Cytotoxic effect of Carvacrol on human cervical cancer cells

Abstract

Carvacrol is a component of numerous aromatic plants which has been evaluated for substantial pharmacological properties. Although the carvacrol induced cytotoxicity has already been reported, but no such study have been made on human cervical cancer, HeLa and SiHa cells. Therefore, in the present study an attempt has been made to investigate the cytotoxic effect of carvacrol on cervical cancer cells. Cytotoxicity induced by carvacrol was determined by different assays like MTT assay and LDH assay. Apoptosis was measured by DNA fragmentation assay. The study clearly showed the dose dependent cytotoxic effect of carvacrol in HeLa and SiHa cells at an IC50 of 50 mg L-1 by both the cytotoxic assays respectively. The dying cells showed characteristics of apoptosis such as, DNA fragmentation. The data in the present study clearly demonstrated cytotoxic effects of carvacrol on human cervical cancer cells. Carvacrol could have a potential therapeutic significance in treating cancer.

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Cytotoxic effect of Carvacrol on human cervical cancer cells
Syed Jafar Mehdi1#, Aijaz Ahmad2#, M Irshad1, Nikhat Manzoor2, *M Moshahid Alam Rizvi1
1 Genome Biology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi-25, India.
2 Enzyme Kinetics Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi-25, India.
# Both the authors have equal contribution
*Corresponding Author: rizvijmi@gmail.com
Abstract
Carvacrol is a component of numerous aromatic plants which has been evaluated for substantial pharmacological
properties. Although the carvacrol induced cytotoxicity has already been reported, but no such study have been made
on human cervical cancer, HeLa and SiHa cells. Therefore, in the present study an attempt has been made to
investigate the cytotoxic effect of carvacrol on cervical cancer cells. Cytotoxicity induced by carvacrol was determined
by different assays like MTT assay and LDH assay. Apoptosis was measured by DNA fragmentation assay. The
study clearly showed the dose dependent cytotoxic effect of carvacrol in HeLa and SiHa cells at an IC50 of 50 mg L-1
by both the cytotoxic assays respectively. The dying cells showed characteristics of apoptosis such as, DNA
fragmentation. The data in the present study clearly demonstrated cytotoxic effects of carvacrol on human cervical
cancer cells. Carvacrol could have a potential therapeutic significance in treating cancer.
Keywords: Carvacrol; Cervical cancer; HeLa cells; SiHa cells; Apoptosis.
Introduction
In recent years, the interest in medicinal plants
and their biologically active derivatives has
increased, in relation to the possible
development of novel potential drugs for several
pathologies of relevant social impact (Hedberg,
1993; Heinrich and Gibbons, 2001). It is well
known that natural products from the extracts of
medicinal plants are used in the treatment of
skin, respiratory, neuromuscular and mental
health disorders and also in obstetrics and
gynecology (Abo et al., 2000; Ahmad et al.,
1998; Ankli et al., 2002; Dutta et al., 1998; Pinn,
2001).The anti-tumor activity and the possible
applications of medicines from medicinal plants
for cancer prevention have been recently
described (Chiu and Wu, 2002; Cragg and
Newman, 1999; Katsube et al., 2003; Mukherjee
et al., 2001; Richardson, 2001; Tatman and Mo,
2002; Wargovich et al., 2001; Popov et al.,
2001; Ruffa et al., 2001).
Essential oils and their components
have been timely honored for their
pharmaceutical properties. Plant-derived
substances have recently become of great
interest owing to their versatile applications
(Baris et al., 2006; Khan et al., 2010). The
development of pharmaceuticals begins with
identification of active principles, detailed
biological assays and dosage formulations,
followed by clinical studies to establish safety,
efficacy and pharmacokinetic profile of new drug
(Iwu et al., 1999). Carvacrol (5-isopropyl-2-
methylphenol) (Figure I) is a natural isopropyl
cresol and is credited with a series of
pharmacological properties including
antimicrobial and antifungal effects (Pina-Vaz et
al., 2004; Braga et al., 2007; Chami et al., 2005).
Carvacrol is generally recognized as a safe food
additive and used as a flavoring agent in baked
foods, sweets, beverages and chewing gum
(Fenaroli, 2002). It is also well known that
essential oils, which are rich in carvacrol,
possess strong antioxidant properties equivalent
to those of ascorbic acid, butyl hydroxyl toluene
(BHT) and vitamin E (Aeschbach et al., 1994;
Mastelic et al., 2008). Since many antioxidants
exert anticarcinogenic effects (Kallistratos et al.,
1994; Evangelou et al., 1997; Liasko et al.,
1998; Karkabounas et al., 2002), it is possible
that carvacrol functions in a similar way and the
anti-proliferative properties of carvacrol on non-
small cell lung cancer cells, A549, chronic
myeloid leukemia cells, K562, murine B16
melanoma cells and human metastatic breast
cancer cell line, MDA-MB 231 have been shown
(He et al., 1997; Horvathova et al., 2007;
Karkabounas et al., 2006; Koparal and
Zeytinoglu, 2003; Lampronti et al., 2006;
Arunasree, 2010).
Based on this hypothesis and reported
data, the present study is aimed to evaluate the
cytotoxic effects of carvacrol on human cervical
cancer HeLa cells and SiHa cells. Quercitin at

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an IC50 value of 50 µM was used as positive
control to assess the efficacy of carvacrol (Wei
et al., 2007).
Figure I: Chemical structure of Carvacrol.
Materials and Methods
Chemicals
Carvacrol (98%) and Quercitin (≥95%) (quercitin
was used as positive control) were purchased
from Sigma–Aldrich (USA). DMEM (Dulbecco’s
modified Eagle’s medium), MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium
bromide) and 0.25% trypsin and 0.02% EDTA
mixture was purchased from Himedia (India).
Cytoscan™-LDH Cytotoxicity Assay Kit was
purchased from GBiosciences (USA). Fetal
bovine serum (FBS) was from Biowest (USA).
All the other chemicals and reagents were
purchased from local companies and were of
molecular biology grade. Carvacrol and
Quercitin were dissolved in DMSO and stored at
4oC.
Figure II: Dose-dependent effect of
carvacrol on SiHa and HeLa cell
proliferation. Both the cells were
cultured in 10% FBS medium and
treated with 25, 50, 100, 200 and
500 mg L-1 carvacrol for 48 h and
cell proliferation was monitored
by MTT assay. The percent
viable cells were calculated
in comparison to untreated
cells taken as 100%. Values
were expressed as mean ± SD
and the experiment was
performed in triplicate (P<0.05).
Cell lines and culture conditions
Human cervical cancer cell lines, HeLa and
SiHa were obtained from National Centre for
Cell Sciences (NCCS) Pune, India. Both the cell
lines were grown as monolayer cultures in
Dulbecco’s modified Eagle’s medium containing
10% fetal bovine serum and antibiotics (100
units/ml penicillin and 100 mg L-1 streptomycin)
in a humidified atmosphere of 5% CO2 at 37°C
in T-75 flasks and were sub cultured twice a
week.
Cytotoxicity assay
The cytotoxic effect of carvacrol was assessed
in human cervical cancer HeLa and SiHa cells
by the MTT assay (Chou and Talalay, 1984).
Briefly, cells were seeded at a number of 2 X 104
per well onto 96-well plates (200µl/well) in
triplicates, allowed to attach and grow for 24 h
and subsequently exposed to different carvacrol
(25 mg L-1-500 mg L-1) concentrations for 48 h.
At the end of the treatment, the medium was
removed and cells were incubated with 20µl of
MTT (5 mg/ml in PBS) in fresh medium for 4 h at
37oC. After four hours, formazan crystals,
formed by mitochondrial reduction of MTT, were
solubilized in DMSO (150 µl/well) and the
absorbance was read at 570 nm after 10 min
incubation on the iMark Microplate Reader
(BioRad, USA). Percent inhibition of cytotoxicity
was calculated as a fraction of control (without
carvacrol) and the cytotoxicity of carvacrol was
expressed as IC50.
0
10
20
30
40
50
60
70
80
90
100
Control 25 50 100 200 500
Viable Cells (%)
Concentration of Carvacrol (µg/ml)
SiHa Cells HeLa Cells

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0
10
20
30
40
50
60
70
80
25 50 100 200 500
Concentration of Carvacrol (µg/ml)
% Cytotoxicity
SiHa
HeLa
LDH assay
Cytotoxicity induced by carvacrol was assessed
by lactate dehydrogenase (LDH) leakage into
the culture medium. Following exposure to the
carvacrol the culture medium was aspirated and
centrifuged at 3000 rpm for 5 min in order to
obtain a cell free supernatant. The activity of
LDH in the medium was determined using a
commercially available Cytoscan™-LDH
Cytotoxicity Assay Kit. Percent inhibition of
cytotoxicity was calculated as a fraction of
control (without carvacrol) and the cytotoxicity of
carvacrol was expressed as IC50.
Detection of apoptotic DNA fragments
DNA fragmentation was detected by agarose gel
electrophoresis. 1X106 HeLa and SiHa cells
were plated in 30 mm culture plate. When the
cells of reached approximately 70% confluency,
increasing concentrations of carvacrol (25 mg L-
1-500 mg L-1) were added and the cells were
incubated for 48 h. After 48 hours, cells were
harvested and pelleted by centrifugation
(Eppendorf 5804R, Germany). Cellular DNA was
extracted by SDS/proteinase K treatment,
phenol–chloroform extraction, and ethanol
precipitation as described previously (Sambrook
and Russell, 2001) and then dissolved and
stored in TE buffer and the DNA samples
obtained were analyzed by 2% agarose gel
electrophoresis. After electrophoresis, the gels
were stained with ethidium bromide, and
visualized as a DNA ladder with UV.
Figure III: Dose-dependent effect
of carvacrol on SiHa and HeLa cell
proliferation. Both the cells were
cultured in 10% FBS medium and
treated with 25, 50, 100, 200 and
500 mg L-1 carvacrol for 48 h and
cytotoxicity was monitored by LDH
assay. The percent cytotoxicity
was calculated in comparison to
untreated cells taken as 100%.
Values were expressed as mean ±
SD and the experiment was
performed in triplicate (P<0.05).
Statistical analysis
Viable cell ratios were evaluated by Student ‘t’
test. The data were presented as arithmetic
mean ± standard deviation. A value of P < 0.05
was considered to be statistically significant.
Results
Carvacrol inhibited proliferation of HeLa and
SiHa cells
Data on the cytotoxic effects of carvacrol using
two human tumor cell lines in vitro are showed in
Figure II and III. Carvacrol has cytotoxic effects
in vitro at clinical acceptable concentrations (IC50
values ≤ 50 mg L-1) by MTT methods and LDH
methods respectively. The cytotoxic effect of
carvacrol was determined using concentrations
ranging 25 mg L-1- 500 mg L-1 for 48 h. After 48
h exposure, carvacrol induced concentration-
dependent cytotoxic effects in cervical cell lines
with IC50 of 50±3.89 mg L-1 and 50±5.95 mg L-1
in SiHa and HeLa cells, respectively using MTT
method and 55±0.15 mg L-1 and 50±0.05 mg L-1
in SiHa and HeLa cells, respectively by LDH
method.
Validation of apoptosis measurement by DNA
laddering
In our results, the cells were treated with
carvacrol, and the DNA was directly extracted
and run on agarose gel. DNA hy-
perfragmentation, if presented, was seen as a
stepwise ladder of DNA fragments.

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Figure IV: Analysis of DNA
fragmentation in human cervical cancer cells
treated with carvacrol. a. HeLa cells treated
with carvacrol. Lane 1: 100 bp DNA ladder;
lane 2: Control cells; lane 3: Cells treated with
25 mg L-1 carvacrol; lane 4: Cells treated with
50 mg L-1 carvacrol; lane 5: Cells treated with
100 mg L-1 carvacrol; lane 6: Cells treated
with 200 mg L-1 carvacrol; lane 7:Cells treated
with 500 mg L-1 carvacrol; lane 8: Cells treated
with 50µM quercitin. b. SiHa cells treated with
carvacrol. Lane 1: 100 bp DNA ladder; lane 2:
Control cells; lane 3: Cells treated with 25
mg L-1 carvacrol; lane 4:Cells treated with 50
mg L-1 carvacrol; lane 5: Cells treated with 100
mg L-1 carvacrol; lane 6: Cells treated with
200 mg L-1 carvacrol; lane 7: Cells treated
with 500 mg L-1 carvacrol; lane 8: Cells treated
with 50µM quercitin.
The data (Figure IV) shows that DNA
laddering is pronounced for carvacrol (50 mg L-1)
in HeLa cells and (50 mg L-1) in SiHa cells
respectively. These results confirm that
carvacrol can induce apoptosis of HeLa and
SiHa cells.
Discussion
Research on biologically active compounds from
essential oils has proved them to be potent anti-
bacterial, antifungal and anti-oxidant agents
(Lampronti et al., 2006; Albuquerque et al.,
2007; Ao et al., 2008; Baik et al., 2008; Bakkali
et al., 2008). Carvacrol (5-isopropyl-2-
methylphenol) is one of main substances of
essential oil possess antimicrobial and
antifungal effects (Pina-Vaz et al., 2004; Braga,
2007; Chami et al., 2005).
Cellular proliferation depends on the
rates of cell division and death and, thus, many
anticancer drugs have been used to prevent
cancer cell division in order to inhibit cancer cell
proliferation. In vitro cytotoxicity assays can be
used to predict human toxicity and for the
general screening of chemicals (Clemedson and
Ekwall, 1999; Scheers et al., 2001). It has been
previously reported that different cytotoxicity
assays can give different results depending on
the test agent used and the cytotoxicity assay
employed (Weyermann et al., 2005). The results
obtained from the cytotoxicity assays indicate
that inhibition of HeLa and SiHa cells was
gradually increased by the addition of carvacrol
in MTT and LDH assays. It reveals that HeLa
and SiHa cell line is susceptible to carvacrol.
Although, the LDH leakage assay is based on
the release of the enzyme into the culture
medium after cell membrane damage whereas
the MTT assay is mainly based on the
enzymatic conversion of MTT in the
mitochondria, both the cytotoxicity assays,
employed to assess carvacrol toxicity in vitro,
showed the similar results.
Apoptosis is a physiological process of
cell elimination, and DNA fragmentation is one
of the hallmarks of cell apoptosis. The results
showed the apoptosis proportion of cells was
increased by treatment of carvacrol in both the
cervical cancer cell lines.
Conclusion
In summary, the present study demonstrated
that carvacrol is a potent anti-cancer compound
with an IC50 of 50 mg L-1at 48 h inducing growth
inhibition in both the human cervical cancer
cells. Further research based on animal models
may resolve in vivo efficacy of carvacrol.
Acknowledgement
The financial assistance provided by the
Defense Research and Development
Organization (DRDO), New Delhi, is gratefully
acknowledged. The assistance of Mr. Irfan
Ahmad is acknowledged with thanks.
b
1
2 3 4 5 6
7
8
1 2 3 4 5
6 7 8
a

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