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Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells.

Significance Statement

The alcohol deterrent disulfiram has more recently been proposed as an anti-cancer therapeutic based predominantly on its capacity to chelate copper ions and thereby inhibit copper dependent processes. Little research has been conducted to determine the relationship between disulfiram and zinc. This is despite knowledge that the drug is also able to bind zinc ions, and substantial evidence showing that zinc is dysregulated in breast cancer. This study investigated the potential role of zinc in disulfiram- mediated activity in breast cancer cells. Disulfiram was toxic to MCF-7 and BT474 breast cancer cell lines and produced a pronounced biphasic toxicity profile. In comparison, non- cancerous breast epithelial MCF-10A cells were only sensitive to the drug at much higher (non-physiologically relevant) concentrations. Further analyses demonstrated that the biphasic cytotoxicity response in breast cancer cells could be reversed by supplementing disulfiram containing culture media with zinc or copper. Using a combination of live cell confocal microscopy and flow cytometry we showed for the first time that disulfiram had a powerful zinc sequestering effect in breast cancer cell lysosomes. This raises the possibility that the effect of this drug on cell viability may be due to acute zinc overload. Zinc challenged cells have previously been shown to internalize these metal ions and sequester them in lysosomes as a protective mechanism. However, when lysosomal storage of zinc is saturated lysosomal function is compromised and the cells undergo apoptosis. We propose that a similar mechanism may underlie our findings and supporting this we also demonstrated that disulfiram causes mislocalisation of lysosomes. Finally, through studying the major disulfiram metabolite, DDC, and synthesizing a novel disulfiram analog, termed FS03EB, we demonstrated a correlation between the ability of these compounds to increase intracellular zinc and influence cytotoxicity. Our study reveals a new mechanism of action for disulfiram based on its zinc ionophore activity and capacity to increase lysosomal levels of this metal. A zinc-rich microenvironment may provide more favorable conditions for this drug to promote intracellular delivery and lysosomal sequestration of zinc to induce cancer cell death. This could have important clinical implications in environments of high extracellular zinc, such as breast and other tumors. Taken in the context of the literature demonstrating the clinical safety and tolerability of disulfiram, our study supports the view that this drug or analogues could be part of a future therapeutic strategy for breast and other cancers.

 

Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells. Global Medical Discovery

 

 

 

 

Journal Reference

Wiggins HL1, Wymant JM1, Solfa F1, Hiscox SE1, Taylor KM1, Westwell AD1, Jones AT2. Biochem Pharmacol. 2015;93(3):332-42.

1Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, Cardiff, Wales CF10 3NB, UK.

2Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, Cardiff, Wales CF10 3NB, UK.

Electronic address: [email protected]

Abstract

Disulfiram, a clinically used alcohol-deterrent has gained prominence as a potential anti-cancer agent due to its impact on copper-dependent processes. Few studies have investigated zinc effects on disulfiram action, despite it having high affinity for this metal. Here we studied the cytotoxic effects of disulfiram in breast cancer cells, and its relationship with both intra and extracellular zinc. MCF-7 and BT474 cancer cell lines gave a striking time-dependent biphasic cytotoxic response between 0.01 and 10 μM disulfiram. Co-incubation of disulfiram with low-level zinc removed this effect, suggesting that availability of extracellular zinc significantly influences disulfiram efficacy. Live-cell confocal microscopy using fluorescent endocytic probes and the zinc dye Fluozin-3 revealed that disulfiram selectively and rapidly increased  zinc levels in endo-lysosomes. Disulfiram also caused spatial disorganization of late endosomes and lysosomes, suggesting they are novel targets for this drug. This relationship between disulfiram toxicity and ionophore activity was consolidated via synthesis of a new disulfiram analog and overall we demonstrate a novel mechanism of disulfiram-cytotoxicity with significant clinical implications for future use as a cancer therapeutic.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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