在LoVo大腸癌細胞株大量表現雌激素受體進而探討雌激素及其接受體誘導LoVo大腸癌細胞凋亡的機制

The mechanism of estrogen induces apoptosis in overexpression ER-a or ER-β of LoVo colon cancer cell line

許希賢(Hsi-Hsien Hsu)

指導教授 ： 林中生 ； 黃志揚

中山醫學大學/醫學院/醫學研究所/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00083

摘要

Part-I:
在大腸癌症發生的程序中，被認為與多種基因的突變有關，其中包括了：APC、K-RAS、SMAD2/4和p53，而在流行病學的報告中，指出HNPCC (Hereditary Non-Polyposis Colon Cancer)的病患中，男性病患為女性病患的1.5倍，其中在女性的病患中，被認為雌激素(Estrogen;E2)能降低罹患大腸癌的比例，但若循環系統中雌激素的量下降時，會造成雌激素接受體表現下降，進而增加罹患大腸癌的危險性，並且發現在大腸癌症的檢體中，雌激素接受體的表現量比起正常人有明顯下降，顯示出雌激素及其接受體對於罹患大腸癌具有保護的影響,並由已知的文獻中得知，Tumor Necrosis Factor-alpha (TNF-alpha)具有促進大腸癌細胞凋亡的能力。於是在本篇文章中，我們除了進一步探討雌激素及其接受體對於LoVo大腸癌細胞是否具有促凋亡的影響，並進一步研究此凋亡現象是否與活化TNF- alpha具有相關性以及對於細胞增生是否有著抑制作用存在，而對於細胞週期相關蛋白和Wnt訊息路徑是否存在著影響?本實驗使用的方法包括了: 短暫轉殖技術、DNA斷裂、Western blotting和Luciferase assay。而由實驗結果中得知，在短暫轉殖ER-alpha並加入E2的情況下，能經由活化TNF- alpha而造成LoVo大腸癌細胞的凋亡現象，並抑制beta-catein的蛋白量。而此發現或許能進一步用於治療大腸癌。

Part-II:
在大腸癌症發生的程序中，被認為與多種基因的突變有關，其中包括了：APC、K-RAS、SMAD2/4和p53，而在流行病學的報告中，指出HNPCC (Hereditary Non-Polyposis Colon Cancer)的病患中，男性病患為女性病患的1.5倍，其中在女性的病患中，被認為雌激素(Estrogen;E2)能降低罹患大腸癌的比例，但若循環系統中雌激素的量下降時，會造成雌激素接受體表現下降，進而增加罹患大腸癌的危險性，並且發現在大腸癌症的檢體中，雌激素接受體的表現量比起正常人有明顯下降，顯示出雌激素及其接受體對於罹患大腸癌具有保護的影響,然而對於雌激素及其接受體是否具有促進大腸癌細胞凋亡、抑制其增生的功能及其相關的分子機制並未有所探討，於是本篇使用LoVo大腸癌細胞，以短暫轉殖的方式分別大量表現雌激素接受體beta (ER-beta)，然後單獨或共同處理雌激素，並依三個方向來探討對於LoVo大腸癌細胞的影響: ①凋亡路徑( TNF-alpha和Caspase )；Cell Cycle (Rb、Cyclin D1、Cyclin E、c-myc、p21和p27)；Wnt訊息路徑(APC、beta-catenin和Cyclin D1)，使用的方法包括了: 短暫轉殖技術、DNA斷裂、Western Blotting和Luciferase Assay。而由初步的實驗結果中發現到，單獨處理E2與控制組相比即有些許DNA斷裂的現象，而大量表現ER-beta並無顯著DNA斷裂的現象，只有在同時加入E2的情形下，才出現顯著DNA斷裂的現象(Ligand-Dependent)。並同時出現Caspase 8和Caspase 9蛋白活化，但ER-beta無法活化TNF-alpha，故TNF-alpha與ER-beta所造成的凋亡並無相關性。而在細胞週期方面，ER-beta依著Ligand-Independent的方式均負調節著Rb和Cyclin D1的蛋白表現量，且單獨處理E2和同時大量表現ER-beta能使的p21和p27蛋白表現量上升；另外在Wnt訊息路徑中，亦發現ER-beta (Ligand-Dependent) 均能抑制beta-catenin蛋白表現量，其結果與下游調節基因Cyclin D1的表現量相符合。綜合上述的實驗結果，證實雌激素透過ER-beta，促進LoVo大腸癌細胞的凋亡和抑制其增生的現象，而此發現或許能進一步用於治療大腸癌。

關鍵字

LoVo 細胞 ； 雌激素接受體- alpha ； 腫瘤壞死因子- alpha ； beta-catenin ； 凋亡的影響 ； 雌激素 ； 雌激素接受體-beta ； 抗增生

並列摘要

Part-I:
Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estrogen (E2) have been reported to downregulate the expression of E2 receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E2 replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. Evidences strongly suggest the protective roles of E2 and ER against colorectal cancer. However, the mechanisms of ER-alpha effects on colorectal cancer cells remained un-clear after LoVo cells were transiently transfected to overexpress ER-alpha, DNA fragmentation and the activated caspases measurements were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the hTNF- alpha promoter activity. The results clearly demonstrated that overexpressed ER-alpha with or without E2 (10-8M) treatment could activate caspase-8, -9, and -3 and induce DNA fragmentation in LoVo cell. At the same time, overexpressed ER-alpha plus E2 significantly increases the expression and promoter activity of hTNF- alpha, and the DNA fragmentation effect induced by E2 plus ER-alpha were reduced by the addition of hTNF antibody (0.1μg/ml). In addition, E2 plus ER-alpha significantly upregulated p21 and p27 levels and downregulated the beta-catenin and its target genes, cyclin D1 and Rb, which regulate the cell cycle and cell proliferation. The results indicate that E2 plus overexpressed ER-alpha induce LoVo cell apoptosis might mediate through the increase of hTNF-alpha gene expression, which in turn activate caspase-8, -9 and caspase-3 and lead to the DNA fragmentation and apoptosis. E2 plus ER-alpha also showed the downregulation of beta-catenin signalings implicating the suppression of proliferation and metastasis of colorectal cells. Efforts aiming at enhancing ER-alpha expression and/or activity may be proved to be an alternative therapy against colorectal cancer.

Part-II:
Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estrogen (E2) have been reported to downregulate the expression of E2 receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E2 replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. These data strongly suggest the protective roles of E2 and ER against colorectal cancer. However, the mechanisms remain unexplored. LoVo cells were transient transfected to overexpress ER-beta, DNA fragmentation and caspase activity assay were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the TNF-alpha promoter activity. Our data clearly demonstrated that E2 and ER-beta alone could upregulate p21 and p27 proteins, which further activate caspase-8 and caspase-9 to induce apoptosis in LoVo cell, and the ER-beta effects were enhanced by E2. However, overexpressed ER-beta did not influence the expression and promoter activity of TNF-alpha. In addition, E2 and overexpressed ER-beta downregulated the beta-catenin proteins which cause the downregulation of its target genes, cyclin D1 and Rb, to inhibit the cell cycle and cell proliferation. The results indicate that overexpressed ER-beta may induce LoVo cell apoptosis and anti-proliferation by increasing p53 signaling in a ligand-dependent manner, and without hTNF-alpha involvement. Efforts aiming at enhancing ER-beta expression and/or activity may prove to be an attractive alternative therapy against colorectal cancer.

並列關鍵字

LoVo cells ； E2 ； estrogen receptor-alpha ； tumor necrosis factor-alpha ； beta-catenin ； apoptotic effects ； estrogen receptor-beta ； anti-proliferation.

參考文獻

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