Lívia Carvalho Ferreira

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.

Glutathione and glutathione peroxidase expression in breast cancer: An immunohistochemical and molecular study

The use of prognostic markers for breast cancer allows therapeutic strategies to be defined more efficiently. The expression of glutathione (GSH) and glutathione peroxidase (GPX) in tumor cells has been evaluated as a predictor of prognosis and response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results showed that high GSH expression was associated with tumors negative for the estrogen receptor (ER) (P<0.05), and GPX expression was associated with tumors negative for the progesterone receptor (PR) and patient mortality. Focusing on the 37 patients who received adjuvant chemotherapy/radiotherapy (Group I), high expression of GPX was associated with a high rate of patient mortality (P<0.05). The 19 patients who received only adjuvant chemotherapy (Group II) showed high expression of GSH in relation to metastasis (P<0.05). In addition, high levels of GPX expression were significantly associated with a shorter overall survival (P<0.05). To confirm this, the expression of precursor genes of GSH [glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS)] and the GPX gene was analyzed using quantitative PCR in cultured neoplastic mammary cells treated with doxorubicin. Doxorubicin treatment was able to eliminate tumor cells without alterations in the gene expression of GSS, but led to underexpression of the GCLC and GPX genes. Our results suggest that high levels of GPX may be related to the development of resistance to chemotherapy in these tumors, response to treatment and the clinical course of the breast cancer patients.

An immunohistochemical study of interleukin-8 (IL-8) in breast cancer

The use of prognostic markers for breast cancer is important for routine diagnosis and research. Interleukin-8 is a chemotactic cytokine produced by several cell types in response to inflammation, however, its expression, regulation and function are poorly understood. Recent studies have associated angiogenesis and inflammatory processes with tumor malignancy. The present study investigated the correlation between interleukin-8 expression and breast cancer prognosis. Interleukin-8 expression was assessed in 72 women with mammary neoplasia by immunohistochemistry and the results were statistically correlated with clinical-pathological findings. There was an inverse correlation between interleukin-8 expression and metastasis (p=0.03) and/or local recurrence (p=0.02). In the patient group that received post-surgery chemotherapy and radiotherapy, a lower interleukin-8 expression was found in those women that showed local recurrence (p=0.01). Multivariate logistic regression showed estrogen receptor negativity, progesterone positivity and metastasis with increased risk of death (p<0.05). The data reflect the complexity of the role of interleukin-8 in tumor microenvironment and support its classification as a possible prognostic marker, although more studies are necessary for its inclusion in clinical practice.

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.

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.

Abstract A03: Effectiveness of melatonin on the epithelial mesenchymal transition after induction with transforming growth factor beta (TGF-β)

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 Citation Format: Naiane do Nascimento Goncalves, Livia 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

Abstract A02: Effect of curcumin on the tumor growth and angiogenesis of breast cancer

Background: Angiogenesis plays an important role in the pathogenesis of breast cancer. For growth, tumor requires the formation of new blood vessels, which are stimulated by angiogenic factors to initiate the proliferation of endothelial cells. The major contributors to angiogenesis are the vascular endothelial growth factor (VEGF) and its receptors, which promote cell proliferation, migration, permeability and survival. Controlling tumor-associated angiogenesis and metastasis are promising tactics in limiting cancer progression. Curcumin, a polyphenolic compound derived from the spice plant Curcuma longa, displays multiple actions on solid tumours including anti-angiogenic effects. In this study we evaluated the benefits of curcumin treatment on tumor growth and angiogenesis in breast cancer. Methods: MDA-MB-231 breast cancer cell line was cultured and cell viability was measured by MTT assay after incubation with different concentrations of curcumin (25 uM, 50 uM, 100 uM). We performed an in vivo study implanting cells in athymic nude mice. Mice were treated with 300 mg/kg of curcumin (n = 5) or vehicle (n = 8) daily, starting in the same day as tumor implantation and continued for 21 days. Tumors were measured with a digital caliper on day 7, 14 and 21 and the animals underwent SPECT scanning with Tc-99m tagged VEGF-c to detect in vivo angiogenesis (expression of VEGFR2/3). In addition, the markers of angiogenesis (VEGF-A, VEGF-C, VEGFR2, VEGFR3 and von Willebrand Factor (vWF)) and cell proliferation (Ki-67) were evaluated by immunohistochemistry in mammary tumor tissues. Results: Curcumin treatment in vitro was able to significantly decrease cell viability, after 4 hours of exposure, maintaining its efficacy at higher doses after 24 hours exposure (p 0.05). However, decrease of VEGF-A, VEGF-C and VEGFR2 in curcumin treated mice was confirmed by immunohistochemistry (p Citation Format: Livia Carvalho Ferreira, Ali Syed Arbab, Bruna Victorasso Jardim-Perassi, Thaiz Ferraz Borin, Naiane Nascimento Goncalves, Ravi Sankara Varma Nadimpalli, Debora Aparecida Pires de Campos Zuccari. Effect of curcumin on the tumor growth and angiogenesis of breast cancer. [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 A02. doi:10.1158/1538-7445.CHTME14-A02