Optimization and validation of mitochondria-based functional assay as a useful tool to identify BH3-like molecules selectively targeting anti-apoptotic Bcl-2 proteins

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

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.

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.