FLT3-ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

The associations between anthropometric characteristics and nutritional parameters in male elite rugby union players.

The objective was to analyse the associations between anthropometric characteristics and diet in male rugby players according to the playing position. A cross-sectional study was developed. The forwards had higher body weight (107 kg) and fat mass (FM; 12%) than the backs (87.8 kg and 8.47%, respectively) (p < 0.05). The quality of diet needs to improve (KIDMED value of 5.87 and 6.36 for forwards and backs, respectively). Nutritional imbalances, such as deficits in carbohydrates, fibre, calcium, magnesium and vitamin D, and excess of fats, saturated fatty acid, cholesterol and sugars were found. Carbohydrates and proteins intake were significant associated (p < 0.05) with a minor FM. Forwards with a KIDMED index of less than 8 had a significantly higher FM than those who maintained an optimal diet (p < 0.05). The diet of rugby players should be more in line with dietary recommendations and take into account the player position to optimise sports performance.

Calcium acts as a central player in melatonin antitumor activity in sarcoma cells.

PURPOSE: Chondrosarcoma and osteosarcoma are the most frequently occurring bone cancers. Although surgery and chemotherapy are currently clinically applied, improved treatment options are urgently needed. Melatonin is known to inhibit cell proliferation in both tumor types. Although the underlying mechanisms are not clear yet, calcium homeostasis has been reported to be a key factor in cancer biology. Here, we set out to investigate whether regulation of calcium by this indolamine may be involved in its antitumor effect. METHODS: Cell viability was measured using a MTT assay and flow cytometry was used to measure levels of cytosolic calcium, intracellular oxidants, mitochondrial membrane potential and cell cycle progression. Mitochondrial calcium was analyzed by fluorimetry. Cell migration was determined using a scratch wound-healing assay. Western blot analysis was used to assess the expression of proteins related to cell cycle progression, epithelial to mesenchymal transition (EMT), Ac-CoA synthesis and intracellular signaling pathways. RESULTS: We found that melatonin decreases cytosolic and mitochondrial Ca2+ levels, intracellular oxidant levels, mitochondrial function and the expression of the E1 subunit of the pyruvate dehydrogenase complex. These changes were found to be accompanied by decreases in cell proliferation, cell migration and EMT marker expression. The addition of CaCl2 prevented the changes mentioned above, while co-treatment with the calcium chelator BAPTA enhanced the effects. CONCLUSIONS: Our data indicate that regulation of calcium homeostasis is a key factor in the inhibition of cell proliferation and migration by melatonin. This effect should be taken into consideration in combined therapies with traditional or new antitumor compounds, since it may circumvent therapy resistance.

Regulation of cancer cell glucose metabolism is determinant for cancer cell fate after melatonin administration.

Several oncogenic pathways plus local microenvironmental conditions, such as hypoxia, converge on the regulation of cancer cells metabolism. The major metabolic alteration consists of a shift from oxidative phosphorylation as the major glucose consumer to aerobic glycolysis, although most of cancer cells utilize both pathways to a greater or lesser extent. Aerobic glycolysis, together with the directly related metabolic pathways such as the tricarboxylic acid cycle, the pentose phosphate pathway, or gluconeogenesis are currently considered as therapeutic targets in cancer research. Melatonin has been reported to present numerous antitumor effects, which result in a reduced cell growth. This is achieved with both low and high concentrations with no relevant side effects. Indeed, high concentrations of this indolamine reduce proliferation of cancer types resistant to low concentrations and induce cell death in some types of tumors. Previous work suggest that regulation of glucose metabolism and other related pathways play an important role in the antitumoral effects of high concentration of melatonin. In the present review, we analyze recent work on the regulation by such concentrations of this indolamine on aerobic glycolysis, gluconeogenesis, the tricarboxylic acid cycle and the pentose phosphate pathways of cancer cells.

Role of glucose metabolism in the differential antileukemic effect of melatonin on wild­type and FLT3­ITD mutant cells.

The FMS­like tyrosine kinase 3 internal tandem duplication (FLT3­ITD) mutation represents the most frequent genetic alteration in acute myeloid leukemia (AML) and is associated with poor prognosis. The mutation promotes cancer cell survival and proliferation, and shifts their glucose metabolism towards aerobic glycolysis, a frequent alteration in cancer. In the present study, the impact of melatonin on the viability of AML cell lines with (MV­4­11 and MOLM­13) or without the FLT3­ITD mutation (OCI­AML3 and U­937) was evaluated. Melatonin induces cell death in AML cells carrying the FLT3­ITD mutation, but only inhibits the proliferation of AML cells without this mutation. Consistently, melatonin decreases tumor growth and increases animal survival in a xenograft model of FLT3­ITD AML. Toxicity is related to a decrease in glucose uptake, lactate dehydrogenase activity, lactate production and hypoxia­inducible factor­1α activation. Melatonin also regulates the expression of glucose metabolism­related genes, impairing the balance between anaplerosis and cataplerosis, through the upregulation of the expression of phosphoenolpyruvate carboxykinase 2 (PCK2). Collectively, the present findings highlight the regulation of glucose metabolism, currently considered a possible therapeutic target in cancer, as a key event in melatonin­induced cytotoxicity, suggesting its potential as a therapeutic tool for the treatment of patients with AML, particularly those carrying the FLT3­ITD mutation that results in low basal expression levels of PCK2.

Inhibition of FLT3 and PIM Kinases by EC-70124 Exerts Potent Activity in Preclinical Models of Acute Myeloid Leukemia.

Internal tandem duplication (ITD) or tyrosine kinase domain mutations of FLT3 is the most frequent genetic alteration in acute myelogenous leukemia (AML) and are associated with poor disease outcome. Despite considerable efforts to develop single-target FLT3 drugs, so far, the most promising clinical response has been achieved using the multikinase inhibitor midostaurin. Here, we explore the activity of the indolocarbazole EC-70124, from the same chemical space as midostaurin, in preclinical models of AML, focusing on those bearing FLT3-ITD mutations. EC-70124 potently inhibits wild-type and mutant FLT3, and also other important kinases such as PIM kinases. EC-70124 inhibits proliferation of AML cell lines, inducing cell-cycle arrest and apoptosis. EC-70124 is orally bioavailable and displays higher metabolic stability and lower human protein plasma binding compared with midostaurin. Both in vitro and in vivo pharmacodynamic analyses demonstrate inhibition of FLT3-STAT5, Akt-mTOR-S6, and PIM-BAD pathways. Oral administration of EC-70124 in FLT3-ITD xenograft models demonstrates high efficacy, reaching complete tumor regression. Ex vivo, EC-70124 impaired cell viability in leukemic blasts, especially from FLT3-ITD patients. Our results demonstrate the ability of EC-70124 to reduce proliferation and induce cell death in AML cell lines, patient-derived leukemic blast and xenograft animal models, reaching best results in FLT3 mutants that carry other molecular pathways' alterations. Thus, its unique inhibition profile warrants EC-70124 as a promising agent for AML treatment based on its ability to interfere the complex oncogenic events activated in AML at several levels. Mol Cancer Ther; 17(3); 614-24. ©2018 AACR.

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

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.

Intracellular redox state as determinant for melatonin antiproliferative vs cytotoxic effects in cancer cells.

Melatonin is an endogenous indolamine, classically known as a light/dark regulator. Besides classical functions, melatonin has also showed to have a wide range of antitumoral effects in numerous cancer experimental models. However, no definite mechanism has been described to explain the whole range of antineoplasic effects. Here we describe a dual effect of melatonin on intracellular redox state in relation to its antiproliferative vs cytotoxic actions in cancer cells. Thus, inhibition of proliferation correlates with a decrease on intracellular reactive oxygen species (ROS) and increase of antioxidant defences (antioxidant enzymes and intracellular gluthation,GSH levels), while induction of cell death correlates with an increase on intracellular ROS and decrease of antioxidant defences. Moreover, cell death can be prevented by other well-known antioxidants or can be increased by hydrogen peroxide. Thus, tumour cell fate will depend on the ability of melatonin to induce either an antioxidant environment--related to the antiproliferative effect or a prooxidant environment related to the cytotoxic effect.

Regulation of the expression of death receptors and their ligands by melatonin in haematological cancer cell lines and in leukaemia cells from patients.

Incorporation of new therapeutic agents remains as a major challenge for treatment of patients with malignant haematological disorders. Melatonin is an indolamine without relevant side effects. It has been shown previously to exhibit synergism with several chemotherapeutic drugs in Ewing sarcoma cells by potentiating the extrinsic pathway of apoptosis. It also sensitizes human glioma cells against TRAIL by increasing DR5 expression. Here, we report the induction of cell death by melatonin in several human malignant haematological cell lines through the activation of the extrinsic pathway of apoptosis. Such activation was mediated by the increase in the expression of the death receptors Fas, DR4 and DR5 and their ligands Fas L and TRAIL, with a remarkable rise in the expression of Fas and Fas L. The cytotoxic effect and the increase in Fas and Fas L were dependent on Akt activation. Results were corroborated in blasts from bone marrow and peripheral blood of acute myeloid leukaemia patients, where melatonin induced cell death and increased both Fas and Fas L expressions. We conclude that melatonin may be considered as a potential antileukaemic agent and its therapeutic use, either alone or in combination with current chemotherapeutic drugs, should be taken into consideration for further research.

Synergistic antitumor effect of melatonin with several chemotherapeutic drugs on human Ewing sarcoma cancer cells: potentiation of the extrinsic apoptotic pathway.

Ewing sarcoma, the second most frequent bone cancer type, affects mainly adolescents, who have a survival of 50% 5 yr after diagnosis. Current treatments include a combination of surgery, radiotherapy and chemotherapy, which present potential serious side effects. Melatonin, a natural molecule without relevant side effects, has been previously shown to induce cytotoxicity in SK-N-MC cells, a Ewing sarcoma cell line. Here, we found that there is a synergy in the antitumor effect when melatonin (50 mum-1 mm) is combined with vincristine at the concentration of 5-10 nm or with ifosfamide at the range of 100 mum-1 mm. This synergism is due to the potentiation of cell death, particularly to the potentiation of apoptosis, i.e., mainly the extrinsic apoptotic pathway. There is a significant increase in the activation of caspase-3, -8, -9 and Bid when melatonin is combined with vincristine or ifosfamide compared to the individual treatments. Finally, there is also a potentiation of the early free radical production, likely dependent on the extrinsic apoptosis pathway activation, when the drugs are combined with melatonin. Other proteins which are related to this pathway including mitogen-activated protein kinase or protein kinase B/Akt are not involved in apoptosis induced by these agents separately or when combined. The results shown here together with the facts that: (i) no relevant side effects have been reported for melatonin and (ii) melatonin has a cytoprotective effect on noncancer cells, opens the door for a new approach in the treatment of the Ewing sarcoma family of tumors.