| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 96 === Tumor necrosis factor-α (TNF-α) is mainly produced by activated macrophage. This factor regulates inflammation and the activities of immune cells. It has been reported that chronic stimulation with low-dose of TNF-α may lead to carcinogenic changes. We have demonstrated that the invasiveness of 786-O cells was significantly promoted by TNF-α in a dose-dependent manner. Moreover, TNF-α induced the epithelial-mesenchymal transition (EMT) of 786-O cells by repressing E-cadherin, promoting vimentin expression, and activating MMP9 activity. After TNF-α treatment, we observed the activation of PI3K/Akt and suppression of glycogen synthase kinase-3β (GSK-3β), one of the PI3K/Akt downstream substrates. GSK-3β is a serine/threonine kinase that was first identified as a regulator of glycogen synthesis. Since it is a negative regulator of Wnt/β-catenin signaling, GSK-3β may also suppress tumor cell growth. In addition, GSK-3β phosphorylates Snail, promotes its degradation, and then inhibits the EMT. In this study, we determine the role of TNF-α in the invasiveness of 786-O cells and whether the EMT of 786-O cells is through the activation of PI3K/Akt and repression of GSK-3β. The PI3K inhibitor (LY294002) and GSK-3 inhibitor (LiCl) were used to transfect the constitutive active and inactive GSK-3β. After pre-treating LY294002 to prevent GSK-3β from the suppressive effects of TNF-α stimulation, the EMT of 786-O cells was demonstrated to be attenuated. However, inhibition of GSK-3β by LiCl increased the EMT of 786-O cells. Cells stably overexpessing active GSK-3β were found to be more resistant to TNF-α-induced EMT whereas those overexpressing inactive GSK-3β�烢romote the EMT even in the absence of TNF-α. GSK-3β may involve in the regulation of E-cadherin repressor (Snail and Slug). However, there were no significant differences in the MMP9 expression between stable clones and the control cells after TNF-α treatment. Based on these findings, GSK-3β may mediate TNF-α-induced EMT and may not involve in MMP9 regulation. Moreover, GSK-3β may play an important role in TNF-α-induced advanced renal cell carcinoma.
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