Molecular Cancer Therapeutics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, Y.
Right arrow Articles by Ip, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, Y.
Right arrow Articles by Ip, C.
Mol Cancer Ther. 2005;4:1047-1055
© 2005 American Association for Cancer Research

Androgen receptor signaling intensity is a key factor in determining the sensitivity of prostate cancer cells to selenium inhibition of growth and cancer-specific biomarkers

Yan Dong1, Haitao Zhang1, Allen C. Gao2, James R. Marshall1 and Clement Ip1

1 Division of Cancer Prevention and Population Sciences and 2 Departments of Medicine, Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Yan Dong, Department of Cancer Chemoprevention, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-1583. E-mail: yan.dong{at}roswellpark.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous report showed that methylseleninic acid (MSA) significantly decreases the expression of androgen receptor and prostate-specific antigen (PSA) in LNCaP cells. The present study extended the above observations by showing the universality of this phenomenon and that the inhibitory effect of MSA on prostate cancer cell growth and cancer-specific biomarkers is mediated through androgen receptor down-regulation. First, MSA decreases the expression of androgen receptor and PSA in five human prostate cancer cell lines (LNCaP, LAPC-4, CWR22Rv1, LNCaP-C81, and LNCaP-LN3), irrespective of their androgen receptor genotype (wild type versus mutant) or sensitivity to androgen-stimulated growth. Second, by using the ARE-luciferase reporter gene assay, we found that MSA suppression of androgen receptor transactivation is accounted for primarily by the reduction of androgen receptor protein level. Third, MSA inhibition of five androgen receptor–regulated genes implicated in prostate carcinogenesis (PSA, KLK2, ABCC4, DHCR24, and GUCY1A3) is significantly attenuated by androgen receptor overexpression. Fourth, transfection of androgen receptor in LNCaP cells weakened noticeably the inhibitory effect of MSA on cell growth and proliferation. Androgen receptor signaling has been documented extensively to play an important role in the development of both androgen-dependent and -independent prostate cancer. Our finding that MSA reduces androgen receptor availability by blocking androgen receptor transcription provides justification for a mechanism-driven intervention strategy in using selenium to control prostate cancer progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is the second most common cancer and the second leading cause of cancer death in men in the United States. Androgen plays an important role not only in maintaining the function of the prostate but also in promoting the development of prostate cancer (1). Androgen binds to the androgen receptor, which subsequently translocates to the nucleus and interacts with specific androgen-responsive elements (ARE) on the promoters of target genes. The interaction leads to the activation or repression of genes involved in the proliferation and differentiation of the prostate cells (2). Prostate-specific antigen (PSA) and kallikrein 2 (KLK2) are two well-accepted targets of androgen receptor. PSA, also known as kallikrein 3, is an established serum marker for the diagnosis and prognosis of prostate cancer. Although KLK2 is not as widely used as PSA, it is increasingly recognized to provide added information to disease staging (3, 4).

The randomized, placebo-controlled Nutritional Prevention of Cancer trial showed that selenium supplementation reduced the incidence of prostate cancer by 50% (5, 6). This trial was designed initially to assess the effect of selenium on nonmelanoma skin cancer. Because men accounted for a sizable proportion of the cohort (974 of a total of 1,312), there was sufficient power to analyze the changes in prostate cancer risk. When the prostate cancer data were further stratified, there was evidence of a greater reduction in risk from selenium supplementation among men who had low baseline plasma PSA levels (6). Early-stage prostate cancer is mostly responsive to androgen stimulation. The inference that the protective effect of selenium might be more pronounced in early-stage prostate cancer, as reflected by low PSA secretion, lends credence to the idea that selenium might affect androgen signaling.

Recently, we reported that a selenium metabolite, in the form of methylseleninic acid (MSA), greatly down-regulates the expression of androgen receptor and PSA in the androgen-responsive LNCaP human prostate cancer cells (7, 8). The suppression of androgen receptor signaling occurs well before any significant growth inhibition, which is accompanied by correlative changes in numerous cell cycle and apoptosis regulatory molecules (9–13). Androgen receptor signaling involves multiple steps, the receptor itself is just one of many effectors that participate in the process. For example, heat shock proteins are known to modulate the stability of androgen receptor as well as its affinity to androgen (14, 15). The transactivating activity of androgen receptor can be affected markedly by a large number of coactivators and corepressors (16). Our microarray analysis suggests that MSA alters the expression of several heat shock proteins, coactivators, and corepressors of the superfamily of steroid hormone receptors (17). In view of these confounding effects, the present study was designed to determine the role of androgen receptor down-regulation per se in MSA interference of androgen receptor signaling. Our approach was to use the ARE-luciferase reporter gene assay to find out the extent to which selenium suppression of androgen receptor transactivation could be reversed when the luciferase activity is normalized based on androgen receptor protein level. We also investigated whether androgen receptor transfection might attenuate selenium-mediated down-regulation of five androgen receptor targets: PSA, KLK2, ATP-binding cassette C4 (ABCC4, also known as MRP4), 24-dehydrocholesterol reductase (DHCR24, also known as seladin-1), and soluble guanylate cyclase 1 {alpha} 3 (GUCY1A3). These five androgen-inducible genes were selected based on the criteria that they are consistently overexpressed in prostate cancer compared with normal prostate tissue (18). Finally, in an effort to evaluate the biological significance of the selenium-androgen receptor signaling axis, we investigated whether androgen receptor overexpression might block the growth inhibitory effect of selenium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Selenium Reagent, Prostate Cancer Cell Lines, 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay, and Bromodeoxyuridine-Labeling Analysis
MSA was synthesized as previously described (19). The LNCaP and CWR22Rv1 human prostate cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). The LAPC-4 cell line was provided by Dr. Charles L. Sawyers at the University of California at Los Angeles Jonsson Comprehensive Cancer Center. The two androgen-unresponsive LNCaP sublines, LNCaP-LN3 and LNCaP-C81, were obtained from Dr. Curtis A. Pettaway (University of Texas M.D. Anderson Cancer Center) and Dr. Ming-Fong Lin (University of Nebraska Medical Center), respectively. The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 unit/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell growth and the bromodeoxyuridine (BrdUrd) labeling for DNA synthesis were done as described in our previous publication (17).

Transient Transfection of Androgen Receptor
The procedure was carried out using the LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA) per instruction of the manufacturer. At 24 hours before transfection, cells were plated in growth medium without antibiotics at a density to reach 90% to 95% confluency at transfection. The pSG5hAR androgen receptor expression vector (20) or the pSG5 mock plasmid (Stratagene, La Jolla, CA) was introduced into LNCaP cells with or without the cotransfection of the pEGFP-F membrane-GFP-encoding construct (BD Biosciences, San Jose, CA). The purpose of the green fluorescent protein (GFP) was to enable us to enrich for the subset of positively androgen receptor–transfected cells. During cotransfection, the two plasmids were added at 1:1 molar ratio. The amount of DNA transfected was 12 µg per 10-cm culture dish. The DNA/liposome mixture was removed at 3 hours after transfection. For the MTT assay, the cells were trypsinized 16 hours later and plated in triplicate onto a 96-well plate. Cells were allowed to recover for an additional 24 hours before exposure to 10 µmol/L MSA. The MTT assay was conducted at 48 hours post-MSA treatment. For the BrdUrd-labeling analysis, the cells were subjected to MSA treatment at 24 hours posttransfection and labeled with BrdUrd after 24 hours of MSA treatment.

Reporter Gene Assay
The ARE-luciferase reporter plasmid, containing three repeats of the ARE region ligated in tandem to the luciferase reporter (20), was transiently transfected into cells at a concentration of 9 µg per 10-cm culture dish. After incubating with the transfection mixture for 3 hours, the cells were trypsinized, resuspended in medium containing charcoal-stripped serum and 10 nmol/L dihydrotestosterone (Sigma, St. Louis, MO), and plated in triplicate onto 6-well plates. Cells were allowed to recover for an additional 24 hours before exposure to 10 µmol/L MSA. After 6 or 16 hours of MSA treatment, cells were lysed in reporter lysis buffer (Promega, Madison WI), and the luciferase activity was assayed using the Luciferase Assay System (Promega). Protein concentration in cell extracts was determined by the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Luciferase activities were normalized by the protein concentration of the sample. The transfection experiments were repeated thrice.

Western Blot Analysis
Details of the procedure for Western blot analysis were described previously (17). Immunoreactive bands were quantitated by volume densitometry and normalized to glyceraldehyde-3-phosphate dehydrogenase. The following monoclonal antibodies were used in this study (source): anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon, Temecula, CA), anti–androgen receptor (BD Biosciences), and anti-PSA (Lab Vision, Fremont, CA).

Real-time Reverse Transcription-PCR
Real-time reverse transcription-PCR analysis was done as described previously (21). The PCR primers and Taqman probes for ß-actin, androgen receptor, PSA, KLK2, ABCC4, DHCR24, and GUCY1A3 were Assays-on-Demand products from Applied Biosystems (Foster City, CA). The PCR conditions were as follows: an initial incubation at 50°C for 2 minutes, then a denaturation at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The relative quantitation of gene expression was done using the comparative CT ({Delta}{Delta}CT) method (22).

Androgen Receptor mRNA Stability Assay
Actinomycin D (5 µg/mL) was added to the cultures to stop new RNA synthesis at the time of MSA treatment, and androgen receptor mRNA levels were measured by real-time reverse transcription-PCR at hourly intervals for the next 6 hours. The turnover of androgen receptor mRNA was determined by comparing mRNA levels over time in cells treated with or without MSA.

Statistical Analysis
The Student's two-tailed t test was used to determine significant differences between treatment and control values, and P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MSA Depresses Androgen Receptor Transcription
Figure 1 shows the effect of MSA on androgen receptor transcript and protein levels as well as androgen receptor mRNA stability in LNCaP cells. The decrease in androgen receptor transcript, as determined by real-time reverse transcription-PCR, occurred very quickly (Fig. 1A). On the average, there was about a 50% reduction in the first three hours after treatment with 10 µmol/L MSA; by 6 hours, the magnitude of inhibition rose to 80%. At the protein level, there was no change in androgen receptor in the first two hours (Fig. 1B). A modest decrease began to appear at 3 hours, and the inhibition became very pronounced at 6 hours (Fig. 1B). The observation is consistent with the time-dependent sequence of reduced mRNA leading to decreased protein expression. To determine whether the down-regulation of androgen receptor mRNA was due to decreased transcription or increased mRNA degradation, we did an mRNA stability assay under the condition in which new RNA synthesis was blocked. Actinomycin D was added to the culture at the time of MSA treatment, and androgen receptor mRNA levels were followed in a 6-hour time course experiment. Because actinomycin D could be cytotoxic, we also monitored cell growth for up to 8 hours and did not observe cell death or significant growth inhibition during this period. Our results showed that treatment with MSA actually increased the stability of androgen receptor mRNA (Fig. 1C). This observation rules out increased mRNA degradation as a contributing factor. Therefore, the decrease in androgen receptor mRNA level by MSA is likely to be accounted for by a vigorous block of androgen receptor transcription.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Effect of MSA on androgen receptor (AR) expression in LNCaP cells. A, inhibition of androgen receptor mRNA level as determined by real-time RT-PCR. B, inhibition of androgen receptor protein level as determined by Western blot analysis. C, androgen receptor mRNA stability in the presence or absence of MSA. Bars, SE. *, P < 0.05, statistically different compared with untreated control.

 
We next examined the effect of MSA on the expression of androgen receptor and PSA in four additional human prostate cancer cell lines: LAPC-4, CWR22Rv1, LNCaP-C81, and LNCaP-LN3. The LAPC-4 cells are androgen responsive and express a wild-type androgen receptor (23), as opposed to LNCaP cells that are also androgen responsive but express a mutant, although functional, androgen receptor. The other three cell lines are all androgen-unresponsive and express a mutant but functional androgen receptor (24–27). As shown in Fig. 2 (left), MSA decreased androgen receptor and PSA transcript levels progressively as a function of time in all four cell lines examined. The reduction in androgen receptor and PSA proteins (right) paralleled the drop in the transcripts. In LAPC-4, CWR22Rv1, and LNCaP-C81 cells, a decrease in PSA transcript was already detectable as early as 3 hours, at a time when there was no apparent loss of the androgen receptor protein. The data suggest that MSA disrupts androgen receptor signaling through additional mechanism(s) beyond reducing the availability of the androgen receptor protein.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 2. Effect of MSA on androgen receptor (AR) and PSA expression in LAPC-4, CWR22Rv1, LNCaP-C81, and LNCaP-LN3 cells. Left, mRNA levels as determined by real-time RT-PCR; right, protein levels as determined by Western blot analysis. Columns, % inhibition; bars, SE. *, P < 0.05, statistically different compared with untreated control.

 
MSA-Mediated Androgen Receptor Down-Regulation Leads to a Reduction of Androgen Receptor Transactivating Activity
The transactivation of androgen receptor is an indicator of androgen receptor signaling and can be quantified readily by a reporter gene assay. To investigate whether the reduced availability of androgen receptor by MSA is a major factor in modulating androgen receptor transcriptional activity, we transiently transfected LNCaP cells with the ARE-luciferase reporter plasmid and normalized the luciferase activity based on the level of the androgen receptor protein. This normalization step eliminates the level of androgen receptor expression as a determinant of androgen receptor transactivation. The luciferase reporter assay was carried out at 6 and 16 hours after treatment with 10 µmol/L MSA. At these two time points, androgen receptor protein level was inhibited by 60% and 77%, respectively (Fig. 3B, inset). As can be seen in Fig. 3A, without normalizing for the difference in androgen receptor protein level between the MSA-treated and -untreated samples, the ARE-promoter activity was decreased by 65% or 75%, respectively, after 6 or 16 hours of MSA treatment. However, after normalization, the ARE-promoter activity was inhibited by a meager 15% at the 6-hour time point, and the inhibition disappeared completely at 16 hours (Fig. 3B). These findings suggest that the reduced availability of the androgen receptor protein is the major factor in contributing to MSA disruption of androgen receptor signaling.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Effect of MSA on ARE-promoter activity before (A) and after (B) normalizing by androgen receptor (AR) protein level. B, representative Western blot analysis of androgen receptor protein level in the cell extracts (inset). Androgen receptor protein level was depressed by 60% or 77% at 6 or 16 h, respectively. Columns, % inhibition; bars, SE. *, P < 0.05, statistically different compared with untreated control.

 
Overexpression of Androgen Receptor Attenuates the Effect of MSA on the Down-Regulation of Androgen Receptor–Regulated Genes
To delineate the role of low androgen receptor abundance as a cause of reduced PSA expression by selenium, we transiently transfected LNCaP cells with a wild-type androgen receptor construct and determined the response of PSA to MSA. After 3 hours of MSA exposure, PSA transcript was depressed by about 75% in the mock-transfected cells but only by about 45% in the androgen receptor–transfected cells (Fig. 4A). Based on our routine experience of a 40% transfection efficiency as determined by GFP cotransfection analysis (described below), we believe that the inhibitory effect of MSA on PSA mRNA might have been reversed completely in positive androgen receptor transfectants. Our conclusion was derived from the following theoretical calculation: 40% of (1 – x) + 60% of (1 – a) = 1 – b, where x = % inhibition in positive androgen receptor transfectants, a = 75% inhibition in mock transfectants, and b = 45% inhibition in the mixed population of androgen receptor–transfected cells. Solving for x in the above equation gave a value of 0% inhibition. In other words, there was no inhibition of PSA expression by MSA in the positive androgen receptor transfectants (i.e., complete reversal). The difference between the mock- and androgen receptor–transfected cells, although still apparent, was not as great at 4 and 6 hours compared with that at 3 hours. The fact that a robust androgen receptor presence was not sufficient to completely counteract the suppressive effect of MSA on the transcription of PSA at the later time points suggests that there could be a delay in the recruitment of additional mechanisms by which MSA might diminish androgen receptor signaling. We also studied the protein level of PSA by Western blotting. The Western analysis was done at 24 hours after MSA treatment. As shown in Fig. 4B, PSA protein was depressed by about 70% in the mock-transfected cells but only by about 40% in the androgen receptor–transfected cells. The protein levels of androgen receptor in the mock- and androgen receptor–transfected cells are also shown in Fig. 4B for confirmation purposes.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Effect of androgen receptor (AR) overexpression on MSA down-regulation of PSA. A, PSA mRNA expression as determined by real-time RT-PCR, in androgen receptor– or mock-transfected LNCaP cells treated with MSA. Columns, % inhibition. *, P < 0.05, statistically different compared with the value from the mock transfectant. B, PSA Western blot analysis in androgen receptor– or mock-transfected LNCaP cells treated with MSA.

 
Using a bioinformatic data mining approach, we recently identified five additional androgen-inducible genes that are expressed at a higher level in prostate cancer compared with normal prostate tissue; furthermore, their expression is repressed by MSA (18). These genes are KLK2, ABCC4 (also known as MRP4), DHCR24 (also known as seladin-1), GUCY1A3, and long-chain fatty acid CoA ligase 3 (FACL3). MSA down-regulation of their expression only occurs in LNCaP cells but not in the androgen-unresponsive PC-3 cells that express an extremely low level of androgen receptor (18). To verify that the decreased expression of these genes is a direct consequence of MSA suppression of androgen receptor signaling, we applied the same androgen receptor overexpression protocol as described above and used real-time reverse transcription-PCR to quantitate their transcript levels. The FACL3 gene was not included in this study as no Assays-on-Demand primers and probes are available for this gene. The results are shown in Fig. 5. Androgen receptor transfection significantly muted the inhibition of gene expression by MSA. In general, the difference in % inhibition between the mock and androgen receptor transfectants was greatest at 3 hours and narrowed gradually with time. The overall pattern was very similar for KLK2, ABCC4, DHCR24, and GUCY1A3. The data thus show a key role of androgen receptor down-regulation in mediating the inhibitory effects of MSA on the expression of androgen receptor–regulated genes.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. Effect of androgen receptor (AR) overexpression on MSA down-regulation of KLK2, ABCC4, DHCR24, and GUCY1A3 mRNA expression as determined by real-time RT-PCR, in androgen receptor– or mock-transfected LNCaP cells treated with MSA. Columns, % inhibition. *, P < 0.05, statistically different compared with the value from the mock transfectant.

 
Overexpression of Androgen Receptor Interferes with MSA-Mediated Growth Inhibition
In an effort to evaluate the biological significance of MSA suppression of androgen receptor signaling, we transiently transfected LNCaP cells with a wild-type androgen receptor and assessed the response of the androgen receptor–overexpressing cells to MSA-induced growth inhibition. The MTT assay was conducted at 48 hours post-MSA, and the data are presented in Fig. 6A. In the absence of MSA, cell growth was not altered by the transfection of androgen receptor (data not shown), indicating that the endogenous level of androgen receptor is not a limiting factor for the growth of these cells. MSA treatment inhibited growth by 40% in the mock transfectants, as opposed to 27% in the androgen receptor transfectants. The difference is statistically significant (P = 0.003). Thus, androgen receptor overexpression was able to weaken the growth suppressive activity of MSA. One reason that the difference was seemingly compressed was due to the fact that only a fraction of cells was successfully transfected, and in this study, cell growth was assessed using the whole cell population. To address the last problem, we cotransfected cells with the androgen receptor expression vector and a membrane-GFP-encoding construct. The cells were then subjected to BrdUrd labeling, and the data were analyzed by gating just the GFP-positive cells. As shown in Fig. 6B, after selecting for the subset of GFP-positive cells, we found that MSA inhibited DNA synthesis by a very modest 16% in the androgen receptor transfectants, as opposed to 72% in the mock transfectants. Because the GFP and androgen receptor cDNAs are not located in the same plasmid construct, it is possible that not all the cells positive for GFP are also positive for the transfected androgen receptor. Thus, our selection process only led to an enrichment, rather than an exclusive selection, of double-positive cells. Therefore, the difference between the mock transfectants and the androgen receptor transfectants might have been even more pronounced if all the cells used in the experiment were successfully transfected with androgen receptor. Figure 6B also shows that when we did the BrdUrd labeling experiment with the nonenriched androgen receptor–transfected cells, the inhibition by MSA was about 45%, a value half-way between that achieved by the mock transfectants and the enriched androgen receptor transfectants.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 6. Effect of androgen receptor (AR) overexpression on MSA inhibition of cell growth. A, MTT cell growth assay in androgen receptor– or mock-transfected LNCaP cells treated with MSA. Western blot confirmation of androgen receptor protein level (inset). B, BrdUrd labeling of selected GFP-positive or nonselected androgen receptor–transfected LNCaP cells treated with MSA. Columns, % inhibition compared with untreated control. *, P < 0.05, statistically different from mock transfectant. **, P < 0.05, statistically different from mock transfectant and nonselected androgen receptor transfectant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous report showed that selenium significantly decreases the expression and the transactivating activity of androgen receptor in LNCaP cells (21). The present study extended the above observations by showing the universality of this phenomenon and a key role of androgen receptor down-regulation in mediating the inhibitory effects of selenium on prostate cancer cell growth and the expression of cancer-specific biomarkers. First, selenium decreases the expression of androgen receptor and PSA in five human prostate cancer cell lines, irrespective of their androgen receptor genotype (wild type versus mutant) or sensitivity to androgen-stimulated growth. Second, a reporter gene assay with the ARE-luciferase construct indicated that depletion of the androgen receptor protein is a major factor for selenium depression of androgen receptor transactivating activity. Third, overexpression of androgen receptor greatly weakens the inhibitory effects of selenium on prostate cancer cell proliferation as well as the expression of five androgen receptor–regulated genes implicated in prostate carcinogenesis: PSA, KLK2, ABCC4, DHCR24, and GUCY1A3. These findings, however, do not necessarily exclude additional mechanisms by which selenium diminishes androgen receptor signaling (e.g., via modulation of ligand binding, androgen receptor dimerization, nuclear translocation, and the interaction of androgen receptor with its coregulators). In fact, our previous report provided some evidence that selenium is able to inhibit the binding of androgen receptor to the ARE in the absence of a drop in the androgen receptor level (21).

A selenium intervention strategy aimed at diminishing the expression of androgen receptor could be helpful not only for reducing prostate cancer incidence but also for preventing relapses after endocrine therapy. Almost all patients with advanced prostate cancer respond initially to treatments that interfere with the androgen receptor–signaling process. However, these treatments often fail after prolonged use and recurrence becomes a major clinical issue (28). The development of hormone refractory prostate cancer is not associated with loss of androgen receptor (29, 30). Instead, the appearance of several molecular alterations frequently leads to a lower threshold requirement of androgens for the proliferation and survival of prostate cancer cells. Androgen receptor gene mutations could result in a promiscuous receptor with a broad ligand-binding and transactivation spectrum (31). Amplification and/or overexpression of androgen receptor may hypersensitize cells to subphysiologic levels of androgens (32–35). A recent report by Chen et al. (35) claimed that increased androgen receptor expression is both necessary and sufficient to convert prostate cancer growth from androgen-dependent to -independent and that androgen receptor antagonists may display agonistic activity in cells with elevated androgen receptor expression. On the other hand, several studies showed that knocking down the expression of androgen receptor inhibits the growth of prostate cancer cells, both in vitro and in vivo, and induces apoptosis (36–39). Because selenium blocks the transcription of androgen receptor (see Fig. 1), this treatment modality may prove to be effective in prostate cancer intervention.

The down-regulation of androgen receptor targets by selenium has important clinical implication. We have studied PSA, KLK2, ABCC4, DHCR24, and GUCY1A3. All these genes are expressed at a higher level in prostate cancer compared with normal prostate tissue (18). PSA and KLK2 are prostate-specific differentiation markers. They belong to the serine protease family and are both secretory proteins. PSA is the most useful serum marker for the diagnosis and prognosis of prostate cancer. The combined use of PSA and KLK2 has been shown to improve the specificity of biochemical detection of prostate cancer (40–44) and the accuracy in predicting tumor grade and stage (3, 4). ABCC4 (also known as MRP4) is a member of the multidrug resistance-associated protein family of transporters. Overexpression of ABCC4 in neuroblastoma is associated with poor prognosis and resistance to the topoisomerase I poison irinotecan and its active metabolite SN-38 (45). Thus, the down-regulation of MRP4 by selenium might represent a potential mechanism by which selenium enhances the therapeutic efficacy of a number of anticancer drugs, including irinotecan (46). DHCR24 (also known as seladin-1) is an antiapoptotic protein, it inhibits the activity of caspase 3 (47). The overexpression of this gene has also been reported in adrenocortical adenoma cells compared with adjacent nontumor cells (48). GUCY1A3 catalyzes the conversion of GTP to the second messenger cyclic guanosine 3',5'-monophosphate, which regulates the activity of protein kinases, phosphodiesterases, and ion channels (49). Future selenium intervention trial may consider monitoring androgen receptor, PSA, KLK2, ABCC4, DHCR24, and GUCY1A3 in biopsied prostate samples, to obtain a more comprehensive picture of an individual's responsiveness to selenium. Recent data also showed that cellular PSA is more sensitive than secretory PSA to selenium intervention (50). This is one more reason why it is preferable to do the analysis in biopsied prostate tissue.


   Acknowledgments
 
We thank Hai Wang, Dorothy Donovan, Tamora Loftus, Todd Parsons, and Cathy Russin for their excellent technical assistance.


   Footnotes
 
Grant support: Department of Defense Postdoctoral Fellowship Award and New Investigator Award W81XWH-04-1-0009 (Y. Dong); Roswell Park Alliance Foundation (C. Ip); National Cancer Institute grant CA91990 (C. Ip); and National Cancer Institute, Cancer Center Support Grant P30 CA16056 (Roswell Park Cancer Institute).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/25/05; accepted 5/11/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Culig Z, Hobisch A, Hittmair A, et al. Expression, structure, and function of androgen receptor in advanced prostatic carcinoma. Prostate 1998;35:63–70.[CrossRef][Medline]
  2. Koivisto P, Kolmer M, Visakorpi T, Kallioniemi OP. Androgen receptor gene and hormonal therapy failure of prostate cancer. Am J Pathol 1998;152:1–9.[Abstract]
  3. Haese A, Becker C, Noldus J, et al. Human glandular kallikrein 2: a potential serum marker for predicting the organ confined versus non-organ confined growth of prostate cancer. J Urol 2000;163:1491–7.[CrossRef][Medline]
  4. Recker F, Kwiatkowski MK, Piironen T, et al. Human glandular kallikrein as a tool to improve discrimination of poorly differentiated and non-organ confined prostate cancer compared with prostate-specific antigen. Urology 2000;55:481–5.[CrossRef][Medline]
  5. Clark LC, Combs GF Jr, Turnbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 1996;276:1957–63.[Abstract]
  6. Duffield-Lillico AJ, Reid ME, Turnbull BW, et al. Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer Epidemiol Biomarkers Prev 2002;11:630–9.[Abstract/Free Full Text]
  7. Dong Y, Lee SO, Zhang H, et al. Prostate specific antigen expression is down-regulated by selenium through disruption of androgen receptor signaling. Cancer Res 2004;64:19–22.[Abstract/Free Full Text]
  8. Cho SD, Jiang C, Malewicz B, et al. Methyl selenium metabolites decrease prostate specific antigen expression by inducing protein degradation and suppressing androgen-stimulated transcription. Mol Cancer Ther 2004;3:605–12.[Abstract/Free Full Text]
  9. Dong Y, Zhang H, Hawthorn L, Ganther HE, Ip C. Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array. Cancer Res 2003;63:52–9.[Abstract/Free Full Text]
  10. Jiang C, Wang Z, Ganther H, Lu J. Caspases as key executors of methyl selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res 2001;61:3062–70.[Abstract/Free Full Text]
  11. Jiang C, Wang Z, Ganther H, Lu J. Distinct effects of methylseleninic acid versus selenite on apoptosis, cell cycle, and protein kinase pathways in DU145 human prostate cancer cells. Mol Cancer Ther 2002;1:1059–66.[Abstract/Free Full Text]
  12. Wang Z, Jiang C, Lu J. Induction of caspase-mediated apoptosis and cell-cycle G1 arrest by selenium metabolite methylselenol. Mol Carcinog 2002;34:113–20.[CrossRef][Medline]
  13. Zu K, Ip C. Synergy between selenium and vitamin E in apoptosis induction is associated with activation of distinctive initiator caspases in human prostate cancer cells. Cancer Res 2003;63:6988–95.[Abstract/Free Full Text]
  14. Neckers L, Ivy SP. Heat shock protein 90. Curr Opin Oncol 2003;15:419–24.[CrossRef][Medline]
  15. Georget V, Terouanne N, Nicolas J-C, Sultan C. Mechanism of antiandrogen action: key role of Hsp90 in conformational change and transcriptional activity of the androgen receptor. Biochemistry 2002;41:11824–31.[CrossRef][Medline]
  16. Heinlein CA, Chang C. Androgen receptor (AR) coregulators: an overview. Endocr Rev 2002;23:175–200.[Abstract/Free Full Text]
  17. Dong Y, Zhang H, Hawthorn L, Ganther HE, Ip C. Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array. Cancer Res 2003;63:52–9.
  18. Zhang H, Dong Y, Zhao H, et al. Microarray data mining for potential selenium targets in chemoprevention of prostate cancer. Cancer Genomics and Proteomics 2005;2:97–114.
  19. Ip C, Thompson HJ, Zhu Z, Ganther HE. In vitro and in vivo studies of methylseleninic acid: evidence that a monomethylated selenium metabolite is critical for cancer chemoprevention. Cancer Res 2000;60:2882–6.[Abstract/Free Full Text]
  20. Yeh S, Chang C. Cloning and characterization of a specific co-activator, ARA70, for the androgen receptor in human prostate cells. Proc Natl Acad Sci U S A 1996;93:5517–21.[Abstract/Free Full Text]
  21. Dong Y, Lee SO, Zhang H, et al. Prostate specific antigen (PSA) expression is down-regulated by selenium through disruption of androgen receptor signaling. Cancer Res 2004;64:19–22.
  22. Livak LJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-{Delta}{Delta}CT method. Methods 2001;25:402–8.[CrossRef][Medline]
  23. Klein KA, Reiter RE, Redula J, et al. Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice. Nat Med 1997;3:402–8.[CrossRef][Medline]
  24. Igawa T, Lin FF, Lee MS, et al. Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model. Prostate 2002;50:222–35.[CrossRef][Medline]
  25. Nakashiro K, Hara S, Shinohara Y, et al. Phenotypic switch from paracrine to autocrine role of hepatocyte growth factor in an androgen-independent human prostatic carcinoma cell line, CWR22R. Am J Pathol 2004;165:533–40.[Abstract/Free Full Text]
  26. Pettaway CA, Pathak S, Greene G, et al. Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin Cancer Res 1996;2:1627–36.[Abstract]
  27. Veldscholte J, Berrevoets CA, Ris-Stalpers C, et al. The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J Steroid Biochem Mol Biol 1992;41:665–9.[CrossRef][Medline]
  28. Koivisto P, Kolmer M, Visakorpi T, Kallioniemi OP. Androgen receptor gene and hormonal therapy failure of prostate cancer. Am J Pathol 1998;152:1–9.
  29. Hobisch A, Culig Z, Radmayr C, et al. Androgen receptor status of lymph node metastases from prostate cancer. Prostate 1996;28:129–35.[CrossRef][Medline]
  30. Sadi MV, Walsh PC, Barrack ER. Immunohistochemical study of androgen receptors in metastatic prostate cancer-comparison of receptor content and response to hormonal therapy. Cancer 1991;67:3057–64.[CrossRef][Medline]
  31. Zhao XY, Malloy PJ, Krishnan AV, et al. Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor. Nat Med 2000;6:703–6.[CrossRef][Medline]
  32. Koivisto P, Kononen J, Palmberg C, et al. Androgen receptor gene amplification: a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res 1997;57:314–9.[Abstract/Free Full Text]
  33. Kokontis J, Takakura K, Hay N, Liao S. Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. Cancer Res 1994;54:1566–73.[Abstract/Free Full Text]
  34. Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet 1995;9:401–6.[CrossRef][Medline]
  35. Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33–9.[CrossRef][Medline]
  36. Eder IE, Culig Z, Ramoner R, et al. Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides. Cancer Gene Ther 2000;7:997–1007.[CrossRef][Medline]
  37. Eder IE, Hoffmann J, Rogatsch H, et al. Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor. Cancer Gene Ther 2002;9:117–25.[CrossRef][Medline]
  38. Liao X, Tang S, Thrasher JB, Griebling TL, Li B. Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther 2005;4:505–15.[Abstract/Free Full Text]
  39. Zegarra-Moro OL, Schmidt LJ, Huang H, Tindall DJ. Disruption of androgen receptor function inhibits proliferation of androgen-refractory prostate cancer cells. Cancer Res 2002;62:1008–13.[Abstract/Free Full Text]
  40. Hecht SS. Metabolic activation and detoxification of tobacco-specific nitrosoamines: a model for cancer prevention strategies. Drug Metab Rev 1994;26:373–90.[Medline]
  41. Magklara A, Scorilas A, Catalona WJ, Diamandis EP. The combination of human glandular kallikrein and free prostate-specific antigen (PSA) enhances discrimination between prostate cancer and benign prostatic hyperplasia in patients with moderately increased total PSA. Clin Chem 1999;45:1960–6.[Abstract/Free Full Text]
  42. Nam RK, Diamandis EP, Toi A, et al. Serum human glandular kallikrein-2 protease levels predict the presence of prostate cancer among men with elevated prostate-specific antigen. J Clin Oncol 2000;18:1036–42.[Abstract/Free Full Text]
  43. Partin AW, Catalona WJ, Finlay JA, et al. Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis. Urology 1999;54:839–45.[CrossRef][Medline]
  44. Recker F, Kwiatkowski MK, Piironen T, et al. The importance of human glandular kallikrein and its correlation with different prostate specific antigen serum forms in the detection of prostate carcinoma. Cancer 1998;83:2540–7.[CrossRef][Medline]
  45. Norris MD, Smith J, Tanabe K, et al. Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro. Mol Cancer Ther 2005;4:547–53.[Abstract/Free Full Text]
  46. Cao S, Durrani FA, Rustum YM. Selective modulation of the therapeutic efficacy of anticancer drugs by selenium containing compounds against human tumor xenografts. Clin Cancer Res 2004;10:2561–9.[Abstract/Free Full Text]
  47. Greeve I, Hermans-Borgmeyer I, Brellinger C, et al. The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress. J Neurosci 2000;20:7345–52.[Abstract/Free Full Text]
  48. Sarkar D, Imai T, Kambe F, et al. The human homolog of Diminuto/Dwarf1 gene (hDiminuto): a novel ACTH-responsive gene overexpressed in benign cortisol-producing adrenocortical adenomas. J Clin Endocrinol Metab 2001;86:5130–7.[Abstract/Free Full Text]
  49. Zabel U, Weeger M, La M, Schmidt HH. Human soluble guanylate cyclase: functional expression and revised isoenzyme family. Biochem J 1998;335:51–7.
  50. Cho SD, Jiang C, Malewicz B, et al. Methyl selenium metabolites decrease prostate-specific antigen expression by inducing protein degradation and suppressing androgen-stimulated transcription. Mol Cancer Ther 2004;3:605–11.



This article has been cited by other articles:


Home page
 Mol Cancer ResHome page
S. Liu, H. Zhang, L. Zhu, L. Zhao, and Y. Dong
Kruppel-Like Factor 4 Is a Novel Mediator of Selenium in Growth Inhibition
Mol. Cancer Res., February 1, 2008; 6(2): 306 - 313.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
B. Husbeck, R. S. Bhattacharyya, D. Feldman, and S. J. Knox
Inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action.
Mol. Cancer Ther., August 1, 2006; 5(8): 2078 - 2085.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, Y.
Right arrow Articles by Ip, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, Y.
Right arrow Articles by Ip, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online