Abstract
β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation. Exposure of astrocytes to β-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The β-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to β-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of β-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of β-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.
Highlights
The quinone beta-lapachone (β-lap, clinical names: ARQ761 or ARQ501) has been extracted from the bark of the lapacho tree and is known to have various beneficial effects [1, 2]
In order to test whether β-lap has adverse consequences on astrocytes, cultured rat astrocytes were exposed to this compound in concentrations of up to 100 μM for up to 4 h and the membrane integrity as well as the lactate accumulation in the incubation medium were determined
In order to test whether an application of β-lap affects the astrocytic GSH metabolism and the ratio of GSH to GSSG, cultured astrocytes were exposed to β-lap in concentrations of up to 100 μM and the cellular and extracellular contents of GSx and GSSG were determined
Summary
The quinone beta-lapachone (β-lap, clinical names: ARQ761 or ARQ501) has been extracted from the bark of the lapacho tree and is known to have various beneficial effects [1, 2]. The hydroquinone form of β-lap (β-lapachol, Fig. 1) that is generated by NQO1-mediated reduction is labile and autooxidizes quickly in two distinct one-electron steps [1, 10], thereby starting a futile cycle that regenerates the quinone β-lap by producing intracellular ROS which leads to oxidative stress and cell toxicity [2, 13] This NQO1-dependent cycling of β-lap appears to be especially prominent in cancer cells, since such cells are reported to contain higher activities of NQO1 than non-cancerous cells, which supports the potential use of β-lap as an anti-cancer drug [1, 14]
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