Fluoxetine (FLX)是一種常用的抗抑鬱藥用於口服和屬於選擇性血清素再攝取抑製劑(SSRI)，用於中重度抑鬱症的治療(MDD)、強迫症(OCD)和恐慌症。最近研究發現FLX在不同癌症對於細胞生長，細胞凋亡和細胞週期具有影響。因此，我們想了解FLX對於人類胰臟癌是否具有抗腫瘤效果。 我們發現當FLX 處理在人類胰臟癌細胞中，會顯著性的降低細胞增殖/存活和透過細胞週期分析發現FLX會增加Sub G1的表現。另外，西方墨點法分析結果發現增加PARP裂解和caspase 3活化，證實FLX是透過誘導細胞凋亡而改變蛋白質的表現。利用即時聚合酶連鎖反應分析，表明FLX降低cyclin D、cyclin B1、CDK1和CDK2的表現，並增加P27和p19的表現。接著，我們利用in vivo實驗將FLX治療於胰臟癌的基因轉殖鼠，觀察到FLX在PDAC腫瘤誘導細胞凋亡和抑制腫瘤生長。免疫組織化學染色的結果顯示，FLX抑制表皮生長因子受體(EGFR)和Notch-1和累積P27的表現。最後，我們將FLX和抗癌藥物合併使用發現協同抑制腫瘤細胞生長。因此，我們建議，FLX介導的抗腫瘤效果可能是改變腫瘤細胞增殖和細胞凋亡進而使胰臟腫瘤進展受到抑制。

Fluoxetine (FLX) is a commonly antidepressant for oral administration and belonging to the selective serotonin reuptake inhibitors (SSRI), which is used for the treatment of Major Depressive Disorder (MDD), Obsessive Compulsive Disorder (OCD) and panic disorder. Recently has been reported that fluoxetine affects on the cell growth, apoptosis and cell cycle in various types of cancer. Hence, we want to investigate whether FLX has anti-tumor effects on human pancreatic ductal adenocarcinoma. We found that of pancreatic cancer cells treatment with FLX significantly reduced cell proliferation/viability, and cell cycle analysis revealed that exposure of pancreatic cancer cells to FLX resulted in increased Sub G1 peak. Further, western blot analysis found that an increase of PARP cleavage and caspase 3 activity after FLX treatment that confirmed fluoxetine induced apoptosis at molecular level. Quantitative real-time PCR(q-PCR) analysis indicated that FLX down-regulate expression of cyclinD, cyclin B1,CDK1 and CDK2, and up-regulate expression of p27 and P19. Next, we confirm the in vivo efficiency of fluoxetine therapy on our GEM mouse model of human pancreatic cancer in vivo and we observed FLX induces apoptosis in mice PDAC tumors and inhibits tumor growth in vivo. In addition, immunohistochemistry stain (IHC) data showed that FLX suppress EGFR and Notch-1 and accumulates P27 protein expression. Finally, we combine the treatment of Fluoxetine and anticancer drugs to have synergistic effects on the inhibition cancer cell growth in vitro. Therefore, we propose that effects of Fluoxetine mediated antitumor effect may be contributing to the alterations of tumor cell proliferation and apoptosis thus resulting in the inhibition of PDAC progression.

論文審定書 .................................................................................................................... i
中文摘要 ....................................................................................................................... ii
Abstract ....................................................................................................................... iii
Abbreviations ............................................................................................................... v
Contents ....................................................................................................................... vi
Figures And Tables ..................................................................................................... vii
Introductions ................................................................................................................ 1
Material And Methods ................................................................................................. 6
Results ......................................................................................................................... 13
Discussion.................................................................................................................... 20
References ................................................................................................................... 39

Abdul, M., C. J. Logothetis and N. M. Hoosein (1995). "Growth-inhibitory effects of serotonin uptake inhibitors on human prostate carcinoma cell lines." J Urol 154(1): 247-250.
Arulanandam, R., M. Geletu, H. Feracci and L. Raptis (2010). "Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration." Exp Cell Res 316(5): 875-886.
Banerjee, S. K., M. N. Zoubine, M. Mullick, A. P. Weston, R. Cherian and D. R. Campbell (2000). "Tumor angiogenesis in chronic pancreatitis and pancreatic adenocarcinoma: impact of K-ras mutations." Pancreas 20(3): 248-255.
Bardeesy, N., A. J. Aguirre, G. C. Chu, K. H. Cheng, L. V. Lopez, A. F. Hezel, B. Feng, C. Brennan, R. Weissleder, U. Mahmood, D. Hanahan, M. S. Redston, L. Chin and R. A. Depinho (2006). "Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse." Proc Natl Acad Sci U S A 103(15): 5947-5952.
Carver, C. S., S. L. Johnson and J. Joormann (2008). "Serotonergic function, two-mode models of self-regulation, and vulnerability to depression: what depression has in common with impulsive aggression." Psychol Bull 134(6): 912-943.
Cools, R., A. C. Roberts and T. W. Robbins (2008). "Serotoninergic regulation of emotional and behavioural control processes." Trends Cogn Sci 12(1): 31-40.
Corcoran, R. B., G. Contino, V. Deshpande, A. Tzatsos, C. Conrad, C. H. Benes, D. E. Levy, J. Settleman, J. A. Engelman and N. Bardeesy (2011). "STAT3 plays a critical role in KRAS-induced pancreatic tumorigenesis." Cancer Res 71(14): 5020-5029.
De La, O. J., L. L. Emerson, J. L. Goodman, S. C. Froebe, B. E. Illum, A. B. Curtis and L. C. Murtaugh (2008). "Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia." Proc Natl Acad Sci U S A 105(48): 18907-18912.
Decker, G. A., M. J. Batheja, J. M. Collins, A. C. Silva, K. L. Mekeel, A. A. Moss, C. C. Nguyen, D. F. Lake and L. J. Miller (2010). "Risk factors for pancreatic adenocarcinoma and prospects for screening." Gastroenterol Hepatol (N Y) 6(4): 246-254.
Elmore, S. (2007). "Apoptosis: a review of programmed cell death." Toxicol Pathol 35(4): 495-516.
Hingorani, S. R., E. F. Petricoin, A. Maitra, V. Rajapakse, C. King, M. A. Jacobetz, S. Ross, T. P. Conrads, T. D. Veenstra, B. A. Hitt, Y. Kawaguchi, D. Johann, L. A. Liotta, H. C. Crawford, M. E. Putt, T. Jacks, C. V. Wright, R. H. Hruban, A. M. Lowy and D. A. Tuveson (2003). "Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse." Cancer Cell 4(6): 437-450.
Hingorani, S. R., L. Wang, A. S. Multani, C. Combs, T. B. Deramaudt, R. H. Hruban, A. K. Rustgi, S. Chang and D. A. Tuveson (2005). "Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice." Cancer Cell 7(5): 469-483.
Hong, S. M., J. Y. Park, R. H. Hruban and M. Goggins (2011). "Molecular signatures of pancreatic cancer." Arch Pathol Lab Med 135(6): 716-727.
Horvitz, H. R. (1999). "Genetic control of programmed cell death in the nematode Caenorhabditis elegans." Cancer Res 59(7 Suppl): 1701s-1706s.
Hristova, N. R., K. E. Tagscherer, A. Fassl, J. Kopitz and W. Roth (2013). "Notch1-dependent regulation of p27 determines cell fate in colorectal cancer." Int J Oncol 43(6): 1967-1975.
Hu, H., L. Zhou, A. Awadallah and W. Xin (2013). "Significance of Notch1-signaling pathway in human pancreatic development and carcinogenesis." Appl Immunohistochem Mol Morphol 21(3): 242-247.
Huang, S. (2007). "Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications." Clin Cancer Res 13(5): 1362-1366.
Hunter, T. and J. Pines (1994). "Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age." Cell 79(4): 573-582.
Iacobuzio-Donahue, C. A. (2012). "Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project." Gut 61(7): 1085-1094.
Jarpe, M. B., C. Widmann, C. Knall, T. K. Schlesinger, S. Gibson, T. Yujiri, G. R. Fanger, E. W. Gelfand and G. L. Johnson (1998). "Anti-apoptotic versus pro-apoptotic signal transduction: checkpoints and stop signs along the road to death." Oncogene 17(11 Reviews): 1475-1482.
Kannen, V., H. Hintzsche, D. L. Zanette, W. A. Silva, Jr., S. B. Garcia, A. M. Waaga-Gasser and H. Stopper (2012). "Antiproliferative effects of fluoxetine on colon cancer cells and in a colonic carcinogen mouse model." PLoS One 7(11): e50043.
Kannen, V., T. Marini, A. Turatti, M. C. Carvalho, M. L. Brandao, V. A. Jabor, P. S. Bonato, F. R. Ferreira, D. L. Zanette, W. A. Silva, Jr. and S. B. Garcia (2011). "Fluoxetine induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats." Toxicol Lett 204(2-3): 134-140.
Koh, S. J., J. M. Kim, I. K. Kim, N. Kim, H. C. Jung, I. S. Song and J. S. Kim (2011). "Fluoxetine inhibits NF-kappaB signaling in intestinal epithelial cells and ameliorates experimental colitis and colitis-associated colon cancer in mice
" Am J Physiol Gastrointest Liver Physiol 301(1): G9-19.
Komlosi, G., G. Molnar, M. Rozsa, S. Olah, P. Barzo and G. Tamas (2012). "Fluoxetine (prozac) and serotonin act on excitatory synaptic transmission to suppress single layer 2/3 pyramidal neuron-triggered cell assemblies in the human prefrontal cortex." J Neurosci 32(46): 16369-16378.
Lee, J., K. T. Jang, C. S. Ki, T. Lim, Y. S. Park, H. Y. Lim, D. W. Choi, W. K. Kang, K. Park and J. O. Park (2007). "Impact of epidermal growth factor receptor (EGFR) kinase mutations, EGFR gene amplifications, and KRAS mutations on survival of pancreatic adenocarcinoma." Cancer 109(8): 1561-1569.
Lohr, M., G. Kloppel, P. Maisonneuve, A. B. Lowenfels and J. Luttges (2005). "Frequency of K-ras mutations in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis." Neoplasia 7(1): 17-23.
Long, J., Y. Zhang, X. Yu, J. Yang, D. G. LeBrun, C. Chen, Q. Yao and M. Li (2011). "Overcoming drug resistance in pancreatic cancer." Expert Opin Ther Targets 15(7): 817-828.
Miyamoto, Y., A. Maitra, B. Ghosh, U. Zechner, P. Argani, C. A. Iacobuzio-Donahue, V. Sriuranpong, T. Iso, I. M. Meszoely, M. S. Wolfe, R. H. Hruban, D. W. Ball, R. M. Schmid and S. D. Leach (2003). "Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis." Cancer Cell 3(6): 565-576.
Mohammed, A., N. B. Janakiram, Q. Li, V. Madka, M. Ely, S. Lightfoot, H. Crawford, V. E. Steele and C. V. Rao (2010). "The epidermal growth factor receptor inhibitor gefitinib prevents the progression of pancreatic lesions to carcinoma in a conditional LSL-KrasG12D/+ transgenic mouse model." Cancer Prev Res (Phila) 3(11): 1417-1426.
Normanno, N., A. De Luca, C. Bianco, L. Strizzi, M. Mancino, M. R. Maiello, A. Carotenuto, G. De Feo, F. Caponigro and D. S. Salomon (2006). "Epidermal growth factor receptor (EGFR) signaling in cancer." Gene 366(1): 2-16.
Offield, M. F. (1996). "<PDX_PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.Development-1996-Offield-983-95.pdf>." Development 122: 983-995.
Peer, D., Y. Dekel, D. Melikhov and R. Margalit (2004). "Fluoxetine inhibits multidrug resistance extrusion pumps and enhances responses to chemotherapy in syngeneic and in human xenograft mouse tumor models." Cancer Res 64(20): 7562-7569.
Scarpa, A., P. Capelli, K. Mukai, G. Zamboni, T. Oda, C. Iacono and S. Hirohashi (1993). "Pancreatic adenocarcinomas frequently show p53 gene mutations." Am J Pathol 142(5): 1534-1543.
Shangary, S. and D. E. Johnson (2002). "Peptides derived from BH3 domains of Bcl-2 family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and Bax oligomerization, induction of cytochrome c release, and activation of cell death." Biochemistry 41(30): 9485-9495.
Sherr, C. J. and J. M. Roberts (1999). "CDK inhibitors: positive and negative regulators of G1-phase progression." Genes Dev 13(12): 1501-1512.
Shin, E. J. and M. I. Canto (2012). "Pancreatic cancer screening." Gastroenterol Clin North Am 41(1): 143-157.
Siegel, R., J. Ma, Z. Zou and A. Jemal (2014). "Cancer statistics, 2014." CA Cancer J Clin 64(1): 9-29.
Sordella, R., D. W. Bell, D. A. Haber and J. Settleman (2004). "Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways." Science 305(5687): 1163-1167.
Spano, J. P., R. Fagard, J. C. Soria, O. Rixe, D. Khayat and G. Milano (2005). "Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives." Ann Oncol 16(2): 189-194.
Stepulak, A., W. Rzeski, M. Sifringer, K. Brocke, A. Gratopp, K. Kupisz, L. Turski and C. Ikonomidou (2008). "Fluoxetine inhibits the extracellular signal regulated kinase pathway and suppresses growth of cancer cells." Cancer Biol Ther 7(10): 1685-1693.
Troiani, T., E. Martinelli, A. Capasso, F. Morgillo, M. Orditura, F. De Vita and F. Ciardiello (2012). "Targeting EGFR in pancreatic cancer treatment." Curr Drug Targets 13(6): 802-810.
Tutton, P. J. and D. H. Barkla (1982). "Influence of inhibitors of serotonin uptake on intestinal epithelium and colorectal carcinomas." Br J Cancer 46(2): 260-265.
Venkatasubbarao, K., L. Peterson, S. Zhao, P. Hill, L. Cao, Q. Zhou, S. T. Nawrocki and J. W. Freeman (2013). "Inhibiting signal transducer and activator of transcription-3 increases response to gemcitabine and delays progression of pancreatic cancer." Mol Cancer 12(1): 104.
Verbeek, B. S., S. S. Adriaansen-Slot, T. M. Vroom, T. Beckers and G. Rijksen (1998). "Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts." FEBS Lett 425(1): 145-150.
Wang, Z., Y. Zhang, Y. Li, S. Banerjee, J. Liao and F. H. Sarkar (2006). "Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells." Mol Cancer Ther 5(3): 483-493.
Wong, D. T., K. W. Perry and F. P. Bymaster (2005). "Case history: the discovery of fluoxetine hydrochloride (Prozac)." Nat Rev Drug Discov 4(9): 764-774.
Wong, H. H. and N. R. Lemoine (2009). "Pancreatic cancer: molecular pathogenesis and new therapeutic targets." Nat Rev Gastroenterol Hepatol 6(7): 412-422.
Zhou, T., J. Duan, Y. Wang, X. Chen, G. Zhou, R. Wang, L. Fu and F. Xu (2012). "Fluoxetine synergys with anticancer drugs to overcome multidrug resistance in breast cancer cells." Tumour Biol 33(5): 1299-1306.