Dietary compound α-asarone alleviates ER stress-mediated apoptosis in 7ß-hydroxycholesterol-challenged macrophages.

SCOPE: Prolonged endoplasmic reticulum (ER) stress has lost the function of protein folding capacity and the ER accumulation of unfolded proteins that eventually triggers apoptosis. Oxysterols are emerging as contributing factors in atherogenesis known to involve macrophage apoptosis. This study determined the inhibitory effect of α-asarone present in purple perilla, on 7ß-hydroxycholesterol-induced macrophage apoptosis, targeting against ER stress signaling pathway. METHODS AND RESULTS: J774A1 murine macrophages were exposed to 28 µM 7ß-hydroxycholesterol and treated with 1-10 µM α-asarone. Macrophage apoptosis and ER stress were examined by and α-Asarone blocked 7ß-hydroxycholesterol-induced DNA fragmentation and apoptosome formation. Immunoblotting showed that the oxysterol activated the ER transmembrane resident kinases of IRE1α, PERK and ATF4 and triggered caspase-12 signaling cascades, which was reversed by α-asarone. Additionally, 7ß-hydroxycholesterol activated TRAF2-ASK1-JNK1/2 complex following the IRE1α activation, and α-asarone blunted such IRE1α-mediated pathway. Real-time PCR and dual-luciferase reporter analyses revealed that α-asarone reduced transcriptional activation of ER stress-responsive genes including XBP1 and CHOP by 7ß-hydroxycholesterol. Finally, α-asarone disturbed oxysterol-elicited signaling of PERK and ATF4 responsible for CHOP induction. CONCLUSION: α-Asarone blocked 7ß-hydroxycholesterol-induced macrophage apoptosis through allaying ER stress-specific signaling involving caspase activation and CHOP induction. α-Asarone was an anti-atherosclerotic agent antagonizing ER stress-mediated macrophage apoptosis by 7ß-hydroxycholesterol.

Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress.

Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Next-generation sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5' and 3' tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity.

Enhanced survival of transplanted human adipose-derived stem cells by co-delivery with liposomal apoptosome inhibitor in fibrin gel matrix.

To improve the survival of transplanted human adipose-derived stem cells (ADSCs), a liposome preparation containing the apoptosome inhibitor, NS3694, was formulated and co-delivered with ADSCs in fibrin gel scaffolds. Liposomes provided enhanced effect on ADSC proliferation in vitro as compared to free drug. Exposure of ADSCs to liposomal NS3694 for 7 days did not affect the surface marker expression profile. NS3694 encapsulated in negatively charged liposomes composed of phosphatidylcholine, phosphatidylglycerol, and cholesterol was evaluated in vivo following subcutaneous transplantation in mice. Survival of ADSCs co-delivered with liposomal NS3694 was significantly higher than that of untreated ADSCs or ADSCs treated with free NS3694 or empty liposomes. An immunohistochemical analysis revealed a higher number of human nucleus-positive cells after treatment with liposomal NS3694 than following treatment with free NS3694. Similarly, liposomal NS3694 significantly enhanced survival of transplanted ADSCs in rabbits compared to other treatments. Taken together, our results indicate the potential of liposomal NS3694 co-delivered with ADSCs using fibrin gel systems as an in vivo-survival enhancer.

Synthesis of enantiomerically pure perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives exhibiting potent activity as apoptosis inhibitors.

Apoptosis is the process of programmed cell death and plays a fundamental role in several human diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compounds 1 and 2 showed a potent in vitro and in cellular extracts antiapoptotic activity. In view that the chiral discrimination has been an issue in the development and use of pharmaceutical drugs, the present contribution reports the synthesis of enantiopure peptidomimetics 1 and 2. The biological evaluation of these enantiomers as apoptosis inhibitors is also reported.

Effectors of alcohol-induced cell killing in Drosophila.

Heavy alcohol consumption provokes an array of degenerative pathologies but the signals that couple alcohol exposure to regulated forms of cell death are poorly understood. Using Drosophila as a model, we genetically establish that the severity of ethanol challenge dictates the type of death that occurs. In contrast to responses seen under acute exposure, cytotoxic responses to milder challenges required gene encoding components of the apoptosome, Dronc and Dark. We conducted a genome-wide RNAi screen to capture targets that specifically mediate ethanol-induced cell death. One effector, Drat, encodes a novel protein that contains an ADH domain but lacks essential residues in the catalytic site. In cultured cells and neurons in vivo, depletion of Drat conferred protection from alcohol-induced apoptosis. Adults mutated for Drat showed both improved survival and enhanced propensities toward sedation after alcohol challenge. Together, these findings highlight novel effectors that support regulated cell death incited by alcohol stress in vitro and in vivo.

Ursodeoxycholic acid protects colon cancer HCT116 cells from deoxycholic acid-induced apoptosis by inhibiting apoptosome formation.

We previously demonstrated that ursodeoxycholic acid (UDC) requires prolonged (≥5 h) preincubation to exhibit effective protection of colon cancer HCT116 cells from deoxycholic acid (DC)-induced apoptosis. Although UDC diminished DC-mediated caspase-9 activation, cytochrome c release from the mitochondria was not inhibited, indicating that UDC acts on the steps of caspase-9 activation. In the present study, therefore, we investigated the effects of UDC on the factors involved in caspase-9 activation. We found that UDC had no significant effect on the expression of antiapoptotic XIAP. Furthermore, UDC did not affect the expression or release of proapoptotic Smac/DIABLO, or the association of XIAP and Smac/DIABLO. In contrast, association of Apaf-1 and caspase-9 stimulated by 500 µM DC was inhibited by UDC pretreatment. Although UDC caused remarkable activation of Akt/PKB, phosphatidylinositol-3-kinase (PI3K) inhibitor did not significantly reduce UDC-mediated cytoprotection. Furthermore, phosphorylation of threonine residues on caspase-9 after UDC pretreatment could not be detected. UDC-mediated cytoprotection was independent of the MAPK pathway, and cyclic AMP (cAMP) analogue did not inhibit DC-induced apoptosis. Our results indicate that UDC protects colon cancer cells from apoptosis induced by hydrophobic bile acids, by inhibiting apoptosome formation independently of the survival signals mediated by the PI3K, MAPK, or cAMP pathways.

Integrity of ATP binding site is essential for effective inhibition of the intrinsic apoptosis pathway by NAIP.

The importance of the ATP binding site of human Neuronal Apoptosis Inhibitory Protein (NAIP) on its ability in prevention of intrinsic apoptotic pathway was investigated. Thus, ATP binding lysine 476 of NAIP, which is located at the Nucleotide Binding Oligomerization Domain (NOD) was mutated to threonine and the effect of this mutation on autoproteolysis of procaspase-9 and the cleavage of procaspase-3 by apoptosome was investigated. Formation of apoptosome was induced by the addition of cytochrome c and dATP to lysates of HeLa cells transfected with pcDNA-NAIP or pcDNA-NAIP (K476T). Full length wild type NAIP prevented the cleavage of both procaspase-9 to caspase-9 and procaspase-3 to caspase-3. However, K476T variant of NAIP did not block autocleavage of procaspase-9 efficiently. Furthermore, cleavage pattern of procaspase-9 was altered in the presence of mutant NAIP. Interestingly no effect on the procaspase-3 cleavage by apoptosome was observed. The presence of NOD domain by itself had no effect on autocleavage of procaspase-9 yet slightly reduced the cleavage of procaspase-3 by apoptosome. Pull down experiment showed direct interaction of the NOD domain of NAIP with the CARD-NOD domain of Apoptotic Protease Activating Factor 1 (APAF-1). The physical association of these domains was confirmed by pull-down assays. These observations taken with previous findings indicate that the integrity of the NOD domain is essential for effective inhibition of procaspase-9 and procaspase-3 cleavage by the NAIP protein.

Modulation of cellular apoptosis with apoptotic protease-activating factor 1 (Apaf-1) inhibitors.

The programmed cell death or apoptosis plays both physiological and pathological roles in biology. Anomalous activation of apoptosis has been associated with malignancies. The intrinsic mitochondrial pathway of apoptosis activation occurs through a multiprotein complex named the apoptosome. We have discovered molecules that bind to a central protein component of the apoptosome, Apaf-1, and inhibits its activity. These new first-in-class apoptosome inhibitors have been further improved by modifications directed to enhance their cellular penetration to yield compounds that decrease cell death, both in cellular models of apoptosis and in neonatal rat cardiomyocytes under hypoxic conditions.

Big wheel keeps on turning: apoptosome regulation and its role in chemoresistance.

Apoptosis, a form of programmed cell death, enables organisms to maintain tissue homeostasis through deletion of extraneous cells and also serves as a means to eliminate potentially harmful cells. Numerous stress signals have been shown to engage the intrinsic pathway of apoptosis, with the release from mitochondria of proapoptotic factors such as cytochrome c and the subsequent formation of a cytosolic complex between apoptotic protease-activating factor-1 (Apaf-1) and procaspase-9, known as the apoptosome. Recent studies have led to the identification of an array of factors that control the formation and activation of the apoptosome under physiological conditions. Moreover, deregulation of apoptosome function has been documented in various forms of human cancer, and may play a role in both carcinogenesis and chemoresistance. We discuss how the apoptosome is regulated in normal and disease states, and how targeting of apoptosome-dependent, post-mitochondrial stages of apoptosis may serve as a rational approach to cancer treatment.

Conjugation of a novel Apaf-1 inhibitor to peptide-based cell-membrane transporters: effective methods to improve inhibition of mitochondria-mediated apoptosis.

We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.