鯽魚膽(Pluchea indica (L.) Less.)為一多年生的亞灌木菊科植物，其主要分布位置在東南亞及印度的沿岸濱海地帶，因為具有多種傳統的藥用性質而聞名。本篇論文研究關於已烷分層的鯽魚膽根部萃取物，對人類惡性腦瘤細胞的抗癌效果，以及其所透過的機制。實驗使用的細胞株為惡性腦瘤細胞U87，腦瘤細胞的生長明顯的隨著鯽魚膽萃取物的使用濃度提高而被抑制住，顯示其有抑制癌細胞生長的功效。使用Propidium iodide染色並用流式細胞儀分析細胞週期分布的結果顯示出細胞主要停滯在G1期。採用Annexin V與propidium iodide進行雙重染色以觀察凋亡現象發生的結果，顯示沒有明顯凋亡現象的發生。而以螢光顯微鏡的觀察結果能發現到酸性囊狀胞器(AVO)在實驗組的腦癌細胞中形成，同時也觀察到了微管相關蛋白(LC3-II)、磷酸化JNK、以及磷酸化p38的明顯增表現，證實了自噬現象的發生。綜合以上結果，我們的實驗顯示出已烷分層的鯽魚膽根部萃取物，有著抑制腦瘤細胞的功效，且在過程中對細胞引發自噬現象。

Pluchea indica (L.) Less. is a perennial plant known for its versatile uses in traditional oriental medicine. In our previous study, we demonstrate the in vitro anti-cancer activity of Pluchea indica root extract (PIRE) through induction of apoptotic cell death. However, one of the fractional extract, the hexane fraction of PIRE, despite showing the strongest potential of anti-cancer activity, the mechanism of cell death it induced remain unknown. In this study, we investigated the potential anticancer effect of the hexane fraction of Pluchea indica root extract (PIRE) on glioblastoma multiforme U87 cell lin. PIRE significantly suppressed the growth of cells in a dose-dependent manner. Propidium iodide staining result for cell cycle distributi28on shows cell cycle arrest at G1 phase. The result of Annexin V and propidium iodide staining following analysis with flow cytometry shows no significant induction of apoptosis. Fluorescent microscopy observation revealed the formations of acidic vesicular organelle (AVO) in PIRE-treated group. Expression of microtubule-associated protein light chain 3-II (LC3-II) protein also drastically increased, as well as the phosphorylation of JNK and p38 in response to PIRE treatment, suggesting the occurrence of autophagy. Our results provide evidence that through the induction of autophagic cell death, the hexane fraction of PIRE has the potential in its anti-brain cancer activity.

論文審定書……………………………………….…………………...i
致謝…………………………………………………………………...ii
摘要…………………………………………………………......……iii
Abstract…..……………………………………….……………..…..iv
目錄………………………………………………………………...…v
壹、前言……………………………………………………....……..1
一、鯽魚膽……………………………………………...……..….1
二、神經母細胞瘤……………………………………...…………2
三、程序化細胞死亡…………………………………...…...……3
四、細胞凋亡...………………………………………...…………3
五、自噬現象…………………………………………......…..….4
貳、實驗目的………………………………………………...…...…6
參、材料與方法……………………………………………...………7
一、鯽魚膽的來源與萃取方法………..………….……………...7
二、鯽魚膽根部萃取物的基本植物成分分析…….........…….…7
三、細胞的培養……...……………………………...……….…..9
四、細胞活性分析…………………………………..…….…..…11
五、細胞週期分析……………………………………..….…..…11
六、細胞凋亡分析………………………………………..…...…12
七、螢光顯微鏡觀察…………………………………….……....13
八、西方式墨點法………………………………………..…..….14
肆、結果………………………………….…………….………...…16
一、鯽魚膽根部萃取物的初步成分分析………………....……16
二、鯽魚膽根部萃取物抑制腦癌細胞活性…………….…..... 16
三、鯽魚膽根部萃取物抑制腦癌細胞的生長於G1時期……...17
四、鯽魚膽根部萃取物引發的細胞死亡並非凋亡現象…….…18
五、鯽魚膽根部萃取物促使腦癌細胞產生自噬體………….…18
六、鯽魚膽根部萃取物引發腦癌細胞產生自噬現象…….…...19
伍、討論……………………………………………………………..21
陸、參考文獻……………………………………………………..…26
圖表…………...……………………………………………..………30
附錄…………………………………………………………………..38

(1) 《中國高等植物資料庫全庫》中國科學院微生物研究所. 2009.
(2) Pacific Island Ecosystems at Risk (PIER) http://www.hear.org/pier/
(3) 李甯漢、劉啟文. 1986. 欒樨 Pluchea indica (L.) Less. 常見中草藥第五冊
(4) Sen T, Dhara AK, Bhattacharjee S, Pal S, Nag Chaudhuri AK. Antioxidant activity of the methanol fraction of Pluchea indica root extract. Phytother Res. 2002.
(5) Mahapatra PK, Chaudhuri AK. Neuropharmacological Studies on Pluchea indica. Planta Med. 1986.
(6) Thongpraditchote S, Matsumoto K, Temsiririrkkul R, Tohda M, Murakami Y, Watanabe H. Neuropharmacological actions of Pluchea indica Less root extract in socially isolated mice. Biol Pharm Bull. 1996.
(7) Biswas R, Dutta PK, Achari B, Bandyopadhyay D, Mishra M, Pramanik KC, Chatterjee TK. Isolation of pure compound R/J/3 from Pluchea indica (L.) Less. and its anti-amoebic activities against Entamoeba histolytica. Phytomedicine. 2007.
(8) Sen T, Nag Chaudhuri AK. Antiinflammatory evaluation of a Pluchea indica root extract. J Ethnopharmacol. 1991.
(9) Sen T, Ghosh TK, Chaudhuri AK. Studies on the mechanism of anti-inflammatory and anti-ulcer activity of Pluchea indica--probable involvement of 5-lipooxygenase pathway. Life Sci. 1993.
(10) Mohamad S, Zin NM, Wahab HA, Ibrahim P, Sulaiman SF, Zahariluddin AS, Noor SS. Antituberculosis potential of some ethnobotanically selected malaysian plants. J Ethnopharmacol. 2011.
(11) Locher CP, Witvrouw M, De Béthune MP, Burch MT, Mower HF, Davis H, Lasure A, Pauwels R, De Clercq E, Vlietinck AJ. Antiviral activity of Hawaiian medicinal plants against human immunodeficiency Virus Type-1 (HIV-1). Phytomedicine. 1996.
(12) Gomes A, Saha A, Chatterjee I, Chakravarty AK. Viper and cobra venom neutralization by beta-sitosterol and stigmasterol isolated from the root extract of Pluchea indica Less. (Asteraceae). Phytomedicine. 2007 Sep;14(9):637-43. Epub 2007.
(13) Cho JJ, Cho CL, Kao CL, Chen CM, Tseng CN, Lee YZ, Liao LJ, Hong YR. Crude aqueous extracts of Pluchea indica (L.) Less. inhibit proliferation and migration of cancer cells through induction of p53-dependent cell death. BMC Complement Altern Med. 2012.
(14) Kao CL, Cho J, Lee YZ, Cheng YB, Chien CY, Hwang CF, Hong YR, Tseng CN, Cho CL. Ethanolic Extracts of Pluchea indica Induce Apoptosis and Antiproliferation Effects in Human Nasopharyngeal Carcinoma Cells. Molecules. 2015.
(15) Ellis HP, Greenslade M, Powell B, Spiteri I, Sottoriva A, Kurian KM. Current Challenges in Glioblastoma: Intratumour Heterogeneity, Residual Disease, and Models to Predict Disease Recurrence. Front Oncol. 2015.
(16) Johnson DR, O'Neill BP. Glioblastoma survival in the United States before and during the temozolomide era. J Neurooncol. 2012.
(17) National Comprehensive Cancer Network (nccn) NCCN Clinical Practice Guidelines in Oncology: Central Nervous System Cancers. Ver ns Fort Washington PA: NCCN; 2014.
(18) Temozolomide – birth of a blockbuster. Clare Sansom for Chemistry World. July 2009.
(19) Weller M, Cloughesy T, Perry JR, Wick W. Standards of care for treatment of recurrent glioblastoma—are we there yet? Neuro Oncol. 2013.
(20) Torok JA, Wegner RE, Mintz AH, Heron DE, Burton SA. Re-irradiation with radiosurgery for recurrent glioblastoma multiforme. Technol Cancer Res Treat. 2011.
(21) Friedman HS, Prados MD, Wen PY, Mikkelsen T, Schiff D, Abrey LE, Yung WK, Paleologos N, Nicholas MK, Jensen R, Vredenburgh J, Huang J, Zheng M, Cloughesy T. Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. J Clin Oncol. 2009.
(22) Batchelor TT, Mulholland P, Neyns B, Nabors LB, Campone M, Wick A, Mason W, Mikkelsen T, Phuphanich S, Ashby LS, Degroot J, Gattamaneni R, Cher L, Rosenthal M, Payer F, Jürgensmeier JM, Jain RK, Sorensen AG, Xu J, Liu Q, van den Bent M. Phase III randomized trial comparing the efficacy of cediranib as monotherapy, and in combination with lomustine, versus lomustine alone in patients with recurrent glioblastoma. J Clin Oncol. 2013.
(23) Mellinghoff IK, Wang MY, Vivanco I, Haas-Kogan DA, Zhu S, Dia EQ, Lu KV, Yoshimoto K, Huang JH, Chute DJ, Riggs BL, Horvath S, Liau LM, Cavenee WK, Rao PN, Beroukhim R, Peck TC, Lee JC, Sellers WR, Stokoe D, Prados M, Cloughesy TF, Sawyers CL, Mischel PS. Molecular determinants of the response of glioblastoma to egfr kinase inhibitors. N Engl J Med. 2005.
(24) Bregy A, Wong TM, Shah AH, Goldberg JM, Komotar RJ. Active immunotherapy using dendritic cells in the treatment of glioblastoma multiforme. Cancer Treat Rev. 2013.
(25) Mulpur BH, Nabors LB, Thompson RC, Olson JJ, LaRocca RV, Thompson Z, Egan KM. Complementary therapy and survival in glioblastoma. Neurooncol Pract. 2015.
(26) Levin GT, Greenwood KM, Singh F, Tsoi D, Newton RU. Exercise Improves Physical Function and Mental Health of Brain Cancer Survivors: Two Exploratory Case Studies. Integr Cancer Ther. 2015.
(27) Gillett J, Ientile C, Hiscock J, Plank A, Martin JM. Complementary and alternative medicine use in radiotherapy: what are patients using? J Altern Complement Med. 2012.
(28) Chrystal K, Allan S, Forgeson G, Isaacs R. The use of complementary/alternative medicine by cancer patients in a New Zealand regional cancer treatment centre. N Z Med J. 2003.
(29) Green, Douglas. Means To An End. New York: Cold Spring Harbor Laboratory Press. ISBN 978-0-87969-887-4. 2011.
(30) Sun Y, Peng ZL. Programmed cell death and cancer. Postgrad Med J. 2009.
(31) Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, Bao JK. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 2012.
(32) Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res. 2011.
(33) Gasco M, Shami S, Crook T. The p53 pathway in breast cancer. Breast Cancer Res. 2002.
(34) Rodrigues NR, Rowan A, Smith ME, Kerr IB, Bodmer WF, Gannon JV, Lane DP. p53 mutations in colorectal cancers. Proc Natl Acad Sci U S A. 1990.
(35) Morton JP, Timpson P, Karim SA, Ridgway RA, Athineos D, Doyle B, Jamieson NB, Oien KA, Lowy AM, Brunton VG, Frame MC, Evans TR, Sansom OJ. Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc Natl Acad Sci U S A. 2010.
(36) "Chapter 18 Apoptosis: Programmed Cell Death Eliminates Unwanted Cells". Molecular Biology of the Cell (textbook) (5th ed.).
(37) Yamabe K, Shimizu S, Ito T, Yoshioka Y, Nomura M, Narita M, Saito I, Kanegae Y, Matsuda H. Cancer gene therapy using a pro-apoptotic gene, caspase-3. Gene Ther. 1999.
(38) Jensen M, Engert A, Weissinger F, Knauf W, Kimby E, Poynton C, Oliff IA, Rummel MJ, Osterborg A. Phase I study of a novel pro-apoptotic drug R-etodolac in patients with B-cell chronic lymphocytic leukaemia. Invest New Drugs. 2008.
(39) Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994.
(40) Kobayashi S. Choose Delicately and Reuse Adequately: The Newly Revealed Process of Autophagy. Biol Pharm Bull. 2015.
(41) Lu Z, Luo RZ, Lu Y, Zhang X, Yu Q, Khare S, Kondo S, Kondo Y, Yu Y, Mills GB, Liao WS, Bast RC Jr. The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells. J Clin Invest. 2008.
(42) Yang ZJ, Chee CE, Huang S, Sinicrope FA. The Role of Autophagy in Cancer: Therapeutic Implications. Mol Cancer Ther. 2011.
(43) Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi Y, Gélinas C, Fan Y, Nelson DA, Jin S, White E. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006.
(44) Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, Thomas-Tikhonenko A, Thompson CB. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007.
(45) Ding WX, Ni HM, Gao W, Chen X, Kang JH, Stolz DB, Liu J, Yin XM. Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy. Mol Cancer Ther. 2009.
(46) Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen EL, Mizushima N, Ohsumi Y, Cattoretti G, Levine B. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003.
(47) White E, DiPaola RS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res. 2009.
(48) Patel AS, Lin L, Geyer A, Haspel JA, An CH, Cao J, Rosas IO, Morse D. Autophagy in idiopathic pulmonary fibrosis. PLoS One. 2012.
(49) Dynamics of Cell Fate Decision Mediated by the Interplay of Autophagy and Apoptosis in Cancer Cells. 2015.
(50) Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther. 2011.
(51) Mizushima N. Autophagy: process and function. Genes Dev. 2007.
(52) Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol. 2009.
(53) Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I. Induction of Autophagic Cell Death in Malignant Glioma Cells by Arsenic Trioxide. Cancer Res. 2003.
(54) Harborne JB. Phytochemical methods: a guide to modern techniques of plant analysis. 3. London, New York: Chapman and Hall; 1998.
(55) Butler LG, Price mL, Brotherton JE. Vanillin assay for proanthocyanidins (condensed tannins): modification of the solvent for estimation of the degree of polymerization. J Agric Food Chem. 1982.
(56) Wang Y, Wu J, Lin B, Li X, Zhang H, Ding H, Chen X, Lan L, Luo H. Galangin suppresses HepG2 cell proliferation by activating the TGF-β receptor/Smad pathway. Toxicology. 2014.