(Received for publication, July 13, 1995; and in revised form, August 11, 1995)
From the
When activated, NF-B, a ubiquitous transcription factor,
binds DNA as a heterodimeric complex composed of members of the
Rel/NF-
B family of polypeptides. Because of its intimate
involvement in host defense against disease, this transcription factor
is an important target for therapeutic intervention. In the present
report we demonstrate that curcumin (diferuloylmethane), a known
anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of
NF-
B activation. Treatment of human myeloid ML-1a cells with tumor
necrosis factor (TNF) rapidly activated NF-
B, which consists of
p50 and p65 subunits, and this activation was inhibited by curcumin.
AP-1 binding factors were also found to be down-modulated by curcumin,
whereas the Sp1 binding factor was unaffected.
Besides TNF, curcumin
also blocked phorbol ester- and hydrogen peroxide-mediated activation
of NF-B. The TNF-dependent phosphorylation and degradation of
I
B
was not observed in curcumin-treated cells; the
translocation of p65 subunit to the nucleus was inhibited at the same
time. The mechanism of action of curcumin was found to be different
from that of protein tyrosine phosphatase inhibitors. Our results
indicate that curcumin inhibits NF-
B activation pathway at a step
before I
B
phosphorylation but after the convergence of
various stimuli.
Members of the transcription factor NF-B family play a
central role in various responses leading to host defense, activating a
rapid progression of gene expression. These transcription factors are
dimeric complexes composed of different members of the Rel/NF-
B
family of polypeptides. This family is distinguished by the presence of
a Rel homology domain of about 300 amino acids that displays a 35 to
61% identity between various family members (for review, see (1) ). Although NF-
B is a ubiquitous transcription factor,
it plays a critical role in the cells of the immune system, where it
controls the expression of various cytokines and the major
histocompatibility complex genes. The inappropriate regulation of
NF-
B and its dependent genes have been associated with various
pathological conditions including toxic/septic shock, graft versus host reaction, acute inflammatory conditions, acute-phase
response, viral replication, radiation damage, atherosclerosis, and
cancer(1, 2) . No wonder NF-
B is an important
target for therapeutic intervention.
Unlike other transcription
factors, the NF-B proteins and other members of the Rel family
reside in the cytoplasm in an inactive state but upon activation, they
are translocated to the nucleus. The nuclear translocation of Rel
proteins is induced by many agents, including inflammatory cytokines (e.g. tumor necrosis factor (TNF), (
)lymphotoxin,
and interleukin-1), mitogens, bacterial products, protein synthesis
inhibitors, oxidative stress (H
O
), ultraviolet
light, and phorbol esters(3, 4) . Upon activation of
NF-
B, a large number of genes are induced including various
inflammatory cytokines, adhesion molecules, and Rel proteins (for
review, see (3) and (4) ).
Curcumin
(diferuloylmethane) has been shown to block many reactions in which
NF-B plays a major role. This agent is a major active component of
turmeric (Curcuma longa) and it gives specific flavor and
yellow color to curry. The compound has been shown to display
anticarcinogenic properties in animals as indicated by its ability to
inhibit both tumor initiation induced by benz(
)pyrene and
7,12-dimethylbenz(
)anthracene (5, 6, 7, 8) and tumor promotion
induced by phorbol esters(9, 10) , which are known to
activate NF-
B. Curcumin has also been shown to inhibit type 1
human immunodeficiency virus long terminal repeat (HIV-LTR) directed
gene expression and virus replication stimulated by TNF and phorbol
ester(11) , which likewise require NF-
B activation. The
anti-inflammatory and antioxidant properties of curcumin have been well
documented(12, 13, 14) . How these inhibitory
responses are modulated by curcumin is not understood.
In the
present report we show that curcumin is a potent inhibitor of NF-B
activation induced by various agents. The results also indicate that
curcumin inhibits at a step in the signal transduction cascade of
NF-
B activation that occurs before I
B
phosphorylation
but after the point at which various signals transduced by different
stimuli converge. This study shows that curcumin is a potential
candidate for modulation of NF-
B-dependent pathological
conditions.
Electrophoretic mobility shift assays (EMSA)
were performed by incubating 4 µg of nuclear extract (NE), with 16
fmol of P-end-labeled 45-mer double-stranded NF-
B
oligonucleotide from the HIV-LTR,
5`-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3` (17) , for 15 min at 37 °C. The incubation mixture included
2-3 µg of poly(dI-dC) in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 1%
Nonidet P-40, 5% glycerol, and 50 mM
NaCl)(18, 19) . The DNA-protein complex formed was
separated from free oligonucleotide on 4.5% native polyacrylamide gel
using buffer containing 50 mM Tris, 200 mM glycine,
pH 8.5, and 1 mM EDTA(20) , and then the gel was
dried. A double-stranded mutated oligonucleotide,
5`-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3`, was
used to examine the specificity of binding of NF-
B to the DNA. The
specificity of binding was also examined by competition with the
unlabeled oligonucleotide.
For supershift assays, nuclear extracts
prepared from TNF-treated cells were incubated with the antibodies
against either p50 or p65 subunits of NF-B for 30 min at room
temperature before the complex was analyzed by EMSA(21) .
Antibodies against cyclin D1 and preimmune serum were included as
negative controls.
The EMSAs for AP-1 and Sp1 were performed as
described for NF-B using
P-end-labeled
double-stranded oligonucleotides. Specificity of binding was determined
routinely by using an excess of unlabeled oligonucleotide for
competition as described earlier(21) .
Visualization and quantitation of radioactive bands was carried out by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ``Image-quant'' software.
In this report we examined the effect of curcumin on the
activation of transcription factor NF-B. We used human ML-1a cells
for these studies because their response to NF-
B activation by
various stimuli has been well
characterized(21, 22, 23) . The time of
incubation and the concentration of the drugs used in our studies had
no effect on the cell viability (data not shown).
Figure 1:
Dose
response and kinetics of inhibition of TNF-dependent NF-B
activation by curcumin. a, ML-1a cells (2
10
/ml) were preincubated at 37 °C for 60 min with
different concentrations (2-60 µM) of curcumin
followed by 30 min incubation with 0.1 nM TNF. b,
ML-1a cells (2
10
/ml) were preincubated at 37
°C with 20 µM curcumin for different times and then
tested for NF-
B activation at 37 °C for 30 min either with or
without 0.1 nM TNF. - indicates time curcumin was
present before the addition of TNF; 0 indicates co-incubation with TNF;
and + indicates time curcumin was added after TNF. For panel
c, ML-1a cells (2
10
/ml) were incubated at 37
°C with 50 µM curcumin for 60 min followed by
treatment with 10 nM TNF for different times. After these
treatments nuclear extracts were prepared and then assayed for
NF-
B as described under ``Experimental Procedures.'' The
arbitrary units represent the relative amounts of the radioactivity
present in respective bands.
Previous studies from our laboratory
have shown that a high concentration of TNF (10 nM) can
activate NF-B within 5 min and this induction is higher in its
intensity than that obtained with cells using 100-fold lower
concentration of TNF for longer time(23) . To determine the
effect of curcumin on NF-
B activation at higher TNF concentration
and its effect on kinetics of TNF-mediated activation of NF-
B,
curcumin-pretreated cells were exposed to 10 nM TNF for
various times (Fig. 1c). In agreement with our previous
results, the induction of NF-
B by 10 nM TNF was very high
and occurred within 5 min. Curcumin could completely inhibit the
activation of NF-
B induced by 10 nM as efficiently as it
did with 0.1 nM TNF. This suggests that curcumin is a very
potent inhibitor of NF-
B activation.
To show that the retarded
band observed by EMSA in TNF-treated cells was indeed NF-B we
incubated the nuclear extracts with antibody to either p50 (NF-
B1)
or p65 (Rel A) subunits and then carried out EMSA. The results from
this experiment (Fig. 2a) show that antibodies to
either subunit of NF-
B shifted the band to higher molecular
weight, thus suggesting that the TNF-activated complex consisted of p50
and p65 subunits. Neither preimmune serum nor irrelevant antibody
against cyclin Di had any affect on the mobility of NF-
B.
Figure 2:
Supershift analysis and specificity of the
effect of curcumin on the NF-B activation. For panel a,
nuclear extracts were prepared from untreated or TNF (0.1
nM)-treated ML-1a cells (2
10
/ml),
incubated for 30 min with antibodies and then assayed for NF-
B as
described under ``Experimental Procedures.'' For panel
b, nuclear extract prepared from TNF pretreated cells were
incubated with different concentrations of curcumin for 15 min and then
analyzed for NF-
B by EMSA. DMSO, dimethyl sulfoxide; PIS,
preimmune serum.
Both
TPCK and herbimycin A have been shown to interfere with the binding of
NF-B to the DNA(25, 52) . To determine the effect
of curcumin on the binding of NF-
B to the DNA, the nuclear
extracts from TNF-preactivated cells were incubated with curcumin and
then EMSA was performed. The results of this experiment (Fig. 2b) show that curcumin did not modify the ability
of NF-
B to bind to the DNA.
Figure 3:
Effect of curcumin on PMA- and
HO
-mediated activation of NF-
B. ML-1a
cells (2
10
/ml) were preincubated for 60 min at 37
°C with curcumin (50 µM) followed by PMA (25 ng/ml) or
H
O
(0.5 mM) or the indicated
combinations for 30 min and then tested for NF-
B activation as
described under ``Experimental
Procedures.''
Figure 4: Effect of curcumin on AP-1 and Sp1 transcription factors. Cells were treated with different concentrations of curcumin for 60 min at 37 °C, and nuclear extract were then prepared and used for EMSA of AP-1 and Sp1 transcription factors as described.
Figure 5:
Effect of DTT and DMP on the curcumin and
phenylarsine oxide (PAO)-induced inhibition of NF-B
activation. ML-1a (2
10
/ml) were incubated at 37
°C for 60 min with DTT (100 µM) or DMP (100
µM) in the presence of curcumin (50 µM) or
phenylarsine oxide (2.4 µM) or the indicated combinations
followed by 30 min incubation with 0.1 nM TNF and then assayed
for NF-
B activation as described under ``Experimental
Procedures.''
Figure 6:
Effect of curcumin on TNF-induced
phosphorylation and degradation of IB
and on level of p65 in
cytoplasm and nucleus. ML-1a (2
10
/ml) pretreated
(for 60 min at 37 °C) with or without curcumin (50 µM)
were incubated for different times with TNF (0.1 nM), and then
assayed for I
B
(panel A) and for p65 (panel
B) in cytosolic fractions by Western blot analysis as described
under ``Experimental Procedures.'' For panel C,
ML-1a (2
10
/ml) pretreated (for 60 min at 37
°C) with curcumin were incubated with TNF (0.1 nM) for 30
min. Nuclear and cytoplasmic extracts were assayed by Western blot
analysis for p65. The arbitrary units represent the relative amounts of
the respective proteins as described under ``Experimental
Procedures.''
We also
measured the level of p65 in the cytoplasm and nucleus. As expected
upon TNF treatment, the level of p65 declined in the cytoplasm with a
concurrent increase in the nucleus (Fig. 6, B and C). The treatment of cells with curcumin abolished the
TNF-dependent change in the nuclear and cytoplasmic p65 levels. These
results show that curcumin inhibits the TNF-induced translocation of
p65 to the nucleus and this is consistent with the inhibition of
TNF-dependent degrdation of IB
by curcumin.
Curcumin is a pharmacologically safe compound with known
anti-inflammatory, anticarcinogenic, and free radical scavenger
properties(6, 10, 27, 28, 29, 30) .
However, how curcumin carries out these functions is not very clear. We
investigated curcumin's effect on NF-B activation because
NF-
B is involved in so many of the activities that curcumin is
known to block. NF-
B plays a pivotal role in cells of the immune
system because it is rapidly activated by a wide variety of pathogenic
signals and functions as a potent and pleiotropic transcriptional
activator. Intervention in NF-
B activation may be beneficial in
suppressing toxic/septic shock, graft versus host reactions,
acute inflammatory reactions, HIV replication, acute-phase response,
and radiation damage.
Our results show that curcumin completely
blocked the TNF-dependent activation of NF-B. The activation
induced by various other agents including phorbol ester and
H
O
was also inhibited by curcumin. As has been
shown with other inhibitors, the effect of curcumin was not due to the
chemical modification of NF-
B proteins (25, 31, 52) . The inhibition of NF-
B
activation was accompanied by the inhibition of p65 translocation to
the nucleus and of I
B
degradation.
Identifying how
curcumin blocks the activation of NF-B requires an understanding
of the mechanism by which various inducers activate this important
transcription factor. The role of different TNF-activated signals
including acidic and neutral sphingomyelinase-generated ceramides,
proteases, serine/threonine protein kinase, protein tyrosine kinase,
protein tyrosine phosphatase, and superoxide radicals in the activation
of NF-
B have been
implicated(1, 21, 22, 32, 33, 34, 35) .
Whether these signals are generated by TNF sequentially or
independently of each other, however, is not understood.
All three
inducers of NF-B used in our studies are known to produce reactive
oxygen intermediates (ROI). Therefore, it is possible that the effect
of curcumin is through quenching of ROI production. The inhibitors of
mitochondrial electron transport have been shown to impair the
TNF-induced activation of NF-
B(36) , thus also suggesting
the role of ROI. Several additional, indirect lines of evidence suggest
a role for ROI as a common and critical
denominator(40, 41) , including evidence that cellular
levels of ROI increase in response to TNF, interleukin-1, PMA,
lipopolysaccharide, UV light, and
-irradiation (for review, see (1) ). But among the various ROI administered to cells in
culture, only hydrogen peroxide was found to be an effective activator
of NF-
B(42) .
Curcumin may also block NF-B
activation by inhibiting a protein kinase. In vitro, curcumin
has been shown to inhibit both serine/threonine protein kinase and
protein tyrosine kinase(44) . The protein kinase needed for the
activation of NF-
B has not, however, been identified. Although PMA
is an activator of protein kinase C, both TNF and H
O
have been shown to activate both protein kinase C and protein
tyrosine kinase. NF-
B activation by TNF and H
O
has been shown to be blocked by inhibitors of both protein kinase
C and protein tyrosine kinase(50) . The role of a protein
tyrosine kinase has also been implicated in NF-
B activation by
ultraviolet light, lipopolysaccharide, hypoxia, and
v-src(37, 38, 39, 40, 51) .
We have shown that TNF-dependent activation of NF-
B is dependent
on erbstatin-sensitive protein tyrosine kinase(22) . Studies of
Schievien et al.(43) showed that protein tyrosine
kinase inhibitors block
-irradiation-induced NF-
B activation,
a stimulant thought to work through the immediate generation of ROI,
which suggest that protein tyrosine kinase activation may precede ROI
generation. Thus there are different early events involved in
activation of NF-
B but all of them may converge to phosphorylate
the I
B
which precedes its degradation and the subsequent
translocation of p65 into the nucleus.
It has been shown that
curcumin not only inhibits the DNA binding of c-jun/AP-1
transcription factor but it also down-modulates c-jun level by
preventing its transcription(24) . Our data are in agreement,
but this raises the question of what other transcription factors
curcumin inhibits. We found that curcumin did not inhibit the Sp1
transcription factor under the same conditions in which it inhibited
NF-B and AP-1 transcription factors. Curcumin has also been shown
to inhibit TNF and phorbol ester-stimulated type 1 HIV-LTR-directed
gene expression and virus replication(11) , and this may be
mediated through the inhibition of NF-
B. Recently it has been
reported that curcumin can also inhibit nitric oxide
synthase(45, 46, 47) . These observations can
be explained based on our results since the expression of this enzyme
is NF-
B dependent. This is consistent with the observation that
TPCK, a protease inhibitor that blocks NF-
B activation, also
blocks the expression of nitric oxide synthase(48) . TPCK,
however, may exert its effect by a different mechanism than curcumin
does. It has been shown that TPCK chemically modifies NF-
B, thus
altering its release from I
B
(25) . Curcumin, however,
does not chemically modify the DNA binding properties of NF-
B.
Another level of modification that could prevent formation of
p50/p65 heterodimer is down-modulation of the cytoplasmic pool of p65
subunit of NF-B. Our results, however, show that p65 was not
down-modulated by curcumin but its translocation to the nucleus was
inhibited, most likely through inhibition of degradation of
I
B
.
The observation that TNF-induced phosphorylation and
degradation of IB
is abolished by curcumin indicate that the
step in the signal transduction pathway of NF-
B activation
inhibited by this agent is at or before the phosphorylation step of
NF-
B (Fig. 7). That it can inhibit NF-
B activation by
diverse agents indicate that this step is after or at the step where
the diverse signals converge. Overall we conclude that because of its
very low pharmacological toxicity and its ability to modulate
activation of NF-
B by various agents, curcumin has a high
potential for use in modulating expression of genes regulated by
NF-
B.
Figure 7:
Possible site of action of curcumin on
TNF- PMA- and HO
-induced NF-kB
activation.