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 . 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 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 [ 57 ] .
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 [ 1416 ] . 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 .
biologically active , nonsteroidal , androgenic , and estrogenic 
 agents are found primarily in soy products , legumes , and whole grains .
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 . 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 .
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 .
lncap or du145 cells ( 1  10 ) 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 80c until used .
the 
 ether extract of bizzy nut ( biz-2 ) was prepared as previously described . 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 plated in 96-well plates ( 1  10 cells / well ) and allowed to attach overnight .
the biz-2 extract in 0.1% dmso was added in a series of concentrations 
 ( 01000 ppm ) to a 
 96-well plate . as a control and a reference ,
10 m genistein ( ge ) 
 or 10 m dht was added to separate wells of each plate .
, 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 . ,
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 .
total rna was obtained from cells treated with 01000 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 .
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 25c for 10 minutes then at 37c 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 table 1 ) , 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 : 50c for 2 minutes , 
 95c for 10 minutes , 40 cycles at 95c for 15 seconds , and 60c for 1 minute . 

an increase in fluorescence was obtained at the annealing and extension step at 
 60c .
the relative level of expression of each gene 
 in the samples was determined using the relative 2 expression   method as described in detail in the abi prism sequence detection system 
 user bulletin 2 .
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 .
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 [ h]-r1881 in the presence or absence of a 10-fold serial 
 dilution of 10 000 ppm biz-2 , for 18 hours at 4c .
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   using the prism 
 iii software program ( graphpad , san diego , calif , usa ) . for microscopy , 5  10 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 l 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 .
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  10 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 37c 
 for 510 minutes .
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 .
immunoblot of the lncap protein fraction was performed as 
 previously described [ 28 , 29 ] .
briefly , lncap cells grown 
 under the indicated condition were lysed at 4c 
 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 420% 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 ) .
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 .
briefly , 1  10 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 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 ( 050 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 .
briefly , cells 
 were plated at a density of 1  10 cells / well in 96-well plates , 
 and incubated overnight .
biz-2 in 0.1% 
 dimethyl sulfoxide was added at a series of concentrations ( 01000 ppm ) to 
 eight wells each of a 96-well plate , and incubated for 24 hours . after adding 0.5 uci / well of [ methyl - h]thymidine
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 .
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 ) .
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 0.45 m 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 .
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 .
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 .
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 .
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 ) . 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 ) 
 ( figure 1(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
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 .
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 ( figure 2 ) . in the present study , the mrna 
 expression of psa was determined using the 2 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 ) . 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 . 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 ) [ 3335 ] and three steroid receptor genes ( er- , er , 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 ( er : 1.4  0.3 ; er : 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 . 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 with [ h]r1881 for binding to ar using 
 purified , recombinant - expressed ar , lncap cells , which contained high levels of 
 the mutated form of ar , 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 ] .
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 [ h]r1881 
 by 50% ( ic50 ) was determined by incubating protein obtained from 
 transfected and nontransfected cells with 2  nm [ h]r1881 in the 
 absence and presence of increasing concentration of biz-2 .
biz-2 was able to displace [ 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 [ 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 .
the ic50 was determined from 
 nonlinear regression of the data fitted to a one - site competition binding , 
 where the kd for [ 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 <
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 at high concentrations reduced psa secretion by lncap cells , 
 but increased psa mrna levels . at these high concentrations , components of 
 biz-2
to test this hypothesis , we determined 
 whether biz-2 exerted any cytotoxic effects using mtt viability and a [ 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 [ 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 ,
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 [ h ] 
 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 .
our current data suggest that 
 in ar - positive lncap cells , biz-2 is cytotoxic , which presumably leads to 
 inhibition of cell proliferation .
we investigated whether the observed cytotoxicity was a result of 
 a chemically induced cellular cytotoxicity or cell - induced apoptosis .
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  10 ) 
 were grown , induced with increasing concentration ( 0.1100 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 ( 7060 cells ) then 
 dividing by the total number of cells per field ( 120140 cells ) . in 
 the case of lncap cells ,
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 2025  3% apoptosis in lncap as compared to 1012  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 ) ) . to gain an understanding of the relationship 
 between biz-2-induced cytotoxicity and apoptosis
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 
 ( figure 7(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 ( 0.51.3-fold ) in bax 
 protein and a dramatic increase ( 9.97-fold as compared to control ) in its mrna 
 expression within the first 1224 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 ,
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 ( 0.110 ppm ) as 
 evidenced by the 2.5-fold increase in caspase 3/7 activity relative to the 
 untreated control ( figure 7(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 ) .
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 ( figure 8) .
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 ,
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 ) . 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 ( figure 8) . 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 .
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 .
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 .
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 ,
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 , 4345 ] .
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 . 

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 .
. 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
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 .
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 .
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 .
 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 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion