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R E S E A R C H A R T I C L E Open Access
Cellular effects of a turmeric root and
rosemary leaf extract on canine neoplastic
cell lines
Corri B. Levine
1
, Julie Bayle
2
, Vincent Biourge
2
and Joseph J. Wakshlag
1*
Abstract
Background: The use of nutraceuticals is gaining in popularity in human and canine oncology with a relatively
limited understanding of the effects in the vastly different tumor types seen in canine oncology. We have previously
shown that turmeric root (TE) and rosemary leaf (RE) extracts can work synergistically to reduce neoplastic cell growth,
but the mechanisms are poorly understood and require further elucidation.
Results: Three different canine cell lines (C2 mastocytoma, and CMT-12 mammary carcinoma, D17 osteosarcoma) were
treated with 6.3 μgmL
−1
extract individually, or 3.1 μgmL
−1
of each extract in combination based on studies showing
synergy of these two extracts. Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of
signaling pathways were assessed. The TE + RE combination treatment resulted in Caspase 3/7 activation and apoptosis
in all cell lines, beyond the effects of TE alone with the CMT-12 cell line being most susceptible. Both extracts had
antioxidant effects with RE reducing reactive oxygen species (ROS) by 40–50% and TE reducing ROS by 80–90%. In
addition RE treatment enhanced the c-jun N-terminal kinase (JNK) activity in the C2 cell line and TE + RE exposure
increased activated JNK by 4–5 times in the CMT-12 cell line. Upon further examination, it was found that RE treatment
caused a significant increase in the cellular accumulation of curcumin by approximately 30% in the C2 and D17 cell
lines, and by 4.8-fold in the CMT-12 cell line. This increase in intracellular curcumin levels may play a role in the synergy
exhibited when using TE and RE in combination.
Conclusions: The use of RE in combination with TE induces a synergistic response to induce apoptosis which is better
than either extract alone. This appears to be related to a variable increased TE uptake in cells and activation of
pathways involved in the apoptotic response.
Keywords: Apoptosis, Canine cancer, Mammary carcinoma, Osteosarcoma, Mastocytoma, Curcumin, Rosemary
Background
The use of natural remedies, or nutraceuticals, in the
treatment of cancer and a variety of other diseases ap-
pears prevalent in human and veterinary medicine. The
use of plant extracts has been around for centuries, but
investigations into the mechanisms of action across vari-
ous cancer cell lines are more recent, and appear to be
highly variable in cell culture systems [1–3]. The effect-
ive compounds of interest have been purified from a var-
iety of plants and are used in treating various diseases,
including cancer [4]. The benefit of using these plant
extracts to treat cancer is the potential synergy of mul-
tiple compounds found within a single extract whereby
the major compound may have one or more targets,
while other molecules in the extract may be affecting
other targets or influencing absorption kinetics [5].
The effects of these purified compounds have been ex-
amined in vitro in a variety of human cell lines derived
from tumors of the colon, skin, and breast tissue [6], but
only a few studies have looked at the effects in canine
cancer cells lines [7–9]. The major types of cancer found
in the dog differ from humans with lymphoma, mast cell
disease, osteosarcoma, and mammary neoplasia being
most often diagnosed in canine oncology [10]. We previ-
ously identified two extracts, turmeric extract rich in
curcuminoids (TE) and rosemary leaf extract rich in
* Correspondence: jw37@cornell.edu
1
Department of Clinical Sciences,Veterinary Medical Center C2-009, Cornell
University College of Veterinary Medicine, Ithaca, NY 14853, USA
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Levine et al. BMC Veterinary Research (2017) 13:388
DOI 10.1186/s12917-017-1302-2
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
carnosic acid (RE), which were shown to be cytotoxic
and reduce proliferation in a synergistic manner in ca-
nine mastocytoma, mammary carcinoma, and osteosar-
coma cell lines [11]. The use of TE and its major
compound of interest, curcumin, has been extensively
studied to treat a variety of diseases and ailments, per-
haps due to its ability to bind and interact with a variety
of cellular proteins [12]. Unfortunately, the use of TE in
vivo has been limited by its poor bioavailability and ef-
forts are still underway to increase the absorption and
bioavailability of the curcuminoids found in this extract
[13]. This obstacle may be overcome through the use of
combination treatments with other extracts that improve
bioavailability or hinder additional pathways [14–16]. In
our previous study, RE worked in a synergistic manner
with TE to decrease cellular proliferation when used in
combination. Carnosic acid, the compound of interest in
RE, can target a variety of signaling pathways, many of
which overlap with those targeted by curcumin. The
effects of these two compounds in combination have
been examined in acute myeloid leukemia cells and
breast cancer cells [17, 18], showing synergy in anti-
proliferative effects and increased pro-apoptotic signal-
ing. The safety of these commonly used feed ingredients
and continual synergy between the extracts make them
candidates for inclusion in the diet as a potential adju-
vant treatment for dogs diagnosed with neoplasia, if ap-
propriate serum concentrations can be achieved.
The objective of this in vitro study was to determine
the effects on canine cancer cell death and possible
mechanisms by which TE and RE exert anti-proliferative
and cytotoxic effects individually and in combination on
canine mastocytoma, mammary carcinoma, and osteo-
sarcoma cell lines. Concentrations were chosen based on
our prior publication surrounding the effective concen-
trations for synergy between the two extracts of interest
[11]. Markers of apoptosis, antioxidant capabilities, and
changes in the activation of common cell signaling path-
ways were analyzed after treatment.
Methods
Natural extracts
Turmeric extract (TE; 88% total curcuminoids, #DA251471
Naturex, Avignon, France) and rosemary extract (RE; 67%
carnosic acid, #302036 Vitiva, Markovcih, Slovenia) were
solubilized in 100% dimethyl sulfoxide (DMSO; Sigma-
Aldrich, St. Louis, MO, USA) at 20 mg mL
−1
.Freshextract
stock solutions were prepared and used for every
experiment.
Cell culture
Three canine neoplastic established cell lines, represent-
ing hematopoietic, epithelial, and mesenchymal tumor
types were used for all experiments; mastocytoma C2
(Dr. Warren Gold, University of California, San Fran-
cisco, USA), mammary gland carcinoma CMT-12 (Dr. R.
Curtis Bird, Auburn University, Alabama, USA), and
osteosarcoma D17 (#CCL-183; ATCC, Manassas, VA,
USA). These cell lines were chosen for initial screening
as representative cell lines of the three major cell line-
ages of cancer in dogs in hopes of finding a similar glo-
bal effect across different cell lineages. Cell lines were
grown on tissue culture-treated plates (Laboratory Prod-
uct Sales, Rochester, NY, USA) at 37 °C and 5% CO
2
for
all experiments and passage of cells, unless otherwise
noted. Cell lines were cultured in appropriate complete
medium as previously described.
11
All culture reagents
were purchased from Invitrogen, Carlsbad, CA, USA,
unless otherwise indicated.
Apoptosis–associated caspase 3/7 activation assay
Cells were plated at a density of 4 × 10
3
cells per well on
white walled 96-well tissue culture-treated plates (Ther-
moFisher Scientific, Waltham, MA, USA) and incubated
overnight in complete medium. Cells were treated the
following day with DMSO vehicle control, 6.3 μgmL
−1
extract alone, or 3.1 μgmL
−1
each extract in combin-
ation for 36 h. Chemotherapeutic drugs at a 50% inhibi-
tory concentration (IC
50
) were used as a positive
control; 12.5 nM toceranib phosphate (Palladia™, Zoetis
Animal Health, Florham Park, NJ) was used for the C2
cell line, and 0.3 or 0.5 μM doxorubicin hydrochloride
(Sigma Aldrich, St Louis, MO) was used for the CMT-12
and D17 cell lines, respectively. Background fluorescence
and luminescence was measured in wells containing
treatments but no cells. Caspase 3/7 activation was
quantified using the ApoLive-Glo™Multiplex Assay
(Promega, Madison, WI, USA) following manufacturer’s
instructions. Briefly, after 36 h of treatment, viability re-
agent was added to the wells and incubated at 37 °C for
30 m and fluorescence was measured at 400
Ex
/505
Em
.
Next, Caspase-Glo 3/7 Reagent was added to all wells,
incubated for 30 m at room temperature, and lumines-
cence was measured. Fluorescence and luminescence
was measured using SpectraMax M3 Microplate Reader
(Molecular Devices, Sunnyvale, CA, USA).
Flow Cytometry
Cells were plated on 60 mm tissue culture-treated plates
(LPS, Rochester, NY) and incubated in complete medium
until 60% confluent. Cells were then treated with medium,
DMSO vehicle control, extract alone, or extracts in com-
bination. Cells were treated for 12 h (reactive oxygen spe-
cies generation), 24 h (curcumin accumulation), or 48 h
(Apoptosis/Necrosis, Cell Cycle). All flow cytometric ana-
lysis was performed on BD FACSCalibur (BD Biosciences,
San Jose, CA, USA).
Levine et al. BMC Veterinary Research (2017) 13:388 Page 2 of 12
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Cell cycle analysis
Cell cycle effects were analyzed after 24 h (data not
shown) and 48 h treatment using propidium iodide
staining to label DNA content. Briefly, cells were de-
tached with Accumax cell dissociation solution (Innova-
tive Cell Technologies, San Diego, CA USA), collected
in tubes with 1% fetal bovine serum (FBS) in Phosphate
Buffered Saline (PBS) and centrifuged for 5 m at 300 rcf
at 4 °C. The cell pellet was washed twice with 1% FBS in
PBS, filtered, and resuspended in 70% cold ethanol for
overnight fixation. The following day, samples were cen-
trifuged for 10 m at 500 rcf at 4 °C, resuspended in cold
PBS. Samples were centrifuged again for 5 m at 300 rcf
at 4 °C and resuspended in DNA staining solution [2%
propidium iodide (Sigma Aldrich), 0.1% Triton X-100
(Sigma Aldrich), in PBS]. Samples were then incubated for
30 m at room temperature and analyzed with an excita-
tion of wavelength of 488 nm and emission of 617 nm.
Only C2 and D17 cell lines were analyzed due to the pres-
ence of frequent doublets with CMT-12 cells resulting in
an artificial accumulation in the G2/M phase.
Apoptosis and necrosis assay
Apoptosis and necrosis was measured after 48 h treat-
ment using Annexin-V and 7-AAD staining. Briefly, cells
were detached with Accumax dissociation solutions (In-
novative Cell Technologies, San Diego, CA, USA), col-
lected and centrifuged for 10 m at 500 rcf at 4 °C. The
cell pellet was washed once with PBS before resuspen-
sion in Annexin Binding Buffer (ABB; 10 mM HEPES,
140 mM NaCl, 2.5 mM CaCl
2
, pH 7.4) at a density of
1×10
6
cell mL
−1
. Annexin-V 488 conjugate and 7-
Aminoactinomycin D (7-AAD) were added to the cell
suspensions and incubated for 15 m at room
temperature. After the incubation, ABB was added to
the cell suspension and kept on ice until fluorescence
analysis. Events labeled only Annexin-V positive were
considered to represent apoptotic cells; events labeled
Annexin-V positive and 7-AAD positive were considered
to represent necrotic cells.
Intracellular reactive oxygen species (ROS) analysis
Since the main constituents of TE and RE (curcumin
and carnosic acid, respectively) have been implicated as
antioxidants, Dihydrorhodamine123 (DHR123; Invitro-
gen, Carlsbad, CA, USA) assay was used to determine
the amount of reactive oxygen species (ROS) present
after 12 h treatment with each extract according to lit-
erature [19]. Briefly, cells were detached using Accumax
dissociation solution (Innovative Cell Technologies), col-
lected and centrifuged for 10 m at 500 rcf at 4 °C. The
pellet was washed once with PBS before resuspension in
1 mL of stain (30 μM DHR123 in DMEM). The cell sus-
pension was then incubated at 37 °C for 30 m, pelleted,
and resuspended in 1 mL DMEM and filtered before
fluorescence analysis of cells.
Cellular accumulation of curcumin
The cellular accumulation of curcumin was measured by
exploiting the auto-fluorescent properties of this com-
pound [20]. After 24 h treatment, cells were detached
with Accumax dissociation solution (Innovative Cell
Technologies), collected and centrifuged for 10 m at 500
rcf at 4 °C. The cell pellet was washed once with PBS be-
fore resuspension in DMEM, and filtered before fluores-
cence analysis when excited at a wavelength of 488 nm
and then measuring emission using a 530/30 filter.
Western blotting assessment of affected signaling pathways
Cells were plated on 100 mm tissue culture-treated
plates (LPS) and incubated overnight in complete
medium until 60% confluency was reached. Cells were
treated the following day with DMSO vehicle control,
6.3 μgmL
−1
extract alone, or 3.1 μgmL
−1
each extract
in combination. Cells were harvested and lysed at 12 h
and 24 h after treatment using Mammalian Lysis Buffer
(MLB; 25 mM Tris, 100 mM NaCL, 1 mM EDTA, 1%
Triton X-100, 0.004% NaF, 1 mM NaVO4, 25 mM -gly-
cerophosphoric acid, 100 μg/ml phenylmethanesulfonyl
fluoride, and 1 μg/ml each aprotinin and leupeptin,
pH 7.4) and sonication, and then centrifuged for 5 m at
14,000 rcf at 4 °C. The supernatant was collected and
the protein concentration was determined using the
Bradford assay (Coomassie-dye; ThermoFisher Scientific
Pierce, Waltham, MA, USA). Samples were equilibrated
to a common volume (μgμL
−1
) in MLB and 5× laemmili
loading buffer (300 mM Tris-HCl pH 6.8, 10% Sodium
dodecyl sulfate, 50% glycerol, 12.5% β-Mercaptoethanol,
0.025% Bromophenol blue). For each protein of interest,
30 μg total proteins were subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
on gels ranging from 6 to 15% based on the molecular
weight of the protein of interest. The proteins were then
transferred to 0.45 μm pore size polyvinylidene fluoride
membrane (Immobilon-P Membrane; EMD Millipore,
Billerica, MA, USA) for 1 h at 333 mA and then blocked
in 5% milk in Tris-buffered saline/0.05% Tween-20 solu-
tion (TBST). Membranes were incubated overnight in
primary antibody solutions at a dilution of 1:1000 in
TBST on a rocking platform at 4 °C. Primary antibodies
included mouse anti- phosphorylated-gamma H2A.X
and extracellular regulated kinase (ERK) (R&D Biosciences,
Boston, MA, USA); mouse anti- Thr202/Tyr204 phosphor-
ylated p44/42 MAPK (ERK1/2) and STAT3 (Cell Signaling
Technology,Danvers,MA,USA);rabbitanti-proteinkin-
ase B (AKT), Ser473 phosphorylated-AKT, stress-activated
protein kinase/jun-N-terminal kinase (SAPK/JNK),
Thr183/Tyr185 phosphorylated-SAPK/JNK, focal adhesion
Levine et al. BMC Veterinary Research (2017) 13:388 Page 3 of 12
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kinase (FAK), Tyr397 phosphorylated-FAK, Tyr576/Tyr577
phosphorylated-FAK, Tyr925 phosphorylated-FAK, Src,
Tyr416 phosphorylated-Src, Tyr527 phosphorylated-Src,
mammalian target of rapamycin (mTOR), Ser2448
phosphorylated-mTOR, Janus kinase 2 (JAK2), Tyr1007/
Tyr1008 phosphorylated-JAK2, Ser727 phosphorylated-
signal transducer and activator of transcription 3 (STAT3),
Tyr705 phosphorylated-STAT3, B-Cell CLL/Lymphoma 2
(BCL2), and BCL2-Associated X Protein (BAX) (Cell Sig-
naling Technology). Membranes were washed three times
with TBST and incubated at room temperature for 1 h in
the corresponding secondary anti-mouse IgG or anti-
rabbit IgG horseradish peroxidase-conjugated antibody at
a dilution of 1:2000 (Cell Signaling Technology). Mem-
branes were washed three times with TBST and visualized
with a chemi-luminescent reagent (Clarity Western ECL
Substrate; Bio-Rad, Hercules, CA, USA). Digital images
were captured using an imaging system (Biospectrum 410;
UVP, Upland, CA, USA). After images were collected,
membranes were washed three times in TBST and incu-
bated with a 1:10,000 dilution in TBST of the house-
keeping antibody β-Actin (Sigma-Aldrich) for 1 h at room
temperature. Membranes were washed, incubated with
mouse secondary antibody at a dilution of 1:2000, and im-
aged as described.
Data management and statistical analysis
For all flow cytometry experiments, 10,000 events were
collected per sample and then gated based on a forward-
scatter/side-scatter plot. Minimally three independent ex-
periments were examined for each treatment through the
different assays (percent of gated cells in each phase cycle,
percent of apoptotic cells, intracellular ROS and curcumin
level) and analyzed with Cell Quest software (BD Biosci-
ences). DMSO was compared against cells in media alone
showing no significant differences, therefore DMSO
treated cells were used as the control sample for all com-
parisons. The geometric mean fluorescence (GMF) from
each treatment was compared to the DMSO treated sam-
ples and represented as fold change for all experiments
using GMF due to the differences in fluorescence intensity
across cell lines. In addition, for measurements of ROS, an
unstained control was used to determine the baseline
GMF of each extract. This value was subtracted from the
GMF of stained samples to correct for any shift due to
auto-fluorescence of the extract with cells alone. Caspase
3/7 activation was determined as caspase activation per
total viable cells for each treatment. Raw data from the
viability portion of the assay (individual fluorescence
values of each well) were normalized to the vehicle alone
treatment for each cell line, considered to represent 100%
proliferating cells. The ratio of caspase activation to viable
cells is represented as fold increase over DMSO treatment
alone. Each of the treatment conditions were completed
in duplicate and averaged in four independent experi-
ments. Western blots were run in three independent time
course experiments and densitometry was completed
using ImageJ [21]. Values are represented as a ratio of
phosphorylated protein to total protein and standardized
to DMSO vehicle control at every time point examined.
All statistical analyses were performed using JMP Pro
(v. 11.2.1; SAS Institute Inc., Cary, NC, USA). The resid-
uals of all statistical models were found to be normally
distributed therefore parametric statistics were utilized.
The fold-change data from caspase 3/7 activation, per-
cent of apoptotic cells, intracellular ROS level and cur-
cumin accumulation assays and the ratio data from
western blot assay were processed using analysis of vari-
ance with Tukey’s method for multiple comparisons be-
tween all treatment conditions (single, combination and
DMSO control). In the case of cell cycle dynamics, Dun-
nett’s method was used to control for multiple compari-
sons when studying the percent of gated events
difference between single treatment or dual combination
and DMSO control only at each time point. Differences
were considered statistically significant at p< 0.05.
Results
Turmeric and rosemary extracts effects on cell cycle
The effects of TE and/or RE on cell cycle progression were
measured on C2 and D17 tumor cell lines using propi-
dium iodide staining. Cell cycle dynamics were analyzed
after 24 h and 48 h of incubation with the different treat-
ments; no significant difference was seen between these
two time-points therefore only data from the 48 h time
point is shown (Fig. 1A-B). Representative histograms are
shown for C2 (Fig. 1C) and D17 (Fig. 1D) cell lines. Single
treatment with 6.3 μgmL
−1
TE resulted in a significant
decrease in S phase (DNA replication) in the D17 cell line
compared to DMSO control. Treatment with 6.3 μgmL
−1
RE induced a significant decrease in G
1
/G
0
phase in the
D17 cell line, a reduction in S phase in both cell lines, and
an increase in G
2
/M phase (cell division) in the D17 cell
line. The combination treatment using 3.1 μgmL
−1
both
extracts induced a small decrease in S phase in only the
C2 cell line, and a modest increase in G
2
/M phase in only
the D17 cell line. While these differences were significant,
the mild alterations in cell cycle in the D17 and C2 of 5–
10% decrease in the G
1
/G
0
and increase in G
2
/M phases
with RE and less consistent yet similar changes with both
TE and RE combined were not considered large enough
to continue examining pathways related to cell cycle arrest
as observed in prior studies using these cell lines [22].
Cellular apoptosis is induced by turmeric and rosemary
extract treatments.
After treating cells for 36 h, TE alone (6.3 μgmL
−1
) re-
sulted in a significant increase in apoptotic cells in the
Levine et al. BMC Veterinary Research (2017) 13:388 Page 4 of 12
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C2 and CMT-12 cell lines as determined by Caspase 3/7
activation, 2- and 2.5-fold, respectively (Fig. 2), and
Annexin-V staining which increased from 6% to 11%
apoptotic cells in the C2 (Fig. 3E) and from 4% to 13%
in the CMT-12 cell line (represented in Fig. 3A and C
lower right quadrant; full analysis Fig. 3E). A treatment
with 6.3 μgmL
−1
RE alone resulted in a statistically
significant increase of 1.4-fold in Caspase 3/7 activation
in all three cell lines when compared to vehicle control.
When the dual combination treatment (3.1 μgmL
−1
TE
+ 3.1 μgmL
−1
RE) was used, a significant increase in
Annexin-V positive cells compared to vehicle control
was seen in the 3 cell lines, but this was not significant
compared to 6.3 μgmL
−1
TE alone in C2 cell line.
Fig. 1 Effects of turmeric and rosemary extract on cell cycle in C2 and D17 cell lines. Cells were treated with indicated concentrations of extracts
or DMSO for 48 h and the DNA contents were analyzed using propidium iodide staining by flow cytometry. Percentages of cells within each cell
cycle phase (G1, S, and G2/M) were expressed as mean ±standard deviation in (a) C2 and (b) D17 cell lines. Bar graphs represent the average of
three individual experiments performed in duplicate and representative cell cycle histograms of DMSO control treated (c) C2 and (d) D17 cell
lines are shown at 48 h of treatment. All treatments were compared to DMSO vehicle control. Treatments which induced a statistically significant
change from DMSO within each cell cycle phase are indicated
Fig. 2 Caspase 3/7 activation induced by turmeric and rosemary extracts in C2, CMT-12, and D17 cell lines. Cells were treated with indicated
concentrations of extracts or DMSO for 36 h. Activated caspase 3/7 per viable cells was expressed as mean fold change from DMSO control values
± standard deviation from three independent replicates. Within each cell line, values with different letters are significantly different from each
other (C2 p< 0.001; CMT-12 p< 0.005; D17 p< 0.05)
Levine et al. BMC Veterinary Research (2017) 13:388 Page 5 of 12
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However, in the CMT-12 and D17 cell lines, the com-
bination treatment induced a significantly greater per-
centage of apoptotic cells, 40% and 13%, respectively,
compared to 6.3 μgmL
−1
TE alone (13% and 7%) and
6.3 μgmL
−1
RE alone (5%) (Fig. 3). This was further val-
idated with the caspase activation assay in which all
Fig. 3 Apoptosis induction by turmeric and rosemary extracts in C2, CMT-12, and D17 cell lines. Cells were incubated with the indicated treatments
for 48 h and the induction of apoptosis was detected by Annexin V-FITC and 7-AAD staining followed by flow cytometric analysis. Representative
quadrant plots of the CMT-12 cell line treated with (a)DMSO,(b)6.3μgmL
−1
TE, (c)6.3μgmL
−1
RE, or (d)3.1μgmL
−1
TE + 3.1 μgmL
−1
RE are shown.
Each quadrant represents the number of events considered live (lower left), early apoptotic (lower right), or late apoptotic/necrotic (upper right). e
Percent early apoptotic cells (lower right quadrant of Annexin V positive and 7-AAD negative cells) are represented as mean ± standard deviation
(three independent replicates). Within each cell line, means with different letters are significantly different from each other (p<0.05)
Levine et al. BMC Veterinary Research (2017) 13:388 Page 6 of 12
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three cell lines (C2, CMT-12 and D17) showed a signifi-
cant increase in cleaved Caspase 3/7 when the combin-
ation treatment was used (2.8, 5- and 2.2-fold,
respectively), compared to TE alone (2-, 2.5- and 1.2-
fold) or RE alone (1.4-fold across all three cell lines).
Antioxidant activity of TE and RE in cancer cell lines
TE alone was a significantly stronger antioxidant than
RE alone using same extract concentration (6.3 μgmL
−1
) in all the three cell lines (C2, CMT-12 and D17) with
TE reducing ROS by about 75–90-80%, respectively and
RE reducing ROS by about 50–40-40%, respectively. The
dual combination treatment using half the concentration
(3.1 μgmL
−1
each extract) was as effective as 6.3 μgmL
−1
TE alone in all three cancer cell lines (Fig. 4).
Cellular accumulation of curcumin induced by RE
treatment
Observation from previous flow cytometry experiments
showed an unexpected increase in the GMF when cells
were treated with TE alone when excited at a wavelength
of 488 nm, whereas no change was observed when RE
was used alone (Fig. 5D). A similar increase in GMF was
also seen when half the concentration of each extract
was used in combination (data not shown). Therefore,
the possibility that RE could increase the cellular accu-
mulation of the fluorescent compound curcumin was in-
vestigated when these compounds were used in
combination. TE alone (3.1 μgmL
−1
) significantly in-
creased the GMF in the C2 and D17 cell lines, 1.7- and
1.8-fold, respectively; while when using RE with TE the
increase was 2.2 and 2.3 fold, respectively (Fig. 5A, C; p
< 0.0001). The addition of RE at the same concentration
to TE resulted in a significant increase in GMF of 4.8-
fold in the CMT-12 cell line beyond that of TE alone
(Fig. 5B; p< 0.0001).
TE and RE SAPK/JNK activation
After examination of several cell signaling pathways, no
consistent trend was seen in the phosphorylation status
of the variety of signaling proteins, alterations in the
mitochondrial proteins involved in apoptosis or markers
of DNA damage (data not shown). However, changes in
Thr183/Tyr185 phosphorylated-SAPK/JNK (p-SAPK/
JNK; Fig. 6) were detected in the three cancer cell lines.
Treatment with 6.3 μgmL
−1
TE resulted in an increase
from a densitometry value of 1.1 at 12 h to 1.5 at 24 h in
p-SAPK/JNK in the C2 cell line, stable activation from
12 h to 24 h in the CMT-12 cell line (1.5 and 1.8, re-
spectively). In the D17 cell line only a minor non-
significant increase was observed (1.2 at 12 h, 1.1 at
24 h). Activated SAPK/JNK increased from 12 h to 24 h
in the C2 cell line (1.8 and 2.1, respectively) and the
CMT-12 cell line (1.2 at 12 h to 1.5 at 24 h) which was
not significant over time. Minimal change was seen in
the D17 cell lines after treatment with 6.3 μgmL
−1
RE,
1.1 at both time points. The greatest increase in p-
SAPK/JNK was seen with the combination of 3.1 μg
mL
−1
each of TE and RE in the CMT-12 cell line where
densitometry values increased from 1.0 with DMSO
treatment to 4.3 after 12 h incubation and 4.8 after
24 h incubation (p< 0.05 from DMSO treatment). Al-
though there were similar increases in SAPK/JNK acti-
vation with TE and dual treatment of TE/RE (half
doses) in C2 and D17 cell lines, these were not signifi-
cantly different from DMSO control at 12 h or 24 h in-
cubation time points. These results demonstrate
possible mechanisms behind the observed susceptibility
differences across the three cell lines, particularly in
light of the heightened response of lesser doses of RE
and TE in combination when compared to higher con-
centrations each extract independently in the CMT12
cell line.
Fig. 4 Antioxidant effects of turmeric and rosemary extracts in C2, CMT-12 and D17 cell lines. Cells were treated with the indicated concentrations of
extracts for 12 h followed by determination of intracellular levels of reactive oxygen species using Dihydrorhodamine123 staining. Values are expressed
as mean ± standard deviation of four independent replicates. Reported values are represented as fold change compared to DMSO vehicle control.
Within each cell line, means with different letters are significantly different from each other (C2 p <0.05; CMT-12 and D17 p< 0.0001)
Levine et al. BMC Veterinary Research (2017) 13:388 Page 7 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Discussion
Bioactive molecules derived directly from plants, or
modeled after plant compounds, continue to be an ac-
tive area of cancer research. The majority of these stud-
ies have been focused on human and rodent cancer
models and the effects of these plant extracts and select
compounds vary depending on species and cell origin
[23, 24]. Few studies have been completed in dogs or re-
lated cell lines, therefore it is necessary to examine the
effects of these compounds in vitro before using them in
clinical veterinary trials. The aim of the current in vitro
study was to examine the molecular effects of two nat-
ural extracts, turmeric root extract (rich in curcumi-
noids) and rosemary leaf extract (rich in carnosic acid),
previously shown to inhibit proliferation synergistically
in three established canine cancer cell lines [11]. These
experiments were designed to focus on concentrations
that may have utility in vivo and concentrations that
showed synergistic effects of the compounds in our prior
experiments (focusing on synergistic concentrations of
3μg/mL of TE and RE versus 6 μg/mL
−1
of each extract
independently) [11]. In agreement with our previous
proliferation and cytotoxicity results, cell treatment
using TE alone was more potent than RE single treat-
ment using the same extract concentrations and experi-
mental conditions. TE had a greater effect on inducing
cell apoptosis as measured by Caspase 3/7 activation
and Annexin-V staining, and the combination treatment
Fig. 5 Effect of rosemary extract on intracellular accumulation of curcumin in canine tumor cell lines. The C2 (a), CMT-12 (b), and D17 (c) cell lines
were treated with the indicated concentration of extracts for 24 h and then cellular accumulation of curcumin was quantified by flow cytometry. Y-axis
values represent the fold change in geometric mean fluorescence (GMF) of all cells compared to DMSO control. Reported data are expressed as mean
± standard deviation of 4 independent replicates. Within each cell line, means with different letters are significantly different from each other (p
< 0.0001). dRepresentative histogram of emission intensity in CMT-12 cell line after 24 h treatment with DMSO, 3.1 μgmL
−1
TE alone,
3.1 μgmL
−1
RE alone, or 3.1 μgmL
−1
TE + RE combination is shown
Levine et al. BMC Veterinary Research (2017) 13:388 Page 8 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
using only half the concentration of each extract induced
a similar, if not greater, cell response. Varying degrees of
susceptibility were detected across the three cancer cell
lines used, with the CMT-12 cell line being the most
susceptible to these treatments, perhaps due to a greater
increase in intracellular curcumin accumulation as
shown by flow cytometry. These differences across cell
lines suggest the complexity in cellular response and po-
tential susceptibility of various cell lines to treatment,
further clarifying the need to understand what types of
cancer may be more responsive to these interventions.
Prior studies have shown the autofluorescence of cur-
cumin can be examined with flow cytometry [20]. An in-
crease in curcumin fluorescence was seen across the
three cell lines with the greatest signal measured in the
CMT-12 cell line, especially when the two extracts were
used in combination. A previous study in the human
breast cancer cell line, MCF-7, showed an increase in
intracellular accumulation of various chemotherapeutic
drugs which was attributed to competitive inhibition of
transmembrane transport pump P-glycoprotein by rose-
mary extract [25]. As this was not within the scope of
our investigation, further experiments examining P-
glycoprotein inhibition or curcumin utilization of other
channels to enter cells are warranted to better under-
stand the mechanisms by which rosemary enhances the
accumulation of curcumin within cells.
The global effects of both TE, RE, and the two in com-
bination showed no appreciable alteration in cell cycle
kinetics. Of the three cell lines used in our experiments,
only the C2 and D17 cells could be examined as a single
cell suspension for cell cycle dynamics, while the CMT-
Fig. 6 Changes in the protein expression levels of SAPK/JNK pathway in turmeric and rosemary-treated cells. C2, CMT-12 and D17 cell lines were
harvested and lysed after 12 h or 24 h treatment with DMSO vehicle control, or 6.3 μgmL
−1
Turmeric extract (TE) alone, or 6.3 μgmL
−1
Rosemary
extract (RE) alone, or combination of 3.1 μgmL
−1
each of TE + RE. Expression level of Thr183/Tyr185 phosphorylated-SAPK/JNK (p46/p54) and total
SAPK/JNK were determined by Western blot analysis. Each blot is a representative of three independent experiments. Densitometry values
represent a ratio of phosphorylated protein to total protein and normalized to DMSO vehicle control of the same time point (mean of
three separate experiments). Changes in densitometry compared to DMSO control with significance of p< 0.05 represented by *. β-Actin
was used as a loading control for every blot to ensure even loading of samples
Levine et al. BMC Veterinary Research (2017) 13:388 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
12 cells displayed an artificial accumulation of cells in
the G
2
/M phase. This was attributed to cell clumping in
this cell line due to the fixation method used and use of
propidium iodide causing cell-doublets to be inappropri-
ately represented as G
2
M phase. There were some mild
alterations in cell cycle in the D17 and C2 cell lines
showing decreases in the G
1
/G
0
and increases in G
2
/M
phases with RE and the combination treatment. There
were minimal to no cell cycle changes with TE extract
alone, therefore further examination of cell cycle path-
way analysis was not pursued. Prior literature has shown
that curcumin can have significant effects on cell cycle
dynamics through the upregulation of cyclins or cyclin
dependent kinase activity [26–28]. In this previous lit-
erature, there was complete loss or greater than 50% re-
duction or increase in various portions of the cell cycle
warranting further examination of cellular pathways in-
volved. Generally, these differences may be due to the
lower concentrations utilized in our experiments com-
pared to the prior studies.
In line with pathway disruption and cell cycle dynam-
ics, TE and RE are often thought to be antioxidants,
however curcumin has been shown to cause oxidative
damage to DNA and induction of the DNA damage re-
sponse pathways that are intimately involved in cell cycle
alteration or apoptosis [29, 30]. Our assessment of anti-
oxidant status after treatment unanimously indicated
that the cells are under less oxidative stress after treat-
ment with either TE or RE alone or in dual combination
within 12 h of incubation. To further assess the oxida-
tive status, western blot analysis for gamma-histone
H2A.X phosphorylation status was assessed in the three
cell lines with TE, RE or dual treatment showing no
phosphorylation in DMSO control or treated cells.
Gamma-histone H2A.X is a marker of DNA oxidation
and initiation of repair and was not detected when com-
pared to UV irradiation as a positive control for DNA
damage (data not shown). Under our cell culture condi-
tions and extract concentrations used, we could not
elicit a pro-oxidative response from TE or RE in any cell
line used. This anti-oxidant property has been thought
to be involved in cell survival and possible resistance to
chemotherapeutic intervention [31]. Our data at these
concentrations only suggest pro-apoptotic responses,
suggesting that the mechanisms are unlikely to rely on
oxidative damage.
Further examination of the cellular effects into cell sig-
naling pathways previously implicated in TE and RE
treatment were performed [25–35]. Concentrations that
appeared to be most synergistic at inhibiting prolifera-
tion from our prior publication [11] were used to ob-
serve enhanced or diminished signaling events over
extended periods of time from 12 to 24 h that might
provide insights into the modest apoptotic response.
Apoptosis could be, in part, due to overlapping effects
on various signaling pathways including SAPK/JNK,
ERK 1/2, STAT3, FAK, Src, mTOR, and membrane per-
meability proteins Bcl-2 and Bax [36, 37]. Previous lit-
erature has shown a synergistic effect between these two
extracts, specifically the cleavage of poly ADP-ribose
polymerase and Caspase-8, −9, and −3 on human cell
lines [17]. Though there is relatively little primary litera-
ture on canine cell lines, one study has shown that a
curcumin analog effectively alters STAT phosphorylation
and activation in canine osteosarcoma cells [38]. After
screening several signaling pathways, a consistent in-
crease in the phosphorylated, or active, form of SAPK/
JNK was detected with no consistent alterations in any
other pathways examined via western blotting. This
pathway has been implicated in driving cells to apoptosis
when faced with environmental stressors such as oxida-
tive stress, inhibition of protein synthesis, changes in the
cell-matrix interaction, or signaling from inflammatory
cytokines [39–41]. Consistent with our results, studies
have shown that the downstream effects of SAPK/JNK
activation are both cell and context dependent: pathway
activation can be either pro-apoptotic or pro-
proliferative. [42, 43] Changes in activation of MAPK/
ERK were not observed after treatment in any of the cell
lines examined. In general, early, transient activation of
JNK may lead to cell survival, while sustained activation
can induce apoptosis and curcumin or rosemary extracts
appear to be involved in this constitutive activation of
SAPK/JNK [44–46].
Differences in treatment responses were observed in
the three cell lines. Our results showed an increase in
phosphorylated SAPK/JNK after 12 h and 24 h of treat-
ment with TE alone. RE induced a significant increase in
phosphorylation after 12 h and 24 h of treatment in the
CMT-12 cell line, while in the C2 cell line this increase
was only seen at 12 h and returned to baseline by 24 h.
The dual combination treatment had the greatest effect
in the CMT-12 cell line, resulting in phosphorylated
SAPK/JNK at levels greater than either extract alone
(even using twice the concentration). Only the CMT-12
cell line showed sustained activation of SAPK/JNK with
the combination treatment which may be the underlying
reason behind the increased susceptibility of this cell
line. SAPK/JNK has been implicated as a therapeutic tar-
get in certain contexts and patterns of activation
whereby constitutive activation appears to be beneficial
towards a pro-apoptotic response in a variety of cell
lines and animal models [47–49]. The transient nature
of activated SAPK/JNK in the C2 and D17 cell lines lead
us to believe this may be involved in the diminished pro-
liferation, however other pathways may be involved in
the induction of apoptosis in these cancer cell lines. This
data further demonstrates that the cell line and context
Levine et al. BMC Veterinary Research (2017) 13:388 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
specific effects of these extracts are vastly different and
other approaches are needed to completely understand
the complex interaction of the pathways involved during
apoptosis induction.
Although the results of these experiments are promis-
ing, the clinical utility is complex due to the absorption,
transformation and elimination kinetics of these com-
pounds in general. The use of highly bioavailable curcu-
min is currently being examined and has 10–15%
bioavailability [50]. Carnosic acid bioavailability from
rosemary extract, though good, has not been studied ex-
tensively; however there is rapid glucuronidation, methy-
lation, and oxidation of carnosic acid and related
molecules from rosemary extract, and the bioactivity of
these modified derivatives are unknown at this time [51].
Conclusions
The results of this study provides some insights into
possible mechanisms by which TE and RE induce apop-
tosis across three canine neoplastic cell lines. Our results
indicate that different tumor types are likely to have a
differential response to such interventions. The en-
hanced susceptibility found in the CMT-12 mammary
cancer cell line may be due to the increased accumula-
tion of curcumin when the combination treatment was
used. In addition, sustained activation and signaling
through the SAPK/JNK pathway may play a role in this
cell line’s increased sensitivity to apoptosis. The results
of this study warrant further investigations into the
pharmacodynamics and pharmacokinetics of these ex-
tracts in dogs when incorporated into feed to determine
if clinical trials are feasible.
Abbreviations
AKT: Protein kinase B; BAX: BCL2-Associated X Protein; BCL2: B-Cell CLL/
Lymphoma 2; DMSO: Dimethyl sulfoxide; ERK: Extracellular related kinase;
FAK: Focal adhesion kinase; FBS: Fetal bovine serum; GMF: Geometric mean
fluorescence; JAK: Janus kinase; mTOR: Mammalian target of rapamycin;
PBS: Phosphate buffered saline; RE: Rosemary extract; ROS: Reactive oxygen
species; SAPK/JNK: Stress activated kinase/jun-N-terminal kinase; STAT: Signal
transducer and activator of transcription; TBST: Tris buffered saline tween;
TE: Turmeric extract
Acknowledgements
We are grateful to the University of California San Francisco, especially Dr.
Warren Gold and Dr. George Caughey, for supplying the C2 canine
mastocytoma cell lines and to Auburn University, especially Dr. R. Curtis Bird,
for supplying CMT-12 canine mammary gland carcinoma cell line.
Funding
The aforementioned study was funded by Royal Canin (JB and VB)
collaborated on the study design and interpretation of data collected.
Availability of data and materials
Datasets used and analyzed during this study are available from the
corresponding author upon reasonable request.
Author contributions
CBL carried out the technical experimentation, performed statistical analysis,
and was primary author in the manuscript. JB and VB conceived the study
and participated in its design and participated in manuscript editing. JW
helped conceive the study, supervised the study and helped in manuscript
drafting and editing. All authors have read and accepted the final manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The research leading to these results was supported by Royal Canin SAS.
Royal Canin participated in writing the protocol, analyzing the data,
contributing compounds, revising the manuscript for publication. JB and VB
are employed by Royal Canin. JJW has received compensation from Nestle
Purina, Mars, Annamaet Pet Food Company, and Veterinary Recommend
Solutions for consultation.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Clinical Sciences,Veterinary Medical Center C2-009, Cornell
University College of Veterinary Medicine, Ithaca, NY 14853, USA.
2
Royal
Canin Research Center, Airmargues, France.
Received: 9 June 2017 Accepted: 27 November 2017
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