CANCER PREVENTION RESEARCH ORIGINAL ARTICLE Bcl-2 과발현인체혈구암세포의인진쑥유래 Esculetin 에의한 Apoptosis 유발에서 Extracellular-regulated Kinase 의역할 1 동의대학교블루바이오소재개발센터, 2 자연과학대학생명응용학과, 3 생활과학대학식품영양학과, 한의과대학 4 생화학교실, 5 생리학교실및 6 대학원바이오물질제어학과, 7 부산대학교한의학전문대학원경락구조의학부 박철 1 ㆍ권현주 1,2,6 ㆍ황혜진 1,3 ㆍ이용태 1,5 ㆍ최병태 7 ㆍ김병우 1,2,6 ㆍ최영현 1,4,6 Involvement of Extracellular-Regulated Kinase in Esculetin-Induced Apoptosis of Bcl-2 Overexpressing Human Leukemia U937 Cells Cheol Park 1, Hyun Ju Kwon 1,2,6, Hye Jin Hwang 1,3, Yong Tae Lee 1,5, Byung Tae Choi 7, Byung-Woo Kim 1,2,6 and Yung Hyun Choi 1,4,6 1 Blue-Bio Industry RIC, 2 Department of Life Science and Biotechnology, College of Natural Science, 3 Department of Food and Nutrition, College of Human Ecology, Departments of 4 Biochemistry and 5 Physiology, College of Oriental Medicine, 6 Department of Biomaterial Control, Graduate School, Dongeui University, Busan 614-714, 7 Division of Meridian and Structural Medicine, School of Oriental Medicine, Pusan National University, Busan 609-735, Korea Bcl-2 is the prototypic anti-apoptotic protein involved in the regulation of apoptosis. Commonly, overexpression of Bcl-2 confers resistance to the apoptotic effect of chemo- and radiotherapy. Esculetin is a coumarin compound that is found in various natural plant products and induces apoptosis in several types of human cancer cells. However, the underlying mechanisms of its action are not completely understood. In the present study, we observed overexpressing Bcl-2 attenuated esculetin-induced up-regulation of death receptor 4 (DR4), down-regulation of X-linked inhibitor of apoptosis protein (XIAP) and β-catenin, and degradation of DNA fragmentation factor 45/inhibitor of caspase-activated DNase (DFF45/ICAD). However, HA14-1, a small molecule Bcl-2 antagonist, increased sensitivity to the apoptotic effect of the esculetin in Bcl-2 overexpressing U937/Bcl-2 cells that correlated with the loss of mitochondrial membrane potential (MMP). Furthermore, inactivation of extracellular-regulated kinase (ERK) by PD98059 significantly decreased esculetin-induced cell death and restored XIAP, β-catenin, phospholipase Cγ-1 (PLCγ1) and DFF45/ICAD in pretreatment with HA14-1 in Bcl-2 overexpressing U937/Bcl-2 cells. These results demonstrate an additional mechanism of regulation of cell survival mediated by Bcl-2, namely through ERK. Therefore, directed inhibition of Bcl-2 may alter diverse pathways controlling cell survival and overcome the apoptotic resistance that is the hallmark of human leukemia cells. (Cancer Prev Res 14, 40-47, 2009) Key Words: Esculetin, Apoptosis, Bcl-2, ERK 책임저자 : 최영현, 614-052, 부산시진구양정동산 45 동의대학교한의과대학생화학교실 Tel: 051-850-7413, Fax: 051-853-4037 E-mail: choiyh@deu.ac.kr 접수일 :2009 년 3 월 5 일, 게재승인일 :2009 년 3 월 16 일 Correspondence to:yung Hyun Choi Department of Biochemistry, College of Oriental Medicine, Graduate School, Dongeui University, Yangjung-dong, Busanjin-gu, Busan 614-714, Korea Tel: +82-51-850-7413, Fax: +82-51-853-4037 E-mail: choiyh@deu.ac.kr 40
박철외 6 인 :Esculetin 에의한 Apoptosis 유발에서 ERK 의역할 41 서 Apoptosis는배아의형성, 면역과신경계의발달및암발생등과같은생체내의중요한생리및병리학적인상태에서흔히관찰되는세포죽음의한기전으로중요한역할을한다. 1,2) 특히종양의자연치유나항암제에의해암세포의죽음이유발될때이러한 apoptosis가많이관찰되므로항암제의항종양기전연구에있어서많은연구자들의관심을받고있다. Apoptosis 과정은 extrinsic pathway 및 intrinsic pathway로크게구별되어지는데, extrinsic pathway는 mitochondria 비의존적인 apoptosis 과정으로서 apoptotic ligand가 Fas, TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 (TRAIL-R1, DR4), TRAIL-R2 (DR5) 등과같은 death receptor와결합함으로서유발되어진다. 3,4) 이와반대로 intrinsic pathway는 mitochondria 의존적인과정으로서여러가지외부자극에의하여 mitochondrial membrane permeability (MMP, Δψm) 의변화가유발되어 mitochondria 내에존재하는여러종류의 apoptotic molecule들이세포질로방출됨으로서시작된다. 5,6) MMP 변화를유발하는데있어서 Bcl-2 family가중요한역할을하는것으로알려져있는데, 특히 apoptosis 유발을억제하는것으로알려진 anti-apoptotic 유전자인 Bcl-2는대부분의암세포에서과발현되어있으며항암제내성의원인이된다고알려져있다. 7 9) 따라서 Bcl-2의발현을조절하는것은암치료의효율을높이는한방법으로유용하게사용될수있다. 한편 mitogen-activating protein kinases (MAPKs) 는 serine/ threonine protein kinases로서세포의성장, 분화, 증식, 사멸및스트레스반응등과같은여러가지세포반응을조절하는것으로알려져있으며, c-jun NH2-terminal kinase (JNK), p38-mapk 및 extracellular signal-regulated kinase (ERK) 로구성되어있다. 10 12) 일반적으로 JNK 및 p38-mapk는 proinflammatory cytokines, UV irradiation, heat, osmotic shock, hydrogen peroxide 및 DNA 손상등에의하여활성화되어스트레스반응, 성장억제및 apoptosis 유발에관여하는반면에, ERK의경우는 growth factors, cytokines 및 phorbol esters 등과같은 mitogenic stimuli에의하여활성화되어세포의성장및분화에관여하는것으로알려져있다. 10 14) 하지만최근연구에따르면세포가처한환경변화에따라이들 MAPKs 모두성장억제및 apoptosis 유발과세포의성장및분화에각기다르게관여할수있다고보고되어지고있다. 15 17) 론 Lipoxygenase inhibitor 로알려진바있는 esculetin (6,7-dihydroxycoumarin) 은인진쑥 (Artemisia scoparia), 비쑥 (A. capillaries), 갯질경이 (Ceratostigma willmottianum) 및레몬 (Citrus limonia) 의잎등에많이함유되어있는페놀계화합물이다. 18) 최근연구들에의하면 esculetin은 Chinese hamster lung fibroblast에서 H 2O 2 에의하여유도되는손상에대한항산화작용을가지는것으로알려진바있으며, croton oil ear test에서 anti-inflammatory effect 및 peripheral analgesic activity를가지며 3T3-L1 세포에서 adipogenesis 억제효과를가지는것으로보고된바있다. 19 21) 또한인체혈구암세포인 U937 세포에서 G1기에서세포주기억제및 apoptosis를유발하는것으로확인되었지만, 16,22) 항암제내성의가장큰원인중의하나인 Bcl-2 과발현암세포에서 esculetin의항암성에대해서는알려진바없다. 따라서본연구에서는선행연구결과에준하여 16,22) 인진쑥에서유래된 esculetin이인체혈구암세포인 U937 세포및 Bcl-2가과발현된 U937/Bcl-2 세포에 16) 미치는 apoptosis 유발정도를비교분석하였고, Bcl-2 및 MAPKs 가 esculetin에의하여유발되는 apoptosis에어떠한영향을미치는지를조사하여유의적인결과를얻었기에이를보고하는바이다. 1. 실험재료 재료및방법 Esculetin 및 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) 은 Sigma-Aldrich (St. Louis, MO, USA) 에서구입하였다. Bcl-2, FasL, death receptor (DR) 4, DR5, XIAP, β-catenin, DNA fragmentation factor 45/ inhibitor of caspase-activated DNase (DFF45/ICAD), phospholipase Cγ-1 (PLCγ1) 및 actin 항체는 Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) 및 CalBiochem (San Diego, CA, USA) 에서구입하였으며, 2차항체로사용된 peroxidase-labeled donkey anti-rabbit 및 peroxidase-labeled sheep anti-mouse immunoglobulin은 Amersham Life Science Corp. (Arlington Heights, IL, USA) 에서구입하였다. 2. 세포배양및형태관찰 인체혈구암 U937 세포는 American Type Culture Collection (ATCC, Rockville, MD, USA) 에서분양받았으며 90% 의 RPMI-1640 배지 (Gibco BRL, Grand Island, NY, USA), 10% fetal bovine serum (FBS) 에 2 mm glutamine, 100 U/ml penicillin 및 100μg/ml streptomycin (Gibco BRL) 이포함된배지를사용하여 5% CO 2, 37 C의조건하에서배양하였다. 또한 Bcl-2가과발현된 U937/Bcl-2 세포는 0.7μg/ml geneticin
42 Cancer Prevention Research Vol. 14, No. 1, 2009 (G418 sulfate, Calbiochem) 을첨가하여배양하였다. 16) Esculetin 처리에의한 U937 및 U937/Bcl-2 세포의형태변화는적정시간동안 esculetin을처리하여배양한후, 도립현미경 (inverted microscope, Carl Zeiss, Germany) 을이용하여 200배의배율로관찰하였다. 3. Flow cytometry 분석 Esculetin이처리된 U937 및 U937/Bcl-2 세포를모은다음 CycleTEST PLUS DNA REAGENT Kit (Becton Dickinson, San Jose, CA, USA) 를이용하여 4 C, 암실에서 30분동안고정및염색을하였다. 염색된세포를 35-mm mesh를이용하여단일세포로분리한후 FACSCalibur (Becton Dickinson) 를이용하여형광반응에따른 Cellular DNA content 및 histogram을 CellQuest software 및 ModiFit LT (Becton Dickinson) 프로그램을이용하여분석하였다. 4. Mitochondrial membrane potential (MMP, Δψm) 분석 Esculetin에의한 MMP 변화정도의측정은선행방법에준하여 16) 500μl의 PBS에 10μM JC-1을처리하여 20 분간 37 C에서반응시킨다음 2,000 rpm으로원심분리하여상층액을제거하고다시 500μl의차가운 PBS를첨가하여 35-mm mesh를이용하여단일세포로분리한후 FACSCalibur를이용하여분석하였다. 5. Protein extraction and western blot analysis 준비된암세포에 lysis buffer [25 mm Tris-Cl (ph 7.5), 250 mm NaCl, 5 mm EDTA, 1% NP-40, 1 mm phenymethylsulfonyl fluoride (PMSF), 5 mm dithiothreitol (DTT)] 를처리하여단백질을분리한다음 Bio-Rad 단백질정량시약 (Bio-Rad, Hercules, CA, USA) 을이용하여정량하였다. 동량의단백질을 sodium dodecyl sulphate (SDS)-polyacrylamide gel을이용하여전기영동으로분리한다음 nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA) 으로 electroblotting에의해전이시켰다. 단백질이전이된 membrane에 1차및 2차항체를반응시킨다음 Enhanced Chemiluminoesence (ECL) 용액 (Amersham Life Science Corp., Arlington Heights, IL, USA) 을처리하여암실에서 X-ray film 에감광시켜특정단백질의양을분석하였다. 결과 1. Bcl-2 과발현이 esculetin에의한 MMP 변화및형태에미치는영향인체혈구암세포인 U937 세포에서 esculetin에의한 apoptosis 유발에있어서 Bcl-2 과발현이미치는영향을 Fig. 1. Bcl-2 overexpression protects against esculetin-induced loss of MMP and morphological changes in U937 cells. U937 and U937/Bcl-2 cells were seeded at 2 10 5 cells/ml and then incubated with 30μg/ml of esculetin for 6 h. (A) The MMP was measured with the lipophilic cationic probe, JC-1. The cells were exposed esculetin, after which flow cytometric analyses were performed as described in materials and methods. Data are expressed as means of three independent experiments. (B) Morphological changes of U937 and U937/Bcl-2 cells after treatment with esculetin were visualized by an inverted microscope. Magnification, 200.
박철외 6 인 :Esculetin 에의한 Apoptosis 유발에서 ERK 의역할 43 먼저비교하였다. Fig. 1A에나타낸바와같이 U937 세포에서는 esculetin 처리에의하여 MMP의 loss가유발되는것으로나타났지만 Bcl-2가과발현된세포에서는 MMP 의 loss가현저하게억제되는것으로나타났으며, esculetin 처리에따른형태변화도 Bcl-2 과발현에의하여억제되는것으로관찰되었다. 아울러 esculetin이처리된 U937 세포의경우전반적인세포의응축과단편화현상이관찰되었으나, Bcl-2가과발현된세포 (U937/Bcl-2) 에서는이러한현상이나타나지않았다 (Fig. 1B). 따라서 Bcl-2가 esculetin에의하여유발되는 mitochondria의기능손상을억제함으로서 apoptosis 유발을막아주는것으로생각된다. 2. Bcl-2 과발현이 apoptosis 관련단백질의발현에미치는영향 U937 및 U937/Bcl-2 세포에서 esculetin이 apoptosis 관련단백질들의발현에어떠한영향을미치는조사한결과는 Fig. 2에나타난바와같다. 결과에서알수있듯이 Bcl-2, FasL 및 DR5 단백질의발현에는두세포주모두에 서큰변화가관찰되지않았지만 DR4, XIAP, β-catenin 및 DFF45/ICAD 단백질들은 esculetin 처리에의한발현변화가 Bcl-2 과발현에의하여억제되는것으로나타났다. 따라서 U937/Bcl-2 세포에서의 apoptosis 억제는이들 Fig. 2. Bcl-2 overexpression protects against esculetin-induced down-regulation or proteolytic cleavage of DR4, XIAP, β-catenin and DFF45/ICAD. After treatment with esculetin for 6 h, the cells were lysed, and cellular proteins were separated by SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control. Fig. 3. Co-treatment with esculetin and HA14-1 restores apoptosis in U937/Bcl-2 cells. (A) After co-treatment with esculetin and HA14-1 for 6 h, the cells were collected and stained with CycleTEST PLUS DNA REAGENT Kit for flow cytometry analysis. The percentage of cells with hypodiploid DNA (sub- G1 phase) content represent the fractions undergoing apoptotic DNA degradation. (B) Morphological changes of U937/Bcl-2 cells after co-treatment with esculetin and HA14-1 were visualized by an inverted microscope. Magnification, x200. (C) The cells were exposed esculetin and HA14-1, after which the levels of MMP were measured with the lipophilic cationic probe, JC-1, using flow cytometric analyses as described in Materials and methods. All data are expressed as means of three independent experiments.
44 Cancer Prevention Research Vol. 14, No. 1, 2009 단백질들의발현변화억제와관련성이있음을추정할수있었다. 3. U937/Bcl-2 세포에서 esculetin 및 HA14-1 처리에의한 apoptosis 유발 U937 세포에서 esculetin에의하여유발되었던 MMP 및 apoptosis 관련단백질들의변화가 Bcl-2의과발현에의하여억제되는것으로관찰되었기에, 이러한현상들을재확인하기위하여 Bcl-2 억제제인 HA14-1을이용하여 U937/ Bcl-2 세포에서 Bcl-2를억제하였을경우 esculetin에의한 apoptosis가다시유발되는지를확인하였다. 먼저 Fig. 3A 및 B에나타난바와같이 U937/Bcl-2 세포에서는 esculetin 에의한 sub-g1기의증가및형태변화가관찰되지않았지만 HA14-1을처리하였을경우에는 sub-g1기가현저하게증가하였으며, 세포의형태도현저하게변하는것으로나타났다. 이러한 sub-g11기의증가및형태변화에 mitochondria의연관성을확인한결과, Fig. 3C에나타난바와같이 HA14-1 처리에의하여 MMP의 loss도증가하 는것으로나타났으므로 U937/Bcl-2 세포에서 esculetin 및 HA14-1 처리에의한 apoptosis 유발에있어서 mitochondria 가중요한역할을하는것으로생각된다. 4. ERK pathway가 esculetin 및 HA14-1에의한 sub-g1 및 MMP 변화에미치는영향 U937/Bcl-2 세포에서 esculetin 및 HA14-1 처리에의하여유발되는 apoptosis에있어서 MAPKs pathway가어떠한영향을미치는지를확인한결과는 Fig. 4에나타난바와같다. 먼저 Fig. 4A에서와같이 U937/Bcl-2 세포에 esculetin을단독으로처리하였을경우에는 sub-g1기의증가가관찰되지않았지만 esculetin 및 HA14-1을같이처리하였을경우에는 sub-g1기가현저하게증가하여약 26% 정도로나타났다. 하지만 ERK 억제제인 PD98059를선처리후 esculetin 및 HA14-1을처리하였을경우에는 sub-g1기에해당하는세포가약 8.8% 로매우억제되었다. 또한 Fig. 4B에서와같이 MMP의 loss를관찰한결과도 PD9 8059의선처리에의하여현저하게억제되었음을알수 Fig. 4. Involvement of ERK pathway in esculetin plus HA14-1- induced apoptosis in U937/Bcl-2 cells. Cells were pretreated with PD98059 (100μM) or SP600125 (40μM) for 1 h before treatment with 30μg/ml of esculetin and 15 μm of HA14-1 for 6 h. (A) The cells were collected and stained with CycleTEST PLUS DNA REAG- ENT Kit for flow cytometry analysis. The percentage of cells with hypodiploid DNA (sub-g1 phase) content represent the fractions undergoing apoptotic DNA degradation. (B) The MMP was measured with the lipophilic cationic probe, JC-1. All data are expressed as means of three independent experiments.
박철외 6 인 :Esculetin 에의한 Apoptosis 유발에서 ERK 의역할 45 Fig. 5. The ERK pathway plays important roles in esculetin plus HA14-1-induced down-regulation and proteolytic cleavage of XIAP, β-catenin, PLCγ1 and DFF45/ICAD in U937/Bcl-2 cells. Cells were pretreated with PD98059 (100μM) for 1 h before treatment with 30μg/ml of esculetin and 15μM of HA14-1 for 6 h. The cells were lysed, and cellular proteins were separated by SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control. 있었다. 그러나 p38-mapk 억제제인 SP600125의선처리는 esculetin 및 HA14-1의동시처리에의한 apoptosis 유발은크게억제하지못하여, U937/Bcl-2 세포에서 ERK pathway가 esculetin 및 HA14-1에의하여유발되는 apoptosis 에중요하게관여하고있음을알수있었다. 5. ERK pathway가 esculetin 및 HA14-1에의한 apoptosis 관련단백질의발현에미치는영향 U937/Bcl-2 세포에서 esculetin 및 HA14-1 처리에의하여유발되는 apoptosis 관련단백질들의변화와 ERK pathway와의관계를조사한결과는 Fig. 5에나타난바와같다. 결과에서알수있듯이 esculetin 및 HA14-1 처리에의하여유발되었던 XIAP와 caspase-3의기질단백질들인 β-catenin, PLCγ1 및 DFF45/ICAD의발현감소및단편화현상이 PD98059 처리에의한 ERK pathway의억제에의하여원래대로회복되는것으로나타났다. 이는 Bcl-2 과발현 U937 세포에서 esculetin 및 HA14-1에의하여유도되는 apoptosis에 ERK pathway가관여하고있음을확인시켜주는결과라고할수있다. 고 본연구팀은선행연구에서 esculetin이인체혈구암세포인 U937 세포의증식을억제하였으며, 이는세포주기 찰 G1 arrest 및 apoptosis의유발과연관성이있음을제시한바있다. 16,22) 본연구에서는이를바탕으로하여대부분의인체암세포에과발현되어있으며여러가지항암제에의한암치료에있어서저항성을가지는원인중하나인 Bcl-2를과발현시켰을경우 esculetin에의하여유발되는 apoptosis가억제되는지를확인하였다. 이를위하여 U937 세포및 Bcl-2가과발현된 U937/Bcl-2 세포에각각 30μg/ml 농도의 esculetin을 6시간동안처리한다음 MMP의변화및형태변화의정도를조사한결과, U937 세포에서는 MMP의 loss가증가하면서심한형태적변형이관찰되었지만 U937/Bcl-2 세포에서는 MMP의 loss가현저하게억제되었고형태적변형도거의나타나지않는것으로관찰되었다 (Fig. 1). 그러나 Bcl-2 저해제인 HA14-1을 esculetin과동시에처리하였을경우 Bcl-2가과발현된 U937 세포에서도 apoptosis가유도되었으며, 이때 ERK pathway과관여하는것으로나타났다. Apoptosis 유발에는여러가지단백질들이관여하지만특히 DR4의경우는 TRAIL과의결합을통하여 extrinsic pathway를통한 apoptosis 유발에중요한역할을하며, IAP family 중가장강력한유전자인 XIAP의경우에는활성화된 caspase와의높은친화력을가지며 apoptosis를억제하는것으로알려져있다. 23,24) 또한 caspase가활성화되면여러가지기질단백질들을분해시킴으로서 apoptosis 를유발하게되는데대표적인기질단백질로는세포내골격의유지와다양한부착성세포의전사조절에관여하는 β-catenin과 DNA 단편화에관여하는 DFF45/ICAD 등이있다. 25 27) 따라서 U937 및 U937/Bcl-2 세포에서 esculetin이이러한 apoptosis 유발관련단백질들의발현에어떠한영향을미치는지를확인한결과, U937 세포에서는 DR4, XIAP, β-catenin 및 DFF45/ICAD의발현감소와단편화현상이관찰되었지만 U937/Bcl-2 세포에서는아무런변화가나타나지않았다 (Fig. 2). 이는 Bcl-2의과발현에의하여 esculetin에의한 U937 세포의 apoptosis 유도가억제되었음을직접적으로보여주는증거로서 esculetin에의한 apoptosis에 Bcl-2가중심적인역할을할수있음을의미하며, U937 세포에서 Bcl-2의과발현으로 esculetin에대한세포독성저항성이획득되었음을의미한다. 아울러 U937/Bcl-2 세포에서의 apoptosis 억제가 Bcl-2의과발현과직접적인연관이있는지를재확인하기위하여 HA14-1을이용하여확인하였는데, HA14-1은 Bcl-2와결합함으로서 Bcl-2의기능을억제하여 apoptosis 유발을막는것으로보고된바있는강력한 Bcl-2 억제제이다. 28,29) Fig. 3에나타난바와같이 esculetin 단독처리군에비하여 HA14-1과 esculetin을동시에처리하였을경우에는
46 Cancer Prevention Research Vol. 14, No. 1, 2009 sub-g1기세포빈도, 형태변화및 MMP loss의정도가현저하게증가되어 Bcl-2의과발현으로항암제저항성을지닐수있는암세포에서 Bcl-2 활성억제제의동시처리는항암제내성을극복할수있는새로운방법이될수있음을알수보여주었다. 한편이러한현상들이세포의성장, 분화, 증식및사멸등과같은여러가지세포반응조절에관여하는 MAPKs와어떠한관련이있는지를확인하기위하여몇가지 MAPK 특이적저해제를이용하여조사한결과, U937/Bcl-2 세포에서 HA14-1 및 esculetin 처리에의해유발되었던 apoptosis가 ERK inhibitor인 PD 98059의선처리에의하여다시억제되었으며 MMP loss 의정도도현저하게감소하는것으로나타났다 (Fig. 4). 또한 Bcl-2의과발현에의하여변화가억제되었던 apoptosis 유발관련단백질들인 XIAP, β-catenin, PLCγ1 및 DFF45/ICAD의발현감소와단편화현상이다시나타남을알수있었다 (Fig. 5). 이는 Bcl-2 과발현에 esculetin의항암내성극복을위한 HA14-1과의동시처리에 ERK pathway가직접적으로관여하고있음을보여주는결과이다. 이상의결과들을살펴볼때 esculetin은 U937 세포에서 DR4의발현증가, 미토콘드리아막전위의변화및 caspase 기질단백질들의분해등을통하여 apoptosis를유발하였음을알수있었다. 또한 Bcl-2 과발현에따른 esculetin 내성극복을위한 Bcl-2 저해제의혼용처리에의한 apoptosis 유발에는 ERK pathway가관여하고있음을알수있었다. 결론 Apoptosis 조절에중요한역할을하는 Bcl-2는 prototypic anti-apoptotic 단백질로서작용하며, 일반적으로 Bcl-2 유전자의과발현은화학요법및방사선조사에의한암세포의 apoptosis 유발에저항인자로작용한다. 인진쑥에서유래된 esculetin에의한인체혈구암 U937 세포의 apoptosis 유발은 DR4의발현증가, XIAP와 β-catenin 발현의감소및 DFF45/ICAD 단백질의단편화를수반하였으나, Bcl-2의과발현은 esculetin에의한 apoptosis 유발효과를차단시켰다. 그러나 Bcl-2 antagonist인 HA14-1을 Bcl-2가과발현된 U937/Bcl-2 세포에 esculetin과동시에처리했을경우 apoptosis가다시유발되었으며, 이는 MMP의감소와연관성이있었다. 또한 ERK 저해제처리는 U937/ Bcl-2 세포에서 apoptosis 유발효과를더욱증가시켰다. 본연구의결과는 Bcl-2 과발현에의한생존전략에 ERK 경로가관여함을보여주는것이며, Bcl-2 발현의조절을 통하여암세포의약재저항성을극복할수있음을시사하여준다. 감사의글 이연구는지식경제부 부산광역시지원지역혁신센터사업동의대학교블루바이오소재개발및실용화지원센터 (RIC08-06-07) 및 2008년도부산테크노파크지원에의하여이루어진결과입니다. 참고문헌 1) Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239-257, 1972. 2) Evans VG. Multiple pathways to apoptosis. Cell Biol Int 17, 461-476, 1993. 3) Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2, 420-430, 2002. 4) Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, Ashkenazi A. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611-620, 2000. 5) Schultz DR, Harrington WJ Jr. Apoptosis: programmed cell death at a molecular level. Semin Arthritis Rheum 32, 345-369, 2003. 6) Zamzami N, Susin SA, Marchetti P, Hirsch T, Gómez-Monterrey I, Castedo M, Kroemer G. Mitochondrial control of nuclear apoptosis. J Exp Med 183, 1533-1544, 1996. 7) Jeong SY, Seol DW. The role of mitochondria in apoptosis. BMB Rep 41, 11-22, 2008. 8) Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590-8607, 2003. 9) Bouillet P, Huang DC, O'Reilly LA, Puthalakath H, O'Connor L, Cory S, Adams JM, Strasser A. The role of the pro-apoptotic Bcl-2 family member bim in physiological cell death. Ann N Y Acad Sci 926, 83-89, 2000. 10) Sigoillot FD, Evans DR, Guy HI. Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades. J Biol Chem 277, 15745-15751, 2002. 11) Chang L, Karin M. Mammalian MAP kinase signaling cascades. Nature 410, 37-40, 2001. 12) Geilen CC, Wieprecht M, Orfanos CE. The mitogen-activated protein kinases system (MAP kinase cascade): its role in skin signal transduction. A review. J Dermatol Sci 12, 255-262, 1996. 13) Ichijo H. From receptors to stress-activated MAP kinases. Oncogene 18, 6087-6093, 1999.
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