title: Optimization and validation of mitochondria-based functional assay as a useful tool to identify BH3-like molecules selectively targeting anti-apoptotic Bcl-2 proteins.
abstract: BACKGROUND
  Mitochondrial outer membrane permeabilization (MOMP) is a crucial step leading to apoptotic destruction of cancer cells. Bcl-2 family proteins delicately regulate mitochondrial outer membrane integrity through protein-protein interactions, which makes the mitochondrion an ideal cell-free system for screening molecules targeting the Bcl-2 anti-apoptotic proteins. But assay conditions need to be optimized for more reliable results. In this study, we aimed at establishing a reliable functional assay using mitochondria isolated from breast cancer cells to decipher the mode of action of BH3 peptides derived from BH3-only proteins. In this study, high ionic strength buffer was adopted during the initiation of MOMP. Mitochondria isolated from human breast cancer cell lines with distinct expression patterns of Bcl-2 anti-apoptotic proteins were permeabilized by different BH3 peptides alone or in combination, with or without the presence of recombinant anti-apoptotic Bcl-2 family proteins. Cytochrome C and Smac/Diablo were tested in both supernatants and mitochondrial pellets by Western blotting.
  
  
  RESULTS
  Sufficient ionic strength was required for optimal release of Cytochrome C. Bad and Noxa BH3 peptides exhibited their bona fide antagonistic effects against Bcl-2/Bcl-xL and Mcl-1 proteins, respectively, whereas Bim BH3 peptide antagonized all three anti-apoptotic Bcl-2 members. Bad and Noxa peptides synergized with each other in the induction of MOMP when mitochondria were dually protected by both Bcl-2/Bcl-xL and Mcl-1.
  
  
  CONCLUSIONS
  This method based on MOMP is a useful screening tool for identifying BH3 mimetics with selective toxicity against breast cancer cell mitochondria protected by the three major Bcl-2 anti-apoptotic proteins.
authors.affiliation: Department of Internal Medicine, Hematology/Oncology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Jianting
authors.initials: J
authors.lastname: Long
authors.forename: Liu
authors.initials: L
authors.lastname: Liu
authors.forename: Zaneta
authors.initials: Z
authors.lastname: Nikolovska-Coleska
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Shangary
authors.forename: Han
authors.initials: H
authors.lastname: Yi
authors.forename: Shenming
authors.initials: S
authors.lastname: Wang
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002474:Cell-Free System; D045304:Cytochromes c / Q000378:metabolism; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000037:antagonists & inhibitors*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D051025:bcl-Associated Death Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000037:antagonists & inhibitors*
chemical_list: D051017:Apoptosis Regulatory Proteins; C494819:BAD protein, human; C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; C579095:MCL1 protein, human; D024101:Mitochondrial Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; C498702:PMAIP1 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D045304:Cytochromes c