• 대한한방내과학회지
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• 제24권1호
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• pp.123-133
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• 2003

Objectives : This study was designed to investigate the effects of Chungganhaeju-tang on expression of alcohol metabolizing enzymes, cell viability and alcohol-induced apoptosis. Materials and Methods : For this study, the human hepatoma cell line HepG2 was used. HepG2 cells were treated with ethanol-or acetaldehyde, chungganhaeju-tang, anti-Fas neutralizing antibody and were investigated by using quantitative RT-PCR, MTT and Trypan blue exclusion assays. Results : The results are summarized as follows: 1. Quantitative RT-PCR analysis demonstrated that ethanol-or acetaldehyde-mediated increase of ALDH gene expression was not affected by Chungganhaeju-tang treatment. 2, Ethanol-or acetaldehyde-induced apoptosis was remarkably inhibited by Chungganhaeju-tang in a dose-dependent manner. 3, Ethanol-or acetaldehyde-induced apoptosis was significantly blocked by anti-FasL neutralizing antibody, suggesting apoptosis induced by alcohol might be mediated by FasL/Fas signaling pathway. Conclusions : Taken all together, these results indicate that the FasL/Fas signaling plays a critical role in alcohol-induced apoptosis and Chungganhaeju-tang increases viability of liver cells by suppression of the FasL/Fas-mediated apoptosis-signaling pathway.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.756-766
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• 2005

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

• Radiation Oncology Journal
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• 제19권2호
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• pp.153-162
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• 2001

목적 : 방사선 조사에 의하여 유발되는 세포고사의 신호전달기전, 특히 caspase계 cysteine protease의 활성화, Bcl2 및 Bax 단백질, cytochrome c의 세포질내로의 방출, Fas 와 Fas-L 단백질의 발현양상 등의 조사를 통하여 방사선 조사에 의하여 유발되는 세포고사기전을 규명하고자 하였다. 대상 및 방법 : HL-60 세포주에 6 MV의 X-선을 조사하고 세포생존율, Caspase의 활성도, $Bcl_2$ 및 Bax 단백질, cytochrome c의 세포질내로의 방출여부, 및 Fas 와 Fas-L 단백질의 발현양상을 조사하였다. 결과 : 방사선조사 후 세포의 생존율은 조사선량과 조사 후 시간경과에 따라 감소되었다. 세포고사의 특징인 사다리형 DNA 분절은 방사선조사 4시간 후부터 시간경과에 따라 증가하였으며, 조사선량이 증가할수록 더욱 현저하였다. 방사선조사 후 caspase계 cysteine proteases 중 caspase-2, 3, 6, 8 및 9의 활성화가 시간경과에 따라 증가하였으며, 16 Gy의 방사선량조사 4시간 후에 poly (ADP-ribosyl) polymerase (PARP)의 분절과 Western blot을 이용한 procaspase-3의 분절을 확인함으로서 caspase-3의 활성을 간접적으로 증명할 수 있었다. $Bcl_2$ 단백질은 방사선조사 후 시간경과에 따라 감소하였으며, Bax 단백질은 시간경과에 따라 발현이 증가하는 양상을 관찰할 수 있었다. 방사선조사 후 cytochrome c의 세포질내로의 방출을 확인하였다. 또한 Fas 및 Fas-L 단백질 모두 방사선조사 후 발현이 증가하는 양상을 관찰할 수 있었다. 결론 : HL-60 세포주에서 방사선 조사에 의해 유발되는 세포사멸이 세포고사기전에 의해서 매개됨을 확인하였으며, 이는 세포내 caspase계 cysteine proteases, $Bcl_2$, Bax, 세포질내로의 cytochrome c 방출 그리고 Fas, Fas-L가 관여하는 신호전달경로의 활성화에 의한 것임을 의미하였다.

• Molecules and Cells
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• 제26권2호
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• pp.165-170
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• 2008

Procaspase-8 is activated by forming a death-inducing signaling complex (DISC) with the Fas-associated death domain (FADD) and the Fas receptor, but the mechanism of its activation is not well understood. Procaspase-8 devoid of the death effector domain at its N-terminus (${\Delta}nprocaspase-8$) was reported to be activated by kosmotropic salts, but it has not been induced to form a DISC in vitro because it cannot interact with FADD. Here, we report the production of full-length procaspase-8 and show that it is activated by adding the Fas death domain (Fas-DD) and the FADD forming the cytoplasmic part of the DISC (cDISC). Furthermore, mutations known to affect DISC formation in vivo were shown to have the same effect on procaspase-8 activation in vitro. An antibody that induces Fas-DD association enhanced procaspase-8 activation, suggesting that the Fas ligand is not required for low-level activation of procaspase-8, but that Fas receptor clustering is needed for high-level activation of procaspase-8 leading to cell death. In vitro activation of procaspase-8 by forming a cDISC will be invaluable for investigating activation of ligand-mediated apoptosis and the numerous interactions affecting procaspase-8 activation.

• Toxicological Research
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• 제18권2호
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• pp.183-190
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• 2002

The mechanism by which catechin-mediated cytotoxicity against tumor cells remains to be elusive. To elucidate the mechanical mights of anti-tumor effects, (-)epigallocatechin-gallate (EGCG) of catechin was applied to human prostate cancer DU 145 cells. Cell viability was measured by crystal violet staining. Cell lysates were wed to measure the catalytic activity of caspases by using fluorogenic peptide: Ac-DEVD-AMC for caspase-3 protease, Z-IETD-AFC for caspase-8 protease, Ac-LEHD-AFC for caspase-9 protease as substrates. The equal amounts of protein from cell lysate was separated on SDS-PAGE and analyzed by western blotting with anti-Fas antibody, anti-FasL antibody, anti-BCL2 antibody and anti-Bax antibody. (-)EGCG induced the death of DUl45 cells, which was revealed as apoptosis shown by DNA fragmentation. (-)EGCG induced the activation of caspase family cysteine proteases including caspase-3, -8 and -9 proteases in DU145 cells. Also, (-)EGCG increased the expression of Fas and Fas ligand (FasL) protein in DU145 colls. The expression level of BCL2 was decreased in (-)EGCG treated DU145 cells, whereas Bax protein was increased in a time-dependent manner. We suggest that (-)EGCG-induced apoptosis of DU145 cells is mediated by signaling pathway involving caspase family cysteine protease, mitochondrial BCL2-family protein and Fas/FasL.

• 한국발생생물학회지:발생과생식
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• 제9권2호
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• pp.115-121
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• 2005

본 연구는 Leydig 세포주와 Sertoli 세포주상에 bisphenol A(BPA)와 diethylstilbestrol(DES)의 영향을 알아보고자 수행하였다. 세포 종류에 따른 BPA의 영향을 알아보기 위해, BPA의 농도별로 두 세포주에 처리하여 세포생존율을 비교하였다. Sertoli 세포주가 Leydig 세포주에 비해서 저농도의 BPA에서 생존율이 유의하게 감소되는 것을 확인할 수 있어, Sertoli 세포가 Leydig 세포주에 비해 BPA에 더욱 민감한 것을 알 수 있었다. 또한 BPA나 DES를 처리했을 때 세포 내 분화 및 사멸 신호 전달에 관여하는 phospholipase D(PLD)의 활성이 현저하게 저하되는 것을 확인하였다. 역전사효소 연쇄 반응을 이용하여 세포막상의 세포자연사 신호전달자인 fas 와 fas ligand의 mRNA 발현을 확인해 본 결과, BPA의 처리에 의해 fas ligand의 발현이 다른 실험군에 비해 유의하게 증가하는 것을 확인할 수 있었다. 이상의 결과들을 정리해 볼 때, 내분비계 교란물질인 BPA는 Sertoli 세포 내 fas/fasL 신호전달계를 자극하여 PLD 활성을 저하시킴으로서, Sertoli 세포 내 세포자연사를 유발시키는 것으로 사료된다.

• Toxicological Research
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• 제19권3호
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• pp.197-203
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• 2003

Apicidin, a histone-deacetylase inhibitor, has been successfully used to inhibit the growth of cancer cells. In this study, the apoptotic potential and mechanistic insights of apicidin were investigated in human myeloid leukemia U937 cells. Treatment of U937 cells with apicidin resulted in a decrease of cell viability with apoptotic characteristics, including chromatin condensation and ladder-pattern fragmentation of genomic DNA. Apicidin converted the procaspase-3 protease to catalytically active effector protease, resulting in subsequent cleavage of poly (ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated deoxyribonuclease (ICAD). In addition, apicidin induced the activation of caspase-9 protease and the cytosolic release of mitochondrial cytochrome c with mitochon-drial membrane potential transition. Moreover, apicidin transiently increased the expression of Fas and Fas ligand proteins. Taken together, the results suggest that apicidin induces apoptosis of U937 cells through activation of intrinsic caspase cascades and Fas/FasL system with mitochondrial dysfunction.

• 대한수의학회지
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• 제43권3호
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• pp.485-492
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• 2003

The pathological mechanism of impaired spermatogenesis after vasectomy has not been completely investigated. In this study, we examined pathological changes of the testis and the Fas-Fas ligand (FasL) mediated signaling pathway in apoptotic germ cell death after vasectomy in rats. Ten-weeks old Sprague-Dawley rats were underwent bilateral vasectomy and sacrificed after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, and 4 weeks of surgery and the testes were removed. Histopathological evaluation of spermatogenesis was performed by hematoxylin-eosin and periodic acid-Schiff-hematoxylin staining. To elucidate the pathophysiology of seminiferous tubule damage, terminal dUTP nick end labeling staining, electrophoresis assay of DNA fragmentation, and Western blotting analysis for Fas-FasL were performed. Relative weights of testes were decreased from 5 days after vasectomy. Germ cell degeneration were first found in the spermatogonia and spermatocytes at stages I-VI, and XII-XIV seminiferous tubules. Mean incidence of apoptotic germ cells after vasectomy progressively increased to peak in 5 days, and then gradually decreased to the control levels in 2 weeks after vasectomy. The expression of Fas-FasL reached maximum level at 5 days after vasectomy and then declined. In conclusion, impaired spermatogenesis after vasectomy associated with an increase in germ cell apoptasis, which is partly mediated by the activation of Fas-FasL.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2005년도 제48차 춘계 학술대회
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• pp.111-111
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• 2005

• Asian Pacific Journal of Cancer Prevention
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• 제15권21호
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• pp.9319-9325
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• 2014

Alkaloids are the most extensively featured compounds of natural anti-tumor herbs, which have attracted much attention in pharmaceutical research. In our previous studies, a mixture of major three alkaloid components (5, 6-dihydrobicolorine, 7-deoxy-trans-dihydronarciclasine, littoraline) from Hymenocallis littoralis were extracted, analyzed and designated as AHL. In this paper, AHL extracts were added to human liver hepatocellular cells HepG-2, human gastric cancer cell SGC-7901, human breast adenocarcinoma cell MCF-7 and human umbilical vein endothelial cell EVC-304, to screen one or more AHL-sensitive tumor cell. Among these cells, HepG-2 was the most sensitive to AHL treatment, a very low dose ($0.8{\mu}g/ml$) significantly inhibiting proliferation. The non-tumor cell EVC-304, however, was not apparently affected. Effect of AHL on HepG-2 cells was then explored. We found that the AHL could cause HepG-2 cycle arrest at G2/M checkpoint, induce apoptosis, and interrupt polymerization of microtubules. In addition, expression of two cell cycle-regulated proteins, CyclinB1 and CDK1, was up-regulated upon AHL treatment. Up-regulation of the Fas, Fas ligand, Caspase-8 and Caspase-3 was observed as well, which might imply roles for the Fas/FsaL signaling pathway in the AHL-induced apoptosis of HepG-2 cells.