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系統識別號 U0026-1706201915390400
論文名稱(中文) 探討物理微環境刺激對替莫唑胺誘導抗性神經膠質母細胞瘤的影響
論文名稱(英文) The effects of physical microenvironment stimuli on Temozolomide-induced resistant glioblastoma
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 107
學期 2
出版年 108
研究生(中文) 張謦讌
研究生(英文) Cing-Yan Jhang
學號 T96054028
學位類別 碩士
語文別 英文
論文頁數 39頁
口試委員 指導教授-司君一
口試委員-王仰高
口試委員-鄧志娟
中文關鍵字 膠質母細胞瘤  替莫唑胺誘導抗性  微環境  醛脫氫酶1  自噬  間質分化 
英文關鍵字 Glioblastoma  Temozolomide-induced resistant  microenvironment  ALDH1  autophagy  mesenchymal differentiation 
學科別分類
中文摘要 神經膠質母細胞瘤(GBM)是原發型惡性腦腫瘤,在2016年世界衛生組織(WHO)定義為第四級的膠質瘤,目前標準治療神經膠質母細胞瘤病人的方式,是以手術摘除後給予放射線及化療藥物輔助治療。帝盟多(TMZ)是口服烷基化試劑和神經膠質母細胞瘤的標準化療藥劑。帝盟多會甲基化去氧核醣核酸(DNA)並影響其複製機制進而導致細胞死亡。在神經膠質母細胞瘤的治療中會產生帝盟多的抗性。醛脫氫酶1(ALDH1)屬醛脫氫酶酵素家族之一,負責催化細胞內醛的氧化,進而成為神經膠質母細胞瘤治療的主要障礙之一。醛脫氫酶1直接參與神經膠質母細胞瘤治療抗性的產生,並在化療期間受自噬調節。自噬有助於癌細胞克服癌症惡化過程中的壓力。到目前為止,少有論文研究神經膠質母細胞瘤在治療抵抗過程涉及自噬和上皮-間質轉化(EMT)的過程。上皮-間質轉化或所謂的間質分化(MD)過程使上皮細胞轉化為間質細胞。間質分化的可逆性顯示環境信號與侵襲性腫瘤表型有強烈相關性。在化學抗性和惡性進展的過程中,腫瘤微環境有著關鍵作用。最近的研究發現,細胞外基質硬度的提高可獨立促進神經膠質母細胞瘤的侵襲。因此,我們假設當神經膠質母細胞瘤接受微環境的物理環境改變時,ALDH1、自噬和間質分化可能在帝盟多誘導的抗性過程中發生改變。本實驗我們使用了U87MG及1306MG兩種人類神經膠質母細胞瘤細胞株,並探討兩種細胞株的不同。首先,結果發現在具有帝盟多抗性的神經膠質母細胞瘤細胞株中ALDH1、自噬和間質分化都有增強的表現。然後我們建立了兩種不同硬度的聚丙烯酰胺(PA)凝膠來培養親代神經膠質母細胞瘤細胞株和具有帝盟多抗性的神經膠質母細胞瘤細胞株。我們使用原子力顯微鏡(AFM)測量建立的PA凝膠及控制組的玻片,其量測結果硬的凝膠為10.5 kPa、軟的為0.1 kPa及玻片為181.8 kPa。在軟凝膠的柔軟度相當於人類腦組織的軟度,然而在硬凝膠的硬度相當於人類皮膚或肌肉組織。我們觀察到兩種皆具有帝盟多抗性的神經膠質母細胞瘤(TIR-GBM)細胞株在軟凝膠中培養後,細胞形態從紡錘狀變為圓形,並且尺寸變得更小。在硬凝膠中培養發現,具有抗性的U87MG細胞面積皆顯著變小;在具有抗性的1306MG方面,細胞型態則有顯著拉長。最後,我們證明了具有帝盟多抗性的神經膠質母細胞瘤對於環境的反應不同。在親代細胞系中,自噬標記物Beclin和LC3顯示低表達水平,然而,它在抗性細胞系中增加。當TIR-U87MG在軟凝膠上生長時,LC3陽性細胞增加,然而,TIR-1306MG中陽性細胞的數量減少。當TIR細胞系在硬凝膠中生長時,自噬標記物沒有顯著的差異。當具有TMZ抗性的U87MG在軟凝膠中生長時,ALDH1,自噬,間充質標記物 - 纖連蛋白和波形蛋白的螢光強度增強,但在硬凝膠中未發現。當TIR-U87MG在軟凝膠中生長時,在螢光強度ALDH1、自噬、間質標誌-的都有增強的現象,在硬凝膠中則無此現象。此外,我們發現在上皮標誌 β-catenin的螢光強度降低,而Fibronectin和Vimentin則增加。結果顯示當環境變為軟凝膠時,某些細胞表型會發生變化,ALDH1、自噬、間質標誌-Fibronectin和Vimentin的螢光強度在TIR-1306MG呈現下降趨勢,且在硬介質也有一樣的發現。在TIR-1306MG培養在軟凝膠後,β-catenin表現顯著增加,但在硬介質無此現象。這些結果也清楚地表明,當U87MG與1306MG相比時,對培養的微環境表現出不同的反應。總結,當神經膠質母細胞瘤在化學抗性的軟環境或硬環境中生長時,確實產生ALDH1不同的表現並且可能促使或削弱自噬及間質分化的細胞反應。
英文摘要 Glioblastoma (GBM) is the primary malignant brain tumor. In 2016, the World Health Organization (WHO) define GBM as the grade IV glioma. The current standard of care for GBM patients are surgical resection followed by adjuvant radiation therapy and chemotherapy. Temozolomide (TMZ) is the oral alkylating agent and GBM chemotherapy standard agent. TMZ methylates DNA and interferes with replication that results in cell death. Resistance to TMZ is one of the major barrier for GBM therapy. Aldehyde dehydrogenase 1 (ALDH1) is a member of the ALDH enzyme family, which catalyzes the oxidation of intracellular aldehydes. ALDH1 is directly involved in therapy resistance of GBM, and is regulated by autophagy during chemotherapy. Autophagy helps cancer cells to overcome the stressful conditions during cancer progression. Up to data, a few papers show that in the development of therapeutic resistant GBM involve the autophagy and epithelial-mesenchymal transition (EMT) processes. EMT or so called mesenchymal differentiation (MD) process in gliomas that makes epithelial cells transform to mesenchymal cells. The reversibility of MD suggests that environmental signals strongly correlate with aggressive tumor phenotypes. Tumor microenvironment plays important role in the development of chemoresistance and in malignant progression. The recent studies showed that elevated extracellular matrix stiffness independently foster GBM aggression. Thus, we hypothesize that when GBM grows in altered microenvironment with different stiffness, ALDH1, autophagy, and MD maybe altered in parental and TMZ-induced resistant GBM (TIR-GBM) cell lines. In this experiment, we used U87MG and 1306MG human GBM cell lines to develop TIR-GBM. First, we found that autophagy and MD markers were enhanced in TIR-GBM cell lines. Then we established two different stiffness polyacrylamide (PA) gels to culture parental and TIR-GBM cell lines. By using atomic force microscope (AFM) to measure gel stiffness and control glass, we demonstrated that the stiffness of hard matrix was 10.5 kPa, soft PA gel was 0.1 kPa, and glass was 181.8 kPa. The soft matrix softness was similar to human brain tissue, and the hard substrate stiffness was similar to human skin or muscle tissue. We observed that TIR-GBM cell lines cultured on soft substrate, morphology of the cells changed from spindle to round shape and have smaller size. In hard substrate, the cellular area of resistant U87MG became smaller, and length of resistant 1306MG became longer. Finally, we demonstrated that TIR-U87MG showed different responses with microenvironment. In parental cell lines, autophagy markers Beclin and LC3 showed low expression levels, however, it was increased in resistant cell lines. When TIR-U87MG grew on soft gel, LC3 positive cells were increased, however, numbers of positive cells in TIR-1306MG were reduced. There was no significant alternation in autophagy markers when TIR cell lines grew on hard gel. When U87MG with TMZ-resistance was grew on a soft matrix, the fluorescence intensity of ALDH1, autophagy, mesenchymal markers-Fibronectin and Vimentin were enhanced, but it not found in hard gels. On the contrary, epithelium marker β-catenin, fluorescence intensity was reduced. However, Fibronectin and Vimentin were increased. The results indicated when environment changed to soft gel certain cellular phenotypes are altered, such as ALDH1, autophagy, Fibronectin and Vimentin intensity were decreasd in TIR-1306MG. It also found in hard gels. β-catenin was significant increase after TIR-1306MG cultured on soft matrix, but it not found in hard gels. These results also clearly demonstrated that when U87MG compared with 1306MG showed different responses to the cultured microenvironments. In summary, when GBM grow on soft or hard environmental in chemoresistant, it indeed resulted in changes in ALDH1 expression, and may either promote or diminished autophagy and MD processes.
論文目次 Abstract I
Chinese abstract III
Acknowledgement V
Table of Contents VI
Introduction 1
Glioblastoma (GBM) 1
Temozolomide (TMZ) resistant in GBM 1
Aldehyde dehydrogenase 1 (ALDH1) in GBM 2
Autophagy in GBM 2
Mesenchymal differentiation (MD) in GBM 3
Autophagy and MD processes in GBM 3
The tumor microenvironment and extracellular matrix (ECM) 4
Gliomas develop within microenvironment 4
Objective and specific aims 6
Materials and Methods 7
Cell culture 7
Establishment of TMZ-induced resistant GBM (TIR-GBM) cell lines. 7
Establishment and functionalization of polyacrylamide (PA) gel 7
Trypan Blue Exclusion Assay 8
Western blotting analysis 8
Immunofluorescent staining 9
Statistical Analysis 9
Results 10
Different parental GBM cell lines display different protein expression patterns 10
ALDH1 expression levels in parental and TMZ induce resistant GBM (TIR-GBM) 10
Autophagic status of TIR-GBM 11
Mesenchymal differentiation (MD) status of TIR-GBM 11
Establishment of different stiffness microenvironment for GBM cell 11
The survival of parental and TIR-GBM cell on PA gels with different stiffness 12
The morphological changes of the parental and TIR-GBM cell lines grew on different stiffness PA gels 12
The altered expression of ALDH1, autophagy, and MD markers in parental and TIR-U87MG cultured on PA gels with different stiffness 13
The altered expression of ALDH1, autophagy, and MD markers in parental and TIR-1306MG cultured on different stiffness PA gels 14
Discussion 15
Conclusion 18
References 19
Figures 23
Figure 1. U87MG and 1306MG GBM cell lines displayed different protein expression patterns. 23
Figure 2. GBM cells with TMZ resistant progression altered ALDH1 expression. 24
Figure 3. GBM cells with TMZ resistant altered autophagy. 25
Figure 4. Observed GBM cells with TMZ resistant progression that altered mesenchymal differentiation. 27
Figure 5. Established stiffness microenvironment that cultured GBM cells with TMZ resistant progression. 29
Figure 6. The morphological changes of the parental and TIR-GBM cell lines grew on different stiffness PA gels. 31
Figure 7. The expression patterns in U87MG cell with TMZ-resistant was cultured on PA gels with different stiffness. 34
Figure 8. The expression patterns in 1306MG cell with TMZ resistant was cultured on PA gels with different stiffness. 36
Figure 9. Illustrated the effects of physical microenvironment stimuli on Temozolomide-induced resistant glioblastoma 37
參考文獻 1. Tian. T, Mingyi. M, Qiu. X, Qiu. Y. MicroRNA-101 reverses temozolomide resistance by inhibition of GSK3β in glioblastoma. Oncotarget. 2016;7(48).
2. Lim E-J, Suh Y, Kim S, Kang S-G, Lee S-J. Force-mediated proinvasive matrix remodeling driven by tumor-associated mesenchymal stem-like cells in glioblastoma. BMB Reports. 2018;51(4):182-7.
3. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016;131(6):803-20.
4. Yu F, Li G, Gao J, Sun Y, Liu P, Gao H, et al. SPOCK1 is upregulated in recurrent glioblastoma and contributes to metastasis and Temozolomide resistance. Cell Prolif. 2016;49(2):195-206.
5. Ashizawa T, Akiyama Y, Miyata H, Iizuka A, Komiyama M, Kume A, et al. Effect of the STAT3 inhibitor STX-0119 on the proliferation of a temozolomide-resistant glioblastoma cell line. Int J Oncol. 2014;45(1):411-8.
6. Rasper M, Schafer A, Piontek G, Teufel J, Brockhoff G, Ringel F, et al. Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity. Neuro Oncol. 2010;12(10):1024-33.
7. Huang R, Li X, Holm R, Trope CG, Nesland JM, Suo Z. The expression of aldehyde dehydrogenase 1 (ALDH1) in ovarian carcinomas and its clinicopathological associations: a retrospective study. BMC Cancer. 2015;15:502.
8. Wu W, Schecker J, Wurstle S, Schneider F, Schonfelder M, Schlegel J. Aldehyde dehydrogenase 1A3 (ALDH1A3) is regulated by autophagy in human glioblastoma cells. Cancer Lett. 2018;417:112-23.
9. Zhao. Z, Zhao. J, Xue. J, Zhao. X, Liu P. Autophagy inhibition promotes epithelial-mesenchymal transition through ROS/HO-1 pathway in ovarian cancer cells. Am J Cancer Res. 2016;6(10):2162-77.
10. Piao S, Ojha R, Rebecca VW, Samanta A, Ma XH, McAfee Q, et al. ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells. Autophagy. 2017;13(12):2056-71.
11. Lu Y, Xiao L, Liu Y, Wang H, Li H, Zhou Q, et al. MIR517C inhibits autophagy and the epithelial-to-mesenchymal (-like) transition phenotype in human glioblastoma through KPNA2-dependent disruption of TP53 nuclear translocation. Autophagy. 2015;11(12):2213-32.
12. Zhang P, Sun S, Li N, Ho ASW, Kiang KMY, Zhang X, et al. Rutin increases the cytotoxicity of temozolomide in glioblastoma via autophagy inhibition. J Neurooncol. 2017;132(3):393-400.
13. Yoshida GJ. Therapeutic strategies of drug repositioning targeting autophagy to induce cancer cell death: from pathophysiology to treatment. J Hematol Oncol. 2017;10(1):67.
14. Gugnoni M, Sancisi V, Gandolfi G, Manzotti G, Ragazzi M, Giordano D, et al. Cadherin-6 promotes EMT and cancer metastasis by restraining autophagy. Oncogene. 2017;36(5):667-77.
15. Gugnoni M, Sancisi V, Manzotti G, Gandolfi G, Ciarrocchi A. Autophagy and epithelial-mesenchymal transition: an intricate interplay in cancer. Cell Death Dis. 2016;7(12):e2520.
16. Yamini B. NF-kappaB, Mesenchymal Differentiation and Glioblastoma. Cells. 2018;7(9).
17. Bartis D, Mise N, Mahida RY, Eickelberg O, Thickett DR. Epithelial-mesenchymal transition in lung development and disease: does it exist and is it important? Thorax. 2014;69(8):760-5.
18. Moreno M, Pedrosa L, Pare L, Pineda E, Bejarano L, Martinez J, et al. GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma. Cell Rep. 2017;21(8):2183-97.
19. Mathias RA, Gopal SK, Simpson RJ. Contribution of cells undergoing epithelial-mesenchymal transition to the tumour microenvironment. J Proteomics. 2013;78:545-57.
20. Morandi A, Taddei ML, Chiarugi P, Giannoni E. Targeting the Metabolic Reprogramming That Controls Epithelial-to-Mesenchymal Transition in Aggressive Tumors. Front Oncol. 2017;7:40.
21. Chen HT, Liu H, Mao MJ, Tan Y, Mo XQ, Meng XJ, et al. Crosstalk between autophagy and epithelial-mesenchymal transition and its application in cancer therapy. Mol Cancer. 2019;18(1):101.
22. Colella B, Faienza F, Di Bartolomeo S. EMT Regulation by Autophagy: A New Perspective in Glioblastoma Biology. Cancers (Basel). 2019;11(3):312.
23. Barnes JM, Przybyla L, Weaver VM. Tissue mechanics regulate brain development, homeostasis and disease. J Cell Sci. 2017;130(1):71-82.
24. Janji B, Berchem G, Chouaib S. Targeting Autophagy in the Tumor Microenvironment: New Challenges and Opportunities for Regulating Tumor Immunity. Front Immunol. 2018;9:887.
25. Koh I, Cha J, Park J, Choi J, Kang SG, Kim P. The mode and dynamics of glioblastoma cell invasion into a decellularized tissue-derived extracellular matrix-based three-dimensional tumor model. Sci Rep. 2018;8(1):4608.
26. Cha J, Kim P. Biomimetic Strategies for the Glioblastoma Microenvironment. Frontiers in Materials. 2017;4.
27. Mehta S, Lo Cascio C. Developmentally regulated signaling pathways in glioma invasion. Cell Mol Life Sci. 2018;75(3):385-402.
28. Lin H-H, Lin H-K, Lin I-H, Chiou Y-W, Chen H-W, Liu C-Y, et al. Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing. Oncotarget. 2015;6(25):20946.
29. Senthebane DA, Rowe A, Thomford NE, Shipanga H, Munro D, Mazeedi M, et al. The Role of Tumor Microenvironment in Chemoresistance: To Survive, Keep Your Enemies Closer. Int J Mol Sci. 2017;18(7).
30. Park KM, Gerecht S. Polymeric hydrogels as artificial extracellular microenvironments for cancer research. Eur Polym J. 2015;72:507-13.
31. Miroshnikova YA, Mouw JK, Barnes JM, Pickup MW, Lakins JN, Kim Y, et al. Tissue mechanics promote IDH1-dependent HIF1alpha-tenascin C feedback to regulate glioblastoma aggression. Nat Cell Biol. 2016;18(12):1336-45.
32. Jawhari S, Bessette B, Hombourger S, Durand K, Lacroix A, Labrousse F, et al. Autophagy and TrkC/NT-3 signaling joined forces boost the hypoxic glioblastoma cell survival. Carcinogenesis. 2017;38(6):592-603.
33. Inukai M, Hara A, Yasui Y, Kumabe T, Matsumoto T, Saegusa M. Hypoxia-mediated cancer stem cells in pseudopalisades with activation of hypoxia-inducible factor-1alpha/Akt axis in glioblastoma. Hum Pathol. 2015;46(10):1496-505.
34. Xiao W, Zhang R, Sohrabi A, Ehsanipour A, Sun S, Liang J, et al. Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma. Cancer Res. 2018;78(5):1358-70.
35. Shen X, Kan S, Liu Z, Lu G, Zhang X, Chen Y, et al. EVA1A inhibits GBM cell proliferation by inducing autophagy and apoptosis. Exp Cell Res. 2017;352(1):130-8.
36. Ciechomska IA, Przanowski P, Jackl J, Wojtas B, Kaminska B. BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells. Sci Rep. 2016;6:38723.
37. Colella B, Faienza F, Carinci M, D'Alessandro G, Catalano M, Santoro A, et al. Autophagy induction impairs Wnt/beta-catenin signalling through beta-catenin relocalisation in glioblastoma cells. Cell Signal. 2019;53:357-64.
38. Ulrich TA, de Juan Pardo EM, Kumar S. The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells. Cancer Res. 2009;69(10):4167-74.
39. Mattern R-H, Read SB, Pierschbacher MD, Sze C-I, Eliceiri BP, Kruse CA. Glioma cell integrin expression and their interactions with integrin antagonists. Cancer Ther. 2005;3:325.
40. Xiao W, Sohrabi A, Seidlits SK. Integrating the glioblastoma microenvironment into engineered experimental models. Future science OA. 2017;3(3):FSO189.
41. Mierke CT, Frey B, Fellner M, Herrmann M, Fabry B. Integrin alpha5beta1 facilitates cancer cell invasion through enhanced contractile forces. J Cell Sci. 2011;124(Pt 3):369-83.
42. Ulbricht A, Eppler FJ, Tapia VE, van der Ven PF, Hampe N, Hersch N, et al. Cellular mechanotransduction relies on tension-induced and chaperone-assisted autophagy. Curr Biol. 2013;23(5):430-5.
43. Corsi L, Mescola A, Alessandrini A. Glutamate Receptors and Glioblastoma Multiforme: An Old "Route" for New Perspectives. Int J Mol Sci. 2019;20(7).
44. Fernandez-Fuente G, Mollinedo P, Grande L, Vazquez-Barquero A, Fernandez-Luna JL. Culture dimensionality influences the resistance of glioblastoma stem-like cells to multikinase inhibitors. Mol Cancer Ther. 2014;13(6):1664-72.
45. Erickson AE, Lan Levengood SK, Sun J, Chang FC, Zhang M. Fabrication and Characterization of Chitosan-Hyaluronic Acid Scaffolds with Varying Stiffness for Glioblastoma Cell Culture. Adv Healthc Mater. 2018;7(15):e1800295.
46. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO Classification of Tumours of the Central Nervous System. Acta Neuropathol. 2007;114(2):97-109.
47. Young RM, Jamshidi A, Davis G, Sherman JH. Current trends in the surgical management and treatment of adult glioblastoma. Ann Transl Med. 2015;3(9):121.
48. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature. 2009;462(7274):739-44.
49. Li S, Chou AP, Chen W, Chen R, Deng Y, Phillips HS, et al. Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation. Neuro Oncol. 2013;15(1):57-68.
50. Andronesi OC, Kim GS, Gerstner E, Batchelor T, Tzika AA, Fantin VR, et al. Detection of 2-Hydroxyglutarate in IDH-Mutated Glioma Patients by In Vivo Spectral-Editing and 2D Correlation Magnetic Resonance Spectroscopy. Sci Transl Med. 2012;4(116):116ra4-ra4.
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