• Nuclear Medicine and Molecular Imaging
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• 제41권1호
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• pp.54-58
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• 2007

목적: Firefly luciferase (이하 Fluc)는 분자영상 분야에 가장 널리 쓰이는 리포터 유전자 중 하나이다. 발광반응의 기질로 사용되는 D-luciferin 은 가격이 비싸고 실험동물의 무게에 비례해서 기질을 주입 하므로 마우스나 렛트와 같은 소동물을 대상으로 전임상 연구가 이루어지고 있다. 본 실험실에서는 중동물인 토끼의 관절강에 D-luciferin을 국소 주입하여 발광영상을 획득하였다. 대상 및 방법: 연골세포를 일주일 동안 배양한 후 Fluc 아데노바이러스에 감염시켰다. 감염된 연골세포를 토끼의 관절강에 주입 또는 이식하였다. 착상된 무릎의 관절강부위에 D-luciferin을 국소 주입한 후 본 실험실서 보유하고 있는 CCD 카메라가 장착된 실시간 영상장비를 이용하여 날짜 별로 분자영상을 획득하였다. 결과: 착상되어진 토끼의 관절강 부위에 기질을 국소주입하여 영상을 성공적으로 획득하였다. 연골세포 주입 및 이식 후 1일째부터 토끼의 관절강에서 빛이 방출되었으며 토끼의 관절강에 주입하는 것보다 이식하는 방법이 강한 빛을 방출함을 알 수 있었다. 또한 7일째까지 토끼의 관절강에 연골세포를 이식한 것이 주입한 것보다 총 광량이 5배에서 10배까지 강하게 나타남을 확인하였고 9일째에는 약 10배정도 강하게 나타났다. 결론: 중동물인 토끼를 이용하여 Fluc을 발현하는 연골세포를 주입 또는 이식한 관절강에 D-luciferin을 국소 주입하여 영상을 성공적으로 획득하였으며, 이러한 결과를 통해 중동물에 소량의 D-luciferin 국소주입하여도 발광영상을 얻는데 충분함을 알 수 있었다.

• 생명과학회지
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• 제17권7호통권87호
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• pp.923-930
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• 2007

성체흰쥐의 경우 항암제인 싸이클로포스파마이드 (CY)처리로 퇴축된 가슴샘은 2주 후에 정상조직으로 재생된다. 가슴샘 발생과정에서 이미 알려진 Wnt신호전달의 중요성과는 달리 성체의 가슴샘 재생과정에서 그 역할에 관해서는 알려진 바 전혀 없다. 본 연구의 목적은 발생중인 가슴샘 상피세포에서 발현이 증가된다고 이미 알려져 있는 Wnt7b가 성체의 가슴샘재생과정에서 어떤 발현 양상을 보이는지를 조사하는 것이다. Wnt7b는 가슴샘의 급성 퇴축 이후 3일째 되는 시기에 mRNA와 단백질의 양이 급격히 증가 하였으며, 이중 면역 염색 형광법을 통해 큰포식 세포와 위치적 분포가 일치함을 확인하였다. 또한, Wnt7b유전자의 발현 조절 기전을 밝히기 위해 Wnt7b의 Reporter Vector를 제작하여 Luciferase assay를 이용하여 상위의 신호를 분석하였고, 그 결과 Wnt7b는 RANKL에 의해 그 발현이 감소된다는 사실을 처음으로 밝혔다. 따라서, 본 연구 결과들을 통해 Wnt 7b는 가슴샘의 급성 퇴축 초기 과정에서 나타나는 손상된 세포를 처리하는 큰포식 세포의 기능 조절에 관여할 것으로 생각된다.

• Molecules and Cells
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• 제37권2호
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• pp.178-186
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• 2014

Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) bound directly to these sites and activated transcription. Exposure to the hypoxia-mimetic compound $CoCl_2$, incubation under 1% $O_2$ conditions, or overexpression of HIF-$1{\alpha}$ enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with $CoCl_2$ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-$1{\alpha}$ to HRE sites and is epigenetically controlled by methylation of its promoter region.

• Biomolecules & Therapeutics
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• 제22권4호
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• pp.347-354
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• 2014

Larrea nitida is a plant that belongs to the Zygophyllaceae family and is widely used in South America to treat inflammatory diseases, tumors and menstrual pain. However, its pharmacological activity remains unclear. In this study we evaluated the property of selective estrogen receptor modulator (SERM) of Larrea nitida extracts (LNE) as a phytoestrogen that can mimic, modulate or disrupt the actions of endogenous estrogens, depending on the tissue and relative amount of other SERMs. To investigate the property of SERM of LNE, we performed MCF-7 cell proliferation assays, estrogen response element (ERE)-luciferase reporter gene assay, human estrogen receptor (hER) binding assays and in vivo uterotrophic assay. To gain insight into the active principles, we performed a bioassay-guided analysis of LNE employing solvents of various polarities and using classical column chromatography, which yielded 16 fractions (LNs). LNE showed high binding affinities for $hER{\alpha}$ and $hER{\beta}$ with $IC_{50}$ values of $1.20{\times}10^{-7}$ g/ml and $1.00{\times}10^{-7}$ g/ml, respectively. LNE induced $17{\beta}$-estradiol (E2)-induced MCF-7 cell proliferation, however, it reduced the proliferation in the presence of E2. Furthermore, LNE had an atrophic effect in the uterus of immature rats through reducing the expression level of progesterone receptor (PR) proteins. LN08 and LN10 had more potent affinities for binding on $hER{\alpha}$ and ${\beta}$ than other fractions. Our results indicate that LNE had higher binding affinities for $hER{\beta}$ than $hER{\alpha}$, and showed SERM properties in MCF-7 breast cancer cells and the rat uterus. LNE may be useful for the treatment of estrogen-related conditions, such as female cancers and menopause.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7849-7855
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• 2014

Purpose: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. Materials and Methods: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ${\beta}$-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ${\beta}$-catenin, HDAC1and HDAC3 was tested by q-PCR. ${\beta}$-catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. Results: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was $6.22{\pm}0.25%$, which increased to $7.17{\pm}0.20%$ and $18.1{\pm}0.42%$ in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ${\beta}$-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ${\beta}$-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ${\beta}$-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. Conclusions: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/${\beta}$-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

• 한국발생생물학회지:발생과생식
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• 제17권4호
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• pp.379-387
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• 2013

Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.

• 세라미스트
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• 제22권3호
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• pp.281-300
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• 2019

Surface enhanced Raman scattering (SERS) was first discovered in 1974 by an unexpected Raman signal increase from Pyridine adsorbed on rough Ag electrode surfaces by the M. Fleishmann group. M. Moskovits group suggested that this phenomenon could be caused by surface plasmon resonance (SPR), which is a collective oscillation of free electrons at the surface of metal nanostructures by an external light source. After about 40 years, the SERS study has attracted great attention as a biomolecule analysis technology, and more than 2500 new papers and 500 review papers related to SERS topic have been published each year in recently. The advantages of biomaterials analysis using SERS are as follows; ① Molecular level analysis is possible based on unique fingerprint information of biomolecule, ② There is no photo-bleaching effect of the Raman reporters, allowing long-term monitoring of biomaterials compared to fluorescence microscopy, ③ SERS peak bandwidth is approximately 10 to 100 times narrower than fluorescence emission from organic phosphor or quantum dot, resulting in higher analysis accuracy, ④ Single excitation wavelength allows analysis of various biomaterials, ⑤ By utilizing near-infrared (NIR) SERS-activated nanostructures and NIR excitation lasers, auto-fluorescence noise in the visible wavelength range can be avoided from in vivo experiment and light damage in living cells can be minimized compared to visible lasers, ⑥ The weak Raman signal of the water molecule makes it easy to analyze biomaterials in aqueous solutions. For this reason, SERS is attracting attention as a next-generation non-invasive medical diagnostic device as well as substance analysis. In this review, the principles of SERS and various biomaterial analysis principles using SERS analysis will be introduced through recent research papers.

• Molecules and Cells
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• 제39권8호
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• pp.611-618
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• 2016

Fibroblast-like synoviocytes (FLS) with aberrant expression of microRNA (miRNA) are critical pathogenic regulators in rheumatoid arthritis (RA). Previous studies have found that overexpression or silencing of miRNA can contribute to the development of miRNA-based therapeutics in arthritis models. In this study, we explored the effects of miR-27a on cell migration and invasion in cultured FLS from RA patients. We found that miR-27a was markedly downregulated in the serum, synovial tissue, and FLS of RA patients. Meanwhile, the expression of follistatin-like protein 1 (FSTL1) was upregulated, which suggests that FSTL1 plays a key role in RA development. The results of a Transwell assay showed that miR-27a inhibited FLS migration and invasion. However, miR-27a inhibition promoted the migration and invasion of FLS. In addition, the down-regulated expression of matrix metalloproteinases (MMP2, MMP9, and MMP13) and Rho family proteins (Rac1, Cdc42, and RhoA) was detected after treatment with miR-27a in RA-FLS by quantitative reverse transcription-PCR and western blot analysis. Then, a luciferase reporter assay validated that miR-27a targeted the 3-untranslated region (3'-UTR) of FSTL1. Moreover, miR-27a caused a significant decrease of FSTL1. In addition, the expression of TLR4 and $NF{\kappa}B$ was inhibited by miR-27a but increased by FSTL1 overexpression. In conclusion, we found that miR-27a inhibited cell migration and invasion of RA-FLS by targeting FSTL1 and restraining the $TLR4/NF{\kappa}B$ pathway.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7943-7957
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• 2015

Background and Aims: Colorectal cancer is one of the leading causes of death in the world. The aim of this study was to investigate the growth-suppression potentiality of a crude saponin extract (CSENS) prepared from medicinal herb, Nigella sativa, on human colon cancer cells, HCT116. Materials and Methods: HCT116 cells were subjected to increasing doses of CSENS for 24, 48 and 72 h, and then harvested and assayed for cell viability by WST-1. Flow cytometry analyses, cell death detection ELISA, fluorescent stains (Hoechst 33342 and acridine orange/ethidium bromide), DNA laddering and comet assays were carried out to confirm the apoptogenic effects of CSENS. Luciferase reporter gene assays, quantitative reverse transcription-polymerase chain reaction and Western blot analyses were performed to assess the impact of CAERS and CFEZO on the expression levels of key regulatory proteins in HCT116 cells. Results: The results demonstrated that CSENS inhibited proliferation and induced apoptosis. Apoptosis was confirmed by flow cytometry analyses, while CSENS-treated cells exhibited morphological hallmarks of apoptosis including cell shrinkage, irregularity in cellular shape, cellular detachment and chromatin condensation. Biochemical signs of apoptosis, such as DNA degradation, were observed by comet assay and gel electrophoresis. The pro-apoptotic effect of CSENS was caspase-3-independent and associated with increase of the Bax/Bcl-2 ratio. CSENS treatment down-regulated transcriptional and DNA-binding activities of NF-${\kappa}B$ and AP-1 proteins, associated with down-regulation of their target oncogenes, c-Myc, cyclin D1 and survivin. On the other hand, CSENS up-regulated transcriptional and DNA-binding activities of Nrf2 and expression of cytoprotective genes. In addition, CSENS modulated the expression levels of ERK1/2 MAPK, p53 and p21. Conclusions: These findings suggest that CSENS may be a valuable agent for treatment of colon cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제17권4호
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• pp.1947-1959
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• 2016

Background: Colorectal cancer (CRC) is a major cause of morbidity and mortality, being the second most common type of cancer worldwide in both men and women. It accounts yearly for approximately 9% of all new cases of cancers. Furthermore, the current chemotherapeutic regimens seem unsatisfactory, so that exploration of novel therapeutic modalities is needed. The present study was undertaken to investigate the inhibitory effects of a crude alkaloid extract (CAERS) of a medicinal herb, Rhazya stricta, on proliferation of CRC HCT116 cells and to elucidate mechanisms of action. To achieve these aims, we utilized MTT, comet, DNA laddering and gene reporter assays, along with Western blot and RT-PCR analyses. Results: We found that CAERS inhibited cell proliferation and induced apoptotic cell death in HCT116 cells. Hallmarks of morphological and biochemical signs of apoptosis were clearly evident. CAERS down-regulated DNA-binding and transcriptional activities of NF-${\kappa}B$ and AP-1 proteins, while up-regulating expression of the Nrf-2 protein. It also down-regulated expression levels of the ERK MAPK, Bcl-2, cyclin D1, CDK-4, survivin and VEGF and up-regulated levels of Bax, caspase-3/7 and -9, p53, p21, Nrf-2. Markedly, it promoted mRNA expression levels of cytoprotective genes including the hemeoxygenase-1, NAD(P)H quinine oxidoreductase 1 and UDP-glucuronyltransferase. Conclusions: These findings indicate that CAERS exerts antiproliferative action on CRC cells through induction of apoptotic mechanisms, and suggest CAERS could be a promising agent for studying and developing novel chemotherapeutic agents aimed at novel molecular targets for the treatment of CRC.