title: Doxorubicin activates nuclear factor of activated T-lymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium.
abstract: Doxorubicin (DOX), a widely used antitumour drug, causes dose-dependent cardiotoxicity. Cardiac mitochondria represent a critical target organelle of toxicity during DOX chemotherapy. Proposed mechanisms include generation of ROS (reactive oxygen species) and disturbances in mitochondrial calcium homoeostasis. In the present study, we probed the mechanistic link between mitochondrial ROS and calcium in the embryonic rat heart-derived H9c2 cell line and in adult rat cardiomyocytes. The results show that DOX stimulates calcium/calcineurin-dependent activation of the transcription factor NFAT (nuclear factor of activated T-lymphocytes). Pre-treatment of cells with an intracellular calcium chelator abrogated DOX-induced nuclear NFAT translocation, Fas L (Fas ligand) expression and caspase activation, as did pre-treatment of cells with a mitochondria-targeted antioxidant, Mito-Q (a mitochondria-targeted antioxidant consisting of a mixture of mitoquinol and mitoquinone), or with adenoviral-over-expressed antioxidant enzymes. Treatment with GPx-1 (glutathione peroxidase 1), MnSOD (manganese superoxide dismutase) or a peptide inhibitor of NFAT also inhibited DOX-induced nuclear NFAT translocation. Pre-treatment of cells with a Fas L neutralizing antibody abrogated DOX-induced caspase-8- and -3-like activities during the initial stages of apoptosis. We conclude that mitochondria-derived ROS and calcium play a key role in stimulating DOX-induced 'intrinsic and extrinsic forms' of apoptosis in cardiac cells with Fas L expression via the NFAT signalling mechanism. Implications of ROS- and calcium-dependent NFAT signalling in DOX-induced apoptosis are discussed.
authors.affiliation: Department of Biophysics, Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Shasi V
authors.initials: SV
authors.lastname: Kalivendi
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Konorev
authors.forename: Sonya
authors.initials: S
authors.lastname: Cunningham
authors.forename: Sravan K
authors.initials: SK
authors.lastname: Vanamala
authors.forename: Eugene H
authors.initials: EH
authors.lastname: Kaji
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D053222:Fas Ligand Protein; D005786:Gene Expression Regulation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects*; D048069:Tumor Necrosis Factors / Q000235:genetics*; D015854:Up-Regulation
chemical_list: D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D050778:NFATC Transcription Factors; D009419:Nerve Tissue Proteins; C477593:Nfatc4 protein, rat; D017382:Reactive Oxygen Species; D048069:Tumor Necrosis Factors; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D020169:Caspases; D002118:Calcium