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

Abstract

Abstract 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). Among other proteins evaluated, melatonin treatment under hypoxia resulted in a decrease of VEGF-C, VEGFR2, VEGFR3, matrix metalloproteinase 9 (MMP-9) and angiogenin in MCF-7 cell line (p < 0.05). For MDA-MB-231, a significant reduction was observed for VEGFR2 protein, epidermal growth receptor (EGFR), and angiogenin (p <0.05). In vivo study, mice treated with melatonin showed reduced tumor growth compared to control animals (144.90 ± 38.38 mm3 vs 282.00 ± 88.53 mm3, p < 0.05). Furthermore, one animal showed tumor regression during melatonin treatment (Day 7 = 27.38 mm3, 8.79 mm3 Day = 14, mm3 Day 21 = 4.8). SPECT detect less radioactivity of Tc-99m-HYNIC-VEGF-c and consequent reduced expression of VEGFR2/3 in tumors treated with melatonin (150,46 ± 17,06 % vs 183,55 ± 20,92 %) but statistical significance was not achieved (p > 0.05). The reduction of VEGFR2 in tumors treated with melatonin was confirmed by immunohistochemistry (p <0.05), as well as the reduction of micro-vessel density (vWF) and cell proliferation (ki-67) (p < 0.05). There was no change in the expression of the other proteins evaluated, however a significant increase in EGFR and Insulin-like growth factor (IGF-I) was observed in tumors treated with melatonin (p< 0.05). Taken together, our results showed that melatonin has an important anti-angiogenic effect, suggesting its potential therapeutic action in breast cancer. Support: FAPESP. Citation Format: Debora C Zuccari, Bruna V Jardim-Perassi, Mateus R Lourenço, Gabriel M Doho, Gabriela B Gelaleti, Lívia 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.