• Toxicological Research
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• v.18 no.2
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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.

• Biomedical Science Letters
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• v.26 no.2
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• pp.57-65
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• 2020

Excessive drinking of alcohol is known to be one of the main causes of various neurological diseases, such as Alzheimer's disease. Scopoletin is known to have anti-inflammatory and antioxidative properties, and to protect nerve cells. This study examined whether scopoletin inhibits the alcohol-induced apoptosis of primary hippocampal neurons, and how scopoletin regulates several factors associated with the caspase-mediated pathway. To achieve this, the cell viability and apoptosis rate of primary hippocampal neurons were measured by Cell Counting Kit-8 and flow cytometry, respectively. Apoptosis-related protein expressions (Bax, Bid, caspase-3, caspase-9, and Poly (ADP-ribose) polymerase (PARP)) were analyzed by Western blotting, and the ANOVA method was used to confirm the significance of the measured results. As a result, scopoletin inhibited the expressions of alcohol-induced apoptosis and apoptosis-related proteins in primary hippocampal neurons. These results suggest that down-regulation of Bid, Bax, and cleaved caspase-9 expression induced by scopoletin down-regulates the expression of cleaved caspase-3, inhibits the expression of cleaved PARP, and finally, inhibits mitochondrial apoptotic pathways. The study suggests that scopoletin is worth developing as a candidate for neuroprotective agent.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2637-2641
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• 2013

6,8-Dihydroxy-7-methoxy-1-methyl-azafluorenone (DMMA), a purified compound from Polyalthia cerasoides roots, is cytotoxic to various cancer cell lines. The aims of this study were to demonstrate the type of cancer cell death and the mechanism(s) involved. DMMA inhibited cell growth and induced apoptotic death in human leukemic cells (HL-60, U937, MOLT-4), human breast cancer MDA-MB231 cells and human hepatocellular carcinoma HepG2 cells in a dose dependent manner, with $IC_{50}$ values ranging between 20-55 ${\mu}M$. DMMA also decreased cell viability of human peripheral blood mononuclear cells. The morphology of cancer cells induced by the compound after staining with propidium iodide and examined under a fluorescence microscope was condensed nuclei and apoptotic bodies. Mitochondrial transmembrane potential (MTP) was decreased after 24h exposure in all five types of cancer cells. DMMA-induced caspase-3, -8, and -9 activity was strongly induced in human leukemic HL-60 and MOLT-4 cells, while in U937-, MDA-MB231- and HepG2-treated cells there was partial induction of caspase. In conclusion, DMMA-induced activation of caspase-8 and -9 resulted in execution of apoptotic cell death in human leukemic HL-60 and MOLT-4 cell lines via extrinsic and intrinsic pathways.

• Journal of Life Science
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• v.29 no.12
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• pp.1393-1400
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• 2019

In the present study, we examined the effect of mitochondrial K⁺ channel opener diazoxide on the mitochondria-dependent apoptotic signaling in endothelial cells exposed to high glucose (HG) media. Endothelial cells derived from human umbilical veins were exposed to HG media containing 30 mM glucose, and the degree of apoptotic cell death associated with activation of the mitochondria-dependent apoptotic signaling pathway was determined. Exposure to HG media was seen to enhance apoptotic cell death in a time-dependent manner. In these cells, activation of caspases 3, 8, and 9 was observed, and while caspase-3 and -9 inhibitors suppressed the HG-induced apoptotic cell death, a caspase-8 inhibitor did not. The HG-treated cells exhibited disruption of mitochondrial membrane potential, formation of permeability transition pores, and cytosolic release of cytochrome c. Subsequently, diazoxide was seen to attenuate the HG-induced apoptotic cell death; caspase-9 activation was suppressed but caspase 8 was not. Diazoxide also suppressed the depolarization of mitochondrial membrane potential, the formation of mitochondrial permeability transition pores, and the release of cytochrome c. These effects were significantly inhibited by 5-hydroxydecanoate, a selective blocker of ATP-sensitive K⁺ channels (K_ATP). The present results demonstrate that diazoxide exhibits a beneficial effect to ameliorate HG-induced endothelial cell apoptosis. Opening the K_ATP could help preserve the functional integrity of mitochondria and provide an underlying mechanism to suppress HG-triggered apoptotic signaling.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.5
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• pp.1771-1779
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• 2015

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.5
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• pp.516-523
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• 2006

The mushroom Inonotus obliquue (IO) has been traditionally used for the treatment of gastrointestinal cancer in Russia, Poland, and most of Baltic countries. To explore the possibility that IO has chemoprevention effects, we examined whether or not the aqueous extract of IO inhibits HT-29 cell growth and investigated tile mechanism for this effect. Cells were incubated in the presence of increasing concentrations of the aqueous extract of IO. The extract substantially inhibited the viable HT-29 cell number in a dose-dependent manner and inhibited 5-bromo-2'-deoxyuridine incorporation into DNA of HT-29 cells. Annexin-V staining followed by flow cytometry revealed that the extract induced apoptosis of HT-29 cells in a dose-dependent manner. Western blot analysis of total cell lysates revealed that the extract induced cleavage of caspase-8, -9 and -3 and poly (ADP-ribose) polymerase, but did not affect the protein levels of Bax and Bcl-2. In addition, the extract dose-dependently increased the activity of caspase-8, -9 and -3. We have demonstrated that the aqueous extract of IO inhibits cell proliferation and induces apoptosis in HT-29 cells, which may be mediated by its ability to activate the caspase pathway.

• Journal of Life Science
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• v.27 no.11
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• pp.1340-1348
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• 2017

Sanguinarine is a benzophenanthridine alkaloid derived from the roots of Sanguinaria canadensis L., which is used for the purpose of treating various diseases. Although studies of anticancer activities have been performed using various cancer cell lines, the phenomenon of inducing apoptosis in cancer cells by using sanguinarine requires more research. Therefore, this study investigated the anti-cancer activities and related mechanisms of sanguinarine used with Hep3B human hepatocellular carcinoma cells in terms of the regulation of apoptosis. Sanguinarine inhibited the proliferation of Hep3B cells in a concentration-dependent manner, which was associated with the induction of apoptosis. Sanguinarine also increased the activity of caspase-3, which is a typical effector caspase, and the activities of caspase-8 and caspase-9, which are key when initiating extrinsic and intrinsic apoptosis pathways, respectively. In addition, sanguinarine increased the expression of death receptor-related genes and pro-apoptotic BAX, which belongs to the Bcl-2 family, while suppressing the expression of anti-apoptotic Bcl-2. Sanguinarine promoted the truncation of Bid and enhanced the release of cytochrome c from the mitochondria to the cytoplasm due to a loss of mitochondrial membrane potential. Furthermore, the reduction of a survival rate that was induced by sanguinarine and the induction of apoptosis disappeared with the inhibition of artificial caspase activity. Therefore, the results of the study indicated that sanguinarine-induced apoptosis in Hep3B cells involves both extrinsic and intrinsic pathways; such apoptosis is a caspase-dependent phenomenon.

• Natural Product Sciences
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• v.22 no.4
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• pp.293-298
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• 2016

Plant-derived triterpenoids commonly possesses biological properties such as anti-inflammatory, antimicrobial, anti-viral and anti-cancer. Luvunga scandens is one of the plant that produced triterpenoids. The aims of the study was to analyze cell cycle profile and to determine the expression of p53 unregulated modulator of apoptosis (PUMA), caspase-8 and caspase-9 genes at mRNA level in MCF-7 cell line treated with two triterpenoids, flindissol (1) and 3-oxotirucalla-7,24-dien-21-oic-acid (2) isolated from L. scandens. The compounds were tested for cell cycle analysis using flow cytometer and mRNA expression level using quantitative RT-PCR. The number of MCF-7 cells population which distributed in Sub G1 phase after treated with compound 1 and 2 were 7.7 and 9.3% respectively. The evaluation of the expression of genes showed that both compounds exhibited high level of expression of PUMA, caspase-8 and caspase-9 as normalized to ${\beta}-actin$ via activation of those genes. In summary, the isolated compounds of L. scandens plant showed promising anticancer properties in MCF-7 cell lines.

• Korean Journal of Head & Neck Oncology
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• v.28 no.2
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• pp.107-113
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• 2012

배 경 편평상피암은 두경부의 악성종양 중 가장 흔하며, 임상적인 경과가 불량하다. 따라서 나쁜 예후를 가지는 환자군을 조기에 선별하여 더 적극적인 치료의 시행을 결정짓는 표지자의 필요성이 대두된다. 우리는 일련의 두경부 편평세포암 검체에서 몇몇 분자 표지자의 예후적 유용성을 평가하고자 하였다. 방 법 23예의 두경부 편평세포암 검체를 대상으로 VEGF, p53, Apaf-1, caspase-9의 발현과 몇몇 임상병리학적 지표들간의 연관성을 면역조직화학염색을 통해 조사하였다. 결 과 환자군은 남성이 더 많았으며 평균연령은 63.7세였다. 1기가 5예, 2기가 2예, 3기가 8예, 4기가 8예였다. 평균생존기간은 37.3개월이었다. VEGF단백 발현은 종양의 크기와 통계적으로 유의한 연관성을 보였다. 이와 더불어 VEGF단백 발현은 병기, 그리고 림프관 침습과 연관적인 경향성을 보였다. 그러나 VEGF단백 발현과 생존기간과는 관련성이 없었다. 또한, Apaf-1과 caspase-9의 단백발현은 다른 임상지표, 환자의 생존기간과는 관련이 없었다. 결 론 VEGF단백 발현은 두경부 편평세포암 환자에서 나쁜 임상경과를 예측할 수 있게 하는 표지자로서의 역할을 할 수 있다. 또한 본 연구는 두경부 편평세포암에서 별로 연구되지 않은 Apaf-1과 caspase-9의 발현상태를 밝힌 점에서 의의가 있다.

• 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.