論文名稱:

APE1/Ref-1在口腔癌化中角色之研究

 

The Role of Apurinic/Apyrimidinic Endonuclease/Redox Factor-1(APE1/Ref-1) in Oral Carcinogenesis

研究生:

鄭惠文  Huei-Wen Jeng

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

夏堪臺  Kan-Tai Hsia

 

楊世芳  Shih-Fang Yang

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39217010

          學年度:

93

          語文別:

中文

          出版年:

94

關鍵字:

口腔癌化  oral carcinogenesis

 

  APE1

 

  Ref-1

全文說明:

(本論文未授權公開)

 

電子全文

        論文頁數:

102

摘要:

    鹼基刪除修復[ base excision repair (BER) pathway ]是細胞受到烷化物(alkylation agent)及氧化性傷害的主要修復機制,當BER pathway啟動時,其中參與BER的蛋白質— apurinic/apyrimidinic 1 endonuclease/redox factor-1 (APE/Ref-1)會辨識DNA上的AP-site,並且在AP-site5’端進行切割,之後再由其他BER蛋白質去繼續進行DNA修復。此外,APE1/Ref-1也是一個reduction-oxidation (redox) factor,可幫助轉錄因子維持活化的還原形式,並能提高轉錄因子結合到DNA的能力,進而去引發細胞產生不同的反應,諸如:細胞生長、分化及細胞凋亡。過去的研究指出,在子宮頸癌,卵巢癌、前列腺癌,均發現APE1/Ref-1有高度的表現量,且其在細胞核質的比例亦有異常改變,是以推論APE1/Ref-1和口腔癌之間亦有一定的相關性。另外,也有研究發現,APE1/Ref-1基因在不同位置的多型性(polymorphism)差異會影響個體之間對cancer的感受性。因此,本研究要更進一步去探討在口腔癌化中APE1/Ref-1表現及其功能為何。

 

    本研究利用免疫組織化學法(immunohistochemistry),觀察口腔癌組織及正常口腔上皮組織中APE1/Ref-1蛋白的表現及表現位置,結果顯示口腔癌組織中APE1/Ref-1表現上升,且表現位置會從細胞核逐漸轉移到細胞質上,但是轉移到細胞質的APE1/Ref-1其影響功能尚未確定。另外,APE1/Ref-1表現程度和癌症組織分化情形具有關聯性。在APE1/Ref-1 Asp148Glu多型性研究上,利用高效能液相層析(denaturing high performance liquid chromatography)、序列定序及polymerase chain reaction with confronting two-pair primers (PCR-CTPP)來分析,已發現正常樣本的TG allele frequencies分別為0.5930.406;口腔癌樣本的TG allele frequencies分別為0.5480.452。另外,以黃樟素(safrole)濃度0~3000μM 處理SCC25OECM-1 24小時後,發現在1000μM 時會達到50%的致死情況。而在Safrole影響APE1/Ref-1 mRNA及蛋白表現上,發現APE1/Ref-1可能不是決定safrole修復的主要蛋白。

 

綜觀本研究之結果,在口腔癌中,APE1/Ref-1表現過多,其可能會改變其修復或是氧化還原的功能,而影響癌症細胞,因此推測APE1/Ref-1表現的異常可能為一癌前特徵,將可用來預防癌症發生。

   
 

    The DNA base excision repair (BER) pathway is responsible for the cellular and oxidative DNA damage. In the BER pathway, APE1/Ref-1 (apurinic/apyrimidinic endonuclease/redox effector factor) acts as an 5’-endonuclease, and it can remove apurinic/apyrimidinic (AP) site. APE1/Ref-1 is a multifunctional protein that is not only responsible for repair of AP sites, but also functions as a reduction-oxidation (redox) factor maintaining transcription factors in an active reduced state. It has been shown to stimulate the DNA binding activity of numerous transcription factors that are involved in cell proliferation, differentiation and so on. APE1/Ref-1 levels have been found to be elevated in a number of cancers such as cervical cancer, ovarian cancer and prostate cancer. Also, the cellular locations of APE1/Ref-1 in many cancers distribute abnormally. Furthermore, APE1/Ref-1 (Asp148Glu) polymorphism site has been reported to be associated with human cancer. Therefore, APE1/Ref-1 may be as a candidate for disease-susceptibility gene in oral carcinogenesis.

 

    In the study, forty-five samples of oral cancer tissues and forty-seven samples of normal gingival tissue were analyzed using immunohistochmeistry. APE1/Ref-1 protein was elevated in oral cancer tissue. Among those with known clinical outcome, there was a significant correlation between high APE1/Ref-1 expression levels and tissue moderately and poorly differentiation. Also, the cellular location of APE1/Ref-1 in the cancer tissue distribute from nucleus to cytoplasm. In the APE1/Ref-1 Asp148Glu polymorphism, the T allele frequencies ratio is 0.593 and 0.548, respectively, in normal blood samples and oral cancer cases; the G allele frequencies ratio is 0.406 and 0.452. Finally, safrole, a component of piper betel inflorescence, was as an oxidative stimulus to observe the expression of APE1/Ref-1 in oral cancer cell lines. This study demonstrated that the ID50 with safrole treatment is 1000μM. APE1/Ref-1 expression did not show remarkable changes after safrole treatment in RT-PCR and western blot. However, the mRNA expression of NTH1 has been elevated, and it is assumed that the DNA repair after safrole treatment may be through another mechanism.

 

    In conclusion, the APE1/Ref-1 has been elevated in oral cancer and there is increase in the amount of APE1/Ref-1 in the cytoplasm. It can suggest that APE1/Ref-1 may be as a diagnostic marker for oral cancer.