• Nutrition Research and Practice
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• 제8권2호
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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.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6469-6473
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• 2013

The aim of this study was to detect the efficiency of arsenic trioxide (ATO) alone or together with bortezomib to inhibit proliferation and induce apoptosis in a multiple myeloma (MM) RPMI 8266 cells. Mechanisms of action were also investigated. RPMI 8266 cells were treated with ATO alone and in combination with bortezomib for 24 hours, and cell viability was assessed by modified MTT. Annexin V-F1TC and PI staining was used to detect the apoptosis rate and cell cycling was investigated by flow cytometry, along with expression of cell surface death receptor-4(DR4) and death receptor-5 (DR5). Western blotting was applied to detect the expression of bcl-2, caspase-3, caspase-8, and caspase-9. As a result, the ATO combined with bortezomib group showed more inhibition of RPMI 8266 cell viability than theATO group. Expression of DR4 and DR5 on the cell surfaces, and the apoptosis rate were increased after treatment by ATO alone or combined with bortezomib. The cells appeared to arrest in G2/M phase after treatment. Expression of bcl-2 was more significantly decreased in the combination group, and that of caspase-3, caspase-8 and caspase-9 was significantly increased as well. Therefore, bortezomib can enhance ATO actions to induce apoptosis in RPMI 8266 cells, with decrease in expression of bcl-2 and increase of caspase-3, caspase-8 and caspase-9 proteins.

• 대한수의학회지
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• 제45권4호
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• pp.495-500
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• 2005

The nucleoside analogue gemcitabine (2', 2-difluorideoxycytide) is potential against a wide variety of solid tumors and considered to be one of the most active drugs in the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated the signals of gemcitabine-induced apoptosis, especially in point of caspase pathway in A549. We exposed A549 cells to gemcitabine for dose/time dependent manner and the results showed that gemcitabine induced apoptotic cell death in a time/dose-dependent manner. We also treated to gemcitabine and Z-VAD-fmk as a pan-caspase inhibitor for 24 hours. Gemcitabine alone induced 35.3% cell death, and co-treatment with gemcitabine and Z-VAD-fmk induced 15.1% apoptotic cell death. Our results demonstrated that Z-VAD-fmk as a pan-caspase did not completely block the gemcitabine-induced apoptosis. Western blotting analysis showed that gemcitabine increased caspase-3, active caspase-8, p21 and p53 protein expressions in A549. Co-treatment with Z-VAD-fmk completely blocked caspase-3 and active caspase-8 protein expressions, but did not change the level of p21 and p53 protein expressions. Our data indicate that gemcitabine induced apoptosis through caspase-dependent and -independent pathways in A549.

• Molecules and Cells
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• 제24권1호
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• pp.95-104
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• 2007

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition ($IC_{50}$) of MCF-7 cells at $26.4{\pm}0.7{\mu}M$ over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with $100{\mu}M$ acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun $NH_4$-terminal kinase 1/2 (SAPK/JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.

• 생명과학회지
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• 제21권12호
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• pp.1678-1688
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• 2011

다양한 식용식물에 함유되어 있는 것으로 알려진 phenolic acids의 일종인 p-coumaric acid의 항암활성을 규명하고자, 인체 급성백혈병 T 세포주인 Jurkat T 세포에 대한 p-coumaric acid의 에폽토시스 유도기전을 조사하였다. Jurkat T 세포를 p-coumaric acid (50-$150{\mu}M$)로 처리한 결과, 세포독성, 에폽토시스-관련 DNA fragmentation, 및 pro-apoptotic multidomain Bcl-2 family member인 Bak의 활성화, ${\Delta}{\psi}m$ loss, caspase-9, -3, -7, 및 -8의 활성화, 그리고 PARP 분해 등의 여러 에폽토시스-관련 생화학적 현상들이 농도의존적으로 나타났다. 그러나 이러한 에폽토시스-관련 생화학적 현상들은 Jurkat T 세포에 anti-apoptotic Bcl-2 단백질을 과발현할 경우에는 나타나지 않았다. 또한 p-coumaric acid처리에 의해 유도되는 Jurkat T 세포의 에폽토시스에는 necrosis가 수반되지 않는 것으로 확인되었다. Jurkat T 세포를 pan-caspase inhibitor인 z-VAD-fmk를 전처리할 경우, p-coumaric acid 처리에 의해 유도되는 apoptotic sub-$G_1$ peak는 차단되어 나타나지 않았으나 ${\Delta}{\psi}m$ loss는 여전히 나타났는데, 이는 p-coumaric acid처리에 의한 에폽토시스의 유도에 caspase cascade 활성화가 필수적이며 ${\Delta}{\psi}m$ loss의 downstream 현상임을 나타낸다. 한편, FADD 및 caspase-8을 함께 발현하는 Jurkat T 세포주 A3, FADD-결손 Jurkat T 세포주 I2.1, 그리고 caspase-8-결손 Jurkat T 세포주 I9.2의 p-coumaric acid의 세포독성에 대한 감수성은 서로 유사하게 나타났는데, 이는 p-coumaric acid처리에 의한 에폽토시스의 유도가 Fas와 FasL간의 상호작용에 의해 개시되지 않음을 시사한다. p-Coumaric acid의 세포독성은 Jurkat T 세포에 비해 인체 정상 말초혈액 T 세포에서 훨씬 낮게 나타났다. 이러한 결과들은 p-coumaric acid 처리에 의해 유도되는 Jurkat T 세포의 에폽토시스가 Bak 활성화, ${\Delta}{\psi}m$ loss, caspase-9, -3, -7, 및 -8로 이루어진 caspase cascade의 활성화, 그리고 PARP 분해에 의해 유도되며, 또한 anti-apoptotic 단백질인 Bcl-2의 과발현에 의해서 음성적으로 조절됨을 나타낸다.

• Toxicological Research
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• 제20권1호
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• pp.63-69
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• 2004

Casapse-3 protease is known as a key role of apoptotic enzyme, and caspase-3 activity is a central event that occurs upstream of DNA fragmentation during apoptosis. This study demonstrates that chitosan pretreatment inhibits cadmium-induced apoptosis by attenuating the activity of caspase-3. We also analyzed the protective effect of chitosan on DNA fragmentation induced by cadmium. Cadmium toxicity was examined by DNA fragmentation and nuclear condensation with Hoechst stain. Caspase-3 activities were increased cadmium treated group for 3 hours compared with control. When chitosan (150 mg/ml) was pretreated at 30 min before cadmium treatment, cadmium cytotoxicity was suppressed in a dose-dependent manner evaluated by DNA fragmentation and caspase activity. From these results, it is suggest that the protective effect of chitosan pretreatment against cadmium-induced cytotoxicity is mediated through inhibition of caspase-3 protease activation and DNA fragmentation.

• 약학회지
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• 제43권3호
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• pp.385-390
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• 1999

Camptothecin (CPT) has been known to induce apoptosis in various cancer cell lines. To examine the intracellular apoptotic death signal initiated by CPT, we investigated the possible connection between caspase-3 activation and GSH depletion during CPT-induced apoptosis in HL-60 cells. Treatment of cells with $1{\;}{\mu}M$ CPT induced PARP cleavage accompanied by DNA fragmentation. z-VAD-fmk, a caspase-3 inhibitor, blocked the CPT-induced DNA fragmentation. Pretreatment of cells with N-acetylcysteine, a precursor of GSH biosynthesis, failed to inhibit CPT-induced PARP celavage and DNA gragmenatation. No significant changes in GSH depletion is not essential for caspase activation during CPT-induced apoptosis. We also investigated whether CPT-induced apoptosis is associated with changes of the levels of Bax and Bcl-2, two proteins involved in the control of apoptosis. Bcl-2 levels exhibited a late decrease compared with the kinetics of DNA fragmentation, whereas Bax levels increased more rapidly after CPT treatment. These results suggest that Bax plays more important role than Bcl-2 in inducing DNA fragmentation and may function upsteam of proteolytic activation of caspase-3 pathway in CPT-induced apoptosis.

• 한국식품영양과학회지
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• 제39권8호
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• pp.1119-1125
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• 2010

본 연구에서는 resveratrol을 전립선 암 치료제로의 활용 가능성을 조사하기 위하여 primary 인체 전립선 암세포에 대한 resveratrol의 성장억제 효과 및 그 기전에 대하여 조사 하였다. Resveratrol은 RC-58T/h/SA#4 세포에서 농도 및 시간에 의존적으로 세포의 증식을 억제하였으며, $IC_{50}$ 값은 암세포인 RC-58T/h/SA#4, LNCaP, PC-3에서는 각각 245, 320, $340\;{\mu}M$, 전립선 정상세포인 RWPE-1에서는 $982\;{\mu}M$로 나타나 정상세포에서보다는 암세포에서 그 독성이 크게 나타났다. 또한 resveratrol에 의해 유도된 세포 사멸은 핵 응축, sub-G1 함량 증가 및 DNA 분절 현상이 나타나 apoptosis를 유도함을 알 수 있었다. Resveatrol은 caspase-8, -9 및 effector casapse-3 활성을 농도 의존적으로 증가시켰으며, caspase 저해제인 z-VAD-fmk로 caspase의 처리 시 resveratrol에 의한 apoptosis 유도 현상이 유의적으로 감소되어 resveratrol에 의한 RC-58T/h/SA#4 세포의 apoptosis 유도에 caspase가 중요한 역할을 하고 있음을 확인하였다. Resveratrol에 의해 anti-apoptotic 인자인 Bcl-2 및 Bid 단백질의 발현은 감소하였으나, pro-apoptotic 인자인 Bax 단백질 발현은 변화가 없었다. 따라서 본 연구는 resveratrol이 RC-58T/h/SA#4세포에서 caspase 의존형 미토콘드리아 경로에 의해 유도되며, resveratrol은 전립선암 치료제로서 사용 가능성을 시사한다.

• BMB Reports
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• 제53권11호
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• pp.588-593
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• 2020

The accumulation of triglycerides (TGs) in macrophages induces cell death, a risk factor in the pathogenesis of atherosclerosis. We had previously reported that TG-induced macrophage death is triggered by caspase-1 and -2, therefore we investigated the mechanism underlying this phenomenon. We found that potassium efflux is increased in TG-treated THP-1 macrophages and that the inhibition of potassium efflux blocks TG-induced cell death as well as caspase-1 and -2 activation. Furthermore, reducing ATP concentration (known to induce potassium efflux), restored cell viability and caspase-1 and -2 activity. The activation of pannexin-1 (a channel that releases ATP), was increased after TG treatment in THP-1 macrophages. Inhibition of pannexin-1 activity using its inhibitor, probenecid, recovered cell viability and blocked the activation of caspase-1 and -2 in TG-treated macrophages. These results suggest that TG-induced THP-1 macrophage cell death is induced via pannexin-1 activation, which increases extracellular ATP, leading to an increase in potassium efflux.

• 한국자원식물학회지
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• 제33권6호
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• pp.659-665
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• 2020

Adenophorae Radix (AR) has been used as a traditional medicine for various diseases. However, the regulatory mechanisms of AR in allergic inflammation are not yet understood. The present study was conducted to investigate the effect and mechanisms of AR on the mast cell-mediated allergic response. To determine the pharmacological mechanisms of AR in allergic inflammation, we evaluated the effects of AR on the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and IL-8 as well as the activation of nuclear factor-κB (NF-κB) and caspase-1 in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cells (HMC-1). Our results demonstrated that AR effectively attenuated the PMACI-induced production of TNF-α, IL-6, IL-1β and IL-8 in stimulated HMC-1. Additionally, we showed that the inhibitory effect of AR on inflammatory cytokines in PMACI-stimulated HMC-1 cells involved the suppression of the activation NF-kB/caspase-1 in PMACI-stimulated HMC-1. Collectively, these findings provide experimental evidence that AR may be a useful candidate for the treatment of allergic inflammation.