砷化物對人類乳腺腫瘤MCF-7細胞增生及其動情素受體表現量的影響

Effects of Arsenics on Cell Growth and Estrogen Receptor-αExpression in MCF-7 Breast Cancer Cells

許玉青(Hsu-Yu Ching)

指導教授 ： 童麗珠

國立臺灣師範大學/理學院/生命科學研究所/碩士(2004年)

摘要

無機砷是一種環境中常見的污染物，廣泛的分佈在環境中。有關無機砷化物的毒害已有許多報導，它不僅為人類的致癌物質，與皮膚癌、肺癌、膀胱癌致癌相關，且可誘發細胞毒性、染色體異常、氧化性傷害、影響DNA的修復及遺傳毒性。
近年來，少數的報導認為無機砷是藉由動情素的模式引起生理變化，而有類似環境荷爾蒙的作用。本研究目的在證明亞砷酸鈉及砷酸鈉是否具有干擾細胞對動情素作用的特性。研究先以MTT Test進行亞砷酸鈉與砷酸鈉對MCF-7細胞毒性及細胞增生測定。結果顯示：當藥物處理的濃度小於1μM亞砷酸鈉、小於10μM砷酸鈉時，不會影響細胞生長，細胞可以增生；高於這二濃度則可抑制細胞增生。0.1~1μM亞砷酸鈉、1~10μM砷酸鈉處理細胞24小時，長達6天的觀察，發現細胞有持續增生的趨勢。在協同處理動情素拮抗劑(ICI 182,780)時，發現單獨處理亞砷酸鈉、砷酸鈉及E2所誘發的細胞增生都被抑制。以Western blotting分析，發現低劑量的亞砷酸鈉及砷酸鈉處理均會使細胞的動情素受器ERα蛋白質的表現量下降，但對動情素受體ERβ影響則不顯著。細胞以ERα抗體免疫螢光染色，在以100倍的顯微鏡下觀察，可發現亞砷酸鈉及17β-estrodiol (E2)處理組的ERα亮度較控制組弱；放大倍率觀察單一細胞，發現亞砷酸鈉及E2處理組的ERα螢光呈現較控制組少，而ERα分布以單獨E2與control組二者較為相似，細胞膜與細胞質都有。在亞砷酸鈉處理組，ERα主要分佈於細胞質中，在細胞核則無法發現。在進行亞砷酸鈉與動情素競爭結合動情素受體之實驗，發現0.1、0.5、1μM亞砷酸鈉與動情素受體的結合比為28%、35%、48%，顯示亞砷酸能與動情素受體結合。
由上述實驗證實低劑量的亞砷酸鈉及砷酸鈉處理MCF-7人類乳腺腫瘤細胞，證明會造成細胞增生、影響動情素受體α的表現及與動情素受體的結合，顯示無機砷是藉由動情素的模式引起細胞生理作用。

關鍵字

砷化物 ； 環境荷爾蒙 ； 動情素 ； 動情素受體 ； HASH(0x9138bc0)

並列摘要

Inorganic arsenics, the common environmental pollutants, are widely dispersed and the well-documented human carcinogens associated with cancers of skin, lung and bladder and can induce cytotoxicity, chromosomal abnormalities, oxidative stress, altered DNA repair, altered growth factors etc.
Several reports indicated that inorganic arsenic might somehow act through an estrogenic mode of action. In this study, effects of sodium arsenite and sodium arsenate on cytotoxicity, cell proliferation and the expression of estrogen receptorα(ERα) in breast adenocarcinoma MCF-7 cells are determinated. The preliminary results show that the inhibiting threshold concentrations of sodium arsenite and sodium arsenate on cell growth are at 1μM and 10μM, respectively, whereas their concentrations lower than inhibiting threshold concentrations promoted cell growth instead of cell toxicity. Time course studies on cells treated with 0.1~1μM sodium arsenite and 1~10μM sodium arsenate for 24 h, and observed consecutive six days without drugs, MCF-7 cells proliferation exhibited continuous increase. Cotreated with drugs and estrogen antagoist (ICI 182,780), MCF-7 cell proliferations were completely inhibited. Data of Western blotting show that the expression of ERα were decreased in cells treated with low dose of sodium arsenite and sodium arsenate, but expression of estrogen receptorβ were not affected. Immunocytochemical studies with fluorescent microscopy illustrated that ERα displayed a diffusion distribution in cytoplasm and were more concentrated within nucleus of control cell and E2-treated cells. In arsenic-treated cells, the ERαonly displayed in cytoplasm. Whole-cell competitive estrogen-receptor binding assay demonstrate that 0.1, 0.5 and 1μM sodium arsenite could bound with 28%, 35% and 48% in ER respectively. In this study it was concluded that arsenite could bind with estrogen receptor.
Based on results above, MCF-7 cells treated with a low dose sodium arsenite and sodium arsenate affect cell proliferation, ERαexpression, and competition of estrogen receptor binding；and indicate that inorganic arsenics do have the characteristics of environmental hormone for inducing physiological effect, through a estrogen model.

並列關鍵字

arsenite ； arsenate ； endocrine disruption ； estrogen ； estrogen receptor