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대한한의학방제학회지제 28 권제 1 호 Herbal Formula Science 2020;28(1):63~70 pissn 1229-1218, eissn 2288-5641 https://doi.org/10.14374/hfs.2020.28.1.63 Official Journal of The Korean Medicine Society For The Herbal Formula Study Available at http://www.

com HFS Original Article / 원저 18α-Glycyrrhetinic acid 의위암세포사멸효과에관한연구 김정남, 김병주 * 부산대학교한의학전문대학원양생기능의학부 18α-Glycyrrhetinic acid induces apoptosis of AGS human gastric cancer cells Jeong Nam Kim, Byung

1 대한한의학방제학회지제 28 권제 1 호 Herbal Formula Science 2020;28(1):63~70 pissn , eissn Official Journal of The Korean Medicine Society For The Herbal Formula Study Available at HFS Original Article / 원저 18α-Glycyrrhetinic acid 의위암세포사멸효과에관한연구 김정남, 김병주 * 부산대학교한의학전문대학원양생기능의학부 18α-Glycyrrhetinic acid induces apoptosis of AGS human gastric cancer cells Jeong Nam Kim, Byung Joo Kim * Division of Longevity and Biofunctional Medicine School of Korean Medicine, Pusan National University ABSTRACT Objectives : The purpose of this study was to investigate the anti-cancer effects of 18α-Glycyrrhetinic acid (18α -GA), a hydrolyzed metabolite of glycyrrhizin, in AGS human gastric adenocarcinoma cells. Methods : We used human gastric adenocarcinoma cell line, AGS cells. We examined cell death by MTT assay and caspase 3 and 9 assay with 18α-GA. To examine the inhibitory effects of 18α-GA, sub-g1 analysis was done the AGS cells after 24 hours with 18α-GA. Also, to investigate the inhibitory mechanisms of 18α-GA, mitogen-activated protein kinase pathways and reactive oxygen species (ROS) generation were examined. Results : 1. 18α-GA inhibited the growth of AGS cells in a dose-dependent fashion. 2. Sub-G1 fractions were significantly and dose-dependently increased by 18α-GA α-GA increased the caspase 3 and 9 activities in AGS cells α-GA inhibited proliferation of AGS cells via the modulation of c Jun N terminal kinase (JNK) signaling pathways, which results in the induction of apoptosis α-GA enhanced ROS accumulation in AGS cells. Conclusions : Our findings provide insight into unraveling the effects of 18α-GA in human gastric adenocarcinoma cells and developing therapeutic agents against gastric cancer. c 2020 The Korean Medicine Society For The Herbal Formula Study This paper is available at which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 63

2 대한한의학방제학회지제 28 권제 1 호 (2020 년 2 월 ) Herb. Formula Sci. 2020;28(1):63~70 Key words : 18α-Glycyrrhetinic acid, Human gastric adenocarcinoma cell, AGS, Anti-cancer. Ⅰ. 서론 a) 암은전세계에서가장생명을위협하는질병중하나이며그중에서위암은세계에서두번째로암과관련된사망원인이다 1,2). 위암은매년거의 100만명의새로운환자에게서진단되고있으며, 동아시아, 동유럽, 중남미일부지역에서가장높은발병률을보이고있다 3). 위암치료법은수술만이유일한치료법이라고알려져있지만최근에는위암수술후각종화학요법으로위암치료결과를좋게하고있다 4). 따라서, 새롭고더효과적인항암제를연구하고개발해야한다. 감초 (Glycyrrhiza uralensis) 의성분인 Glycyrrhetic acid (GA) 은간보호같은여러가지질병의치료에자주처방되어온잘알려진한약제제이다 5,6). GA의항암활동메커니즘은세포증식감소, 세포전이억제, 세포주기변화등을통해나타나는것으로알려져왔다 7,8). 18α-Glycyrrhetinic acid (18α-GA; Glycyrrhizin 의가수분해대사물 ) 는항염증, 항바이러스, 항바이러스, 항암효능등을보이는것으로나타났다 9,10). 최근에는 18α-GA 가 gingival fibroblast 의성장을억제해서세포사멸을일으키고, 인간백혈병세포주인 HL-60세포를카스파제 (caspase) 와미토콘드리아 (mitochondria) 의존적으로세포사멸을일으키는것으로보고되었다 11,12). 하지만, 18α-GA 가인간위암세포에미치는작용에대한연구는많이부족한현실이다. 본연구에서는 18α-GA 의위암세포에서의효능및작용기전을확인하는기초적연구를수행하였다. -GA) 는시그마알드리치 (Sigma-Aldrich, St. Louis, MO, USA) 에서구입한후사용하였다. (2) 대상세포주이실험에사용한인체위암세포는서울대학교암세포주은행에서구입한위암세포주 AGS이며, adenocarcinoma 의 histopathology 특징을나타내고있다. 2. 방법 (1) 암세포의배양암세포주는 10% fetal bovine serum (Gibco BRL, MD, USA), 1% broad-spectrum antibiotics (Gibco BRL, MD, USA) 가함유된 RPMI-1640 (Gibco BRL, MD, USA) 배지를이용하여 37, 5% CO 2 incubator (Precision Scientific Inc. NY, USA) 에서배양하였고, 세포는 0.1% Trypsin-EDTA (Sigma, MD, USA) 를이용하여 37 에서 5분간처리한후회수하였다. (2) MTT assay 세포생존율측정은세포배양판 (12-well) 에암세포를 cells/well 씩분주하여 24시간이상배양후 18α-GA 를다양한농도로처리한다음, 37, 5% CO 2 하에서 48시간또는 72시간배양한후, MTT용액 (5 mg/ml) 을배양액최종부피의 1/10되게첨가하였다. 3시간후배양액을제거하고형성된 formazan 침전물을 dimethyl sulfoxide (DMSO) 1 ml로녹여서 540 nm에서흡광도를측정하였다. Ⅱ. 재료및방법 1. 재료 (1) 약재실험에사용한 18α-Glycyrrhetinic acid (18α (3) Caspase 3과 9 assay 측정 Caspase 3 과 9 assay kits를 BioMol (Plymouth, PA, USA) 에서구입하여측정하였다. 405 nm의흡광도에서여러번측정하였다. *Corresponding author : Byung Joo Kim. Division of Longevity and Biofunctional Medicine School of Korean Medicine, Pusan National University, 49, Busandaehak-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 50612, Republic of Korea. Tel : , Fax : , vision@pusan.ac.kr Received : November 7, 2019 / Revised : November 18, 2019 / Accepted : February 25,

18α-GA 을첨가한배지에서 3일간배양된 AGS세포를 PBS용액으로세척한후, 4 에서 30분간 70% ethanol로고정하였다. 고정후 propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) 로 DNA를염색하여 Becton Dickinson FACStar Flow Cytometry 를이용하여세포주기를비교분석하였다.

3 김정남외 1 인 : 18α-Glycyrrhetinic acid 의위암세포사멸효과에관한연구 Kim et al.,18α-glycyrrhetinic acid induces apoptosis of AGS human gastric cancer cells (4) Flow cytometry를이용한세포주기분석 18α-GA 이 AGS세포의증식을억제하는데있어서세포주기의어느단계를지연시키는지 flow cytometry 를이용하여분석하였다. 18α-GA 을첨가한배지에서 3일간배양된 AGS세포를 PBS용액으로세척한후, 4 에서 30분간 70% ethanol로고정하였다. 고정후 propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) 로 DNA를염색하여 Becton Dickinson FACStar Flow Cytometry 를이용하여세포주기를비교분석하였다. (5) 활성산소 (Reactive Oxygen Species; ROS) 측정 AGS세포의 ROS 생성은 DCF-DA (2,7 - dichlorodihydrofluorescein diacetate; Molecular Probes, Eugene, OR, USA) 를사용하여측정하였다. 이세포들은 37 C에서 30분동안 20 μl DCF-DA 로처리되었고 PBS로세척하였다. 488/525 nm의 excitation/emission 파장에서 FACS (Becton- Dickinson, Mountain View, CA, USA) 를사용하여측정하였다. (6) 통계분석실험성적은평균 ± 표준편차 (mean ± SD) 로나 타내었으며, 결과의차이를검정할때에는 Student's t-test로검정하여 p 값이 0.05 미만일때유의한차이가있는것으로판정하였다. Ⅲ. 결과 1. 위암세포사멸효과위암세포사멸효과를측정하기위하여 18α-GA 50, 100, 150, 200 μm 농도로 AGS세포에시험한결과 24시간후인 (A) 에서는 150 μm이상에서뚜렷한위암세포사멸효과를볼수있었으며, 48시간후인 (B) 에서는 50 μm 이상에서뚜렷한위암세포사멸효과를볼수있었다 (Fig. 1). MTT assay로 24 시간후인 (A) 경우에는 50 μm에서 76.5 ± 2.7% (P<0.01), 100 μm 에서 81.4 ± 3.6% (P<0.01), 150 μm에서 56.6 ± 4.6% (P<0.01), 200 μm에서 26.8 ± 2.3% (P<0.01) 의세포생존결과를보였다. 48시간후인 (B) 경우에는 50 μm에서 67.3 ± 4.0% (P<0.01), 100 μm 에서 67.6 ± 3.2% (P<0.01), 150 μm에서 35.7 ± 0.7% (P<0.01), 200 μm에서 2.6 ± 0.1% (P<0.01) 의세포생존결과를보였다. Fig α-GA inhibited the cell viability in AGS cells. Cell viabilities were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) aassay. (A) 18α-GA reduced cell viabilities in a dose-dependent manner for 24 h. (B) 18α-GA reduced cell viabilities in a dose-dependent manner for 48 h. Bars represent mean values±s.d. **P < CTRL: Control. 2. Flow cytometry 를이용한세포주기분석 18α-GA 에의한세포사가 apoptosis 에의한것인 지확인하기위하여 flow cytometry 를이용한세포 주기 sub-g1 분석을실시하였다. 18α-GA 에의한 65

대한한의학방제학회지제 28 권제 1 호 (2020 년 2 월 ) Herb. Formula Sci. 2020;28(1):63~70 Sub-G1 분석결과 18α-GA 농도에비례해서 sub-g1 peak 가증가함을알수있다. Sub-G1 비율 은 50 μm 에서 3.9 ± 1.6%, 100 μm 에서 5.5 ± 2.1%, 150 μm 에서 6.3 ± 0.

4 대한한의학방제학회지제 28 권제 1 호 (2020 년 2 월 ) Herb. Formula Sci. 2020;28(1):63~70 Sub-G1 분석결과 18α-GA 농도에비례해서 sub-g1 peak 가증가함을알수있다. Sub-G1 비율 은 50 μm 에서 3.9 ± 1.6%, 100 μm 에서 5.5 ± 2.1%, 150 μm 에서 6.3 ± 0.8% (P<0.05), 200 μ M 에서 18.1 ± 3.1% (P<0.01) 로나타났다 (Fig. 2). Fig. 2. Effect of 18α-GA on AGS cell apoptosis. (A) Cells were treated with 18α-GA for 24 h. Cell cycle analysis was conducted by flow cytometry. (B) Sub-G1 fractions were significantly and dose-dependently increased by 18α-GA. Sub-G1 fractions are expressed as percentages. Bars represent the mean values ± S.D. **P < CTRL: Control. 3. Caspase 3과 caspase 9를통한암세포사멸확인 18α-GA 에의한세포사가 apoptosis 에의한것인지확인하기위하여농도별로세포사멸의진행정도를알아보았다. Caspase 3과 9의활성도를측정한 결과 18α-GA에의해 caspase 3과 9의활성도가농도에의존적으로증가함을알수있고이러한활성도는 pan-caspase inhibitor 인 zvad-fmk 에의해서억제됨도알수있었다 (Fig. 3). Fig α-GA increased the caspase 3 and 9 activities in AGS cells. Caspase assays were performed following the addition of the indicated 18α-GA concentration for 24 h. 18α-GA and zvad fmk as a pan-caspase inhibitor were simultaneously treated. Bars represent the mean values ± S.D. *P < **P < CTRL: Control. 66

억제제인 PD98059 을이용하여 MTT 방법으로세포사멸변화여부를알아보았다. 18α-GA 와 SP600125 를함께투여할경우 18α-GA 단독투여시에비해서세포사멸효과가감소함을보였지만, PD98059 인경우는세포사멸변화 가없었다. MTT assay로 SP600125 처치시 50 μm 에서 79.9 ± 4.9%, 100 μm에서 76.6 ± 0.

5 김정남외 1 인 : 18α-Glycyrrhetinic acid 의위암세포사멸효과에관한연구 Kim et al.,18α-glycyrrhetinic acid induces apoptosis of AGS human gastric cancer cells 4. 18α-GA 에의한 apoptosis 에서 c Jun N terminal kinase (JNK) 와 p42/44 mitogen-activated protein kinase (MAPK) 의관련성확인 18α-GA 에의한 AGS 세포사멸에서 JNK 와 p42/44 MAPK 기전관련성을알아보기위해서 JNK 억제제인 SP 와 p42/44 MAPK 억제제인 PD98059 을이용하여 MTT 방법으로세포사멸변화여부를알아보았다. 18α-GA 와 SP 를함께투여할경우 18α-GA 단독투여시에비해서세포사멸효과가감소함을보였지만, PD98059 인경우는세포사멸변화 가없었다. MTT assay로 SP 처치시 50 μm 에서 79.9 ± 4.9%, 100 μm에서 76.6 ± 0.2%, 150 μm에서 58.8 ± 4.3%, 200 μm에서 35.5 ± 2.1% (P<0.05) 의세포생존결과를보였다 (Fig. 4A). 또한 PD98059 처치시에는 50 μm에서 76.5 ± 2.1%, 100 μm에서 62.8 ± 1.6%, 150 μm에서 35.9 ± 3.9%, 200 μm에서 21.1 ± 4.7% 의세포생존결과를보였다 (Fig. 4B). 이러한결과는통계적으로유의하게 JNK기전이 18α-GA 에의한 apoptosis 에관여함을알수있다. Fig. 4. Effect of 18α-GA on the JNK and p42/44 MAPK pathway in AGS cells. MTT assays were used to determine cell viabilities in the presence of (A) SP (JNK II inhibitor) or (B) PD98059 (a p42/44 MAPK inhibitor). Cells were treated with the indicated concentrations of 18α-GA with SP or PD98059 for 24 h. Bars represent the mean values ± S.D. **P < # p<0.05 compared with each other. CTRL: Control. 18α-GA: 18α-Glycyrrhetinic acid α-GA 에의한 apoptosis 에서세포내활성산소의관련성확인 18α-GA 에의한 AGS세포사멸에서활성산소발생여부를알아보기위해서형광염색제 DCF-DA 를 이용하였다. AGS 세포에 18α-GA 를주고 flow cytometry 를이용하여활성산소를측정해보면 18α-GA 농도 의존적으로활성산소발생이증가하였다 (Fig. 5). Fig α-GA enhanced ROS accumulation in AGS cells. (A) Cells were stained with DCF-DA and intracellular ROS levels were measured. (B) Intracellular ROS was detected in AGS cells treated with the indicated concentrations of 18α-GA for 24 h. Bars represent the mean values ± S.D. *P < **P < CTRL: Control. ROS: Reactive Oxygen Species. 67

6 대한한의학방제학회지제 28 권제 1 호 (2020 년 2 월 ) Herb. Formula Sci. 2020;28(1):63~70 Ⅳ. 고찰전통의학은다양한질병을치료하기위한다양한많은약초들의조합으로구성되어있다. 많은질병들중에서, 특히암에대한유용한보완적인치료법으로서이러한약초들과구성성분들이효과적이라고인식되고있다 13). 감초추출물은여러가지질병의치료에가장자주처방되어온잘알려진한약제제이다 14). 감초는부상이나붓기치료, 해독작용에사용되고, 약제, 담배, 껌, 사탕, 음료등에서도향신료와감미료로사용된다 15). 감초의주요활성성분은 glycyrrhizin 이다 16). Glycyrrhizin 은항염증, 면역조절, 항암작용등여러가지약리활동을하고있는것으로보고되고있다 17-19). Glycyrrhizin 은장에서 glycyrrhetinic acid 로바뀌고더좋은활성을나타낸다 20). Glycyrrhetinic acid는 trans 형태의 18α-glycyrrhetinic acid (18 α-ga) 와 cis 형태의 18β-glycyrrhetinic acid (18 β-ga) 가있다 21-23). 18α-GA 는 gingival fibroblast 의성장을억제해서세포사멸을일으키고, 인간백혈병세포주인 HL-60 세포에서도 caspase 와 mitochondria 의존적으로세포사멸을일으키는것으로보고되었다 11,12). 또염증관련유전자를조절해서전립선암세포사멸효능을나타내고있다 24). 18β-GA 는 COX-2 발현을억제하고, mir-149-3p-wnt-1 기전을활성화시켜위암세포의성장을억제한다는보고가있고, ROS/protein kinase C (PKC)-α/extracellularsignal-regulated kinase (ERK) 기전을통해서위암세포의이동과전이를억제한다고알려지고있다 25,26). 하지만 18α-GA 에의한위암세포사멸에대한연구는거의되어있지않다. 이번연구에서위암세포주에대한 18α-GA 의효능을연구하여 caspase, JNK에의존적이며활성산소를일으켜세포사멸을일으키는것을확인하였다. 세포사멸은발달과노화단계에서정상적으로일어나는항상성유지메커니즘의하나로면역반응과같은방어기제로서도발생할수있고, 세포가질병이나유해물질에의해손상되었을때도발생할수있다 27,28). 이러한세포사멸의이해는항암효능을나타내는다양한물질의반응과작용기전을이해할수있으며, 새로운항암제개발에중요한아이디어를제공할수있다고생각된다. 세포내부또는세포상호간신호를담당하는막단백질중하나인이온채널또한암세포사멸에중요한역할을한다 29). 이온채널중일시적수용체전압멜라스타틴타입 7 (transient receptor potential melastatin 7; TRPM7) 채널은위암세포의생존에필수적이며, 따라서위암치료타겟중하나이다 30,31). TRPM2 채널도 AKT 신호경로를통한위암이동및전이에중요한역할을한다 32). 또한 human ether à-go-go-related gene 1 (herg1) 은인간의위암에서비정상적으로표현되며, 위암의예측마커로이용될수있는것으로알려지고있다 33). 따라서이러한이온통로가 18α-GA 에의한위암성장억제효과에관여하고있는지여부를연구할필요가있다고생각된다. 본연구에서는 18α-GA 에의한위암세포인 AGS 세포에서의항암작용을조사해보았다. 24시간후에 18α-GA 농도의존적으로 AGS세포가사멸되는효과를보았고, sub-g1 analysis 와 caspase 3 또는 9 를통해서세포사멸에 apoptosis가관여함을알수있었다. 또 18α-GA 에의한세포사멸에 JNK 기전이관여함을확인하였고, 활성산소가발생되어세포사멸을조절할수있음을알수있었다. 따라서 18α-GA 에의한인체위암세포에서의효과를확인할수있었고, 이런결과를바탕으로한의학에서사용하는감초의성분중하나인 18α-GA 효능에대한적응증을확대시킬수있는좋은자료가될수있을것으로생각된다. 감사의글이성과는 2017년도정부 ( 과학기술정보통신부 ) 의재원으로한국연구재단의지원을받아수행된연구임 (No. 2017R1A2B ). References 1. Luo Z, Zeng H, Ye Y, Liu L, Li S, Zhang J, et al. Safflower polysaccharide inhibits the proliferation and metastasis of MCF-7 breast cancer cell. Mol Med Rep. 2015;11: Sitarz R, Skierucha M, Mielko J, Offerhaus GJA, Maciejewski R, Polkowski WP. Gastric 68

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical