title: Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells.
abstract: Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: Villanueva-Paz
authors.affiliation: Facultad de Odontología, Universidad de Sevilla, Sevilla, Spain.
authors.forename: Mario D
authors.initials: MD
authors.lastname: Cordero
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Ana Delgado
authors.initials: AD
authors.lastname: Pavón
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Beatriz Castejón
authors.initials: BC
authors.lastname: Vega
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Cotán
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: De la Mata
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Oropesa-Ávila
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Elizabet
authors.initials: E
authors.lastname: Alcocer-Gomez
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: de Lavera
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Garrido-Maraver
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Carrascosa
authors.affiliation: Sistemas Físicos, Químicos y Naturales-Universidad Pablo de Olavide, Sevilla, Spain.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Zaderenko
authors.affiliation: Departmento de Cirugía General y Aparato Digestivo, Hospital Universitario Virgen del Rocío/Instituto de Biomedicina de Sevilla (IBiS)/CSIC/Universidad de Sevilla, Sevilla, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Muntané
authors.affiliation: Departamento de Citología e Histología Normal y Patológica, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: de Miguel
authors.affiliation: Centro Andaluz de Biología de Desarrollo (CABD), Universidad Pablo de Olavide/CSIC/, Sevilla, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Sánchez-Alcázar
keywords: HepG2 cells; amitriptyline; apoptosis; mitophagy; oxidative stress