Under both physiological and pathological conditions, cell death is an inevitable and critical step in the life cycle of a cell. Traditionally, cell death has been categorized into two groups: apoptosis and necrosis.

Cell death occurs when cells are no longer able to sustain their essential life functions. Based on the conventional classification, cell death is divided into Accidental Cell Death (ACD) and Regulated Cell Death (RCD). ACD is an uncontrolled, accidental biological process, whereas RCD involves a signal transduction process with the participation of effector molecules. In this context, RCD is also referred to as Programmed Cell Death (PCD) and typically occurs under physiological conditions.

Depending on the morphological structure, enzymatic activity, or immunological features, various types of cell death have been identified. To date, studies have mainly focused on apoptosis, pyroptosis, necroptosis, and autophagy, all of which fall under the umbrella of PCD. In 2012, Dixon introduced the concept of ferroptosis for the first time. Ferroptosis is a form of cell death dependent on iron and independent of apoptosis, characterized by the accumulation of lipid reactive oxygen species (ROS). In terms of morphology and function, ferroptosis differs significantly from necrosis, apoptosis, and autophagy.

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Apoptosis

Cells undergoing apoptosis exhibit morphological changes such as membrane blebbing, chromatin condensation, formation of apoptotic bodies, and disintegration of the cytoskeleton—among which nuclear alterations are the most critical. Upon receiving a stimulus (e.g., DNA damage, growth factors), pro-apoptotic proteins like Bad/Bax form oligomeric complexes and translocate from the cytoplasm to the outer mitochondrial membrane. This process alters the permeability and transmembrane potential of the outer membrane, allowing the release of pro-apoptotic factors.

These factors include Apoptotic Protease Activating Factor-1 (Apaf-1), cytochrome c, and dATP, which together form the apoptosome, leading to the activation of caspase-9. Subsequently, executioner caspases such as caspase-3/7 are activated by caspase-9, initiating the proteolytic cascade and executing apoptosis.

Besides the classical caspase-dependent pathway involving cytochrome c, there is an alternative mitochondrial apoptotic pathway driven by Apoptosis-Inducing Factor (AIF). Normally located within mitochondria, AIF translocates to the cytoplasm and eventually to the nucleus upon exposure to intrinsic apoptotic stimuli, leading to DNA damage and cell death.

The extrinsic pathway of apoptosis involves two types of plasma membrane receptors: dependence receptors and death receptors. Death receptors are characterized by a cysteine-rich extracellular domain and an intracellular death domain. Typical death receptors include DR3/4/5, Fas, and TNFR1/2.

Upon ligand binding, these receptors recruit adaptor proteins (e.g., caspase-8) to form Death-Inducing Signaling Complexes (DISC). DISC acts similarly to apoptosomes by activating apoptotic proteases like caspase-3, which then triggers downstream reactions leading to cell death.

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Two classical signaling pathways for apoptosis: the extrinsic apoptotic pathway and the intrinsic apoptotic pathway.

 

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Pyroptosis

Pyroptosis is divided into classical and non-classical pathways. The classical pathway generally occurs in two steps. First is the “priming” step, in which many proteins are upregulated through activation of the NF-κB pathway. These proteins become components of the inflammasome complex, which includes a cytoplasmic pattern recognition receptor (PRR), an adaptor protein, and pro-caspase-1.

In the second step, caspase-1 becomes activated and cleaves the N-terminal portion of gasdermin D, which then inserts into the plasma membrane forming pores—thus initiating pyroptosis. Simultaneously, pro-IL-1β and pro-IL-18 are hydrolyzed into their active, pro-inflammatory forms (IL-1β and IL-18), triggering inflammation and immune responses.

Pyroptosis can also occur via a non-classical LPS-mediated pathway, in which caspase-11 in mice and caspase-4/5 in humans cleave gasdermin D to initiate the process.

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Pyroptotic signaling pathways: caspase-1-dependent and caspase-1-independent pyroptosis.

 

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Necroptosis

Necroptosis is a regulated form of necrosis that is triggered when apoptotic pathways are inhibited. Unlike apoptosis and other necrotic pathways, necroptosis proceeds independently of caspase activity and instead relies on phosphorylation of MLKL by RIPK3.

This phosphorylation enables MLKL to form pore-like structures in the plasma membrane, leading to DAMP (Damage-Associated Molecular Patterns) release, cell swelling, and membrane rupture. During necroptosis, various stages of cellular degradation are observed, such as organelle swelling, membrane disintegration, and eventual collapse of cytoplasmic and nuclear contents.

Necroptosis is typically triggered by extracellular signals such as TNF-α binding to death receptors on the cell surface. Receptors of the TNF superfamily—including TNFR1, Fas/CD95, DR4/TRAIL-R1, and DR5/TRAIL-R2—activate adaptor proteins TRADD and TRAF2, initiating downstream activation of RIP kinases.

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Molecular mechanism of apoptosis and necroptosis: Death receptors can initiate both extrinsic apoptosis and necroptosis, with RIPK1 playing a key regulatory role in both.

 

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Ferroptosis

Unlike necrosis, ferroptosis is not characterized by cell or organelle swelling, or membrane rupture. Likewise, features of apoptosis such as membrane blebbing, chromatin condensation, and apoptotic body formation are absent. Moreover, autophagic double-membrane vesicles are not observed.

Ferroptosis is a newly recognized form of iron-dependent regulated cell death. It plays a critical regulatory role in various diseases including tumors, neurological disorders, acute kidney injury, and ischemia/reperfusion injury. Modulating the ferroptosis pathway—either activating or inhibiting it—offers a promising therapeutic approach for these diseases.

Iron is a key factor in driving lipid peroxidation and the ferroptotic process. Ferroptosis can be inhibited by iron chelators (e.g., deferoxamine) or accelerated by iron supplementation (e.g., ferric ammonium citrate). Studies have shown that regulation of genes involved in iron metabolism can influence ferroptosis. These genes include transferrin, NFS1, IREB2, and NCOA4. Therefore, intracellular iron overload is considered a major indicator of ferroptosis.

To detect iron level fluctuations during ferroptosis, the fluorescent probe FIP-1 (FRET Iron Probe 1) is widely used. Iron concentration can also be measured using ICP-MS (Inductively Coupled Plasma-MS) or Perls’ Prussian Blue staining.

Lipid peroxidation is the hallmark biochemical marker of ferroptosis. Many models show elevated levels of peroxidized phospholipids. BODIPY-C11 and LiperFluo are widely used probes to measure lipid peroxidation during ferroptosis, with LiperFluo being more specific. Additional assays include GPx4 enzyme activity, or LC-MS detection of phosphatidylcholine hydroperoxides.

 

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Schematic of the ferroptosis signaling pathway: Various steps controlling susceptibility to ferroptosis through lipid ROS production. PE: phosphatidylethanolamines; PLH: phospholipid; PL-O·: phospholipid alkoxyl radical; PL-OO·: phospholipid peroxyl radical; PL-OOH: phospholipid hydroperoxide; TF: transferrin.

 

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References

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Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26(1):99-114. doi:10.1038/s41418-018-0212-61.

Yang WS, Stockwell BR. Ferroptosis: Death by Lipid Peroxidation. Trends Cell Biol. 2016;26(3):165-176. doi:10.1016/j.tcb.2015.10.014

Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ. 2012;19:107.

He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell 137, 1100–1111 (2009).