Disulfiram anti-cancer efficacy without copper overload is enhanced by extracellular H2O2 generation: antagonism by tetrathiomolybdate

Ali Calderon-Aparicio,Mary Strasberg-Rieber,Manuel Rieber

doi:10.18632/oncotarget.4833

Ali Calderon-Aparicio, Mary Strasberg-Rieber + Show 1 more

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https://doi.org/10.18632/oncotarget.4833

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Abstract

Cu/Zn superoxide dismutases (SODs) like the extracellular SOD3 and cytoplasmic SOD1 regulate cell proliferation by generating hydrogen peroxide (H2O2). This pro-oxidant inactivates essential cysteine residues in protein tyrosine phosphatases (PTP) helping receptor tyrosine kinase activation by growth factor signaling, and further promoting downstream MEK/ERK linked cell proliferation. Disulfiram (DSF), currently in clinical cancer trials is activated by copper chelation, being potentially capable of diminishing the copper dependent activation of MEK1/2 and SOD1/SOD3 and promoting reactive oxygen species (ROS) toxicity. However, copper (Cu) overload may occur when co-administered with DSF, resulting in toxicity and mutagenicity against normal tissue, through generation of the hydroxyl radical (•OH) by the Fenton reaction. To investigate: a) whether sub-toxic DSF efficacy can be increased without Cu overload against human melanoma cells with unequal BRAF(V600E) mutant status and Her2-overexpressing SKBR3 breast cancer cells, by increasing H2O2 from exogenous SOD; b) to compare the anti-tumor efficacy of DSF with that of another clinically used copper chelator, tetrathiomolybdate (TTM). a) without copper supplementation, exogenous SOD potentiated sub-toxic DSF toxicity antagonized by sub-toxic TTM or by the anti-oxidant N-acetylcysteine; b) exogenous glucose oxidase, another H2O2 generator resembled exogenous SOD in potentiating sub-toxic DSF. potentiation of sub-lethal DSF toxicity by extracellular H2O2 against the human tumor cell lines investigated, only requires basal Cu and increased ROS production, being unrelated to non-specific or TTM copper chelator sequestration. These findings emphasize the relevance of extracellular H2O2 as a novel mechanism to improve disulfiram anticancer effects minimizing copper toxicity.

Highlights

Copper (Cu) is an essential trace element in living systems due to its requirement in a number of enzymes like mitochondrial cytochrome c oxidase, important in fueling cell proliferation [1], copper-zinc dependent SODs required to modulate oxidative stress [2], and copper-activated MAP kinase kinase MEK1 responsible for phosphorylating the mitogen-activated proteín kinase ERK [3]
Exogenous SOD promotes sublethal DSF toxicity antagonized by thiomolybdate or N-acetylcysteine in human melanoma cell lines irrespective of V600E-BRAF status
Apoptosisassociated PARP cleavage normalized to actin levels was used to extend the live-dead studies, confirming PARP fragmentation only in SKBR3 cells exposed to DSF+SOD, effect inhibited by concomittant TTM addition (Figure 4B) extending the results shown in Figure 2 for human melanoma cell lines

Summary

Introduction

Copper (Cu) is an essential trace element in living systems due to its requirement in a number of enzymes like mitochondrial cytochrome c oxidase, important in fueling cell proliferation [1], copper-zinc dependent SODs required to modulate oxidative stress [2], and copper-activated MAP kinase kinase MEK1 responsible for phosphorylating the mitogen-activated proteín kinase ERK [3]. One of the purposes of this study was to take advantage of the frequent higher levels of mitogenic ROS in cancer cells, to further increase their ROS and promote their preferential cell death [16] For this purpose, we used disulfiram (DSF), a Cu chelator which has been shown to have an important potential as an anti-cancer agent [24,25,26,27]. Cu/Zn superoxide dismutases (SODs) like the extracellular SOD3 and cytoplasmic SOD1 regulate cell proliferation by generating hydrogen peroxide (H2O2) This pro-oxidant inactivates essential cysteine residues in protein tyrosine phosphatases (PTP) helping receptor tyrosine kinase activation by growth factor signaling, and further promoting downstream MEK/ERK linked cell proliferation. Disulfiram (DSF), currently in clinical cancer trials is activated by copper chelation, being potentially capable of diminishing the copper dependent activation of MEK1/2 and SOD1/SOD3 and promoting reactive oxygen species (ROS) toxicity. Purpose: To investigate: a) whether sub-toxic DSF efficacy can be increased without Cu overload against human melanoma cells with unequal BRAF(V600E) mutant status and Her2-overexpressing SKBR3 breast cancer cells, by increasing H2O2from exogenous SOD; b) to compare the anti-tumor efficacy of DSF with that of another clinically used copper chelator, tetrathiomolybdate (TTM)

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