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

Science Note

[Feb. 13, 2024]                                                                                                                                                                                                                            Previous Science Note

Ferroptosis Activation, Inhibition, and Sensitization 

Ferroptosis is a form of programmed cell death characterized by the accumulation of lipid peroxides to lethal levels and is distinct from other forms of cell death such as apoptosis or necrosis. In cancer, ferroptosis can act as a double-edged sword: on the one hand, inducing ferroptosis in cancer cells may be a promising therapeutic strategy, as many cancers are resistant to traditional forms of cell death such as apoptosis. On the other hand, cancer cells can develop resistance to ferroptosis, which contributes to therapy resistance and tumor progression. Understanding and manipulating the pathways that regulate ferroptosis in cancer cells holds the potential for developing novel cancer treatments and overcoming resistance to existing therapies.

Targeted activation of ferroptosis in colorectal cancer via LGR4 targeting overcomes acquired drug resistance
Click here for the original article: Hao Zheng, et. al., Nature Cancer, 2024.

7-Dehydrocholesterol is an endogenous suppressor of ferroptosis
Click here for the original article: Florencio Porto Freitas et. al., Nature, 2024.

Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest
Click here for the original article: Jason Rodencal et. al., Cell Chemical Biology, 2023.

Point of Interest
- Chemoresistant cancer-derived organoids exhibited increased expression of LGR4 and activation of the Wnt signaling pathway.

- Inhibition of LGR4-Wnt signaling sensitized drug-induced ferroptosis.

- LGR4-dependent Wnt signaling upregulated SLC7A11, a key inhibitor of ferroptosis, leading to drug resistance.

Point of Interest
- High concentrations of 7-DHC are cytotoxic to developing neurons by promoting lipid peroxidation.

- On the other hand, 7-DHC accumulation confers a prosurvival function in cancer cells.

- 7-DHC effectively protects (phospho)lipids from autoxidation and subsequent fragmentation due to its superior reactivity with peroxyl radicals.

- Accumulation of 7-DHC induces a shift to a ferroptosis-resistant state in tumors.

Point of Interest
- Stabilization of p53 and inhibition of CDK4/6 enhance ferroptosis induced by GPX4 inhibitors.

- Stabilization of p53 and inhibition of CDK4/6 decrease the expression of MBOAT1 and EMP2.

- Loss of EMP2 increases cell sensitivity to GPX4 inhibitors by altering lipid metabolism.

- An orally bioavailable GPX4 inhibitor shows in vivo activity.

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 MitoBright ROS 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
           Antibody/Protein labeling with fast and
           high recovery
Fluorescein, Biotin, and Peroxidase Labeling Kit - NH2
Related Applications

The simultaneous detection of lysosomal function with Mitochondrial ROS and intracellular Fe2+

Lysosomal Function and Iron Homeostasis

 

 

Recent reports suggest that lysosomal neutralization can result in iron depletion, consequently leading to the disruption of cell viability. To verify this, HeLa cells were labeled with FerroOrange for Fe2+ detection, and the lysosomal mass and pH were separately detected with LysoPrime DeepRed and pHLys Green (a product currently under development). Co-staining with FerroOrange and Lysosomal dyes demonstrated that Bafilomycin A1 (Baf. A1), an inhibitor of lysosomal acidification, causes iron depletion consistent with the findings reported in the article. Interestingly, the iron chelator, Deferiprone (DFP), did not impact lysosomal pH, suggesting that lysosomal function plays a key role in managing iron homeostasis.

Reference: Ross A Weber, et. al., Mol Cell (2020)

Products in Use
   - FerroOrange
   - pHLys Green*
   - LysoPrime Deep Red

*pHLys Green is available as the "Lysosomal Acidic pH Detection Kit-Green/Deep Red". 


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

 


 

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.", 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 & 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 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 News2021, 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

Product Classification

Product Classification