Activation of voltage-sensitive sodium channels during oxygen deprivation leads to apoptotic neuronal death

Sodium (Na(+)) entry into neurons during hypoxia is known to be associated with cell death. However, it is not clear whether Na(+) entry causes cell death and by what mechanisms this increased Na(+) entry induces death. In this study we used cultures of rat neocortical neurons to show that an increase in intracellular sodium (Na(i)(+)) through voltage-sensitive sodium channels (VSSCs), during hypoxia contributes to apoptosis. Hypoxia increased Na(i)(+) and induced neuronal apoptosis, as assessed by electron microscopy, annexin V staining, and terminal UDP nick end labeling staining. Reducing Na(+) entry with the VSSC blocker, tetrodotoxin (TTX), attenuated apoptotic neuronal death via a reduction in caspase-3 activation. Since the attenuation of apoptosis by TTX during hypoxia suggested that the activation of VSSCs and Na(+) entry are crucial events in hypoxia-induced cell death, we also determined whether the activation of VSSCs per se could lead to apoptosis under resting conditions. Increasing Na(+) entry with the VSSC activator veratridine also induced neuronal apoptosis and caspase-3 activation. These data indicate that a) Na(+) entry via VSSCs during hypoxia leads to apoptotic cell death which is mediated, in part, by caspase-3 and b) activation of VSSCs during oxygen deprivation is a major event by which hypoxia induces cell death.     

谷氨酸诱导海马神经元凋亡涉及线粒体释放细胞色素C

目的：建立体外谷氨酸诱导神经元兴奋损伤模型，探索其凋亡发生是否通过线粒体信号转导途径介导的细胞色素C（Cyt C）释放而实现，为今后干预性使用神经保护剂提供依据。方法:分离及培养新生Wistar大鼠海马神经元，选用合适谷氨酸浓度建立神经元损伤模型；利用LDH测定及流式细胞仪Annexin V/PI双染色法检测谷氨酸暴露后不同时点神经元凋亡及坏死的动态改变；采用Western blotting法检测caspase-3活性及线粒体内和胞浆内Cyt C水平动态变化。结果:谷氨酸诱导神经元损伤呈明显浓度及时间依赖性，50 μmol/L浓度可使LDH释放量明显增加 (18.4%,P<0.05)，暴露后6 h凋亡率显著增加；凋亡发生前，神经元caspase-3活性已明显增高（3 h），6 h达高峰；线粒体Cyt C释放发生在caspase-3增高前，30 min时胞浆内Cyt C水平即明显增加（P<0.05），3 h胞浆内Cyt C水平超过线粒体内，而线粒体内Cyt C水平进行性减少。结论:50 μmol/L谷氨酸可诱导海马神经元凋亡，凋亡机制可能是通过损伤线粒体膜，使Cyt C易位释放入胞浆激活caspase级联反应而致。     

3,4-dihydroxybenzoic acid from Smilacis chinae rhizome protects amyloid beta protein (25-35)-induced neurotoxicity in cultured rat cortical neurons

The neuroprotective effect of 3,4-dihydroxybenzoic acid (3,4-DHBA) isolated from Smilacis chinae rhizome against Abeta (25-35)-induced neurotoxicity on cultured rat cortical neurons was found in this study. The protective effect of 3,4-DHBA against Abeta (25-35)-induced neuronal cell death was investigated by measuring cell viability via a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. 3,4-DHBA (1 and 10 microM) concentration-dependently inhibited 10 microM Abeta (25-35)-induced neuronal apoptotic death. 3,4-DHBA (1 and 10 microM) inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fluo-4 AM. 3,4-DHBA also inhibited glutamate release into medium, reactive oxygen species (ROS) generation, and caspase-3 activation, which were induced by 10 microM Abeta (25-35). These results suggest that 3,4-DHBA prevents Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.     

Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective as well as angiogenic properties, but the pathways involved in VEGF-mediated neuronal survival have not been identified. We found previously that VEGF protects cultured neural cells from death induced by serum withdrawal or hypoxia via the activation of VEGF-2/fetal liver kinase-1 receptors, phosphatidylinositol 3'-kinase, Akt and nuclear factor-kappa B. We now report that in mouse cortical neuron cultures subjected to hypoxia, the neuroprotective effect of VEGF involves suppression of cell-death pathways mediated by caspase-3. Exposure to hypoxia for 24 h caused the death of 71+/-4% of cultured neurons; this was reduced to 40+/-1% by VEGF (n=3, P<0.005) and to 44+/-1% by the caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (n=3, P<0.005). VEGF inhibited the activation of caspase-3 as measured by the 17-20-kDa caspase-3 cleavage product, and immunolocalization of VEGF and activated caspase-3 showed segregated expression in separate neuronal populations. An antisense, but not sense, oligodeoxyribonucleotide directed against VEGF increased the proportion of neurons expressing activated caspase-3, and correspondingly reduced the viability of hypoxic neurons by 37+/-2% (n=3, P<0.005).These findings suggest that VEGF protects neurons from hypoxic injury by inhibiting the activation of caspase-3, and could therefore act as an endogenous neuroprotective factor in cerebral ischemia.     

Increased oxidative stress is associated with chronic intermittent hypoxia-mediated brain cortical neuronal cell apoptosis in a mouse model of sleep apnea

Chronic intermittent hypoxia (CIH), as occurs in obstructive sleep apnea (SA), is associated with substantial cortico-hippocampal damage leading to impairments of neurocognitive, respiratory and cardiovascular functions. Previous studies in a rat model have shown that CIH increases brain cortical neuronal cell death. However, the molecular events leading to CIH-mediated neuronal cell death remain largely undefined. The oscillation of O2 concentrations during CIH remarkably mimics the processes of ischemia/re-oxygenation and could therefore increase cellular production of reactive oxygen species (ROS). We extended the CIH paradigm to a mouse model of SA to identify the molecular mechanisms underlying cortical neuronal cell death. A significant increase of ROS production in mouse brain cortex and cortical neuronal cells was detected by fluorescent oxidation assays upon exposure of mice to CIH, followed by increased expression of oxidative stress response markers, c-Fos, c-Jun and NF-kappaB in mouse brain cortex, as revealed by immunohistochemical and LacZ reporter assays respectively. Long-term exposure of mice to CIH increased the levels of protein oxidation, lipid peroxidation and nucleic acid oxidation in mouse brain cortex. Furthermore, exposure of mice to CIH induced caspase-3 activation and increased some cortical neuronal cell apoptosis. On the other hand, transgenic mice overexpressing Cu,Zn-superoxide dismutase exposed to CIH conditions had a lower level of steady-state ROS production and reduced neuronal apoptosis in brain cortex compared with that of normal control mice. Taken together, these findings suggest that the increased ROS production and oxidative stress propagation contribute, at least partially, to CIH-mediated cortical neuronal apoptosis and neurocognitive dysfunction.     

Blockade of nitric oxide formation does not prevent glutamate-induced neurotoxicity in neuronal cultures from rat hippocampus.

This study examined the role of nitric oxide (NO) in glutamate-induced, N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat hippocampal neuronal cultures grown under serum-free conditions. Formation of cGMP was used as an indirect measure of NO formation. Neuronal cell degeneration was monitored by measuring the release of lactate dehydrogenase (LDH). Neuronal cells showed a 4-fold increase in cGMP formation and release of LDH upon exposure to 30 microM glutamate. cGMP formation was fully inhibited by 1 microM nitro-arginine (N-Arg), 100 microM hemoglobin or 1 microM MK-801. In the presence of 1 microM MK-801, glutamate induced neither cGMP formation nor neuronal cell degeneration. However, when NO formation was inhibited by means of 100 microM N-Arg, glutamate still induced neurotoxicity. Therefore, in serum-free hippocampal cultures glutamate neurotoxicity occurs notwithstanding complete inhibition of the NO-synthase enzyme by N-Arg. Our data provide evidence that NO, synthesized upon glutamate exposure, has not a primary toxic action in pure hippocampal neuronal cultures.     

Aryl hydrocarbon receptor-mediated apoptosis of neuronal cells: A possible interaction with estrogen receptor signaling

Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, β-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. β-Naphthoflavone (0.1–100 μM) enhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. α-Naphthoflavone inhibited the action of β-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced β-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERα antagonist, methyl-piperidino-pyrazole, did not affect β-naphthoflavone-induced caspase-3 activity. However, they inhibited β-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of β-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERβ as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.     

Effects of estrone on N-methyl-D-aspartic acid- and staurosporine-induced changes in caspase-3-like protease activity and lactate dehydrogenase-release: time- and tissue-dependent effects in neuronal primary cultures

A growing body of evidence indicates that estrogens affect apoptotic processes in neuronal cells. However, their effects seem to depend on type of neuronal tissue, stage of development and apoptosis inducing factors. In the present study we compared effects of estrone (100 and 500 nM) on N-methyl-D-aspartic acid (NMDA) (1 mM)- and staurosporine (1 microM)-induced caspase-3-like activity and lactate dehydrogenase (LDH)-release in primary cultures of rat hippocampal and neocortical neurons. Fluorometric and colorimetric determination of enzyme activity was performed 6 h, 14 h, and 24 h after exposure to apoptotic agents. In the hippocampal cell cultures on 7 days in vitro (DIV), a time-dependent NMDA-induced activation of caspase-3-like proteases was accompanied by increased LDH-release. In neocortical cell cultures on 7 DIV NMDA did not affect caspase activity and decreased LDH-release. In neocortical cell cultures on 12 DIV NMDA inhibited spontaneous caspase activity, but was toxic to neurons after 24 h exposure suggesting that these cells underwent necrotic rather than apoptotic death. Estrone has attenuated both pro- and anti-apoptotic NMDA-induced changes in rat primary neuronal cultures acting independently of estrogen receptors, as detected with ICI 182, 780. In hippocampal neurons estrone antagonized not only the NMDA-induced caspase-3-like activity, but also NMDA-mediated LDH-release. However, in neocortical neurons estrone either attenuated NMDA-induced inhibition of caspase-3-like activity (12 DIV) or partly blocked NMDA-mediated decrease in LDH-release (7 DIV). In contrast to NMDA, staurosporine elevated caspase-3-like activity and LDH-release in a time-dependent manner in all used culture systems. Estrone inhibited pro-apoptotic effects of staurosporine in neocortical neurons, but only at later stage of development in vitro, which points to the protective role of estrogens during the brain tissue maturation. Since estrone triggered its effects via non-genomic mechanisms, it suggests that the other estradiol metabolites exhibiting low affinity to hormone receptors may be potent neuroprotective agents, which could retain the favorable and minimize the adverse side effects of estrogens.     

肝细胞生长因子减轻皮质神经元的缺氧/复氧损伤

目的研究肝细胞生长因子(HGF)对缺氧/复氧诱导大鼠皮质神经元凋亡的保护。方法分离培养SD大鼠皮质神经元,经HGF(15、30和60μg/L)预处理后经缺氧/复氧诱导凋亡,用MTT比色法检测细胞存活率;用Hoechst33258染色法和流式细胞仪检测神经元的凋亡;用比色法检测细胞乳酸脱氢酶(LDH)和caspase-3活性变化。结果与正常对照组相比,缺氧/复氧组神经元的细胞存活率显著下降,细胞凋亡率和caspase-3活性升高(均P<0.05);而HGF预处理12 h可显著逆转缺氧/复氧所致的细胞存活率降低、凋亡率增加、LDH活性上升、caspase-3活性升高的改变(均P<0.05),且这些效应与HGF剂量正相关。此外,HGF的抗凋亡效应可被PI3K/Akt通路抑制剂LY294002阻断。结论HGF减轻缺氧/复氧所诱导的神经元凋亡可能与其激活神经元的PI3K/Akt信号通路、减少caspase-3表达有关。     

The protection of Bcl-2 overexpression on rat cortical neuronal injury caused by analogous ischemia/reperfusion in vitro

Recent studies have suggested that neuronal apoptosis in cerebral ischemia could arise from dysfunction of endoplasmic reticulum (ER) and mitochondria. B-cell lymphoma/leukemia-2 gene (Bcl-2) has been described as an inhibitor both in programmed cell death (PCD) and ER dysfunction during apoptosis, and the Bcl-2 family play a key role in regulating the PCD, both locally at the ER and from a distance at the mitochondrial membrane. However, its signal pathways and concrete mechanisms in endoplasmic reticulum-initiated apoptosis remain incompletely understood. We therefore investigate whether ischemia/reperfusion (I/R) causes neuronal apoptosis in part via cross-talk between ER and mitochondria or not, and how the overexpression of Bcl-2 prevents this form of cell death. Here we show that analogous I/R-induced cell death occurs consequent to interactions of ER stress and mitochondrial death pathways. The participation of the mitochondrial pathway was demonstrated by the release of cytochrome C (cyt C) from mitochondrial into cytoplasmic fractions and caspase-9 cleavage. The involvement of ER stress was further supported by the observable increase of glucose-regulated protein 78(GRP78)/BiP expression and caspase-12 activity. Furthermore, prior to these changes, swelling of the ER lumen and dissociation of ribosomes from rough ER were detected by electron microscopy. Bcl-2 overexpression inhibits the release of cyt C and the activation of caspase-9/-8/-3 but not caspase-12 based on the results of Western blot. These suggest that cross-talk between ER and mitochondria participate in neuronal damage after ischemia/reperfusion. Bcl-2 overexpression could suppress I/R-induced neuronal apoptosis via influencing mitochondrial integrity.     

A peptide derived from acetylcholinesterase induces neuronal cell death: characterisation of possible mechanisms

Acetylcholinesterase (AChE) exhibits functions unrelated to the catalysis of acetylcholine (ACh) in particular during development. Although the underlying mechanism(s) is presently unknown, a candidate peptide fragment (AChE-peptide) has recently been identified, and been shown to induce a continuum of apoptotic and necrotic neuronal cell death in rat hippocampal organotypic cultures. The aim of this study was to trace the cell death pathway initiated by AChE-peptide. Using specific antagonists, it was possible to track a series of cellular events following application of 1 nM AChE-peptide: NMDA receptor activation, opening of the L-type voltage gated calcium channel, activation of calcium/calmodulin kinase II, generation of reactive oxygen species and caspase activation. Pharmacological interception at any stage of this cascade blocked the effect of 1 nM AChE-peptide on neurite retraction. Lactate dehydrogenase (LDH) release, a marker for cell lysis, was unaffected by 1 nM AChE-peptide. In contrast, cell death induced by 1 mM AChE-peptide, monitored as neurite retraction and increased LDH efflux, was not offset by any drug treatment. These data suggest that nanomolar concentrations of AChE-peptide exhibit pathophysiological activity via an apoptotic pathway that could play an important role in neuronal development and neurodegeneration.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1.We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.     

Protective role of N-trans-feruloyltyramine against β-amyloid peptide-induced neurotoxicity in rat cultured cortical neurons

Enhanced oxidative stress and inflammation play important roles in the pathogenesis of Alzheimer's disease (AD). Amyloid β-peptide (Aβ), a major component of amyloid plaques, is considered to have a causal role in the development and progress of AD by being the initiator of a pathological cascade leading to oxidative stress. The present study investigated the effect of N-trans-feruloyltyramine (NTF) purified from Polyalthia suberosa, an alkaloid shown to protect against oxidative stress and cell death. Pre-treatment of rat primary cortical cell cultures with 25-250μM NTF significantly attenuated 10μM Aβ(1-42)-induced neuronal death in a dose-dependent manner. Apoptotic cell death was demonstrated morphologically as well as by detection of the presence of activated caspase-3 and Bax, levels of which could be reduced by NTF pre-treatment. NTF also reduced production of reactive oxygen species induced by Aβ(1-42). These findings suggest that the protective effect of NTF against Aβ(1-42)-induced neuronal death might be due to its antioxidative property.     

Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ

Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in na?ve macrophages infected with Yersinia enterocolitica. Na?ve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.     

Exacerbation of apoptosis of cortical neurons following traumatic brain injury in par-4 transgenic mice

Traumatic brain injury (TBI) is a significant clinical problem, yet few effective strategies for treating it have emerged. People that sustain and survive a TBI are left with significant cognitive, behavioral, and communicative disabilities. Apoptotic neuronal death occurs following TBI. Prostate apoptosis response-4 (Par-4) is a death domain-containing protein initially characterized as a critical regulator of apoptosis in prostate cancer cells. We have recently generated and characterized Par-4 transgenic mice in which the expression of the par-4 transgene was limited to cells of neuronal lineage. We now provide evidence that, in cortical neurons from these mice, Par-4 drastically increases apoptotic neuronal death in both in vitro and in vivo models of TBI. In vitro experiments were performed in 7-day-old primary cultures of cortical neurons using a previously published, scratch-induced mechanical trauma model. Neurons that overexpress Par-4 showed not only a significant decrease in overall neuron survival after TBI compared to wild-type cells, but also exhibited a sharper decrease in mitochondrial transmembrane potential, a higher degree of free radical accumulation, and earlier activation of caspase-3 than wild-type cells did. In vivo experiments were performed utilizing a weight drop TBI model. A significantly increased volume of cortical injury and exacerbated activation of caspase-3 were observed in Par-4 transgenic mice when compared to those in wild-type mice. These data suggests that aberrant Par-4 expression exacerbates neuronal cell death following TBI by altering mitochondrial function, enhancing oxidative damage, and execution of apoptosis via caspase activation.     

Rho mediates calcium-dependent activation of p38alpha and subsequent excitotoxic cell death

Excitotoxic neuronal death contributes to many neurological disorders, and involves calcium influx and stress-activated protein kinases (SAPKs) such as p38alpha. There is indirect evidence that the small Rho-family GTPases Rac and cdc42 are involved in neuronal death subsequent to the withdrawal of nerve growth factor (NGF), whereas Rho is involved in the inhibition of neurite regeneration and the release of the amyloidogenic Abeta(42) peptide. Here we show that Rho is activated in rat neurons by conditions that elevate intracellular calcium and in the mouse cerebral cortex during ischemia. Rho is required for the rapid glutamate-induced activation of p38alpha and ensuing neuronal death. The ability of RhoA to activate p38alpha was not expected, and it was specific to primary neuronal cultures. The expression of active RhoA alone not only activated p38alpha but also induced neuronal death that was sensitive to the anti-apoptotic protein Bcl-2, showing that RhoA was sufficient to induce the excitotoxic pathway. Therefore, Rho is an essential component of the excitotoxic cell death pathway.     

Effects of noradrenaline on neuronal NOS2 expression and viability

The authors previously showed that conditioned media (CM) from activated microglia increased inducible nitric oxide synthase (NOS2) in cortical neurons. Here they examined the ability of noradrenaline (NA) to reduce neuronal NOS2 or cell death. Primary mouse cortical neurons were activated using CM from microglia incubated with lipopolysaccharide (LPS). Neuronal NOS2 was assessed by increases in nitrite accumulation, and increases in NOS2 mRNA levels and fluorescence of the NO-sensitive probe DAF-2 DA. NOS2 induction was associated with an increase in neuronal LDH release. When NA was added during microglial activation, neuronal NOS2 was significantly reduced (by approximately 70%); in contrast if NA was added to the neurons along with CM, there was less reduction (about 35% decrease) in NOS2 expression. NA added to either microglia or to neurons reduced neuronal LDH release comparably. Pretreatment of CM with blocking antibody to TNFalpha, alone or with IL1-receptor antagonist, partially reduced neuronal cell death and NOS2. Incubation of neurons with NA increased IkBalpha, which could reduce NOS2. These results demonstrate that NA modulates neuronal NOS2 expression and damage, and that these effects are primarily due to inhibition of microglia released factors. Perturbations of NA could exacerbate neuronal damage by allowing for increased inflammatory responses.     

Pertussis toxin B-oligomer suppresses human immunodeficiency virus-1 Tat-induced neuronal apoptosis through feedback inhibition of phospholipase C-beta by protein kinase C

Human immunodeficiency virus (HIV)-1 Tat is a multifunctional protein involved in viral replication, inflammation and apoptosis. Tat activates phospholipase C-beta (PLC-beta), presumably via a pertussis toxin (PTX) sensitive G(i) protein, which is critical for neuronal apoptosis. In this study, we show that Tat-mediated intracellular Ca(2+) release in rat pheochromocytoma (PC-12) cells and rat primary cortical neuronal cultures was abrogated by pretreatment with either pertussis toxin and/or its B-oligomer subunit (PTX-B), devoid of ADP ribosyltransferase activity. PTX-B pretreatment also inhibited intracellular Ca(2+) release by bradykinin and 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (m-3M3FBS), a director activator of phospholipase C. Activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PdBu) mimicked the PTX-B-mediated inhibition of m-3M3FBS-stimulated intracellular Ca(2+) increase, while inhibition of PKC by bisindolylmaleimide I hydrochloride (BIM) reversed the inhibitory action of PTX-B. Functionally, PTX-B reduced Tat-induced Bax and caspase-3 proteins and reduced cell apoptosis. We conclude that PTX inhibition of Tat-mediated intracellular Ca(2+) release is independent of ADP ribosylation of the G(i) protein via the A protomer, but mediated by the B-oligomer. Furthermore, PTX-B suppresses HIV-1 Tat-mediated apoptosis by reducing its activation of PLC-beta through a PKC activation pathway.     

Taxol induces oxidative neuronal cell death by enhancing the activity of NADPH oxidase in mouse cortical cultures

We examined the involvement of oxidative stress in neuronal cell death induced by taxol, a microtubule-stabilizing anti-cancer drug and investigated whether NADPH oxidase plays a role in taxol-induced neuronal cell death in mouse cortical cultures. Cell death was assessed by measuring lactate dehydrogenase in the bathing media after 24-h exposure to taxol. Taxol (30-1000nM) induced the concentration-dependent neuronal death with apoptotic features. The neuronal death induced by taxol was significantly attenuated not only by anti-apoptotic drugs such as z-VAD-fmk and cycloheximide but also by antioxidants such as trolox, ascorbic acid and tempol. Vinblastine, a microtubule-depolymerizing anti-cancer drug, also induced neuronal death. The neuronal cell death induced by vinblastine was also attenuated by z-VAD-fmk, but not by antioxidants and NADPH oxidase inhibitors. Exposure the cortical cultures to taxol for 80min formed neurite beadings visualized by fluorescence immunocytochemistry for tubulin. Treatment with either trolox or apocynin, an NADPH oxidase inhibitor, did not affect formation of the neurite beadings. RT-PCR and Western blot analysis revealed that exposure to taxol increased the expression of p47(phox) and gp91(phox) and induced translocation of the p47(phox) to the membrane in cortical cultures. Exposure to taxol markedly increased cellular 2,7-dichlorofluorescin diacetate fluorescence, an indicator for reactive oxygen species. Apocynin and trolox markedly inhibited the taxol-induced increase of the fluorescence. Moreover, treatment with NADPH oxidase inhibitors or suppression of gp91(phox) by siRNA significantly attenuated the taxol-induced neuronal death. These results indicate that taxol induces oxidative neuronal apoptosis by enhancing the activity of NADPH oxidase.     

RETRACTED: Resibufogenin suppresses growth and metastasis through inducing caspase-1-dependent pyroptosis via ROS-mediated NF-κB suppression in non-small cell lung cancer

The purpose of this study is to explore the antitumor properties of resibufogenin (RB) in non-small cell lung cancer (NSCLC) and elucidate its underlying mechanism. A549 and H520 cells were treated with various concentrations of RB with or without NLRP3 inhibitor (MCC950), caspase-1 inhibitor (VX765), or N-acetyl-l -cysteine (an ROS scavenger). Cell counting kit-8 and colony formation assays were conducted to determine cell viability. Cell invasion was detected by using the transwell assay. The release of lactate dehydrogenase (LDH) was determined by the LDH detection assay. The protein expression levels of related genes were measured by western blotting and immunohistochemistry. The reactive oxygen species (ROS) level was detected by using a 2,7-dichlorodihydrofluorescein diacetate ROS Assay Kit. The in vivo effects of RB were evaluated in a xenograft mouse model. RB treatment reduced cell viability and invasion in a dose-dependent manner. Furthermore, RB also enhanced pyroptosis levels in A549 and H520 cells, as indicated by the increased release of LDH and pyroptosis-related proteins. Interestingly, we also found that the antiproliferative and antimetastatic effects of RB were alleviated by the blockade of pyroptosis using NLRP3 inhibitor MCC950. Further study demonstrated that RB induced pyroptosis in a caspase-1-dependent manner, as evidenced by the finding that VX765 effectively reversed the effects of RB on A549 and H520 cells. We also found that RB could trigger caspase-1-dependent pyroptosis through ROS-mediated NF-κB suppression. In summary, our findings provide a potential antitumor agent and a novel insight into the mechanism of RB treatment of NSCLC.