Neuroprotective Function of Rasagiline and Selegiline, Inhibitors of Type B Monoamine Oxidase, and Role of Monoamine Oxidases in Synucleinopathies.

Synucleinopathies are a group of neurodegenerative disorders caused by the accumulation of toxic species of α-synuclein. The common clinical features are chronic progressive decline of motor, cognitive, behavioral, and autonomic functions. They include Parkinson's disease, dementia with Lewy body, and multiple system atrophy. Their etiology has not been clarified and multiple pathogenic factors include oxidative stress, mitochondrial dysfunction, impaired protein degradation systems, and neuroinflammation. Current available therapy cannot prevent progressive neurodegeneration and "disease-modifying or neuroprotective" therapy has been proposed. This paper presents the molecular mechanisms of neuroprotection by the inhibitors of type B monoamine oxidase, rasagiline and selegiline. They prevent mitochondrial apoptosis, induce anti-apoptotic Bcl-2 protein family, and pro-survival brain- and glial cell line-derived neurotrophic factors. They also prevent toxic oligomerization and aggregation of α-synuclein. Monoamine oxidase is involved in neurodegeneration and neuroprotection, independently of the catalytic activity. Type A monoamine oxidases mediates rasagiline-activated signaling pathways to induce neuroprotective genes in neuronal cells. Multi-targeting propargylamine derivatives have been developed for therapy in various neurodegenerative diseases. Preclinical studies have presented neuroprotection of rasagiline and selegiline, but beneficial effects have been scarcely presented. Strategy to improve clinical trials is discussed to achieve disease-modification in synucleinopathies.

Mitochondria in Neuroprotection by Phytochemicals: Bioactive Polyphenols Modulate Mitochondrial Apoptosis System, Function and Structure.

In aging and neurodegenerative diseases, loss of distinct type of neurons characterizes disease-specific pathological and clinical features, and mitochondria play a pivotal role in neuronal survival and death. Mitochondria are now considered as the organelle to modulate cellular signal pathways and functions, not only to produce energy and reactive oxygen species. Oxidative stress, deficit of neurotrophic factors, and multiple other factors impair mitochondrial function and induce cell death. Multi-functional plant polyphenols, major groups of phytochemicals, are proposed as one of most promising mitochondria-targeting medicine to preserve the activity and structure of mitochondria and neurons. Polyphenols can scavenge reactive oxygen and nitrogen species and activate redox-responsible transcription factors to regulate expression of genes, coding antioxidants, anti-apoptotic Bcl-2 protein family, and pro-survival neurotrophic factors. In mitochondria, polyphenols can directly regulate the mitochondrial apoptosis system either in preventing or promoting way. Polyphenols also modulate mitochondrial biogenesis, dynamics (fission and fusion), and autophagic degradation to keep the quality and number. This review presents the role of polyphenols in regulation of mitochondrial redox state, death signal system, and homeostasis. The dualistic redox properties of polyphenols are associated with controversial regulation of mitochondrial apoptosis system involved in the neuroprotective and anti-carcinogenic functions. Mitochondria-targeted phytochemical derivatives were synthesized based on the phenolic structure to develop a novel series of neuroprotective and anticancer compounds, which promote the bioavailability and effectiveness. Phytochemicals have shown the multiple beneficial effects in mitochondria, but further investigation is required for the clinical application.

Modification of α-synuclein by lipid peroxidation products derived from polyunsaturated fatty acids promotes toxic oligomerization: its relevance to Parkinson disease.

Recently, toxic α-synuclein oligomer, which can mediate cell-to-cell propagation is suggested to cause sporadic Parkinson disease. α-Synuclein interacts with membrane lipids especially polyunsaturated fatty acids to stabilize its three-dementional structure. Peroxidation of polyunsaturated fatty acids may reduce their affinity to α-synuclein and peroxidation byproducts might modify α-synuclein. 4-Hydroxy-2-nonenal derived from n-6 polyunsaturated fatty acids was reported to modify α-synuclein to produce a toxic oligomer. Moreover, the accumulation of 4-hydroxy-2-nonenal, which could induce oligomeriztion of α-synuclein, was found in parkinsonian brains. Docosahexaenoic acid, an n-3 polyunsaturated fatty acids abundant in the neuronal membrane, was also found to enhance α-synuclein oligomerization; however, the precise details of the chemical reaction involved are unclear. Propanoylated lysine, a specific indicator of docosahexaenoic acid oxidation, was increased in neuronal differentiated human neuroblastoma SH-SY5Y cells overexpressing α-synuclein. α-Synuclein might be modified by the peroxidation products and then, is degraded by the autophagy-lysosome system. In addition, in the cells overexpressing α-synuclein, the mitochondrial electrone transfer chain was found to be inhibited. Accumulation of abnormal α-synuclein modified by lipid radicals derived from polyunsaturated fatty acids may be not only an indicator of brain oxidative stress but also causative of neurodegeneration such as Parkinson disease by impairing mitochondrial function.

Type A and B monoamine oxidases distinctly modulate signal transduction pathway and gene expression to regulate brain function and survival of neurons.

Type A and B monoamine oxidases (MAO-A, -B) mediate and modulate intracellular signal pathways for survival or death of neuronal cells. MAO-A is associated with development of neuronal architecture, synaptic activity, and onset of psychiatric disorders, including depression, and antisocial aggressive impulsive behaviors. MAO-B produces hydrogen peroxide and plays a vital role in neuronal loss of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. This review presents a novel role of MAO-A and B, their substrates and inhibitors, and hydrogen peroxide in brain function and neuronal survival and death. MAO-A activity is regulated not only by genetic factor, but also by environmental factors, including stress, hormonal deregulation, and food factors. MAO-A activity fluctuates by genetic-environmental factors, modulates the neuronal response to the stimuli, and affects behavior and emotional activities. MAO-B inhibitors selegiline and rasagiline protect neurons via increase expression of anti-apoptotic Bcl-2 and pro-survival neurotrophic factors in human neuroblastoma SH-SY5Y and glioblastoma U118MG cell lines. MAO-A knockdown suppressed the rasagiline-induced gene expression in SH-SY5Y cells, whereas MAO-B silencing enhanced the basal- and selegiline-induced gene expression in U118MG cells. MAO-A and B were shown to function as a mediator or repressor of gene expression, respectively. Further study on cellular mechanism underlying regulation of signal pathways by MAO-A and B may bring us a new insight on the role of MAOs in decision of neuronal fate and the development of novel therapeutic strategy may be expected for neuropsychiatric disorders.

Neurotrophic function of phytochemicals for neuroprotection in aging and neurodegenerative disorders: modulation of intracellular signaling and gene expression.

Bioactive compounds in food and beverages have been reported to promote health and prevent age-associated decline in cognitive, motor and sensory activities, and emotional function. Phytochemicals, a ubiquitous class of plant secondary metabolites, protect neuronal cells by interaction with cellular activities, in addition to the antioxidant and anti-inflammatory function. In aging and age-associated neurodegenerative disorders, phytochemicals protect neuronal cells by neurotrophic factor-mimic activity, in addition to suppression of apoptosis signaling in mitochondria. This review presents the cellular mechanisms underlying anti-apoptotic function and neurotrophic function of phytochemicals in the brain. Phytochemicals bind to receptors of neurotrophic factors, and also receptors for γ-aminobutyric acid, acetylcholine, serotonin, and glutamate and estrogen, and activate downstream signal pathways. Phytochemicals also directly intervene intracellular signaling molecules to modify the brain function. Finally, phytochemicals enhance the endogenous biosynthesis of genes coding anti-apoptotic Bcl-2 and neurotrophic factors, such as brain-derived and glial cell line-derived neurotrophic factor. The gene induction may play a major role in the neuroprotective function of dietary compounds shown by epidemiological studies. Quantitative measurement of neurotrophic factors induced by phytochemicals in the serum, cerebrospinal fluid, and other clinical samples is proposed as a surrogate assay method to evaluate the neuroprotective potency. Development of novel neuroprotective compounds is expected among compounds chemically synthesized from the brain-permeable basic structure of phytochemicals.

Type B and A monoamine oxidase and their inhibitors regulate the gene expression of Bcl-2 and neurotrophic factors in human glioblastoma U118MG cells: different signal pathways for neuroprotection by selegiline and rasagiline.

Type B monoamine oxidase (MAO-B) in glial cells has been considered to be associated with neuronal death in Parkinson's disease. MAO-B inhibitors, rasagiline and selegiline [(-)deprenyl], protect neurons in animal and cellular models of neurodegeneration. However, the role of MAO-B itself in the regulation of cell death processing remains elusive, whereas type A MAO (MAO-A) mediates the induction of anti-apoptotic Bcl-2 genes by rasagiline and selegiline. In this paper, the involvement of MAOs in the induction of neuroprotective genes by MAO inhibitors was investigated in human glioblastoma U118MG cells expressing mainly MAO-B. Selegiline significantly increased Mao-B, which was suppressed by Mao-A knockdown with short interfering (si)RNA, whereas rasagiline less markedly increased Mao-B, which was not affected by Mao-A knockdown. Mao-A mRNA was also markedly increased by rasagiline and selegiline, and Mao-B knockdown significantly enhanced the induction by selegiline, but not by rasagiline. Mao-B knockdown also significantly increased mRNA levels of Bcl-2, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Selegiline synergistically enhanced the expression of these genes in Mao-B knockdown cells, but Mao-A knockdown suppressed the increase. Rasagiline increased BDNF and GDNF, which Mao-B and Mao-A knockdown inhibited. These results show that MAO-B might function as a repressor and MAO-A as a mediator in the constitutional expression of pro-survival genes, and that MAO-B and MAO-A might regulate different signal pathways for rasagiline and selegiline to induce neuroprotective genes. The novel role of glial MAOs in the regulation of gene expression is discussed.

Phytochemicals prevent mitochondrial membrane permeabilization and protect SH-SY5Y cells against apoptosis induced by PK11195, a ligand for outer membrane translocator protein.

Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure-activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

Rasagiline prevents cyclosporine A-sensitive superoxide flashes induced by PK11195, the initial signal of mitochondrial membrane permeabilization and apoptosis.

Rasagiline, a neuroprotective inhibitor of type B monoamine oxidase, prevented PK111195-induced apoptosis in SH-SY5Y cells through inhibition of mitochondrial apoptosis signaling (J Neural Transm 120:1539-1551, 2013, J Neural Transm 122:1399-1407, 2015). This paper presents that PK11195 induced superoxide flashes, the transit production burst, mediated by cyclosporine A-sensitive membrane permeability transition. Rasagiline prevented superoxide flashes, calcium efflux, and cell death by PK11195. Regulation of the initial pore formation at the inner mitochondrial membrane was confirmed as the decisive mechanism of neuroprotection by rasagiline.

Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection.

Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed.

Revelation in the neuroprotective functions of rasagiline and selegiline: the induction of distinct genes by different mechanisms.

In Parkinson's disease, cell death of dopamine neurons in the substantia nigra progresses and neuroprotective therapy is required to halt neuronal loss. In cellular and animal models, selegiline [(-)deprenyl] and rasagiline, inhibitors of type B monoamine oxidase (MAO)-B, protect neuronal cells from programmed cell death. In this paper, the authors review their recent results on the molecular mechanisms by which MAO inhibitors prevent the cell death through the induction of antiapoptotic, prosurvival genes. MAO-A mediates the induction of antiapoptotic bcl-2 and mao-a itself by rasagiline, whereas a different mechanism is associated with selegiline. Rasagiline and selegiline preferentially increase GDNF and BDNF in nonhuman primates and Parkinsonian patients, respectively. Enhanced neurotrophic factors might be applicable to monitor the neurorescuing activity of neuroprotection.

Rasagiline prevents apoptosis induced by PK11195, a ligand of the outer membrane translocator protein (18 kDa), in SH-SY5Y cells through suppression of cytochrome c release from mitochondria.

Rasagiline protects neuronal cells from cell death caused by various lines of insults. Its neuroprotective function is due to suppression of mitochondrial apoptosis signaling and induction of neuroprotective genes, including Bcl-2 and neurotrophic factors. Rasagiline inhibits the mitochondrial membrane permeabilization, an initial stage in apoptosis, but the mechanism has been elusive. In this paper, it was investigated how rasagiline regulates mitochondrial death cascade in apoptosis induced in SH-SY5Y cells by PK11195, a ligand of the outer membrane translocator protein of 18 kDa. Rasagiline prevented release of cytochrome c (Cyt-c), and the following caspase 3 activation, ATP depletion and apoptosis, but did not inhibit the mitochondrial membrane potential collapse, in contrast to Bcl-2 overexpression. Rasagiline stabilized the mitochondrial contact site and suppressed Cyt-c release into cytoplasm, which should be the critical point for the regulation of apoptosis. Monoamine oxidase was not associated with anti-apoptotic activity of rasagiline in PK11195-induced apoptosis.

Type A monoamine oxidase is associated with induction of neuroprotective Bcl-2 by rasagiline, an inhibitor of type B monoamine oxidase.

Rasagiline and (-)deprenyl (selegiline), irreversible type B monoamine oxidase (MAO-B) inhibitors, protect neuronal cells through gene induction of pro-survival Bcl-2 and neurotrophic factors in the cellular models of neurodegenerative disorders. In this paper, the role of MAO in the up-regulation of neuroprotective Bcl-2 gene by these inhibitors was studied using type A MAO (MAO-A) expressing wild SH-SY5Y cells and the transfection-enforced MAO-B overexpressed cells. Rasagiline and (-)deprenyl, and also befloxatone, a reversible MAO-A inhibitor, increased Bcl-2 mRNA and protein in SH-SY5Y cells. Silencing MAO-A expression with short interfering (si) RNA suppressed Bcl-2 induction by rasagiline, but not by (-)deprenyl. MAO-B overexpression inhibited Bcl-2 induction by rasagiline and befloxatone, but did not affect that by (-)deprenyl, suggesting the different mechanisms behind Bcl-2 gene induction by these MAO-B inhibitors. The novel role of MAO-A in Bcl-2 induction by rasagiline is discussed with regard to the molecular mechanism underlying neuroprotection by the MAO inhibitors.

Mitochondria in neurodegenerative disorders: regulation of the redox state and death signaling leading to neuronal death and survival.

In Parkinson's disease, impaired function of mitochondrial complex I is involved in selective degeneration of dopamine neurons in the substantia nigra. Mitochondria are now considered to play an active role in neuronal death process through activating "intrinsic" apoptotic signaling, in addition to production of reactive oxygen species. This paper presents our recent findings on new functions of mitochondria in regulation of their redox state and function through reversible "S-glutathionylation", a mixed disulfide binding between sulfhydryl groups of GSH and protein cysteine in complex I subunits. Type A monoamine oxidase (MAO-A) localized at the mitochondrial outer membrane is a binding site of neurotoxins leading to apoptosis. Rasagiline and (-)deprenyl, type B MAO inhibitors of propagylamine-derivatives, bind to MAO-A to protect neuronal cells against apoptosis through induction of pro-survival Bcl-2 and neurotrophic factors. This review discusses the new role of mitochondria in regulation of neuronal cell death of neurodegenerative disorders.

Glutathione redox status in mitochondria and cytoplasm differentially and sequentially activates apoptosis cascade in dopamine-melanin-treated SH-SY5Y cells.

Neuromelanin (NM)-containing dopaminergic neurons in the substantia nigra are selectively vulnerable in Parkinson's disease (PD), suggesting the involvement of NM in the pathogenesis. NM is composed of protein, lipid, trace metals and melanin component, a mixture of eumelanin produced from dopamine (DA)-quinone and pheomelanin containing 5-S-cyteinyl-DA-quinone. We reported that NM induces mitochondria-mediated apoptosis in human dopaminergic SH-SY5Y cells, which was suppressed completely by Protease K-treatment, suggesting the essential requirement for the protein component. In this paper, the role of the melanin component in NM-dependent apoptosis was studied using SH-SY5Y cells and synthesized DA-melanin (DAM) and L-cysteinyl-DAM (Cys-DAM). DAM oxidatively decreased glutathione (GSH) and sulfhydryl (SH) content in mitochondria, whereas NM increased GSH by de-S-glutathionylation of complex I. DAM induced mitochondrial permeability transition (mPT), leading to membrane potential collapse and cytochrome c release, whereas Cys-DAM did not. However, the cytotoxicity of DAM itself was rather mild and thiol-targeting reducing reagents, including GSH, dithiothreitol and N-acetyl-cysteine, increased apoptosis significantly. The reducing SH reagents activated caspase 3 and induced apoptosis, but did not affect mPT. On the other hand, NM itself activated mitochondria-initiated apoptotic cascade, which GSH suppressed completely. The results indicate that DAM induces apoptosis through the sequential activation by oxidation of SH status in mitochondria and reduction in cytoplasm, in contrast to the case with NM. The regulation of apoptotic processing by SH redox state is discussed in relation to degeneration of nigra-striatal DA neurons in aging and PD, where oxidative stress is increased with impaired antioxidant capacity.

Metabolites of sesamin, a major lignan in sesame seeds, induce neuronal differentiation in PC12 cells through activation of ERK1/2 signaling pathway.

Sesamin, a major lignan in sesame seeds, exhibits various health benefits. Here, we investigated effects of sesamin, its stereoisomer episesamin, and their metabolites on neuronal differentiation in rat pheochromocytoma PC12 cells. Among all compounds tested, primary metabolites of sesamin and episesamin, SC-1 and EC-1 {S- and R-epimer of 2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane}, were the most potent to induce neuronal differentiation. SC-1 alone induced neuronal differentiation through extracellular signal-regulated kinase (ERK) 1/2 activation that is essential for nerve growth factor (NGF)-induced neuronal differentiation, as shown by the suppression with MEK1/2 inhibitors, PD98059 and U0126. However, SC-1 did not increase phosphorylation of TrkA, a high-affinity NGF receptor, and a TrkA inhibitor, K252a, did not affect SC-1-induced neuronal differentiation. Furthermore, SC-1 potentiated neuronal differentiation in cells co-treated with NGF, which was associated with enhanced ERK1/2 activation and increased expression of neuronal differentiation markers. Interestingly, when treated with SC-1 and a high dose of NGF, formation of synaptic connections and synaptophysin accumulation at the neurite terminals were markedly enhanced. These results indicate that (1) SC-1 alone induces neuronal differentiation, (2) SC-1 potentiates neuronal differentiation in NGF-treated cells, (3) SC-1 enhances formation of synaptic connections in cells treated with a high dose of NGF, all of which are associated with ERK1/2 activation. It is therefore concluded that SC-1 may promote neuronal differentiation by tapping into the ERK1/2-MAPK (mitogen-activated protein kinase) signaling pathway downstream from the TrkA receptor in PC12 cells.

Neuromelanin selectively induces apoptosis in dopaminergic SH-SY5Y cells by deglutathionylation in mitochondria: involvement of the protein and melanin component.

Parkinson's disease (PD) is characterized by selective depletion of nigral dopamine (DA) neurons containing neuromelanin (NM), suggesting the involvement of NM in the pathogenesis. This study reports induction of apoptosis by NM in SH-SY5Y cells, whereas protease-K-treated NM, synthesized DA- and cysteinyl dopamine melanin showed much less cytotoxicity. Cell death was mediated by mitochondria-mediated apoptotic pathway, namely collapse of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 3, but Bcl-2 over-expression did not suppress apoptosis. NM increased sulfhydryl content in mitochondria, and a major part of it was identified as GSH, whereas dopamine melanin significantly reduced sulfhydryl levels. Western blot analysis for protein-bound GSH demonstrated that only NM reduced S-glutathionylated proteins in mitochondria and dissociated macromolecular structure of complex I. Reactive oxygen and nitrogen species were required for the deglutathionylation by NM, which antioxidants reduced significantly with prevention of apoptosis. These results suggest that NM may be related to cell death of DA neurons in PD and aging through regulation of mitochondrial redox state and S-glutathionylation, for which NM-associated protein is absolutely required. The novel function of NM is discussed in relation to the pathogenesis of PD.

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells.

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.

Type A monoamine oxidase is the target of an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, leading to apoptosis in SH-SY5Y cells.

Mitochondrial monoamine oxidase (MAO) has been considered to be involved in neuronal degeneration either by increased oxidative stress or protection with the inhibitors of type B MAO (MAO-B). In this paper, the role of type A MAO (MAO-A) in apoptosis was studied using human neuroblastoma SH-SY5Y cells, where only MAO-A is expressed. An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, an MAO-A inhibitor, reduced membrane potential, DeltaPsim, in isolated mitochondria, and induced apoptosis in the cells, which 5-hydroxytryptamine, an MAO-A substrate, prevented. In contrast, beta-phenylethylamine, an MAO-B substrate, did not suppress the DeltaPsim decline by N-methyl(R)salsolinol. The binding of N-methyl(R)salsolinol to mitochondria was inhibited by clorgyline, a MOA-A inhibitor, but not by (-)deprenyl, an MAO-B inhibitor. RNA interference targeting MAO-A significantly reduced the binding of N-methyl(R)salsolinol with simultaneous reduction in the MAO activity. To examine the intervention of MAO-B in the apoptotic process, human MAO-B was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even although the activity and protein of MAO increased markedly. These results demonstrate a novel function of MAO-A in the binding of neurotoxins and the induction of apoptosis, which may account for neuronal cell death in neurodegenerative disorders, including Parkinson's disease.

A potent apoptosis-inducing activity of a sesquiterpene lactone, arucanolide, in HL60 cells: a crucial role of apoptosis-inducing factor.

Six main sesquiterpene lactones (germacranolides) from Calea urticifolia were evaluated for in vitro cytotoxicity against human tumor cell lines HL60 and SW480 cells. Among them, arucanolide and parthenolide displayed marked cytotoxicity against both cell lines. Arucanolide exhibited a low IC(50) in HL60 cells. The cytotoxic activity of arucanolide was observed at lower concentrations compared to that of parthenolide, which has been reported to be a typical and simple germacranolide. The activity was found to be mainly due to apoptosis that was assessed by morphological findings, DNA ladder formation (24 - 36 h), and flow cytometric analysis in HL60 cells. Western blotting and an apoptosis inhibition assay using caspase inhibitors did not demonstrate the activation of any caspases tested. However, the mitochondrial membrane potential of HL60 cells was lost after 24-h treatment with arucanolide, and concurrently apoptosis-inducing factor (AIF) released from mitochondria was detected by Western blot analysis. The inactivation of nuclear factor-kappaB, which has been commonly shown in parthenolide-induced apoptosis, did not occur in arucanolide-induced apoptosis. Taken together, the findings presented here indicate that arucanolide induced marked apoptosis in HL60 cells mainly by dissipating mitochondrial membrane potential, which would trigger AIF-induced apoptosis.

A highly bioactive lignophenol derivative from bamboo lignin exhibits a potent activity to suppress apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y cells.

Approaches to protection against neurodegenerative diseases, in which oxidative stress and inflammation are implicated, should be based on the current concept on the etiology of these diseases. Recently, a new therapeutic strategy has been proposed to protect neurons from cell death by attenuating the apoptotic signal transduction. Lignin, a durable aromatic network polymer second to cellulose in abundance, was able to be converted into highly active lignophenol derivatives with antioxidant activity by using our newly developed phase-separation technique. These lignophenol derivatives were found to show the potent neuroprotective activity against oxidative stress. Among the compounds examined, a lignocresol derivative from bamboo (lig-8) exhibited the most potent neuroprotective activity against hydrogen peroxide (H(2)O(2))-induced apoptosis in human neuroblastoma cell line SH-SY5Y by preventing the caspase-3 activation via either caspase-8 or caspase-9. Furthermore, it was found that lig-8 exerted the antiapoptotic effect by inhibiting dissipation of the mitochondrial membrane permeability transition induced by H(2)O(2) or by the peripheral benzodiazepin receptor ligand PK11195. Lig-8 was also shown to be potent in the antioxidant activity in the cells exposed to H(2)O(2), as assessed by flow cytometry using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and in vitro reactive oxygen species-scavenging potency. These data suggest that lig-8 is a promising neuroprotector, which affects the signaling pathway of neuronal cell death and that it would be of benefit to delay the progress of neurodegenerative diseases.