Erved in other tumor cells, it suggests, according to our results, that the disruption of mitochondrial membrane potential is not essential for melatonin-induced antiproliferative actions. Despite this, P19 cells relying on oxidative metabolism showed a decrease in cell respiration after buy Triptolide treatment with melatonin, an effect also identified in Glu-CSCs when maximal respiration is induced with FCCP. Although GluCSCs present depolarized mitochondria and decreased oxygen consumption, mitochondrial respiration is not completely impaired as cells possess functional OXPHOS machinery but probably decouple respiration from ATP production. Then, when cells undergo differentiation, mitochondria became polarized suggesting a different modulation of mitochondrial 
potential between stem and differentiated cells. While mitochondrial membrane potential in differentiated cells is maintained by the electron transport chain, in undifferentiated cells this process depends mostly on glycolysis and on ATP hydrolase activity of the F1Fo-ATPase. Our results suggest a direct effect of melatonin in the electron transport chain, although the observed increase of mitochondrial membrane potential may not result from an inhibition of the PTK/ZK respiratory chain alone but instead from effects on ATP synthase. Previous works showed an effect of melatonin in inhibiting the increased respiration resulting from stimulation of Krebs’ cycle, protecting mitochondria from oxidative damage. In another study, it was reported that melatonin decreased OXPHOS through complex IV inhibition while increasing the glycolytic efficiency, an effect that might explain the resistance of Glu-CSCs to melatonin, as well as the observed decrease in respiration and increased ATP levels. In our previous report, we demonstrated, by blocking OXPHOS with oligomycin, that glycolysis is the primary pathway for ATP production in P19 cells. Although we have found here that 1mM melatonin also produced an increase in the ATP content in Gal-CSCs and Gal-dCCs, we have not found differences regarding the percentage of ATP on the total adenine nucleotide pool between controls and melatonin-treated cells. Thus, the energetic balance was not affected by the treatment with melatonin. It was described that melatonin maintains mitochondrial homeostasis in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19858355 normal cells since it is able to reduce oxygen consumption while maintaining OXPHOS activity and ATP synthesis. Despite this, the higher amount of adenine nucleotides observed after the treatment with melatonin could also be related with an action of melatonin in triggering and/or directing cell differentiation as previously described. This action would be mediated by its interaction with the retinoicrelated orphan receptor alpha whose expression is temporally regulated during differentiation of P19 cells into neural lineages, as probably occurs in P19 cells grown in galactose that express higher amounts of the neuron-specific marker betaIII-tubulin. In general, our data indicate that the predictable outcome of melatonin Oncotarget treatment depends on the involvement of mitochondrial bioenergetics to cell metabolism. It is known that melatonin-induced apoptosis in cancer cells is associated with ROS production, and that oxidative stress determines cancer cells fate in response to melatonin. Here, we found a link between impaired oxygen consumption, mitochondrial hyperpolarization and oxidative stress generation in cells grown in the modified galactose media and treat.Erved in other tumor cells, it suggests, according to our results, that the disruption of mitochondrial membrane potential is not essential for melatonin-induced antiproliferative actions. Despite this, P19 cells relying on oxidative metabolism showed a decrease in cell respiration after treatment with melatonin, an effect also identified in Glu-CSCs when maximal respiration is induced with FCCP. Although GluCSCs present depolarized mitochondria and decreased oxygen consumption, mitochondrial respiration is not completely impaired as cells possess functional OXPHOS machinery but probably decouple respiration from ATP production. Then, when cells undergo differentiation, mitochondria became polarized suggesting a different modulation of mitochondrial potential between stem and differentiated cells. While mitochondrial membrane potential in differentiated cells is maintained by the electron transport chain, in undifferentiated cells this process depends mostly on glycolysis and on ATP hydrolase activity of the F1Fo-ATPase. Our results suggest a direct effect of melatonin in the electron transport chain, although the observed increase of mitochondrial membrane potential may not result from an inhibition of the respiratory chain alone but instead from effects on ATP synthase. Previous works showed an effect of melatonin in inhibiting the increased respiration resulting from stimulation of Krebs’ cycle, protecting mitochondria from oxidative damage. In another study, it was reported that melatonin decreased OXPHOS through complex IV inhibition while increasing the glycolytic efficiency, an effect that might explain the resistance of Glu-CSCs to melatonin, as well as the observed decrease in respiration and increased ATP levels. In our previous report, we demonstrated, by blocking OXPHOS with oligomycin, that glycolysis is the primary pathway for ATP production in P19 cells. Although we have found here that 1mM melatonin also produced an increase in the ATP content in Gal-CSCs and Gal-dCCs, we have not found differences regarding the percentage of ATP on the total adenine nucleotide pool between controls and melatonin-treated cells. Thus, the energetic balance was not affected by the treatment with melatonin. It was described that melatonin maintains mitochondrial homeostasis in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19858355 normal cells since it is able to reduce oxygen consumption while maintaining OXPHOS activity and ATP synthesis. Despite this, the higher amount of adenine nucleotides observed after the treatment with melatonin could also be related with an action of melatonin in triggering and/or directing cell differentiation as 
previously described. This action would be mediated by its interaction with the retinoicrelated orphan receptor alpha whose expression is temporally regulated during differentiation of P19 cells into neural lineages, as probably occurs in P19 cells grown in galactose that express higher amounts of the neuron-specific marker betaIII-tubulin. In general, our data indicate that the predictable outcome of melatonin Oncotarget treatment depends on the involvement of mitochondrial bioenergetics to cell metabolism. It is known that melatonin-induced apoptosis in cancer cells is associated with ROS production, and that oxidative stress determines cancer cells fate in response to melatonin. Here, we found a link between impaired oxygen consumption, mitochondrial hyperpolarization and oxidative stress generation in cells grown in the modified galactose media and treat.