FINO2 initiates ferroptosis through GPX4 inactivation and iron oxidation

Abstract

Ferroptosis is a non-apoptotic form of regulated cell death caused by the failure of the glutathione-dependent lipid-peroxide-scavenging network. FINO₂ is an endoperoxide-containing 1,2-dioxolane that can initiate ferroptosis selectively in engineered cancer cells. We investigated the mechanism and structural features necessary for ferroptosis initiation by FINO₂. We found that FINO₂ requires both an endoperoxide moiety and a nearby hydroxyl head group to initiate ferroptosis. In contrast to previously described ferroptosis inducers, FINO₂ does not inhibit system x_c^- or directly target the reducing enzyme GPX4, as do erastin and RSL3, respectively, nor does it deplete GPX4 protein, as does FIN56. Instead, FINO₂ both indirectly inhibits GPX4 enzymatic function and directly oxidizes iron, ultimately causing widespread lipid peroxidation. These findings suggest that endoperoxides such as FINO₂ can initiate a multipronged mechanism of ferroptosis.

Figure 1. FINO₂ induces ferroptotic cell death

(A) Organic peroxides and FINO₂ (B) The dose-dependent effect of cell death-suppressing compounds on ferroptosis triggered by FINO₂ (10 μM) in HT-1080 cells. Viability measured 24 h after compound treatment. Experiments were performed with triplicate cell cultures. Data are plotted as the mean ± s.d., n=3. (C) Ability of iron chelator deferoxamine (DFO) to prevent ferroptosis-dependent C11-BODIPY oxidation when incubated together for 6 h. Three independent experiments were performed with similar results. (D) Ability of ferrostatin-1 (Fer-1) (2 μM) to prevent accumulation of thiobarbituric acid reactive substances (TBARS) when co-treated with erastin (5 μM) or FINO₂ (10 μM) for 6 h. Data are plotted as the mean ± s.d., n=5. P values were determined using one-way ANOVA; *P=0.003, **P < 0.001 versus DMSO control. (E) Changes in oxidized phosphatidylethanolamine abundance as detected by LC-MS after treatment with FINO₂ (10 μM) for 6 h. Individual lipid species are plotted based on their Log₂ fold change in abundance (horizontal axis) and the statistical significance of the change (Log₁₀ P-value) on the vertical axis. P values were determined using two-sided t test. Lipid species with significant change upon FINO₂ treatment were plotted above the dot line (p<0.05). Experiments were performed in triplicate with biologically independent samples.

Figure 2. FINO₂ does not alter glutathione homeostasis

(A) Effect of FINO₂ (10 μM) and system x_c⁻ inhibitors erastin (10 μM) and sulfasalazine (1 mM) on glutamate release after 1 h incubation. Data are plotted as the mean ± s.d., n=3 biologically independent samples. **P and ^#P < 0.001 versus DMSO control and Erastin, respectively. (B) Intracellular GSH levels in HT-1080 cells treated with ferroptosis inducers RSL3 (0.5 μM) for 90 m or erastin (5 μM), and FINO₂ (10 μM) for 6 h, data are plotted as the mean ± s.d., n=8 biologically independent samples. *P=0.016, **P<0.001 versus DMSO control. (C) CHAC1 mRNA levels following erastin (10 μM) or FINO₂ (10 μM) treatment for 6 h. Data are plotted as the mean ± s.d., n=3 biologically independent samples. *P=0.007, **P<0.001 versus DMSO control; ^#P < 0.001 versus erastin. All P values were determined using one-way ANOVA.

Figure 3. FINO₂ indirectly inhibits GPX4 activity

(A) Effect of ferroptosis inducers on GPX4 activity within the GPX4-containing cell lysates. Cell lysates were treated with PCOOH and GSH and the abundance of PCOOH was measured by LC-MS. Data are plotted as the mean ± s.d., n=3 biologically independent samples. *P=0.009, **P < 0.001 versus DMSO control (B) HSQC spectrum of GPX4 protein (black; 50 μM) overlaid with the spectrum of GPX4 incubated with FINO₂ for 6 h (red; 50 μM and 500 μM, respectively) (C) PTGS2 mRNA levels following treatment with RSL3 (0.5 μM), erastin (10 μM), and FINO₂ (10μM) for 6 h. Data are plotted as the mean ± s.d., n=3 biologically independent samples. ^#P=0.021, ^##P=0.017 versus RSL3. (D) Ability of β-mercaptoethanol to prevent ferroptosis initiated by different ferroptosis inducers. Viability measured 24 h after co-treatment. Data are plotted as the mean ± s.d., n=12 biologically independent samples. ^##P < 0.001 versus RSL3. (E) GPX4 protein abundance in HT-1080 cells co-treated with RLS3 (1 μM), erastin (10 μM), FIN56 (5 μM), or FINO₂ (10 μM) and 100 μM α-tocopherol for 10 h, data are plotted as the mean ± s.d., n=3 biologically independent samples. ^#P=0.002, ^##P<0.001 versus FIN56. Representative blot image is shown in Supplementary Figure 7. All P values were determined using unpaired two-tailed Student’s t-test.

Figure 4. Potency of non-peroxide analogs (A) and peroxide analogs (B) of FINO₂

BJ-eLR, BJ-hTERT, or CAKI-1 cells were treated with either vehicle (DMSO), FINO₂, or a FINO₂ analogue at 5, 10, 25, 50, or 100 μM concentrations for 48 h. Cell viability was then measured and normalized to the DMSO vehicle to extract EC₅₀ values. EC₅₀ values are shown in parentheses. Compounds 15, 31, 32 and 37 were tested as a mixture of diastereomers

Figure 5. FINO₂ directly oxidizes ferrous ion

(A) Oxidation of ferrous iron (500 μM) in the presence of different oxidizing agents (500 μM). Data are plotted as the mean ± s.d., n=4 biologically independent samples. P values were determined using unpaired two-tailed Student’s t-test. *P = 0.035, **P<0.001 versus DMSO control. (B) ¹H-NMR showing degradation of FINO₂ following incubation with FeSO₄•7H₂O in CD₃CN/D₂O (1:1) for 12 h. (C) Ability of iron chelator deferoxamine (DFO) to inhibit ferroptosis initiated by different ferroptosis inducers. Experiments were performed in triplicate with three biologically independent samples. Data are plotted as the mean ± s.d..

Figure 6. Ferroptosis initiated by FINO₂ oxidizes a large subset of the lipidome independent of lipoxygenase activity

(A) Schematic of lipoxygenase inhibition by deuterated arachidonic acid. (B) Effect of deuterated arachidonic acid incubation (80 μM for 24 h) on HT-1080 sensitivity to ferroptosis inducers. Experiments were performed in triplicate with biologically independent samples. Data are plotted as the mean ± s.d.. (C) Volcano plots showing the change in abundance of oxidized phosphatidylethanolamine species in HT-1080 cells following incubation with FINO₂ (10 μM) or erastin (5 μM) for 6 h. Red circles indicate significant increase in abundance, blue circles indicate a significant depletion following treatment. Experiments were performed in triplicate with biologically independent samples. P values were determined using two-sided t test. Lipid species with significant change upon FINO₂ treatment were plotted above the dot line (p<0.05). (D) Number of oxidized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin (CL) species upregulated in HT-1080 cells following treatment with FINO₂ (10 μM) or erastin (5 μM) for 6 h.