系統識別號 U0026-1308201917591900
論文名稱(中文) 建立IRSp53S可調控式表達的大腸癌細胞株SW480以研究IRSp53S如何對大腸癌細胞生長的影響
論文名稱(英文) Establishing IRSp53S-inducible SW480 colorectal cancer cell lines and studying how IRSp53S affects cell proliferation in colon cancer cells
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 107
學期 2
出版年 108
研究生(中文) 王毓華
研究生(英文) Yu-Hua Wang
學號 S26061062
學位類別 碩士
語文別 英文
論文頁數 56頁
口試委員 指導教授-呂增宏
中文關鍵字 大腸直腸癌  Eps8  IRSp53S  SW480  細胞凋亡 
英文關鍵字 colorectal cancer  Eps8  IRSp53S  SW480  cell apoptosis 
中文摘要 在人類的大腸直腸癌中,除了epidermal growth factor receptor (EGFR),其下游的受體EGFR pathway substrate number 8 (Eps8)也有過度表現的情況。Eps8的過度表現會促進Src以及FAK kinase的活性,進而去促進癌細胞的生長以及爬行能力。Insulin Receptor tyrosine kinase Substrate Protein of 53 kDa (IRSp53)是一種adaptor蛋白,他可以連結 Rho-family small GTPases 例如Rac 和Cdc42,進而去促進肌動蛋白細胞骨架重組。在我們過去的研究中發現Eps8-IRSp53S複合物參與了v-Src介導的腫瘤發生。為了證實Eps8-IRSp53之間的相互作用在人類大腸直腸癌中的重要性,我們建立了在SW480細胞中同時表達Eps8和IRSp53S的細胞。而SW480與其相對應的轉移的SW620細胞相比,SW480細胞的Eps8以及IRSp53S蛋白質表現都來的較少。不幸的是,我們並無法在SW480細胞中獲得同時表達Eps8和IRSp53的細胞株,我們推測可能是在SW480細胞中同時表達Eps8和IRSp53會造成細胞生長停滯或凋亡。為了研究和證實這個現象,我們首先建立了Tet-Off control和Tet-Off調控的IRSp53S-expressing 的SW480細胞。當我們將IRSp53S-inducible細胞轉染EGFP或EGFP-Eps8的質體DNA時,我們發現IRSp53S/EGFP-Eps8-overexpressing細胞會有染色質濃縮的現象,而IRSp53S/EGFP-expressing或IRSp53S/EGFP-Eps8 overexpressing加doxycycline的細胞則沒有這個現象。我們也在MTT試驗中看到同時表達Eps8及IRSp53S的調控式細胞會降低細胞生存率。在使用流式細胞儀研究中我們也發現同時表達Eps8及IRSp53S的細胞會增加G1 phase以及減少S phase的細胞比例,進而延遲細胞週期的進展。然而,在我們初步的異種移植動物實驗的結果,由於細胞株同時真正表達Eps8與IRSp53S的細胞比例非常的低(低於10%),這些細胞所產生的腫瘤大小和IRSp53S-expressing cells或是表達質體控制組的SW480細胞所產生的腫瘤相比,在統計分析上並沒有差異。我們的結果顯示IRSp53S-Eps8相互作用可能在調節結腸癌進展中發揮獨特作用而值得進一步研究。
英文摘要 In addition to epidermal growth factor receptor (EGFR), overexpression of its downstream substrate EGFR pathway substrate number 8 (Eps8) occurs in human colorectal cancer (CRC). Eps8 overexpression promotes the kinase activity of Src and FAK, leading to the enhancement of cell proliferation and motility in cancer cells. Insulin Receptor tyrosine kinase Substrate Protein of 53 kDa (IRSp53) is an adaptor protein that links Rho-family small GTPases such as Rac and Cdc42 to the actin cytoskeleton reorganization. Previously, our study indicated that Eps8-IRSp53S complex participated in v-Src-mediated tumorigenesis. To substantiate the importance of Eps8-IRSp53 interaction in CRC, we generated cells expressing both Eps8 and IRSp53S in SW480 cells that exhibits much lower level of these proteins as compared to their metastasized counterpart SW620 cells. Unfortunately, we were unable to obtain cell lines constitutively expressing both Eps8 and IRSp53 in SW480 cells suggestting that Eps8-IRSp53 interaction might cause growth arrest and/or apoptosis in SW480 cells. To confirm this, we generate Tet-Off control and Tet-Off regulated IRSp53S-expressing SW480 cells first. Then, IRSp53S-inducible cells were transfected with EGFP-expressing or EGFP-Eps8 epressing plasmid DNA. In this way, we observed enhanced chromatin condensation only in IRSp53S/EGFP-Eps8-overexpressing cells, but not in IRSp53S/EGFP-expressing cells, nor in doxycycline-treated IRSp53S/EGFP-Eps8 overexpressing cells. In addition, MTT assay revealed that double-overexpressing Eps8 and IRSp53S reduced cell viability/proliferation in SW480 cells. Flow cytometry studies also showed that double-expressing Eps8 and IRSp53S retarded cell cycle progression as indicated by increased G1 phase and reduced S phase population. However, our prelimery study in animal xenografted tumor model revealed that due to low percentage (less than 10%) of cells really expressing both Eps8 and IRSp53S at the same time, tumors derived from this double overexpressing cell line were not statistically different from those derived from IRSp53S-expressing cells nor from vector control cells. Nevertheless, our data highlighted IRSp53S-Eps8 interaction might play a sophisticated role in regulating colon cancer progression, which deserves further study.
論文目次 Abstract in English i
Abstract in Chinese iii
Acknowledgement v
List of Figures ix
Abbreviations x
1. Introduction 1
1.1. Colorectal cancer 1
1.2. Eps8 2
1.3. IRSp53 3
1.4. Ways of Cell Death : Apoptosis, Necrosis, and Autophagy 4
1.5. Specific aim 9
2. Materials and Methods 10
3. Results 18
3.1. Analyze Eps8, IRSp53, FAK and Src protein expressions of five colon cancer cell lines 18
3.3. IRSp53S overexpression increases Src Pi-Y416, but not FAK Pi-Tyr86l in SW480 cells 19
3.4. Transient Eps8-IRSp53S double overexpression induces chromatin condensation in SW480 cells 19
3.5. Inducible Eps8-IRSp53S double overexpressing SW480 cells exhibits decreased cell proliferation 20
3.6. Induction of both Eps8 and IRSp53S suppressed the activity of Src and FAK Pi-Tyr861 in SW480 cells 21
3.7. Eps8-IRSp53S double expression promotes cell cycle arrest in SW480 cells 21
3.8. IRSp53S overexpression does not promote tumor growth in mice with SW480 xenograft 22
4. Discussion 23
5. References 28
Figures 37
Appendix 53

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