Ferroptosis: Mechanisms in Disease and Kit Selection

Science Note

[Oct. 29, 2024] 

Exploring Ferroptosis Resistance Mechanisms for Cancer Treatment 

In recent cancer research, ferroptosis, a type of regulated cell death involving iron-dependent lipid peroxidation, has shown promise in cancer treatment. Here are some of the papers that have identified mechanisms of ferroptosis resistance in cancer cells and suggested that they may be therapeutic targets.

Ferroptosis, a type of regulated cell death involving iron-dependent lipid peroxidation, has shown promise in cancer treatment. It provides an alternative pathway to eliminate cancer cells that are resistant to traditional therapies such as apoptosis. By targeting ferroptosis, researchers can exploit metabolic weaknesses in cancer cells, particularly when antioxidant defenses are disrupted. This approach is being explored to improve the efficacy of existing treatments and potentially overcome drug resistance in several types of cancer.

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

Tumor suppressor Par-4 activates autophagy-dependent ferroptosis
Click here for the original article: Karthikeyan Subburayan et. al., Communications Biology, 2024.

BRCA1-Mediated Dual Regulation of Ferroptosis Exposes a Vulnerability to GPX4 and PARP Co-Inhibition in BRCA1-Deficient Cancers
Click here for the original article: Jonathan Sun et. al., Cancer Discovery, 2024.

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
- Point o- Par-4/PAWR activation is critical during ferroptosis, with its depletion blocking and overexpression enhancing this ferroptosis process.

- Upregulation of Par-4 promotes NCOA4-dependent ferritinophagy, a selective autophagy process that degrades ferritin, increasing free iron release, lipid peroxidation, and ROS production.

- Par-4 inhibition blocks ferroptosis-driven tumor suppression, highlighting its potential for cancer therapy.

Point of Interest
- BRCA1 deficiency in cancers confers resistance to erastin-induced ferroptosis but increases susceptibility to GPX4 inhibitor-induced ferroptosis.

- Combined PARP and GPX4 inhibition in BRCA1-deficient cancers promotes ferroptosis, providing a strategy to overcome PARP inhibitor resistance.

- Reduced GPX4 expression and NCOA4-induced ferritinophagy in BRCA1-mutant cancers increases sensitivity to co-treatment with PARP and GPX4 inhibitors.
Related Techniques
Intracellular / mitochondrial ferrous ion (Fe2+) detection FerroOrange(intracellular), Mito-FerroGreen(mitochondrial)
Lipid peroxidation detection Liperfluo(intracellular), MitoPeDPP(mitochondrial)
First-time autophagy research 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
Cellular senescence detection SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples
Glutathione Quantification GSSG/GSH Quantification Kit
Cystine Uptake detection Cystine Uptake Assay Kit
Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit 
Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit,  MT-1 MitoMP Detection Kit
Apoptosis detection in multiple samples Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST
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

Previous Science Note

- Diseases Associated with Ferroptosis: Cancer, Neurodegeneration, and Age-Related Diseases  [Jul. 9, 2024] 
- 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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Product Classification

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