Melatonin and Its Role in the Epithelial-to-Mesenchymal Transition (EMT) in Cancer.

The epithelial-to-mesenchymal transition (EMT) is a cell-biological program that occurs during the progression of several physiological processes and that can also take place during pathological situations such as carcinogenesis. The EMT program consists of the sequential activation of a number of intracellular signaling pathways aimed at driving epithelial cells toward the acquisition of a series of intermediate phenotypic states arrayed along the epithelial-mesenchymal axis. These phenotypic features include changes in the motility, conformation, polarity and functionality of cancer cells, ultimately leading cells to stemness, increased invasiveness, chemo- and radioresistance and the formation of cancer metastasis. Amongst the different existing types of the EMT, type 3 is directly involved in carcinogenesis. A type 3 EMT occurs in neoplastic cells that have previously acquired genetic and epigenetic alterations, specifically affecting genes involved in promoting clonal outgrowth and invasion. Markers such as E-cadherin; N-cadherin; vimentin; and transcription factors (TFs) like Twist, Snail and ZEB are considered key molecules in the transition. The EMT process is also regulated by microRNA expression. Many miRNAs have been reported to repress EMT-TFs. Thus, Snail 1 is repressed by miR-29, miR-30a and miR-34a; miR-200b downregulates Slug; and ZEB1 and ZEB2 are repressed by miR-200 and miR-205, respectively. Occasionally, some microRNA target genes act downstream of the EMT master TFs; thus, Twist1 upregulates the levels of miR-10b. Melatonin is an endogenously produced hormone released mainly by the pineal gland. It is widely accepted that melatonin exerts oncostatic actions in a large variety of tumors, inhibiting the initiation, progression and invasion phases of tumorigenesis. The molecular mechanisms underlying these inhibitory actions are complex and involve a great number of processes. In this review, we will focus our attention on the ability of melatonin to regulate some key EMT-related markers, transcription factors and micro-RNAs, summarizing the multiple ways by which this hormone can regulate the EMT. Since melatonin has no known toxic side effects and is also known to help overcome drug resistance, it is a good candidate to be considered as an adjuvant drug to conventional cancer therapies.

Melatonin Modulation of Radiation-Induced Molecular Changes in MCF-7 Human Breast Cancer Cells.

Radiation therapy is an important component of cancer treatment scheduled for cancer patients, although it can cause numerous deleterious effects. The use of adjuvant molecules aims to limit the damage in normal surrounding tissues and enhance the effects of radiation therapy, either killing tumor cells or slowing down their growth. Melatonin, an indoleamine released by the pineal gland, behaves as a radiosensitizer in breast cancer, since it enhances the therapeutic effects of ionizing radiation and mitigates side effects on normal cells. However, the molecular mechanisms through which melatonin modulates the molecular changes triggered by radiotherapy remain mostly unknown. Here, we report that melatonin potentiated the anti-proliferative effect of radiation in MCF-7 cells. Treatment with ionizing radiation induced changes in the expression of many genes. Out of a total of 25 genes altered by radiation, melatonin potentiated changes in 13 of them, whereas the effect was reverted in another 10 cases. Among them, melatonin elevated the levels of PTEN and NME1, and decreased the levels of SNAI2, ERBB2, AKT, SERPINE1, SFN, PLAU, ATM and N3RC1. We also analyzed the expression of several microRNAs and found that melatonin enhanced the effect of radiation on the levels of miR-20a, miR-19a, miR-93, miR-20b and miR-29a. Rather surprisingly, radiation induced miR-17, miR-141 and miR-15a but melatonin treatment prior to radiation counteracted this stimulatory effect. Radiation alone enhanced the expression of the cancer suppressor miR-34a, and melatonin strongly stimulated this effect. Melatonin further enhanced the radiation-mediated inhibition of Akt. Finally, in an in vivo assay, melatonin restrained new vascularization in combination with ionizing radiation. Our results confirm that melatonin blocks many of the undesirable effects of ionizing radiation in MCF-7 cells and enhances changes that lead to optimized treatment results. This article highlights the effectiveness of melatonin as both a radiosensitizer and a radioprotector in breast cancer. Melatonin is an effective adjuvant molecule to radiotherapy, promoting anti-cancer therapeutic effects in cancer treatment. Melatonin modulates molecular pathways altered by radiation, and its use in clinic might lead to improved therapeutic outcomes by enhancing the sensitivity of cancerous cells to radiation and, in general, reversing their resistance toward currently applied therapeutic modalities.

Melatonin as an Adjuvant to Antiangiogenic Cancer Treatments.

Melatonin is a hormone with different functions, antitumor actions being one of the most studied. Among its antitumor mechanisms is its ability to inhibit angiogenesis. Melatonin shows antiangiogenic effects in several types of tumors. Combination of melatonin and chemotherapeutic agents have a synergistic effect inhibiting angiogenesis. One of the undesirable effects of chemotherapy is the induction of pro-angiogenic factors, whilst the addition of melatonin is able to overcome these undesirable effects. This protective effect of the pineal hormone against angiogenesis might be one of the mechanisms underlying its anticancer effect, explaining, at least in part, why melatonin administration increases the sensitivity of tumors to the inhibitory effects exerted by ordinary chemotherapeutic agents. Melatonin has the ability to turn cancer totally resistant to chemotherapeutic agents into a more sensitive chemotherapy state. Definitely, melatonin regulates the expression and/or activity of many factors involved in angiogenesis which levels are affected (either positively or negatively) by chemotherapeutic agents. In addition, the pineal hormone has been proposed as a radiosensitizer, increasing the oncostatic effects of radiation on tumor cells. This review serves as a synopsis of the interaction between melatonin and angiogenesis, and we will outline some antiangiogenic mechanisms through which melatonin sensitizes cancer cells to treatments, such as radiotherapy or chemotherapy.

Melatonin as a Radio-Sensitizer in Cancer.

Radiotherapy is one of the treatments of choice in many types of cancer. Adjuvant treatments to radiotherapy try, on one hand, to enhance the response of tumor cells to radiation and, on the other hand, to reduce the side effects to normal cells. Radiosensitizers are agents that increase the effect of radiation in tumor cells by trying not to increase side effects in normal tissues. Melatonin is a hormone produced mainly by the pineal gland which has an important role in the regulation of cancer growth, especially in hormone-dependent mammary tumors. Different studies have showed that melatonin administered with radiotherapy is able to enhance its therapeutic effects and can protect normal cells against side effects of this treatment. Several mechanisms are involved in the radiosensitization induced by melatonin: increase of reactive oxygen species production, modulation of proteins involved in estrogen biosynthesis, impairment of tumor cells to DNA repair, modulation of angiogenesis, abolition of inflammation, induction of apoptosis, stimulation of preadipocytes differentiation and modulation of metabolism. At this moment, there are very few clinical trials that study the therapeutic usefulness to associate melatonin and radiotherapy in humans. All findings point to melatonin as an effective adjuvant molecule to radiotherapy in cancer treatment.

Editorial for the Special Issue "New Strategies in Cancer Pharmacotherapy: Development of Hormonal Antineoplastic Drugs, Cytotoxic Drugs and Targeted Therapies".

The Special Issue entitled "New Strategies in Cancer Pharmacotherapy: Development of Hormonal Antineoplastic Drugs, Cytotoxic Drugs and Targeted Therapies" was conceived with the idea of compiling information on the latest advances in the treatment of both hormone-dependent and hormone-independent cancers [...].

Usefulness of melatonin as complementary to chemotherapeutic agents at different stages of the angiogenic process.

Chemotherapeutics are sometimes administered with drugs, like antiangiogenic compounds, to increase their effectiveness. Melatonin exerts antitumoral actions through antiangiogenic actions. We studied if melatonin regulates the response of HUVECs to chemotherapeutics (docetaxel and vinorelbine). The inhibition that these agents exert on some of the processes involved in angiogenesis, such as, cell proliferation, migratory capacity or vessel formation, was enhanced by melatonin. Regarding to estrogen biosynthesis, melatonin impeded the negative effect of vinorelbine, by decreasing the activity and expression of aromatase and sulfatase. Docetaxel and vinorelbine increased the expression of VEGF-A, VEGF-B, VEGF-C, VEGFR-1, VEGFR-3, ANG1 and/or ANG-2 and melatonin inhibited these actions. Besides, melatonin prevented the positive actions that docetaxel exerts on the expression of other factors related to angiogenesis like JAG1, ANPEP, IGF-1, CXCL6, AKT1, ERK1, ERK2, MMP14 and NOS3 and neutralized the stimulating actions of vinorelbine on the expression of FIGF, FGFR3, CXCL6, CCL2, ERK1, ERK2, AKT1, NOS3 and MMP14. In CAM assay melatonin inhibited new vascularization in combination with chemotherapeutics. Melatonin further enhanced the chemotherapeutics-induced inhibition of p-AKT and p-ERK and neutralized the chemotherapeutics-caused stimulatory effect on HUVECs permeability by modifying the distribution of VE cadherin. Our results confirm that melatonin blocks proangiogenic and potentiates antiangiogenic effects induced by docetaxel and vinorelbine enhancing their antitumor effectiveness.

Melatonin Modulation of Radiation and Chemotherapeutics-induced Changes on Differentiation of Breast Fibroblasts.

Melatonin exerts oncostatic actions and sensitizes tumor cells to chemotherapeutics or radiation. In our study, we investigated the effects of docetaxel, vinorelbine, and radiation on human breast fibroblasts and its modulation by melatonin. Docetaxel or vinorelbine inhibits proliferation and stimulates the differentiation of breast preadipocytes, by increasing C/EBPα and PPARγ expression and by downregulating tumor necrosis factor α (TNFα), interleukin 6 (IL-6), and IL-11 expression. Radiation inhibits both proliferation and differentiation through the downregulation of C/EBPα and PPARγ and by stimulating TNFα expression. In addition, docetaxel and radiation decrease aromatase activity and expression by decreasing aromatase promoter II and cyclooxygenases 1 and 2 (COX-1 and COX-2) expression. Melatonin potentiates the stimulatory effect of docetaxel and vinorelbine on differentiation and their inhibitory effects on aromatase activity and expression, by increasing the stimulatory effect on C/EBPα and PPARγ expression and the downregulation of antiadipogenic cytokines and COX expression. Melatonin also counteracts the inhibitory effect of radiation on differentiation of preadipocytes, by increasing C/EBPα and PPARγ expression and by decreasing TNFα expression. Melatonin also potentiates the inhibitory effect exerted by radiation on aromatase activity and expression by increasing the downregulation of promoter II, and COX-1 and COX-2 expression. Our findings suggest that melatonin modulates regulatory effects induced by chemotherapeutic drugs or radiation on preadipocytes, which makes it a promising adjuvant for chemotherapy and radiotherapy sensibilization.

Deciphering the Molecular Basis of Melatonin Protective Effects on Breast Cells Treated with Doxorubicin: TWIST1 a Transcription Factor Involved in EMT and Metastasis, a Novel Target of Melatonin.

Melatonin mitigates cancer initiation, progression and metastasis through inhibition of both the synthesis of estrogens and the transcriptional activity of the estradiol-ER (Estrogen receptor) complex in the estrogen-dependent breast cancer cell line MCF-7. Moreover, melatonin improves the sensitivity of MCF-7 to chemotherapeutic agents and protects against their side effects. It has been described that melatonin potentiates the anti-proliferative effects of doxorubicin; however, the molecular changes involving gene expression and the activation/inhibition of intracellular signaling pathways remain largely unknown. Here we found that melatonin enhanced the anti-proliferative effect of doxorubicin in MCF-7 but not in MDA-MB-231 cells. Strikingly, doxorubicin treatment induced cell migration and invasion, and melatonin effectively counteracted these effects in MCF-7 but not in estrogen-independent MDA-MB-231 cells. Importantly, we describe for the first time the ability of melatonin to downregulate TWIST1 (Twist-related protein 1) in estrogen-dependent but not in estrogen-independent breast cancer cells. Combined with doxorubicin, melatonin inhibited the activation of p70S6K and modulated the expression of breast cancer, angiogenesis and clock genes. Moreover, melatonin regulates the levels of TWIST1-related microRNAs, such as miR-10a, miR-10b and miR-34a. Since TWIST1 plays a pivotal role in the epithelial to mesenchymal transition, acquisition of metastatic phenotype and angiogenesis, our results suggest that inhibition of TWIST1 by melatonin might be a crucial mechanism of overcoming resistance and improving the oncostatic potential of doxorubicin in estrogen-dependent breast cancer cells.

Melatonin Enhances the Usefulness of Ionizing Radiation: Involving the Regulation of Different Steps of the Angiogenic Process.

Radiotherapy is a part of cancer treatment. To improve its efficacy has been combined with radiosensitizers such as antiangiogenic agents. Among the mechanisms of the antitumor action of melatonin are antiangiogenic effects. Our goal was to investigate whether melatonin may modulate the sensitivity of endothelial cells (HUVECs) to ionizing radiation. Melatonin (1 mM) enhanced the inhibition induced by radiation on different steps of the angiogenic process, cell proliferation, migration, and tubular network formation. In relation with the activity and expression of enzymes implicated in estrogen synthesis, in co-cultures HUVECs/MCF-7, radiation down-regulated aromatase mRNA expression, aromatase endothelial-specific promoter I.7, sulfatase activity and expression and 17ß-HSD1 activity and expression and melatonin enhanced these effects. Radiation and melatonin induced a significant decrease in VEGF, ANG-1, and ANG-2 mRNA expression. In ANG-2 and VEGF mRNA expression melatonin potentiated the inhibitory effect induced by radiation. In addition, melatonin counteracted the stimulatory effect of radiation on FGFR3, TGFα, JAG1, IGF-1, and KDR mRNA expression and reduced ANPEP expression. In relation with extracellular matrix molecules, radiation increased MMP14 mRNA expression and melatonin counteracted the stimulatory effect of radiation on MMP14 mRNA expression and increased TIMP1 expression, an angiogenesis inhibitor. Melatonin also counteracted the stimulatory effect of radiation on CXCL6, CCL2, ERK1, ERK2, and AKT1 mRNA expression and increased the inhibitory effect of radiation on NOS3 expression. In CAM assay, melatonin enhanced the reduction of the vascular area induced by radiation. Melatonin potentiated the inhibitory effect on the activation of p-AKT and p-ERK exerted by radiation. Antiangiogenic effect of melatonin could be mediated through AKT and ERK pathways, proteins involved in vascular endothelial (VE) cell growth, cell proliferation, survival, migration, and angiogenesis. In addition, radiation increased endothelial cell permeability and melatonin counteracted it by regulating the internalization of VE-cadherin. Radiation has some side effects on angiogenesis that may reduce its effectiveness against tumor growth and melatonin is able to neutralize these negative actions of radiation. Additionally, melatonin potentiated radiation-induced antiangiogenic actions on several steps of the angiogenic process and enhanced its antitumor action. Our findings point to melatonin as a useful molecule as adjuvant to radiotherapy in cancer treatment.

Melatonin enhances the apoptotic effects and modulates the changes in gene expression induced by docetaxel in MCF­7 human breast cancer cells.

Results from clinical trials and multiple in vivo and in vitro studies point to melatonin as a promising adjuvant molecule with many beneficial effects when concomitantly administered with chemotherapy. Melatonin palliates side­effects and enhances the efficacy of chemotherapeutic agents. However, the mechanisms through which melatonin regulates molecular changes induced by chemotherapeutic agents remain largely unknown. In this study, we demonstrated that melatonin enhanced the anti-proliferative and apoptotic responses to low doses of docetaxel in breast cancer cells. Importantly, these effects were more potent when melatonin was added prior to docetaxel. Treatment with 1 µM docetaxel (equivalent to the therapeutic dosage) induced changes in gene expression profiles and melatonin modulated these changes. Specifically, docetaxel downregulated TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cadherin 13 (CDH13), and upregulated mucin 1 (MUC1), GATA binding protein 3 (GATA3) and c-MYC, whereas melatonin counteracted these effects. Melatonin further stimulated the expression of the pro-apoptotic BAD and BAX genes, and enhanced the inhibition of the anti-apoptotic gene BCL-2 induced by docetaxel. The findings of this study suggest that melatonin is a molecule with potential for use as an adjuvant in cancer chemotherapy, which may have implications for designing clinical trials using chemotherapeutic drugs in combination with melatonin.

Complementary actions of melatonin on angiogenic factors, the angiopoietin/Tie2 axis and VEGF, in co­cultures of human endothelial and breast cancer cells.

Melatonin exerts oncostatic activity in breast cancer through antiangiogenic actions. There, the aim of the present study was to ascertain whether melatonin modulates, in a coordinated action, angiopoietin-1 (ANG-1), ANG-2, their cognate Tie2 receptor and VEGF in co-cultures of human endothelial cells (HUVECs) and breast cancer (MCF-7) cells. To accomplish this we used co-cultures of human breast cancer cells (MCF-7) or non-malignant human mammary epithelial cells (MCF­10A) with endothelial cells (HUVECs). The presence of breast cancer cells increased HUVEC proliferation and 1 mM melatonin prevented this effect. ANG-1, ANG-2 and VEGF levels in co-culture media and mRNA expression were upregulated and Tie2 mRNA expression was downregulated in the HUVECs and MCF-7. Melatonin (1 mM) downregulated ANG-1, ANG-2 and VEGF levels in the co-culture media and mRNA expression in both types of cells and upregulated Tie2 mRNA expression in HUVECs. ANG-1, ANG-2, Tie2 and VEGF mRNA expression were not modified during HUVEC/MCF-10A co-culture. Estradiol (10 nM) increased ANG-1, ANG-2 and VEGF mRNA expression in HUVECs and melatonin (1 mM) counteracted this effect. We conclude that melatonin simultaneously coordinates downregulation of angiopoietins with a reduction in VEGF, which could be an effective therapeutic strategy for blocking tumor angiogenesis.

What is known about melatonin, chemotherapy and altered gene expression in breast cancer.

Melatonin, synthesized in and released from the pineal gland, has been demonstrated by multiple in vivo and in vitro studies to have an oncostatic role in hormone-dependent tumors. Furthermore, several clinical trials point to melatonin as a promising adjuvant molecule to be considered for cancer treatment. In the past few years, evidence of a broader spectrum of action of melatonin as an antitumor agent has arisen; thus, melatonin appears to also have therapeutic effects in several types of hormone-independent cancer, including ovarian, leukemic, pancreatic, gastric and non-small cell lung carcinoma. In the present study, the latest findings regarding melatonin molecular actions when concomitantly administered with either radiotherapy or chemotherapy in cancer were reviewed, with a particular focus on hormone-dependent breast cancer. Finally, the present study discusses which direction should be followed in the next years to definitely clarify whether or not melatonin administration could protect against non-desirable effects (such as altered gene expression and post-translational protein modifications) caused by chemotherapy or radiotherapy treatments. As treatments move towards personalized medicine, comparative gene expression profiling with and without melatonin may be a powerful tool to better understand the antitumor effects of melatonin, the pineal gland hormone.

Melatonin inhibits angiogenesis in SH-SY5Y human neuroblastoma cells by downregulation of VEGF.

Vascular endothelial growth factor (VEGF) produced from tumor cells plays a crucial role in the pathogenesis and neovascularization of neuroblastoma. Inhibition of VEGF secretion by tumor cells, as well as VEGF-regulated signaling in endothelial cells, are important to reduce the angiogenesis and growth of neuroblastoma. Since melatonin has anti-angiogenic effects in tumor cell lines, the aim of the present study was to study melatonin modulation of the pro-angiogenic effects of VEGF in neuroblastoma cells (SH-SY5Y). We used co-cultures of SH-SY5Y and endothelial cells. VEGF expression and protein levels were analyzed by quantitative RT-PCR and ELISA, respectively. Endothelial cell migration was assessed by wound-healing assay and endothelial angiogenesis by a tube formation assay. Melatonin inhibited the pro-angiogenic effects of SH-SY5Y cells. The conditioned medium collected from the neuroblastoma cells was angiogenically active and stimulated proliferation, migration and tube formation in endothelial cells. This effect was significantly counteracted by the addition of either anti-VEGF or melatonin. Melatonin inhibited VEGF expression and secretion in SH-SY5Y cells, decreasing the levels of VEGF available for endothelial cells. Melatonin has anti-angiogenic effects at different steps of the angiogenic process in SH-SY5Y neuroblastoma cells, through the downregulation of VEGF.

Melatonin enhancement of the radiosensitivity of human breast cancer cells is associated with the modulation of proteins involved in estrogen biosynthesis.

Enhancing the radiosensitivity of cancer cells is one of the most important tasks in clinical radiobiology. Endocrine therapy and radiotherapy are two cancer treatment modalities which are often given together in patients with locally-advanced breast cancer and positive hormone-receptor status. Oncostatic actions of melatonin are relevant on estrogen-dependent mammary tumors. In the present study, we wanted to evaluate the effects of the combination of ionizing radiation and melatonin on proteins involved in estrogen biosynthesis in breast cancer cells. We demonstrated a role of melatonin in mediating the sensitization of human breast cancer cells to the ionizing radiation by decreasing around 50% the activity and expression of proteins involved in the synthesis of estrogens in these cells. Thus, melatonin pretreatment before radiation reduces the amount of active estrogens at cancer cell level. Melatonin 1 nM induced a 2-fold change in p53 expression as compared to radiation alone. The regulatory action of melatonin on p53 could be a link between melatonin and its modulatory action on the sensitivity of breast cancer cells to ionizing radiation. These findings may have implications for designing clinical trials using melatonin and radiotherapy.

Melatonin affects the dynamic steady-state equilibrium of estrogen sulfates in human umbilical vein endothelial cells by regulating the balance between estrogen sulfatase and sulfotransferase.

Melatonin is known to reduce the growth of endocrine-responsive breast cancers by interacting with estrogen signaling pathways. Estrogens play an important role in breast cancer, but also in various types of tissues, including vascular tissue. Estrogen sulfatase (STS) converts inactive estrogen sulfates into active estrogens, whereas estrogen sulfotransferase (EST) sulfonates estrogens to estrogen sulfates. Therefore, STS and EST are considered to be involved in the regulation of local estrogen levels in hormone­dependent tumors and in non-pathologic tissues, such as those of the vascular system. Estrogens have a major impact on the vasculature, influencing vascular function, the expression of adhesion proteins, angiogenesis and the inflammatory state. In this study, we investigated the status of STS and EST in human umbilical vein endothelial cells (HUVECs) and the modulatory effects of melatonin. Both STS and EST were highly expressed in the HUVECs. The enzymatic activity correlated with the expression levels in these cells. Our findings also demonstrated that melatonin, at physiological concentrations, modulated the synthesis and transformation of biologically active estrogens in HUVECs through the inhibition of STS activity and expression, and the stimulation of EST activity and expression. Since melatonin decreased the STS levels and increased the EST levels, it modified the dynamic steady­state equilibrium of estrogen sulfates by increasing the inactive estrogen levels and decreasing the active estrogen levels. Therefore, melatonin may modulate the known different biological actions of estrogens in endothelial cells, as well as in estrogen-dependent tumors and non-pathologic tissues.

Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double-strand DNA break repair.

Radiation and adjuvant endocrine therapy are nowadays considered a standard treatment option after surgery in breast cancer. Melatonin exerts oncostatic actions on human breast cancer cells. In the current study, we investigated the effects of a combination of radiotherapy and melatonin on human breast cancer cells. Melatonin (1 mm, 10 µm and 1 nm) significantly inhibited the proliferation of MCF-7 cells. Radiation alone inhibited the MCF-7 cell proliferation in a dose-dependent manner. Pretreatment of breast cancer cells with melatonin 1 wk before radiation led to a significantly greater decrease of MCF-7 cell proliferation compared with radiation alone. Melatonin pretreatment before radiation also decreased G2 -M phase arrest compared with irradiation alone, with a higher percentage of cells in the G0 -G1 phase and a lower percentage of cells in S phase. Radiation alone diminished RAD51 and DNA-protein kinase (PKcs) mRNA expression, two main proteins involved in double-strand DNA break repair. Treatment with melatonin for 7 days before radiation led to a significantly greater decrease in RAD51 and DNA-PKcs mRNA expression compared with radiation alone. Our findings suggest that melatonin pretreatment before radiation sensitizes breast cancer cells to the ionizing effects of radiation by decreasing cell proliferation, inducing cell cycle arrest and downregulating proteins involved in double-strand DNA break repair. These findings may have implications for designing clinical trials using melatonin and radiotherapy.

Melatonin modulation of crosstalk among malignant epithelial, endothelial and adipose cells in breast cancer (Review).

Melatonin, the main secretory product of the pineal gland, is an oncostatic agent that reduces the growth and development of various types of tumors, particularly mammary tumors whose growth is dependent on estrogens. Previous in vivo and in vitro studies point to the hypothesis that melatonin interplays with estrogen signaling pathways at three different levels: i) an indirect mechanism, by interfering with the hypothalamic-pituitary-reproductive axis in such way that the level of plasma estrogens synthesized by the gonadal glands are downregulated; ii) a direct mechanism of the pineal gland at the cell cancer level, disrupting the activation of estradiol receptors, therefore behaving as a selective estrogen receptor modulator; and iii) by regulating the enzymes involved in the biosynthesis of estrogens in other tissues, thus behaving as a selective estrogen enzyme modulator. The intratumoral metabolism and synthesis of estrogens, as a result of the interactions of various enzymes, is more important than blood uptake to maintain mammary gland estrogen levels in menopausal females. Additionally, estrogens are considered to play an important role in the pathogenesis and development of hormone-dependent breast carcinoma. Paracrine interactions among malignant epithelial cells and proximal adipose and endothelial cells, through cytokines and growth factors produced by breast tumor cells, modulate estrogen production at the mammary tumor level and, as a consequence, the genesis and development of mammary tumors. The aim of the present review is to summarize the recent findings describing the mechanisms by which melatonin is able to modulate the crosstalk among malignant epithelial, endothelial and adipose cells in breast cancer.

Melatonin modulates aromatase activity and expression in endothelial cells.

Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real­time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. We conclude that melatonin inhibits aromatase activity and expression in HUVECs by regulating gene expression of specific aromatase promoter regions, thereby reducing the local production of estrogens.

Antiangiogenic effects of melatonin in endothelial cell cultures.

Endothelial cells represent one of the critical cellular elements in tumor microenvironment playing a crucial role in the growth and progression of cancer through controlling angiogenesis. Vascular endothelial growth factor (VEGF) produced from tumor cells is essential for the expansion of breast cancer and may function in both paracrine and autocrine manners to promote proliferation, growth, survival and migration of endothelial cells. Since melatonin regulates tumor microenvironment by decreasing the secretion of VEGF by malignant epithelial cells and also regulates VEGF expression in human breast cancer cells, the aim of the present study was to investigate the anti-angiogenic activity of melatonin against the pro-angiogenic effects of breast cancer cells. In this work, we demonstrate that melatonin strongly inhibited the proliferation as well as invasion/migration of human umbilical vein endothelial cells (HUVECs). Melatonin disrupted tube formation and counteracted the VEGF-stimulated tubular network formation by HUVEC. In addition, conditioned media collected from human breast cancer cells were angiogenically active and stimulated tubule length formation and this effect was significantly counteracted by the addition of anti-VEGF or melatonin. Melatonin also disintegrated preformed capillary network. All these findings demonstrate that melatonin may play a role in the paracrine interactions that take place between malignant epithelial cells and proximal endothelial cells. Melatonin could be important in reducing endothelial cell proliferation, invasion, migration and tube formation, through a downregulatory action on VEGF. Taken together, our findings suggest that melatonin could potentially be beneficial as an antiangiogenic agent in breast cancer with possible future clinical applications.

Regulation of vascular endothelial growth factor by melatonin in human breast cancer cells.

Melatonin exerts oncostatic effects on breast cancer by interfering with the estrogen-signaling pathways. Melatonin reduces estrogen biosynthesis in human breast cancer cells, surrounding fibroblasts and peritumoral endothelial cells by regulating cytokines that influence tumor microenvironment. This hormone also exerts antiangiogenic activity in tumoral tissue. In this work, our objective was to study the role of melatonin on the regulation of the vascular endothelial growth factor (VEGF) in breast cancer cells. To accomplish this, we cocultured human breast cancer cells (MCF-7) with human umbilical vein endothelial cells (HUVECs). VEGF added to the cultures stimulated the proliferation of HUVECs and melatonin (1 mM) counteracted this effect. Melatonin reduced VEGF production and VEGF mRNA expression in MCF-7 cells. MCF-7 cells cocultured with HUVECs stimulated the endothelial cells proliferation and increased VEGF levels in the culture media. Melatonin counteracted both stimulatory effects on HUVECs proliferation and on VEGF protein levels in the coculture media. Conditioned media from MCF-7 cells increased HUVECs proliferation, and this effect was significantly counteracted by anti-VEGF and 1 mM melatonin. All these findings suggest that melatonin may play a role in the paracrine interactions between malignant epithelial cells and proximal endothelial cells through a downregulatory action on VEGF expression in human breast cancer cells, which decrease the levels of VEGF around endothelial cells. Lower levels of VEGF could be important in reducing the number of estrogen-producing cells proximal to malignant cells as well as decreasing tumoral angiogenesis.