Ferroptosis DOJINDO LABORATORIES

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., Cell, 2012, 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.", Cell, 2017, 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: Cystine Uptake Assay Kit

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

Research on Related Diseases

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 & Disease, 2019, 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 ATP, a-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”)

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 & Disease, 2019, 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 ATP, a-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 ferroptosisIn 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 Neuroscience, 2021, 24, 1020

				Cancer
			
				Regulation of cancer immunity via ferroptosisCD8+ 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", Nature, 2019, 569, 270

				Ferroptosis
			
				Ferroptosis – a newly identified, iron-dependent form of programmed cell deathA 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 News, 2021, 178, 1

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 Neuroscience, 2021, 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", Nature, 2019, 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 News, 2021, 178, 1

Ferroptosis-Related Reagent Selection Guide

Lipid Peroxide and Iron (Fe²⁺) Detection Reagents

Product name	Liperfluo	MitoPeDPP	Mito-FerroGreen	FerroOrange	MDA Assay Kit
Target	Lipid Peroxidation	Lipid Peroxidation	Ferrous Ion(Fe²⁺)	Ferrous Ion(Fe²⁺)	Malondialdehyde
Localization	Intracellular	Mitochondria	Mitochondria	Intracellular	Intracellular
Detection (Fluorescence:Ex/Em)	Fluorescence (524 nm/535 nm)	Fluorescence (452 nm/470 nm)	Fluorescence (505 nm/580 nm)	Fluorescence (543 nm/580 nm)	Fluorescence (540 nm/590 nm) Colorimetric(532 nm)
Instrument	Fluorescence Microscope, FCM	Fluorescence Microscope, FCM	Fluorescence Microscope, Microplate Reader	Fluorescence Microscope, Microplate Reader	Microplate Reader
Sample	Live Cell	Live Cell	Live Cell	Live Cell	Cell or Tissue

Oxidative Stress and Metabolism-Related Reagents and Kits

Product name	ROS Assay Kit -Highly Sensitive DCFH-DA-	GSSG/GSH Quantification Kit	Cystine Uptake Assay Kit	Glutamate Assay Kit-WST
Target	ROS (Reactive oxygen species)	Glutathione (oxidized/reduced)	Cystine Uptake	Glutamate
Localization	Intracellular	Intracellular	-	Intracellular/Extracellular
Detection (Fluorescence:Ex/Em	Fluorescence (505 nm/525 nm)	Colorimetric (412 nm)	Fluorescence (490 nm/535 nm)	Colorimetric (450 nm)
Instrument	Fluorescence Microscope, FCM, Microplate Reader	Microplate Reader	Microplate Reader	Microplate Reader
Sample	Live Cell	Cell, Tissue, Blood Plasma, Red Blood Cell	Live Cell	Cell, Cell Culture

Ferroptosis Research in Asia

Year	Journal	Title	Products
2022	Oxid. Med. Cell. Longev.	5-Lipoxygenase Inhibition Protects Retinal Pigment Epithelium from Sodium Iodate-Induced Ferroptosis and Prevents Retinal Degeneration	Cell Counting Kit-8 FerroOrange
2022	Redox Biol.	Poly(rC)-binding protein 1 represses ferritinophagy-mediated ferroptosis in head and neck cancer	FerroOrange Mito-FerroGreen
2021	Theranostics	An innovative NRF2 nano-modulator induces lung cancer ferroptosis and elicits an immunostimulatory tumor microenvironment	Cell Counting Kit-8 Liperfluo
2021	Biomolecules	Esophageal Cancer Stem-like Cells Resist Ferroptosis-Induced Cell Death by Active Hsp27-GPX4 Pathway	Cell Counting Kit-8
2021	Cancer Lett.	Mitochondrial pyruvate carrier 1 regulates ferroptosis in drug-tolerant persister head and neck cancer cells via epithelial-mesenchymal transition	Cell Counting Kit-8
2021	Antioxidants	Histochrome Attenuates Myocardial Ischemia-Reperfusion Injury by Inhibiting Ferroptosis-Induced Cardiomyocyte Death	Cell Counting Kit-8 FerroOrange

Ferroptosis

What is Ferroptosis?

How Does Ferroptosis Cause Cell Death?

Induction of Ferroptosis by Erastin?

＜Experimental example＞

Research on Related Diseases

Suppression of hepatitis via ferroptosis

Confirmation of the link between lysosomal disorders and ferroptosis

Regulation of cancer immunity via ferroptosis

Ferroptosis – a newly identified, iron-dependent form of programmed cell death

Ferroptosis-Related Reagent Selection Guide

Ferroptosis Research in Asia

Product Classification

Product Classification

Cell Proliferation / Cell Cytotoxicity Assay

Cell Staining

Intracellular Fluorescent Probes

Labeling Chemistry

Oxidative Stress

-Bacstain- Series

Molecular Biology

Detergents

Cross-Linking Reagents

Redox Dyes

Ion Analysis

Organic Scintillator

Buffers

Metal Chelates

Chromogen/Metal Indicator

Water Analysis

Extraction Reagent

High Purity Solvent

Biochemicals

Functional Organic Material