CANCER PREVENTION RESEARCH ORIGINAL ARTICLE 수용성된장추출물의대장암세포주 HT-29 와 CaCo-2 에대한항암효과 숙명여자대학교생명과학과, 1 한국식품연구원 김화영ㆍ정유선ㆍ구민선 1 ㆍ최순영 Anticancer Effects by the Water Soluble Factor of Doenjang Extracts on Human Colon Cancer HT-29 and CaCo-2 Cells Hwa Young Kim, You Sun Jung, Min Seon Koo 1 and Soon Young Choi Division of Biological Science, Sookmyung Women s University, Seoul 140-742, 1 Korea Food Research Institute, Sungnam 463-746, Korea Doenjang is one of the important fermented foods in Korea. The traditionally fermented soypaste, Doenjang, have higher anti-mutagenic activity than the raw soybeans, cooked soybeans, meju and other fermented soybeans. In this study, we investigated the anticancer effects of the water soluble factor of Doenjang extracts on human colon cancer HT-29 and CaCo-2 cells for a time. To identity the effects of the water soluble factor of Doenjang extracts, we treated the water soluble factor of Doenjang extracts on HT-29 and CaCo-2 cells for 24 h and 48 h. These results showed that HT-29 and CaCo-2 cells were slightly inhibited cell growth compared to control by WST-1 assay. The treatment of the water soluble factor of Doenjang extracts down-regulated gene expression of colon cancer bio-marker such as cox-2, tgf-beta receptor II and smad-4. It also down-regulated the expression of oncogenes related with cell proliferation, survival and metastasis such as her-2, cyclind-1, myc, mmp14. Our data indicated that the water soluble factor of Doenjang extracts has anticancer effect on human colon cancer. (Cancer Prev Res 13, 324-329, 2008) Key Words: Doenjang extracts, Colon cancer, Proliferation, Bio-marker, Anti-cancer 서론발효식품은세계적으로중요한식이요법으로잘알려져있다. 우리나라의대표적인전통발효식품중의하나인된장은대두를찐다음으깨어메주를만들면자연에서의발효균이콩의단백질을먹으며대두를발효시킨다. 1) 대두단백질은발효시미생물들이생산하는가수분해효소에의해여러종류의펩타이드로분해된다. 이 러한수용성된장추출물은다양한생리활성을나타내는것으로알려지고있는데 aflatoxin과같은다양한 carcinogen과 mutagen에대한강력한항돌연변이물질로작용하는것이확인되었으며, 2) 세포분자수준과생체수준에서의암형성과전이를억제하는항암효과도확인되었다. 3 5) 또한된장은혈전용해활성, glucose 및 bile acid 투과억제효과, ACE 저해활성, 장내세균의증식억제효과및면역력의증가등이효과를나타내는것으로보고되고있다. 6) 책임저자 : 최순영, 140-742, 서울시용산구효창원길 52 숙명여자대학교이과대학생명과학과 Tel: 02-710-9510, Fax: 02-2077-7322 E-mail: sychoi@sookmyung.ac.kr 접수일 :2008 년 11 월 11 일, 게재승인일 :2008 년 11 월 28 일 Correspondence to:soon Young Choi Division of Biological Science, Sookmyung Women s University, Hyochangwon-gil 52, Yongsan-gu, Seoul 140-742, Korea Tel: +82-2-710-9510, Fax: +82-2-2077-7322 E-mail: sychoi@sookmyung.ac.kr 324
김화영외 3 인 : 수용성된장추출물의대장암세포주 HT-29 와 CaCo-2 에대한항암효과 325 최근우리나라의경제성장발전에따른생활수준의향상과다양한외식문화의보급으로인해식생활의형태가변화되고있으며복잡한사회생활에따른스트레스와함께각종질환을가져오게되었다. 7) 특히암의발생으로인한사망률이점차증가하는추세로암의원인의 70% 정도는흡연, 감염, 음식등의환경요인이주이며유전적인원인이 5% 정도이다. 8) 우리나라에서암발병률은 2001년위암, 폐암, 간암, 대장암순이었으나, 현재대장암의발병률은 2위까지올랐다. 대장암사망자는미국, 영국, 일본등에서는줄어들고있는추세이나, 우리나라를포함한일부국가에선빠르게증가하고있다. 9) 대장암은직장, 결장, 맹장에생기는악성종양중하나로많은세포들이응축된형태로써혈관을중심으로성장하는대표적인고형암이다. 또한약물이제대로투과하지못한환경때문에대장암세포를완벽하게제거하기는어렵다. 10 12) 현재대장암치료법은외과수술, 방사선치료, 화학요법등이이용되고있다. 13) 특히치료에사용되는항암제는암세포뿐만아니라정상세포에도세포사멸등을일으켜다양한부작용을일으키고있으며지속적인항암제투입은내성을생기게한다. 14) 따라서본연구에서는정상세포에대해독성이없으면서암세포에항암효과를나타내는생리활성물질을개발하기위해숙성된기간에따른된장의수용성추출물이대장암세포주 HT-29와 CaCo-2의성장에어떠한영향을미치는지살펴보고자하였다. 이번실험에서는대두자체가아닌발효되어생성된물질의영향을살펴보기위함이고또한이미많은연구가된지용성추출물혹은 methanol 추출물과비교하여수용성추출물에대한효과를알아보고자하였다. 이러한연구를바탕으로천연생리활성물질인수용성된장추출물이대장암세포주의증식억제에미치는영향에대해알아보고자하였다. 재료및방법 1. 된장의수용성추출물제조 생리활성을평가하기위하여시료는다음과같은방법으로조제하여생리활성평가용시험액을조제하였다. 즉, 균질화된된장 20 g에 80 ml의증류수를넣고, 1분간균질기 (Pulsifier) 로충분히혼합한후, 3,000 rpm으로 10분간원심분리하였다. 상등액을다시 4 o C 하에서 12,000 g에서 10분간원심분리하여, 상등액을 0.2μm membrane filter로여과하여그여액을생리활성평가를위한시험액으로사용하였다. 2. 세포배양인간대장암유래의세포주 HT-29와 CaCo-2는한국세포주은행 (KCLB, Seoul) 으로부터분양받아본실험에사용하였다. 세포배양은 HT-29는 RPMI1640 medium (Wel- GENE Inc., Daegu, Korea) 에, CaCo-2는 MEM medium (WelGENE Inc., Daegu, Korea) 에 10% fetal bovine serum (FBS) (Gibco BRL, NY, USA) 및 1% penicillin/streptomycin (WelGENE Inc., Daegu, Korea) 을첨가하여사용하였다. 95% 의습도가유지되는 37 o C, 5% CO 2 incubator (ASTEC, model SCA-165DS, Fukuoka, Japan) 에서배양하였다. 3. 세포증식차이관찰 HT-29와 CaCo-2 세포주를 60 mm plate에 6 10 5 cells/ plate로 24시간동안배양하고, 된장을숙성시킨기간별 (1년, 2년, 3년숙성 ) 에따른각각의수용성된장추출물을 50 ul/ml로 24시간동안처리한후, micoscope (OLYM- PUS, DP70, Tokyo, Japan) 를이용하여세포증식의차이를관찰하였다. 4. WST-1 assay를통한세포증식억제측정수용성된장추출물에의한세포증식을측정하기위하여 WST-1 Proliferation assay (Roche Diagnostics, GmbH Mannheim, Germany) 를이용하였다. 96 well plate에 HT-29 세포는 6 10 3 cells/well을, CaCo-2 세포는 8 10 3 cells/well을 200 ul medium에서 24시간동안배양하고수용성된장추출물을숙성기간별로 10 ul/200 ul을 24시간, 48시간동안처리하였다. 각 well에 WST-1 reagent를 20 ul씩첨가하여다시 2시간동안배양시킨후, ELISA Reader (Molecular Devices, model SpectraMaxM5 e ) 를이용하여 450 nm에서흡광도를측정하였다. 오차를줄이기위해각 sample 당 5개의 well에서수행하였고평균값을구하여결과값으로결정하였다. 5. Reverse transcriptase-polymerase chain reaction (RT-PCR) 분석동일조건에서준비된세포를 PBS로세척하고 TRIzol reagent (Invitrogen Co., CA, USA) 를이용하여 total RNA를분리하였다. 분리된 RNA를정량한후, total RNA 1 ug로부터 ImProm-II TM Reverse Transcription System (Promega, WI, USA) 을이용하여 cdna를합성하였다. 이 cdna를 template로하여관련된유전자특이적 primer를사용하여 polymerase chain reaction (PCR) 방법으로증폭하였다 (Table 1). 이때 housekeeping 유전자인 β-actin 유전자를
326 Cancer Prevention Research Vol. 13, No. 4, 2008 internal control로사용하였다. 각 PCR 산물들은 1.3% agarose gel을이용하여전기영동하고 ethidium bromide (EtBr, BiONEER, Daejeon, Korea) 를이용하여염색한후 UV 하에서 gel-doc program (DNR bio-imaging system, Jerusalem, Israel) 으로촬영하였다. Table 1. Gene-specific primers for RT-PCR Gene name COX-2 TGF-β R II Smad-4 HER-2 CCND-1 MYC MMP 14 Beta-actin Sequence CGC TTT ATG CTG AAG CCC TA GAA AAC CCA CTT CTC CAC CA CAG TTT GCC ATG ACC CCA AG CAT TGC ACT CAT CAG AGC TAC AGG AAG GTG AAG GTG ATG TTT G TAA GGG CCC CAA CGG TAA A CCC AAC CAG GCG CAG AT AGG GAT CCA GAT GCC CTT GTA TCC TCT CCA AAA TGC CAG AG GGC GGA TTG GAA ATG AAC T TGC TCC TGA GGA GAC ACC CTC TGA CCT TTT GCC AGG AG CCC CGT TGT CTC CTG CTC GGG AGG CAG GTA GCC ATA TT CTC GGA CGA CAT GGA GAA GGA TCT TCA TGA GGT AGT C 결과및고찰 1. 수용성된장추출물에의한대장암 bio-maker들의발현억제효과 대장암세포주에대한수용성된장추출물의효과를확인하기위하여배양된대장암세포에수용성된장추출물을처리한후 RT-PCR을실행하였다. Bio-marker는 cox-2, tgf-beta receptor II, smad-4를사용하였으며이 gene들은대장암세포주에서과발현되는것으로널리알려져있다. 특히, cox-2는대장암세포주에서 80% 이상과발현이되며대장암세포주의 apoptosis 억제와 angiogenesis 유발그리고세포증식에관여를하는것으로밝혀져있다. 15) Tgf-beta receptor II의유발은 smad-4의발현과연관이되어있으며대장암세포주의진행정도를알려주는지표로쓰이고있다. 16,17) 수용성된장추출물을 24시간동안처리한후대장암세포주에서과발현되는 bio-marker로 RT-PCR을이용해유전자의발현정도를확인하였다 (Fig. 1). 아무것도처리하지않은암세포와일반적인각된장수용성물질들의염농도가 2% 이므로 2% NaCl 처리한암세포를대조군으로사용하였으며대조군과비교하였을때수용성된장추출물을처리한세포주들은전체적으로 bio-marker들의발현이감소하였다. 수용성된장추출물은각각의숙성연도에따라 #1, #2, #3으로표기하였다. Fig. 1에서보듯이대장암 bio-marker인 cox-2, tgf-beta receptor II, smad-4의발현은 HT-29에서모두감소되었으며, CaCo-2의경우에는 bio-marker의발현이 HT-29에비하여미미하지만감소하는것이확인되었다. 이러한결과로수용성된장추출물이대장암세포에서과발현되는유전자들의발현을전사수준에서억제하는것을확인하였다. Fig. 1. Inhibition of colon cancer overexpression genes by the water soluble factor of Doenjang extracts treatment for 24 hr in human colon cancer HT-29 (A) and CaCo-2 cells (B) at transcription level. Data showed that overexpression genes were decreased to ripening periods of Doenjang compared with untreated cells and 2% NaCl treated cells.
김화영외 3 인 : 수용성된장추출물의대장암세포주 HT-29 와 CaCo-2 에대한항암효과 327 2. 수용성된장추출물에의한대장암세포주의증식억제대장암세포주의증식에미치는숙성기간별수용성된장추출물의영향을조사하기위하여대장암세포주 HT-29와 CaCo-2에수용성된장추출물을처리하였다. 24 시간후, 현미경으로확인결과수용성된장추출물을처리한대장암세포주들은대조군에비해세포증식이억제가됨을알수있었다 (Fig. 2). 육안으로관찰된세포증식억제효과를확인하기위하여대장암세포주에 24시간, 48시간동안수용성된장추출물을처리한후 WST-1 reagent assay를실시하였다. 암세포에테트라졸륨성분 (WST-1) 을넣어주게되면세포내의미토콘드리아가가진탈수소효소에의해특정색깔을타나내는물질로변하게된다. 이러한색깔의변화를측정함으로써세포내의미토콘드리아가얼마나활성을가지는지알수있고, 미토콘드리아가살아있다는것은곧세포가살아있다는뜻이되기때문에세포의수나증식정도, 활성등을알수있다. 18) 배양된대장암세포주에수용성된장추출물을첨가하는시점을 0시간으로정하였으며, 처리후 24시간과 48시간이되는시점에 WST-1 reagent를첨가하여 assay를실시하였다. Fig. 3 에서보듯이대장암세포 HT-29와 CaCo-2에수용성된장추출물을처리하였을때모두세포증식억제의효과 Fig. 2. Proliferation inhibition by the water soluble factor of Doenjang extracts treatment for 24 hr in human colon cancer HT-29 (A) and CaCo-2 (B) cells. Data showed that proliferation was decreased to ripening periods of Doenjang compared with untreated cells and 2% NaCl treated cells. Fig. 3. Growth inhibitory effect of the water soluble factor of Doenjang extracts from various ripening periods of Doenjang on human colon cancer HT-29 (A) and CaCo-2 (B) cells by WST-1 assay.
328 Cancer Prevention Research Vol. 13, No. 4, 2008 Fig. 4. Suppression of oncogene expression by the water soluble factor of Doenjang extracts treatment for 24 hr in human colon cancer HT-29 (A) and CaCo-2 cells (B) at transcription level. Data showed that oncogene expression was decreased to ripening periods of Doenjang compared with untreated cells and 2% NaCl treated cells. 를보였다. HT-29세포는 48시간처리하였을때세포증식억제의효과가나타났으며, CaCo-2세포는 24시간시점부터세포증식에큰차이를보였다. 이상의결과로부터수용성된장추출물이 HT-29와 CaCo-2 세포주에대해암세포증식억제의효과를보이는것을확인할수있었다. 3. 대장암세포주에서수용성된장추출물에의한 oncogene의발현억제 세포내 oncogene으로알려진유전자들은암유발, 암세포성장, 증식, 전이와관련되어과발현되어있는것이일반적이다. 대장암의 oncogene은 HER-2, CycilnD-1, Myc, TOP2A, EGFR 등이있다. 19) Her-2는대장암뿐만아니라유방암에서도특히과발현을보인다. Egfr이발현이되면 ccnd-1, myc, bcl-x L 등의 transcription이일어나며이러한유전자들은암세포에서과발현되어암세포의 proliferation, differentiation, survival을촉진하며 angiogenesis를일으켜 metastasis를촉진하기도한다. 20) 앞의결과에서수용성된장추출물에의한대장암세포의세포증식억제효과를확인하였다. 이러한세포증식억제효과가세포내 oncogene으로알려진유전자들의발현변화와연관되어있는지확인하기위하여수용성된장추출물을처리한세포의 total RNA를추출하여 RT-PCR을실행하였다. Fig. 4에서수용성된장추출물을대장암세포에처리하였을때암세포증식과전이에연관된유전자들의발현이모두감소하였다. HT-29세포에서 her-2와 mmp14의발현은숙성기간 1년부터현저히감소되었으며, ccnd1의발현은숙성기간에따라더욱감소하였다. CaCo-2 세포의경우 ccnd1 유전자의발현은 HT-29와마찬가지로숙성기간에의존적으로감소하였으며 mmp14와 myc 유전자의발현도의미있게감소됨을확인하였다. 이러한결과로부터수 용성된장추출물의대장암세포주 HT-29와 CaCo-2에서종양유전자인 oncogene들을감소시키며된장숙성기간이길어짐에따라억제의정도가강해지는것을증명하였다. 결론우리나라의대표적인발효식품으로숙성시킨된장의기간에따른대장암세포주의항암효과에대해살펴보았다. 대장암세포주에서과발현되는유전자들은암세포증식에기여를하는데대조군에비해수용성된장추출물을처리한암세포에서과발현되는유전자들이감소하는것을확인하였다. 이를대장암세포주 HT-29와 CaCo-2의현미경으로관찰한결과, 수용성된장추출물을처리한대장암세포주는대조군에비해세포증식이느린것으로관찰되었다. 더나아가 WST-1 assay를통해암세포의성장억제효과를관찰한결과, 수용성된장추출물을처리한암세포들은대조군에비해미토콘드리아생존율이감소되었음을확인하였다. 암세포의 proliferation, differentiation, survival, angiogenesis 그리고 metastasis를조절하는 marker로알려진 oncogene의발현을 RT-PCR로확인한결과수용성된장추출물을처리한암세포에서된장의숙성시킨기간에따라이러한유전자들의발현이감소되는것을알수있었다. 이상의결과를바탕으로된장의수용성추출물에함유된생리활성물질이대장암의증식과분화그리고전이를억제하는기능이있으며또한이러한기능은된장의숙성기간에따라다르게나타날것으로보인다. 따라서된장의수용성추출물과숙성기간에따른생리활성물질의항암효과에관한연구가계속진행되어야할것이다.
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