Javier Espino

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.

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.

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.

High endogenous melatonin concentrations enhance sperm quality and short‐term in vitro exposure to melatonin improves aspects of sperm motility

Abstract:  Although human seminal fluid contains melatonin and spermatozoa reportedly possess membrane melatonin receptors, there are no experimental studies that have ascertained the relationship between melatonin and male infertility. This study evaluated whether urinary 6‐sulfatoxymelatonin and urinary total antioxidant capacity correlate with different seminal parameters including sperm concentration, motility and morphology. Also, the in vitro effects of melatonin on human sperm motility were investigated. Semen samples from 52 men who were counselled for infertility were obtained. Sperm concentration was determined using the haemocytometer method, motility kinematic parameters were assessed using a computer‐aided semen analysis system, while morphology and vitality were evaluated after Diff‐Quick and Eosin–Nigrosin vital staining, respectively. For the quantification of urinary 6‐sulfatoxymelatonin, a commercial ELISA kit was used, and urinary total antioxidant capacity was evaluated by means of a colorimetric assay kit. For the in vitro effects of melatonin, samples were incubated for 30 min in the presence or absence of 1 mm melatonin. Both urinary 6‐sulfatoxymelatonin and total antioxidant capacity levels positively correlated with sperm concentration, motility and morphology, as well as negatively correlated with the number of round cells. Additionally, 30‐min exposure of sperm to 1 mm melatonin improved the percentage of motile and progressively motile cells and decreased the number of static cells, thereby promoting the proportion of rapid cells. Therefore, melatonin improves semen quality, which is important because melatonin supplementation may be potentially used to obtain a successful assisted reproductive technique outcome.

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.

Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

BackgroundAsthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia.MethodsHuman ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4–5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2.ResultsTreatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia.ConclusionOur results suggest that spermatozoa from asthenozoospermic patients present a reduced responsiveness to progesterone.

Melatonin sensitizes human cervical cancer HeLa cells to cisplatin-induced cytotoxicity and apoptosis: effects on oxidative stress and DNA fragmentation

Melatonin has antitumor activity via several mechanisms including its antiproliferative and pro‐apoptotic effects as well as its potent antioxidant actions, although recent evidence has indicated that melatonin may perform pro‐oxidant actions in tumor cells. Therefore, melatonin may be useful in the treatment of tumors in association with chemotherapy drugs. This study was intended to evaluate the in vitro effect of melatonin on the cytotoxic and pro‐apoptotic actions of various chemotherapeutic agents in cervical cancer HeLa cells. Herein, we found that both melatonin and three of the chemotherapeutic drugs tested, namely cisplatin (CIS), 5‐fluorouracil (5‐FU), and doxorubicin, induced a decrease in HeLa cell viability. Furthermore, melatonin significantly increased the cytotoxic effect of such chemotherapeutic agents. Consistently, costimulation of HeLa cells with any chemotherapeutic agent in the presence of melatonin further increased caspase‐3 activation, particularly in CIS‐ and 5‐FU‐challenged cells. Likewise, concomitant treatments with melatonin and CIS significantly enhanced the ratio of cells entering mitochondrial apoptosis due to reactive oxygen species (ROS) overproduction, substantially augmented the population of apoptotic cells, and markedly enlarged DNA fragmentation compared to the treatments with CIS alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in 5‐FU‐stimulated HeLa cells, as suggested by slight increments in the percentage of cells stimulated for ROS production and in the proportion of early apoptotic cells compared to the treatments with 5‐FU alone. In summary, our findings provided evidence that in vitro melatonin strongly enhances CIS‐induced cytotoxicity and apoptosis in HeLa cells and, hence, the indoleamine could be potentially applied to cervical cancer treatment as a powerful synergistic agent.