Synthetic ceramides induce growth arrest or apoptosis by altering cellular redox status

Reactive oxygen species (ROS) and ceramide are each partly responsible for the signal transduction of a variety of extracellular agents. Furthermore, the application of synthetic, short-chain ceramides mimics the cellular responses to these extracellular agents. However, the significance of ROS involvement in ceramide signaling pathways is poorly understood. Here we describe that the (cellular responses to C2-/C6-ceramide of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells are preceded by a rise in mitochondrial peroxide production. In Jurkat T-cells, this is associated with a large time- and dose-dependent loss of cellular glutathione. However, in U937 monocytes, glutathione loss is transient. Differences in the magnitude and kinetics of this alteration in cellular redox state associate with discrete outcomes, namely growth arrest or apoptosis. © 2002 Elsevier Science (USA). All rights reserved.

Nuclear translocation of cytochrome c during apoptosis

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.

Apoptosis of neurons and oligodendrocytes in the spinal cord of spinal hyperostotic mouse (twy/twy):possible pathomechanism of human cervical compressive myelopathy

Cervical compressive myelopathy is the most serious complication of cervical spondylosis or ossification of the posterior longitudinal ligament (OPLL) and the most frequent cause of spinal cord dysfunction. There is little information on the exact pathophysiological mechanism responsible for the progressive loss of neural tissue in the spinal cord of such patients. In this study, we used the spinal hyperostotic mouse (twy/twy) as a suitable model of human spondylosis, and OPLL to investigate the cellular and molecular changes in the spinal cord. Mutant twy/twy mouse developed ossification of the ligamentum flavum at C2-C3 and exhibited progressive paralysis.

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage. We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O2 in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP. In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.

Effects of oxidative stress and neuroprotection in apoptosis in neuronal cell models

The PC12 and SH-SY5Y cell models have been proposed as potentially realistic models to investigate neuronal cell toxicity. The effects of oxidative stress (OS) caused by both H2O2 and Aβ on both cell models were assessed by several methods. Cell toxicity was quantitated by measuring cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) viability assay, an indicator of the integrity of the electron transfer chain (ETC), and cell morphology by fluorescence and video microscopy, both of which showed OS to cause decreased viability and changes in morphology. Levels of intracellular peroxide production, and changes in glutathione and carbonyl levels were also assessed, which showed OS to cause increases in intracellular peroxide production, glutathione and carbonyl levels. Differentiated SH-SY5y cells were also employed and observed to exhibit the greatest sensitivity to toxicity. The neurotrophic factor, nerve growth factor (NGF) was shown to cause protection against OS. Cells pre-treated with NGF showed higher viability after OS, generally less apoptotic morphology, recorded less apoptotic nucleiods, generally lower levels of intracellular peroxides and changes in gene expression. The neutrophic factor, brain derived growth factor (BDNF) and ascorbic acid (AA) were also investigated. BDNF showed no specific neuroprotection, however the preliminary data does warrant further investigation. AA showed a 'janus face' showing either anti-oxidant action and neuroprotection or pro-oxidant action depending on the situation. Results showed that the toxic effects of compounds such as Aβ and H2O2 are cell type dependent, and that OS alters glutathione metabolism in neuronal cells. Following toxic insult, glutathione levels are depleted to low levels. It is herein suggested that this lowering triggers an adaptive response causing alterations in glutathione metabolism as assessed by evaluation of glutathione mRNA biosynthetic enzyme expression and the subsequent increase in glutathione peroxidase (GPX) levels.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice

STUDY DESIGN: The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. OBJECTIVE: To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. SUMMARY OF BACKGROUND DATA: Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. METHODS: AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of ß-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. CONCLUSION: Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

HOCl-modified phosphatidylcholines induce apoptosis and redox imbalance in HUVEC-ST cells

Electrophilic attack of hypochlorous acid on unsaturated bonds of fatty acyl chains is known to result mostly in chlorinated products that show cytotoxicity to some cell lines and were found in biological systems exposed to HOCl. This study aimed to investigate more deeply the products and the mechanism underlying cytotoxicity of phospholipid-HOCl oxidation products, synthesized by the reaction of HOCl with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonyl-phosphatidylcholine. Phospholipid chlorohydrins were found to be the most abundant among obtained products. HOCl-modified lipids were cytotoxic towards HUVEC-ST (endothelial cells), leading to a decrease of mitochondrial potential and an increase in the number of apoptotic cells. These effects were accompanied by an increase of the level of active caspase-3 and caspase-7, while the caspase-3/-7 inhibitor Ac-DEVD-CHO dramatically decreased the number of apoptotic cells. Phospholipid-HOCl oxidation products were shown to affect cell proliferation by a concentration-dependent cell cycle arrest in the G/G phase and activating redox sensitive p38 kinase. The redox imbalance observed in HUVEC-ST cells exposed to modified phosphatidylcholines was accompanied by an increase in ROS level, and a decrease in glutathione content and antioxidant capacity of cell extracts. © 2014 Elsevier Inc. All rights reserved.

Induction of apoptosis by a cachectic-factor in murine myotubes and inhibition by eicosapentaenoic acid

Treatment of C2C12 myotubes with a tumour-derived proteolysis-inducing factor (PIF) at concentrations between 1 and 10 nM was shown to stimulate the activity of the apoptotic initiator caspases-8 and -9 and the apoptotic effector caspases-2,-3 and -6. This increased caspase activity was attenuated in myotubes pretreated with 50 μM eicosapentaenoic acid (EPA). At least part of the increase in caspase activity may be related to the increased proteasome proteolytic activity, since a caspase-3 inhibitor completely attenuated the PIF-induced increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome. However, Western blot analysis showed that PIF induced an increase in expression of the active form of caspase-3, which was also attenuated by EPA. Further Western blot analysis showed PIF increased the cytosolic content of cytochrome c, as well as expression of the pro-apoptotic protein bax but not the antiapoptotic protein bcl-2, which were both attenuated by 50 μM EPA. Induction of apoptosis by PIF in murine myotubes was confirmed by an increase in free nucleasomes formation and increased DNA fragmentation evidenced by a nucleasomal ladder typical of apoptotic cells. This process was again inhibited by pre-incubation with EPA. These results suggest that in addition to activating the proteasome, PIF induces apoptosis in C2C12 myotubes, possibly through the common intermediate arachidonic acid. Both of these processes would contribute to the loss of skeletal muscle in cancer cachexia.

Leptin decreases apoptosis and alters BCL-2:bax ratio in clonal rodent pancreatic beta-cells

The adipocyte derived peptide hormone leptin is known to regulate apoptosis and cell viability in several cells and tissues, as well as having several pancreatic islet beta-cell specific effects such as inhibition of glucose-stimulated insulin secretion. This study investigated the effects of leptin upon apoptosis induced by serum depletion and on expression of the apoptotic regulators B-cell leukaemia 2 gene product (BCL-2) and BCL2-associated X protein (Bax) in the glucose-responsive BRIN-BD11 beta-cell line.

Apoptosis induction in gastric mucous cells in vitro:lesser potency of Helicobacter pylori than Escherichia coli lipopolysaccharide, but positive interaction with ibuprofen

Non-steroidal anti-inflammatory drugs (NSAIDs) cause peptic ulcer disease, but whether they interact with Helicobacter pylori to promote damage is controversial. Moreover, the reported induction of apoptosis in gastric cells by H. pylori lipopolysaccharide (LPS) (10-9 g /ml) contrasts with studies showing low immunological potency of this LPS. Therefore, the effects of LPS from H. pylori NCTC 11637 and Escherichia coli 0111:B4 on apoptosis in a primary culture of guinea-pig gastric mucous cells were investigated in the presence and absence of the NSAID, ibuprofen. Cell loss was estimated by a crystal violet assay, and apoptosis determined from caspase activity and from condensation and fragmentation of nuclei. Exposure to E. coli LPS for 24 h caused cell loss and enhanced apoptotic activity at concentrations ≥ 10-9 g/ml, but similar effects were only obtained with H. pylori LPS at concentrations ≥10-6 g/ml. Although ibuprofen (250 μM) caused cell loss and apoptosis, addition of either E. coli or H. pylori LPSs further enhanced these effects. In conclusion, LPS and ibuprofen interact to enhance gastric cell loss and apoptosis. In such interactions, E. coli LPS is more potent than that of H. pylori. The low potency of H. pylori LPS may contribute to a chronic low-grade gastritis that can be enhanced by the use of NSAIDs. © W. S. Maney & Son Ltd.

Innate immunity and apoptosis:CD14-dependent clearance of apoptotic cells

This is the first comprehensive book about the relationship between apoptosis and autoimmune diseases. It offers a unique up–to–date overview on research results on the defective execution of apoptosis and the incomplete clearance of apoptotic cells. The molecular and cellular mechanisms involved are described in detail. As a possible consequence of apoptotic dysfunction, the development of severe autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus) is discussed. An outlook on future research topics includes the evaluation of novel therapeutic strategies.