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From the * Departments of Urology Research,
Molecular Pharmacology and Experimental
Therapeutics, and Biochemistry and Molecular
Biology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester,
Minnesota.
| Correspondence to: Dr Donald J Tindall, Department of Urology Research, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN 55905 (e-mail: tindall{at}mayo.edu). |
| Received for publication March 29, 2004; accepted for publication June 20, 2004. |
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Abstract |
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)
apoptotic and cell-survival pathways, correlating with the growth and survival
effects in the LNCaP cells. Real-time polymerase chain reaction confirmed
expression level changes seen by microarray analysis of candidate genes such
as PLA2G2A, CDK8, CASP7, MDK, and NKX3.1. Collectively, our findings delineate
the cellular and molecular effects of dutasteride in androgen-responsive PCa
cells in vitro and may lead to its better therapeutic and chemopreventive use
in PCa.
Key words: LNCaP, gene-expression profiling, REDUCE trial, apoptosis
The prostate gland requires androgens for development and growth
(Cunha, 1985). The natural
ligands for the androgen receptor (AR) are testosterone and
dihydrotestosterone (DHT). The majority of testosterone (95%) is produced by
the testes, with the rest (5%) being produced by the adrenal glands
(Partin and Rodriguez, 2002). Testosterone diffuses from the capillary bed in the prostatic stroma, across
the basement membrane, and into the prostate basal epithelial cells. The basal
cells express both 5-reductase (5-R) isoenzymes, 5-R1 and
5-R2, that convert testosterone to the more potent DHT steroid. DHT
binds the AR with up to 10 times greater affinity than testosterone and
activates gene transcription of androgen-regulated genes and cellular
proliferation (Grossmann et al,
2001).
5-R enzymatic activity converts 90% of testosterone to DHT in the
prostate, and inhibition of this activity drastically reduces the amount of
the more potent ligand available to the AR. 5-R2 is the predominant
isoenzyme in the human prostate, being expressed in both epithelial and
stromal cells. Lesser amounts of 5-R1 are also present in both types of
prostate cells (Habib et al,
1998). Mutations in codon 49 of the gene encoding 5-R2
(SRD5A2) have been shown to be associated with high-risk populations and are
more prevalent in PCa than normal tissue
(Ross et al, 1998; Jaffe et al, 2000).
Additionally, these mutations are correlated with high enzymatic activity
(Makridakis et al, 1997,
2000). Recently, it has been
confirmed that a polymorphism in the SRD5A2 gene (specifically the V89L
variant) may influence the risk of developing prostate cancer in men diagnosed
at a younger age or with more aggressive disease
(Cicek et al, 2004).
The Prostate Cancer Prevention Trial (PCPT), a 7-year chemoprevention trial
with 18 882 men taking the drug finasteride, was the first successful
demonstration of PCa prevention using finasteride, an inhibitor of 5-R2
(18.4% of those receiving finasteride developed PCa compared with 24.8% on
placebo; Thompson et al,
2003). A surprising finding from the PCPT involved an association
between those taking finasteride and a greater incidence of higher Gleason
grade tumors than those on placebo
(Reynolds, 2003; Thompson et al, 2003). It
remains unclear what led to the finasteride-associated higher grade cancers.
One factor to be considered is the increase in bioavailable intraprostatic
testosterone that occurs with finasteride treatment
(Uygur et al, 1998). This
unexpected finding supports the need to better delineate the cellular and
molecular basis at work in prostate cancer cells during this type of
antiandrogen therapy.
Dutasteride, a dual inhibitor of 5-R1 and 5-R2, has been
approved for use in men with benign prostatic hyperplasia (BPH). Dutasteride
suppresses serum DHT more effectively than finasteride
(Bartsch et al, 2002). However,
the clinical benefits of inhibiting both isoenzymes remain to be defined. The
Reduction by Dutasteride of Prostate Cancer Events trial
(REDUCE)1 has been
initiated and will involve 8000 men taking dutasteride for 5 years. The
purpose of the study is to evaluate the safety and effectiveness of
dutasteride in reducing the risk of prostate cancer. It is anticipated that
inhibiting both 5-R isoenzymes will result in a better clinical
outcome. In addition to its use in the prevention of PCa, dutasteride could
potentially be used in the early treatment of PCa because of its ability to
reduce DHT levels in the prostate. However, results of clinical trials using
dutasteride for treatment of BPH indicated that treatment with this drug can
also result in increased levels of intraprostatic testosterone
(Foley and Kirby, 2003).
The molecular effects of dutasteride on androgen-responsive PCa cells are
unknown. Given the importance of mechanistic insights in the rational design
and targeting of important biomolecules and their cellular pathways, here we
present preclinical studies of dutasteride effects on the growth and
proliferation of the androgen-responsive PCa cell line LNCaP. Time and
dose-response treatment of LNCaP cells with dutasteride revealed a strong
inhibition of cell viability and proliferation at doses comparable to those
used in experimental animal models in vivo. Microarray gene-expression
analysis under these conditions identified important genes and cellular
pathways involved in metabolism, cell cycle, and apoptotic pathways, which are
disrupted by dutasteride, in addition to androgen-signaling pathways.
Real-time polymerase chain reaction confirmed expression level changes seen by
microarray analysis of candidate genes such as PLA2G2A, CDK8, MDK, and NKX3.1.
In addition, dutasteride affected several genes involved in the
FasL/TNF- apoptotic pathway and cell-survival pathways correlating with
the viability and proliferation effects seen in LNCaP cells. Collectively, our
findings delineate the cellular and molecular effects of dutasteride in
androgen-responsive PCa cells in vitro. These findings pave the way for
understanding the molecular basis of its effects in vivo and may lead to
better use in the chemoprevention trials and possible treatment of PCa.
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Materials and Methods |
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Viability and Proliferation Assays
Cells were seeded in 96-well plates at 3 x 103 cells per
well. After 48 hours media was changed to contain DMSO (vehicle) or
concentrations ranging from 1 to 100 µM dutasteride for the indicated time
periods. Viability was assayed using the CellTiter 96 Aqueous nonradioactive
cell proliferation assay (Promega, Madison, Wis). Proliferation was assayed
using the Cell Proliferation ELISA (colorimetric), BrdU incorporation assay
(Roche, Indianapolis, Ind), both following the manufacturer's
instructions.
Caspase Assays
Caspase 7 activity was assessed using a caspase-glo 3/7 kit (Promega).
Cells were seeded at 3 x 103 per well in 96-well plates and
treated for the indicated times with 0 to 50 µM dutasteride. Assays were
performed following the manufacturer's instructions.
Microarray Gene-Expression Analysis
Total RNA was isolated from LNCaP cells after treatment with either vehicle
alone (DMSO) or 10 µM dutasteride in DMSO for 48 hours using Trizol
(InVitrogen, Carlsbad, Calif) followed by further cleaning with Rneasy kit
(Qiagen, Valencia, Calif). Triplicate samples of each were quality checked
using Agilent, labeled, and hybridized to U95Av2 microchip arrays following
the manufacturer's instructions (Affymetrix, Santa Clara, Calif). The
microarray data were normalized using cyclic loess normalization
(Dudoit et al, 2002). Genes
were identified as being differentially expressed between the untreated group
and the dutasteride-treated group with a linear mixed model, similar to that
proposed by Chu et al (Chu et al,
2002). The genes were ranked according to their P value
(smallest to largest). An arbitrary decision was made to focus attention on
the top 200 differentially expressed genes as measured by their P
value.
Real-Time Polymerase Chain Reaction
Two-step real-time polymerase chain reaction was performed using cDNA
prepared from RNA isolated as described above using first strand cDNA
synthesis kit (Roche, Indianapolis, Ind) and SYBR Green polymerase chain
reaction (PCR) Master Mix (Applied Biosystems, Foster City, Calif) on an ABI
PRISM 7700 SDS following the manufacturer's instructions. The primers for SYBR
green amplification were designed using Primer3 software
(Rozen and Skaletsky, 2000), and both forward and reverse primers were used at a final concentration of 900
nM. PCR products (120–150 bp) were run on 1.2% agarose gels to check for
nonspecific amplification. Relative quantitation was used to determine fold
change in expression levels by the comparative CT method
using the formula 2-
CT, where
CT is the threshold cycle of amplification.
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Results |
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Cellular and Genetic Pathways Affected by Dutasteride in LNCaP Cells
RNA was isolated from vehicle-treated and 10 µM dutasteride-treated
cells at 48 hours and used to prepare probes for hybridizing Affymetrix U95Av2
microchip gene arrays. RNA from this time point was chosen based on the fact
that after 48 hours of treatment with 10 µM dutasteride, cell proliferation
was reduced by approximately 50%; therefore, pathways being affected by the
drug were likely to be fully engaged. For the purpose of this communication,
we focused our analysis on the top 200 differentially regulated gene
transcripts (P 
.001) and used the DAVID gene ontology annotation
tool to group genes by function (Figure 2A
through C) (Chu et al,
2002; Dudoit et al,
2002; Dennis et al,
2003). Subsequent ontological analysis revealed metabolism and
catalytic activity gene pathways as the predominant divergences between
treated and nontreated cells (Figure
2C). These were followed by cell growth/maintenance and protein
metabolism, which together with the first 2 groups are consistent with the
major anabolic effects of androgens in responsive cells. Only genes involved
in signal transduction functions were more numerous than other cell and
nucleic acid metabolism. Apparently less numerous, but critically important,
were gene groups involving stress, phosphorylation, and cell death, which we
further analyzed given the cell stress and death effects of dutasteride on
LNCaP cells.
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We chose genes from 3 of these ontological groups plus several genes known to be involved in androgen signaling and confirmed the array results using real-time PCR (RTPCR; Figure 3A through C). Of the 17 genes chosen, 11 generated RTPCR profiles consistent with the array data (Figure 3C). Four genes showed no differences in expression levels between untreated and treated samples, and 2 were expressed at extremely low or undetectable levels (data not shown).
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We next sought to identify the apoptotic and survival pathways that were
affected in dutasteride-treated cells and the cell death genes activated. Two
components of the FasL/TNF- apoptotic signaling pathway, caspase 7 and
caspase 8, were found to be up-regulated in cells treated with dutasteride. To
further determine the functional significance of the gene-expression changes
of caspase 7 and correlations with cell death seen, we used a DEVD cleavage
assay to detect enzymatic activity of caspase 7. The enzymatic activity of
caspase 7 increased in a dose-dependent manner at 48 hours for the treated
cells (Figure 4C), providing
functional significance and further confirming that this pathway is being
activated by dutasteride treatment in LNCaP cells.
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Discussion |
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-R1 and -R2
isoenzymes, thus greatly reducing the amount of DHT available to bind the AR
and direct proliferation (Bartsch et al,
2002). It may be potentially advantageous to effect this dual
inhibition for preventing or treating PCa. However, as the PCPT findings
suggested, as yet unknown mechanisms may come into play during steroid
5-R inhibition (Reynolds,
2003; Thompson et al,
2003). The REDUCE trial has been initiated, further supporting the
logic of better delineating the cellular and molecular effects of such dual
inhibition on androgen-responsive PCa cells. Understanding such mechanisms may
aid in better use of dutasteride as a chemopreventive and treatment drug for
PCa. As in the PCPT, the present findings revealed a more complex picture than expected. However, overall, dutasteride treatment of LNCaP PCa cells results in cells that phenotypically resemble LNCaP cells undergoing androgen deprivation in vitro (Murillo et al, 2001). In those studies, LNCaP cells underwent apoptosis and neuroendocrine differentiation. If the androgen-deprivation conditions are maintained chronically, there is an eventual rise of androgen-independent LNCaP cell sublines (Murillo et al, 2001). In the present studies, dutasteride induces these changes, including induction of neurogenesis genes such as MDK.
Dutasteride effectively inhibits both viability and proliferation of LNCaP
prostate cancer cells within 48 hours of treatment with 1–10 µM,
consistent with DHT's importance in these cells' growth and survival. However,
these cells are in whole media containing less than or equal to 1 pmol
testosterone, so observed effects cannot totally be explained by inhibition of
conversion of testosterone to DHT. Our array data have revealed up-regulation
of 2 genes, UGT2B15 and UGT2B17, in LNCaP cells treated with dutasteride
(Figures 2B and
3A and C). These
UDP-glucuronosyltransferases specifically recognize DHT and its metabolites,
androstane-3, 17ß-dial, and androsterone, leading to inactivation
and subsequent secretion by the cells of the respective inactive derivatives
(Turgeon et al, 2001). It has
been suggested that these enzymes have an important role in inactivating
androgens in steroid target tissues
(Turgeon et al, 2003). Increased expression of these genes with dutasteride treatment is potentially
another mode of action for this drug in prostate cells. In addition to
blocking conversion of testosterone to DHT, dutasteride can further reduce the
amount of androgen available to prostate cells by up-regulating enzymes that
degrade DHT or any of its metabolites that may be present in the cells. A
recent report examining the effects of dutasteride on the growth and
proliferation of LNCaP cells has noted that after treatment with exogenous DHT
in combination with dutasteride, cell proliferation is still dramatically
decreased (Lazier et al,
2004). This is consistent with our findings that the effects
dutasteride is having on LNCaP cells are not solely related to inhibition of
testosterone to DHT conversion.
We first focused on genes involved in the androgen-signaling pathway since
it has been shown that inhibition of 5-R in LNCaP cells affects
androgen-regulated genes (Zhu et al,
2003). RTPCR findings confirmed the microchip gene-expression
data, showing up-regulation of AR mRNA and down-regulation of NKX3.1 and PSA
(KLK3) after 48 hours of dutasteride treatment. Previous studies have
demonstrated that PSA gene expression in LNCaP cells is mediated via
conversion of testosterone to DHT (Zhu et
al, 2003); thus, blocking this conversion with dutasteride is
affecting this and possibly other AR-regulated genes. These findings suggest
that dutasteride stimulates LNCaP cells to rapidly respond to the decreased
DHT levels and AR action by up-regulating AR transcription. Under
androgen-deprived conditions the AR can bind other ligands or function in a
ligand-independent manner to promote growth and proliferation; therefore,
expression levels of the AR can be critical to cell survival under such
conditions (Culig et al, 2003).
Additionally, LNCaP cells contain a mutation in the ligand-binding domain of
the AR, leading to speculation that 5-R inhibitors may be working
through this mutation in these cells. However, Long et al tested several novel
androgen-synthesis and/or 5-R and inhibitors, along with finasteride,
for their effects on cell growth and their ability to bind AR, specifically
LNCaP mutant AR vs wild type AR in transfected PC-3 cells. They reported that
while finasteride's growth inhibitory properties are specific for the LNCaP
AR, the other dual inhibitors they tested interacted equally with both
receptors. In binding assays, finasteride competed to a small degree with
synthetic androgen R1881 equally well for both mutated and wild type AR
(Long et al, 2000).
There is evidence that in LNCaP cells treated with finasteride,
down-regulation of PSA is a result of the inhibition of the complex formation
between nuclear proteins and the steroid receptor-binding consensus (SRBC)
site in the PSA promoter (Wang et al,
1997), although in those experiments finasteride was used at
higher concentrations, 25–100 µM, than the dutasteride concentration
we have used (1–10 µM). In our studies, in agreement with those of
Zhu et al (Zhu et al, 2003), both 5-R1 and 5-R2 can be RTPCR amplified in LNCaP cells (data
not shown). It is unclear whether it matters which isoenzyme is inhibited in
LNCaP cells; however, dutasteride inhibits both and thus diminishes the
potential of any testosterone conversion to DHT. Nevertheless, it would be
important to further define additional dutasteride interactions, if any, with
other cellular proteins.
More importantly, the DNA microarray gene-expression analysis of LNCaP
cells under these conditions revealed genes involved in apoptotic, metabolic,
and cell cycle pathways, which are in addition to the expected
androgen-signaling pathway. Specifically, several genes in the
FasL/TNF- apoptotic pathway were found to be up-regulated with
dutasteride treatment (Figure
4A), pointing to a possible engagement of this cell death pathway
by dutasteride in PCa cells. Moreover, several genes involved in cell survival
or resistance to apoptosis, such as BIRC1 (baculoviral IAP repeat-containing
1), showed increased levels of expression.
Studies have shown that caspase 8 activation is necessary for
TNF-–related apoptosis inducing ligand (TRAIL)-mediated apoptosis
in LNCaP cells (Rokhlin et al,
2002). Although we did not formally test this possibility, it is
likely that this is one of the pathways by which apoptosis is occurring in
LNCaP cells treated with dutasteride. In support of this possibility were our
findings showing several key players of this pathway being affected by
dutasteride. For example, mRNA levels of TRADD, caspase 7, caspase 8, and
BIRC1 were increased, as was caspase-dependent, DEVD-cleavage activity.
Strictly speaking, the latter activity can represent caspase 3 and 7 enzymatic
activity; however, caspase 3 is considered to act upstream of caspase 7 and
enzymatic separation of the two is not possible
(Thornberry et al, 1997).
Regardless of caspase 3 contributions to our assayed DEVD-activity, both
caspases are downstream effectors of cell death
(Nunez et al, 1998). Of note,
prior studies of LNCaP cells undergoing apoptosis have also shown induction
and activation of caspase 7 (Marcelli et
al, 1998; Marcelli et al,
1999). Little change in message levels was seen for FasL or
TNF-; however, their exquisite regulation is primarily
posttranscriptional and their engagement does not necessitate new mRNA
(Beyaert et al, 2002;
Schultz and Harrington,
2003).
The phospholipase A2 gene (PLA2G2A) was found to be one of the most highly up-regulated genes in dutasteride-treated cells. Activation of phospholipases such as PLA2 results in accumulation of arachidonic acid (AA) (Seilhamer et al, 1989). Accumulation of AA and inhibition of AA metabolism, leading to increased apoptosis, has been implicated as a chemopreventive mechanism for anti-inflammatory drugs (Kelloff, 2000). Activation of PLA2, resulting in increased apoptosis, is possibly another mode of action for dutasteride-induced cell death and potential chemopreventive action in prostate cells. However, more studies are needed to explore this hypothesis in PCa cells.
Another gene found to be up-regulated in dutasteride-treated cells was CDK8, a gene involved in the regulation of transcription. CDK8 has been shown to regulate transcription by targeting the CDK7/cyclinH subunits of TFllH and providing a link between mediator complexes and basal transcription (Di Pietro et al, 1999; Akoulitchev et al, 2000). It is possible that under the dutasteride-treatment conditions, the dramatic switch in the LNCaP cell's transcription program may be aided by induction of such genes as CDK8.
The genes we have described illustrate the variety of cellular responses
taking place in LNCaP cells treated with dutasteride. Our studies were
performed in a human PCa cell line in vitro; however, LNCaP cells have been
shown to exhibit most of the characteristics of human, androgen-responsive,
PCa. Although the doses we have used in our in vitro studies correspond to
those that have been used in vivo in animal studies (both rat and dog), they
are significantly higher than levels achieved in human clinical trials; the
highest concentration reported for dutasteride in prostate tissue was 457
ng/ml (approximately 1 µM) after treatment with 5 mg per day (Roger
Rittmaster, personal communication). We are currently examining the effects of
dutasteride on LNCaP cells at the levels being used in the REDUCE trial (0.5
mg dose per day) and have observed some of the same genes being regulated at
the RNA level as early as 24 hours after treatment (preliminary data not
shown); hence, we believe that these in vitro data represent a valid starting
point for assessing dutasteride's effects on PCa cells. The in vivo findings
of the REDUCE trial could further aid our understanding of dutasteride effects
in prostate cells. Collectively, our findings delineate the cellular and
molecular effects of dutasteride in androgen-responsive PCa cells in vitro.
Further analysis of those changes that are important with regard to cell death
vs cell survival will result in a better understanding and potential use of
dutasteride in the prevention or treatment of prostate
cancer.
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Acknowledgments |
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Footnotes |
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1 Information on the REDUCE trial can be found at
www.reducestudy.com/agi.
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