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Artemisia absinthium (AA): a novel potential complementary
and alternative medicine for breast cancer
Gowhar Shafi •Tarique N. Hasan •Naveed Ahmed Syed •
Amal A. Al-Hazzani •Ali A. Alshatwi •
A. Jyothi •Anjana Munshi
Received: 16 May 2011 / Accepted: 25 January 2012
ÓSpringer Science+Business Media B.V. 2012
Abstract Natural products have become increasingly
important in pharmaceutical discoveries, and traditional her-
balism has been a pioneering specialty in biomedical science.
The search for effective plant-derived anticancer agents has
continued to gain momentum in recent years. The present
study aimed to investigate the role of crude extracts of the
aerial parts of Artemisia absinthium (AA) extract in modu-
lating intracellular signaling mechanisms, in particular its
ability to inhibit cell proliferation and promote apoptosis in a
human breast carcinoma estrogenic-unresponsive cell line,
MDA-MB-231, and an estrogenic-responsive cell line, MCF-
7. Cells were incubated with various concentrations of AA,
and anti-proliferative activity was assessed by MTT assays,
fluorescence microscopy after propidium iodide staining,
western blotting and cell cycle analysis. Cell survival assays
indicated that AA was cytotoxic to both MDA-MB-231 and
MCF-7 cells. The morphological features typical of nucleic
staining and the accumulation of sub-G1 peak revealed that
the extract triggered apoptosis. Treatment with 25 lg/mL AA
resulted in activation of caspase-7 and upregulation of Bad in
MCF-7 cells, while exposure to 20 lg/mL AA induced
upregulation of Bcl-2 protein in a time-dependent response in
MDA-MB-231 cells. Both MEK1/2 and ERK1/2 was inacti-
vated in both cell lines after AA treatment in a time-dependent
manner. These results suggest that AA-induced anti-prolif-
erative effects on human breast cancer cells could possibly
trigger apoptosis in both cell lines through the modulation of
Bcl-2 familyproteinsand the MEK/ERK pathway.This might
lead to its possible development as a therapeutic agent for
breast cancer following further investigations.
Keywords Artemisia absinthium Caspase-7 Bad
Bcl-2 MEK/ERK MDA-MB-231 MCF-7
Apoptosis Cancer therapy
Introduction
Artemisia absinthium (AA) is commonly called worm-
wood, and is locally known as ‘Tethwen’ in the Kashmir
Valley, India. It is used in indigenous medicine as a ver-
mifuge, an insecticide, an antispasmodic, an antiseptic, and
in the treatment of chronic fevers and inflammation of the
liver [1]. Its essential oil has antimicrobial [2] and anti-
fungal activity [3]. Chemical analysis of AA extracts has
shown that its volatile oil is rich in thujone, which has been
reported as an anthelmintic [4]. In Turkish folk medicine,
AA has been used as an antipyretic, antiseptic, anthel-
mintic, tonic, diuretic, and for the treatment of stomach-
aches [5].
Breast cancer is the most common oncological disease in
women worldwide, and its incidence and mortality rates
G. Shafi A. Jyothi A. Munshi (&)
Department of Molecular Biology, Institute of Genetics and
Hospital for Genetic Diseases, Osmania University, Begumpet,
Hyderabad 500016, Andhra Pradesh, India
e-mail: anjanadurani@yahoo.co.in
G. Shafi A. Munshi
Dr. NTR University of Health Sciences, Vijayawada,
Andhra Pradesh, India
G. Shafi T. N. Hasan N. A. Syed A. A. Alshatwi
A. Munshi
Molecular Cancer Biology Research Lab (MCBRL),
Department of Food Sciences and Nutrition, College of Food
and Agricultural Sciences, King Saud University, Riyadh,
Saudi Arabia
A. A. Al-Hazzani
Department of Botany and Microbiology,
King Saud University, Riyadh, Saudi Arabia
123
Mol Biol Rep
DOI 10.1007/s11033-012-1569-0
may be explained by differences in the relative risk or
prevalence of risk factors, including dietary factors [6].
However, the efficacy of currently available drugs is very
limited, and anticancer agents that can target multiple points
in the apoptotic cascade to achieve synergistic actions are
urgently required. Chinese herbs have obtained considerable
attention for the prevention and treatment of certain cancer
types in clinical studies [7–10]. In many cases however,
extracts obtained from plants are not highly effective and
require chemical modification for improved potency and
toxicity profile [11–13]. Several phytochemicals that have
been used in clinical cancer chemotherapy were originally
derived from herbs and plants, such as paclitaxel [9,14],
etoposide [15], camptothecin [8] and vinca alkaloids [16].
Thus, studies of naturally-occurring plant-based agents
could lead to the development of new strategies for the
management of cancer and related diseases. Specific com-
pounds in certain foods have been shown to reduce human
breast cancer cell proliferation through apoptosis and cell
cycle arrest [17]. Interestingly, the apoptosis pathway was a
novel target for cancer chemoprevention in recent studies of
several chemopreventive agents [18]. The current study was
designed to investigate the in vitro anticancer activity of
crude extracts of the aerial parts of AA on estrogen-
responsive MCF-7 and estrogen-unresponsive MDA-MB-
231 human breast cancer cell lines.
Materials and methods
Preparation of plant material
Plants were selected on the basis of ethnopharmacology,
and 3 kg of plant material was collected locally. It was
identified by a botanist from the Department of Botany,
Osmania University, and a specimen was deposited at the
herbarium. The aerial parts of AA were air-dried in shade,
then powdered using a milling machine. The powdered
plant material was extracted with methanol as described
previously [19]. Briefly, powdered plant material was
soaked in ten times the volume of methanol for extraction.
Extraction was performed three times, and each extraction
was performed for 24 h. Methanolic filtrate was then
evaporated under reduced pressure to obtain a residue
(500 g of AA yielded 29.15 g of residue). The residue was
dried using a rotary evaporator to obtain the powder/paste,
and the required quantity was dissolved in dimethyl sulf-
oxide (DMSO).
Preparation of drug
A stock of plant extract was prepared to a concentration of
1 mg/mL in DMSO and sterilized by autoclaving at 121°C
and 15 lb for 15 min. Then, five concentrations of test
drug (5, 20, 50, and 100 lg/mL) were prepared by dilut-
ing stock with DMEM. DMEM alone was used as a
vehicle control.
Maintenance of cell lines
MDA-MB-231 and MCF-7 cells were procured from the
National Centre for Cell Science (Pune, India). The cell
lines were maintained and propagated in 90% Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and 1% penicillin/strepto-
mycin. Cells were cultured as adherent monolayers to
approximately 70–80% confluence, and maintained at 37°C
in a humidified atmosphere of 5% CO
2
. Cells were har-
vested after brief trypsinization. All chemicals used were of
research grade.
Cell toxicity and viability assays
MDA-MB-231 and MCF-7 cells were grown in DMEM at
37°C under 5% CO2 in a humidified incubator. Cells were
harvested, counted and transferred to 96-well plates, and
incubated for 24 h prior to addition of the test compounds.
The extracted compounds were processed and applied in
various concentrations, and the treated cells were incubated
for 72 h. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide) (5 mg) was dissolved in 1 mL
phosphate-buffered saline (PBS), and 25 lL MTT solution
was added to each of the wells. The plates were wrapped in
aluminum foil and incubated at 37°C for 4 h. The solution
in each well, containing media, unbound MTT and dead
cells, was removed by suction, and replaced with 200 lL
DMSO. The plates were then shaken, and the optical
density was measured using a microplate reader at 575 nm.
Three independent experiments were performed for each
study and all measurements were performed in triplicate.
Results were expressed as the percentage proliferation with
respect to vehicle-treated cells. Cells were further treated
with IC
50
concentrations of AA (25 lg/mL for MCF-7
cells and 20 lg/mL for MDA-MB-231 cells) or vehicle
(0.1%) for 24, 48 or 72 h.
Apoptotic assays
The apoptotic effects of AA on MDA-MB-231 and MCF-7
cells were analyzed by nuclear DNA staining assays. AA-
treated and untreated cells were fixed in 4% paraformal-
dehyde in PBS for 20 min, washed twice with PBS, and
stained with 1 lg/mL propidium iodide (PI) (Sigma) for
15 min. Stained cells were then washed twice with PBS.
The changes in nuclei were observed under an ultraviolet
fluorescent microscope (Carl Zeiss).
Mol Biol Rep
123
Flow cytometry analysis
MDA-MB-231 and MCF-7 cells in the exponential phase
of growth were treated with AA extract (20 lg/mL for
MDA-MB-231 cells and 25 lg/mL for MCF-7 cells) for 24
and 48 h, then harvested by trypsinization, washed twice
with ice-cold PBS and fixed by 70% ethanol at -20°C for
at least 30 min. The fixed cells were then washed twice
with ice-cold PBS and stained with 50 lg/mL PI for
30 min. Cell cycle distribution was analyzed by using a
FACSCalibur (Becton–Dickinson, USA). Data from
10,000 cells per sample were collected and analyzed using
the Cell Fit Cell analysis program.
Western blot analysis
MDA-MB-231 and MCF-7 cells were grown in 6-well
plates, and when cell density reached 80–90% confluence,
cells were treated with AA (20 lg/mL for MDA-MB-231
cells and 25 lg/mL for MCF-7 cells) for 24, 48 or 72 h.
After treatment, cells were collected and washed twice
with cold PBS. The cells were then lysed in lysis buffer
(50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet
P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM
NaF, 1 mM DTT, 1 mM PMSF, 25 lg/mL aprotinin, and
25 lg/mL leupeptin) and kept on ice for 30 min. The lysates
were then centrifuged at 12,0009gat 4°Cfor20minand
the supernatants were stored at -70°C until use. Protein
concentration was determined by the Bradford method.
Aliquots of the lysates (30 lgofprotein)wereseparatedin
12% SDS-PAGE and transferred onto a nitrocellulose
membrane using transfer buffer (192 mM glycine, 25 mM
Tris–HCl, pH 8.8, and 20% methanol [v/v]). After blocking
with 5% non-fat dried milk, the membranes were subse-
quently incubated with the corresponding primary antibod-
ies, as indicated: a rabbit anti-PARP p85 fragment, anti-
active ERK1/2, anti-active MEK1/2 and anti-MEK1/2
polyclonal antibody, a mouse anti-caspase-7, anti-Bcl-2 and
anti-Bad monoclonal antibody, rabbit anti-ERK 1/2 poly-
clonal antibody (Abcam Inc, USA); Antibody recognition
was detected with the respective secondary antibody, anti-
mouse IgG antibodies linked to horseradish peroxidase
(Abcam Inc, USA). Primary antibodies were used at a
1:1,000 dilution, while horseradish peroxidase-conjugated
horse anti-rabbit IgG (Sigma Chemicals, USA) was used at a
1:5,000 dilution as the secondary antibody. The membrane
was then exposed and protein bands were detected using
Enhanced Chemiluminescence (Abcam Inc, USA).
Statistical analysis
The data were expressed as the mean (±SD). Comparisons
between groups were performed by Student’s ttest and
one-way analysis of variance (ANOVA). All statistical
analyses were performed using the statistical software
PASW 18.0. Pvalues of \0.05 were regarded as statisti-
cally significant.
Results
Inhibition of cell proliferation in AA-treated MCF-7
and MDA-MB-231 cells
To investigate the effects of AA methanolic extract on
MDA-MB-231 and MCF-7 cell proliferation, the cells were
treated with various concentrations of AA for 72 h. AA at
25 lg/mL caused almost 50% inhibition of cell prolifera-
tion in MCF-7 cells compared with controls, while 20 lg/
mL caused 50% inhibition in MDA-MB-231 cells with
(Figs. 1,2a, b). The anti-proliferative activity was time-
dependent in both cell types. Therefore, we used respective
concentrations to perform the downstream experiments.
Microscopic signs of apoptosis in AA-treated cells
The ability of AA to alter cell morphology indicative of
apoptosis in the cancer cell lines was assessed using PI
staining (Fig. 3). After incubation for 48 h with AA, the
cells became rounded in appearance, exhibited nuclear
condensation and significant nuclei fragmentation indicat-
ing apoptosis.
Flow cytometry analysis
Cell cycle analysis by flow cytometry was used to confirm
the AA-induced cell death in MDA-MD-231 and MCF-7
Fig. 1 Cell viability was determined by MTT assays. Cells were
treated with various concentrations of AA, and results are expressed
as percentages of proliferation compared with untreated control
(mean ±SD, n=3)
Mol Biol Rep
123
cells. Indeed, both cell lines accumulated in the sub-G1
phase gradually from 24 to 72 h after treatment with AA,
whereas the number of cells in the G2/M phase decreased
in a similar fashion. These results indicate that apoptosis
was induced in MDA-MD-231 (Fig. 4a) and MCF-7 cells
(Fig. 4b).
Effects of AA on PARP cleavage and caspase-7
dependency
MDA-MB-231 and MCF-7 cells were treated with 25 and
20 lg/ml AA, respectively, and were harvested after 24, 48
Fig. 2 Typical effects of AA in reducing the viability of MCF-7
(a) and MDA-MB-231 cells (b). Cells were treated with 25 lg/mL of
AA for 24, 48 and 72 h (mean ±SD, n=3)
Fig. 3 Detection of apoptotic morphological changes in MDA-MB-
231 and MCF-7 cells treated with AA for 72 h. Nuclei were stained
with PI and examined by fluorescence microscopy. Untreated MCF-7
cells (left) and MCF-7 cells treated with 25 lg/mL AA (right);
untreated MDA-MB-231 cells (left) and MDA-MB-231 cells treated
with 20 lg/mL AA (right)
Fig. 4 MDA-MB-231 (a) and MCF-7 (b) cells were treated with 20
and 25 lg/mL AA, respectively for 24, 48 and 72 h. Cell cycle
distribution was determined in samples stained with propidium iodide
and measured by flow cytometry; percentage of cells in sub-G1, G1/S,
and G2/M phase was then calculated. Data shown are from a
representative experiment repeated three times
Mol Biol Rep
123
and 72 h of exposure. Caspase-7 cleavage was detected
after 24 h of exposure to AA, and PARP was cleaved to its
active 85 kDa form in both cell lines (Fig. 5a, b). These
results showed that AA induced apoptosis through the
cleavage of PARP, a substrate of several interleukin-1b-
converting enzyme-like proteases.
Modulation of regulatory apoptotic proteins Bad
and Bcl-2
Cell lines were treated with their respective IC
50
concen-
tration of AA, harvested after 24, 48 and 72 h of exposure,
and analyzed as described above. Pro-apoptotic Bad was
upregulated in MCF-7 cells treated for 24 h (Fig. 5c) but
anti-apoptotic Bcl-2 protein was decreased in MDA-MB-
231 cells after treatment (Fig. 5d). No changes were
observed Bcl-2 protein levels in MCF-7 cells or Bad pro-
tein levels in MBA-MB-231 cells after treatment (data not
included). The results suggest that AA-induced apoptosis
in human breast cancer cells might be mediated through the
modulation of the Bcl-2/Bad pathway.
Effects of AA on the MEK/ERK pathway
The involvement of mitogen-activated protein kinases
(MAPKs) in AA-induced apoptosis in MCF-7 and MDA-
MB-231 cells was analyzed by assessing the time-depen-
dent effect on the ERK1/2 MAPK pathway. As shown in
Fig. 6a, AA-induced inactivation of ERK1/2 began at 48 h
and lasted up to 72 h in MCF-7 cells. In contrast, phospho-
ERK1/2 was slowly downregulated by AA over 72 h in
MDA-MB-231 cells (Fig. 6b). The same blots were sub-
sequently stripped and re-blotted with an antibody that
recognized total ERK to verify equal amounts of the pro-
tein in various samples. MEK1/2 phosphorylation was
decreased in a time-dependent fashion following AA
treatment over the same timeframe, as seen for phosphor-
ylated ERK1/2 (Fig. 6c, d). Given that ERK1/2 activation
is considered to be a determining factor in cell survival and
apoptosis, these results suggest that AA-induced apoptosis
in both cell lines may be associated with activation of the
MEK/ERK pathway.
Discussion
The drug discovery process is becoming more complex and
capital-intensive, and systematic and critical review of the
methods and approach towards the entire process is
required to rediscover the discovery process afresh.
Screening of medicinal plants for potential anticancer
properties has increased greatly over the past few decades.
For instance, the US National Cancer Institute has imple-
mented a large-scale project of acquisition and screening of
compounds isolated from medicinal plants originating from
various regions of the world. These medicinal plants are
identified based on ethnopharmacological, chemosystemic
and ecological information. In the present study, irrevers-
ible inhibition of proliferation was observed in AA-treated
Fig. 5 Effects of AA on cleaved PARP and caspase-7 proteins.
MCF-7 cells were treated with 25 lg/mL AA (a) and MDA-MB-231
cells were treated with 20 lg/mL AA (b) for indicated times. Total
cell lysates were prepared and western blotting was performed with
antibodies specific for corresponding proteins. Effects of AA on Bad
protein; MCF-7 cells were treated with 25 lg/mL AA for indicated
times (c). Effects of AA on Bcl-2 protein; MDA-MB-231 cells were
treated with 20 lg/mL AA for indicated times (d)
Fig. 6 Effects of AA on the levels of ERK1/2, pERK1/2, MEK1/2
and pMEK1/2 in MCF-7 (a,c) and MDA-MD-231 (b,d) cells. Cells
were cultured as described in the ‘‘Materials and methods’’ section.
MCF-7 and MDA-MB-231 cells were treated with 25 and 20 lg/mL
AA, respectively, for 24, 48 and 72 h. Cells were harvested and total
cell lysates were prepared. Western blotting was performed with
antibodies specific for corresponding proteins. Experiments were
repeated three times with similar results
Mol Biol Rep
123
breast cancer cells in a concentration and time-dependent
fashion. Thus, it is clear that AA inhibits cell proliferation
via apoptotic death, as visualized by nuclear fragmentation
and condensation, as well as increased sub-G1 phase.
Apoptosis is an active physiological process resulting in
cellular self-destruction that involves specific morphological
and biochemical changes in the nucleus and cytoplasm [20].
Agents that suppress the proliferation of malignant cells by
inducing apoptosis may represent a useful mechanistic
approach to both cancer chemoprevention and chemotherapy.
While many anticancer agents have been developed, unfa-
vorable side-effects and resistance are serious problems [21].
Thus, there is growing interest in the use of plant materials for
the treatment of various cancers, and for the development of
safer and more effective therapeutic agents [22]. AA has been
used as a folk remedy for several diseases without any
understanding of the underlying mechanisms. Therefore, we
chose to investigate the role of AA in the inhibition of pro-
liferation and promotion of apoptosis in human breast cancer
MDA-MB-231 and MCF-7 cells. AA treatment induced the
activation of caspase-7 in these cell lines. An inactive caspase-
7 precursor was cleaved to form the active protease during
apoptosis, resulting in PARP degradation.
In general, apoptosis is controlled by the complex inter-
play between regulatory proteins of the Bcl-2 family [23].
These pro- and anti-apoptotic proteins are key regulators of
the intrinsic apoptotic pathway, controlling the point of no
return and setting the threshold for engagement of the death
machinery [24]. Previous reports have shown that the ratio
of Bax to Bcl-2 determines, in part, the susceptibility of cells
to death signals [25]. Therefore, Bcl-2 proteins have
emerged as attractive targets for the development of novel
anticancer drugs [26]. Changes in the Bcl-2/Bax ratio have
been reported to be caused by downregulation of Bcl-2 and
slight downregulation of Bax [27], downregulation of Bcl-2
and upregulation of Bax [28,29], and downregulation of
Bcl-2 with no change in the level of Bax [30]. However,
other members of the Bcl-2 family, Bax, Bad, Bak, Bik and
Bid, promote cell death [31]. Anti-apoptotic Bcl-2 expres-
sion was significantly downregulated after AA exposure for
48 h in MDA-MB-231 cells. However, our results clearly
showed upregulation of Bad after treatment of MCF-7 for
24 h. Thus it is reasonable to conclude that AA treatment
may induce apoptosis by regulating the Bcl-2 family.
In addition, MAPK cascades transmit and amplify sig-
nals involved in cell proliferation and apoptosis. Three
major MAPK pathways exist in human tissues, but the
ERK1/2 cascade is most relevant to breast cancer because
if the appropriate receptors are present, upregulation of
ligands may be responsible for increased MAPKs in these
cancers [32]. Exposure of MDA-MB-231 and MCF-7 cells
to AA resulted in highly significant inhibition of MEK and
ERK1/2. The mechanism of action of many anticancer
drugs is based on their ability to induce apoptosis [33,34].
Hence, we were interested in identifying if cancer cells
treated with methanolic extracts of AA utilized apoptosis
as their mode of cell death. This was approached by
studying distinct morphological features (nuclear chroma-
tin condensation, fragmentation of nuclear material) and
molecular features (expression of certain crucial genes).
The MAPK pathway represents a cascade of phosphor-
ylation events including three pivotal kinases, namely Raf,
MEK and ERK; activated MEK1/2 can activate ERK1/2.
This concurs with the observation of Agarwal et al. [35]
who showed that a polyphenolic fraction isolated from
grape seeds induced growth inhibition of breast MDA-MB-
468 carcinoma cells by inhibiting MAPK activation. Taken
together, we therefore suggest that AA plays critical roles
in apoptotic and MAPK pathways. The benefit of herbal
decoctions is that they can nourish the body as a whole by
supporting various organ systems. More importantly as
they have no synthetic elements, there is very less likeli-
hood of an unexpected adverse effect.
In conclusion, AA inhibits proliferation of human breast
cancer MDA-MB-231 and MCF-7 cells through the
induction of apoptosis by regulating Bcl-2 family proteins
and MEK/MAPK signaling. We also found in this study
that p53-independent cell death induced by AA is regulated
Fig. 7 Schematic summary of our findings. On the basis of the results
of the current study, we propose that AA treatment triggers activation
of the MEK/ERK pathway, which then initiates the mitochondrial
pathway of caspase activation, and regulates Bad and Bcl-2 family
proteins, culminating in the apoptotic death of MCF-7 and MDA-MB-
231 cells
Mol Biol Rep
123
through an MEK–ERK–mitochondria–caspase cascade that
may be critical to improvement of the clinical outcome
(Fig. 7). This suggests AA extract has an anticancer effect
that is mediated via the apoptotic pathway in human breast
cancer cell lines. However, the potent extracts need to be
tested on several other cancer cell lines, and animal models
should be used to establish the therapeutic efficacy and
toxicity of this substance. In addition, extracts must be
subjected to HPLC and GC–MS analyses to identify and
characterize the efficacious phytotherapeutic and/or bio-
active compound(s) in AA.
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