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.     

Cytokine action and oxidative stress response in differentiated neuroblastoma SH-SY5Y cells

In the retinoic acid-differentiated neuroblastoma SH-SY5Y cells, IL-1 induced binding activity of NFkappaB and up-regulated the expression and activity of MnSOD. The IL-1-elicited effects were partly reversed by IL-4 and IL-6. It is proposed that IL-4 and IL-6 may participate in the regulation of the imbalanced oxidant status induced by IL-1 in differentiated neuroblastoma cells. In the SH-SY5Y cell line, TNFalpha neither activated NFkappaB nor induced MnSOD expression and activity, but was capable of modulating the IL-1 effects. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NFkappaB activation, down-regulated the expression and activity of MnSOD, which may suggest that the regulation of MnSOD by IL-1 in retinoic acid-differentiated neuroblastoma cells was mediated by the nuclear factor kappaB.     

Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells

We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).     

Neurotoxicity mediated by oxidative stress caused by titanium dioxide nanoparticles in human neuroblastoma (SH-SY5Y) cells

BackgroundTitanium is widely used in biomedicine. Due to biotribocorrosion, titanium dioxide (TiO₂) nanoparticles (NPs) can be released from the titanium implant surface, enter the systemic circulation, and migrate to various organs and tissues including the brain. A previous study showed that 5 nm TiO₂ NPs reached the highest concentration in the brain. Even though TiO₂ NPs are believed to possess low toxicity, little is known about their neurotoxic effects. The aim of the study was to evaluate in vitro the effects of 5 nm TiO₂ NPs on a human neuroblastoma (SH-SY5Y) cell line.MethodsCell cultures were divided into non-exposed and exposed to TiO₂ NPs for 24 h. The following were evaluated: reactive oxygen species (ROS) generation, apoptosis, cellular antioxidant response, endoplasmic reticulum stress and autophagy.ResultsExposure to TiO₂ NPs induced ROS generation in a dose dependent manner, with values reaching up to 10 fold those of controls (p < 0.001). Nrf2 nuclear localization and autophagy, also increased in a dose dependent manner. Apoptosis increased by 4- to 10-fold compared to the control group, depending on the dose employed.ConclusionsOur results show that TiO₂ NPs cause ROS increase, induction of ER stress, Nrf2 cytoplasmic translocation to the nucleus and apoptosis. Thus, neuroblastoma cell response to TiO₂ NPs may be associated with an imbalance of the oxidative metabolism where endoplasmic reticulum-mediated signal pathway seems to be the main neurotoxic mechanism.     

Quercetin mitigates the deoxynivalenol mycotoxin induced apoptosis in SH-SY5Y cells by modulating the oxidative stress mediators

Deoxynivalenol (DON) is Fusarium mycotoxin that is frequently found in many cereal-based foods, and its ingestion has a deleterious impact on human health. In this investigation, we studied the mechanism of DON-induced neurotoxicity and followed by cytoprotective efficacy of quercetin (QUE) in contradiction of DON-induced neurotoxicity through assessing the oxidative stress and apoptotic demise in the human neuronal model, i.e. SH-SY5Y cells. DON diminished the proliferation of cells in the manner of dose and time-dependent as revealed by cell viability investigations, i.e. MTT and lactate dehydrogenase assays. Additional studies, such as intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential (MMP), DNA damage, cell cycle, and neuronal biomarkers (amino acid decarboxylase, tyrosine hydroxylase, and brain-derived neurotrophic factor) demonstrated that DON induces apoptotic demise in neuronal cells through oxidative stress intermediaries. On another hand, pre-treatment of neuronal cells with 1 mM of quercetin (QUE) showed decent viability upon exposure to 100 µM of DON. In detailed studies demonstrated that QUE (1 mM) pre-treated cells show strong attenuation efficiency against DON-induced ROS generation, LPO, MMP loss, DNA impairment, cell cycle arrest, and down-regulation of neuronal biomarkers. The consequences of the investigation concluded that QUE mitigates the DON-induced stress viz., decreased ROS production and LPO generation, upholding MMP and DNA integrity and regulation of neuronal biomarker gene expression in SH-SY5Y cells.     

Angelicin induces apoptosis through intrinsic caspase-dependent pathway in human SH-SY5Y neuroblastoma cells

Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for its antiproliferative activity in treatment of different skin diseases. To verify the activity of angelicin, we employed human SH-SY5Y neuroblastoma cells to investigate its cytotoxicity, although its mechanism of action has not yet been fully elucidated. Here, we examined the cellular cytotoxicity of angelicin by cell viability assay, DNA fragmentation by DNA ladder assay, and activation of caspases and Bcl-2 family proteins by western blot analyses. The results of our investigation suggest that angelicin increased cellular cytotoxicity in a dose- and time-dependent manner with IC(50) of 49.56?μM at 48?h of incubation. In addition, angelicin dose-dependently downregulated the expression of anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1 suggesting the involvement of the intrinsic mitochondria-mediated apoptotic pathway which did not participate in Fas/FasL-induced caspase-8-mediated extrinsic, MAP kinases, and PI3K/AKT/GSK-3β pathway. Furthermore, we clarified the dose-dependent upregulation of caspase-9 and caspase-3 which indicated that angelicin-induced apoptosis is mediated primarily through the intrinsic caspase-mediated pathway. In particular, the caspase-3 inhibitor, DEVD-fmk, induced a reduction in angelicin-induced cytotoxicity which confirmed that the intrinsic caspase-dependent pathway during this apoptosis which did not prevent cytotoxicity using MAP kinases and GSK-3 inhibitor. Taken together, our data shows that angelicin is an effective apoptosis-inducing natural compound of human SH-SY5Y neuroblastoma cells which suggests that this compound may have a role in future therapies for human neuroblastoma cancer.     

Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 μM DAS and 50 μM DADS but slightly affected with 100 μM DADS. Treatment with 50 and 100 μM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 μM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca²⁺]_i, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFκB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.     

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using (32)P-5'-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H(2)O(2) generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.     

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using ³²P-5′-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H₂O₂ generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.     

SH-SY5Y细胞α-突触核蛋白的过表达可部分抵抗鱼藤酮诱导的氧化应激

帕金森病（Parkinson’s disease,PD）的发病机制涉及到遗传和环境因素。环境因素通过线粒休导致氧化应激和α-突触核蛋白（α—synuclein）聚集,但其确切的作用机制尚不明确。本文利用过表达α-突触核蛋白-增强型绿色荧光蛋白（enhanced green fluorescent protein．EGFP）的人多巴胺能神经母细胞瘤细胞株SH—SY5Y为模型,研究α-突触核蛋白对鱼藤酮诱导氧化应激的影响,从而进一步了解α-突触核蛋白和细胞存活之间的关系。（1）用荧光显微镜观察融合绿色荧光蛋白的α-突触核蛋白的表达情况;（2）用实时定量PCR检测α-突触核蛋白基因的表达;（3）用免疫细胞化学测定α-突触核蛋白的分布;（4）用不同浓度的鱼藤酮作用细胞后,以MTT法测细胞的活力、DCF法检测细胞的氧化应激状态、黄嘌呤氧化酶法检测超氧化物歧化酶的活力,并用流式细胞仪分析细胞的凋亡。实时定量PCR结果显示,α-突触核蛋白基因表达量在α-突触核蛋白过表达的细胞要高于SH—SY5Y细胞,在荧光显微镜下可见绿色荧光蛋白和α-突触核蛋白的表达。鱼藤酮可使细胞活力下降、线粒体complex Ⅰ的活性降低,诱导细胞内氧化应激,而过表达α-突触核蛋白的细胞可以部分抵抗鱼藤酮的毒性作用,表现为细胞抗氧化能力迅速增高（P〈0．05）和鱼藤酮诱导的细胞凋亡数目明显降低。本研究证明α-突触核蛋白对鱼藤酮产生的氧化应激有部分抵抗作用,而使过表达α-突触核蛋白的SH—SY5Y细胞对鱼藤酮的毒性作用表现出一定的耐受性。这种耐受性也可能是细胞对外界损害的一种代偿反应,从而促进细胞的存活。     

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.     

The radical scavenger CR-6 protects SH-SY5Y neuroblastoma cells from oxidative stress-induced apoptosis: effect on survival pathways

Reactive oxygen species (ROS) and oxidative stress have long been linked to cell death of neurons in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are at present unclear. In the current work we have used the human neuroblastoma SH-SY5Y cell line as a model for studying the molecular events occurring after inducing apoptosis with H2O2. We show that treatment of SH-SY5Y cells with H2O2 up-regulates survival pathways during early stages of apoptosis. Subsequently, the decline of anti-apoptotic protein levels leads to the activation of the calcium-dependent proteases calpains and the cysteine proteases caspases. Additionally, we demonstrate that CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acts as a scavenger of ROS and prevents apoptosis by enhancing and prolonging up-regulation of survival pathways. Furthermore, we show that pre-treatment of SH-SY5Y cells with a cocktail containing CR-6, the pan-caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methylketone) and the calpain inhibitor SJA6017 confers almost total protection against apoptosis. In summary, the present work characterizes the molecular mechanisms involved in oxidative stress-induced apoptosis in SH-SY5Y cells. Our findings highlight the relevance of CR-6, alone or in combination with other drugs, as potential therapeutic strategy for the treatment of neurodegenerative diseases.     

EV71诱导人神经细胞SH-SY5Y自噬的分子机制

【背景】EV71感染所致的重症手足口病易导致神经系统并发症,使患儿预后较差,甚至死亡。【目的】从EV71可诱导神经细胞自噬这一现象出发,探索该病毒诱导神经细胞自噬的miRNA机制,探讨EV71损伤神经细胞可能的分子机制。【方法】通过RT-PCR及Westernblot技术,在感染EV71病毒的人神经母细胞瘤细胞SH-SY5Y中检测细胞自噬变化;通过芯片分析细胞感染前后差异表达的miRNA分子,再使用miRNA mimics调节工具明确与EV71诱导神经细胞自噬有关的miRNA分子。【结果】EV71可诱导SH-SY5Y细胞自噬增加,下调细胞内miRNA29b(miR29b)分子的表达水平;当上调细胞内miR29b的表达后,EV71诱导细胞自噬增加的现象可被逆转,病毒复制水平下降。【结论】EV71诱导神经细胞自噬是通过下调miR29b分子的表达水平实现;miR29b不仅与自噬相关,它与EV71病毒复制也存在密切关系。因此,该研究不仅有助于阐明EV71导致神经系统损伤的具体分子机制,还为miR29b成为治疗EV71感染可能的新药物靶点奠定了理论基础。     

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.     

Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells

Endoplasmic reticulum (ER) stress has increasingly come into focus as a factor contributing to neuronal injury. Although caspase-dependent mechanisms have been implicated in ER stress, the signaling pathways involved remain unclear. In this study, we examined the role of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase pathway that is highly conserved in many systems for balancing cell survival and death. Prolonged treatment of the human neuroblastoma cell line SH-SY5Y with thapsigargin, an inducer of ER stress, increased cell death over 24-48 h, as measured by LDH release. Caspases were involved; increased levels of active caspase-3 and cleaved caspase substrate PARP were detected, and treatment with Z-VAD-FMK reduced thapsigargin-induced cytotoxicity. In contrast, inhibition of calpain was not protective, although calpain was activated following thapsigargin treatment. An early and transient phosphorylation of ERK1/2 occurred after thapsigargin-induced ER stress, and targeting this pathway with the MEK inhibitors U0126 or PD98059 significantly reduced cell death. Similar cytoprotection was obtained against brefeldin A, another ER stress agent. However, protection against ER stress via ERK inhibition was not accompanied by amelioration of caspase-3 activation, PARP cleavage, or DNA laddering. These data indicate that ERK may contribute to non-caspase-dependent pathways of injury after ER stress.     

Role of viscoelastic properties of differentiated SH-SY5Y human neuroblastoma cells in cyclic shear stress injury

Shear stress and strain lead to neurodegeneration in vivo during head injury, glaucoma, and certain repetitive motion disorders. In vitro, shear stress and strain have been shown to lead to cell injury in a number of models using neurons and neuron-like cells. In the present study we examined the relationship between shear stress, strain, and the extent of cell injury in a cyclic shear stress induced model of cell injury using differentiated SH-SY5Y (human neuroblastoma) cells. Shear stress led to cell strain that increased with increasing stress and diminished upon cessation of shear. Strain rate during cyclic application of shear stress increased by over an order of magnitude from the first to all subsequent cycles, suggesting that the cell and/or its polymer network became more elastic upon cyclic shear stress application. To support this conclusion we measured the degree of cytoskeletal polymerization before and after exposure of cells to cyclic shear stress and found that the fraction of polymerized tubulin in the cell relative to total tubulin decreased by a factor of 2 after six cycles of shear stress. The extent of injury, as indicated by the fraction of cells with fragmented DNA, was three times higher for cyclic shear stress than for steady shear stress and may be related to the change in strain rate and/or cytoskeletal reorganization associated with cyclic stress. These findings may aid in understanding the mechanism by which neurons and neuron-like cells respond to cyclic shear stress and strain and lead to new treatments for disease or injury arising from the exposure of neurons to abnormal cyclic or repetitive stress and strain.     

Effect of lycopene on As2O3 induced oxidative stress in SH-SY5Y cells

It is known that oxidative stress may cause neuronal injury and several experimental models showed that As₂O₃ exposure causes oxidative stress. Lycopene, a carotenoid, has been shown to have protective effect in neurological disease models due to antioxidant activity, but its effect on As₂O₃-induced neurotoxicity is not identified yet. The aim of this study is to investigate the effects of lycopene on As₂O₃-induced neuronal damage and the related mechanisms. Cell viability was determined by the MTT assay. Lycopene was administrated with different concentrations (2, 4, 6 and 8 µM) one hour before 2 µM As₂O₃ exposure in SH-SY5Y human neuroblastoma cells. The anti-oxidant effect of lycopene was determined by measuring superoxide dismutase (SOD), catalase (CAT) hydrogen peroxide (H₂O₂), malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS). MTT results and LDH cytotoxicity analyses showed that pretreatment with 8 µM lycopene significantly improved the toxicity due to As₂O₃ exposure in SH?SY5Y neuroblastoma cells. Pretreatment with lycopene significantly increased the activities of anti?oxidative enzymes as well as total antioxidant status and decreased total oxidative status in As₂O₃ exposed cells. The results of this study indicate that lycopene may be a potent neuroprotective against oxidative stress and could be used to prevent neuronal injury or death in several neurological diseases.

     

Resveratrol interference with the cell cycle protects human neuroblastoma SH-SY5Y cell from paclitaxel-induced apoptosis

In previous studies we demonstrated that resveratrol acts in an antiapoptotic manner on the paclitaxel-treated human neuroblastoma (HN) SH-SY5Y cell line inhibiting the apoptotic pathways induced by the antineoplastic drug. In the present study we evaluated the antiapoptotic effect of resveratrol, studying its activity on cell cycle progression. We determined the mitotic index of cultures exposed to resveratrol and paclitaxel alone or in combination, the cell cycle distribution by flow cytometric analysis (FACS), and the modulation of some relevant cell cycle regulatory proteins. Resveratrol is able to induce S-phase cell arrest and this interference with the cell cycle is associated with an increase of cyclin E and cyclin A, a downregulation of cyclin D1, and no alteration in cyclin B1 and cdk 1 activation. The resveratrol-induced S-phase block prevents SH-SY5Y from entering into mitosis, the phase of the cell cycle in which paclitaxel exerts its activity, explaining the antiapoptotic effect of resveratrol.