Ferroptosis Inducer RSL3 Inhibits Cell Death by Camptothecin-Induced Apoptosis

Abstract

Ferroptosis, an iron-dependent type of cell death, is being considered for new clinical treatments of malignant tumors that are difficult to treat with apoptosis inducers. Although several reports have attempted to increase the sensitivity of cells to cell death by combining ferroptosis and apoptosis inducers using a single treatment, detailed elucidation of the respective mechanisms of ferroptosis and apoptosis during cell death remains unclear. Here, we evaluated combined treatment effectiveness using the apoptosis-sensitive rat insulinoma INS-1 cell lines. DNA laddering, an indicator of camptothecin (CPT)-induced apoptosis, was abolished by adding RSL3 and ML-162, but not erastin. We found that when the cells were treated with the apoptosis inducer CPT or the ferroptosis inducer RSL3, respectively, the degree of cytotoxicity observed increased dose-dependently. However, a combined CPT and RSL3 treatment did not show a synergistic decrease in cell viability. Camptothecin did not significantly affect increases in intracellular lipid peroxidation and reactive oxygen species or increases in mitochondrial and cytoplasmic free iron levels that were induced by treatment with RSL3 alone. Moreover, deferoxamine and α -tocopherol were found to inhibit RSL3-induced cytotoxicity but did not protect against CPT or CPT and RSL3-induced cytotoxicity. Finally, the exogenous addition of tert -butyl hydroperoxide inhibited DNA ladder formation that is induced by CPT, while the addition of hydrogen peroxide or ferrous ammonium sulfate had no effect. Taken together, these results suggest that lipid peroxides generated during ferroptosis may suppress cell death induced by apoptotic mechanisms.