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Actas Urol Esp. 2014;38(6):397---404
Actas Urológicas Españolas
www.elsevier.es/actasuro
ORIGINAL ARTICLE
Effects of resveratrol and other wine polyphenols on
the proliferation, apoptosis and androgen receptor
expression in LNCaP cells夽
A. Ferruelo a , I. Romero b , P.M. Cabrera b , I. Arance b , G. Andrés b , J.C. Angulo b,∗
a
Unidad de Investigación, Fundación para la Investigación Biomédica, Hospital Universitario de Getafe, Madrid, Spain
Servicio de Urología, Hospital Universitario de Getafe, Departamento Clínico, Facultad de Ciencias Biomédicas, Universidad
Europea de Madrid, Madrid, Spain
b
Received 10 February 2014; accepted 19 February 2014
Available online 22 May 2014
KEYWORDS
Cell proliferation;
Apoptosis;
Androgen receptor;
LNCaP;
Resveratrol;
Polyphenols
Abstract
Purpose: To address the effect of resveratrol and other red wine polyphenols on cell proliferation, apoptosis and androgen receptor (AR) expression in human prostate cancer LNCaP
cells.
Materials and methods: LNCaP cells (5 × 102) were cultured in microtiter plate modules and
treated with gallic acid, tannic acid and quercetin (1, 5 and 10 ␮M), rutin and morin (25, 50
and 75 ␮M) and resveratrol (5, 10 and 25 ␮M). To address the extent of proliferation at 24, 48,
72 and 96 h, a colorimetric immunoassay method was used. An activity caspase 3/7 detection
assay was used to disclose apoptosis at 24, 48 and 72 h. AR mARN levels were determined by
real time RT-PCR.
Results: All polyphenols studied significantly inhibited (P < .05) cell proliferation compared
to control. However, there were moderate differences between them. Resveratrol was the
strongest inhibitor at different times and doses. Also, caspase-3 and caspase-7 activity was
significantly higher (P < .05) than control in the presence of all the compounds, but the earlier
response was achieved by resveratrol. Resveratrol, quercetin and morin were the only nutrients
that significantly inhibited AR mRNA expression. Again resveratrol produced the highest inhibition (90---250 times less than control), followed by morin (67---100 times) and quercetin (55---91
times).
夽 Please cite this article as: Ferruelo A, Romero I, Cabrera PM, Arance I, Andrés G, Angulo JC. Los efectos de resveratrol y otros polifenoles
del vino sobre la proliferación, apoptosis y expresión de receptor androgénico en células LNCaP. Actas Urol Esp. 2014;38:397---404.
∗ Corresponding author.
E-mail address: javier.angulo@salud.madrid.org (J.C. Angulo).
2173-5786/$ – see front matter © 2014 AEU. Published by Elsevier España, S.L. All rights reserved.
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398
A. Ferruelo et al.
Conclusions: All polyphenols studied showed important antiproliferative effects and induced
apoptosis when added to LNCaP cells culture. We confirm that resveratrol, morin and quercetin
may achieve such effect through reduced expression of AR. The synergistic effects of these
compounds and their potential to prevent progression of hormone-dependent prostate cancer
merit further study.
© 2014 AEU. Published by Elsevier España, S.L. All rights reserved.
PALABRAS CLAVE
Proliferación celular;
Apoptosis;
Receptor
androgénico;
LNCaP;
Resveratrol;
Polifenoles
Los efectos de resveratrol y otros polifenoles del vino sobre la proliferación,
apoptosis y expresión de receptor androgénico en células LNCaP
Resumen
Propósito: Abordar el efecto del resveratrol y otros polifenoles del vino tinto sobre la proliferación celular, la apoptosis y la expresión del receptor androgénico (RA) en células LNCaP de
cáncer de próstata humano.
Materiales y métodos: Las células LNCaP (5 × 102) se cultivaron en módulos de placas de
microtitulación y se trataron con ácido gálico, ácido tánico y quercetina (1, 5 y 10 ␮M), rutina
y morina (25, 50 y 75 ␮M) y resveratrol (5, 10 y 25 ␮M). Para abordar el alcance de la proliferación a las 24, 48, 72 y 96 h se utilizó un método de inmunoanálisis colorimétrico. Se utilizó
un ensayo de detección de actividad de caspasa 3/7 para revelar la apoptosis a las 24, 48 y
72 h. Se determinaron niveles de ARNm de RA mediante RT-PCR a tiempo real.
Resultados: Todos los polifenoles estudiados inhibieron significativamente (p<0,05) la proliferación celular en comparación con el control. Sin embargo, hubo diferencias moderadas entre
ellos. El resveratrol fue el inhibidor más fuerte en diferentes momentos y dosis. También la
actividad de caspasa-3 y caspasa-7 era significativamente mayor (p<0,05) que el control en la
presencia de todos los compuestos, pero la respuesta anterior se logró por el resveratrol. El
resveratrol, la quercetina y la morina fueron los únicos nutrientes que inhibían significativamente la expresión de ARNm de RA. Una vez más el resveratrol produjo la inhibición más alta
(90-250 veces menor que el control), seguido por morina (67-100 veces) y quercetina (55-91
veces).
Conclusiones: Todos los polifenoles estudiados mostraron importantes efectos antiproliferativos y apoptosis inducida cuando se añadieron al cultivo de células LNCaP. Confirmamos que el
resveratrol, la morina y la quercetina pueden lograr tal efecto a través de la reducción de expresión de RA. Los efectos sinérgicos de estos compuestos y su potencial para prevenir la progresión
del cáncer de próstata hormono-dependiente merecen ser estudiados en profundidad.
© 2014 AEU. Publicado por Elsevier España, S.L. Todos los derechos reservados.
Introduction
Prostate cancer is the most common neoplasm diagnosed
in men in industrialized countries.1 Since the introduction of prostate-specific antigen (PSA) screening test in the
late 1980s and because of the increasing public awareness
with respect to this disease in the early 1990s, the number of new cases has increased dramatically during the last
decade.1 Besides PCa screening,2 other possible causes for
its increasing incidence are aging of the population, the
high consumption of meat and fat and the higher rates of
obesity.3
In Mediterranean countries, with a high intake of
olive oil, fruits, vegetables and wine, the incidence of
prostate cancer is much lower than that in other western countries.4,5 All those food products contain important
amounts of phenolic compounds, commonly referred to as
polyphenols.6 Flavonoids are the most common polyphenolic compounds present in vegetables. An important effect
of flavonoids is their strong antioxidant activity: they
are scavengers of oxygen-derived free radicals which are
involved in tumor promotion.2 Flavonoids have also shown
many biological properties that may account for cancer
chemoprevention as apoptosis enhancement, cell growth
arrest, inhibition of DNA synthesis, and modulation of signal
transduction pathways.7 The authors conducted a previous study that showed that some red wine polyphenols
inhibited the proliferation and induced apoptosis in human
prostate cancer LNCaP cells cultured in medium containing androgens8 and also in murine bladder cancer cell line
MB-49.9
Polyphenols are usually absorbed from the upper gastrointestinal tract, showing an increased uptake at the liver,
heart and kidney.10 A regular ingestion of polyphenols is necessary to obtain biologically important concentrations.11 Red
wine is a rich source of polyphenolic substances and more
than 200 individual phenolic compounds have been identified catechin, quercetin, phenolic acid, and resveratrol
being the most important.11,12
On the other hand, evidence has shown that androgens
are involved in the development and progression of prostate
cancer,13 and their biological effects in the prostate are
mediated by the AR, a ligand-activated transcription factor
of the nuclear receptor superfamily.14 Most of the molecular
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Resveratrol and LNCaP cell
mechanisms responsible for the development of recurrent
hormone-refractory tumors involve alterations in the function of the AR and its complex signaling pathways.14 Thus,
the AR could still play a role in the development of prostatic
malignancy and strategies to minimize the AR function are
expected to play a beneficial role. We investigate the effect
of some red wine polyphenols on cell proliferation, apoptosis, and androgen receptor expression in human prostate
cancer LNCaP cells.
Materials and methods
Cell cultures and treatments
The androgen-responsive LNCaP human prostate cancer cell
line (American Type Culture Collection, Rockville, MD, USA)
was used. This cell line shows functional AR.15 It was
maintained in RPMI 1640 (Gibco BRL, Barcelona, Spain)
supplemented with 5% heat-inactivated fetal bovine serum
(FCS, LINUS, Cultek, Madrid, Spain), 100 U/ml penicillin G,
0.1 mg/ml streptomycin, 50 ␮g/ml gentamicin, and 2 mM
l-glutamine (Gibco BRL, Barcelona, Spain). All cells were
cultured in a humidified atmosphere of 95% air and 5%
CO2 at 37 ◦ C. For the assays, the FCS in the medium was
replaced by 5% charcoal-stripped FCS (cFCS, LINUS, Cultek, Madrid, Spain) and the cells were treated with several
concentrations of the following polyphenols: tannic acid
(1---10 ␮M), gallic acid (1---10 ␮M), quercetin (1---10 ␮M), rutin
(25---75 ␮M), morin (25---75 ␮M) and resveratrol (5---25 ␮M)
during 24, 48, 72 and 96 h. All compounds were purchased
from Sigma---Aldrich (St. Louis, MO, USA) and dissolved
in either ethanol (tannic acid, gallic acid, quercetin and
resveratrol) or DMSO (rutin and morin) and stored at −20 ◦ C.
The final concentrations used for different experiments
were prepared by diluting the stock with RPMI before use.
Non-treated LNCaP cells in a carrier solvent (0.1% DMSO or
ethanol) were used as control.
Assay for cell proliferation
Cells were subcultured into a 96-well plate with 1 × 103
cells per well in 100 ␮l medium. After overnight incubation, the medium in each well was discarded and replaced
by fresh medium with the above-mentioned polyphenols at
the indicated concentrations. The medium was changed and
the same polyphenol concentration added every 24 h. The
results were obtained after 24, 48, 72 and 96 h of incubation.
The extent of proliferation was assessed using a colorimetric immunoassay (Cell Proliferation ELISA BrdU, Roche,
Barcelona, Spain). This technique is based on the incorporation of the pyridine analog bromodeoxyuride (BrdU)
instead of thymidine into the DNA of proliferating cells.
This BrdU is detected by immunoassay, by measuring the
absorbance at 450 nm in an ELISA microplate auto reader
(Bio-Tek Instruments, Winoonski, USA). Absorbance values
directly correlate with the amount of DNA synthesis and
therefore with the number of proliferating cells. Experiments were repeated a minimum of three times and the
results expressed in absorbance units (AU).
399
Caspase-3/7 activity assays
The cells were grown in 6-well plates at a density of 1 × 105
cells per well and treated with the highest doses of each
polyphenol used in the proliferation study. At 24, 48, 72
and 96 h of treatment, the media was aspirated and the
cells were washed once with PBS. Then we added M-PER
mammalian protein extraction reagent (Pierce, Rockford,
IL, USA) with fresh protease inhibitor cocktail (Boehringer
Mannheim, Germany). The cells were gently stirred for 5 min
and the lysate was collected in a microfuge tube. The lysate
was cleared by centrifugation at 13,000 rpm for 10 min and
the supernatant was either used or immediately stored at
−80 ◦ C. The protein concentration was determined by BCA
Protein Assay using the manufacturer’s protocol (Pierce,
Rockford, IL, USA).
For apoptosis quantification we used a luminescent assay
that measures caspase-3 and -7 activities (Caspase-Glo 3/7
Assay. Promega, Madison, WI, USA). Briefly, the assay provides a proluminescent substrate for caspase 3/7 in a
reagent optimized for caspase activity, luciferase activity, and cell lysis. The addition of reagent results in cell
lysis, caspase cleavage of the substrate, and generation of
a ‘‘glow-type’’ luminescent signal produced by luciferase.
Luminescence is proportional to the amount of caspase
activity present. For this assay we used 1 ␮g protein for each
determination.
Androgen receptor real-time RT-PCR
RNA was prepared from cell samples using Rneasy (Qiagen, Valencia, CA, USA) according to the manufacturer’s
instructions. The RNA was then quantitated using a spectrophotometer (absorbance to 260/280 nm). To analyze the
expression of AR by real-time PCR, 1 ␮g of total RNA from
each sample was used for first-strand cDNA using random
primers and Moloney murine leukemia virus (MMLV) reverse
transcriptase (Sigma, St. Louis, MO, USA). All PCR reactions were performed using a fluorescence temperature
cycler (iCycler, Bio-Rad Laboratories, Hercules, CA, USA)
with 25 ␮l reaction mixture containing 12.5 ␮l Universal PCR
Master Mix (Applied Biosystems, Madrid, Spain), 1.25 ␮l of
a mix of unlabelled PCR primers (AR or 18S) and Taqman
MGB probe (Assays-on-Demand, Applied Biosystems, Madrid,
Spain). PCR reactions were cycled according to the manufacturer’s instructions. To determine the relative expression
of AR in each sample, the results were normalized to 18S
(used as an internal standard) and the comparative CT
method was used (User Bulletin #2 (2001), Applied Biosystem, Madrid, Spain). For the comparative CT calculation to
be valid, the efficiency of the target amplification should
approximately equal the reference amplification. Finally,
template dilution was made for AR (range: 0.025---1 ng) and
18S (range: 0.001---0.025 ng) and the PCR efficiency of the
target amplification was approximately equal to the reference amplification.
Cells were treated with control and the highest concentrations of different polyphenols for 24 and 48 h. At
the end of each incubation period, total RNA was reversetranscripted to cDNA and quantitative real-time PCR was
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400
A. Ferruelo et al.
b
800
800
Control
700
1µM
700
600
5µM
10µM
600
AU (450-630 nm)
AU (540-630 nm)
a
500
400
300
Control
25µM
50µM
75µM
500
400
300
200
200
100
100
0
0
24H
48H
72H
96H
24H
800
d
Control
800
1µM
700
25µM
600
5µM
10µM
600
50µM
75µM
500
400
300
100
48H
72H
24H
96H
Incubation time (h)
48H
96H
Incubation time (h)
f
Control
1µM
5µM
10µM
AU (450-630 nm)
AU (450-630 nm)
72H
0
24H
600
96H
300
200
700
72H
400
100
800
96H
500
200
0
e
72H
Control
700
AU (450-630 nm)
AU (450-630 nm)
c
48H
Incubation time (h)
Incubation time (h)
500
400
300
800
5µM
600
10µM
25µM
500
400
300
200
200
100
100
0
Control
700
0
24H
48H
72H
96H
24H
Incubation time (h)
48H
Incubation time (h)
Figure 1 Effect of polyphenols on cell proliferation of human prostate cancer cells (LNCaP) over 96 h of incubation. Cells were
treated with different concentrations of polyphenols (␮M) and proliferation cell determined by the cell proliferation ELISA BrdU
assay. (a) Tannic acid, (b) rutin, (c) gallic acid, (d) morin, (e) quercetin and (f) resveratrol. Values are means ± SE of a typical
experiment in triplicate. *P < 0.05 and **p < 0.01 vs. control.
performed. Values were calculated as mean ± SE of a typical
experiment performed in triplicate.
and 7 activity were carried out with Student’s unpaired t
test. Significance level was reached when p < 0.05.
Results
Statistical analysis
Effect of polyphenols on cell proliferation
One-way ANOVA with Dunnett t-test post hoc analysis was
used to compare and AR expression analysis between treated
and control groups. Comparisons with regard to caspase 3
Fig. 1 shows the effect of different polyphenols on LNCaP
cells proliferation. Although all the tested polyphenols
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Resveratrol and LNCaP cell
401
a
b
250
250
Control
10µM
200
RLU X 1000
RLU X 1000
200
150
100
50
48h
72h
c
24h
48h
72h
48h
72h
48h
72h
d
160
140
Control
10µM
140
120
RLU X 1000
120
RLU X 1000
100
0
24h
100
80
60
80
60
40
20
20
0
24h
140
120
Control
75µM
100
40
0
48h
72h
24h
f
Control
10µM
300
Control
25µM
250
100
RLU X 10000
RLU X 1000
150
50
0
e
Control
75µM
80
60
40
200
150
100
50
20
0
0
24h
48h
72h
24h
Figure 2 Polyphenol-induced apoptotic cell death in LNCaP cells. Cells were treated with the highest concentrations of polyphenols for 24, 48 and 72 h. At the end of the different incubation period, cells were lysated and analyzed for activity of caspase-3 and
-7 with the aid of a luminometric assay. (a) Tannic acid, (b) rutin, (c) gallic acid, (d) morin, (e) quercetin and (f) resveratrol. Values
are means ± SE of a typical experiment performed in triplicate. *P < 0.05 and **p < 0.01 vs. control.
successfully inhibited cell proliferation, there were moderate differences between them. Resveratrol was the
strongest inhibitor at different times and doses. Cell proliferation was significantly inhibited by tannic and gallic
acid at 5 and 10 ␮M at any incubation time. The weakest effect was produced by quercetin; however, it was
able to inhibit cell proliferation at 96 h (as the rest of
nutrients).
Effect on caspase-3 and caspase-7 activity
We added flavonoids at highest concentrations that inhibited
proliferation in this study, during 24, 48, and 72 h. The
cell lysates were then analyzed for caspase-3/7 activity by
means of a luminometric assay (Fig. 2). All the polyphenols
tested significantly increased the caspase-3/7 activity. At
24 h, the activity of both enzymes increased and was highest at 72 h after the addition of resveratrol. With the rest
of polyphenols, the activity of caspase-3/7 increased at 48 h
and it also reached its maximum at 72 h.
Effect on androgen receptor expression
To determine changes in AR mRNA expression after polyphenol exposure, real time RT-PCR was used. LNCaP cells
were exposed to a nutrient concentration known to induce
caspase-3/7 activity as revealed in the previous phase
of the present study. As shown in Table 1, quercetin,
morin and resveratrol were the only nutrients that significantly inhibited AR mRNA expression. Again, resveratrol
produced the highest inhibition (90---250 times less than control). Quercetin and morin also produced inhibition, but
the lower magnitude (55---91 and 67---100 times, respectively).
Discussion
Epidemiological studies have demonstrated a considerable
geographical variation in the age-adjusted incidence of
prostate cancer and environmental agents play a very important role.5,16 Many studies have addressed the relationship
between nutrients (some of them included in the present
study) and anticancer protection.4,17 Nutritional agents as
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402
A. Ferruelo et al.
Table 1 Relative quantification of androgen receptor mARN by real-time RT-PCR in LNCaP cells treated with the highest
concentrations of polyphenols for 24 and 48 h.
Nutrients
48 h
Mean
CT RA
Mean
CT 18S
C
T10
M75
G10
R75
Q10
RV25
28.4
26.83
32.9
27.53
27.87
32.62
33.4
±
±
±
±
±
±
±
0.1
0.21
0.1
0.06
0.15
0.15
0.1
25.63
24.47
24.07
23.93
25.7
24.07
24.17
±
±
±
±
±
±
±
0.21
± 0.21
0.21
0.15
0.17
0.15
0.1
C
T10
M75
G10
R75
Q10
RV25
28.07
27.97
34.2
28.57
28.17
34.53
36.07
±
±
±
±
±
±
±
0.12
0.06
0.2
0.06
0.06
0.21
0.15
24.8
24.8
24.3
24.93
24.8
24.77
24.7
±
±
±
±
±
±
±
0.1
0.1
0.2
0.06
0.1
0.15
0.1
CT
(AR-18S)
CT
CT −CT (control)
RA levels
(2−CT )
2.77
2.34
8.84
3.57
2.17
8.57
9.23
±
±
±
±
±
±
±
0.23
0.29
0.23
0.16
0.23
0.22
0.12
0.0
−0.4
6.06
0.83
−0.6
5.8
6.46
±
±
±
±
±
±
±
0.23
0.29
0.23
0.16
0.23
0.22
0.12
1.0
1.32
0.015
0.58
1.52
0.018
0.011
3.27
3.17
9.9
3.63
3.37
9.77
11.37
±
±
±
±
±
±
±
0.15
0.12
0.28
0.08
0.12
0.26
0.18
0.0
0.1
6.63
0.36
0.1
6.5
8.1
±
±
±
±
±
±
±
0.15
0.12
0.28
0.08
0.12
0.26
0.18
1.0
1.07
0.01
0.78
0.94
0.011
0.004
CT ; cycle threshold; C: control; T: tannic acid; M: morin; G: gallic acid; R: rutin; Q: quercetin; RV: resveratrol.
soy isoflavones, lycopene, vitamin E, selenium, vitamin D,
and other polyphenols have been found to inhibit proliferation or to induce apoptosis in some prostate cancer cell
lines. Grapes and wine are known natural sources of dietary
polyphenols, and tannins as tannic and gallic acid, both
present in red wine, have also been found to have antiinitiating and antipromoting properties.18 In a previous study
the authors demonstrated the inhibitory effect of some of
the mentioned polyphenols on the proliferation of LNCaP
cells.8
In our study we focussed on six polyphenols (quercetin,
rutin, morin, resveratrol, gallic acid and tannic acid) that
are invariably present in red wine, an important constituent
of Mediterranean diet. We have demonstrated a growth inhibition of LNCaP cells at different concentrations and time
of exposure to those nutrients. Resveratrol, tannic and gallic acid were the most successful inhibitors of the growth
of LNCaP cells, while quercetin, rutin and morin required
higher concentrations and longer exposure times to inhibit
cell proliferation. Our findings were consistent with reports
available in the literature.19,20 Suppression of cell proliferation through alterations in division and apoptosis has been
documented as well.21 Caspase-3/7 ultimately appears to
function downstream in the apoptosis pathway and is associated with the cleavage of many critical cellular substrates.
In our experiment we found an increase in the activity of
both enzymes in LNCaP cells after exposure to the studied polyphenols. This finding supports apoptosis as a major
mechanism of polyphenol-induced antiproliferative effects
in our study.8,20
On the other hand, we used a real-time RT-PCR method
for relative quantification of AR gene expression. We
observed a reduced expression of AR mRNA in LNCaP
cells induced by the flavonoids rutin and morin and the
phytoalexin resveratrol at 24 and 48 h after exposure.
Other studies have demonstrated the inhibition capacity of
resveratrol and quercetin over AR mRNA expression, but
not by the flavonoid morin.22 LNCaP cells are known to
be androgen sensitive and proliferate in response to AR
activation, and down-regulation of the AR protein using
antisense knockdown produced an inhibition of growth
and an increase of the apoptosis rate.23 This could indicate that the effects of resveratrol, quercetin and morin
could be, at least in part, mediated by regulation of
the expression of the AR gene. On the other hand, such
hypothesis is not likely to be valid for the other studied nutrients. Androgens are involved in the development
and progression of prostatic neoplasia and AR is the
essential mediator for the androgen action. This nuclear
receptor is activated by its ligands, testosterone and 5␣-dihydrotestosterone, and consequent dissociation by the
heat shock proteins. The activation process includes recruitment of coactivators. Finally, activation of AR upregulates
the transcription of genes containing androgen-response
elements in their promoters. So, the suppression of androgen signaling is a therapeutic target for prostate cancer.
Resveratrol inhibits the function of the androgen receptor and also modulates AR transcriptional activity,24 but
this is not the only nutrient to downregulate expression
of androgen synthesizing genes as revealed by studies conducted with pomegranate and green tea polyphenols in
LNCaP-AR, LNCaP 104-S, LNCaP R1Ad and LNCaP 104-R1
cells.25,26
Apart from the reduction of AR gene expression, cell
cycle arrest and promotion of apoptosis, many other mechanisms are likely to mediate the action of polyphenols
on prostate cancer cell lines. Resveratrol, quercetin and
other nutrients have also been reported to inhibit the
hedgehog cascade and GLI-1 expression and suppress the
in vitro growth of androgen-dependent LNCaP and androgenindependent AI PC3 cell lines.27
According to the National Cancer Institute, about 400
compounds are considered potentially chemopreventive
agents.28 Data regarding the accumulated evidence of
polyphenolic substances and flavonoids limiting presentation and or progression of prostate cancer in humans
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Resveratrol and LNCaP cell
are very scarce, although resveratrol, epigallocatechin3-gallate, curcumin, genistein, and quercetin are under
clinical evaluation.29 Preclinical investigations have established the anticarcinogenic effects induced by some dietary
compounds using TRAMP and PTEN knockdown transgenic
mouse models of prostate cancer and also the potential to
reverse treatment resistance.30---32
In conclusion, the polyphenols studied have important
antiproliferative effects and can induce apoptosis when
added to LNCaP cells in culture. Resveratrol appears to
be the most potent of these compounds and one of the
mechanisms involved in the effect of this substance is
mediated through a reduced expression of the androgen
receptor. Very likely the effect of resveratrol is synergistic with that of other polyphenols, such as morin and
quercetin. The results presented provide a rationale to more
deeply study the in vivo effects of these nutrients. Possibly, with the aid of animal and epidemiological studies,
more firm recommendations about the intake of these substances as chemopreventive agents could be made in the
future.
Funding
This work was funded by the Beca Fundación para la
Investigación en Urología (FIU) of the Spanish Urological
Association.
Conflict of interest
The authors declare that they have no conflict of interest.
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