Caspase-3 Processing Research Articles

Ally or traitor: the dual role of p62 in caspase-2 regulation

Caspase-2 is a unique and conserved cysteine protease that is involved in several cellular processes, including different forms of cell death, maintenance of genomic stability, and the response to reactive oxygen species. Despite advances in caspase-2 research in recent years, the mechanisms underlying its activation remain largely unclear. Although caspase-2 is activated in the PIDDosome complex, its processing could occur even in the absence of PIDD1 and/or RAIDD, suggesting the existence of an alternative platform for caspase-2 activation. Here, we show that caspase-2 undergoes ubiquitination and interacts with scaffolding protein p62/sequestosome-1 (SQSTM1) under normal conditions and in response to DNA damage. p62 promotes proteasomal but not autophagic caspase-2 degradation as well as its dimerization and activation that triggers the caspase cascade and, subsequently, cell death. Inhibition of p62 expression attenuates cisplatin-induced caspase-2 processing and apoptosis. Notably, the ZZ domain of p62 is critical for caspase-2 binding, whereas the UBA domain is seemingly required to stabilize the p62–caspase-2 complex. Thus, we have uncovered the dual role of p62 in regulating caspase-2 activity: it can foster the degradation of caspase-2 in the proteasome or facilitate its activation by acting as a scaffold platform.

The apoptotic effects of soybean agglutinin were induced through three different signal pathways by down-regulating cytoskeleton proteins in IPEC-J2 cells

Soybean agglutinin (SBA) is a main anti-nutritional factor in soybean. SBA exhibits its anti-nutritional functions by binding to intestinal epithelial cells. Keratin8 (KRT8), Keratin18 (KRT18) and Actin (ACTA) are the representative SBA-specific binding proteins. Such cytoskeletal proteins act a crucial role in different cell activities. However, limited reports reveal what the signal transduction pathway of apoptosis caused by SBA when binding to KRT8, KRT18 and ACTA. We aimed to evaluate the effects of SBA on cell apoptosis and the expression of the cytoskeletal protein (KRT8, KRT18 and ACTA), reveal the roles of these cytoskeletal proteins or their combinations on SBA-induced cell apoptosis in IPEC-J2 cell line, evaluate the influences of SBA on the mitochondria, endoplasmic reticulum stress and death receptor-mediated apoptosis signal pathway and to show the roles of KRT8, KRT18 and ACTA in different apoptosis signal pathways induced by SBA. The results showed that SBA induced the IPEC-J2 cell apoptosis and decreased the mRNA expression of KRT8, KRT18 and ACTA (p < 0.05). The degree of effect of three cytoskeleton proteins on cell apoptosis was ACTA > KRT8 > KRT18. The roles of these three cytoskeletal proteins on IPEC-J2 apoptotic rates had a certain accumulation effect. SBA up-regulated mitochondrial fission variant protein (FIS1) and fusion protein (Mfn2) promoted CytC and AIF in mitochondria to enter the cytoplasm, activated caspase-9 and caspase-3, damaged or declined mitochondrial function and reduced ATP synthesis (p < 0.05). Also, SBA up-regulated the expression of GRP78, XBP-1, eIF2α, p-eIF2α and CHOP (p < 0.05), down-regulated the expression level of ASK1 protein (p < 0.05). SBA led to the recruitment of FADD to the cytoplasmic membrane and increased the expression of FasL, resulting in caspase-8 processing. SBA up-regulated the expression level of Bax protein and decreased cytosolic Bcl-2 and Bid (p < 0.05). In addition, there was a significant negative correlation between the gene expression of cytoskeleton proteins and apoptosis, as well as the expression of key proteins of apoptosis-related signal transduction pathways. In conclusion, SBA induced the activation of the mitochondria, endoplasmic reticulum stress and the death receptor-mediated apoptosis signal pathway and the crosstalk between them. The effect of SBA on these three pathways was mainly exhibited via down-regulation of the mRNA expression of the three cytoskeletal expressions. This study elucidates the molecular mechanism and signaling pathway of SBA that lead to apoptosis from the perspective of cell biology and molecular biology and provides a new perspective on the toxicity mechanism of other food-derived anti-nutrients, medical gastrointestinal health and related cancer treatment.

F-actin-rich territories coordinate apoptosome assembly and caspase activation during DNA damage-induced intrinsic apoptosis.

The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death is not well understood. Here we show that in response to DNA damage, human cells form a juxtanuclear F-actin-rich territory that coordinates the organized progression of apoptosome assembly to caspase activation. This cytoskeletal compartment is created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and it excludes proteins that inhibit JMY and WHAMM activity. Within the territory, mitochondria undergo outer membrane permeabilization and JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome c and Apaf-1. The F-actin-rich area also encompasses initiator caspase-9 and clusters of a cleaved form of executioner caspase-3 but restricts accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases. [Media: see text].

Caspase-2 is a mediator of apoptotic signaling in response to gemtuzumab ozogamicin in acute myeloid leukemia

The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg®) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.

The deubiquitinase Usp27x as a novel regulator of cFLIPL protein expression and sensitizer to death-receptor-induced apoptosis

Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.

An organometallic analogue of combretastatin A-4 and its apoptosis-inducing effects on lymphoma, leukemia and other tumor cells in vitro.

Hexacarbonyl[1,3-dimethoxy-5-((4'-methoxyphenyl)ethynyl)benzene]dicobalt (NAHO27), an organometallic analogue of combretastatin A-4, has been synthesized and its activity against lymphoma, leukemia, breast cancer and melanoma cells has been investigated. It was shown that NAHO27 specifically induces apoptosis in BJAB lymphoma and Nalm-6 leukemia cells at low micromolar concentration and does not affect normal leukocytes in vitro. It also proved to be active against vincristine and daunorubicin resistant leukemia cell lines with p-glycoprotein-caused multidrug resistance and showed a pronounced (550%) synergistic effect when co-applied with vincristine at very low concentrations. Mechanistic investigations revealed NAHO27 to induce apoptosis via the mitochondrial (intrinsic) pathway as reflected by the processing of caspases 3 and 9, the involvement of Bcl-2 and smac/DIABLO, and the reduction of mitochondrial membrane potential. Gene expression analysis and protein expression analysis via western blot showed an upregulation of the proapoptotic protein harakiri by 9%.

Abstract 13544: Inflammasome Activation In Kawasaki Disease And Multisystem Inflammatory Syndrome In Children

Introduction: Multisystem Inflammatory Syndrome in Children (MIS-C) is a newly identified syndrome elicited by infection of acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although MIS-C shares several clinical features with Kawasaki disease (KD), a greater magnitude of systemic inflammation is usually associated with MIS-C. Inflammasome activation induces the secretion of pro-inflammatory cytokines, IL-1β and IL-18 and the inflammatory form of cell death, pyroptosis. To provide mechanistic insights into MIS-C and KD, we compared the expression and activation of the inflammasome in blood samples from MIS-C and KD patients. Methods: Expression levels of canonical and non-canonical inflammasome components, including NLRP3 , CASP1 , CASP4 , CASP5 , IL1B , and the inflammatory mediator, TIFA in whole blood from KD and convalescent patients were analyzed from microarray datasets. The expression of these inflammasome-related genes was further examined in whole blood samples from MIS-C, KD, KD shock syndrome (KDSS) and convalescent patients using RT-qPCR. Inflammasome activation and TIFA expression were validated in granulocytes of febrile control, KD and MIS-C patients by Western blotting. Results: TIFA , NLRP3 , CASP1 , CASP4 , CASP5 , IL1B were upregulated in whole blood from MIS-C, KD, and KDSS patients as compared to convalescent patients. However, the differences were not significant among diseases. Although gene expression profiles were similar in KD, KDSS and MIS-C whole blood RNA, the processing of canonical and non-canonical inflammasome caspases, caspase-1, and caspase-4 were only observed in granulocytes isolated from MIS-C patients, but not KD and febrile controls. Moreover, TIFA was upregulated along with the activation of the inflammasome in granulocytes of MIS-C patients. Conclusions: Our results suggest that activation of inflammasomes, especially non-canonical inflammasome induction in granulocytes, is a hallmark of MIS-C, and differentiates it from KD.

Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells.

Mast cells are multifunctional immune cells scattered in tissues near blood vessels and mucosal surfaces where they mediate important reactions against parasites and contribute to the pathogenesis of allergic reactions. Serine proteases released from secretory granules upon mast cell activation contribute to these functions by modulating cytokine activity, platelet activation and proteolytic neutralization of toxins. The forced release of granule proteases into the cytosol of mast cells to induce cell suicide has recently been proposed as a therapeutic approach to reduce mast cell numbers in allergic diseases, but the molecular pathways involved in granule-mediated mast cell suicide are incompletely defined. To identify intrinsic granule proteases that can cause mast cell death, we used mice deficient in cytosolic serine protease inhibitors and their respective target proteases. We found that deficiency in Serpinb1a, Serpinb6a, and Serpinb9a or in their target proteases did not alter the kinetics of apoptosis induced by growth factor deprivation in vitro or the number of peritoneal mast cells in vivo. The serine protease cathepsin G induced marginal cell death upon mast cell granule permeabilization only when its inhibitors Serpinb1a or Serpinb6a were deleted. In contrast, the serine protease granzyme B was essential for driving apoptosis in mast cells. On granule permeabilization, granzyme B was required for caspase-3 processing and cell death. Moreover, cytosolic granzyme B inhibitor Serpinb9a prevented caspase-3 processing and mast cell death in a granzyme B-dependent manner. Together, our findings demonstrate that cytosolic serpins provide an inhibitory shield preventing granule protease-induced mast cell apoptosis, and that the granzyme B-Serpinb9a-caspase-3 axis is critical in mast cell survival and could be targeted in the context of allergic diseases.

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

VRK1 Depletion Facilitates the Synthetic Lethality of Temozolomide and Olaparib in Glioblastoma Cells.

BackgroundGlioblastomas treated with temozolomide frequently develop resistance to pharmacological treatments. Therefore, there is a need to find alternative drug targets to reduce treatment resistance based on tumor dependencies. A possibility is to target simultaneously two proteins from different DNA-damage repair pathways to facilitate tumor cell death. Therefore, we tested whether targeting the human chromatin kinase VRK1 by RNA interference can identify this protein as a novel molecular target to reduce the dependence on temozolomide in combination with olaparib, based on synthetic lethality.Materials and MethodsDepletion of VRK1, an enzyme that regulates chromatin dynamic reorganization and facilitates resistance to DNA damage, was performed in glioblastoma cells treated with temozolomide, an alkylating agent used for GBM treatment; and olaparib, an inhibitor of PARP-1, used as sensitizer. Two genetically different human glioblastoma cell lines, LN-18 and LN-229, were used for these experiments. The effect on the DNA-damage response was followed by determination of sequential steps in this process: H4K16ac, γH2AX, H4K20me2, and 53BP1.ResultsThe combination of temozolomide and olaparib increased DNA damage detected by labeling free DNA ends, and chromatin relaxation detected by H4K16ac. The combination of both drugs, at lower doses, resulted in an increase in the DNA damage response detected by the formation of γH2AX and 53BP1 foci. VRK1 depletion did not prevent the generation of DNA damage in TUNEL assays, but significantly impaired the DNA damage response induced by temozolomide and olaparib, and mediated by γH2AX, H4K20me2, and 53BP1. The combination of these drugs in VRK1 depleted cells resulted in an increase of glioblastoma cell death detected by annexin V and the processing of PARP-1 and caspase-3.ConclusionDepletion of the chromatin kinase VRK1 promotes tumor cell death at lower doses of a combination of temozolomide and olaparib treatments, and can be a novel alternative target for therapies based on synthetic lethality.

Modeling hypoxia facilitates cancer cell survival through downregulation of p53 expression

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP.

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting.

Determination of Caspase Activation by Western Blot.

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.

Requirement for Serine-384 in Caspase-2 processing and activity

Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.

Mcl-1 targeting strategies unlock the proapoptotic potential of TRAIL in melanoma cells.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in cancer cells. For melanoma, the targeting of TRAIL signaling appears highly attractive, due to pronounced TRAIL receptor expression in tumor tissue. However, mechanisms of TRAIL resistance observed in melanoma cells may limit its clinical use. The Bcl-2 family members are critical regulators of cell-intrinsic apoptotic pathways. Thus, the antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1) is overexpressed in many tumor types and was linked to chemotherapy resistance in melanoma. In this study, we evaluated the involvement of antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Bcl-w, Mcl-1, Bcl-A1, and Bcl-B) in TRAIL resistance. They were targeted by small interfering RNA-mediated silencing in TRAIL-sensitive (A-375, Mel-HO) and in TRAIL-resistant melanoma cell lines (Mel-2a, MeWo). This highlighted Mcl-1 as the most efficient target to overcome TRAIL resistance. In this context, we investigated the effects of Mcl-1-targeting microRNAs as well as the Mcl-1-selective inhibitor S63845. Both miR-193b and S63845 resulted in significant enhancement of TRAIL-induced apoptosis, associated with decreased cell viability. Apoptosis induction was mediated by caspase-3 processing as well as by Bax and Bak activation, indicating the critical involvement of intrinsic apoptosis pathways. These data may indicate a high relevance of Mcl-1 targeting also in melanoma therapy. Furthermore, the data may suggest to consider the use of the tumor suppressor miR-193b as a strategy for countering TRAIL resistance in melanoma.

Cytotoxicity of the Sesquiterpene Lactone Spiciformin and Its Acetyl Derivative against the Human Leukemia Cell Lines U-937 and HL-60.

Spiciformin (1) is a sesquiterpene lactone with a germacrane skeleton that is found in two Tanacetum species endemic to the Canary Islands. In this study, the cytotoxicities of 1 and its acetyl derivative (2) were evaluated against human tumor cells. These sesquiterpene lactones were cytotoxic against human acute myeloid leukemia (U-937 and HL-60) cells, even in cells over-expressing the pro-survival protein Bcl-2, but melanoma (SK-MEL-1) and human mononuclear cells isolated from blood of healthy donors were more resistant. Both compounds are apoptotic inducers in human leukemia U-937 cells. Cell death was mediated by the processing and activation of initiator and effector caspases and the cleavage of poly(ADP-ribose) polymerase, and it was blocked by a broad-spectrum caspase inhibitor and (in the case of sesquiterpene lactone 2) by the selective caspase-3/7, -8, and -9 inhibitors. In addition, certainly in the case of compound 2, this was found to be associated with a decrease in mitochondrial membrane potential, downregulation of the anti-apoptotic protein Bcl-2, activation of the mitogen-activated protein kinases signaling pathway, and generation of reactive oxygen species. It will, therefore, be relevant to continue characterization of this class of compounds.

FLIP(L): the pseudo-caspase.

Possessing structural homology with their active enzyme counterparts but lacking catalytic activity, pseudoenzymes have been identified for all major enzyme groups. Caspases are a family of cysteine‐dependent aspartate‐directed proteases that play essential roles in regulating cell death and inflammation. Here, we discuss the only human pseudo‐caspase, FLIP(L), a paralog of the apoptosis‐initiating caspases, caspase‐8 and caspase‐10. FLIP(L) has been shown to play a key role in regulating the processing and activity of caspase‐8, thereby modulating apoptotic signaling mediated by death receptors (such as TRAIL‐R1/R2), TNF receptor‐1 (TNFR1), and Toll‐like receptors. In this review, these canonical roles of FLIP(L) are discussed. Additionally, a range of nonclassical pseudoenzyme roles are described, in which FLIP(L) functions independently of caspase‐8. These nonclassical pseudoenzyme functions enable FLIP(L) to play key roles in the regulation of a wide range of biological processes beyond its canonical roles as a modulator of cell death.

Biodegradable Porous Silicon Nanocontainers as an Effective Drug Carrier for Regulation of the Tumor Cell Death Pathways.

Nanocontainers based on solid materials have great potential for drug delivery applications. However, since nanocontainer-mediated delivery can alter the drug internalization pathways and metabolism, it is important to find out what are the mechanisms of cancer cell death induced by nanocontainers and, moreover, is it possible to regulate them. Here, we report on the detailed investigation of the internalization kinetics and intracellular spatial distribution of porous silicon nanoparticles (PSi NPs) loaded with doxorubicin (DOX) and response of cancer cells to treatment with DOX-PSi NPs as well as studies of nanocontainer biodegradation by applying various microscopy methods, Raman microspectroscopy and biological experiments with cancer cells of different etiology. The obtained results revealed the absence of toxicity of unloaded PSi NPs to cancer cells up to a concentration of 700 μg/mL during the prolonged incubation time. Thus, given the fact that the nanocontainers themselves are not toxic, it is easy to adjust the dose of the drug that they deliver to the cells. It is shown, that the treatment with DOX-loaded PSi NPs more efficiently eliminates cancer cells in comparison with the free DOX. At the same time, the obtained results demonstrate the possibility of regulating the initiation of apoptosis or necrosis in tumor cells after treatment with different concentrations of DOX-PSi NPs, as revealed by the analysis of the caspase-3 processing, the accumulation of sub-G1 cell fraction, and morphological changes determined by electron and light microscopy. The obtained results are important for future applications of porous silicon nanocontainers in drug delivery for apoptotic pathway-targeted cancer therapy.

TRAF2 Controls Death Receptor-Induced Caspase-8 Processing and Facilitates Proinflammatory Signaling.

Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity.