Ana B. Rodríguez

Melatonin induces mitochondrial‐mediated apoptosis in human myeloid HL‐60 cells

Abstract:  The role of melatonin in the mediation of apoptotic events has recently gained attention, especially after recent studies have reported that melatonin exerts antiapoptotic actions in normal cells but may activate proapoptotic pathways in some tumor cells. Here, we have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL‐60. Melatonin treatment (1 mm) induced a significant increase in caspase‐3 and ‐9 activities. The effect of melatonin on the activation of caspases was time dependent, reaching a maximum after 12 hr of stimulation, and then decreasing to a minimum after 72 hr. Treatment with melatonin also evoked mitochondrial membrane depolarization and permeability transition pore induction, which caused loss of mitochondrial staining by calcein, and increased cell death by apoptosis/necrosis as demonstrated by propidium iodide positive‐staining of cells after 72 hr of stimulation. In addition, the exposure of cells to melatonin resulted in an activation and association of the proapoptotic proteins Bax and Bid, as well as promoting detectable increases in the expression of both proteins. We conclude that melatonin has proapoptotic and/or oncostatic effects in the human myeloid cell line HL‐60.

Melatonin potentiates chemotherapy-induced cytotoxicity and apoptosis in rat pancreatic tumor cells

Abstract:  Melatonin has antitumor activity via several mechanisms including its antiproliferative and proapoptotic effects in addition to its potent antioxidant action. Thus, melatonin has proven useful in the treatment of tumors in association with chemotherapeutic drugs. This study was performed to evaluate the effect of melatonin on the cytotoxicity and apoptosis induced by three different chemotherapeutic agents, namely 5‐fluorouracil (5‐FU), cisplatin, and doxorubicin in the rat pancreatic tumor cell line AR42J. We found that both melatonin and the three chemotherapeutic drugs induce a time‐dependent decrease in AR42J cell viability, reaching the highest cytotoxic effect after 48 hr of incubation. Furthermore, melatonin significantly augmented the cytotoxicity of the chemotherapeutic agents. Consistently, cotreatment of AR42J cells with each of the chemotherapeutic agents in the presence of melatonin increased the population of apoptotic cells, elevated mitochondrial membrane depolarization, and augmented intracellular reactive oxygen species (ROS) production compared to treatment with each chemotherapeutic agent alone. These results provide evidence that in vitro melatonin enhances chemotherapy‐induced cytotoxicity and apoptosis in rat pancreatic tumor AR42J cells and, therefore, melatonin may be potentially applied to pancreatic tumor treatment as a powerful synergistic agent in combination with chemotherapeutic drugs.

Circadian rhythm of melatonin, corticosterone and phagocytosis: effect of stress

Melatonin has a functional connection with the immune system. Phagocyte function is altered by extirpation of the pineal gland, one source of melatonin, or by in vitro incubation of phagocytes with pharmacological concentrations of melatonin. Given that its synthesis by pinealocytes is under the control of the noradrenaline released by the sympathetic postganglionaric nerve endings, the present work was aimed at evaluating the circadian rhythm of melatonin, corticosterone, and phagocytosis in BALB/c mice in basal and stress situations. Peritoneal macrophages were used as phagocytes, latex beads as the particles to be ingested, and forced swimming to exhaustion as the stress situation. Radioimmunoassay was used to determine the animals’ serum hormone levels. Samples were taken every 3 hr in the period from 04:00 to 22:00 hr, and every 30 min during the remaining period from 22:00 to 04:00 hr. Control mice presented a short‐term melatonin peak at 23:30 hr, while the maximum inert‐particle ingestion capacity of the peritoneal macrophages also occurred during the night but at 03:30 hr. The corticosterone levels in control mice presented a circadian rhythm with a day‐time maximum peak (16:00 hr). Compared with the controls, the animals subjected to stress maintained, although at lower values, the melatonin peak at 23:30 hr, but they presented a loss of the rhythm of serum corticosterone levels, and the corticosterone levels and the macrophage phagocytic capacity were greater at all hours of the day.

Melatonin Reduces Apoptosis Induced by Calcium Signaling in Human Leukocytes: Evidence for the Involvement of Mitochondria and Bax Activation

We have evaluated the effect of melatonin on apoptosis evoked by increases in [Ca2+]c in human leukocytes. Our results show that treatment of neutrophils with the calcium mobilizing agonist FMLP or the specific inhibitor of calcium reuptake thapsigargin induced a transient increase in [Ca2+]c. Our results also show that FMLP and thapsigargin increased caspase-9 and -3 activities and the active forms of both caspases. The effect of FMLP and thapsigargin on caspase activation was time-dependent. Similar results were obtained when lymphocytes were stimulated with thapsigargin. This stimulatory effect was accompanied by induction of mPTP, activation of the proapoptotic protein Bax and release of cytochrome c. However, when leukocytes were pretreated with melatonin, all of the apoptotic features indicated above were significantly reversed. Our results suggest that melatonin reduces caspase-9 and -3 activities induced by increases in [Ca2+]c in human leukocytes, which are produced through the inhibition of both mPTP and Bax activation.

Protective effect of melatonin against human leukocyte apoptosis induced by intracellular calcium overload: relation with its antioxidant actions

Abstract:  Apoptosis or programmed cell death plays a critical role in both inflammatory and immune responses. Recent evidence demonstrates that control of leukocyte apoptosis is one of the most striking immune system‐related roles of melatonin. For this reason, this study evaluated the protective effects of melatonin on human leukocyte apoptosis induced by sustained cytosolic calcium increases. Such protective effects are likely mediated by melatonin’s free‐radical scavenging actions. Treatments with the specific inhibitor of cytosolic calcium re‐uptake, thapsigargin (TG), and/or the calcium‐mobilizing agonist, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), induced intracellular reactive oxygen species (ROS) production, caspase activation as well as DNA fragmentation in human leukocytes. Also, TG‐ and/or FMLP‐induced apoptosis was dependent on both cytosolic calcium increases and calcium uptake into mitochondria, because when cells were preincubated with the cytosolic calcium chelator, dimethyl BAPTA, and the inhibitor of mitochondrial calcium uptake, Ru360, TG‐ and FMLP‐induced apoptosis was largely inhibited. Importantly, melatonin treatment substantially prevented intracellular ROS production, reversed caspase activation, and forestalled DNA fragmentation induced by TG and FMLP. Similar results were obtained by preincubating the cells with another well‐known antioxidant, i.e., N‐acetyl‐l‐cysteine. To sum up, depletion of intracellular calcium stores induced by TG and/or FMLP triggers different apoptotic events in human leukocytes that are dependent on calcium signaling. The protective effects resulting from melatonin administration on leukocyte apoptosis likely depend on melatonin’s antioxidant action because we proved that this protection is melatonin receptor independent. These findings help to understand how melatonin controls apoptosis in cells of immune/inflammatory relevance.

Effect of orally administered l-tryptophan on serotonin, melatonin, and the innate immune response in the rat

To assess the effects of external administration of L-tryptophan on the synthesis of serotonin and melatonin as well as on the immune function of Wistar rats, 300 mg of the amino acid were administered through an oral cannula either during daylight (08:00) or at night (20:00) for 5 days. Brain, plasma, and peritoneal macrophage samples were collected 4 h after the administration. The accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used to measure the rate of tryptophan hydroxylation in vivo. Circulating melatonin levels were determined by radioimmunoassay, and the phagocytic activity of macrophages was measured by counting, under oil-immersion phase-contrast microscopy, the number of particles ingested. The results showed a diurnal increase (p < 0.05) in the brain 5-HTP, serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindolacetic acid (5-HIAA) of the animals which had received tryptophan at 08:00 and were killed 4 h later. In the animals which received tryptophan during the dark period, the 5-HT declined but the 5-HT/5-HIAA ratio remained unchanged. There was also a significant increase (p < 0.05) in nocturnal circulating melatonin levels and in the innate immune response of the peritoneal macrophages in the animals which had received tryptophan at 20:00. The results indicated that the synthesis of serotonin and melatonin, as well as the innate immune response, can be modulated by oral ingestion of tryptophan. (Mol Cell Biochem 267: 39–46, 2004)

Jerte Valley Cherry-Enriched Diets Improve Nocturnal Rest and Increase 6-Sulfatoxymelatonin and Total Antioxidant Capacity in the Urine of Middle-Aged and Elderly Humans

Tryptophan, serotonin, and melatonin, present in Jerte Valley cherries, participate in sleep regulation and exhibit antioxidant properties. The effect of the intake of seven different Jerte Valley cherry cultivars on the sleep-wake cycle, 6-sulfatoxymelatonin levels, and urinary total antioxidant capacity in middle-aged and elderly participants was evaluated. Volunteers were subjected to actigraphic monitoring to record and display the temporal patterns of their nocturnal activity and rest. 6-sulfatoxymelatonin and total antioxidant capacity were quantified by enzyme-linked immunosorbent assay and colorimetric assay kits, respectively. The intake of each of the cherry cultivars produced beneficial effects on actual sleep time, total nocturnal activity, assumed sleep, and immobility. Also, there were significant increases in 6-sulfatoxymelatonin levels and total antioxidant capacity in urine after the intake of each cultivar. These findings suggested that the intake of Jerte Valley cherries exerted positive effect on sleep and may be seen as a potential nutraceutical tool to counteract oxidation.

Melatonin protects human spermatozoa from apoptosis via melatonin receptor– and extracellular signal–regulated kinase-mediated pathways

OBJECTIVE To evaluate whether the protective effect of melatonin on H2O2-induced caspase activation and DNA fragmentation depends on the interaction between melatonin and its surface receptors. DESIGN Laboratory study. SETTING Center for assisted human reproduction at a Spanish hospital. PATIENT(S) Twenty-one healthy donors. INTERVENTION(S) Human spermatozoa were treated with increasing concentrations of hydrogen peroxide (H2O2; 1 μM, 10 μM, 100 μM, 1 mM) and preincubated with 1 mM melatonin. MAIN OUTCOMES MEASURE(S) Activation of caspase-3 and -9 as well as DNA fragmentation were examined by fluorescence methods. RESULT(S) Our findings showed that H2O2 induced a significant increase in caspase-9 and caspase-3, which was dose independent. Conversely, pretreatment with melatonin reduced H2O2-mediated caspase activation in a dose-dependent way. Moreover, the antiapoptotic effects of melatonin in ejaculated human spermatozoa may involve membrane melatonin receptor MT1. In addition, we found that the survival-promoting pathway extracellular signal-regulated kinase (ERK) is likely to have a role in the protective actions of melatonin in ejaculated human spermatozoa. Finally, we confirmed these results further by demonstrating that melatonin prevention of H2O2-induced DNA fragmentation is dependent on both MT1 receptor and ERK signaling. CONCLUSION(S) These results indicate that the stimulation with melatonin triggers a set of events culminating in cell death prevention in ejaculated human spermatozoa.

Pro‐Oxidant Effect of Melatonin in Tumour Leucocytes: Relation with its Cytotoxic and Pro‐Apoptotic Effects

Melatonin has many effects on a wide range of physiological functions and is involved in a number of pathological events including oncostatic and neoplastic processes. The tissue protective actions of melatonin are attributed to its well-known antioxidant activity though melatonin might also exert pro-oxidant effects, particularly in tumour cells. This study evaluated the pro-oxidant effects of melatonin in tumour cell lines of human haematopoietic origin. Melatonin treatment is able to stimulate production of intracellular reactive oxygen species (ROS), as revealed by the increase in rhodamine-123 fluorescence, which was associated with significant cytotoxicity and activation of caspase activities. Furthermore, pre-treatment of cells with well-known antioxidants, such as N-acetyl-L-cysteine (NAC), trolox, PEG-catalase and reduced glutathione (GSH), reversed the effects of melatonin on both intracellular ROS production, as on the cytotoxicity and caspase activation. This pro-oxidant action of melatonin may assist in limiting tumour cell growth.

Melatonin as a potential tool against oxidative damage and apoptosis in ejaculated human spermatozoa

It is assumed somatic cells can die in the apoptotic, the autophagic, or the necrotic way; however, the mechanisms of sperm death are not clear. Here, ejaculated human spermatozoa were evaluated for apoptosis and reactive oxygen species production in the absence or presence of melatonin, and we concluded that melatonin reverses sperm apoptosis due to its free radical scavenging actions.