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.

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.

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: A Molecule for Reducing Breast Cancer Risk.

The objective of this article is to review the basis supporting the usefulness of melatonin as an adjuvant therapy for breast cancer (BC) prevention in several groups of individuals at high risk for this disease. Melatonin, as a result of its antiestrogenic and antioxidant properties, as well as its ability to improve the efficacy and reduce the side effects of conventional antiestrogens, could safely be associated with the antiestrogenic drugs presently in use. In individuals at risk of BC due to night shift work, the light-induced inhibition of melatonin secretion, with the consequent loss of its antiestrogenic effects, would be countered by administering this neurohormone. BC risk from exposure to metalloestrogens, such as cadmium, could be treated with melatonin supplements to individuals at risk of BC due to exposure to this xenoestrogen. The BC risk related to obesity may be reduced by melatonin which decrease body fat mass, inhibits the enhanced aromatase expression in obese women, increases adiponectin secretion, counteracts the oncogenic effects of elevated concentrations of leptin; and decreases blood glucose levels and insulin resistance. Despite compelling experimental evidence of melatonin's oncostatic actions being susceptible to lowering BC risk, there is still a paucity of clinical trials focused on this subject.

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 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.

Intake of cooked tomato sauce preserves coronary endothelial function and improves apolipoprotein A-I and apolipoprotein J protein profile in high-density lipoproteins.

Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remains unknown whether tomato intake exerts protective effects on the vasculature. The aim of this study was to determine whether medium-term supplementation with cooked tomato sauce (CTS) Mediterranean style (sofrito) attenuates diet-induced coronary endothelial dysfunction in an animal model with clinical impact and explore the mechanisms behind the effects. Pigs (N = 18) were fed a 10-day hypercholesterolemic diet. Half of the animals were given a supplement of 100 g/d of CTS (21.5 mg lycopene per day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide synthase [eNOS] inhibitor) were measured using flow Doppler. In the coronary arteries, we investigated eNOS gene expression and activation, monocyte chemoattractant protein 1 (MCP-1) expression, and oxidative DNA damage. In the circulation, we investigated lipoprotein resistance to oxidation and the differential proteomic protein profile. In dyslipidemic animals, CTS intake prevented diet-induced impairment of receptor-operated and nonreceptor-operated endothelial-dependent coronary vasodilation. These beneficial effects were associated with enhanced eNOS transcription and activation and diminished DNA damage in the coronary arteries. CTS-fed animals showed lower lipid peroxidation, higher high-density lipoprotein (HDL) antioxidant potential and plasma lycopene levels of 0.16 mg/L. Interestingly, improved HDL functionality was associated with protein profile changes in apolipoprotein A-I and apolipoprotein J. Lipids levels and MCP-1 expression were not affected by CTS. We report that CTS intake protects against low-density lipoprotein-induced coronary endothelial dysfunction by reducing oxidative damage, enhancing eNOS expression and activity, and improving HDL functionality.