Stress In Neuroblastoma SH-SY5Y Cells Research Articles

Exendin-4 protects against high glucose-induced mitochondrial dysfunction and oxidative stress in SH-SY5Y neuroblastoma cells through GLP-1 receptor/Epac/Akt signaling

Mitochondrial dysfunction under diabetic condition leads to the development and progression of neurodegenerative complications. Recently, the beneficial effects of glucagon-like peptide-1 (GLP-1) receptor agonists on diabetic neuropathies have been widely recognized. However, molecular mechanisms underlying the neuroprotective effects of GLP-1 receptor agonists against high glucose (HG)-induced neuronal damages is not completely elucidated. Here, we investigated the underlying mechanisms of GLP-1 receptor agonist treatment against oxidative stress, mitochondrial dysfunction, and neuronal damages under HG conditions mimicking a diabetic hyperglycemic state in SH-SY5Y neuroblastoma cells. We revealed that treatment with exendin-4, a GLP-1 receptor agonist, not only increased the expression of survival markers, phospho-Akt/Akt and Bcl-2, but also decreased the expression of pro-apoptotic marker, Bax, and reduced the levels of reactive oxygen species (ROS) defense markers (catalase, SOD-2, and HO-1) under HG conditions. The expressions of mitochondrial function associated genes, MCU and UCP3, and mitochondrial fission genes, DRP1 and FIS1, were decreased by exendin-4 compared to non-treated levels, while the protein expression levels of mitochondrial homeostasis regulators, Parkin and PINK1, were enhanced. In addition, blockade of Epac and Akt activities was able to antagonize these neuroprotective effects of exendin-4. Collectively, we demonstrated that stimulation of GLP-1 receptor propagates a neuroprotective cascade against the oxidative stress and mitochondrial dysfunction as well as augments survival through the Epac/Akt-dependent pathway. Therefore, the revealed mechanisms underlying GLP-1 receptor pathway by preserving mitochondrial homeostasis would be a therapeutic candidate to alleviate neuronal dysfunctions and delay the progression of diabetic neuropathies.

Design and biological evaluation of Repaglinide loaded polymeric nanocarriers for diabetes linked neurodegenerative disorder: QbD-driven optimization, in situ, in vitro and in vivo investigation

Diabetes mellitus is a metabolic disorder characterized by inadequate insulin secretion and signaling dysfunction, leading to a vast spectrum of systemic complications. These complications trigger cascades of events that result in amyloid-beta plaque formation and lead to neurodegenerative disorders such as Alzheimer’s. Repaglinide (REP) an insulinotropic agent, suppresses the down regulatory element antagonist modulator (DREAM) and enhances the ATF6 expression to provide neuroprotection following the DREAM/ATF6/apoptotic pathway. However, oral administration of REP for brain delivery becomes more complicated due to its physicochemical characteristics (high protein binding (>98%), low permeability, short half-life (∼1 h), low bioavailability). Therefore, to circumvent these problems, we develop a polymeric nanocarrier system (PNPs) by in-house synthesized di-block copolymer (PEG-PCL). PNPs were optimized using quality by design approach response surface methodology and characterized by particle size (112.53 ± 5.91 nm), PDI (0.157 ± 0.08), and zeta potential (−6.20 ± 0.82 mV). In vitro release study revealed that PNPs (∼70% in 48 h) followed the Korsmeyer-Peppas model with a Fickian diffusion release pattern, and in intestinal absorption assay PNPs showed increment of ∼1.3 folds compared of REP. Moreover, cellular studies confirmed that REP-loaded PNPs significantly enhance the cellular viability, uptake and reduce the peroxide-induced stress in neuroblastoma SHSY-5Y cells. Further, pharmacokinetic parameters of PNPs showed an increment in tmax (2.46-fold), and Cmax (1.25-fold) associated with REP. In the brain biodistribution study, REP loaded PNPs was sustained for 24 h whereas free REP sustained only for12 h. In DM induced neurodegenerative murine model, a significantly (p < 0.01) enhanced pharmacodynamic was observed in PNP treated group by estimating biochemical and behavioral parameters. Hence, oral administration of REP-loaded PNPs promotes efficient brain uptake and improved efficacy of REP in the diseased model.

Development and in vitro safety evaluation of pramipexole-loaded hollow mesoporous silica (HMS) particles

In this study, spherical hollow mesoporous silica (HMS) particles with average size of 550 nm were loaded with dopamine-receptor agonist pramipexole, as a model drug. High drug loading (55%) was achieved by its incorporation in the reservoir structure of HMS. The newly developed drug-delivery systems were further double-coated with bioadhesive polymers chitosan and sodium alginate to achieve sustained release. The double chitosan/sodium alginate coating diminished the initial burst effect and lowered pramipexole release at two pH values (1.2 and 6.8) as shown by in vitro release studies. The haemolytic assay and MTT-test in human neuroblastoma SH-SY5Y cells showed good safety profile of the novel pramipexole-loaded HMS particles. Moreover, a more pronounced protection of human neuroblastoma SH-SY5Y cells against H2O2-induced oxidative damage was observed upon treatment with pramipexole-loaded (non-coated or chitosan-coated) HMS particles compared to the free drug. In conclusion, we found that HMS particles might be of great interest as a promising drug-delivery system for pramipexole. The coating with biopolymer chitosan modified both the drug-delivery process and the in vitro protection against oxidative stress in neuroblastoma SH-SY5Y cells.

Bradykinin-potentiating PEPTIDE-10C, an argininosuccinate synthetase activator, protects against H2O2-induced oxidative stress in SH-SY5Y neuroblastoma cells

Bradykinin-potentiating peptides (BPPs – 5a, 7a, 9a, 10c, 11e, and 12b) of Bothrops jararaca (Bj) were described as argininosuccinate synthase (AsS) activators, improving l-arginine availability. Agmatine and polyamines, which are l-arginine metabolism products, have neuroprotective properties. Here, we investigated the neuroprotective effects of low molecular mass fraction from Bj venom (LMMF) and two synthetic BPPs (BPP-10c, <ENWPHPQIPP; BPP-12b, <EWGRPPGPPIPP) in the SH-SY5Y cell line against H2O2-induced oxidative stress. The neuroprotective effects against H2O2-induced were analyzed by reactive oxygen species (ROS – DCFH) production; lipid peroxidation (TBARS); intracellular GSH; AsS, iNOS, and NF-kB expressions; nitrite levels (Griess); mitochondrial membrane potential (TMRM); and antioxidant activity (DPPH). Analysis of variance followed by Tukey’s post hoc test were calculated for statistical comparisons. Pre-treatment with both BPPs significantly reduced cell death induced by H2O2, but BPP-10c showed higher protective capacity than BPP-12b. LMMF pretreatment was unable to prevent the reduction of cell viability caused by H2O2. The neuroprotective mechanism of BPP-10c against oxidative stress was investigated. BPP-10c reduced ROS generation and lipid peroxidation in relation to cells treated only with H2O2. BBP-10c increased AsS expression and was not neuroprotective in the presence of MDLA, a specific inhibitor of AsS. BPP-10c reduced iNOS expression and nitrate levels but decreased NF-kB expression. Furthermore, BPP-10c protected the mitochondrial membrane against oxidation. Overall, we demonstrated for the first time neuroprotective mechanisms of BPPs against oxidative stress, opening new perspectives to the study and application of these peptides for the treatment of neurodegenerative diseases.

Differentiation impairs Bach1 dependent HO-1 activation and increases sensitivity to oxidative stress in SH-SY5Y neuroblastoma cells

Neuronal adaptation to oxidative stress is crucially important in order to prevent degenerative diseases. The role played by the Nrf2/HO-1 system in favoring cell survival of neuroblastoma (NB) cells exposed to hydrogen peroxide (H2O2) has been investigated using undifferentiated or all-trans retinoic acid (ATRA) differentiated SH-SY5Y cells. While undifferentiated cells were basically resistant to the oxidative stimulus, ATRA treatment progressively decreased cell viability in response to H2O2. HO-1 silencing decreased undifferentiated cell viability when exposed to H2O2, proving the role of HO-1 in cell survival. Conversely, ATRA differentiated cells exposed to H2O2 showed a significantly lower induction of HO-1, and only the supplementation with low doses of bilirubin (0,5–1 μM) restored viability. Moreover, the nuclear level of Bach1, repressor of HO-1 transcription, strongly decreased in undifferentiated cells exposed to oxidative stress, while did not change in ATRA differentiated cells. Furthermore, Bach1 was displaced from HO-1 promoter in undifferentiated cells exposed to H2O2, enabling the binding of Nrf2. On the contrary, in ATRA differentiated cells treated with H2O2, Bach1 displacement was impaired, preventing Nrf2 binding and limiting HO-1 transcription. In conclusion, our findings highlight the central role of Bach1 in HO-1-dependent neuronal response to oxidative stress.

In vitro protective effects of encapsulated quercetin in neuronal models of oxidative stress injury

ABSTRACTThe aim of the present study was to evaluate the protective effects of free and encapsulated quercetin against oxidative stress injury in neuronal SH-SY5Y cells and isolated rat brain synaptosomes. Quercetin (QR) was encapsulated in nanoparticles consisting of sodium alginate and chitosan and the ratio between both biopolymers was 10:1 or 1:10, respectively. The nanoparticles formulated with higher amount of sodium alginate were negatively charged, whereas those with higher chitosan amount were positively charged. The protective effects of free and encapsulated quercetin were studied in two in vitro models: H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells and 6-hydroxydopamine (6-OHDA) induced neurotoxicity in isolated rat brain synaptosomes. Comparing to free quercetin, quercetin encapsulated in the nanoparticles formulated with higher chitosan concentration exerted more pronounced neuroprotective activity in a model of H2O2-induced (1 mmol/L H2O2, 15 min) oxidative stress in neuroblastoma SH-SY5Y cells. A similar trend was observed in a model of 6-OHDA induced neurotoxicity in rat brain synaptosomes. In conclusion, encapsulation of quercetin in nanoparticles based predominantly on chitosan improved its neuroprotective activity in vitro.

Evaluation of the Protective Effects of Sarains on H2O2-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Neuroblastoma Cells.

Sarains are diamide alkaloids isolated from the Mediterranean sponge Haliclona (Rhizoniera) sarai that have previously shown antibacterial, insecticidal and anti-fouling activities. In this study, we examined for the first time the neuroprotective effects of sarains 1, 2 and A against oxidative stress in a human neuronal model. SH-SY5Y cells were co-incubated with sarains at concentrations ranging from 0.01 to 10μM, and the well-known oxidant hydrogen peroxide at 150μM for 6h and the protective effects of the compounds were evaluated. Among the sarains tested, sarain A was the most promising compound, improving mitochondrial function and decreasing reactive oxygen species levels in human neuroblastoma cells treated with the compound at 0.01, 0.1 and 1μM. This compound was also able to increase the activity of the antioxidant enzymes superoxide dismutases by inducing the translocation of the nuclear factor E2-related factor 2 (Nrf2) to the nucleus at the lower concentrations tested (0.01 and 0.1μM). Moreover, sarain A at 0.1 and 1μM blocked the mitochondrial permeability transition pore (mPTP) opening through cyclophilin D inhibition. These results suggest that the protective effects produced by the treatment with sarain A are related with its ability to block the mPTP and to enhance the Nrf2 pathway, indicating that sarain A may be a candidate compound for further studies in neurodegenerative diseases.

200 - Aggravation of Oxidative Stress after Thioredoxin Reductase Deficiency Interrupts Starvation-Induced Autophagy through Oxidative Modification of Lysosomal Cathepsins

Oxidative damage and aggregation of cellular proteins is a hallmark of neurodegenerative diseases. Autophagy induction as a means for degradation of protein aggregates in lysosomes is an important vital process in normal conditions, and its interruption has been linked to neuronal cell death in these diseases. The importance of redox status of the cell in induction of autophagy has been linked to oxidative modification of cysteine groups in key molecular proteins. The cellular redox milieu is regulated by the availability of reduced glutathione and Thioredoxin system and is important in fine tuning of many cellular function including the autophagy machinery. Amongst the intricate network of many autophagy related proteins (ATGs), only ATG4 has been identified as a potential target of Trx; we therefore aimed to further investigate the role of Trx system in regulation of autophagy process. Using pharmacological and RNA-editing technology we show that exacerbation of oxidative stress after Trx-reductase deficiency in a model of nutritional stress in neuroblastoma SH-SY5Y cells interferes with autophagy progression and promotes apoptosis. This was associated with decreased protein-degradation in lysosomes due to altered lysosomal acidification and accumulation of autophagosomes. Previous studies have shown the critical role of low levels of ROS for normal induction of autophagy. Our study provides novel evidence that aggravated oxidative stress affects the final stages of autophagy by inhibiting lysosome maturation by modification of cathepsin processing. This provides a novel mechanistic link between protein aggregation and conversion of autophagy to apoptosis in SH-SY5Y cells.

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

Antiglycative activity of sulforaphane: a new avenue to counteract neurodegeneration?

L-Carnitine attenuates H2O2-induced neuron apoptosis via inhibition of endoplasmic reticulum stress

Both oxidative stress and endoplasmic reticulum stress (ER stress) have been linked to pathogenesis of neurodegenerative diseases. Our previous study has shown that l-carnitine may function as an antioxidant to inhibit H2O2-induced oxidative stress in neuroblastoma SH-SY5Y cells. To further explore the neuroprotection of l-carnitine, here we study the effects of l-carnitine on the ER stress response in H2O2-induced SH-SY5Y cell injury. Our results showed that l-carnitine pretreatment could increase cell viability; inhibit apoptosis and ROS accumulation caused by H2O2 or tunicamycin (TM). l-carnitine suppress the endoplasmic reticulum dilation and activation of ER stress-associated proteins including glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), JNK, Bax and Bim induced by H2O2 or TM. In addition, H2O2-induced cell apoptosis and activation of ER stress can also be attenuated by antioxidant N-acetylcysteine (NAC), CHOP siRNA and the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, our results demonstrated that H2O2 could trigger both oxidative stress and ER stress in SH-SY5Y cells, and ER stress participated in SH-SY5Y apoptosis mediated by H2O2-induced oxidative stress. CHOP/Bim or JNK/Bim-dependent ER stress signaling pathways maybe related to the neuroprotective effects of l-carnitine against H2O2-induced apoptosis and oxidative injury.

PEP-1–ribosomal protein S3 protects dopaminergic neurons in an MPTP-induced Parkinson's disease mouse model

Parkinson's disease (PD) is a neurodegenerative disease characterized by a gradual loss of dopaminergic (DA) neurons in the substantia nigra (SN) of the brain. Ribosomal protein S3 (rpS3) has multiple functions related to protein synthesis, antioxidative activity, and UV endonuclease III activity. We have previously shown that PEP-1–rpS3 inhibits skin inflammation and provides neuroprotection against experimental cerebral ischemic damage. In this study, we examined whether PEP-1–rpS3 can protect DA neurons against oxidative stress in SH-SY5Y neuroblastoma cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1–rpS3 was efficiently delivered to SH-SY5Y cells and the SN of the brain as confirmed by Western blot and immunohistochemical analysis. PEP-1–rpS3 significantly inhibited reactive oxygen species generation and DNA fragmentation induced by 1-methyl-4-phenylpyridinium, consequently leading to the survival of SH-SY5Y cells. The neuroprotection was related to the antiapoptotic activity of PEP-1–rpS3 that affected the levels of proapoptotic and antiapoptotic mediators. In addition, immunohistochemical data collected using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that PEP-1–rpS3 markedly protected DA cells in the SN against MPTP-induced oxidative stress. Therefore, our results suggest that PEP-1–rpS3 may be a potential therapy for PD.

Biological Effects of MC2050, a Quinazoline‐Based PARP‐1 Inhibitor, in Human Neuroblastoma and EBV‐Positive Burkitt′s Lymphoma Cells

The master of ceremonies! MC2050, a new water-soluble quinazoline-based PARP-1 inhibitor, exerts a cytoprotective effect against oxidative stress in SH-SY5Y neuroblastoma cells by abolishing the toxic effect of H2O2 and counteracting PARP-1 overactivation. Moreover, pretreatment of lytic-cycle-induced Raji cells with MC2050 leads to the modulation of EBNA1 protein expression.

Protective Effects of Flavonoids Against Oxidative Stress Induced by Simulated Microgravity in SH-SY5Y Cells

Many lines of evidence suggest that microgravity results in increased oxidative stress in the nervous system. In order to protect neuronal cells from oxidative damage induced by microgravity, we selected some flavonoids that might prevent oxidative stress because of their antioxidant activities. Among the 20 flavonoids we examined, we found that isorhamnetin and luteolin had the best protective effects against H(2)O(2) or SIN-1-induced cytotoxicity in SH-SY5Y cells. Using a clinostat to simulate microgravity, we found that isorhamnetin and luteolin treatment protected SH-SY5Y cells by preventing microgravity-induced increases in reactive oxygen species (ROS), nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels, and a decrease in antioxidant power (AP). Moreover, isorhamnetin and luteolin treatment downregulated the expression of inducible nitric oxide synthase (iNOS), and oxidative stress was significantly inhibited by an iNOS inhibitor in SH-SY5Y cells exposed to simulated microgravity (SMG). These results indicate that isorhamnetin and luteolin could protect against microgravity-induced oxidative stress in neuroblastoma SH-SY5Y cells by inhibiting the ROS-NO pathway. These two flavonoids may have potential for preventing oxidative stress induced by space flight or microgravity.

Synergistic neuroprotective effect of combined low concentrations of galantamine and melatonin against oxidative stress in SH-SY5Y neuroblastoma cells

Melatonin is a potent free radical scavenger, antioxidant and neuroprotective drug. On the other hand, galantamine is a cholinergic drug with antioxidant and neuroprotective properties linked to inhibition of acetylcholinesterase and allosteric modulation of nicotinic receptors. This investigation evaluated a possible synergistic neuroprotective effect of subeffective concentrations of combined galantamine and melatonin. Human neuroblastoma SH-SY5Y cells were subjected to a mitochondrial oxidative stress, by blockade of mitochondrial complexes I and V with rotenone and oligomycin-A (R/O); cells were treated for 24 hr with R/O. This caused 40% of the cell to die as measured by lactate dehydrogenase (LDH) release. Cell incubation with increasing concentrations of galantamine (10-300 nm) or melatonin (0.3-10 nm) for 24 hr, followed by a 24-hr period with R/O, caused a concentration-dependent protection; maximum protection was achieved with 300 nm galantamine (56% protection) and 10 nm melatonin (50% protection). Combination of subeffective concentrations of melatonin (0.3 nm) and galantamine (30 nm) caused a synergistic and significant protection that was similar to the maximum protection afforded by effective concentrations of melatonin or galantamine alone. This protective effect was completely reversed when nicotinic and melatonin receptors were blocked respectively by mecamylamine and luzindole. The neuroprotective effect was prevented by chelerythrine, LY294002, and Sn (IV) protoporphyrin IX dichloride (SnPP), indicating the participation of the PKC/PI3K/Akt activation and induction of the antioxidant enzyme heme oxygenase-1. The synthesis of novel multitarget compounds having in a single molecule the combined neuroprotective properties of galantamine and melatonin could be a new strategy for potential therapeutic agents in neurodegenerative diseases.

Neuroprotection by Spirulina platensis protean extract and phycocyanin against iron-induced toxicity in SH-SY5Y neuroblastoma cells

We investigated the effect of Spirulina platensis protean extract and the biliprotein phycocyanin isolated from this microalga, on the activities of the antioxidant enzymes SOD, CAT, GPx, and GR, lipid peroxidation inhibitory activity and glutathione levels after the iron induced oxidative stress in SH-SY5Y neuroblastoma cells. Iron is one of the most important agents that produce oxidative stress and decline of neuronal functions. S. platensis protean extract and phycocyanin exert the antioxidant activity by protecting the activity of the cellular antioxidant enzymes total GPx, GPx-Se and GR and by increasing reduced glutathione in cells against oxidative stress induced by iron. These results suggested that S. platensis protean extract is a powerful antioxidant through a mechanism related to antioxidant activity, capable of interfering with radical-mediated cell death. S. platensis may be useful in diseases known to be aggravated by reactive oxygen species and in the development of novel treatments for neurodegenerative disorders as long as iron has been implicated in the neuropathology of several neurodegenerative disorders such as Alzheimer's or Parkinson diseases.

Impairment of glutamate transport and increased vulnerability to oxidative stress in neuroblastoma SH-SY5Y cells expressing a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis

Human neuroblastoma SH-SY5Y cells transfected with either familial amyotrophic lateral sclerosis-typical G93A mutant or wild-type copper/zinc superoxide dismutase were compared to untransfected cells in term of glutamate transport. V max of glutamate uptake was reduced in mutant cells, with no change in K m. No difference in EAAT1, EAAT2 and EAAT3 glutamate transporter mRNAs and immunoreactive proteins was found, suggesting that one or more transporters are functionally inactivated, possibly due to increased oxidative stress induced by the G93A mutation. Mutant cells showed a marked sensitivity to oxidants, resulting in a more pronounced reduction of glutamate uptake. Short-term antioxidant treatment did not reverse the impairment of glutamate uptake in G93A cells. Interestlingly, N-acetylcysteine was partially effective in preventing glutamate uptake reduction due to exogenous oxidative insults. Since the inhibition of the EAAT2 transporter subtype had no effect on glutamate re-uptake in this model, our study suggests an impaired function of the EAAT1/3 transporter subtypes, possibly due to oxidative inactivation, in the presence of mutant copper/zinc superoxide dismutase. Therefore, this model might prove to be a valuable tool to study the effects of mutant copper/zinc superoxide dismutase associated with amyotrophic lateral sclerosis on glutamate transport in neuronal cells, without the specific contribution of glial cells. These findings might lead to the identification of new therapeutic strategies aimed at preventing the damage associated with ALS.

Increase of antioxidative potential by tert-butylhydroquinone protects against cell death associated with 6-hydroxydopamine-induced oxidative stress in neuroblastoma SH-SY5Y cells

Recent findings suggest that oxidative stress caused by dopamine could be closely involved in the pathogenesis of Parkinson's disease (PD). tert-Butylhydroquinone (tBHQ) is known as a strong inducer of phase II detoxification enzymes which have antioxidative functions. In this study, we investigated the neuroprotective effect of tBHQ against 6-hydroxydopamine (6-OHDA)-induced cell death using human neuroblastoma SH-SY5Y cells. The pretreatment of SH-SY5Y cells with tBHQ significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS), the phosphorylation of c-Jun N-terminal kinase (JNK), and subsequent cell death. We also observed that tBHQ increased the intracellular glutathione levels and induced the expression of NAD(P)H:quinone oxidoreductase (NQO1) mRNA. In addition, tBHQ dose-dependently activated the antioxidant responsive element (ARE), which plays a key role in the transcriptional activation of phase II detoxification enzymes including NQO1. These results indicate that an increase of intracellular antioxidative potential in SH-SY5Y cells by tBHQ treatment protects cells from 6-OHDA-induced oxidative stress.