Topical curcumin-based cream is equivalent to dietary curcumin in a skin cancer model.
ABSTRACT Skin squamous cell carcinoma (SCC), the most common cancer in the USA, is a growing problem with the use of tanning booths causing sun-damaged skin. Antiproliferative effects of curcumin were demonstrated in an aggressive skin cancer cell line SRB12-p9 (P < 0.05 compared to control). Topical formulation was as effective as oral curcumin at suppressing tumor growth in a mouse skin cancer model. Curcumin at 15 mg administered by oral, topical, or combined formulation significantly reduced tumor growth compared to control (P = 0.004). Inhibition of pAKT, pS6, p-4EBP1, pSTAT3, and pERK1/2 was noted in SRB12-p9 cells post-curcumin treatment compared to control (P < 0.05). Inhibition of pSTAT3 and pERK1/2 was also noted in curcumin-treated groups in vivo. IHC analysis revealed human tumor specimens that expressed significantly more activated pERK (P = 0.006) and pS6 (P < 0.0001) than normal skin samples. This is the first study to compare topical curcumin to oral curcumin. Our data supports the use of curcumin as a chemopreventive for skin SCC where condemned skin is a significant problem. Prevention strategies offer the best hope of future health care costs in a disease that is increasing in incidence due to increased sun exposure.
- SourceAvailable from: Jillian Wong[Show abstract] [Hide abstract]
ABSTRACT: Nonmelanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma, are common neoplasms worldwide and are the most common cancers in the United States. Standard therapy for cutaneous neoplasms typically involves surgical removal. However, there is increasing interest in the use of topical alternatives for the prevention and treatment of nonmelanoma skin cancer, particularly superficial variants. Botanicals are compounds derived from herbs, spices, stems, roots, and other substances of plant origin and may be used in the form of dried or fresh plants, extracted plant material, or specific plant-derived chemicals. They possess multiple properties including antioxidant, anti-inflammatory, and immunomodulatory properties and are, therefore, believed to be possible chemopreventive agents or substances that may suppress or reverse the process of carcinogenesis. Here, we provide a review of botanical agents studied for the treatment and prevention of nonmelanoma skin cancers.Dermatology Research and Practice 01/2013; 2013:837152.
Hindawi Publishing Corporation
Journal of Skin Cancer
Volume 2012, Article ID 147863, 9 pages
TopicalCurcumin-BasedCreamIs Equivalentto Dietary
KunalSonavane,1Jeffrey Phillips,1Oleksandr Ekshyyan,1,2TaraMoore-Medlin,1,2
JenniferRoberts Gill,3XiaohuaRong,1,2RaghunathaReddy Lakshmaiah,1FleuretteAbreo,4
DouglasBoudreaux,5John L.Clifford,3and Cherie-AnnO.Nathan1,2,6
1Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport,
LA 71130-3932, USA
2Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA
3Department of Biochemistry, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA
4Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA
5Boudreaux’s Compounding Pharmacy, Shreveport, LA 71130-3932, USA
6Department of Surgery, Overton Brooks VA Medical Center, Shreveport, LA 71130-3932, USA
Correspondence should be addressed to Cherie-Ann O. Nathan, firstname.lastname@example.org
Received 7 September 2012; Accepted 20 November 2012
Academic Editor: Ajit K. Verma
Copyright © 2012 Kunal Sonavane et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Skin squamous cell carcinoma (SCC), the most common cancer in the USA, is a growing problem with the use of tanning booths
(P < 0.05 compared to control). Topical formulation was as effective as oral curcumin at suppressing tumor growth in a mouse
compared to control (P = 0.004). Inhibition of pAKT, pS6, p-4EBP1, pSTAT3, and pERK1/2 was noted in SRB12-p9 cells post-
curcumin treatment compared to control (P < 0.05). Inhibition of pSTAT3 and pERK1/2 was also noted in curcumin-treated
groups in vivo. IHC analysis revealed human tumor specimens that expressed significantly more activated pERK (P = 0.006)
and pS6 (P < 0.0001) than normal skin samples. This is the first study to compare topical curcumin to oral curcumin. Our data
supports the use of curcumin as a chemopreventive for skin SCC where condemned skin is a significant problem. Prevention
strategies offer the best hope of future health care costs in a disease that is increasing in incidence due to increased sun exposure.
The American Cancer Society estimates that 1–1.3 million
cases of nonmelanoma skin cancer (NMSC) will be detected
annually. Cutaneous SCC accounts for nearly 20% of all
skin cancers, and excluding melanoma, 75% of all deaths
attributed to skin cancers . Unlike the more prevalent
basal cell carcinoma (BCC), SCC is an aggressive tumor
that metastasizes with a frequency as high as 12.5% .
Prevalence is common in fair complexion Caucasians with
lower reported rates in individuals with darker complexions
including Asians and Africans. Cutaneous SCC of the face
often metastasizes to parotid lymph nodes, which can be
detrimental to the facial nerve during treatment and nodes
in the neck, as the head and neck are rich in lymphatic
networks. Treatment for NMSC may include cryotherapy,
electrosurgery, topical 5-fluorouracil, photodynamic ther-
apy, imiquimod, and radiation therapy; however, surgical
intervention is the primary treatment modality. When
treated early, the five-year cure rate is greater than 90%
. NMSC recurrence varies from 8–16%, second lesion
recurrence rates are as high as 75% within the first two
years and 95% within five years . This suggests a window
of opportunity for chemopreventive agents to delay or
prevent a recurrence or metastatic spread. Lymph node
metastasis in NMSC varies from 0.1 to 28%, with a resulting
2Journal of Skin Cancer
mortality from 50–75% . Overall five-year survival rates
for regional lymph node metastasis are 25–35% [3, 5–7] and
less than 20% at ten years . Early cancer detection offers
the best window of opportunity for treatment. Early stage
skin cancer has a high cure rate, whereas advanced stage
cutaneous SCC often develops resistance to chemotherapy.
Therefore, research has focused on developing these novel
chemopreventive agents to delay or prevent cutaneous SCC
been investigated in a variety of human cancers including
pancreatic, prostate, breast, and head and neck cancer.
The first published report demonstrating the topical use
of curcumin in cancer reported a sustainable reduction in
lesion size and pain . Curcumin has antioxidant, anti-
inflammatory, antiangiogenic and anticarcinogenic activity,
although its clinical use is limited by low bioavailability .
More recently, several studies have examined curcumin’s
effect in inhibiting skin carcinogenesis. Additionally, numer-
ous reports have identified signaling pathways related to
epidermal growth factor receptor (EGFR) that are essential
to formation and progression of cutaneous malignancy. The
MTOR and MEK/ERK signaling cascades are two of the
most well-studied pathways . In a prior study by our
group  we subcutaneously injected immunodeficient
mice with SRB12-p9 skin SCC and demonstrated that
curcumin administered by oral gavage significantly inhibited
tumor growth and downregulated pS6, a well-established
downstream biomarker of the MTOR and MEK/ERK path-
ways. Curcumin’s anti-carcinogenic effects have been linked
to inhibition of the MEK/ERK signaling pathway in breast
carcinogenesis, and researchers continue to explore these
potential biomarkers in other cancers . However, ERKs
activity in cutaneous malignancy is not well defined in
the literature. Hence, we wanted to determine if topical
curcumin was as efficacious as oral curcumin in a SCC
skin xenograft model and elucidate the pathways down-
regulated by curcumin as potential biomarkers for future
chemopreventive studies with our topical curcumin cream.
In addition, we wanted to observe the potentially additive
effects of topical application and oral dosing. We also wished
to explore whether the MEK/ERK pathway is overexpressed
a novel intracellular target at which curcumin may act to
inhibit tumorigenesis. We hypothesized that pERK and its
downstream target pS6 would be overexpressed in cutaneous
skin cancers given its role in promoting cellular proliferation
in aggressivemalignancy. Identifying intermediate endpoints
is necessary to assess intervention results for primary cancer
prevention and address problems with feasibility posed by
large patient numbers, length of study, and cost when cancer
occurrence or recurrence is an endpoint .
2.1. Curcumin. Curcumin C3 Complex (>98% pure) was
obtained from Sabinsa Corp. In vivo studies were conducted
with curcumin (15mg) suspended in vehicle (100μL corn
oil) for oral gavage feeding or suspended in a vanishing
provided by our study compounding pharmacist (DB).
2.2. Cell Lines and Xenografts. The human skin SCC cell
line SRB12-p9 was derived by single-cell cloning from
aggressive skin SCC SRB12 cells (a gift from Dr. Reuben
Lotan, Department of Thoracic Head and Neck Medical
Oncology, University of Texas M.D. Anderson Cancer Center
chosen due to its sensitivity to curcumin as evidenced in cell
culture studies. DNA was isolated from the cell lines using a
commercially available DNA purification kit (Qiagen). DNA
sample was sent to Genetica (Cincinnati, OH, USA), and the
cell line was validated by DNA profiling.
2.3. Cell Proliferation. 2,000 SRB12-p9 cells per well were
seeded in triplicate onto 96 well plates in complete media
at 37◦C with 5% CO2. After adherence, cells were treated
with curcumin (0–40μM) for 0–72 hours. Cell viability was
measured using MTS (Promega).
2.4. Subcutaneous HNSCC Xenograft Model. Studies were
conducted in accordance with the Declaration of Helsinki
(1964) and in compliance with Louisiana State University
Health Sciences Center Institutional Animal Care and Use
Committee guidelines. Animals housed in a barrier facility
were maintained on a normal diet ad lib. Forty 6–8-
week-old Severe Combined Immunodeficiency (SCID) mice
were shaved and pretreated with either 0mg (corn oil),
15mg curcumin by oral gavage, 15mg curcumin topical
paste, or combined 15mg oral gavage and 15mg curcumin
topical paste once daily for 3 days prior to squamous cell
carcinoma xenograft injection (n = 10 per group). Mice
were then injected subcutaneously with 1 × 106SRB12-p9
cells suspended in sterile PBS (Day 0). All mice continued
daily treatment with either 0mg or 15mg curcumin by
gavage, topical, or both, and tumors were measured daily
with digital calipers. Xenograft tumors did not form in
one animal per group and were excluded (n = 9 per
group). Tumor volume (mm3) was calculated using the
following formula: (0.52 × length2× width). Body weight
was measured daily, and mice were monitored for adverse
effects from the experiment. Daily oral gavage and tumor
volume measurement continued through day 29, at which
time tumors were harvested after the mice were anesthetized
with isoflurane and sacrificed. Ex vivo tumor volume was
calculated using the following formula: (4/3π0.5 × length ×
0.5 × width × 0.5 × height). The study pathologist (FA)
measured maximum skin thickness, including the stratum
corneum but not the granular layer.
2.5. ELISA. Pooled serum from mice (n = 3/group) was
analyzed by enzyme-linked immunosorbent assay (ELISA,
to assess expression of human and murine IL6. Samples
were analyzed in duplicate for IL-6 expression with a
spectrophotometric plate reader.
Journal of Skin Cancer3
2.6. Immunohistochemical Analysis of Molecular Markers in
Skin Squamous Cell Carcinoma. Tumors harvested on day 29
were embedded in paraffin, sectioned, and H&E stained for
confirmation of squamous cell carcinoma presence by our
study pathologist (FA). Tumors (n = 3 per group) were then
stained with phospho-ERK (cell signaling, Thr202/Tyr204;
1:600) and phospho-STAT3 (cell signaling, Tyr705; 1:200)
as previously described [15, 16]. Subcellular localization
was determined by immunofluorescence. Paraffin sections
of tumors with overlying mouse skin were probed with
pERK1/2 and pSTAT3 antibodies (Cell Signaling) followed
by an Alexa-546-labeled secondary antibody.
Human actinic keratosis, skin SCC, and BCC paraffin-
embedded blocks were sectioned and stained with phospho-
p44/42 MAPK (ERK 1/2) rabbit monoclonal antibody
(Thr202/Tyr204, 1:600) and phospho-S6 ribosomal protein
rabbit monoclonal antibody (Ser235/236, 1:100) as previ-
ously described [17–19] and read by our study pathologist
(FA). Specimens were scored based on the intensity of
antibody nuclear and cytoplasmic staining in each slide, with
absence of staining scored as a , weak or focal staining
scored as a [+], and strong staining with a [++].
2.7. Western Blot Analysis. Soluble proteins extracted from
SRB12-p9 cell lysates treated with 0μM or 20μM curcumin
for 24 hours or xenograft tumors were analyzed by western
blot as previously described . Proteins were detected
using enhanced chemiluminescence (Amersham Pharmacia
Biotech, Piscataway, NJ, USA) and analyzed with Image-
Quant TL7.0 (GE Healthcare) software (n = 6/group).
The following antibodies from cell signaling were used:
AKT (1:200), phospho-AKT (Ser473; 1:100), S6 ribosomal
protein (1:500), phospho-S6 ribosomal protein (Ser235/236;
1:500), STAT3 (1:200), phospho-STAT3 (Tyr705; 1:200),
4EBP1 (1:200), phospho-4EBP1 (Ser65; 1:200), ERK1/2
(1:200), phospho-ERK1/2 (Thr202/Tyr204; 1:200), and actin
2.8. Patient Tissue Samples and Controls. All BCC and SCC
tissue samples were obtained from patients recently diag-
nosed with nonmelanoma skin cancer of the face or
neck, after obtaining approval by the institutional review
board and obtaining informed consent from all subjects.
Patients were treated primarily with surgical resection at
Louisiana State University Health Shreveport and the Over-
ton Brooks Veterans Administration Hospital from 2009
to 2011. Formalin-fixed, paraffin-embedded tissue blocks
were obtained from 27 BCC tissue samples, 4 Actinic
Keratosis (AK) tissue samples, and 17 SCC tissue samples
(from 16 SCC patients). Normal human skin samples were
undergoing resection for skin cancer. Total of 25 normal
(noncancer) skin samples were analyzed in the study. Several
5μm slides were cut from each tissue block, and one
slide was stained with hematoxylin and eosin (H&E) and
reviewed by a pathologist to confirm pathologic findings
and assess surgical margins. All other slides were used for
2.9. Statistics Applied for the Analysis. Proliferating cell per-
centages were compared using one-way analysis of variance
(ANOVA). One-way ANOVA was also used to determine
significant differences in skin thickness and the differences
between individual treatment groups. A Tukey’s multiple
comparison as a post hoc test was performed to eval-
uate differences between treatment groups. Tukey’s post-
hoc testing, Chi-square test for independence, or Fisher’s
exact probability test was used to determine the ability
of pERK and pS6 expression to correlate with cutaneous
SCC, differentiate tumor types from normal skin and BCC,
and determine if there was a significant difference between
pERK and pS6 staining and the different types of histologic
cutaneous lesions. Paired t-test was used to determine
significant difference in biomarker expression by western
3.1. Growth Inhibitory Effects of Curcumin In Vitro and In
Vivo. To determine whether a skin SCC cell line is sensitive
to curcumin, a cell proliferation assay was performed on
SRB12-p9 SCC cell line. Curcumin’s growth inhibitory
effects in the aggressive skin cancer cell line (SRB12-p9)
were noted as early as day 2 at 20μM (P
20μM and 40μM was significantly effective in inhibiting the
proliferation of SRB12-p9 cells compared to control on days
2 and 3 (P < 0.05; Figure 1(a)).
in SRB12-p9 xenograft tumors after tumor cells had a
chance to engraft (Figure 1(b)). There was a significant effect
for curcumin treatment (F(3,96) = 11.58, P < 0.001)
in suppressing growth of the SRB12-p9 xenograft tumors.
Tukey’s post hoc comparisons of the four groups indicate
tumor volume from the gavage group (M = 44.55, 95%
CI [35.77, 53.77]) and the combined group (M = 88.81 CI
[71.73, 105.89]) was significantly smaller than the control
group tumor volume (M
= 191.35, 95% CI [127.12,
255.59]), P < 0.001. The topical group (M = 130.66, 95% CI
[95.29, 166.04]) tumor volume was also statistically smaller
than the control group tumor volume (P = 0.02). There was
no difference between the gavage group tumor volume and
the topical group tumor volume (P = 0.19).
Because invasive tumors could give inaccurate measure-
ments and overlying skin could influence in vivo tumor mea-
surements, we also measured tumors ex vivo and measured
skin thickness (Figure 1(c)). There was a significant effect
of curcumin on ex vivo tumor volume (F(3,32) = 5.49,
P = 0.004). Tukey’s post hoc comparisons of the four groups
72.06, 95% CI [37.78, 106.35]), topical group (M = 195.82,
95% CI [71.59, 320.05]), and combined group (M = 152.32,
control (M = 416.77, 95% CI [161.48, 672.06]), P < 0.001,
P = 0.006 and 0.02, respectively. There was a significant
effect for curcumin treatment on tumor mass (F(3,32) =
5.79, P = 0.003), where the gavage group (M = 0.043, 95%
4Journal of Skin Cancer
Day 0 Day 1
Day 2Day 3
Days after tumor injection
Tumor volume (mm3)
Figure 1: Curcumin inhibits skin SCC cell growth in vitro and in vivo. (a) Cell proliferation of the aggressive skin cancer cell line SRB12-p9
after treatment with 0–40μM curcumin.∗P < 0.05 versus control group;∗∗P < 0.01 versus control group;∗∗∗P < 0.001 versus control
group. (b) Mice were pretreated with the indicated dose of curcumin for 3 days prior to injection with 1 × 106SRB12-p9 tumor cells in the
dorsal region (day 0) and continued receiving daily curcumin treatment (9 mice per group, mean tumor volume ± SD). Tukey’s post hoc test:
∗P < 0.05 versus control group;∗∗∗P < 0.001 versus control group. (c) Representative images of xenograft tumors at harvest and ex vivo
from the indicated treatment groups.
CI [0.02, 0.07]), topical group (M = 0.112, 95% CI [0.041,
0.184]), and combined treatment group tumors (M = 0.076,
95% CI [M = 0.050, 0.101]) were significantly smaller
than that of the control group (M = 0.244, 95% CI [0.09,
0.39]) tumors, P < 0.001, P = 0.02, and 0.003, respectively.
There was no difference in skin thickness in mice treated
with curcumin by gavage, topical, and combined groups
compared to the control group (P = 0.73).
3.2. Curcumin’s Effects on Signaling Pathways. We next eval-
uated curcumin’s effects on signaling pathways in the aggres-
siveskin cancercellline (SRB12-p9) invitro. Usinga concen-
tration that significantly inhibited cell growth (20μM), there
was significant inhibition of pAKT, pS6, p-4EBP1, pSTAT3,
and pERK1/2 (Figure 2). As can be seen in Figure 2 there
was about twofold inhibition in the phosphorylation of the
aforementioned markers in SRB12-p9 cells after curcumin
We next evaluated curcumin’s effects on signaling path-
ways in xenograft tumors using western blot analysis
(Figure 3). Among the tested biomarkers an inhibition of
inhibition of pSTAT3 was only noted in the combined
curcumin group (Figure 3(a)).
As western blot analysis involves homogenization of
total tumor tissue, such as stroma and infiltrating host
inflammatory cells, we also evaluated curcumin’s effects
on signaling pathways by immunohistochemistry, which
can distinguish nonviable and nontumor components, such
as stroma, that are not included in the scoring of the
biomarker analyzed. IHC results revealed strong positive
pERK staining throughout tumors in the control group
and weaker, focal staining in the curcumin-treated tumors
(Figure 3(b)). Immunofluorescence confirmed curcumin’s
effects on pERK and a shift in the subcellular localization of
the control group (Figure 3(c)). Curcumin is known for its
anti-inflammatory effects. Therefore, we evaluated its effects
on the inflammatory marker IL6 in all curcumin treatment
groups using pooled serum samples. The levels of soluble
Journal of Skin Cancer5
Expression of biomarkers normalized to actin (a.u.)
Figure 2: Curcumin’s effects on AKT/MTOR and ERK pathways in vitro. (a) Western blot of SRB12-p9 tumor cells treated with (+) or
without (−) 20μM curcumin for 24 hours and probed with the indicated antibody. Representative Western blots for two analyzed sets
are shown. (b) Band densities of indicated biomarkers (n = 6) were quantified using ImageQuant software and normalized to actin protein
level. Data presented as Mean ± SE.∗Indicates P < 0.05 versus vehicle-treated control. A significant inhibition ofexpression of the following
biomarkers was observed: pAKT (P = 0.0368); pS6 (P = 0.0182); p4EBP1 (P = 0.0098); pSTAT3 (P < 0.0001); pERK1/2 (P = 0.0313). a.u.:
IL6 were the lowest in the topical curcumin group, while
curcumin did not affect IL6 levels in the gavage or combined
groups (Figure 3(d)).
3.3. Patient Characteristics. Patient demographics and clini-
cal characteristics are summarized in Table 1. Tissue samples
from 46 male patients and 4 female patients were analyzed.
Age ranged from 39 to 93 with a mean age of 66 ± 14
years. There was no difference in age between the groups by
ANOVA(F = 1.272,P = 0.29).Thelargemajorityofpatients
were white, except for one African American patient with
albinism. Nonmelanoma skin cancers analyzed were excised
from the external nasal skin (14), cheeks (14), ears (9), scalp
and forehead (13), neck, chin, and lip (6). No skin site was
overrepresented in analysis.
3.4. IHC Analysis of Patient Tissues. The presence and inten-
sity of pERK and pS6 staining in all SCC, BCC, and normal
tissue samples were compared (Table 2). All SCC specimens
(n = 17, 100%) stained positive for phosphorylated ERK,
while only 10 of 27 (37%) BCC samples stained positive.
Although all the normal skin samples stained weakly positive
(grade 1+) for activated pERK in the stroma, palisading
cells, and epithelium (n = 24, 100%), significantly more
SCC specimens showed strong staining with pERK (grade
2+) than normal skin (P = 0.0028, Table 2 and Figure 4).
However, the majority of BCC specimens (17/27, 63%)
showed no pERK staining (P < 0.0001 compared to normal
Most specimens containing SCC (n = 13, 81%) and
BCC (n = 16; 64%) showed strong staining (grade 2+) for
activated pS6, while all the analyzed normal skin specimens
(n = 8; 100%) demonstrated negative pS6 staining. Tumor
specimens expressed significantly more activated pS6 than
normal skin samples (1+ score and above; P < 0.0001;
Figure 4). Skin cancer type significantly predicted intensity
of pERK staining, as SCC tumors stained more intensely for
pERK than the background stroma in normal skin and BCC
tumor cells (P < 0.0001; Figure 4). When pERK expression
was analyzed and compared to other demographic factors,
the variance in pERK expression scores correlated signifi-
cantly with tumor type, R2= 0.25, P = 0.0007. Patient
age (P = 0.85) and gender (P = 0.35) did not explain the
variance in pERK staining.
6Journal of Skin Cancer
Figure 3: Curcumin’s effects on the ERK pathway in vivo. (a) Western blot of pooled xenograft tumors (n = 6/group) of the indicated
antibody. (b) The presence and intensity of pERK staining (brown) in the control group compared to the presence and intensity of pERK
of tumors were probed with STAT3 phospho-Tyr705 (pSTAT3, top row) or ERK1/2 phospho-Thr202/Tyr204 (pERK, bottom row), followed
by an Alexa546-labeled secondary antibody (400x). (d) IL-6 ELISA of pooled mouse serum (n = 3/group) in duplicate.
Identifying consistent intracellular biomarkers at which a
potential chemopreventive may act is essential prior to
initiating clinical trials. As curcumin acts on many different
biomolecular targets in a variety of different cell types it is
important to determine if curcumin directly affects either
a few major downstream biomarkers or a multiplicity of
downstream targets which may serve to explain curcumin’s
varying effects in different cell types. Aberrant signaling
through the epidermal growth factor receptor (EGFR) plays
a major role in cutaneous skin cancer progression. EGFR
inhibitors have been used for SCC therapy to downregulate
aberrant EGFR signaling with little change in overall survival
, possibly due to compensating mutations downstream
of EGFR. One of these signaling pathways is PI3K/AKT
that plays a role in skin carcinogenesis and in chemotherapy
Activated Ras/Raf signaling has also been implicated in a
activation of ERK1 and ERK2, which are constitutively active
in 70% of malignant melanoma due to RAS or BRAF
activating mutations . Activated ERK1/2 is rarely seen in
normal skin specimens but is shown in all cases of SCC with
a positive association with the degree of malignancy and
proliferative activity of SCC . In this study, Zhang et al.
looked at 10 well-differentiated and 10 poorly differentiated
human head and neck squamous carcinoma specimens .
Therefore, inhibiting ERK may be a promising approach
in targeted cutaneous skin SCC therapy. Having previously
Journal of Skin Cancer7
Table 1: Clinical and demographic patient characteristics.
AK SCC BCCP value
Scalp and forehead
∗Normal skin samples were surgically obtained from uninvolved adjacent skin in patients undergoing resection for skin cancer.
∗∗Some patients had more than one type of cancer and are counted in both groups.
∗∗∗No significant difference in number of males and females, race, age, or skin site distribution per group by Fisher’s exact test.
Table 2: Summary of pERK and pS6 IHC staining in normal (noncancer), AK, BCC, and SCC skin samples.
∗Compared to normal skin by Fisher’s exact test. P values for overall comparison are shown. See text for a subset analysis.
determined curcumin’s growth inhibitory effects in skin SCC
, we sought to determine whether these effects were
similar to our observations in upper aerodigestive head
and neck SCC (HNSCC) where curcumin inhibited the
AKT/MTOR pathway through rapid curcumin-dependent
inhibition of MTOR’s downstream target pS6 and 4EBP1
In this study we found significant and complete inhi-
bition of SRB12-p9 cell proliferation after treatment with
curcumin at a dose 20μM or higher (Figure 1(a)) suggesting
a highly potent anticarcinogenic effect of curcumin in skin
cancer. Additionally, we found that the inhibitory effect of
curcumin on skin cancer proliferation was associated with
inhibition of AKT/mTOR and ERK signaling (Figure 2).
In our in vivo study, curcumin paste was formulated to
penetrate human skin epidermis and dermis. However, given
the thin nature of mouse skin, topical curcumin penetration
was much greater such that curcumin possibly did not
remain in the epidermis for a prolonged period, leading
to prolonged contact with the cancer cells. The irritant
nature of the cream caused the skin overlying the tumor
to thicken, although this was not statistically significantly
different from control (P = 0.73). The SRB12-p9 cell line
is invasive in this model , producing inaccurate tumor
caliper measurements due to the inability to account for
the portion of the tumor that invaded into the abdominal
wall. Therefore, the ex vivo tumor weight provided a more
accurate tumor size endpoint. In human skin, SCC emerges
directly from the epidermal layer, unlike in our xenograft
therefore anticipate a more pronounced tumor-suppressive
effect of topical curcumin in humans.
The SRB12-p9 xenograft cells were more sensitive to
curcumin-induced cell death and apoptosis than the sur-
rounding normal mouse skin and grew at a much slower
8Journal of Skin Cancer
Normal AK BCCSCC
Figure 4: IHC analysis of pS6 and pERK expression in patients with negative (blue) staining and strong positive (brown) staining of tumor
cells with pERK and pS6. Normal patient skin samples with minimal background staining and normal appearing cells. Representative
and few scattered positive (brown) staining of tumor cells. Representative SCC patient samples with strong positive (brown) nuclear and
cytoplasmic staining with pERK and pS6. Note that the stroma stains positive (brown) in BCC, whereas the tumor stains negative (blue).
inflammatory response . Because IL-6 may contribute
to angiogenesis and metastasis , inhibition of IL-6 with
Although curcumin has previously been shown to inhibit
IL-6 in HNSCC cell lines , this is the first skin cancer
model investigating curcumin’s inhibition of systemic IL-
6. The present study demonstrates that topical curcumin
reduces skin SCC tumor growth, and this effect might be
explained, by the inhibition of IL-6.
In this study we demonstrated significant inhibition of
several biomarkers of the AKT/mTOR pathway as well as
STAT3 and ERK1/2 in SRB12-p9 cells after treatment with
20μM of curcumin. In our in vivo experimentation, we
and inhibition of pSTAT3 in the combined curcumin group.
However, tumor heterogeneity and degree of dysplasia can
often confound immunohistochemistry results, depending
on where in the lesion the biopsy was taken. Therefore,
it is important to develop serum biomarkers that can be
obtained with a simple blood draw. As curcumin is a well-
known anti-inflammatory agent, we measured its effects on
pooled serum of treated mice and noted a decrease in IL-
6 in the topical group compared to the control group. We
observed that systemic curcumin did not cause a decrease
in serum IL-6 levels. However, only three mice in each
group were analyzed, and it is possible that statistically
significant differences in IL6 levels could be detected upon
analysis of greater numbers of mice in the topical and
combined curcumin-treated groups compared to control
As curcumin slowed progression of aggressive skin SCC
xenografts and inhibited pERK expression, the ERK pathway
may prove to be a key biomarker in developing topical
pharmaceutical agents that prevent skin SCC tumor growth
or recurrence. We observed that the overall reduction in
pERK staining in the curcumin-treated tumors was not cell
autonomous but rather manifested as an expansion in areas
of very low or no expression, such that focal regions of
intense staining remained. Alternatively, control tumors had
smaller regions of low staining and a higher number of
of pERK staining was achieved with curcumin treatment,
rather than a complete shutdown.  confirmed that
phosphorylated ERK is overexpressed in patient skin SCC in
a Caucasian population, which further supports our findings
and suggests that pERK may be a useful chemoprevention
Chronic inflammation is linked to both cancer and
angiogenesis. The anti-inflammatory properties of curcumin
may contribute to its potential as an effective chemopreven-
tive agent. However, curcumin’s systemic anti-inflammatory
effects (reduced serum IL-6 levels) were more pronounced
in topical curcumin group compared to gavage. Given these
findings, it was unexpected that tumor growth was inhibited
more effectively in the gavage group than in the topical
group. However, there was no statistically significant differ-
ence in tumor volume between the two treatment groups.
Despite this data, we speculate that local anti-inflammatory
activity of topically applied curcumin contributes signifi-
As curcumin continues to be explored as a chemopre-
ventive and therapeutic agent for skin cancer treatment,
establishing defined biomarkers upon which curcumin acts
to inhibit tumorigenesis is essential. The ERK pathway is
an important protein kinase signaling cascade involved in
cellular proliferation and is activated in carcinogenesis. In
this study, activated pERK expression significantly increased
in SCC compared to the less aggressive BCC and AK. As
curcumin has been shown to inhibit activated ERKs in
carcinogenesis, the present data suggests that components
of the ERK pathway may prove to be key biomarkers for
curcumin chemopreventive efficacy in cutaneous SCC.
Journal of Skin Cancer9
Conflict of Interests
The authors declare that they have no conflict of interests.
 M. Alam and D. Ratner, “Cutaneous squamous-cell carci-
noma,” The New England Journal of Medicine, vol. 344, no. 13,
pp. 975–983, 2001.
 C. L. Green and P. A. Khavari, “Targets for molecular therapy
of skin cancer,” Seminars in Cancer Biology, vol. 14, no. 1, pp.
 D. E. Rowe, R. J. Carroll, and C. L. Day, “Prognostic factors for
local recurrence, metastasis, and survival rates in squamous
cell carcinoma of the skin, ear, and lip: implications for treat-
ment modality selection,” Journal of the American Academy of
Dermatology, vol. 26, no. 6, pp. 976–990, 1992.
 V. de Lima Vazquez, T. Sachetto, N. M. Perpetuo, and A. L.
Carvalho, “Prognostic factors for lymph node metastasis from
advanced squamous cell carcinoma of the skin of the trunk
and extremities,” World Journal of Surgical Oncology, vol. 6,
article 73, 2008.
 M. G. Joseph, W. P. Zulueta, and P. J. Kennedy, “Squamous cell
carcinoma of the skin of the trunk and limbs: the incidence of
metastases and their outcome,” Australian and New Zealand
Journal of Surgery, vol. 62, no. 9, pp. 697–701, 1992.
 R. E. Kwa, K. Campana, and R. L. Moy, “Biology of cutaneous
squamous cell carcinoma,” Journal of the American Academy of
Dermatology, vol. 26, no. 1, pp. 1–26, 1992.
 D. H. Kraus, J. F. Carew, and L. B. Horrison, “Regional lymph
node metastasis from cutaneous squamous cell carcinoma,”
Archives of Otolaryngology—Head and Neck Surgery, vol. 124,
no. 5, pp. 582–587, 1998.
 R. Kuttan, P. C. Sudheeran, and C. D. Josph, “Turmeric and
curcumin as topical agents in cancer therapy,” Tumori, vol. 73,
no. 1, pp. 29–31, 1987.
 C. H. Hsu and A. L. Cheng, “Clinical studies with curcumin,”
Advances in Experimental Medicine and Biology, vol. 595, pp.
 J. Dujic, S. Kippenberger, A. Ramirez-Bosca et al., “Curcumin
in combination with visible light inhibits tumor growth in a
xenograft tumor model,” International Journal of Cancer, vol.
124, no. 6, pp. 1422–1428, 2009.
 J. M. Phillips, C. Clark, L. Herman-Ferdinandez et al.,
“Curcumin inhibits skin squamous cell carcinoma tumor
growth in vivo,” Otolaryngology—Head and Neck Surgery, vol.
145, pp. 58–63, 2011.
 W. F. Hua, Y. S. Fu, Y. J. Liao et al., “Curcumin induces down-
regulation of EZH2 expression through the MAPK pathway in
Pharmacology, vol. 637, no. 1–3, pp. 16–21, 2010.
 J. S. Vourlekis and E. Szabo, “Predicting success in cancer
prevention trials,” Journal of the National Cancer Institute, vol.
95, no. 3, pp. 178–179, 2003.
 J. L. Clifford, X. Yang, E. Walch, M. Wang, and S. M.
Lippman, “Dominant negative signal transducer and activator
of transcription 2 (STAT2) protein: stable expression blocks
interferonalphaactioninskinsquamouscell carcinoma cells,”
Molecular Cancer Therapeutics, vol. 2, pp. 453–459, 2003.
 C. A. O. Nathan, N. Amirghahari, F. Abreo et al., “Overex-
and neck cancer patients via activation of the Akt/mammalian
no. 17, pp. 5820–5827, 2004.
 H. Kleiner-Hancock, R. Shi, A. Remeika et al., “Effects of
ATRA combined with citrus and ginger-derived compounds
in human SCC xenografts,” BMC Cancer, vol. 10, article 394,
 Z. Syed, S. B. Cheepala, J. N. Gill et al., “All-trans retinoic acid
suppresses Stat3 signaling during skin carcinogenesis,” Cancer
Prevention Research, vol. 2, no. 10, pp. 903–911, 2009.
 C. A. Clark, M. D. McEachern, S. H. Shah et al., “Curcumin
inhibits carcinogen and nicotine-induced mammalian target
of rapamycin pathway activation in head and neck squamous
cell carcinoma,” Cancer Prevention Research, vol. 3, no. 12, pp.
 W. Yin, S. Cheepala, J. N. Roberts, K. Syson-Chan, J.
DiGiovanni, and J. L. Clifford, “Active Stat3 is required for
survival of human squamous cell carcinoma cells in serum-
free conditions,” Molecular Cancer, vol. 5, article 15, 2006.
 D. L. Wheeler, E. F. Dunn, and P. M. Harari, “Understanding
resistance to EGFR inhibitors-impact on future treatment
strategies,” Nature Reviews Clinical Oncology, vol. 7, no. 9, pp.
 S. Claerhout, L. Verschooten, S. Van Kelst et al., “Concomitant
inhibition of AKT and autophagy is required for efficient
cisplatin-induced apoptosis of metastatic skin carcinoma,”
International Journal of Cancer, vol. 127, no. 12, pp. 2790–
 W. E. Pierceall, L. H. Goldberg, M. A. Tainsky, T. Mukhopad-
hyay, and H. N. Ananthaswamy, “ras Gene mutation and
amplification in human nonmelanoma skin cancers,” Molec-
ular Carcinogenesis, vol. 4, no. 3, pp. 196–202, 1991.
 X. Zhang, T. Makino, F. C. Muchemwa et al., “Activation of
the extracellular signal-regulated kinases signaling pathway in
squamous cell carcinoma oftheskin,”BioscienceTrends,vol.1,
no. 3, pp. 156–160, 2007.
 J. Albanell, J. Codony-Servat, F. Rojo et al., “Activated extra-
cellular signal-regulated kinases: association with epidermal
growth factor receptor/transforming growth factor α expres-
sion in head and neck squamous carcinoma and inhibition
by anti-epidermal growth factor receptor treatments,” Cancer
Research, vol. 61, no. 17, pp. 6500–6510, 2001.
 S. Rose-John, J. Scheller, G. Elson, and S. A. Jones,
“Interleukin-6 biology is coordinated by membrane-bound
and soluble receptors: role in inflammation and cancer,”
Journal of Leukocyte Biology, vol. 80, no. 2, pp. 227–236, 2006.
and L. Y. Dirix, “Serum interleukin 6, plasma VEGF, serum
VEGF, and VEGF platelet load in breast cancer patients,”
Clinical Breast Cancer, vol. 2, no. 4, pp. 311–315, 2002.
 A. N. Cohen, M. S. Veena, E. S. Srivatsan, and M. B. Wang,
“Suppression of interleukin 6 and 8 production in head and
neck cancer cells with curcumin via inhibition of Iκβ kinase,”
Archives of Otolaryngology—Head and Neck Surgery, vol. 135,
no. 2, pp. 190–197, 2009.