Diversity of Cell Death

Cell death is the ultimate fate of all cells. The modes of cell death are a major research focus in the biomedical field, involving a variety of complex biological mechanisms. The primary function of cell death is to maintain tissue homeostasis by eliminating dysfunctional, damaged, and harmful cells. In 2005, the first Nomenclature Committee on Cell Death provided a relatively clear definition, describing cell death as a state distinct from the reversible condition of dying cells, a dead cell is defined as one that has reached the end of its life cycle and is in an irreversible state.

Figure 1. The discovery timeline of cell death

1. Types of Cell Death

There are two main forms of cell death: necrosis and programmed cell death (PCD). Necrosis is a non-programmed form of cell death typically triggered by traumatic injury, while PCD is a regulated process initiated by a cascade of molecular events in response to various physiological or developmental signals. Apoptosis is a well-characterized mechanism of PCD. Other types of PCD include: autophagic cell death, lysosomal cell death, mitoptosis, paraptosis, pyroptosis, NETosis, necroptosis, immunogenic cell death, entosis, methuosis, parthanatos, ferroptosis, autosis and so on.

Figure 2. Types of cell death

2. Mechanisms of Cell Death

2.1 Apoptosis

Apoptosis is activated through two primary pathways: the extrinsic and intrinsic pathways. The extrinsic pathway is triggered by the binding of extracellular ligands, such as TNF-α and Fas ligand (FasL), to their respective death receptors—namely, TNF receptor and Fas receptor. Upon ligand-receptor binding, the death-inducing signaling complex (DISC) is formed, which recruits and activates caspases, including caspase-8 and caspase-10. The intrinsic pathway is activated by intracellular stress factors, such as DNA damage, oxidative stress, and loss of survival signals, leading to increased permeability of the mitochondrial outer membrane. This pathway is regulated by the Bcl-2 family of proteins, including anti-apoptotic proteins (e.g., Bcl-2 and Bcl-xL), pro-apoptotic proteins (e.g., Bax and Bak), and BH3-only proteins (e.g., Bim and Bid). In response to intracellular stress, activated pro-apoptotic BH3-only proteins inhibit the anti-apoptotic proteins, thereby enabling Bax and Bak to form mitochondrial pores and release cytochrome c into the cytoplasm. The released cytochrome c then binds to apoptotic protease-activating factor-1 (Apaf-1) to form the apoptosome, which activates caspase-9.

Figure 3. Mechanisms of apoptosis

2.2 Necroptosis

Necroptosis is a cell lytic form of PCD that can trigger inflammation. It is primarily mediated through the RIPK1-RIPK3-MLKL signaling pathway. When caspase-8 activity is inhibited by drugs or viral inhibitors, stimulation of receptors such as TNFR1 or TLR can induce necroptosis. This process involves auto-phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Activated RIPK1 then activates RIPK3, which in turn phosphorylates and activates the downstream MLKL (mixed lineage kinase domain-like protein). MLKL serves as the terminal executor of necroptosis, leading to plasma membrane rupture and release of inflammatory factors. Although it exhibits regulatory features similar to apoptosis, its morphology presents characteristics of necrosis. FASL, TNF-related apoptosis-inducing ligand (TRAIL), TNF, and type I interferon (IFN-1) activate their respective receptors, recruiting MLKL, RIPK1, and RIPK3 to assemble into the necrosome through phosphorylation. Phosphorylation-mediated activation of MLKL and subsequent MLKL-driven pore formation in the membrane are critical steps in necroptosis. In response to TNF-α induced necroptosis, phosphoglycerate mutase 5 (PGAM5) is recruited to the RIPK1/RIPK3 complex on the outer mitochondrial membrane. This recruitment activates dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and triggers the release of substantial amounts of reactive oxygen species (ROS). And further activates poly (ADP-ribose) polymerase 1 (PARP-1), leading to decreased NAD⁺ levels and exacerbation of the process, which is considered an essential step in the execution of necroptosis.

Figure 4. Mechanisms of necroptosis

2.3 Autophagic cell death

The term “autophagy” is derived from the Greek word “auto”, meaning self-eating or phage. It describes an orderly process by which cells break down and recycle their own components to carry out various cellular functions. Autophagy is a naturally occurring physiological process that maintains homeostasis through the rational allocation of resources and the clearance of harmful or useless substances, thereby regulating the cell life cycle and natural development. However, when cells are subjected to starvation, oxidative stress, or exposure to toxic substances, the autophagic process may become dysregulated, leading to cell death, specifically referred to as autophagic cell death or Type II cell death. This dual functionality demonstrates the critical role of autophagy in determining cell fate, it can function as a survival mechanism, and under specific conditions, it can also drive cell death, depending on the cellular environment and external stimuli. The essential nature of autophagy lies in the rearrangement of intracellular membranes. The initial step of autophagy activation is the formation of an isolation membrane, also known as a autophagosome, which is a double-membrane structure that sequesters cytoplasmic components for degradation. The primary driving factor of the autophagic process is the UNC-51-like kinase 1 (ULK1) complex, while the mammalian target of rapamycin (mTOR) pathway serves as a key regulatory route that either promotes or inhibits the formation of this complex. Under normal conditions, mTOR activation suppresses autophagy by inhibiting the formation of the ULK1 complex, however, under stress or nutrient deprivation, the inhibition of mTOR promotes the assembly of the ULK1 complex, which initiates the formation of a double-membrane structure called the phagophore. The phagophore expands and encloses intracellular components to form the autophagosome, which subsequently fuses with a lysosome to create an autolysosome, where cellular components are digested by lysosomal enzymes. Multiple proteins, including those of the autophagy-related gene (ATG) family and Beclin-1, play crucial roles in the autophagy process by participating in phagophore formation and the recruitment of autophagy-related proteins. Microtubule-associated protein light chain 3 (LC3) is involved in elongation and closure of the phagophore, and maturation of the autophagosome. Autophagy activation leads to phagophore formation, which in turn can result in autophagic cell death.

Figure 5. Mechanisms of autophagic cell death

2.4 Lysosomal Cell Death

Lysosomal cell death, also known as lysosome-dependent cell death (LCD), occurs due to lysosomal membrane permeabilization, which leads to the release of lysosomal enzymes into the cytoplasm and activation of cell death pathways. LCD is typically triggered by reactive oxygen species (ROS) or other external stimuli. A sharp increase in ROS is a key factor leading to increased calcium ion (Ca²⁺) concentration. This process is mediated by the over activation of the Transient Receptor Potential Melastatin 2 (TRPM2) channel and the efflux of Ca²⁺ from lysosomes, which in turn induces lysosomal membrane permeabilization (LMP) and the release of lysosomal proteases (such as cathepsins) into the cytoplasm. Lysosomal proteases catalyze the degradation of various substrates, including Bid and other apoptotic proteins, thereby initiating caspase-dependent cell death pathways. Additionally, the occurrence of LCD involves the activation of Ca²⁺-dependent adenylate cyclase 1 (ADCY1), followed by an increase in cyclic adenosine monophosphate (cAMP), which ultimately inhibits the activity of lysosomal acid sphingomyelinase (SMase). Concurrently, endoplasmic reticulum stress can also elevate cytoplasm Ca²⁺ level. Excess cytoplasm calcium stimulates calpain activation, leading to the degradation of lysosomal membrane proteins (such as LAMP1/2), which in turn induces lysosomal membrane permeabilization and ultimately results in LCD.

Figure 6. Mechanisms of lysosomal cell death

2.5 Immunogenic Cell Death

Immunogenic cell death (ICD) is a form of PCD, a type of regulated cell death (RCD) capable of activating adaptive immunity in immunocompetent hosts. ICD can be triggered by a limited set of stimuli, including viral infections, certain FDA-approved drugs (such as anthracyclines), specific forms of radiotherapy, photodynamic therapy and so on. When dying cells release damage-associated molecular patterns (DAMPs), then trigger an immune response. These DAMPs attract immune cells to the site of cell death. During ICD, dying tumor cells express calreticulin (CALR) on their surface, which acts as an "eat me" signal for dendritic cells (DCs) and other phagocytes. This signal enables DCs to engulf the dead cells, thereby activating an immune response. DAMPs include surface-exposed CALR and heat shock proteins (HSPs), as well as extracellularly released adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1). These DAMPs function as danger signals that are recognized by pattern recognition receptors (PRRs) of the innate immune system, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), consequently activating an anti-tumor immune response.

Figure 7. Mechanisms of immunogenic cell death

2.6 Alkaliptosis

Alkaliptosis is a form of PCD triggered by elevated intracellular pH. It is typically initiated by NF-κB activation, which leads to the down regulation of carbonic anhydrase 9 (CA9) or the upregulation of ATPase H+ transporting V0 subunit D1 (ATP6V0D1), thereby disrupting lysosomal pH homeostasis. CA9, an enzyme that maintains pH balance, catalyzes the reversible hydration of carbon dioxide to facilitate the transport of bicarbonate ions and protons across the cell membrane. When CA9 is significantly downregulated, impaired H⁺ efflux activates other transporters or regulators to elevate intracellular pH as an adaptive response. This alkalinization disrupts intracellular homeostasis and can activate signaling pathways that induce cell death.

Figure 8. Mechanisms of alkaliptosis

2.7 Oxeiptosis

Oxeiptosis is a caspase-independent, apoptosis-like cell death pathway first described in 2018. It is a non-classical form of cell death induced by oxidative stress, characterized by its non-inflammatory nature and close association with the KEAP1-PGAM5-AIF signaling axis. Under oxidative stress, elevated intracellular ROS levels trigger conformational changes in KEAP1 (Kelch-like ECH-associated protein 1), leading to its dissociation from NRF2. KEAP1 then translocates to the mitochondria, where it mediates the release of AIF (apoptosis-inducing factor) from PGAM5 (phosphoglycerate mutase family member 5) and facilitates AIF translocation to the nucleus. Inside the nucleus, dephosphorylation of AIF at serine-116 (S116) ultimately initiates cell death.

Figure 9. Mechanisms of oxeiptosis 

2.8 Pyroptosis

Pyroptosis is a form of inflammatory PCD mediated by caspase-1, typically triggered by the activation of intracellular inflammasomes. This process leads to plasma membrane rupture and the release of pro-inflammatory cytokines, and is widely observed during infections and immune responses. Upon recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), canonical inflammasomes (e.g., NLRP3, NLRP1, NLRC4, AIM2) in the cytoplasm are activated in response to microbial infections (e.g., microbial toxins) or danger signals (e.g., double-stranded DNA and crystals), leading to caspase-1 activation. In contrast, non-canonical inflammasomes directly respond to LPS or other stimuli by activating caspase-4/5/11. Following inflammatory caspase activation, pro-IL-1β, pro-IL-18, and GSDMD are cleaved. This cleavage releases the N-terminal fragment of GSDMD (GSDMD-N), which forms pores in the plasma membrane, thereby facilitating the release of inflammatory mediators such as IL-1β and IL-18. In addition to these pathways, pyroptosis can also be initiated through the activation of caspase-3, caspase-8, and caspase-9, as well as the cleavage of gasdermin E, B, and C (GSDME, GSDMB, and GSDMC, respectively). Furthermore, the influx of sodium ions and the accompanying water entry lead to cellular swelling. As this process continues, the plasma membrane may become unable to withstand the increasing internal pressure, resulting in membrane rupture. Following rupture, all remaining soluble cytoplasmic contents are rapidly—almost instantaneously—released, ultimately culminating in pyroptosis.

Figure 10. Mechanisms of pyroptosis

2.9 Ferroptosis

Ferroptosis is an iron-dependent form of PCD characterized by the accumulation of lipid peroxides and oxidative stress, ultimately leading to membrane damage.

Ferroptosis is triggered by the accumulation of lipid peroxides, which are produced through the oxidation of polyunsaturated fatty acids by lipoxygenases or other enzymes. The buildup of lipid peroxides via polyunsaturated fatty acids oxidation can be further amplified by the iron-catalyzed Fenton reaction, producing reactive oxygen species (ROS) and hydroxyl radicals. These radicals attack and damage cellular components, particularly cell membranes, ultimately leading to cell death. A hallmark of ferroptosis is the depletion of intracellular glutathione (GSH) and reduced activity of glutathione peroxidase 4 (GPX4), resulting in the accumulation of unmetabolized lipid peroxides and the generation of high levels of ROS.

Figure 11. Mechanisms of ferroptosis

2.10 NETosis

NETosis is a form of inflammatory cell death in neutrophils that involves the release of web-like structures called neutrophil extracellular traps (NETs) to capture and eliminate pathogens. It is characterized by the extrusion of NETs into the extracellular space, NETs are composed of chromatin, histones, and granular proteins.

A key event in NETosis is the activation of the NADPH oxidase complex, which depends on increased cytosolic Ca²⁺ levels and subsequent ROS production. Excessive ROS leads to the degradation of cytoplasmic granules and promotes the translocation of neutrophil elastase (NE), myeloperoxidase (MPO), and peptidylarginine deiminase 4 (PAD4) into the nucleus, resulting in chromatin decondensation and nuclear envelope disintegration. Another critical factor is the translocation of PAD4 from the cytoplasm to the nucleus, where it catalyzes histone citrullination, leading to chromatin decondensation. In the final stage of NETosis, pores form in the plasma membrane, allowing the release of chromatin into the extracellular environment to form NETs.

Figure 12. Mechanisms of NETosis

2.11 Parthanatos

Parthanatos is a form of PCD mediated by the hyperactivation of PARP. It is characterized by the accumulation of PAR polymers and the release of AIF, ultimately leading to DNA fragmentation. This process can be triggered by various stimuli, including ROS, hydrogen peroxide, ionizing radiation, and alkylating agents. In cases of mild DNA damage, PARP-1 recruits DNA repair proteins to restore damaged DNA. However, severe DNA damage leads to excessive activation of PARP-1 and the formation of PAR polymers. The accumulated PAR polymers bind to AIF and facilitate its release from mitochondria. AIF interacts with macrophage migration inhibitory factor (MIF) to form the AIF/MIF complex, which translocates to the nucleus, resulting in DNA degradation and the induction of Parthanatos.

Figure 13. Mechanisms of parthanatos

2.12 Cuproptosis

Cuproptosis is a copper-induced form of PCD characterized by mitochondrial stress due to the aggregation of lipoylated mitochondrial enzymes and loss of iron-sulfur cluster proteins. Excessive copper is toxic to cells and tissues, leading to a condition known as copper overload or copper toxicity. The mitochondrial matrix reductase ferredoxin 1 (FDX1) reduces Cu²⁺ to Cu⁺ and releases it into the mitochondrial matrix. Furthermore, FDX1 has been identified as a novel lipoylation effector that promotes the accumulation of toxic lipoylated Dihydrolipoamide S-Acetyltransferase (DLAT), thereby triggering cuproptosis. This cell death process depends on intracellular copper levels and the lipoylation status of tricarboxylic acid (TCA) cycle enzymes.

Figure 14. Mechanisms of cuproptosis

2.13 Disulfidptosis

Disulfidptosis is a novel form of cell death triggered by disulfide stress, with its core mechanism initiating from NADPH depletion due to glucose starvation. This leads to a collapse of the intracellular reducing capacity, rendering cells unable to maintain protein thiols in a reduced state. A key step involves cells with high SLC7A11 expression, which take up large amounts of cystine but cannot reduce it. This results in aberrant disulfide bond formation between cysteine-rich proteins such as those in the actin cytoskeleton, directly causing actin filament cross-linking, contraction, and collapse. The process ultimately leads to loss of cellular adhesion and death. This pathway is distinct from known forms of cell death such as apoptosis, necroptosis, pyroptosis, and ferroptosis, and is unique in that disulfide stress directly disrupts the cytoskeletal architecture.

Figure 15. Mechanisms of disulfidptosis

3. Proteins Involved in Cell Death

3.1 Major proteins involved in apoptosis

3.2 Major proteins involved in Necroptosis

3.3 Major proteins involved in autophagic cell death

3.4 Major proteins involved in lysosomal cell death

3.5 Major proteins involved in alkaliptosis

3.6 Major proteins involved in oxeiptosis

3.7 Major proteins involved in pyroptosis

3.8 Major proteins involved in ferroptosis

3.9 Major proteins involved in NETosis

3.10 Major proteins involved in parthanatos

3.11 Major proteins involved in cuproptosis

3.12 Major proteins involved in disulfidptosis

Cell death is an actively regulated programmed process orchestrated by a series of key proteins. The activation and interaction of its core executor molecules, such as the caspase family, RIPK, MLKL, and the Gasdermin family, collectively determine the fate of different modes of cell death. These cell death pathways not only play essential roles in normal physiological processes but are also critically involved in the pathogenesis and progression of various diseases.

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