論文名稱:

檳榔子水萃取物對人類口腔上皮細胞表型及相關抑癌基因活化影響之探討

 

Investigation of the effects on cellular phenotypes and activation of relative tumor suppressor genes by areca nut extract in human oral keratinocytes.

研究生:

呂思逸  Suu-Yi Lu

 

(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:

張國威  Kuo-Wei Chang

        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:

38917002

          學年度:

91

          語文別:

中文

          出版年:

92

關鍵字:

檳榔子水萃取物  Areca nut extract

 

表型  Phenotype

 

口腔上皮細胞  Oral keratinocyte

 

細胞週期  Cell cycle

 

老化  Senescence

 

抑癌基因  Tumor supressor gene

 

突變  mutation

 

p53  p53

        論文頁數:

90

摘要:

全球約有六億人口有嚼食檳榔習慣。在印度、台灣、非洲南部以及其他東南亞國家中,嚼食檳榔塊為罹患口腔癌之主要危險因子。近幾年在台灣,口腔癌於男性癌症發生率排名已躍升第五位,所有癌症死亡率之第七位。檳榔子水萃取物(ANE)為檳榔嚼塊之主要成分,且對於多種型態細胞具有細胞毒性以及基因毒性。然而目前檳榔參與口腔癌化過程之分子證據仍未明瞭。因此,我們首先探討p53表現基因狀態於兩種口腔鱗狀上皮細胞癌細胞株(OSCC)—OECM-1以及OC3OC3是從一位長期嚼食檳榔但不抽菸之口腔癌患者所新建立之細胞株。結果發現,OC3細胞具有野生型p53表現序列且p53p21蛋白表現量與正常人類口腔上皮細胞(NHOK)相類似。但在OECM-1細胞株中於密碼子173位置上有一個點突變(V to L)因而造成p53蛋白過度表現及p21蛋白表現量降低。接下來我們利用NHOK以及OC3細胞來探討ANE對於細胞表型以及相關抑癌基因活化之影響。ANE會促使NHOK細胞之細胞週期停滯於G1時期並且隨濃度與時間增加趨勢而愈明顯。然而OC3細胞則是在ANE處理開始24小時短暫於G1時期停留,之後2472小時細胞族群有轉向G2/M時期停滯之趨勢。在BrdU嵌合試驗中證實ANE隨濃度增加對於NHOKOC3細胞抑制其增殖能力愈明顯。以ANE持續處理連續繼代之NHOK細胞發現隨繼代數增加其細胞提早老化表型愈明顯。接著我們偵測ANE對於細胞週期相關基因之調控。細胞在ANE處理後624小時可發現p53蛋白於Ser15位置磷酸化程度增加,證實p53蛋白之活化。此外,ANE對於NHOK細胞中p21在轉錄與轉譯層次均有明顯上升調控,但對於OC3細胞則只在轉譯層次明顯增加表現量。Rb蛋白透過磷酸化程度之降低而表現其抑制功能。因此,ANE促使細胞週期停滯以及提早老化表型之影響可能是經由活化這些抑制蛋白而造成。最後我們偵測ANE對於壓力活化蛋白激酶其中之一,p38-MAPK之調控作用。ANE增加p38-MAPK活性於長時間處理愈明顯,此訊息路徑之活化也可能與細胞提早老化相關。綜合以上本研究發現也許可以對於檳榔相關疾病提供一些線索。

 

There are around 600 million Areca quid (AQ) chewers in the world. AQ chewing is the major risk factor of oral cancer in India, Taiwan, South Africa and other Far Eastern countries. In recent years, the incidence of oropharyngeal cancer is the 5th most common cancer in males and the 7th highest cause for cancer death in Taiwan. Areca nut extract (ANE), the main component of AQ, exerts cytotoxicity and genotoxicity to several types of cells. However the molecular evidence of areca in oral tumorigenesis is still not clear. First we investigated the p53 status in two OSCC cell lines, OECM-1 and OC3, a new cell line established from an OSCC in a long-term areca user who does not smoke. OC3 cells bear wild type p53 coding sequence and have a p53 and p21 protein expression level similar to normal human oral keratinocyte (NHOK), But OECM-1 cells contain a point mutation at codon 173(V to L) leading to p53 overexpression and a reduction in p21 expression. Next, we used NHOK and OC3 cells to investigate the effects of ANE on cellular phenotypes and associating with tumor suppressor genes activation. NHOK cells exhibited a G1 cell cycle arrest modulated by ANE in a dose and time dependent manner. However OC3 cells were sustained in G1 arrest for the initial 24h then transited to G2/M arrest during 24~72h following ANE treatment. The BrdU incorporation assay demonstrated that ANE inhibits the proliferation of NHOK and OC3 cells in a dose dependent manner. Persistent treated of ANE in a serially-passages to NHOK results in a progressive occurrence of pre-mature senescence phenotype. Further, we examined the regulation of ANE on genes associated with cell cycle. Activation of p53 protein after exposure to ANE for 6~24h was identified with increasing the phosphorylation level at Ser 15. ANE significantly increases p21 expression both on transcription and translation levels in NHOK cells but only on translation level in OC3 cells. pRb exerts inhibitive function by decreasing the phosphorylation level. It is likely that the activation of these suppressor genes may contribute to cell cycle arrest and premature-senescence phenotype. Finally, the effect of ANE on stress-activated protein kinase (SAPK)-p38 MAPK was also examined. ANE activates p38-MAPK in a long period treatment appears to correlate with long-term effect of ANE on cell pre-senescence phenotype. Overall, the findings in the present study may provide clues about areca-associated pathogenesis.