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. 2018 Mar 28;4(3):387-396.
doi: 10.1021/acscentsci.7b00589. Epub 2018 Feb 7.

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis (VSports)

Ron Shah  1 , Mikhail S Shchepinov  2 , Derek A Pratt  1
Affiliations

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis

Ron Shah et al. ACS Cent Sci. .

Abstract

Lipoxygenases (LOXs) have been implicated as central players in ferroptosis, a recently characterized cell death modality associated with the accumulation of lipid hydroperoxides: the products of LOX catalysis. To provide insight on their role, human embryonic kidney cells were transfected to overexpress each of the human isoforms associated with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell lines that were demonstrably sensitized to ferroptosis. Interestingly, the cells could be rescued by less than half of a diverse collection of known LOX inhibitors. Furthermore, the cytoprotective compounds were similarly potent in each of the cell lines even though some were clearly isoform-selective LOX inhibitors. The cytoprotective compounds were subsequently demonstrated to be effective radical-trapping antioxidants, which protect lipids from autoxidation, the autocatalytic radical chain reaction that produces lipid hydroperoxides. From these data (and others reported herein), a picture emerges wherein LOX activity may contribute to the cellular pool of lipid hydroperoxides that initiate ferroptosis, but lipid autoxidation drives the cell death process. VSports手机版.

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Conflict of interest statement

The authors declare the following competing financial interest(s): M. S V体育安卓版. S. owns shares in Retrotope, Inc.

"V体育ios版" Figures

Figure 1
Figure 1
(A) Formation of cellular lipid hydroperoxides (LOOH) occurs primarily by iron-accelerated free radical autoxidation and LOX-catalyzed oxidation of polyunsaturated fatty acids. (B) Overexpression of 5-LOX, platelet 12-LOX, and 15-LOX-1 in HEK293 cells. (C) MS/MS transitions for the 5-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs) produced by the overexpressed LOXs. HETEs were analyzed following reduction of the corresponding hydroperoxyeicosatetraenoic acids (HPETEs) with TCEP. (D) LC/MS/MS chromatograms of organic extracts of cell lysates following incubation with 70 μM arachidonic acid for 10 min. (E, F) RSL3- and erastin-induced ferroptosis in each cell line following 4 and 24 h incubation, respectively. Data represent the mean ± SD of 3 independent experiments (P < 0.0001 and P ≤ 0.001 for LD50 values of RSL3 and erastin as determined by one-way ANOVA followed by Dunnett’s multiple comparisons test). (G) Flow cytograms of C11-BODIPY-treated cells supplemented with 70 μM AA.
Figure 2
Figure 2
Structures of some LOX inhibitors (A), their isoform-selectivity (B), and potency as inhibitors of RSL3-induced ferroptosis in the four different cell lines (C). Structures of common RTAs (D), their activity as inhibitors of the various LOX isoforms (E), and potency as inhibitors of RSL3-induced ferroptosis in the four different cell lines (F).
Figure 3
Figure 3
STY-BODIPY (A) serves as the signal carrier in inhibited autoxidations, enabling determination of inhibition rate constants (kinh) and stoichiometries (n) for reactions of inhibitors with chain-carrying peroxyl radicals (eqs 1 and 2, respectively). Coautoxidations of cumene (3.6 M) and STY-BODIPY (10 μM) initiated by AIBN (6 mM) in chlorobenzene at 37 °C (black) and inhibited by 2 μM of NDGA, zileuton, PD146176, CAY10649, CJ-13610, Lip-1, Fer-1, C15-THN, α-TOH, and PMC (B). Coautoxidations of egg phosphatidylcholine liposomes (1.0 mM) and STY-BODIPY (10 μM) suspended in phosphate-buffered saline (10 mM) at pH 7.4 initiated by MeOAMVN (0.2 mM) at 37 °C (black) and inhibited by 2 μM of NDGA, zileuton, PD146176, CAY10649, CJ-13610, Lip-1, Fer-1, C15-THN, α-TOH, and PMC (C). Inhibition rate constants obtained from coautoxidations of cumene (D) or phosphatidylcholine liposomes (E) with STY-BODIPY at 37 °C. *Rate constants were determined from styrene/PBD-BODIPY co-autoxidations, see ref. (20).
Figure 4
Figure 4
(A) Structures of deuterated arachidonic acids. HPETE formation in cells overexpressing 5-LOX (B), p12-LOX (C), and 15-LOX-1 (D) incubated with 70 μM AA (black), 7,7,10,10,13,13-d6-AA (red), 7,7-d2-AA (blue), 10,10-d2-AA (green), and 13,13-d2-AA (magenta). RSL3-induced ferroptosis in nontransfected HEK293 cells (E) and transfected cells overexpressing 5-LOX (F), p12-LOX (G), and 15-LOX-1 (H) preincubated with 40 μM arachidonic acid (black, ●), 7,7-d2-AA (blue, ▲), 10,10-d2-AA (green, ◆), 13,13-d2-AA (magenta, ▼), and 7,7,10,10,13,13-d6-AA (red, ■).
Figure 5
Figure 5
Formation of 5-HETE (blue, ●), 12-HETE (red, ■), and 15-HETE (green, ▲) in nontransfected HEK293 cells (A), and cells overexpressing 5-LOX (B), p12-LOX (C), and 15-LOX-1 (D) treated with 5 μM RSL3 and 40 μM arachidonic acid. Cell viability is overlaid on each graph (red ×). (E) Sensitization of wild-type cells to ferroptosis with 0.5 μM RSL3 in the presence of various concentrations of exogenously prepared hydroperoxides: oleic 10-OOH (green), cholesterol-7-OOH (red), tert-dodecyl-OOH (blue), or PC-oleate 10-OOH (black). (F) Viability of HEK293 cells exposed to oleic-10-OOH in the presence of 0.5 μM RSL3 (black, ●) inhibited with 3.1 (red, ■), 6.3 (blue, ▲), 12.5 (green, ◆), 25 (magenta, ●), 50 (cyan, ▼), or 100 (yellow, ★) nM Lip-1.
Figure 6
Figure 6
Kinetic expressions for uninhibited and inhibited lipid autoxidation (A). Schematic demonstrating the interplay between small molecule and enzymatic inducers and inhibitors of ferroptotic cell death associated with the accumulation of (phospho)lipid hydroperoxides (B).
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