Current trends in luminescence-based assessment of apoptosis

Abstract

Apoptosis, the most extensively studied type of cell death, is known to play a crucial role in numerous processes such as elimination of unwanted cells or cellular debris, growth, control of the immune system, and prevention of malignancies. Defective regulation of apoptosis can trigger various diseases and disorders including cancer, neurological conditions, autoimmune diseases and developmental disorders. Knowing the nuances of the cell death type induced by a compound can help decipher which therapy is more effective for specific diseases. The detection of apoptotic cells using classic methods has brought significant contribution over the years, but innovative methods are quickly emerging and allow more in-depth understanding of the mechanisms, aside from a simple quantification. Due to increased sensitivity, time efficiency, pathway specificity and negligible cytotoxicity, these innovative approaches have great potential for both in vitro and in vivo studies. This review aims to shed light on the importance of developing and using novel nanoscale methods as an alternative to the classic apoptosis detection techniques.

Fig. 1. Schematic representation of apoptosis. There are two main pathways that conduct to the installation of programmed cell death: extrinsic pathway and intrinsic pathway. The extrinsic one starts with a stress stimuli outside of cell (1) that determines the secretion and binding of specific ligands to death receptors. The chemical messengers can be secreted by neighbouring cells (2) and are often represented by FAS and TRAIL (3) that bind their specific receptors on the targeted cell (e.g. FASR, TRAILR) (4). The ligand–receptor interaction determines modification within the intracellular domain which activates FADD protein inside the cell (5). Activated FADD interacts with Pro-caspase-8 and pro-caspase-10, causing their activation through cleavage of proteins that keep them inactive (6). The active caspases (Caspase 8 and Caspase 10) are further dispersed within the cell cytoplasm and trigger the activation of specific molecules, including proteins involved in DNA breakdown (7), finally causing the installation of apoptosis (8). Activated Caspase 8 and Caspase 10 are also interacting with BID that is transformed in tBID (9). tBID moves into the mitochondria causing the activation of BAX and BAK (9) – this step is also the intersection point between the extrinsic pathway and the intrinsic one. The last ones are favouring the installation of mitochondrial outer membrane permeability (MOMP) that is allowing the release of cytochrome C from the mitochondria into the cytoplasm (10). The next steps involve the formation of complex of proteins termed “apoptosome” (11), complex that is managing the activation of Pro-caspase-9 (12). Activated Caspase-9 is triggering the activation of Caspase-3 and Caspase-7 (among other functions) (13), enzymes that begin the process of cellular materials breakdown that finally concludes to installation of apoptosis (14). The extrinsic pathway is triggered by an internal stress stimulus (e.g. DNA damage, hypoxia) (1*) which activates a class of proteins called BH3-only proteins (2*). Pro-apoptotic BH3-only proteins are managing the same activation of BAX and BAK, point which represents the intersection between the extrinsic and intrinsic pathway. This figure was created using https://www.biorender.com/.