Effect of cyclopamine inhabited hedgehog signaling pathway on the reversion of epithelial-mesenchymal transition in esophageal cancer cells and its mechanism

Abstract:

Objective：To explore the effect of cyclopamine on epithelial-mesenchymal transition in human esophageal cancer EC109 cells and the possible mechanism. Methods：EC109 cells were cultured in vitro. After adding cyclopamine for 48 h，the expression of Gli1 was tested by real-time PCR，and morphological change of cells was observed by inverted microscope. The transwell chamber assay was used to examine the invasive and metastatic ability of EC109 cells treated with cyclopamine for 48 h. The ability of vascu－larization was observed by the method of vascilogenic mimicry and the ability of adhension was detected by adhension experiment. Real-time PCR and Western blot were used to detect the expressions of such EMT markers as E-cadherin，β-catenin and vimentin and such transcription factors as Snail，Twist1. Results：The inhibition of hedgehog signaling significantly reduced the mRNA expression of Gli1（41.819±20.150）%，and the invasion[（24.800±2.588） vs. （55.400±4.879）]，migration capacities[（23.200±1.924） vs.（65.400±4.775）] of EC109 cells in vitro were decreased（t=-12.390，P=0.000；t=-18.331，P=0.000）. Homogeneous cells intercellular adhesion was increased（F=9.327，P=0.009），while heterogeneous cells intercellular adhesion was decreased（F=20.459，P=0.000）. Angiogenesis capacity[（2.780±0.424） vs. （5.080±0.634）] was decreased（t=-16.919，P=0.000）. The expression of E-cadherin increased[（0.38８±0.56５） vs. （0.22８±0.582）] significantly（t=3.421，P=0.027），while Vimentin and β-catenin in the cyclopamine treatment group[（0.58８±0.109） vs. （0.507±0.05１）；（0.998±0.128） vs. （0.756±0.03８）] were significantly down-regulated（t=4.221，P=0.013；t=6.781，P=0.002）. Compared to those in control group，the expressions of Snail were significantly reduced in the cyclopamine treatment group[（0.756±0.03８） vs. （0.401±0.02１）]（t=-6.455，P=0.023）. Also，the mRNA expressions of Twist1 were significantly reduced（74.987±9.031）%. Conclusion：The inhibi－tion of hedgehog signaling pathway in vitro can effectually suppress the process of EMT，even reverse this process. The mechanism is partially correlated with the down-regulations of Snail and Twist1.