Melatonin, experimental basis for a possible application in breast cancer prevention and treatment

The role of the pineal as an oncostatic gland has been studied in animal models of tumorigenesis, especially on those concerning the mammary gland. The general conclusion is that experimental manipulations activating pineal gland, or the administration of melatonin, reduce the incidence and growth rate of chemically-induced murine mammary tumors, while pinealectomy or situations which implicate a reduction of melatonin production usually stimulate mammary carcinogenesis. The direct actions of melatonin on mammary tumors have been suggested because of its ability to inhibit, at physiological doses (1nM), the in vitro proliferation of MCF-7 human breast cancer cells. In this article we review the outstanding findings related to melatonin actions on mammary which, taken together, support a possible usefulness of this indoleamine in the prevention and treatment of mammary gland malignancy.     

Blood pressure modulation and cardiovascular protection by melatonin: potential mechanisms behind

The production of the pineal hormone melatonin is synchronized with day-night cycle via multisynaptic pathway including suprachiasmatic nucleus linking several physiological functions to diurnal cycle. The recent data indicate that impaired melatonin production is involved in several cardiovascular pathologies including hypertension and ischemic heart disease. However, the mechanisms of melatonin effect on cardiovascular system are still not completely understood. The activation of melatonin receptors on endothelial and vascular smooth muscle cells and antioxidant properties of melatonin could be responsible for the melatonin effects on vascular tone. However, the data from in vitro studies are controversial making the explanation of the melatonin effect on blood pressure in vivo difficult. In vivo, melatonin also attenuates sympathetic tone by direct activation of melatonin receptors, scavenging free radicals or increasing NO availability in the central nervous system. The central and peripheral antiadrenergic action of chronic melatonin treatment might eliminate the mechanisms counter-regulating decreased blood pressure, providing thus additional cardioprotective mechanism. The extraordinary antioxidant activity and antilipidemic effects of melatonin may enhance the modulation of blood pressure by melatonin and probably play the most important role in the amelioration of target organ damage by chronic melatonin treatment. Further investigation of these mechanisms should provide novel knowledge about pathophysiological mechanisms of cardiovascular diseases, additional explanation for their circadian and seasonal variability and potentially generate new impulses for the development of therapeutic arsenal.     

Involvement of cGMP in cellular melatonin responses.

Melatonin can enhance and suppress constitutive protein secretion from murine melanoma M2R cells in vitro in a cholera-toxin (CTX) sensitive process. In a number of tissues melatonin has been shown to modulate cGMP levels. The involvement of cGMP in melatonin responses in the melanoma cells was investigated. The effects of melatonin on melanoma cells cGMP and cGMP-phosphodiesterase activity and the effects of cGMP analogs on the melatonin-mediated modulation of protein secretion were studied. Melatonin reduced cGMP levels in the melanoma cells. CTX treatment had a similar and non-additive effect. The effects of melatonin on protein secretion were abrogated by activation of cGMP-dependent protein kinases. In addition, melatonin inhibited cGMP phosphodiesterase activity in these cells. The data presented indicate that inhibition of cGMP via a CTX sensitive G protein may be a major signal transduction pathway used by melatonin in melanoma cells.     

Effect of the preventive-therapeutic administration of melatonin on mammary tumour-bearing animals

Melatonin has been reported to be involved in the feedback between neuroendocrine and immune functions and to exert oncostatic actions. Likewise, this hormone seems to lengthen life span in healthy animals. As of present, most studies have analysed the therapeutic effect of melatonin on cancer growth, but few have tested the preventive effect of melatonin in reducing the risk of cancer. Thus, the aim of this study was to evaluate the preventive-therapeutic effects of melatonin on rats with DMBA-induced mammary tumours, and to examine the effect of melatonin on the first line of cell defence against cancer (macrophages and NK cells) and on some of the neuroendocrine factors that are involved in the development of tumours (prolactin and catecholamines). Melatonin treatment (5 mg/day/animal) began one month prior to DMBA (9,10-dimethyl-1,2-benzanthracene) administration to females Sprague Dawley rats. It was found that the treatment led to an increase in survival and in latency time in the tumour-bearing rats. Although the melatonin treatment did not influence either the phagocytic capacity of macrophages or the number of peripheral blood NK cells, it did stabilise the levels of prolactin by returning the concentrations of this hormone to those of the healthy animals. We conclude that melatonin can exert an oncostatic action, lengthening the survival time of mammary tumour-bearing animals, and suggest that this effect is due, at least in part, to regulating the neuroendocrine parameters of tumour-bearing animals, bringing them closer to their optimal physiological status. (Mol Cell Biochem 268: 25–31, 2005)     

Melatonin: neuritogenesis and neuroprotective effects in crustacean x-organ cells

Melatonin has both neuritogenic and neuroprotective effects in mammalian cell lines such as neuroblastoma cells. The mechanisms of action include receptor-coupled processes, direct binding and modulation of calmodulin and protein kinase C, and direct scavenging of free radicals. While melatonin is produced in invertebrates and has influences on their physiology and behavior, little is known about its mechanisms of action. We studied the influence of melatonin on neuritogenesis in well-differentiated, extensively-arborized crustacean x-organ neurosecretory neurons. Melatonin significantly increased neurite area in the first 24 h of culture. The more physiological concentrations, 1 nM and 1 pM, increased area at 48 h also, whereas the pharmacological 1 μM concentration appeared to have desensitizing effects by this time. Luzindole, a vertebrate melatonin receptor antagonist, had surprising and significant agonist-like effects in these invertebrate cells. Melatonin receptors have not yet been studied in invertebrates. However, the presence of membrane-bound receptors in this population of crustacean neurons is indicated by this study. Melatonin also has significant neuroprotective effects, reversing the inhibition of neuritogenesis by 200 and 500 μM hydrogen peroxide. Because this is at least in part a direct action not requiring a receptor, melatonin's protection from oxidative stress is not surprisingly phylogenetically-conserved.     

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells,lifetime and histopathology in Swiss mice

Aims

One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy.

Main methods

Animals were inoculated with 5 × 10⁶ cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 μg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage.

Key findings

Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration.

Significance

Thus, we can conclude that doses of 150 and 300 μg/30 g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.     

Melatonin inhibits pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP accumulation and [Ca2+]i in cultured cells of neonatal rat pituitary

The effects of melatonin on pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP and [Ca2+]i were studied in neonatal rat pituitary cells. The polypeptide increased cyclic AMP accumulation. In the presence of melatonin the increase of cyclic AMP was inhibited in a dose-dependent manner, the maximal inhibition was achieved with 1-10 nM melatonin. Pituitary adenylyl cyclase-activating polypeptide also increased [Ca2+]i in 30% of the pituitary cells and melatonin inhibited the effect. Most of the cells sensitive to adenylyl cyclase-activating polypeptide (77%) were also sensitive to GnRH, suggesting they are gonadotrophs. The remaining cells were not identified. The polypeptide-induced [Ca2+]i increase was inhibited in Ca2+-free medium in 2/3 of the cells indicating that Ca2+ influx was involved. To examine causal relationship between cyclic AMP and [Ca2+]i increase, we have studied the effect of adenylyl cyclase activation by forskolin on intracellular Ca2+ concentration. Forskolin had similar effects as adenylyl cyclase-activating polypeptide: it increased [Ca2+]i in the pituitary cells and the increase was dependent on presence of Ca2+ in the medium. Melatonin inhibited the forskolin induced [Ca2+]i increase. Our observations indicate that increase of cyclic AMP stimulates Ca2+ influx in the pituitary cells of neonatal rat and that this mechanism is involved in [Ca2+]i increase induced by the pituitary adenylyl cyclase-activating polypeptide. Because melatonin inhibits increase of cyclic AMP induced by pituitary adenylyl cyclase-activating polypeptide or forskolin, the inhibitory effect of melatonin on Ca2+-influx may be mediated by the decrease of cyclic AMP concentration. This mechanism of melatonin action has not been described previously. Because melatonin inhibits the polypeptide- or forskolin-induced [Ca2+]i also in the cells not sensitive to GnRH, melatonin receptors seem to be present on both gonadotrophs and non-gonadotrophic pituitary cells.     

Long-acting somatostatin analogues in pancreatic islet cell carcinoma

In secreting islet cell carcinoma, the long-acting somatostatin analogue, Sandostatin, reduces symptoms of endocrine secretion both by inhibiting peptide secretion and by acting on the target organs. It could be used during the initial evaluation of patients with such tumors and thereafter when the tumor cannot be found or is metastatic. Its efficacy depends upon the tumor type and probably the presence of somatostatin receptors on the tumoral cells. It could decrease with time, especially in VIPomas. Side effects are few and usually mild. Hypoglycemia attacks in insulinoma could be worsened during treatment by the complete inhibition of hyperglycemic hormones. The inhibition of tumoral growth, based on animal models, appears infrequent in clinical practice.     

Light during darkness,melatonin suppression and cancer progression

Over the past few years, we have shown that the surge of melatonin in the circulation during darkness represents a potent oncostatic signal to tissue-isolated rat hepatoma 7288CTC, which is an ER+ adenocarcinoma of the liver. This oncostatic effect occurs via a melatonin receptor-mediated suppression of tumor cAMP production that leads to a suppression of the tumor uptake of linoleic acid (LA), an essential fatty acid with substantial oncogenic properties. The ability of LA to promote cancer progression is accomplished by its intracellular metabolism to 13-hydroxyoctadecadienoic acid (13-HODE) which amplifies the activity of the epidermal growth factor receptor/mitogen-activated protein kinase pathway leading to cell proliferation. By blocking tumor LA uptake, melatonin effectively blocks the production of 13-HODE and thus, markedly attenuates tumor growth. A similar effect of melatonin is observed in tissue-isolated, ER+ MCF-7 human breast cancer xenografts and nitrosomethylurea (NMU)-induced rat mammary cancers. When male rats bearing tissue-isolated hepatomas are exposed either to constant bright light (300 lux) or dim light (0.25 lux) during the dark phase of a 12L:12D photoperiod, the latency to onset was significantly reduced while the growth of tumors was markedly increased over a 4 wk period as compared with control tumors in 12L:12D-exposed rats. In constant light- and dim light during darkness-exposed rats, melatonin levels were completely suppressed while tumor growth, LA uptake and 13-HODE production were markedly increased. Similar results were obtained in constant bright light-exposed female rats bearing tissue-isolated NMU-induced mammary cancers or MCF-7 human breast cancer xenografts. To date, these studies provide the most definitive experimental evidence that light exposure during darkness increases the risk of cancer progression via elimination of the nocturnal melatonin signal and its suppression of tumor LA uptake and metabolism to 13-HODE.     

Characteristics of the NK cells in Syrian hamsters carrying tumors with varying metastatic and resistance-suppressing activity

Experiments in vivo and in vitro were made to study the effects of HETR-MLN-8 and HETR tumor cells differing in metastatic ability and inhibition of the natural host resistance to tumor on cytotoxic activity of NK from Syrian hamsters. Marked inhibition of cytotoxicity and ability for interferon activation was detected in NK isolated from tumors (as compared with blood), with that inhibition being far more pronounced in highly malignant HETR-MLN-8 tumors. This may indicate a direct inhibitory action of the tumor or its products on NK cytotoxicity. The in-vitro competition inhibition test yielded results showing that HETR-MLN-8 cells capable of in-vivo inhibition of the natural host resistance to the tumor also display much more demonstrable ability for in-vitro inhibition of NK cytotoxicity as compared to HETR cells.     

Melatonin action in neonatal gonadotrophs

Neonatal pituitary cells express MT1 and MT2 subtype of melatonin receptors that are coupled to pertussis toxin-sensitive G proteins. Their activation by melatonin leads to a decrease in cAMP production and activity of protein kinase A, and attenuation of gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion. Single cell calcium and electrophysiological recordings have revealed that a reduction in gonadotropin release results from melatonin-induced inhibition of GnRH-stimulated calcium signaling. Melatonin inhibits both calcium influx through voltage-dependent calcium channels and calcium mobilization from intracellular stores. Inhibition of calcium influx, probably in a cAMP/protein kinase C-dependent manner, and the accompanying calcium-induced calcium release from ryanodine-sensitive intracellular pools by melatonin results in a delay of GnRH-induced calcium signaling. Melatonin-induced attenuation of GnRH-induced and inositol (1,4,5)-trisphosphate-mediated calcium release from intracellular pools attenuates the amplitude of calcium signal. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on plasma gonadotropin levels. During the development, such tonic inhibitory effects of melatonin on GnRH action gradually decline due to a decrease in expression of functional melatonin receptors. In adult animals, melatonin does not have obvious direct effects on pituitary functions, whereas the connections between melatonin release and hypothalamic functions, including GnRH release, are preserved, and are critically important in synchronizing the external photoperiods and reproductive functions through still not well characterized mechanisms.     

CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.     

Biology of mammalian photoperiodism and the critical role of the pineal gland and melatonin

In mammals, photoperiodic information is transformed into a melatonin secretory rhythm in the pineal gland (high levels at night, low levels during the day). Melatonin exerts its effects in discrete hypothalamic areas, most likely through MT1 melatonin receptors. Whether melatonin is brought to the hypothalamus from the cerebrospinal fluid or the blood is still unclear. The final action of this indoleamine at the level of the central nervous system is a modulation of GnRH secretion but it does not act directly on GnRH neurones; rather, its action involves a complex neural circuit of interneurones that includes at least dopaminergic, serotoninergic and aminoacidergic neurones. In addition, this network appears to undergo morphological changes between seasons.     

mt(1) Receptor-mediated antiproliferative effects of melatonin on the rat uterine antimesometrial stromal cells.

It has been shown that melatonin regulates uterine function. Our previous studies have demonstrated the presence of melatonin receptors in the rat uterine endometrium, indicating that melatonin may act directly on the uterus. In the present study, the histological localization of the rat uterine melatonin binding was revealed by autoradiography and the molecular subtyping was studied by in situ hybridization in the stromal cells. The signal transduction process and effects of melatonin on stromal cell proliferation was also investigated. Our autoradiograms showed that 2[(125)I]iodomelatonin binding sites were localized in the antimesometrial endometrial stroma. In situ hybridization with specific mt(1) receptor cDNA probe in the primary culture of antimesometrial stromal cells demonstrated the expression of mt(1) receptor mRNAs. Melatonin dose-dependently inhibited forskolin-stimulated cAMP accumulation, which was reversed by pertussis toxin. This indicates that the rat uterine melatonin receptors are negatively coupled to adenylate cyclase via pertussis toxin sensitive G(i) protein. Melatonin also inhibited the incorporation of [(3)H]thymidine in the rat uterine antimesometrial stromal cells, showing that melatonin has an anti-proliferative effect on the uterus. Our results suggest that melatonin may act directly on the mt(1) melatonin receptors in the rat uterine antimesometrial stromal cells to inhibit their proliferation. Its action may be mediated through a pertussis toxin-sensitive adenylate cyclase coupled G(i)-protein.     

Somatostatin receptors in human cancer: incidence, characteristics, functional correlates and clinical implications.

Somatostatin receptors (SS-R) have been identified in membrane homogenates or tissue sections from several hundred tumors. SS-R were found in most neuroendocrine tumors, i.e. GH and TSH producing pituitary tumors, endocrine gastroenteropancreatic (GEP) tumors, paragangliomas, pheochromocytomas, medullary thyroid carcinomas (MTC) and small cell lung carcinomas. SS-R were also expressed in a majority of malignant lymphomas, in several brain tumors (all meningiomas, most astrocytomas) and in breast tumors. The majority of tumors expressing SS-R are rather differentiated (i.e. astrocytomas vs glioblastomas), but exceptions exist (high grade malignant lymphomas). An inverse relationship exists between SS-R and receptors for epidermal growth factor (EGF-R) incidence in lung tumors, glial tumors and most breast tumors, whereas meningiomas express simultaneously both receptors. A minority of tumors (ovarian tumors, MTC, insulinomas) express a subtype of SS-R, characterized by low affinity for the octapeptide SS analog octreotide. The function mediated by SS-R in human tumors may differ according to the tumor type. SS-R in pituitary and GEP tumor mediate hormone secretion inhibition with, in addition, possibly some antiproliferative effects. In meningiomas, however, activation of SS-R inhibits forskolin-stimulated adenylate cyclase activity, and weakly stimulates proliferation. Whereas SS-R seem to mediate antiproliferative effects in animal models and cell lines of lymphomas, breast and lung tumors, such an effect has not yet been convincingly documented in human primary tumors. The clinical implications of the presence of SS-R in tumors are manyfold: (1) as a predictive marker for efficient therapy with octreotide in pituitary and GEP tumors; (2) as a diagnostic marker: for pathobiochemical classification of tumors, using in vitro detection methods; for clinical evaluation using in vivo scanning techniques; (3) as a prognostic marker; and (4) as a potential radiotherapeutic target.     

Targeting Gbetagamma signaling to inhibit prostate tumor formation and growth

Prostate cancer starts as androgen-dependent malignancy and responds initially to androgen ablative therapy. Beneficial effects of androgen ablation, however, are often temporary and the cancer reappears as androgen-independent tumor, suggesting the existence of additional factors responsible for progression of the disease. Attention has focused on receptor tyrosine kinases as the growth mediators of androgen-independent prostate cancer; overexpression of epidermal growth factor receptors or their ligand heparin-bound epidermal growth factor, for example, promotes transition to androgen independence. Emerging data demonstrate involvement of another class of cell membrane-anchored receptors, the heterotrimeric guanine-binding (G) protein-coupled receptors (GPCRs) in prostate cancer. In vitro, stimulation of many endogenous GPCRs induces mitogenic signaling and growth of prostate cancer cells. The GPCRs transduce mitogenic signals via activated G proteins in the form of Galpha-GTP and Gbetagamma subunits. Here, we show that expression of a Gbetagamma inhibitor peptide derived from carboxy terminus of G protein-coupled receptor kinase 2 obliterates serum-regulated prostate cancer cell growth in vitro and prevents prostate tumor formation in vivo. We also demonstrate that inhibition of Gbetagamma signaling retards growth of existing prostate tumors by inducing cell death. These data establish a central role for heterotrimeric G proteins in prostate cancer and suggest targeted inhibition of Gbetagamma signaling may serve as specific molecular therapy tool to limit pathologic growth of advanced prostate cancer.