SH-SY5Y Cell Death Research Articles

Neuroprotective Effect of Demethylsuberosin, a Proteasome Activator, against MPP+-induced Cell Death in Human Neuroblastoma SH-SY5Y Cells

Demethylsuberosin isolated from the roots of Cudrania tricuspidata demonstrated a potent proteasome activator by enhancing all three chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in a 20S proteasome activity assay. It also attenuated the 1-methyl-4-phenylpyridinium-induced dysfunction of the chymotrypsin-like and caspase-like activities of proteasome in SH-SY5Y cells with EC 50 values of 0.76 µM and 0.82 µM, respectively. Additionally, demethylsuberosin protected neuronal cells against 1-methyl-4-phenylpyridinium-induced cell death with an EC 50 value of 0.17 µM, while the EC 50 value of betulinic acid was 4.29 µM. We are reporting that demethylsuberosin is a potent proteasome activator with a neuroprotective effect, suggesting a possible candidate for the protection or treatment of neurodegenerative diseases such as Parkinsonʼs disease.

Protective effects of notoginsenoside R1 against amyloid-β(1-42) induced mitochondrial apopototic death in SH-SY5Y cells

To investigate the effects and underlying mechanism of notoginsenoside R1 on amyloid-β (1-42) (Aβ(1-42)) induced mitochondrial apoptotic death in SH-SY5Y cells. Cell viability was assayed by MTT, apoptotic rates were analyzed with PI/Annexin V flow cytometry, Bax and Bcl-2 expression were detected with Western blotting, enzymatic activity of caspase-3, caspase-8 and caspase-9 were measured by ELISA assay. The 6.25-100 nmol x L(-1) of notoginsenoside R1 attenuate Aβ(1-42) induced apoptotic death of SH-SY5Y in dose dependent manner. The ratio of Bcl-2/Bax was elevated in SH-SY5Y with notoginsenoside R1 treatment. Caspase-3 and caspase-9 were activated with notoginsenoside R1 treatment while caspase-8 was not affected. Notoginsenoside R1 could protect SH-SY5Y cells from Aβ(1-42) induced apoptosis via mitochondria related apoptotic pathway.

Neural stem and progenitor cell fate transition requires regulation of Musashi1 function.

BackgroundThere is increasing evidence of a pivotal role for regulated mRNA translation in control of developmental cell fate transitions. Physiological and pathological stem and progenitor cell self-renewal is maintained by the mRNA-binding protein, Musashi1 through repression of translation of key mRNAs encoding cell cycle inhibitory proteins. The mechanism by which Musashi1 function is modified to allow translation of these target mRNAs under conditions that require inhibition of cell cycle progression, is unknown.ResultsIn this study, we demonstrate that differentiation of primary embryonic rat neural stem/progenitor cells (NSPCs) or human neuroblastoma SH-SY5Y cells results in the rapid phosphorylation of Musashi1 on the evolutionarily conserved site serine 337 (S337). Phosphorylation of this site has been shown to be required for cell cycle control during the maturation of Xenopus oocytes. S337 phosphorylation in mammalian NSPCs and human SH-SY5Y cells correlates with the de-repression and translation of a Musashi reporter mRNA and with accumulation of protein from the endogenous Musashi target mRNA, p21WAF1/CIP1. Inhibition of Musashi regulatory phosphorylation, through expression of a phospho-inhibitory mutant Musashi1 S337A or over-expression of the wild-type Musashi, blocked differentiation of both NSPCs and SH-SY5Y cells. Musashi1 was similarly phosphorylated in NSPCs and SH-SY5Y cells under conditions of nutrient deprivation-induced cell cycle arrest. Expression of the Musashi1 S337A mutant protein attenuated nutrient deprivation-induced NSPC and SH-SY5Y cell death.ConclusionsOur data suggest that in response to environmental cues that oppose cell cycle progression, regulation of Musashi function is required to promote target mRNA translation and cell fate transition. Forced modulation of Musashi1 function may present a novel therapeutic strategy to oppose pathological stem cell self-renewal.

Anti-apoptotic effect of fermentedCitrus sunkipeel extract on chemical hypoxia-induced neuronal injury

Purpose: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimers disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride (CoCl₂)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. Methods: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2,7- dichlorofluorescin diacetate (DCF-DA) and 4,6-diamidino-2-phenylindole (DAPI) staining. Results: Exposure to CoCl₂, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the CoCl₂-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. Conclusion: Based on these data, fermented C. sunki peel extract might have a protective effect against CoCl₂-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.

Neurotoxicity effects of atrazine-induced SH-SY5Y human dopaminergic neuroblastoma cells via microglial activation.

Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine; ATR) is a broad-spectrum herbicide with a wide range of applications worldwide. However, ATR is neurotoxic; it reduces dopamine levels in the substantia nigra and corpus striatum in the midbrain, affects the absorption of synaptic vesicles and synaptic bodies, and interferes with dopamine storage and uptake in synaptic vesicles, leading to neurodegenerative disorders. Microglia are resident immunocompetent and phagocytic cells that regulate and participate in the microenvironment in the central nervous system. They demonstrate macrophage characteristics after activation by releasing inflammatory cytokines and neurotoxic substances to increase the inflammatory response, and are thus involved in neurodegeneration. The aim of this study was to investigate the neurotoxic effects of ATR-activated microglia-mediated neuronal damage in terms of human dopaminergic neuroblastoma SH-SY5Y cell death. ATR was administered to BV-2 microglial cells at 12.5, 25, and 50 μM for 1, 6, 12, 24 and 48 h, respectively. ATR increased activated-microglia-induced overexpression of reactive oxygen species, inducible nitric oxide synthase, nitric oxide, gp91(phox), p47(phox), and the inflammatory cytokines tumor necrosis factor α and interleukin-1β, thus reducing SH-SY5Y cell viability. These results suggest that activated microglia may play a critical role in inflammation-mediated dopaminergic neuronal death, and provide the basis for further studies on the mechanisms of ATR-induced dopaminergic system toxicity.

Isoflavones with neuroprotective activities from fruits of Cudrania tricuspidata

Ten isoflavones, cudraisoflavones B–K (1–10), together with 27 known isoflavones, were isolated from the EtOAc soluble extract of fruits of Cudrania tricuspidata. The structures of compounds 1–10 were elucidated on the basis of MS and NMR spectroscopic data, including 2D NMR experiments. Compounds 7–9 and three known (11–13) compounds showed neuroprotective activity against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells, with EC50 values of 0.5–9.2μM.

Hypothermia Reduces but Hyperthermia Augments T Cell-Derived Release of Interleukin-17 and Granzyme B that Mediate Neuronal Cell Death

T cells infiltrate into the infarcted brain within days after cerebral ischemia and play essential roles in exacerbating the delayed phase of the brain injury by producing pro-inflammatory factors. However, the involvement of these factors in brain damage is also demonstrated systemically. Such periphery-brain abnormalities are interesting because they may constitute a pathway to the central nervous system (CNS), which may be a target of therapeutic hypothermia. Although this therapy protects neurons after severe brain damage, the underlying mechanisms are partly understood. We examined the effects of hypothermic and hyperthermic cultures on peripheral T cell-derived release of interleukin (IL)-17 and granzyme B (GrB) and evaluated whether and how these factors induced neurotoxicity and activated brain endothelial cells. We determined levels of IL-17 and GrB produced by several activated, IL-1β/IL-23-treated activated T cells (naïve CD4(+), CD4(+), CD8(+), and γδ T cells obtained from healthy humans) under hypothermia, normothermia, and hyperthermia. The viability of neuronal SH-SY5Y cells treated with IL-17 or GrB and mRNA expression of adhesion molecules/chemokines by brain endothelial bEND.3 cells treated with IL-17 were also measured. Compared with normothermia, IL-17 and GrB release in these T cells was reduced by hypothermia but augmented by hyperthermia. IL-17 and GrB caused the death of neuronal SH-SY5Y cells, and IL-17 upregulated mRNA expression of several adhesion molecules/chemokines in bEND.3 cells; both effects were concentration-dependent. Hypothermia reduced but hyperthermia augmented T cell-derived release of IL-17 and GrB that mediate neuronal cell death, suggesting that the attenuation of T cell-derived release of these factors by therapeutic hypothermia leads to the inhibition of neuronal cell death in the delayed phase of brain injury. Moreover, hypothermia may suppress but hyperthermia may promote the recruitment of inflammatory cells to CNS by regulating brain endothelial activation of IL-17.

Neuroprotective effect from stem bark extracts of Knema laurina against H2O2- and Aβ1–42-induced cell death in human SH-SY5Y cells

The stem bark extracts of Knema laurina inhibited the hydrogen peroxide (H2O2)- and aggregated amyloid β-peptide 1–42 length (Aβ1–42)-induced cell death in differentiated SH-SY5Y cells. Exposure of 250 μM H2O2 or 20 μM Aβ1–42 to the cells for 24 h reduced 50% of cell viability. Pretreatment of cells with ethyl acetate extract (EAE) or n-butanol extract (BE) at 300 μg/mL and then exposure to H2O2 protected the cells against the neurotoxic effects of H2O2. Besides, methanolic extract (ME) at 1 and 10 μg/mL exerted neuroprotective effect on Aβ1–42-induced toxicity to the cells. These results showed that EAE, BE and ME exhibited neuroprotective activities against H2O2- and Aβ1–42-induced cell death. Flavonoids (3–6) and β-sitosterol glucoside (8) were isolated from the EAE. Compound 1 was isolated from hexane extract, and compounds 2 and 7 were isolated from dichloromethane extract. All these observations provide the possible evidence for contribution in the neuroprotective effects.

Novel Platinum(II) compounds modulate insulin-degrading enzyme activity and induce cell death in neuroblastoma cells.

The properties of three novel Platinum(II) compounds toward the insulin-degrading enzyme (IDE) enzymatic activity have been investigated under physiological conditions. The rationale of this study resides on previous observations that these compounds, specifically designed and synthesized by some of us, induce apoptosis in various cancer cell lines, whereas IDE has been proposed as a putative oncogene involved in neuroblastoma onset and progression. Two of these compounds, namely [PtCl(O,O'-acac)(DMSO)] and [Pt(O,O'-acac)(γ-acac)(DMS)], display a modulatory behavior, wherefore activation or inhibition of IDE activity occurs over different concentration ranges (suggesting the existence of two binding sites on the enzyme). On the other hand, [Pt(O,O'-acac)(γ-acac)(DMSO)] shows a typical competitive inhibitory pattern, characterized by a meaningful affinity constant (K i =0.95±0.21μM). Although all three compounds induce cell death in neuroblastoma SHSY5Y cells at concentrations exceeding 2μM, the two modulators facilitate cells' proliferation at concentrations≤1.5μM, whereas the competitive inhibitor [Pt(O,O'-acac)(γ-acac)(DMSO)] only shows a pro-apoptotic activity at all investigated concentrations. These features render the [Pt(O,O'-acac)(γ-acac)(DMSO)] a promising "lead compound" for the synthesis of IDE-specific inhibitors (not characterized yet) with therapeutic potentiality.

Pinocembrin attenuates 6-OHDA-induced neuronal cell death through Nrf2/ARE pathway in SH-SY5Y cells.

Pinocembrin (PB), the most abundant flavonoid in propolis, has been known to display antioxidant activity. However, the mechanism as how PB can induce antioxidant activity remains elusive. The purpose of the present study was to investigate the potential neuroprotective role of PB and to delineate its mechanism of action against the Parkinson's disease-related neurotoxin 6-hydroxydopamine(6-OHDA)-induced cell death in neuroblastoma SH-SY5Y cells. Results indicate that pretreatment with PB for 4h significantly reduced the 6-OHDA-induced cell viability loss, apoptotic rate and decreased Bcl-2/Bax ratio. In addition, PB inhibited 6-OHDA-induced oxidative stress as measured by the formation of reactive oxygen species, the level of malondialdehyde, mitochondrial membrane potential, and superoxide dismutase. Moreover, we have revealed the PB treatment resulted in an increase in nuclear factor E2-related factor 2 (Nrf2) protein levels and subsequent activation of antioxidant response element (ARE) pathway genes of heme oxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS) in SH-SY5Y cells. Treatment of SH-SY5Y cells with Nrf2 small interference RNA abolished PB-induced HO-1 and γ-GCS expression and its protective effects. Taken together, these findings suggest that PB can protect the SH-SY5Y cells from 6-OHDA-induced oxidative cell death via Nrf2/ARE pathway. Thus, our study indicates that PB has a partial cytoprotective role in dopaminergic cell culture systems.

Edible bird's nest ameliorates oxidative stress-induced apoptosis in SH-SY5Y human neuroblastoma cells.

BackgroundParkinson’s disease (PD) is the second most common neurodegenerative disorder affecting the senile population with manifestation of motor disability and cognitive impairment. Reactive oxygen species (ROS) is implicated in the progression of oxidative stress-related apoptosis and cell death of the midbrain dopaminergic neurons. Its interplay with mitochondrial functionality constitutes an important aspect of neuronal survival in the perspective of PD. Edible bird’s nest (EBN) is an animal-derived natural food product made of saliva secreted by swiftlets from the Aerodamus genus. It contains bioactive compounds which might confer neuroprotective effects to the neurons. Hence this study aims to investigate the neuroprotective effect of EBN extracts in the neurotoxin-induced in vitro PD model.MethodsEBN was first prepared into pancreatin-digested crude extract and water extract. In vitro PD model was generated by exposing SH-SY5Y cells to neurotoxin 6-hydroxydopamine (6-OHDA). Cytotoxicity of the extracts on SH-SY5Y cells was tested using MTT assay. Then, microscopic morphological and nuclear examination, cell viability test and ROS assay were performed to assess the protective effect of EBN extracts against 6-OHDA-induced cellular injury. Apoptotic event was later analysed with Annexin V-propidium iodide flow cytometry. To understand whether the mechanism underlying the neuroprotective effect of EBN was mediated via mitochondrial or caspase-dependent pathway, mitochondrial membrane potential (MMP) measurement and caspase-3 quantification were carried out.ResultsCytotoxicity results showed that crude EBN extract did not cause SH-SY5Y cell death at concentrations up to 75 μg/ml while the maximum non-toxic dose (MNTD) of water extract was double of that of crude extract. Morphological observation and nuclear staining suggested that EBN treatment reduced the level of 6-OHDA-induced apoptotic changes in SH-SY5Y cells. MTT study further confirmed that cell viability was better improved with crude EBN extract. However, water extract exhibited higher efficacy in ameliorating ROS build up, early apoptotic membrane phosphatidylserine externalization as well as inhibition of caspase-3 cleavage. None of the EBN treatment had any effect on MMP.ConclusionsCurrent findings suggest that EBN extracts might confer neuroprotective effect against 6-OHDA-induced degeneration of dopaminergic neurons, particularly through inhibition of apoptosis. Thus EBN may be a viable nutraceutical option to protect against oxidative stress-related neurodegenerative disorders such as PD.

Abstract 2622: New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK

Abstract The anaplastic lymphoma kinase (ALK), a tyrosine kinase receptor involved in the genesis of several human cancers, is a promising therapeutic target in Neuroblastoma (NB). Many studies on sporadic cases with advanced NB have shown ALK mutated or highly expressed independently of its genetic status (mutated, amplified, wild-type) and its pivotal role in NB growth and survival. As ALK is currently considered a master driver of NB oncogenesis, the use of therapies inhibiting ALK represents a suitable treatment option. The ALK inhibitor Crizotinib was recently approved for the treatment of advanced NSCLC patient. However, as often observed with kinase inhibitors, some patients develop acquired resistance in part due to genetic ALK mutations. To overcome this limitation a selective and more potent ALK inhibitor, X-396, was developed that is currently in phase I clinical trial in adult patients with advanced solid tumors, but it has not been tested in preclinical animal models of NB. We have successfully employed a RNAi-mediated therapeutic approach to selectively knockdown ALK expression by using nanoliposomes entrapping siRNA and coupled with anti-GD2 antibodies to target NB cells. Our data demonstrate for the first time a role of ALK in NB-induced angiogenesis and in tumor invasion. Here, we aimed to test whether the combination of the novel ALK kinase inhibitor X-396 with the liposome-based silencing of ALK could represent a powerful synergistic and or additive effect in NB xenografts to promote long-term survival. In vitro, X-396 showed to be more effective than Crizotinib in inhibiting cell proliferation and in inducing cell death in both LAN-5 (R1275Q ALK mut, Crizotinib-sensitive) and SH-SY5Y (F1174L ALK mut, Crizotinib-resistant) cells. Pharmacokinetic profiles of X-396 after multiple PO administration in NB-xenografts revealed a good bioavailability and a moderate half-life. Biodistribution study clearly indicated that the concentration of X-396 8h post-administration was 35 fold higher in the tumor site than in plasma. X-396 reduced the tumor volume in subcutaneous SH-SY5Y NB-model in a dose-dependent manner and it was more effective than Crizotinib when administered at the same concentration. In orthotopic xenografts of SH-SY5Y and LAN-5 cells, X-396 significantly increased life span. In combination studies, all effects were significantly improved in the mice treated with targeted liposomal formulation of ALK-siRNA and X-396 compared to untreated control mice or mice receiving the single agent treatments. In the latter studies, further enhancement of life span was obtained by increasing the X-396 dose. Moreover, the combined treatment was also proven to be effective in the SH-SY5Y-pseudometastatic model in determining long-term survivors. Our findings provide a rational basis to design innovative molecular-based combination of treatments for clinical application in NB. Citation Format: Daniela Di Paolo, Laura Emionite, George Liu, Michele Cilli, Annarita Di Fiore, Chiara Brignole, Chris Liang, Fabio Pastorino, Jay Gibbons, Mirco Ponzoni, Patrizia Perri. New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2622. doi:10.1158/1538-7445.AM2014-2622

Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism

Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson's disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperitoneal administration of Alda-1 significantly reduced rotenone- or MPTP-induced death of SN tyrosine hydroxylase (TH)-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that ALDH2 plays a crucial role in maintaining normal mitochondrial function to protect against neurotoxicity and that Alda-1 is effective in ameliorating mitochondrial dysfunction and inhibiting mitochondria-mediated apoptotic pathway. These results indicate that ALDH2 activation could be a neuroprotective therapy for PD.

Neuroprotective effects of melittin on hydrogen peroxide-induced apoptotic cell death in neuroblastoma SH-SY5Y cells.

BackgroundFree radicals are involved in neuronal cell death in human neurodegenerative diseases. Since ancient times, honeybee venom has been used in a complementary medicine to treat various diseases and neurologic disorders. Melittin, the main component of honeybee venom, has various biologic effects, including anti-bacterial, anti-viral, and anti-inflammatory activities.MethodsWe investigated the neuroprotective effects of melittin against H2O2-induced apoptosis in the human neuroblastoma cell line SH-SY5Y. The neuroprotective effects of melittin on H2O2-induced apoptosis were investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide assay, caspase 3 activity, 4,6-diamidino-2-phenylindole staining, a lactate dehydrogenase release assay, Western blots, and reverse transcription-polymerase chain reaction.ResultsThe H2O2-treated cells had decreased cell viability with apoptotic features and increased production of caspase-3. On the other hand, melittin treatment increased cell viability and decreased apoptotic DNA fragmentation. Melittin attenuated the H2O2-induced decrease in mRNA and protein production of the anti-apoptotic factor Bcl-2. In addition, melittin inhibited both the H2O2-induced mRNA and protein expression of Bax-associated pro-apoptotic factor and caspase-3.ConclusionsThese findings suggest that melittin has potential therapeutic effects as an agent for the prevention of neurodegenerative diseases.

Chlorpyrifos and cypermethrin induce apoptosis in human neuroblastoma cell line SH-SY5Y.

Our previous in vivo studies showed that chlorpyrifos (CPF) and cypermethrin (CM) in a mixture dermally administered, strongly inhibited cholinesterase activity in plasma and the brain and were very toxic to the rat central nervous system. In this work, the mechanisms of neurotoxicity have not been elucidated. We used human undifferentiated SH-SY5Y cells to study mechanisms of pesticide-induced neuronal cell death. It was found that chlorpyrifos (CPF) and its mixture with cypermethrin (CPF+CM) induced cell death of SH-SY5Y cells in a dose- and time-dependent manner, as shown by MTT assays. Pesticide-induced SH-SY5Y cell death was characterized by concentration-dependent down-regulation of Bcl-2 and Bcl-xL as well as an increase in the caspase 3 activation. Pan-caspase inhibitor Q-VD-OPh produced a slight but significant reversal effect of pesticide-induced toxicity indicating that the major caspase pathways are not integral to CPF- and CPF+CM-induced cell death. Furthermore, signal transduction inhibitors PD98059, SL-327, SB202190, SP600125 and mecamylamine failed to attenuate pesticides effect. Atropine exhibited minimal ability to reverse toxicity. Finally, it was shown that inhibition of TNF-α by pomalidomide attenuated CPF-/CPF+CM-induced apoptosis. Overall, our data suggest that FAS/TNF signalling pathways may participate in CPF and CPF+CM toxicity.

Neuroprotective Xanthones from the Root Bark of Cudrania tricuspidata.

Seventeen new prenylated xanthones (1-17) were isolated from an ethyl acetate-soluble extract of root bark of Cudrania tricuspidata together with 17 previously identified xanthones. The structures of the new compounds were elucidated by spectroscopic methods. Six new compounds (3, 7, 8, 9, 15, and 16) and six known compounds (18-23) showed neuroprotective effects against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells, with EC50 values of 0.7-16.6 μM.

Involvement of activation of the Nrf2/ARE pathway in protection against 6-OHDA-induced SH-SY5Y cell death by α-iso-cubebenol

Free radical-mediated neurodegeneration is one of the many causes of Parkinson's disease (PD). As part of our ongoing studies on the identification of biologically active Schisandra chinensis components, we have isolated and structurally elucidated α-iso-cubebenol. This study was carried out in an attempt to clarify the neuroprotective effect of α-iso-cubebenol on toxin-insulted dopaminergic neuronal death using 6-hydroxy-dopamine (6-OHDA)-induced dopaminergic SH-SY5Y cells. α-iso-cubebenol significantly attenuated the loss of mitochondrial function (MTT assay) and membrane integrity (lactate dehydrogenase assay) associated with 6-OHDA-induced neurotoxicity. Pretreatment of the cells with α-iso-cubebenol diminished the intracellular accumulation of reactive oxygen species (ROS) and calcium in response to 6-OHDA. Moreover, α-iso-cubebenol protected against 6-OHDA-induced neurotoxicity through inhibition of SH-SY5Y cell apoptosis. In addition, JC-1 staining, which is a well-established measure of mitochondrial damage, was decreased after treatment with α-iso-cubebenol. Notably, α-iso-cubebenol inhibited the release of mitochondrial flavoprotein apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus following 6-OHDA treatment. In addition, α-iso-cubebenol reduced the 6-OHDA-induced phosphorylation of ERK and induced the phosphorylation of PKA, PKB, and CREB in a dose-dependent manner. Moreover, α-iso-cubebenol stimulated the activation of Nrf2, a downstream target of CREB. Furthermore, α-iso-cubebenol stimulated the expression of multiple antioxidant response genes (NQO-1 and HO-1). Finally, CREB and Nrf2 siRNA transfection diminished α-iso-cubebenol-mediated neuroprotection.

Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP+-induced oxidative stress and cell death in SH-SY5Y cells

Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced damage in SH-SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP+-induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP+-induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP+-induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling.

Lignans from the fruit of Schisandra glaucescens with antioxidant and neuroprotective properties.

Two rare 7,8-seco-lignans (1, 2), three new lignan glycosides (3, 4a, 4b), and 10 known lignans (5-14) were isolated from the fruit of Schisandra glaucescens Diels. The absolute configurations of 1 and 2 were determined by comparing their experimental and calculated electronic circular dichroism spectra. The molecular structures of the new compounds (3, 4a, and 4b), including their absolute configurations, were determined using various spectroscopic methods and hydrolysis reactions. The antioxidant activities of the isolated compounds were tested using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays. Compounds 4, 7, 8, 10, 11, and 12 exhibited antioxidant activities of varying potential in both assays. Of these compounds, 7 showed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, with IC50 values of 15.7 (150 μM DPPH) and 34.6 μM (300 μM DPPH), respectively, and 4, 12, and 7 displayed higher total antioxidant activities than Trolox in the ferric reducing antioxidant power assay. The neuroprotective effects of these compounds against Aβ25-35-induced cell death in SH-SY5Y cells were also investigated. Compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant neuroprotective effects against Aβ25-35-induced SH-SY5Y cell death compared with the group treated only with Aβ25-35.

4-Hydroxy-trans-2-nonenal (4-HNE) induces neuronal SH-SY5Y cell death via hampering ATP binding at kinase domain of Akt1.

Inhibition mechanism(s) of protein kinase B/Akt1 and its consequences on related cell signaling were investigated in human neuroblastoma SH-SY5Y cells exposed to 4-hydroxy-trans-2-nonenal (4-HNE), one of the most reactive aldehyde by-products of lipid peroxidation. In silico data indicate that 4-HNE interacts with kinase domain of Akt1 with the total docking score of 6.0577 and also forms H-bond to Glu234 residue similar to highly potent Akt1 inhibitor imidazopiperidine analog 8b, in which the protonated imidazole nitrogen involves in two hydrogen bonds between Glu234 and Asp292. The strong hydrogen bonding with Glu234 and hydrophobic interactions with several residues, namely Leu156, Gly157, Val164, Ala177, Tyr229, Ala230, Met281 and Thr291, at the vicinity which is normally occupied by the ribose of ATP, appear to be the main causes of Akt1 inhibition and lead to the significant conformational change on this region of protein. Results of mutational docking prove that Glu234 plays a major role in 4-HNE-mediated Akt1 inhibition. In silico data on Akt inhibition were further validated by observing the down-regulated levels of phosphorylated (Thr308/Ser493) Akt1 as well as the altered levels of the downstream targets of pAkt, namely downregulated levels of pGSK3β (Ser9), β-catenin, Bcl2 and upregulated levels of pro-apoptotic markers, namely Bad, Bax, P(53) and caspase-9/3. The cellular fate of such pAkt inhibition was evidenced by increased reactive oxygen species, degraded nuclei, transferase dUTP nick end labeling positive cells and upregulated levels of pJNK1/2. We identified that 4-HNE-mediated Akt1 inhibition was due to the competitive inhibition of ATP by 4-HNE at the kinase domain of ATP binding sites.