E differences may come as consequence of the heterogeneity buy JW-55 within cell populations. Both types of cells, Gal-CSCs and Gal-dCCs, are composed of a range of different cell populations with different cell cycle regulation, morphology, growth rate, differentiation patterns and metabolism that may lead to divergent responses upon melatonin treatment. In glycolytic and resistant cells, melatonin induced alternative types of cell Oncotarget cycle arrest, namely at phases G2/M and G1/G0 in GluCSCs and Glu-dCCs respectively. Recent studies reported different results regarding cell cycle progression in cancer cells treated with melatonin. In human colorectal cancer cells, melatonin decreased the S-phase population and induced cell death, whereas in osteosarcoma and leukemia cells, melatonin reduced the proportion of cells in the S- and G2/M-phases while increasing cells in G1/G0 phase, an effect also observed by us in Glu-dCCs. However, in this work, melatonin-induced cell cycle arrests seem to be incomplete in several cases, probably as a consequence of the observed heterogeneity within cell populations. On the other hand, high concentrations of melatonin increased the number of hepatoma cells in S-phase, showing an antiproliferative effect which was also observed by us in P19 cells cultured in galactose, glutamine/pyruvate- containing medium. Therefore, although melatonin induced alterations in cell cycle progression, those effects depend on the overall metabolic and differentiation state of the cancer cells. Even so, in the more resistant P19 Glu-CSCs, melatonin was able to induce 
an arrest at G2/M albeit without exerting significant cytostatic effects. Thus, in the P19 CSCs model, melatonin was only able to reduce cell proliferation when the cells lost pluripotency and cell cycle was modified to the canonical structure. Spontaneous Ca2+ oscillations are restricted to the G1/S phase transition in P19 CSCs, suggesting a role for Ca2+ in stem cell cycle progression. In fact, we have order STA 4783 recently reported that P19 stem cells present low cytosolic calcium levels, which are increased during cell differentiation. The higher concentrations of free calcium found in P19 cells grown in galactose medium are correlated with the changes found on cell cycle progression. Accordingly, the effect of melatonin on free Ca2+ was also dissimilar. Glu-CSCs 
showed increased Ca2+ levels upon melatonin treatment while having no effect on cell viability. Conversely, melatonin-treated cells grown in galactose, glutamine/pyruvatecontaining medium showed cell cycle arrest at S-phase, decreased cell viability and low intracellular free Ca2+ concentrations which, according with our previous results, seem to be required at high levels during the process of cell differentiation and mitochondrial maturation. These different results, which depend on the cellular metabolic state, are probably influenced by the lower calcium retention ability of mitochondria from differentiated cells than those from Glu-CSCs. Thus, the effects of melatonin PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19860992 on calcium signaling may play a role in mediating its growth-inhibitory action in cells with an active mitochondrial metabolism. Melatonin mitigates mitochondrial dysfunction in healthy cells, maintaining membrane potential and optimizing electron transport within the respiratory chain. However, in some melatonin-sensitive cancer cells, mitochondrial depolarization is observed. www.impactjournals.com/oncotarget 17089 Although this effect is not obs.E differences may come as consequence of the heterogeneity within cell populations. Both types of cells, Gal-CSCs and Gal-dCCs, are composed of a range of different cell populations with different cell cycle regulation, morphology, growth rate, differentiation patterns and metabolism that may lead to divergent responses upon melatonin treatment. In glycolytic and resistant cells, melatonin induced alternative types of cell Oncotarget cycle arrest, namely at phases G2/M and G1/G0 in GluCSCs and Glu-dCCs respectively. Recent studies reported different results regarding cell cycle progression in cancer cells treated with melatonin. In human colorectal cancer cells, melatonin decreased the S-phase population and induced cell death, whereas in osteosarcoma and leukemia cells, melatonin reduced the proportion of cells in the S- and G2/M-phases while increasing cells in G1/G0 phase, an effect also observed by us in Glu-dCCs. However, in this work, melatonin-induced cell cycle arrests seem to be incomplete in several cases, probably as a consequence of the observed heterogeneity within cell populations. On the other hand, high concentrations of melatonin increased the number of hepatoma cells in S-phase, showing an antiproliferative effect which was also observed by us in P19 cells cultured in galactose, glutamine/pyruvate- containing medium. Therefore, although melatonin induced alterations in cell cycle progression, those effects depend on the overall metabolic and differentiation state of the cancer cells. Even so, in the more resistant P19 Glu-CSCs, melatonin was able to induce an arrest at G2/M albeit without exerting significant cytostatic effects. Thus, in the P19 CSCs model, melatonin was only able to reduce cell proliferation when the cells lost pluripotency and cell cycle was modified to the canonical structure. Spontaneous Ca2+ oscillations are restricted to the G1/S phase transition in P19 CSCs, suggesting a role for Ca2+ in stem cell cycle progression. In fact, we have recently reported that P19 stem cells present low cytosolic calcium levels, which are increased during cell differentiation. The higher concentrations of free calcium found in P19 cells grown in galactose medium are correlated with the changes found on cell cycle progression. Accordingly, the effect of melatonin on free Ca2+ was also dissimilar. Glu-CSCs showed increased Ca2+ levels upon melatonin treatment while having no effect on cell viability. Conversely, melatonin-treated cells grown in galactose, glutamine/pyruvatecontaining medium showed cell cycle arrest at S-phase, decreased cell viability and low intracellular free Ca2+ concentrations which, according with our previous results, seem to be required at high levels during the process of cell differentiation and mitochondrial maturation. These different results, which depend on the cellular metabolic state, are probably influenced by the lower calcium retention ability of mitochondria from differentiated cells than those from Glu-CSCs. Thus, the effects of melatonin PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19860992 on calcium signaling may play a role in mediating its growth-inhibitory action in cells with an active mitochondrial metabolism. Melatonin mitigates mitochondrial dysfunction in healthy cells, maintaining membrane potential and optimizing electron transport within the respiratory chain. However, in some melatonin-sensitive cancer cells, mitochondrial depolarization is observed. www.impactjournals.com/oncotarget 17089 Although this effect is not obs.
