Nature reviews the pathogenesis, biology and role of iron death in diseases

Since the term iron death was proposed in 2012, iron death has attracted widespread attention

Most organ damage and degenerative diseases are caused by iron death

Therefore, the induction and inhibition of iron death through pharmacological regulation has great potential in the treatment of drug-resistant tumors, ischemic organ damage and other degenerative diseases related to extensive lipid peroxidation

In 2021, a review published in NATURE REVIEWS | MOLECULAR CELL BIOLOGY systematically explained the molecular mechanism and regulatory network of iron death, as well as the physiological function, pathological effect and targeted therapy potential of iron death in tumor suppression and immune monitoring.

Among the factors that cause cell oxidative stress, lipid oxidative modification in the lipid bilayer, especially lipid peroxidation, has become an important regulator of cell fate

Lipid peroxidation is affected by the environment and genes, including heat, radiation, metabolism, redox homeostasis and cell-to-cell contact, as well as carcinogenic and tumor suppressor signals

At the same time, iron death also affects the development of certain fungi and the senescence of nematodes

From 1950 to 1960, Harry Eagle observed this iron-like death phenomenon

For the synthesis of glutathione, cysteine ​​can be passed through a neutral amino acid transporter or Xc-cystine/glutamate antiporter (a transmembrane protein complex containing SLC7A11 and SLC3A2 subunits, hereinafter referred to as Xc-system) is oxidized and absorbed from the environment, or synthesized through the transsulfurization pathway of methionine and glucose

Existing studies have shown that GSH synthesis or enhanced Xc-system or active GPX4 can protect a variety of cells from oxidative stress, especially in the absence of cell death caused by thiols.

The mechanism of iron death

Early studies have shown that the iron death pathway of Xc-System-GSH-GPX4 is inhibited, and phospholipids peroxides (PLOOHs) are a type of lipid based on reactive oxygen species (ROS) and are the key to inhibiting iron death

An iron death monitoring pathway that relies on GPX4 has been discovered, and the synthesis mechanism of PLOOH, especially the synthesis and activation of PLOOH precursor polyunsaturated fatty acids (PUFAs), has also received extensive attention

These studies focused on cell metabolism and revealed the close connection between iron death and metabolic pathways

This mechanism indicates that the inactivation of GPX4-RSL3 directly induces iron death, while erastin inhibits the transfer of cystine and makes cells lose cysteine.

The accumulation of PLOOHs can cause rapid and irreparable damage to the cell membrane, leading to cell death

Unrestricted lipid peroxidation is characteristic of iron death
.
Early studies in 1950 showed that the trace elements selenium, vitamin E and cysteine ​​may inhibit lipid peroxidation
.

The activation of lipid peroxidation requires the removal of a diallyl hydrogen atom (located between two carbon-carbon double bonds) from the phospholipids (PUFA-PLs), which contain polyunsaturated fatty acid acyl groups in the lipid bilayer
.
Then a carbon-centered phospholipid group (PL • ) is formed, which reacts with oxygen to form a phospholipid hydrogen peroxide radical (PLOO • ), and removes a hydrogen from another PUFA to form PLOOH
.

If GPX4, PLOOH and lipid radicals (especially PLOO • and alkoxyphospholipid radicals PLO • ) cannot be converted to the corresponding alcohols (PLOH), these substances will be removed by removing PUFA-PLs at the remote end
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Hydrogen atoms react to form PLOOH, and react with oxygen molecules to form PLOOHs
.

Ultimately, this produces many products, including the breakdown products of lipid peroxides (such as 4-hydroxynonenoic acid and malondialdehyde) as well as oxidized and modified proteins
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This chain reaction may eventually destroy the integrity of the cell membrane, leading to the rupture of organelles and/or cell membranes
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Neuron-related studies have shown that membranes with higher PUFA-PL content are prone to peroxidation
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Existing research is not clear which lipid peroxidation of cell membranes (such as mitochondria, endoplasmic reticulum, peroxisomes, lysosomes and cell membranes) is related to iron death
.

The sensitivity of cells to iron death depends on the degree of unsaturation of the lipid bilayer, but how lipid peroxidation occurs is still unclear
.
The generation of lipid radicals or hydroxyl radicals ( • OH) can trigger the non-enzymatic lipid peroxidation reaction, which may be driven by the Fenton reaction with iron as a catalyst
.

Some lipoxygenases (LOXs) are independent heme dioxygenases for PUFAs, which can directly oxidize PUFAs and pufa-containing lipids on biofilms, suggesting that LOXs may induce iron death
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The role of LOXs in iron death is still unclear, and the latest research shows that the pan-expressed cytochrome P450 oxidoreductase (POR) also plays a role in lipid peroxidation
.

NADPH provides electrons through POR, and downstream electron acceptors (such as cytochrome P450 and CYB5A) accept electrons and decrease accordingly
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This may be due to the dehydrogenation of PUFAs or the reduction of ferric iron to divalent iron directly or indirectly triggering lipid peroxidation
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Iron death metabolism

The role of iron, lipids, ROS and cysteine ​​in iron death indicates that the mode of cell death is closely related to cell metabolism
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Professor Xuejun Jiang’s research attempts to explore how metabolism determines cell fate to further reveal the complex relationship between iron death and metabolism
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The catabolic process of autophagy is an important survival mechanism in response to various stresses, but whether autophagy can promote cell death (ie, "autophagic cell death") and how to promote cell death has always been the focus of debate
.

Studies have found that when there is complete serum in the medium and lack of amino acids (a state that triggers autophagy), autophagy promotes non-apoptotic, non-necrotic cell death
.

The transferrin and glutamine in the serum are necessary for this form of cell death, especially deprivation of cysteine ​​in the cell culture medium can trigger cell death
.
The iron- and cysteine-dependent protective effects indicate that iron death is a mechanism of cell death under these conditions
.

In iron death caused by lack of cysteine, autophagy is achieved by autophagic degradation of ferritin (also called iron autophagy protein), which increases the unstable iron content in cells and makes iron death more sensitive
.

Due to lack of cysteine, glutamine metabolism or glutamine decomposition is a necessary condition for iron death, and cysteine ​​links iron death with oxidative metabolism
.

Glutamine is a key supplemental metabolite that promotes the mitochondrial tricarboxylic acid cycle (TCA).
TCA increases the respiratory frequency of mitochondria and promotes the production of ROS
.

Therefore, the normal metabolic function of mitochondria is related to iron death-this conclusion has been confirmed by pharmacological, cytological and genetic analysis
.

Does not rely on the monitoring channel of GPX4

Recently, a genome-wide screening revealed an iron death monitoring mechanism that does not rely on GPX4
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The first mechanism involves iron death suppression protein 1 (FSP1; also known as AIFM2)
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AIFM1 was originally thought to be a homolog of FSP1, which can promote cell apoptosis (such as FSP1/AIFM2)
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It is now believed that this is related to the transport and correct folding of proteins between mitochondrial membranes
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FSP1 lacks substantial pro-apoptotic function, but can actually protect cells from iron death caused by GPX4 gene suppression or deletion
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FSP1 is myristoylated and is related to a variety of cell membrane structures (including cell membrane, Golgi apparatus and perinuclear structures)
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Mutations in the myristoylation site will lose the anti-iron death function
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Due to the ubiquitin oxidoreductase activity of NADH, FSP1 reduces lipid peroxidation and iron death by reducing ubiquitin (or its partial oxidation product semihydroquinone) to produce ubiquitin to inhibit lipid peroxidation and iron death, thereby reducing lipid peroxidation and iron death.
Directly reduce lipid free radicals and stop lipid auto-oxidation, or indirectly prevent lipid auto-oxidation by regenerating and oxidizing vitamin E (a powerful natural antioxidant) (see Figure 2 above)
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Another study showed that GTP cyclohydrolase 1 (GCH1) prevents iron death through its metabolites, tetrahydrobiological terophyllin (BH4) and dihydrobiological terocancer (BH2)
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BH4 has anti-oxidative degradation effects on phospholipids containing two PUFA tails, which may involve a dual mechanism: directly capture antioxidant free radicals and participate in the synthesis of ubiquitinone (see Figure 2c above)
.

Although the role of GCH1 in protecting tissues and organs from iron death is unclear, gene knockout studies have shown that mice lacking the GCH1 gene develop bradycardia and embryonic death in the second trimester
.

In addition to peroxides that act directly on the lipid bilayer or free radicals that act on phospholipids by capturing antioxidant free radicals, there may be other mechanisms that can protect cells from lipid peroxidation damage
.

Squalene is a metabolite of cholesterol metabolism
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It has anti-iron death effects on cholesterol-deficient lymphoma cell lines and primary tumors
.
However, whether this is a tumor subtype-specific effect or a common protective mechanism remains to be verified (Figure 2c)
.

The role of Hippo–YAP signaling pathway in iron death

The Hippo–YAP signaling pathway is involved in a variety of biological functions, including the control of cell proliferation and organ size
.
Researchers have studied the role of this pathway in iron death and have observed that high-density cells tend to be more resistant to iron death caused by lack of cysteine ​​and GPX4 inhibition
.

In mechanism, the cell density effect of epithelial cell iron death is mediated by E-cadherin-mediated cell-cell contact, which activates the Hippo signaling pathway through NF2 (also known as Merlin) tumor suppressor protein
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Therefore, inhibition of nuclear translocation and transcription jointly regulate the activity of YAP factor
.

YAP targets a variety of iron death regulators (ACSL4, transferrin receptor, and other regulatory factors), and the susceptibility to iron death depends on the activity of the Hippo pathway.
The activity of Hippo varies with the inhibition of Hippo and the activation of YAP.
Increase (article Figure 4)
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In kidney cancer cells that mainly express TAZ instead of YAP, the YAP analog TAZ was found to promote iron death by regulating cell density
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The E-cadherin–NF2–Hippo–YAP/TAZ pathway plays an important role in determining the sensitivity to iron death
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First, multiple components of this pathway are frequently mutated in tumors, which can enhance the expression and/or activity of YAP/TAZ, and inducing iron death may become a potential treatment for certain specific tumors
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Second, cell density-dependent iron death was also observed in non-epithelial cells that did not express E-cadherin
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Researchers believe that other cadherins or cell adhesion molecules may also inhibit iron death through similar mechanisms
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Third, the Hippo–YAP pathway is very important during development
.

It interacts with a variety of signaling pathways, and there may be a link between iron death and normal cell biology
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Finally, the researchers speculated that the original function of cadherin might be to protect cells from oxidative stress and iron death
.

The role of AMPK signaling pathway in iron death

Cellular metabolic stress and glucose deficiency increase the production of ROS, indicating that glucose deficiency promotes iron death
.
However, some studies believe that lack of glucose can inhibit iron death
.
This protection depends on the activity of adenylate activated protein kinase (AMPK)
.
In the absence of glucose, AMPK is activated, the energy stress protection program is activated, and the biosynthesis of PUFAs is disturbed to fight iron death, which is a necessary condition for lipid peroxidation to cause iron death (article Figure 4)
.

Iron death in tumor suppression

Studies have shown that a variety of tumor suppressor factors can make cells susceptible to iron death
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After careful analysis of p53-specific lysine acetylation sites, the researchers found that p53 enhances the transcription of the iron death subunit SLC7A11 system by inhibiting the X-c-system, which may be involved in the tumor suppressor function of p53 in vivo and in vitro
.

Tumors are susceptible to p53 single nucleotide polymorphisms leading to the substitution of P47S amino acids, which resist iron death of tumor cells
.

It is not clear whether the loss of iron death activity caused by p53 is the only functional consequence of these specific mutations
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Conversely, p53 also prevents iron death by regulating other transcription targets
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Since p53 regulates target genes and participates in a variety of biological processes, its exact role in iron death may be related to the environment
.

Similar to p53, the tumor suppressor and epigenetic regulator BAP1 promotes iron death by down-regulating the expression of SLC7A11
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Unlike p53, the iron death-promoting activity of p53 has been proven to inhibit the occurrence of tumors in the body, but it is not clear whether the iron death activity of BAP1 can produce tumor suppressor function
.

Iron death in immune surveillance

Studies have shown that iron death plays an important role in cell death induced by immune cells
.
IFN-γ (IFNγ) inhibits the X-c-system, CD8 + T cells produce cytokine iron and cause tumor cell death
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Whether the mechanism by which immune cells induce iron death is related to its physiological function is still unclear
.
IL-4 and IL-13 inhibit the expression of GPX4 in certain cells (kidney, lung, spleen, heart), increase the expression of ALOX15, and produce a large number of important inflammatory intermediates-arachidonic acid metabolites
.

By reducing lipid peroxidation, GPX4 inhibits the activity of LOXs and cyclooxygenase, weakens the activity of GPX4, which may affect the secretion of immunoregulatory lipid mediators, and then informs the immune system that cells are in a state of iron death sensitivity, which helps immunity Surveillance (detection of injury or malignancy)
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Potential anticancer therapy to induce iron death

Currently, cancer treatment methods based on iron-induced death are being actively explored
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People have tried to use non-targeting strategies based on nanoparticles to deliver iron, peroxides and other toxic substances to kill tumor cells
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The existence of a variety of enzymes that regulate iron death makes the development of targeted therapy possible
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One of the most important targets is GPX4, which is expressed in most cancer cell lines and is essential for the survival of cancer cells
.
GPX4 lacks the classic small molecule binding pocket and the existing GPX4 inhibitors covalently modify the selenocysteine ​​residues of GPX4 and other selenoproteins, which has specificity and potential toxicity
.

These inhibitors are highly active and unstable, but they can be overcome by the development of pre-encapsulated drugs, which are metabolized in the cell to convert them into an active form
.
GPX4 is essential for various peripheral tissues, such as mouse renal tubular cells and certain neuron subgroups
.

Therefore, unless a treatment that targets tumor cells is used, targeting GPX4 may have side effects
.

GPX4 targets differently.
Considering that the knock-out gene Slc7a11 mice will not cause major pathological changes, and the expression of SLC3A2 and/or Slc7a11 genes is negatively correlated with the clinical outcomes of patients with melanoma and glioma, by inhibiting the Xc system, The cellular cysteine ​​method is very promising
.

In fact, inhibiting the growth and metastasis of various tumors in mice or by pharmacologically inhibiting genetic Xc- has been a very promising system result, which is both effective and low-toxic
.

Compared with the normal Xc-system, it has a stronger inhibitory effect on tumor tissues because it is more susceptible to changes in active metabolism and other aspects during oxidative stress, so it is more dependent on the ROS detoxification function of the Xc system
.

Expert Opinion

Using a system based on inhibition of Xc-therapy, the needs of tumor patients are divided into a clear Xc-expression system (for example, SLC7A11 overexpressed cysteine-dependent cancer cells show clear reactive oxygen species), and the decision to Xc- discussed before Tumor suppression because the system is sensitive to other biomarkers
.

Similar to the lack of Slc7a11, knocking out Fsp1 will not cause embryo death or produce obvious pathological changes, indicating that targeting Fsp1 has a broad therapeutic window
.

FSP1 is abundantly expressed in most cancer cell lines and is the highest-ranked gene among 860 cancer cell lines related to GPX4 inhibitor resistance
.
GPX4 cancer cells lacking a gene may be a specific FSP-iFSP1 inactivation inhibitor and stored in the oncogene GPX4 death induced by iron and iFSP1-RSL3
.

Therefore, FSP1 inhibitors can be used clinically, especially for the treatment of drug-resistant tumors or tumors with differentiation characteristics
.

Expert comment

As a unique method of cell death, iron death integrates different components of previous cell metabolism into a tight network, including iron, selenium, amino acids, lipids, and redox reactions (Figure 1)
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With the progress of iron death research, we have begun to realize that this network plays a broad role in biological processes (physiology and pathology)
.

The definition of iron death is the process of iron depletion and lipophilic free radical antioxidants (such as iron inhibin 1, lipoprotein 1, vitamin E or ubiquitin) inhibiting cell death
.

There are two main mechanisms.
One is the iron-dependent lethal mechanism, which is not completely equivalent to iron death.
Iron death may be related to lysosomal toxicity; the second is the iron-independent oxidative stress mechanism
.

The researchers also discovered another possibility: direct detection of lipid peroxidation (using mass spectrometry, fluorescent dyes, or antibodies such as 1F83)
.
The latest research shows that the mobilization and up-regulation of transferrin receptor is another potential sign of iron death, which can distinguish oxidative stress from iron death
.

Other iron death markers still have high value in this field
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Another focus of the field is that the use of experimental methods is not suitable for dealing with the expected problems
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This is an inevitable problem in any emerging field
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Although considerable progress has been made in the regulation of iron death, the exact molecular events of iron death that lead to cell death are still unclear
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In the next few years, the pathogenesis of iron death is expected to be clarified
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These studies will shed light on the physiological and pathological effects of iron death
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Under the guidance of the use of specific biomarkers and accurate assessment of the patient's background, new iron-based death-based therapies will be discovered and applied in the clinic
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