Study of potent cytotoxic activity of Helleborus cyclophyllus Boiss against a human adenocarcinoma cell line

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.