Human Immunodeficiency Virus (HIV) protease initiates apoptosis of HIV-infected cells by proteolytic cleavage of procaspase 8, creating a novel peptide termed casp8p41. Expression of casp8p41 alone is sufficient to initiate caspase-dependent cell death associated with mitochondrial depolarization. Since casp8p41 does not contain the catalytic cysteine at position 360, the mechanism by which casp8p41 initiates apoptosis is unclear. We demonstrate that casp8p41 directly causes mitochondrial depolarization and release of cytochrome c with downstream caspase 9 activation. Moreover, death induced by casp8p41 requires the presence of mitochondria, and in intact cells, casp8p41 colocalizes with mitochondria. These results illuminate a novel mechanism of cell death induced by a caspase 8 cleavage fragment whereby mitochondrial interaction leads to depolarization and cytochrome c release.

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival (P = 0.008 and P = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086-1.670; P = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups (P = 0.001) and in non-basal like subgroup (P = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort (P = 0.005) and basal-like subgroup (P = 0.034), as indicated by Kaplan-Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts.

Caspase-8 is an initiator of apoptotic signalling, and aberrations in procaspase-8 have been verified to be associated with malignant tumours. In previous studies, various procaspase-8 mutants were identified in AML patients, such as Q482H and M476W/Q482H mutations. The Q482H mutation can abolish caspase-8-mediated apoptosis by attenuating the dimerization of procaspase-8 protein monomers, causing AML patients carrying the Q482H mutation to develop resistance to chemotherapeutics. The patients with the M476W/Q482H mutation were sensitive to chemotherapy. Nevertheless, the underlying molecular mechanism is still unclear in regard to how the M476W/Q482H mutation restored caspase-8-mediated apoptosis. In this study, apoptosis was detected in the cells overexpressing the WT or mutant procaspase-8 with or without TRAIL treatment. Western blotting was devoted to detect the cleavage of procaspase-8 or the expression of proteins downstream in the apoptotic cascade and CO-IP was employed to analyze the dimerization of WT and mutant procaspase-8 proteins. Results demonstrated cells carrying the M476W/Q482H mutation restored caspase-8-mediated and TRAIL-induced apoptosis. The M476W/Q482H mutation recovered the dimerization of procaspase-8. Taken together, the M476W/Q482H mutation have restored caspase-8-mediated apoptosis resulting from the recovery of procaspase-8 dimerization.

Previous studies show that caspase-6 and caspase-8 are involved in neuronal apoptosis and regenerative failure after trauma of the adult central nervous system (CNS). In this study, we evaluated whether caspase-6 or -8 inhibitors can reduce cerebral or retinal injury after ischemia. Cerebral infarct volume, relative to appropriate controls, was significantly reduced in groups treated with caspase-6 or -8 inhibitors. Concomitantly, these treatments also reduced neurological deficits, reduced edema, increased cell proliferation, and increased neurofilament levels in the injured cerebrum. Caspase-6 and -8 inhibitors, or siRNAs, also increased retinal ganglion cell survival at 14 days after ischemic injury. Caspase-6 or -8 inhibition also decreased caspase-3, -6, and caspase-8 cleavage when assayed by western blot and reduced caspase-3 and -6 activities in colorimetric assays. We have shown that caspase-6 or caspase-8 inhibition decreases the neuropathological consequences of cerebral or retinal infarction, thereby emphasizing their importance in ischemic neuronal degeneration. As such, caspase-6 and -8 are potential targets for future therapies aimed at attenuating the devastating functional losses that result from retinal or cerebral stroke.

Caspase 8 initiates apoptosis downstream of TNF death receptors by undergoing autocleavage and processing the executioner caspase 3 (ref. 1). However, the dominant function of caspase 8 is to transmit a pro-survival signal that suppresses programmed necrosis (or necroptosis) mediated by RIPK1 and RIPK3 (refs 2-6) during embryogenesis and haematopoiesis(7-9). Suppression of necrotic cell death by caspase 8 requires its catalytic activity but not the autocleavage essential for apoptosis(10); however, the key substrate processed by caspase 8 to block necrosis has been elusive. A key substrate must meet three criteria: it must be essential for programmed necrosis; it must be cleaved by caspase 8 in situations where caspase 8 is blocking necrosis; and mutation of the caspase 8 processing site on the substrate should convert a pro-survival response to necrotic death without the need for caspase 8 inhibition. We now identify CYLD as a substrate for caspase 8 that satisfies these criteria. Following TNF stimulation, caspase 8 cleaves CYLD to generate a survival signal. In contrast, loss of caspase 8 prevented CYLD degradation, resulting in necrotic death. A CYLD substitution mutation at Asp 215 that cannot be cleaved by caspase 8 switches cell survival to necrotic cell death in response to TNF.

Sarcopenia has become a leading cause of disability and mortality in the elderly. It has been reported that programmed cell death (PCD) is associated with the development of sarcopenia that is characterized by reduction of muscle fiber size and number. TNF-α is also validated to play a prominent role in sarcopenia through its complex signaling pathways including cell death signaling. However, it is still unclear whether TNF-α contributes to sarcopenia by mediating pyroptosis, one type of PCD. Here, we first established naturally aged mice with sarcopenia model and confirmed an inflammatory state represented by TNF-α in aged mice. Evidence of GSDME-mediated pyroptosis and activation of apoptotic caspase-8/-3 were also found in skeletal muscle cells of aged mice with sarcopenia. We demonstrated that TNF-α triggered GSDME-mediated pyroptosis in myotubes through activating caspase-8 and caspase-3 by using caspase-8 and caspase-3 inhibitors. Comparing the activation of caspase-8 and GSDME expression between TNF Complex IIa and TNF Complex IIb, TNF-α was found to be more inclined to assemble TNF Complex IIb in activating caspase-8 and triggering pyroptosis. Moreover, pyroptotic myotubes were validated to result in decreased expression of MHC1 and finally loss of myotubes by knockdown of GSDME. Our work reveals a novel mechanism that TNF-ɑ/caspase-8/caspase-3/GSDME signaling-mediated pyroptosis contributes to the development of sarcopenia. Caspase-3/GSDME signaling-mediated pyroptosis may be a promising therapeutic target for sarcopenia.

Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway.

Osteosarcoma is the most common primary malignant bone tumor. Cancer cells employ a host of mechanisms to develop resistance to adriamycin (ADM) or other chemotherapeutic drugs. Shikonin (SK), an active constituent extracted from a Chinese medicinal herb, has been shown to cooperate with ADM in the treatment of osteosarcoma and certain other types of cancer by contributing to the response rate of chemotherapy and the side effects. The aim of the present study was to investigate the role and underlying mechanism of SK in chemotherapy for osteosarcoma. In the present study, a CCK-8 assay was performed to assess cell survival rate in vitro. Western blot analysis was performed to determine the expression levels of B‑cell lymphoma 2‑associated X protein (Bax), caspase‑3, caspase‑8, and poly (ADP‑ribose) polymerase (PARP). Flow cytometry was used to analyze cell cycle and cell death. The survival rate of cells decreased significantly in a dose‑ and time‑dependent manner when treated with a combination of SK and ADM. Western blot analysis revealed increased expression levels of Bax, caspase‑3, caspase‑8 and PARP in U2OS and MG63 cells 48 h following treatment with SK and ADM. Flow cytometric analysis showed that the combined treatment of SK and ADM significantly induced apoptosis in the osteosarcoma cells. Taken together SK cooperated with ADM to promote apoptosis, possibly by inducing caspase‑3‑ and caspase‑8‑dependent apoptosis. SK may be a potential enhancer in the treatment of drug‑resistant primary osteosarcoma.

Caspase-8 is a key initiator of death receptor-induced apoptosis. Here we report a novel short isoform of caspase-8 (caspase-8s), which encodes the first (Death Effector Domain) DED and part of the second DED, missing the C-terminal caspase domain. In vivo binding assays showed that transfected caspase-8s bound to (Fas-associated death domain protein) FADD, the adaptor protein in (death-induced signal complex) DISC. To investigate the potential effects of caspase-8s on cell apoptosis, Jurkat cells were stably transfected with caspase-8s. Overexpression of caspase-8s increased sensitivity to the apoptotic stimuli, Fas-agonistic antibody CH11. These results suggest that caspase-8s may act as a promoter of apoptosis through binding to FADD and is involved in the regulation of apoptosis. In addition, the results also indicate that the first DED was an important structure mediating combination between caspase-8 and FADD.

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

Millions of patients suffer from silicosis, but it remains an uncurable disease due to its unclear pathogenic mechanisms. Though the Nlrp3 inflammasome is involved in silicosis pathogenesis, inhibition of its classic downstream factors, Caspase-1 and Gsdmd, fails to block pyroptosis and cytokine release. To clarify the molecular mechanism of silicosis pathogenesis for new therapy, we examined samples from silicosis patients and genetic mouse models. We discovered an alternative pyroptotic pathway which requires cleavage of Gsdme by Caspases-3/8 in addition to Caspase-1/Gsdmd. Consistently, Gsdmd-/-Gsdme-/- mice showed markedly attenuated silicosis pathology, and Gsdmd-/-Gsdme-/- macrophages were resistant to silica-induced pyroptosis. Furthermore, we found that in addition to Caspase 1, Caspase-8 cleaved IL-1β in silicosis, explaining why Caspase-1-/- mice also suffered from silicosis. Finally, we found that inhibitors of Caspase-1, -3, -8 or an FDA approved drug, dimethyl fumarate, could dramatically alleviate silicosis pathology through blocking cleavage of Gsdmd and Gsdme. This study highlights that Caspase-1/Gsdmd and Caspase-3/8/Gsdme-dependent pyroptosis is essential for the development of silicosis, implicating new potential targets and drug for silicosis treatment.

Caspase 8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway in a manner dependent upon association with early endosomes. Previously, we identified caspase 8 as an effector of migration, promoting motility in a manner dependent upon phosphorylation on Tyr-380 by Src family kinases and its subsequent association with Src homology 2 domain-containing proteins. Here we demonstrate the regulation of the small GTPase Rab5, which mediates early endosome formation, homotypic fusion, and maturation by caspase 8. Regulation requires the Tyr-380 phosphorylation site but not caspase proteolytic activity. Tyr-380 is essential for interaction with the Src homology 2 domains of p85alpha, a multifunctional adaptor for phosphatidylinositol 3-kinase, that possesses Rab-GAP activity. Interaction between caspase 8 and p85alpha promotes Rab5 GTP loading, alters endosomal trafficking, and results in the accumulation of Rab5-positive endosomes at the edge of the cell. Conversely, caspase 8-dependent GTP loading of Rab5 is overcome by increased expression of p85alpha in a Rab-GAP-dependent manner. Thus, we demonstrate a novel function for caspase 8 as a modulator of p85alpha Rab-GAP activity and endosomal trafficking.

TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials.

In the death receptor induced apoptotic pathway, caspase-8 autocatalytically cleaves itself at specific cleavage sites. To better understand the regulatory mechanisms behind caspase-8 activation, we compared active wild-type caspase-8 (wtC8) and an uncleavable form of procaspase-8 (uncleavable C8). We demonstrate that wtC8 predominantly exists as a monomer and dimerizes in a concentration and inhibitor binding-dependent fashion. The K(D) for dimeric wtC8 is approximately 50 micro M and decreases when inhibitor bound. Uncleavable C8 is mainly monomeric, but a small amount that dimerizes is as active as wtC8. Inhibitor binding does not favor dimerization but induces active site rearrangements in uncleavable C8. Our findings suggest that dimerization is the crucial factor for caspase-8 activation.

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation, and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8-dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins, and activation of Rac, in a caspase-8-dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5-mediated internalization and the recycling of beta1 integrins, increasing cell migration independently of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8-mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8-driven cell migration in vivo, because knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8-mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.

Lung cancer is one of the leading causes of cancer death worldwide. Despite advances in therapy, conventional therapy is still the main treatment and has a high risk of chemotherapy resistance. Caspase-8 is involved in cell death and is a recognized marker for poor patient prognosis.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/β expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/β release.

Monocytes are peripheral leukocytes that function in innate immunity. Excessive triglyceride (TG) accumulation causes monocyte death and thus can compromise innate immunity. However, the mechanisms by which TG mediates monocyte death remain unclear to date. Thus, this study aimed to elucidate the mechanisms by which TG induces monocyte death. Results showed that TG induced monocyte death by activating caspase-3/7 and promoting poly (ADP-ribose) polymerase (PARP) cleavage. In addition, TG induced DNA damage and activated the ataxia telangiectasia mutated (ATM)/checkpoint kinase 2 and ATM-and Rad3-related (ATR)/checkpoint kinase 1 pathways, leading to the cell death. Furthermore, TG-induced DNA damage and monocyte death were mediated by caspase-2 and -8, and caspase-8 acted as an upstream molecule of caspase-2. Taken together, these results suggest that TG-induced monocyte death is mediated via the caspase-8/caspase-2/DNA damage/executioner caspase/PARP pathways. [BMB Reports 2023; 56(3): 166-171].

Cardiomyocyte death is an important reason for the cardiac syndromes, such as heart failure (HF) and myocardial infarction (MI). In the heart diseases, necrosis is one of the main forms of cell death. MicroRNAs (miRNAs) are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. Hitherto, it is not yet clear whether miRNA can regulate necrosis in cardiomyocyte. In this work, we performed a microarray to detect miRNAs in response to H2O2 treatment, and the results showed that miR-874 was substantially increased. We further studied the function of miR-874, and observed that knockdown of miR-874 attenuated necrosis in the cellular model and also MI in the animal model. We searched for the downstream mediator of miR-874 and identified that caspase-8 was a target of miR-874. Caspase-8 was able to antagonize necrosis. When suppressed by miR-874, caspase-8 lost the ability to repress necrotic program. In exploring the molecular mechanism by which miR-874 expression is regulated, we identified that Foxo3a could transcriptionally repress miR-874 expression. Foxo3a transgenic or knockout mice exhibited a low or high expression level of miR-874, and a reduced or enhanced necrosis and MI. Our present study reveals a novel myocardial necrotic regulating model, which is composed of Foxo3a, miR-874 and caspase-8. Modulation of their levels may provide a new approach for tackling myocardial necrosis.