Mol Biol Rep (2008) 35:107–117
DOI 10.1007/s11033-007-9059-5
ORIGINAL PAPER
Coprinus comatus and Ganoderma lucidum interfere
with androgen receptor function in LNCaP prostate cancer cells
Ben-Zion Zaidman Æ Solomon P. Wasser Æ
Eviatar Nevo Æ Jamal Mahajna
Received: 5 October 2006 / Accepted: 17 January 2007 / Published online: 13 March 2007

Springer Science+Business Media B.V. 2007
Abstract In this study, we screened a total of 201
diethyl ether, ethanol, and ethyl acetate fungal
Basidiomycetes extracts for anti-androgenic activity.
Based on our screened results in combination with the
selective inhibition of prostate cancer LNCaP cells, we
selected Coprinus comatus and Ganoderma lucidum for
further evaluation. We demonstrated that ethanol and
ethyl acetate extracts from C. comatus and G. lucidum,
respectively, selectively inhibit dihydrotestosterone-in-
duced LNCaP cell viability, suppress levels of secreted
prostate-specific antigen in a dose-dependent manner,
and cause a G1 phase arrest in LNCaP, but not in DU
145 and PC-3 cells. For the first time, to the best of our
knowledge, we demonstrated that C. comatus and
G. lucidum decreased androgen and glucocorticoide
receptors transcriptional activity in breast cancer
MDA-kb2 cells in a dose-dependent manner, and sup-
pressed androgen receptor (AR) protein level in
LNCaP and MDA-kb2 cells. Our findings suggest that
AR and non-AR mediated mechanisms underlie the
effects of C. comatus and G. lucidum.
Keywords Androgen receptor
Coprinus comatus

Ganoderma lucidum
LNCaP
Prostate cancer
B.-Z. Zaidman
S. P. Wasser
E. Nevo
Institute of Evolution, University of Haifa, Mount Carmel,
Haifa 31905, Israel
B.-Z. Zaidman
J. Mahajna (&)
Cancer Drug Discovery Program, Migal, Galilee
Technology Center, P.O. Box 831, Kiryat Shmona 11016,
Israel
e-mail: jamalm@migal.org.il
Introduction
The human prostate gland, a male sexual accessory
tissue involved in seminal fluid production, has a
remarkably high incidence of neoplastic disease [20].
Prostate cancer (PCa) tends to be a disease of older
men, with more than 70% of the diagnoses found in
men over 65 years of age. The aging population, cou-
pled with early detection programs and improved
screening, has affected the incidence rate of PCa to
increase dramatically. The age-standardized incidence
rate of PCa is highest in the USA (137 per 100,000 in
black men), lower in European countries (40 and 30
per 100,000 in England and Denmark, respectively),
and lowest in Asia (11 and 2.2 per 100,000 in Japan and
China, respectively) (adapted from [25]). Recent
studies based on epidemiological data suggest that
environmental factors, such as diet, can be a partial
contributor to the difference between Western and
Eastern statistics [24, 39] and therefore can signifi-
cantly influence the prevention or progression of PCa.
Of special interest to us is the growing body of research
that corroborates the anti-cancer properties of certain
fungi that were used for centuries in traditional medi-
cine, especially in Eastern countries [31].
Over the last three to four decades scientific and
medical studies carried out in Japan, China, Korea, and
more recently the USA have increasingly demonstrated
the potent and unique properties of compounds
extracted from a range of medicinal fungi [31]. Initially
this research focused on high-molecular-weight
polysaccharides and polysaccharide–protein complexes
that are used to enhance various immune responses
and act as immunomodulators or biological response
modifiers [35]. However, in recent years, there is a shift
123
108
in interest toward low-molecular-weight fungal
secondary metabolites that can target specific cellular
processes at the molecular level and have a clear
mechanism of action [44].
Androgens play an important role in the prolifera-
tion, differentiation, maintenance, and function of the
prostate [23]. Evidence shows that androgens are also
involved in the development and progression of PCa
[5]. Androgen action is mediated by the androgen
receptor (AR), which, upon binding to the active
androgen dihydrotestosterone (DHT), translocates
into the nucleus and regulates androgen responsive
genes implicated in the development of PCa [9].
Androgen deprivation is the only effective systemic
therapy available for advanced and metastatic PCa [2].
This therapy has been achieved using anti-androgens
[28]. However, all anti-androgens developed so far
display moderate affinity for the AR, and thus mod-
erate efficacy in vitro and in vivo. There is a need for
second-generation anti-androgens, which could display
an equal or even higher affinity for AR compared to
the natural androgens and, at the same time, maintain
its pure anti-androgenic activity thereby providing an
improved androgen blockade [28].
The present study was undertaken to screen
Hymenomycetes fungi as AR modulators and eluci-
date their mechanism of action. We hypothesize that
many fungal secondary metabolites are excellent can-
didates as AR modulators. This is supported by four
decades of research on anti-cancer activities of fungal
extracts and isolated compounds [44], and the sugges-
tion that the pharmacophoric structural features of
some of these compounds are consistent with quanti-
tative structure–activity relationship models and AR
binding studies.
In this report, we show the anti-androgenic activity
of Coprinus comatus and Ganoderma lucidum organic
extracts. These extracts selectively decrease viability
and induce a G1 phase arrest in androgen-dependent
LNCaP cells; decrease luciferase reporter activity in
DHT-induced MDA-kb2 breast cancer cells; decrease
secreted levels of AR-regulated prostate-specific anti-
gen (PSA) in DHT-induced LNCaP cells; and decrease
AR protein level in LNCaP and MDA-kb2 cells.
Materials and methods
Chemicals and media
Dexamethasone (DEX), DHT, 17b-estradiol (E2),
flutamide, and all other fine chemicals were purchased
from Sigma-Aldrich (Rehovot, ISR). Tissue culture
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Mol Biol Rep (2008) 35:107–117
media and reagents were purchased from Biological
Industries (Beit Haemeq, ISR). Yeast extract and
peptone were purchased from BD Diagnostic Systems
(Sparks, MD, USA).
Fungi growth conditions
Pre-inocula for cultures were prepared by transferring
ten 5-mm diameter disks from the Higher Basidiomy-
cetes Culture Collection at the Institute of Evolution,
University of Haifa, to flasks containing 100 ml of YE
medium (yeast extract 1 g/l, peptone 1 g/l, glucose
20 g/l, KH2PO4 1 g/l, MgSO4 0.5 g/l, supplemented
with FeSO4
7H2O 2.5 mg/l, MnCl2
4H2O 0.35 mg/l,
ZnSO4
7H2O 0.3 mg/l, CuSO4
5H2O 0.5 mg/l), and
incubated for 14 days. Contents of the pre-inocula
flasks were homogenized and transferred to flasks
containing 1 l of YE medium. Inoculated flasks were
shaken at 160 rpm at 27C for 12 days, then filtered
and washed to remove culture broth. Wet mycelia were
dried at 37C.
Fungi extraction
Dry mycelia were ground to fine powder and extracted
with ethyl acetate, diethyl ether, or ethanol for 7 days
on a rotary shaker at 160 rpm at room temperature.
Flask content was paper filtered, and solvents were
removed under reduced pressure. Stock solutions were
prepared in dimethylsulfoxide (DMSO) and stored at
–70C.
Cell lines and cell culture
Human LNCaP (AR positive, androgen-dependent
PCa), DU 145 (AR negative, androgen-independent
PCa), PC-3 (AR negative, androgen-independent PCa),
and MDA-kb2 (AR positive, androgen-independent
breast cancer) cell lines (ATCC, Rockville, MD, USA)
were grown in RPMI 1640 containing 2 mM L-gluta-
mine, 10% fetal bovine serum (FBS), 100 IU/ml
penicillin, and 100 lg/ml streptomycin. All cell lines
were grown at 37C in a humidified atmosphere with
5% CO2. Cells were transferred with 0.025% trypsin
and 0.02% EDTA.
Trypan blue exclusion assay
Cells were plated (3 · 105 cells/well) in six-well plates
containing 3 ml of phenol red-free medium supple-
mented with charcoal-stripped serum. After 48 h,
fungal extracts in increasing concentrations were ad-
ded in addition to 5 nM DHT. After 24 h, cells were
Mol Biol Rep (2008) 35:107–117
collected, stained with 0.4% trypan blue solution (1:1),
and counted using a hemacytometer.
Luciferase reporter assay
To study the effects of fungal extracts on AR tran-
scriptional activity with minimal effects on cells pro-
liferation, we used the MDA-kb2 cell line. This cell
line expresses firefly luciferase under control of the
mouse mammary tumor virus (MMTV) promoter that
contains response elements for both GR and AR [12].
MDA-kb2 cells were plated at 5 · 105 cells/well in 12-
well plates. After 48 h, cells were treated as indicated.
After an additional 24 h, cells were collected and wa-
shed twice in phosphate-buffered saline (PBS) at
3,000 rpm for 6 min. PBS was removed, and into each
tube 50 ll lysis buffer (Promega, Madison, WI, USA)
was added. After 30 min, tubes were centrifuged at
12,000 rpm for 8 min, and the supernatant was used for
luciferase readings. Luciferase activity was determined
using a microtiter plate luminometer (Anthos Labtec
Instruments, Wals, Austria) and quantified as relative
light units (RLU). RLU units were normalized against
total cell lysate protein concentration as determined
with Bio-Rad DC protein assay procedure and re-
agents (Bio-Rad, Hercules, CA, USA).
Western blot analysis
Cells were plated at 5 · 105 cells in 25 cm2 flasks. After
48 h, cells were treated as indicated. After an addi-
tional 48 h, incubation cells were collected and washed
twice in PBS at 3,000 rpm for 6 min at 4C. PBS was
removed and cells were lysed in 60 ll of lysis buffer
(10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA,
1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM
Ma3VO4, 1% Triton X-100, 10% glycerol, 1 mM
PMSF, 0.1% SDS, and 0.5% sodium deoxycholate,
supplemented with protease and phosphatase inhibitor
cocktails). Cell lysates were centrifuged at 13,000 rpm
for 10 min at 4C, and the supernatant was collected.
Protein concentration in the cell lysate was determined
as described in the previous section. Equal amounts of
protein (40 lg) were fractionated on 8% SDS-PAGE.
The PAGE-fractionated protein extracts were then
transferred to nitrocellulose membranes and incubated
overnight at 4C with HRP-conjugated mouse anti-
human AR monoclonal antibodies (Santa Cruz Bio-
technology Inc., Santa Cruz, CA, USA). Membranes
were incubated with Western blotting detection re-
agents (Bio-Rad, Hercules, CA, USA), and visualized
on Kodak BIOMAX MR films (Sigma-Aldrich, Re-
hovot, ISR).
109
PSA ELISA assay
LNCaP cells were plated at 5 · 105 cells in 25 cm2
flasks. After 48 h, cells were treated as indicated and
after an additional 48 h growth medium was collected
and concentrated 17-fold using Microcon YM-10
(Millipore, Bedford, MA, USA) centrifugal filter at
11,000 rpm at 4C for 60 min. Total secreted PSA was
determined using a PSA ELISA kit (MP Biomedicals,
Irvine, CA, USA) following the manufacturer’s pro-
tocol. PSA levels were normalized to protein concen-
tration of each sample.
Cell cycle analysis
LNCaP, PC-3, and DU 145 cells were plated at
5 · 105 cells in 25 cm2 flasks. After 48 h, cells were
treated as indicated, and after an additional 48 h, cells
were trypsinized and washed twice in cold PBS at
1,200 rpm at 4C for 5 min. After washing, 70% cold
ethanol was added to vials while gently vortexing. Cells
were incubated at 4C for 24 h. After fixing, cells were
washed twice with PBS and cell pellets were re-sus-
pended in 1 ml PBS containing 100 lg/ml of RNase A.
After 5 min propidium iodide was added to a final
concentration of 50 lg/ml and left for 15 min at room
temperature. Analysis was performed on the BD
FACSCalibur system with CellQuest software (BD
Biosciences, Franklin Lakes, NJ, USA). Data was
analyzed using the ModFit software (Verity Software
House, Topsham, ME, USA).
Results
Validation of the transactivation assay
The human breast cancer cell line MDA-kb2 was used
in our transactivation assay. MDA-kb2 cell line was
generated by stably transfecting the MMTV luciferase-
neo reporter gene construct into MDA-MB-453, a
breast cancer cell line [12]. The parental MDA-MB-
453 is known to express significant amounts of endog-
enous AR as well as other steroid hormone receptors.
The effect of the AR ligand, DHT, on the AR-
dependent transactivation activity was assayed over a
broad range of DHT concentrations ranging from
1 pM to 1 lM (Fig. 1a). Presence of DHT induced
luciferase activity in a concentration-dependent man-
ner showing that the presence of DHT levels as low as
0.1 nM were efficient in causing a significant increase
in the luciferase activity, and DHT at 0.1 lM caused
maximum induction of reporter activity and remained
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110 Mol Biol Rep (2008) 35:107–117

16 in future experiments it will allow us the ability to

14
A monitor changes in reporter activity in response to
12 treatment with the appropriate extracts.
Fold induction

10 Since the MDA-MB-453 parental cell line is known

8 to contain significant amounts of AR and GR, but not
6 PR, estrogen receptor (ER)a or ERb [38], we tested
4
the ability of different steroid hormones to activate
2
reporter activity from the MMTV promoter. The AR
ligand DHT at 5 nM induced luciferase activity by 7.7-
0
fold, and the GR agonist DEX at 10 nM induced
1.0E-12

1.0E-11

1.0E-10

1.0E-09

1.0E-08

1.0E-07

1.0E-06

1.0E-05
luciferase activity by 13.8-fold (Fig. 1b). In addition,
DHT concentration (M) the ER ligand, E2 at 10 lM, induced luciferase activity
18 by 7.5-fold (Fig. 1b). Reporter activity induced by E2
16 B believed to be mediated by the AR and not the ER due
14 to the fact that MDA-kb2 cell line lacks ERa and has
very weak ERb expression. In addition, E2-induced
Fo l d i n d u c t i o n

12

10
luciferase activity can be blocked by hydroxyflutamide
8
(an AR antagonist), and not by the ER antagonist ICI
6
182, 780. Therefore, it was concluded by Hartig et al.
4
[12] that E2 acts as an AR agonist in MDA-kb2 cells.
2
Next, we monitored the ability of known anti-
0
androgens to affect DHT-induced reporter activity
None DHT (5 nM) DEX (10 nM) E2 (10 µM) from MDA-kb2 cells. The herbicide linuron (structur-
Steroid hormone ally related to the non-steroidal anti-androgen,
10 flutamide) and the pesticide p,p¢-dichlorodiphenyldi-
9 C chloroethylene (p,p¢-DDE), a stable metabolite of
8
dichlorodiphenyltrichloroethane, have been shown to
Fo l d i n d u c t i o n

7
6 possess anti-androgenic activity [16, 33, 34], together
5
with anti-androgen flutamide, were tested for their
4
3 ability to affect DHT-induced reporter activity in
2 MDA-kb2 cells. All anti-androgens used caused a sig-
1
0
nificant reduction in DHT-induced reporter activity by
DMSO + - - - - more than 59% as compared to DHT-treated cells
DHT (5 nM) - + + + + (Fig. 1c). Thus, the above data provide sufficient evi-
Flutamide (1 µM) - - + - -
dence for the ability of the described system to serve as
a reliable screening tool for the identification of fungal
Linuron (10 µM) - - - + -
extracts capable of modulating AR function.
p ,p’-DDE (10 µM) - - - - +
Screening for anti-androgenic activity
Fig. 1 Induction of luciferase reporter in MDA-kb2 cells. (a)
Concentration-dependent induction by DHT. Luciferase activity
was calculated against DMSO-treated control wells. (b) Cells A total of 201 diethyl ether, ethanol, and ethyl acetate
were treated with AR and ER natural ligands DHT and E2, fungal extracts were screened in the AR transactiva-
respectively, and the GR agonist DEX. (c) Inhibition of reporter tion assay. This screening allows us to select fungal
activity by the anti-androgens flutamide, linuron, or p,p¢-DDE. extracts that interfere with AR activity. Our criteria for
Cells were treated for 24 h. Luciferase RLU readings were
normalized against total cell lysate protein concentration. Values selecting active extracts were 40% or more reduction
are represented the fold induction compared to the control of in the AR transcriptional activity. Results from a rep-
three replicates ± standard deviation resentative experiment are shown in Fig. 2. In this
experiment we screened ethyl acetate extracts of fungi
level thereafter when higher DHT concentrations were from 5 of the 12 orders in the class Basidiomycetes;
used. Figure 1a shows that DHT at a concentration of Polyporales, Agaricales, Stereales, Hymenochaetales,
5 nM causes induction of reporter activity of about and Russulales. Positive numbers illustrate inhibition
50% of the maximal induction rate. Thus, we used of AR transactivation, whereas negative numbers
DHT at a concentration of 5 nM for our screening, and indicate an AR stimulating activity probably due to the

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Mol Biol Rep (2008) 35:107–117 111

Ganoderma lucidum
Coprinus comatus A 100
Omphalotus olearius
Fomitopsis pinicola 90

Percent reduction of luciferase

Armillariella mellea
Flammulina velutipes 80
Gymnopilus spectabilis
Leucoagaricus leucothites
Bjerkandera adusta 70
Schizophyllum commune
Lentinus edodes
60

activity
Pleurotus ostreatus
Hypsizygus marmoreus
Piptoporus betulinus 50
Inonotus tamarias
Grifola frondosa
Trametes versicolor 40
Hericium erinaceus
Oudemansiella radicata 30
Rigidoporus ulmarius
Volvariella volvacea
Daedalea quercina 20 C. comatus
Funalia trogii
Merulius tremellosus
Psilocybe cubensis 10 G. lucidum
Laetiporus sulphureus
-60 -40 -20 0 20 40 60 80 0
Percent reduction of luciferase activity 0 50 100 150 200
Concentration (µg/ml)
Fig. 2 Treatment of MDA-kb2 cells with ethyl acetate fungal
extracts. Cells were treated with 25 lg/ml fungal extracts for
24 h. Percent reduction in 5 nM DHT-induced luciferase activity B 100

was calculated against DHT-treated control. Luciferase RLU 90

Percent reduction of luciferase

readings were normalized against total cell lysate protein 80
concentration. Fungi that were found strong antagonists inhib- 70
iting reporter activity by more than 40% were repeated several
60
times with similar results

activity
50
40
30 C. comatus
presence of AR agonist activity. G. lucidum and C.
20 G. lucidum
comatus extracts reduced luciferase activity by 65 and
45%, respectively (Fig. 2). On the other hand, extracts 10

from Laetiporus sulphureus and Psilocybe cubensis 0

increased reporter activity by 50 and 40%, respectively, 0 50 100 150 200

suggesting androgenic activity. Concentration (µg/ml)

Fig. 3 Coprinus comatus and G. lucidum extracts inhibit

C. comatus and G. lucidum fungal extracts decrease
AR and GR transcriptional activity in MDA-kb2
cells in a dose-dependent manner
The ability of different concentrations of both extracts
to affect DHT-induced reporter activity was evaluated,
and results are summarized in Fig. 3a showing that C.
comatus extract decreased luciferase activity by 51% at
a concentration of 160 lg/ml. Interestingly, G. lucidum
extract was much more potent than C. comatus,
decreasing luciferase activity by 93% at 160 lg/ml
(Fig. 3a).
Our group and others confirmed that MDA-kb2 cell
lines express both the GR and AR genes (data not
shown). Thus, we wanted to test the selectivity of the
selected extracts toward the AR by monitoring their
effect on DEX-induced reporter activity. Results in
Fig. 3b show that C. comatus and G. lucidum extracts
inhibit DEX-induced luciferase activity in a concen-
tration-dependent manner with comparable potency to
their effect against DHT-induced reporter activity. For
example, at a concentration of 160 lg/ml C. comatus
and G. lucidum extracts inhibit DEX-induced lucifer-
ase activity by 92 and 96%, respectively (Fig. 3b).
luciferase reporter activity in MDA-kb2 cells. (a) Effect of
fungal extracts on 5 nM DHT-induced cells. (b) Effect of fungal
extracts on 10 nM DEX-induced cells. Cells were treated with
increasing concentrations of fungal extracts for 24 h. Reduction
of luciferase activity was calculated against inducer-only treated
cells that were regarded as 100%. Luciferase RLU readings were
normalized against total cell lysate protein concentration. Values
are represented as the mean of two replicates ± standard
deviation
C. comatus and G. lucidum extracts selectively
decrease LNCaP cells viability
Androgen receptor is known to modulate proliferation
and viability of LNCaP PCa cell lines [43]. We tested
the ability of the selected two extracts to affect the
viability of LNCaP cells. We employed the trypan blue
dye exclusion membrane integrity assay to assess cell
viability and to estimate IC50 values for the two
selected extracts. Data shown in Table 1 illustrated
that both extracts inhibited proliferation of LNCaP
cells at IC50 values of 28.3 and 44.8 lg/ml for
C. comatus and G. lucidum, respectively. Interestingly,
on one hand, a significantly higher concentration of the
C. comatus extract was needed to cause 50% growth

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112 Mol Biol Rep (2008) 35:107–117

Table 1 Effectiveness of inhibiting cancer cell viability by G. lucidum and C. comatus extracts
Species Cell line
LNCaP DU 145 PC-3 MDA-kb2

C. comatus 28.3 ± 12.6 560.0 ± 69.7 220.0 ± 42.4 245.0 ± 65.4

G. lucidum 44.8 ± 16.7 77.0 ± 13.0 176.4 ± 30.7 118.0 ± 10.2
Cells were treated with increasing concentrations of fungal extracts. After 24 h, cells were collected, stained with 0.4% trypan blue
solution (1:1), and counted using a hemacytometer. Percent inhibition of cells’ viability was calculated against DHT-treated control.
Values (lg/ml) are represented as the mean IC50 of three independent experiments ± standard deviation

inhibition of the other lines tested, suggesting a selec-
tivity toward LNCaP cell lines. On the other hand, the
G. lucidum extract exhibited reduced selectivity com-
pared to the C. comatus extract.
C. comatus and G. lucidum induce G1 arrest
in LNCaP but not in PC-3 or DU 145 cells
We wanted to evaluate the ability of the selected
fungal extracts to affect cell cycle progression of PCa
cell lines. The three PCa cell lines LNCaP, PC-3, and
DU 145 were treated with C. comatus extract at con-
centrations of 30 and 120 lg/ml, and G. lucidum ex-
tract at concentrations of 40 and 120 lg/ml for 48 h, at
which time cell cycle distributions were studied by
FACS analysis.
Dihydrotestosterone treatment of LNCaP cells
caused a decrease in the percentage of cells in the G1
phase from 66 to 43%. The decrease in the percentage
of cells at the G1 phase was accompanied with an in-
crease in the percentage of cells in the S phase from 16
to 38% (Fig. 4a). This illustrates that LNCaP cells are
androgen-dependent for their growth. Flutamide, an
AR inhibitor, increased the G1 phase population of
LNCaP cells from 43 to 65% and decreased the S phase
population from 38 to 16% (Fig. 4a). In contrast, no
effect on cell cycle distribution of the cells PC-3 and
DU 145 was observed in the presence of flutamide
(Fig. 4b, c). Treatment of LNCaP cells with C. comatus
and G. lucidum extracts caused a concentration-
dependent increase in the percentage of cells in the G1
phase (69 and 77% at high concentration, respectively,
compared to 43% in the control). The increase in the
percentage of cells in the G1 phase population was also
accompanied with a concentration-dependent decrease
in the S phase population (10 and 3%, respectively, vs.

Fig. 4 Effects of C. comatus A 100 DHT B 100

DMSO DMSO DHT
and G. lucidum extracts on 90 Flutamide C. comatus Low 90 Flutamide C. comatus Low
C. comatus High G. lucidum Low
cell cycle distribution of G. lucidum High 80
C. comatus High G. lucidum Low
80 G. lucidum High
LNCaP (a), DU 145 (b), and
70 70
PC-3 (c) cells. Cells were
Percent cells
Percent cells

treated with 30 lg/ml C. 60 60

comatus low, 120 lg/ml C. 50 50
comatus high, 40 lg/ml G. 40 40
lucidum low, 120 lg/ml G. 30 30
lucidum high, and flutamide
20 20
5 lM for 48 h. Cells were co-
10 10
administered with 5 nM
DHT. Propidium iodide- 0 0
stained cells were analyzed by G1 S G2 G1 S G2
FACS. Experiment was
C 100
repeated twice with similar DMSO DHT
outcome 90 Flutamide C. comatus Low
C. comatus High G. lucidum Low
80 G. lucidum High
70
Percent cells

60

50
40
30

20
10
0
G1 S G2

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Mol Biol Rep (2008) 35:107–117
38% in the control) (Fig. 4a). In contrast, C. comatus
and G. lucidum extracts had no effect on the cell cycle
distribution of DU 145 and PC-3 cells (Fig. 4b, c). In
conclusion, fungi-treated cells show an increase in the
percentage of cell population in the G1 phase accom-
panied with a decrease in the percentage of cell pop-
ulation in the S phase arguing for a G1 phase arrest
caused by both the C. comatus and G. lucidum in
LNCaP cells with minimal effects on PC-3 and DU 145
cells (Fig. 4b, c).
C. comatus and G. lucidum extracts decrease levels
of secreted PSA in a dose-dependent manner
Prostate-specific antigen is an AR-regulated 34-kDa
glycoprotein, which has been used as a serum marker
for screening and staging of PCa [30]. LNCaP cells are
known to express and secrete PSA [18]. Therefore, to
characterize the effects of fungal extracts on the
function of the AR, we measured levels of secreted
PSA from LNCaP cells in response to treatment with
different concentrations of the selected extracts. DHT
treatment of LNCaP cells cause an increase of secreted
PSA by 2.47-fold. In addition, presence of flutamide at
1 lM decreased the level of secreted PSA by 53%
(data not shown). Treatment of LNCaP cells with both
extracts caused a concentration-dependent decrease in
the secreted PSA levels. The level of secreted PSA was
decreased by 77 and 66% following treatment with G.
lucidum and C. comatus extracts at concentrations of
60 and 160 lg/ml, respectively (Fig. 5).
C. comatus and G. lucidum extracts suppress AR
expression in LNCaP and MDA-kb2 cells
Prostate-specific antigen is an AR-responsive gene and
interfering with AR function might cause its down-
regulation by the two fungal extracts. Thus, AR pro-
tein levels in treated LNCaP cells were monitored.
Results presented in Fig. 6 showed that DHT treat-
ment of LNCaP cells cause an increase of steady-state
levels of the AR protein, while treatment with
flutamide (1 lM) caused a moderate decrease in the
AR protein levels. However, treatment with C. coma-
tus and G. lucidum extracts caused a significant de-
crease of the AR protein levels in a concentration-
dependent manner.
It is well documented that LNCaP cells are AR
dependent for their growth and proliferation [43].
Thus, the decrease in the AR protein level shown in
Fig. 6a could have resulted from an inhibition of
LNCaP cell proliferation by the fungal extracts. To
clarify this issue, we tested the effect of fungal extracts
113
100
90 G. lucidum C. comatus
Percent reduction of secreted
80
70
60
PSA
50
40
30
20
10
0
0 20 40 60 80 100 120 140 160 180
Concentration (µg/ml)
Fig. 5 Suppression of secreted PSA in LNCaP cells medium
using G. lucidum and C. comatus extracts. LNCaP cells were
incubated in phenol red-free RPMI 1640 containing 10%
charcoal/dextran-stripped FBS for 48 h and then treated with
DHT (5 nM) and/or fungal extracts. DHT (5 nM) serves as
control. After 48 h, growth medium was collected and concen-
trated 17-fold. Aliquots of 50 ll were subjected to ELISA assay
as described in ‘‘Materials and methods’’. Values represent
percent reduction in secreted PSA compared to the control
normalized against total cell lysate protein concentration
on the AR protein level in MDA-kb2 breast cancer
cells. MDA-MB-453 cells, the parental cell line of
MDA-kb2 cells, are known to be an androgen-inde-
pendent breast cancer cell line. Our data show that the
proliferation of the MDA-kb2 breast cancer cells was
not inhibited by both extracts at the concentrations
used. However, C. comatus and G. lucidum extracts
caused a reduction in the AR protein level in
MDA-kb2 cells (Fig. 6b). In contrast, flutamide and
DHT caused only minor changes in the AR protein
levels in MDA-kb2 cells (Fig. 6b).
Discussion
In this study, we utilized the MMTV-luciferase stably
transfected breast cancer cell line MDA-MB-453 to
follow the effect of fungal extracts on AR transacti-
vation function by monitoring luciferase reporter
activity. In addition, we utilized three PCa cell lines;
LNCaP as an androgen-dependent cell line, PC-3, and
DU 145 as androgen-independent cell lines to study
the effect of selected fungal extracts on cells prolifer-
ation and viability. We screened 68 Basidiomycetes
species from five orders for anti-androgenic activity.
Out of 201 ethanol, ethyl acetate, and diethyl ether
extracts screened, 11 (5.5%) exhibited anti-androgenic
123
114
A C. comatus
80 µ g/ ml
AR
Flutamide - - + -
DHT - + + +
DMSO + + + +
B C. comatus
100 µ g /ml
200 µ g/ ml
AR
Flutamide - - + -
DHT - + + +
DMSO + + + +
Fig. 6 Effect of C. comatus and G. lucidum extracts on
intracellular AR levels in LNCaP (a) and MDA-kb2 cells (b).
Cells were incubated in phenol red-free RPMI 1640 containing
10% charcoal/dextran-stripped FBS for 48 h and then treated
with DHT (5 nM) and/or fungal extracts. DMSO (0.5%) and/or
flutamide (1 lM) serve as controls. After 48 h, cells were lysed to
prepare cellular protein extracts. Aliquots of 40 lg total cellular
proteins were separated on 8% SDS gels and transferred to
nitrocellulose membranes. AR expression was determined by
incubating with HRP-conjugated mouse anti-human AR mono-
clonal antibody (1:5,000). Immunoreactive signals were detected
using a chemiluminescent kit
activity inhibiting AR-mediated reporter activity by
more than 40% at a concentration of 25 lg/ml.
Extracts prepared from C. comatus and G. lucidum
showed the most promising results and thus were
selected for further evaluation.
The AR is known to exist as a monomer in the
cytoplasm of responsive cells, sequestered in a complex
with heat-shock proteins (HSPs), and its activation
includes several sequential steps. On administration
and binding of androgens, the AR changes conforma-
tion, and the HSPs are released followed by a second
conformational change. The receptor dimerizes, binds
to DNA, and interacts with the transcription initiation
complex to regulate transcription of responsive genes
[17]. Thus, interfering with one or more of the steps
leading to AR activation might lead to effective anti-
androgenic intervention. We suggest that the observed
decrease in the AR transcriptional activity by C.
comatus and G. lucidum could possibly result from one
or more of the above steps. Furthermore, the reduction
123
Mol Biol Rep (2008) 35:107–117
G. lucidum in the transcriptional activity of AR could result from a
decrease of the AR protein stability and turnover.
160 µ g/ml
6 0 µ g /m l
1 5 µ g/ml
30 µ g/ml
However, the nature of the fungal active moieties and
their exact mode of action remain unknown.
Ganoderma lucidum is known for its anti-cancer
properties [29]. This fungus contains an abundance of
- - - - various low-molecular-weight secondary metabolites,
+ + + + many of which are responsible for mediating the bio-
logical effects on specific signaling pathways. For
+ + + +
example, Stanley et al. [32] recently demonstrated that
a mixture of polysaccharides and triterpenes from G.
G. lucidum
lucidum modulates MAPK and Akt signaling in PC-3
PCa cells, which, in turn, inhibits the activity of AP-1
12 0 µ g /m l
30 µ g/ ml
50 µ g/ ml
60 µ g /m l
resulting in the down-regulation of the secreted VEGF
and TGF-b1 factors. Furthermore, more than 130
pharmacological active triterpenoids have been iso-
lated from species of the genus Ganoderma [8]. We
- - - - -
speculate that some of these triterpenoids include ste-
+ + + + + roids that could possibly interfere or compete with the
+ + + + +
DHT binding to the AR, hence, its pronounced activity
in the transactivation assay. C. comatus, on the other
hand, was studied to a lesser extent in comparison to
G. lucidum. However, in recent studies it was found
that members of the Coprinaceae family contain illudin
sesquiterpenes [10, 26]. Illudins and illudin-like com-
pounds obtained from Omphalotus illudens and
Lampteromyces japonicus have been extensively stud-
ied for their cytotoxic and anti-tumor properties [44].
Coprinus comatus and G. lucidum extracts also
exhibited anti-GR activities (Fig. 3b). It is possible that
one chemical entity is responsible for these effects as
exemplified with some non-specific steroid hormone
antagonists. For example, it has been shown that
mifepristone is able to antagonize the activity of GR,
PR, and AR [3]. The anti-androgenic activity of this
compound was attributed to the induction of a non-
functional conformation of the LBD rendering an
inactive AR [17]. Other examples include HSP90
inhibitors like geldanamycine inhibiting the GR, PR,
ER, and AR [36], and cisplatin inhibiting the GR and
AR [27, 36]. Alternatively, it is possible that the ex-
tracts contain several structurally related entities, each
of which affects different steroid hormone receptor.
The structure and the nature of the active moieties
found in the two fungal extracts remains to be eluci-
dated.
We speculated that fungal substances capable of
interfering with AR activity, as demonstrated in
Fig. 3a, might also influence the viability of LNCaP
cells with minimal effect on the androgen-independent
cell lines. Data shown in Table 1 illustrate that the two
extracts decreased LNCaP cell viability with minimal
effect against androgen-independent cell lines. AR
Mol Biol Rep (2008) 35:107–117
regulates many cellular processes in prostate cells
including growth and survival of androgen-dependent
cell lines, such as LNCaP. Yang et al. [43], elegantly
demonstrated, using small interfering RNA to suppress
the AR, that LNCaP cells require the AR to support
cell proliferation and viability. Thus, active substances
from C. comatus and G. lucidum that interfere with the
AR function are expected to be more effective in
inhibiting proliferation and viability of androgen-
dependent and not androgen-independent cell lines.
This suggestion is supported by the observation that
both extracts decreased AR protein levels in AR-
expressing MDA-kb2 cells (Fig. 6b), with minimal ef-
fect on viability (Table 1). This result is not surprising
since MDA-kb2 cells, unlike LNCaP cells, do not de-
pend on AR for growth and proliferation.
Although C. comatus and G. lucidum extracts were
highly potent in inhibition proliferation of AR-depen-
dent cell line, such as LNCaP, they also exhibited
varying cytotoxicity against other cell lines tested. It is
plausible to speculate that the two activities are caused
by different moieties. The anti-proliferative effect ob-
served against LNCaP cells is probably caused by
fungal substances that interfere with the AR function,
while different fungal moiety mediate the cytotoxic
activity observed against other cell lines. Thus, AR-
independent mechanisms, like cytotoxicity, may play a
role in the inhibitory effects of both fungi, especially G.
lucidum. It is well documented that G. lucidum con-
tains some highly cytotoxic compounds. For example,
it was found that lucidenic acids N and A are some of
the most cytotoxic constituents of G. lucidum [41]. The
researchers used the Kenacid Blue cytotoxicity assay to
establish IC50 values of 0.21 and 0.16 nM, respectively,
toward human hepatoma Hep G2 cells [41]. This may
point toward the possibility of a cell type specific
cytotoxicity, especially from G. lucidum, as demon-
strated by Wu et al. [41]. For example, the authors
show that cytotoxicity toward hepatoma Hep G2 was,
on the average, higher by five orders of magnitude in
comparison to human mouth epidermal carcinoma KB
cells, and CCM2 cells, and by two orders of magnitude
in comparison to murine lymphocytic leukemia P388
cells [41].
Recently, Fujita et al. [7] demonstrated anti-andro-
genic activity of G. lucidum extracts on the rat ventral
prostate model via inhibition of the enzyme 5-a
reductase, the enzyme that converts testosterone to its
biologically active form, DHT. This report is of little
relevance to our results since we are using exogenous
DHT as an AR inducer and thus bypassing the need
for 5-a reductase activity in the tested cell lines. Thus,
fungal substances reported here are likely to interfere
115
with AR function and not with testosterone conver-
sion. In addition, these extracts appear to be devoid of
the dual agonist/antagonist property of some AR
antagonists, like flutamide.
Coprinus comatus and G. lucidum extracts also
down-regulate the AR levels in LNCaP and MDA-kb2
cell lines (Fig. 6). The observed decrease in protein
levels could result from protein degradation. In
androgen-dependent cells like LNCaP cells, hormone
responsiveness is under tight control. One of the
mechanisms that control this responsiveness is modu-
lation of AR protein half-life. Androgens have been
reported to stabilize the AR protein. For example, the
half-life of the AR protein in LNCaP cells is approxi-
mately 3 h in the absence of androgens and is longer
than 10 h in the presence of 10 nM DHT [11]. Thus, it
is possible that by decreasing DHT binding to the AR,
fungal extracts increased the rate of AR degradation
by the proteasome-ubiquitin pathway.
DNA content (as an indicator of cell proliferation)
analysis showed that C. comatus and G. lucidum ex-
tracts blocked LNCaP cell cycle progression at the
transition from G1 to S phase. These results are in
accordance with several recent reports showing a G1
phase arrest [13, 14, 42, 45] or a G2 phase arrest [15, 19,
21] following treatment with G. lucidum. It has been
shown that androgen-dependent, but not androgen-
independent, cell lines require AR activity for pro-
gression from the G1 into the S phase [4]. Thus, we
suggest that the selective G1 arrest caused by
C. comatus and G. lucidum extracts in LNCaP cells is
due to interference with the AR function.
Like C. comatus and G. lucidum extracts, flutamide
also caused a G1 phase arrest in LNCaP cells with no
effect on DU 145 and PC-3 cells. Flutamide at 5 lM
decreased DHT-induced AR-mediated reporter activ-
ity by 52% in MDA-kb2 cells (data not shown).
Moreover, flutamide at 1 lM decreased the level of
secreted PSA by 53% in LNCaP cells (data not
shown). These results are consistent with published
data on the activities of flutamide and other structur-
ally related pharmacophores, such as bicalutamide and
nilutamide. Flutamide is a competitive antagonist of
the wild-type AR and the AR mutant T877A [6, 38, 40].
For example, hydroxyflutamide (a potent derivative of
flutamide) inhibited reporter activity in DHT-induced
MDA-kb2 cells in a dose-dependent manner [38], and
in transiently transfected CV-1 cells with the pCMV-
hAR expression vector [40]. In a competitive binding
assay it was found that the antagonist had higher
affinity to the T877A mutant AR in comparison to the
wild-type AR [40]. Hydroxyflutamide has been shown
to both increase and decrease PSA production in
123
116
DHT-induced LNCaP cells [22, 37]. This phenomenon
can be explained by the partial agonist/antagonist
nature of the compound. These effects have been ob-
served in both the wild-type and the T877A mutant of
AR [38, 40].
Coprinus comatus and G. lucidum extracts that
interfere with AR function were found to inhibit
secretion of AR-regulated PSA. The effect of both
extracts was a concentration-dependent phenomenon.
The down-regulation of secreted PSA by C. comatus
and G. lucidum extracts is very important because of
the association of PSA with PCa. First, PSA is the most
commonly used biochemical marker for detection and
monitoring of PCa, and decreases in PSA levels are
associated with better PCa prognosis [30]. Second, it
has been suggested that PSA regulate PCa growth and
metastasis by modulating expression of genes involved
in prostate tumor growth [1]. Thus, down-regulation of
PSA expression may be important in the anti-prolif-
erative effects of C. comatus and G. lucidum extracts in
LNCaP cells.
Finally, the two extracts exhibited anti-androgenic
activity in both LNCaP and MDA-MB-453 cell lines.
MDA-MB-453 cell line contains wild-type AR, while
LNCaP cell line contains a mutated AR (T877A
mutation). The fact that the selected extracts were
active against both the wild-type AR and the T877A
AR mutation suggest that they have the potential to
serve as an effective therapy for primary and refractory
PCa. It is of great interest for us to isolate and eluci-
date the chemical structure of active moieties from
both extracts. In addition, we are interested in
exploiting the natural diversity of the active moieties in
quantity and composition found in different species of
the same genus and within strains of the selected spe-
cies.
Altogether our results illustrate the ability of natural
product-derived substances to modulate molecular
targets implicated in carcinogenesis and thus to
potentially be used in cancer therapeutics.
Acknowledgments We thank R. Permut (Israel) for the English
editing. This research was supported by the Ministry of Science
and Technology of Israel, grant no. 3-0561.
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