Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA.
Int J Cancer. 2013 Mar 1;132(5):1004-12. doi: 10.1002/ijc.27762. Epub 2012 Aug 24.
Endothelin-1 (ET-1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET-2 and ET-3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET-2 and ET-3 expression and determine the underlying mechanisms. Human primary colon cancers and carcinogen-induced rat colon tumors were subjected to real-time RT-PCR, immunoblotting and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation-specific PCR, bisulfite sequencing and pyrosequencing; and forced expression of ET-2 and ET-3 was conducted in human colon cancer cells followed by real-time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET-2 and ET-3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers and in a panel of human colon cancer cell lines. Forced expression of ET-2 and ET-3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET-2 and ET-3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re-expression of ET-2 and ET-3 as natural antagonists of ET-1 in colon cancer.
内皮素-1（ET-1）及其受体在人类癌症中过度表达，但关于 ET-2 和 ET-3 在癌症发病机制中的作用知之甚少。我们试图研究人类和大鼠结肠肿瘤中 ET-2 和 ET-3 表达的失调，并确定潜在的机制。对人原发性结肠癌和化学诱发性大鼠结肠癌进行实时 RT-PCR、免疫印迹和免疫组织化学检测；通过甲基化特异性 PCR、亚硫酸氢盐测序和焦磷酸测序检测 EDN2 和 EDN3 基因；在人结肠癌细胞中强制表达 ET-2 和 ET-3，然后进行实时细胞迁移和侵袭测定。与匹配的对照相比，大鼠和人类结肠肿瘤的 ET-2 和 ET-3 mRNA 和蛋白表达明显降低。机制研究表明，人原发性结肠癌和一系列人结肠癌细胞系中 EDN2 和 EDN3 基因发生高度甲基化。ET-2 和 ET-3 的强制表达显著减弱了人结肠癌细胞的迁移和侵袭。我们得出结论，在大鼠和人类结肠癌中经常发生 ET-2 和 ET-3 的表观遗传失活。目前的治疗策略通过小分子抑制剂和受体拮抗剂靶向 ET 轴的过度表达成员，但这项工作支持基于 ET-2 和 ET-3 作为 ET-1 在结肠癌中的天然拮抗剂的重新表达的互补方法。
