Carcinogenesis vol.29 no.10 pp.2011–2018, 2008
Advance Access publication August 5, 2008
Guggulsterone modulates MAPK and NF-kB pathways and inhibits skin tumorigenesis
in SENCAR mice
Sami Sarfaraz1, Imtiaz A.Siddiqui, Deeba N.Syed, Farrukh
Afaq and Hasan Mukhtar?
Chemoprevention Program Paul P Carbone Comprehensive Cancer Center
and Department of Dermatology, School of Medicine and Public Health,
University of Wisconsin, 1300 University Avenue, Medical Sciences Center,
B-25, Madison, WI 53706, USA
1Present address: Medical Oncology Branch, National Cancer Institute,
Building 37, Room 1136, 37 Convent Drive, Bethesda, MD 20892, USA
?To whom correspondence should be addressed. Tel: þ1 608 263 3927;
Fax: þ1 608 263 5223;
Guggulsterone (GUG), a resin of the Commiphora mukul tree, has
been used in ayurvedic medicine for centuries to treat a variety of
ailments. Recent studies have suggested that GUG may also pos-
sess anticancer effects. In the present study, we show that GUG
possesses antitumor-promoting effects in SENCAR mouse skin
tumorigenesis model. We first determined the effect of topical
application of GUG to mice against 12-O-tetradecanoylphorbol-
13-acetate (TPA)-induced conventional markers and other novel
markers of skin tumor promotion. We found that topical applica-
tion of GUG (1.6 mmol per mouse) 30 min prior to TPA (3.2 nmol
per mouse) application onto the skin of mice afforded significant
inhibition against TPA-mediated increase in skin edema and hy-
perplasia. Topical application of GUG was also found to result in
substantial inhibition against TPA-induced epidermal (i) orni-
thine decarboxylase (ODC) activity; (ii) ODC, cyclooxygenase-2
and inducible nitric oxide synthase protein expressions; (iii) phos-
phorylation of extracellular signal-regulated kinase1/2, c-jun
N-terminal kinases and p38; (iv) activation of NF-kB/p65 and
IKKa/b and (v) phosphorylation and degradation of IkBa. We
next assessed the effect of topically applied GUG on TPA-induced
skin tumor promotion in 7,12-dimethyl benz[a]anthracene-initi-
ated mice. Compared with non-GUG-pretreated mice, animals
pretreated with GUG showed significantly reduced tumor inci-
dence, lower tumor body burden and a significant delay in the
latency period for tumor appearance from 5 to 11 weeks. These
results provide the first evidence that GUG possesses anti-skin
conventional as well as novel biomarkers of tumor promotion.
In summary, GUG could be useful for delaying tumor growth in
SENCARmice and inhibits
is a plant polyphenol obtained from the gum resin of the Commiphora
mukul tree; it has been used in ayurvedic medicine for centuries to treat
a variety of ailments like obesity, diabetes, hyperlipidemia, atheroscle-
rosis and osteoarthritis (1–5). The anti-inflammatory activity of gum
guggul is well known (6), and recent studies have suggested that
GUG may alsopossess anticancer effects (7,8) but the molecular mech-
anisms underlying the anticancer effects of GUG are beginning to
emerge (7,8). We considered the possibilitythat GUG may also possess
The multistage mouse skin carcinogenesis model, although an
artificial one, is an ideal system to study a number of biochemical
alterations, changes in cellular functions and histologic changes that take
place during the different stages of chemical carcinogenesis (9,10). This
system has also served as a useful model for initial screen for cancer
chemopreventive effects of most dietary substances (11). Studies have
shown that skin application of tumor-promoting agents results in inflam-
matory responses, such as development of edema, hyperplasia, induction
of proinflammatory cytokine interleukin-1a, induction of epidermal or-
nithine decarboxylase (ODC) and cyclooxygenase-2 (COX-2) protein
expression and activity, as well as activation of NF-jB (11–14). Activa-
tion of mitogen-activated protein kinases (MAPKs)/NF-jB pathways has
been shown to be involved in tumor growth and development (15,16). In
the present study, we show that topical application of GUG to SENCAR
mice possesses antitumor-promoting effects and these effects are medi-
ated via the ability of GUG to modulate MAPK and NF-jB pathways.
Materials and methods
GUG, [4,17(20)-pregnadiene-3,16-dione], was purchased from Steraloids
(Newport, RI). Extracellular signal-regulated kinase (ERK)1/2 (phospho-
p44/42), c-jun N-terminal kinase (JNK) (phospo-p54/46), p38 (phospho-
p38), IjBa and IjBa (phospho) antibodies were obtained from New England
Biolabs (Beverly, MA). NF-jB/p65 antibody was procured from Geneka Bio-
technology (Montreal, Canada). IKKa and ODC, COX-2 and inducible nitric
oxide synthase (iNOS) antibodies were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA). Anti-mouse or anti-rabbit secondary antibody
horseradish peroxidase conjugate was obtained from Amersham Life Sci-
ence (Arlington Height, IL). 7,12-Dimethyl benz[a]anthracene (DMBA) and
12-O-tetradecanoyl-phorbal-13-acetate (TPA) were purchased from Sigma
Chemicals (St Louis, MO). The DC BioRad Protein assay kit was purchased
from Bio-Rad Laboratories (Hercules, CA). Novex pre-cast Tris-Glycine
gels were obtained from Invitrogen (Carlsbad, CA).
Animals and treatment for biomarker studies
Female SENCAR mice (5–6 weeks old) obtained from Charles River Labora-
tories were housed four per cage and were acclimatized for 1 week before use
in both short-term and long-term experiments. Animals were subjected to a 12 h
light–dark cycle, housed at 24 ± 2?C and 50 ± 10% relative humidity and fed
a Purina chow diet and water ad libitum. For short-term biomarker studies,
mice were divided into four groups, shaved on the dorsal side of the skin and
treated topically on the shaved area. The mice in the first group received
a topical application of 0.2 ml acetone and 0.1 ml dimethyl sulfoxide (DMSO),
and those in the second group received 1.6 lmol GUG/0.1 ml DMSO per
mouse. The mice in the third group received a topical application of 0.1 ml
acetone alone, and those in the fourth group received 0.8 lmol GUG/0.1 ml
DMSO per mouse. Thirty minutes after these treatments, the mice in group 3
and group 4 were treated with a single topical application of TPA (3.2 nmol/
0.1 ml acetone per mouse). At desired times after these treatments, the mice
Edema and epidermal hyperplasia
To assess the inhibitory effect of preapplication of GUG on TPA-induced
edema, 1 cm diameter punches of skin from vehicle-, GUG-, TPA- or GUG-
and TPA-treated animals were removed, made free of fat pads and weighed
quickly. After drying for 24 h at 50?C, the skin punches were reweighed, and
the loss of water content was determined. The difference in the amount of
water gain between the control (vehicle treated) and TPA treated represented
the extent of edema induced by TPA, whereas that between the control vehicle
and GUG plus TPA represented the inhibitory effect of GUG. For the hyper-
plasia study, skin was removed, fixed in 10% formalin and embedded in par-
affin. Vertical sections (5 lm) were cut, mounted on a glass slide and stained
with hematoxylin and eosin.
Preparation of cytosolic and nuclear lysates
Epidermis from the whole skin was separated as described earlier (17) and
was homogenized in ice-cold lysis buffer [50 mM Tris–HCl, 150 mM NaCl,
Abbreviations: COX-2, cyclooxygenase; DMBA, 7,12-dimethyl benz[a]an-
thracene; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic
acid; ERK, extracellular signal-regulated kinase; GUG, guggulsterone; iNOS,
inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; MAPK,
mitogen-activated protein kinases; ODC, ornithine decarboxylase; TPA, 12-
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1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1 mM ethylene-
diaminetetraacetic acid (EDTA), 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40,
1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride (pH 7.4)] with
freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III;
Calbiochem, La Jolla, CA). The homogenate was then centrifuged at 14 000g
for25min at4?C andthe supernatant (total celllysate)wascollected, aliquoted
and stored at ?80?C. For the preparation of nuclear and cytosolic lysates, 0.2 g
of the epidermis was suspended in 1 ml of cold buffer [10 mM N-2-hydrox-
yethylpiperazine-N#-2-ethanesulfonic acid (pH 7.9), 2 mM MgCl2, 10 mM
KCI, 1 mM dithiothreitol, 0.1 mM EDTA and 0.1 mM phenylmethylsulfonyl
fluoride] with freshly added protease inhibitor cocktail (Protease Inhibitor
Cocktail Set III; Calbiochem). After homogenization in a tight-fitting Dounce
homogenizer, the homogenateswere left on ice for10 min and thencentrifuged
at 25000g for 10 min. The supernatant was collected as cytosolic lysate and
stored at ?80?C. The nuclear pellet was resuspended in 0.1 ml of the buffer
containing 10 mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid
(pH 7.9), 300 mM NaCI, 50 mM KCI, 0.1 mM EDTA, 1 mM dithiothreitol,
0.1 mM phenylmethylsulfonyl fluoride and 10% glycerol with freshly added
protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbio-
chem). The suspension was gently shaken for 20 min at 4?C. After centri-
fugation at 25000g for 10 min, the nuclear extracts (supernatants) were
collected and quickly frozen at ?80?C. The protein content in the lysates
was measured by DC BioRad assay (Bio-Rad Laboratories) as per the man-
ODC enzyme activity
The epidermis from the dissected skin was separated as described earlier (17)
and homogenized at 4?C in a glass-to-glass homogenizer in 10 volumes of
ODC buffer [50 mM Tris–HCl buffer (pH 7.5) containing 0.1 mM EDTA,
and 0.1% Tween 80]. The homogenate was centrifuged at 100 000g at 4?C and
the supernatant was used for enzyme determination. ODC enzyme activity was
determined in epidermal cytosolic fraction by measuring the release of14CO2
fromthe D,L-[14C] ornithine by the method described earlier (17). Briefly, 400 ll
of the supernatant was added to 0.95 ml of the assay mixture [35 mM sodium
phosphate (pH 7.2), 0.2 mM pyridoxal phosphate, 4 mM dithiothreitol, 1 mM
EDTA and 0.4 mM L-ornithine containing 0.5 lCi of D,L-[1-14C] ornithine
hydrochloride] in 15 ml corex centrifuge tube equipped with rubber stoppers
in 2:1(vol/vol) ratio. After incubation at 37?C for 60 min, the reaction was
terminated by the addition of 1.0 ml of 2 M citric acid, using a 21G needle
per syringe. The incubation was continued for 1 h. Finally, the central well
containing the ethanolamine–methoxyethanol mixture to which14CO2has been
trapped was transferred to a vial containing 10 ml of toluene-based scintillation
SC liquid scintillation counter. Enzyme activity was expressed as picomoles
Western blot analysis
For western analysis, 25–50 lg of the protein was resolved .8–12% poly-
acrylamide gels and transferred to a nitrocellulose membrane. The blot con-
taining the transferred protein was blocked in blocking buffer for 1 h at room
temperature followedby incubationwithappropriatemonoclonalor polyclonal
primary antibody in blocking buffer for 1 h 30 min to overnight at 4?C. This
was followed by incubation with anti-mouse, anti-rabbit or anti-sheep second-
ary antibodies horseradish peroxidase for 1 h 30 min and then washed four
times with wash buffer and detected by chemiluminescence (ECL kit, Amer-
sham Life Sciences) and autoradiography using XAR-5 film obtained from
Eastman Kodak Co. (Rochester, NY).
Immunostaining for iNOS and COX-2
Mouse skin was fixed in 10% neutralized formalin and embedded in paraffin.
Five micormeter sections were cut, deparaffinized in xylol and rehydrated to
70% ethanol and washed in phosphate-buffered saline. For antigen retrieval,
sections were heated at 95?C for 30 min in citrate buffer (pH 6.0) and then
cooled for 20 min and washed in phosphate-buffered saline. Endogenous per-
oxidase was quenched by incubation in 0.3% hydrogen peroxide, for 20 min
and washed in washing buffer (phosphate-buffered saline plus Tween). Non-
specific binding sites were blocked by incubating the sections with goat serum
blocking solution for 1 h. Sections were incubated with primary antibody
against iNOS and COX-2 overnight at 4?C followed by incubation with spe-
cific horseradish peroxidase-labeled secondary antibody for 1 h at room tem-
perature. After washing in wash buffer, the sections were incubated with
diaminobenzidene peroxidase substrate solution for 2 min at room temperature
and rinsed with distilled water followed by counterstaining with Mayer’s
Hematoxylin solution. Sections were rinsed in tap water, dehydrated through
70–100% graded alcohol cleared in xylene and finally mounted in permanent
Female SENCAR mice were used in DMBA- and TPA-induced, two-stage
skin tumorigenesis protocol. The dorsal side of the skin was shaved using
electric clippers, and the mice with hair cycles in the resting phase were used
for tumor studies. In each group, 20 animals were used. Tumor induction was
initiated by a single topical application of 50 nmol DMBA in 200 ll acetone,
and 1 week later, the tumor growth was promoted with twice-weekly topical
applications of 3.2 nmol TPA in 200 ll acetone. Treatment with TPA alone or
with GUG plus TPA was repeated twice weekly up to the termination of the
experiments at 20 weeks. Animals in both the groups were watched for any
apparent signs of toxicity, such as weight loss or mortality during the entire
period of study. Skin tumor formation was recorded weekly, and tumors .1
mm in diameter were included in the cumulative number if they persisted for
2 weeks or more.
Microscopy and photography
Images from immunostaining experiments were obtained using a Zeiss Axioplot
microscope (Thornwood, NY) and Kodak Ektachrome 160T film (Rochester,
NY). These images were scanned (SprintScan; Polaroid, Cambridge, MA) and
Fig. 1. (A) Structure of GUG. Inhibitory effect of GUG on TPA-induced
skin edema and hyperplasia in SENCAR mice. (B) Twenty-four and 48 h
after TPA treatment, the skin edema was determined by weighing 1 cm
diameter punch skin as described in text. At least four determinations were
made at different dorsal skin sites per mouse in each group. The data
represent the mean ± SE of eight mice (?P , 0.01 versus TPA). (C) Twenty-
four and 48 h after treatment, the animals were killed; skin biopsies were
processed for hematoxylin and eosin staining. Representative pictures are
shown. Sections were photographed using an ?80 objective.
S.Sarfaraz et al.
formatted as Tag Image File Format images in Adobe Photoshop 6.0 software to
make the composite figures.
A two-tailed Student’s t-test was used to assess the statistical significance be-
tweentheTPA-treatedand GUGplus TPA-treated groups.A P value,0.05was
considered statistically significant. In tumorigenesis experiments, the statistical
significance of difference in terms of tumor incidence and multiplicity between
TPA and GUG plus TPA groups was evaluated by the Wilcoxon rank-sum test
and chi-square analysis. An advantage of Wilcoxon rank-sum test is that its
validity does not depend on any assumption about the shape of the distribution
of tumor multiplicities.
Inhibitory effect of GUG on TPA-induced cutaneous edema
Studies from our laboratory and others have shown that TPA appli-
cation to mouse skin results in cutaneous edema (17,18). In the
present study, we evaluated the protective effects of topical applica-
tion of GUG in TPA-mediated cutaneous edema in SENCAR mice.
We tested two doses 0.8 and 1.6 lmol of GUG per animal in our
preliminary studies. Since 0.8 lmol GUG did not exhibit any sig-
nificant effect on primary biomarkers of tumor promotion (data not
shown), therefore we selected dose of 1.6 lmol of GUG for further
studies. The SENCAR mice were topically treated with GUG (1.6
lmol per mouse) and 30 min later were topically treated with TPA
(3.2 nmol per mouse). As determined by theweight of 1 cm diameter
punch of the dorsal skin, application of TPA to SENCAR mouse skin
resulted in significant development of skin edema at 24 and 48 h
post-TPA treatment compared with control and GUG-treated groups
(Figure 1B). The skin application of GUG 30 min prior to that of
TPA application showed significant protection against TPA-induced
skin edema measured at 24 (51%; P , 0.01) and 48 (49%;
P , 0.01) h posttreatment. We found that topical application of
GUG alone to mice did not result in increase in skin edema at 24
and 48 h posttreatment (Figure 1B).
Inhibitory effect of GUG on TPA-induced epidermal hyperplasia
The effect of topical application of GUG on TPA-mediated induction
of epidermal hyperplasia was then assessed. As shown in Figure 1C,
topical application of TPA resulted in an increase in epidermal hy-
perplasia at 24 and 48 h after treatment when compared with control-
treated animals. The topical application of GUG, however, prior to
that of TPA application to mouse skin resulted in inhibition in the
induction of epidermal hyperplasia (Figure 1C). GUG alone did not
induce any epidermal hyperplasia as the histology of these animals
was comparable with that of control mice (Figure 1C).
Inhibitory effect of GUG on TPA-induced ODC activity
activity in SENCAR mice, groups of animals were treated topically
with GUG (1.6 lmol per animal) 30 min prior to topical application
of TPA (3.2 nmol per animal). TPA was applied in 0.2 ml acetone
and GUG was applied in 0.1 ml DMSO. As shown in Figure 2A,
pretreatment of animals with GUG resulted in inhibition of the
TPA-caused induction of epidermal ODC activity. GUG at a dose of
1.6 lmol per animal caused a 53% inhibition (P , 0.005) in the
epidermal ODC activity in mice treated with TPA. Topical application
of GUG alone (1.6 lmol per animal) was without any effect on basal
epidermal ODC activity.
Inhibitory effect of GUG on TPA-induced epidermal ODC protein
Next, we assessed the effect of skin application of GUG on TPA-
caused enhanced expression of ODC protein in the epidermis. West-
ern blotting revealed that at 24 h posttreatment of TPA, there was
maximum expression of epidermal ODC protein expression and it
gradually declined with the passage of time, (48 h post-TPA treat-
ment) (Figure 2B). Treatment of TPA caused a 3- to 4-fold increase in
epidermal ODC protein level as compared with acetone-treated con-
trol, whereas pretreatment of animals with GUG resulted in a signif-
icant inhibition against TPA-caused induction of epidermal ODC
protein expression at all time points investigated (Figure 2B). Densi-
tometric analysis of these blots indicated that, under experimental
conditions used, the inhibition varied from 45 to 50% in GUG-
Inhibitory effect of GUG on TPA-induced epidermal COX-2 and iNOS
COX-2 and iNOS are well-established biomarkers of inflammation
and tumor promotion. We next assessed the effect of skin applica-
tion of GUG on TPA-induced epidermal iNOS and COX-2 protein
expression. We found that topical application of TPA to SENCAR
mice resulted in an increase in epidermal COX-2 protein expres-
sion, which was maximum (5.1-fold) at 6 h post-TPA treatment
when compared with the control (Figure 3A). The TPA-caused in-
duction in the expression level of epidermal COX-2 gradually
declined with time, that is, at 24 and 48 h post-TPA treatment.
However, at all time points, the expression of COX-2 in mouse
skin following TPA application remained higher than correspond-
ing control group.
We further observed that topical application of TPA to SENCAR
mice resulted in a significant increase in the expression of epidermal
iNOS protein (Figure 3A). The expression of iNOS was observed to
reach its peak at 24 h post-TPA treatment. Topical application of
TPA alone caused 3- to 4-fold increase in iNOS protein expression in
Fig. 2. Inhibitory effect of GUG on TPA-induced epidermal ODC activity
and protein expression in SENCAR mice. (A) The animals were killed at 6 h
after TPA treatment, epidermal cytosolic fraction was prepared and ODC
activity was determined. The data are shown as ODC activity (pmol/h/mg
protein) and represent the mean ± SE of eight mice; each assay was
performed in duplicate (?P , 0.01 versus TPA). (B) At different time after
treatment, the animals were killed, epidermal lysates were prepared and
protein expression was determined as described in text. Equal loading was
confirmed by stripping the immunoblot and reprobing it for b-actin. The
immunoblots shown here are representative of three independent
experiments with similar results. The values above the figures represent
relative density of the band normalized to b-actin.
Guggulsterone inhibits skin tumorigenesis in SENCAR mice
mouse skin as compared with vehicle-treated controls at 6 and 24 h
posttreatment; however, pretreatment of GUG to the skin caused
inhibition against TPA-caused increases of iNOS protein expression.
Densitometric analysis of blots revealed that mice pretreated with
GUG (1.6 lmol per animal) showed 68% inhibition against TPA-
induced epidermal iNOS protein expression at 48 h posttreatment
(Figure 3A). The application of GUG alone at the dose of 1.6 lmol
did not produce any change in epidermal COX-2 and iNOS protein
expression when compared with vehicle-treated control animals.
Immunostaining analysis further validated these observations. As
evident from Figure 3B and C, there was a significant increase in
protein expressions of COX-2 and iNOS in the epidermis of TPA-
treated mice as compared with the untreated controls. In contrast,
GUG-treated mice showed a significant decrease in the expression
level of both proteins at the selected time points. This suggests that
GUG has the ability to suppress the tumor promoter activity of TPA
in the murine skin.
Inhibitory effect of GUG on TPA-mediated phosphorylation of
To determine whether TPA could induce activation of MAPKs un-
der in vivo condition in SENCAR mice, western blot analysis was
performed using phospho-specific MAPKs antibodies. In the pres-
ent study, as evident from western blot analysis, we found that
topical application of TPA resulted in an increased phosphorylation
of ERK1/2, JNK1/2 and p38 (Figure 4A). There was no effect on
the total amount of ERK1/2, JNK1/2 and p38 proteins after TPA or
GUG treatment (Figure 4A). We found that topical application of
TPA resulted in an increased phosphorylation of ERK1/2 (p44 and
p42) at 6 h post-TPA application that gradually decreases. The
effect of TPA application was more pronounced on phosphoryla-
tion of p38 at 24 h and JNK at 6 h for given post-TPA application;
then it gradually subsides but was still higher when compared with
control. Topical application of GUG prior to TPA application
Fig. 3. Inhibitory effect of GUG on TPA-induced epidermal COX-2 and iNOS protein expression in SENCAR mice. (A) At different time after treatment, the
animals were killed, epidermal lysates were prepared and protein expression was determined as described in text. Equal loading was confirmed by stripping the
immunoblot and reprobing it for b-actin. The immunoblots shown here are representative of three independent experiments with similar results. The values above
the figures represent relative density of the band. Immunostaining of (B) COX-2 and (C) iNOS. At different time after treatment, the animals were killed, the skin
punchbiopsieswere fixed in10%neutralizedformalinandembeddedin paraffin.Five micrometersectionswerecut, deparaffinizedin xylolandrehydratedto 70%
ethanol and washed in phosphate-buffered saline and immunostaining of COX-2 and iNOS was performed as detailed in text. A representative picture from three
independent immunostaining is shown. Scale bar 5 50 lm.
S.Sarfaraz et al.
resulted in inhibition of TPA-mediated phosphorylation of MAPKs
protein (Figure 4A).
Inhibitory effect of GUG on TPA-induced activation of NF-jB and
IKKa and phosphorylation and degradation of IjBa protein
Studies have shown that one of the critical events in NF-jB acti-
vation is its dissociation with subsequent degradation of inhibitory
protein IjBa via phosphorylation and ubiquitination (19–21). Ac-
tivation and nuclear translocation of NF-jB is preceded by the
phosphorylation and proteolytic degradation of IjBa (20,22). To
determine whether the inhibitory effect of GUG was attributable to
an effect on IjB degradation, we examined the cytoplasmic level of
IjBa protein expression by western blot analysis. We found that
TPA application to mouse skin resulted in the degradation of IjBa
protein expression at 6, 12 and 24 h after treatment. However,
topical application of GUG 30 min prior to TPA application re-
sulted in inhibition of TPA-induced degradation of IjBa protein
(Figure 4B). We next assessed whether TPA application affects the
phosphorylation of IjBa protein. As shown by western blot, TPA
induced a marked increase in the phosphorylation level of IjBa
protein at Ser (23) after treatment, which was inhibited by topical
application of GUG prior to TPA application (Figure 4B). Studies
have shown that IKKa activity is necessary for IjBa protein phos-
phorylation/degradation (24,25). To determine whether inhibition
of TPA-induced IKKa activation by GUG is attributable to sup-
pression of IjBa phosphorylation/degradation, we also measured
IKKa protein level. We found that TPA application resulted in the
activation of IKKa protein that in turn phosphorylated and
degraded IjBa protein. Topical application of GUG prior to TPA
application inhibited TPA-induced increase expression of IKKa
and phosphorylation of IKKa/b (Figure 4B). Next, we investigated
whether topical application of GUG inhibited TPA-induced activa-
tion and nuclear translocation of NF-jB/p65, the functionally
active subunit of NF-jB in mouse skin. In the nuclear fraction,
we found that TPA application onto the skin of SENCAR mice
resulted in the activation and nuclear translocation of NF-jB/p65.
However, topical application of GUG prior to TPA application in-
hibited TPA-induced NF-jB/p65 activation and nuclear translocation
Inhibitory effect of GUG on TPA-induced skin tumor promotion
We next assessed the effect of skin application of GUG on TPA-
induced skin tumor promotion in DMBA-initiated SENCAR mice.
As shown by data in Figure 5, topical application of GUG prior to
that of TPA in DMBA-initiated SENCAR mice skin resulted in an
inhibition of skin tumorigenesis. This inhibition was evident when
tumor data were considered as the percentage of mice with tumors
(Figure 5A), the number of tumors per group (Figure 5B) and the
number of tumors per mouse (Figure 5C). The animals pretreated
with GUG showed substantially reduced tumor incidence and lower
tumor body burden when assessed as total number of tumors per
group, percent of micewith tumors and number of tumors per animal
as compared with animals that did not receive GUG (Figure 5A). In
TPA-treated group, 100% of the mice developed tumors at 14 weeks
on test, whereas at this time in GUG-treated group, only 30% of the
mice exhibited tumors (Figure 5A). Skin application of GUG prior to
TPA application resulted in a delay in the latency period from 5 to 11
weeks and afforded protection when tumor data were considered in
terms of tumor incidence and tumor multiplicity throughout the
treatment period (P , 0.05, chi-square test). At the termination of
the experiment at 20 weeks, 50% of the mice were tumor free in the
group that received skin application of GUG prior to each TPA
application. At the termination of the experiment at 20 weeks on
test, compared with a total of 212 tumors in TPA-treated group
of animals, only 66 tumors in GUG-treated group were recorded
(Figure 5B). Compared with the non-GUG-treated group, such
decrease in the total number of tumors in the GUG-treated group
corresponded to 69% inhibition. When these tumor data were con-
sidered in terms of number of tumors per mice, at the termination
of the experiment at 20 weeks on test, compared with 10.6 tumors
per mouse in TPA-treated group of animals, only 3.3 tumors per
mouse in GUG-treated group were recorded (Figure 5C). Compared
with the non-GUG-treated group, such decrease in the number of
tumor per mouse in the GUG-treated group corresponded to 69%
Fig. 4. Inhibitory effect of GUG on TPA-induced phosphorylation of
MAPKs, activation of NF-jB and IKKa/b, phosphorylation and degradation
of IjBa in SENCAR mice. (A and B) At different time after treatment, the
animals were killed, epidermal cytosolic and nuclear lysates were prepared
and protein expression was determined as described in text. Equal loading
was confirmed by stripping the immunoblot and reprobing it for b-actin. The
immunoblots shown here are representative of three independent
experiments with similar results. The values above the figures represent
relative density of the band normalized to b-actin.
Guggulsterone inhibits skin tumorigenesis in SENCAR mice
Cancer chemoprevention has become an important area of cancer
research, which, in addition to providing a practical approach to iden-
tifying potentially useful inhibitors of cancer development, also af-
carcinogenesis (26,27). One excitement of chemoprevention is that
agents can be targeted for intervention either at the initiation, pro-
motion or progression stage of multistage carcinogenesis. The inter-
vention of cancer at the promotion stage appears to be the most
appropriate and practical. The major reason for this relates to the fact
that tumor promotion is a reversible event at least in early stages and
requires repeated and prolonged exposure of a promoting agent (28).
For this reason, it is important to identify mechanism-based effective
novel antitumor-promoting agents. It is appreciated that those agents,
which have the ability to intervene at more than one critical pathway
in the carcinogenic process, will have greater advantage over other
single-target agents. This study was designed to show the chemopre-
ventive potential of GUG by using carcinogenesis-associated bio-
chemical endpoints in a mouse skin tumorigenesis model. The
topical application of TPA to mouse skin or its treatment in certain
epidermal cells is known to result in a number of biochemical alter-
ations, changes in cellular functions and histologic changes leading
to skin tumor promotion (13,24,29). Our data clearly demonstrate
that topical application of GUG prior to TPA application affords
significant inhibition of TPA-induced skin edema and hyperplasia
(Figure 1B and C).
ODC, the first and the rate-limiting enzyme in the biosynthesis of
polyamines, plays an important role in the regulation of cell prolifer-
ation and development of cancer (30). Studies with the mouse skin
model have shown an excellent correlation between the induction of
ODC activity and the tumor-promoting ability of a variety of substan-
ces (31,32). Several lines of evidence indicate that aberrations in ODC
regulation and subsequent polyamine accumulation are intimately
associated with neoplastic transformation (33,34). Elevated levels of
ODC gene products are consistently detected in transformed cell
lines, virtually all animal tumors and in certain tissues predisposed
to tumorigenesis (33). Agents that block induction of ODC can pre-
vent tumor formation; therefore, its inhibition was shown to be
a promising tool for screening inhibitors of tumorigenesis (35,36).
In the present study, topical application of GUG prior to that of
TPA resulted in a significant inhibition of TPA-mediated induction
of epidermal ODC activity (Figure 2A). It is reasonable to believe that
GUG application inhibited the action ofthe tumorpromoter and/or the
enzymatic pathways that regulates the ODC induction rather than
interacting directly with the enzyme. In addition, our data obtained
from western blot analysis demonstrate that prior application of GUG
to that of TPA showed an inhibitory effect of GUG against TPA-
induced increases inthe levelsof epidermal ODCprotein inthe mouse
skin (Figure 2B). The magnitude of the inhibitory effect of topical
application of GUG on TPA-induced increases in ODC protein ex-
pression seems to be similar to that for inhibition of TPA-induced
increases in ODC enzyme activity.
Tumor promotion is closely linked to inflammation and oxidative
stress, and it is probable that compounds that have anti-inflammatory
and antioxidative properties act as antitumor promoters as well (37).
COX-2 isoform and iNOS are important enzymes involved in medi-
ating the inflammatory process (38,39). COX-2 and iNOS have been
reported to play an important role in cutaneous inflammation, cell
proliferation and skin tumor promotion (40,41). There is considerable
body of compelling evidence that inhibition of COX-2 and iNOS
expression or activity is important for not only alleviating inflamma-
tion but also for the prevention of cancer (41). Previous studies have
demonstrated that GUG inhibits cytokine-induced COX-2 expression
and NF-jB activation (42). In this study, we showed the inhibitory
effects of GUG against TPA-caused induction of epidermal COX-2
and iNOS protein expression in SENCAR mice (Figure 3). These
inhibitory effects also correlate with the inhibitory effect of GUG
against TPA-caused induction of skin edema (Figure 1B) and hyper-
plasia (Figure 1C). These inhibitory effects of GUG against TPA-
mediated responses in the mouse skin suggest that the primary effect
of GUG may be against inflammatory responses, which may then
result in the inhibition of tumor promotion.
Fig. 5. Inhibitory effect of GUG on DMBA-initiated and TPA-promoted
tumor formation in SENCAR mice. In each group, 20 animals were used.
Tumorigenesis was initiated in the animals by a single topical application of
50 nmol DMBA in 0.2 ml vehicle on the dorsal shaved skin, and 1 week later,
the tumor growth was promoted with twice-weekly applications of 3.2 nmol
TPA in 0.2 ml vehicle. To assess its anti-skin tumor-promoting effect, GUG
at a dose of0.5 mg peranimal wasappliedtopically30 min prior to each TPA
application in different groups. Treatment with TPA alone or GUG plus TPA
was repeated twice weekly up to the termination of the experiments at 20
weeks. Animals in all the groups were watched for any apparent signs of
toxicity, such as weight loss or mortality during the entire period of study.
Skin tumor formation was recorded weekly, and tumors .1 mm in diameter
were included in the cumulative number only if they persisted for 2 weeks or
more. The tumor data are represented as (A) the percentage of mice with
tumors, (B) the number of tumors per group and (C) the number of tumors
per mouse. The data were analyzed by Wilcoxon rank-sum test and chi-
S.Sarfaraz et al.
MAPKs constitute a superfamily of proteins that include ERK1/2,
JNK1/2 and p38 kinase (15,27). The involvement of MAPKs pathway
in tumor proliferation is well documented. Activation of the MAPKs
pathway occurs in response to integrin-mediated cellular adhesion to
the extracellular matrix, which plays a critical role in both tumor
metastasis and angiogenesis (43,44). In the present study, employing
western blot analysis, we found that topical application of TPA re-
sulted in a marked increase in the phosphorylated form of ERK1/2,
JNK1/2 and p38 protein expression. Importantly, topical application
of GUG prior to TPA application was found to inhibit TPA-mediated
phosphorylation of MAPKs (Figure 4A). Several studies have shown
that JNK pathway plays a major role in cellular function, such as cell
proliferation and transformation (45), whereas the ERK pathway sup-
presses apoptosis and enhances cell survival or tumorigenesis (46).
Studies have shown that ERK1/2 and p38 are involved in the tran-
scriptional activation of NF-jB (47,48). NF-jB has emerged as one of
the most promising molecular targets in the prevention of cancer. We
nextinvestigated the effect ofGUG on the pattern ofNF-jB activation
and its nuclear translocation by TPA in SENCAR mice skin. NF-jB
resides in the inactive state in the cytoplasm as a heterotrimer con-
sisting of p50, p65 and IjBa subunits. An IjBa kinase, IKKa, phos-
phorylates serine residues in IjBa at position 32 and 36 (49). Upon
phosphorylation and subsequent degradation of IjBa, NF-jB acti-
vates and translocates to the nucleus, where it binds to DNA and
activates the transcription of various genes (49,50). Several lines of
evidence suggest that proteins from the NF-jB and IjB families are
involved in carcinogenesis. Studies have also shown that NF-jB ac-
tivity affects cell survival and determines the sensitivity of cancer
cells to cytotoxic agents as well as ionizing radiation (51). Shishodia
et al. (42) have shown that GUG inhibits tumor necrosis factor-
induced IKK activity and also suppresses inducible and constitutive
NF-jB activation without directly affecting binding of NF-jB to
DNA. In the present study, we have demonstrated that topical appli-
cation of TPA to mouse skin resulted in activation and nuclear trans-
location of NF-jB/p65 (Figure 4B). We also found that TPA
application to mouse skin resulted in an increased expression of IKKa
and phosphorylation and degradation of IjBa protein (Figure 4B).
Interestingly, we observed that topical application of GUG prior to
TPA application to mouse skin inhibited TPA-induced NF-jB/p65
and IKKa activation and phosphorylation and degradation of IjBa
protein (Figure 4B). Because GUG inhibited IjBa phosphorylation
and degradation,this study suggests that the effect of GUG on NF-jB/
p65 is through inhibition of phosphorylation and subsequent proteol-
ysis of IjBa. Since GUG contains two a,b-unsaturated carbonyl moi-
ety, it seems that the compound may cause cysteine thiol modification
in key molecule such as IKK of the NF-jB-signaling pathway (52).
The results in Figure 5 show the protective effects of skin applica-
tion of GUG on TPA-caused tumor promotion in DMBA-initiated
SENCAR mouse skin. The preapplication of GUG to that of TPA
showed substantially reduced tumor incidence and lower tumor body
burden when assessed as total number of tumors per group, percent of
micewith tumors and number of tumors per animal, as compared with
animals that did not receive GUG (Figure 5). These chemopreventive
and antitumor promotion observations in murine skin by GUG can be
explained by the biochemical mechanisms examined in the present
In summary, our results suggest that topical application of GUG
prior to TPA application to SENCAR mice resulted in a significant
decrease in skin edema, hyperplasia, epidermal ODC activity and
protein expression of ODC, COX-2 and iNOS classical markers of
inflammation and tumor promotion. In addition, our results also sug-
gest that topical application of GUG prior to TPA application also
resulted in inhibition of phosphorylation of MAPKs, activation of
NF-jB/p65 and IKKa/b and degradation and phosphorylation of
IjBa. Our data clearly demonstrate that GUG could be a potent
antitumor-promoting agent because it inhibits several biomarkers of
TPA-induced tumor promotion in an invivo animal model. One might
envision the use of chemopreventive agents such as GUG in an
emollient or patch for chemoprevention or treatment of skin cancer.
United States Public Health Service (R01 CA 78809, R01CA 101039,
R01 CA 120451, R21 AT 002429).
Conflict of Interest Statement: None declared.
1.Gujral,M.L. et al. (1960) Antiarthritic and anti-inflammatory activity of
gum guggul (Balsamodendron mukul Hook). Indian J. Physiol. Pharma-
col., 4, 267–273.
2.Sharma,J.N. et al. (1977) Comparison of the anti-inflammatory activity of
Commiphora mukul (an indigenous drug) with those of phenylbutazone and
ibuprofen in experimental arthritis induced by mycobacterial adjuvant.
Arzneimittelforschung, 27, 1455–1457.
3.Sinal,C.J. et al. (2002) Guggulsterone: an old approach to a new problem.
Trends Endocrinol. Metab., 13, 275–276.
4.Urizar,N.L. et al. (2003) GUGULIPID: a natural cholesterol-lowering
agent. Annu. Rev. Nutr., 23, 303–313.
5.Tripathi,Y.B. et al. (1988) Thyroid stimulatoryaction of (Z)-guggulsterone:
mechanism of action. Planta. Med., 4, 271–277.
6.Singh,B.B. et al. (2003) The effectiveness of Commiphora mukul for
osteoarthritis of the knee: an outcomes study. Altern. Ther. Health
Med., 9, 74–79.
7.Singh,S.V. et al. (2007) Guggulsterone-induced apoptosis in human
prostate cancer cells is caused by reactive oxygen intermediate depen-
dent activation of c-Jun NH2-terminal kinase. Cancer Res., 67, 7439–
8.Shishodia,S. et al. (2007) Guggulsterone inhibits tumor cell proliferation,
induces S-phase arrest, and promotes apoptosis through activation of c-Jun
N-terminal kinase, suppression of Akt pathway, and downregulation of
antiapoptotic gene products. Biochem. Pharmacol., 74, 118–130.
9.Slaga,T.J. et al. (1982) Studies on the mechanisms involved in multistage
carcinogenesis in mouse skin. J. Cell. Biochem., 18, 99–119.
10.Katiyar,S.K. et al. (1997) Inhibition of phorbol ester tumor promoter 12-
O-tetradecanoylphorbol-13-acetate-caused inflammatory responses in
SENCAR mouse skin by black tea polyphenols. Carcinogenesis, 18,
11.Katiyar,S.K. et al. (1996) Inhibition of tumor promotion in SENCAR
mouse skin by ethanol extract of Zingiber officinale rhizome. Cancer
Res., 56, 1023–1030.
12.Katiyar,S.K. et al. (1995) Inhibition of 12-O-tetradecanoylphorbol-13-ac-
etate and other skin tumor-promoter-caused induction of epidermal inter-
leukin-1 alpha mRNA and protein expression in SENCAR mice by green
tea polyphenols. J. Invest. Dermatol., 105, 394–398.
13.Chun,K.S. et al. (2002) Effects of yakuchinone A and yakuchinone B
on the phorbol ester-induced expression of COX-2 and iNOS and activa-
tion of NF-jB in mouse skin. J. Environ. Pathol. Toxicol. Oncol., 21, 131–
14.Seo,H.J. et al. (2002) Inhibitory effects of the standardized extract (DA-
9601) of Artemisia asiatica Nakai on phorbol ester-induced ornithine de-
carboxylase activity, papilloma formation, cyclooxygenase-2 expression,
inducible nitric oxide synthase expression and nuclear transcription factor
kappa B activation in mouse skin. Int. J. Cancer, 100, 456–462.
15.Afaq,F. et al. (2003) Suppression of UVB-induced phosphorylation of mi-
togen-activated protein kinases and nuclear factor kappa B by green tea
polyphenol in SKH-1 hairless mice. Oncogene, 22, 9254–9264.
16.Shishodia,S. et al. (2003) Ursolic acid inhibits nuclear factor-kappaB acti-
vation induced by carcinogenic agents through suppression of IkappaBal-
pha kinase and p65 phosphorylation: correlation with down-regulation of
cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1. Cancer Res.,
17.Afaq,F. et al. (2005) Anthocyanin-and hydrolyzable tannin-rich pomegran-
ate fruit extract modulates MAPK and NF-jB pathways and inhibits skin
tumorigenesis in CD-1 mice. Int. J. Cancer, 113, 423–433.
18.Saleem,M. et al. (2004) Lupeol modulates NF-jB and PI3K/Akt pathways
and inhibits skin cancer in CD-1 mice. Oncogene, 23, 5203–5214.
19.Takada,Y. et al. (2004) Flavopiridol inhibits NF-jB activation induced
by various carcinogens and inflammatory agents through inhibition of
Ikappa Balpha kinase and p65 phosphorylation: abrogation of cyclin
D1, cyclooxygenase-2 and matrix metalloprotease-9. J. Biol. Chem.,
Guggulsterone inhibits skin tumorigenesis in SENCAR mice
20.Bharti,A.C. et al. (2002) Chemopreventive agents induce suppression of Download full-text
nuclear factor-kappaB leading to chemosensitization. Ann. N. Y. Acad. Sci.,
21.Israel,A. (1995) A role for phosphorylation and degradation in the control
of NF-kappa B activity. Trends Genet., 11, 203–205.
22.Afaq,F. et al. (2003) Inhibition of ultraviolet B-mediated activation of nu-
clear factor kappaB in normal human epidermal keratinocytes by green tea
constituent (-)-epigallocatechin-3-gallate. Oncogene, 22, 1035–1044.
23.Tanaka,T. et al. (1986) Tannins and related compounds: XLI, isolation and
characterization of novel ellagitannins, puicacorteins A, B, C and D, and
punigluconin from the bark of Punicum granatum. L. Chem. Pharm. Bull.,
24.Baldwin,A.S.Jr (1996) The NF-kappa B and I kappa B proteins: new dis-
coveries and insights. Annu. Rev. Immunol., 14, 649–683.
25.Maniatis,T. (1997) Catalysis by a multiprotein IkappaB kinase complex.
Science, 278, 818–819.
26.Bickers,D.R. et al. (2000) Novel approaches to chemoprevention of skin
cancer. J. Dermatol., 27, 691–695.
27.Ding,M. et al. (2004) Inhibition of AP-1 and neoplastic transformation by
fresh apple peel extract. J. Biol. Chem., 279, 10670–10676.
28.DiGiovanni,J. (1991) Modification of multistage carcinogenesis. In Ito,N.
and Sugano,H. (eds.) Modification of Tumor Development in Rodents:
Progress in Experimental Tumor Research. Vol. 33. Karger, Basel, Switzer-
land, pp. 192–229.
29.Katiyar,S.K. et al. (1997) Protective effects of silymarin against photocar-
cinogenesis in a mouse skin model. J. Natl Cancer Inst., 89, 556–566.
30.Thomas,T. et al. (2003) Polyamine metabolism and cancer. J. Cell. Mol.
Med., 7, 113–126.
31.Einspahr,J.G. et al. (2003) Skin cancer chemoprevention: strategies to save
our skin. Recent Results Cancer Res., 163, 151–164.
32.Ahmad,N. et al. (2001) A definitive role of ornithine decarboxylase in
photocarcinogenesis. Am. J. Pathol., 159, 885–892.
33.Auvinen,M. (1997) Cell transformation, invasion, and angiogenesis: a reg-
ulatory role for ornithine decarboxylase and polyamines? J. Natl Cancer
Inst., 89, 533–537.
34.Mohan,R.R. et al. (1999) Overexpression of ornithine decarboxylase in
prostate cancerandprostatic fluid inhumans.Clin.CancerRes., 5,143–147.
35.Verma,A.K. et al. (1979) Correlation of the inhibition by retinoids of tumor
promoter-induced mouse epidermal ornithine decarboxylase activity and of
skin tumor promotion. Cancer Res., 39, 419–425.
36.Nakadate,T. et al. (1985) Inhibition of teleocidin-caused epidermal orni-
thine decarboxylase induction by phospholipase A2-, cyclooxygenase- and
lipoxygenase-inhibitors. Jpn. J. Pharmacol., 37, 253–258.
37.Bhimani,R.S. et al. (1993) Inhibition of oxidative stress in HeLa cells by
chemopreventive agents. Cancer Res., 53, 4528–4533.
38.Herschman,H.R. (1994) Regulation of prostaglandin synthase-1 and pros-
taglandin synthase-2. Cancer Metastasis Rev., 13, 241–256.
39.Smith,W.L. et al. (1996) Prostaglandin endoperoxide H synthases (cyclo-
oxygenases)-1 and -2. J. Biol. Chem., 271, 33157–33160.
40.Furstenberger,G. et al. (1985) In Fischer,S.M. and Slaga,T.J. (eds.) Arach-
idonic Acid Metabolism and Tumor Promotion. Martinus Nijhoff Publish-
ing, Boston, MA, pp. 49–72.
41.Kim,D.J. et al. (2003) Chemoprevention of colon cancer by Korean food
plant components. Mutat. Res., 523–524, 99–107.
42.Shishodia,S. et al. (2004) Guggulsterone inhibits NF-jB and IkappaBalpha
kinase activation, suppresses expression of anti-apoptotic gene products,
and enhances apoptosis. J. Biol. Chem., 279, 47148–47158.
43.Chen,W. et al. (1998) UVB irradiation-induced activator protein-1 activa-
tion correlates with increased c-fos gene expression in a human keratino-
cyte cell line. J. Biol. Chem., 273, 32176–32181.
44.Zhu,W.H. et al. (2002) Regulation of angiogenesis by vascular endothelial
growth factor and angiopoietin-1 in the rat aorta model: distinct temporal
patterns of intracellular signaling correlate with induction of angiogenic
sprouting. Am. J. Pathol., 161, 823–830.
45.Potapova,O. et al. (2000) c-Jun N-terminal kinase is essential for growth of
human T98G glioblastoma cells. J. Biol. Chem., 275, 24767–24775.
46.Huang,C. et al. (1998) Shortage of mitogen-activated protein kinase is
responsible for resistance to AP-1 transactivation and transformation in
mouse JB6 cells. Proc. Natl Acad. Sci. USA, 95, 156–161.
47.Adderley,S.R. et al. (1999) Oxidative damage of cardiomyocytes is limited
by extracellular regulated kinases 1/2-mediated induction of cyclooxyge-
nase-2. J. Biol. Chem., 274, 5038–5046.
48.Carter,A.B. et al. (1999) The p38 mitogen-activated protein kinase is re-
quired for NF-jB-dependent gene expression: the role of TATA-binding
protein (TBP). J. Biol. Chem., 274, 30858–30863.
49.Karin,M. et al. (2000) Phosphorylation meets ubiquitination: the control of
NF-[kappa]B activity. Annu. Rev. Immunol., 18, 621–663.
50.Garg,A. et al. (2002) Nuclear transcription factor-kappaB as a target for
cancer drug development. Leukemia, 16, 1053–1068.
51.Epinat,J.C. et al. (1999) Diverse agents act at multiple levels to inhibit the
Rel/NF-jB signal transduction pathway. Oncogene, 18, 6896–6909.
52.Na,H.K. et al. (2006) Transcriptional regulation via cysteine thiol modifi-
cation: a novel molecular strategy for chemoprevention and cytoprotection.
Mol. Carcinog., 45, 368–380.
Received March 25, 2008; revised July 29, 2008; accepted July 30, 2008
S.Sarfaraz et al.