title: Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: Identification of phenolic acids with cytotoxic potential.
abstract: 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; 24.8μg/ml) for 48h 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.
authors.affiliation: Cancer Drug Resistance Group, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Portugal; CIMO - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal; CEQUIMED-UP - Center of Medicinal Chemistry-University of Porto, Portugal; Health School, Polytechnic Institute of Bragança, Av. D. Afonso V, 5300-121 Bragança, Portugal; Department of Biological Sciences, Laboratory of Microbiology, Faculty of Pharmacy, University of Porto, Portugal.
authors.forename: Josiana A
authors.initials: JA
authors.lastname: Vaz
authors.affiliation: Cancer Drug Resistance Group, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Portugal.
authors.forename: Gabriela M
authors.initials: GM
authors.lastname: Almeida
authors.affiliation: CIMO - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal. Electronic address: iferreira@ipb.pt.
authors.forename: Isabel C F R
authors.initials: IC
authors.lastname: Ferreira
authors.affiliation: CIMO - Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal.
authors.forename: Anabela
authors.initials: A
authors.lastname: Martins
authors.affiliation: Cancer Drug Resistance Group, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Portugal; Department of Biological Sciences, Laboratory of Microbiology, Faculty of Pharmacy, University of Porto, Portugal.
authors.forename: M Helena
authors.initials: MH
authors.lastname: Vasconcelos
mesh_terms: D000363:Agaricales / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects*; D001688:Biological Products / Q000737:chemistry*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000401:mortality; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003602:Cytotoxicity, Immunologic / Q000235:genetics*; D006801:Humans; D062385:Hydroxybenzoates / Q000737:chemistry*; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000401:mortality; D015996:Survival Rate
chemical_list: D001688:Biological Products; D062385:Hydroxybenzoates; D053148:Caspase 3; C017616:phenolic acid
keywords: Apoptosis; Cell cycle; Citotoxicity; Clitocybe alexandri; Phenolic acids