Beyond Lipoxygenase: Studying the Initiation of Ferroptosis & On the Mechanism Behind α-Eleostearic Acid Autoxidation

Abstract

Ferroptosis is a recently characterized cell death pathway associated with the iron-dependent accumulation of lipid hydroperoxides in phospholipid bilayers. The origin of these hydroperoxides has been an ongoing topic of debate and many researchers argue for a lipoxygenase (LOX) enzyme-controlled mechanism of initiation, given their known role as dioxygenases of polyunsaturated fatty acids (PUFAs). In response to this, our lab investigated the induction and inhibition of ferroptosis in human embryonic kidney (HEK-293) cells transfected to overexpress the three most prevalent LOX isoforms, 5-LOX, p12-LOX, and 15-LOX-1. These studies did not support a role for LOX in the execution of ferroptosis; LOX inhibition was not associated with ferroptosis suppression and in fact, anti-ferroptotic activity was directly tied to purported LOX inhibitors’ ability to act as radical-trapping antioxidants (RTAs). We have investigated the effects of LOX inhibitors on ferroptosis in human fibrosarcoma (HT-1080) cells, the cell line in which ferroptosis was initially characterized, and mouse hippocampal neuronal (HT-22) cells, the cell line in which the closely related cell death modality oxytosis was characterized. In sum, our findings mirror those obtained in HEK-293 cells, and the effectiveness of an inhibitor is tied to its off-target RTA activity, not inhibition of LOX. Moreover, we observed suppression of ferroptosis via necrostatin-1 (Nec-1), a known receptor-interacting serine/threonine-protein kinase 1 (RIPK1) (and necroptosis) inhibitor. Herein, we show that Nec-1 is not an RTA and exerts its effects by a yet unknown mechanism which we investigate in a series of exploratory experiments. Conjugated fatty acids – particularly α-ESA – have recently been reported to induce ferroptosis by an unclear mechanism. Theorizing this phenomenon was tied to the autoxidation of α-ESA’s conjugated trienic unit, we aimed to investigate the kinetic and biological properties of natural α-ESA alongside a deuterated isotopologue. Herein, we report preliminary work to derive biologically relevant rate constants for addition and hydrogen-atom transfer (HAT) of α-ESA. Moreover, we report our progress towards the synthesis of a deuterated α-ESA which will facilitate future study alongside its natural counterpart.