From the Hormel Institute, University of Minnesota, Austin, Minnesota 55912
Received for publication, March 13, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Cyclooxygenases (COX) are rate-limiting enzymes
that catalyze the conversion of arachidonic acid to prostaglandins,
which are involved in many physiological and pathophysiological
responses. COX-2, one of two isoforms of COX, was recently found to
play an important role in carcinogenesis in many cell and tissue types. COX-2 inhibitors, which belong to the family of nonsteroidal
anti-inflammatory drugs, are believed to be effective in many
biological activities such as tumor chemoprevention because of their
inhibition of COX-2. However, in the present study we found that both
piroxicam, a general COX inhibitor, and NS-398, a COX-2 selective
inhibitor, effectively suppressed the activation of transcription
factor activator protein 1 (AP-1) induced by ultraviolet B (UVB) or
12-O-tetradecanoylphorbol-13-acetate in mouse epidermal JB6
cells. These COX-2 inhibitors could also inhibit
12-O-tetradecanoylphorbol-13-acetate-induced cell
transformation. UVB significantly increased AP-1 activity in
Cox-2 Cyclooxygenases (COX)1
are rate-limiting enzymes that catalyze the conversion of arachidonic
acid to prostaglandins, which are involved in many normal and
pathophysiological responses (1-3). Of the two known COX enzymes,
COX-1 is expressed in nearly all cells, whereas COX-2 is primarily
considered an inducible immediate-early gene product (4). COX-2 is
suggested to play an important role in tumorigenesis because of
epidemiological evidence showing that various types of cancers and
transformed cells tend to overexpress COX-2 constitutively (5-10).
Moreover, the tumor promoter
12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth
factor, and ultraviolet (UV) irradiation can induce COX-2 protein
expression in many tissues including the epidermis of skin and also in
epidermal keratinocytes (11).
COX inhibitors are known to function through the inhibition of the COX
enzymes. However, other potential mechanisms are still being considered
(12). COX inhibitors were reported to protect neurons against
hypoxia/reperfusion through mechanisms independent of COX (13). We and
others have shown that nonsteroidal anti-inflammatory drugs can block
mitogen-induced transcription activator protein 1 (AP-1) activity and
transactivation (14). Considering the important role of AP-1 in
tumoroigensis, the inhibition of AP-1 is likely to be one of the major
mechanisms involved in nonsteroidal anti-inflammatory drugs
chemopreventive effects.
The aim of this study was to determine whether a COX-2 highly selective
inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl)
methanesulfonamide (NS-398), or a general inhibitor of COX-2,
4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2-benzothiazine-3-carboxamid-1,1-dioxid (piroxicam), is effective in blocking AP-1 activation and whether COX-2
is involved in the inhibitory effects.
Cell Culture and Reagents--
AP-1 luciferase reporter plasmid
stably transfected mouse epidermal JB6 P+1 Luciferase Assay for AP-1 Transactivation--
Confluent
monolayers of cells were trypsinized, and 8000 viable cells suspended
in 100 µl of 5% FBS MEM were added to each well of a 96-well plate.
Plates were incubated at 37 °C in a humidified atmosphere of 5%
CO2, and 12-24 h later, cells were starved by culturing in
0.1% FBS MEM for 24 h before being treated or not treated with
different concentrations of NS-398 or piroxicam for 30 min. The cells
were then exposed to UVB (4 kJ/m2) or TPA (20 ng/ml)
independently. After an additional 12 h (for AP-1 treated with
UVB) or 24 h (for AP-1 treated with TPA) of culturing, the cells
were extracted with lysis buffer (0.1 M potassium phosphate buffer (pH
7.8), 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA), and luciferase activity was measured using a
luminometer (Monolight 2010). The results are expressed as relative
AP-1 (17).
Anchorage-independent Transformation Assay--
The effect of
NS-398, piroxicam, or SP600125 on TPA-induced cell transformation was
investigated in JB6 C1 41 cells. Cells (8 × 103/ml)
were exposed to TPA with or without the chemicals at the concentration
indicated in 1 ml of 0.33% basal medium Eagle (BME) agar containing
10% FBS over 3.5 ml of 0.5% BME agar medium containing 10% FBS. The
cultures were maintained in a 37 °C, 5% CO2 incubator for 4 weeks, and the cell colonies were scored by the methods described
by Colburn et al. (20). The effect of NS-398, piroxicam, or
SP600125 on transformation of JB6 C1 41 cells is presented as a
percentage inhibition compared with control.
Assay of AP-1 Activity in Vivo--
AP-1-luciferase
reporter transgenic mice were established as previously described (18).
All the mice were characterized by testing both the basal level and
UVB-induced level of luciferase activity. The AP-1-luciferase reporter
gene bearing male and female mice (6-9 weeks old) were randomly
divided into six groups (22 mice/group) for the treatment indicated in
the relevant figure legend. Five topical doses of NS-398 or piroxicam
dissolved in 300 µl of acetone were applied to the dorsal skin of the
mice over 8 days. The last of the five topical doses of NS-398 or
piroxicam was given 3 h before 10 kJ/m2 of UVB
irradiation. 48 h after UVB exposure the level of AP-1 luciferase
activity was measured from a skin biopsy. Negative control mice were
treated with acetone alone, and the luciferase activity of the biopsied
epidermis was measured as described previously (19).
DNA Binding Studies--
Electrophoretic mobility shift assays
were performed essentially as described (21). Nuclear protein extracts
were prepared by the modified method of Monick et al. (22).
Briefly, cells were harvested and disrupted in 500 µl of lysis buffer
(25 mM HEPES, pH 7.8, 50 mM KCl, 0.5% Nonidet
P-40, 100 µM dithiothreitol, 10 µg/ml leupeptin, 25 µg/ml aprotinin, and 1 mM phenylmethanesulfonyl fluoride). After centrifugation for 1 min (16,000 × g,
4 °C), the pelleted nuclei were washed once with 500 µl of wash
buffer (25 mM HEPES, pH 7.8, 50 mM KCl, 100 µM dithiothreitol, 10 µg/ml leupeptin, 25 µg/ml
aprotinin, and 1 mM phenylmethanesulfonyl fluoride). The
pelleted nuclei were resuspended in 150 µl of extraction buffer (25 mM HEPES, pH 7.8, 500 mM KCl, 100 µM dithiothreitol, 10 µg/ml leupeptin, 25 µg/ml
aprotinin, 1 mM phenylmethanesulfonyl fluoride, and 10%
glycerol) and shaken at 4 °C for 30 min. Nuclear extracts were
stored at Western Immunoblotting--
Cells growing in 100-mm cell culture
plates were lysed with 0.6 ml radioimmune precipitation assay buffer
(1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, and freshly added 100 µg/ml
phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 1 mM
sodium orthovanadate), and the samples for electrophoresis were
prepared according to the manufacturer's instructions (Santa Cruz
Biotechnology, Santa Cruz, CA). The protein concentration of each
sample was determined, and equal amounts of protein were loaded for SDS
gel electrophoresis. Immunoblotting for the proteins of COX-2, total
JNKs, and phosphorylated JNKs was carried out using antibodies against
COX-2 (Upstate Biotechnology, Lake Placid, NY), total JNKs, and the
phosphorylated sites of JNKs, respectively (Cell Signaling Technology,
Beverly, CA).
Transient Transfection--
Plasmids of COX-2,
COX-2-S516M, and COX-2-S516Q mutants in pOSML
were kind gifts from Dr. David L. DeWitt (Michigan State University)
(23). The AP-1 luciferase reporter plasmid ( Prostaglandin E2 (PGE2) Assay--
Cells
were treated with 30 µM arachidonic acid for 15 min, and
PGE2 release in the culture medium was determined using the PGE2 enzyme immunoassay (EIA) system (Amersham Biosciences)
according to the protocol provided.
Kinase Assay--
In vitro phosphorylation of JNK1
and JNK2 by active MKK4 was determined according to the manufacturer's
protocol (Upstate Biotechnology) except with the modification that
NS-398 or piroxicam at the concentration indicated was added to the
samples before incubation for 30 min at 30 °C. For JNK kinase
activity assay, Cl 41 cells growing in 100-mm cell culture plates were
exposed to UVB (4 kJ/m2). After a 30-min incubation at
37 °C, 5% CO2, the cells were lysed, and JNKs were
selectively pulled down by N-terminal c-Jun fusion protein bound to
glutathione-Sepharose beads (stress-activated protein kinase (SAPK)/JNK
assay kit from Cell Signaling). In vitro analysis of
phosphorylation of c-Jun by pull-down JNK kinase assay was determined
according to the manufacturer's protocol except with the modification
that NS-398 or piroxicam at the concentration indicated was added to
the samples before incubation for 30 min at 30 °C.
Statistical Analysis--
Significant differences in AP-1
activity were determined with the Student's t test. The
results are expressed as the means ± S.D.
NS-398 or Piroxicam Inhibits TPA-induced AP-1 Activity and Cell
Transformation--
Increased AP-1 activity leads to malignant
transformation, and thus, the suppression of AP-1 activity is suggested
to be involved in the mechanisms of action of many potential
chemopreventive agents (25-27). We therefore investigated whether
NS-398 or piroxicam could block malignant cell transformation. We used
TPA, one of the most potent experimental stimuli used to activate AP-1
activity and tumor promotion in skin, to test the effects of the COX-2 inhibitors on AP-1 activity and cell transformation. NS-398 or piroxicam significantly suppressed both TPA-induced AP-1 activity (Fig.
1A) and TPA-induced cell
transformation on soft agar (Fig. 1B) in a
concentration-dependent manner. As we have previously reported, increased AP-1 activity is required for tumor
promoter-induced transformation (24, 28). Therefore, the inhibition of
AP-1 activity may be functionally linked to the anti-cancer effect of
these chemicals.
NS-398 or Piroxicam Suppresses UVB-induced AP-1
Transactivation Both in Vitro and in Vivo--
Solar UVB is a strong
etiological factor in human skin cancer (29). UVB-induced AP-1 and
other signal transduction pathways play a role in skin carcinogenesis
(23). To determine the effect of NS-398 or piroxicam on UVB-induced
AP-1 transactivation, AP-1-luciferase reporter gene-bearing JB6 C1 41 cells were incubated with the COX-2 inhibitors and then exposed to UVB.
Both NS-398 and piroxicam markedly inhibited UVB-induced AP-1
luciferase activity, and the inhibitory effects appeared to be
dose-dependent (Fig.
2A). The doses of NS-398 and
piroxicam used in the experiment were below the observed cytotoxic
range (30, 31).
NS-398 or Piroxicam Blocks UVB-activated JNKs and AP-1 DNA Binding
Activity--
Mitogen-activated protein kinases comprise the most
common pathways known to mediate AP-1 function (32). Many reports
indicated that JNK, a member of the mitogen-activated protein kinase
family, is critical in mediating AP-1 transactivation and malignant
transformation (25, 33-36). Inhibition of JNK leads to suppression of
AP-1 activity and cell transformation (23). In our results, both NS-398
and piroxicam inhibited UVB-induced phosphorylation of JNKs (Fig. 3A), indicating that the
blocking of JNK activation is involved in the inhibitory effects of
NS-398 or piroxicam on these signal transduction pathways.
AP-1 functions as a transcription factor by binding to the
transactivation promoter region (TPA response elements) of specific genes. To determine whether NS-398 or piroxicam functions through the
attenuation of AP-1 binding to its target DNA, we performed electrophoretic mobility shift assay as an indicator of AP-1 DNA binding activity. UVB (4 kJ/m2) induced a significant
increase in AP-1 DNA binding activity (Fig. 3B, lane
3) compared with the unexposed control (Fig. 3B, lane 2). The elevated AP-1 DNA binding was suppressed by
NS-398 or piroxicam in a dose-dependent manner (Fig.
3B, lanes 4-7). The DNA binding was specific for
AP-1 because a 10-fold excess of unlabeled AP-1 probe successfully
competed with the labeled probe (Fig. 3B, lane
1).
The Inhibitory Effects of NS-398 or Piroxicam on AP-1 Are
Independent of COX-2--
Although we have observed that both NS-398
and piroxicam could efficiently block AP-1 activation,
whether the effects of the COX-2 inhibitors on AP-1
occurred through their inhibition of COX-2 is important to determine
because of the key role that AP-1 plays in tumorigenesis. Cells
transfected with the AP-1 luciferase reporter gene showed that an
increase in AP-1 activity could also be induced by UVB exposure in
Cox-2
COX inhibitors block the catalysis of arachidonic acid to
PGE2 (39-41). Several studies suggest that
PGE2 signals through AP-1 and, thus, may play a definitive
role in tumor development (42-44). To elucidate whether NS-389 or
piroxicam blocks AP-1 activation by attenuating PGE2
production, we transfected two acetylation active site mutants of
COX-2, S516 M and S516Q, together with the AP-1 luciferase reporter
gene into Cox-2 The Blocking of JNK Kinase Activation Inhibits
TPA-induced Cell Transformation--
Although COX-2 has been well
recognized as an important target for therapy/prevention of cancer and
COX-2 selective inhibitors are already applied as an approach for
prevention or treatment of cancer, many reports suggest that some
tumors can still occur or grow in COX-2-negative mice or cell lines.
COX-2 inhibitors exhibit identical tumor growth inhibitory effects on
either Cox-2+/+,
Cox-2+/ NS-398 and Piroxicam Block JNK Phosphorylation in Vitro--
To
further confirm that the COX-2 inhibitors act on JNK
function, we studied the effect of NS-398 or piroxicam on active
MKK4-induced JNK1/2 phosphorylation in vitro. Our results
indicated that the COX-2 inhibitors effectively suppressed JNK1/2
phosphorylation in vitro (Fig.
9A). In another investigation
as to whether the COX-2 inhibitors target mediators other than JNKs
existing in the pathway, we used N-terminal c-Jun fusion protein-bound
glutathione-Sepharose beads to pull down the JNK proteins from
UVB-irradiated cells and incubated them with ATP at the presence of
NS-398 or piroxicam in vitro. Piroxicam at the high
concentration of 10 µM in vitro had a weak
inhibitory effect on active JNK-induced phosphorylation of c-Jun, the
downstream oncogene target of JNKs and a component of AP-1,
whereas NS-398 had little effect (Fig. 8B). This result combined with the observation that NS-398 or piroxicam blocks JNK
phosphorylation, indicates that the chemicals may work on JNK signaling
from within the JNKs themselves.
In summary, our data indicated that the COX-2 "selective"
inhibitors NS-398 or piroxicam effectively blocked UVB-induced AP-1 activity both in vivo and in vitro. They could
also suppress TPA-induced AP-1 activity and cell transformation. The
blocking of JNK activation by the chemicals appears to be involved in
their inhibition of AP-1 activity. We further demonstrated that the
inhibition of AP-1 by these compounds was COX-2-independent.
Considering the critical role that AP-1 plays in tumorigenesis, these
results may supply some novel insights regarding the mechanism of COX-2 inhibitory effects and provide a biological basis for the development of new chemopreventive agents for cancer.
/
fibroblasts transfected with an AP-1
luciferase reporter gene, and this increase was blocked by NS-389 or
piroxicam. In JB6, Cox-2
/
, or wild-type
Cox-2+/+ cells, both NS-398 and piroxicam
inhibited UVB-induced phosphorylation of c-Jun NH2-terminal
kinases, the kinases that activate the AP-1/c-Jun complex. Based on our
results, we propose that the inhibition of AP-1 activity by COX-2
inhibitors NS-398 or piroxicam may occur by a mechanism that is
independent of COX-2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1 cells and the JB6 P+
mouse epidermal cell line, C1 41, were cultured in monolayers at
37 °C, 5% CO2 using minimum essential medium (MEM)
containing 5% fetal bovine serum (FBS), 2 mM
L-glutamine, and 25 µg of gentamicin/ml.
Cox-2
/
murine embryo fibroblasts (MEFs) and
wild-type Cox-2+/+ MEFs were kind gifts from
Drs. Jeff Reese and Sudhansu K. Dey (University of Kansas Medical
Center) (15). The cells were derived from COX-2 knockout mice supplied
by Drs. Joseph E. Dinchuk and James M. Trzaskos (DuPont Merck
Pharmaceutical Co.) (16). The cells were cultured in monolayers at
37 °C, 5% CO2 using Dulbecco's modified Eagle's
medium containing 10% FBS, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids, and 50 IU/ml
penicillin/streptomycin. FBS and MEM were from BioWhittaker, Inc.
(Walkersville, MD), TPA, aprotinin, and leupeptin were from Sigma, and
the luciferase assay substrate was from Promega (Madison, WI).
70 °C. The DNA binding reaction (electrophoretic mobility shift assay) was performed for 30 min at room temperature in a
mixture containing 4 µg of nuclear proteins, 1 µg of
polydeoxyinosinic-deoxycytidylic acid (dI·dC), and 15,000 cpm of
32P-labeled double-stranded oligonucleotide probe. The
samples were fractionated through a 5% polyacrylamide gel. The
sequence of the AP-1 oligonucleotide probe was
5'-CGCTTGATGAGTCAGCCGGAA-3'. Gels were dried and analyzed using the
Storm 840 Phospho-Image System (Molecular Dynamics).
73/+63 collagenase-luciferase) and cytomegalovirus-neo marker vector plasmid
were constructed as previously reported (24). Transient transfection
was carried out using LipofectAMINE PLUSTM Reagent
(Invitrogen) according to the protocol supplied.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
NS-398 or piroxicam suppresses
TPA-induced AP-1 activity and cell transformation in JB6 cells.
A, 8 × 103 mouse epidermal JB6 Cl 41 AP-1
luciferase reporter stably transfected P+1 1 (Cl 41 P+1
1) cells
suspended in 5% FBS MEM were added to each well of a 96-well plate.
After an overnight culture at 37 °C, the cells were starved by
replacing the medium with 0.1% FBS MEM for 24 h. The cells were
then treated for 30 min with NS-398 or piroxicam at the concentrations
indicated before exposure to TPA (20 ng/ml). After another 48 h,
AP-1 luciferase activity was measured as described previously. The
results are presented as relative AP-1 activity. Each bar
indicates the mean and S.D. of six assay wells from three independent
experiments. *, a significant (p < 0.01) inhibition
was observed compared with TPA treatment and no inhibitor present.
B, 104 JB6 Cl 41 cells in 0.33% agar were
exposed for 4 weeks simultaneously to TPA with or without NS-398 or
piroxicam at the concentration indicated and scored for colonies at the
end of the experiment. Results are expressed as the mean and S.D. of
triplicate experiments. A significant (*, p < 0.01)
inhibition of AP-1 activity was observed in cells treated with TPA plus
inhibitors compared with cells treated only with TPA.
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Fig. 2.
NS-398 or piroxicam suppresses UVB-induced
AP-1 activity in JB6 cells in vitro and in
vivo. A, Cl 41 P+1 1 cells were prepared
and serum-starved as in Fig. 1. The cells were then treated with NS-398
or piroxicam for 30 min at the concentrations indicated before exposure
to UVB (4 kJ/m2). After another 12 h, the AP-1
luciferase activity was measured as described previously. The results
are presented as relative AP-1 activity. Each bar indicates
the mean and S.D. of six assay wells from three independent
experiments. A significant (*, p < 0.01) inhibition of
AP-1 activity was observed in cells treated with UVB plus inhibitors
compared with cells treated only with UVB. B,
AP-1-luciferase reporter gene-bearing mice were grouped and treated
with NS-398 or piroxicam as described under "Materials and
Methods." Each bar indicates the mean and S.D. of the AP-1
activity for each group of 22 mice. A significant (*, p < 0.01) inhibition of AP-1 activity was observed in mice treated with
UVB plus inhibitors compared with mice treated only with UVB.
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Fig. 3.
NS-398 or piroxicam suppresses UVB-induced
JNKs phosphorylation and AP-1 DNA binding activity in JB6 cells.
A, C1 41 cells were serum-starved 24 h and then treated
for 30 min with NS-398 or piroxicam at the concentration indicated. The
cells were then exposed to UVB (4 kJ/m2) followed by
culturing for another 30 min. Western immunoblotting for detection of
phosphorylation of JNKs was carried out using phospho-specific
mitogen-activated protein kinase antibodies against phosphorylated
sites of JNKs. Levels of JNKs non-phosphorylated protein indicates
equal protein loading. B, Cl 41 cells were serum-starved for
24 h and treated or not treated for 30 min with NS-398 or
piroxicam at the concentrations indicated. The cells were then exposed
to UVB at a dose of 4 kJ/m2. After exposure, the cells were
cultured for another 8-10 h. The cells were then harvested, and
gel-shift assays were performed as described under "Materials and
Methods." Each bar indicates the mean and S.D. of the
densitometry analysis of the gels from three independent experiments. A
significant (*, p < 0.01) inhibition of AP-1 DNA
binding activity was observed. Incubation with an excess of unlabeled
probe indicates that the AP-1 DNA binding was specific.
/
MEFs (Fig.
4). NS-398 and piroxicam suppressed the
UVB-induced increase in AP-1 activity in these MEFs (Fig. 4). We
observed that similar to Cox-2+/+ cells, either
UVB irradiation or TPA exposure could still induce a significant
increase in AP-1 DNA binding in Cox-2
/
MEFs
(Fig. 5, A and B),
and NS-398 and piroxicam suppressed the increases in AP-1 DNA binding
in these cells (Fig. 5, A and B). These results
indicated that COX-2 is not required in the signaling pathways
mediating UVB-induced AP-1 activation because the COX-2 inhibitors
NS-398 and piroxicam suppressed AP-1 activity by mechanisms independent
of their inhibition of COX-2. To further investigate whether the
absence of COX-2 affects JNK activation, we exposed Cox-2
/
MEFs to different doses of UVB
irradiation and harvested the cells at different time points after UVB
treatment. The results showed that UVB activated JNKs in
Cox-2
/
as well as in
Cox-2+/+ MEFs in a time (Fig.
6A)- and dose (Fig.
6B)-dependent manner, and both NS-398 and
piroxicam effectively blocked UVB-induced JNK activation in both cell
types (Fig. 6C). These results indicated that COX-2 is not
involved in the inhibitory mechanism of NS-398 or piroxicam affecting
UVB-induced AP-1 activity. Some reports suggest that JNKs and the
downstream AP-1 were required in mediating COX-2 expression (37, 38),
but whether COX-2 is involved in the regulation of JNKs or AP-1
activation is still unclear.
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Fig. 4.
NS-398 or piroxicam suppresses
UVB-induced AP-1 activity in Cox-2-null MEFs.
8 × 103 Cox-2 /
MEFs
transfected with the AP-1 luciferase reported plasmid were added to
each well of a 96-well plate. After an overnight culture at 37 °C,
the cells were starved by replacing the medium with serum-free
Dulbecco's modified Eagle's medium for 12 h. The cells were then
treated for 30 min with NS-398 or piroxicam at the concentration
indicated before exposure to UVB (4 kJ/m2). After another
12 h, AP-1 luciferase activity was measured as described
previously. The results are presented as relative AP-1 activity.
Each bar indicates the mean and S.D. of three assay wells
from three independent experiments. A significant (*,
p < 0.01) inhibition of AP-1 activity was observed in
cells treated with UVB plus inhibitors compared with cells treated with
UVB only.
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Fig. 5.
NS-398 or piroxicam suppresses UVB-induced
AP-1 DNA binding activity in Cox-2-null MEFs.
Cox-2+/+ and Cox-2 /
MEFs were serum-starved for 6 h and either treated or not treated
for 30 min with NS-398 or piroxicam at the concentrations indicated.
The cells were then exposed to UVB (4 kJ/m2) (A)
or TPA (B). After exposure, the cells were cultured for
another 8-10 h. The cells were then harvested, and gel-shift assays
were performed as described under "Materials and Methods."
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Fig. 6.
NS-398 or piroxicam suppresses UVB-induced
JNKs phosphorylation in Cox-2-null MEFs.
Cox-2 /
and Cox-2+/+
MEFs were serum-starved for 12 h and then exposed to UVB (4 kJ/m2) and harvested at the different time points indicated
(A), or the cells were exposed to different doses of UVB as
indicated and then harvested 30 min after treatment (B). The
cells were also treated for 30 min with NS-398 or piroxicam at the
concentrations indicated and then exposed to UVB (4 kJ/m2)
followed by culturing for another 30 min (C). Western
immunoblotting for phosphorylation of JNKs was carried out using
phospho-specific mitogen-activated protein kinase antibodies against
phosphorylated sites of JNKs. JNKs non-phosphorylated protein level
indicates that an equal amount of protein was loaded.
/
cells. The two mutant cell
lines did not produce PGE2 (Fig.
7A), although they could
encode the COX-2 protein (Fig. 7B). NS-398 and piroxicam
blocked UVB-induced AP-1 activation in both COX-2 mutant-transfected
Cox-2
/
cells (Fig. 7C). The
results indicated that PGE2 production was not the major
effect involved in the inhibition of AP-1 by NS-398 or piroxicam.
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Fig. 7.
Mutants (S516M or S516Q) of COX-2 do not
attenuate the inhibitory effects of NS-398 or piroxicam on AP-1
activity. Cox-2 /
and
Cox-2+/+ MEFs were transiently transfected with
the AP-1 luciferase reporter plasmid. Cox-2
/
MEFs bearing the AP-1 luciferase reporter gene were also transfected
with COX-2, COX-2-S516M, or COX-2-S516Q mutant plasmids as described
under "Materials and Methods." A, the transfected cells
were treated and assessed for PGE2 production as described
under "Materials and Methods." The asterisks indicate a
significantly (p < 0.01) lower production of
PGE2 in COX-2 null cells compared with cells having COX-2
gene expression. B, COX-2 protein expression in each cell
type was assessed as described under "Materials and Methods."
-Actin was applied as an inner control of the equal protein volume
loaded. C, the transfected cells were also treated with
NS-398 or piroxicam 30 min before UVB exposure. The results are
presented as relative AP-1 activity. Each bar indicates the
mean and S.D. of three assay wells from three independent experiments.
The asterisks indicate that a significant (p < 0.01) inhibition of AP-1 activity occurred in cells treated with UVB
plus inhibitors compared with cells treated with UVB only.
, or Cox-2
/
mice or cell lines (45-48), indicating that alternative pathways exist. So far in this paper, we have demonstrated that NS-398 or
piroxicam inhibits AP-1 transactivation independently of COX-2, and
blocking of JNK kinase phosphorylation likely plays a role in the
function of the chemicals. However, we still don't know if JNKs are
really important in mediating mitogen-induced cell transformation. To
answer the question we utilized a highly selective JNK inhibitor,
SP600125, to observe its effect on TPA-induced JB6 cell transformation
on soft agar. SP600125 inhibits JNKs with more than a 20-fold
selectivity versus other kinases such as extracellular signal-regulated kinases, p38 kinase, or protein kinase C, etc. (49),
and it is not cytotoxic for JB6 cells, as indicated by the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay at the concentration range used in our experiment (CellTiter 96® AQueous One Solution Cell
Proliferation assay kit, Promega) (data not shown). Our results showed
that SP600125 significantly suppressed TPA-induced JB6 cell
transformation (Fig. 8A),
indicating that the activation of JNKs is required in the signaling
pathway leading to cell transformation. Similar observations have also
been reported previously (23). However, further investigation revealed
a synergistic inhibition when both SP600125 and NS-398 or piroxicam
were included with TPA on soft agar (Fig. 8B). The results
indicated that although JNKs might be a major target of COX-2
inhibitors, other molecules may play a role in the mechanisms of COX-2
inhibition of cell transformation.
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[in a new window]
Fig. 8.
JNK selective inhibitor SP600125 and NS-398
or piroxicam synergistically suppress TPA-induced cell transformation
in JB6 cells. A, 104 JB6 Cl 41 cells in
0.33% agar for 4 weeks were exposed simultaneously to TPA with or
without SP600125 at the concentration indicated and scored for colonies
at the end of the experiment. Results are expressed as the mean and
S.D. of triplicate experiments. The asterisks indicate that
a significant (p < 0.01) inhibition of cell growth
occurred in cells exposed to TPA plus SP600125 compared with cells
treated with TPA only. DMSO, Me2SO.
B, the same cell type and soft agar procedure as for
A, except that the cells were exposed to TPA with or without
SP600125 plus NS-398 or piroxicam at the concentrations indicated. The
double asterisks indicate that a significant
(p < 0.01) inhibition of cell growth occurred in cells
treated with TPA plus SP600125 and COX-2 inhibitors compared with cells
treated with TPA plus SP600125 indicated by *.
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[in a new window]
Fig. 9.
NS-398 or piroxicam suppresses JNK1/2
phosphorylation but not activation in vitro.
A, active MKK4 was incubated with unactive JNK1/2 and
[ -32P]ATP in the presence of NS-398 or piroxicam at
the concentration indicated. The isotope labeled JNK1/2 proteins were
separated by 8% SDS-PAGE, and autoradiography was carried out as
described under "Materials and Methods." Either NS-398 or piroxicam
significantly blocked active MKK4-activated JNK1/2 phosphorylation
in vitro. B, Cl 41 cells were treated or not
treated with UVB, and the JNKs kinase assay was carried out as
described under "Materials and Methods." NS398 or piroxicam at the
concentration indicated did not significantly block the phosphorylation
of c-Jun induced by UVB-activated JNKs kinase in vitro.
"Beads" indicate N-terminal c-Jun fusion protein-bound
glutathione-Sepharose beads alone.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jeff Reese and Dr. Sudhansuk Dey
for providing Cox-2+/+ and
Cox-2/
cells and Dr. David L. DeWitt for
providing plasmids of COX-2, COX-2-S516M, and
COX-2-S516Q mutants. We thank Andria Hansen for secretarial assistance.
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FOOTNOTES |
---|
* This work was supported by The Hormel Foundation and National Institutes of Health Grants CA74916 and CA77646.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: The Hormel Institute,
University of Minnesota, 801 16th Ave. N.E., Austin, MN 55912. Tel.:
507-437-9600; Fax: 507-437-9606; E-mail: zgdong@hi.umn.edu.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M202443200
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ABBREVIATIONS |
---|
The abbreviations used are: COX, cyclooxygenase; TPA, 12-O-tetradecanoylphorbol-13-acetate; UVB, ultraviolet B; AP-1, transcription factor activator protein 1; MEM, minimum essential medium; FBS, fetal bovine serum; MEF, murine embryo fibroblast; JNK, c-Jun NH2-terminal kinase; PGE2, prostaglandin E2.
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