• Biomolecules & Therapeutics
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• v.11 no.2
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• pp.112-118
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• 2003

1-Methylated $\beta$-carbolines (harmaline and harmalol) and antioxidants (N-acetylcysteine and ascorbate) reduced the loss of cell viability in differentiated PC 12 cells treated with 5 mM glutamate. $\beta$-Carbolines prevented the glutamate-induced decrease in mitochondrial membrane potential, cytochrome c release and caspase-3 activation in PC 12 cells. $\beta$-Carbolines reduced the formation of reactive oxygen species and depletion of glutathione due to glutamate in PC12 cells. $\beta$-Carbolines revealed a scavenging action on hydrogen peroxide and reduced the iron and EDTA-mediated degradation of 2-deoxy-D-ribose. The results suggest that I-methylated $\beta$-carbolines attenuate the cytotoxic effect of glutamate on PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.

• Korean Journal of Pharmacognosy
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• v.27 no.2
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• pp.155-158
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• 1996

The methanol extract of Albizzia julibrissin Durazz. (Leguminosae) was successively partitioned into dichloromethane, ethylacetate, butanol (BuOH) and water fractions, and the effects of the each solvent extract on catecholamine biosynthesis in PC12 cells were investigated. Among them, the BuOH fraction $(5{\mu}g/ml\;medium)$ showed 68.8% and 63.6% inhibition on dopamine and norepinephrine content in PC12 cells, respectively. Tyrosine hydroxylase (TH) activity was also reduced markedly by treatment of the BuOH fraction (41.8% inhibition at $5{\mu}g/ml$ in the medium). Each solvent fraction did not show cytotoxicity towards PC12 cells by trypan blue exclusion test. This result suggests that the BuOH fraction has an inhibitory effect on catecholamine biosynthesis by reducing TH activity in PC12 cells.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.33-37
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• 2003

Oxidative damage to mitochondria is a critical mechanism in necrotic or apoptotic cell death induced by many kinds of toxic chemicals. Thioredoxin (Trx) family proteins are known to play protective roles in organisms under oxidative stress through redox reaction by using reducing equivalents of cysteines at a conserved active site, Cys-X-X-Cys. Whereas biological and physiological properties of Trx1 are well characterized, significance of mitochondrial thioredoxin (Trx2) is not well known. Therefore, we addressed physiological role of Trx2 in PC12 cells under oxidative stress. In PC12 cells, transiently overexpressed Trx2 significantly reduced cell death induced by hydrogen peroxide, whereas mutant Trx2, having serine residues instead of two cysteine residues at the active site did not. In addition, stably expressed Trx2 protected PC12 cells from serum deprivation. These results suggest that Trx2 may play defensive roles in PC12 cells by reducing oxidative stress to mitochondria.

• Archives of Pharmacal Research
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• v.29 no.8
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• pp.645-650
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• 2006

Tributyltin chloride (TBTC) at concentrations of $0.5-1.0\;{\mu}M$ inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of TBTC on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were investigated. TBTC at concentrations up to $1.0\;{\mu}M$ neither affected cell viability, nor induced apoptosis after 24 or 48 h in PC12 cells. However, TBTC at concentrations higher than $2.0\;{\mu}M$ caused cytotoxicity through an apoptotic process. In addition, exposure of PC12 cells to non-cytotoxic (0.5 and $1.0\;{\mu}M$) or cytotoxic $(2.0\;{\mu}M)$ concentrations of TBTC in combination with L-DOPA (20, 50 and $100\;{\mu}M$) resulted in a significant increase in cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTC or L-DOPA alone. The enhancing effects of TBTC on L-DOPA-induced cytotoxicity were concentration- and treatment time-dependent. These data demonstrate that TBTC enhances L-DOPA-induced cytotoxicity in PC 12 cells.

• BMB Reports
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• v.31 no.5
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• pp.468-474
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• 1998

Membrane depolarization in PC12 cells induces calcium influx via an L-type voltage-sensitive calcium channel (L-VSCC) and increases intracellular free calcium, which leads to tyrosine phosphorylation of epidermal growth factor (EGF) receptor and the associated adaptor protein, She. This activated EGF receptor complex then can activate mitogen-activated protein (MAP) kinase, as in nerve growth factor (NGF) receptor activation. In the present study, we investigated the role of EGF receptor in the signaling pathway initiated by membrane depolarization of PC12 cells. Prolonged membrane depolarization induced phosphorylation of extracellular signal-regulated kinase (ERK) within 1 min in undifferentiated PC12 cells. Pretreatment of PC12 cells with the calcium chelator EGTA abolished depolarization-stimulated ERK phosphorylation, but NGF-induced phosphorylation of ERK was not affected. The chronic treatment of phorbol ester, which down-regulated the activity of protein kinase C (PKC), did not affect the phosphorylation of ERK upon depolarization. In the presence of an inhibitor of EGF receptor, neither depolarization nor calcium ionophore increased the level of ERK phosphorylation. These data imply that the EGF receptor is functionally necessary to activate ERK and neurite outgrowth in response to the prolonged depolarization in PC12 cells, and also that PKC is apparently not involved in this signaling pathway.

• BMB Reports
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• v.35 no.3
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• pp.267-272
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• 2002

TNF-$\alpha$ elicits various responses including apoptosis, proliferation, and differentiation according to cell type. In neuronal PC12 cells, TNF-$\alpha$ induces moderate apoptosis while lipopolysarccaharide or trophic factor deprivation can potentiate apoptosis that is induced by TNF-$\alpha$. TNF-$\alpha$ initiates various signal transduction pathways leading to the activation of the caspase family, NF-${\kappa}B$, Jun N-terminal kinase, and p38 MAPK via the death domain that contains the TNF-$\alpha$ receptor. Inhibition of translation using cycloheximide greatly enhanced the apoptotic effect of TNF-$\alpha$. This implies that the induction of anti-apoptotic genes for survival by TNF-$\alpha$ may be able to protect PC12 cells from apoptosis. Accordingly, Bcl-2, an anti-apoptotic genes for survival by TNF-$\alpha$ may be able to protect PC12 cells from apoptosis. Accordingly, Bcl-2, an anti-apoptotic Bcl-2 family member, was highly expressed in response to TNF-$\alpha$. In this study, we examined the anti-apoptotic role of p38 MAPK that is activated by TNF-$\alpha$ in neuronal PC12 cells. The phosphorylation of p38 MAPK in response to TNF-$\alpha$ slowly increased and lasted several hours in the PC12 cell and DRG neuron. This specific inhibitor of p38 MAPK, SB202190, significantly enhanced the apoptosis that was induced by TNF-$\alpha$ in PC12 cells. This indicates that the activation of p38 MAPK could protect PC12 cells from apoptosis since there is no known role of p38 MAPK in resoonse to TNF-$\alpha$ in neuron. This discovery could be evidence for the neuroprotective role of the p38 MAPK.

• YAKHAK HOEJI
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• v.49 no.2
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• pp.156-161
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• 2005

Tetrahydropapaveroline (THP) at 5-15 ${\mu}$M has been found to induce L-DOPA-induced oxidative apoptosis in PC12 cells. In this study, the inhibitory effects of THP on dopamine bios ynthesis in PC12 cells and tyrosine hydroxylase (TH) activity in bovine adrenal were investigated. Treatment of PC12 cells with THP at 2.5-10 ${\mu}$M significantly decreased the intracellular dopamine content in a concentration-dependent manner (18.3% inhibition at 10 ${\mu}$M THP). In these conditions, TH activity was markedly inhibited by the treatment with THP at 2.5-10 ${\mu}$M in PC12 cells (23.4% inhibition at 10 $\mu$ M THP). In addition, THP had an inhibitory effect on bovine adrenal TH activity IC50 value, 153.9${\mu}$M). THP exhibited uncompetitive inhibition on bovine adrenal TH activity with a substrate L-tyrosine with the KI value of 0.30 mM. Treatment with L-DOPA at 20~50 ${\mu}$M increased the intracellular dopamine content in PC12 cells, and the increase in dopamine content by L-DOPA was inhibited in part when THP at non-cytotoxic (5-10 ${\mu}$M) or cytotoxic (15${\mu}$M) concentrations was associated with L-DOPA (20 and 50 ${\mu}$M) for 24 h incubation. These results suggest that THP at 5-10${\mu}$M decreases the basal dopamine content and reduces the increased dopamine content induced by L-DOPA in part by the inhibition of TH activity, and that THP at 15${\mu}$M also decreases dopamine content by oxidative stress in PC12 cells.

• Natural Product Sciences
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• v.10 no.3
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• pp.124-128
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• 2004

Previously, $(1R,9S)-{\beta}-Hydrastine$ hydrochloride has been found to lower dopamine content in PC12 cells (Kim et al., 20001). In this study, the effects of $(1R,9S)-{\beta}-Hydrastine$ hydrochloride on L-DOPA-induced cytotoxicity in PC12 cells were investigated. Treatment with $(1R,9S)-{\beta}-Hydrastine$ hydrochloride at concentrations higher than $500\;{\mu}M$ caused cytotoxicity in PC12 cells. In addition, $(1R,9S)-{\beta}-Hydrastine$ hydrochloride at non-cytotoxic or cytotoxic concentrations significantly enhanced L-DOPA-induced cytotoxicity (L-DOPA concentration, $50\;{\mu}M$). Treatment of PC12 cells with $750\;{\mu}M$ $-1R,9S)-{\beta}-Hydrastine$ hydrochloride and $50\;{\mu}M$ L-DOPA, alone or in combination, also induced cell death via a mechanism which exhibited morphological and biochemical characteristics of apoptosis, including chromatin condensation and membrane blebbing. Exposure of PC12 cells to $(1R,9S)-{\beta}-Hydrastine$ hydrochloride, L-DOPA and $(1R,9S)-{\beta}-Hydrastine$ hydrochloride plus L-DOPA for 48 h resulted in a marked increase in the cell loss and percentage of apoptotic cells compared with exposure for 24 h. These data indicate that $(1R,9S)-{\beta}-Hydrastine$hydrochloride at higher concentration ranges aggravates L-DOPA-induced neurotoxicity cytotoxicity in PC12 cells. Therefore, it is proposed that the long-term L-DOPA therapeutic patients with $(1R,9S)-{\beta}-Hydrastine$ hydrochloride could be checked for the adverse symptoms.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.1
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• pp.117-125
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• 2007

Neuronal ischemia is a pathological process caused by a lack of oxygen (anoxia) and glucose (hypoglycemia), resulting in neuronal death. It is believed that apoptosis is one of the mechanisms involved in ischemic cell death. Neuronal apoptosis is a process characterized by nuclear DNA fragmentation, changes of plasma membrane organization. To elucidate the mechanism of neuronal death following ischemic insult and to develop neuroprotective effects of Daebowonjeon(DBWJ) against ischemic damage, in vitro models are used. In vitro models of cell death have been devloped with pheochromocytoma (PC12) cell, which have become widely used as neuronal models of oxidative stress, trophic factor, serum deprivation and chemical hypoxia. Using a special ischemic device and PC12 cultures, we investigated an in vitro model of ischemia based on combined Oxygen and Glucose Deprivation (OGD) insult, followed by reoxygenation, mimicking the pathological conditions of ischemia. In this study, Daebowonjeon rescued PC12 cells from Oxygen-Glucose Deprivation (OGD)-induced cell death in a dose-dependent manner The nuclear staining of PC12 cells clearly showed that DBWJ attenuated nuclear condensation and fragmentation which represent typical neuronal apoptotic characteristics. DBWJ also prevents the LDH release and induction of Hypoxia Inducing Factor (HIF)-1 by OGD-exposed PC12 cells. Furthermore, DBWJ reduced the activation of polyADP-ribose polymerase (PARP) by OGO-exposed PC12 cells. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that oriental medicine, such as DBWJ, may prevent PC12 cell from OG D-induced neuronal death by inhibiting the apoptotic process.

• Molecules and Cells
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• v.20 no.2
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• pp.280-287
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• 2005

The insulin-mediated Ras/mitogen-activated protein (MAP) kinase cascade was examined in SK-N-BE(2) and PC12 cells, which can and cannot produce reactive oxygen species (ROS), respectively. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) was much lower in SK-N-BE(2) cells than in PC12 cells when the cells were treated with insulin. The insulin-mediated interaction of IRS-1 with Grb2 was observed in PC12 but not in SK-N-BE(2) cells. Moreover, the activity of extracellular-signal-related kinase (ERK) was much lower in SK-N-BE(2) than in PC12 cells when the cells were treated with insulin. Application of exogenous $H_2O_2$ caused increased tyrosine phosphorylation and Grb2 binding to IRS-1 in SK-N-BE(2) cells, while exposure to an $H_2O_2$ scavenger (N-acetylcysteine) or to a phophatidylinositol-3 kinase inhibitor (wortmannin), and expression of a dominant negative Rac1, decreased the activation of ERK in insulin-stimulated PC12 cells. These results indicate that the transient increase of ROS is needed to activate ERK in insulin-mediated signaling and that an inability to generate ROS is the reason for the insulin insensitivity of SK-N-BE(2) cells.