TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin

Article abstract of DOI:10.1016/j.bcp.2006.07.007

The activator protein-1 (AP-1) family of transcription factors, including the most common member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple activities reported for curcumin, including inhibition of the stress-induced activation of AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present study, a library of analogs of curcumin was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for screening potential inhibitors. Numerous analogs were identified that were more active than curcumin, including analogs that were not anti-oxidants and analogs that were not Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an essential feature of active compounds. In addition, a number of analogs were identified that enhanced the TPA-induced activation of AP-1. The results from screening were confirmed using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced COX-2 expression; analogs identified as more potent than curcumin in the screening assay were also more potent than curcumin in preventing COX-2 expression.

Full text of DOI:10.1016/j.bcp.2006.07.007

b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biochempharm
TPA-induced up-regulation of activator protein-1
can be inhibited or enhanced by analogs of the
natural product curcumin
Waylon M. Weber a, Lucy A. Hunsaker b, Amanda M. Gonzales b, Justin J. Heynekamp a,
Robert A. Orlando b, Lorraine M. Deck a, , David L. Vander Jagt
*
b,**
a Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA
b Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The activator protein-1 (AP-1) family of transcription factors, including the most common
member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in
proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including
pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of
plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been
shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is
potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple
activities reported for curcumin, including inhibition of the stress-induced activation of
AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present
study, a library of analogs of curcumin was screened for activity against the TPA-induced
activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for
screening potential inhibitors. Numerous analogs were identified that were more active
than curcumin, including analogs that were not anti-oxidants and analogs that were not
Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an
essential feature of active compounds. In addition, a number of analogs were identified that
enhanced the TPA-induced activation of AP-1. The results from screening were confirmed
using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced
COX-2 expression; analogs identified as more potent than curcumin in the screening assay
were also more potent than curcumin in preventing COX-2 expression.
Received 21 March 2006
Accepted 14 July 2006
Keywords:
AP-1
Curcumin
Inhibitors
Activators
Anti-oxidants
Abbreviations:
AP-1, activator protein-1
TPA, 12-O-tetradecanoylphorbol-
13-acetate
TRE, TPA-responsive element
# 2006 Elsevier Inc. All rights reserved.
1.
Introduction
proliferation, cell cycle regulation, differentiation, and apop-
tosis [1–6]. The major AP-1 proteins in mammalian cells are
Activator protein-1 (AP-1) is a family of dimeric transcription
factors containing homo- or heterodimers of members of the
Jun, Fos, ATF and Maf families of proteins. AP-1-mediated gene
transcription contributes to many cellular processes such as
Jun-Fos dimers, mainly c-Jun-c-Fos. Active AP-1 dimers can
bind to TPA-responsive elements (TREs) in the promoters of
AP-1 responsive genes. In addition to tumor promoters, AP-1
binding to TREs is rapidly induced by growth factors,
* Corresponding author. Tel.: +1 505 277 5438.
** Corresponding author. Tel.: +1 505 272 5788; fax: +1 505 272 3518.
E-mail addresses: ldeck@unm.edu (L.M. Deck), dlvanderjagt@salud.unm.edu (D.L. Vander Jagt).
0006-2952/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2006.07.007

b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
929
cytokines, and oncoproteins. This has contributed to the
general view that activation of AP-1 is oncogenic by con-
tributing to proliferation, survival and transformation of cells.
This view is supported by the observation that several AP-1
proteins, including c-Jun and c-Fos, can transform cells in
culture [7-9]. Development of inhibitors of activation of AP-1
may be a promising approach to development of new anti-
cancer therapeutics [10,11]. It is noteworthy, however, that
certain AP-1 dimers can be anti-oncogenic [4,12,13]. Whether
or not AP-1 is oncogenic depends upon cell type, genetic
background, nature of the stimulus, and state of differentia-
tion [4,5]. This makes it difficult to make generalized
statements about the physiological roles of AP-1 in the
absence of specific information concerning cell type and
environmental conditions.
well as analogs that are not Michael acceptors can retain or
even exhibit increased activity compared to curcumin as
inhibitors of the TPA-induced activation of AP-1. Moreover,
some analogs can function as enhancers of the TPA-induced
activation of AP-1.
2.
Materials and methods
2.1.
Synthesis of analogs of curcumin
Curcumin is a relatively simple bis-phenolic compound that
can be prepared in a single reaction. The procedures used to
prepare curcumin analogs in the 7-carbon, 5-carbon, and 3-
carbon series were described previously, and are summarized
in Fig. 2 [28–38]. The following describes the synthesis of
analogs of curcumin that were not included in our previous
studies [28,29] but were included in this study. The bold,
underlined numbers refer to the numbering of the analogs as
they appear in Figs. 3–8.
Compounds that have been shown to inhibit the activation
of AP-1 include
a number of phytochemicals such as
curcumin, resveratrol, epigallocatechin gallate, and theafla-
vins [6,14–16]. Curcumin is especially of interest. This
phytochemical, obtained from the rhizome of the herb
Curcuma longa, is a yellow compound in the spice turmeric,
which has been used for centuries in Asia as a herbal
medicinal to treat a wide range of health problems. Numerous
studies of the anti-inflammatory and anti-cancer properties of
curcumin have been reported (reviewed in refs. [16–19]).
Curcumin is considered a non-toxic phytochemical. Doses as
high as 12 g/day have been used in recent clinical studies of
curcumin for the treatment of a wide variety of malignancies
[20–22]. Curcumin (Fig. 1) is also a strong anti-oxidant by virtue
of its phenolic groups [23–25]. In the multiple studies of the
activities ascribed to curcumin, it is often suggested that the
anti-oxidant properties of curcumin contribute to the
observed biological activities. However, experiments designed
specifically to answer this question are rarely included in
these studies. In spite of reports indicating that curcumin is an
unstable compound [26] and is especially reactive as a Michael
acceptor involving its a,b-unsaturated ketone functionalies
[27], the intrinsic reactivity of curcumin is generally over-
looked in many of the studies of its biological properties.
In the present study, a library of analogs of curcumin was
examined for activity as inhibitors of the TPA-induced
activation of AP-1 in HEK293 cells transfected with an AP-1-
dependent luciferase construct. This study compared analogs
that retained the 7-carbon dienone spacer between the
aromatic rings, as in curcumin, but with various ring
substituents including non-phenolic groups; this provided
analogs that were no longer active anti-oxidants and could be
used to address the question as to whether the anti-oxidant
activity is required for biological activity. In addition, analogs
were included that contained 5-carbon or 3-carbon enone
spacers as well as analogs devoid of the enone functionality.
We report here that analogs devoid of anti-oxidant activity as
1,5-Bis(2-thienyl)-1,4-pentadien-3-one (59). Yellow solid:
mp 115–117 8C [literature [39] 115–117 8C]; 13C NMR: d 124.4,
128.2, 128.7, 131.7, 135.5, 140.2, 187.5.
4-Benzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane-
3,5-dione (20). The compound was prepared [31] by reaction of
4-benzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hepta-
diene-3,5-dione (0.25 g, 0.5 mmol) with hydrogen in the
presence of palladium on activated carbon (0.20 g, 10%) to
give 0.12 g (48%) of compound 20 as a pale yellow oil; 1H NMR d
enol form: 2.66 (m, 8H), 3.53 (s, 2H), 3.77 (s, 6H), 5.55 (s, 2H), 6.56
(m, 4H), 6.77 (d, 2H, J = 7.6 Hz), 7.06 (m, 6H), 7.20 (m, 2H); keto
form: 2.66 (m, 8H), 3.08 (d, 2H, J = 7.3 Hz), 3.82 (s, 6H), 3.92 (t, 1H,
J = 8.3 Hz), 5.55 (s, 2H), 6.56 (m, 4H), 6.77 (d, 2H, J = 7.6 Hz), 7.06
(m, 6H), 7.20 (m, 2H); 13C NMR: d 29.0, 31.1, 31.8, 34.3, 37.7, 44.6,
55.8, 69.2, 111.0, 114.2, 120.7, 120.8, 126.6, 127.4, 128.5, 128.6,
132.3, 132.6, 137.9, 143.8, 146.3, 193.4, 204.5. Exact mass calcd.
for C28H30O6: 462.2042, observed (M + H) 463.2073.
1,5-Bis(4-dimethylaminophenyl)-1,4-pentadien-3-one (41).
Orange solid: mp 179–181 8C [literature [40] 174–176 8C]; 1H
NMR: d 3.01 (s, 12H), 6.69 (d, 4H, J = 8.7 Hz), 6.87 (d, 2H,
J = 15.7 Hz), 7.50 (d, 4H, J = 8.7 Hz), 7.67 (d, 2H, J = 15.7 Hz); 13C
NMR: d 40.2, 98.9, 111.8, 121.2, 122.9, 129.9, 142.8, 151.6.
1,5-Bis(1-naphthyl)-1,4-pentadien-3-one (39). Yellow solid:
mp 132–133 8C [literature [41] 128 8C]; 13C NMR: d 123.4, 125.1,
125.4, 126.2, 126.9, 128.1, 128.7, 130.7, 131.7, 132.2, 133.7, 140.3,
188.5.
2,6-Bis(4-methylphenyl)-1-methyl-4-piperidone (83). White
solid: mp 120–121 8C [literature [42] 105–107 8C]; 1H NMR d 1.79 (s,
3H), 2.36 (s, 6H), 3.10 (d, 2H, J = 14.9 Hz), 2.79 (t, 2H, J = 13.1 Hz),
3.35 (dd, 2H, J = 11.9, 2.4 Hz), 7.15 (d, 4H, J = 8.0 Hz), 7.21 (d, 4H,
J = 7.9 Hz); 13C NMR: d 21.2, 40.7, 50.9, 70.0, 126.9, 129.4, 137.2,
140.2, 207.1.
Fig. 1 – Curcumin exists as an equilibrium mixture of the keto and enol forms.

930
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
Fig. 2 – Synthesis of enone analogs of curcumin. Series I analogs, maintaining the 7-carbon dienone spacer between the
aromatic rings as in curcumin, were synthesized from aromatic aldehydes by condensation with 2,4-pentanedione in an
aldol type reaction [30–33]. This involves base-catalyzed condensation in the presence of a trialkylborate to complex with
the carbonyl groups, which prevents enolization and guides the reaction. If a mixture of two different aldehydes is used,
unsymmetrical analogues are formed. Series II analogs, containing a 5-carbon enone spacer, were synthesized either by
base-catalyzed condensation of the appropriate aldehyde with acetone or by acid-catalyzed condensation with 3-oxo-
glutaric acid [34,35]. Series III analogs, containing a 3-carbon enone spacer, were synthesized by base-catalyzed
condensation of the appropriate aldehyde with a substituted acetophenone [36–38].
2,6-Bis(4-methoxyphenyl)-1-methyl-4-piperidone
(84).
J = 11.7 Hz), 7.49 (m, 4H), 7.81 (m, 10H); 13C NMR: d 41.1, 50.7,
70.3, 124.6, 125.9, 126.0, 126.2, 127.6, 127.7, 128.9, 133.0, 133.4,
140.4, 206.6. Exact mass calcd. for C26H23NO: 365.1780,
observed (M + H) 366.1852.
White solid: mp 141–143 8C [literature [42] 129-130 8C]; 1H
NMR d 1.77 (s, 3H), 2.45 (d, 2H, J = 14.5 Hz), 2.78 (t, 2H,
J = 12.9 Hz), 3.33 (d, 2H, J = 11.9 Hz), 3.79 (s, 6H), 6.88 (d, 4H,
J = 8.5 Hz), 7.32 (d, 4H, J = 8.5 Hz); 13C NMR: d 40.6, 50.9, 55.3,
69.5, 114.1, 128.0, 135.3, 158.9, 207.1.
2,6-Bis(2,5-dimethoxyphenyl)-1-methyl-4-piperidone (86).
The compound was prepared [42] by the reaction of 1,5-bis(2,5-
dimethoxyphenyl)-1,4-pentadien-3-one (0.85 g, 2.20 mmol)
with methylamine (1.85 mL, 24 mmol) to give 0.68 g (68%) of
compound 86 as white needles: mp 169–170 8C; 1H NMR: d 1.90
(s, 3H), 2.56 (m, 4H), 3.75 (s, 6H), 3.80 (s, 6H), 4.05 (dd, 2H, J = 11.7,
3.2 Hz), 6.76 (m, 4H), 7.28 (d, 2H, J = 2.8 Hz); 13C NMR: d 40.3, 49.1,
55.7, 56.1, 61.3, 112.0, 113.6, 132.7, 150.7, 154.1, 207.5. Exact
mass calcd. for C22H27NO5: 385.1889, observed (M + H)
386.1967.
2,6-Bis(2-methylphenyl)-1-methyl-4-piperidone (85). The
compound was prepared [42] by the reaction of 1,5-bis(2-
methylphenyl)-1,4-pentadien-3-one (0.50 g, 1.9 mmol) with
methylamine (1.0 mL, 11.6 mmol) to give 0.29 g (52%) of
compound 85 as a white solid: mp 155–157 8C; 1H NMR d
1.82 (s, 3H), 2.40 (s, 6H), 2.44 (d, 2H, J = 11.9 Hz), 2.77 (t, 2H,
J = 13.3 Hz), 3.74 (d, 2H, J = 11.9 Hz), 7.16 (m, 6H), 7.67 (d, 2H,
J = 7.0 Hz); 13C NMR: d 19.5, 39.9, 49.3, 65.8, 126.7, 126.9, 130.6,
134.8, 140.9, 207.2. Exact mass calcd. for C20H23NO: 293.1779,
observed (M + H) 294.1856.
2,6-Bis(2,4-dimethoxyphenyl)-1-methyl-4-piperidone (87).
The compound was prepared [42] by the reaction of 1,5-bis(2,4-
dimethoxyphenyl)-1,4-pentadien-3-one (0.85 g, 2.20 mmol)
with methylamine (1.85 mL, 24 mmol) to give 0.61 g (66%) of
compound 87 as white needles: mp 164–165 8C; 1H NMR: d 1.84
(s, 3H), 2.45 (d, 2H, J = 12.9), 2.63 (t, 2H, J = 12.1 Hz) 3.77 (s, 6H),
3.80 (s, 6H), 4.96 (dd, 2H, J = 11.3, 3.2 Hz,), 6.74 (d, 2H, J = 2.4 Hz),
6.54 (dd, 2H, J = 7.7, 2.2 Hz,), 7.54 (d, 2H, J = 8.6 Hz); 13C NMR: d
39.9, 49.4, 55.3, 55.5, 60.9, 98.2, 100.8, 105.2, 124.0, 128.2, 157.4,
159.5, 208.2. Exact mass calcd. for C22H27NO5: 385.1889,
observed (M + H) 386.1967.
2,6-Bis(2-methoxyphenyl)-1-methyl-4-piperidone (80). The
compound was prepared [42] by the reaction of 1,5-bis(2-
methoxyphenyl)-1,4-pentadien-3-one (0.26 g, 0.9 mmol) with
methylamine (0.40 mL, 4.6 mmol) to give 0.16 g (55%) of
compound 80 as a white solid: mp 146–148 8C; 1H NMR d
1.89 (s, 3H), 2.50 (d, 2H, J = 13.7 Hz), 2.65 (t, 2H, J = 11.9 Hz), 3.82
(s, 6H), 4.11 (d, 2H, J = 11.5 Hz), 6.87 (d, 2H, J = 8.3 Hz), 7.03 (t, 2H,
J = 7.2 Hz), 7.23 (t, 2H, J = 5.8 Hz), 7.72 (d, 2H, J = 7.6 Hz); 13C
NMR: d 40.3, 49.2, 55.4, 61.2, 110.7, 121.0, 127.6, 127.8, 131.5,
156.3, 208.1. Exact mass calcd. for C20H23NO3: 325.1678,
observed (M + H) 326.1754.
4-Benzyl-1,7-diphenylheptane-3,5-dione (17). The com-
pound was prepared [31] by reaction of 4-benzyl-1,7-diphe-
nyl-1,6-heptadiene-3,5-dione (0.26 g, 0.7 mmol) with hydrogen
in the presence of palladium on activated carbon (0.25 g, 10%)
to give 0.18 g (69%) of compound 17 as white needles: mp 74–
75 8C; 1H NMR d enol form: 2.61 (m, 10H), 7.14 (m, 15H); keto
form: 2.61 (m, 8H), 3.07 (d, 2H, J = 7.2 Hz), 3.90 (t, 1H, J = 7.6 Hz),
7.14 (m, 15H); 13C NMR: d 29.3, 34.3, 44.3, 69.2, 126.1, 126.7, 128.3,
2,6-Bis(2-naphthyl)-1-methyl-4-piperidone (82). The com-
pound was prepared [42] by the reaction of 1,5-bis(2-
naphthyl)-1,4-pentadien-3-one (0.82 g, 2.5 mmol) with methy-
lamine (1.30 mL, 15.1 mmol) to give 0.20 g (22%) of compound
22 as a white solid: mp 209–212 8C; 1H NMR d 1.89 (s, 3H), 2.59
(d, 2H, J = 13.9 Hz), 2.97 (t, 2H, J = 11.3 Hz), 3.66 (d, 2H,

b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
931
Fig. 3 – Inhibition of the TPA-induced activation of AP-1 by curcumin and analogs in the 7-carbon series. Curcumin and
analogs were screened in triplicate at 15 mM concentrations. Analogs 2 and 3 were selected for further study (Table 1).
128.4, 128.6, 128.7, 137.9, 140.4, 204.3. Exact mass calcd. for
C26H26O2: 370.1933, observed (M + H) 371.2014.
trapping anti-oxidant parameter assay (TRAP assay) measures
the ability of an analog to react with the pre-formed radical
monocation of 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfo-
nic acid) (ABTSꢀ+) [44]. ABTS was reacted with potassium
persulfate in the dark, overnight, to generate the colored
ABTSꢀ+ radical cation, which has an absorption maximum at
734 nm. The activities of curcumin and analogs were
determined by their abilities to quench the color of the radical
cation. The ferric reducing/anti-oxidant power assay (FRAP assay)
4,4-Dimethyl-1,7-diphenylheptane-3,5-dione (18). The
compound was prepared [31] by reaction of 4,4-dimethyl-
1,7-diphenyl-1,6-heptadiene-3,5-dione (0.15 g, 0.5 mmol) with
hydrogen in the presence of palladium on activated carbon
(0.2 g, 10%) to give 0.12 g (80%) of compound 18 as a pale yellow
oil; 1H NMR d 1.25 (s, 6H), 2.60 (t, 4H, J = 7.4 Hz), 2.80 (t, 4H,
J = 7.0 Hz), 7.18 (m, 10H); 13C NMR: d 21.1, 29.8, 40.2, 62.4, 126.1,
128.3, 140.7, 208.4. Exact mass calcd. for C21H24O2: 308.1776,
observed (M + H) 309.1843.
measures the ability of an analog to reduce
a ferric
tripyridyltriazine complex [45]. The ferric complex of 2,4,6-
tripyridyl-s-triazine was prepared at acidic pH, and the anti-
oxidant activities of curcumin and analogs were determined
by their abilities to reduce the ferric complex to the ferrous
complex, monitored by formation of the ferrous complex at
593 nm. In both colorimetric assays, the Vitamin E analog
Trolox was used as a control.
2.2.
Assay of the anti-oxidant activities of curcumin and
analogs
The anti-oxidant activities of curcumin and analogs were
determined using two standard assays [43]. The total radical-

932
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
2.3.
Assay of the activities of curcumin and analogs as
cycling conditions and used to normalize values obtained for
COX-2 expression.
inhibitors of the TPA-induced activation of AP-1
An AP-1 reporter stable cell line derived from human 293T
embryonic kidney cells transfected with a luciferase reporter
construct containing three AP-1 binding sites in the promoter
(293T/AP-1-luc, Panomics, Inc., Redwood City, CA) was grown
in a humidified atmosphere at 37 8C in 5% CO2/95% air. The
cells were maintained in Dulbecco’s Modified Eagle’s Medium
(DMEM—high glucose containing 4 mM glutamine) supple-
mented with 10% fetal bovine serum (FBS), 1 mM sodium
pyruvate, 100 units/mL penicillin, 100 mg/mL streptomycin
and 100 mg/mL hygromycin (Gibco/Invitrogen, Carlsbad, CA) to
maintain cell selection. One day prior to treatment, the 293T/
AP-1-luc cells were plated into 24-well cell culture plates
(Costar, Cambridge, MA) in the above media without hygro-
mycin. The following day, the cells, which were at approxi-
mately 60% confluency, were fed fresh media with or without
TPA, 10 ng/mL (Calbiochem) and immediately treated with
curcumin or analog prepared in DMSO stock solutions. The
cells were placed again in a humidified atmosphere at 37 8C in
5% CO2/95% air for 24 h. Plate wells were gently washed with
phosphate buffered saline, pH 7.4, and lysed with 1ꢁ passive
lysis buffer (Promega, Madison, WI). The subsequent lysates
were analyzed with the Luciferase Assay System (Promega)
utilizing a TD-20/20 luminometer (Turner Designs, Sunnyvale,
CA). The firefly luciferase relative light units were normalized
to protein (mg/mL) with BCATM Protein Assay Kit (Pierce,
Rockford, IL) and standardized to percent of control (TPA
control).
3.
3.1.
Results
Inhibition or enhancement of the TPA-induced
activation of AP-1 by curcumin analogs in the 7-carbon series
Curcumin (1) and 23 analogs of curcumin in the 7-carbon
series were compared for their effects on the TPA-induced
activation of AP-1 in an initial screening at 15 mM concentra-
tion of the analogs. Analogs that inhibited the activation of AP-
1 and showed activities comparable to or better than curcumin
(analogs 2–7) are shown in Fig. 3 along with the structures of
analogs (8–18) that showed little or no activity (data not
shown). The most active compounds, analogs 2 and 3, retain
the same ring substituents as curcumin but contain either an
alkyl or arylalkyl group attached to the central carbon in the 7-
carbon spacer. Both 2 and 3 were shown previously to possess
anti-oxidant activities [29]. However, analogs 4 and 7, which
exhibit activities comparable to curcumin, do not show anti-
oxidant activities in the FRAP or TRAP assays. We conclude,
therefore, that anti-oxidant activity is not an essential
property of analogs that inhibit the TPA-induced activation
of AP-1.
2.4.
Inhibition of COX-2 expression by curcumin and
analogs
Mouse microglial cells (BV-2) were kindly provided by Dr. Paul
M. Stemmer (Institute of Environmental Health Sciences,
Wayne State University, Detroit, MI). Cells were cultured in
RPMI-1640 (Cellgro, Herndon, VA) supplemented with 10% FBS,
1 mM sodium pyruvate, 2 mM L-glutamine, 100 mg/mL strep-
tomycin sulfate and 100 units/mL penicillin. Cells were grown
on culture plates, pre-treated with 1% gelatin for 30 min, at
37 8C and passaged twice weekly.
BV-2 cells were incubated in the absence or presence of
0.2 mg/mL lipopolysaccharide (LPS) (Sigma, St. Louis, MO) to
induce the inflammatory gene response. Those cells that
were treated with LPS were incubated in parallel with
curcumin or curcumin analogs 27 or 80 for 24 h at the
indicated concentrations. Total RNA was purified using
RNeasy (Qiagen, Valencia, CA) and converted to cDNA using
TaqMan Reverse Transcriptase (Applied Biosystems, Branch-
burg, NJ). Cyclooxygenase-2 (COX-2) mRNA levels were
measured using quantitative Real Time PCR analysis (qRT-
PCR) of cDNA samples. Primers specific for COX-2 were
designed to amplify a 132 base pair sequence flanking intron
7. Primer sequences for COX-2 were: upstream, TGGGGTGAT-
GAGCAACTATT; downstream, AAGGAGCTCTGGGTCAAACT.
qRT-PCR was performed using ABsolute QPCR SYBR Green
Mix (Fisher Scientific, Atlanta, GA) with the following cycling
parameters: 1 cycle, 95 8C, 15 min; 40 cycles, 95 8C, 15 s, 60 8C,
1 min. b-Actin mRNA levels were quantitated using identical
Fig. 4 – Enhancement of the TPA-induced activation of AP-1
by analogs in the 7-carbon series. Conditions were as
described in Fig. 3. Analog 24 gave the strongest
enhancement.

b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
933
Fig. 5 – Inhibition of the TPA-induced activation of AP-1 by analogs in the 5-carbon series. Conditions were as described in
Fig. 3. Analogs 25–35 were selected for further study (Table 1).
A number of analogs in the 7-carbon series (19–24, Fig. 4)
enhance the activation of AP-1 in response to stimulation with
TPA. For analog 24, which is devoid of anti-oxidant activity,
the enhancement was almost four-fold. Analog 23, which gave
a two-fold enhancement, is an isomer of curcumin, differing
only in the locations of the ring substituents. Clearly, small
structural changes can convert an inhibitor into an enhancer.
The enhancer activities of the analogs 19–24 are only observed
when TPA is present to stimulate the cells. These analogs have
no effect on AP-1 expression in the absence of TPA.
3.2.
Inhibition or enhancement of the TPA-induced
activation of AP-1 by curcumin analogs in the 5-carbon series
Forty-three compounds in the 5-carbon series, analogs 25–67,
were compared with curcumin as inhibitors or enhancers of
TPA-induced activation of AP-1. Analogs 25–41 inhibited
activation, with many showing greater activity than curcumin
(Fig. 5). Most of these analogs were devoid of anti-oxidant
activity, which further supports the conclusion that anti-
oxidant activity is not an essential property of analogs that

934
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
Fig. 6 – Enhancement of the TPA-induced activation of AP-1 by analogs in the 5-carbon series. Conditions were as described
in Fig. 3. Analogs 64–67 gave the strongest enhancement.
effectively inhibit activation of AP-1. Analogs 42–49, whose
structures are shown in Fig. 5, exhibited little or no activity
(data not shown).
activity. Analogs 77–79 showed little or no activity (data not
shown). Most of the analogs in the 3-carbon series were devoid
of anti-oxidant activity.
Analogs 50–67 of the 5-carbon series were enhancers of the
TPA-induced activation of AP-1, with enhancement activities
up to three-fold for analogs 65–67 (Fig. 6). As was observed
with the 7-carbon series, anti-oxidant activity is not essential
for enhancement activity. For example, analog 66 exhibits
anti-oxidant activity whereas analog 67 does not.
3.4.
Michael adducts retain activity as inhibitors or
enhancers of the TPA-induced activation of AP-1
Curcumin and most of the analogs in Figs. 3–7 are a,b-
unsaturated ketones that are potential Michael acceptors in
reactions with nucleophiles such as sulfhydryl groups or
amino groups. Therefore, the question was addressed as to
whether activity, either as inhibitors or as enhancers of the
TPA-induced activation of AP-1, requires retention of this
enone functionality. A series of analogs in the 5-carbon series
was reacted with methylamine to produce piperidone deri-
vatives, which resulted from a double Michael addition. The
activities of these analogs (80–87) are shown in Fig. 8. Analogs
3.3.
Inhibition or enhancement of the TPA-induced
activation of AP-1 by curcumin analogs in the 3-carbon series
Twelve analogs in the 3-carbon series were compared with
curcumin, as shown in Fig. 7. Analog 68 showed activity
comparable to curcumin as an inhibitor of the TPA-induced
activation of AP-1, while analogs 69–76 exhibited some

b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
935
Fig. 7 – Inhibition of the TPA-induced activation of AP-1 by analogs in the 3-carbon series. Conditions were as described in
Fig. 3.
80 and 81 were more active than curcumin; analog 83 was
inactive. By comparison, analogs 84–87 produced modest
enhancements of activity. Clearly, the enone functionality is
not essential for biological activity.
We conclude, therefore, that biological activity can be retained
with wide variations in structures, some of which are only
remotely related to curcumin. Numerous analogs can be
viewed as lead compounds for the development of inhibitors
of the activation of AP-1.
3.5.
Dose–response curves and determination of IC50
values
3.6.
Inhibition of COX-2 expression in microglial cells
Analogs of curcumin in the 7-carbon and 5-carbon series that
were more active than curcumin were analyzed further by
dose–response studies to obtain IC50 values. Representative
dose–response plots are shown in Fig. 9 and the IC50 values are
summarized in Table 1 along with the anti-oxidant properties
of the active analogs. Many of the active analogs do not show
anti-oxidant activity. The most active analogs, such as 25, are
an order of magnitude more potent than curcumin as
inhibitors of the TPA-induced activation of AP-1. Michael
adduct 80 is one of the more active analogs. Even analogs with
heterocyclic rings such as 27 are more active than curcumin.
To determine whether the effects of curcumin and its analogs
in inhibiting the activation of AP-1 extend beyond the 293/AP-
1-luc reporter cell line used for screening, curcumin and
analogs 27 and 80 were compared using microglial BV-2 cells.
This cell line has been shown to express COX-2 in response to
LPS stimulation by an AP-1-dependent pathway that is
inhibited by curcumin [46]. BV-2 cells stimulated with LPS
showed a strong induction of COX-2 mRNA (Fig. 10) that was
markedly suppressed by 15 mM curcumin and 50% inhibited
by 3 mM curcumin. Analog 27 and analog 80 at 3 mM
concentrations were as effective as 15 mM curcumin, con-

936
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
Fig. 9 – Representative dose–response plots. Curcumin and
analogs were analyzed in triplicate. Error bars represent
standard deviations.
Fig. 8 – Inhibition and enhancement of the TPA-induced
activation of AP-1 by Michael adducts. Conditions were as
described in Fig. 3. The piperidones are the products of
double Michael addition of methylamine to selected
analogs from the 5-carbon series. Analogs 80 and 81 were
selected for further study (Table 1).
sistent with the conclusions from Table 1 that these two
analogs are more potent than curcumin. Moreover, these
results indicate that analogs lacking anti-oxidant activity and
analogs that are not Michael acceptors are potent inhibitors of
AP-1-regulated endogenous gene expression. We conclude,
therefore, that the results from the screening studies that use
293/AP-1-luc cells are applicable to other cells and that
analogs 27 and 80 can be viewed as promising lead
compounds for development of inhibitors of the activation
of AP-1.
4.
Discussion
The ability of curcumin to suppress the constitutive activation
of AP-1 or to inhibit the stress-induced activation of AP-1 has
been reported in
a
number of studies. In addition to
Fig. 10 – Representative plot of the inhibitory effects of
curcumin and analogs 27 and 80 on LPS-induced
expression of COX-2 mRNA in BV-2 microglial cells.
suppression of LPS-induced COX-2 expression in microglial
cells by inhibiton of the activation of AP-1 [46], curcumin

b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
937
Table 1 – IC50 values and anti-oxidant activities for inhibition of the TPA-induced activation of AP-1 by curcumin and
analogs
Structure
IC50 (mm)
Anti-oxidant assay
TRAP
FRAP
1
12.8 ꢂ 0.5
+
+
+
+
2
3
5.3 ꢂ 0.7
6.0 ꢂ 0.2
ꢃ
+
25
1.4 ꢂ 0.2
ꢃ
ꢃ
26
27
28
29
30
31
8.3 ꢂ 0.6
4.1 ꢂ 0.02
6.6 ꢂ 0.2
7.1 ꢂ 0.3
7.3 ꢂ 0.4
8.2 ꢂ 0.3
ꢃ
ꢃ
ꢃ
ꢃ
+
ꢃ
ꢃ
ꢃ
ꢃ
+
ꢃ
ꢃ
32
33
4.1 ꢂ 0.3
4.8 ꢂ 0.2
ꢃ
ꢃ
ꢃ
+
34
35
11.4 ꢂ 1.0
6.0 ꢂ 0.4
+
ꢃ
ꢃ
ꢃ
80
81
3.8 ꢂ 0.5
5.7 ꢂ 0.5
ꢃ
ꢃ
ꢃ
ꢃ

938
b i o c h e m i c a l p h a r m a c o l o g y 7 2 ( 2 0 0 6 ) 9 2 8 – 9 4 0
suppresses AP-1 activity in HeLa cells [47], inhibits the
expression of COX-2 in UVB-irradiated keratinocytes by
inhibiting activation of AP-1 [48], suppresses the constitutive
activation of AP-1 in HTLV-1-infected T-cells [49] and
abolishes bile acid-induced hepatocyte apoptosis by inhibiting
activation of AP-1 [50]. Moreover, synthetic curcumin analogs
have been reported to exhibit biological activities and to be
active as inhibitors of the activation of AP-1. For example,
curcumin analogs that inhibit the activation of AP-1 have been
shown to reduce tumor angiogenesis [51]. Many studies of
analogs of curcumin have reported biological activities of
these analogs, including anti-inflammatory activity, inhibition
of angiogenesis, induction of apoptosis, anti-tumor activity,
anti-oxidant activity and antibacterial activity [52–65]. These
studies did not focus on a possible role for AP-1 in these
multiple biological activities, although it is reasonable to
suggest that AP-1 may play a role.
enhancement of AP-1 activity in response to TPA-induced
activation of the 293T/AP-1-luc cells. There are numerous
upstream targets, especially protein kinases, that might be
targets, as well as AP-1 itself or AP-1-DNA interactions.
Curcumin is known to inhibit the activation of transcription
factor NF-kB as well as AP-1 [73]. Our earlier studies of
curcumin and analogs as inhibitors of the TNFa-induced
activation of the transcription factor NF-kB [29], which used a
similar Panomics cell line for screening and included many of
the analogs used in the present study, suggested that there
may be multiple targets, based upon pharmacophore analysis
of the data. Interestingly, a number of the most active analogs
identified in the current study (Table 1) were also identified in
our study of NF-kB, suggesting that there may be common
targets for both the AP-1 and NF-kB pathways that are
inhibited by some of these analogs. These targets remain to
be identified. It also remains to be explained how some of the
analogs function as enhancers of the TPA-induced activation
of AP-1.
These multiple studies of the biological activities of
curcumin and its analogs do not address the question
whether the anti-oxidant activity of curcumin or its analogs
is required for the reported biological activity or whether the
intrinsic reactivity of curcumin and many of its analogs as
Michael acceptors is essential for activity. AP-1 is one of a
number of redox-sensitive transcription factors whose
activity appears to be regulated either directly or indirectly
through reversible oxidation-reduction of critical cysteine
residues [66,67]. c-Jun and c-fos have single conserved
cysteine residues in their DNA-binding domains that undergo
reversible redox reactions that alter their DNA-binding
properties [68]. In addition, there are a number of AP-1-
associated proteins, such as thioredoxin, jun activation
domain-binding protein 1 (Jab1), and redox factor-1 (Ref-1),
that contribute to regulation of the redox state of AP-1 [69,70].
Other studies suggest that direct covalent modification of the
reactive cysteine residues in AP-1 may be important. For
example, direct inhibition of AP-1 by 15-deoxy-delta-12,14-
prostaglandin J2, which is a cyclopentenone prostaglandin
capable of Michael addition, has been demonstrated [71].
Recent studies with curcumin and analogs, however, suggest
that inhibition of AP-1 by these Michael acceptors does not
involve direct modification of AP-1 [72].
Acknowledgment
This work was supported by grant BC043125 from the US
Army/DOD Breast Cancer Program.
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