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Mammary neoplasias are the most common tumors observed in female dogs. Identification of these tumors is valuable in order to identify beneficial therapeutic agents as alternative treatments for this tumor type. Oral administration of melatonin appears to exert an oncostatic effect on mammary neoplasia and may have a possible mechanism of action through its interaction with estrogen receptors on epithelial cells. Hence, we analyzed the potential therapeutic value of melatonin in tumors that are estrogen-dependent or -independent, and established a relationship of its action with the expression of the melatonin receptors MT1 and MT2. Furthermore, we analyzed the rate of cell proliferation and apoptosis after treatment with melatonin. Cell cultures were performed using 10 canine mammary tumor fragments and were divided into estrogen receptor (ER)-positive and ER-negative tumors. The results showed that both ER-positive and ER-negative tumors had decreased cell viability and proliferation after treatment with melatonin (p<0.05), although treatment was more effective in the ER-positive tumors. Analysis of the relative expression of the MT1 and MT2 genes by quantitative PCR was performed and the data were compared with the expression of ER in 24 canine mammary tumors and the cellular response to melatonin in 10 samples. MT1 was overexpressed in ER-positive tumors (p<0.05), whereas MT2 was not expressed. Furthermore, melatonin treatment in ER-positive tumors showed an efficient oncostatic effect by inhibiting cell viability and proliferation and inducing apoptosis. These results suggest that melatonin decreased neoplastic mammary cell proliferation and viability and induced apoptosis, with greater efficacy in ER-positive tumors that have a high expression of melatonin receptor MT1. This is a strong evidence for the use of melatonin as a therapeutic agent for estrogen-dependent canine mammary tumors.

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The metastasis occurrence, an important prognostic factor, depends on peculiarities such as cellular invasiveness and cell migration, mechanisms controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). An increased expression of this protein promotes tumor growth and metastasis, a mechanism which can be restricted by the use of the effector's inhibitors. Melatonin, a hormone secreted by the pineal gland, has shown oncostatic action and anti-metastatic effects and can modulate the ROCK-1expression. The objective of this study was to investigate the anti-metastatic response mediated by ROCK-1 and through the action of melatonin and its specific inhibitor (Y27632) in vitro and in vivobreast cancer models. Cells from metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer lines were treated with melatonin and Y27632. Cell viability was verified by MTT assay, cell migration/invasion assays in Boyden chamber, ROCK-1 protein and gene expression by western blot and quantitative real time PCR, respectively. In addition, the in vivometastasis study was performed using female athymic nude mice induced by injection of 2x105 MDA-MB-231 viable cells by tail veinfor lung metastasis and byintracardiac for bone metastasis, during 3 weeks. The animals were treated with melatonin and Y27632 for 2 weeks. The metastasis developments were evaluated by single photon emission computed tomography (SPECT). Treatment with melatonin reduced cell viability and migration of both cell lines (p&lt;0.05). The use of melatonin and Y27632, in association or not, decreased the ROCK-1 protein expression in metastatic cells, not significantly altering its expression in the non-metastatic line (p&gt;0.05). An statistically significant reduction of ROCK-1 gene expression was observed in all treatment groups (p&lt;0.05). ROCK-1 downexpression was more efficient in the group with associated treatments for both lines (p &lt; 0.05). In vivoSPECT images showed multiple foci in the lungs (on Tc-99-tetrofomin images) and in the vertebrae (on Tc-99-MDP images). The numbers of "hot" spots were significantly higher in lung metastasis of control animals compared to treated groups. Semi quantitative analysis showed significantly lower activity in animal lungs that received treatment (p&lt;0.05). The bone metastasis did not show difference between control and treatment groups. Melatonin and Y27632 are effective drugs of metastatic breast cancerin vitro treatment, confirming their effects in decreasing cell viability, invasion, migration and protein expression of ROCK-1 in these cells. In vivo, melatonin and Y27632 treatments seem to be effective reducing lung metastasis but not bone metastasis. Melatonin, in particular, appears to be more effective when combined to ROCK-1 inhibitor. Support: FAPESP. Citation Format: Debora C Zuccari, Thaiz F Borin, Ali S Arbab, Lívia C Ferreira, Marina G Moschetta, Gustavo R Martins, Larissa B Maschio, Vanessa A Fabri, Verena B Coimbra. Melatonin's inhibitory effect on breast cancer metastasis mediated by ROCK-1 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-20.

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Background: The mammary tumors are the most frequent in women in Brazil and worldwide. The high mortality rate in breast cancer is mainly due to invasiveness and metastatic. The metastatic process involves a mechanism called epithelial-mesenchymal transition (EMT). The EMT mechanism can be induced by growth factors such as transforming growth factor beta (TGF-β), which alters the epithelial phenotype (E-cadherin) to a mesenchymal phenotype (n-caderina/vimentina). Studies have shown significant importance of the epithelial mesenchymal transition in breast cancer, which makes the study of new therapeutic agents that act in the process, very important. Melatonin, a hormone naturally secreted by the pineal gland, has shown oncostatic properties. For these reasons, the aim of this study was to evaluate the anti-metastatic potential of melatonin by the epithelial mesenchymal transition induced by TGF-β in breast cancer cell line. Methods: The breast cancer cell line MCF-7 was cultured at 37 ° C and 5% CO2 in DMEM High Glucose culture medium. Once established, the best concentration of TGF-β (0.5 ng / mL) to induce epithelial mesenchymal transition the cells were divided into four groups (Group I - Control, Group II - TGF-β, Group III - Melatonin and Group IV - TGF- β + Melatonin) to evaluate the protein expression of E-cadherin, N-cadherin and vimentin by immunocytochemistry. Quantitation of immunoreactivity was performed by densitometry (ImageJ software). The results were compared by ANOVA followed by Bonferroni. Results: The results showed that there were statistically significant increased protein e-cadherin in cells treated with melatonin (P &lt; 0.05) when compared to cells treated with TGF-β. Furthermore, immunostaining of proteins N-cadherin and vimentin was lower in cells treated with TGF-B + melatonin (P &lt; 0.05) compared to cells treated only with TGF-β. Conclusion: Melatonin interfered in the expression of proteins related to epithelial mesenchymal transition after induction with TGF-B which may suggested a possible anti-metastatic role of this hormone. Citation Format: Naiane do Nascimento Gonçalves, Lívia Carvalho Ferreira, Juliana Ramos Lopes, Larissa Bazela Maschio, Marina Gobbe Moschetta, Gabriela Bottaro Gelaleti, Gustavo Rodrigues Martins, Debora Aparecida Pires de Campos Zuccari. Effectiveness of melatonin on the epithelial mesenchymal transition after induction with transforming growth factor beta (TGF-β). [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A03. doi:10.1158/1538-7445.CHTME14-A03