Owing to the high levels of antiapoptotic B-cell lymphoma 2 (BCL-2) family members observed in several cancers there has been a major effort to develop inhibitors of the BCL2-family as chemotherapeutic brokers. for other nonapoptotic roles of the BCL-2 family ranging from ionic homeostasis and autophagy to the regulation of fission-fusion dynamics in subcellular organelles including the endoplasmic reticulum and mitochondria. In this study we characterize the specificity of two novel putative MCL-1 inhibitors BI97C1 (Sabutoclax) and BI112D1 in inducing apoptosis in a BAX/BAK-dependent manner and in an MCL-1-dependent system. In addition to their being proapoptotic these inhibitors also cause enhanced mitochondrial fragmentation that accompanies a time-dependent loss of optic atrophy 1 (OPA1) suggesting an impairment of mitochondrial fusion. This mitochondrial fragmentation occurs independently of dynamin-related protein 1 (DRP1)-mediated fission activity and unlike most apoptotic stimuli occurs upstream of and/or impartial of BAX BAK and other BH3-only proteins. Furthermore this mitochondrial fragmentation occurred rapidly and preceded other hallmarks of apoptosis including the loss in mitochondrial membrane potential and the release of cytochrome and efficacy and inhibits tumorigenesis in various models of prostate cancer [23 24 In addition one optically real apogossypolone derivative BI112D1 ((-)BI97D6) is also a potent pan-active BCL-2 family MifaMurtide inhibitor and exerts antitumor activity in a prostate cancer xenograft model in mice [25 26 Both BI97C1 and BI112D1 induced apoptosis in a BAX/BAK-dependent manner and in MCL-1-dependent cells. These inhibitors also caused a time-dependent loss of optic atrophy 1 (OPA1) that accompanied enhanced mitochondrial fragmentation as well as an increased mitochondrial accumulation of reactive oxygen species (ROS). Materials and Methods Cell Culture Wild-type (WT) and BAX/BAK double knockout (DKO) mouse embryonic fibroblasts (MEFs) from Dr A. Strasser (Walter and Eliza Hall Institute Melbourne Australia) were cultured in Dulbecco’s altered Eagle’s medium supplemented with 5 mM l-glutamine and 10% fetal calf serum (all from Life Technologies Inc Paisley United Kingdom). H23 cells from Prof. C. Pritchard (University of Leicester Leicester United Kingdom) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 5 mM l-glutamine. Reagents and Plasmids BI97C1 and BI112D1 were synthesized as described [22 26 ABT-263 was obtained from Selleck MifaMurtide Chemicals Co (Houston TX). Antibodies against cytochrome Release and Western Blot Analysis Cytochrome release experiments were carried out in cells exposed to different drugs for the indicated occasions and assessed as previously described . Western blots were carried out according to standard protocols . Briefly 50 μg of total protein lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently proteins were transferred to nitrocellulose membrane and protein bands were visualized with ECL reagents (GE Healthcare Bucks United Kingdom). Microscopy For immunofluorescent staining cells produced on coverslips were fixed with 4% (vol/vol) paraformaldehyde permeabilized with 0.5% (vol/vol) Triton X-100 in phosphate-buffered Rabbit Polyclonal to ALPK1. saline and followed by incubations with primary antibodies and analyzed as previously described . For monitoring mitochondrial fragmentation and changes in mitochondrial membrane potential cells were stained for 30 minutes with 200 nM MitoTracker Deep Red and 500 MifaMurtide nM TMRE before image acquisition. For electron microscopy cells were fixed and processed as previously described . Electron micrographs were recorded using a Megaview 3 digital camera and iTEM software (Olympus Soft Imaging Solutions GmbH Münster Germany) in a Jeol 100-CXII electron microscope (Jeol UK Ltd Welwyn Garden City United Kingdom). Flow Cytometry Loss in mitochondrial membrane potential (ψm) was assessed as described previously by staining cells with TMRE a lipophilic fluorescent dye that accumulates in the mitochondria in proportion to the membrane potential . Cell death MifaMurtide was assessed by phosphatidylserine (PS).