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Koppikar et al. BMC Cancer 2010, 10:210
http://www.biomedcentral.com/1471-2407/10/210
Open Access
RESEARCH ARTICLE
© 2010 Koppikar et al; licensee BioMed Cen tral Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
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Research article
Aqueous Cinnamon Extract (ACE-
c
) from the bark
of
Cinnamomum cassia
causes apoptosis in human
cervical cancer cell line (SiHa) through loss of
mitochondrial membrane potential
SoumyaJKoppikar
†1
, Amit S Choudhari
†1
, Snehal A Suryavanshi
1
, Shweta Kumari
1
, Samit Chattopadhyay
2
and
Ruchika Kaul-Ghanekar*
1
Abstract
Background: Chemoprevention, which includes the use of synthetic or natural agents (alone or in combination) to
block the development of cancer in human beings, is an extremely promising strategy for cancer prevention.
Cinnamon is one of the most widely used herbal medicines with diverse biological activities including anti-tumor
activity. In the present study, we have reported the anti-neoplastic activity of cinnamon in cervical cancer cell line, SiHa.
Methods: The aqueous cinnamon extract (ACE-c) was analyzed for its cinnamaldehyde content by HPTLC analysis. The
polyphenol content of ACE-c was measured by Folin-Ciocalteau method. Cytotoxicity analysis was performed by MTT
assay. We studied the effect of cinnamon on growth kinetics by performing growth curve, colony formation and soft
agar assays. The cells treated with ACE-c were analyzed for wound healing assay as well as for matrix metalloproteinase-
2 (MMP-2) expression at mRNA and protein level by RT-PCR and zymography, respectively. Her-2 protein expression
was analyzed in the control and ACE-c treated samples by immunoblotting as well as confocal microscopy. Apoptosis
studies and calcium signaling assays were analyzed by FACS. Loss of mitochondrial membrane potential (Δψm) in
cinnamon treated cells was studied by JC-1 staining and analyzed by confocal microscopy as well as FACS.
Results: Cinnamon alters the growth kinetics of SiHa cells in a dose-dependent manner. Cells treated with ACE-c
exhibited reduced number of colonies compared to the control cells. The treated cells exhibited reduced migration
potential that could be explained due to downregulation of MMP-2 expression. Interestingly, the expression of Her-2
oncoprotein was significantly reduced in the presence of ACE-c. Cinnamon extract induced apoptosis in the cervical
cancer cells through increase in intracellular calcium signaling as well as loss of mitochondrial membrane potential.
Conclusion: Cinnamon could be used as a potent chemopreventive drug in cervical cancer.
Background
Cervical cancer, which accounts for the second most
common malignancy among women worldwide, is highly
radio-resistant, often resulting in local treatment failure
[1]. For locally advanced disease, radiation is combined
with low-dose chemotherapy; however, this modality
often leads to severe toxicity. Complementary and Alter-
native Medicine (CAM) is recently becoming a popular
treatment for various cancers among which herbal medi-
cine is one of the methods used in cancer therapy [2,3].
Currently, plants, vegetables, herbs and spices used in
folk and traditional medicine have been accepted as one
of the main sources of chemopreventive drugs [4-8]. Tra-
ditional medicine that includes herbal medicine has been
used from time immemorial to treat chronic ailments
such as cancer. Recently, scientific studies support herbal
medicine as potent anti-cancer drug candidates [9-13].
Cinnamon, a widely used food spice, has been shown to
exhibit diverse biological functions including anti-inflam-
* Correspondence: ruchika.kaulghanekar@gmail.com
1 Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth
University Medical College Campus, Pune, Maharashtra, India
† Contributed equally
Full list of author information is available at the end of the article
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matory [14], anti-oxidant [15,16], anti-microbial [15,17],
and anti-diabetic effects [18-20]. Recently, the anti-tumor
activity of cinnamon has been shown both in vitro [21-
23] and in vivo [10]. Cinnamaldehyde, the bioactive com-
ponent of cinnamon, has been shown to inhibit prolifera-
tion of several human cancer cell lines including breast,
leukemia, ovarian, and lung tumor cells [24]. Recently, we
reported a comparative analysis of cytotoxic effect of
aqueous extract of cinnamon (ACE) from C. zeylanicum
with that of commercial cinnamaldehyde on a variety of
cell lines [23]. Compared to the commercial cinnamalde-
hyde, ACE proved to be more cytotoxic owing to the
presence of polyphenolic compounds, besides cinnamal-
dehyde, that may act synergistically to induce enhanced
cytotoxicity.
In the present work, we have reported the putative
mechanism of cancer cell growth inhibition by aqueous
cinnamon extract (ACE-c), from the bark of Cinnamo-
mum cassia L. family Lauraceae, in a human cervical can-
cer cell line, SiHa. We observed that cinnamon altered
the growth kinetics of cells in a dose-dependent manner.
Our colony formation and soft agar assays demonstrated
that the number of colonies in cells treated with ACE-c
was less compared to the untreated control cells. The
ACE-c treated cells exhibited slow migration potential
compared to the control cells that could be explained due
to reduced MMP-2 expression in the former. Cinnamon
extract increased the intracellular calcium that might be
responsible for the loss of mitochondrial membrane
potential (Δψm), finally leading to cellular apoptosis.
Methods
Reagents
Tissue culture plasticware was purchased from BD Bio-
sciences, CA, USA; Axygen Scientific Inc, CA, USA and
Nunc, Roskilde, Denmark. Dulbecco's Modified Eagles
Medium (DMEM) was obtained from Himedia Corpora-
tion, Mumbai, India. Penicillin and streptomycin were
obtained from Gibco BRL, CA, USA. Fetal bovine serum
was purchased from Moregate Biotech, Australia, N. Z
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylthiazolium
bromide (MTT), FCCP, JC-1 and TMRE were purchased
from Sigma-Aldrich (St. Louis, MO). Her-2 antibody was
purchased from Santa Cruz Biotechnology, CA, USA,
Donkey anti-Mouse IgG Cy-3conjugate (Millipore, MA)
and Annexin V-FITC apoptosis kit #3 from Invitrogen
(CA, USA). All other common reagents were procured
from Qualigens fine chemicals (Mumbai, India).
ACE-c preparation and characterization
The bark of Cinnamomum cassia was purchased from
Shivam Ayurvedics Pune, Maharashtra, India with
voucher specimen number for Cinnamomum cassia bark
was 104. The sample was authenticated from Regional
Research Institute (AY) Kothrud, Pune (ref no.1045). The
bark was weighed, powdered and extracted in double dis-
tilled water (the ratio of cinnamon: water used was 1:16)
in a hot water extractor [25]. The resulting extract was
centrifuged at 13000 rpm for 15 min to remove the par-
ticulate matter. The supernatant was further filter-steril-
ized using swiney filter (pore size, 0.45 μm) and the
resultant filtrate was stored in aliquots at -80°C until use.
The bark identity was further confirmed by detecting the
marker molecule cinnamaldehyde in ACE-c by HPTLC
analysis as described previously [23,26]. The total poly-
phenol content of ACE-c was measured by Folin-Ciocal-
teau method as described previously [23,26].
Cell culture
The human cervical carcinoma cell line, SiHa, used in the
study was obtained from National Centre for Cell Science
(NCCS), Pune, India. The cells were grown in DMEM
containing 2 mM L-glutamine supplemented with 10%
fetal bovine serum and 100 U/ml of penicillin-streptomy-
cin. The cells were incubated in a humidified 5% CO2
incubator at 37°C.
Cell viability
The cell viability was determined by MTT dye uptake as
described previously [23]. Briefly, SiHa cells were seeded
at a density of 1 × 105 cells/ml density in 96-well plates.
An untreated group was kept as a negative control. The
aqueous cinnamon extract (ACE-c) was added at follow-
ing concentrations: 10, 20, 40, 80, 160 and 320 μg/ml, in
wells in triplicates. The MTT solution (5 mg/ml) was
added to each well, and the cells were cultured for
another 4 h at 37°C in 5% CO2 incubator. The formazan
crystals formed were dissolved by addition of 90 μl of
SDS-DMF (20% SDS in 50% DMF). After 15 min, the
amount of colored formazan derivative was determined
by measuring optical density (OD) using the ELISA
microplate reader (Biorad, Hercules, CA) at 570 nm
(OD570-630 nm). The percentage viability was calculated as:
% Viability = [OD of treated cells/OD of control cells] ×
100
Cell growth analysis
SiHa cells were seeded at a density of 1 × 105 cells/ml in
24-well plates in triplicates. Next day, the cells were dosed
with different concentrations of ACE-c (0, 10, 20, 40 and
80 μg/ml) and grown for 24, 48 and 72 h. The cells were
harvested and counted for viability using trypan blue dye
exclusion method.
Colony formation assay
The cells were plated at a seeding density of 1 × 103 cells/
ml in 6-well plates. After 24 h, the cells were exposed to
various concentrations of ACE-c: 0, 10, 20, 40, and 80 μg/
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ml. Plates were incubated at 37°C in a 5% CO2 incubator
for one week. This was followed by fixing the colonies
with 4% paraformaldehyde and staining with 0.5% crystal
violet [27]. The colonies were photographed with Sony
DSC-S75 cyber-shot camera.
Soft agar assay
Control SiHa cells (5 × 103 cells/ml) as well as cells treated
with different concentrations of ACE-c (10-80 μg/ml)
were mixed at 40°C with 0.35% agarose (DNA grade,
GIBCO BRL, CA, USA) in culture medium and gelled at
room temperature for 20 min over a previously gelled
layer of 0.5% agarose in culture medium in 6-well plates.
After incubation for 10 days, colonies were photographed
directly using an Axiovert 200 M microscope (Carl Zeiss,
Germany) and counted [27].
Wound healing assay
Cells were plated at a seeding density of 4 × 105 cells/ml in
24-well plates and grown overnight at 37°C in 5% CO2
incubator. An artificial wound was made with 10 μl
micropipette after 6 h serum starvation in control cells as
well as cells treated with different concentrations of ACE-
c (10-80 μg/ml). Time-lapse imaging of migrating cells
was performed on Nikon Eclipse TE2000-E microscope
(Nikon, Tokyo, Japan) over 15 h in serum containing
medium in a humidified chamber at 37°C and 5% CO2
atmosphere. Images were obtained every 20 min using a
10× phase objective of NA 0.25 and analyzed by image
analysis software Metamorph Universal Imaging, USA.
The average migration rate in μm/h was calculated and
graphs were plotted using Microsoft Excel and Sigma plot
program.
RT-PCR analysis
The total cellular RNA from control as well as cells
treated with different doses of ACE-c (0-80 μg/ml) was
extracted by a one-step acid guanidine isothiocyanate-
phenol method using TRI reagent (Sigma, St. Louis, MO).
RNA was precipitated with isopropanol and the concen-
tration was estimated by spectrophotometer (Biorad,
SmartSpec™ 3000). Ten microgram of total RNA was used
for each RT-PCR reaction. Fifty units of Moloney murine
leukemia virus reverse transcriptase (MMuLV) (Banga-
lore Genei, Banglore, India) were added in a typical 50 μl
reaction (10 μg RNA, 1× first-strand buffer, 1 mM DTT,
2.5 mM dNTPs, 50 ng/μl random primers and 15 U/μl
RNAse i) and incubated for 1 h at 42°C followed by incu-
bation at 95°C for 5 min. The purified cDNA template
was amplified using different sets of primers. The primers
used were as follows: β-actin-F: 5'-taccactggcatcgtgatg-
gact-3'; β-actin-R: 5'-tttctgcatcctgtcggaaat-3'; MMP-2-F:
5'-ggctggtcagtggcttggggta-3'; MMP-2-R: 5'-agatcttcttct-
tcaaggaccggtt-3'. PCRs were performed in 25 μl volume
in which 1× PCR buffer, 2.5 mM dNTPs, 1.5 mM MgCl2,
1 U of Taq polymerase and 100 ng of the specific primers
were added. A brief initial denaturation at 95°C for 5 min
was followed by 35 cycles with the following steps: 95°C
for 1 min, annealing at 55-55.2°C for 1 min and extension
at 72°C for 1 min. RT-PCR products were then separated
on a 1.2% agarose gel and visualized by staining with
ethidium bromide. The intensities of the bands corre-
sponding to the RT-PCR products were quantified using
phosphorimager (Alpha Imager using Alpha Ease FC
software, Alpha Innotech) and normalized with respect
to the β-actin product.
Gelatin zymography
The Gelatin zymography was performed to detect the
presence of extracellular MMP-2 [28]. The conditioned
medium of control cells as well as cells treated with 80 μg/
ml ACE-c was collected and concentrated in Centricon
YM-30 tubes (Millipore, MA). Both the control as well as
the treated samples containing equal amount of total pro-
teins were mixed with sample buffer (2% SDS, 25% glyc-
erol, 0.1% bromophenol blue and 60 mM Tris- HCl pH
6.8) and loaded onto 7.5% SDS-polyacrylamide gel con-
taining gelatin (0.5 mg/ml). The gel was washed with
0.25% Triton X-100 and incubated overnight in incuba-
tion buffer (150 mM NaCl, 100 mM CaCl2, 50 mM Tris-
HCl pH 7.5, 1% Triton X- 100, 0.02% NaN3) at 37°C. The
gel was stained with staining solution (0.1% Coomassie
Brilliant blue R-250 in 40% isopropanol) and destained in
7% acetic acid. Gelatinolytic activity was detected as
unstained bands on a blue background. The quantitation
of bands in control and treated samples was performed
by densitometric analysis on Alpha Imager using Alpha
Ease FC software, Alpha Innotech.
Immunoblotting
Cell extracts were prepared from control as well as cells
treated with different concentrations of ACE-c: (0-80 μg/
ml). Briefly, the cell pellet was resuspended in 80 μl lysis
buffer containing 50 mM Tris (pH 7.4), 5 mM EDTA,
0.5% NP40, 50 mM NaF, 1 mM DTT, 0.1 mM PMSF, 0.5
μg/ml leupeptin (Pro-pure Amersco, Solon, USA), 1 μg/
ml pepstatin (Amresco, Solon, USA), 150 mM NaCl, 0.5
μg/ml aprotinin (Amersco, Solon, USA) and protease
inhibitor cocktail (Roche, Lewes, UK) and incubated on
ice for 1 h with intermittent mixing. The extract was cen-
trifuged for 20 min at 4°C at 12000 rpm. The protein was
estimated using Bradford reagent (Biorad Laboratories
Inc, CA, USA). Equal amount of protein was loaded on a
10% SDS-polyacrylamide gel and transferred electropho-
retically to Amersham Hybond-P PVDF membrane (GE
Healthcare, UK) in sodium phosphate buffer (pH 6.8).
The membrane was blocked in 5% BSA in TST and incu-
bated at room temperature for 1 h with mouse monoclo-
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nal antibody for Her-2 and tubulin (Santacruz, CA, USA)
at a 1:1000 and 1:2500 dilution, respectively. The mem-
brane was washed in TST and incubated with donkey anti
mouse IgG HRP conjugate at 1:5000 (for Her-2) and
1:3000 (for tubulin) dilutions. Proteins were visualized
using a chemiluminescence kit (Amersham ECL Advance
western blotting detection kit, GE Healthcare, UK) and
densitometric analysis of X-ray films was performed on
Alpha Imager using Alpha Ease FC software, Alpha Inno-
tech.
Measurement of Apoptosis
The cells were plated at a seeding density of 5 × 105 cells/
well and treated with different concentrations of ACE-c:
(0-80 μg/ml). After 24 h of treatment, the cells were har-
vested and washed with PBS twice. Cells were stained
with Annexin V-FITC following the manufacturer's
instructions (Annexin V-FITC apoptosis kit #3, Invitro-
gen) and analyzed for apoptosis by FACS using CellQuest
Software.
Intracellular calcium measurement by flow cytometry
Intracellular Ca2+ levels were analyzed in control cells as
well as cells treated with different doses of ACE-c: (0-80
μg/ml) by flow cytometry [29]. Cells were loaded with 5
μM Fluo-3/AM (Sigma, St. Louis, MO) and 100 μg/ml of
Pluronic F127 (Sigma, St. Louis, MO) in centrifuge tubes
and incubated at 37°C, 5% CO2 for 1 h in the dark. The
cells were resuspended after every 20 min to ensure even
dye loading. The cell pellets were washed twice with 0.9%
saline and finally resuspended in 3 ml Hank's Balanced
Salt Solution (HBSS) in FACS tubes. Ionomycin (30 μM)
was used as a positive control. Fluorescence intensities
were measured at 525 nm by FACS Calibur (Becton Dick-
inson Immunocytometry Systems, San Jose, CA) to
obtain baseline readings. Mean channel fluorescence
intensities were calculated using CellQuest software.
Detection of Mitochondrial Membrane Potential (Δψm)
using JC-1
Mitochondrial membrane potential was estimated using
2.5 μg/ml JC-1 fluorescent dye either by confocal micros-
copy or by flow cytometry. For confocal studies, cells
were seeded at a density of 1 × 105 cells/ml on coverslips
in 6-well plates. After 24 h, cells were treated with differ-
ent concentrations of ACE-c: (0-80 μg/ml). Next day, the
media was removed and the cells were incubated with
fresh culture medium containing JC-1 dye for 30 min at
37°C in the dark. Cells on coverslips were washed with
PBS twice and fixed with 2.5% paraformaldehyde made in
200 mM HEPES buffer for 15 min at room temperature
followed by PBS wash. The coveslips were mounted in
antifade mounting medium containing DAPI (Ultracruz
mounting medium, Santacruz) on glass slides. The cells
were then analyzed for JC-1 uptake by using Zeiss
LSM510 META confocal laser scanning microscope
(Zeiss, Thornwood, NY) having LSM Image Examine
software. For Δψm detection by flow cytometry [30], 5 ×
105 cells were plated in 6-well plates. The cells were
treated with different concentrations of ACE-c (0-80 μg/
ml). Twenty four hours post treatment; the cells were har-
vested, washed with PBS and incubated with culture
medium containing JC-1 for 30 min at 37°C in the dark.
Cells were washed in PBS twice and analyzed for Δψm
using FACS. FCCP (10 μM) was used as a positive con-
trol. The fluorescence intensities were measured at 527
nm (green) and 590 nm (red). Analysis was done by Cell-
Quest software.
Immunofluorescence microscopy
For immunostaining SiHa cells were plated on coverslips
in 6-well plates at a seeding density of 2 × 105 cells/ml.
After 24 h, the cells were dosed with different concentra-
tions of ACE-c (0-80 μg/ml). Twenty four hours post-
treatment; the cells were washed with PBS and fixed in
2.5% paraformaldehyde made in 200 mM HEPES buffer
for 15 min at room temperature. Cells were washed for 5
min in PBS, permeabilized with 0.1% Triton X-100 in PBS
for 5 min and blocked in 10% FBS (made in PBS) for 1 h.
For detection, the cells were incubated with Her-2 anti-
body (Santa Cruz Biotechnology, Santa Cruz, CA) that
was diluted in blocking buffer at 1:100 dilution. After
washing with PBS, the cells were incubated with the sec-
ondary antibody [CY3-conjugated antimouse immuno-
globulin (Millipore)] at a dilution of 1:300. Slides were
then mounted in antifade mounting medium (Ultracruz
mounting medium, Santacruz) and analyzed with a Zeiss
LSM 510 META confocal laser scanning microscope
(Zeiss, Thornwood, NY) using LSM Image Examine soft-
ware.
Statistical analysis
All experiments were performed in triplicates and
repeated at least five times and the data were presented as
mean ± SD. Statistical analysis was conducted with the
SigmaStat 3.5 program (Systat Software, Inc.) using one-
way ANOVA. The α level used for comparisons was α =
0.05.
Results
Cinnamon treatment alters growth kinetics of SiHa cells
Aqueous extract of cinnamon prepared from C. cassia
(ACE-c) was analyzed for the presence of cinnamalde-
hyde as well as polyphenols to ensure the quality and
purity of the preparation [see Additional file 1: Figs. S1 A
and S1 B]. We initially performed MTT assay to define
the optimal concentration at which cinnamon was non-
toxic to cells. Up till 0.32 μg/ml ACE-c concentration, the
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cells exhibited 100% survival within 24 h [see Additional
file 1: Fig. S2].
To test the effect of cinnamon on the growth kinetics,
SiHa cells were treated with different concentrations of
ACE-c: 0, 10, 20, 40, and 80 μg/ml and were grown for 24,
48 and 72 h. At the end of each treatment, the cells were
stained with trypan blue, and the viable cells that
excluded the dye were counted. It was observed that there
was a dose- dependent decrease in the growth kinetics of
ACE-c-treated cells compared to the untreated control
cells (Fig. 1A). Moreover, it was found that at around 80
μg/ml concentration of ACE-c treatment, there was a sig-
nificant decrease (~2-fold) in the growth kinetics com-
pared to that observed in the untreated control cells (p ≤
0.05 for 24 h; p ≤ 0.001 for 48 h and 72 h). This was fur-
ther confirmed by colony forming assay wherein at a
lower seeding density, cells were treated with different
concentrations of ACE-c for one week. At 80 μg/ml con-
centration of ACE-c, the cells exhibited relatively lesser
colonies compared to the control cells (Fig. 1B). Consis-
tent with the slow growth rate, it was observed that cin-
namon extract induced a dose-dependent decrease in the
number of soft agar colonies. Interestingly, at 80 μg/ml
ACE-c treatment, the number of soft agar colonies was
reduced by ~3-fold (p ≤ 0.001) compared to the untreated
control cells (Fig. 1C). All these data indicated that cinna-
mon altered the growth kinetics of SiHa cells in a signifi-
cant manner that could be a positive indicator for testing
its antineoplastic activity in cervical cancer cells.
Figure 1 Cinnamon alters growth kinetics of cervical cancer cells. (A) The cells were treated with various concentrations (0-80 μg/ml) of ACE-c for
24, 48 and 72 h, and the number of viable cells were counted using the trypan blue dye exclusion assay. The growth kinetics has been presented in
the figure taken at different time points. Data represent mean ± SD of five different experiments. (B) The cells (1 × 103/ml) were grown in 6-well plates
and treated with various concentrations (0-80 μg/ml) of ACE-c for one week. The cells were then stained with crystal violet and photographed. The
experiments were repeated five times. (C) The cells (5 × 103) were treated with various concentrations (0-80 μg/ml) of ACE-c and grown in soft agar
for 10 days, and the colonies were counted. Colonies were counted from at least 10 different areas, and the average of each is plotted. The data rep-
resent mean ± SD of five independent experiments.
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Cinnamon extract decreases cell migration through
reduction in MMP-2 expression
To examine the effect of ACE-c on cell migration, we per-
formed wound healing assay on confluent monolayers of
SiHa cells. After making the wound with a pipette tip, the
cells were cultured in presence or absence of different
concentrations of the aqueous cinnamon extract and
imaged with real time-lapse video for a period of 15 h. It
was observed that ACE-c effectively inhibited the migra-
tion of cells in a dose- and time-dependent manner com-
pared to the untreated control cells (Fig. 2A and 2B). The
control cells filled-up the wound gap completely after 15
h whereas in cells treated with ACE-c, particularly at 40
and 80 μg/ml concentrations, the wound gap was not
completely filled. Interestingly, at 80 μg/ml, cinnamon
treatment significantly decreased (~1.5-fold; p ≤ 0.001)
the migration rate of SiHa cells thereby affecting their
migration capability.
Since MMP-2 is known to play a significant role in the
invasive property of tumor cells, we investigated the
mechanism behind the delay in wound healing exhibited
by ACE-c treated cells. We tested the expression of
MMP-2 in cells treated with/without cinnamon extract. It
was observed that the expression of MMP-2 was signifi-
cantly down-regulated both at mRNA (Fig. 2C) as well as
protein level (Fig. 2D) in a dose-dependent manner com-
pared to the untreated control cells. Interestingly, at 80
μg/ml concentration of ACE-c, there was a ~1.6-fold (p ≤
Figure 2 Cinnamon reduces migration potential.(A) Photomicrographs of time-lapse image at 0 and 15 h in a wound-healing assay in cells treated
with different concentrations (0-80 μg/ml) of ACE-c. The upper panel of the image shows the wound made at 0 h. The lower panel shows cell move-
ment correspond ing to the dist ance traveled by the cells at 15 h of time-lapse imaging. (B) Rate of migration of cells during the wound healing assay
analyzed by the time-lapse imaging of SiHa cells. Migration rate (μm/h) for each sample from five different fields was calculated. Error bars represent
standard deviation and the data is representative of five independent experiments. (C) ACE- c treatment reduces the MMP-2 expression at mRNA level
that has been shown by RT-PCR. β-actin was used as the loading control. Densitometric analysis of MMP-2 expression was performed using phospho-
rimag er. The data rep resents m ean ± SD of five independent experiments. (D) Gelatin zymography showing downregulation of MMP-2 expression in
SiHa cells at 80 μg/ml ACE-c treatment compared to the untreated control cells. The bands were quantified by densitometry using phosphorimager
and the data represents mean ± SD of five independent experiments.
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0.001) decrease in MMP-2 transcript and ~4-fold (p ≤
0.001) down regulation in the expression of MMP-2 pro-
tein. These data suggested that ACE-c induced decrease
in the migration of cervical cancer cells through down-
regulation of MMP-2 expression.
Cinnamon treatment downregulates the expression of Her-
2 oncoprotein
Various studies have shown that a variable proportion of
cervical carcinoma tumors overexpress Her-2 oncopro-
tein [31]. Moreover, there are reports suggesting a corre-
lation between Her-2 overexpression and upregulation of
MMP-2 and MMP-9 expression [32,33]. Since we found
that cinnamon downregulated the expression of MMP-2,
we wanted to examine the status of Her-2 in cinnamon-
treated cells and correlate its expression with that of
MMP-2. Thus SiHa cells were treated with different con-
centrations of ACE-c (0-80 μg/ml) followed by immunob-
lotting of the extracted proteins using Her-2 antibody.
Interestingly, we observed that the cinnamon extract
down regulated the expression of Her-2 protein in a dose-
dependent manner compared to the control cells (Fig.
3A), the maximum reduction being at 80 μg/ml (~2.6-
fold, p ≤ 0.001). This was further proved by confocal stud-
ies wherein at 80 μg/ml of ACE-c treatment, a significant
reduction in the expression of Her-2 could be observed
(Fig. 3B). These results correlated with decreased MMP-2
expression observed in cinnamon-treated cells, thereby
elucidating the potential antineoplastic role of cinnamon
in cervical cancer through reduction of Her-2 and MMP-
2 expression.
Cinnamon extract induces apoptosis through increase in
intracellular calcium as well as loss of mitochondrial
membrane potential
To further elucidate the anti-cancer mechanism of cinna-
mon in cervical cancer cells, we performed apoptosis
studies. After treating the cells with different doses of
ACE-c, the percent apoptotic cells were assessed by
Annexin V-FITC and propidium iodide staining, followed
by flow cytometric analysis (Fig. 4A). It was observed that
at concentrations of 40 and 80 μg/ml ACE-c, there was a
significant increase in the percentage of cells undergoing
apoptosis. Interestingly at 80 μg/ml ACE-c concentration,
there was ~2.6-fold (p ≤ 0.001) increase in the population
of cells undergoing apoptosis compared to the untreated
control cells.
Since intracellular Ca2+ is a powerful activator of apop-
tosis [34,35], we studied the Ca2+ signaling mechanism in
cells treated with ACE-c to elucidate the cause of apopto-
sis. It was observed that after treatment of SiHa cells with
various concentrations of ACE-c (0-80 μg/ml), there was
a dose-dependent increase in the intracellular levels of
calcium. It was noted that the calcium increase was maxi-
mal (~2.64-fold; p ≤ 0.001) at the concentration of 80 μg/
ml (Fig. 4B) compared to the control cells. Ionomycin was
used as a positive control.
It is known that increase in intracellular calcium might
be one of the factors responsible for disrupting the mito-
chondrial membrane potential resulting finally into cell
apoptosis [36-39]. To measure the collapse of electro-
chemical gradient across the mitochondrial membrane,
we stained the cells with JC-1 dye that aggregates into
healthy mitochondria and fluoresces red. By confocal as
well as flow cytometry assays, we observed that the cells
exposed to ACE-c exhibited a dose-dependent decrease
in JC-1 staining (Fig. 4C and 4D, respectively) compared
to the untreated control cells. This indicated that there
was a loss of mitochondrial membrane potential in cinna-
mon-treated cells, which approached the loss of potential
observed after treating the cells with the positive control
agent, FCCP (Fig. 4D). As clearly observed from the fig-
ure, cinnamon induced significant depolarization at 80
μg/ml ACE-c concentration wherein there was ~5-fold
reduction in the ratio of red-green fluorescence intensity
(p ≤ 0.001). Taken together, all these results suggested
that cinnamon extract exhibited a potent antineoplastic
effect in cervical cancer cells through increase in intracel-
lular calcium flux as well as through loss of mitochondrial
membrane potential, ultimately leading to apoptosis.
Discussion
Dietary constituents may display promising chemopre-
ventive and chemotherapeutic potential and thus amelio-
rate the side effects associated with conventional
chemotherapy. Recently, more attention is being focused
on complementary and alternative medicine (CAM) as an
alternative therapeutic modality for treatment of cancer
patients [2,3,9-13,40].
In the present study, we have reported the anti-cancer
potential of cinnamon extract in vitro in a human cervical
cancer cell line and have elucidated the possible underly-
ing mechanism. The anti-tumor activity of cinnamon has
been reported in vitro [21-23] as well as in vivo [10]; how-
ever, its role in cervical cancer remained to be elucidated.
We found that the aqueous cinnamon extract signifi-
cantly affected the growth rate of SiHa cells in a dose-
dependent manner. This data was further supported by
results from colony formation and soft agar assays, which
demonstrated statistically significant reduction in the
number of colonies in ACE-c treated cells compared to
the untreated control cells. Thus, cinnamon could be pro-
posed to be a promising candidate for restricting the
growth of cervical cancer cells.
It is well known that metastasis, being one of the major
causes of mortality in cancer, involves various steps such
as cancer cell adhesion, invasion, and migration [41].
Thus, to examine the effect of cinnamon extract on
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migration of SiHa cells, wound healing assays were per-
formed on untreated control and ACE-c treated cells.
Interestingly, cinnamon reduced the migration of cancer
cells in a significant manner, further strengthening its
potential use as an anti-cancer drug in cervical cancer.
One of the key steps in the invasive progress of cancer
cells is the degradation of extracellular matrix (ECM)
proteins by a family of zinc-binding enzymes called as
matrix metalloproteinases [42]. To elucidate the reason
behind the poor migration of ACE-c treated cells, we
tested the expression of MMP-2 (gelatinase) in control as
well as cinnamon-treated cells. A significant decrease in
the expression of MMP-2 was observed both at mRNA as
well as protein levels in ACE-c treated cells that could be
the reason for their reduced migration capability com-
pared to the control cells. Thus, downregulation of
MMP-2 expression by cinnamon could be regarded as a
rational approach towards metastatic disease therapy in
cervical cancer.
It is well-known that Her-2/Erb2, a transmembrane
receptor protein with tyrosine kinase activity from EGF3-
receptor family, is a critical marker of cervical and breast
cancer. Moreover, it has been shown that Her2 overex-
pression is related with the invasion capacity of the tumor
cells that is related partly with the up-regulation of MMP-
2 and MMP-9 expression as well as proteolytic activity
[32,33]. Interestingly, we found that cinnamon could
effectively and significantly down-regulate the expression
of Her-2 in SiHa cells. Thus, downregulation of Her-2
oncoprotein expression by cinnamon could be correlated
with the reduction in the expression of MMP-2 protein.
These leads could be explored in detail to further estab-
lish the antineoplastic activity of cinnamon in cervical
Figure 3 Cinnamon decreased the expression of Her-2 oncoprotein. (A) Western blot analysis showing decrease in Her-2 expression in SiHa cells
treated with different concentrations of ACE-c (0-80 μg/ml). Equal amounts of protein were loaded on 10% SDS-gel and immunoblotted with anti-
Her-2 antibody. Tubulin was used as a loading control. Densitometric analysis of Her-2 expression was performed using phosphorimager. The data
represents mean ± SD of five independent experiments. (B) Confocal images of the cells treated with indicated concentrations of ACE-c showing de-
crease in Her-2 expression. The cells were stained indirectly for Her-2 using Cy3 antibody (Panel II) and counterstained with DAPI (Panel I). Panel III
represents the merge images.
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cancer that would in turn emphasize the chemopreven-
tive potential of natural products.
Apoptosis plays a key role in the regulation of normal
tissue homeostasis and participates in the elimination of
abnormal cells. Most of the antitumor drugs kill the can-
cer cells by stimulating the apoptotic pathway [43]. To
test whether cinnamon could induce apoptosis in cervical
cancer cell line SiHa, we carried out apoptosis studies. At
an effective ACE-c concentration of 80 μg/ml, a signifi-
cant proportion of cells were observed to undergo apop-
tosis compared to the control cells. To further elucidate
the mechanism of apoptosis, we tested whether cinna-
mon could modulate calcium flux as the latter is known
to be one of the major mediators of apoptosis.
Intracellular Ca2+ trafficking is known to govern a num-
ber of vital cellular functions that affect cell survival.
Cytosolic calcium, (Ca2+)c, is usually maintained at lower
level (~100 nmol/L) compared to the extracellular con-
centration (~1 mmol/L). The cells regulate (Ca2+)c pri-
marily by regulating the Ca
2+ trafficking across the
plasma membrane and in and out of key organelles, such
as the endoplasmic reticulum and the mitochondria [42].
The endoplasmic reticulum is the largest reservoir of
Figure 4 Cinnamon induces apoptosis in SiHa cells through dysregulation of mitochondrial membrane potential. (A) SiHa cells were treated
with different concentrations of ACE-c (0-80 μg/ml) followed by Annexin V-FITC and PI staining to analyze the effect of cinnamon in apoptosis. This
was determined by FACS analysis showing the percentage of early (lower right quadrant) and late (upper right quadrant) apoptotic cells. (B) Flow
cytometric analysis of the rapid calcium release in SiHa cells after treatment with cinnamon. Cells (5 × 103 cells) were treated with different doses (0-
80 μg/ml) of ACE-c for 24 h. This was followed by loading the cells with Fluo-3/AM for 1 h before analyzing in calcium-free HBSS. Ionomycin was used
as a positive control. Fluorescence intensities were measured with FACS Calibur flowcytometer. The data represents mean ± SD of five independent
experiments. (C) Confocal images showing mitochondrial membrane depolarization induced by cinnamon. Control and cinnamon-treated SiHa cells
were stained with JC-1 and the staining pattern w as monitored by confocal laser scanning microscopy. For dete ction of J-aggregate form (red) (Panel
II) and J-monomer alone (green) (Panel I), Argon-Krypton laser line was excited at 590 nm and 527 nm, respectively. Panel III represents the merge
images. (D) Flow cytometric analysis with JC -1 dy e sho wing dec reas e in r ed t o gre en fl uor esce nce r ati o. Co ntro l (5 × 105) and cells trea ted with various
concentrations (0-80 μg/ml) of ACE-c were stained with JC-1 dye for 30 min. Fluorescence intensities were measured with FACS Calibur flowcytome-
ter. The data represents mean ± SD of five independent experiments.
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Ca2+ in normal cells whereas mitochondrial levels of Ca2+
are quite low. But during apoptosis, mitochondria are
known to accumulate Ca2+, especially when the (Ca2+)c
level is high [35,37-39]. Increase in mitochondrial cal-
cium, (Ca2+)m, induces apoptosis resulting into loss of
Δψm, expansion of the matrix, and the rupture of the
outer mitochondrial membrane [36]. Interestingly, at 80
μg/ml concentration of cinnamon extract, there was a sig-
nificant increase in the levels of intracellular calcium in
SiHa cells that could result into their apoptosis.
Since cinnamon increased intracellular calcium levels
as well as induced apoptosis in cervical cancer cells, we
wanted to know the status of mitochondrial membrane
potential in these cells. Moreover, mitochondria are
known to accumulate Ca2+ during apoptosis especially
when the cytosolic calcium level is high [37-39,44]. Thus,
we tested the Δψm in cinnamon treated cells by using the
fluorescent dye, JC-1 that aggregates into healthy mito-
chondria and fluoresces red. Upon mitochondrial col-
lapse in apoptotic cells, JC-1 dye no longer accumulates
and instead is distributed throughout the cell resulting
into decrease in red fluorescence. In accordance with
this, we found that ACE-c indeed disrupted the mito-
chondrial membrane potential as observed by decrease in
the intensity of red fluorescence. FCCP, a drug known to
disrupt the transmembrane potential of mitochondria
[45], was used as a positive control. Thus, the increased
intracellular calcium induced by ACE-c might be associ-
ated with the observed decline in mitochondrial mem-
brane potential.
Dietary polyphenols have recently invited a great deal
of attention owing to their chemopreventive properties
[46-48]. They can modulate the process of carcinogenesis
through several mechanisms. Interestingly, polyphenols
seem to play dual roles (either protective or therapeutic)
under different situations. For example, cinnamon poly-
phenols have been recently shown to play a protective
role by attenuating the decline in mitochondrial mem-
brane potential induced by ischemic injury in C6 glioma
cells [49]. On the other hand, in our case, we observe that
cinnamon extract, which contains a mixture of polyphe-
nols together with cinnamaldehyde as the major bioactive
component, plays a therapeutic role in cervical cancer
cells through depolarization of the mitochondrial mem-
brane potential resulting into cellular apoptosis. These
natural products seem to work in a tightly regulated man-
ner wherein they switch their roles either towards protec-
tive or therapeutic side depending upon either the
amount of the drug being used or upon the cellular phe-
notype [50]. For example, resveratrol, another polyphe-
nol, has also been shown to play a dual role, either
protective [51-53] or therapeutic [54]. Based on all these
observations, our data strongly implicates that cinnamon
could be proposed to be a potent antineoplastic agent in
cervical cancer wherein it could induce apoptosis
through increase in calcium flux as well as through loss of
mitochondrial membrane potential.
Conclusion
The failure of conventional chemotherapy to reduce mor-
tality invites attention towards new alternative
approaches that would reduce morbidity as well as side
effects conferred by conventional chemotherapy. Plants
have played a significant role as a source of effective anti-
cancer agents and 60% of currently used anti-cancer
drugs are derived from natural sources such as plants,
marine organisms and microorganisms [55,56]. Recently,
a greater emphasis has been given towards the research
involving complementary and alternative medicine in
cancer management. Several studies have been con-
ducted on herbs that possess anticancer properties and
have been used as potent anticancer drugs [57]. The pres-
ent work has addressed the antineoplastic potential of the
spice cinnamon in cervical cancer. Cinnamon besides
altering the growth kinetics of cells induces apoptosis
through loss of mitochondrial membrane potential. Cin-
namon reduces the expression of prognostic marker, Her-
2, of cervical cancer that calls attention for further studies
in this area. Collectively, these data suggest that cinna-
mon extract could be proposed to be a potent anticancer
drug candidate in cervical cancer.
Additional material
Abbreviations
ACE-c: Aqueous Cinnamon Extract from C. cassia; CAM: Complementary and
Alternative Medicine; Δψm: Mitochondrial membrane potential; MMP-2: Matrix
Metalloproteinase-2; MTT: 4,5-dimethylthiazol-2-yl-2,5-diphenylthiazolium bro-
mide; FCCP: Carbonyl Cyanide p-(trifluoromethoxy) Phenylhydrazone; TMRE:
Tetramethylrhodamine ethyl ester; Her-2: Human epithelial receptor 2; JC-1:
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl enzamidazolocarbocyanin iodide
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RKG designed the study and drafted the manuscript. SJK and ASC have carried
out all the experiments. SK had participated in cell growth analysis and RT-PCR
experiments. SAS contributed to the FACS analysis. SC has helped in time-lapse
study. All the authors read and approved the final version of the manuscript.
Acknowledgements
This work was supported by funding from the Interactive Research School for
Health Affairs (IRSHA), Bharati Vidyapeeth University. We thank our Director, Dr
PK Ranjekar for encouraging us to complete this work. We would like to thank
Dr MK Bhat (NCCS) for helping us in zymography experiment. We thank Dr GC
Mishra, Director, NCCS, for allowing us to use Confocal and FACS facilities with-
out which completion of this work was highly impossible. In this regard, we
Additional file 1 Biochemical Analysis and Cytotoxic Activity of Aque-
ous Cinnamon Extract (ACE-c). Data providing HPTLC analysis and poly-
phenol content of ACE-c as well as cytotoxic activity of the extract on SiHa
cells. It includes supplementary figs. S1 and S2.
Koppikar et al. BMC Cancer 2010, 10:210
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Page 11 of 12
would like to thank Dr Limaye, Ms Hemangini Shikhara, Mr Swapnil Walke, Mrs
Pratibha and Mrs Ashwini Atre for technical assistance at NCCS.
Author Details
1Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth
University Medical College Campus, Pune, Maharashtra, India and 2National
Center for Cell Science (NCCS), Pune University Campus, Ganeshkhind, Pune,
Maharashtra, India
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Published: 18 May 2010
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Cite this article as: Koppikar et al., Aqueous Cinnamon Extract (ACE-c) from
the bark of Cinnamomum cassia causes apoptosis in human cervical cancer
cell line (SiHa) through loss of mitochondrial membrane potential BMC Can-
cer 2010, 10:210
