• The Journal of Korean Medicine
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• v.29 no.4
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• pp.171-179
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• 2008

Objective: Bufonis Venenum is the traditional Korean medicine Chan Su, which is obtained from the skin and parotid venom gland of the toads. It has been used for myocardial diseases, inflammation diseases, pain relief, cancer and others. The main components of BV are cinobufotoxin, cinobufalin, bufalin and others. Of these, bufalin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti-tumor effects. There was no report of anti-tumor screening of BV on hepatic cancer and which signaling pathway can be involved. In order to examine the effect of BV on hepatic cancer and the related signaling pathway with BV-induced apoptosis, human Hep G2 cells were used. Methods: Analysis of apoptosis was confirmed by MTT assay. BV decreased cell viability in a dose and duration dependent manner. To observe which signaling molecules will be activated by BV, phosphorylation of MAPK (p38, ERK, JNK), caspase 8 and caspase 9 were examined by Western blot analysis. Results: The phosphorylation levels of p38 started to increase at 5 min after addition of 5 ${\mu}g$/ml of BV and sustained to increase until 48 hours. The phosphorylation levels of other MAPK (ERK and JNK), caspase 8 and caspase 9 increased in a time-dependent manner. These imply that BV may activate different signaling pathways, MAPK, caspase 8 and caspase 9. These results propose that BV may induce apoptosis on Hep G2 cells through the activation of MAPK, caspase 8 and caspase 9.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4095-4099
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• 2013

Oxidative stress induces apoptosis in many cellular systems including glioblastoma cells, with caspase-8 activation was regarded as a major contribution to $H_2O_2$-induced cell death. This study focused on the role of the autophagic protein p62 in $H_2O_2$-induced apoptosis in U87MG cells. Oxidative stress was applied with $H_2O_2$, and cell apoptosis and viability were measured with use of caspase inhibitors or autophagic mediators or siRNA p62, GFP-p62 and GFP-p62-UBA (del) transfection. We found that $H_2O_2$-induced U87MG cell death was correlated with caspase-8. To understand the role of p62 in MG132-induced cell death, the levels of p62/SQSTM1 or autophagy in U87MG cells were modulated with biochemical or genetic methods. The results showed that the over-expression of wild type p62/SQSTM1 significantly reduced $H_2O_2$ induced cell death, but knockdown of p62 aggravated the process. In addition, inhibition of autophagy promoted p62 and active caspase-8 increasing $H_2O_2$-induced apoptosis while induction of autophagy manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 required its C-terminus UBA domain to attenuate $H_2O_2$ cytotoxity by inhibition of caspase-8 activity. Our results indicated that p62/SQSTM1 was a potential contributor to mediate caspase-8 activation by autophagy in oxidative stress process.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.12
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• pp.4849-4852
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• 2015

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been investigated as an effective agent to treat various cancers. Cancer stem cells are resistant to TRAIL treatment, but the mechanism of TRAIL resistance remains unknown. In this study, brain cancer stem cells were isolated by CD133 magnetic sorting, and the number of CD133 positive cells detected by flow cytometry. The self-renewing capacity of brain cancer stem cells was examined by a neurosphere formation assay, and the percentage of cell death after TRAIL treatment was examined by an MTS assay. Expression of DR5, FADD, caspase 8 and BCL2 proteins was detected by western blot. The amount of CD133 positive cells was enriched to 71% after CD133 magnetic sorting. Brain cancer stem cell neurosphere formation was significantly increased after TRAIL treatment. TRAIL treatment also reduced the amount of viable cells and this decrease was inhibited by a caspase 8 inhibitor or by the pan-caspase inhibitor z-VAD (P<0.05). Brain cancer stem cells expressed lower levels caspase 8 protein and higher levels of BCL2 protein when compared with CD133 negative cells (P<0.05). Our data suggest that TRAIL resistance is related to overexpression of BCL2 and low expression of caspase 8 which limit activation of caspase 8 in brain cancer stem cells.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.5
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• pp.389-396
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• 2001

Neuroprotective properties of lithium were investigated by using in vivo NMDA excitotoxicity model. The appearance of TUNEL positive cells was prominent within 24 h of NMDA (70 mg/kg, i.p.) injection in the regions of the cortex, hippocampal formation, and thalamus of mouse cerebrum. NMDA treatment resulted in the extensive enhancement of Bax immunoreactivity in the cortical and hippocampal regions. NMDA also increased the immunoreactivity of caspase 8 in the similar regions of the mouse cerebrum. However, the increased immunoreactivity of Bax and caspase 8 were dramatically attenuated by chronic lithium pretreatment (lithium chloride, 300 mg/kg/d, i.p. for $7{\sim}10$ days). At the same time, lithium ion blocked the appearance of TUNEL positive cells, and the morphological assessment indicated an effective neuroprotection by lithium against NMDA excitotoxicity. Although the exact action mechanism of lithium is not straightforward at this time, we propose that the inhibition of Bax and caspase cascade is involved in the neuroprotective action of lithium.

• 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.

• Journal of Life Science
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• v.13 no.1
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• pp.118-127
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• 2003

$\rho$-Fluorophenylalanine (FPA), a phenylalanine analog, is able to induce apoptotic cell death of human acute leukemia Jurkat T cells. To better understand the mechanism by which FPA induces apoptotic cell death, the effect of ectopic expression of antiapoptotic proteins, Bcl-2 and Bcl-xL, on FPA-induced apoptosis was investigated by employing lurkat T cells transfected with Bcl-2 gene (JT/Bcl-2) or Bcl-xL gene (1/Bcl-xL) and Jurkat T cells transfected with vector (JT/Neo or J/Neo). When Jurkat T cells, JT/Neo or J/Neo, were exposed to FPA at concentrations ranging from 0.63 to 5.0 mM, the cell viability determined by MTT assay declined in a dose-dependent manner. In addition, apoptotic DNA fragmentation along with several apoptotic events such as caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, caspase-9 activation, caspase-3 activation, and degradation of PARP was induced. However, the FPA-induced cytotoxic effect, activation of caspase-8, and cleavage of Bid were significantly abrogated by ectopic expression of Bcl-2 or Bcl-xL. At the same time, there was marked reduction in the level of cytochrome c release from mitorhondria, caspase-9 activation, caspase-3 activation, and degradation of PARP. These results indicate that caspase-8 activation, Bid cleavage, and mitochondrial cytochrome c release with subsequent activation of the caspase cascade are negatively regulated by Bcl-2 or Bcl-xL, and are thus required for FPA-induced apoptosis in Jurkat T cells

• Journal of Integrative Natural Science
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• v.7 no.2
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• pp.87-91
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• 2014

Amyloid beta ($A{\beta}$) peptide has been implicated in the pathogenesis of Alzheimer's disease and has been reported to induce apoptotic death in cell culture. Cysteine Proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. In the present study, we examined the caspase activity and cell death in $A{\beta}$-treated SHSY5Y cells, as an attempt to elucidate the relationship between the type of caspase and $A{\beta}$-induced cell death. $A{\beta}$ at 20 ${\mu}M$ induce activation of caspase-3, 8 and 9 activity, but not the caspase-1. Caspase-3, 8 and 9 were processed by Ab treatment, consistent with the activity assay. Inhibition of the caspase activities by the selective inhibitors, however, marginally affected the cell death induced by $A{\beta}$. Taken together, the results indicate that $A{\beta}$-induced cell death may be independent of caspase activity and rather, the enzymes might be activated as a result of the cell death.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.3
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• pp.802-810
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• 2005

The tetrahydroisoquinolines included potent cytotoxic agents that showed antitumor activity,antimicrobial activity, and other biological properties. We studied the effect of CDST, 1-Chloromethyl-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-sulfonic acid amide, a newly synthesized anti-cancer agent. The cytotoxic activity of CDST in HL-60 cells was increased in a dose-dependent manner. CDST, tetrahydroisoquinolines derivative, was cytotoxic to HL-60 cells, with IC50 of $80{\mu}g/ml$. Treatment of CDST to HL-60 cells showed the fragmentation of DNA in a dose- and time dependent manner, suggesting that thesecells underwent apoptosis. Treatment of HL-60 cells with CDST was induced in a dose- and time-dependent activation of caspase-3, caspase-8 and proteolytic cleavage of poly(ADP-ribose) polymerase. In caspase activity assay, caspase-3 and -8 was activated after 12 h and 6 h posttreatment, respectively. CDST also caused the release of cytochrome c from mitochondria into the cytosol. CDST-induced cytochrome c release was mediated by caspase-8-dependent cleavage of Bid and Bax translocation. These results suggest that caspase-8 induced Bid cleavage and Bax translocation, caused mitochondrial cytochrome c release, and induce caspase-3 activationduring CDST-induced apoptosis in HL-60 cells.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.11
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• pp.4589-4592
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• 2015

Background: Missense and frame-shift mutations within the dimer forming domain of the caspase 8 gene have been identified in several cancers. However, the genetic status of this region in precancerous lesions, like oral submucous fibrosis (OSMF), and well differentiated oral squamous cell carcinomas (OSCCs) in patients from southern region of India is not known, and hence the present study was designed to address this issue. Materials and Methods: Genomic DNA isolated from biopsy tissues of thirty one oral submucous fibrosis and twenty five OSCC samples were subjected to PCR amplification with intronic primers flanking exon 7 of the caspase 8 gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the status of mutation. Results: Sequence analysis identified a frame-shift and a novel missense mutation in two out of twenty five OSCC samples. The frame-shift mutation was due to a two base pair deletion (c.1225_1226delTG), while the missense mutation was due to substitution of wild type cysteine residue with phenylalanine at codon 426 (C426F). The missense mutation, however, was found to be heterozygous as the wild type C426C codon was also present. None of the OSMF samples carried mutations. Conclusions: The identification of mutations in OSCC lesions but not OSMF suggests that dimer forming domain mutations in caspase 8 may be limited to malignant lesions. The absence of mutations in OSMF also suggests that the samples analyzed in the present study may not have acquired transforming potential. To the best of our knowledge this is the first study to have explored and identified frame-shift and novel missense mutations in OSCC tissue samples.

• 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.