Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by Smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/ DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with downregulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

The radiosensitizing activity of the SMAC-mimetic, Debio 1143, is TNFα-mediated in head and neck squamous cell carcinoma.

BACKGROUND AND PURPOSE: Second mitochondria-derived activator of caspase (SMAC)-mimetics are a new class of targeted drugs that specifically induce apoptotic cancer cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. MATERIAL AND METHODS: The present study was designed to investigate the radiosensitizing effect and optimal sequence of administration of the novel SMAC-mimetic Debio 1143 in vitro and in vivo. Apoptosis, alteration of DNA damage repair (DDR), and tumor necrosis factor-alpha (TNF-α) signaling were examined. RESULTS: In vitro, Debio 1143 displayed anti-proliferative activity and enhanced intrinsic radiation sensitivity in 5/6 head and neck squamous cell carcinoma (HNSCC) cell lines in a synergistic manner. In vivo, Debio 1143 dose-dependently radio-sensitized FaDu and SQ20B xenografts, resulting in complete tumor regression in 8/10 FaDu-xenografted mice at the high dose level. At the molecular level, Debio 1143 combined with radiotherapy (RT) induced enhancement of caspase-3 activity, increase in Annexin V-positive cells and karyopyknosis, and increase in TNF-α mRNA levels. Finally, in a neutralization experiment using a TNF-α-blocking antibody and a caspase inhibitor, it was shown that the radiosensitizing effect of Debio 1143 is mediated by caspases and TNF-α. CONCLUSIONS: These results demonstrate that the novel SMAC-mimetic Debio 1143 is a radiosensitizing agent that is worthy of further investigation in clinical trials in combination with radiotherapy.

SMAC multi-hop mesh routing protocol using IEEE 802.15.4

This paper discusses some aspects related to Wireless Sensor Networks over the IEEE 802.15.4 standard, and proposes, for the very first time, a mesh network topology with geographic routing integrated to the open Freescale protocol (SMAC - Simple Medium Access Control). For this is proposed the SMAC routing protocol. Before this work the SMAC protocol was suitable to perform one hop communications only. However, with the developed mechanisms, it is possible to use multi-hop communication. Performance results from the implemented protocol are presented and analyzed in order to define important requirements for wireless sensor networks, such as robustness, self-healing property and low latency. (c) 2011 Elsevier Ltd. All rights reserved.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella

The X‐linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti‐apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase‐mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP‐mediated immune response by inducing the BID‐dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain‐dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Revision of Simulation Model of Automobile Collisions (SMAC) computer program: investigation of new integration algorithm. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

SMAC Atmopsheric Correction

Resources created at the University of Southampton for the module Remote Sensing for Earth Observation

Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation.

Interleukin-1β (IL-1β) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1β activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1β that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1β by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1β and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis.

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.

A mechanistic view of mitochondrial death decision pores

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells

Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway.

Survival of Shiga toxin-producing Escherichia coli O157:H7 in Minas frescal cheese

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains (isolated by cattle’s faeces and a reference strain, EDL933), were inoculated into pasteurized milk (102 and 103 cells.mL–1) to prepare the Minas frescal cheese. As control was used uninfected milk. Physicochemical and microbiological analyses were performed to milk and elaborated cheese. The O157:H7 strains were quantified in the stages of cheese processing and during 0, 2, 4, 5, 7, 10 and 15 storage days at 8 °C onto Sorbitol MacConkey Agar supplemented with potassium tellurite and cefixime (CT-SMAC). O157:H7 was not present in the pasteurised milk prior to the artificial inoculation. At the end of the processing the cheese had 10 to 100 times more STEC O157:H7 than the initial inoculum. During the storage, the Minas frescal cheese exhibited the largest population increase on the 4th and 5th day when inoculated with 102 and 103 cells.mL–1, respectively. Additionally, viable cells were found up to the 10th and 15th day, according to the amount of initial inoculum. This number of cells is able to cause infection in humans, and therefore, Minas frescal cheese, even when stored under refrigeration, is a potential vehicle of disease caused by STEC O157:H7.

Conception, synthèse et diversification de l'azaGly-Pro : un mime de tour beta, outil d'étude structure-activité : application au développement d'un nouvel agent tocolytique

Les accouchements prématurés constituent un problème médical majeur en constante augmentation et ce, malgré tous les efforts mis en œuvre afin de contrer le déclenchement des contractions avant terme. Cette thèse relate du ''design'' rationnel d'un nouvel agent thérapeutique (i.e., tocolytique) qui serait capable de 1) arrêter les contractions, et 2) prolonger la gestation. Pour ce faire, une nouvelle cible, la prostaglandine F2α et son récepteur ont été sélectionnés et le peptidomimétisme a été choisi afin de résoudre cette problématique. L'introduction contient un historique rapide de la conception à la synthèse (''drug design'') du peptide parent, le PDC113, premier peptide a avoir démontré des aptitudes tocolytiques suffisantes pour faire du peptidomimétisme. La deuxième partie de l'introduction présente les concepts du peptidomimétisme appliqués au PDC113 qui ont permis d'accéder au PDC113.824, inhibiteur allostérique du récepteur de la prostaglandine F2α, et explique comment ce mime nous a permis d'élucider les mécanismes de signalisation intracellulaire impliqués dans la contraction musculaire lisse. Cette thèse présente la conception, la synthèse et l'étude structure-activité de mimes de repliement de tour β au sein du mime peptidique original (PDC113.824) dans lequel nous avons remplacé l'azabicycloalkane central (l'indolizidin-2-one) par une série d'autres azabicycloalcanes connus et des acides aza-aminés dont nous avons élaboré la synthèse. Dans un premier temps, une nouvelle stratégie de synthèse en solution de l'aza-glycyl-proline à partir de la diphényle hydrazone et du chloroformate de p-nitrophényle a été réalisée. Cette stratégie a permis d'éliminer les réactions secondaires de cyclisation intramoléculaires communément obtenues lors de l'introduction d'acides aza-aminés avec les protections traditionnelles de type carbamate en présence de phosgène, mais aussi de faciliter l'accès en une étape à des dérivés peptidiques du type aza-glycyle. L'élongation de l'aza-glycyl-proline en solution nous a permis d'accéder à un nouveau mime tetrapeptidique du Smac, un activateur potentiel de l'apoptose au sein de cellules cancéreuses. Par la suite, nous avons développé une stratégie de diversification sélective de l'azote α du résidu azaglycine en utilisant différents types d'halogénures d'alkyle en présence de tert-butoxyde de potassium. Afin de valider le protocole d'alkylation de l'aza-dipeptide, différents halogénures d'alkyle ont été testés. Nous avons également démontré l'utilité des aza-dipeptides résultants en tant que ''building block'' afin d'accéder à une variété d'azapeptides. En effet, l'aza-dipeptide a été déprotégée sélectivement soit en N-terminal soit en C-terminal, respectivement. D'autre part, la libération de l'amine de l'ester méthylique de l'aza-alkylglycyl-proline a conduit à une catégorie de composés à potentiel thérapeutique, les azadicétopipérazines (aza-DKP) par cyclisation intramoléculaire. Enfin, notre intérêt quant au développement d'un nouvel agent tocolytique nous a amené à développer une nouvelle voie de synthèse en solution du PDC113.824 permettant ainsi d'élucider les voies de signalisation intracellulaires du récepteur de la prostaglandine F2α. Afin de valider l'importance de la stéréochimie et d'étudier la relation structure/ activité du mime, nous avons remplacé l'indolizidin-2-one (I2aa) centrale du PDC113.824 par une série d'autres azabicycloalcanes et azadipeptides. Les azabicycloalcanes D-I2aa, quinolizidinone, et indolizidin-9-one ont été synthétisés et incorporés au sein du dit peptide ne donnant aucune activité ni in vitro ni ex vivo, validant ainsi l'importance du tour β de type II' pour le maintien de l'activité biologique du PDC113.824. Finalement, l'insertion d'une série de dérivés aza(alkyl)glycyl-prolyles a mené à de nouveaux inhibiteurs allostériques du récepteur de la PGF2α, l'un contenant l'azaglycine et l'autre, l'azaphénylalanine. Cette thèse a ainsi contribué, grâce à la conception et l'application de nouvelles méthodes de synthèse d'aza-peptides, au développement de nouveaux composés à potentiel thérapeutique afin d'inhiber le travail prématuré.

Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) , a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.