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

BMC Biotechnology

Shaomeng Wang³  Shenming Wang²  Han Yi³  Sanjeev Shangary³  Zaneta Nikolovska-Coleska¹  Liu Liu³  Jianting Long³ 

[1]Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA

[2]Breast Disease Center, Department of Surgery, The First Affiliated Hospital, Sun Yat-Sen University, 58, Zhongshan No.2 Road, Guangzhou 510080, China

[3]Department of Internal Medicine, Hematology/Oncology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA

关键词: Mitochondrial outer membrane permeabilization (MOMP);    Bcl-2 homolog domain 3 (BH3);    B cell lymphoma 2 (Bcl-2);    Mitochondrion;   

Others  :  1123168
DOI  :  10.1186/1472-6750-13-45

 received in 2012-09-05, accepted in 2013-05-21,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Long et al.; licensee BioMed Central Ltd.

【 图 表 】

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