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As an uncontrolled inflammatory response to infection, sepsis and sepsis induced organ dysfunction are great threats to the lives of septic patients. Unfortunately, the pathogenesis of sepsis is complex and multifactorial, which still needs to be elucidated. Pyroptosis is a newly discovered atypical form of inflammatory programmed cell death, which depends on the Caspase-1 dependent classical pathway or the non-classical Caspase-11 (mouse) or Caspase-4/5 (human) dependent pathway. Many studies have shown that pyroptosis is related to sepsis. The Gasdermin proteins are the key molecules in the membrane pores formation in pyroptosis. After cut by inflammatory caspase, the Gasdermin N-terminal fragments with perforation activity are released to cause pyroptosis. Pyroptosis is closely related to the occurrence and development of sepsis induced organ dysfunction. In this review, we summarized the molecular mechanism of pyroptosis, the key role of pyroptosis in sepsis and sepsis induced organ dysfunction, with the aim to bring new diagnostic biomarkers and potential therapeutic targets to improve sepsis clinical treatments., Competing Interests: Declaration of Competing Interest Authors declared no competing interests., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

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., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Caspase-11 is a pro-inflammatory enzyme that is stringently regulated during its expression and activation. As caspase-11 is not constitutively expressed in cells, it requires a priming step for its upregulation, which occurs following the stimulation of pathogen and cytokine receptors. Once expressed, caspase-11 activation is triggered by its interaction with lipopolysaccharide (LPS) from Gram-negative bacteria. Being an initiator caspase, activated caspase-11 functions primarily through its cleavage of key substrates. Gasdermin D (GSDMD) is the primary substrate of caspase-11, and the GSDMD cleavage fragment generated is responsible for the inflammatory form of cell death, pyroptosis, via its formation of pores in the plasma membrane. Thus, caspase-11 functions as an intracellular sensor for LPS and an immune effector. This review provides an overview of caspase-11-describing its structure and the transcriptional mechanisms that govern its expression, in addition to its activation, which is reported to be regulated by factors such as guanylate-binding proteins (GBPs), high mobility group box 1 (HMGB1) protein, and oxidized phospholipids. We also discuss the functional outcomes of caspase-11 activation, which include the non-canonical inflammasome, modulation of actin dynamics, and the initiation of blood coagulation, highlighting the importance of inflammatory caspase-11 during infection and disease.

Plants usually employ resistance (R) genes to defend against the infection of pathogens, and most R genes encode intracellular nucleotide-binding, leucine-rich repeat (NLR) proteins. The recognition between R proteins and their cognate pathogens often triggers a rapid localized cell death at the pathogen infection sites, termed the hypersensitive response (HR). Metacaspases (MCs) belong to a cysteine protease family, structurally related to metazoan caspases. MCs play crucial roles in plant immunity. However, the underlying molecular mechanism and the link between MCs and NLR-mediated HR are not clear. In this study, we systematically investigated the MC gene family in maize and identified 11 ZmMCs belonging to two types. Further functional analysis showed that the type I ZmMC1 and ZmMC2, but not the type II ZmMC9, suppress the HR-inducing activity of the autoactive NLR protein Rp1-D21 and of its N-terminal coiled-coil (CC D21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CC D21 in vivo. We further showed that ZmMC1 and ZmMC2, but not ZmMC9, are predominantly localized in a punctate distribution in both N. benthamiana and maize (Zea mays) protoplasts. Furthermore, the co-expression of ZmMC1 and ZmMC2 with Rp1-D21 and CC D21 causes their re-distribution from being uniformly distributed in the nucleocytoplasm to a punctate distribution co-localizing with ZmMC1 and ZmMC2. We reveal a novel role of plant MCs in modulating the NLR-mediated defense response and derive a model to explain it., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Breast cancer accounts for 1.4 million new cases every year. Triple-negative breast cancer (TNBC) is one the leading cause of mortality in developing countries and is associated with early age onset (under 40 years old). Chemotherapy has a poor success rate in patients with TNBC as compared to other types of breast cancers. It is due to the lack of expression of three validated molecular markers for breast cancer, the estrogen and progesterone receptors, and the amplification of HER-2/Neu. Therefore, a clear need exists for a greater understanding of TNBC at all levels and for the development of better therapies. We have studied the anti-tumor effects of a potential drug, maslinic acid, which can be extracted from olive oil industry waste. This natural product showed inhibitory effect at concentrations ranging from 30 to 50 µM within 24 h. It exhibited divergent effects in cell cycle progression for the MCF7 (estrogen positive) cell line when compared with TNBCs like MDA-MB-231 and MDA-MB-468. Also, maslinic acid treatment altered the mitochondrial membrane electrochemical potential and the reactive oxygen species (ROS) levels to cause a caspase-independent programmed cell death. In silico approaches and immunoblotting suggested the involvement of the MAPK pathway explaining the variability in cell cycle progression along with the apoptotic cell death caused by maslinic acid.

Diffuse large B-cell lymphoma (DLBCL), the most common type of aggressive non-Hodgkin lymphoma (NHL), has highly heterogeneous molecular characteristics. Although some patients initially respond to standard R-CHOP therapy, 30-40% develop refractory disease or suffer relapse. Signal transducer and activator of transcription 3 (STAT3), which regulates multiple oncogenic processes, has been found to be constitutively activated in various cancers, including DLBCL, suggesting its potential as a therapeutic target. In this study, we determined that 34% (23/69) of DLBCL patients expressed pSTAT3 (Y705) in tumour tissues. Napabucasin, a novel STAT3 inhibitor, exhibited potent cytotoxicity against NHL cell lines in a dose-dependent manner. Mechanistic studies demonstrated that napabucasin induced intrinsic and extrinsic cell apoptosis, downregulated the expression of STAT3 target genes, including the antiapoptotic protein Mcl-1, and regulated the ROS-mediated mitogen-activated protein kinase (MAPK) pathway. Most importantly, in vivo studies revealed the suppressive efficacy of napabucasin as a monotherapy without obvious toxicity. Furthermore, preliminary combination studies of napabucasin with doxorubicin showed significant synergism both in vitro and in vivo. Thus, our studies provide evidence that napabucasin alone or in combination is a promising therapeutic candidate for DLBCL patients., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. We found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wg RNAi , shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. We found that upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. Our data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development.

Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses., (©2020 Society for Leukocyte Biology.)

Targeting the epigenome of cancer cells with the combination of Bromodomain and Extra Terminal (BET) protein inhibitors and histone deacetylase (HDAC) inhibitors has shown synergistic antitumor effects in several cancer types. In this study, we investigate the antitumor potential of the novel dual BET/HDAC inhibitor TW09 in rhabdomyosarcoma (RMS) cells. TW09 reduces cell viability, suppresses long-term clonogenic survival and induces cell death in RMS cells in a dose-dependent manner. Compared to BET/HDAC co-inhibition using JQ1 and MS-275, TW09 induces similar cell death at equimolar concentrations and regulates BET and HDAC target proteins (e.g. c-MYC, H3 acetylation). Mechanistic studies revealed that TW09 upregulates BIM, NOXA, PUMA and BMF, while downregulating BCL-X L , leading to proapoptotic rebalancing of BCL-2 proteins. This results in BAK and BAX activation and caspase-dependent apoptosis, since individual genetic silencing of BIM, NOXA, PUMA, BMF, BAK or BAX, overexpression of BCL-2 or the caspase inhibition with zVAD.fmk all rescue JQ1/BYL719-induced cell death. In conclusion, TW09 shows potent antitumor activity in RMS cells in vitro by inducing mitochondrial apoptosis and may represent a promising new therapeutic option for the treatment of RMS., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The interaction between apoptosis-inducing factor (AIF) and cyclophilin A (CypA) has been shown to contribute to caspase-independent apoptosis. Blocking the AIF/CypA interaction protects against glutamate-induced neuronal cell death in vitro, and the purpose of this study was to determine the in vivo effect of an AIF/CypA interaction blocking peptide (AIF(370-394)-TAT) on neonatal mouse brain injury after hypoxia-ischemia (HI). The pups were treated with AIF (370-394)-TAT peptide intranasally prior to HI. Brain injury was significantly reduced at 72 h after HI in the AIF(370-394)-TAT peptide treatment group compared to vehicle-only treatment for both the gray matter and the subcortical white matter, and the neuroprotection was more pronounced in males than in females. Neuronal cell death was evaluated in males at 8 h and 24 h post-HI, and it was decreased significantly in the CA1 region of the hippocampus and the nucleus habenularis region after AIF(370-394)-TAT treatment. Caspase-independent apoptosis was decreased in the cortex, striatum, and nucleus habenularis after AIF(370-394)-TAT treatment, but no significant change was found on caspase-dependent apoptosis as indicated by the number of active caspase-3-labeled cells. Further analysis showed that both AIF and CypA nuclear accumulation were decreased after treatment with the AIF(370-394)-TAT peptide. These results suggest that AIF(370-394)-TAT inhibited AIF/CypA translocation to the nucleus and reduced HI-induced caspase-independent apoptosis and brain injury in young male mice, suggesting that blocking AIF/CypA might be a potential therapeutic target for neonatal brain injury., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report a role for the C. elegans separase SEP-1, primarily known for its essential activity in cell division and cortical granule exocytosis, in developmentally programmed cell death when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2 implicating SEP-1 in execution of apoptosis. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation, meiotic chromosome disjunction, egg shell formation, and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors result in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

The granulomatous lesion resulting from infection with the fungus Paracoccidioides brasiliensis is characterized by a compact aggregate of mature cells, surrounded by a fibroblast- and collagen-rich content. Granuloma formation requires signaling elicited by inflammatory molecules such as members of the interleukin-1 family. Two members of this family have been thoroughly studied, namely IL-1α and IL-1β. In this study, we addressed the mechanisms underlying IL-1α secretion and its functional role on the host resistance to fungal infection. We found that, the expression of caspase-11 triggered by P. brasiliensis infection of macrophages depends on IFN-β production, because its inhibition reduced procaspase-11 levels. Curiously, caspase-11 deficiency did not impair IL-1β production, however caspase-11 was required for a rapid pore-mediated cell lysis. The plasma membrane rupture facilitated the release of IL-1α, which was necessary to induce NO production and restrict fungal replication. Furthermore, P. brasiliensis-infected macrophages required IL-1α to produce optimal levels of IL-6, a major component of Th17 lymphocyte differentiation. Indeed, IL-1α deficiency accounted for a significant reduction of Th17 lymphocytes in lungs of infected mice, correlating with diminished neutrophil infiltration in the lungs. Strikingly, we identified that IL-1α directly reprograms the transcriptional profile of Th17-committed lymphocytes, increasing cellular proliferation, as for boosting IL-17 production by these cells. Beyond neutrophil chemotaxis in vivo, IL-17 also amplified IL-1α production by infected macrophages in vitro, endorsing a critical amplification loop of the inflammatory response. Therefore, our data suggest that the IFN-β/caspase-11/IL-1α pathway shapes a protective antifungal Th17 immunity, revealing a molecular mechanism underlying the cross-talk between innate and adaptive immunity., Competing Interests: The authors have declared that no competing interests exist.

It has been shown that caspase-1, but not its upstream activator, ASC, contributes to oviduct pathology during mouse genital Chlamydia muridarum infection. We hypothesized that this dichotomy is due to the inadvertent absence of caspase-11 in previously used caspase-1-deficient mice. To address this, we studied the independent contributions of caspase-1 and -11 during genital Chlamydia infection. Our results show that caspase-11 deficiency was sufficient to recapitulate the effect of the combined absence of both caspase-1 and caspase-11 on oviduct pathology. Further, mice that were deficient for both caspase-1 and -11 but that expressed caspase-11 as a transgene (essentially, caspase-1-deficient mice) had no significant difference in oviduct pathology from control mice. Caspase-11-deficient mice showed reduced dilation in both the oviducts and uterus. To determine the mechanism by which caspase-11-deficient mice developed reduced pathology, the chlamydial burden and immune cell infiltration were determined in the oviducts. In the caspase-11-deficient mice, we observed increased chlamydial burdens in the upper genital tract, which correlated with increased CD4 T cell recruitment, suggesting a contribution of caspase-11 in infection control. Additionally, there were significantly fewer neutrophils in the oviducts of caspase-11-deficient mice, supporting the observed decrease in the incidence of oviduct pathology. Therefore, caspase-11 activation contributes to pathogen control and oviduct disease independently of caspase-1 activation., (Copyright © 2019 American Society for Microbiology.)

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitans A. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the β 2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent., (Copyright © 2019 American Society for Microbiology.)

Members of the mammalian inflammatory caspase family, including caspase-1, caspase-4, caspase-5, caspase-11, and caspase-12, are key regulators of the innate immune response. Most studies to date have focused on the role of caspase-1 in the maturation of the proinflammatory cytokine interleukin-1β and its upstream regulation by the inflammasome signaling complexes. However, an emerging body of research has supported a role for caspase-4, caspase-5, and caspase-11 in both regulating caspase-1 activation and inducing the inflammatory form of cell death called pyroptosis. This inflammatory caspase pathway appears essential for the regulation of cytokine processing. Consequently, insight into this noncanonical pathway may reveal important and, to date, understudied targets for the treatment of autoinflammatory disorders where the inflammasome pathway is dysregulated. Here, we will discuss the mechanisms of inflammasome and inflammatory caspase activation and how these pathways intersect to promote pathogen clearance., (© 2019 Federation of European Biochemical Societies.)

Proinflammatory immune responses to Gram-negative bacterial lipopolysaccharides (LPS) are crucial to innate host defenses but can also contribute to pathology. How host cells sensitively detect structural features of LPS was a mystery for years, especially given that a portion of the molecule essential for its potent proinflammatory properties-lipid A-is buried in the bacterial membrane. Studies of responses to extracellular and vacuolar LPS revealed a crucial role for accessory proteins that specifically bind LPS-rich membranes and extract LPS monomers to generate a complex of LPS, MD-2, and TLR4. These insights provided means to understand better both the remarkable host sensitivity to LPS and the means whereby specific LPS structural features are discerned. More recently, the noncanonical inflammasome, consisting of caspases-4/5 in humans and caspase-11 in mice, has been demonstrated to mediate responses to LPS that has reached the host cytosol. Precisely how LPS gains access to cytosolic caspases-and in what form-is not well characterized, and understanding this process will provide crucial insights into how the noncanonical inflammasome is regulated during infection. Herein, we briefly review what is known about LPS detection by cytosolic caspases-4/5/11, focusing on lessons derived from studies of the better-characterized TLR4 system that might direct future mechanistic questions., (©2019 Society for Leukocyte Biology.)

Brucellosis, caused by the intracellular bacterial pathogen Brucella, is a globally important zoonotic disease for which arthritis is the most common focal complication in humans. Wild-type mice infected systemically with Brucella typically do not exhibit arthritis, but mice lacking IFN-γ develop arthritis regardless of the route of Brucella infection. Here, we investigated mechanisms by which IFN-γ suppresses Brucella-induced arthritis. Several cell types, including innate lymphoid cells, contributed to IFN-γ production and suppression of joint swelling. IFN-γ deficiency resulted in elevated joint IL-1β levels, and severe joint inflammation that was entirely inflammasome dependent, and in particular, reliant on the NLRP3 inflammasome. IFN-γ was vital for induction of the nitric oxide producing enzyme, iNOS, in infected joints, and nitric oxide directly inhibited IL-1β production and inflammasome activation in Brucella-infected macrophages in vitro. During in vivo infection, iNOS deficiency resulted in an increase in IL-1β and inflammation in Brucella-infected joints. Collectively, this data indicate that IFN-γ prevents arthritis both by limiting Brucella infection, and by inhibiting excessive inflammasome activation through the induction of nitric oxide., (©2019 Society for Leukocyte Biology.)

The aim of this study was to explain the possible mechanisms by which melatonin deficiency results in cardiovascular injury and to investigate the effects of melatonin administration on important signalling pathways and element equilibrium in the thoracic aorta (TA). For this purpose, we analysed the cellular and molecular effects of melatonin deficiency or administration on oxidative stress, DNA damage, molecular chaperone response, and apoptosis induction in TA tissues of pinealectomised rats using ELISA, RAPD, qRT-PCR, and Western blot assays. The results showed that melatonin deficiency led to an imbalance in essential element levels, unfolded or misfolded proteins, increased lipid peroxidation, and selectively induced caspase-dependent apoptosis in TA tissues without significantly affecting the Bcl-2/BAX ratio (2.28 in pinealectomised rats, 2.73 in pinealectomised rats treated with melatonin). In pinealectomised rats, the genomic template stability (80.22%) was disrupted by the significantly increased oxidative stress, and heat shock protein 70 (20.96-fold), TNF-α (1.73-fold), caspase-8 (2.03-fold), and caspase-3 (2.87-fold) were markedly overexpressed compared with the sham group. Melatonin treatment was protective against apoptosis and inhibited oxidative damage. In addition, melatonin increased the survivin level and improved the regulation of element equilibrium in TA tissues. The results of the study indicate that melatonin deficiency induces TNF-α-related extrinsic apoptosis signals and that the administration of pharmacological doses of melatonin attenuates cardiovascular toxicity by regulating the increase in the rate of apoptosis caused by melatonin deficiency in TA tissue of Sprague-Dawley rats.

Background: Human and mouse studies implicate the inflammasome in the pathogenesis of inflammatory bowel diseases, though the effects in mice are variable. The noncanonical inflammasome activator caspase-11 (Casp11) reportedly attenuates acute dextran sodium sulfate (DSS) colitis in mice. However, the effects of Casp11 on chronic experimental colitis and factors that influence the impact of Casp11 on acute DSS colitis are unknown., Methods: We studied the role of Casp11 in Il10-/- mice and acute and chronic DSS colitis mouse models. We quantified colonic Casp11 mRNA using quantative polymerase chain reaction and colitis using weight loss, blinded histological scoring, IL-12/23p40 secretion by colonic explants, and fecal lipocalin-2. We determined fecal microbial composition using 16S amplicon sequencing., Results: We detected increased colonic Casp11 mRNA in Il10-/- mice with chronic colitis, but not in mice with DSS colitis. The presence of Casp11 did not alter the severity of chronic colitis in DSS-treated or Il10-/- mice. Contrary to prior reports, we initially observed that Casp11 exacerbates acute DSS colitis. Subsequent experiments in the same animal facility revealed no effect of Casp11 on acute DSS colitis. There were pronounced stochastic changes in the fecal microbiome over this time. The majority of bacterial taxa that changed over time in wild-type vs Casp11-/- mice belong to the Clostridiales., Conclusions: Casp11 does not impact chronic experimental colitis, and its effects on acute DSS colitis vary with environmental factors including the microbiota, particularly Clostridiales. Stochastic drifts in intestinal microbiota composition, even in mice in the same housing facility, should be considered when interpreting studies of acute DSS colitis models.

Guanylate-binding proteins (GBPs) are conserved family of IFN-inducible GTPases that play an important role in the host immunity against bacterial, viral, and protozoan pathogens. GBPs protect the host by associating with intracellular microbes, their vacuolar niche or, in the case of viruses, with their replication complex. This association results in a restriction of the respective pathogen, yet the exact molecular mechanisms of the antimicrobial functions of GBPs are still unclear. Recent work has linked the GBPs with the activation of inflammasomes, multi-protein complexes that assemble upon recognition of pathogen- or host-derived signals and that drive the release of cytokines and host cell death. Here, we will focus on the most recent findings that have started to unravel the manifold restriction mechanism controlled by GBPs in mouse and human cells, and that shed light on the molecular cues that control GBP recruitment to bacterial membranes., (©2018 Society for Leukocyte Biology.)

Brucellosis, caused by the intracellular bacterial pathogen Brucella , is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we investigated the role of inflammasomes and their effectors, including interleukin-1 (IL-1), IL-18, and pyroptosis, on inflammation and control of infection during Brucella -induced arthritis. Early in infection, both caspase-1 and caspase-11 were found to initiate joint inflammation and proinflammatory cytokine production. However, by 1 week postinfection, caspase-1 and caspase-11 also contributed to control of Brucella joint infection. Inflammasome-dependent restriction of Brucella joint burdens did not require AIM2 (absent in melanoma 2) or NLRP3 (NLR family, pyrin domain containing 3). IL-1R had a modest effect on Brucella -induced joint swelling, but mice lacking IL-1R were not impaired in their ability to control infection of the joint by Brucella In contrast, IL-18 contributed to the initiation of joint swelling and control of joint Brucella infection. Caspase1/11-dependent cell death was observed in vivo , and in vitro studies demonstrated that both caspase-1 and caspase-11 induce pyroptosis, which limited Brucella infection in macrophages. Brucella lipopolysaccharide alone was also able to induce caspase-11-dependent pyroptosis. Collectively, these data demonstrate that inflammasomes induce inflammation in an IL-18-dependent manner and that inflammasome-dependent IL-18 and pyroptosis restrict Brucella infection., (Copyright © 2018 American Society for Microbiology.)

The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, therefore, plays a key role in apoptosis execution. However, the detailed molecular mechanisms of these events remain unclear. To get more insights into the mechanisms of nuclear events during apoptosis we set up a rapid fractionation protocol for the separation of the cytoplasmic and nuclear fractions of cells undergoing cisplatin-induced apoptosis. Importantly, nuclear accumulation of effector caspase-3 as well as initiator caspase-2, -8 and -9 was observed using the developed protocol and immunofluorescence microscopy. The detection of caspases and their cleavage products in the nucleus occurred within the same time interval after cisplatin treatment and took place shortly before nuclear fragmentation. The entry of initiator caspases to the nucleus was independent of caspase-3. Given that all three initiator caspases had catalytic activity in the nuclei, our findings indicate that initiator caspases might participate in the proteolysis of nuclear components during apoptosis, promoting its disintegration and apoptotic cell death.

Glioblastoma is the most common and aggressive glioma, characterized by brain invasion capability. Being very resistant to the current therapies, since even under treatment, surgery, and chemotherapy with temozolomide (TMZ), patients achieve a median survival of one year. In the search for more effective therapies, new molecules have been designed. For nervous system cancers, molecules able to cross the blood-brain barrier are handled with priority. Accordingly, tacrine was chosen for this study and the inclusion of spiro-heterocyclic rings was done in its structure resulting in new compounds. Cytotoxic activity of tacrine derivatives was assayed using glioblastoma cell line (SF295) as well as analyzing cell death mechanism. Increased caspases activities were observed, confirming apoptosis as cell death type. Some derivatives also increased reactive oxygen species formation and decreased the mitochondrial membrane potential. Moreover, compounds acted on several glioblastoma-related proteins including p53, HLA-DR, beta-catenin, Iba-1, MAP2c, Olig-2, and IDH1. Therefore, tacrine derivatives presented promising results for the development of new glioblastoma therapy, particularly to treat those patients resistant to TMZ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1β and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1β and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1β and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18's protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells., Competing Interests: The authors have declared that no competing interests exist.

Purpose: The changes in molecular structure and the physiological properties of a gamma-irradiated aloe-emodin were examined., Materials and Methods: Aloe-emodin was gamma-irradiated at doses ranging from 0 to 150 kGy, and the molecular structure was then analyzed using high-performance liquid chromatography (HPLC). AGS cells were cultured in RPMI medium and treated gamma irradiated aloe-emodin. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis efficiency was investigated by cell cycle arrest, cell morphology, and signaling pathway. The structure of new radiolytic peak was identified by the hydrogen-nuclear magnetic resonance ( 1 H NMR)., Results: HPLC results showed that gamma irradiation induced new radiolytic peaks that were distinguishable from the aloe-emodin standard, and the area of new peaks was increased as the radiation dose increased. Gamma-irradiated aloe-emodin treatment significantly increased the cytotoxicity in AGS tumor cells. We also found that 150 kGy aloe-emodin increased the expression of Bax, cytosolic cytochrome c, PARP cleavage, and the activation of caspases-8, -9, -3, Bid, and Bcl-2. Treatment of 150 kGy aloe-emodin induced ROS production, DNA fragmentation, alterations of cell morphology, and the migration in AGS cells. Gamma-irradiated aloe-emodin induced an increase of sub-G1 phase and depolarization of mitochondrial membrane potential in AGS cells. We also confirmed that fractionated AEF1 (new radiolytic peak) induce the cell death, migration, an increase of sub-G1 phase and cytochrome c in a ROS-dependent manner., Conclusions: The radiolysis product (AEF1) of aloe-emodin transformed by gamma-irradiation strongly induced apoptotic cell death in AGS cells, indicating AEF1 is a potential candidate drug for use in anti-cancer drug.

Pyroptosis is a form of inflammatory programmed cell death activated by caspase-1 and caspase-4/5/11, and involves in the pathogenesis of infectious diseases and nervous system diseases. Pyroptosis is mediated by canonical inflammasome pathway and non-canonical inflammasome pathway. The canonical inflammasome pathway is activated in stroke and aggravates brain injury. Inhibition of inflammasome, caspase-1, IL-1β and IL-18 ameliorates brain injury. These studies indicate that canonical inflammasome pathway contributes to post-stroke brain injury, therefore, pyroptosis has become a potential therapeutic target for preventing excessive cell death during stroke. We reviewed the relationship between pyroptosis and stroke to provide some perspectives on future researches in this field.

Understanding the key regulators which impact the innate immune response during initial phases of tissue injury, can advance the use of therapeutic approaches which aim at attenuating inflammation and organ damage. Recognition of microbial components by TLRs, initiates the transcription of innate immune signal pathways, that induce the expression of key inflammatory mediators: cytokines, chemokines and adhesion molecules. Beside regulating apoptotic cell death, recent studies have revealed distinct roles for caspases in the optimal production of inflammatory cytokines and host defense against injurious infections. Whether caspases can play an immune regulatory role in vivo has not been sufficiently investigated. This study aims to explore whether the pan caspase inhibitor z-VAD-fmk can control inflammation and cytokine production subsequent to challenging the innate immunity of the exocrine secretory tissues in vivo. Submandibular glands (SMGs) of the C57BL/6 mice were challenged with the TLR3 stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Results obtained from the current study provide evidence that caspases can control immune responses downstream of TLR3 ligation. The present work proposes a novel mechanism that can prevent overactivation of the innate immunity, which typically leads to fatal immune disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Regulation of the amyloid precursor protein (APP) processing by α- and β-secretases is of special interest to Alzheimer's disease (AD), as these proteases prevent or mediate amyloid beta formation, respectively. Neuroinflammation is also implicated in AD. Our data demonstrate that the endogenous mediator of inflammation prostaglandin J2 (PGJ2) promotes full-length APP (FL-APP) processing by α- and β-secretases. The decrease in FL-APP was independent of proteasomal, lysosomal, calpain, caspase, and γ-secretase activities. Moreover, PGJ2-treatment promoted cleavage of secreted APP, specifically sAPPα and sAPPβ, generated by α and β-secretase, respectively. Notably, PGJ2-treatment induced caspase-dependent cleavage of sAPPβ. Mechanistically, PGJ2-treatment selectively diminished mature (O- and N-glycosylated) but not immature (N-glycosylated only) FL-APP. PGJ2-treatment also increased the overall levels of protein O-GlcNAcylation, which occurs within the nucleocytoplasmic compartment. It is known that APP undergoes O-GlcNAcylation and that the latter protects proteins from proteasomal degradation. Our results suggest that by increasing protein O-GlcNAcylation levels, PGJ2 renders mature APP less prone to proteasomal degradation, thus shunting APP toward processing by α- and β-secretases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

Human induced pluripotent stem cell (iPSC) lines are a promising model for the early phase of human embryonic development. Here, their contribution to the still incompletely understood pathogenesis of congenital virus infections was evaluated. The infection of iPSC lines with miscarriage-associated coxsackievirus B3 (CVB3) and measles virus (MV) was compared to the efficient teratogen rubella virus (RV). While CVB3 and MV were found to be cytopathogenic on iPSC lines, RV replicated without impairment of iPSC colony morphology and integrity. This so far outstanding course of infection enabled maintenance of RV-infected iPSC cultures over several passages and their subsequent differentiation to ectoderm, endoderm, and mesoderm. A modification of the metabolic profile of infected iPSC lines was the only common aspect for all three viruses. This study points toward two important aspects. First, iPSC lines represent a suitable cell culture model for early embryonic virus infection. Second, metabolic activity represents an important means for evaluation of pathogen-associated alterations in iPSC lines.

Pyroptosis is a lytic type of programmed cell death that was traditionally associated with the involvement of inflammatory caspases, such as caspase-1. These inflammatory caspases are activated within multi-protein complexes called inflammasomes that are assembled in response to invading pathogens and/or danger signals. Pyroptotic cell death was suggested to evolve via the formation of pores in the plasma membrane, but the exact mechanism underlying the formation of these pores remained unclear. Recently, gasdermin D, a member of the gasdermin protein family was identified as a caspase substrate and essential effector of pyroptosis, being identified as the protagonist of membrane pore formation. Gasdermins have emerged as a family of new class of cell death inducers, but many questions remain unanswered. Here, we present an overview of recent work being done in the area of programmed cell death and the latest evidence regarding the role and participation of gasdermin D as an effector of pyroptosis., (Copyright © 2017 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Acute lung injury is a leading cause of death in bacterial sepsis due to the wholesale destruction of the lung endothelial barrier, which results in protein-rich lung edema, influx of proinflammatory leukocytes, and intractable hypoxemia. Pyroptosis is a form of programmed lytic cell death that is triggered by inflammatory caspases, but little is known about its role in EC death and acute lung injury. Here, we show that systemic exposure to the bacterial endotoxin lipopolysaccharide (LPS) causes severe endothelial pyroptosis that is mediated by the inflammatory caspases, human caspases 4/5 in human ECs, or the murine homolog caspase-11 in mice in vivo. In caspase-11-deficient mice, BM transplantation with WT hematopoietic cells did not abrogate endotoxemia-induced acute lung injury, indicating a central role for nonhematopoietic caspase-11 in endotoxemia. Additionally, conditional deletion of caspase-11 in ECs reduced endotoxemia-induced lung edema, neutrophil accumulation, and death. These results establish the requisite role of endothelial pyroptosis in endotoxemic tissue injury and suggest that endothelial inflammatory caspases are an important therapeutic target for acute lung injury.

Phagocytosis-induced cell death (PICD) is diminished in cord blood monocytes (CBMO) as compared to cells from adults (PBMO) due to differences in the CD95-pathway. This may support a prolonged pro-inflammatory response with sequels of sustained inflammation as seen in neonatal sepsis. Here we hypothesized that TNF-α mediated induction of apoptosis is impaired in CBMO due to differences in the TNFR1-dependent internalization. Monocytes were infected with Escherichia coli-GFP (E. coli-GFP). Monocyte phenotype, phagocytic activity, induction of apoptosis, and TNF-α/TNF-receptor (TNFR) -expression were analysed. In the course of infection TNF-α-secretion of CBMO was reduced to 40% as compared to PBMO (p<0.05). Neutralization of TNF-α by an αTNF-α antibody reduced apoptotic PICD in PBMO four-fold (p < 0.05 vs. infection with E. coli). PICD in CBMO was reduced 5-fold compared to PBMO and showed less responsiveness to αTNF-α antibody. CBMO expressed less pro-apoptotic TNFR1, which, after administration of TNF-α or infection with E. coli was internalized to a lesser extent. With similar phagocytic capacity, reduced TNFR1 internalization in CBMO was accompanied by lower activation of caspase-8 (p < 0.05 vs. PBMO). Stronger caspase-8 activation in PBMO caused more activation of effector caspase-3 and apoptosis (all p < 0.05 vs. PBMO). Our results demonstrate that TNFR1 internalization is critical in mediating PICD in monocytes after infection with E.coli and is reduced in CBMO.

Centchroman (CC) or Ormeloxifene has been shown to induce apoptosis and cell cycle arrest in various types of cancer cells. This has, however, not been addressed for endometrial cancer cells where its (CC) mechanism of action remains unclear. This study focuses on the basis of antineoplasticity of CC by blocking the targets involved in the cell cycle, survival and apoptosis in endometrial cancer cells. Ishikawa Human Endometrial Cancer Cells were cultured under estrogen deprived medium, exposed to CC and analyzed for proliferation and apoptosis. Additionally, we also analyzed oxidative stress induced by CC. Cell viability studies confirmed the IC 50 of CC in Ishikawa cells to be 20 µM after 48 h treatment. CC arrests the cells in G0/G1 phase through cyclin D1 and cyclin E mediated pathways. Phosphatidylserine externalization, nuclear morphology changes, DNA fragmentation, PARP cleavage, and alteration of Bcl-2 family protein expression clearly suggest ongoing apoptosis in the CC treated cells. Activation of caspase 3 & 9, up-regulation of AIF and inhibition of apoptosis by z-VAD-fmk clearly explains the participation of the intrinsic pathway of programmed cell death. Further, the increase of ROS, loss of MMP, inhibition of antioxidant (MnSOD, Cu/Zn-SOD and GST) and inhibition of apoptosis with L-NAC suggests CC induced oxidative stress leading to apoptosis via mitochondria mediated pathway. Therefore, CC could be a potential therapeutic agent for the treatment of Endometrial Cancer adjunct to its utility as a contraceptive and an anti-breast cancer agent.

Stroke is one of the leading causes of death and disability worldwide. Inflammation plays a key role across the time course of stroke, from onset to the post-injury reparative phase days to months later. Several regulatory molecules are implicated in inflammation, but the most established inflammatory mediator of acute brain injury is the cytokine interleukin-1. Interleukin-1 is regulated by large, macromolecular complexes called inflammasomes, which play a central role in cytokine release and cell death. In this review we highlight recent advances in inflammasome research and propose key roles for inflammasome components in the progression of stroke damage., (© 2016 International Society of Neuropathology.)

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor clinical outcome. Recent studies revealed the importance of B-cell receptor (BCR) signaling in maintaining MCL survival. However, it remains unclear which role MALT1, an essential component of the CARD11-BCL10-MALT1 complex that links BCR signaling to the NF-κB pathway, plays in the biology of MCL. Here we show that a subset of MCLs is addicted to MALT1, as its inhibition by either RNA or pharmacologic interference induced cytotoxicity both in vitro and in vivo. Gene expression profiling following MALT1 inhibition demonstrated that MALT1 controls an MYC-driven gene expression network predominantly through increasing MYC protein stability. Thus, our analyses identify a previously unappreciated regulatory mechanism of MYC expression. Investigating primary mouse splenocytes, we could demonstrate that MALT1-induced MYC regulation is not restricted to MCL, but represents a common mechanism. MYC itself is pivotal for MCL survival because its downregulation and pharmacologic inhibition induced cytotoxicity in all MCL models. Collectively, these results provide a strong mechanistic rationale to investigate the therapeutic efficacy of targeting the MALT1-MYC axis in MCL patients., (© 2017 by The American Society of Hematology.)

Hepatocellular carcinoma is a lethal cancer with high recurrence ratio and lacks effective therapeutics. In the past few years, it has been reported that increased intake of vegetables and fruits could reduce the cancer incidence, which suggests dietary agents might possess anticancer effects. Eriocitrin is a flavonoid isolated from lemon, which is known as a strong antioxidant agent. We here for the first time demonstrated that eriocitrin could inhibit the proliferation of hepatocellular carcinoma cell lines by arresting cell cycle in S phase through up-regulation of p53, cyclin A, cyclin D3 and CDK6. Furthermore, we found that eriocitrin could trigger apoptosis by activating mitochondria-involved intrinsic signaling pathway. Thus, eriocitrin might be regarded as a potential chemopreventive natural product to inhibit the early malignant transformation of hepatocellular carcinoma.

Prostate cancer is the most frequently diagnosed malignancy in men and the second highest contributor of male cancer mortality. The crude extract of Euphorbia formosana (CEEF) has been used for treatment of different diseases but the cytotoxic effects of CEEF on human cancer cells have not been reported. The purpose of the present experiments was to determine effects of CEEF on cell cycle distribution and induction of apoptosis in DU145 human prostate cancer cells in vitro. Contrast-phase microscope was used for examining cell morphological changes. Flow cytometric assays were used for cell viability, cell cycle, apoptosis, reactive oxygen species, and Ca 2+ production and mitochondria membrane potential (ΔΨ m ). Western blotting was used for examining protein expression of cell cycle and apoptosis associated proteins. Real-time PCR was used for examining mRNA levels of caspase-3, -8, and -9, AIF, and Endo G. Confocal laser microscope was used to examine the translocation of AIF, Endo G, and cytochrome in DU145 cells after CEEF exposure. CEEF-induced cell morphological changes, decreased the percentage of viable cells, and induced S phase arrest and apoptosis in DU145 cells. Furthermore, CEEF promoted RAS and Ca 2+ production and reduced ΔΨ m levels. Real-time QPCR confirmed that CEEF promoted the mRNA expression of caspase-3 and -9, AIF and Endo G and we found that AIF and Endo G and cytochrome c were released from mitochondria. Taken together, CEEF-induced cytotoxic effects via ROS production, induced S phase arrest and induction of apoptosis through caspase-dependent and independent and mitochondria-dependent pathways in DU245 cancer cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1600-1611, 2016., (© 2015 Wiley Periodicals, Inc.)

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) - a novel sphingolipid-like marine compound - was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

This review aims to highlight the proteases required for regulated cell death mechanisms in animals and plants. The aim is to be incisive, and not inclusive of all the animal proteases that have been implicated in various publications. The review also aims to focus on instances when several publications from disparate groups have demonstrated the involvement of an animal protease, and also when there is substantial biochemical, mechanistic and genetic evidence. In doing so, the literature can be culled to a handful of proteases, covering most of the known regulated cell death mechanisms: apoptosis, regulated necrosis, necroptosis, pyroptosis and NETosis in animals. In plants, the literature is younger and not as extensive as for mammals, although the molecular drivers of vacuolar death, necrosis and the hypersensitive response in plants are becoming clearer. Each of these death mechanisms has at least one proteolytic component that plays a major role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes. Some similarities are found among animal and plant cell death proteases but, overall, the pathways that they govern are kingdom-specific with very little overlap., (© 2015 FEBS.)

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in preclinical animal models, and in a phase I clinical trial in patients with advanced cancers without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting endoplasmic reticulum stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival, and angiogenesis. To enhance cancer-selective expression and targeted anticancer activity of mda-7/IL-24, we created a tropism-modified cancer terminator virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24 We now show that Ad.5/3-CTV induces profound neuroblastoma antiproliferative activity and apoptosis in a caspase-3/9-independent manner, both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis-inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV-induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ-H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small-molecule inhibitors that attenuated PARP cleavage by inhibiting γ-H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV-infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24-induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM, and γ-H2AX. Cancer Res; 76(12); 3572-82. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: The modus operandi for an anti-cancer drug must allow for an efficient discrimination system between tumorigenic and non-tumorigenic cells. Targeting ER stress and mitochondrial function in cancer cells appears to be a suitable option, as these processes are dysregulated in tumor cells. AECHL-1, a novel triterpenoid, exhibits potent anticancer activity against an array of cancer cell lines however, its mechanism of action remains elusive., Methods: Molecular targets of AECHL-1 were investigated using breast adenocarcinoma cells MCF-7, MDA-MB-231 and mammary epithelial cell line MCF 10A in vitro and xenograft tumors in SCID mice in vivo. Western blotting, flow cytometry, and immunohistochemical studies were employed to delineate the molecular pathways., Results: AECHL-1 caused a transient elevation of ER stress proteins along with a prolonged phosphorylation of eIF2α in breast cancer cells. This was accompanied by a simultaneous release of calcium from ER stores and subsequent mitochondrial accumulation. These effects could be reversed by using ER stress inhibitors. AECHL-1 brings about mitochondria mediated, caspase independent cell death via AIF in MCF-7 cells; MDA-MB-231 succumbed to caspase dependent extrinsic pathway. Xenograft studies closely echoed our in vitro results. AECHL-1 did not alter cellular and molecular parameters in MCF 10A., Conclusion: These findings reveal that, AECHL-1 targets the Achilles Heel of cancer cell, namely dysfunctional ER and mitochondria while being non toxic to normal parenchyma and can thus be further explored as a potential chemotherapeutic intervention., General Significance: Aggravation of ER stress by AECHL-1 uncovers a novel pathway for selective elimination of cancer cells., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor onset and progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors. However, the role of Karyopherin α2 (KPNA2) in human tongue squamous cell carcinoma (TSCC) remains unknown. We assessed the proliferation, apoptosis and migration of TSCC CAL-27 cells using wound healing, Transwell and MTT assays, western blotting, electron microscopy and acridine orange/ethidium bromide staining following knockdown of KPNA2. The results revealed the antiproliferative, proapoptotic and anti-migratory effects of KPNA2 silencing on the TSCC CAL-27 cells. Moreover, the knockdown of KPNA2 proved to be accompanied by the upregulation of active caspase-3, cytochrome c, Bax, Bad and decreased expression of Bcl-2, p-Bad and XIAP. KPNA2 activated the caspase-dependent pathway in the CAL-27 cells with upregulation of p53, p21Cip1/Waf1 and p16INK4a. Thus, the present study demonstrated that p53/p21Cip1/Waf1/p16INK4a may be an important pathway involved in the function of KPNA2 in TSCC CAL-27 cells.

The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)-κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)-expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression., (© International & American Associations for Dental Research 2015.)

Background & Aims: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation., Methods: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies., Results: Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV., Conclusion: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Some forms of regulated cell death, such as apoptosis, are precipitated by the activation of cysteine proteases of the caspase family, including caspase 8, 9, and 3. Other caspases, such as caspase 1 and 4, are well known for their pro-inflammatory functions but regulate cell death in a limited number of pathophysiological settings. Accumulating evidence suggests that the most conserved function of mammalian caspases is not to control cell death sensu stricto, but to regulate inflammatory and immune reactions to dying cells and infectious challenges. Here, we review the molecular and cellular mechanisms though which mammalian caspases connect cell-death signaling to the maintenance of organismal homeostasis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The paracaspase MALT1 has a central role in the activation of lymphocytes and other immune cells including myeloid cells, mast cells and NK cells. MALT1 activity is required not only for the immune response, but also for the development of natural Treg cells that keep the immune response in check. Exaggerated MALT1 activity has been associated with the development of lymphoid malignancies, and recently developed MALT1 inhibitors show promising anti-tumor effects in xenograft models of diffuse large B cell lymphoma. In this review, we provide an overview of the present understanding of MALT1's function, and discuss possibilities for its therapeutic targeting based on recently developed inhibitors and animal models.