흑마늘 추출물에 의한 AGS 인체 위암세포의 Apoptosis 유도에 관한 연구

신동역,윤무경,최영환,권오천,김정인,최영현 대한암예방학회 2009 Journal of cancer prevention Vol.14 No.4

Garlic is generally used as a therapeutic reagent against various diseases, and many studies have indicated that garlic and its derivatives can reduce the risk of various types of human cancer. In this study, the potential of water extract of raw garlic (RG) and water extract of aged black garlic (ABG) to induce apoptosis in human gastric carcinoma AGS cells was investigated. Treatment of AGS cells to ABG, but not RG, resulted in the inhibition of cell growth and viability and the induction of apoptosis, which could be proved by MTT assay, DAPI staining and flow cytometry analysis. The increase in apoptosis that was induced by ABG was correlated with down-regulation of anti-apoptotic Bcl-2 and Bcl-xL expression, up-regulation of pro-apoptotic Bax, Fas and Fas ligand, and a decrease in the mitochondrial membrane potential. ABG treatment induced the proteolytic activation of caspases and a concomitant degradation of poly (ADP-ribose)-polymerase. Cleavage of Bid and a down-regulation of the inhibitor of apoptosis family proteins were also observed in ABG-treated AGS cells. Taken together, the data from this study indicates that ABG induces the apoptosis of AGS cells through a signaling cascade of death receptor-mediated extrinsic and mitochondria-mediated intrinsic caspase pathways.

글루타민 결핍에 의한 LnCap 인체 전립선암세포의 증식억제 현상

신동역,최영현 대한암예방학회 2011 Journal of cancer prevention Vol.16 No.1

While the amino acid L-glutamine, a most abundant amino acid in the blood, is known to play a role in the survival and death of several cell types, the underlying molecular mechanisms are still poorly defined. The purpose of this study is to determine whether glutamine deprivation induces growth inhibition and apoptosis in human prostate carcinoma LnCap cells. Our data indicated that glutamine deprivation triggered a progressive reduction in the cell viability through arresting the cell cycle in the S phase. This process involved the increase of sub-G1 phase DNA contents, however chromatin condensation as well as DNA fragmentation were not detected in cells cultured in the absence of glutamine indicating that glutamine deprivation did not induce apoptosis in LnCap cells. Supporting the data, the several important apoptosis-related gene products such as death receptor-related genes, IAP and Bcl-2 family members, and caspases, were not changed or activated. Moreover, the level poly-(ADP-ribose) polymerase, β-catenin and phospholipase C-γ1 expression, activated caspase-3 target proteins, remained unchanged in response to glutamine deprivation. This study provides biochemical evidence that glutamine deprivation suppresses cell viability without induction of apoptosis in human prostate carcinoma LnCap cells. (Cancer Prev Res 16, 41-50, 2011)

인체 혈구암세포 U937에서 해양해면동물에서 추출된 Pectenotoxin-2에 의한 Apoptosis의 유발에 관한 연구

신동역,강호성,배송자,정지형,최영현,Shin, Dong Yeok,Kang, Ho Sung,Bae, Song-Ja,Jung, Jee H.,Choi, Yung Hyun 한국해양바이오학회 2006 한국해양바이오학회지 Vol.1 No.2

본 연구에서는 U937 인체 백혈병 세포의 증식에 미치는 PTX-2의 영향을 조사한 결과, PTX-2의 처리에 따라 U937 세포는 처리 농도 및 처리 시간 의존적으로 심한 형태적 변형과 함께 증식이 억제되었다. 이러한 PTX-2 처리에 의한 U937 세포의 증식억제는 apoptosis 유발과 관련이 있었으며, 이를 DAPI staining에 의한 apoptotic body 형성, flow cytometry를 이용한 sub-G1 세포 빈도의 정량적 분석을 통하여 확인하였다. 이러한 PTX-2 처리에 의한 U937 세포의 apoptosis 유발은 Bcl-2 family에 속하는 anti-apoptotic 인자인 Bcl-$X_L$의 발현 감소 및 IAPs family에 속하는 유전자들의 선택적 발현 감소와 연관성이 있음을 알 수 있었다. 이상의 결과들은 인체 암세포에서 PTX-2의 항암작용을 이해하는데 중요한 자료가 될 것이고 나아가 PTX-2을 포함한 그와 유사한 항암제 후보물질들의 연구에 있어서 기초 자료로서 사용될 수 있을 것으로 생각된다. Natural product compounds are the source of numerous therapeutic agents. The marine environment produces natural products from a variety of structural classes exhibiting activity against numerous disease targets including anticancer agents. Among these, pectenotoxin-2 (PTX-2), which was first identified as a cytotoxic entity in marine sponges, which depolymerizes actin filaments, was found to be highly effective and more potent to activate an intrinsic pathway of apoptosis in p53-deficient tumor cells compared to those with functional p53 both in vitro and in vivo. However, the anti-proliferative mechanism of the compound at non-cytotoxic concentrations has not yet been explored. In the current study, we sought to investigate anti-proliferation and apoptosis of PTX-2 against U937 human leukemic cells and its underlying molecular mechanism. Exposure of U937 cells to PTX-2 resulted in growth inhibition and induction of apoptosis in dose- and time-dependent manner as measured by MTT assay, fluorescent microscopy and flow cytometric analysis. The anti-proliferative effect of PTX-2 was associated with a marked increase in the expression of cyclin-dependent kinase p21 (WAF1/CIP1) mRNA which was tumor suppressor p53-independent. The increase in apoptosis was connected with a time-dependent down-regulation of anti-apoptotic Bcl-XL and inhibitor of apoptosis proteins (IAPs) family such as XIAP and cIAP-2. Though additional studies are needed, these findings suggested that PTX-2-induced inhibition of U937 cells was associated with the induction of apoptotic cell death and the results provided important new insights into the possible molecular mechanisms of the anti-cancer activity of PTX-2.

번역판례 : 베를린 장벽경비판결 (BGH NStZ 1993. 129)

신동운(역) ( Sin Dong Un ),김재봉(역) ( Kim Jae Bong ) 서울대학교 법학연구소 1993 서울대학교 法學 Vol.34 No.3

자료 : 정당화와 면책 -독일의 범죄개념의 발전에 관하여-

신동운(역) ( Sin Dong Un ),( Albin Eser ) 서울대학교 법학연구소 1985 서울대학교 法學 Vol.26 No.2,3

종양억제유전자 p53 결손 인체간암세포에서 Pectenotoxin-2에 의한 Apoptosis 유도

신동역(Dong Yeok Shin),김기영(Gi Young Kim),최병태(Byung Tae Choi),강호성(Ho Sung Kang),정지형(Jee H. Jung),최영현(Yung Hyun Choi) 한국생명과학회 2007 생명과학회지 Vol.17 No.10

해양생물 유래 항암활성을 가지는 천연물의 탐색과정에서 해면동물에서 유래된 PTX-2는 p53 결손 암세포에서 세포독성 효과가 높은 것으로 보고된 바 있다. 본 연구에서는 인체 간암세포 모델을 이용하여 PTX-2의 효능을 조사한 결과는 p53 결손 Hep3B 세포에서 p53 정상 HepG2에 비하여 항암활성이 매우 높았으며, 이는 apoptosis 유발과 연관성이 있음을 확인하였다. PTX-2에 의한 Hep3B 세포의 apoptosis 유발은 DFF family의 발현 변화, pro-apoptotic Bax 및 Bcl-xS 단백질의 발현 증가, caspases (-3, -8 및 -9)의 활성화 등이 관여함을 알 수 있었다. PTX-2는 또한 Hep3B 세포에서 AKT 및 ERK1/2의 활성화를 유도하였으며, caspase-3, AKT 및 ERK1/2의 특이적 저해제에 의하여 PTX-2에 의한 세포증식 억제 효능이 유의적으로 감소되었다. 본 연구는 PTX-2에 의한 Hep3B 세포에서의 apoptosis 유도에 AKT 및 ERK1/2 신호 전달 경로가 중요한 역할을 하고 있음을 보여주는 결과이다. Through the screening of marine natural compounds that inhibit cancer cell proliferation, we previously reported that pectenotoxin-2 (PTX-2) isolated from marine sponges exhibits selective cytotoxicity against several cell lines in p53-deficient tumor cells compared to those with functional p53. However, the molecular mechanisms of its anti-proliferative action on malignant cell growth are not completely known. To further explore the mechanisms of its anti-cancer activity and to test whether the status of p53 in liver cancer cells correlates with their chemosensitivities to PTX-2, we used two well-known hepatocarcinoma cell lines, p53-deficient Hep3B and p53-wild type HepG2. We have demonstrated that PTX-2 markedly inhibits Hep3B cell growth and induces apoptosis whereas HepG2 cells are much more resistant to PTX-2 suggesting that PTX-2 seems to act by p53-independent cytotoxic mechanism. The apoptosis induced by PTX-2 in Hep3B cells was associated with the modulation of DNA fragmentation factor (DFF) family proteins, up-regulation of pro-apoptotic Bcl-2 family members such as Bax and Bcl-xS and activation of caspases (caspase-3, -8 and -9). Blockade of the caspase-3 activity by caspase-3 inhibitor, z-DEVD-fmk, prevented the PTX-2-induced growth inhibition in Hep3B cells. Moreover, treatment with PTX-2 also induced phosphorylation of AKT and extracellular-signal regulating kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MARK). Specific inhibitors of PI3K inhibitor (LY294002) and ERK1/2 inhibitor (PD98059) significantly blocks PTX-2-induced-anti-proliferative effects, whereas a JNK inhibitor (SP600125) and a p38 MAPK inhibitor (SB203580) have no significant effects demonstrating that the pro-apoptotic effect of PTX-2 mediated through activation of AKT and ERK signal pathway in Hep3B cells.

글루타민 결핍에 의한 PC3 인체 전립선 암세포의 G2/M 세포주기 억제 유발

신동역(Dong Yeok Shin),최성현(Sung Hyun Choi),박동일(Dong Il Park),최영현(Yung Hyun Choi) 한국생명과학회 2013 생명과학회지 Vol.23 No.6

본 연구에서는 생체 내 구성요소 및 에너지원으로서 중요한 역할을 하는 글루타민 결핍에 의한 인체 전립선 PC3 암세포의 증식에 관한 기전 연구를 실시하였다. 글루타민 결핍에 의한 PC3 세포의 증식억제는 세포주기 G2/M arrest와 연관성이 있었으나, apoptosis 유발 현상은 관찰되지 않았다. 글루타민 결핍에 의한 G2/M arrest는 전사 및 번역 수준에서 Cdc2, cyclin A 및 cyclin B1의 발현 억제 및 p53 비의존적인 p21(WAF1/CIP1)의 발현증가와 연관성이 있었다. 아울러 글루타민 결핍은 Chk1 및 Chk2의 인산화를 증가시켰으나, Cdc25C의 인산화는 감소시켰다. 본 연구의 결과는 글루타민 결핍에 의한 PC3 세포의 증식억제가 apoptosis 유발과는 상관없이 G2/M arrest를 유발시킨다는 첫 번째 증거이다. In this study, it was investigated the possible mechanisms by which glutamine deprivation exerts its anti-proliferative action in cultured human prostate carcinoma PC3 cells. Glutamine deprivation resulted in inhibition of growth and G2/M arrest of the cell cycle in a time-dependent manner without apoptosis induction, as determined by MTT assay, DAPI staining and flow cytometry analyses. The induction of G2/M arrest by glutamine deprivation was associated with the inhibition of expression of Cdc2, cyclin A and cyclin B1, and up-regulation of the expression of cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/CIP1) in both transcriptional and translational levels. Moreover, glutamine deprivation increased the phosphorylation of checkpoint kinase (Chk)1 and Chk2; however, the levels of Cdc25C phosphorylation were decreased in response to glutamine deprivation in a time-dependent manner. Our data provide a first biochemical evidence that glutamine deprivation suppresses cell viability through G2/M phase arrest without induction of apoptosis in PC3 cells.

글루타민 결핍에 따른 Tight Junction 및 MMPs 활성 조절을 통한 전립선 암세포의 침윤 억제 현상

신동역(Dong Yeok Shin),최영현(Yung Hyun Choi) 한국식품영양과학회 2013 한국식품영양과학회지 Vol.42 No.8

암세포를 포함한 생체 내 빠른 분열을 요구하는 세포 집단에서 세포 내 구성요소 및 에너지원으로서 글루타민의 요구량이 증대되지만, 종양세포의 글루타민 의존적 대사작용에 관한 기전은 여전히 잘 알려진 바 없다. 본 연구에서는 LnCaP 전립선 암세포의 이동성 및 침윤성에 미치는 글루타민 결핍효능을 조사하였다. 본 연구의 결과에 의하면 LnCaP 세포에서 글루타민 결핍에 의하여 세포의 이동성 및 세포의 침윤성이 현저하게 억제되었으며, 이러한 이동성 및 침윤성 억제는 TIMPs의 발현 증대에 의한 MMPs의 발현 감소 및 그들의 효소적 활성 저하와 연관성이 있었다. 또한 글루타민이 결핍된 조건에서 배양된 LnCaP 세포에서 TER의 현저한 증가가 관찰되었는데, 이는 TJs의 조절인자인 claudin family 발현의 차단에 의한 것으로 생각되어진다. 본 연구의 결과에 의하면 암세포의 증식에서 글루타민의 결핍은 TJ의 결합력 증대와 MMPs의 활성을 저하시킴으로써 암세포 전이에 가장 기본적인 과정인 암세포의 이동성과 침윤성을 억제시킬 수 있을 것으로 생각된다. Cancer cells exhibit increased demand for glutamine-derived carbons to support anabolic processes. Indeed, the spectrum of glutamine-dependent tumors and the mechanisms through which glutamine supports cancer metabolism remain areas of active investigation. In the present study, we investigated the effects of glutamine deprivation on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in human prostate carcinoma LnCap cells. Glutamine deprivation markedly inhibited cell motility and invasiveness in a time-dependent manner. The anti-invasive activity of glutamine deprivation was associated with an increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were inhibited in a time-dependent fashion by glutamine deprivation, which was correlated with a decrease in expression of their mRNA and proteins and up-regulation of tissue inhibitors of metalloproteinases (TIMPs) expression. Furthermore, glutamine deprivation repressed the levels of the claudin family members, which are major components of TJs that play a key role in the control and selectivity of paracellular transport. Moreover, the levels of E-cadherin, a type I transmembrane glycoprotein, and snail, an epithelial to mesenchymal transition regulator and zinc finger transcription factor, were markedly modulated by glutamine deprivation. Taken together, these findings suggest that TJs and MMPs are critical targets of glutamine deprivation-induced anti-invasion in human prostate carcinoma LnCap cells.

흑마늘 추출물이 인체위암세포의 tight junction 투과성 조절과 세포 침윤성 억제에 미치는 영향

Dong Yeok Shin(신동역),Moo Kyoung Yoon(윤무경),Young-Whan Choi(최영환),Oh-Cheon Gweon(권오천),Jung-In Kim(김정인),Tae Hyun Choi(최태현),Yung Hyun Choi(최영현) 한국생명과학회 2010 생명과학회지 Vol.20 No.4

본 연구에서는 흑마늘 열수 추출물(ABG)의 암세포 전이억제 효능을 검정하였다. AGS AGS 인체위암세포의 이동성과 침윤성을 억제하였으며, 이는 TER의 증가와 연관성이 있었다. 또한 ABG에 의한 AGS 위암세포의 침윤성 억제는 TIMPs 발현 증가에 의한 MMPs의 발현 및 활성 저하에 의한 것임을 알 수 있었다. 아울러 AGS 위암세포에서 과발현을 나타내는 TJ 단백질인 claudins의 발현 저하 등이 ABG의 항전이 효과에 연관되어 있음을 알 수 있었다. 이상의 결과는 흑마늘 열수 추출물이 단순한 암세포의 증식억제를 통한 암예방 및 항암활성 뿐만 아니라 암세포의 전이 또한 효과적으로 억제할 수 있음을 보여주며, 이와 연관된 보다 구체적인 분자세포생물학적 접근 및 in vivo 연구의 필요성이 요구됨을 의미한다. Garlic (Allium sativum) has been well-known as a folk remedy for a variety of ailments since ancient times, and it is well documented that enhanced garlic consumption leads to a decrease in incidences of cancer. Tight junctions (TJs) are critical structures for the maintenance of cellular polarity, acting as paracellular permeability barriers and playing an essential role in regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. Matrix metalloproteinases (MMPs) have been implicated as possible mediators of invasiveness and metastasis in some cancers. In this study, we investigated the potential effects of water extract of aged black garlic (ABG) on the correlation between tightening of TJs and anti-invasive activity in human gastric carcinoma AGS cells. The inhibitory effects of ABG on cell motility and invasiveness were found to be associated with increased tightness of TJs, which was demonstrated by an increase in transepithelial electrical resistance. Additionally, the activities of MMP-2 and -9 in AGS cells were inhibited by treatment with ABG, and this was also correlated with a decrease in the expression of their mRNA and proteins. Furthermore, RT-PCR and immunoblotting results indicated that ABG repressed the levels of the claudin proteins, major components of TJs that playa key role in the control and selectivity of paracellular transport. In conclusion, these results suggest that ABG treatment may inhibit tumor metastasis and invasion, and therefore may act as a dietary source to decrease the risk of developing cancer.

보중익기가미길경탕에 의한 A549 인체 폐암세포의 Apoptosis 유도

배종훈,신동역,박철,최영현,박동일 대한암예방학회 2011 Journal of cancer prevention Vol.16 No.4

Bojungikkigamikilkyung-tang (BJIKTP) is a traditional oriental herbal formulation consisting of crude ingredients extracted from nine herbs. This prescription has been widely used for patients suffering from a lung disease in Oriental medicine. In the present study, it was examined the biochemical mechanisms of apoptosis induced by BJIKTP in human non-small-cell lung cancer (NSCLC) cells. It was found that exposure to BJIKTP was found to result in a concentration-dependent growth inhibition by induction of apoptosis, as evidenced by chromatin chondensation, DNA fragmentation and the accumulation of sub-G1phase, in NSCLC A549 cells, but not in NCI-H460 cells. Apoptosis induction of A549 cells by BJIKTP was connected with a modulation of Bcl-2 and inhibitors of apoptosis (IAP) family members, and up-regulation of death-receptor related members. BJIKTP treatment also induced proteolytic activation of caspase-3, -8 and -9, and degradation of caspase-3 substrate proteins including poly (ADP-ribose)polymerase and phospholipase C γ-1. In addition, BJIKTP-induced apoptosis is associated with the reduction of mitochondrial membrane potential (MMP) and cleavage of Bid proteins. Taken together, the results indicate that BJIKTP may be a potential chemotherapeutic agent for use in the control of human lung cancer and that further studies are needed for the identification of the active compounds. (Cancer Prev Res 16, 358-370, 2011)