氯化鎳誘導人類支氣管上皮細胞進行上皮-間質轉換及其作用機制之研究

細胞進行上皮-間質型轉換過程 (epithelial-mesenchymal transition, EMT) 是肺纖維化和腫瘤轉移前的重要程序，在此過程中，細胞與細胞間結合的標記蛋白E-cadherin會減少，間質型標記蛋白fibronectin和vimentin的表現會增加，並且許多不同的訊息傳遞路徑參與其中。已知鎳化合物能夠造成肺部癌化以及對健康產生不良影響，然而目前仍不清楚鎳是否會誘導細胞進行上皮-間質轉換過程。我們的研究主要探討鎳是否誘發人類支氣管上皮細胞產生上皮-間質轉換現象以及相關基因表現，並且釐清氯化鎳啟動細胞進行上皮-間質轉換之可能分子機制。首先將BEAS-2B細胞暴露於鎳化合物，運用RT-PCR和免疫轉漬法觀察E-cadherin和fibronectin的表現量，觀察到氯化鎳誘導E-cadherin減少以及fibronectin增加，造成細胞產生上皮-間質轉換的現象。另外，在一維電泳分析偵測出，氯化鎳可減少Annexin 2的蛋白表現；NF-κB抑制劑BAY11-7082可降低氯化鎳所誘導的fibronectin表現量，但較不影響E-cadherin的變化。運用ROS抑制劑、DNA甲基轉移酶抑制劑、histone去乙醯基酶抑制劑以及蛋白酶體抑制劑等配合使用流式細胞儀、免疫轉漬法，研究氯化鎳調控E-cadherin的作用機制，觀察到ROS抑制劑NAC能部分減緩氯化鎳所誘導的EMT現象，並且抑制氯化鎳所誘導的HIF-1α和LCN2表現；DNA甲基轉移酶抑制劑5-Aza-dc也可減緩氯化鎳降低E-cadherin表現，並且藉由甲基特異性連鎖反應分析，發現氯化鎳可增進E-cadherin的promoter高度甲基化；histone去乙醯基酶抑制劑TSA有些微回復氯化鎳所減少的E-cadherin表現；同時蛋白酶體抑制劑MG132也降低氯化鎳所誘導的E-cadherin蛋白水解作用。因此，氯化鎳會透過ROS的活化、造成E-cadherin promoter高度甲基化、histone去乙醯基化和蛋白酶體的降解作用等，來減少E-cadheirn的表現。本研究發現鎳會誘導細胞進行上皮-間質轉換，這可能參與鎳所造成的組織纖維化和細胞癌化的過程。

關鍵字

人類支氣管上皮細胞；上皮-間質轉換；氯化鎳；活性氧化物；高度甲基化

並列摘要

Epithelial-mesenchymal transition (EMT) has been considered as a critical event in the pathogenesis of lung fibrosis and tumor metastasis. In EMT, the expression of differentiation markers switched from cell-cell junction proteins such as E-cadherin to mesenchymal markers including fibronectin and vimentin, and the mechanism underlying EMT involves coordination of multiple signaling pathways. Nickel containing compounds have been associated with lung carcinogenesis and other adverse health effects. However, the relationship between nickel and EMT in cells is not clear. This study mainly explored whether nickel affect the expression of gene durning EMT in humen bronchial epithelial cells. We also clarified the mechemisms of nickel chloride induced EMT. BEAS-2B cells were exposed to nickel compounds. E-cadherin and fibronectin expression levels were examined by RT-PCR and immunoblot. We found that nickel chloride induced the EMT phenotype markers alterations such as up-regulation of fibronectin and down-regulation of E-cadherin. The results of 1-D gel electrophoresis/SDS-PAGE indicated that nickel chloride decreased the expression of Annexin 2. The NF-κB inhibitor, BAY 11-7082, abolished the up-regulation of fibronectin induced by nickel chloride, but no effect in E-cadherin. Reactive oxygen species (ROS) inhibitor, DNA methyltransferase (DNMT) inhibitor, histone deacetylase (HDAC) inhibitor and proteasome inhibitor were used with folw cytometry and immunoblot to determine that mechanisms of nickel chloride regulated E-cadherin in BEAS-2B cells. The ROS inhibitor, N-Acetyl Cysteine (NAC), could block EMT and the expression of HIF-1α and LCN2 induced by nickel chloride. DNMT inhibitor, 5-aza-2’-deoxycytidine (5-Aza-dc), could restore the down-regulation of E-cadherin induced by nickel chloride. The hypermethylation of E-cadherin promoter was also induced by nickel chloride using methyl-specific polymerase chain reaction (MSP) to ditect. The HDAC inhibitor, trichostatin A (TSA), could slightly reverse the down-regulation of E-cadherin by nickel chloride. We also found that proteasome inhibitor, MG132, could diminish degradation of E-cadherin by nickel chloride. Our conclusion is that nickel chloride induced down-regulation of E-cadherin by possibly involving in ROS induction, hypermethylation in E-cadherin promoter, histone deacetylation and proteasome degradation. These findings demonstrate for the first time that nickel could induce EMT, which may participate in the processes of tissue fibrosis and carcinogenesis.

並列關鍵字

BEAS-2B cells ； EMT ； nickel chloride ； ROS ； hypermethylation

參考文獻

Acevedo F, Serra MA, Ermolli M, Clerici L, Vesterberg O (2001) Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase. Toxicology 159(1-2): 33-41

Andrew A, Barchowsky A (2000) Nickel-induced plasminogen activator inhibitor-1 expression inhibits the fibrinolytic activity of human airway epithelial cells. Toxicol Appl Pharmacol 168(1): 50-57

Andrew AS, Klei LR, Barchowsky A (2001) AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen. Am J Physiol Lung Cell Mol Physiol 281(3): L616-623

Andrew AS, Warren AJ, Barchowsky A, Temple KA, Klei L, Soucy NV, O'Hara KA, Hamilton JW (2003) Genomic and proteomic profiling of responses to toxic metals in human lung cells. Environ Health Perspect 111(6): 825-835

Arora P, Kim EO, Jung JK, Jang KL (2008) Hepatitis C virus core protein downregulates E-cadherin expression via activation of DNA methyltransferase 1 and 3b. Cancer Lett 261(2): 244-252

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氯化鎳誘導人類支氣管上皮細胞進行上皮-間質轉換及其作用機制之研究

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