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 anticancer potential, and clinical trials are currently underway for patupilone (epothilone B) in various cancer indications. In this study, we characterized novel aspects of patupilone's cellular action that may underlie 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 first 2 h, patupilone increased the generation of reactive oxygen species (ROS; i.e., superoxides and hydrogen peroxide, 33 Ϯ 6 and 51 Ϯ 3% increase, respectively), specifically from mitochondria. ROS scavengers and mitochondrial DNA depletion [ (Ϫ) cells] significantly protected cells against patupilone cytotoxicity, indicating that ROS generation is a key event in the initial phase of apoptosis. Although the Bim expression level was not modified by patupilone, this proapoptotic protein accumulated in the mitochondrial compartment (2.4-fold increase at IC 70 ) after only a 6-h treatment. In contrast, Bax and Bcl-2 mitochondrial levels were not changed during treatment. It is noteworthy that 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 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.

Transcriptional down-regulation of Bcl-2 by vinorelbine: Identification of a novel binding site of p53 on Bcl-2 promoter

“…It is well-known that p53 expression is induced by different anti-cancer drugs, including MTAs [6][7]. Consistently with this and our previous data [20], the present study showed that VRL increased the amount of cellular p53 and its target gene p21 that reflects p53 functionality.…”

“…It depolymerises the microtubule network and leads to apoptosis after cell cycle arrest. Like other MTAs, it activates the intrinsic mitochondrial apoptotic pathway [6][7][8].…”

Gold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications

“…After 30 minutes of H 2 -DCF-DA (10 µM) incubation in white medium, the formation of DCF fluorescent product was measured in a Fluoroskan Ascent (Thermo Fisher Scientific) microplate fluorometer (510 nm excitation, 580 nm emission), as previously described. 44 …”

Epothilones

“…6,54 Treatment of human cancer cells with low nanomolar concentrations of Epo B produces aberrant mitotic spindles, results in cell cycle arrest in mitosis, and eventually leads to apoptotic cell death. 4,16 IC 50 s for cancer cell growth inhibition are in the nanomolar or even sub-nanomolar range, depending on the specific cell type, 4,16,18 In addition, apoptosis induction by Epo B has been suggested to involve the formation of reactive oxygen species in mitochondria; 55 it has also been linked to the activation of the nuclear translocation of the transcription factor NFkB in a process that would be independent of the microtubule effects of the compound. 56 The true significance of these findings for the antitumor activity of Epo B still needs to be established.…”