Publication of the International Union Against Cancer
Int. J. Cancer: 104, 204 –212 (2003)
© 2003 Wiley-Liss, Inc.
DIFFERENTIAL EXPRESSION OF GENES INDUCED BY RESVERATROL IN
LNCAP CELLS: P53-MEDIATED MOLECULAR TARGETS
Bhagavathi A. NARAYANAN1,2*, Narayanan K. NARAYANAN2, Gian G. RE3 and Daniel W. NIXON4
1
Division of Nutritional Carcinogenesis, American Health Foundation, Valhalla, NY, USA
2
Microarray Unit, Molecular Pathology and Bioinformatics, American Health Foundation, Valhalla, NY, USA
3
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA
4
American Health Foundation, Valhalla, NY, USA
Prostate cancer prevention by key elements present in
human nutrients derived from plants and fruits has been
confirmed in various cell cultures and tumor models. Resveratrol (RE), a phytoalexin, induces remarkable inhibitory
effects in prostate carcinogenesis via diverse cellular mechanisms associated with tumor initiation, promotion and progression. Earlier studies have shown that RE alters the expression of genes involved in cell cycle regulation and
apoptosis, including cyclins, cdks, p53 and cdk inhibitors.
However, most of the p53-controlled effects related to the
role of RE in transcription either by activation or repression
of a sizable number of primary and secondary target genes
have not been investigated. Our study examined whether RE
activates a cascade of p53-directed genes that are involved in
apoptosis mechanism(s) or whether it modifies the androgen
receptor and its co-activators directly or indirectly and induces cell growth inhibition. We demonstrate by DNA microarray, RT-PCR, Western blot and immunofluorescence
analyses that treatment of androgen-sensitive prostate cancer cells (LNCaP) with 10-5 M RE for 48 hr downregulates
prostate-specific antigen (PSA), AR co-activator ARA 24 and
NF-kB p65. Altered expression of these genes is associated
with an activation of p53-responsive genes such as p53, PIG 7,
p21Waf1-Cip1, p300/CBP and Apaf-1. The effect of RE on p300/
CBP plays a central role in its cancer preventive mechanisms
in LNCaP cells. Our results implicate activation of more than
one set of functionally related molecular targets. At this
point we have identified some of the key molecular targets
associated with AR and p53 target genes. These findings
point to the need for further extensive studies on AR coactivators, such as p300, its central role in post-translational
modifications such as acetylation of p53 and/or AR by RE in a
time- and dose-dependent manner at different stages of prostate cancer that will fully elucidate the role of RE as a chemopreventive agent for prostate cancer in humans.
© 2003 Wiley-Liss, Inc.
Key words: resveratrol; cDNA microarray; gene expression; RTPCR; p300/CBP; p53 signaling; phenolic-antioxidant
Phenolic antioxidants attract increasing attention for their potential as cancer chemopreventive agents.1–10 Resveratrol (RE), a
phytoalexin present in grapes and berries, is one of the most
promising agents for prostate cancer prevention.11–18 RE has antioxidant and antimutagenic activities that induce phase II drugmetabolizing enzymes; it mediates anti-inflammatory effects and
inhibits cyclooxygenase and hydroperoxidase functions.19 –22 In
vitro research and preclinical studies strongly support the anticancer effects of RE. RE induces cell cycle arrest, inhibits DNA
synthesis and induces apoptosis in prostate cancer cells and thus
holds great promise for development as a chemopreventive agent
for prostate cancer.17,23–30 RE is strongly linked with androgen
receptor regulation. Recent studies by Mitchell et al.23 and Hsieh
et al.31 have shown that RE represses several classes of androgenupregulated genes at the mRNA level in LNCaP cells. Our earlier
studies with RE indicated an inhibition of the AR co-activator
ARA-24 in LNCaP cells consistent with previous reports.15 Although these cited studies point to the importance of RE as a
potential chemopreventive agent, the effect of RE on transcriptional activation or repression of primary and secondary targets of
p53 and AR in prostate cancer has not been investigated.
Numerous studies provide evidence for the anticarcinogenic
activity of RE,17,19,23,24,32– 45 but the precise mechanisms involved
in the modulation of oncogenic precursors of prostate carcinogenesis remain to be elucidated. Earlier studies indicated that the p53
protein is stabilized and activated after exposure of mammalian
cells to DNA-damaging agents.46 However, it is not known
whether p53 activation by RE follows the same pathway as that
initiated by other agents that induce G1 arrest and apoptosis. There
are indications that the nature of p53 response depends on the
levels of the p53 protein, the type of inducing agent and the cell
type employed. Significant cellular death was observed only when
several of the genes controlled by p53 were expressed in concert,
suggesting that p53 needs to activate parallel apoptotic pathways
to induce programmed cell death.47,48
To delineate the complete cascade of molecular events in response to RE treatment of prostate cancer requires a comprehensive study on gene expression at the transcription level. In our
study, we employed human cDNA microarray analysis to obtain a
genetic profiling of p53-targeted genes. We focused on determining whether RE activates a cascade of p53-directed genes and
transcription factors that are involved in apoptosis mechanism(s)
in prostate cancer cells. In addition, we examined the p53-activated apoptotic pathway in conjunction with the modification of
acetyltransferase p300 and caspase activator Apaf-1 that is induced
by RE in the androgen-sensitive prostate cancer cell line (LNCaP).
MATERIAL AND METHODS
Chemical
RE obtained from CALBIOCHEM (La Jolla, CA) was purified
by crystallization from ethanol until a purity of 99% was demonstrated in high performance liquid chromatography (HPLC) as
described by Palomino et al.49 The stock solution of RE (10-3 M)
was prepared in DMSO. After the HPLC purification, aliquots of
RE were protected from light and stored at ⫺20°C until use.
Abbreviations: Apaf-1, apoptotic protease activating factor-1; AR, androgen receptor; ARA 24, androgen receptor-associated-protein 24; ASP,
apoptosis-specific protein; DAPI, 4⬘,6-diamidine-2-phenylindole dihydrochloride; PSA, prostate-specific antigen; RE, resveratrol.
Grant sponsor: NCI Cancer Center; Grant number: CA-17613; Grant
sponsor: AICR; Grant number: 01A015.
*Correspondence to: Division of Nutritional Carcinogenesis, and Microarray Unit, Molecular Pathology and Bioinformatics, 1 Dana Road,
Valhalla, NY 10595, USA. Fax: ⫹914-592-6317;
E-mail: [email protected]
Received 10 June 2002; Revised 6 September 2002; Accepted 21 October 2002
DOI 10.1002/ijc.10932
RESVERATROL-INDUCED APOPTOSIS AND MOLECULAR TARGETS
Cell culture and treatment
Prostate cancer cells (LNCaP) were grown at 37°C in RPMI,
GIBCO brand (Invitrogen Life Technologies, Carlsbad, CA) supplemented with L-glutamine, 7.5% fetal bovine serum (FBS) and
antibiotics in a water-saturated atmosphere of 5% CO2. To determine the effect of RE over time, 75% confluent cells were treated
with 10-5 M and observed for 24 hr and 48 hr. This concentration
at these time points were selected on the basis of our earlier
observations that showed irreversible cell growth inhibition associated with apoptosis in a nontoxic manner. In our study,
trypsinized and PBS-washed cells were counted with a Coulter
particle counter to quantify cell growth in response to RE. Cells
harvested from a parallel set of experiments were used for cell
cycle analysis by means of flow cytometry and RNA extractions
for gene expression analysis. We used DMSO as the solvent
control appropriately in our experiments to compare the efficacy
and gene expression altered after RE treatment.
Detection of apoptotic cells and DNA fragmentation analysis
Apoptosis induced by RE (10-5 M) over 24 hr and 48 hr in
treated LNCaP cells was localized with DAPI staining. Cells
showing fragmented and condensed nuclear material characteristic
of apoptosis were localized at 40⫻ magnifications under a research
grade fluorescence microscope (AX70-Olympus). The percentage
of apoptotic cells with characteristic morphologic changes was
determined from 3 identical experiments with RE and compared to
that in control experiments. To further confirm the induction of
apoptosis, a DNA fragmentation analysis was conducted simultaneously by extracting DNA from a similar set of experiments at
different time periods as described by us earlier.13,15
Flow cytometric analysis
LNCaP cells treated with 10-5 M of RE or with control were
harvested at 24 hr and 48 hr time periods, washed in PBS and fixed
in 1% formaldehyde for 15 min on ice. After rewashing with PBS,
the cells were fixed in 80% ethanol for 30 min. Cell suspensions
having approximately 3 ⫻ 106 cells were centrifuged and the
pellets of cells were resuspended with PBS and further treated with
1 mg/mL of RNase at room temperature for 30 min. Propidium
iodide (1 mg/mL, final concentration in PBS) was added and flow
cytometric analysis was performed after 30 min. For cell cycle
analysis, we employed Epics XL MCL (Phoenix Flow Systems;
San Diego, CA).
Immunofluorescence detection of p300
LNCaP cells grown in 35 mm cell culture dishes were treated
with 10-5 M of RE for 48 hr. Cells washed with 1⫻ PBS were then
fixed in formalin for 15 min before incubation with appropriate
concentrations of anti-p300 antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) for 1 hr. After washing with PBS twice, cells
were again incubated with FITC-conjugated goat anti-rabbit IgG
(Jackson ImmunoResearch Laboratories, West Grove, PA) for 45
min, followed by a final wash with PBS and mounted in DAPI/
Antifade (ONCOR, Gaithersburg, MD). Fluorescence signals for
p300-positive cells were visualized under 40⫻ magnification using
the fluorescence microscope (AX-70 Olympus). The percentages
of p300-positive cells determined from 3 identical experiments
treated with RE were compared to control experiments.
cDNA microarray analysis
Human cDNA microarrays purchased from Perkin Elmer-NEN
Life Sciences (Boston, MA) spotted with 2,400 genes per array
(known genes and ESTs), sequence-verified by Alpha Gene, were
used for gene expression profiling. More than 60% of the genes are
full-length copies of known genes, which include two types of
control genes, a complement of 3 nonmammalian (plant) genes and
35 housekeeping genes (human).
Total RNA was isolated from RE-treated and control LNCaP
cells using Trizol reagent and Qiagen columns (Invitrogen Life
Technologies and Qiagen, Valencia, CA, respectively). RNA from
205
control cells was labeled with cyanine cy3-dUTP and RNA from
RE-treated cells was labeled with cy5-dUTP. Briefly, an aliquot of
RNA (20 ␮g) for each labeling was precipitated with 2.5 volumes
of cold ethanol and 0.1 volume of 3 M sodium acetate. The
precipitated RNA was then harvested by centrifugation and resuspended in 17 ␮l of DEPC (diethylpyrocarbonate)-treated water.
This was added to 2 ␮l of dNTP primer mix (PE/NEN Life
Sciences). The reaction mixture was kept at 65°C for 10 min to
denature the nucleic acid, then cooled to 25°C for 5 min. It was
then reacted with 2.4 ␮l of 10⫻ RT buffer, 2 ␮l of AMV RT/
RNase inhibitor mix, 4 ␮l of cy3-dUTP and 2 ␮l of cy5-dUTP
(PE/NEN Life Sciences) for the reverse transcription reaction that
was carried out for 1 hr at 42°C. The probes were cooled down to
4°C for 10 min, and 2.5 ␮l of 0.5 M EDTA as well as 2.5 ␮l of 1
N NaOH were added before incubation at 65°C for another 30 min.
After cooling the above mixture to 4°C for 5 min, 6.5 ␮l of 1 M
Tris-HCl was added. The cDNA was purified by isopropanol
precipitation or by using a spin column. The dried pellet was
resuspended in 13 ␮l of RNase-free water; the cy3-dUTP-labeled
control and cy5-dUTP-labeled products were combined and 10 ␮g
of carrier DNA was added. After adding 2 ␮l of 5 M ammonium
acetate and 50 ␮l of isopropanol, the probe mixture was pelleted at
14,000 rpm for 10 min at 4°C. The pellets were washed again with
70% and 95% ethanol respectively and were stored at ⫺20°C for
further hybridization.
The microarray to the probe was hybridized according to manufacturer’s instructions (PE/NEN Life Sciences) with appropriate
modifications as described in our earlier study.15 The mixed probe
pellet was resuspended in 25 ␮l of hybridization buffer and immediately added to human cDNA arrays: a 22 ⫻ 22 cover slip was
placed carefully on the arrays that were then transferred into
hybridization cassettes especially made for submerging the arrays
in a 65°C water bath for 12 hr. After hybridization, the slides were
washed with microarray wash buffer (0.5 ⫻ SSC, 0.01% SDS)
until the cover slip fell off. The slides were washed twice more,
once with 0.5 ⫻ SSC, 0.01% SDS and again with 0.06 ⫻ SSC,
0.01% SDS for 15 min. A final wash of the slides were with 0.06 ⫻
SSC for 15 min and the cleaned slides were spin-dried before
scanning.
Scanning and data analysis
The Axon GenePix 4000B scanner, a nonconfocal scanning
instrument containing 2 lasers that excite cyanine dyes at 635 nm
for Cy5, and 532 nm for Cy3, respectively, and high-resolution (10
␮m pixel size) photo multiplier tubes that detect fluorochrome
emission, were used for scanning the hybridized cDNA microarrays (Axon Instruments, Foster City, CA). GenPix Pro software
was used to analyze the images and to extract the data sets into a
Microsoft Excel spreadsheet. The data sets consist of signal and
background intensity, standard deviation of signal and background
intensity and ratio of median and/or mean of total intensity, including flags. A macroprogram in Microsoft Excel enabled normalization; we used the intensity of each spot for variations in the
overall intensity of the image with respect to control image to
perform normalization. A GeneSpring Bioinformatics software
package was used for data analysis (Silicon Genetics, Redwood
City, CA) as described earlier.15
Validation of cDNA microarray results
To verify the gene expression pattern, experiments were repeated with 3 similar sets of arrays containing similar sets of genes
and prosite motifs. The level of expression pattern was compared
to similar spots present in the respective quadrants in all slides
simultaneously; 87% of the spots showed similarity in their expression in all arrays that were hybridized with similar samples.
High- and low-expressed genes were confirmed with RT-PCR
using sequence-specific primers. RT-PCR conditions and a detailed protocol are described elsewhere.15
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NARAYANAN ET AL.
Western blot analysis
LNCaP cells treated with RE (10-5 M) for 48 hr were harvested
by trypsinization. Cellular protein was extracted and Western
blotting for p53 and p300/CBP protein was carried out using
mouse monoclonal antibody for p53 (DO-1) and p300/CBP purchased from Santa Cruz Biotechnology. The reactive protein bands
were developed with chemiluminescent detection reagents (ECL
Kit, Amersham Biosciences, Piscataway, NJ).
RESULTS
RE-induced G1 arrest and apoptosis
The anticancer effect of RE was studied on exponentially growing LNCaP cells that were treated with the agent (10-5 M) for 24
hr and 48 hr periods. RE-induced cell death via apoptosis was
examined by DAPI staining (Fig. 1a) and DNA fragmentation
analysis (Fig. 1b). We demonstrated by DAPI staining a remarkable decrease in cell growth inhibition by RE via apoptotic cell
death after 48 hr. Cell death analysis indicated 30% apoptotic cells
(Fig. 1c) with characteristic morphologic and nuclear material
changes.
Cell cycle analysis showed a minor G1 arrest after 24 hr as
determined by flow cytometry (Fig. 2). Figure 2a and b indicate
that there was a pronounced G1 (84%) peak after 48 hr with far
fewer cells in the S phase (8%). The reduced number of cells in the
S phase in RE-treated cells may indicate a possible cell cycle arrest
at the G2/M phase. Control cells did not exhibit much of an effect
on the G1 fraction; on the other hand, it exhibited a higher DNA
content with 26.0% of cells in the G2/M phase after 24 hr and
31.6% after 48 hr (Fig. 2c,d), indicating a 3- to 4-fold increase in
the cells in G2/M phase.
FIGURE 1 – DAPI staining and localization of apoptotic cells after 48 hr treatment with RE compared to control cells. (a) LNCaP cells
exhibiting fragmented and condensed nuclei, characteristic of apoptosis. Original magnification 40⫻. (b) DNA fragmentation analysis: Agarose
gel (1.8%) shows the laddering of DNA fragmentation after 24 hr and 48 hr. Lane 1, molecular marker; lane 2, control; lanes 3 and 4, RE treated.
(c) Quantification of apoptotic cells at 24 hr and 48 hr (as described in Material and Methods).
RESVERATROL-INDUCED APOPTOSIS AND MOLECULAR TARGETS
207
total of 2,400 genes. To get reproducible results, the experiments
were repeated with the same source of RNA from LNCaP cells
treated with RE and were compared with control cells. Genes
expressed were confirmed with RT-PCR (Fig. 4), using sequencespecific primers designed for selected genes.
The results presented here are the averages from triplicate
experiments. A list of differentially expressed genes shows at least
a moderate level of expression, varying more than 2-fold between
the control and RE-treated cells (Table I). A 2-fold difference in
the gene expression was preferred because (i) the signal intensities
of nonhomologous plant genes as internal controls present in the
array were used; (ii) with respect to the intensity ratio of plant
genes in our analysis, we decided to keep the signal:background
ratio as 1.0. This ratio was used to normalize the entire array so
that all the slides could be compared directly for a 2-fold increase.
Although many of our microarray data analyses were performed
with a cutoff for 5-fold increase as preferred by other investigators,50 in our study we aimed to separate the genes of interest; for
instance, with respect to p53-responsive genes, a 2-fold increase
permitted us to identify a large number of transcription factors
related to their activation. Although a 5-fold increase is a highly
significant signal for the expressed genes, a 2-fold increase is an
acceptable level of difference in expression as indicated by several
earlier studies51,52 within a 95% confidence interval as in our
present study.
FIGURE 2 – Flow cytometry analysis with propidium iodide for DNA
content as described in Material and Methods indicates the effect of
RE (10-5 M) on LNCaP cells inducing G1 arrest after 24 hr and 48 hr.
(a) Control after 24 hr; (b) RE-treated cells exhibiting G1 peak after 24
hr; (c) control after 48 hr; (d) RE-treated cells exhibiting a pronounced
G1 peak after 48 hr.
Effect of RE on gene expression profile
Measurements on the expression levels of differentially expressed genes are presented in Figure 3 as a simple bivariate
scatter plot. Upregulated genes amounted to 5.25% and downregulated genes accounted for 17.88% of all expressed genes out of a
Differential expression of primary and secondary targets of p53
genes
Given the significant role that the tumor suppressor gene p53
appears to play in human cancer, we analyzed whether RE activates more than a few known p53 targets or whether there may be
expression of other gene transcripts that contributes to p53-mediated apoptosis. Surprisingly, this approach revealed for the first
time the differential expression of more than 34 transcripts that are
either primary or secondary targets of p53 and are either activated
or repressed by RE (Table I). Sixteen of these 34 transcripts,
including the programmed cell death factor 2 (PDCD-2), p300,
Apaf-1, CPP32, PIG 7 and PIG 8, BAK protein, p57 (Kip2) and an
isoform of zinc finger protein 135, that were expressed at significantly higher levels (increased ⬎ 2-fold, p ⬍ 0.01) are involved
in apoptosis. Likewise, a significant fraction of genes that are
involved in DNA damage, cell cycle, oxidative stress and microtubule-disturbing activity were downregulated.
RE-induced apoptotic genes
RE has been shown to induce apoptosis and to inhibit the
growth of certain cancer cells in vitro. In our study, using
LNCaP cells, we demonstrated activation of several pro-apoptotic genes that are listed in Table I. The results presented here
are consistent with the activation of p53 target genes that are
FIGURE 3 – Scatter plot view showing the distribution of differentially expressed genes after 48 hr of resveratrol treatment (10-5 M) in
LNCaP cells. Among the differentially expressed genes, upregulated
(⬎2-fold) genes (5.25%) are shown above the median diagonal line
and downregulated (⬍2-fold) genes (17.8%) are shown below the
median diagonal line.
FIGURE 4 – Validation of microarray data by RT-PCR. Agarose gel
(2.5%) stained with ethidium bromide showing the amplified RT-PCR
product with equal amount of RNA (2 ␮g). GAPDH was used as the
internal control. Total RNA isolated from RE treated (10-5 M) for 48
hr and control cells were used for RT-PCR reactions using sequencespecific primers as described in Material and Methods. ⫹, treated; ⫺,
control.
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TABLE I – DIFFERENTIALLY EXPRESSED GENES IN LNCAP CELLS EXPOSED TO RESVERATROL RELATED TO P53-MEDIATED MOLECULAR TARGETS
Accession no.
Gene description
Mean Cy5/Cy3
ratio
U01877
AF013263
p300 protein1
Apoptotic protease activating factor 1 (Apaf-1); cytochrome c-dependent
activation of caspase-31
Transcription factor TFIIE beta
Receptor protein-tyrosine kinase (HEK8)
Insulin-like growth factor binding protein 2 (IGFBP2)
Seryl-tRNA synthetase
Protein tyrosine phosphatase receptor pi (PTPRP)
Apoptosis-specific protein1
Actin-binding double-zinc-finger protein (abLIM)
Putative tumor suppressor ST13 (ST13)
Zinc finger protein
Neuronal PAS2 (NPAS2)1
Glutathione reductase
Nicotinic acetylcholine receptor alpha4
p21 (WAF1/CIP1)
Cysteine protease CPP32 isoform beta; interleukin 1-beta converting
enzyme; apoptotic protein
Zinc finger protein 198
Zinc finger protein 135
IFp53
PDCD-2 factor1
p53 induced Pig 71
CdK-inhibitor p57KIP21
Bak protein; induction of apoptosis1
Cysteine protease CPP32 isoform alpha; interleukin 1-beta converting
enzyme; apoptotic protein1
DNA fragmentation factor-45; triggers DNA fragmentation during
apoptosis
p53-induced Pig 8
Pig10 (PIG10)
TRAIL receptor 2; binds cytotoxic ligand TRAIL; mediates apoptosis
Bc12 phosphorylated1
Inhibitor of apoptosis protein 2; inhibition of apoptosis
Bcl-xS mRNA
Aac11 (aac11); anti-apoptosis
14-3-3 protein epsilon isoform
Secreted apoptosis-related protein 2 (SARP2)
Serine/threonine protein kinase
Phospholipase C-gamma
Cyclin D protein kinase (CDPK)
Checkpoint suppressor
NF-kappa B transcription factor p651
TGFB1-anti-apoptotic factor1
ts11-G1 progression protein
K-ras oncogene protein
Zinc finger DNA binding motifs
Zinc finger protein RIZ
Transcription factor TFIIIB (hTFIIIB90)
Zinc finger protein RES4-26
cGMP-stimulated phosphodiesterase PDE2A3
Regulator of G protein signaling (RGS5)
Replication protein
Microtubule-associated protein 4
RBP1, retinoblastoma binding protein 1
Microtubule-associated protein 1A
Neuronal apoptosis inhibitory protein
Phospholipase A2
BRCA1 protein
Zinc finger protein 216splice variant
PTP ase alpha mRNA
Microtubule-associated protein tau
Peroxisome proliferator-activated receptor (PPAR)
Prostate-specific antigen
Androgen receptor-associated protein 24 (ARA24)1
5.09 ⫾ 1.21
4.40 ⫾ 2.44
X63469
L36645
M35410
X91257
U81561
Y11588
AF005654
U17714
Z21943
U77970
X15722
U62433
L13738
U13738
AF012126
U09413
X62570
S78085
AF010312
U22398
U23765
U13737
U91985
AF010313
AF010314
AF016266
U15173
U45879
Z23116
U83857
U43399
AF017987
D86550
M34667
U79269
U68723
L19067
D86970
M15798
M54968
M93119
U17838
U28838
AB000468
U67733
AF030108
M63488
M64571
S66427
U14577
U19251
M86400
AF045581
AF062347
M34668
J03778
L07592
U17040
AF052578
1
3.85 ⫾ 0.15
3.82 ⫾ 0.37
3.69 ⫾ 0.20
3.60 ⫾ 0.40
3.44 ⫾ 0.52
3.41 ⫾ 1.01
3.40 ⫾ 0.46
3.38 ⫾ 0.41
3.29 ⫾ 0.30
2.96 ⫾ 0.66
2.94 ⫾ 0.49
2.87 ⫾ 0.23
2.70 ⫾ 0.52
2.67 ⫾ 0.90
2.64 ⫾ 0.58
2.46 ⫾ 0.72
2.40 ⫾ 0.40
2.38 ⫾ 0.94
2.23 ⫾ 0.24
2.16 ⫾ 0.74
2.14 ⫾ 0.56
2.13 ⫾ 0.80
2.13 ⫾ 3.46
2.11 ⫾ 1.09
1.90 ⫾ 1.25
1.87 ⫾ 0.63
1.80 ⫾ 0.98
1.57 ⫾ 0.40
1.11 ⫾ 0.20
0.82 ⫾ 0.49
0.78 ⫾ 0.30
0.68 ⫾ 0.30
0.50 ⫾ 0.10
0.50 ⫾ 0.17
0.49 ⫾ 0.20
0.48 ⫾ 0.14
0.47 ⫾ 0.13
0.46 ⫾ 0.20
0.46 ⫾ 0.41
0.46 ⫾ 0.23
0.46 ⫾ 0.32
0.45 ⫾ 0.35
0.45 ⫾ 0.39
0.44 ⫾ 0.37
0.44 ⫾ 0.14
0.41 ⫾ 0.16
0.41 ⫾ 0.26
0.41 ⫾ 0.17
0.41 ⫾ 0.28
0.41 ⫾ 0.20
0.39 ⫾ 0.19
0.38 ⫾ 0.20
0.37 ⫾ 0.60
0.33 ⫾ 0.24
0.33 ⫾ 0.14
0.31 ⫾ 0.20
0.30 ⫾ 0.17
0.10 ⫾ 0.09
0.01 ⫾ 0.02
RT-PCR confirmed.
involved in cell growth inhibition and apoptosis. Activation of
Apaf-1, BAK protein, cystein protease CPP32 isoforms, DNA
fragmentation factor and apoptosis-specific proteins provides
additional evidence for an effect of RE on cell death induction
in LNCaP cells.
Effect of RE on transcription factors
As shown in Table I, we observed activation of acetyltransferase
p300 associated with alterations in the expression of other transcription factors, including transcription activator TFIIE ␤, zinc
RESVERATROL-INDUCED APOPTOSIS AND MOLECULAR TARGETS
finger protein, seryl tRNA synthetase and putative tumor suppressor ST13. Changes in the expression of these genes seem to be
consistent with the shutdown of several other oncogenic precursors, such as serine threonine kinase, phospholipase C and phospholipase A2, K-ras oncogene, androgen receptor-associated protein and prostate-specific antigen precursor protein. Detection of
another set of pro-apoptotic genes, namely Apaf-1, 14-3-3 and
IGF-binding protein 2, suggests a potential role of RE in the
modification of these gene transcripts in G1-arrested LNCaP cells.
Expression of p300 and Apaf-1
In addition to microarray and RT-PCR analysis, we observed an
increase in the total protein for p300 in RE-treated LNCaP cells
(Fig. 5). Expression of transcriptional co-activator p300 that could
acetylate human p5333,34 was high along with that of the apoptosisspecific protein Apaf-1. Nuclear localization by immunofluorescence detection (Fig. 5a) and quantification (Fig. 5b) corresponds
with the results of Western blot analysis (Fig. 5c) for total protein
expression. Overall, these findings support the expression and
activation of p53 and p300, the key transcription factors that are
involved in cell growth arrest and apoptosis.
Effect of RE on androgen receptor and PSA
Proteins that are known to interact with several steroid receptors
also influence the functional activity of the wild-type AR.53–56
209
Two reference genes, namely androgen receptor-associated protein
(ARA 24) and PSA, were first identified by microarray and then
confirmed by repeated RT-PCR (Fig. 4). Results from microarray
and RT-PCR analyses indicated a downregulation of androgen
receptor-associated protein ARA-24 and PSA, suggesting a potential role for RE in inducing cell growth inhibition via anti-androgenic mechanisms.
NF-kB regulation by RE
Results from the microarray analyses indicated significant
downregulation of NF-kB p65 (Table I), while the level of NF-kB
p50 expression remained unchanged. RT-PCR analyses confirmed
a lower expression level for PPAR␣ and NF-kB p65 (Fig. 4).
However, an inverse association between the p300 and NF-kB p65
confirmed a possible p53 targeted transcriptional inactivation of
nuclear factors that may be cell or organ specific. This requires
further investigation. Delineation of the differential regulation of
p300, PPAR␣ and the NF-kB family of genes by RE in LNCaP
cells is in progress. Comparing the level of expression of NF-kB
and the anti-apoptotic Bcl2 family of genes shown in Table I, there
is differential expression within the Bcl2 family of genes. For
example, there was a higher activation of BAK and another phosphorylated isoform of Bcl2 (identified by Western blot, data not
shown) that suggests a possible reprogramming of these genes
by RE.
FIGURE 5 – Expression of p300 in LNCaP cells treated with RE (10-5 M) for 48 hr. (a) Immunofluorescence detection of p300 using anti-p300
monoclonal antibody conjugated with FITC. (b) Quantification of p300-positive cells at 24 hr and 48 hr as described in Material and Methods.
Original magnification 40⫻. (c) Western blot analysis showing the protein p300/CBP and p53 in LNCaP cells after 48 hr RE treatment indicates
activation of p300 and p53 when compared to the protein level in the control.
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DISCUSSION
Gene expression analysis by DNA microarray presented here is
part of our ongoing research on antioxidant-regulated genes in
human prostate cancer. To determine the effect of RE on genes
involved in apoptosis and to study the associated cascade of
molecular events in LNCaP cells, we used a human cDNA microarray with 2,400 genes. Results of our study with respect to
gene expression analysis are limited due to the single time-point
measurement in one cell type (LNCaP) at the specified 48 hr point
after RE treatment. Although deeper biologic insight with respect
to transcriptional regulation of specific genes is likely to develop
from cDNA microarray analyses at multiple time points with
multiple cell lines, our aim in this study was to gain a general
understanding of the genetic response of LNCaP cells to RE
treatment. Toward this aim, we used a time (48 hr) and dose (10-5
M) evaluation for RE that had already been tested in the induction
of cell cycle arrest and apoptosis in androgen-sensitive LNCaP
cells. We preferred such a focused approach mainly to identify
those molecular targets with a specific dose and time regimen that
may lead to a different phenotype. Although studies with LNCaP
cells generally describe a poor correlation between RNA levels
and the corresponding proteins,50 findings from this study support
the hypothesis that RE induces differential expression of genes at
the transcription level that are directly or indirectly involved in cell
cycle control and apoptosis mediated by p53-dependent mechanisms. Exploring transcriptional and post-translational modifications of target genes and proteins modified by RE in a time- and
dose-dependent manner may provide more insight into differences
in the RNA vs. protein expression. Overcoming these limitations
may be possible via further perfection in technologies, such as
aDNA microarray and 2D gel/MS analysis, to detect mRNA and
the corresponding protein interactions in the same sample.
RE exerts chemopreventive activity by inhibiting cellular events
during tumor initiation, promotion and progression.17,19,28,37,41,44
Using cDNA microarray as well as RT-PCR, immunofluorescence
and Western blot analyses, we have demonstrated alterations in the
expression of p53 target genes associated with cell cycle arrest and
apoptosis as a result of RE treatment in androgen-dependent LNCaP cells. Activation of pro-apoptotic genes including p53,
p21(waf1/Cip1), PIG 7 and PIG 8, p300/CBP, Apaf-1, BAK and
zinc finger proteins by RE was consistent with the downregulation
of ARA 24 and PSA, NF-kB p65 and Bcl2. This demonstrates a
potential role of RE in inducing apoptosis while downregulating
the expression of prostate cancer precursor genes either by activating p53 signaling mechanisms and/or by acting in synergy with
blocking other androgen-signaling pathways. In our earlier studies
with human cancer cells, we have found a remarkable increase in
the level of p53 mRNA and protein, in conjunction with an
increase in the level of p21 (Waf1/Cip1) after exposure to phenolic
antioxidants.13,15 The current study indicates involvement of more
than one apoptotic pathway in LNCaP cells in response to RE.
Activation of Apaf-1, a novel protein induced by RE, has been
reported to participate in the cytochrome-C-dependent activation
of caspase 3,57 which triggers a cascade of events in apoptosis.
More than 30 genes in this category, specifically proteases and
protease inhibitors, are stabilized and activated when mammalian
cells are exposed to a variety of stressors including DNA-damaging agents.58 Expression and activation of p300 and the tumor
suppressor gene p53 in LNCaP cells suggests a possible role for
RE in inducing acetylation at the carboxyl terminus of p53; this
type of modification is known to increase the level of sequencespecific DNA binding of p53.48,59 It is also important to note that
RE may induce the antiproliferative effects in LNCaP cells
through post-translational modifications of p53, followed by a
post-transcriptional activation of p300 after DNA damage in G1arrested cells.60 Direct evidence demonstrating a role for p300 in
human tumors was lacking until recently, but now several studies
strongly support a role for p300/CBP as a tumor suppressor.61 The
results from our present study underscore the tumor suppressor
effect of highly activated p300 in G1-arrested LNCaP cells exposed to RE. In addition, AR transactivation activity been shown
to be triggered by androgens or by growth factors; moreover,
CBP/p300 has been reported to play a major role in the liganddependent AR transactivation activity.62 Cell culture studies have
shown that p300 and CBP can act as transcriptional co-activators
for a large number of transcription factors. The co-activation
potential of p300 and CBP is in part brought about by an acetyltransferase (AT) activity located in the central part of these 2
proteins. In vitro, histones and certain transcription factors, such as
p53, are acetylation substrates of p300.48,63 However, the biologic
significance of RE-induced p300 in ligand-dependent or -independent AR transactivation is as yet unknown. Recent studies indicate
that AR can be modified by acetylation both in vitro and in vivo;64
acetyltransferase co-activators, such as RE, have important roles in
targeting AR function and the associated signaling mechanisms
involved in prostate carcinogenesis. RE also activates ERKs and
p38 kinase, which, in turn, mediate apoptosis through phosphorylation of p53 at serine 15.28 MEKKI-activated p300-mediated
transcription may be involved in RE-induced apoptosis; however,
this effect may be due to modulation of more than one signaling
pathway. A mechanistic model developed on the basis of currently
available comprehensive data generated from this study, by microarray analysis, RT-PCR, Western blot and immunofluorescence
detection in LNCaP cells with or without RE treatment for 48 hr,
together with information from earlier studies,65 is presented in
Figure 6.
In summary, a number of the p53 target genes revealed in our
microarray analysis provide new molecular targets that may also
serve to assess the efficacy of other potential chemopreventive
agents for prostate cancer besides RE. There are indications that
the nature of p53 response depends on the levels of p53 protein, the
type of inducing agent and the cell type employed. Significant
FIGURE 6 – Mechanistic model illustrating the predicted cascade of
molecular events elicited by RE in LNCaP cells. AR conveys its
transcription function by recruiting several co-activators shown in the
left box and induces cell proliferation. However, the functions of these
co-activators are diverse and depend on the status of AR or p53 (wild
type or mutant). RE-mediated transcriptional repression and activation
of AR and p53 target genes is predicted to be centered around p300.
Consistent with earlier reports on AR acetylation and our observation,
we predict that RE-mediated p300 and p53 activation is associated
with the expression of several pro-apoptotic genes. Dashed lines
indicate the pathway yet to be confirmed (see Discussion for further
explanation).
RESVERATROL-INDUCED APOPTOSIS AND MOLECULAR TARGETS
cellular death was observed only when several of the genes controlled by p53 were expressed in concert, suggesting that p53
appears to activate parallel apoptotic pathways to induce programmed cell death.47,48 The full delineation of the cascade of
molecular events in response to RE treatment of prostate cancer
requires a comprehensive study on gene expression at the transcription level.
211
ACKNOWLEDGEMENTS
This study was presented at the American Association for Cancer Research 93rd Annual Meeting (Abstract #830, Cancer Prevention III, Prevention and Survivorship Research 2, Poster Discussion Session), 2002. We thank Ms. I. Hoffmann for editing the
manuscript.
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