• Journal of Life Science
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• v.28 no.8
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• pp.945-954
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• 2018

Omega-3 polyunsaturated fatty acids (${\omega}3$-fatty acid) have been found to possess anticancer properties in a variety of cancer cell lines and animal models, but their effects in human tongue squamous cell carcinomas (SCCs) remain unclear. This study was designed to examine the effect of ${\omega}3$-fatty acid desaturase (fat-1) gene expression on invasion and tumorigenicity in human tongue SCC cells and the molecular mechanism of its action. Docosahexaenoic acid (DHA) treatment inhibited in vitro invasion in a dose-dependent manner. In zymography, matrix metalloproteinase-9 (MMP-9) and Matrix metallopeptidase-2 (MMP-2) activities were reduced, and MMP-9 and MMP-2 promoter activities were inhibited by the DHA treatment. In addition, cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) promoter reporter activities were inhibited in SCC-4 and SCC-9 cells after the DHA treatment. To investigate the effect of a high level of endogenous ${\omega}3$ fatty acids, a stable SCC-9 cell line expressing the ${\omega}3$-desaturase gene (fSCC-9sc) was generated. The growth rate and colony-forming capacity of fSCC-9sc were remarkably decreased as compared with those of fSCC-9cc. Likewise, the tumor size and volume of fSCC-9sc implanted into nude mice were significantly inhibited, with increases in the cell death index. Furthermore, a transwell chamber invasion assay showed a reduction in cell invasion of the fSCC-9sc lines when compared with that of the fSCC-9cc line. These findings suggested that fat-1 gene expression inhibited tumorigenicity, as well as invasion in human tongue SCC cells. Thus, utilization of ${\omega}3$ fatty acids may represent a promising therapeutic approach for chemoprevention and the treatment of human tongue SCCs.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.95-103
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• 2001

연구목적: 본 연구는 생쥐 preantral follicles의 체외 배양 조건을 확립하고 이를 기초로 높은 체외 발달률 그리고 산자 생산률을 얻고자 하였다. 연구재료 및 방법 : Preantral follicles의 oocyte-granulosa cell complexes (OGCs)는 생후 12일된 FI ($C57BL{\times}CBA$)으로부터 난소를 적출하여 효소를 이용한 방법을 통해 획득하였다. 회수된 complexes는 10일 또는 12일 동안 체외 성장을 위해 Transwell-COL membrane insert로 옮겨졌고 5% FBS, 100 mIU/ml FSH, 100 mIU/ml hMC가 첨가된 ${\alpha}MEM$에서 배양되었다. 체외 성숙을 위해 1.5 IU/ml hCG가 첨가된 ${\alpha}MEM$에서 18 hrs 배양을 실시하였다. 그 후 M16에서 수정능력이 획득된 정자와 수정을 하여 4 hrs, 7 hts, 9 hrs 후에 10% FBS가 첨가된 modified M16 배양액에서 4일간 배양하거나 또는 bovine cumulus cell과 co-culture를 실시하였다. 그리고 형태적으로 정상적인 22개의 상실배와 포배를 2마리의 위임신 대리모 (ICR)의 자궁에 이식하여 산자 생산을 유도하였다. 결과: 1) OGCs 크기가 mouse preantral follicles의 핵 및 세포질 성숙에 미치는 영향을 조사하였을 때 $120{\sim}150{\mu}m$의 preantral follicles (MII: 33.0%, 난할률: 36.7%, 상실배 이상; 20.9%)은 핵 및 세포질 성숙에 있어서 $70{\sim}110{\mu}m$ (MII: 12.2%, 난할률: 10.2%, 상실배 이상: 4.8%)보다 더 높았다(p<0.001). 2)체외 성장기간의 연장이 mouse preantral follicles의 핵 및 세포질 성숙에 미치는 영향을 조사하였을 때 10일 (난할률: 38.2%)은 12일 (난할률: 20.0%)보다 난할률에서만 더 높았다 (p<0.01). 3) 체외 수정 시간의 연장이 mouse preantral follicle의 세포질 성숙에 미치는 영향을 조사하였을 때 9 hrs (난할룰 31.5%, 상실배 이상: 14.3%)은 4 hrs (난할률: 17.5%, 상실배 이상: 4.8%), 7 hrs (난할률: 20.4%, 상실배 이상: 6.1%) 보다 세포질성숙에 있어서 유의하게 높은 발달률을 나타냈다 (p<0.01). 4) 공배양이 mouse preantral follicle의 세포질성숙에 미치는 영향을 조사하였을 때 공배양 (상실배 이상: 17.4%)을 실시했을 때와 M16 (상실배 이상17.4%)에서 배양되었을 때는 차이가 없었다. 5)preantral follicle의 크기 ($120{\sim}150{\mu}m$), 체외 성장기간 (10일), 체외 수정 기간 (9시간), 배양 환경 (단지 medium만 존재)의 적절한 결과들을 종합하여 수행하였을 때 MII 성숙률, 난할률, 상실배 이상의 발달률은 30.2%, 39.3%, 22.5%이었고 총 22개의 상실배 및 포배를 2마리의 대리모에 이식했을 때 1마리가 임신했고 1마리의 산자를 생산했다. 결론: 따라서, 본 실험은 preantral follicle을 이용한 체외 배양 시스템이 생쥐 oocyte를 공급하는 또 다른 방법으로 효과적으로 이용될 수 있다는 것을 시사한다.

• Journal of Periodontal and Implant Science
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• v.27 no.4
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• pp.785-804
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• 1997

It was well known that calcium sulfate was biocompatible, resorbed rapidly in the body, had potential as a good barrier membrane. Platelet-derived growth factor(PDGF) was one of polypeptide growth factor that had been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. The purpose of this study was to evaluate the effects of a combination of calcium sulfate and PDGF on periodontal ligament cells in vitro to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the premolar tooth extracted for the orthodontic treatment. Cells were cultured in ${\alpha}-MEM$ contained with 20% FBS, at the $37^{\circ}C$, 100% of humidity, 5% $Co_2$ incubator. Cells were inoculated and cultured into 96 well culture plate with $1{\times}10^4cells/well$ of ${\alpha}-MEM$ for 1 day. After discarding the medium, those cells were cultured in ${\alpha}-MEM$ contained with 10% FBS alone(control group), in calcium sulfate(calcium sulfate group), in calcium sulfate treated with 15ng/ml of PDGF-BB(calcium sulfate+PDGF group), in ${\alpha}-MEM$ contained with 10% FBS treated with 15ng/ml of PDGF-BB(PDGF group) for 1, 2, 3 day respectively. And then each group was characterized by examining of the cell counting, MTT assay, collagen synthesis. The results were as follows. 1. In the analysis of cell proliferation by cell counting, both calcium sulfate group and calcium sulfate plus PDGF group showed no stastically significant difference compared to control group, but there was stastically significant difference between PDGF group and calcium sulfate group at 1, 2 day(P<0.05). 2. In the analysis of cell proliferation by MTT assay in calcium sulfate extracts, both calcium sulfate group and calcium sulfate plus PDGF group showed no stastically significant difference compared to control group, but there was stastically significant difference between PDGF group and calcium sulfate group at 2, 3 day, and between calcium sulfate plus PDGF group and calcium sulfate group at 2 day(P

• Korean Journal of Clinical Laboratory Science
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• v.51 no.2
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• pp.245-251
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• 2019

This study examined the effects of 2-methoxy-1,4-naphthoquinone (MQ) on the monocyte chemoattractant protein-1 (MCP-1)-induced migration of monocytes, which is an important phenomenon for the body defense and immune response. MQ is a major component extracted from Impatiens balsamina leaves, which have been used for many years in Asian medicine for the treatment of a range of diseases and pain. The cytotoxicity of MQ began to appear at a concentration of $10{\mu}M$, and approximately 50% cytotoxicity was confirmed at $100{\mu}M$. The MCP-1 induced migration of the THP-1 monocyte cell line increased after MQ treatment in a dose dependent manner and the largest increase was observed at $0.1{\mu}M$. The level of cAMP expression decreased after a treatment with $0.1{\mu}M$ MQ. The phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), a key signaling protein involved in the signaling pathway of C-C motif chemokine receptor 2 (CCR2), a receptor for MCP-1, was increased by the simultaneous treatment of $0.1{\mu}M$ MQ. These results show that MQ increases the MCP-1-induced migration of THP-1, decreases the level of cAMP expression, and increases the level of Erk1/2 phosphorylation.

• Journal of Life Science
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• v.32 no.2
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• pp.135-141
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• 2022

Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.

• Radiation Oncology Journal
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• v.19 no.2
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• pp.171-180
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• 2001

Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.