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Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes—ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types. In inflammasomes, caspase-1 activation leads to pyroptosis mediated by gasdermin D, but cells lacking gasdermin-D still initiate caspase-dependent cell death. Here, Tsuchiya et al. show that these cells undergo Bid- and caspase-3-dependent apoptosis.

Head and neck cancer (HNC) is an ascending and agressive disease. The search for new molecular markers is emerging to solve difficulties in diagnosis, risk management, prognosis and effectiveness of treatments. Proteins related to apoptotic machinery have been identified as potential biomarkers. Caspase 3 is the main effector caspase and has a key role in apoptosis. The objective of this systematic review and meta-analysis is to review studies that analyze changes in Caspase 3 and Cleaved Caspase 3 expression both in oral premalignant disorders (OPMD) as well as in head and neck cancer (HNC). This study also proposes to review the prognostic values associated with HNC according to the expression of Caspase 3. Medline (via PubMed), EMBASE, Scopus, Cochrane, Web of Science and Grey Literature Database were screened from inception to june of 2022 and 18 studies were selected and 8 were included in the prognostic meta-analysis. Results related to the comparison of Caspase 3 expression demonstrated similar expression of Caspase 3 in HNC, with an average of 51.9% (9.5–98.1) showing high/moderate expression compared to 45.7% (14.6–84.7) in OPMD. Of interest, Cleaved Caspase 3 resulted incresed in HNC when compared with OPMD, being 73.3% (38.6–88.3) versus 22.9% (7.1–38.7). Pooled Fixed effect of HR values (95% CI) for OS related to Caspase 3 IHC expression in HNC patients was 1.48 (95% CI 0.95–2.28); also, the rate of heterogeneity was low, as revealed by I2 = 31%. For DFS was 1.07 (95% CI 0.79–1.45) with I2 = 0% and DSS showed a HR of 0.88 (95% CI 0.69–1.12) with I2 = 37%. Caspase 3 and Cleaved Caspase 3 expression could be linked with malignancy progression, but the expression of Caspase 3 did not influence the prognosis of patients with HNC.

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors. The limited ability of cancer therapeutics in crossing the cancer cell membrane hampers their therapeutic potential. Here, the authors report Salmonella-based system for intracellular delivery of protein drugs, e.g. caspase-3, and show reduction of tumors in mouse models of breast and liver cancer.

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme–inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

Members of the caspase family of cysteine proteases play central roles in coordinating the stereotypical events that occur during apoptosis. Because the major executioner caspases, caspase-3 and caspase-7, exhibit almost indistinguishable activity toward certain synthetic peptide substrates, this has led to the widespread view that these proteases occupy functionally redundant roles within the cell death machinery. However, the distinct phenotypes of mice deficient in either of these caspases, as well as mice deficient in both, is at odds with this view. These distinct phenotypes could be related to differences in the relative expression levels of caspase-3 and caspase-7 in vivo, or due to more fundamental differences between these proteases in terms of their ability to cleave natural substrates. Here we show that caspase-3 and caspase-7 exhibit differential activity toward multiple substrate proteins, including Bid, XIAP, gelsolin, caspase-6, and cochaperone p23. Caspase-3 was found to be generally more promiscuous than caspase-7 and appears to be the major executioner caspase during the demolition phase of apoptosis. Our observations provide a molecular basis for the different phenotypes seen in mice lacking either caspase and indicate that these proteases occupy nonredundant roles within the cell death machinery.

Activation of microglia and inflammation-mediated neurotoxicity are suggested to play a decisive role in the pathogenesis of several neurodegenerative disorders. Activated microglia release pro-inflammatory factors that may be neurotoxic. Here we show that the orderly activation of caspase-8 and caspase-3/7, known executioners of apoptotic cell death, regulate microglia activation through a protein kinase C (PKC)-δ-dependent pathway. We find that stimulation of microglia with various inflammogens activates caspase-8 and caspase-3/7 in microglia without triggering cell death in vitro and in vivo . Knockdown or chemical inhibition of each of these caspases hindered microglia activation and consequently reduced neurotoxicity. We observe that these caspases are activated in microglia in the ventral mesencephalon of Parkinson’s disease (PD) and the frontal cortex of individuals with Alzheimer’s disease (AD). Taken together, we show that caspase-8 and caspase-3/7 are involved in regulating microglia activation. We conclude that inhibition of these caspases could be neuroprotective by targeting the microglia rather than the neurons themselves. Caspases and neurotoxicity Brain inflammation is a typical feature of neurodegenerative diseases and acute forms of brain injury. Microglia are thought to play a part in the pathogenesis of such disorders by secreting neurotoxic cytokines. Experiments in cell and animal models of inflammation show that microglia activation requires the orderly activation of caspase-8 and caspase-3/7 — well known as agents of cell death. Inhibition of the caspase cascade prevents activation of microglia and protects against neurotoxicity. Caspase activation also occurs in microglia in the brains of patients with Parkinson's disease and Alzheimer's disease, raising the prospect that caspase inhibitors may have therapeutic potential.

Taurine is a ubiquitous sulfur-containing amino acid found in high concentration in most tissues. Because of its involvement in fundamental physiological functions, such as regulating respiratory chain activity, modulating cation transport, controlling inflammation, altering protein phosphorylation and prolonging lifespan, taurine is an important nutrient whose deficiency leads to severe pathology and cell death. However, the mechanism by which taurine deficiency causes cell death is inadequately understood. Therefore, the present study examined the hypothesis that overproduction of reactive oxygen species (ROS) by complex I of the respiratory chain triggers mitochondria-dependent apoptosis in hearts of taurine transporter knockout (TauTKO) mice. In support of the hypothesis, a 60% decrease in mitochondrial taurine content of 3-month-old TauTKO hearts was observed, which was associated with diminished complex I activity and the onset of mitochondrial oxidative stress. Oxidative damage to stressed mitochondria led to activation of a caspase cascade, with stimulation of caspases 9 and 3 prevented by treatment of 3-month-old TauTKO mice with the mitochondria specific antioxidant, MitoTempo. In 12 month-old, but not 3-month-old, TauTKO hearts, caspase 12 activation contributes to cell death, revealing a pathological role for endoplasmic reticulum (ER) stress in taurine deficient, aging mice. Thus, taurine is a cytoprotective nutrient that ensures normal mitochondrial and ER function, which is important for the reduction of risk for apoptosis and premature death.

Induction of apoptosis is often necessary for successful cancer therapy, and the non-invasive monitoring of apoptosis post-therapy could assist in clinical decision making. Isatins are a class of compounds that target activated caspase-3 during apoptosis. Here we report the synthesis of the 5-iodo-1,2,3-triazole (FITI) analog of the PET tracer [ 18 F]ICMT11 as a candidate tracer for imaging of apoptosis with SPECT, as well as PET. Labelling with radioiodine ( 123,125 I) was achieved in 55 ± 12% radiochemical yield through a chelator-accelerated one-pot cycloaddition reaction mediated by copper(I) catalysis. The caspase-3 binding affinity and selectivity of FITI compares favourably to that of [ 18 F]ICMT11 (K i = 6.1 ± 0.9 nM and 12.4 ± 4.7 nM, respectively). In biodistribution studies, etoposide-induced cell death in a SW1222 xenograft model resulted in a 2-fold increase in tumour uptake of the tracer. However, the tumour uptake was too low to allow in vivo imaging of apoptosis with SPECT.

Executioner-caspase activation has been considered a point-of-no-return in apoptosis. However, numerous studies report survival from caspase activation after treatment with drugs or radiation. An open question is whether cells can recover from direct caspase activation without pro-survival stress responses induced by drugs. To address this question, we engineered a HeLa cell line to express caspase-3 inducibly and combined it with a quantitative caspase activity reporter. While high caspase activity levels killed all cells and very low levels allowed all cells to live, doses of caspase activity sufficient to kill 15 to 30% of cells nevertheless allowed 70 to 85% to survive. At these doses, neither the rate, nor the peak level, nor the total amount of caspase activity could accurately predict cell death versus survival. Thus, cells can survive direct executioner-caspase activation, and variations in cellular state modify the outcome of potentially lethal caspase activity. Such heterogeneities may underlie incomplete tumor cell killing in response to apoptosis-inducing cancer treatments.