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1 ORIGINAL ARTICLE Journal of Breast Cancer J Breast Cancer 21 December; 13(4): DOI: 1.448/jbc 유방암세포주 ( 와 MDA-MB231) 에서 Genistein 의세포주기 G2/M 정지기전 박지영ㆍ강구성ㆍ손윤경 경북대학교의학전문대학원병리학교실 Molecular Mechanism of the G2/M Arrest in Breast Cancer Cell Lines ( and MDA-MB231) Induced by Genistein Ji-Young Park, Ku-Seong Kang, Yoon Kyung Sohn Department of Pathology, Kyungpook National University School of Medicine, Daegu, Korea Purpose: To analyze the effect of the growth control on human breast cancer cells with genistein treatment and to investigate the mechanism of genistein-induced G2/M arrest in and MDA-MB231 breast carcinoma cells by Cdc25C expression. Methods: We analysed the proliferartion of the two cell lines by using MTT proliferation assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting and investigated the effect of genistein on cell survival, cellular toxicity, cell cycle progression-related genes and their mrna and protein alterations. Results: The DNA flow cytometric analysis of both cell lines treated with genistein showed a dose-dependent growth inhibition and accumulation in the G2/M phase of cell cycle. The expression of mrna and protein increased in both cell lines following genistein treatment but expression was unchanged. Furthermore, decreased Cdc25C expression with decreased polo-like kinase (PLK) 1 expression and increased expression were observed after genistein treatment. The decreased level of Cdc25C in the nucleus was associated with decreased phosphorylation of Cdc25C by. The expression of was increased with a dose-dependent and a time-dependent manner and was associated with decreased Cdc25C expression. Check point kinase (CHK) 1 and CHK2 revealed different expression patterns each other. The CHK1 expression was independent of the presence of genestein. CHK2 expression increased in MDA-MB231 cells associated with decreased Cdc25C expression but not in. Conclusion: These results suggest that genistein induces a G2/M arrest in human breast cancer cells, the mechanism of which is due, in part, to decreased in Cdc25C phosphatase by a regulatory effect of,, and CHK2 as well as increased expression of the cyclin dependent kinase inhibitor (WAF1/CIP1). Key Words: Breast neoplasms, Cell cycle, Genistein 중심단어 : 유방암, 세포주기, 게니스틴 서 Genistein은콩에존재하는대표적인 isoflavone으로서유방암세포를비롯한많은암세포에서 G2/M arrest와세포자멸사를 론 책임저자 : 손윤경 대구광역시중구동덕로 2, 경북대병원병리과 Tel: , Fax: yksohn@knu.ac.kr 접수일 : 21 년 6 월 18 일게재승인일 : 21 년 11 월 12 일 조절하는유전인자에관여하여암세포의성장을억제하는등의항암효과가있는것으로알려져있다.(1-8) 1987년에 Akiyama 등 (9) 이 genistein이 protein tyrosine kinase (PTK) 의활성을억제하는효과가있음을처음으로기술하였는데, PTKs는종양발생뿐아니라분화에도결정적인역할을미치는것으로알려져있어서 (1) genistein이암을억제하는역할을설명하는근거가되고있다. 또다른연구는 genistein에의한 DNA topoisomerase II의억제 (11) 와 ribosomal S6 kinase 활성의억제를보고하였 382

2 Genistein Induced G2/M Arrest in Breast Cancer Cells 383 다.(12) 또한, 시험관연구에서 genistein은혈관생성을억제하고,(13) 종양세포의전이를억제하였다.(14) Genistein의작용에 p53 단백이나에스트로겐수용체와안드로겐수용체등은유의한관계가없는것으로알려져있다.(15) 지금까지연구결과들을토대로할때, genistein이암세포의성장을억제하고, 분화를유도하며세포자멸사를초래하여암세포의성장을억제하는역할을하는것으로보이지만, 이러한작용의분자생물학적기전은아직명확히규명되지않았다. Genistein을유방암세포에처리했을때 G2/M arrest를유도하여암세포의성장을억제하는데, 그기전으로 genistein에의한 단백의증가와 cyclin B1의감소, 그리고 cdc2의활성억제를들수있다.(1,4,15) 다양한종류의암세포주에 genistein 을처리하였을때 단백의증가와 G2/M arrest 유도가대부분관련이있었으나, 단백의변화와상관없이 G2/M arrest 가일어나기도하였다.(2,16) 따라서 genistein이 G2/M arrest 를유도하는데있어서 단백을경유하는경로외에다른기전이관여하였을것으로생각된다. Cdc25C 단백은 CDK/cyclin 복합체를탈인산화하여 G2 에서 M주기로넘어가는것을촉진하는데중요한역할을한다. Cdc25C의활성은인산화된 serine의위치에따라달라지는데, 인산화를조절하는데에 polo-like kinase (PLK) 와 check point kinase (CHK) 가역할을하는것으로알려져있다.(17-22) 본연구에서는 genistein이유방암세포에서 G2/M arrest를유도하는과정에서 과같은 cyclin dependent kinase inhibitor를통하지않는다른경로, 특히 Cdc25C 단백과그조절인자들과관련된경로와연관되었는지를알아보고자하였다. 방법세포배양과 genistein 처리유방암세포주는 p53 단백돌연변이세포주이면서에스트로겐수용체가있는 와에스트로겐수용체가없는 MDA-MB231 (American Type Culture Collection, Rockville, USA) 를이용하였고, genistein (Sigma Chemical Co., St. Louis, USA) 은 dimethyl sulfoxide (DMSO; Sigma Chemical Co., St. Louis, USA) 에용해하여사용하였다. 세포성장정도분석 Genistein 처리후의세포성장정도를알아보기위해서 와 MDA-MB231을한 well당 2,개의세포를 24시간동안배양한후 genistein을각각,.1, 1, 1, 2, 5, 1 μm 로처리하여 72 시간동안배양하였다. Triazolyl blue (MTT; Sigma Chemical Co., St. Louis, USA) 용액 (2 mg/ml) 을한 well당 5 μl 씩첨가하여 4시간동안 37 에서반응시킨후 glycine: DMSO (1:8) 혼합액을한 well당 15 μl 씩첨가하여반응을종료하고 57 nm에서의흡광도를측정하였다. 이때대조군으로서최종농도가.1% 가되도록 DMSO를첨가하였다. DNA 유세포분석 1 mm 배양용기에 개의세포를배양하고 24 시간후최종농도가 1 μm 가되게 genistein을처리하였다. 3일후 PBS 완충용액으로수세하고 Trypsin/EDTA를처리하여세포를모은후 7% 에탄올로 4 에서 1시간동안처리하여세포를고정하고 PBS 완충용액으로두번수세하였다. 5 μg/ml의 RNase A를 37 에서 3 분동안반응시킨후첨가한 RNase A 와같은부피로 5 μg/ml의 propidium iodide 용액을첨가한후 FACScan flow cytometer (Becton-Dickinson, San Jose, USA) 를이용하여분석하였다. 세포주기관련유전자의 mrna 정량분석 Total RNA 분리 6 mm 배양용기에배양된각각의세포에 TRIzol (Invitrogen, Carlsbad, USA) 을 1. ml 넣고, ml 튜브에옮긴후,.2 ml 의클로로포름을넣고잘섞어준후 3분간상온에서반응시켰다. 12, g 에서 7분간원심분리하여생긴상층액을새튜브로옮긴후.8 배의이소프로판올을첨가하여섞어주고상온에서 1 분간반응시킨후 12, g 에서 1 분간원심분리하였다. 원심분리후생긴침전물을 75% 의알코올을첨가하여 vortex한후 7,5 g 에서 5분간원심분리하여용액을덜어낸후침전물을공기중에서말리고각각의튜브마다 3 μl 의 RNase free water 를첨가하여녹인후원액을 1 배희석하여 26 nm 파장에서정량을하였다. RNA 를분리하는데사용된용기와용액은모두.1% 4-diethyl pyrocarbonate를처리하였다. First strand cdna 합성 First strand cdna 합성 kit는 TaqMan Reverse Transcription Reagent (Applied Biosystems, Foster City, USA) 를이용하였다. 각각의튜브에전체 RNA 의양이 5 μg, random hexamer를 μg, MgCl 2 을 5.5 mm, deoxy NTP를 5 μm, RNase inhibitor를.8 U, 1X RT buffer를 2 μl, multiscribe reverse transcriptase를.5 U 넣은다음최종부피가 2 μl 가되게한후, 25 에서 1 분, 48 에서 3 분, 95 에서 5분반응시켜 cdna를합성하였다.

3 384 Ji-Yong Park, et al. 실시간정량분석 (Real time quantitative RT-PCR) 주형으로사용된 cdna는위에서만든 cdna를 1 배희석하여사용하였다. 반응액의조성은 2X SYBR green master mix 1. μl, 주형 4 μl, 각각의 primer를 2 nm 되게넣고물로최종부피를 2 μl 되게하였다. 반응조건은 5 에서 2분, 95 에서 1 분, 그리고 95 에서 15 초와 6 에서 1분을 4 회반응시켰다. 이때각 cycle마다의형광신호를 SDS 7 (Applied Biosystems, Foster City, USA) 로모니터링하여나타나는 threshold cycle (CT) 을분석한후 2 - CT 방법을이용하여그래프로나타내었다.(23) 이때사용한각유전자의프라이머염기서열은 Table 1과같다. 5% skim milk) 으로상온에서 2시간동안반응시킨후 TTBS 용액으로수세하고 1차항체,,,, Cdc25C, p-s-216-cdc25c, CHK1, alpha-tublin (Santa Cruz Biotechnology, Santa Cruz, USA), p-serine-198 Cdc25C 항체 (Cell Signaling Technologies, Beverly, USA) 을 1:2-1:1,으로항체반응용액 (25 mm Tris [ph 7.5], 15 mm NaCl,.1% Tween 2, 1% skim milk) 으로희석하여 4 에서 12 시간동안반응시켰다. TTBS 용액으로수세한후 2차항체를 1:1,으로희석하여상온에서 2시간동안반응시킨후 TTBS 용액으로수세하고 Immobilon Western 키트 (Millipore, Billerica, USA) 를이용하여 X-ray 필름에감광시켰다. Western blot 분석 1 mm 배양용기에 개의세포를배양한후 genistein 1 μm 되게첨가하고 1일, 2일, 3일후세포를모아서 lysis buffer (15 mm NaCl, 1.% NP-4, 5 mm Tris [ph 8.], 1 mm PMSF, 1 μg/ml aprotinin, 1 μg/ml Leupeptin,.1 mm sodium orthovanadate, 1 mm NaF) 를첨가하여얼음에서 1분간반응시킨후 12, rpm에서 1 분간원심분리하여상층액을모아서 BCA 방법으로단백질의양을결정하였다. 약 3-1 μg 의단백질을 8-12% acrylamide gel에전기영동하고, nitrocellulose membrane에블로팅한후 Ponceau 용액으로단백질의블로팅을확인하고 TTBS 용액 (25 mm Tris [ph 7.5], 15 mm NaCl,.1% Tween 2) 으로수세한후 blocking 용액 (25 mm Tris [ph 7.5], 15 mm NaCl,.1% Tween 2, Table 1. Primer sequence of cell cycle regulatory genes Gene GAPDH Cdc25C CHK1 CHK2 Primer sequence (sense/antisense) 5 -TGG GCT ACACTG AGC ACC AG-3 5 -GGG TGT CGC TGT TGA AGT CA-3 5 -CAA AGG CCC GCT CTA CAT CTT-3 5 -AGG AAC CTC TCA TTC AAC CGC-3 5 -GGA ATA AGG AAG CGA CCT GCA-3 5 -TCC ACA GAA CCG GCA TTT G-3 5 -ATA GAG CGT GAC GGC ACT GAG T-3 5 -TGC TCG CTC ATG TAA TTG CG-3 5 -TGT CAA AGT CAT CCC GCA GAG-3 5 -TGC GAA AAA CGC ACG ATG T-3 5 -TTT TTC CAA GGT ATG TGC GCT G-3 5 -TGG AAC TTC CCC GAC AGT AAG G-3 5 -CGT GAG CGT TTG TTG AAC AAG A-3 5 -CAC AGG ACC AAA CAT CAA CTG G-3 5 -GTG CCT GTG GAG AGG TAA AGC T-3 5 -TGC CTC TCT TGC TGA ACC AAT A-3 GAPDH=glyceraldehyde3-phosphatedehydrogenase; PLK=polo-like kinase; CHK=check point kinase. 결 과 Genistein 에의한 와 MDA-MB231 의성장억제 Genistein이유방암세포주 와 MDA-MB231의성장에미치는영향을알아보기위해서각각의세포주에 genistein을.1, 1, 1, 2, 5, 1 μm 되게처리하여 MTT 분석을하였다. 각각의세포는 1 μm 까지는성장에변화를보이지않지만, 2 μm 부터농도에비례하여세포의성장이억제되었다 (Figure 1). 이때대조군에비해 genistein 처리후세포의밀도가줄었다 (Figure 2). Genistein 에의한 G2/M arrest 유도 Genistein이각각의세포분열주기에미치는영향을알아보기위해서 FACS 분석을하였다. 는 G2/M에해당하는세포수가대조군은 21.94% 이었고 genistein을처리하였을때 69.26% 로약 3배증가하였다. MDA-MB231은대조군이 13.2% 이었고 Viable cells (%) Figure 1. Concentration dependent growth inhibition of estrogen receptor (ER)-positive () and ER-negative (MDA-MB231) breast cancer cells by genistein. Twenty-four hours after inoculation, cells were cultured for 3 days in the absence or presence of genistein at concentratins ranging from.1 to 1 μm. The data represent the mean±sd of six independent experiments.

Genistein Induced G2/M Arrest in Breast Cancer Cells 385 genistein을처리하였을때에는 55.55% 로서약 4배증가하였다 (Figure 3).

CDK 저해제인 의 mrna는처리한 genistein의농도가높을수록발현이두세포주모두에서증가하였고, 1 μm 의 genistein을처리한후시간에비례하여 24 시간에서 48 시간사이에최대치로발현하였다 (Figure 4).

1% DMSO (control) or 1 μm of genistein for 3 days. Genistein-treated cells are decreased in cellularity than control.

그러나 Cdc25C의활성을억제하는데에관여하는것으로알려진, CHK1, CHK2의 mrna 는서로다른발현양상을나타내었다. 은 genistein의농도와 genistein 처리후배양시간에비례하여 mrna의발현이두세포주모두에서증가하였다 (Figure 7).

4 Genistein Induced G2/M Arrest in Breast Cancer Cells 385 genistein을처리하였을때에는 55.55% 로서약 4배증가하였다 (Figure 3). MDA-MB231 Genistein 처리후세포주기관련유전자의 mrna 발현 mrna와 mrna의발현세포분열주기에관여하는유전자들의발현양상을알아보기위하여 mrna 실시간정량분석을하였다. CDK 저해제인 의 mrna는처리한 genistein의농도가높을수록발현이두세포주모두에서증가하였고, 1 μm 의 genistein을처리한후시간에비례하여 24 시간에서 48 시간사이에최대치로발현하였다 (Figure 4). 반면 mrna는 genistein 처리를한군과하지않은군사이의발현이거의차이가없었고, 배양시간과 genistein 의농도와도무관하였다 (Figure 5). A C Figure 2. Changes of cell densitiy in cells (A, C) and MDA- MB231cells (B, D). Each cells were treated with.1% DMSO (control) or 1 μm of genistein for 3 days. Genistein-treated cells are decreased in cellularity than control. B D Cdc25C와 polo-like kinase (, ), check point kinase (CHK1, CHK2) mrna의발현 Cdc25C의활성을증가시킨다고알려져있는 의 mrna 는 genistein 처리후시간이지남에따라두세포주모두에서그발현이감소하였다 (Figure 6). 그러나 Cdc25C의활성을억제하는데에관여하는것으로알려진, CHK1, CHK2의 mrna 는서로다른발현양상을나타내었다. 은 genistein의농도와 genistein 처리후배양시간에비례하여 mrna의발현이두세포주모두에서증가하였다 (Figure 7). 반면 CHK1은 genistein 처리후시간에관계없이두세포주모두에서 mrna의발현양상에변화가없었고 (Figure 6), CHK2는 MDA-MB % 58.19% 12.4% 21.94% CON % 25.48% 3.52% 55.6% Gen 1 μm 36 Counts Counts FL2-A FL2-A A % 73.9% 7.2% 13.2% CON % 2.7% 1.6% 55.55% GEN 1 μm 16 Counts Counts FL2-A FL2-A B Figure 3. DNA-fluorescence histogram of and MDA-MB231 cells after genistein treatment. (A) and MDA-MB231 (B) cells were treated with.1% DMSO (CON) or 1 μm of genistein (GEN) for 72 hr. Three days after genistein treatment, there was 3.1-fold increase in the percent of cells in G2/M and 4.2-fold increase in the percent of MDA-MB231cells in G2/M, respectively.

5 386 Ji-Yong Park, et al Figure 4. Real time quantitative analysis for mrna of genistein-treated and MDA-MB231 cells. Each cell (5 1 5 cells) was treated with -1 μm genistein for 3 days or 1 μm of genistein for.5, 1, 2, 3 days Figure 5. Real time quantitative analysis for mrna of genistein-treated and MDA-MB231 cells. Each cell (5 1 5 cells) treated with -1 μm genistein for 3 days or 1 μm of genistein for.5, 1, 2, 3 days.

6 Genistein Induced G2/M Arrest in Breast Cancer Cells Control 12 hr 24 hr 45 hr 72 hr. Control 12 hr 24 hr 45 hr 72 hr Figure 6. Real time quantitative analysis for mrna and CHK1 mrna of genistein-treated and MDA-MB231 cells. Each cell (5 1 5 cells) treated with 1 μm of genistein for.5, 1, 2, 3 days Figure 7. Real time quantitative analysis for of genistein-treated and MDA-MB231 cells. Each cell (5 1 5 cells) treated with -1 μm genistein for 3 days or 1 μm of genistein for.5, 1, 2, 3 days.

7 388 Ji-Yong Park, et al CHK CHK CHK CHK2.. Figure 8. Real time quantitative analysis for CHK2 mrna of genistein-treated and MDA-MB231 cells. Each cell (5 1 5 cells) treated with -1 μm genistein for 3 days or 1 μm of genistein for.5, 1, 2, 3 days cdc25c cdc25c.. Figure 9. Real time quantitative analysis for Cdc25C mrna of genistein-treated and MDA-MB231 cells. Each cell (5 1 5 cells) treated with -1 μm genistein for 3 days or 1 μm of genistein for.5, 1, 2, 3 days. 세포에서는 genistein의농도와배양시간에따라서 mrna의발현이증가하였으나, 세포에서는유의한변화가없었다 (Figure 8). 그리고 G2/M 전환에중심역할을하는 Cdc25C는세포주의종류와상관없이 genistein 처리후배양시간에비례하여 mrna의발현이줄었다 (Figure 9). Genistein 처리후세포주기관련유전자의단백발현양상 Genistein 1 μm 을처리한후시간에따른세포주기관련 유전자의단백발현양상을비교하기위하여 western blot을수행하였다 (Figure 1). CDK 저해제인 은두세포주모두에서시간에비례하여단백의발현이증가하였고, 은시간에관계없이단백의발현양상에변화가없었다. 은두세포주모두시간에비례하여단백의발현이감소하였고, 은시간에비례하여발현이증가하였다. 그러나, CHK1은발현양상에변화가없었다. CDC25C는두세포주모두시간에비례하여단백의발현이감소하였는데, 198-serine과 216-serine 아미노산이인산화

8 Genistein Induced G2/M Arrest in Breast Cancer Cells 389 CHK1 cdc25c p-cdc25c- S198 p-cdc25c- S216 a-tublin C 1D 2D 3D Figure 1. Western blot analysis of cell cycle-related genes. Each cell was treated with 1 μm of genistein for 1, 2, 3 days. C;.1% DMSO treated cells. 된 Cdc25C 단백의양도각각감소하였다. 고 본연구에서 genistein에의한세포성장의억제와 G2/M arrest 는세포의에스트로겐수용체의유무와상관없었다. Genistein 처리후 CDK 저해제인 의 mrna와단백발현은시간과농도에비례하여증가하였으며이는이미보고된다른연구들과일치하는결과이다.(3-5,15) 반면, genistein 처리후농도에의존하여유의하게증가함을보고한 Yoon 등 (24) 의연구결과와는달리 단백은변화가거의없었다. 이는 MDA-MB231과 MCF-7 유방암세포주, MCF-1A 유방상피세포주, 그리고 DU145 전립선암세포주에서 genistein 처리후 단백의발현이증가하였다는보고와는다소다르지만,(6,24-26) B16-F1 흑색종세포주에서 genistein 처리후 단백의발현이변화가없었다는보고와는일치한다.(8),, CHK1, CHK2는 Cdc25C를인산화시키는효소로서 DNA 손상후에일어나는 G2/M arrest와관계가있다. 은 Cdc25C의 198번 serine을인산화하여핵내로이동하는것을촉진하여세포분열을활성화시키는데관여한다.(27,28) 본연구에서는 genistein 처리후 단백의발현이낮아졌고, 이는 Cdc25C의 198번 serine의인산화의감소를초래하여결국핵에존재하는 Cdc25C의양이줄어들므로 G2/M arrest유도에관여할것으로생각된다. 반면, genistein을두유방암세포주에처리하였을때시간과농도에비례하여 단백발현은증가하였다. Xie 등 (29) 의보고에의하면암세포주에 DNA 손상을주었을때, 의발현은억제되었지만 의발현은오히려증가하였다. 따라서 DNA 손상후일어나는수복과정에 과 찰 MB231 C 1D 2D 3D 은서로반대의역할을할것이라고주장하였다. 두유방암세포주에 genistein 처리후 과 의서로다른발현양상은 DNA 손상후일어나는 G2/M arrest와관계되어있음을시사한다. 하지만 이 Cdc25C를유일한기질로사용하지않기때문에 genistein에의해증가된 이어떻게 G2/M arrest를유도하는가에대한기전은더많은연구가필요할것이다. Cdc25C 의 216번 serine을인산화시켜 Cdc25C의핵으로의이동을억제하는데관여하는 CHK1과 CHK2는서로다른발현양상을나타내었다. Genistein 처리후두세포주모두 CHK1 단백발현은변화가없었다. 반면 CHK2은 세포주에서는단백발현에변화가없었으나 MDA-MB231 세포주에서는발현이증가하였다. Ismail 등 (3) 은신경모세포암세포주를이용한연구에서 genistein 처리후 mediator of DNA damage check point protein 1 (MDC1) 의발현이증가되고, 이와연관하여 CHK2 가활성화되며, PLK-1의발현의저하와연관된세포자멸사가유도되는것을보고하였고, 이과정에 MDC1이중요한역할을한다고하였다. 그러나본연구에서유방암세포주에 genistein을처리하였을때 Cdc25C 단백의발현양도줄어들었고또한 216 번 serine이인산화된 Cdc25C의양도함께감소하였다. 뿐만아니라 198 번 serine이인산화된 Cdc25C의양도감소하였다. 이는 Cdc25C 의전체양이줄어들었기때문에인산화된 Cdc25C의양도적은것으로생각한다. Genistein 처리에따른 Cdc25C 단백발현과관련하여 Chang 등 (3) 은 genistein 처리에의해 Cdc25C의발현은줄어들지만, 216 번 serine에인산화된 Cdc25C는증가한다고보고하였다. 이는본연구의결과와상반되는결과이며, 어떻게서로다르게발현될수있는지는좀더많은연구가필요하지만, genistein 처리후 CHK2, 과 이서로복합적으로상호작용하여 Cdc25C의인산화를조절하여 G2/M arrest를일으키는데관계할것으로생각한다. 결론지금까지의결과를요약하면 genistein 처리에의해서일어나는 G2/M arrest는 CDK 억제단백질의증가와 Cdc25C의감소, 그리고 CDC25C를인산화시키는 의발현증가와 의감소가관계되는것으로보이며, 여기에 CHK2와 CHK1 이 DNA 손상에의한 checkpoint pathway에영향을미치는것으로보인다. 참고문헌 1. Cappelletti V, Fioravanti L, Miodini P, Di Fronzo G. Genistein blocks

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