Effects And Mechanism Of Dehydroepiandrosterone On Epithelial-mesenchymal Transition In Bronchial Epithelial Cells

Objectives:The human bronchial epithelial cells (16HBE-14o) were stimulated with TGF-β1to imitate the process called epithelial-mesenchymal transition (EMT) in vitro, in order to investigate the effects of Dehydroepiandrosterone (DHEA) on TGF-β1-induced EMT and discuss the possible mechanism.Methods:1.16HBE-14o cells were cultured with MEM medium, and then were plated at a denisity of approximately5×104cells per milliliter. When the cells were incubated with5ng/ml TGF-β1for72h, the changes of cells morphology were observed and cells were harvested for E-cadherin, a-SMA protein detection with Western Blot (WB).2. Cells were preincubated with DHEA at different concentrations (1,10,100μM) for24h, and then co-treated with TGF-β1for72hours. Detect cells as method1above.3. Cells were preincubated with or without100uM DHEA for24h, and then co-treated with5ng/ml TGF-(31for different times (0,0.5h,1h,2h). Cells were harvested for p-AKT, AKT protein with WB.4. Cells were grouped and cultured with different interventions:(1)control group,(2)TGF-β1group,(3)DHEA100(the terminal concentration was100μM)+TGF-β1group,(4)DHEA100+TGF-β1+flutamide group,(5)TGF-β1+LY294002(LY) group,(6)TGF-β1+IGF-1group,(7)TGF-β1+DHEA100+LY group,(8)TGF-β1+DHEA100+ IGF-1group. When cells were co-treated with5ng/ml TGF-β1for1h, Cells were harvested for p-AKT, AKT protein with WB. When co-treated for72h, Cells were harvested for E-cadherin, a-SMA protein with WB.Results:1. Untreated16HBE-14o cells showed a pebble-like shape and cell-cell adhesions were clearly observed. TGF-β1-treated cells showed a decrease in cell-cell contacts and adopted a more elongated morphologic shape, in combination with a decreased expression of E-cadherin and a increased expression of a-SMA.2. DHEA inhibited the EMT induced by TGF-β1, and the effect of DHEA was concentration dependent, with DHEA concentrations of100μM inducing maximal inhibitive effect.3. TGF-β1stimulated a rapid AKT phosphorylation in cells, with maximal activation at lh incubation. The activation of AKT phosphorylation by TGF-β1was inhibited by the preincubation of DHEA. Total AKT expression was unaltered over the time course.4. Preincubation of cells with Flutamide, an AR antagonist, fully attenuated the inhibition of AKT phosphorylation, as well as EMT induced by DHEA. Pharmacological inhibition of PI3K, the upstream kinase of AKT with LY294002, completely prevented the phosphorylation of AKT and EMT induced by TGF-β1. Accordingly, activation of PI3K by IGF-1promoted TGF-β1-mediated activation of AKT and EMT.Conclusions:1. TGF-β1can induce EMT in16HBE-14o cells.2. The presence of DHEA was effective in blocking the TGF-β1-induced EMT, and the effect of DHEA is concentration dependent.3. DHEA exerted the inhibition of TGF-β1-induced EMT through AR.4. The inhibitive role of DHEA to EMT involves PI3K/AKT signaling pathway.