Iran J Cancer Preven. 2015 May; 8(3):e2331. DOI: 10.17795/ijcp2331
Published online 2015 May 25. Research Article
Anticancer Activity of Curcumin on Human Breast Adenocarcinoma:
Role of Mcl-1 Gene
Zeinab Khazaei Koohpar
; Maliheh Entezari
; Abolfazl Movafagh
; Mehrdad Hashemi
Department of Herbal Medicine, Institute of Islamic and Complementary Medicine, Iran University of Medical Sciences, Tehran, IR Iran
Department of Biology, Tonekabon Branch, Islamic Azad University, Tonekabon, IR Iran
Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, IR Iran
Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
*Corresponding author: Mehrdad Hashemi, Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, IR Iran. Tel: +98-2122006664,
Received: April 17, 2015; Revised: April 25, 2015; Accepted: May 8, 2015
Background: Breast cancer is the second leading cause of cancer-related death among females in the world. To date, chemotherapy
has been the most frequently used treatment for breast cancer and other cancers. However, some natural products have been used, as
alternative treatments for cancers including breast cancer, due to their wide range of biological activities and low toxicity in animal
Objectives: The present study examined the anti-proliferative activity of curcumin and its eﬀect(s) on the apoptosis of breast cancer cells.
Materials and Methods: This study was performed by an in vitro assay and the anticancer eﬀects of curcumin were determined by MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide). We used quantitative real time Polymerase Chain Reaction (PCR) for
detection of Mcl-1 gene expression in treated groups and then compared them to control samples.
Results: In the treatment group, there were higher levels of cell death changes than the control group. The results also showed that the
Mcl-1 gene expression declined in the tested group as compared to the control group.
Conclusions: Our present ﬁndings indicated that curcumin signiﬁcantly inhibited the growth of human breast cancer cell MCF-7 by
inducing apoptosis in a dose- and time- dependent manner, accompanied by a decrease in MCF-7 cell viability. Furthermore, our results
showed that quantitative real-time PCR could be used as a direct method for detection Mcl-1 gene expression in tested samples and normal
Keywords: Breast Cancer; Curcumin; Mcl-1 gene; Apoptosis
Copyright © 2015, Iranian Journal of Cancer Prevention. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Com-
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ages, provided the original work is properly cited.
It is known that breast cancer is the most common
cancer for women worldwide, and accounts for approxi-
mately 25% of all female malignancies with a higher prev-
alence in developed countries. Breast cancer is the second
leading cause of cancer-related death among females in
the world (1). The discovery of novel natural compounds
with low toxicity and high selectivity for killing cancer
cells is an important area in cancer research (2).
To date, chemotherapy has been the most frequently
used treatment for breast cancer and other cancers.
However, this method of treatment also destroys some
normal cells as well. Due to their wide range of biological
activities and low toxicity in animal models, some natu-
ral products have been used as alternative treatments for
cancers including breast cancer (2).
Curcumin or diferuloylmethane is the major yellow
pigment extracted from turmeric (Curcuma longa) and is
commonly used as a ﬂavoring agent in food (3).
Curcumin has been widely studied for its anti-inﬂam-
matory, anti-angiogenic, antioxidant, wound healing,
and anti-cancer eﬀects because of its medicinal proper-
ties in Indian and Chinese medicine (2). Moreover, exten-
sive research has shown that curcumin possesses anti-
proliferative and anti-carcinogenic properties in a wide
variety of cell lines and animals (4).
In addition, recent studies have shown that curcumin,
either alone or in combination with other anticancer
agents, can eﬃciently induce apoptosis (2).
Apoptosis is a tightly regulated process of programmed
cell death, including the activation of various molecules
for initiating cell death.
Speciﬁc activation of apoptosis in tumor cells oﬀers a
promising approach for cancer therapy. However, the
speciﬁc mechanisms of curcumin-induced cytotoxicity
remain controversial due to the variable anti-and pro-
apoptotic signaling pathways in diﬀerent cell types.
Oxidative stress derived from curcumin through pro-
duction of reactive oxygen species and oxidative dam-
age causes DNA modiﬁcation. This process continues
and carries on proteolytic events and induces apoptosis
Khazaei Koohpar Z et al.
Iran J Cancer Preven. 2015;8(3):e2331
(4). Plenty of proteins and a number of genes regulate
apoptosis, in which two pairs of proteins play an impor-
tant part. Bcl-2 is a member of a family of proteins in-
volved both in preventing apoptosis (pro-survival) and
in promoting it (pro-apoptotic). Anti-apoptotic proteins
include the Bcl-2 family (such as Bcl-2, Bcl-XL, Mcl-1 etc.).
Pro-apoptotic proteins are sub-grouped into the Bax fam-
ily (such as Bax, Bak, etc.) and the BH3-only family (such as
Bid, Bad, Bim, etc.) (5).
The Tumor Necrosis Factor (TNF)-Related Apoptosis In-
ducing Ligand (TRAIL) has promising anticancer activity.
Curcumin enhances TRAIL-induced apoptosis of breast
cancer cells by regulating apoptosis-related proteins
(such as Mcl-1, ERK and Akt) (6, 7).
The exact mechanism by which curcumin exerts its
apoptotic eﬀects in breast cancer cells still remains un-
clear. The present study examined the anti-proliferative
activity of curcumin on breast cancer cells.
3. Materials and Methods
Curcumin was purchased from Sigma-Aldrich Corpora-
tion and was prepared with Dimethyl Sulfoxide (DMSO)
at a concentration of 10 mM, stored as small aliquots at
-20°C, and thawed and diluted as needed in cell culture
medium. Dimethyl Sulfoxide, Propidium Iodide (PI) and
trypsin were purchased from sigma (St, Louis, Mo, USA).
Roswell Park Memorial Institute (RPMI) 1640, penicillin,
streptomycin and other cell culture supplies were from
Gibco BRL (Grand Island, NT, USA). Fetal bovine serum
was from Hyclone (Logan, UT, USA). MTT (3-(4,5-dimeth-
ylthiazol-2-yl)- 2,5- diphenyl trazolium bromide) was ob-
tained from Fluka (Ron Konkoma, NY, USA).
3.2. Cell Line and Culture
Human breast cancer cell line, MCF7, was obtained from
the Pasteur institute of Iran. These cells were cultivated
in T75 tissue culture ﬂasks in RPMI-1640 supplemented
with 10% fetal calf serum, 100 μg/mL penicillin, 100 μg/mL
streptomycin, 2 mM L-glutamine, and 20 mM hydroxy-
ethyl piperazine ethanesulfonic acid, and incubated in a
humidiﬁed incubator containing 5% CO
3.3. MTT Assay
Cell viability was assessed using the MTT assay. Breast
cancer (5 × 10
) cells were seeded in 200 μL of RPMI-1640
medium in 96-well plates, and cultured overnight. Next,
the medium was replaced with fresh RPMI-1640 or the
same media containing diﬀerent concentrations of cur-
cumin. After a further incubation for 24 or 48 hours, 50
μL of MTT (2 mg/mL) was added to each well followed by
4 hours of incubation. The medium was discarded and
150 μL of dimethyl sulfoxide was added to each well, and
incubated for 20 minutes. The OD was measured at 490
nm. The cell viability index was calculated according to
the following Equations:
Cytotoxicity% =1 −
Mean absorbance of toxicant
Mean absorbance of negative control
Viability% = 100 − Cytotoxicity%
To diminish test error level, the MTT strain was added
to wells without cells and along with other wells, absor-
bance level was read and ultimately subtracted from the
3.4. Real-Time Polymerase Chain Reaction with
SYBR Green I
Total RNA was extracted from cells using an RNA isola-
tion reagent (sigma) as recommended by the manufac-
turer and the extracted RNA was puriﬁed using RNeasy
Mini Kit (Qiagen), and cDNA was synthesized using Quan-
titect Reverse Transcription Kit (Qiagen) according to the
manufacturer’s instructions. Reverse transcription was
carried out as follows: 42°C for two minutes, 42°C for 15
minutes, and 95°C for three minutes (one cycle). cDNA
was stored at -20°C for PCR.
Real-time PCR was performed in a 25 μL reaction solution.
The following sequences were used as primers (Table 1).
Real time PCR was carried out in optical grade 96-well
plates (Micro amp, Applied Biosystems, Singapore) at re-
action volume of 25 μL, including 12.5 SYBR Green Master
Mix (Primer design), 300 nm of each primer and 5 ng of
template DNA. All samples were run in duplicates. Ther-
mal cycling was performed on the Applied Biosystems
7300 real-time PCR system. Threshold cycle (Ct) data were
collected using ABI Prism 7300 sequence detection sys-
tem version 1.2.3 (Applied Biosystems, UK).
The relative gene expression was analyzed by the 2
method. The fold change in target gene cDNA relative to
the HPRT (Hypoxanthine-guanine phosphoribosyltrans-
ferase) internal control was determined by:
Fold change= 2
Where ΔΔCt = (Ct
Table 1. Characteristics of the Primers Used in the Real-Time
Polymerase Chain Reaction
3.5. Statistical Analysis
Statistical signiﬁcances were calculated using the Stu-
dent’s t-test and one-way analysis of variance.
Khazaei Koohpar Z et al.
Iran J Cancer Preven. 2015;8(3):e2331 www.ijcancerprevention.com
4.1. The Eﬀects of Curcumin on Inhibition and Pro-
liferation of MCF7 Cell Line
The eﬀect of curcumin was studied as a dose-response
experiment. Proliferation of MCF7 cells was signiﬁcantly
inhibited by curcumin in a concentration-dependent
manner during 48 hours (P < 0.01). Diﬀerent concentra-
tions of curcumin at 48 hours had diﬀerent cytotoxicity
eﬀects on MCF7 cell line (Figure 1).
The 50% inhibition concentration (IC50) values of cur-
cumin on MCF7 cells was determined (Figure 2). Further-
more, IC50 was determined by probit analysis using the
Pharm PCS (Pharmacologic Calculation System) statisti-
cal package (Springer-Verlag, USA). There were signiﬁcant
diﬀerences in IC50 curcumin (P < 0.05).
Cell viability (%)
Concentration (µ g/ml)
Figure 1. Eﬀects of Curcumin on MCF-7 Cell Viability
Figure 2. The IC50 of Curcumin for MCF-7 Cell Line
Fluorescence - d(F1)/dT
07.0 03.0 70.0 72.0 74.0 76.0 73.0 80.0 82.0 84.0 85.0 88.0 00.0
Figure 3. Melting Curve Analysis of the Mcl-1 and HPRT Genes
As expected, there was a signiﬁcant diﬀerence between
the tested and normal samples regarding Mcl-1 gene ex-
pression changes. To optimize and validate the real-time
PCR assay before using the ΔΔCT method for gene expres-
sion, a validation experiment was performed to deter-
mine the PCR eﬃciencies of the target and the reference
genes. Melting curve analysis was performed for every
single reaction to exclude ampliﬁcation of non-speciﬁc
products. Each valid ampliﬁcation reaction displayed a
single peak at the expected Tm (melting temperature).
The results also showed that Mcl-1 gene expression de-
clined in the control group as compared to the experi-
mental group. The mean ratio was determined for both
groups as follows: 1.04 ± 0.13 for control group, and 0.49 ±
0.12 for the experimental group.
For centuries, curcumin has been consumed in the
diet and used as a herbal medicine in several Far Eastern
Countries (2). Curcumin has cancer chemopreventive
properties in a variety of animal models of chemical car-
cinogenesis, including those resulting in tumors of the
mammary gland (6, 7).
Oxidative stress and oxidative damage are involved in
the pathophysiology of many chronic inﬂammatory and
degenerative disorders, particularly cancer. The genera-
tion of Reactive Oxygen Species (ROS), particularly O
and OH, play important roles in the development of can-
cer (8, 9). Curcumin has been shown to scavenge O
OH radicals (10, 11).
As evident curcumin expresses anti-oxidant, anti-in-
ﬂammatory, anti-antigenic, anti-mitotic and anti-meta-
static activities in vitro and in animal experiments, thus
it might be a promising molecule for the prevention and
treatment of cancer in humans. Curcumin has antiprolif-
erative eﬀects in diﬀerent types of cell lines in vitro. One
of the initial reported descriptions of curcumin cyto-
toxicity occurred in Dalton’s lymphoma ascites cells, in
which curcumin at a concentration of 4 μg/mL produced
50% cytotoxicity. Curcumin also inhibited the growth of
Chinese hamster ovary cells and human leukemic lym-
phocytes in culture (12). At a concentration of 20 μg/mL,
curcumin produced 50% growth arrest in K-562 human
chronic myelogenous leukemia cells (13).
Curcumin has also been shown to inhibit the growth
of human breast cancer cell lines in vitro (5), including
HL60, k562, MCF- 7 and Hela cells (14). Also, to date, no
curcumin-related toxicity was observed in either ex-
perimental animals or humans, even at very high doses
(15). Our data are consistent with previous studies that
reported curcumin exerts its anticancer eﬀects via pro-
liferation inhibition and apoptosis induction in breast
cancer cells (15). In this study, the eﬀect of diﬀerent cur-
cumin doses (0 - 100 μm) on MCF-7 cell morphology was
examined. After a 24-hour period, curcumin treatment
caused MCF-7 cell shrinkage, rounding and partial de-
tachment, thus demonstrating the cytotoxic eﬀects of
Khazaei Koohpar Z et al.
Iran J Cancer Preven. 2015;8(3):e2331
curcumin on MCF-7 cells. On assaying the eﬀect of cur-
cumin on cell viability by the MTT assay, we observed a
decrease in cell viability. At curcumin concentration of
40 μm, signiﬁcant loss of viability can be detected during
the 0-24 hour treatment period.
It is important to mention that curcumin-mediated
reduction of cell viability was dose- and time- depen-
dent. Our present ﬁndings indicate that curcumin sig-
niﬁcantly inhibited the growth of human breast cancer
cell MCF-7 by inducing apoptosis in a dose- and time-
dependent manner, accompanied by a decrease in MCF-
7 cell viability.
Curcumin is highly cytotoxic toward several colon can-
cer cell lines. Curcumin blocked the entry to cell cycle
from G2 to M by inhibiting expression of cdc2/cyclin B
(16). The proapoptotic members of the Bcl-2 family, such
as Bax, were activated, and antiapoptotic genes such as
Bcl-XL were inhibited by curcumin (17).
Curcumin also triggers caspase-3-mediated cell death. It
activated GADD153, which in turn acts as an activator of
apoptosis (18). Curcumin decreases the expression of an-
tiapoptotic members of the Bcl-2 family and elevates the
expression of p53, Bax, and procaspases-3, -8 and -9 (19).
We selected the Mcl-1 gene, because the bcl-2 family of
proteins functions as pro- and anti-apoptotic members
(20). The bcl-2 members such as bax, bak, bad or bcl-Xs
promote apoptosis, whereas other members such as bcl-
2 and bcl-Xl prevent apoptosis by blocking the transloca-
tion of cytochrome c, and subsequent caspase activation.
Mitochondria are involved in excitotoxic injury during
cerebral ischemia and the release of cytochrome C, an
apoptogenic factor that propagates death signals by trig-
gering caspases leading to cell death. Using these assay,
status of all subjects was successfully determined (5). We
expanded the coverage of the detectable Mcl-1 gene by
SYBR Green assay for Mcl-1 gene expression.
Given the potential and safety of curcumin, it is a prom-
ising candidate for therapy of breast cancer, although ad-
ditional studies are needed.
We sincerely thank the department of herbal medicine,
Institute for Islamic and Complementary Medicine of
Iran for their ﬁnancial supports.
Zeinab Khazaei Koohpar, Maliheh Entezari and Abolfazl
Movafagh: performed the laboratory experiments and
prepared the manuscript. Mehrdad Hashemi: designed
the study, provided technical support, performed the lab-
oratory experiments, analyzed the data and revised the
The Department of herbal medicine, Institute for Islam-
ic and Complementary Medicine of Iran provided fund-
ing for this research.
Conﬂict of Interest
The authors made no disclosures.
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