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J Korean Med Ophthalmol Otolaryngol Dermatol 2014;27(4):158-176 pissn 1738-6640 eissn 2234-4020 http://www.ood.or.kr http://dx.doi.org/10.6114/jkood.2014.27.4.158 Original Article / 원저인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한 세포사멸효과 주현아 배현진 황충연 원광대학교한의과대학안이비인후피부과학교실 Apoptotic Effect of ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts on MIA PaCa-2 Cells Hyun-A Joo Hyeon-Jin Bae Chung-Yeon Hwang Won-Kwang University Oriental medicine Opthalmology Otolaryngology Dermatology Abstract Purpose : The purpose of this study is to investigate the effect of ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts on cell death in pancreatic cancer cells. Method : Human-derived pancreatic cancer cell line, MIA PaCa-2 cells were treated by Prescription A with various concentrations and the cytotoxicity was determined by MTT assay. To investigate the effects of Prescription A on pancreatic cancer cells, the staining of Annexin V/PI, cell cycle arrest, nuclear chromatin condensation and the production of reactive oxygen species (ROS) were examined. The effect of Prescription A's effective components, ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts on cell death were also observed. Results : The viability of MIA PaCa-2 cells treated with Prescription A were decreased in a dose dependent manner. Prescription A induced cell death in MIA PaCa-2 cells as shown by result of Annexin V/PI double staining, chromatin condensation and cell cycle arrest. In addition, production of ROS was increased by Prescription A treatment, suggesting that ROS induced by Prescription A mediated cell death. Furthermore, Prescription A's effective components, ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts were also induced apoptosis of MIA PaCa-2 cells through ROS production. c 2014 the Society of Korean Medicine Ophthalmology & Otolaryngology & Dermatology This is an Open Access journal distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 158

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 Conclusion : These results suggest that Prescription A's effective components, ARTEMISIAE CAPILLARIS HERBA and COPTIDIS RHIZOMA Extracts induced death of MIA PaCa-2 through ROS production. Key words : ARTEMISIAE CAPILLARIS HERBA; COPTIDIS RHIZOMA; Prescription A; pancreatic cancer; MIA PaCa-2; death; apoptosis; ROS Ⅰ. 緖論 췌장암은발생빈도는높지않으나사망률이높아 발생수와사망수가대체로일치하여 5 년생존율이 8% 로보고되며특히 2 년생존율이 10% 내외에불과 한예후가극히불량한암이다 1,2). 현재완치를기대 할수있는유일한방법은수술적치료이지만, 대부분 의췌장암환자는진행된상태에서발견되어수술적 제거를받지못하는경우가많다. 이는췌장암을의심 할수있는상복부통증, 폐쇄성황달, 체중감소, 위기 능장애등의증상이이미암이진행된상태에서주 로나타나고, 암세포의파급이빠르며, 췌장에국한된 조기췌장암의발견을위한민감한검진방법이아직 까지없기때문이다 1,3,4). 또한근치적절제술후재발률은 80~90% 에이르 는데, 췌장암은특징적으로후복막조직으로침윤하는 경우가빈번하고림프절전이가많기때문에국소재 발이 50~80% 나될정도로예후가불량하다 5). 이러한 불량한예후의한계점을극복하고절제가불가능한 췌장암환자에서예후를향상시키려는목적으로보조 적치료의필요성이절실히요구되고있다. 유일하게 생존율과임상증상의개선을유도한다고알려진 gemcitabine 의경우에도 5-fluorouracil (FU) 와비교 연구에서한달남짓의연장을가져올뿐으로이러한 현실은새로운약물의출현을필요로하는실정이다 1,5). 한의학에서췌장암은黃疸 6-12), 伏梁 7-9,11,12), 腹痛 8,11,12), 癥積 6), 癥瘕 7,9,10), 積聚 7,9,10), 痞块 7,11,12) 7), 結胸 교신저자 : Chung-Yeon Hwang Won-Kwang University Kwang-Ju Oriental Medical Hospital Opthalmology Otolaryngology Dermatology (Tel : 062-670-6434, E-mail: hwangida@wonkwang.ac.kr) 접수 2014/10/14 수정 2014/11/5 채택 2014/11/12 등의범주에속하며, 그주요病因病機로는七情內傷, 飮食不節하여肝脾損傷, 臟腑失和, 濕濁阻遏, 氣血瘀滯가日久하여형성된것이다 8). 치료는주로健脾和胃, 淸熱利濕, 理氣活血, 軟堅消癥이원칙으로울금에해당하는 curcumin 13) 이나꾸지뽕잎추출물 14), 황백물추출물 15) 외에도扶正抗癌湯 16), 八珍抗癌丹 17) 등의췌장암에대한항암효과가보고되고있다 8). 또한茵蔯蒿湯의사람간암세포주인 Hep G2에대한항암효과와황련에들어있는 berberine 이구강암에서세포사멸을유도한다고보고되고있다 18,19). 이를바탕으로서적들을조사하여저자는우선췌장암세포주인 MIA PaCa-2 세포에영향을미칠것으로예상되는처방 4가지 (Prescription A 20), B 21), C 22), D 23) ) 를선택한후유의한결과를나타내었던 Prescription A의 MIA PaCa-2 세포에대한생존률및 apoptosis 에관여하는 reactive oxygen species (ROS) 의활성을관찰하여유의한결과를얻었다. 또한이유의한처방을구성하는약물들이약재별로 MIA PaCa-2 세포에대해미치는영향을조사한결과인진 (Artemisiae capillaris Herba) 과황련 (Coptidis Rhizoma) 이 MIA PaCa-2 세포에대하여유효한효과를보여주었기에이를보고하는바이다. Ⅱ. 實驗材料및方法 1. 재료 1) 약재실험에사용한한약재는옴니허브 ( 영천, 경북, 대한민국 ) 에서구입후원광대학교한의과대학본초학교실에서精選하여사용하였다. 159

2) 세포주 Human-derived pancreatic adenocarcinoma cell line인 MIA PaCa-2 와 pancreatic acinar cell인 266-6 은한국세포주은행 ( 서울, 대한민국 ) 으로부터분양받아사용하였다. 3) 시약 Fetal bovine serum (FBS), DMEM medium, penicillin-streptomycin 등의세포배양용시약들은 Gibco BRL (NY, USA) 사에서구입하였다. 실험에사용된시약중 MTT, Annexin V detection kit, DAPI, DCF-DA, n-acetyl cystein (NAC) 등은 Sigma (St. Louis, USA) 사에서구입하였으며, 실험에사용된모든시약은분석용등급이상으로사용하였다. 2. 방법 1) 시료제조한약재를 3차증류수 1 L에넣고 2 시간 30 분동 안전탕한액을 -80 에서동결건조시켜얻어진분말을 3차증류수에녹여서 filter 로여과하여사용하였다. 2) 세포배양 5% CO 2, 37 incubator 조건하에서 10% FBS와 penicillin-streptomycin 이함유된 DMEM 배지에배양하였으며 2일마다 100 mm dish에분주하였다. 3) MTT Assay MIA PaCa-2 세포와 266-6 세포의생존율은밀집세포의미토콘드리아탈수소효소에의해자줏빛 formazan 생성물로변하는 MTT 환원을바탕으로 MTT 분석법으로측정했다. 세포들을 DMEM 배지에서 2 10 5 cells/well 의밀도로현탁하여농도별로약물을처리하였다. 배양후 5 mg/ml 의농도로 MTT 용액을첨가하고다시 30 분동안배양하였다. MTT-formazan 생성물은 DMSO 200 μl를첨가함으로써용해했다. formazan의양은해액 ( 解液 ) 을 96-well plate에 loading하여 spectrophotometer Table 1. Components and Yield rate of Prescription A, B, C, D Components (g) Powder(g) Yield Rate (%) Prescription A 20) Artemisiae capillaris Herba 30, Gentianae Radix 15, Coptidis Rhizoma 6, Gleditsiae Spina 2 11.2 21.13 Prescription B 21) Chelidonii Herba 30, Euonymi Rmulus 30 10.8 18 Prescription C 22) Coicis Semen 30, Sparganii Rhizoma 15, Zedoariae Rhizoma 15, Scutellariae barbatae Herba 15 13.6 18.13 Prescription D 23) Taraxaci Herba 40, Hedyotis diffusae Herba 40, Salviae miltiorrhizae Radix 30, Coicis Semen 30, Paeoniae Radix Rubra 15 23.69 15.28 Table 2. Yield rate of Prescription A's components Components (g) Powder (g) Yield Rate (%) Artemisiae capillaris Herba 100 11.36 11.36 Gentianae Radix 100 15.69 15.69 Coptidis Rhizoma 100 13.62 13.62 Gleditsiae Spina 100 17.63 17.63 160

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 (Molecular devices, Palo Alto, USA) 를이용하여 540 nm에흡수되는양을측정함으로서결정했다. 세포의생존율은어떠한처치도가하지않은 control cells과의비율로나타내었다. [ 즉, viability(%) = 100 (absorbance of treated sample)/(absorbance of control)] 4) FACS analysis 1 Annexin V and PI Annexin V와 PI의발현은 Annexin V detection kit으로확인하였다. MIA PaCa-2 세포에 Prescription A를농도별로처리하여 24 시간배양한후, 부착된세포를 trypsin 으로처치하고회수하여, PBS (Phosphate buffered saline ph 7.4) 로 1회세척하였다. Binding buffer (10 mm HEPES/NaOH, ph 7.4, 140 mm NaCl, 2.5 mm CaCl 2 ) 85 μl에세포를부유시킨후 10 μl의 Annexin V-FITC 와 PI-PE 로 15분간암반응시켰다. Binding buffer 를 300 μl를첨가하여 flow cytometer (FACSCalibur, BD Biosciences, San Jose, USA) 로분석하였다. 2 PI (propidium iodide) 세포주기에미치는영향을알아보기위해 PI로 DNA를염색한후에 flow cytometry 를이용하여형광의세기를측정하였다. MIA PaCa-2 세포에 Prescription A를처리하여 24 시간배양후포집하여 PBS로두번세척하였다. 세척후, 세포를에탄올에넣어고정시킨후, 세포는 PI 용액 (0.1 % Triton X-100, 20 μg/ml PI, 200 μg/ml RNase) 600 μl로 20분반응하였다. Flow cytometry 를통해 DNA 용적히스토그램을얻고 sub G1기의세포수백분율을구하여세포주기를분석하였다. 3 DCF-DA (dichlorofluorescein diacetate) 약재에의한세포내활성화산소의생성을측 정하기위하여형광 probe 2',7'-dichlorofluorescein diacetate (DCF-DA) 를이용하였다. 비형광물질인 DCF-DA 는세포내 hydrogen peroxide (H 2 O 2 ) 와관련된 peroxides 존재시형광의 DCF로변환되어녹색의형광을발한다. MIA PaCa-2 세포에약재를처리한후 24시간동안배양하였다. 세포를수확하기전에 10 μ M DCF-DA 를처리하여 37 에서 20분동안배양하였다. 배양한세포는 PBS로세척하여 1 % trypsin-edta 용액을처리하여세포를수확하고다시 PBS로세척하여 flow cytometry 로형광을측정하였다. 정보의분석은 Cell Quest software (BD Biosciences, San Jose, USA) 를이용하였다. 5) Fluorescence microscopy method 1 DAPI MIA PaCa-2 세포에 Prescription A를처리하여 24시간배양후포집하여 PBS로두번세척하였다. 세척후, 세포를 4% 포름알데하이드에넣어고정시킨후, 세포는 DAPI (0.5 mg/ml) 로 5분반응하였다. 그후, Fluorescence microscopy 로관찰하였다. 3. 통계처리모든실험결과는 3회이상실시하여그평균값을기초로 Mean ± S.D. 로나타내었다. 실험결과에대한통계처리는 Student's t-test 혹은 ANOVA 에준하였고, 사후검정은 post hoc으로시행하였다. p-value 가 0.05 혹은 0.001 미만일경우유의한것으로판정하였다 161

Ⅲ. 결과 1. Prescription A, B, C, D가 MIA PaCa-2 cell에미치는생존율감소및 MIA PaCa-2 cell과 266-6 cell에서증식억제의차이 Prescription A, B, C, D가췌장암세포주의생존율에미치는영향을알아보기위하여 MIA PaCa-2 세포에 Prescription A, B, C, D를농도의존적으로처리하여 24 시간후에세포의생존율을 MTT 방법으로측정하였다. 그결과 Prescription A, B, C, D 중 Prescription A가 0.1 mg/ml 투여군 (99.16 ± 0.65), 0.5 mg/ml 투여군 (86.31 ± 0.68), 1 mg/ml 투여군 (58.55 ± 1.46) 의순으로농도의존적으로 MIA PaCa-2 세포의생존율을감소시켰다 (Fig. 1A). 또한 MIA PaCa-2 세포에작용하는 Prescription A가췌장선방세포주인 266-6 세포에미치는영향을알아보기위하여 Prescription A를농도의존적으로 266-6 세포에처리하여 24시간후에세포의생존율을계산하였다. 그결과 Prescription A는췌장의정상세포에는영향을미치지않으면서췌장암세포에서만농도의존적으로세포증식을억제하였다 (Fig. 1B). 2. Prescription A가 MIA PaCa-2 cell에서 Annexin V와 PI의 staining에미치는영향 Prescription A가 MIA PaCa-2 세포에대한 apoptosis 인지 necrosis 인지를알아보기위하여 Annexin V와 PI의염색을이용하여조사하였다. MIA PaCa-2 세포에 Prescription A를 24 시간동안농도별로처리하여배양한후세포를순수하게분리하여 10μl 의 Annexin V-FITC 와 PI-PE 로 15분간암반응시켰다. 이후 flow cytometer 로분석하였다. 그결과 Prescription A는농도의존적으로 Annexin V의염색양이증가하였고 PI의염색양은변화하지않았다 (Fig. 2). 이는 Annexin V는 positive 상태이면서 PI는 negative 상태인 early apoptosis 단계를의미하는것으로 Prescription A는농도의존적으로 early apoptosis 되는 MIA PaCa-2 세포를유의성있게증가시킴을알수있다. 3. Prescription A가 MIA PaCa-2 cell에서세포주기에미치는영향 Prescription A가 MIA PaCa-2 세포의 apoptosis 를유발함에따라서 cell cycle arrest 에영향을주는지알아보았다. Prescription A를 MIA PaCa-2 세포 Fig. 1. The cytotoxic effects of Prescription A, B, C, D on MIA PaCa-2 and pancreatic acinar cell line, 266-6. (A) MIA PaCa-2 cells were incubated with or without Prescription A, B, C, and D as indicated concentration for 24 hrs. (B) MIA PaCa-2 cells and 266-6 cells were incubated with or without Prescription A as indicated concentration for 24 hrs. Cell viability was measured by MTT assay as described in materials and methods. Data are means of three independent experiments. #p<.001 : in comparison with saline. The similar results were obtained from three additional experiments. 162

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 Fig. 2. The effects of Prescription A on Annexin V/PI expression in MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Prescription A as indicated concentration for 24 hrs and stained with Annexin V/PI. The cells were measured by flow cytometery analysis as described in materials and methods. Data are means of three independent experiments. The similar results were obtained from three additional experiments. Fig. 3. The effects of Prescription A on cell cycle arrest in MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Prescription A as indicated concentration for 24 hrs and stained with PI. (A) The number of sub G1 phase cell was measured by flow cytometery analysis. (B) And the percentage of sub G1 phase cell number was expressed in graph. Data are means of three independent experiments. *p<.05 : in comparison with saline. The similar results were obtained from three additional experiments. 163

에 24 시간처리한후순수한세포를얻어세포를고정시키고, 고정후 PI로염색하여 cell cycle 을측정하였다. Prescription A를처리한군에서는그렇지않은군에비해서 sub-g1 구간이농도의존적으로증가하는것을확인할수있었다 (Fig. 3A and B). 이는 Prescription A가 MIA PaCa-2 세포의 apoptosis 를유발하여 cell cycle arrest 에영향을주어 proliferation 을억제하였음을알수있다. 4. Prescription A가 MIA PaCa-2 cell에서 nuclear morphology 에미치는영향 Apoptosis 에서는세포질과 nuclear chromatin 이응축되는데 Prescription A가 MIA PaCa-2 세포의 apoptosis 를유도함을확인하기위해서세포내핵의 변화를관찰하였다. Prescription A를 MIA PaCa-2 세포와 24시간동안배양하여세포의핵을 DAPI 로염색한후그모양을관찰하였다 (Fig. 4A). 그결과 Prescription A를처리한군에서는핵의응축이농도의존적으로진하게일어났다 (Fig. 4B). 이는 Prescription A가농도의존적으로 MIA PaCa-2 세포의 apoptosis 를유발함을보여준다. 5. Prescription A가 MIA PaCa-2 cell에서 ROS 생성에미치는영향 Prescription A가유발하는 apoptosis 가세포내의 ROS 발생증가와연관이있는지알아보기위해 ROS 를측정하였다. Apoptosis 를유도하기위해서 ROS의활성이중요한요인으로작용한다고알려져있다. Fig. 4. The nuclear morphology of MIA PaCa-2 cells treated with Prescription A. MIA PaCa-2 cells were incubated with or without Prescription A as indicated concentrations for 24 hrs and stained with DAPI. (A) The morphological change of nuclear chromatin observed by fluorescent microscopy as described in materials and methods. (B) And the relative intensity of flourescense was expressed in graph. Data are means of three independent experiments. *p<.05 : in comparison with saline. The similar results were obtained from three additional experiments. 164

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 Prescription A를 24시간동안 MIA PaCa-2 세포와배양한뒤, 세포를순수하게분리하여 DCF-DA 염색후 ROS의변화를 flow cytometer 로분석하였다. 그결과 Prescription A는농도의존적으로 ROS의발현을증가시켰다 (Fig. 5A and B). 또한 Prescription A에의한 MIA PaCa-2 세포의 apoptosis 발생이 ROS를매개로함을알아보기위하여, ROS scavenger 인 NAC를처리하여세포사멸에변화가있는지를측정하였다. ROS scavenger인 NAC를 1시간전에전처리하고 Prescription A를 24시간동안 MIA PaCa-2 세포와배양한후세포생존율을측정한결과 ROS 억제시 Prescription A로 인해일어난세포사멸이거의회복되어있음을알수있었다 (Fig. 5C). 이는 Prescription A가유도한 ROS 가 MIA PaCa-2 세포의 apoptosis 에유의한작용을한다는것을보여준다. 6. Prescription A의약재별 MIA PaCa-2 cell에미치는생존율감소 Prescription A를구성하는약재 4가지중에서어떤약재가 MIA PaCa-2 세포의생존율에영향을미치는지알아보기위하여 MIA PaCa-2 세포에 Artemisiae capillaris Herba, Gentianae Radix, Fig. 5. The effects of Prescription A on ROS production in MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Prescription A as indicated concentration for 24 hrs and stained with DCF-DA. (A) The number of DCF-DA positive cells was measured by flow cytometery analysis. (B) The percentage of DCF-DA positive cells number was expressed in graph. (C) MIA PaCa-2 cells were pre-treated with or without NAC for 1 hr, and incubated with or without Prescription A for 24 hrs. NAC(10 mm) was used as ROS scavenger. Cell viability was measured by MTT assay as described in materials and methods. Data are means of three independent experiments. *p<.05, #p<.001 : in comparison with saline. The similar results were obtained from three additional experiments. 165

Coptidis Rhizoma, Gleditsiae Spina를각각농도의존적으로처리하여 24시간후에세포의생존율을측정하였다. 그결과, Artemisiae capillaris Herba 를투여한군과 Coptidis Rhizoma 를투여한군에서농도의존적으로 MIA PaCa-2 세포의생존율을감소시켰다 (Fig. 6). 7. Artemisiae capillaris Herba가 MIA PaCa-2 cell에서 ROS 생성에미치는영향 Prescription A 중 Artemisiae capillaris Herba 가유발하는 apoptosis 가세포내의 ROS 발생증가와연관이있는지알아보기위해 ROS를측정하였다. Fig. 6. The cytotoxic effects of Artemisiae capillaris Herba, Gentianae Radix, Coptidis Rhizoma and Gleditsiae Spina on MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Artemisiae capillaris Herba, Gentianae Radix, Coptidis Rhizoma and Gleditsiae Spina as indicated concentration for 24 hrs. Cell viability was measured by MTT assay as described in materials and methods. Data are means of three independent experiments. #p<.001 : in comparison with saline. The similar results were obtained from three additional experiments. 166

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 Artemisiae capillaris Herba 를 24시간동안 MIA PaCa-2 세포와배양한뒤세포를순수하게분리하여 DCF-DA 염색후 ROS의변화를 flow cytometer 로분석하였다. 이를통해 Artemisiae capillaris Herba 는농도의존적으로 ROS의발현을증가시킴을알수있었다 (Fig. 7A). 또한 ROS 억제시세포사멸에변화가있는지알아보기위해 NAC를처리하여 apoptosis 에변화가있는지를측정하였다. 그결과 ROS 억제시 Artemisiae capillaris Herba 로인한세포사멸이거의회복되어있음을알수있었다 (Fig. 7B). 이는 Prescription A를구성하는약물중 Artemisiae capillaris Herba 가유도한 ROS가 MIA PaCa-2 세포의 apoptosis 에주요한작용을함을보여준다. 와배양한뒤세포를순수하게분리하여 DCF-DA 염색후 ROS의변화를 flow cytometer 로분석하였다. 그결과 Coptidis Rhizoma 는 ROS 발현을증가시킴을알수있었으며 (Fig. 8A), 또한 NAC를처리하여 ROS 억제시 Coptidis Rhizoma 로인한세포사멸이거의회복되어있음을알수있었다 (Fig. 8B). 이는 Prescription A를구성하는약물중 Artemisiae capillaris Herba 와더불어 Coptidis Rhizoma 역시 Coptidis Rhizoma 가유도한 ROS가 MIA PaCa-2 세포의 apoptosis 에주요한작용을함을알수있었다. 8. Coptidis Rhizoma 가 MIA PaCa-2 cell에서 ROS 생성에미치는영향 Prescription A를구성하는약물중 Artemisiae capillaris Herba 와마찬가지로세포생존율에영향을미친 Coptidis Rhizoma 또한 ROS를측정하였다. Coptidis Rhizoma 를 24시간동안 MIA PaCa-2 세포 Fig. 7. The effects of Artemisiae capillaris Herba on ROS production in MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Artemisiae capillaris Herba as indicated concentration for 24 hrs and stained with DCF-DA. (A) The percentage of DCF-DA positive cells number was expressed in graph. (B) MIA PaCa-2 cells were pre-treated with or without NAC for 1 hr, and incubated with or without Artemisiae capillaris Herba for 24 hrs. Cell viability was measured by MTT assay as described in materials and methods. Data are means of three independent experiments. *p<.05, #p<.001 : in comparison with saline. The similar results were obtained from three additional experiments. 167

Fig. 8. The effects of Coptidis Rhizoma on ROS production in MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Coptidis Rhizoma as indicated concentration for 24 hrs and stained with DCF-DA. (A) The percentage of DCF-DA positive cells number was expressed in graph. (B) MIA PaCa-2 cells were pre-treated with or without NAC for 1 hr, and incubated with or without Coptidis Rhizoma for 24 hrs. Cell viability was measured by MTT assay as described in materials and methods. Data are means of three independent experiments. *p<.05, #p<.001 : in comparison with saline. The similar results were obtained from three additional experiments. Fig. 9. The cytotoxic effects depend on combination ratio of Artemisiae capillaris Herba (ACH) and Coptidis Rhizoma (CR) in MIA PaCa-2 cells. MIA PaCa-2 cells were incubated with or without Prescription A, 10:1 (ACH:CR), 5:1 (ACH:CR), and 1:1 (ACH:CR) as indicated concentration for 24 hrs. Cell viability was measured by MTT assay as described in materials and methods. Data are means of three independent experiments. #p<.001 : in comparison with saline. The similar results were obtained from three additional experiments. 168

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 9. Artemisiae capillaris Herba와 Coptidis Rhizoma 의배율별 MIA PaCa-2 cell에미치는생존율감소 Artemisiae capillaris Herba와 Coptidis Rhizoma 가 Prescription A의주요한효능을보이는약재임에착안하여, Prescription A의농도별생존율과 Artemisiae capillaris Herba 와 Coptidis Rhizoma 의혼합비율이각각 10:1, 5:1, 1:1인경우를비교하여세포생존율에미치는영향을측정하였다. 그결과 Artemisiae capillaris Herba 대비 Coptidis Rhizoma 의비율이증가할수록 MIA PaCa-2 세포의생존율을감소시켰다 (Fig. 9). 특히기존처방대비, Artemisiae capillaris Herba 및 Coptidis Rhizoma 만을처리한그룹에서더유효한세포사멸효과를보인것은 Artemisiae capillaris Herba 및 Coptidis Rhizoma 외의약물은 MIA PaCa-2 세포사멸을억제하는효과를보인것으로도보인다. 이는 Artemisiae capillaris Herba 와 Coptidis Rhizoma 가단미및혼합제제로 MIA PaCa-2 세포의 apoptosis 에유효함을보여준다. Ⅳ. 고찰최근암진단과치료에많은발전이있었으나전세계적으로발생률이증가하고있는췌장암은조기진단과치료가어려워불량한예후를보이는질환이다 24). 췌장암의위험군으로는고령, 흑인, 남자, 흡연, 만성췌장염이나오래지속된당뇨병등이잘알려져있으며, 유발하는원인으로는화학물질, 바이러스감염, 고칼로리식이, 과도한포화지방산의섭취, 알코올섭취, 유전소인등이관련한다 1,25-28). 진단은복부초음파, 복부전산화단층촬영, 내시경역행췌담관조영술, 내시경초음파검사및다양한종양표지자가이용되고있음에도불구하고췌장암이조기에발견되는경우는드물다 29). 게다가췌장암은항암화학요법에제대로반응하지않는대표적인종양이어서반응률이 20% 에미치지못하고, 반응지속기간은 3~6개월정도에불과한현실은치료효과개선을위한다양한약물요법이필요하다 5). 한의학에서췌장암은七情內傷, 飮食不節하여肝脾損傷, 臟腑失和, 濕濁阻遏, 氣血瘀滯가日久하여형성된것으로보고치료는주로健脾和胃, 淸熱利濕, 理氣活血, 軟堅消癥을원칙으로한다 8). 이를바탕으로저자는健脾和胃, 淸熱利濕, 理氣活血, 軟堅消癥에유효한방제를 Prescription A( 茵陳 30g, 龍膽草 15g, 黃連 6g, 皂角刺 2g) 20), Prescription B( 白屈菜 30g, 鬼箭羽 30g) 21), Prescription C( 生薏苡仁 30g, 三棱 15g, 蓬莪朮 15g, 半枝蓮 15g) 22), Prescription D( 蒲公英 40g, 白花蛇舌草 40g, 丹蔘 30g, 薏苡仁 30g, 赤芍藥 15g) 23) 로선정하였다. 그후유의한결과를나타내었던 Prescription A의약물중인진 (Artemisiae capillaris Herba) 과황련 (Coptidis Rhizoma) 이 MIA PaCa-2 세포에미치는영향을연구해보고자하였다. 간암세포에대한항암효과가보고되고있는 Artemisiae capillaris Herba 는사철쑥의幼嫩莖葉으로性味는苦, 微寒하고淸利濕熱, 退黃疸의효능이있어濕熱의熏蒸으로인한黃疸, 身目黃色鮮明, 發熱, 小便短赤또는發黃등의병증을다스리는要藥으로주성분은 coumarin, chlorogenic acid, caffeic acid, essential oil이다 18,30,31). Artemisiae capillaris Herba 는담즙분비를촉진시키고간장해에보호효과를나타내고간세포의괴사를저해하는등의간기능에대한효과, 지질저하, 관상동맥확장및피브린용해효과, 혈압저하효과, 항미생물효과, 해열, 진통및항염증효과가보고되었다 32). Coptidis Rhizoma 는苦寒한性味로淸熱燥濕, 淸心除煩, 瀉火解毒등의효능이있으며주성분은 berberine 으로腸胃의濕熱로인한嘔吐, 瀉痢, 痔瘡, 心火亢盛, 煩躁不眠, 火毒癰瘍, 耳目腫痛, 口舌生瘡등의병증을치료한다 30,31). 또한 169

항염증작용, 항균작용, 항말라리아작용등의약리작용이실험을통해확인되었다 32). 특히 berberine 은구강암, 유방암, 전립선암에대한항암효능이보고되고있다 19,33,34). 암을치료하기위한여러가지방법중에서암세포의죽음을유발시키는것이효율적인치료방법으로대두되고있다 35). 이러한암세포의죽음은감염이나조직손상에의하여발생하는비정상적인과정인세포괴사 (necrosis) 와프로그램된세포죽음의한기전으로개체의발생단계나 DNA 손상, 바이러스감염등에따른유전적조절하에서일어나는정교한개체의생존을위한손상된세포들의제거를위한중요방어기전인세포사멸 (apoptosis) 로구별될수있다 36-40). 그러므로 apoptosis 의균형은개체유지에중요한역할을하지만 apoptosis 과정이실패하게되면암을유발할수있어서최근에는 apoptosis 과정을통하여비정상적인세포나암세포를제거하는치료제개발이중요시되고있다 41,42). 또한 apoptosis 의정도를정량적으로측정함으로써항암화학치료제의투여전감수성지표로이용하는연구들도시행되었다 43-45). 이에저자는췌장암세포주 MIA PaCa-2 세포에있어서 Prescription A의항암효과와그기전을실험적으로알아보고 Prescription A에서주요한작용을한약물인 Artemisiae capillaris Herba 와 Coptidis Rhizoma 가 MIA PaCa-2 세포에서어떻게 apoptosis 를유발하는지에대하여연구하였다. 먼저유효한영향을나타낼것으로예상되는 Prescription A, B, C, D의 MIA PaCa-2 세포에미치는생존율의영향에대해알아보았다. MIA PaCa-2 세포에 Prescription A, B, C, D를각각 0.1, 0.5, 1mg/ml 로처리하여 24시간후에세포증식을관찰한결과 Prescription A 만이농도의존적으로 MIA PaCa-2 세포의생존율을감소시켰다 (Fig. 1A). 또한정상세포에대한독성여부를파악하기위해췌장선방세포주인 266-6 세포에대한증식억제를측정한결과 Prescription A는췌장의정상세포에는영향 을미치지않으면서 MIA PaCa-2 세포에서만농도의존적으로세포증식을억제함을알수있었다 (Fig. 1B). 다음으로이러한생존율및증식율의감소가 apoptosis 에의한것인지확인하기위하여 Annexin V/PI의발현여부, cell cycle arrest, chromatin condensation 등을측정지표로사용하였다. Apoptosis 의특징으로는세포질및염색질의응축, DNA 단편화, 세포표면에 phosphatidylserine 의발현및세포막의수포화현상등과같은형태적또는생태학적변화를동반한다 36,37,46). 여기에서세포막의변화는세포가완전히파괴되기전탐식세포에의해인지되어처리되도록하는결정적인과정으로그변화중한가지가세포막내측에위치하는 phosphatidylserine (PS) 이세포막바깥쪽으로전위되며, 이것이표적으로작용하여식작용을일으킨다 47,48). 전위된 PS는 Ca 2+ 의존성인지질결합특성을가지고있는단백질인 Annexin V와높은친화력을가지고결합하므로이를 early apoptosis 의지표로사용될수있으나 Annexin V는 apoptosis 세포와 necrosis 세포에모두결합할수있으므로이것을구분하기위해핵속의 DNA 에 intercalation 되는 PI를동시에염색한다. Propidium iodide (PI) 는세포괴사에의해손상된세포막을통과하여세포핵에대한표지자로서핵을염색시키므로세포막파괴여부를확인함으로써 apoptosis 와 necrosis 를구분할수있다 45,49,50). Flow cytometry 를이용하여전위된 PS와결합하는 Annexin V와 PI의이중염색으로 necrosis 및 apoptosis 의감별측정이가능하다 51-54). 이러한 Annexin V/PI의이중형광염색에서 Annexin V와 PI 가모두 negative 인상태는정상세포이며, Annexin V만이단독 positive 이고 PI는 negative 인상태는 apoptosis 의초기단계로이미 apoptosis signal로진입된세포를의미한다. Annexin V와 PI가모두 positive 인경우는완전히죽은세포로세포막까지파괴되어핵이노출된상태인 late apoptosis 또는 170

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 ncerosis 를의미하며, Annexin V는 negative 이고 PI 는 positive 인상태는 apoptosis 가아니라외부의심한충격으로죽은 necrosis 단계를의미하며세포막의 PS 노출이없는것이특징이다 49). 본연구에서 Prescription A는농도의존적으로 Annexin V의염색양이증가하였고 PI의염색양은변화하지않았다 (Fig. 2). 이는 Annexin V는 positive 상태이면서 PI는 negative 상태인 early apoptosis 단계를의미하는것으로 Prescription A는농도의존적으로 MIA PaCa-2 세포에대해 early apoptosis 를유도시킨다고볼수있다. Cell cycle analysis 에서 apoptosis 가일어나는동안에는 calcium and magnesium dependent nuclease 들이활성화되어 DNA 를절단하게되고, 이로인해잘려진작은 DNA 절편들은 PBS와같은 washing buffer 에녹아나와 washing 단계에서제거되게된다. 그러므로 apoptosis 가일어난세포를정량적인 DNA-binding dye인 PI를사용하여염색해보면 apoptosis 에의한 DNA fragmentation 현상에의해세포내에존재하는 DNA 양이줄어들어 histogram 에서 G0/G1 기의세포들보다더왼쪽에나타나게되는데이것을 sub-g1 이라고한다 55). Prescription A를 MIA PaCa-2 세포에 24 시간처리한후순수한세포를얻어세포를고정시키고, 고정후 PI로염색하여 cell cycle을측정하였을때 Prescription A를처리한군에서는그렇지않은군에비해서 sub-g1 구간이 Prescription A 0.1 mg/ml 투여군 (5.76 ± 0.12), 0.5 mg/ml 투여군 (14.64 ± 1.20), 1 mg/ml 투여군 (36.42 ± 2.66) 의순으로농도의존적으로증가하는것을확인할수있었다 (Fig. 3A and B). 이는 Prescription A가 MIA PaCa-2 세포의 apoptosis 를유발하여 cell cycle arrest에영향을주어 proliferation 을억제하였음을알수있다. 또한 apoptosis 가유발되었을때특이적으로나타나는핵의형태적변화를관찰하기위해 Prescription A를 MIA PaCa-2 세포와 24시간동안배양하여세포 의핵을 DAPI 로염색한후그모양을관찰하였다 (Fig. 4A). 그결과 Prescription A를처리한군에서는농도의존적으로세포의밀도가감소하였고, apoptosis 가일어난세포에서전형적으로관찰되는 chromatin condensation 에의한 apoptotic body 의형성이증가하는것을확인하였다 (Fig. 4B). 이는 Prescription A가농도의존적으로 MIA PaCa-2 세포의 apoptosis 를유발함을보여준다. 이러한 Annexin V/PI의발현여부, cell cycle arrest, chromatin condensation 의결과들로미루어보아 Prescription A에의한 MIA PaCa-2 세포의생존율및증식율의감소가 apoptosis 에의한것으로보여진다. 한편 ROS는산소원자를포함한화학적으로반응성있는분자로반응성있는산소는짝지어지지않은전자때문에매우반응성이있으며, 산소의정상적인대사작용에의해서자연스럽게생기고세포신호와항상성에중요한역할을한다 56). UV, inflammatory cytokine, ionizing radiation, 다양한항암활성화합물이나대사과정중미토콘드리아에의해서도생성되며, 생체에서는 ROS의생성과제거사이에서세포기능을유지하면서도여러가지자극에의한 ROS의과도한생산은산화스트레스 (oxidative stress) 를일으켜 cell cycle arrest와 apoptosis 를유도하는핵심적인역할을한다 57-60). 이러한 ROS 발생증가가 Prescription A에의해유발된 MIA PaCa-2 세포의 apoptosis 와연관이있는지를알아보기위하여본연구에서는 Prescription A 를 24시간동안 MIA PaCa-2 세포와배양한뒤, 세포를순수하게분리하여 DCF-DA 염색후 ROS의변화를 flow cytometer 로분석하였다. 그결과 Prescription A는농도의존적으로 ROS의발현을증가시켰다 (Fig. 5A and B). 또한 Prescription A에의한 MIA PaCa-2 세포의 apoptosis 발생이 ROS를매개로함을알아보기위하여, ROS scavenger 인 NAC 를 1시간전에전처리하고 Prescription A를 24시간 171

동안 MIA PaCa-2 세포와배양한후세포생존율을측정한결과 ROS 억제시 Prescription A로인해일어난세포사멸이거의회복되어있음을알수있었다 (Fig. 5C). 이는 Prescription A가유도한 ROS가 MIA PaCa-2 세포의 apoptosis 에유의한작용을한다는것을보여준다. 이상의결과를종합하면 Prescription A는 ROS 생성을유도하여 apoptosis 를일으킴으로써 MIA PaCa-2 세포에대하여항암효과가있음을알수있다. 그렇다면 Prescription A를구성하는약재 4가지중에서어떤약재가 MIA PaCa-2 세포의생존율에영향을미치는지알아보기위하여 MIA PaCa-2 세포에 Artemisiae capillaris Herba, Gentianae Radix, Coptidis Rhizoma, Gleditsiae Spina를각각농도의존적으로처리하여 24시간후에세포의생존율을측정하였다. 그결과, Artemisiae capillaris Herba 를투여한군과 Coptidis Rhizoma 를투여한군에서농도의존적으로 MIA PaCa-2 세포의생존율을감소시켰다 (Fig. 6). 다음으로이러한생존율및증식율의감소에영향을미친 Artemisiae capillaris Herba 와 Coptidis Rhizoma 가 ROS 발생증가로인한 apoptosis 를유발하는것인지확인하기위하여 Artemisiae capillaris Herba 와 Coptidis Rhizoma 를각각 24시간동안 MIA PaCa-2 세포와배양한뒤세포를순수하게분리하여 DCF-DA 염색후 ROS의변화를 flow cytometer 로분석하였다. 이를통해 Artemisiae capillaris Herba 는농도의존적으로 ROS의발현을증가시킴을알수있었으며 (Fig. 7A), Coptidis Rhizoma 또한 ROS 발현을증가시킴을알수있었다 (Fig. 8A). 또한 ROS 억제시세포사멸에변화가있는지알아보기위해 NAC를처리하여 apoptosis 에변화가있는지를측정하였는데, 그결과 ROS 억제시 Artemisiae capillaris Herba 로인한세포사멸이거의회복되어있었고 (Fig. 7B), Coptidis Rhizoma 또한 세포사멸이거의회복되어있음을알수있었다 (Fig. 8B). 이는 Prescription A를구성하는약물중 Artemisiae capillaris Herba 와더불어 Coptidis Rhizoma 도 ROS 생성을유도하여 MIA PaCa-2 세포의 apoptosis 에주요한작용을함을보여준다. 더불어 Artemisiae capillaris Herba 와 Coptidis Rhizoma 가 Prescription A의주요한효능을보이는약재임에착안하여, Prescription A의농도별생존율과 Artemisiae capillaris Herba 와 Coptidis Rhizoma 의혼합비율이각각 10:1, 5:1, 1:1인경우를비교하여세포생존율에미치는영향을측정하였다. 그결과 Artemisiae capillaris Herba 대비 Coptidis Rhizoma 의비율이증가할수록 MIA PaCa-2 세포의생존율을감소시켰다 (Fig. 9). 특히기존처방대비, Artemisiae capillaris Herba 및 Coptidis Rhizoma 만을처리한그룹에서더유효한세포사멸효과를보인것은 Artemisiae capillaris Herba 및 Coptidis Rhizoma 외의약물은 MIA PaCa-2 세포사멸을억제하는효과를보인것으로도보인다. 이는 Artemisiae capillaris Herba 와 Coptidis Rhizoma 가단미및혼합제제로 MIA PaCa-2 세포의 apoptosis 에유효함을보여주며, Coptidis Rhizoma 의비율이증가할수록효과가증대됨을미루어볼때 Artemisiae capillaris Herba 와 Coptidis Rhizoma 의각각단미제제로써 apoptosis 에관한추가적실험이앞으로필요할것으로보인다. Ⅴ. 결론 Prescription A(Artemisiae capillaris Herba 30g, Gentianae Radix 15g, Coptidis Rhizoma 6g, Gleditsiae Spina) 를인간유래췌장암세포주인 MIA PaCa-2 세포에처치하여이들의 apoptosis 과정을 MTT assay, FACS analysis, Fluorescence microscopy 등으로분석한결과다음과같은결론을얻었다. 172

주현아외 2 인 : 인진과황련추출물의췌장암세포주 MIA PaCa-2 에대한세포사멸효과 1. Prescription A는농도 (0.1mg/ml, 0.5mg/ml, 1mg/ml) 의존적으로인간유래췌장암세포주 MIA PaCa-2 세포의생존율을유의하게감소시켰다. 2. Prescription A는 Annexin V/PI 이중형광염색에서농도의존적으로 MIA PaCa-2 세포에대해 early apoptosis 를유도시킨다. 3. Prescription A는 MIA PaCa-2 세포에서 sub-g1 구간을농도의존적으로증가시켜 cell cycle arrest 에영향을주어 proliferation 을억제하였다. 4. Prescription A는 MIA PaCa-2 세포에서 chromatin condensation 에의한 apoptotic body 의형성을농도의존적으로증가시켰다. 5. Prescription A는농도의존적으로 ROS 생성을증가시켰고이는 apoptosis 를유도하였다. 6. Prescription A 중에서 Artemisiae capillaris Herba 와 Coptidis Rhizoma 는농도의존적으로 MIA PaCa-2 세포의생존율을감소시켰다. 7. Artemisiae capillaris Herba와 Coptidis Rhizoma 는각각 ROS 생성을유도하여 MIA PaCa-2 세포의 apoptosis 에주요한작용을하였다. 8. Artemisiae capillaris Herba 대비 Coptidis Rhizoma 의비율이증가할수록 MIA PaCa-2 세포의생존율을감소시켰다. 이상의실험결과로볼때 Prescription A를구성하는약물중 Artemisiae capillaris Herba 와 Coptidis Rhizoma 는 ROS 생성을증가시켜 apoptosis 를유도함으로써인간유래췌장암세포주 MIA PaCa-2 세포에유의한효과를나타냈음을알수있다. 감사의글본연구는 2012 년원광대학교교비지원에의해수행되었습니다. References 1. Chung JB. Pancreatic cancer. Seoul:Koon Ja Publishing Inc. 2006:23, 131, 135, 142, 283-4, 287. 2. Cho JH. Recent Update of Molecular Targeted Therapy in Pancreatic Cancer. Korean J Gastroenterol. 2013;61(3):147-54. 3. Barkin JS, Goldstein JA. Diagnostic and therapeutic approach to pancreatic cancer. Biomed Pharmacother. 2000;54:400-9. 4. Sikora SS, Mital BR, Prasad KR, Das BK, Kaushik SP. Functional gastric impairment in carcinoma of the pancreas. Br J Surg 1995;82(8):1112-3. 5. Park JG, Bang YJ, Ha SH. Oncology. Seoul:Iljogak. 2012:317-8, 327-8, 330-1, 329. 6. Cho JG. Herbal Clinical Oncology. Daejeon:Ju Min Publishing Inc. 2005:503. 7. Chen Y, Cong Z. Tumor single prescription Daquan. Beijing:Chinese Medicine Publishing Inc. 1998:508. 8. Moon G, Jeong BH, Kim BJ. Cancer- Combined treatment of East and West 2. Iksan:Wonkwang University Publishing. 1999:356, 377. 9. Ling DQ. Zong liu bian bing zhuan fang zhi liao. Beijing:Ren min wei sheng Publishing Inc. 2000:245. 10. Hu XM. Chinese medicine secret recipe(2). Seoul:Hagyongsa. 1994:699. 11. Zheng YL, Han XW. Zhong xi yi zhong liu zhen liao da quan. Beijing:Zhongguo Zhong yi yao Publishing Inc. 1996:484. 12. Xia HD, Zhang LJ. Zhong liu bing zheng zhi 173

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