In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii

Abstract

BackgroundThe 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 Černjavski & Soška (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).MethodsThe 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.ResultsThe 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.ConclusionExtracts 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.

Evaluation of the anti-cancer potential of <i>Mahonia aquifolium</i> extracts via apoptosis and anti-angiogenesis

“…Then cells were stained with propidium iodide (40 μg/mL). Cell cycle phase distribution was assessed using a FACSCalibur flow cytometer (BD Biosciences Franklin Lakes, NJ, USA) and analysis was performed using CELLQuest software (BD Biosciences), on a 10,000 cells per each sample (Matić et al, 2013a). The results were obtained from three independent experiments and presented with standard deviations.…”

“…After incubation, the cells were collected by trypsinization, fixed in 70% ethanol on ice and stored at -20ºC for one week (Matić et al, 2013a). Before staining, the cells were washed and treated with RNAse A (100 μg/mL) at 37ºC for 30 min.…”

“…Somatic human umbilical vein endothelial (EA.hy926) cell line was grown in DMEM with high glucose (4.5 g/L). The conditions for cell culture growth were described previously (Matić et al, 2013a;Matić et al, 2013b). The cell lines used in the study were obtained from the American Type Culture Collection (USA).…”

“…The next day, the extracts and cisplatin, as a positive control, were added to cells at 2IC50 concentrations. After 24 hours of incubation, the cells were stained with a mixture of acridine orange and ethidium bromide dyes (3 μg/mL acridine orange and 10 μg/mL ethidium bromide in phosphate buffered saline (PBS), and visualized under a fluorescence microscope -Carl Zeiss PALM MicroBeam with Axio Observer.Z1 using AxioCam MRm (filters Alexa 488 and 568), as already described (Matić et al, 2013a;Živković et al, 2016).…”

“…HeLa, LS-174T, MRC-5 and EA.hy926 were seeded into 96-well microtiter plate (the seeding densities for each cell line were: 2000, 7000, 5000 and 5000 cells/well, respectively). 20 hours later five concentrations of extracts and berberine were added to the cells, except to the control cell samples, according to established procedure (Matić et al, 2013a;Matić et al, 2013b). Tested concentrations ranged from 12.5 to 200 μg/mL for extracts and from 2.1 to 67 μg/ mL for berberine.…”