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 G 1 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.
Keywords: Hedgehog signaling pathway; cell growth; cell migration; cyclopamine; uveal melanoma.