乳癌病人Cytochrome P4502E1 及Glutathione
S-transferase M1 基因多型性及其蛋白質表現

乳癌在歐美佔女性癌症的第一位，為國內女性癌症罹患率之第二位，僅次於子宮頸癌，在過去20 年呈現穩定的上升且逐年增加。雖然環境、基因、營養和荷爾蒙一般被認為與乳癌的形成有關，但是乳癌的發生學在現在仍未完全的被釐清。本研究主要探討去除環境致癌物的代謝酵素之多型性與乳癌發生率的相關性。同時，觀察其蛋白質表現與乳癌的相關性。人體內的去毒代謝性機制主要是依靠第一相與第二相去毒性酵素的交互作用。這些去毒性代謝酵素基因大多都具有多型性，個體間對於癌症的適應性不同可能就是因為這樣的基因多型性與外在環境暴露所互相影響的關係。第一相酵素:細胞色素 P4502E1(cytochrome P450 2E1;以下簡稱CYP2E1)與酒精的代謝有關，同時也負責代謝一些潛在的致癌物，如:N-nitrosamine， benzene， urethane 和styrene。第二相酵素榖胱苷肽轉移酶M1(glutathione s-transferase;以下簡稱GSTM1) 主要可代謝致癌物及一些致癌物的中間產物。因為這些去毒性代謝酵素被認為與個體對乳癌的適應性有關，所以，本實驗使用聚合酶鍊鎖反應與限制片段長度多型性來對 CYP2E1 與GSTM1 作分析，探討其多型性與乳癌之間的關係。這個病例對照研究包含了262 女性乳癌病人及225 位對照組。病例與對照組各抽取5mL 的EDTA 血液作多型性的研究。結果顯示;在乳癌患者中，CYP2E1 A 型(C1/C1)者為162 名(61.8%)，B 型(C1/C2)者為96 名 (36.6%)，C 型者(C2/C2)為4 名(1.5%)。在正常對照組中，CYP2E1 A 型佔134 名(59.6%)，B 型佔44 名(34.2%)，C 型者佔14(6.2%)。乳癌患者中，GSTM1 表現型者為139 人(53.1%)，缺損型者為123 人(46.9%); 正常對照組之GSTM1 表現型者為122 人(54.2%)，缺損型者為103 人(45.8%)。統計比較發現CYP2E1 C 型對比於A 型達到顯著性差異 (OR = 0.238， 95% CI = 0.077-0.738)，表示具CYP2E1 C 型基因型的人群罹患乳癌之危險性為具A 型之0.238 倍。GSTM1 表現型與不表現型對於乳癌罹患的相關，以GSTM1 表現型 ( wildtype )為基準，具 GSTM1 缺損型的人口，罹患乳癌的相對危險比為表現型的1.048 倍，但未達統計意義(OR 值為1.048，95%信賴區間0.733-1.498)。於262 名病例中，具組織病理學資料者有164 名，依其分级，I+II 型者有120 名，其中，CYP2E1 A 型者佔80 ( 66.7%) B 型者佔37 名( 30.8% )，C 型者佔3 名( 2.5% )；GSTM1 表現型者佔65 名( 54.2% )，缺損型者佔55 名( 45.8% )。組織病理學分级III-IV 者有44 人， CYP2E1 A 型者佔24( 55.6%) B 型者佔20 名( 44.4% )，C 型者佔0 名( 0% )； GSTM1 表現型者佔22 名( 55.1% )，缺損型者佔23 名( 48.9% )。與 CYP2E1 作統計，顯示乳癌病人中具CYP2E1 B 型或C 型者其乳癌之組織學上較嚴重者之危險性為相對於A型為0.6 倍但不具有顯著性差異， (OR = 0.6 ， 95%CI = ( 0.2966-1.2136 )。依照乳癌病人組織學分類對於GSTM1 作統計，得知GSTM1 缺損型之乳癌病人，其乳癌組織學上較嚴重者的危險性為GSTM1 表現型之0.809 倍，然而其並未達到統計上之顯著性差異， (OR = 0.809， 95%CI = 0.408-1.607 ) 將乳癌病人與正常人以年齡作分類，以平均數約50 歲為基準分為兩群。50 歲以上佔248 人，50 歲以下佔239 名。50 歲以下者，CYP2E1 B+C 型對比於A 型之相對危險比並無達到顯著意義(OR = 1.337， 95%CI = 0.791-2.261)。GSTM1 缺損型對比於表現亦無達到統計上的顯著差異(OR =1.506， 95%CI = 0.901-2.518)。但是對於50 歲以上的族群而言， CYP2E1 B+C 型對比於A 型之相對危險比達到統計上顯著意義(OR = 0.583， 95%CI = 0.342-0.994)。GSTM1 缺損型對比於表現亦無達到統計上的顯著差異(OR = 1.199， 95%CI = 0.720-1.995)。採用西方點墨法來研究CYP2E1 的蛋白質表現，結果顯示:CYP2E1 在腫瘤組織之中的表現比在正常組織中為低。顯示了或許腫瘤的發生生會抑制CYP2E1 之表現，這可能是因為腫瘤組織為了要順利發展而抑制毒性代謝酵素CYP450 家族之表現，或者是因為 CYP2E1 的下降導致了腫瘤的發生，這在未來上有討論之空間。。

關鍵字

細胞色素p4502E1 ；乳癌；榖胱??轉移?M1

並列摘要

Breast cancer has become one of the main cancers in Taiwan. The incidence rate of breast cancer has increased in last two decades. The etiology of breast cancer is still unknown, and environmental, genetic, nutritional and hormonal factors are known to contribute to the breast cancer risk. This study focuses on some enzymes encoded by genes with polymorphisms that detoxify some kinds of environmental carcinogens. Phase I detoxification enzymes results in activation of carcinogens, usually mediated through enzymes encode by CYP family of genes. The detoxification phase (Phase II) occurs when the activated carcinogen is processed and rendered more hydrophilic, thus excretable. Genes encoding for xenobiotic metabolizing enzymes are mainly polymorphic. Individuals susceptibility to cancer may be due to the cross reaction of this kind of genetic variations and the exposures to some environmental carcinogens. Cytochrome p4502E1 (CYP2E1), one of the phase I enzymes, is involved in the ethanol metabolism and in the metabolic activation of some potential carcinogens, such as N-nitrosamine, benzene, urethane and styrene. The glutathione S-transferase family are phase II enzymes that detoxify carcinogens and their intermediates. Glutathione S-transferase M1 (GSTM1) is a member of the glutathione S-transferase family. Because genetic differences in such carcinogen metabolism genes are considered to be associated with the individual susceptibility to develop breast cancer, in this study, we evaluate the association between the polymorphism of CYP2E1 and GSTM1 and breast cancer. Besides, we also analyzed the expression of CYP2E1 by Western blot. This case-control study encompassed 262 patients with breast cancer and 225 cancer-free controls. EDTA blood (5ml) was taken from patients admitted to breast cancer surgery and from 225 controls recruited among volunteers at Kaohsiung Medical University Hospital between July 2001 and October 2003. The breast tissues, including cancer tissues and cancer-free tissues, were taken from the patients with breast cancer during the surgery. They all provided written informed consent. In this study, we found that significant relationship was observed in CYP2E1 genotype C2/C2 compared with CYP2E1 C1/C1 (OR = 0.238. 95% CI = 0.077-0.738). However, there was no significant difference in the polymorphism of GSTM1 in the case-control study. The expression of CYP2E1 was significantly lower in the tumor tissue than that in the adjacent cancer-free tissue. This means that the occurrence of breast cancer may inhibit the expression of CYP2E1 or the deficiency of CYP2E1 results in the occurrence of breast cancer. The mechanism is unknown, and there will be more studies to confirm it.