Bruna Victorasso Jardim-Perassi

Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho‐associated kinase protein (ROCK‐1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK‐1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti‐angiogenic effects and can modulate ROCK‐1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK‐1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK‐1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X‐ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK‐1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK‐1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of ‘hot’ spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK‐1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK‐1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

Molecular Markers of Angiogenesis and Metastasis in Lines of Oral Carcinoma after Treatment with Melatonin

BACKGROUND Oral cancer is the most common type of head and neck cancer and its high rate of mortality and morbidity is closely related to the processes of angiogenesis and tumor metastasis. The overexpression of the pro-angiogenic genes, HIF-1α and VEGF, and pro-metastatic gene, ROCK-1, are associated with unfavorable prognosis in oral carcinoma. Melatonin has oncostatic, antiangiogenic and antimetastatic properties in several types of neoplasms, although its relationship with oral cancer has been little explored. This study aims to analyze the expression of the genes HIF-1α, VEGF and ROCK-1 in cell lines of squamous cell carcinoma of the tongue, after treatment with melatonin. METHODS SCC9 and SCC25 cells were cultured and cell viability was assessed by MTT assay, after treatment with 100 μM of CoCl2 to induce hypoxia and with melatonin at different concentrations. The analysis of quantitative RT-PCR and the immunocytochemical analysis were performed to verify the action of melatonin under conditions of normoxia and hypoxia, on gene and protein expression of HIF-1α, VEGF and ROCK-1. RESULTS The MTT assay showed a decrease in cell viability in both cell lines, after the treatment with melatonin. The analysis of quantitative RT-PCR indicated an inhibition of the expression of the pro-angiogenic genes HIF-1α (P < 0.001) and VEGF (P < 0.001) under hypoxic conditions, and of the pro-metastatic gene ROCK-1 (P < 0.0001) in the cell line SCC9, after treatment with 1 mM of melatonin. In the immunocytochemical analysis, there was a positive correlation with gene expression data, validating the quantitative RT-PCR results for cell line SCC9. Treatment with melatonin did not demonstrate inhibition of the expression of genes HIF-1α, VEGF and ROCK-1 in line SCC25, which has different molecular characteristics and greater degree of malignancy when compared to the line SCC9. CONCLUSION Melatonin affects cell viability in the SCC9 and SCC25 lines and inhibits the expression of the genes HIF-1α, VEGF and ROCK-1 in SCC9 line. Additional studies may confirm the potential therapeutic effect of melatonin in some subtypes of oral carcinoma.

HET0016, a selective inhibitor of 20-HETE synthesis, decreases pro-angiogenic factors and inhibits growth of triple negative breast cancer in mice.

A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals’ right flank and randomly assigned to early (1 and 2), starting treatments on day 0, or delayed groups (3 and 4) on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg) treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05). Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found in vitro at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05) compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day’s data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.

Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines.

Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p < .05). This treatment also decreased both gene and protein expression of HIF-1α and VEGF-A under hypoxic conditions (p < .05). Among the proteins evaluated by protein array, melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p < .05). Taken together, these results showed that melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions.

Abstract P6-04-01: Melatonin on angiogenesis in breast cancer

The rapid tumor growth results in hypoxia on tumor microenvironment, leading to a cascade of events that induce angiogenesis and subsequent cancer progression. Thus, the identification of therapeutic agents that can inhibit angiogenesis is essential for the control of tumor progression. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. The aim of this study was to evaluate the effectiveness of melatonin treatment on angiogenesis in breast cancer, in the in vitro and in vivo studies. In the in vitro study, breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with melatonin under cobalt chloride (CoCl2)-induced hypoxic conditions. Cell viability was measured by MTT assay, the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF-A) was assessed by real-time PCR and immunocytochemistry. Additionally, other proteins involved in angiogenesis were evaluated by the Protein Array. In the in vivo study, the MDA-MB-231 cells were implanted in athymic nude mice, which were treated with melatonin (40 mg/kg) for 21 days. The tumor was measured weekly and evaluation of angiogenesis was performed by single-photon emission computed tomography (SPECT) with Tc-99m-HYNIC-VEGF-c, which is specific for VEGF receptors (VEGFR2/VEGF3). Moreover, VEGFR2, VEGFR3, von Willebrand factor (vWF) and cell proliferation marker (Ki-67) were evaluated in tumor tissue by immunohistochemistry, and other angiogenic proteins by Protein Array. Results from the in vitro study showed that 1 mM of melatonin under hypoxic conditions (200 µM CoCl2) led to decreased cell viability, protein levels of HIF-1α and gene and protein expression of VEGF-A in both cell lines (p 0.05). The reduction of VEGFR2 in tumors treated with melatonin was confirmed by immunohistochemistry (p Support: FAPESP. Citation Format: Debora C Zuccari, Bruna V Jardim-Perassi, Mateus R Lourenco, Gabriel M Doho, Gabriela B Gelaleti, Livia C Ferreira, Thaiz F Borin, Marina G Moschetta. Melatonin on angiogenesis in breast cancer [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 P6-04-01.

Immunoexpression of ROCK-1 and MMP-9 as prognostic markers in breast cancer

Breast cancer is the most common tumor in women and it has high mortality mainly due to the occurrence of tumor metastasis. Both the processes of cell migration and anchorage to the substrate are essential for the development of metastasis. These processes occur by rearrangements of the actin cytoskeleton, regulated by Rho-associated protein kinase 1 (ROCK-1). The degradation of the extracellular matrix, influenced by metalloproteinase 9 (MMP-9) also exerts greater cell invasiveness. The present study evaluated the ROCK-1 and MMP-9 proteins using an immunohistochemical method through the selection of invasive ductal breast carcinoma. The protein expression was correlated to clinicopathological parameters and overall survival of the patients. High expression of the ROCK-1 protein was correlated statistically to the status of lymph nodes (p=0.007) and showed variable expression in different clinical stages of the tumor. MMP-9 showed a strong immunostaining in patients with metastasis that had died, whereas there was no marker in normal breast tissues. In addition, 46.6% of patients classified as poor prognosis showed high expression of ROCK-1 and MMP-9 protein and another 40.0% just showed high expression of MMP-9. Thus, the differential expression of ROCK-1 and MMP-9 proteins suggests their potential use as prognostic markers in breast cancer.

Melatonin Regulates Angiogenic and Inflammatory Proteins in MDA-MB-231 Cell Line and in Co-culture with Cancer-associated Fibroblasts.

BACKGROUND Cancer-associated fibroblast (CAFs) are the most abundant cells in the tumor microenvironment, able to secrete growth factors and act on tumor progression. Melatonin is associated with several mechanisms of action with oncostatics and oncoprotectors effects, and also participate in the reduction of synthesis of surrounding fibroblasts and endothelial cells in breast cancer. OBJECTIVE The objectives of this study were to determine the effectiveness of melatonin in cell viability and expression of proteins involved in angiogenesis and inflammation in triplenegative mammary tumor cell line (MDA-MB-231) and in co-culture with CAFs. METHOD Cell viability was measured by MTT assay and the protein expression was evaluated by Membrane Antibody Array after melatonin treatment. RESULTS Melatonin treatment (1 mM) for 48 hours reduced the cell viability of MDA-MB-231, CAFs and co-culture (p < 0.05). The semi-quantitative protein analysis showed that when monoculture of tumor cells were compared with co-culture of CAFs, there was a regulation of angiogenic and inflammatory proteins (p < 0.05). Melatonin treatment also leads a differential expression of angiogenic and inflammatory proteins in both monoculture and co-culture of tumor cells and CAFs (p < 0.05). CONCLUSION The influence of CAFs under the tumor microenvironment was confirmed, increasing the malignancy of the tumor. In addition, melatonin is effective in both monoculture and co-culture, regulating angiogenic and inflammatory proteins that contribute to tumor progression. This study show an overview of melatonin ability in regulating angiogenic and inflammatory proteins, and opens the way for exploration of each individual protein in further studies.

Evaluation of melatonin treatment in primary culture of canine mammary tumors

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.

Efficacy of melatonin, IL-25 and siIL-17B in tumorigenesis-associated properties of breast cancer cell lines

ABSTRACT Mammary tumorigenesis can be modulated by melatonin, which has oncostatic action mediated by multiple mechanisms, including the inhibition of the activity of transcription factors such as NF‐&kgr;B and modulation of interleukins (ILs) expression. IL‐25 is an active cytokine that induces apoptosis in tumor cells due to differential expression of its receptor (IL‐17RB). IL‐17B competes with IL‐25 for binding to IL‐17RB in tumor cells, promoting tumorigenesis. This study purpose is to address the possibility of engaging IL‐25/IL‐17RB signaling to enhance the effect of melatonin on breast cancer cells. Breast cancer cell lines were cultured monolayers and 3D structures and treated with melatonin, IL‐25, siIL‐17B, each alone or in combination. Cell viability, gene and protein expression of caspase‐3, cleaved caspase‐3 and VEGF‐A were performed by qPCR and immunofluorescence. In addition, an apoptosis membrane array was performed in metastatic cells. Treatments with melatonin and IL‐25 significantly reduced tumor cells viability at 1 mM and 1 ng/mL, respectively, but did not alter cell viability of a non‐tumorigenic epithelial cell line (MCF‐10A). All treatments, alone and combined, significantly increased cleaved caspase‐3 in tumor cells grown as monolayers and 3D structures (p < 0.05). Semi‐quantitative analysis of apoptosis pathway proteins showed an increase of CYTO‐C, DR6, IGFBP‐3, IGFBP‐5, IGFPB‐6, IGF‐1, IGF‐1R, Livin, P21, P53, TNFRII, XIAP and hTRA proteins and reduction of caspase‐3 (p < 0.05) after melatonin treatment. All treatments reduced VEGF‐A protein expression in tumor cells (p < 0.05). Our results suggest therapeutic potential, with oncostatic effectiveness, pro‐apoptotic and anti‐angiogenic properties for melatonin and IL‐25‐driven signaling in breast cancer cells.