Curcumin Suppresses Activation of NF-kB and AP-1 Induced by Phorbol Ester in Cultured Human Promyelocytic Leukemia Cells

Journal of Biochemistry and Molecular Biology, Vol. 35, No. 3, May 2002, pp. 337-342
© BSRK & Springer-Verlag 2002
Curcumin Suppresses Activation of NF-?B and AP-1 Induced by
Phorbol Ester in Cultured Human Promyelocytic Leukemia Cells
Seong-Su Han, Young-Sam Keum, Hyo-Joung Seo and Young-Joon Surh*
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
Received 21 February 2002, Accepted 19 March 2002
Many components that are derived from medicinal or
DNA damage in mouse epidermis (Huang et al., 1997) and
dietary plants possess potential chemopreventive
cultured mouse fibroblast cells (Shih and Lin, 1993), as well
properties. Curcumin, a yellow coloring agent from
as superoxide production in macrophages (Ruby et al., 1995).
turmeric (Curcuma longa Linn, Zingiberaceae), possesses
It also suppresses the expression of phospholipase,
strong antimutagenic and anticarcinogenic activities. In
cyclooxygenase, and inducible nitric oxide synthase that are
this study, we have found that curcumin inhibits the 12-O-
involved in mediating inflammatory responses (Huang et al.,
tetradecanoylphorbol-13-acetate (TPA)-induced nuclear
1991; Rao et al., 1995; Pan et al., 2000). Moreover, curcumin
factor ?B (NF-?B) activation by preventing the
preferentially causes the apoptosis of several types of cancer
degradation of the inhibitory protein I?B? and the
cells (Jiang et al., 1996a,b; Kuo et al., 1996; Samaha et al.,
subsequent translocation of the p65 subunit in cultured
1997; Shim et al., 2001). For instance, curcumin inhibits the
human promyelocytic leukemia (HL-60) cells.
proliferation/growth of Jurkat T leukemia cells (Sikora et al.,
Alternatively, curcumin repressed the TPA-induced
1997) and BKS-2 B lymphoma cells (Han et al., 1999) more
activation of NF-?B through direct interruption of the
effectively than the primary thymocytes and normal splenic B
binding of NF-?B to its consensus DNA sequences.
cells, respectively.
Likewise, the TPA-induced DNA binding of the activator
The nuclear transcription factor kappa-B (NF-?B) is one of
protein-1 (AP-1) was inhibited by curcumin pretreatment.
the most ubiquitous transcription factors that regulates the
expression of distinct sets of genes that encode proteins
Keywords: AP-1, Curcumin, HL-60 cells, NF-?B, 12-O-
involved in mediating cellular proliferation, inflammatory
Tetradecanoylphorbol-13-acetate
responses, cell adhesion, etc. The functionally active NF-?B
exists mainly as a hetero-dimer consisting of subunits of the
Rel family, which are normally sequestered in the cytosol as
Introduction
an inactive complex by binding to the inhibitory protein I?B.
A wide variety of naturally-occurring substances in edible
Phosphorylation and subsequent ubiquitination of I?B upon
plants possess substantial chemopreventive or
exposure of the cells to various extracellular stimuli causes
chemoprotective activities. These are often attributed to their
rapid degradation of this inhibitory subunit by proteosomes.
antioxidative and anti-inflammatory properties (Surh, 1999;
The resulting free NF-?B translocates to the nucleus, where it
Surh et al., 2001). Curcumin (diferuloylmethane; structure
binds to the specific ?B binding sites that are located in the
shown in Fig. 1), a yellow pigment that is derived from
promoter region of the target genes, thereby controlling their
turmeric (Curcuma longa L., Zingiberaceae), is protective
against a wide range of experimentally-induced tumors. These
include mammary, forestomach, duodenal, skin, and colon
cancers (Nagabhushan et al., 1992; Huang et al., 1997;
Samaha et al., 1997; Verma et al., 1997; Huang et al., 1998;
Dorai et al., 2000). The compound alleviates the 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced oxidative
*To whom correspondence should be addressed.
Tel: 82-2-880-7845; Fax: 82-2-874-9775
E-mail: surh@plaza.snu.ac.kr
Fig. 1. Structure of curcumin.

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Seong-Su Han et al.
expression (Sen and Packer, 1996; Barnes et al., 1997). NF-
double-strand NF-?B oligonucleotide was labeled with [?-32P]ATP by
?B has dual functions in terms of regulating cell survival and
T4 polynucleotide kinase and purified on a Nick column (Pharmacia
apoptosis (Shishodia and Aggarwal, 2002). Another
Biotech Inc., Buckinghamshire, UK). The binding reaction was
transcription factor, activator protein-1 (AP-1), also has a
carried out in 25 µl of a mixture that contained 5 µl of an incubation
central role in controlling the eukaryotic gene expression. AP-
buffer [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM
1 is composed of Jun and Fos proteins, which interact via a
EDTA, 4% (v/v) glycerol, and 0.1 mg/ml sonicated salmon sperm
leucine-zipper domain. Like NF-?B, DNA binding of AP-1 is
DNA], 10 µg of nuclear extract, and 100,000 cpm of the labeled
influenced by the cellular redox state (Abate et al., 1990; Sen
probe. To verify the specificity of NF-?B, fifty-fold excess of
and Packer, 1996). AP-1 activation is required for TPA-
unlabeled NF-?B oligonucleotide was added to the reaction mixture
as a competitor. For the supershift assay, 2 µg of p50 or p65 antibody
stimulated cellular proliferation and transformation. It is
was added. After a 20-min incubation at room temperature, 2 µl of
considered to be essential in tumor promotion (Angel and
0.1% bromophenol blue was added. The samples were then
Karin, 1991; Huang et al., 1991; Dong et al., 1994; Li et al.,
electrophoresed through a 6% nondenaturing polyacrylamide gel at
1997).
150 V at room temperature. Finally, the gel was dried and exposed to
Curcumin has multifaceted functions in influencing the
x-ray film. EMSA for AP-1 was carried out in the same manner as
expression of proteins that are involved in cellular
that for NF-?B, except that the AP-1 oligonucleotide (Promega,
proliferation, inflammation, adhesion, malignant
Madison, USA) was used as a probe (Kwon et al., 2001).
transformation, etc. As part of our research program to
elucidate the molecular mechanisms that underlie the
Western blot analysis of p65 and I?B? Both the nuclear and
pleiotropic actions of this chemopreventive phytochemical, we
cytosolic extracts that were prepared from the HL-60 cells were
investigated its effects on the activation of two prototype
subjected to 12% SDS-polyacrylamide gel electrophoresis for
eukaryotic transcription factors, NF-?B and AP-1.
measuring p65 and I?B? levels. After a 3-h transfer of the gel to the
PVDF membrane (Amersham Life Sciences, Arlington Heights,
USA), the blots were blocked with 5% fat-free dry milk in
Materials and Methods
phosphate-buffered saline that contained 0.1% Tween-20 for 2 h at
Chemicals
Curcumin and gentamycin were purchased from the
room temperature, then washed in the same buffer. The p65 protein
Sigma Chemical Co. (St. Louis, USA). TPA was a product of
was detected with a rabbit p65 polyclonal antibody (Santa Cruz
Alexis Biochemicals (San Diego, USA). RPMI 1640 and fetal
Biotech., Santa Cruz, USA) that was diluted 1:2000. I?B? protein
bovine serum were supplied from Gibco-BRL (Rockville, USA).
was detected with a rabbit I?B? polyclonal antibody (Santa Cruz
Biotech, Santa Cruz, USA) that was diluted 1 : 1000. Goat anti-rabbit
Preparation of cytosolic and nuclear extracts from HL-60 cells
immunoglobulin G-conjugated horseradish peroxidase (diluted
Unless otherwise specified, the HL-60 cells (1 × 107) were grown in
1 : 5000) was used as a secondary antibody. The transferred proteins
a RPMI 1640 medium that was supplemented with 10% heat-
were visualized with an enhanced chemiluminescence (ECL)
inactivated fetal bovine serum and gentamycin (5 µg). Cells were
detection kit (Amersham Life Sciences, Arlington Heights, USA).
treated with dimethyl sulfoxide (DMSO) or TPA (10 nM) for 1 h.
When necessary, varying concentrations of curcumin were added
Results
30 min before the TPA treatment. Curcumin was dissolved in
DMSO. The proportion of DMSO in the culture media did not
TPA induces the activation of NF-?B in HL-60 cells in a
exceed 0.5%. The control cells were treated with the same volume
dose- and time-related manner
In order to investigate the
of solvent. The cells were lysed by incubation at 4oC for 10 min in
effect of TPA on the activation of NF-?B, the HL-60 cells
400 µl of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.2 mM
were incubated with various concentrations of TPA for 1 h,
EDTA, 1.5 mM MgCl , 0.5 mM DTT, 0.2 mM
2
and EMSA was performed. The activation of NF-?B, as
phenylmethylsulfonyl fluoride (PMSF)]. The cell lysate was
assessed in terms of its DNA binding activity, was evident
centrifuged for 6 min. The resulting pellet was resuspended in
when the cells were incubated with 1 nM TPA. The maximal
100 µl of ice-cold buffer C [20 mM HEPES (pH 7.9), 420 mM
NF-?B DNA binding was observed with 10 nM TPA (Fig.
NaCl, 1.5 mM MgCl , 20% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM
2
2A). We then examined the kinetics of the TPA-induced
DTT, 0.2 mM PMSF], followed by incubation at 4oC for 20 min.
activation of NF-?B in the same cell line after treatment with
After centrifugation, the supernatant was collected, aliquoted, and
10 nM TPA. As shown in Figure 2B, NF-?B activation peaked
stored at ?70oC (Dent and Latchman, 1993). The protein content of
the final extracts was estimated using the BCA kit that was supplied
at 1 h, and decreased to the baseline level in 6 h.
from Bio-Rad (Richmond, USA), according to the manufacturers
One of the most predominant forms of NF-?B/Rel proteins
protocol.
is a heterodimer of p50 and p65 (Thanos and Maniatis, 1995).
In order to ascertain the specificity, as well as the identity of
Electrophoretic mobility shift assay (EMSA)
EMSA was
NF-?B in HL-60 cells, EMSA was performed with excess
performed using a DNA-protein binding detection kit (Gibco-BRL;
amounts of unlabeled NF-?B oligonucleotide for the
Rockville, USA) for the measurement of NF-?B binding, according
competition assay and with antibodies against typical NF-?B
to the manufacturer’s protocol with minor modifications. Briefly, the
subunits, p50 and p65, for the super-shift assay. As illustrated

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Inhibition of Phorbol Ester-Induced Activation of NF-?B and AP-1 by Curcumin
339
Fig. 2. (A) Concentration-dependent activation of NF-?B in HL-60 cells treated with TPA. HL-60 cells (1 × 106/ml) were treated with
0, 1, 5, 10, or 20 nM of TPA for 1 h. (B) Kinetics of NF-?B DNA binding in HL-60 cells treated with 10 nM TPA. HL-60 cells
(1 × 106/ml) were treated with 10 nM TPA for various time periods. Lane 1, probe only; lane 2, DMSO control; lane 3, 0.5 h; lane 4,
1 h; lane 5, 1.5 h; lane 6, 2 h; lane 7, 4 h; lane 8, 6 h; lane 9, 8 h. Nuclear extracts (10 µg) were incubated with radiolabeled NF-?B
oligonucleotide at room temperature for 20 min. (C) Competition and super-shift assays for NF-?B DNA binding. Nuclear extracts
(10 µg) from TPA (10 nM)-treated HL-60 cells were incubated with 50-fold excess of unlabeled NF-?B oligonucleotide (lane 3), 2 µg
of p50 antibody (lane 4), and 2 µg of p65 antibody (lane 5). Lane 1, probe only. Lane 2, nuclear extract from TPA-treated cells alone.
EMSA was performed as described in Materials and Methods.
in Figure 2C, incubation of the TPA-stimulated nuclear extract
with the 50-fold excess unlabeled NF-?B oligonucleotide
before EMSA abolished the NF-?B DNA binding that was
induced by TPA. This indicates that the retarded band that was
observed in EMSA is indeed NF-?B. Incubation of TPA-
stimulated nuclear extracts with an antibody against either p50
or p65 shifted the band with the higher molecular weight (Fig.
2C). These results indicate that the TPA-activated NF-?B
complex in HL-60 cells exists as a heterodimer that consists of
at least two typical NF-?B subunits, p50 and p65 proteins.
Curcumin inhibits TPA-induced NF-?B activation by
blocking the degradation of I?B? and nuclear
translocation of p65 subunits
To examine whether
curcumin could modulate TPA-induced NF-?B activation in
the HL-60 cells, the cells were treated with various
concentrations of curcumin for 30 min prior to the stimulation
with 10 nM TPA for 1 h. TPA-induced NF-?B activation was
significantly inhibited when the HL-60 cells were pretreated
Fig. 3. (A) Effects of curcumin on TPA-induced NF-?B
with 5 µM or 10 µM curcumin, while no significant effect
activation in HL-60 cells. HL-60 cells (1 × 106/ml) were treated
was observed at 1 µM (Fig. 3A). In an attempt to elucidate the
with DMSO alone (lane 2) or 10 nM TPA for 1 h in the absence
mechanism that underlies the inhibitory effects of curcumin
(lane 3) or presence of 1 µM (lane 4), 5 µM (lane 5), or 10 µM
on TPA-induced NF-?B activation, we tested whether
(lane 6) of curcumin. Experimental details are described in
curcumin could block the TPA-induced degradation of I?B?
Materials and Methods. Lane 1 represents the probe only. (B)
and nuclear translocation of p65. As shown in Figure 3B,
Effects of curcumin on the levels of I?B? in cytosol and p65 in
curcumin inhibited both processes in a concentration-
nucleus. HL-60 cells (1 × 106/ml) were treated with DMSO or
curcumin (5 or 10 µM) for 30 min prior to stimulation with TPA
dependent manner.
(10 nM). Nuclear and cytosolic fractions were prepared 1 h later
and subjected to an immunoblot analysis to detect p65 and
Curcumin suppresses the activation of AP-1 induced by
I?B?, respectively. Lane 1, DMSO as a control; lane 2, TPA
TPA in HL-60 cells
Besides NF-?B, AP-1 also plays a
alone; lane 3, 1 µM curcumin + TPA; lane 4, 5 µM curcumin +
crucial role in the regulation of a vast variety of genes that are
TPA; lane 5, 10 µM curcumin + TPA. Abbreviations: NE, nuclear
responsible for cell proliferation and differentiation.
extract; CE, cytosolic extract.

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340
Seong-Su Han et al.
Fig. 5. Effect of curcumin on DNA-binding activity of NF-?B
(A) and AP-1 (B) in HL-60 cells. HL-60 cells were treated with
Fig. 4. Effect of curcumin on TPA-induced AP-1 activation in
DMSO (lane 2) or 10 nM TPA in the absence (lane 3) or
HL-60 cells. HL-60 cells (1 × 106/ml) were treated with 0 µM
presence (lane 4) of 10 µM curcumin. For lane 5, the nuclear
(lane 3), 1 µM (lane 4), 5 µM (lane 5), or 10 µM (lane 6) of
extracts from TPA-stimulated HL-60 cells were incubated with
curcumin for 30 min prior to TPA as described in the legend to
10 µM curcumin for 20 min at room temperature before EMSA
Fig. 3. The control cells were treated with DMSO alone (lane 2).
with an oligonucleotide probe for NF-?B or AP-1. Lane 1
The nuclear extract (10 µg) was subjected to EMSA, described
represents the probe only.
under Materials and Methods. Lane 1 represents the probe only.
activation of NF-?B and AP-1 may differ from one cell type
Therefore, we also examined the effect of curcumin on the
to another, and depend on the types and duration of stimuli
DNA binding of AP-1. When the HL-60 cells were
(Angel and Karin, 1991; Johnson et al., 1996), we initially
preincubated with various concentrations of curcumin, TPA-
tried to assess whether or not the classical tumor promoter
induced activation of AP-1 was inhibited in a concentration-
TPA can activate NF-?B and AP-1 in the HL-60 cells, as it
dependent manner (Fig. 4). An almost complete inhibition of
does in other cell lines. Our results show that TPA rapidly and
AP-1 activation was achieved with curcumin at a
transiently induces the activation of the former transcription
concentration as low as 1 µM (Fig. 4).
factor. NF-?B that was activated in the TPA-stimulated HL-60
cells was found to consist of p65 and p50 subunits.
Curcumin can also directly interrupt DNA binding of NF-
There is accumulating evidence that the activation of NF-
?B and AP-1 to their consensus sequences In the previous
?B is pivotal in regulating the expression of proteins that are
experiment, curcumin was found to inhibit NF-?B activation
associated with tumor promotion, which is suppressed by
by blocking I?B? degradation in the cytoplasm and
some chemopreventive agents, including curcumin (Singh and
translocation of p65 to the nucleus. Alternatively, curcumin
Aggarwal, 1995; Natarajan et al., 1996; Bierhous et al., 1997;
could suppress NF-?B activation by directly interfering with
Plummer et al., 1999; Surh et al., 2000). Therefore, we found
the DNA binding of the functionally active subunit of NF-?B.
that curcumin inhibits the activation of this transcription factor
To test this possibility, the nuclear extract that was isolated
in TPA-treated HL-60 cells. Since the translocation of p65 to
from the TPA-stimulated HL-60 cells was treated with 10 µM
the nucleus is preceded by the phosphorylation and
curcumin in vitro, and EMSA was conducted. Curcumin
degradation of I?B? in the cytoplasm (Remacle et al., 1995;
directly inhibited the ability of NF-?B to bind DNA (Fig. 5A).
Thanos and Maniatis, 1995), we also determined whether or
Likewise, the direct DNA binding capability of AP-1 was
not curcumin could inhibit the nuclear translocation of p65 by
repressed by addition of curcumin to the EMSA mixture
preventing the degradation of the inhibitory protein I?B?. Our
containing preactivated nuclear extracts (Fig. 5B).
results reveal that curcumin does inhibit the TPA-induced NF-
?B activation by preventing the degradation of I?B? and
Discussion
subsequent translocation of the p65 subunit, which agrees
with previously reported findings (Singh et al., 1996; Jobin et
Recent advances in our understanding of the biochemical and
al., 1999). Curcumin completely blocks the TPA- and
molecular basis of inflammatory processes reveal that the
bacterial lipopolysaccharide-induced activation of NF-?B in
transcription factors NF-?B and AP-1 are implicated in the
endothelial cells (Pendurthi et al., 1997). Curcumin also
inducible expression of a wide array of genes in response to
prevents the tumor necrosis factor-? (TNF-?)-induced NF-?B
proinflammatory cytokines, reactive oxygen species, and
activation by inhibiting the phosphorylation and degradation
mitogens (Chabot-Fletcher, 1996; Sen and Packer, 1996).
of I?B in human myelomonoblastic leukemia cells (Singh and
Since intracellular signaling pathways that lead to the
Aggarwal, 1995). According to Pendurthi et al. (1997), the

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Inhibition of Phorbol Ester-Induced Activation of NF-?B and AP-1 by Curcumin
341
TNF-?-induced activation of NF-?B was suppressed by
sites of NF-?B and AP-1 transcription factors through the
curcumin through the blockade of the degradation of I?B?
Michael addition, thereby hampering their DNA binding.
and subsequent activation of p65 in endothelial cells.
In conclusion, our findings indicate that curcumin
Likewise, curcumin inhibited the interleukin-1?-mediated
suppresses the TPA-stimulated activation of NF-?B and AP-1.
phosphorylation and subsequent degradation of I?B? in rat
This may contribute to the anti-tumor promoting properties
intestinal epithelial cells, which led to the inactivation of NF-
this chemopreventive phytochemical retains.
?B (Jobin et al., 1999). Therefore, it seems likely that
curcumin regulates the upstream pathway(s) of I?B?
Acknowledgments
This work was supported by a grant
phosphorylation, thereby preventing I?B? degradation, which
(2000-2-20800-003-5) from the Basic Research Program of
results in the repression of NF-?B activation (Jobin et al.,
the Korea Science and Engineering Foundation (KOSEF).
1999; Pan et al., 2000). Additional studies will be necessary in
order to determine whether or not curcumin can suppress the
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