N active mitochondrial metabolism are susceptible to the anticancer agent dichloroacetate.

N active get Crenolanib mitochondrial metabolism are susceptible to the anticancer agent dichloroacetate. Surprisingly, when we combined 1 mM melatonin and 10 mM dichloroacetate, cytotoxicity in the highly MedChemExpress SB203580 resistant Glu-CSCs was observed. These results are of great importance considering that to our knowledge, this is the only treatment showing an efficient and viable effect against P19 Glu-CSCs. Furthermore, the synergistic capability of this treatment combination was observed in P19 cells with the most active mitochondrial metabolism. The mechanism of action of dichloroacetate ultimately concerns the activity of PDH that, according to our previous results, seems to be deregulated and overexpressed in P19 CSCs. However, upon melatonin treatment, no significant changes in the content of PDH on its active form were found, which would allow us to explain its synergistic effect with dichloroacetate. The absence of correlation between the phosphorylation status of PDH and the observed synergistic effect reinforce the hypothesis about a deregulation in the PDK-PDH axis, at least in the more undifferentiated P19 cells. Thus, this deregulation might be related with the preference of primitive cells for a more glycolytic metabolism. In agreement, the group composed of more differentiated and oxidative cells showed significant changes in phosphoPDH when treated with melatonin and dichloroacetate. Since other mechanisms of PDH regulation may involve other phosphorylation sites, our results suggest the contribution of alternative mechanisms which might also be independent of the phosphorylation status of PDH, thus explaining part of the synergistic effect of the combination. For example, it was recently described that dichloroacetate is able to suppress mTOR activity specifically through pyruvate dehydrogenase kinase 4 and independently of PDH. Curiously, it was also described that Pdk4 gene expression can be increased by melatonin in mice. Oncotarget ascribes an anti-tumor effect for melatonin only in differentiated cancer cells with an active oxidative metabolism, triggering a type of mitochondrial-mediated cell death which is likely to be characterized by an arrest at S-phase, reduction of the mitochondrial electron transport chain, generation of reactive oxygen species, BCL-2 down-regulation and AIF release. Thus, the treatment with melatonin and the stimulation of mitochondrial metabolism constitute promising strategies against resistant cancer stem cells. Nonetheless, the decrease of phospho-PDH observed in Gal-dCCs after treatment with melatonin, reinforces the idea that the anti-tumor actions of melatonin occur at a mitochondrial level. Accordingly, our results suggest that melatonin exerts its anticancer effects in P19 cells with an active oxidative metabolism, triggering a type of mitochondrial cell death which is caspase-3-independent and is probably AIF-mediated. These results are in accordance with previous works developed in other types of cancer cells such as MCF-7. Mitochondrial activity, function and differentiation strongly impact the antitumoral ability of melatonin which involves, among others, the activation of intrinsic apoptotic pathways, in P19 embryonal carcinoma cells as well as in other types of cancer PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19859838 cells. It is known that AIF controls programmed cells death during early morphogenesis and that its genetic inactivation renders embryonic stem cells resistant to cell death. Consequently, the mechanism of caspase-3-independent cell deat.N active mitochondrial metabolism are susceptible to the anticancer agent dichloroacetate. Surprisingly, when we combined 1 mM melatonin and 10 mM dichloroacetate, cytotoxicity in the highly resistant Glu-CSCs was observed. These results are of great importance considering that to our knowledge, this is the only treatment showing an efficient and viable effect against P19 Glu-CSCs. Furthermore, the synergistic capability of this treatment combination was observed in P19 cells with the most active mitochondrial metabolism. The mechanism of action of dichloroacetate ultimately concerns the activity of PDH that, according to our previous results, seems to be deregulated and overexpressed in P19 CSCs. However, upon melatonin treatment, no significant changes in the content of PDH on its active form were found, which would allow us to explain its synergistic effect with dichloroacetate. The absence of correlation between the phosphorylation status of PDH and the observed synergistic effect reinforce the hypothesis about a deregulation in the PDK-PDH axis, at least in the more undifferentiated P19 cells. Thus, this deregulation might be related with the preference of primitive cells for a more glycolytic metabolism. In agreement, the group composed of more differentiated and oxidative cells showed significant changes in phosphoPDH when treated with melatonin and dichloroacetate. Since other mechanisms of PDH regulation may involve other phosphorylation sites, our results suggest the contribution of alternative mechanisms which might also be independent of the phosphorylation status of PDH, thus explaining part of the synergistic effect of the combination. For example, it was recently described that dichloroacetate is able to suppress mTOR activity specifically through pyruvate dehydrogenase kinase 4 and independently of PDH. Curiously, it was also described that Pdk4 gene expression can be increased by melatonin in mice. Oncotarget ascribes an anti-tumor effect for melatonin only in differentiated cancer cells with an active oxidative metabolism, triggering a type of mitochondrial-mediated cell death which is likely to be characterized by an arrest at S-phase, reduction of the mitochondrial electron transport chain, generation of reactive oxygen species, BCL-2 down-regulation and AIF release. Thus, the treatment with melatonin and the stimulation of mitochondrial metabolism constitute promising strategies against resistant cancer stem cells. Nonetheless, the decrease of phospho-PDH observed in Gal-dCCs after treatment with melatonin, reinforces the idea that the anti-tumor actions of melatonin occur at a mitochondrial level. Accordingly, our results suggest that melatonin exerts its anticancer effects in P19 cells with an active oxidative metabolism, triggering a type of mitochondrial cell death which is caspase-3-independent and is probably AIF-mediated. These results are in accordance with previous works developed in other types of cancer cells such as MCF-7. Mitochondrial activity, function and differentiation strongly impact the antitumoral ability of melatonin which involves, among others, the activation of intrinsic apoptotic pathways, in P19 embryonal carcinoma cells as well as in other types of cancer PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19859838 cells. It is known that AIF controls programmed cells death during early morphogenesis and that its genetic inactivation renders embryonic stem cells resistant to cell death. Consequently, the mechanism of caspase-3-independent cell deat.