High glucose and insulin enhance uPA expression, ROS formation and invasiveness in breast cancer-derived cells

Accumulating evidence indicates that type 2 diabetes is associated with an increased risk to develop breast cancer. This risk has been attributed to hyperglycemia, hyperinsulinemia and chronic inflammation. As yet, however, the mechanisms underlying this association are poorly understood. Here, we studied the effect of high glucose and insulin on breast cancer-derived cell proliferation, migration, epithelial-mesenchymal transition (EMT) and invasiveness, as well as its relationship to reactive oxygen species (ROS) production and the plasminogen activation system. MDA-MB-231 cell proliferation, migration and invasion were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), scratch-wound and matrigel transwell assays, respectively. ROS production was determined using 2' 7'-dichlorodihydrofluorescein diacetate. The expression of E-cadherin, vimentin, fibronectin, urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) were assessed using qRT-PCR and/or Western blotting assays, respectively. uPA activity was determined using gel zymography. We found that high glucose stimulated MDA-MB-231 cell proliferation, migration and invasion, together with an increased expression of mesenchymal markers (i.e., vimentin and fibronectin). These effects were further enhanced by the simultaneous administration of insulin. In both cases, the invasion and growth responses were found to be associated with an increased expression of uPA, uPAR and PAI-1, as well as an increase in active uPA. An osmolality effect of high glucose was excluded by using mannitol at an equimolar concentration. We also found that all changes induced by high glucose and insulin were attenuated by the anti-oxidant N-acetylcysteine (NAC) and, thus, depended on ROS production. From our data we conclude that hyperglycemia and hyperinsulinemia can promote breast cancer cell proliferation, migration and invasion. We found that these features were associated with increased expression of the mesenchymal markers vimentin and fibronectin, as well as increased uPA expression and activation through a mechanism mediated by ROS.