Effects of inhibition of hedgehog signaling on cell growth and migration of uveal melanoma cells
Effects of inhibition of hedgehog signaling on cell growth and migration of uveal melanoma cells
复制标题
抑制hedgehog信号对葡萄膜黑色素瘤细胞生长和迁移的影响
DOI：
10.4161/cbt.28157
复制
发表时间：
2014-05-01
影响因子：
3.6
通讯作者：
Li, Jin
中科院分区：
文献类型：
--
作者：
Duan, Fei;Lin, Ming;Li, Jin
Hedgehog (Hh) 信号通路已被证明在控制胚胎发育、组织模式、伤口愈合和多种细胞功能中发挥着关键作用。 Hh 信号传导的异常激活与许多人类癌症的发病机制和血管生成有关。然而，该途径在葡萄膜黑色素瘤（UM）癌变中的作用仍不清楚。在这项研究中，我们使用特异性 Smoothened (Smo) 拮抗剂环杷明来研究 Hh 抑制的效果，以阻断表达 Hh 信号成分的培养人 UM 细胞系中的 Hh 信号。环巴明治疗通过下调 Hh 最终仲裁者胶质母细胞瘤 1 (Gli1)（调节细胞核中靶基因的转录），有效增加细胞凋亡并抑制细胞增殖、迁移和上皮间质转化 (EMT)。分别通过实时PCR、蛋白质印迹和免疫细胞化学检测基因和蛋白质表达水平的变化。通过流式细胞术证明了环杷明诱导的细胞周期和细胞凋亡调节。此外，Transwell 迁移和划痕实验证明，UM 细胞的迁移能力降低。通过管形成实验检测Hh抑制对UM细胞分泌的血管生成因子水平的影响。结论：环巴明阻断 Hh 通路可降低 UM 细胞的活力、迁移、EMT 和血管生成，增加细胞凋亡，并诱导 G1 期细胞周期停滞。总的来说，这些结果首次证明了 Smo 下游 Gli1 激活作为治疗靶点的重要性以及环巴明治疗人类 UM 的潜在价值。
The Hedgehog (Hh) signaling pathway has been demonstrated to play a critical role in controlling embryonic development, tissue patterning, wound healing and a variety of cell functions. Aberrant activation of Hh signaling is implicated in the pathogenesis of many human cancers, and in angiogenesis. However, the role of this pathway in uveal melanoma (UM) carcinogenesis remains unknown. In this study, we investigated the effects of Hh inhibition using the specific Smoothened (Smo) antagonist cyclopamine to block Hh signaling in cultured human UM cell lines expressing Hh signaling components. Cyclopamine treatment effectively increased apoptosis and inhibited cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT) by downregulating the Hh final arbiter glioblastoma 1 (Gli1), which regulates the transcription of target genes in the nucleus. Changes in gene and protein expression levels were detected by real-time PCR and by western blotting and immunocytochemistry, respectively. Cell cycle and apoptosis regulation induced by cyclopamine were demonstrated by flow cytometry. In addition, the migration capability of UM cells was reduced, as demonstrated by transwell migration and scratch assays. The effects of Hh inhibition on the levels of angiogenesis factors secreted by UM cells were examined by tube-formation assay. Conclusion: Blocking the Hh pathway by cyclopamine decreased cell viability, migration, EMT, and angiogenesis, increased apoptosis, and induced G1 phase cell cycle arrest in UM cells. Collectively, these results provide the first evidence of the significance of Gli1 activation downstream of Smo as a therapeutic target and the potential value of cyclopamine for the treatment of human UM.