Carcinogenesis vol.29 no.10 pp.2001–2010, 2008
Advance Access publication June 26, 2008
Differential effects of resveratrol on androgen-responsive LNCaP human prostate
cancer cells in vitro and in vivo
Thomas T.Y.Wang?, Tamaro S.Hudson1, Tien-Chung
Wang2, Connie M.Remsberg3, Neal M.Davies3, Yoko
Takahashi4, Young S.Kim5, Harold Seifried5, Bryan
T.Vinyard6, Susan N.Perkins7and Stephen D.Hursting7,8
Diet, Genomics and Immunology Laboratory, Beltsville Human Nutrition
Research Center, Agriculture Research Service, United States Department of
Agriculture, 10300 Baltimore Avenue, Building 307C, Room 132, Beltsville,
MD 20705, USA,1Laboratory of Cellular Regulation and Carcinogenesis,
National Cancer Institutes, National Institutes of Health, Bethesda, MD
20892, USA,2Department of Nutrition and Food Science, University of
Maryland, College Park, MD 20742, USA,3Department of Pharmaceutical
Sciences and Pharmacology and Toxicology Graduate Program College of
Pharmacy, Washington State University, Pullman, WA 99164-6534, USA,
4National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan,
5Nutritional Sciences Research Group, Division of Cancer Prevention,
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892,
USA,6Biometrical Consulting Service, Beltsville Area, Agriculture Research
Service, United States Department of Agriculture, Beltsville, MD 20705,
USA,7Division of Nutritional Sciences, University of Texas at Austin, Austin,
TX 78712, USA and8Department of Carcinogenesis, MD Anderson Cancer
Center, Smithville, TX 78957, USA
?To whom correspondence should be addressed. Tel: þ1 301 504 8459;
Fax: þ1 301 504 9456;
use as a prostate cancer chemopreventive agent. However, the effi-
cacy, as well as the mechanisms of action of resveratrol on prostate
cancer prevention, remains largely unknown. This study seeks to
address these questions and examine the cancer preventive effects
of resveratrol using complementaryhuman LNCaP prostate cancer
that resveratrol inhibited cell growth. The growth inhibitory effects
microarrays identified androgen-responsive genes as a group of
genes universally affected by resveratrol in LNCaP cells in vitro.
The effect of resveratrol on expression of these genes appeared to
scription. In a xenograft model, resveratrol delayed LNCaP tumor
growth and inhibited expression of a marker for steroid hormone
responses. However, exposure to resveratrol also led to increased
angiogenesis and inhibition of apoptosis in the xenograft. In sum-
mary, resveratrol may act through modulation of steroid hormone-
dependent pathways to inhibit prostate cancer cell growth in both
culture and xenografts, but exposure in vivo may be of concern.
Prostate cancer is the second leading causeof cancer death inAmerican
men (1). Although the etiology of prostate cancer remains unknown,
elevated levels of steroid hormones such as androgens and estrogens, as
well as growth factors such as insulin-like growth factor-1 (IGF-1), are
considered to be important risk factors (2–4). These hormones and
growth factors have been shown to promote proliferation of prostate
cancer cells through the activation of receptor-mediated signaling path-
ways (2–4). Therapeutic as well as preventive strategies have explored
modulation of these pathways as potential approaches to prevent or
control prostate cancer (5–7).
Bioactive food components, in particular, are increasingly being
evaluated as potential prostate cancer chemopreventive agents because
of their presumed safety (8). One such agent is resveratrol (9), a poly-
phenol (trans-3,5,4#-trihydroxystilbene) categorized as a phytoalexin
(10), found principally in the skin of grapes, but also in peanuts and
otherplantspecies(11).Red wine,oftenmentionedasa goodsourceof
resveratrol, contains 1–10 mg of resveratrol/l (4–40 lM) (12). Recent
studies attributed a variety of health benefits to consumption of foods
containing resveratrol, including protection against cancers, cardiovas-
cular disease and aging (13).
inhibit initiation, promotion and progression (14). Moreover, molecular
studies show that resveratrol possesses anticancer activities, including
acting as an antioxidant (15), possessing anti-inflammatory properties
(11,13) and functioning as a weak estrogen (16). In vitro experiments
using prostate cancer cell lines provide support for resveratrol to serve
as a candidate prostate cancer preventive agent. Resveratrol has been
showntoinhibit prostate cancer cell growthinculture (17,18), to inhibit
DNA synthesis (19) and to increase apoptosis in LNCaP cells, a human
prostate cancer cell line (20). It has been reported that resveratrol may
increase expression and serine phosphorylation levels of the tumor sup-
pressor protein p53 (21), thereby affecting activation of p53-dependent
signaling pathways, such as inhibition of cell cycle progression and
induction of apoptosis. Resveratrol has also been found to decrease
expression of prostate-specific antigen (PSA), an androgen-responsive
gene (ARG) that is often used as a marker for prostate cancer cell
growth (17). Moreover, a recent microarray study revealed that resver-
atrol may exert global effects on ARG expression in LNCaP cells (22).
ARGs such as PSA play important roles in cellular functions, including
cell cycle regulation, transcription, cell proliferation and differentiation,
as well as metabolism (23,24). We have shown previously that
estrogen as well as androgen can regulate ARG expression (25), sug-
gesting that the effects of resveratrol on ARGs may be through modu-
lation of steroid hormone-mediated pathways. Given the roles of
of these pathways may contribute to resveratrol’s protective effects
against prostate cancer. However, despite the in vitro work suggesting
that resveratrol shows promise as a prostate cancer chemopreventive
agent, the in vivo effects of resveratrol, as well as the mechanisms un-
derlying those effects on prostate cancer, remain largely unknown.
graft models to test the hypothesis that resveratrol is protective against
prostate cancer. We also test the hypothesis that resveratrol exerts its
ways.Wereportherethatresveratrolinvitro appeared to affect multiple
pathways that impact prostate cancer cell growth, and that the effects of
resveratrol are mediated in part by modulation of androgen receptor
(AR)- and estrogen receptor-dependent signaling pathways. In vivo,
resveratrol initially delayed tumor growth but was also found to
decrease tumor apoptosis and increase tumor angiogenesis.
Materials and methods
Resveratrol, 17b-estradiol, dihydrotestosterone and dimethylsulfoxide were
from Sigma Chemical Co. (St Louis, MO). The synthetic androgen R1881
was from NEN Life Science Products (Boston, MA).
Abbreviations: AR, androgen receptor; ARG, androgen-responsive gene;
B2M, beta-2-microglobulin; CDKN1A, cyclin-dependent kinase inhibitor
1A/p21WAF1/CIP1; CDS, charcoal–dextran-treated FBS; FBS, fetal bovine se-
rum; FU, fluorescence units; HPLC, high-performance liquid chromatography;
IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1
receptor; IHC, immunohistochemical; IS, Internal standard; mRNA, messen-
ger RNA; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear
antigen; PECAM-1, platelet/endothelial cell adhesion molecule-1; PSA, pros-
tate-specific antigen; RT–PCR, reverse transcription–polymerase chain reac-
tion; SEPP1, selenoprotein P, plasma, 1; STK39, serine threonine kinase 39;
VEGF, vascular endothelial growth factor.
? The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org 2001
Cells and cell culture
LNCaP human prostate cancer cells were obtained from the American Type
Culture Collection (Manassas, VA) and maintained in Media A [RPMI 1640
medium with phenol red (Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Sigma,
St. Louis, MO), 100 U/ml penicillin and 100 lg/ml streptomycin (BioSource
International, Camarillo, CA) with 10% fetal bovine serum (FBS) (Invitrogen)].
Cells were incubated in the presence of 5% CO2in air at 37?C.
Cell growth assay
LNCaP cells (5 ? 104cells per well) were plated in 24-well plates (Costar,
Corning Incorporated, Corning, NY); 24 h later, treatments were begun. Cells
were treated with 0, 1, 5, 10 or 25 lM resveratrol (dimethylsulfoxide as vehicle)
for 0–96 h, and the medium containing resveratrol was replaced every 24 h. Cell
growth was analyzed using the sulforhodamine B assay as described previously
(25). Forexperiments using the syntheticandrogenR1881 or 17b-estradiol, cells
were switchedtoMedia B[RPMI 1640mediumwithoutphenolred(Invitrogen),
2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 lg/ml streptomycin
with 10% charcoal–dextran-treated FBS (CDS; Hyclone, Logan, UT)] 24 h after
plating to minimize the effect of serum steroid hormones. The cells were then
incubated in Media B for an additional 24 h before the treatments were begun.
Microarray analysis of the effect of resveratrol on global gene expression in
previously (26). Briefly, LNCaP cells (2 ? 107cells per T-175 flask) were
exposed to 0, 1, 5 or 25 lM resveratrol for 48 h, total RNA was isolated and
microarray analysis was performed as described previously using the Affyme-
trix (Affymetrix, Santa Clara, CA) platform (26). The Affymetrix U133 chip
was used for gene expression analysis. Resveratrol-responsive genes were
identified using the MAS 5.0 suite (Affymetrix) following criteria of a 1.5-fold
increase and P , 0.01 as a cutoff as described previously (26). Triplicate
treatments were performed for each concentration. Microarray data are avail-
able upon request and also will be deposited in public database.
Determination of the effects of resveratrol on gene expression in LNCaP cells
using reverse transcription–polymerase chain reaction
LNCaP cells were plated in six-well plates (1 ? 106cells per well) in Media A
and switched to Media B containing 10% CDS 24 h after plating to minimize
the effect of serum hormones. Twenty-four hours later, the medium was re-
placed with fresh medium containing 1 nM R1881 or 17b-estradiol with or
without 25 lM resveratrol. Fresh medium containing the test compounds was
changed daily and cells were harvested for total RNA isolation using the Trizol
method (Invitrogen) after 48 h as described previously (25,26). Real-time re-
verse transcription–polymerase chain reaction (RT–PCR) was used to quantify
expression of the following genes as described previously (25,26): PSA; beta-
2-microglobulin (B2M); selenoprotein P, plasma, 1 (SEPP1); serine threonine
kinase 39 (STK39); cyclin-dependent kinase inhibitor 1A/p21WAF1/CIP1
(CDKN1A) and insulin-like growth factor-1 receptor (IGF-1R).
Western analysis of the effects of resveratrol on PSA expression in LNCaP cells
LNCaP cells were plated on 100 mm Falcon tissue culture plates (2 ? 106cells
per plate) in Media A. After 24 h, the medium was changed to Media B with
10% CDS, and the cells were incubated for an additional 24 h. After this
incubation, the cells were then treated with either 1 nM R1881 or 1 nM
17b-estradiol in the presence or absence of 25 lM resveratrol. Medium con-
tainingthe testcompoundswas replacedevery 24 h. Cells were treated with the
test compounds for a total of 96 h. Immunodetection of PSA was performed
following the protocol described previously (27).
AR binding assays
To assess the affinity of resveratrol for AR, the Androgen Receptor Competitor
Assay Kit, Green (Invitrogen), a fluorescence polarization AR binding assay,
was used according to the manufacturer’s protocol. Dihydrotestosterone
(0–1.5 lM) was used as a positive control. The resveratrol concentration range
used was 0–50 lM. Fluorescence polarization was detected using a TECAN
ULTRA fluorescence plate reader (Tecan Systems, San Jose, CA) set at 485 nm
excitation and 535 nm emission wavelengths. Results were calculated as percent
(no compound control FU ? with 1.5 lM dihydrotestosterone FU)] ? 100%.
Triplicate assays were performed and results expressed as percent control ± SD.
Effects of resveratrol on LNCaP cancer cell xenografts in athymic nude mice
To determine the prostate cancer protective effects of resveratrol in vivo, a nude
mouse xenograft model (28) was used. Five-week-old male athymic nude mice
(BALB/cAnNCr-nu/nu, 20–22 g; Charles River, Frederick, MD) were individu-
ally housed in filter-top cages at the Beltsville Human Nutrition Research Center
animal facility. Animals were randomly assigned to the following diet groups: (i)
control (AIN-93M) diet, (ii) AIN-93M supplemented with 50 mg resveratrol/kg
diet (RES50) or (iii) AIN-93M supplemented with 100 mg resveratrol/kg diet
(RES100). Pellet diets were prepared by Research Diets (New Brunswick, NJ).
The concentrations of resveratrol were selected based on published effective
doses for cancer prevention and those used in prior animal studies (13,14,29).
Twenty-two micewere used per treatment group. After 2 weeks of adaptation on
the diets, human LNCaP prostate tumors were established in the animals by
a subcutaneous injection of LNCaP cells (2 ? 106cells) resuspended in 50 ll
of phosphate-buffered saline (PBS) (pH 7.4) plus 50 ll of Matrigel (BD Bio-
of tumor volume began on the third week after injection. The cancer preventive
efficacy of resveratrol was assessed by twice-weekly measurements of tumor
volume, which was calculated using the equation: tumor volume (cm3) 5
0.523 ? [length (cm) ? width2(cm2)] (30). Mice were fed the diets described
above until 7 weeks after the injection of cells, at which time the animals were
was fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 5
lm sections for in situ immunohistochemical (IHC) analysis. Remaining tumor
samples were flash frozen under liquid nitrogen and stored at ?80?C for resver-
atrol analysis as described below. Plasma samples were collected and stored at
?80?C using the BDVacutainer? PPTTMPlasma Preparation Tube Procedure as
provided by the manufacturer (BD Biosciences, San Jose, CA).
IHC determination of steroid hormone-responsive pathway response,
proliferation, apoptosis and angiogenesis
IHC staining protocol for PSA PSA,aclassicARGresponsivetobothandrogen
and estrogen (25), was used to assess the effect of resveratrol on steroid hor-
ethanol. Endogenous peroxidase activity was blocked using 0.6% H202in meth-
anol. Slides were briefly rinsed in PBS prior to application of the serum block
(Rabbit Elite ABC Kit, Vector Laboratories, Burlingame, CA, Kit# PK-6101).
Slides were incubated with 250 lg/ml of rabbit polyclonal anti-Human PSA
(Dako, Carpinteria, CA, Ref. A0562, Lot 00012666, 3.0 g/l) in 0.1% bovine
serum albumin–PBS overnight at 4?C and rinsed in PBS; secondary antibody
(Rabbit Elite Kit) was then applied as directed. Slides were again rinsed in PBS,
ABC reagent (Rabbit Elite Kit) was applied, a final PBS wash was performed
and slides were stained with 3,3-diaminobenzidine tetrahydrochloride (Sigma–
Aldrich, St. Louis, MO, 10 mg of substrate/tablet). Slides were counterstained
with hematoxylin. Human prostate tissue was used as the positive control.
IHC staining protocol for proliferating cell nuclear antigen Proliferating cell
nuclear antigen (PCNA) was used as a proliferation marker (31). Freshly cut
paraffin-embedded sections were deparaffinized into ethanol. Endogenous per-
oxidase activity was blocked using 0.6% H2O2in methanol. Antigen retrieval
was performed by microwaving in deionized water for two 5 min cycles. Slides
were cooled for 20 min and rinsed in deionized water. Monoclonal mouse anti-
ml. Following a final PBS wash, slides were stained with 3,3-diaminobenzidine
tetrahydrochloride (Sigma–Aldrich, 10 mg of substrate/tablet). Slides were
counterstained with hematoxylin. Mouse small intestine tissue was used as a
IHC staining protocol for apoptosis Paraffin-embedded sections were depar-
affinized into PBS. An apoptosis detection kit using the terminal deoxynucleo-
tidyl transferase dUTP nick and labeling assay (32) was used (ApopTag
PeroxidaseIn Situ Apoptosis Detection Kit, Chemicon International, Temecula,
CA) according to the manufacturer’s instructions. Mouse testes tissue was used
as a positive control.
IHC staining protocol for platelet/endothelial cell adhesion molecule-1 Micro
(PECAM-1) staining (33). Paraffin-embedded sections were deparaffinized and
placedin water. Slides were postfixed in zinc fixative (BD Pharmigen, San Jose,
CA) for 10 min followed by a deionized water and PBS rinse. Endogenous
peroxidase activity was blocked using 0.6% H2O2in methanol. Antigen re-
trieval was performed by microwaving the slide for two 10 min cycles in
1 mM ethylenediaminetetraacetic acid buffer. Slides were cooled for 20 min
at room temperature, followed by a 30 min avidin–biotin block (Vector Avidin–
Biotin Blocking Kit). Slides were briefly rinsed in PBS prior to application of
the serum block (Goat Elite ABC Kit, Vector Laboratories, Kit# PK-6105).
Slides were incubated with 1 lg/ml of anti-PECAM-1 antibody (M-20, sc-
1506, goat polyclonal IgG, 200 lg/ml, Santa Cruz Biotechnologies, Santa Cruz,
CA) in 0.1% bovine serum albumin–PBS for 30 min at room temperature and
rinsed in PBS;secondaryantibody (GoatEliteKit) was then appliedas directed.
Slides were rinsed in PBS, ABC reagent (Goat Elite Kit) was applied, a final
PBSwashwasperformedandVectorNova Red(VectorNova RedSubstrateKit,
for Peroxidase, sk-4800) was applied. Slides were counterstained with hema-
toxylin. Mouse kidney and heart tissues were used as positive controls.
T.T.Y.Wang et al.
IHC staining protocol for vascular endothelial growth factor Paraffin-embedded
sections were deparaffinized into ethanol. Endogenous peroxidase activity was
blocked using 0.6% H2O2 in methanol. Antigen retrieval was performed
by microwaving for two 10 min cycles in citrate buffer. Slides were cooled for
20 min at room temperature, followed by a 20 min serum block (Goat Elite ABC
Kit,Vector Laboratories,Kit#PK-6105). Anti-mousevascular endothelial growth
factor (VEGF) antibody, P20 from Santa Cruz Biotechnologies (sc1836, lot
A1405, goat polyclonal IgG, 200 lg/ml), was used. Slides were incubated with
0.2 lg/ml of P20 in 0.1% bovine serum albumin–PBS for 60 min at room
temperature. Slides were rinsed in PBS (0.05% Tween); secondary antibody
(Goat Elite Kit) was then applied as directed. Slides were again rinsed in PBS
(0.05% Tween), ABC reagent (Goat Elite Kit) wasapplied, a final PBS wash was
performed and slides were stained with 3,3-diaminobenzidine tetrahydrochloride
(Sigma–Aldrich, 10 mg of substrate/tablet). Slides were counterstained with
hematoxylin. Mouse kidney tissue was used as a positive control. Five samples
measurement of VEGF expression.
Quantitation of IHC results using image analysis
All IHC slides, other than VEGF, were quantitated using the following pro-
tocol. The image was acquired using a Nikon DXM1200F Digital Camera
(Nikon Eclipse 80i Microscope, Nikon ACT-1 v2.70 software) and analyzed
using Image-Pro Plus v5.0 with custom-designed macros. Parameters for im-
age analysis were as follows: (i) All images were taken at a constant exposure
associated with a particular group of stains. (ii) Images were acquired pseu-
dorandomly with no images overlapping the same areas. (iii) All macros were
constructed from a random set of images from the associated stain to compen-
sate for variability within the group. (iv) All images were acquired at ?20
(?200 total magnification). (v) Ten areas were randomly taken for each slide,
and data are presented as an average. Special notes per stain are as follows. For
PSA analysis, no post-capture modification was done to the images in order to
preserve the color intensity of each sample. A 3-tier intensity scale was arbi-
trarily constructed to sort the varying degree of positive cells into groups. Data
supplied included raw area in millimeter square and percent composition of
each group of cells that make up the particular image and were expressed as
PSA expression indices. For PCNA analysis, all images were calibrated to
a master (single image) color profile to maximize accuracy. Total cells were
counted as well as total tissue area (excluding white space). Data were pre-
sented as cells per area of tissue in millimeter square and expressed as pro-
liferationindices. Forapoptosisanalysis, all images were calibrated to a master
(single image)color profile tomaximize accuracy.Apoptotic cells were analyzed
through contrast enhancement algorithms. Data were presented as raw apoptotic
were calibrated to a master (single image) color profile to maximize accuracy.
Vessels were analyzed through contrast enhancement algorithms. Non-specific/
background signal was filtered out via morphological algorithms. Data were pre-
sented as raw vessel counts and expressed as angiogenesis indices.
Determination of resveratrol content in mouse plasma and tumor samples
Plasma and tumor resveratrol concentrations were determined using liquid
chromatography–electrospray ionization–mass spectrometry following the
protocol described below.
Liquid chromatography–electrospray ionization–mass spectrometry system
and conditions The liquid chromatography–electrospray ionization–mass
spectrometry system used was a Shimadzu LCMS-2010 EV liquid chromato-
graph mass spectrometer system (Kyoto, Japan) connected to the LC portion
consisting of two LC-10AD pumps, a SIL-10AD VP auto injector, a SPD-10A
VP ultraviolet detector and a SCL-10AVP system controller. Data qualitation
and quantitation were accomplished using Shimadzu LCMS Solutions Version
3 software (Kyoto, Japan). The analytical column used was a Phenomenex
Luna C18(2) (150 ? 4.6 mm intradermally, 5 lm particle size). The mobile
phase consisted of (i) acetonitrile and (ii) 0.5% aqueous acetic acid (vol/vol),
filtered and degassed under reduced pressure prior to use. Separation was
achieved using gradient elutions of 18–31% A at 0–10 min and 31–48% A
at 10–20 min at a flow rate of 1.0 ml/min at ambient temperature (25 ± 1?C).
This was followed by a 5 min equilibration period with initial conditions prior
to injection of the next sample. Ultraviolet detection was set at 320 nm.
The mass spectrometer conditions consisted of a curved desolvation line
temperature of 200?C and a block temperature of 200?C. The curved desolva-
tion line, interface and detector voltages were ?20.0 V, 4.5 kV and 1.2 kV,
respectively. Vacuum was maintained by an Edwards?E2M30 rotary vacuum
pump (Edwards, UK). Liquid nitrogen (Washington State University Central
Stores, Pullman, WA) was used as a source of nebulizer gas (1.5 l/min).
Resveratrol and chlorogenic acid internal standard (IS) were qualitated in
selected ion monitoring negative mode. The monitored single plot transitions
were resveratrol at m/z 227 and chlorogenic acid at m/z 353.
Stock and working standard solution Methanolic stock solutions of resveratrol
(100 lg/ml) and the IS, chlorogenic acid (100 lg/ml), were prepared. These
solutions were protected from light and stored at ?20?C between uses, for no
longer than 3 months. Calibration standards in serum were prepared daily from
the stock solution of resveratrol by sequential dilution with blank rat serum,
yielding a series of concentrations, namely, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0
and 100.0 g/ml.
Sample preparation Plasma samples (0.1 ml) were aliquoted and 25 ll of
chlorogenic acid (IS) was added to each sample. The proteins present were
precipitated using 1 ml of ice-cold high-performance liquid chromatography
(HPLC)-grade acetonitrile, vortexed for 1 min (Vortex Genie-2, VWR Scien-
tific, West Chester, PA) and centrifuged at 5 000 r.p.m. for 5 min (Beckman
Microfuge centrifuge, Beckman Coulter, Fullerton, CA). The supernatants
were evaporated to dryness under compressed nitrogen gas. The residue was
reconstituted with 100 ll of mobile phase, vortexed for 1 min and centrifuged
at 5000 r.p.m. for 5 min; the supernatant was transferred to HPLC vials, and
25 ll of it was injected into the LC/MS system. Weighed tumors were rapidly
frozen in liquid nitrogen and pulverized to a fine powder with a mortar and
pestle under liquid nitrogen. To each sample, 25 ll of chlorogenic acid (IS) and
1 ml ice-cold HPLC-grade acetonitrile were added. Samples were vortexed for
1 min and centrifuged at 5000 r.p.m. for 5 min. The supernatants were evap-
orated to dryness under compressed nitrogen gas. The residue was reconsti-
tuted with 100 ll of mobile phase, vortexed for 1 min and centrifuged at 5000
r.p.m. for 5 min; the supernatant was transferred to HPLC vials, and 25 ll of it
was injected into the LC/MS system.
Forinvitro experiments,StatView (SASInstitute,Cary,NC) softwarewasused
for statistical analysis. Multiple group data were analyzed using analysis of
variance followed by post hoc analysis with Fisher’s PLSD test. The unpaired
Student’st-test wasused to compare experimentswithtwo groups.Values were
considered significant at P , 0.05. The animal tumor data for each treatment
group were analyzed using SAS? Proc MIXED (SAS Institute). Data mea-
sured on animals that did not survive the entire experiment were not included
in statistical analyses. A random coefficients model (34) was fit to the data to
model the linear relationships between tumor size and time after injection for
each treatment group and to make comparisons among rates of tumor growth
and average tumor size at specific times. Correlation among the weekly mea-
sured tumor sizes in each animal was modeled using a Toeplitz covariance
structure with heterogeneous variances to accurately represent the increasing
variation in tumor size relative to time (35). Only prespecified comparisons
for comparing treatment group means without risking inflation of the Type I
error (P 5 0.05).
Effects of resveratrol on LNCaP cell growth in vitro
As shown in Figure 1A, in vitro exposure of androgen-responsive
LNCaP human prostate cancer cells to medium containing 10%
FBS with various concentrations of resveratrol (0–25 lM), relative
to medium without resveratrol, resulted in concentration-dependent
growth inhibition. The inhibitory effects of resveratrol occurred at
concentrations as low as 5 lM (Figure 1A). As both androgen and
estrogen can contribute to cell growth in LNCaP cells cultured in 10%
FBS (25), we also examined the interactive effects of resveratrol,
androgen and estrogen on growth to elucidate the underlying mech-
anisms. LNCaP cells were cultured in 10% CDS with 1 nM synthetic
androgen R1881 or 17b-estradiol in the presence of 0, 1, 5 or 25 lM
cell growth induced by R1881 or 17b-estradiol in a concentration-
dependent manner. The effects of resveratrol occurred at concentra-
tions as low as 1 lM for androgen (Figure 1B) and as low as 5 lM
for 17b-estradiol (Figure 1C).
Microarray and RT–PCR analysis of the effects of resveratrol on gene
expression in LNCaP cells in vitro
To further elucidate the molecular mechanisms underlying the growth
inhibitory effects of resveratrol, we performed microarray analysis to
study the effects of resveratrol on global gene expression in LNCaP
cells. As shown in Figure 2A, ARGs appeared to be globally affected
by treatment with resveratrol. Expression of classic ARGs such as
Differential effects of resveratrol on androgen-responsive LNCaP human prostate cancer cells in vitro and in vivo
kallikrein 3 [commonly known as PSA (23,25)] was downregulated,
whereas androgen-downregulated genes such as BCHE (23,25) were
upregulated by resveratrol. In addition, several messenger RNAs
(mRNAs) coding for genes involved in the AKT-mediated pathway
(36,37) were also affected by exposure to resveratrol. These included
IGF-1R, PIK3R3, FRAP/mTOR and FOX3A (Figure 2A). Of these
genes, IGF-1R, PIK3R3 and FRAP/mTOR are known to be ARGs
(23,38). Selected ARGs were subjected to RT–PCR confirmation, and
consistent with the microarray results, expression of PSA and STK39
was upregulated in response to resveratrol, whereas no changes were
observed for B2M and SEPP1 (Figure 2B). Additionally, IGF-1R,
a receptor for IGF-1, was found to be downregulated by resveratrol
in microarray analysis (Figure 2A), which was confirmed by RT–PCR
(Figure 2C). Our microarray results also indicated that resveratrol
induced expression of CDKN1A. CDKN1A is a cyclin inhibitor reg-
ulated by p53, a tumor suppressor protein involved in cell cycle arrest
and apoptosis (39). Using RT–PCR, we found that in LNCaP cells
exposed to resveratrol for 48 h, CDKN1A mRNA increased signifi-
cantly after 24 h exposure to 25 lM resveratrol, but not at lower
resveratrol concentrations (Figure 2D).
Because resveratrol has been shown to be estrogenic but appears to
have low affinity for estrogen receptors (16), we also examined the
affinityofresveratrolforARsinLNCaPcells.AsshowninFigure 2E, in
a competition assay, resveratrol appeared to have little affinity for ARs,
as indicated by its inability to displace fluorescent ligand from ARs.
Since expression of selected ARG mRNAs, as with cell growth, is
subject to regulation by androgen and 17b-estradiol (25), the effects
of resveratrol on both androgen- and estrogen-mediated changes in
ARGs were further examined using RT–PCR. As shown in Figure 3A,
treatment of LNCaP cells with resveratrol effectively inhibited the
R1881-induced increase in several known ARGs: B2M, PSA, SEPP1,
STK39 and IGF-1R (23,25). Resveratrol also inhibited 17b-estradiol
induction of PSA, STK39 and IGF-1R (Figure 3B). Expression of
B2M and SEPP1 was not 17b-estradiol inducible. Consistent with
changes at the message level, the effect of resveratrol on the expres-
sion of the classic ARG, PSA, was also reflected at the protein ex-
pression level. As shown in Figure 3C by western analysis, 25 lM
resveratrol effectively inhibited the induction of PSA by 1 nM R1881
Effects of resveratrol on a LNCaP prostate cancer cell xenograft
Having established the in vitro effects of resveratrol, we sought to
extend the findings invivo using a LNCaP cell tumor xenograft model
(28,30).Resveratrolinthe diet didnotsignificantly affect bodyweight
of the animals (Figure 4A). As shown in Figure 4B, tumor volume
Effects of resveratrol on 17β-estradiol-induced
LNCaP cell growth.
Relative growth index
Relative growth index
Effects of resveratrol on R1881-induced
Relative growth index
Fig. 1. Effects of resveratrol on LNCaP cell growth in culture. (A) Concentration-dependent effects of resveratrol on LNCaP cells cultured in 10% FBS. LNCaP
cells were plated in 24-well plates; 24 h after plating, cells were treated with 0, 1, 5 or 25 lM resveratrol for 96 h and cell growth determined as described in
Materials and Methods. Results are expressed as mean ± SD (n 5 4). Bars with?are significantly different from vehicle control (0) at P , 0.05. (B) Effects of
resveratrol on R1881-induced cell growth. LNCaP cells were plated in 24-well plates; 24 h after plating, the medium was switched to Media B, which contains
10% CDS, for an additional 24 h. Cells were then treated with and without R1881 (1 nM) in the presence of 0, 1, 5 or 25 lM resveratrol for 96 h and cell growth
determined as described in Materials and Methods. Results are expressed as percent inhibition (mean ± SD, n 5 4). Bars with different letters are significantly
different from each other at P , 0.05. (C) Effects of resveratrol on 17b-estradiol-induced growth. LNCaP cells were seeded in 24-well plates; 24 h after plating,
medium was switched to Media B, which contains 10% CDS, for an additional 24 h. Cells were then treated with and without 17b-estradiol (1 nM) in the presence
of0, 1, 5 and 25 lM resveratrol for 96 h, and cell growthwas determined as describedin Materials and Methods. Results areexpressed aspercent inhibition (mean±
SD, n 5 4). Bars with different letters are significantly different from each other at P , 0.05.
T.T.Y.Wang et al.
Fig. 2. EffectsofresveratrolonARGexpressionandaffinityofresveratrolforAR.(A)MicroarrayanalysisofARGgene expression.LNCaPcellsculturedin10%FBS
were treated with 0, 1, 5 or 25 lM resveratrol for 48 h, and total RNA isolation and microarray analysis for ARG expression were performed as described in Materials
and Methods. Triplicate treatments and analyses were conducted for each concentration. Results are expressed as a heat map. (Red color: upregulated gene and green
color: downregulated gene.) Intensity of the color is proportional to the magnitude of effects on a log 2 scale, ?3 to þ3. (B) RT–PCR analysis of effects of resveratrol
on ARG expression.LNCaP cellscultured in 10% FBS were treated with or without resveratrol (25 lM) for 48 h,totalRNAwasisolated and mRNA levels of selected
ARGs (B2M, PSA, SEPP1 and STK39) were determined as described in Materials and Methods. Results are expressed as mean± SD (n 5 3). Bar with?is significantly
different from vehicle control at P , 0.05. (C) RT–PCR analysis of effects of resveratrol on IGF-1R expression. LNCaP cells cultured in 10% FBS were treated with
mean± SD (n 5 3).Bars with?are significantly different from vehicle control (0) atP , 0.05. (D) RT–PCR analysis ofeffects of resveratrol on CDKN1Aexpression.
LNCaP cells cultured in 10% FBS were treated with 0, 1, 5 or 25 lM resveratrol for 48 h, and total RNAwas isolated and mRNA levels of CDKN1A determined as
described in Materials and Methods. Results are expressed asmean± SD (n 5 3). Bar with?is significantly different from vehicle control (0) at P , 0.05. (E) Affinity
of resveratrol for AR. AR binding assays were performed asdescribedin Materials and Methods. Dihydrotestosterone (DHT) wasused as positive control. Results are
expressed as percent control ± SD (n 5 3). Points with?are significantly different from vehicle control (0) at P , 0.05.
Differential effects of resveratrol on androgen-responsive LNCaP human prostate cancer cells in vitro and in vivo
increased in all diet groups (Figure 4B). However, as shown in Figure
4C and D, there was a significant delay in tumor growth in animals
consuming the RES50 diet (week 3 after injection of LNCaP cells) or
RES100 diet (weeks 3 and 4). By the seventh week, there were no
differences in tumor volume. The rate of tumor growth was not sta-
tistically different across weeks 3–7 for control versus RES50 versus
RES100. By IHC analysis, expression of PSAwas significantly less in
tumors from mice fed resveratrol, and this effect was dose dependent
(Figure 5A). The expression of PSAwithin the tumors appeared to be
heterogeneous, with some cells containing more intense PSA staining
than others. The IHC analysis showed no differences between the treat-
mentsinoveralllevelsofPCNA,amarkerforproliferation (Figure 5B).
Interestingly, we observed significantly lower levels of apoptosis (as
assessed by ApopTag) in tumors from the RES50- and RES100-fed
animals compared with control (Figure 5C). Moreover, we also
observed an increase in microvessel formation in RES100-fed animals
as assessed by PECAM-1 staining (Figure 5D), suggesting an increase
in angiogenesis in this group. However, we did not observe quali-
tative differences in VEGF staining (data not shown) between the diet
Resveratrol concentrations in plasma and tumor samples were de-
termined. We found plasma resveratrol concentrations for animals fed
with resveratrol averaged 0.78 ± 0.0326 lM (mean ± SE, n 5 12) and
1.30 ± 0.336 lM (mean ± SE, n 5 12) for the RES50 and RES100
groups, respectively, with concentrations ranging from 0.7 lM to as
high as 5.25 lM was observed. Resveratrol was also detectable in
tumors, and the average concentrations were 0.33 ± 0.051 lM (mean ±
SE, n 5 12) and 0.33 ± 0.045 lM (mean ± SE, n 5 12) for the RES50
and RES100 groups, respectively, with concentrations as high as 0.7
lM in some tumor samples. We did not detect modified forms of
resveratrol metabolites using the described analytical method.
In the present study, we examined the chemopreventive effects and
mechanisms of action of resveratrol on the androgen-responsive hu-
man prostate cancer cell LNCaP in cell culture and in a xenograft
model. Our results suggest that resveratrol exerts differential effects in
these in vitro and in vivo models.
Resveratrol exerted growth inhibitory effects in our cell culture
model (Figure 1). This is consistent with observations for resveratrol
in similar in vitro models (17–22) and appeared to be due in part to
modulation of androgen- and estrogen-responsive pathways, as we
found that resveratrol inhibited both synthetic androgen R1881-
induced and 17b-estradiol-induced LNCaP cell growth (Figure 1B
and C). The actions of resveratrol on these two pathways were further
supported by our microarray and RT–PCR analyses of gene expres-
sion. Our microarray results indicated that resveratrol exerts a global
effect on ARG mRNA expression (Figure 2A and B), confirming
findings by Jones et al. (22). As in the cell growth experiment, resver-
atrol’s action on ARG mRNAs appeared to involve modulation of
androgen- and estrogen-responsive pathways. Resveratrol inhibited
both androgen and estrogen induction of ARG mRNA in LNCaP cells
(Figure 3A and B). The effect of resveratrol on ARG mRNAs corre-
lated with expression at the protein level, as we showed resveratrol
inhibited the R1881- and 17b-estradiol-induced increases in PSA pro-
tein levels (Figure 3C), similar to those reported by Hsieh et al. (40).
These molecular results are consistent with recent reports of in vitro
effects of resveratrol (41–43) and provide further support for modu-
lation of steroid hormone-dependent events as potential mechanisms
that contribute to the overall growth inhibitory effects of resveratrol
on LNCaP cells. Resveratrol is known to have weak estrogenic activ-
ity (16), but in our experiments resveratrol appeared to act mainly as
an antiestrogen, as resveratrol inhibited both 17b-estradiol-induced
growth and increases in ARG mRNA levels. Interestingly, our binding
study result (Figure 2E), as well as that reported by others (16),
suggests that resveratrol may not exerts its actions on androgen-
and estrogen-mediated effects through direct competition of binding
Fig. 3. Effects of resveratrol on R1881- and 17b-estradiol-induced ARG
mRNA and protein levels. (A) Effects of resveratrol on the R1881-induced
increase in ARG mRNA levels. LNCaP cells were plated in six-well plates;
24 h after plating, the mediumwas switched to Media B, which contains 10%
CDS, for an additional 24 h. Cells were then treated with or without R1881
(1 nM) in the presence or absence of resveratrol (25 lM) for 48 h, total RNA
was isolated and mRNA levels of selected ARGs (B2M, PSA, SEPP1 and
STK39) were determined as described in Materials and Methods. Results are
expressed as mean ± SD (n 5 3). Bars with different letters are significantly
different from each other at P , 0.05. (B) Effects of resveratrol on the
17b-estradiol-induced increase in ARG mRNA levels. LNCaP cells were
plated in six-well plates; 24 h after plating, the medium was switched to
Media B, which contains 10% CDS, for an additional 24 h. Cells were then
treated with or without 17b-estradiol (1 nM) in the presence or absence of
resveratrol (25 lM) for 48 h, total RNA was isolated and mRNA levels of
selected ARGs (PSA and STK39) were determined as described in Materials
and Methods. Results are expressed as mean ± SD (n 5 3). Bars with
different letters are significantly different from each other at P , 0.05. (C)
Effects of resveratrol on PSA protein levels. LNCaP cells were plated in 100
mm plates; 24 h after plating, the medium was switched to Media B, which
contains 10% CDS, for an additional 24 h. Cells were then treated with or
without R1881 or 17b-estradiol (1 nM) in the presence or absence of
resveratrol (25 lM) for 48 h, protein was isolated and immunodetection of
PSA was performed as described in Materials and Methods.
T.T.Y.Wang et al.
of steroid hormones to their receptors. Additional research is needed
to elucidate the precise mechanisms of action.
Data presented here also show a novel effect of resveratrol on
expression of steroid hormone-regulated genes in the LNCaP model.
Resveratrol selectively reduced PSA, STK39 and IGF-1R (Figure 2B
and C) mRNAs in cells cultured in 10% FBS, but not expression of
two other ARGs, B2M and SEPP1 (Figure 2B). We reason that the
presence of estrogen-like activity in cell culture medium may explain
of effects on expression of B2M and SEPP1 in the presence of exter-
nally added 17b-estradiol but not in the presence of the synthetic an-
drogen R1881 in cells cultured in medium that is free of phenol red,
greatly affect the interpretation of resveratrol’s biological activities.
In addition to the steroid hormone-responsive pathways, modula-
tion of p53- and IGF-1-dependent events by resveratrol was also
observed, as we found by microarray and confirmed by RT–PCR that
resveratrol modulated both CDKN1A and IGF-1R mRNA levels.
However, activation of the p53 pathway appeared to require a higher
concentration (25 lM), whereas regulation of the steroid hormone and
IGF-1 pathways appeared to have a lower threshold (1–5 lM). Thus,
resveratrol may exert effects on multiple pathways in a concentration-
dependent fashion. Given that, we measured circulating levels of
resveratrol no higher than ?5 lM in the plasma of our animals, it is
probably that modulation of steroid hormone–IGF-1-mediated events
may be the more physiologically relevant mechanisms.
As illustrated in Figure 2A, we observed that exposure to resvera-
trol can lead to downregulation of IGF-1R and FRAP1–mTOR
mRNAs but to upregulation of PIK3R3 mRNA. All three genes have
been reported to be upstream regulators of AKT activity (36–38).
Activation of IGF-1R and FRAP/mTOR upregulates AKT activity
(36,37), whereas PIK3R3 downregulates AKT activity (37). Modula-
tion of IGF-1R, PIK3R3 and FRAP1–mTOR by resveratrol as re-
ported here is consistent with recent reports of inhibitory effects of
resveratrol on AKT-mediated pathways (41–43). These results and
our findings on modulation of androgen- and estrogen-mediated ex-
pression by resveratrol (Figure 3A and B) lend further support for
a recent report on modulation of androgen- and estrogen-mediated
activation of AKT by resveratrol (41). More importantly, we provide
evidence that additional upstream molecular targets of AKT pathways
such as IGF-1R, FRAP/mTOR and PIK3R3 are also modulated by
resveratrol through androgen–estrogen-related events.
Our IHC results on expression of PSA, an ARG–estrogen-
responsive gene, in xenograft tumors confirm the cell culture results
and support our hypothesis that modulation of androgen- and estro-
gen-mediated pathways may contribute to resveratrol’s prostate
Week 3 tumor volume.
Tumor volume (mm3)
Tumor volume (mm3)
Tumor volume (mm3)
Body weight (g)
Week 4 tumor volume.
Fig. 4. Effects of resveratrol on LNCaP cell-derived tumor growth in athymic nude mice. LNCaP cell tumor xenografts were established in athymic nude mice
(n 5 22 perdiet group) as described in Materialsand Methods.Tumor volumes were measured twice a weekand calculatedas described in Materials and Methods.
The linear coefficients and standard errors from the random effects model are shown in the graph. (A) Body weights of control, RES50 and RES100 groups during
the study. (B) Temporal effects on tumor growth in animals fed control (filled circles), RES50 (open circles) or RES100 (filled triangles) diets. (C) Comparison of
tumor volumes between animals fed control, RES50 or RES100 diet in week 3. Week 3 results from Panel B were regraphed, and?indicates significantly different
from control at P , 0.05. (D) Comparison of tumor volumes between animals fed control, RES50 or RES100 diets in week 4. Week 4 results from Panel B were
regraphed, and?indicates significantly different from control at P , 0.05.
Differential effects of resveratrol on androgen-responsive LNCaP human prostate cancer cells in vitro and in vivo
Fig. 5. IHC analysis of markers for the steroid hormone-responsive pathway, proliferation, apoptosis and angiogenesis. IHC analyses for the steroid hormone-
responsive gene PSA, the proliferation marker PCNA, apoptosis and the angiogenesis marker PECAM-1 were performed on paraffin-embedded tumor samples as
described in Materials and Methods. (A) PSA. PSA expression index 5 average PSA-positive cells/mm2(B) PCNA. Proliferation index 5 average PCNA-positive
cells/mm2(C) ApopTag. Apoptosis index 5 average apoptotic cells per field (D) PECAM-1. Angiogenesis index 5 average vessel counts per field?indicates
significantly different from control at P , 0.05.
T.T.Y.Wang et al.
cancer preventive effect in vivo. Tumors from animals fed RES50 and
RES100 diets had lower numbers of cells expressing PSA, corre-
sponding to the observed delay in tumor growth in animals consuming
these two diets. The effects on tumor growth and PSA protein expres-
sion appeared to be dose dependent (Figure 4A and Figure 5A). How-
ever, we also observed that not all cells within a tumor expressed
similar levels of PSA. One possible explanation is that tumor cells
in the xenograft may differ from the parent LNCaP cells, as LNCaP
cells are known to undergo changes with different passages (44).
Interestingly, our animal experiment results are only partially corre-
lated with the cell culture results. The lag in growth of tumor volume
for animals consuming resveratrol appeared to ‘catch up’ to control
animals by the seventh week (Figure 4A). Hence, modulation of the
steroid hormone-dependent pathways may afford only partial protec-
tion against tumor development. Our IHC data on apoptosis and an-
giogenesis appear to suggest that resveratrol may not be entirely
beneficial against tumor development. Tumors from animals fed re-
sveratrol appeared to have lower apoptosis frequencies than animals
on control diet. Moreover, tumors from the animals fed the higher
dose of resveratrol (100 mg resveratrol/kg diet) also had significantly
higher blood vessel counts, suggesting increased angiogenesis in the
RES100 group compared with the control diet group. This effect of
resveratrol on angiogenesis appeared notto be related toproduction of
VEGF by tumor cells, as we did not observe qualitative differences in
VEGF levels using IHC analysis.
The amount of resveratrol ingested by the animals per day (3–6 mg/
day) in the present experiment is equivalent to consumption of 500–
1000 ml of red wine per day and is readily achievable in humans (45).
By body surface area calculations (46), our experimental animals
consumed a dose of 12–24 mg/m2of resveratrol and appeared to be
at the lower end of pharmacological doses used in a recent human trial
(47). However, our dose was ?10? lower than that used in a recent
study with TRAMP mice (48). Interestingly, the plasma levels in our
animals (1.3 ± 0.336 lM, RES100) were higher than those in Harper’s
study [52 ± 18 nM, (48)]. Hence, additional work would be necessary
to extrapolate our results, as well as those from other animal models,
to human populations.
In summary, we report here that resveratrol differentially affects
in vivo and in vitro models of prostate cancer. In vitro, resveratrol
appeared to exert growth inhibitory effects on cultured LNCaP cells
through multiple pathways, including steroid hormone-dependent
pathways. In vivo, resveratrol delayed the initial development of
xenograft LNCaP cell tumors, consistent with an effect on steroid
hormone-mediated events. However, exposure to resveratrol appeared
to lead to promotion of angiogenesis and inhibition of apoptosis in
LNCaP cell-derived tumors, as assessed by IHC markers.
USA appropriated funds to United States Department of Agriculture
project number 1235-51530-052-00 to T.T.Y.W., T.-C. W and B.V.;
National Cancer Institute to T.S.H., Y.S.K. and H.S.; National Insti-
tute of Environmental Health Sciences to S.D.H. and S.N.P.
T.S.H. is a National Cancer Institute cancer prevention fellow. The authors
wouldliketo thankDr Diana Haines, MaureenKennedyand ScottLawrence of
the Pathology/Histotechnology Laboratory, SAIC-Frederick, for their assis-
tance in IHC analysis. The authors would also like to thank Drs Chang-hee
Kim of SAIC-Frederick and Gadisetti V.R.Chandramouli of the National
Cancer Institute for their assistance in microarray analysis.
Conflict of Interest Statement: None declared.
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Received October 18, 2007; revised May 29, 2008; accepted May 29, 2008
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