Osmotic stress induces loss of glutathione and increases the sensitivity to oxidative stress in H9c2 cardiac myocytes

It has been observed that H9c2 cardiac cells cultured in physiologic solutions exhibit delayed cell death after repeated medium replacements, of which the cause was the relatively mild osmotic challenges during the renewal of the culture medium. Interestingly, the cell damage was associated with altered intracellular GSH homeostasis. Therefore, this study attempted to elucidate the effects of osmotic stress on GSH metabolism. In cells subjected to osmotic stress by lowering the NaCl concentration of the medium, the cell swelling was rapidly counterbalanced, but the intracellular GSH content was significantly lower in 3 h. Meanwhile, the ratio of GSH-to-GSSG was not affected. As expected, osmotic stress also increased the sensitivity to H₂O₂, which was attributable to the decrease of GSH content. The decrease of GSH content was similarly evident when the synthetic pathways of GSH were blocked by BSO or acivicin. It was concluded that osmotic stress induced the decrease of intracellular GSH content by increased consumption and this loss of GSH rendered the cells susceptible to a subsequent oxidative stress.     

Oxidative-stress-dependent up-regulation of Bcl-2 expression in the central nervous system of aged Fisher-344 rats

Oxidative stress has been shown to play a role in aging and in neurodegenerative disorders. Some of the consequences of oxidative stress are DNA base modifications, lipid peroxidation, and protein modifications such as formation of carbonyls and nitrotyrosine. These events may play a role in apoptosis, another factor in aging and neurodegeneration, in response to uncompensated oxidative stress. Bcl-2 is a mitochondrial protein that protects neurons from apoptotic stimuli including oxidative stress. Using immunohistochemistry and western blot analysis, here we show that Bcl-2 is up-regulated in the hippocampus and cerebellum of aged (24 months) Fisher 344 rats. Treatment with the free radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN) effectively reverses this age-dependent Bcl-2 up-regulation indicating that this response is redox sensitive. This conclusion was further supported by inducing the same regional Bcl-2 up-regulation in young (3 months) Fisher 344 rats exposed to 100% normobaric O(2) for 48 h. Our results indicate that Bcl-2 expression is increased in the aged brain, possibly as a consequence of oxidative stress challenges. These results also illustrate the effectiveness of antioxidants in reversing age-related changes in the CNS and support further research to investigate their use in aging and in age-related neurodegenerative disorders.     

The retina as a novel in vivo model for studying the role of molecules of the Bcl-2 family in relation to MPTP neurotoxicity

To determine the roles of different members of the family of B cell lymphoma protooncogene (Bcl-2) in relation to neurotoxin-induced neuronal degeneration, the pattern of the expression of a number of molecules of the Bcl-2 family was studied immunocytochemically in the retinas of C57BL/6J mice after intraperitoneal (IP) injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three days to 12 weeks after MPTP treatment, a detectable reduction of tyrosine hydroxylase immunoreactivity in the amacrine cells was observed, with an increase of Bcl-2 expression in the Müller glial cells, and a de novo expression of Bad and Bax in the retinal ganglion cells, optic nerve fibers and plexiform layers. In contrast, a slight decrease of Bcl-x_L immunoreactivity in the retinal ganglion cells was observed, whereas Bcl-x_S/L immunoreactivity was increased slightly in the retinas of MPTP-treated mice compared with that of the controls. In animals that received MPTP injection, an increase in immunostaining of GFAP, glutamine synthetase, and Mac-1 (CD11b) in astrocytes, Müller cells, and microglia was invariably observed, indicating an activation or dysfunction of retinal glial cells. These findings are consistent with the current view that glial dysfunction is important in mediating the cytotoxic effect of a variety of neurotoxic molecules, including MPTP, and that different members of Bcl-2 family may have different roles as far as neuronal degeneration or neuroprotection is concerned.     

Unknotting the roles of Bcl-2 and Bcl-xL in cell death

The antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL play important roles in inhibiting mitochondria-dependent extrinsic and intrinsic cell death pathways. It seems that these two proteins have distinct functions for inhibiting extrinsic and intrinsic cell death pathways. The overexpression of Bcl-2 is able to inhibit not only apoptotic cell death but also in part nonapoptotic cell death, which has the role of cell cycle arrest in the G1 phase, which may promote cellular senescence. The overexpression of Bcl-2 may also have the ability to enhance cell death in the interaction of Bcl-xL with other factors. The overexpression of Bcl-xL enhances autophagic cell death when apoptotic cell death is inhibited in Bax(-/-)/Bak(-/-) double knockout cells. This review discusses the previously unexplained aspects of Bcl-2 and Bcl-xL functions associated with cell death, for better understanding of their functions in the regulation.     

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.     

Full-length expanded ataxin-3 enhances mitochondrial-mediated cell death and decreases Bcl-2 expression in human neuroblastoma cells

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. An unstable CAG trinucleotide repeat expansion in MJD gene on long arm of chromosome 14 has been identified as the pathologic mutation of MJD and apoptosis was previously shown to be responsible for the neuronal cell death of the disease. In this study, we utilized human neuronal SK-N-SH cells stably transfected with HA-tagged full-length MJD with 78 polyglutamine repeats to examine the effects of polyglutamine expansion on neuronal cell survival in the early stage of disease. Various pro-apoptotic agents were used to assess the tolerance of the mutant cells and to compare the differences between cells with and without mutant ataxin-3. Concentration- and time-dependent experiments showed that the increase in staurosporine-induced cell death was more pronounced and accelerated in cells containing expanded ataxin-3 via MTS assays. Interestingly, under basal conditions, Western blot and immunocytochemical analyses showed a significant decrease of Bcl-2 protein expression and an increase of cytochrome c in cells containing expanded ataxin-3 when compared with those of the parental cells. The same reduction of Bcl-2 was further confirmed in fibroblast cells with mutant ataxin-3. In addition, exogenous expression of Bcl-2 desensitized SK-N-SH-MJD78 cells to poly-Q toxicity. These results indicated that mitochondrial-mediated cell death plays a role in the pathogenesis of MJD. In our cellular model, full-length expanded ataxin-3 that leads to neurodegenerative disorders significantly impaired the expression of Bcl-2 protein, which may be, at least in part, responsible for the weak tolerance to polyglutamine toxicity at the early stage of disease and ultimately resulted in an increase of stress-induced cell death upon apoptotic stress.     

Bcl-2-regulated cell death signalling in the prevention of autoimmunity

Cell death mediated through the intrinsic, Bcl-2-regulated mitochondrial apoptosis signalling pathway is critical for lymphocyte development and the establishment of central and maintenance of peripheral tolerance. Defects in Bcl-2-regulated cell death signalling have been reported to cause or correlate with autoimmunity in mice and men. This review focuses on the role of Bcl-2 family proteins implicated in the development of autoimmune disorders and their potential as targets for therapeutic intervention.     

Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative stress and cell death induced by hydrogen peroxide, 4-hydroxynonenal or serum deprivation: potentiation of injury by ALS-related mutant superoxide dismutases and protection by Bcl-2

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE). Overexpression of Bcl-2 had little effect on levels of protein carbonyls, lipid peroxidation, 8-hydroxyguanine (8-OHG) or 3-nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8-OHG and 3-nitrotyrosine, changes that were attenuated in cells overexpressing Bcl-2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl-2. By contrast, expression of wild-type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild-type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.     

Down-regulation of Bcl-2 and Bcl-xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death

The functions of the antiapoptotic proteins Bcl-2 and Bcl-xL were examined in glioblastoma cells. Expression of both Bcl-2 and Bcl-xL were found to be elevated in protein lysates from seven early passage cell lines derived from human glioblastoma tumors compared with non-neoplastic glial cells. Down-regulation of both bcl-2 and bcl-xL expression in glioblastoma cell lines U87 and NS008 with bcl-2/bcl-xL bispecific antisense oligonucleotide resulted in spontaneous cell death. The mechanism of cell death was partially caspase-dependent. Executioner caspase 6 and caspase 7, but not caspase 3, were involved in apoptosis induced by bcl-2/bcl-xL antisense treatment. Interestingly, western blots failed to demonstrate expression of caspase 3 in two of the seven glioblastoma cell lines examined. The data support the hypothesis that Bcl-2 and Bcl-xL are important in preventing cell death in glioblastoma cells. It also suggests that there are functional pathways capable of successful completion of caspase-dependent cell death in gliomas. These findings support a potential role of bcl-2/bcl-xL bispecifc antisense oligonucleotide therapy as a treatment strategy to enhance caspase-dependent cell death in patients with glioblastoma.     

Induction of Bcl-2 by functional regulation of G-protein coupled receptors protects from oxidative glutamate toxicity by increasing glutathione

Glutamate treatment depletes hippocampal HT22 cells of glutathione, which renders the cells incapable to reduce reactive oxygen species and ultimately cumulates in cell death by oxidative stress. HT22 cells resistant to glutamate displayed increased phosphorylation of cAMP-response-element binding (CREB) and decreased ERK1/2 suggestive of differences in signal transmission. We investigated the amount of candidate G-protein-coupled receptors involved in this resistance and found an increase in mRNA for receptors activated by the vasoactive intestinal peptide VIP (VPAC₂, 12.6-fold) and glutamate like the metabotropic glutamate receptor mGlu₁ (5.3-fold). Treating cells with VIP and glutamate led to the same changes in protein phosphorylation observed in resistant cells and induced the proto-oncogene Bcl-2. Bcl-2 overexpression protected by increasing the amount of intracellular glutathione and Bcl-2 knockdown by small interfering RNAs (siRNA) increased glutamate susceptibility of resistant cells. Other receptors upregulated in this paradigm might represent useful targets in the treatment of neurological diseases associated with oxidative stress.     

Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.     

Induction of Bcl-2 by functional regulation of G-protein coupled receptors protects from oxidative glutamate toxicity by increasing glutathione

Glutamate treatment depletes hippocampal HT22 cells of glutathione, which renders the cells incapable to reduce reactive oxygen species and ultimately cumulates in cell death by oxidative stress. HT22 cells resistant to glutamate displayed increased phosphorylation of cAMP-response-element binding (CREB) and decreased ERK1/2 suggestive of differences in signal transmission. We investigated the amount of candidate G-protein-coupled receptors involved in this resistance and found an increase in mRNA for receptors activated by the vasoactive intestinal peptide VIP (VPAC₂, 12.6-fold) and glutamate like the metabotropic glutamate receptor mGlu₁ (5.3-fold). Treating cells with VIP and glutamate led to the same changes in protein phosphorylation observed in resistant cells and induced the proto-oncogene Bcl-2. Bcl-2 overexpression protected by increasing the amount of intracellular glutathione and Bcl-2 knockdown by small interfering RNAs (siRNA) increased glutamate susceptibility of resistant cells. Other receptors upregulated in this paradigm might represent useful targets in the treatment of neurological diseases associated with oxidative stress.     

Protective effects of 15-deoxy-Delta12,14-prostaglandin J2 against glutamate-induced cell death in primary cortical neuron cultures: induction of adaptive response and enhancement of cell tolerance primarily through up-regulation of cellular glutathione

There is increasing evidence to suggest that reactive oxygen species, including a variety of lipid oxidation products and other physiologically existing oxidative stimuli, can induce an adaptive response and enhance cell tolerance. In the present study, by using cultured cortical neurons, we investigated the effect of electrophilic lipids, such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and 4-hydroxy-2-nonenal (4-HNE) against the cell death induced by H(2)O(2) and glutamate. Pre-treatment with both 15d-PGJ(2) and 4-HNE at sublethal concentrations resulted in a significant protective effect against oxidative stress, and 15d-PGJ(2), in particular, exhibited a complete protective effect against glutamate-induced neuronal cell death. Pre-treatment with 15d-PGJ(2) increased the intracellular glutathione (GSH) as well as the gene expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. 15d-PGJ(2) protected cells from glutamate-induced GSH depletion, while the inhibition of cellular GSH synthesis by buthionine sulfoximine abolished the adaptive response induced by 15d-PGJ(2). These findings indicate that at low levels, 15d-PGJ(2) acts as a potent survival mediator against glutamate-induced insults via the induction of an adaptive response primarily through the up-regulation of the intracellular GSH synthesis.     

Sulforaphane as an inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma cell line

The total GSH depletion observed in the substantia nigra (SN) appears to be responsible for subsequent oxidative stress (OS), mitochondrial dysfunction, and dopaminergic cell loss in patients with Parkinson's disease. A strategy to prevent the OS of dopaminergic cells in the SN may be the use of chemopreventive agents as inducers of endogenous GSH, antioxidant and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with sulforaphane (SF), a cruciferous vegetables inducer, resulted in significant increases of total GSH level, NAD(P)H : quinone oxidoreductase-1, GSH-transferase and -reductase, but not GSH-peroxidase, catalase and superoxide dismutase activities. Further, the elevation of GSH levels, GSH-transferase and NAD(P)H:quinone oxidoreductase-1 activities was correlated to an increase of the resistance of SH-SY5Y cells to toxicity induced by H₂O₂ or 6-hydroxydopamine (6-OHDA). The pre-treatment of SH-SY5Y cells with SF was also shown to prevent various apoptotic events (mitochondrial depolarization, caspase 9 and 3 activation and DNA fragmentation) and necrosis elicited by 6-OHDA. Further, the impairment of antioxidant capacity and reactive oxygen species formation at intracellular level after exposure to 6-OHDA was effectively counteracted by pre-treatment with SF. Last, both the cytoprotective and antioxidant effects of SF were abolished by the addition of buthionine sulfoximine supporting the main role of GSH in the neuroprotective effects displayed by SF. These findings show that SF may play a role in preventing Parkinson's disease.     

Enhanced thioredoxin,glutathione and Nrf2 antioxidant systems by safflower extract and aceglutamide attenuate cerebral ischaemia/reperfusion injury

A large number of reactive oxygen species (ROS) aggravate cerebral damage after ischaemia/reperfusion (I/R). Glutathione (GSH), thioredoxin (Trx) and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) represent three major antioxidant systems and play vital roles in affecting each other in eliminating ROS. Identification of drugs targeting triple antioxidant systems simultaneously is vital for inhibiting oxidative damage after cerebral I/R. This study investigated the protective effect of safflower extract and aceglutamide (SAAG) against cerebral I/R injury through modulating multiple antioxidant systems of GSH, Trx and Nrf2 and identified each role of its component acegluatminde (AG) and safflower extract (SA) on these systems. Safflower extract and aceglutamide and its two components decreased neurological deficit scores, infarction rate, apoptosis and oxidative damage after cerebral I/R while enhanced cell viability, decreased reactive oxygen species and nitric oxide level in H₂O₂‐induced PC12 cell model. Importantly, compared to its two components, SAAG demonstrated more effective enhancement of GSH, Nrf2 and Trx systems and a better protection against cerebral I/R injury. The enhanced antioxidant systems prevented ASK1 activation and suppressed subsequent p38 and JNK cascade‐mediated apoptosis. Moreover, inhibition of Trx and Nrf2 systems by auranofin and ML385 abolished SAAG‐mediated protection, respectively. Thus, enhanced triple systems by SAAG played a better protective role than those by SA or AG via inhibition of ASK1 cascades. This research provided evidence for the necessity of combination drugs from the perspective of multiple antioxidant systems. Furthermore, it also offers references for the study of combination drugs and inspires novel treatments for ischaemic stroke.     

Differential regulation of nuclear and mitochondrial Bcl-2 in T cell apoptosis

Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis. D. Scheel-Toellner and K. Raza have contributed equally to this study.     

Free radical-dependent nuclear localization of Bcl-2 in the central nervous system of aged rats is not associated with Bcl-2-mediated protection from apoptosis

We have previously reported that Bcl-2 is up-regulated in the CNS of aged F344 rats as a consequence of oxidative stress. In addition to increased levels of expression, we now report that there is a subcellular redistribution of Bcl-2 in the CNS of aged F344 rats. Using western blotting, we found Bcl-2 predominantly located in the cytosol of young rats. However, in aged rats Bcl-2 was found primarily in the nucleus. This distribution, in the hippocampus and cerebellum, was reversed by treatment with the nitrone spin trap N-tert-butyl-alpha-phenylnitrone (PBN). Paradoxically, PBN treatment in young rats had the opposite effect, changing Bcl-2 from predominantly cytosolic to nuclear. We also detected an increase in Bax in aged hippocampal samples (both nuclear and cytosolic), which was reversed by treatment with PBN. The distribution of Bcl-2 and Bax in the cytosol of aged rats dramatically decreased the Bcl-2/Bax ratio, a probable indicator of neuronal vulnerability, which was restored upon treatment with PBN. In order to assess the effect of nuclear association of Bcl-2 we used PC12 cells stably transfected with a Bcl-2 construct to which we added the nuclear localization sequence of the SV40 large T antigen to the N-terminus which resulted in nuclear targeting of Bcl-2. Measurement of cell death using lactate dehydrogenase assays showed that, contrary to wild-type Bcl-2, Bcl-2 localized to the nucleus was not effective in protecting cells from treatment with 250 microm H2O2. These results suggest that nuclear localization of Bcl-2 observed in the aged CNS may not reflect a protective mechanism against oxidative stress, a major component of age-associated CNS impairments.     

Bcl-2和Bax在吗啡依赖雄性大鼠生殖细胞中的表达

目的：探讨B细胞淋巴瘤/白血病-2和人Bcl-2相关x蛋白（Bcl-2、Bax）在吗啡依赖大鼠睾丸生殖细胞中的表达及细胞凋亡可能机制,为治疗阿片类毒品造成的男性性功能减退提供理论依据。方法：以递增法每日给予雄性大鼠皮下注射盐酸吗啡针剂,建立吗啡依赖组。空白对照组注射等量生理盐水。实验成功后将两组大鼠睾丸组织作常规HE染色和免疫组化染色。结果：吗啡依赖组大鼠生精管壁细胞明显地出现上皮层次减少,仅有2~3层,细胞排列疏松,界限模糊,精子细胞和精子数目减少,并发现曲细精管腔内有脱落的生精细胞;免疫组化结果：吗啡依赖组大鼠生殖细胞中bcl-2的阳性表达率明显低于对照组（P〈0.01）,而生殖细胞中bax蛋白的阳性表达率明显高于对照组（P〈0.01）。结论：吗啡依赖可造成雄性大鼠生殖细胞凋亡数量显著增加,其机制可能是通过下调抑凋亡因子Bcl-2,上调促凋亡因子Bax,促进生殖细胞凋亡来实现的。