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1 CANCER PREVENTION RESEARCH ORIGINAL ARTICLE Mouse 악성흑색종에서 Lipoic Acid 가항산화효소계에미치는영향 숙명여자대학교약학대학약학부 정수연ㆍ김안근 The Effect of Lipoic Acid on Antioxidant Enzyme System in Murine Melanoma Cells Suyeon Jung and An Keun Kim Department of Pharmacy, College of Pharmacy, Sookmyung Women's University, Seoul , Korea Reactive oxygen species (ROS) has been reported to cause DNA damage, lipid peroxidation, or protein denaturation and to be related also to the various diseases such as cancer, aging, cardiovascular disease, inflammation radiation injury, etc. through the different mechanisms. Being produced in the process of natural metabolism, ROS is controlled in the cell by the antioxidative enzymes of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), etc. Belonging to the sulfur containing compound, α-lipoic acid is a physioactive substance from the mitochondria energy metabolism, and its oxidized form (disulfide bonding in a molecule) can be reduced to two SH-free radicals that are distinguished in antioxidative effect. We have measured MTT assay, enzyme activity assay, and ROS level in order to examine antioxidative effect of the lipoic acid in the B16F10 murine melenoma cell. B16F10 cells were exposed to medium containing lipoic acid for a period of 24 h. Lipoic acid increased the activities of superoxide dismutase and catalase compared to those of the control, but decresed the activity of glutathione peroxidase. Lipoic acid also reduced ROS content in a dose-dependent manner. (Cancer Prev Res 15, , 2010) Key Words: Lipoic acid, Antioxidant enzyme, Murine melanoma (B16F10) cells, ROS 서 Reactive oxygen species (ROS) 는다양한기전을통해 DNA damage 나 lipid peroxidation, protein denaturation 등 1) 을일으키며 cancer aging cardiovascular disease 2) inflammation radiation injury 등의여러가지질병과관련이있는것으로알려져왔다. 3) 산소를이용하는생물체에서는 론 정상적인대사를하는동안에도지속적으로 superoxide radical anion (O 2 ㆍ ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OHㆍ) 등과같은 reactive oxygen species (ROS) 가만들어진다. 이런 free oxygen radicals 이생성되면항산화적방어기전과의균형에의해조직의손상도가정해진다. 4) Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) 와같은항산화효소들은 membrane의 lipid peroxidase damage, sulfhydryl-containning enzymes의 inactiva- 책임저자 : 김안근, , 서울시용산구청파동 52 번지숙명여자대학교약학대학 Tel: , Fax: akkim@sookmyung.ac.kr 접수일 :2010 년 5 월 10 일, 1 차수정일 : 2010 년 5 월 13 일, 2 차수정일 : 2010 년 5 월 18 일, 게재승인일 :2010 년 5 월 24 일 Correspondence to:an Keun Kim College of Pharmacy, Sookmyung Women's University, 52, Chungpadong, Yongsan-gu, Seoul , Korea Tel: , Fax: akkim@sookmyung.ac.kr 152

2 정수연ㆍ김안근 :Mouse 악성흑색종에서 Lipoic Acid 가항산화효소계에미치는영향 153 tion, integral protein의 cross-linking 등에관련된 oxygen species를불활성화시키거나제거함으로써항산화작용을하게된다. 진핵세포에서는 CuZnSOD (SOD-1), MnSOD (SOD-2) 와 extracellular SOD (EC-SOD) 의세가지형태의 SOD가존재하며각각은세포의 cytosol, mitochondria, extracellular space에존재한다. 5) SOD는 free radical superoxide 을 hydrogen peroxide로전환시킨다. SOD 반응에의해생성된 hydrogen peroxide는 Fenton reaction을통해더욱반응성이큰 hydroxyl radical로전환될수있다. 이렇게생성된 hydrogen peroxide는주로 peroxisomes에존재하며간세포와적혈구에풍부한 catalase에의해 O 2 와 H 2O로전환되며 6) 또한 cytoplasm과 mitochondria에존재하는 GPx에의해서 H 2O로무독화된다 (Fig. 1). 7,8) 이런항산화효소를조절하는식이적항산화제들이 vitamin C, E, A, 9-11) carotenoid 등이있으며이들에관한많은연구가되어지고있고천연물로부터보다안전하고강한활성을지닌항산화제의개발이요구되어지고있다. 이에따라본연구에서는 sulfur containing amino acid의한종류인 lipoic acid 을 mouse의 melanoma cell에처리하여항산화효과를나타내는지여부를알아보고자하였다. 특히 sulfur는 amino acid과 proteins, 특히 iron-containing flavoenzymes에도중요한역할이알려져있다. 채식주의자, 어린이, AIDS 환자에게는 sulfur-containing amino acid가치료용으로사용되기도한다. 12,13) Lipoic acid는 pyruvate dehydrogenase 복합체, Fig. 1. Antioxidant enzyme system. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of the superoxide radical anion to hydrogen peroxide and oxygen. The catalase (CAT) and glutathione peroxidase (GPx) convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are convert into the harmless product water. GPx requires several secondary enzyme (GR and G-6-PDH) and cofactors (GSH, NADPH, and glucose 6-phosphate) to function. In this scheme, GR and G-6-PDH are considered secondary antioxidant enzyme, because they do not act on ROS directly but enable GPx to function. α-ketoglutarate dehydrogenase 복합체, α-keto acid dehydrogenase의 보조효소로생체대사에관여하며 vitamin E와 C를재순환시키고세포내 glutathione을증가시켜다른항산화제의대사에영향을주기도한다. 14) 또한 ischemia-reperfusion injury diabetes diabetic neuropathy neuro degeneration radiation injury HIV activation 등에영향이있다고알려져있다. 15) 본실험에서는천연으로부터보다안전하고강한활성을지닌항산화제의개발의일환으로항산화관련연구에많이사용되고있는침습성이강하고전이가빠른피부암세포인악성흑색종세포에서 lipoic acid의 (Fig. 2) MTT assay, 항산화효소활성, reactive oxygen species (ROS) level를측정하여항산화효소의활성변화와 ROS level 변화를검토함으로써 lipoic acid의항산화작용을평가하고자하였다. 1. 시약및기기 재료및방법 RPMI 1640 powder medium, antibiotics (10,000 units/ml penicillin G sodium, 10,000μg/ml, streptomycin sulfate), trypan blue는 Gibco BRL life Technologies Inc. (Carlsbod, CA, USA) 제품을사용하였고, fetal bovine serum (FBS) 은 Bio Whittaker TM (Walkersville, MD, USA), protease inhibitor cocktail tablets은 Roche에서구입하여사용하였다. KCl/ KH 2 PO 4 /K 2 HPO 4 /Boric acid (Junsei Co.), Na 2 HPO4 (Amresco Co.), 2,7 -dicholrofluorescein diacetate (DCF-DA; Eastman KodakMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], bicinchonic acid protein assay kit, β-nicotinamide adenine dinucleotide phosphate reduced form (β- NADPH), glutathione reduced form, (±)-α-lipoic acid, Tris[hydroxyl-methyl]aminomethane, hematoxylin는 Sigma Co. (St. Louis, USA) 에서구입하여사용하였다. ELISA reader는 Bio-Tek instrument Inc. (Washington, USA), cytofluor 2350 plate reader는 Millipore (Bedford, MA, USA), CO 2 incubator (Forma science), autoclave (Sanyo), tab- Fig. 2. Chemical structure of Lipoic acid.

3 154 Cancer Prevention Research Vol. 15, No. 2, 2010 letop centrifuge (Hanil scienceindustrial Co. Ltd.), digital ph meter (Mettler Delta 340) inverted microscope는 Olympus CK2(Massachusetts, USA), UV/visible spectrophotometer는 Ultraspec 2000(Pharmacia Biotech.) 제품을사용하였다. 2. 세포배양 Mouse melanoma cell로부터유래된 B16F10 cell은 ATCC (American Type Culture Collection) 으로부터분양받았다. 10% heat-inactivated fetal bovine serum, 항생제 (10,000 units/ml penicillin G sodium, 10,000μg/ml streptomycin sulfate), 1 mm sodium pyruvate를포함하는 RPMI 1640 배지를배양액으로하여 37 o C, humidified 5% CO 2 incubator에서배양하였다. 25 cm 3 tissue culture flask나 75 cm 3 tissue culture flask에서계대배양하고 confluent 되었을때 cell dissociation solution을처리하여실험에이용하였다. 3. 시료의조제 Lipoic acid는 dimethyl sulfoxide (DMSO) 에희석하여사용하였다. 시료는 0.2μm pore size syringe filter로여과하여 stock solution을만들었다. 실험에이용하기직전에 DMSO 의최종농도가 0.1% 가되도록 RPMI 1640 세포배양배지로희석하여사용하였다. 4. 세포생존율측정 B16F10 cell suspension을 cells/ml의농도로 96-well plate의 well에 100μl씩가하여배양기에서 24시간동안안정화시켰다. Lipoic acid를농도별로처리, 24시간배양한후 5.0 mg/ml MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide) 용액을 well당 50μl씩넣어 4시간동안배양기에방치했다. 이후상층액을제거하고 DMSO를 well당 100μl씩가하여 1분간 shaking하여 formazan을완전히용해시킨후 ELISA plate reader를이용하여 570 nm에서흡광도를측정하였다. 5. 항산화효소의활성측정 1) 시료의처리와단백질정량 : cells/ml의 B16F10 cell suspension을 100π tissue culture dish에가하여배양기에서 24시간동안안정화시킨후 lipoic acid 농도별로처리하여 3, 6, 12, 24시간배양하였다. Culture dish에서배지를제거한후 PBS로세척하고 trypsin 처리하여농도별 sample을얻고 PBS로 2회세척후상층액을제거한 pellet 에 lysis buffer 1 ml을가했다. Lysis buffer를가한각각의 sample을 14,000 rpm, 4 o C에서 5분간원심분리하고상층액만을취하여 enzyme assay sample로사용하였다. Sample protein 의정량은 bovine serum albumin (BSA) 을 standard 로사용하여 BCA (bicinchoninic acid) protein assay 을하였다. 16) 2) Superoxide dismutase (SOD) 의활성측정 : SOD 활성측정은 hematoxylin을이용한 Martin의방법을사용하였다. 17) Hematoxylin은자연상태에서붉은색인 hematin으로자가산화한다. 이러한과정에 SOD가관여하면자동산화를억제하게된다. 0.1 mm EDTA가함유된 50 mm potassium phosphate buffer (ph 7.5) 1 ml에각각의 sample을 100μl씩가하고 5분동안미리배양시켰다. 여기에 5 mm hematoxylin을 50μl을가한후 phosphate buffer를 blank로하여 UV/visible spectrophotometer를이용하여 568 nm에서흡광도를측정하였다. 1분간격으로 5분동안흡광도의변화를측정하고다음식에의해효소활성을계산하였다. SOD activity = a = standard의흡광도변화 b = sample의흡광도변화 3) Glutathione peroxidase (GPx) 의활성측정 : GPx의활성은 Paglia와 Valentine의방법에의해 spectrophotometer 를사용하여측정하였다. 7) GPx의반응동안 glutathione (GSSG) 은 GSH의일정농도에대해서제공되는과잉의 glutathione reductase (GR) 에의해환원되며이때 reduced nicotinamide adenine dinucleotide phosphate (NADPH) 의산화를관찰하였다. 0.1 M potassium phosphate buffer (ph 7.0, 1 mm EDTA) 500μl에 10 mm GSH 100μl, 각 sample 100μl, 0.5 unit/ml GR 100μl, 1 mm sodium azide 100μl 을가한후 37 o C에서 10분간미리배양시켰다. 여기에 1.5 mm NADPH 100μl 가한후 3분동안 NADPH 소비를관찰하였다미리가온한 15 mm H 2O 2 100μl를넣고 NADPH의감소한흡광도가직선이나타나도록 340 nm 에서 1분간격으로 5분간흡광도를측정하고다음식에의해효소활성을계산하였다. A=0.868 NADPH/t = 1분당 NADPH의흡광도변화 [GSH] = GSH의초기농도 Vi = incubation mixture의 volume Vs = enzyme sample의 volume 4) Catalase (CAT) 의활성측정 : CAT 의활성측정은

4 정수연ㆍ김안근 :Mouse 악성흑색종에서 Lipoic Acid 가항산화효소계에미치는영향 155 hydrogen peroxide의분해에따라흡광도차를측정하는 Aebi 방법을이용하였다. 6) 50 mm phosphate buffer (ph 7.0) 에 30% H 2O 2 를넣어 10.5 mm substrate solution (A240=0.5) 을만든다. 이 substrate solution 1 ml에각 sample 100μl를가한후 43.6 M -1 cm -1 의 exitinction coefficient를사용하여 UV/visible spectrophotometer로 240 nm에서 phosphate buffer를 blank로하여 1분간격으로 5분동안흡광도를측정하고다음식에의해효소활성을계산하였다. CAT activity = 6. Reactive oxygen species level 측정 세포내의 ROS를측정하는방법으로 DCF-DA는세포내에서 esterase에의해 DCFH로가수분해된다. 이 DCFH는 H 2 O 2 나 superoxide에의해형광을나타내는 DCF로산화되는데산화된 DCF의형광성은 485 nm의 excitation wavelength와 538 nm의 emission wavelength에서측정한다. 18) B16F10 cell suspension을 96 well plate에각 well 당 cells로하여 24시간동안배양하였다. 시료를농도별로처리하고 30분미리배양한후배지를제거하고 PBS로 2번세척해준다. 상온에서 50μM DCF-DA 100μl를처리한후 Cytofluor 2350 plate reader를이용하여 5분간격으로측정하였다. 7. 통계처리 본연구의그래프와표의모든수치는각실험횟수에대한평균과표준오차로표시하였으며, 모두 triplicate set 로세차례이상수행하였다. 각 sample의통계적유의성에대한검증은 student t-test를시행하여계산하였다. 1. 세포생존율 결과및고찰 B16F10 murine melanoma cell에서 lipoic acid의농도별세포생존율을알아보기위해 MTT assay를실행하였다. 배양기간은 2일로고정하였으며 lipoic acid의농도는 mm 범위에서실험을시행하였다. 1 mm 이하에서는세포생존율에크게영향을미치지못하였으나 (88%), lipoic acid의 2.5 mm 이상의농도에서농도가증가함에따라세포생존율이농도의존적으로감소하여 lipoic acid의농도가 5 mm과 7.5 mm에서는세포생존율이각각 51%, 44% 로나타났다. Lipoic acid의농도가 0.75 mm일때세포생존율이 95% 로독성을거의나타내지않았으 Fig. 3. Cell viability of B16F10 melanoma cells after treatment with lipoic acid. The cells were exposed to various concentrations of lipoic acid for 24 hr. Percentage of cell viability was determined by using MTT assay. Results are expressed as percentage of control. Values are means±s.d. and were obtained from three different experiments. *p<0.05, **p<0.001 compared with control. 므로 0.75 mm을최고농도로하여실험을시행하였다 (Fig. 3). 2. 항산화효소활성에미치는영향 항산화효소계는산화적인스트레스로부터생체를보호하는중요한역할을하며 SOD, GPx, CAT 등은유해한활성산소를제거하는대표적인항산화효소이다. 이들항산화효소들이 lipoic acid의농도와처리시간에따라효소활성에어떠한영향을미치는지를알아보기위해 lipoic acid를다양한농도와시간으로처리하여 SOD, GPx, CAT의활성변화를측정하였다. Fig. 4 6에나타난효소활성측정결과를보면시간과농도에따른활성변화가다양하게나타나비교검토하기가어렵지만 24시간경과후에는 SOD, GPx, CAT 활성변화양상을잘알수있다. Lipoic acid 처리후 24시간경과후효소활성을비교해보면 SOD의활성은 control (100%) 에비해 0.75 mm (164%) 에서, CAT의활성은 0.25 mm (227%), 0.5 mm (268%), 0.75 mm (309%) 에서모두유의적으로증가하였고 (Fig. 4, 6), 특히 CAT의활성은농도의존적으로증가하였다. GPx의활성은 0.25 mm (83%), 0.5 mm (83%), 0.75 mm (78%) 에서유의적으로활성이저하되었다 (Fig. 5).

5 156 Cancer Prevention Research Vol. 15, No. 2, 2010 Fig. 4. Effect of lipoic acid on superoxide dismutase (SOD) activities in B16F10 murine melanoma cells. The cells were exposed to various time and concentrations of lipoic acid. The control cells and lipoic acid-treated cells were harvested and protein was isolated from the cells. Absorbance of each enzyme sample was read at 568 nm. Results are expressed as average of triplicate samples. *Significantly different from the control at the p<0.05 compared with control. Fig. 6. Effect of lipoic acid on catalase (CAT) activities in B16F10 murine melanoma cells. The cells were exposed to various time and concentrations (mm) of lipoic acid. The control cells and lipoic acid-treated cells were harvested and protein was isolated from the cells. Absorbance of each enzyme sample was read at 240 nm. Results are expressed as average of triplicate samples. *Significantly different from the control at the p<0.05 compared with control. Fig. 5. Effect of lipoic acid on glutathione peroxidase (GPx) activities in B16F10 murine melanoma cells. The cells were exposed to various time and concentrations (mm) of lipoic acid. The control cells and lipoic acid-treated cells were harvested and protein was isolated from the cells. Absorbance of each enzyme sample was read at 340 nm. Results are expressed as average of triplicate samples. *Significantly different from the control at the p<0.05 compared with control. Fig. 7. Measurement of reactive oxygen species (ROS) level after lipoic acid treatment. The melanoma cells ( /well) were incubated with lipoic acid at various concentration (mm). After incubation for 30 min, 100μl DCFH-DA (50μM) was added as a substrate for ROS. ROS levels were measured by spectrofluoremeter (excitation: 485 nm, emission: 538 nm). Results are expressed as average of triplicate samples with ±S.D. *p<0.001 compared with control. 3. Reactive oxygen species (ROS) level에미치는영향활성산소종 (reactive oxygen species; ROS) 은 O 2 가전자전달계에의해 H 2O까지환원되어가는중에전자 (e ) 한 개씩에의해순차적으로환원되면서생성되는 OH, O 2, H 2O 2 등의파생물로과잉생성될경우세포의퇴화를촉진시키며, DNA의돌연변이를유발해다양한종류의암을비롯한난치질환의근원이된다. 이에따라 lipoic acid 가 B16F10 cell에서직접활성산소를제거할수있는지

6 정수연ㆍ김안근 :Mouse 악성흑색종에서 Lipoic Acid 가항산화효소계에미치는영향 157 를알아보기위해세포내 ROS level을측정결과 ROS의 level이감소하는것을확인할수있었다. Lipoic acid의 0.5 mm와 0.75 mm 농도에서 ROS level이 control에비해 32%, 35% 가감소하였다 (Fig. 7). 이러한결과를볼때 lipoic acid의농도증가에따라 ROS level이감소하였다는것은 lipoic acid가항산화능을가지고있다고할수있다. 이러한항산화작용은처리후 6시간에서는 GPx도활성이 control 보다높은것으로보아 ROS제거과정에관여된것으로볼수있으나 24시간경과에서볼때 SOD와 CAT에의해 ROS가제거되는것으로볼수있다. 그중에서도 CAT가크게작용한것으로나타났다. Fig. 1에서보는바와같이 SOD는 O 2 ㆍ 를 H 2O 2 로변화시키고그다음 CAT가 H 2O 2 에작용하여무독한 H 2O와 O 2 로변화시킴으로써 ROS를제거시키는경로에의한것이라볼수있다. 이와같이항상화효소활성의변화는투여하는항산화물질에따라다르게나타나는것으로보고되어있다. 19,20) 이런면을고려할때세포환경에따른항산화효소계에대한다른영향이있을것으로사료되며 lipoic acid의효율성확인이요구되어지기때문에앞으로더많은연구가선행되어져야할것이다. 결 본연구에서는 lipoic acid 처리하여항산화효소의활성, ROS level에대해살펴본결과, 다음과같은결론을얻을수있었다. 1. Lipoic acid로처리하고 24시간경과했을때의 SOD의활성은 lipoic acid 농도 0.75 mm에서, CAT의활성은 0.25 mm, 0.5 mm, 0.75 mm에서유의적으로증가하였다. GPx 의활성은 0.25 mm, 0.5 mm, 0.75 mm에서유의적으로활성이저하되었다. 2. ROS level은 lipoic acid 농도를 0.25 mm, 0.5 mm, 0.75 mm로처리하였을때농도가증가할수록농도의존적으로감소하는것으로나타났다. 론 참고문헌 1) Mates JM, Perez-Gomez C, Nunez de Castro I. Antioxidant enzymes and human disease. Clin Biochem 32, , ) Gavat V, Voroniuc O. Oxidative stress and antioxidants in the diet in pathological processes at the level of the cardiovascular system. Rev Med Chir Soc Med Nat Iasi 103, 37-41, ) Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med 91, 14-22, ) Machlin LJ, Bendich A. Free radical tissue damage: Protective role of antioxidant nutrients. FASEB J 1, 441, ) McCord JM. Superoxide, superoxide dismutase and oxygen toxicity. In: eds, by Hodgson E, Bend JR, Philpot RM, Reviews in Biochemical Toxicology 1, , ) Aebi Hugo. Catalase in vitro. Method in Enzymology 105, , ) Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70, , ) Li S, Yan T, Yang JQ, Oberley TD, Oberley LW. The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res 60, , ) Bendich A, Machlin LJ, Scadurra O, Burton GW, Wayner DDM. The antioxidant role of vitamin C. Adv. Free Radical Biol Med 2, , ) Krajcovicova-Kudlackova M, Paukova V, Bacekova M, Dusinska M Cent. Lipid peroxidation in relation to vitamin C and vitamin E levels. Eur J Public Health 12, 46-48, ) Enesco H, Verdone-Smith C. α-tocopherol increase life span in the rotifer Philodina. Exp Gerontol 15, , )Paecell S. Sulfur in human nutrition and applications in medicine. Altern Med Rev 7, 22-44, ) Grimble RF, Grimble GK. Immunonutrition: Role of sulfur amino acid, related amino acid and polyamines. Nut 14, , ) Navari-Izzo F, Quartacci MF, Sgherri C. Lipoic acid : a unique antioxidant in the detoxification of activated oxygen species. Plant Physiol Biochem 40, , ) Bustamante J, Ladge JK, Marcocci L, Tritschler HJ, Parker L, Rihn BH. Lipoic acid in the liver metabolism and disease. Free Radic Biol Med 24, , ) Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76-85, ) Martin JP, Dailey M, Sugarman E. Negative and Positive ass superoxide dismutase based on hematoxylin autoxidation. Arch. Biochem. Biophys 255, , ) Sattier M, Winkler T, Verma S, Byrne CH, Shrinkhande G, Sagila R, Griffin JD. Hematopoieticd growth factors signal through the formation of reactive oxygen species. Blood 93, 2928, ) Yu JS, Kim AK. Effect of taurine on antioxidant enzme system in B16F10 melanoma cells. Advanced in Experimental Medicine and Biology 643, , ) Cha EJ, Pyo MY, Yang KS, Kim AK. Effect of Phellinus Linteus extract on the activity of antioxidant enzyme. Cancer Prevention Research 13, , 2008.

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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