Abstract
The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.
Highlights
BH3 mimetics are promising chemotherapeutics, but their full mechanisms of action are underexplored
To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple B cell lymphoma-2 (BCL-2) family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization
To determine how BH3 mimetic responses are influenced by proapoptotic BCL-2 proteins, we employed a biochemically defined Large unilamellar vesicle (LUV) model system that faithfully recapitulates BAXdependent mitochondrial outer membrane permeabilization (MOMP) [5, 28]
Summary
BH3 mimetics are promising chemotherapeutics, but their full mechanisms of action are underexplored. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. The relatively recent development of small molecules that directly inhibit the anti-apoptotic BCL-2 members allows for the pharmacological regulation of MOMP and apoptosis [11,12,13] These molecules, termed BH3 mimetics, function similar to the sensitizer/derepressor BH3-only proteins by binding within the hydrophobic groove of anti-apoptotic proteins [12, 14, 15]. We directly examined the relationships between BH3 mimetics and various BCL-2 family protein functional complexes and reveal that proapoptotic BCL-2 proteins impose additional specificities for responses to these drugs
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