The concept of intrinsic versus extrinsic apoptosis.

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer.

Interleukin-6 sensitizes TNF-α and TRAIL/Apo2L dependent cell death through upregulation of death receptors in human cancer cells.

Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.

Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways.

BACKGROUND Hypoxia induces cell apoptosis in the uterosacral ligaments of patients with pelvic organ prolapse by upregulation of hypoxia-inducible factor-1α (HIF-1α). This study aimed to investigate the effects of HIF-1α on human uterosacral ligament fibroblasts (hUSLFs) following treatment with the chemical inducer of hypoxia, cobalt chloride (CoCl2), and to explore the underlying mechanisms. MATERIAL AND METHODS Ten women who underwent hysterectomy for benign disease provided uterosacral ligament tissue for cell extraction. Following CoCl2 treatment, cell viability of isolated and cultured hUSLFs was evaluated by the MTT assay. JC-1 fluorescence mitochondrial imaging was used to study the change in mitochondrial membrane potential. Cell apoptosis and expression of apoptosis-associated proteins and collagen type I alpha 1 (COL1A1) were measured by flow cytometry, TUNEL and Western blot, respectively. RESULTS Hypoxia increased the expression of HIF-1a and increased cell apoptosis, decreased cell viability and expression levels of COL1A1. The JC-1 assay showed that the mitochondrial membrane potential was reduced and caspase-8, and -9 inhibitors partly reduced hUSLF apoptosis. HIF-1α treatment downregulated the expression of cellular FLICE inhibitory protein (c-FLIP), decoy receptor 2 (DcR2), and the ratio of Bcl-2 to Bax, and upregulated the expression tumor necrosis factor related apoptosis-inducing ligand (TRAIL), death receptor 5 (DR5) or TRAIL-R2, Fas, Bcl-2 interacting protein 3 (BNIP3), and cytochrome C, and increased the activation of caspase-3, caspase-8, and caspase-9, all of which were reversed by knockdown of HIF-1α. CONCLUSIONS HIF-1α significantly induced apoptosis of hUSLFs through both the cell death receptor and the mitochondrial-associated apoptosis pathways.

Naringin Protects Pancreatic ß-Cells Against Oxidative Stress-Induced Apoptosis by Inhibiting Both Intrinsic and Extrinsic Pathways in Insulin-Deficient Diabetic Mice.

SCOPE: Oxidative stress has been suggested to play a central role in the pathogenesis of diabetes, as well as other metabolic disorders. Naringin, a major flavanone glycoside in citrus species, has been shown to display strong antioxidant potential in in vitro and in vivo models of oxidative stress; however, the underlying protective mechanisms in diabetes are unclear. METHODS AND RESULTS: To study the protective effects and molecular mechanisms of naringin in preventing islet dysfunction and diabetes, we examined glucose homeostasis, ß-cell apoptosis, and inflammatory response in insulin-deficient diabetic mice exposed to acute oxidative stress with streptozotocin (STZ). Naringin dose-dependently ameliorated hyperglycemia and islet dysfunction in insulin-deficient diabetic mice. Naringin counteracted STZ-induced ß-cell apoptosis by inhibiting both the intrinsic (mitochondria-mediated) and extrinsic (death receptor-mediated) pathways. Furthermore, these protective effects were associated with suppression of DNA damage response and nuclear factor-kappa B- and mitogen-activated protein kinase-mediated signaling pathways, as well as reduction of reactive oxygen species accumulation and pro-inflammatory cytokine production in the pancreas. CONCLUSION: Taken together, our study provides insights into the underlying mechanisms through which naringin protects the pancreatic ß-cells against oxidative stress-induced apoptosis.

Apoptotic Caspases in Promoting Cancer: Implications from Their Roles in Development and Tissue Homeostasis.

Apoptosis, a major form of programmed cell death, is an important mechanism to remove extra or unwanted cells during development. In tissue homeostasis apoptosis also acts as a monitoring machinery to eliminate damaged cells in response to environmental stresses. During these processes, caspases, a group of proteases, have been well defined as key drivers of cell death. However, a wealth of evidence is emerging which supports the existence of many other non-apoptotic functions of these caspases, which are essential not only in proper organism development but also in tissue homeostasis and post-injury recovery. In particular, apoptotic caspases in stress-induced dying cells can activate mitogenic signals leading to proliferation of neighbouring cells, a phenomenon termed apoptosis-induced proliferation. Apparently, such non-apoptotic functions of caspases need to be controlled and restrained in a context-dependent manner during development to prevent their detrimental effects. Intriguingly, accumulating studies suggest that cancer cells are able to utilise these functions of caspases to their advantage to enable their survival, proliferation and metastasis in order to grow and progress. This book chapter will review non-apoptotic functions of the caspases in development and tissue homeostasis with focus on how these cellular processes can be hijacked by cancer cells and contribute to tumourigenesis.

Molecular Cell Biology of Apoptosis and Necroptosis in Cancer.

Cell death is a major mechanism to eliminate cells in which DNA is damaged, organelles are stressed, or oncogenes are overexpressed, all events that would otherwise predispose cells to oncogenic transformation. The pathways that initiate and execute cell death are complex, genetically encoded, and subject to significant regulation. Consequently, while these pathways are often mutated in malignancy, there is considerable interest in inducing cell death in tumor cells as therapy. This chapter addresses our current understanding of molecular mechanisms contributing to two cell death pathways, apoptotic cell death and necroptosis, a regulated form of necrotic cell death. Apoptosis can be induced by a wide variety of signals, leading to protease activation that dismantles the cell. We discuss the physiological importance of each apoptosis pathway and summarize their known roles in cancer suppression and the current efforts at targeting each pathway therapeutically. The intricate mechanistic link between death receptor-mediated apoptosis and necroptosis is described, as well as the potential opportunities for utilizing necroptosis in the treatment of malignancy.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

The role of necroptosis in neurosurgical diseases

Programmed necrosis or necroptosis is an alternative form of cell death that is executed through a caspase-independent pathway. Necroptosis has been implicated in many pathological conditions. Genetic or pharmacological inhibition of necroptotic signaling has been shown to confer neuroprotection after traumatic and ischemic brain injury. Therefore, the necroptotic pathway represents a potential target for neurological diseases that are managed by neurosurgeons. In this review, we summarize recent advances in the understanding of necroptotic signaling pathways and explore the role of necroptotic cell death in craniocerebral trauma, brain tumors, and cerebrovascular diseases.

The role of necroptosis in neurosurgical diseases.

Programmed necrosis or necroptosis is an alternative form of cell death that is executed through a caspase-independent pathway. Necroptosis has been implicated in many pathological conditions. Genetic or pharmacological inhibition of necroptotic signaling has been shown to confer neuroprotection after traumatic and ischemic brain injury. Therefore, the necroptotic pathway represents a potential target for neurological diseases that are managed by neurosurgeons. In this review, we summarize recent advances in the understanding of necroptotic signaling pathways and explore the role of necroptotic cell death in craniocerebral trauma, brain tumors, and cerebrovascular diseases.

Carboxylation of multiwalled carbon nanotube attenuated the cytotoxicity by limiting the oxidative stress initiated cell membrane integrity damage, cell cycle arrestment, and death receptor mediated apoptotic pathway.

In this study, the effects of carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) on human normal liver cell line L02 was compared with that of pristine multiwalled carbon nanotubes (p-MWCNTs). It was shown that compared with MWCNTs-COOH, p-MWCNTs induced apoptosis, reduced the level of intracellular antioxidant glutathione more significantly, and caused severer cell membrane damage as demonstrated by lactate dehydrogenase leakage. Cell cycles were arrested by both MWCNTs, while p-MWCNTs induced higher ratio of G0/G1 phase arrestment as compared with MWCNTs-COOH. Caspase-8 was also activated after both MWCNTs exposure, indicating extrinsic apoptotic pathway was involved in the apoptosis induced by MWCNTs exposure, more importantly, MWCNTs-COOH significantly reduced the activation of caspase-8 as compared with p-MWCNTs. All these results suggested that MWCNTs-COOH might be safer for in vivo application as compared with p-MWCNTs.

The versatile roles of CARDs in regulating apoptosis, inflammation, and NF-κB signaling.

CARD subfamily is the second largest subfamily in the DD superfamily that plays important roles in regulating various signaling pathways, including but not limited to NF-kB activation signaling, apoptosis signaling and inflammatory signaling. The CARD subfamily contains 33 human CARD-containing proteins, regulating the assembly of many signaling complexes, including apoptosome, inflammsome, nodosome, the CBM complex, PIDDosome, the TRAF2 complex, and the MAVS signalosome, by homotypic CARD-CARD interactions. The mechanism of how CARDs find the right binding partner to form a specific complex remains unclear. This review uses different classification schemes to update the classification of CARD-containing proteins. Combining the classification based on domain structures, functions, associated signaling complexes, and roles would help better understand the structural and function diversity of CARD-containing proteins. This review also summarizes recent structural studies on CARDs. Especially, the CARD-containing complexes can be divided into the homodimeric, heterodimeric, oligomeric, filamentous CARD complexes and the CARD-ubiquitin complex. This review will give an overview of the versatile roles of CARDs in regulating signaling transduction, as well as the therapeutic drugs targeting CARD-containing proteins.

Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4(+) T cell-mediated autoimmune diabetes. METHODS: We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4(+) T cell-mediated attack using an in vivo graft model. RESULTS: Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4(+) T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either. CONCLUSIONS/INTERPRETATION: Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

Hijacking of death receptor signaling by bacterial pathogen effectors.

Death receptors such as Tumor necrosis factor receptor 1, FAS and TNF-associated apoptosis-inducing ligand-R1/2 play a major role in counteracting with bacterial pathogen infection through regulation of inflammation and programmed cell death. The highly regulated death receptor signaling is frequently targeted by gram-negative bacterial pathogens such as Salmonella, Shigella, enteropathogenic Escherichia coli and enterohamorrhagic Escherichia coli, which harbor a conserved type III secretion system that delivers a repertoire of effector proteins to manipulate host signal transductions for their own benefit. This review focuses on how bacterial gut pathogens hijack death receptor signaling to inhibit host NF-κB and programmed cell death pathways.

BID mediates selective killing of APC-deficient cells in intestinal tumor suppression by nonsteroidal antiinflammatory drugs.

Colorectal tumorigenesis is driven by genetic alterations in the adenomatous polyposis coli (APC) tumor suppressor pathway and effectively inhibited by nonsteroidal antiinflammatory drugs (NSAIDs). However, how NSAIDs prevent colorectal tumorigenesis has remained obscure. We found that the extrinsic apoptotic pathway and the BH3 interacting-domain death agonist (BID) are activated in adenomas from NSAID-treated patients. Loss of BID abolishes NSAID-mediated tumor suppression, survival benefit, and apoptosis in tumor-initiating stem cells in APC(Min/+) mice. BID-mediated cross-talk between the extrinsic and intrinsic apoptotic pathways is responsible for selective killing of neoplastic cells by NSAIDs. We further demonstrate that NSAIDs induce death receptor signaling in both cancer and normal cells, but only activate BID in cells with APC deficiency and ensuing c-Myc activation. Our results suggest that NSAIDs suppress intestinal tumorigenesis through BID-mediated synthetic lethality triggered by death receptor signaling and gatekeeper mutations, and provide a rationale for developing more effective cancer prevention strategies and agents.

Regulated cell death: signaling and mechanisms.

Cell turnover is a fundamental feature in metazoans. Cells can die passively, as a consequence of severe damage to their structural integrity, or actively, owing to a more confined biological disruption such as DNA damage. Passive cell death is uncontrolled and often harmful to the organism. In contrast, active cell death is tightly regulated and serves to support the organism's life. Apoptosis-the primary form of regulated cell death-is relatively well defined. Necroptosis-an alternative, distinct kind of regulated cell death discovered more recently-is less well understood. Apoptosis and necroptosis can be triggered either from within the cell or by extracellular stimuli. Certain signaling components, including several death ligands and receptors, can regulate both processes. Whereas apoptosis is triggered and executed via intracellular proteases called caspases, necroptosis is suppressed by caspase activity. Here we highlight current understanding of the key signaling mechanisms that control regulated cell death.

Silencing of JMJD2B induces cell apoptosis via mitochondria-mediated and death receptor-mediated pathway activation in colorectal cancer.

OBJECTIVE: To investigate the molecular mechanism of colorectal cancer (CRC) cell apoptosis induced by the Jumonji domain containing 2B (JMJD2B) silencing. METHODS: Both HCT116 and LoVo CRC cell lines were used for analyses. Cell apoptosis after JMJD2B silencing was determined by flow cytometry. JC-1 fluorescence probe was applied to measure the mitochondrial outer membrane permeabilization by flow cytometry and fluorescence microscopy. Immunofluorescence was used to detect cytochrome C translocation from mitochondria to cytosol after JMJD2B silencing. The efficacy of JMJD2B silencing on the protein levels of Bcl-2 family, caspase proteins, CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were detected by Western blot. RESULTS: JMJD2B silencing induced CRC cell apoptosis via a decrease of the anti-apoptotic gene Bcl-2 family expression, leading to the translocation of Bak and Bax proteins and the promotion of mitochondrial membrane disruption, resulting in the release of cytochrome C from mitochondria and subsequent caspase-9 and caspase-3 cleavage. It also increased the amount of cleaved caspase-8 involved in the death receptor-related apoptotic pathway. Bcl-2 homology 3 interacting-domain death agonist (Bid), a specific caspase-8 substrate involved in the Fas signaling pathway, subsequently induced cleavage via caspase-8 activation. However, levels of CHOP and GRP78 remained unchanged after JMJD2B silencing. CONCLUSIONS: JMJD2B silencing induced CRC cell apoptosis via both mitochondria-related and death receptor-related pathways. The cleavage of Bid activated by caspase-8 might serve as a crosstalk mediator between these two pathways in CRC.

Cell death in the myocardium: my heart won't go on.

Loss of cardiomyocytes plays a critical role in the pathogenesis of heart failure. With fewer myocytes, the heart is unable to sustain efficient contraction. Much attention has been focused on understanding mechanisms of cell death in myocytes with the ultimate goal being to reduce the extent of injury and improve function in the failing myocardium. Both necrosis and apoptosis contribute to loss of myocytes, and this loss of cells is a hallmark of cardiac pathologies, including ischemia/reperfusion, myocardial infarction, and heart failure. Apoptosis is a highly regulated process that is activated via death receptors in the plasma membrane or via permeabilization of the mitochondria. Necrosis is generally viewed as an uncontrolled process that leads to mitochondrial swelling, cell rupture, and subsequent inflammation. However, recent studies have uncovered a signaling pathway that mediates regulated necrosis or necroptosis. Mitochondria play an important role in both apoptosis and necrosis, and changes in their morphology can affect the cells' susceptibility to stress. This review focuses on the various modes of cell death in the myocardium and highlights how they contribute to loss of myocytes in response to stress.

Death receptor-ligand systems in cancer, cell death, and inflammation.

The discovery of tumor necrosis factor (TNF) marked the beginning of one of the most fascinating journeys in modern biomedical research. For the moment, this journey has culminated in the development of drugs that inhibit TNF. TNF blockers have revolutionized the treatment of many chronic inflammatory diseases. Yet, the journey seems far from over. TNF is the founding member of a family of cytokines with crucial functions in cell death, inflammation, and cancer. Some of these factors, most prominently TNF, CD95L, and TRAIL, can induce cell death. The receptors that mediate this signal are therefore referred to as death receptors, even though they also activate other signals. Here I will take you on a journey into the discovery and study of death receptor-ligand systems and how this inspired new concepts in cancer therapy and our current understanding of the interplay between cell death and inflammation.

Non-canonical kinase signaling by the death ligand TRAIL in cancer cells: discord in the death receptor family.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based therapy is currently evaluated in clinical studies as a tumor cell selective pro-apoptotic approach. However, besides activating canonical caspase-dependent apoptosis by binding to TRAIL-specific death receptors, the TRAIL ligand can activate non-canonical cell survival or proliferation pathways in resistant tumor cells through the same death receptors, which is counterproductive for therapy. Even more, recent studies indicate metastases-promoting activity of TRAIL. In this review, the remarkable dichotomy in TRAIL signaling is highlighted. An overview of the currently known mechanisms involved in non-canonical TRAIL signaling and the subsequent activation of various kinases is provided. These kinases include RIP1, IκB/ NF-κB, MAPK p38, JNK, ERK1/2, MAP3K TAK1, PKC, PI3K/Akt and Src. The functional consequences of their activation, often being stimulation of tumor cell survival and in some cases enhancement of their invasive behavior, are discussed. Interestingly, the non-canonical responses triggered by TRAIL in resistant tumor cells resemble that of TRAIL-induced signals in non-transformed cells. Better knowledge of the mechanism underlying the dichotomy in TRAIL receptor signaling may provide markers for selecting patients who will likely benefit from TRAIL-based therapy and could provide a rationalized basis for combination therapies with TRAIL death receptor-targeting drugs.