• 생명과학회지
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• 제29권3호
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• pp.295-302
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• 2019

Liquiritigenin (LG)은 licorice 뿌리에서 분리된 chiral flavonoid이다. LG는 항산화, 항암 및 항염증 효과를 포함하여 다양한 생물학적 활성을 가지고 있다. 구강편평세포암종에서 LG의 항암 활성은 아직 밝혀지지 않았다. 본 연구에서는 구강편평상피암 세포(HN22)에서 LG의 항암 효능을 시험하였다. HN22 세포에 LG를 처리하여 MTT 분석으로 세포 생존율을 평가하였으며, Annecin V/7-Aminactinomycin D 염색, 세포주기 및 Multi-caspase 활성을 $Muse^{TM}$ cell Analyzer로 분석하여 세포사멸 유도를 확인하였다. 분석결과, 구강편평상피암 HN22 세포에 LG를 처리시 G2/M 세포주기 정지를 일으켰으며, Western blotting 통하여 cyclin B1 및 CDC2 발현 감소와 p27 발현 증가를 확인하였다. LG는 활성산소종의 생성을 유발하고, CCAAT/enhancer-binding protein homologous protein (CHOP) 및 78-kDa glucose regulated protein (GRP78)의 발현을 농도의존적으로 유도하였다. HN22 세포에 LG의 처리는 미토콘드리아 막전위의 손실(${\Delta}{\Psi}m$)을 일으켰다. LG를 처리한 HN22 세포의 단백질 분석결과 apoptotic protease activating factor-1 (Apaf-1), cleaved Poly (ADP-Ribose) Polymerase (C-PARP) 및 Bax의 발현을 증가함을 확인하였다. 따라서 우리의 결과는 LG이 구강편평상피암 세포의 세포사멸을 유도하여 항암제 역할을 할 수 있는 천연 화합물임을 시사한다.

• 생명과학회지
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• 제32권4호
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• pp.271-278
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• 2022

신규 종양 유전자 Cancer Upregulated Gene (CUG) 2가 어떻게 유사-종양줄기세포 표현형을 유도하는지 잘 알려져 있지 않다. Cyclin E, c-Myc, Notch, 그리고 Yap1와 같은 종양단백질를 분해하여 그 발현을 조절하는 FBXW7 E3 ligase의 발현이 대장암, 자궁경부암, 그리고 위암 등 여러 암조직에서 낮아져 있음이 보고되고 있다. 그래서 우리는 이 FBXW7 단백질이 CUG2에 의한 종양형성에 관여할 수 있다는 가설을 세웠다. 이 연구에서 우리는 각 대조구 세포주보다 CUG2가 과발현된 A549 폐암 세포주와 BEAS-2B 기관지 세포주에서 FBXW7 단백질 발현이 낮게 나왔다. 여기서 MG132를 처리하게 되면 감소된 FBXW7과 FBXW7 기질로 알려진 Yap1 단백질 발현이 증가되는 결과를 관찰하였다. 종양줄기세포 현상에서 FBXW7의 역할을 규명하기 위하여, FBXW7 siRNA를 처리하였다. 대조구 세포주에서 감소된 FBXW7의 조건은 세포 이동 침습, 그리고 구형 형성이 증가되는 종양줄기세포 현상이 촉진되는 것을 관찰하였고, CUG2가 과발현된 두 세포주에서 FBXW7 발현 벡타 도입으로 FBXW7 발현 증가는 종양줄기세포 현상이 억제됨을 알 수 있었다. 또한 FBXW7의 감소는 EGFR-Akt-ERK1/2와 β-catenin-Yap1-NEK2 신호 경로를 활성화시키고, 반대로 FBXW7 발현 증가는 이 두 경로의 활성이 억제됨을 알 수 있었다. 이들 결과를 종합해 보면, CUG2 과발현은 FBXW7의 발현 감소로 이어지고, 이는 EGFR-Akt-ERK1/2와 β-catenin-Yap1-NEK2 신호경로를 활성화시켜 유사-종양줄기세포 현상을 촉진하는 것으로 생각된다.

• 구강회복응용과학지
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• 제38권3호
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• pp.150-161
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• 2022

목적:인간 치은섬유아세포의 세포활동에 관련된 다양한 유전자들의 발현에 마이크로그루브-파이브로넥틴 복합 표면이 미치는 영향을 확인하고자 하였다. 연구 재료 및 방법: 평활한 티타늄시편(NE0), 산처리만 시행한 티타늄시편(E0), 마이크로그루브 및 산처리된 티타늄시편(E60/10), 파이브로넥틴을 고정시킨 평활한 티타늄시편(NE0FN), 산처리 및 파이브로넥틴을 고정시킨 티타늄시편(E0FN), 그리고 마이크로그루브 및 산처리 후 파이브로넥틴을 고정시킨 티타늄시편(E60/10FN) 실험군 제작 후 인간 치은섬유아세포의 세포행동 관련 44개 유전자에 대한 실시간 중합효소연쇄반응 실험을 진행하였다. 결과: 인간치은섬유아세포 부착과 증식 등에 관여하는 4종류 신호전달 경로가 활성화되었다. Focal adhesion 경로에 속하는 Integrin α5, Integrin β1, Integrin β3, Talin-2 유전자들, PI3K-AKT 신호 전달 경로에 속하는 AKT1, AKT2, NF-κB 유전자들, MAPK 신호전달 경로에 속하는 MEK2, ERK1, ERK2 유전자들, 세포주기 신호 전달 경로에 속하는 cyclin D1, CDK4, CDK6 유전자들이 마이크로그루브-파이브로넥틴 복합 티타늄표면(E60/10FN)에서 상향조절되었다. 결론: 마이크로그루브-파이브로넥틴 복합 티타늄표면이 세포행동에 관여하는 다양한 유전자들을 상향조절할 수 있다.

• International Journal of Oral Biology
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• 제32권1호
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• Asian Pacific Journal of Cancer Prevention
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• 제15권21호
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• pp.9319-9325
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• 2014

Alkaloids are the most extensively featured compounds of natural anti-tumor herbs, which have attracted much attention in pharmaceutical research. In our previous studies, a mixture of major three alkaloid components (5, 6-dihydrobicolorine, 7-deoxy-trans-dihydronarciclasine, littoraline) from Hymenocallis littoralis were extracted, analyzed and designated as AHL. In this paper, AHL extracts were added to human liver hepatocellular cells HepG-2, human gastric cancer cell SGC-7901, human breast adenocarcinoma cell MCF-7 and human umbilical vein endothelial cell EVC-304, to screen one or more AHL-sensitive tumor cell. Among these cells, HepG-2 was the most sensitive to AHL treatment, a very low dose ($0.8{\mu}g/ml$) significantly inhibiting proliferation. The non-tumor cell EVC-304, however, was not apparently affected. Effect of AHL on HepG-2 cells was then explored. We found that the AHL could cause HepG-2 cycle arrest at G2/M checkpoint, induce apoptosis, and interrupt polymerization of microtubules. In addition, expression of two cell cycle-regulated proteins, CyclinB1 and CDK1, was up-regulated upon AHL treatment. Up-regulation of the Fas, Fas ligand, Caspase-8 and Caspase-3 was observed as well, which might imply roles for the Fas/FsaL signaling pathway in the AHL-induced apoptosis of HepG-2 cells.

• Archives of Pharmacal Research
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• 제29권3호
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• pp.224-234
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• 2006

We employed human SK-MEL-28 cells as a model system to identify cellular proteins that accompany N-(4-methyl)phenyl-O-(4-methoxy)phenyl-thionocarbamate (MMTC)-induced apoptosis based on a proteomic approach. Cell viability tests revealed that SK-MEL-28 skin cancer cells underwent more cell death than normal HaCaT cells in a dose-dependent manner after treatment with MMTC. Two-dimensional electrophoresis in conjunction with matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analysis or computer matching with a protein database further revealed that the MMTC-induced apoptosis is accompanied by increased levels of caspase-1, checkpoint suppressor-1, caspase-4, NF-kB inhibitor, AP-2, c-Jun-N-terminal kinase, melanoma inhibitor, granzyme K, G1/S specific cyclin D3, cystein rich protein, Ras-related protein Rab-37 or Ras-related protein Rab-13, and reduced levels of EMS (oncogene), ATP synthase, tyrosine-phosphatase, Cdc25c, 14-3-3 protein or specific structure of nuclear receptor. The migration suppressing effect of MMTC on SK-MEL-28 cell was tested. MMTC suppressed the metastasis of SK-MEL-8 cells. It was also identified that MMTC had little angiogenic effect because it did not suppress the proliferation of HUVEC cell line. These results suggest that MMTC is a novel chemotherapeutic and metastatic agents against the SK-MEL-28 human melanoma cell line.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.6159-6164
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• 2013

Background: Phytochemical compounds are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. The purpose of this study was to investigate the potential effcts of thymoquinone, caffeic acid phenylester (CAPE) and resveratrol on inflammatory markers, oxidative stress parameters, mRNA expression levels of proteins and survival of lung cancer cells in Vitro. Materials and Methods: The A549 cell line was treated with benzo(a)pyrene, benzo(a)pyrene plus caffeic acid phenylester (CAPE), benzo(a)pyrene plus resveratrol (RES), and benzo(a)pyrene plus thymoquinone (TQ). Inflammatory markers, oxidative stress parameters, mRNA expression levels of apoptotic and anti-apoptotic proteins and cell viability were assessed and results were compared among study groups. Results: TQ treatment up-regulated Bax and down-regulated Bcl2 proteins and increased the Bax/Bcl2 ratio. CAPE and TQ also up-regulated Bax expression. RES and TQ down-regulated the expression of Bcl-2. All three agents decreased the expression of cyclin D and increased the expression of p21. However, the most significant up-regulation of p21 expression was observed in TQ treated cells. CAPE, RES and TQ up-regulated TRAIL receptor 1 and 2 expression. RES and TQ down-regulated the expression of NF-kappa B and IKK1. Viability of CAPE, RES and TQ treated cells was found to be significantly decreased when compared with the control group (p=0.004). Conclusions: Our results revealed up-regulation of the key upstream signaling factors, which ultimately cause increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall these results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ, RES and CAPE.

• Journal of Microbiology and Biotechnology
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• 제28권6호
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• pp.1007-1021
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• 2018

Cancer represents one of the most significant threats to human health on a global scale. Hence, the development of effective cancer prevention strategies, as well as the discovery of novel therapeutic agents against cancer, is urgently required. In light of this challenge, this research aimed to evaluate the effects of several potent bioactive peptides and proteins contained in crocodile white blood cell extract (cWBC) against LU-1, LNCaP, PC-3, MCF-7, and CaCo-2 cancer cell lines. The results demonstrate that 25, 50, 100, and $200{\mu}g/ml$ cWBC exhibits a strong cytotoxic effect against all investigated cell lines ($IC_{50}$ $70.34-101.0{\mu}g/ml$), while showing no signs of cytotoxicity towards noncancerous Vero and HaCaT cells. Specifically, cWBC treatment caused a significant reduction in the cancerous cells' colony forming ability. A remarkable suppression of cancerous cell migration was observed after treatment with cWBC, indicating potent antimetastatic properties. The mechanism involved in the cancer cell cytotoxicity of cWBC may be related to apoptosis induction, as evidenced by typical apoptotic morphology features. Moreover, certain cWBC concentrations induced significant overproduction of ROS and significantly inhibited the $S-G_2/M$ transition in the cancer cell. The molecular mechanisms of cWBC in apoptosis induction were to decrease Bcl-2 and XIAP expression levels and increase the expression levels of caspase-3, caspase-8, and p53. These led to a decrease in the expression level of the cell cycle-associated gene cyclin-B1 and the arrest of cell population growth. Consequently, these findings demonstrate the prospect of the use of cWBC for cancer therapy.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.540-549
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• 2021

The Wnt/β-catenin signaling pathway is involved in breast cancer and Myxococcus fulvus KYC4048 is a myxobacterial strain that can produce a variety of bioactive secondary metabolites. Although a previous study revealed that KYC4048 metabolites exhibit anti-proliferative effects on breast cancer, the biochemical mechanism involved in their effects remains unclear. In the present study, KYC4048 metabolites were separated into polar and non-polar (ethyl acetate and n-hexane) fractions via liquid-liquid extraction. The effects of these polar and non-polar KYC4048 metabolites on the viability of breast cancer cells were then determined by MTT assay. Expression levels of Wnt/β-catenin pathway proteins were determined by Western blot analysis. Cell cycle and apoptosis were measured via fluorescence-activated cell sorting (FACS). The results revealed that non-polar KYC4048 metabolites induced cell death of breast cancer cells and decreased expression levels of WNT2B, β-catenin, and Wnt target genes (c-Myc and cyclin D1). Moreover, the n-hexane fraction of non-polar KYC4048 metabolites was found most effective in inducing apoptosis, necrosis, and cell cycle arrest, leading us to conclude that it can induce apoptosis of breast cancer cells through the Wnt/β-catenin pathway. These findings provide evidence that the n-hexane fraction of non-polar KYC4048 metabolites can be developed as a potential therapeutic agent for breast cancer via inhibition of the Wnt/β-catenin pathway.

• Animal Bioscience
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• 제34권4호
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.