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Original Research Article
Medicinal properties of Angelica archangelica root extract: Cytotoxicity
in breast cancer cells and its protective effects against in vivo tumor
development
Carlos R. Oliveira
a,b,
⇑
,1
, Daniel G. Spindola
a,b
, Daniel M. Garcia
a
, Adolfo Erustes
a
, Alexandre Bechara
a
,
Caroline Palmeira-dos-Santos
a
, Soraya S. Smaili
a
, Gustavo J.S. Pereira
a
, André Hinsberger
b
,
Ezequiel P. Viriato
c
, Maria Cristina Marcucci
d
, Alexandra C.H.F. Sawaya
e
, Samantha L. Tomaz
f
,
Elaine G. Rodrigues
f
, Claudia Bincoletto
a,
⇑
,1
a
Departamento de Farmacologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP 04044-020, Brazil
b
Grupo de Fitocomplexos e Sinalização Celular, Escola de Ciências da Saúde, Universidade Anhembi Morumbi, São Paulo, SP 03164-000, Brazil
c
Faculdade de Ciências Farmacêuticas e Bioquímicas Oswaldo Cruz, São Paulo, SP 01151-000, Brazil
d
Laboratório de Produtos Naturais e Quimiometria, Programa de pós-graduação em Farmácia e Biotecnologia, Universidade Anhanguera de São Paulo, São Paulo, SP 05145-200, Brazil
e
Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-871, Brazil
f
Unidade de Oncologia Experimental, EPM, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP 04023-901, Brazil
article info
Article history:
Received 16 May 2018
Accepted 2 November 2018
Available online 08 February 2019
Keywords:
Angelica archangelica
Umbelliferae
Apoptosis
Cell death
Breast cancer
Angelicin
abstract
Objective: Although Angelica archangelica is a medicinal and aromatic plant with a long history of use for
both medicinal and food purposes, there are no studies regarding the antineoplastic activity of its root.
This study aimed to evaluate the cytotoxicity and antitumor effects of the crude extract of A. archangelica
root (CEAA) on breast cancer.
Methods: The cytotoxicity of CEAA against breast adenocarcinoma cells (4T1 and MCF-7) was evaluated
by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Morphological
and biochemical changes were detected by Hoechst 33342/propidium iodide (PI) and annexin V/PI stain-
ing. Cytosolic calcium mobilization was evaluated in cells staining with FURA-4NW. Immunoblotting was
used to determine the effect of CEAA on anti- and pro-apoptotic proteins (Bcl-2 and Bax, respectively).
The 4T1 cell-challenged mice were used for in vivo assay.
Results: Using ultra-high-performance liquid chromatography–mass spectrometry analysis, angelicin, a
constituent of the roots and leaves of A. archangelica, was found to be the major constituent of the
CEAA evaluated in this study (73 mg/mL). The CEAA was cytotoxic for both breast cancer cell lines studied
but not for human fibroblasts. Treatment of 4T1 cells with the CEAA increased Bax protein levels accom-
panied by decreased Bcl-2 expression, in the presence of cleaved caspase-3 and cytosolic calcium mobi-
lization, suggesting mitochondrial involvement in breast cancer cell death induced by the CEAA in this
cell line. No changes on the Bcl-2/Bax ratio were observed in CEAA-treated MCF7 cells. Gavage adminis-
tration of the CEAA (500 mg/kg) to 4T1 cell-challenged mice significantly decreased tumor growth when
compared with untreated animals.
Conclusion: Altogether, our data show the antitumor potential of the CEAA against breast cancer cells
in vitro and in vivo. Further research is necessary to better elucidate the pharmacological application of
the CEAA in breast cancer therapy.
Please cite this article as: Oliveira CR, Spindola DG, Garcia DM, Erustes A, Bechara A, Palmeira-dos-
Santos C, Smaili SS, Pereira GJS, Hinsberger A, Viriato EP, Cristina Marcucci M, Sawaya ACHF, Tomaz
https://doi.org/10.1016/j.joim.2019.02.001
2095-4964/Ó2019 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.
⇑
Corresponding authors at: Departamento de Farmacologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, SP 04044-020,
Brazil (C.R. Oliveira).
E-mail addresses: carlos.oliveira@anhembimorumbi.edu.br (C.R. Oliveira), claudia.bincoletto@unifesp.br (C. Bincoletto).
1
Claudia Bincoletto and Carlos R. Oliveira contributed equally to the study.
Journal of Integrative Medicine 17 (2019) 132–140
Contents lists available at ScienceDirect
Journal of Integrative Medicine
journal homepage: www.jcimjournal.com/jim
www.journals.elsevier.com/journal-of-integrative-medicine
SL, Rodrigues EG, Bincoletto C. Medicinal properties of Angelica archangelica root extract: Cytotoxicity
in breast cancer cells and its protective effects against in vivo tumor development. J Integr Med. 2019;
17(2): 132–140.
Ó2019 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.
1. Introduction
Angelica archangelica (Linn.) is a member of the Apiaceae family
(Umbelliferae) [1]. Several studies have demonstrated that all parts
of the plant can be used for pharmacological and food purposes,
although the main medicinal part is the root [1–5]. The major con-
stituents of A. archangelica are coumarins, such as angelicin, osthol,
bergapten, imperatorin, oreoselone, oxypeucedanin, umbellifer-
one, xanthotoxin, and xanthotoxol. Other constituents in the plant,
including archangelenone (flavonoid), palmitic acid, and sugars
(fructose, glucose, sucrose and umbelliferose), have also been
reported [1,6].
Sigurdsson et al. [7] demonstrated that A. archangelica tincture
fruits, as well as its components (imperatorin and xanthotoxin),
inhibited the proliferative potential of human pancreas cancer
cells PANC-1. The essential oils of A. archangelica fruits were also
cytotoxic to the PANC-1 cells [8]. The leaf extract from
A. archangelica also inhibited the proliferation of mouse mammary
carcinoma cells in vitro and protected mice from tumor develop-
ment in vivo [9].
Several studies have shown that the active components of
A. archangelica are of pharmacological interest. Imperatonin
induced apoptosis in leukemia HL60 cells [10], and inhibited the
proliferation of lung carcinoma (A549), melanin pigment-
producing mouse melanoma (B16 melanoma 4A5), human T-cell
leukemia (CCRF-HSB-2), and human gastric carcinoma derived
from metastatic lymph node (TGBC11TKB) [11]. In combination
with quercetin, imperatorin was antiproliferative for HeLa and
Hep-2 cells, inducing apoptosis accompanied by decreased Hsp27
and Hsp72 protein levels with cleaved caspases and no effects on
the autophagy process [12]. Imperatorin also inhibited the
biological activation of the carcinogens benzo[a]pyrene and
7,12-dimethylbenz[a]anthracene in MCF7 cells [13]. Angelicin, a
furocoumarin also found in A. archangelica, enhanced human
tumor necrosis factor-related apoptosis in renal carcinoma Caki
cells but not in normal cells [14]. The antitumor effects of angelicin
were also observed in mice bearing liver tumor xenografts, without
producing significant secondary adverse effects [15].
In this study, our goal was to elucidate the anticancer effects
of crude extract of A. archangelica root (CEAA). For this purpose,
using two breast cancer cell lines, human MCF7 and mouse 4T1
breast tumor cells, as a model for in vitro and in vivo studies,
respectively, we demonstrated that the CEAA administered by
gavage decreased tumor growth in a temporal dependent manner.
In vitro analyses have demonstrated cytotoxic activity and cell
death-inducing properties of the CEAA on breast cancer cells.
These in vitro effects are probably involved in the antitumor
activity of A. archangelica.
2. Materials and methods
2.1. CEAA
The A. archangelica root and rhizome (20 g) crude extract was
manufactured by Almeida Prado Homeopathic Laboratories (batch
# 039tintura, on 16th June, 2014), São Paulo/SP, Brazil. This extract
was kindly provided by the mentioned laboratory for this study.
After authentication at the laboratory, the extract was prepared
according to the Brazilian Homoeopathic Pharmacopoeia and pro-
vided as a 65% ethanolic extract after a technical report attesting to
its origin. The CEAA was extracted using a hydroethanolic mixture
(55%) to a final volume of 100 mL [16].
2.2. Ultra-high-performance liquid chromatography–mass
spectrometry analysis
Aiming to quantify the angelicin in the CEAA, main marker of the
plant, chromatographic analysis of the crude extract was performed
using an ultra-high-performance liquid chromatography–mass
spectrometry (UHPLC–MS) with a C
18
BEH Waters column
(1.7 mm2.1 mm 50 mm), an oven temperature of 30 °C, and
7mL of each sample was injected at 20 °C (standard and sample).
The elution was performed using a gradient with a flow of
200 mL/min, mobile phase of water (Milli-Q) with 0.1% of formic acid
(A) and acetonitrile (Merck Darsmstadt, Germany, chromatographic
grade) (B), beginning with 10% B, ramping to 25% B in 4 min
and to 100% B by 8 min, held at 100% B until 8.5 min then returning
to the initial conditions and re-equilibrating until 10 min. The com-
ponents were detected using a triple-quadrupolar mass spectrome-
ter (TQD Acquity Waters) with electrospray ionization, in the
positive ion mode, under the following conditions: capillary of
3000 V, cone 35 V, extractor of 1.0 V, source temperature of 150 °C,
and desolvation temperature of 300 °C. An external calibration curve
of angelicin in the concentration range from 10 to 1000 mg/mL was
used to quantify this compound in the extract of A. archangelica.
2.3. Culture and cytotoxicity evaluation of CEAA by MTT assay
MCF7 and 4T1 cell lines were generously donated by Dr. Elaine
G. Rodrigues, Experimental Oncology Unit (UNONEX-Universidade
Federal de São Paulo). The CCD1072Sk new-born foreskin fibrob-
last (nontumor cells) was obtained from the Rio de Janeiro Cell
Bank (UFRJ, RJ, Brazil). The human breast cancer cells MCF7 and
the mouse mammary carcinoma cells 4T1 were cultured in a
monolayer in RPMI-1640 medium supplemented with 10% fetal
bovine serum, 100 UI/mL penicillin, and 100
l
g/mL streptomycin
in a humidified atmosphere at 37 °Cin5%CO
2
. CCD1072Sk cells
were cultured in Iscove’s modified Dulbecco’s medium with the
same supplementation and under the same conditions. The cells
were trypsinized every 72 h using 0.01% trypsin and 1 mmol/L
ethylene diamine tetraacetic acid (EDTA). For all the experiments,
the CEAA was dissolved in the culture medium in appropriate
concentrations.
The cell viability of control and CEAA (50–250
l
g/mL)-treated
MCF7, 4T1, and CCD1072Sk cells was measured using a standard
MTT assay. Briefly, 5 10
4
viable cells were seeded into clear
96-well flat-bottom plates (Corning) in RPMI medium supple-
mented with 10% fetal bovine serum and incubated with different
concentrations of the extract for 24 h. Then, 10
l
L/well of MTT
(5 mg/mL) was added and the cells were incubated for 4 h.
Following incubation, 100
l
L of 10% sodium dodecyl sulfate
(SDS) solution in deionized water was added to each well and left
overnight. The absorbance was measured at 595 nm in a benchtop
multimode reader (Molecular Device).
C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140 133
2.4. Cell death evaluation using propidium iodide in MCF7 and 4T1
cells treated with CEAA
For sub-G1 cell fraction determination (cell death), MCF7 and
4T1 cells treated for 24 h with the CEAA (250 mg/mL) were washed
in phosphate buffered saline (PBS), adjusted (10
6
cells/mL) and
fixed in 50% ethanol solution for 30 min. The fixed cells were resus-
pended in PBS containing propidium iodide (PI) (50 mg/mL) and
RNase (100 mg/mL) for 30 min and then analyzed by flow cytome-
try. The data were collected (10
4
events) in a FACSCalibur flow
cytometer (Becton-Dickinson) and acquired with Cell Quest soft-
ware (Becton-Dickinson). The acquired data were analyzed using
FlowJo V10.7 (FlowJo Enterprise).
2.5. Biochemical and morphological changes induced by CEAA in
cancer cells
MCF7 and 4T1 cells treated with the CEAA (250 mg/mL) for 24 h
were stained with fluorescein isothiocyanate (FITC)-conjugated
annexin V/PI according to the manufacturer’s instructions
(Annexin V/FITC Apoptosis Detection Kit, BD Pharmingen). The
population of annexin V
/PI
(viable cells), annexin V
+
/PI
(apoptotic cells), annexin V
/PI
+
(necrotic cells), and annexin
V
+
/PI
+
(secondary necrosis/late apoptosis) cells was evaluated
by flow cytometry. The data were collected (10
4
events) in a
FACSCalibur flow cytometer (Becton-Dickinson) and were acquired
with Cell Quest software (Becton-Dickinson). The acquired data
were analyzed using FlowJo V10.7 (FlowJo Enterprise).
Nuclear morphology of the treated and untreated MCF7 and
4T1 cells was evaluated by fluorescence microscopy with Hoechst
33342 DNA staining. The cells were fixed in ice-cold 50% ethanol
for 30 min. After fixation, the samples were incubated with
Hoechst 33342 for 15 min. The samples were visualized on a Zeiss
Axioskop fluorescence microscope (Zeiss) fitted with G365, FT395,
and LP420 bandpass filters.
The cleaved caspase-3 was evaluated in 4T1 cells treated with
the CEAA for 24 h using the flow cytometric analysis, according
to the manufacturer’s instructions (Cell Signaling). Briefly, CEAA-
treated 4T1 cells were stained with cleaved caspase-3 (Asp175)
and Alexa Fluor 488-conjugated antibody for 1 h in the dark. Then,
10
5
events were acquired in FACSCalibur (Becton-Dickinson). The
data were obtained through the Cell Quest software, and the
results were analyzed using FlowJo V10.7 (FlowJo enterprise).
2.6. Western blotting for evaluation of Bcl-2/Bax protein levels after
exposure of 4T1 cells to CEAA
The CEAA-treated 4T1 and MCF7 cells (250 mg/mL) were lysed in
NP-40 lysis buffer (50 mmol/L Tris-HCl (pH 7.4), 1% NP-40,
150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 30 mmol/L
Na
4
P
2
O
7
, 1 mmol/L Na
3
VO
4
) supplemented with protease and
phosphatase inhibitors (protease inhibitor cocktail plus 5 mmol/L
sodium fluoride, 0.5 mmol/L sodium orthovanadate, 1 mmol/L
sodium molybdate, 50 mmol/L 2-chloroacetamide, 2 mmol/L
1,10-phenanthroline monohydrate, and 0.5 mmol/L phenyl-
methanesulfonyl fluoride (Sigma). The lysates were incubated at
4°C for 30 min in ice. After centrifugation at 4 °C for 10 min at
13,000 r/min in a 5.5 cm rotor radius microcentrifuge, the protein
concentration was determined using a Bradford assay (Biorad).
From the total proteins, 50
l
g was resolved using SDS–polyacry-
lamide gel electrophoresis gels (Life Technologies) and electroblot-
ted onto polyvinylidene fluoride (Millipore) membranes. The blots
were incubated with primary antibodies (anti-Bcl-2 1:500 and
anti-Bax 1:500), suspended in 5% nonfat dry milk in PBS plus
0.1% Tween-20 overnight at 4 °C. The detection was achieved using
a horseradish peroxidase-conjugated secondary antibody (1:5000)
in 5% nonfat dry milk in PBS plus 0.1% Tween-20, Jackson Immuno
Research Laboratories, and visualized with electrochemical lumi-
nescence (ECL, GE Healthcare). The images were recorded using a
Chemidoc apparatus (Uvitec). The antitubulin was used as an inter-
nal control.
2.7. Cytosolic Ca
2+
measurements after exposure of 4T1 cells to CEAA
To evaluate Ca
2+
handling, the 4T1 cells were treated with the
CEAA (250 mg/mL) for 24 h in 96-well plaques in RPMI medium
supplemented with 10% fetal bovine serum. Then, the culture
medium was removed, and 50 mL of a buffer containing Fluo-4
NW (Molecular Probes, USA) was added to each well. The plaque
was incubated for 35 min, and cytosolic Ca
2+
levels were assessed
by a FlexStation-3 benchtop multimode reader (Molecular
Devices). All incubations were performed in fluorescence buffer
with the following composition (mmol/L): NaCl 138, KCl 5.7, CaCl
2
1.8, NaH
2
PO
4
0.36, NaHCO
3
15 and glucose 5.5 (pH 7.4). Data were
expressed as an increase in the amount of relative fluorescence
units. Using the SoftMax Pro software 5.3, we converted the
expressed data to a percentage and then performed the statistical
analysis.
2.8. Antitumor activity of CEAA expressed by decreased tumor size
For in vivo studies, 10 female Balb/c mice, 6–8 weeks old, were
purchased from the Center for Development of Experimental
Models, at Universidade Federal de São Paulo. All animal
experiments were approved by the Animal Experimentation Ethics
Committee at Universidade Federal de São Paulo, under protocol
number 341644 approved on July 10th, 2013. The mice were
inoculated with 10
4
4T1 cells subcutaneously and 24 h later, they
were separated into two groups of five mice each (untreated and
treated) to evaluate the anticancer effects of the CEAA. The treated
group received 500 mg/kg of the CEAA by gavage (0.1 mL, final
volume), while the untreated group was fed with saline, which
was the vehicle of delivery. After 33 days of treatment on alternate
days, the mammary tumors were removed, weighted and measured.
2.9. Statistical analysis
All data were expressed as mean ± standard error values, and
statistical analyses were done using Prism version 5.0 (GraphPad
Software, Inc., La Jolla, CA, USA). Data were analyzed using
Student’s ttest and a two-way analysis of variance (ANOVA),
followed by Bonferroni’s test for in vivo data. Differences with
P < 0.05 were considered significant. At least two independent
experiments were done to confirm that the results were
representative.
3. Results
3.1. Phytochemical analysis of the CEAA
In the present study, the chromatographic analysis of the CEAA
in comparison with an angelicin standard (calibration curve,
R= 0.9942) was first done; it indicated the presence of this com-
pound in the CEAA composition (73 mg/mL). In general, methanolic
and ethanol–water mixtures are used to evaluate the total content
of furanocoumarins by HPLC, including angelicin [17,18]. In our
study, the UHPLC–MS chromatogram in the positive ion mode of
the CEAA showed peaks with retention times around 6.0 min
(Rt = 6.08 min.) for angelicin. The CEAA chromatograms, as well
as the internal standard, are shown in Fig. 1 (A–C).
134 C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140
3.2. CEAA decreased the viability of MCF-7 and 4T1 cancer cells, and
increased the sub-G1 events with induced-phosphatidylserine
externalization in cells treated with CEAA for 24 h
Next, the effects of the CEAA on MCF7 and 4T1 cell viability
were evaluated using the MTT assay. As shown in Fig. 2A, the CEAA
was cytotoxic against MCF7 and 4T1 cells, in which 250
l
g/mL
inhibited 50% of cell viability in both cell lines studied. Impor-
tantly, the same extract did not exhibit cytotoxicity against the
normal CCD1072Sk fibroblasts as shown in Fig. 2B, suggesting a
selective effect of the plant on cancer cells. Flow cytometric
analysis using PI showed an increase in MCF7 and 4T1 cells in
the sub-G1 fraction (Fig. 2C and D, respectively) following the
CEAA treatment, suggesting cell death.
The subsequent aim of this study was to investigate whether
the CEAA was, in fact, able to induce apoptosis in MCF7 and
4T1 cells using annexin V/PI by flow cytometric analysis. Our
results showed a greater proportion of annexin V
+
/PI
(apopto-
sis) and annexin V
+
/PI
+
(late apoptosis)-stained MCF7 and
4T1 cells treated with the CEAA (250 mg/mL) (Fig. 3A and
Fig. 3B, respectively) in relation to untreated cells. The exposure
of these cells to the CEAA for 24 h also decreased the viability of
both the cell lines (annexin V
/PI
) and induced approximately
15% necrosis in 4T1 cells stained (annexin V
/PI
+
). In Fig. 3C,
considerable nuclear fragmentation, one of the characteristic
features of apoptotic cell death [19], was observed in the CEAA
(250 mg/mL)-treated breast cancer cells, as assessed by Hoechst
33342 staining and fluorescence microscopy [20], highlighting
the occurrence of apoptosis in both breast cancer cells treated
with the CEAA. Based on this preliminary result showing
that both 4T1 and MCF7 cell lines were responsive to the
CEAA in a similar way, 4T1 cells were chosen to evaluate the
molecular events involved in the CEAA cytotoxicity and for
in vivo assay.
3.3. CEAA exposure of 4T1, but not MCF7 cells decreased Bcl-2, and
increased Bax, caspase-3 and cytosolic calcium
To determine whether the CEAA-induced apoptosis occurred
through alterations in expression of the pro- or anti-apoptotic
proteins Bax and Bcl-2, respectively, Western Blot was carried
out. Although no changes on Bcl-2/Bax ratio were observed in
MCF7 cells treated with CEAA for 12 h (Fig. 4A), as expected, this
ratio was increased in CEAA-treated 4T1 cells (250 mg/mL)
(Fig. 4B). Caspase-3, which can be activated by a mitochondrial
apoptotic pathway contributing to cell death by apoptosis [21],
was also verified in 4T1 cells exposed to the CEAA for 24 h, as
evidenced by an increase in the relative fluorescence intensity in
the CEAA-treated group as shown in Fig. 4C.
Our results showed that the CEAA increased cytosolic calcium
concentration Ca
2+
, in 4T1 cancer cells (Fig. 4D), which strongly
suggests that calcium is involved in the CEAA-induced death of
breast cancer cells.
3.4. CEAA administration to 4T1-challenged mice decreased tumor
development
The anticancer effects of the CEAA were also assessed in 4T1
breast cancer cell-challenged mice, a classical model for in vivo
studies [22]. Administration of the CEAA (500 mg/kg) for 33 days
on alternate days significantly reduced the tumor growth in a
temporal dependent manner in the CEAA-treated mice
(Fig. 5A). Tumor weight was also significantly reduced by the
treatment as shown on Fig. 5B. From these results, we showed
that CEAA conferred a significant protection against tumor devel-
opment, as the tumor grew faster in the control group and
slower in the CEAA-treated animals. Importantly, no signs of tox-
icity, such as piloerection, irritability and ocular irritation were
observed in treated animals.
Time (min)
Percentages of peaks (%)
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
0
100
0
100
0
100 A
B
C
Angelicin
Angelicin
Angelicin
Fig. 1. UHPLC–MS extracted ion chromatogram of m/z 187. (A) Angelicin standard; (B) Angelica archangelica root extract in the BPI chromatogram; (C) A. archangelica root
extract analyzed in the TIC chromatogram. UHPLC–MS: ultra-high-performance liquid chromatography–mass spectrometry; BPI: base peak intensity; TIC: total ion current.
C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140 135
4. Discussion
Data from the literature have reported the beneficial effects of
medicinal plants on the quality of life of cancer patients when used
in combination with conventional anticancer therapies [23]. In line
with these data, further studies are necessary to establish the new
medicinal applications for a plant based on its folk uses.
Although several studies have experimentally evaluated the
anticancer activity of A. archangelica as well as its components
[7,12,24], no studies were found regarding the antineoplastic prop-
Concentrations (μg/mL)
Cell viability (%)
Cell viability (%)
Events
MCF7 sub-G1 (%)
Events
Concentrations (μg/mL)
Propidium iodide
Control CEAA
Control CEAA Propidium iodide
A
C
D
B
0
20
40
60
80
100
4T1 sub-G1 (%)
0
20
40
60
80
100
Sub-G1
11.5%
Sub-G1
23.5%
Sub-G1
8.5%
Sub-G1
58.5%
Fig. 2. CEAA decreases the viability of MCF-7 and 4T1 cancer cells and increases percentage of sub-G1 events. Effects of CEAA on the viabilities of MCF7 and 4T1 cancer cells
(A) and human CCD1072Sk fibroblasts (B). Cell death is also demonstrated by flow cytometry analyses, showing an increase in the sub-G1 cell populations for both MCF7 (C)
and 4T1 (D) cells following treatment with CEAA for 24 h.
*
P< 0.05,
**
P< 0.01,
***
P< 0.001, vs untreated control groups (CTL) (Student’s ttest). CEAA: crude extract of
A. archangelica root. CTL: control; EtOH: ethyl alcohol.
136 C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140
Annexin V/FITC
Control cells CEAA (250 μg/mL) treated cells
Annexin V/FITC
C
AB
Fig. 3. CEAA induced phosphatidylserine externalization in cells treated for 24 h. Annexin V-FITC/PI double staining shows phosphatidylserine externalization in breast
cancer cells MCF-7 (A) and 4T1 (B) treated with CEAA (250
l
g/mL) for 24 h. A significant increase in annexin V
+
/PI
and annexin V
+
/PI
+
cells was observed in both cell lines,
suggesting apoptosis and late apoptosis, respectively.
*
P< 0.05,
**
P< 0.01,
***
P< 0.001, vs untreated control groups (Student’s ttest). Exposure of MCF7 and 4T1 cells to CEAA
for 24 h induced chromatin condensation in both breast cancer cell lines, evaluated using Hoechst 33342 staining (C). CEAA: crude extract of A. archangelica root; FITC:
fluorescein isothiocyanate; PI: propidium iodide.
C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140 137
erty of its root, the main part of the plant involved in medicinal
uses. In this study, we set out to evaluate the biological potential
of A. archangelica in in vitro and in vivo experiments. Using MTT
reduction test, it was verified that CEAA is cytotoxic for both breast
cancer cell lines, but not to normal human fibroblasts. CEAA
increased the percentage of MCF-7 and 4T1 cells in the sub-G1
fraction (23.5% and 58.5%, respectively), suggesting for CEAA death
induction properties by apoptosis. This hypothesis was confirmed
by flow cytometry and double staining with annexin V/PI, which
determine whether cells are in the process of apoptosis. The CEAA
treatment increased the number of annexin V
+
/PI
cells when com-
pared with the untreated control cells. Nuclear fragmentation, one
of the characteristic features of apoptotic cell death [19], was
observed in both breast cancer cells staining with Hoechst
33342, a fluorescent DNA-binding agent [20].
In order to determine whether CEAA-induced apoptosis
occurred through alterations in expression of the pro- or anti-
apoptotic proteins Bax and Bcl-2, respectively, Western blot analy-
ses were carried out. As expected, reduced expression of Bcl-2 and
increased expression of Bax protein levels were observed in
4T1 cells treated with CEAA for 12 h. Caspase-3 can be activated
by a mitochondrial apoptotic pathway involving caspase-9, termed
Fig. 4. CEAA induced a significant increase in Bax accompanied by a decrease in Bcl-2 protein levels after 12 h incubation, in 4T1, but not in MCF7 cells. (A and B) MCF7 and
4T1, respectively, treated by CEAA for 12 h; (C) cleaved caspase-3 was also detected in 4T1 cells treated with CEAA for 24 h; (D) a pronounced increase in cytosolic calcium
mobilization was also observed in CEAA-treated 4T1 cells.
*
P< 0.05,
***
P< 0.001, vs untreated buffer control (Student’s ttest). CEAA: crude extract of A. archangelica root.
138 C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140
the intrinsic pathway, or by a death receptor pathway involving
caspase-8, the extrinsic pathway. Both pathways may converge,
contributing to cell apoptosis [25]. The results obtained revealed
that treatment of 4T1 cells with CEAA resulted in cleavage of
caspase-3. Furthermore, exposure of this cell line to CEAA
(250 mg/mL) for 12 h increased cytosolic calcium, suggesting an
involvement of this element on CEAA cytotoxicity.
Mitochondria participate in apoptosis through a range of
mechanisms [21,25]. It is well known that the release of
cytochrome cpromotes caspase activation, a process that can be
regulated by proteins from the Bcl-2 family, which are subdivided
into groups of pro-Bax, Bak, and Bok/Mtd or anti-apoptotic
members, such as Bcl-2, Bcl-x
L
, Bcl-w, and Mcl-1. Our results
suggest that CEAA triggers apoptosis through the intrinsic pathway
in 4T1 cells and death in MCF7 cell line by a caspase-independent
mechanism, as these last cells are deficient in caspase-3 expression
[26] and no changes on the Bcl-2/Bax ratio were observed in our
study after treatment.
Data from the literature have demonstrated that various forms
of cell death share molecular effectors and signaling pathways. In
this scenario, a clear role is played by calcium (Ca
2+
), a secondary
messenger involved in several physiological and pathological
processes [27–29], including activation of caspase-dependent and
independent cell death [28]. Our results showed that the CEAA
increases cytosolic Ca
2+
, in 4T1 cancer cells, which strongly
suggests that calcium is involved in the CEAA-induced death of
breast cancer cells.
The anticancer effects of the CEAA were also assessed in
4T1 breast cancer cell-challenged mice, a classical model for
in vivo studies [22]. Administration of the CEAA (500 mg/kg) for
33 alternate days significantly reduced the tumor growth in a tem-
poral dependent manner. No signs of acute toxicity were observed
in the CEAA-treated mice. Importantly, acute toxicity study
conducted by Kumar & Bhat [17] demonstrated no abnormality or
mortality in rats orally treated with the root, stem, leaf and fruits
of, or the whole A. archangelica extract (1600 and 3200 mg/kg),
suggesting that the plant is safe in the doses used in this study
(500 mg/kg).
Several explanations for this in vivo antitumor effect of the
CEAA must be considered. First of all, as observed in our in vitro
studies, there is a direct cytotoxic effect of the CEAA, which can
be due to, at least in part, the presence of furanocoumarins such
as angelicin, and other secondary metabolites such as flavonoids
in the A. archangelica extract composition [1]. Supporting our sug-
gestion is the fact that angelicin was the major compound found in
the CEAA root, as shown by UHPLC analysis. Angelicin has
apoptosis-inducing effects in human neuroblastoma cells [30],
and is commonly used for its antiproliferative activity in the treat-
ment of different skin diseases [31]. Angelicin as well as its analogs
exert potent cytotoxic activity in human lung carcinoma, inhibiting
growth and metastasis by targeting extracellular regulated protein
kinases and c-Jun N-terminal kinase signaling [32,33]. Angelicin
also inhibits liver cancer cell growth in vitro and in vivo [15].
Second, the antiproliferative properties of the A. archangelica
compounds described in the current literature [1,10,11,15] may
contribute to the delay in breast tumor development, which was
verified in this study.
5. Conclusion
Altogether, we have reported for the first time the potential
of the CEAA as a cell death inducer in breast cancer cells. This
activity might be partially attributed to the angelicin content
of the studied extract. As the formation of tumors in mice
injected with 4T1 cells was significantly reduced in those treated
with the CEAA, our findings illustrate the potential use of this
extract to control breast cancer as a form of complementary
medicine in conjunction with standard treatments. Further stud-
ies are needed to elucidate the precise molecular mechanism
involved in the results presented here to improve the clinical
application of A. archangelica.
Acknowledgments
The authors would like to thank Almeida Prado Homeopathic
Laboratories for kindly donating the CEAA evaluated in this
research and P.M. for the UHPLC–MS equipment (FAPESP
2008/58035-6). The authors thank the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES),
FAPESP and CNPq for the financial support.
Competing interests
The authors declare that there is no competing interest.
Fig. 5. CEAA administration to 4T1-challenged mice decreased tumor development. After 24 h, the mice were separated into two groups of five animals each. One group was
treated with CEAA (500 mg/kg), and the other (control group) received saline only. Treatment was done by gavage for 33 alternate days. The average of tumor size (A) and
mass tumor weight (B) in CEAA-treated mice was significantly reduced in comparison with the saline-treated control group.
*
P< 0.05, vs untreated (control) groups. CEAA:
crude extract of A. archangelica root.
C.R. Oliveira et al. / Journal of Integrative Medicine 17 (2019) 132–140 139
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