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Life Sciences 70 (2002) 1821 1839

Interactive gene expression pattern in prostate cancer cells


exposed to phenolic antioxidants
Bhagavathi A. Narayanana,*, Narayanan K. Narayanana,
Gary D. Stonerb, Bryan P. Bullockc
a
Microarray Systems Laboratory, American Health Foundation,
1 Dana Road, Valhalla, NY, 10595, USA
b
Ohio State University Comprehensive Cancer Center, Columbus, OH, 43210, USA
c
PE/NEN Life Sciences, 549 Albany Street, Boston, MA, 02119, USA

Received 27 June 2001; accepted 13 November 2001

Abstract

Dietary phenolic compounds are known to elicite vital cellular responses such as cell cycle arrest,
apoptosis and differentiation by activating a cascade of molecular events. As there is an increasing
interest to improve the efficacy of these compounds for use as potential chemopreventive agents, we
wanted to understand the impact of phenolic compounds on target genes in prostate cancer. In this
study we used human cDNA microarrays with 2400 clones consisting of 17 prosite motifs to
characterize alterations in gene expression pattern in response to the phenolic antioxidants ellagic
acid (EA) and resveratrol (RE). Over a 48-hr exposure of androgen - sensitive LNCaP cells to EA
and RE, a total of 593 and 555 genes respectively, showed more than a two fold difference in
expression. A distinct set of genes in both EA-and RE-treated cells may represent the signature
profile of phenolic antioxidant-induced gene expression in LNCaP cells. Although extensive
similarity was found between effects of EA - and RE - responsive genes in prostate cancer cells, out
of 246 genes with overlapping responses, 25 genes showed an opposite effect. Quantitative RT
PCR was used to verify and validate the differential expression of selected genes identified from
cDNA microarrays. In-depth analysis of the data from this study provided insight into the alterations
in the p53 - responsive genes, p300, Apaf 1, NF kBp50 and p65 and PPAR families of genes,
suggesting the activation of multiple signaling pathways that leads to growth inhibition of LNCaP

* Corresponding author. Microarray Systems Laboratory, American Health Foundation, 1 Dana Road,
Valhalla, NY 10595. Tel: +1-914-789-7247; fax: +1-914-592-6317.
E-mail address: bhagavat@mindspring.com (B.A. Narayanan).

0024-3205/02/$ see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 4 8 1 - 9
1822 B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839

cells. This is a first study to look for changes in a large number of human genes in response to
dietary compounds. D 2002 Elsevier Science Inc. All rights reserved.

Keywords: LNCaP; Resveratrol; Ellagic acid; Androgen; Prostate; cDNA microarrays; Gene expression profiling;
Transcription factors; Transmembrane proteins

Introduction

The development of prostate cancer is accompanied by genetic alterations that involve a


diverse group of gene products including tumor suppressor genes, oncogenes, cell cycle
regulators, transcription factors, growth factors, DNA repair genes and cell death regulators.
Although multiple mechanisms appear to be responsible for the tumor inhibitory properties
of dietary polyphenols, information from earlier studies [19] on the genetic response to
phytochemicals is very limited. EA has been shown [1014] to inhibit tumor growth in
animal assays with chemical carcinogens such as polycyclic aromatic hydrocarbons,
Nnitrosoamines, aflatoxins, and aromatic amines. Similarly, resveratrol, a phytolaxin
phenolic compound inhibited human prostate cancer cell growth by regulating prostate-
specific antigen (PSA) and androgen receptor (AR) expression levels through modulation of
different signal transduction pathways [1519]. EA and RE are structurally different (Fig. 1),
but both are present in fruits such as grapes and berries at fairly high levels. However, the
similarity and the difference in the mechanism of action of these two phenolic compounds in
cancer cells are not fully understood. This begs the question whether the cancer preventive
potential is due to one agent - specific pharmacological effect or whether it represents a
synergistic effect of both EA and RE in modulating more than one mechanism. Our earlier
observations [20,21] indicated that ellagic acid inhibits cell proliferation by activating Waf1/
Cip1 and by down regulating IGF II in a p53 - independent manner in p53-mutated human
cancer cells. Thus one would expect multiple mechanistic pathways for EA to induce cell

Fig. 1. Structure of EA and RE.


B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1823

growth arrest and apoptosis. On the other hand, resveratrol, a closely related phenolic
compound has been implicated in altering the function of cyclooxygenases in cancer cells
by unknown mechanisms [22]. To address the above questions, we have chosen LNCaP cells
that are known to produce human prostatic acid phosphatase and prostate specific antigen,
have androgen receptors and are tumorigenic in nude mice [2325]. LNCaP cells have been
used extensively to study the anticancer property and efficacy of several chemopreventive
agents [19,2629]. Our rationale for the present study is that monitoring the regulation of
genes and their transcription profiles in response to dietary phenolic compounds will lead us to
a have clear understanding of the beneficial pharmacodynamic and possible adverse toxico-
logical responses.
The advent of high-density DNA microarray technology has enabled the simultaneous
analysis of the expression profiles of thousands of genes in yeast and man [3039]. High
throughput analysis of gene expression profiles has been used to understand the mechanism
of action of chemopreventive agents in colonic epithelial cells [30,31]. A few recent studies
connect gene expression patterns to therapeutic groups in prostate and breast cancer and
demonstrate the potential of high throughput technology in the characterization of gene
expression changes [3238]. Studies by Ross et al. [39] have demonstrated how gene
expression profiles characterize patterns of phenotypic variation in 60 different cancer cell
types; however, the alterations of gene expression in response to drugs, and their cellular
effects are yet to be clarified. In this study, we used human cDNA microarrays to understand
the expression profiles of phenolic antioxidant - responsive genes in LNCaP cells. A sig-
nature profile of phenolic antioxidant target genes is discussed.

Materials and Methods

Cell growth and treatment

LNCaP cells were grown under cell culture conditions maintained by serial passage in
RMPI containing 10% FBS. LNCaP cells were treated with HPLC - purified EA (105 M
final concentration) or RE, (Calbiochem) for 48 hrs. Stock solutions of RE and EA were made
in DMSO. Control cells were was treated with DMSO alone.

cDNA Microarrays

The human cDNA microarrays (MICROMAX) purchased from PerkinElmer-NEN Life


Science Products, Boston, MA were used for this study. The Micromax System was
provided with two human cDNA microarrays, spotted with 2,400 genes per array, (known
genes and ESTs); sequence verified by Alpha Gene. The genes were derived from diverse
human tissue sources covering a broad spectrum of functionality consisting of 17 prosite
categories (Table 1). More than 60% of the genes are full-length copies of known genes,
that also include two types of control genes, a complement of three non-mammalian (plant)
genes, and 35 housekeeping genes (human).
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Table 1
Prosite motifs of human cDNA microarray with a total number of 2400 genes (Listing provided by Alpha Gene/
PerkinElmer-NEN Life Sciences)
Prosite Motifs Category Number of Genes
Cytokines and growth factors 20
DNA or RNA associated proteins 270
Domains 353
Electron transport proteins 39
Hormones and active peptides 14
Hydrolases 159
Inhibitors 18
Isomerases 24
Ligases 43
Lyases 25
Oxidoreductases 72
Post-translational modifications 339
Protein secretion and chaperones 22
Receptors 63
Structural proteins 95
Transferases 164
Other Transport proteins 55
Others/ESTs and controls 625
TOTAL 2400

RNA isolation and probe preparation

Cultured LNCaP cells with appropriate treatments were harvested and total RNA was
isolated using Trizol reagent, and Qiagen columns (Life Technologies, MD and Qiagen,
Valencia, CA.). One control probe for untreated LNCaP cells and two test probes, one for EA
treated and another one for RE treated were made using total RNA extracted from the respective
parallel experiments with similar conditions. RNA from the untreated cells was labeled with
cyanine 3dUTP (cy3dUTP) and used as the control probe. RNA from EA- and RE - exposed
LNCaP cells was labeled separately with cyanine 5 (cy5dUTP) and was used as test probe.
Aliquots of RNA (100mg) were precipitated by adding 2.5 volumes of cold ethanol 0.1 volume
of 3 M sodium acetate. RNA was then harvested by centrifugation, briefly lyophilized, and
resuspended in 17 ml of DEPC (diethylpyrocarbonate)- treated water. This was added to 2 ml of
dNTP primer mix (NEN Life sciences product). This mixture was heated at and kept at 65 C
for 10 minutes to denature the nucleic acid then cooled to 25 C for 5 minutes. Then, following
reagents were added: 2.4 ml of 10X RT buffer, 2 ml of AMV RT/Rnase inhibitor mix, and 4 ml of
cy3dUTP and 2 ml of cy5dUTP (NEN Life Science Products). The reverse transcription
reaction was carried out for 1 hr at 42 C. The probes were cooled down to 4 C for 10 minutes,
and 2.5 ml of 0.5 M EDTA as well as 2.5 ml of 1N NaOH were added before incubation at 65 C
for another 30 minutes. After cooling the above mixture to 4 C for 5 minutes, 6.5 ml of 1M
TrisHCl was added. The cDNA was purified with isopropanol precipitation or by using a spin
column. The dried pellet was resuspended in a small volume (13 ml) of Rnase-free water; the
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1825

cy3dUTP labeled control and cy5dUTP labeled treated reactions were combined and 10 mg
of carrier DNA was added. After adding 2 ml of 5M ammonium acetate and 50 ml of
isopropanol, the probe mixture was pelleted at 14,000 rpm for 10 minutes at 4 C. The pellets
were washed again with 70 % and 95% ethanol respectively, and were stored at 20 C for
further hybridization.

Microarray Hybridization

Hybridizations were carried out at the American Health Foundation Microarray Labor-
atory. The mixed probe pellet of EA was resuspended in 25 ml of hybridization buffer and
immediately added to human cDNA arrays: a 2222 cover slip was placed carefully on the
arrays and transferred into hybridization cassettes especially made for submerging the arrays
in a 65 C water bath for 12 hrs. 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 0.5XSSC, 0.01% SDS and again with 0.06 SSC, 0.01% SDS for
15 minutes. A final wash of the slides were with 0.06  SSC for 15 minutes and the cleaned
slides were spin-dried before scanning. The probe hybridization and washing process were
carried out for RE probe strictly adhering the above protocols. The probe hybridization and
washing processes was repeated for both EA and RE probes on a second set of cDNA arrays
that was provided with the PE/NEN MICROMAX microarray kit.

Scanning and image analysis

The Axon GenePix 4000A scanner, a non-confocal scanning instrument containing two
lasers, that excite cyanine dyes at appropriate wavelengths, 635 nm for cy5 and 532 nm for
cy3 respectively, with high-resolution (10 micron pixel size) photo multiplier tubes that detect
fluorochrome emission, was used for scanning the hybridized cDNA microarrays (Axon
Instruments Inc., Foster City, CA). The PMT levels of the two channels at 635 nm and
532 nm were balanced (100 to 1000 volts) to limit the number of saturated pixels for
generating a gray scale TIFF image file. The microarray images were analyzed using Axon
GenPix Pro-3.0 software, a completely automated image processing system, that is capable of
processing image files in a batch-mode and allows high-throughput of microarray image
analyses. The specific features include grid-placement and spot finding, elimination of noise
signals and minimizing operators involvement in the data analysis process. The microarray
data sets and color images were generated on Microsoft Excel spreadsheets and JPEG images,
respectively. The normalization factor, arrived based on the average value of the ratio of the
medians of the housekeeping genes, was used to adjust expression ratio calculations to
determine whether gene expression differs significantly between the red and green channels.

Data analysis and molecular bioinformatics

The GeneSpring2 bioinformatics software package (Silicon Genetics, Inc.) was used to
explore the microarray data sets generated from this study for proprietary clustering
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applications, and multivariate analysis (principal component analysis) in search for consistent
patterns and/or systematic relationships between genes, and then to validate the findings by
applying the detected patterns to new subsets of data [4043].

Cluster analysis

The following novel statistical methods were carried out for the comparison of gene
expression profiles: (a) Hierarchical clustering: The data points were forced into a strict
hierarchy of nested subsets. The closest pair of points was grouped and replaced by a
single point representing their average; the next closest pair of points was treated similarly,
and so on [44,45]. (b) K-means (non-hierarchical): an unstructured approach, which
proceeds in an entirely local fashion can facilitate visualization by interpreting similarities
between the two sets of cDNA array data and experiments involving ellagic acid and
resveratrol treatment [46,47]. (c) Principal Component Analysis (PCA): an exploratory
multivariate statistical technique was used for simplifying the complex microarray data sets
generated from this study. Given m observations on n variances, the goal of PCA was to
reduce the dimensionality of the data matrix by finding r new variables, where r was less
than n. Termed principal components, these r new variables together account for as much
of variance in the original n variables as possible while remaining mutually uncorrelated
and orthogonal. Each principal component was a linear combination of the original
variables, and so it was often possible to ascribe meaning to what the component re-
present [48,49].

Table 2
Primer sequences used for semiquantitative RT PCR
EA Ratio RE Ratio
Genes Accession # Primers (50 30) (Cy5/Cy3) (Cy5/Cy3) RT-PCR
Glyceraldehyde 3 phosphate M33197 Sense: cat ctc tgc ccc 1.07 1.16 No Change
dehydrogenase (GAPDH). ctc tgc tga
Anti-sense: gga tga cct
tgc cca cag cct
p300 protein U01877 Sense: gaa tta atc aac 2.98 5.09 Induced by
tct aca ga > 3 fold
Anti-sense: caa ttt atc increase
aaa cct aat cc
Androgen receptor AF052578 Sense: att gtc ttc cac 0.54 0.01 Repressed by
associated protein 24. cga aag aa < 1 fold
Anti-sense: gag tgc agt
tgt ctg agc aa
Neuronal PAS2 U77970 Sense: ccg cca agt ctg 2.64 2.96 Induced by
aat ctg a > 3 fold
Anti-sense: agg gct gcc increase
ttg cgt cac ag
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1827

Validation of gene expression by semiquantitative RTPCR

As RTPCR of mRNA provides maximum sensitivity, a standardized measurement of


expressed genes were carried out using a semi-quantitative RTPCR. RTPCR was carried
out using 33 cycles for selected gene specific primer sequences (Table 2). All templates were
initially denatured for 2 minutes at 94 C and the amplification of amplicon was extended at a
final extension temperature of 72 C for 7 minutes. A separate set of RTPCR reactions with
an increasing amount of RNA were carried out in order to show a linear increase in the band
intensity of the amplified PCR product. PCR amplification with GAPDH from normal
prostate epithelial cells (PrEC, Clonetics) with constant amount of total RNA was used in each
set of reactions to document no variability of amplification efficiency from experiment to
experiment. Primers for a few highly expressed genes such as p300 and NAPS2 and AR for
repressed genes were used to show the linearity between RNA input and the amount of PCR
product as part of the validation of gene expression (Fig. 2A and 2B). In addition, based on
the normalized ratiometric data (cy5/cy3 ratios), the top ten highly expressed and the lowest
ten under expressed genes were confirmed by standard quantitative RTPCR. Densitometric
analysis for quantification of the PCR bands was carried out using Gel Pro 3.0 software
(Media Cybernetics).
As the DNA microarray technology involves complex data analysis, the authors like to
address a few details on the complex microarray data analysis. Several key factors were
considered for effective data processing and most of the details are already described in
the methods. Equal loading of labeled probes of control (cy3dUTP) and (cy5dUTP) ex-
periments were performed according the manufacturers recommendations (Micromax- PE/
NEN-direct labeling). The experiments were repeated twice on two similar 2400 cDNA
microarrays. The average of the two data points was considered for calculating the cy5/cy3
ratios. Cluster analysis was done in two different ways, one was by using the clustering
protocol for gene expression studies designed by Eisen et al (tree view clustering) and the other
was by using the software from silicon genetics Genespring. for K-mean clustering. The
level of gene expression using cDNA microarrays were confirmed by RTPCR by three
individual separate experiments in addition to the two arrays to show reproducibility and
confirmation of the gene expression analysis.

Results

Differential Expression of Genes in Response to EA and RE

Although a deeper biological insight is likely to develop from microarray analysis using
multiple time points with different cell lines treated independently or in combination, in this
study gene expression analysis was done for two samples: a) RE or EA treated vs. b) untreated.
Results from the scanned images of identical experiments demonstrated alterations in
expression of cDNAs after 48 hrs of induction. Analysis of differentially expressed genes
revealed remarkable alterations in the level of expression. Induced genes (2 fold increase) with
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Fig. 2. Quantitative RT-PCR. 2A: Gel (2% agarose) showing the RT-PCR products amplified for selected genes.
Note the linearity in the size of the PCR bands with the increase in the amount of RNA. Total RNA isolated from
LNCaP cells exposed to EA and RE for 48 hrs was used for PCR amplification using specific primers designed for
p300, NPAS2 (Neuronal specific antigen 2) and AR (Androgen receptor associated protein). Simultaneously, a
constant amount of total RNA (2mg) isolated from normal prostate cells (PrEC - Clonetics) was used to amplify
GAPDH using specific primers for normalization. 2B: Quantification of the PCR products was carried out by
densitometry analysis and the data (pixels) generated were compared against cy5/cy3 ratios of the respective genes.
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1829

RE and EA were found to be 5.25 % and 10.67% respectively of the total expressed genes from
the 2,400 gene array used in this study. Repressed genes by 2 fold with RE and EA were 17.88%
and 14.04% respectively suggesting significant number of genes repressed by resveratrol
compared to that of ellagic acid. A comparative ratiometric (cy5/cy3) and densitometric
analysis in terms of pixels for RTPCR products of top ten highly expressed and lowest 10
repressed genes for EA and RE treated samples are presented in Table 3 and Table 4.

Identification of Major Clusters of Responsive Genes

We identified six major clusters of highly responsive genes that are differentially
expressed. Interestingly, a few clusters of genes that come under the category of oncogenes
and protein kinases are co-regulated with a two-fold increase by both EA and RE. In contrast,
a higher percentage of similar set of genes from the above two categories were down

Table 3
List of genes showing at least more than two fold difference in the average expression between EA treated vs.
untreated LNCaP cells
(Cy5/Cy3) Number of
Accession # Gene Name Ratio* Pixels**
M33197 Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) 1.07 447
Upregulated ( >2.0 folds)
U01877 p300 protein 2.98 1475
U51903 RasGAP-related protein (IQGAP2) 2.89 1431
U81561 Protein tyrosine phosphatase receptor pi (PTPRP) 2.86 1417
X53605 Prostatic acid phosphatase (EC 3.1.3.2) 2.66 1318
U77970 Neuronal PAS2 (NPAS2) 2.64 1307
AF012126 Zinc finger protein (ZNF198) 2.64 1307
U25033 Neuronatin alpha 2.63 1302
X63469 Transcription factor TFIIE beta 2.54 1259
U50040 Signaling inositol polyphosphate 5 phosphatase SIP-110 2.51 1243
Y11588 Apoptosis specific protein 2.51 1243
Downregulated ( < 0.5 folds)
AF052578 Androgen receptor associated protein 24 (ARA24) 0.54 267
L07592 Peroxisome proliferator activated receptor (PPAR) 0.53 262
AF030108 Regulator of G protein signaling (RGS5) 0.47 230
U14577 Microtubule-associated protein 1A (MAP1A) 0.46 228
U17040 Prostate specific antigen 0.42 208
L11701 Phospholipase D 0.40 199
J02947 Extracellular-superoxide dismutase (SOD3) 0.31 155
Y07512 Beta cGMP-dependent protein kinase 0.23 114
AF013263 Apoptotic protease activating factor 1 (Apaf. 1) 0.13 64
S76638 p65 NF kappa B homolog 0.01 05
Relative expression levels (cy5/cy3) ratios vs. densitometric analyses (pixels) of semiquantitative RT PCR bands
are displayed for each gene.
* Gene expression levels.
** Densitometric measurement of RT PCR band.
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Table 4
List of genes showing at least more than two fold differences in the average expression between RE treated vs.
untreated LNCaP cells
(Cy5/Cy3) Number of
Accession # Gene Name Ratio* Pixels**
M33197 Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) 1.07 447
Upregulated ( >2.0 folds)
U01877 p300 protein 5.09 2384
X63469 Transcription factor TFIIE beta 3.85 1805
M35410 Insulin-like growth factor binding protein 2 (IGFBP2) 3.69 1730
Y11588 Apoptosis specific protein 3.41 1597
U77970 Neuronal PAS2 (NPAS2) 2.96 1387
X15722 Glutathione reductase 2.94 1379
AF053304 Mitotic checkpoint component Bub3 (BUB3) 2.54 1188
U62589 Glutathione S transferase P1c (GSTp1c) 2.47 1158
U23765 Bak protein 2.14 1002
M68867 Cellular retinoic acid-binding protein II (CRABP) 2.09 981
Downregulated ( < 0.5 folds)
S76638 p65 NF kappa B homolog 0.47 221
M54968 K ras oncogene protein 0.46 217
U67733 cGMP-stimulated 30,50-cyclic nucleotide phosphodiesterase PDE2A3 0.44 206
AF010314 Pig10 (PIG10) 0.41 192
U77352 MAP kinase-activating death domain protein (MADD) 0.33 153
L11701 Phospholipase D 0.31 147
L07592 Peroxisome proliferator activated receptor (PPAR) 0.30 143
U28838 Transcription factor TFIIIB 90 kDa subunit (hTFIIIB90) 0.29 134
AF013263 Apoptotic protease activating factor 1 (Apaf 1) 0.07 33
AF052578 Androgen receptor associated protein 24 (ARA24) 0.01 05
Relative expression levels (cy5/cy3) ratios vs. densitometric analyses (pixels) of semiqunatitative RT PCR bands
are displayed for each gene.
* Expression levels.
** Densitometric measurement of RT PCR band.

regulated simultaneously by both EA and RE, suggesting a selective inducing and repressing
effect in these two clusters of genes in the same cell type. Notably, a large number of
glycoproteins and transglutaminses showed a remarkable inductive effect by RE but not by
EA. A detailed analysis of the data as shown in Table 5 revealed a few set of oncogenes,
protein kinases, transmembrane proteins, and transcription factors that are down regulated
(two fold) by both RE and EA are co-regulated.

Effects on Transmembrane Proteins, Transglutaminases and Transcription Factors

Among a total of 246 expressed genes (transcripts induced by both EA and RE), 18.29% of
the genes in the microarray, were transmembrane proteins comprising subsets of genes in the
category of G protein-coupled receptors, endothelial cell protein receptors, integral membrane
proteins, transmembrane secretory components, transmembrane glycoproteins, and cell
surface glycoproteins. Interestingly, very few genes are showing a greater than two-fold
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1831

increase in expression after exposure to EA or RE for 48 hrs. A larger number of


transmembrane proteins were down regulated by both EA and RE. However, the antioxidant
functions of EA and RE in conjunction with their effect on transmembrane proteins rich in
tyrosine and tryptophan-containing peptides and their effect on oxidative stress need to be
investigated. Transglutaminases include intracellular and extracellular enzymes that catalyze
Ca2-dependent reactions resulting in the covalent incorporation of di- and polyamines and
histamines. At least four transglutaminases (Tgases 1,2, 3 and X) are expressed in mammalian
cells during terminal differentiation and death of epidermal keratinocytes. Our study indicates
a two-fold increase in the expression of Tgase3 in LNCaP cells (Gene ID L10386) by RE
and a one-fold increase by EA. This suggests a possible role for RE and EA in apoptosis and
differentiation. A total of 48 transcription factors were expressed in both EA - and
RE - treated cells. Although more than 80% of the transcription factors behaved in the same
way for both EA - and RE - treatment, a few of them, e.g., transcription factor TFIIB, mRNA
for zinc finger protein, and Retinoid X receptor gamma, were upregulated by RE only but not
by EA. Among the transcription factors a higher percentage of clones were found to be
upregulated by RE, compared to EA, which showed equal numbers of clones that are up or
down regulated. Among the families of genes in the nuclear factors, both EA and RE
repressed NFkBp50 and NFkBp65 two-folds (Table 5 and Fig. 3).

Effects on Growth Factors and Hormones

Data on the expression patterns of growth factors due to the effect of EA and RE indicate
that apoptosis in prostate cancer could have been induced by the altered expression pattern of
TGFb signaling pathways. The differential expression of TGFb and IGF-like growth factors,
receptors, and binding proteins, in conjunction with the co-regulation of several related
growth factors, suggests their positive role in inducing cell cycle arrest and apoptosis in a
p53- independent manner without showing any dramatic effect on the level of p53 (supported
by data shown in our earlier publications). Even so, the present study indicates altered
expression of a few p53-responsive genes (Table 5). Induction of the TGFb- pathway by
phenolic compounds is strengthened by the observed upregulation of cell cycle regulatory
and apoptosis-inducing genes, along with an increase in the expression of TGF b, IGF I and II
growth factors and binding proteins. Plant phenol-mediated cell cycle arrest in concert with
the induction of p21, and downregulation of IGFII in EA-treated cells [20,21] is likely
connected to the gene expression pattern of growth factors and their orchestrated activation
and downregulation that leads to growth arrest in human prostate cancer cells. Among the
12 hormonal receptors expression of androgen receptor-associated protein and TR3 orphan
receptors were down regulated significantly by both EA and RE (Table 5).

Effects on Phase II Drug-Metabolizing Enzymes and Antioxidants

Phase II drug metabolizing enzymes consist of many super families of enzymes including
glutathione S-transferases (GST). In the present study, RE was found to activate four out of six
GST genes compared to activation of two by EA. The effects of EA and RE on the antioxidant
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Table 5
List of expressed genes identified from different functional groups in response to EA and RE in LNCaP cells after
48 hrs of treatment
Accession # EA Ratio RE Ratio Gene Description
Apoptotic genes
Y11588 2.51 3.41 Apoptosis specific protein
AF013263 0.13 0.07 Apoptotic protease activating factor 1
U13738 2.25 2.67 Cysteine protease CPP32 isoform beta
U19251 0.68 0.39 Neuronal apoptosis inhibitory protein
U23765 0.62 2.14 Bak protein
U91985 0.44 0.13 DNA fragmentation factor-45
AF039136 0.35 0.25 Fas binding protein
U13737 0.28 0.01 Cysteine protease CPP32 isoform alpha

Genes involved in differentiation


L09604 2.74 3.41 Differentiation-dependent A4 protein
X85750 2.56 2.34 Transcript associated with macrophage differentiation
Y11588 2.51 3.41 Apoptosis specific protein
D28540 2.30 1.37 Diff6, H5, CDC10 homologue
S72493 1.93 1.90 Keratin, keratin 16 homolog
AB004047 1.85 2.17 Beta actin
M33509 1.84 2.14 HLA-B-associated transcript 2
AJ002962 1.16 2.55 hB FABP
AF031647 1.08 2.34 JAB1-containing signalosome subunit 3
D14811 1.01 0.11 KIAA0110 gene
U49187 1.00 0.43 Placenta (Diff48)
M62302 0.65 0.37 Growth/differentiation factor 1
Y10313 0.62 0.29 interferon-related developmental regulator
L12261 0.58 0.32 Recombinant glial growth factor
U29943 0.54 0.43 ELAV-like neuronal protein 2 Hel N2
X64229 0.54 0.50 Dek mRNA
X53416 0.49 0.82 Actin-binding protein (filamin)
Y12065 0.45 0.17 Nucleolar protein hNop56
U27460 0.33 0.01 Uridine diphosphoglucose pyrophosphorylase

Transcription factors
U50315 2.84 2.06 Enhancer of zeste homolog 1
M83233 2.73 3.03 Transcription factor (HTF4A)
X63469 2.54 3.85 Transcription factor TFIIE beta
U87269 2.46 2.48 p120E4F transcription factor
L08424 2.41 2.74 Achaete scute homologous protein
Y07595 2.37 3.04 52 KD subunit of transcription factor TFIIH
M62831 2.29 7.21 Transcription factor ETR101
L19871 2.20 2.82 Activating transcription factor 3
M87503 2.08 2.65 IFN-responsive transcription factor
L29277 2.05 1.27 DNA-binding protein (APRF)
X78710 1.60 2.01 MTF 1 metal-regulatory transcription factor
M76766 1.41 2.62 Transcription factor (TFIIB)
Z21943 1.41 3.29 Zinc finger protein
U11701 1.32 2.17 LIM-homeobox domain protein
(continued on next page)
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1833

Table 5 (continued )
Accession # EA Ratio RE Ratio Gene Description
Transcription factors
L08895 0.92 3.03 MADS/MEF2-family transcription factor
L31881 0.90 2.01 Nuclear factor I X
X59268 0.61 0.39 General transcription factor IIB
D32257 0.53 0.22 GTF3A Xenopus transcription factor IIIA
Z19002 0.50 0.23 PLZF - kruppel-like zinc finger protein
U28838 0.49 0.29 Transcription factor TFIIIB 90 kDa
L39059 0.49 0.62 Transcription factor SL1
L19067 0.47 0.21 NF kappa B transcription factor p65
M73778 0.47 1.61 PML 1 protein
U75276 0.47 0.13 TFIIB related factor hBRF
U22431 0.46 0.47 Hypoxia-inducible factor 1 alpha
X63741 0.44 0.99 Pilot mRNA
M84739 0.38 0.31 Autoantigen calcreticulin
M62810 0.27 0.04 Mitochondrial transcription factor 1
S76638 0.01 0.47 P65 NF kappa B homolog.

Cell cycle related genes


M15798 2.51 2.38 ts11 G 1 progression protein
D14878 1.32 0.02 Protein D123
U01877 2.98 5.09 p300 protein
L34075 0.59 5.16 FKBP rapamycin associated protein

Genes related to p53 responsive genes


X62570 2.40 2.39 IFP53
AF010312 2.23 2.27 Pig7
U93162 1.07 2.27 C4 asterol methyl oxidase homolog
AF010313 1.00 1.77 Pig8
AF010314 0.58 0.41 Pig10

Genes related to GST


AF020918 2.44 2.91 Glutathione transferase
M63509 1.95 1.94 Glutathione transferase M2
U62589 0.88 2.47 Glutathione S transferase P1c
X17644 0.58 0.46 GST1 Hs mRNA for GTP-binding protein.
AF017305 0.00 2.16 Deubiquitinating enzyme UnpEL

Genes related to growth factors


X02812 2.46 1.11 Transforming growth factor-beta
M35410 2.15 3.69 Insulin-like growth factor binding protein 2
M57399 2.13 1.51 Never growth factor
U07707 2.02 2.14 Epidermal growth factor receptor substrate
M19154 1.84 2.13 Transforming growth factor-beta-2
L10844 1.40 1.61 Cellular growth-regulating protein
L13740 1.36 0.55 TR3 orphan receptor
M12783 1.28 2.13 C sis/platelet-derived growth factor 2
Y00285 1.21 14.60 Insulin-like growth factor II receptor
(continued on next page)
1834 B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839

Table 5 (continued )
Accession # EA Ratio RE Ratio Gene Description
Genes related to growth factors
X57025 0.98 2.48 IGF I mRNA for insulin-like growth factor I
S82451 0.96 0.70 Latent transforming growth factor-beta-binding protein -2
S82024 0.93 1.51 Neuron-specific growth-associated protein/stathmin homolog
M60278 0.91 0.67 Heparin-binding EGF-like growth factor
M96995 0.80 0.28 Epidermal growth factor receptor-binding protein GRB2
M62302 0.65 0.37 Growth/differentiation factor 1
AF089750 0.64 4.37 flotillin 1
Y10313 0.62 0.29 IFRD1 (PC4) interferon-related developmental regulator
M25667 0.58 0.48 Neuronal growth protein 43 (GAP 43)
D16431 0.52 0.14 Hepatoma-derived growth factor
M60315 0.47 0.12 Transforming growth factor-beta
M34186 0.01 3.04 Fibroblast growth factor receptor (FGFr) transmembrane
M62403 0.01 1.83 Insulin-like growth factor binding protein 4
X52946 0.01 1.23 Pleiotrophin (PTN) mRNA

enzyme transcripts indicate an upregulation of superoxide dismutases by a two-fold increase in


extracellular superoxide dismutase (SOD3) and of the mRNA for Cu/Zn superoxide dismutase
and manganese-containing superoxide dismutase. The gene transcription for antioxidant
enzyme catalase (Gene IDX04076) is repressed by both EA and RE (Table 5).

Effects on Cell Cycle and Apoptosis and Differentiation

Among the specific responses by the seven functional categories those falling under the
cluster of apoptosis and cell cycle behaved similarly (induced or repressed) in their response
to EA and RE. On the other hand, the genes under the cluster of growth factors responded
significantly to EA within 48 hrs by comparison to their response to RE. Results from our
earlier studies [21] on the mRNA expression of IGFI and IGFII in colon cancer cells reflects a
similar downregulatory effect in response to EA similar to that observed here in prostate
cancer cells. Because EA and RE are both anti-proliferative agents, we hypothesized that the
greater similarity between EA and RE may in part be attributable to their mechanism of action
in tumor inhibition. We therefore investigated whether we could identify a population of
genes for which altered expression by EA or RE was attributable to an altered effect on
p53-responsive genes, or cell cycle regulatory genes or proapoptotic genes. We observed the
expression of a similar set of genes that are p53 - responsive or proapoptotic, in both EA and
RE treated cells. Although these gene clusters showed similar responses to EA and RE, the
data were characterized by extensive quantitative differences. This is important for functional
classes of genes involved in signaling pathways and in cell cycle progression. Inhibition of
cell proliferation induced by phenolic compounds reportedly exerts positive effect on the
induction of differentiation. The analyses of the expression of differentiation-related genes
showed more than two-fold increases. Phenolic extracts affect cell growth arrest and
apoptosis and induce differentiation in breast cancer cells. In the present study more than
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1835

Fig. 3. Histogram indicates the induction or repression of gene clusters after 48 hrs of treatment with EA and RE in
LNCaP cells. 3A: EA treatment and 3B: RE treatment (OG: Oncogenes, PK: Protein kinases, GP: Glycoproteins,
TMP: Transmembrane proteins, TF: Transcription factors, and TG: Transglutaminases).

10 genes that are directly or indirectly involved in differentiation were found to show more
than two fold increase in expression (Table 5).
1836 B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839

Discussion

Results from this study is a first report on the changes in a large number of human genes
in response to dietary compounds, revealed a regulated pattern of expression of different
clusters of genes that have similar functional end points, such as inhibition of cell
proliferation and induction of apoptosis. Induction of cell death seems to be orchestrated
by a modified expression of several genes. Among a total of 246 expressed genes 18.29%
of genes were transmembrane proteins, comprising of sub- sets of genes in the category of
G protein coupled receptors (GPCRs), endothelial cell protein receptors, integral membrane
proteins, transmembrane secretary components, transmembrane glycoproteins, and cell
surface glycoproteins. Differential expression of the subset of genes in both EA- and
RE- treated LNCaP cells may represent the molecular profile of phenolic antioxidants.
Several distinct signaling mechanisms have been reported to contribute to GPCR mediated-
MAPK activation [50]. Although a pathway involved in the inactivation of Ras MAPK, Src
and Stat3 by G proteins has been strongly implicated in the results obtained in this study, the
specific role of RE and EA in the modulation of downstream factors involved in G protein
signaling needs further study.
A group of transmembrane proteins found to upregulated by both EA and RE are expressed
more than 2 fold. However, the antioxidant functions of EA and RE in conjunction with their
effect on transmembrane proteins that are rich in tyrosine and tryptophan containing peptides
and their effect on oxidative stress need to be investigated. The effects of EA and RE on the
antioxidant enzyme transcripts indicated an upregulation of superoxide dismutases by a
two-fold increase in extracellular superoxide dismutase (SOD3), mRNA for Cu/Zn superoxide
dismutase, and manganese-containing superoxide dismutase. Gene transcription for antiox-
idant enzyme catalase (Gene IDX04076) was repressed by both EA and RE. Several
molecular mechanisms appear to be responsible for the tumor inhibitory properties of the
polyphenols, including enhancement of the antioxidant (glutathione peroxidase, catalase and
quinone reductases), and phase II (glutathione S transferase) enzyme activities; inhibition of
lipid peroxidation, inhibition of TPA- induced epidermal ornithine decarboxylase (ODC), and
cyclooxygenase activities, inhibition of protein kinases, and cellular proliferation [8,1921].
Although lower expression of antioxidant enzyme expression in PIN and prostatic carcinoma
has been reported in human prostate tissues [51], results from this study indicate a trend toward
an increase in the antioxidant enzyme level. However, an earlier report on the effect of
androgen in LNCaP indicated a shift in the balance between pro and antioxidants enzymes [52].
It is very important to mention here that the extensive alteration in the expression of
transglutaminases (Tgases) in prostate cancer cells by resveratrol is a first observation. At least
seven distinct transglutaminases have been characterized in mammals [53], and four trans-
glutaminases are expressed and synthesized during terminal differentiation and death of
human epidermal keratinocytes [54,55]. That activation of transglutaminase-3 in LNCaP by
RE occurs to a greater extent than by EA supports its role in inducing apoptosis and
differentiation. Indeed, during apoptosis de novo transcription of the Tgase gene is induced
by several factors (e.g. retinoic acid, prostaglandin E2, interleukin 6, and tumor growth factor
B). In addition to transcriptional regulation, Tgase can also be modulated post translationally
B.A. Narayanan et al. / Life Sciences 70 (2002) 18211839 1837

during apoptosis, which irreversibly modifies cell organization before clearance by phagocyt-
osis, thus contributing to the prevention of inflammation in the surrounding tissues [5658].
Both EA and RE activated apoptosis- specific protein, apoptotic protease activating factor 1,
and cysteine protease CPP32 isoform, showing a greater than two-fold increase in LNCaP
cells. Results from this study revealed an activation of p300 by both EA and RE with almost
the same level of expression, suggesting a potential role for cell growth regulation in these
cells. This is in agreement with other studies [59], indicating p300 as an important component
of p53 signaling, and thus providing new insight into the mechanism of cellular proliferation.

Acknowledgments

This study was supported in part by AICR 01A015R and NCI CA17613 grants. We
thank Axon Instruments and Silicon Genetics for providing technical guidance in scanning
and on microarray data analysis, respectively. We thank Ilsa Hoffmann, and Joanne Braley for
editing the manuscript.

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