Open Access is an initiative that aims to make scientific research freely available to all. To date our community has made over 100 million downloads. It’s based on principles of collaboration, unobstructed discovery, and, most importantly, scientific progression. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. How? By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers.
We are a community of more than 103,000 authors and editors from 3,291 institutions spanning 160 countries, including Nobel Prize winners and some of the world’s most-cited researchers. Publishing on IntechOpen allows authors to earn citations and find new collaborators, meaning more people see your work not only from your own field of study, but from other related fields too.
Apoptosis, a highly regulated form of programmed cell death (PCD), is essential for development, tissue homeostasis, and the immune response. This self-destructive process is characterized by distinct cellular changes, including membrane blebbing, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies. Apoptosis can be triggered by two primary signaling pathways: the intrinsic (mitochondrial) pathway, responding to internal cellular stress, and the extrinsic (death receptor) pathway, initiated by external signals. Both pathways ultimately activate caspases, proteolytic enzymes that dismantle the cell in an orderly manner, preventing inflammation. The intrinsic pathway is regulated by the Bcl-2 protein family, balancing pro-apoptotic and anti-apoptotic signals to maintain cellular integrity, while mitochondrial disruptions lead to the release of cytochrome c and activation of downstream apoptotic machinery. Dysregulation of apoptosis is linked to various diseases, including cancer, where defective apoptotic processes allow uncontrolled cell proliferation, and neurodegenerative disorders, where excessive apoptosis leads to cell loss. This review offers an in-depth understanding of apoptosis, and its regulatory mechanisms offer crucial insights for therapeutic approaches targeting apoptosis in diseases characterized by abnormal cell survival or death.
Pharmacology Division, Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
Aalim Maqsood Bhat
Pharmacology Division, Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
Sheikh Tasduq Abdullah
*
Pharmacology Division, Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
*Address all correspondence to: stabdullah@iiim.res.in, tasduq11@gmail.com
1. Introduction to apoptosis
The concept of apoptosis has deep historical roots, though early observations of programmed cell death predate its modern understanding. The term “apoptosis” itself was formally introduced in 1972 by John F. R. Kerr, Andrew H. Wyllie, and Alastair R. Currie in their seminal paper, which described the distinct morphological changes observed in cells undergoing this regulated death process. They choose the term “apoptosis” from the Greek word for “falling off,” as in the falling of leaves from trees, symbolizing the natural and orderly process of cell removal [1]. This was a pivotal moment that distinguished apoptosis from necrosis and paved the way for understanding cell death as an active, regulated process rather than a passive consequence of injury. Even before this formal introduction, researchers observed the phenomenon of controlled cell death in various biological contexts [2]. In the early nineteenth century, scientists noted the natural process of tissue sculpting and cell removal during embryonic development, such as the disappearance of the tail in tadpole metamorphosis and the formation of individual fingers and toes in human embryos [3]. However, these observations lacked a mechanistic framework and were often seen as isolated phenomena. In the mid-twentieth century, advancements in microscopy and cell biology allowed for more detailed observation of cellular processes. Researchers began to note consistent patterns of cell death that were not attributable to injury or infection by the 1980s and 1990s. Research in the field had exploded, with scientists identifying caspases, the Bcl-2 family of proteins, and various other regulators of apoptosis. This period also highlighted the importance of apoptosis in disease contexts, particularly cancer, where defective apoptotic pathways allow cells to evade death and proliferate uncontrollably [4].
Apoptosis is a form of programmed cell death (PCD) characterized by an organized and tightly regulated sequence of biochemical events, leading to distinct morphological changes and, ultimately, cell death without causing inflammation or damage to surrounding tissues [5]. This process is critical for maintaining cellular homeostasis by eliminating damaged, aged, or unnecessary cells. Unlike necrosis, which is typically a response to acute injury and results in cell rupture and inflammation, apoptosis proceeds in an orderly manner [6]. Apoptotic cells undergo specific changes, including cell shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing, and the formation of apoptotic bodies, which are then phagocytosed by neighboring cells or immune cells, preventing inflammatory response [7].
Two primary pathways mediate apoptosis: the intrinsic (mitochondrial) pathway, triggered by internal stress signals, and the extrinsic pathway, which is activated by external signals from the cellular environment [8]. Both pathways ultimately lead to the activation of caspases, a family of cysteine proteases that systematically dismantle the cell [9]. This self-destructive process is essential in numerous physiological contexts, including development, immune response, and tissue remodeling, as well as in the prevention of cancer and other diseases associated with uncontrolled cell growth [10].
Apoptosis is fundamental to the survival and health of multicellular organisms [11]. It is an essential, highly regulated process that helps organisms manage cellular turnover, tissue remodeling, immune response, and the elimination of potentially dangerous cells [5]. Here are the key aspects of apoptosis and why it is critical for biological systems. Apoptosis plays a central role in maintaining cellular balance within tissues [5]. In most adult tissues, the rate of cell death is closely matched by the rate of cell division. Apoptosis removes aged, damaged, or dysfunctional cells that could otherwise accumulate and impair tissue function [12]. By preventing the over-accumulation of cells, apoptosis maintains a healthy cellular environment, ensuring tissues and organs operate efficiently. For example, epithelial cells in the intestines and skin continuously regenerate, with older cells being replaced by new ones [5]. Apoptosis is responsible for clearing away these old cells, allowing tissues to remain functional and preventing overcrowding, which could lead to structural abnormalities [13]. During embryonic development, apoptosis is crucial for shaping the developing organism. It helps to sculpt tissues, forming organs and structures by removing cells in specific patterns [14]. Apoptosis enables the precise removal of cells, ensuring that organs and limbs form correctly. Examples include formation of fingers and toes: In human and vertebrate embryonic development, apoptosis eliminates the webbing between fingers and toes, leading to the formation of distinct digits. Neural development: In the developing brain, excess neurons are produced initially [15]. Apoptosis selectively removes neurons that do not make proper connections, refining neural circuits and ensuring only properly connected neurons survive, which is critical for proper brain function [16]. Immune System Maturation: Apoptosis is also involved in eliminating self-reactive immune cells during immune system development, which helps prevent autoimmune diseases [14]. Without apoptosis during development, organisms could have structural malformations, impaired organ function, or dysfunctional immune responses [17]. Defense against disease and cellular damage: Apoptosis acts as a protective mechanism, eliminating cells that may be harmful to the organism [18]. Cells with irreparable DNA damage, oxidative stress, or exposure to harmful agents like radiation or toxins are often targeted for apoptosis [19]. This selective elimination helps prevent the accumulation of potentially cancerous or dysfunctional cells. Prevention of cancer: Apoptosis acts as a defense against cancer by eliminating cells with damaged DNA or other oncogenic mutations [20]. When cells accumulate mutations that could lead to uncontrolled proliferation, apoptosis mechanisms typically remove these cells before they become malignant [21]. In many cancers, apoptosis is dysregulated, allowing abnormal cells to evade death, leading to tumor development [22]. Response to infection: During infection, apoptosis plays a role in containing pathogens [23]. Infected cells often undergo apoptosis to prevent the spread of viruses or bacteria. For example, cytotoxic T cells and natural killer cells can induce apoptosis in infected cells, limiting pathogen proliferation [24]. By destroying infected cells, apoptosis helps control infections and minimize the damage to surrounding tissues. It helps in the regulation of immune system function. The immune system relies on apoptosis for both its development and its regulated response to infections and foreign invaders [25]. Apoptosis shapes immune cell populations and prevents autoimmunity by eliminating immune cells that might attack the body’s own tissues [26]. In the thymus, apoptosis helps remove self-reactive T cells, a process known as clonal deletion. This process is critical for establishing immune tolerance, preventing the immune system from attacking the body’s own cells. Without apoptosis in immune cell maturation, autoimmune diseases would become more common [27]. Apoptosis is also known to cause resolution of immune responses: After an infection is cleared, apoptosis reduces the number of active immune cells to return the immune system to its baseline state, preventing excessive inflammation or tissue damage. If immune cells persist inappropriately, they can cause chronic inflammation, contributing to diseases like rheumatoid arthritis [27]. The role of apoptosis has been identified in neurodegeneration and aging. In neurons, which are highly specialized and often non-dividing cells, apoptosis is carefully regulated, as excessive apoptosis in the brain can lead to neurodegenerative diseases. During aging, an imbalance in apoptosis can lead to cell loss in tissues with limited regenerative capacity [28]. Excessive apoptosis is implicated in neurodegenerative conditions such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. In these diseases, neurons in specific brain regions undergo excessive apoptosis, contributing to the progressive loss of brain function [29]. The other role of apoptosis includes aging and tissue atrophy. As organisms age, apoptosis can contribute to tissue and organ atrophy if cellular replenishment cannot keep up with cell loss. For instance, excessive apoptosis in muscle or bone cells contributes to conditions like sarcopenia (muscle wasting) and osteoporosis [30]. Unlike necrosis, which typically results from injury and leads to cell rupture and inflammation, apoptosis is a controlled, contained process. The cell’s contents are packaged into apoptotic bodies and phagocytosed by neighboring cells or immune cells, preventing the release of potentially harmful intracellular substances that could trigger inflammation. This aspect of apoptosis is particularly important in preventing inflammatory diseases and maintaining tissue integrity. For example, when heart cells die from ischemia (lack of blood flow), apoptosis limits inflammatory damage compared to necrosis, which can exacerbate injury to surrounding cells [31]. The controlled nature of apoptosis makes it a promising target for therapies in diseases where abnormal cell survival or cell death occurs. In cancer, for example, where apoptosis is often impaired, therapeutic agents that can restore apoptotic signaling pathways are under development to trigger death in cancer cells selectively. Apoptosis modulation also holds potential for treating autoimmune diseases, neurodegenerative diseases, and infections by targeting pathways that either enhance or inhibit apoptotic mechanisms as needed. Examples of therapeutic approaches include cancer therapy. Drugs like BH3 mimetics and proteasome inhibitors are designed to stimulate apoptosis selectively in cancer cells, exploiting the apoptotic machinery to overcome resistance. In neurodegenerative diseases, strategies to inhibit excessive apoptosis are being explored to protect neurons and slow disease progression [32].
3. Decoding apoptosis: A comparison with other cell death pathways
Apoptosis is a tightly regulated, programmed process of cell death. It is orchestrated by specific signaling pathways that ensure cell death occurs in an orderly, controlled manner. This control prevents damage to surrounding cells and allows efficient removal of the dying cell by immune cells without inflammation [12]. Necrosis, in contrast, is typically an uncontrolled, accidental form of cell death caused by external injury or trauma, such as physical damage, lack of oxygen, or exposure to toxins. Unlike apoptosis, necrosis lacks any regulation and generally occurs due to severe cellular stress or injury that the cell cannot repair [33].
Autophagy is primarily a survival mechanism. Unlike apoptosis and necrosis, it is an intracellular recycling process, allowing cells to break down and reuse damaged components under stress [34]. However, excessive or dysfunctional autophagy can lead to a self-destructive process known as autophagic cell death, although this process is less organized and regulated than apoptosis [30, 35].
Pyroptosis is a highly inflammatory form of programmed cell death that plays a critical role in the immune response. Unlike apoptosis, which is non-inflammatory, pyroptosis results in the release of pro-inflammatory cytokines such as IL-1β and IL-18, along with cellular contents, leading to localized inflammation. This process is primarily triggered by infections or cellular stress and involves the activation of inflammasomes, which are intracellular multiprotein complexes. Key molecular players in pyroptosis include caspase-1 and the gasdermin family, particularly gasdermin D (GSDMD). Upon activation by inflammasomes, caspase-1 cleaves GSDMD, releasing its N-terminal fragment. This fragment forms pores in the plasma membrane, leading to cell lysis and the release of inflammatory mediators. Pyroptosis is critical for combating infections by eliminating infected cells and recruiting immune cells to the site of infection. However, dysregulated pyroptosis is implicated in various diseases, including chronic inflammation, autoimmune disorders, and sepsis. As a result, it is a significant focus of research in immunology and therapeutic development [36]. Cells respond to death-inducing stimuli by activating distinct pathways critical for maintaining balance in multicellular organisms illustrated in Figure 1.
Figure 1.
This figure illustrates that how cells respond to death-inducing stimuli by activating distinct pathways critical for maintaining balance in multicellular organisms. Failure to execute these pathways can result in developmental defects, organ dysfunction, cancer, or inappropriate immune responses. Apoptosis involves caspase activation, nuclear condensation, apoptotic body formation, and phagocytic clearance; without clearance, secondary necrosis may occur. Autophagy degrades cellular components within autophagic vacuoles, characterized by vacuolization and slight chromatin condensation. Oncosis is marked by cellular swelling, membrane breakdown, and the release of inflammatory contents [37]. Pyroptosis, a caspase-1-mediated process, activates proinflammatory cytokines IL-1β and IL-18, causing cell lysis and inflammation. Susa. Fink et al. [2] … https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=1087413_zii0040547300002.jpg.
Table 1 provides a detailed comparison of apoptosis, necrosis, and autophagy based on key characteristics, including the type of cell death, effects on surrounding tissue, underlying mechanisms, triggers, and their roles in health and disease. Apoptosis is a regulated form of programmed cell death essential for tissue homeostasis, while necrosis is an uncontrolled, pathological process often associated with inflammation and tissue damage. Autophagy primarily functions as a survival mechanism by degrading and recycling cellular components but can also contribute to cell death under certain conditions. Understanding these processes is critical for deciphering their implications in various physiological and pathological contexts, including cancer, neurodegeneration, and immune responses.
Feature
Apoptosis
Necrosis
Autophagy
Type of cell death
Programmed cell death (regulated)
Unregulated cell death (often pathological)
Survival mechanism, but can lead to cell death
Trigger
Internal/external signals (e.g., DNA damage, death signals)
Physical injury, toxins, lack of oxygen
Nutrient deprivation, cellular stress
Key mechanism
Caspase activation, mitochondrial and death receptor pathways
Loss of plasma membrane integrity
Formation of autophagosomes, lysosomal degradation
5. Morphological and structural changes in apoptosis
Light and electron microscopy have revealed various morphological changes characteristic of apoptosis [38]. In the initial stages of apoptosis, light microscopy reveals cell shrinkage and pyknosis [39]. Shrinking cells appear smaller, with a dense cytoplasm and more tightly packed organelles. Pyknosis, caused by chromatin condensation, is a hallmark of apoptosis. On histological examination with hematoxylin and eosin stain, apoptotic cells are observed as single cells or small clusters [40]. They typically appear as round or oval structures with dark eosinophilic cytoplasm and dense purple nuclear chromatin fragments. Electron microscopy provides a more detailed view of subcellular changes. During early chromatin condensation, electron-dense nuclear material aggregates near the nuclear membrane, though nuclei may also appear uniformly dense [40]. Plasma membrane blebbing intensifies, leading to karyorrhexis and the formation of apoptotic bodies through a process called “budding.” These apoptotic bodies, containing cytoplasm with tightly packed organelles and sometimes nuclear fragments, maintain organelle integrity and are enclosed within an intact plasma membrane. Macrophages, parenchymal cells, or neoplastic cells subsequently phagocytose and degrade these bodies in phagolysosomes [41]. Macrophages that engulf apoptotic cells, termed “tingible body macrophages,” are often found in the reactive germinal centres of lymphoid follicles or occasionally in the thymic cortex. Tingible bodies represent nuclear debris from apoptotic cells [5].
Notably, apoptosis is not associated with inflammation or tissue damage, as apoptotic cells do not release their contents into surrounding tissue. They are rapidly engulfed, preventing secondary necrosis. Furthermore, phagocytic cells do not release pro-inflammatory cytokines [42]. Apoptosis is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation, leading to the formation of membrane-bound apoptotic bodies that sequester cellular components. In contrast, necrosis involves cell swelling (oncosis), membrane rupture, and uncontrolled release of cellular contents, which can trigger inflammation and damage nearby cells. Autophagy, distinct from both, features the formation of autophagosomes—double-membrane vesicles that encapsulate and degrade cellular components.
Autophagy lacks the characteristic nuclear changes of apoptosis and the membrane rupture seen in necrosis and instead involves extensive cytoplasmic vacuolization [2]. Apoptosis, a highly regulated form of programmed cell death, is marked by specific and distinctive morphological changes that distinguish it from other forms of cell death, such as necrosis. These changes include membrane blebbing, chromatin condensation, nuclear fragmentation, and the formation of apoptotic bodies. Such features are essential not only for recognizing apoptosis under a microscope but also for understanding the sequence and significance of events leading to cellular disassembly.
5.1 Membrane blebbing
Membrane blebbing is one of the most characteristic early morphological changes in apoptosis leading to the formation of spherical protrusions on the cell surface, characteristic of dynamic cytoskeletal changes represented in Figure 2. This process involves the formation of dynamic, bulging protrusions on the plasma membrane. Membrane blebbing begins as the cytoskeleton underneath the cell membrane reorganizes and becomes destabilized. The actin-myosin network contracts, driven by the activity of enzymes like rho-associated protein kinase (ROCK), which phosphorylates myosin light chains, promoting actin-myosin interactions. This contraction exerts tension on the plasma membrane, causing it to protrude outwards, forming “blebs. Blebbing plays a role in the later steps of apoptosis, contributing to cell disassembly [43]. By fragmenting into smaller parts, the cell prepares for the formation of apoptotic bodies. This blebbing helps prevent the release of cellular contents into the surrounding environment, thereby reducing the risk of inflammation, a key distinction between apoptosis and necrosis [44].
Figure 2.
Scanning electron microscopy (SEM) image performed in our study illustrating membrane blebbing in a cell. The image highlights the formation of spherical protrusions on the cell surface, characteristic of dynamic cytoskeletal changes. These blebs are indicative of cellular stress or a programmed cell death process such as apoptosis, where membrane integrity is maintained during early stages.
5.2 Chromatin condensation
Chromatin condensation (pyknosis), where chromatin undergoes profound changes in structure as shown in Figure 3. Chromatin condensation is initiated by the activation of caspases, particularly caspase-3 and caspase-6, which target proteins involved in nuclear integrity and chromatin structure. These caspases activate endonucleases, such as caspase-activated DNase (CAD), which cleaves DNA at specific internucleosomal sites. Consequently, chromatin condenses and aggregates at the periphery of the nuclear membrane. Under a microscope, chromatin condensation appears as a dense, dark staining of nuclear material in apoptotic cells, often forming crescent-shaped patches against the nuclear envelope [47]. Eventually, the condensed chromatin fragments, a feature that is easily distinguishable from the nuclear swelling and random DNA fragmentation typically observed in necrosis. Chromatin condensation serves two main purposes. First, it compacts the DNA and prepares it for packaging into apoptotic bodies, aiding in the non-inflammatory disposal of nuclear material. Second, chromatin condensation signals to phagocytic cells that the apoptotic cell is ready for engulfment, ensuring that cellular remnants are cleared without spurring an immune response.
Nuclear fragmentation (karyorrhexis) is the process by which the nucleus breaks down into smaller, distinct fragments, further aiding in cellular dismantling represented in Figure 4. Following chromatin condensation, CAD and other nucleases continue to break down nuclear DNA, fragmenting the nucleus itself. Caspase-activated nucleases degrade nuclear Lamins, proteins that maintain the nuclear envelope’s structure, leading to the nuclear envelope’s disintegration. The nuclear contents fragment into smaller pieces, each enveloped by a segment of the nuclear membrane [48]. Nuclear fragmentation ensures that the cell’s genetic material is partitioned into manageable units, which can be engulfed by phagocytes. This compartmentalization of DNA prevents the exposure of nuclear material to the extracellular environment, thus reducing the risk of autoimmunity or inflammatory responses.
Figure 4.
Nuclear structure alterations during apoptosis visualized with DAPI staining. The figure shows the progressive distortion of nuclear architecture during apoptosis, visualized using DAPI (4′,6-diamidino-2-phenylindole) staining. DAPI binds to DNA and emits blue fluorescence under UV light. In healthy cells, the nuclei appear intact and uniformly stained. During early apoptosis, chromatin condensation is observed as intense, localized fluorescence [45].
5.4 Formation of apoptotic bodies
The final stage of apoptosis involves the formation of apoptotic bodies, small membrane-bound vesicles that contain cellular components, including cytoplasm, organelles, and nuclear fragments. Apoptotic bodies form as the cell undergoes further cytoskeletal contraction and membrane blebbing. Cytoskeletal elements continue to fragment the cell into discrete sections, each surrounded by plasma membrane. Apoptotic bodies are often heterogeneous in size and may contain intact organelles, portions of the nucleus, or cytoplasmic proteins. Apoptotic bodies expose specific “eat-me” signals on their surface, such as phosphatidylserine, which is normally found on the inner leaflet of the plasma membrane but flips to the outer surface during apoptosis. This signal is recognized by receptors on phagocytes, allowing these immune cells to engulf and digest apoptotic bodies without releasing their contents into the surrounding tissue [49]. The formation of apoptotic bodies ensures that cellular debris is neatly packaged, allowing it to be efficiently cleared by phagocytes. This organized packaging minimizes the risk of inflammation, as it prevents the release of pro-inflammatory intracellular components, which would otherwise occur in cases of cell rupture, as seen in necrosis.
In addition to its distinct morphological characteristics, apoptosis is also characterized by specific biochemical changes. These changes serve as crucial indicators of apoptosis and distinguish it from other forms of cell death, such as necrosis and autophagy. Two of the most significant biochemical hallmarks of apoptosis are DNA fragmentation and phosphatidylserine (PS) exposure [50]. These hallmarks reflect the regulated and orderly dismantling of cellular components during apoptosis, enabling apoptotic cells to be efficiently recognized and removed by phagocytes, thereby avoiding inflammation. Below is an in-depth look at these biochemical markers and their roles in apoptosis.
6.1 DNA fragmentation
DNA fragmentation is one of the most definitive biochemical indicators of apoptosis, marked by the cleavage of genomic DNA into characteristic fragments shown in Figure 5. DNA fragmentation in apoptosis is largely mediated by a family of enzymes known as caspases, specifically caspase-activated DNase (CAD). Under normal conditions, CAD is kept inactive by an inhibitor, ICAD (inhibitor of caspase-activated DNase). During apoptosis, caspase-3 cleaves ICAD, releasing CAD to enter the nucleus and cleave DNA at internucleosomal sites. This cleavage results in fragments that are multiples of approximately 180-200 base pairs, corresponding to the DNA wrapped around each nucleosome [52]. The fragmented DNA generated during apoptosis often forms a characteristic “DNA ladder” pattern when analyzed by gel electrophoresis. This laddering is due to the precise and regular cleavage by CAD, which is distinct from the random and extensive DNA degradation that occurs in necrosis. Additionally, DNA fragmentation can be detected in situ using techniques like the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labelling) assay, which labels DNA breaks to visualize apoptotic cells [53].
Figure 5.
DNA fragmentation during apoptosis assessed by agarose gel electrophoresis. DNA isolated from apoptotic cells was subjected to agarose gel electrophoresis to analyze the characteristic DNA fragmentation. The gel shows a DNA laddering pattern, indicative of internucleosomal cleavage, a hallmark of apoptosis. Lanes represent samples from untreated control cells, showing intact genomic DNA, and cells treated with an apoptosis-inducing agent, displaying the fragmented DNA pattern [51].
DNA fragmentation is a key step in the irreversible progression of apoptosis, ensuring that the genetic material is non-functional and preparing the cell for complete disassembly. By condensing and fragmenting the DNA, the cell also facilitates the efficient packaging of nuclear material into apoptotic bodies, allowing for their safe engulfment by phagocytes without exposing intact DNA to the extracellular environment [46, 53].
6.2 Phosphatidylserine (PS) exposure
Phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane is another essential hallmark of apoptosis. This event serves as an “eat-me” signal for phagocytes, marking the apoptotic cell for recognition and clearance as shown in Figure 6. Under normal circumstances, PS resides exclusively on the inner leaflet of the plasma membrane. During apoptosis, caspase activation leads to the inactivation of flippases, enzymes responsible for maintaining membrane asymmetry, and the activation of scramblases, which move PS from the inner to the outer membrane leaflet. One such scramblase, Xkr8, is directly activated by caspase-3, facilitating the exposure of PS on the cell surface [54]. PS exposure can be detected using annexin V, a protein that specifically binds to PS in the presence of calcium ions. Annexin V staining, in combination with propidium iodide (a dye that enters cells with compromised membranes), allows researchers to differentiate between early apoptotic cells (annexin V-positive, PI-negative) and late apoptotic or necrotic cells (annexin V-positive, PI-positive). The exposure of PS on the outer leaflet is crucial for the immunologically silent clearance of apoptotic cells. Phagocytic cells, such as macrophages, have receptors that recognize PS, facilitating the engulfment of apoptotic cells or apoptotic bodies. This process of “silent phagocytosis” prevents the release of inflammatory signals, distinguishing apoptosis from necrosis, where cellular contents are released and can stimulate an immune response. By enabling rapid and efficient clearance, PS exposure plays a key role in maintaining tissue homeostasis and preventing autoimmune reactions [55].
Figure 6.
This figure illustrates the binding of Annexin V conjugated with FITC (fluorescein isothiocyanate) to phosphatidylserine (PS) molecules translocated to the outer leaflet of the plasma membrane during apoptosis. Normal cells retain PS on the inner leaflet of their membrane and remain unstained (research gate by nacalai.com).
In addition to DNA fragmentation and PS exposure, apoptosis is accompanied by several other biochemical changes that contribute to its orderly execution: caspase activation. Caspases are a family of proteases that serve as central regulators of apoptosis. Initiator caspases (like caspase-8 and caspase-9) activate effector caspases (such as caspase-3, −6, and − 7), which then cleave various substrates in the cell, leading to the characteristic morphological and biochemical changes of apoptosis. Caspase activation is tightly regulated, ensuring that apoptosis proceeds in a controlled manner [56]. In addition to this, mitochondrial outer membrane permeabilization (MOMP) is a pivotal event in intrinsic apoptosis. The release of cytochrome c and other pro-apoptotic factors from the mitochondria into the cytosol activates apoptotic signaling pathways, including the formation of the apoptosome, a multiprotein complex that activates caspase-9. This leads to the downstream activation of effector caspases, amplifying the apoptotic cascade. MOMP finally leads to the loss of mitochondrial membrane potential. The mitochondrial membrane potential (ΔΨm) is crucial for mitochondrial function, and its disruption is an early event in apoptosis. The loss of ΔΨm can be detected using specific dyes that respond to changes in mitochondrial potential, indicating the onset of apoptosis [57]. The biochemical hallmarks of apoptosis—specifically, DNA fragmentation and phosphatidylserine exposure—are fundamental to its role as a non-inflammatory form of programmed cell death. DNA fragmentation ensures the inactivation of genetic material and signals the cell’s progression through apoptosis, while PS exposure facilitates efficient phagocytic recognition and clearance. These markers enable the precise identification of apoptotic cells and underscore the controlled, immunologically silent nature of apoptosis. This level of regulation distinguishes apoptosis from other cell death forms and highlights its essential role in maintaining tissue homeostasis and preventing disease [58].
Apoptosis is mediated by a cascade of caspases, especially executioner caspases like caspase-3 and caspase-7, which dismantle cellular structures. The Bcl-2 protein family plays a key role, balancing pro-apoptotic and anti-apoptotic signals, while proteins like cytochrome c initiate the cascade from the mitochondria. Necrosis lacks specific molecular regulation. However, certain types, like necroptosis (a regulated necrosis form), involve proteins like RIPK1 and RIPK3 that lead to cell rupture [59]. The loss of ATP, build-up of calcium ions, and production of reactive oxygen species (ROS) are also characteristic of necrotic death. Autophagy relies on unique proteins, like LC3 (microtubule-associated protein 1A/1B-light chain 3) and Beclin-1, which form autophagosomes and recruit components for degradation. This pathway is regulated by nutrient-sensitive signaling cascades like mTOR and AMPK, which determine when cells need to enter survival mode [60]. The effect on surrounding tissue and inflammation apoptosis is non-inflammatory. The cell contents are enclosed in apoptotic bodies, which are then phagocytosed by immune cells, preventing any inflammatory response. This “clean” form of cell death minimizes harm to surrounding tissue. Necrosis, however, is highly inflammatory. The rupture of the cell membrane releases damage-associated molecular patterns (DAMPs) that activate the immune system, leading to an inflammatory response and often causing further tissue damage. This inflammatory environment can exacerbate diseases, such as during a heart attack or in chronic inflammatory conditions [61].
Apoptosis, or programmed cell death, is governed by two primary pathways: the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway. These pathways, though distinct in their initiation, are highly interconnected and converge to execute apoptosis, ensuring the elimination of damaged or unnecessary cells in a controlled and non-inflammatory manner.
8.1 The intrinsic pathway
The intrinsic pathway is primarily triggered by internal stress signals, such as DNA damage, oxidative stress, or metabolic dysfunction. These stressors lead to mitochondrial outer membrane permeabilization (MOMP), a crucial event mediated by the Bcl-2 protein family. Pro-apoptotic members of this family, such as Bax and Bak, oligomerize to form pores in the mitochondrial outer membrane, enabling the release of apoptotic factors, including cytochrome c, into the cytosol. Cytochrome c, upon release, binds to Apaf-1 and ATP, forming the apoptosome complex. This complex activates initiator caspase-9, which subsequently activates executioner caspases, such as caspase-3 and caspase-7 shown in Figure 7. These effector caspases dismantle the cell by cleaving structural proteins, enzymes, and other cellular components, resulting in chromatin condensation, DNA fragmentation, membrane blebbing, and cellular disassembly [62].
Figure 7.
Cell apoptosis determined by Annexin V/PI double-staining assay followed by flow cytometer analysis in our study. (a) Control cells without any treatment. (b) Induction of apoptosis in cells using capmptothecin.
Anti-apoptotic Bcl-2 family members, such as Bcl-2 and Bcl-xL, counteract this process by sequestering Bax and Bak, thereby preventing MOMP and cytochrome c release. This balance between pro- and anti-apoptotic proteins is critical for determining cell fate and is tightly regulated [58]. Dysregulation of this balance can lead to pathological conditions, such as cancer, where anti-apoptotic proteins are often overexpressed, allowing cells to evade death.
8.2 The extrinsic pathway
The extrinsic pathway is initiated by external signals through the binding of death ligands, such as FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), or TNF-α, to their respective death receptors on the cell surface. These receptors, part of the tumor necrosis factor (TNF) receptor superfamily, include Fas (CD95), DR4, DR5, and TNFR1 shown in Figure 8. Ligand binding induces receptor oligomerization and the recruitment of adaptor proteins like FADD (Fas-associated death domain), leading to the formation of the death-inducing signaling complex (DISC). Within the DISC, procaspase-8 is cleaved to its active form, caspase-8, which either directly activates downstream executioner caspases or cleaves Bid, linking the extrinsic and intrinsic pathways. Caspases, a family of cysteine proteases, are central to the execution of apoptosis [64]. They exist as inactive precursors (zymogens) and are activated in response to apoptotic signals. Initiator caspases, such as caspase-8 and caspase-9, serve as the initial response elements, activating effector caspases, such as caspase-3 and caspase-7. Effector caspases dismantle the cell by cleaving substrates like PARP (poly ADP-ribose polymerase) and nuclear lamins, which are crucial for maintaining nuclear structure and DNA integrity. This cascade ensures the orderly dismantling of the cell while preserving surrounding tissue integrity. The regulation of caspase activity is essential for preventing excessive or premature apoptosis. Inhibitor of apoptosis proteins (IAPs), such as XIAP (X-linked inhibitor of apoptosis protein), suppresses caspase activation to maintain cellular survival under non-stress conditions. Additionally, SMAC/DIABLO, a mitochondrial protein released during apoptosis, counteracts IAPs to promote caspase activation. This intricate balance of pro- and anti-apoptotic regulators ensures that apoptosis occurs only under appropriate conditions [65].
Figure 8.
Diagram of intrinsic and extrinsic pathways of apoptosis. (A) In the intrinsic pathway, the proapoptotic BH3-only family members activate Bax or Bak, leading to mitochrondrial outer membrane permeabilization, which drives formation of the apoptosome, activation of the executioner caspases, 3 and 7, and subsequent apoptosis. The proapoptotic BH3-only proteins are inhibited via interactions with the anti-apoptotic Bcl-2 family of proteins. (B) In the extrinsic pathway, ligands such as Fas, tumor necrosis factor (TNF), or tumor necrosis factor-related apoptosis-inducing (TRAIL) ligand bind to death receptors. This results in the recruitment of Fas-associated death domain protein (FADD) and activation of caspase 8. Caspase 8 directly activates caspase 3 and 7. The two pathways interact via caspase 8-mediated cleavage of Bid [63].
8.3 Crosstalk between intrinsic and extrinsic pathways
Although the intrinsic and extrinsic pathways are triggered by different stimuli, they are highly interconnected. Bid, a member of the Bcl-2 family, serves as a key mediator of this crosstalk. Bid and tBid caspase-8, activated in the extrinsic pathway, cleaves Bid into its active form, tBid. tBid translocates to the mitochondria, where it activates Bax and Bak, promoting MOMP and integrating the extrinsic and intrinsic pathways. Amplification of apoptosis: this cross-talk ensures that signals from external stimuli are amplified through mitochondrial involvement, leading to a robust and irreversible apoptotic response [66].
8.4 Mechanisms of MOMP and the apoptosome
MOMP represents the point of no return in apoptosis, marking the release of mitochondrial proteins that drive the caspase cascade. Formation of the apoptosome due to cytochrome c, released during MOMP, binds to Apaf-1 and dATP, forming the apoptosome. This complex recruits and activates procaspase-9, which in turn activates effector caspases. Effector caspases cleave numerous cellular components, including cytoskeletal proteins, nuclear Lamins, and enzymes like PARP, ultimately resulting in the orderly disassembly of the cell. The balance between pro-apoptotic and anti-apoptotic members of the Bcl-2 family determines whether a cell will undergo apoptosis. Pro-apoptotic proteins: Bax and Bak form oligomers that create pores in the mitochondrial membrane, releasing apoptotic factors. Bid enhances this process by bridging extrinsic and intrinsic pathways’ – apoptotic proteins: Bcl-2 and Bcl-xL prevent Bax and Bak activation, maintaining mitochondrial integrity and blocking the initiation of apoptosis [67].
Apoptosis is a highly regulated process involving the intrinsic and extrinsic pathways, both of which culminate in the activation of caspases that execute cell death. The intrinsic pathway is driven by mitochondrial changes, while the extrinsic pathway responds to external death signals. Crosstalk between these pathways ensures a robust apoptotic response. The Bcl-2 protein family serves as a crucial regulator, balancing pro-apoptotic and anti-apoptotic signals. This intricate regulation of apoptosis is critical for maintaining tissue homeostasis and has profound implications for understanding and treating diseases characterized by aberrant cell death, such as cancer, neurodegenerative disorders, and autoimmune conditions [68].
9. The role of apoptosis in pathogenesis regulation
Apoptosis is a vital process in the body that maintains cellular balance, prevents disease, and facilitates proper tissue development. Its precise control over cell death ensures that harmful, damaged, or excess cells are removed in a way that avoids inflammation or damage to surrounding cells. In health, apoptosis is essential for development, immune function, and tissue homeostasis. However, when dysregulated, apoptosis contributes significantly to numerous diseases, such as cancer, neurodegeneration, and autoimmune conditions. In this section, the importance of apoptosis in maintaining health, along with its pathological consequences when its regulatory mechanisms fail will be discussed. During development, apoptosis plays a pivotal role in sculpting tissues and organs by selectively removing unnecessary or redundant cells. One well-documented example is the elimination of cells in the webbing between the developing digits of embryos, which shapes fingers and toes. This process is tightly controlled and ensures that tissues achieve their proper form and function. Similarly, apoptosis is vital for neural development, where it removes surplus neurons to refine synaptic connections and optimize neural networks. This pruning ensures the brain’s structural and functional integrity, enabling efficient information processing and adaptability. Studies have shown that insufficient or excessive neuronal apoptosis during development can lead to neurodevelopmental disorders such as autism and schizophrenia, underscoring the critical balance required in this process [69]. Apoptosis also underpins immune system development and regulation. Autoreactive immune cells, which have the potential to attack the body’s own tissues, are eliminated during maturation in a process known as negative selection. This ensures self-tolerance and prevents autoimmune disorders. Following an immune response, apoptosis clears immune cells, such as neutrophils and lymphocytes, once they have fulfilled their function. This resolution of inflammation is critical to avoid prolonged immune activity, which could otherwise lead to tissue damage or chronic inflammation.
In adult tissues, apoptosis maintains tissue homeostasis by balancing cell turnover, particularly in high-turnover tissues such as the gut epithelium, skin, and blood. By removing aged, damaged, or dysfunctional cells, apoptosis prevents the accumulation of potentially harmful cells that could impair organ function or promote malignancy. For example, apoptotic pathways are activated in response to DNA damage or oxidative stress, preventing the survival of cells with mutations that could progress to cancer [70]. The importance of apoptosis in maintaining cellular integrity is evident in its role in protecting tissues from hyperplasia or hypertrophy, which could disrupt normal organ function.
When apoptotic pathways are dysregulated, they contribute significantly to the onset and progression of various diseases. In this section, we will specifically look into the intricate role of apoptosis in disease pathogenesis like cancer neurodegeneration diseases, autoimmune disease, and inflammatory diseases, emphasizing the molecular mechanisms and pathological consequences of its dysregulation [12].
9.1 Role of apoptosis in cancer pathology
In normal tissues, apoptosis serves as a protective mechanism to eliminate cells with genetic abnormalities or oncogenic mutations. By doing so, it acts as a barrier to tumor initiation and progression. For example, elimination of mutated cells apoptosis removes cells that acquire DNA damage or undergo oncogene activation, thereby preventing their clonal expansion. Apoptotic processes are integral to immune system function, facilitating the removal of potentially malignant cells through phagocytosis by macrophages and other immune cells. In tissues with high turnover rates, apoptosis ensures a balance between cell proliferation and cell death, preventing hyperplasia and subsequent tumorigenesis [71].
Evasion of Apoptosis in Cancer Development.
In cancer, cells acquire the ability to evade apoptosis, contributing to uncontrolled growth and survival. This evasion is a hallmark of cancer and occurs through various mechanisms that collectively enable tumor progression [72]. Key consequences include the following:
Sustained proliferation: The inability of cancer cells to undergo apoptosis allows them to continue dividing despite genetic aberrations and unfavorable microenvironmental conditions.
Increased resistance to cellular stress: Cancer cells often face hypoxia, oxidative stress, and metabolic challenges in the tumor microenvironment. Resistance to apoptosis enables them to survive and adapt under these conditions.
Immune evasion: By avoiding apoptosis, cancer cells may evade detection and destruction by the immune system. This is particularly significant in metastatic cancer, where immune evasion facilitates dissemination to distant sites [73].
9.2 Role in tumor heterogeneity and progression
Evasion of apoptosis contributes to the development of tumor heterogeneity—a critical factor in cancer progression and therapeutic resistance. Cancer cells that survive apoptotic triggers such as DNA damage or chemotherapy become the dominant population within the tumor. This selection pressure promotes the emergence of more aggressive and treatment-resistant clones. The failure of apoptosis to remove genetically unstable cells allows the accumulation of mutations, fostering a more heterogeneous tumor cell population with diverse phenotypic and genotypic trait. Apoptosis resistance is essential for cancer cells to survive during detachment, circulation, and colonization at secondary sites. Cells with enhanced survival capabilities are more likely to establish metastases. The tumor microenvironment (TME) plays a significant role in modulating apoptosis in cancer cells. Interactions between cancer cells and their surrounding stroma, immune cells, and extracellular matrix can either suppress or promote apoptotic pathways. The hypoxic regions of tumors promote resistance to apoptosis by stabilizing hypoxia-inducible factors (HIFs), which regulate survival pathways. Pro-survival signals from cytokines and growth factors in the TME, such as interleukin-6 (IL-6) or vascular endothelial growth factor (VEGF), can inhibit apoptotic processes in cancer cells [74]. Tumor-associated immune cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), can suppress apoptosis-inducing immune responses, further promoting tumor cell survival [75]. Resistance to apoptosis is a significant contributor to cancer therapy failure and disease relapse. As cancer cells evade programmed cell death, they become less responsive to traditional treatments such as chemotherapy and radiation, which rely on inducing apoptosis to eradicate tumors. Furthermore, this resistance facilitates the survival of minimal residual disease and the re-establishment of tumors after treatment. Apoptosis plays a pivotal role in the pathology of cancer, with its disruption contributing to tumor initiation, progression, and resistance. Understanding the intricate relationship between apoptosis and cancer pathology highlights the complexity of tumor biology and underscores the importance of addressing apoptotic dysregulation in cancer research and treatment strategies [76].
In cancer, one of the hallmarks of tumorigenesis is the ability of cells to evade apoptosis, enabling unchecked proliferation and survival under otherwise lethal conditions. This evasion is achieved through several mechanisms such as mutations in tumor suppressor genes, overexpression of anti-apoptotic proteins, and the downregulation of pro-apoptotic factors. The TP53 gene, encoding the tumor suppressor protein p53, plays a vital role in initiating apoptosis in response to cellular stress, particularly DNA damage. Mutations in TP53, observed in many cancers, disrupt this function, allowing damaged cells to evade apoptosis. Proteins like BCL2 inhibit mitochondrial outer membrane permeabilization (MOMP), blocking the release of cytochrome c and subsequent activation of caspases. This anti-apoptotic shift promotes cancer cell survival under stress. Proto-oncogenes like c-MYC exhibit paradoxical roles, promoting proliferation in nutrient-rich environments while triggering apoptosis under growth-limiting conditions [77]. Cancer cells circumvent this apoptotic pressure through additional mutations or signaling alterations. Alterations in Fas/FasL signaling, critical for extrinsic apoptosis, allow cancer cells to escape immune-mediated apoptosis, contributing to immune evasion [78].
Spontaneous apoptosis occurs in untreated tumors and is influenced by factors such as mild ischemia, immune cell infiltration, and intrinsic tumor properties. While insufficient to control tumor growth, this process reflects the dynamic interplay between proliferation and cell death in the tumor microenvironment [79]. Understanding these mechanisms provides insights into tumor biology and therapeutic opportunities.
9.3 Apoptosis and neurodegenerative diseases
While insufficient apoptosis underlies cancer progression, excessive apoptosis is a hallmark of neurodegenerative diseases. In conditions such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), chronic neuronal apoptosis contributes to the progressive loss of neural populations and associated functional decline. The accumulation of amyloid-beta plaques and hyperphosphorylated tau proteins in Alzheimer’s disease induces oxidative stress and mitochondrial dysfunction, activating the intrinsic apoptotic pathway. Caspase-3, a key executioner caspase, is upregulated in affected brain regions such as the hippocampus and cortex, leading to synaptic dysfunction and neuronal loss [80]. In Parkinson’s disease, misfolded alpha-synuclein proteins form toxic aggregates that disrupt mitochondrial function and initiate apoptosis in dopaminergic neurons of the substantia nigra, causing motor dysfunction. Similarly, the mutant huntingtin protein in Huntington’s disease disrupts mitochondrial dynamics and induces oxidative stress, activating apoptotic cascades that result in striatal neuron loss and progressive motor and cognitive impairments. Therapeutic strategies in neurodegeneration focus on inhibiting excessive apoptosis, with interventions targeting caspases, mitochondrial integrity, and oxidative stress showing promise in preclinical models [81].
Apoptosis, is critical for maintaining homeostasis and eliminating damaged or dysfunctional cells in the nervous system. In neurodegenerative diseases, dysregulated apoptosis contributes significantly to neuronal loss, which underpins the progression of these conditions.
In neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS), excessive apoptosis leads to the loss of neurons in specific brain regions. Different neuronal populations show varying susceptibility to apoptosis. For instance, dopaminergic neurons in the substantia nigra are predominantly affected in PD, while hippocampal and cortical neurons are targeted in AD. Apoptotic neuronal loss disrupts neural circuits essential for cognitive, motor, and sensory functions, directly causing disease-specific symptoms. Neurodegenerative diseases are marked by persistent inflammation that exacerbates apoptosis [82]. Overactive microglia release inflammatory cytokines like TNF-α and IL-1β, which induce apoptosis in neurons and glial cells, amplifying neurodegeneration. Astrocytes, crucial for neuroprotection, may lose their supportive role and contribute to apoptosis through oxidative stress and inflammatory signaling. Misfolded and aggregated proteins characteristic of neurodegenerative diseases activate apoptotic pathways. Amyloid-β plaques and tau tangles cause synaptic dysfunction and neuronal apoptosis, leading to progressive memory loss. Aggregates of α-synuclein disrupt mitochondrial integrity and promote apoptosis in dopaminergic neurons. Mutant huntingtin protein induces transcriptional dysregulation and mitochondrial stress, triggering apoptosis in striatal and cortical neurons [83].
Oxidative stress, a common feature in neurodegenerative diseases, directly contributes to apoptosis. Neurons rely heavily on mitochondrial energy. Mitochondrial dysfunction leads to the release of pro-apoptotic factors like cytochrome c, promoting cell death. Oxidative damage to cellular components, including DNA and lipids, activates apoptotic signaling, further exacerbating neuronal loss. The failure to efficiently remove apoptotic cells intensifies neurodegeneration. Inefficient clearance by microglia results in the release of cellular debris and inflammatory mediators, perpetuating neuro-inflammation [84]. The accumulation of apoptotic remnants disrupts the extracellular environment, impairing neighboring cell functions and contributing to disease progression. Apoptosis disrupts synaptic integrity, leading to network-level impairments. Apoptotic loss of neurons and glial cells weakens synaptic connections, reducing the brain’s ability to transmit and process information. Diminished ability to form new connections or repair damaged ones worsens cognitive and motor symptoms in neurodegenerative diseases. Apoptosis in the hippocampus and cortex leads to progressive memory impairment and cognitive decline. Parkinson’s disease: Loss of dopaminergic neurons in the substantia nigra due to apoptosis causes motor dysfunction, including tremors and rigidity. Apoptosis in the striatum disrupts movement coordination and contributes to psychiatric symptoms. Motor neuron apoptosis results in muscle weakness and eventual paralysis [16].
The role of apoptosis extends beyond the central nervous system in neurodegenerative diseases. Apoptosis in peripheral nerves may contribute to sensory and autonomic dysfunction. Endothelial cell apoptosis can compromise the blood-brain barrier, intensifying neuroinflammation and neuronal damage. Apoptosis is intricately linked to the pathology of neurodegenerative diseases, where its dysregulation contributes to neuronal loss, chronic inflammation, and systemic manifestations. Understanding how apoptosis shapes the course of these conditions provides critical insights into their progression and potential management strategies [85].
9.4 Apoptosis and autoimmune diseases
In autoimmune diseases, apoptosis plays a paradoxical role, contributing to both the prevention and exacerbation of immune-mediated damage. During immune development, apoptosis ensures the removal of self-reactive T and B cells in a process called negative selection. Defective apoptosis in this context allows autoreactive cells to persist, leading to autoimmune conditions such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Conversely, in affected tissues, excessive apoptosis can release intracellular antigens, perpetuating a self-amplifying cycle of immune activation and chronic inflammation. For instance, in RA, apoptotic debris can stimulate further immune responses, exacerbating joint destruction. Restoring apoptotic balance is critical in autoimmune diseases [86]. Approaches include promoting the apoptosis of autoreactive immune cells while minimizing excessive apoptotic activity in target tissues. In healthy individuals, apoptosis is critical for the establishment and maintenance of immune tolerance. It ensures the elimination of autoreactive lymphocytes during both central and peripheral immune tolerance processes. During the development of T and B cells in the thymus and bone marrow, apoptosis removes immature lymphocytes that strongly react to self-antigens. This prevents the release of self-reactive cells into the peripheral immune system. In the periphery, apoptosis eliminates autoreactive lymphocytes that escape central tolerance through mechanisms such as activation-induced cell death (AICD). This prevents inappropriate immune activation against self-antigens. In autoimmune diseases, the regulation of apoptosis is often impaired, leading to the survival of autoreactive immune cells or inappropriate death of essential immune-regulating cells. This dysregulation contributes to chronic inflammation and tissue damage. Key roles include the following: impaired apoptotic pathways allow autoreactive T and B cells to persist, leading to the production of autoantibodies and the activation of inflammatory cascades. For instance, in systemic lupus erythematosus (SLE), defective clearance of apoptotic cells results in the accumulation of cellular debris, which acts as a source of autoantigens. Regulatory T cells (Tregs) are crucial for suppressing autoimmune responses. Aberrant apoptosis leading to the depletion of Tregs can disrupt immune homeostasis and contribute to disease pathogenesis [87].
Apoptosis dysregulation in autoimmune diseases often results in chronic inflammation, which perpetuates tissue damage. This can occur as follows: when apoptotic cells are not efficiently cleared by phagocytes, they undergo secondary necrosis, releasing pro-inflammatory cytokines and autoantigens. This is a prominent feature in diseases such as rheumatoid arthritis (RA) and SLE. The persistence of apoptotic cell-derived debris can activate dendritic cells and other antigen-presenting cells, leading to the stimulation of autoreactive T and B cells and amplifying the autoimmune response. The impact of apoptosis in autoimmune diseases often varies depending on the affected tissue: In RA, resistance to apoptosis in synovial fibroblasts leads to their hyperplasia and contributes to joint destruction. Simultaneously, defective clearance of apoptotic cells in the synovium enhances local inflammation. Apoptosis of insulin-producing beta cells in the pancreas, triggered by autoreactive T cells, is a hallmark of type 1 diabetes. This targeted cell death results in the loss of insulin production and the development of hyperglycemia [88]. Abnormal apoptosis in keratinocytes and immune cells contributes to the characteristic skin lesions of psoriasis, with hyperproliferation of keratinocytes and persistent inflammation. Beyond localized tissue effects, apoptosis dysregulation in autoimmune diseases can have systemic consequences [88]. In diseases like SLE, the failure to clear apoptotic debris leads to the generation of autoantibodies against nuclear antigens, exacerbating systemic inflammation. Chronic inflammation driven by apoptosis defects can result in widespread organ damage, as seen in lupus nephritis or autoimmune hepatitis. The microenvironment in autoimmune diseases influences apoptotic processes. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) can modulate apoptotic pathways, either promoting or inhibiting cell death in specific contexts. Interactions between dendritic cells, macrophages, and lymphocytes can either exacerbate or mitigate apoptosis-related dysregulation, depending on the signaling milieu [89].
9.5 Apoptosis and chronic inflammatory diseases
Chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD) and Crohn’s disease, are characterized by impaired apoptotic clearance, leading to persistent inflammation and tissue damage. The failure of macrophages to effectively clear apoptotic cells prolong inflammatory responses and contributes to the accumulation of necrotic debris, exacerbating tissue injury. Chronic inflammation can drive fibrotic changes in tissues, as seen in COPD, where prolonged immune cell activity damages alveolar structures, impairing lung function. Therapies aimed at enhancing efferocytosis—the phagocytic clearance of apoptotic cells—represent a promising avenue for reducing inflammation and tissue remodeling in these conditions [90]. One of the hallmark features of COPD is emphysema, characterized by the destruction of alveolar walls and loss of elastic recoil in the lungs. Apoptosis significantly contributes to this process. Increased apoptosis of alveolar epithelial cells and endothelial cells leads to the breakdown of alveolar walls. This disrupts the architecture of the gas exchange surface and contributes to airflow limitation. In COPD, excessive apoptosis is not adequately counterbalanced by tissue repair mechanisms, resulting in progressive loss of lung parenchyma. COPD is characterized by and persistent inflammation, which interacts with apoptosis to exacerbate tissue damage. Apoptosis is critical for regulating the lifespan of inflammatory cells such as neutrophils, macrophages, and T lymphocytes. In COPD, impaired clearance of apoptotic immune cells (efferocytosis) leads to secondary necrosis and the release of pro-inflammatory mediators, perpetuating inflammation. Chronic exposure to inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) promotes apoptosis in structural lung cells, contributing to tissue damage [91].
Small airway remodeling, another key feature of COPD, involves structural changes in the bronchioles, leading to airflow obstruction. Apoptosis contributes to these changes in several ways. Increased apoptosis of airway epithelial cells weakens the protective barrier, making the airways more susceptible to damage and inflammation. Dysregulated apoptosis of fibroblasts and myofibroblasts can lead to abnormal extracellular matrix deposition and airway wall thickening, contributing to remodeling and obstruction.
COPD is strongly associated with oxidative stress due to chronic exposure to cigarette smoke and environmental pollutants. Oxidative stress amplifies apoptosis through various pathways, exacerbating tissue damage. Oxidative stress increases the susceptibility of alveolar epithelial and endothelial cells to apoptosis, accelerating lung tissue destruction. Oxidative stress-induced mitochondrial damage in lung cells promotes apoptotic cell death, contributing to both emphysema and airway remodeling [92].
Cellular senescence is a prominent feature in COPD, where senescent cells accumulate in the lungs due to impaired apoptosis. Senescent cells secrete pro-inflammatory mediators, including interleukins and matrix metalloproteinases, which contribute to chronic inflammation and tissue degradation. The persistence of senescent cells in the lung microenvironment hinders regenerative processes, exacerbating the structural and functional decline in COPD [93].
The interplay between apoptosis and the lung microenvironment significantly influences COPD pathology. Inefficient clearance of apoptotic cells by macrophages in COPD leads to secondary necrosis, further amplifying inflammation and tissue injury. Apoptosis of lung structural cells contributes to ECM degradation and loss of lung elasticity, hallmarks of COPD progression. Dysregulated apoptosis alters the behavior of immune cells in the lung microenvironment, perpetuating a cycle of inflammation and cell death. COPD is a systemic disease, and apoptosis dysregulation in the lungs can have widespread effects. Apoptosis in skeletal muscle cells contributes to muscle weakness and reduced exercise capacity in COPD patients. Apoptosis of endothelial cells in systemic circulation may contribute to the increased risk of cardiovascular disease in COPD. Apoptosis plays a multifaceted role in the pathology of COPD, influencing alveolar destruction, chronic inflammation, airway remodeling, and systemic manifestations. The dysregulation of apoptotic processes is central to disease progression, highlighting its critical role in shaping the structural and functional decline observed in COPD [94].
Pathogens often exploit apoptotic pathways to enhance their survival and replication within the host. Viruses such as HIV and Epstein-Barr virus inhibit host cell apoptosis by upregulating anti-apoptotic proteins or directly interfering with caspase activation, prolonging the survival of infected cells. Conversely, pathogens like influenza virus and certain bacterial toxins actively induce apoptosis to disseminate infection and evade immune responses. For example, Shiga toxin-producing bacteria trigger endothelial cell apoptosis, contributing to vascular damage. Understanding pathogen-induced modulation of apoptosis has led to novel therapeutic strategies aimed at restoring host cell apoptotic pathways or blocking pathogen-driven apoptotic triggers [95].
9.6 Apoptosis and cardiovascular diseases
Dysregulated apoptosis is implicated not only in neurodegenerative diseases but also in disorders affecting the cardiovascular and immune systems, where both excessive and insufficient apoptosis can exacerbate disease progression.
In myocardial infarction, ischemia caused by blocked coronary arteries compromises oxygen supply to the heart, leading to mitochondrial dysfunction and increased production of reactive oxygen species (ROS). These events activate the intrinsic apoptotic pathway, resulting in cardiomyocyte apoptosis. The loss of heart muscle cells contributes to tissue damage and scar formation, impairing the heart’s ability to contract and pump blood effectively. Therapeutic strategies to mitigate this damage focus on inhibiting apoptosis in cardiomyocytes through interventions targeting Bcl-2 family proteins, caspase activity, or oxidative stress.
In summary, dysregulation of apoptosis—whether through suppression or overactivation—is central to the pathogenesis of many diseases. While insufficient apoptosis drives cancer progression, excessive apoptosis underpins neurodegeneration and chronic inflammation. Therapeutic modulation of apoptotic pathways holds significant potential for addressing these pathological states, offering hope for improved outcomes in conditions ranging from malignancies to autoimmune and infectious diseases. As research continues, the ability to fine-tune apoptotic responses presents an opportunity to develop more targeted and effective treatments for a wide array of disorders, paving the way for advances in precision medicine and personalized therapeutic approaches.
Small molecules and gene therapy approaches targeting apoptosis pathways offer promising strategies for treating cancer and diseases involving apoptosis dysregulation. The Bcl-2 family of proteins plays a critical role in regulating the intrinsic apoptosis pathway by balancing pro-apoptotic and anti-apoptotic signals at the mitochondria. Targeting these proteins with small molecules, such as BH3 mimetics, has shown therapeutic potential. BH3 mimetics mimic the function of pro-apoptotic BH3-only proteins like Bim, Bid, Puma, and Bad, which neutralize anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1. This interaction activates pro-apoptotic proteins like Bax and Bak, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Venetoclax, a BH3 mimetic specifically targeting Bcl-2, has demonstrated efficacy in hematologic malignancies like chronic lymphocytic leukemia. Similarly, molecules like Navitoclax, which target multiple anti-apoptotic Bcl-2 family members, show promise in restoring apoptosis in cancer cells that evade death through overexpression of these proteins [96].
Caspases, central executioners of apoptosis, represent another therapeutic target. Activating caspases can promote apoptosis in cancer cells, while inhibiting caspases can prevent excessive apoptosis in conditions like neurodegenerative diseases. Caspase activators stimulate initiator caspases such as caspase-8 and caspase-9 or executioner caspases like caspase-3 and caspase-7, either directly or through upstream signaling pathways. For instance, Smac mimetics activate caspases by inhibiting inhibitor of apoptosis proteins (IAPs) that block caspase activity. Conversely, caspase inhibitors aim to protect neurons from apoptosis in diseases such as Alzheimer’s and Parkinson’s, though careful design is essential to avoid promoting cancer cell survival in other contexts [97].
Understanding the mechanisms of apoptosis provides insights into potential treatments for diseases where apoptosis is dysregulated. Cancer therapies aim to restore apoptotic pathways to kill cancer cells [98]. Drugs that inhibit anti-apoptotic proteins or activate pro-apoptotic signaling are designed to selectively induce apoptosis in tumor cells, minimizing harm to healthy cells. In neurodegenerative conditions, inhibitors of apoptotic pathways, like caspase inhibitors, are explored to slow neuronal loss and potentially preserve cognitive and motor function in diseases like Alzheimer’s and Parkinson’s and for autoimmune diseases therapies focus on reducing autoreactive immune cells by promoting their apoptosis, helping to alleviate autoimmune symptoms by preventing attacks on the body’s own tissues [99]. During chronic inflammatory conditions, regulating apoptosis in immune cells can resolve inflammation and could prevent tissue damage, offering therapeutic potential in conditions like chronic obstructive pulmonary disease (COPD) [100]. Gene therapy offers precise control over apoptotic pathways by delivering genes that either promote or inhibit apoptosis. In cancer therapy, reactivating or restoring mutant p53 function through gene delivery can re-enable apoptosis in tumor cells. Viral vectors, such as adenoviruses, have been engineered to deliver genes like TRAIL that activate the extrinsic apoptotic pathway, inducing tumor cell death. Gene therapy approaches can also introduce pro-apoptotic genes, such as Bax, Puma, or Bak, to promote apoptosis selectively in cancer cells. For example, adenovirus-mediated gene transfer of cytochrome c can activate caspases and enhance tumor cell apoptosis. In immunotherapy, genetically modified T cells expressing pro-apoptotic proteins represent another innovative approach to inducing apoptosis in cancer cells. These strategies collectively highlight the potential of targeting apoptosis pathways to treat a wide range of diseases [101].
11. Challenges and future directions in apoptosis-based therapies
Dysregulation of apoptosis contributes to a range of pathological conditions. In cancer, resistance to apoptosis enables tumor cells to evade immune surveillance and persist in the body. Conversely, excessive apoptosis in neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s, leads to neuronal loss, contributing to cognitive and motor impairments. In autoimmune diseases, inappropriate apoptosis regulation exacerbates immune responses, resulting in tissue damage [102].Targeting apoptotic pathways for therapeutic intervention has emerged as a promising approach for treating these disorders. Strategies include the use of BH3 mimetics, caspase activators, gene therapies, and small molecules that modulate apoptotic regulators like the Bcl-2 family and death receptors. BH3 mimetics, for instance, mimic pro-apoptotic BH3-only proteins to neutralize anti-apoptotic Bcl-2 family members, promoting mitochondrial outer membrane permeabilization (MOMP) and subsequent apoptosis in cancer cells. While progress has been made, significant challenges persist in apoptosis-based therapies. Ensuring selective targeting of diseased cells, such as cancer cells, while sparing healthy tissue remains a critical hurdle. Resistance to apoptosis-targeting therapies is common in cancer cells, which can upregulate anti-apoptotic proteins or acquire mutations in pro-apoptotic genes. Overcoming this resistance is essential for improving therapeutic efficacy. Additionally, many apoptosis-inducing therapies have off-target effects that can lead to toxicity in healthy tissues. The challenge in neurodegenerative diseases lies in preventing excessive apoptosis in neurons without inadvertently promoting cancer cell survival [103].
Advancements in combination therapies, where apoptosis-inducing drugs are paired with chemotherapy, radiotherapy, or immunotherapy, offer a promising solution to overcome resistance and improve treatment outcomes. Personalized medicine is another key area of development, enabling tailored interventions based on the genetic and molecular profiles of patients, particularly in cancers with mutations in apoptotic regulators like p53. Nanotechnology and novel drug delivery systems, including nanoparticles and liposomes, are also being explored to enhance the precision and bioavailability of apoptosis-targeting agents, minimizing off-target effects and improving therapeutic outcomes [104].
Future research in apoptosis-based therapies is poised to uncover novel apoptotic pathways, such as autophagic cell death and ferroptosis, which could offer new therapeutic targets. Furthermore, more advanced targeting of specific apoptotic molecules, like Mcl-1 or Bax, through protein-protein interactions, may lead to more selective and effective treatments. The integration of apoptosis modulation with other therapeutic approaches holds the potential for synergistic effects, particularly in treating cancers and chronic degenerative diseases. As advancements in genomic and proteomic technologies continue, personalized approaches to apoptosis-based therapies are expected to refine treatment strategies, enhancing their specificity and efficacy.
In conclusion, while significant challenges remain, apoptosis-based therapies hold immense potential for treating a wide array of diseases, from cancer to neurodegenerative and autoimmune disorders. Future developments in small molecules, gene therapy, combination treatments, and targeted delivery systems promise to overcome current limitations, offering new hope for patients. As our understanding of apoptosis deepens, these therapies are set to become a cornerstone of precision medicine, providing more effective and less toxic treatments for a range of conditions.
12. Detection and measurement of apoptosis
Identifying apoptotic cells and quantifying apoptosis are critical for understanding cell death processes in both physiological and pathological contexts. Various methods are used to detect apoptosis, each targeting specific biochemical or morphological hallmarks, such as DNA fragmentation, phosphatidylserine exposure, and nuclear changes. Below is an overview of some of the most widely used techniques in apoptosis detection: TUNEL assay, Annexin V staining, and DAPI staining.
12.1 TUNEL assay (terminal deoxynucleotidyl transferase dUTP Nick end labelling)
The TUNEL assay is a widely used technique for detecting DNA fragmentation, a key biochemical hallmark of apoptosis. The TUNEL assay detects DNA strand breaks by labelling the free 3’-OH ends of fragmented DNA. During apoptosis, CAD (caspase-activated DNase) cleaves DNA at internucleosomal regions, creating numerous 3’-OH termini. The enzyme terminal deoxynucleotidyl transferase (TdT) adds modified nucleotides, typically conjugated with a fluorophore or a chromogen, to these ends, allowing visualization of apoptotic cells under a microscope. Cells or tissue sections are fixed and incubated with TdT and labeled nucleotides. After labelling, apoptotic cells can be detected by fluorescence microscopy or flow cytometry. The presence of fluorescent or colored cells indicates DNA fragmentation typical of apoptosis. The TUNEL assay is sensitive and allows for the localization of apoptosis within tissue architecture, making it valuable for histological studies. However, it can also detect DNA damage unrelated to apoptosis, such as that caused by necrosis or autophagy, so results should be corroborated with additional assays [105].
12.2 Annexin V staining
Annexin V staining is one of the most specific and commonly used methods for identifying early apoptotic cells by detecting phosphatidylserine (PS) exposure on the cell membrane. During apoptosis, PS translocates from the inner to the outer leaflet of the plasma membrane, exposing it to the extracellular environment. Annexin V is a protein that binds specifically to PS in the presence of calcium, allowing for the identification of apoptotic cells. Annexin V is typically conjugated to a fluorescent dye (e.g., FITC or Alexa Fluor) for easy detection. Cells are incubated with labeled Annexin V and a viability dye, such as propidium iodide (PI), which is excluded by intact cell membranes. Cells that are Annexin V-positive and PI-negative are considered early apoptotic, while cells positive for both Annexin V and PI indicate late apoptosis or necrosis, as membrane integrity has been lost. Annexin V staining is highly specific for apoptosis and can be performed on live cells, allowing real-time analysis by flowcytometry or fluorescence microscopy shown in Figure 7. However, PS exposure is reversible in some contexts, and caution is needed to avoid false positives from cells undergoing reversible damage [106].
DAPI staining is commonly used to detect nuclear changes associated with apoptosis, such as chromatin condensation and nuclear fragmentation. DAPI is a fluorescent stain that binds specifically to A-T rich regions of double-stranded DNA, emitting blue fluorescence when bound. During apoptosis, nuclear chromatin condenses and becomes more densely packed, which intensifies DAPI staining. DAPI can also detect nuclear fragmentation, as fragmented apoptotic nuclei appear as multiple DAPI-stained bodies within a cell. Fixed cells or tissue sections are incubated with DAPI solution, followed by washing to remove excess stain. The stained cells are observed under a fluorescence microscope. Condensed or fragmented nuclei, indicative of apoptosis, appear brighter than normal nuclei. DAPI staining is quick, inexpensive, and effective for identifying apoptotic nuclei [58]. However, it is not specific to apoptosis; other types of cell death can also show chromatin changes, so DAPI is often used in combination with other assays (e.g., TUNEL or Annexin V staining) for accurate apoptosis detection. The accurate detection of apoptosis relies on recognizing its specific biochemical hallmarks. The TUNEL assay, Annexin V staining, and DAPI staining each provide unique insights into different aspects of apoptosis—DNA fragmentation, phosphatidylserine exposure, and nuclear morphology, respectively. When used in combination, these assays allow researchers to robustly and accurately identify apoptotic cells, distinguish apoptosis from other forms of cell death, and gain insights into cellular responses in both health and disease contexts [107].
13. Conclusion
Apoptosis presents transformative potential for therapeutic advancements. In cancer, therapies are being designed to restore apoptotic processes that tumors evade. Agents such as BH3 mimetics mimic the function of pro-apoptotic proteins, directly targeting cancer cells with overexpressed anti-apoptotic factors like Bcl-2. By reactivating apoptotic pathways, these therapies promote the selective destruction of malignant cells while sparing healthy ones. Another promising approach involves the use of death receptor agonists, which activate the extrinsic apoptotic pathway, inducing cell death in tumors. These treatments are particularly effective in overcoming resistance to conventional therapies, such as chemotherapy and radiation, which often fail due to the cancer cells’ ability to suppress apoptosis. In neurodegenerative diseases, apoptosis-modulating strategies focus on inhibiting excessive neuronal death. The gradual loss of neurons in conditions like Alzheimer’s or Parkinson’s is a direct result of oxidative stress, mitochondrial dysfunction, and protein aggregation, all of which activate apoptotic cascades. Therapeutic interventions aim to block key apoptotic players, such as caspases, or stabilize mitochondrial integrity to preserve neuronal populations. Although these approaches are still in experimental stages, they hold the potential to slow disease progression and maintain cognitive and motor functions for longer periods. Autoimmune disorders also benefit from apoptosis-centered therapies. Dysregulated apoptosis in immune cells leads to chronic inflammation and tissue destruction, as seen in diseases like systemic lupus erythematosus. Therapies are being developed to restore normal apoptotic processes, ensuring the elimination of autoreactive immune cells while preventing the accumulation of apoptotic debris that exacerbates autoimmune responses. Similar principles are applied in chronic inflammatory diseases, where regulating apoptosis in immune cells can resolve inflammation and prevent further tissue damage.
Despite these advances, challenges persist. Achieving therapeutic specificity is a significant hurdle. Therapies must selectively target diseased cells without inducing unwanted apoptosis in healthy tissues, which could lead to side effects such as immunosuppression or damage to normal tissues. Resistance to apoptosis-based therapies, particularly in cancer, is another obstacle. Tumors often adapt by mutating key apoptotic regulators or upregulating survival pathways, necessitating combination therapies that simultaneously target multiple apoptotic and survival mechanisms.
Future directions in apoptosis research promise to address these challenges. Advances in genomic and proteomic technologies pave the way for personalized medicine, allowing treatments to be tailored to the specific molecular profile of a patient’s disease. For example, understanding mutations in apoptotic regulators like p53 or Bcl-2 can guide the use of targeted therapies in cancers with such alterations. Nanotechnology offers solutions for precise drug delivery, using nanoparticles to transport apoptosis-inducing agents directly to diseased tissues, minimizing off-target effects.
Moreover, the integration of apoptosis modulation with other therapeutic modalities, such as immunotherapy, is an exciting frontier. By combining apoptosis-based treatments with approaches that enhance the immune system’s ability to recognize and attack diseased cells, it may be possible to achieve synergistic effects that improve therapeutic outcomes. In summary, apoptosis is more than a mechanism of cell death; it is a fundamental process that defines health and disease. Understanding its pathways and players has revolutionized our approach to treating diseases characterized by its dysregulation. From targeting cancer cells that evade death to protecting neurons from excessive apoptosis, therapeutic strategies are evolving to harness the power of apoptosis. As research continues to unravel its complexities, the potential for breakthroughs in treating some of the most challenging diseases of our time becomes ever more tangible. The future of apoptosis-based therapies lies in their precision, adaptability, and integration with cutting-edge medical advancements, offering renewed hope for improved patient outcomes and quality of life.
Acknowledgments
The author acknowledges the use of openai.com for language polishing of the manuscript.
References
1.Duque-Parra JE. Note on the origin and history of the term? Apoptosis? The Anatomical Record Part B: The New Anatomist. 2005;283B(1):2-4. DOI: 10.1002/ar.b.20047
2.Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: Mechanistic description of dead and dying eukaryotic cells. Infection and Immunity. 2005;73(4):1907-1916. DOI: 10.1128/IAI.73.4.1907-1916.2005
3.Yaoita Y. Tail resorption during metamorphosis in Xenopus tadpoles. Frontiers in Endocrinology (Lausanne). 2019;10. DOI: 10.3389/fendo.2019.00143
4.Ola MS, Nawaz M, Ahsan H. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Molecular and Cellular Biochemistry. 2011;351(1-2):41-58. DOI: 10.1007/s11010-010-0709-x
5.Elmore S. Apoptosis: A review of programmed cell death. Toxicologic Pathology. 2007;35(4):495-516. DOI: 10.1080/01926230701320337
6.Park W, Wei S, Kim B-S, Kim B, Bae S-J, Chae YC, et al. Diversity and complexity of cell death: A historical review. Experimental & Molecular Medicine. 2023;55(8):1573-1594. DOI: 10.1038/s12276-023-01078-x
7.Núñez R, Sancho-Martínez SM, Novoa JML, López-Hernández FJ. Apoptotic volume decrease as a geometric determinant for cell dismantling into apoptotic bodies. Cell Death and Differentiation. 2010;17(11):1665-1671. DOI: 10.1038/cdd.2010.96
8.Jan R, Chaudhry G-S. Understanding apoptosis and apoptotic pathways targeted cancer therapeutics. Advanced Pharmaceutical Bulletin. 2019;9(2):205-218. DOI: 10.15171/apb.2019.024
9.Van Opdenbosch N, Lamkanfi M. Caspases in cell death, inflammation, and disease. Immunity. 2019;50(6):1352-1364. DOI: 10.1016/j.immuni.2019.05.020
10.Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: The balancing act of BCL-2 family proteins. Nature Reviews. Molecular Cell Biology. 2019;20(3):175-193. DOI: 10.1038/s41580-018-0089-8
11.Kalimuthu S, Se-Kwon K. Cell survival and apoptosis signaling as therapeutic target for cancer: Marine bioactive compounds. International Journal of Molecular Sciences. 2013;14(2):2334-2354. DOI: 10.3390/ijms14022334
12.Mustafa M, Ahmad R, Tantry IQ, Ahmad W, Siddiqui S, Alam M, et al. Apoptosis: A comprehensive overview of signaling pathways, morphological changes, and physiological significance and therapeutic implications. Cells. 2024;13(22):1838. DOI: 10.3390/cells13221838
13.Coletti DJ et al. Abstracts from the 2017 Society of General Internal Medicine Annual Meeting. Journal of General Internal Medicine. 2017;32(S2):83-808. DOI: 10.1007/s11606-017-4028-8
14.Suzanne M, Steller H. Shaping organisms with apoptosis. Cell Death and Differentiation. 2013;20(5):669-675. DOI: 10.1038/cdd.2013.11
15.Zhu Y, Crowley SC, Latimer AJ, Lewis GM, Nash R, Kucenas S. Migratory neural crest cells phagocytose dead cells in the developing nervous system. Cell. 2019;179(1):74-89.e10. DOI: 10.1016/j.cell.2019.08.001
16.Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC. Neuronal cell death. Physiological Reviews. 2018;98(2):813-880. DOI: 10.1152/physrev.00011.2017
17.Zhang N, Hartig H, Dzhagalov I, Draper D, He YW. The role of apoptosis in the development and function of T lymphocytes. Cell Research. 2005;15(10):749-769. DOI: 10.1038/sj.cr.7290345
18.Portt L, Norman G, Clapp C, Greenwood M, Greenwood MT. Anti-apoptosis and cell survival: A review. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research. 2011;1813(1):238-259. DOI: 10.1016/j.bbamcr.2010.10.010
19.Franco R, Cidlowski JA. Apoptosis and glutathione: Beyond an antioxidant. Cell Death and Differentiation. 2009;16(10):1303-1314. DOI: 10.1038/cdd.2009.107
20.Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D’Orazi G. Apoptosis as anticancer mechanism: Function and dysfunction of its modulators and targeted therapeutic strategies. Aging. 2016;8(4):603-619. DOI: 10.18632/aging.100934
21.Zhang S, Xiao X, Yi Y, Wang X, Zhu L, Shen Y, et al. Tumor initiation and early tumorigenesis: Molecular mechanisms and interventional targets. Signal Transduction and Targeted Therapy. 2024;9(1):149. DOI: 10.1038/s41392-024-01848-7
22.Tong X, Tang R, Xiao M, Xu J, Wang W, Zhang B, et al. Targeting cell death pathways for cancer therapy: Recent developments in necroptosis, pyroptosis, ferroptosis, and cuproptosis research. Journal of Hematology & Oncology. 2022;15(1):174. DOI: 10.1186/s13045-022-01392-3
23.Labbé K, Saleh M. Cell death in the host response to infection. Cell Death and Differentiation. 2008;15(9):1339-1349. DOI: 10.1038/cdd.2008.91
24.Belizário JE, Neyra JM, Setúbal Destro Rodrigues MF. When and how NK cell-induced programmed cell death benefits immunological protection against intracellular pathogen infection. Innate Immunity. 2018;24(8):452-465. DOI: 10.1177/1753425918800200
25.Buckner JH, Ziegler SF. Regulating the immune system: The induction of regulatory T cells in the periphery. Arthritis Research & Therapy. 2004;6(5):215. DOI: 10.1186/ar1226
26.Mahoney JA, Rosen A. Apoptosis and autoimmunity. Current Opinion in Immunology. 2005;17(6):583-588. DOI: 10.1016/j.coi.2005.09.018
27.Thapa P, Farber DL. The role of the thymus in the immune response. Thoracic Surgery Clinics. 2019;29(2):123-131. DOI: 10.1016/j.thorsurg.2018.12.001
28.Radi E, Formichi P, Battisti C, Federico A. Apoptosis and oxidative stress in neurodegenerative diseases. Journal of Alzheimer’s Disease. 2014;42(s3):S125-S152. DOI: 10.3233/JAD-132738
29.Gorman AM. Neuronal cell death in neurodegenerative diseases: Recurring themes around protein handling. Journal of Cellular and Molecular Medicine. 2008;12(6a):2263-2280. DOI: 10.1111/j.1582-4934.2008.00402.x
30.Sharma AR, Chatterjee S, Lee YH, Lee SS. Targeting crosstalk of signaling pathways among muscles-bone-adipose tissue: A promising therapeutic approach for sarcopenia. Aging and Disease. 1 Aug 2024;15(4):1619-1645. DOI: 10.14336/AD.2023.00903. PMID: 37815907; PMCID: PMC11272187
31.Yang Y, Jiang G, Zhang P, Fan J. Programmed cell death and its role in inflammation. Military Medical Research. 2015;2(1):12. DOI: 10.1186/s40779-015-0039-0
32.Wong RS. Apoptosis in cancer: From pathogenesis to treatment. Journal of Experimental & Clinical Cancer Research. 2011;30(1):87. DOI: 10.1186/1756-9966-30-87
34.Iba T, Helms J, Maier CL, Ferrer R, Levy JH. Autophagy and Autophagic cell death in sepsis: Friend or foe? Journal of Intensive Care. 2024;12(1):41. DOI: 10.1186/s40560-024-00754-y
35.Fan Y-J, Zong W-X. The cellular decision between apoptosis and autophagy. Chinese Journal of Cancer. 2012. DOI: 10.5732/cjc.012.10106
36.Yu P, Zhang X, Liu N, Tang L, Peng C, Chen X. Pyroptosis: Mechanisms and diseases. Signal Transduction and Targeted Therapy. 2021;6(1):128. DOI: 10.1038/s41392-021-00507-5
37.Lifshitz A, Butron L, Ariza A. Immunoblastic lymphadenopathy and toxacariasis. Report of a case. La Prensa Médica Mexicana. 1976;41(9-10):323-327
38.Häcker G. The morphology of apoptosis. Cell and Tissue Research. 2000;301(1):5-17. DOI: 10.1007/s004410000193
39.Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wideranging implications in tissue kinetics. British Journal of Cancer. 1972;26(4):239-257. DOI: 10.1038/bjc.1972.33
41.Velasquez EV, Ríos M, Ortiz ME, Lizama C, Nuñez E, Abramovich D, et al. Concanavalin-a induces granulosa cell death and inhibits FSH-mediated follicular growth and ovarian maturation in female rats. Endocrinology. 2013;154(5):1885-1896. DOI: 10.1210/en.2012-1945
42.Lauber K, Blumenthal SG, Waibel M, Wesselborg S. Clearance of apoptotic cells. Molecular Cell. 2004;14(3):277-287. DOI: 10.1016/S1097-2765(04)00237-0
43.Jia W, Czabanka M, Broggini T. Cell blebbing novel therapeutic possibilities to counter metastasis. Clinical & Experimental Metastasis. 2024. DOI: 10.1007/s10585-024-10308-z
44.Wickman GR, Julian L, Mardilovich K, Schumacher S, Munro J, Rath N, et al. Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs. Cell Death and Differentiation. 2013;20(10):1293-1305. DOI: 10.1038/cdd.2013.69
45.Rahman Md A, Sahabjada S, Akhtar J. Evaluation of anticancer activity of Cordia dichotoma leaves against a human prostate carcinoma cell line, PC3. Journal of Traditional and Complementary Medicine. 2017;7(3):315-321. DOI: 10.1016/j.jtcme.2016.11.002
46.Yan B, Wang H, Peng Y, Hu Y, Wang H, Zhang X, et al. A unique role of the DNA fragmentation factor in maintaining genomic stability. Proceedings of the National Academy of Sciences. 2006;103(5):1504-1509. DOI: 10.1073/pnas.0507779103
47.Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto Y. Acinus is a Caspase-3-activated protein required for apoptotic chromatin condensation. Nature. 1999;401(6749):168-173. DOI: 10.1038/43678
48.Toné S, Sugimoto K, Tanda K, Suda T, Uehira K, Kanouchi H, et al. Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis. Experimental Cell Research. 2007;313(16):3635-3644. DOI: 10.1016/j.yexcr.2007.06.018
49.Battistelli M, Falcieri E. Apoptotic bodies: Particular extracellular vesicles involved in intercellular communication. Biology (Basel). 2020;9(1):21. DOI: 10.3390/biology9010021
50.Saraste A. Morphologic and biochemical hallmarks of apoptosis. Cardiovascular Research. 2000;45(3):528-537. DOI: 10.1016/S0008-6363(99)00384-3
51.Rafiq RA, Quadri A, Nazir LA, Peerzada K, Ganai BA, Tasduq SA. A potent inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, quercetin (3, 3′, 4′, 5, 7-Pentahydroxyflavone) promotes cell death in ultraviolet (UV)-B-irradiated B16F10 melanoma cells. PLoS One. 2015;10(7):e0131253. DOI: 10.1371/journal.pone.0131253
52.Kitazumi I, Tsukahara M. Regulation of DNA fragmentation: The role of caspases and phosphorylation. The FEBS Journal. 2011;278(3):427-441. DOI: 10.1111/j.1742-4658.2010.07975.x
53.Ungerer V, Bronkhorst AJ, Uhlig C, Holdenrieder S. Cell-free DNA fragmentation patterns in a cancer cell line. Diagnostics. 2022;12(8):1896. DOI: 10.3390/diagnostics12081896
54.Shlomovitz I, Speir M, Gerlic M. Flipping the dogma – Phosphatidylserine in non-apoptotic cell death. Cell Communication and Signaling. 2019;17:139. DOI: 10.1186/s12964-019-0437-0
55.Crowley LC, Marfell BJ, Scott AP, Waterhouse NJ. Quantitation of apoptosis and necrosis by Annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols. 2016;2016(11). DOI: 10.1101/pdb.prot087288
56.Parrish AB, Freel CD, Kornbluth S. Cellular mechanisms controlling caspase activation and function. Cold Spring Harbor Perspectives in Biology. 2013;5(6):a008672-a008672. DOI: 10.1101/cshperspect.a008672
57.Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G. Mechanisms of cytochrome c release from mitochondria. Cell Death and Differentiation. 2006;13(9):1423-1433. DOI: 10.1038/sj.cdd.4401950
58.Kari S, Subramanian K, Altomonte IA, Murugesan A, Yli-Harja O, Kandhavelu M. Programmed cell death detection methods: A systematic review and a categorical comparison. Apoptosis. Aug 2022;27(7-8):482-508. DOI: 10.1007/s10495-022-01735-y. Epub 2022 Jun 17. PMID: 35713779; PMCID: PMC9308588
59.Hussar P. Apoptosis regulators Bcl-2 and Caspase-3. Encyclopedia. 2022;2(4):1624-1636. DOI: 10.3390/encyclopedia2040111
60.Heckmann BL, Boada-Romero E, Cunha LD, Magne J, Green DR. LC3-associated phagocytosis and inflammation. Journal of Molecular Biology. 2017;429(23):3561-3576. DOI: 10.1016/j.jmb.2017.08.012
61.Downey J, Pernet E, Coulombe F, Divangahi M. Dissecting host cell death programs in the pathogenesis of influenza. Microbes and Infection. 2018;20(9-10):560-569. DOI: 10.1016/j.micinf.2018.03.005
62.Kalkavan H, Green DR. MOMP, cell suicide as a BCL-2 family business. Cell Death and Differentiation. 2018;25(1):46-55. DOI: 10.1038/cdd.2017.179
63.Ho J. The regulation of apoptosis in kidney development: Implications for nephron number and pattern? Frontiers in Pediatrics. 2014;2. DOI: 10.3389/fped.2014.00128
64.Diaz Arguello OA, Haisma HJ. Apoptosis-inducing TNF superfamily ligands for cancer therapy. Cancers (Basel). 2021;13(7):1543. DOI: 10.3390/cancers13071543
65.Guerrache A, Micheau O. TNF-related apoptosis-inducing ligand: Non-apoptotic signalling. Cells. 2024;13(6):521. DOI: 10.3390/cells13060521
66.Roy S, Nicholson DW. Cross-talk in cell death Signaling. The Journal of Experimental Medicine. 2000;192(8):F21-F25
67.Tait SWG, Green DR. Mitochondrial regulation of cell death. Cold Spring Harbor Perspectives in Biology. 2013;5(9):a008706. DOI: 10.1101/cshperspect.a008706
68.Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. Cell Research. May 2019;29(5):347-364. DOI: 10.1038/s41422-019-0164-5. Epub 2019 Apr 4. PMID: 30948788; PMCID: PMC6796845
69.Dekkers MPJ, Nikoletopoulou V, Barde Y-A. Death of developing neurons: New insights and implications for connectivity. Journal of Cell Biology. 2013;203(3):385-393. DOI: 10.1083/jcb.201306136
70.Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000;21(3):485-495. DOI: 10.1093/carcin/21.3.485
71.Ravichandran KS. Find-me and eat-me signals in apoptotic cell clearance: Progress and conundrums. Journal of Experimental Medicine. 2010;207(9):1807-1817. DOI: 10.1084/jem.20101157
72.Dandoti S. Mechanisms adopted by cancer cells to escape apoptosis–A review. Biocell. 2021;45(4):863-884. DOI: 10.32604/biocell.2021.013993
73.Feitelson MA, Arzumanyan A, Kulathinal RJ, Blain SW, Holcombe RF, Mahajna J, et al. Sustained proliferation in cancer: Mechanisms and novel therapeutic targets. Seminars in Cancer Biology. 2015;35:S25-S54. DOI: 10.1016/j.semcancer.2015.02.006
74.Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;144(5):646-674. DOI: 10.1016/j.cell.2011.02.013
75.Lindau D, Gielen P, Kroesen M, Wesseling P, Adema GJ. The immunosuppressive tumour network: Myeloid-derived suppressor cells, regulatory T cells and natural killer T cells. Immunology. 2013;138(2):105-115. DOI: 10.1111/imm.12036
76.Mohammad RM, Muqbil I, Lowe L, Yedjou C, Hsu H-Y, Lin L-T, et al. Broad targeting of resistance to apoptosis in cancer. Seminars in Cancer Biology. 2015;35:S78-S103. DOI: 10.1016/j.semcancer.2015.03.001
77.Hernández Borrero LJ, El-Deiry WS. Tumor suppressor P53: Biology, signaling pathways, and therapeutic targeting. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 2021;1876(1):188556. DOI: 10.1016/j.bbcan.2021.188556
78.Nagao M, Nakajima Y, Hisanaga M, Kayagaki N, Kanehiro H, Aomatsu Y, et al. The alteration of Fas receptor and ligand system in hepatocellular carcinomas: How do Hepatoma cells escape from the host immune surveillance in vivo? Hepatology. 1999;30(2):413-421. DOI: 10.1002/hep.510300237
79.Peng F, Liao M, Qin R, Zhu S, Peng C, Fu L, et al. Regulated cell death (RCD) in cancer: Key pathways and targeted therapies. Signal Transduction and Targeted Therapy. 2022;7(1):286. DOI: 10.1038/s41392-022-01110-y
80.Yuan J, Lipinski M, Degterev A. Diversity in the mechanisms of neuronal cell death. Neuron. 2003;40(2):401-413. DOI: 10.1016/S0896-6273(03)00601-9
81.Stefanis L. Synuclein in Parkinson’s disease. Cold Spring Harbor Perspectives in Medicine. 2012;2(2):a009399. DOI: 10.1101/cshperspect.a009399
82.Ruffini N, Klingenberg S, Schweiger S, Gerber S. Common factors in neurodegeneration: A meta-study revealing shared patterns on a multi-omics scale. Cells. 2020;9(12):2642. DOI: 10.3390/cells9122642
84.Cenini G, Lloret A, Cascella R. Oxidative stress in neurodegenerative diseases: From a mitochondrial point of view. Oxidative Medicine and Cellular Longevity. 2019;2019:1-18. DOI: 10.1155/2019/2105607
85.Yuan J, Yankner BA. Apoptosis in the nervous system. Nature. 2000;407(6805):802-809. DOI: 10.1038/35037739
86.Eguchi K. Apoptosis in autoimmune diseases. Internal Medicine. 2001;40(4):275-284. DOI: 10.2169/internalmedicine.40.275
87.Campisi L, Cummings RJ, Blander JM. Death-defining immune responses after apoptosis. American Journal of Transplantation. 2014;14(7):1488-1498. DOI: 10.1111/ajt.12736
88.Lleo A, Selmi C, Invernizzi P, Podda M, Gershwin ME. The consequences of apoptosis in autoimmunity. Journal of Autoimmunity. 2008;31(3):257-262. DOI: 10.1016/j.jaut.2008.04.009
89.Tsai F, Perlman H, Cuda CM. The contribution of the programmed cell death machinery in innate immune cells to lupus nephritis. Clinical Immunology. 2017;185:74-85. DOI: 10.1016/j.clim.2016.10.007
90.Szondy Z, Garabuczi Ã, Joós G, Tsay GJ, Sarang Z. Impaired clearance of apoptotic cells in chronic inflammatory diseases: Therapeutic implications. Frontiers in Immunology. 2014;5. DOI: 10.3389/fimmu.2014.00354
91.Demedts IK, Demoor T, Bracke KR, Joos GF, Brusselle GG. Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema. Respiratory Research. 2006;7(1):53. DOI: 10.1186/1465-9921-7-53
92.Barnes PJ, Shapiro SD, Pauwels RA. Chronic obstructive pulmonary disease: Molecular and cellular mechanisms. European Respiratory Journal. 2003;22(4):672-688. DOI: 10.1183/09031936.03.00040703
93.Hansel C, Jendrossek V, Klein D. Cellular senescence in the lung: The central role of senescent epithelial cells. International Journal of Molecular Sciences. 2020;21(9):3279. DOI: 10.3390/ijms21093279
94.Schmidt EP, Tuder RM. Role of apoptosis in amplifying inflammatory responses in lung diseases. Journal of Cell Death. 2010;3. DOI: 10.4137/JCD.S5375
95.Wyżewski Z, Mielcarska MB, Gregorczyk-Zboroch KP, Myszka A. Virus-mediated inhibition of apoptosis in the context of EBV-associated diseases: Molecular mechanisms and therapeutic perspectives. International Journal of Molecular Sciences. 2022;23(13):7265. DOI: 10.3390/ijms23137265
96.Fischer U, Schulze-Osthoff K. Apoptosis-based therapies and drug targets. Cell Death and Differentiation. 2005;12(S1):942-961. DOI: 10.1038/sj.cdd.4401556
97.Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1867;2020(6):118688. DOI: 10.1016/j.bbamcr.2020.118688
98.Tian X, Srinivasan PR, Tajiknia V, Sanchez Sevilla Uruchurtu AF, Seyhan AA, Carneiro BA, et al. Targeting apoptotic pathways for cancer therapy. Journal of Clinical Investigation. 2024;134(14). DOI: 10.1172/JCI179570
99.Moujalled D, Strasser A, Liddell JR. Molecular mechanisms of cell death in neurological diseases. Cell Death and Differentiation. 2021;28(7):2029-2044. DOI: 10.1038/s41418-021-00814-y
100.Yao H, Rahman I. Current concepts on the role of inflammation in COPD and lung cancer. Current Opinion in Pharmacology. 2009;9(4):375-383. DOI: 10.1016/j.coph.2009.06.009
101.Das SK, Menezes ME, Bhatia S, Wang X, Emdad L, Sarkar D, et al. Gene therapies for cancer: Strategies, challenges and successes. Journal of Cellular Physiology. 2015;230(2):259-271. DOI: 10.1002/jcp.24791
102.Neophytou CM, Trougakos IP, Erin N, Papageorgis P. Apoptosis deregulation and the development of cancer multi-drug resistance. Cancers (Basel). 2021;13(17):4363. DOI: 10.3390/cancers13174363
103.Baig S, Seevasant I, Mohamad J, Mukheem A, Huri HZ, Kamarul T. Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand? Cell Death & Disease. 2016;7(1):e2058. DOI: 10.1038/cddis.2015.275
104.Krzyszczyk P, Acevedo A, Davidoff EJ, Timmins LM, Marrero-Berrios I, Patel M, et al. The growing role of precision and personalized medicine for cancer treatment. Technology (Singapore : World Scientific Publishing Co. Pte. Ltd.). 2018;6(3-4):79-100. DOI: 10.1142/S2339547818300020
105.Mirzayans R, Murray D. Do TUNEL and other apoptosis assays detect cell death in preclinical studies? International Journal of Molecular Sciences. 2020;21(23):9090. DOI: 10.3390/ijms21239090
106.Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C. A novel assay for apoptosis flow Cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunological Methods. 1995;184(1):39-51. DOI: 10.1016/0022-1759(95)00072-I
107.Bugger H, Pfeil K. Mitochondrial ROS in myocardial ischemia reperfusion and Remodeling. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2020;1866(7):165768. DOI: 10.1016/j.bbadis.2020.165768
Written By
Irshad Ahmad Bhat, Aalim Maqsood Bhat and Sheikh Tasduq Abdullah
Submitted: 05 December 2024Reviewed: 30 December 2024Published: 02 April 2025
Open access peer-reviewed chapter - ONLINE FIRST
