亞砷酸鈉在人類正常泌尿道上皮細胞(SV-HUC-1)之p53、COX-2、Bcl-6和JAK-STAT訊息傳遞路徑之影響

Effects of sodium arsenite on p53,COX-2,Bcl-6,and JAK-STAT signaling transduction pathway in SV-40 immortailzed human uroepithelial cells (SV-HUC-1)

黃雅君(Ya-Chun Huang)

指導教授 ： 蔡志仁

高雄醫學大學/醫學院/醫學研究所/碩士(2007年)

摘要

無機砷廣泛存在土壤及水中之類金屬化合物，並在工業上大量的使用，使得環境長期受到砷污染，因此常可見到無機砷高暴露之人類族群。在流行病學的研究報告中充分顯示無機砷為人類致癌物，長期無機砷攝取除主要誘引皮膚癌外也會有膀胱癌、肝癌和腎癌的發生。然而，飲水中慢性砷中毒已知與台灣西南部的烏腳病(blackfoot disease; BFD) 流行區之泌尿道上皮細胞癌的高發生率有密切的關係，並且許多國內外的研究也證實砷是造成當地泌尿道上皮細胞癌和其他癌症的主要原因。雖然如此，至今砷是如何造成細胞癌化之機轉仍不清楚，並且在動物實驗尚未直接證實砷化物的致癌性。然而，在許多人類癌症中可時常發現突變型p53、COX-2、Bcl-6、p-STAT3有過度表現的情形。因此，本研究的目的是要去探討砷是否會造成人類泌尿道上皮細胞基因毒性和突變型p53、COX-2、Bcl-6、p-STAT3致癌蛋白質表現之影響。本研究中，首先利用Comet assay來評估亞砷酸鈉是否對於已被SV-40病毒轉染過的人類輸尿管泌尿道上皮細胞(SV-HUC-1)造成基因毒性。接著，利用免疫細胞化學染色和西方墨點法來觀察突變型p53、COX-2、Bcl-6、p-STAT3蛋白質的表現。由實驗結果顯示亞砷酸鈉會造成SV-HUC-1細胞DNA damage，並且其基因毒性的程度與砷的濃度是呈正相關。同時，亞砷酸鈉也會誘發突變型p53蛋白質的表現，其最大表現量是在4 μM的時候。突變型p53蛋白質的表現與Comet srcoe是呈平行關係。一樣地，亞砷酸鈉也會誘發COX-2、Bcl-6、p-STAT3蛋白質表現，且是呈dose-dependent的增加。綜合以上的結果，可以證明亞砷酸鈉會造成泌尿上皮細胞發生基因毒性，並且會誘發突變型p53、COX-2、Bcl-6、p-STAT3蛋白質的表現。意指這些致癌蛋白質的參與有可能是砷致癌的機轉之一。藉由這個研究可得知砷會造成泌尿道上皮細胞過度表現突變型p53、COX-2、Bcl-6、p-STAT3，這也表示COX-2和p-STAT3的抑制劑可能具有治療人類泌尿道上皮細胞癌的能力。希望這些成果對於目前研究砷在泌尿道上皮細胞癌之致病機轉方面有所益處。

關鍵字

亞砷酸鈉 ； SV-HUC-1 ； p53 ； COX-2 ； Bcl-6 ； JAK-STAT訊息傳遞路徑

並列摘要

Arsenic is a naturally occurring constituent of soil and water, and arsenic metalloid is widely used in industry. The combination of lengthy environmental persistence with widespread arsenic contamination contributes to a high exposure incidence among human populations. Arsenic is a human carcinogen. With ingestion of inorganic forms of this metalloid causes primarily skin cancer, but also cancers of the bladder, liver, and kidney. However, environmental or occupational exposure to arsenic was associated with a greatly increased risk of skin, urinary bladder, and respiratory tract cancers, especially in arsenic-endemic areas throughout the world. Chronic arseniasis was considered responsible for the high incidence of urothelial carcinoma (UC) in the blackfoot disease (BFD) endemic area in Southwest Taiwan. Although there are many studies have demonstrated an association between arsenic and the incidence of UC and others cancers, but the molecular mechanisms of arsenic-mediated UC carcinogenesis has not yet been defined. And inorganic arsenic fails to induce tumors in most laboratory animals. In addition, overexpression of mutant-type p53, COX-2, Bcl-6, and p-STAT3 frequency occur in a variety of human malignancies. It is therefore necessary to study the genotoxicity of arsenic salts on human uroepithelial cells and the expression of the oncoproteins p53, COX-2, Bcl-6, and p-STAT3. In this study, the relative genotoxicity of sodium arsenite was evaluated in SV-40 immortalized human urothelium cells (SV-HUC-1) using alkaline Comet assay. The expression of mutant-type p53, COX-2, Bcl-6, and p-STAT3 were also evaluated by immunocytochemistry and Western blotting. Our results revealed that the sodium arsenite was able to induce DNA damage, and the genotoxicity is correlated with its concentration. In addition, the expression of mutant-type p53 was parallelly correlated with comet scores, and the maximal expression of mutant-type p53 was observed at 4 μM arsenite. Similarly, sodium arsenite a stimulated dose-dependent increase in COX-2, Bcl-6, and p-STAT3 expression. In conclusion, this study demonstrated that sodium arsenite is genotoxic to uroepithelium , and that it will induce the expression of mutant-type p53, COX-2, Bcl-6, and p-STAT3 proteins, indicating its possible mechanism of carcinogenesis. This study provides us with knowledge of the relationship between p53, COX-2, Bcl-6, and p-STAT3 ovexpression in arsenite treated urothelial cells and suggests a potential therapeutic role of COX-2 and STAT3 inhibitors in human urothelial malignancies. We expect the results of the project will a better understanding of the carcinogenesis of arsenic-induces urothelial malignancies.

並列關鍵字

sodium arsemite ； SV-HUC-1 ； p53 ； COX-2 ； Bcl-6 ； JAK-STAT signaling transduction paythway

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