• Journal of Korean Neurosurgical Society
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• 제38권6호
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• pp.456-464
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• 2005

Objective : The authors investigated whether rotenone induces cellular death also in non-dopaminergic neurons and high concentration of potassium ion can show protective effect for non-dopaminergic neuron in case of rotenone-induced cytotoxicity. Methods : Neuro 2A cells was treated with rotenone, and their survival as well as cell death mechanism was estimated using 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium[MTT] assay, Lactate dehydrogenase[LDH] release assay, fluorescence microscopy, and agarose gel electrophoresis. The changes in rotenone-treated cells was also studied after co-treatment of 50mM KCl. And the protective effect of KCl was evaluated by mitochondrial membrane potential assay and compared with the effects of various antioxidants. Results : Neuro 2A cells treated with rotenone underwent apoptotic death showing chromosome condensation and fragmentation as well as DNA laddering. Co-incubation of neuro 2A cells with 50mM KCl prevented it from the cytotoxicity induced by rotenone. Intracellular accumulation of reactive oxygen species[ROS] resulting by rotenone were significantly reduced by 50mM KCl. Potassium exhibited significantly similar potency compared to the antioxidants. Conclusion : The present findings showed that potassium attenuated rotenone-induced cytotoxicity, intracellular accumulation of ROS, and fragmentation of DNA in Neuro 2A cells. These findings suggest the therapeutic potential of potassium ion in neuronal apoptosis, but the practical application of high concentration of potassium ion remains to be settled.

• Biomolecules & Therapeutics
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• 제27권1호
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• pp.48-53
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• 2019

Reactive oxygen species (ROS) are widely generated in biological processes such as normal metabolism and response to xenobiotic exposure. While ROS can be beneficial or harmful to cells and tissues, generation of ROS by diverse anti-cancer drugs or phytochemicals plays an important role in the induction of apoptosis. We recently identified a derivative of naphthalene, MS-5, that induces apoptosis of an ovarian cell, CAOV-3. Interestingly, MS-5 induced apoptosis by down-regulating the ROS. Cell viability was evaluated by water-soluble tetrazolium salt (WST-1) assay. Apoptosis was evaluated by flow cytometry analysis. Intracellular ROS ($H_2O_2$), mitochondrial superoxide, mitochondrial membrane potential (MMP) and effect on cycle were determined by flow cytometry. Protein expression was assessed by western blotting. The level of ATP was measured using ATP Colorimetric/Fluorometric Assay kit. MS-5 inhibited growth of ovarian cancer cell lines, CAOV-3, in a concentration- and time-dependent manner. MS-5 also induced G1 cell cycle arrest in CAOV-3 cells, while MS-5 decreased intracellular ROS generation. In addition, cells treated with MS-5 showed the decrease in MMP and ATP production. In this study, we found that treatment with MS-5 in CAOV-3 cells induced apoptosis but decreased ROS level. We suspect that MS-5 might interfere with the minimum requirements of ROS for survival. These perturbations appear to be concentration-dependent, suggesting that MS-5 may induce apoptosis by interfering with ROS generation. We propose that MS-5 may be a potent therapeutic agent for inducing apoptosis in ovarian cancer cell through regulation of ROS.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.775-783
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• 2021

Sea cucumber, Holothuria scabra, is a well-known traditional Asian medicine that has been used for suppressing inflammation, promoting wound healing, and improving immunity. Moreover, previous studies demonstrated that the extract from H. scabra contains many bioactive compounds with potent inhibitory effect on tumor cell survival and progression. However, the effect of the methanolic extract from the body wall of H. scabra (BWMT) on human prostate cancer cells has not yet been investigated. In this study, we aimed to investigate the effects and underlying mechanism of BWMT on prostate cancer cell viability and metastasis. BWMT was obtained by maceration with methanol. The effect of BWMT on cell viability was assessed by MTT and colony formation assays. The intracellular ROS accumulation was evaluated using a DCFH-DA fluorescence probe. Hoechst 33342 staining and Annexin V-FITC/PI staining were used to examine the apoptotic-inducing effect of the extract. A transwell migration assay was performed to determine the anti-metastasis effect. BWMT significantly reduced cell viability and triggered cellular apoptosis by accumulating intracellular ROS resulting in the upregulation of JNK and p38 signaling pathways. In addition, BWMT also inhibited the invasion of PC3 cells by downregulating MMP-2/-9 expression via the ERK pathway. Consequently, our study provides BWMT from H. scabra as a putative therapeutic agent that could be applicable against prostate cancer progression.

• Molecules and Cells
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• 제45권7호
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• pp.454-464
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• 2022

DJ-1 is one of the causative genes of early-onset familial Parkinson's disease (PD). As a result, DJ-1 influences the pathogenesis of sporadic PD. DJ-1 has various physiological functions that converge to control the levels of intracellular reactive oxygen species (ROS). Based on genetic analyses that sought to investigate novel antioxidant DJ-1 downstream genes, pyruvate dehydrogenase (PDH) kinase (PDK) was demonstrated to increase survival rates and decrease dopaminergic (DA) neuron loss in DJ-1 mutant flies under oxidative stress. PDK phosphorylates and inhibits the PDH complex (PDC), subsequently downregulating glucose metabolism in the mitochondria, which is a major source of intracellular ROS. A loss-of-function mutation in PDK was not found to have a significant effect on fly development and reproduction, but severely ameliorated oxidative stress resistance. Thus, PDK plays a critical role in the protection against oxidative stress. Loss of PDH phosphatase (PDP), which dephosphorylates and activates PDH, was also shown to protect DJ-1 mutants from oxidative stress, ultimately supporting our findings. Further genetic analyses suggested that DJ-1 controls PDK expression through hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator of the adaptive response to hypoxia and oxidative stress. Furthermore, CPI-613, an inhibitor of PDH, protected DJ-1 null flies from oxidative stress, suggesting that the genetic and pharmacological inhibition of PDH may be a novel treatment strategy for PD associated with DJ-1 dysfunction.

• 치위생과학회지
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• 제23권4호
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• pp.264-270
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• 2023

Background: Resin-based dental materials release residual monomers or other substances from incomplete polymerization into the oral cavity, thereby causing adverse biological effects on oral tissue. 10-Methacryloyloxydecyl dihydrogen phosphate (10-MDP), an acidic monomer containing dihydrogen phosphate and methacrylate groups, is the most commonly used component of resin-based dental materials, such as restorative composite resins, dentin adhesives, and resin cements. Although previous studies have reported the cytotoxicity and biocompatibility in various cultured cells, the effects of resin monomers on cellular aging have not been reported to date. Therefore, this study aimed to investigate the effects of the resin monomer 10-MDP on cellular senescence and inflamm-aging in vitro. Methods: After stimulation with 10-MDP, MC3T3-E1 osteoblast-like cells were examined for cell viability by WST-8 assay and reactive oxygen species (ROS) production by flow cytometry. The protein and mRNA levels of molecular markers of aging were determined by western blotting and RT-PCR analysis, respectively. Results: Treatment with 0.05 to 1 mM 10-MDP for 24 hours reduced the survival of MC3T3-E1 cells in a concentration-dependent manner. The intracellular ROS levels in the 10-MDP-treated experimental group were significantly higher than those in the control group. 10-MDP at a concentration of 0.1 mM increased p53, p16, and p21 protein levels. Additionally, an aging pattern was observed with blue staining due to intracellular senescence-associated beta-galactosidase activity. Treatment with 10-MDP increased the levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8, however their expression was decreased by mitogen-activated-protein-kinase (MAPK) inhibitors. Conclusion: Taken together, these results suggest that the exposure of osteoblast-like cells to the dental resin monomer 10-MDP, increases the level of cellular senescence and the inflammatory response is mediated by the MAPK pathway.

• 생명과학회지
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• 제30권7호
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• pp.581-591
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• 2020

본 연구에서는 한국의 전통발효식품인 식해, 열무김치, 비지에서 분리한 유산균의 프로바이오틱스로써의 사용 가능성을 확인하였다. 분리된 유산균 중 pH 2.5의 산에서 60% 이상의 생존율을 나타내는 Pediococcus inopinatus BZ4, Lactobacillus plantarum SH1, Lactobacillus brevis SH14, Pediococcus pentosaceus YMT1, Leuconostoc mesenteroides YMT2는 0.3% 담즙산에서도 모두 우수한 생존율을 나타내어 이 5종을 선별하여 실험을 진행하였다. 간접적으로 유용미생물의 군집화 및 병원성 세균의 부착을 저해하는 자가 응집 및 상호 응집 실험에서 다섯 개의 유산균은 강력한 응집능을 나타내었다. 유기용매를 이용한 세포 표면 소수성 실험에서 3가지 용매에 모두 부착성을 나타내어 세포 표면의 높은 소수성을 보여주었으며 이는 간접적으로 장세포에 부착할 수 있는 세포 부착능이 우수하다는 것을 보여준다. 또한, DPPH, ABTS 라디컬 소거능 측정, 지질 과산화억제능 실험에서도 선별된 유산균의 cell-free supernatant 및 intracellular cell-free extract는 항산화 활성을 나타내었다. 마지막으로 진균인 C. albicans ATCC 10231를 제외한 4가지 병원성세균(E. coli KCTC 2571, H. pylori HPKCTC B0150, L. monocytogenes KCTC 13064, S. aureus KCTC 1916)에서 모두 항균활성이 나타남을 확인하였다. 상기 실험결과를 바탕으로, 분리된 유산균은 항산화, 항균활성을 보유하고 있는 프로바이오틱스 제제로써 활용이 가능할 것으로 기대되며 이는 기초적인 실험으로써 산업화를 위한 임상검증 등의 추가적인 연구가 필요하다고 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제26권6호
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• pp.531-540
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• 2022

Group 1 metabotropic glutamate receptors (mGluRs) can positively affect postsynaptic neuronal excitability and epileptogenesis. The objective of the present study was to determine whether group 1 mGluRs might be involved in synaptically-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) spikes and neuronal cell death induced by 0.1 mM Mg²⁺ and 10 µM glycine in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague-Dawley rats using imaging methods for Ca²⁺ and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays for cell survival. Reduction of extracellular Mg²⁺ concentration ([Mg²⁺]_o) to 0.1 mM induced repetitive [Ca²⁺]_i spikes within 30 sec at day 11.5. The mGluR5 antagonist 6-Methyl2-(phenylethynyl) pyridine (MPEP) almost completely inhibited the [Ca²⁺]_i spikes, but the mGluR1 antagonist LY367385 did not. The group 1 mGluRs agonist, 3,5-dihydroxyphenylglycine (DHPG), significantly increased the [Ca²⁺]_i spikes. The phospholipase C inhibitor U73122 significantly inhibited the [Ca²⁺]_i spikes in the absence or presence of DHPG. The IP₃ receptor antagonist 2-aminoethoxydiphenyl borate or the ryanodine receptor antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate also significantly inhibited the [Ca²⁺]_i spikes in the absence or presence of DHPG. The TRPC channel inhibitors SKF96365 and flufenamic acid significantly inhibited the [Ca²⁺]_i spikes in the absence or presence of DHPG. The mGluR5 antagonist MPEP significantly increased the neuronal cell survival, but mGluR1 antagonist LY367385 did not. These results suggest a possibility that mGluR5 is involved in synaptically-induced [Ca²⁺]_i spikes and neuronal cell death in cultured rat hippocampal neurons by releasing Ca²⁺ from IP₃ and ryanodine-sensitive intracellular stores and activating TRPC channels.

• 대한수의학회지
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• 제39권1호
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• pp.34-44
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• 1999

Ischemic damage in the selectively vulnerable populations of neurons is thought to be caused by an abnormal accumulation of intracellular calcium. It has been reported that the neurons, expressing specific calcium binding proteins, might effectively control intracellular calcium concentrations because of a high capacity to buffer intracellular calcium in the brain ischemic condition. It is uncertain that parvalbumin, one of the calcium binding proteins, can protect the neurons from the cerebral ischemic damage. Recently, treatment of kappa opioid agonists increased survival rate, improved neurological function, and decreased tissue damage under the cerebral ischemic condition. Many evidences indicate that these therapeutic effects might result from regulation of calcium concentration. This study was designed to analyze the changes of number in parvalbumin-positive neurons after cerebral ischemic damage according to timepoints after cerebral ischemic induction. In addition, we evaluated the effect of GR89696 (kappa opioid agonist) or naltrexone(non selective opioid antagonist) on the changes of number in parvalbumin expressing neurons under ischemic condition. Cerebral ischemia was induced by occluding the common carotid artery of experimental animals. The hippocampal areas were morphometrically analyzed at different time point after ischemic induction(1, 3, 5 days) by using immuno-histochemical technique and imaging analysis system. The number of parvalbumin-positive neurons in hippocampus was significantly reduced at 1 day after ischemia(p<0.05). Furthermore, the number of parvalbumin-immunoreactive neurons was dramatically reduced at 3 and 5 days after cerebral ischemic induction(p<0.05) as compared to 1 day group after ischemia, as well as sham control group. Significant reduction of parvalbumin positive neurons in CA1 region of hippocampus was observed at 1 day after cerebral ischemic induction. However, significant loss of MAP2 immunoreactivity was observed at 3 day after cerebral ischemia. The loss of parvalbumin-positive neurons and MAP2 immunoreactivity in CA1 region was prevented by pre-administration of GR89696 compared to that of saline-treated ischemic group. Furthermore, protective effect of GR89696 partially reversed by pre-treatment of naltrexone. These data indicate that parvalbumin-positive neurons more sensitively responded to cerebral ischemic damage than MAP2 protein. Moreover, this loss of parvalbumin-positive neurons was effectively prevented by the pretreatment of kappa opioid agonist. It was also suggested that the changes of number in parvalbumin-positive neurons could be used as the specific marker to analyze the degree of ischemic neuronal damage.

• 한국발생생물학회지:발생과생식
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• 제18권2호
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• pp.117-125
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• 2014

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.101-108
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• 2005

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a well-known inducer of apoptotic cell death in many tumor cells. 1RAIL is expressed in human placenta, and cytotrophoblast cells express 1RAIL receptors. However, the role of TRAIL in human placentas and cytotrophoblast cells is not. well understood. In this study a trophoblast cell line, JEG-3, was used as a model system to examine the effect of TRAIL. on key intracellular signaling pathways involved in the control of trophoblastic cell apoptosis and survival JEG-3 cells expressed receptors for 1RAIL, death receptor (DR) 4, DR5, decoy receptor (OcR) 1 and DeR2. Recombinant human TRAIL (rhTRAIL) did not have a cytotoxic effect determined by MIT assay and did not induce apoptotic cell death determined by poly-(ADP-ribose) polymerase cleavage assay. rhTRAIL induced a rapid and transient nuclear translocation of nuclear $factor-{\kappa}B(NF-{\kappa}B)$ determined by immunoblotting using nuclear protein extracts. rhTRAIL rapidly activated extracellular signal-regulated protein kinase (ERK) 1/2 as determined by immnoblotting for phospho-ERK1/2. However, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK) and Akt (protein kinase B) were not activated by rhTRAIL. The ability of 1RAIL to induce $NF-{\kappa}B$ and ERK1/2 suggests that interaction between TRAIL and its receptors may play an important role in trophoblast cell function during pregnancy.