Protective effects of pinostilbene,a resveratrol methylated derivative,against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

Resveratrol (3,4′,5-trans-trihydroxystilbene) is a phytoalexin with emerging lines of evidence supporting its beneficial effects on cardiovascular systems and inhibition of carcinogenesis. It has also been reported that certain methylated resveratrol derivatives are more effective than resveratrol in the prevention/treatment of cancer. However, little is known about the impact of resveratrol and its derivatives on the development of Parkinson's disease. In this study, we compared the neuroprotective effects of resveratrol with four methylated (fully or partially) resveratrol derivatives against parkinsonian mimetic 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. Release of lactate dehydrogenase and activity of caspase-3 triggered by 6-OHDA were significantly reduced by resveratrol and one of the methylated derivatives, pinostilbene (3,4′-dihydroxy-5-methoxystilbene), in a dose-dependent manner. In addition, pinostilbene exerted a potent neuroprotective effect with a wider effective concentration range than resveratrol. By using high-performance liquid chromatography, we found that uptake of pinostilbene into SH-SY5Y cells was significantly higher than that of resveratrol. Enhanced bioavailability may thus be a major factor contributing to the neuroprotective activity of pinostilbene. Moreover, Western blot analysis demonstrated that pinostilbene markedly attenuated the phosphorylation of JNK and c-Jun triggered by 6-OHDA. Besides, mammalian target of rapamycin kinase may be an intracellular target accounting for the neuroprotective effects of pinostilbene. Our findings demonstrate the potential of methylated stilbenes in neuroprotection and provide important information for further research in this field.     

Protective effects of riluzole on dopamine neurons: involvement of oxidative stress and cellular energy metabolism

Riluzole is neuroprotective in patients with amyotrophic lateral sclerosis and may also protect dopamine (DA) neurons in Parkinson's disease. We examined the neuroprotective potential of riluzole on DA neurons using primary rat mesencephalic cultures and human dopaminergic neuroblastoma SH-SY5Y cells. Riluzole (up to 10 microM:) alone affected neither the survival of DA neurons in primary cultures nor the growth of SH-SY5Y cells after up to 72 h. Riluzole (1-10 microM:) dose-dependently reduced DA cell loss caused by exposure to MPP(+) in both types of cultures. These protective effects were accompanied by a dose-dependent decrease of intracellular ATP depletion caused by MPP(+) (30-300 microM:) in SH-SY5Y cells without affecting intracellular net NADH content, suggesting a reduction of cellular ATP consumption rather than normalization of mitochondrial ATP production. Riluzole (1-10 microM:) also attenuated oxidative injury in both cell types induced by exposure to L-DOPA and 6-hydroxydopamine, respectively. Consistent with its antioxidative effects, riluzole reduced lipid peroxidation induced by Fe(3+) and L-DOPA in primary mesencephalic cultures. Riluzole (10 microM) did not alter high-affinity uptake of either DA or MPP(+). However, in the same cell systems, riluzole induced neuronal and glial cell death with concentrations higher than those needed for maximal protective effects (> or =100 microM:). These data demonstrate that riluzole has protective effects on DA neurons in vitro against neuronal injuries induced by (a) impairment of cellular energy metabolism and/or (b) oxidative stress. These results provide further impetus to explore the neuroprotective potential of riluzole in Parkinson's disease.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Inhibition of tyrosinase reduces cell viability in catecholaminergic neuronal cells

The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3, 4-dihydroxyphenylalanine (L-DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L-DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH-SY5Y cells using tyrosinase inhibitors-specifically, phenylthiourea (PTU) and 5-hydroxyindole (5-HI)-and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose-dependent manner. PTU also specifically enhanced DA-induced cell death, but 5-HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5-HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L-DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA-induced cell death of SH-SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA-induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.     

Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.     

Carnosine Protects Against the Neurotoxic Effects of a Serotonin-Derived Melanoid

Anesthesia-related postoperative cognitive dysfunction (POCD) leads to morbidity in the elderly. Lipid peroxidative byproducts (i.e. acrolein) accumulate in aging and may play a role. Sevoflurane, an inhaled anesthetic, sequesters acrolein and enhances the formation of a serotonin-derived melanoid (SDM). SDM may be a biologically relevant polymeric melanoid that we previously showed exhibits redox activity and disrupts lipid bilayers. In this study, we examined the toxicity of SDM in cell culture and looked at protection using L-carnosine. SDM’s toxic effects were tested on neuronal-like SH-SY5Y cells, causing an exponential decrease in viability, while human dermal fibroblasts were completely resistant to the toxic effects. SDM brought about morphological changes to differentiated SH-SY5Y cells, particularly to neuronal processes. Co- but not pre-treatment with L-carnosine protected differentiated SH-SY5Y cells exposed to SDM. Our mechanism suggests focal sevoflurane-induced sequestration of age-related acrolein leading to SDM synthesis and neuronal impairment, which is prevented by L-carnosine.     

Compensatory activation of ERK1/2 in Atg5-deficient mouse embryo fibroblasts suppresses oxidative stress-induced cell death

Despite of the increasing evidence that oxidative stress may induce non-apoptotic cell death or autophagic cell death, the mechanism of this process is unclear. Here, we report a role and a down-stream molecular event of Atg5 during oxidative stress-induced cell death. Compared to wild type (WT) cells, Atg5-deficient mouse embryo fibroblasts (Atg5-/- MEFs) and Atg5 knockdown HT22 neuronal cells were more resistant to cell death induced by H2O2. On the contrary, Atg5-/- MEFs were as sensitive to tumor necrosis factor (TNF)-alpha and cycloheximide as WT cells, and were more sensitive to cell death triggered by amino acid-deprivation than WT MEFs. Treatment with H2O2 induced the recruitment of a GFP-LC3 fusion protein and conversion of LC3 I to LC3 II, correlated with the extent of autophagosome formation in WT cells, but much less in Atg5-deficient cells. Among stress kinases, ERK1/2 was markedly activated in Atg5-/- MEFs and Atg5 knockdown HT22 and SH-SY5Y neuronal cells. The inhibition of ERK1/2 by MEK1 inhibitor (PD98059) or dominant negative ERK2 enhanced the susceptibility of Atg5-/- MEFs to H2O2-induced cell death. Further, reconstitution of Atg5 sensitized Atg5-/- MEFs to H2O2 and suppressed the activation of ERK1/2. These results suggest that the inhibitory effect of Atg5 deficiency on cell death is attributable by the compensatory activation of ERK1/2 in Atg5-/- MEFs during oxidative stress-induced cell death.     

Growth Inhibition and Apoptosis of Neuroblastoma Cells Through ROS-Independent MEK/ERK Activation by Sulforaphane

Deregulation of apoptosis alters the balance of cell proliferation and cell death, resulting in a variety of diseases, including cancer. In recent studies, sulforaphane (SFN) has demonstrated potent anti-tumor and chemopreventive activities. A possible signal transduction pathway has also been elucidated for SFN-induced apoptosis in human neuroblastoma SH-SY5Y cells. The present study further investigates the anti-proliferation activities of SFN through induced apoptosis in SH-SY5Y cells. We found that treating SH-SY5Y cells with SFN resulted in the depletion of mitochondrial membrane potential (Δψ), which in turn increased caspase 9, caspase 3, and the up-regulation of phosphorylated MEK/ERK without generating reactive oxygen species. Results were confirmed by MTT assay, which demonstrated the cytotoxic activity of SFN against SH-SY5Y cells (IC₅₀ values of 20 μM).     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.     

Oxygen Glucose Deprivation (OGD)/Re-Oxygenation-Induced In Vitro Neuronal Cell Death Involves Mitochondrial Cyclophilin-D/P53 Signaling Axis

Oxidative stress-induced neuronal cell death requires opening of the mitochondrial permeability transition pore. P53 mitochondrial translocation and association with Cyclophilin D (Cyp-D) is required for the pore opening. Here we tested this signaling axis in oxygen glucose deprivation (OGD)/re-oxygenation-induced in vitro neuronal death. Using mitochondrion immunoprecipitation, we found that p53 translocated to mitochondrion and associated with Cyp-D in SH-SY5Y cells exposed to (OGD)/re-oxygenation. Disruption of this complex by Cyp-D inhibitor Cyclosporine A (CsA), or by Cyp-D or p53 deficiency, significantly inhibited OGD/re-oxygenation-induced apoptosis-independent cell death. Conversely, over-expression of Cyp-D in SH-SY5Y cells caused spontaneous cell death, and these cells were more vulnerable to OGD/re-oxygenation. Finally, CsA or Cyp-D RNAi suppressed OGD/re-oxygenation-induced neuronal cell death in primary cultures. Together, our study suggests that OGD/re-oxygenation-induced in vitro cell death involves a mitochondrial Cyp-D/p53 signaling axis.     

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depleted of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.     

Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [(3)H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired.     

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.     

MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes

MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0–G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells.     

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.