Download Peracetylated (−)-epigallocatechin-3-gallate

Carcinogenesis vol.34 no.6 pp.1315–1322, 2013
doi:10.1093/carcin/bgt042
Advance Access publication February 5, 2013
Peracetylated (−)-epigallocatechin-3-gallate (AcEGCG) potently prevents skin
carcinogenesis by suppressing the PKD1-dependent signaling pathway in CD34+ skin
stem cells and skin tumors
Yi-Shiou Chiou1,5, Shengmin Sang2, Kuang-Hung Cheng3,
Chi-Tang Ho4, Ying-Jan Wang5 and Min-Hsiung Pan1,*
1
Department of Seafood Science, National Kaohsiung Marine University,
Kaohsiung 811, Taiwan, 2Center for Excellence in Post-Harvest
Technologies, North Carolina Agricultural and Technical State University,
North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081,
USA, 3Institute of Biomedical Sciences, National Sun Yat-Sen University,
Kaohsiung 80424, Taiwan, 4Department of Food Science, Rutgers University,
New Brunswick, NJ 08901-8520, USA and 5Department of Environmental
and Occupational Health, National Cheng Kung University Medical College,
Tainan 704, Taiwan
*To whom correspondence should be addressed. Department of Seafood
Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd,
Nan-Tzu, Kaohsiung, Taiwan. Tel: +886-7-361-7141; Fax: +886-7-361-1261;
Email: [email protected]
Correspondence may also be addressed to Ying-Jan Wang. Department of
Environmental and Occupational Health, National Cheng Kung University
Medical College, 138 Sheng-Li Road, Tainan 70428, Taiwan. Tel: +886-6235-3535 ext. 5804; Fax: +886-6-2752484;
Email: [email protected]
During the process of skin tumor promotion, expression of the
cutaneous cancer stem cell (CSC) marker CD34+ is required for
stem cell activation and tumor formation. A previous study has
shown that activation of protein kinase D1 (PKD1) is involved in
epidermal tumor promotion; however, the signals that regulate
CSCs in skin carcinogenesis have not been characterized. This
study was designed to investigate the chemopreventive potential of peracetylated (−)-epigallocatechin-3-gallate (AcEGCG)
on 7,12-dimethylbenz[a]-anthracene (DMBA)-initiated and
12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin
tumorigenesis in ICR mice and to elucidate the possible mechanisms involved in the inhibitory action of PKD1 on CSCs.
We demonstrated that topical application of AcEGCG before
TPA treatment can be more effective than EGCG in reducing
DMBA/TPA-induced tumor incidence and multiplicity. Notably,
AcEGCG not only inhibited the expression of p53, p21, c-Myc,
cyclin B, p-CDK1 and Cdc25A but also restored the activation
of extracellular signal-regulated kinase 1/2 (ERK1/2), which
decreased DMBA/TPA-induced increases in tumor proliferation
and mitotic index. To clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the expression and activation
of PKD1 in CD34+ skin stem cells and skin tumors. We found
that PKD1 was strongly expressed in CD34+ cells and that pretreatment with AcEGCG markedly inhibited PKD1 activation
and CD34+ expression. More importantly, pretreatment with
AcEGCG remarkably suppressed nuclear factor-kappaB, cyclic
adenosine 3′,5′-monophosphate-responsive element-binding protein (CREB) and CCAAT-enhancer-binding protein (C/EBPs)
activation by inhibiting the phosphorylation of c-Jun-N-terminal
kinase 1/2, p38 and phosphatidylinositol 3-kinase (PI3K)/Akt
and by attenuating downstream target gene expression, including inducible nitric oxide synthase, cyclooxygenase-2, ornithine
Abbreviations: AcEGCG, peracetylated (−)-epigallocatechin-3-gallate;
COX-2, cyclooxygenase-2; CREB, cAMP-responsive element-binding protein; CSC, cancer stem cell; DMBA, 7,12-dimethylbenz[a]anthracene; EBP,
enhancer-binding protein; EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; H&E, hematoxylin and eosin; iNOS, inducible
nitric oxide synthase; IκB, inhibitor κB; NF-κB, nuclear factor-κB; ODC,
ornithine decarboxylase; PI3K, phosphatidylinositol 3-kinase; PKD1, protein kinase D-1; TPA, 12-O-tetradecanoylphorbol-13-acetate; VEGF, vascular
endothelial growth factor.
decarboxylase and vascular endothelial growth factor. Moreover,
this is the first study to demonstrate that AcEGCG is a CD34+
and PKD1 inhibitor in the multistage mouse skin carcinogenesis
model. Overall, our results powerfully suggest that AcEGCG
could be developed into a novel chemopreventive agent and that
PKD1 may be a preventive and therapeutic target for skin cancer
in clinical settings.
Introduction
The incidence of skin cancer is rising steadily and presents a growing health problem. The development of skin cancer is a multistage
process including initiation, promotion and progression. The functions of cancer stem cells (CSCs) and keratinocyte stem cells play
critical roles during the process of skin carcinogenesis (1). Recent
evidence suggests that the cell surface glycoprotein, CD34+, a stem
and progenitor cell marker, is expressed in tumor epithelial cells and
is required for skin tumor development in mice (2). In addition to
CD34, PKD1 (protein kinase D1) also plays an important role in
keratinocyte functions that regulate proliferative and antidifferentiative processes (3). Similarly, Ristich et al. (4) found that aberrant
distribution and upregulation of PKD1 are associated with neoplastic
mouse keratinocyte and basal cell carcinoma development, suggesting a putative tumorigenic role for PKD1 in the skin. The PKD1 gene
is a novel serine/threonine kinase originally categorized as a member
of the protein kinase C (PKC) family (and designated PKCμ) because
of its two cysteine-rich domains that bind diacylglycerol and phorbol
esters (5). Since its discovery, PKD1 has been shown to interact with
and modulate the functions of proteins implicated in variety of signaling pathways, including proliferation, survival, apoptosis, angiogenesis and motility (6). Although PKD1 is known to regulate skin tumor
promotion, its role in regulating cancer stemness and the resulting
skin tumor progression remains unknown. In this study, we sought
to identify the expression machinery critical for PKD1 activation and
skin CSC proliferation.
The two-stage model of mouse skin carcinogenesis has been
used for studying mechanisms of chemical carcinogenesis and
evaluating novel skin cancer prevention strategies. In the mouse
model of skin cancer, tumor initiation can be induced by a single
topical application of 7,12-dimethylbenz[a]-anthracene (DMBA), a
mutagenic chemical initiator that acts on the H-Ras proto-oncogene.
Tumor promotion follows and is produced by the repeated application
of 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter
that leads to tumor development (7). In this model, all of the stages
of carcinogenesis can be targeted for chemopreventive intervention
(8). Epidemiological and clinical studies have suggested that the
consumption of green tea lowers the risk of cancer. (−)-Epicatechin3-gallate (EGCG), the major natural polyphenolic compound in
green tea, exhibits skin cancer-preventive activity in various in vitro
and in vivo models (9). However, EGCG is hydrophilic and exhibits
poor cellular absorption, low bioavailability and low stability, which
lead to a reduction in its cancer-preventive activity. To increase the
lipophilicity and membrane permeability and improve the bioefficacy
of EGCG, peracetylated EGCG (AcEGCG) was synthesized (10).
Previous studies have demonstrated that AcEGCG acts as a prodrug of
EGCG with greater potency than EGCG in the inhibition of proteasome
activity, tumor proliferation and inflammation. In addition, AcEGCG
has been shown to induce apoptosis in vitro and in vivo (11–13). Studies
from our laboratory have demonstrated that AcEGCG is more potent
than EGCG for the treatment of colitis-induced colon carcinogenesis
© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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(14). However, the specific regulatory mechanisms of AcEGCG at the
level of target proteins (CD34 or PKD1) are not known for the skin
carcinogenesis process. In this research, we compared the effects of
EGCG and AcEGCG on DMBA/TPA-mediated skin tumorigenesis
and explored the underlying molecular mechanisms of signaling. We
examined whether AcEGCG can be used as a non-selective PKD1 and
CD34 inhibitor and also investigated the role of PKD1 in proliferative
CD34-expressing CSCs.
In summary, our results reveal for the first time that AcEGCG is a
more potent chemopreventive agent than EGCG for the prevention of
DMBA/TPA-induced skin carcinogenesis. This activity appears to be
mediated through a blockade of nuclear factor-κB (NF-κB), CCAATenhancer-binding protein β and δ (C/EBPβ and C/EBPδ) activation
via the downregulation of PKD1 signaling, as well as several downstream targets, including c-Jun-N-terminal kinase 1/2, p38, PI3K/Akt
and cell cycle-regulated signaling pathways. To our knowledge, this
is the first study to show that PKD1 is a novel regulator in cutaneous
CD34+ CSCs during DMBA/TPA-mediated tumorigenesis. Further
understanding of the role of PKD1 may contribute to new preventive
and therapeutic strategies in inflammation-related tumorigenesis.
Materials and methods
Reagents and antibodies
TPA and DMBA were purchased from Sigma Chemical Co. (St Louis, MO).
EGCG (100% pure) was isolated from a crude green tea polyphenol extract
and provided by Dr Chi-Tang Ho (Department of Food Science, Rutgers
University). AcEGCG was provided by Dr Shengmin Sang (North Carolina
Agricultural and Technical State University). Briefly, AcEGCG was synthesized using a pyridine catalyzed reaction of EGCG with acetic anhydride
(15). Antibodies against iNOS, IκBα, p-IκBα, p65, p50, vascular endothelial
growth factor (VEGF), MMP-9, C/EBPβ, C/EBPδ, phospho-PI3K (Tyr508)
and β-actin were purchased from Santa Cruz Biotechnology, CA. Anti-COX-2
and ornithine decarboxylase (ODC) monoclonal antibodies were purchased
from BD Transduction Laboratories, Lexington, KY, Taiwan. Phospho-p65
(Ser536), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-JNK1/2
(Thr183/Tyr185), ERK, JNK1/2, p38, p-PKD1 (Ser916 and 744/748), P53,
P21, c-Myc, cyclin B, CDK-1, cdc25A and PKD1 were purchased from Cell
Signaling Technology, Beverly, MA. p-IκBα (Ser32/Ser36), p-Akt (Ser473)
and Akt polyclonal antibodies were purchased from Upstate Biotechnology,
Lake Placid, NY. CD34 and PKD1 polyclonal antibodies were purchased from
Bioss.
Animals care and two-stage tumorigenesis in mouse skin
Male ICR mice at 6 weeks of age were purchased from the BioLASCO
Experimental Animal Center (Taiwan Co., Ltd). After 1 week of acclimation, animals were randomly distributed into control and experimental groups.
All animals were housed in a controlled atmosphere (25 ± 1°C at 50% relative humidity) under a 12 h light/12 h dark cycle. Animals had free access
to food and water at all times. Food cups were replenished with fresh diet
every day. All animal experimental protocol used in this study was approved
by Institutional Animal Care and Use Committee of the National Kaohsiung
Marine University (IAC UC, NKMU). One group was composed of 12 female
ICR mice. These mice were given commercial rodent pellets and fresh tap
water ad libitum, both of which were changed twice a week. The dorsal region
of each mouse was shaved with an electric clipper 2 days before initiation.
Mice at 6 weeks old were started on 200 nM DMBA in 200 μl acetone (Ac)
and control mice received 200 μl acetone alone. One week after initiation, the
mice were topically treated with 200 μl acetone or promoted with TPA (5 nM
in 200 μl acetone) twice a week for 20 weeks. For the other two groups, the
mice were treated with EGCG or AcEGCG (1 and 5 μΜ in 200 μl acetone)
30 min before each TPA treatment. Tumors of at least 1 mm of diameter in an
electronic digital caliper were counted and recorded twice every week and the
diameters of skin tumors were measured at the same time. The results were
expressed as the average number of tumors per mouse, percentage of tumorbearing mice, weight of tumors per mouse and size distribution per mouse.
Western blot analysis
For protein isolation from mouse skin, the dorsal skins of mice derived from different experiments were excised. After the fat from the dorsal skin was removed
on ice, the skin samples were immediately placed in liquid nitrogen. The epidermal and tumor protein were homogenized on ice for 15 s using a Polytron tissue
homogenizer and then lysed in 0.5 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4,
1 mM NaF, 150 mM NaCl, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic
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acid, 1 mM phenylmethane-sulfonyl fluoride, 1% NP-40 and 10 μg/ml leupeptin) on ice for 30 min, followed by centrifugation at 10 000g for 30 min at 4°C.
The samples (50 μg of protein) were mixed with 5× sample buffer containing
0.3 M Tris-HCl (pH 6.8), 25% 2-mercapto-ethanol, 12% sodium dodecyl sulfate,
25 mM ethylenediaminetetraacetic acid, 20% glycerol and 0.1% bromophenol blue. The mixtures were boiled at 100°C for 5 min and were then subjected
to stacking gel, following which, they were resolved by 12% sodium dodecyl
sulfate–polyacrylamide minigels at a constant current of 20 mA. Subsequently,
electrophoresis was carried out on sodium dodecyl sulfate–polyacrylamide gels.
For western blot analysis, proteins on the gel were electrotransferred onto a 45 μ
immobile membrane (polyvinylidene difluoride; Millipore Corp., Bedford, MA)
with transfer buffer composed of 25 mM Tris-HCl (pH 8.9), 192 mM glycine and
20% methanol. The membranes were blocked with a solution of 20 mM Tris-HCl
(pH 7.4), 0.2% Tween 20, 1% bovine serum albumin and 0.1% sodium azide.
The membrane was further incubated with specific antibodies, at appropriate dilution (1: 1000) using blocking solution with the primary antibodies overnight at
4°C. The membranes were subsequently probed with anti-mouse or anti-rabbit
IgG antibody conjugated to horseradish peroxidase (Transduction Laboratories,
Lexington, KY) and visualized using enhanced chemiluminescence (Amersham).
The densities of the bands were quantified using a computer densitometer
(AlphaImagerTM 2200 System). All membranes were stripped and reprobed
for β-actin, glyceraldehyde 3-phosphate dehydrogenase and Lamin B (Sigma
Chemical Co.) as loading control.
Electrophoretic mobility shift assay
Cytosolic and nuclear proteins were extracted as described previously (16).
The electrophoretic mobility shift assay analysis was performed with a nonradioactive (biotin label) gel shift assay according to the manufacturer’s protocol. The NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) and CREB
(5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′) consensus oligonucleotide
probes were end labeled with biotin (Pierce, Rockford, IL) with terminal deoxynucleotidyl transferase. For the binding reaction, 6 μg of nuclear extract protein
was incubated in a total volume of 20 μl with binding buffer containing 50 fmol
of biotin end-labeled oligonucleotide. The mixture was incubated at room temperature for 20 min. The specificity was determined by adding a 100-fold excess
of unlabeled double-stranded consensus oligonucleotide to the reaction mixture
to act as a competition reaction. Following addition of 5 μl of sample buffer, the
DNA–protein complexes were resolved on a 6% non-denaturing polyacrylamide
gel in a 0.5× Tris-borate-ethylenediaminetetraacetic acid buffer at 100 V for 2 h
and then transferred to nylon membrane. Finally, the biotin-labeled DNA was
detected by chemiluminescence using the LightShift Chemiluminescent EMSA
Kit (Pierce, Rockford, IL) and exposed to X-ray film.
Measurement of mitotic index
Multiple formalin-fixed skin and tumor sections were stained with hematoxylin and eosin (H&E) and analyzed for mitosis using a method described previously (17). To determine the mitotic index, all various mitotic stages including
prophase, metaphase, anaphase, telophase and cytokinesis were calculated
with a 400-fold magnification. In this way, a total of 1000 cells (~50 cells
per microscopic field) from epidermis and papillomas were counted for each
lesion, and results were presented as a percentage of mitosis per total cells.
Immunohistochemical analysis
Three micrometer sections of skin and tumor were deparaffinized, rehydrated and
treated with 0.3% hydrogen peroxide (H2O2) for 15 min to block endogenous
peroxidase. Sections were pressure cooked (4 × 7 min) in 10 mM citrate buffer,
pH 6.0 (Immuno DNA retriever with citrate, BIO SB, Santa Barbara, CA) to
unmask epitopes. Sections were incubated with primary antibody to CD34 and
PKD1 (1:200 dilutions in phosphate-buffered saline) for 1 h. Immunoreactivity
was determined using biotin-labeled secondary antibody and streptavidin–biotin
peroxidase for 30 min each. 3,3′-Diaminobenzidine tetrahydrochloride (0.05%)
was used as the substrate, and positive signal was detected as a brown color under
a light microscope. The detailed procedures for the stained tissue analysis method
were reported previously (18). For PKD1, the criterion for positive expression
was membrane staining. For the immunoreactive score, the scores for the percentage of positive cells and the staining intensity were multiplied.
PKD1 kinase activity assay
The PKD kinase activity assay was performed as in Triantafyllou et al. (19),
following immunoprecipitation of PKD using the Bioss PKD antibodies.
Transient transfection
The wild-type HA-PKD1 plasmid have been described previously (17) and
acquired from Addgene (http://Addgene.org). A431 cells were transient
transfections and harvested 24 h after transfection.
AcEGCG prevents skin carcinogenesis in mice
Statistical analysis
Relative expression values are given as the mean ± SE for the indicated fold of
expression in the skin and papillomas of mice. SigmaPlot 10.0 software was
used for illustration and analysis. Statistical analyses were performed using
two-way analysis of variance with subsequent Bonferroni post hoc test for
pairwise comparisons between two particular groups. A P value of <0.05 was
considered statistically significant.
Results
AcEGCG prevented DMBA-initiated and TPA-promoted mouse skin
tumorigenesis more effectively than EGCG
We first investigated the antitumor-promoting activity of EGCG and
AcEGCG on DMBA/TPA-mediated skin tumorigenesis in female ICR
mice (Figure 1). Throughout the experiment, there was no noticeable
difference in weight gain between the mice treated with two doses of
EGCG or AcEGCG and those not treated, indicating that the topical
application of EGCG or AcEGCG did not result in systemic toxicity. Topical pretreatment with AcEGCG at the higher dose resulted
in a lower tumor incidence (percentage of tumor-bearing mice;
Figure 1A) and lower tumor multiplicity (average number of tumors
per mouse; Figure 1B). The tumor promotion data were also analyzed in terms of the papilloma size distribution and compared with
the DMBA + TPA-treated group. The number of papillomas (1–3 mm
in diameter) per mouse was inhibited in a dose-dependent manner in
the EGCG- and AcEGCG-pretreated groups (Figure 1C). Compared
with the EGCG-pretreatment groups, AcEGCG-pretreatment groups
showed a significant inhibition in the number of tumors that are
3–5 mm and ≥5 mm in size (Figure 1C); tumor weight was also inhibited by AcEGCG pretreatment (Figure 1D).
AcEGCG inhibited DMBA/TPA-promoted tumor proliferation by
reducing the mitotic index
Increases in the mitotic index and induction of cell cycle regulators have been well demonstrated during DMBA/TPA-induced skin
tumor formation (20). To determine whether the observed hyperplasia was caused by increased mitosis, the mitotic index was calculated using H&E-stained sections of dorsal skin and papillomas.
Figure 2A shows representative H&E-stained sections of dorsal
skin and papillomas after DMBA treatment followed by acetone,
TPA, 1 μΜ AcEGCG or 5 μΜ AcEGCG treatment. Histological
examination of the tissue sections revealed that AcEGCG significantly reduced hyperplasia in the papillomas and their mitotic
index (Figure 2B; P < 0.05). We next analyzed the expression of
cell cycle regulators. Although many studies have shown that p53
and p21 are downregulated in cancer cells (18), it has also been
reported that p53 expression is increased in keratoacanthomas
(19) and DMBA/TPA-treated tissues (20). In addition, deregulation and elevated expression of Myc have also been observed to
lead to significant increases in cell proliferation and tumorigenesis
(21). In this study, topical pretreatment of AcEGCG at both doses,
30 min prior to TPA application, markedly inhibited the expression
of p53, p21 and c-Myc (Figure 2C). Indeed, progression through
mitosis requires the coordinated regulation of cyclin B1/cyclindependent kinase (Cdk) 1 activity. Previous studies have suggested
that cyclin B1/CDK1 complex assembly and dephosphorylation of
Cdk1 during G2/M phase is tightly coupled and regulated by Cdc25
phosphatases (22). Thus, downregulation of Cdc25A leads to an
induction of G2/M phase arrest. Cdc25A has been shown to be
degraded by the activation of the ERK pathway and phosphorylation of Cdc25A, resulting in cell cycle arrest (23). As illustrated in
Fig. 1. Inhibitory effects of EGCG and AcEGCG on DMBA-initiated and TPA-promoted mouse skin tumorigenesis. Groups of 12 female ICR mice, 6 weeks of
age, were first treated with 200 nM DMBA. After 1 week of DMBA initiation, mice were topically treated twice weekly with EGCG, AcEGCG or acetone 30 min
before each topical application of TPA (5 nM) for 20 weeks. (A) Tumor incidence (percentage of mice with papillomas). (B) Tumor multiplicity (average number
of papillomas per mouse ± SD). (C) Average numbers of papillomas per mouse in different tumor diameter groups. (D) Weight of papillomas per mouse. Tumors
of at least 1 mm diameter were counted and recorded weekly. Each bar represents the mean ± SD of the average of the 12 mice scored. Statistical significance was
done by analysis of variance, *P < 0.05; **P < 0.01; ***P < 0.001 compared with the DMBA + TPA-treated mice or DMBA + EGCG + TPA versus DMBA +
AcEGCG + TPA; #P < 0.001 compared with the DMBA + Ac group. Results were statistically analyzed by analysis of variance. aP < 0.05; bP < 0.01; cP < 0.001
EGCG (1 μM) versus EGCG (5 μM), and AcEGCG (1 μM) versus AcEGCG (5 μM).
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Fig. 2. The effects of AcEGCG on the DMBA/TPA-induced mitotic index and cell cycle regulators of the G2/M transition in mouse skin and papillomas. (A)
Histochemical analysis of epidermal and tumor sections stained with H&E (200× magnification). (B) Quantification of the mitotic index. The mitotic index was
evaluated as described in the Materials and methods. Papillomas of 1–3 mm size were selected and analyzed in each experiment. Each bar represents the mean ±
SE of 3–6 mice scored. Statistical significance, *P < 0.05 compared with the DMBA + TPA-treated group. Equal amounts of cell lysates (50 μg of protein) were
subjected to western blot analysis in C and D to determine the levels of cell cycle regulators. C: DMBA treatment. Quantification of protein level was normalized
to β-actin using densitometry. The results are representative of three independent experiments.
Figure 2D, topical pretreatment with AcEGCG dramatically inhibited the levels of cyclin B and Cdc25 proteins and enhanced the
levels of p-ERK1/2 and Tyr15 p-CDK1. These results suggest that
pretreatment with AcEGCG could lead to the activation of ERK,
the degradation of Cdc25A and the inhibition of cyclin B1/CDK1
complex assembly; these effects cause G2/M phase arrest and block
mitotic progression.
AcEGCG suppressed DMBA/TPA-induced PKD1 activation and
CD34 expression
PKD1 can be activated by TPA, which initiates keratinocyte hyperproliferation, abnormal differentiation and inflammation through
the increased expression of PKD1; these effects occur as the result
of the autophosphorylation of ser 916 and transphosphorylation of
ser744/748 (24,25). Recent evidence also suggests an important
function for CD34 in DMBA/TPA-induced skin tumorigenesis (26).
This research indicated that CD34 is required for TPA-induced skin
stem cell activation and tumor formation in mice (2). As illustrated
in Supplementary Figure 1A and B, available at Carcinogenesis
Online, topical application of TPA (A) or DMBA (B) over 12 h
greatly increased the protein levels of PKD1 and CD34. To understand the correlation between PKD1 activation and CD34 expression
in DMBA/TPA-induced skin tumor initiation and progression, the
PDK1 and CD34 levels were examined using immunohistochemical
analysis. The levels of PKD1 and the skin CSC marker, CD34, were
significantly higher in DMBA/TPA-induced mice compared with the
untreated group (Figure 3). AcEGCG pretreatments significantly lowered DMBA and/or TPA-induced levels of PKD1 and CD34 (Figure 3
and Supplementary Figure 1C, available at Carcinogenesis Online).
We next examined whether AcEGCG inhibited activation of PKD1
using a co-immunoprecipitation strategy, in which we immunoprecipitated PKD1 and analyzed the resulting kinase activity. Western blots
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and kinase activity assays demonstrated that AcEGCG significantly
suppressed DMBA/TPA-induced PKD1 levels, serine phosphorylation (at ser916 and ser744/748) and active PKD1 levels (Figure 4A
and B). To determine whether AcEGCG directly inhibited PKD1, we
measured purified PKD1 (2 ng) activity in the presence of 5 and 25 μM
AcEGCG. Surprisingly, we found that the kinase activity of purified
PKD1 was inhibited by AcEGCG in a dose-dependent manner to a
level of 70% (Figure 4C). These data support the direct inhibition of
PKD1 by AcEGCG. Our results strongly suggest that the overexpression and activation of PKD1 in CD34+ skin stem cells and skin tumors
are potential targets for the treatment of skin carcinogenesis.
AcEGCG blocked DMBA/TPA-induced activation of the p38,
JNK1/2 and PI3K/Akt signaling pathways and downregulated target protein expression
Although we demonstrated that PKD1 is a key regulator of the
inhibition of tumor formation by AcEGCG, the downstream signaling
targets of the MAPKs and PI3K/Akt pathways in skin tumorigenesis
remain to be identified. Studies have shown that inflammatory and
proliferative protein expression is controlled by the MAPKs and
PI3K/Akt pathways. We and others have previously also shown that
the transcription of iNOS, COX-2, VEGF and ODC is regulated by
C/EBPs, NF-κB and CREB, which are required for the DMBA/TPAinduced tumor inflammation and proliferation (27–39). To further
explore the potential mechanisms by which AcEGCG inhibited
DMBA/TPA-induced epidermal hyperproliferation and skin tumor
promotion, experiments were conducted to evaluate the changes
in various members of the epidermal and papilloma signaling
pathways (p38, JNK1/2 and PI3K/Akt) and well-known biomarkers
of inflammation and tumor promotion (iNOS, COX-2, ODC and
VEGF). Pretreatment with AcEGCG at a dose of 1 or 5 μΜ resulted
in a decrease in the levels of phosphorylated JNK1/2, p38 and PI3K/
AcEGCG prevents skin carcinogenesis in mice
Fig. 3. Inhibitory effect of AcEGCG on DMBA/TPA-induced activation of PKD1 in CD34+ skin stem cells and CSCs. (A) Staining and (B) quantification
patterns of PKD1 and CD34 protein levels in mouse skin and papillomas (100× magnification). Papillomas of 1–3 mm size were selected and analyzed in each
experiment. Positive expressing cells are stained brown. Each bar represents the mean ± SE of 3–6 mice scored. Statistical significance, *P < 0.05; **P < 0.01
compared with the DMBA + TPA-treated group.
Fig. 4. Inhibitory effect of AcEGCG on TPA and DMBA/TPA-induced activation of PKD1 in mouse skin and papillomas. (A) PKD1 immunoprecipitation
from control, DMBA/TPA or DMBA + AcEGCG + TPA-treated skin and papilloma lysates. The concentration of active PKD1 was measured by enzyme-linked
immunosorbent assay-based assay from Enzo Life Sciences. Each bar represents the mean ± SE of three mice scored. Statistical significance, *P < 0.05 compared
with the DMBA + TPA-treated group. (B) Equal amounts of cell lysate (50 μg of protein) were subjected to western blot analysis to determine the activation of
PKD1. Papillomas of 1–3 mm size were selected and analyzed in each experiment. Quantification of protein level was normalized to β-actin using densitometry.
A431 cells were transfected with HA-PKD1 and used as a positive control to verify PKD1 band. Endogenous (PKD, down band) and expressed PKD (HA-PKD1,
up band) were detected on anti-PKD 1 immunoblot. The results are representative of three independent experiments. C: DMBA treatment. (C) Inhibitory
activity of AcEGCG was determined in the presence of 2 ng purified PKD1. Each bar represents the average of triplicate determinations. Statistical significance,
**P < 0.01 compared with the active PKD1-treated group.
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Fig. 5. Inhibitory effect of AcEGCG on DMBA/TPA-stimulated nuclear translocation of NF-κB, C/EBPβ and C/EBPδ and the activation of the MAPKs and
PI3K/Akt signaling pathways. Western blot analysis of PI3K/Akt and MAPKs signaling molecules (A and B) and downstream transcription factors (C). Total
tissue lysates or nuclear and cytosolic extracts were prepared from mouse skin and papillomas. C: DMBA treatment. Papillomas of 1–3 mm size were selected
and analyzed in each experiment. The western bolt is representative of at least three independent experiments.
Akt compared with the levels in DMBA/TPA-mediated tumors
(Figure 5A and B). The protein levels of NF-κB, C/EBPβ and C/
EBPδ in nuclear fractions were markedly inhibited by AcEGCG
pretreatment (Figure 5C). Previous studies indicate that PKD1 is a
mediator of NF-κB and CREB induction (30–32). Activation of
NF-κB can act via the phosphorylation and degradation of IκBα,
resulting in the nuclear translocation and DNA binding of NF-κB.
It has been reported that histone protein substrate acetylation by
histone acetyltransferases is crucial for the generation of a chromatinrelaxation state and gene transcription (33). We also observed that the
DMBA/TPA stimulation of NF-κB and CREB-DNA-binding activity
was attenuated by pretreatment with AcEGCG in a dose-dependent
manner (Figure 6A and B). Topical pretreatment with AcEGCG
not only decreased the expression of the p-p65, IκBα and acetylhistone 3 lysine 9 proteins but also significantly increased the level
of IκBα in mouse papillomas (Figure 6C). In addition, preapplication
of AcEGCG markedly suppressed the expression of iNOS, COX-2,
ODC and VEGF by DMBA/TPA treatment (Figure 6D). Overall,
we speculate that AcEGCG exerts antiproliferative and/or antiinflammatory effects in CD34+ skin stem cells and skin tumors and
that the suppression of PKD1 activity and its downstream signaling
pathways may be involved in the prevention of skin carcinogenesis.
Discussion
In this study, we investigated the differences in chemopreventive
potency of AcEGCG and EGCG for DMBA/TPA-induced murine skin
tumorigenesis (Figure 1). The mitotic index, a measure of the percentage of cells undergoing mitosis, is an established indicator of tumor
proliferation. Earlier findings have shown mitosis to be an important
target for anticancer therapies. The cyclin B1/CDK1 complex controls
mitotic entrance from G2 to M phase. In fact, inhibiting aberrant activity of cyclin B1/CDK1 in tumor cells leads to mitotic arrest and consequently, although not always, to cell death (34). Previous studies have
1320
reported that DMBA-initiated skin tissue has significantly increased
the levels of mitochondrial p53 and the survival response mediated by
cyclin B1/CDK1 phosphorylation at ser-315 of p53 (35,36). However,
our present data cannot prove that ser-315 of p53 is phosphorylated
by mitochondrial cyclin B1/CDK1 in DMBA/TPA-induced mouse
papillomas, thus avoiding the pathway toward apoptosis. Analysis of
H&E-stained dorsal skin and papillomas showed that topical pretreatment with AcEGCG could significantly decrease the proliferation of
epidermal cells and induce G2/M phase arrest by inhibition of cyclin
B1/CDK1 activity (Figure 2).
Previous studies have reported that aberrant activation and upregulation of PKD1 might be involved in various cellular functions that
are potentially significant in skin tumor development, such as proliferation, survival, apoptosis and differentiation (4,37,38). In vitro
analysis of keratinocytes exposed to ultraviolet B radiation revealed
the inhibition of apoptosis due to the overexpression of wild-type
PKD1, suggesting a putative tumorigenic role of PKD1 in the skin
(39). Since their discovery, the non-selective PKD1 inhibitor (resveratrol) and selective PKD1 inhibitor, Gö6976, have been proposed to
act as antiproliferative and prodifferentiative signals in TPA-treated
keratinocytes in vitro and epidermis in vivo (40). Furthermore, PKD1
can serve as a marker of sensitivity to skin carcinogenesis.
In addition, PKD1 and CD34 have also been defined and isolated
based on their locations within cutaneous stem cells, where they
potentially mediate CSC activation and promote skin carcinogenesis
(26). The results of this study further confirmed and provided new
evidence that DMBA/TPA-induced skin tumorigenesis occurs via the
induction of CD34 expression and the activation of PKD1-regulated
downstream signaling, including the p38, JNK1/2 and PI3K/Akt
pathways (Figures 3–5). Activation of multiple kinases leads to the
phosphorylation and upregulation of downstream transcription factor
activity, including C/EBPs, NF-κB and CREB, which transcribe
proinflammatory and proproliferative genes, including iNOS, COX2, ODC and VEGF (Figures 5C and 6). In addition to transcription
factors, acetylation of acetyl-histone 3 lysine 9 has been shown to
AcEGCG prevents skin carcinogenesis in mice
Fig. 6. Inhibitory effect of AcEGCG on DMBA/TPA-regulated DNA-binding activity of NF-κB and C/EBP and transcription of downstream target proteins.
For analysis by the electrophoretic mobility shift assay, nuclear extracts were prepared and incubated with biotin-labeled (non-radioactive) oligonucleotides
containing the (A) NF-κB and (B) CREB sequences. (C and D) Equal amounts of cell lysates (50 μg of protein) were subjected to western blot analysis to
determine the levels of p-p65, p-IκBα, IκBα, iNOS, COX-2, ODC and VEGF. C: DMBA treatment. Quantification of protein level was normalized to β-actin
using densitometry. The results are representative of three independent experiments. Papillomas of 1–3 mm size were selected and analyzed in each experiment.
be involved in promoting the transcriptional activity of target genes
in DMBA/TPA-treated mouse papillomas (Figure 6C). In an in
vitro kinase assay, we first demonstrated that treatment with 5 and
25 μM AcEGCG could directly inhibit PKD1 activity (Figure 4D).
Additionally, our study clearly showed that topical pretreatment of
AcEGCG not only abated CD34 expression and the activation of
multiple kinases but also suppressed C/EBPs, NF-κB and CREBmediated target gene expression in DMBA/TPA-treated mouse
papillomas (Supplementary Figure 2, available at Carcinogenesis
Online). In this study, we found that there is little dose-dependent
response of AcEGCG in inhibiting PKD1 activity at an early stage
of signaling pathways. We suggest that AcEGCG could be quite
successful in improving the biological activity and bioavailability
of EGCG. Therefore, AcEGCG more effectively exerts cancer
chemopreventive ability in lower concentration.
In this study, we also analyzed CD34 and PKD1 expression levels in human keratinocytes (HaCaT) and human epithelial carcinoma
cells (A431) and found that A431 cells had an ~4-fold induction of
CD34 and PKD1 expression compared with HaCaT cells. In addition, CD34 and PKD1 upregulation correlated with a fast proliferative potential (Supplementary Figure 3, available at Carcinogenesis
Online). This is also the first study to find that AcEGCG could suppress cell proliferation by decreasing PKD1 and CD34 levels in A431
cells (Supplementary Figure 3, available at Carcinogenesis Online).
Thus, we identified CD34 and PKD1 as proproliferative genes in epithelial carcinoma cells and as prognostic factors for the prediction
of clinical outcome. Although numerous studies support the importance of PKD1 in processes related to proliferation in many tumor
cell types, including keratinocytes, there are currently no specific,
effective and safe inhibitors of PKD1 available. Targeting CD34 in
conjunction with PKD1 inhibitors could become a viable option for
the development of new and more effective multitarget strategies for
preventing skin carcinogenesis. Taken together, our findings have substantial implications because AcEGCG may serve as a novel chemopreventive and therapeutic agent that could inhibit PKD1 activity and
the inflammation associated with tumorigenesis.
Supplementary material
Supplementary Figures 1–3 can be found at http://carcin.oxfordjournals.org/
Funding
National Science Council (NSC 98-2313-B-022-002-MY3, 101-2628B-022-001-MY4).
Conflict of Interest Statement: None declared.
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Received September 14, 2012; revised January 14, 2013; accepted January
29, 2013