title: Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts.
abstract: The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Portugal.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Moreira
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Portugal.
authors.forename: Ana F
authors.initials: AF
authors.lastname: Branco
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Portugal.
authors.forename: Susana F
authors.initials: SF
authors.lastname: Sampaio
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Cunha-Oliveira
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Department of Life Sciences, University of Coimbra, Portugal.
authors.forename: Tatiana R
authors.initials: TR
authors.lastname: Martins
authors.affiliation: Department of Biomedical Sciences, University of Minnesota, Medical School-Duluth, Duluth, MN, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Holy
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal. Electronic address: pauloliv@ci.uc.pt.
authors.forename: Paulo J
authors.initials: PJ
authors.lastname: Oliveira
authors.affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.
authors.forename: Vilma A
authors.initials: VA
authors.lastname: Sardão
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D061945:Caspase Inhibitors / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D013997:Time Factors
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000903:Antibiotics, Antineoplastic; D051033:Apoptosis Inducing Factor; D061945:Caspase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004317:Doxorubicin; D020169:Caspases
keywords: Apoptosis; Apoptosis-inducing factor; Cardiomyoblast; Doxorubicin