褐藻醣膠 (Fucoidan) 為從褐藻 (Fucus vesiculosus or Laminaria japonica) 萃取之天然硫化多醣。目前褐藻醣膠已知有調節免疫與抗癌的功效。吉非替尼(Gefitinib)，治療非小細胞肺癌標靶藥物，干擾表皮生長因子受體酪氨酸激酶結構域與競爭ATP結合位點，進而影響細胞的生長及促進細胞的凋亡。順鉑(Cisplatin)，是一種含鉑的抗癌藥物，與DNA形成共價鍵的複體，干擾細胞的複製、轉錄，最終走向細胞死亡。而此篇研究，同時處理褐藻醣膠與兩種化療藥物，具有協同性抑制非小細胞肺癌細胞株生長，並促進誘導細胞凋亡路徑的活化。同時，發現褐藻醣膠結合吉非替尼會降低Rad51(DNA修復蛋白)的表現量。此外，我們也利用順序處理褐藻醣膠及化療藥物的實驗方式，發現前處理褐藻醣膠能夠增加後處理化療藥物的效果在非小細胞肺癌細胞株。並且利用離心分離裝置，分離出不同單元的褐藻醣膠，而在50~3 KD單元的褐藻醣膠抑制非小細胞肺癌細胞株的效果最好。綜合以上結果，小分子褐藻醣膠結合化療藥物合併策略，可以增強在非小細胞肺癌抗癌的效果。

Fucoidan, a sulfated polysaccharide, is extracted from brown algae, Fucus vesiculosus or Laminaria japonica. Recent studies show that fucoidan exhibits immunomodulatory and anti-cancer activities. In this study, we showed that the combination of fucoidan and therapeutic agents (include cisplatin and gefitinib) synergistically inhibited cell viability in non-small cell lung cancer. Specifically, we demonstrated that the effect of simultaneous treatment, induced apoptosis in the testing cells partially contributed from caspase 3 activation. Moreover, we found that combination of fucoidan and gefitinib induces down-regulation of Rad51 via ER stress in A549 cells, a DNA repair protein involves in lung tumorigenesis. In addition, we adopted the sequential therapy method and examined the effect of a sequential treatment of fucoidan and therapeutic agents in NSCLCs. We found that the sequential therapy (fucoidan followed by therapeutic agents) could enhanced inhibition of viability in NSCLCs. We further examine the efficacy of various molecular-weight of fucoidan, we utilized centrifugal filter tubes to separate the fractions of fucoidan. We found that low molecular-weight (50~3 KD) of fucoidan presented highly inhibition of cell viability, compared to original fucoidan. Our results suggested that small molecule of fucoidan combination with therapeutic agents might be a promising therapeutic strategy for the treatment of NSCLCs.

誌謝 i
Abbreviations ii
Abstract iii
中文摘要 iv
Content v
Introduction 1
Materials and methods 4
Cell culture 4
Reagents and antibodies 4
Western blot analysis 5
Cell viability assay 5
Cell cycle analysis 5
Apoptosis analysis 6
Statistical analysis 7
Results 8
Fucoidan and therapeutic agents inhibit cell viability in non-small cell lung cancer cells (NSCLCs) 8
Simultaneous treatment of fucoidan with cisplatin/gefitinib enhances induced-cytotoxicity in NSCLCs 9
Fucoidan enhances the effect of cisplatin/gefitinib with a sequential treatment in NSCLCs 10
Fucoidan enhances cisplatin/gefitinib -induced apoptosis in NSCLCs 11
Discussion 14
References 17
Figure legends 21
Figure 1. Fucoidan and therapeutic agents inhibit cell viability in non-small cell lung cancer cells (NSCLCs) 21
Figure 2. Fucoidan enhances cisplatin-induced cytotoxicity in NSCLCs 24
Figure 3. Pre-treatment of fucoidan enhances cisplatin-inhibited viability in NSCLCs 27
Figure 4. Fucoidan inhibits cell viability after cisplatin treatment in NSCLCs 29
Figure 5. Fucoidan enhances gefitinib-induced cytotoxicity in NSCLCs 31
Figure 6. Pre-treatment of fucoidan enhances gefitinib-inhibited viability in NSCLCs 34
Figure 7. Fucoidan inhibits cell viability after gefitinib treatment in NSCLCs 35
Figure 8. Fucoidan enhances cisplatin-induced apoptosis in NSCLCs 38
Figure 9. Fucoidan enhances gefitinib-induced apoptosis in NSCLCs 42
Figure 10. Fucoidan induces down-regulation of Rad51 via ER stress in NSCLCs 46
Figure 11. Fucoidan enhances gefitinib induced-cytotoxicity down-regulation of Rad51 in NSCLCs 48
Figure 12. HiQ-fucoidan enhances therapeutic agents-induced cytotoxicity in NSCLCs 50
Figure 13. Fraction of HiQ-fucoidan enhances therapeutic agents-induced cytotoxicity in NSCLCs 53
Appendices 56
Table I. Established single drug IC50 (half maximal inhibitory concentration) 56
Established drug combos (combination index) 57
Table II. The IC50, CI and DRI of fucoidan and cisplatin/gefitinib in NSCLC 58
The IC50, CI and DRI of fucoidan and gefitinib in NSCLC 59

Boo, H. J., J. Y. Hong, S. C. Kim, J. I. Kang, M. K. Kim, E. J. Kim, J. W. Hyun, Y. S. Koh, E. S. Yoo, J. M. Kwon and H. K. Kang (2013). "The anticancer effect of fucoidan in PC-3 prostate cancer cells." Mar Drugs 11(8): 2982-2999.
Chou, T. C. (2006). "Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies." Pharmacol Rev 58(3): 621-681.
Chou, T. C. (2008). "Preclinical versus clinical drug combination studies." Leuk Lymphoma 49(11): 2059-2080.
Deben, C., A. Wouters, K. Op de Beeck, J. van Den Bossche, J. Jacobs, K. Zwaenepoel, M. Peeters, J. Van Meerbeeck, F. Lardon, C. Rolfo, V. Deschoolmeester and P. Pauwels (2015). "The MDM2-inhibitor Nutlin-3 synergizes with cisplatin to induce p53 dependent tumor cell apoptosis in non-small cell lung cancer." Oncotarget 6(26): 22666-22679.
Fulda, S. (2014). "Targeting apoptosis for anticancer therapy." Semin Cancer Biol.
Geisen, U., M. Zenthoefer, M. Peipp, J. Kerber, J. Plenge, A. Manago, M. Fuhrmann, R. Geyer, S. Hennig, D. Adam, L. Piker, G. Rimbach and H. Kalthoff (2015). "Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells." Mar Drugs 13(7): 4470-4491.
Hsu, H. Y., T. Y. Lin, Y. C. Wu, S. M. Tsao, P. A. Hwang, Y. W. Shih and J. Hsu (2014). "Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFbeta receptor degradation." Oncotarget 5(17): 7870-7885.
Imai, K. and A. Takaoka (2006). "Comparing antibody and small-molecule therapies for cancer." Nat Rev Cancer 6(9): 714-727.
Klein, H. L. (2008). "The consequences of Rad51 overexpression for normal and tumor cells." DNA Repair (Amst) 7(5): 686-693.
Ko, J. C., J. H. Hong, L. H. Wang, C. M. Cheng, S. C. Ciou, S. T. Lin, M. Y. Jheng and Y. W. Lin (2008). "Role of repair protein Rad51 in regulating the response to gefitinib in human non-small cell lung cancer cells." Mol Cancer Ther 7(11): 3632-3641.
Kwak, J. Y. (2014). "Fucoidan as a marine anticancer agent in preclinical development." Mar Drugs 12(2): 851-870.
Lee, H., J. S. Kim and E. Kim (2012). "Fucoidan from seaweed Fucus vesiculosus inhibits migration and invasion of human lung cancer cell via PI3K-Akt-mTOR pathways." PLoS One 7(11): e50624.
Li, B., F. Lu, X. Wei and R. Zhao (2008). "Fucoidan: structure and bioactivity." Molecules 13(8): 1671-1695.
Li, J., N. Hou, A. Faried, S. Tsutsumi, T. Takeuchi and H. Kuwano (2009). "Inhibition of autophagy by 3-MA enhances the effect of 5-FU-induced apoptosis in colon cancer cells." Ann Surg Oncol 16(3): 761-771.
Li, Y. Y., S. K. Lam, J. C. Mak, C. Y. Zheng and J. C. Ho (2013). "Erlotinib-induced autophagy in epidermal growth factor receptor mutated non-small cell lung cancer." Lung Cancer 81(3): 354-361.
Marino, G., M. Niso-Santano, E. H. Baehrecke and G. Kroemer (2014). "Self-consumption: the interplay of autophagy and apoptosis." Nat Rev Mol Cell Biol 15(2): 81-94.
Molina, J. R., P. Yang, S. D. Cassivi, S. E. Schild and A. A. Adjei (2008). "Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship." Mayo Clin Proc 83(5): 584-594.
Oh, B., J. Kim, W. Lu and D. Rosenthal (2014). "Anticancer effect of fucoidan in combination with tyrosine kinase inhibitor lapatinib." Evid Based Complement Alternat Med 2014: 865375.
Park, S. H., H. S. Park, J. H. Lee, G. Y. Chi, G. Y. Kim, S. K. Moon, Y. C. Chang, J. W. Hyun, W. J. Kim and Y. H. Choi (2013). "Induction of endoplasmic reticulum stress-mediated apoptosis and non-canonical autophagy by luteolin in NCI-H460 lung carcinoma cells." Food Chem Toxicol 56: 100-109.
Pu, F., F. Chen, S. Lin, S. Chen, Z. Zhang, B. Wang and Z. Shao (2016). "The synergistic anticancer effect of cisplatin combined with Oldenlandia diffusa in osteosarcoma MG-63 cell line in vitro." Onco Targets Ther 9: 255-263.
Qiao, G. B., Y. L. Wu, X. N. Yang, W. Z. Zhong, D. Xie, X. Y. Guan, D. Fischer, H. C. Kolberg, S. Kruger and H. W. Stuerzbecher (2005). "High-level expression of Rad51 is an independent prognostic marker of survival in non-small-cell lung cancer patients." Br J Cancer 93(1): 137-143.
Qiao, H., T. Y. Wang, W. Yan, A. Qin, Q. M. Fan, X. G. Han, Y. G. Wang and T. T. Tang (2015). "Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro." Acta Pharmacol Sin 36(9): 1085-1098.
Richardson, C., J. M. Stark, M. Ommundsen and M. Jasin (2004). "Rad51 overexpression promotes alternative double-strand break repair pathways and genome instability." Oncogene 23(2): 546-553.
Sharma, S. V., D. W. Bell, J. Settleman and D. A. Haber (2007). "Epidermal growth factor receptor mutations in lung cancer." Nat Rev Cancer 7(3): 169-181.
Siddik, Z. H. (2003). "Cisplatin: mode of cytotoxic action and molecular basis of resistance." Oncogene 22(47): 7265-7279.
Siegel, R. L., K. D. Miller and A. Jemal (2016). "Cancer statistics, 2016." CA Cancer J Clin 66(1): 7-30.
Su, Y. J., M. S. Tsai, Y. H. Kuo, Y. F. Chiu, C. M. Cheng, S. T. Lin and Y. W. Lin (2010). "Role of Rad51 down-regulation and extracellular signal-regulated kinases 1 and 2 inactivation in emodin and mitomycin C-induced synergistic cytotoxicity in human non-small-cell lung cancer cells." Mol Pharmacol 77(4): 633-643.
Wang, D. and S. J. Lippard (2005). "Cellular processing of platinum anticancer drugs." Nat Rev Drug Discov 4(4): 307-320.
Wong, R. S. (2011). "Apoptosis in cancer: from pathogenesis to treatment." J Exp Clin Cancer Res 30: 87.
Wu, J. X., L. Y. Zhang, Y. L. Chen, S. S. Yu, Y. Zhao and J. Zhao (2015). "Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation." Neural Regen Res 10(3): 481-489.
Yamamori, T., S. Meike, M. Nagane, H. Yasui and O. Inanami (2013). "ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51." FEBS Lett 587(20): 3348-3353.
Yatabe, Y. and T. Mitsudomi (2007). "Epidermal growth factor receptor mutations in lung cancers." Pathol Int 57(5): 233-244.
Zhang, Z., K. Teruya, T. Yoshida, H. Eto and S. Shirahata (2013). "Fucoidan extract enhances the anti-cancer activity of chemotherapeutic agents in MDA-MB-231 and MCF-7 breast cancer cells." Mar Drugs 11(1): 81-98.