Caspase

The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 express...

Tacrolimus (FK506) is widely used as an immunosuppres-sor in clinical practice; nonetheless a great part of its mecha-nisms of action and the processes triggering side effects remain unknown.1) A wide spectrum of adverse effects including neurotoxic, nephrotoxic, ...

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.

It has been increasingly recognized that cell death phenotypes and their molecular mechanisms are highly diverse. Necrosis is no longer considered a single entity, passively mediated by energy failure. Moreover, caspase-dependent apoptosis is not the only pathway involved in programmed cell death or even the only apoptotic mechanism. Recent experimental work emphasizes the diverse and interrelated nature of cell death mechanisms. Thus, there are both caspase-dependent and caspase-independent forms of apoptosis, which may differ morphologically as well as mechanistically. There are also necrotic-like phenotypes that requirede novo protein synthesis and are, therefore, forms of programmed cell death. In addition, forms of cell death showing certain morphological features of both necrosis and apoptosis have been identified, leading to the term aponecrosis. Considerable experimental evidence also shows that modulation of one form of cell death may lead to another. Together, these observations underscore the need to substantially revise our conceptions about neuroprotection strategies. Use of multiple treatments that target different cell death cascades, or single agents that moderate multiple cell death pathways, is likely to lead to more effective neuroprotection for clinical disorders.

The in vivo response to radiotherapy is not well understood but appears to involve the p53 tumor suppressor protein. We investigated the expression of apoptosis-inducing p53 target genes during ␥-irradiation-induced cell death in p53 ؉/؉ or p53 ؊/؊ mouse tissues using in situ hybridization. Our results reveal striking tissue specificity with distinct regulation of target p53-induced genes in different cells and tissue compartments, as well as variations in dependence on p53 for basal expression. p53-dependent induction of Puma occurred in the splenic white pulp, whereas Noxa and Bid were induced in the red pulp. These patterns correlated with activation of caspase-3 in both compartments. All apoptotic targets of p53 studied here (DR5, Bid, Puma, Noxa) were induced in the jejunum and ileum, which appeared to be the tissues most sensitive to irradiation. We also observed unexpected differences in p53 target gene activation between the transverse and descending colon. Finally, in the liver where irradiation did not lead to caspase-3 activation, we primarily observed p21 WAF1 induction as the major p53-dependent target gene response. Our findings indicate that the selectivity of p53 in transactivation following DNA damage in vivo results in unique tissue and cell type specificity, which may correlate with growth arrest or variable sensitivity to ␥-irradiation.

Apoptotic myocyte cell death, diastolic dysfunction and progressive deterioration in LV pump function, characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG)-transmits danger signals in cardiac muscle cells (CMC). The growth factor adapter protein, p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here, we demonstrate interventions that attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser-36), inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes (ARVM) obtained by enzymatic dissociation were transduced with mutant36 p66ShcA (mu-36) dominant negative expression vector and plated in serum free media (SFM) containing 5 mM or 25 mM glucose. At HG, ARVM exhibit marked increase in reactive oxygen species (ROS) production, upregulation of phospho-Ser-36, collapse of mitochondrial transmembrane potential (Δψm) and increased formation of p66ShcA/cytochrome c complexes. These indices of oxidative stress were accompanied by 40% increase in apoptosis and upregulation of cleaved caspase 3 and the apoptosis related proteins p53 and Bax. To test if p66ShcA functions as a redox sensitive molecular switch in vivo, we examined hearts of male Akita diabtic nonobese (C57BL/6J) mice. Immunoblot analysis detected upregulation of phospho-Ser-36, translocation of p66ShcA to mitochondria and formation of p66ShcA/cytochrome c complexes. Conversely, correction of HG by adenoassociated viral delivery of leptin

LINGO-1 is a CNS-specific protein and a functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 signaling complexes that mediate inhibition of axonal outgrowth. These receptor complexes mediate the axonal growth inhibitory effects of Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) via RhoA activation. Soluble LINGO-1 (LINGO-1-Fc), which acts as an antagonist of these pathways by blocking LINGO-1 binding to NgR1, was administered to rats after dorsal or lateral hemisection of the spinal cord. LINGO-1-Fc treatment significantly improved functional recovery, promoted axonal sprouting and decreased RhoA activation and increased oligodendrocyte and neuronal survival after either rubrospinal or corticospinal tract transection. These experiments demonstrate an important role for LINGO-1 in modulating axonal outgrowth in vivo and that treatment with LINGO-1-Fc can significantly enhance recovery after spinal cord injury.

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDN...

It is generally agreed that physiological levels of melatonin, a hormone secreted by the pineal gland, are important in protecting against oxidative stress-induced tissue damage. We investigated the effects that pinealectomy and the administration of exogenous melatonin have on the brains, testes, duodena and stomachs of rats. Pinealectomized (Px) and sham-operated (non-Px) rats were used. We evaluated structural changes, and catalase (CAT), reduced glutathione (GSH), super oxide dismutase (SOD) and malondialdehyde (MDA) levels. The rats were divided into the following five groups (eight rats in each group): sham (non-Px), Px+ vehicle, Px+ melatonin (10 mg/kg given daily intraperitoneally for a week), melatonin and ethyl alcohol. The antioxidant levels in the tissue of Px rats were significantly lower than in those of the sham group. Administering melatonin significantly increased antioxidant levels (p < 0.05). The Px rats also showed a significant increase in MDA levels when com...

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.

Abstract:  The pancreas is highly susceptible to the oxidative stress induced by ischemia/reperfusion (IR) injury leading to the generation of acute pancreatitis. Melatonin has been shown to be useful in the prevention of the damage by ischemia-reperfusion in liver, brain, myocardium, gut and kidney. The aim of the study was to evaluate the cytoprotective properties of melatonin against injury induced by IR in pancreas. The obstruction of gastro-duodenal and inferior splenic arteries induced pancreatic IR in male Wistar rats. Melatonin was intraperitoneally administered before or/and after IR injury. The animals were killed at 24 and 48 hr after reperfusion and there were evaluated parameters of oxidative stress (lipoperoxides, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione), glandular endocrine and exocrine function (lipase, amylase, insulin) and cell injury (apoptosis and necrosis). The IR induced a marked enhancement of oxidative stress and impaired pancreatic function. The histological analysis showed that IR induced acute pancreatitis with the accumulation of inflammatory infiltrate, disruption of tissue structure, cell necrosis and hemorrhage. Melatonin administration before or after pancreatic IR prevented all tissue markers of oxidative stress, biochemical and histological signs of apoptosis and necrosis, and restored glandular function. No histological signs of pancreatitis were observed 48 hr after reperfusion in 80% of the animals treated with melatonin, with only a mild edematous pancreatitis being observed in the remaining rats. Preventive or therapeutic administration of melatonin protected against the induction of oxidative stress and tissue injury, and restored cell function in experimental pancreatic IR in rats.

Epidemiological studies of the Guamanian variants of amyotrophic lateral sclerosis (ALS) and parkinsonism, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC), have shown a positive correlation between consumption of washed cycad seed flour and disease occurrence. Previous in vivo studies by our group have shown that the same seed flour induces ALS and PDC phenotypes in out bred adult male mice. In vitro studies using isolated cycad compounds have also demonstrated that several of these are neurotoxic, specifically, a number of water insoluble phytosterol glucosides of which β-sitosterol β-D-glucoside (BSSG) forms the largest fraction. BSSG is neurotoxic to motor neurons and other neuronal populations in culture. The present study shows that an in vitro hybrid motor neuron (NSC-34) culture treated with BSSG undergoes a dose-dependent cell loss. Surviving cells show increased expression of HSP70, decreased cytosolic heavy neurofilament expression, and have various morphological abnormalities. CD-1 mice fed mouse chow pellets containing BSSG for 15 weeks showed motor deficits and motor neuron loss in the lumbar and thoracic spinal cord, along with decreased glutamate transporter labelling, and increased glial fibrillary acid protein reactivity. Other pathological outcomes included increased caspase-3 labelling in the striatum and decreased tyrosine-hydroxylase labelling in the striatum and substantia nigra. C57BL/6 mice fed BSSG-treated pellets for 10 weeks exhibited progressive loss of motor neurons in the lumbar spinal cord that continued to worsen even after the BSSG exposure ended. These results provide further support implicating sterol glucosides as one potential causal factor in the motor neuron pathology previously associated with cycad consumption and ALS-PDC.

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC 50 . Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC 50 doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent. ' 2006 Wiley-Liss, Inc.

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson's disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H 2 O 2 , 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson's disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.

Rotenone, a mitochondrial complex 1 inhibitor, causes oxidative damage via production of reactive oxygen species. We examined the pathophysiology of neuronal and glial cells of the nigrostriatal pathway following unilateral infusion of varying doses of rotenone into the substantia nigra or medial forebrain bundle of adult male Sprague-Dawley rats, sacrificed 14 and 60 days after infusion. Immunofluorescence techniques were used to qualitatively and quantitatively assay dopaminergic neurons, their projections, glial cells, synapses, and oxidative stress. Rotenone infusion into the substantia nigra at all concentrations caused extensive damage and tissue necrosis, therefore of limited relevance for producing a Parkinson disease model. Infusion of 0.5 μg of rotenone targeting the medial forebrain bundle induced oxidative stress in dopaminergic neurons causing ongoing cell stress as defined by an elevation of stress granule and oxidative stress markers. This treatment resulted in the loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra (p ≤ 0.01) and loss of tyrosine hydroxylase immunoreactive nerve fibres and synaptic specialisations in the striatum (p ≤ 0.01). The infusion of 0.5 μg of rotenone also caused an increase in astrocytes and microglial cells in the substantia nigra in comparison to control (p ≤ 0.01). We examined the time-dependent reduction of tyrosine hydroxylase-positive nerve fibres and cell bodies in the striatum and substantia nigra respectively, with a progressive reduction evident 60 days after infusion (p ≤ 0.01, p ≤ 0.05). Dopaminergic axons exposed to low-dose rotenone undergo oxidative stress, with a resultant ongoing loss of dopaminergic neurons, providing an animal model relevant to Parkinson disease.

Attenuated salmonella have been reported to be capable of both selectively growing in tumors and expressing exogenous genes for tumor-targeted therapy. As 6-methoxypurine 2′ ′ ′ ′-deoxyriboside (MoPdR) is similar to 6-methylpurine 2′ ′ ′ ′-deoxyriboside in structure, we aimed to evaluate the antitumoral effect of the Escherichia coli purine nucleoside phosphorylase (ePNP) gene, using an attenuated salmonella-mediated delivery system, in combination with MoPdR. A novel mutant serovar Typhimurium (SC36) was used to carry the pEGFP-C1-ePNP vector that contains an enhanced green fluorescent protein and an ePNP gene under the control of the cytomegalovirus promoter. The function of the ePNP expression vector was confirmed in vitro using the enzymic conversion of MoPdR into methoxypurine. We also observed a high bystander effect induced by the ePNP/MoPdR system with a very low proportion (1%) of ePNP-positive cells and 5 µ µ µ µg/mL MoPdR, although the growth of parental cells was affected appreciably by MoPdR. The killing effect and increased apoptosis induced by SC36 carrying the ePNP expression vector (SC/ePNP) were detected by cytotoxicity assay and propidium iodide staining flow cytometry analysis, in combination with MoPdR. SC/ePNP was given orally to mice bearing mammary carcinomas, and its antitumor effect was evaluated. SC/ ePNP plus MoPdR significantly inhibited tumor growth by approximately 86.6-88.7% and prolonged the survival of tumorhosting mice. Our data support the view that MoPdR combined with the ePNP gene could be used in gene-directed enzyme prodrug therapy. Attenuated salmonella could be a promising strategy to improve ePNP/MoPdR bystander killing due to its preferential accumulation and anticancer activity in tumors. (Cancer Sci 2008; 99: 1172-1179) Bruce A.. A novel purine-auxotrophic mutant SC36 (his G aro A cys pur I) with a low residual virulence was gained by diethyl sulfate mutagenesis of SL3261, as previously described. Bacterial strains were routinely grown at 37°C in Luria broth (LB) or agar supplemented with kanamycin (50 µg/mL). pEGFP-C1 and pEGFP-C1-ePNP were first transformed into LB5000 using electrotransformation in cuvettes (0.2 cm electrode gap; Eppendorf) with a single pulse of 12.5 kV/cm (2.5 kV, 200 Ω, 25 µF). After amplification of the colony, the plasmids were extracted from the transferred LB5000 (3S Spin Plasmid Miniprep Kit V3.1; Shanghai Shenergy Biocolors Bioscience & Technology Co. Ltd.) then introduced into SC36 (as carried out for LB5000) to generate SC/pEGFP and SC/ePNP. Cells, cell culture, and bacterial infection. Murine mammary carcinoma 4T1 cell lines were obtained from Shanghai No.1 People's Branch Hospital (China). The cells (10 5 cells/well) were cultured in 12-well plates overnight in antibiotic-free RPMI-1640 media (Gibco BRL) in a 5% CO 2 atmosphere at 37°C. Bacteria (SC/pEGFP or SC/ePNP) were shaken in LB broth with kanamycin (50 µg/mL) overnight at 37°C to reach the late logarithmic phase of growth (OD 600 of approximately 4) and suspended in antibiotic-free media. Then 2.5 × 10 7 c.f.u. of bacteria (at a multiplicity of infection of 1:250) were directly added into 12-well plates and co-incubated with tumor cells for 3 h. Subsequently, the cells were washed three times with phosphate-buffered saline (PBS) and replenished with complete media (containing 100 U/mL penicillin and 100 µg/mL streptomycin) and further cultured for 1 or 2 days. The infected cells were collected and some of them were used to monitor the gene expression by reverse transcription (RT)-PCR (AccessQuick;

inflammatory factor 1-3 . It reduces organ damage in animal models of sepsis, ischemic injury and stroke 1,4,5 and substantially reduces mortality in patients with severe sepsis 6 . It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect 5,7 is secondary to its anticoagulant and anti-inflammatory effects 1-3 . We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells 8,9 . Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR 10 . This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.

Staphylococcal brain infections may cause mental deterioration and epileptic seizures, suggesting interference with normal neurotransmission in the brain. We injected Staphylococcus aureus into rat striatum and found an initial 76% reduction in the extracellular level of glutamate as detected by microdialysis at 2 hr after staphylococcal infection. At 8 hr after staphylococcal infection, however, the extracellular level of glutamate had increased 12-fold, and at 20 hr it had increased >30-fold. The extracellular level of aspartate and γ-aminobutyric acid (GABA) also increased greatly. Extracellular Zn(2+) , which was estimated at ∼2.6 µmol/liter in the control situation, was increased by 330% 1-2.5 hr after staphylococcal infection and by 100% at 8 and 20 hr. The increase in extracellular glutamate, aspartate, and GABA appeared to reflect the degree of tissue damage. The area of tissue damage greatly exceeded the area of staphylococcal infiltration, pointing to soluble factors be...

Objective. Ovarian cancer retains a poor prognosis among the female gynaecological malignancies. It constitutes about 3% of all malignancies in women and accounts for 5% of all female cancer related deaths. A standard treatment is cytoreductive surgery followed by adjuvant chemotherapy, and re-treatment with platinum based chemotherapy at the time of relapse. In order to improve cisplatin response in ovarian cancer cells, we utilized a high-throughput RNAi screening to identify kinase modulators.

Introduction: The aim of this study was to evaluate the cellular toxicity of different pH levels on the R3230AC mammary tumour cell line (clone-D) in vitro and to determine in what way the pH affects the tumour cells. The results could be used to interpret the cell damaging effects seen in electrochemical treatment of tumours (EChT), where pH alteration in tissue is the major event. Methods: Tumour cells were treated with pH 3.5, 5, 7, 9, 10 and 11 for 10, 20 or 30 min, respectively, followed by studies with the viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (methyltetrazolium (MTT)), morphological observation in phase contrast microscope (PCM) and light microscope, nucleotide analogue incorporation (BrdU; 5-Brdmo-2V -deoxyuridine), Caspase-3 activity measurement and detection of DNA fragmentation by an agarose gel electrophoresis. Results: In the viability assay, it was found that different pH levels had cytotoxic effects; these effects were dependent on the pH value and on the time of exposure at a given pH. Morphologically, cells in pH 3.5 and 5 had shrunk, were rounded and had condensed chromatin, whereas prominent cell swelling and nuclear expansion were seen in the pH 9-and 10treated cells. Gross cytolysis was found in pH 11. A BrdU incorporation assay indicated that proliferation rate is inhibited markedly both with decreasing and increasing pH. Significant Caspase-3 activity was found in pH 3.5 and 5 groups. Caspase-3 levels for the alkaline exposure were equal or below the normal control. DNA ladder formation, a characteristic of apoptosis, was only visualised in the treatment of pH 3.5 for 30 min. Conclusions: pH changes inhibit cell proliferation and decrease cell viability. The pathway of killing tumour cell in low pH probably has at least two directions: apoptosis and cell necrosis, whereas high pH results in only cell necrosis. The study suggests that low pH environment can induce apoptosis in unphysiological condition comparable with tissue pH at EChT. In addition, it seems that R3230AC mammary tumour cells are more tolerant to high pH than to acidic changes. This supports the theory that anodic EChT should be more efficient than cathodic. D

Objectives: The neurobiological basis for autism remains poorly understood. However, research suggests that environmentalfactors and neuroinflammation, as well as genetic factors, are contributors. This study aims to test the role that might be played by heat shock protein (HSP)70, transforming growth factor (TGF)-β 2 , Caspase 7 and interferon-γ (IFN-γ)in the pathophysiology of autism. Materials and methods: HSP70, TGF-β 2, Caspase 7 and INF-γ as biochemical parameters related to inflammation were determined in plasma of 20 Saudi autistic male patients and compared to 19 age-and gender-matched control samples. Results: The obtained data recorded that Saudi autistic patients have remarkably higher plasma HSP70, TGF-β 2, Caspase 7 and INF-γ compared to age and gender-matched controls. INF-γ recorded the highest (67.8%) while TGF-β recorded the lowest increase (49.04%). Receiver Operating Characteristics (ROC) analysis together with predictiveness diagrams proved that the measured parameters recorded satisfactory levels of specificity and sensitivity and all could be used as predictive biomarkers. Conclusion: Alteration of the selected parameters confirm the role of neuroinflammation and apoptosis mechanisms in the etiology of autism together with the possibility of the use of HSP70, TGF-β 2, Caspase 7 and INF-γ as predictive biomarkers that could be used to predict safety, efficacy of a specific suggested therapy or natural supplements, thereby providing guidance in selecting it for patients or tailoring its dose.

Objectives: To investigate the frequency of chondrocyte apoptosis in equine articular cartilage (AC) specimens and to examine the relationship between the process of cell death and the degree of cartilage degradation using a direct quantification of numbers of apoptotic cells and expression of active caspase-3.

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.

The mammalian thioredoxin (Trx) system, composed of Trx, Trx reductase (TrxR), and NADPH, is the most important thiol system involved in the redox control of signaling and regulatory proteins in apoptosis and cell proliferation. Here we addressed the inhibition of the Trx system by 13-hydroxy-15-oxo-zoapatlin (OZ), a nor-kaurane diterpene previously shown to possess proapoptotic potential and to cause cell cycle arrest in leukemia cells. OZ was found, by both biochemical and mass spectrometry-based approaches, to target Trx1 and TrxR in a cell-free system. In particular, the formation of reversible OZ adducts to Trx1 Cys35, Cys62, and Cys73 was demonstrated. We next showed that OZ efficiently inhibited Trx and TrxR catalytic activity in Molt4 cells. The occurrence of oxidative modifications of Trx molecules was assessed by "redox Western blot" analyses. OZ-mediated Trx oxidation resulted in apoptosis signaling kinase-1 release and activation of downstream JNK and p38 pathways. By means of specific inhibitors of these two stress-activated protein kinases, we demonstrated that the JNK pathway plays a major role in determining the apoptotic fate of OZexposed cells, whereas p38 activation seems to be involved mainly in OZ-induced G2/M block.

Ephedra, a herb reported to suppress appetite and stimulate the sympathetic nervous system as well as cardiac performance, has recently been related to several adverse events, including seizure, stroke, hypertension, myocardial infarction, and sudden death. Here, we describe the case of a 45-year-old woman who died of cardiovascular collapse while taking ephedra. Tissue analysis revealed non-specific degenerative alterations in the myocardium (lipofuscin accumulation, basophilic degeneration and vacuolation of myocytes, as well as myofibrillary loss), associated with myocyte apoptosis, caspase activation, and extensive cleavage of miofibrillary proteins a-actin, a-actinin, and cardiac troponin T. Healthcare professionals are therefore urged to warn their patients about the risk of serious adverse effects, which may follow ephedra intake.

Fagonia indica is a small spiny shrub of great ethnopharmacological importance in folk medicine. The aqueous decoction of aerial parts is a popular remedy against various skin lesions, including cancer. We used a biological activity-guided fractionation approach to isolate the most potent fraction of the crude extract on three cancer cell lines: MCF-7 oestrogen-dependent breast cancer, MDA-MB-468 oestrogen-independent breast cancer, and Caco-2 colon cancer cells. A series of chromatographic and spectroscopic procedures were utilised on the EtOAc fraction, which resulted in the isolation of a new steroidal saponin glycoside. The cytotoxic activity of the saponin glycoside was determined in cancer cells using the MTT and neutral red uptake assays. After 24 h treatment, the observed IC 50 values of the saponin glycoside were 12.5 lM on MDA-MB-468 and Caco-2 cells, but 100 lM on MCF-7 cells. Several lines of evidence: PARP cleavage, caspase-3 cleavage, DNA ladder assays, and reversal of growth inhibition with the pan-caspase inhibitor Z-VAD-fmk, suggested stimulation of apoptosis in MDA-MB-468 and Caco-2 cells, but not in MCF-7 cells, which do not express caspase-3. The haemolytic activity of the saponin glycoside was confirmed in sheep red blood cells, with cell lysis observed at >100 lM, suggesting that, at this concentration, the saponin glycoside caused necrosis through cell lysis in MCF-7 cells. Using the DNA ladder assay, the saponin glycoside (12.5 lM) was not toxic to HUVEC (human umbilical vein endothelial cells) or U937 cells, indicating some selectivity between malignant and normal cells. We conclude that the steroidal saponin glycoside isolated from F. indica is able to induce apoptosis or necrosis in cancer cells depending on the cell type.

Abnormal patterns of cell death, including increased apoptosis, can influence homeostasis of ligaments and could be involved in the pathogenesis of cranial cruciate ligament (CCL) rupture. Increased nitric oxide (NO) production has been implicated as a stimulus to increased apoptosis in articular cartilage. This study investigated apoptotic cell death in ruptured canine CCL (CCL group, n = 15), in ruptured CCL of dogs treated with oral

Teratospermia (.60% morphologically abnormal sperm/ejaculate) is associated with increased sperm output in the domestic cat. The objective of this study was to determine whether increased sperm production in teratospermic donors was associated with disturbances in germ cell apoptosis, the usual mechanism for sperm cell elimination. Apoptosis was measured by evaluating DNA fragmentation, expression of Caspase-3, and anti-apoptosis repressor with caspase recruitment domain (ARC) in the testes of normospermic compared with teratospermic cats. Testes (n 5 6 males/group) were obtained by bilateral castration and immediately fixed in Bouin solution. Results revealed that greater than 97% of cells labeled as DNA fragmented were tubular regardless of male type. Fewer (P , .05) apoptotic spermatogenic cells per tubule (0.52 6 0.11 cells/tubule, x 6 SEM) and per 100 Sertoli cells (3.79 cells/ 100 Sertoli cells) were observed in teratospermic compared with normospermic (1.25 6 0.36 cells/tubule and 6.44 cells/100 Sertoli cells) cats. Among the spermatogenic cells, fewer (P , .03) spermatocytes were positively labeled in teratospermic (0.3 6 0.07/ tubule) compared with normospermic (0.83 6 0.28/tubule) counterparts. Neither donor type differed in Caspase-3 or ARC expression activity. However, each factor was both cell-and stage-specific in expression. Specifically, Caspase-3 was located in Sertoli cells, Aspermatogonia, and round spermatids at stage V. The ARC was found in primary spermatocytes at each stage of the spermatogenic cycle. These results demonstrate that the high incidence of morphologically abnormal sperm in teratospermic male cats is accompanied by a reduced elimination of defective spermatogenic cells via apoptosis.

PC12 cells are used to study the signaling mechanisms underlying the neurotrophic and neuroprotective activities of pituitary adenylate cyclase-activating polypeptide (PACAP) and nerve growth factor (NGF). Previous microarray experiments indicated that serpinb1a was the most induced gene after 6 h of treatment with PACAP or NGF. This study confirmed that serpinb1a is strongly activated by PACAP and NGF in a time-dependent manner with a maximum induction (~50-fold over control) observed after 6 h of treatment. Coincubation with PACAP and NGF resulted in a synergistic up-regulation of serpinb1a expression (200-fold over control), suggesting that PACAP and NGF act through complementary mechanisms. Consistently, PACAP-induced serpinb1a expression was not blocked by TrkA receptor inhibition. Nevertheless, the stimulation of serpinb1a expression by PACAP and NGF was significantly reduced in the presence of extracellular signal-regulated kinase, calcineurin, protein kinase A, p38, and PI3K inhibitors, indicating that the two trophic factors share some common pathways in the regulation of serpinb1a. Finally, functional investigations conducted with siRNA revealed that serpinb1a is not involved in 1 These authors contributed equally to this work. Abbreviations used: Akt, v-akt murine thymoma viral oncogene homolog; AP-1, activator protein 1; cAMP, cyclic adenosine monophosphate; caspases, cysteine-dependent aspartate-directed proteases; dbcAMP, N 6 , 2 0 -O-dibutyryladenosine 3 0 -5 0 -cyclic monophosphate; DCC, deleted in colorectal cancer; DISC-1, disrupted-in-schizophrenia 1; DMEM, Dulbecco's modified Eagle's medium; Egr1, early growth response 1; ERK, extracellular signal-regulated kinase; FEZ-1, fasciculation and elongation protein zeta-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun NH 2 -terminal kinase; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor-kappa B; NFATs, nuclear factor of activated T cells; NGF, nerve growth factor; p75-NTR, p75 neurotrophin receptor; PAC1, specific PACAP receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; PBS, phosphate-buffered saline; PC12, rat adrenal pheochromocytoma cell; PEDF, pigment epithelium-derived factor; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol-12-myristate-13-acetate; PN-1, protease nexin-1; serpin, serine (or cysteine) peptidase inhibitor; siRNA, small interfering RNA; TrkA, tyrosine kinase member A receptor; VIP, vasoactice intestinal polypeptide; VPAC1, VIP and PACAP receptor 1; VPAC2, VIP and PACAP receptor 2. the effects of PACAP and NGF on PC12 cell neuritogenesis, proliferation or body cell volume but mediates their ability to block caspases 3/7 activity and to promote PC12 cell survival.

The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000 ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48 h of Roundup exposure Leydig cells are damaged. Within 24-48 h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1 ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests.

Venom of some species of scorpions induces apoptosis and arrests proliferation in cancer cells. This is an important property that can be harnessed and can lead to isolation of compounds of therapeutic importance in cancer research. Cytotoxicity was investigated using MTT reduction and confirmed with lactate dehydrogenase release following venom exposure. Apoptosis was evaluated with determination of mitochondrial membrane potential, reactive nitrogen species assay, measurement of Caspase-3 activity and DNA fragmentation analysis. To confirm that venom can inhibit DNA synthesis in proliferating breast cancer cells, immunocytochemical detection of BrdU incorporation was done. Our results demonstrated that venom of Odontobuthus doriae not only induced apoptosis but lead to the inhibition of DNA synthesis in human breast cancer cells (MCF-7). Cell viability decreased with parallel increment of LDH release in dose dependent manner after treatment with varying concentrations of venom. Moreover, venom depleted cellular antioxidants evidenced by depression of GSH and Catalases and concomitantly increased reactive nitrogen intermediates (RNI). These events were related to the depolarization of mitochondria and associated Caspase-3 activation following venom treatment in a concentration dependent manner. Finally, fragmentation of nuclear DNA following venom treatment confirmed the apoptotic property of the said venom. These results suggest that venom of O. doriae can be potential source for the isolation of effective anti-proliferative and apoptotic molecules.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.

Chromium(III) oxide (Cr 2 O 3 ) is used for industrial applications such as catalysts and pigments. In the classical form, namely the fine particle, Cr 2 O 3 is insoluble and chemically stable. It is classified as a low-toxicity chromium compound. Recently, industrial application of nanoparticles (a new form composed of small particles with a diameter of 100 nm, in at least one dimension) has been increasing. Cellular effects induced by Cr 2 O 3 nanoparticles are not known. To shed light upon this, the release of soluble chromium from Cr 2 O 3 nano-and fine-particles in culture medium was compared. Fine Cr 2 O 3 particles were insoluble in the culture medium; on the contrary, Cr 2 O 3 nanoparticles released soluble hexavalent chromium into the culture medium. Cr 2 O 3 nanoparticles showed severe cytotoxicity. The effect of Cr 2 O 3 nanoparticles on cell viability was higher than that of fine particles. Cr 2 O 3 nanoparticles showed cytotoxicity equal to that of hexavalent chromium (K 2 Cr 2 O 7 ). Human lung carcinoma A549 cells and human keratinocyte HaCaT cells showed an increase in intracellular reactive oxygen species (ROS) level and activation of antioxidant defense systems on exposure to Cr 2 O 3 nanoparticles. Exposure of Cr 2 O 3 nanoparticles led to caspase-3 activation, showing that the decrease in cell viability by exposure to Cr 2 O 3 nanoparticles was caused by apoptosis. Cellular responses were stronger in the Cr 2 O 3 nanoparticles-exposed cells than in fine Cr 2 O 3 -and CrCl 3 -exposed cells. Cellular uptake of Cr 2 O 3 particles were observed in nano-and fine-particles. The cellular influence of the extracellular soluble trivalent chromium was lower than that of Cr 2 O 3 nanoparticles. Cr 2 O 3 nanoparticles showed cytotoxicity by hexavalent chromium released at outside and inside of cells. The cellular influences of Cr 2 O 3 nanoparticles matched those of hexavalent chromium. In conclusion, Cr 2 O 3 nanoparticles have a high cytotoxic potential. # 2011 Wiley Periodicals, Inc. Environ Toxicol 00: 000-000, 2011.