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

Cell Mol Biol (Noisy-le-grand). 2007 Jan 21:52 Suppl:OL915-22.


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.

MeSH terms

  • Apoptosis / drug effects
  • Apoptosis Regulatory Proteins
  • BH3 Interacting Domain Death Agonist Protein / genetics
  • BH3 Interacting Domain Death Agonist Protein / metabolism*
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology
  • Camptothecin / pharmacology*
  • Caspase 8 / metabolism*
  • Cell Line, Tumor
  • Enzyme Inhibitors / pharmacology
  • Female
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Kinetics
  • Mitochondrial Proteins / antagonists & inhibitors
  • Mitochondrial Proteins / metabolism*
  • bcl-2-Associated X Protein / metabolism*


  • Apoptosis Regulatory Proteins
  • BH3 Interacting Domain Death Agonist Protein
  • DIABLO protein, human
  • Enzyme Inhibitors
  • Intracellular Signaling Peptides and Proteins
  • Mitochondrial Proteins
  • bcl-2-Associated X Protein
  • Green Fluorescent Proteins
  • Caspase 8
  • Camptothecin