The effect of estradiol, progesterone, and melatonin on estrous cycling and ovarian aromatase expression in intact female mice.

OBJECTIVE: Melatonin and progesterone levels decline during the perimenopause. Both hormones inhibit estrogen action and endometrial cancer, but little is known about how they act in combination. Therefore, the interplay of progesterone (P4) and melatonin was investigated in intact female mice. STUDY DESIGN: Three P4 doses, low (25mg), mid (50mg), and high (100mg), combined with 0.5mg 17ß-estradiol (E), were administered in the diet (per 1800kcal) for 30 days. Hormone therapy (HT) with the low P4 dose (estradiol/low progesterone replacement therapy (EPLRT)) was used to create an excess estrogen environment to mimic perimenopause. Half the mice were treated with melatonin (M) 15mg/L in the drinking water at night. RESULTS: The unbalanced EPLRT treatment increased estrogen-regulated responses. Specifically, mice treated with EPLRT had significantly higher levels of ovarian aromatase mRNA versus control, which was prevented in the presence of higher doses of P4 and/or the addition of melatonin. The number of days in estrus also increased in EPLRT-treated versus control mice with no change in the length or number of complete estrous cycles. Melatonin, combined with all doses of P4, increased the number of days spent in estrus, but not the length or number of estrous cycles compared to melatonin alone; however, two-way ANOVA revealed a significant interaction between melatonin and P4 dose for days in estrus and for number of cycles. Although none of the E2 and P4 combinations significantly affected uterine weight compared to control, melatonin addition to the low or mid P4 HT resulted in slightly higher uterine weights compared to melatonin-treated mice. Melatonin significantly increased uterine estrogen receptor alpha (ERα) and progesterone receptor A levels compared to control animals. HT, added in combination with melatonin, reduced ERα levels back to control levels, but PR levels remained elevated albeit intermediary to those achieved with melatonin alone. CONCLUSION: The findings that melatonin supplementation inhibits ovarian aromatase expression and increases uterine receptors in mice given an HT that mimics perimenopause may have important clinical applications for the improvement of menopause-related conditions, like menorrhagia, associated with high levels of E2 and low levels of P4.

Basic mechanisms involved in the anti-cancer effects of melatonin.

It is commonly accepted that melatonin (N-acetyl-5-methoxytryptamine), the most relevant pineal secretory product, has oncostatic properties in a wide variety of tumors and, especially, in those identified as being hormonedependent. The objective of the present article is to offer a global and integrative view of the mechanisms involved in the oncostatic actions of this indoleamine. Due to the wide spectrum of melatonin's actions, the mechanisms that may be involved in its ability to counteract tumor growth are varied. These include: a) antioxidant effects; b) regulation of the estrogen receptor expression and transactivation; c) modulation of the enzymes involved in the local synthesis of estrogens; d) modulation of cell cycle and induction of apoptosis; e) inhibition of telomerase activity; f) inhibition of metastasis; g) prevention of circadian disruption; h) antiangiogenesis; i) epigenetic effects; j) stimulation of cell differentiation; and k) activation of the immune system. The data supporting each of these oncostatic actions of melatonin are summarized in this review. Moreover, the list of actions described may not be exhaustive in terms of how melatonin modulates tumor growth.

Scientific basis for the potential use of melatonin in bone diseases: osteoporosis and adolescent idiopathic scoliosis.

The objective of this paper was to analyze the data supporting the possible role of melatonin on bone metabolism and its repercussion in the etiology and treatment of bone pathologies such as the osteoporosis and the adolescent idiopathic scoliosis (AIS). Melatonin may prevent bone degradation and promote bone formation through mechanisms involving both melatonin receptor-mediated and receptor-independent actions. The three principal mechanisms of melatonin effects on bone function could be: (a) the promotion of the osteoblast differentiation and activity; (b) an increase in the osteoprotegerin expression by osteoblasts, thereby preventing the differentiation of osteoclasts; (c) scavenging of free radicals generated by osteoclast activity and responsible for bone resorption. A variety of in vitro and in vivo experimental studies, although with some controversial results, point toward a possible role of melatonin deficits in the etiology of osteoporosis and AIS and open a new field related to the possible therapeutic use of melatonin in these bone diseases.

Clinical uses of melatonin: evaluation of human trials.

During the last 20 years, numerous clinical trials have examined the therapeutic usefulness of melatonin in different fields of medicine. The objective of this article is to review, in depth, the science regarding clinical trials performed to date. The efficacy of melatonin has been assessed as a treatment of ocular diseases, blood diseases, gastrointestinal tract diseases, cardiovascular diseases, diabetes, rheumatoid arthritis, fibromyalgia, chronic fatigue syndrome, infectious diseases, neurological diseases, sleep disturbances, aging and depression. Melatonin has been also used as a complementary treatment in anaesthesia, hemodialysis, in vitro fertilization and neonatal care. The conclusion of the current review is that the use of melatonin as an adjuvant therapy seems to be well funded for macular degeneration, glaucoma, protection of the gastric mucosa, irritable bowel syndrome, arterial hypertension, diabetes, side effects of chemotherapy and radiation in cancer patients or hemodialysis in patients with renal insufficiency and, especially, for sleep disorders of circadian etiology (jet lag, delayed sleep phase syndrome, sleep deterioration associated with aging, etc.) as well as in those related with neurological degenerative diseases (Alzheimer, etc.,) or Smith-Magenis syndrome. The utility of melatonin in anesthetic procedures has been also confirmed. More clinical studies are required to clarify whether, as the preliminary data suggest, melatonin is useful for treatment of fibromyalgia, chronic fatigue syndrome, infectious diseases, neoplasias or neonatal care. Preliminary data regarding the utility of melatonin in the treatment of ulcerative colitis, Crohn's disease, rheumatoid arthritis are either ambiguous or negative. Although in a few cases melatonin seems to aggravate some conditions, the vast majority of studies document the very low toxicity of melatonin over a wide range of doses.

Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells.

BACKGROUND: Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. Our objective was to study the promoters through which melatonin modifies aromatase expression, evaluate the ability of melatonin to regulate cyclooxygenases and assess whether the effects of melatonin are related to its effects on intracellular cAMP, in MCF-7 cells. METHODS: Total aromatase mRNA, aromatase mRNA promoter regions and cyclooxygenases mRNA expression were determined by real-time RT-PCR. PGE(2) and cAMP were measured by kits. RESULTS: Melatonin downregulated the gene expression of the two major specific aromatase promoter regions, pII and pI.3, and also that of the aromatase promoter region pI.4. Melatonin 1 nM was able to counteract the stimulatory effect of tetradecanoyl phorbol acetate on PGE(2) production and inhibit COX-2 and COX-1 mRNA expression. Melatonin 1 nM elicited a parallel time-dependent decrease in both cyclic AMP formation and aromatase mRNA expression. CONCLUSIONS: This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.

Melatonin as a selective estrogen enzyme modulator.

Melatonin exerts oncostatic effects on different kinds of tumors, especially on hormone-dependent breast cancer. The general conclusion is that melatonin, in vivo, reduces the incidence and growth of chemically-induced mammary tumors in rodents, and, in vitro, inhibits the proliferation and invasiveness of human breast cancer cells. Both studies support the hypothesis that melatonin inhibits the growth of breast cancer by interacting with estrogen-signaling pathways through three different mechanisms: (a) the indirect neuroendocrine mechanism which includes the melatonin down-regulation of the hypothalamic-pituitary-reproductive axis and the consequent reduction of circulating levels of gonadal estrogens, (b) direct melatonin actions at tumor cell level by interacting with the activation of the estrogen receptor, thus behaving as a selective estrogen receptor modulator (SERM), and (c) the regulation of the enzymes involved in the biosynthesis of estrogens in peripheral tissues, thus behaving as a selective estrogen enzyme modulator (SEEM). As melatonin reduces the activity and expression of aromatase, sulfatase and 17beta-hydroxysteroid dehydrogenase and increases the activity and expression of estrogen sulfotransferase, it may protect mammary tissue from excessive estrogenic effects. Thus, a single molecule has both SERM and SEEM properties, one of the main objectives desired for the breast antitumoral drugs. Since the inhibition of enzymes involved in the biosynthesis of estrogens is currently one of the first therapeutic strategies used against the growth of breast cancer, melatonin modulation of different enzymes involved in the synthesis of steroid hormones makes, collectively, this indolamine an interesting anticancer drug in the prevention and treatment of estrogen-dependent mammary tumors.

Selective estrogen enzyme modulator actions of melatonin in human breast cancer cells.

Melatonin exerts oncostatic effects on different kinds of neoplasias, especially on estrogen-dependent mammary tumors. Current knowledge about the mechanisms by which melatonin inhibits the growth of breast cancer cells point to an interaction of melatonin with estrogen-responsive pathways. The intratumoral production of estrogens in breast carcinoma tissue plays a pivotal role in the proliferation of mammary tumoral cells and its blockade is one of the main objectives of the treatment of breast cancer. The aim of the present work is centered on the study of the role of melatonin in the control of some enzymes involved in the formation and transformation of estrogens in human breast cancer cells. The present study demonstrates that melatonin, at physiologic concentrations, modulates the synthesis and transformation of biologically active estrogens in MCF-7 cells, through the inhibition of sulfatase (STS) and 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) activity and expression, enzymes involved in the estradiol formation in breast cancer cells. Physiologic concentrations of melatonin also stimulate the activity and expression of estrogen sulfotransferase (EST), the enzyme responsible for the formation of the biologically inactive estrogen sulfates. The level of EST mRNA steady-state of cells treated with melatonin was three times higher than that in control cells. These findings which document that melatonin has an inhibitory effect on STS and 17beta-HSD1 and a stimulatory effect on EST, in combination with its previously described antiaromatase effect, can open up new and interesting possibilities in clinical applications of melatonin in breast cancer.

Inhibitory effects of pharmacological doses of melatonin on aromatase activity and expression in rat glioma cells.

Melatonin exerts oncostatic effects on different kinds of neoplasias, especially on oestrogen-dependent tumours. Recently, it has been described that melatonin, on the basis of its antioxidant properties, inhibits the growth of glioma cells. Glioma cells express oestrogen receptors and have the ability to synthesise oestrogens from androgens. In the present study, we demonstrate that pharmacological concentrations of melatonin decreases the growth of C6 glioma cells and reduces the local biosynthesis of oestrogens, through the inhibition of aromatase, the enzyme that catalyses the conversion of androgens into oestrogens. These results are supported by three types of evidence. Firstly, melatonin counteracts the growth stimulatory effects of testosterone on glioma cells, which is dependent on the local synthesis of oestrogens from testosterone. Secondly, we found that melatonin reduces the aromatase activity of C6 cells, measured by the tritiated water release assay. Finally, by (RT)-PCR, we found that melatonin downregulates aromatase mRNA steady-state levels in these glioma cells. We conclude that melatonin inhibits the local production of oestrogens decreasing aromatase activity and expression. By analogy to the implications of aromatase in other forms of oestrogen-sensitive tumours, it is conceivable that the modulation of the aromatase by pharmacological melatonin may play a role in the growth of glioblastomas.

Melatonin and estradiol effects on food intake, body weight, and leptin in ovariectomized rats.

OBJECTIVE: The study in ovariectomized (Ovx) rats, as a model of menopausal status, of the effects of melatonin (M) and/or estradiol (E), associated or not with food restriction, on body weight (BW) and serum leptin levels. METHODS: Female SD rats (200-250 g) were Ovx and treated with E, M, E+M or its diluents. Control sham-Ovx rats were treated with E-M diluents. After 7 weeks being fed ad libitum, the animals were exposed for 7 more weeks to a 30% food restriction. We measured: food intake, BW, nocturnal and diurnal urinary excretion of sulphatoxymelatonin (aMT6s), leptin in midday and midnight blood samples, glucose, total cholesterol, LDL, HDL and triglycerides. RESULTS: Day/night rhythm of aMT6s excretion was preserved in all cases. The increase of aMT6s excretion in M-treated animals basically affected the nocturnal period. In animals fed ad libitum, E fully prevented Ovx-induced increase of BW, leptin and cholesterol. Melatonin reduced food intake and partially prevented the increase of BW and cholesterol, without changing leptin levels. Under food restriction, M was the most effective treatment in reducing BW and cholesterol. Leptin levels were similar in M, E or E+M treated rats, and lower than in untreated Ovx rats. CONCLUSIONS: Our result gives a preliminary experimental basis for a post-menopausal co-treatment with estradiol and melatonin. It could combine the effectiveness of estradiol (not modified by melatonin) with the positive effects of melatonin (improvement of sleep quality, prevention of breast cancer, etc.). The possible beneficial effects of melatonin which could justify its use, need to be demonstrated in clinical trials.

Melatonin prevents the estrogenic effects of sub-chronic administration of cadmium on mice mammary glands and uterus.

Cadmium (Cd) is a heavy metal classified as a human carcinogen. Occupational exposure, dietary consumption and cigarette smoking are sources of Cd contamination. Cd-induced carcinogenicity depends on its oxidative and estrogenic actions. A possible role of Cd in breast cancer etiology has been recently suggested. Melatonin, because of its antioxidant and antiestrogenic properties could counteract the toxic effects of this metalloestrogen. Our aim was both to determine the effects of relevant doses of Cd on mice mammary glands and uterus and to test whether melatonin would counteract its effects. Female mice of different ages and estrogenic status (prepuberal, adult intact, adult ovariectomized) were treated with CdCl(2) (2-3 mg/kg, i.p.), melatonin (10 microg/mL in drinking water), CdCl(2) + melatonin, or diluents. Whereas in prepuberal animals Cd disturbs mammary ductal growth and reduces the number of terminal end buds, in adults, regardless of the steroidal milieu, Cd exerts estrogenic effects on mammary glands, increasing lobuloalveolar development and ductal branching. Uterine weight also increased as a result of Cd treatment. The effects of Cd are partially inhibited by melatonin. In adult ovariectomized mice, Cd concentration in blood of animals treated with CdCl(2) + melatonin was lower than in mice receiving only Cd; the opposite effects were found in non-castrated animals. As Cd mimics the effect of estrogens, the high incidence of breast cancer in tobacco smokers and women working in industries related with Cd could be explained because of the properties of this metal. The effects of melatonin point to a possible role of this indoleamine as a preventive agent for environmental or occupational Cd contamination.

Effects of MT1 melatonin receptor overexpression on the aromatase-suppressive effect of melatonin in MCF-7 human breast cancer cells.

A major mechanism through which melatonin reduces the development of breast cancer is based on its anti-estrogenic actions by interfering at different levels with the estrogen-signalling pathways. Melatonin inhibits both aromatase activity and expression in vitro (MCF-7 cells) as well as in vivo, thus behaving as a selective estrogen enzyme modulator. The objective of this study was to study the effect of MT1 melatonin receptor overexpression in MCF-7 breast cancer cells on the aromatase-suppressive effects of melatonin. Transfection of the MT1 melatonin receptor in MCF-7 cells significantly decreased aromatase activity of the cells and MT1-transfected cells showed a level of aromatase activity that was 50% of vector-transfected MCF-7 cells. The proliferation of estrogen-sensitive MCF-7 cells in an estradiol-free media but in the presence of testosterone (an indirect measure of aromatase activity) was strongly inhibited by melatonin in those cells overexpressing the MT1 receptor. This inhibitory effect of melatonin on cell growth was higher on MT1 transfected cells than in vector transfected ones. In MT1-transfected cells, aromatase activity (measured by the tritiated water release assay) was inhibited by melatonin (20% at 1 nM; 40% at 10 microM concentrations). The same concentrations of melatonin did not significantly influence the aromatase activity of vector-transfected cells. MT1 melatonin receptor transfection also induced a significant 55% inhibition of aromatase steady-state mRNA expression in comparison to vector-transfected MCF-7 cells (p<0.001). In addition, in MT1-transfected cells melatonin treatment inhibited aromatase mRNA expression and 1 nM melatonin induced a higher and significant down-regulation of aromatase mRNA expression (p<0.05) than in vector-transfected cells. The findings presented herein point to the importance of MT1 melatonin receptor in mediating the oncostatic action of melatonin in MCF-7 human breast cancer cells and confirm MT1 melatonin receptor as a major mediator in the melatonin signalling pathway in breast cancer.

Melatonin inhibits both ER alpha activation and breast cancer cell proliferation induced by a metalloestrogen, cadmium.

Cadmium (Cd) is a heavy metal affecting human health both through environmental and occupational exposure. There is evidence that Cd accumulates in several organs and is carcinogenic to humans. In vivo, Cd mimics the effect of estrogens in the uterus and mammary gland. In estrogen-responsive breast cancer cell lines, Cd stimulates proliferation and can also activate the estrogen receptor independent of estradiol. The ability of this metalloestrogen to increase gene expression in MCF7 cells is blocked by anti-estrogens suggesting that the activity of these compounds is mediated by ER alpha. The aims of this work were to test whether melatonin inhibits Cd-induced proliferation in MCF7 cells, and also to study whether melatonin specifically inhibits Cd-induced ER alpha transactivation. We show that melatonin prevents the Cd-induced growth of synchronized MCF7 breast cancer cells. In transient transfection experiments, we prove that both ER alpha- and ER beta-mediated transcription are stimulated by Cd. Melatonin is a specific inhibitor of Cd-induced ER alpha-mediated transcription in both estrogen response elements (ERE)- and AP1-containing promoters, whereas ER beta-mediated transcription is not inhibited by the pineal indole. Moreover, the mutant ER alpha-(K302G, K303G), unable to bind calmodulin, is activated by Cd but becomes insensitive to melatonin treatment. These results proved that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the stimulatory effect of the heavy metal in cells expressing ER alpha at both ERE-luc and AP1-luc sites. We can infer from these experiments that melatonin regulates Cd-induced transcription in both ERE- and AP1 pathways. These results also reinforce the hypothesis of the anti-estrogenic properties of melatonin as a valuable tool in breast cancer therapies.

Melatonin and mammary cancer: a short review.

Melatonin is an indolic hormone produced mainly by the pineal gland. The former hypothesis of its possible role in mammary cancer development was based on the evidence that melatonin down-regulates some of the pituitary and gonadal hormones that control mammary gland development and which are also responsible for the growth of hormone-dependent mammary tumors. Furthermore, melatonin could act directly on tumoral cells, as a naturally occurring antiestrogen, thereby influencing their proliferative rate. The first reports revealed a low plasmatic melatonin concentration in women with estrogen receptor (ER)-positive breast tumors. However, later studies on the possible role of melatonin on human breast cancer have been scarce and mostly of an epidemiological type. These studies described a low incidence of breast tumors in blind women as well as an inverse relationship between breast cancer incidence and the degree of visual impairment. Since light inhibits melatonin secretion, the relative increase in the melatonin circulating levels in women with a decreased light input could be interpreted as proof of the protective role of melatonin on mammary carcinogenesis. From in vivo studies on animal models of chemically induced mammary tumorigenesis, the general conclusion is that experimental manipulations activating the pineal gland or the administration of melatonin lengthens the latency and reduces the incidence and growth rate of mammary tumors, while pinealectomy usually has the opposite effects. Melatonin also reduces the incidence of spontaneous mammary tumors in different kinds of transgenic mice (c-neu and N-ras) and mice from strains with a high tumoral incidence. In vitro experiments, carried out with the ER-positive MCF-7 human breast cancer cells, demonstrated that melatonin, at a physiological concentration (1 nM) and in the presence of serum or estradiol: (a) inhibits, in a reversible way, cell proliferation, (b) increases the expression of p53 and p21WAF1 proteins and modulates the length of the cell cycle, and (c) reduces the metastasic capacity of these cells and counteracts the stimulatory effect of estradiol on cell invasiveness; this effect is mediated, at least in part, by a melatonin-induced increase in the expression of the cell surface adhesion proteins E-cadherin and beta(1)-integrin. The direct oncostatic effects of melatonin depends on its interaction with the tumor cell estrogen-responsive pathway. In this sense it has been demonstrated that melatonin down-regulates the expression of ERalpha and inhibits the binding of the estradiol-ER complex to the estrogen response element (ERE) in the DNA. The characteristics of melatonin's oncostatic actions, comprising different aspects of tumor biology as well as the physiological doses at which the effect is accomplished, give special value to these findings and encourage clinical studies on the possible therapeutic value of melatonin on breast cancer.

Decreased sleep quality in patients suffering from retinitis pigmentosa.

The purpose of this study was to assess the prevalence of sleep disturbance in subjects diagnosed with retinitis pigmentosa (RP), as well as the influence of age and gender. Sleep quality was assessed, by means of the Pittsburgh Sleep Quality Index (PSQI), in people with RP (n=177) and gender-and age-matched normally sighted individuals (n=491). The population was divided, according to their age, in eight decade groups. People on shift-work, with affective disorders or with visual impairment other than RP, were excluded. The influence of cataracts in sleep quality was also studied in non-RP people (n=57), with cataracts significantly impairing visual acuity. Another group of healthy controls (n=190) was studied in different seasons of the year for a possible seasonality in sleep disturbance. Global sleep quality decreased in an age-dependent manner in RP-patients, especially from the second decade of life. Retinitis pigmentosa-patients showed, in relation to age-matched controls: lower subjective sleep quality and efficiency, longer sleep latency, shorter sleep duration, higher daytime dysfunction and a higher use of sleeping medication. No significant differences in sleep quality were found among RP-patients or controls depending either on their gender or on the presence of cataracts. Normal sighted individuals did not show seasonality in their sleep quality. We conclude that the sleep quality of RP-patients decreases in an age-dependent manner and points to the probably degeneration of photoreceptors mediating the photic input to the suprachiasmatic nuclei of the hypothalamus in this disease.

Direct antiproliferative effects of melatonin on two metastatic cell sublines of mouse melanoma (B16BL6 and PG19).

The effects of melatonin on the growth of two highly tumorigenic rodent melanoma cells were studied in vitro. PG19, an amelanotic mouse melanoma cell line, and B16BL6, a melanotic melanoma cell line selected for its invasive potential in vitro, were cultured in the presence of different concentrations of melatonin (10 microM to 0.1 pM). Five days later, viable cells were determined in a haemocytometer by the trypan blue exclusion test. Melatonin at concentrations of 1 nM and 10 pM (within the range of concentrations that correspond to physiological night-time and daytime levels in human blood) significantly inhibited proliferation in both melanoma cell lines. Subphysiological (0.1 pM) or supraphysiological (10 microM to 100 nM) concentrations of melatonin lacked this effect. These results support the hypothesis that, at physiological concentrations, melatonin exerts a direct inhibitory effect on PG19 and B16BL6 cells proliferation.

Doses and time-dependent effects of 3'-azido-3'-deoxythymidine on T47D human breast cancer cells in vitro.

The objective of the present work was to study the effects of 3'-azido-3'-deoxythymidine (azidothymidine, Zidovudine) on human breast cancer cells by using, as a model, the T47D cell line (typified as oestrogen-dependent and p53-mutated). Low azidothymidine doses (3.125 microM) increase the percentage of cells in S-phase, with the effect reversing after 24 hr of incubation; as azidothymidine doses increase, the magnitude and duration of its effect increase proportionally, although, even with the highest concentrations (50-100 microM) the effects decline after 48 hr of incubation. If media (containing azidothymidine or vehicle) are daily renewed, the azidothymidine effects (accumulation of cells in S-phase) are higher and decline later than when media and drug are not changed during the whole culture period, thereby suggesting that the reversion of azidothymidine effects could be related with a degradation of the drug or accumulation in media of substances which counteract its effects. Azidothymidine inhibits T47D cell proliferation at concentrations higher than 50 microM. The exposure to 50 or 100 microM azidothymidine induced cell apoptosis after 48 hr or more of incubation. We conclude that: a) azidothymidine, with appropriate doses and duration of treatment, synchronizes cells in S-phase, inhibits proliferation, and induces apoptosis, b) the discontinuous application of the drug rather than continuous exposure to it increases its efficiency to synchronize the T47D cell cycle. This in vitro anti-breast cancer activity suggests that a possible clinical usefulness of azidothymidine, either alone or associated with other drugs with cycle-specific antitumoural activity circumscribed to the S-phase of cell cycle, is worthy of investigation.

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.

Influence of serum from healthy or breast tumor-bearing women on the growth of MCF-7 human breast cancer cells.

Sera from women healthy (HW) or with breast (BCW), ovarian or endometrial cancer, were added (10%) to the culture media of MCF-7 cells and cell proliferation assessed 4 days later to verify: a) whether sera from BCW, obtained before or 8 days after tumor ablaction, influence the proliferation of these cells, b) whether the effects of serum from BCW are specific for mammary tumor cells. Sera from BCW, but not sera from women with ovarian or endometrial cancer, increased MCF-7 cell proliferation in comparison with sera from HW. After surgical ablation of the breast tumors, serum's ability to increase MCF-7 cell proliferation decreased significantly. These effects cannot be explained by differences on serum levels of estradiol or melatonin. These results suggest the presence of growth-promoting substances of possible tumoral origin in serum of BCW, a fact that should be considered as support for the surgical treatment of tumor masses.

Melatonin and mammary pathological growth.

In this article we review the state of the art on the role of the pineal gland and melatonin in mammary cancer tumorigenesis in vivo as well as in vitro. The former hypothesis of a possible role of the pineal gland in mammary cancer development was based on the evidence that the pineal, via its main secretory product, melatonin, downregulates some of the pituitary and gonadal hormones which control mammary gland development and are also responsible for the growth of hormone-dependent mammary tumors. Furthermore, melatonin could act directly on tumoral cells, thereby influencing their proliferative rate. Other possible origins of melatonin's antitumoral actions could be found in its antioxidant or immunoenhancing properties. The working hypotheses of most experiments were that the activation of the pineal gland, or the administration of melatonin, should give rise to antitumoral behavior; conversely, suppression of the pineal gland or melatonin deficits should stimulate mammary tumorigenesis. From in vivo studies on animal models of tumorigenesis, the general conclusion is that experimental manipulations activating the pineal gland, or the administration of melatonin, enlarge the latency and reduce the incidence and growth rate of chemically induced mammary tumors, while pinealectomy usually has the opposite effects. The direct actions of melatonin on mammary tumors have been suggested because of its ability to inhibit, at physiological doses (1 nM), the in vitro proliferation and invasiveness of MCF-7 human breast cancer cells. The fact that most studies have been performed on two models, chemically induced mammary adenocarcinoma in rats (in vivo studies) and the cell tumor line MCF-7 (in vitro studies), makes the generalization of the results somewhat difficult. However, the characteristics of these actions, comprising different aspects of tumor biology such as initiation, proliferation, and metastasis, as well as the doses (physiological range) at which the effect is accomplished, give special value to these findings. On the strength of these data, the small number of clinical studies focusing on the possible therapeutic value of melatonin on breast cancer is surprising.

Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro.

The aim of the present work was to study whether melatonin, at physiological concentrations, exerts its antiproliferative effects on MCF-7 human breast cancer cells by inducing the expression of some of the proteins involved in the control of the cell cycle. MCF-7 cells were cultured for 48 h in DMEM media containing either melatonin (1 nM) or the diluent (0.001% ethanol). At this concentration, after 48 hours of incubation, melatonin reduced the number of viable cells in relation to controls. The decreased cell proliferation was coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins. These results demonstrate that melatonin inhibits MCF-7 cell proliferation by inducing an arrest of cell cycle dependent on an increased expression of p21WAF1 protein, which is mediated by the p53 pathway.