Journal of Ethnopharmacology 190 (2016) 362371

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Chelidonium majus crude extract inhibits migration and induces cell

cycle arrest and apoptosis in tumor cell lines
Milena Deljanin a, Mladen Nikolic b, Dejan Baskic c, Danijela Todorovic d,
Predrag Djurdjevic e, Milan Zaric f, Milan Stankovic g, Milos Todorovic h, Dusko Avramovic i,
Suzana Popovic c,n
a
College for Preschool Teachers, Cirila i Metodija 22, 37000 Krusevac, Serbia
b
College of Applied Technical Studies and Technology, Kosanciceva 36, 37000 Krusevac, Serbia
c
Department of Immunology and Microbiology, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia
d
Department of Genetics, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia
e
Department of Pathophysiology, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia
f
Department of Biochemistry, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia
g
Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovica 12, 34000 Kragujevac, Serbia
h
Department of Forensic Medicine, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia
i
Special Hospital for Internal Desease, Vojvode Misica 2, 11400 Mladenovac, Serbia

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Chelidonium majus L (Papaveraceae) is widely used in alternative med-
Received 14 March 2016 icine for treatment of various disorders. Antitumor activities of alkaloids isolated from this plant have
Received in revised form been reviewed, while there are only a few studies that examine properties of the whole extract.
22 June 2016
Aim of the study: The aim of the present study was to investigate direct cytotoxic effects, as well as
Accepted 24 June 2016
Available online 25 June 2016
indirect antitumor effects of Chelidonium majus ethanolic extract against different tumor cell lines,.
Materials and methods: MTT and SRB assays were performed to estimate cytotoxic effects of Chelidonium
Keywords: majus extract against human tumor cell lines A549, H460, HCT 116, SW480, MDA-MB 231 and MCF-7 and
Chelidonium majus L peripheral blood mononuclear cells from healthy individuals. Cell cycle analysis was performed by ow
Crude extract
cytometry. Type of cell death induced by extract was determined by ow cytometry and cell morphology
Cytotoxicity
assessment. Inhibitory effect on migration of cancer cells was assessed by wound healing assay.
Apoptosis
Cancer Results: Chelidonium majus extract showed selective time- and dose-dependent increase of cytotoxicity
in all six cell lines, with individual cell line sensitivities. Extract promoted cell cycle arrest and induced
apoptosis. Cotreatment with doxorubicin enhanced cytotoxicity of the drug. Also, inhibitory effect on
migration was shown with non-toxic extract concentration.
Conclusions: These results indicate possible usefulness of Chelidonium majus crude extract in antitumor
therapy, whether through its direct cytotoxic effect, by prevention of metastasis, or as adjuvant therapy.
& 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Searching for antitumor agents is the most intensive segment
in the development of new drugs. Modern approach to the re-
search is focused on nding compounds which affect important
processes in tumor development, progression and metastasis. Key
n
Corresponding author.
E-mail addresses: milena.nikolic.1987@gmail.com (M. Deljanin),
mladennikolic2603@yahoo.com (M. Nikolic), dejan.baskic@gmail.com (D. Baskic),
dtodorovic@medf.kg.ac.rs (D. Todorovic), pdjurdjevic@sbb.rs (P. Djurdjevic),
zaricmilan@gmail.com (M. Zaric), mstankovic@kg.ac.rs (M. Stankovic),
mtodorovickg@gmail.com (M. Todorovic), dr.damv@gmail.com (D. Avramovic),
suzana.popovic@medf.kg.ac.rs (S. Popovic).
molecular mechanisms that are being explored are apoptosis ac-
tivation, metastasis inhibition, inhibition of angiogenesis and tar-
geting tumor specic molecules. Very important source of new
drugs represent bioactive components of natural products.
Nowadays we know that plants are especially rich source of
compounds with antioxidative and immunomodulatory proper-
ties, as well as ingredients that have inuence on gene regulation,
proliferation and apoptosis of malignant cells (Strissel and Strick,
2005).
Chelidonium majus L. (greater celandine, local name rosopas)
belongs to Papaveraceae family and has been used since the middle
ages for treatment of many diseases in Europe and Western Asia
(Colombo and Bosisio, 1996). Description and medicinal uses of
this plant are monographed in the European Pharmacopoeia and

http://dx.doi.org/10.1016/j.jep.2016.06.056
0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371
in a national pharmacopoeia, and reported in well established
documents (EMA/HMPC, 2011). A number of pharmacological
properties, e.g. spasmolitic (Hiller et al., 1998), anti-inammatory
(Lee et al., 2007), antioxidant (Jakovljevi et al., 2013), antiulcer
(Khayyal et al., 2001), antibacterial (Zuo et al., 2008), antiviral
(Gerencer et al., 2006), immunomodulatory (Chung et al., 2004;
Song et al., 2002) and antitumor (Aljuraisy et al., 2012; Mahmoud
et al., 2013) have been reviewed and supported by clinical data
(Knpfel, 1991; Ritter et al., 1993). By virtue of these properties C.
majus is widely used in alternative medicine to treat liver diseases,
gastric ulcer and spasm, oral infections, rheumatic diseases, tu-
berculosis and jaundice (Abu, 1956; Amirdowlat, 1990; Mills and
Bone, 2000; Pelagi, 1893; Zadorozhnyj, 1990). Topically, the plant
is used against several skin disorders including warts, eczema and
corns (Van Wyk and Wink, 2004). Vast array of biological activities
have been achieved due to variety of alkaloids (chelidonine,
homochelidonine, chelerytrine, sanguinarine, berberine, coptizine,
protopine), avonoids, phenolic acids, vitamin C and carotene
(Jakovljevi et al., 2013).
Antitumor actions of this plant have been studied in many in
vitro and in vivo experiments. Also, a few clinical trials suggested
benecial effect of C. majus in the treatment of human cancer
(Habermehl et al., 2006; Musianowycz et al., 1992; Panzer et al.,
2000; Uglyanitsa et al., 2000). However, most investigations of
antitumor properties of this plant were focused on the assessment
of individual isolated alkaloids (Avijit et al., 2013; Slunska et al.,
2010; Safaa et al., 2012; Noureini and Esmaili, 2014), while there
are only a few studies that examine properties of the whole extract
(Aljuraisy et al., 2012; Capistrano et al., 2015; Dae et al., 1997;
Nadova et al., 2008).
In the present study, ethanol extract of Chelidonium majus was
evaluated for its anti-cancer properties on six human cancer cell
lines, including MCF-7, MDA MB-231, A549, H460, HCT116 and
SW480. We have investigated the effect of the extract on pro-
liferation and viability, cell cycle, migration and combined use
with doxorubicin on selected cancer cell lines. The cytotoxic effect
on normal human peripheral blood mononuclear cells was also
estimated.
2. Materials and methods
2.1. Plant material and preparation of extract
Chelidonium majus plants (greater celandine, local name roso-
pas) with all plant parts were collected from natural population in
the territory of Kragujevac central Serbia, during June 2015. The
voucher specimen were conrmed and deposited at the Herbar-
ium of the Faculty of Science, University of Kragujevac. Sampled
material was dried at ambient temperature in a dark place. Air-
dried material was milled in a grinder. Prepared plant material
(10 g) was transferred into dark-coloured ask, lled with 200 ml
of ethanol and stored at the room temperature. After 24 h, infusion
was ltered using Whatman No. 1 lter paper and residue was re-
extracted with equal volume of solvent. After 48 h, the process was
repeated. Combined supernatants were evaporated to dryness
under vacuum at 40 C. The obtained extract was kept in sterile
sample tube and stored at 4 C until the analysis. Stock solution
was prepared as 100 mg/ml DMSO and kept on 4 C. The content
of chelidonine and berberine alkaloids, polyphenols and sterols in
C. majus ethanol extract was dened by Parvu et al. (2013).
2.2. Cell culture
Human lung (A549 and H460), breast (MDA-MB 231 and MCF-
7) and colon (HCT116 and SW480) tumor cell lines were obtained
363
from the American Type Culture Collection (ATCC). The cells were
maintained in DMEM or RPMI 1640 medium supplemented with
10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml strep-
tomycin (all from Sigma, Germany). Cells were cultivated at 37 C
in an atmosphere of 5% CO2 and absolute humidity.
Peripheral blood mononuclear cells (PBMNC) were obtained
from heparinized venous blood of healthy volunteers. The study
was approved by local Ethics Committee and prior to initiation
written informed consent was obtained from all subjects accord-
ing to the Declaration of Helsinki. PBMNC were isolated from
peripheral blood by density gradient centrifugation (Histopaque
1077, Sigma, Germany). Separated cells were washed three times
and nally suspended in supplemented RPMI 1640 culture med-
ium. Cell number and viability were determined using acridine
orange/ethidium bromide staining. Peripheral blood mononuclear
cells were used in cell viability assay as healthy cells.
2.3. Cytotoxicity assays
2.3.1. MTT assay
To estimate cytotoxicity of extract against six cell lines MTT
assay was performed according to manufacturer's instructions
(Sigma-Aldrich, Germany). In brief, cells were seeded in 96-well
plates (Nunc A/S, Rockilde, Denmark) and incubated overnight for
adherence. After 24 h medium was replaced with medium con-
taining a range concentration of extract: 16, 32, 62.5, 125, 250 and
500 mg/ml, and with fresh medium as a control. Freshly isolated
PBMNC (10  105/well) were treated with the same concentrations
of extract. Cells were incubated at 37 C in an atmosphere of 5%
CO2 and absolute humidity for 24 and 48 h. After incubation MTT
(0.5 mg/ml) was added in each well and cells were incubated 4 h
under culture conditions. For that time, in metabolically active
viable cells, yellow MTT was reduced to purple formazan, due to
activity of mitochondrial dehydrogenase. After dissolving for-
mazan crystals in DMSO, absorbance was measured at 550 nm
with a multiplate reader (Zenith 3100, Anthos Labtec Instruments
GmbH, Austria). Experiments were performed in triplicates and
repeated in three independent series.
2.3.2. SRB assay
Cells were seeded in 96-well plates (5  103 per well) and in-
cubated overnight for adherence. After 24 h medium was replaced
with medium containing a range concentration of extract: 16, 32,
62.5, 125, 250 and 500 mg/ml, and fresh medium as a control. Cells
were incubated at 37 C in an atmosphere of 5% CO2 and absolute
humidity. After 24 and 48 h incubation supernatants were dis-
carded and cells were xed with 100 l ice-cold 15% tri-
chloroacetic acid (TCA, Aldrich Chemical). After 1 h incubation at
4 C plates were washed ve times with 200 l distillated cold
water and air-dried. SRB stain (Sigma, Aldrich) was added into
each well and left for 1 h. Wells were then washed with 1% acetic
acid, and rinsed until only dye bound to the cells was left. Tris base
(10 mM unbuffered, pH 10.5, Sigma) was added to each well to
dissolve the dye. Absorbance was measured at 550 nm. Experi-
ments were performed in triplicates and repeated in three in-
dependent series.
2.4. Determination of apoptosis/necrosis by ow cytometry
A549, HCT116 and MDA-MB 231 cells were treated with C.
majus extract in concentrations corresponding to IC50 value for
each cell line, or media alone (control), and incubated for 24 h at
37 C, 5% CO2. After treatment cells were harvested and analyzed
by ow cytometer Cytomics FC500 (Beckman Coulter).
364 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371
2.4.1. Annexin V-FITC/7-AAD apoptosis assay
Cells were washed with PBS and resuspended in 500 l of ice
cold binding buffer. One hundred l of cell suspension (5  105
cells) was transferred to ow cytometric tubes and stained with
10 l of Annexin V-FITC and 20 l of 7-AAD (Annexin V-FITC/7-
AAD Kit, Beckman Coulter, USA). After 15 min incubation in dark,
400 l of binding buffer was added to each tube and samples were
analyzed by ow cytometry. The percent of early apoptotic, late
apoptotic and necrotic cells was determined using Flowing Soft-
ware (http://www.owingsoftware.com/) and the results were
presented by dot plots.
2.4.2. Analysis of apoptosis-related proteins by ow cytometry
In order to conrm the induction of apoptosis, treated and
untreated A549 cells were harvested, washed with PBS, xed and
permeabilized using Fixation and Permeabilization Kit
(eBioscience), and then stained separately for Bcl-2, Bax and
cleaved-caspase-3. For Bcl-2 staining permeabilized cells were
incubated with FITC-conjugated anti-Bcl-2 monoclonal antibody
(Life technologies) for 20 min at room temperature. For other
proteins staining was performed by incubation with primary anti-
Bax antibody (Santa Cruz Biotech. Inc) or anti-cleaved caspase-3
antibody (Cell signaling echnology) for 30 min at room tem-
perature. Cells were than washed and incubated with secondary
FITC-conjugated antibody (ab6785, Abcam) for 20 min. After
washing cells were analyzed by ow cytometry. The Bcl-2 and Bax
levels, expressed as mean uorescence index (MFI), and percent of
cells displaying cleaved caspase-3 were evaluated using Flowing
Software and the results were presented by histograms.
2.5. Apoptosis detection by cell morphology assessment
A549 cells were cultured in DMEM medium with 250 g/ml of
C. majus extract for 24 h at 37 C in an atmosphere of 5% CO2 and
absolute humidity. After incubation cells were harvested and
stained with uorescent dyes Acridine orange (AO) and Ethidium
bromide (EB). Nine microliters of cell suspension was mixed with
1 l of dye mixture (100 mg/ml AO and 100 mg/ml EB) and im-
mediately analyzed for cell morphology with uorescence micro-
scope (Polywar, Reinchard Jung) at 400  magnication. Images
were taken with Canon PC 1089 camera.
2.6. Cell cycle analysis
A549, HCT116 and MDA-MB 231 cells were cultured with C.
majus extract in concentrations corresponding to IC50 value for
each cell line, or media alone (control) at 37 C in an atmosphere
of 5% CO2 and absolute humidity. After 24 h both attached and
detached cells were harvested, washed in PBS and xed in 70% ice
cold ethanol overnight at 4 C. Fixed cells were pelleted, re-
suspended in 1 ml PBS with RNAse A (500 g/ml) and incubated
for 30 min at 37 C. After adding 5 l of staining solution (10 mg
propidium iodide/ml PBS) cells were incubated for 15 min in dark.
Cells were analyzed by ow cytometer Cytomics FC500. DNA
content was determined using Flowing Software and cell cycle
distribution was presented by histograms.
2.7. Analysis of doxorubicin and C. majus extract interactions
To investigate whether C. majus extract has synergistic effect
with doxorubicin, their interactions were analyzed with combi-
nation-index (CI) method for drug combination studies developed
by Chou and Talalay (1984). A549, HCT 116 and MDA-MB 231 cells
were treated with raising concentration of doxorubicin (1, 2, 4 and
6 mM) alone or in combination with 16, 32 and 62.5 mg/ml C. majus
extract, mixed at a xed ratio (1:1, v/v). After 24 h incubation MTT
assay was performed and cytotoxicity was calculated. To evaluate
the nature of interaction between compounds combination index
(CI) was calculated using CompuSyn Software (ComboSyn, Inc.,
Paramus, NJ., USA).
2.8. Wound-healing motility assay
A549, HCT116 and MDA-MB 231 cells were seeded in 12-well
plates in duplicate and cultured to form a conuent monolayer. A
micropipette tip was used to make a stretch. Detached cells and
cell debris were removed by gentle washing with PBS. DMEM (for
control) and 16 g/ml extract (non-toxic concentration) were ad-
ded into the wells and plates were incubated at 37 C, 5% CO2.
Images of cell movement were captured at 0, 8 h and 20 h after
wounding with inverted microscope and Canon PC 1089 camera.
Images were analyzed with ISCapture Software (http://iscapture.
software.informer.com/download/).
2.9. Statistical analysis
The data were expressed as mean 7standard error of the mean
(SE) from three indenpendent experiments performed in triplicate.
Commercial SPSS version 20.0 was used for statistical analysis. The
distributions of data were evaluated for normality using the Sha-
piro-Wilk test. Depending on data distribution statistical evalua-
tion was performed by Student's t-test for paired observations, or
by one-way ANOVA. P values less than 0.05 were considered sig-
nicant. IC50 was calculated by non-linear regression analysis of
the data with the ED50plus v1.0 Software (http://www.softlookup.
com/download.asp? ID 2972).
3. Results
3.1. C. majus extract induces cytotoxicity in tumor cell lines
Treatment with C. majus extract resulted in time- and dose-
dependent increase in cytotoxicity in all six cell lines, as determined
by MTT assay. The data revealed that the percentage of cell viability
was signicantly reduced in treated cells, with individual cell line
sensitivities. Calculated IC50 values (Table 1) showed moderate to
high cytotoxic activity depending on the cell type. IC50 value for
PBMNC was above 500 g/ml, the highest tested concentration.
Similar values for cytotoxic activity of extract were obtained by
SRB assay with minor deviations (Fig. 1).
3.2. C. majus extract induces apoptosis
In accordance with results obtained by MTT cell viability assay
Annexin V-FITC/7-AAD apoptosis assay showed that treatment
with IC50 concentrations of extract induced cell death in high
percent (A549 39,62%; HCT116 38,10%; MDA-MB 231 37,23%),
while a negligible percent of treated cells were necrotic (Fig. 2).
The apoptotic type of cell death was further conrmed by ow
cytometric analysis of apoptosis-related protein expression in
A549 cells. The expression of anti-apoptotic Bcl-2 decreased in
treated cells, while that of Bax, the pro-apoptotic protein, in-
creased (Fig. 3). The Bcl-2/Bax ratio consequently declined. Fur-
thermore, the percent of cells expressing cleaved, active form of
caspase-3 increased after treatment.
Fluorescence microscopy of AO/EB stained cells demonstrated
alterations in cell morphology typical for apoptosis: cell rounding
and shrinkage, nuclear condensation and fragmentation and for-
mation of apoptotic bodies (Fig. 4). This observation provided
additional conrmation that extract-induced cell death is occur-
ring via apoptosis.
 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371 365

Table 1 of cell percentage in G0/G1 phase (Fig. 5). The percentage of cells
In vitro growth inhibitory activity (IC50 mg/ml) of C.majus extract against various accumulated in G2/M phase after treatment with extract increased
human cancer cell lines and PBMNC after 24 h and 48 h treatment (X mean 7S.E).
Doxorubicin was used as positive control.
from 48,10% (control) to 73,38% in A549, from 58,68% to 72,28% in
HCT116 and from 42,81% to 69,03% in MDA-MB 231 cells.
Cell line C. majus extract (g/ml) Doxorubicin (g/ml)

24 h 48 h 24 h 48 h 3.4. C. majus extract potentiates cytotoxicity of doxorubicin

A549 213,357 3,74 59,277 2,01 7,05 7 0,35 4,93 7 0,78 In order to investigate the nature of interaction between C.
H460 177,69 7 5,14 112,64 71,40 1,85 7 0,05 0,117 0,02
majus extract and doxorubicin, cells were treated with combina-
HCT116 186,317 8,47 134,667 9,63 3,067 0,13 2,137 0,53
SW480 174,58 7 2,81 143,55 7 5,10 7,22 7 0,68 5,56 7 0,76 tion of 4 doses of the drug and three concentrations of extract. The
MDA-MB 231 143,617 5,87 68,46 72,86 2,477 0,12 1,687 0,33 cytotoxic effect of drug and extract alone and their combinations
MCF-7 179,357 9,92 44,65 7 3,66 1,50 7 0,20 1,067 0,05 were used for calculating combination index (CI). CI values 41
PBMNC 4 500 4500 82,357 4,67 55,077 5,03
indicate antagonism, CI 1 additive effect and CI o1 synergy. The
synergistic cytotoxic effect was observed with various combina-
3.3. C. majus extract induces cell cycle arrest tions depending on cell line (Fig. 6). In MDA-MB 231 cells extract
increased cytotoxicity of doxorubicin in all tested combinations.
In all three cell lines 24 h incubation with extract resulted in However, in A549 and HCT116 extract showed synergistic effect
accumulation of cells in G2/M phase, with concomitant reduction only with lower doxorubicin concentrations (1 and 2 M); while

Fig. 1. Comparison between dose-response curves obtained by MTT and SRB assay after 24 h and 48 h treatment with various concentration of C. majus extract. Results are
presented as mean 7 SD.
366 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371

Fig. 2. Flow cytometric analysis of Annexin V-FITC/7-AAD staining. Dot plots present percentage of viable, early apoptotic, late apoptotic and necrotic cells in untreated
(control) and treated (CM) A549 (A), HCT116 (B) and MDA-MB231 (C) cells. Lower left quadrant represents viable cells (Annexin V  7-AAD  ), lower right early apoptotic
(Annexin V 7-AAD  ), upper right late apoptotic (Annexin V 7-AAD ) and upper left necrotic cells (Annexin V  7-AAD ).

in combination with high doxorubicin concentrations (4 and
6 M) their mutual effect was antagonistic. These results suggest
that synergistic cytotoxic effects of extract and doxorubicin de-
pend on cell line and doxorubicin concentration.
3.5. C. majus extract inhibits tumor cells migration in vitro
extract, at non-toxic concentration, had inhibitory effect on mi-
gration. The extent of inhibition depended on cell line. In HCT116
and MDA-MB 231 20 h incubation with extract restrained wound
closure almost entirely, while in A549 cell movement was not
completely inhibited since about 50% of wound was lled up by
migrating cells.

The effect of C. majus extract on the migratory potential of 4. Discussion

A549, HCT116 and MDA-MB 231 cells was determined by wound-
healing assay (Fig. 7). In absence of extract (control) cells migrated Previous studies have shown that alkaloids isolated from C.
and after 20 h almost completely covered the wound, whereas majus induce cell death in a wide range of tumor cell lines.
 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371 367

Fig. 3. Expression of apoptosis-related proteins in untreated (control) and treated (CM) A549 cells presented by histograms. The mean uorescence intensity (MFI) of Bcl-2
(A) and Bax staining (B) and the percent of cells expressing cleaved caspase-3 (C) are indicated on histograms. (D) Chart showing Bcl-2/Bax ratio in untreated and treated
cells.
368 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371
Fig. 4. Changes in cell morphology observed under uorescent microscope at 400x magnication in A549 cells treated with 250 g/ml extract for 24 h. Viable cells have
green nuclei with organized structure. Early apoptotic cells have bright green and late apoptotic cells bright orange to red nuclei with condensed or fragmented chromatin.
(For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
Cytotoxic effect of chelidonine and stylopine has been demon-
strated in cancer cell lines such as prostate, breast, lung, liver and
colon (Jun et al., 2005; Noureini and Esmaili, 2014). Sanguilutine
and chelilutine showed antiproliferative effect on leukemia, cervix
and ovarian tumor cells (Slunska et al., 2010). Also, Ukrain, an al-
kaloid derivative of C. majus, in numerous in vitro and in vivo
studies was veried as an effective chemotherapeutic drug. As a
result of selective toxicity toward malignant cells only, this drug
causes minimal side effects (Musianowycz et al., 1992). Since
majority of investigations have been focused on isolated alkaloids
of this plant, the aim of this study was to evaluate antitumor ef-
fects of crude extract, considering additive and synergistic effects
of individual bioactive molecules. Furthermore, utilization of ex-
tract is more accessible and economically acceptable than isolation
of solitary alkaloids.
To evaluate extract-induced growth inhibition, MTT and SRB
assays were used. MTT assay is based on reduction of MTT to
formazan and therefore detects metabolically active viable cells.
Dye Sulforodamine B (SRB) stains total cellular proteins and thus
indirectly estimates cell number (Skehan et al., 1990). Both tests
were conducted in order to determine whether the extract kills or
stops the metabolic activity of cells. The results of MTT and SRB
assays on six cell lines showed approximate values. The cell before
being killed becomes metabolic inactive, so the obtained deroga-
tions are expected.
Our results demonstrated that C. majus extract decreased via-
bility of tumor cells, as shown in earlier studies (Aljuraisy et al.,
2012; Capistrano et al.,2015; Dae et al., 1997; Nadova et al., 2008).
Treatment with C. majus extract resulted in time- and dose-de-
pendent increase in cytotoxicity in all six tumor cell lines. The
cytotoxic effect of extract on MCF-7 has been previously reported
by Fatemeh et al. (2013) with the IC50 values of 95,38 mg/ml after
24 h treatment. Our result is about two times higher and amounts
179,35 mg/ml. The differences in these values might be due to
different protocols, cell culture conditions or difference in the
composition of alkaloids in two extracts. Capistrano et al. (2015)
after 48 h treatment on MDA-MB 231 reported IC50 value that is
close to our nding.
PBMNCs are immunocompetent cells that have signicant role
in antitumor immune response. Concerning their importance and
possible effect of antitumor drugs on their viability, we have in-
vestigated activity of extract against these cells. Also, PBMNCs
were considered as healthy non-cancerous control. Importantly,
we showed that extract did not affect viability of PBMNC since
IC50 for these cells was above the highest tested concentration of
extract.
It is known that necrosis is a type of cell death that induces
inammation, which can damage local healthy tissue. In contrast
to necrosis, apoptosis is a highly regulated and controlled process
which is limited to individual cells and causes no damage to the
other cells. Therefore, induction of apoptosis is the most eligible
strategy in anticancer therapy (Russo et al., 2006). Numerous
methods available for apoptosis detection have its advantages and
disadvantages. Therefore, more than one method has to be em-
ployed to conrm apoptosis. Results obtained from Annexin
V-FITC/7-AAD assay showed that C. majus extract is strong inducer
of apoptosis with high percent of apoptotic cells, while negligible
percent of necrotic cells. The expression of Bcl-2 and Bax, im-
portant regulators of apoptosis, changed after treatment with ex-
tract. More importantly, the balance between these proteins was
disturbed, favouring activation of apoptotic machinery. The cas-
pase-3 cleavage/activation refers to induction of apoptosis. The
morphological criterion is still the most specic method for dis-
criminating apoptosis (Baskic et al., 2006). Here, the results of ow
cytometric analysis were conrmed with assessment of morpho-
logical changes in extract-treated cells.
Each phase of cell cycle has checkpoints that can promote cell
cycle arrest, enabling activation of repair mechanisms and xing
the damage. If the damage cannot be xed, apoptosis program is
activated. Our results showed that in selected tumor cell lines
extract induces cell cycle arrest in G2/M phase. G2 checkpoint
abrogation may be a mechanism of extract-induced cytotoxicity
since cancer cells, in contrast to normal cells, are more depend on
it for DNA damage repair (Kawabe, 2004). Furthermore, chelido-
nine and sanguinarine, alkaloids isolated from C. majus, have been
shown to inhibit tubulin polymerization and initiate mitotic arrest
 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371 369

Fig. 5. Effect of C. majus extract on cell cycle distribution. In two independent experiments treatment with extract caused similar increase in percentage of cells in G2/M
phase. Histograms are representative of two experiments and present cell cycle distribution in untreated (control) and treated (CM) A549 (A), HCT116 (B) and MDA-MB 231
(C) cells.

Fig. 6. Combination Index Plots presenting the combined effects of C. majus extract and doxorubicin on A549, HCT116 and MDA-MB 231 cells. Extract in concentrations 16,
32 and 62,5 g/ml was combined with various doxorubicin concentrations: 1 M (D1 CM), 2 M (D2 CM), 4 M (D4 CM) and 6 M (D6 CM).
370 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371

Fig. 7. The effect of C. majus extract on migration of cancer cells. (A) Images of the wound at time 0 h, 8 h and 20 h in untreated (control) and treated (CM 16 g/ml) A549,
HCT116 and MDA-MB 231 cells. Charts present percent of area non-overlayed with cells comparatively at time 0 h and 8 h (B) and at time 0 h and 20 h (C) for untreated
(control) and treated cells (CM 16 g/ml).

(Lopus and Panda, 2006; Wolff and Knipling, 1993). Anyhow, G2/M
arrest can be a possible mechanism for growth inhibition and
apoptosis induced by C. majus extract.
Doxorubicin is a potent antineoplastic drug, which is widely
used in treatment of various types of carcinomas. As well as other
cytostatic drugs used in modern therapy, it causes various side
effects, including cardiomyopathy, alopecia, bone marrow de-
pression and nausea. These side effects and multidrug resistance,
which can occur during the therapy with doxorubicin, limits its
use. Safaa et al. (2012) have shown that certain secondary plant
metabolites, including sanguinarin, can be useful in combination
with doxorubicin by decreasing IC50 of this drug. The results of
our study showed that the combination of C. majus extract and
doxorubicin is more effective than doxorubicin alone, depending
on cell line and doxorubicin concentration. This nding suggests
that C. majus extract, in correctly adjusted quantities of both re-
medials, could be useful adjuvant in cancer therapy by reducing
the dose and therefore the side effects of doxorubicin.
In addition to these properties, wound-healing assay on three
cell lines showed that extract in non-toxic concentration induces
inhibition of cancer cell migration, i.e. movement of cells on 2D
surfaces. Cell migration into surrounding tissue is the main process
that promotes tumor metastasis, the foremost cause of death in
cancer patients. Recent in vivo study (Capistrano et al., 2015)
showed a signicant reduction in number of metastases in tumor-
bearing mice after intraperitoneal administration of C. majus crude
extract. These results uphold the use of this plant in prevention or
treatment of metastasis.
5. Conclusion
Chelidonium majus crude extract showed cytotoxic activity
against various types of cancer cell lines and inhibitory effect on
migration of cancer cells in vitro. Also, extract enhanced cyto-
toxicity of doxorubicin indicating that the extract could be used as
adjuvant therapy, to minimize side effects of antineoplastics.
However, we examined only a part of possibilities of this plant. It is
necessary to further investigate other indirect antitumor proper-
ties of crude extract, e.g. antioxidative and immunomodulatory
activity.
Authors contributions
SP designed the study, MD, MN, DB, DT, PD and MT were in-
vestigators in this study, MZ performed SP analysis, MS prepared
the extract, DA contributed to data analysis and interpretation. All
 M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371
authors revised the manuscript critically for intellectual content,
and have approved the nal version.
Acknowledgements
This study was funded by the Ministry of Science and Tech-
nology of the Republic of Serbia (III41010).
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