Abstract B38: Melatonin efficacy in breast cancer microenvironment under acidosis
Authors: Sonehara Nathália Martins , Jardim-Perassi Bruna Victorasso , Mota André de Lima , Moschetta Marina Gobbe , Zuccari Debora Aparecida Pires de Campos , Paula Rubens de 
Published: May 2016

Breast cancer is the most common neoplasia among women and has been frequently associated to high morbidities and mortalities rates. Cancer progression is strongly influenced by conditions of tumor microenvironment. Rapid tumor growth results in decreased of oxygen intake, restricting metabolic demands in specific regions of tumor. This poor perfusion coupled with preferential anaerobic glycolysis leads to acidification of tumor microenvironment. Survival mechanisms of tumor cells under acidic conditions are not well characterized, but has been shown that adverse conditions in tumor microenvironment can select phenotypes that are able to adapted to survival and growth in low pH conditions, hence resulting in tumor progression. Thus, controlling these adverse conditions in tumor microenvironment is a promising tactic in limiting cancer development. Melatonin, a hormone naturally produced and secreted by pineal gland, appears to play multiple roles in cancer and it is proposed as a possible keeper of an extreme environment hindering the survival of these cells. In this study, the benefits of melatonin treatment on cell viability of breast cancer cell lines under acidosis condition were evaluated. Breast cancer cell lines estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37°C in 5% CO2. Cell viability was measured using an MTT assay. Individual wells of a 96-well plate were seeded with 100 µL of medium containing 0.05 x 106 cells and cells were separated into the following groups: Group 1) Cells cultured in pH 7.4 with DMEM (control); Group 2) Cells cultured in pH 6.7 with DMEM supplemented with 25 mmol/L buffer 2- (N-Morpholino) ethanesulfonic acid (MES) for 24 hours, to mimic an acute acidosis in tumor microenvironment; Group 3) Cells cultured with DMEM supplemented with 25 mmol/L MES and treated with 1 mM of melatonin for 24 hours; Group 4) Cells cultured with DMEM supplemented with 25 mmol/L MES for 12 hours and treated with 1 mM of melatonin for an addition 12 hours. After treatments, MTT solution were added to each well, and the plates were incubated at 37°C for an additional 4 hour. To solubilize the MTT formazan crystals, the cells were incubated with dimethyl sulfoxide (DMSO) for 10 minutes and absorbance was measured at 540 nm. Cell viability (%) was calculated for all groups compared to control samples. The results were compared by ANOVA, followed by the Bonferroni test. The results showed that there was a decrease of both MCF-7 and MDA-MB-231 cells viability under acute acidosis conditions (Group 1 vs Group 2, p &lt; 0.05). MCF-7 cells showed 65.36 ± 1.16 % of cell viability while MDA-MB-231 cells showed 52.98 ± 0.90 of cell viability under acidosis conditions (Group 2). Melatonin treatment for 24 hours was effective in reducing cell viability in acidosis conditions, leading to a decrease in MCF-7 cells (Group 3: 52.41 ± 1.32 %) and MDA-MB-231 cells (Group 3: 47.83 ± 0.67 %) when compared with cells under acidosis without treatment (Group 2 vs Group 3, p &lt; 0.05). Furthermore, in both cell lines there was a decrease in cell viability in Group 4 (MCF-7 = 54.24 ± 1.22 %; MDA-MB-231 = 45.60 ± 0.45 %) compared to cells under acidosis (Group 2 vs Group 4, p &lt; 0.05), and no difference was observed when compared with cells under acidosis and treated with melatonin for 24 hours (Group 3 vs Group 4, p &gt; 0.05). In conclusion, our results show that acute acidosis reduces the viability of breast cancer cells and melatonin treatment is effective, leading to decrease the cell viability under acute acidosis.In addition, studies are being madeto evaluate the effectiveness of melatonin also in chronic acidosis conditions, and to investigate its signaling pathways in the extreme tumor microenvironment, in order to confirm potential effectiveness of melatonin in breast cancer in these conditions.

Citation Format: Nathália Martins Sonehara, Bruna Victorasso Jardim-Perassi, André de Lima Mota, Marina Gobbe Moschetta, Debora Aparecida Pires de Campos Zuccari, Rubens de Paula, Jr. Melatonin efficacy in breast cancer microenvironment under acidosis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B38.