• Molecules and Cells
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• 제38권4호
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• pp.349-355
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• 2015

EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 co-precipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinase-inactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptor-like protein acts as a biochemical linker between the Eph receptor and caspase-8.

• Archives of Pharmacal Research
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• 제23권3호
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• pp.246-251
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• 2000

In most tissues, apoptosis plays a pivotal role in normal development and for regulating cell number, thus inappropriate apoptosis underlies a variety of diseases. Caspase-3 is one of a family of caspases that are mainly involved in the apoptotic signal transduction pathway, where caspase-3 acts as an effect molecule to proteolytically cleave intracellular substrates that are necessary for maintaining cell survival. Recent evidences show that apoptotic cell death can be blocked by inhibiting caspase-3, suggesting its inhibitors have potential to be therapeutic drugs for the diseases related with inappropriate apoptosis. We have established a screening system to search caspase-3 inhibitors from chemical libraries stocked in our institute. The enzyme assay is configured entirely in 96-well format, which is easily adapted for high throughput screening. Before performing mass screening, 80 in-house compounds were screened as a preliminary experiment, and we found that morin hydrate inhibited caspase-3 by 66.4 % at the final concentration of 20 ${\mu}g/m{\ell}$.

• Food Science and Biotechnology
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• 제17권2호
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• pp.308-312
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• 2008

The flower buds of Tussilago farfara (TF) have been traditionally used in oriental medicine for the treatment of bronchitis and asthma. In our study, the primary objective was to determine the mechanisms that are inherent to TF-induced cytotoxicity and apoptosis, using the methanolic extract of TF (TFM) in HT-29 human colon cancer cells. We found that TFM-induced induced cytotoxicity in HT-29 cells in a dose-dependent manner. This effect was verified via an MTT reduction assay, an lactate dehydrogenase (LDH) release assay, and a colony formation assay. Interestingly, we also detected apoptotic bodies on Hoechst staining, and attempted to determine whether TFM-induced apoptosis involved the caspase pathway using a caspase-3/7 activity assay. Overall, the results indicate that TFM contain chemotherapeutic agents and potential candidates use for against human colon cancer cells.

• 대한한방내과학회지
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• 제32권4호
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• pp.504-518
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• 2011

Objectives : This study was performed to investigate the anti-fibrogenic effect of greater celandine on cultured rat hepatic stellate cells. Materials and Methods : Hepatic stellate cells (HSC-T6) were treated with various concentrations of greater celandine extract for 24, 48, and 72 hours. The extraction was done with distilled water. After the treatment, cell viability, proliferation, mRNA of the ${\alpha}SMA$, TIMP-1, TIMP-2, collagen I ${\alpha}$ 1, MMP-2, IL-6, TGF-${\beta}1$, PDGFr-${\beta}1$, Bcl-2, Bax, Bcl-xl, caspase-3, caspase-9 and the activities of SOD and catalase were measured by using MTT assay, BrdU assay, real-time PCR, superoxide dismutase assay and catalase assay. Results : The viability, proliferation, mRNA expression and synthesis of collagen of the hepatic stellate cells were inhibited as the concentration increased, which indicates the herb has an inhibitory effect on fibrogenesis of the liver by regulating the fibrosis associated genes in transcription. Conclusions : These results suggest that greater celandine would be beneficial in the treatment of fibrotic patients as well as for patients with chronic hepatitis.

• 한국식품영양과학회지
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• 제46권2호
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• pp.161-168
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• 2017

본 연구에서는 산화적 스트레스로 유도한 시신경 세포사멸에 대한 차조기 물 추출물(PFE)의 효과를 확인하였다. Staurosporine으로 분화된 ssdRGC-5 세포에 buthionine과 glutamate(B/G)로 산화적 스트레스를 유도하였으며, LDH release assay, MTT reduction assay를 통하여 PFE가 농도 의존적으로 B/G에 의한 세포사멸을 억제함을 관찰하였다. 세포사멸의 기전을 연구하기 위해 caspase 활성, 세포 내 ROS 생성량, 세포고사 관련 단백질 발현을 관찰한 결과, B/G에 의해 증가한 ROS 생성량, caspase 활성을 PFE가 억제하였고, 세포질로 방출된 cytochrome c와 미토콘드리아로 이동한 Bax도 감소함을 확인하였다. 이상의 결과로부터 차조기는 산화적 스트레스로 유도된 시신경 세포사멸 과정에서 항산화 효과와 미토콘드리아성 세포사멸을 완화함으로써 세포 보호 작용을 나타냄을 확인하였다.

• 한국식품영양과학회지
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• 제45권9호
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• pp.1257-1264
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• 2016

개똥쑥은 예로부터 항암, 항바이러스 및 항균의 효능을 지니는 것으로 알려져 왔지만 작용 기작에 대한 내용이 많이 알려지지 않았다. 본 연구에서는 AGS 인체 위암 세포를 대상으로 개똥쑥 추출물(AAE)에 의한 apoptosis 효과와 신호경로 연구를 시행하였다. AAE의 암세포 성장에 미치는 영향을 확인하기 위하여 AGS cell에 AAE를 처리하고 MTT assay와 LDH assay를 수행한 결과 AAE 농도 의존적으로 나타난 세포 성장 억제가 세포 손상에 의한 것임을 확인하였다. 또한, AAE에 의한 암세포 증식 억제 효과가 apoptosis에 의한 것인지 확인하기 위하여 Hoechst 33342 staining과 Annexin V-PI staining을 수행한 결과, Hoechst 33342 staining에서 apoptotic body와 세포질 응축이 농도 의존적으로 증가하는 것을 확인하였고, Annexin V-PI staining에서 apoptotic cells의 변화가 농도 의존적으로 증가함을 확인하였다. Western blotting의 결과 AAE가 농도 의존적으로 세포 생장에 관여하는 신호 단백질인 p-Akt, p-TSC2, p-mTOR, p-GSK-$3{\beta}$의 발현이 감소함을 확인하였고, anti-apoptotic 단백질인 Bcl-2의 발현이 억제됨으로써 proapoptotic 단백질인 Bax, Bak의 발현이 증가하는 일련의 신호경로를 조절할 수 있다는 것을 확인하였다. 미토콘드리아 막 전위의 탈분극 유도를 확인하기 위한 JC-1 assay 수행 결과, AAE 농도 의존적으로 미토콘드리아 막 전위의 탈분극이 유도됨을 확인하였다. 탈분극에 의한 caspase 활성을 확인하기 위해 caspase-3/7 activity assay를 수행한 결과, AAE 농도 의존적으로 caspase activity 증가를 확인하였다. 또한, apoptosis가 일어나는 일련의 신호경로를 확인하기 위해 apoptosis 상위 단백질인 Akt, mTOR, GSK-$3{\beta}$의 활성을 억제하는 LY294002, Rapamycin, BIO를 각각 AGS cell에 처리하고 세포증식에 미치는 영향과 신호 단백질의 발현 양상을 알아보기 위해 MTT assay, LDH assay, western blotting을 수행하였다. 그 결과 AAE와 LY294002, Rapamycin 처리군에서 세포증식 억제와 LDH 방출량 증가뿐만 아니라 세포 생장 신호 단백질인 p-mTOR, p-TSC2, p-Akt, p-GSK-$3{\beta}$의 발현이 감소하는 것을 확인하였고, Bcl-2의 발현이 억제됨으로써 Bax와 Bak의 발현을 증가시키는 신호경로를 조절할 수 있다는 것을 확인하였다. 따라서 AGS cell에 개똥쑥 추출물을 처리하였을 때 유도되는 apoptosis 효과는 Akt/mTOR/GSK-$3{\beta}$ 경로 활성 억제를 통해 Bcl-2 발현이 감소함에 따라 Bax, Bak를 활성화해 세포질로의 cytochrome C 유리에 따른 caspase 활성으로 이루어진다는 것을 알 수 있었다.

• 생명과학회지
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• 제20권4호
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• pp.519-527
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• 2010

Paclitaxel은 미세소관의 탈중합을 억제하는 시약으로 알려져 있다. Paclitaxel은 다양한 세포에서 세포 내 방추체를 안정화시킴으로써 유사분열 억제 및 세포사멸을 유도한다. 본 실험에서는 토끼 관절 연골세포에서 paclitaxel이 연골세포의 증식과 사멸에 미치는 효과에 대한 연구를 수행하였다. MTT assay를 수행한 결과 paclitaxel은 연골세포에서 농도 의존적으로 세포 증식을 억제한다는 것을 확인 할 수 있었으며, FACS analysis와 Western blot analysis를 수행한 결과, paclitaxel이 G2/M 정지를 유도하는 것을 확인하였다. 또한, paclitaxel이 비정상적인 세포 분열유도와 핵 단편분절 유도없이 일어나는 mitotic catastrophe 즉, caspase-3 비의존적인 세포사멸을 유도하였다. Paclitaxel을 처리한 세포에서 일어나는 이러한 mitotic catastrophe에 의한 세포 죽음은 G1/S기의 진행을 억제하는 시약인 thymidine을 처리하는 것에 의해 억제되는 것을 확인할 수 있었다. 이러한 결과를 종합해 볼 때, paclitaxel에 의한 토끼 관절 연골 세포에서의 세포 죽음은 caspase-3 비의존적인 mitotic catastrophe에 의해 일어나는 것으로 사료되어진다.

• Advances in Traditional Medicine
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• 제10권4호
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• pp.254-262
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• 2010

Artemisia capillaris (A. capillaries) is known to play roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We investigated the antifibrogenic efficacy of A. capillaris in the immortalized human hepatic stellate cell line LX2. Cell proliferation was determined by the MTT assay. Cell cycle was analyzed by the flow cytometry. Apoptotic cells were measured using a cell death detection ELISA. Caspase activity was detected by a colorimetric assay. The mRNA level of Bcl-2 and Bax mRNA were measured by real-time PCR. MEK and ERK protein were detected by Western blot analysis. We provide evidence that A. capillaris induces cell cycle arrest, apoptosis, and potently inhibits the mitogen-activated protein kinase pathway. A. capillaris inhibited cell proliferation of LX2 cells in a dose- and time-dependent manner, increased the apoptosis fraction at cell cycle analysis with an accompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 mRNA levels and an increase in Bax expression. Exposure of LX2 cells to A. capillaris induced caspase-3 activation, but co-treatment of A. capillaris with the pan-caspase inhibitor Z-VAD-FMK, and the caspase-3 inhibitor Z-DEVE-FMK, blocked apoptosis. A. capillaris down-regulated Mcl-1 protein levels and inhibited phosphorylation of MEK/ERK, suggesting that it mediates cell death in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independent pathway.

• 대한한방내과학회지
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• 제24권1호
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• pp.104-112
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• 2003

Objectives : The purpose of this study is to evaluate the effects of Sohaphyang-won and is to study the mechanism for neuronal death protection in hypoxia with Embryonic day 20(E20) cortical cells of a guinea pig(Sprague Dawley). Methods : E20 cortical cells, used in this investigation were dissociated in Neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Sohaphyang-won was added to the culture media for 72 hours. On 17 DIV, cells were given a hypoxic shock and further incubated in normoxia for another three days. On 20 DIV, Sohaphyang-won's effects for neuronal death protection were evaluated by LDH assay and the mechanism was studied by Bcl-2, Bak, Bax, caspase family. Results : This study indicates that Sohaphyang-won's effects for neuronal death protection in hypoxia is confirmed by LDH assay by the method of Embryonic day 20(E20) cortical neuroblast. Conclusions : Sohaphyang-won's mechanism for neuronal death protection in hypoxia restrains inflow of cytochrome C into cellularity caused by Bcl-2 increase and reduces the caspase cascade initiator caspase-10 and the effector caspase-3.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1617-1620
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• 2012

Background: Gold nanoparticles have recently been investigated with respect to biocompatibility according to their interactions with cells. The purpose of this study was to examine cytotoxicity and apoptosis induction by well-characterized gold nanoparticles in human breast epithelial MCF-7 cells. Methods: Apoptosis was assessed by TUNEL, cytotoxicity by MTT assay and caspase 3, 9, p53, Bax and Bcl expression by real-time PCR assays. Results: Gold nanoparticles at up to $200\;{\mu}g/mL$ for 24 hours exerted concentration-dependent cytotoxicity and significant upregulation of mRNA expression of p53, bax, caspase-3 & caspase-9, whereas expression of antiapoptotic bcl-2 was down-regulated. Conclusion: To the best of our knowledge this is the first report showing that gold nanoparticles induce apoptosis in MCF-7cells via p53, bax/bcl-2 and caspase pathways.