前言：多型性膠質母細胞瘤 (GBM)在世界衛生組織的分級中屬於第四級的星狀細胞瘤 (astrocytoma)，是一種非常惡性的腦瘤。儘管目前在治療上有手術切除、放射線治療與化療等方式，但仍無法完全治癒而且產生抗藥性的機率非常高，使得GBM更加難以治療。近年來，GBM幹細胞 (GSCs) 的存在被認為是抗藥性產生的一大主因。其中，文獻證實幹細中的特定基因如Bmi-1的表現量會大幅提升並且參與在藥物抗藥性能力的發展。
方法：在本篇研究中，我們將目標放在乙酰苯胺羥肟酸 SAHA上，SAHA是一個組蛋白去乙醯酶抑制劑。為了探討SAHA如何抑制Bmi-1的表現，我們利用螢光素酶檢測法、 DNA親和性沉澱測定法 (DAPA)和免疫沉澱分析法 (IP) 來探討其中的分子機轉。在動物實驗中，我們將具有TMZ抗藥性的GBM細胞植入老鼠 (SCID mice)背部的皮下並分成兩組，一組給予TMZ另一組給予SAHA加上TMZ。我們觀察這兩組之間的腫瘤大小差異。
結果：在確認所建立的具有TMZ抗藥性的GBM細胞確實對TMZ不敏感後，我們發現SAHA可以有效的抑制這些具有抗藥性的細胞的生長。另一方面，我們發覺相較於一般的GBM細胞，一些在癌症幹細胞中大量表現的特定幹細胞標記也在這群具有TMZ抗藥性的GBM中有增加的趨勢。因此我們認定這些抗藥性的細胞可能具有較多GSCs的能力。所以我們建立了幹細胞的模式並給予SAHA做治療。我們發現SAHA可以抑制GSCs的生長和其幹細胞標記的蛋白質表現量。由於文獻證實轉錄因子Sp1可以調控Bmi-1的表現，我們利用DAPA assay發現SAHA可以抑制Sp1連接到Bmi-1的啟動子。而且IP assay的結果顯示SAHA可以抑制組蛋白去乙醯酶而使Sp1的蛋白質維持在高度乙醯化的狀態。另有文獻證實 Sp1蛋白質的乙醯化程度增加會抑制其連接到DNA的能力。 因此，我們透過以上實驗及文獻證實SAHA增加Sp1蛋白質的乙醯化程度會抑制Sp1連結到Bmi-1的能力進而降低了Bmi-1的表現量。在動物模式中，我們發現經過 SAHA搭配上TMZ的治療後，腫瘤的大小相較於只給予TMZ的組別有比較小的趨勢。
結論：透過SAHA增加Sp1蛋白質的乙醯化程度，可以抑制Sp1連結到Bmi-1的能力進而降低了Bmi-1的表現量。我們的結果提供了在治療具有TMZ抗藥性的GBM腫瘤中，SAHA搭配上TMZ或許是一種新的治療方法。

Introduction: GBM, known as a grade IV astrocytoma, is the most aggressive brain tumor. Although GBM patients receive the standard treatment including surgery, radiation therapy, and chemotherapy, they are never considered curable and the tumors become more resistant to therapy. Recently, GSCs have been suggested to correlate with therapeutic resistance. Bmi-1 is upregulated in GSCs and controls multi-drug resistance.
Method: In this study, we focused on SAHA with functions in altering gene expression by the inhibition of histone deacetylases. We used reporter assay, DAPA assay, and IP assay to investigate the molecular mechanisms. In vivo, we injected TMZ resistant GBM cells subcutaneously into SCID mice, and separated into two groups, control (TMZ) and experimental (TMZ+SAHA). We compared tumor sized between two groups.
Results: TMZ resistant GBM cells were less responsive to TMZ, and SAHA inhibited the cell growth of TMZ resistant GBM cells. We found that the expressions of stem cell markers such as Bmi-1, CD133, and SOX2 were higher in TMZ resistant GBM cells than wild type GBM cells. Therefore, we considered TMZ resistant GBM cells as GSCs. We found that SAHA inhibited the expressions of Bmi-1 and other stem cell markers in GSCs. Transcription factor Sp1 acted as an essential upstream regulator of Bmi-1, and we used DAPA assay to prove that SAHA inhibited the binding ability of Sp1 to Bmi-1. Furthermore, we used IP assay to confirm that SAHA induced Sp1 acetylation. Induction of Sp1 acetylation reduced the DNA binding ability of Sp1. Taken together, we found that SAHA reduced the expression of Bmi-1 by inducing Sp1 protein acetylation. In xenograft mouse model, we found SAHA combined with TMZ had smaller tumor sizes than TMZ only group.
Conclusion: Sp1 acted as an essential upstream regulator of Bmi-1, and SAHA induced Sp1 acetylation and attenuated its expression resulting in the reduction of Bmi-1 transcription. Our results provide a perspective on combining SAHA with TMZ to inhibit GCSs and to enhance TMZ sensitivity in drug-resistant GBM.

1. Acknowledgments….………………………………………………....4
2. Abbreviation……………………………………….………………….5
3. Abstract
3.1 English abstract………………………………………………………………6
3.2 Chinese abstract……………………………………………………………...7
4. Introduction
4.1 GBM…………………….……………………………………………………8
4.2 Drug resistance of GBM……………………………………………………9
4.3 Cancer stem cells……………………………………………………………10
4.4 SAHA.........................................................…………………………………11
4.5 Transcription factor Sp1……………………….…...………………………12
5. Materials and Methods
5.1 Material resources…………………………………………………………..15
5.2 The preparation of chemicals……………………………………………….17
5.3 Cell culture………………………………………………………………….17
5.4 Plasmid DNA construction………………………………………………….18
5.5 Transfection…………………………………………………………………24
5.6 Western blotting…………………………………………………………….24
5.7 DAPA assay…………………………..……………………………………..25
5.8 IP assay……………………………………………………………………..26
5.9 MTT assay…………………………………………………………………..26
5.10 Reporter assay……………………………………………………………..26
5.11 PCR………………………………………………………………………27
5.12 SiRNA knockdown………………………………………………………...28
5.13 Nuclear extraction…………………………………………………………28
5.14 Animal model……………………………………………………………...29
5.15 IHC stain……………………….………………………………………..30
5.16 Statistical analysis ………………………………………………………...30
6. Results
6.1 TMZ resistant GBM cells are less responsive to TMZ treatment than wild type GBM cells. ……………………………………………………………31
6.2 SAHA inhibits cell growth of wild type and TMZ resistant GBM cells. …..31
6.3 Stem cell markers increase in the TMZ resistant GBM cells and GSCs. …..32
6.4 SAHA inhibits not only the expression of stem cell markers but also the cell growth of GSCs and TMZ resistant cells. ………………………………….32
6.5 SAHA regulates the transcriptional level of Bmi-1 through Sp1. ………….33
6.6 SAHA reduces the binding ability of Sp1 to Bmi-1 promoter region. ……..33
6.7 SAHA treatment attenuates Sp1 protein expression and its acetylation. …..34
6.8 Acetylation level of Sp1 in TMZ resistant GBM cells is lower than wild type GBM cells. …………………………………………………………………34
6.9 Sp1 and Bmi-1 are important in TMZ drug resistance. ……………………35
6.10 The inhibition of Sp1 and Bmi-1 suppress the sphere formation of GSCs. 35
6.11 SAHA combining with TMZ inhibits the tumor growth and the expression of Sp1 and Bmi-1 of TMZ resistant GBM tumor in vivo. ………………….36
7. Discussion……………………………………………………...........37
8. Reference……………………………………………………………39
9. Data…………………………………………………………….……42
10. Appendix……………………………………………………….……55
11. Personal Information………………………………………………...61
12. Awards in Graduate School………………………………………….62

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