Recent evidence show that stress hormones derived from the activation of Sympathetic Nervous System (SNS) and Hypothalamic-Pituitary-Adrenal (HPA) axis can promote DNA damage and, consequently, influence the tumorigenicity of somatic cells. The main risk factors for oral cancer are smoking and alcoholism. However, there are no studies that have evaluated the effects of stress hormones' exposure in the malignant transformation of human oral keratinocytes or as a cofactor of chemical carcinogenesis. The aim of this study will be to evaluate the effects of norepinephrine and cortisol on DNA damage and malignant transformation of human oral keratinocytes in the presence and absence of carcinogen derived from tobacco 4- (N-methyl-N-nitrosamino) -1- (3-pyridyl) -butan-1-one) (NNK). Keratinocytes will be stimulated with different concentrations of norepinephrine or cortisol in the presence or absence of NNK. Non-stimulated cells will be used as control. After 24 hours or 7 days of exposure to hormones, the epithelial cells will be evaluated for the presence of DNA damage by the comet and TUNEL assays. Cellular levels of 8-hydroxy-2-deoxyguanosine (8OHdG) will be measured by ELISA, and the cellular activity of caspases 3 and 7 will be assessed by immunofluorescence. Soft agar colony formation assays will be used to evaluate the effects of stress hormones on the malignant transformation of keratinocytes. The participation of ²-adrenergic receptors types 1, 2 and 3, glucocorticoid receptor and signaling pathways of the protein kinases PKA and SGK1 in DNA damage and malignant transformation of keratinocytes (induced by stress-related hormones) will be evaluated by Western Blotting, inhibition assays and assays for evaluation of the malignant phenotype. Specific tests will be used to determine if there are significant differences between the groups treated with the hormones in the presence or absence of NNK and specific inhibitors.
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