CI-1010 induced opening of the mitochondrial permeability transition pore precedes oxidative stress and apoptosis in SY5Y neuroblastoma cells

• Published in: Brain research 2003-02, Vol.963 (1), p.43-56
• Main Authors: Miller, Terry J., Phelka, Amanda D., Tjalkens, Ronald B., Dethloff, Lloyd A., Philbert, Martin A.
• Format: Article
• Language: English
• Subjects: 2-Nitroimidazole; Antineoplastic agents; Antioxidants - pharmacology; Apoptosis; Apoptosis - drug effects; Biological and medical sciences; Brain Neoplasms - metabolism; Brain Neoplasms - ultrastructure; Cell Death - drug effects; Cell Survival - drug effects; Chemotherapy; CI-1010; DNA Fragmentation - drug effects; Flow Cytometry; Humans; In Situ Nick-End Labeling; Medical sciences; Membrane Potentials - drug effects; Microscopy, Electron; Mitochondria - drug effects; Mitochondria - ultrastructure; Mitochondrial permeability transition; Neuroblastoma - metabolism; Neuroblastoma - ultrastructure; Nitroimidazoles - pharmacology; Oxidative stress; Oxidative Stress - physiology; Permeability - drug effects; Pharmacology. Drug treatments; Photoreceptor Cells - drug effects; Photoreceptor Cells - physiology; Radiation-Sensitizing Agents - pharmacology; Reactive Oxygen Species - metabolism; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Vitamin E - pharmacology
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/S0006-8993(02)03838-6

The hetero-bifunctional nitroimidazole radiosensitizer CI-1010, R-α-{[(2-bromoethyl)-amino]methyl}-2-nitro-1H-imidazole-1-ethanol monohydrobromide, causes selective irreversible apoptotic loss of retinal photoreceptor cells in vivo. The human neuroblastoma cell line, SH-SY5Y, was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in neuroblastoma cells, as determined by histopathological and ultrastructural analysis and by TUNEL technique. CI-1010 causes a dose-dependent decrease in cell viability in SY5Y cells, as measured by the reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Superoxide dismutase reduced loss of cell viability following CI-1010 treatment suggesting an oxidative stress-mediated mechanism of toxicity. The effects of CI-1010 on mitochondrial membrane potential and intracellular levels of reactive oxygen species were assessed in live SY5Y cells by confocal microscopy using the fluorescent dyes, tetramethylrhodamine methyl ester and 5,6-carboxy-2′,7′-dihydrodichlorofluorescein diacetate. CI-1010 caused a rapid depolarization of mitochondria in SY5Y cells followed by an increase in ROS. Both CI-1010-induced mitochondrial depolarization and subsequent increases in ROS were prevented by pretreatment with either the permeability transition pore inhibitor, cyclosporin A (CsA), and by the antioxidant, α-tocopherol. However, CsA and α-tocopherol were unable to prevent apoptosis in CI-1010-treated cells, suggesting the influence of additional mechanism(s) of CI-1010-induced toxicity. This study evaluates intracellular oxidative stress associated with pore opening prior to apoptosis and provides evidence in support of a mitochondrial mechanism of CI-1010-induced neuronal cell death.