ORIGINAL ARTICLE Korean J Clin Lab Sci. 2018;50(3):337-344 https://doi.org/10.15324/kjcls.2018.50.3.337 pissn 1738-3544 eissn 2288-1662 Korean J Clin Lab Sci. Vol. 50, No. 3, September 2018 337 Silymarin Attenuates Invasion and Migration through the Regulation of Epithelial-mesenchymal Transition in Huh7 Cells Do-Hoon Kim 1, So-Jeong Park 1, Seung-Yeon Lee 1, Hyun-Seo Yoon 2, Chung Mu Park 1 1 Department of Clinical Laboratory Science, Dong-Eui University, Busan, Korea 2 Department of Dental Hygiene, Dong-Eui University, Busan, Korea 간암세포주에서상피간엽전환억제를통한 Silymarin 의침윤및전이억제효과 김도훈 1, 박소정 1, 이승연 1, 윤현서 2, 박충무 1 1 동의대학교임상병리학과, 2 동의대학교치위생학과 Hepatocellular carcinoma (HCC), a major type of hepatoma, is associated with high recurrence and mortality because of its uncontrolled metastatic feature. Silymarin is a polyphenolic flavonoid from Silybum marianun (milk thistle) and exhibits anti-carcinogenic activity through modulation of the epithelial-mesenchymal transition (EMT) in several cancer cells. In this study, the inhibitory mechanism of silymarin against migration and invasion was investigated in the Huh7 HCC cell line. Wound healing and in vitro invasion assays were conducted to examine the effects of silymarin on migration and invasion. Western blot analysis was also applied to evaluate the inhibitory effects of silymarin on the EMT-related genes and their upstream signaling molecules. Silymarin inhibited the migratory and invasive activities of Huh7 cells. In addition, silymarin attenuated the protein expression levels of vimentin and matrix metalloproteinase (MMP)-9 as well as their transcription factors, Snail, and nuclear factor (NF)- B, while the expression of E-cadherin was increased by the silymarin treatment. Among the upstream signaling molecules, the phosphorylation of Akt was inhibited by the silymarin treatment, which was confirmed by the selective inhibitor, LY294002. Consequently, silymarin inhibited the invasive and migratory activities in Huh7 cells through the modulation of EMT-related gene expression by the PI3K/Akt signaling pathway, which may have potential as a chemopreventive agent against HCC metastasis. Key words: Epithelial-mesenchymal transition, NF-kappa B, Silymarin, Snail family transcription factors Corresponding author: Chung Mu Park Department of Clinical Laboratory Science, Dong-Eui University, 176 Eomgwang-ro, Busanjin-gu, Busan 47340, Korea Tel: 82-51-890-2685 Fax: 82-505-182-6877 E-mail: cmpark@deu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2018 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: July 18, 2018 Revised: August 2, 2018 Accepted: August 2, 2018 서론 원발암에서전이를시도하는암세포의중요한특징중하나는상피간엽전환 (epithelial-mesenchymal transition, EMT) 으로상피세포가간엽세포로바뀌는것을말한다. 그과정에서상피세포는세포간부착능력을잃고유리세포로바뀌면서침투성을동시에얻어간엽세포로분화하게된다 [1]. EMT는정상적인조직의발달과염증의유발, 암세포의전이에관련된것으로
338 Do-Hoon Kim, et al. Anti-invasive and Anti-migratory Effects of Silymarin in Huh7 Cells 알려져있다 [2]. 특히, EMT는전이가발생하는암의진행단계에서나타나는두드러진특징으로상피세포는상피-상피간혹은상피-기질간부착단백질이존재하고극성이보이지만중간엽세포는개개의세포로떨어져서움직이고극성을보이지않는다. 이러한중간엽세포의특성을보이는세포는상피내암세포보다항암제에강한저항성을보이고전이능또한높아지게된다 [3]. EMT의중요한과정중하나는 E-cadherin 이 N-cadherin 으로전환되는 cadherin switch로세포간부착단백질인 E-cadherin 이소실되고 N-cadherin 이세포표면에활성화된다. 그리고 cadherin 의활성화와관련된 matrix metalloproteinase (MMP)-9과중간엽줄기세포의특징인 vimentin 또한발현이증가하는것으로알려져있다 [4]. 이들의발현조절과정에서전사인자인 Snail과 nuclear factor (NF)- B가증가하였고, 특히 Snail은 E-cadherin 을억제하고침윤도를높이는것으로밝혀졌다 [5]. 따라서 Snail과 NF- B의조절을통해 EMT 관련유전자의발현을조절하면암세포의전이능을조절할수있을것으로생각된다. 실리마린은엉겅퀴 (Silybum marianun, milk thistle) 에서추출한 polyphenolic flavonoid로서 silybinin을주성분으로하며기타성분으로는 silybin, dihydrosibilyn, silydianin, silychristin 등이있는것으로알려져있다 [6-9]. Silymarin은항산화효과, 지질과산화억제효과등으로인해간보호효과가있고, 간세포막의독성물질이수용체와결합후세포막을파괴하는작용을방해함으로써세포를안정시켜간세포를보호하기도하며혈중지단백의대사를정상화함으로써혈중콜레스테롤수치를낮추는효과가있는것으로보고되었다 [10]. 특히, silymarin은 non-small cell lung carcinoma, prostate cancer, bladder cancer 등다양한암세포에서 EMT 조절을통해항암효과를보이는것으로보고되었으나간세포암에서는그효과가보고되지않았으므로본논문에서는그활성을분석하고자하였다 [7, 9, 11]. 재료및방법 1. 세포배양및시료실험에사용한 Huh7 cell (human hepatocellular carcinoma) 은한국세포주은행 (Seoul, Korea) 에서분양받았고, 10% 의 fetal bovine serum (FBS, Hyclone, South Logan, UT, USA), Penicillin/Streptomycin (Hyclone) 이포함된 DMEM배지를사용하여 37 C, 5% CO 2, 가습된조건에서배양하였다. Silymarin, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) 는 Sigma-Aldrich (St. Louis, MO, USA) 에서구입하였다. Western blot을위한 E-cadherin (clone 24E10, mab), vimentin (clone D21H3, mab), MMP-9 (clone D6O3H, mab), Snail (clone C15D3, mab), p-p65 (clone 93H1, mab), phospho-akt (clone D9E, mab), Akt (clone C67E7, mab), phospho-extracellular signal-regulated kinase (p-erk, clone D13.14.4E, mab), ERK (clone 137F5, mab), phospho-c-jun NH 2 -protein kinase (p-jnk, clone 81E11, mab), JNK (pab), phospho-p38 (clone D3F9, mab), p38 (clone D13E1, mab), actin (clone 13E5, mab) 등에대한 1차항체는토끼를호스트로하여제조된것을, 2차항체인 horseradish peroxidase (HRP)-conjugated anti-rabbit IgG는 Cell Signaling Technology (Boston, MA, USA) 에서모두구입하여 1:1,000의비율로희석하여분석에사용하였다. 2. 세포독성평가세포생존율은 EZ-Cytox cell viability assay kit (Daeil lab service, Seoul, Korea) 을사용하여측정하였다. Huh7 cell을 24 well plate에 1 10 5 cell/well 의농도로파종한후 24시간동안배양하고 silymarin을농도별로처리하였다. 그후 24시간배양하고 EZ-Cytox 시약 10 L를첨가하여 1시간동안배양한후 microplate reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA) 로 480 nm에서흡광도를측정하였다. 3. Western blot 분석 Huh7 cell은 100 mm dish에 5 10 6 cells/dish 의농도로파종하고 24시간동안배양한후 silymarin 을농도별로처리한후다시 24시간동안배양하였다. 시료처리가끝난세포는 PBS로 2회세척하고 0.5 ml의단백질추출용액 (PRO-PREP, Intron Biotechnology, Seongnam, Korea) 으로수확한후얼음에 10 분동안정치하여세포를용해하였다. 용해된세포가포함된단백질추출용액은 13,000 g에서 5분동안원심분리한후분리된상층을새튜브로옮겨분석에사용하였다. 단백질농도는 Bradford 법으로정량하였다. 분리한단백질 50 g이포함된시료를준비하여 10% SDS-polyacrylamide gel에전기영동한후 polyvinylidene fluoride membrane (PVDF, Bio-rad) 으로이동시켰다. 단백질이이동된 PVDF membrane 은 5% 탈지분유를 TBST에녹인용액으로 1시간동안실온에서블로킹을진행하였다. 블로킹반응이끝난 membrane 은 1:1,000 으로희석한 1차항체와섞은후 4 C에서 24시간동안보합반응을하였다. 1 차항체와의보합반응이끝난 membrane 은 TBST로 3회세척
Korean J Clin Lab Sci. Vol. 50, No. 3, September 2018 339 후다시 1:1,000으로희석한 2차항체와실온에서 2시간동안보합반응을하였다. 2차항체까지반응이끝난후 enhanced chemiluminescence solution (ECL, Santa Cruz Biotechnology, Dallas, TX, USA) 을이용하여반응을유도한후 ECL sensitive film에감광시켜단백질의발현변화를측정하였고 Gel Doc EQ system (Biorad) 으로정량분석하였다. 4. Wound healing 분석 Huh7 cell을 6 well plate에 3 10 5 cell/well 의농도로파종후 24시간동안배양하여세포가 plate에부착되도록하였다. 그후 10 L pipet tip을이용하여 plate의바닥을일자로긁어 wound를만들었다. Silymarin을농도별로처리한후 24, 48시간동안배양하면서시간별로세포가이동한정도를현미경으로관찰하여결과를비교하였다. 5. In vitro invasion 분석 Huh7 cell을 invasion이가능한용기의 transwell (8 μm pore, Corning Inc, Corning, NY, USA) 위쪽에 silymarin과함께 1 10 5 cell/well 의농도로분주후 24시간동안배양하였다. 24시간후위쪽에남아있는세포는면봉으로제거후 transwell membrane 의하단으로침투한세포는 crystal violet 으로염색하여확인하였다. 6. 통계분석 3회반복시행한모든실험결과는 SPSS 통계프로그램 (version 25.0, SPSS Inc., Chicago, IL, USA) 을이용하여평균 ± 표준편차 (mean ±SD) 로나타내었다. 실험군간의유의성검증에는일원배치분산분석 (one-way ANOVA) 을이용하였고, Duncan s multiple range test 방법으로사후검증을시행하였다. 대조군과실험군사이의비교에는 t-test를사용하였고, P 값이 0.05 미만인경우통계적으로유의한것으로판단하였다. 결과 1. Silymarin 에의한 Huh7 cell 의 cell migration 억제효과 Silymarin이간세포암세포주인 Huh7 cell의침윤과전이에미치는영향을알아보기위하여 silymarin 이 cell migration에미치는효과를분석하였다. 먼저 silymarin이 Huh7 cell에미치는세포독성효과를알아본결과 Figure 1에서보는바와같이 silymarin 을 12.5, 25, 50, 100 g/ml의농도로 24시간처리했을때 50, 100 g/ml에서 10, 20% 가량세포생존율이감소하는것을확인할수있었다. 그러므로 wound healing assay, in vitro invasion assay의 silymarin 50, 100 g/ml 실험군에는 Huh7 cell을 10, 20% 더파종하여세포독성에의한효과를상쇄하고자하였다. Huh7 cell은세포자체가가지는침윤ㆍ전이능으로인해강한 cell migration과 invasion activity를보였다 [12]. Huh7 cell이파종된 6-well plate에 pipet tip으로 scratch를만든후 silymarin을처리한결과 Figure 2A에서보는것과같이 Huh7 cell의 migration 에의해 24시간, 48시간이경과함에따라 wound healing이이루어졌으나 25, 50, 100 g/ml 의농도로 silymarin 을처리한세포의경우에는 migration 이현저히떨어져 wound healing이제대로일어나지않는것을확인할수있었다. 그리고이결과를바탕으로 in vitro에서침윤을억제하는효과가있는지의여부를 8 m의 pore size를가진 transwell membrane 을이용하여분석하였다. Figure 2B 에서보는바와같이시료를처리하지않은세포의경우 transwell membrane을통과하여 chamber 의아래쪽으로많은수의세포가이동한것과달리 silymarin을처리한세포의경우이동한세포의수가현저히줄어드는것을확인할수있었다. 이결과로미루어보아 silymarin 은 Huh7 cell의 cell migration 과 in vitro에서의침윤을억제할수있는능력이있음을알수있었다. Figure 1. Cytotoxic effect of silymarin in Huh7 cells. Huh7 cells were treated with indicated concentrations of silymarin for 24 hours. Cell viability was estimated by a WST assay. Data represent the mean±sd of triplicate experiments. *P<0.05 vs untreated control. 2. Silymarin 에의한 EMT 관련유전자발현조절 Wound healing assay와 in vitro invasion assay 결과, silymarin은 Huh7 cell의침윤과전이를억제하는활성이있는
340 Do-Hoon Kim, et al. Anti-invasive and Anti-migratory Effects of Silymarin in Huh7 Cells Figure 2. Inhibitory effect of silymarin on cell migration and in vitro invasion in Huh7 cells. Wound healing and in vitro invasion assays were conducted with indicated concentrations of silymarin for 24 and 48 hours in Huh7 cells. 것으로 판단할 수 있었다. 그러므로 silymarin이 EMT 관련 유 전자의 조절을 통해 이러한 활성을 보이는지를 확인하기 위하 여 Huh7 cell내에서 발현되는 관련 단백질의 발현을 western blot analysis를 이용하여 분석하였다. 본 연구에서는 EMT 관 련 유전자 중 세포 표면 단백질인 E-cadherin, 세포외 기질을 분해하는 효소인 MMP-9과 중간엽세포의 지표인 vimentin을 분석하였다. E-cadherin은 인접한 세포의 E-cadherin 분자와 부착하여 세포간 강한 결합을 유지하고 세포의 극성을 유지하 는 역할을 한다. 그러나 종양세포에서 EMT과정이 진행되면 E-cadherin의 발현이 줄어들고 상피세포간 이음부가 느슨해 지면서 MMP의 활성에 의해 세포외기질이 분해됨에 따라 상피 세포가 간질로 이동할 수 있게 된다[13]. 더불어 EMT가 진행될 수록 종양세포는 상피세포보다 전이에 유리한 줄기세포의 특성 을 강하게 보이게 되고 이 때 특징적으로 발현되는 단백질이 vimentin이다[14]. 이들을 분석한 결과 Figure 3에서 보는 바 와 같이 silymarin의 처리에 의해 농도의존적으로 E-cadherin 의 발현은 증가하고 vimentin은 감소하는 결과를 볼 수 있었다. Figure 3. Silymarin inhibited expression of E-cadherin, vimentin, and MMP-9 in Huh7 cells. Panel A shows the protein expression levels of E-cadherin, vimentin, and MMP-9 by silymarin. All signals were normalized to the protein level of actin, an internal control, and expressed as a ratio (Panel B). Data represent the mean±sd of triplicate experiments. *P 0.05, **P 0.005 vs untreated control. 또한, 세포외기질을 분해하여 종양세포의 전이를 돕는 MMP-9 의 활성 또한 silymarin의 처리에 의해 억제되는 것을 확인할 수 있었다. 그리고 세포 내 여러 전사인자 중 Snail과 NF- B는 EMT가 진행되는 동안 활성이 증가함으로써 종양세포의 침윤 과 전이활성을 높이는 것으로 보고되고 있고[5], 본 실험에서 이
Korean J Clin Lab Sci. Vol. 50, No. 3, September 2018 341 두전사인자의활성은 silymarin에의해유의적으로억제되었다 (Figure 4). 이러한결과로미루어보아 silymarin 은 E- cadherin 증가와 vimentin의활성을억제하여 Huh7 cell의 EMT 유도를효과적으로저해하고, 종양세포주변세포외기질의재구성에관여하는효소인 MMP-9의활성을억제함으로써종양세포의침윤과전이를억제하는것으로나타났다. 그리고이러한 EMT 관련유전자들의발현조절은전사인자인 Snail과 NF- B의활성억제를통한것임을알수있었다. 3. 상위신호전달체계의조절을통한 Silymarin의 EMT 유전자발현조절다양한세포외부의자극을세포내부로전달하는역할을하는신호전달물질중 mitogen activated protein kinases (MAPKs) 와 PI3K/Akt는세포의증식, 분화, 사멸을포함한여러가지세포의활성을매개하는 serine/threonine kinase이다. 특히이들은 leptin으로유도된간세포암의성장, 침윤과전이에관련된것으로보고되기도하였다 [15]. 따라서본연구에서 는 Huh7 cell의 EMT관련유전자조절에어떤신호전달물질이관여하고, silymarin 에의해조절되는지의여부를분석하였다. 실험결과 Figure 5에서보는바와같이 silymarin이 ERK, JNK, p38의인산화에는영향을주지못하였으나 Akt의인산화는유의적으로억제하는것을확인할수있었다. 또한 silymarin 이 PI3K/Akt 신호전달경로를통해 EMT 관련유전자를조절하는지검증하기위하여 PI3K의선택적저해제인 LY294002를 20 M로처리한후 E-cadherin, vimentin, MMP-9의발현정도를분석한결과 Figure 6에서보는것과같이 EMT 관련유전자들의발현이억제되는것을확인할수있었다. 이상의결과로 silymarin은 PI3K/Akt 경로를통해 EMT 관련유전자를조절함으로써 Huh7 cell의전이와침윤을억제하는것으로생각된다. Figure 4. Silymarin inhibited the expression of Snail and p-p65 in Huh7 cells. Panel A shows the protein expression levels of Snail and p-p65, one subunit of NF- B, by silymarin. All signals were normalized to the protein level of Actin, an internal control, and expressed as a ratio (Panel B). Data represent the mean±sd of triplicate experiments. Values sharing the same superscript are not significantly different at P<0.05 by Duncan s multiple range tests. *P<0.05, **P<0.005 vs untreated control. Figure 5. Silymarin inhibited phosphorylation of Akt in Huh7 cells. Panel A shows the protein expression levels of p-akt, p-erk, p-jnk and p-p38 by silymarin. All signals were normalized to protein levels of Akt, ERK, JNK and p38, internal controls, and expressed as a ratio (Panel B). Data represent the mean±sd of triplicate experiments. *P<0.05, **P<0.005 vs untreated control.
342 Do-Hoon Kim, et al. Anti-invasive and Anti-migratory Effects of Silymarin in Huh7 Cells Figure 6. Silymarin inhibited EMT-related genes expression depends on PI3K/Akt signaling pathway in Huh7 cells. Twenty microliters of a selective inhibitor of PI3K was treated in Huh7 cells. Protein expression of E-cadherin, vimentin, and MMP-9 was analyzed by Western blot analysis. The data are representative of three independent experiments. The relative induction of EMT-related genes expression was quantified by densitometry and actin was used as an internal control. The data represent the mean±standard deviation of triplicate experiments. *P<0.05, **P<0.005 vs untreated control. 고찰 우리나라에서발생하는악성신생물중간암은인구 10만명당 30.9명이발생하여 6위에해당하지만, 사망률은폐암에이어두번째로높은 21.5명에이르는것으로보고되고있다 [16]. 조직학적으로는간암전체발생건수가운데암종이 96.6%, 육종이 0.2% 를차지하고암종중에서간세포암이 76.2% 로가장많았다 [17]. 특히간세포암은간절제술등의치료후에높은재발율로인해예후가매우불량한것으로알려져있다 [18]. 이러한높은재발율은간암의전치절제후남아있는간조직에서발생하고이렇게발생한암은전체재발암의 75% 를차지한다. 절제후초기에재발하는암은주로간내전이로부터발생되고후기재발암은다발성발암과정으로인해발생하는것으로보고되고있다 [19, 20]. 따라서, 본연구에서는간염의치료에효과적인것으로알려진 silymarin 이 EMT 조절을통해간세포암의침윤과전이를억제하는지를밝히고자하였다. 분석결과 Huh7 cell의운동성에의해시간이경과함에따라 wound healing이이루어졌으나 silymarin을처리한세포는운동성이현저히떨 어져 wound healing이제대로일어나지않았다. In vitro invasion assay에서도 silymarin 을처리한세포의이동이현저히억제되는것을볼수있었고이결과를바탕으로 silymarin이 HCC인 Huh7 cell의세포운동성을억제하는활성이있음을알수있었다. 그리고 silymarin 에의해억제된 Huh7 cell의운동성이침윤과전이를억제하는지를확인하기위하여 EMT와관련된전이와침윤단백질의발현을분석하였다. EMT의중요한과정중하나는 E-cadherin 이 N-cadherin 으로전환되는 cadherin switch로서세포간의부착을유도하는세포표면단백질인 E-cadherin 이소실되면서 N-cadherin 이활성화되는과정이다. 이 cadherin switch를조절하는데 MMP의활성이중요한것으로알려져있고, E-cadherin 의활성을잃은암줄기세포유사세포는 MMP에의해분해된세포외기질을통해간질로이동할수있게된다 [13]. MMP는종양미세환경조절에중요한역할을하는효소로서세포외기질을분해하여조직내에서종양세포가움직일수있는공간을확보하고, 세포간부착단백질인 E-cadherin 을분해하여종괴에서종양세포가떨어질수있도록할뿐만아니라 integrin 을활성화하여다른조직에부착할수있는능력을높이는역할을한다. 암줄기세포 (cancer stem cell) 의기원은정확하게알려져있지않지만, 상피세포를 tumor growth factor (TGF)- 자극하여 EMT를유도하였을때종양줄기세포와유사한표현형을보이므로 EMT의진행이암줄기세포의생성에관여되어있는것으로생각하고있다 [21]. 또한중간엽줄기세포의주요한특성중하나이고상피세포에서는발현되지않는것으로알려져있는 vimentin은흑색종에서처음으로과발현과암의전이가관련이있음이보고된이후유방암, 자궁경부암, 신장암, 전립선암, 간세포암에서도동일한결과가보고되었다 [22]. EMT 과정에서발현되는 vimentin은세포골격단백질인 actin과다른중간섬유들과의상호작용을통한연속적인형태변화와재구성에필수적인단계로이해되고있고, 이러한이유로인해 vimentin이전이능과관련이있을것으로생각된다 [14]. EMT가유도될때발현이증가되는전사인자로 Snail, Twist, zinc-finger E-box-binding (ZEB) 이있고이들은상피세포의표현형인 E-cadherin 을억제하고중간엽세포의표현형인 vimentin을활성화할뿐만아니라종양주변의 ECM을재구성하는데관여하는 MMP의활성도촉진하는것으로보고되고있다 [5]. 뿐만아니라염증과관련된인자들을조절하는데핵심적인역할을하는 NF- B 또한 EMT과정에서상기의전사인자들과같은역할을하는것으로알려지고있다 [4]. 특히, Hsieh 등 [12] 의연구에의하면간세포암세포주에서 NF- B와 activator
Korean J Clin Lab Sci. Vol. 50, No. 3, September 2018 343 protein (AP)-1의조절을통해 MMP-9의활성을억제함으로써침윤과전이를억제한다고보고하였다. 본연구에서 E-cadherin 과 vimentin을분석한결과 silymarin에의해농도의존적으로 E-cadherin 은증가하고 vimentin과 MMP-9는억제되었다. 그리고이들의전사인자인 Snail과 NF- B의활성또한 silymarin에의해억제되는것을확인하였다. 이를바탕으로 silymarin 은 Huh7 cell의 EMT 유도와 MMP-9의활성을조절함으로써 HCC의침윤을억제하였고이것은 Snail과 NF- B를통한것임을알수있었다. MAPKs는현재까지비교적잘알려진신호전달기전으로 ERK, JNK, p38 MAPK 세효소들이 superfamily 에포함된다. 이들 MAPKs는세포의증식, 분화, 침윤, 전이와세포사멸등과같은다양한세포외자극을세포내부의반응으로전달하거나매개하는역할을한다. 최근의연구에서는암세포의전이와침윤을조절과관련있는것으로보고되기도하였다 [23, 24]. 특히 silymarin의주요활성성분중하나인 silibinin 은 human lung cancer cell인 A549 cell line, human breast cancer cell인 MCF-7 cell line, human osteosarcoma cell인 MG63 cell line, prostate cancer cell 등의다양한암세포주에서 MAPKs 조절을통해전이와침윤에영향을주는여러단백질들의활성을억제하는결과를보여왔다 [6, 25, 26]. Silymarin의침윤과전이억제기전을분석한결과 Akt의인산화가억제되는것을확인하였고이는선택적저해제를활용한분석에서검증되었다. 이결과로미루어보아 silymarin은 E-cadherin, vimentin, MMP-9 의발현과이들의전사인자인 Snail과 NF- B의활성을억제하여간세포암인 Huh7 cell의침윤과전이를저해하는것으로나타났다. 그리로이들의상위신호전달물질을분석한결과 PI3K/Akt 신호전달체계로인한것임을알수있었다. 요약발생하는간암중가장주요한형태인간세포암은강한전이특성으로인해높은재발율과사망률을보인다. Silymarin은엉겅퀴에서추출한플라보노이드성분으로여러암세포주에서상피간엽전환 (epithelial mesenchymal transition, EMT) 조절을통해항암효과를보이는것으로보고되었다. 본연구에서는 silymarin이 EMT의조절을통해간세포암세포주인 Huh7 cell 의침윤과전이를억제하는지를분석하고자하였다. Huh7 cell 의침윤과전이활성을분석하기위하여 wound healing assay 와 in vitro invasion assay를시행하였고 EMT 관련유전자와상위신호전달물질의발현분석을위해 Western blot assay를 실시하였다. 그결과 silymarin은농도의존적으로 Huh7 cell의침윤과전이를억제하였다. EMT 관련유전자중세포부착단백질인 E-cadherin 은증가하였으나, 중간엽세포의지표인 vimentin, 종양미세환경조절에관여하는 MMP-9 의발현은억제되었고이들의활성에관여하는전사인자인 Snail과 nuclear factor (NF)- B 또한농도의존적으로활성이감소하는것을확인할수있었다. 특히, 상위신호전달물질중 silymarin은 phosphoinositide-3-kinase (PI3K)/Akt 의인산화억제를통해 EMT 관련유전자들을조절하는것으로나타났고이것은 selective inhibitor인 LY294002의처리결과로확인할수있었다. 결과적으로, silymarin 은 PI3K/Akt 경로를통해 EMT 관련유전자의발현을조절함으로써 Huh7 cell의침윤과전이를억제하는것으로생각된다. 이를통해 silymarin 이간세포암의전이억제에효과적인항암물질의후보가될수있는잠재력을가진후보물질이될수있음을보여주었다. Acknowledgements: The research was supported by Undergraduate Research Program through the Korean Foundation for the Advancement of Science and Creativity funded by the Ministry of Education in 2017. Conflict of interest: None REFERENCES 1. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009;119:1429-1437. 2. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009; 139:871-890. 3. van Zijl F, Zulehner G, Petz M, Schneller D, Kornauth C, Hau M, et al. Epithelial-mesenchymal transition in hepatocellular carcinoma. Future Oncol. 2009;5:1169-1179. 4. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014; 15:178-196. 5. Kim SM, Han JH, Park SM. The role of epithelial-mesenchymal transition in the gastroenterology. Kor J Gastroenterol. 2010; 56:69-77. 6. Chen PN, Hsieh YS, Chiou HL Chu SC. Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chem Biol Interact. 2005;156:141-150. 7. Cufí S, Bonavia R, Vazquez-Martin A, Corominas-Faja B, Oliveras-Ferraros C, Cuyàs E, et al. Silibinin meglumine, a water-soluble form of milk thistle silymarin, is an orally active anti-cancer agent that impedes the epithelial-to-mesenchymal transition (EMT) in EGFR-mutant non-small-cell lung carcinoma cells. Food Chem Toxicol. 2013;60:360-368.
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