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1 大韓本草學會誌제 30 권제 2 호 (2015 년 3 월 ) Kor. J. Herbology 2015;30(2):25-30 ISSN (Print), ISSN (Online) 구강암세포주에서김추출물에의한세포자멸사유도 김상찬 1#, 이종록 2, 박숙자 1* 1 : 대구한의대학교한의과대학방제과학글로벌연구센터 2 : 대구한의대학교제약공학과 Porphyra tenera induces apoptosis of oral cancer cells Sang Chan Kim 1#, Jong Rok Lee 2, Sook Jahr Park 1* 1 : MRC-GHF, College of Korean Medicine, Daegu Haany University 2 : Department of Pharmaceutical Engineering, Daegu Haany University ABSTRACT Objectives : Laver (Porphyra tenera), a red algae species, is one of the most widely consumed edible seaweed in Korea. Laver contains various substances such as essential amino acid, fiber, minerals and polyphenols that benefit human health. In the present study, we prepared ethanol extracts from commercially processed product of Porphyra tenera, and evaluated the growth inhibitory effect against human oral squamous carcinoma YD-10B cells. Methods : Cell viability was measured by MTT assay. Apoptosis was confirmed by TUNEL assay and flow cytometry with the green fluorescent dye FITC annexin V entering apoptotic cells and the red fluorescent dye PI not entering. The expression of the relevant proteins was detected using Western blot. Results : Ethanol extracts of Porphyra tenera (PTE, μg / ml ) caused a significant decrease of cell viability in a dose dependant manner. The cell death occurred as a result of apoptotic process as determined by TUNEL assay and flow cytometric analysis. In line with this observation, decrease in procaspase proteins and increase in cytosolic cytochrome c were observed in cells treated with PTE. In addition, exposure to PTE decreased the expression levels of Bcl-2, and induced PARP cleavage and AIF translocation from mitochondria to nucleus. Conclusions : In conclusion, PTE exerts anti-cancer effects by inducing apoptosis via caspase activation and AIF nuclear translocation in YD-10B cells. These results provide evidence for the possible therapeutic effect of Porphyra tenera in oral cancer cells. Key words : Porphyra tenera, apoptosis, oral cancer cells 서론 1) 식용으로널리사용되고있는김은우리나라연안에서다량으로서식하는보라털과 (Bangiaceae) 의홍조류로서해태 ( 海苔 ), 감태 ( 甘苔 ) 라고도하며한의학에서는치질, 토사곽란 ( 吐瀉霍亂 ) 을치료하는약재로도사용되어왔다 1,2). 다른해조류와달리김은트레오닌 (threonine), 트립토판 (tryptophan), 메티오닌 (methionine) 등필수아미노산과단백질이많이들 어있으며, 칼슘, 요오드를비롯한무기질과비타민도풍부하게함유되어있어양질의영양소를가진건강식품으로알려져있다 3,4). 또한칼로리가거의없고식이섬유소가풍부하여다이어트에효과적이며, 당뇨병과비만을비롯한만성질환의발병을낮춰주는것으로보고되었다 5,6). 김에대한연구로는아질산염소거효과 7), N-Nitrosodimethylamine 생성억제효과 8), collagen 합성및 pyridinoline 의생성을증가시키는효과 9), 항균효과 10) 등 *Corresponding author : Sook Jahr Park. MRC-GHF, College of Korean Medicine, Daegu Haany University Tel : haany@dhu.ac.kr #First author : Sang Chan Kim. MRC-GHF, College of Korean Medicine, Daegu Haany University Tel : sckim@dhu.ac.kr Received:24 February 2015 Revised:12 March 2015 Accepted:16 March 2015

2 26 大韓本草學會誌 Vol. 30 No. 2, 2015 이수행되었으며, 동맥경화와고혈압을일으키는원인으로알려진콜레스테롤을감소시키는효과도보고되었다 11). 그리고김에는 polyphenols, carotenoids, tocopherols과같은성분이다량함유되어있으며, 김의항산화작용에이러한성분들이기여하는것으로여겨지고있다 12). 김에서추출 분리한 porphyran 은고지혈증쥐에서무기질조절효과가보고되어졌다 13). 과학과의학기술의발전으로암 ( 癌 ) 은완치율이점차높아지고있지만, 여전히한국인의대표적인사망원인중하나이다. 2014년발표된중앙암등록본부자료에따르면 2011년우리나라에서발생한전체암중에서구강암은약 0.2% 의비율을차지하고있는희귀암으로, 그예후가좋지못하며 5년동안생존율도 50% 에불과하다고알려져있다 14). 치료를위해외과수술, 방사선치료, 화학요법등이사용되고있으나치료과정에서나타날수있는부작용과낮은완치율이문제가되고있다. 기존항암제가암세포뿐만아니라정상세포의기능도억제하기때문에생길수있는많은부작용을극복하기위해다양한표적치료항암제개발이시도되고있다. 표적치료제개발이란암세포에서특이적으로확인되는분자를표적으로하여효과를나타내는물질들로부터치료제를개발하는것으로세포주기조절 (cell cycle regulation), 혈관신생 (angiogenesis), 세포자멸사 (apoptosis) 등과같은암발생과관련된신호전달체계에대해이해가우선되어야한다. 외부로부터의손상에의해세포가사멸하는괴사 (necrosis) 와달리세포자멸사 (apoptosis) 는일반적이고예정된자기파괴의과정으로조절에이상이생길경우양성종양에서악성종양으로의형질전환에관여하는것으로알려져있다 15). 암세포에서는흔히세포자멸사과정이비정상적으로억제되어있어, 세포가계획대로사멸되지못하고과도한증식이촉진되어암의진행을가속화하게된다 15,16). 이에, 암치료를위한전략으로암세포의세포자멸사를일으킬수있는물질들에대한연구가많이수행되어지고있다. 하지만구강암에대한연구성과는미비한상황으로구강암의예방및치료에효과를나타낼수있는물질들에대한더많은연구가요구되고있다. 본연구에서는식생활속에서쉽게접할수있는김을활용하여그추출물이인체구강암세포주의세포자멸사와관련분자들에대해어떠한영향을미치는지평가하고자하였다. 재료및방법 1. 시약 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoleum (MTT), dimethyl sulfoxide (DMSO) 는 Sigma (St. Louis, MO, USA) 에서구입하였고, DMEM, fetal bovine serum (FBS), antibiotics (penicillin-streptomycin) 는 Gibco/BRL (Eggenstein, Germany) 로부터구입하였다. 1차 antibody 인 actin, AIF, Bcl-2, procaspase-3 는 Santa Cruz Biotechnology (Bergheimer, Germany) 에서구입하였고, cytochrome c, cleaved PARP, procaspase-9 은 Cell Signalling Technology (Irvine, CA, USA) 에서구입하였다. 2차 antibody 는 1차 antibody 의 host에따라 anti-rabbit IgG-HRP (Actin, procaspase-3, procaspase-9, cleaved PARP) 와 anti-mouse IgG-HRP (AIF, Bcl-2, cytochrome c) 를 Cell Signalling Technology에서구입하여사용하였다. 2. 추출물의제조김 ( 海苔 ; Porphyra tenera) 은대구시내칠성시장에서구입하여관능평가후, 수도수로세척하여이물질을제거한다음음건하여사용하였다. 에탄올추출물은건조중량 (405 g) 의 10배에해당되는에탄올에침지하여 72시간동안추출한후에회전감압농축기 (EYELA, Tokyo, Japan) 로감압농축하고 freezer dryer(labconco, MO, USA) 로동결건조하여최종추출물시료 (0.933 g) 를획득하였으며, 실험에사용할때까지 -20 에서보관하였다. 김에탄올추출물 (Porphyra tenera extracts, PTE) 에대한최종수율은 0.23% 였으며, DMEM 배지에녹여실험에사용하였다. 3. 세포배양및시료처치 Human유래 oral squamous cell carcinoma cell line인 YD-10B cell은한국세포주은행 (Korea Cell Line Bank, Seoul, Korea) 으로부터구입하였으며, 10% fetal bovine serum, 100 units/ml penicillin 과 100 mg/ml streptomycin 이포함된 DMEM 배지에서 37 의온도와 5% 의 CO 2 가유지되는환경에서배양하였다. 실험과정의모든 cells은 80~90% 의 confluence 에서실험하였고, 20 passages 를넘기지않은 cell만사용하였다. 세포생존율실험을수행하기위해 PTE를처리하지않고배지만주입한세포를대조군으로하였고, 실험군은 3개로분류하여각각 50, 100, 200 μg / ml의 PTE를처치하였다. 이후의실험에서는대조군 (0 μg / ml ) 과한농도의 PTE 실험군 (100 μg / ml ) 을지정하여세포자멸사에대한효과를살펴보았다. 4. MTT assay YD-10B cell 을 12 well plate 에 cells/well로분주한다음, PTE를농도별 (50, 100, 200 μg / ml ) 로처치하여세포의생존율을구하였다. 배양세포에 MTT 용액 (1 mg / ml ) 를 500 μl넣고 4시간배양하여생성된 formazan crystals 을 DMSO에녹여 Titertek Multiskan Automatic ELISA microplate reader (Model MCC/340, Huntsville, AL) 를사용하여 570 nm에서흡광도를측정하였다. 세포생존율은시료를처치하지않은대조군에대한백분율로나타내었다. [viability (%) = 100 (absorbance of treated sample) / (absorbance of untreated control)]. 5. TUNEL (TdT-mediated dutp-biotin nick end-labeling) assay 4 well chamber slide의 well당 개의농도가되게세포를배양하고 PTE를 24시간처리하였다. Cell은 PBS로 2 회 washing 한후에 4% paraformaldehyde 로고정하여 In

구강암세포주에서김추출물에의한세포자멸사유도 27 Situ cell death detection kit-pod (Roche, Mannheim, Germany) 를사용하여 kit에포함된 protocol에맞춰분석하였다. 즉, paraformaldehyde 로고정된 cell을 0.3% H 2O 2 를사용하여 blocking 시키고 0.

3 구강암세포주에서김추출물에의한세포자멸사유도 27 Situ cell death detection kit-pod (Roche, Mannheim, Germany) 를사용하여 kit에포함된 protocol에맞춰분석하였다. 즉, paraformaldehyde 로고정된 cell을 0.3% H 2O 2 를사용하여 blocking 시키고 0.1% triton X-100/0.1% sodium citrate로 permeabilisation 상태로만든후에 TdT-enzyme 이처리된용액으로 labeling 하였다. PBS로 2회 washing 한후에 Converter-peroxisase(POD) 로 37 에서 30분간처리하고 DAB로발색하여 light microscope로관찰하였다. 6. Flow cytometric analysis 붉은색형광염색약인 propidium iodide (PI) 와초록색형광염색약인 FITC annexin V (Molecular Probes, Eugene, OR) 를사용하여 apoptotic cell을분석하였다 의 cells 을 10cm 2 plastic plate에깔고 70-80% 의 confluency 를유지하였다. 먼저 cells에 24시간배지고갈을하고, 다음 24시간동안 PTE를처치하였다. 부착된 cells을 trypsin 을처치하여회수하였고부유 cells과 trypsin 처치 cells은모두 70% ethanol로 resuspension하여 -20 에서 overnight하여고정시켰다. 고정된 cell 은 개 / ml의농도로 PBS 에 suspension 시킨후, FITC annexin V와 PI를처리하여얼음위에서 20 분간반응시켜 flow cytometer (Particle Analysis System, Partec GmbH, Mnster, Germany) 로분석하였다. 10. 통계분석모든실험은 3회이상반복실시하였으며실험결과는 mean ± S.D. 로나타내었다. 유의성검정은윈도우용 SPSS 17.0을사용하여 one way analysis of varience (ANOVA), Turkey test (multiple comparison) 방식으로분석하였으며, p값이 0.05 미만일때통계적으로유의하다고판정하였다. 결과 1. PTE가 YD-10B cell의세포증식에미치는영향 PTE가구강암세포주인 YD-10B cell의세포증식에미치는영향을조사하기위하여다양한농도 (50, 100, 200 μg / ml ) 로처치하고 24시간혹은 48시간동안배양하여 MTT assay로 cell viability를측정하였다. 24시간배양후, PTE를처치하지않은대조군세포의생존율을 100% 로하였을때 PTE 50, 100, 200 μg / ml은각각 85.0%, 40.7%, 9.2% 의세포생존율을나타내었으며, 48시간배양에서 70.9%, 35.2%, 9.1% 의생존율을나타내어농도별, 시간별세포사멸및세포증식억제효과가확인되었다 (Fig. 1). 7. Total cell lysates의준비배양세포는배지를제거하고 PBS로세척한후에 protease inhibitor cocktail 이포함된 RIPA 용액으로 lysis하였다. Lysis된세포는 15,000 g에서 15 분간원심분리하여찌꺼기를제거하고상등액을취하여 total cell lysate 로사용하였다. 8. Cell fractionation PARP cleavage 확인을위한세포핵분획물은시판되는 nuclear extraction kit (Thermo scientific, Rockford, IL, USA) 를사용하여제조사의매뉴얼에따라배양세포로부터추출하였다. 미토콘드리아의 AIF protein 과세포질의 cytochrome c protein 의발현을조사하기위한세포분획은 Mitochondria Isolation Kit (Thermo scientific, Rockford, IL, USA) 를이용하여준비하였다. 9. Immunoblot analysis 준비된단백질시료의정량은 BCA protein assay kit (Thermo scientific, Rockford, IL, USA) 를이용하였다. 동일한양의단백질을 SDS-PAGE로분리하여 nitrocellulose membrane 으로옮기고 1차 antibody 로 2시간반응시켰다. TBST 로 3회 washing 하고 2차 antibody 로 1시간더반응시킨후에각각의 protein band 는 ECL western blotting detection reagents (Amersham) 를사용하여발색하였다. 발색후각단백질의발현량을평가하기위하여 image analyzing system (Ultra-Violet Products Ltd., Upland, CA, USA) 을이용하여 densitometric analysis 를실시하였다. Fig. 1. Effects of PTE on the cell viability of YD-10B cells. YD-10B cells were exposed to PTE (50, 100, 200 μg / ml ) for 24 or 48 h. Cell viability was assessed by MTT assay. Data represent the mean ± S.D. with three separate experiments (#; significant as compared to control; ## p<0.01). 2. YD-10B cell 에서 PTE 에의한세포자멸사 (apoptosis) 유도효과 일반적으로생존세포수는세포증식및세포사멸의균형에의해조절되는것으로, Fig. 1에서본바와같이 PTE는 YD-10B cell의세포사멸을유의하게 (p<0.01) 유도함으로써세포증식을억제하였다. PTE에의한세포사멸이 apoptosis 에기인하는지를확인하기위해 FITC annexin V/PI 이중염색을실시하여 flow cytometry 로분석하였다. Fig. 2에서보는바와같이세포자멸사가진행된세포는 FITC annexin V에염색된상태이며 (Q2와 Q4), 특히 PI 염색여부에따라 PI(+) 는 late apoptosis(q2), PI(-) 는 early apoptosis(q4) 가진행중임을의미한다. PTE(100 μg / ml ) 를 24시간동안반응시킨결과, control 세포에비해증가된세포자멸사세포를확인할수있었다 (Fig. 2A). Control 세포는 24시간배양후에두가지염색시약에모두반응하지않는 live cell(q1) 의형

28 大韓本草學會誌 Vol. 30 No. 2, 2015 태로대부분존재하였고 apoptotic cell은 2.5% 였으나, PTE로처치된세포는 29.5% 의 apoptotic cell population을나타내어 PTE에의한 YD-10B 세포사멸에 apoptosis signaling 이유의하게 (p<0.01) 관여함을알수있었다 (Fig. 2B).

YD-10B cells were treated with 100 μg / ml of PTE for 24 h and applied to flow cytometer after stain with FITC Annexin V (FL1-green) and PI (FL3-red).

4 28 大韓本草學會誌 Vol. 30 No. 2, 2015 태로대부분존재하였고 apoptotic cell은 2.5% 였으나, PTE로처치된세포는 29.5% 의 apoptotic cell population을나타내어 PTE에의한 YD-10B 세포사멸에 apoptosis signaling 이유의하게 (p<0.01) 관여함을알수있었다 (Fig. 2B). (A) Fig. 2. Flow cytometric profile of YD-10B cells stained with FITC Annexin V and PI. YD-10B cells were treated with 100 μg / ml of PTE for 24 h and applied to flow cytometer after stain with FITC Annexin V (FL1-green) and PI (FL3-red). The population was separated into three groups: live cells (Q3, left low) showing only a low level of fluorescence, apoptotic cells (Q4, right low) showing green fluorescence and necrotic cells (Q2, right upper) showing both red and green fluorescence (A), and the % population of apoptotic cell was calculated (B). Data represent the mean ± S.D. of three separate experiments ( #, significant as compared to control; ## p <0.01). (B) 효과를좀더상세히이해하기위하여 apoptosis 신호전달과정에서주요한역할을담당하고있는단백질들의발현을조사하였다. Proenzyme 의형태로존재하며 proteolysis 를통해활성화되는 caspase 와활성화된 caspase 에의해절단되는 PARP(poly ADP ribose polymerase) 단백질은손상된세포를인지하고복구할수있는능력을소실하여 apoptosis를유도하게된다. Western blot 결과를보면, PTE는 procaspase 3/9의발현을농도의존적으로감소시켰으며절단된 (cleaved) PARP 단백질의발현은증가시켰다 (Fig. 4). 이결과는 caspase 의 activation 과 PARP 단백질의파괴가세포의 DNA 복제및유전자발현과관련된신호전달에영향을주어 apoptosis 를통한세포사멸에기여했음을의미한다. 또한미토콘드리아에의한 apoptosis 신호경로에관여하는 Bcl-2, AIF (apoptosis - inducing factor), cytochrome c 단백질의발현도 PTE의처리에의해농도의존적으로변화하였다. PTE에의해 anti - apoptotic regulation 을하는 Bc l- 2와미토콘드리아분획속의 AIF 단백질발현은감소되었으며, 세포질로분비된 cytochrome c의발현은증가되었음을확인하였다 (Fig. 4). 구강암세포주 YD-10B 에대한 PTE의 apoptosis 유도효과는 TUNEL assay 를실시하여좀더명확히규명하고자하였다. 24시간동안의 PTE 처리결과, control에비하여진한갈색의핵 ( 화살표 ) 을가진세포들이뚜렷하게관찰되었다 (Fig. 3A). 이것은 apoptosis 에의해 DNA 분절 (fragmentation) 이일어난세포의핵이 TUNEL staining에반응하여나타난것으로 PTE가 YD-10B 세포의 apoptosis 를유도하였음을의미한다. TUNEL stain에양성인세포가거의확인되지않은 control(6%) 에비하여 PTE는 TUNEL positive 세포의수를 64% 로증가시켰다 (Fig. 3B). 이러한결과는 flow cytometry 결과 (Fig. 2) 와더불어 PTE가구강암세포주인 YD-10B 세포에서 apoptotic cell death에관여함을명확하게보여준다. (A) (B) Fig. 4. Effect of PTE on the expression of apoptosis regulator proteins. Apoptosis-related proteins were assayed by Western blot with specific antibodies. Procaspase 3/9, cleaved PARP and Bcl-2 were analyzed in total cell lysates. The mitochondrial and cytosolic fractions were prepared by differential centrifugation and used to detect the protein levels of AIF and cytochrome c, respectively. Actin protein expression was measured as an internal control. 고찰 Fig. 3. Induction of apoptosis by PTE in YD-10B cells. Apoptotic cells were detected by TUNEL staining after the treatment with 100 μg / ml of PTE for 24 h. Arrows indicate TUNEL-positive nuclei stained dark brown (A). TUNEL-positive cells in random microscope fields were determined, and apoptotic cells were expressed as the percentage of TUNEL-positive cells over total cells (B). Data represent the mean ± S.D. of three separate experiments ( #, significant as compared to control; ## p<0.01). 3. 세포자멸사관련단백질의발현에 PTE 가미 치는영향 구강암세포주에서 PTE 에의해나타나는 apoptosis 유도 현대사회는다양한직업군의발생으로그에따른불규칙한생활패턴과정신적스트레스로인하여암과같은만성질환의발생이증가하고있는추세이다. 이에, 국민건강증진을위하여다양한치료제개발연구가수행되고있으며, 합성화학물질에비해부작용이적다고인식되고있는천연물소재가관심을받고있다. 김은우리나라에서가장많이소비되는해조류중의하나로칼로리가낮고필수아미노산이풍부하며, 겨울철비타민공급원역할을하는고급식품이다. 최근의연구들에따르면김은항균효과 10) 와항콜레스테롤효과 11) 를비롯한다양한생리활성을지닌것으로보고되어식품이상의가치를지닌천연물로대두되어지고있다. 본연구에서는김에탄올추출물을준비하여인체구강암세포주인 YD-10B 세포에대한세포사멸효과를조사한결과 apoptosis 를통한

5 구강암세포주에서김추출물에의한세포자멸사유도 29 구강암세포의사멸이진행됨을확인하였다. 김추출물은 caspase, PARP, Bcl-2와같은 apoptosis 관련단백질들의발현을조절함으로써암세포성장억제및사멸을유도하였다. Apoptosis 는주변조직에상해를주지않으며손상된조직및세포들을제거하는정상적인과정으로체계적인분자신호전달에의해진행되는 programmed cell death (PCD) 이다 15). 이러한 apoptosis 과정의회피는암세포의무한증식을가능하게하여암의발생및진행에기여함으로, 특정약물에의한 apoptosis 의유도는암을치료할수있는표적이될수있다 15,16). 실제로항암제후보발굴을위한연구결과들을살펴보면, 세포사멸의방법으로항암활성을발휘하는물질들은 target cell에서 apoptosis 를유도함으로써그활성을나타내고있다 17,18). Apoptosis 작용에의해나타나는대표적인현상들로는 plasma membrane의변화에의한 phosphatidylserine (PS) 의 flip-flop 과 DNA 의분절화 (fragmentation) 등을들수있으며 15,16), 본실험에서는김추출물처치에의해 YD-10B 세포에서이두가지현상이일어남을확인할수있었다. 인지질의 flip-flop은 PS와결합하는형광염색약 (annexin-v) 을활용하여 flow cytometry 로확인하였으며, 손상된 DNA 는 TUNEL staining을통해관찰하였다. Protease 의일종인 caspase 는 apoptosis 를위한도구역할을담당하는단백질로서, apoptosis 에관한많은연구들이 caspase 의활성도에의존하는 pathway에초점을두고진행되어왔다 19,20). Caspase 의활성화는세포의에너지공장인미토콘드리아의존적으로진행될수있다. Apoptosis 신호가미토콘드리아에작용하게되면 bcl-2와같은 anti-apoptotic regulator 들의발현은감소하고, 미토콘드리아외막의투과성은증대되어 cytochrome c가 cytosol 로이동되게된다 21). Cytochrome c의방출은 caspase-9을활성화시키고, 활성화된 caspase-9 은연속적으로 caspase-3 의활성을증가시켜 apoptosis 를유도하게된다. 이과정에서핵에서손상된 DNA 를수선 (repair) 하여세포의생존에기여하는 PARP는 caspase-3 에의해절단되어비활성화됨으로써, apoptosis 를진행시키는데역할을담당하게된다 22). 본연구에서김추출물은 apoptosis 억제단백질인 bcl-2와 procaspase-3/9 의발현을저해하였고, cytosol 분획속의 cytochrome c와 cleaved PARP 의발현을증가시켰다. 이결과는김추출물이 cytochrome c의방출, caspase activation, PARP cleavage 로연결되는미토콘드리아를통한 caspase 활성화경로에관여하였음을의미한다. 더불어김추출물은미토콘드리아분획의 AIF 단백질발현도저해하였다. 손상된미토콘드리아로부터유출된 AIF 는 apoptosis 신호를핵내로확산시키는역할을하게된다. 즉, 정상세포의미토콘드리아에존재하는 AIF는 apoptosis 과정에서핵으로이동하여 DNA fragmentation 과 nuclear condensation 을유발시킨다 23). 최근의연구들에의하면, AIF의 nuclear translocation( 핵내이동 ) 은 PARP inhibitor 에의해저해될수있으나 caspase inhibitor 에의해서는저해되지않음이보고되었다 24,25). 이는 AIF가 caspase 비의존적 apoptosis pathway에관여함을의미하는것으로김추출물이여기에부분적으로관여할것으로사료되나, 미토콘드리아의 AIF 단백질발현감소와 PARP 활성화를연결하는신호과정에서김추출물의정확한역할은좀더연구를진행하여야설명될수있을것이다. 이상의결과를종합하여, 인체구강암세포주인 YD-10B 세포에서김추출물이미토콘드리아를통한 apoptosis pathway 를통해세포사멸을유도한다는것이확인되었다. 시험관에서자란암세포에서 apoptosis 를일으킬수있다는것은생체내에서도그러한효과를발휘할수있음을의미하는것으로, 본연구는항암효능을지닌천연물의약품으로발전할수있는본초로서의김에대한약리효능을잘보여주는결과라할수있겠다. 결론 본연구에서는인체구강암세포주인 YD-10B cell에대한김에탄올추출물의세포성장억제및사멸에미치는영향을조사하여다음과같은결론을얻었다. 1. 김에탄올추출물은구강암세포주인 YD-10B cell에대하여시간별, 농도별세포사멸효과를나타내었다. 2. Apoptotic cell을염색할수있는 TUNEL assay와 Flow cytometry를이용한분석결과, 김에탄올추출물에의한세포사멸이 apoptosis 에기인함을알수있었다. 3. 세포자멸사에서주요한역할을담당하는단백질들의발현조사를통해, 김추출물이미토콘드리아의장애 (dysfunction) 와연관된 caspase 활성화및미토콘드리아내의 AIF 발현감소등에관여함으로써 apoptosis 를유도함을알수있었다. 이러한결과들은구강암세포의 apoptosis 신호전달과정에서김추출물이작용하는기전을이해하게함으로써암세포치료제로서의김에대한활용가능성을제시하고있다. 감사의글 이논문은 2014년도정부 ( 미래창조과학부 ) 의재원으로한국연구재단의지원을받아수행된연구입니다 (No. 2012R1A5A2A ). References 1. Jimenez-Escrig A, Goni Cambrodon I. Nutritional evaluation and physiological effects of edible seaweeds. Arch Latinoam Nutr ; 49(2) : The korean association of university professors of herbology. Herbology. Seoul : Younglimsa : 471-4, Park CK, Kang TJ. The nutritional and functional constituents of laver. Bull Fish Sci Inst Yosu Nat'l Univ ; 9 : 133-7

6 30 大韓本草學會誌 Vol. 30 No. 2, Kim D, Park J, Lee TK. Analysis of biochemical compositions and nutritive values of six species of seaweeds. J Life Sci ; 23(8) : Lee HS, Choi MS, Lee YK, Park SH, Kim YJ. A Study on the development of high-fiber supplements for the diabetic patients - Effect of seaweed supplementation on the lipid and glucose metabolism in streptozotocin-induced diabetic rats. Korean J Nutr ; 29(3) : Han HS, Bae SJ, Kim MH. The effect of porphyra tenera extract on serum lipid content in ovariectomized rats. J Life Sci ; 14(2) : Lee SJ, Chung MJ, Shin JH, Sung NJ. Effect of natural plant components on the nitrite-scavenging. J Food Hyg Saf ; 15(2) : Lee SJ, Shin JH, Chung MJ, Sung NJ. Effect of natural foods on the inhibition of N-nitrosodimethylamine formation. J Food Hyg Saf ; 15(2) : Han HS, Bae SJ, Kim MH. Effects of porphyra tenera extracts on formation of collagen cross - link in ovariectomized rats. J Korean Soc Food Nutr ; 22(2) : Park SJ, Park CI, Kim SC. Antimicrobial activities of ethanolic extracts of marine resources against Propionibacterium acnes. Kor J Herbology ; 25(2) : Hong YK, Park IS, Jung YH, Song SH, Hong SY. Effect of the seaweed porphyra yezoensis extract on triton WR-1339 induced hypercholesterolemia in mouse. J Korean Fish Soc ; 31(4) : Kwak CS, Kim SA, Lee MS. The correlation of antioxidative effects of 5 korean common edible seaweeds and total polyphenol content. J Korean Soc Food Nutr ; 34(8) : Jung BM, Shin TS, Kim HR, Jung KJ, Kim SB. Effect of porphyran isolated from laver, porphyra yezoensis on calcium, magnesium and potassium contents in hyperlipidemic rats. J Korean Fish Soc ; 36(3) : Choi SW. Clinical diagnosis of oral cancer. J Korean Dent Assoc ; 49(3) : Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer ; 73(8) : Kang MC, Lee KS. Expression of apoptosis related genes from HaCaT cell after UVB irradiation. Korean J Dermatol ; 47(7) : Safarzadeh E, Sandoghchian Shotorbani S, Baradaran B. Herbal medicine as inducers of apoptosis in cancer treatment. Adv Pharm Bull ; 4(Suppl 1) : Kim JU, Goo M, Park C, Lee JH, Ji HM. Studies on the anti-cancer effect and the mechanism of apoptosis by Baekduong-tang in human colon cancer cell line HCT-11. Korean J Orient Int Med ; 31(2) : Cohen GM. Caspases: the executioners of apoptosis. Biochem J ; 326(Pt1) : Ola MS, Nawaz M, Ahsan H. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol Cell Biochem ; 351(1-2) : Shimizu S. Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature ; 399(6735) : Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S, Smulson M. Role of poly (ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Bio Chem ; 274(33) : Joza N, SUsin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan L, Ferri KF, Zamzami N, Wakeham A, Hakem R, Yoshida H, Kong YY, Mak TW, Zuniga-Pflucker JC, Kremer G, Penninger JM. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature ; 410(6828) : Lorenzo HK, Susin SA, Penninger J, Kroemer G. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ ; 6(6) : Yu SW, Wang H, Poitras MF, Coombs C, Bowers WJ, Federoff HJ, Poirer GG, Dawson TM, Dawson VL. Mediation of poly(adp-ribose) polymerase-1- dependent cell death by apoptosis-inducing factor. Science ; 297(5579) :

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Jkbcs016(92-97).hwp Expression of bcl-2 and Apoptosis and Its Relationship to Clinicopathological Prognostic Factors in Breast Cancer - A Study with Long Term Follow-up correlated with the survival rate.(journal of Korean