• Clinical and Experimental Pediatrics
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• 제49권2호
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• pp.198-202
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• 2006

목 적 : Fas는 세포표면 수용체로 세포사멸 신호를 전도한다. 많은 질환에서 세포표면의 Fas가 Fas ligand(FasL)와 결합하여 세포사멸 과정을 유발하게 된다. 연구자들은 7일된 신생 흰쥐에 저산소성 허혈성 손상을 유발한 후 뇌 해마에서 Fas와 FasL의 발현을 관찰하고자 하였다. 방 법 : 7일된 신생 흰쥐를 오른쪽 총 경동맥 영구 결찰 후 8% 산소에 2시간 노출시켰다. 저산소성 허혈성 손상 후 12, 24, 48시간에 뇌를 적출 냉동 보관하였다. Western blotting 방법과 면역형광염색 방법으로 냉동 보관된 뇌의 경동맥을 결찰한 오른 쪽 해마에서 Fas와 FasL의 발현을 관찰하였다. 결 과 : Fas와 FasL의 발현은 저산소성 허혈성 손상 후 12시간에 경동맥이 결찰된 오른쪽 해마에서 크게 증가하고 이후 감소하는 것을 western blotting 방법에 의해 관찰하였다. Fas와 FasL의 면역형광발현은 오른쪽 해마의 CA1 영역에서 손상 후 12시간과 24시간에 대조군에 비해 증가하였다. Fas의 면역형광 발현은 손상 후 48시간에 감소하였으나 FasL의 면역형광발현은 손상 후 48시간에도 지속되었다. 결 론 : 세포표면에서 Fas와 FasL의 발현과 그들의 결합은 저산소성 허혈성 손상을 받은 미성숙 뇌의 신경세포 손상에 기여할 것으로 추측되었다.

• 생명과학회지
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• 제21권2호
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• pp.286-291
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• 2011

본 연구는 인간 폐암세포의 저산소 상태를 재현하기 위한 $CoCl_2$의 처리 조건을 최적화 하였고, 최적화 된 저산소 상태에서 인간 폐암세포의 암화 과정 및 기전을 규명하였다. 인간 폐암세포, A549와 H460에 500 ${\mu}M$ $CoCl_2$를 24시간 처리하였을 때 저산소 상태의 대표적인 전사인자, HIF-$1{\alpha}$의 발현이 증가함을 확인하였고 인간 폐암세포들의 성장에는 전혀 영향을 미치지 않음을 확인하였다. 또한 $CoCl_2$를 처리한 인간 폐암 세포에서 상피-중간엽 이행(epithelial-to-mesenchymal-like transition)의 대표적인 마커인 E-cadherin 발현의 감소와 ${\alpha}$-SMA의 증가를 확인하였고, 세포-세포 간 junction 부위가 깨어짐을 E-cadherin 형광염색 실험을 통하여 확인하였다. 더 나아가 $CoCl_2$를 처리한 인간 폐암 세포에서 상피-중간엽 이행의 분자적 기전을 밝히기 위해 세포벽에 존재하는 인테그린(integrin)의 발현을 웨스턴 블랏팅과 FACS분석을 통하여 알아본 결과, $CoCl_2$를 처리한 인간 폐암세포에서 인테그린 ${\beta}3$발현의 증가를 확인하였다. 뿐만 아니라, $CoCl_2$를 처리한 인간 폐암세포에서 인테그린 ${\beta}3$의 하부 신호전달 물질인 국소부착 카이네이즈(FAK)의 인산화가 증가함을 확인하였다. 상기의 결과로서, 국소부착 카이네이즈의 인산화를 저해함으로써 인간 폐암세포가 악성세포로 전이되는 것을 저해할 수 있을 것으로 기대 되어진다.

• Journal of Ginseng Research
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• 제48권1호
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• pp.31-39
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• 2024

Background: Ginsenoside Rg3, a primary bioactive component of red ginseng, has anti-cancer effects. However, the effects of Rg3-enriched ginseng extract (Rg3RGE) on apoptosis and autophagy in breast cancer have not yet been investigated. In the present study, we explored the anti-tumor effects of Rg3RGE on breast cancer cells stimulated CoCl₂, a mimetic of the chronic hypoxic response, and determined the operative mechanisms of action. Methods: The inhibitory mechanisms of Rg3RGE on breast cancer cells, such as apoptosis, autophagy and ROS levels, were detected both in vitro. To determine the anti-cancer effects of Rg3RGE in vivo, the cancer xenograft model was used. Results: Rg3RGE suppressed CoCl₂-induced spheroid formation and cell viability in 3D culture of breast cancer cells. Rg3RGE promoted apoptosis by increasing cleaved caspase 3 and cleaved PARP and decreasing Bcl2 under the hypoxia mimetic conditions. Further, we identified that Rg3RGE promoted apoptosis by inhibiting lysosomal degradation of autophagosome contents in CoCl₂-induced autophagy. We further identified that Rg3RGE-induced apoptotic cell death and autophagy inhibition was mediated by increased intracellular ROS levels. Similarly, in the in vivo xenograft model, Rg3RGE induced apoptosis and inhibited cell proliferation and autophagy. Conclusion: Rg3RGE-stimulated ROS production promotes apoptosis and inhibits protective autophagy under hypoxic conditions. Autophagosome accumulation is critical to the apoptotic effects of Rg3RGE. The in vivo findings also demonstrate that Rg3RGE inhibits breast cancer cell growth, suggesting that Rg3RGE has potential as potential as a putative breast cancer therapeutic.

• The Korean Journal of Physiology and Pharmacology
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• 제14권2호
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• pp.91-97
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• 2010

Epidermal keratinocytes overgrow in response to ultraviolet-B (UVB), which may be associated with skin photoaging and cancer development. Recently, we found that HIF-$1{\alpha}$ controls the keratinocyte cell cycle and thereby contributes to epidermal homeostasis. A further study demonstrated that HIF-$1{\alpha}$ is down-regulated by UVB and that this process is involved in UVB-induce skin hyperplasia. Therefore, we hypothesized that the forced expression of HIF-$1{\alpha}$ in keratinocytes would prevent UVB-induced keratinocyte overgrowth. Among several agents known to induce HIF-$1{\alpha}$, pyrithione-zinc (Py-Zn) overcame the UVB suppression of HIF-$1{\alpha}$ in cultured keratinocytes. Mechanistically, Py-Zn blocked the degradation of HIF-$1{\alpha}$ protein in keratinocytes, while it did not affect the synthesis of HIF-$1{\alpha}$. Moreover, the p21 cell cycle inhibitor was down-regulated after UVB exposure, but was robustly induced by Py-Zn. In mice repeatedly irradiated with UVB, the epidermis became hyperplastic and HIF-$1{\alpha}$ disappeared from nuclei of epidermal keratinocytes. However, a cream containing Py-Zn effectively prevented the skin thickening and up-regulated HIF-$1{\alpha}$ to the normal level. These results suggest that Py-Zn is a potential agent to prevent UVB-induced photoaging and skin cancer development. This work also provides insight into a molecular target for treatment of UVB-induced skin diseases.

• Biomolecules & Therapeutics
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• 제29권5호
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• pp.506-518
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• 2021

The imprinted tumour suppressor NOEY2 is downregulated in various cancer types, including ovarian cancers. Recent data suggest that NOEY2 plays an essential role in regulating the cell cycle, angiogenesis and autophagy in tumorigenesis. However, its detailed molecular function and mechanisms in ovarian tumours remain unclear. In this report, we initially demonstrated the inhibitory effect of NOEY2 on tumour growth by utilising a xenograft tumour model. NOEY2 attenuated the cell growth approximately fourfold and significantly reduced tumour vascularity. NOEY2 inhibited the phosphorylation of the signalling components downstream of phosphatidylinositol-3'-kinase (PI3K), including phosphoinositide-dependent protein kinase 1 (PDK-1), tuberous sclerosis complex 2 (TSC-2) and p70 ribosomal protein S6 kinase (p70S6K), during ovarian tumour progression via direct binding to vascular endothelial growth factor receptor-2 (VEGFR-2). Particularly, the N-terminal domain of NOEY2 (NOEY2-N) had a potent anti-angiogenic activity and dramatically downregulated VEGF and hypoxia-inducible factor-1α (HIF-1α), key regulators of angiogenesis. Since no X-ray or nuclear magnetic resonance structures is available for NOEY2, we constructed the three-dimensional structure of this protein via molecular modelling methods, such as homology modelling and molecular dynamic simulations. Thereby, Lys15 and Arg16 appeared as key residues in the N-terminal domain. We also found that NOEY2-N acts as a potent inhibitor of tumorigenesis and angiogenesis. These findings provide convincing evidence that NOEY2-N regulates endothelial cell function and angiogenesis by interrupting the VEGFR-2/PDK-1/GSK-3β signal transduction and thus strongly suggest that NOEY2-N might serve as a novel anti-tumour and anti-angiogenic agent against many diseases, including ovarian cancer.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• The Korean Journal of Physiology and Pharmacology
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• 제9권4호
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• pp.231-238
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• 2005

Neuronal apoptotic events, which result in cell death, are occurred in hypoxic/ischemic conditions. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway of $CoCl_2$-induced neuronal cell death and the inhibitory effects of estradiol. Administration of $CoCl_2$ decreased cell viability in both a dose- and time-dependent manner in PC12 cells. $CoCl_2$-induced cell death produced genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. It was found that $CoCl_2$-treated cells increased the reactive oxygen species (ROS) as well as caspase-8, -9 and -3 activities. However, pretreatment with estradiol before exposure to $CoCl_2$ prevented the reduction in cell viability reduction and attenuated DNA fragmentation and morphologic changes caused by $CoCl_2$. Furthermore, the $CoCl_2$-induced increases of ROS levels and caspases activities were attenuated by estradiol. Gene expression analysis revealed that estradiol blocked the underexpression of the Bcl-2 and ameliorated the increase in the release of cytochrome c from mitochondria into cytoplasm and Fas-ligand (Fas-L) upregulated by $CoCl_2$. These results suggest that $CoCl_2$ induce apoptosis in PC12 cells through both mitochondria- and death receptor-mediated cell death pathway. Estradiol was found to have a neuroprotective effect against $CoCl_2$-induced apoptosis through the inhibition of ROS production and by modulating apoptotic effectors associated with the mitochondria- and death-dependent pathway in PC12 cells.

• 한국미생물·생명공학회지
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• 제40권2호
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• pp.117-127
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• 2012

피부재생에 대한 지방줄기세포 배양상등액(ADSC-CM)의 효능에 대한 연구를 진행하였다. ADSC-CM이 피부재생에 기여하는 기작은 명확하지 못하지만, ADSC-CM은 다양한 분비물을 포함하고 있고 따라서 피부트러블 처리를 위한 훌륭한 재료이다. 저 산소 상태에서 생산된 ADSC-CM, 즉 advanced adipose-derived stem cell protein extract (AAPE)는 피부재생에 보다 좋은 재료이다. 본 연구는 피부 재생에 결정적 역할을 하는 인체 primary 세포인 섬유아세포(HDF)와 케라티노사이트(HK)를 이용하여 AAPE의 효능을 검증하였다. 0.32 ${\mu}g/ml$ AAPE에서 콜라겐 합성이 관찰 되었으며 AAPE는 stress fiber 형성을 강화하였다. DNA microarray 결과에서는 세포증식, 세포이동, 세포부착, 상처반응에 관여하는 133개의 유전자 발현이 조절되는 것을 알았다. Antibody array를 통해 CD54, FGF-2, GM-CSF, IL-4, IL-6, VEGF, TGF-${\beta}2$, TGF-${\beta}3$, MMP-1, MMP-10, 그리고 MMP-19와 같은 MMP, 성장인자, 사이토카인등 25개의 알려진 단백질이 포함되어 있다는 것을 알았다. 따라서, AAPE는 HK의 세포생물학적 기능을 활성화 할 수 있다고 사료되며 HDF에서는 콜라겐 합성을 유도하였다. 이러한 결과는 AAPE가 피부재생에 임상적 적용이 가능하리라는 것을 의미한다.

• Radiation Oncology Journal
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• 제13권3호
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• pp.205-214
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• 1995

목적 : C3H 마우스의 섬유육종에 있어서 저산소 분획 및 대사상태에 미치는 Ginkgo biloba extract (GBE)의 영향을 알아보기 위해 본 실험을 시행하였다. 방법 : C3H 마우스의 우측 대퇴부에 이식한 섬유육종이 6mm가 되었을 때 $TCD_50$를 이용하여 저산소 세포 분획을 계산하였다. 방사선은 GBE (100mg/kg) 1회 투여시는 1시간 후에, 2회 투여시는 24시간 간격으로 2회 투여후 1시간 경과후 방사선을 조사하였으며, 산소공급이 충분한 상태와 결찰에 의한 저산소 상태에서 각각 조사하였다. 종양의 대사 상태 변화를 알아보기 위해 GBE 투여 전과 투여 후 1시간에 각각 ^{31}P$ 핵자기 공명 분광분석법을 시행하였다. 결과 : 종양 치료방사선량은 결찰에 의한 저산소 상태시 81.7 (77.7 - 86.0) Gy, 정상 혈류상태시는 69.6 (66.8 - 72.5) Gy 였으며, GBE 1회 투여한 경우에는 67.5 (64.1 - 71.1) Gy, GBE 2회 투여시는 62.2 (59.1 - 65.5) Gy 로서, 2회 투여시 현저히 감소하였다. 이로부터 계산한 저산소 세포 분획은 정상 혈류 상태에서는 $10.6{\%}$, GBE 1회 투여시는 $7.2{\%}$로 다소 감소하였고, GBE 2회 투여시는 $2.7{\%}$로 현저히 감소하였다. ^{31}P$ 핵자기공명분광분석결과 PCr/Pi 비는 대조군 $0.27{\pm}0.04$, GBE 1회 투여시는 $0.40{\pm}0.04$, 2회 투여시는 $0.71{\pm}0.04$로 GBE 2회 투여시 종양의 대사상태가 현저히 호전되었다. 결론 : 이상의 결과를 종합해 볼 때, GBE 의 투여에 의해 혈류량과 산소 및 영양분의 공급이 증가함에 따라, 저산소세포분획이 감소하였고, 종양의 대사 상태가 호전되어, 종양의 방사선에 대한 감수성이 높아졌다고 판단되었다.

• 대한한방내과학회지
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• 제26권2호
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• pp.420-430
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• 2005

The water extract of Omijatang(OMJT) has been traditionally used for treatment of abscess and heart palpitation in oriental medicine, However, little is known about the mechanism by which the water extract of OMJT rescues cells from these damages. This study was designed to investigate the protective mechanisms of OMJT in H9c2 cardiomyoblasts on oxidative stress-induced cytotoxicity including $H_2O_2,\;ZnCl_2$, hypoxia, and reoxygenation. Oxidative stress markedly decreased the viability of H9c2 cells. This was characterized with apparent apoptotic features such as chromatin condensation as well as fragmentation of genomic DNA and nuclei. However, OMJT significantly reduced $H_2O_2$-induced cell death and apoptotic characteristics as well as $ZnCl_2$, hypoxialreoxygenation. Taken together, this study suggests that the water extract of OMJT has the protective effects against oxidative injuries.