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Plant-derived 3,3ⴕ-Diindolylmethane Is a Strong Androgen
Antagonist in Human Prostate Cancer Cells*
Received for publication, January 19, 2003, and in revised form, March 26, 2003
Published, JBC Papers in Press, March 27, 2003, DOI 10.1074/jbc.M300588200
Hien T. Le‡, Charlene M. Schaldach§, Gary L. Firestone¶, and Leonard F. Bjeldanes‡储
From the ‡Department of Nutritional Sciences and Toxicology and ¶Department of Molecular and Cell Biology,
The University of California, Berkeley, California 94720-3104 and §Lawrence Livermore National Laboratory,
Livermore, California 94550
3,3ⴕ-Diindolylmethane (DIM) is a major digestive prod-
uct of indole-3-carbinol, a potential anticancer compo-
nent of cruciferous vegetables. Our results indicate that
DIM exhibits potent antiproliferative and antiandro-
genic properties in androgen-dependent human pros-
tate cancer cells. DIM suppresses cell proliferation of
LNCaP cells and inhibits dihydrotestosterone (DHT)
stimulation of DNA synthesis. These activities were not
produced in androgen-independent PC-3 cells. More-
over, DIM inhibited endogenous PSA transcription and
reduced intracellular and secreted PSA protein levels
induced by DHT in LNCaP cells. Also, DIM inhibited, in
a concentration-dependent manner, the DHT-induced
expression of a prostate-specific antigen promoter-reg-
ulated reporter gene construct in transiently trans-
fected LNCaP cells. Similar effects of DIM were ob-
served in PC-3 cells only when these cells were co-
transfected with a wild-type androgen receptor
expression plasmid. Using fluorescence imaging with
green fluorescent protein androgen receptor and West-
ern blot analysis, we demonstrated that DIM inhibited
androgen-induced androgen receptor (AR) transloca-
tion into the nucleus. Results of receptor binding assays
indicated further that DIM is a strong competitive in-
hibitor of DHT binding to the AR. Results of structural
modeling studies showed that DIM is remarkably simi-
lar in conformational geometry and surface charge dis-
tribution to an established synthetic AR antagonist, al-
though the atomic compositions of the two substances
are quite different. Taken together with our published
reports of the estrogen agonist activities of DIM, the
present results establish DIM as a unique bifunctional
hormone disrupter. To our knowledge, DIM is the first
example of a pure androgen receptor antagonist from
plants.
Prostate cancer is the second leading cause of cancer-related
mortality in American men, with more than 40,000 deaths in
1997 (1). One of every four cancers diagnosed is of prostatic
origin, making prostate cancer the most commonly diagnosed
cancer (2). Although the incidence of prostate cancer in Japa-
nese and Chinese men is remarkably low compared with the
incidence in American males, after migration to the US, the
risk of later generations of Asian immigrants rises to levels
that are similar to American males (3, 4). The differences in
prostate cancer diagnosed among various population groups
suggest that factors in the environment, lifestyles, and diet
play a role in prostate cancer initiation and/or progression.
One possible contributor to the lower prostate cancer rates in
Asian men is the higher consumption of phytochemical-rich
vegetables that is typical of this population (5, 6). Consumption
of cruciferous vegetables, including broccoli, Brussels sprouts,
kale, and cauliflower, has been associated with a decreased risk
of various human cancers. The strongest associations are with
cancers of the breast, endometrium, colon, and prostate (7–10).
Incorporation of Brassica plants in feed reduces spontaneous
and carcinogen-induced tumorigenesis in experimental ani-
mals, with the greatest protective effects seen in mammary
tumors (11–13). A major active compound in cruciferous vege-
tables, indole-3-carbinol, along with its primary digestive de-
rivative, 3,3⬘-diindolylmethane (DIM),
1
exhibit promising can-
cer-protective properties in vivo and in vitro. These compounds
reduced the incidence of dimethylbenzanthracene-induced
mammary tumors in rats, benzo(a)pyrene-induced tumors of
the forestomach in mice, and benzo(a)pyrene-induced pulmo-
nary adenomas in mice (14, 15). Indole-3-carbinol has been
shown to inhibit proliferation of both breast (16, 17) and pros-
tate cancer cells (18, 19) by blocking the cell cycle and inducing
apoptosis. In addition, DIM inhibited proliferation and induced
programmed cell death in human breast tumor cells in culture
(20, 21). The cancer-preventive effects of DIM, especially on
hormone-mediated breast cancer, and the effects of indole-3-
carbinol on prostate cancer cells led us to investigate the effects
and mechanism of action of DIM against proliferation of pros-
tate tumor cells.
To examine the androgen antagonist effects of DIM, we con-
ducted a series of cell proliferation and gene activation studies
in androgen-dependent (LNCaP) and androgen-independent
(PC-3) human prostate cancer cell lines. LNCaP cells were
derived from lymph node metastasis, and PC-3 cells were de-
rived from bone metastasis (22–25). We found that DIM is a
strong antiandrogen that inhibited androgen-dependent tumor
cell growth and competitively inhibited androgen receptor
translocation and signal transduction. In addition, DIM down-
regulated prostate-specific antigen (PSA) expression at the
transcriptional level. Results from androgen receptor (AR) com-
* This work was supported in part by the California Cancer Research
Program sc09147V-10010 and by NIEHS, National Institutes of Health,
Grant P30-ESO1896. The costs of publication of this article were de-
frayed 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.
储To whom correspondence should be addressed: University of Cali-
fornia, Berkeley, Dept. of Nutritional Sciences and Toxicology, Berke-
ley, CA 94720-3104. Tel.: 510-642-5202; Fax: 510-642-0535; E-mail:
lfb@nature.berkeley.edu.
1
The abbreviations used are: DIM, 3,3⬘-diindolylmethane; AR, an-
drogen receptor; ARE, androgen response element; DHT, 5
␣
-dihy-
drotestosterone; GFP, green fluorescent protein; MMTV-Luc, murine
mammary tumor virus-luciferase; PSA, prostate-specific antigen;
CREB, cAMP-response element-binding protein; DMEM, Dulbecco’s
modified Eagle’s medium; FBS, fetal bovine serum; DCC, dextran-
coated charcoal.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 23, Issue of June 6, pp. 21136–21145, 2003
Printed in U.S.A.
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petitive binding assays, nuclear translocation studies, and
structural modeling computations suggest that DIM disrupts
AR function in a manner similar to a chemically dissimilar
synthetic antiandrogen, Casodex. Our results identify DIM as a
structurally novel, naturally occurring, pure androgen antago-
nist of potential cancer preventive and therapeutic usefulness
for prostate cancer.
EXPERIMENTAL PROCEDURES
Materials—Dulbecco’s modified Eagle’s medium (DMEM), Opti-
MEM, and LipofectAMINE reagent were supplied by Invitrogen. Phe-
nol red-free DMEM base, fetal bovine serum (FBS), calf serum, cypro-
terone acetate (6-chloro-1

,2

-dihydro-17-hydroxy-3⬘H-cyclopropa-(1,
2)-pregna-1,4,6-triene-3,20-dione acetate) and 5
␣
-dihydrotestosterone
were supplied by Sigma. Casodex was provided as a gift from Astra-
Zeneca. Dextran-coated charcoal-FBS (DCC-FBS) was from Hyclone
(Logan, UT). [
␥
-
32
P]ATP, [
3
H]DHT, and [
3
H]thymidine were supplied by
PerkinElmer Life Sciences. AR rabbit (sc-816, sc-815) polyclonal IgGs
and PSA mouse (sc-7316) and goat (sc-7638) mono- and polyclonal IgGs
were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PSA total
(M86506M) and free (M86806M) monoclonal antibodies were from
Biodesign International (Saco, ME). DIM was prepared from indole-3-
carbinol as described (26–28) and recrystallized in toluene. All other
reagents were of the highest grade available.
Cell Culture—The human prostate adenocarcinoma cell lines LN-
CaP-FGC and PC-3 were obtained from the American Type Culture
Collection (Manassas VA). They were grown as adherent monolayers in
10% FBS-DMEM, supplemented with 4.0 g/liter glucose and 3.7 g/liter
sodium bicarbonate in a humidified incubator at 37 °C and 5% CO
2
, and
passaged at ⬃80% confluency. Cultures used in subsequent experi-
ments were at less than 40 passages. Cells grown in stripped conditions
were in 5% DCC-FBS-DMEM base supplemented with 4.0 g/liter glu-
cose, 3.7 g/liter sodium bicarbonate, and 0.293 g/liter L-glutamine.
Cell Growth—Before the beginning of the treatments, cells were
depleted of androgen for 4–7 days in medium composed of DMEM base
without phenol red and with 4.0 g/liter glucose and 3.7 g/liter sodium
bicarbonate. During the depletion period, medium was changed every
48 h. Treatments were administered by the addition of 1
l of a 1,000-
fold concentrated solution of DIM in Me
2
SO/ml of medium. Once the
treatment period started, medium was changed daily to counter possi-
ble loss of readily metabolized compounds.
Cell Counting—Cells were harvested by trypsinization and resus-
pended in culture medium. Aliquots were diluted 50-fold in Isoton II
(Coulter Corp., Miami, FL), and 200-
l duplicates were counted in a
model Z1 Coulter particle counter and averaged.
[
3
H]Thymidine Incorporation—LNCaP cells were plated onto 24-well
plates (Corning) with 2 ⫻10
4
cells/well and treated with varying
concentrations of DIM with and without 1 nMDHT for 24–48 h.
[
3
H]Thymidine (3
Ci) was then added to each well and incubated at
37 °C for 2–3 h. Medium was removed, and the cells were washed 3
times with 2 ml of ice-cold 10% trichloroacetic acid followed by the
addition of 300
lof0.3NNaOH to each well and then incubated at
room temperature for 30 min. Aliquots (150
l) were transferred into
the scintillation vials with 4 ml of ScintiVerse BD scintillation fluid
(Fisher) and counted for radioactivity by a Beckman liquid scintillation
counter.
Plasmid Reporters and Expression Vectors—The ARE-responsive lu-
ciferase reporter plasmid, pPSA-630 luciferase (pPSA-Luc), was a gift
from Dr. M. D. Sadar (29). pPSA-Luc contains the PSA promoter region
(⫺630 to 12) with three AREs, all of which are critical to the activity of
the pPSA-Luc promoter. The MMTV-Luc, containing one consensus
ARE, and the expression vector, pCMV-hAR, which constitutively ex-
presses a fully functional human androgen receptor, were also gener-
ously provided by Dr. M. D. Sadar. The pCMV-GFP-rAR was a gift from
Dr. A. K. Roy (30).
RNA Extraction, mRNA Purification, and Northern Hybridization—
mRNA isolation and Northern blot analyses were conducted as de-
scribed previously (20, 31). PSA cDNA was generously provided by Dr.
M. D. Sadar, and the cDNA probes were biotinylated using NEBlot
Phototope kit (New England Biolabs, Beverly, MA), purified via precip-
itation with 3 Msodium acetate, pH 5.2, and washed with 70% ethanol.
After hybridization with cDNA probes, the membrane was incubated
with streptavidin then biotinylated with alkaline phosphatase followed
by the Phototope-CDP-Star assay (New England Biolabs) and autora-
diographed. The amount of mRNA was quantified by Gel Densitometer
(Bio-Rad) and normalized with

-actin as an internal control.
Analysis of Intracellular and Secreted PSA—LNCaP cells growing on
100-mm plates were treated as indicated for 24 h. Cells were lysed as
previously described (20) for intracellular PSA analysis. For secreted
proteins, spent medium was collected and concentrated 18-fold using
Millipore Centriprep YM-10 following the manufacturer’s protocol
(Bedford, MA). Protease inhibitors (10
g/ml aprotinin, 10
g/ml leu-
peptin, 5
g/ml pepstatin, 50
g/ml phenylmethylsulfonyl fluoride)
were added, and the proteins were immunoprecipitated with 3
g/ml
monoclonal free PSA antibody (Biodesign International) for2hand
co-immunoprecipitated overnight at 4 °C with protein A/G-agarose
(Santa Cruz Biotechnology) on a rotator. The samples were then sub-
jected to Western blot analysis as described previously (20) using a
monoclonal PSA (sc-7316) primary antibody and a goat anti-mouse-
IgG-AP secondary antibody (sc-2008) from Santa Cruz Biotechnology.
Transient Transfections with Reporters and Luciferase Assay—LN-
CaP and PC-3 cells were transfected with some modifications, as pre-
viously described (20). For AR transactivation, cells were transfected
with 0.1
g of MMTV-Luc or pPSA-Luc per plate. Co-transfection ex-
periments with pCMV-hAR or pCMV-GFP-rAR, 0.1
g/plate, was also
used. For experiments involving GFP fluorescence imaging, treatments
were not added until 30 h after transfection.
Hormone Binding Assay—LNCaP cells were grown in 5% DCC-FBS-
FIG.1.The effect of DIM on human prostate cancer cell pro-
liferation and DNA synthesis. A, to determine the effect of DIM on
cell proliferation, LNCaP (●) and PC-3 (f) cells grown in 5% DCC-FBS
DMEM were plated at a density of 10
5
cells/well in 6-well plates for 6
days. They were treated with DIM at varying concentrations, and cells
from individual wells were counted after 4 days. The data are repre-
sentative of three individual experiments done in triplicate. B, LNCaP
cells grown in 5% DCC-FBS DMEM were plated at a density of 2 ⫻10
4
cells/well in 24-well plates for 6 days. They were treated with increasing
concentrations of DIM in the presence and absence of 1 nMDHT, and
DNA synthesis was determined using thymidine uptake. The figure is
representative of three individual experiments done in triplicate.
DIM Is a Potent Androgen Antagonist in Prostate Cancer Cells 21137
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DMEM medium supplemented with 4.0 g/liter glucose and 3.7 g/liter
sodium bicarbonate and harvested in Hepes-buffered saline containing
1.5 mMEDTA by scraping with a rubber policeman. The cells were
placed on ice, collected by centrifugation, washed with ice-cold TKEG
buffer (20 mMTris-HCl, pH 7.4, 50 mMKCl, 1 mMEDTA, 0.1 mM
phenylmethylsulfonyl fluoride, and 10% glycerol) and resuspended in
250
l/plate homogenization buffer (50 mMTris-HCl, pH 7.4, 1.5 mM
EDTA, 10 mMsodium molybdate, 2.5 mM

-mercaptoethanol, 50 mM
KCl, 0.1 mMphenylmethylsulfonyl fluoride, and 10% glycerol). Cells
were homogenized using a Polytron apparatus at medium speed for 1
min on ice. The homogenates were centrifuged at 50,000 rpm in 4 °C for
60 min. The supernatant solution was divided into 1.0-ml aliquots,
quickly frozen in a dry-ice/ethanol bath, and stored at ⫺80 °C. Protein
concentration was determined by the Bradford assay using bovine se-
rum albumin as the standard. For each competitive binding assay, 5
l
of 20 nM[
3
H]DHT in 50% ethanol, 10 mMTris, pH 7.5, 10% glycerol, 1
mg/ml BSA, and 1 mMdithiothreitol was placed in a 1.5-ml microcen-
trifuge tube. Competitive ligands were added as 1.0
lof100⫻solution
in Me
2
SO. After mixing, 95
l of either LNCaP cell extracts or recom-
binant AR protein (PanVera, Madison, WI) was added, and the solu-
tions were vortexed and incubated at room temperature for 2–3h.
Proteins were precipitated by the addition of 100
l of 50% hydroxyl-
apatite slurry equilibrated in TE (50 mMTris, pH 7.4, 1 mMEDTA) and
incubated on ice for 15 min with vortexing every 5 min to resuspend the
hydroxylapatite. The pellet was washed with 1.0 ml of ice-cold wash
buffer (40 mMTris, pH 7.4, 100 mMKCl) and centrifuged for 5 min at
10,000 ⫻gat 4 °C. The supernatant was carefully aspirated, and the
pellet was washed 2 more times with 1.0 ml of wash buffer. The final
pellet was resuspended in 200
l of ethanol and transferred to a scin-
tillation vial. The tube was washed with another 200
l of ethanol,
which was then added to the same counting vial. A negative control
contained no protein, and nonspecific binding was determined using
100-fold (0.1
M) excess unlabeled DHT.
Subcellular Fractionation—Three near confluent (80–90%) cultures
of LNCaP cells in 100-mm Petri dishes were used for each treatment.
Treatments were added as 1
l of a 1,000-fold concentrated solution of
DIM in Me
2
SO/ml of medium for the indicated time. After incubation
with treatments at 37 °C, cytosolic and nuclear proteins were prepared
as described (32, 33) with modifications. Briefly, cells were lysed in
hypotonic buffer (10 mMHepes, pH 7.5) and harvested in MDH buffer (3
mMMgCl
2
,1mMdithiothreitol, 25 mMHepes, pH 7.5). After homoge-
nization, supernatant was saved for cytosolic proteins, and nuclear
proteins were extracted from the pellets using MDHK buffer (3 mM
MgCl
2
,1mMdithiothreitol, 0.1 MKCl, 25 mMHepes, pH 7.5) followed by
FIG.2.The effect of DIM on expression of endogenous PSA gene. A, Northern blot of PSA mRNA in LNCaP cells treated with 1, 10, and
50
MDIM for 24 h. B, time course of PSA down-regulation by treatment with 10
MDIM. C, cells grown in either complete (C) or stripped (S)
medium were treated with 1 nMDHT for increasing times. Greatest induction of PSA mRNA by 1 nMDHT at 72 h was inhibited with 24 h of 50
MDIM co-treatment.

-Actin was used as the internal control for Northern blot analysis. Regulation of PSA gene expression was quantified by
scanning with a gel densitometer and is represented as -fold change over control.
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HDK buffer (25 mMHepes, pH 7.5, 1 mMdithiothreitol, 0.4 MKCl).
Cytosolic and nuclear extracts were subsequently analyzed by Western
blot analysis.
Western Blot Analysis—After the indicated treatment, Western im-
munoblot analyses of androgen receptor from LNCaP cells were per-
formed as described previously (20). In short, polyclonal AR antibodies,
sc-816 and sc-815, from Santa Cruz Biotechnology were used as pri-
mary antibodies with a chemiluminescence protein detection method.
Blotted membranes were stained with Coomassie Blue to determine
protein loading, or

-actin (sc-8432, Santa Cruz Biotechnology) was
used as an internal control. The amount of protein was quantified by
Gel Densitometer (Bio-Rad) and normalized with

-actin when used as
an internal control.
Fluorescence Imaging—PC-3 cells were plated on cover slips in 6-well
culture plates at 1.5 ⫻10
5
cells/well in 5% DCC-FBS-DMEM medium.
Cells were co-transfected with pCMV-GFP-rAR and pPSA-Luc or
MMTV-Luc as indicated above. Cover slips were placed on microscope
slides, and images were taken at 1000⫻. Fluorescence imaging of GFP
was performed using a Zeiss Axiophot 381 and Q-imaging MicroPub-
lisher at the College of Natural Resources Biological Imaging Facility of
the University of California, Berkeley, CA.
Modeling of DIM Binding to the AR Ligand Binding Domain—
Quantum mechanical geometry optimizations were performed at a high
level of theory, 6–31G**/MP2, for DHT, DIM, Casodex, and R1881.
Using these molecular coordinates, a solvent-accessible surface was
constructed surrounding each molecule; such a surface enables coupling
of the ab initio electronic structure calculations to the solution of the
Poisson-Boltzmann equation (34). The coupling was accomplished
through the single and double layers of charge at the boundary and
allowed for relaxation of the quantum electronic charge distribution in
response to these surrounding layers. This first principles approach
eliminated the need to assign fractional charges to the atoms. The
induced polarization charge at the interface was then mapped onto the
nodes (“dots”) of the elements of the solvent accessible surface. A com-
parison was then made between these molecules.
The atomic configuration of DHT determined experimentally, i.e.
obtained from the crystal structure of the molecule in the androgen
receptor, provided a template for comparison of the feasibility of the
androgen receptor binding a different ligand (35). We, therefore, again
FIG.3.DIM inhibits expression of secreted and intracellular
PSA protein. A, Western blot analysis of intracellular PSA protein
level in cells treated with Me
2
SO (DMSO) control and 50
MDIM in the
presence and absence of 1 nMDHT. B, DHT (1 nM) induced the expres-
sion of secreted PSA, and co-treatment with 50
MDIM inhibited the
expression of the secreted protein. Coomassie Blue staining was used to
verify equal protein loading.
FIG.4. Transcriptional activation of reporter genes in the
presence of DIM and DHT. A, LNCaP cells were transiently trans-
fected with the MMTV-Luc promoter containing a single androgen
response element. After transfection, the cells were treated with in-
creasing concentrations of DIM with and without 1 nMDHT for 24 h. B,
LNCaP cells were transiently transfected with the pPSA-Luc promoter
containing three androgen response elements. After transfection, the
cells were treated with increasing concentrations of DIM with and
without 1 nMDHT for 24 h. For both experiments at the completion of
treatment luciferase analysis was performed, and luciferase activity in
cytosol preparations from individual plates was normalized for protein
concentration as determined by Bradford assay. The graph is represent-
ative of three different experiments.
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constructed a solvent-accessible surface, SR, surrounding the DHT
molecule (crystal structure) and used this surface as a reference “stand-
ard”or template for the androgen receptor’s ligand binding site. The
center-of-mass of each androgen receptor ligand, DHT, Casodex, R1881
(optimized coordinates), and DIM, was translated to the center-of-mass
of the template (crystal structure coordinates) and then rotated about
the x,y, and zaxes through the center-of-mass. We calculated the
fractional surface area of the ligand, which did not fit into the binding
site template, as follows. For each element, i, of the ligand surface, SL,
we then found its nearest neighbor element j on SR, allowing us to form
the vector rij ⴝri-rj from element j (nearest neighbor to i of SL) on SR
to element i. By forming the dot product of rij with the normal to the
element at j on SR, rij ⴛnj, we could determine whether element i of
SL is inside or outside of SR. In this way we calculated a ⌬SL, the
fractional surface area of each ligand that lies outside the template
DHT surface SR. This method was repeated using the crystal structure
of R1881 as the binding site template (36).
RESULTS
DIM Inhibits the Proliferation and DNA Synthesis of Unin-
duced and DHT-induced LNCaP Cells—The effects of DIM on
human prostate cancer cell growth were examined using LN-
CaP and PC-3 cells. After a 96-h treatment, DIM produced a
concentration-dependent inhibition of LNCaP cell proliferation
with maximal inhibition of 70% at 50
M. At these concentra-
tions, DIM had no observable effects on the growth of PC-3 cells
(Fig. 1A). In addition, we examined the effects of varying con-
FIG.5. Androgen receptor-mediated transcriptional activa-
tion of the MMTV-Luc reporter gene in PC-3 cells. PC-3 cells were
transiently transfected with the MMTV-Luc promoter containing a
single androgen response element and pCMV-hAR, an androgen recep-
tor expression plasmid. After transfection, the cells were treated with
increasing concentrations of DIM with and without 1 nMDHT for 24 h.
Luciferase analysis was performed after treatment, and luciferase ac-
tivity in cytosol preparations from individual plates was normalized for
protein concentration as determined by Bradford assay. The figure is
representative of three different experiments.
FIG.6. Relative binding affinity to
the androgen receptor. A, whole cell
extracts from LNCaP cells were used in
the competitive binding assay. [
3
H]DHT
(20 nM) was competed by unlabeled DHT,
cyproterone acetate, Casodex, and DIM.
B, recombinant androgen receptor (Pan-
Vera) at 6 pmol/reaction was used in the
competitive binding assay. [
3
H]DHT (5
nM) was competed by unlabeled DHT,
cyproterone acetate, Casodex, and DIM.
The figure is representative of experi-
ments that were repeated three times.
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centrations of DIM with and without 1 nMDHT on DNA syn-
thesis in LNCaP cells (Fig. 1B). Our results showed a concen-
tration-dependent inhibition of DNA synthesis of these cells of
up to 90% under both uninduced and androgen-induced growth
conditions.
Inhibition of Endogenous PSA Expression by DIM—North-
ern blot analysis was used to examine the effect of DIM on
endogenous PSA gene expression. Fig. 2 shows concentration-
dependent (A) and time-dependent (B) decreases of up to 70%
in PSA mRNA levels after DIM treatments. In addition, PSA
mRNA induction by DHT with increasing time of treatment
was inhibited by up to 80% by 24 h of co-treatment with DIM
(Fig. 2C). Furthermore, Western immunoblot analysis showed
that DIM reduced levels of intracellular and secreted PSA
protein to background concentrations (Figs. 3, Aand B) after
DHT co-treatments. The reduction of PSA expression was com-
parable with the reduction in DHT-induced mRNA expression
determined by Northern blot analysis. These results are con-
sistent with DIM regulation of PSA expression occurring at the
transcriptional level and consistent with the antiandrogenic
activity of DIM observed in the cell proliferation experiments.
DIM Down-regulates the Activities of DHT-induced Reporter
Genes—The antiandrogenic effects of DIM were further exam-
ined with reporter assays using a MMTV-Luc promoter con-
struct that contains one ARE and a pPSA-Luc promoter con-
struct containing three AREs. These plasmids were transiently
transfected into LNCaP cells and, by luciferase analysis,
showed that DIM strongly inhibited DHT induction of andro-
gen-responsive genes by more than 50% at 1
Mand more than
90% at 10
Min both promoter constructs (Fig. 4, Aand B).
Treatment with DIM alone failed to induce transactivation of
these reporter genes. These results further confirm that DIM
inhibition of AR-responsive gene expression occurs at the tran-
scriptional level.
The AR Is the Central Modulator of DIM Inhibitory Effects on
Androgen-regulated Gene Expression—To confirm the impor-
tance of the AR in the transcriptional activation of the ARE
promoters, we employed PC-3 cells, which exhibit little or no
AR expression. We transfected these cells with the pPSA-Luc
promoter and performed luciferase analysis to show that with-
out co-transfection of an AR expression vector, DIM has no
effect (data not shown). In contrast, co-transfection of an AR
expression vector with the pPSA-Luc reporter construct led to
a concentration-dependent inhibition of DHT-induced transac-
tivation by DIM that was similar to the effect we had observed
in LNCaP cells (Fig. 5). The same results were seen with the
MMTV-Luc promoter (data not shown). Moreover, DIM by it-
self did not induce transactivation of these reporter genes in
either cell line with or without co-transfection of the wild-type
androgen receptor.
DIM Competes with Androgen for Binding to the AR in LN-
CaP Cells and in Recombinant AR Protein—Because our re-
sults strongly implicate the AR as the focus of the DIM mode of
action in prostate cells, we assessed directly the ability of DIM
to bind to this receptor. Our results of competitive binding
assays with both the mutant AR of LNCaP cells and a wild-type
recombinant human AR demonstrate that DIM, in the micro-
molar concentration range, competes with labeled DHT for
binding to the AR (Fig. 6). Cyproterone acetate and Casodex,
two well known antiandrogens, were used as positive controls.
DIM and Casodex exhibited similar binding affinity for the AR.
Biochemical Analysis of AR Cytoplasmic/Nuclear Distribu-
tion in Cells Treated with DHT and DIM—To examine the
effect of DIM on nuclear translocation of the AR, both Western
blot analysis and fluorescence imaging of tagged AR were con-
ducted. LNCaP cells were treated with DIM in the presence
and absence of 1 nMDHT. Cytoplasmic and nuclear protein
fractions were extracted and subjected to Western blot analysis
for the AR. The results show that DIM by itself had no effect on
nuclear translocation and that 1 nMDHT produced a strong
translocation of the AR into the nucleus. However, DHT-in-
duced AR translocation was blocked up to 75% when cells were
co-treated with DIM (Fig. 7).
Fluorescence imaging using a pCMV-GFP-rAR co-trans-
fected with pPSA-Luc was used to confirm and extend the
results of our Western blot analyses of endogenous AR trans-
location. Cells treated with 1 nMDHT showed hormone-in-
duced trafficking of the AR to be predominantly nuclear within
1 h of treatment (Fig. 8A). However, co-treatment with 50
M
DIM partially inhibited the translocation of AR induced by
FIG.7. Cellular localization of the
AR in LNCaP cells treated with DIM
and DHT. LNCaP cells grown in 5%
DCC-FBS DMEM for 5 days were treated
for 24 h before extraction of cytosolic and
nuclear proteins with increasing concen-
trations of DIM in the presence and ab-
sence of 1 nMDHT. Western blot analysis
was performed using an antibody to the
AR (Santa Cruz Biotechnology).
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DHT treatment and showed distribution of the AR to be both
cytoplasmic and nuclear (Fig. 8B). Furthermore, DIM treat-
ment prevented the formation of AR foci in the nucleus. DIM
alone produced a predominantly cytoplasmic distribution of
fluorescence.
In a control experiment, the expression vector for the
GFP-AR was co-transfected with the androgen-regulated re-
porter genes, pPSA-Luc and MMTV-Luc, to investigate the
activity of the pCMV-GFP-rAR. The results verified that the
activity of the chimeric receptor construct in the presence of
DHT and DIM was similar to activity of the simple pCMV-AR-
derived receptor (data not shown). These results show that
DIM both inhibits the nuclear translocation of the liganded AR
and prevents the formation of nuclear AR foci.
Structural Modeling of DIM Binding to the AR and Compar-
ison with DHT, R1881, and Casodex—Because DIM is a strong
antagonist of AR function but exhibits less than obvious struc-
tural similarity to the endogenous AR ligand, DHT, we com-
pared the structure of DIM to DHT and other AR ligands.
Results from these calculations showed similarities among
DIM, DHT, the AR agonist R1881, and the AR antagonist
Casodex, structural representations of which are included in
Fig. 9. A comparison of the dimensions of all the ligands with-
out their hydrogen atoms showed the experimentally deter-
mined structures of DHT and R1881 to be ⬃20 Åin the long
axis versus 18 Åfor the optimized DIM and 15 Åfor Casodex.
All of the ligands exhibit the same width, but DIM and Casodex
are twice the height of the other ligands. In addition, compar-
ison of the crystal structure of DHT with its computationally
optimized conformation showed a slight bending upward of the
3-OH end in the optimized molecule versus a more planar,
slightly downward-pointing 3-OH end in the crystal structure.
The same change is seen in R1881 (data not shown). This result
suggests a slight conformational change in the ligand when it
binds to the receptor binding site.
We then compared the solvent-induced polarization charges
for the AR ligands. We compared solvent-accessible surfaces for
DIM, DHT, R1881, and Casodex. The results indicated a sim-
ilar charge pattern and ellipsoid shapes for all of the ligands,
with positive surface charge above the oxygen, fluorine, or
nitrogen atoms on both ends of the molecules (data not shown).
Because both DIM and Casodex act as pure antiandrogens,
we compared the structures of these ligands more closely. As
shown in Fig. 10, the two ligands are remarkably similar in
conformation despite their considerable difference in atomic
compositions. Both molecules have a planar region (Fig. 10A)
containing a polar atom (nitrogen for DIM, fluorine for Caso-
dex) that can bind into the known AR binding site in a manner
comparable with the 3-OH group of DHT, combined with a
bulky region at the opposite end of the ligand. When rotated by
90 °, to look directly down the bulky end (Fig. 10B), we observe
that this end of each of the molecules tilts 30–45 °relative to
the distal aromatic rings, suggesting a similar fit into the
androgen receptor ligand binding site. These conformations are
in contrast to the more planar structures of the AR agonists,
DHT and R1881.
DISCUSSION
The present study characterized the antiandrogenic activity
of DIM and investigated the mechanism of its action in human
prostate tumor cells. This study is the first to reveal that (a)
DIM suppresses DHT-induced cell growth and PSA expression
and exhibits no AR agonist activity, (b) DIM has a strong
affinity for both the mutant AR inLNCaP cells and for recom-
binant wild-type human AR, (c) nuclear translocation and foci
formation of DHT-bound AR are inhibited by DIM, and (d)
modeling studies showed that DIM is remarkably similar in
molecular geometry and surface charge distribution to the well
established synthetic antiandrogen, Casodex. Our investiga-
tion, leads to the conclusion that DIM is a strong, pure andro-
gen antagonist.
Considerable progress has been made in recent years in
elucidating the sub-cellular mode of action of the AR. The
unliganded AR resides predominantly in the cytoplasm where
it is sequestered as a multiprotein complex with heat shock
proteins and immunophilins. Upon ligand binding, the AR dis-
sociates from the multiprotein complex, homodimerizes, and is
transported into the nucleus, resulting in stimulation or inhi-
bition of androgen receptor-mediated gene expression (30, 37).
Our Western blot analysis showed that treatment with DHT
alone induced nuclear translocation of the AR and that co-
treatment with DIM inhibited DHT-induced translocation in a
concentration-dependent manner. To confirm our Western
analysis, we used fluorescence imaging with GFP-AR to show
that unliganded GFP-AR is primarily localized in the cyto-
plasm, and upon androgen treatment, it migrates into the
nucleus. Translocation of AR induced by DHT is inhibited by
DIM co-treatment.
FIG.8.Cytoplasmic and nuclear distribution of the AR in the
presence of DHT and DIM. PC-3 cells were co-transfected with
pCMV-GFP-rAR and pPSA-Luc and treated as indicated. A, fluores-
cence imaging of a time course distribution of AR in cells treated with
1n
MDHT shows predominantly nuclear translocation by 60 min. B,
co-treating cells with 50
MDIM inhibited DHT-induced translocation
of the AR. Treatment with DIM alone shows predominantly cytoplasmic
fluorescence.
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Distinct patterns of nuclear distribution of the GFP-AR exist
for different ligands, and these patterns correlate with the
transactivation activity of the ligand of the AR. Exposure to
DHT causes a punctate distribution pattern that is indicative
of the association of the translocated receptor within a sub-
nuclear compartment. The formation of these nuclear foci is
thought to provide platforms for the interaction of nuclear
receptor and co-activators (38). Liganded steroid hormone re-
ceptors are transferred to common compartments located in the
euchromatin region and form a complex with co-activators,
such as steroid receptor coactivator 1, transcriptional interme-
diary factor 2, and CREB-binding protein, which are also ac-
cumulated in the same subnuclear compartments. For the AR,
CREB-binding protein was found to be essential for foci forma-
tion, and the process of compartmentalization is essential for
full transactivation (39). Furthermore, foci formation was
shown to be closely linked to transcriptional activation by the
AR. It has been reported that a homogeneous pattern of nuclear
distribution correlates with an inactive receptor (30, 39). How-
ever, patterns of subnuclear compartmentalization vary among
different antiandrogens. Well known antiandrogens such as
cyproterone acetate and hydroxyflutamide can inhibit andro-
gen activity at relatively high concentrations, whereas they
exhibit AR agonist activity at low concentrations (40). Cyprot-
erone acetate induces formation of nuclear foci at low concen-
trations, also. However, in cells treated with the pure AR
antagonist, Casodex, the translocated receptor showed an
evenly distributed pattern (37, 41). Other chemicals that have
antiandrogen activities and that are well known environmental
endocrine disrupters, were also reported to follow this correla-
tion of AR transactivation function and subnuclear clustering.
The agricultural fungicide vinclozolin and the insecticide nitro-
fen have been shown to disrupt formation of intranuclear flu-
orescence foci while inhibiting AR transactivation (30, 42).
Similarly, we show that DIM exerts no agonistic activity while
strongly inhibiting DHT-mediated AR transactivation and
DHT-induced formation of nuclear foci. Co-treatment with DIM
and DHT inhibited AR translocation and produced a homoge-
neous pattern of fluorescence distribution.
Further comparisons of DIM to known antiandrogens show
clear differences in modes of action. The partial androgen ago-
nists, cyproterone acetate and hydroxyflutamide, have been
shown to induce partial nuclear translocation of AR at concen-
trations as low as 1
M(43). Casodex, which is reported to be a
pure AR antagonist, could also stimulate AR nuclear translo-
cation (41). In contrast, DIM did not stimulate AR nuclear
FIG.9.Comparison of the quantum mechanically optimized conformations of DIM (A) and Casodex (D) with the crystal structures
of DHT (B) and R1881 (C). The crystal structures do not contain hydrogen atoms.
DIM Is a Potent Androgen Antagonist in Prostate Cancer Cells 21143
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translocation, even at the highest concentration used in the
present studies. These results indicate that DIM might medi-
ate an early block in androgen action, including the inhibition
of heat shock protein dissociation from the AR and/or a mask-
ing of the nuclear translocation signal.
A comparison of the expected lowest energy conformation of
DIM with conformations of other androgen receptor ligands,
DHT, R1881 and Casodex, indicated several similarities.
Quantum mechanical comparisons showed that all of these
ligands exhibit similar solvent-induced polarization charge dis-
tributions around the region of the molecule that fits into the
3-OH end of the AR binding site, thought to be important for
ligand stabilization (35). Although generally comparable in
overall size, DIM is a bulkier ligand than either of the two
agonists DHT or R1881, which may increase the pocket vol-
ume, decreasing hydrogen bonding at the 17-

-OH end of the
binding site. This disturbance could interfere with helix posi-
tion or orientation of the bound AR, ultimately affecting down-
stream actions of the AR. Because it has been suggested that
the precise positioning of helix 12 is required for the activation
of the AR (35), the possibility exists that DIM causes a mis-
placement of this helix, which contributes to DIM antagonist
activity. Furthermore, the structural similarities of DIM and
Casodex support the notion that these two ligands may affect
their antagonistic effects through a similar steric mechanism.
The down-regulation of PSA by DIM is important because of
the association of PSA expression with prostate cancer. PSA is
a 240-amino acid glycoprotein with a molecular mass of ⬃34
kDa that is secreted by prostatic epithelial cells. PSA has been
reported to promote the proliferation, migration, and metasta-
sis of prostate cancer cells through several mechanisms, includ-
ing cleavage of insulin-like growth factor-binding protein-3 and
degradation of extracellular matrix proteins fibronectin and
laminin (44, 45). PSA expression is regulated by the AR and is
thought to function as a growth factor in LNCaP cells (46–49).
Thus, down-regulation of PSA expression may be important in
the antiproliferative effects of DIM in LNCaP cells. In addition,
PSA is the most commonly used biochemical marker for detec-
tion and monitoring of prostate cancer, and decreases in PSA
levels are associated with better prostate cancer prognosis (50,
51). Thus, these results indicate a possible role of DIM in
prostate cancer therapy.
It is interesting to note that the antiproliferative and anti-
androgenic activity of DIM in LNCaP cells were observed at
physiologically relevant concentrations. A man of average
weight who consumes 200 g of broccoli daily will obtain ⬃12 mg
of DIM. With maximum absorption of DIM, the blood concen-
tration of DIM would be as high as ⬃10
M. Therefore, in vivo
concentrations of DIM from dietary Brassica vegetables repre-
sent the effective levels of DIM in vitro.
In conclusion, our study establishes DIM as a pure androgen
antagonist that blocks expression of androgen-responsive
genes and inhibits AR nuclear translocation and nuclear foci
formation. The discovery of DIM as the first pure androgen
receptor antagonist from plants establishes this substance as a
new class of hormonally active agents with potential both as
environmental androgen disrupters and as prostate tumor pre-
ventive and therapeutic agents.
Acknowledgments—We extend our gratitude to Dr. Marianne D.
Sadar for the pPSA-Luc and MMTV-Luc plasmids, pCMV-hAR expres-
sion vector, and PSA cDNA. We thank Dr. Arun Roy for the pCMV-
GFP-rAR expression vector. We also thank Dr. Zhongdong Liu for
assistance with the Northern analysis.
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Hien T. Le, Charlene M. Schaldach, Gary L. Firestone and Leonard F. Bjeldanes
Prostate Cancer Cells
-Diindolylmethane Is a Strong Androgen Antagonist in Human′Plant-derived 3,3
doi: 10.1074/jbc.M300588200 originally published online March 27, 2003
2003, 278:21136-21145.J. Biol. Chem.
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