Function and evolution of vertebrate globins

Globins are haem‐proteins that bind O₂ and thus play an important role in the animal's respiration and oxidative energy production. However, globins may also have other functions such as the decomposition or production of NO, the detoxification of reactive oxygen species or intracellular signalling. In addition to the well‐investigated haemoglobins and myoglobins, genome sequence analyses have led to the identification of six further globin types in vertebrates: androglobin, cytoglobin, globin E, globin X, globin Y and neuroglobin. Here, we review the present state of knowledge on the functions, the taxonomic distribution and evolution of vertebrate globins, drawing conclusions about the functional changes underlying present‐day globin diversity.     

p62 protects SH-SY5Y neuroblastoma cells against H2O2-induced injury through the PDK1/Akt pathway

The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H₂O₂ treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H₂O₂-induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H₂O₂-induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinase-defective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3θ protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival.     

Protective effects of quercetin against hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species that has been implicated in various neurodegenerative diseases. Quercetin, one of the plant flavonoids, has been reported to harbor various physiological properties including antioxidant activity. In this study, we investigated the neuroprotective effects of quercetin against H₂O₂-induced apoptosis in human neuronal SH-SY5Y cells. H₂O₂-mediated cytotoxicity and lactate dehydrogenase release were suppressed in a quercetin concentration-dependent manner. In addition, quercetin repressed the expression of the pro-apoptotic Bax gene and enhanced that of the anti-apoptotic Bcl-2 gene in SH-SY5Y cells. Moreover, quercetin effectively inhibited the activation of the caspase cascade that leads to DNA fragmentation, a key feature of apoptosis, and subsequent cell death. These results indicate the importance of quercetin in protecting against H₂O₂-mediated neuronal cell death. Thus, quercetin might potentially serve as an agent for prevention of neurodegenerative diseases caused by oxidative stress and apoptosis.     

Reactive oxygen species production and MAPK activation are implicated in tetrahydrobiopterin-induced SH-SY5Y cell death

Tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine (DA) synthesis, has been shown to produce reactive oxygen species (ROS) upon its autoxidation and induce selective dopaminergic cell death in many in vivo and in vitro models of Parkinson's disease (PD). The precise molecular mechanisms underlying neuronal death upon BH4 exposure, however, have not yet been well elucidated. The present study aims to examine the intracellular ROS production and the signal transduction pathways underlying the toxic effects of BH4 on human dopaminergic SH-SY5Y cells. The results show that BH4 treatment at concentrations ranging from 50 μM to 400 μM induces neuronal death in a dose-dependent manner. In concomitant with the elevation of intracellular ROS formation, BH4-induced activation of MAPK, p38 and ERK1/2 in SH-SY5Y cells is attenuated by pretreatment with MAPK inhibitors, SB203580 or PD98059. These data indicate that MAPK activation and oxidative stress are involved in BH4-induced dopaminergic cell death, possibly through the autoxidation of BH4 and subsequent ROS production.     

Lithium and oxidative stress lessons from the MPTP model of Parkinson's disease

Lithium has been successfully employed therapeutically for treatment of bipolar depressive illness; however, its mechanism of action is poorly understood. Recently, it has been demonstrated by us that lithium can prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) dopaminergic neurotoxicity in mice. From analyzing the pattern of protection in various parameters, we suggest that lithium protects against MPTP-induced depletion of striatal dopamine (DA) by preventing free radical-induced inactivation of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. Possible neuroprotective effect of lithium against H₂O₂-induced cell death was assessed in human neuroblastoma; SH-SY5Y cell line. Pretreatment with LiCl (2 mM and 4 mM) for 7 days protected against H₂O₂ neurotoxicity in a dose-dependent manner. However, this protection could not be achieved through short-term incubation with LiCl. In agreement; we found that lithium lacks immediate antioxidant activity using the in vitro lipid peroxidation essay indicating that not acute but chronic treatment with lithium allows cells to deal better with oxidative stress.     

Opposing role of JNK-p38 kinase and ERK1/2 in hydrogen peroxide-induced oxidative damage of human trophoblast-like JEG-3 cells

Trophoblasts play a crucial role in embryo implantation and maintenance of normal pregnancy. Recently, oxidative stress has been considered as one important factor in the pathogenesis of spontaneous abortion and preeclampsia. Many studies have reported that the plasma levels of hydrogen peroxide (H₂O₂) are significantly increased in women with preeclampsia, but the mechanisms involved in H₂O₂-induced cell cytotoxicity in trophoblasts are still not completely explained. Our present study was undertaken to provide a united understanding of the role of oxidative stress generated by H₂O₂ on human trophoblasts and the underlying intracellular signaling pathways. Exposure to H₂O₂ resulted in a concentration-dependent growth decrease and apoptosis in human trophoblast-like JEG-3 cells. H₂O₂ treatment also caused intracellular reactive oxygen species (ROS) production and concomitant dissipation of the mitochondrial membrane potential. The three MAPK subfamilies, ERK1/2, JNK and p38 kinase, were all activated under H₂O₂-induced oxidative stress. Blocking the activation of JNK and p38 kinase increased cell viability and decreased apoptosis induced by H₂O₂ with their respective inhibitors, SP600125 and SB203580. However, preventing ERK1/2 activation further increased H₂O₂-induced cell death with U0126, an inhibitor of ERK upstream kinase MEK1/2. Taken together, these findings suggest that the mitochondria-dependent pathways and JNK-p38 kinase pathways are involved in H₂O₂-induced oxidative damage of human trophoblast-like JEG-3 cells, while ERK1/2 pathway may play an active role in cell survival following oxidant injury.     

Purified high molecular weight synthetic Aβ(1–42) and biological Aβ oligomers are equipotent in rapidly inducing MTT formazan exocytosis

Synthetic soluble Aβ oligomers are often used as a surrogate for biologic material in a number of model systems. We compared the activity of Aβ oligomers (synthetic and cell culture media derived) on the human SH-SY5Y neuroblastoma and C2C12 mouse myoblast cell lines in a novel, modified MTT assay. Separating oligomers from monomeric peptide by size exclusion chromatography produced effects at peptide concentrations approaching physiologic levels (10–100 nM). Purified oligomers, but not monomers or fibrils, elicited an increase of a detergent-insoluble form of MTT formazan within 2 h as opposed to a control toxin (H₂O₂). This effect was comparable for biological and synthetic peptide in both cell types. Monomeric Aβ attenuated the effect of soluble oligomers. This study suggests that the activities of biological and synthetic oligomers are indistinguishable during early stages of Aβ oligomer–cell interaction.     

Phospholipids block nuclear factor-kappa B and tau phosphorylation and inhibit amyloid-beta secretion in human neuroblastoma cells

Inflammation and oxidative stress have been shown to play a critical role in the pathophysiology that leads to neurodegeneration. Omega-6 phospholipids, e.g. dilinoleoylphosphatidylcholine (DLPC), have been shown to have anti-inflammatory properties and therefore experiments were undertaken to determine whether DLPC can prevent inflammatory neurodegenerative events in the model neuronal cell line, SH-SY5Y. Tumor necrosis factor (TNF-α) and H₂O₂ activate mitogen-activated protein kinase (MAPK) in SH-SY5Y cells within 5 min and this activation is completely blocked by DLPC (12 μM). DLPC blocks IκBα phosphorylation in the SH-SY5Y cells and prevents the phosphorylation and activation of nuclear factor-kappa B (NF-κB). The phospholipid inhibits induction of MAPK and NF-κB in similar fashion to the MEK1/2-inhibitor, U0126 (10 μM). DLPC completely abolishes TNF-α, H₂O₂ and lipopolysaccaride (LPS)-induced neuronal tau phosphorylation. Cellular amyloid precursor protein levels are reduced by DLPC and LPS-induced amyloid-β expression and secretion in SH-SY5Y cells are completely blocked by DLPC. Taken together, these data suggest that DLPC can act through MAPK to block neuronal inflammatory cascades and prevent potential pathological consequences in the neuronal metabolism of amyloid and tau proteins.     

Protective effect of pyrroloquinoline quinone against Aβ-induced neurotoxicity in human neuroblastoma SH-SY5Y cells

The neurotoxicity of aggregated β-amyloid (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease (AD). It can cause neurotoxicity in AD by evoking a cascade of oxidative damage-dependent apoptosis to neurons. In the present study, we for the first time investigated the protective effect of pyrroloquinoline quinone (PQQ), an anionic, water soluble compound that acts as a redox cofactor of bacterial dehydrogenases, on Aβ-induced SH-SY5Y cytotoxicity. Aβ_25–35 significantly reduced cell viability, increased the number of apoptotic-like cells, and increased ROS production. All of these phenotypes induced by Aβ_25–35 were markedly reversed by PQQ. PQQ pretreatment recovered cells from Aβ_25–35-induced cell death, prevented Aβ_25–35-induced apoptosis, and decreased ROS production. PQQ strikingly decreased Bax/Bcl-2 ratio, and suppressed the cleavage of caspase-3. These results indicated that PQQ could protect SH-SY5Y cells against β-amyloid induced neurotoxicity.     

Antioxidative Potential of Duranta Repens (Linn.) Fruits Against H2O2 Induced Cell Death In Vitro

The effects of Duranta repens fruits were investigated on H₂O₂ induced oxidative cell death to evaluate its antioxidative potential in vitro. HEK293T cells were treated with different concentrations [0–1000 µg/ ml] of ethanol extract (E-Ex) and methanol extract (M-Ex) of D. repens for 24h, and then treated with 100 µM H₂O₂ for 24h. Cell viability, antioxidant parameters of cells, and antioxidant constituents of the extracts were determined. Treatment with limited dose of E-Ex or M-Ex increased the survival rate of H₂O₂-treated HEK293T cells, however the extra-high dose showed growth inhibitory effect. Treatment with E-Ex or M-Ex protected cellular lipid per-oxidation. In vitro analyses showed the 2,2-diphenyl-1-picrylhydrazyl and H₂O₂ scavenging activities as well as reducing potential of the extracts. We report here that the limited dose of E-Ex and M-Ex possess antioxidative potential, which can protect H₂O₂-induced oxidative cell damage.     

Quercetin protects neuroblastoma SH-SY5Y cells against oxidative stress by inhibiting expression of Krüppel-like factor 4

Quercetin is one of flavonoids with cyto-protective activities. It has been demonstrated that quercetin inhibits oxidative stress in some animal models and specific cells, but the particular mechanism is known a little. In the present study, we found that quercetin could decrease the expression of Krüppel-like factor 4 (KLF4) induced by hydrogen peroxide (H₂O₂) in human neuroblastoma SH-SY5Y cells, further increase the expression ratio of bcl-2 to bax, which were apoptotic-related target genes of KLF4, thus alleviate the apoptotic rate and caspase-3 enzyme activity of SH-5YSY cells; the overexpression or inhibition of KLF4 demonstrated the mediated role of KLF4 for the protective effect of quercetin on cell damage induced by H₂O₂. All results suggest a potential molecular mechanism of quercetin protecting against the oxidative damage, which may be applied in the treatment of oxidative related diseases, such as neurodegeneration diseases.     

Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway

Oxidative stress by exposure to H₂O₂ induces various types of cell death depending on cell type and conditions. We report herein on a study of the mechanisms underlying H₂O₂-induced cell death in C6 glioma cells. The findings show that H₂O₂ triggers a caspase-independent autophagic cell death in these cells. The findings also show that H₂O₂ induces the dephosphorylation of the mammalian target of rapamycin (mTOR) at Ser 2481 and the p70 ribosomal protein S6 kinase (p70S6K) at Thr389 in a Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3)-dependent manner. BNIP3 has the capacity to inhibit mTOR activity and mTOR inhibition plays a role in autophagic induction. This suggests that BNIP3 may mediate H₂O₂-induced autophagic cell death through the suppression of mTOR. The findings show that the down-regulation of BNIP3 by BNIP3 siRNA prevents C6 cells from undergoing H₂O₂-induced autophagic cell death. Collectively, these results suggest that H₂O₂ induces autophagic cell death in C6 cells via the BNIP3-mediated suppression of the mTOR pathway.     

Buffalo casein derived peptide can alleviates H2O2 induced cellular damage and necrosis in fibroblast cells

Oxidative stress is one of a critical pathogenic factor in the progression of aging and chronic diseases such as cancer, myocardial inflammation and diabetes. In the present scenario, peptides with short half life and more biological specificities are gaining much attention as prodrugs. Thus, the present investigation carried out to screen potential antioxidative peptide, VLPVPQK to cope with the cellular oxidative damage. Our results showed that treatment of rat fibroblast cells with 0.2 mM H₂O₂ for 6 h significantly declined different oxidative stress biomarkers such as SOD, CAT, GSH, and promoted LDH activity. In addition, ROS and TNF-α levels were also increased upon H₂O₂ exposure for 6 h and thereby, it induced cell death. Amazingly, pretreatment of the peptide (VLPVPQK) significantly elevated cell survivability, by reversing all H₂O₂ induced alterations in fibroblast cells. Therefore, our results indicated that, the peptide (VLPVPQK) acted as a potential cytoprotective agent, who restored redox balance and cell homeostasis in cultured fibroblast cells, even after H₂O₂ exposure, suggesting that the peptide can be valuable as an effective remedy in treatment of oxidative stress related diseases and skin inflammation related disorders.     

Lipid Peroxidation in the Liver of Mice during Development of Granulomatous Inflammation after Combined Infection with <Emphasis Type="Italic">C. albicans</Emphasis> and <Emphasis Type="Italic">M. tuberculosis</Emphasis>

Programmed death of peripheral blood mononuclear cells from healthy donors was studied during culturing with various concentrations of H₂O₂ and selective inhibitors of JNK (SP600125) and p38 MAPK (ML3403). In vitro incubation of mononuclear leukocytes with 1 mM H₂O₂ stimulated apoptotic cell death. Treatment with inhibitors (SP600125 and ML3403) during in vitro oxidative stress prevented the increase in the number of annexin-positive mononuclear cells. Our results indicate that MAP kinases JNK and p38 are involved in the mechanisms of oxidative dysregulation of apoptosis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 505–508, May, 2008     

Homocysteine induced SH-SY5Y apoptosis through activation of NADPH oxidase in U251MG cells

Epidemiological studies have indicated a correlation between homocysteinemia and dementia, including Alzheimer's disease. However, the mechanism by which homocysteine (Hcy) induces neuronal cell death remains unknown. We found that micromolar concentrations of Hcy induced neuroblastoma SH-SY5Y cell death only when co-cultured with glioblastoma U251MG cells. In this culture system, cysteine had no effect on SH-SY5Y cell death. There was an increase in TUNEL-positive cells and loss of mitochondrial membrane potential following treatment with 100 μM Hcy. Addition of conditioned medium prepared from U251MG cells in the presence of 100 μM Hcy also reduced SH-SY5Y cell viability, while this effect was prevented when using conditioned medium from U251MG cells exposed to 100 μM Hcy + apocynin, a specific NADPH oxidase inhibitor. Following exposure to 100 μM Hcy in U251MG cells, expression of Rac1, a compartment of NADPH oxidase, was translocated to the plasma membrane, and the active form of Rac1 was increased. There was no change in peroxide concentration in the medium of U251MG cells after addition of Hcy. Overall, these data suggest that Hcy stimulates Rac1 activation and NADPH oxidase, resulting in superoxide anion production that may induce SH-SY5Y cell apoptosis.     

Protective effects of the citrus flavanones to PC12 cells against cytotoxicity induced by hydrogen peroxide

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities. One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the protective actions of citrus flavanones naringin and nobiletin against the cytotoxicity induced by exposure to hydrogen peroxide (H₂O₂) (150 μM, 3 h) in PC12 cells. The results showed that naringin and nobiletin inhibited the decrease of cell viability (MTT reduction), prevented membrane damage (LDH release), scavenged ROS formation, reduced caspase-3 activity, and attenuated the decrease of mitochondrial membrane potential (MMP), respectively, in H₂O₂-induced PC12 cells. Meanwhile, naringin and nobiletin increased superoxide dismutase (SOD) and glutathione (GSH) activity, while decreased malondialdehyde (MDA), the production of lipid peroxidation, in H₂O₂-induced PC12 cells. In addition, the percentage of cells undergoing H₂O₂-induced apoptosis was decreased in the presence of naringin and nobiletin. These results first demonstrate that naringin and nobiletin, even at physiological concentrations, have neuroprotective effects against H₂O₂-induced cytotoxicity in PC12 cells. All the above results suggest that these dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.     

Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stress-induced hydroxyl radical formation in mitochondria

Although nitric oxide (NO) and oxidative stress both contribute to proinflammatory cytokine toxicity in pancreatic β-cells during type 1 diabetes mellitus (T1DM) development, the interactions between NO and reactive oxygen species (ROS) in cytokine-mediated β-cell death have not been clarified. Exposure of insulin-producing RINm5F cells to IL-1β generated NO, while exposure to a combination of IL-1β, TNF-α, and IFN-γ, which simulates T1DM conditions, generated both NO and ROS. In theory, two reactions between NO and ROS are possible, one with the superoxide radical yielding peroxynitrite, and the other with hydrogen peroxide (H₂O₂) yielding hydroxyl radicals. Results of the present work exclude peroxynitrite involvement in cytokine toxicity to β-cells because its generation did not correlate with the toxic action of cytokines. On the other hand, we show that H₂O₂, produced upon exposure of insulin-producing cell clones and primary rat islet cells to cytokines almost exclusively in the mitochondria, reacted in the presence of trace metal (Fe⁺⁺) with NO forming highly toxic hydroxyl radicals, thus explaining the severe toxicity that causes apoptotic β-cell death. Expression of the H₂O₂-inactivating enzyme catalase in mitochondria protected against cytokine toxicity by preventing hydroxyl radical formation. We therefore conclude that proinflammatory cytokine-mediated β-cell death is due to nitro-oxidative stress-mediated hydroxyl radical formation in the mitochondria.     

Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by amyloid-β peptide (1–42)

Accumulation and deposition of amyloid β peptide (Aβ) in the brain causes neuronal apoptosis and eventually leads to Alzheimer's disease (AD). A therapeutic target for AD is to block the cascade reaction induced by Aβ. It has been demonstrated that glucagon-like peptide-1 (GLP-1), which is an endogenous insulinotropic peptide secreted from the gut, binds to its receptor in the brain and possesses neuroprotective effects. Using site-directed mutagenesis and gene recombination techniques, we generated a mutated recombinant human glucagon-like peptide-1 (mGLP-1) which has longer half-life as compared with native GLP-1. This present work aims to examine whether mGLP-1 attenuates Aβ_1–42-mediated cytotoxicity in SH-SY5Y cells and to explore the possible mechanisms. Our data indicate that ≥0.02 ng/ml of mGLP-1 facilitated cell proliferation and 0.1 ng/ml and 0.5 ng/ml of mGLP-1 rescued SH-SY5Y cells from Aβ_1–42-induced apoptosis. Moreover, Aβ_1–42 treatment dramatically stimulated the release of Ca²⁺ from internal calcium stores in SH-SY5Y cells, while mGLP-1 helped to maintain the intracellular Ca²⁺ homeostasis. Aβ_1–42 also significantly increased the expression level of TP53 and Bax genes which are involved in apoptotic pathways, and mGLP-1 decreased Aβ_1–42-induced up-regulation of TP53 and Bax. Since mGLP-1 treatment elevated cytosolic cAMP concentration in SH-SY5Y cells, we postulate that mGLP-1 may exert its influence via binding to GLP-1 receptors in SH-SY5Y cells and stimulating the production of cAMP. These results suggest that mGLP-1 exhibited neuronal protection properties, and could potentially be a novel therapeutic agent for intervention in Alzheimer's disease.