• Bulletin of the Korean Chemical Society
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• v.27 no.1
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• pp.87-92
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• 2006

Fas-associated death domain protein (FADD) recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins. Cellular FLICE-inhibitory protein (c-FLIP) was identified as a molecule with sequence homology to caspase-8. It has been postulated that c-FLIP prevents formation of the competent death-inducing signaling complex in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. However, the interaction of FADD and $c-FLIP_s$ (short form) in apoptosis signaling has been controversially discussed. We show the purification and the characterization of human full-length FADD and $c-FLIP_s$ expressed in Escherichia coli. The purified FADD and $c-FLIP_s$ are shown as homogeneity, respectively, in SDS-PAGE analysis and light-scattering measurements. The folding properties of the $\alpha$-helical structure of FADD and the super-secondary structure of $c-FLIP_s$ proteins were characterized by circular dichroism spectroscopy. Furthermore, we report here a series of biochemical and biophysical data for FADD-$c-FLIP_s$ binding in vitro. The binding of both FADD and $c-FLIP_s$ proteins was detected by BIAcore biosensor, fluorescence measurement, and size-exclusion column (SEC).

• The Korea Journal of Herbology
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• v.21 no.2
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• pp.37-46
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• 2006

Objectives : The purpose of this study was to investigate the effect of Dangguibohyultang (DB) and its combination (DB-I; Astragali membraneus BUNGE : Angelica gigas NAKAI=5:1, DB-II; Astragali membraneus BUNGE:Angelica gigas NAKAI=1:1, DB-III; Astragali membraneus BUNGE:Angelica gigas NAKAI=1:5,) on apoptosis in human colorectal adenocarcinoma HCT116 cells. Methods : To study the cytotoxic effect of methanol extract of DB-I, DB-II and DB-III on HCT116 cells, the cell viability was determined by XTT reduction method and ttypan blue exclusion assay. To confirm the induction of apoptosis, the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of procaspase-3, -8 and -9 were examined by western blot analysis. Furthermore, DB-induced apoptosis was confirmed by DNA fragmentation. The release of cytochrome C from mitochondria to cytosol, the level of Bcl-2 and Bax, and the expressions of Raf/MEK/ERK were examined by western blot analysis. Results : DB-I and DB-II reduced proliferation of HCT116 cells in a dose-dependent manner. DB-I and DB-II decreased procaspase-3, -8, -9 levels in a dose-dependent manner and induced the clevage of PARP. DB-I and DB-II also triggered the mitochondrial apoptotic signaling by increasing the release of cytochrome C from mitochondria to cytosol, decreasing of anti-apoptotic Bcl-2, and increasing of pro-apoptotic Bax. DB-I and DB-II decreased the activation of Ras/Raf/MEK/ERK cascade in a dose-dependent manner. Conclusion : These results suggest that DB-I and DB-II induce apoptosis via mitochondrial pathway in HCT116 cells. Furthermore, Raf/MEK/ERK cascade is involved in DB-induced apoptosis. These results suggest that DB is potentially useful as a chemotherapeutic agent in human liver cancer.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.5
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• pp.555-560
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• 2008

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the $Na^+$-independent transport of neutral amino acids including several essential amino acids. The L-type amino acid transporter 1 (LAT1), an isoform of system L amino acid transporter, is highly expressed in cancer cells to support their continuous growth and proliferation. 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is a model compound for the study of amino acid transporter as a system L selective inhibitor. We have examined the effect and mechanism of BCH on cell growth suppression in HEp2 human head and neck squamous cell carcinoma. The BCH inhibited the L-leucine transport in a concentration-dependent manner with a $IC_{50}$ value of $51.2{\pm}3.8{\mu}M$ in HEp2 cells. The growth of HEp2 cells was inhibited by BCH in the timeand concentration-dependent manners. The formation of DNA ladder was not observed with BCH treatment in the cells. Furthermore, the proteolytic processing of caspase-3 and caspase-7 in the cells were not detected by BCH treatment. These results suggest that the BCH inhibits the growth of HEp2 human head and neck squamous cell carcinoma through the intracellular depletion of neutral amino acids for cell growth without apoptotic processing.

• BMB Reports
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• v.47 no.9
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• pp.488-493
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• 2014

Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED.

• International Journal of Oral Biology
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• v.41 no.4
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• pp.175-181
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• 2016

Tyrosol, a phenylethanoid and a derivative of phenethyl alcohol, possesses various biological properties, such as anti-oxidative and cardioprotective activity. Olive oil is the principal source of tyrosol in the human diet. However, so far the anti-cancer activity of tyrosol has not yet been well defined. This study therefore undertakes to examine the cytotoxic activity and the mechanism of cell death exhibited by tyrosol in KB human oral cancer cells. Treatment of KB cells with tyrosol induced the cell growth inhibition in a concentration- and a time-dependent manner. Furthermore, the treatment of tyrosol induced nuclear condensation and fragmentation of KB cells. Tyrosol also promoted proteolytic cleavage of procaspase-3, -7, -8 and -9, increasing the amounts of cleaved caspase-3, -7, -8 and -9. In addition, tyrosol increased the levels of cleaved PARP in KB cells. These results suggest that tyrosol induces the suppression of cell growth and cell apoptosis in KB human oral cancer cells, and is therefore a potential candidate for anti-cancer drug discovery.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.6
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• pp.1636-1639
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• 2005

To develop novel anti-leukemic medicine, we have prepared a Korean traditional medicine, named Hang-baek-Tang, which is composed of 8 kinds of anti-leukemic medicinal plants. The water extracts was examined anti-leukemic activity using the human leukemia cell line, HL-60 cells. HL-60 cells showed the growth inhibition and several apoptotic features, including DNA ladders, morphological changes, by treatment of the cells with Hang-Daek-Tang. We have observed that Hang-baek-Tang induced the activation of caspase-3, caspase-8 and caspase-9. Further molecular analysis demonstrated that Hang-baek-Tang induced cleavage of PARP and increase of hypodiploid (Sub-G1) population in flow cytometric analysis. These results indicate that Hang-baek-Tang has been considered to exert anti-leukemic activity through the procaspase-3 activation pathway.

• Development and Reproduction
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• v.26 no.3
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• pp.99-105
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• 2022

Styrene is the precursor of polystyrene. Human exposure to styrene could occur in occupational and residential settings and via food intake. Styrene is metabolized to styrene-7,8-oxide by cytochrome P450 enzyme. In the present study, we investigated the cytotoxicity mediated by styrene and styrene-7,8-oxide in TM3 testicular Leydig cells in vitro. We first monitored the nuclear fragmentation in Leydig cells after exposure to styrene or styrene-7,8-oxide. Hoechst 33258 cell staining showed that styrene exposure in TM3 Leydig cells did not exhibit nuclear fragmentation at any concentration. In contrast, nuclear fragmentation was seen in styrene-7,8-oxide-exposed cells. These results indicate that cytotoxicity-mediated cell death in Leydig cells is more susceptible to styrene-7,8-oxide than to styrene. Following styrene treatment, procaspase-3 and XIAP protein levels did not show significant changes, and cleaved (active) forms of caspase-3 were not detected. Consistent with the western blot results, the active forms of caspase-3 and XIAP proteins were not prominently altered in the cytoplasm of cells treated with styrene. In contrast to styrene, styrene-7,8-oxide induced cell death in an apoptotic fashion, as seen in caspase-3 activation and increased the expression of XIAP proteins. Taken together, the results obtained in this study demonstrate a fundamental idea that Leydig cells are capable of protecting themselves from cytotoxicity-mediated apoptosis as a result of styrene exposure in vitro. It remains unclear whether the steroid-producing function, i.e., steroidogenesis, of Leydig cells is also unaffected by exposure to styrene. Therefore, further studies are needed to elucidate the endocrine disrupting potential of styrene in Leydig cells.

• International Journal of Oral Biology
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• v.45 no.1
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• pp.8-14
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• 2020

Bilobalide isolated from the leaves of Ginkgo biloba has several pharmacological activities such as neuroprotective, anti-inflammatory, and anticonvulsant. However, the effect of bilobalide on cancer has not been clearly established. The main purpose of this study was to investigate the effect of bilobalide on cell growth and apoptosis induction in FaDu human pharyngeal squamous cell carcinoma. This was examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, nuclear 4′,6-diamidino-2-phenylindole dihydrochloride staining, DNA fragmentation analysis, and immunoblotting. Bilobalide inhibited the growth of FaDu cells in dose- and time-dependent manners. Treatment with bilobalide resulted in nuclear condensation and DNA fragmentation in FaDu cells. Furthermore, it promoted the proteolytic cleavage of procaspase-3/-7/-8/-9 with increase in the amount of cleaved caspase-3/-7/-8/-9. Bilobalide-induced apoptosis in FaDu cells was mediated by the expression of Fas and the activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting revealed that the antiapoptotic mitochondrial protein Bcl-2 was downregulated, but the proapoptotic protein Bax was upregulated by bilobalide in FaDu cells. Bilobalide significantly increased Bax/Bcl-2 ratio. These results suggest that bilobalide inhibits cell proliferation and induces apoptosis in FaDu human pharyngeal squamous cell carcinoma via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondrial-mediated intrinsic apoptotic pathway.

• The Journal of the Korean Society for Microbiology
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• v.34 no.6
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• pp.501-512
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• 1999

Gliotoxin, a fungal metabolite, is one of the epipolythiodioxopiperazine classes and has a variety of effects including immunomodulatory and apoptotic agents. This study is designed to evaluate the effect of zinc on gliotoxin-induced death of HL-60 cells. Here, we demonstrated that treatment of gliotoxin decreased cell viability in a dose and time-dependent manner. Gliotoxin-induced cell death was confirmed as apoptosis characterized by chromatin margination, fragmentation and ladder-pattern digestion of genomic DNA. Gliotoxin increased the proteolytic activities of caspase 3, 6, 8, and 9. Caspase-3 activation was further confirmed by the degradation of procaspase-3 and PARP in gliotoxin-treated HL-60 cells. Zinc compounds including $ZnCl_2$ and $ZnSO_4$ markedly inhibited gliotoxin-induced apoptosis in HL-60 cells (from 30% to 90%). Consistent with anti-apoptotic effects, zinc also suppressed the enzymatic activities of caspase-3 and -9 proteases. In addition, cleavage of both PARP and procaspase 3 in gliotoxin-treated HL-60 cells was inhibited by the addition of zinc compounds. We further demonstrated that expression of Fas ligand by gliotoxin was suppressed by zinc compounds. These data suggest that zinc may prevent gliotoxin-induced apoptosis via inhibition of Fas ligand expression as well as suppression of caspase family cysteine proteases-3 and -9 in HL-60 cells.

• Nutrition Research and Practice
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• v.8 no.4
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• pp.391-397
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• 2014

BACKGROUND/OBJECTIVE: Myocardial cell death due to occlusion of the coronary arteries leads to myocardial infarction, a subset of coronary heart disease (CHD). Dietary fiber is known to be associated with a reduced risk of CHD, the underlying mechanisms of which were suggested to delay the onset of occlusion by ameliorating risk factors. In this study, we tested a hypothesis that a beneficial role of dietary fiber could arise from protection of myocardial cells against ischemic injury, manifested after occlusion of the arteries. MATERIALS/METHODS: Three days after rats were fed apple pectin (AP) (with 10, 40, 100, and 400 mg/kg/day), myocardial ischemic injury was induced by 30 min-ligation of the left anterior descending coronary artery, followed by 3 hr-reperfusion. The area at risk and infarct area were evaluated using Evans blue dye and 2,3,5-triphenyltetrazolium chloride (TTC) staining, respectively. DNA nicks reflecting the extent of myocardial apoptosis were assessed by TUNEL assay. Levels of cleaved caspase-3, Bcl-2, and Bax were assessed by immunohistochemistry. RESULTS: Supplementation of AP (with 100 and 400 mg/kg/day) resulted in significantly attenuated infarct size (IS) (ratio of infarct area to area at risk) by 21.9 and 22.4%, respectively, in the AP-treated group, compared with that in the control group. This attenuation in IS showed correlation with improvement in biomarkers involved in the apoptotic cascades: reduction of apoptotic cells, inhibition of conversion of procaspase-3 to caspase-3, and increase of Bcl-2/Bax ratio, a determinant of cell fate. CONCLUSIONS: The findings indicate that supplementation of AP results in amelioration of myocardial infarction by inhibition of apoptosis. Thus, the current study suggests that intake of dietary fiber reduces the risk of CHD, not only by blocking steps leading to occlusion, but also by protecting against ischemic injury caused by occlusion of the arteries.