Mechanistically, NBBS binds to AR and inhibits its translocation to the cell nucleus. Furthermore, using a battery of chemically synthesized derivatives of NBBS we revealed important structural aspects for androgen antagonism and have identified more potent AR antagonistic compounds. Our data suggest that NBBS is one of the active compounds of P. africanum bark and may serve as a naturally occurring, novel therapeutic agent for treatment of prostatic diseases. Thus, NBBS and its derivatives may serve as novel chemical platform for treatment prostatitis, BPH and PCa.

Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells

Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.

Transcriptional Repression and Inhibition of Nuclear Translocation of Androgen Receptor by Diallyl Trisulfide in Human Prostate Cancer Cells

The present study was undertaken to determine the effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, on androgen receptor (AR) protein expression and function using prostate cancer cells. The protein levels of AR and prostate-specific antigen (PSA) were determined by immunoblotting. The effect of DATS treatment on AR mRNA level and AR promoter activity was determined by quantitative reverse transcription-PCR and luciferase reporter assay, respectively. Expression of AR protein in poorly differentiated carcinoma and normal prostate of transgenic adenocarcinoma of mouse prostate (TRAMP) mice was determined by immunohistochemistry. Confocal microscopy was done to determine nuclear translocation of AR. Cell viability was determined by trypan blue dye exclusion assay. Exposure of prostate cancer cells (LNCaP, C4-2, and TRAMP-C1) to DATS resulted in a concentration-dependent decrease in protein level of AR, which was accompanied by suppression of intracellular and secreted levels of PSA. Structure-activity studies revealed critical roles for allyl groups and the oligosulfide chain length in DATS-mediated down-modulation of AR protein. Quantitative reverse transcription-PCR showed a dose-dependent decrease in AR mRNA level, which correlated with inhibition of AR promoter activity. DATS treatment inhibited synthetic androgen (R1881)-stimulated nuclear translocation of AR in LNCaP/C4-2 cells and proliferation of LNCaP cells. Oral gavage of 2 mg/day DATS (three times per week for 13 weeks) markedly suppressed AR protein level in poorly differentiated prostate cancer in TRAMP mice. The present study shows, for the first time, that DATS treatment suppresses AR function in prostate cancer cells.

Inhibition of prostate cancer cell growth by second-site androgen receptor antagonists

The impact of ligand binding on nuclear receptor (NR) structure and the ability of target cells to distinguish between different receptor-ligand complexes are key determinants of the pharmacological activity of NR ligands. However, until relatively recently, these mechanistic insights have not been used in a prospective manner to develop screens for NR modulators with specific therapeutic activities. Driven by the need for unique androgen receptor (AR) antagonists that retain activity in hormone-refractory prostate cancer, we developed and applied a conformation-based screen to identify AR antagonists that were mechanistically distinct from existing drugs of this class. Two molecules were identified by using this approach, D36 and D80, which interact with AR in a unique manner and allosterically inhibit AR agonist activity. Unlike the clinically important antiandrogens, casodex and hydroxyflutamide, both D36 and D80 block androgen action in cellular models of hormone-refractory prostate cancer. Mechanistically, these compounds further distinguish themselves from classical AR antagonists in that they do not promote AR nuclear translocation and quantitatively inhibit the association of AR with DNA even under conditions of overexpression. Although the therapeutic potential of these antiandrogens is apparent, it is the demonstration that it is possible, to modulate the interaction of cofactors with agonist-activated AR, using second-site modulators, that has the greatest potential with respect to the therapeutic exploitation of AR and other NRs.

D,l-Sulforaphane causes transcriptional repression of androgen receptor in human prostate cancer cells

D,L-Sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived L-isomer, inhibits the growth of human prostate cancer cells in culture and in vivo and retards cancer development in a transgenic mouse model of prostate cancer. We now show that SFN treatment causes transcriptional repression of androgen receptor (AR) in LNCaP and C4-2 human prostate cancer cells at pharmacologic concentrations. Exposure of LNCaP and C4-2 cells to SFN resulted in a concentration-dependent and time-dependent decrease in protein levels of total AR as well as Ser(210/213)-phosphorylated AR. The SFN-mediated decline in AR protein level was accompanied by a decrease in intracellular as well as secreted levels of prostate-specific antigen, an AR-regulated gene product. The decrease in AR protein level resulting from SFN exposure was not reversed in the presence of the protein synthesis inhibitor cycloheximide. Reverse transcription-PCR analysis revealed a dose-dependent decrease in AR mRNA levels, indicating transcriptional repression of this ligand-activated transcription factor. The SFN treatment inhibited AR promoter activity as revealed by luciferase reporter assay. Synthetic androgen (R1881)-stimulated nuclear translocation of AR was markedly suppressed in the presence of SFN in both cell lines. The SFN treatment also inhibited R1881-stimulated proliferation of LNCaP cells. Naturally occurring thio analogues (iberverin, erucin, and berteroin), but not the sulfonyl analogues (cheirolin, erysolin, and alyssin sulfone), of SFN were also effective in reducing protein levels of AR in LNCaP cells. In conclusion, the present study shows for the first time that SFN treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells.

Activin A Enhances Prostate Cancer Cell Migration Through Activation of Androgen Receptor and Is Overexpressed in Metastatic Prostate Cancer

Bone metastasis is the major cause of mortality associated with prostate cancer. Whereas activin A is known to inhibit prostate cancer cell growth and promote apoptosis, the correlation of elevated activin A with increasing serum prostate-specific antigen (PSA) levels in bone metastatic stages of prostate cancer is well documented. The molecular mechanisms explaining these paradoxical effects of activin A and how activin A influences the progression of prostate cancer with bone metastasis remain unclear. By comparing expression profiles of primary prostate cancer biopsies, with and without bone metastasis, we discovered that the expression of activin A is increased in cases with bone metastatic propensity and correlates with increased androgen receptor (AR), PSA expression, and Gleason scores. Activin A promotes migration of prostate cancer cells to osteoblasts, elevates the AR gene transcription through Smads through binding to AR promoter, and induces nuclear translocation of AR to interact with Smad3. Knockdown of Smad3 by siRNA decreases activin A-promoted AR expression and cancer cell migration. Overexpression of AR reversed Smad3-siRNA suppression on activin A-mediated cell migration to osteoblasts. These data suggest that activation of the AR through Smads is required for activin A-promoted prostate cancer cell migration to bone matrix, thereby promoting the bone metastatic phenotype, and the activin A-Smad-AR axis may be considered a therapeutic target in bone metastatic diseases.

Development of Androgen Receptor Antagonists with Promising Activity in Castration-Resistant Prostate Cancer

Androgen receptor (AR) continues to play a central role in prostate cancers that relapse after androgen deprivation therapy, but these tumors are refractory to available AR antagonists. In a recent issue of Science, Tran et al. describe an antagonist that prevents AR recruitment to chromatin and shows efficacy in relapsed prostate cancer.

Interleukin-6 increases prostate cancer cells resistance to bicalutamide via TIF2

The standard treatment for advanced, androgen-responsive prostate cancer is androgen deprivation therapy with or without a nonsteroidal antiandrogen, such as bicalutamide. Although maximal androgen blockade exhibits favorable responses in the majority of patients, prostate cancer eventually progresses to an androgen-refractory stage. The mechanism underlying bicalutamide resistance in the course of prostate cancer progression is incompletely understood. However, interleukin-6 (IL-6) plays a critical role in the development and progression of CRPC. Herein, we explored an association between IL-6 and bicalutamide resistance. To study this, series of lower and higher passages of LNCaP cell sublines generated by long-term exposure to IL-6 were used. The cells from higher passages of LNCaP treated with IL-6 developed resistance to bicalutamide treatment compared with parental LNCaP cells. The levels of transcriptional intermediary factor 2 (TIF2) in IL-6-treated LNCaP cells were found to be significantly higher than parental LNCaP cells. Down-regulation of TIF2 expression via short hairpin RNA in IL-6-treated LNCaP cells sensitized these cells to bicalutamide treatment, whereas overexpression of TIF2 in the parental LNCaP cells increased resistance to bicalutamide. Furthermore, overexpression of IL-6 attenuated bicalutamide-mediated blockage of androgen-induced androgen receptor nuclear translocation and recruitment. These results show that overexpression of IL-6 increases the resistance of prostate cancer cells to bicalutamide via TIF2. Overexpression of IL-6 not only plays an important role in prostate cancer progression but also contributes to bicalutamide resistance. Our studies suggest that bicalutamide-IL-6-targeted adjunctive therapy may lead to a more effective intervention than bicalutamide alone.

Inhibition of Androgen Receptor Transcriptional Activity as a Novel Mechanism of Action of Arsenic

Environmental sodium arsenite is a toxin that is associated with male infertility due to decreased and abnormal sperm production. Arsenic trioxide (ATO), another inorganic trivalent semimetal, is an effective therapy for acute promyelocytic leukemia, and there is investigation of its possible efficacy in prostate cancer. However, the mechanism of arsenic action in male urogenital tract tissues is not clear. Because the androgen receptor (AR) plays an important role in spermatogenesis and prostate cancer, we explored the possibility that trivalent arsenic regulates AR function. We found that arsenic inhibited AR transcriptional activity in prostate cancer and Sertoli cells using reporter gene assays testing several androgen response element-containing regions and by assessing native target gene expression. Arsenic inhibition of AR activity was not due to down-regulation of AR protein levels, decreased hormone binding to AR, disruption of AR nuclear translocation, or interference with AR-DNA binding in vitro. However, chromatin immunoprecipitation studies revealed that arsenic inhibited AR recruitment to an AR target gene enhancer in vivo. Consistent with a deficiency in AR-chromatin binding, arsenic disrupted AR amino and carboxyl termini interaction. Furthermore, ATO caused a significant decrease in prostate cancer cell proliferation that was more pronounced in cells expressing AR compared with cells depleted of AR. In addition, inhibition of AR activity by ATO and by the AR antagonist, bicalutamide, was additive. Thus, arsenic-induced male infertility may be due to inhibition of AR activity. Further, because AR is an important target in prostate cancer therapy, arsenic may serve as an effective therapeutic option.

Regulation of Myogenic Differentiation by Androgens: Cross Talk between Androgen Receptor/ β-Catenin and Follistatin/Transforming Growth Factor-β Signaling Pathways

Androgens are important regulators of body composition and promote myogenic differentiation and inhibit adipogenesis of mesenchymal, multipotent cells. Here, we investigated the mechanisms by which androgens induce myogenic differentiation of mesenchymal multipotent cells. Incubation of mesenchymal multipotent C3H 10T1/2 cells with testosterone and dihydrotestosterone promoted nuclear translocation of androgen receptor (AR)/beta-catenin complex and physical interaction of AR, beta-catenin, and T-cell factor-4 (TCF-4). Inhibition of beta-catenin by small inhibitory RNAs significantly decreased testosterone-induced stimulation of myogenic differentiation. Overexpression of TCF-4, a molecule downstream of beta-catenin in Wnt signaling cascade, in C3H 10T1/2 cells significantly up-regulated expression of myoD and myosin heavy chain II proteins and of follistatin (Fst), which binds and antagonizes native ligands of the TGF-beta/Smad pathway. Gene array analysis of C3H 10T1/2 cells treated with testosterone revealed that testosterone up-regulated the expression of Fst and modified the expression of several signaling molecules involved in the TGF-beta/Smad pathway, including Smad7. Lowering of testosterone levels in mice by orchidectomy led to a significant decrease in Fst and Smad7 expression; conversely, testosterone supplementation in castrated mice up-regulated Fst and Smad7 mRNA expression in androgen-responsive levator ani muscle. Testosterone-induced up-regulation of MyoD and myosin heavy chain II proteins in C3H 10T1/2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. In conclusion, our data suggest the involvement of AR, beta-catenin, and TCF-4 pathway during androgen action to activate a number of Wnt target genes, including Fst, and cross communication with the Smad signaling pathway.

Regulation of Akt/FOXO3a/GSK-3β/AR Signaling Network by Isoflavone in Prostate Cancer Cells

We have previously shown that genistein could inhibit Akt activation and down-regulate AR (androgen receptor) and PSA (prostate-specific antigen) expression in prostate cancer (PCa) cells. However, pure genistein showed increased lymph node metastasis in an animal model, but such an adverse effect was not seen with isoflavone, suggesting that further mechanistic studies are needed for elucidating the role of isoflavone in PCa. It is known that FOXO3a and GSK-3beta, targets of Akt, regulate cell proliferation and apoptosis. Moreover, FOXO3a, GSK-3beta, and Src are AR regulators and regulate transactivation of AR, mediating the development and progression of PCa. Therefore, we investigated the molecular effects of isoflavone on the Akt/FOXO3a/GSK-3beta/AR signaling network in hormone-sensitive LNCaP and hormone-insensitive C4-2B PCa cells. We found that isoflavone inhibited the phosphorylation of Akt and FOXO3a, regulated the phosphorylation of Src, and increased the expression of GSK-3beta, leading to the down-regulation of AR and its target gene PSA. We also found that isoflavone inhibited AR nuclear translocation and promoted FOXO3a translocation to the nucleus. By electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we found that isoflavone inhibited FOXO3a binding to the promoter of AR and increased FOXO3a binding to the p27(KIP1) promoter, resulting in the alteration of AR and p27(KIP1) expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive PCa cells. These results suggest that isoflavone-induced inhibition of cell proliferation and induction of apoptosis are partly mediated through the regulation of the Akt/FOXO3a/GSK-3beta/AR signaling network. In conclusion, our data suggest that isoflavone could be useful for the prevention and/or treatment of PCa.

Abstract LB-203: Phase I/II study of the androgen receptor antagonist MDV3100 in castration-resistant prostate cancer

Abstract Introduction: Overexpression of the androgen receptor (AR) is believed to contribute to the progression of CRPC. MDV3100 is a small molecule AR antagonist with a novel mechanism of action that blocks nuclear translocation of AR, DNA binding, and has no agonist activity when AR is overexpressed. MDV3100 has been shown to be effective in preclinical models of CRPC. In July 2007, a multi-center first-in-man Phase 1-2 trial was initiated to determine safety, pharmacokinetics (PK), and antitumor activity including effects on prostate-specific antigen (PSA), bone and soft tissue metastases, and in selected pts fluorodeoxyglucose (FDG) and fluorodihydrotestosterone (FDHT) uptake by positron emission tomography (PET). Methods: Pts were administered MDV3100 orally, once daily, starting at 30 mg with sequential escalations in cohorts of 3 pts. Enrollment was expanded at 60 mg and above to include up to 12 chemotherapy-naïve and 12 post-chemotherapy pts, once the safety of a dose was established. PK parameters for the dose-escalation cohorts were estimated using a 2-compartment model. Results: 85 pts have been enrolled to date on doses up to 240 mg. 21 pts at 60 mg, and 22 pts at 150 mg have been followed for 12+ and 8+ weeks respectively. MDV3100 has been well-tolerated without dose-limiting toxicity. PK were dose-linear with a half-life of approximately one week. At doses of 30, 60, 150, and 240 mg, geometric mean steady state Cmax values were 2.8, 6.1, 17.3, and 23.7 µg/mL and AUC24hr were 61, 120, 361, and 480 µg*hr/mL, respectively. Effective AR blockade by MDV3100 has been shown by PSA declines and by PET findings. Evaluation at the 12 week timepoint for 21 pts at 60 mg showed 9 pts (43%) had >50% decline in PSA from baseline. To date, 8 pts have had FDG and FDHT PET at baseline of whom 4 have had 1 or more follow-up scans. All 8 had abnormal FDHT accumulation at baseline while 4 of 4 with follow-up scans had no FDHT accumulation. Conclusions: MDV3100 is a novel AR antagonist in clinical investigation. The observed PSA declines are consistent with the inhibition of AR signaling. The inhibition of FDHT accumulation demonstrates receptor blockade in vivo. MDV3100 has been well-tolerated to date and appears to be a promising candidate for the treatment of CRPC. Pt follow-up and dose escalation are continuing. The analysis of the association between PSA declines and clinical outcomes is ongoing and will be reported at a future date.

Oriental herbs as a source of novel anti-androgen and prostate cancer chemopreventive agents

Androgen and androgen receptor (AR) signaling are crucial for the genesis of prostate cancer (PCa), which can often develop into androgen-ligand-independent diseases that are lethal to the patients. Recent studies show that even these hormone-refractory PCa require ligand-independent AR signaling for survival. As current chemotherapy is largely ineffective for PCa and has serious toxic sideeffects, we have initiated a collaborative effort to identify and develop novel, safe and naturally occurring agents that target AR signaling from Oriental medicinal herbs for the chemoprevention and treatment of PCa. We highlight our discovery of decursin from an Oriental formula containing Korean Angelica gigas Nakai (Dang Gui) root as a novel anti-androgen/AR agent. We have identified the following mechanisms to account for the specific anti-AR actions: rapid block of AR nuclear translocation, inhibition of binding of 5alpha-dihydrotestesterone to AR and increased proteasomal degradation of AR protein. Furthermore, decursin lacks the agonist activity of the "pure" anti-androgen bicalutamide and is more potent than bicalutamide in inducing PCa apoptosis. Structure-activity analyses reveal a critical requirement of the side-chain on decursin or its structural isomer decursinol angelate for anti-AR, cell cycle arrest and proapoptotic activities. This work demonstrates the feasibility of using activity-guided fractionation in cell culture assays combined with mechanistic studies to identify novel anti-androgen/ AR agents from complex herbal mixtures.

A novel class of pyranocoumarin anti–androgen receptor signaling compounds

Androgen and the androgen receptor (AR)-mediated signaling are crucial for prostate cancer development. Novel agents that can inhibit AR signaling in ligand-dependent and ligand-independent manners are desirable for the chemoprevention of prostate carcinogenesis and for the treatment of advanced prostate cancer. We have shown recently that the pyranocoumarin compound decursin from the herb Angelica gigas possesses potent anti-AR activities distinct from the anti-androgen bicalutamide. Here, we compared the anti-AR activities and the cell cycle arrest and apoptotic effects of decursin and two natural analogues in the androgen-dependent LNCaP human prostate cancer cell culture model to identify structure-activity relationships and mechanisms. Decursin and its isomer decursinol angelate decreased prostate-specific antigen expression with IC(50) of approximately 1 mumol/L. Both inhibited the androgen-stimulated AR nuclear translocation and transactivation, decreased AR protein abundance through proteasomal degradation, and induced G(0/1) arrest and morphologic differentiation. They also induced caspase-mediated apoptosis and reactive oxygen species at higher concentrations. Furthermore, they lacked the agonist activity of bicalutamide in the absence of androgen and were more potent than bicalutamide for suppressing androgen-stimulated cell growth. Decursinol, which does not contain a side chain, lacked the reactive oxygen species induction and apoptotic activities and exerted paradoxically an inhibitory and a stimulatory effect on AR signaling and cell growth. In conclusion, decursin and decursinol angelate are members of a novel class of nonsteroidal compounds that exert a long-lasting inhibition of both ligand-dependent and ligand-independent AR signaling. The side chain is critical for sustaining the anti-AR activities and the growth arrest and apoptotic effects.

Down-regulation of androgen receptor by 3,3′-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in both hormone-sensitive LNCaP and insensitive C4-2B prostate cancer cells: Bhuiyan MM, Li Y, Banerjee S, Ahmed F, Wang Z, Ali S, Sarkar FH, Departments of Pathology and Internal Medicine, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI

Despite the initial efficacy of androgen deprivation therapy, most patients with advanced prostate cancer eventually progress to hormone-refractory prostate cancer, for which there is no curative therapy. Previous studies from our laboratory and others have shown the antiproliferative and proapoptotic effects of 3,3′-diindolylmethane (DIM) in prostate cancer cells. However, the molecular mechanism of action of DIM has not been investigated in androgen receptor (AR)-positive hormone-responsive and -nonresponsive prostate cancer cells. Therefore, we investigated the effects of B-DIM, a formulated DIM with greater bioavailability, on AR, Akt, and nuclear factor κB (NF-κB) signaling in hormone-sensitive LNCaP (AR+) and hormone-insensitive C4-2B (AR+) prostate cancer cells. We found that B-DIM significantly inhibited cell proliferation and induced apoptosis in both cell lines. By Akt gene transfection, reverse transcription-PCR, Western blot analysis, and electrophoretic mobility shift assay, we found a potential crosstalk between Akt, NF-κB, and AR. Importantly, B-DIM significantly inhibited Akt activation, NF-κB DNA binding activity, AR phosphorylation, and the expressions of AR and prostate-specific antigen, suggesting that B-DIM could interrupt the crosstalk. Confocal studies revealed that B-DIM inhibited AR nuclear translocation, leading to the down-regulation of AR target genes. Moreover, B-DIM significantly inhibited C4-2B cell growth in a severe combined immunodeficiency-human model of experimental prostate cancer bone metastasis. These results suggest that B-DIM-induced cell proliferation inhibition and apoptosis induction are partly mediated through the down-regulation of AR, Akt, and NF-κB signaling. These observations provide a rationale for devising novel therapeutic approaches for the treatment of hormone-sensitive, but more importantly, hormone-refractory prostate cancer by using B-DIM alone or in combination with other therapeutics.

Essential Role for Co-chaperone Fkbp52 but Not Fkbp51 in Androgen Receptor-mediated Signaling and Physiology

Fkbp52 and Fkbp51 are tetratricopeptide repeat proteins found in steroid receptor complexes, and Fkbp51 is an androgen receptor (AR) target gene. Although in vitro studies suggest that Fkbp52 and Fkbp51 regulate hormone binding and/or subcellular trafficking of receptors, the roles of Fkbp52 and Fkbp51 in vivo have not been extensively investigated. Here, we evaluate their physiological roles in Fkbp52-deficient and Fkbp51-deficient mice. Fkbp52-deficient males developed defects in select reproductive organs (e.g. penile hypospadias and prostate dysgenesis but normal testis), pointing to a role for Fkbp52 in AR-mediated signaling and function. Surprisingly, ablation of Fkbp52 did not affect AR hormone binding or nuclear translocation in vivo and in vitro. Molecular studies in mouse embryonic fibroblast cells uncovered that Fkbp52 is critical to AR transcriptional activity. Interestingly, Fkbp51 expression was down-regulated in Fkbp52-deficient males but only in affected tissues, providing further evidence of tissue-specific loss of AR activity and suggesting that Fkbp51 is an AR target gene essential to penile and prostate development. However, Fkbp51-deficient mice were normal, showing no defects in AR-mediated reproductive function. Our work demonstrates that Fkbp52 but not Fkbp51 is essential to AR-mediated signaling and provides evidence for an unprecedented Fkbp52 function, direct control of steroid receptor transcriptional activity.

Down-regulation of Androgen Receptor by 3,3′-Diindolylmethane Contributes to Inhibition of Cell Proliferation and Induction of Apoptosis in Both Hormone-Sensitive LNCaP and Insensitive C4-2B Prostate Cancer Cells

Despite the initial efficacy of androgen deprivation therapy, most patients with advanced prostate cancer eventually progress to hormone-refractory prostate cancer, for which there is no curative therapy. Previous studies from our laboratory and others have shown the antiproliferative and proapoptotic effects of 3,3'-diindolylmethane (DIM) in prostate cancer cells. However, the molecular mechanism of action of DIM has not been investigated in androgen receptor (AR)-positive hormone-responsive and -nonresponsive prostate cancer cells. Therefore, we investigated the effects of B-DIM, a formulated DIM with greater bioavailability, on AR, Akt, and nuclear factor kappaB (NF-kappaB) signaling in hormone-sensitive LNCaP (AR+) and hormone-insensitive C4-2B (AR+) prostate cancer cells. We found that B-DIM significantly inhibited cell proliferation and induced apoptosis in both cell lines. By Akt gene transfection, reverse transcription-PCR, Western blot analysis, and electrophoretic mobility shift assay, we found a potential crosstalk between Akt, NF-kappaB, and AR. Importantly, B-DIM significantly inhibited Akt activation, NF-kappaB DNA binding activity, AR phosphorylation, and the expressions of AR and prostate-specific antigen, suggesting that B-DIM could interrupt the crosstalk. Confocal studies revealed that B-DIM inhibited AR nuclear translocation, leading to the down-regulation of AR target genes. Moreover, B-DIM significantly inhibited C4-2B cell growth in a severe combined immunodeficiency-human model of experimental prostate cancer bone metastasis. These results suggest that B-DIM-induced cell proliferation inhibition and apoptosis induction are partly mediated through the down-regulation of AR, Akt, and NF-kappaB signaling. These observations provide a rationale for devising novel therapeutic approaches for the treatment of hormone-sensitive, but more importantly, hormone-refractory prostate cancer by using B-DIM alone or in combination with other therapeutics.