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¹ Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, Texas; ² National Cancer Institute, Frederick, Maryland; and ³ MGI Pharma, Inc., Bloomington, Minnesota

Requests for reprints: Jan M. Woynarowski, Department of Radiation Oncology, University of Texas Health Science Center, IDD Building, 14960 Omicron Drive, San Antonio, TX 78245. Phone: 210-677-3832; Fax: 210-677-0058. E-mail: jmw1{at}saci.org, Phone: 210-677-3846; Fax: 210-677-0058. E-mail: bwoynar{at}saci.org

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 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 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.

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Note: A preliminary account of this study has been presented in part at the 92nd Annual Meeting of AACR, March 24–28, 2001, New Orleans, Proc. AACR 42, 2001, 640.

⁴ Liang and Woynarowska, unpublished data.

Received 7/ 8/04; revised 8/23/04; accepted 9/15/04.