• 한국발생생물학회지:발생과생식
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• 제15권2호
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• pp.113-120
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• 2011

BCL-2 family 단백질들은 세포사멸 신호전달 체계에서 중추적인 역할을 수행하는 것으로 알려져 있으며, BCL2L10 단백질은 그 중 하나로 세포의 사멸과 생존을 조절하는 것으로 알려져 있다. 특이하게도 BCL2L10 단백질은 세포 또는 조직 특이적으로 서로 상반되는 친 세포사멸 또는 항 세포사멸 효과가 각각 보고되어 있다. 현재까지 난소세포에서의 BCL2L10의 발현 여부 및 기능은 알려져 있지 않다. 따라서 본 연구에서 인간 난소 과립세포주인 KGN 세포에서의 BCL2L10 단백질의 발현 여부를 확인한 결과, 상당한 양의 단백질이 발현함을 확인할 수 있었으며, 또한 세포사멸효과를 확인하기 위해서 BCL2L10 단백질을 dose-dependent하게 과발현시킨 후 세포의 생존에 미치는 영향을 분석한 결과,BCL2L10은 KGN 세포에서 과발현 시 세포사멸을 유도함을 관찰하였다. BCL2L10 단백질을 과발현 시 Caspase 9와 3를 활성화 하였으며, 면역염색법을 통해서 BCL2L10 단백질이 미토콘드리아에 위치하는 것을 확인하였다. 또한 BCL2L10단백질의 과발현에 의해 미토콘드리아에서 cytochrome c가 세포질로 분비되는 것을 확인하였다. 이상의 결과로서 본 연구는 BCL2L10의 과발현이 KGN 세포에서 세포사멸을 유도하고, 또한 미토콘드리아에 위치하여 세포질로 cytochrome c를 분비하여 Caspase 9와 3을 활성화 시키는 메커니즘으로 세포사멸을 유도함을 확인하였다. 이러한 연구결과는 BCL2L10단백질이 인간 난소과립세포의 생존과 사멸을 조절하는 인자임을 최초로 규명한 것으로서, 추후 난소에서의 BCL2L10단백질의 생리적인 기능 및 신호 조절 연구의 기반 데이터로서 그 의의가 있으며 활용될 수 있다.

• The Korean Journal of Physiology and Pharmacology
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• 제18권1호
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• pp.25-32
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• 2014

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.

• 대한약침학회지
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• 제19권1호
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• pp.51-58
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• 2016

Objectives: Oldenlandia diffusa is traditionally used to relieve the symptoms of and to treat various diseases, but its anti-cancer activity has not been well studied. In the present study, the authors investigated the anti-cancer effects of an ethanol extract of Oldenlandia diffusa (EOD) on HT-29 human adenocarcinoma cells. Methods: Cells were treated with different concentrations of an EOD, and cell death was assessed by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Analyses of the sub G1 peak, the caspase-3 and -9 activities, and the mitochondrial membrane depolarizations were conducted to confirm cell death by apoptosis. Also, intracellular reactive oxygen species (ROS) generation was determined using carboxy-H2DCFDA (5-(and-6)-carboxy-20,70-dichlorodihydrofluorescein diacetate). Results: EOD inhibited the proliferation of HT-29 cells for 24 hours by $78.6%{\pm}8.1%$ at $50{\mu}g/mL$, $74.4%{\pm}4.6%$ at $100{\mu}g/mL$, $65.9%{\pm}5.2%$ at $200{\mu}g/mL$, $51.4%{\pm}6.2%$ at $300{\mu}g/mL$, and by $41.7%{\pm}8.9%$ at $400{\mu}g/mL$, and treatment for 72 hours reduced the proliferation at the corresponding concentrations by $43.3%{\pm}8.8%$, $24.3{\pm}5.1mV$, $13.5{\pm}3.2mV$, $6.5{\pm}2.3mV$, and by $2.6{\pm}2.3mV$. EOD increased the number of cells in the sub-G1 peak in a dose-dependent manner. The mitochondrial membrane depolarization was elevated by EOD. Also, caspase activities were dose-dependently elevated in the presence of EOD, and these activities were repressed by a pan-caspase inhibitor (zVAD-fmk). The ROS generation was significantly increased by EOD and N-acetyl-L-cysteine (NAC; a ROS scavenger) remarkably abolished EOD-induced cell death. In addition, a combination of sub-optimal doses of EOD and chemotherapeutic agents noticeably suppressed the growth of HT-29 cancer cells. Conclusion: These results indicate that EOD might be an effective chemotherapeutic for the treatment of human colorectal cancer.

• 전자공학회논문지
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• 제50권10호
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• pp.181-187
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• 2013

인간의 청각은 청각 장면 분석을 통해 배경 잡음이나 여러 사람들이 동시에 말하는 상황에서도 특정 목적을 가지는 음성 신호를 청취할 수 있는 능력을 가지고 있다. 인간의 청각 능력 시스템을 잘 반영한 CASA 시스템을 이용해 음성을 분리를 할 수 있다. 그러나 CASA 세그먼트에서 음성의 위치를 잘못 결정 했을 때 CASA 시스템의 성능은 감소된다. 본 논문에서는 CASA 시스템에서 잘못된 음성 영역 위치로 인해 발생되는 성능 감소를 개선하기 위하여 청각 장면, 그리고 주기 성분과 비주기 성분의 비율(PAR)을 결합한 음성 영역 검출 알고리즘을 제안한다. 음성 영역 검출의 성능을 평가하기 위하여 백색 잡음과 자동차 잡음 환경에서 신호 대 잡음비의 변화에 따라 실험을 수행하였다. 본 논문에서는 신호 대 잡음비 15~0dB에서 기존의 알고리즘(Pitch 와 Guoning Hu)과 제안한 알고리즘을 비교한 결과, 음성 영역 검출의 정확도가 백색잡음과 자동차 잡음에서 신호 대 잡음비 15dB 에서 최대 4%, 0dB에서 최대 34% 씩 각각 향상되었다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.6115-6120
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• 2013

Introduction: Some non-small cell lung cancer (NSCLC) tumor cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -based therapy. This study was conducted to examine the effect of embelin on the sensitivity of the A549 NSCLC cell line to TRAIL receptor2 (TRAILR2) monoclonal antibodies and to investigate the potential mechanisms. Materials and Methods: A549 cells were treated with embelin, TRAILR2 mAb or a combination of both. Cell viability was measured using ATPlite assay and apoptosis rates were determined by flow cytometry with AnnexinV-FITC and propidium iodide staining, with the expression levels of proteins analyzed by Western blotting. Results: The cell survival rate of separate treatments with 100 ng/ml TRAILR2 antibody or 25 uM embelin were $81.5{\pm}1.57%$ and $61.7{\pm}2.84%$, respectively. Their combined use markedly decreased cell viability in A549 cells to $28.1{\pm}1.97%$ (P<0.05). The general caspase inhibitor Z-VAD-FMK could inhibit the embelin-enhanced sensitivity of A549 cells to TRAILR2 mAb ($75.97{\pm}3.17%$)(P<0.05). Both flow cytometry and cell morphological analysis showed that embelin was able to increase TRAIL-induced apoptosis in A549 cells. Combined treatment with embelin and TRAILR2 mAb augmented the activation of initiator caspases and effector caspase. In addition, A549 cells showed increasing levels of TRAILR2 protein and decreasing levels of Bcl-2, survivin and c-FLIP following the treatment with embelin+TRAILR2 mAb. Conclusions: Embelin could enhance TRAIL-induced apoptosis in A549 cells. The synergistic effect of the combination treatment might be due to modulation of multiple components in the TRAIL receptor-mediated apoptotic signaling pathway, including TRAILR2, XIAP, survivin, Bcl-2 and c-FLIP.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5291-5298
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• 2012

Beta-asarone is one of the main bioactive constituents in traditional Chinese medicine Acorus calamu. Previous studies have shown that it has antifungal and anthelmintic activities. However, little is known about its anticancer effects. This study aimed to determine inhibitory effects on LoVo colon cancer cell proliferation and to clarify the underlying mechanisms in vitro and in vivo. Dose-response and time-course anti-proliferation effects were examined by MTT assay. Our results demonstrated that LoVo cell viability showed dose- and time-dependence on ${\beta}$-asarone. We further assessed anti-proliferation effects as ${\beta}$-asarone-induced apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide assay usinga flow cytometer and observed characteristic nuclear fragmentation and chromatin condensation of apoptosis by microscopy. Moreover, we found the apoptosis to be induced through the mitochondrial/caspase pathway by decreasing mitochondrial membrane potential (MMP) and reducing the Bcl-2-to-Bax ratio, in addition to activating the caspase-9 and caspase-3 cascades. Additionally, the apoptosis could be inhibited by a pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). When nude mice bearing LoVo tumor xenografts were treated with ${\beta}$-asarone, tumor volumes were reduced and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays of excised tissue also demonstrated apoptotic changes. Taken together, these findings for the first time provide evidence that ${\beta}$-asarone can suppress the growth of colon cancer and the induced apoptosis is possibly mediated through mitochondria/caspase pathways.

• Biomolecules & Therapeutics
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• 제21권1호
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• pp.29-34
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• 2013

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines. TRAIL selectively induces apoptotic cell death in various tumors and cancer cells, but it has little or no toxicity in normal cells. Agonism of TRAIL receptors has been considered to be a valuable cancer-therapeutic strategy. However, more than 85% of primary tumors are resistant to TRAIL, emphasizing the importance of investigating how to overcome TRAIL resistance. In this report, we have found that nemadipine-A, a cell-permeable L-type calcium channel inhibitor, sensitizes TRAIL-resistant cancer cells to this ligand. Combination treatments using TRAIL with nemadipine-A synergistically induced both the caspase cascade and apoptotic cell death, which were blocked by a pan caspase inhibitor (zVAD) but not by autophagy or a necrosis inhibitor. We further found that nemadipine-A, either alone or in combination with TRAIL, notably reduced the expression of survivin, an inhibitor of the apoptosis protein (IAP) family of proteins. Depletion of survivin by small RNA interference (siRNA) resulted in increased cell death and caspase activation by TRAIL treatment. These results suggest that nemadipine-A potentiates TRAIL-induced apoptosis by down-regulation of survivin expression in TRAIL resistant cells. Thus, combination of TRAIL with nemadipine-A may serve a new therapeutic scheme for the treatment of TRAIL resistant cancer cells, suggesting that a detailed study of this combination would be useful.

• Biomolecules & Therapeutics
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• 제22권3호
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• pp.184-192
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• 2014

${\beta}$-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of ${\beta}$-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. ${\beta}$-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in ${\beta}$-lapachone-treated AGS cells. Treatment with ${\beta}$-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished ${\beta}$-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by ${\beta}$-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased ${\beta}$-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of ${\beta}$-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to ${\beta}$-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

• Archives of Pharmacal Research
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• 제24권2호
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• pp.126-135
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• 2001

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of $DiOC_6$/ demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (${\blacktriangle}{\Psi}_m$). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector caspase-3, Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDCA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDCA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

• 생명과학회지
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• 제17권7호통권87호
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• pp.948-955
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• 2007

생체 면역반응에 중요한 역할을 하는 호중구의 세포사멸은 자연적으로 일어나거나 여러 외부자극에 의한 신호의 전달에 의해 증가하거나 지연된다. 또한 사이토카인과 같은 분화제에 의해 세포사멸이 지연되고 항원 제시 기능을 가진 수지상 세포로 분화되기도 한다. 본 연구에서는 세포사멸 억제제와 사이토카인을 이용한 시스템에서 호중구가 수지상 세포로 분화되는가를 조사하였다. Pancaspase와 serine protease의 억제제인 zVAD-fmk와 AEBSF를 처리하였을 때 호중구의 세포사멸은 현저히 감소되며 AEBSF는 caspase-3와 serine protease활성을 모두 억제하였다. 호중구의 세포사멸을 효과적으로 억제하는 AEBSF와 함께 분화제로 널리 쓰이는 CM-CSF를 같이 처리하여 3일 동안 배양하면 수지상 세포에서 높이 발현되는 CD8O, CD83 및 MHC class ll의 세포표면 마커의 발현이 증가하였다. AEBSF와 CM-CSF를 처리한 호중구를 T-세포와 함께 배양하였을 때 SEB가 존재할 경우 T-세포가 증식되었으며 SEB가 없이도 $IFN{\gamma}$는 생성되었다. 이들 결과들로 부터 serine protease 억제제인 AEBSF를 호중구에 처리하여 세포사멸을 효과적으로 억제하는 것과 수지상 세포로의 분화를 촉진하는 사이토카인인 CM-CSF의작용을 나타내게 하는 조건과는 서로 상호적으로 연관되어 작용할 수 있다는 것을 제시하고 있다.