Endoplasmic reticulum stress mediates the arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells

Arsenic trioxide has been proven to trigger apoptosis in human hepatocellular carcinoma cells. Endoplasmic reticulum stress has been known to be involved in apoptosis through the induction of CCAAT/enhancer-binding protein homologous protein. However, it is unknown whether endoplasmic reticulum stress mediates arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells. Our data showed that arsenic trioxide significantly induced apoptosis in human hepatocellular carcinoma cells. Furthermore, arsenic trioxide triggered endoplasmic reticulum stress, as indicated by endoplasmic reticulum dilation, upregulation of glucose-regulated protein 78 and CCAAT/enhancer-binding protein homologous protein. We further found that 4-phenylbutyric acid, an inhibitor of endoplasmic reticulum stress, alleviated arsenic trioxide-induced expression of CCAAT/enhancer-binding protein homologous protein. More important, knockdown of CCAAT/enhancer-binding protein homologous protein by siRNA or inhibition of endoplasmic reticulum stress by 4-phenylbutyric acid alleviated apoptosis induced by arsenic trioxide. Consequently, our results suggested that arsenic trioxide could induce endoplasmic reticulum stress-mediated apoptosis in hepatocellular carcinoma cells, and that CCAAT/enhancer-binding protein homologous protein might play an important role in this process.     

Characteristics of lysophosphatidylcholine-induced Ca2+ response in human neuroblastoma SH-SY5Y cells

Lysophosphatidylcholine (LPC) is an important bioactive lipid. In the nervous system, elevated levels of LPC have been shown to produce demyelination. In the present study, we examined the effect of exogenous LPC on intracellular Ca2+ mobilization in human neuroblastoma SH-SY5Y cells. In Ca2+-containing medium, introduction of LPC induced a steady rise in cytosolic Ca2+ levels ([Ca2+]i) in a dose-dependent manner, and this rise was provoked by LPC itself, not by its hydrolysis product produced by lysophospholipase. The increase in [Ca2+]i was reduced by 36% by removal of extracellular Ca2+, while preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, inhibited the response by 23%, part of the Ca2+ influx. Conversely, Ni2+, which inhibits the Na+-Ca2+ exchanger, or Na+-deprivation did not affect LPC-induced Ca2+ influx. In Ca2+-free medium, depletion of Ca2+ stores in the endoplasmic reticulum (ER) by thapsigargin, an ER Ca2+-ATPase inhibitor, abolished the Ca2+ increase. Moreover, LPC-induced [Ca2+]i increase was fully blocked by ruthenium red and procaine, inhibitors of ryanodine receptor (RyR), but was not affected by 2-aminoethoxydiphenyl borate, an inhibitor of inositol triphosphate receptor, or by pertussis toxin, a G(i/o) protein inhibitor. Combined treatment with verapamil plus thapsigargin markedly inhibited but did not abolish the LPC-induced Ca2+ response. These findings indicate that LPC-induced [Ca2+]i increase depends on both external Ca2+ influx and Ca2+ release from ER Ca2+ stores, in which L-type Ca2+ channels and RyRs may be involved. However, in digitonin-permeabilized SH-SY5Y cells, LPC could not induce any [Ca2+]i increase in Ca2+-free medium, suggesting that LPC may act indirectly on RyRs of ER.     

NELL2 Function in the Protection of Cells against Endoplasmic Reticulum Stress

Continuous intra- and extracellular stresses induce disorder of Ca²⁺ homeostasis and accumulation of unfolded protein in the endoplasmic reticulum (ER), which results in ER stress. Severe long-term ER stress triggers apoptosis signaling pathways, resulting in cell death. Neural epidermal growth factor-like like protein 2 (NELL2) has been reported to be important in protection of cells from cell death-inducing environments. In this study, we investigated the cytoprotective effect of NELL2 in the context of ER stress induced by thapsigargin, a strong ER stress inducer, in Cos7 cells. Overexpression of NELL2 prevented ER stress-mediated apoptosis by decreasing expression of ER stress-induced C/EBP homologous protein (CHOP) and increasing ER chaperones. In this context, expression of anti-apoptotic Bcl-xL was increased by NELL2, whereas NELL2 decreased expression of pro-apoptotic proteins, such as cleaved caspases 3 and 7. This anti-apoptotic effect of NELL2 is likely mediated by extracellular signal-regulated kinase (ERK) signaling, because its inhibitor, U0126, inhibited effects of NELL2 on the expression of anti- and pro-apoptotic proteins and on the protection from ER stress-induced cell death.     

Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.     

Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.     

Treatment of human neuroblastoma cell line SH-SY5Y with HSP27 siRNA tagged-exosomes decreased differentiation rate into mature neurons

Heat shock proteins (HSPs) participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 small interfering RNA (siRNA)-tagged exosomes for the inhibition of Hsp27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Exosomes were isolated, characterized by scanning electron microscope (SEM) and CD63 then enriched with siRNA against Hsp27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hr. Exosome uptake was monitored by immunofluorescence assay. The cell viability and proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine/5-bromo-2′-deoxyuridine incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. The cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to Hsp27 siRNA tagged exosomes. The cell viability and proliferation were reduced compared to cells received nude exosomes ( p < 0.05). Clonogenic activity of cells was diminished by the inhibition of Hsp27. Flow cytometry analysis revealed that the inhibition of Hsp27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control. These data confirmed that exosomes could be used as appropriate bio-shuttles for the inhibition of Hsp27-aborted cell differentiation toward mature neuron.     

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.     

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.     

Epigenetic Silencing of CHOP Expression by the Histone Methyltransferase EHMT1 Regulates Apoptosis in Colorectal Cancer Cells

Colorectal cancer (CRC) has a high mortality rate among cancers worldwide. To reduce this mortality rate, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan) or targeted therapy (bevacizumab, cetuximab, and panitumumab) has been used to treat CRC. However, due to various side effects and poor responses to CRC treatment, novel therapeutic targets for drug development are needed. In this study, we identified the overexpression of EHMT1 in CRC using RNA sequencing (RNA-seq) data derived from TCGA, and we observed that knocking down EHMT1 expression suppressed cell growth by inducing cell apoptosis in CRC cell lines. In Gene Ontology (GO) term analysis using RNA-seq data, apoptosis-related terms were enriched after EHMT1 knockdown. Moreover, we identified the CHOP gene as a direct target of EHMT1 using a ChIP (chromatin immunoprecipitation) assay with an anti-histone 3 lysine 9 dimethylation (H3K9me2) antibody. Finally, after cotransfection with siEHMT1 and siCHOP, we again confirmed that CHOP-mediated cell apoptosis was induced by EHMT1 knockdown. Our findings reveal that EHMT1 plays a key role in regulating CRC cell apoptosis, suggesting that EHMT1 may be a therapeutic target for the development of cancer inhibitors.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: Function of arachidonic acid

Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 μM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 μM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A₂, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4α-phorbol 12, 13-didecanoate (4α-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4α-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells. © 1996 Wiley-Liss, Inc.     

Degradation of the type I inositol 1,4,5-trisphosphate receptor by caspase-3 in SH-SY5Y neuroblastoma cells undergoing apoptosis

The type I inositol 1,4,5-trisphosphate (IP(3)) receptor is selectively down-regulated in several neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and ischemia, all conditions in which apoptotic neuronal loss occurs. In the present study, we used a neuronal cell line, human neuroblastoma SH-SY5Y cells, to investigate whether the levels of IP(3) receptor are changed during apoptosis in these cells. Following induction of apoptosis by staurosporine, the immunoreactivity of the type I IP(3) receptor in microsome preparations from SH-SY5Y cells was reduced within 2 h, with a further reduction during subsequent hours. Immunoblot analyses, using antibodies to poly(ADP-ribose) polymerase and spectrin breakdown products, revealed proteolysis of these caspase-3 substrates within 3 h, confirming that IP(3) receptor cleavage is an early consequence of apoptosis. In vitro incubation of SH-SY5Y microsomes or immunopurified IP(3) receptor from rat cerebellum with recombinant caspase-3 led to generation of immunoreactive breakdown products similar to those observed in intact cells, suggesting that the type I IP(3) receptor is a potential substrate for caspase-3. Preincubation of the neuroblastoma cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone prevented IP(3) receptor degradation. These results show that the type I IP(3) receptor is a substrate for caspase-3 in neuronal cells and indicate that apoptotic down-regulation of IP(3) receptor levels may contribute to the pathology of neurodegenerative conditions.     

Activation of muscarinic cholinergic receptors enhances the volume-sensitive efflux of myo-inositol from SH-SY5Y neuroblastoma cells

A mechanism used by cells to regulate their volume under hypo-osmotic conditions is the release of organic osmolytes, one of which is myo-inositol. The possibility that activation of phospholipase-C-linked receptors can regulate this process has been examined for SH-SY5Y neuroblastoma cells. Incubation of cells with hypo-osmolar buffers (160-250 mOsm) led to a biphasic release of inositol which persisted for up to 4 h and could be inhibited by inclusion of anion channel blockers - results which indicate the involvement of a volume-sensitive organic anion channel. Inclusion of oxotremorine-M, a muscarinic cholinergic agonist, resulted in a marked increase (80-100%) in inositol efflux under hypo-osmotic, but not isotonic, conditions. This enhanced release, which was observed under all conditions of hypo-osmolarity tested, could be prevented by inclusion of atropine. Incubation of the cells with either the calcium ionophore, ionomycin, or the phorbol ester, phorbol 12-myristate 13-acetate, partially mimicked the stimulatory effect of muscarinic receptor activation when added singly, and fully when added together. The ability of oxotremorine-M to facilitate inositol release was inhibited by removal of extracellular calcium, depletion of intracellular calcium or down-regulation of protein kinase C. These results indicate that activation of muscarinic cholinergic receptors can regulate osmolyte release in this cell line.     

Celastrol protects human neuroblastoma SH-SY5Y cells from rotenone-induced injury through induction of autophagy

Celastrol, an active component found in the Chinese herb tripterygium wilfordii has been identified as a neuroprotective agent for neurodegenerative diseases including Parkinson’s disease (PD) through unknown mechanism. Celastrol can induce autophagy, which plays a neuroprotective role in PD. We tested the protective effect of celastrol on rotenone-induced injury and investigated the underlying mechanism using human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were treated with celastrol before rotenone exposure. The cells survival, apoptosis, accumulation of α-synuclein, oxidative stress and mitochondrial function, and autophagy production were analyzed. We found celastrol (500 nM) pre-treatment enhanced cell viability (by 28.99%, P < 0.001), decreased cell apoptosis (by 54.38%, P < 0.001), increased SOD and GSH (by 120.53% and 90.46%, P < 0.01), reduced accumulation of α-synuclein (by 35.93%, P < 0.001) and ROS generation (by 33.99%, P < 0.001), preserved MMP (33.93 ± 3.62%, vs. 15.10 ± 0.71% of JC-1 monomer, P < 0.001) and reduced the level of cytochrome C in cytosol (by 45.57%, P < 0.001) in rotenone treated SH-SY5Y cells. Moreover, celastrol increased LC3-II/LC3 I ratio by 60.92% (P < 0.001), indicating that celastrol activated autophagic pathways. Inhibiting autophagy by 3-methyladenine (3-MA) abolished the protective effects of celastrol. Our results suggested that celastrol protects SH-SY5Y cells from rotenone induced injuries and autophagic pathway is involved in celastrol neuroprotective effects.     

Protective effect of orexin-A on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by progressive and selective death of midbrain dopaminergic neurons. Pharmacologic treatment of PD can be divided into symptomatic and neuroprotective therapies.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

The essential role of ERK in 4-oxo-2-nonenal-mediated cytotoxicity in SH-SY5Y human neuroblastoma cells

Lipid peroxidation byproducts, such as 4-hydroxynonenal (HNE) and 4-oxo-2-nonenal (ONE), induce cell death in a wide variety of cell types, partly by modulating intracellular signaling pathways. However, the specific mechanisms involved, particularly for ONE, are unclear while c-Jun N-terminal kinase (JNK) has been shown to be essential in HNE-mediated cytotoxicity. In this study, we examined the role of mitogen-activated protein kinases signaling pathways in ONE-induced cytotoxicity in SH-SY5Y human neuroblastoma cells and found that ONE strongly induces the phosphorylation of extracellular signal-regulated kinase (ERK) and JNK, but not p38 MAPK. Interestingly, a transient exposure of the cells to ONE resulted in cell death, which contrasts with HNE-mediated toxicity. Importantly, blocking the ERK pathway, but not the JNK pathway, protected cells against ONE-induced cytotoxicity indicating a striking difference between the ONE- and HNE-mediated cytotoxicity mechanisms. Furthermore, inhibition of ERK reduced ONE-induced phosphorylation of p53, a key modulator of the cellular stress response, and the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), a hallmark of apoptosis. Overall, these data strongly suggest that ERK plays an essential role in ONE-mediated cytotoxicity and that ERK is an upstream component of p53-mediated apoptosis.     

A novel reciprocal and biphasic relationship between membrane cholesterol and β-secretase activity in SH-SY5Y cells and in human platelets

Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the Aβ peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane β-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane β-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment ( n  = 172) were significantly more likely to lie within the negative correlation zone than control platelets ( n  = 171). Pharmacological inhibition of SH-SY5Y β-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane β-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.