結直腸癌腫瘤組織中 AhR 與代謝蛋白表現對於 BPDE-like DNA 鍵結物含量與 p53 蛋白高度表現之相關性研究

Association of AhR and metabolic proteins expression with BPDE-like DNA adduct levels and p53 protein overexpression in colorectal tumour tissues

廖尹群(Yin-Qun Liao)

指導教授 ： 李輝 ； 鄭雅文

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00031

摘要

惡性腫瘤一直是臺灣民眾的首位死因，而結直腸癌已躍居癌症死因的第三位。流行病學研究指出長期吸菸和紅肉攝取可能增加結直腸癌的危險；推測可能暴露較多致癌性的芳香烴類化合物 (polycyclic aromatic hydrocarbons, PAHs)。當 Benzo[a]pyrene (BaP) 類 PAHs 進入體內，會經由 AhR 路徑轉錄活化代謝基因之表現，例如 CYP1A1 會將 BaP 代謝為 BPDE 活化代謝物，攻擊 DNA 而形成 BPDE- DNA 鍵結物。另外 BaP 可經由 AhR/Nrf2 路徑交互作用而誘導 GSTM1、DDH 與 NQO1 的轉錄活性。為了解這些代謝基因之表現是否經由 AhR 路徑之調控，本研究共收集 152 位結直腸癌患者之腺癌腫瘤病理切片，以免疫組織化學染色法 (immunohistochemistry, IHC) 分析腺癌腫瘤組織中 AhR 蛋白與 CYP1A1、GSTM1、NQO1、DDH 四種代謝蛋白之相關性，結果發現四種蛋白表現均與 AhR 蛋白表現呈正相關性。這結果顯示結直腸組織中之 AhR 路徑確實參與調控代謝基因之轉錄活化。為了解該鍵結物之形成是否與結直腸組織中之代謝基因表現有關？進一步分析四種代謝基因蛋白與 BPDE-like DNA 鍵結物含量之相關性，結果發現除了 GSTM1 之外，其他三種代謝蛋白之表現，即 CYP1A1、NQO1、DDH 與 DNA 鍵結物之形成都具有正相關性，都達到統計上之顯著相關。
過去有研究顯示 AhR 會直接調控 p53 蛋白之磷酸化，而穩定正常 p53 蛋白之表現。因此本研究亦分析兩者間之相關性，結果發現結直腸癌患者正常 p53 蛋白之表現，與 AhR 蛋白表現呈顯著正相關性 (P=0.028)，但結直腸癌患者突變 p53 蛋白之表現，亦與 AhR 蛋白表現呈正相關之趨勢，但沒有達到統計意義 (P=0.080)。又已知 p53 蛋白過度表現與結直腸癌之腫瘤發展與惡化有關，並可作為患者之預後指標。為了解結直腸癌形成是否與 PAHs 類致癌物暴露，代謝活化產生 BPDE-like DNA 鍵結物，而引起 p53 蛋白異常表現。進一步分析 p53 蛋白和 BPDE-like DNA 鍵結物含量之相關性，結果發現結直腸癌患者腫瘤組織中 BPDE-like DNA 鍵結物含量較高者，具有較高 p53 蛋白表現之趨勢，但沒有達到統計意義 (P=0.094)。同時發現 p53 蛋白高度表現者具有較低的存活率 (P=0.076)。因此 BPDE-like DNA 鍵結物似乎參與結直腸腫瘤之 p53 蛋白的異常表現，而參與其腫瘤發展。
同時本研究亦發現 AhR 蛋白表現會隨著腫瘤之期別愈高，其蛋白之表現頻率愈高。總之，本研究之結果顯示，結直腸組織暴露環境中 BaP 類致癌物，可能會藉由活化 AhR 訊息傳遞路徑，活化 CYP1A1、DDH、NQO1 代謝基因之表現，而產生 BPDE-like DNA 鍵結物，而此鍵結物的累積可能參與 p53 蛋白過度表現。另外亦有可能直接經由 BaP 類致癌物之誘導，而活化 AhR 蛋白之表現，促使正常 p53 蛋白之磷酸化，而穩定該蛋白之表現。因此本研究提出相關數據，支持多吃燒烤之肉類食品，會增加罹患結直腸癌之危險性。

關鍵字

結直腸癌 ； 多環芳香烴化合物 ； 代謝酵素 ； AhR ； BaP ； BPDE-like DNA 鍵結物 ； p53

並列摘要

Neoplasm is the leading death of Taiwanese since 1982 and colorectal cancer is the third cause of cancer death. Epidemiological studies indicated that cigarette smoking and the consumption of red meat may increase the risk of colorectal cancer. They might consume relatively higher amounts of genotoxic or carcinogenic polycyclic aromatic hydrocarbons (PAHs) which can up-regulate the metabolic genes including CYP1A1, GSTM1, NQO1, and DDH through AhR/Nrf2 signaling pathways. The BaP active metabolite BPDE may then attack DNA to form BPDE-N2dG DNA adduct and to cause tumor suppressor gene p53 mutation, p53 genetic mutation has been demonstrated to be correlated with p53 overexpression in colorectal cancer. In this study, we investigated (1) whether p53 overexpression was a poor prognostic factor in Taiwanese colorectal cancer patients to suggest that p53 overexpression may play an important role in colorectal tumor progression, (2) whether BPDE-like DNA adducts was associated with p53 overexpression, (3) whether the metabolic enzymes of CYP1A1, GSTM1, NQO1, and DDH were induced by PAHs to involve in the formation of BPDE-like DNA adducts, (4) whether the induction of these metabolic genes was mediated through AhR signaling pathway, and (5) whether AhR protein expression was associated with p53 overexpression in colorectal tumors. The above gene expressions and BPDE-like DNA adducts in 152 colorectal tumors were all examined by immunohistochemistry (IHC). Our data indicated that the four metabolic protein expressions were significantly correlated with AhR protein expression such finding suggest that AhR pathway was markedly induced by the inducer such as PAHs. In addition, BPDE-like DNA adduct levels were positively correlated with CYP1A1, NQO1, and DDH protein expressions, and the adduct levels were also positively correlated with p53 overexpression. More interestingly, AhR protein expression was significantly correlated with p53 overexpression and this observation seems to support previous report indicated that AhR may promote the phosphorylation of p53 to stabilize p53 protein expression in tumor cells. In summary, p53 overexpression in colorectal tumors might associate with the induction of AhR signaling pathway by PAHs derived from cigarette smokes and cooked meats and BPDE-like DNA adducts were also associated with p53 overexpression. Thus, our data may support the possibility that the consumption of cigarette smokes and cooked meats may increase the risk of colorectal cancer.

並列關鍵字

Colorectal cancer ； Polycyclic aromatic hydrocarbons ； metabolic enzymes ； Aryl hydrocarbon receptor ； Benzo[a]pyrene ； BPDE-like DNA adducts ； p53

參考文獻

Alexandrov K, Rojas M, Kadlubar FF, Lang NP, Bartsch H. (1996). Evidence of anti-benzo[a]pyrene diolepoxide-DNA adduct formation in human colon mucosa. Carcinogenesis. 17(9):2081-3.

Asher G, Lotem J, Sachs L, Kahana C, Shaul Y. (2002). Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1. Proc Natl Acad Sci U S A. 99(20):13125-30.

Belinsky M, Jaiswal AK. (1993). NAD(P)H:quinone oxidoreductase1 (DT-diaphorase) expression in normal and tumor tissues. Cancer Metastasis Rev. 12(2):103-17.

Bieler CA, Arlt VM, Wiessler M, Schmeiser HH. (2003). DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes. Cancer Lett. 200(1):9-18.

Bostick RM, Fosdick L, Wood JR, Grambsch P, Grandits GA, Lillemoe TJ, Louis TA, Potter JD. (1995). Calcium and colorectal epithelial cell proliferation in sporadic adenoma patients: a randomized, double-blinded, placebo-controlled clinical trial. J Natl Cancer Inst. 87(17):1307-15.