Irofulven (hydroxymethylacylfulvene) is a novel
antitumor drug, which acts by alkylating cellular macromolecular targets. The
drug is a potent inducer of apoptosis in various types of
tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to
irofulven involving
mitochondrial dysfunction and leading to death of prostate
tumor LNCaP-Pro5 cells.
Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and
cytochrome c release at 4 to 12 hours. These effects preceded
caspase activation and during the first 6 hours were not affected by
caspase inhibitors. Processing of
caspase-9 initiated the
caspase cascade at approximately 6 hours and progressed over time. The activation of the
caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and
cytochrome c release. General and specific
caspase inhibitors abrogated
irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-
caspase > or =
caspase-9 >
caspase-8/6 >
caspase-2 >
caspase-3/7 >
caspase-1/4. Abrogation of
caspase-mediated DNA fragmentation failed to salvage
irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a
caspase-independent cell death.
Monobromobimane, an inhibitor of alternative
caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as
phosphatidylserine externalization, and cytotoxicity of
irofulven. Collectively, the results indicate that
irofulven-induced signaling is integrated at the level of
mitochondrial dysfunction. The induction of both
caspase-dependent and
caspase-independent death pathways is consistent with pleiotropic effects of
irofulven, which include targeting of cellular
DNA and
proteins.