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1 동의생리병리학회지제 21 권 6 호 Korean J. Oriental Physiology & Pathology 21(6): , 2007 六鬱湯에의한인체자궁경부암세포의증식억제에관한연구 최영현 * 최병태 2 이용태 1 동의대학교한의과대학생화학교실 대학원바이오물질제어학과, 1 : 한의과대학생리학교실, 2: 부산대학교한의학전문대학원해부학교실 Induction of Apoptosis by Yukwool-tang in Human Cervical Carcinoma HeLa Cells Yung Hyun Choi*, Byung Tae Choi 2, Yong Tae Lee 1 Department of Biochemistry, College of Oriental Medicine and Department of Biomaterials Control, Graduate School, 1:Department of Physiology, College of Oriental Medicine, Dongeui University, 2:Department of Anatomy, Graduate School of Oriental Medicine, Pusan National University Yukwool-tang (YWT) is a traditional Chinese medicine, which has been used for patients suffering from a uterine disease in Oriental medicine. In the present study, it was examined the biochemical mechanisms of apoptosis by YWT in human cervical carcinoma HeLa cells. It was found that YWT could inhibit the cell growth of HeLa cells in a dose-dependent manner, which was associated with apoptotic cell death such as formation of apoptotic bodies and DNA fragmentation. Flow cytometry analysis confirmed that YWT treatment increased populations of apoptotic-sub-g1 phase of the cell cycle. We observed the p53-independent induction of p21 proteins, down-regulation of anti apoptotic Bcl-2 expression and proteolytic activation of caspase-3 in YWT-treated HeLa cells. YWT treatment also concomitant degradation and/or inhibition of poly (ADP-ribose) polymerase (PARP), phospholipase C-1 (PLCγ1), β-catenin and DNA fragmentation factor 45/inhibitor of caspase-activated DNase (DFF45/ICAD). Taken together, these findings partially provide novel insights into the possible molecular mechanism of the anti-cancer activity of YWT. Key words : Yukwool-tang( 六鬱湯 ), cervical carcinoma HeLa cells, apoptosis 서론 세포의증식과암의발생측면에서세포주기조절의교란에의해비정상적인세포주기반복을통한증식이계속되는것을암세포이다. 따라서세포주기와연관된특정유전자의발현조절을통한 apoptosis 유발기전의해석은암을포함한특정약제의개발에매우중요한의의를가질수있다. 특히 apoptosis는개체의발생단계나 DNA 손상, 바이러스감염등에의한유전적조절하에서일어나는정교한인체방어기전이라는점에서 necrosis 와구별된다 1,2). 또한 apoptosis는개체보존수준에서손상된세포들의제거를위한중요한수단이며, 정상적인세포주기의이탈이나특정세포주기조절인자활성의변화가 apoptosis의주원인이 * 교신저자 : 최영현, 부산시진구양정동산 45-1 동의대학교한의과대학 choiyh@deu.ac.kr, Tel : 접수 : 2007/08/08 채택 : 2007/11/13 될수있다 3). Apoptosis의유발에는세포증식억제에중요한역할을하는종양적제유전자인 p53이나 cyclin-dependent kinase (Cdk) inhibitor인 p21(cip1/waf1) 뿐만아니라, apoptosis를직접적으로조절하는유전자가관여한다는사실이알려지면서 apoptosis와연관된분자적기전이최근많이밝혀지고있다 4,5). 그중잘알려진유전자는 Bcl-2/Bax family 단백질로서 Bcl-2는 apoptosis를억제하면서세포의증식을유도하는반면, Bax는과발현되었을때 apoptosis를유도한다 4). 이들두단백질은서로 dimer를형성하면서 mitochodria에서세포질내로 cytochrome c 의유리같은 apoptosis 유발에관여하는인자들을조절한다. 특히 DNA 손상에의한 p53의발현증가는 p21과연관되어세포주기의억제뿐만아니라 Bax 유전자의활성을유도하고 Bcl-2 유전자의발현을억제함으로서 apoptosis에관여하는것으로알려져있다 4,5). 또한 capase라고이름붙여진 ICE/CED-like protease family 역시 apoptosis 유발에중요한역할을하는데, 이들은

2 최영현 최병태 이용태 proenzyme 형태로존재하다가 apoptosis 유도를활성화시키는신호에의하여활성화된 cystein-related proteases로되어직접또는간접적으로세포내에존재하는많은표적단백질들의분해에관여한다 2,4). 본연구진은최근울병에사용하는대표적인처방인육울탕 6,7) 의생리 생화학적기전연구의일환으로구속스트레스부여에다른육울탕의영향을병리조직화학및면역조직화학적수준에서조사한바있다 8). 선행연구의결과에의하면육울탕은면역강화및항염증효능이매우우수할것으로평가되어, 부가적인다양한효능의검색필요성을제시하여주었다. 따라서본연구에서는육울탕의효능에관한연속적연구의하나로서인체자궁경부암 HeLa 세포모델을이용하여암세포의증식에미치는육울탕의영향을조사하였고, 이와연관된 apoptosis의유발에관여하는중요한유전인자들의발현변화를조사하였다. 재료및방법 1. 시료준비, 암세포주및배양조건 실험에사용한육울탕 (Table 1) 수용액추출물을얻기위하여약재 1 g당증류수 1 ml을가하여환류냉각장치가장착된가열기에서 180~200 의온도로 2시간동안끓였다. 이를 3,000 rpm에서 20분간원심분리시킨후, 그상층액을 Whatman 필터 (No. 2) 로걸러내고감압농축과가열을통해고형성분을얻어내어잘게마쇄하고밀봉시켜초저온냉동고에보관하였다. 실험에사용한 HeLa 인체자궁경부암세포는생명공학연구소 (KRIBB, Korea) 에서분주받아 90% 의 DMEM 배지 (Gibco BRL, Grand Island, NY) 와 10% 의우태아혈청 (fetal bovine serum, Gibco BRL) 과 1% 의 penicillin 및 streptomycin (Gibco BRL) 등이포함된배지를사용하여 37, 5% CO 2 조건하에서배양하였다. Table 1. Prescription of Yukwool-tang used in the present study 韓藥名生藥名重量 (g) 향부자 CYPERI RHIZOMA 7.5 창출 ATRACTYLODIS RHIZOMA 6.0 천궁 LIGUSTICI RHIZOMA 6.0 진피 CITRI RETICULATAE PERICARPIUM 4.0 생강 ZINGIBERIS RHIZOMA 3.0 반하 ( 제 ) PINELLIAE RHIZOMA 2.0 적복령 PORIA 2.8 치자 GARDENIAE FRUCTUS 2.8 사인 AMOMI FRUCTUS 2.0 감초 GLYCYRRHIZAE RADIX 2.0 總重量 MTT assay 에의한세포증식억제조사 세포배양용 6 well plate에 HeLa 세포를 3 X 10 4 개 /ml로분주하고 24시간동안안정화시킨다음육울탕을배지에희석하여처리하였다. 72시간후배지를제거하고 tetrazolium bromide salt (MTT, Sigma, St. Louis, MO) 를첨가하고반응시킨후, dimethylsulfoxide (DMSO) 으로 well에생성된 formazin을녹인후 ELISA reader (Molecular Devices, Sunnyvale, CA) 로 540 nm에 서흡광도를측정하였다. 측정은모두세번을하였으며, 그에대한평균값과표준오차를 Microsoft Excel 프로그램으로구하였다. 3. 세포형태변화관찰세포전체의모양변화관찰은육울탕처리후, 도립현미경 (inverted microscope, Carl Zeiss, Germany) 을이용하여 200배의배율로관찰하였다. 육울탕처리에의한 apoptosis 유발여부확인을위한핵의형태적변화관찰은준비된세포를 3.7% formaldehyde 용액으로고정후 4',6-diamidino-2-phenylindole (DAPI, Sigma) 용액으로염색하고, 형광현미경 (Carl Zeiss) 을이용하여 400배의배율로실시하였다. 4. DNA flow cytometry에의한분석준비된세포를차가운 ethanol로 4 에서고정시킨후, 1% bovine serum albumin (BSA, Sigma) 이함유된 PBS로 2 ~ 3회수세하였다. 여기에 DNA intercalating dye인 50 μg/ml의 propidium iodide (PI, Sigma) 및 0.1 mg/ml의 RNase (Sigma, St. Louis, MO, USA) 를처리하여암실, 4 에서 1시간동안염색시켰다. PBS로수세과정을거쳐단일세포로분리시킨후 DNA flow cytometry (Becton Dickinson, San Jose, CA) 에적용시켜형광반응에따른 histogram을 ModiFit LT program을사용하여분석하였다. 5. DNA fragmentation의분석정상및육울탕이처리된세포를모아 lysis buffer [5 mm Tris-HCl (ph 7.5), 5 mm EDTA, 0.5% Triton X-100] 를 4 에서 30분간처리후, 14,000 rpm에서 20분간원심분리하여얻어진상층액에 0.5 mg/ml의 proteinase K (Sigma) 를처리한다음 60 에서 3시간동안반응시켰다. 그후 phenol : chloroform : isoamyl alcohol 혼합용액 (25 : 24 : 1, Sigma) 을첨가하고 30분간 rotate시킨다음 14,000 rpm에서 10분간원심분리하였다. 여기서얻어진상층액에적정량의 isopropanol과 5 M NaCl를첨가하였다. 24시간정도냉장보관한후, 14,000 rpm, 4 에서 30 분간원심분리시킨후상층액을버리고, RNase A가적당량들어있는 TE buffer를이용하여 pellet을녹이고 gel loading dye (Bioneer, Korea) 를섞었다. 이를 1.5% agarose gel을이용하여분리후 ethidium bromide (EtBr, Sigma) 로염색하고 ultra violet (UV) 하에서관찰하였다. 6. RT-PCR에의한 mrna 발현의분석준비된세포에 TRIzol reagent (Invitrogen Co., Carlsbad, CA) 를 4 에서 1시간동안처리하여 total RNA를분리및정량한후, 각각의 primer (Table 2), DEPC water 그리고 ONE-STEP RT-PCR PreMix Kit (Intron, Korea) 를넣고 Mastercycler gradient (Eppendorf, Hamburg, Germany) 를이용하여증폭하였다. 각 PCR 산물들을양적차이를확인하기위하여 1x TAE buffer로 1% agarose gel을만들고 well 당각각의 primer에해당하는 PCR 산물에 DNA gel loading solution을섞어서

六鬱湯에의한인체자궁경부암세포의증식억제에관한연구 loading 한후 100 V에서전기영동을하였다. 전기영동으로 DNA 분리가끝난 gel을 EtBr을이용하여염색한후 UV 하에서확인하였다. RT-PCT 분석과정에서 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 를 internal control로사용하였다. Table 2.

3 六鬱湯에의한인체자궁경부암세포의증식억제에관한연구 loading 한후 100 V에서전기영동을하였다. 전기영동으로 DNA 분리가끝난 gel을 EtBr을이용하여염색한후 UV 하에서확인하였다. RT-PCT 분석과정에서 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 를 internal control로사용하였다. Table 2. Sequence of primers used for RT-PCR Gene name GAPDH p53 p21 Fas FasL Bax Bcl-2 Bcl-XL XIAP Sequence 5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' 5'-AGC CTT CTC CAT GGT GGT GAA GAC-3' 5'-GCT CTG ACT GTA CCA CCA TCC-3' 5'-CTC TCG GAA CAT CTC GAA GCG-3' 5'-CTC AGA GGA GGC GCC ATG-3' 5'-GGG CGG ATT AGG GCT TCC-3' 5'-TCT AAC TTG GGG TGG CTT TGT CTT C-3' 5'-GTG TCA TAC GCT TTC TTT CCA T-3' 5'-GGA TTG GGC CTG GGG ATG TTT CA-3' 5'-AGC CCA GTT TCA TTG ATC ACA AGG-3' 5'-ATG GAC GGG TCC GGG GAG-3' 5'-TCA GCC CAT CTT CTT CCA-3' 5'-CAG CTG CAC CTG ACG-3' 5'-GCT GGG TAG GTG CAT-3' 5'-CAG CTG CAC CTG ACG-3' 5'-GCT GGG TAG GTG CAT-3' 5'-GAA GAC CCT TGG GAA CAA CA-3' 5'-CGC CTT AGC TGC TCT CTT CAG T-3' 7. Western blot analysis 에의한단백질발현의분석 정상및육울탕이처리된배지에서자란세포들을 PBS로씻어내고적당량의 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)] 를첨가하여 4 에서 30분간반응시킨후, 14,000 rpm으로 30분간원심분리하여그상층액을취하였다. 상층액의단백질농도를 Bio-Rad 단백질정량시약 (Bio-Rad, Hercules, CA) 사용방법에따라정량한다음동량의 Laemmli sample buffer (Bio-Rad) 를섞어서 sample을만들었다. 이렇게만든동량의단백질을 SDS-polyacrylamide gel을이용하여전기영동으로분리하고, 분리된단백질을함유한 gel을 nitrocellulose membrane (Schleicher and Schuell, Keene, NH) 으로 electroblotting에의해전이시켰다. 준비된 membrane에해당단백질의 1차항체를적정시간처리하고 2차항체를사용하여반응시킨후, enhanced chemiluminoesence (ECL) 용액 (Amersham Life Science Corp., Arlington Heights, IL) 을적용시킨다음암실에서 X-ray film에감광시켜특정단백질의양을분석하였다. 본실험에사용된항체들은 Santa Cruz Biotechnology Inc. (Santa Cruz, CA) 및 Calbiochem (Cambridge, MA) 에서구입하였으며, 2차항체로사용된 peroxidase-labeled donkey anti-rabbit 및 peroxidase-labeled sheep anti-mouse immunoglobulin은 Amersham에서구입하였다. 8. in vitro caspase-3, -8 및 -9의 activity 측정 Caspases의 in vitro 활성측정을위한 colorimetric assay kits는 R&D Systems (Minneapolis, MN) 에서구입하였으며, 제시된방법에준하여활성의증감여부를조사하였다. 이를위하여준비된세포의단백질을추출하고정량하여각각적정단백질을 fluorogenic peptide 기질이함유된 extraction buffer [40 mm HEPES (ph 7.4), 20% glycerol (v/v), 1 mm EDTA, 0.2% NP-40 and 10 mm DL-DTT] 에혼합하였다. 실험에사용된기질 은 caspase-3의 경우에는 Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pna), caspase-8은 Ile-Glu-Thr-Asp (IETD)-p-nitroaniline (pna), caspase-9는 Leu-Glu-His-Asp (LEHD)-pNA였다. 준비된 plate를 37 에서 반응시킨 후 VERSAmax tunable microplate reader를이용하여 405 nm의흡 광도에서반응의정도를측정하였다. 결과및고찰 1. 육울탕에의한 HeLa 세포의증식억제및 apoptosis 유발 인체자궁경부암세포주인 HeLa 세포의증식에미치는육울탕의영향을알아보기위하여이를 72시간동안농도별로처리한후 MTT assay를이용하여조사한결과는 Fig. 1A에서나타낸바와같다. 결과에서알수있듯이처리농도가증가함에 HeLa 세포의증식이억제되었으며다양한세포의형태적변형이동반되었다. 특히농도가증가할수록세포의밀도가감소하면서길고분지를형성하는듯한 dendrite-like한구조로바뀌었으며, 부착력을상실하고배지위로부유되는경향성이증가되었다 (Fig. 1B). Fig. 1. Inhibition on the cell viability and morphological changes by Yukwool-tang (YWT) treatment in HeLa human cervical carcinoma cells. (A) Cells were seeds as described in materials and methods, and treated with various concentrations of YWT. After 72 h incubation, MTT assay was performed. The data shown are mean +/- SD of three independent experiments. (B and C) Exponentially growing cells were incubated with either vehicle alone various concentrations of YWT for 72 h. Cell morphology was visualized by inverted microscopy (Magnification, X200, B). The cells were stained with DAPI solution and then photographed by fluorescent microscope using blue filter. (Magnification, X400, C). 다음은육울탕의처리에의한생존율감소및형태변화가 apoptosis의유발과연관성이있을것으로기대되어육울탕처리

최영현 최병태 이용태 후 HeLa 세포핵의형태변화를조사하였다. Fig. 1C에제시된바와같이정상배지에서자란대조군에서는핵의형태에아무런변화가보이지않았지만육울탕을처리했을경우는처리농도가증가할수록 apoptosis 유발특이적인핵내 DNA 단편화에의한염색질의응축된형태인 apoptotic body의형성정도가증가되었다.

따라서육울탕처리에따른 apoptosis 유발의정도를정량적으로비교평가하기위하여동일한조건으로배양된세포들을대상으로세포주기 sub-g1기에해당하는세포의빈도변화를조사한결과, 육울탕의처리농도의증가에따라 sub-g1기에해당하는세포의빈도가증가하여 3 mg/ml 처리군에서는대조군에비하여약 4배증가되었음을알수있었다 (Fig. 2C).

4 최영현 최병태 이용태 후 HeLa 세포핵의형태변화를조사하였다. Fig. 1C에제시된바와같이정상배지에서자란대조군에서는핵의형태에아무런변화가보이지않았지만육울탕을처리했을경우는처리농도가증가할수록 apoptosis 유발특이적인핵내 DNA 단편화에의한염색질의응축된형태인 apoptotic body의형성정도가증가되었다. 또한 apoptotic body 형성과함께 apoptosis 유발의직접적인증거에해당하는 DNA 단편화여부를조사한결과, Fig. 2B의결과에서처럼육울탕의처리에따라단편화의정도역시증가되었다. 따라서육울탕처리에따른 apoptosis 유발의정도를정량적으로비교평가하기위하여동일한조건으로배양된세포들을대상으로세포주기 sub-g1기에해당하는세포의빈도변화를조사한결과, 육울탕의처리농도의증가에따라 sub-g1기에해당하는세포의빈도가증가하여 3 mg/ml 처리군에서는대조군에비하여약 4배증가되었음을알수있었다 (Fig. 2C). 이상의결과는육울탕처리에따른 HeLa 세포의생존율의감소및형태변화가 apoptosis 유발과밀접한연관성이있음을보여주는결과이다. 살펴본결과 p21은 p53에비해 2.5배정도증가하는것으로확인되었다. Cdk inhibitor인 p21은 p53의발현증가에의하여조절될수있고또한 p53 비의존적으로발현이조절될수도있으며 9,10), 암세포의증식억제, apoptosis 및분화유도에중요한역할을하는세포주기전반에걸친가장중요한조절인자란점에서 11-13) 육울탕의처리에의하여 p53의발현과상관없이 p21 유전자가증가되었다는점은매우흥미로운결과라고사료된다. 그러나본연구의결과만으로육울탕에의한인체자궁경부암세포의세포주기교란에관한명확한증거는제시할수없으며, 세포주기분석과관련유전자들의 kinase 활성도검사등을통한후속적인연구가진행되어야할것이다. Fig. 2. Induction of DNA fragmentation and increase of sub-g1 cell population by YWT treatment in HeLa cells. (A and C) The cells were exposed for 72 h to an increasing concentration of YWT. Then the cells were collected and stained with PI for flow cytometry analysis. The percentage of cells with hypodiploid DNA contents represent the fractions undergoing apoptotic DNA degradation. Each point represents the average of three independent experiments. (B) After YWT treatment for 72 h, the cells were collected and DNA was extracted. The fragmented DNA was separated on 1.5% agarose gel electrophoresis and visualized under UV light after stained with EtBr. 2. p53 및 p21 의발현에미치는육울탕의영향 다음은육울탕처리에의한 HeLa 자궁경부암세포의증식억제현상이종양억제유전자또는세포주기조절억제인자들의발현변화와상관성이있는지의여부를조사하기위하여현재까지알려진종양억제유전자중가장중요한 p53 및전체적인세포주기에중요한조절역할을하는 Cdk inhibitor p21의발현에미치는육울탕의영향을조사하였다. Fig. 3의결과에서볼수있듯이 p53 유전자의경우는전사및번역수준에서변화가관찰되지않았지만 p21 유전자의발현은육울탕의처리에따라전사수준에서는변화가없었지만번역수준에서증가하였음을알수있었다. 또한번역수준에서 p53과 p21의상대적인발현비율을 Fig. 3. Induction of cyclin-dependent kinase p21(cip1/waf1) in YWT-treated HeLa cells. (A) After 72 h incubation with YWT, total RNAs were isolated and reverse-transcribed. The resulting cdnas were subjected to PCR with p53 and p21 primers and the reaction products were subjected to electrophoresis in a 1% agarose gel and visualized by EtBr staining. GAPDH was used as an internal control. (B) The cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with anti-p53 and anti-p21 antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control. (C) Density of protein expression was determined by image analysis software. Each value indicates the mean of two separate experiments. 3. Caspase의발현및활성에미치는육울탕의영향 Caspase는세포의 apoptosis 유발에핵심적인역할을하는인자로서세포내의핵과 mitochondria의외막에불활성상태인 proenzyme 형태로존재하다가 apoptosis를유도하는자극에의하여활성화된 protease로전환되어직접또는간접적으로세포내에존재하는많은표적단백질의분해에관여한다 14). 따라서 caspase의활성화는 apoptosis의유발에대한또다른증거가될수있으며많은선행연구등에서검증되어왔다 15). 지금까지알려진 caspase 중에서 apoptosis가유발에상대적으로중요한 caspase-3, -8 및 -9의활성에미치는육울탕의영향을 Western bolt analysis를통하여조사하였다. Fig. 4A의결과에서볼수있듯이육울탕의처리에의한 caspase-8 단백질발현의변화가없

六鬱湯에의한인체자궁경부암세포의증식억제에관한연구 었지만 caspase-3 및 -9의경우는육울탕처리농도의존적으로활성형단백질의발현이증가하였다. 이러한단백질수준에서의결과를재확인하기위하여 in vitro caspase activity assay를통하여 caspases의활성정도를직접분석한결과, Fig.

diacylglycerol 및세포내 Ca 2+ 조절에중요한역할을하는 inositol 1,4,5-trisphosphate (IP3) 를생산한다 20).

5 六鬱湯에의한인체자궁경부암세포의증식억제에관한연구 었지만 caspase-3 및 -9의경우는육울탕처리농도의존적으로활성형단백질의발현이증가하였다. 이러한단백질수준에서의결과를재확인하기위하여 in vitro caspase activity assay를통하여 caspases의활성정도를직접분석한결과, Fig. 4B에서나타난바와같이육울탕처리농도의존적으로 caspase-3의활성이증가하는것을확인할수있었다. 하지만 caspase-8과 caspase-9의활성정도는큰변화가관찰되지않았으므로육울탕에의한 HeLa 세포의 apoptosis 유발에 caspase-3의활성화가중요한역할을함을알수있었다. diacylglycerol 및세포내 Ca 2+ 조절에중요한역할을하는 inositol 1,4,5-trisphosphate (IP3) 를생산한다 20). 따라서 phosphatidylinositol 3-kinase나 Ras와같은세포성장신호분자와같이 PLC-γ1 역시세포의증식에중심적인역할을하는것으로알려져있으나, apoptosis가유발될경우활성화된 caspase 효소에의하여 PLC-γ1 단백질은분해될수있기때문에상기두종류의단백질과함께 apoptosis 유발의생화학적표식자로사용이가능하다 21). 따라서육울탕에의한 HeLa 세포의 apoptosis 유발시이러한단백질들의단편화가동반되는지의여부를조사한결과, Fig. 5A에서나타낸바와같이 PARP의경우는정상배지에서배양된세포들은 116 kda 위치에강한주 band를볼수있었고육울탕의처리농도의존적으로 85 kda의단편이증가되는것을관찰할수있었다. β-catenin의경우는육울탕의처리에의한 kda의단편은관찰되지않았으나주 band의발현정도가현저히감소하는것으로관찰되었다. 이는육울탕에의한 HeLa 세포의 apoptosis가세포내골격및부착능력의상실과연관이있음을알수있었다. 또한육울탕의처리농도가증가될수록 PLC-γ 1 단백질의발현도매우빨리감소되었음을알수있었으며, 처리농도증가에따른발현의감소정도는 β-catenin과매우유사하였다. 이상의결과로육울탕에의한인체자궁경부암세포의 apoptosis 유발에는 caspase의활성화에따른이들표적단백질의분해및발현감소가중요한역할을하는것으로생각된다. Fig. 4. Activation of caspase-3 by YWT treatment in HeLa cells. (A) The cells were treated with the indicated concentrations of YWT for 72 h and collected. Western blots were detected with indicated antibodies and ECL detection. (B) After 72 h incubation with YWT, aliquots (150 g protein) were incubated with substrates, DEVD-pNA, IETD-pNA and LEHD-pNA, for in vitro caspase-3, -8 and -9 activity, respectively, at 37 for 3 h. The released fluorescent products were measured. Data are means average of two separate experiments. 4. PARP, β-catenin 및 PLC-γ1의발현에미치는육울탕의영향한편 Poly(ADP-ribose) polymerase (PARP) 단백질은 DNA repair나 genomic stability의유지에매우중요한역할을하지만, apoptosis 유발과정중활성화된 caspase에의하여분해가일어나면 PARP의효소적기능의상실로인하여정상적인 DNA repair 과정이억제되어진다. 정상적인세포의경우 PARP 단백질은 116 kda의분자량을가지지만 apoptosis가일어난경우 85 kda 크기의단편이관찰되거나주 band의발현이감소된다 16,17). 또한 catenin family에속하는단백질 (α, β 및 γ) 은모든세포에서발현되는세포연접기능에중요한역할을하는것으로알려져있는데, 특히 β-catenin은세포내골격의유지와다양한부착성세포의전사조절에중요하며세포유착과관계된 apoptosis 조절과연관성을가지고있다. 정상세포의경우 β-catenin은 92 kda의분자량을가지나세포유착성 apoptosis가일어나면활성화된 caspase에의하여 kda로단편화가일어난다 18,19). 그리고 phospholipase C-γ1 (PLC-γ1) 은 phosphatidylinositol 4,5-bisphosphate를 hydrolyze시켜 protein kinase C activator인 Fig. 5. Effects of YWT treatment on the expression of poly(adp-ribose) polymerase, β-catenin, phospholipase C-γ1 and DNA fragmentation factor family proteins in HeLa cells. (A) The cells were treated with the indicated concentrations of YWT for 72 h and collected. Western blots were detected with indicated antibodies and ECL detection. (B) Density of protein expression was determined by image analysis software. Each value indicates the mean of two separate experiments. 5. DFF40/CAD 및 DFF45/IACD의발현에미치는육울탕의영향 Apoptosis의가장중요한현상중하나가 DNA 단편화이며여기에관여하는중요한인자가 DNA fragmentation factor

6 최영현 최병태 이용태 (DFF) 이다. DFF family는 caspase-activated DNase인 DFF40/CAD와 inhibitor of caspase-activated DNase인 DFF45/ICAD로구성되어있다. DFF40/CAD와 DFF45/ICAD는서로 complex를형성하고있으며, apoptosis가유발되면 caspases에의하여 ICAD의두군데 Asp 잔기에서단편화가일어나는것으로알려져있다 22,23). 따라서 DFF40/CAD 및 DFF45/ICAD가육울탕의처리에따른 apoptosis와연관이있는지를조사한결과, Fig. 5A에서보는바와같이 DFF40/CAD의경우는고농도처리군에서약간증가되었다. 그러나 DFF45/ICAD는육울탕처리농도의존적으로발현양의감소가관찰되었으며, DFF40/CAD와 DFF45/ICAD의상대적인발현정도를조사해본결과최고농도에서약 4.8배정도의차이가나타났다 (Fig. 5B). 즉육울탕의처리에의한 HeLa 세포의 apoptosis 유발은 caspase의활성화와연관된 DFF45/ICAD의상대적인발현양의차이가중요한역할을할것으로생각된다. 의존적으로감소되었다. 이는육울탕처리에의한 apoptosis이유발에는최소한 Bcl-2 family가중요한역할을하고있음을의미하는것이며, Bcl-2의발현감소로인한 apoptosis 유발관련효소들의활성화가이루어지고있음을시사하여주는것이다. 6. Fas/FasL system 및 Bcl-2 family의발현에미치는육울탕의영향 Apoptosis를유발하는경로는크게 mitochondrial pathway 와 death receptor pathway의두가지로구분할수있는데, death receptor는세포사멸의기능을갖는 tumor necrosis factor (TNF) 수용체군을의미한다 24). 현재가장잘알려진다섯종류중하나가 APO-1 또는 CD95라고도알려진 Fas에의한신호전달계이다. 그리고 TNF 수용체군에작용하는대표적인 ligand 중의하나가 FasL이다 25). FasL는 type II transmembrane protein으로서 TNF 수용체 super-family인 Fas에결합하여 apoptosis를유발한다. 따라서육울탕에의한 apoptosis 유발에 death receptor가관여하는지의여부를 RT-PCR 및 Western blotting으로조사한결과는 Fig. 6에나타낸바와같다. 결과에서볼수있듯이 Fas 및 FasL 모두에서전사및번역수준에서의아무런변화가관찰되지않았다. 이는 HeLa 세포에육울탕을처리하였을경우 death receptor pathway 중 Fas 및 FasL를통한 apoptosis는일어나지않았음을보여주는결과라고생각된다. Bcl-2 family에속하는몇가지중요한인자들은 apoptosis 유발조절에가장대표적인유전자인데그중 Bcl-2는 anti-apoptotic 분자로서 apoptosis의유발을억제하는기능을가지며, Bax는 pro-apoptotic 분자로 Bax 단백질발현의증가는 apoptosis의유발과관계가있는것으로알려져있다 26). 이들두유전자는세포내소기관중 mitochondria로부터의 cytochrome c를유리시켜 cysteine-related protease인 caspase, 종양억제유전자인 p53, DNA의단편화와연관된 endonuclease 등의활성을조절한다 27). 이들은서로 dimer의형태로존재하며그들의발현수준에변화가초래되면 apoptosis가유발되는것으로알려져있다. 따라서 HeLa 세포에서육울탕에의한 apoptosis 유발에이들유전자가관련되어있는지의여부를 RT-PCR 및 Western blotting으로조사한결과는 Fig. 6에나타낸바와같이, Bax 및 Bcl-XL 유전자의발현은전사및번역수준에서는거의변화가관찰되지않은반면, Bcl-2의발현은번역수준에서만처리농도 Fig. 6. Effects of YWT treatment on the expression of Fas/FasL system and Bcl-2 family members in HeLa cells. (A) After 72 h incubation with YWT, total RNAs were isolated and reverse-transcribed. The resulting cdnas were subjected to PCR with indicated primers and the reaction products were subjected to electrophoresis in a 1% agarose gel and visualized by EtBr staining. GAPDH was used as an internal control. (B) The cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with indicated antibodies. Proteins were visualized using an ECL detection system. Actin was used as an internal control. 결 론 본연구에서는인체자궁경부암 HeLa 세포를대상으로육울탕에의하여항암효능의기전을조사하였다. 육울탕처리에의한 HeLa 세포의증식억제는 p53 비의존적인 p21 단백질발현의증가와연관성이있었으며, 육울탕의처리에의한 HeLa 세포의증식억제는 apoptosis 유발과밀접한연관성이있음을핵의응측화, DNA의단편화및 sub-g1기세포빈도의증가등으로확인하였다. 육울탕의처리는 HeLa 세포에서특히 caspase-3의활성화를유의적으로유도하였으며, 이에따른 caspase-3의표적기질단백질인 PARP, β-catenin 및 PLC-γ1의단편화 / 분해촉진이관찰되었다. 육울탕처리에의한 apoptosis 유발은또한 DFF 및 Bcl-2 family에속하는유전자들의변화를초래하였으나, Fas/FasL system에는큰변화가없었다. 감사의글 본논문은부산테크노파크공모과제로지원되는연구비 ( 과제번호 2005QB015) 로조성되었습니다

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