• Biomolecules & Therapeutics
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• 제7권1호
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• pp.22-28
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• 1999

Induction of apoptosis is considered to be the underlying mechanism that accounts for the efficiency of chemotherapeutic drugs. It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human leukemic cells via the Fas/Fas Ligand (FasL) system. Comparison of Fas and FasL mRNA expression between drug- and anti-Fas antibody(Fas-Ab)- induced apoptosis was analyzed for examining the role of Fas/FasL system in the mediation of drug-induced apoptosis. After HL-60 cells were routinely cultured, MTT assay was performed for cytotoxicity test. Giemsa staining was carried out to monitor the apoptosis morphologically. By semiquantitative RT-PCR analysis, the expression of Fas and FasL at 4, 10, 24 hours was determined after DOX and Fas-Ab treatment. Dose-dependent cytotoxicity was induced by DOX-treatment, while Fas-Ab treatment showed the similar dose-dependent pattern but the cytotoxicity is not reached at LD$_{50}$ at 100 ng/ml concentration of Fas-Ab. In the 10ng/m1 DOX and 10ng/m1 Fas-Ab treated group, typical apoptotic cell morphology was shown such as fragmented nuclei and cell membrane budding in the Giemsa-stained slide. Fas mRNA expression was not changed significantly in the both groups. But, FasL mRNA expression was induced significantly at initial period of apoptosis. In this study, Fas/FasL interaction assumed to be involved in drug-induced apoptosis.s.

• 대한한방내과학회지
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• 제22권3호
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• pp.353-360
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• 2001

Objectives: Recently, it was known that the major cause of hepatitis is apoptosis reaction mediated by Fas-FasL. Since Artemisia Capillaris Fructus has long been applied to cure the jaundice in oriental medicine. Therefore, this study was carried out to examine the effect of fractions of Artemisia Capillaris Fructus on Fas-FasL-mediated apoptosis in hepatocytes. Methods: This study employed propidium iodide negative cell count assay and some the other biochemical assays. Results : This study confirms that hepatitis has been occured by apoptosis mediated by Fas-FasL in cultured hepatocyte and fractions of Artemisia Capillaris Fructus restrain apoptosis induced Fas-FasL. Conclusions : Water-extracted fraction, methanol extracts, ether-soluble fraction, and buthanol-soluble fractions of Artemisia Capillaris Fructus restrain Fas-FasL-mediated apoptosis in hepatocyte. Silica gel chromatograph of Buthanol-soluble fraction of Artemisia Capillaris Fructus restrain Fas-FasL-mediated apoptosis in hepatocyte. Artemisia Capillaris Fructus could be applied to cure hepatitis.

• 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.

• Archives of Pharmacal Research
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• 제27권4호
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• pp.402-406
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• 2004

This research team found in previous studies, that the ginseng saponin metabolite IH901 induces apoptosis in HepG2 cells via a mitochondrial-mediated pathway, which resulted in the activation of caspase-9 and subsequently of caspase-3 and -8. Based on these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in IH901-induced apoptosis in HepG2 cells, was investigated. Levels of Fas and the Fas ligand (FasL) mRNA or protein were not increased by IH901, rather they were decreased significantly at 18 h post treatment. Soluble FasL (sFasL) was detectable by immunoprecipitation analysis En the medium of HepG2 cells treated with IH901. Increased levels of sFasL were inversely correlated with the levels of FasL. Preincubation of HepG2 cells with antagonistic anti-Fas antibody showed little protective effect, if any, on IH901-induced cell death. At a $30{\mu}M$ (24 and 48 h) and $40{\mu}M$ (24 h) concentration of IH901, the cytotoxic effect of IH901 was less then $50\%$, anti-Fas antibody prevented IH901-induced cell death. However, at a $60{\mu}M$ (24 and 48 h) and $40{\mu}M$ (48 h) concentration of IH901, cell death rates were about $80\%$ or more and most of the chemopreventive and chemotherapeutic effects of IH901 were manifested. Blocking the Fas receptor did not influence IH901-induced cell death. These results indicate that the Fas/FasL system is engaged, but not required for IH901-induced cell death, at pharmacologically significant concentrations.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.281-285
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• 2013

Purpose: To probe the role of FasL in cell apoptosis in oral squamous cell carcinomas (OSCCs). Methods: The expression of Fas/FasL was assessed in 10 cases of normal oral epithelium, 38 cases of OSCC and tumor infiltrating lymphocytes (TIL), and 11 cases of metastatic lymph nodes by immunohistochemistry. Apoptosis of tumor cells and TIL was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). FasL-induction of T cell apoptosis was tested by co-culture assay in vitro with SCC-9 and Jurkat T cells. Results: The 10 cases of normal oral epithelium all demonstrated extensive expression of Fas, the positive rate being largely down-regulated in OSCC (21/38) (P<0.05) compared to the normal (10/10). At the same time, the positive rate of FasL significantly increased in OSCC (P<0.05) especially those with lymph node metastasis (P<0.05). The positive rates of Fas in well and middle differentiated OSCC were higher than those in poor differentiated OSCC (P<0.05). The AI of tumor cells in Fas-positive OSCC was remarkably higher than that in Fas-negative OSCC (P<0.01), with a positive correlation between Fas expression and cell differentiation as well as apoptosis (r=0.68, P<0.01). The AI of tumor cells in FasL positive OSCC was remarkably lower than that in control while the AI of TIL was higher than in FasL negative OSCC (P<0.05). The AI of tumor cells reversely correlated with that of TIL (r = -0. 72, P<0.05). It was found that SCC-9 cells expressing functional FasL could induce apoptosis of Jurkat cells as demonstrated by co-culture assays. As a conclusion, it is evident that OSCC cells expressing FasL can induce apoptosis in Fas-expressing T cells. Conclusions: In progression of OSCC, expression of the Fas/FasL changes significantly. The results suggest that FasL is a mediator of immune privilege in OSCC and may serve as an marker for predicting malignant change in oral tissues.

• 한국발생생물학회지:발생과생식
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• 제20권4호
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• pp.315-329
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• 2016

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-${\gamma}$) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-${\gamma}$ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-${\gamma}$ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-${\gamma}$ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-${\gamma}$ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (${\geq}1,000U/mL$) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-${\gamma}$ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-${\gamma}$ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.167-178
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• 2002

Objective : The present study was performed to investigate whether apoptosis occur in human embryos by annexin staining and detect the expression of Fas, Fas-ligand (FasL), Bax, and Bcl-2 in human fragmented embryos derived from IVF-ET by immunofluorescence and Western blot analysis. Materials and Methods: Using annexin staining, immunofluorescence and Western blot analysis on normal and fragmented embryos, we were able to detect apoptotsis and apoptotic gene products in fragmented embryos. Result: Phosphatidylserine (PS) translocation, the marker for apoptosis, were detected frequently in fragmented embryos. Bcl-2 and Bax protein were detected in both fragmented and non-fragmented embryos. When fragmented embryos compared to normal embryos, immunofluorescent intensity of Bcl-2 tended to be lower in fragmented embryos. Bax gene expression increased in the fragmented embryos compared to the normal embryos. This result supports a model in which the molar ratio of Bcl-2 to Bax determines whether apoptosis induced or inhibited in human embryo. Fas was highly expressed in human preimplantation embryos but not FasL. It suggests that embryo may undergo apoptosis by binding with FasL produced by follicular or immune cells. Conclusion: The over expression of Bax and Fas will trigger apoptosis to lead embryo fragmentation and change embryo to be nonviable.

• Radiation Oncology Journal
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• 제21권3호
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• pp.222-226
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• 2003

목적: 변이를 보이는 lpr 마우스와 Fas ligand 변이를 보이는 gld 마우스를 이용하여 in vivo에서 Fas와 Fas ligand의 발현이 전리 방사선에 의해 유도되는 apoptosis에서 어떤 역할을 하는지 조사하고자 하였다. 대상 및 방법: Fas의 변이를 보이는 $C57BL/6J-Fas^{lpr}$ 마우스와 대조군인 C57BL/6J 마우스, Fas ligand 변이를 보이는 $C3H/HeJ-Fas^{gld}$ 마우스와 대조군인 C3H/HeJ 마우스를 대상으로 하였다. 마우스는 8주령 웅성으로서 전신 방사선 조사하여 일정 시간 후 비장을 적출하였다. 조직을 hematoxylin-eosin 염색하여 apoptosis 유도 수준을 비교 분석하였다. 또한 apoptosis 조절 물질인 p53, Bcl-2, Bax, Bcl-X_L,\;Bcl-X_S$에 대하여 Western Western blotting을 시행하고 발현수준을 densitometry로 분석하여 관련된 기전을 연구하였다. 결과: 2.5 Gydh k10 Gy 조사시에 $C57BL/6J-Fas^{lpr}$ 마우스와 $C3H/HeJ-Fas^{gld}$ 마우스에서 대조군 비하여 방사선에 의한 apoptosis가 유의하게 감소되는 것으로 나타났다(p<0.05). C57BL/6J 마우스와, C3H/HeJ 마우스에서 10 Gy 방사선 조사 후 Bax가 8시간 째에 각각 3배, 3.3배의 증가를 보였으나 $C57BL/6J-Fas^{lpr}$ 마우스와, $C3H/HeJ-Fas^{gld}$ 마우스에서는 뚜렷한 발현증가가 관찰되지 않았다. 결과: Fas의 변이가 있는 lpr 마우스와 Fas ligand의 변이가 있는 gld 마우스에서 방사선에 의한 apoptosis가 대조군 보다 현저하게 낮으며 이는 방사선에 의한 Bax의 유도가 미약한 것과 연관된 것으로 나타났다. 방사선에 의한 apoptosis 유도에 Fas의 역할이 매우 중요한 것으로 보인다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2613-2618
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• 2014

Aims: Dysfunction of the host immune system in cancer patients can be due to a number of factors, including lymphocyte apoptosis. Several studies showed that $Foxp3^+T$ cells take part in inducing this process by expressing FasL in tumor patients. However, the relationship between apoptosis, $CD8^+T$ cells and $Foxp3^+T$ cells in HCC patients is still unclear. The present study was designed to investigate the correlation between apoptosis levels and Fas/FasL expression in $CD8^+T$ lymphocytes and $Foxp3^+T$ cells in patients with HCC. Methods: $CD8^+T$ cells and $CD3^+Foxp3^+T$ cells were tested from peripheral blood of HCC patients and normal controls and subjected to multicolor flow cytometry. The expression of an apoptosis marker (annexin V) and the death receptor Fas in $CD8^+T$ cells and FasL in $CD3^+Foxp3^+T$ cells were evaluated. Serum TGF-${\beta}1$ levels in patients with HCC were measured by enzyme-linked immunosorbent assay. The relationship between apoptosis and Fas expression, as well as FasL expression in $CD3^+Foxp3^+T$ cells was then evaluated. Results: The frequency of $CD8^+T$ cells binding annexin V and Fas expression in $CD8^+T$ cells, were all higher in HCC patients than normal controls and the proportion of apoptotic $CD8^+T$ cells correlated with their Fas expression. Serum TGF-${\beta}1$ levels correlated inversely with $CD3^+Foxp3^+T$ cells. Conclusions: Fas/FasL interactions might lead to excessive turnover of $CD8^+T$ cells and reduce anti-tumor immune responses in patients with HCC. Further investigations of apoptosis induction in $Fas^+CD8^+T$ cells in vitro are required.

• 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.