Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid

Different classes of anticancer drugs may trigger apoptosis by acting on di fferent subcellular targets and by activating distinct signaling pathways, Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent t hat triggers apoptosis by a direct effect on mitochondria. In isolated mito chondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase . This is inhibited by bongkrekic acid, an agent that stabilizes the permea bility transition pore complex. Mitochondria undergoing BetA-induced permea bility transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspa se-8 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochrome c to cytosolic extracts results in cleavage of caspase-8, but n ot of caspase-8. However, supernatants of mitochondria, which have undergon e permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mito chondrial permeability transition alone is sufficient to trigger the full a poptosis program and that some cytotoxic drugs such as BetA may induce apop tosis via a direct effect on mitochondria.