I.
甲狀腺素受體作用蛋白質（tyroid receptor interacting protein 6, TRIP6）作為溶血磷脂酸（lysophosphatidic acid, LPA）受體下游的影響目標，會調節細胞肌動蛋白質（actin），進一步影響細胞移動及細胞內部多重訊號傳遞。TRIP6目前已知在多型性神經膠質母細胞瘤 （glioblastoma, GBM）有過度表現的現象，可能與GBM高度侵襲性有關。干擾素誘導四肽重複蛋白質5（interferon induced protein with tetratricopeptide repeats 5, IFIT5）為干擾誘導蛋白質家族的一員，參與人體先天性免疫反應。但有學者發現有部份獨立的IFIT5會與肌動蛋白質連結並表現在細胞膜上，可能與調控細胞移動有關。我們檢測IFIT5在多型性膠質母細胞瘤（glioblastoma, GBM）細胞中是否參與TRIP6調控的細胞遷移與腫瘤形成。本研究透過免疫共沉澱證實TIPR6與IFIT5在細胞中的確存在交互作用，並利用活細胞顯微影像追蹤兩者對細胞遷移的影響。我們發現IFIT5本身並不會增加細胞泡足的產生，但會增進TRIP6造成的細胞動態變化。

II.
多型性神經膠質母細胞瘤（Glioblastoma multiforme, GBM）是成人原發性腦癌中最常見的惡性腦瘤，被世界衛生組織（WHO）歸列為第四級神經膠質瘤。其具有高度侵潤性與異質性，對傳統化療及放射療法皆有抗性，因此許多病患在術後仍有很高的機率再復發，其存活率不超過14個月。部分學者認為，癌症復發與抗藥性可能與癌細胞中少部分的癌幹細胞有關，因此針對癌幹細胞作為治療策略亦是一重要方法。現今有諸多研究嘗試從植物中萃取成分，這些成分具有抗發炎、抗病蟲害或抗癌症之功能，進而研發出更多相似結構分子作為治療疾病的藥物。本文研究發現海檬果萃取物及neriifolin會抑制細胞生長及細胞遷移，並造成細胞週期停滯與細胞凋亡的發生。我們進一步探討neriifolin對神經膠質母細胞瘤類癌幹細胞的訊息路徑，發現neriifolin主要透過抑制Akt路徑活化而使細胞生長能力降低並抑制神經膠質母細胞瘤癌幹細胞的維持。

I
Thyroid receptor interacting protein 6（TRIP6）is a downstream signaling molecule of lysophosphatidic acid（LPA）receptor. It enhances activation of signaling molecules regulating actin to induce cell migration, division and proliferation. TRIP6 is overexpressed in glioblastoma multiform（GBM）and may be a characteristic of aggressiveness of GBM. Interferon induced protein with tetratricopeptide repeats 5（IFIT5）is a member in interferon induced protein family, and involves in the regulation of human innate immune response. It has been reported that IFIT5 interaction with actin on the cell cortex, and may involve in the regulation of cell migration. To examine whether IFIT5 plays a role in the TRIP6 regulated cell migration and glioblastoma tumorigenesis, we investigated the interaction of these two proteins. Here, we demonstrated the interaction of IFIT5 and TRIP6 in cells by co-immunoprecipitation. Moreover, we elucidated the effect of these two proteins on cell migration by live cell imaging, and found that IFIT-5 itself does not enhance the cell blebbing, but promotes TRIP6-mediated cell dynamics.

II
Glioblastoma multiforme （GBM）, classified as the grade IV astrocytoma, is the most common malignant brain cancer in adult. GBM possess the characters of invasiveness, heterogeneity and resistance to chemical and radiation therapy. The GBM patients have poor prognosis, the median survival of the patients is about 14 months. Some studies have showed that the glioblastoma stem cells（GSCs）, a small population of cells, may play the critical role in cancer relapse. Therefore, targeting GSCs is one of the potential therapeutic strategies for GBM. Recent researches studied the plant extracts and found the anti-inflammatory, anti-virus and anti-cancer activity of compositions in the plants. These components can be developed further to other structurally similar molecules for treatment of diseases. In this study, we detection that two extracts and neriifolin inhibited viability and movement of GBM cells and GSCs, and induced cell cycle arrest and apoptosis of GSCs. Stimulation of GSCs with neriifolin inhibited activity of Akt pathway to reduce cell viability and maintain of GSCs.

1. Ohgaki, H., Epidemiology of brain tumors. Methods Mol Biol, 2009. 472: p. 323-42.
2. Jain, R.K., et al., Angiogenesis in brain tumours. Nat Rev Neurosci, 2007. 8(8): p. 610-22.
3. Sizoo, E.M., et al., Symptoms and problems in the end-of-life phase of high-grade glioma patients. Neuro Oncol, 2010. 12(11): p. 1162-6.
4. Correa, D.D., Cognitive functions in brain tumor patients. Hematol Oncol Clin North Am, 2006. 20(6): p. 1363-76.
5. Davis, M.E., Glioblastoma: Overview of Disease and Treatment. Clin J Oncol Nurs, 2016. 20(5 Suppl): p. S2-8.
6. Blanchette, M. and D. Fortin, Blood-brain barrier disruption in the treatment of brain tumors. Methods Mol Biol, 2011. 686: p. 447-63.
7. van Tellingen, O., et al., Overcoming the blood-brain tumor barrier for effective glioblastoma treatment. Drug Resist Updat, 2015. 19: p. 1-12.
8. Jain, R.K., et al., Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol, 2006. 3(1): p. 24-40.
9. Pope, W.B., et al., MRI in patients with high-grade gliomas treated with bevacizumab and chemotherapy. Neurology, 2006. 66(8): p. 1258-60.
10. Ohgaki, H. and P. Kleihues, Epidemiology and etiology of gliomas. Acta Neuropathol, 2005. 109(1): p. 93-108.
11. Mao, H., et al., Deregulated signaling pathways in glioblastoma multiforme: molecular mechanisms and therapeutic targets. Cancer Invest, 2012. 30(1): p. 48-56.
12. Johnson, D.R. and B.P. O'Neill, Glioblastoma survival in the United States before and during the temozolomide era. J Neurooncol, 2012. 107(2): p. 359-64.
13. Krex, D., et al., Long-term survival with glioblastoma multiforme. Brain, 2007. 130(Pt 10): p. 2596-606.
14. Nizamutdinov, D., et al., Prognostication of Survival Outcomes in Patients Diagnosed with Glioblastoma. World Neurosurg, 2018. 109: p. e67-e74.
15. Verhaak, R.G., et al., Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell, 2010. 17(1): p. 98-110.
16. Yung, Y.C., N.C. Stoddard, and J. Chun, LPA receptor signaling: pharmacology, physiology, and pathophysiology. J Lipid Res, 2014. 55(7): p. 1192-214.
17. Ballestrem, C., et al., Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer. J Cell Sci, 2006. 119(Pt 5): p. 866-75.
18. Xu, J., et al., TRIP6 enhances lysophosphatidic acid-induced cell migration by interacting with the lysophosphatidic acid 2 receptor. J Biol Chem, 2004. 279(11): p. 10459-68.
19. Kim, D.S., et al., Lysophosphatidic acid inhibits melanocyte proliferation via cell cycle arrest. Arch Pharm Res, 2003. 26(12): p. 1055-60.
20. Lin, V.T., et al., TRIP6 regulates p27 KIP1 to promote tumorigenesis. Mol Cell Biol, 2013. 33(7): p. 1394-409.
21. Lindholm, P.F. and Y.S. Hwang, LPA Increases Tumor Growth and Bone Destruction Through Enhancement of Osteoclastogenic Cytokines. Anticancer Res, 2016. 36(1): p. 61-70.
22. Chen, Y., D.P. Ramakrishnan, and B. Ren, Regulation of angiogenesis by phospholipid lysophosphatidic acid. Front Biosci (Landmark Ed), 2013. 18: p. 852-61.
23. Beckerle, M.C., Zyxin: zinc fingers at sites of cell adhesion. Bioessays, 1997. 19(11): p. 949-57.
24. Petit, M.M., et al., LPP, the preferred fusion partner gene of HMGIC in lipomas, is a novel member of the LIM protein gene family. Genomics, 1996. 36(1): p. 118-29.
25. Kanungo, J., et al., Ajuba, a cytosolic LIM protein, shuttles into the nucleus and affects embryonal cell proliferation and fate decisions. Mol Biol Cell, 2000. 11(10): p. 3299-313.
26. Bach, I., The LIM domain: regulation by association. Mech Dev, 2000. 91(1-2): p. 5-17.
27. Lai, Y.J., et al., c-Src-mediated phosphorylation of TRIP6 regulates its function in lysophosphatidic acid-induced cell migration. Mol Cell Biol, 2005. 25(14): p. 5859-68.
28. Yi, J., et al., Members of the Zyxin family of LIM proteins interact with members of the p130Cas family of signal transducers. J Biol Chem, 2002. 277(11): p. 9580-9.
29. Lai, Y.J., et al., The adaptor protein TRIP6 antagonizes Fas-induced apoptosis but promotes its effect on cell migration. Mol Cell Biol, 2010. 30(23): p. 5582-96.
30. Lai, Y.J., et al., TRIP6 regulates neural stem cell maintenance in the postnatal mammalian subventricular zone. Dev Dyn, 2014. 243(9): p. 1130-42.
31. Zhou, X., et al., Interferon induced IFIT family genes in host antiviral defense. Int J Biol Sci, 2013. 9(2): p. 200-8.
32. Kumar, P., et al., Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs. Nucleic Acids Res, 2014. 42(5): p. 3228-45.
33. Feng, F., et al., Crystal structure and nucleotide selectivity of human IFIT5/ISG58. Cell Res, 2013. 23(8): p. 1055-8.
34. Harrison, D.A., The Jak/STAT pathway. Cold Spring Harb Perspect Biol, 2012. 4(3).
35. Fensterl, V. and G.C. Sen, The ISG56/IFIT1 Gene Family. J Interferon Cytokine Res, 2011. 31(1): p. 71-8.
36. Lai, K.C., et al., IFN-induced protein with tetratricopeptide repeats 2 inhibits migration activity and increases survival of oral squamous cell carcinoma. Mol Cancer Res, 2008. 6(9): p. 1431-9.
37. Zheng, C., et al., IFIT5 positively regulates NF-kappaB signaling through synergizing the recruitment of IkappaB kinase (IKK) to TGF-beta-activated kinase 1 (TAK1). Cell Signal, 2015. 27(12): p. 2343-54.
38. Katibah, G.E., et al., tRNA binding, structure, and localization of the human interferon-induced protein IFIT5. Mol Cell, 2013. 49(4): p. 743-50.
39. Li, J.R., et al., Cancer RNA-Seq Nexus: a database of phenotype-specific transcriptome profiling in cancer cells. Nucleic Acids Res, 2016. 44(D1): p. D944-51.
40. Anaya, J., OncoLnc: linking TCGA survival data to mRNAs, miRNAs, and lncRNAs. PeerJ Computer Science, 2016. 2.
41. Charras, G. and E. Paluch, Blebs lead the way: how to migrate without lamellipodia. Nat Rev Mol Cell Biol, 2008. 9(9): p. 730-6.
42. Fackler, O.T. and R. Grosse, Cell motility through plasma membrane blebbing. The Journal of Cell Biology, 2008. 181(6): p. 879-884.
43. Laffaire, J., et al., Methylation profiling identifies 2 groups of gliomas according to their tumorigenesis. Neuro-Oncology, 2011. 13(1): p. 84-98.
44. Lv, K., et al., Trip6 promotes dendritic morphogenesis through dephosphorylated GRIP1-dependent myosin VI and F-actin organization. J Neurosci, 2015. 35(6): p. 2559-71.
45. Giese, A. and M. Westphal, Treatment of malignant glioma: a problem beyond the margins of resection. J Cancer Res Clin Oncol, 2001. 127(4): p. 217-25.
46. Holmen, S.L. and B.O. Williams, Essential role for Ras signaling in glioblastoma maintenance. Cancer Res, 2005. 65(18): p. 8250-5.
47. Cancer Genome Atlas Research, N., Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature, 2008. 455(7216): p. 1061-8.
48. Rahaman, S.O., et al., Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells. Oncogene, 2002. 21(55): p. 8404-13.
49. Galli, R., et al., Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res, 2004. 64(19): p. 7011-21.
50. Frank, N.Y., T. Schatton, and M.H. Frank, The therapeutic promise of the cancer stem cell concept. J Clin Invest, 2010. 120(1): p. 41-50.
51. Schneider, M., et al., A paired comparison between glioblastoma "stem cells" and differentiated cells. Int J Cancer, 2016. 138(7): p. 1709-18.
52. Lapidot, T., et al., A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 1994. 367(6464): p. 645-8.
53. Singh, S.K., et al., Identification of human brain tumour initiating cells. Nature, 2004. 432(7015): p. 396-401.
54. Owens, T.W. and M.J. Naylor, Breast cancer stem cells. Front Physiol, 2013. 4: p. 225.
55. Wang, C., et al., Evaluation of CD44 and CD133 as cancer stem cell markers for colorectal cancer. Oncol Rep, 2012. 28(4): p. 1301-8.
56. Jaiswal, K.R., et al., Comparative testing of various pancreatic cancer stem cells results in a novel class of pancreatic-cancer-initiating cells. Stem Cell Res, 2012. 9(3): p. 249-60.
57. Li, L. and W.B. Neaves, Normal stem cells and cancer stem cells: the niche matters. Cancer Res, 2006. 66(9): p. 4553-7.
58. Gupta, P.B., et al., Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. Cell, 2011. 146(4): p. 633-44.
59. Blagosklonny, M.V., Cancer stem cell and cancer stemloids: From biology to therapy. Cancer Biology & Therapy, 2014. 6(11): p. 1684-1690.
60. Giese, A., Glioma invasion--pattern of dissemination by mechanisms of invasion and surgical intervention, pattern of gene expression and its regulatory control by tumorsuppressor p53 and proto-oncogene ETS-1. Acta Neurochir Suppl, 2003. 88: p. 153-62.
61. Yu, Y., G. Ramena, and R.C. Elble, The role of cancer stem cells in relapse of solid tumors. Front Biosci (Elite Ed), 2012. 4: p. 1528-41.
62. Bao, S., et al., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature, 2006. 444(7120): p. 756-60.
63. Piccirillo, S.G., et al., Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature, 2006. 444(7120): p. 761-5.
64. Hu, Y. and L. Fu, Targeting cancer stem cells: a new therapy to cure cancer patients. Am J Cancer Res, 2012. 2(3): p. 340-56.
65. You, L., X. Guo, and Y. Huang, Correlation of Cancer Stem-Cell Markers OCT4, SOX2, and NANOG with Clinicopathological Features and Prognosis in Operative Patients with Rectal Cancer. Yonsei Med J, 2018. 59(1): p. 35-42.
66. Klonisch, T., et al., Cancer stem cell markers in common cancers - therapeutic implications. Trends Mol Med, 2008. 14(10): p. 450-60.
67. Kim, W.T. and C.J. Ryu, Cancer stem cell surface markers on normal stem cells. BMB Rep, 2017. 50(6): p. 285-298.
68. Carlier, J., et al., The principal toxic glycosidic steroids in Cerbera manghas L. seeds: identification of cerberin, neriifolin, tanghinin and deacetyltanghinin by UHPLC-HRMS/MS, quantification by UHPLC-PDA-MS. J Chromatogr B Analyt Technol Biomed Life Sci, 2014. 962: p. 1-8.
69. Indigenous Drugs of India. British Medical Journal, 1940. 2(4162): p. 491-492.
70. Feng, B., et al., beta-D-Glucosyl-(1-4)-alpha-L-thevetosides of 17beta-digitoxigenin from seeds of Cerbera manghas L. induces apoptosis in human hepatocellular carcinoma HepG2 cells. Exp Toxicol Pathol, 2012. 64(5): p. 403-10.
71. Wang, G.F., et al., Tanghinigenin from seeds of Cerbera manghas L. induces apoptosis in human promyelocytic leukemia HL-60 cells. Environ Toxicol Pharmacol, 2010. 30(1): p. 31-6.
72. Cheenpracha, S., et al., New cytotoxic cardenolide glycoside from the seeds of Cerbera manghas. Chem Pharm Bull (Tokyo), 2004. 52(8): p. 1023-5.
73. Zhao, Q., et al., Neriifolin from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Fitoterapia, 2011. 82(5): p. 735-41.
74. Deng, Y., et al., Acaricidal activity against Panonychus citri and active ingredient of the mangrove plant Cerbera manghas. Nat Prod Commun, 2014. 9(9): p. 1265-8.
75. Mans, D.R., A.B. da Rocha, and G. Schwartsmann, Anti-cancer drug discovery and development in Brazil: targeted plant collection as a rational strategy to acquire candidate anti-cancer compounds. Oncologist, 2000. 5(3): p. 185-98.
76. Lee, D.H., et al., Cardiac glycosides suppress the maintenance of stemness and malignancy via inhibiting HIF-1alpha in human glioma stem cells. Oncotarget, 2017. 8(25): p. 40233-40245.
77. Abe, F. and T. Yamauchi, Studies on Cerbera. I. Cardiac Glycosides in the Seeds, Bark, and Leaves of Cerbera manghas L. CHEMICAL & PHARMACEUTICAL BULLETIN, 1977. 25(10): p. 2744-2748.
78. Giacinti, C. and A. Giordano, RB and cell cycle progression. Oncogene, 2006. 25(38): p. 5220-7.
79. Schutte, B., et al., Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J Neurosci Methods, 1998. 86(1): p. 63-9.
80. Chaitanya, G.V., J.S. Alexander, and P.P. Babu, PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal, 2010. 8: p. 31.
81. McIlwain, D.R., T. Berger, and T.W. Mak, Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol, 2013. 5(4): p. a008656.
82. Lin, V.T. and F.T. Lin, TRIP6: an adaptor protein that regulates cell motility, antiapoptotic signaling and transcriptional activity. Cell Signal, 2011. 23(11): p. 1691-7.
83. Wang, B., et al., Identification and expression analysis of the interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) gene in duck (Anas platyrhynchos domesticus). PLoS One, 2015. 10(3): p. e0121065.