論文名稱:

黃樟素對於人類口腔癌上皮細胞中藥物代謝酵素之影響及其

 

調控機制

 

Effects of safrole on drug metabolizing-enzymes and its

 

regulatory mechanisms in human oral epidermal cancer cells

研究生:

顏伊安  I-Ann Yen

指導教授:

翁芸芳  Yune-Fang Ueng

學位類別(Degree):

碩士

學校名稱(School):

國立陽明大學

系所名稱(Department):

口腔生物研究所

學號(Student Number):

39517004

學年度(Academic Year):

96

語文別(Language):

中文

出版年(Thesis Year):

97

關鍵字:

黃樟素  OECM-1

 

細胞色素P450  safrole

 

  AhR

 

  Nrf2

全文說明(Fulltext description):

(本論文未授權公開)

  論文頁數(Page):

62

摘要:

2007年行政院衛生署的統計資料指出,口腔癌已經位居國人十大癌症死因中的第六位,在男性部分更是高居第四位,並且逐年口腔癌的死亡率都有逐漸攀升的趨勢,顯示口腔癌危害國人健康的問題日益嚴重。黃樟素(safrole)為廣泛存在於自然界中的化合物,多種香料及飲料中都含有此成分,而台灣地區特有的檳榔嚼塊,所含的荖花有高濃度黃樟素的存在,使得嚼食檳榔者的口腔中黃樟素的暴露濃度高達420 μM。另外,過去研究也証實黃樟素為動物性致肝癌物,且會造成多種毒性傷害,因此黃樟素可能與口腔癌的形成有密切相關性。黃樟素在體內主要是經由細胞色素P450CYP)的氧化代謝,而活化生成多種致癌性產物,進而與DNA共價鍵結形成DNA adducts,導致基因突變,促使癌症發生,此為黃樟素致癌的主要原因之一。藥物代謝酵素在致癌物的活化代謝及解毒扮演著重要的角色,因此外來物對代謝酵素之誘導或抑制對於癌症的生成及藥物的藥理作用都會造成很大的影響。本篇研究則是探討黃樟素在藥物代謝酵素之影響對於導致口腔癌發生的可能機轉為何,而分別測定了NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylation (EROD), glutathione S-transferase (GST)NAD(P)H-quinone oxidoreductase 1 (NQO1)。另外,在過去有研究指出黃樟素所造成的氧化傷害亦為致癌的原因之一,因此,口腔癌細胞OECM-1在活性氧化物(reactive oxygen species, ROS)測定及脂質過氧化物(lipid hydroperoxides)的實驗中發現,黃樟素會增加約80%脂質過氧化物的生成,但在ROS生成則無顯著的變化。在時間及黃樟素劑量分別對代謝酵素活性影響之實驗中,經過420 μM黃樟素處理30小時後會增加ERODNQO1的活性,分別上升了3.6倍及0.2倍,而NADPH-cytochrome P450 reductaseGST的活性則是沒有變化。利用免疫染色和RT-PCR的方法證實,黃樟素會促進CYP1ANQO1的蛋白質與mRNA表現,進而誘導其酵素活性增加,而GSTP的蛋白質表現則是沒有變化。目前已知,phase I酵素中的CYP1phase II酵素的轉錄調控路徑主要是以aryl hydrocarbon receptorAhR);而phase II酵素及部分抗氧化酵素則是由nuclear factor erythroid 2 related factor-2Nrf2)所負責。利用免疫染色方法來觀察黃樟素是否會誘導轉錄因子的轉位作用發生,使AhRNrf2進入至細胞核內。結果顯示,隨著黃樟素處理時間的增加,會使細胞質中AhR Nrf2的表現下降,分別減少約20%和60%;而在細胞核,AhR Nrf2的表現則有顯著上升的趨勢,分別增加約27%和270%。因此,黃樟素會促使細胞質中的AhR Nrf2的活化,使其轉入至細胞核內,可能藉由此訊息傳遞路徑而促進下游CYP1ANQO1的表現。綜合上述實驗結果,顯示黃樟素會經由AhRNrf2路徑去誘導CYP1ANQO1之作用,此可幫助細胞對於黃樟素的代謝,但已知有多種致癌物及環境汙染物等多會藉由CYP1A的代謝活化而增加其致癌毒性,因此,黃樟素誘導口腔癌細胞中CYP1A的活性增加也可能是加強了香菸或其他有毒物導致口腔癌的原因之一。

 

In 2007, Department of Health in Taiwan has documented that oral cancer is the sixth leading causes of cancer death, and the fourth for man. The mortality of oral cancer is gradually increased every year, so oral cancer is an important health problem in Taiwan. Safrole is a nature plant constituent of a number of spices, such as nutmeg, anise, cinnamon and black pepper, and also in drinks. Piper betle inflorescence which contains high concentration of safrole, about 15 mg/g wet weight, is a unique ingredient of betel quidBQin Taiwan. Chewing such prepared betel quid could result in safrole concentration as high as 420 μM in saliva. Safrole is an animal hempatocarcinogen. Safrole has various toxicities including genotoxicity, cytotocixity, mutagenicity and tumorigenicity, indicating a possible correlation between safrole and oral cancer. The metabolic bioactivation of safrole are mediated through cytochrome P450, and then produce a number of proximate carcinogens which can give rise to DNA adducts and cause carcinogenesis. Drug metabolizing-enzymes play an important role in bioactivation and detoxification of carcinogens, so regulation of these enzymes could affect carcinogenesis and pharmacology. This study aims to know the effects of safrole on drug metabolizing-enzymes including NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylation (EROD), glutathione S-transferase (GST) and NAD(P)H-quinone oxidoreductase 1 (NQO1). In oxidative stress analysis of reactive oxygen species and lipid hydroperoxides, OECM-1 are treated by 420 µM safrole for 6 hours can increase 80% lipid hydroxyperoxides production, but doesnt significant increase reactive oxygen species production. In time-course and dose-dependent of effects of safrole on drug metabolizing-enzymes activities, at a concentration of 420 µM safrole for thirty hours result in 3.6-fold and 0.2-fold increases of EROD and NQO1, respectively. But activities of NADPH-cytochrome P450 reductase and GST are no effects. Immunoblot analysis and RT-PCR reveal that safrole increases both protein and mRNA expressions of CYP1A and NQO1, but no effect in GSTP1 protein expression. Immunoblot analysis of cytosolic proteins and nuclear extracts showed that safrole-exposure caused 20% and 60% decreases in the level of cytosolic AhR and Nrf2, respectively. Safrole-exposure caused 27% and 270% increases in the level of nuclear AhR and Nrf2, respectively. These results suggest that safrole can activate cytosolic AhR and Nrf2, and make them translocate to nucleus and further induce CYP1A and NQO1 expression. These results demonstrated that safrole induced CYP1A and NQO1 through AhR and Nrf2. However, many carcinogens and pollutants are bioactived through CYP1A, so safrole maybe a possible factor to enhance other toxic compounds like tobacco smoke to cause oral cancer.

論文目次
Table of Content
:

中文摘要..................................................1

 

英文摘要..................................................3

 

緒論......................................................5

 

實驗材料與方法...........................................14

 

壹、實驗材料.............................................14

 

貳、實驗方法.............................................17

 

實驗結果.................................................24

 

討論.....................................................29

 

參考文獻.................................................34

 

圖表.....................................................42

 

Figure 1. Cytotoxicity of various concentrations of safrole and time in OECM-1 was measured by MTT assay.............43

 

Figure 2. Time-course of effects of safrole on intracellular ROS production in OECM-1...................44

 

Figure 3. Measurement of lipid peroxidation products in OECM-1 was treated with safrole..........................45

 

Figure 4. Dose-response of effects of safrole on NADPH-P450 reductase and 7-ethoxyresorufin O-deethylation activities in OECM-1................................................46

 

Figure 5. Dose-response of effects of safrole on GST and NQO1 activities in OECM-1................................47

 

Figure 6. Time-course of effects of safrole on NADPH-P450 reductase and 7-ethoxyresorufin O-deethylation activities in OECM-1................................................48

 

Figure 7. Time-course of effects of safrole on GST and NQO1 activities in OECM-1.....................................49

 

Figure 8. Immunoblot analysis of CYP1A1/2 and CYP1B1 protein levels in OECM-1.................................50

 

Figure 9. Immunoblot analysis of GSTP and NQO1 protein levels in OECM-1.........................................51

 

Figure 10. Dose-response of safrole on CYP1A1, CYP1A2 and NQO1 mRNA by using RT-PCR analysis.......................52

 

Figure 11. Immunoblot analysis of AhR in cytoplasmic proteins.    ................................................53

 

Figure 12. Immunoblot analysis of AhR in nuclear extacts..................................................54

 

Figure 13. Immunoblot analysis of Nrf2 in cytoplasmic proteins.    ................................................55

 

Figure 14. Immunoblot analysis of Nrf2 in nuclear extracts.................................................56

 

Table 1. Effects of safrole on drug-metabolizing enzymes in OECM-1...................................................57