Cephalostatin 1 is a bis-steroidal marine
natural product with a unique cytotoxicity profile in the in vitro screen system of the National Cancer Institute, suggesting that it may affect novel molecular target(s). Here we show that
cephalostatin 1 induces a novel pathway of receptor-independent apoptosis that selectively uses Smac/DIABLO (second mitochondria-derived activator of
caspases/direct inhibitor of apoptosis-
binding protein with a low isoelectric point) as a mitochondrial signaling molecule. At nanomolar concentrations,
cephalostatin 1 triggers dose- and time-dependent DNA fragmentation in
leukemia Jurkat T cells. Apoptosis was found to be dependent on
caspase activity because the pan-
caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone blocks cephalostatin 1-mediated DNA fragmentation. The CD95
death receptor as well as other caspase-8-requiring
death receptors were not involved because Jurkat T cells lacking the CD95 receptor or
caspase-8 and control cells responded equally to
cephalostatin 1. Although
cephalostatin 1 affects mitochondria by dissipating the mitochondrial membrane potential, neither
cytochrome c nor
apoptosis-inducing factor is released, as shown by Western blot analysis. Interestingly,
cephalostatin 1 selectively triggers the mitochondrial release of the inhibitor of apoptosis antagonist Smac/DIABLO. Overexpression of the antiapoptotic
protein Bcl-x(L) delayed both Smac/DIABLO release and onset of apoptosis, suggesting that Smac/DIABLO is required for cephalostatin 1-induced apoptosis. This new mitochondrial pathway is accompanied by marked structural changes of mitochondria as shown by transmission electron microscopy.