ed by the fact that blockage of glycogen breakdown enhances the toxicity of melatonin and is highly lethal for these cells. Inhibition of glycogen phosphorylase- enzyme that breaks down glycogen into glucose subunits- triggers apoptotic cell death due to the absence of energetic substrate in some tumor types, like melatonin does. Blockage of glycolytic metabolism currently constitutes a major target to prevent cancer growth, with various experimental treatments being already tested in preclinical studies. These include genetic or pharmacological inhibition of glycolytic enzymes or LDH; or the use of non-metabolizable analogues, such as 2-Deoxyglucose or 3-Bromopyruvate. Many of these treatments prevent tumor progression, induce an increase in ROS production and kill cancer cells, again consistent with melatonin effects in Ewing sarcoma cells. Melatonin inactivates HIF-1 in Ewing sarcoma cells, which could account for the decrease in aerobic glycolysis. This transcription factor is primarily responsible for the increase in glycolytic activity in most cancer cells, allowing them to survive. Its activation is essential for cancer 11 / 15 Melatonin Regulation of Warburg Effect in Cancer Cells Fig 5. Melatonin inhibits HIF-1 in a PI3K/AKT/mTOR independent pathway. Western blot analyses were carried out to identify the effect of melatonin on the activation of HIF-1 in TC-71 cells and A-4573 and A-673 cells. AKT and mTOR activation in TC-71 and 12 / 15 Melatonin Regulation of Warburg 
Effect in Cancer Cells A-4573 and A-673 cells was evaluated using specific phosphoantibodies. GAPDH was used as a loading control in all cases. A representative blot is showed. Optical density of bands was measured and values of the hydroxy-HIF-1, p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells. Left panel, cell viability was evaluated by MTT reduction assay after treatment of TC-71 cells with 1 mM melatonin alone or in combination with 10 nM Oleandrin supplier rapamycin or 10 M LY294002 for 48 hours. Data are expressed as the percentage of vehicle-treated cells. Right panel, Representative western blot showing the relative protein level of p-AKT, p-mTOR and hydroxy-HIF-1 after 10 nM rapamycin or 10 M LY294002 treatment during 24 hours in TC-71 cell line. p0.05 vs. vehicle-treated cells. doi:10.1371/journal.pone.0135420.g005 cells to exhibit Warburg effect, since it increases the activity of the vast majority of the enzymes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723632 involved in aerobic glycolysis even under normoxic conditions. Inhibition of the transcription factor HIF-1 has been repeatedly suggested as a therapeutic target against cancer, because of its key role in the Warburg effect could allow fine control of tumor growth. In this sense melatonin has been described to decrease invasion and cell migration by blocking HIF-1 in some tumor types. Furthermore, in hypoxic environments, melatonin is able to decrease PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723666 HIF-1 expression levels in several tumor types. Finally, in vivo antitumoral and antiangiogenic effects of melatonin have been also suggested to be mediated by a decrease in HIF1 expression. Although there are a number of studies relating the activation of PI3K/ AKT/mTOR pathway with the activation of HIF-1, and melatonin regulates the PI3K/AKT/mTOR pathway in several models, our results show that these events are not connected in Ewing sarcoma cells. Taken together, the data presented in this work demonstrate the ability of melat