• 대한임상검사과학회지
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• 제51권1호
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• pp.71-77
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• 2019

Menadione은 종양 억제 물질로 알려진 바 있다. 현재 많은 연구에서 다양한 암세포주에 대하여 Menadione의 잠재적인 항암물질로서의 가능성이 보고되었다. 본 연구에서는 Menadione의 항암효과와 세포사멸작용에 연관된 분자신호를 위암세포주에서 확인하였다. Menadione 처리는 위암세포인 MKN45의 세포생존능을 감소시켰다. 감소된 세포생존능은 Western blotting을 통해 caspase-3 과 caspase-7의 활성화와 PARP가 cleavage 된 것을 확인함으로써 세포사멸작용이 유도되었다는 것을 확인했다. 위세포사멸단백질들의 저해제로 작용하는 survivin의 발현을 menadione이 억제한다는 것을 확인함으로써, 세포사멸과정에 포함된 상위조절인자를 확인했다. 우리는 survivin 발현을 조절하는 전사인자로 알려진 ${\beta}$-catenin 또한 menadione에 의해 하향 조절된다는 것을 확인했다. 이전 연구에서 우리는 menadione이 세포사멸유도를 저해는 XIAP의 발현을 억제한다는 것을 확인했으며, menadione이 AGS세포에서 G2/M 세포주기 정체를 유도한다는 것을 확인하였다. 우리는 또 다른 위암세포주인 MKM45 세포에서 menadione의 이전과 다른 항암 기전을 밝혀냈다. 비록 더 자세한 연구가 필요하겠지만, 이 연구를 통해 증명된 억제기전은 menadione에 의한 항암효과를 이해하는 데 도움이 될 것으로 사료된다.

• Tuberculosis and Respiratory Diseases
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• 제65권6호
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• pp.476-486
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• 2008

연구배경: 정상세포는 보호되고 종양세포에 독성을 보인다고 알려진 TNF유전자족으로 새로이 확인된 TRAIL이 폐암세포에서 보이는 아포프토시스 효과를 확인하고, 아포프토시스로부터 세포를 보호하는 전사인자 $NF-{\kappa}B$가 TRAIL에 의하여 활성화 되는 정도를 평가하여 MG132의 $NF-{\kappa}B$활성억제가 TRAIL 유도성 아포프토시스를 감작시키는지를 확인하기 위하여 본 연구를 시행하였다. 방법: A549(wt p53) 및 NCI-H1299(null p53) 폐암세포주를 사용하였다. 세포독성 검사는 MTT assay를 이용하였고 아포프토시스는 Annexin V assay와 FACS 분석을 이용하였다. $NF-{\kappa}B$ 전사활성은 luciferase reporter gene assay를 이용하였고 $I{\kappa}B{\alpha}$ 분해는 western blot을 이용하였으며, TRAIL에 의해 활성화된 $NF-{\kappa}B$와 DNA 결합은 electromobility shift assay와 anti-p65 antibody를 이용한 supershift assay로 확인하였다. 결과: 1) TRAIL 100 ng/ml 농도에서 wild-type p53인 A549 폐암세포는 34.4%, p53 null인 NCI-H1299 폐암세포는 26.4%의 세포사를 관찰하였다. 2) Luciferase reporter gene assay로서 TRAIL에 의한 $NF-{\kappa}B$의 활성이 A549 $IgG{\kappa}B-luc$세포에서 2.45배 증가하고 NCI-H1299 $IgG{\kappa}B-luc$세포에서는 1.47배 증가함을 관찰하여 TRAIL에 의하여 $NF-{\kappa}B$가 활성화됨을 확인하였다. 3) MG132의 전처치로 TRAIL에 의한 $NF-{\kappa}B$의 활성이 A549 세포와 NCI-H1299 세포에서 각각 기저수준의 0.24, 0.21배로 강력히 억제되었다. 4) TRAIL단독으로 30% 전후의 세포독성이 MG132 전처치 후 TRAIL을 투여하면 두 세포주 모두에서 80% 이상의 세포독성이 관찰되어 MG132가 TRAIL유도성 아포프토시스에 감작효과가 있음을 확인하였다. 결론: 이상의 결과로 TRAIL에 상대적인 내성을 보이는 폐암세포주에서 MG132가 $NF-{\kappa}B$ 활성억제로서 TRAIL유도성 아포프토시스를 강화시키는 효과가 있음을 확인할 수 있었다. 따라서 본 연구는 향후 폐암치료에 있어서 TRAIL유도성 아포프토시스가 이용될 수 있는 가능성을 확인한 기초자료가 된다고 생각된다.

• Tuberculosis and Respiratory Diseases
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• 제55권5호
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• pp.488-498
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• 2003

연구배경 : 세포에 방사선을 조사하면 세포사멸과 함께 AP-l, NF-${\kappa}B$와 같은 여러 전사인자가 활성화 되는 것으로 알려져 있다. 이중 염증과 면역반응의 중요한 전사인자인 NF-${\kappa}B$는 항아포프토시스의 기능이 있으며 NF-${\kappa}B$ 활성화를 차단함으로써 TNF-${\alpha}$나 daunorubicine 등에 의 한 항암효과를 상승시킬 수 있음이 밝혀져 있다. NF-${\kappa}B$의 항아포프토시스 기전은 NF-${\kappa}B$ 의존성 단백질인 cIAPI, 2의 전사발현 유도에 의한 것으로 cIAPl, 2는 caspase 3, 7, pro-caspase-9의 활성을 차단함으로써 아포프토시스를 억제하는 것으로 알려져 있다. 이에 저자들은 방사선 유도성 세포사멸에 비교적 내성을 보이는 A549 세포주에서 방사선에 의한 NF-${\kappa}B$ 활성화와 그에 따른 cIAP 발현유도를 조사하고 NF-${\kappa}B$ 활성화를 차단하여 방사선 유도성 세포사멸의 감작효과를 확인하기 위하여 본 연구를 시행하였다. 방 법 : 세포주는 A549 폐암세포주를 이용하였고, 방사선 조사는 Varian사의 Clinac 1800C 선형가속기를 이용하였으며 조사량은 10GY를 사용하였다. 세포독성 검사는 MTT Assay를 이용하였고, NF-${\kappa}B$ 활성화 검사는 luciferase reporter gene assay, electromobility shift assay, $I{\kappa}B-{\alpha}$ degradation에 대한 westem blot을 이용하였다. NF-${\kappa}B$활성을 차단하기 위하여 proteosome inhibitor인 MGI32와 $I{\kappa}B{\alpha}$-superrepressor plasmid를 transfection한 안정적 세포주 A549-$I{\kappa}B{\alpha}$-superrepressor를 이용하였다. cIAP의 발현은 RT-PCR을 이용하였고, cIAP2 promoter 활성은 NF-${\kappa}B$ site를 포함한 cIAP2 유전자 5' f1anking region(1.4kb)을 pGL2-Basic luciferase vector에 cloning 한 construct를 사용하여 transfection 후 luciferase assay를 시행하였다. 결 과 : A549 cell에서 10Gy 방사선 조사에 의한 세포독성은 24hr, 48hr에 각각 $10.82{\pm}.3%$, $17.7{\pm}6.4%$로 비교적 내성이 있음을 확인하였다. 방사선에 의한 NF-${\kappa}B$의 활성은 $I{\kappa}B-{\alpha}$ 분해에 대한 western blot과 EMSA로 확인하였으며 luciferase assay에서도 약 1.6 배 정도의 NF-${\kappa}B$ 활성화가 있었다. NF-${\kappa}B$활성을 차단하기 위해 사용한 MG132는 방사선 유도성 세포사멸에 영향을 주지 않았으며 A549-$I{\kappa}B{\alpha}$-superrepressor 세포주에서도 세포사멸의 감작효과는 없었다. 또한 RT-PCR 결과 방사선에 의한 cIAP1,2 mRNA 발현유도는 관찰되지 않았고 cIAP2 promoter luciferase assay에서도 cIAP2 전사활성 유도는 없었다. 결 론 : A549 폐암세포주에서 방사선에 의해 NF-${\kappa}B$의 활성화는 확인하였으나 활성화 정도가 미약하였고 NF-${\kappa}B$ 의존성 항아포프토시스 유전자인 cIAP가 방사선에 의해 발현유도 되지 않았으며, NF-${\kappa}B$ 활성을 차단함에도 세포독성에 감작효과가 없었으므로 A549 폐암세포주에서 방사선 유도성 세포사멸에 내성을 보이는 기전에는 NF-${\kappa}B$의 역할이 미미하리라고 사료된다.

• 한국식품영양과학회지
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• 제45권11호
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• pp.1589-1594
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• 2016

본 연구는 후추과 식물의 주성분인 piperine이 위암세포 AGS의 항암 효과에 미치는 영향을 확인하기 위해 수행되었다. Piperine에 의한 위암세포 AGS의 생존율을 확인하기 위해 MTT assay를 수행한 결과 농도 의존적으로 암세포의 생존율이 감소하는 것을 확인하였다. 이러한 암세포의 생존율 억제가 apoptosis에 의한 효과인지를 확인하기 위해 DAPI staining을 실시한 결과 piperine을 처치한 군에서 apoptotic body와 염색질 응축이 증가하는 것을 확인하였다. MTT assay와 DAPI staining의 결과를 바탕으로 piperine이 위암세포 AGS에서 apoptosis와 관련한 단백질 발현 양상에 미치는 영향을 확인하기 위해 western blotting을 실시하였다. 그 결과 piperine에 의해 AGS 세포에서 apoptosis를 유도하는 p53, Bax의 발현은 농도 의존적으로 증가하였고, apoptosis를 억제하는 Bcl-2, XIAP의 발현은 농도 의존적으로 감소하였다. Apoptosis의 궁극적 단계인 caspase-9와 손상된 DNA를 복구하는 PARP의 분절은 증가하였으며, apoptosis를 방해하는 인자인 Akt의 인산화는 농도 의존적으로 감소하였다. Akt 억제제인 LY294002와 piperine을 병행 또는 단독으로 처치하여 western blotting을 진행한 결과 Bcl-2의 발현은 piperine을 단독으로 처치했을 때와 유사한 감소 경향을 보였지만, p-Akt의 발현은 대조군과 비교해 LY294002와 piperine을 단독으로 처치했을 때보다 병행했을 때 더욱 감소하였다. 이와 반대로 Bax와 cleaved-PARP의 발현은 강하게 증가하였다. 본 연구의 결과를 종합해볼 때 piperine이 위암세포 AGS에서 Akt 경로를 통해 apoptosis를 유도하는 것으로 여겨지며, 향후 위암 예방제나 치료제로의 개발 가능성이 있을 것으로 생각한다.

• 동의생리병리학회지
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• 제18권4호
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• pp.1055-1060
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• 2004

Apoptosis is the ability of cells to self-destruct by the activation of an intrinsic cellular suicide program when the cells are no longer needed or when they are seriously damaged. Morphologically, apoptosis is characterized by the appearance of membrane blebbing, cell shrinkage, chromatin condensation, DNA cleavage, and the fragmentation of the cell membrane-bound apoptotic bodies. Siegesbeckia glabrescens Makino (Siegesbeckiae Herba, SG) has been widely used as treatments for arthritis, and fever, as well as detoxification properties. The present studies were undertaken to evaluate if SG has an anti-apoptotic property. Cell viability was measured by XTT and tryphan blue stain. Morphological characteristic of human aortic smooth muscle cells(HASMC) were visualized with a phase-contrast microscope. SG significantly reduced HASMC, but not human umbilical vein endothelial cell(HUVEC), viability in a dose-dependent manner. Confluent untreated cells at 24hrs showed normal morphology, flat with a uniform polygonal shape. SG-treated cells (0.5㎎/㎖) at 24hrs showed apoptotic morphology. Cells became irregular with elongated lamellipodia, and exhibited condensed chromatin in nuclei with occasional endoucleation. There was an increase in the number of apoptotic cells rounding-up and being detached from the substrate. TUNEL staining of SG-treated cells showed dark-brown stains in nuclei and cytosol. Caspases are central components of the machinery responsible for apoptosis and are generally divided into two categories; the initiator caspases, which include caspases-2,-8,-9, and -10, and the effector caspases, which include caspases-3,-6, and -7. SG decreased anti-caspase-3 protein expression, which means activation of caspases-3 activity. It has been reported that there is a link between NO formation and apoptosis. NO production was accelerated by SG treatment in HASMC. L-NNA, NOS inhibitor, inhibited SG-induced apoptosis. These results, therefore, indicated that both caspases-3 and NO production are involved in apoptosis in smooth muscle cells. According to these results, SG may have a potential effect in the treatment of hypertensive atherosclerosis.

• Biomolecules & Therapeutics
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• 제26권2호
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• pp.121-129
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• 2018

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.

• 대한한방내과학회지
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• 제26권1호
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• pp.182-198
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• 2005

Object : This study was designed to research whether the protection and inhibitory effects of cardiovascular diseases in L-NAME induced rat or ECV 304 cell lines through the Cell morphological pattern, Tunel assay, LDH activity, heart rate, blood pressure and immunohistochemistric analysis by Boonsimgieum water extract Methods : Nitric oxide(NO) play an important role in normal and pathophysiological cells including as a messenger molecule, neurotransmitter, microbiocidal agent, or dilator of blood vessels and artheriosclerosis, hypertension, myocardial infarction, respectively. Endothelial cell products can modulate the magnitude of a response to a vasoconstrictor, as evinced by the greater constriction after endothelium removal or NO synthesis blockade. To investigate that Boonsimgieum in the potential contribution of the levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against NG-nitro-L-arginine methyl ester (L-NAME), human ECV 304 cells, which normally do not express eNOS, were expressed by L-NAME. L-NAME stimulated rat or cells were found to be resistant to injury and delayed death following the Boonsimgieum. Inhibition of nitric oxide synthesis abolished the protective effect against L-NAME, thrombin and collagen exposure. Interestingly, such effects have been observed during stimulation with agents such as phenylephrine and KCl on L-NAME mediate rats, were damaged by the NOS inhibitor L-NAME. Result : As the result of this study, In group, the anti-apoptosis and necrosis in the cardiovascular system have a potential capacity for prevented, protected and treating the diseases of cardiovascular system, against the necrosis of rat and ECV 304 cells with Caspase 3 and calpain expression by L-NAME is promoted. Conclusion : these results demonstrate neuroprotective and memory enhancing effects of ZIBU, suggesting its beneficial actions for the treatment of AD.

• 대한두경부종양학회지
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• 제35권2호
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• pp.19-25
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• 2019

Background/Objectives: Although anaplastic thyroid carcinoma (ATC) is rare, it is one of the deadliest forms of thyroid cancer. The fatality rate for ATC is high, and the survival rate at one year after diagnosis is <20%. The present study aimed to investigate the anti-tumor activities of paclitaxel, radiation, and tyrosine kinase inhibitor (TKI) combined therapy in anaplastic thyroid cancer cells both in vitro and in vivo and explore its effects on apoptotic cell death pathways. Materials & Methods: ATC cell line was exposed to TKI, lenvatinib in the presence or absence of paclitaxel with radiation, and cell viability was determined by MTT assay. Effects of the combined treatment on cell cycle and intracellular signaling pathways were assessed by flow cytometry and western blot analysis. The ATC cell line xenograft model was used to examine the anti-tumor activity in vivo. Results: Our data revealed that the combined administration of paclitaxel, TKI, and radiation decreased cell viability in ATC cells, and also significantly increased apoptotic cell death in these cells, as demonstrated by the cleavage of caspase-3 and DNA fragmentation. This combination therapy reduced anti-apoptotic factor levels in ATC cells, while significantly decreasing tumor volume and increasing survival in ATC xenografts. Conclusion: These results indicate that administering the combination of paclitaxel, TKI, and radiation therapy may exert significant anticancer effects in preclinical models, potentially suggesting a new clinical approach for treating patients with ATC.

• Journal of Ginseng Research
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• 제44권4호
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• pp.664-671
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• 2020

Background: Ginsenoside compound-Mc1 (Mc1) is a member of the deglycosylated ginsenosides obtained from ginseng extract. Although several ginsenosides have a cardioprotective effect, this has not been demonstrated in ginsenoside Mc1. Methods: We treated H9c2 cells with hydrogen peroxide (H₂O₂) and ginsenoside Mc1 to evaluate the antioxidant effects of Mc1. The levels of antioxidant molecules, catalase, and superoxide dismutase 2 (SOD2) were measured, and cell viability was determined using the Bcl2-associated X protein (Bax):B-cell lymphoma-extra large ratio, a cytotoxicity assay, and flow cytometry. We generated mice with high-fat diet (HFD)-induced obesity using ginsenoside Mc1 and assessed their heart tissues to evaluate the antioxidant effect and the fibrosis-reducing capability of ginsenoside Mc1. Results: Ginsenoside Mc1 significantly increased the level of phosphorylated AMP-activated protein kinase (AMPK) in the H9c2 cells. The expression levels of catalase and SOD2 increased significantly after treatment with ginsenoside Mc1, resulting in a decrease in the production of H₂O₂-mediated reactive oxygen species. Treatment with ginsenoside Mc1 also significantly reduced the H₂O₂-mediated elevation of the Bax:Bcl2 ratio and the number of DNA-damaged cells, which was significantly attenuated by treatment with an AMPK inhibitor. Consistent with the in vitro data, ginsenoside Mc1 upregulated the levels of catalase and SOD2 and decreased the Bax:B-cell lymphoma-extra large ratio and caspase-3 activity in the heart tissues of HFD-induced obese mice, resulting in reduced collagen deposition. Conclusion: Ginsenoside Mc1 decreases oxidative stress and increases cell viability in H9c2 cells and the heart tissue isolated from HFD-fed mice via an AMPK-dependent mechanism, suggesting its potential as a novel therapeutic agent for oxidative stress-related cardiac diseases.

• Journal of Ginseng Research
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• 제41권3호
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• pp.392-402
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• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.