Decane-1,2-diol derivatives as potential antitumor agents for the treatment of glioblastoma.

Glioblastoma remains the most common and aggressive type of malignant brain tumor among adults thus, considerable attention has been given to discovery of novel anti-tumor drugs for its treatment. This study reports the synthesis of a series of twelve novel decane-1,2-diol derivatives and evaluation of its anti-tumor activity in mammalian glioblastoma cell lines, U87 and LN229. Starting from decane-1,2-diol, several derivatives were prepared using a diversity oriented synthesis approach through which a small library composed of esters, silyl ethers, sulfonates, sulfites, sulfates, ketals, and phosphonates was built. The decane-1,2-diol ditosylated derivative, DBT, found to have higher cytotoxicity than the standard drug cisplatin, has IC50 value of 52 µM in U87 and 270 µM in LN229. Migration analysis of U87 cell line treated with the DBT indicated its ability to effectively suppress proliferation during initial hours of treatment and decrease anti-proliferative property over time. Additionally, DBT was assessed for its role in apoptosis, oxidative stress and caspase 3/7 activation in U87. Interestingly, our experiments indicated that its cytotoxicity is independent of Reactive oxygen species induced caspase 3/7 activity. The compound also exhibited caspase independent apoptosis activity in U87. DBT treatment led to G1/S cell cycle arrest and apoptosis induction of glioma cell lines. In addition, we identified 1533 genes with significant changes at the transcriptional level, in response to DBT. A molecular docking study accounting for the interaction of DBT with NMDA receptor disclosed several hydrogen bonds and charged residue interactions with 17 amino acids, which might be the basis of the DBT cytotoxicity observed. We conclude that this molecule exerts its cytotoxicity via caspase 3/7 independent pathways in glioblastoma cells. Concisely, simple decane-1,2-diol derivatives might serve as scaffolds for the development of effective anti-glioblastoma agents.

Loss of the cone-enriched caspase-7 does not affect secondary cone death in retinitis pigmentosa.

Purpose: The apoptotic mechanisms responsible for secondary cone death in retinitis pigmentosa (RP) remain largely unknown. The cone-enriched apoptotic protease caspase-7 (Casp7) is thought to be triggered by endoplasmic reticulum (ER) stress and plays a pivotal role in mice deficient in the cone cyclic nucleotide-gated channels, a deficiency that causes achromatopsia in humans and in mice with autosomal dominant rhodopsin mutations, in particular the T17M mutation. Thus, we tested in two mouse models of RP whether the cone-enriched Casp7 plays a role during secondary cone death. Methods: Casp7 knockout mice were crossed to two different RP mouse models with significantly different rod and cone death kinetics: the rd1 mouse model, which carries a mutation in the Pde6b gene, and the rhodopsin knockout mouse model (Rho-KO or Rho-/- ). In both models, cone survival was assessed on retinal flat mounts by quantifying the percentage of cone arrestin staining over the retinal surface area. The analyses were performed at two different time points for each model. Results: Loss of Casp7 did not alter cone survival in either of the two mouse models tested regardless of the time point analyzed. Rod survival was also not affected in either model nor did loss of Casp7 affect rod or cone function in a wild-type background as assessed with electroretinogram analyses. Conclusions: Secondary cone death in retinitis pigmentosa is unlikely to be triggered by ER stress and is likely independent of Casp7 activity.

Role and Association of Inflammatory and Apoptotic Caspases in Renal Tubulointerstitial Fibrosis.

BACKGROUND/AIMS: Caspases, an evolutionary conserved family of aspartate-specific cystein proteases, play pivotal roles in apoptotic and inflammatory signaling. Thus far, 14 mammalian caspases are identified and categorized into 3 distinct sub-types: inflammatory caspases, apoptotic initiator and apoptotic executioner. Caspase-1 is an inflammatory caspase, while caspase-7 belongs to apoptotic executioner. The roles and association of these two distinct types of caspases in renal tubulointerstitial fibrosis (TIF) have not been well recognized. METHODS: Caspase-1 inhibitor Z-YVAD-FMK and caspase-7 siRNA were used in tubular epithelial cell line NRK-52E (TECs) to test their effects on transforming growth factor-beta1 (TGF-ß1) stimulation. In vivo, Unilateral ureteral obstruction (UUO) animal model was employed in wild-type (WT) and caspase-1 knock out (KO) (caspase-1-/-) mice. RESULTS: In current study, we found that caspase-7 was obviously activated in cultured TECs stimulated by TGF-ß1 and in UUO model of WT mice. While in UUO model of caspase-1 KO mice, the increased caspase-7 activation was suppressed significantly along with reduced trans-differentiation and minimized extracellular matrix (ECM) accumulation, as demonstrated by western blot, Masson trichrome staining and immunohistochemistry. In addition, pharmacological inhibition of caspase-1 dampened caspase-7 activation and TECs' transdifferentiation induced by TGF-ß1 exposure, which was consistent with in vivo study. Notably, caspase-7 gene knock down by specific siRNA abrogated TGF-ß1 driven TECs' trans-differentiation and reduced ECM accumulation. CONCLUSIONS: Our study associated inflammatory and apoptotic caspases in TIF for the first time and we further confirmed that caspase-1 activation is an upstream event of apoptotic caspase-7 induction in TIF triggered by UUO and in TECs mediated by TGF-ß1 induced transdifferentiation.

Caspase-3/7-mediated Cleavage of ß2-spectrin is Required for Acetaminophen-induced Liver Damage.

UNLABELLED: The ubiquitously expressed ß2-spectrin (ß2SP, SPTBN1) is the most common non-erythrocytic member of the ß-spectrin gene family. Loss of ß2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of ß2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that ß2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of ß2-spectrin cleavage robustly attenuated ß2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of ß2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of ß2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of ß2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration. CONCLUSIONS: ß2-Spectrin, a TGF-ß mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of ß2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-ß in liver damage.

Caspase-7: a critical mediator of optic nerve injury-induced retinal ganglion cell death.

BACKGROUND: Axonal injury of the optic nerve (ON) is involved in various ocular diseases, such as glaucoma and traumatic optic neuropathy, which leads to apoptotic death of retinal ganglion cells (RGCs) and loss of vision. Caspases have been implicated in RGC pathogenesis. However, the role of caspase-7, a functionally unique caspase, in ON injury and RGC apoptosis has not been reported previously. The purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis. RESULTS: C57BL/6 (wildtype, WT) and caspase-7 knockout (Casp7(-/-)) mice were used. We show that ON crush activated caspase-7 and calpain-1, an upstream activator of caspase-7, in mouse RGCs, as well as hydrolysis of kinectin and co-chaperone P23, specific substrates of caspase-7. ON crush caused a progressive loss of RGCs to 28 days after injury. Knockout of caspase-7 partially and significantly protected against the ON injury-induced RGC loss; RGC density at 28 days post ON crush in Casp7(-/-) mice was approximately twice of that in WT ON injured retinas. Consistent with changes in RGC counts, spectral-domain optical coherence tomography analysis revealed that ON crush significantly reduced the in vivo thickness of the ganglion cell complex layer (including ganglion cell layer, nerve fiber layer, and inner plexiform layer) in the retina. The ON crush-induced thinning of retinal layer was significantly ameliorated in Casp7(-/-) mice when compared to WT mice. Moreover, electroretinography analysis demonstrated a decline in the positive component of scotopic threshold response amplitude in ON crushed eyes of the WT mice, whereas this RGC functional response was significantly higher in Casp7(-/-) mice at 28 days post injury. CONCLUSION: Altogether, our findings indicate that caspase-7 plays a critical role in ON injury-induced RGC death, and inhibition of caspase-7 activity may be a novel therapeutic strategy for glaucoma and other neurodegenerative diseases of the retina.

Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis.

BACKGROUND: Apoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochrome c release is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined. RESULTS: Here we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochrome c, but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM. CONCLUSIONS: Taken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.

Escherichia coli induces bovine neutrophil cell death independent from caspase-3/-7/-1, but with phosphatidylserine exposure prior to membrane rupture.

Neutrophils are essential for the innate immune response against bacterial pathogens and play a key role during the early phases of infection, including mastitis and endometritis in cows. When directly challenged with bacteria, neutrophils undergo phagocytosis induced cell death (PICD). The molecular mechanisms of this cell death modality are poorly understood, especially for bovine neutrophils. Therefore, this study aimed to determine the mechanisms and hallmarks of PICD in bovine neutrophils after in vitro challenge with Escherichia coli (E. coli). Our data show that various apoptotic hallmarks such as blebbing, chromatin condensation and executioner caspase (C)-3/-7 activity are only observed during constitutive bovine neutrophil apoptosis. In contrast, bovine neutrophil PICD is characterized by production of reactive oxygen species (ROS), pro-inflammatory C-1 activation, nuclear factor (NF)-κB activation, and interleukin (IL)-1ß and IL-6 secretion. Nevertheless, under both conditions these phagocytes undergo cell death with the exposure of phosphatidylserine (PS). Although PS exposure is generally attributed to the anti-inflammatory features of executioner caspase-dependent apoptosis, it surprisingly preceded plasma membrane rupture during bovine neutrophil PICD. Moreover, C-1 inhibition strongly affected IL-1ß production but not the PICD kinetics. This indicates that the secretion of the latter pro-inflammatory cytokine is a bystander effect rather than a regulator of PICD in bovine neutrophils, in marked contrast to the IL-1ß-dependent pyroptosis reported for macrophages.

Inflammasome-activated caspase 7 cleaves PARP1 to enhance the expression of a subset of NF-κB target genes.

Caspase 1 is part of the inflammasome, which is assembled upon pathogen recognition, while caspases 3 and/or 7 are mediators of apoptotic and nonapoptotic functions. PARP1 cleavage is a hallmark of apoptosis yet not essential, suggesting it has another physiological role. Here we show that after LPS stimulation, caspase 7 is activated by caspase 1, translocates to the nucleus, and cleaves PARP1 at the promoters of a subset of NF-κB target genes negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes. These findings propose an apoptosis-independent regulatory role for caspase 7-mediated PARP1 cleavage in proinflammatory gene expression and provide insight into inflammasome signaling.

Effects of down-regulation of microRNA-23a on TNF-α-induced endothelial cell apoptosis through caspase-dependent pathways.

AIMS: Endothelial cell injury induced by inflammatory factors plays a critical role in the pathogenesis of numerous vascular diseases. MicroRNAs are well known to be implicated in cell proliferation and apoptosis in inflammatory responses; however, it remains to be determined whether microRNAs are associated with tumour necrosis factor (TNF)-α-mediated endothelial cell injury. The aim of the present study was to investigate the role of microRNAs in TNF-α-induced endothelial cell apoptosis. METHODS AND RESULTS: Microarrays were used to analyse the global expression of microRNAs in TNF-α-stimulated human primary endothelial cells. Expression profiles of the microRNAs were verified using qRT-PCR. After TNF-α treatment, 12 miRNAs were dramatically up-regulated and nine were down-regulated. LNA-anti-miR-23a and pre-miR-23a were found to modulate one of the markedly down-regulated miRNAs, miR-23a, which could in turn increase or attenuate TNF-α-induced endothelial cell apoptosis. Bioinformatics analysis suggested that caspase-7 and serine/threonine kinase 4 are potential targets of miR-23a. LNA-anti-miR-23a enhanced but pre-miR-23a inhibited the activation of caspase-7, serine/threonine kinase 4, and its related signalling caspase-3 after TNF-α treatment; however, neither pre-miR-23a nor LNA-anti-miR-23a had an effect on TNF-α-induced Bcl-2 activation. CONCLUSION: Our results suggest that miR-23a may be involved in TNF-α-induced endothelial cell apoptosis through regulation of the caspase-7 and serine/threonine kinase 4-caspase-3 pathways.

Cutting edge: proteolytic inactivation of poly(ADP-ribose) polymerase 1 by the Nlrp3 and Nlrc4 inflammasomes.

Caspase-mediated cleavage of the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP1) is a hallmark of apoptosis. However, it remains unclear whether PARP1 is processed during pyroptosis, a specialized cell-death program that occurs upon activation of caspase-1 in inflammasome complexes. In this article, we show that activation of the Nlrp3 and Nlrc4 inflammasomes induces processing of full-length PARP1 into a fragment of 89 kDa in a stimulus-dependent manner. Macrophages deficient for caspase-1 and those lacking the inflammasome adaptors Nlrp3, Nlrc4, and ASC were highly resistant to cleavage, whereas macrophages lacking the downstream inflammasome effector caspase-7 were partially protected. A modest, but statistically significant, reduction in Nlrp3 inflammasome-induced pyroptosis was observed in PARP1 knockout macrophages. Thus, protease-mediated inactivation of PARP1 is a shared feature of apoptotic, necrotic, and pyroptotic cells.

Oxidative stress triggers neuronal caspase-independent death: endonuclease G involvement in programmed cell death-type III.

To characterize neuronal death, primary cortical neurons (C57/Black 6 J mice) were exposed to hydrogen peroxide (H2O2) and staurosporine. Both caused cell shrinkage, nuclear condensation, DNA fragmentation and loss of plasma membrane integrity. Neither treatment induced caspase-7 activity, but caspase-3 was activated by staurosporine but not H2O2. Each treatment caused redistribution from mitochondria of both endonuclease G (Endo G) and cytochrome c. Neurons knocked down for Endo G expression using siRNA showed reduction in both nuclear condensation and DNA fragmentation after treatment with H2O2, but not staurosporine. Endo G suppression protected cells against H2O2-induced cell death, while staurosporine-induced death was merely delayed. We conclude that staurosporine induces apoptosis in these neurons, but severe oxidative stress leads to Endo G-dependent death, in the absence of caspase activation (programmed cell death-type III). Therefore, oxidative stress triggers in neurons a form of necrosis that is a systematic cellular response subject to molecular regulation.

Caspase-7 deficiency protects from endotoxin-induced lymphocyte apoptosis and improves survival.

Extensive apoptosis of leukocytes during sepsis and endotoxic shock constitutes an important mechanism linked to the excessive mortality associated with these disorders. Caspase inhibitors confer protection from endotoxin-induced lymphocyte apoptosis and improve survival, but it is not clear which caspases mediate lipopolysaccharide (LPS)-induced lymphocyte apoptosis and mortality. We report here that the apoptotic executioner caspase-7 was activated in the splenocytes of LPS-injected mice, suggesting a role for caspase-7 in lymphocyte apoptosis. Indeed, caspase-7-deficient mice were resistant to LPS-induced lymphocyte apoptosis and were markedly protected from LPS-induced lethality independently of the excessive production of serum cytokines. These results reveal for the first time a nonredundant role for caspase-7 in vivo and identify caspase-7 inhibition as a component of the mechanism by which caspase inhibitors protect from endotoxin-induced mortality.

[Pt(O,O'-acac)(gamma-acac)(DMS)], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway.

BACKGROUND AND PURPOSE: We showed previously that a new Pt complex containing an O,O'-chelated acetylacetonate ligand (acac) and a dimethylsulphide in the Pt coordination sphere, [Pt(O,O'-acac)(gamma-acac)(DMS)], induces apoptosis in HeLa cells. The objective of this study was to investigate the hypothesis that [Pt(O,O'-acac)(gamma-acac)(DMS)] is also cytotoxic in a MCF-7 breast cancer cell line relatively insensitive to cisplatin, and to gain a more detailed analysis of the cell death pathways. EXPERIMENTAL APPROACH: Cells were treated with Pt compounds and cytotoxicity tests were performed, together with Western blotting of various proteins involved in apoptosis. The mitochondrial membrane potential was assessed by fluorescence microscopy and spectrofluorometry and the Pt bound to cell fractions was measured by atomic absorption spectrometry. KEY RESULTS: In contrast to cisplatin, the cytotoxicity of [Pt(O,O'-acac)(gamma-acac)(DMS)] correlated with cellular accumulation but not with DNA binding. Also, the Pt content in DNA bases was considerably higher for cisplatin than for [Pt(O,O'-acac)(gamma-acac)(DMS)], thus excluding DNA as a target of [Pt(O,O'-acac)(gamma-acac)(DMS)]. [Pt(O,O'-acac)(gamma-acac)(DMS)] exerted high and fast apoptotic processes in MCF-7 cells since it provoked: (a) mitochondria depolarization; (b) cytochrome c accumulation in the cytosol; (c) translocation of Bax and truncated-Bid from cytosol to mitochondria and decreased expression of Bcl-2; (d) cleavage of caspases -7 and -9, and PARP degradation; (e) chromatin condensation and DNA fragmentation. CONCLUSIONS AND IMPLICATIONS: [Pt(O,O'-acac)(gamma-acac)(DMS)] is highly cytotoxic for MCF-7 cells, cells relatively resistant to many chemotherapeutic agents, as it activates the mitochondrial apoptotic pathway. Hence, [Pt(O,O'-acac)(gamma-acac)(DMS)] has the potential to provide us with new opportunities for therapeutic intervention.

Endoplasmic reticulum stress during the embryonic development of the central nervous system in the mouse.

In the present study, we have found evidence for ER stress occurring during development of the central nervous system in the mouse. Several ER-resident stress-regulated chaperones, such as calreticulin, glucose regulated protein 78, glucose regulated protein 94, ER protein 57 and protein disulfide isomerase, were expressed at higher levels in embryonic brain and retina, compared with adult tissues. In contrast, calnexin, a chaperone that is not regulated by stress was equally abundant in embryonic and adult tissues. We also detected unfolded protein response during embryonic development. Both eukaryotic translation initiation factor 2 alpha and its phosphorylated form were more abundant in embryonic brain and retina than in adult tissues. Spliced X-box binding protein-1 mRNA was detected in embryonic brain and retina, while it was absent in adult counterparts. Partially glycosylated form of activating transcription factor 6 alpha, another ER stress indicator, was detected predominantly in embryonic brain. Finally, apoptotic pathway components, caspase-7 and -12, were more abundant in embryonic brain than in adult. The pattern of expression of chaperones together with activation of the unfolded protein response factors suggests the presence of ER stress during development of brain and retina. Furthermore, our data suggest that ER stress-like mechanism may induce apoptosis via activation of the caspases during embryonic development of the central nervous system.

Endoplasmic reticulum stress-induced death of mouse embryonic fibroblasts requires the intrinsic pathway of apoptosis.

Members of the caspase family are essential for many apoptotic programs. We studied mouse embryonic fibroblasts (MEFs) deficient in caspases 3 and 7 and in caspase 9 to determine the role of these proteases in endoplasmic reticulum (ER) stress-induced apoptosis. Both caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs were resistant to cytotoxicity induced via ER stress and failed to exhibit apoptotic morphology. Specifically, apoptosis induced by increased intracellular calcium was shown to depend only on caspases 3 and 9, whereas apoptosis induced by disruption of ER function depended additionally on caspase 7. Caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs also exhibited decreased loss of mitochondrial membrane potential, which correlated with altered caspase 9 processing, increased induction of procaspase 11, and decreased processing of caspase 12 in caspase 3(-/-)/caspase 7(-/-) cells. Furthermore, disruption of ER function was sufficient to induce accumulation of cleaved caspase 3 and 7 in a heavy membrane compartment, suggesting a potential mechanism for caspase 12 processing and its role as an amplifier in the death pathway. Caspase 8(-/-) MEFs were not resistant to ER stress-induced cytotoxicity, and processing of caspase 8 was not observed upon induction of ER stress. This study thus demonstrates a requirement for caspases 3 and 9 and a key role for the intrinsic pathway in ER stress-induced apoptosis.

What are caspases 3 and 7 doing upstream of the mitochondria?

Apoptosis is a cell suicide program that is initiated after cells are exposed to cytotoxic stresses including UV, IR irradiation, chemotherapeutic drugs, hypoxia, serum deprivation and TRAIL. Caspases are the central components of this process. In mammals, caspases involved in apoptotic responses are classified into two groups according to their function and structure. The first group is termed initiator caspases (caspase-2, 8, 9, 10) that contain N-terminal adapter domains which allow for auto-cleavage and activation of downstream caspases. The second group is termed effector or executioner caspases (caspase-3, 6, 7) that lack N-terminal adapter domains and are cleaved and activated by initiator caspases. Lakhani et al. (Science 2006, 311:847-51) have reported that caspase-3 and -7 regulate mitochondrial events in the apoptotic pathway. In this journal club, we summarize the results of the article and include some open questions left in the study.