• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.2
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• pp.296-302
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• 2002

Bohyulmyunyuk-dan is an Oriental herbal formulation to enhance the general body conditions as well as immune response against both endogenous and exogenous harmful challenges. This study was designed to investigate the effect of Bohyulmyunyuk-dan on the cisplatin-induced toxicity of primary rat astrocytes and C6 glioma cells. After trestment of astrocytes and C6 glioma cells with cisplatin, MTT assay was carried out to measure cytotoxicity of brain cells. To explore the mechanism of cytotoxicity, astrocytes were treated with Bohyulmyunyuk-dan and followed by the addition of cisplatin. Then, the protective effects of Bohyulmyunyuk-dan were investigated in apoptosis signaling pathway. The results were obtained as follows ; Bohyulmyunyuk-dan protected the death of astrocytes by cisplatin, which decreased the viability of astrocytes and C6 glioma cells in a time- and dose-dependent manner. Bohyulmyunyuk-dan protected the apoptotic death of astrocytes from cisplatin induced cell apoptosis. Bohyulmyunyuk-dan inhitited the activation of caspase-3 and -9 protease in astrocytes by cisplatin. Bohyulmyunyuk-dan inhibited the deavage of PARP in astrocytes by cisplatin. According to above results, Bohyulmyunyuk-dan may prevent brain cells from cytotoxicity induced cell apoptosis induced by chemotherapeatic agents induding displatin.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.4
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• pp.888-894
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• 2009

It has been reported that silibinin, a natural polyphenolic flavonoid, induces cell death in various cancer cell types. However, the underlying mechanisms by which silibinin induces apoptosis in human glioma cells are poorly understood. The present study was therefore undertaken to examine the effect of silibinin on glioma cell apoptosis and to determine its underlying mechanism in human glioma cells. Apoptosis was estimated by FACS analysis. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (${\Psi}m$) were measured using fluorescence dyes DCFH-DA and $DiOC_6$(3), respectively. Cytochrome c release from mitochondria and caspase-3 activation were estimated by Western blot analysis using specific antibodies. Exposure of cells to 30 mM silibinin induced apoptosis starting at 6 h, with increasing effects after 12-48h in a time-dependent manner. Silibinin caused ROS generation and disruption of ym, which were associated with the silibinin-induced apoptosis. The silibinin-induced ROS generation and disruption in ym were prevented by inhibitors of mitochondrial electron transport chain. The hydrogen peroxide scavenger catalase blocked ROS generation and apoptosis induced by silibinin. Silibinin induced cytochrome c release into cytosolic fraction and its effect was prevented by catalase and cyclosporine A. Silibinin treatment caused caspase-3 activation, which was inhibited by DVED-CHO and cyclosporine A. Pretreatment of caspase inhibitors also protected against the silibinin-induced apoptosis. These findings indicate that ROS generation plays a critical role in the initiation of the silibinin-induced apoptotic cascade by mediation of the mitochondrial apoptotic pathway including the disruption of ${\Psi}m$, cytochrome c release, and caspase-3 activation.

• Toxicological Research
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• v.9 no.2
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• pp.195-206
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• 1993

The expression of proenkephalin (proENK) mRNA and Met-enkephalin (ME) secretion in C6 rat glioma cells and bovine adrenal medullary chromaffin (BAMC) cells were elucidated in the present study. The levels of proENK mRNA and ME secreted into the media in BAMC cells were measured in the presence of cycloheximide and 12-tetrade-canoylphorbol-13-acetate (TPA). Cycloheximide (20 nM) abolished the induction of proENK mRNA expression, protein synthesis and ME secretion by TPA (1nM), indicating that de novo protein synthesis was necessary for proENK gene expression and ME secretion.

• Journal of Life Science
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• v.27 no.3
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• pp.267-274
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• 2017

The patient with end-stage renal disease show several nervous complications. The factors contributing to the nervous complications are still incompletely characterized. Cyanate, known as one of the uremic toxins, is derived spontaneously from urea. To investigate the mechanism of cyanate-induced effect on C6 glioma cells, the glioma cells were treated with 0, 1, 5, 10, 20 and 40 mM cyanate. There was a dose-dependent decrease in cell viability and the decreased number of cell was observed in glioma cells by treatment with cyanate. Western blot showed the down- regulation of procaspase-3, which means up-regulation of caspase-3, and the up-regulation of caspase-8, but the down-regulation by cyanate. In addition, cDNA microarray showed 934 down-regulated genes and 165 up-regulated genes on 1,099 genes in cyanate treated group. Treatment with cyanate led to 16 down-regulated genes and 6 up-regulated genes on apoptosis category, and especially heat shock 70 kD protein 1A gene on the category of apoptosis was significantly up-regulated. These results suggest that cyanate can induce apoptosis through caspase-8 and caspase-3 in glioma cells and decrease of gene expression including apoptosis category in glioma cells. These effects of cyanate may play a role in the nervous complications of patient with end-stage renal disease.

• Korean Journal of Clinical Laboratory Science
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• v.43 no.2
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• pp.75-81
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• 2011

To clarify the oxidative stress of reactive oxygen species (ROS) and the effect of Chrysanthemum morifolium L. (CM) flower extract on the cultured neuroglial cells (C6 glioma) damaged by ROS, cell adhesion effect was measured by colorimetric assay after cultured C6 glioma cells were treated with various concentrations of glucose oxidase (GO) for 5 hours. For the antioxidative effect of CM flower extract, cell adhesion activity (CAA), superoxide dismutase (SOD)-like activity and lactate dehydrogenase (LDH) activity were assessed against GO-induced cytotoxicity on same cultures. In this study, GO remarkably decreased CAA dose-dependently, and the $XTT_{90}$ and $XTT_{50}$ values were measured at 15 mU/mL and 50 mU/mL following the treatment of C6 glioma cells with 5~60 mU/mL of GO. The CM flower extract significantly increased cell adhesion activity damaged by GO-induced cytotoxicity, and it also showed the SOD-like activity and the decrease of LDH activity. From these results, it is suggested that GO was cytotoxic on cultured C6 glioma cells, and CM flower extract showed antioxidative effects as shown by the increased CAA, SOD-like activity and the decrease of LDH activity on GO-induced cytotoxicity on the same cultures.

• Journal of Yeungnam Medical Science
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• v.36 no.2
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• pp.105-108
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• 2019

Background: Although kidney transplantation outcomes have improved dramatically after using calcineurin inhibitors (CNIs), CNI toxicity continues to be reported and the mechanism remains uncertain. Here, we investigated the neurotoxicity of CNIs by focusing on the viability of glioma cells. Methods: Glioma cells were treated with several concentrations of CNIs for 24 hours at $37^{\circ}C$ and their cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: Exposure to 0, 0.25, 0.5, 2.5, 5.0, and 10.0 mM concentrations respectively showed 100%, 64.3%, 61.3%, 68.1%, 62.4%, and 68.6% cell viability for cyclosporine and 100%, 38.6%, 40.8%, 43.7%, 37.8%, and 43.0% for tacrolimus. The direct toxic effect of tacrolimus on glioma cell viability was stronger than that of cyclosporine at the same concentration. Conclusion: CNIs can cause neurological side effects by directly exerting cytotoxic effects on brain cells. Therefore, we should carefully monitor the neurologic symptoms and level of CNIs in kidney transplant patients.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.1
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• pp.19-25
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• 2001

We have studied the effects of excitatory amino acids on the expression of the c-fos and c-jun mRNA in rat C6 glioma cells. The glutamate, $N-methyl-_D-aspartate$ (NMDA), and kainic acid (KA) increased c-fos mRNA level in a concentration-dependent manner. However, they did not affect c-jun mRNA level. In addition, forskolin and phorbol 12-myristate 13-acetate (PMA) increased c-fos mRNA level. Furthermore, PMA increased c-jun mRNA level whereas forskolin downregulated c-jun mRNA level. The glutamate, NMDA and KA, at a concentration of 0.25 mM, did not affect the basal c-fos and c-jun mRNA levels, and also did not affect forskolin- and PMA-induced responses. Furthermore, both forskolin and PMA itself increased the phosphorylation of ERK (extracellular signal regulated kinase) and CREB (cyclicAMP responsible element binding protein) proteins. The KA, NMDA, and glutamate did not affect forskolin- induced increase of ERK and CREB phosphorylation. The KA decreased PMA-induced increase of phosphorylation of ERK and CREB proteins, whereas glutamate and NMDA did not affect the phosphorylation of ERK and CREB proteins induced by PMA. These findings suggest that, in C6 glioma cells, c-fos mRNA induction induced by EAAs is not mediated by phosphorylation of ERK and CREB proteins.

• Biomolecules & Therapeutics
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• v.14 no.1
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• pp.25-29
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• 2006

We tested effects of bioactive lysophospholipids including lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosylphosphorylcholine (SPC), and sphingosine I-phosphate (S1P) on membrane potential in C6 glioma cells to understand action mechanism of the lysophospholipids. Membrane potential was estimated by measuring fluorescence change of DiBAC-loaded glioma cells. LPA largely increased membrane potential and the increase was gradually diminished. LPC also increased the membrane potential, however, the increase sustained. SPC induced smaller increase of membrane potential than LPC. SIP was not able to change the membrane potential. We tested effects of suramin and pertussis toxin on lysophospholipid-induced membrane potential increase. However, there wasn't any effect. The membrane potential increase was partially diminished in $Na^+$-free media, suggesting $Na^+$ influx as a component of membrane potential changes. Thus, involvement of $Na^+$ influx in the increase of membrane potential by lysophospholipids and independence of suramin-sensitive GPCRs and pertussis toxin-sensitive G proteins are found in this study.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.448-453
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• 1999

In ginsenoside Rh2-treated rat glioma C6Bu-1 cells, apoptotic morphological changes, such as cell shrinkage, chromatin condensation and pyknosis were confirmed by means of electron microscopy. To evaluate whether induction of apoptosis by ginsenoside Rh2 is mediated by the members of Bcl-2 family, we first established C6Bu-1 cells overexpressing Bcl-2. It was demonstrated that the expression of Bcl-2, Bcl-xL, and Bax was not altered in ginsenoside Rh2-treated C6Bu-1 overexpressing C6Bu-1 cells failed to prevent from ginsenoside Rh2-induced cell death. These results suggest the existence of other apoptotic pathway that requires induction of apoptosis by ginsenoside Rh2 rather than the pathway through Bcl-2, $Bcl-x_{L}$ or Bax in C6Bu-1 cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.5
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• pp.1322-1329
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• 2008

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruits on cell viability in human glioma cells. The present study was undertaken to examine the effect of mulberry fruit (Mar; Fructus) on cell viability and to determine its underlying mechanism in human glioma cells. Cell viability and cell death were estimated by MTT assay and trypanblue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with $DiOC_6$(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Mori Fructus resulted in apoptotic cell death in a dose- and time-dependent manner. Mori Fructus increased ROS generation and the Mori Fructus-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in Mari Fructus-induced cell death. Western blot analysis showed that Mori Fructus treatment caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). Mori Fructus induced depolarization of mitochondrial membrane potential and its effect was inhibited by the antioxidants NAC and catalase. Mori Fructus induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by Mori Fructus and caspase inhibitors prevented the Mori Fructus-induced cell death. These findings suggest that Mori Fructus results in human glioma cell death through ROS-dependent mitochondrial pathway in human glioma cells.