Analysis of EPR/ESR (electron paramagnetic/spin resonance spectroscopy) in Ferroptosis
In 2003, Dolma et al. discovered that the small molecule erastin (an xCT inhibitor) kills cells. However, compared with camptothecin, erastin does not cause DNA fragmentation, caspase 3 activation and other classical characteristics of apoptosis 1). Instead, erastin was the first compound used to induce ferroptosis.
Ferroptosis, first named by American scientist Stockwell in 2012 2, 3), is a type of iron-dependent non-apoptotic cell death characterized by the accumulation of lipid peroxides in cell membranes. Mechanistically, several proteins regulate ferroptosis by limiting the production of lipid peroxides, including xCT 2), GPX4 4), FSP1 5, 6) and GCH1 7).
However, further research revealed that limitations exist within the current biological technology and some controversies remain. Ferroptosis is ROS (reactive oxygen species)-dependent and there are different kinds of ROS, such as peroxide, superoxide, hydroxyl radical and singlet oxygen. Identification of the ROS associated with the corresponding protein mechanism is required. It can readily catalyze the formation of ROS through processes such as the Fenton reaction because it can coordinate iron ions, which makes this compound highly toxic (Fig. 1) 11). Therefore, the hydroxyl radical and superoxide anion are the focus of research.
Although numerous reports on the cellular reduction system (antioxidant system) have been published, there remains a paucity of information about the oxidation system in the process of lipid peroxidation that causes ferroptosis. These missing data continue to drive controversy in this research field. Thus, determining the ability of drugs to produce free radicals is important when screening for ferroptosis inducers.
EPR/ESR (electron paramagnetic/spin resonance spectroscopy) is commonly used in environmental and catalytic chemistry research. In recent years, EPR/ESR has gained increasing popularity in biological research. Spin trapping is the mutual transformation of free radicals. Active free radicals are transformed into stable or semi-stable free radical addition products by using trapping agents. The use of spin trapping reagents such as DMPO and BMPO can facilitate classification and quantification of free radicals by using an electron spin resonance paramagnetic spectrometer. There is a large body of published work that has used this method in ferroptosis research journals 8, 9, 10).
Of note, the molecular weight (MW) of the spin trapping reagent is generally large (DMPO MW: 113; BMPO MW: 119) and cannot easily pass across the cell membrane. Moreover, the large number of different redox proteins in the organism reduce the stability of the DMPO/BMPO-radical in a cellular environment. Therefore, at present, DMPO/BMPO is primarily used for the analysis of free radicals in drugs, and there are only a few reports describing the direct detection of these reagents in cells or cell lysates.
[参考文献]
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1） S. Dolma et al., “Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells”, Cancer Cell, 2003, 3, 285-296.
2） B. R. Stockwell et al., “Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death”, Cell, 2012, 149, 1060-1072.
3） B. R. Stockwell et al., “Ferroptosis: Death by Lipid Peroxidation”, Trends Cell Biol., 2016, 26(3), 165-176.
4） W. S. Yang et al., “Regulation of Ferroptotic Cancer Cell Death by GPX4”, Cell, 2014, 156, 317-331.
5） K. Bersuker et al., “The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis”, Nature, 2019, 575, 688-692.
6） S. Doll et al., “FSP1 is a glutathione-independent ferroptosis suppressor”, Nature, 2019, 575, 693-698.
7） V. A. N. Kraft et al., “GTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling”, ACS Cent. Sci., 2019, 6(1), 41-53.
8） B. Yan et al., “Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1”, Molecular Cell, 2021, 81(2), 355-369.
9） X. Meng et al., “High-Performance Self-Cascade Pyrite Nanozymes for Apoptosis-Ferroptosis Synergistic Tumor Therapy”, ACS Nano, 2021,15(3), 5735-5751.
10） H. Ren et al., “Self-assembled FeS-based cascade bioreactor with enhanced tumor penetration and synergistic treatments to trigger robust cancer immunotherapy”, Acta Pharmaceutica Sinica B, 2021, in press.
11） J.-H. Su, J.-F. Du, “Electron Paramagnetic Resonance Spectroscopy: Principles and Applications”, 2021.
