Science Note: Ferroptosis

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, necroptosis, and autophagy. In the context of cancer, ferroptosis may act as a tumor suppressor mechanism, as cancer cells often have an increased susceptibility to ferroptosis due to their altered metabolism and increased levels of reactive oxygen species (ROS). Therapeutically, inducing ferroptosis in cancer cells has emerged as a promising strategy for cancer treatment, particularly for tumors that are resistant to traditional therapies such as chemotherapy and radiation. In addition, understanding the specific vulnerabilities of cancer cells to ferroptosis may aid in the design of targeted therapies that exploit these weaknesses, providing a potential avenue to overcome drug resistance and improve patient outcomes.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism
Click here for the original article: Pavan S. Upadhyayula, et. al., Nature Communications, 2023.

Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response
Click here for the original article: Robert V. Swanda et. al., Molecular Cell, 2023.

Mitochondria regulate intracellular coenzyme Q transport and ferroptotic resistance via STARD7
Click here for the original article: Soni Deshwal et. al., Nature Cell Biology, 2023.

Point of Interest
- Cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation.

- A cysteine-depleted, methionine-restricted diet can improve the therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model.

- This CMD diet profoundly alters in vivo metabolome, proteome, and lipidome.

Point of Interest
- Depletion of cysteine induces adaptive ATF4 expression at the transcriptional level.

- A shortage of cystine in lysosomes stimulates ATF4 expression through the AhR signaling pathway.

- A weakened cysteine stress response increases sensitivity to ferroptosis during cysteine deprivation.

- CysRx promotes cancer cell ferroptosis through intracellular nutrient reprogramming.

Point of Interest
- The rhomboid protease PARL cleaves STARD7, allowing its dual localization to the mitochondrial intermembrane space and cytosol.

- The mitochondrial STARD7 supports coenzyme Q synthesis, promotes oxidative phosphorylation, and maintains cristae morphology.

- Its cytosolic counterpart facilitates the transport of coenzyme Q to the plasma membrane and protects against ferroptosis.

- Overexpression of cytosolic STARD7 increases the resistance of cells to ferroptosis and reduces the availability of coenzyme Q in the mitochondria.

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.

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.

Erastin-Induced Ferroptosis: Evaluating Intracellular Uptake and Redox Balance

Scientists have unveiled that in comparison to young mice, one-third of old microglia show Lipofustin-related autofluorescence (AF), characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological removal of microglia in older mice successfully eliminated AF-microglia, following the repopulation of new functional microglia leads to an improvement in age-related neurological impairments and reduces neurodegeneration after traumatic brain injury. Learn more about how the authors phenotyped AF-microglia using Lipi-Blue* for Lipid droplet labeling, and FerroOrange* for iron labeling. (Please refer to Fig. 1E, 5F, 9E for FerrOrange, Fig. 5D, 7E, for Lipi-Blue)

*Listed at 2023 Summer Promotion! Click here.

Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration    Click here for the original article: Rodney M Ritzel, et. al., Sci Adv (2023)

Point of Interest
- Lipofuscin accumulates in the brain's old microglia characterized by profound changes in lipid and iron content, phagocytosis, and oxidative stress.
- Increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia are chronically driven by phagocyte-mediated oxidative stress.
- Pharmacological removal of lipofuscin-laden microglia in older mice leads to an improvement in age-related neurological impairments.

Measurement of intracellular iron changes and lysosomal pH changes

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis    Ruilin Tian, et. al., Nature Neuroscience (2022)

Point of Interest
- The study reveals pathways that govern neuronal response to chronic oxidative stress, a factor in neurodegenerative diseases.
- Suppression of the lysosomal protein prosaposin makes neurons highly susceptible to oxidative stress. 
- This happens through the induction of lipofuscin formation which sequesters iron.
- Iron accumulation contributes to creating reactive oxygen species and lipid peroxidation, triggering ferroptosis, exclusively in neurons.

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

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.

• - iPS cell-derived tri-culture system that contains microglia, neurons, and astrocytes are used in this study
• - Microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis
• - Iron overload causes a marked shift in the microglial transcriptional state 
• - This microglial response contributes to neurodegeneration and is regulated by a novel ferroptosis susceptibility gene, SEC24B

• - Iron release from ferritin storage is through NCOA4-mediated autophagic degradation, known as ferritinophagy
• - Deletion of Ncoa4 in mouse hearts improved cardiac function along with the attenuation of the upregulation of ferritinophagy-mediated ferritin degradation 4 weeks after pressure overload
• - Free ferrous iron overload and increased lipid peroxidation were suppressed in NCOA4-deficient hearts
• - Inhibition of lipid peroxidation significantly mitigated the development of pressure overload-induced dilated cardiomyopathy in wild-type mice

• - The level of the anabolic reductant NADPH is a biomarker of ferroptosis sensitivity
• - The transmembrane endoplasmic reticulum MARCHF6 E3 ubiquitin ligase recognizes NADPH through its C-terminal regulatory region
• - This interaction upregulates the E3 ligase activity of MARCHF6, thus downregulating ferroptosis
• - Inhibiting ferroptosis rescued the growth of MARCHF6-deficient tumours and peri-natal lethality of Marchf6^–/– mice.