We are unable to create an online viewer for this document. Please download the document instead.
Journal of Biochemistry and Molecular Biology, Vol. 38, No. 4, July 2005, pp. 474-480
Curcumin Derivatives Inhibit the Formation of Jun-Fos-DNA Complex
Independently of their Conserved Cysteine Residues
Chi Hoon Park, Ju Hyung Lee and Chul Hak Yang*
Division of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Korea
Received 9 December 2004, Accepted 26 April 2005
Curcumin, a major active component of turmeric, has
been identified as an inhibitor of the transcriptional
activity of activator protein-1 (AP-1). Recently, it was also
Curcumin, a dietary pigment responsible for the yellow color
found that curcumin and synthetic curcumin derivatives
of cur y, is used as a traditionalmedicine for the treatment of
can inhibit the binding of Jun-Fos, which are the members
inflammatory conditions (Ammon et al., 1991). Further,
of the AP-1 family, to DNA. However, the mechanism of
curcumin has been reported to have anti-inflammatory and
this inhibition by curcumin and its derivatives was not
anti-oxidant activities, and chemopreventive effects (Srimal et
disclosed. Since the binding of Jun-Fos dimer to DNA can
al., 1973; Sharma, 1976; Toda et al., 1985; Satoskar et al.,
be modulated by redox control involving conserved cysteine
1986). In vivo, the administration of curcumin to mice treated
residues, we studied whether curcumin and its derivatives
with skin and colon carcinogens was found to reduce the
inhibit Jun-Fos DNA binding activity via these residues.
incidence and size of tumors produced (Conney et al., 1991;
However, the inhibitory mechanism of curcumin and its
Huang et al., 1992; Huang et al., 1994; Rao et al., 1995).
derivatives, unlike that of other Jun-Fos inhibitors, was
Many molecular investigations related to curcumin have been
found to be independent of these conserved cysteine
performed. Curcumin was found to inhibit cyclooxygenase-2
residues. In addition, we investigated whether curcumin
transcription (Zhang et al., 1999) and to mediate apoptosis in
derivatives can inhibit AP-1 transcriptional activity in vivo
AK-5 tumor cells (Bhaumik et al., 1999). Moreover, curcumin
using a luciferase assay. We found that, among the
was reported to inhibit proliferation and cell cycle progression
curcumin derivatives examined, only inhibitors shown to
and to inhibit the angiogenic response stimulated by
inhibit the binding of Jun-Fos to DNA by Electrophoretic
Fibroblast Growth Factor-2 (Mohan et al., 2000). In addition,
Mobility Shift Assay (EMSA) inhibited AP-1 transcriptional
it is well known to inhibit the transcriptional activity of AP-1
activity in vivo. Moreover, RT-PCR revealed that curcumin
(Huang et al., 1991; Han et al., 2002).
derivatives, like curcumin, downregulated c-jun mRNA in
Jun and Fos proteins are members of the AP-1 family, a family
JB6 cells. These results suggest that the suppression of the
of eukaryotic transcription factors that bind DNA in a sequence
formation of DNA-Jun-Fos complex is the main cause of
specific manner. The AP-1 binding site was first identified in the
reduced AP-1 transcriptional activity by curcuminoids,
enhancer regions of simian virus 40, human metallothionein (Lee
and that EMSA is a suitable tool for identifying inhibitors
et al., 1987). And, it is now apparent that similar nucleotide
of transcriptional activation.
sequence motifs are present in the negative and positive
regulatory regions of several genes (Distel et al., 1987). The
Keywords: AP-1, Curcumin, Cysteine, EMSA, Jun-Fos
sequence involved is 5'-TGAG/CTCA-3', termed TRE
(tetradecanoyl phorbol acetate[TPA] response elements). To form
the Jun-Fos-DNA complex, two domains of Jun and Fos are
important. One is a leucine zipper domain which is responsible
for the dimerization of Jun and Fos (Kouzarides et al., 1988),
whilst the other is a DNA binding domain containing clustered
basic amino acids, which is located immediately upstream of the
leucine zipper and is characterized by an abundance of positively
charged residues (Bohmann et al., 1989).
*To whom correspondence should be addressed.
Tel: +82-2-878-8545; Fax: +82-2-889-1568
No direct clue explains the relationship between AP-1
transcriptional activator and cancer promotion. However,
The Inhibition of AP-1 by Curcuminoids
some investigators have identified a variety of different tumor
Expression of wild and mutant truncated Jun and Fos proteins
promoters, including phorbol esters, UV irradiation, and trace
Truncated polypeptides corresponding to amino acid residues 116-
ion like As3+ that stimulate AP-1 transcriptional activity (Angel
211 of Fos and 224-334 of Jun, were expressed as hexahistidine
et al., 1991; Devary et al., 1991; Devary et al., 1992).
fusion proteins in E. coli and purified by nickel chelate af inity
Therefore, chemicals capable of inhibiting the transcriptional
chromatography (Park et al., 2002). Cysteine154 was replaced by
activity of AP-1 are important from the viewpoint of tumor
serine in the Fos mutant (FosM), and cysteine272 was replaced by
suppression. Recently, we showed that curcumin derivatives
serine in the Jun mutant (JunM) (Abate et al., 1990a).
can inhibit the formation of Jun-Fos-DNA complex as
efficiently as curcumin (Hahm et al., 2002); however, the
Electrophoretic mobility-shift assay (EMSA) Nuclear extract
inhibitory mechanism has not been elucidated. To disclose
(5 µg), prepared as described above, or truncated Jun and Fos
this mechanism we searched for residues of Jun-Fos dimer
proteins were incubated for 30min at room temperature in binding
that interact with curcumin and curcuminoids. It is believe
buf er [50 mM sodium phosphate (pH 7.3), 5% glycerol, 5 mM
that reduced cysteine residues in Jun and Fos proteins enhance
MgCl , 0.01% BSA] with curcumin or curcuminoids and a 32P-
DNA-binding whereas oxidized residues inhibit binding
labeled probe. The inhibitory effect of curcumin and some
curcuminoids were analyzed by was analyzed by nondenaturing 5%
et al., 1990b). We noticed cysteine residues (Fos-
polyacrylamide gel electrophoresis in 0.5 × TBE buf er at 100 V for
154 and Jun-Cys272 ) in the DNA-binding domains of the
40 min, and gels were visualized by autoradiography. For quantitative
two proteins, and presumed that these curcumin and
study, the Fos-Jun-DNA complex band on autoradiographed film
curcuminoids could inhibit Jun-Fos-DNA formation by
was scanned and interpreted quantitatively using the TotalLabTM
affecting the redox states of these cysteine residues. After
program from Phoretix (v. 1.0, NonLinear Dynamics Ltd., UK).
performing in vitro experiments, we examined whether the
inhibition of Jun-Fos-DNA complex formation by curcuminoids
Luciferase assay A confluent monolayer of JB6 cells was
is related to the intracellular suppression of AP-1 transcriptional
trypsinized and 3×105 cells were seeded in 60mm cell-culture plates,
activity by curcumin and curcuminoids. Through these
which were incubated at 37oC in a humidified atmosphere of 5% CO2
studies, it was possible to uncover the inhibitory mechanism
for 2 days. Cells were then starved for 24 h and treated for another 5 h
by which curcuminoids inhibit AP-1 in cells, and to
by culturing them in 0.5% FBS MEM containing the indicated
demonstrate that EMSA is a suitable tool for identifying
curcuminoids (Table 1). The cells were then exposed to TPA (20 ng/
inhibitors of transcription activators.
ml). After 24 h in culture, the cells were extracted with 300 µl of lysis
buf er and luciferase activity was measured using a luminometer.
Luciferase assays were performed using the Luciferase reporter assay
Materials and Methods
system as per the manufacturer’s instructions (Promega, Madison,
USA). For normalization, the Bradford assay was used.
Materials Curcumin was purchased from Acros Organics (2440
Geel, Belgium). Wild and mutant wbjun (224-334), and wild and
Synthesis and identification of curcumin derivatives Curcumin
mutant wbfos (116-211) genes were kindly provided by Dr. T.
derivatives were synthesized according to the method of Pabon
Curran (St Jude’s Children’s Research Hospital, USA). JB6 cells,
(Pabon, 1964), and identified by 1H NMR, melting points, Mass
stably transfected with the AP-1 luciferase gene (Ding et al., 2001),
Spectrometry, IR and by elemental analysis (Hahm et al., 2002).
were kindly provided by Dr. V. Vallyathan (National Institute for
Occupational Safety and Health, USA).
RNA-isolation and RT-PCR Total RNA was isolated from cells
using the easy-BLUETM RNA Extraction Kit (iNtRON, Korea),
Cell culture JB6 cells were cultured in MEM supplemented with
according to the manufacturer’s instructions. For cDNA synthesis,
penicillin, streptomycin, neomycin and 10% FBS in a humidified 5%
4 µg of total RNA was placed in a 25 µl reaction volume containing
CO atmosphere. AGS (gastric adenocarcinoma), HCT116, or
oligo (dT) primers and reverse transcriptase (Promega, USA),
SW480 (colon cancer) cells were grown in RPMI1640 supplemented
following the manufacturer’s instructions. For PCR, 1/25 of the
with penicillin, streptomycin, and 10% FBS. To verify the effect of
reverse transcription reaction mixture was amplified using 35
curcuminoids on AP-1, JB6 cells were treated with curcuminoids and
cycles for c-jun, 30 cycles for c-fos or 17 cycles for â-actin. To
harvested for luciferase assays and RT-PCR. SW480, HCT116, and
amplify c-jun, c-fos, and ?-actin fragments 15, 20 and 30 cycles
AGS cel s were harvested for MTT assays and EMSA.
were used to determine whether the DNA amplification was linear.
The PCR conditions were as follows: at 94oC for 30 s, at 55oC for
Isolation of AGS nuclear extracts Nuclear pellets were prepared
1 min, at 72oC for 1 min. All PCR products were analyzed by
by resuspending cells in 400 µl of lysis buffer [10 mM HEPES, pH
electrophoresis on 1.5% agarose gels and photographed. The
7.8, 10 mM KCl, 2 mM MgCl , 0.1 mM EDTA], placing them on
sequences of the primers used from RT-PCR were as follows:
ice for 15 min, and then by vigorous mixing after adding 25 µl of
?-actin: 5'-TCATGAAGTGTGACGTTGACATCCGT-3' (sense) and
10% Nonidet P-40. After 30-s centrifugation (16,000 × g, 4oC), the
5'-CCTAGAAGCATTTGCGGTGCACGATG-3' (antisense); c-jun:
pelleted nuclei were resuspended in 50ìl of extraction buf er
5'-GCATGAGGAACCGCATCGCTGCCTCCAAGT-3' (sense) and
[50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM
5'-GCGACCAAGTCCTTCCCACTCGTGCACACT-3' (antisense); c-
EDTA, 10% glycerol] and incubated on ice for 20 min. Nuclear
fos: 5'-AAGGAGAATCCGAAGGGAAAGGAATAAGATGGCT (sense)
extracts were stored at ?70oC.
and 5'-CCTAGAAGCATTTGCGGTGCACGATG (antisense)
Chi Hoon Park et al.
MTT assay The MTT assay is based on the ability of mitochondria
0.38 mM, and 0.64 mM respectively (Hahm et al., 2002). In
in live cells to oxidize thiazolyl blue, a tetrazolium salt (MTT;
our experiment, excessive inhibitor quantities were used to
Sigma, St. Louis, USA), to an insoluble blue formazan product
produce complete inhibition. The ability of these curcuminoids
(Shim et al., 2002). To determine cell growth rates, cells were
to inhibit the formation of AP-1-DNA complexes was tested
counted, and 7 × 103 were plated in 96-well culture plates (in
using AGS (a gastric cancer cell-line) nuclear extracts,
octuplicate) in RPMI-1640 medium 24 h prior to being treated with
which contained endogenous AP-1. In was observed that
curcumin or curcuminoids for 3 days. Cells were then washed with
thesecurcuminoids inhibited the formation of endogenous AP-
PBS, and incubated with MTT (0.1 mg) at 37oC for 3 h. The
1-DNA completely, as did the truncated proteins (Fig. 1B).
reagent was then removed, and 150 µl DMSO was added to each
well. The optical density of the solution was read at 540 nm in an
enzyme-linked immunosorbent assay (enzyme-linked immunosorbent
The binding of mutated Jun-Fos dimer to DNA was
assay) plate reader. Since the generation of the blue product is
inhibited by curcumin and curcuminoids, but not by
proportional to mitochondrial dehydrogenase activity, decreases in
selenium The inhibitory effects of curcumin and curcuminoids
absorbance at 540 nm provided a direct measure of cell death.
were tested using mutated Jun (JunM) and Fos (FosM)
truncated polypeptides. Cys272 was replaced by Ser272 in JunM,
and Cys154 was replaced by Ser154 in FosM. JunM and FosM
were able to dimerize like native Jun and Fos. Moreover, the
binding of cysteine-to-serine mutants to DNA is known not to
Curcuminoids can inhibit the formations of Jun-Fos-DNA
be abolished by oxidizing agents, unlike that of the native
and endogenous AP-1-DNA Eleven curcuminoids were
synthesized using Pabon’s method (Pabon, 1964); their
structures are shown in Table 1. We examined whether these
curcuminoids could inhibit the binding of a Jun-Fos dimer to
DNA, using Jun and Fos truncated polypeptides, which both
contained a leucine zipper domain and a DNA binding
domain. These were expressed in E. coli(BL21) and purified
by nickel affinity chromatography. Curcumin and three of the
11 curcuminoids (CHC007, CHC009 and CHC010) inhibited
the formation of Jun-Fos-DNA (Fig. 1A). It has already been
reported that the IC of curcumin, CHC007, and CHC009
with respect to Jun-Fos dimmer DNA bindingare 0.48 mM,
Table 1. The functional groups of curcumin derivatives
Fig. 1. The inhibitory ef ects of curcumin and curcuminoids on
truncated Jun-Fos dimer, endogenous AP-1, and truncated JunM-
FosM dimer. EMSA was performed by incubating truncated
proteins (A,C,D) or AGS nuclear extracts (B) and 32P-labeled
doubled-stranded AP-1 oligonucleotide with the inhibitors
indicated above the gel picture for 30 min at room temperature.
All inhibitors were dissolved in DMSO. Arrowheads indicate the
positions of the retarded protein-DNA complexes representing
the truncated Jun-Fos dimer(A) or Ed-confirm OK -please take
care truncated JunM-FosM dimer(C,D) or AP-1 endogenous
proteins (B). Curcumin and its derivatives inhibited the formation
of JunM-FosM-DNA complex (D), and of Jun-Fos-DNA
complex (A). They also suppressed the binding of endogenous
AP-1 to DNA(B), indicating that the inhibitory mechanism
involved is independent of cysteine residues. Cont; treated with
The Inhibition of AP-1 by Curcuminoids
Fig. 2. Curcumin and Curcuminoids inhibit TPA-induced AP-1 transcriptional activity and downregulate c-jun mRNA, not c-fos
mRNA, in JB6 cells. (A)JB6 cells, which were stably transfected with AP-1/luciferase, were treated with curcuminoids. Ed-pl.
consider - only altered here in the paper. Bradford assay was used for normalization. JB6 cells were cultured and treated as described in
Materials and Methods. Data are means±SD of triplicate experiments. Species that suppressed Jun-Fos-DNA complex formation in
vitro, efficiently downregulated the transcriptional activity of AP-1. (B,C,D) A confluent monolayer of JB6 cells was trypsinized and
3 × 105 cells were seeded in 60mm cell-culture plates. The RT-PCR procedure is described in detail in Materials and Methods.
Curcumin and C007 downregulated c-jun, but not c-fos transcripts levels. Lane 1, in Fig. 2 represent the DNA marker.
proteins (Abate et al., 1990b). Initially, we investigated whether
The IC ’s of CHC005 and CHC006 against Jun-Fos dimer were
that the band observed on EMSA gel was that of a JunM-FosM
>2.50 mM. The amount of luciferase expressed in the cells
heterodimer or a JunM-JunM homodimer. As shown in Fig. 1C,
treated with CHC007, C009 and C010 decreased markedly in a
no band appeared in the presence of only JunM (lane 2) or
concentration dependent manner. On the other hand, CHC005
FosM (lane 3); however, in the presence of both JunM and
and CHC006 did not reduce the amount of luciferase (Fig. 2A),
FosM, the band appeared, thus indicating that the band in lane1
indicating that the transcriptional activity of AP-1 in cel s was
was of JunM-FosM heterodimer and not JunM-JunM
reduced only by curcuminoids known to efficiently inhibit Jun-
homodimer. Excessive amounts of inhibitors (curcumin, C007,
Fos activity in vitro.
C009 and C010), i.e., exceeding their inhibitory IC ’s, were
added to JunM-FosM dimer. Although selenium, a well known
Curcumin or CHC007 reduced TPA-induced c-jun
Jun-Fos inhibitor, did not inhibit JunM-FosM dimer formation,
mRNA levels To investigate the effect of CHC007 on c-jun
the curcuminoids inhibited JunM-FosM-DNA complex
and c-fos transcription, we used RT-PCR (Fig. 2). It is known
formation, as did curcumin (Fig. 1D).
that curcumin downregulates c-jun mRNA and that c-fos
mRNA is not affected by curcumin in NIH3T3 cells (Huang
Inhibition of the transcriptional activity of AP-1 by
et al., 1991). As shown in Fig. 2B, curcumin inhibited c-jun
curcuminoids in JB6 cel s Luciferase assays were performed
transcription in JB6 cells. To verify the effect of CHC007 on
to determine whether curcuminoids can act as AP-1 inhibitors
c-jun and c-fos mRNA, JB6 cells were treated with CHC007
intracellulary JB6 cel s, stably transfected with the AP-1luciferase
for 5 h prior to TPA treatment. After 2 h or 5 h, RNA was
gene, were treated with CHC007, CHC009 or CHC010,
isolated for RT-PCR. Figure 2C shows that the effect of
inhibitors of Jun-Fos dimer in an in vitro experiment, and with
CHC007 on c-jun mRNA was manifest after 5 h, that is,
CHC005 and CHC006, which had much higher IC ’s that the
c-jun transcription. However, CHC007
former inhibitors, 24 h prior to performing the luciferase assay.
did not affect c-fos mRNA levels (Fig. 2D).
Chi Hoon Park et al.
Cytotoxicity of curcuminoids in SW480, HCT116, and
AGS cancer cel To investigate the potential of using
curcuminoids as candidate anti-cancer agents, we adopted an
MTT assay approach (Fig. 3). Cancer cells (AGS, SW480, or
HCT116) were incubated with curcumin or curcuminoids for
72hr. Curcumin, CHC009, or CHC010 showed high cytotoxicities
in SW480 (a colon cancer cell-line) cells at 20 µM; however,
most SW480 cells remained viable in the presence of
CHC007 even at 50 µM. Similar results were obtained for
AGS gastric cancer cells and HCT116 colon cancer cells.
Recently, there has been a marked increase in interest in
curcumin derivatives. Ohtsu et al. reported that curcumin
derivatives possessing a methoxy group are novel androgen
receptor antagonists with anti-prostate cancer potential (Ohtsu
et al., 2002). In addition, it has been reported that some
curcuminoids can inhibit the binding of Jun-Fos dimer to
DNA more efficiently than curcumin. To understand their
inhibitory mechanism in vitro, JunM(Cysteine ? Serine) and
FosM (Cysteine ? Serine) were used. Cysteine residues in
the DNA binding domains of Jun and Fos proteins are known
to play important roles in DNA binding by Jun-Fos dimers
(Abate et al., 1990b), which bind to DNA even in the absence
of a reducing agent. Curcumin and its derivatives were
reported to have anti-oxidant effects in vitro and in vivo
(Sugiyama et al., 1996). However, other reports claim that
curcumin has a pro-oxidizing nature and that it is capable of
oxidative DNA cleavage, particularly in the presence of
transition metal ions such as copper (Rahman et al., 1989). In
addition, Nikitovic et al. and Handel et al. reported that nitric
oxide, gold, or selenium have an AP-1 inhibitory effect that is
associated with cysteine residues, suggesting the thiol group
oxidation. In these papers, the presence of this inhibitory
Fig. 3. Curcumin and curcuminoids had high cytotoxicity but
mechanism was demonstrated by showing that NO, gold (I)
CHC007 had little cytotoxicity in cancer cell-lines. To evaluate
thiomalate (AuTM), and Na SeO all inhibited the formation
cell growth rates, cells were counted, and 7 × 103 were plated in
96-well culture plates in RPMI-1640 medium 24 h before being
of a Jun-Fos-DNA complex, but not the formation of a JunM-
treated with curcumin or curcuminoids for 72 h. MTT assays
FosM-DNA complex (Handel et al., 1995; Nikitovic et al.,
was performed as described in Materials and Methods. Curcumin
1998). Based on these results, we presumed that curcumin
and the curcuminoids tested showed high cytotoxicity against
could oxidize the thiol group in the reduced cysteine residues
cancer cells, except CHC007. Data are the means ± SD of
in Jun and Fos proteins and thus inhibit the binding of Jun-Fos
dimer to DNA. To investigate this presumption, curcumin and
some curcuminoids, including CHC007, CHC009, and
curcuminoids inhibit Jun-Fos dimer differs from that of other
CHC010, which have been reported to inhibit the formation of
well known AP-1 inhibitors. In order to clarify the details of
Jun-Fos-DNA in vitro, were investigated to determine
this inhibitory mechanism, further studies are needed.
whether they have an inhibitory ef ect on JunM-FosM dimer
Next, we tested the inhibitory effect of curcuminoids on the
and on Jun-Fos dimer. We found that curcumin and the
oncogenic transcriptional activity of AP-1 in vivo. For this
curcuminoids, C007, C009 and C010, inhibited JunM-FosM-
study, JB6 cells stably transfected with AP-1luciferase gene
DNA formation (Fig. 1). This result indicates that curcumin
(Ding et al., 2001) were used. Cells were treated with
and these curcuminoids, unlike nitric oxide, gold, or selenium,
curcuminoids and then with TPA, and luciferase expressions
block the ability of Jun-Fos dimer to bind DNA regardless of
were measured. The levels of luciferase in C007, C009, and
the presence of conserved cysteine residues. This finding
C010 treated cells were significantly lower than that in
indicates that the mechanism whereby curcumin or
DMSO treated cells. On the other hand, C005 and C006,
The Inhibition of AP-1 by Curcuminoids
Fig. 4. Mechanism of AP-1 inhibition by curcuminoids.
which had little inhibitory effect on Jun-Fos dimer by EMSA,
curcuminoids on entering a cell (observed by fluorescence
did not inhibit AP-1 transcriptional activity (Fig. 2A). This
microscopy - data not shown) interfere with AP-1/DNA
result has two important implications; first, the inhibition of
binding inducedby TPA activation, regardless of cysteine
AP-1 transcriptional activity by curcumin or curcuminoids, in
residues in Jun and Fos proteins. This reduced DNA/AP-1
vivo, is due to interference with the formation of the Jun-Fos-
binding leads to inhibited AP-1 transcriptional activity and to
DNA complex. Huang et al. showed that curcumin suppresses
the suppression of AP-1 target genes, including c-jun, which
AP-1 activity in NIH3T3 cells (Huang et al., 1991), and
reduces the level of c-Jun product, which reinforces reduced
Hahm et al. reported that curcumin inhibits Jun-Fos-DNA
AP-1 transcriptional activity (Fig. 4).
complex formation in vitro (Hahm et al., 2002). The present
study shows for the first time an important relationship
Acknowledgments We thank Dr. T. Curran (St. Jude’s
between these two earlier studies, that is to say, it
Children’s Research Hospital, USA) for generously donating
demonstrates that the inhibition of Jun-Fos to DNA binding
the wild and mutant wbjunand wbfos genes, and Dr. V.
by curcumin or curcuminoids is a direct cause of AP-1
Vallyathan (National Institute for Occupational Safety and
inhibition in vivo. Secondly, the study also demonstrates that
Health, USA) for the JB6 cells stably transfected with AP-1
EMSA is an excellent tool for determining the identities of
luciferase. This research was supported by the Korean
species that inhibit transcriptional activators, because EMSA
Ministry of Science and Technology (Grant no., KRF 2001-
can test protein to DNA binding, which is the primary
015-DP0344). We also gratefully acknowledge the financial
function of a transcription activator.
support provided by the Brain Korea 21 program.
To determine the cytotoxicities of curcuminoids that inhibit
AP-1 transcriptional activity, we used the MTT assay (Fig. 3).
Gastric and colon cancer cell lines treated with curcumin died
at a curcumin concentration of 10ìM, and other curcumin
derivatives also showed excellent cytotoxicity at ca. 20ìM.
Abate, C., Luk, D., Gentz, R., Rauscher, F. J. III. and Curran, T.
These data indicate that curcumin and curcumin derivatives
(1990a) Expression and purification of the leucine zipper and
have anti-cancer potential.
DNA-binding domains of Fos and Jun: Both Fos and Jun
In addition, RT-PCR showed that one of the curcuminoids,
contact DNA directly. Proc. Natl. Acad. Sci. USA 87, 1032-
CHC007, like curcumin, suppressed c-jun transcription (Fig.
2C). Since c-jun is the target gene of AP-1 (Angel et al.,
Abate, C., Patel, L., Rauscher, F. J. III. and Curran, T. (1990b)
1988), c-jun mRNA attenuation was attributed to suppressed
Redox regulation of fos and jun DNA-binding activity in vitro.
AP-1 transcriptional activity by curcumin or curcuminoids.
Science 249, 1157-1161.
Because it is known that
Ammon, H. P. and Wahl, M. A. (1991) Pharmacology of
c-jun is positively autoregulated by
its product c-Jun/AP-1(Angel
Plata. Med. 57, 1-7.
et al., 1988), we were able to
Angel, P., Hattori, K., Smeal, T. and Karin, M. (1988) The jun
summarize the mechanism underlying AP-1 inhibitory by
proto-oncogene is positively autoregulated by its product, Jun/
curcuminoids, as shown in Fig. 4. Summarizing, curcumin or
AP-1. Cell 55, 875-885.
Chi Hoon Park et al.
Angel, P. and Karin, M. (1991) The role of Jun, Fos and the AP-1
Cell 49, 741-752.
complex in cell-proliferation and transformation. Biochim.
Mohan, R., Sivak, J., Ashton, P., Russo, L. A., Pham, B. Q.,
Biophys. Acta. 1072, 129-157.
Kasahara, N., Raizman, M. B. and Fini, M. E. (2000)
Bhaumik, S., Anjum, R., Rangaraj, N., Pardhasaradhi, B. V. and
Curcuminoids inhibit the angiogenic response stimulated by
Khar, A. (1999) Curcumin mediated apoptosis in AK-5 tumor
fibroblast growth factor-2, including expression of matrix
cells involves the production of reactive oxygen intermediates.
metalloproteinase gelatinase B. J. Biol. Chem. 275, 10405-
FEBS Lett. 456, 311-314.
Bohmann, D. and Tjian, R. (1989) Biochemical Analysis of
Nikitovic, D., Holmgren, A. and Spyrou, G. (1998) Inhibition of
Transcriptional Activation by Jun: dif erential activity of C-
AP-1 DNA binding by nitric oxide involving conserved
and v-Jun. Cell 59, 709-717.
cysteine residues in Jun and Fos. Biochem. Biophys. Res.
Conney, A. H., Lysz, T., Ferraro, T., Abidi, T. F., Manchand, P. S.,
Commun. 242, 109-112.
Laskin, J. D. and Huang, M. T. (1991) Inhibitory effect of
Ohtsu, H., Xiao, Z., Ishida, J., Nagai, M., Wang, H. K., Itokawa,
curcumin and some related dietary components on tumor
H., Su, C. Y., Shih, C., Chiang, T., Chang, E., Lee, Y., Tsai,
promotion and arachidonic acid metabolism in mouse skin.
M. Y., Chang, C. and Lee, K. H. (2002) Antitumor Agents.
Adv. Enzyme Regul. 31, 385-396.
217. Curcumin analogues as novel androgen receptor
Devary, Y., Gottlieb, R. A., Lau, L. F. and Karin, M. (1991)
antagonists with potential as anti-prostate cancer agents. J.
Rapid and preferential activation of the c-jun gene during the
Med. Chem. 45, 5037-5042.
mammalian UV response. Mol. Cell. Biol. 11, 2804-2811.
Pabon, H. J. J. (1964) A synthesis of curcumin and related
Devary, Y., Gottlieb, R. A., Smeal, T. and Karin, M. (1992) The
compounds. Rec. Trav. Chim. Pays. Bas. 83, 379-386.
Mammalian ultraviolet response is triggered by activation of
Park, J. E., Lee, K. Y., Do, S. I. and Lee, S. S. (2002) Expression
src tyrosine kinases. Cell 71, 1081-1091.
and characterization of ?-1,4-galactosyltransferase from
Ding, M., Shi, X., Lu, Y., Huang, C., Leonard, S., Roberts, J.,
Neisseria meningitidis and Neisseria gonorrhoeae. J. Biochem.
Antonini, J., Castranova, V. and Val yathan, V. (2001)
Mol. Biol. 35, 330-336.
Induction of activator protein-1 through reactive oxygen species
Rahman, A., Shahabuddin, H. S. M., Parish, J. H. and Ainley, K.
by crystalline silica in JB6 cells. J. Biol. Chem. 276, 9108-
(1989) Strand scission in DNA induced by quercetin and
Cu(II): role of Cu(I) and oxygen free radicals. Carcinogenesis
Distel, R. J., Ro, H. S., Rosen, B. S., Groves, D. L. and
Spiegelman, B. M. (1987) Nucleoprotein complexes that
Rao, C. V., Rivenson, A., Simi, B. and Reddy, B. S. (1995)
regulate gene expression in adipocyte differentiation: direct
Chemoprevention of colon carcinogenesis by dietary curcumin,
participation of c-fos. Cell 49, 835-844.
a naturally occurring plant phenolic compound. Cancer Res.
Hahm, E. R., Cheon, G., Lee, J. H., Kim, B. J., Park, C. H. and
Yang, C. H. (2002) New and known symmetrical curcumin
Satoskar, R. R., Shah, S. J. and Shenoy, S. G. (1986) Evaluation
derivatives inhibit the formation of Fos-Jun-DNA complex.
of anti-inflammatory property of curcumin (dieferuloyl
Cancer Lett. 184, 89-96.
methane) in patients with postoperative inflammation. Int. J.
Han, S. S., Keum, Y. S., Soe, H. J. and Surh, Y. J. (2002)
Clin. Pharmacol. Ther. Toxicol. 24, 651-654.
Curcumin suppresses activation of NF-?B and AP-1 induced
Sharma, O. P. (1976) Antioxidant activity of curcumin and related
by phorbol ester in cultured human promyelocytic leukemia
compounds. Biochem. Pharmacol. 25, 1811-1812.
cells. J. Biochem. Mol. Biol. 35, 337-342.
Shim, M. J., Kim, H. J., Yang S. J., Lee, I. S., Choi, H. I. and
Handel, M. L., Watts, C. K., DeFazio, A., Day, R. O. and
Kim, T. U. (2002) Arsenic trioxide induces apoptosis in
Sutherland, R. L. (1995) Inhibition of AP-1 binding and
chronic myelogenous leukemia K562 cells: possible
transcription by gold and selenium involving conserved
involvement of p38 MAP kinase. J. Biochem. Mol. Biol. 35,
cysteine residues in Jun and Fos. Proc. Natl. Acad. Sci. USA
Singh, A. K., Sidhu, G. S., Deepa, T. and Maheshwari, R. K.
Huang, M. T., Deschner, E. E., Newmark, H. L., Wang, Z. Y.,
(1996) Curcumin inhibits the proliferation and cell cycle
Ferraro, T. A. and Conney, A. H. (1992) Effect of dietary
progression of human umbilical vein endothelial cel . Cancer
curcumin and ascorbyl palmitate on azoxymethanol-induced
Let . 107, 109-115.
colonic epithelial cell proliferation and focal areas of dysplasia.
Srimal, R. C. and Dhawan, B. N. (1973) Pharmacology of
Cancer Lett. 64, 117-121.
diferuloyl methane (curcumin), a non-steroidal anti-inflammatory
Huang, M. T., Lou, Y. R., Ma, W., Newmark, H. L., Reuhl, K. R.
agent. J. Pharm. Pharmacol. 25, 447-452.
and Conney, A. R. (1994) Inhibitory effects of dietary
Sugiyama, Y., Kawakishi, S. and Osawa, T. (1996) Involvement of
curcumin on forestomach, duodenal, and colon carcinogenesis
the beta-diketone moiety in the antioxidative mechanism of
in mice. Cancer Res. 54, 5841-5847.
tetrahydrocurcumin. Biochem. Pharmacol. 52, 519-525.
Huang, T. S., Lee, S. C. and Lin, J. K. (1991) Suppression of c-
Toda, S., Miyase, T., Arichi, H., Tanizawa, H. and Takiyano, Y.
jun/AP-1 activation by an inhibitor of tumor promotion in
(1985) Natural antioxidants, III: antioxidative components
mouse fibroblast cells. Proc. Natl. Acad. Sci. USA 88, 5292-
isolated from rhizome of curcuma longa L. Chem. Pharm.
Bull. 33, 1725-1728.
Kouzarides, T. and Zif , E. (1988) The role of the leucine zipper
Zhang, F., Altorki, N. K., Mestre, J. R., Subbaramaiah, K. and
in the fos-jun interaction. Nature 336, 646-651.
Dannenberg, A. J. (1999) Curcumin inhibits cyclooxygenase-2
Lee, W., Mitchell, P. and Tjian, R. (1987) Purified transcription
transcription in bile acid - and phorbol ester-treated human
factor AP-1 interacts with TPA-inducible enhancer elements.
gastrointestinal epithelial cells. Carcinogenesis 20, 445-451.