, 2007), thereby contributing to the development of these tumors. There are few studies investigating the influence of stress hormones on HNSCC. Recently, the presence of β-adrenergic receptors (β-AR) for NE and E has been identified in oral (Shang et al., 2009) and esophagus cancer (Liu et al., 2008) cell lines. These investigations also showed that the proliferation of these cell lines is high throughput screening stimulated
by NE and E, respectively. Nevertheless, there is no evidence that IL-6 expression in oral cancer can be influenced by stress hormones. In this study, we have evaluated the effects of stress-related hormones on IL-6 expression and proliferation of OSCC cells, and evidence that OSCC biopsies express β-ARs is provided. The OSCC-derived
cell lines SCC9, SCC15, and SCC25 selleck were used in the evaluation of the effects of stress hormones. The cell lines were kindly provided by Dr. Ricardo Della Coletta (School of Dentistry, State University of Campinas, Piracicaba, São Paulo, Brazil). These cells were maintained and propagated in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (DMEM/F12; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, 100 μg/mL streptomycin, and 0.1% gentamicin, at 37 °C, in 5% CO2 humidified atmosphere. Experiments were carried out with 80% confluent cultures. SCC9, SCC15, and SCC25 cells were seeded in 24-well plates (1.0 × 105 cells per well) Astemizole and cultured
for 24 h in serum-reduced medium (0.1% FBS). The following hormones were tested: NE (Calbiochemical Co, La Jolla, CA), cortisol (Sigma–Aldrich, St. Louis, MO) and isoproterenol (Sigma–Aldrich, St. Louis, MO), a β-adrenergic agonist. The cells SCC9 and SCC25 were then treated with NE or isoproterenol at 0, 0.1, 1, and 10 μM, or cortisol at 0, 1, 10, 100, and 1000 nM. These concentrations were used in the subsequent experiments. The cells SCC15 were treated with NE and cortisol. For blocking experiments, 1 μM propranolol was added to the cell cultures 1 h before addition of 10 μM NE. Cell-free supernatants and cells were collected at 1, 6, and 24 h, and kept at − 80 °C until the assays were performed. The hormone concentrations employed were defined by taking the physiological levels that usually reaching in the tumor microenvironment. NE basal circulating levels range between 10 pM and 1 nM (Sood et al., 2006), and studies have suggested that stress increases these levels to approximately 100 nM, and they may reach 10 μM in the microenvironment of some types of tumors (Antoni et al., 2006 and Sood et al., 2006). The concentrations of 10 and 100 nM cortisol reflect similar levels to those found in stress conditions, and higher concentrations (1000 nM) simulate pharmacological doses of glucocorticoids (Miller and O’Callaghan, 2002).