• Molecules and Cells
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• v.38 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.950-956
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• 2002

The antitumor activity of the insect pathogenic fungus Paecilomyces japonica has been attributed to apoptotic cell death. However, the mechanism underlying the induced apoptosis has not yet been elucidated. In this study, we for the first time show that mitochondria-dependent caspase-3 activation were associated with the apoptotic activity of P. japonica in human acute leukemia Jurkat T cells. When Jurkat T cells were treated with the ethyl acetate extract of P japonica at concentrations ranging from $2-6{\mu}g/ml$, apoptotic cell death. accompanied by several biochemical events such as caspase-9 activation, caspase-3 activation, degradation of poly (ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation, was induced in a dose-dependent manner. In addition, the release of cytochrome c from mitochondria was detected. Under these conditions, the expression of Fas and Fas-ligand (FasL) remained unchanged. Ethyl acetate extract-induced mitochondrial cytochrome c release, caspase-3 activation, PARP cleavage, and apoptotic DNA fragmentation were suppressed by the ectopic expression of Bcl-2, which is known to block mitochondrial cytochrorme c release. Accordingly, these results demonstrate that P. japonica-induced apoptotic cell death is mediated by a cytochrome c-dependent caspase-3 activation pathway that can be interrupted by Bcl-2.

• Neonatal Medicine
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• v.17 no.1
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• pp.44-52
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• 2010

Purpose : The aim of this study was to develop a model for necrotizing enterocolitis (NEC) in the neonatal rat using endotoxin and hypoxia, a plausible insult in a neonatal intensive care and to investigate the role of apoptosis as the underlying mechanism. Methods : Newborn rats were given oral endotoxin and intermittent 8% hypoxia$\pm$caspase inhibitor. The intestinal histology was evaluated using hematoxylin-eosin staining. Apoptosis was analyzed with TUNEL staining and by measuring the caspase 3 activity in the intestinal lysates. IEC-6 cells were assessed for apoptosis and the expression of Bax, Bcl-2, Fas and FasL was measured after treatment with endotoxin and hypoxia. Results : Oral endotoxin (5 mg/kg) and exposure to 8% hypoxia of 60-min duration twice induced human NEC-like lesions in the rat intestine. Intestinal tissue revealed increased apoptosis and caspase-3 activity. After caspase inhibitor treatment, the grades of both apoptosis and NEC were significantly reduced. IEC-6 cells exhibited increased apoptosis and caspase 3 activity after endotoxin and hypoxia treatment and significantly increased Bax/Bcl- 2 ratio compared to control cells. Conclusion : This neonatal rat model of NEC which was induced by oral endotoxin and intermittent hypoxia showed increased apoptosis of intestinal epithelial cells that was mediated by caspase 3 activation. Our model has a advantage in the study of NEC because the use of much more clinically plausible insults may provide a suitable model for the investigation of its pathophysiology and therapeutic trials.

• Food Science and Biotechnology
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• v.17 no.6
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• pp.1305-1309
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• 2008

Baicalein, one of the major flavonoid in Scutellaria baicalensis, has been known for its effects on proliferation and apoptosis of many tumor cell lines. Most biological effects of baicalein are thought to be from its antioxidant and prooxidant activities. In this report, baicalein was found to induce apoptosis in HL60 human promyelocytic leukemia cell line. Baicalein treatment induced DNA fragmentation and typical morphological features of apoptosis. To elucidate the mechanism of baicalein-induced apoptosis, the activities of the members of caspase family were measured. Interestingly caspase 2, 3, and 6 were significantly activated whereas caspase 1, 8, and 9 were not activated, suggesting selective involvement of specific caspases. Further, treatment with caspase inhibitors also supports the involvement of caspase 2 in apoptosis process. Although it has been reported that baicalein can induce apoptosis through many caspase pathways, the present study indicates that caspase 2 not caspase 9 pathway may be the important step in apoptosis on HL60 cell line.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.3
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• pp.772-778
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• 2005

The chloromethyl-2-dihydroxyphosphinyl-6,7-dimethoxy-1,2,3,4-tetrahydro- isoquinoline (CDDT) is a newly synthesized derivative from 1,2,3,4-Tetra- hydroisoquinoline (THIQ). The THIQs include potent cytotoxic agents that display a range of antitumor activities, antimicrobial activity, and other biological properties. In this study, we investigated the effect of CDDT on the cytotoxicity, induction of apoptosis in human promyelocytic leukemia cells (HL-60 cells). CDDT showed a significant cytotoxic activity in HL-60 cells ($IC_{50}$ = approximately $37\;{\mu}g/ml$) at a 24 hr incubation. Treatment of HL-60 cells with CDDT displayed several features of apoptosis, including formation of DNA ladders in agarose gel electrophoresis, morphological changes of HL-60 cells with DAPI stain. Here we observed that CDDT caused activation of caspase-3, caspase-8, and caspase-9. The most efficacious time on the activation of caspases-3 was achieved at 12 hr. Further molecular analysis demonstrated that CDDT led to cleavage of poly(ADP-ribose) polymerase (PARP), increase of hypodiploid (Sub-G1) population in the flow cytometric analysis. In conclusion, these above results indicate that CDDT dramatically suppresses HL-60 cell growth by activation of caspase-3 with caspase-8, -9 activity. These data may support a pivotal mechanism for the use of CDDT in the prevention and treatment of leukemia.

• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.3
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• pp.25-40
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• 2006

Purpose : This study was undertaken to evaluate the inhibitory effects of Arisaematis rhizoma on the cell proliferation in HeLa cells. Methods : The cultured cell after treatment in the different duration in 24, 48, 72 hours with solution of 1%. 5%, 10% Arisaematis rhizoma was quantified by trypan blue exclusin method. The control group was treated with 2% FBS in the different duration in 24, 48, 72 hours. We examined DNA of activated caspase by FACS analysis, caspase-3 activity, DNA fragmentation by DNA laddering, activity of HeLa Cells by the XTT assay, activity of MAP kinase by RT-PCR analysis. Results : After 72 hours culture, the growth activities of 1%, 5%, 10% Arisaematis rhizoma-treated Hela cell were significantly reduced with control group, respectively. After 24 hours culture, the ratio of cells showing caspase activity by FACS analysis were increased in 1%, 5%, 10% Arisaematis rhizoma-treated Hela cell. It were also increased in 48 hours culture of 10% and 72 hours culture of 5%, 10% Arisaematis rhizoma-treated Hela cell. In 24, 48 and 72 hours culture, DNA fragmentations of 5%, 10% Arisaematis rhizoma-treated Hela cell were obviously observed. These results meaned that Arisaematis rhizoma induces apoptosis of HeLa cells. It was supported by increased caspase-3 activity and decreased MAP kinase activity according to time periods and concentrations of Arisaematis rhizoma solution. Conclusion : The study shows that Arisaematis rhizoma has inhibitory effect on cell proliferation and induction capacity of apoptosis of human cevical carcinoma cell line, HeLa cells, in vitro. These results suggest that Arisaematis rhizoma should be useful for treatment of human cevical carcinoma.

• Korean Journal of Head & Neck Oncology
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• v.18 no.2
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• pp.142-149
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• 2002

Objectve : Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 and 2A. In order to know the mechanism of apoptosis induced by okadaic acid, we treated FRTL-5 thyroid cells with okadaic acid and measured the changes of important proteins that are involved in apoptosis. Materials and Methods: We measured caspase 3 activity, $PLC-{\gamma}1$ degradation, the expression of XIAP, cIAP1, cIAP2, and cytochrome c release in okadaic acid-treated FRTL-5 thyroid cells. Results: Okadaic acid-induced caspase 3 activation and $PLC-{\gamma}1$ degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 80 nmol and time-dependent with a maximal effect at 24 hours after treatment. The elevated caspase 3 activity in okadaic acid treated FRTL-5 thyroid cells are correlated with down-regulation of XIAP and cIAP1, but not cIAP2. General and potent inhibitor of caspases, z-VAD-fmk. abolished okadaic acid-induced caspase 3 activity and $PLC-{\gamma}1$ degradation. The release of cytochrome c in okadaic acid-induced FRTL-5 thyroid cells was dose-dependent with a maximal effect at a concentration of 80 nmol. Conclusions: These findings suggest that mechanism of okadaic acid-induced apoptosis is associated with cytochrome c release and increase of caspase 3 activation in FRTL-5 thyroid cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.15
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• pp.6075-6080
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• 2014

Lung cancer is the leading cause of cancer-related death worldwide. Here we investigated the antitumor effect and mechanism of Zhejiang (Huzhou and Jiande) saffron against lung cancer cell lines, A549 and H446. Using high performance liquid chromatography (HPLC), the contents of crocin I and II were determined. In vitro, MTT assay and annexin-V FITC/PI staining showed cell proliferation activity and apoptosis to be changed in a dose- and time-dependent manner. The inhibition effect of Jiande saffron was the strongest. In vivo, when mice were orally administered saffron extracts at dose of 100mg/kg/d for 28 days, xenograft tumor size was reduced, and ELISA and Western blotting analysis of caspase-3, -8 and -9 exhibited stronger expression and activity than in the control. In summary, saffron from Zhejiang has significant antitumor effects in vitro and in vivo through caspase-8-caspase-9-caspase-3 mediated cell apoptosis. It thus appears to have more potential as a therapeutic agent.

• Journal of Radiation Protection and Research
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• v.32 no.4
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• pp.134-139
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• 2007

In order to find out the Radioprotective effect of algin-oligosaccharide(AOS), this study, with a mouse of which whole frame irradiated by 3 Gy radiation once, measured caspase-3 and caspase-9 amid cell signaling connected to apoptosis in order to observe cell activation. In Caspase-3 and Caspase-9 test for observing cell activation, both of Caspase-3 and Caspase-9 showed highly increased O.D. value in the irradiation control group, while the whole groups treated with algin-oligosaccharide before or after irradiation indicated lower O.D. value than the irradiation control group, especially showed big difference in 7 day's treatment group of before irradiation (P<0.001). It confirmed that Caspase generation was restrained in AOS treatment group. Consequently, this study inquired into the fact that algin-oligosaccharide with superior antioxidant activity performed radiation protection by inducing restraint of Caspase generation and confirmed that natural product with less chemical toxicity was able to be applied as radioprotector.

• Nutrition Research and Practice
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• v.8 no.2
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• pp.132-137
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• 2014

BACKGROUND/OBJECTIVES: In this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells. MATERIALS/METHODS: Cytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. RESULTS: Exposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade. CONCLUSIONS: Based on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.