Off-Target Activities of Lipoxygenase Inhibitors Confound the Role of Enzyme-Catalyzed (Phospho)Lipid Peroxidation in Ferroptosis

Abstract

Ferroptosis is a recently characterized iron-dependent form of regulated cell death associated with the accumulation of (phospho)lipid hydroperoxides. Since its characterization, there has been a spirited debate in the literature over the origin of the lipid hydroperoxides in ferroptotic cells. Many investigators have implicated lipoxygenases (LOXs), enzymes known to catalyze the oxidation of polyunsaturated fatty acids (especially linoleate and arachidonate) to yield lipid hydroperoxides. Previous work by our group, investigated the induction and suppression of ferroptosis in human embryonic kidney (HEK-293) cells transfected to overexpress the three most widespread isoforms of LOX (5-LOX, 15-LOX-1 and p12-LOX). The results suggested that LOX catalysis is not required for ferroptosis. Our previous work did not include investigations into cells transfected to overexpress 15-LOX-2. However, a series of recent publications has since implicated the 15-LOX-2/PEPB1 complex as a key player in ferroptotic cell death. Therefore, in this work, HEK-293 cells were transfected to overexpress the 15-LOX-2 isoform, as confirmed by immunodetection, and were subject to induction and suppression of ferroptosis pharmacologically. A library of small molecules was assembled consisting of LOX inhibitors, radical-traping antioxidants (RTAs) and LOX inhibitors that display off-target RTA activity. Consistent with our previous investigations, only LOX inhibitors with radical trapping activity or iron chelators were effective at suppressing ferroptosis. Furthermore, the poor performance of 15-LOX-2 inhibitors at rescuing cells transfected to overexpress 15-LOX-2 from ferroptosis does not support the role of the 15-LOX-2/PEBP1 complex as a central mediator of ferroptotic lipid peroxidation. We also report the details of corresponding investigations in cell lines that are reported to express high levels of LOXs and that have been used to establish characteristics of ferroptosis, including HT-22 mouse hippocampal cells (15-LOX-1 and/or 15-LOX-2) and HT-1080 human fibrosarcoma cells (all LOXs). The two cellular models were also subject to cell-rescue studies with our small molecule library. Again, only LOX inhibitors that possess radical-trapping antioxidant activity or which are good iron chelators could rescue cells from ferroptosis. These results underscore our previous conclusion that although lipoxygenase activity may contribute to the pool of cellular lipid hydroperoxides, autoxidation drives ferroptotic cell death.