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Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells.

Sanchez-Sanchez AM, Antolin I, Puente-Moncada N, Suarez S, Gomez-Lobo M, Rodriguez C, Martin V - PLoS ONE (2015)

Bottom Line: Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively.Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis).Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina, c/Julian Claveria, 33006 Oviedo, University of Oviedo, Oviedo, Spain; Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Oviedo, Spain.

ABSTRACT
Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.

No MeSH data available.


Related in: MedlinePlus

Melatonin inhibits HIF-1α in a PI3K/AKT/mTOR independent pathway.Western blot analyses were carried out to identify the effect of melatonin (1 mM, 4 and 8 hours) on the activation of HIF-1α in TC-71 cells (A) and A-4573 and A-673 (1mM, 4 hours) cells (B). AKT and mTOR activation in TC-71 (C) and A-4573 and A-673 cells (D) 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-1α (inactivated form), p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells (dotted line). (E) 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 rapamycin or 10 μM 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-1α after 10 nM rapamycin or 10 μM LY294002 treatment during 24 hours in TC-71 cell line. p*≤0.05 vs. vehicle-treated cells.
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pone.0135420.g005: Melatonin inhibits HIF-1α in a PI3K/AKT/mTOR independent pathway.Western blot analyses were carried out to identify the effect of melatonin (1 mM, 4 and 8 hours) on the activation of HIF-1α in TC-71 cells (A) and A-4573 and A-673 (1mM, 4 hours) cells (B). AKT and mTOR activation in TC-71 (C) and A-4573 and A-673 cells (D) 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-1α (inactivated form), p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells (dotted line). (E) 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 rapamycin or 10 μM 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-1α after 10 nM rapamycin or 10 μM LY294002 treatment during 24 hours in TC-71 cell line. p*≤0.05 vs. vehicle-treated cells.

Mentions: Activation of the master regulator of oxygen homeostasis HIF-1α is essential for maintenance of Warburg effect [23]. As shown in Fig 5, melatonin induced a significant increase in the inactivated (hydroxylated) form of HIF-1α in TC-71 (Fig 5A), A-4573 and A-673 cells (Fig 5B), which is consistent with its inhibition of Warburg effect. PI3K/AKT/mTOR signaling can increase HIF-1α activity by inducing its stabilization, and we and others have shown that melatonin regulates the PI3K/AKT/mTOR pathway in some tumor types [5,24]. However, melatonin activated AKT and mTOR in TC-71 (Fig 5C), A-4573 and A-673 cells (Fig 5D), which is not consistent with the inactivation of HIF-1α. On the other hand, rapamycin or LY294002, specific inhibitors of mTOR and PI3K respectively, did not prevent melatonin-induced decrease in TC-71 cell viability (Fig 5E, left panel). Both inhibitors reduced the phosphorylation of their target proteins, as expected, and none of them modified HIF-1α expression (Fig 5E, right panel). These data suggest that PI3K/AKT/mTOR activation may not be responsible for HIF-1α regulation by melatonin in Ewing sarcoma cells, and therefore this pathway is probably not involved in melatonin toxicity.


Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells.

Sanchez-Sanchez AM, Antolin I, Puente-Moncada N, Suarez S, Gomez-Lobo M, Rodriguez C, Martin V - PLoS ONE (2015)

Melatonin inhibits HIF-1α in a PI3K/AKT/mTOR independent pathway.Western blot analyses were carried out to identify the effect of melatonin (1 mM, 4 and 8 hours) on the activation of HIF-1α in TC-71 cells (A) and A-4573 and A-673 (1mM, 4 hours) cells (B). AKT and mTOR activation in TC-71 (C) and A-4573 and A-673 cells (D) 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-1α (inactivated form), p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells (dotted line). (E) 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 rapamycin or 10 μM 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-1α after 10 nM rapamycin or 10 μM LY294002 treatment during 24 hours in TC-71 cell line. p*≤0.05 vs. vehicle-treated cells.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4529102&req=5

pone.0135420.g005: Melatonin inhibits HIF-1α in a PI3K/AKT/mTOR independent pathway.Western blot analyses were carried out to identify the effect of melatonin (1 mM, 4 and 8 hours) on the activation of HIF-1α in TC-71 cells (A) and A-4573 and A-673 (1mM, 4 hours) cells (B). AKT and mTOR activation in TC-71 (C) and A-4573 and A-673 cells (D) 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-1α (inactivated form), p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells (dotted line). (E) 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 rapamycin or 10 μM 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-1α after 10 nM rapamycin or 10 μM LY294002 treatment during 24 hours in TC-71 cell line. p*≤0.05 vs. vehicle-treated cells.
Mentions: Activation of the master regulator of oxygen homeostasis HIF-1α is essential for maintenance of Warburg effect [23]. As shown in Fig 5, melatonin induced a significant increase in the inactivated (hydroxylated) form of HIF-1α in TC-71 (Fig 5A), A-4573 and A-673 cells (Fig 5B), which is consistent with its inhibition of Warburg effect. PI3K/AKT/mTOR signaling can increase HIF-1α activity by inducing its stabilization, and we and others have shown that melatonin regulates the PI3K/AKT/mTOR pathway in some tumor types [5,24]. However, melatonin activated AKT and mTOR in TC-71 (Fig 5C), A-4573 and A-673 cells (Fig 5D), which is not consistent with the inactivation of HIF-1α. On the other hand, rapamycin or LY294002, specific inhibitors of mTOR and PI3K respectively, did not prevent melatonin-induced decrease in TC-71 cell viability (Fig 5E, left panel). Both inhibitors reduced the phosphorylation of their target proteins, as expected, and none of them modified HIF-1α expression (Fig 5E, right panel). These data suggest that PI3K/AKT/mTOR activation may not be responsible for HIF-1α regulation by melatonin in Ewing sarcoma cells, and therefore this pathway is probably not involved in melatonin toxicity.

Bottom Line: Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively.Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis).Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Morfología y Biología Celular, Facultad de Medicina, c/Julian Claveria, 33006 Oviedo, University of Oviedo, Oviedo, Spain; Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Oviedo, Spain.

ABSTRACT
Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis). Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells) and in cells where it inhibits proliferation (chondrosarcoma cells). Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.

No MeSH data available.


Related in: MedlinePlus