Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells.
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.
Gorka
Magdalena
M
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland.
Godlewski
Michal Marek
MM
Gajkowska
Barbara
B
Wojewodzka
Urszula
U
Motyl
Tomasz
T
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D002154:Calpain / Q000037:antagonists & inhibitors; D002166:Camptothecin / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D047569:Laser Scanning Cytometry; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D018384:Oxidative Stress; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D002352:Carrier Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D045304:Cytochromes c; D002154:Calpain; C449000:m-calpain; D002166:Camptothecin