MCP-1 Induced Epithelial-mesenchymal Transition And Migration Of Human Breast Cancer MCF-7 Cells Through Mek/erk/gsk-3β Signaling Pathway

Monocyte chemoattractant protein-1(MCP-1) belongs to the chemotactic cytokines family, which is highly converved in many species, such as human and mouse. MCP-1 can bind to its receptor cysteine-cysteine chemokine receptor 2(CCR2), and plays an important role in breast cancer cell metastasis. However, the underlying molecular mechanisms are still not well understood. The aim of the present study is to elucidate how MCP-1 regulates the migration and epithelial-mesenchymal transition(EMT) of human breast carcinoma MCF-7 cells. Wound healing assay and transwell assay were used to measure the migration and invasion of MCF-7 cells induced by MCP-1(50ng/ml). It was found that, as compared with control, the migration and invasion enhanced approximately 2-fold(migration) and 1.4-fold(invasion) after MCP-1 treatment, respectively. Next we determined whether MCP-1 is sufficient to induce EMT by observing morphology changes and examining expression of the EMT markers. The results showed that the cell morphology changes from cobblestone morphology to mesenchymal spindle-like and fusiform features. Immunofluorescence and western blotting analysis showed that this morphological change was associated with the down regulation of E-cadherin, and the upregulation of Fibronectin and Vimentin. These results indicated that MCP-1 could promote the migration and invasion of MCF-7 cells undergoing EMT. Moreover, our studies also demonstrated that MCP-1 can not only regulate stabilization and subcellular localization of Snail, but also increase the expression and activity of MMP-2/9, VEGF. In addition, MCP-1 treatment inhibited glycogen synthase kinase 3β(GSK-3β) activity through serine-9 phosphorylation and then elevated snail stability mediated by MEK/ERK signaling pathway in MCF-7 cells. Inhibition of MEK/ERK by U0126 attenuated the MCP-1-induced phosphorylation of GSK-3β and altered the protein levels of Snail, EMT markers, migration and invasion of MCF-7 cells. Inactivation of GSK-3β by LiCl significantly increased MMP-2/9 activity, VEGF expression and EMT of MCF-7 cells. These findings revealed that MCP-1-induced EMT and migration is mediated by ERK/ GSK-3β/Snail pathway.