Guillermo García-Santos

Intracellular signaling pathways involved in the cell growth inhibition of glioma cells by melatonin.

Melatonin is an indolamine mostly produced in the pineal gland, soluble in water, and highly lipophilic, which allows it to readily cross the blood-brain barrier. Melatonin possesses antioxidant properties and its long-term administration in rodents has not been found to cause noteworthy side effects. In the present work, we found that millimolar concentrations of this indolamine reduced cell growth of C6 glioma cells by 70% after 72 hours of treatment, inhibiting cell progression from G(1) to S phase of the cell cycle. Intraperitoneal administration of 15 mg/kg body weight of melatonin to rats previously injected in the flank with C6 glioma cells reduces tumor growth by 50% 2 weeks after the implant. Inhibition of cell growth does not depend on melatonin membrane receptor activation whereas it seemingly relates to the reduction of intracellular basal free radical levels by 30%. Increase of basal redox state of the cells and constitutive activation of tyrosine kinase receptor [receptor tyrosine kinase (RTK)] pathways, including the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Akt and protein kinase C (PKC) signaling pathways, contribute to the progression of the gliomas leading to the constitutive activation of the redox-dependent survival transcription factor nuclear factor kappaB (NF-kappaB). The antioxidant effect of melatonin in C6 cells is associated to inhibition of NF-kappaB and Akt, but not of ERK1/2. The antiproliferative effect of the indolamine on these cells is partially abolished when coincubated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate, thus indicating that the ability of melatonin to change cellular redox state may be inactivating the pathway RTK/PKC/Akt/NF-kappaB.

Melatonin induces apoptosis in human neuroblastoma cancer cells.

Abstract:  Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK‐N‐MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6‐diamidino‐2‐phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase‐3 activation. Cell death was dose and time‐dependent; the lowest effective concentration of melatonin was 100 μm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.

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

Abstract:  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 μm–1 mm) is combined with vincristine at the concentration of 5–10 nm or with ifosfamide at the range of 100 μm–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.

Melatonin prevents glutamate-induced oxytosis in the HT22 mouse hippocampal cell line through an antioxidant effect specifically targeting mitochondria

The pineal hormone melatonin has neuroprotective effects in a large number of models of neurodegeneration. Melatonin crosses the blood–brain barrier, shows a decrease in its nocturnal peaks in blood with age that has been associated with the development of neurodegenerative disorders, and has been shown to be harmless at high concentrations. These properties make melatonin a potential therapeutic agent against neurodegenerative disorders but the pathways involved in such neuroprotective effects remain unknown. In the present report we study the intracellular pathways implicated in the complete neuroprotection provided by melatonin against glutamate‐induced oxytosis in the HT22 mouse hippocampal cell line. Our results strongly suggest that melatonin prevents oxytosis through a direct antioxidant effect specifically targeted at the mitochondria. Firstly, none of the described transducers of melatonin signalling seems to be implicated in the neuroprotection provided by this indole. Secondly, melatonin does not prevent cytosolic GSH depletion‐dependent increase in reactive oxygen species (ROS), but it totally prevents mitochondrial ROS production despite the fact that the latter is much higher than the former. And finally, there is a high correlation between the concentration at which melatonin and closely related indoles exert a direct antioxidant effect in vitro and a neuroprotective effect against glutamate‐induced oxytosis.

Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine.

Oxidative stress has been shown to mediate neuron damage in Parkinson’s disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post‐mitotic PC12 cells treated with the neurotoxin 6‐hydroxydopamine (6‐OHDA). The use of post‐mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re‐entry of neurons (post‐mitotic cells) in the cell cycle. We find that 6‐OHDA sequentially increases intracellular oxidants, functional cell damage and caspase‐3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress‐dependent phosphorylation of the MAPK JNK and oxidative stress‐independent PKB/Akt dephosphorylation are involved in 6‐OHDA neurotoxicity. Decrease in p21WAF1/CIP1 and cyclin‐D1 expression, disappearance of the non‐phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post‐mitotic cells, suggest a re‐entry of differentiated cells into cell cycle. Our results indicate that such a re‐entry is mediated by oxidative stress and is involved in 6‐OHDA‐induced cell death. We conclude that at least three intracellular pathways are involved in 6‐OHDA‐induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress‐dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.

Melatonin sensitizes human malignant glioma cells against TRAIL-induced cell death.

Despite the common expression of death receptors, many types of cancer including gliomas are resistant to the death receptor ligand (TRAIL). Melatonin antitumoral actions have been extensively described, including oncostatic properties on several tumor types and improvement of chemotherapeutic regimens. Here, we found that melatonin effectively increase cell sensitivity to TRAIL-induced cell apoptosis in A172 and U87 human glioma cells. The effect seems to be related to a modulation of PKC activity which in turns decreases Akt activation leading to an increase in death receptor 5 (DR5) levels and a decrease in the antiapoptotic proteins survivin and bcl-2 levels.

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

Abstract 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

Abstract:  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.

Involvement of protein kinase C in melatonin’s oncostatic effect in C6 glioma cells

Abstract:  Classical anticancer therapies often are ineffective in patients with malignant glioma who have a survival of <1 year. Our previous studies showed a potent inhibitory effect of melatonin on glioma cell proliferation. This effect seems to be mediated by the well‐known antioxidant properties of this molecule and the negative regulation of some intracellular effectors, such as the kinase Akt or the transcription factor nuclear factor (NF)‐κB. Finally, protein kinase C (PKC) also seems to be implicated in this effect although the intracellular pathways involved have not been elucidated. In this study, we analyzed the role of PKC in the regulation by melatonin of intracellular effectors leading to inhibition of cell proliferation. Activation of PKC by incubation with triphorbol ester acetate (TPA) blocks the inhibitory effect of melatonin on Akt and NF‐κB activity. Moreover, incubation with melatonin induces a decrease in p21 expression in these cells that is partially blocked by co‐incubation with TPA. Taken together, these results suggest that melatonin’s oncostatic effect on glioma cells is mediated, at least in part, by the inhibition of PKC activity which, in turn, results in Akt and NF‐κB activity inhibition and modulation of cell cycle‐related gene expression.

Cooperative action of JNK and AKT/mTOR in 1-methyl-4-phenylpyridinium-induced autophagy of neuronal PC12 cells

Parkinsons disease has been widely related to both apoptosis and oxidative stress. Many publications relate the loss of mitochondrial potential to an apoptosis‐mediated cell death in different in vivo and in vitro models of this pathology. The present study used the dopaminegic specific neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP+) on neuron‐like PC12 cells, which is a well‐accepted model of Parkinsons disease. Results showed an early increase in oxidants, which drives the modulation of c‐Jun N‐terminal kinase (JNK) and AKT/mammalian target of rapamycin (mTOR) pathways, mimicking peroxide treatment. However, the cell death found in neuronal PC12 cells treated with MPP+ was not a caspase‐associated apoptosis. Electron microscopic images illustrated autophagic cell death, which was confirmed by a Beclin‐1 and ATG expression increase, accumulation of acidic vesicles, and rescue by an autophagy inhibitor. In conclusion, the boost in oxidants from MPP+ treatment in neuronal PC12 is modulating both survival (AKT/mTOR) and death (JNK) pathways, which are the perpetrators of an autophagic cell death.