Department of Pharmacology (C.-C.C., K.-T.C., S.-T.C.) and School
of Pharmacy (J.-W.C.), College of Medicine, National Taiwan University,
Taipei, Taiwan
 |
Introduction |
Cyclooxygenase
(COX) is the rate-limiting enzyme in the conversion of arachidonic acid
into prostaglandin H2
(PGH2), the precursor of a large group of
biologically active mediators, such as PGE2, prostacyclin, and thromboxane A2, which are
involved in several biological processes, including inflammation,
immune responses, cell growth, ovulation, and regulation of vascular
tone (Williams and DuBois, 1996
). The two COX isoforms, COX-1 and
COX-2, are encoded by separate genes (Fletcher et al., 1992
; Hla and
Neilson, 1992
; Kraemer et al., 1992
). Although their enzymatic function is similar, regulation of their cellular expression differs. COX-1 gene
expression is largely constitutive, whereas COX-2 gene expression is
negligible under basal conditions but can increase dramatically in many
cell types in response to mitogenic and inflammatory stimuli (Smith and
Dewitt, 1996
). COX-2 is expressed in activated macrophages, monocytes,
and several other cell types and has been identified in chronic
inflammatory conditions in vivo (Vane et al., 1994
). It is implicated
in physiological processes, such as ovulation and delivery (Lim et al.,
1997
), and in pathological states, such as colorectal cancer,
Alzheimer's disease, heart failure, and even hypertension (Levy, 1997
;
Oka and Takashima, 1997
; Hartner et al., 1998
; Wong et al., 1998
). Much
evidence suggests that COX-2 is an important therapeutic target for the
prevention and treatment of arthritis and cancer. Reducing the levels
of COX-2 will be an effective strategy for inhibiting inflammation and carcinogenesis (Anderson et al., 1996
; Kawamori et al., 1998
). Because of this, there has been great interest in the role(s) of COX-2
and the usefulness of drugs that can selectively block this isozyme
(Frolich, 1997
).
COX-2 is an early gene expressed in response to many cytokines,
and its transcriptional regulation is, at least in part, under the
control of NF-
B (Newton et al., 1997
). NF-
B activation is tightly
regulated by its endogenous inhibitor I
B, which complexes with and
sequesters NF-
B in the cytoplasm. After cytokine stimulation, I
B
is phosphorylated at serines 32 and 36, initiating the
selective ubiquitination and rapid degradation of this inhibitor by the nonlysosomal, ATP-dependent 26S proteolytic complex composed of a
700-kDa proteasome (Stancovski and Baltimore, 1997
). I
B
phosphorylation involves the successive participation of various
kinases linked to cytokine-specific membrane receptor complexes and
adapter proteins, which converge on NF-
B-inducing kinase (NIK)
(Malinin et al., 1997
). Activated NIK then phosphorylates and activates
the I
B kinase (IKK) complex (Ling et al., 1998
; Lin et al., 1998
).
IKK is part of a multiprotein complex that contains IKK
and IKK
subunits (Woronicz et al., 1997
). Activation of the IKK complex leads
to specific I
B
phosphorylation/degradation and the subsequent release of NF-
B, which then translocates to the nucleus and
activates the transcription of multiple
B-dependent genes, including
COX-2 (Barnes and Karin, 1997
). Because NF-
B plays a central role in regulating the genes involved in the initiation of immune, acute phase,
and inflammatory responses, there is growing interest in modulating its
activity. The pathways leading to NF-
B activation are therefore
frequent targets for a variety of anti-inflammatory drugs. The
anti-inflammatory drugs aspirin and salicylate suppress inducible COX-2
gene transcription (Xu et al., 1999
) and inhibit I
B kinase-
(Yin
et al., 1998
). We have demonstrated that, in NCI-H292 human alveolar
epithelial cells, TNF-
induces COX-2 expression via the PKC- and
mitogen-activated protein kinase-dependent IKK1/2 and NF-
B
activation pathway (Chen et al., 2000
, 2001
). In this study, we have
identified two conjugated polyhydroxybenzene derivatives,
2-(3',4'-dihydroxyphenyl)5-hydroxybenzo[b]furan (GF-015) and 2,3-di(3',4'-dihydroxy-transstyryl) pyridine (GF-90) (Fig. 1) that block TNF-
-induced COX-2
expression through NF-
B inhibition by targeting the IKK complex.

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Fig. 1.
Effect of nine conjugated polyhydroxybenzene
derivatives and curcumin on TPA-induced COX-2 expression in NCI-H292
cells (A and B) and chemical structure of GF-015 and GF-90 (C). A,
cells were pretreated with 100 or 5 µM different derivatives; B,
cells were pretreated with 50 µM GF-015, GF-90, or curcumin. After
pretreatment for 30 min, 1 µM TPA was added for 16 h. Whole-cell
lysates were prepared and subjected to Western blotting using antibody
specific for COX-2, as described under Materials and
Methods.
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|
 |
Experimental Procedures |
Materials.
The NF-
B probe, goat polyclonal antibodies
specific for COX-2, and rabbit polyclonal antibodies specific for
IKK
and I
B
were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). MG132 was from Sigma (St. Louis, MO). Recombinant
human TNF-
was purchased from R & D Systems, Inc. (Minneapolis, MN).
RPMI 1640 medium, fetal calf serum (FCS), penicillin, and streptomycin were from Invitrogen (Gaithersburg, MD). T4 polynucleotide
kinase and rabbit polyclonal antibody specific for the phosphorylated form of I
B
(Ser 32) were from New England Biolabs (Beverly, MA).
Poly (dI/dC), horseradish peroxidase-labeled donkey anti-goat or
anti-rabbit second antibody and the ECL detection reagent were from
Amersham Pharmacia Biotech (Piscataway, NJ).
[
-32P]ATP (3000 Ci/mmol) and
[
-32P]dCTP (3000 Ci/mmol) were from
PerkinElmer Life Sciences (Boston, MA). Tfx-50 and the
luciferase assay kit were from Promega (Madison, WI). GF-015 and GF-90
were synthesized (J.-W. Chern, unpublished observations) and dissolved
as stock solutions (50 mM) in dimethyl sulfoxide.
Plasmids.
The COX-2 promoter construct (
459/+9) was a
generous gift from Dr. L. H. Wang (University of Texas-Houston,
Houston, TX). Plasmids containing wild-type NIK, IKK
, and IKK
were provided by Dr. M. Karin (University of California, San Diego,
CA). pGEX-I
B
(1-100) and pGEX-I
B
(1-100) (S32A, S36A)
were gifts from Dr. H. Nakano (Juntendo University, Tokyo, Japan).
Cell Culture.
NCI-H292 cells, a human alveolar epithelial
cell carcinoma, were obtained from the American Type Culture Collection
(Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10%
FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin in either
six-well plates (COX-2 protein expression, PGE2
production, and transfection) or 10-cm dishes (COX-2 mRNA expression,
NF-
B gel shift assay, and IKK activation).
Northern Blot Analysis.
Total cellular RNA was isolated from
cultured cells using TRIzol (Invitrogen). A sample of the RNA (20-30
µg/lane) was fractionated on a formaldehyde-containing 1% agarose
gel and transferred to a positively charged Immobilon-N (Millipore
Corporation, Bedford, MA) membrane. After UV cross-linking, the
membranes were prehybridized for 1 h at 60°C and then probed for
12 h at 60°C with the human COX-2 cDNA probe (1.9 kilobases)
labeled with [
-32P]dCTP by random primer
using Rediprime (Amersham Pharmacia Biotech). Hybridization reactions
were performed in Church buffer consisting of 7% SDS, 1% bovine serum
albumin, 10 mM EDTA, and 0.4 M
NaH2PO4, pH 7.2. The
membranes were washed at room temperature for 60 min once in 2× SSC
containing 1% SDS and again in 0.2× SSC containing 1% SDS before
exposure to film. The membranes were then stripped in 0.2× SSC
containing 1% SDS at room temperature for 60 min and rehybridized with
human glyceraldehyde-3-phosphate dehydrogenase cDNA (1 kilobase). Blots
were quantitated using a computer densitometer and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Preparation of Cell Extracts and Western Blot Analysis of
COX-2.
After 16-h treatment with TNF-
or TPA, the cells were
harvested and collected, and cell lysates were prepared and subjected to SDS-PAGE using 7.5% running gels as described previously (Chen et
al., 2000
). The proteins were then transferred to nitrocellulose membranes, which were incubated successively at room temperature with
0.1% nonfat dry milk in Tris-buffered saline containing Tween 20 (TTBS) for 1 h, with goat antibody specific for COX-2 for
1 h, and with horseradish peroxidase-labeled anti-goat antibody for 30 min. After each incubation, the membrane was washed extensively with TTBS. The immunoreactive band was detected using ECL detection reagent and visualized using Hyperfilm ECL (Amersham Pharmacia Biotech). Quantitative data were obtained using a computing
densitometer and ImageQuant software (Molecular Dynamics). In
pretreatment experiments, cells were incubated for 30 min with various
concentrations of GF-015, GF-90, or curcumin before addition of TNF-
or TPA. These inhibitors (except 100 µM curcumin) had no cytotoxic
effect on NCI-H292 cells, and the 0.001% dimethyl sulfoxide (vehicle) used as a control had no effect on TNF-
- or TPA-induced COX-2 expression.
Determination of PGE2 Concentration.
Cells were
pretreated with 10 or 50 µM GF-015 or GF-90 for 30 min; then, TNF-
or TPA was added for 16 h. PGE2 levels in
the culture medium were measured using an enzyme immunoassay kit from Amersham Pharmacia Biotech.
Transient Transfection and Luciferase Assay.
NCI-H292 cells
grown in six-well plates were transfected with the human COX-2 firefly
luciferase plasmid, pGS459 (
459/+9), using Tfx-50 (Promega) according
to the manufacturer's recommendations. Briefly, reporter DNA (0.3 µg) and
-galactosidase DNA (0.1 µg) were mixed with 1.8 µl of
Tfx-50 in 1 ml of serum-free RPMI 1640 medium. The plasmid pRK,
containing the
-galactosidase gene driven by the constitutively
active SV40 promoter, was used to normalize transfection efficiency.
After 10 to 15 min of incubation at room temperature, the mixture was
applied to the cells. One hour later, 1 ml of RPMI 1640 medium
containing 20% FCS was added; then the cells were grown in a medium
containing 10% FCS. On the following day, they were pretreated with
various concentrations of GF-015 or GF-90 before exposure to 30 ng/ml
TNF-
or 1 µM TPA for 6 h; then cell extracts were prepared,
luciferase (Promega) and
-galactosidase activity measured, and the
luciferase activity of each well normalized to the
-galactosidase
activity. In experiments involving overexpression of wild-type
plasmids, cells were cotransfected with reporter,
-galactosidase,
and either wild-type NIK, IKK
, IKK
(0.3 µg of DNA), or the
empty vector.
In Vitro IKK Activity Assay.
After 10 min of treatment with
TNF-
or TPA or 30 min of pretreatment with GF-015 or GF-90 before
addition of TNF-
or TPA, cells were washed rapidly with PBS and then
lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA,
150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 5 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM NaF, and 1 mM
Na3VO4), and the IKK
proteins were immunoprecipitated. Fifty micrograms of total cell
extract was incubated for 1 h at 4°C with 0.5 µg of
anti-IKK
antibody and the antibody-bound protein was collected using
protein A-Sepharose CL-4B beads (Sigma). The beads were then washed
three times with lysis buffer without Triton X-100 and incubated for 30 min at 30°C in 20 µl of kinase reaction mixture consisting of 20 mM
HEPES, pH 7.4, 5 mM MgCl2, 5 mM
MnCl2, 0.1 mM
Na3VO4, 1 mM DTT, 1 µg of
bacterially expressed GST-I
B
(1-100), and 10 µM
[
32-P]ATP. The reaction was stopped by the
addition of an equal volume of Laemmli buffer and the material
subjected to 10% SDS-PAGE, and phosphorylated GST-I
B
(1-100)
was visualized by autoradiography.
Preparation of Cell Extracts and Western Blot Analysis of
Phosphorylated I
B
and I
B
.
After 5, 10, 30, and 60 min
of treatment with TNF-
or 60 min of pretreatment with MG132, GF-015,
GF-90, or curcumin before challenge with TNF-
for 5 or 10 min, the
cells were washed rapidly with PBS and then lysed with fresh ice-cold
lysis buffer as described previously (Chen et al., 2001
). The lysates
were then subjected to SDS-PAGE using a 10% running gel. The proteins
were transferred to nitrocellulose paper, and immunoblot analysis was
performed as described above, except that rabbit antibodies specific
for phosphorylated I
B
or nonphosphorylated I
B
were used.
Preparation of Nuclear Extracts and the Electrophoretic Mobility
Shift Assay.
Cells were pretreated with 10 or 50 µM GF-015 or
GF-90 for 30 min before addition of TNF-
or TPA for 1 h; then
nuclear extracts were prepared as described previously (Chen et al.,
2000
). Briefly, cells were washed with ice-cold PBS and pelleted; then
the cell pellet was resuspended in hypotonic buffer (10 mM HEPES, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 mM
NaF, and 1 mM Na3VO4),
incubated for 15 min on ice, and the cells lysed by the addition of
0.5% Nonidet P-40, followed by vigorous vortexing for 10 s. The
nuclei were pelleted and resuspended in extraction buffer (20 mM HEPES,
pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM
NaF, and 1 mM Na3VO4); then the tube was vigorously shaken for 15 min at 4°C on a shaking platform. The nuclear extracts were then centrifuged, and the supernatants were aliquoted and stored at
80°C.
Oligonucleotides corresponding to the NF-
B consensus sequences in
the human COX-2 promoter
(5'-AGAGTGGGGACTACCCCCTCT-3') were synthesized,
annealed, and end-labeled with [
-32P]ATP
using T4 polynucleotide kinase. The nuclear extract (6-10 µg) was
incubated at 30°C for 20 min with 1 ng of
32P-labeled NF-
B probe (40,000-60,000 cpm) in
10 µl of binding buffer containing 1 µg of poly (dI/dC), 15 mM
HEPES, pH 7.6, 80 mM NaCl, 1 mM EGTA, 1 mM DTT, and 10% glycerol, as
described previously (Chen et al., 2000
). DNA-nuclear protein complexes
were separated from the DNA probe by electrophoresis on a native 6%
polyacrylamide gel; then the gel was vacuum dried and subjected to
autoradiography at
80°C using an intensifying screen. Quantitative
data were obtained using a computing densitometer and ImageQuant
software (Molecular Dynamics).
Statistical Analyses.
All data are expressed as the
mean ± S.E.M. Statistical analyses were performed using
Student's t test.
 |
Results |
Testing of Nine Conjugated Polyhydroxybenzene Derivatives.
Nine conjugated polyhydroxybenzene derivatives are tested for their
inhibitory effect on TPA-induced COX-2 expression in NCI-H292 cells. As
shown in Fig. 1A, six of them at 100 µM had no cytotoxic effect on
the cells and only two (GF-015 and GF-90) of these six showed
inhibitory effect on TPA-induced COX-2 expression. Because three
(GF-56, EO-161, and GG-062) of the nine derivatives had cytotoxicity on
cells at 100 µM, the concentration was reduced to 5 µM. They had no
inhibitory effect on TPA-induced COX-2 expression at this concentration
(Fig. 1A). Therefore, the mechanism of action of GF-015 and GF-90 in
inhibiting COX-2 induction was further studied. The synthesis of GF-015
was based on stilbene, whereas that of GF-90 was based on curcumin. The
purity and structure of these two compounds were proved by NMR spectrum
and elemental analysis. Potency of curcumin and these two compounds was
compared. As shown in Fig. 1B, GF-90 at 50 µM was more potent than
curcumin in inhibiting COX-2 induction. Concentration of 100 µM was
not applied, because curcumin at this concentration showed serious cytotoxicity to NCI-H292 cells.
Effects of GF-015 and GF-90 on TNF-
- or TPA-Induced Expression
of COX-2 mRNA and Protein and PGE2 Release.
The
effects of the conjugated polyhydroxybenzene derivatives GF-015 and
GF-90 on COX-2 gene expression and the IKK/NF-
B pathway were
investigated using NCI-H292 epithelial cells, in which TNF-
has been
shown to induce a dose- and time-dependent increase in COX-2 protein
expression and PGE2 formation, the maximal effect being obtained by treatment with 30 ng/ml TNF-
for 16 h. This COX-2 expression has been demonstrated to involve the PKC-dependent activation pathway; therefore, direct activation of PKC by TPA also
induced COX-2 protein expression (Chen et al., 2000
). TNF-
(30 ng/ml) also induced the expression of COX-2 mRNA in a time-dependent manner; this effect was significant and maximal at 1 h, declined slightly after 3 and 6 h, and was no longer seen after 9 h of treatment (Fig. 2A). To determine the
effects of GF-015 and GF-90 on TNF-
-induced COX-2 gene expression,
cells were pretreated for 30 min with each of the compounds at
concentrations of 10 or 50 µM before stimulation with TNF-
for
3 h. When COX-2 mRNA expression was analyzed by Northern blotting,
GF-015 and GF-90 both inhibited TNF-
-induced COX-2 mRNA expression
in a dose-dependent manner (Fig. 2B).

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Fig. 2.
Time-dependent TNF- -induced COX-2 mRNA expression
in NCI-H292 epithelial cells and inhibitory effect of GF-015 and GF-90.
Cells were incubated at 37°C with 30 ng/ml TNF- for various time
intervals (A) or pretreated with 10 or 50 µM GF-015 or GF-90 for 30 min before incubation with 30 ng/ml TNF- for 3 h (B). Total RNA
was analyzed by Northern blotting, as described under Materials
and Methods.
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Inhibition of COX-2 gene expression was confirmed by measuring COX-2
protein expression. To determine the effect of GF-015 and GF-90 on
TNF-
- or TPA-induced COX-2 protein expression, cells were incubated
for 30 min with or without the test compounds at concentrations varying
from 3 to 30 µM before induction for 16 h with either TNF-
(30 ng/ml) or TPA (1 µM). Both compounds led to a significant
concentration-dependent reduction in TNF-
- or TPA-induced COX-2
expression (Fig. 3). The
IC50 values for GF-015 for the inhibition of
TNF-
- and TPA-induced COX-2 expression were 13.3 and 12.1 µM,
respectively (Fig. 3A), whereas the corresponding values for GF-90 were
5.2 and 7.1 µM (Fig. 3B).

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Fig. 3.
Inhibitory effect of GF-015 and GF-90 on TNF- - or
TPA-induced COX-2 protein expression in NCI-H292 epithelial cells.
Cells were pretreated with 3, 10, or 30 µM GF-015 (A) or GF-90 (B)
for 30 min before incubation with 30 ng/ml TNF- or 1 µM TPA for
16 h. Whole-cell lysates were prepared and subjected to Western
blotting using antibody specific for COX-2, as described under
Materials and Methods. COX-2 expression was quantified
using a densitometer with ImageQuant software. The results are
expressed as the mean ± S.E.M. of three independent experiments.
*, P < 0.05 compared with TNF- or TPA
alone.
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Because the major prostaglandin synthesized by COX in alveolar
epithelial cells is PGE2, we next examined
whether TNF-
- and TPA-induced PGE2 release was
inhibited by these two compounds. Both TNF-
(30 ng/ml) and TPA (1 µM) cause PGE2 release in NCI-H292 cells, and
these effects are blocked by 10 µM NS-398 (a COX-2 inhibitor) (Chen
et al., 2000
; data not shown). After pretreatment of cells with 10 or
50 µM GF-015, TNF-
-induced PGE2 production was inhibited by 24 or 71%, respectively, whereas TPA-induced PGE2 production was inhibited by 47 or 75% (Fig.
4A). Similarly, using 10 or 50 µM
GF-90, TNF-
-induced PGE2 production was
inhibited by 63 or 88%, respectively, whereas TPA-induced
PGE2 production was inhibited by 54 or 92% (Fig.
4B).

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Fig. 4.
Inhibitory effect of GF-015 and GF-90 on TNF- - or
TPA-induced PGE2 production in NCI-H292 epithelial cells.
Cells were pretreated with 10 or 50 µM GF-015 (A) or GF-90 (B) for 30 min before incubation with 30 ng/ml TNF- or 1 µM TPA for 16 h; then the medium was removed and analyzed for PGE2
production. The results are expressed as mean ± S.E.M. of three
independent experiments performed in triplicate. *,
P < 0.05 compared with TNF- or TPA alone.
|
|
GF-015 and GF-90 Inhibit TNF-
- and TPA-Induced COX-2 Promoter
Activity.
To elucidate the mechanism involved in the GF-015- and
GF-90-mediated inhibition of COX-2 expression, transient transfections were performed using a human COX-2 promoter-luciferase construct, pGS459. As reported previously (Chen et al., 2000
), treatment with
TNF-
or TPA led to a 4.1- or 5-fold increase, respectively, in COX-2
promoter activity, and these effects were inhibited by GF-015 and GF-90
in a dose-dependent manner (Fig. 5). The
IC50 values for GF-015 for inhibition of TNF-
-
and TPA-induced COX-2 promoter activity were 12.5 and 11 µM,
respectively, whereas the corresponding values for GF-90 were 5.9 and
6.4 µM (Fig. 5).

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Fig. 5.
Inhibitory effect of GF-015 and GF-90 on TNF- - or
TPA-induced COX-2 promoter activity in NCI-H292 epithelial cells. Cells
were transfected with the pGS459 luciferase expression vector and then
pretreated with 3, 10, or 50 µM GF-015 (A) or GF-90 (B) for 30 min
before incubation with 30 ng/ml TNF- or 1 µM TPA for 6 h.
Luciferase activity was assayed as described under Materials and
Methods. The results were normalized to the -galactosidase
activity and expressed as the mean ± S.E.M. of three independent
experiments performed in triplicate. *, P < 0.05 compared with TNF- or TPA alone.
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Cytokine-mediated I
B phosphorylation/degradation and NF-
B
activation involve the activation of at least two sequential proximal kinases, NIK and IKK (Maniatis, 1997
). IKK
/
binds NIK, a member of the mitogen-activated protein kinase kinase kinase family, to link
I
B degradation and NF-
B activation to the TNF-
receptor complex (Malinin et al., 1997
). TNF-
-induced COX-2 expression involves the PKC-dependent NIK, IKK1/2, and NF-
B activation pathway (Chen et al., 2000
). To investigate the effect of both compounds on
NF-
B activation-induced COX-2 expression, cotransfection with wild-type NIK, IKK
, or IKK
and the COX-2 promoter was performed. Overexpression of wild-type NIK, IKK
, or IKK
DNAs resulted in a
3.5-, 2.6-, or 2.6-fold increase, respectively, in COX-2 promoter activity, and these effects were suppressed by 10 or 50 µM GF-015 or
GF-90 (Fig. 6). Pretreatment with 10 or
50 µM GF-015 resulted in 34 or 65% inhibition, respectively, of
COX-2 promoter activity induced by NIK, 64 or 87% inhibition of that
induced by IKK
, and 36 or 77% of that induced by IKK
(Fig. 6A);
the corresponding values for 10 and 50 µM GF-90 were 48 and 100% for
NIK, 78 and 100% for IKK
, and 58 and 100% for IKK
(Fig. 6B).

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Fig. 6.
Inhibitory effect of GF-015 and GF-90 on wild-type
NIK-, IKK -, or IKK -induced COX-2 promoter activity. Cells were
cotransfected with plasmids coded for wild-type NIK, IKK , IKK , or
empty vector and the pGS459 construct and then treated with 10 or 50 µM GF-015 (A) or GF-90 (B) for 6 h. Luciferase activity was
assayed as described under Materials and Methods. The
results were normalized to -galactosidase activity and expressed as
the mean ± S.E.M. of three independent experiments performed in
triplicate. *, P < 0.05 compared with wild-type
NIK, IKK , or IKK alone.
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GF-015 and GF-90 Inhibit TNF-
- or TPA-Induced IKK Activation,
I
B
Phosphorylation, and NF-
B DNA-Protein Binding
Activity.
Because COX-2 promoter activity induced by
TNF-
, TPA, or wild-type NIK, IKK
, or IKK
was inhibited by
GF-015 and GF-90, indicating that both compounds targeted IKK
/
to
inhibit TNF-
- and TPA-induced COX-2 gene expression; the effect of
the test compounds on TNF-
- and TPA-induced IKK activity was
examined. As reported previously (Chen et al., 2000
), both TNF-
and
TPA induced IKK activation after 10 min of treatment. The IKK complex kinase activity was specific for Ser 32/Ser 36, because it was not
observed when both serines in I
B
substrate were substituted with
alanines (Fig. 7A, lanes 4-6). Equal
amount of I
B
substrate present in the in vitro kinase assay or
the amount of IKK complex immunoprecipitated (Fig. 7A, lanes 1-6).
GF-015 and GF-90 (10 or 50 µM) inhibited the TNF-
and TPA-induced
IKK activations in a dose-dependent manner (Fig. 7, B and C).

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Fig. 7.
Activation of IKK activity by TNF- and TPA in
NCI-H292 epithelial cells and inhibitory effects of GF-015 and GF-90.
A, cells were treated with 30 ng/ml TNF- or 1 µM TPA for 10 min;
then whole cell lysates were immunoprecipitated with anti-IKK
antibody followed by antoradiography for phosphorylated GST-I B
(1-100) or GST-I B (1-100) mutant (S32A; S36A). The level of
immunoprecipitated IKK were detected using IKK specific antibody
and levels of GST-I B were stained by Coomassie Brilliant
blue. B and C, cell were pretreated with 10 or 50 µM GF-015 or
GF-90, respectively, for 30 min before incubation with 30 ng/ml TNF-
or 1 µM TPA for 10 min; then whole cell lysates were
immunoprecipitated with anti-IKK antibody followed by
electrophoresis and autoradiography of phosphorylated GST-I B
(1-100) as described under Materials and Methods.
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|
Phosphorylation of I
B
on serines 32 and 36 by IKK is
necessary for its degradation and subsequent NF-
B activation (Chen et al., 1995
). Because TNF-
- or TPA-induced IKK activity was inhibited by GF-015 and GF-90, and NF-
B activation is essential for
TNF-
-induced COX-2 expression in NCI-H292 cells (Chen et al.,
2000
), we next examined the effect of GF-015 and GF-90 on TNF-
-
induced I
B
phosphorylation and degradation. Endogenous I
B
phosphorylation was assessed using Western blot with a specific I
B
phospho-serine 32 antibody. As shown in Fig.
8A, when cells were stimulated with
TNF-
for 5, 10, 30, or 60 min, phosphorylation of I
B
was seen
at 5 min of treatment, whereas it disappeared at 10 or 30 min and
reappeared at 60 min. Almost complete degradation of I
B
was seen
after 10 min of treatment with TNF-
, and full restoration was seen
at 60 min, as reported previously (Chen et al., 2000
). The lack of
phosphorylated I
B
at 10 min was caused by degradation of I
B
protein (Fig. 8A, lane 3), because phosphorylated I
B
was seen at
10 min in the presence of proteasome inhibitor MG132, which allowed
accumulation of the unstable phosphorylated I
B
(compare Fig. 8C,
lane 8, with 8B, lane 2) (Chen et al., 1995
). GF-015, GF90, and
curcumin but not MG132 inhibited the accumulation of phosphorylated
I
B
protein seen at 5 min of treatment with TNF-
(compare Fig.
8B, lanes 3-5 and 8 with lane 2). The inhibitory effect of GF-015 and
GF90 on I
B
phosphorylation was further demonstrated by
combination of these two compounds with MG132. As shown in Fig. 8, B
and C, the phosphorylated I
B
seen in the presence of MG132 at 5 or 10 min of treatment with TNF-
was decreased by either GF-015 or
GF-90 (compare Fig. 8B, lanes 6-7 with lane 8, and Fig. 8C, lanes 6-7
with lane 8). These results indicated that these two compounds
inhibited the phosphorylation of I
B
protein but not the activity
of proteasome complex. However, the TNF-
-induced I
B
degradation seen at 10 min of treatment was not prevented by GF-015 and
GF-90 but prevented by curcumin and MG132 (Fig. 8C, lanes 3-5 and 8).

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Fig. 8.
Time-dependent phosphorylation of I B serine 32 and I B degradation by TNF- in NCI-H292 cells and the effects
of GF-15, GF-90, curcumin, and MG132. A, cells were stimulated with 30 ng/ml TNF- for 5, 10, 30, or 60 min. B and C, cells were pretreated
with 50 µM GF-015, GF-90, curcumin or 30 µM MG132 or MG132 plus
GF-015 or GF-90 for 60 min before stimulation with TNF- for 5 min
(B) or 10 min (B). Cell lysates were prepared and subjected to Western
blotting using antibody specific for phosphorylated form of I B ;
then membranes were striped and reprobed with anti-I B antibody as
described under Materials and Methods.
|
|
To test whether the inhibitory effect of GF-015 and GF-90 on IKK
activity led to NF-
B inhibition, the effect of both compounds on
TNF-
- and TPA-stimulated NF-
B-specific DNA-protein binding activity was examined. Both compounds at concentrations of 10 or 50 µM inhibited TNF-
- or TPA-induced NF-
B-specific DNA-protein binding in a dose-dependent manner (Fig.
9).

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Fig. 9.
Inhibitory effect of GF-015 and GF-90 on TNF- - or
TPA-induced NF- B-specific DNA-protein complex formation in nuclear
extracts of NCI-H292 epithelial cells. Cells were pretreated with 10 or
50 µM GF-015 (A) or GF-90 (B) for 30 min before incubation with 30 ng/ml TNF- or 1 µM TPA for 1 h; then nuclear extracts were
prepared and NF- B DNA-protein binding activity was determined by
electrophoretic mobility shift assay as described under
Materials and Methods.
|
|
 |
Discussion |
An expanding body of evidence indicates that COX-2 inhibitors are
useful for treating inflammation and preventing cancer (Anderson et
al., 1996
; Oshima et al., 1996
; Kawamori et al., 1998
). The search for
selective COX-2 inhibitors started after the identification of this
cytokine-inducible isoform of COX (Fu et al., 1990
). So far, more than
a dozen such compounds have been described (Frolich, 1997
; DeWitt,
1999
), all of which act as competitive inhibitors of the enzyme. An
alternative approach chosen by some research groups is to develop
irreversible COX-2-selective inhibitors (Kalgutkar et al., 1998
) that
could prevent the resumption of prostaglandin production once drug
plasma levels fall, similar to the effect of aspirin on COX-1 and, to a
lesser extent, on COX-2. Moreover, because of the rapid induction of
COX-2 by proinflammatory stimuli, another interesting approach would be
to identify drugs that selectively block the expression of the enzyme
(Pennisi, 1998
). In this study, we analyzed the effect of nine
conjugated polyhydroxybenzene derivatives on TPA-induced COX-2
expression and found that two, GF-015 and GF-90, had inhibitory
effects. We then studied the effect of GF-015 and GF-90 on the
IKK/NF-
B pathway and their mechanisms of action. We report here that
pretreatment with either compound resulted in inhibition of
TNF-
-mediated NF-
B activation with concomitant down-regulation
of COX-2 gene expression and that NF-
B blockade by both compounds
involved inhibition of TNF-
-mediated IKK activation.
It is rational to study COX-2 gene expression and the accompanying
signaling pathways in alveolar epithelial cells. Epithelial cells play
an active role in inflammation by producing various cytokines that are
involved in the late asthmatic response (Barnes, 1994
).
The respiratory epithelium contributes to normal pulmonary function by removing inhaled particulates by its mucociliary action, ensures alveolar patency through surfactant secretion, and facilitates bacterial opsonization by the production of secretory immunoglobulin. There is now considerable evidence in support of an additional role for
airway epithelial cells in amplifying cytokine signals, including the
activation of alveolar macrophages and the secretion of chemokines,
arachidonic acid metabolites, and phospholipids, which recruit
additional inflammatory cells into the airway mucosa (Standiford et
al., 1990
; Stadnyk, 1994
). Of the potent alveolar macrophage-derived
cytokines studied, TNF-
in particular has been implicated in the
pathophysiology of neutrophilic infiltrating disorders, including acute
lung injury from sepsis, silica-induced pulmonary fibrosis, allograft
rejection, and acute respiratory tract infection (Piguet et al., 1990
;
Suter et al., 1992
; Beutler, 1995
). Upon binding to its cell surface
receptors, TNF activates the cytoplasmic form of NF-
B (Baeuerle and
Henkel, 1994
; Baldwin, 1996
).
Some of the conjugated polyhydroxybenzene derivatives tested in this
study were based on the structure of stilbene, one of the major
structural classes of COX-2 inhibitors (DeWitt, 1999
), whereas others
were based on curcumin, which has anti-inflammatory and chemopreventive
activity (Ammon and Wahl, 1991
; Rao et al., 1993
). On the nine tested,
only GF-015 and GF-90 had an inhibitory effect on TPA-induced COX-2
expression (Fig. 1A). The potency of GF-90 is greater than that of
curcumin (Fig. 1B). Further experiments demonstrated that TNF-
- or
TPA-induced expression of COX-2 mRNA and protein,
PGE2 production, and COX-2 promoter activity were attenuated by pretreatment with GF-015 and GF-90. Using molecular biological approaches, ectopic expression of NIK or IKK
/
allowed us to bypass the TNF-
receptor and specifically address the
effect of these two compounds on more proximal NF-
B-inducing
signals. Using this approach, we found that GF-015 and GF-90 interfere directly with NIK- or IKK-induced COX-2 promoter activity, indicating that these two compounds act at the level of IKK in TNF-
-mediated NF-
B activation, leading to COX-2 expression. This was further confirmed by the finding that TNF-
-induced IKK activation was inhibited by GF-015 and GF-90 (Fig. 7). Thus, these two conjugated polyhydroxybenzene derivatives inhibit the signal going to the IKK
complex by directly interfering with this complex and then inhibit
NF-
B-specific DNA-protein binding activity. Because phosphorylated I
B
protein was seen at 5 min of treatment with TNF-
and
disappeared at 10 min due to I
B
degradation (Fig. 8A), cells were
treated with TNF-
for 5 min to examine the phosphorylation of
I
B
(Fig. 8B) or 10 min to examine the degradation of I
B
(Fig. 8C). Accumulation of phosphorylated I
B
protein was
inhibited by GF-015 and GF-90, but not by MG132 (Fig. 8B), indicating
that these two compounds inhibited IKK activity but not the proteasome
complex. This finding was further demonstrated by the result that the
phosphorylated I
B
protein seen in the presence of MG132 at 10 min
of treatment with TNF-
was inhibited by GF-015 and GF-90 (Fig. 8C).
TNF-
-induced degradation of I
B
protein was prevented by
curcumin, which also inhibited accumulation of phosphorylated I
B
protein (Fig. 8B) and MG132, but not by GF-015 and GF-90 (Fig. 8C). The
discrepancy that GF-015 and GF-90 inhibit IKK activity but do not
prevent I
B
degradation is unknown at present and requires further
investigation. The ability to inhibit the NIK-IKK signaling complex may
be common to other anti-inflammatory chemopreventive agents. Aspirin
and salicylate, shown previously to inhibit I
B phosphorylation and degradation (Kopp and Ghosh, 1994
), inhibit I
B phosphorylation by
specifically reducing the binding of ATP to IKK
(Yin et al., 1998
).
Salicylate also inhibits COX-2 induction by lipopolysaccharide in
macrophages (Tordjman et al., 1995
) and by interleukin-1
and TPA in
endothelial cells (Xu et al., 1999
). Similar interference at the
IKK
/
level, leading to the inhibition of NF-
B activation and
COX-2 expression, is seen with curcumin (Plummer et al., 1999
). Overexpression of COX-2 in colon epithelial cells may promote tumor
development (Tsujii and Dubois, 1995
), and nonsteroidal anti-inflammatory drugs (NSAIDs), which directly inhibit COX-2 activity
(Vane and Botting, 1995
), cause regression of adenomatous polyps
(Giardiello et al., 1995
). COX-2 has therefore been suggested as an
important target for the chemopreventive effects of these agents
(Giardiello et al., 1997
). However, chronic administration of NSAIDs
results in serious side effects because of concomitant inhibition of
COX-1, making the development of selective COX-2 inhibitors highly
desirable. Such agents could act either by direct inhibition of COX-2
and/or inhibition of COX-2 gene expression (Subbaramaiah et al., 1997
).
COX-1 enzyme activity is inhibited only 30% by 10 µM GF-015 or
GF-90, whereas 10 nM indomethacin produces 58% inhibition (our
unpublished observations). In addition, COX-2 enzyme activity is not
inhibited but increased 18 or 42%, respectively, by 10 µM GF-015 or
GF-90 compared with the 51% inhibition seen using the specific COX-2
inhibitor celecoxib at 0.3 µM. Thus, GF-015 and GF-90 inhibit COX-2
gene expression and also have the advantage of having less of an
inhibitory effect on COX-1 enzyme activity compared with NSAIDs.
The development of new drugs that inhibit the NF-
B activation
pathway will require pharmacokinetic and toxicity studies in conjunction with clinical verification of in vivo activity. In this
study, the inhibitory effect of GF-015 and GF-90 on TNF-
- and
TPA-induced COX-2 protein expression was caused by suppression of IKK
activity and NF-
B activation in the COX-2 promoter, resulting in
attenuation of COX-2 gene expression and PGE2
production. Because these compounds have less of an effect on COX-1
enzyme activity than NSAIDs, these two conjugated polyhydroxybenzene
derivatives could serve as lead compounds for the development of new
drugs in preventing or treating inflammation and cancer.
This work was supported by a research grant from the National
Science Council of Taiwan.
Dr. Ching-Chow Chen, Department
of Pharmacology, College of Medicine, National Taiwan University, No.1,
Jen-Ai Road, 1st Section, Taipei, 10018, Taiwan. E-mail:
ccchen{at}ha.mc.ntu.edu.tw