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

Cell death plays a crucial role for a myriad of physiological processes, and several human diseases such as cancer are characterized by its deregulation. There are many methods available for both quantifying and qualifying the accurate process of cell death which occurs. Choosing the right assay tool is essential to generate meaningful data, provide sufficient information for clinical applications, and understand cell death processes. In vitro cell death assays are important steps in the search for new therapies against cancer as the ultimate goal remains the elaboration of drugs that interfere with specific cell death mechanisms. However, choosing a cell viability or cytotoxicity assay among the many available options is a daunting task. Indeed, cell death can be approached by several viewpoints and require a more holistic approach. This review provides an overview of cell death assays usually used in vitro for assessing cell death so as to elaborate new potential chemotherapeutics and discusses considerations for using each assay.

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Mitochondrion-Dependent Cell Death in TDP-43 Proteinopathies

Biomedicines ◽

10.3390/biomedicines9040376 ◽

2021 ◽

Vol 9 (4) ◽

pp. 376

Author(s):

Chantal B. Lucini ◽

Ralf J. Braun

Keyword(s):

Cell Death ◽

Oxygen Species ◽

In Vivo Models ◽

Dependent Cell ◽

Cell Death Mechanisms ◽

Sting Pathway ◽

Mitochondrial Membrane Permeabilization

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

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Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00098.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. H1730-H1739 ◽

Cited By ~ 37

Author(s):

Ron Zohar ◽

Baoqian Zhu ◽

Peter Liu ◽

Jaro Sodek ◽

C. A. McCulloch

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Cardiac Fibroblasts ◽

Chromatin Condensation ◽

Terminal Deoxynucleotidyl Transferase ◽

Tunel Staining ◽

Wild Type ◽

Nuclear Fragmentation ◽

A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

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Implication of caspases during maedi–visna virus-induced apoptosis

Journal of General Virology ◽

10.1099/0022-1317-83-12-3153 ◽

2002 ◽

Vol 83 (12) ◽

pp. 3153-3161 ◽

Cited By ~ 20

Author(s):

R. Duval ◽

V. Bellet ◽

S. Delebassée ◽

C. Bosgiraud

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Specific Cell ◽

Caspase 9 ◽

Virus Growth ◽

Caspase Inhibitors ◽

Visna Virus ◽

Maedi Visna Virus ◽

Cellular Lysis

Maedi–visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep. In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infection in vitro induces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diverse in vitro and in situ strategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, during in vitro infection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated: significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infection in vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPC in vitro infection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infection in vitro induces SCPC cell death by a mechanism that is strongly dependent on active caspases.

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Comparison of Cellular Death Pathways after mTHPC-mediated Photodynamic Therapy (PDT) in Five Human Cancer Cell Lines

Cancers ◽

10.3390/cancers11050702 ◽

2019 ◽

Vol 11 (5) ◽

pp. 702 ◽

Cited By ~ 9

Author(s):

Carsten Lange ◽

Christiane Lehmann ◽

Martin Mahler ◽

Patrick J. Bednarski

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Photodynamic Therapy ◽

Cell Lines ◽

Cancer Cell ◽

Human Cancer ◽

Human Cancer Cell ◽

Human Cancer Cell Lines ◽

Cell Death Mechanisms

One of the most promising photosensitizers (PS) used in photodynamic therapy (PDT) is the porphyrin derivative 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC, temoporfin), marketed in Europe under the trade name Foscan®. A set of five human cancer cell lines from head and neck and other PDT-relevant tissues was used to investigate oxidative stress and underlying cell death mechanisms of mTHPC-mediated PDT in vitro. Cells were treated with mTHPC in equitoxic concentrations and illuminated with light doses of 1.8–7.0 J/cm2 and harvested immediately, 6, 24, or 48 h post illumination for analyses. Our results confirm the induction of oxidative stress after mTHPC-based PDT by detecting a total loss of mitochondrial membrane potential (Δψm) and increased formation of ROS. However, lipid peroxidation (LPO) and loss of cell membrane integrity play only a minor role in cell death in most cell lines. Based on our results, apoptosis is the predominant death mechanism following mTHPC-mediated PDT. Autophagy can occur in parallel to apoptosis or the former can be dominant first, yet ultimately leading to autophagy-associated apoptosis. The death of the cells is in some cases accompanied by DNA fragmentation and a G2/M phase arrest. In general, the overall phototoxic effects and the concentrations as well as the time to establish these effects varies between cell lines, suggesting that the cancer cells are not all dying by one defined mechanism, but rather succumb to an individual interplay of different cell death mechanisms. Besides the evaluation of the underlying cell death mechanisms, we focused on the comparison of results in a set of five identically treated cell lines in this study. Although cells were treated under equitoxic conditions and PDT acts via a rather unspecific ROS formation, very heterogeneous results were obtained with different cell lines. This study shows that general conclusions after PDT in vitro require testing on several cell lines to be reliable, which has too often been ignored in the past.

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Neuroprotective Effect of Bergamot Juice in 6-OHDA-Induced SH-SY5Y Cell Death, an In Vitro Model of Parkinson’s Disease

Pharmaceutics ◽

10.3390/pharmaceutics12040326 ◽

2020 ◽

Vol 12 (4) ◽

pp. 326 ◽

Cited By ~ 3

Author(s):

Nadia Ferlazzo ◽

Santa Cirmi ◽

Alessandro Maugeri ◽

Caterina Russo ◽

Giovanni Enrico Lombardo ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Cell Death ◽

Nuclear Translocation ◽

Neuroprotective Effect ◽

Neuroblastoma Cells ◽

Specific Cell ◽

Protective Effects ◽

Human Neuroblastoma

Much evidence suggests that both oxidative stress and apoptosis play a key role in the pathogenesis of Parkinson’s disease (PD). The present study aims to evaluate the protective effect of bergamot juice (BJ) against 6-hydroxydopamine (6-OHDA)- or H2O2-induced cell death. Treatment of differentiated SH-SY5Y human neuroblastoma cells with 6-OHDA or H2O2 resulted in cell death that was significantly reduced by the pre-treatment with BJ. The protective effects of BJ seem to correlate with the reduction of intracellular reactive oxygen species and nitric oxide generation caused by 6-OHDA or H2O2. BJ also attenuated mitochondrial dysfunction, caspase-3 activation, imbalance of pro- and anti-apoptotic proteins, MAPKs activation and reduced NF-ĸB nuclear translocation evoked by neurotoxic agents. Additionally, BJ exhibited excellent antioxidant capability in cell-free assays. Collectively, our results suggest that BJ exerts neuroprotective effect through the interplay with specific cell targets and its antioxidant activity, making it worthy of consideration for the management of neurodegenerative diseases.

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Mortalin (GRP75/HSPA9) Promotes Survival and Proliferation of Thyroid Carcinoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20092069 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2069 ◽

Cited By ~ 9

Author(s):

Dmytro Starenki ◽

Nadiya Sosonkina ◽

Seung-Keun Hong ◽

Ricardo V. Lloyd ◽

Jong-In Park

Keyword(s):

Cell Death ◽

Thyroid Carcinoma ◽

Thyroid Tumor ◽

Anaplastic Thyroid Carcinoma ◽

Mitochondrial Activity ◽

Phase Arrest ◽

M Phase ◽

A Cell ◽

Mitochondrial Hsp70

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.

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Preclinical photodynamic therapy research in Spain 3: Localization of photosensitizers and mechanisms of cell deathin vitro

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424609000516 ◽

2009 ◽

Vol 13 (04n05) ◽

pp. 544-551 ◽

Cited By ~ 11

Author(s):

Magdalena Cañete ◽

Juan C. Stockert ◽

Angeles Villanueva

Keyword(s):

Cell Death ◽

Photodynamic Therapy ◽

Cell Cultures ◽

Cellular Localization ◽

Cell Damage ◽

Therapeutic Modality ◽

Action Mechanisms ◽

Cell Death Mechanisms

Photodynamic therapy (PDT) is a subject of increasing biomedical research and represents a very promising therapeutic modality for palliative or even curative treatment of some superficial or endoscopically accessible tumors. In addition to the first photosensitizers (PSs) applied (hematoporphyrin-based drugs), second generation PSs with improved photophysical and photobiological properties are now studied using cell cultures, experimental tumors and clinical trials. On the other hand, there is a growing interest in the analysis of cell death mechanisms by apoptosis, which is especially relevant in oncology, because many anticancer drugs work, at least in part, by triggering apoptosis in neoplastic cells both in vitro and in vivo. The evaluation of cell death mechanisms is an important parameter to determine the efficacy and the potential toxicity of a treatment, allowing better adjustment of protocol. Using cell cultures, our research team has studied the mechanisms of cell damage and death implicated in the photodynamic processes, as well as the relationship between the cellular localization of the PS and the organelle damage during photosensitization. The results obtained in our laboratory provide a deeper understanding on the action mechanisms that lead to cell inactivation by PDT, and also allow selection of PSs with higher potential for clinical application than those currently in use.

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New in vitro insights on a cell death pathway induced by magnolol and honokiol in aristolochic acid tubulotoxicity

Food and Chemical Toxicology ◽

10.1016/j.fct.2015.11.020 ◽

2016 ◽

Vol 87 ◽

pp. 77-87 ◽

Cited By ~ 11

Author(s):

Valérian Bunel ◽

Marie-Hélène Antoine ◽

Caroline Stévigny ◽

Joëlle Nortier ◽

Pierre Duez

Keyword(s):

Cell Death ◽

Aristolochic Acid ◽

Cell Death Pathway ◽

A Cell

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A Priori Activation of Apoptosis Pathways of Tumor (AAAPT) Technology: Development of Targeted Apoptosis Initiators for Cancer Treatment

10.1101/844605 ◽

2019 ◽

Author(s):

Raghu Pandurangi ◽

Marco Tomasetti ◽

Thillai Verapazham Sekar ◽

Ramasamy Paulmurugan ◽

Cynthia Ma ◽

...

Keyword(s):

Cell Death ◽

Cancer Cells ◽

Technology Development ◽

Tumor Regression ◽

A Priori ◽

Specific Cell ◽

Cell Death Pathways ◽

Survival Pathways ◽

Apoptosis Pathways

AbstractCancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. The principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral damaging effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept “A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (ling cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro. At higher dose, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AMP-001 as a neoadjuvant to chemotherapy to achieve a better efficacy and reduced off-target toxicity by the current treatments.Summary StatementA Priori Activation of Apoptosis Pathways of Tumor often referred to as “AAAPT” is a novel targeted tumor sensitizing technology which synergizes with chemotherapy to enhance the treatment efficacy.

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Mechanisms of Human intelligence — From RNA and Synapse to Broadband

10.31234/osf.io/7w63s ◽

2019 ◽

Author(s):

Robert Traill

Keyword(s):

Underlying Mechanism ◽

Long Term Memory ◽

Optical Signals ◽

A Cell ◽

Impossible Task ◽

The Many ◽

The Right ◽

Label Sequence

Simple thought has been explained by the action-potential (AP) system with its synapses. In contrast, in-depth details for “Declarative” intellectual thought have been a complete mystery because (it is argued here) its main underlying mechanism is fundamentally different. Declarative thinking depends heavily on linear coding based on digit-like elements — something which an unaided AP system could never offer......Looking instead to psychology, Piaget (1920s) proposed basic units of action-sequences (“schèmes” whereby one could mentally construct object-concepts). There is now evidence that some ncRNA serves this verb-like action-coding role. — (Other ncRNA demonstrably serves as adjectival/adverbial “regulators” — while the remaining ≈3% of RNA encodes physical structures, the traditional noun-like role). If valid, then:–•NEW FOCUS ONTO ULTRAMICRO: — The whole Piagetian structure-coding for a concept could fit into one of the many 125nm capsids (“granules”). Moreover, many more concepts (and duplicates) could fit into a cell-body. — The vast abundance of coding-sites would allow comprehensive “wasteful” rapid use of Jerneian/Darwinian selection instead of problematic “writing down” of new learnings. — Estimates of memory-capacity increase vastly. — And hereditary-schèmes obviously explain inherited behaviour-traits. — Piaget’s other theory about develop¬mental stages also seems compatible.•Quantum-constraints ensure that such micro-sites would USE OPTICAL FREQUENCY signalling. That opens the way to greatly enhanced “Gigabit” rates, and optical-interference tricks.•MYELIN gets the EXTRA ROLE OF OPTIC-CABLE.So nerve-fibres become seen as simultan¬eous paths for two different types of signal (also demonstrated by Sun-et-al, 2010), with AP still dominant in some roles, but subservient to “UPE” optical signals elsewhere.•LOCATING MEMORIES? Choosing the right address means selecting some sort of “phone-number or numbered plug-socket.” That is best provided as an “address-label” sequence on the transmitted version of the schème-coding — in which case, actual destination-location may be less important.•“Moving-house” TO CORTICAL LONG-TERM MEMORY. The memory-move must preserve existing (i) memory-structure, and (ii) links to distant static archives. This “impossible” task would seem feasible if memories are actually held within individual cells (as above). There is indeed lifelong flow of such neurons in some mammals; but these flows seem to cease in adulthood for humans and dolphins! So the search continues.

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