Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells |
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Authors: | Gorka Magdalena Godlewski Michal Marek Gajkowska Barbara Wojewodzka Urszula Motyl Tomasz |
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Institution: | Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland. |
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Abstract: | Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c. |
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Keywords: | Smac/DIABLO Bax Apoptosis Homeostatic confocal microscopy |
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