Ferroptosis: Mechanisms in Disease and Kit Selection

Science Note

[Jul. 9, 2024]                                                                                                                                                                                                                             Previous Science Note

Diseases Associated with Ferroptosis: Cancer, Neurodegeneration, and Age-Related Diseases 

Ferroptosis and senescence are two distinct cellular processes that significantly impact disease initiation and progression. Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxidation that plays a critical role in neurodegenerative diseases by promoting neuronal death and in cancer where it can either inhibit or promote tumor growth depending on the context. Senescence involves a state of permanent cell cycle arrest that acts as a tumor suppressor by preventing the proliferation of damaged cells, but also contributes to aging and tissue dysfunction. Both processes are integral to the pathophysiology of several diseases and provide potential targets for therapeutic intervention.

Aging promotes metabolic dysfunction-associated steatotic liver disease by inducing ferroptotic stress
Click here for the original article: Kuo Du, et. al., Nature Aging, 2024.

Microglial ferroptotic stress causes non-cell autonomous neuronal death
Click here for the original article: Jeffrey R. Liddell et. al., Mol Neurodegener, 2024.

Fatty acid binding protein 5 suppression attenuates obesity-induced hepatocellular carcinoma by promoting ferroptosis and intratumoral immune rewiring
Click here for the original article: Jonathan Sun et. al., Nature Metabolism, 2024.

Point of Interest
- A gene cluster characteristic of aging hepatocytes is enriched in diseased livers and includes genes that induce ferroptosis.

- Aged mice show increased hepatocyte ferroptosis and liver degeneration under conditions that induce metabolic stress.

- Inhibition of ferroptosis in aged mice reverses age-related liver damage.

Point of Interest
- Ferroptotic stress in microglia triggers inflammatory cascades, leading to neuronal death through neurotoxic astrocytes.

- Human amyotrophic lateral sclerosis (ALS) spinal cord tissue shows ferroptosis signatures, that are also observed in the ALS mouse model, where ferroptosis inhibition is neuroprotective.

- Ferroptosis in microglia can cause non-cell autonomous neuronal death, suggesting a novel pathophysiological role in neurodegenerative diseases.

Point of Interest
- Fatty acid binding protein 5 (FABP5) drives obesity-induced hepatocellular carcinoma (HCC), and its inhibition induces lipid peroxidation and ferroptosis in cancer cells.

- FABP5 inhibition reduces HCC burden in mice by promoting a pro-inflammatory tumor microenvironment.

- FABP5 inhibition activates CD8+ T cells and increases expression of CD80 and CD86, T cell activation molecules in tumor-associated macrophages.
Related Techniques
Ferrous ion (Fe2+) detection FerroOrange(intracellular), Mito-FerroGreen(mitochondrial)
Lipid peroxidation detection Liperfluo(intracellular), MitoPeDPP(mitochondrial)
Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples
Mitophagy or autophagy detection Mitophagy Detection Kit, Autophagic Flux Assay Kit
Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit,  MT-1 MitoMP Detection Kit
Lysosomal function Lysosomal Acidic pH Detection Kit -Green/Red and Green/Deep Red
Glutathione Quantification GSSG/GSH Quantification Kit
Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit 
Related Applications

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.

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 Glu            

Glutamate Assay Kit-WST

③Intracellular GSH     

GSSG/GSH Quantification Kit

④Intracellular Fe2+     

FerroOrange

⑤Intracellular ROS     

Highly Sensitive DCFH-DA

⑥Intracellular Lipid   

Liperfluo

 

 

Previous Science Note

- Protein Upregulation to Protect Against Ferroptosis [Jun. 4, 2024]
- Cancer Ferroptosis: Relationship on Metabolism, Lysosome, and Mitochondria [Mar. 12, 2024]
- Ferroptosis Activation, Inhibition, and Sensitization [Feb. 13, 2024]
- Ferroptosis Induced by FSP1-dependent Phase Separation [July 25, 2023]
- Age-related Microglial Phenotype Characterized by Lipid and Iron Contents [July 5, 2023]
- Link Lysosomal Failure to Ferroptosis in Human Neurons [May 9, 2023]
- Mechanisms and role of ferroptosis in disease [Jan. 17, 2023]

 

 

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 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?

Open / Close the Article

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

Open / Close the Article
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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