Abstract

Introduction. Ferroptosis, an iron-dependent form of nonapoptotic cell death, has been identified as a type of death that could be activated in certain types of cancer. In addition, a popular hypothesis suggests that mega doses of the reduced form of vitamin C, ascorbic acid (AA), can act as an anti-cancer pro-drug. However, several cell types are resistant to mega doses of AA, such as glioblastoma. Interestingly, treatment with the oxidized form of vitamin C, dehydroascorbic acid (DHA), induces cell death by consumption of glutathione (GSH) and increasing reactive oxygen species (ROS) and lipid ROS in certain cell types. To date, it is unknown how to sensitize cells resistant to ferroptosis to this type of death. Here, we show that glioblastoma cells treated with erastin-DHA cotreatment are sensitized to death by ferroptosis. Materials and methods. Human glioblastoma cells (U87) and normal primary rat astrocytes were treated with erastin, RSL-3, iron overload, or glutamate and were cotreated with DHA. Ferrostain and -mercaptoethanol were used as ferroptosis inhibitors. Additionally, we evaluated the participation of autophagy in ferroptosis sensitization by DHA with bafilomycin A, chloroquine, and NH4Cl. Cell death was monitored by the incorporation of Sytox green with real-time live-cell microscopy using an IncuCyte S3 system. Total (CellRox), mitochondrial (mitosox) and lipid (Image-iT lipid perdoxidation) ROS levels were determined by FACS. We also evaluated the expression of GPX4, ACSL4, IREB2 and PTGS2 by qPCR, and mitochondrial fragmentation using superresolution microscopy and mitotracker. Results. We found that human glioblastoma cells are resistant to conventional ferroptosis induction therapies (Erastin, RSL3, iron overload or glutamate treatment) despite expressing ACSL4. Interestingly, DHA-erastin co-treatment sensitizes glioblastoma cells to ferroptosis. While in primary astrocytes, treatment with DHA inhibits erastin-induced ferroptosis, suggesting that DHA-erastin would be specific for tumor cells. Acknowledgements: Fondecyt 1181243; CMA BIO BIO, PIA-CONICYT, ECM-12.