Impaired kinetics of Bax-GFP and Smac/DIABLO-GFP in caspase-8- and bid-silenced and Bcl-2 overexpressed breast cancer MCF-7 cells exposed to camptothecin.
Smac/DIABLO, a proapoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspases activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Present study is focused on the role of Bid in the control of Bax-GFP and Smac/DIABLO-GFP kinetics in breast cancer MCF-7 cells stimulated to apoptosis with camptothecin (CPT). Minute kinetics of proteins was examined by homeostatic confocal microscopy. The release of Smac/DIABLO-GFP from mitochondria comprised two phases: initial-rapid, lasting 20-30 min and subsequent 30 min-plateau phase, followed by the decrease of Smac/DIABLO-related fluorescence due to cell destruction. The kinetics of Bax-GFP aggregation on mitochondria coincided in time with Smac/DIABLO-GFP release from these organelles. Bid knock down and Bcl-2 overexpression delayed Bax-GFP aggregation and completely inhibited Smac/DIABLO-GFP release from mitochondria. Knock down of caspase 8 (activator of Bid) delayed both Bax-GFP aggregation and Smac/DIABLO-GFP release in CPT-treated cells. In conclusion, Bid protein is crucial for the control of the release of Smac/DIABLO from mitochondria in breast cancer MCF-7 stimulated to apoptosis with CPT.
Górka
M
M
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland.
Lamparska-Przybysz
M
M
Motyl
T
T
D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002166:Camptothecin / Q000494:pharmacology*; D053181:Caspase 8 / Q000378:metabolism*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D051028:bcl-2-Associated X Protein; D049452:Green Fluorescent Proteins; D053181:Caspase 8; D002166:Camptothecin