因為神經膠母細胞瘤的侵略性高而且對於放射線療法和化學療法的敏感性很低，神經膠母細胞瘤(GBM)是許多難治療的腫瘤之一。在以前的研究中顯示，轉化生長因子誘導的抗凋亡因子1(TIAF1)會頻繁的表達在生長中的腫瘤中和增殖的神經膠母細胞瘤中。神經膠母細胞瘤會對放射線療法和化學療法產生抗性。為了研究TIAF1，是否在有壓力的環境中參與調控腫瘤生長，我的實驗將使用抗癌藥物紫杉醇(Paclitaxel,Taxol)使神經膠母細胞瘤的細胞週期停滯。調查TIAF1在紫杉醇誘導的神經膠母細胞瘤的細胞週期停滯中所扮演的角色。
TIAF1具有轉化生長因子(TGF-β)的下游效應。我們使用細胞存活率分析、流式細胞儀、免疫細胞化學染色、細胞增殖分析和西方墨點法。分別檢測紫杉醇亦或TGF-β1處理之下細胞存活率、細胞週期、細胞型態、細胞的增殖和細胞週期相關蛋白質的表現。我們的結果顯示，在紫杉醇處理36小時之下，共同處理TGF-β1容易使大鼠的神經膠母細胞瘤細胞株CNS-1離開G0/G1時期，進入S與G2時期，而TGF-β1是藉由在第24小時的時候增加細胞週期相關蛋白質cyclin A、cdc25C和cyclin B1使細胞週期改變。而濃度500nM的紫杉醇共同處理TGF-β1 48小時之後，會誘導更多的細胞進入G2/M時期，這是藉由TGF-β1在第24或36小時的時候所增加的細胞週期相關蛋白質p53、cdc25C、cyclin B1和CDK1使細胞週期改變。而在人類的神經膠母細胞瘤細胞株1306-MG中，紫杉醇共同處理TGF-β1 36小時之後，會使更多的細胞離開G0/G1時期，這是藉由TGF-β1在第24或36小時的時候所增加的細胞週期相關蛋白質cyclin E使細胞週期改變。接著我們將TIAF1的質體DNA轉染到神經膠母細胞瘤細胞株中。我們的結果顯示，TIAF1共同處理濃度100或500nM紫杉醇在CNS-1細胞株之後，會抑制細胞的subG1和S時期而且會使細胞週期停滯在G2/M時期。而在1306-MG細胞株中，TIAF1共同處理濃度100或500nM紫杉醇，細胞的G0/G1時期會下降而且細胞分裂會被抑制。
本篇的結論是，TGF-β1在紫杉醇誘導的細胞週期停滯中所扮演的角色，為減少CNS-1的G0/G1時期，藉由增加cyclin A、cdc25C和cyclin B1使細胞週期離開G0/G1時期。而TGF-β1在紫杉醇誘導的細胞週期停滯中所扮演的角色，也會藉由增加p53、cdc25C、cyclin B1和CDK1而使得CNS-1的細胞週期停滯在G2/M時期。另外，1306-MG細胞中，TGF-β1在紫杉醇誘導的細胞週期停滯中所扮演的角色，TGF-β1會藉由增加cyclin E而使得細胞週期離開G0/G1時期。TIAF1在紫杉醇誘導的細胞週期停滯中所扮演的角色，是下調CNS-1的subG1和S時期的比例，和誘導CNS-1的細胞週期停滯在G2/M時期。而在1306-MG中，TIAF1在紫杉醇誘導的細胞週期停滯中所扮演的角色，是下調G0/G1時期和抑制細胞分裂。

Glioblastoma (GBM) is one of most difficult to treat tumors, due to its invasiveness and low sensitivity to radiotherapy and chemotherapy. The expression of transforming growth factor β 1-induced anti-apoptotic factor 1 (TIAF1) is upregulated in the developing tumor and proliferating GBM. GBM is frequently resistant to radiotherapy or chemotherapy. In order to study the potential role of TIAF1 in tumor growth under the stress. We used the anticancer drug Paclitaxel (Taxol) to arrest the cell cycle in GBM cells. We were investigated the role of TIAF1 in paclitaxel-induced cell cycle arrest in Glioblastoma.
TIAF1 is a downstream effector of the Transforming growth factor β (TGF-β). We used the MTT assay, flow cytometry, immunocytochemistry, bromodeoxyuridine (BrdU) incorporation assay, and Western blot to examine the cell viability, cell cycle distribution, proliferation, and cell cycle related proteins following Taxol and/or TGF-β1 treatment in rat GBM CNS-1 and human GBM 1306-MG cells. Our data showed that TGF-β1 allow cells to leave G0/G1 phase and enter S and G2/M phase, under the 100nM Taxol treatment for 36 hours in CNS-1 cells. The TGF-β1 was via upregulated cell cycle related proteins cyclin A, cdc25C, and cyclin B1 in CNS-1 cells under the 100nM Taxol treatment for 24 hours. In CNS-1 cells, TGF-β1 also induced G2/M phase arrest under 500nM Taxol treatment for 48 hours. The TGF-β1 was via upregulated cell cycle related proteins p53, cdc25C, cyclin B1, and CDK1 in CNS-1 cells under the 500nM Taxol treatment for 24 or 36 hours. In 1306-MG cells, TGF-β1 allows more cells leave G1 phase under the Taxol treatment for 36 hours. The TGF-β1 was via upregulated cell cycle related cyclin E in 1306-MG cells under the Taxol treatment for 24 and 36 hours. Then, we transfected the TIAF1 plasmid DNA into GBM cells. Our data showed that TIAF1 downregulated subG1 and S phase, and arrested CNS-1 cells in G2/M phase following 100 or 500 nM Taxol treatment. In 1306-MG cells, TIAF1 arrested cells in G0/G1 phase and inhibited cells division following 100 or 500 nM Taxol treatment.
In conclusion, our findings indicate that the role of TGF-β1 in Taxol induce cell cycle arrest, may through upregulated cyclin A, cdc25C, and cyclin B to induce G0/G1 phase downregulation or through upregulated p53, cdc25C, cyclin B, and CDK1 to induced G2/M phase arrest in CNS-1 cells. The role of TGF-β1 in Taxol induce cell cycle arrest, may through upregulated cyclinE to induce G0/G1 phase downregulation in 1306-MG cells. Then, TIAF1 in Taxol-induced cell cycle arrest may downregulate subG1 and S phase, and induced G2/M arrest in CNS-1 cells. In 1306-MG cells, the role of TIAF1 in Taxol induces cell cycle arrest may downregulate G0/G1 phase and inhibite the cells division.

Agarwal, M. L., Agarwal, A., Taylor, W. R., & Stark, G. R. (1995). p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proc Natl Acad Sci U S A, 92(18), 8493-8497.
Bhola, N. E., Balko, J. M., Dugger, T. C., Kuba, M. G., Sanchez, V., Sanders, M., Stanford, J., Cook, R. S., Arteaga, C. L. (2013). TGF-beta inhibition enhances chemotherapy action against triple-negative breast cancer. J Clin Invest, 123(3), 1348-1358. doi: 10.1172/jci65416
Chang, J. Y., Chiang, M. F., Lin, S. R., Lee, M. H., He, H., Chou, P. Y., Chen, S. J., Chen, Y. A., Yang, L. Y., Lai, F. J., Hsieh, C. C., Hsieh, T. H., Sheu, H. M., Sze, C. I., Chang, N. S. (2012). TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death. Cell Death Dis, 3, e302. doi: 10.1038/cddis.2012.36
Chang, N. S., Mattison, J., Cao, H., Pratt, N., Zhao, Y., & Lee, C. (1998). Cloning and characterization of a novel transforming growth factor-beta1-induced TIAF1 protein that inhibits tumor necrosis factor cytotoxicity. Biochem Biophys Res Commun, 253(3), 743-749. doi: 10.1006/bbrc.1998.9846
Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., & Andreu, J. M. (2000). Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site. J Biol Chem, 275(34), 26265-26276. doi: 10.1074/jbc.M003120200
Elliott., R. L., & Blobe., G. C. (2005). Role of Transforming Growth Factor Beta in Human Cancer. Clinical Oncology. doi: 10.1200/JCO.2005.02.047
Eyler, C. E., Wu, Q., Yan, K., MacSwords, J. M., Chandler-Militello, D., Misuraca, K. L., Lathia, J. D., Forrester, M. T., Lee, J., Stamler, J. S., Goldman, S. A., Bredel, M., McLendon, R. E., Sloan, A. E., Hjelmeland, A. B. Rich, J. N. (2011). Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2. Cell, 146(1), 53-66. doi: 10.1016/j.cell.2011.06.006
George, J., Banik, N. L., & Ray, S. K. (2010). Molecular Mechanisms of Taxol for Induction of Cell Death in Glioblastomas. Glioblastoma: Molecular Mechanisms of Pathogenesis and Current Therapeutic Strategies, doi: 10.1007/978-1-4419-0410-2_14
Karmakar., S., Banik., N. L., & Ray., S. K. (2010). Current Endeavors for Enhancing Efficacy of Paclitaxel for Treatment of Glioblastoma. Glioblastoma: Molecular Mechanisms of Pathogenesis and Current Therapeutic Strategies, doi: 10.1007/978-1-4419-0410-2_15
Khera, S., & Chang, N. S. (2003). TIAF1 participates in the transforming growth factor beta1--mediated growth regulation. Ann N Y Acad Sci, 995, 11-21.
Lee, J., Choi, J. H., & Joo, C. K. (2013). TGF-beta1 regulates cell fate during epithelial-mesenchymal transition by upregulating survivin. Cell Death Dis, 4, e714. doi: 10.1038/cddis.2013.244
Lee, M. H., Lin, S. R., Chang, J. Y., Schultz, L., Heath, J., Hsu, L. J., Kuo, Y. M., Hong, Q., Chiang, M. F., Gong, C. X., Sze, C. I., Chang, N. S. (2010). TGF-beta induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid beta plaques in Alzheimer's disease. Cell Death Dis, 1, e110. doi: 10.1038/cddis.2010.83
Nilsson, I., & Hoffmann, I. (2000). Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res, 4, 107-114.
Ohtsubo, M., Theodoras, A. M., Schumacher, J., Roberts, J. M., & Pagano, M. (1995). Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol, 15(5), 2612-2624.
Piacibello, W., Severino, A., Stacchini, A., & Aglietta, M. (1991). Differential effect of transforming growth factor beta 1 on the proliferation of human lymphoid and myeloid leukemia cells. Haematologica, 76(6), 460-466.
Schultz, L., Khera, S., Sleve, D., Heath, J., & Chang, N. S. (2004). TIAF1 and p53 functionally interact in mediating apoptosis and silencing of TIAF1 abolishes nuclear translocation of serine 15-phosphorylated p53. DNA Cell Biol, 23(1), 67-74. doi: 10.1089/104454904322745943
Stupp, R., Hegi, M. E., Mason, W. P., van den Bent, M. J., Taphoorn, M. J., Janzer, R. C., Ludwin, S. K., Allgeier, A., Fisher, B., Belanger, K., Hau, P., Brandes, A. A., Gijtenbeek, J., Marosi, C., Vecht, C. J., Mokhtari, K., Wesseling, P., Villa, S., Eisenhauer, E., Gorlia, T., Weller, M., Lacombe, D., Cairncross, J. G., Mirimanoff, R. O. (2009). Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol, 10(5), 459-466. doi: 10.1016/s1470-2045(09)70025-7
Sze, C. I., Su, W. P., Chiang, M. F., Lu, C. Y., Chen, Y. A., & Chang, N. S. (2013). Assessing current therapeutic approaches to decode potential resistance mechanisms in glioblastomas. Front Oncol, 3, 59. doi: 10.3389/fonc.2013.00059
Villapol, S., Logan, T. T., & Symes, A. J. (2013). Role of TGF-β Signaling in Neurogenic Regions After Brain Injury. doi: 10.5772/53941
Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., & McPhail, A. T. (1971). Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc, 93(9), 2325-2327.
Yam, C. H., Fung, T. K., & Poon, R. Y. (2002). Cyclin A in cell cycle control and cancer. Cell Mol Life Sci, 59(8), 1317-1326.
Yue, J., & Mulder, K. M. (2001). Transforming growth factor-beta signal transduction in epithelial cells. Pharmacol Ther, 91(1), 1-34.
Zetterberg, A., Larsson, O., & Wiman, K. G. (1995). What is the restriction point? Curr Opin Cell Biol, 7(6), 835-842.
Zhang, D., Yang, R., Wang, S., & Dong, Z. (2014). Paclitaxel: new uses for an old drug. Drug Des Devel Ther, 8, 279-284. doi: 10.2147/dddt.s56801