Review: Ferroptosis Mechanisms in Disease (NASH, Neurodegenerative disease, and Cancer)

Science Note

[July 25, 2023]                                                                                                                                                                                                                            Previous Science Note

Ferroptosis Induced by FSP1-dependent Phase Separation 

Ferroptosis suppressor protein-1 (FSP1) has recently been identified as a second system that suppresses ferroptosis, preventing lipid peroxidation independently of the glutathione-GPX4 axis. Researchers have used a small molecule library screen to discover 3-phenylquinazolinones, represented by icFSP1, as potent inhibitors of FSP1. Unlike previous inhibitors, icFSP1 does not directly inhibit FSP1 enzyme activity, but causes FSP1 to relocate from the membrane and condense, working in synergy with GPX4 inhibition.

Learn more about how the authors detected Lipid peroxide, one of the ferroptosis markers, using Liperfluo. We also offer ferroptosis-related products: FerroOrange, Mito-FerroGreen. 

Phase separation of FSP1 promotes ferroptosis    Click here for the original article: T. Nakamura, et. al., Nature (2023)

Point of Interest
- Ferroptosis suppressor protein-1 (FSP1) suppresses ferroptosis, preventing lipid peroxidation independently of the glutathione-GPX4 axis.
- Using a small molecule library screen, 3-phenylquinazolinones, represented as icFSP1, are potent inhibitors of FSP1.  
- icFSP1 does not directly inhibit FSP1 enzyme activity, but causes FSP1 to relocate from the membrane and condense, working in synergy with GPX4 inhibition.
- icFSP1 has been found to reduce tumor growth and induce FSP1 condensates in tumors in vivo.
Related Techniques
           Intracellular lipid peroxidation measurement Liperfluo
           Mitochondria lipid peroxidation measurement MitoPeDPP
           Intracellular ferrous ion (Fe2+) detection FerroOrange
           Mitochondria ferrous ion (Fe2+) detection Mito-FerroGreen
​​           Mitochondrial superoxide detection mtSOX Deep red - Mitochondrial superoxide Detection
           ​​Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA 
           Lipid droplets detection Lipi-Blue / Green / Red / Deep Red
           NAD(H) and NADP(H) redox couples assay NAD/NADH and NADP/NADPH Assay Kit 
 
Related Applications

Erastin-Induced Ferroptosis: Evaluating Intracellular Uptake and Redox Balance

We investigated the transition of cellular metabolisms in A549 cells treated with erastin, a known ferroptosis inducer. Our results revealed the following.

- The inhibition of cystine uptake by erastin led to a depletion of cysteine, which in turn increased the compensatory uptake of other amino acids.

- Glucose uptake, which typically promotes ferroptosis*, was found to decrease upon erastin treatment, suggesting a potential cellular self-defense mechanism.

- The depletion of cysteine resulted in a decrease in glutathione levels and an increase in Fe2+, ROS, and lipid peroxides, all of which are recognized markers of ferroptosis.

Cell Line: A549
Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h

Reference: *Xinxin Song, et al., Cell Reports, (2021)

Products in Use

① Amino Acid Uptake : Amino Acid Uptake Assay Kit
② Glucose Uptake : Glucose Uptake Assay Kit-Green
③ Cystine Uptake : Cystine Uptake Assay Kit
④ Intracellular glutathione : GSSG/GSH Quantification Kit
⑤ Intracellular labile Fe : FerroOrange
⑥ Intracellular total ROS : ROS Assay Kit -Highly Sensitive DCFH-DA-
⑦ Lipid Peroxides : Liperfluo

 

 

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.

 

 

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., "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 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

Name Liperfluo MitoPeDPP Mito-FerroGreen FerroOrange
Target Lipid Peroxidation Lipid Peroxidation Ferrous Ion(Fe2+) Ferrous Ion(Fe2+)
Localization Intracellular Mitochondria Mitochondria 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)
Instrument Fluorescence Microscope,
FCM		 Fluorescence Microscope,
FCM		 Fluorescence Microscope,
Microplate Reader		 Fluorescence Microscope
Sample Live Cell Live Cell Live Cell Live Cell

Oxidative Stress- and Metabolism-Related Reagents and Kits

Name ROS Assay Kit
-Highly Sensitive DCFH-DA-		 GSSG/GSH Quantification Kit Glutamine Assay Kit-WST Glutamate Assay Kit-WST
Target ROS (Reactive oxygen species) Glutathione (oxidized/reduced) Glutamine Glutamine
Localization Intracellular Intracellular Intracellular/Extracellular Intracellular/Extracellular
Detection
(Fluorescence:Ex/Em)		 Fluorescence
(505 nm/525 nm)		 Colorimetric:412 nm Colorimetric:450 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 Cell, Cell Culture Cell, Cell Culture

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