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系統識別號 U0026-2207201117513100
論文名稱(中文) 探討在急性淋巴白血病中Aurora kinases抑制劑VE-465的作用機制
論文名稱(英文) Studying the molecular mechanism of Aurora kinase inhibitor, VE-465, in acute lymphoblastic leukemia (ALL)
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 99
學期 2
出版年 100
研究生(中文) 林慧如
研究生(英文) Hui-Ju Lin
學號 s26981050
學位類別 碩士
語文別 英文
論文頁數 82頁
口試委員 指導教授-洪良宜
指導教授-張文昌
口試委員-陳炳焜
中文關鍵字 VE-465 
英文關鍵字 VE-465 
學科別分類
中文摘要  許多文獻報導Aurora kinases 會在人類癌症中過度表現,包括肝癌、乳癌、直腸癌、胰臟癌、卵巢癌、胃癌和血癌。 Aurora kinases 的成員在細胞週期中扮演一個重要的調控角色,當Aurora kinases 有異常表現時,會造成基因的不穩定和細胞質分裂失敗,進而導致癌症。近幾年來,Aurora kinases已經成為具有展望性的小分子抑制劑的標的, 被用在癌症的標靶治療上。在我的論文當中,將急性淋巴白血病的細胞處理VE-465 – 一種Aurora kinases 抑制劑, 用以評估VE-465 在急性淋巴細胞中的效力及其分子作用機制。實驗成果發現, 細胞在處理 VE-465 後, 皆能有效地抑制Aurora kinases的活性,但是在九株急性淋巴白血病的細胞中,卻觀察到他們對藥物有不同的感受性。其中,RS4;11是對VE-465 有感受性的細胞,在處理VE-465之後,會使得細胞周期停留在G2/M,最後導致細胞死亡;而RPMI8402為對VE-465具抗藥性的細胞,細胞不會有死亡的現象,但是可觀察到多倍體細胞的產生。分析這些ALL細胞株Aurora kinases 及其活化因子,發現並無顯著性的差異。有趣的是,在藥物處理後,RS4;11細胞中CDKN1A的表現有顯著性地上升;相對地, RPMI8402 則維持在極低的表現。最後,利用在RS4;11中knockdown,以及在RPMI8402中overexpression CDKN1A 的實驗,我們證實了CDKN1A 的確參與在細胞對於VE-465不同的感受性中。
英文摘要  Overexpression of Aurora kinases has been reported in a variety of human cancers, including hepatoma, breast, colon, pancreatic, ovarian, stomach and leukemia. Members of Aurora kinases play important roles in cell-cycle control. Aberrant expression of Aurora kinases result in genomic instability, cytokinesis failure, and finally lead to tumorigenesis. Recently, Aurora kinases have been greaty expected to be a target for therapeutic purpose in cancer therapy. In this study, we used VE-465, a pan-Aurora kinases inhibitor, to treat human acute lymphoblastic leukemia (ALL) cancer cell lines and then to assess its efficacy and investigate the underline mechanism. Cells treated with VE-465 can inhibit the activation of Aurora kinases effectively. Among nine ALL cell lines, some of them are resistant to VE-465 treatment and others are sensitive. In the VE-465-sentive cell lines, the addition of VE-465 can result in an increased G2/M and sub-G1 populations. Analyzing the expression levels of Aurora kinases and their activators, it exists no difference between the two kinds of cells. Importantly, the mRNA and protein expression levels of CDKN1A are up-regulated upon VE-465 treatment in the drug-sensitive cell – RS4;11, but keep in a very low levels and no significance change under VE-465 treatment in the drug-resistance cell – RPMI8402. Here, we uncover the role of CDKN1A involved in the susceptibility for VE-465 treatment by the manner of overexpression and knockdown CDKN1A.
論文目次 中文摘要 I
ABSTRACT II
ACKNOWLEDGEMENT III
BIBLIOGRAPHY IV
LIST OF FIGURES VIII
LIST OF APPENDICES X
ABBREVIATIONS XI
I. INTRODUCTION 1
1-1. Cell cycle progression 1
1-2. Entry into each phase of the cell cycle is controlled 1
1-3. Cancer is a disease of uncontrolled cell proliferation 2
1-4. Aurora kinase family 2
1-5. Aurora kinases aberrations in human cancer 4
1-6. The potential of Aurora kinase inhibitors as a targeted cancer therapeutics 5
1-7. Philadelphia chromosome 6
1-8. Inhibitors of BCR/ABL 6
1-9. VX-680 (MK-0457) 7
1-10. Objectives of the present study 8
II. MATERIALS AND METHODS 9
2-1. Cell culture 9
2-2. Drug treatment 11
2-3. Oligonucleotide transfection 11
2-4. Transduction with lentivirus vectors 13
2-5. Cell proliferation assay 14
2-6. RNA extraction and Reverse transcriptase polymerase chain reaction 15
2-7. Western blotting 21
2-8. In vitro measurement of apoptosis 25
2-9. Cell cycle assay 26
2-10. Functional analyses 27
2-11. Statistical analysis 27
III. RESULTS 28
3-1. Expression levels of Aurora kinases in human acute lymphoblastic leukemia (ALL) cell lines 28
3-2. The cytotoxicity of VE-465 in nine human acute lymphoblastic leukemia (ALL) cancer cell lines 28
3-3. Lower poliferative cells have high sensitivity to VE-465 29
3-4. Activation status of Aurora kinases and expression levels of their activator 30
3-5. VE-465 can efficiently inhibit the Aurora kinases’ activity in ALL cell lines 31
3-5. VE-465 suppresses of the activities of Aurora kinases in RS4;11 VE-465-sensitive cell line and RPMI8402 VE-465-resistant cell line 31
3-6. Effect of VE-465 on growth arrest 32
3-7. VE-465 induces G2/M arrest in RS4;11 and polypoidy in RPMI 8402 32
3-8. VE-465 induces a cell cycle distribution change 33
3-9. Role of Aurora kinase inhibition-mediated apoptosis in the response to VE-465 34
3-10. Identification of genes that confer cell sensitivity to VE-465 35
3-11. RS4;11 expresses a higher level of CDKN1A (p21) than RPMI8402 35
3-12. Down-regulation of p21 by a shRNA blunts the ability of VE-465 to induce apoptosis of RS4;11 cells 36
3-13. Overexpression of CDKN1A can modulate the drug susceptibility of cells 37
3-14. Role of p53 in the response to Aurora kinase inhibition by VE-465 37
3-15. VE-465 has activity in a solid tumor 38
3-16. The increased expression of CDN1A might contribute to VE-465 sensitivity also observed in colon cancer cells 39
IV. DISCUSSION 40
4-1. VE-465 is a candidate in MLL-AF4 acute lymphocytic leukemia 40
4-2. VE-465 induces apoptosis in Aurora A-low cells 40
4-3. The potential anticancer effects of VE-465 42
4-4. The molecular mechanism of Aurora kinase inhibitor, VE–465, in acute lymphoblastic leukemia (ALL) 43
V. CONCLUSIONS 45
VI. PROSPECT 46
VII. REFERENCE 47
Fig. 1 The expression levels of Aurora kinases in the tested cell lines 54
Fig. 2 The effects of VE-465 on human acute lymphoblastic leukemia (ALL) cancer cell lines. 55
Fig. 3 Antiproliferative activity of VE-465 versus VX-680. 56
Fig. 4 The poliferative rate in RS4;11 and RPMI8402 cells. 57
Fig. 5 The expression status of phosphorylated Aurora A and B and the activators of Aurora kinases. 58
Fig. 6 Efficacy of VE-465 on Aurora kinases. 59
Fig. 7 Evaluation of the inhibition of Aurora kinase 60
Fig. 8 VE-465 induces the phosphorylation of Histone H3. 61
Fig. 9 VE-465 affects Cell-cycle distribution. 62
Fig. 10 VE-465 affects Cell-cycle distribution. 63
Fig. 11 VE-465 induces caspase 3 activation. 64
Fig. 12 Functional analysis from IPA in dataset-1 65
Fig. 13 Functional analysis from IPA in dataset-2 66
Fig. 14 Integrated miRNA and mRNA expression by bioinformatics analyses. 67
Fig. 15 Analysis of TP53 downstream genes. 68
Fig. 16 Effect of CDKN1A silencing. 69
Fig. 17 Effect on ectopic p21 expression. 70
Fig. 18 Analysis of TP53 expression. 71
Fig. 19 The different VE465 sensitivity of human colorectal cancer cell lines – SW480 and SW620 is due to the different expression levels of CDKN1A. 72
Fig. 20 Overexpression of CDKN1A. 73
Appendix I: The development of Aurora kinase inhibitors 74
Appendix II: The information of the nine cell lines, by which cell viability assay, was carried out. 75
Appendix III: Status of TP53 in RS4;11 and RPMI8402 cells and comparision of RS4;11 and RPMI8402 cells 76
Appendix IV: TP53 regulated signaling pathway 77
Appendix V: (A) The effect of VE-465 and VX-680 as well as (B) the expression level of p21 in MLL-AF4 leukemia cells 78
Appendix VI: Inhibition of Aurora A in RS4;11 cells 79
Appendix VII: Inhibition of Aurora B in RPMI8402 cells 80
Appendix VIII: The potential anticancer effects of VE-465 81
自述 82

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