• 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.

• Toxicological Research
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• 제20권1호
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• pp.71-82
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• 2004

Changes in cell cycle control in the lungs and liver of the B6C3F1 mice (20 males per each group) exposed to ozone (0.5 ppm), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1.0 mg/kg), and dibutyl phthalate (DBP, 5,000 ppm) after 52 weeks were examined through Western, Northern blot, and immunohistochemistry based on alterations in protein expression levels of G1/S checkpoints (cyclin D1, cyclin E, and PCNA), G2/M checkpoints (cyclin B1, cyclin G, and cyclin A), negative regulators (p53, p21, GADD45, and p27), and positive regulator (mdm2). Expression levels of cyclins D1, E, G, PCNA, mutant p53, and mdm2 proteins were higher in the lungs and livers treated with combination of toxicants than in those treated with ozone only. Expression levels of the wild-type and mutant p53, p21, GADD45, p27, and mdm2 proteins and mRNAs were higher in toxicant-treated groups than those of the control. Immunohistochemical analysis revealed staining intensities of the PCNA, cyclin D1, c-myc and mdm2 protein- treated lungs and livers were stronger than those of the control group. Our results showed that combined treatment of ozone with NNK/DBP altered the cell cycle control through instability of the wild-type p53 gene. Such pivotal p53-mediated cell cycle alterations may be responsible for the toxicity observed under our experimental condition. These results may be applied to risk assessment of mixture-induced toxicity.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2006년도 Principles and Practice of Microarray for Biomedical Researchers
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• pp.83-89
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• 2006

Cellular functions are carried out by a concerted action of biochemical pathways whose components have genetic interactions. Abnormalities in the activity of the genes that constitute or modulate these pathways frequently have oncogenic implications. Therefore, identifying the upstream regulatory genes for major biochemical pathways and defining their roles in carcinogenesis can have important consequences in establishing an effective target-oriented antitumor strategy We have analyzed the gene expression profiles of human liver cancer samples using cDNA microarray chips enriched in liver and/or stomach-expressed cDNA elements, and identified groups of genes that can tell tumors from non-tumors or normal liver, or classify tumors according to clinical parameters such as tumor grade, age, and inflammation grade. We also set up a high-throughput cell-based assay system (cell chip) that can monitor the activity of major biochemical pathways through a reporter assay. Then, we applied the cell chip platform for the analysis of the HCC-associated genes discovered from transcriptome profiling, and found a number of cancer marker genes having a potential of modulating the activity of cancer-related biochemical pathways such as E2F, TCF, p53, Stat, Smad, AP-1, c-Myc, HIF and NF-kB. Some of these marker genes were previously blown to modulate these pathways, while most of the others not. Upon a fast-track phenotype analysis, a subset of the genes showed increased colony forming abilities in soft agar and altered cell morphology or adherence characteristics in the presence of purified matrix proteins. We are currently analyzing these selected marker genes in more detail for their effects on various biological Processes and for Possible clinical roles in liver cancer development.

• Journal of Acupuncture Research
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• 제17권2호
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• pp.169-186
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• Animal Bioscience
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• 제35권7호
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• pp.964-974
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• 2022

Objective: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines. Methods: Resistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing. Results: A gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens. Conclusion: Resistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

• 생명과학회지
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• 제32권3호
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• pp.210-221
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• 2022

비스테로이드소염제(NSAID)와 γ-secretase 저해제(DAPT) 또는 SIRT1저해제(MHY2245)의 병용 효과를 인간 대장암(KM12) 및 간암(SNU475) 세포를 대상으로 조사한 결과, celecoxib (CCB) 및 2, 5-dimethyl celecoxib (DMC)를 포함하는 NSAID는 DAPT 또는 MHY2245와의 병용에 의하여 COX-2활성과 상관없이 NSAID의 암세포 증식 억제능이 현저히 증강되었다. DAPT와 MHY2245는 p62단백질 감소와 동시에 Notch1, CD44, CD133, octamer- binding transcription factor 4 (Oct4) 등의 다수의 종양 줄기세포 표지자 및 NICD1 발현 양을 감소시켰지만, activating transcription factor 4 (ATF4) 발현은 증강시켰다. 또한 NSAID 단독처리 보다 NSAID/DAPT 및 NSAID/MHY2245 병용 처리에 의하여 오토파지가 촉진되므로서 종양 줄기세포 표지자의 발현 및 단백질양의 감소가 가속화되고, 이에 따라 PARP 활성화 및 세포사멸이 현저히 증강 되었다. 결론적으로 NSAID/DAPT 및 NSAID/MHY2245의 병용 투여는 종양 줄기세포 표지자를 발현하는 인간 암세포의 증식 억제 및 제거에 효과적인 처리방법으로, 임상에 적용시킬 수 있는 학문적 근거로서 제공 될 수 있다.

• 대한한방내과학회지
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• 제22권4호
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• pp.579-587
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• 2001

The efficacy of Sagunja-tang and Sagunja-tang plus Mylabris phalerata against the human stomach cancer was examined and molecular biological fight of its actions was studied. In the efficacy test of anti-stomach cancer cells growth using the MTT assay, administration of Sagunja-tang resulted in no significant change of stomach cancer cells growth, with the control group. Administration of Sagunja-tang plus Mylabris phalerata resulted in a decrease of stomach cancer cells growth in proportion to the concentration of mylabris phalerata and time, which was significantly different from the control group(significance recognized when p<0.05). In the test using the apoptosis assay, administration of Sagunja-tang showed an increase in apoptosis of human stomach cancer cells, with no significant difference from the control group. Administrating Sagunja-tang plus Mylabris phalerata showed an increase in apoptosis of stomach cancer cells in proportion to the concentration of mylabris phalerata and time, which was significantly different from the control group(significance recognized when p<0.05). In the test using the quantitative RT-PCR to examine stomach cancer cells growth and revelation of apoptosis related genes, administrating Sagunja-tang plus Mylabris phalerata resulted in a decrease of Bcl-2, an anti-apoptosis gene, in proportiong to concentration. No significant change was examined in the revelation of CDK1, Cdc2, Cyclin D1, PCNA, c-myc, which are genes related to the stomach cancer cells growth, and Bax, Bel-XL, the genes related to apoptosis, and p53. Referring to the results above, Sagunja-tang plus Mylabris phalerata may be considered to have an anti-growth efficacy against human stomach cancer cells, and an inducement efficacy. Therefore, it can be clinically implemented in the human stomach cancer.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권6호
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• pp.483-490
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• 2001

There were many controversies in the cause and progress of tumorigenesis. Recently, studies on the mutation of genes related to the tumor have extensively been performed due to development of molecular biology. Structural and morphological changes of chromosomes, which are related to the abnormal activation of oncogenes or inactivation of tumor suppression genes, transform the normal cells into the tumor cells. p53 and Rb are well known tumor suppressor genes, while oncogenes include c-myc, bcl-2 and ras, etc. When exposed to cell damaging agents, p53 inhibits cell growth by inducing transcription of p21. Especially p73, which is homo-logy of p53, frequently deleted in melanoma, neuroblastoma, colon cancer, and breast cancer, when over produced, p73 activates the transcription of p21, bax-1 and inhibits cell growth by inducing apoptosis. For study on mRNA expression of p21 and p73, normal oral keratinocytes, and cell lines of primary and metastatic oral squamous cell carcinoma were cultured and then electrophoresis and RT-PCR(reverse transcription-polymerase chain reaction) were performed. 1. The mRNA of p21 and p73 in normal oral keratinocyte expressed lower than that of primary squamous cell carcinoma. 2. The mRNA of p21 in metastatic oral squamous carcinoma cell lines was expressed as various patterns compared with that of normal oral keratinocyte. 3. In the metastatic oral squamous cell lines, the mRNA of HN8 expressed higher than that of HN12 or HN19. 4. The mRNA of p73 in primary oral squamous cell lines expressed 4-5 times higher than that of normal keratinocyte. 5. In metastatic oral squamous cell lines, there was no significant expression of p73 mRNA compared with that of normal oral keratinocyte. From the results obtained in this study, mRNA expression of p73 in primary oral squamous cell lines was remarkable, while mRNA expression of p21 and p73 in metastatic oral squamous cell lines were statistically insignificant.

• Molecules and Cells
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• 제41권5호
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• pp.423-435
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• 2018

This investigation was aimed at working out the combined role of lncRNA H19, miR-29b and Wnt signaling in the development of colorectal cancer (CRC). In the aggregate, 185 CRC tissues and corresponding para-carcinoma tissues were gathered. The human CRC cell lines (i.e. HT29, HCT116, SW480 and SW620) and normal colorectal mucosa cell line (NCM460) were also purchased. Si-H19, si-NC, miR-29b-3p mimics, miR-29b-3p inhibitor, si-PGRN and negative control (NC) were, respectively, transfected into the CRC cells. Luciferase reporter plasmids were prepared to evaluate the transduction activity of $Wnt/{\beta}-catenin$ signaling pathway, and dual-luciferase reporter gene assay was arranged to confirm the targeted relationship between H19 and miR-29b-3p, as well as between miR-29b-3p and PGRN. Finally, the proliferative and invasive capacities of CRC cells were appraised through transwell, MTT and scratch assays. As a result, overexpressed H19 and down-expressed miR-29b-3p displayed close associations with the CRC patients' poor prognosis (P < 0.05). Besides, transfection with si-H19, miR-29b-3p mimic or si-PGRN were correlated with elevated E-cadherin expression, decreased snail and vimentin expressions, as well as less-motivated cell proliferation and cell metastasis (P < 0.05). Moreover, H19 was verified to directly target miR-29b-3p based on the luciferase reporter gene assay (P < 0.05), and miR-29b-3p also bound to PGRN in a direct manner (P < 0.05). Finally, addition of LiCl ($Wnt/{\beta}-catenin$ pathway activator) or XAV93920 ($Wnt/{\beta}-catenin$ pathway inhibitor) would cause remarkably altered E-cadherin, c-Myc, vimentin and snail expressions, as well as significantly changed transcriptional activity of ${\beta}-catenin/Tcf$ reporter plasmid (P < 0.05). In conclusion, the lncRNA H19/miR-29b-3p/PGRN/Wnt axis counted a great deal for seeking appropriate diagnostic biomarkers and treatment targets for CRC.

• 대한수의학회지
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• 제60권1호
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• pp.25-32
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• 2020

Natural killer (NK) cells play have a crucial role in the early phase of immune responses against various pathogens. We compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. REM134 showed higher expression of progesterone receptor, proliferative cell nuclear antigen, Ki67, multiple drug resistance, Bmi-1, c-myc, E-cadherin, and human epidermal growth factor receptor type-2 than that of CF41.Mg. For specific expansion and activation of NK cells, we isolated CD5 negative cells from canine peripheral blood mononuclear cells and co-cultured K562 cells in the presence of interleukin (IL)-2, IL-15, and IL-21 for 21 days. As a result, we found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found there was upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Specifically, we observed that cytotoxicity of NK cells against target cells was more sensitively reacted to CF41.Mg than REM134. Based on the results of this study, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research.