• Biomolecules & Therapeutics
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• v.21 no.5
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• pp.358-363
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• 2013

In the present study, we investigated the effect of intracellular glutathione (GSH) depletion in heart-derived H9c2 cells and its mechanism. L-buthionine-S,R-sulfoximine (BSO) induced the depletion of cellular GSH, and BSO-induced reactive oxygen species (ROS) production was inhibited by glutathione monoethyl ester (GME). Additionally, GME inhibited BSO-induced caspase-3 activation, annexin V-positive cells, and annexin V-negative/propidium iodide (PI)-positive cells. Treatment with rottlerin completely blocked BSO-induced cell death and ROS generation. BSO-induced GSH depletion caused a translocation of PKC-${\delta}$ from the cytosol to the membrane fraction, which was inhibited by treatment with GME. From these results, it is suggested that BSO-induced depletion of cellular GSH causes an activation of PKC-${\delta}$ and, subsequently, generation of ROS, thereby inducing H9c2 cell death.

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2129-2140
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• 2017

Silibinin is the major active component of silymarin, extracted from the medicinal plant Silybum marianum. Silibinin has potent antibacterial activity; however, the exact mechanism underlying its activity has not been elucidated. Here, we investigated the novel mechanism of silibinin against Escherichia coli. Time-kill kinetic assay showed that silibinin possess a bactericidal effect at minimal inhibitory concentration (MIC) and higher concentrations (2-and 4-fold MIC). At the membrane, depolarization and increased intracellular $Ca^{2+}$ levels were observed, considered as characteristics of bacterial apoptosis. Additionally, cells treated with MIC and higher concentrations showed apoptotic features like DNA fragmentation, phosphatidylserine exposure, and caspase-like protein expression. Generally, apoptotic death is closely related with ROS generation; however, silibinin did not induce ROS generation but acted as a scavenger of intracellular ROS. These results indicate that silibinin dose-dependently induces bacterial apoptosis-like death, which was affected by ROS depletion, suggesting that silibinin is a potential candidate for controlling bacteria.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.6
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• pp.519-528
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• 2019

Mitochondrial dysfunction is closely associated with reactive oxygen species (ROS) generation and oxidative stress in cells. On the other hand, modulation of the cellular antioxidant defense system by changes in the mitochondrial DNA (mtDNA) content is largely unknown. To determine the relationship between the cellular mtDNA content and defense system against oxidative stress, this study examined a set of myoblasts containing a depleted or reverted mtDNA content. A change in the cellular mtDNA content modulated the expression of antioxidant enzymes in myoblasts. In particular, the expression and activity of glutathione peroxidase (GPx) and catalase were inversely correlated with the mtDNA content in myoblasts. The depletion of mtDNA decreased both the reduced glutathione (GSH) and oxidized glutathione (GSSG) slightly, whereas the cellular redox status, as assessed by the GSH/GSSG ratio, was similar to that of the control. Interestingly, the steady-state level of the intracellular ROS, which depends on the reciprocal actions between ROS generation and detoxification, was reduced significantly and the lethality induced by $H_2O_2$ was alleviated by mtDNA depletion in myoblasts. Therefore, these results suggest that the ROS homeostasis and antioxidant enzymes are modulated by the cellular mtDNA content and that the increased expression and activity of GPx and catalase through the depletion of mtDNA are closely associated with an alleviation of the oxidative stress in myoblasts.

• The Korean Journal of Food & Health Convergence
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• v.10 no.2
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• pp.13-17
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• 2024

Oxygen is necessary to sustain life, but reactive oxygen species (ROS) produced by oxygen metabolism can cause mutations and toxicity. ROS can damage cellular macromolecules, leading to oxidative stress, which can accelerate cell death and aging. ROS generated in food affect the taste, color, and aroma of food, and high levels of ROS in meat can cause spoilage. Superoxide dismutase (SOD) plays an important role in scavenging ROS in food and reducing their toxicity to organisms. SOD exerts its antioxidant effect by catalyzing the breakdown of O₂-• to H₂O₂. As a natural antioxidant, SOD has the ability to regenerate and maintain its activity over a long period of time without depletion, unlike chemical antioxidants that may have side effects or stability issues. This antioxidant effect of SOD has great potential in a variety of industries, and in the food industry it can be utilized to improve product quality and provide safe and healthy products to consumers. By disrupting the SOD2 and SOD3 genes in Candida albicans, we studied the effects of SOD2 and SOD3 genes on the antioxidant system, suggesting its potential as a natural antioxidant.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.493-502
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• 2020

Apigenin, a naturally occurring flavonoid, is known to exhibit significant anticancer activity. This study was designed to determine the effects of apigenin on two malignant mesothelioma cell lines, MSTO-211H and H2452, and to explore the underlying mechanism(s). Apigenin significantly inhibited cell viability with a concomitant increase in intracellular reactive oxygen species (ROS) and caused the loss of mitochondrial membrane potential (ΔΨm), and ATP depletion, resulting in apoptosis and necroptosis in monolayer cell culture. Apigenin upregulated DNA damage response proteins, including the DNA double strand break marker phospho (p)-histone H2A.X. and caused a transition delay at the G₂/M phase of cell cycle. Western blot analysis showed that apigenin treatment upregulated protein levels of cleaved caspase-3, cleaved PARP, p-MLKL, and p-RIP3 along with an increased Bax/Bcl-2 ratio. ATP supplementation restored cell viability and levels of DNA damage-, apoptosisand necroptosis-related proteins that apigenin caused. In addition, N-acetylcysteine reduced ROS production and improved ΔΨm loss and cell death that were caused by apigenin. In a 3D spheroid culture model, ROS-dependent necroptosis was found to be a mechanism involved in the anti-cancer activity of apigenin against malignant mesothelioma cells. Taken together, our findings suggest that apigenin can induce ROS-dependent necroptotic cell death due to ATP depletion through mitochondrial dysfunction. This study provides us a possible mechanism underlying why apigenin could be used as a therapeutic candidate for treating malignant mesothelioma.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.427-433
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• 1998

In brain hypoxic-ischemia, an excess release of glutamate and a marked production of reactive oxygen species (ROS) occur in neuronal and non-neuronal cells. The present study investigated the effect of the biological antioxidants dihydrolipoic acid (DHLA) and lipoic acid (LA) on N-methyl-D-aspartate (NMDA)- and ROS-induced neurotoxicity in cultured rat cortical neurons. DHLA enhanced NMDA-evoked rises in intracellular calcium concentration ($[Ca^{2+}]_i$). In contrast, LA did not alter the NMDA-evoked calcium responses but decreased after a brief treatment of dithiothreitol (DTT), which possesses a strong reducing potential. Despite the modulation of NMDA receptor-mediated rises in $[Ca^{2+}]_i$, neither DHLA nor LA altered the NMDA receptor-mediated neurotoxicity, as assessed by measuring the amount of lactate dehydrogenase released from dead or injured cells. DHLA, but not LA, prevented the neurotoxicity induced by xanthine/xanthine oxidase-generated superoxide radicals. Both DHLA and LA decreased the glutathione depletion-induced neurotoxicity. The present data may indicate that biological antioxidants DHLA and LA protect neurons from ischemic injuries via scavenging oxygen free radicals rather than modulating the redox modulatory site(s) of NMDA receptor.

• Biomolecules & Therapeutics
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• v.13 no.4
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• pp.256-262
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• 2005

We examined the signaling molecules involved in the 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and increase in cellular glutathione (GSH) level in SK-N-SH cells. The 6-OH-DA-induced cell death was significantly prevented by the pretreatment with N-acetylcysteine (NAC), a thiol antioxidant, and BAPTA, an intracellular $Ca^{2+}$ chelator. Although 6-OHDA induced ERK phosphorylation, the pretreatment with PD98059, an ERK inhibitor, did not block 6-OHDA-induced cell death. In addition, the 6-OHDA-induced activation of caspase-3, a key signal for apoptosis, was blocked by the pretreatment with NAC and BAPTA. While the level of reactive oxygen species (ROS) was significantly increased in the 6-OHDA-treated cells, the cellular GSH level was not altered for the first 6-hr exposure to 6-OHDA, but after then, the level was significantly increased, which was also blocked by the pretreatment with NAC and BAPTA, but not by PD98059. Depletion of GSH by pretreating the cells with DL-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor, rather significantly potentiated the 6-OHDA-induced death. In contrast to the pretreatment with NAC, 6-OHDA-induced cell death was not prevented by the post-treatment with NAC 30 min after 6-OHDA treatment. The results indicate that the GSH level which is increased adaptively by the 6-OHDA-induced ROS and intracellular $Ca^{2+}$ is not enough to overcome the death signal mediated through ROS-$Ca^{2+}$ -caspase pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.2
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• pp.121-130
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• 2019

Glutamate toxicity-mediated mitochondrial dysfunction and neuronal cell death are involved in the pathogenesis of several neurodegenerative diseases as well as acute brain ischemia/stroke. In this study, we investigated the neuroprotective mechanism of dieckol (DEK), one of the phlorotannins isolated from the marine brown alga Ecklonia cava, against glutamate toxicity. Primary cortical neurons ($100{\mu}M$, 24 h) and HT22 neurons (5 mM, 12 h) were stimulated with glutamate to induce glutamate toxic condition. The results demonstrated that DEK treatment significantly increased cell viability in a dose-dependent manner ($1-50{\mu}M$) and recovered morphological deterioration in glutamate-stimulated neurons. In addition, DEK strongly attenuated intracellular reactive oxygen species (ROS) levels, mitochondrial overload of $Ca^{2+}$ and ROS, mitochondrial membrane potential (${\Delta}{\Psi}_m$) disruption, adenine triphosphate depletion. DEK showed free radical scavenging activity in the cell-free system. Furthermore, DEK enhanced protein expression of heme oxygenase-1 (HO-1), an important anti-oxidant enzyme, via the nuclear translocation of nuclear factor-like 2 (Nrf2). Taken together, we conclude that DEK exerts neuroprotective activities against glutamate toxicity through its direct free radical scavenging property and the Nrf-2/HO-1 pathway activation.

• Biomolecules & Therapeutics
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• v.12 no.2
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• pp.92-100
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• 2004

Effect of the depletion or oxidation of GSH on mitomycin c (MMC)-induced mitochondrial damage and cell death was assessed in small cell lung cancer (SCLC) cells. MMC induced cell death and the decrease in the GSH contents in SCLC cells, which were inhibited by z-LEHD.fmk (a cell permeable inhibitor of caspase-9), z-DQMD.fmk (a cell permeable inhibitor of caspase-3) and thiol compound, N-acetylcysteine. MMC caused nuclear damage, release of cytochrome c and activation of caspase-3, which were reduced by N-acetylcysteine. The depletion of GSH due to L-butionine-sulfoximine enhanced the MMC-induced cell death and formation of reactive oxygen species in SCLC cells, whereas the oxidation of GSH due to diamide or $NH_2Cl$ did not affect cytotoxicity of MMC. The results show that MMC may cause cell death in SCLC cells by inducing mitochondrial dysfunction, leading to activation of caspase-9 and -3. The MMC-induced change in the mitochondrial membrane permeability, followed by cell death, in SCLC cells may be significantly enhanced by the depletion of GSH. In contrast, the oxidation of GSH may not affect cytotoxicity of MMC.

• International Journal of Oral Biology
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• v.35 no.3
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• pp.129-135
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• 2010

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ($[Ca^{2+}]i$) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the $[Ca^{2+}]i$, which was inhibited by pretreatment with phenyl-N-tertbuthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced $[Ca^{2+}]i$ increase was suppressed both in a calcium free solution and after depletion of the intracellular $Ca^{2+}$ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red ($50\;{\mu}M$) and capsazepine ($10\;{\mu}M$). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.