頭頸鱗狀細胞癌是全世界第六大常見癌症，包括上呼吸道、口腔、咽、喉，每年診斷出超過60萬新發病例。口腔癌在台灣男性中發生率排名第五名，死亡率排名第四名。根據實驗室之前的研究結果，由粗毛金星蕨( Thelypteris torresiana )所分離純化的黃酮類化合物AN30，在口腔癌細胞( Ca9-22 ) 具有選擇性毒殺及誘導口腔癌細胞自噬作用的產生。因為在其他癌症中，已經有作AN30對其癌症機制試驗。在本篇研究中，進一步研究AN30對口腔癌細胞誘導細胞凋亡的機制。以流式細胞儀染Annexin V/7AAD，探討AN30對誘導口腔癌細胞細胞凋亡的影響，發現隨著劑量增加誘導細胞凋亡的情形也增加，接著檢測口腔癌細胞內ROS的濃度，發現隨著AN30的劑量增加，ROS的濃度也被誘導增加，接著加入ROS的抑制劑NAC後，發現抑制ROS的產生後，口腔癌細胞細胞凋亡的現象也被抑制住了，染細胞凋亡蛋白pan-caspase也有相同的表現結果。可以證實AN30是透過ROS去調節細胞走向細胞凋亡。以生物資訊的方式去探討AN30對於癌細胞中基因表現的影響性，發現ROS會去誘導gene FoxM1、TXNRD1表現量上升、自噬作用會去誘導gene DUSP1表現量下量、細胞凋亡會去誘導gene MAP2K6、MYLK表現量下降。綜合之前實驗室對細胞自噬的結果，結合流式細胞儀及生物資訊的結果、誘導細胞自噬及細胞凋亡與基因之間的關係。結果將對AN30在治療癌症的應用提供進一步的基礎。

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide,including the oral cavity, pharynx, and larynx ,with more than 600,000 new cases reported each year. The incidence of oral cancer ranked fifth, and the mortality rate ranked fourth of top 10 cancer in Taiwan. Accroding to previous study, the extract of the whole plant Thelypteris torresiana (Gaud.) has the select killing effect and induce autophagy on oral cancer cell in vitro.AN30 has been tested in many cancers except in oral cancer. In this study, we tested and summarized the effects concerning about ROS, apoptosis and autophagy of AN30 treatment on oral cancer cells. The results show levels of ROS and apoptosis are raised in dose dependent mode. After NAC treatment, the level of apoptosis is decreased in annexin V and pan-caspase flow cytometry assay. We also analyze gene expression of cancer cells under AN30 treatment with bioinformation and microarray data, and found FoxM1, TXNRD1 expressions are induced by ROS, DUSP1 expression decreases under autophagy, and MAP2K6, MYLK expression decreases under apoptosis. These results will provide bases for further application of AN30 on cancer therapy.

學位論文審定書 i
致謝 ii
摘要 iii
ABSTRACT iv
英文縮寫表 v
第一章 、序言 (Introduction) 1
1.1口腔癌背景介紹 1
1.2口腔癌成因 1
1.3口腔癌症狀、分期 2
1.4口腔癌治療 3
1.5口腔癌五年存活率 5
1.6口腔癌前病變 5
1.7藥物介紹AN30 6
1.7.1 AN30的發現 6
1.7.2 AN30對autophagy跟apoptosis 7
1.7.3 AN30 跟ROS相關研究 8
1.8 細胞計畫性死亡 ( Programmed Cell Death, PCD ) 8
1.9自噬作用( Autophagy ) 8
1.9.1 自噬作用的功能 9
1.9.2 自噬作用路徑 ( Autophagy pathway ) 9
1.9.3 追蹤自噬作用 10
1.9.4 自噬作用抑制劑合併口腔癌治療藥物 11
1.9.5 自噬作用調控路徑 - Akt / mTOR路徑 11
1.10 細胞凋亡 ( Apoptosis ) 12
1.10.1 細胞凋亡傳遞訊息路徑 12
1.11 活性氧 ( Reactive Oxidative Species, ROS ) 13
1.11.1 活性氧( Reactive Oxygen Species, ROS ) 和細胞凋亡基因的關係 14
第二章 、實驗目的 ( Specific Aims ) 15
第三章 、實驗流程 ( Experiment Flowchart ) 15
第四章 、實驗方法 16
4.1細胞株 16
4.2藥物 16
4.3藥品與試劑 16
4.4.細胞株培養液配置 17
4.4.1 Ca9-22細胞株培養液配置 17
4.4.2 Phosphate-Buffered Saline ( PBS ) Buffer 配方 17
4.4.3 Trypsin-EDTA 配方 17
4.4.4 細胞凍存液配方 17
4.5. 細胞繼代培養 18
4.5.1 解凍細胞 18
4.5.2 繼代細胞 18
4.5.3 冷凍保存細胞 19
4.6 細胞存活率試驗 MTT assay 19
4.7 Acridine orange染色 20
4.7.1步驟 20
4.8 西方墨點法 ( Western blot ) 20
4.8.1 口腔癌細胞株Ca9-22蛋白萃取 20
4.8.2 蛋白定量 21
4.8.3 聚丙烯醯胺膠體電泳SDS-PAGE ( Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis ) 22
4.8.3.1溶液配方 22
4.8.3.2 實驗流程 23
4.9 流式細胞儀試驗 ( Flow cytometry ) 25
4.10 基因微陣列 ( Gene microarray ) 25
4.10.1 抽RNA 25
4.10.2 RNA濃度定量 26
第五章 、結果與討論 27
5.1 AN30對口腔細胞Ca922誘導細胞凋亡的分析 27
5.2 AN30對口腔細胞Ca9-22誘導細胞自噬的分析 27
5.3 AN30對口腔細胞Ca9-22誘導ROS及相關基因之研究 28
5.4 AN30誘導細胞自噬及細胞凋亡基因表現及機制研究 29
第六章 、參考文獻 31
第七章、實驗結果圖 40
圖7.1 (A) Ca9-22 cell treated AN30 for 24 hr stain with AnnexinV/7AAD 40
圖7.1 (B) Annexin V/7AAD定量圖 41
圖7.1 (C) CAL27 cell treated AN30 for 24 hr stain with Annexin V/7AAD 42
圖7.1 (D) AnnexinV/7AAD 定量圖 43
圖7.2 (A) Ca9-22 ROS detection w/wo NAC 44
圖7.2 (B) Ca9-22 ROS detection定量圖 44
圖7.3 (A) Ca9-22 treated with AN30/NAC, Annexin V/7AAD flow chart 45
圖7.3 (B) Ca9-22 treated AN30/NAC stain定量圖 45
圖7.4 (A) Ca9-22 treated AN30 and NAC, Pan-caspase flow chart 46
圖7.4 (B) Ca9-22 treated AN30 and NAC, Pan-caspase定量圖 46
表7.5、Flavonoids及AN30 derivatives結構 47
表7.6、AN30相關論文研究整理表 49
表7.7、AN30衍生物相關研究整理表 50
第八章、附錄 53
圖8.1 (A) AN30 induce Ca9-22 Autophagy with Acridine orange stain 53
圖8.1 (B) Ca9-22 induce Ca9-22 Autophagy with Acridine orange stain定量圖 53
圖8.2 氧化壓力在細胞內不同濃度所代表的意義 54
表8.3、以gene chip分析藥物影響Hela細胞內基因表現研究整理 55
表8.4、六種不同癌症中氧化壓力相關基因與預後差的關係(Array圖) 58
表8.5、六個癌症中表現量高的氧化壓力基因 59
8.6 pBABE – puro – mCherry – EGFP – LC3B 61
8.6.2使用 VIOGENE研發的Mini-MTM Plasmid DNA Extraction System的套組萃取質體 62
8.6.3轉染 ( Transfection ) 63

Allen, R. G. and M. Tresini (2000). "Oxidative stress and gene regulation." Free Radic Biol Med 28(3): 463-499.
Andersen, J. K. (2004). "Oxidative stress in neurodegeneration: cause or consequence?" Nat Med 10 Suppl: S18-25.
Ashford, T. P. and K. R. Porter (1962). "CYTOPLASMIC COMPONENTS IN HEPATIC CELL LYSOSOMES." The Journal of Cell Biology 12(1): 198-202.
Bishayee, K., S. Ghosh, A. Mukherjee, R. Sadhukhan, J. Mondal and A. R. Khuda-Bukhsh (2013). "Quercetin induces cytochrome-c release and ROS accumulation to promote apoptosis and arrest the cell cycle in G2/M, in cervical carcinoma: signal cascade and drug-DNA interaction." Cell Prolif 46(2): 153-163.
Caccamo, A., M. A. Maldonado, S. Majumder, D. X. Medina, W. Holbein, A. Magri and S. Oddo (2011). "Naturally secreted amyloid-beta increases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediated mechanism." J Biol Chem 286(11): 8924-8932.
Chang, H. L., Y. C. Wu, J. H. Su, Y. T. Yeh and S. S. Yuan (2008). "Protoapigenone, a novel flavonoid, induces apoptosis in human prostate cancer cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase 1/2." J Pharmacol Exp Ther 325(3): 841-849.
Chen, H. M., F. R. Chang, Y. C. Hsieh, Y. J. Cheng, K. C. Hsieh, L. M. Tsai, A. S. Lin, Y. C. Wu and S. S. Yuan (2011). "A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species." Free Radic Biol Med 50(9): 1151-1162.
Chen, W. Y., Y. A. Hsieh, C. I. Tsai, Y. F. Kang, F. R. Chang, Y. C. Wu and C. C. Wu (2011). "Protoapigenone, a natural derivative of apigenin, induces mitogen-activated protein kinase-dependent apoptosis in human breast cancer cells associated with induction of oxidative stress and inhibition of glutathione S-transferase pi." Invest New Drugs 29(6): 1347-1359.
Chen, Y. J., Y. J. Cheng, A. C. Hung, Y. C. Wu, M. F. Hou, Y. C. Tyan and S. S. Yuan (2013). "The synthetic flavonoid WYC02-9 inhibits cervical cancer cell migration/invasion and angiogenesis via MAPK14 signaling." Gynecol Oncol 131(3): 734-743.
Chiu, C. C., H. W. Chang, D. W. Chuang, F. R. Chang, Y. C. Chang, Y. S. Cheng, M. T. Tsai, W. Y. Chen, S. S. Lee, C. K. Wang, J. Y. Chen, H. M. Wang, C. C. Chen, Y. C. Liu and Y. C. Wu (2009). "Fern plant-derived protoapigenone leads to DNA damage, apoptosis, and G(2)/m arrest in lung cancer cell line H1299." DNA Cell Biol 28(10): 501-506.
Cohen, G. M., X. M. Sun, H. Fearnhead, M. MacFarlane, D. G. Brown, R. T. Snowden and D. Dinsdale (1994). "Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes." J Immunol 153(2): 507-516.
Cohen, J. J. and R. C. Duke (1984). "Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death." J Immunol 132(1): 38-42.
Elmore, S. (2007). "Apoptosis: a review of programmed cell death." Toxicol Pathol 35(4): 495-516.
Gump, J. M. and A. Thorburn (2011). "Autophagy and apoptosis: what is the connection?" Trends Cell Biol 21(7): 387-392.
Haigis, M. C. and B. A. Yankner (2010). "The aging stress response." Mol Cell 40(2): 333-344.
Hansen, T. E. and T. Johansen (2011). "Following autophagy step by step." BMC Biol 9: 39.
He, C. and D. J. Klionsky (2009). "Regulation Mechanisms and Signaling Pathways of Autophagy." Annual review of genetics 43: 67-93.
Hundeshagen, P., A. Hamacher-Brady, R. Eils and N. R. Brady (2011). "Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy." BMC Biol 9: 38.
Hunyadi, A., A. Martins, B. Danko, D.-W. Chuang, P. Trouillas, F.-R. Chang, Y.-C. Wu and G. Falkay (2013). "Discovery of the first non-planar flavonoid that can strongly inhibit xanthine oxidase: protoapigenone 1′-O-propargyl ether." Tetrahedron Letters 54(48): 6529-6532.
Jiang, K., W. Wang, X. Jin, Z. Wang, Z. Ji and G. Meng (2015). "Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells." Oncol Rep 33(6): 2711-2718.
Kabuta, T., Y. Suzuki and K. Wada (2006). "Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-superoxide dismutase proteins by macroautophagy and the proteasome." J Biol Chem 281(41): 30524-30533.
Kamata, H. and H. Hirata (1999). "Redox regulation of cellular signalling." Cell Signal 11(1): 1-14.
Kerr, J. F., A. H. Wyllie and A. R. Currie (1972). "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics." Br J Cancer 26(4): 239-257.
Klionsky, D. J., Z. Elazar, P. O. Seglen and D. C. Rubinsztein (2008). "Does bafilomycin A1 block the fusion of autophagosomes with lysosomes?" Autophagy 4(7): 849-850.
Kohler, C., S. Orrenius and B. Zhivotovsky (2002). "Evaluation of caspase activity in apoptotic cells." J Immunol Methods 265(1-2): 97-110.
Lander, H. M. (1997). "An essential role for free radicals and derived species in signal transduction." Faseb j 11(2): 118-124.
Leone, A., M. S. Roca, C. Ciardiello, S. Costantini and A. Budillon (2017). "Oxidative Stress Gene Expression Profile Correlates with Cancer Patient Poor Prognosis: Identification of Crucial Pathways Might Select Novel Therapeutic Approaches." Oxidative Medicine and Cellular Longevity 2017: 18.
Lillo, C., U. S. Lea and P. Ruoff (2008). "Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway." Plant Cell Environ 31(5): 587-601.
Lin, A. S., F. R. Chang, C. C. Wu, C. C. Liaw and Y. C. Wu (2005). "New cytotoxic flavonoids from Thelypteris torresiana." Planta Med 71(9): 867-870.
Liu, Z., Z. Liu, X. Zhang, P. Xue and H. Zhang (2014). "RY10-4 suppressed metastasis of MDA-MB-231 by stabilizing ECM and E-cadherin." Biomed Pharmacother 68(4): 439-445.
Maiuri, M. C., E. Zalckvar, A. Kimchi and G. Kroemer (2007). "Self-eating and self-killing: crosstalk between autophagy and apoptosis." Nat Rev Mol Cell Biol 8(9): 741-752.
Mizushima, N. (2007). "Autophagy: process and function." Genes Dev 21(22): 2861-2873.
Mohapatra, S., B. Chu, X. Zhao and W. J. Pledger (2005). "Accumulation of p53 and reductions in XIAP abundance promote the apoptosis of prostate cancer cells." Cancer Res 65(17): 7717-7723.
Nordberg, J. and E. S. Arner (2001). "Reactive oxygen species, antioxidants, and the mammalian thioredoxin system." Free Radic Biol Med 31(11): 1287-1312.
Poór, M., Y. Li, S. Kunsági-Máté, Z. Varga, A. Hunyadi, B. Dankó, F.-R. Chang, Y.-C. Wu and T. Kőszegi (2013). "Protoapigenone derivatives: Albumin binding properties and effects on HepG2 cells." Journal of Photochemistry and Photobiology B: Biology 124: 20-26.
Psahoulia, F. H., S. Moumtzi, M. L. Roberts, T. Sasazuki, S. Shirasawa and A. Pintzas (2007). "Quercetin mediates preferential degradation of oncogenic Ras and causes autophagy in Ha-RAS-transformed human colon cells." Carcinogenesis 28(5): 1021-1031.
Ravikumar, B., C. Vacher, Z. Berger, J. E. Davies, S. Luo, L. G. Oroz, F. Scaravilli, D. F. Easton, R. Duden, C. J. O'Kane and D. C. Rubinsztein (2004). "Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease." Nat Genet 36(6): 585-595.
S. Zappavigna, A., G. V. Luce, S. N. Merola and M. C. Facchini (2013). "Autophagic cell death: A new frontier in cancer research." Advances in Bioscience and Biotechnology 4: 250-262.
Shibutani, S. T. and T. Yoshimori (2014). "A current perspective of autophagosome biogenesis." Cell Res 24(1): 58-68.
Shimizu, S., T. Yoshida, M. Tsujioka and S. Arakawa (2014). "Autophagic cell death and cancer." Int J Mol Sci 15(2): 3145-3153.
Shintani, T. and D. J. Klionsky (2004). "Autophagy in health and disease: a double-edged sword." Science 306(5698): 990-995.
Singh, R. P., K. Raina, G. Sharma and R. Agarwal (2008). "Silibinin Inhibits Established Prostate Tumor Growth, Progression, Invasion and Metastasis, and Suppresses Tumor Angiogenesis and EMT in TRAMP Mice." Clinical cancer research : an official journal of the American Association for Cancer Research 14(23): 7773-7780.
Su, F., S. Zhu, J. Ruan, Y. Muftuoglu, L. Zhang and Q. Yuan (2016). "Combination therapy of RY10-4 with the γ-secretase inhibitor DAPT shows promise in treating HER2-amplified breast cancer." Oncotarget 7(4): 4142-4154.
Takeuchi, H., Y. Kondo, K. Fujiwara, T. Kanzawa, H. Aoki, G. B. Mills and S. Kondo (2005). "Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors." Cancer Res 65(8): 3336-3346.
Tan, S., Y. Sagara, Y. Liu, P. Maher and D. Schubert (1998). "The regulation of reactive oxygen species production during programmed cell death." J Cell Biol 141(6): 1423-1432.
Trachootham, D., J. Alexandre and P. Huang (2009). "Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?" Nat Rev Drug Discov 8(7): 579-591.
Turrens, J. F. (2003). "Mitochondrial formation of reactive oxygen species." J Physiol 552(Pt 2): 335-344.
Vidya Priyadarsini, R., R. Senthil Murugan, S. Maitreyi, K. Ramalingam, D. Karunagaran and S. Nagini (2010). "The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-kappaB inhibition." Eur J Pharmacol 649(1-3): 84-91.
Wang, H. C., A. Y. Lee, W. C. Chou, C. C. Wu, C. N. Tseng, K. Y. Liu, W. L. Lin, F. R. Chang, D. W. Chuang, A. Hunyadi and Y. C. Wu (2012). "Inhibition of ATR-dependent signaling by protoapigenone and its derivative sensitizes cancer cells to interstrand cross-link-generating agents in vitro and in vivo." Mol Cancer Ther 11(7): 1443-1453.
Wang, J., J.-Y. Zhou, D. Kho, J. J. Reiners and G. S. Wu (2016). "Role for DUSP1 (dual-specificity protein phosphatase 1) in the regulation of autophagy." Autophagy 12(10): 1791-1803.
Wang, Q., L. Xue, X. Zhang, S. Bu, X. Zhu and D. Lai (2014). "The machinery of macroautophagy." Cell Research 24: 0.
Wu, W., P. Liu and J. Li (2012). "Necroptosis: an emerging form of programmed cell death." Crit Rev Oncol Hematol 82(3): 249-258.
Wu, Y. T., H. L. Tan, G. Shui, C. Bauvy, Q. Huang, M. R. Wenk, C. N. Ong, P. Codogno and H. M. Shen (2010). "Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase." J Biol Chem 285(14): 10850-10861.
Xue, P., X. Yang, Y. Liu, C. Xiong and J. Ruan (2014). "A novel compound RY10-4 downregulates P-glycoprotein expression and reverses multidrug-resistant phenotype in human breast cancer MCF-7/ADR cells." Biomedicine & Pharmacotherapy 68(8): 1049-1056.
Xue, P., Y. Zhao, Y. Liu, Q. Yuan, C. Xiong and J. Ruan (2014). "A novel compound RY10-4 induces apoptosis and inhibits invasion via inhibiting STAT3 through ERK-, p38-dependent pathways in human lung adenocarcinoma A549 cells." Chem Biol Interact 209: 25-34.
Yang, Z. J., C. E. Chee, S. Huang and F. A. Sinicrope (2011). "The role of autophagy in cancer: therapeutic implications." Mol Cancer Ther 10(9): 1533-1541.
Yuan, Q., S. Cai, X. Zhang, Z. Liu, Z. Li, X. Luo, C. Xiong, J. Wang, J. Hu and J. Ruan (2012). "A new protoapigenone analog RY10-4 induces apoptosis and suppresses invasion through the PI3K/Akt pathway in human breast cancer." Cancer Lett 324(2): 210-220.
Zhang, X., Y. Wang, S. Han, H. Xiang, Y. Peng, Y. Wu, S. Pan, Y. Zhang and J. Ruan (2016). "RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis In Vitro and In Vivo." PLoS One 11(3): e0151679.
Zhang, X., H. Wei, Z. Liu, Q. Yuan, A. Wei, D. Shi, X. Yang and J. Ruan (2013). "A novel protoapigenone analog RY10-4 induces breast cancer MCF-7 cell death through autophagy via the Akt/mTOR pathway." Toxicol Appl Pharmacol 270(2): 122-128.
Zhang, X., Q. Xu, Z. Zhang, W. Cheng, W. Cao, C. Jiang, C. Han, J. Li and Z. Hua (2016). "Chloroquine enhanced the anticancer capacity of VNP20009 by inhibiting autophagy." Sci Rep 6: 29774.
Kopan, R., & Ilagan, M. X. G. (2009). The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism. Cell, 137(2), 216–233.
Kim, G. T., Lee, S. H., & Kim, Y. M. (2013). Quercetin Regulates Sestrin 2-AMPK-mTOR Signaling Pathway and Induces Apoptosis via Increased Intracellular ROS in HCT116 Colon Cancer Cells. Journal of Cancer Prevention, 18(3), 264–270.
WHO. World Cancer Report 2014.
袁行修 (2010) “ Protoapigenone 及其衍生物在婦女癌症上的應用 ” 國家衛生研究院整合性醫藥衛生科技研究計畫,1-38.
陽光社會福利基金會－認識口腔癌 http://teacher.sunshine.org.tw/oral/cure.asp
蘇連瓔、呂孟儒、鄭凱芸、陳羿瑾（2002）”希望之路面對口腔癌”台北：中華民國癌症希望協會。
葉坤輝、張獻崑、許派州（2008）”希望之路面對化學治療”中華民國癌症希望 協會編印，1-58。
成佳憲、李百勛（2008）”希望之路面對放射線治療”中華民國癌症希望協會編 印，1-30。
行政院衛生署國民健康局（2007）”化學治療自我照顧手冊”馬偕紀念醫院癌症 防治中心及護理部編印，1-12。
林佳蓉（2005）一位口腔癌患者接受口顎重建手術術後之護理經驗 嘉基護理， 6（2），21-30。
田必潔、賴惠玲、許文林、彭遠、李茹萍（2005）”護理衛教對頭頸部病人口腔 照顧認知健康信念及自我效能之成效”實證護理，3（3），215-224。