The Mechanism Study On The Inhibition Effect Of Halofuginone On Tumor Cell Epithelial-Mesenchymal Transition Induced By Irradiation

Objective:To investigate the inhibition effect of halofuginone on tumor cell epithelial-mesenchymal transition induced by irradiation and try to explore the underlying mechanisms.Methods:We investigated the effects of halofuginone on inhibition of epithelial-mesenchymal transition induced by irradiation by using Lewis lung cancer cells in this research. The toxicity of halofuginone on Lewis lung cancer cells was observed by the MTT method, and the appropriate dose of halofuginone was then determined before experiments started. Lewis lung cancer cells were divideded into4groups. Control group, without any treatment; halofuginone group, with management of cell culture media containing constant concentration of halofuginone; irradiation group,6MV X-ray irradiation with10Gy/f X1f; combination group, with halofuginone administration as in the halofuginone group, and irradiation was given as in the irradiation group. Western blotting was used to test the expression of P-Smad3in4groups at the time of2days and5days respectively in order to determine the appropriate detecting time of the TGF-P signaling activity. Changes of the4groups, including morphology, migration and invasion were observed by microscope, scratch assay and transwell chamber assay respectively. Western blotting was used to test the expression of EMT markers, downstream proteins of TGF-β signal transduction pathway, such as P-Smad2, P-Smad3and Smad7in4groups.Results:We drawn inhibition curves of Lewis lung cancer cells according to the results of MTT in24and48hours and the appropriate halofuginone concentration used in this experiment was determined as31.17ng/ml. Compared to detected at2days after irradiation, the expressions of P-Smad3in irradiation group were increased after5days of irradiation, and for this reason, the time of following experiment was chosen as5days after irradiation. Compared with that in the control group, in the halofuginone group, the areas of scratch wound healing were reduced and the numbers of the cells transferred to the lower surface in transwell chamber were decreased, the expressions of epithelial markers E-Cadherin was enhanced and mesenchymal markers N-Cadherin was descended after the administration of halofuginone. In the irradiation group, the Lewis lung cancer cells presented with the mesenchymal phenotype, and compared with the control group, the areas of scratch wound healing were enlarged and the numbers of the cells transferred to the lower surface in transwell chamber were increased, the expressions of epithelial markers E-Cadherin was descended and mesenchymal markers N-Cadherin was enhanced. In the combination group, compared to that in the irradiation group, the mesenchymal phenotype was reversed, and the areas of scratch wound healing were reduced and the numbers of the cells transferred to the lower surface in transwell chamber were decreased. The expressions of epithelial marker E-Cadherin was up-regulated and the mesenchymal marker N-Cadherin was down-regulated. Compared with control group, the expressions of Smad2and Smad3were down-regulated and the expression of Smad7was up-regulated after the administration of halofuginone. After irradiation, the expressions of Smad2and Smad3were up-regulated and the expression of Smad7was down-regulated when comparing with the control group. For combination group, the expressions of Smad2and Smad3were down-regulated and the expression of Smad7was up-regulated when comparing with the irradiation group.Conclusion:Halofuginone could inhibit the epithelial-mesenchymal transition of Lewis lung cancer cells induced by irradiation. The possible mechanism may be due to the inhibition effect on TGF-β signal transduction.