Melatonin and ovarian cancer: evaluation of energy metabolism, oxidative stress, and mesenchymal stem cell modulation

Abstract

Ovarian cancer (OC) is the second gynecological cancer that affects women and, due to its generic symptoms, it has high rates of recurrence with rapid progression and most patients develop chemoresistance to conventional treatments. Uncontrolled cell proliferation represents one of the characteristics of neoplastic disease and, tumor cells may adjust their energy metabolism in favor of rapid cell growth and progression; this metabolic pattern is very different from that found in healthy cells. Melatonin is a hormone produced and secreted by the pineal gland in the dark and, more recently, its production has been documented in the mitochondria of cells - the concentration of melatonin is at low levels in OC. Experiments involving animal models and OC cell culture have already demonstrated the antitumor and oncostatic properties of melatonin. Therefore, the objective of the present study is to evaluate the action of melatonin on energy metabolism and oxidative stress in high-grade (SKOV-3) and low-grade (CAISMOV24) human ovarian carcinoma cells. For in vitro experiment, the two strains will be divided into control groups and melatonin-treated groups (doses will be established based on IC50 assay) in the presence of luzindole. The cell cytotoxicity test will be performed by MTT, and cell migration and invasion will be tested using inserts. To verify the effects of melatonin on cellular energy metabolism, the activities of the following enzymes will be quantified: glucose-6 phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49), related to the pentose-phosphate pathway; phosphofructokinase 1 (PFK-1, E.C. 2.7.1.11), related to the glycolytic pathway; pyruvate dehydrogenase complex (PDH, E.C. 1.2.4.1) and its regulator pyruvate dehydrogenase kinase (PDK, E.C. 2.7.11.2), both related to the flow of intermediates for mitochondrial metabolism; citrate synthase (CS, E.C. 4.1.3.7), related to the citrate cycle; beta-hydroxyl acyl-CoA dehydrogenase (OHADH, E.C. 1.1.1.35); related to the oxidation of fatty acids; lactate dehydrogenase (LDH, E.C. 1.1.1.27), related to cellular anaerobic metabolism. The levels of GLUT1, HIF-1±, G6PDH, PDK and PDH proteins will also be measured by Western blot. The profiles of glucose consumption and lactate production will be analyzed, by spectrophotometry, as well as the metabolism of glutamine (glutaminolysis). Enzymes linked to oxidative stress such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.9), as well as reduced glutathione (GSH) and oxidized (GSSG), and the levels of lipid peroxidation, through the formation of malondialdehyde, will be investigated by spectrophotometry. The profile of cell signaling molecules involved with tumor aggressiveness will also be evaluated through a multiplex assay, and melatonin levels will be assayed by ELISA. It will also be investigated the effect of the conditioned medium of each cell line on the other, accompanied or not by melatonin treatments and paclitaxel, in the presence of MSCs, in an independent culture system or indirect co-culture. After treatments, the exosomes will be evaluated by mass spectrometry. Immunophenotyping assays involving markers for differentiation in CTC and FACs will be performed by flow cytometry in each cell line. Based on these findings, we hope to understand the effects of melatonin on the cellular energy metabolism of OC cells, as well as the role of oxidative stress in this scenario. It will be possible to elucidate possible mechanisms by which melatonin regulates the pattern of cell proliferation in a high and low-grade OC cell model, thereby evidencing its role on tumor development and progression and its likely benefit for the treatment of OC. (AU)