• Asian Pacific Journal of Cancer Prevention
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• 제13권2호
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• pp.473-477
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• 2012

Introduction: The esophagus squamous cell carcinoma (ESCC) is one of the most deadly malignances, and a current challenge is the development of effective therapeutic agents. Our present work addressed the effect of HIF-$1{\alpha}$ siRNA alone or in combination with cisplatin on the growth of ESCC in nude mice. Materials and Methods: Xenografts were established by inoculating ESCC TE-1 cells in nude mice, and transplanted tumors were treated with HIF-$1{\alpha}$ siRNA, cisplatin alone or together. Growth was assessed by measuring tumor volume. HIF-$1{\alpha}$ mRNA and protein expression were detected using RT-PCR and immunohistochemistry, respectively. Apoptosis of ESCC TE-1 cells was analyzed by flow cytometry. Results: In our nude mice model, HIF-$1{\alpha}$ siRNA effectively inhibited the growth of transplanted ESCC, downregulating HIF-$1{\alpha}$ mRNA and protein expression, and inducing ESCC TE-1 cell apoptosis. Notably when combinated with cisplatin, HIF-$1{\alpha}$ siRNA showed synergistic interaction in suppressing tumor growth. Furthermore, the proportion of apoptotic cells in HIF-$1{\alpha}$ siRNA plus cisplatin group was significantly higher than that in cisplatin or HIF-$1{\alpha}$ siRNA-treated groups (P<0.05). Conclusions: Down-regulated HIF-$1{\alpha}$ expression induced by siRNA could effectively suppress the growth of transplanted ESCC $in$ $vivo$. HIF-$1{\alpha}$ siRNA could enhance the cytotoxicity of cisplatin, which suggests that a combination of these two agents may have potential for therapy of advanced ESCC.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.165-171
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• 1998

p62는 임파구에 특이적으로 발현하는 단백질 티로신 키나제인 p56$^{lck}$의 SH2 doamin과 결합하는 세포질 단백질로서 두 단백질의 결합에는 지금까지 알려진 바와 다르게 인산화된 티로신이 필요없다. p62는 기능이 다른 여러 조직에서 공통적으로 발현되며 유비퀴틴, 단백질 키나제 C 이성질체 둥 다양한 단백질과 결합하는 것이 알려져 있다. 이와 같은 현상으로 p62가 다양한 생물학적 기능을 수행할 수 있음을 예측할 수 있으나 그 자세한 기작은 잘 알려져 있지 않다. 본 연구에서는 p62가 T-세포에 특이적으로 발현하는 14-3-3 $ au$ 이성질체와 결합하는 것을 확인하였으며, p62를 인위적으로 T-세포에 다량으로 발현시키면 세포예정사 (apoptosis)의 시작이 지연되는 현상을 조사하였다. 이때 세포사멸과정에서 전형적으로 나타나는 DNA 절단현상 (DNA fragmentaion)과 poly (ADP-ribose) polymerase의 분해가 지연됨을 알 수 있었다. 최근 14-3-3 단백질이 임파구에서 세포예정사를 촉진시키는 기능을 가진 Bad와 결합함으로써 세포의 생존 신호 전달에 중요한 역할을 한다는 것이 보고된 바 있다. 따라서 본 연구의 결과는 T-세포의 활성으로 일어나는 사멸예정사 과정 중에 p62와 14-3-3 단백질에 의해 수행되는 조절 기작이 있음을 시사하고 있다.다.

• 생명과학회지
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• 제14권3호
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• pp.445-452
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• 2004

본 연구에서 국내 연근해에 서식하는 해면 Sarcotragus sp. (Dictyoceratida속)에서 분리 추출된 7종의 furanoterpenoid계 화합물〔sarcotin A, epi-sarcotin A, ircinin-1, epi-sarcotrine B, sarcotin I, (8E,13Z,20Z) -strobilinin/ (7E,13Z,20Z) -felixinin and (7E,12E,18R,20Z)-variabilin〕의 항암 활성을 비교하기 위하여 A549 인체폐암세포를 대상으로 그들의 세포독성을 조사하였고, 이와 연관된 세포증식 억제 및 apoptosis 유발에 관여할 것으로 예상되는 중요한 유전자 몇 가지의 발현에 미치는 영향을 조사하였다. 조사된 7종의 화합물 모두 처리 농도 의존적으로 A549 폐암세포의 증식을 억제하였는데, 그중 sarcotin A 및 (7E,12E,18R,20Z)-variabilin이 비교적 높은 세포독성을 나타내었다. 이러한 세포증식의 억제는 종양억제 유전자 p53 의존적 또는 비의존적으로 Bcl-2 유전자에 대한 Bax의 발현 증가와 연관된 apoptosis 유발과 관련이 있었으며, epi-sarcotin A, ircinin-1 및 epi-sarcotrine B 처리군에서 이러한 현상은 두드러지게 관찰되었다. 또한 epi-sarcotin A와 ircinin-1은 COX-1의 발현에는 아무런 영향을 미치지 않았으나, COX-2의 발현은 선택적으로 저해하였다. 이러한 결과는 해양 해면동물에서 유래된 furanoterpenoid계 화합물이 선택 적으로 강력한 항암효과를 가질 수 있다는 것을 의미한다.

• 한국식품위생안전성학회지
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• 제29권3호
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• pp.248-251
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• 2014

천연유래물질 중의 하나인 관중은 양치식물 고사리목으로 항 염증 작용, 항 바이러스 작용 및 구충작용과 자궁 수축 작용 같은 효능이 있다고 알려져 있지만, 종양에서의 구강암 예방효능에 대한 연구는 보고된 바가 없다. 본 연구에서는 사람 구강암세포주인 MC3와 HN22세포에서 관중 메탄올추출물의 증식억제 효능 및 관련 분자표적을 확인하고자 하였다. 관중메탄올추출물은 세포증식을 유의성 있게 억제하고, Western blot분석법, DAPI염색법 결과에서 알 수 있듯이 세포사멸을 유도하는 것으로 나타났다. 또한, MC3세포에서는 Bak의 발현을 농도의존적으로 증가시켰고, HN22세포에서는 Bid의 발현에 영향을 주어 truncation을 유도하였다. 하지만, Bak과 Bid 이외의 다른 Bcl-2 family에는 영향을 주지 못했다. 따라서, 이러한 결과를 종합해 볼 때, 관중메탄올추출물은 구강암에서 암 예방효능을 가진 잠재성 있는 천연추출물이 될 수 있을 것으로 사료된다.

• 한국식품영양과학회지
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• 제33권8호
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• pp.1237-1245
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• 2004

브라질 기원인 신령 버섯 (A. blazei murill)은 강력한 항암 및 면역강화 작용을 가진 것으로 알려져 있다. 본 연구에서는 신령버섯 수용성 추출물(water extracted A. blazei Murill, WEAB)이 A549 인체 폐암세포 증식에 미치는 영향을 조사하였으며, 증식억제와 연관된 기전 해석을 시도하였다. WEAB가 처리된 A549 세포는 처리 농도 의존적으로 생존율이 감소되었으며, WEAB 처리는 암세포의 다양한 형태적 변형을 유발하였다. Flow cytometry 분석 결과로서 WEAB 처리에 의한 A549 폐암세포의 증식억제는 세포주기 G2/M arrest 및 apoptosis 유발과 직접적으로 연관성이 있음을 알 수 있었다. WEAB가 처리된 암세포에서 전사 및 번역 수준에서 cyclin A 발현의 감소 및 Cdk inhibitor p21 발현의 증가 현상이 관찰되었으나, cyclin B1, Cdk2, Cdc2, Wee1, Cdc25c 및 p53 등의 발현에는 큰 변화가 관찰되지 못하였다. 또한 WEAB의 처리는 COX-2 선택적 발현 저하를 유발하였으나, telomere 조절 관련 유전자들의 발현에는 큰 영향을 주지 못하였다. 이상의 결과는 신령버섯 추출물이 강력한 항암 및 암 예방 효능의 잠재력을 가지고 있음을 의미하며, 이에 관한 지속적인 연구가 필요할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.926-936
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• 2003

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.

• 한국안광학회지
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• 제12권2호
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• pp.79-86
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• 2007

본 연구는 mycoplasma가 오염된 배양 각막 상피세포에 anti-FAS and anti-FAS ligand antibody를 노출시킨 후 세포고사 메커니즘을 조사하기 위해 시행하였다. 배양각막 상피세포에 anti-FAS antibody 와 anti-FAS ligand antibody를 2일과 4일 동안 처리하여 주어진 기간 동안 배양하였다. 배양 중인 각막상피세포가 mycoplasma sp.에 의해 오염된 것이 밝혀졌다. mycoplasma removal agent(MRA)가 세포주로부터 세균을 제거하기 위해 이용되었다. MRA를 세포주에 첨가하여 1주일 동안 배양하였다. 그 후 각막상피 세포주는 MRA가 포함되지 않는 배양액에서 수세대 동안 배양되어 커버글라스에 계대배양하여 50-80% 채워졌을 때 MRA 제거여부를 확인하기 위한 검사 키트에 제공된 Hoechst staining을 이용하여 염색하였다. 세포고사 실험은 MRA 제거 전과 제거 후에 시행되었다. mycoplasma가 오염된 배양 각막상피세포에 대한 anti-FAS and anti-FAS ligand antibody의 영향을 알아보기 위해 Hoechst 33342 staining과 Annexin V-FITC and Propidium Iodide Staining을 이용하여 세포고사 유도를 확인하였다. 본 연구는 anti-FAS antibody가 배양각막 상피세포에서 시간과 농도에 비례하는 메커니즘으로 세포고사를 유도한다. mycoplasma가 오염된 세포주는 FAS 유도 세포고사의 민감성을 증가시키는 것으로 나타났다.

• 대한약침학회지
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• 제19권1호
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• pp.37-44
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• 2016

Objectives: This study examined the relative efficacies of a derivative of betulinic acid (dBA) and its poly (lactide-co-glycolide) (PLGA) nano-encapsulated form in A549 lung cancer cells in vivo and in co-mutagen [sodium arsenite (SA) + benzo[a]pyrene (BaP)]-induced lung cancer in mice in vivo. Methods: dBA was loaded with PLGA nanoparticles by using the standard solvent displacement method. The sizes and morphologies of nano-dBA (NdBA) were determined by using transmission electron microscopy (TEM), and their intracellular localization was verified by using confocal microscopy. The binding and interaction of NdBA with calf thymus deoxyribonucleic acid (CT-DNA) as a target were analyzed by using conventional circular dichroism (CD) and melting temperature (Tm) profile data. Apoptotic signalling cascades in vitro and in vivo were studied by using an enzyme-linked immunosorbent assay (ELISA); the ability of NdBA to cross the blood-brain barrier (BBB) was also examined. The stage of cell cycle arrest was confirmed by using a fluorescence-activated cell-sorting (FACS) data analysis. Results: The average size of the nanoparticles was ~ 110 nm. Confocal microscopy images confirmed the presence of NdBA in the cellular cytoplasm. The bio-physical properties of dBA and NdBA ascertained from the CD and the Tm profiles revealed that NdBA had greater interaction with the target DNA than dBA did. Both dBA and NdBA arrested cell proliferation at G0/G1, NdBA showing the greater effect. NdBA also induced a greater degree of cytotoxicity in A549 cells, but it had an insignificant cytotoxic effect in normal L6 cells. The results of flow cytometric, cytogenetial and histopathological studies in mice revealed that NdBA caused less nuclear condensation and DNA damage than dBA did. TEM images showed the presence of NdBA in brain samples of NdBA fed mice, indicating its ability to cross the BBB. Conclusion: Thus, compared to dBA, NdBA appears to have greater chemoprotective potential against lung cancer.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.103-103
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• 2018

Oenothera biennis, commonly known as evening primrose, a potential source of natural bioactive substances: flavonoids, steroids, tannins, fatty acids and terpenoids responsible for a diverse range of pharmacological functions. However, whether extract prepared from aerial part of O. biennis (APOB) protects skin against oxidative stress remains unknown. To investigate the protective effects of APOB against oxidative stress-induced cellular damage and elucidated the underlying mechanisms in the HaCaT human skin keratinocytes. Our results revealed that treatment with APOB prior to hydrogen peroxide ($H_2O_2$) exposure significantly increased viability, and the highest DPPH radical-scavenging activities and reducing power of HaCaT cells. APOB also effectively attenuated H2O2-induced comet tail formation and inhibited the $H_2O_2$-induced phosphorylation levels of the histone ${\gamma}H2AX$, as well as the number of apoptotic bodies and Annexin V-positive cells. In addition, APOB exhibited scavenging activity against intracellular reactive oxygen species (ROS) accumulation and restored the mitochondrial membrane potential loss by $H_2O_2$. Moreover, $H_2O_2$ enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase (PARP), a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with APOB. Furthermore, APOB increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, APOB is able to protect HaCaT cells from $H_2O_2$-induced DNA damage and cell death through blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating the Nri2/HO-1 signaling pathway.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2004년도 학술대회지
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• pp.1-20
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• 2004

Polychlorinated biphenyls (PCBs), one a group of persistent and widespread environmental pollutants, have been considered to be involved in immunotoxicity, carcinogenesis, and apoptosis. However, the toxic effects and physical properties of a PCB congener are dependent on the structure. In the present study, we investigate the toxic effects and action mechanisms of PCBs In cells. Among the various congeners tested, 2,2',4,6,6'-PeCB-pentachlorobiphenyl (PeCB), a highly ortho-substituted congener having negligible binding affinity for aryl hydrocarbon receptor (AhR), caused the most potent toxicity and specific effects in several cell types. 2,2',4,6,6'-PeCB induced apoptotic cell death of human monocytic cells, suggesting that PCB-induced apoptosis may be linked to immunotoxicity. In addition, 2,2',4,6,6'-PeCB induced mitotic arrest by interfering with mitotic spindle assembly in NIH3T3 fibroblasts, followed by genetic instability which triggers p53 activation. Which suggests that 2,2',4,6,6'-PeCB may be involved in cancer development by causing genetic instability through mitotic spindle damage. On the other hand, 2,2',4,6,6'-PeCB increased cyclooxygenase-2 (COX-2) involved in cell survival through ERK1/2 MAPK and p53 in Rat-1 fibroblasts and mouse embryonic fibroblasts, triggering compensatory mechanism for abating its toxicity. Taken together, these results demonstrate that PCB congeners of different structure have distinct mechanism of action and 2,2',4,6,6'-PeCB causes several toxicity as well as compensatory mechanism in cells.