Defining a pharmacological inhibitor fingerprint for oxytosis/ferroptosis

Abstract

Ferroptosis was first described in 2012 as an iron- and lipid peroxidation-dependent form of regulated cell death. Since its initial description, these two characteristics have informed numerous cell culture studies where inhibitors of lipid peroxidation and/or iron chelators have been shown to prevent cell death induced by a wide range of insults. However, it is not clear whether these two characteristics are sufficient to distinguish ferroptosis from other forms of regulated cell death. Thus, the primary goal of this study was to determine whether a unique combination of features could be identified that would provide an approach to more clearly separate ferroptosis from other forms of regulated cell death. To this end, multiple pharmacological inhibitors based on a variety of studies were tested. Many of these inhibitors were previously shown to protect cells from oxytosis, a regulated cell death pathway that mechanistically overlaps with ferroptosis and is induced by some of the same chemicals as ferroptosis. These inhibitors were not only tested against both known ferroptosis and oxytosis inducers but also a number of other insults that have been suggested to induce ferroptosis. The results show that a pharmacological fingerprint for ferroptosis can be established and used to categorize toxic insults into those that overlap with oxytosis/ferroptosis and those that do not.

Keywords: Glutathione; Iron chelators; Lipid peroxidation; Mitochondria; Radical trapping antioxidant.

Figure 1:

Oxytosis/Ferroptosis pathway showing the targets of most of the pharmacological inhibitors tested.

Figure 2:. Effect of HIF1α knockdown on protection by deferiprone and CoCl₂.

HIF1α was knocked down using specific siRNA as described in Materials and Methods (A). Cells treated with control siRNA or HIF1α siRNA were then assayed for the ability of either deferiprone (DF) or CoCl₂ (Co) to protect from glutamate (glu, 5 mM), erastin (eras, 500 nM) or RSL3 (250 nM) toxicity (B).

Figure 3:

(A) Increased efficacy of clorgyline against RLS3 toxicity. HT22 cells in 96 well plates were treated overnight with the indicated concentrations of clorgyline and glutamate (5 mM), erastin (500 nM) or RSL3 (250 nM). Cell survival was measured the next day by the MTT assay. (B) Time dependent changes in ATP levels following treatment of HT22 cells with glutamate (5 mM), erastin (500 nM) or RSL3 (250 nM). Cells were treated in 35 mm dishes and then harvested at the indicated times for measurement of ATP levels. (C) Time dependent changes in ATP levels following treatment of HT22 cells with H₂O₂ (750 μM), t-BOOH (5 μM) or IAA (20 μM). (D) Time dependent changes in ATP levels following treatment of HT22 cells with paraquat (PQ, 2.5 mM), 6OHDA (500 μM), CdCl₂ (50 μM) or cisplatin (100 μM). Cells were treated in 35 mm dishes and then harvested at the indicated times for measurement of ATP levels.

Figure 4:. Potentiation of the toxicity of oxytosis/ferroptosis inducers by pCPT-cGMP.

(A) HT22 cells in 96 well plates were treated with vehicle (cGMP alone) or fixed concentrations of glutamate (2 mM), erastin (100 nM) or RSL3 (50 nM) and the indicated concentrations of pCPT-cGMP. Cell survival was measured the next day by the MTT assay. ***p < 0.001 relative to pCPT-cGMP alone. HT22 cells in 96 well plates were treated with (B) glutamate alone or glutamate + 1 mM pCPT-cGMP; (C) erastin alone or erastin + 1 mM pCPT-cGMP; and (D) RSL3 alone or RSL3 + 1 mM pCPT-cGMP. Cell survival was measured the next day by the MTT assay. *p <0.05; **p < 0.01; ***p < 0.001 relative to glutamate, erastin or RSL3 alone.

Figure 5:

(A) Effects of ATG5 knockdown on glutamate (5 mM), erastin (500 nM) or RSL3 (250 nM)-induced cell death in HT22 cells. ATG5 was knocked down with specific siRNA as described in Materials and Methods and then the sensitivity to the oxytosis/ferroptosis inducers was assayed. *p <0.05; ***p < 0.001 relative to glutamate, erastin or RSL3 alone. (B) Effects of ATG5 knockdown on glutamate (50 mM), erastin (2.5 μM) or RSL3 (1 μM)-induced cell death in HT1080 cells. ATG5 was knocked down with specific siRNA as described in Materials and Methods and then the sensitivity to the oxytosis/ferroptosis inducers was assayed. Inset shows level of knockdown. (C) Time dependent changes in the LC3II/LC3I ratio and the levels of ferritin heavy chain (FTH) following treatment of HT22 cells with glutamate or erastin. (D) Time dependent changes in the LC3II/LC3I ratio and the levels of ferritin heavy chain (FTH) following treatment of HT22 cells with RSL3. Cells were treated in 35 mm dishes and then harvested at the indicated times for Western blotting using the indicated antibodies. Inset shows representative blots. Graphed results are the average of 3 independent experiments.

Figure 6:. In vitro radical trapping antioxidant activity of the oxytosis/ferroptosis inhibitors.

Co-autoxidation of STY-BODIPY (1.5 μM) and the polyunsaturated lipids of egg-phosphatidylcholine liposomes (1 mM). The indicated compounds (10 μM) were added to the STY-BODIPY/liposome mix and incubated at 37°C for 10 min. Then, lipid autoxidation was initiated using V-70 (0.5mM) and monitored by measuring the fluorescence increase over time at 37°C. Graph shows the average of n=4 representative experiments.

Figure 7:

Final pharmacological fingerprint and heat map of cell death inducers versus the inhibitors.