전립선암에서조직내 와 βii 발현의임상적의의 Clinical Significance of the Expression of and βii in Prostate Cancer Tae Heung Kim, Tae Hyung Kim, Shin Young Lee, Young Sun Kim, Mi Kyung Kim 1, Soon Chul Myung From the Departments of Urology and 1 Pathology, College of Medicine, Chung- Ang University, Seoul, Korea Purpose: This study examined the significance of tubulin α and βⅡ expression in benign prostatic hyperplasia (BPH) and prostate cancer. Materials and Methods: A total 103 cases diagnosed with either BPH or prostate cancer were divided into 4 groups: BPH (n=25), localized prostate cancer (n=41), locally advanced prostate cancer (n=18), metastatic prostate cancer (n=18). The tissues were taken through a transurethral resection of the prostate, suprapubic prostatectomy, prostatic needle biopsy or radical retropubic prostatectomy. The expression was measured by immunohistochemical staining. The degree of expression was graded on a 4-point scale. The data was compared in terms of stage, the serum prostatic specific antigen (PSA) level and Gleason score. The statistical comparisons were made using one-way ANOVA test and a Fisher's exact test. Results: The levels of tubulin α and βii expression were stronger in the patients with prostate cancer than those with BPH (p<0.001). On the other hand, there was no difference between metastatic prostate cancer and localized prostate cancer (p>0.05). The level of tubulin α expression was stronger in the patients with a Gleason score 8 than in those of Gleason score 7(p<0.05), but there was no such difference in tubulin βii expression. The level of tubulin α and βii expression was stronger in the patients with a serum PSA level 4ng/ml than in those with the serum PSA level <4ng/ml (p<0.001). Conclusions: The expression of tubulin α and βii may be associated with the development of prostate cancer. These might be used as useful markers for the diagnosis, prognosis and treatment response. (Korean J Urol 2008; 49:709-714) Key Words: Tubulin, Prostate, Neoplasms 대한비뇨기과학회지제 49 권제 8 호 2008 중앙대학교의과대학비뇨기과학교실, 1 병리학교실 김태흥ㆍ김태형ㆍ이신영김영선ㆍ김미경 1 ㆍ명순철 접수일자 :2008년채택일자 :2008년 3월 21일 6월 25일 교신저자 : 명순철중앙대학교병원비뇨기과서울시동작구흑석동 224-1 156-755 TEL: 02-6299-1808 FAX: 02-6294-1406 E-mail: uromyung@ yahoo.co.kr 본연구는보건복지부보건의료기술진흥사업의지원에의하여이루어진것임 ( 과제고유번호 : A062254). 서론전립선암은서구에서가장높은발생률을보이고암관련사망의세번째원인이다. 1 우리나라에서도전립선암은 1998년에는남성암중발생빈도가 8위였으나 2002년에는 6 위로발생빈도가증가하였으며다른종양에비해빠르게증가하고있다. 2 전립선암의발생과진행의생체분자학적기전을명확하게하고조기발견할수있는표지자를찾기위한노력이현재까지이렇다할성과를내지못하고있는 실정이다. 따라서전립선암의발생과정중나타나는생화학적혹은세포학적표현형에대한이해가중요하며이는향후전립선암의조기진단과치료를위한새로운방법을제시할것으로생각한다. 미세관 (microtubule) 은필라멘트단백중합체로유사분열과감수분열중염색체의분리, 편모와섬모의운동, 세포기관의운반, 세포형태유지등다양한기능을한다. 3-6 Tubulin은미세관의주요구성단백성분으로정상세포, 신경계세포, 평활근세포등다양한세포에서관찰된다. Tubulin에는 α와 β 아단위가있으며 tubulin β에는 6개의 709
710 대한비뇨기과학회지 : 제 49 권제 8 호 2008 아형이존재한다. 6 는전립선암세포의경우, 정상세포와는다른분자적프로파일을가지기때문에진단적가치를가질것으로기대되며새로운분자생물학적치료방법을제공할수있을것으로기대된다. 7 항암화학요법제중에서미세관기능을억제하는제제가중요한한분류를차지하고있다. 전립선암치료에단독제제의효과는제한적이지만항미세관제제복합요법은효과가있는것으로보고되고있다. 이러한제제는대부분 tubulin β와결합한다. 8-12 Tubulin β는미세관의결합, 안정성, 항미세관제제에대한민감도등에영향을미치며, 13-16 여섯가지아형중에서 tubulin βii가전립선비대증에비해전립선암에서더많이발현이된다는보고가있다. 17 이에전립선비대증과전립선암조직에서면역조직화학염색으로 tubulin α와 βii의발현도를관찰하여전립선암에서 tubulin α, βii 발현의임상적의의에대하여알아보고자하였다. 재료및방법 1. 조직의선택전립선비대증과전립선암으로진단받은환자의조직을전립선비대증 25례, 국소성전립선암 41례, 국소진행성전립선암 18례, 전이성전립선암 18례로네군으로구분하였다. 전립선비대증환자에서경요도내시경하전립선절제술과상치골전립선절제술을통해서얻은조직을사용하고, 국소성전립선암은전립선생검과근치적전립선절제술로조직을얻고, 국소진행성전립선암과타장기로전이가확인된전립선암군에서는전립선생검을시행한조직을사용하였다. 2. 면역조직화학염색각예의파라핀포매조직에서 4-5μm 두께의절편을박절하고탈파라핀하고, 무수알코올, 90%, 75% 및 50% 알코올에서차례대로함수과정을거쳐서증류수에 5분간수세하였다. 내인성과산화효소의활성을억제하기위해 0.5% hydrogen peroxide에 10분간처리후증류수로수세하였다. 항원성회복을위하여 10mM citrate (ph 6.0) 완충액에담가 5분간 2회극초단파처리후실온에서냉각시키고 50mM Tris 완충용액 (TBS, ph 7.5) 으로수세하였다. 면역조직화학염색의비특이성반응을제거하기위해 30분간염소혈청으로처리하고, 여분의용액을제거한다음일차항체인 DM- 1A (cat. No.: MU121-UC, mouse monoclonal, dilution 1:100, Applied Biogenex, San Ramon, USA) 와 JDR3B8 (cat. No.: MU176-UC, mouse monoclonal, dilution 1:50, Applied Biogenex, San Ramon, USA) 을각각실온에서하룻밤반응시킨후 TBS로 5분간 3회수세한다음 biotin이부착된이차항체 (Applied Zymed Co. Carlsbad, USA) 에 20분간작용후, streptoavidin과결합한과산화효소복합체를가하여 20분간반응시킨후 AEC chromogen (3-amino-9-ethylcarbazole) 용액으로발색하고 Mayer hematoxylin으로대조염색을실시하여광학현미경으로관찰하였다. 면역조직화학염색반응의음성대조군으로는전립선조직에서일차항체를처리하지않고음성대조시약으로대체하여이용하였고양성대조군으로는소장조직과폐암조직을이용하였다. 3. 면역조직화학염색의평가 Tubulin에대한염색의판정은전체세포의 10% 이상에서, 세포질에적갈색의염색상을보일때양성으로판정하였고양성률이 10-30% 인경우를 1+ ( 국소성 ), 30-70% 인경우를 2+ ( 다발성 ), 70% 이상인경우를 3+ ( 미만성 ) 로판정하였다 (Fig. 1). 4. 결과분석전립선비대증과전립선암, 국소성전립선암과전이성전립선암, Gleason 점수 7점이하와 8점이상, 그리고혈청 prostate-specific antigen (PSA) 4ng/ml 미만과 4ng/ml 이상인군사이에 tubulin α와 βii의발현도를각각비교하였다. 통계는 SPSS (version 12.0) 를사용하여 one-way ANOVA test, Fisher's exact test를이용하여분석하였으며 p값이 0.05 미만인경우를통계학적으로의미가있다고판정하였다. 결과 1. 대상군의특성전립선비대증환자의연령, 혈청 PSA는 68.5±8.4세, 1.5± 1.0ng/ml였다. 국소성전립선암에서연령, 혈청 PSA, Gleason 점수가 8점이상인경우는 65.6±8.8세, 13.2±14.9ng/ml, 2례 (4.9%) 였다. 국소진행성전랍선암에서연령, 혈청 PSA, Gleason 점수가 8점이상인경우는 67.7±8.0세, 13.4±10.4ng/ ml, 6례 (33.3%) 였다. 전이성전립선암환자에서연령, 혈청 PSA, Gleason 점수가 8점이상인경우는 72.4±6.1세, 345.7± 710.6ng/ml, 12례 (66.7%) 였다. 각군사이에연령차이는없었으나혈청 PSA와 Gleason 점수는암의진행에따라통계적으로유의하게높게관찰되었다 (p<0.05) (Table 1). 2. 전립선비대증과전립선암에서 tubulin α, βii의발현도비교 1) : 전립선비대증에서미만성 (3+) 발현은전
김태흥 외 전립선암에서 와 βii의 발현 711 Fig. 1. Grading system in the expression of tubulin α and βii (immunohistochemical stain, x100). The degree of expression was graded on a 4-point scale: 0 (negative stain), 1 (focal stain), 2 (multifocal stain), 3 (diffuse stain). Negative stain for tubulin α in benign prostatic hyperplasia (BPH) tissue (A) and diffuse stain for tubulin α in metastatic prostate cancer (B). Negative stain for tubulin βii in BPH tissue (C) and diffuse stain for tubulin βii (D) in metastatic prostate cancer. Table 1. Characteristics for benign prostatic hyperplasia and prostate cancer Age (years) PSA (ng/ml) Gleason score 7 8 BPH (n=25) LoCaP (n=41) LACaP (n=18) MetCaP (n=18) 68.5±8.4 1.5±1.0 65.6±8.8 13.2±14.9 39 (95.1%) 2 (4.9%) 67.7±8.0 13.4±10.4 12 (66.7%) 6 (33.3%) 72.4±6.1 345.7±710.6 6 (33.3%) 12 (66.7%) 0.05* 0.0000017 BPH: benign prostatic hyperplasia, LoCaP: localized prostate cancer, LACaP: locally advanced prostate cancer, MetCaP: metastatic prostate cancer, PSA: prostate-specific antigen. *: one-way ANOVA test, : Fisher's exact test 혀 관찰되지 않았으며 전립선암에서 전립선비대증에 비해 강한 발현도를 나타냈다 (p=0.0000034) (Table 2). 2) Tubulin βii: 전립선비대증에서 전혀 발현되지 않았 으나 전립선암에서는 더 많이 발현되어 전립선비대증에 비 해 강한 발현도를 나타냈다 (p=0.000001) (Table 2). 3. 국소성 전립선암과 전이성 전립선암에서 tubulin α, βii의 발현도 비교 1) : 전이성 전립선암에서 국소성 전립선암과 발현도에 차이가 나타나지 않았다 (p=0.115) (Table 3).
712 대한비뇨기과학회지 : 제 49 권제 8 호 2008 Table 2. Expression of tubulin α and βii in BPH and prostate cancer BPH (n=25) CaP (n=77) Tubulin βii 14 (56) 16 (20.7) 10 (40) 15 (19.5) 1(4) 23 (29.9) 23 (29.9) 0.0000034* BPH: benign prostatic hyperplasia, CaP: prostate cancer. *: Fisher's exact test 25 (100) 31 (40.2) 17 (22.1) 16 (20.8) 13 (16.9) 0.000001* Table 3. Expression of tubulin α and βii in localized prostate cancer and metastatic prostate cancer LCaP (n=59) MetCaP (n=18) Tubulin βⅡ 15 (25.4) 1 (5.6) 13 (22.1) 2 (11.2) 15 (25.4) 8 (44.4) 16 (27.1) 7 (38.8) 0.115* 24 (40.7) 7 (38.9) 14 (23.7) 3 (16.6) 9(15.3) 7(38.9) 12 (20.3) 1 (5.6) LCaP: localized prostate cancer (including locally advanced cancer), MetCaP: metastatic prostate cancer. *: Fisher's exact test 0.1503* Table 4. Expression of tubulin α and βii in the group with Gleason scores 7 and Gleason score 8 GS 7 (n=55) GS 8 (n=21) Tubulin βii 13 (23.6) 3 (14.2) 15 (27.3) GS: Gleason score, *: Fisher's exact test 14 (25.5) 9 (42.9) 13 (23.6) 9 (42.9) 0.011* 26 (47.3) 5 (23.8) 11 (20.0) 6 (28.6) 11 (20.0) 4 (19.0) 7 (12.7) 6 (28.6) 0.177* Table 5. Expression of tubulin α and βii in the group with serum PSA<4 and PSA 4 PSA<4 (n=27) PSA 4 (n=76) Tubulin βii 14 (51.9) 16 (21.1) 10 (37.0) 16 (21.1) 3 (11.1) 21 (27.6) PSA: prostate-specific antigen, *: Fisher's exact test 23 (30.1) 0.000067* 25 (92.6) 31 (40.8) 1 (3.7) 17 (22.4) 1 (3.7) 15 (19.7) 13 (17.1) 0.000037* 2) Tubulin βii: 전이성전립선암에서국소성전립선암과발현도에차이가나타나지않았다 (p=0.150) (Table 3). 4. Gleason 점수에따른 tubulin α, βii의발현도비교 1) : Gleason 점수 8 이상인군에서 Gleason 점수가 7 이하인군보다더강하게발현되어 Gleason 점수가높은경우더강한발현도를나타냈다 (p=0.011) (Table 4). 2) Tubulin βii: Gleason 점수 8 이상인군과 Gleason 점수가 7 이하인군사이에발현도의차이를보이지않았다 (p=0.177) (Table 4). 5. 혈청 PSA 농도에따른 tubulin α, βii의발현도비교 1) : 혈청 PSA 4ng/ml 미만인군에서미만성 (3+) 발현은관찰되지않았으며혈청 PSA 4ng/ml 이상인군에서더강하게발현되어, 혈청 PSA가높은경우더강한발현도를나타냈다 (p=0.000067) (Table 5). 2) Tubulin βii: 혈청 PSA 4ng/ml 이상인군에서혈청 PSA 4ng/ml 미만인군보다강하게발현되어혈청 PSA가높
김태흥외 : 전립선암에서 와 βii 의발현 713 은경우더강한발현도를나타냈다 (p=0.000037) (Table 5). 고찰 는전립선암세포에서정상세포와는다른분자적프로파일을갖는다고보고되고있어새로운분자생물학적치료방법을제공할수있을것으로기대된다. 또한 Tubulin α 아세틸레이션의감소는안드로겐수용체소실의지표로서진단적가치를가질것으로기대된다. 7 Tubulin이보통 αβ이량체 (heterodimer) 로존재하고있으며면역형광염색에서 tubulin α, βii가같은패턴으로관찰되어 tubulin α의발현이 tubulin βii의발현과연관이있다는보고가있으며 18,19 본연구에서도실제로 tubulin α는전립선암에서더강한발현도를나타냈으며 tubulin α와 βii 의발현도는비슷한양상으로관찰되었다. 따라서 tubulin α와 βii 모두전립선암의발생과연관이있을것으로생각한다. Ranganathan 등 17 은전립선비대증에비해서전립선암에서 tubulin βii가더많이발현된다고보고하였다. 정상세포의세포질에만존재하는 tubulin βii는전립선암세포의핵내에서관찰된다. 정상세포의핵에서 tubulin βii가관찰되지않는점은핵내 tubulin βii가암과관련있을것으로생각된다. 17 최근 hormone refractory prostate cancer (HRPC) 에 taxol 계열의제제사용이효과가있다고보고되고있다. Taxol은다른 tubulin β 아형보다 tubulin βii에더특이적으로작용하는것으로알려져있다. 20 Taxol은두가지효과를나타내는데세포고사뿐만아니라 tubulin βii를재배열시키고더높은농도에서는 tubulin βii를고갈시킨다. 19 Tubulin βii의발현이이러한항미세관제제에대한저항성을나타낸다는보고가있다. 21,22 따라서전립선암환자에서 tubulin βii의발현정도에따라항암화학요법에대한반응예측및치료효과판정에이용할수있을것으로생각한다. 본연구에서는전립선암으로새롭게진단된환자에서만 tubulin βii의발현도를조사한한계가있다. 따라서항암화학요법등치료전후발현도에대한조사가필요하다. 16례의전립선암에서모두핵내 tubulin βii의발현이관찰되나발현되는위치나정도는병기또는분화도사이에연관성이없다는보고가있으며, 전립선비대증에서는 19례중단 1례를제외한모든예에서핵내 tubulin βii 발현이있었다는보고가있으나핵내와세포질내의전체 tubulin βii의발현은전립선비대증보다전립선암에서높게발현된다. 23 본연구에서도마찬가지로전립선비대증보다전립선암에서통계적으로유의하게강한발현도를나타냈으나 전립선비대증에서는 25례모두에서발현되지않았다. 전립선비대증에서전혀발현되지않은점은 Yeh 등 23 의보고와는상이한결과로그이유는명확하지않다. 병기에따른비교에서국소성전립선암과전이성전립선암사이에는발현도에차이가나타나지않았다. 분화도에따른비교에서는 Gleason 점수 8 이상인군에서더강한발현도를나타냈다. 따라서병기보다는분화도에따라더강한발현도를나타낸것으로생각한다. 다만병기에따른비교의경우보다많은증례에대해실험한다면같은결과가나올것으로생각한다. 혈청 PSA에따른 tubulin α와 βii의발현도에대한이전의보고는없었으나 tubulin α와 βii 모두혈청 PSA 4ng/ml 이상인경우더강한발현도를나타냈다. 따라서 tubulin α 와 βii가전립선암의진단, 예후, 치료에대한반응의표지자가될수있을것으로생각한다. 따라서전립선암의치료후재발률, 생존율등과 tubulin α와 βii의발현정도의변화와의상관성에대한연구가필요할것으로생각한다. 결 와 βii가전립선비대증조직에비해전립선암에서더많이발현되는것은 와 βii의발현이전립선암의발생과연관이있을것으로생각한다. 또한 tubulin α와 βii가 Gleason 점수, 혈청 PSA 농도증가에따라더강한발현도를나타내고항미세관항암화학요법제제의주요표적이되고있어전립선암의진단, 예후, 치료및치료의평가에이용할수있을것으로생각한다. 론 REFERENCES 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106-30 2. Cancer Registration and Biostatistics Branch, National Cancer Center. Cancer Statistics in Korea, 2003 3. Musch A. Microtubule organization and function in epithelial cells. Traffic 2004;5:1-9 4. Sullivan KF, Cleveland DW. Identification of conserved isotype-defining variable region sequences for four vertebrate β- tubulin polypeptide classes. Proc Natl Acad Sci USA 1986; 83:4327-31 5. Sullivan KF. Structure and utilization of tubulin isotypes. Annu Rev Cell Biol 1988;4:687-716 6. Ludueña RF. Multiple forms of tubulin: different gene products and covalent modifications. Int Rev Cytol 1998;178:207-75 7. Soucek K, Kamaid A, Phung AD, Kubala L, Bulinski JC, Harper RW, et al. Normal and prostate cancer cells display
714 대한비뇨기과학회지 : 제 49 권제 8 호 2008 distinct molecular profiles of alpha-tubulin posttranslational modifications. Prostate 2006;66:954-65 8. Hudes GR, Greenberg R, Krigel RL, Fox S, Scher R, Litwin S, et al. Phase II study of estramustine and vinblastin, two microtubule inhibitors, in hormone-refractory prostate cancer. J Clin Oncol 1992;10:1754-61 9. Pienta KJ, Redman B, Hussain M, Cummings G, Esper PS, Appel C, et al. Phase II evaluation of oral estramustine and oral etoposide in hormone-refractory adenocarcinoma of the prostate. J Clin Oncol 1994;12:2005-12 10. Hudes GR, Nathan FE, Khater C, Greenberg R, Gomella L, Stern C, et al. Paclitaxel plus estramustine in metastatic hormone-refractory prostate cancer. Semin Oncol 1995;22(5 Suppl 12):41-5 11. Lee JC, Harrison D, Timasheff SN. Interaction of vinblastine with calf brain microtubule protein. J Biol Chem 1975;250: 9276-82 12. Rao S, Horwitz SB, Ringel I. Direct photoaffinity labeling of tubulin with taxol. J Natl Cancer Inst 1992;84:785-8 13. Banerjee A, Roach MC, Trcka P, Luduena RF. Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of β-tubulin. J Biol Chem 1990;265:1794-9 14. Banerjee A, Luduena RF. Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of β- tubulin. J Biol Chem 1991;266:1689-91 15. Lu Q, Luduena RF. Removal of βiii isotype enhances taxol induced microtubule assembly. Cell Struct Funct 1993;18: 173-82 16. Panda D, Miller HP, Banerjee A, Luduena RF, Wilson L. Microtubule dynamics in vitro are regulated by the tubulin isotype composition. Proc Natl Acad Sci USA 1994;91:11358-62 17. Ranganathan S, Salazar H, Benetatos CA, Hedes GR. Immunohistochemical analysis of β-tubulin isotypes in human prostate carcinoma and benign prostatic hypertrophy. Prostate 1997;30:263-8 18. McKean PG, Vaughan S, Gull K. The extended tubulin superfamily. J Cell Sci 2001;114:2723-33 19. Xu K, Ludueña RF. Characterization of nuclear βii-tubulin in tumor cells: a possible novel target for taxol. Cell Motil Cytoskeleton 2002;53:39-52 20. Derry WB, Wilson L, Khan IA, Ludueña RF, Jordan MA. Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified β-tubulin isotypes. Biochemistry 1997;36:3554-62 21. Sangrajrang S, Denoulet P, Laing NM, Tatoud R, Millot G, Calvo F, et al. Association of estramustine resistance in human prostatic carcinoma cells with modified patterns of tubulin expression. Biochem Pharmacol 1998;55:325-31 22. Burkhart CA, Kavallaris M, Band Horwitz S. The role of beta-tubulin isotypes in resistance to antimitotic drugs. Biochim Biophys Acta 2001;1471:O1-9 23. Yeh IT, Ludueña RF. The βii isotype of tubulin is present in the cell nuclei of a variety of cancers. Cell Motil Cytoskeleton 2004;57:96-106