Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Toxicol Lett. 2011 Feb 25;201(1):62-71. doi: 10.1016/j.toxlet.2010.12.006. Epub 2010 Dec 15.
It has been shown that the ingestion of arsenic-contaminated drinking water is closely correlated with risk of several cancers. The mechanism of arsenic-induced carcinogenesis is still unclear. The RECK, MMP-9, -2, uPA and VEGF are the most common dysregulation in human tumors and cancer cell lines. However, the effect of arsenite on these markers expression and the molecular mechanism are still unclear. The purpose of the study was to investigate the relationship between the expression of RECK, MMP-9, -2, uPA and VEGF in arsenite-treated human and rat uroepithelial cells. In addition, we also observed and compared the expression of these markers in urothelial carcinoma (UC) from Blackfoot disease (BFD) areas and non-Blackfoot disease (non-BFD) areas. We analyzed the arsenite causing cell proliferation, RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, immunocytochemistry (ICC), RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using methylation-specific PCR (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 and VEGF were increased in human uroepithelial cells (SV-HUC-1). However, when the cells were pretreated with inhibitors (5-aza-CdR or U0126) for 24h, the effects of arsenite on RECK, MMP-9, -2, uPA and VEGF expression were suppressed. 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 immunohistochemistry (IHC). In in vivo study, our results showed the RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups. In conclusion, our results support the notion that arsenite might cause the histologic changes, RECK, MMP-9, -2, uPA and VEGF dysregulation through epigenetic inactivation and ERK1/2 activation in SV-HUC-1 cells. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.
已经表明，摄入受砷污染的饮用水与几种癌症的风险密切相关。砷致癌的机制尚不清楚。RECK、MMP-9、-2、uPA 和 VEGF 是人类肿瘤和癌细胞系中最常见的失调标志物。然而，亚砷酸盐对这些标志物表达的影响及其分子机制尚不清楚。本研究的目的是研究亚砷酸盐处理的人及大鼠尿路上皮细胞中 RECK、MMP-9、-2、uPA 和 VEGF 的表达关系。此外，我们还观察并比较了黑脚病（BFD）区和非黑脚病（non-BFD）区尿路上皮癌（UC）中这些标志物的表达。我们通过 Western blot、免疫细胞化学（ICC）、RT-PCR 和明胶酶谱分析研究了亚砷酸盐引起的细胞增殖、RECK、MMP-9、-2、uPA 和 VEGF 的表达。我们通过甲基化特异性 PCR（MSP）分析研究了亚砷酸盐对 RECK 启动子甲基化状态的影响。结果表明，亚砷酸盐通过启动子超甲基化引起 RECK 的表观遗传失活而下调，人尿路上皮细胞（SV-HUC-1）中 MMP-9、-2、uPA 和 VEGF 的水平增加。然而，当细胞用抑制剂（5-氮杂胞苷或 U0126）预处理 24 小时时，亚砷酸盐对 RECK、MMP-9、-2、uPA 和 VEGF 表达的影响被抑制。事实上，我们还发现 BFD 区和非 BFD 区 UC 中 RECK、MMP-9、-2、uPA 和 VEGF 的表达存在显著差异（p=0.006、0.007、0.003、<0.001 和 0.001），通过免疫组织化学（IHC）检测。在体内研究中，我们的结果表明，RECK 蛋白表达减少，MMP-9、-2、uPA 和 VEGF 的表达在亚砷酸盐处理组中增加。总之，我们的结果支持这样一种观点，即亚砷酸盐可能通过 SV-HUC-1 细胞中的表观遗传失活和 ERK1/2 激活引起组织学变化、RECK、MMP-9、-2、uPA 和 VEGF 失调。这些发现可能为更好地理解亚砷酸盐引起的尿路上皮癌变提供依据。
