Вы находитесь на странице: 1из 10

Journal of Ethnopharmacology 190 (2016) 362371

Contents lists available at ScienceDirect

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 molecular mechanisms that are being explored are apoptosis ac-
tivation, metastasis inhibition, inhibition of angiogenesis and tar-
Searching for antitumor agents is the most intensive segment geting tumor specic molecules. Very important source of new
in the development of new drugs. Modern approach to the re- drugs represent bioactive components of natural products.
search is focused on nding compounds which affect important Nowadays we know that plants are especially rich source of
processes in tumor development, progression and metastasis. Key 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,
n
Corresponding author. 2005).
E-mail addresses: milena.nikolic.1987@gmail.com (M. Deljanin), Chelidonium majus L. (greater celandine, local name rosopas)
mladennikolic2603@yahoo.com (M. Nikolic), dejan.baskic@gmail.com (D. Baskic), belongs to Papaveraceae family and has been used since the middle
dtodorovic@medf.kg.ac.rs (D. Todorovic), pdjurdjevic@sbb.rs (P. Djurdjevic),
zaricmilan@gmail.com (M. Zaric), mstankovic@kg.ac.rs (M. Stankovic),
ages for treatment of many diseases in Europe and Western Asia
mtodorovickg@gmail.com (M. Todorovic), dr.damv@gmail.com (D. Avramovic), (Colombo and Bosisio, 1996). Description and medicinal uses of
suzana.popovic@medf.kg.ac.rs (S. Popovic). 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 363

in a national pharmacopoeia, and reported in well established from the American Type Culture Collection (ATCC). The cells were
documents (EMA/HMPC, 2011). A number of pharmacological maintained in DMEM or RPMI 1640 medium supplemented with
properties, e.g. spasmolitic (Hiller et al., 1998), anti-inammatory 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml strep-
(Lee et al., 2007), antioxidant (Jakovljevi et al., 2013), antiulcer tomycin (all from Sigma, Germany). Cells were cultivated at 37 C
(Khayyal et al., 2001), antibacterial (Zuo et al., 2008), antiviral in an atmosphere of 5% CO2 and absolute humidity.
(Gerencer et al., 2006), immunomodulatory (Chung et al., 2004; Peripheral blood mononuclear cells (PBMNC) were obtained
Song et al., 2002) and antitumor (Aljuraisy et al., 2012; Mahmoud from heparinized venous blood of healthy volunteers. The study
et al., 2013) have been reviewed and supported by clinical data was approved by local Ethics Committee and prior to initiation
(Knpfel, 1991; Ritter et al., 1993). By virtue of these properties C. written informed consent was obtained from all subjects accord-
majus is widely used in alternative medicine to treat liver diseases, ing to the Declaration of Helsinki. PBMNC were isolated from
gastric ulcer and spasm, oral infections, rheumatic diseases, tu- peripheral blood by density gradient centrifugation (Histopaque
berculosis and jaundice (Abu, 1956; Amirdowlat, 1990; Mills and 1077, Sigma, Germany). Separated cells were washed three times
Bone, 2000; Pelagi, 1893; Zadorozhnyj, 1990). Topically, the plant and nally suspended in supplemented RPMI 1640 culture med-
is used against several skin disorders including warts, eczema and
ium. Cell number and viability were determined using acridine
corns (Van Wyk and Wink, 2004). Vast array of biological activities
orange/ethidium bromide staining. Peripheral blood mononuclear
have been achieved due to variety of alkaloids (chelidonine,
cells were used in cell viability assay as healthy cells.
homochelidonine, chelerytrine, sanguinarine, berberine, coptizine,
protopine), avonoids, phenolic acids, vitamin C and carotene
(Jakovljevi et al., 2013). 2.3. Cytotoxicity assays
Antitumor actions of this plant have been studied in many in
vitro and in vivo experiments. Also, a few clinical trials suggested 2.3.1. MTT assay
benecial effect of C. majus in the treatment of human cancer To estimate cytotoxicity of extract against six cell lines MTT
(Habermehl et al., 2006; Musianowycz et al., 1992; Panzer et al., assay was performed according to manufacturer's instructions
2000; Uglyanitsa et al., 2000). However, most investigations of (Sigma-Aldrich, Germany). In brief, cells were seeded in 96-well
antitumor properties of this plant were focused on the assessment plates (Nunc A/S, Rockilde, Denmark) and incubated overnight for
of individual isolated alkaloids (Avijit et al., 2013; Slunska et al., adherence. After 24 h medium was replaced with medium con-
2010; Safaa et al., 2012; Noureini and Esmaili, 2014), while there taining a range concentration of extract: 16, 32, 62.5, 125, 250 and
are only a few studies that examine properties of the whole extract 500 mg/ml, and with fresh medium as a control. Freshly isolated
(Aljuraisy et al., 2012; Capistrano et al., 2015; Dae et al., 1997; PBMNC (10  105/well) were treated with the same concentrations
Nadova et al., 2008). of extract. Cells were incubated at 37 C in an atmosphere of 5%
In the present study, ethanol extract of Chelidonium majus was CO2 and absolute humidity for 24 and 48 h. After incubation MTT
evaluated for its anti-cancer properties on six human cancer cell (0.5 mg/ml) was added in each well and cells were incubated 4 h
lines, including MCF-7, MDA MB-231, A549, H460, HCT116 and under culture conditions. For that time, in metabolically active
SW480. We have investigated the effect of the extract on pro- viable cells, yellow MTT was reduced to purple formazan, due to
liferation and viability, cell cycle, migration and combined use activity of mitochondrial dehydrogenase. After dissolving for-
with doxorubicin on selected cancer cell lines. The cytotoxic effect mazan crystals in DMSO, absorbance was measured at 550 nm
on normal human peripheral blood mononuclear cells was also with a multiplate reader (Zenith 3100, Anthos Labtec Instruments
estimated. GmbH, Austria). Experiments were performed in triplicates and
repeated in three independent series.

2. Materials and methods 2.3.2. SRB assay


Cells were seeded in 96-well plates (5  103 per well) and in-
2.1. Plant material and preparation of extract cubated overnight for adherence. After 24 h medium was replaced
with medium containing a range concentration of extract: 16, 32,
Chelidonium majus plants (greater celandine, local name roso- 62.5, 125, 250 and 500 mg/ml, and fresh medium as a control. Cells
pas) with all plant parts were collected from natural population in were incubated at 37 C in an atmosphere of 5% CO2 and absolute
the territory of Kragujevac central Serbia, during June 2015. The humidity. After 24 and 48 h incubation supernatants were dis-
voucher specimen were conrmed and deposited at the Herbar- carded and cells were xed with 100 l ice-cold 15% tri-
ium of the Faculty of Science, University of Kragujevac. Sampled chloroacetic acid (TCA, Aldrich Chemical). After 1 h incubation at
material was dried at ambient temperature in a dark place. Air- 4 C plates were washed ve times with 200 l distillated cold
dried material was milled in a grinder. Prepared plant material water and air-dried. SRB stain (Sigma, Aldrich) was added into
(10 g) was transferred into dark-coloured ask, lled with 200 ml each well and left for 1 h. Wells were then washed with 1% acetic
of ethanol and stored at the room temperature. After 24 h, infusion
acid, and rinsed until only dye bound to the cells was left. Tris base
was ltered using Whatman No. 1 lter paper and residue was re-
(10 mM unbuffered, pH 10.5, Sigma) was added to each well to
extracted with equal volume of solvent. After 48 h, the process was
dissolve the dye. Absorbance was measured at 550 nm. Experi-
repeated. Combined supernatants were evaporated to dryness
ments were performed in triplicates and repeated in three in-
under vacuum at 40 C. The obtained extract was kept in sterile
dependent series.
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 2.4. Determination of apoptosis/necrosis by ow cytometry
C. majus ethanol extract was dened by Parvu et al. (2013).
A549, HCT116 and MDA-MB 231 cells were treated with C.
2.2. Cell culture majus extract in concentrations corresponding to IC50 value for
each cell line, or media alone (control), and incubated for 24 h at
Human lung (A549 and H460), breast (MDA-MB 231 and MCF- 37 C, 5% CO2. After treatment cells were harvested and analyzed
7) and colon (HCT116 and SW480) tumor cell lines were obtained 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 assay was performed and cytotoxicity was calculated. To evaluate
Cells were washed with PBS and resuspended in 500 l of ice the nature of interaction between compounds combination index
cold binding buffer. One hundred l of cell suspension (5  105 (CI) was calculated using CompuSyn Software (ComboSyn, Inc.,
cells) was transferred to ow cytometric tubes and stained with Paramus, NJ., USA).
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, 2.8. Wound-healing motility assay
400 l of binding buffer was added to each tube and samples were
analyzed by ow cytometry. The percent of early apoptotic, late A549, HCT116 and MDA-MB 231 cells were seeded in 12-well
apoptotic and necrotic cells was determined using Flowing Soft- plates in duplicate and cultured to form a conuent monolayer. A
ware (http://www.owingsoftware.com/) and the results were micropipette tip was used to make a stretch. Detached cells and
presented by dot plots. cell debris were removed by gentle washing with PBS. DMEM (for
control) and 16 g/ml extract (non-toxic concentration) were ad-
2.4.2. Analysis of apoptosis-related proteins by ow cytometry ded into the wells and plates were incubated at 37 C, 5% CO2.
In order to conrm the induction of apoptosis, treated and Images of cell movement were captured at 0, 8 h and 20 h after
untreated A549 cells were harvested, washed with PBS, xed and wounding with inverted microscope and Canon PC 1089 camera.
permeabilized using Fixation and Permeabilization Kit Images were analyzed with ISCapture Software (http://iscapture.
(eBioscience), and then stained separately for Bcl-2, Bax and software.informer.com/download/).
cleaved-caspase-3. For Bcl-2 staining permeabilized cells were
incubated with FITC-conjugated anti-Bcl-2 monoclonal antibody 2.9. Statistical analysis
(Life technologies) for 20 min at room temperature. For other
proteins staining was performed by incubation with primary anti- The data were expressed as mean 7standard error of the mean
Bax antibody (Santa Cruz Biotech. Inc) or anti-cleaved caspase-3 (SE) from three indenpendent experiments performed in triplicate.
antibody (Cell signaling echnology) for 30 min at room tem- Commercial SPSS version 20.0 was used for statistical analysis. The
perature. Cells were than washed and incubated with secondary distributions of data were evaluated for normality using the Sha-
FITC-conjugated antibody (ab6785, Abcam) for 20 min. After piro-Wilk test. Depending on data distribution statistical evalua-
washing cells were analyzed by ow cytometry. The Bcl-2 and Bax tion was performed by Student's t-test for paired observations, or
levels, expressed as mean uorescence index (MFI), and percent of by one-way ANOVA. P values less than 0.05 were considered sig-
cells displaying cleaved caspase-3 were evaluated using Flowing nicant. IC50 was calculated by non-linear regression analysis of
Software and the results were presented by histograms. the data with the ED50plus v1.0 Software (http://www.softlookup.
com/download.asp? ID 2972).
2.5. Apoptosis detection by cell morphology assessment

A549 cells were cultured in DMEM medium with 250 g/ml of 3. Results
C. majus extract for 24 h at 37 C in an atmosphere of 5% CO2 and
absolute humidity. After incubation cells were harvested and 3.1. C. majus extract induces cytotoxicity in tumor cell lines
stained with uorescent dyes Acridine orange (AO) and Ethidium
bromide (EB). Nine microliters of cell suspension was mixed with Treatment with C. majus extract resulted in time- and dose-
1 l of dye mixture (100 mg/ml AO and 100 mg/ml EB) and im- dependent increase in cytotoxicity in all six cell lines, as determined
mediately analyzed for cell morphology with uorescence micro- by MTT assay. The data revealed that the percentage of cell viability
scope (Polywar, Reinchard Jung) at 400  magnication. Images was signicantly reduced in treated cells, with individual cell line
were taken with Canon PC 1089 camera. sensitivities. Calculated IC50 values (Table 1) showed moderate to
high cytotoxic activity depending on the cell type. IC50 value for
2.6. Cell cycle analysis PBMNC was above 500 g/ml, the highest tested concentration.
Similar values for cytotoxic activity of extract were obtained by
A549, HCT116 and MDA-MB 231 cells were cultured with C. SRB assay with minor deviations (Fig. 1).
majus extract in concentrations corresponding to IC50 value for
each cell line, or media alone (control) at 37 C in an atmosphere 3.2. C. majus extract induces apoptosis
of 5% CO2 and absolute humidity. After 24 h both attached and
detached cells were harvested, washed in PBS and xed in 70% ice In accordance with results obtained by MTT cell viability assay
cold ethanol overnight at 4 C. Fixed cells were pelleted, re- Annexin V-FITC/7-AAD apoptosis assay showed that treatment
suspended in 1 ml PBS with RNAse A (500 g/ml) and incubated with IC50 concentrations of extract induced cell death in high
for 30 min at 37 C. After adding 5 l of staining solution (10 mg percent (A549 39,62%; HCT116 38,10%; MDA-MB 231 37,23%),
propidium iodide/ml PBS) cells were incubated for 15 min in dark. while a negligible percent of treated cells were necrotic (Fig. 2).
Cells were analyzed by ow cytometer Cytomics FC500. DNA The apoptotic type of cell death was further conrmed by ow
content was determined using Flowing Software and cell cycle cytometric analysis of apoptosis-related protein expression in
distribution was presented by histograms. A549 cells. The expression of anti-apoptotic Bcl-2 decreased in
treated cells, while that of Bax, the pro-apoptotic protein, in-
2.7. Analysis of doxorubicin and C. majus extract interactions creased (Fig. 3). The Bcl-2/Bax ratio consequently declined. Fur-
thermore, the percent of cells expressing cleaved, active form of
To investigate whether C. majus extract has synergistic effect caspase-3 increased after treatment.
with doxorubicin, their interactions were analyzed with combi- Fluorescence microscopy of AO/EB stained cells demonstrated
nation-index (CI) method for drug combination studies developed alterations in cell morphology typical for apoptosis: cell rounding
by Chou and Talalay (1984). A549, HCT 116 and MDA-MB 231 cells and shrinkage, nuclear condensation and fragmentation and for-
were treated with raising concentration of doxorubicin (1, 2, 4 and mation of apoptotic bodies (Fig. 4). This observation provided
6 mM) alone or in combination with 16, 32 and 62.5 mg/ml C. majus additional conrmation that extract-induced cell death is occur-
extract, mixed at a xed ratio (1:1, v/v). After 24 h incubation MTT 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 extract, at non-toxic concentration, had inhibitory effect on mi-
6 M) their mutual effect was antagonistic. These results suggest gration. The extent of inhibition depended on cell line. In HCT116
that synergistic cytotoxic effects of extract and doxorubicin de- and MDA-MB 231 20 h incubation with extract restrained wound
pend on cell line and doxorubicin concentration. closure almost entirely, while in A549 cell movement was not
completely inhibited since about 50% of wound was lled up by
3.5. C. majus extract inhibits tumor cells migration in vitro 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- PBMNCs are immunocompetent cells that have signicant role
strated in cancer cell lines such as prostate, breast, lung, liver and in antitumor immune response. Concerning their importance and
colon (Jun et al., 2005; Noureini and Esmaili, 2014). Sanguilutine possible effect of antitumor drugs on their viability, we have in-
and chelilutine showed antiproliferative effect on leukemia, cervix vestigated activity of extract against these cells. Also, PBMNCs
and ovarian tumor cells (Slunska et al., 2010). Also, Ukrain, an al- were considered as healthy non-cancerous control. Importantly,
kaloid derivative of C. majus, in numerous in vitro and in vivo we showed that extract did not affect viability of PBMNC since
studies was veried as an effective chemotherapeutic drug. As a IC50 for these cells was above the highest tested concentration of
result of selective toxicity toward malignant cells only, this drug extract.
causes minimal side effects (Musianowycz et al., 1992). Since It is known that necrosis is a type of cell death that induces
majority of investigations have been focused on isolated alkaloids inammation, which can damage local healthy tissue. In contrast
of this plant, the aim of this study was to evaluate antitumor ef- to necrosis, apoptosis is a highly regulated and controlled process
fects of crude extract, considering additive and synergistic effects which is limited to individual cells and causes no damage to the
of individual bioactive molecules. Furthermore, utilization of ex- other cells. Therefore, induction of apoptosis is the most eligible
tract is more accessible and economically acceptable than isolation strategy in anticancer therapy (Russo et al., 2006). Numerous
of solitary alkaloids. methods available for apoptosis detection have its advantages and
To evaluate extract-induced growth inhibition, MTT and SRB disadvantages. Therefore, more than one method has to be em-
assays were used. MTT assay is based on reduction of MTT to ployed to conrm apoptosis. Results obtained from Annexin
formazan and therefore detects metabolically active viable cells. V-FITC/7-AAD assay showed that C. majus extract is strong inducer
Dye Sulforodamine B (SRB) stains total cellular proteins and thus of apoptosis with high percent of apoptotic cells, while negligible
indirectly estimates cell number (Skehan et al., 1990). Both tests percent of necrotic cells. The expression of Bcl-2 and Bax, im-
were conducted in order to determine whether the extract kills or portant regulators of apoptosis, changed after treatment with ex-
stops the metabolic activity of cells. The results of MTT and SRB tract. More importantly, the balance between these proteins was
assays on six cell lines showed approximate values. The cell before disturbed, favouring activation of apoptotic machinery. The cas-
being killed becomes metabolic inactive, so the obtained deroga- pase-3 cleavage/activation refers to induction of apoptosis. The
tions are expected. morphological criterion is still the most specic method for dis-
Our results demonstrated that C. majus extract decreased via- criminating apoptosis (Baskic et al., 2006). Here, the results of ow
bility of tumor cells, as shown in earlier studies (Aljuraisy et al., cytometric analysis were conrmed with assessment of morpho-
2012; Capistrano et al.,2015; Dae et al., 1997; Nadova et al., 2008). logical changes in extract-treated cells.
Treatment with C. majus extract resulted in time- and dose-de- Each phase of cell cycle has checkpoints that can promote cell
pendent increase in cytotoxicity in all six tumor cell lines. The cycle arrest, enabling activation of repair mechanisms and xing
cytotoxic effect of extract on MCF-7 has been previously reported the damage. If the damage cannot be xed, apoptosis program is
by Fatemeh et al. (2013) with the IC50 values of 95,38 mg/ml after activated. Our results showed that in selected tumor cell lines
24 h treatment. Our result is about two times higher and amounts extract induces cell cycle arrest in G2/M phase. G2 checkpoint
179,35 mg/ml. The differences in these values might be due to abrogation may be a mechanism of extract-induced cytotoxicity
different protocols, cell culture conditions or difference in the since cancer cells, in contrast to normal cells, are more depend on
composition of alkaloids in two extracts. Capistrano et al. (2015) it for DNA damage repair (Kawabe, 2004). Furthermore, chelido-
after 48 h treatment on MDA-MB 231 reported IC50 value that is nine and sanguinarine, alkaloids isolated from C. majus, have been
close to our nding. 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 showed a signicant reduction in number of metastases in tumor-
arrest can be a possible mechanism for growth inhibition and bearing mice after intraperitoneal administration of C. majus crude
apoptosis induced by C. majus extract. extract. These results uphold the use of this plant in prevention or
Doxorubicin is a potent antineoplastic drug, which is widely treatment of metastasis.
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- 5. Conclusion
pression and nausea. These side effects and multidrug resistance,
which can occur during the therapy with doxorubicin, limits its Chelidonium majus crude extract showed cytotoxic activity
use. Safaa et al. (2012) have shown that certain secondary plant against various types of cancer cell lines and inhibitory effect on
metabolites, including sanguinarin, can be useful in combination migration of cancer cells in vitro. Also, extract enhanced cyto-
with doxorubicin by decreasing IC50 of this drug. The results of toxicity of doxorubicin indicating that the extract could be used as
our study showed that the combination of C. majus extract and adjuvant therapy, to minimize side effects of antineoplastics.
doxorubicin is more effective than doxorubicin alone, depending However, we examined only a part of possibilities of this plant. It is
on cell line and doxorubicin concentration. This nding suggests necessary to further investigate other indirect antitumor proper-
that C. majus extract, in correctly adjusted quantities of both re- ties of crude extract, e.g. antioxidative and immunomodulatory
medials, could be useful adjuvant in cancer therapy by reducing activity.
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 Authors contributions
inhibition of cancer cell migration, i.e. movement of cells on 2D
surfaces. Cell migration into surrounding tissue is the main process SP designed the study, MD, MN, DB, DT, PD and MT were in-
that promotes tumor metastasis, the foremost cause of death in vestigators in this study, MZ performed SP analysis, MS prepared
cancer patients. Recent in vivo study (Capistrano et al., 2015) the extract, DA contributed to data analysis and interpretation. All
M. Deljanin et al. / Journal of Ethnopharmacology 190 (2016) 362371 371

authors revised the manuscript critically for intellectual content, Knpfel, S.A., 1991. Behandlung von Erkrankungen der Gallenwege und der Gal-
and have approved the nal version. lenblase mit dem natrlichen Cholendynamikum Chelidonin. Auch gegen
Gallenleiden ist ein Kraut gewachsen [Treatment of biliary tract and cholecystic
diseases with natural bile-excreting Chelidonine. Also a plant can treat diseases
of the liver]. Therapeutikon 5, 205208.
Acknowledgements Lee, Y.C., Kim, S.H., Roh, S.S., Choi, H.Y., Seo, Y.B., 2007. Suppressive effects of Che-
lidonium majus methanol extract in knee joint, regional lymph nodes, and
spleen on collagen-induced arthritis in mice. J. Ethnopharmacol. 112, 4048.
This study was funded by the Ministry of Science and Tech- Lopus, M., Panda, D., 2006. The benzophenanthridine alkaloid sanguinarine per-
nology of the Republic of Serbia (III41010). turbs microtubule assembly dynamics through tubulin binding. A possible
mechanism for its antiproliferative activity. FEBS J. 273, 21392150.
Mahmoud, Z.E.R., Safaa, Z.E., Mohamed, L.A., Ahmad, T., Michael, W., 2013. Mod-
ulation of multidrug resistance in cancer cells by chelidonine and Chelidonium
References majus alkaloids. Phytomedicine 20, 282294.
Mills, S., Bone, K., 2000. Principles and Practice of Phytotherapy: Modern Herbal
Medicine. Churchill Livingstone, Edinburgh.
Abu, A.I.S., 1956. Kanon Vratchebnoi Nauki. Fan, Tashkent.
Musianowycz, J., Judmajer, F., Manfreda, D., Spangler, P., Albrecht, H., Hoffmann, J.,
Aljuraisy, Y.H., Mahdi, N., Kh, Al-Darraji, M.N.J., 2012. Cytotoxic effect of Chelido-
1992. Clinical studies of Ukrain in terminal cancer patients (phase II). Drugs
nium majus on cancer cell lines. Al-Anbar J. Vet. Sci. 5, 8590.
Amirdowlat, A., 1990. Nenuzhnoe dlja neuczej (Needless for Ignoramus). Nauka, Exp. Clin. Res. 18, 4550.
Moscow. Nadova, S., Miadokova, E., Alfoldiova, L., Kopaskova, M., Hasplova, K., Hudecova, A.,
Avijit, P., Sreemanti, D., Jayeeta, D., Asmita, S., Anisur, R.K.B., 2013. Cytotoxicity and Vaculcikova, D., Gregan, F., Cipak, L., 2008. Potential antioxidant activity, cyto-
apoptotic signaling cascade induced by chelidonine-loaded PLGA nanoparticles toxic and apoptosis-inducing effects of Chelidonium majus L. extract on leuke-
in HepG2 cells in vitro and bioavailability of nano-chelidonine in mice in vivo. mia cells. Neuro Endocrinol. Lett. 29, 649652.
Toxicol. Lett. 222, 1022. Noureini, S.K., Esmaili, H., 2014. Multiple mechanisms of cell death induced by
Baskic, D., Popovic, S., Ristic, P., Arsenijevic, N.N., 2006. Analysis of cycloheximide- chelidonine in MCF-7 breast cancer cell line. Chemico-Biol. Interact. 223,
induced apoptosis in human leukocytes: uorescence microscopy using an- 141149.
nexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Panzer, A., Hamel, E., Joubert, A.M., Bianchi, P.C., Seegers, J.C., 2000. Ukrain(TM), a
Int. 30, 924932. semisynthetic Chelidonium majus alkaloid derivate, acts by inhibition of tubulin
Capistrano, R.I., Wounters, A., Lardon, F., Gravecamp, C., Apers, S., Pieters, L., 2015. polymerization in normal and malignant cell lines. Cancer Lett. 160, 149157.
In vitro and in vivo investigations on the antitumor activity of Chelidonium Parvu, M., Vlase, L., Fodorpataki, L., Parvu, O., Rosca-Casian, O., Bartha, C., Barbu-
majus. Phytomedicine 22, 12791287. Tudoran, L., Parvu, A.E., 2013. Chemical composition of celandine (Chelidonium
Chou, T.C., Talalay, P., 1984. Quantitative analysis of dose-effect relationships: the majus L.) extract and its effects on Botrytis tulipae (Lib.) Lind Fungus and the
combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. Tulip. Not. Bot. Horti Agrobo 41, 414426.
22, 2755. Pelagi, V., 1893. Stvarni narodni uitelj. Srpska knjievna zadruga, Beograd.
Chung, H.S., An, H.J., Jeong, H.J., Won, J.H., Hong, S.H., Kim, H.M., 2004. Water ex- Ritter, R., Schatton, W.F.H., Gessner, B., Willems, M., 1993. Clinical trial on stan-
tract isolated from Chelidonium majus enhances nitric oxide and tumor necrosis dardized celandine extract in patients with functional epigastric complaints:
factor-alpha production via nuclear factor kappa B activation in mouse peri- results of a placebo-controlled doubleblind trial. Complement. Ther. Med. 1,
toneal macrophages. J. Pharm. Pharm. 56, 129134. 189193.
Colombo, M.L., Bosisio, E., 1996. Pharmacological activities of Chelidonium majus L. Russo, A., Terrasi, M., Agnese, V., Santini, D., Bazan, V., 2006. Apoptosis: a relevant
(Papaveraceae). Pharm. Res. 33, 127134. tool for anticancer therapy. Ann. Oncol. 17, 115123.
Dae, J.K., Byeongwoo, A., Beom, S.H., Hiroyuki, T., 1997. Potential preventive effects Safaa, Y.E., Mahmoud, Z.E.R., Michael, W., 2012. Synergism of three-drug combi-
of Chelidonium majus L. (Papaveracea)e) herb extract on glandular stomach nation of sanguinarine and other plant secondary metabolites with digitonin
tumor development in rats treated with N-methyl-N-nitro-N nitrosoguanidine and doxorubicin in multi-drug resistant cancer cells. Phytomedicine 19,
(MNNG) (and hypertonic sodium chloride. Cancer Lett. 112, 203208. 12881297.
European Medicine agency, 2012. Assesment Report on Chelidonium majus L., herba. Skehan, P.I., Storeng, R., Scudiero, D., Monks, A., McMakon, J., Vistica, D., Warren, J.T.,
Available from: http://www.ema.europa.eu/docs/en_GB/document_library/ Bokesh, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity asssay
Herbal_-_HMPC_assessment_report/2012/01/WC500120711.pdf.
for anticancer drug screening. J. Nat. Cancer Inst. 82, 11071112.
Fatemeh, Z.S., Behzad, B., Mona, O., Fatemeh, Z., 2013. In vitro cytotoxic activity of
Slunska, Z., Gelnarova, E., Hammerova, J., Taborska, E., Slaninova, I., 2010. Effect of
four plants used in persian traditional medicine. Adv. Pharm. Bull. 3, 453455.
quaternary benzo[c]phenanthridine alkaloids sanguilutine and chelilutine on
Gerencer, M., Turecek, P.L., Kistner, O., Mitterer, A., Savidis-Dacho, H., Barrett, N.P.,
normal and cancer cells. Toxicol. Vitr. 24, 697706.
2006. In vitro and in vivo anti-retroviral activity of the substance puried from
Song, J.Y., Yang, H.O., Pyo, S.N., Jung, I.S., Yi, S.Y., Yun, Y.S., 2002. Immunomodulatory
the aqueous extract of Chelidonium majus L. Antivir. Res. 72, 153156.
activity of protein-bound polysaccharide extracted from Chelidonium majus.
Habermehl, D., Kammerer, B., Handrick, R., Eldh, T., Gruber, C., Cordes, N., Daniel, P.,
Arch. Pharm. Res. 25, 158164.
Plasswilm, L., Bamberg, M., Belka, C., Jendrossek, V., 2006. Proapoptotic activity
Strissel, P.M., Strick, R., 2005. Multiple effects of bioavonoids on gene regulation,
of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mi-
tochondrial death pathway. BMC Cancer 6, 122. cell proliferation and apoptosis: natural compounds move into the lime light of
Hiller, K.O., Ghorbani, M., Schilcher, H., 1998. Antispasmodic and relaxant activity of cancer research. Leuk. Res., 859861.
chelidonine, protopine, coptisine, and Chelidonium majus extracts on isolated Uglyanitsa, K.N., Nefyodov, L.I., Karayedova, L.M., Nowicky, J.W., Brzosko, W.J., 2000.
guinea-pig ileum. Planta Med 64, 758760. Clinical aspects of cancer treatment and new biochemical mechanisms of the
Jakovljevi, D.Z., Stankovi, M.S., Topuzovi, M.D., 2013. Seasonal variability of drug Ukrain. Drugs Exp. Clin. Res. 26, 239247.
Chelidonium majus L. secondary metabolites content and antioxidant activity. Van Wyk, B.-E., Wink, M., 2004. Medicinal Plants of the World: An Illustraded
EXCLI J. 12, 260268. Scientic Guide to Important Medicinal Plants and Their Uses, rst ed. Timber
Jun, L., Mi, Y.S., Dae, S.Y., Tae, Y.H., Seon, W.H., Sang, H.N., Eun-Kyong, S., Ki, H.P., Press, Portland, Oregon.
Min, S.Y., 2005. Cytotoxic Isoquinoline Alkaloids from Chelidonium majus var. Wolff, J., Knipling, L., 1993. Antimicrotubule properties of benzophenanthridine
asiaticum. Agric. Chem. Biotechnol. 48, 198201. alkaloids. Biochemistry 32, 1333413339.
Kawabe, T., 2004. G2 checkpoint abrogators as anticancer drugs. Mol. Cancer Ther. Zadorozhnyj, A.M., 1990. Spravocznik po lekarstvennim rastenijam. Lesnaja pro-
3, 513519. myshlennost, Moscow.
Khayyal, M.T., El-Ghazaly, M.A., Kenawy, S.A., Seifel-Nasr, M., Mahran, L.G., Kafa, Y. Zuo, G.Y., Meng, F.Y., Hao, X.Y., Zhang, Y.L., Wang, G.C., Xu, G.L., 2008. Antibacterial
A., Okpanyi, S.N., 2001. Antiulcerogenic effect of some gastrointestinally acting alkaloids from Chelidonium majus Linn (Papaveraceae) against clinical isolated
plant extracts and their combination. Arzneimittelforschung 51, 545553. of methicillin-resistant Staphylococcus aureus. J. Pharm. Pharm. Sci. 11, 9094.