TNF-α induces expression of urokinase-type plasminogen activator and β-catenin activation through generation of ROS in human breast epithelial cells

Malignant tumors have a capability to degrade the extracellular matrix (ECM) by controlled proteolysis. One of the important components of the proteolysis system involved in such process is urokinase-type plasminogen activator (uPA). Tumor necrosis factor (TNF)-α was found to stimulate uPA. TNF-α impaired the ability of cells to aggregate and to attain compaction. Dyscohesion (cell-cell dissociation) induced by TNF-α was associated with the disordered expression of cadherin/β-catenin at the sites of cell-cell contact. We observed that human breast epithelial (MCF-10A) cells treated with TNF-α transiently up-regulated expression of uPA and its mRNA transcript. In addition, TNF-α induced activation of β-catenin in MCF-10A cells. Based on these findings, we attempted to examine the role of β-catenin and its partner, Tcf-4 in upregulation of uPA. siRNA knock down of β-catenin abrogated TNF-α-induced uPA expression as well as Tcf-4/β-catenin DNA binding. TNF-α-stimulated MCF-10A cells exhibited increased intracellular accumulation of reactive oxygen species (ROS). TNF-α-induced expression of uPA and activation of β-catenin signaling appear to be mediated by ROS in MCF-10A cells, as both events were blocked by the antioxidant N-acetylcysteine. Eupatilin (5,7-dihydroxy-3',4',6-tri-methoxy-flavone), a pharmacologically active flavone derived from Artemisia asiatica, has been shown to possess strong antioxidative activity. Eupatilin inhibited TNF-α-induced intracellular ROS accumulation, expression of uPA and β-catenin activation. Moreover, eupatilin inhibited the TNF-α-induced invasion of MCF-10A cells. Taken together, the above results suggest that eupatilin has chemopreventive effects on mammary tumorigenesis by targeting the β-catenin-uPA axis stimulated by TNF-α.