Biomol Ther.  2022 Mar;30(2):184-190. 10.4062/biomolther.2021.105.

Extracellular Concentration of L-Cystine Determines the Sensitivity to System x_c - Inhibitors

• Abdullah M
• Lee SJ

• KMID: 2526821
• DOI: http://doi.org/10.4062/biomolther.2021.105

Abstract

Targeting the cystine/glutamate exchange transporter, system x_c-, is a promising anticancer strategy that induces ferroptosis, which is a distinct form of cell death mediated by iron-dependent lipid peroxidation. The concentration of L-cystine in culture medium is higher than the physiological level. This study was aimed to evaluate the effects of L-cystine concentration on the efficacy of ferroptosis inducers in hepatocellular carcinoma cells. This study showed that treatment with sulfasalazine or erastin, a system x_c- inhibitor, decreased the viability of Huh6 and Huh7 cells in a dose-dependent manner, and the degree of growth inhibition was greater in medium containing a physiological L-cystine concentration of 83 μM than in commercial medium with a concentration of 200 μM L-cystine. However, RSL3, a glutathione peroxidase 4 inhibitor, decreased cell viability to a similar extent in media containing both L-cystine concentrations. Sulfasalazine and erastin significantly increased the percentages of propidium iodide-positive cells in media with 83 μM L-cystine, but not in media with 200 μM L-cystine. Sulfasalazine- or erastin-induced accumulation of lipid peroxidation as monitored by C11-BODIPY probe was higher in media with 83 μM L-cystine than in media with 200 μM L-cystine. In contrast, the changes in the percentages of propidium iodide-positive cells and lipid peroxidation by RSL3 were similar in both media. These results showed that sulfasalazine and erastin, but not RSL3, were efficacious under conditions of physiological L-cystine concentration, suggesting that medium conditions would be crucial for the design of a bioassay for system x_c- inhibitors.

Keyword

L-Cystine; Sulfasalazine; Erastin; Ferroptosis; System x_c; Glutathione peroxidase 4