Ferroptosis and Reagent Selection Guide

What is Ferroptosis?

“Ferroptosis” was coined by Stockwell et al. at Columbia University in 2012 and described as a form of iron-dependent cell death. * It was reported to be a form of programmed cell death by the Nomenclature Committee on Cell Death (NCCD) in 2018.
Ferroptosis is a form of programmed cell death that is caused by iron ion-dependent accumulation of lipid peroxides. Ferroptosis has been shown to follow a different cell death pathway from apoptosis and thus is attracting attention as a new target for cancer therapy. It has also been found to be associated with various diseases, such as neurodegenerative diseases, cerebral apoplexy, and hepatitis (NASH).

*S. J. Dixon, B. R. Stockwell et al., Ferroptosis: An iron-dependent form of nonapoptotic cell death.Cell2012, 149(5), 1060.

How Does Ferroptosis Cause Cell Death?

Ferroptosis is characterized by the accumulation of lipid peroxides. Lipid peroxides are formed from oxidation of polyunsaturated fatty acids (PUFA) in membrane phospholipids, with iron suggested to be involved. Intracellular glutathione peroxidase 4 (GPX4) uses reduced glutathione (GSH), an antioxidant, to reduce lipid peroxides generated by reactive oxygen species (ROS).*
However, when lipid peroxides accumulate due to GPX4 disruption or GSH depletion, ferroptosis is triggered.

* Stockwell et al, a leading researcher in the field of ferroptosis, summarized inhibitors, inducers, and detection indicators of ferroptosis in the following review, in which Dojindo’s Liperfluo is introduced for detection of lipid peroxides.

B. R. Stockwell, et al., “Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.”Cell2017, 171, 273.

Induction of Ferroptosis by Erastin?

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

<Experimental example>
Using erastin-treated A549 cells, we measured intracellular Fe2+, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe2+ , ROS, and lipid peroxides.

①Cystine Uptake
(Under Development)
②Released Glutamate
Glutamate Assay Kit-WST
③Intracellular
Glutathione
GSSG/GSH Quantification Kit

④Intracellular Fe2+
FerroOrange
⑤Intracellular ROS
ROS Assay Kit -Highly Sensitive DCFH-DA
⑥Intracellular Lipid Peroxide
Liperfluo
Nonalcoholic steatohepatitis (NASH)
Suppression of hepatitis via ferroptosis
In a study involving the livers of NASH model mice, it was confirmed that necrosis precedes apoptosis in the development of fatty liver. Further experiments showed that ferroptosis is involved within necrosis as a trigger for steatohepatitis and that inhibition of ferroptosis almost completely suppressed the onset of hepatitis.

Minoru Tanaka, et al., “Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis”Cell Death & Disease2019, 10, 449.
Related article: changes in intracellular markers associated with NASH

The article summarizes reports on changes in each indicator of metabolic states and cellular senescence using the NASH model.
(Click on the “NASH” tab in the link)


Experimental example: measurement of intracellular metabolism in NASH model tissue
Measurement of ATPa-KG, and NAD levels in liver tissue of high-fat diet-treated type 1 diabetic model mice. (Please refer to each product’s website for more information, “Experimental Example: Change in Metabolism in Liver Tissue of NASH-Induced Mouse”)
Neurodegenerative disease
Confirmation of the link between lysosomal disorders and ferroptosis
In experiments using human neurons, it is reported that knockdown of the lysosomal protein prosaposin induces formation of lipofuscin, a hallmark of aging. This process involves the iron-catalyzed generation of reactive oxygen species, leading to induction of ferroptosis.

Martin Kampmann, et al., “Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis”Nature Neuroscience2021, 24, 1020
Cancer
Regulation of cancer immunity via ferroptosis
CD8+ T cells activated by immunotherapy were found to confer an anti-tumor effect by promoting lipid peroxidation and inducing ferroptosis. The mechanism of immunotherapy-induced inhibition of cystine uptake and promotion of lipid peroxidation in tumor cells is discussed.

Weiping Zou, et al, “CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy”Nature2019, 569, 270
Ferroptosis
Ferroptosis – a newly identified, iron-dependent form of programmed cell death
A summary of the current progress in studying ferroptosis, as well as its potential applications in the fields of biology and medicine.

Fudi Wang, et al., “Ferroptosis: Beauty or the Beast“, Dojin News2021, 178, 1
Lipid Peroxide and Iron (Fe2+) Detection Reagents
NameLiperfluoMitoPeDPPMito-FerroGreenFerroOrange
TargetLipid PeroxidationLipid PeroxidationFerrous Ion(Fe2+)Ferrous Ion(Fe2+)
LocalizationIntracellularMitochondriaMitochondriaIntracellular
Detection
(Fluorescence:Ex/Em)
Fluorescence
(524 nm/535 nm)
Fluorescence
(452 nm/470 nm)
Fluorescence
(505 nm/580 nm)
Fluorescence
(543 nm/580 nm)
InstrumentFluorescence Microscope,
FCM
Fluorescence Microscope,
FCM
Fluorescence Microscope,
Microplate Reader
Fluorescence Microscope
SampleLive CellLive CellLive CellLive Cell
NameROS Assay Kit -Highly Sensitive DCFH-DA-GSSG/GSH Quantification KitGlutamine Assay Kit-WSTGlutamate Assay Kit-WST
TargetROS (Reactive oxygen species)Glutathione (oxidized/reduced)
GlutamineGlutamine
LocalizationIntracellularIntracellularIntracellular/ExtracellularIntracellular/Extracellular
Detection
(Fluorescence:Ex/Em)
Fluorescence
(505 nm/525 nm)
Colorimetric:412 nmColorimetric:450 nmColorimetric:450 nm
InstrumentFluorescence Microscope,
FCM,
Microplate Reader
Microplate ReaderMicroplate ReaderMicroplate Reader
SampleLive CellCell, Tissue, Blood Plasma, Red Blood CellCell, Cell CultureCell, Cell Culture
Beginner’s Guide to Cellular Metabolism

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