Differential Contributions of Distinct Free Radical Peroxidation Mechanisms to the Induction of Ferroptosis

Ferroptosis is a form of regulated cell death driven
by lipid peroxidation
of polyunsaturated fatty acids (PUFAs). Lipid peroxidation can propagate
through either the hydrogen-atom transfer (HAT) or peroxyl radical
addition (PRA) mechanism. However, the contribution of the PRA mechanism
to the induction of ferroptosis has not been studied. In this study,
we aim to elucidate the relationship between the reactivity and mechanisms
of lipid peroxidation and ferroptosis induction. We found that while
some peroxidation-reactive lipids, such as 7-dehydrocholesterol, vitamins
D3 and A, and coenzyme Q10, suppress ferroptosis, both
nonconjugated and conjugated PUFAs enhanced cell death induced by
RSL3, a ferroptosis inducer. Importantly, we found that conjugated
PUFAs, including conjugated linolenic acid (CLA 18:3) and conjugated
linoleic acid (CLA 18:2), can induce or potentiate ferroptosis much
more potently than nonconjugated PUFAs. We next sought to elucidate
the mechanism underlying the different ferroptosis-inducing potency
of conjugated and nonconjugated PUFAs. Lipidomics revealed that conjugated
and nonconjugated PUFAs are incorporated into distinct cellular lipid
species. The different peroxidation mechanisms predict the formation
of higher levels of reactive electrophilic aldehydes from conjugated
PUFAs than nonconjugated PUFAs, which was confirmed by aldehyde-trapping
and mass spectrometry. RNA sequencing revealed that protein processing
in the endoplasmic reticulum and proteasome are among the most significantly
upregulated pathways in cells treated with CLA 18:3, suggesting increased
ER stress and activation of unfolded protein response. These results
suggest that protein damage by lipid electrophiles is a key step in
ferroptosis.