Apoptosis, or programmed cell death, is a sophisticated process that occurs in multicellular organisms, and it involves an intricate series of biochemical events (Kerr JFR et al, 1972). One million cells in the body die every second as a result of this genetically tightly regulated mechanism. During apoptosis, specific cell death signals are transduced by different biomolecules such as caspases. These initiate a cascade that leads to the orchestrated collapse of a cell. Apoptosis is essential for the survival of the body and plays a critical role in various developmental and immunological processes. Defects in programmed cell death can lead to several diseases, from atrophy to cancer to autoimmune syndromes.
- Necrosis vs. apoptosis
Whereas apoptosis is highly regulated, necrosis happens accidentally following environmental changes. Apoptosis is an active process where cell shrinking and “budding” is followed by phagocytosis of the apoptotic bodies without inflammatory reactions. In contrast, in necrotic processes, the passive death of a cell leads to an uncontrolled release of inflammatory cellular contents. (Fink S and Cookson BT, 2005).
- Apoptosis vs. autophagy
Autophagy describes the programmed degradation of dysfunctional and unnecessary cellular components. During this process, double-membrane enclosed vesicles, called autophagosomes, envelop and then consume cellular components. Autophagy is a catabolic mechanism, which is essential during development and for promoting survival during starvation as it releases energy. Autophagy and apoptosis are distinct cellular processes, but they can be connected, as autophagy is sometimes the first step in the chain leading to apoptosis (Booth LA et al, 2014).
- Apoptosis vs. anoikis
Contact with the extracellular matrix (ECM) is crucial for the proliferation and survival of cells. When cells detach from the surrounding ECM, a form of apoptosis called anoikis is induced. Cancer cells develop mechanisms to overcome anoikis, so they can successfully spread out and metastasize to various tissues and organs (Malagobadan S et Nagoor NH, 2015).
The Process of Apoptosis
- Apoptosis pathway: activation mechanisms
Apoptosis can be triggered by either intracellular or extracellular signals. On the one hand, internal signals, such as biochemical stress or DNA damage, can initiate the mitochondria-dependent intrinsic pathway. On the other hand, the binding of ligands – often produced by T cells in response to infections, to “death receptors” on the cell surface – initiates the extrinsic pathway. Both mechanisms lead to the activation of the caspase cascade, the subsequent cleavage of target proteins, and finally, the death of the cell (Zaman S et al, 2014).
- Apoptosis pathway: caspase cascade and execution phase
As soon as the proteolytic caspases are activated, the execution phase starts. Apoptotic caspases (cysteine-dependent aspartate-specific proteases) can be differentiated in initiator caspases (caspase-2, 8,9, and 10), and effector caspases (caspase-3,6, and 7). Initiator caspases activate effector caspases through proteolytic cleavage. The latter then proteolytically degrade intracellular proteins (Pop C, 2009). During this phase, cells undergo characteristic morphological changes such as membrane blebbing, cell shrinkage and rounding, condensation of cytoplasma, organelles and chromatin (pyknosis), and nuclear fragmentation (karyrrhexis) until they break apart and are quickly removed by phagocytes (Renehan AG et al, 2001).
Since no single parameter fully defines cell death, different methods are used to detect apoptotic cells, or to differentiate between necrosis and apoptosis. Different events occurring in different areas of the cell – such as the plasma membrane, the cytoplasm, the mitochondria, and the nucleus – can be investigated. Various assays can be used to quantify different proteins such as caspases, kinases, cathepsin and calpain, or to detect changes in glutathione level, as well as annexin V binding and DNA fragmentation.
- Caspase assay
Activated caspases can be detected in living cells through the use of fluorometric staining. For this purpose, fluorochrome-conjugated caspase inhibitors are used. Upon entering the apoptotic cells, these inhibitors specifically and irreversibly bind to activated caspases, which can then be visualized by fluorescence microscopy, or analyzed by flow cytometry.
Caspase activity can also be quantified by fluorometric and colorimetric assays. The cleavage of labeled synthetic peptides by activated caspases results in the release of the dye, which is quantified using a fluorometer or spectrophotometer (Boucher D et al, 2014).
- Annexin V assay
In the early stages of apoptosis, changes can be observed on the cell surface: Cells lose their phospholipid membrane asymmetry and display phosphatidylserine (PS) on the exterior plasma membrane. Fluorochrome-coupled Annexin V is very useful for identifying early apoptotic cells, as it selectively binds to PS on the cell surface. This assay allows the detection of apoptosis much earlier than assays based on DNA fragmentation or loss of membrane integrity. The stained cells can be evaluated by flow cytometry or fluorescence microscopy (Crowley LC et al, 2016).
- DNA fragmentation detection
Nuclear DNA fragmentation is a hallmark of apoptosis in eukaryotic cells and can be detected by different assays. The TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling) is considered the “gold standard” for measuring apoptosis because it works on 95 percent of cells. In this assay, a labeled enzyme (deoxynucleotidyl transferase, dUTP) is used to specifically identify apoptotic cells (Kyrylkova K et al, 2012). In the DNA ladder detection assay, DNA fragmentation is visualized by agarose gel electrophoresis (Saadat YR et al, 2015).
- Detecting mitochondrial changes
One of the earliest intracellular events during apoptosis is the disruption of the mitochondrial transmembrane potential. Fluorescent-based in vitro assays or cytochrome c detection allow the monitoring of mitochondrial changes, and therefore the differentiation between healthy and apoptotic cells (Desagher S et al, 1999).
- Differentiation of apoptotic and necrotic cells
Apoptosis and necrosis are the two major processes leading to cell death. The reliable identification of these two processes, as well as the quantification of apoptotic, necrotic and vital cells within the same population, can be performed by flow cytometry or fluorescence microscopy.
The Role of Apoptosis in Cancer
- Role of apoptosis in cancer development and treatment
In cancer, apoptosis is inhibited through a wide variety of mechanisms, resulting in an excessive cellular proliferation and the pathological accumulation of cells. A disbalance between proapoptotic and antiapoptotic signals can cause the deviation from the physiological pathway. In half of human cancers, for example, an overexpression of the BCL-2 survival gene can be detected. As most of the anticancer drugs depend on BCL-2/BAX- to kill cancer cells, disruption of this mechanisms results in a resistance to chemotherapy. Proapoptotic proteins as caspases, on the other hand, are often under-expressed in cancer cells due to mutations in the encoding genes. Altered apoptotic signaling pathways can also promote resistance to immunological defense mechanisms (Pfeffer TM and Singh ATK , 2018). Targeting the apoptotic pathway can represent a successful approach to cancer treatment. New therapeutic strategies that target proteins involved in the apoptotic process BCL-2, or death receptors as DISC (death-inducing signaling complex), are being evaluated in clinical settings (Zaman S et al, 2014 ).
Apoptosis Research Portfolio by PromoCell
Apoptosis is a sophisticated process that involves an intricate series of biochemical events. Specific cell death signals are transduced by different biomolecules such as caspases initiating an apoptotic cascade and finally causing multiple events such as degradation of cellular proteins and chromosomal DNA, mitochondrial disruption or changes in the plasma membrane integrity.
Since no single parameter fully defines cell death, it is recommended to use different methods to detect apoptotic cells or to differentiate between necrosis and apoptosis. Thus, PromoCell provides a selected line of tools for detecting and studying the manifold apoptotic events occurring in different areas of the cell, e.g. the plasma membrane, cytoplasm, mitochondria, and nucleus. A wide range of kits and reagents for detection and quantitation of Caspases, Kinases, Cathepsin and Calpain activity, changes in the glutathione level as well as Annexin V binding and DNA fragmentation are available for apoptosis research. Moreover, apoptosis inducers and a system for apoptotic cell isolation are also provided.