Apoptosis and Necrosis Differences and Comparison

Cell death occurs naturally in multicellular organisms and cells die because of internal and external stimuli. A number of cell death events have been acknowledged (PMID: 29362479; (1)). Their classification is defined by two groups:

  • Programmed or regulated cell death referred to  as apoptosis, and sometimes autophagy & necroptosis
  • Accidental cell death as a result of non-physiological states like infection or injury (necrosis)

Apoptosis and Necrosis Differences and Comparison

Image Credit: Proteintech Group

What is apoptosis?

Apoptosis is the most common form of programmed cell death. It can be induced through a variety of physical, chemical, and biological factors and its cellular response is closely regulated. Caspases, the group of proteases activated throughout the apoptosis process (Caspase family), regulates the controlled degradation of cellular components.

Within healthy cells, caspases are living as proenzymes in their inactive forms. A caspase cascade (caspase 2, 8, 9, and 10 -termed initiator caspases) is activated by apoptotic signaling. In turn, the initiator caspases cleave and activate downstream effector caspases (caspase -3, -6, and -7).

Effector caspases execute apoptosis by cleaving targeted cellular proteins. Stimuli triggering apoptosis can be internal, for example, DNA damage, ER stress, higher  ROS levels, cell defects during mitosis, or external, where extracellular stimuli are identified by cells through plasma membrane receptors (Caspase cascade).

Extrinsic apoptosis pathway

There are two key receptor forms for externally triggered apoptosis process. Death receptors, for example, TNF or Fas receptors, bind their extracellular ligands (TNF-alpha, FasL – Fas ligand, respectively). This encourages their activation and formation into complexes, resulting in the activation of intracellular caspases.

The other apoptotic receptors, termed dependence receptors, such as DCC and PTCH1, depend on an opposite sensing mechanism. In physiological settings, they respond to trophic factors and act as an anti-apoptotic stimulus. However, when their ligand falls under a specific level in the extracellular space, ligand-free receptors induce the apoptotic response.

Intrinsic apoptosis pathway

Intrinsic apoptosis is facilitated by mitochondria-associated BCL-2 family proteins; BAX and BAK. BAK is a transmembrane protein of the outer mitochondrial membrane. When apoptosis is triggered, BAX endures conformational alterations.

This reveals its transmembrane domain, resulting in the insertion of BAX into the outer mitochondrial membrane. BAX-BAK heterodimers create mitochondrial pores, which results in the release of mitochondrial proteins into the cytoplasm, inclusive of cytochrome c and DIABLO proteins, that can trigger caspases.

Changes that appear during apoptosis

Table 1 demonstrates that apoptosis is reflected in significant cell morphological changes. In the earlier stages, a cell undergoing apoptosis drops cell contacts and alters its shape. Chromatin condenses in the nucleus and travels toward the nuclear envelope. Condensation of the nucleus, also known as pyknosis, triggers DNA degradation.

Table 1. Physiological events during apoptosis, autophagy, and necrosis. Source: Proteintech Group

  Apoptosis Autophagy Necrosis

Morphological changes

Cell

Shrinkage and loss of cell-cell contacts; apoptotic cells phagocytose neighboring cells

Extensive vacuolization of the cytoplasm

Swelling; cell lysis

Plasma membrane

Blebbing with intact cell integrity, formation of apoptotic bodies at late stages

Loss of integrity

Loss of integrity; increased permeability

Organelles

No visible changes

In some cases, enlargement of Golgi and ER is observed

Cell fragmentation

Nucleus

Chromatin condensation, fragmentation

No chromatin condensation

Condensation of chromatin and disintegration of the nucleus

Mitochondrion

Potential membrane changes, swelling

Swelling sometimes observed

Non-functional, swelling and fragmentation

Biochemical changes

DNA

Endonuclease-induced cleavage to fragments of specific lengths (DNA laddering)

 

Random degradation of genomic DNA

Proteins

Kinases activation; phosphatases, caspases, and nucleases

Enzymatic degradation in autophagosomes

Unspecific degradation

Anti-apoptotic proteins

Bcl-2 family proteins, Inhibitor of Apoptosis Proteins (IAPs), caspase inhibitors

 

Bcl-2 expression in some cases

Energy

ATP-dependent

Catabolic process

ATP-independent

Tissue response

  • Restricted to individual cells
  • Induced by physiological changes (e.g., Ca2+, free radicals, hormones, toxins, depletion of growth factors)
  • Usually no inflammatory response
  • Induced via TOR signaling and induction of Atg genes
  • Necrosis often affects groups of cells rather than a single cell
  • Induced by external and internal pathological conditions
  • Enhancement of inflammatory response
Post-death clearance Phagocytosis No phagocytosis Cell lysis

 

Water loss leads to significant cell shrinkage and blebbing of the plasma membrane with a small amount or no morphological alterations to the further cellular organelles. Phosphatidylserine, a lipid existing only in the inner layer of the plasma membrane, is now also observable in the outer layer.

Nucleus and cytoplasm splinter into apoptotic bodies. Released cellular proteases result in disintegration of the cellular skeleton, membranes, and proteins. Adjacent macrophages identify, engulf, and digest apoptotic bodies, finishing the process.

Autophagy – A rare type of programmed cell death

Autophagy is a natural process of degradation of cellular contents throughout nutrient stress. In the instance of macro-autophagy, it comprises of the creation of double membrane vesicles termed autophagosomes that fuse with lysosomes to form autolysosomes.

This process is induced by the mechanistic target protein of rapamycin (mTOR) and autophagy-related genes (Atgs) proteins, as shown in Figures 1 and 2. Autophagy encourages cell survival, offering nutrients to starved cells which are acquired through the digestion of non-essential cellular components.

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue slide using 10181-2-AP (ATG5 antibody) at dilution of 1:200 (under 40x lens).

Figure 1. Immunohistochemical analysis of paraffin-embedded human colon cancer tissue slide using 10181-2-AP (ATG5 antibody) at dilution of 1:200 (under 40x lens). Image Credit: Proteintech Group

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue slide using 20986-1-AP (ULK1 Antibody) at dilution of 1:200 (under 40x lens).

Figure 2. Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue slide using 20986-1-AP (ULK1 Antibody) at dilution of 1:200 (under 40x lens). Image Credit: Proteintech Group

Additionally, it was revealed that macro-autophagy can be one of the paths for programmed cell death (PMID: 22052193; (2)). Despite it being substantially less common than apoptosis, it plays a part in regulating some developmental processes. The most renowned is the elimination of specific larval organs, the salivary glands and midgut, in Drosophila melanogaster during larval–pupal transition (PMID:18083103; (3) and 19818615; (4)).

Autophagic cell death was also seen in in vitro cultures of adult hippocampal neural cells as a response to insulin removal (PMID: 18653772; (5)). The key feature of autophagy-dependent cell death is extensive autophagic vacuolization of the cytoplasm, with zero alterations in chromatin organization as seen in apoptosis, shown in Table 1.

Additionally, cell remnants are not cleared by macrophagic phagocytosis as seen in apoptosis. Autophagy results in autophagic cell death that can be blocked by inhibitors or the depletion of Atg proteins, for example, Atg1, Atg5, Atg7. This is shown in Figure 3.

WB result of ATG5 antibody (10181-2-AP; 1:1000; incubated at room temperature for 1.5 hours) with sh-Control and sh-ATG5 transfected HepG2 cells.

Figure 3. WB result of ATG5 antibody (10181-2-AP; 1:1000; incubated at room temperature for 1.5 hours) with sh-Control and sh-ATG5 transfected HepG2 cells. Image Credit: Proteintech Group

What key changes are observed during necrosis?

Necrosis is a form of cell injury with the definition of unregulated cell death that can be caused by internal or external stresses like mechanistic injuries, chemical agents, or pathogens. The process is normally fast and results in cell swelling, known as oncosis, and bursting as a result of loss of osmotic pressure. This is shown in Table 1.

The loss of plasma membrane integrity triggers the escape of cellular contents into the extracellular space, leading to inflammatory responses. Cell disintegration is followed by a series of morphological changes, inclusive of cell organelles disruption, like swelling of the ER and mitochondria, or decay of the Golgi apparatus.

An influx of calcium ions from the extracellular matrix triggers intracellular nucleases that fragment DNA. Unconstrained lysosomal hydrolases are a contribution to the degradation of nucleic acids and proteins. Decay products induce leukocytes, lymphocytes, and macrophages that phagocytose the remnants of dead cells.

Necroptosis: The regulated necrosis

Necroptosis is a form of regulated cell death that generates necrotic phenotype. It occurs in response to stress stimuli (PMID: 19524512; (6), 19524513; (7), and 19498109; (8)) like interferons, death ligands, or Toll-like receptors. Activation is facilitated through protein 3 interacting receptor, RIP3, also known as RIPK3, a serine-threonine kinase, as shown in Figure 4.

IP Result of anti-RIP3 (IP:17563-1-AP, 3ug; Detection:17563-1-AP 1:300) with SW 1990 cells lysate 3000ug.

Figure 4. IP Result of anti-RIP3 (IP:17563-1-AP, 3ug; Detection:17563-1-AP 1:300) with SW 1990 cells lysate 3000 ug. Image Credit: Proteintech Group

Cells in the primary phases of necroptosis are characterized by RIP3 phosphorylation and frequently β-amyloid-like protein complex formation with RIP1, recognized as necrosome. Phosphorylated RIP3 acts downstream by phosphorylation of MLKL, as shown in Figure 5, which leads to necrosis by a mechanism that isn’t yet completely understood (PMID: 30131615; (9)).

Immunofluorescence analysis of (-20 ℃ Ethanol) fixed HepG2 cells using 21066-1-AP (MLKL antibody) at dilution of 1:50 and Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).

Figure 5. Immunofluorescence analysis of (-20 ℃ Ethanol) fixed HepG2 cells using 21066-1-AP (MLKL antibody) at dilution of 1:50 and Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L). Image Credit: Proteintech Group

Final remarks

The two key types of cell death are apoptosis and necrosis. They are different with regards to the stimuli that trigger cell death processes, morphological and biochemical changes, and in the signaling pathways used by cells.

The cause of necrosis is external factors that result in irreversible cell injury, with loss of plasma membrane integrity and fast death frequently leading to the activation of the immune system.

Conversely, apoptosis is triggered by a number of internal and external pathways. It is a highly controlled process that leads to the slow turnover of cell remnants and phagocytosis by neighboring macrophages.

References

  1. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.
  2. Cell death by autophagy: facts and apparent artefacts.
  3. Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila.
  4. Autophagy, not apoptosis, is essential for midgut cell death in Drosophila. Autophagy, not apoptosis, is essential for midgut cell death in Drosophila.
  5. Autophagic death of adult hippocampal neural stem cells following insulin withdrawal.
  6. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.
  7. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.
  8. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis.
  9. RIP kinases as modulators of inflammation and immunity.

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Last updated: Apr 21, 2020 at 4:17 AM

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