Download Synergistic anti-cancer activity of the combination of

Pharmacological Reports
Copyright © 2013
2013, 65, 453–459
by Institute of Pharmacology
ISSN 1734-1140
Polish Academy of Sciences
Synergistic anti-cancer activity of
the combination of dihydroartemisinin
and doxorubicin in breast cancer cells
Guo-Sheng Wu1, Jin-Jian Lu1,2, Jia-Jie Guo1, Ming-Qing Huang3, Li Gan2,
Xiu-Ping Chen1, Yi-Tao Wang1
1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences,
University of Macau, Macao, China
2
College of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, China
3
College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, 350108, China
Correspondence: Jin-Jian
Lu, e-mail: [email protected] and Yi-Tao Wang, e-mail: [email protected]
Abstract:
Background: Dihydroartemisinin (DHA) exhibits potent anti-malarial and anti-cancer activities. This study aimed to investigate the
anti-proliferative effects of a combination of DHA and doxorubicin (DOX) on human breast cancer cells.
Methods: MTT assay and the combination index (CI) were used to show the anti-proliferative effects and calculate the synergism
potential, respectively. Flow cytometry assay was used to detect apoptosis and the intracellular accumulation of DOX. JC-1 staining
was used to determine the mitochondrial membrane potential. Western blot analysis was used to detect the protein expression of
some apoptosis-related molecules.
Results: A synergistic anti-proliferative effect was found, and the enhanced anti-cancer activity was observed to be accompanied by
the prompt onset of apoptosis in MCF-7 cells. The combinative treatment remarkably decreased the mitochondrial membrane potential and activated caspase cascades more than the mono-treatment. Pretreatment with DHA also did not influence the accumulation
of DOX in MCF-7 cells.
Conclusion: This study presented a new opportunity to enhance the effectiveness of future treatment regimens of breast cancer using
DOX.
Key words:
dihydroartemisinin, doxorubicin, synergistic, anti-tumor, apoptosis; MCF-7
Abbreviations: ARTs – artemisinin and its derivatives, CI –
combination index, DHA – dihydroartemisinin, DOX – doxorubicin, JC-1 – 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolylcarbocyanine iodide, MMP – mitochondrial membrane potential, MTT – 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide, PARP – poly (ADP-ribose)
polymerase, PBS – phosphate buffered saline, P-gp – P-glycoprotein, TRAIL – tumor necrosis factor-related apoptosisinducing ligand
Introduction
Breast cancer is one of the most common cancers
worldwide and the most common among women [8].
Doxorubicin (DOX), an anthracycline drug, is widely
used as a chemotherapeutic agent for breast cancer.
Despite the remarkable anti-cancer activity of DOX,
Pharmacological Reports, 2013, 65, 453–459
453
cells. DHA pretreatment was found to enhance DOXmediated apoptosis in MCF-7 cells significantly. This
enhancement was accompanied by an increase in the
cleavage of poly (ADP–ribose) polymerase (PARP)
and the activation of caspase cascades. Our results revealed a new strategy for enhancing the effectiveness
of future treatment regimens using DOX.
Materials and Methods
Reagents
Fig. 1. Chemical
structure of dihydroartemisinin (DHA)
its practical therapeutic use is limited by toxicities
such as cardiotoxicity [19]. Therefore, combined
treatment with other drugs is desirable.
Traditional Chinese medicinal herbs have a long
history and are widely viewed as new, attractive
sources of therapeutic regimens without severe toxicity. The herb Artemisia annua L., which has been
used in China for centuries to treat fever and chills,
contains artemisinin as its active constituent. Artemisinin and its derivatives (ARTs) are extensively used
as anti-malarial drugs without severe side effects and
have been found to inhibit the growth of tumor cells
[4, 11, 17, 18]. One of the main active metabolites of
ARTs is dihydroartemisinin (DHA) (Fig. 1), which is
one of the most effective anti-cancer compounds both
in vitro and in vivo. DHA mediates cell cycle arrest,
induces apoptosis, blocks angiogenesis, and inhibits
metastasis [1, 7, 10–12, 17]. DHA also selectively
kills cancer cells with little effect on normal cells [6,
13]. DHA enhances as well the anti-cancer potential
of gemcitabine in human hepatoma cells and cooperates with tumor necrosis factor-related apoptosisinducing ligand (TRAIL) to induce apoptosis in human prostate cancer cells [5, 6]. Herein, we hypothesized that the combination of DHA and DOX in human breast cancer cells exerts a synergistic anticancer effect.
In this study, we found that the combination DHA
and DOX exerted a synergistic effect on breast cancer
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Pharmacological Reports, 2013, 65, 453–459
DHA was provided by Prof. Ying Li of the Shanghai
Institute of Materia Medica, Chinese Academy of
Sciences (Shanghai, China). DOX was obtained from
Sigma (St. Louis, MO, USA). 3-[4,5-Dimethyl-2thiazolyl]-2,5-diphenyltetrazolium bromide (MTT)
and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were purchased
from USB (OH, USA) and Molecular Probes
(Eugene, OR, USA), respectively.
Cell lines and culture
MCF-7, MDA-MB-231, and T-47D human breast cancer cells were obtained from ATCC (Manassas, VA,
USA). The cells were maintained at 37°C under an atmosphere of 5% CO2 and 95% air in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated
fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin).
MTT assay
Exponentially growing MCF-7, MDA-MB-231, and
T-47D cells were seeded into 96-well plates. The cells
were treated with the indicated compounds, and cell
viability was determined after 48 h of incubation by
adding 20 µl of MTT (5 mg/ml). After slightly aspirating the MTT-containing medium after 4 h, 100 µl
of dimethyl sulfoxide was added to solubilize the formazan followed by shaking 10 min in darkness. The
absorbance at 570 nm was recorded using a Multilabel counter (PerkinElmer, Singapore).
Synergistic activity of the combination of DHA and DOX
Guo-Sheng Wu et al.
Observation of morphologic changes
MCF-7 cells were seeded into six-well plates and
treated with 10 µM DOX for 24 h with or without pretreatment of 20 µM DHA for 6 h. Cellular morphology was observed with an AxioCam HRc CCD camera (Carl Zeiss, Germany).
Flow cytometry assay
MCF-7 cells seeded into six-well plates were treated
with 10 µM DOX for 24 h with or without pretreatment of 20 µM DHA for 6 h. The cells were harvested, fixed in 70% ethanol, and stored at 4°C overnight. Then, the cells were stained in phosphatebuffered saline (PBS) containing 5 µg/ml RNase and
20 µg/ml propidium iodide (PI) in the dark at room
temperature for 30 min. Flow cytometry (Becton
Dickinson FACSCanto™, Franklin Lakes, NJ, USA)
was used to analyze the cells. At least 10,000 events
were counted for each sample, and the sub-G1 was
analyzed.
Mitochondrial membrane potential (MMP) assay
The MMP of intact cells was measured by a fluorescent inverted microscope with the lipophilic probe
JC-1 [15]. MCF-7 cells were seeded into six-well
plates and treated with 10 µM DOX for 24 h with or
without pretreatment of 20 µM DHA for 6 h. JC-1
fluorescence was observed with a fluorescent microscope, and pictures were taken with an Axiovert 200
fluorescent inverted microscope (Carl Zeiss) and an
AxioCam HRc CCD camera (Carl Zeiss).
Western blot analysis
After treatment under desired conditions, the cells
were harvested and total proteins were extracted with
radioimmunoprecipitation assay lysis buffer containing 1% phenylmethanesulfonyl fluoride and 1% protease inhibitor cocktail for 30 min. Equal amounts of
total protein were separated by appropriate SDSPAGE and transferred onto a poly (vinylidene fluoride) membrane. After blocking with 5% non-fat
dried milk for 1 h, the membrane was incubated with
the specific primary antibodies against PARP,
caspase-7, caspase-9, and b-actin (Cell Signaling
Technology, Beverly, MA, USA) followed by incubation with corresponding secondary antibodies at 37°C
for 1 h. Specific protein bands were visualized with
an ECL advanced western blot analysis detection kit.
Intracellular accumulation of DOX
MCF-7 cells were pre-cultured for 6 h with or without
indicated concentrations of DHA and treated with different concentrations of DOX for 1 h. The cells were
then harvested and resuspended in 1 ml of PBS. The
cells were washed three times with PBS, and the
mean fluorescence intensities of the cells were detected with a fluorescence-activated cell sorting
(FACS)-Calibur cytometer (Becton Dickinson, San
Jose, CA, USA).
Statistical analyses
The combination index (CI) is widely used to quantify
drug synergism based on the multiple drug effect
equation of Chou–Talalay [2, 3]. In our study, the CI
values were determined for each concentration of
DHA, DOX, and their combination in cell proliferation assays using CalcuSyn (Biosoft, Cambridge,
UK). CI < 0.9 indicates synergism, CI = 0.9–1.10 indicates additive interaction, and CI > 1.10 indicates
antagonism [20].
Results
DHA synergistically increased the DOXinduced inhibition of cell proliferation
We first examined the synergistic effects of DHA and
DOX on the proliferation of MCF-7, MDA-MB-231,
and T-47D human breast cancer cell lines. Figure 2A
shows a schemate of the experimental protocol for the
combined treatment. Cells in 96-well plates were pretreated with serial concentrations of DHA for 6 h and
further treated with or without indicated DOX for
48 h. The cell proliferation inhibition bars to DHA,
DOX, and DHA combined with DOX from three independent experiments are shown in Figures 2B–D.
DHA exhibited moderate cytotoxicity against MCF-7
and T-47D cancer cells but had little effect on
MDA-MB-231 cells, consistent with our previous reports [11, 12]. After the combined treatment, markedly stronger anti-proliferation abilities were achieved.
Pharmacological Reports, 2013, 65, 453–459
455
Fig. 2.
Cytotoxicity of DOX + DHA in
breast cancer cells. (A) Schematic of
the
experimental
protocol
+ DHA treatment. (B,
C, D)
for
DOX
Dose re-
sponse of MCF-7, MDA-MB-231, and
T-47D cells to DHA, DOX, and DHA
+ DOX. (E) CI values in MCF-7, MDAMB-231and T-47D cells
Subsequently, CI values were calculated using CalcuSyn at fixed-ratio concentrations of DHA and DOX.
DHA plus DOX exhibited synergy in the tested breast
cancer cell lines except at the highest dose in
MDA-MB-231 cells, in which an additive effect was
observed (Fig. 2E).
DHA sensitized DOX-triggered apoptosis
We further investigated the effects of exposure to
DHA, DOX, or their combination on the apoptosisinducing abilities in MCF-7 cells. We first detected
morphological changes after DHA, DOX, and DHA
plus DOX treatment. A significantly higher number of
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Pharmacological Reports, 2013, 65, 453–459
round cells appeared in the combined-treatment group
than in the other groups (Fig. 3A). We then used flow
cytometry analysis with PI staining to detect the subG1 content, which is a characteristic of apoptosis. The
cells treated with both DHA and DOX had the highest
percentage in sub-G1. Given that mitochondria significantly influence DHA-induced apoptotic cell
death [11] and mitochondrial changes including MMP
collapse, the activation of caspases results in apoptosis. Therefore, we detected MMP changes after DHA,
DOX, and DHA + DOX treatment. JC-1, which is
a dual-emission fluorescent dye internalized and concentrated by respiring mitochondria, can reflect MMP
changes in live cells [15]. In 24 h-treated MCF-7
cells, mitochondrial membrane depolarization as de-
Synergistic activity of the combination of DHA and DOX
Guo-Sheng Wu et al.
Fig. 3. DOX + DHA triggers
increased
apoptosis in MCF-7 cells. MCF-7 cells
seeded
into
six-well
plates
were
treated with 20 µM DHA, 10 µM DOX,
or their combination for 24 h; the morphological
changes,
sub-G1,
MMP,
and protein expression were detected.
(A) Classical morphological changes.
(B) Results of flow cytometry with PI
staining. (C) JC-1 mitochondrial probe
for MMP test. (D) Protein extracts were
immunoblotted with the specified antibodies
for
PARP,
caspase-7,
and
caspase-9
termined by JC-1 was moderately induced by DOX or
DHA alone, whereas combined DOX and DHA resulted in a greater additive effect than the individual
agents (Fig. 3C). We investigated further the effect of
DHA, DOX, and their combination on PARP and caspases through western blot analysis. In MCF-7 cells,
treatment with combined DOX (10 µM) and DHA
(20 µM) for 24 h caused a much more significant
cleavage of PARP and activation of caspase-7 than in
mono-treated cells. This finding indicated the involvement of caspase-mediated apoptosis triggered
by DHA + DOX, which favored the sensitizing effects
of DHA on DOX-induced apoptosis (Fig. 3D). Besides, apoptosis induced by DHA + DOX was accompanied by the loss of MMP. This result prompted us to
examine the involvement of caspase-9 in this process.
In MCF-7 cells, DOX combined with DHA activated
caspase-9 to a much higher extent than the monotreatments (Fig. 3D). Collectively, these data showed
that DHA sensitized DOX in activating caspase cascades and triggering apoptosis.
DHA pretreatment did not influence the intracellular concentration of DOX
To determine whether DHA enhanced DOX activity by
increasing the intracellular concentration of DOX,
DOX was examined in the presence or absence of
DHA. Given that DOX exhibited self-fluorescence, the
intensity of intracellular fluorescence was adopted to
reflect the intracellular concentration through FACS
assay. MCF-7 cells were pretreated with DHA for 6 h
and then with DOX for 1 h. We found that the fluoresPharmacological Reports, 2013, 65, 453–459
457
Fig. 4.
Intracellular accumulation of
DOX in MCF-7 cells. (A) Cells were
pretreated with 20 µM DHA for 6 h and
incubated with 10 µM DOX for another
1 h. Intracellular fluorescence was then
measured by flow cytometry analysis,
and the fluorescence intensity indicated the intracellular concentration of
DOX. Typical images of the flow cytometry assay are shown. (B) Semiquantitative analyses of the results in
cence intensity in the DOX group was significantly
higher than that in the blank and DHA-treated groups.
However, the fluorescence intensity in the combinedtreatment group was identical to that in the DOX
group, indicating that DHA did not influence the intracellular concentration of DOX (Figs. 4A and B).
Discussion
The CI is a widely accepted qualitative measure of the
extent of drug interaction. In this study, the CI was used
to evaluate the combination effects of DOX and DHA on
the proliferation of breast cancer MCF-7, MDA-MB-231
and T-47D cells. DHA potentiated DOX-imposed cytotoxicity as revealed by the CI values (Fig. 2E) and the obvious enhancement of proliferation inhibition in the
combined-treatment group (Figs. 2B–D). Compared with
MCF-7 and T-47D cells, MDA-MB-231 cells were much
more susceptible to DOX. However, after being combined with DHA, DOX exhibited similar potent antiproliferative capabilities especially at relatively high concentrations (Figs. 2B–D). More importantly, DHA did not
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Pharmacological Reports, 2013, 65, 453–459
A
enhance intracellular DOX accumulation in MCF-7
cells (Figs. 4A and B). Thus, the same anti-cancer effect may be preferred at low doses of combined DOX
and DHA or high doses of DOX alone. DHA is also
widely used as an anti-malarial drug with few side effects, and the DHA-resistant cancer cell line does not
present a multidrug-resistant phenotype [9]. Thus,
DHA + DOX has tremendous latent capacity for clinical treatment in the future. Artemisinin, the mother
compound of ARTs, reportedly induces resistance to
DOX in human HT29 colon cancer cells and MCF-7
breast cancer cells partially by inducing Pglycoprotein (P-gp) expression [14]. Thus, artemisinin pretreatment reduces the intracellular DOX because of P-gp expression. The discrepancies may be
due to the different compounds used. Although DHA
is one of the main metabolites of ARTs, the pharmacological activities of artemisinin and DHA differ [6].
However, the detailed mechanisms require further investigation.
The PI staining, JC-1 staining, and western blot
analysis data indicated significant effects on apoptosis
in the combined DOX and DHA groups compared
with the mono-treated groups. This improvement partially explained the synergistic anti-proliferation ef-
Synergistic activity of the combination of DHA and DOX
Guo-Sheng Wu et al.
fect of the DHA + DOX treatment. Actually, the enhancement of apoptosis is an effective way to improve the anti-proliferation potential in cancer cells
[16]. However, how does DHA enhance DOXinduced apoptosis still warrants further study.
In summary, DHA + DOX significantly improved
the anti-cancer activity of each component, as revealed by the synergistic inhibitory effects on cancer
cell proliferation. The synergism was partially due to
the sensitized execution of apoptosis. Therefore, the
superior pharmacological activities of DOX + DHA
enabled their use as a promising anti-cancer strategy,
although further research is needed.
8.
9.
10.
11.
12.
Acknowledgments:
This work was supported by Research Fund of Zhejiang Chinese
Medicine University (No. 2009ZZ04), National Natural Science
Foundation of China (No. 81001450), Research Fund of University
of Macau (SRG026-ICMS13-LJJ, UL016/09-Y4/CMS/WYT01/ICMS)
and Science and Technology Development Fund of Macau Special
13.
Administrative Region (029/2007/A2). We greatly thank Prof. Ying Li
form Shanghai Institute of
Materia Medica for providing
DHA and
Dr. Hong Zhu from Zhejiang University for the statistical analyses.
14.
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