The plant material was collected at Tomoros (ca. 1700 altitude), mountain Galičica (Macedonia) during the flowering (17 July 2010) and identified by Vlado Matevski, Institute of Biology, Faculty of Natural Sciences and Mathematics,http://Ss.CyrilandMethodiusUniversityofSkopje, where the voucher specimen is deposited at Macedonian National Herbarium (MKNH) under the number MKNH121335.
Air-dried and powdered aerial parts of Helichrysum zivojinii (330 g) were extracted twice with n-hexane in an ultrasonic bath for 45 min. The combined extracts were concentrated in a vacuum to obtain a hexane extract (4.2 g). The plant material was successively extracted in the same manner with solvents of rising polarity to obtain a dichloromethane extract (1.4 g), an ethyl-acetate extract (0.7 g), a n-butanol extract (5.4 g) and finally a methanol extract (12.4 g).
Stock solutions of the investigated extracts were made in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml.
Instrumentation and chromatographic conditions
1HNMR spectra were recorded with Varian Gemini 200 in CDCl3 and DMSO-d6 with TMS as an internal standard. HPLC-MS analysis was performed with an Agilent 1100 Series chromatography system equipped with a binary pump, degasser, autosampler, column Li Chrospher 100 RP 18 (250 × 4,0 mm i.d. 5 μm), and DAD detector in combination with 6210 Time of Flight MS (Agilent Technologies). The mobile phase consisted of 0.2% formic acid in water (solvent A) and 100% acetonitrile (solvent B) with the following gradient elution: 0–5 min 10–20% B, 5–10 min 20% B, 10–20 min 20–30% B, 20–30 min 30–70% B, 30–35 min 70–100% B, 35–40 min 70% B, 40–41 min 100–10% B, 41–45 min 10% B, at a flow rate of 1 ml/min. The injection volume was 10 μL, the column temperature was 25°C. The effluent was monitored with DAD (190–550 nm) and a mass detector (ESI) which operated in negative mode at atmospheric pressure; the mass range was from m/z 100–2500, with the following ESI parameters: capillary voltage: 4000 V; gas temperature: 350°C; nebulizer pressure: 45 psig; fragmentor voltage: 140 V. Mass Hunter Workstation software was used for data analysis.
Human cervix adenocarcinoma HeLa, human melanoma Fem-x and human breast adenocarcinoma MDA-MB-361 cells were cultured as monolayers. Human chronic myelogenous leukemia K562 cells were grown in a suspension in nutrient medium. Cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The complete nutrient medium was RPMI 1640 supplemented with 3 mM L-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 10% heat-inactivated (56°C) fetal bovine serum and 25 mM Hepes adjusted to pH 7.2 with a bicarbonate solution. The cells were grown at 37°C in an atmosphere of 5% CO2 and humidified air. RPMI 1640, L-glutamine and Hepes were obtained from PAA (Pasching, Austria).
Preparation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were separated from whole heparinized blood of two healthy volunteers by Lymphoprep (Oslo, Norway) gradient centrifugation. Interface cells were washed three times with Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca+, 145 mM Cl- and 35 g/l gelatin polymers, pH 7.4), counted and resuspended in nutrient medium. The protocol of the study was approved by the Ethics Committee of the Institute of Oncology and Radiology of Serbia. Written informed consent was obtained from each healthy donor.
Treatment of cancer cell lines
HeLa (2,000 cells per well), Fem-x (2,000 cells per well), MDA-MB-361 (10,000 cells per well) were seeded into 96-well microtiter plates and 20 h later, after cell adherence, five different concentrations of the tested extracts were added to the wells. Nutrient medium was only added to the cells in the control wells. K562 cells (5,000 cells per well) were seeded 2 h before addition of the extracts. Stock solutions of plant extracts were diluted with complete nutrient medium and applied to target cells at different final concentrations that ranged from 6.25 μg/ml to 100 μg/ml for extracts 1–4, and from 12.5 μg/ml to 150 μg/ml or 200 μg/ml for extract 5. All experiments were done in triplicate. Cisplatin was used as a positive control.
Treatment of PBMC
PBMC (150,000 cells per well) were seeded into nutrient medium or in nutrient medium enriched with (5 μg/ml) (PHA) in 96-well microtiter plates. After 2 h, five different concentrations of the plant extracts were added to the individual wells, in triplicate, except to the control wells where a nutrient medium only was added to the cells. The final concentrations of the tested extracts ranged from 12.5 μg/ml to 200 μg/ml. PHA was obtained from INEP (Belgrade, Serbia). Cisplatin was used as a positive control.
Determination of target cell survival
Cell survival was determined by the MTT test according to the method of Mosmann and modified by Ohno and Abe. Briefly, after the treatment with plant extracts for 72 h, 10 μl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide) was added to each well. Samples were incubated for a further 4 h, followed by the addition of 100 μl of 10% SDS. Absorbance at 570 nm was measured the next day.
To quantify cell survival (S%), the absorbance of a sample with cells grown in the presence of different concentrations of the investigated agents was divided by the absorbance of the control cells grown only in the nutrient medium, and multiplied by 100. It is implied that the absorbance of the blank was always subtracted from the absorbance of the corresponding sample with target cells. The IC50 was defined as the concentration of the agent that inhibited cell survival by 50%, compared to the vehicle-treated control.
Morphological evaluation of HeLa cell death
To evaluate whether the extracts from the endemic plant Helichrysum zivojinii induce apoptosis in HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed. HeLa cells were seeded overnight on coverslips (100,000 cells) in 2 ml of complete medium. The next day, cells were treated with plant extracts for 24 h at concentrations corresponding to IC90 values that were obtained after treatments that lasted 72 h. After this period, the target cells were stained with 18 μl of a mixture of the DNA dyes acridine orange and ethidium bromide (3 μg/ml AO and 10 μg/ml EB in PBS), and visualized under a fluorescence microscope using a fluorescein isothiocyanate (FITC) filter set.
Cell cycle analysis
HeLa cells were incubated in the presence of two different concentrations (corresponding to the IC50 and IC90 values determined after 72 h) of the examined Helichrysum zivojinii extracts for 24, 48 and 72 h. After these incubation times, the target cells were collected, washed and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before staining. HeLa cells were washed in PBS, resuspended in 500 μl of staining solution (PBS containing RNAse A at a final concentration of 200 μg/ml, and propidium iodide (PI) at a final concentration of 20 μg/ml), and incubated for 30 min at 37°C.
Cell cycle phase distribution was determined using a FACSCalibur Flow Cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (10,000 events collected for each sample) were analyzed using CELLQuest software (BD Biosciences).
Determination of target caspases
To identify the caspases involved in the apoptotic cell death pathway induced by the investigated extracts, the percentages of HeLa cells pretreated with caspase inhibitors in the subG1 phase were determined. HeLa cells were preincubated for 2 h with specific caspase inhibitors (at a final concentration of 40 μM). These were: Z-DEVD-FMK, a caspase-3 inhibitor, Z-IETD-FMK, a caspase-8 inhibitor and Z-LEHD-FMK, a caspase-9 inhibitor. Caspase inhibitors were purchased from R&D Systems (Minneapolis, USA). Five tested extracts were applied to the HeLa cells at concentrations that corresponded to the IC90 values obtained after 72 h. For each extract, one sample of HeLa cells was not treated with an inhibitor and served as a reference sample. After 24 h of incubation, cells were harvested and fixed in 70% ethanol on ice. Samples were stored at −20°C for one week before PI staining. Changes in the percentages of cells in the subG1 phase were determined using a FACSCalibur Flow Cytometer and analyzed using CELLQuest software.