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In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.

Matić IZ, Aljančić I, Žižak Ž, Vajs V, Jadranin M, Milosavljević S, Juranić ZD - BMC Complement Altern Med (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia.

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 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).

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.

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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 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).

Mentions: In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure 3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.


In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.

Matić IZ, Aljančić I, Žižak Ž, Vajs V, Jadranin M, Milosavljević S, Juranić ZD - BMC Complement Altern Med (2013)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585823&req=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).
Mentions: In order to determine whether the investigated plant extracts have pro-apoptotic activities, we performed morphological analysis by fluorescent microscopy of acridine orange/ethidium bromide-stained HeLa cells, exposed to the extracts. Microscopic examination revealed that all five extracts applied at IC90 concentrations induced apoptosis in target HeLa cells after 24 h treatment (Figure 3). The morphological characteristics of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange-red stained cells at late stages of apoptosis or secondary necrosis (the latter was observed in extract 1-treated HeLa cells) and apoptotic bodies. These analyses confirmed that the cytotoxicity of the Helichrysum zivojinii extracts is based on their prominent pro-apoptotic effects.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia.

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 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).

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.

Show MeSH
Related in: MedlinePlus