In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

Cell death typically occurs by necrosis, apoptosis, or autophagy. Concerning apoptosis, its characterization implies several criteria, such as changes in the nuclear morphology, especially chromatin fragmentation and condensation, and also the occurrence of apoptotic bodies that contain nuclear material, as well as reduction in cell volume and plasma membrane shrinkage. 9 Intrinsic or extrinsic stimuli can lead to apoptosis, and the 2 different pathways involve specific biochemical manifestations such as the activation of caspases. Caspase 9 and caspase 3 are classically activated in the intracellular pathway with a process of mitochondrial outer membrane permeabilization (MOMP), whereas the extracellular pathway requires the activation of caspases 8 and 3. However, in some cell types, including hepatocytes and pancreatic β cells, the activation of caspase 9 has been described through an MOMP process, leading Galluzzi et al 2 to underline that extrinsic apoptosis is above all activated by extracellular stress signals and is characterized by death receptors that induce a caspase signaling cascade involving caspase 8, caspase 3, or caspase 9. If caspases display a central role in apoptotic mechanism, other families of proteins are also involved, such as SMACs (small mitochondrial-derived activator of caspases), IAPs (inhibitor of apoptosis proteins), and the Bcl-2 family. Death receptor family also plays a central role in the apoptosis machinery through cell surface receptor activation with specific death ligands. 10 The entire process is highly regulated and leads ultimately to massive cell destruction without an inflammatory response, in contrast to immunogenic cell death, a form of cell death also referred as immunogenic apoptosis which can induce an effective antitumor response through the activation of T cells and dendritic cells. Potential immunogenic cell death inducers have been characterized and include some chemotherapies as well as radiotherapy. This cell death variant is defined by the exposure of calreticulin (CALR) on the membrane and the release of other proteins, including members of the heat shock protein family. 11 In parallel, if necrotic cells exhibit common traits in comparison with apoptotic cells, notably some of the cellular changes, including chromatin condensation and apoptotic bodies, are not observed, and necrotic cells stimulate the inflammatory response through the release of intracellular materials into the extracellular milieu. For a long time, necrosis has been regarded only as an uncontrolled cell death process, but the discovery of potential signaling components, such as RIP (receptor-interacting protein) kinases, has led to the concept of programmed necrosis also called necroptosis, a regulated form of necrosis that requires the catalytic activity of receptor-interacting protein kinase 1 (RIPK1) and RIPK3. 12, 13 Regarding autophagy, it is a major, controlled, catabolic mechanism tightly regulated by some autophagy-related genes.