Development and validation of mitochondria-based functional assays to investigate the mechanism of small-molecule inhibitors of Bcl-2/Bcl-xL/Mcl-1 (BH3 mimetics) in cell-free systems

A214

Mitochondrial permeabilization is a crucial step leading to apoptotic destruction of cancer cells. Bcl-2 family proteins anchoring to or associated with the outer membrane of mitochondria delicately regulate this step through protein-protein interactions, which makes the mitochondria an ideal model for studying molecules that target the Bcl-2 pro-survival proteins. Purpose: to establish a reliable and specific functional assay using mitochondria isolated from cancer cells to decipher the mode of action of BH3 peptides derived from BH3-only proteins in the Bcl-2 family as well as the small molecular Bcl-2 inhibitors. Materials and Methods: mitochondria isolated from human breast cancer cell lines with distinct expression of Bcl-2 pro-survival proteins were dually protected by adding exogenous recombinant proteins before incubating with different BH3 peptides alone or in combination at 37°C for 1 hour. Smac and cytochrome c were tested on both supernatants and mitochondrial pellets using immunoblotting. Cell-permeable BH3 peptides were used in a cell-based apoptotic assay to further confirm their selective killing in cells with different Bcl-2 pro-survival proteins status. Non-peptide small-molecule inhibitors were also tested in the same conditions. Results and Conclusions: it is the first time using isolated human cancer cell mitochondria to comprehensively evaluate peptides and non-peptide small molecule inhibitors against three different Bcl-2 members (Bcl-2, Bcl-xL and Mcl-1). Noxa and Bad BH3 peptides exhibit their bona fide antagonistic capability against either Bcl-2/xL or Mcl-1 proteins respectively, whereas Bim BH3 peptide can antagonize all of three anti-apoptotic Bcl-2 members. Bad and Noxa peptides synergize with each other in release of cytochorme c and Smac proteins. These functional assays were then used to characterize our novel small molecule inhibitors of Bcl-2/Bcl-xL/Mcl-1 for their antagonism and specificity on a functional level.