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Inhibitory Effect of Curcumin on TPA (12-O-Tetradecanoyl Phorbol-13-acetate)- Induced Activation of Protein Kinase C Isoenzyme- Epsilon and c-fos Protein Level in Human Keratinocytes

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Protein Kinase C (PKC) is a family of isoenzymes that are normally expressed in human keratinocytes. The expression pattern of PKC under induction of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter, is herein reported. Curcumin was evaluated for its effect on TPA-activated PKC-? because PKC-? has been found to have unique oncogenic potential and substrate specificity. Curcumin was shown previously to inhibit TPA-induced AP-1 activity. c-fos protein is inducible by TPA and thus is associated with c-jun to result in an increased AP-1 activity. In the present study the effect of curcumin on TPA-induced c-fos protein level was determined. Human keratinocytes were treated with 160 nM TPA for 1, 2 and 18 h. Upon stimulation by TPA for 1 or 2 h, PKC-? and -? isoenzymes were translocated from cytosol to membrane by 70 or 85 % and they were completely down regulated after treatment for 18 h. However, effects were not found in PKC-? isoenzyme. Treatment with 20, 40 and 50 µM curcumin inhibited TPA-induced translocation of PKC-? isoenzyme from the cytosol to the membrane in a dose-dependent manner. Curcumin alone did not affect the translocation of PKC-? isoenzyme. The expression level of c-fos protein in human keratinocytes was induced by 160 nM TPA with the maximum result at 2 h, as measured by enhanced chemiluminesence Western blotting detection system. Curcumin pretreated cells decreased TPA-induced c-fos expression in a dose-dependent manner. In conclusion, the induction of c-fos expression and PKC-? activation by TPA occurred in human keratinocytes and this signalling was inhibited by curcumin.
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ScienceAsia 31 (2005): 113-120
Inhibitory Effect of Curcumin on TPA
(12-O-Tetradecanoyl Phorbol-13-acetate)-
Induced Activation of Protein Kinase C Isoenzyme-
Epsilon and c-fos Protein Level in Human Keratinocytes
Pornngarm Limtrakul*, Wanida Chearwae and Sasisopin Luekumharn
Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chaing Mai 50200, Thailand.
* Corresponding author, E-mail: plimtrak@mail.med.cmu.ac.th
Received 17 Sep 2004
Accepted 28 Dec 2004
ABSTRACT: Protein Kinase C (PKC) is a family of isoenzymes that are normally expressed in human keratinocytes.
The expression pattern of PKC under induction of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent
tumor promoter, is herein reported. Curcumin was evaluated for its effect on TPA-activated PKC-? because
PKC-? has been found to have unique oncogenic potential and substrate specificity. Curcumin was shown
previously to inhibit TPA-induced AP-1 activity. c-fos protein is inducible by TPA and thus is associated with
c-jun to result in an increased AP-1 activity. In the present study the effect of curcumin on TPA-induced c-fos
protein level was determined. Human keratinocytes were treated with 160 nM TPA for 1, 2 and 18 h. Upon
stimulation by TPA for 1 or 2 h, PKC-? and -? isoenzymes were translocated from cytosol to membrane by
70 or 85 % and they were completely down regulated after treatment for 18 h. However, effects were not
found in PKC-? isoenzyme. Treatment with 20, 40 and 50 µM curcumin inhibited TPA-induced translocation
of PKC-? isoenzyme from the cytosol to the membrane in a dose-dependent manner. Curcumin alone did not
affect the translocation of PKC-? isoenzyme. The expression level of c-fos protein in human keratinocytes
was induced by 160 nM TPA with the maximum result at 2 h, as measured by enhanced chemiluminesence
Western blotting detection system. Curcumin pretreated cells decreased TPA-induced c-fos expression in a
dose-dependent manner. In conclusion, the induction of c-fos expression and PKC-? activation by TPA
occurred in human keratinocytes and this signalling was inhibited by curcumin.
KEYWORDS: Curcumin, protein kinase C (PKC), human keratinocytes, TPA, c-fos.
INTRODUCTION
induces several biosynthetic processes, namely
enhanced expression of cellular oncogenes such as c-
Turmeric is a perennial herb widely cultivated in
jun, c-fos and c-myc4 and elevation or translocation of
tropical regions of Asia. It has been widely used in many
protein kinase C (PKC)5. PKC is a multigene family
parts of the world as spice, colorant, and cosmetic 1. It
consisting of at least ten distinct serine/threonine
contains about 1-5 % curcumin (diferuloylmethane), a
kinases which play a key role in signal transduction and
phenolic compound that has been identified as the
growth control. PKC isoenzymes are classified into
major pigment in turmeric, and it possesses both anti-
three groups based on activation by Ca2+ and
inflammatory and antioxidant properties2. It has been
phospholipid: Ca2+ dependence or conventional
shown to inhibit mutagenesis, carcinogenesis and DNA-
isoenzymes (PKC-?, ?, ?) , Ca2+ independence or novel
carcinogen adduct formation3. With its perceived
isoenzymes (PKC-?, ?, ?, ?) and not requiring Ca2+ or
human safety, following centuries of use, curcumin
lipid (PKC-?, ?)6. Conventional and novel PKC
becomes a potent phytochemical candidate for cancer
isoenzymes are the major cellular receptors for TPA. In
prevention. However, how curcumin elicits this
addition, PKC isoenzymes display selective organ and
chemopreventive efficacy on carcinogenesis is an
cellular expression. It was found that PKC-?, -? and -?
ongoing research project in many laboratories. A large
isoenzymes are normally expressed in human
body of data has demonstrated that curcumin is
keratinocytes7. Little is known concerning the role of
engaged in multiple anti-tumor promoting pathways. It
individual PKC isoenzymes in human keratinocytes.
has been reported that curcumin inhibited a variety of
Among the PKC isoenzymes it has been reported that
biological activities of 12-O-tetradecanoyl-phorbol-
PKC-? isoenzyme exhibited full oncogenic potential8, 9
13-acetate (TPA), a potent skin tumor promoter. TPA
and is implicated in the regulation of other biological

114
ScienceAsia 31 (2005)
processes, including antiviral resistance, hormonal
as described before12. Briefly, cells were washed twice
secretion, transporter regulation and golgi function
with ice cold Ca2+ and Mg2+ - free phosphate-buffer
modulation10,11. Thus, this study focused on PKC-?
saline (PBS) and detached by adding 0.05% of trypsin-
isoenzyme and determined the effect of curcumin on
EDTA. The reaction was stopped with trypsin-inhibitor.
TPA-induced translocation or downregulation of PKC-
Cells were collected by low speed centrifugation and
? isoenzyme in human keratinocytes. In most studies,
then washed once with PBS. The cell pellets were
the inhibitory effect of curcumin on TPA action has
resuspended in buffer A (25 mM Tris-HCl, pH 7.5, 2
been studied using mouse skin model. The effect of
mM EDTA, 2 mM EGTA, 0.05 mM DTT, 0.02% triton X-
curcumin on TPA actions in human skin model has not
100, 10 µg/ml leupeptin, 25 µg/ml aprotinin, 2 mM
been established. In this paper we demonstrated for
PMSF) and homogenized using a Dounce homogenizer
the first time the protective effect of curcumin on TPA
(pestle A). Cell homogenates were centrifuged at
induced PKC-? activation. The inhibition of TPA-
100,000g for 1 h. The supernatant was collected as a
induced PKC-? by curcumin might explain the decrease
cytosolic PKC fraction. The pellet was washed once in
in c-fos protein level in human keratinocytes.
buffer B (25 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM
EGTA, 2 mM PMSF), then treated on ice for 1 h with
MATERIALS AND METHODS
0.2% triton X-100 in buffer A and centrifuged at
100,000g for 1 h. The supernatant (membrane PKC
Human Keratinocytes Culture
fraction) was collected and concentrated using
Primary keratinocytes were isolated from keratome
Centricon 30 concentrators (Amicon Corp.) and
biopsies of juvenile foreskins (2-3 years old), which
membrane protein concentrations were determined
were obtained from Dr. Aram Limtrakul (Chiang Mai
by Lowry’s method 13. The PKC levels in cytosolic and
Public Health Promotion Center). The skin was placed
membrane fractions were measured by Western-blot
in transport medium (DMEM free serum) and moved
analysis.
to the laboratory, where it was processed as soon as
possible. Using a pair of scissors, all excess dermis and
Preparation of Nuclear Extracts of c-fos Protein
connective tissue were removed, the skin sections were
The nuclear extract was prepared by the method of
sterilized by immersing in absolute ethanol for 1 min
Dignam et al.14 as follows. The pellet obtained from the
and soaked in antibiotic solution (20% penicillin-
low speed centrifugation of the homogenate was
streptomycin and 10% penicillin – streptomycin) at 4
resuspended in buffer C (20 mM HEPES, pH 7.9, 25%
oC for 30 min. The tissue was transferred to dispase
glycerol, 0.42 M NaCl, 1.5 mM MgCl , 0.2 mM EDTA,
2
solution and incubated at 4 oC for 48 h. Then the
0.5 mM PMSF, 0.5 mM DTT) and was stirred gently with
epidermis layer was removed and treated with 2 ml
a magnetic stirring bar for 30 min at 4 oC and then
trypsin-EDTA at 37 oC for 30-45 min, with gentle mixing
centrifuged for 30 min at 25,000g. The supernatant
every 5 min. Addition of 4 ml soybean trypsin-inhibitor
was dialysed against 50 volumes of buffer D (20 mM
stopped the action of trypsin. Then cells were
HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA,
centrifuged at low speed centrifugation (500 rpm) for
0.5 mM PMSF, 0.5 mM DTT) for 5 h. The dialysate was
5 min at room temperature. Supernatant was removed
centrifuged for 20 min at 25,000g and the resulting
and cell pellet resuspended in keratinocyte-SFM 5 ml.
precipitate discarded. The supernatant, designated as
Using syringe with needle size 22G1/2, the suspension
the nuclear extract, was frozen as aliquots in liquid
was mixed very gently to form a single cell suspension.
nitrogen and stored at -80 oC.
The primary keratinocytes were cultured at a density
of 3x106 cells/culture in T-25 flasks in keratinocyte-
Western Blotting for PKC and c-fos Proteins
SFM medium supplemented with epidermal growth
Cells were placed in growth factor-free medium
factor (EGF, 5 ng/ml) and bovine pituitary extract (BPE,
(keratinocyte-SFM without hydrocortisone, insulin,
50 µg/ml). The Ca2+ concentration of the medium was
BPE and EGF) for 48 h. The cells were then treated with
kept at 0.05 mM to maintain undifferentiated
test compounds as described in the text. For detection
keratinocytes. The cells were maintained in a CO -
of PKC protein, proteins (30 µg) from cytosolic and
2
incubator (37 oC, 5% CO ) and the medium was changed
membrane fractions were electrophoresed in
2
every 2-3 days. Passages 3-5 were used in our study.
discontinuous 7.5% SDS-polyacrylamide gels. The
proteins were electroblotted onto Hybond ECL
Preparation of Cytosolic and Membrane Fractions
membrane in a Trans-blot electrophoretic transfer cell
of PKC
(Bio-Rad Laboratories) as described before15. The blots
For detection of PKC, cytosolic and membrane
were blocked with 5% nonfat dry milk dissolved in tris
fractions were prepared from subconfluent cell culture
buffered saline containing 0.2% Tween-20 (TBST). To

ScienceAsia 31 (2005)
115
detect PKC-? (93 kD), the blot was incubated for 2 h
Effect of TPA on the Total Level of the PKC-Epsilon
with anti-PKC-? antibody at a dilution of 1:2500 in
(?) in Whole Cell Lysates
TBST. Whereas for PKC-? (82 kD) and -? (76 kD), the
The activation by TPA may increase the total level
blots were incubated with the anti- PKC-? and -?
of PKC-? isoenzyme in whole cell lysates. To examine
antibodies for 1 h at a dilution of 1:2500. Following
this possibility, whole cell lysates of keratinocytes
washing, immunoreactive proteins were visualized
treated with 160 nM TPA for 0, 1, 2 and 18 h were
using the enhanced chemiluminnescence Western
assayed by Western blot (Fig. 2). No significant
blotting detection system (Amersham). The
difference between the band intensities of the control
autoradiograms were scanned with an Image Master
and 1 and 2 h TPA-treated keratinocytes were observed.
sharp laser densitometric scanner, and the peak areas
This finding suggests that when human keratinocytes
representing PKC bands were determined. Western
were treated with TPA, PKC-? was activated by
blot analysis of the fos protein was performed by the
translocation from cytosol to the membrane but TPA
same protocol as described above except in the
did not affect the total level of PKC protein in the cells.
detection system, the specific antibody to c-fos protein
Long-term treatment with TPA (18 h) induced complete
(p62, 62 kD) was used instead of specific antibody to
downregulation of PKC level.
PKC proteins.
A
82 kDa
Data Manipulation and Statistical Analysis
a
ph
The levels of PKC were presented as percentages of
al
o
l
) 120
C
100
the cytosolic PKC level of DMSO-treated cells (CO,
o
ntr
80
60
control). The levels of c-fos were presented as
i
ve PK
40
(% of c
20
percentages of the c-fos level after 2 hr TPA treatment.
0
Relat
TPA (h)
C0 C1 C2 C18 M0 M1 M2 M18
Results are the means ± SD from three independent
experiments. Differences between the means were
B
93 kDa
analyzed by one-way analysis of variance. Results were
120
considered to be statistically significant when p<0.05.
o
l
) 100
o
ntr 80
60
RESULTS
i
ve PKC epsilon
40
(% of c 20
Relat
0
TPA (h)
Translocation and Downregulation of PKC-Alpha
C0
C1
C2 C18 M0
M1
M2 M18
(?), -Delta (?), and -Epsilon (?) Isoenzymes in
Response to TPA
PKC isoenzyme activation is associated with
C
76 kDa
translocation from cytosol to membrane. The
120
o
l
) 100
translocations of PKC-?, -? and -? isoenzymes, which
ntr
o 80
are TPA responsive isotypes in human keratinocytes,
60
i
ve PKC delta
40
were evaluated in this study. The 80 % confluent human
(% of c 20
keratinocytes at passages 3-5 were placed in growth
Relat
TPA (h)
0
factor-free medium for 48 h. The cells were treated
C0
C1
C2
C18
M0
M1
M2 M18
with 160 nM TPA for 1, 2 and 18 h. Control cells
Cytosol
Membrane
received 0.4% DMSO. Treatment of keratinocytes with
TPA for 1 or 2 h induced translocation of PKC-? and
Fig 1.Translocation and downregulation of PKC-?,?, ?
-? isoenzymes from the cytosol that was associated
isoenzymes in response to TPA. Cytosolic (C) and
with increasing amounts in the membrane by about 70
membrane (M) fractions (30 µg protein) were prepared
or 85 % (Figs.1 A and B). Long-term treatment with TPA
from keratinocytes treated with TPA, separated by SDS-
(18 h) induced complete downregulation (i.e., loss of
PAGE, and immunoblotted as described. Upper:
Western blot analysis of PKC-? (A),? (B) and ?(C)
>99%) of PKC-? and -?. In contrast, densitometric
protein expression using anti PKC-?, ? and ?
analysis of Western blots for PKC-? revealed no
antibodies, respectively. C0 and M0 are cytosolic and
significant reproducible changes in either cytosolic or
membrane fractions of DMSO-treated cells (control).
membrane fractions in response to TPA (Fig.1C). Our
C1 and M1, C2 and M2, C18 and M18 are cytosolic and
data indicated that in human keratinocytes there were
membrane fractions of TPA-treated cells for 1, 2 and 18
two PKC isoenzymes, ? and ?, which were activated by
h respectively. Lower : Relative intensity of the
160 nM TPA and demonstrated the specific activation
immunoblots calculated as means +S.E. Each
of PKC isoenzymes in response to TPA.
histogram obtained from three independent
experiments performed in triplicate.

116
ScienceAsia 31 (2005)
93 kDa
Curcumin treatment (µM)
120
0
20
40
50
100
l
)
1
2
3
4
5
6
7
8
o
80
93 kDa
ntr
o
60
125
40
o
l
) 100
i
ve PKC epsilon
20
(% of c
o
ntr
75
0
TPA (h)
Relat
50
To
T1
T2
T18
i
ve PKC epsilon
(% of c
25
Relat
0
TPA (h)
Fig 2.Effect of TPA on the total level of PKC-? isoenzyme in
C0 M0 C20 M20 C40 M40 C50 M50
human keratinocytes. Whole cell lysates (30 µg
protein) were prepared from keratinocytes treated with
Cytosol
Membrane
160 nM TPA for 1 (T1), 2 (T2) and 18 (T18) h
respectively, separated by SDS-PAGE, and
Fig 3.Effect of curcumin on translocation of PKC-?
immunoblotted as described. Upper: Western blot
isoenzyme. Cytosolic (C) and membrane (M) fractions
analysis of PKC-? protein expression using anti PKC-?
(30 µg protein) were prepared from keratinocytes
antibodies. Lower: Relative intensity of the immunoblots
treated with curcumin (0, 20, 40 and 50 µM) for 1 h,
calculated as means +S.E.. Each histogram was
separated by SDS-PAGE, and analyzed for PKC-?
obtained from three independent experiments per-
isoenzymes. Lanes 1, 3, 5, 7 and 2, 4, 6, 8 are cytosolic
formed in triplicate. The arrow indicates molecular
and membrane fractions, respectively. Lanes 1 and 2,
weight of PKC-? isoenzyme.
control (C0 and M0); lanes 3 and 4, 20 µM curcumin
(C20 and M20); lanes 5 and 6, 40 µM curcumin (C40
Effect of Curcumin on Translocation of PKC-Epsilon
and M40) and lanes 7 and 8, 50 µM curcumin (C50 and
(?)
M50). Upper: Western blot analysis of PKC-?
To explore whether curcumin itself affects
expression using anti-PKC-? antibody. Lower : Relative
translocation of PKC-? in human keratinocytes, the
intensity of the immunoblots calculated as means +S.E.
cells were incubated at 37 oC for 1 h at different
Each histogram was obtained from three independent
concentrations of curcumin (0, 20, 40, 50 µM). It was
experiments performed in triplicate.The arrow indicates
found that the PKC bands were only detected in
molecular weight of PKC-? isoenzyme.
cytosolic fractions, and there were no significant
differences among control and curcumin treated cells
(Fig. 3). Thus, it can be suggested that curcumin itself
from DNA to RNA and protein level (enzyme level).
did not induce translocation of PKC-? enzyme from the
Accordingly, this study examined kinetics of TPA-
cytosol to the membrane in human keratinocytes.
dependent PKC-? activation by curcumin. Primary
culture of human keratinocytes were treated with 50
Effect of Curcumin on TPA-Induced PKC-Epsilon
µM curcumin for 1 h prior to addition of TPA (pre-
(?) Activation
treatment), at the same time as the addition of TPA (co-
We previously reported that curcumin inhibited
treatment), or 1 h after addition of TPA (post-treatment).
TPA-induced tumor promotion on mouse skin19, 20.
After these treatments cytosolic and membrane
However the molecular mechanism of this inhibition
fractions were prepared and then assayed for PKC-? as
remained to be explored in human skin model.
described earlier. It was found that TPA-dependent
Therefore this study examined the effect of curcumin
PKC-? activation was inhibited only when cells were
on translocation of TPA-induced PKC-? which has not
pretreated with curcumin. The co-treatment and post-
yet been reported in other studies. The cells were
treatment were not effective (Fig. 4B). Therefore, this
preincubated for 1 h with different concentrations of
study showed that curcumin inhibited the effect of TPA
curcumin (20, 40, 50 µM) followed by treatment with
in the signal transduction cascade before TPA induced
160 nM TPA for 1 h at 37 oC. The control cells received
the translocation or downregulation of PKC-epsilon.
0.4% DMSO. Cytosolic and membrane fractions were
prepared and analyzed for the level of PKC-? isoenzyme
Effect of Curcumin on TPA-Induced c-fos Gene
by Western blotting. The result indicated that curcumin
Expression
inhibit the TPA induced PKC-? in a dose dependent
One of the important biological activities of TPA
manner (Fig. 4A).
required for tumor promotion is enhanced expression
of cellular oncogenes such as c-jun, c-fos and c-myc.
Effect of Time on Curcumin Addition on TPA-
In this study, the effect of curcumin on TPA-induced c-
Induced PKC-Epsilon (?)
fos gene expression in human keratinocytes was
It appears that the molecular action of curcumin is
evaluated. The 80% confluent human keratinocytes
quite complicated because the targets of its action vary
were incubated with 160 nM TPA for 0, 0.5, 1, 2, 4 and

ScienceAsia 31 (2005)
117
18 h. After these treatments, nuclear extracts were
disappearance of c-fos protein level after long-term
prepared and then assayed for c-fos protein as
treatment with TPA (18 h). The data in Fig. 5 indicated
described. TPA appeared to increase the c-fos protein
that TPA activation of c-fos protein peaked at 2 h;
level after 0.5 h incubation (Fig. 5). This increase in c-
therefore it was chosen for study of the effect of
fos protein level induced by TPA appeared to be
curcumin. Human keratinocytes were preincubated
maximum at 2 h. After 2 h of TPA incubation, the level
for 1 h with different concentrations of curcumin (20,
of c-fos protein declined. We observed complete
40 and 50 µM) followed by treatment with 160 nM TPA
for 2 h. After these treatments nuclear extracts were
prepared and then assayed for c-fos protein as
A
described in Materials and Methods. The result showed
Cytosol
Membrane
that curcumin inhibited TPA-induced expression of c-
93 kDa
fos in human keatinocytes in a dose dependent manner
(Fig. 6). This finding showed for the first time that TPA-
140
120
induced c-fos protein level was suppressed by curcumin
100
in a human skin model.
epsilon
80
o
ntrol)
60
40
DISCUSSION
i
ve PKC
(% of c
20
Relat
0
Con TPA 20 40 50
Con TPA 20 40 50
The previous study from our laboratory indicated
only
only
+ Curcumin (µM)
+ Curcumin (µM)
that topical application of curcumin16 and dietary
Cytosol
Membrane
curcumin15,17 had marked inhibitory effect on TPA
induced tumor promotion in mouse skin , which was
B
consistent with the findings of many previous reports18,
93 kDa
19. In order to further investigate the mechanism by
which curcumin causes inhibition, our study examined
120
the effect of curcumin on the level and distribution of
100
l
)
o
TPA induced PKC-? isoenzyme in primary human
epsilon
80
ntr
o
keratinocytes. The findings of our study demonstrated
60
the chemopreventive potential of curcumin on TPA-
i
ve PKC
40
(% of c
induced PKC-? isoenzyme, i.e., the isoenzyme that could
20
Relat
be a molecular target for tumor promoting phorbol
0
DMSO
Pretreatment
Postreatment
esters in skin chemical carcinogenesis models.
control TPA only
Cotreatment
Curcumin only
Human keratinocytes are the main cell type (>95%)
Cytosol
Membrane
Fig 4.
p62
A:
A: Dose response inhibition of TPA-induced PKC-?
activation by curcumin. Keratinocytes were
l) 120
preincubated for 1 h with different concentrations of
o 100
curcumin followed by treatment with 160 nM TPA for 1
ntr
otein
o 80
h as described in the text. After these treatments
cytosolic (C) and membrane (M) fractions (30 µg
s pr
60
o
protein) were prepared and then analyzed for PKC-?
40
c-f
(% of c
isoenzymes. B:
B: Effect of time on curcumin addition on
20
TPA-induced PKC-? isoenzyme. Human keratinocytes
0
were treated with 50 µM curcumin for 1 h prior to
Cont
0.5
1
2
4
18
addition of TPA (pre-treatment), at the same time as
TPA treatment (h)
the addition of TPA (co-treatment) or 1 h after addition
of TPA (post- treatment). After these treatments
Fig 5.Effect of TPA on c-fos gene expression in human
cytosolic and membrane fractions were prepared and
keratinocytes. Human keratinocytes were incubated
then assayed for PKC-? as described. Upper: Western
with 160 nM TPA for 0, 0.5, 1, 2, 4 and 18 h. After these
blot analysis of PKC-? expression using anti-PKC-?
treatments nuclear extracts were prepared and assayed
antibody from three independent experiments. Lower :
for c-fos protein. Upper: Western blot analysis of c-fos
Relative intensity of the immunoblots calculated as
protein using anti-c-fos antibody. Lower : Relative
means +S.E. Each histogram obtained from three
intensity of the immunoblots calculated as means +S.E.
independent experiments performed in triplicate. The
Each histogram was obtained from three independent
arrow indicates molecular weight of PKC-? isoenzyme.
experiments performed in triplicate.

118
ScienceAsia 31 (2005)
protein which was released into the cytoplasm and
p62
displayed a phospholipid- and Ca2+-independent
protein kinase C activity ( Protein kinase M, PKM), and
120
a 35 KD protein which retained TPA binding activity.
o
l)
100
The role of 35 KD protein in gene regulation has been
otein
o
ntr
80
considered 22, 23, 24. Therefore this study combined with
60
preliminary information can suggest that activation of
o
s pr
40
PKCs is usually associated with their translocation from
c-f
(% of c
20
the cytosol to the membrane. Concomitantly, there is
0
a cleavage of the catalytic domain from the regulatory
Cont TPA only
20
40
50
+ Curcumin ( M
µ )
domain by proteinase, which may occur at the border
of the hinge region and catalytic domain. Then activated
PKC (catalytic domain) functions at the membrane or
Fig 6.Dose response of inhibition of TPA-induced c-fos
may translocate to specific targets such as the nucleus24.
expression by curcumin. Human keratinocytes were
However, exactly how PKC translocates to specific
preincubated for 1 h with different concentrations of
curcumin ( 20, 40 and 50 µM) followed by treatment
targets has not yet been clearly understood. It has been
with 160 nM. TPA for 2 h. After these treatments nuclear
reported that eventually, the activated PKC undergoes
extracts were prepared and then assayed for c-fos
further breakdown. Thus, continuous exposure to
protein by Western blotting. Upper: Western blot analy-
activators lead to activation and down-regulation of
sis of c-fos protein using anti-c-fos antibody. Lower :
PKC, and it is possible that either activation or down-
Scanning densitometry of the relative intensity of the
regulation is responsible for the biological effects.
immunoblots calculated as means +S.E. Each
The roles of PKC-? and -? isoenzymes in human
histogram was obtained from three independent ex-
keratinocytes were linked to epidermal differentiation
periments performed in triplicate.
program and carcinogenesis respectively 25. However,
the other crucial role of PKC isoenzyme in human
of the epidermis which express both mRNA and protein
keratinocytes is apoptosis. A recent study revealed
of PKC-?, -? and -? isoenzymes. Among the first signaling
that in normal human keratinocytes, PKC-? isoenzyme
proteins, PKC isoenzymes have been shown to change
is correlated with the induction of apoptosis by UV
sub-cellular localization upon activation20. Our study
exposure 26, which is a distinct pathway from TPA
found that 160 nM TPA-treatment for 1 or 2 h caused
activation. Those findings were consistent with this
PKC-? and -? isoenzymes to translocate from cytosol
study because this study found that with either short-
to membrane. The levels of cytosolic PKC-? and -?
term TPA treatment (160 nM TPA for 1 and 2 h) or long-
isoenzymes were significantly reduced with a
term treatment (160 nM TPA for 18 h) the PKC-? level
concomitant increase in the membrane fraction,
remained relatively unchanged (Fig. 1C).
indicating specific activation of both isoenzymes (Fig.
Previous studies have shown that PKC-? isoenzyme
1, A and B). For a long time, it has been proven that the
in human keratinocytes was involved with oncogenic
binding site of TPA on PKC isoenzymes is C1 domain
potential. We, therefore, investigate the effect of
whose membrane affinity is increased dramatically
curcumin on TPA-induced PKC-? activation. We found
upon binding to TPA21. This could explain the reason
that TPA did not affect the total level of PKC-?
why the level of PKC-? and - ? isoenzyme on the cell
isoenzymes (Fig. 2), suggesting that TPA had no effect
membrane were higher after 2 h TPA exposure than 1
on new PKC synthesis, but was directly involved with
h. In addition, this study found that PKC-? and -?
the translocation of preexisting PKC-? from the cytosol
isoenzymes underwent downregulation after treatment
to the membrane. Our study showed that curcumin
with 160 nM TPA for 18 h. (Fig. 1, A and B). Translocation
completely inhibited the translocation of PKC-?
of PKC from cytosol to the membrane by TPA seems to
isoenzyme induced by TPA (Fig. 3) in a dose-dependent
activate the enzyme in intact cells and down regulation
manner. In addition, curcumin itself at any
occurs after the translocation. Little is known about
concentration tested (20, 40, 50 µM), did not affect the
the molecular mechanism(s) and in vivo determinants
translocation or down-regulation of PKC-? isoenzyme
of the process of the downregulation through
(Fig. 4A). Therefore, curcumin had no mitogenic activity
proteolysis of PKC. However, one protease cleavage
on human keratinocytes, unlike the TPA actions.
site has been mapped to the border of the hinge region
Moreover TPA-induced translocation of PKC-?
and catalytic domain of the PKC isoenzymes. There is
isoenzyme was inhibited only in case of pretreatment
evidence that the proteolytic process caused by neutral
with curcumin (Fig. 4B). Co- or post-treatment with
Ca2+-dependent proteinase, which was found in the
curcumin was not effective. This result was consistent
membrane, resulted in two fragments of PKC: a 50 KD
with the known effect of curcumin on NF-kB

ScienceAsia 31 (2005)
119
activation27, which was also found that the inhibitory
messenger RNAs in mouse skin. Cancer Lett 87
87, 85-95. Lui
effect of curcumin was accomplished only in case of
JY and Lin JK(1993) Inhibitory effects of curcumin on protein
kinase C activity induced by 12-O-tetradecanoyl-phorbol-
pre-treatment, whereas co- or post-treatment did not.
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This result, combined with the characteristic structure
6. Hug H and Sarre TF (1993) Review article: Protein kinase C
of curcumin, which is exceedingly nonpolar, suggests
isoenzymes: divergence in signal transduction?. Biochem J
that curcumin may be inserted into membrane, bind to
29
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cytosolic PKC at C1 domain, and then inhibit the
7. Nicholas JR, Joseph B and Patricia A (1994) Translocation
molecule. However, this study proposed that the
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expression of c-jun, c-fos and c-myc in JB6 cells and in
125-46.
mouse epidermis28. In the present study, we report the
10. Lehel C, Olah Z, Jakab G and Anderson WB (1995) Protein
suppression of c-fos protein level by curcumin in
kinase C epsilon is localized to the Golgi via its zinc-finger
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92, 1406-10.
inducible by TPA and thus is associated with c-jun to
11. Borner C, Guadagno SN, Fabbro D and Weinstein (1992)
result in an increased AP-1 activity. Curcumin may also
Expression of four protein kinase C isoforms is rat fibroblast.
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12. Ohmori T and Arteaga CL (1998) Protein kinase C epsilon
translocation and phosphorylation by cis-
transcriptional factor AP-1 is believed to play an
diamminedichloroplatinum (II) (CDDP): potential role in
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CDDP-mediated Cytotoxicity. Cell Growth Differ 9, 345-53.
tumor promotion. In conclusion, the data described
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here demonstrated for the first time that the suppression
method of Lowry et al. which is more generally applicable.
of TPA-induced c-fos protein level and PKC-? activation
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83, 346-56.
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FW (2001) Inhibition of carcinogen induced c-Ha-ras and
c-fos protooncogenes expression by dietary curcumin. BMC
Cancer
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This work was supported by Faculty of Medicine
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Endowment Funds, Chiang Mai University. The authors
(1997) Inhibitory effect of curcumin on tumor initiation
wish to express their gratitude to Dr. R.W. Gracy and Dr.
and promotion stages in mouse skin tumorigenesis.
Dan Dmitrijevich of the University of North Texas
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Health Science Center at Fort Worth, Texas, for
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