Received for publication April 30, 2008.
Revised July 1, 2008.
Accepted for publication July 1, 2008.

Patupilone-induced apoptosis is mediated by mitochondrial reactive oxygen species through Bim relocalization to mitochondria

Abstract

Among the new microtubule-targeted agents, the Epothilone family of molecules has shown promising anti-cancer potential, and clinical trials are currently under way for patupilone (epothilone B) in various cancer indications. In this study, we characterized novel aspects of patupilone's cellular action that may underly its potent cytotoxicity in human neuroblastoma cells. Patupilone induced mitochondrial membrane potential collapse, mitochondrial morphological changes and cytochrome c release, leading to apoptosis. Within the two first hours, patupilone increased generation of reactive oxygen species (ROS), i.e. superoxides and hydrogen peroxide (33 ± 6% and 51 ± 3% increase respectively), specifically from mitochondria. ROS scavengers and mitochondrial DNA depletion (^(-) cells) significantly protected cells against patupilone cytotoxicity, indicating that ROS generation is a key event in the initial phase of apoptosis. While Bim expression level was not modified by patupilone, this pro-apoptotic protein accumulated in the mitochondrial compartment (2.4 fold increase at IC₇₀) after only a 6 hr-treatment. In contrast, Bax and Bcl-2 mitochondrial levels were not changed during treatment. Importantly, ROS inhibition prevented Bim relocalization to mitochondria and mitochondrial membrane changes induced by patupilone. Altogether, our data reveal that patupilone-mediated ROS production by mitochondria initiates the intrinsic signaling cascade by inducing Bim accumulation in mitochondria. These results might explain the superior activity of patupilone in tumor cells as compared with paclitaxel that is, until now, the clinical reference among microtubule-stabilizing agents. Furthermore, our data highlight the importance of mitochondria that simultaneously assume the role of activator and integrator of apoptotic signals triggered by patupilone.

Key words: Apoptosis, Oxidative stress/antioxidants, Mechanisms of cell killing/apoptosis, Cytoskeletal targets