Unusual Switch of Cell Death Mode by Docosahexaenoic Acid (DHA) via Enhancing Endoplasmic Reticulum Peroxidation Induced by Lipophilic Iron Complex

Although ferroptosis is a programmed iron-dependent cell death, many lipophilic iron complexes fail to induce ferroptosis through iron overload. The underlying mechanisms remain unclear. Here we found that a typical lipophilic iron complex 8-hydroxyquinoline (8HQ)-Fe(III) (1: 1, 10 μM), can efficiently induce cellular iron overload, lipid peroxidation, and finally cytotoxicity. However, the resulting cell death mode was not typical ferroptosis: it was not inhibited by the classic ferroptosis inhibitors and exhibited mitochondrial swelling rather than shrinkage. Mechanistic studies revealed that 8HQ-Fe(III) triggered both endoplasmic reticulum (ER) peroxidation and mitochondrial damage. The ER-targeted antioxidant (stobadine) inhibited lipid peroxidation but did not suppress cell death. In contrast, mitochondrial protectants effectively blocked 8HQ-Fe-induced cell death by mitigating mitochondrial damage. This indicates that mitochondrial damage plays an overwhelmingly dominant role in driving cell death. Strikingly, pre-incubation with docosahexaenoic acid (DHA) can strongly enrich the ER membrane and increase its peroxidation. As expected, both stobadine and the well-known ferroptosis inhibitor (Fer-1) suppressed 8HQ-Fe(III)-induced cell death via inhibition of ER peroxidation, while mitochondrial protectants failed to prevent cell death. Mitochondrial shrinkage was also observed. Thus, making ER peroxidation the dominant driver of cell death by DHA pretreatment will switch the cell death modality from non-ferroptosis to classical ferroptosis. This study revealed that the balance between ER peroxidation and mitochondrial damage dictated the type of iron-dependent cell death, and established DHA-primed 8HQ-Fe(III) as a novel, tunable strategy for efficiently inducing ferroptosis by deliberately shifting subcellular peroxidation dominance.