3A) and induced a 2.7-fold decrease in IL-6 protein HDAC inhibitor levels (p < 0.001) ( Fig. 3B). In contrast, after 1 h, 10 nM cortisol (simulating physiological stress levels) promoted increase IL-6 mRNA expression (129% compared to control) ( Fig. 3A) and protein levels ( Fig. 3B) in SCC9 cells, but these changes did not reach significance. These cortisol effects were blocked by glucocorticoid inhibitor Mefipristone (data not shown). SCC25 cells

did not exhibit a significant response to cortisol treatment. Specifically, SCC25 cells treated with 1000 nM cortisol at 6 h produced 292.2 ± 17.40 pg/mL of IL-6, resulting in a 1.25-fold decrease compared to the control (p < 0.05) ( Fig. 3D). In these same cells, lower IL-6 mRNA levels were detected at 1 h with 100 nM cortisol (131.1 ± 0.03% compared to the control) and 1000 nM cortisol (152.1 ± 2.7%), while an increase in IL-6 mRNA levels took place at 24 h using 10 nM cortisol (138 ± 12.96%) and 100 nM cortisol (147 ± 28.75%), but these results were not significant ( Fig. 3C). Similar results were found in SCC15 cells, in which lower cortisol concentrations (1 and 10 nM) did not determine large variations in IL-6 mRNA levels, whereas high concentrations simulating pharmacological selleckchem concentrations (e.g., 1000 nM) decreased IL-6 expression (but these results were not significant) (Fig. 3E). To examine

the effects of stress hormones on OSCC cell proliferation, SCC9 and SCC15 cells were treated with different doses of NE and cortisol, and cell proliferation was assayed by MTT at 6, 24, and 48 h. The SCC25 cell line was not assayed by MTT because it did not respond well (absence of cell growth) to culture in serum-reduced medium (0.1% FBS). Stimulation of SCC9 and SCC15 cells with physiological NE stress levels (10 μM) induced an enhancement of 170 ± 17.7% (p < 0.05) and 124 ± 13.7% (p < 0.05) in cell proliferation at 6 h compared with non-treated cells, respectively ( Fig. 4A). These

NE-induced effects of SCC9 and SCC15 cells were not significant at subsequent times (24 and 48 h) (data not shown). In SCC9 cells, treatment with pharmacological levels of cortisol (1000 nM) produced at later time point (48 h) a rise of approximately 200 ± 36.1% in cell proliferation (p < 0.05) ( Fig. 4B). Cortisol doses that simulate stress conditions (10 nM) induced at 48 h an increase Dimethyl sulfoxide in cell proliferation in SCC9 (non-significant) ( Fig. 4B) and in SCC15 cells (135 ± 17.5%; p < 0.05) ( Fig. 4B). There was no significant increase in the cell proliferation index after 6 and 24 h of stimulus with cortisol (data not shown). Real-time PCR assays confirmed that SCC9, SCC15, and SCC25 cells express mRNA for β1- and β2-AR (Fig. 5A). To determine whether the increase in IL-6 expression was mediated through β-adrenergic receptors, the cell lines were pre-treated with a nonspecific β antagonist (propranolol), at the time point of maximum mRNA IL-6 expression (10 μM NE at 1 h).