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研究生:連文慧
研究生(外文):Wen-Hui Lien
論文名稱:探討TNP-470抑制血管新生的分子機轉
論文名稱(外文):Molecular mechanism of angiogenic inhibition by TNP-470 in human endothelial cells
指導教授:吳梨華
指導教授(外文):Li-Wha Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:58
中文關鍵詞:分子機轉
外文關鍵詞:TNP-470cyclin D1p21p53
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腫瘤的生長及轉移是依賴血管新生的,所謂「血管新生」是指由已存在的血管生成新血管的過程。因此,抑制血管新生成為治療癌症的策略。TNP-470是fumagillin的衍生物,具有有效抑制血管新生的活性,並且在動物測試上具有較低的毒性。由先前的研究發現TNP-470能夠透過抑制內皮細胞的生長,而達到抑制腫瘤的生長及轉移;如今,TNP-470已進入人體測試第三期。雖然如此,但對於TNP-470抑制血管新生的分子機制的了解卻是有限的。所以,我們研究的主要目的是要了解TNP-470在基因調控上的作用,及在抑制人類內皮細胞上可能的調控機制。在我們的研究中,我們證明了TNP-470能夠抑制兩個由血管新生因子bFGF所誘導表現的轉錄子,egr-1及junB。當egr-1被過度表現在內皮細胞時,會增加細胞對TNP-470的抵抗作用,意味著egr-1可能在 TNP-470的抑制作用中扮演一個負面調控的角色;不同於egr-1的是,當junB被過度表現在內皮細胞時,會抑制內皮細胞的生長,這與junB在調控細胞生長存活的負面角色是一致的。另外,由西方點墨法的結果得知:p53,p21及cyclin D1的表現受到TNP-470的調控而增加;更加證明TNP-470誘導p21表現在早期是p53-independent的,而在晚期則是p53-dependent的。另一方面,發現TNP-470也能夠誘導cyclin D1累積在細胞核中,且受到誘導的cyclin D1會與不活化的CDK4/6復合體相繫在一起。綜合所有的發現,我們推論TNP-470抑制細胞在G1期生長的機制,不只是透過誘導p53及p21的表現,同時也增加cyclin D1在細胞核的累積。由此可知,cyclin D1在血管新生的調控中是一個相當共通的標的。
Tumor growth and metastasis depends on angiogenesis, formation of new blood vessels from pre-existing capillaries. Inhibition of angiogenesis is emerging as a promising strategy for the treatment of cancer. TNP-470, a synthetic derivative of fumagillin, possesses potent anti-angiogenic activity and has low toxicity in animal studies. Previous studies have shown that TNP-470 inhibits the growth and metastasis of solid tumors through inhibiting endothelial cell growth. TNP-470 is now in the phaseⅢ of human clinical trials. However, the molecular action whereby TNP-470 inhibits angiogenesis is limited. Therefore, the specific aims of our study were to understand the effects of TNP-470 on gene expression and the possible mechanism in the inhibitory effect mediated by TNP-470 on human endothelial cells. In our studies, we demonstrated that TNP-470 repressed bFGF-induced expression of two transcription factors, egr-1 and junB. Overexpression of egr-1 in endothelial cells increased the resistance to TNP-470, suggesting that egr-1 may play a negative regulator in the inhibition of TNP-470. Unlike egr-1, overexpression of junB in endothelial cells decreased HUVEC survival even in the absence of TNP-470. This notion is consistent with that junB behaves as a negative regulator of cell survival. In addition, western blotting analysis indicated that the expressions of p53, p21 and cyclin D1 were increased by TNP-470. Moreover, TNP-470-induced p21 expression was p53- independent at early time points, but p53-dependent at late time points. TNP-470 also induced the accumulation of cyclin D1 in the nucleus. The accumulated cyclin D1 was associated with the inactive form CDK4/6 complex. Altogether, we propose that TNP-470 arrest cell cycle at G1 phase not only through the induction of p53 and p21, but also the accumulation of cyclin D1 in the nucleus. Cyclin D1 may be a common target for angiogenic regulation.
List of Tables...................................................Ⅰ
List of Figures..................................................Ⅱ
List of Abbreviation.............................................Ⅳ
Abstract in Chinese..............................................Ⅴ
Abstract in English..............................................Ⅶ
1. Introduction
1.1 Mechanism and role of neovasculogenesis.....................1
1.2 Regulation of angiogenesis..................................2
1.3 Angiogenic inducers.........................................3
1.4 Angiogenic inhibitors.......................................4
1.5 Tumor angiogenesis..........................................5
1.6 Therapeutic potential of anti-angiogenic therapy............6
1.7 Mechanism of angiogenic inhibition mediated by TNP-470......7
2. Experimental Procedures
2.1 Materials...................................................9
2.2 Methods.....................................................11
2.2.1 Isolation of human umbilical vein endothelial cells
(HUVECs) and cell culture................................11
2.2.2 Cell proliferation assay.................................12
2.2.3 Cell treatment...........................................13
2.2.4 Total RNA & poly (A)+ RNA isolation......................13
2.2.5 cDNA microarray..........................................14
2.2.6 Reverse transcription-PCR & southern blotting analysis...14
2.2.7 Western blot analysis....................................15
2.2.8 Northern blot analysis...................................16
2.2.9 Construction of tetracycline-inducible system............17
2.2.10 Transient transfection and drug-resistant assay..........17
2.2.11 Expression and purification of GST-mRb fusion protein....18
2.2.12 In vitro kinase assay....................................19
2.2.13 Purification of nuclear and cytosolic protein............20
3. Results
3.1 TNP-470 inhibits HUVEC proliferation in the presence of bFGF........................................................23
3.2 Identification of genes modulated by TNP-470 in human
endothelial cells............................................24
3.2.1 Egr-1 and JunB mRNA expression are repressed by TNP-470....24
3.2.2 Overexpression of egr-1 in endothelial cells increases
the resistance to TNP-470.................................25
3.3.3 Overexpression of junB in endothelial cells inhibits
HUVEC proliferation.......................................26
3.3 Genes involving in cell cycle arrest is regulated by
TNP-470......................................................27
3.3.1 TNP-470 induces p53 and p21 gene expression in endothelial cell.......................................................27
3.3.2 TNP-470 induces cyclin D1 protein accumulation in
endothelial cells..........................................28
3.3.3 The accumulated cyclin D1 induced by TNP-470 fails to
form active cyclin D1-dependent kinase.....................29
3.3.4 TNP-470 induces nuclear accumulation of cyclin D1..........30
3.4 TNP-470 deregulates the expression of apoptoic genes........31
4. Discussion...................................................33
5. References...................................................38
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