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Ferroptosis: Mechanisms and Detection Methods

Ferroptosis is a form of programmed cell death (PCD) identified by Brent R. Stockwell in 2012[1]. While studying Erastin’s ability to kill RAS-mutant tumor cells, researchers found that the mode of cell death differed from other PCD, and this cell death could be effectively inhibited by the iron chelator Deferoxamine. Consequently, this iron-dependent form of cell death was named ferroptosis.
Ferroptosis is characterized by disrupted intracellular iron metabolism accompanied by decreased glutathione and accumulation of lipid peroxides, leading to cell membrane rupture and death. Ferroptosis is closely linked to neurodegeneration diseases, cardiovascular disorders, and cancer. Ferroptosis inducers can be used to enhance chemotherapy. Meanwhile, some ferroptosis inhibitors protects from neurodegenerative and cardiovascular diseases by reducing cell damage. Studying ferroptosis provides valuable insights into disease mechanisms and drug development.
In this issue, we will introduce the main types of programmed cell death, the mechanisms of ferroptosis, and the methods used for ferroptosis detection.
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Main Types of Programmed Cell Death (PCD)
Different Mechanisms of Ferroptosis
Detection Methods for Ferroptosis
Main Types of Programmed Cell
Death (PCD)
Different Mechanisms of Ferroptosis
Detection Methods for Ferroptosis
Main Types of Programmed Cell Death (PCD)
Cell death is categorized into two types: accidental cell death (ACD), an uncontrolled process caused by severe physical/chemical damage; and programmed cell death (PCD, also termed regulated cell death, RCD), a genetically regulated mechanism essential for development, homeostasis, and removing damaged cells.
The identification of apoptosis by John Kerr, Andrew Wyllie, and Alastair Currie in 1972 marked a pivotal advancement in PCD research. Currently, PCD is recognized to encompass multiple subtypes—including ferroptosis, cuproptosis, apoptosis, necroptosis, autophagy, and pyroptosis—which are distinguished by their unique morphological features, enzymatic pathways, and immunological signatures. Each subtype contributes distinctively to both physiological processes and pathological conditions, as summarized in Table 1.
Figure 1. Timeline of the terms used in cell death research[1].
Table 1. Classifications of programmed cell death (PCD) and their distinctive characteristics[1-3].
Types of PCD Morphological characteristics Biochemical characteristics Major pathways Key genes
Ferroptosis

● Mitochondrial shrinkage with increased membrane density;

● Reduction or disappearance of mitochondrial cristae;

● Cell membrane rupture

● Accumulation of Fe²⁺;

● Lipid peroxidation;

● Increased levels of MDA and ROS;

● Decreased GSH content

● System Xc⁻-GPX4 pathway;

● Iron metabolism pathway;

● Lipid metabolism pathway;

● Mevalonate (MVA) pathway

● GPX4, SLC7A11;

● ACSL4, ALOXs;

● TFRC
Cuproptosis

● Mitochondrial shrinkage;

● Endoplasmic reticulum (ER) damage;

● Chromatin fragmentation;

● Cell membrane rupture

● Copper accumulation;

● Lipoylated protein aggregation;

● Increased levels of ROS/α-KG;

● Decreased Fe-S protein levels

● Abnormal aggregation of TCA cycle proteins;

● Copper overload mediated by FDX1

● FDX1;

● DLAT;

● LIAS
Apoptosis

● Cell shrinkage and chromatin condensation;

● Nuclear volume reduction and nuclear fragmentation;

● Formation of apoptotic bodies

● DNA fragmentation;

● Caspase activation

● Death receptor (extrinsic) pathway;

● Mitochondrial (intrinsic) pathway;

● Perforin/granzyme pathway

● Caspase family;

● Bcl-2 family;

● Death receptors (DRs)
Necroptosis

● Plasma membrane rupture with release of cellular contents;

● Cytoplasmic and organelle swelling;

● Chromatin condensation

● Decreased ATP levels

● TNF-R1 pathway;

● RIP1/RIP3-MLKL pathway;

● PKC-MAPK-AP-1 pathway;

● ROS-related metabolic regulatory pathways

● RIP1, RIP3
Autophagy

● Formation of double-membrane autophagosomes, including macroautophagy, microautophagy, and chaperone-mediated autophagy

● Increased lysosomal activity

● mTOR pathway;

● Beclin-1 pathway;

● p53 signaling pathway

● ATG5, ATG7;

● LC3;

● Beclin-1;

● DRAM3;

● TFEB
Pyroptosis

● Intact nucleus with cell swelling and deformation;

● Plasma membrane rupture and release of cellular contents;

● Formation of pyroptotic bodies

● Increased expression of GSDM-B/C/D/E, Caspases 1/3/4/5/8/11, and NLRP3;

● Elevated release of IL-1β, IL-18, HMGB1, ATP, and LDH

● Caspase-1-mediated canonical pathway;

● Caspase-11/4/5-mediated non-canonical pathway

● Initiator factors: Caspase-1/3/4/5/11;

● Executioner proteins: GSDMB/C/D/E

Among these distinct PCD subtypes, ferroptosis has garnered significant attention due to its unique molecular underpinnings and its profound implications across multiple disease pathologies.

Different Mechanisms of Ferroptosis
Ferroptosis is primarily triggered by iron-dependent lipid peroxidation, and its molecular mechanisms involve multiple pathways, including the System xc⁻-GSH-GPX4 axis, iron metabolism pathways, lipid metabolism pathways, as well as other mechanisms such as mevalonate pathway, the AIFM2-CoQ10 system, GCH1-BH4 pathway, the ESCRT-III membrane repair system, and the DHODH pathway. (See the detailed diagram of ferroptosis signaling pathways)
System xc⁻-GSH-GPX4 Signaling Pathway
The System xc⁻-GSH-GPX4 axis is a vital intracellular antioxidant system functioning through three key steps. Pharmacological inhibition of System xc⁻ (with Erastin, Sorafenib) or GPX4 (with RSL3, ML-162, ML-210, FIN56) induces ferroptosis[4-5].

1. System xc⁻

System xc⁻ is composed of subunits SLC7A11 (functional) and SLC3A2 (chaperone), mediates a 1:1 exchange of extracellular cystine (Cys2) for intracellular glutamate (Glu).

2. Glutathione (GSH)

GSH is synthesized from cysteine (Cys), glutamate, and glycine via enzymatic reactions involving GCL and GSS. GSH cycles between reduced (G-SH) and oxidized (GS-SG) forms to maintain cellular redox balance.

3. GPX4

Fe²⁺ catalyzes the formation of phospholipid hydroperoxides (PLOOH) via the Fenton reaction. GPX4 uses GSH to reduce lipid hydroperoxides (PLOOH) to non-toxic lipid alcohols (PLOH), preventing ferroptosis. Its Selenol active site is regenerated by GSH and glutathione reductase with NADPH to sustain antioxidant activity.
Figure 2. Mechanisms of System xc⁻-GSH-GPX4 signaling pathway[5].
Iron Metabolism Pathway
Iron accumulation is a key trigger of membrane oxidative damage in ferroptosis. Enhanced iron uptake, reduced storage, or impaired export lead to iron overload and promote ferroptosis. Iron chelators (e.g., Deferoxamine, Deferiprone) can interfere with ferroptosis by reducing the concentration of intracellular free iron[4].

1. Iron Uptake

Fe³⁺ bound to serotransferrin is taken up via TFRC, and is reduced to Fe²⁺ in endosomes, and released into the cytosol by SLC11A2 (DMT1). Other iron sources include lactotransferrin and heme. Ferritin complexes (FTH1/FTL) are degraded through NCOA4-mediated ferritinophagy, increasing cytosolic Fe²⁺. Excess Fe²⁺ generates ROS via the Fenton reaction and activates ALOXs. Activated ALOXs then oxidize polyunsaturated fatty acids that have been incorporated into phospholipids by ACSL4 and LPCAT3, thereby producing lipid hydroperoxides.

2. Iron Export

Fe²⁺ is mainly exported by SLC40A1, and ferritin-bound iron can be secreted via Prominin 2-mediated exosomes, reducing ferroptosis risk.
Figure 3. Mechanisms of iron metabolism pathway[4].
Lipid Metabolism Pathway
Reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂·⁻), and ·OH attack polyunsaturated fatty acids (PUFAs) in membranes, leading to lipid peroxidation and the formation of toxic phospholipid hydroperoxides (PLOOH), which drive ferroptosis.
Besides the ACSL4-LPCAT3-ALOX axis, membrane enzymes like cytochrome P450 oxidoreductase (POR) coupled with CYB5R1 and NADPH oxidases (NOX) promote lipid peroxidation by generating ROS that fuel iron-catalyzed Fenton reactions. These pathways collectively enhance PLOOH production. ACSL4 inhibitors (e.g., PRGL493, Rosiglitazone, Pioglitazone) and ALOX inhibitors (e.g., Zileuton, PD146176) can inhibit ferroptosis by reducing the accumulation of lipid peroxides.
In contrast, antioxidant systems including System xc⁻-GSH-GPX4, AIFM2-CoQ10, and GCH1-BH4 limit lipid peroxidation. PLOOH-induced membrane damage can be repaired by the ESCRT-III membrane repair machinery.
Figure 4. Mechanisms of lipid metabolism pathway[6].
Other Pathways
The mevalonate (MVA) pathway plays a critical role in ferroptosis regulation by producing isopentenyl pyrophosphate (IPP), which is essential for the maturation of selenocysteine-tRNA and subsequent synthesis of GPX4, a key antioxidant enzyme. Additional ferroptosis regulatory mechanisms include the AIFM2-CoQ10 pathway, where AIFM2 (FSP1) regenerates ubiquinol to inhibit lipid peroxidation and activates the ESCRT-III membrane repair system to prevent cell death. The GCH1-BH4 axis controls the synthesis of the antioxidant BH4, which modulates cellular sensitivity to ferroptosis by limiting phospholipid peroxidation. Furthermore, the mitochondrial enzyme DHODH reduces CoQ10 to ubiquinol, independently of cytosolic GPX4 and FSP1, thereby protecting mitochondria from ferroptosis and compensating for GPX4 deficiency. Together, these pathways coordinate to regulate lipid peroxidation and membrane integrity, critically influencing ferroptotic cell death[7-11].
Detection Methods for Ferroptosis
The multi-pathway regulatory mechanisms of ferroptosis provide a targetable foundation for biomarker detection. Methods for detecting ferroptosis include measuring changes in biomarkers related to metabolic processes (such as lipid metabolism, iron metabolism, and GSH-related metabolism), observing alterations in cell morphology, and assessing variations in molecular protein levels or activities.
Table 2. Categories and detection of key ferroptosis biomarkers.
Category Biomarker/Indicator Specific alterations Change direction
Cell
morphology		 Plasma membrane

● Membrane rupture;

● Mitochondrial shrinkage, outer membrane rupture;

● Reduced or absent cristae, increased membrane density;

 /
Cell
metabolism		 Phospholipid hydroperoxides (PLOOH) ● Polyunsaturated fatty acid-containing phospholipids (PUFA-PL) in the lipid bilayer react with oxygen radicals to generate phospholipid hydroperoxyl radicals (PLOO·), which further react to form PLOOH, triggering chain reactions and massive PLOOH production. ↑
Lipid peroxidation byproducts (MDA, 4-HNE) ● Primary lipid peroxidation products are further degraded into a series of complex aldehydes, including malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). ↑
Fe²⁺ ● Accumulation of ferrous ions specifically enhances oxidative stress, promoting lipid peroxidation. ↑
ROS ● Iron catalyzes the Fenton reaction to generate ROS, which directly promote lipid peroxidation. ↑
GSH ● GSH levels decrease during ferroptosis, weakening antioxidant capacity and promoting lipid peroxide accumulation. ↓
Molecular
protein		 GPX4 ● GPX4 expression is typically downregulated or inhibited, leading to lipid peroxide accumulation and triggering ferroptosis. ↓
ACSL4 ● ACSL4 protein is a key regulator of ferroptosis; its increased expression enhances cell sensitivity to ferroptosis. ↑
TFR1 ● TFR1 expression is usually upregulated, facilitating iron uptake and increasing intracellular iron, thereby promoting ferroptosis. ↑
ALOX15 ● ALOX15 expression is usually upregulated, promoting lipid peroxidation. ↑
Summary
Ferroptosis is a distinct form of programmed cell death first described in 2012. It is characterized by iron-dependent lipid peroxidation that leads to cell membrane rupture. Unlike apoptosis, necrosis, or autophagy, ferroptosis is marked by disrupted iron metabolism, reduced GSH levels, and accumulation of lipid peroxides. It plays a significant role in various diseases, including neurodegenerative disorders, cardiovascular diseases, and cancer.
The mechanisms underlying ferroptosis involve several pathways: the System xc⁻-GSH-GPX4 antioxidant axis, iron and lipid metabolism pathways, and regulatory systems such as the mevalonate (MVA) pathway, AIFM2-CoQ10, GCH1-BH4, ESCRT-III, and DHODH pathways. Detection of ferroptosis relies on monitoring changes in metabolic biomarkers (e.g., PLOOH, MDA, Fe²⁺, ROS, GSH), alterations in cell morphology (such as mitochondrial shrinkage and membrane rupture), and shifts in key protein levels (including GPX4, ACSL4, TFR1, and ALOX15). Understanding ferroptosis not only provides insights into disease mechanisms and potential therapeutic targets but also offers new strategies for drug development.
Recommended Compounds and Screening Libraries
Cat. No. Product Name Bioactivity
HY-15763 Erastin Inhibits System xc⁻; blocks cystine uptake; depletes GSH
HY-100218A RSL3 Inhibits GPX4 activity
HY-100002 ML-162 Inhibits GPX4 activity
HY-100003 ML-210 Inhibits GPX4 activity
HY-17386 Rosiglitazone ACSL4 inhibitor; PPARγ agonist
HY-14164 Zileuton 5-LOX (ALOX-5) inhibitor
HY-100579 Ferrostatin-1 Traps lipid peroxyl radicals, prevents free radical chain reactions, inhibits lipid peroxidation
HY-12726 Liproxstatin-1 Traps lipid peroxyl radicals, prevents free radical chain reactions, inhibits lipid peroxidation
HY-136057 iFSP1 Inhibits FSP1 (AIFM2), induces ferroptosis independent of GPX4, increases cancer cell sensitivity to ferroptosis inducers
HY-108325 Brequinar Inhibits DHODH, blocks ubiquinol (CoQ10) regeneration, induces ferroptosis independent of GPX4
HY-L051 Ferroptosis Compound Library 1,300+ ferroptosis-related compounds; a useful tool to study ferroptosis mechanisms and related diseases
HY-L133 Cuproptosis Compound Library 290+ cuproptosis-related compounds; a useful tool for drug research related to cancer, rheumatoid arthritis, and other diseases
HY-L162 Cell Death Library 3,000+ cell death compounds; a useful tool for screening cell death drugs
HY-L003 Apoptosis Compound Library 2,800+ apoptosis-related compounds; can be used in the research of apoptosis signal pathway and related diseases
HY-L059 Pyroptosis Compound Library 1,700+ pyroptosis-related compounds; can be used in the research of pyroptosis signal pathway and related diseases
HY-L029 Autophagy Compound Library 1,600+ autophagy pathway-related compounds; a useful tool for the research of autophagy-related regulation and diseases