# Molecular and Biochemical Effects of a Kola Nut Extract on Androgen Receptor-Mediated Pathways

Authors: Rajasree Solipuram; Sowmya Koppula; Angela Hurst; Kinesha Harris; Srivatcha Naragoni; Krystal  Fontenot; Wesley Gray
Journal: Journal of Toxicology
 (2009)

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## Abstract

The low incidence of prostate cancer in Asians has been attributed to chemopreventative properties of certain chemicals found in their diet. This study characterized the androgenic and chemopreventative properties of the Jamaican bush tea “Bizzy,” using androgen receptor positive and negative cell lines. Exposure of prostate cells to Biz-2 resulted in a growth inhibition (GI50) of 15 ppm in LNCaP cells and 3.6 ppm in DU145 cells. Biz-2 elicited a 2-fold increase in the mRNA of the anti-apoptotic gene Bcl2, with a 10-fold increase in that of the proapoptotic gene Bax. We observed a 2.4- to 7.5-fold change in apoptotic cells in both cell lines. Biz-2 at 10 ppm elicited a time- and dose-dependent stimulation of both the protein and mRNA levels of several androgen-regulated genes. Biz-2 caused a 36% decrease in PSA secretion and a significant increase in PSA mRNA. The relative binding affinity (IC50) of Biz-2 for AR was 2- to 5-fold lower than that of the synthetic androgen R1881.  Biz-2 was found to be a specific ligand for the AR in that the natural ligand, DHT, and the anti-androgen, flutamide, displaced Biz-2 bound to AR and inhibited Biz-2-induced transcription and PSA secretion. This study provided evidence that Biz-2 extract possesses the ability to modulate prostate cancer cell biology in an AR-dependent manner.


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## Body

## 1. Introduction

Nonsteroidal, plant-derived compounds  that  disrupt or  mimic the
normal action of estradiol are referred to as phytoestrogens. Those that
disrupt or mimic the action of testosterone or dihydrotestosterone are referred
to as phytoandrogens. These chemicals influence endocrine activity in animals
and have been shown to play a role in the prevention of certain
hormone-dependent cancers, such as breast and prostate cancers. The American Cancer Society estimated that
218,890 men in USA
would be diagnosed with prostate cancer during 2007. Furthermore, they estimated that prostate
cancer would kill 33,370 men in USA in 2007, making prostate cancer the fourth leading
cause of cancer deaths in American men after lung cancer [1]. At the same time, African Americans have the
highest incidence of prostate cancer and the highest mortality rate due to
prostate cancer of any population studied. In Asian men from China, Japan, and
Thailand, the incidence is much lower [2–4]. The low incidence
of prostate disease in Eastern populations is attributed to dietary
phytochemicals such as isoflavones, flavones, and lignans. Epidemiological
studies suggest that the reduced risk of prostate disease in Asian populations
is associated with consumption of foods rich in soy and vegetable products [5–7].There is growing interest in using natural, dietary plant estrogens
(phytoestrogens), particularly those found in soy products, as a potential
chemopreventive regimen for prostate cancer. Several clinical trials and biochemical studies have been conducted to
ascertain whether diets containing high levels of phytoestrogens may provide a
chemopreventive benefit for prostate cancer patients [3, 8]. Studies analyzing urinary excretion and
prostatic fluid for the presence of phytoestrogens demonstrated that
phytoestrogen values were higher in populations exhibiting a lower prostate
cancer incidence [9, 10]. A phytoestrogen-formulated diet reduces the number of spontaneous
prostate-seminal vesicle tumors in rats predisposed to prostate tumors [11, 12].
In humans, prostate cancer patients who consumed high levels of phytoestrogens
had lower levels of prostate-specific antigen, a common marker for prostate
cancer [6, 13].
In addition, organ culture studies showed that the phytoestrogen genistein
decreased growth of prostate cancer tissue and benign prostatic hypertrophy [14–16]. Taken together,
these  studies suggest that some constituent of phytoestrogen-containing diets
possesses antiandrogenic and antitumorogenic activities, which could provide some beneficial effects
in prostate cancer patients [17].Biologically active, nonsteroidal, androgenic, and estrogenic
agents are found primarily in soy products, legumes, and whole grains. Other
nontraditional sources include medicinal teas and roots. In some cultures,
natural teas are consumed in large portions each day.Kola acuminate, also known as Obi or Bizzy nut
to the Ettu people of Jamaica,
is a “cure-all” herbal medicine. It reportedly affects many biological
processes, many of which are directly, or indirectly, modulated by
hormones. Available ethnobotanical
information suggests that K. acuminate may contain bioactive chemicals
that possess estrogenic and androgenic properties [18, 19]. Anecdotal reports suggest that Bizzy nut may
be useful for a number of medical purposes, such as removal of poisons from the
body, birth control, control of diabetes, weight loss, and relief of menstrual
cramps [20, 21]. Given the hormonal dependency of some of
these biological activities, it is possible that nonsteroidal androgen present
in Bizzy nut extracts be responsible for the medicinal value attributed to
it. Recently, we identified several
hormonally active extracts of Bizzy nut that were capable of inhibiting the
growth of different cancer cell lines [22].In our laboratory, we are interested in
natural products such as Bizzy nut, which contain phytoestrogens,
phytoandrogens, or compounds that are antiandrogenic in nature. This article
describes the putative androgenic effects of a Bizzy nut extract on pathways
mediated by an androgen receptor (AR) in LNCaP cells. Herein, we describe the
ability of a Bizzy nut extract to induce apoptosis in an AR-positive cell line
and to modulate AR-dependent gene expression.


## 2. Materials and Methods

### 2.1. Cell Culture

LNCaP and DU145 cells were obtained from ATCC (Rockville, Md, USA). Cells were maintained in RPMI
1640 (LNCaP) or Kaighn’s modification of Ham’s F-12 (F12-K) medium supplemented
with 10% FBS, 0.2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL
streptomycin. Cells were kept in 5% CO2 in a water-jacketed incubator, and were passaged using a trypsin/EDTA solution
(Sigma-Aldrich, Inc., St. Louis, Mo, USA) when they reached
about 80–90% confluency.


### 2.2. Pharmacological Treatments

LNCaP or DU145 cells (1 × 105) were grown in
poly-d-lysine-coated, 6-well, or 20 cm plates in Cellgro serum-free  medium
(Mediatech, Inc., Herndon, Va, USA) for 24 hours, then induced with varying
amounts of Biz-2 (ether extract) or dihydrotestosterone (DHT).
For time-course studies of induction, cells were induced for 6, 12, and 24 hours
with the appropriate compound after which the medium was collected and
concentrated using a CentriVap concentrator (Labconco,
Kansas City, Mo, USA). The corresponding attached
cells were extracted using RIPA buffer (Pierce Biotechnology, Rockford, Ill, USA), and stored at −80°C until used. The
ether extract of Bizzy nut (Biz-2) was prepared as previously described [22].


### 2.3. Cell Viability Analysis

For experiments involving cell growth and gene induction, LNCaP
cells were grown for five days in RPMI 1640 medium containing 5% FBS that was
stripped three times with dextran-coated charcoal. Cells were then grown for 24
hours in Cellgro serum-free medium. Cells were plated in 96-well plates (1 × 105 cells/well) and allowed to attach overnight. The Biz-2 extract in 0.1% DMSO was added in a series of concentrations
(0–1000 ppm) to a
96-well plate. As a control and a reference, 10-7 M genistein (GE)
or 10-8 M DHT was added to separate wells of each plate. Each
treatment and time point had eight replicates. In each treatment, the final
concentration of vehicle solvent did not exceed 0.01% v/v in the medium. After 24-hour exposure to the test compounds,
the effect on cell viability and gene expression was determined. Cytotoxicity was determined by the CellTiter
96 AQueous One Solution cell proliferation assay (Promega
Corporation, Madison, Wis, USA)
according to the manufacturer’s instructions. After incubation with
3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt (MTS), absorbance at 490 nm was measured using an ELX800UV
universal microplate reader (Bio-Tek, Inc., Winooski, Vt, USA). The cell viability was calculated as [(A490(control)−A490(treatment))/A490(control)]×100. For tryphan blue staining, cells were plated
in 12-well plates (10 000 cells/well) and induced with 100 ppm Biz-2 for 6, 12,
or 18 hours. After induction, cells were harvested and stained with tryphan
blue, and the number of cells was determined using a hemocytometer.


### 2.4. RNA Extraction and Real-Time RT-PCR anAlysis

Total RNA was obtained from cells treated with 0–1000 ppm Biz-2
extract, 10 nM DHT, or 100 nM GE in the presence or absence of 50-fold
flutamide for 24 hours by lysing with 1 mL of TRI reagent (Invitrogen, Carlsbad, Calif., USA). The RNA pellet was resuspended in water,
treated with RNase-free DNase I, reprecipitated, and quantified by reading the
absorbance at 260 and 280 nm on an ND-1000 spectrophotometer (NanoDrop
Tech., Wilmington, Del, USA).For cDNA synthesis, 5 ug of DNase-I-treated
total RNA were reverse-transcribed in a 100 uL reaction mixture using the ABI
High Capacity cDNA Archive Kit according to the manufacturer’s instruction
(Applied Biosystems, Foster City,
Calif, USA). The reaction contained 1X RT buffer, 100 uM
of each deoxynucleoside triphosphate (dNTP), 1X random primer, 5 U of RNase
inhibitor, and 250 U of reverse transcriptase II. The reaction was carried out
at 25°C for 10 minutes then at 37°C for 2 hours. Control reactions were those in which the RT
enzyme or the target RNA was omitted from the reaction.Taqman PCR was performed on the cDNA samples
using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). For each gene tested (see Table1), the PCR
was carried out in a multiplex mode with every 25 uL reaction containing 2 uL
of cDNA reaction, 1X Taqman universal PCR master mix, 250 nM of a gene-specific
primer, 250 nM of an endogenous control primer, 250 nM of FAM-labeled
fluorogenic Taqman probe, a VIC-labeled 18S rRNA fluorogenic control Taqman
probe, and 2.5 U of TaqMan enzyme. The
PCR amplification was conducted using a Taqman Universal PCR Master Mix
(Applied Biosystems) at universal thermal cycling conditions: 50°C for 2 minutes,
95°C for 10 minutes, 40 cycles at −95°C for 15 seconds, and 60°C for 1 minute.
An increase in fluorescence was obtained at the annealing and extension step at
60°C.Table 1
Regulation of androgen receptor target genes
by Biz-2.
GenesmRNA expression relative
to controlGene descriptionBiz-2DHTRES10 ppm–24 h10 nM–24 h10 nM–24 hFoldSEMNFoldSEMNFoldSEMNPSA10.552.2638.090.9133.660.183Prostate-specific antigen: prostate tumor
markerNKX3-13.9320.74313.842.2132.510.243NK3 homeobox: a novel human
prostate-specific, androgen-regulated homeobox gene associated with prostate
cancer progressionTSC225.951.4431.380.1632.450.253Tuberous sclerosis 22: a leucine zipper transcription factor
whose expression is induced by TGF-bPART-110.30.350.02———Prostate androgen-regulated transcript 1: androgen-inducible
gene that is regulated by androgens and phytoestrogenERβ1.170.431.080.3231.270.213Estrogen receptorβ: nuclear transcription factor that mediates transcription of nonsteroidal
and steroidal ligandsERα1.360.28131.360.28131.480.353Estrogen receptorα: ligand-inducible nuclear transcription factor that mediates
estrogen-dependent gene expression in femalesAR2.840.2031.2710.1130.440.013Androgen receptor: transcription factor that mediates
transcription of genes required for development of male reproductive tissuesBcl22.682.6830.850.0731.140.123B cell leukemia/lymphoma-2: an antiapoptotic protein known to
regulate apoptotic pathway and protect against cell deathBax9.972.1337.640.0933.450.173Bcl2-associated X-protein: a proapoptotic protein that regulates
apoptotic pathway and promotes cell deathThe relative level of expression of each gene
in the samples was determined using the relative2ΔΔCt expression  method as described in detail in the ABI PRISM Sequence Detection System
User Bulletin 2 [23]. After the linear
range of amplification (threshold cycle, Ct) was determined for the
genes of interest, it was normalized against an endogenous 18S rRNA control and
then against the untreated control sample, which served as the calibrator. The
value of the relative level of expression for the gene of interest represents
two independent inductions performed in triplicate.


### 2.5. Ligand Binding Analysis

The binding affinity of Biz-2 to the Androgen receptor was
evaluated on whole-cell, LNCaP extract or on 10 nM of the baculovirus-expressed
ligand binding domain of the AR (PanVara, Madison, Wis, USA) [24, 25]. Samples (100 uL) containing
250 ug of total protein in 1X binding buffer (10 mM HEPES, pH 7.9, 10%
glycerol, 0.02% Ficoll 400, 100 mM  NaCl, 0.1 mM EDTA, 2.5 mM for each of MgCl2,
CaCl2, and DTT, and 1 g/mL for each of leupeptin, pepstatin,
antipain, aprotinin, soybean trypsin inhibitor, and chymostatin) were incubated
with 2 nM [3H]-R1881 in the presence or absence of a 10-fold serial
dilution of 10 000 ppm Biz-2, for 18 hours at 4°C. Free and bound radioligands
were separated using the hydroxyapatite assay. Bound radioligand was measured
by scintillation counting as previously described [26]. Specific binding was determined by
subtraction of nonspecific binding from total binding. The IC50 (the
concentration of competitor that results in 50% of maximal binding) and Ki (the competitor binding constant assuming 1 nM Kd for radiolabeled
ligand) were determined by fitting the data to the Cheng-Prusoff equation [27] using the Prism
III software program (GraphPad, San Diego, Calif, USA).


### 2.6. Fluorescence Microscopy

For microscopy, 5 × 105 cells (LNCaP or DU145) were
grown on microscope slides and induced with 0, 100, and 500 ppm of Biz-2 for 24
hours. On each slide, cells were stained for 5 minutes with 5 μL of a 0.1 ug/uL
solution of acridine orange and ethidium bromide. Two fluorescence parameters, green emission
from acridine orange (525 nm) and red emission from ethidium bromide (620 nm),
were examined under a fluorescence light microscope (Nikon Optiphot, Melville, NY, USA) for the nuclear changes
that are typically associated with apoptosis. An index of apoptosis was
calculated as the ratio of the number of cells per microscopic field with early
and late apoptosis characteristics in treated samples relative to the total
number of cells per microscopic field.


### 2.7. Analysis of Caspase 3/7 Activation

Caspase 3/7 activity was determined using an
Apo-One Homogeneous Caspase-3/7 Assay kit (Promega Corporation) according to
the manufacturer’s instructions. Briefly, cells were grown in test medium to a density of 1 × 106 cells/mL. The cells were then treated with Biz-2, antigen, or a combination of
the two, as indicated, for 12 or 24 hours. Following induction, cells were harvested by incubation in 5 mL of
harvesting solution at 37°C
for 5–10 minutes. The cells were collected by
centrifugation. The pellets were
resuspended in 100 uL of hypotonic cell lysis buffer (25 mM HEPES, pH 7.5, 5 mM
MgCl2, 5 mM EDTA, 5 mM DTT, and 2 mM PMSF). The samples were subjected to two rounds of
freezing and thawing in liquid N2, then centrifuged at 14000 × g for 15 minutes. Caspase 3/7-like activity was determined
based on proteolytic cleavage of rhodamine 110,
bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide, Z-DEVD-R110).
The free rhodamine 110 was quantified on a SpectraMax Gemini Fluorescence
Spectrophotometer with excitation at 499 nm and emission at 521 nm using the
SoftMax Pro program in static mode. The
Caspase 3/7-like activity in each sample was determined from the
maximum fluorescence after 3 hours. Recombinant Caspase 3 served as a positive control for the assay.


### 2.8. Western Blot Analysis

Immunoblot of the LNCaP protein fraction was performed as
previously described [28, 29]. Briefly, LNCaP cells grown
under the indicated condition were lysed at 4°C
in RIPA buffer supplemented with 1X sigma protease inhibitor cocktail
(Sigma-Aldrich, Inc.). A BCA protein
assay kit (Pierce Biotechnology) was used to determine protein
concentration. An aliquot of the total
protein sample (30 ug) was size-fractionated using a 10% or a 4–20% precast
(SDS-PAGE) gel (Pierce Biotechnology), then the fractionated proteins were
transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilien,
Millipore, Bedford, Mass, USA). The membrane was blocked with 5% nonfat dry
milk in TBST buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.1% Tween-20) for 1
hour at room temperature, and immunostained with the following antibodies: 0.5 ug/mL anti-Bcl2 and 0.5 ug/mL anti-Bax (Santa Cruz Biotechnology Inc., Santa
Cruz, Calif, USA), 1.5 ug/mL anti-AR and 0.5 ug/mL anti-GDPH (Cell Signaling,
Danvers, Mass, USA). The blots were
washed three times with TBST buffer, and immunoreactive proteins were detected
using a 1:20,000 dilution of antirabbit IgG horseradish peroxidase (Amersham, Arlington Heights, Ill,
USA) at room
temperature for 1 hour. The bands were
visualized using an enhanced chemiluminescence detection system (Pierce
Biotechnology) according to the instructions of the manufacturer. The immunoblot signal was captured using an
AlphaInnotech Fluorochem HD 9900 (AlphaInnotech, San Leandro, Calif, USA) equipped with a CDD camera.
The images were analyzed with the AlphaEaseFC software (AlphaInnotech), and
curves and graphs were fitted with GraphPad Prism 3.0 software (GraphPad) [30].


### 2.9. Analysis of PSA Secretion by Immunoassay

The total amount of prostate-specific antigen (PSA) secreted by
LNCaP cells was quantified under different experimental conditions using a
two-site sandwich, colorimetric base ELISA kit according to the manufacturer’s
instructions (CBI PSA ELISA kit, Calbiotech, Inc.,  Spring Valley, CA, USA). Briefly, 1 × 107 cells were treated with Biz-2,
phytoestrogen, or DHT as indicated. The conditioned medium was removed and
centrifuged at 12000 rpm for 5 minutes to remove any detached cells. An aliquot of the medium (1 or 10 uL) was
added to each well of a 96-well plate, precoated with anti-PSA antibody. The unbound PSA in the sample was allowed to
bind. The unbound PSA was then removed
by washing. The horseradish peroxidase-labeled PSA enzyme conjugate was added
for 30 minutes followed by the addition of the colorimetric terminating message
buffer substrate, and the color change was read at an absorbance of 570 nm
using an ELX800UV universal microplate reader (Bio-Tek, Inc.). To quantify the relationship between
absorbance and the concentration of PSA, a standard curve (0–50 ng/mL) was
prepared from PSA solutions of known concentration. The results were expressed
as nanograms of PSA per mL of conditioned medium, and were adjusted to
nanograms per mg of secreted protein.


### 2.10. Determination of
Cell Proliferation Using Thymidine Incorporation

Thymidine incorporation was measured as previously described [28]. Briefly, cells
were plated at a density of 1 × 104 cells/well in 96-well plates,
and incubated overnight. Biz-2 in 0.1%
dimethyl sulfoxide was added at a series of concentrations (0–1000 ppm) to
eight wells each of a 96-well plate, and incubated for 24 hours. After adding 0.5 uCi/well of [methyl-3H]Thymidine,
plates were incubated for 6 hours. The medium was removed and cells were
harvested onto a Whatman 934-AH glass microfiber filter (Fisher Scientific, Pittsburgh, Pa, USA) using
a 96-well filtration manifold. To lyse
the cells, filters were washed with 0.2 mM NaOH. Filters were then washed with
70% ethanol and dried with 95% ethanol. The filter paper was cut into 96 distinct pieces, placed in
scintillation vials containing 3 mL of liquid scintillation fluid (MP Biomedicals, Solon, Ohio, USA), and
counted in a TriCarb 2200CA scintillation counter (Packard, Medien, Conn, USA).


### 2.11. Solid-Liquid Extraction and TLC Analysis

A 10 g sample of finely ground Bizzy root was sequentially
extracted in a Soxhlet apparatus using 500 mL of 100% hexane, ether, acetone,
methane, or water, solvents with increasing polarity. The extraction mixture was allowed to reflux
for 24 hours at temperatures corresponding to the boiling point of the
respective solvent and the extraction monitored by TLC chromatography.
Following extraction, particulate matter was removed by filtering the samples
through a 0.45μm glass-fritted
filter, and the extract was
evaporated to dryness using a combination of simple distillation and rotary
evaporation. The residue of each extract was resuspended in DMSO to a
concentration of 10000 ppm. Aliquots (100 ppm) of each extract were run on
thin-layer chromatography plates (Silica Gel Hl, Analtech, Newark, DE, USA; ca. 10 cm wide × 20 cm high) using a
solvent of 40% v/v Ether-60% v/v Hexane. The analytes were visualized under
ultraviolet light, and the image was captured digitally using a FluorChem HD
Alpha Innotech System.


### 2.12. Statistical Analysis

All numerical data were expressed as mean ±SEM. In each assay,
three or four measurements were made. Means for the treatment groups were compared using analysis of variance
and Duncan’s
multiple range test (P<.05). To analyze the absorbance density from western blot data, a two-tailed t-test
(P<.05) was used to compare the mean (n = 3) for each treatment group
with the mean for the untreated control group. The GraphPad Prism 3.0 software program (GraphPad) was used for the
statistical analyses [30].


## 2.1. Cell Culture

LNCaP and DU145 cells were obtained from ATCC (Rockville, Md, USA). Cells were maintained in RPMI
1640 (LNCaP) or Kaighn’s modification of Ham’s F-12 (F12-K) medium supplemented
with 10% FBS, 0.2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL
streptomycin. Cells were kept in 5% CO2 in a water-jacketed incubator, and were passaged using a trypsin/EDTA solution
(Sigma-Aldrich, Inc., St. Louis, Mo, USA) when they reached
about 80–90% confluency.


## 2.2. Pharmacological Treatments

LNCaP or DU145 cells (1 × 105) were grown in
poly-d-lysine-coated, 6-well, or 20 cm plates in Cellgro serum-free  medium
(Mediatech, Inc., Herndon, Va, USA) for 24 hours, then induced with varying
amounts of Biz-2 (ether extract) or dihydrotestosterone (DHT).
For time-course studies of induction, cells were induced for 6, 12, and 24 hours
with the appropriate compound after which the medium was collected and
concentrated using a CentriVap concentrator (Labconco,
Kansas City, Mo, USA). The corresponding attached
cells were extracted using RIPA buffer (Pierce Biotechnology, Rockford, Ill, USA), and stored at −80°C until used. The
ether extract of Bizzy nut (Biz-2) was prepared as previously described [22].


## 2.3. Cell Viability Analysis

For experiments involving cell growth and gene induction, LNCaP
cells were grown for five days in RPMI 1640 medium containing 5% FBS that was
stripped three times with dextran-coated charcoal. Cells were then grown for 24
hours in Cellgro serum-free medium. Cells were plated in 96-well plates (1 × 105 cells/well) and allowed to attach overnight. The Biz-2 extract in 0.1% DMSO was added in a series of concentrations
(0–1000 ppm) to a
96-well plate. As a control and a reference, 10-7 M genistein (GE)
or 10-8 M DHT was added to separate wells of each plate. Each
treatment and time point had eight replicates. In each treatment, the final
concentration of vehicle solvent did not exceed 0.01% v/v in the medium. After 24-hour exposure to the test compounds,
the effect on cell viability and gene expression was determined. Cytotoxicity was determined by the CellTiter
96 AQueous One Solution cell proliferation assay (Promega
Corporation, Madison, Wis, USA)
according to the manufacturer’s instructions. After incubation with
3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt (MTS), absorbance at 490 nm was measured using an ELX800UV
universal microplate reader (Bio-Tek, Inc., Winooski, Vt, USA). The cell viability was calculated as [(A490(control)−A490(treatment))/A490(control)]×100. For tryphan blue staining, cells were plated
in 12-well plates (10 000 cells/well) and induced with 100 ppm Biz-2 for 6, 12,
or 18 hours. After induction, cells were harvested and stained with tryphan
blue, and the number of cells was determined using a hemocytometer.


## 2.4. RNA Extraction and Real-Time RT-PCR anAlysis

Total RNA was obtained from cells treated with 0–1000 ppm Biz-2
extract, 10 nM DHT, or 100 nM GE in the presence or absence of 50-fold
flutamide for 24 hours by lysing with 1 mL of TRI reagent (Invitrogen, Carlsbad, Calif., USA). The RNA pellet was resuspended in water,
treated with RNase-free DNase I, reprecipitated, and quantified by reading the
absorbance at 260 and 280 nm on an ND-1000 spectrophotometer (NanoDrop
Tech., Wilmington, Del, USA).For cDNA synthesis, 5 ug of DNase-I-treated
total RNA were reverse-transcribed in a 100 uL reaction mixture using the ABI
High Capacity cDNA Archive Kit according to the manufacturer’s instruction
(Applied Biosystems, Foster City,
Calif, USA). The reaction contained 1X RT buffer, 100 uM
of each deoxynucleoside triphosphate (dNTP), 1X random primer, 5 U of RNase
inhibitor, and 250 U of reverse transcriptase II. The reaction was carried out
at 25°C for 10 minutes then at 37°C for 2 hours. Control reactions were those in which the RT
enzyme or the target RNA was omitted from the reaction.Taqman PCR was performed on the cDNA samples
using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). For each gene tested (see Table1), the PCR
was carried out in a multiplex mode with every 25 uL reaction containing 2 uL
of cDNA reaction, 1X Taqman universal PCR master mix, 250 nM of a gene-specific
primer, 250 nM of an endogenous control primer, 250 nM of FAM-labeled
fluorogenic Taqman probe, a VIC-labeled 18S rRNA fluorogenic control Taqman
probe, and 2.5 U of TaqMan enzyme. The
PCR amplification was conducted using a Taqman Universal PCR Master Mix
(Applied Biosystems) at universal thermal cycling conditions: 50°C for 2 minutes,
95°C for 10 minutes, 40 cycles at −95°C for 15 seconds, and 60°C for 1 minute.
An increase in fluorescence was obtained at the annealing and extension step at
60°C.Table 1
Regulation of androgen receptor target genes
by Biz-2.
GenesmRNA expression relative
to controlGene descriptionBiz-2DHTRES10 ppm–24 h10 nM–24 h10 nM–24 hFoldSEMNFoldSEMNFoldSEMNPSA10.552.2638.090.9133.660.183Prostate-specific antigen: prostate tumor
markerNKX3-13.9320.74313.842.2132.510.243NK3 homeobox: a novel human
prostate-specific, androgen-regulated homeobox gene associated with prostate
cancer progressionTSC225.951.4431.380.1632.450.253Tuberous sclerosis 22: a leucine zipper transcription factor
whose expression is induced by TGF-bPART-110.30.350.02———Prostate androgen-regulated transcript 1: androgen-inducible
gene that is regulated by androgens and phytoestrogenERβ1.170.431.080.3231.270.213Estrogen receptorβ: nuclear transcription factor that mediates transcription of nonsteroidal
and steroidal ligandsERα1.360.28131.360.28131.480.353Estrogen receptorα: ligand-inducible nuclear transcription factor that mediates
estrogen-dependent gene expression in femalesAR2.840.2031.2710.1130.440.013Androgen receptor: transcription factor that mediates
transcription of genes required for development of male reproductive tissuesBcl22.682.6830.850.0731.140.123B cell leukemia/lymphoma-2: an antiapoptotic protein known to
regulate apoptotic pathway and protect against cell deathBax9.972.1337.640.0933.450.173Bcl2-associated X-protein: a proapoptotic protein that regulates
apoptotic pathway and promotes cell deathThe relative level of expression of each gene
in the samples was determined using the relative2ΔΔCt expression  method as described in detail in the ABI PRISM Sequence Detection System
User Bulletin 2 [23]. After the linear
range of amplification (threshold cycle, Ct) was determined for the
genes of interest, it was normalized against an endogenous 18S rRNA control and
then against the untreated control sample, which served as the calibrator. The
value of the relative level of expression for the gene of interest represents
two independent inductions performed in triplicate.


## 2.5. Ligand Binding Analysis

The binding affinity of Biz-2 to the Androgen receptor was
evaluated on whole-cell, LNCaP extract or on 10 nM of the baculovirus-expressed
ligand binding domain of the AR (PanVara, Madison, Wis, USA) [24, 25]. Samples (100 uL) containing
250 ug of total protein in 1X binding buffer (10 mM HEPES, pH 7.9, 10%
glycerol, 0.02% Ficoll 400, 100 mM  NaCl, 0.1 mM EDTA, 2.5 mM for each of MgCl2,
CaCl2, and DTT, and 1 g/mL for each of leupeptin, pepstatin,
antipain, aprotinin, soybean trypsin inhibitor, and chymostatin) were incubated
with 2 nM [3H]-R1881 in the presence or absence of a 10-fold serial
dilution of 10 000 ppm Biz-2, for 18 hours at 4°C. Free and bound radioligands
were separated using the hydroxyapatite assay. Bound radioligand was measured
by scintillation counting as previously described [26]. Specific binding was determined by
subtraction of nonspecific binding from total binding. The IC50 (the
concentration of competitor that results in 50% of maximal binding) and Ki (the competitor binding constant assuming 1 nM Kd for radiolabeled
ligand) were determined by fitting the data to the Cheng-Prusoff equation [27] using the Prism
III software program (GraphPad, San Diego, Calif, USA).


## 2.6. Fluorescence Microscopy

For microscopy, 5 × 105 cells (LNCaP or DU145) were
grown on microscope slides and induced with 0, 100, and 500 ppm of Biz-2 for 24
hours. On each slide, cells were stained for 5 minutes with 5 μL of a 0.1 ug/uL
solution of acridine orange and ethidium bromide. Two fluorescence parameters, green emission
from acridine orange (525 nm) and red emission from ethidium bromide (620 nm),
were examined under a fluorescence light microscope (Nikon Optiphot, Melville, NY, USA) for the nuclear changes
that are typically associated with apoptosis. An index of apoptosis was
calculated as the ratio of the number of cells per microscopic field with early
and late apoptosis characteristics in treated samples relative to the total
number of cells per microscopic field.


## 2.7. Analysis of Caspase 3/7 Activation

Caspase 3/7 activity was determined using an
Apo-One Homogeneous Caspase-3/7 Assay kit (Promega Corporation) according to
the manufacturer’s instructions. Briefly, cells were grown in test medium to a density of 1 × 106 cells/mL. The cells were then treated with Biz-2, antigen, or a combination of
the two, as indicated, for 12 or 24 hours. Following induction, cells were harvested by incubation in 5 mL of
harvesting solution at 37°C
for 5–10 minutes. The cells were collected by
centrifugation. The pellets were
resuspended in 100 uL of hypotonic cell lysis buffer (25 mM HEPES, pH 7.5, 5 mM
MgCl2, 5 mM EDTA, 5 mM DTT, and 2 mM PMSF). The samples were subjected to two rounds of
freezing and thawing in liquid N2, then centrifuged at 14000 × g for 15 minutes. Caspase 3/7-like activity was determined
based on proteolytic cleavage of rhodamine 110,
bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide, Z-DEVD-R110).
The free rhodamine 110 was quantified on a SpectraMax Gemini Fluorescence
Spectrophotometer with excitation at 499 nm and emission at 521 nm using the
SoftMax Pro program in static mode. The
Caspase 3/7-like activity in each sample was determined from the
maximum fluorescence after 3 hours. Recombinant Caspase 3 served as a positive control for the assay.


## 2.8. Western Blot Analysis

Immunoblot of the LNCaP protein fraction was performed as
previously described [28, 29]. Briefly, LNCaP cells grown
under the indicated condition were lysed at 4°C
in RIPA buffer supplemented with 1X sigma protease inhibitor cocktail
(Sigma-Aldrich, Inc.). A BCA protein
assay kit (Pierce Biotechnology) was used to determine protein
concentration. An aliquot of the total
protein sample (30 ug) was size-fractionated using a 10% or a 4–20% precast
(SDS-PAGE) gel (Pierce Biotechnology), then the fractionated proteins were
transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilien,
Millipore, Bedford, Mass, USA). The membrane was blocked with 5% nonfat dry
milk in TBST buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.1% Tween-20) for 1
hour at room temperature, and immunostained with the following antibodies: 0.5 ug/mL anti-Bcl2 and 0.5 ug/mL anti-Bax (Santa Cruz Biotechnology Inc., Santa
Cruz, Calif, USA), 1.5 ug/mL anti-AR and 0.5 ug/mL anti-GDPH (Cell Signaling,
Danvers, Mass, USA). The blots were
washed three times with TBST buffer, and immunoreactive proteins were detected
using a 1:20,000 dilution of antirabbit IgG horseradish peroxidase (Amersham, Arlington Heights, Ill,
USA) at room
temperature for 1 hour. The bands were
visualized using an enhanced chemiluminescence detection system (Pierce
Biotechnology) according to the instructions of the manufacturer. The immunoblot signal was captured using an
AlphaInnotech Fluorochem HD 9900 (AlphaInnotech, San Leandro, Calif, USA) equipped with a CDD camera.
The images were analyzed with the AlphaEaseFC software (AlphaInnotech), and
curves and graphs were fitted with GraphPad Prism 3.0 software (GraphPad) [30].


## 2.9. Analysis of PSA Secretion by Immunoassay

The total amount of prostate-specific antigen (PSA) secreted by
LNCaP cells was quantified under different experimental conditions using a
two-site sandwich, colorimetric base ELISA kit according to the manufacturer’s
instructions (CBI PSA ELISA kit, Calbiotech, Inc.,  Spring Valley, CA, USA). Briefly, 1 × 107 cells were treated with Biz-2,
phytoestrogen, or DHT as indicated. The conditioned medium was removed and
centrifuged at 12000 rpm for 5 minutes to remove any detached cells. An aliquot of the medium (1 or 10 uL) was
added to each well of a 96-well plate, precoated with anti-PSA antibody. The unbound PSA in the sample was allowed to
bind. The unbound PSA was then removed
by washing. The horseradish peroxidase-labeled PSA enzyme conjugate was added
for 30 minutes followed by the addition of the colorimetric terminating message
buffer substrate, and the color change was read at an absorbance of 570 nm
using an ELX800UV universal microplate reader (Bio-Tek, Inc.). To quantify the relationship between
absorbance and the concentration of PSA, a standard curve (0–50 ng/mL) was
prepared from PSA solutions of known concentration. The results were expressed
as nanograms of PSA per mL of conditioned medium, and were adjusted to
nanograms per mg of secreted protein.


## 2.10. Determination of
Cell Proliferation Using Thymidine Incorporation

Thymidine incorporation was measured as previously described [28]. Briefly, cells
were plated at a density of 1 × 104 cells/well in 96-well plates,
and incubated overnight. Biz-2 in 0.1%
dimethyl sulfoxide was added at a series of concentrations (0–1000 ppm) to
eight wells each of a 96-well plate, and incubated for 24 hours. After adding 0.5 uCi/well of [methyl-3H]Thymidine,
plates were incubated for 6 hours. The medium was removed and cells were
harvested onto a Whatman 934-AH glass microfiber filter (Fisher Scientific, Pittsburgh, Pa, USA) using
a 96-well filtration manifold. To lyse
the cells, filters were washed with 0.2 mM NaOH. Filters were then washed with
70% ethanol and dried with 95% ethanol. The filter paper was cut into 96 distinct pieces, placed in
scintillation vials containing 3 mL of liquid scintillation fluid (MP Biomedicals, Solon, Ohio, USA), and
counted in a TriCarb 2200CA scintillation counter (Packard, Medien, Conn, USA).


## 2.11. Solid-Liquid Extraction and TLC Analysis

A 10 g sample of finely ground Bizzy root was sequentially
extracted in a Soxhlet apparatus using 500 mL of 100% hexane, ether, acetone,
methane, or water, solvents with increasing polarity. The extraction mixture was allowed to reflux
for 24 hours at temperatures corresponding to the boiling point of the
respective solvent and the extraction monitored by TLC chromatography.
Following extraction, particulate matter was removed by filtering the samples
through a 0.45μm glass-fritted
filter, and the extract was
evaporated to dryness using a combination of simple distillation and rotary
evaporation. The residue of each extract was resuspended in DMSO to a
concentration of 10000 ppm. Aliquots (100 ppm) of each extract were run on
thin-layer chromatography plates (Silica Gel Hl, Analtech, Newark, DE, USA; ca. 10 cm wide × 20 cm high) using a
solvent of 40% v/v Ether-60% v/v Hexane. The analytes were visualized under
ultraviolet light, and the image was captured digitally using a FluorChem HD
Alpha Innotech System.


## 2.12. Statistical Analysis

All numerical data were expressed as mean ±SEM. In each assay,
three or four measurements were made. Means for the treatment groups were compared using analysis of variance
and Duncan’s
multiple range test (P<.05). To analyze the absorbance density from western blot data, a two-tailed t-test
(P<.05) was used to compare the mean (n = 3) for each treatment group
with the mean for the untreated control group. The GraphPad Prism 3.0 software program (GraphPad) was used for the
statistical analyses [30].


## 3. Results

### 3.1. Activation of an Androgen-Dependent Pathway by Biz-2

The androgenic potential of
natural compounds is manifested in a variety of biological responses including
induction of cell growth, secretion of cellular protein, and receptor-dependent
gene expression. Secretion of PSA by the
prostate is a hallmark of prostatic function. Furthermore, modulation of PSA level is used as a biomarker in assessing
prostate development and function. As an
initial step in identifying and characterizing medicinally relevant, putative,
selective androgen receptor modulators (SARMs) present in Bizzy nut, we
performed solid-liquid extraction using solvents of increasing polarity. We screened each extract for bioactivity
using several in vitro assays that were designed to reveal if any of the
extracts contained androgenic or estrogenic activity [22]. We determined whether an ether extract of Bizzy nut
(Biz-2) was capable of modulating prostate function by exerting its effect on
PSA secretion by LNCaP cells. LNCaP
cells were grown in a serum-free medium for 24 hours, and then induced  with 100 ppm of Biz-2 for 6, 12, or 24 hours. A PSA ELISA was then used to determine the
concentration of PSA in the medium. PSA
secretion by LNCaP cells in the presence of Biz-2 displayed a biphasic response
(Figure 1(a)). Relative to control,
Biz-2 induced an initial 26.59±13.3% increase in PSA after the first 6 hours of
exposure. The level of PSA decreased by
32.6±1.3% during the second six-hour period, and increased by 25.93±3.3% 12 hours
later (Figure 1(a)). In contrast, DHT produced a time-dependent increase
(9.6±6.7% and 130 ± 6% after 12 and 24 hours, resp.) in PSA production under the
same experimental conditions (Figure 1(a); data not shown).Biz-2
modulates PSA secretion by LNCaP cells. LNCaP cells (1E7) were grown in serum-free medium for 24 hours,
then induced with 100 ppm of Biz-2 for 0, 6, 12, and 24 hours. Equal aliquots
(10 uL) of the condition medium were removed and analyzed by PSA ELISA (see Section 2). (a) Time-dependent
secretion of PSA. (b) PSA secretion
induced with 10 ppm Biz-2 for 24 hours in the presence and absence of 10 nM
flutamide. The values are the mean ± SEM of three
separate experiments performed in triplicate. CON: control; DHT:
dihydrotestosterone; Biz-2: ether extract of Bizzy nut; RES: resveratrol.
(a)(b)To understand the relationship between Biz-2
modulation of PSA secretion and the androgen-dependent pathway, we determined
whether the PSA secretion was mediated by a Biz-2/androgen receptor
interaction. To answer this question,
PSA secretion was measured in the presence of the antiandrogen flutamide (FLU)
(Figure1(b)). As a positive control, we measured PSA secretion in the presence
of flutamide with or without the natural androgen, DHT. Consistent with previous studies [31, 32], flutamide blocked
the AR-dependent stimulation of PSA (1.014±0.08 ng/uL for DHT versus 0.549±0.07 ng/uL for DHT/FLU) by LNCaP cells. Treatment of LNCaP cells with 10 ppm Biz-2 plus 1 nM of flutamide
prevented the Biz-2 inhibition of PSA secretion (Figure 1(b)). At 10 ppm Biz-2, PSA levels decreased by 24.80±3.4% of control,
that is, from 0.654±0.01 ng/uL to 0.492±0.022 ng/uL. Addition of flutamide at 1 nM restored the
PSA level to that of control (0.673±0.077 ng/uL Biz-2/FLU versus 0.654±0.03 ng/uL control). In our system, there was
a significant increase (16 ± 2.8%, n = 8, P<.01) in PSA secretion in the
presence of FLU, suggesting that there may have been residual androgen present
in the medium. Alternatively, flutamide may have induced an androgenic effect
on PSA under serum-free conditions (Figure 1(b)). Nevertheless, FLU was able to prevent the
Biz-2-induced decrease in PSA secretion, which was an observation that was
complimentary to what we observed with DHT/FLU treatment. These results strongly suggested that the
Biz-2 inhibition of PSA secretion was dependent on the AR pathway.


### 3.2. Transcriptional Inhibition
of an Endogenous Androgen-Regulated Gene by Biz-2

Modulation of PSA secretion in the presence of
Biz-2 may have resulted from changes in PSA mRNA production, and could have
been independent of the secretion processes that were present in LNCaP
cells. To test this hypothesis, we
measured PSA mRNA levels in cells exposed to Biz-2 using Taqman real-time
RT-PCR. Multiplex real-time PCR was
performed using a primer/probe set specific for PSA and 18S rRNA (PE Applied
Biosystems, Foster City, Calif, USA). The expression levels of PSA were obtained
from DNase-I-treated total RNA, and were normalized to those of the 18S rRNA
gene, which served as the calibrator for our experiments. The logarithm of the fluorescence intensity
of each probe versus the number of cycles and the level of expression of mRNA
of PSA in LNCaP cells was evaluated (Figure2). In the present study, the mRNA
expression of PSA was determined using the 2ΔΔCt method,
and is expressed relative to the control sample which is set to 1. We observed a time-dependent increase in
levels of PSA mRNA between 6 and 24 hours of exposure in the presence of 10 ppm
Biz-2 (Figure 2(b)). Analysis of the
data in Figure 2(b) also indicated that, after treatment with Biz-2 or the
positive control (DHT), levels of PSA mRNA expression showed a statistically
significant increase. Exposure of LNCaP cells to 10 ppm Biz-2 resulted in a
0.87±0.17-fold increase in PSA mRNA, which was
increased by 12-fold (0.87±0.17 to 10.56±2.25) in the second
12-hour period. We determined the
potency of Biz-2 in stimulating PSA mRNA by exposing LNCaP cells to different
amounts of Biz-2 for 24 hours. The
response of PSA mRNA production to Biz-2 concentration was bell-shaped (Figure
2(c)). At concentrations below 10 ppm,
Biz-2 elicited a dose-dependent increase in PSA mRNA expression, whereas at
high concentrations (100 ppm and greater) expression of PSA was inhibited. Biz-2 at low concentrations produced a
similar increase in PSA mRNA at 12 and 24 hours as the endogenous androgen DHT
(Figure 2(b); data not shown).Stimulation of PSA
mRNA expression by Biz-2. LNCaP cells
were induced with Biz-2 or DHT, and 5μg of DNase-I-treated RNA isolated from induced or
uninduced cells were subjected to two-step Taqman real-time RT-PCR (see Section
2). The relative expression of PSA mRNA expression was calculated by the 2ΔΔCt method. First, relative quantitation of
PSA mRNA expression was performed by first normalizing the Ct values
of PSA amplification against the Ct values of endogenous 18S rRNA,
then the resulting Ct values were normalized using the Ct value of the vehicle control sample. (a) Relative amplification plot of mRNA expression (1–NTC. 2–18S rRNA at 24 hours, 3 and 4- CON at 6 and 24 hours, 5 and 6- Biz-2 at 6 and 24 hours). (b) PSA mRNA expression relative to time. (c) Dose-dependent response of PSA to Biz-2. The values are mean ± SEM
of  induction samples analyzed in triplicate.
(a)(b)(c)To substantiate the observation that Biz-2
modulated PSA activity in an AR-dependent manner, we measured Biz-2-induced PSA
mRNA  in the presence of the AR antagonist flutamide. The Biz-2-induced expression of PSA mRNA was
inhibited by 55% (3.46±0.25-fold Biz-2 to 1.55±0.14-fold
Biz-2/Flu) in the presence of 1 nM flutamide (Figure 3) suggesting that Biz-2
mediated an increase in PSA mRNA level through an interaction with the AR
present in LNCaP cells. On the other
hand, resveratrol, a phytoestrogen with bioactivity in LNCaP cells, induced a
1.5-fold increase in PSA mRNA levels, which was independent of the AR (Figure
3). Next we ascertained whether the
stimulatory effects of Biz-2 on PSA expression were a result of RNA stability
by measuring the mRNA levels of PSA in the presence and absence of actinomycin-D,
a known transcription inhibitor. Pretreatment of LNCaP cells with 5 ug/uL of actinomycin-D for  4 hours
resulted in a 26% inhibition of PSA expression relative to Biz-2. At the same time, blocking of protein
synthesis using cycloheximide resulted in an increase in the Biz-2-induced
expression of PSA (Figure 3). Because
actinomycin-D alone could not have induced PSA mRNA synthesis, our data
suggested that Biz-2 regulated PSA expression directly at the level of
transcription.Figure 3
Flutamide
inhibition of Biz-2 stimulation of PSA mRNA expression. LNCaP cells were induced with Biz-2 in the
presence or absence of flutamide, and RNA from induced or uninduced cells was
subjected to two-step Taqman real-time RT-PCR. Relative quantitation of PSA
mRNA expression was preformed as described in Figure2. CON: vehicle control; FLU: flutamide; Biz-2: Bizzy
nut extract-2; RES: resveratrol; Act.D: actinomycin-D; CHX: cycloheximide. The
values are the mean ± SEM
of three separate experiments performed in triplicate.


### 3.3. Modulation of
AR-Dependent Gene Expression by Biz-2

To further characterize the effects of Biz-2 on the AR-dependent
pathway, we examined the androgenic effects of Biz-2 on the transcription
activity of three androgen-regulated genes (PSA, PART-1, and NKX3-1) [33–35] and three steroid receptor genes (ER-β, ERα, and AR) [36, 37] using real-time, quantitative,
two-step RT-PCR (Table 1). Biz-2
produced a robust induction in the three androgen-regulated genes tested (Table 1). We ascertained the specificity and
the strength of induction of androgen-specific genes by Biz-2 by determining
the relative gene expression at 10 ppm Biz-2 or 10 nM DHT. We observed that Biz-2 was as effective as
DHT in inducing the expression of the well-known prostate-specific gene (PSA). Biz-2 at 10 ppm produced 10.55±2.2-, 3.9±0.74-, and 5.95±1.4-fold inductions
of PSA, NKX3-1, and TSC22 mRNA levels, respectively. Relative to DHT, Biz-2 was
six times more effective in stimulating TSC22 expression (Table 1). Biz-2
showed a marginal increase in expression levels of steroid receptor genes (ERα: 1.4±0.3; ERβ: 1.2±0.4; AR: 2.8±0.2) suggesting
differential regulation of androgen-regulated genes by Biz-2 in LNCaP cells
(Table 1). Biz-2 was able to stimulate
each prostate-specific, AR-dependent gene 2-fold to 8-fold relative to
control. Taken together, we surmised
that Biz-2 had a major influence on AR-dependent gene activation in LNCaP
cells.


### 3.4. Components of Bizzy
Nut Extract That Bind to the AR with High Affinity

To corroborate our gene expression data and to provide further
evidence that the effects of Biz-2 are AR-mediated, we measured the ability of
Biz-2 to bind to the AR. We examined the
ability of Biz-2 to compete with3[H]R1881 for binding to AR using
purified, recombinant-expressed AR, LNCaP cells, which contained high levels of
the mutated form of AR [38], or cells transfected with the wild-type pCMV-AR expression
plasmid. A pCMV-AR expression plasmid
was transfected into either LNCaP or DU145 cells, and the ability of the
transfected cells to bind to Biz-2 specifically was measured using a competition
receptor binding assay [28, 39]. LNCaP cells were chosen because they are AR-positive.
The DU145 cells were chosen as a null environment because they are AR-negative.
The concentration of Biz-2 that reduced the specific binding of 3[H]R1881
by 50% (IC50) was determined by incubating protein obtained from
transfected and nontransfected cells with 2 nM 3[H]R1881 in the
absence and presence of increasing concentration of Biz-2. Biz-2 was able to displace 3[H]R1881
bound to the mutant androgen receptor in LNCaP cells, purified recombinant
receptor, or the wild-type receptor protein (Figure 4(a)). Biz-2 displayed high-affinity
binding to both the mutant and the wild-type AR, suggesting that the T877A
mutation of the LNCaP AR was not responsible for the Biz-2/AR biology. The AR binding affinity (IC50 and
Ki) of Biz-2 was determined from the competition assay, because the
assay was performed under conditions where the 3[H]R1881 and the
competing Biz-2 were not depleted. The
IC50 and apparent Ki for Biz-2 binding to AR were
obtained by fitting the radioligand competitive data to the Cheng-Prusoff
equation [27]. The IC50 was determined from
nonlinear regression of the data fitted to a one-site competition binding,
where the Kd for 3[H]R1881 binding to AR was assumed to
be 1 nM (Figure 4(b)). The calculated Ki and IC50 for
Biz-2 binding to the wild-type or LNCaP AR
were 26.45 (CI 17.85 to 39.20)
and 52.91±1.69 ppm (P<.05),
respectively. Comparison of the Ki of Biz-2 to that of R1881 binding to the AR suggested that Biz-2 was only 13X
less potent in binding to the AR.Biz-2 competes for AR binding in LNCaP
cells. AR protein from LNCaP cells,
cells transfected with pCMV-AR, or purified recombinant AR protein was
incubated with 2 nM [3H] R1881 with or without increasing
concentration of Biz-2 for 18 hours at 4°C. Bound and free [3H] R1881 were
separated using the HAP assay (see Section
2). Each data point represents
the mean ± SEM of a representative experiment performed
in triplicate. The IC50 was
calculated from the competition curve using a one-site competition model. Ki was calculated by Ki = IC50/(1 + [ligand]/Kd) in which Kd and ligand
concentration [ligand] were set to 1 and 2 nM, respectively. (a) Single-point assay in the presence of 500X excess R1881 (number above
the bar is % of total binding). (b) Displacement curve of [3H] R1881
by Biz-2 (insert is displacement curve of [3H] R1881 by inert
R1881). N = 2 separate experiments
performed in triplicate.
(a)(b)


### 3.5. Components of Biz-2
Extract are Inhibitory to Proliferation of LNCaP Cells

Biz-2 at high concentrations reduced PSA secretion by LNCaP cells,
but increased PSA mRNA levels. At these high concentrations, components of
Biz-2 may have induced necrotic or apoptotic events in LNCaP cells. To test this hypothesis, we determined
whether Biz-2 exerted any cytotoxic effects using MTT viability and a3[H]-thymidine
proliferation assay. The cytotoxicity of
Biz-2 was determined in both the AR-positive LNCaP and AR-negative DU145 cells.
Biz-2 at less than 20 ppm was capable of inducing cell death (proliferation
inhibition) in more than 50% of LNCaP cells (Figure 5). In addition, Biz-2 was
more effective in inducing death in DU145 cells at the concentrations tested
(Figure 5(a)). Next
we determined the potency of Biz-2 in inhibiting survival of LNCaP and DU-145
cells. The GI50 of Biz-2 was determined by plotting the thymidine
data as a dose-response curve with a variable slope. Maximal inhibition of LNCaP cell growth by
Biz-2 was achieved at 50 ppm with a GI50 of 14.69±1.16 ppm (Figure 5(a)). Biz-2 was five times more potent
in the androgen-independent cell line, DU145, than in the LNCaP cells. Both MTT
and tryphan blue staining demonstrated a significant decrease in cell viability
after 6 hours at 100 ppm Biz-2 (Figure 5(b)). Biz-2 induced  31.44% and 35.63% decreases in LNCaP cell viability and
cell number, respectively (Figure 5(b)). To corroborate the 3[H]-thymidine
incorporation assay, we evaluated the effect of Biz-2 on LNCaP cell
proliferation using MTT exclusion as an index of cell cytotoxicity. Within 24 hours
of exposure to Biz-2, cell viability decreased with dose (Figure 5(c)). This decrease was similar to that observed
with the thymidine incorporation assay (Figure 5(a)). The lethal dose concentration (LD50)
and the growth inhibitory concentration (GI50) of Biz-2 on LNCaP
cells were 14.69±1.16 ppm and 15.24±0.24 ppm in the MTT and [3H]
thymidine incorporation assays, respectively. Taken together, these results
suggested that in LNCaP cells, high concentration of Biz-2 elicits a cytotoxic
effect that is time-dependent and may reduce cell proliferation and increase
the frequency of cell death.Modulation of prostate cell proliferation by Bizzy
nut extracts. LNCaP or DU145 cells were grown in the presence of 0–1000 ppm Biz-2
for 24 hours, and cell growth was determined using (a)3[H] thymidine incorporation, (b) MTT cell viability assay and tryphan
blue dye staining, and (c) cytotoxicity
of Biz-2 in LNCaP cells that was performed as described in Section 2. Growth inhibitory
activity of the extracts was determined as [(A490(control)−A490(extract))/A490(control)]×100. Each value represents mean ± SEM for three experiments performed in
eight wells of a 96-well plate. Values followed by *P<.05 differed
significantly from the controls.
(a)(b)(c)


### 3.6. Biz-2-Induced Apoptosis and Cell Cycle Arrest in
LNCaP Cells

Our current data suggest that
in AR-positive LNCaP cells, Biz-2 is cytotoxic, which presumably leads to
inhibition of cell proliferation. Therefore, we examined the mechanism of Biz-2
cytotoxicity. We investigated whether the observed cytotoxicity was a result of
a chemically induced cellular cytotoxicity or cell-induced apoptosis. We hypothesized that Biz-2 could be cytotoxic
because it ruptures plasma membranes. That is, we hypothesized that a necrotic
reaction, but not programmed cell death, was responsible for the cytotoxity of
Biz-2. Therefore, we examined whether the initial cellular cytotoxicity of
Biz-2 was related to the extent of apoptosis. LNCaP cells (5 × 105)
were grown, induced with increasing concentration (0.1–100 ppm) of
Biz-2, and prepared for microscopic examination as described in Section 2.
Slides were examined under a fluorescent light microscope for the nuclear
changes that are typical of apoptosis. Microscopic examination of apoptotic
cells revealed that there was a dose-dependent increase in apoptosis of both
LNCaP and DU145 cells following 24-hour exposure to Biz-2. Examination of cells treated with Biz-2 by
phase-contrast microscopy showed a dramatic decrease in cell number and
cellular shrinkage (Figures 6(a) and 6(b)). Fluorescence staining with acrydine orange and ethidium bromide revealed
signs of nuclear condensation, nuclei fragmentation, and membrane  budding,
which are all hallmark features of apoptosis (Figures 6(a) and 6(b), bottom
panels). Light microscope analysis of cells indicated that at 1 and 100 ppm Biz-2, 24%–65% of cell death
occurred in LNCaP cells, whereas only 5%–15% cell death
was observed in DU145 cells after a 24-hour exposure. The apoptotic index was determined for both
LNCaP and DU145 by quantifying the relationship between dose of Biz-2 and
apoptosis (Figure 6(c)). We calculate
the apoptotic index by averaging the number of apoptotic cells per field (70–60 cells) then
dividing by the total number of cells per field (120–140 cells). In
the case of LNCaP cells, apoptosis was strongly related to cytotoxicity (Figures
5 and 6(c)). We observed  a greater degree of apoptosis in the AR-positive LNCaP
cells as compared to the AR-negative cell, DU145. A concentration of Biz-2 of 10 ppm induce 20–25±3% apoptosis in LNCaP as compared to 10–12±4% in DU145 cells.
As the concentration of Biz-2 was increased to 10 and 100 ppm, the frequency of
apoptotic cells increased in both cell lines (Figure 6(c)).Cellular and nuclear morphology indicative of
apoptosis induced by Biz-2. LNCaP cells were grown on a microscope slide, then
induced with 1 or 100 ppm of Biz-2 for 24 hours. Cells were stained with a
solution of acridine orange and ethidium bromide for 5 minutes, then examined
by light microscopy. (a) Confocal
laser fluorescent microscope images taken with a 550 nm filter (Top panel: acridine
orange, middle panel: ethidium bromide, bottom panel: both). (b) Apoptotic index was determined. We calculate the apoptotic index by
averaging the number of apoptotic cells per field (70–60 cells), then
dividing by the total number of cells perfield (120–140 cells)  (total number of cells − total number of
apoptotic cells)/(total number of cells).
(a)(b)(c)


### 3.7. Bizzy
Nut Extract Induces Activation of Early Apoptosis Gene

To gain an understanding of the relationship
between Biz-2-induced cytotoxicity and apoptosis, we examined the expression of
several key apoptosis genes. The
temporal dynamics of Bcl2 and Bax during Biz-2-induced cytotoxicity in LNCaP
cells was assessed using western blot analysis and QRT-PCR. Bcl2 protein was
detectable in LNCaP cells grown in serum-free medium for 48 hours. Induction
with 0.1 ppm of Biz-2 resulted in a 4-fold increase in Bcl2 protein and mRNA
(Figure7(a) and Table 1). Following the initial stimulation by Biz-2, the
decrease in Bc12 protein expression was dose-dependent. The decrease in Bcl2 levels with the increase
in Biz-2 probably was not due to metabolism of this compound, because there was
a time-dependent increase in Bcl2 at 10 ppm Biz-2 (data not shown). Similarly,
when we analyzed the dose-dependent expression of the proapoptotic protein Bax
in the presence of Biz-2, there was a slight increase (0.5–1.3-fold) in Bax
protein and a dramatic increase (9.97-fold as compared to control) in its mRNA
expression within the first 12–24 hours of
exposure (Figure 7(a) and Table 1). The
decrease in both Bax and Bcl2 proteins after 24-hour exposure to 100 ppm Biz-2 appeared to be
related to the time-dependent decrease in both proteins (Figure 7(a); data not
shown). Because the Bax and Bcl2 protein levels were important in the release
of a key factor early in the apoptotic cascade, we determined the Bax/Bcl2
ratio in the presence of Biz-2. Bax/Bcl2
levels decreased with increasing concentration of Biz-2. At a high concentration, the LNCaP appeared
to recover from the initial apoptotic event induced by Biz-2.Modulation of apoptotic protein levels in the presence of Biz-2. LNCaP cells were grown in a serum-free medium
in the presence of increasing concentrations of Biz-2 for 24 hours. Whole-cell extracts were isolated with RIPA
buffer, and 30μg
of protein extract were analyzed by immunoblotting for Bcl-2, Bax, AR, and
GDPH expression. (a) Immunoblot of Biz-2-induced
proteins. (b) Ratio of Bcl2/Bax expression. (c) Biz-2 induces increase in Caspase 3/7
activity.
(a)(b)(c)We validated the morphological analysis and
protein assessment of Biz-2-induced apoptosis by ascertaining if the protein
activity of the downstream apoptotic protein Caspase 3/7 had been modulated in
the presence of Biz-2. The level of
active Caspase-3 was measured by proteolytic cleavage of rhodamine 110, from
the bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide)
(Z-DEVD-R110) substrate. There was a dose-dependent increase in Biz-2-induced
cytotoxicity in LNCaP cells (0.1–10 ppm) as
evidenced by the 2.5-fold increase in Caspase 3/7 activity relative to the
untreated control (Figure7(c)). In the analysis of time-dependent Caspase 3/7
activity in the presence of 100 ppm Biz-2, Caspase 3/7 activity decreased
significantly relative to the untreated control within the first 6 hours of
induction, which suggested that a high concentration of Biz-2 inhibited
apoptosis in LNCaP cells (Figure 7(c); data not shown).


### 3.8. Biz-2 Modulation of
Apoptotic Pathway in LNCaP Cells is Mediated through an AR Pathway without New
Protein Synthesis

We used the protein synthesis inhibitor cycloheximide (CHX) and the
AR antagonist flutamide (FLU) to investigate (a) whether Biz-2-induced
apoptosis required protein synthesis and (b) if Biz-2-induced apoptotic
processes use an AR signaling pathway (Figure8). Treatment of LNCaP with 1 ug/mL of CHX for 12 hours after induction with 100 ppm Biz-2 resulted in an inhibition
of Biz-2-induced Caspase 3/7 activity and Bax, and Bcl2 protein expression (Figure
8(b); data not shown). At low concentration, Biz-2 stimulated Caspase 3/7
activity 1-2-fold. CHX completely abolished the Biz-2-stimulated
Caspase levels, which suggested that the effect of Biz-2 on Caspase required
protein synthesis. The analysis of Caspase 3/7 activity and Bax expression in
the presence and absence of actinomycin-D implied that Biz-2 did not elicit a
direct transcription effect on Bax or Caspase levels because pretreatment with actinomycin-D
had no effect on their expression (Figure 8(b); data not shown).Biz-2 increases
Caspase 3/7 levels in LNCaP cells. LNCaP
cells (1 × 106) were grown in test medium and induced with 100 ppm
Biz-2 for 24 hours, and the resulting Caspase 3/7 activity was determined using
the Apo-One Homogeneous Caspase-3/7 assay as described in Section 2. (a) Western blot of Bcl2 and Bax proteins following
induction of LNCaP with Biz-2 in the absence and presence of flutamide. (b) Caspase 3/7 activity was expressed as
n-fold increase relative to untreated control (n = 8, mean ± SEM).
(a)(b)To corroborate the CHX data and to generate
further evidence that Biz-2-modulated apoptosis in LNCaP cells involved
transcriptional regulation through an androgen-dependent pathway, we measured
the expression of Bax and Bcl2 and Caspase 3/7 activity in the presence of the
AR antagonist flutamide. Bcl2 protein
levels and Caspase activity decreased in the presence of 1 nM FLU, which
suggested that the antiandrogen protected prostate cells from apoptotic
assaults (Figure8). At 10 ppm Biz-2,
there was an 80% decrease in Caspase 3/7 activity in the presence of FLU (Figure
8(b)). FLU produced up to a 2-fold
decrease in Bcl2 protein but had a notable 2-fold increase on Bax protein
levels (Figure 8(a)). As with the
Caspase 3/7 activity, cotreatment of Biz-2 with FLU completely blocked
Biz-2-induced increases in Bcl2 and Bax proteins. To substantiate the FLU data, we measured Bcl2-protein levels in the
presence of resveratrol, which induces apoptosis in LNCaP cells [40]. Resveratrol-induced inhibition of Bcl2 and
Bax expression was completely blocked by FLU (Figure 8(a)). Thus, the cytotoxic effect of Biz-2 on LNCaP
cells was decreased by more than 90% in the presence of CHX or the antiandrogen
FLU, and was unaffected by actinomycin-D, which suggested that the mode of
action of Biz-2 involved AR pathways and protein synthesis.


## 3.1. Activation of an Androgen-Dependent Pathway by Biz-2

The androgenic potential of
natural compounds is manifested in a variety of biological responses including
induction of cell growth, secretion of cellular protein, and receptor-dependent
gene expression. Secretion of PSA by the
prostate is a hallmark of prostatic function. Furthermore, modulation of PSA level is used as a biomarker in assessing
prostate development and function. As an
initial step in identifying and characterizing medicinally relevant, putative,
selective androgen receptor modulators (SARMs) present in Bizzy nut, we
performed solid-liquid extraction using solvents of increasing polarity. We screened each extract for bioactivity
using several in vitro assays that were designed to reveal if any of the
extracts contained androgenic or estrogenic activity [22]. We determined whether an ether extract of Bizzy nut
(Biz-2) was capable of modulating prostate function by exerting its effect on
PSA secretion by LNCaP cells. LNCaP
cells were grown in a serum-free medium for 24 hours, and then induced  with 100 ppm of Biz-2 for 6, 12, or 24 hours. A PSA ELISA was then used to determine the
concentration of PSA in the medium. PSA
secretion by LNCaP cells in the presence of Biz-2 displayed a biphasic response
(Figure 1(a)). Relative to control,
Biz-2 induced an initial 26.59±13.3% increase in PSA after the first 6 hours of
exposure. The level of PSA decreased by
32.6±1.3% during the second six-hour period, and increased by 25.93±3.3% 12 hours
later (Figure 1(a)). In contrast, DHT produced a time-dependent increase
(9.6±6.7% and 130 ± 6% after 12 and 24 hours, resp.) in PSA production under the
same experimental conditions (Figure 1(a); data not shown).Biz-2
modulates PSA secretion by LNCaP cells. LNCaP cells (1E7) were grown in serum-free medium for 24 hours,
then induced with 100 ppm of Biz-2 for 0, 6, 12, and 24 hours. Equal aliquots
(10 uL) of the condition medium were removed and analyzed by PSA ELISA (see Section 2). (a) Time-dependent
secretion of PSA. (b) PSA secretion
induced with 10 ppm Biz-2 for 24 hours in the presence and absence of 10 nM
flutamide. The values are the mean ± SEM of three
separate experiments performed in triplicate. CON: control; DHT:
dihydrotestosterone; Biz-2: ether extract of Bizzy nut; RES: resveratrol.
(a)(b)To understand the relationship between Biz-2
modulation of PSA secretion and the androgen-dependent pathway, we determined
whether the PSA secretion was mediated by a Biz-2/androgen receptor
interaction. To answer this question,
PSA secretion was measured in the presence of the antiandrogen flutamide (FLU)
(Figure1(b)). As a positive control, we measured PSA secretion in the presence
of flutamide with or without the natural androgen, DHT. Consistent with previous studies [31, 32], flutamide blocked
the AR-dependent stimulation of PSA (1.014±0.08 ng/uL for DHT versus 0.549±0.07 ng/uL for DHT/FLU) by LNCaP cells. Treatment of LNCaP cells with 10 ppm Biz-2 plus 1 nM of flutamide
prevented the Biz-2 inhibition of PSA secretion (Figure 1(b)). At 10 ppm Biz-2, PSA levels decreased by 24.80±3.4% of control,
that is, from 0.654±0.01 ng/uL to 0.492±0.022 ng/uL. Addition of flutamide at 1 nM restored the
PSA level to that of control (0.673±0.077 ng/uL Biz-2/FLU versus 0.654±0.03 ng/uL control). In our system, there was
a significant increase (16 ± 2.8%, n = 8, P<.01) in PSA secretion in the
presence of FLU, suggesting that there may have been residual androgen present
in the medium. Alternatively, flutamide may have induced an androgenic effect
on PSA under serum-free conditions (Figure 1(b)). Nevertheless, FLU was able to prevent the
Biz-2-induced decrease in PSA secretion, which was an observation that was
complimentary to what we observed with DHT/FLU treatment. These results strongly suggested that the
Biz-2 inhibition of PSA secretion was dependent on the AR pathway.


## 3.2. Transcriptional Inhibition
of an Endogenous Androgen-Regulated Gene by Biz-2

Modulation of PSA secretion in the presence of
Biz-2 may have resulted from changes in PSA mRNA production, and could have
been independent of the secretion processes that were present in LNCaP
cells. To test this hypothesis, we
measured PSA mRNA levels in cells exposed to Biz-2 using Taqman real-time
RT-PCR. Multiplex real-time PCR was
performed using a primer/probe set specific for PSA and 18S rRNA (PE Applied
Biosystems, Foster City, Calif, USA). The expression levels of PSA were obtained
from DNase-I-treated total RNA, and were normalized to those of the 18S rRNA
gene, which served as the calibrator for our experiments. The logarithm of the fluorescence intensity
of each probe versus the number of cycles and the level of expression of mRNA
of PSA in LNCaP cells was evaluated (Figure2). In the present study, the mRNA
expression of PSA was determined using the 2ΔΔCt method,
and is expressed relative to the control sample which is set to 1. We observed a time-dependent increase in
levels of PSA mRNA between 6 and 24 hours of exposure in the presence of 10 ppm
Biz-2 (Figure 2(b)). Analysis of the
data in Figure 2(b) also indicated that, after treatment with Biz-2 or the
positive control (DHT), levels of PSA mRNA expression showed a statistically
significant increase. Exposure of LNCaP cells to 10 ppm Biz-2 resulted in a
0.87±0.17-fold increase in PSA mRNA, which was
increased by 12-fold (0.87±0.17 to 10.56±2.25) in the second
12-hour period. We determined the
potency of Biz-2 in stimulating PSA mRNA by exposing LNCaP cells to different
amounts of Biz-2 for 24 hours. The
response of PSA mRNA production to Biz-2 concentration was bell-shaped (Figure
2(c)). At concentrations below 10 ppm,
Biz-2 elicited a dose-dependent increase in PSA mRNA expression, whereas at
high concentrations (100 ppm and greater) expression of PSA was inhibited. Biz-2 at low concentrations produced a
similar increase in PSA mRNA at 12 and 24 hours as the endogenous androgen DHT
(Figure 2(b); data not shown).Stimulation of PSA
mRNA expression by Biz-2. LNCaP cells
were induced with Biz-2 or DHT, and 5μg of DNase-I-treated RNA isolated from induced or
uninduced cells were subjected to two-step Taqman real-time RT-PCR (see Section
2). The relative expression of PSA mRNA expression was calculated by the 2ΔΔCt method. First, relative quantitation of
PSA mRNA expression was performed by first normalizing the Ct values
of PSA amplification against the Ct values of endogenous 18S rRNA,
then the resulting Ct values were normalized using the Ct value of the vehicle control sample. (a) Relative amplification plot of mRNA expression (1–NTC. 2–18S rRNA at 24 hours, 3 and 4- CON at 6 and 24 hours, 5 and 6- Biz-2 at 6 and 24 hours). (b) PSA mRNA expression relative to time. (c) Dose-dependent response of PSA to Biz-2. The values are mean ± SEM
of  induction samples analyzed in triplicate.
(a)(b)(c)To substantiate the observation that Biz-2
modulated PSA activity in an AR-dependent manner, we measured Biz-2-induced PSA
mRNA  in the presence of the AR antagonist flutamide. The Biz-2-induced expression of PSA mRNA was
inhibited by 55% (3.46±0.25-fold Biz-2 to 1.55±0.14-fold
Biz-2/Flu) in the presence of 1 nM flutamide (Figure 3) suggesting that Biz-2
mediated an increase in PSA mRNA level through an interaction with the AR
present in LNCaP cells. On the other
hand, resveratrol, a phytoestrogen with bioactivity in LNCaP cells, induced a
1.5-fold increase in PSA mRNA levels, which was independent of the AR (Figure
3). Next we ascertained whether the
stimulatory effects of Biz-2 on PSA expression were a result of RNA stability
by measuring the mRNA levels of PSA in the presence and absence of actinomycin-D,
a known transcription inhibitor. Pretreatment of LNCaP cells with 5 ug/uL of actinomycin-D for  4 hours
resulted in a 26% inhibition of PSA expression relative to Biz-2. At the same time, blocking of protein
synthesis using cycloheximide resulted in an increase in the Biz-2-induced
expression of PSA (Figure 3). Because
actinomycin-D alone could not have induced PSA mRNA synthesis, our data
suggested that Biz-2 regulated PSA expression directly at the level of
transcription.Figure 3
Flutamide
inhibition of Biz-2 stimulation of PSA mRNA expression. LNCaP cells were induced with Biz-2 in the
presence or absence of flutamide, and RNA from induced or uninduced cells was
subjected to two-step Taqman real-time RT-PCR. Relative quantitation of PSA
mRNA expression was preformed as described in Figure2. CON: vehicle control; FLU: flutamide; Biz-2: Bizzy
nut extract-2; RES: resveratrol; Act.D: actinomycin-D; CHX: cycloheximide. The
values are the mean ± SEM
of three separate experiments performed in triplicate.


## 3.3. Modulation of
AR-Dependent Gene Expression by Biz-2

To further characterize the effects of Biz-2 on the AR-dependent
pathway, we examined the androgenic effects of Biz-2 on the transcription
activity of three androgen-regulated genes (PSA, PART-1, and NKX3-1) [33–35] and three steroid receptor genes (ER-β, ERα, and AR) [36, 37] using real-time, quantitative,
two-step RT-PCR (Table 1). Biz-2
produced a robust induction in the three androgen-regulated genes tested (Table 1). We ascertained the specificity and
the strength of induction of androgen-specific genes by Biz-2 by determining
the relative gene expression at 10 ppm Biz-2 or 10 nM DHT. We observed that Biz-2 was as effective as
DHT in inducing the expression of the well-known prostate-specific gene (PSA). Biz-2 at 10 ppm produced 10.55±2.2-, 3.9±0.74-, and 5.95±1.4-fold inductions
of PSA, NKX3-1, and TSC22 mRNA levels, respectively. Relative to DHT, Biz-2 was
six times more effective in stimulating TSC22 expression (Table 1). Biz-2
showed a marginal increase in expression levels of steroid receptor genes (ERα: 1.4±0.3; ERβ: 1.2±0.4; AR: 2.8±0.2) suggesting
differential regulation of androgen-regulated genes by Biz-2 in LNCaP cells
(Table 1). Biz-2 was able to stimulate
each prostate-specific, AR-dependent gene 2-fold to 8-fold relative to
control. Taken together, we surmised
that Biz-2 had a major influence on AR-dependent gene activation in LNCaP
cells.


## 3.4. Components of Bizzy
Nut Extract That Bind to the AR with High Affinity

To corroborate our gene expression data and to provide further
evidence that the effects of Biz-2 are AR-mediated, we measured the ability of
Biz-2 to bind to the AR. We examined the
ability of Biz-2 to compete with3[H]R1881 for binding to AR using
purified, recombinant-expressed AR, LNCaP cells, which contained high levels of
the mutated form of AR [38], or cells transfected with the wild-type pCMV-AR expression
plasmid. A pCMV-AR expression plasmid
was transfected into either LNCaP or DU145 cells, and the ability of the
transfected cells to bind to Biz-2 specifically was measured using a competition
receptor binding assay [28, 39]. LNCaP cells were chosen because they are AR-positive.
The DU145 cells were chosen as a null environment because they are AR-negative.
The concentration of Biz-2 that reduced the specific binding of 3[H]R1881
by 50% (IC50) was determined by incubating protein obtained from
transfected and nontransfected cells with 2 nM 3[H]R1881 in the
absence and presence of increasing concentration of Biz-2. Biz-2 was able to displace 3[H]R1881
bound to the mutant androgen receptor in LNCaP cells, purified recombinant
receptor, or the wild-type receptor protein (Figure 4(a)). Biz-2 displayed high-affinity
binding to both the mutant and the wild-type AR, suggesting that the T877A
mutation of the LNCaP AR was not responsible for the Biz-2/AR biology. The AR binding affinity (IC50 and
Ki) of Biz-2 was determined from the competition assay, because the
assay was performed under conditions where the 3[H]R1881 and the
competing Biz-2 were not depleted. The
IC50 and apparent Ki for Biz-2 binding to AR were
obtained by fitting the radioligand competitive data to the Cheng-Prusoff
equation [27]. The IC50 was determined from
nonlinear regression of the data fitted to a one-site competition binding,
where the Kd for 3[H]R1881 binding to AR was assumed to
be 1 nM (Figure 4(b)). The calculated Ki and IC50 for
Biz-2 binding to the wild-type or LNCaP AR
were 26.45 (CI 17.85 to 39.20)
and 52.91±1.69 ppm (P<.05),
respectively. Comparison of the Ki of Biz-2 to that of R1881 binding to the AR suggested that Biz-2 was only 13X
less potent in binding to the AR.Biz-2 competes for AR binding in LNCaP
cells. AR protein from LNCaP cells,
cells transfected with pCMV-AR, or purified recombinant AR protein was
incubated with 2 nM [3H] R1881 with or without increasing
concentration of Biz-2 for 18 hours at 4°C. Bound and free [3H] R1881 were
separated using the HAP assay (see Section
2). Each data point represents
the mean ± SEM of a representative experiment performed
in triplicate. The IC50 was
calculated from the competition curve using a one-site competition model. Ki was calculated by Ki = IC50/(1 + [ligand]/Kd) in which Kd and ligand
concentration [ligand] were set to 1 and 2 nM, respectively. (a) Single-point assay in the presence of 500X excess R1881 (number above
the bar is % of total binding). (b) Displacement curve of [3H] R1881
by Biz-2 (insert is displacement curve of [3H] R1881 by inert
R1881). N = 2 separate experiments
performed in triplicate.
(a)(b)


## 3.5. Components of Biz-2
Extract are Inhibitory to Proliferation of LNCaP Cells

Biz-2 at high concentrations reduced PSA secretion by LNCaP cells,
but increased PSA mRNA levels. At these high concentrations, components of
Biz-2 may have induced necrotic or apoptotic events in LNCaP cells. To test this hypothesis, we determined
whether Biz-2 exerted any cytotoxic effects using MTT viability and a3[H]-thymidine
proliferation assay. The cytotoxicity of
Biz-2 was determined in both the AR-positive LNCaP and AR-negative DU145 cells.
Biz-2 at less than 20 ppm was capable of inducing cell death (proliferation
inhibition) in more than 50% of LNCaP cells (Figure 5). In addition, Biz-2 was
more effective in inducing death in DU145 cells at the concentrations tested
(Figure 5(a)). Next
we determined the potency of Biz-2 in inhibiting survival of LNCaP and DU-145
cells. The GI50 of Biz-2 was determined by plotting the thymidine
data as a dose-response curve with a variable slope. Maximal inhibition of LNCaP cell growth by
Biz-2 was achieved at 50 ppm with a GI50 of 14.69±1.16 ppm (Figure 5(a)). Biz-2 was five times more potent
in the androgen-independent cell line, DU145, than in the LNCaP cells. Both MTT
and tryphan blue staining demonstrated a significant decrease in cell viability
after 6 hours at 100 ppm Biz-2 (Figure 5(b)). Biz-2 induced  31.44% and 35.63% decreases in LNCaP cell viability and
cell number, respectively (Figure 5(b)). To corroborate the 3[H]-thymidine
incorporation assay, we evaluated the effect of Biz-2 on LNCaP cell
proliferation using MTT exclusion as an index of cell cytotoxicity. Within 24 hours
of exposure to Biz-2, cell viability decreased with dose (Figure 5(c)). This decrease was similar to that observed
with the thymidine incorporation assay (Figure 5(a)). The lethal dose concentration (LD50)
and the growth inhibitory concentration (GI50) of Biz-2 on LNCaP
cells were 14.69±1.16 ppm and 15.24±0.24 ppm in the MTT and [3H]
thymidine incorporation assays, respectively. Taken together, these results
suggested that in LNCaP cells, high concentration of Biz-2 elicits a cytotoxic
effect that is time-dependent and may reduce cell proliferation and increase
the frequency of cell death.Modulation of prostate cell proliferation by Bizzy
nut extracts. LNCaP or DU145 cells were grown in the presence of 0–1000 ppm Biz-2
for 24 hours, and cell growth was determined using (a)3[H] thymidine incorporation, (b) MTT cell viability assay and tryphan
blue dye staining, and (c) cytotoxicity
of Biz-2 in LNCaP cells that was performed as described in Section 2. Growth inhibitory
activity of the extracts was determined as [(A490(control)−A490(extract))/A490(control)]×100. Each value represents mean ± SEM for three experiments performed in
eight wells of a 96-well plate. Values followed by *P<.05 differed
significantly from the controls.
(a)(b)(c)


## 3.6. Biz-2-Induced Apoptosis and Cell Cycle Arrest in
LNCaP Cells

Our current data suggest that
in AR-positive LNCaP cells, Biz-2 is cytotoxic, which presumably leads to
inhibition of cell proliferation. Therefore, we examined the mechanism of Biz-2
cytotoxicity. We investigated whether the observed cytotoxicity was a result of
a chemically induced cellular cytotoxicity or cell-induced apoptosis. We hypothesized that Biz-2 could be cytotoxic
because it ruptures plasma membranes. That is, we hypothesized that a necrotic
reaction, but not programmed cell death, was responsible for the cytotoxity of
Biz-2. Therefore, we examined whether the initial cellular cytotoxicity of
Biz-2 was related to the extent of apoptosis. LNCaP cells (5 × 105)
were grown, induced with increasing concentration (0.1–100 ppm) of
Biz-2, and prepared for microscopic examination as described in Section 2.
Slides were examined under a fluorescent light microscope for the nuclear
changes that are typical of apoptosis. Microscopic examination of apoptotic
cells revealed that there was a dose-dependent increase in apoptosis of both
LNCaP and DU145 cells following 24-hour exposure to Biz-2. Examination of cells treated with Biz-2 by
phase-contrast microscopy showed a dramatic decrease in cell number and
cellular shrinkage (Figures 6(a) and 6(b)). Fluorescence staining with acrydine orange and ethidium bromide revealed
signs of nuclear condensation, nuclei fragmentation, and membrane  budding,
which are all hallmark features of apoptosis (Figures 6(a) and 6(b), bottom
panels). Light microscope analysis of cells indicated that at 1 and 100 ppm Biz-2, 24%–65% of cell death
occurred in LNCaP cells, whereas only 5%–15% cell death
was observed in DU145 cells after a 24-hour exposure. The apoptotic index was determined for both
LNCaP and DU145 by quantifying the relationship between dose of Biz-2 and
apoptosis (Figure 6(c)). We calculate
the apoptotic index by averaging the number of apoptotic cells per field (70–60 cells) then
dividing by the total number of cells per field (120–140 cells). In
the case of LNCaP cells, apoptosis was strongly related to cytotoxicity (Figures
5 and 6(c)). We observed  a greater degree of apoptosis in the AR-positive LNCaP
cells as compared to the AR-negative cell, DU145. A concentration of Biz-2 of 10 ppm induce 20–25±3% apoptosis in LNCaP as compared to 10–12±4% in DU145 cells.
As the concentration of Biz-2 was increased to 10 and 100 ppm, the frequency of
apoptotic cells increased in both cell lines (Figure 6(c)).Cellular and nuclear morphology indicative of
apoptosis induced by Biz-2. LNCaP cells were grown on a microscope slide, then
induced with 1 or 100 ppm of Biz-2 for 24 hours. Cells were stained with a
solution of acridine orange and ethidium bromide for 5 minutes, then examined
by light microscopy. (a) Confocal
laser fluorescent microscope images taken with a 550 nm filter (Top panel: acridine
orange, middle panel: ethidium bromide, bottom panel: both). (b) Apoptotic index was determined. We calculate the apoptotic index by
averaging the number of apoptotic cells per field (70–60 cells), then
dividing by the total number of cells perfield (120–140 cells)  (total number of cells − total number of
apoptotic cells)/(total number of cells).
(a)(b)(c)


## 3.7. Bizzy
Nut Extract Induces Activation of Early Apoptosis Gene

To gain an understanding of the relationship
between Biz-2-induced cytotoxicity and apoptosis, we examined the expression of
several key apoptosis genes. The
temporal dynamics of Bcl2 and Bax during Biz-2-induced cytotoxicity in LNCaP
cells was assessed using western blot analysis and QRT-PCR. Bcl2 protein was
detectable in LNCaP cells grown in serum-free medium for 48 hours. Induction
with 0.1 ppm of Biz-2 resulted in a 4-fold increase in Bcl2 protein and mRNA
(Figure7(a) and Table 1). Following the initial stimulation by Biz-2, the
decrease in Bc12 protein expression was dose-dependent. The decrease in Bcl2 levels with the increase
in Biz-2 probably was not due to metabolism of this compound, because there was
a time-dependent increase in Bcl2 at 10 ppm Biz-2 (data not shown). Similarly,
when we analyzed the dose-dependent expression of the proapoptotic protein Bax
in the presence of Biz-2, there was a slight increase (0.5–1.3-fold) in Bax
protein and a dramatic increase (9.97-fold as compared to control) in its mRNA
expression within the first 12–24 hours of
exposure (Figure 7(a) and Table 1). The
decrease in both Bax and Bcl2 proteins after 24-hour exposure to 100 ppm Biz-2 appeared to be
related to the time-dependent decrease in both proteins (Figure 7(a); data not
shown). Because the Bax and Bcl2 protein levels were important in the release
of a key factor early in the apoptotic cascade, we determined the Bax/Bcl2
ratio in the presence of Biz-2. Bax/Bcl2
levels decreased with increasing concentration of Biz-2. At a high concentration, the LNCaP appeared
to recover from the initial apoptotic event induced by Biz-2.Modulation of apoptotic protein levels in the presence of Biz-2. LNCaP cells were grown in a serum-free medium
in the presence of increasing concentrations of Biz-2 for 24 hours. Whole-cell extracts were isolated with RIPA
buffer, and 30μg
of protein extract were analyzed by immunoblotting for Bcl-2, Bax, AR, and
GDPH expression. (a) Immunoblot of Biz-2-induced
proteins. (b) Ratio of Bcl2/Bax expression. (c) Biz-2 induces increase in Caspase 3/7
activity.
(a)(b)(c)We validated the morphological analysis and
protein assessment of Biz-2-induced apoptosis by ascertaining if the protein
activity of the downstream apoptotic protein Caspase 3/7 had been modulated in
the presence of Biz-2. The level of
active Caspase-3 was measured by proteolytic cleavage of rhodamine 110, from
the bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide)
(Z-DEVD-R110) substrate. There was a dose-dependent increase in Biz-2-induced
cytotoxicity in LNCaP cells (0.1–10 ppm) as
evidenced by the 2.5-fold increase in Caspase 3/7 activity relative to the
untreated control (Figure7(c)). In the analysis of time-dependent Caspase 3/7
activity in the presence of 100 ppm Biz-2, Caspase 3/7 activity decreased
significantly relative to the untreated control within the first 6 hours of
induction, which suggested that a high concentration of Biz-2 inhibited
apoptosis in LNCaP cells (Figure 7(c); data not shown).


## 3.8. Biz-2 Modulation of
Apoptotic Pathway in LNCaP Cells is Mediated through an AR Pathway without New
Protein Synthesis

We used the protein synthesis inhibitor cycloheximide (CHX) and the
AR antagonist flutamide (FLU) to investigate (a) whether Biz-2-induced
apoptosis required protein synthesis and (b) if Biz-2-induced apoptotic
processes use an AR signaling pathway (Figure8). Treatment of LNCaP with 1 ug/mL of CHX for 12 hours after induction with 100 ppm Biz-2 resulted in an inhibition
of Biz-2-induced Caspase 3/7 activity and Bax, and Bcl2 protein expression (Figure
8(b); data not shown). At low concentration, Biz-2 stimulated Caspase 3/7
activity 1-2-fold. CHX completely abolished the Biz-2-stimulated
Caspase levels, which suggested that the effect of Biz-2 on Caspase required
protein synthesis. The analysis of Caspase 3/7 activity and Bax expression in
the presence and absence of actinomycin-D implied that Biz-2 did not elicit a
direct transcription effect on Bax or Caspase levels because pretreatment with actinomycin-D
had no effect on their expression (Figure 8(b); data not shown).Biz-2 increases
Caspase 3/7 levels in LNCaP cells. LNCaP
cells (1 × 106) were grown in test medium and induced with 100 ppm
Biz-2 for 24 hours, and the resulting Caspase 3/7 activity was determined using
the Apo-One Homogeneous Caspase-3/7 assay as described in Section 2. (a) Western blot of Bcl2 and Bax proteins following
induction of LNCaP with Biz-2 in the absence and presence of flutamide. (b) Caspase 3/7 activity was expressed as
n-fold increase relative to untreated control (n = 8, mean ± SEM).
(a)(b)To corroborate the CHX data and to generate
further evidence that Biz-2-modulated apoptosis in LNCaP cells involved
transcriptional regulation through an androgen-dependent pathway, we measured
the expression of Bax and Bcl2 and Caspase 3/7 activity in the presence of the
AR antagonist flutamide. Bcl2 protein
levels and Caspase activity decreased in the presence of 1 nM FLU, which
suggested that the antiandrogen protected prostate cells from apoptotic
assaults (Figure8). At 10 ppm Biz-2,
there was an 80% decrease in Caspase 3/7 activity in the presence of FLU (Figure
8(b)). FLU produced up to a 2-fold
decrease in Bcl2 protein but had a notable 2-fold increase on Bax protein
levels (Figure 8(a)). As with the
Caspase 3/7 activity, cotreatment of Biz-2 with FLU completely blocked
Biz-2-induced increases in Bcl2 and Bax proteins. To substantiate the FLU data, we measured Bcl2-protein levels in the
presence of resveratrol, which induces apoptosis in LNCaP cells [40]. Resveratrol-induced inhibition of Bcl2 and
Bax expression was completely blocked by FLU (Figure 8(a)). Thus, the cytotoxic effect of Biz-2 on LNCaP
cells was decreased by more than 90% in the presence of CHX or the antiandrogen
FLU, and was unaffected by actinomycin-D, which suggested that the mode of
action of Biz-2 involved AR pathways and protein synthesis.


## 4. Discussion

Given the high incidence of prostate
cancer, there is an urgent need for treatment paradigms that improve prostate
health. Nonsteroidal compounds are an interesting target for prostate cancer
treatment because they do not produce the undesirable effects associated with
traditional steroidal regimes [12, 39, 41, 42]. The search for
nonsteroidal androgen usually starts by identifying a natural compound that
shows the potential of antiandrogen properties. One group of compounds that has received a tremendous amount of
attention is the naturally occurring phytoestrogens found in fruits, vegetables,
and teas. These compounds have also been
shown to possess antiandrogenic activity due to their ability to inhibit and
delay prostate cancer [11, 43].Growth and normal development of the prostate
gland are under the
control of androgen acting through interaction with the AR. The need for new androgenic therapies to
improve prostate function has centered on agents that alter the AR action. Recently, we reported that the common
Jamaican herbal medicine, Bizzy, contains a putative nonsteroidal compound with
bioactivity in both breast and prostate cancer cells. In this study, we applied
the androgen-dependent LNCaP prostate tumor cell model along with the
androgen-independent DU145 prostate tumor cell model to ascertain the
androgenic effect of this medicinal extract. Screening of several Bizzy
extracts for bioactivity using several in
vitro assays was designed to evaluate whether any of the extracts
contained androgenic or antiandrogenic activity [22]. The study shows that an
ether extract of Bizzy (Biz-2) is the most potent inhibitor of LNCaP cell
growth because it has a high affinity for the AR. At high concentrations, Biz-2
activates AR-dependent gene expression and induces apoptosis in LNCaP cells.We tested the hypothesis that Bizzy
nut extract can modulate prostate activity by using prostate secretion as an
index of prostate function. We observed that the rate of PSA secreted by LNCaP
cells in the presence of Biz-2 was sharply decreased. Biz-2 modulation of PSA
production was AR-dependent, which implied that Biz-2’s biological effect may
serve as an index of putative phytoandrogen in prostate cancer cells.The key to a successfully chemopreventive
agent against prostate and other types of cancer will rely on these agents’
ability to induce cell death in tumor cells in a tissue-dependent and even a
receptor-dependent manner. The search for natural compounds capable of
functioning as potential chemopreventive agents for prostate cancer has met
with mixed results [19, 43–45]. Natural chemopreventive agents as well as
anticancer agents function by modulating one or several major steps in the
carcinogenesis pathway. Naturally occurring agents that could induce apoptosis
in prostate cells in an AR-dependent manner would signal a new generation of
prostate-specific, chemopreventative agents. Our Biz-2 extract appears to possess some of the fundamental activities required for
this type of chemopreventive agent. We demonstrated that in LNCaP cells, Biz-2
inhibits at least one family member of the natural inhibitors of apoptosis
(Bcl2), which suggests that Biz-2 may force cells into the apoptotic pathway.
Bcl2 and Bax are key players in apoptotic events. The ratio of these proteins determines
whether a cell will commit to the apoptosis pathway. The data provided by this
study point to Biz-2 as a putative chemopreventive agent in prostate cells.
Biz-2 showed a time-dependent and dose-dependent increase in the proapoptotic
gene at both the protein and mRNA levels. The increase in proapoptotic protein was coupled to morphological signs
of apoptosis and to an increase in enzyme associated with late apoptotic
events. Furthermore, the ability of the
antiandrogen flutamide to prevent Biz-2 induction of both Caspase activity and
Bcl2 expression supports the role of the AR pathway in this process. It is
interesting to note that in the absence of AR, Bizz-2 may possess
antiproliferative activity. In the AR-negative
cell, DU145, we observed a greater degree of inhibition of cell proliferation
as compared to AR-positive  LNCaP cells. Correspondently, DU145 had a lower apoptotic index in the presence of
Biz-2. Taken together, these observations
suggest that Biz-2 may elicit antiproliferative activity that does not require a functional
androgen receptor pathway.In deciphering the role of Biz-2 in prostate
function, it is important to examine the transcriptional effects of this
extract on both estrogen-responsive and androgen-responsive genes. Androgen,
including phytoandrogen, acts via binding of different types of ligands to the
AR coalescent in gene expression. Antagonists, which inhibit the transcriptional activity of the AR, are
currently being used as potential treatment agents in the fight against
prostate cancer. The involvement of
Biz-2 in AR biology was examined using two parameters: (1) receptor binding and
(2) AR-dependent gene activation. In
classical competition experiments, components of our Biz-2 extract are capable
of displacing androgen from the AR. The
potency of the putative androgenic compound in Biz-2 is only thirteen times
lower than the potency of the synthetic androgen R1881. Examination of the transcriptional response
of several androgen-regulated genes reveals a 5-fold induction in mRNA
expression following a 24-hour exposure to Biz-2. Levels of expression of
NKX3-1 or PSA, both prostate-specific genes, were elevated 5-fold and 10-fold,
respectively, by Biz-2. DHT produced a similar induction in these genes under
identical conditions. We observed that
Biz-2 regulated both androgen-dependent and androgen-independent genes such as
Bcl2 and Bax, but was ineffective in regulating the steroid receptor mRNA
levels. This study clearly demonstrated
that effects of Biz-2 on transcriptional activation of AR-dependent genes
suggested that Biz-2 may be androgenic at the levels of transcription.The biological effects of natural extract
containing putative phytoandrogen or phytoestrogen are multifarious. Several
hypotheses aimed at explaining the diverse action of these compounds have been
proposed. One prevailing hypothesis for the cancer preventative effect of
phytoandrogen or phytoestrogen in prostate cancer is the activation of an
apoptotic cascade by components of the natural extract. In this study, we provide evidence that Biz-2
extract has the ability to eradicate prostate cancer cells by inducing
apoptosis in an apparent AR-dependent manner. Validation of these in vitro
findings with an in vivo model
system is warranted. Our findings highlight the need for a closer examination
of Bizzy nut as a potential source of natural chemicals that may modulate
prostate growth and function.



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*Source: 530279-2008-10-13.xml*