Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells.
Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-L-cysteine (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X(L) and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6h after X irradiation. Furthermore, the O(2)(-*) production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO-OOH was detected. This NADH/succinate-dependent O(2)(-*) production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O(2)(-*) production from mitochondria to trigger cytochrome c release in A549 cells.
Ogura
Aki
A
Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Sapporo, Japan.
Oowada
Shigeru
S
Kon
Yasuhiro
Y
Hirayama
Aki
A
Yasui
Hironobu
H
Meike
Shunsuke
S
Kobayashi
Saori
S
Kuwabara
Mikinori
M
Inanami
Osamu
O
D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis / Q000528:radiation effects; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology / Q000532:radiotherapy*; D053078:Membrane Potential, Mitochondrial / Q000528:radiation effects; D008928:Mitochondria / Q000201:enzymology / Q000528:radiation effects*; D010084:Oxidation-Reduction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000032:analysis; D051020:bcl-X Protein / Q000032:analysis
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D013481:Superoxides; D045304:Cytochromes c; D020169:Caspases; D000111:Acetylcysteine