Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation-induced ferroptosis.

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation-induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation-induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density-induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation-induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma.

Role of ferritinophagy in cystine deprivation-induced cell death in glioblastoma cells.

Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation. Cancer cells increase cystine uptake for the synthesis of glutathione (GSH), which is used by glutathione peroxidase 4 to reduce lipid peroxides. Here, we report that cystine deprivation in glioblastoma cells, but not inhibition of GSH synthesis by l-buthionine sulfoximine (BSO), induces ferroptosis. We found that cystine deprivation decreased the protein levels of ferritin heavy chain FTH1, whereas it was increased by BSO treatment. The lysosome inhibitor bafilomycin A1 or deletion of nuclear receptor coactivator 4 (NCOA4) inhibited cystine deprivation-induced decrease in FTH1 protein levels and cell death. In addition, cystine deprivation induced microtubule-associated protein light chain 3 (LC3)-II protein accumulation, suggesting that cystine deprivation induces ferritinophagy. BSO causes cell death when glioblastoma cells are treated with iron inducers, ferrous ammonium sulfate or hemin. On the other hand, cystine deprivation-induced degradation of FTH1 and cell death required glutamine. This study suggests that ferritinophagy, in addition to GSH depletion, plays an important role in cystine deprivation-induced ferroptosis in glioblastoma cells.