Abstract 2093: Elesclomol-Cu chelate selectively targets mitochondria to induce oxidative stress
Authors: Nagai Masazumi , Vho Nha , Chu John , Wada Yumiko 
Published: July 2012

Introduction: Elesclomol is a first-in-class investigational drug that exerts anticancer activity through the elevation of reactive oxygen species (ROS) and oxidative damage. We recently reported that elesclomol selectively chelates Cu(II) in plasma, which causes a change in conformation that enables its uptake into cells. A cell-free assay system showed that elesclomol-Cu(II) was capable of efficient generation of ROS via the reduction of Cu(II) to Cu(I). A correlation has been observed between redox potential and anticancer activity for Cu chelates of elesclomol and its analogs, suggesting that the ability to promote redox cycling of Cu(II) to Cu(I) is necessary for the anticancer activity. Here, we demonstrate that elesclomol-Cu carries copper to mitochondria, leading to an increase in oxidative stress and apoptosis due to mitochondrial stress.

Results: First we attempted to track subcellular distribution of elesclomol-Cu. Cytosol, nuclear and mitochondrial fractions of HL60 cells were prepared from cells treated with elesclomol-Cu, and total copper levels were determined for each cellular fraction. Increased copper was only observed in the mitochondrial fraction, suggesting that elesclomol-Cu selectively carried copper to the mitochondria. To verify that the copper increased in mitochondria was from elesclomol-Cu, a copper complex of elesclomol was preformed with 65Cu, and this elesclomol-65Cu complex was incubated for 2h with HL60 cells that were previously enriched with 63Cu using 63Cu-supplemented media and subcellular distributions of 63Cu and 65Cu were determined by ICP-MS. Neither 63Cu nor 65Cu was detected in any fractions in DMSO-treated control cells. 65Cu was selectively detected in mitochondrial fraction of elesclomol-65Cu treated cells but not in cytosol and nuclear fractions, demonstrating a selective mitochondrial uptake of copper with elesclomol-Cu. Next we compared the mitochondrial uptake of elesclomol-Cu and disulfiram(DSF)-Cu using isolated mitochondria. DSF is a Cu chelator, and Cu has been shown to enhance DSF-mediated growth inhibition and apoptosis in cancer cells through the generation of ROS. As expected, an increase in the copper level was observed in mitochondria treated with elesclomol-Cu, while there was no increase in copper in the mitochondria treated with DSF-Cu at its cytotoxic concentration, emphasizing the novel mitochondria selectivity of elesclomol-Cu. Lastly, to identify whether the mitochondrial entry of elesclomol-Cu triggers the generation of ROS, ROS in isolated mitochondria was measured. Mitochondrial ROS was immediately increased by adding elesclomol-Cu while no change in mitochondrial ROS was seen using DSF-Cu or free Cu2+. These results indicate that elesclomol-Cu selectively permeates mitochondria and induces mitochondrial ROS, which likely leads to apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2093. doi:10.1158/1538-7445.AM2011-2093