help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1469
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/6/2936    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nguyen, T.-V. V.
Right arrow Articles by Pike, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nguyen, T.-V. V.
Right arrow Articles by Pike, C. J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CYPROTERONE ACETATE
Endocrinology Vol. 148, No. 6 2936-2943
Copyright © 2007 by The Endocrine Society

Flutamide and Cyproterone Acetate Exert Agonist Effects: Induction of Androgen Receptor-Dependent Neuroprotection

Thuy-Vi V. Nguyen, Mingzhong Yao and Christian J. Pike

Neuroscience Graduate Program and Davis School of Gerontology, University of Southern California, Los Angeles, California 90089

Address all correspondence and requests for reprints to: Christian J. Pike, Ph.D., University of Southern California, Davis School of Gerontology, 3715 McClintock Avenue, Los Angeles, California 90089-0191. E-mail: cjpike{at}usc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens can exert profound effects on the organization, development, and function of the nervous system through activation of androgen receptors (ARs). Nonsteroidal and steroidal antiandrogens antagonize AR-mediated, classic genomic actions of androgens. However, emerging studies in nonneuronal cells indicate that antiandrogens can act as partial agonists for the AR. Here we investigated the effects of the antiandrogens flutamide and cyproterone acetate on neuroprotection induced by dihydrotestosterone (DHT). We observed that, although flutamide and cyproterone acetate blocked androgen-induced gene expression, they failed to inhibit DHT protection against apoptotic insults in cultured hippocampal neurons. Interestingly, flutamide and cyproterone acetate alone, like DHT, significantly reduced apoptosis. Furthermore, the protective actions of flutamide and cyproterone acetate were observed specifically in AR-expressing cell lines, suggesting a role for AR in the agonist effects of antiandrogens. Our results indicate that, in contrast to the classic antiandrogen properties of flutamide and cyproterone acetate, these AR modulators display agonist activities at the level of neuroprotection. These findings provide new insight into the agonist vs. antagonist properties of antiandrogens, information that will be crucial to understanding the neural implications of clinically used AR-modulating drugs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS ARE NECESSARY for the proper development and function of numerous tissues, including the central nervous system (1). In addition, accumulating evidence indicates that age-related androgen loss can contribute to neurodegenerative disorders, including Alzheimer’s disease (2, 3, 4, 5). The two primary endogenous androgens are testosterone and its metabolite dihydrotestosterone (DHT) (6), which have broad actions in the central nervous system such as differentiation of neurons in hypothalamus (7) and cerebral cortex (8), promotion of hippocampal sprouting (9) and excitability (10, 11), and enhancement of neurite growth (12) and maintenance of morphology (13, 14) of motor neurons. Although some androgen actions are mediated via estrogen pathways after aromatization of testosterone to estradiol, many androgen effects require direct activation of the androgen receptor (AR). Both AR mRNA and protein are present in select populations of neuronal and glial cells in brain (15, 16, 17). Activated ARs can interact with androgen response elements (AREs) in target genes to regulate transcription (i.e. classic genomic mechanism) (18, 19) and can activate signal transduction pathways (i.e. nongenomic mechanism) and transcription factors (i.e. indirect genomic mechanism) (20, 21, 22).

Antiandrogens were developed to competitively bind to ARs and interfere with androgen-AR association and action (23, 24); however, emerging data indicate that antiandrogens often do not function as pure AR antagonists. There are two general classes of antiandrogens, steroidal and nonsteroidal (25, 26). Steroidal antiandrogens such as cyproterone acetate function largely as AR antagonists, although they can exhibit partial agonist properties with ARs and other members of the steroid receptor family (27). In contrast, the nonsteroidal antiandrogens such as flutamide are generally considered pure antiandrogens that do not exhibit AR agonist activity (23, 27). Yet both steroidal and nonsteroidal antiandrogens can sometimes lack antagonist actions and/or act as partial AR agonists in nonneuronal cells (24, 28, 29). For example, in some paradigms, antiandrogens fail to block AR-dependent, androgen-induced cell signaling (30, 31, 32, 33, 34) and can also mimic such androgen effects (20, 35, 36). The extent to which antiandrogens exhibit agonist vs. antagonist properties in neural tissue is not well understood.

Recent studies have elucidated a role for ARs in androgen neuroprotection. For example, neuroprotection induced by testosterone and DHT is blocked by flutamide in both rat cerebellar and human embryonic neuron cultures, implicating dependence on ARs (37, 38, 39). In contrast, we found that in rat hippocampal neurons, flutamide does not antagonize androgen neuroprotection (40), although AR is implicated in neuroprotection based on AR transfection studies (41). These apparent inconsistencies in the literature may reflect differential actions of antiandrogens on neuroprotection that are dependent on the relevant androgen signaling mechanisms (e.g. genomic vs. nongenomic pathways). To address these issues, in the present study, we evaluated the agonist and antagonist actions of antiandrogens in neural cells with a focus on the androgen neuroprotection. Specifically we determined the abilities of antiandrogens to both antagonize classic (ARE mediated) gene transcription and regulate AR-dependent, androgen-induced neuroprotection. Distinguishing the AR agonist/antagonist properties of antiandrogens, and thus their differing mechanisms, is necessary for the development of AR-modulating ligands to target androgen-related neural disorders, including Alzheimer’s disease (2, 3, 4, 5).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DHT was purchased from Steraloids (Newport, RI). Flutamide, cyproterone acetate, staurosporine, A23187, 3-nitropropionic acid (3-NP), hydrogen peroxide (H2O2), and ferrous/ferric chloride (FeCl2/3) were obtained from Sigma-Aldrich (St. Louis, MO). ß-Amyloid (Aß) peptide 25–35 and apoptosis activator II were acquired from Bachem (Torrance, CA) and Calbiochem (San Diego, CA), respectively.

Neuron cultures
Primary neuronal cultures (~95% neuronal) were generated from the dissected hippocampi of embryonic d 18 Sprague Dawley rat pups (n = 10 per preparation), using a previously described technique (41, 42). In brief, hippocampi were dissociated with 0.125% trypsin-EDTA at 37 C for 10 min and then passaged several times through a flamed-polished Pasteur pipette. The cell suspension was sieved through a 40-µm cell strainer (Falcon, Franklin Lakes, NJ) and then diluted in serum-free DMEM supplemented with 20 mM HEPES, 100 µg/ml transferrin, 5 µg/ml insulin, 100 µM putrescine, and 30 nM selenium. Cells were plated at a density of 3.75 x 104 cells/cm2 in 48-well plates for cell viability experiments and 4 x 105 cells/cm2 in 12-well plates for RT-PCR. Cultures were kept in a humidified incubator with room air supplemented with 5% CO2 at a constant temperature of 37 C. All experiments were performed after 3 d in vitro. Animals were maintained in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and under a protocol approved by the University of Southern California Institutional Animal Care and Use Committee.

AR-transfected cells
PC12 cell lines stably transfected with rat sequence AR (pcDNA3-AR) or empty vector (pcDNA3-ctl) were generated in our laboratory, as previously described (41). Wild-type, pcDNA3-ctl, and pcDNA3-AR PC12 cell lines were expanded in 75-cm2 flasks (Fisher Scientific, Tustin, CA) with RPMI 1640 medium containing 20 mM HEPES, 10 ml/liter penicillin-streptomycin, 10% horse serum/5% fetal bovine serum, and 100 µg/ml G418 (except for wild type). Cells were plated at a density of 1.25 x 104 cells/cm2 in 48-well plates for cell viability experiments and 2 x 105 cells/cm2 in 12-well plates for RT-PCR. After 24 h, medium was replaced with serum-free medium, and cultures were maintained at 37 C in a humidified incubator with room air containing 5% CO2. Three independent clonal lines of pcDNA3-AR and pcDNA3-ctl were evaluated and yielded similar results. For simplicity, results from only a single line are shown.

Cell viability
Cultures were pretreated (0.01, 0.1, 1, or 10 µM) with flutamide, cyproterone acetate, or an equal concentration of ethanol vehicle for 2 h. After antiandrogen pretreatment, cultures were treated with DHT (10 nM) or an equal concentration of ethanol vehicle for 0–72 h before and during 24 h exposure to toxin or equal concentration of ethanol or dimethyl sulfoxide (DMSO) vehicle. Toxins included aggregated Aß peptide 25–35 (0–50 µM) prepared as previously described (43), staurosporine (0–0.5 µM), apoptosis activator II (0–7 µM), H2O2 (0–25 µM), FeCl2/3 (0–2.5 µM), A23187 (0–350 nM), and 3-NP (0–2.5 mM). Antiandrogens, DHT, and toxins were solubilized in 100% ethanol or DMSO, and diluted in culture medium to a final ethanol or DMSO concentration of 0.1% or less. Cell viability was assessed as previously described (41, 42). In brief, live cells were labeled with calcein acetoxymethyl ester (Molecular Probes, Eugene, OR) and then counted in 4 fields per well, 3 wells per condition. At least three independent culture preparations were used for every experiment, and repeated conditions were included in all experiments. The mean number of cells counted per well for vehicle-treated control conditions varied from 100 to 200. Raw cell count data were statistically analyzed as described below.

RT-PCR
Cells were processed for RT-PCR using a standard protocol, as previously described (41). In brief, RNA was extracted with the TRIzol reagent (Invitrogen, Carlsbad, CA) and then quantified with an UV spectrophotometer (A260). Using the SuperScript first-strand synthesis system (Invitrogen), 2 µg RNA were reverse transcribed into cDNA. After transcription, 1 µl reverse transcription product was combined with 2.5 U JumpStart Taq DNA polymerase (Sigma) [20 pmol sense and antisense primers in a buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 2.5 mM MgCl2; 0.2 mM deoxynucleotide triphosphate in 50 µl]. The primers were synthesized chemically (Integrated DNA Technologies, Coralville, IA) and consisted of 5'-TGCTCGACATGCTGGTCTAC-3' and 5'-GGCTGCAGGACGAATGTACT-3' for approximately 194 kb rat 5{alpha}-reductase type I; and 5'-AGCCATGTACGTAGCCATCC-3' and 5'-CTCTCAGCTGTGGTGGTGAA-3' for ß-actin. The PCR cycles included incubation (94 C, 1 min), denaturation (94 C, 30 sec), annnealing (52 C, 30 sec), first extension (72 C, 3.5 min, 30 cycles), and final extension (72 C, 7 min). The PCR products were electrophoresed in 1.5% agarose gels, stained with ethidium bromide, and then sequenced with Gene Wiz (North Brunswick, NJ). Relative levels of 5{alpha}-reductase and ß-actin mRNAs were quantified by band densitometry of gel images using NIH Image 1.61 software. Individual 5{alpha}-reductase bands were expressed as a ratio of their corresponding ß-actin band to control for the possibility of unequal loading and then statistically analyzed.

Statistical analyses
Pooled raw data from cell counts and band densitometry were statistically analyzed using a split-plot ANOVA design, followed by between-group comparisons using the Tukey honestly significant difference (HSD) test. For graphical presentation, both cell viability and 5{alpha}-reductase expression are expressed as percentages of vehicle-treated control values. Statistical significance indicated in graphs reflects statistical analyses of the pooled raw data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antiandrogens antagonize androgen-mediated gene expression
To determine whether classic antiandrogens act as AR antagonists in our paradigm, we tested their abilities to block androgen transcriptional regulation of the ARE-containing gene 5{alpha}-reductase type I, which metabolizes testosterone into DHT in brain (44, 45, 46). We first confirmed that androgens increase expression of 5{alpha}-reductase I in both AR-expressing hippocampal neurons and AR-transfected PC12 cells (pcDNA-AR). We exposed both types of cultures to 10 nM DHT for various times between 0 and 72 h and then collected lysates for RT-PCR. In hippocampal neurons, DHT treatment resulted in 5{alpha}-reductase I mRNA signal that was significantly elevated within 6 h and continued to increase through 72 h (Fig. 1AGo). Similar results were observed in pcDNA-AR cells except that the DHT-induced increase in 5{alpha}-reductase I mRNA was first evident at 24 h (Fig. 1BGo). No between-group changes were observed in the mRNA expression of the control gene ß-actin.


Figure 1
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 1. Androgens up-regulate 5{alpha}-reductase type I gene expression in a time-dependent manner. Cultures of hippocampal neurons and PC12 cells stably transfected with AR (pcDNA3-AR) were treated with vehicle or 10 nM DHT for the indicated times. Agarose gel electrophoresis of RT-PCR products using 5{alpha}-reductase I primers demonstrates a significant increase in 5{alpha}-reductase I mRNA from 6 to 72 h in hippocampal neurons [one-way ANOVA: F (6,18) = 9.6; P < 0.0001] (A) and from 24 to 72 h in pcDNA3-AR cells [one-way ANOVA: F (6,12) = 32.8; P < 0.0001] (B). ß-Actin mRNA for each sample is shown for comparison. Pictured agarose gels are from representative experiments. Graphs show mean values (± SEM) of the combined experiments (n = 3–4) of 5{alpha}-reductase expression corrected with respect to ß-actin and normalized to a percentage of vehicle-treated controls. Statistical significance is based on analysis of pooled raw data using the Tukey HSD. *, P < 0.05 relative to the vehicle-treated control condition.

 
If the antiandrogens, flutamide and cyproterone acetate, act as antagonists of classic genomic AR effects, we would expect them to block DHT-induced increases in 5{alpha}-reductase I expression. To investigate this possibility, we treated hippocampal neuron cultures with increasing concentrations of flutamide or cyproterone acetate for 2 h, followed by exposure to DHT for 48 h, and then collected lysates for RT-PCR. Flutamide blocked the increase in 5{alpha}-reductase I mRNA induced by DHT in a concentration-dependent manner, with inhibition evident at 1 µM and statistically significant at 10 µM (Fig. 2AGo). Cyproterone acetate was observed to be a more potent antiandrogen in our paradigm because it significantly inhibited the DHT-induced 5{alpha}-reductase I mRNA at concentrations of 1 and 10 µM (Fig. 2BGo). In the absence of DHT, neither flutamide (Fig. 2AGo) nor cyproterone acetate (Fig. 2BGo) affected basal levels of 5{alpha}-reductase I mRNA. Similar results were observed in PC12 cells. Flutamide and cyproterone acetate at 10 µM did not affect basal levels of 5{alpha}-reductase I mRNA in the pcDNA3-AR lines but significantly inhibited the DHT-induced increase in 5{alpha}-reductase I mRNA expression (Fig. 3CGo). In wild-type (Fig. 3AGo) and pcDNA3-ctl (Fig. 3BGo) cells, 5{alpha}-reductase I mRNA levels were not altered by any of the treatments. All three cell lines showed similar responses after differentiation with nerve growth factor (data not shown).


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 2. Antiandrogens inhibit androgen-induced 5{alpha}-reductase I gene expression in a concentration-dependent manner. Hippocampal neuron cultures were pretreated with vehicle or increasing concentrations of flutamide (Flut) and cyproterone acetate (Cyp A) for 2 h, followed by exposure to 0 nM (open bars) or 10 nM (solid bars) DHT for 48 h. Agarose gel electrophoresis of RT-PCR products using 5{alpha}-reductase I primers demonstrates significant attenuation of DHT-induced 5{alpha}-reductase I mRNA by 10 µM flutamide [F (9,18) = 19.7; P < 0.0001] (A) and 1–10 µM cyproterone acetate [F (9,18) = 6.1; P = 0.0005] (B). ß-Actin mRNA is shown for comparison. Pictured agarose gels are from representative experiments. Graphs show mean values (± SEM) of the combined experiments (n = 3–4) of 5{alpha}-reductase expression corrected with respect to ß-actin and normalized to a percentage of vehicle-treated controls. Statistical significance is based on analysis of pooled raw data using the Tukey HSD. *, P < 0.05 relative to the vehicle-treated control condition; #, P < 0.05 relative to the vehicle + DHT condition.

 

Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 3. Antiandrogens inhibit androgen-induced 5{alpha}-reductase I gene expression in PC12 cells expressing AR. Agarose gel electrophoresis of RT-PCR products using 5{alpha}-reductase I primers demonstrates that 48 h exposure to 10 nM DHT significantly increased 5{alpha}-reductase I mRNA in the pcDNA-AR cell line [F (5,10) = 18.0; P = 0.0001] (C) but in neither wild-type cells [F (5,10) = 2.0; P = 0.1642] (A) nor pcDNA-ctl cells [F (5,10) = 1.7; P = 0.2321] (B). This increase in 5{alpha}-reductase I mRNA was significantly inhibited by 2 h pretreatment with 10 µM flutamide (Flut) and 10 µM cyproterone acetate (Cyp A). ß-Actin mRNA is shown for comparison. Pictured agarose gels are from representative experiments. Graphs show mean values (± SEM) of the combined experiments (n = 3–4) of 5{alpha}-reductase expression corrected with respect to ß-actin and normalized to a percentage of vehicle-treated controls. Statistical significance is based on analysis of pooled raw data using the Tukey HSD. *, P < 0.05 relative to the vehicle-treated control condition; #, P < 0.05 relative to the vehicle + DHT condition.

 
Antiandrogens mimic rather than antagonize androgen neuroprotection
We previously demonstrated that testosterone and DHT are neuroprotective against Aß toxicity by a rapid cell signaling mechanism rather than a classic genomic pathway (40, 41). To determine whether the antiandrogens flutamide and cyproterone acetate antagonize this form of androgen neuroprotection, we compared cell viability in neurons challenged with Aß peptide in the presence and absence of DHT, flutamide, and cyproterone acetate. Exposure of hippocampal neuron cultures to 50 µM aggregated Aß25–35 for 24 h decreased cell viability by approximately 40–50%, compared with vehicle-treated controls (Fig. 4Go). Treatment of cultures with 10 nM DHT beginning 2 h before Aß exposure significantly reduced Aß toxicity by approximately 20–25% (Fig. 4Go, A and C). Interestingly, pretreatment with 0.01–10 µM flutamide (Fig. 4AGo) or cyproterone acetate (Fig. 4CGo) 2 h before and during addition of androgen and Aß failed to inhibit DHT neuroprotection. In fact, flutamide (Fig. 4BGo) and cyproterone acetate (Fig. 4DGo) alone at 1 and 10 µM significantly reduced Aß toxicity as effectively as DHT. There was no additive protection observed in combinations of DHT with flutamide or cyproterone acetate.


Figure 4
View larger version (30K):
[in this window]
[in a new window]

  		FIG. 4. Androgen neuroprotection is insensitive to antiandrogens. Hippocampal neuron cultures were treated with 50 µM Aß25–35 and 0–10 µM flutamide (Flut) in the presence of 10 nM DHT cotreatment [F (7,73) = 8.7; P < 0.0001] (A) or absence of DHT cotreatment [F (6,138) = 18.4; P < 0.0001] (B). In parallel experiments, cultures were treated with 50 µM Aß25–35 and 0–10 µM cyproterone acetate (Cyp A) in the presence of 10 nM DHT cotreatment [F (7,73) = 24.7; P < 0.0001] (C) or absence of 10 nM DHT cotreatment [F (6,205) = 22.3; P < 0.0001] (D). Cell viability graphs show means (± SEM) of combined independent experiments (n = 3–12). Cell viability is presented as the percentage of live cells normalized to the vehicle-treated control condition. Statistical significance is based on analysis of pooled raw data using the Tukey HSD. *, P < 0.05 relative to the vehicle + Aß condition.

 
Antiandrogen neuroprotection is AR dependent
To determine whether the mechanism of antiandrogen protection is AR dependent, we evaluated the protective properties of flutamide and cyproterone acetate in our PC12 cell lines. We found that 24 h exposure to 50 µM Aß25–35 decreased cell viability of all three PC12 cell lines at similar levels that ranged from 50 to 60% in comparison with vehicle-treated controls across independent experiments (Fig. 5Go, C and D). In the AR-expressing pcDNA3-AR cells, pretreatment with 10 nM DHT significantly attenuated Aß toxicity by 20–30% (Fig. 5Go, A and B). As observed with cultured hippocampal neurons, exposure of pcDNA3-AR cultures with 10 µM flutamide (Fig. 5AGo) or cyproterone acetate (Fig. 5BGo) 2 h before and during addition of androgen and Aß failed to inhibit DHT protection. However, flutamide (Fig. 5CGo) and cyproterone acetate (Fig. 5DGo) delivered alone induced significant protection in pcDNA3-AR cells that was equivalent in magnitude to DHT. In empty vector pcDNA3-ctl and wild-type PC12 cells, both flutamide (Fig. 5CGo) and cyproterone acetate (Fig. 5DGo) failed to significantly affect viability of cultures challenged with Aß.


Figure 5
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 5. Antiandrogen protection is AR dependent. Cultures of pcDNA3-AR PC12 cells were treated with 10 µM flutamide (Flut) or 10 µM cyproterone acetate (Cyp A) for 2 h, followed with 10 nM DHT for 2 h and then exposed to 50 µM Aß25–35. After 24 h, cultures were processed for cell viability. Flutamide [F (4,49) = 9.4; P < 0.0001] (A) and cyproterone acetate [F (4,49) = 30.7; P < 0.0001] (B) did not inhibit DHT protection. Cultures of wild-type, pcDNA3-ctl, and pcDNA3-AR cells were treated with the antiandrogens flutamide [wild-type, F (2,22) = 25.8; P < 0.0001; pcDNA3-ctl, F (2,23) = 13.4; P < 0.0001; pcDNA3-AR, F (2,22) = 45.3; P < 0.0001] (C) and cyproterone acetate [wild-type, F (2,22) = 63.8; P < 0.0001; pcDNA3-ctl, F (2,23) = 13.4; P = 0.0001; pcDNA3-AR, F (2,22) = 54.2; P < 0.0001] (D), followed 2 h later with exposure to Aß. Cell viability graphs show means (± SEM) of combined independent experiments (n = 3). Cell viability is presented as the percentage of live cells normalized to the vehicle-treated control condition. Statistical significance is based on analysis of pooled raw data using the Tukey HSD. *, P < 0.05 relative to the vehicle + Aß condition.

 
Antiandrogen neuroprotection is insult specific
To further investigate similarities in the protective actions of androgens and antiandrogens, we compared the specificity of protection induced by DHT with that induced by flutamide and cyproterone acetate against a panel of neurotoxins. Our recent work has found that androgens selectively protect against toxins that induce apoptosis but not toxins that cause nonapoptotic death (Nguyen, T. V., and C. J. Pike, unpublished observations). If antiandrogens activate a neuroprotective pathway similar to that induced by androgens, then we would predict that, like DHT, antiandrogens would selectively protect against apoptotic insults. To investigate this possibility, hippocampal neuron cultures were pretreated with 10 nM DHT, 10 µM flutamide, or 10 µM cyproterone acetate for 2 h before and during exposure to toxic levels of insults previously determined to induce neuron death in our paradigm by either apoptotic (Aß, staurosporine, apoptosis activator II) or nonapoptotic (H2O2, FeCl2/3, A23187, 3-NP) mechanisms. Consistent with our prior data (Nguyen, T. V., and C. J. Pike, unpublished observations), DHT significantly reduced cell death induced by Aß, staurosporine, and apoptosis activator II (Fig. 6AGo) but not H2O2, FeCl2/3, A23187, and 3-NP (Fig. 6BGo). Similar patterns of selective neuroprotection were induced by flutamide (Fig. 6Go, C and D) and cyproterone acetate (Fig. 6Go, E and F).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 6. Antiandrogens are neuroprotective against apoptotic insults. Hippocampal neuron cultures were treated with 10 nM DHT, 10 µM flutamide (Flut), 10 µM cyproterone acetate (Cyp A), or vehicle for 2 h and then exposed to 50 µM Aß, 0.4 µM staurosporine (STS), 3 µM apoptosis activator II (AAII), 25 µM H2O2, 2.5 µM FeCl2/3, 200 nM A23187, 2.5 mM 3-NP, or vehicle for 24 h and processed for cell viability. DHT was significantly protective against neuronal death induced by apoptotic insults Aß, staurosporine, and apoptosis activator II [F (7,85) = 40.1; P < 0.0001] (A) but not the nonapoptotic insults H2O2, FeCl2/3, A23187, and 3-NP [F (9,107) = 33.2; P < 0.0001] (B). Flutamide [apoptotic insults, F (7,182) = 39.9; P < 0.0001; nonapoptotic insults, F (9,120) = 42.4; P < 0.0001] (C and D) and cyproterone acetate [apoptotic insults, F (7,176) = 31.0; P < 0.0001; non apoptotic insults, F (9,97) = 26.6; P < 0.0001] (E and F) showed similar patterns of neuroprotection. Cell viability graphs show means (± SEM) of combined independent experiments (n = 4–16). Cell viability is presented as the percentage of live cells normalized to the vehicle-treated control condition. Statistical significance is based on analysis of pooled raw data using the Tukey HSD. *, P < 0.05 relative to the paired vehicle + Aß, vehicle + STS, or vehicle + AAII condition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show in cultured neuronal type cells that antiandrogens function as classic AR antagonists by inhibiting classic genomic regulation but as AR agonists by protecting against cell death. Specifically we observed that flutamide and cyproterone acetate behaved as AR antagonists by blocking DHT-induced 5{alpha}-reductase type I gene expression in a concentration-dependent manner. However, rather than blocking DHT neuroprotection, we found that flutamide and cyproterone acetate mimicked DHT neuroprotection with a dose-response relationship that paralleled their antagonist actions. Furthermore, we found that flutamide and cyproterone acetate selectively protected cells against specific insults, a pattern of neuroprotection shared by DHT. This protection by flutamide and cyproterone acetate, like androgens (37, 38, 39), occurred via AR because only PC12 cells expressing AR showed androgen protection against Aß toxicity. These data represent an important new characterization of antiandrogens’ agonist (i.e. neuroprotection) vs. antagonist (i.e. inhibition of ARE-mediated gene expression) activities on androgen-mediated actions in neurons.

In terms of androgen-mediated neuroprotection, the antiandrogens flutamide and cyproterone acetate functioned as AR agonists. Like the androgen DHT, antiandrogens reduced neuronal cell death in an AR-dependent manner against insults that induce apoptosis: Aß, staurosporine, and apoptosis activator II. Findings by other research groups also indicate that androgens can protect against neuronal death induced by apoptotic insults (37, 39, 40). Neither DHT nor the antiandrogens protected against nonapoptotic insults. This largely shared neuroprotective profile may indicate common mechanisms of neuroprotection by androgens and antiandrogens. Consistent with this possibility is our observation of an absence of additive neuroprotection when flutamide and cyproterone acetate were combined with DHT.

It is interesting that androgen neuroprotection in some paradigms is antagonized by antiandrogens (37, 38, 39). For example, Ahlbom et al. (38) found that flutamide blocks the protective effect of testosterone against oxidative stress-induced cell death in cerebellar granule cells. Similarly, Hammond et al. (39) observed that flutamide inhibits testosterone protection against apoptosis induced by serum deprivation in human brain neurons. In a later study, this research group reported similar flutamide-sensitive androgen neuroprotection against Aß toxicity (37). In contrast, previous data from our laboratory showed that flutamide does not block androgen protection against Aß toxicity in hippocampal neuron cultures (40). Instead, flutamide alone is neuroprotective with efficacy equal to testosterone and DHT (40). Perhaps flutamide and other antiandrogens behave as an AR agonists vs. antagonists depending on the androgen effect and mechanism that they influence. That is, Ahlbom et al. (38) and Zhang et al. (37) presented evidence in their paradigms of genomic mechanisms of neuroprotection involving increased expression of catalase and heat shock protein 70, respectively. In contrast, androgen neuroprotection in our paradigm appears to be mediated by a nongenomic, rapid cell signaling mechanism (40, 41). Like estrogen (47, 48, 49), androgens may induce neuroprotection by genomic as well as nongenomic pathways. Thus, both sets of observations may be correct; antiandrogens may block androgen neuroprotection induced by genomic but not nongenomic mechanisms.

Our observations in neurons of agonist actions by antiandrogens parallel findings in nonneuronal cells. Flutamide and other antiandrogens have been observed to induce, rather than block, some AR-dependent androgen actions (20, 35, 36). For example, Peterziel et al. (20) observed that DHT rapidly induced MAPK signaling in prostate cancer cells, an effect that was mimicked rather than blocked by the antiandrogens hydroxyflutamide and biculatamide. Similar to our observation of AR-dependent antiandrogen protection, the MAPK signaling observed by Peterziel et al. (20) also appears to be AR dependent because activation occurred in prostate cancer PC3 cells transfected with AR but not an empty expression vector. Similarly, Zhu et al. (36) found that flutamide did not block ERK signaling induced by the androgen R1881 in a breast cancer cell line but rather activated it to an equivalent extent. Together with our findings, these studies in nonneuronal cells (20, 36, 50) suggest that in at least some cell types, antiandrogens can exhibit agonist properties for rapid cell signaling events induced by androgens.

The reasons that antiandrogens sometimes fail to exhibit antagonist properties and/or exert agonist actions across various situations are unclear. In some cases, agonist activities of antiandrogens may involve promotion of AR association with coactivators such as androgen receptor coactivator 70 (35, 51, 52, 53). Additional evidence suggests that antiandrogens can act AR agonists in the absence of androgens but AR antagonists in the presence of androgens (28). In our paradigm, we found that the agonist vs. antagonist actions of flutamide and cyproterone acetate was affected not by the presence of androgen but rather by the end point evaluated: agonists of AR-dependent neuroprotection, antagonists of AR-dependent gene expression. Thus, antiandrogens may behave as AR agonists selectively for activation of cell signaling pathways, which do not require stabilization of the AR for translocation to the nucleus, as required for transcription of ARE-specific genes (23, 54, 55). However, antiandrogens have been observed to both antagonize (56, 57) and fail to antagonize AR-mediated, rapid cell signaling (30, 31, 32, 33, 34), suggesting that the antagonist-agonist relationship for antiandrogens is likely not explained by a simple dichotomy between classic genomic and rapid cell signaling mechanisms.

Although emerging data generated from nonneuronal tissues demonstrate AR agonist activities of antiandrogens, the established function of flutamide and cyproterone acetate is to interfere with AR-ARE association and consequently block androgen-induced gene transcription via the classic genomic pathway (28, 58). Here we showed that flutamide and cyproterone acetate behave as classic AR antagonists by preventing androgen transcriptional regulation of an ARE-containing gene, 5{alpha}-reductase I (59). The time course of our androgen-induced up-regulation of 5{alpha}-reductase I parallels that demonstrated in previous neural studies (44, 45, 46). Furthermore, the abilities of flutamide and cyproterone acetate to block androgen-induced 5{alpha}-reductase I expression at competitively active concentrations (28, 29) indicate selective antagonism for classic genomic AR mechanisms. Interestingly, we observed that within the same concentration range, flutamide and cyproterone acetate induced both AR antagonist and agonist effects. Indeed, selective estrogen receptor modulators (60) such as tamoxifen, raloxifene, and ICI 182,780 are antiestrogens that can differentially display agonist (bone, uterus) and antagonist (breast, hippocampus) actions under similar treatment conditions depending on tissue type (61, 62).

This paper provides an initial characterization of AR-dependent agonist and antagonist properties of antiandrogens in neuronal cells. Our findings are consistent with recent work in adult male rodents by MacLusky et al. (63), demonstrating that flutamide mimics rather than inhibits DHT-induced increases in hippocampal spine density; however, this effect may be independent of classic AR-dependent mechanisms (64). The combination of both agonist and antagonist properties in antiandrogen compounds is increasingly acknowledged by use of the term selective androgen receptor modulators (SARMs) (27, 65). The therapeutic use of SARMs (e.g. prostate cancer treatment) paired with the recent development of clinical guidelines for long-term testosterone therapy in aging men (66) indicate an increasing importance of androgen-related therapies in aging men. Whereas accumulating data show that androgens regulate neuron viability and vulnerability to neurodegenerative disorders such as Alzheimer’s disease (2), comparatively little is known about the mechanisms by which androgens and SARMs affect neuronal cells. Continued investigation of this issue will not only increase our understanding of the neural actions of SARMs but also may provide a framework for the development of increasingly specific and effective SARMs for the treatment of age-related neurological disorders.


   Footnotes
 
First Published Online March 8, 2007

Abbreviations: Aß, ß-Amyloid; AR, androgen receptor; ARE, androgen response element; DHT, dihydrotestosterone; DMSO, dimethyl sulfoxide; HSD, honestly significant difference; 3-NP, 3-nitropropionic acid; SARM, selective androgen receptor modulator.

This work was supported by National Institutes of Health Grant AG23739.

Disclosure Summary: The authors have nothing to disclose.

Received November 3, 2006.

Accepted for publication March 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morley JE 2001 Androgens and aging. Maturitas 38:61–71; discussion 71–73[CrossRef][Medline]
  2. Pike CJ, Rosario ER, Nguyen TV 2006 Androgens, aging, and Alzheimer’s disease. Endocrine 29:233–241[CrossRef][Medline]
  3. Rosario ER, Chang L, Stanczyk FZ, Pike CJ 2004 Age-related testosterone depletion and the development of Alzheimer disease. JAMA 292:1431–1432[Free Full Text]
  4. Rosario ER, Carroll JC, Oddo S, LaFerla FM, Pike CJ 2006 Androgens regulate the development of neuropathology in a triple transgenic mouse model of Alzheimer’s disease. J Neurosci 26:13384–13389[Abstract/Free Full Text]
  5. Hogervorst E, Williams J, Budge M, Barnetson L, Combrinck M, Smith AD 2001 Serum total testosterone is lower in men with Alzheimer’s disease. Neuroendocrinol Lett 22:163–168[Medline]
  6. Mooradian AD, Morley JE, Korenman SG 1987 Biological actions of androgens. Endocr Rev 8:1–28[Medline]
  7. Beyer C, Hutchison JB 1997 Androgens stimulate the morphological maturation of embryonic hypothalamic aromatase-immunoreactive neurons in the mouse. Dev Brain Res 98:74–81[CrossRef][Medline]
  8. Zhang L, Chang YH, Barker JL, Hu Q, Maric D, Li BS, Rubinow DR 2000 Testosterone and estrogen affect neuronal differentiation but not proliferation in early embryonic cortex of the rat: the possible roles of androgen and estrogen receptors. Neurosci Lett 281:57–60[CrossRef][Medline]
  9. Leranth C, Hajszan T, MacLusky NJ 2004 Androgens increase spine synapse density in the CA1 hippocampal subfield of ovariectomized female rats. J Neurosci 24:495–499[Abstract/Free Full Text]
  10. Pouliot WA, Handa RJ, Beck SG 1996 Androgen modulates N-methyl-D-aspartate-mediated depolarization in CA1 hippocampal pyramidal cells. Synapse 23:10–19[CrossRef][Medline]
  11. Smith MD, Jones LS, Wilson MA 2002 Sex differences in hippocampal slice excitability: role of testosterone. Neuroscience 109:517–530[CrossRef][Medline]
  12. Marron TU, Guerini V, Rusmini P, Sau D, Brevini TA, Martini L, Poletti A 2005 Androgen-induced neurite outgrowth is mediated by neuritin in motor neurones. J Neurochem 92:10–20[CrossRef][Medline]
  13. Brooks BP, Merry DE, Paulson HL, Lieberman AP, Kolson DL, Fischbeck KH 1998 A cell culture model for androgen effects in motor neurons. J Neurochem 70:1054–1060[Medline]
  14. Matsumoto A 1997 Hormonally induced neuronal plasticity in the adult motoneurons. Brain Res Bull 44:539–547[CrossRef][Medline]
  15. Kerr JE, Allore RJ, Beck SG, Handa RJ 1995 Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus. Endocrinology 136:3213–3221[Abstract]
  16. Garcia-Ovejero D, Veiga S, Garcia-Segura LM, Doncarlos LL 2002 Glial expression of estrogen and androgen receptors after rat brain injury. J Comp Neurol 450:256–271[CrossRef][Medline]
  17. Pelletier G 2000 Localization of androgen and estrogen receptors in rat and primate tissues. Histol Histopathol 15:1261–1270[Medline]
  18. Kumar MV, Tindall DJ 1998 Transcriptional regulation of the steroid receptor genes. Prog Nucleic Acids Res Mol Biol 59:289–306[Medline]
  19. MacLean HE, Warne GL, Zajac JD 1997 Localization of functional domains in the androgen receptor. J Steroid Biochem Mol Biol 62:233–242[CrossRef][Medline]
  20. Peterziel H, Mink S, Schonert A, Becker M, Klocker H, Cato AC 1999 Rapid signalling by androgen receptor in prostate cancer cells. Oncogene 18:6322–6329[CrossRef][Medline]
  21. Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, Manolagas SC 2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104:719–730[Medline]
  22. Fronsdal K, Engedal N, Slagsvold T, Saatcioglu F 1998 CREB binding protein is a coactivator for the androgen receptor and mediates cross-talk with AP-1. J Biol Chem 273:31853–31859[Abstract/Free Full Text]
  23. Kemppainen JA, Lane MV, Sar M, Wilson EM 1992 Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem 267:968–974[Abstract/Free Full Text]
  24. Kemppainen JA, Wilson EM 1996 Agonist and antagonist activities of hydroxyflutamide and Casodex relate to androgen receptor stabilization. Urology 48:157–163[CrossRef][Medline]
  25. Gaillard-Moguilewsky M 1991 Pharmacology of antiandrogens and value of combining androgen suppression with antiandrogen therapy. Urology 37:5–12[CrossRef][Medline]
  26. Singh SM, Gauthier S, Labrie F 2000 Androgen receptor antagonists (antiandrogens): structure-activity relationships. Curr Med Chem 7:211–247[Medline]
  27. Berrevoets CA, Umar A, Brinkmann AO 2002 Antiandrogens: selective androgen receptor modulators. Mol Cell Endocrinol 198:97–103[CrossRef][Medline]
  28. Wong C, Kelce WR, Sar M, Wilson EM 1995 Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem 270:19998–20003[Abstract/Free Full Text]
  29. Lu S, Simon NG, Wang Y, Hu S 1999 Neural androgen receptor regulation: effects of androgen and antiandrogen. J Neurobiol 41:505–512[CrossRef][Medline]
  30. Lieberherr M, Grosse B 1994 Androgens increase intracellular calcium concentration and inositol 1,4,5-trisphosphate and diacylglycerol formation via a pertussis toxin-sensitive G-protein. J Biol Chem 269:7217–7223[Abstract/Free Full Text]
  31. Benten WP, Lieberherr M, Giese G, Wrehlke C, Stamm O, Sekeris CE, Mossmann H, Wunderlich F 1999 Functional testosterone receptors in plasma membranes of T cells. FASEB J 13:123–133[Abstract/Free Full Text]
  32. Baron S, Manin M, Beaudoin C, Leotoing L, Communal Y, Veyssiere G, Morel L 2004 Androgen receptor mediates non-genomic activation of phosphatidylinositol 3-OH kinase in androgen-sensitive epithelial cells. J Biol Chem 279:14579–14586[Abstract/Free Full Text]
  33. Estrada M, Espinosa A, Muller M, Jaimovich E 2003 Testosterone stimulates intracellular calcium release and mitogen-activated protein kinases via a G protein-coupled receptor in skeletal muscle cells. Endocrinology 144:3586–3597[Abstract/Free Full Text]
  34. Fix C, Jordan C, Cano P, Walker WH 2004 Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells. Proc Natl Acad Sci USA 101:10919–10924[Abstract/Free Full Text]
  35. Evangelou A, Jindal SK, Brown TJ, Letarte M 2000 Down-regulation of transforming growth factor ß receptors by androgen in ovarian cancer cells. Cancer Res 60:929–935[Abstract/Free Full Text]
  36. Zhu X, Li H, Liu JP, Funder JW 1999 Androgen stimulates mitogen-activated protein kinase in human breast cancer cells. Mol Cell Endocrinol 152:199–206[CrossRef][Medline]
  37. Zhang Y, Champagne N, Beitel LK, Goodyer CG, Trifiro M, LeBlanc A 2004 Estrogen and androgen protection of human neurons against intracellular amyloid ß1–42 toxicity through heat shock protein 70. J Neurosci 24:5315–5321[Abstract/Free Full Text]
  38. Ahlbom E, Prins GS, Ceccatelli S 2001 Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mechanism. Brain Res 892:255–262[CrossRef][Medline]
  39. Hammond J, Le Q, Goodyer C, Gelfand M, Trifiro M, LeBlanc A 2001 Testosterone-mediated neuroprotection through the androgen receptor in human primary neurons. J Neurochem 77:1319–1326[CrossRef][Medline]
  40. Pike CJ 2001 Testosterone attenuates ß-amyloid toxicity in cultured hippocampal neurons. Brain Res 919:160–165[CrossRef][Medline]
  41. Nguyen TV, Yao M, Pike CJ 2005 Androgens activate mitogen-activated protein kinase signaling: role in neuroprotection. J Neurochem 94:1639–1651[CrossRef][Medline]
  42. Pike CJ 1999 Estrogen modulates neuronal Bcl-xL expression and ß-amyloid-induced apoptosis: relevance to Alzheimer’s disease. J Neurochem 72:1552–1563[CrossRef][Medline]
  43. Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW 1993 Neurodegeneration induced by ß-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci 13:1676–1687[Abstract]
  44. Poletti A, Negri-Cesi P, Melcangi RC, Colciago A, Martini L, Celotti F 1997 Expression of androgen-activating enzymes in cultured cells of developing rat brain. J Neurochem 68:1298–1303[Medline]
  45. Melcangi RC, Celotti F, Castano P, Martini L 1992 Intracellular signalling systems controlling the 5{alpha}-reductase in glial cell cultures. Brain Res 585:411–415[CrossRef][Medline]
  46. Martini L, Celotti F, Melcangi RC 1996 Testosterone and progesterone metabolism in the central nervous system: cellular localization and mechanism of control of the enzymes involved. Cell Mol Neurobiol 16:271–282[CrossRef][Medline]
  47. Green PS, Simpkins JW 2000 Neuroprotective effects of estrogens: potential mechanisms of action. Int J Dev Neurosci 18:347–358[CrossRef][Medline]
  48. Linford N, Wade C, Dorsa D 2000 The rapid effects of estrogen are implicated in estrogen-mediated neuroprotection. J Neurocytol 29:367–374[CrossRef][Medline]
  49. Garcia-Segura LM, Azcoitia I, DonCarlos LL 2001 Neuroprotection by estradiol. Prog Neurobiol 63:29–60[CrossRef][Medline]
  50. Lee SR, Ramos SM, Ko A, Masiello D, Swanson KD, Lu ML, Balk SP 2002 AR and ER interaction with a p21-activated kinase (PAK6). Mol Endocrinol 16:85–99[Abstract/Free Full Text]
  51. Miyamoto H, Yeh S, Wilding G, Chang C 1998 Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells. Proc Natl Acad Sci USA 95:7379–7384[Abstract/Free Full Text]
  52. Rahman MM, Miyamoto H, Takatera H, Yeh S, Altuwaijri S, Chang C 2003 Reducing the agonist activity of antiandrogens by a dominant-negative androgen receptor coregulator ARA70 in prostate cancer cells. J Biol Chem 278:19619–19626[Abstract/Free Full Text]
  53. Yeh S, Chang HC, Miyamoto H, Takatera H, Rahman M, Kang HY, Thin TH, Lin HK, Chang C 1999 Differential induction of the androgen receptor transcriptional activity by selective androgen receptor coactivators. Keio J Med 48:87–92[Medline]
  54. Gregory CW, Johnson RTJ, Mohler JL, French FS, Wilson EM 2001 Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. Cancer Res 61:2892–2898[Abstract/Free Full Text]
  55. Zhou ZX, Lane MV, Kemppainen JA, French FS, Wilson EM 1995 Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability. Mol Endocrinol 9:208–218[Abstract]
  56. Gatson JW, Kaur P, Singh M 2006 Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology 147:2028–2034[Abstract/Free Full Text]
  57. Zagar Y, Chaumaz G, Lieberherr M 2004 Signaling cross-talk from Gß4 subunit to Elk-1 in the rapid action of androgens. J Biol Chem 279:2403–2413[Abstract/Free Full Text]
  58. Kuil CW, Mulder E 1996 Deoxyribonucleic acid-binding ability of androgen receptors in whole cells: implications for the actions of androgens and antiandrogens. Endocrinology 137:1870–1877[Abstract]
  59. Russell DW, Wilson JD 1994 Steroid 5{alpha}-reductase: two genes/two enzymes. Annu Rev Biochem 63:25–61[Medline]
  60. Gustafsson JA 1998 Therapeutic potential of selective estrogen receptor modulators. Curr Opin Chem Biol 2:508–511[CrossRef][Medline]
  61. Mize AL, Shapiro RA, Dorsa DM 2003 Estrogen receptor-mediated neuroprotection from oxidative stress requires activation of the mitogen-activated protein kinase pathway. Endocrinology 144:306–312[Abstract/Free Full Text]
  62. Yin C, Chapman J, Tawfik O 2003 Invasive mucinous (colloid) adenocarcinoma of ectopic breast tissue in the vulva: a case report. Breast J 9:113–115[CrossRef][Medline]
  63. MacLusky NJ, Hajszan T, Leranth C 2004 Effects of dehydroepiandrosterone and flutamide on hippocampal CA1 spine synapse density in male and female rats: implications for the role of androgens in maintenance of hippocampal structure. Endocrinology 145:4154–4161[Abstract/Free Full Text]
  64. MacLusky NJ, Hajszan T, Johansen JA, Jordan CL, Leranth C 2006 Androgen effects on hippocampal CA1 spine synapse numbers are retained in Tfm male rats with defective androgen receptors. Endocrinology 147:2392–2398[Abstract/Free Full Text]
  65. Brown TR 2004 Nonsteroidal selective androgen receptors modulators (SARMs): designer androgens with flexible structures provide clinical promise. Endocrinology 145:5417–5419[Free Full Text]
  66. Bhasin S, Calof OM, Storer TW, Lee ML, Mazer NA, Jasuja R, Montori VM, Gao W, Dalton JT 2006 Drug insight: testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging. Nat Clin Pract Endocrinol Metab 2:146–159[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/6/2936    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nguyen, T.-V. V.
Right arrow Articles by Pike, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nguyen, T.-V. V.
Right arrow Articles by Pike, C. J.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CYPROTERONE ACETATE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals