Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: Identification of phenolic acids with cytotoxic potential

Mushrooms are a possible rich source of biologically active compounds with the potential for drug discovery. The aim of this work was to gain further insight into the cytotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterised regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the C. alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI₅₀ concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 μg/ml) for 48 h caused an increase in the levels of wt. p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, evidence was found for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.