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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Unidad de Morfofisiología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, Estado de México, CP, 54090, México.
Luis Antonio
LA
Flores-López
Unidad de Morfofisiología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, Estado de México, CP, 54090, México.
María Guadalupe
MG
Martínez-Hernández
Unidad de Morfofisiología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, Estado de México, CP, 54090, México.
Rubí
R
Viedma-Rodríguez
Unidad de Investigación Médica en Bioquímica, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Av. Cuauhtémoc 330, Col. Doctores, Del, Cuauhtémoc, DF, 06720, México.
Margarita
M
Díaz-Flores
Unidad de Morfofisiología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios 1, Los Reyes Ixtacala, Tlalnepantla, Estado de México, CP, 54090, México. labaiza@unam.mx.
Luis Arturo
LA
Baiza-Gutman
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000502:physiology; D005260:Female; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D006943:Hyperglycemia / Q000503:physiopathology; D006946:Hyperinsulinism / Q000503:physiopathology; D007328:Insulin / Q000494:pharmacology*; D009361:Neoplasm Invasiveness / Q000473:pathology* / Q000503:physiopathology; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis*
D007328:Insulin; D017382:Reactive Oxygen Species; D014568:Urokinase-Type Plasminogen Activator; D005947:Glucose
Breast cancer; Cancer cell invasion; High glucose; Insulin; ROS; uPA