Cell death assays for drug discovery

Cell death is required for many normal physiological functions for both development and homeostasis. The ability of tumor cells to elude programmed cell death, also known as apoptosis, is a hallmark of most types of cancer. A wide variety of cellular proteins, including cell surface receptors, adaptors, proteases and mitochondrial components, regulate a fine balance between cell survival and death by apoptosis. Mutations of these sensor and/or effector proteins may tip the balance, resulting in the uncontrolled survival and proliferation of cancer cells. Uncovering apoptosis mechanisms will result in new strategies to exploit apoptosis for therapeutic benefit. Email:marketing@medicilon.com.cn web:www.medicilon.com

Abstract | Cell death assay has an important role in many human diseases, and strategies aimed at modulating the associated pathways have been successfully applied to treat various disorders. Indeed, several clinically promising cytotoxic and cytoprotective agents with potential applications in cancer, ischaemic and neurodegenerative diseases have recently been identified by high-throughput screening (HTS), based on appropriate cell death assays. Given that different cell death modalities may be dysregulated in different diseases, it is becoming increasingly clear that such assays need to not only quantify the extent of cell death, but they must also be able to distinguish between the various pathways. Here, we systematically describe approaches to accurately quantify distinct cell death pathways, discuss their advantages and pitfalls, and focus on those techniques that are amenable to HTS. Dysregulated cell death is a common feature of many human diseases, including cancer, stroke and neurodegeneration, and modulation of this cellular response has proved to be an effective therapeutic strategy. For example, many cytotoxic agents are potent anticancer therapeutics1, whereas cytoprotective compounds may be used to avoid unwanted cell death in the context of stroke, myocardial infarction or neurodegenerative disorders. The complex mechanisms and pathways that control cell death are increasingly becoming understood and it is now clear that different cell death subroutines have a critical role in multiple diseases. Unwarranted apoptotic and necrotic death of postmitotic cells contributes to the aetiology of many acute and chronic diseases, including ischaemic, toxic, neurodegenerative and infectious syndromes. Conversely, impaired apoptosis is frequently associated with hyperproliferative conditions such as autoimmune diseases and cancer. Defective autophagy has been associated with developmental disorders and muscular dystrophy. In many instances, the modality by which cells die is critical for the outcome of cell death assay at the organismal level. Necrosis has long been recognized as a pro-inflammatory process, whereas apoptosis was considered as a non-immunogenic (if not tolerogenic) cell death subroutine. Recently, immunogenic instances of apoptosis have been unveiled, and it has clearly been shown that immunogenic cell death plays an important part in the response to anticancer therapy in vivo. Cell-based tests that measure cell death-related phenomena are broadly used for drug development. In addition to quantifying cell death, it is critical that such tests accurately determine how potential drugs may be modulating cell death. That is, they should identify the cell death subroutine that is implicated, as well as allow the deconvolution of the biochemical cascades and the identification of the molecular targets that are being inhibited or enhanced by the investigated compounds. Thus, drug discovery assays must first address two critical questions: do the cells die and, if so, through which lethal pathway do they die? Following this, target deconvolution and identification must be undertaken to develop novel drugs from promising candidates. Novel cell death subroutine-specific inducers or inhibitors may provide great advantages over nonspecific cytotoxic or cytoprotective agents for at least two reasons. First, highly selective agents (that is, agents with one target or a few targets) are less prone to provoke side effects than compounds that target whole classes of proteins. Second, there are several instances in which the activation or inhibition of one specific cell death modality over others might be clinically desirable. One example is the induction of necrosis in apoptosis-resistant cancer cells. During the past decade, owing to major technological advances in the field of combinatorial chemistry in addition to the sequencing of an ever-increasing number of genomes, high-content chemical and genetic libraries have become available, raising the need for high-throughput screening (HTS) approaches. In response to this demand, multiple conventional cell death detection methods have been adapted to HTS and many novel HTSamenable techniques have been developed. Several articles have recently been published with general guidelines for the use and interpretation of tests that measure specific cell death modalities. Here, we provide a systematic overview of the methodologies that can accurately quantify cell death and precisely identify lethal biochemical cascades from a drug discovery-oriented perspective. We review the design and use of biosensors of apoptosis, autophagy and necrosis, as well as the implementation of cell death assays on HTS platforms. Cell death assays Cells should be considered as dead when they fulfil at least one of the following criteria: the plasma membrane has lost its integrity, the cell has fragmented into apoptotic bodies or the corpse or its fragments have been taken up by neighbouring cells . Additionally, as dying cells cease all functions, a decrease in metabolism can be observed at the level of the cell population. Thus, cell death assays fall into two major groups: assays that measure bona fide cell death and tests that quantify biochemical processes that are viewed as surrogate viability markers3 . Dozens—if not hundreds—of techniques are currently available to assess cell death, either directly or indirectly through surrogate markers. The detailed description of these methods, their advantages and their drawbacks have recently been reviewed in two comprehensive sets of guidelines. Vital dyes. The most common means of assessing the death of cultured cells is provided by vital dyes, which are fluorescent or coloured molecules that discriminate between living and dead cells. The vital dyes most frequently used in cytofluorometry (owing to its intrinsic statistical power) include so-called exclusion dyes, which cannot cross intact plasma membranes (for example, propidium iodide or 4ʹ,6-diamidino-2-phenylindole (DAPI)) and hence only label dead cells. By contrast, fluorogenic esterase substrates (for example, calcein acetoxymethylester (calcein-AM)) can be used to selectively label living cells. This is possible because these lipophilic, non-fluorescent compounds, which readily penetrate into cells, are hydrolysed by intracellular esterases to generate fluorescent and plasma membrane-impermeant products, which are then retained exclusively by living cells24. However, although exclusion dyes provide robust, artefact-free information, the enzymatic activity of intracellular esterases may be affected by cell death-unrelated phenomena. Intracellular proteins. Plasma membrane rupture can also be biochemically quantified by measuring the spillage of intracellular proteins (most often enzymes) into cell culture supernatants. For example, kits for the fluorometric or colorimetric detection of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) are commercially available. However, a major drawback of these techniques is that the activity of these enzymes may be affected by physicochemical parameters (for example, changes in the pH of the culture medium or side effects of pharmacological inhibitors) and enzyme activity may decay with time in the extracellular milieu10. Nevertheless, the quantification of G6PD or LDH release is suitable for HTS protocols, as it is relatively inexpensive and can easily be implemented in 96-or 384-well plates. Cellular metabolism.The most widely used surrogate biochemical marker for viability is ATP, based on the assumptions that living cells produce ATP and it is indispensable for cellular life. Luciferase-based assays allow for the sensitive quantification of intracellular ATP and are amenable to HTS studies. However, decreased intracellular ATP concentrations may result from non-lethal perturbations, including cessation of proliferation (for example, owing to senescence, starvation or contact inhibition) and inhibited mitochondrial respiration. Thus, measuring ATP does not always directly correlate with cell viability10. Several other assays that measure specific facets of cellular metabolism have been extensively used to monitor viability, including the well-known 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. MTT and other tetrazolium derivatives are colourless salts that are readily taken up by living cells and converted by mitochondrial reductases into coloured compounds that can be easily quantified by measuring their absorbance using a spectrophotometer. Unfortunately, similarly to the ATP-based assays, a number of other factors may inhibit mitochondrial reductases, thus implying that the conversion of tetrazolium derivatives per se cannot provide unequivocal information on cell viability. ATP-based and MTT-based assays are highly susceptible to metabolic interference; consequently, they may generate false positive results. Thus, although metabolism-oriented tests are very useful to obtain preliminary information, their results must be validated in secondary screens based on bona fide cell death markers. Irrespective of these issues, cell metabolism has recently been used to successfully screen chemical and genetic libraries.