Dithiolethiones D3T and ACDT Protect Against Ferroptosis Induced by Erastin and RSL3

Abstract

Ferroptosis is an iron‐dependent and non‐apoptotic form of cell death characterized by increased lipid peroxidation. It is morphologically, biochemically, and genetically different from other types of cell death. Erastin and RSL3 are the canonical inducers of ferroptosis. While erastin inhibits the cystine/glutamate antiporter (xCT) leading to intracellular glutathione (GSH) depletion, RSL3 directly inhibits the glutathione peroxidase‐4 (GPX4) enzyme, resulting in increased lipid peroxidation and ferroptosis. Dithiolethiones 3H‐1,2‐dithiole‐3‐thione (D3T) and 5‐amino‐3‐thioxo‐3H‐(1,2) dithiole‐4‐carboxylic acid ethyl ester (ACDT) are lipophilic, organosulfur compounds that are known to activate the Nrf2 transcription factor and induce the cellular antioxidant machinery. They have previously been shown to be cytoprotective against various oxidative stressors both in vitro and in vivo. Furthermore, as lipophilic antioxidants are known to oppose ferroptosis and the activation of Nrf2 has been reported to inhibit this form of cell death, the present study was aimed to evaluate the protective effect of dithiolethiones against erastin‐ and RSL3‐induced ferroptosis in U‐87 MG human astrocytoma cells. Ferrostatin‐1, a known ferroptosis inhibitor was used as a standard comparator. Cells were pretreated with either 50 µM D3T, ACDT, or 10 µM ferrostatin‐1 for 24 hours followed by another 24 hour exposure to either 20 µM erastin or 10 µM RSL3. Pretreatment with dithiolethiones reversed erastin‐induced cell death measured by MTS cell viability assay and upregulated xCT transporter expression indicated by western blotting. Dithiolethiones increased intracellular GSH (GSH‐Glo luminescence assay) and alleviated lipid peroxidation (TBARS assay) against erastin. RSL3‐induced ferroptosis and lipid peroxidation were also counteracted by both the dithiolethiones, however no effect was observed on GPX4 enzyme levels. These effects of dithiolethiones were comparable to those of the standard ferroptosis inhibitor ferrostatin‐1. Collectively, our data indicate a strong ability of dithiolethiones to counteract ferroptosis, and is the first such study in astrocytes. D3T and ACDT can therefore be potential therapeutic candidates in the treatment of ferroptosis‐related brain disorders.