臺灣博碩士論文加值系統

Sorafenib，索拉非尼，是一種治療腎細胞癌及肝癌藥物，可誘導細胞自噬，抑制腫瘤生長。 Cytochrome P450 1A1 ( CYP1A1 ) 是受 aryl hydrocarbon receptor ( AhR ) 調節的主要調節基因之一，用於代謝外來物及類固醇的合成主要基因之一，在生理上扮演重要的角色。本研究探討 sorafenib 在小鼠肝癌細胞 ( Hepa-1c1c7 ) 和人類肝癌細胞 (HepG2)，如何干擾CYP1A1 的表現及其調節路徑為何。β-Naphthoflavone (β-NF) 是AhR誘導劑；2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester ( ITE )是人體必需胺基酸—色胺酸的代謝產物之一。結果顯示sorafenib會抑制β-NF 和 ITE 誘導的 CYP1A1 mRNA 表現，Western blot的結果也證明sorafenib 會抑制 β-NF 和 ITE 誘導的 CYP1A1 蛋白產生。同時，免疫螢光實驗中，證實 sorafenib 會抑制 β-NF 和 ITE 誘導的 CYP1A1 蛋白產生。 sorafenib會抑制 β-NF 和 ITE 誘導的 aryl hydrocarbon response element ( AhRE ) 的轉錄活性。免疫螢光實驗證實了 sorafenib 會誘導 AhR進入細胞核內。在細胞活力試驗中，β-NF 和 ITE分別與sorafenib共同處理細胞，培養細胞24小時後，只有在高劑量下，細胞活力有比較明顯著的下降，剩40-50 %的細胞活力。但是，在相同的高劑量下，sorafenib卻幾乎完全抑制β-NF 和 ITE 誘導 CYP1A1蛋白產生，證明sorafenib抑制CYP1A1的表現，並非是由於它對細胞的毒性。本研究證實 sorafenib 會阻止AhR被活化，並抑制 CYP1A1 的表現，可以預期sorafenib可能會干擾 AhR 的生理活性。因此，本論文提供了在臨床用藥上重要的資訊。

Sorafenib is an agent for the therapy of kidney and liver cancers. It inhibits tumor growth by inducing autophagy. Cytochrome P450 1A1 ( CYP1A1 ) is one of the major genes regulated by aryl hydrocarbon receptor ( AhR ) and one of the genes for the metabolism of foreign substances and steroids. It plays an important role in physiology. This study investigated how sorafenib interrupted CYP1A1 expression and its regulatory pathway in mouse hepatoma cells (Hepa-1c1c7) and human hepatocellular carcinoma (HepG2) cells. β-Naphthoflavone (β-NF) is an AhR inducer, and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester ( ITE ) is one of the metabolites of human essential amino acid-tryptophan. Sorafenib inhibits β-NF and ITE induced CYP1A1 mRNA expression, and results derived from Western blot also demonstrated that sorafenib inhibits the β-NF and ITE-induced protein of CYP1A1. Immunofluorescence experiments also confirmed that sorafenib inhibits β-NF and ITE induced protein of CYP1A1. Sorafenib inhibited the transcriptional activity of the β-NF and ITE induced aryl hydrocarbon response element (AHRE). Immunofluorescence experiments confirmed that sorafenib induced AhR into the nucleus. In the cell viability assay, it was found that combination treatment of high dose of sorafenib plus β-NF or ITE for 24 hours, significant decreased cell viability, 40-50% cell viability left. However, at the same high dose of treatment, sorafenib almost completely blocked the β-NF- and ITE-induced CYP1A1 expression. Accordingly, the blocking CYP1A1 of expression was not due to the cytotoxicity of sorafenib.
This study confirmed that sorafenib interferes with AhR, and then consequently inhibits the expression of CYP1A1. According the results, we can predict that sorafenib interferes with the physiological activity of AhR. This thesis provides valuable information for clinical application of sorafenib.

摘要 I
Abstract III
致謝 V
總目錄 VI
第一章 文獻回顧 1
1-1. Sorafenib (索拉非尼) 1
1-2. 芳香烴受體及細胞色素CYP450 1
1-3. AhR對生理的重要性 2
1-4. 肝臟的代謝路徑，Phase I與Phase II 2
1-5. 實驗目的 3
第二章 材料與方法 4
2-1. 試劑與抗體 4
I. 藥品與材料 4
II. 抗體 5
III.儀器 6
2-2. 細胞株核細胞培養 7
2-3. 質體的建構和報告子活性檢測 7
2-4. 反轉錄聚合酶鍊式反應(RT-PCR) 和定量PCR 8
2-5. 西方點墨法 9
I. 原理 9
II. 細胞處理 9
III. 蛋白質定量 9
IV. 西方點墨法步驟 10
2-6. 細胞免疫螢光染色 10
2-7. 細胞活力檢測 11
2-8. 流式細胞儀 11
2-9. 統計分析 12
第三章 結果與討論 13
3-1. 索拉非尼對芳香烴反應元件（AHRE）反式激活活性的影響。13
3-2. 索拉非尼降低ITE和β-NF誘導的CYP1A1 mRNA表現。 13
3-3. 索拉非尼降低β-NF-和ITE誘導的CYP1A1蛋白表現。 14
3-4. 索拉非尼誘導芳烴受體（AHR）的入核定位。 14
3-5. 索拉非尼，β-NF和ITE處理的HepG2細胞和Hepa-1c1c7細胞的活力測定。 15
3-6. 索拉非尼和ITE處理的Hepa-1c1c7細胞的細胞週期。 15
圖1.索拉非尼對芳香烴反應元件（AHRE）反式激活活性的影響。18
圖2.索拉非尼降低ITE和β-NF誘導的CYP1A1 mRNA表現。 19
圖3.索拉非尼降低β-NF-和ITE誘導的CYP1A1蛋白表現。 24
圖4.免疫螢光成像顯示索拉非尼對CYP1A1表達的激活作用。25
圖5.索拉非尼誘導芳烴受體（AHR）的核定位。 28
圖6.索拉非尼，β-NF和ITE處理的HepG2細胞和Hepa-1c1c7細胞的活力測定。 29
圖7.索拉非尼和ITE處理的Hepa-1c1c7細胞的細胞週期。 31
第四章 結論 32
參考文獻 33
附錄 36
附錄一、 Sorafenib之化學結構式 (Selleckchem , Houston , USA) 36
附錄二、活化AhR的路徑 37
附錄三、外來物被代謝的路徑 38
附錄四、報告基因結構示意圖 39

Al-Anati, L., Viluksela, M., Strid, A., Bergman, Å., Andersson, P. L., Stenius, U., & Högberg, J. (2015). Hydroxyl metabolite of PCB 180 induces DNA damage signaling and enhances the DNA damaging effect of benzo [a] pyrene. Chemico-biological interactions, 239, 164-173.
Baird, W. M., Hooven, L. A., & Mahadevan, B. (2005). Carcinogenic polycyclic aromatic hydrocarbon‐DNA adducts and mechanism of action. Environmental and molecular mutagenesis, 45(2‐3), 106-114.
Burczynski, M. E., & Penning, T. M. (2000). Genotoxic polycyclic aromatic hydrocarbon ortho-quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor. Cancer research, 60(4), 908-915.
Hao, N., & Whitelaw, M. L. (2013). The emerging roles of AhR in physiology and immunity. Biochemical pharmacology, 86(5), 561-570.
Indra, R., Moserova, M., Kroftova, N., Sulc, M., Martinkova, M., Adam, V., . . . Stiborova, M. (2014). Modulation of human cytochrome P450 1A1-mediated oxidation of benzo [a] pyrene by NADPH: cytochrome P450 oxidoreductase and cytochrome b5. Neuro Endocrinol. Lett, 35, 105-113.
Jin, Y., & Penning, T. M. (2007). Aldo-keto reductases and bioactivation/detoxication. Annu. Rev. Pharmacol. Toxicol., 47, 263-292.
Katz, S. C., Burga, R. A., McCormack, E., Wang, L. J., Mooring, J. W., Point, G., . . . Stainken, B. F. (2015). Phase I hepatic immunotherapy for metastases study of intra-arterial chimeric antigen receptor modified T cell therapy for CEA+ liver metastases. Clinical Cancer Research, clincanres. 1421.2014.
Kawajiri, K., & Fujii-Kuriyama, Y. (2007). Cytochrome P450 gene regulation and physiological functions mediated by the aryl hydrocarbon receptor. Archives of biochemistry and biophysics, 464(2), 207-212.
Keating, G. M., & Santoro, A. (2009). Sorafenib: a review of its use in advanced hepatocellular carcinoma. Drugs, 69(2), 223-240. doi:10.2165/00003495-200969020-00006
Nakamura, M., Tomita, A., Nakatani, H., Matsuda, T., & Nadano, D. (2006). Antioxidant and antibacterial genes are upregulated in early involution of the mouse mammary gland: sharp increase of ceruloplasmin and lactoferrin in accumulating breast milk. DNA and cell biology, 25(9), 491-500.
Plotnikova, M. A., Klotchenko, S. A., & Vasin, A. V. (2016). Development of a multiplex quantitative PCR assay for the analysis of human cytokine gene expression in influenza A virus-infected cells. Journal of immunological methods, 430, 51-55.
Shimada, T. (2006). Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons. Drug metabolism and pharmacokinetics, 21(4), 257-276.
Su, J.-G. J., Huang, M.-C., & Chen, F.-Y. (2011). 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin’s suppression of 1-nitropyrene-induced p53 expression is mediated by cytochrome P450 1A1. Chemical research in toxicology, 24(12), 2167-2175.
Wang, S.-H., Liang, C.-T., Liu, Y.-W., Huang, M.-C., Huang, S.-C., Hong, W.-F., & Su, J.-G. J. (2009). Crosstalk between activated forms of the aryl hydrocarbon receptor and glucocorticoid receptor. Toxicology, 262(2), 87-97.
Wilhelm, S. M., Adnane, L., Newell, P., Villanueva, A., Llovet, J. M., & Lynch, M. (2008). Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Molecular cancer therapeutics, 7(10), 3129-3140.
Zhang, Y., Xu, D., Wang, X., Lu, M., Gao, B., & Qiao, X. (2014). Screening of kinase inhibitors targeting BRAF for regulating autophagy based on kinase pathways. Molecular medicine reports, 9(1), 83-90.
Zheng, Y., Jia, L., Liu, P., Yang, D., Hu, W., Chen, S., . . . Ge, L. (2016). Insight into the maintenance of odontogenic potential in mouse dental mesenchymal cells based on transcriptomic analysis. PeerJ, 4, e1684.