Influence of distress hormones on the oral cancer cells resistance to chemotherapy

Recent findings have shown that norepinephrine (NE) exerts fundamental influence in the process of resistance to the chemotherapy of some types of cancer. However, this topic remains poorly explored and has not been studied in oral cancer yet. Thus, our aim is to investigate the effects and mechanisms involved in the NE actions over the oral cancer cells resistance to the chemotherapeutics in vitro. Three oral squamous cell carcinoma (OSCC) cell lines, SCC-9, SCC-15, and Cal27, will be stimulated with 10¼M of NE one hour prior their treatment with the chemotherapeutics cisplatin and 5-fluorouracil, individually. Twenty-four hours later, the cellular viability will be assessed by MTT and alamar blue. Then, two mechanisms possibly responsible for the NE-induced OSCC chemoresistance will be investigated: the participation of the proteins MDR-1 and ABCG2 in the efflux of the drugs outside the cells and the role of the cancer stem cells in the maintenance and spread of the resistant tumoral population. To study the first mechanism, the gene and protein expressions of MDR-1 and ABCG2 will be verified in the OSCC cells stimulated or not with NE and posteriorly treated with the chemotherapeutics by qRT-PCR and western blotting, respectively. To confirm the role of such proteins, MDR-1 and ABCG2 RNA interference will be introduced, individually, and the cellular viability and gene and protein expressions will be once more verified. To investigate the second mechanism, the chemoresistant OSCC cells stimulated or not by the NE will be characterized by the assessment of ALDH activity, the side population identification by the Hoechst dye efflux activity, and the immunostaining of the surface proteins CD44 and CD133. Additionally, the sphere formation assay will be performed. Finally, to verify the migratory and invasive capabilities of the OSCC cells stimulated or not with NE and treated with the chemotherapeutics posteriorly, the scratching assay and the transwell assay will be performed. Data regarding the cell culture experiments will be confronted through the variance analysis (ANOVA) or "t" student's test. The qRT-PCR data will be presented as fold-change compared to the control (non treated) cells. Level of significance will be set at 5% for all the tests. Protein expressions exhibited by the western blotting will be qualitatively analyzed. (AU)