Abstract
Helleborus cyclophyllus Boiss is a rhizomatous plant species, with strong allelochemical properties, that has been used since ancient times for its therapeutic properties. In the present study we investigated the ability of an aqueous-soluble fraction of the methanol extract of H. cyclophyllus Boiss leaves, to induce apoptotic cell death on A549 human bronchial epithelial adenocarcinoma cells. A primary human lung fibroblasts’ cell line was used as a model of normal-healthy cells for comparison. Cell morphology was examined after appropriate staining, cytotoxic activity of the extract was determined by the MTT assay, the type of cell death was analyzed by flow cytometry, confirmation of apoptosis was evaluated with the analysis of caspase-3, PARP1 by western blotting, while the chemical composition was assessed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). H. cyclophyllus Boiss extract was selectively active on A549 cells inducing significant morphological changes, even at low concentrations. Characteristic morphological alterations included the release of vesicular formations from A549 cell membranes (ectosomes), detachment of cells from their substrate, generation of a large vesicle into the cytoplasm (thanatosome) and the formation of apoptotic bodies. The selective apoptotic action on treated cells was also confirmed by biochemical criteria. Low concentrations, however, did not affect normal cells. The phytochemical analysis of the extract revealed the presence of cardiac glucosides, bufadienolides and phytoecdysteroids. To the best of our knowledge, the above-mentioned sequences of events leading selectively cancer cells to apoptosis, has not been reported before.
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Acknowledgements
The authors would like to acknowledge the support of this work by the project “An Open-Access Research Infrastructure of Chemical Biology and Target-Based Screening Technologies for Human and Animal Health, Agriculture and the Environment (OPENSCREEN-GR)” (MIS 5002691) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund). The authors would also like to thank the “Unit for characterization and testing of bioactive compounds” and the “Unit of high resolution analysis ORBITRAPLC-MSn” of the University of Ioannina for the access to the facilities. Acknowledgments are due to Dr D. Kletsas, Research Director in the National Center for Scientific Research “Democritos” for his kind remarks and the donation of primary human lung fibroblasts.
Funding
The work was supported by the project “An Open-Access Research Infrastructure of Chemical Biology and Target-Based Screening Technologies for Human and Animal Health, Agriculture and the Environment (OPENSCREEN-GR)” (MIS 5002691) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund).
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Yfanti, P., Karkabounas, A., Batistatou, A. et al. Study of potent cytotoxic activity of Helleborus cyclophyllus Boiss against a human adenocarcinoma cell line. Cytotechnology 72, 819–831 (2020). https://doi.org/10.1007/s10616-020-00425-4
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DOI: https://doi.org/10.1007/s10616-020-00425-4