• 한국발생생물학회지:발생과생식
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• 제16권1호
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• pp.59-67
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• 2012

Previously, we found that $Zap70$ (Zeta-chain-associated protein kinase) expressed in the mouse oocytes and played significant role in completion of meiosis specifically at MI-MII (metaphase I-II) transition. Microinjection of $Zap70$ dsRNA into the cytoplasm of germinal vesicle oocyte resulted in MI arrest, and exhibited abnormalities in their spindles and chromosome configurations. The purpose of this study was to determine the mechanisms of action of $Zap70$ in oocyte maturation by evaluating downstream signal networking after $Zap70$ RNAi (RNA interference). The probe hybridization and data analysis were used by Affymetrix Gene Chip Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Total 1,152 genes were up (n=366) and down (n=786) regulated after $Zap70$ RNAi. Among those genes changed, we confirmed the expressional changes of the genes involved in the regulation of actin cytoskeleton and MAPK (mitogen-activated protein kinase) signaling pathway, since the phenotypes of $Zap70$ RNAi in oocytes were found in the changes in the chromosome separation and spindle structures. We confirmed the changes in gene expression in the actin skeletal system as well as in the MAPK signaling pathway, and concluded that these changes are main cause of the aberrant chromosome arrangement and abnormal spindles after $Zap70$ RNAi.

• Journal of Periodontal and Implant Science
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• 제38권sup2호
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• pp.343-354
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect. Materials and Methods: We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion. Results: The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions $Mg^{2+}$ and $Ca^{2+}$. Conclusion: We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.

• Archives of Pharmacal Research
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• 제25권2호
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• pp.191-196
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• 2002

The water extract from the root bark of Cortex Mori (CM, Morus alba L.: Sangbaikpi), a mulberry tree, has been known in Chinese traditional medicine to have antiphlogistic, diuretic, and expectorant properties. In this study, the cytotoxicity of CM against tumor cells and its mechanism was examined . CM exhibited cytotoxic activity on K-562, B38O human leukemia cells and B16 mouse melanoma cells at concentrations of > 1 mg/ml. A DNA fragmentation, PARP cleavage, and nuclear condensation assay showed that those cells exposed to CM underwent apoptosis. The water extract of Scutellarie Radix (SR) was used as a negative control and showed no cytotoxicity in those cells. The flow cytometric profiles of the CM-treated cells were also indicative of apoptosis. However, they did not appear to exert the G1 arrest, which is observed in other tubulin inhibitor agents such as vincristine, taxol. The protein-binding test using Biacore and a microtubule assembly-disassembly assay provided evidence showing that CM bound to the tubulins resulting in 3 markets inhibition of the assembly, but not the disassembly of microtubules. The possible nonspecific effect of the CM extract could be excluded due to the results using SR, which did not affect the assembly process. Overall, the water extract of CM induces apoptosis of tumor cells by inhibiting microtubule assembly.

• Molecules and Cells
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• 제42권11호
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• pp.773-782
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• 2019

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKT-mTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53-dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, $p21^{Cip1/WAF1}$ is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of $p21^{Cip1/WAF}$. Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of $p21^{Cip1/WAF1}$, leading to H3K9me3 and SAHFs formation.

• Molecules and Cells
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• 제42권3호
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• pp.252-261
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• 2019

The omega-3 fatty acid docosahexaenoic acid (DHA) is known to induce apoptosis and cell cycle arrest via the induction of reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stress in many types of cancers. However, the roles of DHA in drug-resistant cancer cells have not been elucidated. In this study, we investigated the effects of DHA in cisplatin-resistant gastric cancer SNU-601/cis2 cells. DHA was found to induce ROS-dependent apoptosis in these cells. The inositol 1,4,5-triphosphate receptor ($IP_3R$) blocker 2-aminoethyl diphenylboninate (2-APB) reduced DHA-induced ROS production, consequently reducing apoptosis. We also found that G-protein-coupled receptor 120 (GPR120), a receptor of long-chain fatty acids, is expressed in SNU-601/cis2 cells, and the knockdown of GPR120 using specific shRNAs alleviated DHA-mediated ROS production and apoptosis. GPR120 knockdown reduced the expression of ER stress response genes, similar to the case for the pre-treatment of the cells with N-acetyl-L-cysteine (NAC), an ROS scavenger, or 2-APB. Indeed, the knockdown of C/EBP homologous protein (CHOP), a transcription factor that functions under ER stress conditions, markedly reduced DHA-mediated apoptosis, indicating that CHOP plays an essential role in the anti-cancer activity of DHA. These results suggest that GPR120 mediates DHA-induced apoptosis by regulating $IP_3R$, ROS, and ER stress levels in cisplatin-resistant cancer cells, and that GPR120 is an effective chemotherapeutic target for cisplatin resistance.

• 대한한방내과학회지
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• 제43권3호
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• pp.344-362
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• 2022

Objective: In this study, the antioxidant activity of Ojayeonjong-hwan extracts was compared, and the following results were obtained. Methods: For hydrothermal and ethanol extracts, DPPH free radical and ABTS cationic radical erasing activity and reducing power using the FRAP method were compared, and the association between the antioxidant power of each extract and total phenol content was investigated. Significant results were obtained through in vitro apoptosis analysis through FFITC staining, mitochondrial membrane potential analysis, and ROS level measurement using C2C12 myoblastoma. Results: 1. In a comparison of DPPH free radical and ABTS cationic radical scavenging activity, water, and 70% ethanol extracts of Ojayeonjong-hwan (WEO and EEO) showed superior radical scavenging ability. 2. In the results of reducing power using the FRAP method, WEO and EEO showed antioxidant activity, which was shown to be dependent on the total phenol content contained in the extracts. 3. In comparison to the protective effect against H₂O₂-induced oxidative stress in C2C12 myoblasts, water extracts had no significant effect, but 70% ethanol extracts inhibited H₂O₂-mediated cytotoxicity in a concentration-dependent manner. 4. The cytotoxic protective effect of EEO against oxidative stress in C2C12 myoblasts was correlated with its inhibitory effects on H₂O₂-induced apoptosis and cell-cycle arrest. 5. In H₂O₂-treated C2C12 myoblasts, the apoptosis inhibitory effects of EEO were associated with the suppression of mitochondrial dysfunction and DNA damage. 6. The protective effects of EEO against H₂O₂-induced oxidative stress in C2C12 myoblasts were directly related to the inhibition of ROS generation. Conclusions: Ojayeonjong-hwan extracts all have protective potential against oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• 제26권5호
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• pp.367-375
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• 2022

Gastric cancer stem cells (GCSCs) are a major cause of radioresistance and chemoresistance in gastric cancer (GC). Therefore, targeting GCSCs is regarded as a powerful strategy for the effective treatment of GC. Atorvastatin is a widely prescribed cholesterol-lowering drug that inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting enzyme in the mevalonate pathway. The anticancer activity of atorvastatin, a repurposed drug, is being investigated; however, its therapeutic effect and molecular mechanism of action against GCSCs remain unknown. In this study, we evaluated the anticancer effects of atorvastatin on MKN45-derived GCSCs. Atorvastatin significantly inhibited the proliferative and tumorsphere-forming abilities of MKN45 GCSCs in a mevalonate pathway-independent manner. Atorvastatin induced cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade. Furthermore, atorvastatin exerted an antiproliferative effect against MKN45 GCSCs by inhibiting the expression of cancer stemness markers, such as CD133, CD44, integrin α6, aldehyde dehydrogenase 1A1, Oct4, Sox2, and Nanog, through the downregulation of β-catenin, signal transducer and activator of transcription 3, and protein kinase B activities. Additionally, the combined treatment of atorvastatin and sorafenib, a multi-kinase targeted anticancer drug, synergistically suppressed not only the proliferation and tumorsphere formation of MKN45 GCSCs but also the in vivo tumor growth in a chick chorioallantoic membrane model implanted with MKN45 GCSCs. These findings suggest that atorvastatin can therapeutically eliminate GCSCs.

• Clinical and Experimental Reproductive Medicine
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• 제50권3호
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• pp.177-184
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• 2023

Objective: Reconstructed oocytes after polar body genome transfer constitute a potential therapeutic option for patients with a history of embryo fragmentation and advanced maternal age. However, the rescue of genetic material from the first polar body (PB1) through introduction into the donor cytoplasm is not yet ready for clinical application. Methods: Eighty-five oocytes were obtained following in vitro maturation (IVM) and divided into two groups: PB1 nuclear transfer (PB1NT; n=54) and control (n=31). Following enucleation and PB1 genomic transfer, PB1 fusion was assessed. Subsequently, all fused oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured in an incubator under a time-lapse monitoring system to evaluate fertilization, embryonic morphokinetic parameters, and cleavage patterns. Results: Following enucleation and fusion, 77.14% of oocytes survived, and 92.59% of polar bodies (PBs) fused. However, the normal fertilization rate was lower in the PB1NT group than in the control group (56.41% vs. 92%, p=0.002). No significant differences were observed in embryo kinetics between the groups, but a significant difference was detected in embryo developmental arrest after the four-cell stage, along with abnormal cleavage division in the PB1NT group. This was followed by significant between-group differences in the implantation potential rate and euploidy status. Most embryos in the PB1NT group had at least one abnormal cleavage division (93.3%, p=0.001). Conclusion: Fresh PB1NT oocytes successfully produced normal zygotes following PB fusion and ICSI in IVM oocytes. However, this was accompanied by low efficiency in developing into cleavage embryos, along with an increase in abnormal cleavage patterns.

• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.249-261
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• 2024

New anti-lung cancer therapies are urgently required to improve clinical outcomes. Since ganodermanontriol (GDNT) has been identified as a potential antineoplastic agent, its role in lung adenocarcinoma (LUAD) is investigated in this study. Concretely, lung cancer cells were treated with GDNT and/or mycophenolate mofetil (MMF), after which MTT assay, flow cytometry and Western blot were conducted. Following bioinformatics analysis, carboxylesterase 2 (CES2) was knocked down and rescue assays were carried out in vitro. Xenograft experiment was performed on mice, followed by drug administration, measurement of tumor growth and determination of CES2, IMPDH1 and IMPDH2 expressions. As a result, the viability of lung cancer cells was reduced by GDNT or MMF. GDNT enhanced the effects of MMF on suppressing viability, promoting apoptosis and inducing cell cycle arrest in lung cancer cells. GDNT up-regulated CES2 level, and strengthened the effects of MMF on down-regulating IMPDH1 and IMPDH2 levels in the cells. IMPDH1 and IMPDH2 were highly expressed in LUAD samples. CES2 was a potential target for GDNT. CES2 knockdown reversed the synergistic effect of GDNT and MMF against lung cancer in vitro. GDNT potentiated the role of MMF in inhibiting tumor growth and expressions of CES2 and IMPDH1/2 in lung cancer in vivo. Collectively, GDNT suppresses the progression of LUAD by activating CES2 to enhance the metabolism of MMF.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10407-10412
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• 2015

Background: ${\beta}$-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ${\beta}$-elemene against glioma cells remains unclear. In the present study, we assessed effects of ${\beta}$-elemene on human glioma cells and explored the underlying mechanism. Materials and Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ${\beta}$-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ${\beta}$-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ${\beta}$-elemne treatment group. Results: With increase in the concentration of ${\beta}$-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability ($IC_{50}$) was $48.5{\mu}g/mL$ for 24h. ${\beta}$-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ${\beta}$-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ${\beta}$-elemene in a time and does-dependent manner. Conclusions: Our results indicate that ${\beta}$-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.