砷於人類泌尿道上皮細胞之細胞訊息傳遞路徑之影響

The effect of arsenic on cell signaling transduction pathway in human uroepithelial cells

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/博士(2012年)

摘要

砷廣泛地存在於自然界，而污染的水源是危害健康最主要的原因之一，砷的高度污染和人類疾病具有相當程度的關聯性，而泌尿道上皮癌是一個與砷暴露有很重要相關性的疾病。例如：烏腳病地區屬於台灣西南沿海地區砷高度污染的區域，此區域泌尿道上皮癌的罹患機率遠比其他地區高出許多。雖然許多研究已證實砷與泌尿道上皮癌及其它癌症之相關性，但至今砷是如何造成細胞癌化之機轉仍不清楚。
第一：細胞自噬是一種處在惡劣環境或壓力狀況下，重新利用營養物質以維持能量恆定的生存機制。它也參與在許多生理及病理過程中。之前，我們研究已證實亞砷酸鈉會造成人類泌尿道上皮細胞(SV-HUC-1)細胞自噬及DAPK啟動子過度甲基化。此外，在人類腫瘤組織部份，MSP結果發現烏腳病地區病人DAPK啟動子過度甲基化比率顯著高於非烏腳病地區 (p=0.018)。免疫組織化學染色結果也顯示來DAPK啟動子過度甲基化與DAPK蛋白質表現減少具有顯著性差異 (p=0.037)。然而，砷是如何造成細胞自噬其相關機轉至今仍未知。在本研究，我們首先藉由免疫細胞化學組織及西方墨點法實驗證實，砷會刺激ERK1/2訊息傳遞路徑活化。另外，利用電子顯微鏡觀察發現砷會造成SV-HUC-1細胞形成雙層膜結構的細胞自噬小體，其細胞自噬數目隨砷濃度增加而增加。接著，我們也發現在細胞自噬小體形成過程中扮演重要的調控者的LC3B及Beclin-1，其表現則隨砷濃度增加而相對增加。並且，砷會藉由DAPK啟動子過度甲基化來抑制DAPK蛋白質之表現。
第二：Cyclin-D1和p16蛋白是細胞週期重要的調控者。此外，Cyclin-D1過度表現及p16表現減少已被證實與癌症病人的預後較差是有相關聯性。在本研究中，我們的免疫化學組織結果發現Cyclin-D1過度表現在烏腳病地區的泌尿道上皮癌病人約為56.3% (9/16)，來自非烏腳病地區則約為6.3% (1/16)(p=0.002)。而p16低度表現在烏腳病地區約為81.2% (13/16)，在非烏腳病地區則為25.0% (4/16)(p=0.001)。此外，砷會造成SV-HUC-1細胞，Cyclin-D1表現增加及經由p16啟動子過度甲基化來使得p16蛋白質表現受抑制。在動物實驗中，我們的實驗結果顯示經砷暴露的大鼠膀胱上皮細胞相較於控制組，Cyclin-D1表現會增加及p16表現則是呈現減少的情形。
第三：RECK通常表現在正常組織和未轉型的細胞，但在大部分的轉型或惡性細胞，其RECK表現呈現抑制或不表現的情形。目前許多研究發現RECK可藉由調控MMP-9和-2進而抑制細胞侵犯性、轉移及血管新生的能力。我們藉由西方墨點法、免疫細胞化學染色、RT-PCR及gelatine zymography來分析砷對於RECK、MMP-9、-2、uPA及VEGF表現之影響。此外，我們利用MSP分析砷對於SV-HUC-1細胞RECK啟動子甲基化之影響。我們實驗結果顯示砷會經由RECK啟動子過度甲基化造成RECK表現減少，且會造成MMP-9、-2、uPA及VEGF表現增加。事實上，經由免疫組織化學染色結果也發現RECK、MMP-9、-2、uPA及VEGF表現在烏腳病地區和非烏腳病地區是具有顯著性差異的 (分別為p=0.006、0.007、0.003、＜0.001和0.001)。在體內研究中，我們結果顯示經砷暴露的大鼠膀胱上皮細胞相較於控制組，RECK表現會減少，但MMP-9、-2、uPA和VEGF表現則是呈現增加的情形。
然而，當SV-HUC-1細胞先以DNMT或MEK抑制劑 (5-aza-CdR或U0216)作用24時，結果顯示抑制劑會抑制砷對於SV-HUC-1細胞內細胞自噬小體數目、p-ERK1/2、Beclin-1、LC3B、DAPK、Cyclin-D1、p16、RECK、MMP-9、-2、uPA及VEGF表現之影響。總結，綜合以上實驗結果發現砷可能藉由外遺傳變異和ERK1/2訊息傳遞路徑之活化，進而造成細胞自噬和上述這些分子表現失去調控。期望這些初步實驗結果有助於砷所造成泌尿道上皮癌相關機制更進一步了解。

關鍵字

砷 ； 泌尿道上皮細胞 ； 細胞自噬 ； 細胞訊息傳遞路徑 ； DNA甲基化

並列摘要

Arsenic is widely distributed in the nature. Its contamination to the water source is considered as one of the health hazards for human being. As we know, many human diseases, for example, urothelial carcinoma is related with heavy contamination of arsenic. In the southern west coast of Taiwan, an endemic area of arsenicism (called blackfoot disease; BFD), is found high incidence of urothelial carcinoma (UC). 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.
First, autophagy is frequently activated and in response to adverse environmental conditions or stress and has been shown to be involved in many physiological and pathological processes. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). We also found the DAPK hypermethylation status was significantly higher in UC in BFD areas when compared with tumours from patients from non-BFD areas using by methylation-specific PCR (MSP) (p=0.018). A close correlation was also found between DAPK promoter methylation and low-intensity DAPK expression, as detected by IHC (p=0.037). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry (ICC) and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes was determined by immunohistochemistry (IHC) and Western blotting. The arsenite causes reduced DAPK protein expression through DAPK promoter hypermethylation in SV-HUC-1 cells.
Second, the Cyclin-D1 and p16 proteins are important indicators in control of cell cycle progression. Moreover, the high expression of Cyclin-D1 and lost of p16 has been associated with worse prognosis in a variety of human cancers. In this study, our results showed the Cyclin-D1 high expression was found in 56.3% (9/16) UC from a BFD area and 6.3% (1/16) UC from a non-BFD area (p=0.002). The p16 low expression in 81.2% (13/16) UC from BFD areas was significantly lower than in non-BFD areas (25.0%; 4/16)(p=0.001). In addition, the Cyclin-D1 increased expression but decrease of p16 expression through promoter hypermethylation in arsenite-treated SV-HUC-1 cells. For in vivo study, our result showed Cyclin-D1 protein expression increased and the p16 expression was reduced in arsenite treatment groups.
Third, the RECK was widely expressed in most normal tissue and cells. But it was down-regulated in many tumor cells. The RECK was a tumor invasion, metastasis and angiogenesis suppressor via negatively regulate matrix-metalloproteinases (MMP-9 and -2). We analyzed the arsenite causing RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, ICC, RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using MSP. Our results show that arsenite downregulation of RECK is caused by epigenetic inactivation via promoter hypermethylation, and that levels of MMP-9, -2, uPA are VEGF were increased in human uroepithelial cells (SV-HUC-1). Indeed, we also found significant differences between the expression of RECK, MMP-9, -2, uPA and VEGF in UC from the BFD areas and non-BFD areas (p=0.006、0.007、0.003、＜0.001 and 0.001, respectively), as detected by IHC. In vivo study, our results showed RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups.
However, when the cells were pretreated with DNMT or MEK inhibitors (5-aza-CdR or U0126) for 24h, the effect of arsenite on p-ERK1/2, Beclin-1, LC3B, DAPK, Cyclin-D1, p16, RECK, MMP-9, -2, uPA and VEGF proteins expression is suppressed. In conclusion, our results support the notion that arsenite might cause autophagy activation and aberrant expression of multiple molecules through epigenetic inactivation and ERK1/2 activation. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.

並列關鍵字

arsenic ； uroepithelial cells ； autophagy ； cell signaling transduction pathway ； DNA methylation

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