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研究生：	黃怡靜 Huang, Yi-Jing
論文名稱：	活化的雌激素受體貝他促進4T1/Luc+乳癌細胞於小鼠體內擴散 Acceleration of tumor spreading by activated ERβ in 4T1/Luc+ breast tumor bearing-mice
指導教授：	蔡美玲 Tsai, Mei-Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 生理學研究所 Department of Physiology
論文出版年：	2012
畢業學年度：	101
語文別：	英文
論文頁數：	75
中文關鍵詞：	乳癌、雌激素、雌激素受體、細胞爬行、微管蛋白、細胞擴散
外文關鍵詞：	17β-estradiol, ERβ, breast tumor spreading, migration, microtubul, α-tubulin
相關次數：	點閱：93 下載：0
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乳癌的發生率受到雌激素(E2)濃度的影響。表現雌激素受體(ERβ)的乳癌細胞具有較高的侵略性而且在人體內造成高度的轉移以及不良的預後。然而，目前並不清楚ERβ在乳癌中所扮演的角色。因此本篇研究的目的是探討雌激素如何促進乳癌的發展以及ERβ在乳癌發展中所調控的機制。首先，我們發現4T1乳癌細胞只表現ERβ。我們將乳癌細胞4T1植入小鼠的背側以建立乳癌自動轉移的動物模式。實驗結果顯示持續二到三周腹腔注射E2至小鼠體內加速原位乳癌細胞的發展。而持續三周同時注射ERβ拮抗劑PHTPP至小鼠體內抑制E2所引起的原位乳癌發展。然而, 持續五周注射E2至小鼠體內不影響原位的乳癌發展以及乳癌細胞的轉移。在細胞實驗中，E2不影響細胞數目但是透過增加細胞面積以及促進細胞突出而改變細胞型態，而PHTPP抑制由E2所促進的細胞突出。此外, PHTPP和ERβ sh-RNA 抑制由E2和DPN (an ERβ agonist) 所引發的4T1乳癌細胞爬行。另一方面，太平洋紫杉醇 (Taxol)、秋水仙素( colchicine )皆可抑制由E2所增加的4T1乳癌細胞爬行。E2處理三小時促進4T1乳癌細胞內的微管 (microtubule)呈放射線狀的散佈於細胞質且延伸至細胞突出處，而這些由E2所誘發的現象都可被PHTPP所抑制。E2處理三小時不影響4T1乳癌細胞聚合態的α-微管蛋白(polymeric α-tubulin)和非聚合態的α-微管蛋白(soluble α-tubulin)的表現。此外, E2處理三小時不影響4T1乳癌細胞α-微管蛋白 (α-tubulin) 的表現量和乙醯化的α-微管蛋白 (acetyl α-tubulin)的表現量以及乙醯化的α-微管蛋白/α-微管蛋白的比率。因為活化態的ERβ造成α-微管蛋白的重新分布以及細胞型態的改變和增加細胞爬行還有促進乳癌的發展，所以由這些結果顯示活化態的ERβ透過造成微管蛋白的重新分布而促進微管動態以及改變細胞型態促進細胞爬行，而這些因素最後促進乳癌細胞的擴散。

The higher incidence of breast cancer is positively associated with the sex hormone estrogen. Estrogen receptor β –positive breast cancer cells are more aggressive that show greater frequency of metastasis and poorer prognosis. However, the role of estrogen receptor β in the development of breast cancer remains largely unclear. The purpose of this study is to examine estrogen’s contribution to the full development of breast tumor and ERβ-mediated mechanisms in breast tumor. Here, we showed that 4T1 breast tumor cells only expressed ERβ. 4T1 cells were subcutaneous implanted into the dorsal flank of BALB/c mice to establish the spontaneous metastasis animal model. Daily intraperitoneal injection of 17β-estradiol (E2) for 2-3 wks accelerated the increase tumor mass of breast tumor at primary sites in 4T1-bearing mice. E2-induced tumor mass and area of 4T1 breast tumor at primary sites was inhibited by 3-wk co-treatment with ERβ antagonist PHTPP. Further exposure to E2 for 5 wks did not affect the development of 4T1 breast tumor at primary site and metastatic site. In 4T1 cells, E2 did not affect the cell number but caused cell morphological changes by increasing cell area and cell protrusion. PHTPP blocked the E2-induced cell protrusion. Furthermore, PHTPP or ERβ sh-RNA inhibited the E2- and DPN- (an ERβ agonist)- increased migration in 4T1 cells. Disturbance of microtubule dynamics by paclitaxel and colchicine counteracted E2-increased migration. 3-hr treatment of E2 induced the formation of long thick microtubule bundles scattered throughout the cytoplasm toward the membrane protruding regions, which was reversed by co-treatment with PHTPP. The treatment of E2 for 3 hrs did not affect the polymeric α-tubulin and solubleα-tubulin. Moreover, the treatment of E2 for 3 hrs did not affect the abundance of α-tubulin, acetylated α-tubulin, and the ratio of acetyl α-tubulin to α-tubulin. Since activated ERβ caused re-distribution of α-tubulin and morphological changes, increased cell migration, and enhancing the development of breast tumor, these results suggest that activated ERβ increases breast tumor cell migration by enhancing microtubule dynamics, specifically accelerating the redistribution of tubulin to protruding regions and morphological changes which ultimately contributes to breast tumor spreading.

Index
中文摘要		I
Abstract		III
致謝		V
List of table		X
List of figures	XI
List of supplementary figures	XIII
Chapter 1.  Literature Review	1
1	Current studies of breast cancer	1
1.1	Epidemiology of Breast Cancer	1
1.2	Breast cancer cell proliferation	2
1.3	Breast cancer cell migration and spreading	3
1.4	Breast cancer cell invasion and metastasis	4
2	Estrogen and breast development	4
2.1	Estrogen biosynthesis	4
2.2	Estrogen receptors	5
2.3	Estrogen in mammary gland development	6
3	Molecular mechanism of Estrogen on breast tumor formation	7
3.1	Estrogen receptors in breast cancer	7
3.2	Role of ERs in breast cancer cell proliferation	7
3.3	Role of ERs in breast cancer cell migration	8
3.4	The role of ERs in breast cancer cell invasion	9
Chapter 2. 	Characterization of breast tumor development in 4T1-bearing mice	10
1	Introduction	10
1.1	Unique features of breast tumor in Asia women	10
1.2	Estrogen on breast tumor development in animal model			10
1.3	The purpose and design of this study	11
2	Material and methods	12
2.1	Reagents	12
2.2	Cell line and cell culture	12
2.3	Animal care	12
2.4	In vivo tumor xenograft model	13
2.5	In vivo imaging	13
3	Results	14
3.1	The influence of gender on the development of breast tumors			14
3.2	The influence of gender on the response of tumor tissues to E2		14
4	Summary and conclusion	14
Chapter 3. Activation of ERβ by estrogens promotes microtubule dynamics, enhances cell migration, and enlarges the ERα-tumors in mice			16
1	Introduction	16
1.1	Clinical studies of estrogen receptors in breast cancer	16
1.2	The role of estrogen and its receptors in breast tumor cell migration	17
1.3	The role of estrogen on the development of breast tumor in animal model	17
1.4	The purpose and designs of this study	18
2	Material and methods	20
2.1	Reagents and antibodies	20
2.2	Cell line and cell culture	20
2.3	Animal care	21
2.4	In vivo tumor xenograft model	21
2.5	In vivo imaging	21
2.6	Cell counting assay	22
2.7	Counting of cell with morphological changes and measuring the cell area	22
2.8	MTT assay	23
2.9	Migration assay	23
2.10	Transfection	23
2.11	Immunofluorescence staining	24
2.12	Whole cell lysate	24
2.13	Isolation of insoluble polymerized microtubules and soluble tubulin dimers	25
2.14	Western blot analysis	25
2.15	Data analysis and statistical evaluation	26
3	Results	27
3.1	4T1 cells express ERβ	27
3.2	Estrogen accelerates the development of tumor tissue at the primary site in 4T1 breast tumor-bearing mice	27
3.3	Activated ERβ by E2 contributes to the development of tumor tissue at primary sites but not in secondary sites in 4T1 breast tumor-bearing mice	28
3.4	Activated ERβ by E2 changed cell morphology only	28
3.5	Activated ERβ increased 4T1 breast tumor cell migration			30
3.6	Polymerization and depolymerization of microtubule response for E2 -increased 4T1 cell migration	31
4	Discussion	34
4.1	Summary of this study	34
4.2	Effect of ERβ on cell proliferation and cell migration in breast cancer cells	35
4.3	Relation between tubulin and aggressiveness in different breast tumor cells	36
4.4	Comparison of spontaneous and experimental metastasis in 4T1-bearing mice	37
Chapter 4. Significance of this study	39
Chapter 5. References	40
Chapter 6. Tables	45
Table 1. List of primary and secondary antibodies used in this study			45
Table 2. List of drugs used in this study	47
Chapter 7. Figures	49
Chapter 8. Supplementary data	68
Curriculum vita	75

                                    

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