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1 J. Exp. Biomed. Sci. 12 (2006) Anticancer Effect of Ferulic Acid on Cultured Human Skin Melanoma Cells Byoung-Kwan Son, Yu-Sun Choi and Young-Woo Sohn Sanbon Medical Center, Wonkwang University, Gyeonggido, Gunpo , Korea It is demonstrated that phenolic compound has cytotoxic effect on cancer cells. Recently, ferulic acid is involved in anticancer activity by showing the decrease of cell viability in cancer cells. But, the anticancer mechanism of ferulic acid is left unknown. The purpose of this study was to examine the anticancer activity of ferulic acid on NIH3T3 fibroblasts and human skin melanoma cells (SK-MEL-3). The anticancer activity was measured by determining the cytotoxicy of ferulic acid on these cells. The cytotoxicity was measured by cell viability via XTT assay in these cells. In this study, ferulic acid decreased cell viability according to the dose-dependent manners after human skin melanoma cells were treated with various concentrations of ferulic acid for 48 hours. especially, ferulic acid remarkably decreased cell viability at a concentration of 120 µm compared with control in human skin melanoma cells. While, ferulic acid did not show the significant decrease of cell viability at concentrations of 30~120 µm in NIH3T3 fibroblasts. These results suggest that ferulic acid showed anticancer activity in cancer cells such as human skin melanoma cells by the decrease of cell viability significantly. Key Words: Anticancer activity, NIH3T3 fibroblasts, Human skin melanoma cells 서 암은현대의학이풀어야할과제중의하나로아직도암에대한자세한기전이잘밝혀져있지않다 (Wattenberg, 1985). 암의발생은갑자기인체내의암유전자 (oncogene) 가작동함으로써암이유발하게된다는것이밝혀지면서암유전자의발견에많은연구가진행되어왔다 (Takemura et al., 2001). 특히, erba 암유전자를비롯하여 fos 암유전자, jun 암유전와같은다양한암유전자들이밝혀져있으며이들이독립적으로또는복합적으로작용함으로써암이발생한다고알려져있다 (Lewin, 1991). 암의치료에있어서현재로서는수술을비롯하여항암제및방사선요법을시행하고있으며암의진행상태에따라이들을복합치료하고있다. 특히, 암은빠르게진행할뿐만아니라이의전이또한매우위험하다 (Sharma et al., 1994). 따라서세계각국은암의정복을위하여새로운항암물질을개발에많은노력과투자를아끼지않고있다 (Husniye & Kucukali, 1998). 지금까지항암제는암치료에있어가장널리사용되고있는약재로서암세포의 론 * 논문접수 : 2006년 11월 15일수정재접수 : 2006년 12월 14일 교신저자 : 손영우, ( 우 ) 경기도군포시산본동 1142, 원광대학교의과대학산본병원 Tel: , giyoung@wonkwang.ac.kr DNA나 RNA의합성을저해함으로써항암작용을나타내는가하면암세포내의단백질합성의저해나또는암세포내신호전달체계를차단함으로써항암작용을나타내기도한다 (Lewin, 1991; Jung et al., 1997). 그러나항암제들은암세포뿐만아니라정상세포에도손상을주기때문에항암제에의하여치료가된다할지라도이의후유증으로환자는심한부작용과고통으로많은어려움을겪게된다 (Goodman et al., 1987). 따라서오래전부터세계각국은정상세포에는독성이없으면서암세포만선택적으로죽일수있는항암제의개발에많은관심이집중되었다. 최근에식물에서추출한페놀화합물이항암효과가뛰어난약리활성성분을가지고있다는것이제시되면서이에대한연구가지속적으로이루어져왔다 (Goldberg et al., 1999; Kamada et al., 2001). 페놀화합물은치환할수있는수산기 (OH) 기를가지고있는방향족고리구조를가지고있는화합물로서과일을비롯하여채소나포도주나녹차와같은음식물에다량함유되어있다 (Khan & Hadi, 1998). 지금까지추출된페놀화합물에는탄닌산을비롯하여갈릭산, 피크린산, 페루릭산및시린직산등여러종류들이추출정제되었다 (Haslam, 1979; Duthie & Alan, 2000). 이들은모두수산기를비롯하여카르복실기, 메틸기및질산기와같은구조를모두가지고있거나아니면이중몇개만을가지고있어이들의작용에따라만약이들이다른물질과치환되면항암작용이나항산화작용을나타낸다고

2 한다 (Li et al., 1999; Heilmann et al., 2000). 페놀화합물중탄닌산은수산기와같은자유산소를생산하여세포내 DNA와같은핵산물질의분절을일으킨다고알려져있다 (Khan & Hadi, 1998). 또한피크린산 (picric acid) 은하나의수산기를가지고있으며카르복실기를갖고있지않는페놀화합물인데비하여갈릭산 (gallic acid) 이나시린직산 (syringic acid) 및페루릭산 (ferulic acid) 들은모두수산기와카르복실기를가지고있으며, 또한두개이상의자유산소기를가지고있어피크린산에비하여항암효과가훨씬강하다고알려져있다 (Yang, 2003). Li et al. (1999) 은수산기와카르복실기가동시에작용할경우정상적인세포에는독성을나타내지않으나암세포에는선택적으로독성을나타낸다고하였다. 이중페루릭산은위에서살펴본바와같이수산기와카르복실기를다같이가지고있는페놀화합물의일종으로이의구조식에수산기하나와카르복실기하나및메틸기하나의구조식을이루고있어매우유용한항암효과가있다고알려져있으나아직까지이의항암작용에대해서는많은연구가되어있지않았다 (De Heredia et al., 2001; Yang, 2003). 근래에세포배양기술이발달되면서멜라닌세포를비롯한각종세포의배양이가능하게되면서배양세포를재료로한항암실험이많이행하여지고있다 (Mosmann, 1983). 특히, 세포배양에있어서멜라닌세포는다른세포들과는달리특히, 태양의자외선이나방사선등에매우민감하기때문에이의배양이다소어렵고동시에여러화학물질이나독성물질에도민감하게반응하기때문에이들에의한영향을정확하게측정할수있는잇점도있다 (Michikawa et al., 1994). 특히, 인체멜라닌흑색종은각종항암제를비롯한화학약제및독성물질에대한효과를정량적으로검정하는데매우적합한재료로선택되고있으며동시에약재의독성이나효능을단시간내에정확히검색하는데널리이용되고있다 (Borenfreund & Puerner, 1984). 본연구는페놀화합물의일종인페루릭산의항암효과에대한기전을규명하기위하여페루릭산이정상세포인 NIH3T3 섬유모세포와인체피부흑색종에미치는영향을 colorimetric assay에의하여정량적으로분석조사하여유의한결과를얻었기에이에보고하고자한다. 재료및방법 1. 약제제조본실험에사용한페루릭산 (ferulic acid, Sigma Co.) 은각각 1 M, 100 mm, 10 mm, 1 mm의저장액을만들어냉암소에보관한다음실험당일최종농도로희석사용하거나필요한양을직접배양액에첨가하여사용하였다. 2. 세포배양배양용기에배양중인섬유모세포 (NIH3T3 fibroblast) 와인체피부흑색종세포 (SK-MEL-3) 의분리는효소해리술에의하여행하였다. 세포는혈구계산기를이용하여배양액에 cells/well의밀도로 96-multiwell에넣어분주하였다. 분주가완료된세포는 CO 2 배양기에서 5일동안배양한후실험에사용하였으며, ferulic acid가포함되지않은배양액을대조군으로하여실험군과비교조사하였다. 3. 페루릭산의처리페루릭산이배양 NIH3T3 섬유모세포와인체흑색종세포에미치는영향을조사하기위하여 30 µm에서 120 µm 까지의농도로페루릭산이각각포함된배양액에서두세포종을 48시간동안배양한후독성효과를세포생존율측면에서조사하였다. 4. 세포생존율조사배양 NIH3T3 섬유모세포와인체피부흑색종세포에여러농도의페루릭산을각각 48시간동안처리한후약재가세포들에미치는영향을 Mosmann (1983) 의 XTT분석법에의하여조사하였다. 즉, PBS (phosphate buffered saline) 에녹인 1 mg laminin의저장액을냉장고에보관한후실험당일필요한양을희석한다음 24 well plate에 200 µl씩분주하여하룻밤동안건조시켰다. 건조완료후 PBS로 3회세척한다음 3% BSA (Bovine serum albumin, Sigma Co.) 를각 well당 200 µl씩첨가하여잘진탕한후다시이를 PBS로두세번세척하였다. 배양된두종류의세포를 cells/ml 밀도로배양용기에넣고 24 시간동안배양하였다. 배양완료후페루릭산을각각의농도를처리하여 48시간동안배양한다음 PBS로 3회세척하였다. 세척완료후 XTT와혼합후각배양용기에 200 µl씩을주입하여 4시간동안배양한다음 450 nm에서 ELISA reader로흡광도를측정하였다. 분석방법의개요는 Fig. 1과같다. 5. 통계처리대조군과실험군의차이에대한통계적유의성은 Student's t-test로비교하였다. 통계적유의수준은 P값이 0.05 미만인경우로하였다. 결과 1. 페루릭산의독성효과측정 1) NIH3T3 세포섬유모세포에대한세포생존율 NIH3T3 섬유모세포에페루릭산이 30~120 µm의농도로

3 Coating Laminin 24 hrs (incubation) Seeding cells 24 hrs (incubation) Adding ferulic acid 48 hrs (incubation) Adding XTT 4 hrs (incubation) Reading XTT absorbance at 450 nm Fig. 1. Flow Scheme of XTT assay Table 1. The cell viability of ferulic acid on NIH3T3 fibroblasts by XTT assay Concentration of Group ferulic acid (µm) Mean ± SD (% of control) Control 9.58± ± ± ± ± IH3T3 fibroblasts were incubated with or without 30~120 µm ferulic acid for 48 hours. The value represent the mean ± SD for triplicate experiments 각각포함된배양액에서 48시간동안배양한후페루릭산의독성효과를 XTT 분석법에의하여조사하였다. 결과 30 µm 페루릭산의처리에서는세포생존율이대조군인 100% (9.58 ±0.83) 비하여 98.3% (9.42±0.76) 로나타났으며 60 µm의처리에서는 97.2% (9.31±0.89) 로나타났다. 또한 90 µm 페루릭산의처리에서는세포의생존율이 96.6% (9.25±0.91) 로나타났으며 120 µm 페루릭산의처리에서는세포생존율이대조군에비하여 96.7% (9.26±0.74) 로나타났다. 이들은모두대조군에비하여유의성이없는것으로나타났다 (Table 1). 2) 인체피부흑색종세포에대한세포생존율인체피부흑색종세포에페루릭산이 30~120 µm의농도로각각포함된배양액에서 48시간동안배양한후페루릭산의독성효과를 XTT 분석법에의하여조사하였다. 결과 30 µm 페루릭산의처리에서는세포생존율이대조군인 100% (5.19 ±0.47) 비하여 95.5% (4.93±0.36) 로나타났으며 60 µm의처리에서는 89.6% (4.65±0.51) 로나타났다. 또한 90 µm 페루릭산의처리에서는세포의생존율이 81.9% (4.25±0.40) 로나타났으며 120 µm 페루릭산의처리에서는세포생존율이대조군에비하여 74.8% (3.88±0.27) 로나타났다. 특히, 120 µm 의페루릭산의처리에서는대조군에비하여세포생존율이유의하게감소한것으로나타났다 (Table 2). Table 2. The cell viability of ferulic acid on human skin melanoma cells (SK-MEL-3) by XTT assay Concentration of Group ferulic acid (µm) Mean ± SD (% of control) Control 5.19± ± ± ± ± * Human skin melanoma cells were incubated with or without 30~ 120 µm ferulic acid for 48 hours. The value represent the mean ± SD for triplicate experiments. *P<0.05 (Student's t-test) Table 3. The cytotoxicity in NIH3T3 fibroblasts and human skin melanoma cells (SK-MEL-3) by XTT assay XTT assay (cell viability) Ferulic acid NIH3T3 SK-MEL µm 96.7% 74.8% * NIH3T3 fibroblasts and human skin melanoma cells (SK-MEL-3) were incubated with 120 µm ferulic acid, respectively. The values were determined by XTT assay. *P<0.05 (Student's t-test) 3) 세포종에대한페루릭산의세포독성 NIH3T3 섬유모세포와인체피부흑색종세포에대한페루릭산의세포독성에있어서각각 120 µm 페루릭산을 48시간동안처리한결과 NIH3T3 섬유모세포에서의세포생존율은대조군인 100% (9.58±0.83) 에비하여 96.7% (9.26±0.74) 로나타난반면인체피부흑색종세포의처리에서는세포생존율이대조군인 100% (5.19±0.47) 비하여 74.8% (3.88±0.27) 로나타났다 (Table 3). 고 최근각국선진국들은천연물로부터독성이적은항암물질을개발하려는목적으로 1990년대부터수많은식물의성분추출조사에착수하였다 (Li et al., 1999; Takemura et al., 2001). 식물성추출성분으로는주로페놀산을비롯하여알카로이드, 카로텐및스테로이드등다양한물질들이포함되었으며특히, 분리된페놀성분이약 8,000종이상되었다고보고되었다 (Duthie & Alan, 2000). 그밖에도항암작용을비롯하여항산화작용및항염증성작용과관련된다양한추출물에관한연구가계속진행되어왔다 (Haslam, 1979; Goldberg et al., 1999). 특히, 페놀화합물의추출물중페루릭산을비롯한시린직산, 갈릭산등은이의분자구조에다른물질과치환할수있는수산기를가지고있으며이들이 carboxyl group의에스테르화와 hydroxyl group의메틸화됨으로써암세포에대하여항암작용을나타낸다고알려져있다 (Sakaguchi et 찰

4 al., 1998; Kawada et al., 2001). 시린직산의경우이에대한약리작용의연구는그리많지않으며단지항산화작용과 DPPH radical-scavenging 활성에관한연구가보고된바있다 (Goldberg et al., 1999, Yang et al., 2003). 갈릭산의경우에는 phenolic hydroxyl group의메틸화와 carboxyl group의에스테르화에의하여세포독성을경감하는약리활성을나타낸다고알려진바있다 (Sakaguchi et al., 1998; Li et al., 1999). 특히, 페루릭산은이의분자구조에수산기와카르복실기를가지고있어에스테르화와메틸화를할수있는능력이있어갈릭산과함께뛰어난항암작용이있다고제시된바있다 (Khan & Hadi, 1998). 그러나페루릭산의약리활성에대한자세한기전은잘밝혀져있지않으며더욱이항암작용에관한연구는소수에불과하다 (Haslam, 1979). 본연구는페루릭산의항암효과를조사하기위하여암세포종인인체피부흑색종세포를배양하여이에독성을정상세포인 NIH3T3 섬유모세포와비교조사하였다. 먼저페루릭산이정상세포에미치는영향을조사하기위하여페루릭산이 30~120 µm 농도로각각포함된배양액내에서 NIH3T3 섬유모세포를 48시간동안배양한후이에대한영향을세포생존율측면에서조사하였다. 그결과페루릭산을처리한농도에따라약간의세포생존율의감소가있었으나 120 µm의갈릭산의처리에서세포생존율은대조군인 100% 에비하여 95.0% 로나타남으로써유의한세포생존율의감소를보이지않았다. 이러한본실험결과는페루릭산이 NIH3T3 섬유모세포에는아무런세포독성을나타내지않음으로써정상세포에대해페루릭산은세포손상을초래하지않은것으로나타났다. 이러한결과는아마도 Li et al. (1999) 과 Sagaguchi et al. (1998) 에근거하여카르복실기와수산기가동시에작용하면암세포에만선택적인독성효과를보인다는연구결과와일치한다고생각된다. 따라서본연구에서는페루릭산이암세포에미치는영향을조사하기위하여 NIH3T3 섬유모세포에처리하였던동일농도를처리하였던바처리농도에비례하여세포생존율의감소를보였으며특히, 120 µm 페루릭산의처리에서는세포생존율이대조군인 100% 에비하여 74.8% 로나타나유의한감소를나타냈다. 본실험결과는페루릭산이인체피부흑색종세포에세포독성효과를나타낸것을알수있었으며이러한현상은페루릭산이선택적으로세포독성을나타낸것을알수있다. 본연구결과는 Yang (2003) 이식물이나견과류에서다량함유되어있는페루릭산을정상세포와암종세포에처리한결과정상세포에는무독성으로나타난데비하여암종세포에는고독성을나타냈다는실험결과와일치함을알수있었다. Heilmann et al. (2000) 은페루릭산과같은페놀화합물이정상세포에는독성효과를보이지않으면서암종세포에선택적으로독성효과를보인것은이들의구조식을이루고있는수산기와카르복실기의상호작용이며또한세포내칼슘이온이나활성산 소와같은세포내인자의변화와도밀접한관련이있다고보고한바있다. 그밖에도페놀화합물은항암작용외에도항산화작용을비롯하여항균작용, 항염작용등이알려져있으나이에대한자세한기전에대해서도잘밝혀져있지않다 (Kawada et al., 2001). 그러나이에대한자세한기전규명을위해서는페루릭산의항암효과와단백질합성간의상호작용을비롯하여암유전자를억제하는효소활성에대한생리활성의기전, 세포의핵산물질인 DNA의합성및세포내 protein kinase C와같은신호전달체계와같은측면에서더욱체계적인분석이필요할것으로생각한다. 감사의글이논문은 2004년도원광대학교의교비지원에의해서수행됨. REFERENCES Borenfreund E, Puerner JA. A simple quantitative procedure using monolayer culture for cytotoxicity assay (HTD/NR-90). J Tiss Cult Meth : 7-9. De Heredia JB, Torregrosa J, Dominguez JR, Peres JA. Kietic model for phenolic compound oxidation by Fenton's reagent. Chemosphere : Duthie Garry C, Alan. Plant-derived phenolic antioxidants. Current Opinion in Clinical Nutrition and Metabolic Care. Lippincott williams and wilkins : Goldberg DM, Hoffman B, Yang J, Soleas GJ. Phenolic constituents, furans, and total antioxidant status of distilled spirits. J Agric Food Chem : Goodman GE, Yen YP, Cox TC, Crowley J. Effect of verapamil on in vitro cytotoxicity of adriamycin and vinblastine in human tumor cells. Cancer Res : Haslam E. Vegetable tannins. Res Adv Phytochem : Heilmann J, Calis I, Kirmizibekmez H, Schuhly W, Harput S, Sticher O. Radical scavenger activity of phenylethanoid glycosides in FMLP stimulated human polymorphonuclear leukocytes: structure-activity relationship. Planta Med : Husniye Dilek F, Kucukali T. Mucin production in carcinomas of the uterine cervix. Eur J Obstet Gynecol Reprod Biol : Kawada M, Ohno Y, Ri Y, Ikoma T, Yuugetu H, Asai T, Watanabe M, Yasuda N, Akao S, Takemura G, Minatoguchi S, Gotoh K, Fujiwara H, Fukuda K. Anti-tumor effect of gallic acid on

5 IL-2 lung cancer cells transplanted in mice. Anticancer Drugs : Khan Nelofer S, Hadi SM. Structural features of tannic acid important for DNA degradation in the presence of Cu(II). Mutat Res : Lewin B. Oncogene conversion by regulatory changes in transcription factors. Cell : Li Z, Inou M, Nose M, Kojima K, Sakaguchi N, Isuzugawa K, takeda T, Ogihara Y. Metabolic fate of gallic acid orally administrated to rat. Biol Pharm Bull : Michikawa M, Lim KT, McLarnon JG, Kim SU. Oxygen radicalinduced neurotoxicity in spinal cord neuron cultures. Neursci Res : Momann T. Rapid colorimetric assays for cellular growth and survival: application to proliperation and cytotoxicity assays. J Immunol Meth : Sakaguchi N, Inoue M, Ogihara Y. Reactive oxygen species and intercellular Ca 2+, common signals for apotosis induced by gallic acid. Biochem Pharmacol : Sharma S, Jill DS, Kelloff GJ, Vernon ES. Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res : Takemura G, Minatoguchi S, Gotoh K, Fujiwara H, Fukuda K. Anti-tumor effect of gallic acid on IL-2 lung cancer cells transplanted in mice. Anticancer Drugs : Wattenberg LW. Chemoprevention of cancer. Cancer Res :

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,