The purpose of this study was to investigate the effect of altering the reducing environment in cancer cells by targeting both glutathione (GSH) and thioredoxin (Trx). This was achieved by administering low levels of buthionine sulfoximine (BSO) to deplete GSH and auranofin (AUR) to lower Trx by inhibiting thioredoxin reductase (TrxR). Methylglyoxal (MG), which alters cellular redox environment, was used in combination with BSO and AUR. The experiments were conducted using the breast cancer cells MDA-MB-231 and MCF-7, cell lines known to exhibit differences in metabolism and responses to chemotherapeutic drugs.
GSH was quantified using a luminescence-based assay. As expected, BSO led to a dramatic decrease of cellular GSH. Treatment with AUR and MG enhanced the decrease of GSH by BSO. Surprisingly, in the absence of BSO the combination of AUR and MG evoked an increase in GSH content in both cell lines. This increase might reflect a compensatory mechanism in which the inhibition of TrxR led to an increase in GSH synthesis in order to resist oxidative damage. Levels of reactive oxygen species (ROS) were quantified using redox-sensitive GFP (roGFP). After baculovirus transduction of the cells with roGFP, an increase in ROS was observed in both cell lines within 30 minutes of treatment with the combination of the three compounds.
Cellular viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt in cells treated with various combinations and concentrations of BSO, AUR, and MG. BSO reduced viability of MDA-MB-231 cells at concentrations at or above 50 µM. AUR led to slight reductions in viability in this cell line, but when administered together, BSO at 100 µM and AUR at 1 µM caused an 85% decrease in viability. In the MCF-7 cell line, BSO caused no notable decrease in viability, while AUR likewise had no effect on viability. Administered together, however, BSO at 100 µM and AUR at 1 µM caused a 95% decrease in viability. MG enhanced the effects of BSO in both cell lines, but the reduction in viability was more pronounced in MDA-MB-231 cells. Similar to the effects of the compounds on viability, BSO alone had little effect on colony formation in both MDA-MB-231 and MCF-7 cells (as visualized by crystal violet staining). In addition, there was no effect on colony formation in either cell line when using AUR and MG unless initially treated with BSO. These effects were most significant when the concentration of BSO was greater than 50 µM.
Previous studies have reported metabolic differences between MDA-MB-231 and MCF-7 cells (Gatenby & Gillies, 2004). MDA-MB-231 cells rely on aerobic glycolysis while MCF-7 cells utilize mitochondrial oxidative phosphorylation. Therefore, MCF-7 cells have developed increased protection against ROS as compared to MDA-MB-231 cells (Theodossiou et al., 2017). These findings support the observed differences in response to BSO, MG, and AUR between the two cell lines.
Citation Format: Kaci Keleher, Emma Meetz, Collin Ellenbecker, Radek Buss, Emily Minton, Morris A. Johnson, Michael Smits, Russ Feirer. Effects of GSH depletion and thioredoxin reductase inhibitors on the viability of MDA-MB-231 and MCF-7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2444.