대한병리학회지 : 제 35 권제 4 호 2001 The Korean Journal of Pathology. 2001; 35: 286-90 위선종과위암종에있어서 Caspase 3와 Ki-67 발현및세포자멸사와의관련성 손진희 채승완 최경찬 신형식 한림의대병리학교실 접수 : 2000년 11월 22일게재승인 : 2001년 7월 16일 책임저자 : 손진희우 150-020 서울특별시영등포구영등포동 94-200 한림의대한강성심병원해부병리과전화 : 02-2639-5572 Fax: 02-672-3602 E-mail: jhsohn@www.hallym.or.kr * 본연구는 2000 년한림대의료원연구비 ( 과제번호 01-2000-03) 지원으로이루어졌음. Caspase 3 and Ki-67 Immunoreactivity and Its Correlation with Frequency of Apoptosis in Gastric Adenomas and Carcinomas Jin Hee Sohn, Seoung Wan Chae, Kyung Chan Choi and Hyung Sik Shin Department of Pathology, Hallym University, College of Medicine, Seoul, Korea Background : Apoptosis, also known as programmed cell death, is under genetic control and is mediated by apoptosis-specific genes, certain oncogenes and tumor suppressor genes. Caspase 3, a group of cystein proteases, is involved in the induction of apoptosis and has been considered to be correlated with apoptosis. Therefore, we tried to define whether caspase 3 is expressed in gastric adenoma and carcinoma, and correlated with apoptosis. Methods : The apoptotic index and caspase 3 and Ki-67 immunoreactivity were observed in 25 gastric adenomas, 31 early gastric carcinomas (EGC) and 64 advanced gastric carcinomas (AGC) by in situ labelling and immunohistochemistry. Results : The mean number of apoptotic bodies and caspase 3 immunoreactivity were significantly increased from adenoma through EGC to AGC. Ki-67 immunoreactivity was more increased in AGC than in adenoma and EGC. And the number of apoptotic bodies were positively correlated with caspase 3 and Ki-67 immunoreactivity, and caspase 3 immunoreactivity was negatively correlated with Ki-67 immunoreactivity even though they were statistically insignificant. Conclusions : Our results suggest that caspase 3 activation is important for inducing apoptosis, and both caspase 3 and apoptosis are increased along the tumor progression. Key Words : Apoptosis, Caspases, Ki-67 antigen, Carcinoma, Stomach 위암종은한국의전체암종중 20.9% 를차지하는발생빈도가높은종양으로서, 1 이종양의진단및예후와관련되는인자를찾는것은중요하다. 세포자멸사는세포의수명과관련된계획된사멸과정의하나로정상조직과종양조직모두에서나타나고여러가지종양유전자, 종양억제유전자, 세포증식인자및효소들의영향을받는다고알려져있다. 2-4 그러므로이와같은세포자멸사의과정에결손이생기면세포의분화와증식이부적절하게일어나다양한병적상태가되고종양이형성된다고하며, 종양의성장속도와전이는종양세포의증식능과세포자멸사에의해서좌우된다고알려져있다. 5,6 또한악성종양의경우등급에따라세포자멸사지수가다르고전암병변도세포자멸사지수가달라서, 전암병변과암종의감별진단이어려울경우세포자멸사지수를감별진단에이용할수있다는보고도있다. 7-11 세포 자멸사의유발에는여러가지요소가관여하며최근에는몇가지생화학적반응이관련되어있음이밝혀졌다. 12-14 이와같은생화학적반응중에서단백분해효소의활성화가하나의중요한역할을한다고알려졌다. 13,14 그리고그중하나인 caspase 3가세포자멸사의유발을일으켜서세포자멸사에이르게한다하였다. 15-17 그러나세포자멸사에대한연구는다양한종양에서비교적많이이루어져있으나, caspase 3에대한연구는많지않고결과또한일관성이없게나와여러장기의여러종양에서연구를시행하여야할것으로생각한다. 따라서저자들은위선종, 조기위암종, 진행위암종을대상으로하여세포자멸사와세포자멸사관련인자인 caspase 3와의상관관계를알아보고, 증식능관련인자인 Ki-67과비교함으로써세포증식능과의연관성을알아보고자하였다. 286
위암종의세포자멸사, Caspase 3, Ki-67 287 재료와방법재료포르말린에고정되고파라핀에포매된위선종과위암종의증례중병력과검사실소견이잘보관되어있고슬라이드및파라핀포매괴가잘유지된위선종 25예, 조기위암종 31예, 진행위암종 64예를대상으로하였다. 조기위암과진행위암에대한임상병리학적소견을조사하였고세포자멸사에대한연구는 TdT-mediated d-utp-biotin nick end labeling (TUNEL) 방법에의하여세포자멸사에빠진세포들을염색하여세포자멸사지수를산출하였고, caspase 3과 Ki-67은면역조직화학검사를하여각각의세포표지지수를산출하였다. 면역조직화학검사 Polyclonal rabbit anti-human Caspase 3 antibody (AHP476, Serotec Ltd, Oxford, U.K.) 와, MIB-1 (Monoclonal mouse anti-ki-67 nuclear antigen, Zymed Lab, Ca, U.S.A.) 을사용하여면역조직화학검사를시행하였다. Caspase 3는 32 kd의단백질로서 17 kd과 11 kd의조각으로나누어지면서활성화되어세포자멸사에관여하는것으로되어있다. 제작사에의하면이항체는활성화되기전의 pro-caspase 3 분자와 17 kd의활성화된조각모두에반응한다고되어있다. 간단히염색방법을기술하면, 파라핀블록을 4 m 두께로자른뒤탈파라핀하고에탄올로함수과정을거친다. 항체반응을좋게하기위하여 10 mm sodium citrate buffer (ph 6.0) 에슬라이드를넣어서 8분간 microwave oven에둔다. 다음으로슬라이드를 3% 과산화수소에 30분간반응시키고 5% normal goat serum 에 60분간반응시킨다. 일차항체는 caspase 3의경우 1:200으로희석한뒤 4 에서 18시간 (overnight) 반응시키고 Ki-67의경우는 1:50으로희석하여 37 에서 1시간반응하였으며, biotinylated secondary antibody (Immunotech, marseille, France) 를 45분간반응시킨후 avidin-biotin-peroxidase complex를 45분간반응시킨다. 발색은 3,3 -diaminobenzidine으로하고헤마톡실린으로대조염색하였다. 양성대조군으로는편도선을같은방법으로염색하였고음성대조군으로는항체대신증류수를사용하였다. TdT-mediated d-utp-biotin nick end labeling (TUNEL) In-situ apoptosis detection kit (TaKaRa Shuzo Co., Ltd. Japan) 을사용하였다. 파라핀조직절편을탈파라핀하여 proteinase K (20 g/ml) 로실온에서 20분간처리한후세척액 (0.01M PBS, ph 7.4) 으로세척하였다. 다음으로실온에서 5 분간 3% H2O2 (in methanol) 로내인성과산화효소를차단하 였다. 세척후얼음위에서 2-5분간 permeabilisation buffer를처리한후, 습도가유지되는 37 chamber에서 fluoresceindutp (TdT+labeling safe buffer) 로 90분간반응시켰다. 세척후 Anti-FITC HRP conjugate를습도가유지되는 37 chamber에서 30분간항체반응후, DAB (5 mg/ml, in Tris buffer, ph 7.0) 로실온에서 10-15분간발색하였고헤마톡실린으로대조염색하였다. 양성대조군으로는편도선을같은방법으로검사하였고, 음성대조군으로는 fluorescein-dutp (TdT+ labeling safe buffer) 에서 TdT효소처리를하지않은것을사용하였다. 판독및통계판독은염색된표본중가장염색이뚜렷한부분을골라서세포 1,000개중양성으로나타나는세포의숫자를세어서세포표지지수와세포자멸사지수를산출하였다. Caspase 3의경우양성은배경이깨끗하며뚜렷한갈색으로세포질에염색이된경우를양성으로하였고, Ki-67의경우는핵이뚜렷한갈색으로염색이된경우를양성으로하였다. 통계처리는 Windows용 SPSS version 7.5 (SPSS Inc., Chicago, U.S.A.) 프로그램을이용하여각군을독립표본 T검정중비모수적방법인 Mann-Whitney test와 Kruskal-Wallis test를시행하였고 Spearman의상관관계분석을시행하였다. 통계적유의성은 p값이 0.05 미만인경우로하였다. 결과위선종, 조기위암종, 진행위암종모두남자에서많이발생하였으며남녀성비는 1.7:1이었다 ( 위선종 ; 2.1:1, 조기위암종 ; 2.4:1, 진행위암종 ; 1.3:1). 평균연령은 56.0세였으며위선종 ( 평균 58.8세 ), 조기위암종 ( 평균 53.7세 ), 진행위암종 ( 평균 56.1 세 ) 에따른차이는관찰되지않았다. Caspase 3 면역염색은세포기원에따른특별한분포는없이종양세포에서관찰되었고 (Fig. 1) 주위의일부염증세포에서도반응하였다. Caspase 3 세포표지지수는선종에서 3.53± 3.71%, 조기위암종 11.90±17.51%, 진행위암종 22.05±23.61% 로, 진행위암으로갈수록양성률이높아졌으며이는통계학적으로유의하였다 (p<0.001). 세포자멸사에빠진세포들은종양세포의핵이갈색으로뚜렷하게염색되었고 (Fig. 2), 세포자멸사지수도양성선종 0.98±0.85%, 조기위암종 1.76±1.68%, 진행위암종 2.55±2.08% 로진행위암종으로갈수록유의하게증가하였다 (p<0.001). Ki-67 또한종양세포의핵이갈색으로뚜렷하게염색되었으며 (Fig. 3), Ki-67 세포표지지수는양성선종 7.31±8.14%, 조기위암종 8.07±6.52%, 진행위암종 15.70± 19.18% 로증가하는경향이었으나통계학적으로유의하지는않
288 손진희 채승완 최경찬외 1 인 Fig. 1. Immunohistochemistry of caspase 3 in advanced gastric carcinoma. Many positive cells with cytoplasmic staining are scattered in the malignant glands. Fig. 3. Immunohistochemistry of Ki-67 in advanced gastric carcinoma. Many positive cells with nuclear staining are scattered in the malignant glands. Table 2. Labeling indices of caspase 3, Ki-67 and apoptosis according to the lymph node metastasis in EGC and AGC Type LN metastasis (No. of cases) index EGC Negative (21) 2.00±2.11 8.95±13.62 7.24±7.61 Positive (10) 1.37±0.42 18.10±23.39 9.80±2.84 p value NS p<0.05 0.05 p<1 AGC Negative (13) 3.22±1.78 30.00±28.07 19.00±22.00 Positive (51) 2.36±2.13 20.02±22.20 14.86±18.54 p value p<0.05 NS NS LN: lymph node, LI: labeling index, EGC: early gastric carcinoma, AGC: advanced gastric carcinoma. Table 3. Labeling indices of caspase 3, Ki-67 and apoptosis according to the differentiation and tumor size in gastric carcinomas Fig. 2. In-situ immunostain for apoptosis shows darkly stained apoptotic bodies in carcinoma cells of advanced gastric carcinoma. Table 1. Labeling indices of caspase 3, Ki-67 and apoptosis in gastric adenomas, EGC and AGC No. of samples index Adenoma 25 0.98±0.85 3.53±3.71 7.31±8.14 EGC 31 1.76±1.68 11.90±17.51 8.07±6.52 AGC 64 2.55±2.08 22.05±23.61 15.70±19.18 p value p<0.001 p<0.001 NS LI: labeling index, EGC: early gastric carcinoma, AGC: advanced gastric carcinoma. Differentiated 4 2.10±1.62 19.78±22.39 17.24±19.84 Undifferentiated 49 2.55±2.30 17.76±22.28 9.43±11.65 p value NS NS p<0.05 Size<6 cm 58 2.02±1.57 17.59±22.48 10.50±14.41 Size 6 cm 37 2.73±2.41 20.53±22.04 17.46±18.81 p value NS NS 0.05 p<1 LI: labeling index. No. index 았다 (Table 1). 이들세가지 caspase 3, Ki-67의세포표지지수와세포자멸사지수간의상관관계를보았을때 caspase 3 세포표지지수와세포자멸사지수간에는양의상관관계 (r=0.117), caspase 3와 Ki-67 세포표지지수간에는음의상관관계 (r=
위암종의세포자멸사, Caspase 3, Ki-67 289-0.039), Ki-67 세포표지지수와세포자멸사지수간에는양의상관관계 (r=0.128) 가있었으나모두통계학적으로유의하지는않았다. 또한조기위암종과진행위암종각각에대하여림프절전이의유무에따른지수를보았을때 (Table 2), 조기위암종의경우 caspase 3는전이가없을경우는 8.95±13.62, 전이가있는경우는 18.10±23.39로전이가있을경우의지수가의미있게증가하였다 (p<0.05). Ki-67의경우도림프절전이가있을경우의지수가높은경향이었으나 (0.05 p<1) 통계학적으로유의하지는않았고, 세포자멸사지수의경우는림프절전이의유무에따른차이가없었다. 그러나진행위암종의경우는림프절전이가있는경우가오히려림프절전이가없는경우보다세포자멸사지수가유의하게낮게나오고 (p<0.05), Ki-67이나 caspase 3 의경우도전이가있는경우가더욱낮게나왔으나통계학적으로유의하지는않았다. 종양의분화도에따른차이를보면 (Table 3) 분화가좋은종양에서 caspase 3와 Ki-67의세포표지지수가높았으나, Ki-67의경우만통계학적으로유의하였고 (p<0.05) caspase 3는통계학적으로유의하지않았다. 세포자멸사지수는분화에따른차이가없었다. 종양의크기에따른표지지수를보면역시 6 cm를기준으로나누었을때종양의크기가큰경우 Ki-67 세포표지지수는작은경우에비해높은경향이었으나통계학적으로유의하지않았다 (0.05 p<1). 그러나세포자멸사지수나 caspase 3 세포표지지수는큰차이가없었다. 고찰세포자멸사는괴사와는다른형태와과정을가진세포사멸로조직의정상발달에많은영향을미칠뿐만아니라여러가지병적현상에도영향을미친다. 18-20 따라서세포자멸사는정상세포와종양세포의형태발생, 면역조절및향상성유지에중요한역할을한다. 또한세포자멸사의발생에는여러가지과정과생화학적으로활성화된요소들이필요하다. 12-14,21 이와같은여러과정중 caspase 3는세포자멸사의과정에가장직접적으로작용하여중요한역할을하는것으로알려져있는데이의활성화에의하여세포자멸사가유발된다하였다. 12,17,18,22-24 즉사립체막단백인 bcl-2가사립체의투과성을변화시켜 cytochrome C 와세포자멸사유발인자 (apoptosis-inducing factor, AIF) 의분비를막아세포자멸사를억제하게되는데세포자멸사유발인자가분비되면비활성화된 caspase 3를활성화시켜세포자멸사를유발한다. 3,4,21,25 Caspase 는모두 12가지로알려져있는데그중에서도 caspase 3은인간의 Jurkat T lymphocyte로부터분리된것으로인체의여러조직과세포에서세포자멸사와직접적인관계가있다고알려져있다. 이에따라배양세포를이용한실험뿐만아니라췌장암, 전립선암, 백혈병, 위암을비롯한각종인체종양에서도 caspase 3에대한연구가이루어지고있으나 3,12,17,18,22-24 연 구가많지않아서세포자멸사와의직접적인관계를정확히알기는어렵다. 따라서여러종양에대한연구가더많이이루어지는것이중요하다하겠다. 저자들의연구에서는위선종과조기위암종, 진행위암종을대상으로보았을때 caspase 3의세포표지지수와세포자멸사모두위선종에서조기위암종을거쳐진행위암종으로진행할수록증가한다는것을알수있어 (p<0.001) 종양의발생과진행에이들이관여함을알수있었다. 그러나 caspase 3 세포표지지수와세포자멸사지수사이의상관관계는통계학적으로유의하지않았다. 그이유는본실험에사용된 caspase 3가활성화되기전의 pro-caspase 3 분자와활성화된조각모두에반응하는것이기때문으로생각된다. 저자들의전립선암에대한연구결과와다른여러보고들에서도 capspase 3 세포표지지수와세포자멸사지수가전암병변보다암종에서높게나왔는데이는이번연구의결과와일치하였다. 7,8,10,18,22,26 그러나위암종에서의 caspase 3 세포표지지수가위선종보다낮다는 Hoshi 등의보고 18 와는상반된결과를보여더많은증례에대한연구가이루어져야할것으로생각된다. 세포증식지수는위선종, 조기위암종, 진행위암종으로진행할수록증가하는경향을보였으며진행위암종에서아주높게나왔으나통계학적으로유의하지않았다. 이는 Hoshi 등의 18 결과와도일치하며 Aihara 등의 11 주장을뒷받침한다. 즉세포자멸사가양성병변에서악성으로갈수록증가한다면세포의전체수가적어야함에도불구하고암이진행할수록종양세포가많아지는것과는모순이되는데, 이는세포분열이나세포증식이세포자멸사에비하여훨씬많기때문이라고설명한 Aihara 등의 11 이론을뒷받침한다. 그러나 caspase 3, Ki-67 세포표지지수및세포자멸사지수간의상관관계는어떤경향을알수있는정도로의상관관계만있었고통계학적으로유의하지는않았는데이는다른보고들에서도유사하였다. 3,18,22 따라서위선종에서조기위암종진행위암종으로갈수록세포자멸사지수와 caspase 3 세포표지지수가유의하게증가함으로써 caspase의활성화가세포자멸사의유발에는중요한역할을하나상관관계가뚜렷하지않아, caspase 3 이외의다른많은경로와과정이동시에작용함을짐작할수있었다. 또한조기위암종에서는림프절전이가있는경우 caspase 3의발현빈도가유의하게증가하였으나 (p<0.05), 진행위암에서는림프절전이가있는경우오히려세포자멸사지수가낮게나와 (p<0.05) 림프절전이유무에따른차이는정확히판단하기어려웠다. 이에대하여도더많은증례를가지고연구를함으로써정확한결과를얻을수있으리라생각한다. 결론적으로 caspase 3 세포표지지수와세포자멸사지수는선종에서조기위암종을거쳐진행위암으로갈수록통계학적으로유의하게양성률이높아졌으며, Ki-67 세포표지지수는진행위암종에서양성선종이나조기위암종과비교하여증가하였다. 따라서종양의진행에세포자멸사와 caspase 3 및 Ki-67이관여하며세포자멸사의유발에 caspase 3의활성화가중요한역할
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