Selective Caspase Inhibitors Research Articles

Systemic inflammation exacerbates developmental neurotoxicity induced by sevoflurane in neonatal rats

BackgroundGeneral anaesthesia in the neonatal period has detrimental effects on the developing mammalian brain. The impact of underlying inflammation on anaesthesia-induced developmental neurotoxicity remains largely unknown. MethodsPostnatal day 7 (PND7) rats were randomly assigned to receive sevoflurane (3 vol% for 3 h) or carrier gas 12 h after bacterial lipopolysaccharide (LPS; 1 μg g−1) or vehicle injection. Pharmacological inhibition of caspase-1 by Vx-765 (two doses of 50 μg g−1 body weight) was used to investigate mechanistic pathways of neuronal injury. Histomorphological injury and molecular changes were quantified 2 h after the end of anaesthesia. Long-term functional deficits were tested at 5–8 weeks of age using a battery of behavioural tests in the memory and anxiety domains. ResultsSevoflurane or LPS treatment increased activated caspase-3 and caspase-9 expression in the hippocampal subiculum and CA1, which was greater when sevoflurane was administered in the setting of LPS-induced inflammation. Neuronal injury induced by LPS+sevoflurane treatment resulted in sex-specific behavioural outcomes when rats were tested at 5–8 weeks of age, including learning and memory deficits in males and heightened anxiety-related behaviour in females. Hippocampal caspase-1 and NLRP1 (NLR family pyrin domain containing 1), but not NLRP3, were upregulated by LPS or LPS+sevoflurane treatment, along with related proinflammatory cytokines, interleukin (IL)-1β, and IL-18. Pretreatment with Vx-765, a selective caspase-1 inhibitor, led to reduced IL-1β in LPS and LPS+sevoflurane groups. Caspase-1 inhibition by Vx-765 significantly decreased activated caspase-3 and caspase-9 immunoreactivity in the subiculum. ConclusionsSystemic inflammation promotes developmental neurotoxicity by worsening anaesthesia-induced neuronal damage with sex-specific behavioural outcomes. This highlights the importance of studying anaesthesia-induced neurotoxicity in more clinically relevant settings.

VX-765 ameliorates inflammation and extracellular matrix accumulation by inhibiting the NOX1/ROS/NF-κB pathway in diabetic nephropathy.

This study explores the potential role of a highly selective caspase-1 inhibitor, VX-765, on extracellular matrix (ECM) accumulation and inflammation in diabetic nephropathy (DN) and the underlying mechanisms. DN rats, induced via high-fat diet/streptozotocin, were used to assess the effects of VX-765. Parallel experiments were carried out on rat mesangial cell line HBZY-1 exposed to high glucose (HG) to reveal the molecular mechanism of VX-765 in preventing DN. Survival analysis, biochemical parameters and renal oxidative stress of rats were observed, and Western blotting and immunofluorescence were evaluated. In vitro, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX)1 silencing by RNA interference and quantitative real-time PCR (qPCR) assays were conducted in HBZY-1 cells exposed to HG levels. In vivo, VX-765 significantly reduced the increase in urine albumin excretion and ECM accumulation. The phosphorylation of nuclear factor kappa-B (NF-κB) and the expression of pro-inflammatory cytokines IL-1β, IL-6 and tumor necrosis factor (TNF)-α were significantly down-regulated. Furthermore, the generation of reactive oxygen species (ROS), phosphorylation of NF-κB and the expression of the NOX1 gene or protein were significantly decreased in HBZY-1 with VX-765 (5 μM) treatment in vitro. Our results demonstrated that VX-765 exerts favourable effects on DN via the simultaneous alleviation of systemic metabolic syndrome and down-regulating the renal NOX1/ROS/NF-κB pathway, suggesting that it has therapeutic potential for DN.

Back cover story: Eur. J. Immunol. 5'21

European Journal of ImmunologyVolume 51, Issue 5 Back CoverFree Access Back cover story: Eur. J. Immunol. 5'21 First published: 05 May 2021 https://doi.org/10.1002/eji.202170058AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Our back cover features H&E stainings from the lungs of LPS-induced sepsis mice pretreated with Nitrosonisoldipine (NTS), a photodegradation product of nisoldipine. The mice displayed the alleviated lung injury. The image is taken from Chen et al. (pp. 1234–1245), where the authors show that pretreatment with NTS confers protection against sepsis in mice. NTS blocks both the non-canonical and canonical pyroptotic pathways but does not affect apoptosis. Mechanistically, as a selective caspase inhibitor, NTS could directly and irreversibly inhibit the enzyme activities of inflammatory caspases (i.e. caspase-1/-4/-11). Furthermore, NTS is safe for intraperitoneally injection in mice, suggesting that it may be used as a prototype to develop drugs for sepsis treatments. Volume51, Issue5May 2021 RelatedInformation

Caspases Mediate Retinoic Acid–Induced Degradation of the Acute Promyelocytic Leukemia PML/RARα Fusion Protein

All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARα product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARα and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARα (U937/PR9) and in blasts from APL patients, the PML/RARα fusion protein is cleaved by a caspase 3–like activity induced by RA treatment. In fact, a caspase 3–like activity is detectable in PML/RARα expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARα deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the α-helix region of the PML component of the fusion protein. The extent of PML/RARα cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARα is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARα.

Cannabinoid 1 Receptor Antagonists Play a Neuroprotective Role in Chronic Alcoholic Hippocampal Injury Related to Pyroptosis Pathway.

Alcohol use disorders affect millions of people worldwide, and there is growing evidence that excessive alcohol intake causes severe damage to the brain of both humans and animals. Numerous studies on chronic alcohol exposure in animal models have identified that many functional impairments are associated with the hippocampus, which is a structure exhibiting substantial vulnerability to alcohol exposure. However, the precise mechanisms that lead to structural and functional impairments of the hippocampus are poorly understood. Herein, we report a novel cell death type, namely pyroptosis, which accounts for alcohol neurotoxicity in mice. For this study, we used an in vivo model to induce alcohol-related neurotoxicity in the hippocampus. Adult male C57BL/6 mice were treated with 95% alcohol vapor either alone or in combination with selective cannabinoid receptor antagonists or agonists, and VX765 (Belnacasan), which is a selective caspase-1 inhibitor. Alcohol-induced in vivo pyroptosis occurs because of an increase in the levels of pyroptotic proteins such as nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), caspase-1, gasdermin D (GSDMD), and amplified inflammatory response. Our results indicated that VX765 suppressed the expression of caspase-1 and inhibited the maturation of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Additionally, chronic alcohol intake created an imbalance in the endocannabinoid system and regulated 2 cannabinoid receptors (CB1R and CB2R) in the hippocampus. Specific antagonists of CB1R (AM251 and AM281) significantly ameliorated alcohol-induced pyroptosis signaling and inactivated the inflammatory response. Alcohol induces hippocampal pyroptosis, which leads to neurotoxicity, thereby indicating that pyroptosis may be an essential pathway involved in chronic alcohol-induced hippocampal neurotoxicity. Furthermore, cannabinoid receptors are regulated during this process, which suggests promising therapeutic strategies against alcohol-induced neurotoxicity through pharmacologic inhibition of CB1R.

From the Explored to the Unexplored: Computer-Tailored Drug Design Attempts in the Discovery of Selective Caspase Inhibitors.

The pathophysiological roles of caspases have made them attractive targets in the treatment and amelioration of neurologic diseases. In normal conditions, the expression of caspases is regulated in the brain, while at the onset of neurodegeneration, such as in Alzheimer's disease, they are typically overexpressed. Till date, several therapeutic efforts that include the use of small endogenous binders have been put forward to curtail dysfunctionalities that drive aberrant death in neuronal cells. Caspases are highly homologous, both in structure and in sequence, which leaves us with the question: is it possible to specifically and individually target caspases, while multiple therapeutic attempts to achieve selective targeting have failed! Based on antecedent events, the use of Computer-Aided Drug Design (CADD) methods has significantly contributed to the design of small molecule inhibitors, especially with selective target ability and reduced off-target therapeutic effects. Interestingly, we found out that there still exists an enormous room for the integration of structure/ligand-based drug design techniques towards the development of highly specific reversible and irreversible caspase inhibitors. Therefore, in this review, we highlight drug discovery approaches that have been directed towards caspase inhibition in addition to an insightful focus on applicable CADD techniques for achieving selective targeting in caspase research.

AMPK Activation of Apoptotic Markers in Human Breast Cancer Cell Lines with Different p53 Backgrounds: MCF-7, MDA-MB-231 and T47D Cells.

Background:Downregulation of AMPK has been established as a major contributor to carcinogenesis in many types of human cancer. We sought to investigate the influence of activated AMPK on apoptotic markers in human breast cancer cells differing in their p53 status, as well as estrogen receptor status (MCF-7 (p53+ and ER+), MDA-MB-231 (p53 mutant and ER-) and T47D (p53 mutant and ER+)). Methods:We examined the effect of AICAR-activated AMPK on PARP cleavage, Bax redistribution, the involvement of intrinsic and extrinsic pathways of apoptosis using selective caspase inhibitors and cell cycle progression and p21 levels. Results:PARP cleavage occurred to a greater extent in MCF-7 and MDA-MB-231 cells, whereas Bax translocation was slower in MDA-MB-231 cells. Although there were quantitative differences in the effect of caspase inhibitors, it was clear that AMPK activation predominately affected the intrinsic pathway of apoptosis. Although, p21 was increased in all 3 cell types, there were quantitative and time differences. Apoptosis, as measured by fluorimetry, was increased in all three cell types. Conclusion:The impact of AMPK activation was cell type dependent resulting in differential activation of apoptotic markers, confirming that the genetic background of breast cancer may have an influence on the mode of action of AMPK. Thus, different anti-tumour mechanisms may be elicited depending on the cellular genotype.

Selective and Rapid Cell-Permeable Inhibitor of Human Caspase-3.

Individual roles and overlapping functionalities of 12 human caspases during apoptosis and other cellular processes remain poorly resolved primarily due to a lack of chemical tools. Here we present a new selective caspase-3 inhibitor, termed Ac-ATS010-KE, with rapid and irreversible binding kinetics. Relative to previously designed caspase-3-selective molecules that have tremendously abated inhibitory rates and thus limited use in biological settings, the improved kinetics of Ac-ATS010-KE permits its use in a cell-based capacity. We demonstrate that Ac-ATS010-KE prevents apoptosis with comparable efficacy to the general caspase inhibitor Ac-DEVD-KE and surprisingly does so without side-chain methylation. This observation is in contrast to the well-established peptide modification strategy typically employed for improving cellular permeability. Ac-ATS010-KE protects against extrinsic apoptosis, which demonstrates the utility of a thiophene carboxylate leaving group in biological settings, challenges the requisite neutralization of free carboxylic acids to improve cell permeability, and provides a tool-like compound to interrogate the role of caspase-3 in a variety of cellular processes.

Inhibitory Effects of Simvastatin on Oxidized Low-Density Lipoprotein-Induced Endoplasmic Reticulum Stress and Apoptosis in Vascular Endothelial Cells.

Background:Oxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells.Methods:Human umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P < 0.05 was considered statistically significant.Results:Exposure of HUVECs to ox-LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P < 0.05). Simvastatin (0.1, 0.5, and 2.5 μmol/L) led to a suppression of ox-LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P < 0.05, compared with control group). Ox-LDL significantly increased the expression of PERK (499.5%, P < 0.05) and phosphorylation of eIF2α (451.6%, P < 0.05), if both of which in the control groups were considered as 100%. Simvastatin treatment (0.1, 0.5, and 2.5 μmol/L) blunted ox-LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P < 0.05, compared with control group) and phosphorylation of eIF2α (407.8%, 339.1%, 187.5%, F = 11.430, all P < 0.05, compared with control group). In contrast, DEVD-CHO treatment had no significant effect on ox-LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment.Conclusions:This study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.

Inflammasome activation involved in early inflammation reaction after liver transplantation

Liver transplantation has emerged as a vital therapy for end-stage liver diseases. Acute −phase inflammation play an important role in liver graft injury.Recent studies have revealed that inflammasome are responsible for initiating inflammation in early stage of acute organ rejection in liver transplantation, however the underlying mechanism remains unclear. Here we explored to block inflammasome activation to see whether it can alleviate early inflammation reaction during rejection of allgrafts in a rat model and gain further insights into the mechanism of inhibiting inflammation in allografts. By using Ac-YVAD-CMK, a highly selective caspase-1 inhibitor, to inhibit inflammation reaction involved in allograft rejection in a rat model. Our results showed that the rejection activity index (RAI) of Ac-YVAD-CMK-treated allografts is significantly diminished in similar magnitude to that of isografts. Compared with isografts, the expression of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and IL-1β in allograft group increased significantly with the development of rejection, exhibiting apparent correlation. Expression of IFN-γ mRNA in untreated allografts was maximal on day 3 while in Ac-YVAD-CMK-treated allografts and isografts, IFN-γ mRNA levels remained low over the duration of the time course. ELISA results revealed serum elevation of IL-1β by day 7 after othotopic liver transplantation (OLT) in comparison with isografts. There were no statistically significant differences between isografts and Ac-YVAD-CMK-treated allografts. For the first time, our data reveal that inhibition of the inflammasome activation pathway attenuates inflammation reaction of hepatic transplant rejection.

The Highly Selective Caspase-1 Inhibitor VX-765 Provides Additive Protection Against Myocardial Infarction in Rat Hearts When Combined With a Platelet Inhibitor.

Use of ischemic postconditioning and other related cardioprotective interventions to treat patients with acute myocardial infarction (AMI) has failed to improve outcomes in clinical trials. Because P2Y12 inhibitors are themselves postconditioning mimetics, it has been postulated that the loading dose of platelet inhibitors routinely given to patients treated for AMI masks the anti-infarct effect of other intended cardioprotective interventions. To further improve outcomes of patients with AMI, an intervention must be able to provide additive protection in the presence of a P2Y12 platelet inhibitor. Previous studies reported an anti-infarct effect using a peptide inhibitor of the pro-inflammatory caspase-1 in animal models of AMI. Herein we tested whether a pharmacologic caspase-1 inhibitor can further limit infarct size in open-chest, anesthetized rats treated with a P2Y12 inhibitor. One hour occlusion of a coronary branch followed by 2 hours of reperfusion was used to simulate clinical AMI and reflow. One group of rats received an intravenous bolus of 16 mg/kg of the highly selective caspase-1 inhibitor VX-765 30 minutes prior to onset of ischemia. A second group received a 60 µg/kg intravenous bolus of the P2Y12 inhibitor cangrelor 10 minutes prior to reperfusion followed by 6 µg/kg/min continuous infusion. A third group received treatment with both inhibitors as above. Control animals received no treatment. Infarct size was measured by tetrazolium stain and volume of muscle at risk by fluorescent microspheres. In untreated hearts, 73.7% ± 4.1% of the ischemic zone infarcted. Treatment with either cangrelor or VX-765 alone reduced infarct size to 43.8% ± 2.4% and 39.6% ± 3.6% of the ischemic zone, respectively. Combining cangrelor and VX-765 was highly protective, resulting in only 14.0% ± 2.9% infarction. The ability of VX-765 to provide protection beyond that of a platelet inhibitor alone positions it as an attractive candidate therapy to further improve outcomes in today's patients with AMI.

Role of the apoptosis pathway in cryopreservation-induced cell death in mesenchymal stem cells derived from umbilical cord blood.

Cryopreservation of mesenchymal stem cells (MSCs) is important because of their commercial applications in the clinical sector. MSCs are vulnerable to cryopreservation-induced apoptosis due to activation of apoptosis-related proteins during thawing. But the relationship between cryopreservation and apoptosis is not well understood. MSCs derived from umbilical cord blood were cryopreserved using Me2SO as the cryoprotective agent, with or without pre-treatment with the general caspase inhibitor z-VAD-FMK, or with the more selective caspase inhibitors z-IETD-FMK, z-LEHD-FMK and z-DEVD-FMK. To evaluate the effect of the calcium-mediated pathway, cryopreserved MSCs were tested with and without a calpain inhibitor. FACS was used to measure cell viability, mitochondrial membrane potential, and cell cycle analysis. Processing of the pro-caspases-3, -8, -9, calpain and Bid were determined by Western blotting. Cryopreservation of MSCs resulted in characteristic apoptosis within 24 h after thawing. Results show that intrinsic, extrinsic, and calpain pathways are activated after cryopreserved MSCs are thawed. Compared to selective caspase inhibitors, a general caspase inhibitor blocked DNA degradation more effectively and also inhibited caspases-3 and -8 processing as well as Bid cleavage, showing the beneficial effect of reducing cryopreservation-induced apoptosis. Similarly, calpain inhibition reduced cryopreservation-induced apoptosis. These data indicate that caspase-mediated extrinsic and intrinsic pathways and the proteolytic calpain cascade were activated after cryopreservation using a standard cryopreservation protocol. This activation might play an important role in the process of cryopreservation-induced cell death. Furthermore, the inhibition of calpain activity and caspase-mediated pathways might improve preservation efficacy.

Cytoprotective effect of selective small-molecule caspase inhibitors against staurosporine-induced apoptosis.

Caspases are currently known as the central executioners of the apoptotic pathways. Inhibition of apoptosis and promotion of normal cell survival by caspase inhibitors would be a tremendous benefit for reducing the side effects of cancer therapy and for control of neurodegenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect. We completed the high-throughput screening of Bionet’s 37,500-compound library (Key Organics Limited, Camelford, Cornwall, UK) against caspase-1, -3, and -9 and successfully identified 43 initial hit compounds. The 43 hit compounds were further tested for cytoprotective activity against staurosporine-induced cell death in NIH3T3 cells. Nineteen compounds were found to have significant cytoprotective effects in cell viability assays. One of the compounds, RBC1023, was demonstrated to protect NIH3T3 cells from staurosporine-induced caspase-3 cleavage and activation. RBC1023 was also shown to protect against staurosporine-induced impairment of mitochondrial membrane potential. DNA microarray analysis demonstrated that staurosporine treatment induced broad global gene expression alterations, and RBC1023 co-treatment significantly restored these changes, especially of the genes that are related to cell growth and survival signaling such as Egr1, Cdc25c, cdkn3, Rhob, Nek2, and Taok1. Collectively, RBC1023 protects NIH3T3 cells against staurosporine-induced apoptosis via inhibiting caspase activity, restoring mitochondrial membrane potential, and possibly upregulating some cell survival-related gene expressions and pathways.

Chemical stimulation of the intracranial dura activates NALP3 inflammasome in trigeminal ganglia neurons

Inflammasomes are molecular platforms that upon activation by cellular infection or stress trigger the maturation of proinflammatory cytokines such as interleukin (IL)-1β to engage innate immune defenses. Increased production of IL-1β in pain and inflammation such as headache is well documented. However, limited evidence addresses the participation of inflammasomes in inflammatory pain. The present study used rat inflammatory dural stimulation-induced model of intracranial pain to assess whether headache-related pain can induce the activation of NACHT, LRR, and PYD-containing protein (NALP)-3 inflammasome pathway in the trigeminal ganglia (TG) and which cells express NALP3 inflammasome proteins and IL-1β. Chemical stimulation of the intracranial dura caused a total drug dose- and time-dependent induction of activated caspase-1 and mature IL-1β proteins. Application of a selective caspase-1 inhibitor diminished these effects. Immunohistochemistry revealed that both NALP3 inflammasome and IL-1β immunoreactivity were existed mainly in small to medium-sized C-type neurons and increased over time, with intense cytoplasmic staining after 3 days of dural inflammation. Overall, the present observation indicated that dural inflammation promoted assembly of the multiprotein NALP3 complex, activated caspase-1, and induced processing of IL-1β, which provides an indirect evidence of the participation of NALP3 inflammasome in the cascade of events involved in the genesis of headaches by promoting IL-1β maturation in the TG. This may contribute to strategies for headache control.

IL-1β processing in mechanical ventilation-induced inflammation is dependent on neutrophil factors rather than caspase-1.

PurposeMechanical ventilation can cause ventilator-induced lung injury, characterized by a sterile inflammatory response in the lungs resulting in tissue damage and respiratory failure. The cytokine interleukin-1β (IL-1β) is thought to play an important role in the pathogenesis of ventilator-induced lung injury. Cleavage of the inactive precursor pro-IL-1β to form bioactive IL-1β is mediated by several types of proteases, of which caspase-1, activated within the inflammasome, is the most important. Herein, we studied the roles of IL-1β, caspase-1 and neutrophil factors in the mechanical ventilation-induced inflammatory response in mice.MethodsUntreated wild-type mice, IL-1αβ knockout and caspase-1 knockout mice, pralnacasan (a selective caspase-1 inhibitor)-treated mice, anti-keratinocyte-derived chemokine (KC)-treated mice and cyclophosphamide-treated neutrophil-depleted wild-type mice were ventilated using clinically relevant ventilator settings (tidal volume 8 ml/kg). The lungs and plasma were collected to determine blood gas values, cytokine profiles and neutrophil influx.ResultsMechanical ventilation resulted in increased pulmonary concentrations of IL-1β and KC and increased pulmonary neutrophil influx compared with non-ventilated mice. Ventilated IL-1αβ knockout mice did not demonstrate this increase in cytokines. No significant differences were observed between wild-type and caspase-1-deficient or pralnacasan-treated mice. In contrast, in anti-KC antibody-treated mice and neutropenic mice, inflammatory parameters decreased in comparison with ventilated non-treated mice.ConclusionsOur results illustrate that IL-1 is indeed an important cytokine in the inflammatory cascade induced by mechanical ventilation. However, the inflammasome/caspase-1 appears not to be involved in IL-1β processing in this type of inflammatory response. The attenuated inflammatory response observed in ventilated anti-KC-treated and neutropenic mice suggests that IL-1β processing in mechanical ventilation-induced inflammation is mainly mediated by neutrophil factors.Electronic supplementary materialThe online version of this article (doi:10.1186/2197-425X-1-8) contains supplementary material, which is available to authorized users.

Prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim reduces the association of Bcl-2 with Bak or Bim, provoking Bak activation and mitochondrial apoptosis in nocodazole-treated Jurkat T cells

Treatment of Jurkat T cells with the microtubule-depolymerizing agent nocodazole (NOC) caused prometaphase arrest and apoptosis. NOC-induced mitochondrial apoptotic events including Bak activation, Δψm loss, cytochrome c release, and caspase cascade activation were blocked by Bcl-2 overexpression. However, mitotic arrest, Cdc25C activation, upregulation of cyclin B1 levels, Cdk1 activation, Bcl-2 phosphorylation at Thr-56 and Ser-70, and Bim phosphorylation were retained. The treatment of Jurkat T cells concomitantly with NOC and the G1/S-blocking agent hydroxyurea resulted in G1/S arrest and complete abrogation of all apoptotic events. The association of Bcl-2 with Bim or Bak declined after the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, whereas the association of Bcl-2 with Bax remained relatively constant. Although Bax was redistributed from the cytosol to the mitochondria, resulting in an increase in the mitochondrial level of Bax following NOC treatment, the subcellular localization of Bcl-2, Bim, Bak and apoptosis-inducing factor was confined to the mitochondrial fraction irrespective of NOC treatment. Experiments using selective caspase inhibitors showed that mitochondria-dependent activation of caspase-9 and -3 was crucial for NOC-induced apoptosis. NOC-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and mitochondria-dependent apoptotic events were significantly suppressed by a Cdk1 inhibitor roscovitine, but not by the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580. These results show that the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, which was mediated by Cdk1, could reduce the association of Bcl-2 with Bak or Bim to allow Bak activation and mitochondrial apoptotic events in Jurkat T cells exposed to NOC.

Amino acids derived benzoxazepines: Design, synthesis and antitumor activity

Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride (LAH) reduction, debenzylation, bimolecular nucleophilic substitution (SN2) reaction. The present study investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF-7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hallmarks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depolarization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could serve as a base for the development of a new group of effective breast cancer therapeutics.

Enhancement of long-term depression by soluble amyloid β protein in rat hippocampus is mediated by metabotropic glutamate receptor and involves activation of p38MAPK, STEP and caspase-3

It is reported that the amyloid-β protein (Aβ)-induced impairments in synaptic plasticity coincide with memory decline and dementia. Although Aβ-induced inhibition of hippocampal long-term potentiation has been intensively investigated, the underlying mechanism of Aβ-enhanced long-term depression (LTD) is not clear. Here, we report that acute exposure of rat hippocampal slices to soluble Aβ-enhanced LTD induced by weak low-frequency stimulation (wLFS; 1Hz for 3min, 180 pulses) in granule cells of the dentate gyrus. Application of LY341495 (a non-selective Group I/II metrabotropic glumate receptor (mGluR) antagonist) completely blocked Aβ-enhanced LTD, whereas D-AP5 (a not selective N-methyl-d-aspartate receptor (NMDAR) antagonist) had no effect on Aβ-enhanced LTD compared with controls. In addition, Aβ-enhanced LTD was occluded by pre-application of 3,5-dihydroxyphenylglycine, a Group1 mGluR (mGluR1/5) agonist, suggesting Aβ-enhanced LTD depends on mGluR1/5 but not NMDAR. We also report here that p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580 and postsynaptic protein tyrosine phosphatase inhibitors phenylarsine oxide and sodium orthovanadate prevented the facilitatory effect of Aβ on LTD. Application of striatal-enriched protein tyrosine phosphatase (STEP) activator MG132 facilitated induction of LTD by wLFS, but did not block following Aβ-enhanced LTD induced by another wLFS. On the other hand, Aβ-enhanced LTD blocked following MG132-LTD by wLFS, suggesting Aβ-enhanced hippocampal LTD involves STEP activation. Application of either non-selective caspase inhibitor Z-VAD-FMK or caspase-3 selective inhibitor Z-DEVD-FMK prevented Aβ-enhanced LTD. However, neither the tumor necrosis factor-α converting enzyme inhibitor TAPI-2 nor the mammalian target of rapamycin inhibitor rapamycin prevented the enhancement of Aβ on LTD. Therefore, we conclude that soluble Aβ enhances LTD in the hippocampal dentate gyrus region, and the facilitatory effect of Aβ on LTD involves mGluR1/5, p38MAPK, STEP and caspase-3 activation.

Basal caspase-3 activity promotes migration, invasion, and vasculogenic mimicry formation of melanoma cells

Melanoma is the least common but most serious form of skin cancer. The leading cause of death in melanoma patients is widespread metastasis caused by increased cell motility and a rich blood supply for tumor cells. A unique form of microcirculation called vasculogenic mimicry, which efficiently supplies blood to tumor cells, has been reported recently. Apoptosis-related protein performs a nonapoptotic function to promote migration and invasion of tumor cells. This study focuses on the nonapoptotic role of caspase-3 in melanoma and its effects on the migration, invasion, and vasculogenic mimicry formation of melanoma cells. Human melanoma samples were used to detect active caspase-3 expression and determine its relationship with clinicopathologic parameters. In addition, a human melanoma A375 cell line was used to determine the role of caspase-3 in migration and invasion using z-DEVD-fmk, a selective caspase-3 inhibitor, to inhibit caspase-3 activity. The findings suggest that active caspase-3 is expressed in nonapoptotic melanoma cells and is related to metastasis and vasculogenic mimicry formation in patients with melanoma. Low doses of caspase-3 inhibitor reduced caspase-3 activity without affecting cell apoptosis. Inhibition of caspase-3 activity using low-dose z-DEVD-fmk decreased the migration, invasion, and vasculogenic mimicry formation of melanoma cells in vitro. Similarly, downregulation of caspase-3 by specific small interfering RNA also inhibited the migratory, invasive, and tube-forming potential of melanoma cells. The caspase-3-mediated promotion of melanoma cell motility may be because of the cleavage of matrix metalloproteinase-2.

Antiproliferative Activity of Abietane Diterpenoids against Human Tumor Cells

In the present study, the cytotoxicity of 30 diterpenoids with an abietane or a halimane skeleton was determined against five human tumor cell lines (HL-60, U937, Molt-3, SK-MEL-1, and MCF-7). Diterpenoids containing an abietane skeleton including taxodone (1) and taxodione (2), as well as the semisynthetic derivatives 12, 14, 15, 17, and 22, were the most cytotoxic compounds for human leukemia cells. Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-x(L) did not confer resistance to abietane diterpene-induced cytotoxicity. Studies performed on HL-60 cells indicated that growth inhibition triggered by compounds 1, 12, 14, and 15 was caused by induction of apoptosis. This was prevented by the nonspecific caspase inhibitor Z-VAD-FMK and, in the case of compounds 14 and 15, reduced by the selective caspase-8 inhibitor Z-IETD-FMK. Cell death induced by these abietane diterpenes was found to be associated with the release of mitochondrial proteins, including cytochrome c, Smac/DIABLO, and AIF (apoptosis-inducing factor), accompanied by dissipation of the mitochondrial membrane potential (ΔΨ), and modulated by inhibition of extracellular signal-regulated kinases signaling and the p38 mitogen-activated protein kinase pathway.