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研究生(外文):Deh-Wei Tang
論文名稱(外文):The Induction of COX-2 and DDH by Areca Quid Components and It''s Role in Oral Carcinogenesis
指導教授(外文):Tsung-Yun Liu
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環氧化酵素 (cyclooxygenase, COX) 為合成前列腺素所需的關鍵酵素。近幾年來,許多研究都指出在多種癌症組織中,COX-2 都會大量表現。在台灣,口腔癌發生率高居第七位,流行病學研究也顯示,咀嚼檳榔與罹患口腔癌有很密切的關係。因此,本論文探討 COX-2 在人類口腔癌、癌前期病變以及正常組織中的表現是如何? 進一步,本論文以檳榔萃取或成分物來處理正常口腔細胞,分析 COX-2 的表現,以便進一步建立檳榔成分、COX-2 表現與口腔癌發生之關係。實驗完成了 27 對 (口腔癌組織 vs. 正常組織) 口腔癌病人組織切片的免疫染色,經由影像處理後的結果顯示,口腔癌病人的癌細胞 COX-2 蛋白的表現較周圍正常細胞有增加的趨勢 (P < 0.01)。在 RNA 方面,經由反轉錄聚合酵素鏈鎖反應以及 in situ hybridization 的方法,發現口腔癌病人的組織中 COX-2 mRNA 的表現也較鄰近正常口腔組織來得高。另外,此研究將一些檳榔成分處理正常口腔細胞,結果發現檳榔萃取物 (ANE) 和 hydroxychavicol (HC) 會造成細胞 COX-2 mRNA 及蛋白量的增加,且具有劑量以及時間依附的關係。因此本研究認為,檳榔的確是口腔病變發生過程中一個重要的危險因子,且 COX-2 在口腔癌發生過程中也扮演一個重要的角色。流行病學的研究顯示,抽菸與嚼食檳榔對於罹患口腔癌有加成的作用。先前實驗已知 HC 處理角質細胞可誘發 COX-2 的表現。COX-2 具有催化 B[a]P-7,8-dihydrodiol (B[a]P-diol) 使其轉變成 B[a]P-7,8-diol-9,10-epoxide (BPDE) 的能力。本研究欲了解在口腔癌細胞株中,HC 能否調控香菸主要致癌物 B[a]P 的基因毒性。以32P-postlabeling 的方法來偵測 HC 所誘發出來的 COX-2 對 B[a]P 形成 B[a]P 鍵結物能力的影響。結果顯示前處理 HC 雖可誘發 COX-2 的生成,卻有意義地降低了 B[a]P 所產生的 B[a]P-DNA 鍵結物的量。但相同作用條件下,HC 仍可導致細胞毒性以及 HPRT 基因突變頻率的增加。微陣列分析法顯示 HC 可以誘發許多 phase II 解毒酵素的表現。利用西方墨點法,發現 HC 也可誘發 dihydrodiol dehydrogenase (DDH) 的表現。DDH 是醛酮還原酵素 (aldo-keto reductase, AKR) 家族的一員。已知其會與 P450s 競爭而使 B[a]P-diol 走 B[a]P-7,8-quinone (BPQ) 的代謝活化路徑,進而產生氧化性自由基。藉由流式細胞儀,偵測到細胞前處理 HC 所產生 8-oxoguanine 的含量,較單獨處理 B[a]P 有意義地增加 (P < 0.001)。因而認為 HC 雖會降低 B[a]P 所產生的 B[a]P-DNA 鍵結物,但 DDH 的誘發以及氧化性自由基的產生卻會進一步對細胞產生毒性以及突變的傷害。因此本研究認為,檳榔嚼塊主成分之一的 HC 具有調控香菸主要致癌物 B[a]P 基因毒性的能力。
Elevated expression of cyclooxygenase (COX)-2, a rate-limiting enzyme in the conversion of arachidonic acid to prostaglandin (PG) biosynthesis, has been demonstrated in several human cancers including squamous cell carcinoma of the head and neck. Oral squamous cell carcinoma (OSCC) is an important malignancy in Taiwan, which is highly associated with areca quid (AQ) use. Whether COX-2 is up-regulated in AQ related OSCC is unknown, and the potential of AQ ingredient to induce COX-2 expression has not been studied. COX-2 expression was analyzed by immunohistochemistry, in situ hybridization and reverse transcription-PCR in paired OSCC and adjacent normal tissues. The COX-2 mRNA and protein induction potential of AQ components, such as hydroxychavicol (HC) were quantitatively analyzed by real-time RT-PCR and Western blot analysis in cultured human normal oral keratinocytes and fibroblasts The cytotoxicity and PGE2 production as modulated by betel ingredient was assayed by ELISA assay. Using immunohistochemistry, the COX-2 expression was found to be significantly higher (P < 0.05) in OSCC as compared with their adjacent normal tissue (n = 27). The level of COX-2 mRNA was remarkably elevated in 63% (12 / 19) OSCC carcinomas compared with adjacent normal tissues (n = 11). Using in situ hybridization, the COX-2 mRNA was found to have higher expression in cytoplasm of cancer cells. HC, the unique AQ component in locally chewed AQ, induced COX-2 mRNA and protein expression dose- and time-dependently in oral keratinocytes. These results suggest the early involvement of COX-2 in AQ induced oral cancer carcinogenesis. AQ chewing and smoking have synergistic potential in the development of OSCC. It has been shown that COX-2 was expressed in OSCC and HC induced COX-2 expression. COX-2 is involved in benzo[a]pyrene (B[a]P) metabolism. This study investigated whether HC modulates B[a]P-mediated genotoxicity through COX-2 induction. Pretreatment of HC, with known elevated COX-2 expression, did not increase B[a]P-DNA adduct, instead, it significantly reduced B[a]P-DNA adduct levels. This is demonstrated by 32P-postlabeling analysis (P < 0.05). However, this treatment resulted in higher cytotoxicity and HPRT gene mutation frequency (P < 0.05). Western blot analysis showed that the expression of dihydrodiol dehydrogenase (DDH) was also increased. DDH has been shown to divert B[a]P-diol to B[a]P-7,8-quinone, which resulted in the generation of reactive oxygen species. Using flow cytometry, I showed that the production of 8-oxoguanine was increased following the treatment (P < 0.001). Overall, the results suggest that HC induced DDH is more important than site-by-site up-regulation of COX-2 in B[a]P-induced cytotoxicity and HPRT gene mutation. Furthermore, DDH mediated oxidative DNA damage and not B[a]P adduct formation may be involved in the B[a]P-induced toxic effects.
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