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Carcinogenesis vol.29 no.1 pp.62–69, 2008
doi:10.1093/carcin/bgm239
Advance Access publication November 4, 2007
Resveratrol is pro-apoptotic and thyroid hormone is anti-apoptotic in glioma cells:
both actions are integrin and ERK mediated
Hung-Yun Lin
1,2,
, Heng-Yuan Tang
1
, Travis Keating
1
,
Yun-Hsuan Wu
1
, Ai Shih
2
, Douglas Hammond
2
, Mingzeng
Sun
1
, Aleck Hercbergs
3
, Faith B.Davis
1
and Paul J.Davis
1,2
1
Signal Transduction Laboratory, Ordway Research Institute, 150 New
Scotland Avenue, Albany, NY 12208, USA,
2
Research Service, Stratton
Veterans Affairs Medical Center, Albany, NY, USA and
3
Department of
Radiation Oncology, The Cleveland Clinic, Cleveland, OH, USA
To whom correspondence should be addressed. Tel: þ1 518 641 6428;
Email: hlin@ordwayresearch.org
The stilbene resveratrol (RV) initiates p53-dependent apoptosis
via plasma membrane integrin aVb3 in human cancer cells.
A thyroid hormone (L-thyroxine, T
4
) membrane receptor also
exists on aVb3. Stilbene and T
4
signals are both transduced by
extracellular-regulated kinases 1 and 2 (ERK1/2); however, T
4
promotes cell proliferation in cancer cells, whereas RV is pro-
apoptotic. Thyroid hormone has been shown to interfere with
RV-induced apoptosis. However, the mechanisms involved are
not fully understood. In this study, we examined the mechanism
whereby T
4
inhibits RV-induced apoptosis in glioma cells. RV
activated conventional protein kinase C and ERK1/2 and caused
nuclear localization of cyclooxygenase-2 (COX-2), consequent p53
phosphorylation and apoptosis. RV-induced ERK1/2 activation is
involved in not only COX-2 expression but also nuclear COX-2
accumulation. NS-398, a COX-2 inhibitor, did not affect ERK1/2
activation, but reduced the nuclear abundance of COX-2 protein
and the formation of complexes of nuclear COX-2 and activated
ERK1/2 that are required for p53-dependent apoptosis in RV-
treated cells. T
4
inhibited RV-induced nuclear COX-2 and cyto-
solic pro-apoptotic protein, BcLx-s, accumulation. Furthermore,
T
4
inhibited RV-induced apoptosis by interfering with the inter-
action of nuclear COX-2 and ERK1/2. This effect of T
4
was pre-
vented by tetraiodothyroacetic acid (tetrac), an inhibitor of the
binding of thyroid hormone to its integrin receptor. Tetrac did
not, in the absence of T
4
, affect induction of apoptosis by RV.
Thus, the receptor sites on aVb3 for RV and thyroid hormone
are discrete and activate ERK1/2-dependent downstream effects
on apoptosis that are distinctive.
Introduction
Thyroid hormone (L-thyroxine, T
4
; 3,5,3#-triiodo-L-thyronine) is
a proliferation factor in vitro for a rat glioblastoma cell line (C6)
via a cell-surface receptor for the hormone on integrin aVb3 (1). This
receptor is at or near the arginine–glycine–aspartate (RGD) recogni-
tion site on the integrin that is involved in the interactions of the
integrin with extracellular matrix proteins. The intracellular domain
of the integrin may activate extracellular-regulated kinases 1 and 2
(ERK1/2) (2–4) and we have shown that T
4
rapidly increases cellular
ERK1/2 activity via the integrin (5). Short integrin antagonist peptides
that contain the RGD sequence have been designed as tools to dem-
onstrate the role of integrins in transducing the signals of a number of
extracellular matrix proteins. RGD peptides block thyroid hormone
actions mediated by the integrin (1).
Resveratrol (RV), a polyphenolic antioxidant enriched in grape
skin, has been shown to cause apoptosis in a number of different
cancer cell lines (6,7) and to suppress cancer growth in vivo in animal
models (8). We have recently described a RV receptor on integrin
aVb3 using probes such as antibody to aVb3 and an siRNA of
aVb3 construct to interfere with integrin-mediated actions of the
stilbene (9). RV (1–10 lM) induces p53-dependent apoptosis in breast
(10), prostate (11,12) and thyroid cancer (13) cells in culture. RV
stimulates Ser-15 phosphorylation of p53 by a mitogen-activated pro-
tein kinase-dependent pathway (ERK1/2), and this step in RV-treated
cells is required for apoptosis to occur (13). Other kinases such as
conventional protein kinase C (cPKC), p38 and c-jun N-terminal
kinase are also activated in RV-treated cells (12,14). On the other
hand, nuclear factor-jB is inhibited by RV (15). Growth factors such
as epidermal growth factor (12) and hormones such as estrogen have
also been shown to activate ERK1/2 in selected human cancer cells
(16), but cancer cells treated with these agents may no longer undergo
apoptosis when treated with RV (10). We have shown that thyroid
hormone (T
4
,10
7
M total hormone concentration, 10
10
M free T
4
)
induces ERK1/2 activation in the CV-1 green monkey kidney epithe-
lial cell (17) and RV did not activate ERK1/2 in the same cell line until
the stilbene concentration reached 100 lM (Lin HY et al., unpub-
lished results). On the other hand, thyroid hormone, specifically T
4
,
blocks the pro-apoptotic effect of RV in thyroid cancer cells (18).
However, the mechanisms underlying the inhibitory effect of thyroid
hormone on RV-induced apoptosis are not fully understood.
We show here in glioma cells that while RV and thyroid hormone
both bind to the plasma membrane integrin aVb3 and activate ERK1/2,
they do so via different ligand-binding sites, leading to the disparate
effects seen in these cells. While T
4
stimulates glial cell proliferation,
the p53-dependent action of RV leads to apoptosis. We have shown
elsewhere (19) that RV-associated apoptosis requires a pool of induc-
ible cyclooxygenase-2 (COX-2) in the nucleus, upstream of p53. The
inhibition of apoptosis by thyroid hormone in RV-treated cells is
initiated at the plasma membrane integrin receptor for the hormone;
this leads to suppression of the nuclear interaction of COX-2 protein
and activated ERK1/2, which is essential to the pro-apoptotic action
of RV. That is, this nuclear complex is formed in glioma cells exposed
to RV, alone, but does not occur in T
4
-treated cells or in cells in-
cubated with both RV and T
4
.
Materials and methods
Materials
T
4
, tetraiodothyroacetic acid (tetrac) and RGD and RGE peptides were ob-
tained from Sigma Chemical Co. (St Louis, MO). T
4
and tetrac were prepared
as a stock 10
4
M solution in 0.04 N KOH and 4% propylene glycol. RV was
purchased from Calbiochem (San Diego, CA) and dissolved in ethanol as a 100
mM stock solution. The PKC agonist phorbol 12-myristate 13-acetate (PMA)
was obtained from Sigma Chemical Co. and prepared as a 100 lg/ml PMA in
dimethyl sulfoxide. A specific PKC inhibitor, CGP41251 (CGP) obtained as
a gift from Novartis Pharma (Basel, Switzerland) was prepared as a stock 100
mM solution in dimethyl sulfoxide. The specific ERK1/2 kinase inhibitor,
PD98059, purchased from Calbiochem was prepared as a stock 30 mM solu-
tion in dimethyl sulfoxide. The final concentrations of solvents in which re-
agents were dissolved were tested for activity and did not affect the
experimental outcomes. Polyclonal rabbit anti-phospho-ERK1/2 (pERK12)
was obtained from Cell Signaling (Beverly, MA), monoclonal mouse anti-
proliferating cell nuclear antigen and anti-BcLx-s from Santa Cruz Biotech-
nology (Santa Cruz, CA) and polyclonal rabbit anti-mouse COX-2 from
Cayman (Ann Arbor, MI). Goat anti-rabbit IgG and rabbit anti-mouse IgG
were obtained from Dako (Carpinteria, CA), and the chemiluminescence
reagents (ECL kit) and [
3
H]-thymidine from Amersham (Piscataway, NJ).
Nucleosome enzyme-linked immunoadsorbent assay (ELISA) kit was pur-
chased from Calbiochem.
Abbreviations: COX-2, cyclooxygenase-2; cPKC, conventional protein
kinase C; ELISA, enzyme-linked immunoadsorbent assay; ERK1/2,
extracellular-regulated kinases 1 and 2; FBS, fetal bovine serum; pERK12,
phospho-ERK1/2; PMA, phorbol 12-myristate 13-acetate; RGD, arginine–
glycine–aspartate; RGE, arginine-glycine-glutamate; RV, resveratrol; TCA,
trichloroacetic acid; tetrac, tetraiodothyroacetic acid; T
4
,L-thyroxine.
ÓThe Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 62
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Cell culture
The rat glioma C6 cell line and human glioblastoma U87MG cell line were
purchased from American Type Culture Collection (Manassas, VA). The rat
glioma GL261 cells were obtained from the Roswell Park Cancer Institute,
Buffalo, NY. C6 cells were maintained in F12K medium supplemented with
18% fetal bovine serum (FBS), U87MG cells were maintained in Minimal
essential medium supplemented with 10% FBS and GL261 cells were main-
tained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS.
All cell cultures were maintained in a 5% CO
2
/95% air incubator at 37°C. Prior
to treatment, cells were placed in 0.25% hormone-stripped FBS-containing
medium for 2 days. The free T
4
concentration was 0.7 10
10
M, as directly
measured in aliquots of representative culture media of hormone-treated cells
by the Clinical Chemistry Laboratory of Albany Medical Center Hospital,
Albany, NY (20).
Apoptosis/nucleosomes
An early event in apoptosis is DNA fragmentation followed by release of
nucleosomes into the cytoplasm. The nucleosome is the basic unit of chromatin
and results from the ordered association of histones and DNA (21). The double-
antibody sandwich ELISA is based upon the specific recognition of
nucleosomes by a pair of monoclonal antibodies and detects cytoplasmic
nucleosomes onto the ELISA plate. Cells were treated with different reagents
for 48 h. The medium was harvested, spun down and pellets were washed twice
with phosphate-buffered saline. Pelleted cells were lysed. Supernatants were
collected and stored for at least 18 h at 20°C. From each appropriately diluted
sample, 100 ll were added to a 96-well plate coated with a DNA-binding
protein and incubated at room temperature for 3 h. After three washes with
wash buffer, detector antibody was added for 1 h. Streptavidin conjugate was
added and incubated for 0.5 h before adding substrate. Plates were read at 450 nm.
Immunoblotting and immunoprecipitation
Nuclear and cytosol protein extracts were prepared, quantitated and separated
on discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis,
and then transferred by electroblotting to nitrocellulose membranes (Millipore,
Bedford, MA), as we have described previously (9–13). For immunoprecipi-
tation, 200 lg of each nucleoprotein sample was exposed to anti-pERK1/2
and immunoprecipitated proteins separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, and then transferred by electroblotting to
nitrocellulose membranes (Millipore). Membranes were blocked with 5% milk
in Tris-buffered saline containing 0.1% Tween, and then incubated with se-
lected antibodies overnight. Secondary antibodies were either goat anti-rabbit
IgG or rabbit anti-mouse IgG (1:1000), depending on the origin of the primary
antibody. Immunoreactive proteins were detected by chemiluminescence
(ECL, Amersham Life Science, Arlington Heights, IL) and integrated optical
density of bands was quantitated by phosphoimaging and with ImageQuant
software on a Storm 860 Phosphorimager (Molecular Dynamics, Sunnyvale,
CA) as described previously (19).
Confocal microscopy
Exponentially growing C6 glioma cells were seeded in slide chambers. After
exposure to 0.25% stripped FBS-containing medium for 2 days, cells were
either treated with 20 lM RVin the presence or absence of the cPKC inhibitor,
CGP or the MEK inhibitor, PD for 24 h. In other sets of experiments, U87MG
cells and GL261 cells were treated with 20 lMRV,10
7
MT
4
or both for 24 h.
Cells were fixed with 4% formaldehyde in acetone for 30 min and then per-
meabilized in 0.06% Triton X-100 for 30 min. The cells were incubated with
monoclonal antibody to COX-2 followed by Alexa-488-labeled goat anti-
mouse antibody and the signal revealed using the Histostain SP kit as recom-
mended by the manufacturer (Zymed–Invitrogen, Carlsbad, CA). Nuclear
staining with propidium iodide was also employed. Cells were examined under
250 magnification.
Radiolabeled thymidine incorporation
Aliquots of cells were incubated with 1 lCi [
3
H]-thymidine (final concentra-
tion, 13 nM) in a 24-well culture tray for 16 h in the presence or absence of
reagents as indicated. Cells were then washed twice with cold phosphate-
buffered saline, after which 5% trichloroacetic acid (1 ml) was added and
the plate was kept at 4°C for 30 min. The precipitate was then washed twice
with cold ethanol, and 1 ml of 2% sodium dodecyl sulfate was added to each
well. The trichloroacetic acid-precipitable radioactivity was quantitated in
a liquid scintillation counter.
Quantitation of results and statistical analysis
Immunoblot densities were measured with a Storm 860 Phosphorimager fol-
lowed by analysis with ImageQuant software (Molecular Dynamics). Student’s
t-test, with P,0.05 as the threshold for significance, was used to evaluate
results from three or more experiments.
Results
Thyroid hormone and RV, both ligands of integrin aVb3, have
competitive actions in C6 glial cells
In studies of rat glioma C6 cell proliferation, T
4
treatment increased
radiolabeled thymidine incorporation (Figure 1A). In contrast, RV
Fig. 1. Effects of RV, T
4
and tetrac on cell proliferation and apoptosis. (A)The
effect of 10
7
MT
4
and 1–50 lMRVon[
3
H]-thymidine incorporation was
measured as described in Materials and Methods. T
4
increased thymidine
incorporation by C6 glial cells. This effect was inhibited by RV in
a concentration-dependent manner (P,0.05 with 10 lMandP,0.01 with
50 lM). (B) C6 cells were treated with 10 lM RV in the presence or absence
of T
4
(10
8
and 10
7
M). RV-induced apoptosis (nucleosome ELISA) was
inhibited significantly by co-incubation with 10
8
to 10
7
MT
4
(P,0.01).
(C) The effect of T
4
on RV-induced apoptosis was examined in the presence
and absence of tetrac. The inhibition by T
4
of apoptosis induced by RV (lane 7
compared with lane 5, P,0.05) was blocked by the addition of tetrac (lane 8
versus lane 7, P,0.01), indicating that tetrac blocks the inhibitory effect of
T
4
, but does not block the effect of the stilbene.
Apoptosis in glioma cells
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Fig. 2. RV activates ERK1/2 and cPKC via binding to integrin aVb3 in C6 glioma cells. (A) Cells were treated with 10 lM RV for 4 h, in the presence or absence
of 5–500 nM RGD peptide or 500 nM arginine–glycine–glutamate (RGE) peptide (24 h). Concentration-dependent inhibition of RV-induced cPKC and ERK1/2
activation was seen with the RGD peptide (P,0.01), but no inhibition was seen with arginine–glycine–glutamate peptide. Lamin-B and b-actin immunoblots are
provided in this and subsequent figures as controls for gel loading of nucleoproteins and cytosolic proteins, respectively. The graphs in this and subsequent figures
represent the mean ± SD of band intensities normalized to a value of 1 in untreated cells, in three separate experiments. (B) C6 cells were treated with PMA
(100 ng/ml) for 24 h prior to addition of RV (10 lM) for 4 h. PMA treatment, alone, caused a significant increase in nuclear accumulation of nuclear factor-jB
subunits, both p65 and p50 (P,0.01). RV did not cause nuclear factor-jB (NFjB) accumulation, but did cause a significant increase in ERK1/2 activation and
nuclear translocation (P,0.01), which was reduced by 24 h pre-treatment with PMA (P,0.01). The levels of cytosolic phosphorylated PKCa/bin cells treated
with RV were not seen in cells depleted of PKC with PMA treatment. (C) Cells were treated with RV (10 lM, 4 h), in the presence or absence of the ERK1/2
activation inhibitor, PD98059 (PD, 3 or 30 lM, 4.5 h) or the PKCa/binhibitor, CGP41251 (CGP, 10 or 100 nM, 4.5 h). ERK1/2 activation associated with RV
treatment, shown in nuclear fractions, was inhibited by both PD and CGP. RV-induced phosphorylation of PKCa/bwas inhibited by CGP but not by PD (P,0.05,
comparing RV-treated cell fractions with corresponding samples treated with RVand either inhibitor).
H.-Y.Lin et al.
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(1–50 lM) caused apoptosis and thus suppressed thymidine uptake.
Further, the growth-stimulatory effect of T
4
on C6 cells was pro-
gressively diminished by increased concentrations of RV (Figure
1A). In studies of C6 cell apoptosis, T
4
alone had no appreciable
effect, whereas RV treatment produced apoptosis as expected
(nucleosome ELISA, Figure 1B). The deaminated T
4
analog tetrac
did not inhibit the effect on apoptosis of RV, alone (Figure 1C, lane 6),
but did inhibit T
4
suppression of RV-induced apoptosis (comparing
lanes 7 and 8). This finding suggests that T
4
and RV interact with the
integrin at discrete sites and that tetrac only binds to the T
4
-binding
site.
RV-stimulated ERK1/2 and cPKC activation in C6 cells is inhibited by
an integrin aVb3 recognition site RGD peptide
To illustrate the role of integrin aVb3 and consequent intracellular
signaling in RVaction, we measured the effect of RGD and arginine–
glycine–glutamate peptides on the activation of ERK1/2 and of cPKC
by RV. While control arginine–glycine–glutamate peptide did not
alter ERK1/2 or cPKC phosphorylation by the stilbene, increasing
concentrations of RGD peptide (5–500 nM) suppressed these effects
(Figure 2A). This finding is consistent with displacement of RV from
an integrin-binding site at or near the RGD recognition domain.
Fig. 3. Activation of the ERK1/2 pathway is required for RV-induced COX-2 nuclear accumulation in C6 cells. (A) Cells were treated with RV (10 lM) for 24 h,
with or without PD98059 (PD, 30 lM) or with PD for the last 12 h of RV treatment. Nuclear content of COX-2 increased with RV treatment, and this effect was
inhibited by PD (P,0.05), particularly after 24 h of inhibitor treatment. On the other hand, the accumulation of cytosolic COX-2 in RV-treated cells was
significantly enhanced by PD treatment (P,0.05) for 12 or 24 h. (B) C6 cells were treated with 10 lM RV in the presence or absence of CGP41251 (CGP) or
PD98059 (PD) for 24 h and then examined by confocal microscopy. RV caused nuclear accumulation of COX-2, indicated by the appearance of a yellow color due
to superimposition (co-localization) of COX-2 (green) and nuclear (red) images. In the presence of CGP (two middle panels) and PD (two panels at right), nuclear
COX-2 was not evident, although cytosolic COX-2 is still seen in some cells. Cells were viewed at 250 magnification. Images by differential interference contrast
are shown at the lower left of each group of four images, in order to clarify outlines of cells and nuclei.
Apoptosis in glioma cells
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In cells depleted of PKC by treatment with PMA (100 mg/ml) for 24 h,
RV-induced ERK1/2 activation and phosphorylation of PKCa/bwere
both inhibited (Figure 2B). There was no nuclear factor-jB activation
in RV-treated cells, although PMA alone increased both p50 and p65
subunit accumulation in nuclei (Figure 2B). RV-induced ERK1/2 ac-
tivation and nuclear translocation were inhibited by the ERK1/2 ac-
tivation inhibitor PD (Figure 2C). Treatment of cells with the cPKC
activation inhibitor, CGP, resulted in similar suppression of ERK1/2
phosphorylation; this inhibitor also partially inhibited phosphoryla-
tion of PKCa/b, although PD did not inhibit this latter effect. These
results indicate that ERK1/2 activation in RV-treated C6 cells is de-
pendent on cPKC activation as well as on MEK activation.
Activated ERK is associated in nuclei with COX-2 in C6 cells treated
with RV; this effect is required for apoptosis
We have shown that activated ERK1/2 is essential for RV-induced
COX-2 expression (19). Treatment of C6 glioma cells with RV caused
nuclear accumulation of COX-2, and inhibition of ERK1/2 activation
with PD for 24 h blocked this RV effect (Figure 3A). However, treat-
ment of C6 cells with PD for only the last 12 h of a 24-h RV exposure
did not inhibit COX-2 expression but partially inhibited nuclear ac-
cumulation of COX-2 and there was increased accumulation of COX-
2 in the cytosol. COX-2 levels increased in the cytosol of cells treated
with PD alone and in cells exposed to RV and PD; this suggests that
activation of ERK1/2 is required for nuclear accumulation of COX-2
in the presence of RV.
In confocal microscopy studies, we have also demonstrated ERK1/2
activation-dependent nuclear accumulation of COX-2 (green) in
RV-treated cells (Figure 3B). Untreated cells demonstrate a minimal
amount of COX-2 in cytosol. Cells treated with RV show increased
cellular COX-2, and in particular increased nuclear COX-2, indicated
by a yellow color formed by superimposition of the COX-2 green
color on a red nucleoprotein stain. Inhibitors of cPKC activation
and pERK1/2 activation both blocked nuclear COX-2 accumulation
in the presence of RV. These results confirm that RV-induced cPKC
activation is upstream of ERK1/2 activation and that inhibition of
either cPKC or ERK1/2 activation will inhibit ERK1/2-dependent
nuclear COX-2 accumulation in RV-treated C6 cells.
Demonstration of the interrelationships between COX-2 activity,
pERK1/2 accumulation and Ser-15 phosphorylation of p53
In the presence of the specific COX-2 inhibitor, NS-398 (0.1–10 nM),
RV-induced nuclear pERK1/2 content was unaffected (Figure 4).
However, the formation of co-immunoprecipitated nuclear complexes
of COX-2 and pERK1/2 in RV-treated cells was inhibited by NS-398,
but not by the non-specific COX inhibitor, indomethacin. NS-398 did
not affect pERK1/2 activation per se confirming that RV-induced
ERK1/2 activation is upstream COX-2 expression. On the other hand,
NS-398 reduced RV-induced Ser-15–p53 phosphorylation (Figure 4).
Apoptosis induced by RV was also inhibited by NS-398, but not
affected by indomethacin (Figure 4). These results also appear to
implicate RV-inducible nuclear COX-2 accumulation in the ERK1/2-
dependent activation (phosphorylation) of p53 that leads to cancer cell
apoptosis.
Thyroid hormone blocks RV action by disrupting COX-2–pERK1/2
complexing in C6 glial cells
Thyroid hormone inhibits RV-induced apoptosis in C6 cells (Figure 1),
although the two agonists share the same plasma membrane receptor
and some of the same signaling pathways. At what point in the sig-
naling sequence does the hormone block the action of RV? We have
demonstrated that T
4
stimulates ERK1/2 activation and cell prolifer-
ation in different glioma cell lines (1). In contrast, RV (10 lM) also
stimulates ERK1/2 activation (Figures 2 and 4), but causes nuclear
COX-2 accumulation and apoptosis. We therefore examined whether
T
4
blocks RV-induced nuclear COX-2 accumulation. Studies of
confocal microscopy using two different glioma cell lines, human
glioblastoma U87MG cells (Figure 5A, left panel) and rat glioma
GL261 cells (Figure 5A, right panel) indicate that RV caused nuclear
COX-2 accumulation and that this effect was blocked by T
4
. This
inhibitory effect of T
4
on RV-induced nuclear COX-2 accumulation
is also shown in immunoblots of nuclear fractions with a parallel
decrease in the pre-apoptotic protein, BcLx-s (Figure 5B).
Fig. 4. Inhibition of COX-2 activity suppresses nuclear accumulation of
COX-2, complexing of pERK1/2 and COX-2, nuclearSer-15-phosphorylated
p53 and apoptosis in RV-treated cells. C6 cells were treated with the COX-2
inhibitor NS-398 (0.1–10 lM) and 10 lM RV for 24 h. Aliquots of nuclear
extracts were immunoblotted with antibodies to COX-2, pSer15–p53,
pERK1/2 or ERK1/2, whereas additional nuclear extracts were
immunoprecipitated with anti-pERK1/2 and these solubilized
immunoprecipitates (IP) then separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and immunoblotted with anti-COX-2 to
demonstrate co-immunoprecipitation of pERK1/2 and COX-2. RV increased
nuclear content of COX-2, pERK1/2 and pSer15–p53, as well as co-
immunoprecipitation of pERK1/2 and COX-2 (lane 4). NS-398 had no effect
alone (lane 2) and did not inhibit RV-induced ERK1/2 activation (lanes 5–7).
However, nuclear co-immunoprecipitation of pERK1/2 with COX-2 was
inhibited, as were nuclear accumulation of COX-2 and of Ser-15-
phosphorylated p53 (lanes 5–7 compared with lane 4). The inhibitory effect
of NS-398 on RV-induced nuclear accumulation of COX-2 was significant
(P,0.01). In contrast, indomethacin was not inhibitory (comparing lanes
4 and 8). A representative IgG-H chain immunoblot served as a loading
control for the immunoprecipitates. Apoptosis (nucleosome ELISA) due to
RV treatment was also inhibited by NS-398, but not by indomethacin.
H.-Y.Lin et al.
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The thyroid hormone analog, tetrac, is known to inhibit T
4
binding
to plasma membranes and purified integrin aVb3 (5) and to inhibit
membrane-associated effects of T
4
on signal transduction pathways
(20,22). Results also indicate that tetrac reversed the inhibitory effect
of T
4
on RV-induced apoptosis (Figure 1C). We therefore examined
the actions of tetrac on the inhibitory effect of T
4
on nuclear accu-
mulation of pERK1/2, COX-2 and pSer15–p53 in C6 cells treated
with RV. T
4
treatment alone caused nuclear accumulation of
pERK1/2 and pSer15–p53, but not that of COX-2 (Figure 6, lane 3).
Tetrac partially blocked the hormone effects (lane 4). RV stimulated
nuclear accumulation of pERK1/2, COX-2 and pSer15–p53, as well
as the pro-apoptotic protein BcLx-s (Figure 6, lane 5). Tetrac did not
inhibit the action of RV on these parameters (lane 6 compared with
lane 5). When RV and T
4
were added together to cells (Figure 6, lane 7),
Fig. 5. Effect of T
4
on RV-induced nuclear COX-2 and cytosolic BcLx-s accumulation. (A) U87MG cells (left panel) and GL261 cells (right panel) were treated
with 20 lMRV,10
7
MT
4
or both for 24 h, and then examined by confocal microscopy. RV caused nuclear accumulation of COX-2, indicated by the appearance
of a yellow color due to superimposition of COX-2 (green) and nuclear (red) images. In the presence of T
4
, however, nuclear COX-2 was not evident in RV-treated
cells, although cytosolic COX-2 is still seen in some cells. Cells were viewed at 250 magnification. (B) U87MG cells (left panel) and GL261 cells (right panel)
were treated with 10 lM RV in the presence or absence of 10
7
MT
4
for 24 h. RV induced nuclear COX-2 and cytosolic pro-apoptotic protein BcLx-s
accumulation in both cell lines, and T
4
inhibited those effects of the stilbene (P,0.01).
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the stilbene effects on COX-2, pSer15–p53 and BcLx-s were in-
hibited, although pERK1/2 activation remained. In contrast, when
tetrac was added to both RV and T
4
, the inhibitory effects of T
4
on
RV action were blocked (lane 8), but the changes with RV were
similar to those shown in lane 5 with RV, alone, and with RV and
tetrac (lane 6).
Further, development of the immunoprecipitable nuclear complex of
inducible COX-2 and activated ERK1/2 in RV-treated cells (Figure 6,
lane 5) was inhibited by co-incubation with RV and T
4
(lane 7),
whereas T
4
alone did not induce this nuclear association (lane 3).
With the addition of tetrac, the inhibitory effect of T
4
on COX-2
and pERK1/2 complex formation was reversed (Figure 6, lane 8).
These results suggest that the nuclear immunoprecipitable complex
formation between pERK1/2 and COX-2 plays a crucial role in RV-
induced apoptosis. Further, this complex formation can be disrupted
by thyroid hormone, leading to inhibition of RV-induced pro-
apoptotic protein BcLx-s accumulation (Figure 6, lane 7) and apopto-
sis (Figure 1).
As indicated above, we know that both thyroid hormone and RV have
receptor sites on integrin aVb3 and that both hormone and stilbene
activate ERK1/2. The antagonism by T
4
of the action of RVon signal-
ing events that promote apoptosis and the reversal by tetrac of this
hormone effect, but not the activity of RV, point to the existence of
discrete hormone- and stilbene-binding sites on integrin aVb3.
Discussion
RV is a stilbene that has antitumor properties in glial cells (8). Using
a variety of human cancer cell lines, we have demonstrated that RV is
capable of inducing p53-dependent apoptosis via ERK1/2-mediated
serine phosphorylation at residue 15 of the oncogene suppressor pro-
tein (10–13). This is the case even when the cancer cells contain
mutated p53, as long as residue 15 remains a serine (12). We have
recently shown, however, that this clinically desirable action of
RV may be antagonized by estrogen in estrogen-responsive (ERa-
positive) tumor cells, such as MCF-7 breast cancer cells (10).
We have also determined that the growth of glioma cells in vitro is
importantly thyroid hormone dependent (1). We have shown previ-
ously that both T
4
and RVare ligands of integrin aVb3 (5,9) and both
activate ERK1/2 via the integrin. Both receptors are at or near the
RGD recognition site on the integrin, but our current findings do not
disclose direct competition between thyroid hormone and RV for
a single site on integrin aVb3. That is, tetrac, a specific inhibitor of
binding of T
4
to the integrin and of downstream actions of the hor-
mone initiated at the integrin (1,5) did not block the actions of RV.
We have identified a step downstream of ERK1/2 in the transduc-
tion of the RV and thyroid hormone signals at which a divergence
occurs that appears to account for the different biological effects of T
4
on cell growth and of RV leading to apoptosis. RV increases nuclear
abundance of COX-2, whereas T
4
, alone, has no effect on nuclear
COX-2 (Figures 5 and 6). However, the hormone decreases formation
of nuclear complexes between pERK1/2 and COX-2 in stilbene-
treated cells (Figure 6) and also inhibits RV-induced p53 phosphory-
lation. How this signal transduction step (nuclear complexing of
COX-2 and activated ERK1/2, upstream of activation of p53) is dif-
ferentially affected by T
4
and RV is not yet clear. Among the possible
explanations is that the pools of activated ERK1/2 that result from the
transduction of stilbene and thyroid hormone signals are discrete (23).
For example, the pro-apoptotic response of C6 cells to RV treatment
was blocked in vitro by physiological concentrations of thyroid hor-
mone. In the intact animal test model, normal circulating levels of
thyroid hormone may blunt an apparent response to the stilbene. Since
tetrac does not block stilbene action, however, a potential therapeutic
combination of tetrac—to block endogenous T
4
action at the
integrin—and RV would allow an unopposed pro-apoptotic effect of
the stilbene at its integrin receptor to be obtained.
Funding
Office of Research Development, Medical Research Service, Depart-
ment of Veterans Affairs Merit Award to H.-Y. Lin (5317-002) and P.J.
Davis (4999-0002); Charitable Leadership Foundation and Beltrone
Foundation.
Acknowledgements
The authors appreciate the contributions of Cassie Lin to the work described.
Conflict of Interest Statement: None declared.
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