Matić et al. BMC Complementary and Alternative Medicine 2013, 13:36
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RESEARCH ARTICLE Open Access
In vitro antitumor actions of extracts from
endemic plant Helichrysum zivojinii
Ivana Z Matić1, Ivana Aljan%0ńić2, %7ńeljko %7ńi~
ak1, Vlatka Vajs2, Milka Jadranin2, Slobodan Milosavljević3
and Zorica D Juranić1*
Abstract
Background: The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of
five extracts isolated from the endemic plant species Helichrysum zivojinii 
ernjavski & Soa
ka (family Asteraceae)
against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in
the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood
mononuclear cells (PBMC).
Methods: The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of
increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The
cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human
myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the
MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of
HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the
tested extracts were determined using specific caspase inhibitors.
Results: The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against
selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic
actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger
cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth
noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated
PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both
intrinsic and extrinsic signaling pathways.
Conclusion: Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of
novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.
Keywords: Helichrysum zivojinii, Cytotoxicity, Cancer cells, Peripheral blood mononuclear cells, Apoptosis
Background implicated in cell growth, replication, apoptosis, as well
Bioactive constituents of medicinal plants are in the cen- as in angiogenesis, invasion and metastasis of cancer
ter of attention of modern anticancer research due to cells [1-4]. To evaluate the anticancer properties of novel
their prospective roles in suppressing the different stages chemotherapeutic agents, the selectivity of their actions
of malignant transformation. The antitumor potential of against malignant cells in comparison to healthy non-
plant extracts and compounds could be attributed to transformed cells, especially immunocompetent cells
their ability to induce changes in the regulation of target involved in the immune control of tumor suppression,
molecules in oncogenic signal transduction pathways needs to be carefully examined.
Helichrysum zivojinii 
ernjavski & Soa
ka is an en-
demic plant species that grows in the National Park
* Correspondence: juranicz@ncrc.ac.rs
1 "Gali%0Å„ica" in Macedonia. Some of the plant species from
Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000,
the large genus Helichrysum are used in different regions
Belgrade, Serbia
Full list of author information is available at the end of the article
© 2013 Matić et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Matić et al. BMC Complementary and Alternative Medicine 2013, 13:36 Page 2 of 12
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of the world in traditional medicine for treating wounds, Stock solutions of the investigated extracts were
respiratory tract infections and gastro-intestinal disorders made in dimethyl sulfoxide (DMSO) at a concentration
[5-8]. This plant genus is a valuable source of several of 5 mg/ml.
different secondary metabolites/phytochemicals, such
as flavonoids, acetophenones, phloroglucinols, pyrones, Instrumentation and chromatographic conditions
1
diterpenes and sesquiterpenes [5]. Different morpho- HNMR spectra were recorded with Varian Gemini 200 in
logical groups of Helichrysum species often display unique CDCl3 and DMSO-d6 with TMS as an internal standard.
qualitative and quantitative chemical compositions [5]. It HPLC-MS analysis was performed with an Agilent 1100
has been reported that extracts and individual consti- Series chromatography system equipped with a binary
tuents of these plants possess significant biological and pump, degasser, autosampler, column Li Chrospher 100
pharmacological properties, including antibacterial, anti- RP 18 (250 × 4,0 mm i.d. 5 źm), and DAD detector in
viral, antifungal, antioxidant, anti-inflammatory and anti- combination with 6210 Time of Flight MS (Agilent Tech-
diabetic activities [9-16]. A search through the literature nologies). The mobile phase consisted of 0.2% formic
suggests that plants from the genus Helichrysum could be acid in water (solvent A) and 100% acetonitrile (solvent
a significant source of compounds with potential anti- B) with the following gradient elution: 0 5 min 10 20%
cancer activities [17-20]. B, 5 10 min 20% B, 10 20 min 20 30% B, 20 30 min
The main goal of this research was to investigate the 30 70% B, 30 35 min 70 100% B, 35 40 min 70% B,
cytotoxic activities of five extracts isolated as fractions 40 41 min 100 10% B, 41 45 min 10% B, at a flow rate
from the endemic plant Helichrysum zivojinii towards of 1 ml/min. The injection volume was 10 źL, the col-
selected human malignant cell lines. To assess the sensi- umn temperature was 25°C. The effluent was monitored
tivity of normal immunocompetent cells included in the with DAD (190 550 nm) and a mass detector (ESI)
antitumor immune response, the cytotoxicity of these which operated in negative mode at atmospheric pres-
extracts was also tested against human peripheral blood sure; the mass range was from m/z 100 2500, with the
mononuclear cells (PBMC)  both unstimulated and following ESI parameters: capillary voltage: 4000 V; gas
stimulated to proliferate by the mitogen phytohemagglu- temperature: 350°C; nebulizer pressure: 45 psig; fragmentor
tinin (PHA). To elucidate the molecular mechanisms of voltage: 140 V. Mass Hunter Workstation software was
the cytotoxic effects of the tested extracts, the distribu- used for data analysis.
tion of target HeLa cells at specific phases of the cell
cycle after the actions of these agents was also analyzed. Cell culture
The mode of HeLa cell death induced by the extracts Human cervix adenocarcinoma HeLa, human melan-
was also investigated. Elucidation of the signaling path- oma Fem-x and human breast adenocarcinoma MDA-
ways implicated in the induction of apoptosis by the MB-361 cells were cultured as monolayers. Human
tested extracts was conducted by identification of target chronic myelogenous leukemia K562 cells were grown
caspases. in a suspension in nutrient medium. Cancer cell lines
were obtained from the American Type Culture Collection
Methods (Manassas, VA, USA). The complete nutrient medium
Plant extracts was RPMI 1640 supplemented with 3 mM L-glutamine,
The plant material was collected at Tomoros (ca. 1700 100 źg/ml streptomycin, 100 IU/ml penicillin, 10% heat-
altitude), mountain Gali%0Å„ica (Macedonia) during the inactivated (56°C) fetal bovine serum and 25 mM Hepes
flowering (17 July 2010) and identified by Vlado adjusted to pH 7.2 with a bicarbonate solution. The cells
Matevski, Institute of Biology, Faculty of Natural were grown at 37°C in an atmosphere of 5% CO2 and
Sciences and Mathematics, Ss. Cyril and Methodius humidified air. RPMI 1640, L-glutamine and Hepes were
University of Skopje, where the voucher specimen is obtained from PAA (Pasching, Austria).
deposited at Macedonian National Herbarium (MKNH)
under the number MKNH121335. Preparation of peripheral blood mononuclear cells
Air-dried and powdered aerial parts of Helichrysum Peripheral blood mononuclear cells (PBMC) were se-
zivojinii (330 g) were extracted twice with n-hexane in an parated from whole heparinized blood of two healthy
ultrasonic bath for 45 min. The combined extracts were volunteers by Lymphoprep (Oslo, Norway) gradient cen-
concentrated in a vacuum to obtain a hexane extract trifugation. Interface cells were washed three times with
(4.2 g). The plant material was successively extracted in Haemaccel (aqueous solution supplemented with 145 mM
the same manner with solvents of rising polarity to obtain Na+, 5.1 mMK+, 6.2 mMCa+, 145 mMCl- and 35 g/l gel-
a dichloromethane extract (1.4 g), an ethyl-acetate extract atin polymers, pH 7.4), counted and resuspended in nutri-
(0.7 g), a n-butanol extract (5.4 g) and finally a methanol ent medium. The protocol of the study was approved by
extract (12.4 g). the Ethics Committee of the Institute of Oncology and
Matić et al. BMC Complementary and Alternative Medicine 2013, 13:36 Page 3 of 12
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Radiology of Serbia. Written informed consent was ob- was performed. HeLa cells were seeded overnight on
tained from each healthy donor. coverslips (100,000 cells) in 2 ml of complete medium.
The next day, cells were treated with plant extracts for
Treatment of cancer cell lines 24 h at concentrations corresponding to IC90 values that
HeLa (2,000 cells per well), Fem-x (2,000 cells per well), were obtained after treatments that lasted 72 h. After
MDA-MB-361 (10,000 cells per well) were seeded into this period, the target cells were stained with 18 źl of a
96-well microtiter plates and 20 h later, after cell adherence, mixture of the DNA dyes acridine orange and ethidium
five different concentrations of the tested extracts were bromide (3 źg/ml AO and 10 źg/ml EB in PBS), and
added to the wells. Nutrient medium was only added to the visualized under a fluorescence microscope using a
cells in the control wells. K562 cells (5,000 cells per well) fluorescein isothiocyanate (FITC) filter set.
were seeded 2 h before addition of the extracts. Stock
solutions of plant extracts were diluted with complete nu-
Cell cycle analysis
trient medium and applied to target cells at different final
HeLa cells were incubated in the presence of two different
concentrations that ranged from 6.25 źg/ml to 100 źg/ml
concentrations (corresponding to the IC50 and IC90 values
for extracts 1 4, and from 12.5 źg/ml to 150 źg/ml or
200 źg/ml for extract 5. All experiments were done in trip- determined after 72 h) of the examined Helichrysum
zivojinii extracts for 24, 48 and 72 h. After these incuba-
licate. Cisplatin was used as a positive control.
tion times, the target cells were collected, washed and
Treatment of PBMC
PBMC (150,000 cells per well) were seeded into nutrient
Table 1 Components of five Helichrysum zivojinii extracts
medium or in nutrient medium enriched with (5 źg/ml)
Compounds Extracts
(PHA) in 96-well microtiter plates. After 2 h, five different
Hexane CH2Cl2 EtOAc BuOH MeOH
concentrations of the plant extracts were added to the in-
(1) (2) (3) (4) (5)
dividual wells, in triplicate, except to the control wells
1. C8H6O4 (166)  + + + +
where a nutrient medium only was added to the cells. The Phtalic acid
final concentrations of the tested extracts ranged from
2. C21H20O12 (464)   + + +
O glc or O gal of
12.5 źg/ml to 200 źg/ml. PHA was obtained from INEP
quercetin
(Belgrade, Serbia). Cisplatin was used as a positive control.
3. C25H24O12(516)   + + ++a
chlorogenic acids
Determination of target cell survival
4. C21H20O11(448)   + ++a +
Cell survival was determined by the MTT test according to
O glc of apigenin
the method of Mosmann [21] and modified by Ohno and
5. C21H20O10(432)   + + +
Abe [22]. Briefly, after the treatment with plant extracts for
O glc of kaempferol
72 h, 10 źl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-
or luteolin
2,5-dyphenyl tetrazolium bromide) was added to each well.
6. C21H20O11(448)   + + +
Samples were incubated for a further 4 h, followed by the
O glc of apigenin
addition of 100 źl of 10% SDS. Absorbance at 570 nm was
7. C19H30O14 or + +   
measured the next day. C26H26O9 (482)
To quantify cell survival (S%), the absorbance of a
8. C15H10O6(286)   + + +
O glc of flavonols
sample with cells grown in the presence of different
kaempferol,
concentrations of the investigated agents was divided by
luteolin or
the absorbance of the control cells grown only in the nu-
6-hydroxyapigenin
trient medium, and multiplied by 100. It is implied that
9. C21H24O9 or + +   
the absorbance of the blank was always subtracted from
C14H28O14 (420)
the absorbance of the corresponding sample with target
10. C15H12O5 (272)  + + + +
cells. The IC50 was defined as the concentration of the flavanone
naringenin
agent that inhibited cell survival by 50%, compared to
11. C22H26O9 or + +   
the vehicle-treated control.
C15H30O14 (434)
12. C15H10O5 (270)  + ++a + +
Morphological evaluation of HeLa cell death
apigenin
To evaluate whether the extracts from the endemic plant
13. C21H18O4 (334) +    
Helichrysum zivojinii induce apoptosis in HeLa cells,
14. C18H16O7 (344) +    
morphological analysis by microscopic examination of
a
acridine orange/ethidium bromide-stained target cells More abundant on the expense of other fraction constituents.
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Figure 1 Survival of HeLa (A), Fem-x (B), K562 (C) and MDA-MB-361 cells (D) grown for 72 h in the presence of increasing
concentrations of Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
fixed in 70% ethanol on ice. Samples were stored at -20°C inhibitors in the subG1 phase were determined. HeLa
for one week before staining. HeLa cells were washed in cells were preincubated for 2 h with specific caspase
PBS, resuspended in 500 źl of staining solution (PBS inhibitors (at a final concentration of 40 źM). These were:
containing RNAse A at a final concentration of 200 źg/ Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a
ml, and propidium iodide (PI) at a final concentration of caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibi-
20 źg/ml), and incubated for 30 min at 37°C. tor. Caspase inhibitors were purchased from R&D Systems
Cell cycle phase distribution was determined using a (Minneapolis, USA). Five tested extracts were applied to
FACSCalibur Flow Cytometer (BD Biosciences Franklin the HeLa cells at concentrations that corresponded to the
Lakes, NJ, USA). The data (10,000 events collected for IC90 values obtained after 72 h. For each extract, one sam-
each sample) were analyzed using CELLQuest software ple of HeLa cells was not treated with an inhibitor and
(BD Biosciences). served as a reference sample. After 24 h of incubation,
cells were harvested and fixed in 70% ethanol on ice.
Determination of target caspases Samples were stored at -20°C for one week before PI
To identify the caspases involved in the apoptotic cell staining. Changes in the percentages of cells in the subG1
death pathway induced by the investigated extracts, phase were determined using a FACSCalibur Flow
the percentages of HeLa cells pretreated with caspase Cytometer and analyzed using CELLQuest software.
Table 2 Concentrations of five Helichrysum zivojinii extracts, which induced 50% decrease in the target cancer cell
survival, determined by MTT test
HeLa Fem-x K562 MDA-MB-361
Extract 1 IC50 [źg/ml] 24.63 ą 4.12 28.85 ą 5.49 11.78 ą 0.94 81.74 ą 6.27
Extract 2 IC50 [źg/ml] 20.11 ą 4.49 23.64 ą 1.41 23.82 ą 6.54 81.74 ą 13.31
Extract 3 IC50 [źg/ml] 37.98 ą 2.33 47.04 ą 4.79 27.52 ą 4.96 79.93 ą 13.49
Extract 4 IC50 [źg/ml] 56.70 ą 6.05 74.84 ą 7.55 50.37 ą 3.28 69.96 ą 11.70
Extract 5 IC50 [źg/ml] 84.68 ą 10.39 77.29 ą 6.55 74.88 ą 7.57 94.92 ą 6.85
Cisplatin IC50 [źM] 5.60 ą 1.41 5.02 ą 0.59 5.35 ą 0.70 28.23 ą 5.04
Time of continuous agent s action was 72 h.
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Figure 2 Survival of resting PBMC (A) and PHA-stimulated PBMC (B) grown for 72 h in the presence of increasing concentrations of
Helichrysum zivojinii extracts, determined by MTT test. Representative graphs are shown.
Results (or possibly luteolin), and another glucoside of kaempferol
Chemical analysis of plant extracts (possibly luteolin or 6-hydroxyapigenin), were present. Fla-
The hexane (1) and dichloromethane extracts (2) vonoid aglycones, such as apigenin and naringenin, were
1
presented similar H NMR spectra (recorded in CDCl3). detected. Phthalic acid was found in extracts 2 5.
1
The signals in the H NMR spectra of both extracts
pointed to a complex mixture with prevailing relatively In vitro cytotoxic activity
non-polar substances (region ´ 0.8 2.2). LC/DAD ana- The cytotoxicity of the five isolated extracts was tested
lysis revealed the presence of a flavonoid with the mo- against selected cancer cell lines: human cervix ade-
lecular formula C18H16O7 (344) in the hexane extract, nocarcinoma HeLa, human melanoma Fem-x, human
while the flavanone naringenin and flavone apigenin myelogenous leukemia K562 and human breast adeno-
were confirmed in the dichloromethane extract, also by carcinoma MDA-MB-361 cells. All investigated extracts
LC/DAD analysis. Three compounds found in both exerted selective dose-dependent cytotoxic actions on
extracts are presented in Table 1, with their presumed mo- malignant cells. The decrease in survival of target cancer
lecular formulae and measured m/z values corresponding cells induced by the five Helichrysum zivojinii extracts is
to identified ions obtained by ESI ToF mass spectrometry. shown in Figure 1 and Table 2.
According to LC/DAD analysis, these compounds unfortu- In general, extracts 1 and 2 (as well as cisplatin, which
nately did not absorb in the UV spectrum, suggesting the served as a positive control) exhibited the highest cyto-
presence of structures without a chromophore. The ethyl- toxic actions against target malignant cell lines; extracts
acetate extract (3) and n-butanol extracts (4) presented 3 and 4 displayed less pronounced cytotoxicity; extract 5
1
H NMR spectra (recorded in DMSO-d6) with approxi- had the lowest cytotoxic action.
mately identical groups of signals in the regions ´ 12.5 With regard to the specific sensitivities of the different
13.9 (hydroxy protons), ´ 5.9-8.1 (protons on the aromatic cells to the cytotoxic activities of the extracts, it is im-
ring that is substituted with one or more hydroxy groups), portant to note that K562 cells were the most sensitive
and ´ 4.7 5.5 (group of protons corresponding to a sugar to the cytotoxic actions of extracts 1 and 3. HeLa and
1
unit). The H NMR spectrum of the most abundant metha- Fem-x cells exhibited a lower sensitivity, while the sensi-
nol extract (5), also recorded in DMSO-d6, resembles the tivity of breast cancer MDA-MB-361 cells to the toxic
spectra of the ethyl-acetate and n-butanol extracts by the
presence of signals in the region ´ 5.9 8.1 (aromatic
Table 3 Concentrations of five Helichrysum zivojinii
protons), as well as by the presence of a signal at ´ 13.9 extracts, which induced 50% decrease in target PBMC
survival, determined by MTT test
(hydroxy proton); it differs from the last two spectra by
possessing a more pronouncedregionat ´ 4.7 5.5, which is PBMC PBMC + PHA
responsible for the protons of sugar units. LC/DAD
Extract 1 IC50 [źg/ml] 131.59 ą 9.93 99.07 ą 8.02
analysis and ESI ToF mass spectrometry of these three
Extract 2 IC50 [źg/ml] 81.74 ą 0.50 67.65 ą 11.37
more polar extracts (3, 4 and 5) revealed quite similar
Extract 3 IC50 [źg/ml] 74.82 ą 6.51 61.86 ą 5.53
constituents in each of them. Apart from chlorogenic acid,
Extract 4 IC50 [źg/ml] 185.17 92.20 ą 9.01
two groups of flavonoid compounds were detected: flavon-
Extract 5 IC50 [źg/ml] > 200 128.12 ą 35.46
oid O-glycosides, and flavonoid aglycons. Among the O-
glycosides, the glucoside (or possibly galactoside) of quer- Cisplatin IC50 [źM] > 33.34 > 33.34
cetin, the glucoside of apigenin, the glucoside of kaempferol Time of continuous agent s action was 72 h.
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Table 4 Selectivity in the antitumor action of five actions of the tested extracts was the lowest (being
Helichrysum zivojinii extracts
several times lower than that of the other cell lines to
Selectivity IC50PBMC/ IC50PBMC IC50PBMC/ IC50PBMC extracts 1, 2 and 3 especially).
coefficient in the IC50 HeLa + PHA/ IC50 K562 + PHA/
Considering the possible effects of applied antitumor
antitumor action IC50 HeLa IC50 K562
drugs on normal healthy immunocompetent cells, com-
Extract 1 5.34 4.02 11.17 8.41
ponents of the antitumor immune response, their viabil-
Extract 2 4.06 3.36 3.43 2.84
ity is significant for tumor control. For that reason, the
Extract 3 1.97 1.63 2.78 2.25
activities of the investigated Helichrysum zivojinii ex-
tracts were evaluated against healthy unstimulated and
Extract 4 3.27 1.63 3.68 1.83
PHA-stimulated PBMC (Figure 2 and Table 3). It should
Extract 5 > 2.36 1.51 2.67 1.71
be noted that these extracts overall exhibited weaker cyto-
toxic effects against unstimulated PBMC than against
stimulated PBMC. Moreover, extracts 2 and 3 exerted a
more pronounced cytotoxicity against unstimulated and
PHA-stimulated PBMC than extracts 1, 4 and 5.
Figure 3 Photomicrographs of acridine orange/ethidium bromide-stained control HeLa cells (A), and HeLa cells treated with different
Helichrysum zivojinii extracts for 24 h (extracts 1 5, photomicrographs B-F, consecutively).
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Figure 4 Changes in the cell cycle phase distribution of HeLa cells induced by the Helichrysum zivojinii extracts after 24 (A,B), 48 (C,D)
and 72 h (E) treatment (applied concentrations of tested extracts corresponded to IC50 and IC90 values determined for 72 h). (C- control
HeLa cells, 1  5  corresponding extracts are numbered consecutively). Representative graphs are shown.
In order to further evaluate the anticancer potential of apoptosis in target HeLa cells after 24 h treatment
the extracts, the selectivity in the antitumor action (Figure 3). The morphological characteristics of apoptotic
against specific malignant cell line in comparison to cell death, such as cell shrinkage, condensation and even
healthy PBMC was determined as well. These data are fragmentation of nucleus, as well as the presence of
presented in Table 4 from which it can be observed that orange-red stained cells at late stages of apoptosis or sec-
extract 1 exhibited highly selective antitumor action, es- ondary necrosis (the latter was observed in extract 1-
pecially against K562 cells. Extract 2 also displayed good treated HeLa cells) and apoptotic bodies. These analyses
selectivity in its antitumor action. confirmed that the cytotoxicity of the Helichrysum
zivojinii extracts is based on their prominent pro-
apoptotic effects.
Morphological analysis of HeLa cell death mode
In order to determine whether the investigated plant
extracts have pro-apoptotic activities, we performed mor- Analysis of changes in cell cycle phase distribution
phological analysis by fluorescent microscopy of acridine Examination of changes in the cell cycle phase distribu-
orange/ethidium bromide-stained HeLa cells, exposed to tion of HeLa cells treated with these extracts for 24, 48
the extracts. Microscopic examination revealed that all and 72 h was done to elucidate the mechanisms of the
five extracts applied at IC90 concentrations induced observed cytotoxic actions (Figure 4). Results from this
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Figure 5 Effects of specific caspase inhibitors on the percentages of apoptotic subG1 HeLa cells treated with Helichrysum zivojinii
extracts for 24 h (A - extract 1, B - extract 2, C - extract 3, D - extract 4, E - extract 5). (Z-DEVD-FMK - caspase-3 inhibitor; Z-IETD-FMK -
caspase-8 inhibitor; Z-LEHD-FMK - caspase-9 inhibitor).
analysis showed a time - dependent increase in the per- Discussion
centages of HeLa cells in the subG1 phase after exposure The plant kingdom provides a rich source of compounds
to an IC50 concentration of all of the tested extracts. Add- with promising cancer chemopreventive and cancer thera-
itionally, exposure to extracts at IC90 concentrations peutic potential. The main drugs currently used in clinical
induced significant increases in the percentages of cells practice in the treatment of malignant diseases originate
in the subG1 phase 24 h after exposure. It should be from plants: vinca alkaloids, taxanes, camptothecins and
mentioned that the investigated extracts induced a slight epipodophyllotoxins [23]. Over the past years, the focus of
accumulation of HeLa cells in the S phase after 72 h. modern anticancer drug discovery has been on a wide
Examination of the cell cycle changes that were induced variety of natural compounds, especially on phenolic
after exposure for 72 h to IC90 for each extract was not compounds. Phytochemicals have been reported to affect
performed because at this time point and at this extract different intracellular signaling pathways implicated in
concentration low numbers of mostly dead or dying cells the initiation, promotion and progression of cancer.
were present in the sample. The antitumor effects of plant constituents have been
Since the Helichrysum zivojinii extracts exhibited the associated with the induction of carcinogen detoxifying
pro-apoptotic activities against cervix adenocarcinoma enzymes, the scavenging of free radicals, anti-inflammatory
HeLa cells, the identification of target caspases involved activity, cell cycle arrest, the triggering of apoptosis, inhib-
in the apoptotic pathway was performed. The presence ition of tumor angiogenesis and invasiveness [1-4].
of the specific caspase inhibitors (caspase-3 inhibitor, The antioxidant and anti-inflammatory activities of
caspase-8 inhibitor or caspase-9 inhibitor) significantly extracts and isolated compounds from plants belonging to
reduced the percentages of apoptotic subG1 HeLa cells the large genus Helichrysum have been well documented
treated with each of the five plant extracts, as shown in [9,11,15,18]. Arzanol, a phloroglucinyl Ä…-pyrone, is a con-
Figure 5. The effect of the caspase-3 inhibitor on HeLa stituent of Helichrysum italicum that has been reported to
cells treated with extracts is shown in Figure 6. It can be inhibit the NF-ºB transcription factor, cyclooxygenase and
seen that there is an increase in rounded, but attached lipooxygenase, as well as the release of proinflammatory
and live HeLa cells treated with the caspase-3 inhibitor cytokines [11,18]. Regarding the link between inflam-
before the addition of the extracts in relation to target mation and cancer, chemicals with anti-inflammatory
HeLa cells only exposed to the tested extracts. properties targeting the molecules of signaling cascades
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Figure 6 (See legend on next page.)
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(See figure on previous page.)
Figure 6 Effects of pretreatment of HeLa cells with caspase-3 inhibitor (Z-DEVD-FMK), exposed to Helichrysum zivojinii extracts
(applied concentrations of tested extracts corresponded to IC90 values determined for 72 h). A  control; B  Extract 1, C  Extract 1 + Z-
DEVD-FMK; D  Extract 2, E  Extract 2 + Z-DEVD-FMK; F  Extract 3, G  Extract 3 + Z-DEVD-FMK; H  Extract 4, I  Extract 4 + Z-DEVD-FMK;
J  Extract 5, K  Extract 5 + Z-DEVD-FMK.
implicated in inflammation and carcinogenesis may be comparison to stimulated PBMC. This finding indicates
useful as cancer chemopreventive drugs. that the extracts possess the ability to inhibit the proli-
On the other hand, data about the potential anticancer feration of PHA-stimulated PBMC. Thus, these agents
activity of extracts and phytochemicals of plants from the may even suppress certain immune functions, particu-
genus Helichrysum are scarce. The antiproliferative effect larly non-specific antigen stimulation. Additionally, the
of the ethanol extract from Helichrysum maracandicum observed lower activities against resting PBMC than
towards SENCAR mouse skin transformed cells has been against mitogen-stimulated PBMC point to components
demonstrated [20]. This extract suppressed the expres- in pathways regulating cell proliferation as the possible
sion of p38 MAP kinase. An examination of the arzanol molecular targets of the Helichrysum zivojinii extracts.
properties showed that this compound, which was isolated However, it is very important to note that when extracts 1,
from Helichrysum italicum, did not exert cytotoxic action 2 and 3 were applied at lower concentrations, they
against monkey VERO cells at concentrations up to stimulated the proliferation of resting PBMC. This growth
40 źM [24]. In contrast, another study showed that stimulation effect of lower concentrations of extracts is in
arzanol at a concentration of 50 źM significantly accordance with the well-known effect of very small doses
suppressed the survival of human lung carcinoma A549 of X rays on enhanced proliferation of irradiated cells [25].
cells [18]. It should be mentioned that methanolic extracts The observed effects of low concentrations of these
prepared from different Helichrysum species were found extracts on one of the main components of the immune
to inhibit DNA topoisomerase I [19]. Moreover, the cyto- response point to the possibility of their use to enhance
toxicity of Helichrysum gymnocephalum essential oil to- immunity. It would be interesting to investigate their ac-
wards human breast adenocarcinoma MCF-7 cells has tion towards different PBMC subpopulations and eluci-
been documented [17]. date the potential mechanisms through which they
The results presented herein demonstrate the selective stimulate proliferation. The possible immunostimulatory
dose-dependent cytotoxic actions of the five extracts effects of extracts at lower concentrations might be
isolated from the endemic plant species Helichrysum explained by a modulation in lymphocyte cytokine pro-
zivojinii against target cancer cell lines and against healthy duction, including IL-2, IFN-Å‚, as well as IL-4 and IL-6.
immunocompetent PBMC that have been stimulated to The immunoregulatory actions of phenolic compounds,
proliferate, while their cytotoxic actions were not as such as quercetin, kaempferol and apigenin, have been
pronounced against unstimulated PBMC. The observed reported [26-28]. Considering the presented results, the
selectivity in the antitumor effects of the extracts against effects of the extracts on PBMC might be mediated
specific malignant cell types could be attributed to the through NF-ºB.
actions of different Helichrysum zivojinii constituents on Examination of in vitro cytotoxicity revealed that extracts
target molecules of the signal transduction pathways that 1 and 2 might be a significant source of novel promising
regulate cell proliferation and apoptosis. Furthermore, anticancer compounds in view of their pronounced cyto-
each of the investigated extracts exhibited considerably toxic activities against HeLa, Fem-x and especially against
stronger cytotoxicity to HeLa, Fem-x and K562 cells when K562 cells, as well as their high selectivity in the antitumor
compared to PBMC, both resting and PHA-stimulated, actions against cancer cells in comparison to healthy
which points to the cancer specificity of their actions. It is PBMC. Chemical analyses of the Helichrysum zivojinii
noteworthy that when these extracts were applied at extracts showed the presence of phenolic compounds
concentrations that were highly cytotoxic to malignant whose antitumor potential has already been documented.
cells, they demonstrated very low toxicity towards healthy The bioactive flavone apigenin that was found in extracts
immunocompetent PBMC, the key players in immune 2 5 has been reported to exhibit anticancer activities
defenses against tumors. The good selectivity of their against different types of malignant cells including breast,
antitumor actions highlights the significant anticancer cervical, ovarian, prostate, colon, gastric, liver and lung
potential of Helichrysum zivojinii extracts. The promi- cancers, as well as skin and thyroid cancer, diverse
nent antitumor properties of these extracts need to be hematological malignancies and neuroblastoma [28]
examined further in in vivo studies. and references cited therein]. The cytotoxic activities of
It should be stressed that all of these extracts exhibited extracts 2 5 may be at least in part due to the flavonoid
weaker cytotoxic effects against unstimulated PBMC in naringenin. This flavonoid has been shown to exert
Matić et al. BMC Complementary and Alternative Medicine 2013, 13:36 Page 11 of 12
http://www.biomedcentral.com/1472-6882/13/36
cytotoxicity towards various malignant cell lines, such as Competing interests
The authors declare that they have no competing interests.
breast cancer cell lines (MCF-7, MDA-MB-231), cervix
adenocarcinoma (HeLa), liver cancer (HepG2, Hep3B,
Authors contributions
Huh7), pancreas cancer (PK-1), colon cancer (Caco-2),
IM performed all analyses of the anticancer properties of investigated
stomach cancer (KATOIII, MKN-7) and leukemia cells
extracts, interpreted obtained data and wrote the first and last version of the
(Jurkat, HL-60, U937, NALM-6, THP-1) [29-31]. Add-
manuscript. IA participated in design of the study, prepared extracts,
performed chemical characterization, interpreted data and wrote the part of
itionally, the cancer-preventive and cancer-suppressive
the manuscript. %7Å„%7Å„ participated in acquisition and analysis of data. MJ carried
properties of quercetin, whose O-glycosides were identi-
out chemical analyses of the extracts. VV and SM participated in design of
fied in extracts 3, 4 and 5, have been documented as well
the study and interpreted obtained data. ZJ designed the research on
anticancer properties of tested extracts, interpreted obtained data,
[32]. The antiproliferative and pro-apoptotic effects of
participated in writing the manuscript and critically revised the manuscript.
quercetin were shown against the HeLa cell line [33].
All authors have read and approved the final version of the manuscript.
Morphological analysis of the mode of HeLa cell death,
together with the cell cycle analysis, showed that the treat-
Acknowledgments
ment of HeLa cells with higher concentrations of the
The authors are grateful to the Ministry of Education, Science and
examined extracts induced apoptotic cell death. To con- Technological Development of the Republic of Serbia for the financial
support (Projects 175011 and 172053). Also the authors would like to thank
firm the pro-apoptotic action of the tested Helichrysum
Tatjana Petrović for her excellent technical assistance. For the supply of plant
zivojinii extracts and to identify the caspases implicated in
material, we thank National park Gali%0Å„ica, Ohrid, Macedonia. We thank
the employed apoptotic pathways, specific caspase inhibi- Andon Bojad~
i and Oliver Avramoski, National park Gali%0Å„ica.
tors were used (Z-DEVD-FMK, Z-IETD-FMK, Z-LEHD-
Author details
FMK). A prominent decrease in the percentages of subG1
1
Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000,
2
apoptotic HeLa cells after treatments with each of the
Belgrade, Serbia. Institute for Chemistry, Technology and Metallurgy,
3
University of Belgrade, Njegoa
eva 12, 11000, Belgrade, Serbia. Faculty of
tested extracts in combination with specific caspase inhi-
Chemistry, University of Belgrade, Studentski trg 16, 11000, Belgrade, Serbia.
bitors compared to the percentages of subG1 cells after
treatments with only the corresponding extracts, indicates
Received: 13 September 2012 Accepted: 12 February 2013
Published: 18 February 2013
that each of the five extracts induced apoptosis through
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human leukemia THP-1 cells. Food Chem Toxicol 2008, 46:3684 3690.
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Cite this article as: Matić et al.: In vitro antitumor actions of extracts
from endemic plant Helichrysum zivojinii. BMC Complementary and
Alternative Medicine 2013 13:36.
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