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系統識別號 U0026-2707201516055400
論文名稱(中文) 離子通道活性與間質性分化在具放射線抗性的多型性神經膠母細胞瘤中之關聯性
論文名稱(英文) Ion-Channel Activity and Mesenchymal Differentiation in Radiation Resistance in Glioblastoma Multiforme
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 103
學期 2
出版年 104
研究生(中文) 劉嬋娟
研究生(英文) Chan-Chuan Liu
學號 T96024015
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-司君一
口試委員-吳勝男
口試委員-簡伯武
口試委員-鄧志娟
中文關鍵字 鈣離子活化型鉀離子通道  間質性分化  神經膠母細胞瘤  放射線抗性  轉化生長因子-β 
英文關鍵字 Calcium-activated potassium channel  mesenchymal differentiation  glioblastoma  radiation resistance  Transforming Growth Factor-beta (TGF-β) 
學科別分類
中文摘要 多型性神經膠母細胞瘤是最致命的原生性腦瘤。目前的標準治療方式是以外科手術移除大部分的腫瘤,再配合放射線與化學藥物做治療。但多型性神經膠母細胞瘤易產生對治療的抗性,因此為目前最難治療的腫瘤。根據先前的研究,放射線療法會誘導多型性神經膠母細胞瘤之癌細胞進行間質性分化,也就是上皮細胞間質化,且間質性分化也可能會幫助多型性神經膠母細胞瘤建立對放射線之抗性。此外,目前已知轉化生長因子-β可調控癌細胞的放射線反應,且在細胞轉型的過程中亦扮演相當重要的角色,因此轉化生長因子-β可能藉由促進間質性分化,幫助癌細胞建立放射線抗性。另外,放射線也會影響鈣離子活化型鉀離子通道活性,此現象也可能與放射線抗性相關。
因此,我們將探討鈣離子活化型鉀離子通道活性與間質性分化在多型性神經膠母細胞瘤建立放射線抗性過程中之關聯性。首先,我們利用台盼藍染色法觀察放射線對細胞存活率之影響,並觀察細胞型態之變化。我們發現部分細胞有型態改變,我們稱之為變形細胞。根據結果,我們選用存活率百分之五十之劑量做為連續照射誘導抗性之劑量,存活率由最低點回升的第一個劑量做為高劑量誘導抗性之陽性控制組。當連續照射組的存活率高過當次的陽性控制組,我們定義此細胞具有放射線抗性。因此,人類多型性神經膠母細胞瘤之細胞株1306MG需要3.5Gy照射六次可誘發放射線抗性, U87MG則需要2Gy照射四次才可誘發放射線抗性,而大鼠的多型性神經膠母細胞瘤之細胞株CNS-1則需要7Gy照射四次才可誘發放射線抗性。在放射線抗性建立過程中,變形細胞會逐漸增加。此外,在免疫螢光染色結果中,這些變形細胞表現部分間質性分化之標誌分子的形式與非變形細胞不同,像是在變形細胞中的E-cadherin會有明顯的聚集,β-catenin與pan-cytokeratins的表現密度減少,以及N-cadherin和Fibronectin的表現密度上升。此外,根據西方墨點法的結果,間質性標誌分子N-cadherin會隨著放射線抗性之建立增加,而上皮性標誌分子β-catenin則是逐漸減少。在電生理的實驗中則發現BK 通道 (big potassium channel) 之活性在連續照射組中會逐漸減少,但在高劑量的照射組中則是明顯上升。然而,無論是高劑量或連續照射IK通道 (intermediate potassium channel) 之活性沒有影響。此外,我們亦利用轉化生長因子-β (Transforming growth factor-β, TGF-β) 與其接受器之抑制劑LY364947調控TGF-β/Smad路徑之活性。在西方墨點法之結果中發現,當TGF-β/Smad路徑之活性被抑制時,在具連續性照射誘導之抗性的癌細胞株中會藉由增加β-catenien之表現及減少N-cadherin與Fibronectin之表現,逆轉其間質性分化。根據以上結果,我們認為BK通道活性的減少與間質性分化可能促進多型性神經膠母細胞瘤建立放射線抗性,且TGF-β/Smad路徑可在放射線抗性建立過程中調控部分間質性分化。因此,我們認為放射線抗性建立是一個連續且動態的過程,且除了生化因子之外,電生理因子在此過程亦扮演重要的角色。
英文摘要 Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Current standard therapy is surgery combined with radio-therapy and/or chemo-therapy. Unfortunately, it is still the most difficult-to-treat tumor due to the existence of resistant tumor cells. According to the previous studies, radio-therapy induced mesenchymal differentiation (MD) which is a process of epithelial-mesenchymal transition, and MD may promote GBM to establish radiation resistance. Transforming Growth Factor-beta (TGF-β) is a modifier of radiation response, and plays an important role in cell transformation. Therefore, TGF-β pathway might involve in developing radiation resistance via regulating MD. Also, radiation treatment influences on calcium-activated potassium channel activity, it might relate to radiation resistance.
Thus, we explored the correlation of calcium-activated potassium channel activity and MD in the process of establishing radiation resistance in human GBM cell lines. We first examined the effect of radiation doses on cell viability by trypan blue, and observed cell morphology. According to the observation, the cells showed morphology change were called transformed cells. Also, we chose the radiation dose of 50% cell viability for the established of consecutive-irradiation-induced resistance. The radiation dose of the cell viability greater than 50% was chosen to establish high-dose-irradiation-induced resistance as positive control. When viability of cells recovered from consecutive-irradiation exposures which were higher than positive control, the cells were defined as consecutive-irradiation-induced resistance. To develop radiation resistance, 3.5Gy exposure for 6 times were required for 1306MG, 2Gy exposure for 4 times were needed for U87MG, and 7Gy for 4 times were required for CNS-1. As establishing radiation resistance, the numbers of transformed cells increased. Moreover, immunocytochemistry staining showed expression of certain MD markers on transformed were different from on non-transformed cells, such as E-cadherin forming clusters, reduced the density of the expression of β-catenin and pan-cytokeratins, and increased the density of the expression of N-cadherin and Fibronectin in transformed cells. In addition, immunoblotting results showed mesenchymal marker N-cadherin increased and epithelial marker beta-catenin decreased in radiation resistance. Electrophysiology results indicated consecutive irradiation reduced big potassium channel (BK channel) activity, but high-dose irradiation increased BK channel activity. However, radiation does not impacted on intermediate potassium channel (IK channel) activity. Finally, we used TGF-β and TGF-β receptor inhibitor LY364947 to regulate the activity of TGF-β/Smad pathway. The immunoblotting results revealed that inhibition of TGF-β pathway reversed MD in consecutive-irradiation-induced resistant cell line via increasing the expression of β-catenin and reducing the expression of N-cadherin and Fibronectin. Therefore, our results suggest that mesenchymal differentiation and decrement of BK channel activity may both lead to radiation resistance, and TGF-β/Smad pathway regulated the expression of certain MD markers during developing radiation resistance. Finally, we concluded that the developing of radiation resistance is a continuous and dynamic processes, and not only biochemical factors but also electrophysiological factors play important role in these processes.
論文目次 Abstract in Chinese…………………………………………………………………..…….I
Abstract in English…………………………………………………………………..….III
Acknowledgement…………………………………………………………………..…….V
Table of Contents……………………………………..……………………………..….VI
List of Figures………………………………..…………….……….…………..…….VIII
Introduction……………………………………………..…...…..…………………..…....1
Glioblstoma Multiforme (GBM) ………..…………….……….………………..….....1
Radiation Resistance and Mesenchymal Differentiation (MD) ….………………….1
Calcium-activated Potassium channels……………………………..…………..….….3
Transforming growth factor-β (TGF-β) ………………………………...……………..3
Rationale and Aims………………………………..……………….………………….…...5
Materials and Methods……………………..…………….……….………………..…..7
Cell Cultures………………………...…..…………….……….………………..…...7
Viability Curve of Radiation Exposure………………………………………..……..7
Establish Radiation Resistant Cell Lines…………………….………………..……..8
Trypan Blue Exclusion Assay……………..………….……….………………..……..8
Immunoblotting Assay…………………….………….……….………………..….....8
Immunocytochemistry Staining (ICC).……..………….…….……………….……..9
Electrophysiology Experiment………..……….…….……….………………..……10
Statistical Analysis………………….…………………….….………………...…….11
Results……………..………….……….…………….……….……….………….…..……12
Viability and Morphological Changes in GBM Cell Lines after Different radiation dose exposure……………..………….……….…………...…..…….……….………12
Establish Radiation Resistant GBM cell Lines and Morphological Change in the Processes of Establishing Radiation Resistance…….……….……….……….……12
Protein Expression and Activity of Calcium-activated Potassium Channels in GBM Cell Lines with or without Development of Radiation Resistance……….………13
Changes of MD Markers Expression in the Processes of Radiation Resistance…..14
Altered Expression of Mesenchymal Differentiation Markers in Transformed Cells……………..………….……….………………..……….……….…….……..14
TGF-β/Smad Pathway Partially Regulated Mesenchymal Differentiation in Radiation Resistance……………..………….……….………………….……......…15
Figures…………..………….……….………………..…….……….……….………......17
Conclusion……………..………….…………………….……….……….…………..…47
Discussion……………..………….……….…………….……….……….…………..…48
The Numbers of Transformed Cells increased in Consecutive-irradiation-induced Resistance…..…………………..…..…………….………..…………………………48
High-dose-irradiation-induce Radiation Resistance in GBM……………………..49
BK Channel Activity Increased in High-dose-irradiation-induced Resistance, but Decreased in Consecutive-irradiation-induced Resistance…..…………………..50
The Effect of the Activity of TGF-β/Smad Pathway on Mesenchymal Differentiation in Radiation Resistance……….………………….………………..51
References…………..……………….………….……….………….………………..….53
參考文獻 References
Anthony, B., Khe, H. X., Carpentier, A. F., and Delattre, a. J. Y. (2003). Primary brain tumors in adults. The Lancet 361, 323-331.
Bao, S., Wu, Q., McLendon, R. E., Hao, Y., Shi, Q., Hjelmeland, A. B., Dewhirst, M. W., Bigner, D. D., and Rich, J. N. (2006). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760.
Bhat, K. P., Balasubramaniyan, V., Vaillant, B., Ezhilarasan, R., Hummelink, K., Hollingsworth, F., Wani, K., Heathcock, L., James, J. D., Goodman, L. D., et al. (2013). Mesenchymal differentiation mediated by NF-kappaB promotes radiation resistance in glioblastoma. Cancer Cell 24, 331-346.
Borthwick, L. A., Gardner, A., De Soyza, A., Mann, D. A., and Fisher, A. J. (2012). Transforming Growth Factor-beta1 (TGF-beta1) Driven Epithelial to Mesenchymal Transition (EMT) is Accentuated by Tumour Necrosis Factor alpha (TNFalpha) via Crosstalk Between the SMAD and NF-kappaB Pathways. Cancer Microenvironment : Official Journal of the International Cancer Microenvironment Society 5, 45-57.
Bruna, A., Darken, R. S., Rojo, F., Ocana, A., Penuelas, S., Arias, A., Paris, R., Tortosa, A., Mora, J., Baselga, J., and Seoane, J. (2007). High TGFbeta-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene. Cancer Cell 11, 147-160.
Chang, J. E., Khuntia, D., Robins, H. I., and Mehta, M. P. (2007). Radiotherapy and Radiosensitizers in the Treatment of Glioblastoma Multiforme. Clinical Advances in Hematology and Oncology 5.
Dancea, H. C., Shareef, M. M., and Ahmed, M. M. (2009). Role of Radiation-induced TGF-beta Signaling in Cancer Therapy. Molecular and Cellular Pharmacology 1, 44-56.
Drabsch, Y., and ten Dijke, P. (2012). TGF-beta signalling and its role in cancer progression and metastasis. Cancer Metastasis Reviews 31, 553-568.
Eyler, C. E., and Rich, J. N. (2008). Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology 26, 2839-2845.
Gerstner, L., Jellinger, K., Heiss, W. D., and W6ber, G. (1977). Morphological changes in anaplastic gliomas treated with radiation and chemotherapy. Acta Neurochirurgica 36, 117-138.
Hardee, M. E., Marciscano, A. E., Medina-Ramirez, C. M., Zagzag, D., Narayana, A., Lonning, S. M., and Barcellos-Hoff, M. H. (2012). Resistance of glioblastoma-initiating cells to radiation mediated by the tumor microenvironment can be abolished by inhibiting transforming growth factor-beta. Cancer Research 72, 4119-4129.
Hazawa, M., Hosokawa, Y., Monzen, S., Yoshino, H., and Kashiwakura, I. (2012). Regulation of DNA damage response and cell cycle in radiation-resistant HL60 myeloid leukemia cells. Oncology Reports 28, 55-61.
Huang, X., and Jan, L. Y. (2014). Targeting potassium channels in cancer. The Journal of Cell Biology 206, 151-162.
Jellinger, K. (1978). Glioblastoma Muhiforme Morphology and Biology. Acta Neurochirurgica 48, 5-32.
Johnson, D. R., and O'Neill, B. P. (2012). Glioblastoma survival in the United States before and during the temozolomide era. Journal of Neuro-oncology 107, 359-364.
Mamuya, F. A., and Duncan, M. K. (2012). aV integrins and TGF-beta-induced EMT: a circle of regulation. Journal of Cellular and Molecular Medicine 16, 445-455.
Mani, S. A., Guo, W., Liao, M. J., Eaton, E. N., Ayyanan, A., Zhou, A. Y., Brooks, M., Reinhard, F., Zhang, C. C., Shipitsin, M., et al. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704-715.
Mattern, R. H., Read, S. B., Pierschbacher, M. D., Sze, C. I., Eliceiri, B. P., and Kruse, C. A. (2005). Glioma cell integrin expression and their interactions with integrin antagonists.pdf. Cancer Therapy 3, 325-340.
Michor, F., Iwasa, Y., Vogelstein, B., Lengauer, C., and Nowak, M. A. (2005). Can chromosomal instability initiate tumorigenesis? Seminars in Cancer Biology 15, 43-49.
Olive, P. L. (1998). The Role of DNA Single- and Double-Strand Breaks in Cell Killing by Ionizing Radiatio. Radiation Research 150.
Osoegawa, A., Yoshino, I., Yamaguchi, M., Kameyama, T., Kometani, T., Kumamoto, Y., and Maehara, Y. (2008). Resection of radiation-induced sarcoma of the clavicle. Ann Thorac Cardiovasc Surg 14, 178-180.
Pala, A., Karpel-Massler, G., Kast, R. E., Wirtz, C. R., and Halatsch, M. E. (2012). Epidermal to Mesenchymal Transition and Failure of EGFR-Targeted Therapy in Glioblastoma. Cancers 4, 523-530.
Pardo, L. A., and Stuhmer, W. (2014). The roles of K(+) channels in cancer. Nature Reviews Cancer 14, 39-48.
Polyak, K., and Weinberg, R. A. (2009). Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nature Reviews Cancer 9, 265-273.
Ponnusamy, M. P., Seshacharyulu, P., Lakshmanan, I., Vaz, A. P., Chugh, S., and Batra, S. K. (2013). Emerging Role of Mucins in Epithelial to Mesenchymal Transition. Curr Cancer Drug Targets 13.
Rao, J. S. (2003). Molecular mechanisms of glioma invasiveness: the role of proteases. Nature Reviews Cancer 3, 489-501.
Rich, J. N. (2007). Cancer stem cells in radiation resistance. Cancer Research 67, 8980-8984.
Rich, J. N., and Bigner, D. D. (2004). Development of novel targeted therapies in the treatment of malignant glioma. Nature Reviews Drug discovery 3, 430-446.
Roscher, M., Hormann, I., Leib, O., Marx, S., Moreno, J., Miltner, E., and Friesen, C. (2013). Targeted alpha-therapy using [Bi-213] anti-CD20 as novel treatment option for radio- and chemoresistant non-Hodgkin lymphoma cells. Oncotarget 4, 218-230.
Roth, P., Silginer, M., Goodman, S. L., Hasenbach, K., Thies, S., Maurer, G., Schraml, P., Tabatabai, G., Moch, H., Tritschler, I., and Weller, M. (2013). Integrin control of the transforming growth factor-beta pathway in glioblastoma. Brain : a Journal of Neurology 136, 564-576.
Shieh, C. C., Coghlan, M., Sullivan, J. P., and Gopalakrishnan, M. (2000). Potassium channels: molecular defects, diseases, and therapeutic opportunities. Pharmacological Reviews 52, 557-594.
Singh, A., and Settleman, J. (2010). EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741-4751.
Sontheimer, H. (2008). A role for glutamate in growth and invasion of primary brain tumors. Journal of Neurochemistry 105, 287-295.
Steinle, M., Palme, D., Misovic, M., Rudner, J., Dittmann, K., Lukowski, R., Ruth, P., and Huber, S. M. (2011). Ionizing radiation induces migration of glioblastoma cells by activating BK K(+) channels. Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology 101, 122-126.
Sze, C. I., Su, W. P., Chiang, M. F., Lu, C. Y., Chen, Y. A., and Chang, N. S. (2013). Assessing current therapeutic approaches to decode potential resistance mechanisms in glioblastomas. Frontiers in Oncology 3, 59.
Thiery, J. P., Acloque, H., Huang, R. Y., and Nieto, M. A. (2009). Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890.
Tsai, J. H., and Yang, J. (2013). Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes & Development 27, 2192-2206.
Turner, K. L., and Sontheimer, H. (2014). Cl- and K+ channels and their role in primary brain tumour biology. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 369, 20130095.
Tysnes, B. B., and Mahesparan, R. (2001). Biological mechanisms of glioma invasion and potential therapeutic targets. Journal of Neuro-oncology 53, 129-147.
Wang, Y., and Jiang, T. (2013). Understanding high grade glioma: molecular mechanism, therapy and comprehensive management. Cancer Letters 331, 139-146.
Weller, M., and Wick, W. (2014). Neuro-oncology in 2013: improving outcome in newly diagnosed malignant glioma. Nature Reviews Neurology 10, 68-70.
Yan L, Xu G, Qiao T, Chen W, Yuan S, and X, L. (2013). CpG-ODN 7909 increases radiation sensitivity of radiation-resistant human lung adenocarcinoma cell line by overexpression of Toll-like receptor 9. Cancer Biother Radiopharm 28, 559-564.
Zhang, M., Kleber, S., Rohrich, M., Timke, C., Han, N., Tuettenberg, J., Martin-Villalba, A., Debus, J., Peschke, P., Wirkner, U., et al. (2011). Blockade of TGF-beta signaling by the TGFbetaR-I kinase inhibitor LY2109761 enhances radiation response and prolongs survival in glioblastoma. Cancer Research 71, 7155-7167.
Zhang, X., Fang, B., Mohan, R., and Chang, J. Y. (2012). Coxsackie-adenovirus receptor as a novel marker of stem cells in treatment-resistant non-small cell lung cancer. Radiotherapy and Oncology : journal of the European Society for Therapeutic Radiology and Oncology 105, 250-257.
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