Copper diethyldithiocarbamate as an activator of Nrf2 in cultured vascular endothelial cells

Abstract

The interest in organic–inorganic hybrid molecules as molecular probes for biological systems has been growing rapidly. Such hybrid molecules exhibit unique biological activities. Herein, copper(II) bis(diethyldithiocarbamate) (Cu10) was found to activate the transcription factor NF-E2-related factor 2 (Nrf2), which is responsible for regulating antioxidant and phase II xenobiotic enzymes, in vascular endothelial cells. The copper complex rapidly accumulated within cells and induced nuclear translocation of Nrf2, leading to upregulation of the expression of downstream proteins without cytotoxic effects. However, while copper bis(2-hydroxyethyl)dithiocarbamate activated Nrf2, copper ion, diethyldithiocarbamate ligand with or without zinc or iron failed to exhibit this activity. Intracellular accumulation of Cu10 was higher than that of Cu(II) and Cu(I). While the accumulation of copper(II) bis(dimethyldithiocarbamate) was reduced by small interfering RNA (siRNA)-mediated knockdown of the copper transporter CTR1, the knockdown did not affect Cu10 accumulation, indicating that Cu10 rapidly enters vascular endothelial cells via CTR1-independent mechanisms. In addition, copper and iron complexes with other ligands tested could not activate Nrf2, suggesting that the intramolecular interaction between copper and dithiocarbamate ligand is important for the activation of the transcription factor. Cu10 induced the expression of heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and γ-glutamylcysteine synthetase, downstream proteins of Nrf2. It was suggested that Cu10-induced activation of Nrf2 was due to proteasome inhibition as well as binding to Kelch-like ECH-associated protein 1. Since the effects of Cu10 on vascular endothelial cells are unique and diverse, the copper complex may be a good molecular probe to analyze the functions of the cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-016-1337-z) contains supplementary material, which is available to authorized users.

Synergistic cytotoxicity caused by forming a complex of copper and 2,9-dimethyl-1,10-phenanthroline in cultured vascular endothelial cells

“…Although the mechanisms by which high accumulations of Cu-DMP form within vascular endothelial cells are currently unclear, it appears that a copper transporter (CTR1), which is a transporter for copper(I), is capable of transporting Cu-DMP from the culture medium into the cells because that transporter is also involved in the uptake of cisplatin, which is a platinum complex as well as copper(I) ion Ishida et al, 2002). Additionally, it appears that Cu-DMP may also serve as a donor of copper(I) ion to CTR1; in fact, the vascular endothelial cells used in our system express CTR1 (Fujie et al, 2016e). Taken together, these results indicate that additional experiments should be performed in order to clarify whether CTR1 may be involved in the uptake of Cu-DMP, and to determine whether there are other mechanisms by which Cu-DMP is transported into vascular endothelial cells.…”

Copper diethyldithiocarbamate as an inhibitor of tissue plasminogen activator synthesis in cultured human coronary endothelial cells

“…Such studies should investigate the physical characteristics of Cu10 that are required for the specific biological activities, including endothelial metallothionein induction (Fujie et al, 2016b) and the t-PA synthesis shown in the present study, as well as activation of nuclear factor, erythroid 2-like 2 (Fujie et al, 2016d).…”

“…We hypothesized that copper diethyldithiocarbamate (Cu10), which is a copper complex that exhibits interesting biological activities including endothelial metallothionein induction (Fujie et al, 2016b) and activation of nuclear factor, erythroid 2-like 2 (Fujie et al, 2016d), may provide a useful tool for analyzing new mechanisms that regulate endothelial fibrinolytic activity. Herein, we report on the findings of our experiments, in which we show that Cu10 suppresses the fibrinolytic activity of human coronary endothelial cells in culture.…”

Induction of metallothionein isoforms by copper diethyldithiocarbamate in cultured vascular endothelial cells

“…These compounds are expected to exhibit unique biological activities, different from those of organic and inorganic compounds, which are based on their unique three-dimensional structures and electronic states (Fujiwara et al, 2005;Murakami et al, 2015;Kohri et al, 2015). We have recently found that copper(II) bis(diethyldithiocarbamate), termed Cu10, significantly activates Nrf2 in vascular endothelial cells without exhibiting cytotoxicity (Fujie et al, 2016a). This activity was not observed with copper(II) sulfate, diethyldithiocarbamates of sodium(I), zinc(II) or iron(III), and copper(II) complexes with other types of ligands tested therein.…”

“…AREs exist in the promoter region of MT genes (Ohtsuji et al, 2008). We previously showed that Cu10 increases Nrf2 expression by proteasome inhibition as well as binding to Kelch-like ECH-associated protein 1, which induces heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and gammaglutamate-cysteine ligase, modifier subunit (Fujie et al, 2016a). We postulate that the induction of endothelial MT expression involves the Nrf2-ARE pathway as well as the MTF-1-MRE pathway and that Cu10 is a good agent to analyze the relationship between Nrf2 activation and MT induction.…”