Abstract
BackgroundMitochondrial 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.ResultsSufficient 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.ConclusionsThis 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.
Highlights
Mitochondrial outer membrane permeabilization (MOMP) is a crucial step leading to apoptotic destruction of cancer cells
This method based on MOMP is a useful screening tool for identifying Bcl-2 homolog domain 3 (BH3) mimetics with selective toxicity against breast cancer cell mitochondria protected by the three major B cell lymphoma 2 (Bcl-2) anti-apoptotic proteins
A panel of human normal cell lines and breast cancer cell lines were analyzed by Western blotting for their basal levels of three key anti-apoptotic proteins, Bcl-2, B-cell lymphoma-extra large (Bcl-xL) and Myeloid cell leukemia 1 (Mcl-1) (Figure 1). 2LMP cell line, a subclone of MDA-MB-231, is characterized by its abundant cytoplasm and rapid proliferation which are critical for providing sufficient mitochondria
Summary
Mitochondrial outer membrane permeabilization (MOMP) is a crucial step leading to apoptotic destruction of cancer cells. The Bcl-2 family includes antiapoptotic proteins like Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 containing all four Bcl-2 homology domains (BH1-4), pro-apoptotic proteins like Bax, Bak and Bok lacking the BH4 domain, and the pro-apoptotic BH3-only proteins like Bim, Bid, Puma, Bad and Noxa [3]. Among all of these proteins, Bax and Bak are believed to be the “executors” which will exhibit conformational change and oligomerization upon activation and subsequently induce MOMP and cell death. Bim, Bid and Puma proteins are far more potent than the others, such as Bad and Noxa, because they can engage all the anti-apoptotic proteins, while Bad and Noxa selectively bind only a subset of anti-apoptotic proteins [6,7]
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