DBPIA-NURIMEDIA

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1 Journal of Life Science 2010 Vol. 20. No ~280 cjls / ISSN Effect of Sulforaphane on LPS-Induced Matrix Metalloproteinase-9 (MMP-9) Expression Jung Tae Lee, Kyung Jin Woo and Taeg Kyu Kwon* Department of Immunology, School of Medicine, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu , Korea Received January 5, 2010 /Accepted February 22, 2010 Sulforaphane is a naturally occurring member of the iosothiocyanate family, which reveals chemopreventive capacities including anti-cancer, anti-inflammation and inhibition of MMP-9 activities. In this study, we investigated the effect of sulforaphane on the expression of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide (LPS)-induced Raw cells. Sulforaphane strikingly suppressed the LPS-induced MMP-9 activity and mrna expression in a dose-dependent manner. In addition, sulforaphane inhibited not only the LPS-induced MMP-9 promoter activity but also LPS-mediated activator protein-1 (AP-1) and nuclear factor-κb (NF-κB) promoter activity. Transient transfection by MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of LPS and sulforaphane were mediated via AP-1 and NF-κB response elements. We found that sulforaphane had the ability to suppress LPS-induced invasion in vitro. Taken together, these results demonstrated that sulforaphane effectively suppressed LPS-induced MMP-9 expression via modulation of promoter elements (AP-1 and NF-κB) in MMP-9 transcriptional activation. Key words : Sulforaphane, matrix metalloproteinase-9 (MMP-9), lipopolysaccharide (LPS), nuclear factor-κb (NF-κB), Raw cells 서론암세포의전이는세포의이동, 부착, 침범등일련의복잡한과정을통해이루어진다. 이러한과정중, 초기단계에서일어나는필수적인현상이바로 collagens, proteoglycans 및당단백질등의구조단백질로구성된세포외기질 (Extracelluar matrix, ECM) 의분해이다 [3]. Serine 단백질분해효소, cysteine 단백질분해효소, matrix metalloproteinases (MMPs) 등이이에관련되어있으며, 특히 matrixins이라고도불리는 MMPs는 collagen, fibronetic, laminin 등과같은 ECM의구성성분을분해함으로써, 전이뿐만아니라암세포의성장, 혈관생성에도중요한기능을한다고알려져있다 [11]. 현재 human MMPs 로 20종류가동정되어있고, 분해기질에따라네분류로나누어져있다. Collagenases, gelatinases, stromelysins, membrane- associated MMPs [7]. Human MMPs 중, 기저막의주요성분인 type Ⅳ collagen을분해하는데중요한역할을하는효소인 MMP-2와 MMP-9는여러종류의암세포전이뿐만아니라혈관생성에도관여하고있음이밝혀져있다 [11]. 최근 breast, lung, colon, prostate 등의조직에서형성되는종양에서 MMP-9의과발현이알려졌고 [9,15], 이는전이또는종양형성과정에서 MMP-9가기능적으로중요한역할을하고있음을시사한다. Human 암세포에서 MMP-9 발현조절기전은 아직규명되지않았지만, MMP-9의프로모터에는전사조절인자인 AP-1 (-533 bp, -71 bp), NF-kB (-600 bp), Sp-1 (-588 bp) 의결합부위가존재하고, 이러한전사조절인지들이 MMP-9 발현조절에관여한다고알려져있다 [12]. Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] 은십자화과채소에존재하는 isothiocyanate family에속하는화합물로써, detoxification에관여하는 phase Ⅱ enzyme을활성화시켜세포내항산화능력을촉진시킨다고알려져있다 [4]. 뿐만아니라, sulforaphane은세포사멸을증가시키거나, 세포주기진행의억제, 항염증반응등의효과가관찰됨으로써, chemopreventive agent로각광받고있다 [6]. 최근다양한암세포주에서 sulforaphane에의한암세포주의 invasion, 전이및 MMPs 활성억제효과가보고되어지고있지만 [2,6,16], 명확한기전을밝혀지지않고있다. 본연구에서는대식세포에 LPS에의해서유도되는 MMP-9 의활성증가에 sulforaphane이미치는영향에대해서조사를하였다. 아울러 sulforaphane에의한 MMP-9 활성억제효과가세포 invasion 능력과어떠한연관성이있는지도연구하였으며, sulforaphane이 MMP-9 활성을조절하는데관여하는기전에대한결과를보고하고자한다. 재료및방법 *Corresponding author *Tel: , Fax: * kwontk@dsmc.or.kr 세포배양및재료 대식세포주인 Raw 세포는 American Type Culture

2 276 생명과학회지 2010, Vol. 20. No. 2 Collection (Rockville, MD) 에서구입한것을분양받았으며, 세포배양을위해 RPMI 1640에 100 U/ml penicillin, 100 µg /ml streptomycin, 10% feral bovine serum을첨가한배지를사용하여 37 o C, 5% CO 2 조건하에서배양하였다. 일주일에두번씩계대배양을하였고, 배양중인세포를 6-well plate에 의세포수로분주하고 37 o C, 5% CO 2 incubator에서안정화시킨후시료를처리하였다. LPS를비롯한본실험에서사용된재료는 Sigma-Aldrich에서구입하였다. Sulforaphane 은 dimethyl sulfoxide (DMSO, Sigma Chemical Co., St Louis, MO) 을이용하여 20 mm의 stock solution을만든다음적정농도로배지에희석하여사용하였다. Gelatin substrate gel zymography LPS에의해유도되는 MMP-9의활성에 sulforaphane이미치는영향을알아보기위하여세포에 LPS를전처리후 sulforaphane을처리하여 gelatin zymography를시행하였다. 6-well plate에적당량의세포를분주한후시약을처리하여 24시간배양한후세포배양액을거두어 2% geletain이함유된 10% SDS polyacrylamide gel에서전기영동을실시하였다. 2.5% Triton X-100으로 1시간동안 Gel을세척하여 SDS를제거한다음, 5 mm CaCl 2 과 ZnCl 2 가포함된완충액에넣고 37 o C에서 24-48시간동안배양하였다. 0.25% Coomassie blue를이용하여 30분동안 Gel을염색한후, acetic acid와 methanol이포함된탈색완충액을처리하여흰색밴드를관찰하였다. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) Sulforaphane에의해억제되는 LPS 유도 MMP-9 활성이 MMP-9의 mrna levels에어떠한결과를초래하는알아보기위해서 RT-PCR을이용하여 MMP-9의 mrna 발현을분석하였다. 시료를처리한세포에 Trizol reagent (Life Technologies) 를처리하여 total RNA를분리한후, M-MLV 역전사효소 (Gibco-BRL, Gaithersburg, MD, USA) 를이용하여 cdna를합성하였다. 합성된 cdna를 MMP-9 특이적 primer 와함께반응시켜서유전자를증폭시켰다. 그양적차이를비교하기위해서 1% agarose gel에각각의 PCR 산물을 loading 하여전기영동을한다음 EtBr로염색한후 UV 상에서발현의정도를확인하였다. PCR에사용한 MMP-9 primer sequence는 forward: 5'-CAC TGT CCA CCC CTC AGA GC-3', reverse: 5'-GCC ACT TGT CGG CGA TAA GG-3' 이다. Plasmids, transfections and luciferase gene assays LPS 유도시 MMP-9 유전자의전사제어에대한영향을분석하기위하여 MMP-9-Luc (human MMP-9 promoter construct) 및각종전사인자의 binding site에대한 point mutant promoter construct를사용하였다 [18]. 6-well plate에 24시간 배양한세포에 human MMP-9-Luc 및각종 promoter construct를 lipofectamine 방법에따라 transfection을하였고, LPS와 sulforaphane이처리된배지에서 24시간더배양을한후, luciferase 활성을측정하였다. 또한 AP-1, NF-kB reporter construct는 Clontech (Palo Alto, CA, USA) 으로부터구입하여위와같은방법으로활성을측정하였다. Invasion assay sulforaphane이세포침투력에미치는영향을관찰하기위해서 membrane filter (8 µm pore-size) 가부착된 12 well boyden chamber를사용하여 invasion assay를실행하였다 의 Raw 세포를아무것도처리안한배지, LPS 단독또는 LPS와 sulforaphane을함께처리한배지에각각현탁하여, transwell의 upper chamber에넣고, 24시간동안 37 o C에서배양하였다. Filter의위쪽에위치한이동하지않은세포를면봉으로제거하고, filter 아래쪽으로이동한세포는고정시킨후 methanol, acetic acid, 물이 45:10:45로혼합된 0.125% Comassie Blue로염색하여광학현미경으로 transwell을통과한세포의수를세었다. 결 Raw 세포에서 LPS에의해서유도된 MMP-9의활성에미치는 sulforaphane의영향 Sulforaphane이 LPS에의해증가된 MMP-9 활성에미치는영향을조사하였다. Sulforaphane을농도별로 30분전처리한후 LPS를 24시간동안처리하여세포배양액을분리하여 gelatin zymography를하였다. MMP-9 활성은 LPS 처리에의하여높은활성을보였다. LPS에의해서유도된 MMP-9 활성이 sulforaphane에의해서농도의존적으로억제됨을알수있었다. Sulforaphane의 MMP-9 활성억제효과를다양한염증매개의 cytokine인 IL-1β 나 TNF-α를처리하여확인하였다. IL-1β 와 TNF-α에의한 MMP-9 활성증가는 LPS에비하여약하지만 gelatin zymopraphy에서확인할수있었다. 또한 sulforaphane은 IL-1β와 TNF-α에의한 MMP-9 활성증가를농도의존적으로억제함을확인하였다 (Fig. 1B and 1C). 과 LPS에의해유도된 MMP-9의 mrna 활성증가에미치는 sulforaphane의영향 발현과 promoter Sulforaphane에의한 LPS 유도 MMP-9의활성을감소의원인인 MMP-9 단백질발현의전사단계의조절에기인되는지를확인하기위하여 RT-PCR 방법으로 mrna 발현정도를측정하였다. Sulforaphane을농도별로 30분동안전처리한후, 50 ng/ml LPS를처리하여 MMP-9의 mrna 발현변화를관찰한결과, LPS에의해증가했던 MMP-9의 mrna 발현이 sulforaphane 농도의존적으로현저히감소되는것을알수

Journal of Life Science 2010, Vol. 20. No. 2 277 (A) (A) (B) (B) (C) (C) Fig. 1. Effect of sulforaphane on MMP-9 activity. (A) Raw 264.

Conditional media were collected and LPS-induced MMP-9 activity was analyzed using gelatin zymography.

Conditional media were collected after 24 hr followed by gelatin zymography. 있었다 (Fig. 2A). Sulforaphane에의한 LPS 유도 MMP-9의 mrna 발현감소가 MMP-9 mrna 안정성조절에의하여야기되는지를확인하였다. Raw 264.

3 Journal of Life Science 2010, Vol. 20. No (A) (A) (B) (B) (C) (C) Fig. 1. Effect of sulforaphane on MMP-9 activity. (A) Raw cells were treated with or without various concentrations of sulforaphane in presence of LPS (50 ng/ml) for 24 hr. Conditional media were collected and LPS-induced MMP-9 activity was analyzed using gelatin zymography. (B and C) Cells were pretreated with varying concentration of sulforaphane for 30 min and then treated with IL-1β (40 ng/ml) and TNF-α (40 ng/ml), respectively. Conditional media were collected after 24 hr followed by gelatin zymography. 있었다 (Fig. 2A). Sulforaphane에의한 LPS 유도 MMP-9의 mrna 발현감소가 MMP-9 mrna 안정성조절에의하여야기되는지를확인하였다. Raw 세포를 LPS (50 ng/ml) 가함유된배지에 12시간동안배양한후 RNA 합성억제제인 actinomycin D (Act.D) 단독과 Act.D와 sulforaphane을처리하였다. Sulforaphane의영향에따른 MMP-9 mrna 안정성을시간별로조사해본결과, Act.D 단독과 Act.D와 sulforaphane이혼합처리된시료사이에뚜렷한차이점은관찰할수가없었다 (Fig. 2B). 이결과는 sulforaphane의 MMP-9 mrna 발현조절에있어서 mrna 안정성에는영향을미치지않음을의미하는것이다. Sulforaphane이 LPS로유도된 MMP-9 발현을 promoter 활성화저해를통한발현조절임을확인하기위하여 reporter gene assay를수행하였다. Raw 세포에 MMP-9-Luc reporter gene을 transfection 시킨후 LPS 처리구와무처리구, LPS+sulforaphane 처리구로나누어실험하였다. LPS만처리한경우 luciferase 활성이무처리구와비교하여 3.2배정도증가하였으며, sulforaphane과함께처리한경우에는 LPS 처리구와비교하여논도의존적으로 luciferase 활성이감소하였다 (Fig. 2C). 이상의결과로부터 sulforaphane이 LPS 유도에의한 MMP-9의 mrna 발현및 promoter 활성을억제시킴을알수있으며, MMP-9 promoter 부위에 sulforaphane에의해조절되는반응성분 (response element) 이존재함을유추할수있다. Fig. 2. Repression of LPS-induced MMP-9 mrna levels and promoter activity by sulforaphane treatment. (A) Raw cells were treated with or without various concentrations of sulforaphane in presence of LPS (50 ng/ml) for 24 hr. Total RNA was isolated and RT-PCR analysis was performed using specific primer of MMP-9. (B) Cells were incubated with 50 ng/ml LPS for 12 h and then washed out. LPS-treated cells were treated with or without 5 µm sulforaphane in present 5 µg/ml actinomycin D (Act.D) for indicated times. Total RNA isolated and RT-PCR analysis was performed. (C) WT-MMP-9 promoter-containing reporter vector was transfected, and treated with varying concentrations of sulforaphane in the absence or presence of LPS (50 ng/ml) for 24 hr. The cells were lysed and luciferase activity measured. Data represent the mean±sd from at least 3 independent experiments. Sulforaphane이전사조절인자 NF-kB와 AP-1 활성에미치는영향 Sulforaphane이 LPS에의해증가하는 MMP-9 활성을억제하는조절기전을명확히규명하기위해서 MMP-9 promoter 에존재하는전사조절유전자들 (NF-κB, AP-1) 의결합부위를돌연변이시킨 promoter를사용하여 luciferase 활성정도를조사하였다. 정상 MMP-9-Luc vector (WT-MMP-9) 와다양한돌연변이 MMP-9-Luc vector를각각 transfection 한후 LPS (50 ng/ml) 을 24시간동안처리한후 luciferase 활성을측정하였다 (Fig. 3A). Fig. 3A에서나타낸결과에서알수있듯이 LPS를처리한후정상 MMP-9 프로모터활성이증가한것에비해서전사조절유전자들의결합부위가변이된 MMP-9 프로모터활성은확연히감소하였다. 이결과는 LPS 유도에의해증가하는 MMP-9의프로모터활성에전사조절유전자인 NF-κB와 AP-1이중요한역할을하고있음을나타낸다. Sulforaphane에의한 MMP-9 promoter 활성억제효과에 NF-κB와 AP-1의관

278 생명과학회지 2010, Vol. 20. No. 2 (A) (B) (C) Fig. 3. Effect of sulforaphane on the activities of AP-1 and NF-κ B. (A) Schematic structure of MMP-9 promoter constructs used for luciferase assay.

The cells were lysed and luciferase activity measured.

The cells were treated with or without various concentrations of sulforaphane in presence of LPS (50 ng/ml). Luciferase activity was measured.

4 278 생명과학회지 2010, Vol. 20. No. 2 (A) (B) (C) Fig. 3. Effect of sulforaphane on the activities of AP-1 and NF-κ B. (A) Schematic structure of MMP-9 promoter constructs used for luciferase assay. Mutations were introduced into the AP-1 or NF-κB binding sites of WT-MMP-9. WT- MMP-9 promoter or mutant MMP-9 promoters were transfected and incubated with 50 ng/ml LPS for 24 hr. The cells were lysed and luciferase activity measured. (B, C) To elucidate the effects of sulforaphane on the AP-1 and NF-κB activities, a reporter vector that has AP-1 (B) or NF-κB (C) binding sites was transfected. The cells were treated with or without various concentrations of sulforaphane in presence of LPS (50 ng/ml). Luciferase activity was measured. Data represent the mean±sd from at least 3 independent experiments. 련성을더확인하기위하여 NF-κB와 AP-1 basal element를함유한각각의 luciferase vector를 transfection 한후 sulforaphane과 LPS를앞선조건과동일하게처리하였다. 그결과, LPS에의해서 NF-κB와 AP-1의 promoter 활성이증가함을확인할수있었다. NF-κB와 AP-1의 promoter 활성증가는 sulforaphane 농도의존적으로억제됨을알수있었다. 이상의결과는 sulforaphane이 LPS에의한 MMP-9 활성증가에중요한역할을하는전사조절요소인 NF-κB와 AP-1을효과적으로억제함으로써 MMP-9의활성을조절함을의미하는것이다. In vitro 상태에서 Raw 세포의침투력에 sulforaphane 이미치는영향 Invasion assay를이용하여 sulforaphane이 Raw 264.7의세포침투력에미치는영향을알아보았다. Fig. 4에서보는바와같이 LPS에의해 invasion 능력이 2.5배증가하였으며, sulforaphane를전처리시급격한감소를확인할수있었다. 이러한 sulforaphane의 invasion 억제효과는 MMP-9 활성억제효과와밀접한관련이있을것이라생각된다. 고 Type IV collagenase인 MMP-2와 MMP-9은암세포의전이와침윤시 ECM 및기저막을분해하여암세포의전이, 침윤, 찰 Fig. 4. Effect of sulforaphane on matrigel invasion by Raw cells. For invasion assay, the lower and upper parts of Transwells were coated with matrigel. Raw cells were incubated with in the presence or absence of either LPS (50 ng/ml) or sulforaphane (20 µm) in the upper well. After 24 hr, invasiveness of the cells on the upper side of the filter were removed and the cells on the bottom side of the filter were fixed, stained with 0.125% Comassie blue, and counted. Data represent the mean±sd from at least 3 independent experiments. angiogenesis를활성화시켜암세포성장에중요한역할을하는금속이온을 cofactor로요구하는효소이다 [12]. MMP-9 단백질분해효소의발현은다양한성장인자, cytokine, 그리고 12-myristate 13-acetate (PMA) 와같은 xenobiotics에의해조절되어진다 [8,11,13]. 본연구에서는대식세포에서 LPS에의해증가하는 MMP-9 활성에 sulforaphane이미치는영향과 sulforaphane의처리에의한세포 invasion 능력억제여부에대해서조사하였다. 먼저 LPS 유도에의한 MMP-9 활성에미치는영향을알아보기위해서대식세포에 sulforaphane을전처리한후 LPS를처리하여 zymography를실시하였다. LPS에의해증가하였던 MMP-9 활성이 sulforaphane의농도의존적으로억제가됨을확인할수있었고, LPS 뿐아니라다른종류의 cytokine (IL-1β 와 TNF-α) 에의한 MMP-9 활성증가역시 sulforaphane이효과적으로억제시킴을관찰할수있었다. Sulforaphane의 MMP-9 활성억제기전을조사하기위해서 RT-PCR 및 MMP-9 luciferase reporter assay를통해서조사한결과 sulforaphane의 MMP-9의활성을억제는 MMP-9의전사단계에서조절됨을알수있었다. mrna stability 실험을통해서 sulforaphane이 mrna 안정성을조절과는무관함을확인하였다. MMP-9 발현조절에 mitogen- activating protein kinase (MAPK) 의신호전달계가관련되어있으며이중에 ERK와 JNK 활성이 MMP-9 발현의증가를유도한다 [5]. Simon et al. 는 p38 MAPK의억제가 12-myristate 13-acetate (PMA) 에의해증가되는 MMP-9의발현을감소시킨다는결과를보고하

5 Journal of Life Science 2010, Vol. 20. No 였다 [14]. MMP-9의발현을조절하는전사인자의발현기전은명확하게밝혀지지는않았으나, 최근 chemopreventive agent 로잘알려진 resveratrol이 PMA에의해증가하는 MMP-9의활성을억제하는데있어서전사조절인자 AP-1, NF-κB 활성조절이중요한역할을한다고알려져있다 [17]. Sulforaphane 의 MMP-9 활성억제에전사조절인자들의관련여부를확인하기위해서 MMP-9의 promoter 활성을 wild type과 mutant AP-1 및 mutant NF-κB 비교분석한결과 mutant에서현저히감소됨을확인하였다. 이는 MMP-9의 promoter 활성에 AP-1 과 NF-κB 전사인자의결합이매우중요한요소임을시사한다. LPS와 sulforaphane을혼합처리하였을때 AP-1과 NF-κB의 basal 활성도를확인해본실험에서도 LPS에의해서증가한 AP-1과 NF-κB의 luciferase 활성이 sulforaphane에의해서현저히감소되었다. 이는 sulforaphane이전사조절인자인 AP-1 과 NF-κB의활성억제를통해서 LPS 유도에의한 MMP-9의활성증가를감소시킴을의미하는결과이다. Sulforaphane이 LPS 유도에의해증가하는 MMP-9 활성억제효과에관련하여세포 invasion 또한감소시킴을확인하였다. Human brain microvascular endothelial cell과 human breast cancer cell에서 sulforaphane이 MMP-9 억제를통해세포이동과 tubulogenesis, 세포침윤을방해하는데유의한효과가있음이밝혀지고있지만 [1,10] 그기전을명확히제시하지는못하였다. 이상의결과에서 sulforaphane이 MMP-9의활성을억제하고세포침윤능력을억제하는기전에대해설명하였고, 이는더나아가암세포의전이억제를통한암치료제개발에중요한근거를제시하는바이다. 감사의글 이연구는한국과학재단의 MRC(R ) 지원에의하여이루어진결과입니다. References 1. Annabi, B., S. Rojas-Sutterlin, M. Laroche, M. P. Lachambre, R. Moumdjian, and R. Béliveau The diet-derived sulforaphane inhibits matrix metalloproteinase-9-activated human brain microvascular endothelial cell migration and tubulogenesis. Mol. Nutr. Food Res. 52, Asakage, M., N. H. Tsuno, J. Kitayama, T. Tsuchiya, S. Yoneyama, J. Yamada, Y. Okaji, S. Kaisaki, T. Osada, K. Takahashi, and H. Nagawa Sulforaphane induces inhibition of human umbilical vein endothelial cells proliferation by apoptosis. Angiogenesis 9, Basset, P., A. Okada, M. P. Chenard, R. Kannan, I. Stoll, P. Anglard, J. P. Bellocq, and M. C. Rio Matrix metalloproteinases as stromal effectors of human carcinoma progression: therapeutic implications. Matrix Biol. 15, Brooks, J. D., V. G. Paton, and G. Vidanes Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol. Biomarkers Prev. 10, Gum, R., H. Wang, E. Lengyel, J. Juarez, and D. Boyd Regulation of 92 kda type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene 14, Juge, N., R. F. Mithen, and M. Traka Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cell Mol. Life Sci. 64, Kerkelä, E, and U. Saarialho-Kere Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer. Exp. Dermatol. 12, Lee, P. P., J. J. Hwang, G. Murphy, and M. M. Ip Functional significance of MMP-9 in tumor necrosis factor-induced proliferation and branching morphogenesis of mammary epithelial cells. Endocrinology 141, Nelson, A. R., B. Fingleton, M. L. Rothenberg, and L. M. Matrisian Matrix metalloproteinases: biologic activity and clinical implications. J. Clin. Oncol. 18, Rose, P., Q. Huang, C. N. Ong, and M. Whiteman Broccoli and watercress suppress matrix metalloproteinase- 9 activity and invasiveness of human MDA-MB-231 breast cancer cells. Toxicol. Appl. Pharmacol. 209, Roy, R., J. Yang, and M. A. Moses Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J. Clin. Oncol. 27, Sato, H. and M. Seiki Regulatory mechanism of 92 kda type IV collagenase gene expression which is associated with invasiveness of tumor cells. Oncogene 8, Sato, H., M. Kita, and M. Seiki v-src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements. A mechanism regulating gene expression independent of that by inflammatory cytokines. J. Biol. Chem. 268, Simon, C., H. Goepfert, and D. Boyd Inhibition of the p38 mitogen-activated protein kinase by SB blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion. Cancer Res. 58, Stetler-Stevenson, W. G. and A. E. Yu Proteases in invasion: matrix metalloproteinases. Semin. Cancer Biol. 11, Thejass, P. and G. Kuttan Antimetastatic activity of Sulforaphane. Life Sci. 78, Woo, J. H., J. H. Lim, Y. H. Kim, S. I. Suh, D. S. Min, J. S. Chang, Y. H. Lee, J. W. Park, and T. K. Kwon Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta signal transduction. Oncogene 23, Woo, J. H., J. W. Park, S. H. Lee, Y. H. Kim, I. K. Lee, E. Gabrielson, S. H. Lee, H. J. Lee, Y. H. Kho, and T. K. Kwon Dykellic acid inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting nuclear factor kappa B transcriptional activity. Cancer Res. 63,

6 280 생명과학회지 2010, Vol. 20. No. 2 초록 :Sulfolaphane 이 lipopolysaccharide (LPS) 에의해유도된 matrix metalloproteinase-9 (MMP-9) 발현에미치는영향이정태 우경진 권택규 * ( 계명대학교의과대학면역학교실 ) Sulforaphane은십자가화채소에존재하는화합물로항염증, 항암및신생혈관생성의억제효과가알려짐으로써최근많은연구가활발히이루어지고있으나, LPS에의한 MMP-9 활성조절에대한연구는매우미흡한편이다. 따라서본연구에서 sulforaphane이 LPS 유도에의한 MMP-9 활성에미치는영향에대해서조사해보았다. Raw 세포에 sulforaphane을전처리한후 LPS를처리하여 gelatin zymography를실시해본결과, LPS 에의해유도된 MMP-9 활성증가가 sulforaphane 농도의존적으로감소됨을확인하였다. 또한 RT-PCR과 MMP-9의 luciferase assay를통한실험에서 sulforaphane의 MMP-9 억제효과가전사단계에서조절됨을추측할수있었다. MMP-9 promoter 부위에여러가지의전사조절인자결합부위가존재한다. 특히 AP-1과 NF-κB가중요전사조절인자로작용하여 MMP-9 발현조절에관여한다. 본실험에서 sulforaphane에의한 MMP-9 억제효과기전에이들전사조절인자들의중요한역할을조사하였다. AP-1과 NF-κB 결합부위를변형시킨 vector를 transfection 하여 MMP-9의 promoter 활성을측정한결과, 정상 vector에비해그활성도가현저히떨어짐을확인하였고, LPS에의해증가되는 AP-1과 NF-κB의 basal promoter 활성또한 sulforaphane에의해감소됨을관찰할수있었다. 이상의결과에서 sulforaphane의 MMP-9 활성억제효과는 AP-1과 NF-κB와같은전사인자들이 MMP-9의전사를조절함으로써일어나는것임을알수있었다. 그리고 sulforaphane은세포의 invasion능력또한효과적으로억제시킴을관찰할수있었는데이는 MMP-9 활성억제효과와밀접한관련이있음을추측할수있었다.

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract