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ISSN: 0892-3973 (print), 1532-2513 (electronic)
Immunopharmacol Immunotoxicol, Early Online: 1–8
! 2014 Informa Healthcare USA, Inc.
DOI: 10.3109/08923973.2014.931421

RESEARCH ARTICLE

Chemopreventive effect of a novel, selective TACE inhibitor on

DMBA- and TPA-induced skin carcinogenesis
Manoranjan Sharma1, Jogeswar Mohapatra1, Anil Argade2, Shrikalp S. Deshpande3, Gaurang B. Shah3,
Abhijit Chatterjee1, and Mukul R. Jain1
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by 202.131.108.40 on 06/20/14

1
Department of Pharmacology and 2Department of Medicinal Chemistry, Zydus Research Centre, Moraiya, Ahmedabad, Gujarat, India, and
3
Department of Pharmacology, K.B. Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, India

Abstract Keywords
Context: Tumor necrosis factor (TNF)-a, a potent proinflammatory cytokine, plays a major role in EGFR, inflammation, skin cancer, TNF-a, TNF-a
the pathogenesis of cancer. TNF-a converting enzyme (TACE) mediates processing and release converting enzyme
of biologically active TNF-a.
Objective: We aimed to investigate the effect of a novel, selective TACE inhibitor History
(compound 11p) on skin inflammation and associated tumorigenesis in mice.
Methods: Skin edema was induced in mice by dermal application 12-O-tetradecanoylphorbol- Received 31 March 2014
13-acetate (TPA) solution in acetone on to the ear and the effect of post-treatment of Revised 21 May 2014
compound 11p (topical application) was evaluated. Edema and inflammation was assessed by Accepted 30 May 2014
measuring ear thickness, weight of skin punch and cytokine levels. Skin cancer in mice was Published online 20 June 2014
initiated by single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) and
For personal use only.

promoted by repeated TPA application for 20 weeks. The effect of compound 11p on
papilloma incidence and multiplicity was evaluated.
Results: Treatment with compound 11p strongly suppressed TPA-induced elevation in skin
thickness and weight. A dose-dependent suppression in TPA-mediated TNF-a, IL-6, IFN-g, IL-17
and PGE2 levels which was associated with a decrease in infiltration of inflammatory cells was
also observed with the treatment. Moreover, compound 11p treatment delayed the onset,
markedly reduced the papilloma incidence and multiplicity persuaded by DMBA and TPA.
Discussion and conclusion: These findings suggest that selective blockade of TACE suppresses
TPA-induced epidermal hyperplasia, inflammatory cell infiltration and cytokine level. Inhibition
of inflammatory events related to tumor growth might have led to the anti-tumor effect in
mouse skin cancer model induced by DMBA and TPA.

Introduction to the tissue remodeling and stromal development necessary

for tumor growth and spread4. Additionally, mice deficient
Inflammation has been a striking feature of many epithelial
with TNF receptor shows impaired pre-neoplastic changes
cancers, as it plays a major role in tumor initiation and
and tumor formation5.
progression. Recent evidences elucidate the importance of
TNF-a is initially synthesized as a 26-kDa membrane
cytokines as a link between inflammation and cancer.
protein (mTNF-a) which is then cleaved to its 19-kDa soluble
Cytokine like tumor necrosis factor (TNF)-a is involved in
form (sTNF-a) by TNF-a converting enzyme (TACE)6–8, also
many inflammatory diseases, and its inhibition with specific
called as a disintegrin and metalloproteinase (ADAM)-17.
antibodies has been proven to be highly efficacious in the
The epidermal growth factor receptor (EGFR) is a tyrosine
treatment of autoimmune and inflammatory diseases such as
kinase receptor family, which is frequently activated in many
rheumatoid arthritis, Crohn’s disease and ulcerative colitis1,2.
epithelial tumors, leading to enhanced growth and other
TNF-a plays a conflicting role in malignant diseases. Local
tumor promoting activities9. Targeting this receptor has been
administration of high dose of TNF-a destroys tumor blood
proved as an anti-neoplastic therapeutic tool for breast cancer
vessels and has powerful anti-cancer effect3. However, TNF-a
in clinic. TACE mediates processing and release of EGFR
may also act as an endogenous tumor promoter, contributing
ligands like transforming growth factor alpha (TGF-a),
amphiregulin (AR) and heparin-binding epidermal growth
factor (HB-EGF) leading to cancer cell proliferation, survival
and tumor growth10–13. Using small molecular inhibitor or
Address for correspondence: Abhijit Chatterjee, Ph.D., Department of
siRNA for TACE, a marked regression of malignant breast
Pharmacology and Toxicology, Zydus Research Centre, Ahmedabad
380015, Gujarat, India. Tel: +91 2717 665555. Fax: +91 2717 665355. cancer cell line has been demonstrated14. INCB-3619, a
E-mail: abhijitchatterjee@zyduscadila.com potent inhibitor of ADAM-10 and TACE, was shown to
 2 M. Sharma et al.
inhibit shedding of EGFR ligands AR, heregulin, TGF-a,
HB-EGF and EGF in vitro15. In pre-clinical model,
INCB-3619 reduced levels of circulating TGF-a and inhibited
growth of a series of xenografted cancer cell lines16. A recent
study, using a specific antibody against ADAM-17, demon-
strated a strong inhibition in TGF-a release and ovarian tumor
growth in vivo17. These evidences clearly suggest the role of
TACE in tumor growth via EGFR signaling pathways.
Skin cancer is the most common form of cancer which
comprises of two subtypes such as basal cell cancer and
squamous cell cancer. Squamous cell carcinomas (SCCs) are
the most aggressive and heterogeneous skin cancers. The
multistage mouse skin carcinogenesis model has been widely
studied for the identification of molecular and biochemical
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events of SCCs. In the chemically induced mouse skin
tumorigenesis, tumor initiation is caused by single topical
application of 9,10-dimethylbenz(a)anthracene (DMBA), a
carcinogen that mutates H-Ras proto-oncogene. In this model,
tumor promotion is caused by repeated application of
12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol-type
tumor promoter18,19. Application of TPA induces TNF-a,
which is likely to play a key role in tumor promotion-related
events such as epidermal hyperplasia and leukocyte
infiltration into the dermis19. Involvement of TNF-a in the
pathogenesis of skin cancer is demonstrated by the observa-
tion that mice deficient with TNF-a are resistant to develop
DMBA- and TPA-induced skin carcinogenesis20.
For personal use only.
Furthermore, treatment with anti-TNF antibody inhibits the
progression of DMBA- and TPA-induced skin cancer21.
However, the role of TACE in TNF-a dependent tumor
growth is not explored yet.
We aimed to investigate the involvement of TACE in skin
inflammation and associated tumorigenesis using a novel and
highly selective TACE inhibitor. Our data demonstrated that
selective inhibition of TACE blocks TPA-induced skin
inflammation associated with suppression in various inflam-
matory cytokine levels. Furthermore, our results also
emphasized that the treatment with selective TACE inhibitor
markedly suppresses papilloma development during skin
carcinogenesis.
Materials and methods
Chemicals
DMBA and TPA were purchased from Sigma–Aldrich,
St. Louis, MO. TACE inhibitor Compound 11p, [(R)-2-((R)-
3-Amino-3-(4-((2-(2-methoxyethyl)quinolin-4-yl)methoxy)-
phenyl)-2-oxopyrrolidin-1-yl)-N-hydroxy-4-methylpentana-
mide] with 99% purity was synthesized in-house by Dept. of
Medicinal Chemistry, Zydus Research Centre.
Animals
Female Balb/c mice of 7–9 weeks old were housed in
individually ventilated cages and given pelleted food
(Standard Rodent diet, NIN, Hyderabad, India) and water ad
libitum in a temperature (25 ± 3  C) and humidity (50–70%)
controlled environment with a 12-h/12-h dark–light cycle. All
animal care and experimental procedures were complied with
the guidelines of the Committee for the Purpose of Control
Immunopharmacol Immunotoxicol, Early Online: 1–8
and Supervision of Experiments on Animals as stated by the
National Institutes of Health and were approved by the
Institutional Animal Ethics Committee. Experiments were
performed in an Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC)
International accredited facility.
TPA-induced ear edema
Mice were randomized into six groups (n ¼ 5). Basal ear
thickness was measured with a digital caliper. Edema was
induced in the right ear of each mouse by topical application
of 2 mg TPA dissolved in 20 ml of acetone to both inner and
outer surfaces (10 ml to each side). A group of mice applied
with 20 ml acetone served as normal control. One hour after
the application of TPA, animals were applied topically with
20 ml of acetone or different concentration of compound 11p
(0.5%, 1% or 2% in acetone). A group of mice applied with
1% solution of hydrocortisone acetate (HC) 1-h post-TPA
challenge served as positive control. After 24 h of TPA
challenge, ear thickness was measured, animals were
sacrificed and ear biopsies were taken using punch of 8 mm
diameter, weighed and snap frozen and stored at 80  C till
biochemical estimations. A part of ear tissue was kept for
histological analysis. In another set of experiment, to study
the kinetics of TPA-induced TNF-a, 20 animals were exposed
to TPA. After 0, 3, 6 or 24 h of TPA challenge, five animals
for each time point were subjected to collection of blood for
serum and ear tissue samples.
A separate study was carried out to evaluate the effect of
compound 11p on the soluble TNF-a in the serum, the level
of membrane TNF-a in the tissue. Mice divided into five
groups (n ¼ 5) received either acetone or TPA solution. One-
hour post-TPA challenge, the animals were subjected to
topical application of acetone or different concentrations
(i.e. 0.5%, 1% or 2%) of compound 11p. After 3 h of TPA
challenge, blood for serum and ear biopsies was collected for
biochemical estimation and gene expression.
Measurement of tissue cytokines and PGE2
For cytokine estimations, ear biopsies were homogenized
with lysis buffer (50 mM Tris–HCl buffer of pH 8, 150 mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and
1 mM PMSF) containing protease inhibitor cocktail and
subjected to two freeze-thaw cycles. For measurement of
PGE2, tissue samples were homogenized with PBS contain-
ing 10 mM indomethacin and protease inhibitor cocktail. The
homogenate was centrifuged and the supernatant was stored at
80  C until analyzed. Mouse TNF-a, IL-6 and IFN-g were
quantified in the samples using ELISA kits (BD Biosciences,
San Jose, CA). PGE2 and IL-17 were measure using ELISA
kit from R&D Systems, Minneapolis, MN.
mRNA isolation and gene expression analysis
Total RNA was extracted from the ear samples by TRIzol
reagent (Invitrogen Life Technolgies, Carlsbad, CA) in
accordance with the supplier’s instructions. One microgram
total RNA from each sample was taken for first-strand cDNA
synthesis using High-Capacity cDNA Archive Kit
 DOI: 10.3109/08923973.2014.931421
(Applied Biosystems, Foster City, CA; Part no. 4322171).
An equal amount of cDNA from each sample was taken for
quantitative real-time PCR using ABI-7300 (Applied
Biosystems, Foster City, CA). Gene expression of
mouse TNF-a (F:50 -ACGTCGTAGCAAACCACCAA-30 and
R:50 -ACAAGGTACAACCCATCGGC-30 ), IL-6 (F:50 -CGGC
CTTCCCTACTTCACAA-30 and R:50 -TGCCATTGCACAAC
TCTTTTCT-30 ), IFN-g (F:50 -CGGCACAGTCATTGAAAGC
C-30 and R:50 -TGTCACCATCCTTTTGCCAGT-30 ) and
IL-17 (F:50 -TCATCCCTCAAAGCTCAGCG-30 and R:50 -TT
CATTGCGGTGGAGAGTCC-30 ) was determined using
SYBR Green quantitative real-time PCR and QIAGEN
QuantiFast SYBR Green kit (Cat. no. 204052, Qiagen,
Germantown, MD). Mouse b-actin (F:50 -TACAGCTTCAC
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CACCACAGC-30 and R:50 -TCTCCAGGGAGGAAGAGG
AT-30 ) was used as an internal control for normalization of
the results.
Histological examinations
Ear samples were fixed in 10% buffered formalin, pH 7.4 and
embedded in paraffin. Sections (5 mm) on slides were
deparaffinized in xylene, rehydrated in decreasing concentra-
tions of ethanol and subjected to hematoxylin and eosin
(H&E). The pathological alterations in the stained specimens
were evaluated and confirmed by a certified independent
pathologist.
For personal use only.
DMBA- and TPA-induced mouse skin tumorigenesis
Skin tumorigenesis was initiated by single topical application
of DMBA (100 mg/200 ml in acetone) to the shaved dorsal skin
of each mouse. One week after initiation, all the mice were
treated with topical applications of TPA (2 mg/200 ml in
acetone) twice weekly for 20 weeks. A group of mice received
200 ml of acetone only. Hundred microliters of acetone alone
or 2% solution of compound 11p in acetone was applied 1 h
after each TPA application. The number of suspected
papillomas having the diameter 41 mm was counted weekly.
After 5, 10 and 20 weeks of DMBA application, blood sample
was collected from retro-orbital plexus after 3 h of TPA
challenge.
Statistical analysis
Data are expressed as mean ± SEM. The statistical analysis
was performed using one-way or two-way ANOVA followed
by Dunnett’s multiple comparison tests for comparison
between different groups. For each analysis, p values 50.05
were considered to represent statistical significance. All
analyses were performed using Graph Pad Prism software 5.0
(GraphPad, La Jolla, CA).
Results
Selective TACE inhibitor, compound 11p, attenuates
TPA-induced ear edema
Single topical application of TPA significantly elevated ear
thickness and weight after 24 h with respect to the normal
control group (Figure 1A and B). Topical treatment with
compound 11p solution significantly suppressed the
TPA-induced ear thickness and weight when compared to
Chemopreventive effect of a novel, selective TACE inhibitor 3
the vehicle control group (Figure 1A and B). Histological
analysis of ear tissue samples with H&E staining demon-
strated that TPA challenge markedly elevated epidermal
hyperplasia and infiltration of inflammatory cells into the
dermis (Figure 1C). Application of 2% solution of compound
11p had a striking suppression in epidermal hyperplasia
and inflammatory cell infiltration into the dermis
(Figure 1C). As shown in Figure 1, treatment with 1%
hydrocortisone (HC) had a marked suppression in TPA-
induced edema which was associated with an improvement in
dermal hyperplasia and inflammatory cell infiltration.
The overall efficacy of 2% solution of compound 11p was
comparable to 1% HC treatment. Additionally, we have
also studied the effect of 2% solution of compound 11p
alone applied daily for 7 days for any possible effect on
normal skin and did not find any signs of inflammation (data
not shown).
Effect of compound 11p on TPA-induced TNF-a
We aimed to study the regulation of both serum and tissue
levels of TNF-a at various time intervals after TPA. TPA
challenge elevated serum TNF-a which peaked at 3 h and
gradually restored to the normal level by 24 h (Figure 2A).
A significantly high level was detected at 3 and 6 h of TPA
challenge. TPA-induced tissue TNF-a level gradually
increased from 3 h, peaked at 6 h and was significantly high
in both the time points when compared to the basal level
(Figure 2A). Next, we evaluated the effect of compound 11p
treatment on serum and tissue TNF-a at 3 h of TPA challenge.
Compound 11p treatment dose-dependently reduced
TPA-induced serum TNF-a with a statistical significance
from 0.5% dose and onwards (Figure 2B). However, the
treatment did not affect the tissue TNF-a level up to the
highest dose tested, i.e. 2% (Figure 2C). Similar to the tissue
protein level, TNF-a mRNA expression in the compound 11p
(2%) treated animals was not significantly different from the
vehicle control group (Figure 3D).
Compound 11p treatment inhibits TPA-induced
elevation in IL-6, IFN-g, IL-17 and PGE2
We performed a time course analysis of IL-6, IFN-g, IL-17
and PGE2 level in tissue after TPA challenge and their peak
levels were found at 24 h (data not shown). Hence, in the
present study, we chose 24-h time point for the measurement
of these cytokines. Compound 11p had a dose-dependent
suppression in TPA-induced IL-6, IFN-g and IL-17 level with
a statistical significance from 1% concentration and onwards
(Figure 3A–C). We next evaluated the effect of 2% solution of
compound 11p on mRNA levels of IL-6, IFN-g and IL-17
after 3 h of TPA challenge. The tissue mRNA levels of IL-6,
IFN-g and IL-17 were markedly increased after TPA
challenge by almost 6-, 3.5- and 2.8-fold, respectively, when
compared to the normal control group (Figure 3D). Treatment
with compound 11p at 2% solution significantly suppressed
the TPA-induced mRNA levels of IL-6, IFN-g and IL-17
(Figure 3D). Additionally, tissue level of PGE2 was promin-
ently high after 24 h of TPA challenge and a dose-dependent
suppression was observed with the compound 11p treatment
(Figure 3E).
 4 M. Sharma et al. Immunopharmacol Immunotoxicol, Early Online: 1–8
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Figure 1. Effect of compound 11p on TPA-induced ear edema and hyperplasia. Edema was induced in mice by topical application of 2 mg TPA
dissolved in 20 ml of acetone on to the ear (10 ml to each side). The indicated concentration of compound 11p solution in acetone was applied after 1 h of
TPA challenge. A group of mice receiving 1% HC served as positive control. After 24 h of TPA challenge, ear thickness (A) and a punch weight of
8 mm diameter was measured (B). Data are expressed as mean ± SEM (n ¼ 5). *p50.05 when compared with normal control. #p50.05 when
compared with vehicle control group. Representative images showing H&E stains of ear tissue (C) (magnification 10) collected after 24 h of TPA
challenge.

Effect of compound 11p on DMBA- and TPA-induced

skin tumorigenesis
The effect of compound 11p was evaluated on DMBA-
initiated and TPA-promoted skin tumorigenesis. The onset of
papillomas was evident from 7 weeks, resulting in a 91%
incidence and a mean papilloma of 7.5/mouse at 20th week
(Figure 4A and B). Twice weekly treatment with compound
11p resulted in a delay in the latency period from 7 to
11 weeks with a marked suppression in papilloma incidence
and multiplicity up to 20th week. Compound 11p treatment
lowered the incidence to 55% and the mean no. of papilloma
to 3.2/mouse at 20th week (Figure 4A and B). Additionally,
we have also evaluated the effect of repeated TPA application
on serum TNF-a level after 5, 10 or 20 weeks DMBA
initiation. Serum TNF-a level, measured after 3 h of TPA
challenge, was significantly high when compared to the
DMBA only treated group at 5, 10 or 20 weeks (Figure 4C).
Treatment with compound 11p significantly suppressed the
TPA-induced serum TNF-a level at all the time points
(Figure 4C).
 DOI: 10.3109/08923973.2014.931421
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Figure 2. Kinetics of TPA-induced TNF-a and effect of compound 11p. The level of TNF-a in serum and ear tissue homogenate was measured after
the indicated times (A). Effect of post-treatment with various concentrations of compound 11p on TNF-a level in serum (B) and ear tissue homogenate
(C) was depicted. Data are expressed as mean ± SEM (n ¼ 5). *p50.05 when compared with normal control. #p50.05 when compared with vehicle
control group.
Discussion
Clinical and experimental evidences suggested the essential
contribution of inflammation in skin papilloma development.
The tumor promoters induce secretion of pro-inflammatory
molecules by keratinocytes resulting in the recruitment of
inflammatory cells, such as leukocytes, lymphocytes
and macrophages, into the dermis. These activated cells
then produce growth factors, cytokines and chemokines that
promote cell proliferation, matrix remodeling and angiogen-
esis which ultimately lead to tumor growth22–24. The classical
skin tumor promoter, TPA, has been shown to induce
skin tumorigenesis via activation of various inflammatory
pathways. A recent study demonstrated that mice over-
expressing TACE shows augmented dermal fibrosis and
inflammation in response to TPA25, however, mice lacking
TACE in keratinocytes develops spontaneous dermal
inflammation26,27. The present study was designed to dem-
onstrate the effect of a selective TACE inhibitor on skin
inflammation and tumorigenesis induced by TPA in mice.
We selected a novel TACE inhibitor, compound 11p,
which is potent and highly selective (nearly 500-fold)
for TACE over other MMPs and ADAM family members28.
In a very recent study, we have demonstrated the anti-
inflammatory effect of compound 11p in colon
inflammation29.
Chemopreventive effect of a novel, selective TACE inhibitor 5
Topical application of TPA on to mouse ear induces
inflammation characterized by skin edema and accumulation
of inflammatory cells30. TPA-induced inflammation is also
associated with elevation in cytokine such as TNF-a, IL-1b
and IL-6 levels in the affected skin tissues31. In the present
study, topical application of TPA induced a marked increase
in ear thickness and weight which was associated with
elevated tissue levels of proinflammatory cytokines.
Treatment with compound 11p strongly suppressed skin
edema induced by TPA in a dose-dependent manner.
Treatment with compound 11p strikingly suppressed the
level of TPA-induced serum TNF-a, however, the tissue
TNF-a protein or mRNA level remain unaltered in the
treatment group indicating that the drug has a direct effect on
TACE activity. Interestingly, compound 11p treatment dose-
dependently suppressed the elevated tissue levels of IL-6,
IFN-g and IL-17 due to TPA challenge. Topical application of
TPA induces infiltration of inflammatory cells to the site
leading to production of several pro-inflammatory cytokines.
In the present study, inhibition of several cytokines by
compound 11p treatment clearly demonstrates its potential to
suppress inflammation induced by TPA. This was further
supported by the histological analysis showing a marked
suppression in infiltration of inflammatory cells in the
treatment group with respect to the control animals.
Increased production of eicosanoids such as PGE2 and
 6 M. Sharma et al. Immunopharmacol Immunotoxicol, Early Online: 1–8
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Figure 3. Effect of compound 11p on inflammatory cytokines and PGE2. Protein levels of IL-6 (A), IFN-g (B) and IL-17 (C) in ear tissue homogenate
after 24 h of TPA application were analyzed by ELISA method. mRNA expression levels of IL-6, IFN-g and IL-17 was measured in ear tissue
samples collected after 3 h of TPA challenge by quantitative RT-PCR. After normalized by b-actin, fold change in IL-6, IFN-g and IL-17 with respect
to normal control group is depicted in bar diagram as relative expression (D). PGE2 was estimated in tissue biopsies harvested after 24 h of TPA
challenge (E). Data are expressed as mean ± SEM (n ¼ 5). *p50.05 when compared with normal control. #p50.05 when compared with vehicle
control group.

leukotriene B4 are also associated with TPA-induced inflam-
mation30,32. Although the mechanism of TPA-induced
eicosanoid is not completely known, but TPA is demonstrated
to trigger protein kinase C33, phospholipase A234 and
cyclooxygenase35. Therefore, we aimed to study the effect
of compound 11p treatment on PGE2 level in the inflamed ear
tissue. TPA challenge significantly elevated PGE2 level and a
dose-dependent suppression was observed with compound
11p. It is interesting to note that selective inhibition of TACE
could suppress several cytokines and a diverse inflammatory
mediator like PGE2. This finding is in agreement with several
reports emphasizing the upstream role of TNF-a in inducing
several cytokines and mediators that orchestrate inflammatory
responses36,37.
Having observed that TACE inhibition led to suppression
of TPA-induced inflammation, we then intended to study the
effect of selective TACE inhibitor on inflammation-induced
tumor promotion. To investigate this, we chose to evaluate the
dose of compound 11p that exhibited the maximum suppres-
sion of inflammation (i.e. 2%) in mouse DMBA- and
TPA-induced skin carcinogenesis model. Interestingly, topical
treatment with compound 11p distinctly delayed the onset of
papilloma incidences in comparison to the control group.
Furthermore, the treatment significantly suppressed the tumor
multiplicity and incidences as evaluated by number of
papillomas per mouse and percentage of tumor bearing
mice. One interesting observation made in this study was the
elevation in serum TNF-a after repeated exposure to TPA.
The level of TNF-a was gradually increasing with the number
of TPA applications up to the 20th week, and was effectively
inhibited by the treatment with TACE inhibitor. These data
indicate that the TACE dependent TNF-a shedding might be
 DOI: 10.3109/08923973.2014.931421 Chemopreventive effect of a novel, selective TACE inhibitor 7
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Figure 4. Effect of compound 11 p on DMBA- and TPA-induced skin tumorigenesis. Skin cancer was initiated with single application of DMBA and
promoted with multiple TPA challenge. Topical application of 100 ml of compound 11p (2%) was done after 1 h of each TPA challenge for 20 weeks.
Skin papilloma 41 mm diameter were recorded weekly and each value represents the mean ± SEM of 12 mice (A). Percentage of mice bearing tumor
was calculated (B). Serum TNF-a after 3 h of TPA application was estimated on 5, 10 and 20th weeks and represented as mean ± SEM (n ¼ 5) (C).
*p50.05 when compared with normal control. #p50.05 when compared with vehicle control group.

involved in the TPA-induced skin tumorigenesis. Studies Conclusions

using knockout mouse have established the role of TNF-a in
In conclusion, the present study demonstrates the role of
DMBA- and TPA-induced skin cancer20,38. Additionally,
TACE in skin inflammation and associated carcinogenesis.
treatment with a TNF-a biosynthesis inhibitor (pentoxifyllin)
Our data suggest that selective blockade of TACE suppresses
or a specific antibody against TNF-a have demonstrated
TPA-induced epidermal hyperplasia, infiltration of inflam-
protection against skin tumorigenesis induced by DMBA and
matory cells and cytokine levels leading to protection against
TPA21,39. These two approaches are able to limit both the
skin inflammation. The results also indicate that blockade of
forms of TNF-a, i.e. soluble and the membrane-bound.
inflammatory events facilitated via TACE inhibition may play
However, TACE inhibition only limits the release of soluble
an important role in preventing the skin tumorigenesis in
TNF-a without affecting the membrane-bound form or the
mice. Our findings provide a new approach of topical delivery
TNF-a synthesis. Our findings demonstrate that inhibition of
of TACE inhibitor against skin inflammation and tumorigen-
soluble TNF-a alone, as with the selective TACE inhibitor,
esis which might be helpful in overcoming toxicity associated
can effectively prevent skin carcinogenesis.
with the systemic exposure.
In addition to TNF-a, pro-inflammatory cytokines like
IFN-g and IL-17 also play key role in tumor promotion.
Deletion of either IL-17 or its receptor reduced tumorigenesis Declaration of interest
in DMBA- and TPA-induced skin cancer40,41. Similarly, This work was supported by the Zydus Research Centre,
IFN-g also promotes DMBA- and TPA-mediated skin tumor Ahmedabad, India (Communication Number: ZRC-463).
development by enhancing IL-17 associated inflammatory The authors declare no conflict of interest.
reaction42. In our study of acute TPA-induced inflammation,
compound 11p treatment was able to inhibit both IFN-g and
IL-17 effectively. Here, it can be speculated that inhibition of References
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