Naiane N Gonçalves

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.

Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44+/CD24low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44+/CD24low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.

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

Evaluation of the anti-angiogenic action of melatonin in breast cancer

Background Once a tumor lesion exceeds a few millimeters in diameter, hypoxia triggers a cascade of events to allow angiogenesis and tumor progression. As angiogenesis is essential for tumor growth and metastasis, controlling tumor-associated angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been suggested to inhibit angiogenesis in cancers, although this effect has not been described in breast cancer. We evaluated the effects of melatonin treatment on angiogenesis in breast cancer.

Response of angiogenic factors to the treatment with melatonin in breast cancer cell lines.

120 Background: Tumor growth and metastasis are the leading cause of death in breast cancer patients. The tumor growth requires neoangiogenesis, stimulated by vascular endothelial growth factor (VEGF) expressed under control of hypoxia-inducible factor-1α (HIF-1α). Melatonin, a natural hormone has been suggested as a new therapeutic agent to inhibit angiogenesis factors in several types of cancers. We evaluated the HIF-1α and VEGF expression in breast cancer cell line after hypoxia and treatment with melatonin. METHODS Breast cancer cell lines ER-positive (MCF-7) and ER-negative (MDA-MB-231) were cultured in DMEM at 37°C in 5% CO2. The cells were treated with CoCl2 to mimic hypoxia, and then treated with melatonin. Cell viability was measured by MTT assay with five melatonin concentrations (0.5mM, 1mM, 2mM, 5mM, 10mM). For the pharmacological concentration of melatonin (1mM) the protein and gene expression of HIF-1α and VEGF were detected by immunocytochemistry and real time PCR, respectively. RESULTS There was a significantly decrease in MCF-7 cells viability after treatment with all concentrations of melatonin and in MDA-MB-231 cells just with 1mM (p<0.05). The melatonin treatment did not alter the protein and gene expression of HIF-1α in MCF-7 cells (p>0.05), however, it significantly decreased the VEGF protein expression (p<0.05). In MDA-MB-231 cells there was significant decreased in the HIF-1α protein expression (p<0.05). CONCLUSIONS Our results showed that 1mM of melatonin decreased the cell viability and HIF-1α protein expression in ER-negative cells, and VEGF protein expression in ER-positive cells. This study is the first that evaluated the expression of these angiogenic factors in breast cancer cells after treatment with melatonin that can be a potential therapeutic agent. Thus, we are performing in vivo study to confirm the potential effectiveness of melatonin in the control of angiogenesis in breast cancer.

Prognostic value of vascular endothelial growth factor and hypoxia-inducible factor 1α in canine malignant mammary tumors

Mammary tumors are the most common type of tumor in dogs, with approximately half of these tumors being malignant. Hypoxia, characterized by oxygen levels below normal, is a known adverse factor to cancer treatment. The hypoxia-inducible factor 1α (HIF-1α) is a central regulator of the pathophysiological response of mammalian cells to low oxygen levels. HIF-1α activates the transcription of vascular endothelial growth factor (VEGF), which in turn promotes angiogenesis through its ability to stimulate the growth, migration and invasion of endothelial cells to form new blood vessels, contributing to tumor progression. In this study, we evaluated the serum concentration and gene expression of VEGF and HIF-1α linking them with clinicopathological parameters and survival of dogs with mammary tumors in order to infer the possible prognostic value of these factors. We collected blood and tumor fragments of 24 female dogs with malignant mammary tumors (study group) and 26 non-affected female dogs (control group) to verify the gene expression of VEGF and HIF-1α by quantitative real-time PCR (qPCR) and the serum levels by ELISA (enzyme-linked immunosorbent). The results showed high serum levels of VEGF in the study group and its correlation between abundant vascularization, lymph node involvement, metastasis, death rate and low survival (p<0.05). The serum percentage of HIF-1α in female dogs with mammary neoplasia was lower than that in the control group and higher in female dogs with tumor metastasis and history of tumor recurrence (p<0.05). Regarding gene expression, there was a gene overexpression of VEGFA in female dogs with poor outcome, in contrast to the gene underexpression of HIF-1A. Taken together, these results suggested that VEGF is important in tumor progression and can be used as a potential prognostic marker in the clinic and may be useful in predicting tumor progression in dogs with mammary neoplasia.

Abstract A10: Simultaneous block of angiogenesis through PI3K-AMPK/AKT/mTOR signaling pathways after treatment with metformin and LY294002 in canine mammary tumor cell line

Introduction: The PI3K-AMPK / Akt / mTOR signaling pathways are the most important signaling pathways involved in the angiogenesis process, which is regulated by numerous factors, being the most important the hypoxia-inducible factor - 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Metformin, the prescribed drug for patients with metabolic syndrome, has been suggested as a new therapeutic agent with potential anti-neoplastic action capable to inhibit cell growth by AMPK signaling pathway. In the same way, the LY294002, an important inhibitor of PI3K / Akt / mTOR signaling pathway, has anti- angiogenic properties able to decrease the release of growth factors, such as VEGF. In this context, the aim of this study was to evaluate the effectiveness of treatment with metformin and LY294002 in angiogenesis process. Methods: Canine mammary tumor cell line ER-positive (CMT-U229) was cultured in HAM-F12 culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with varying concentrations of metformin and LY294002. Once stablished the concentration of metformin (1mM) and LY294002 (5µM) treatment, the protein and gene expression of HIF-1α and VEGF were detected by immunocytochemistry and real time PCR, respectively. The immunostaining was quantified by optical densitometry technique, slides were analyzed and photographed in Nikon Eclipse E200 microscope at 40X objective, and proteins were quantified by the ImageJ software analysis. For real time PCR, the relative expression of the genes of interest was determined by DataAssist v3.0 software, using ΔΔCt method. Results: There was a significantly decrease of cell viability after treatment with all concentrations (1mM, 2mM, 3mM and 5mM) of metformin, 5µM and 10µM of LY294002 (p Financial support: FAPESP Citation Format: Marina Gobbe Moschetta, Larissa Bazela Maschio, Bruna Victorasso Jardim-Perassi, Gabriela Bottaro Gelaleti, Camila Leonel, Livia Carvalho Ferreira, Naiane Nascimento Goncalves, Debora Aparecida Pires De Campos Zuccari. Simultaneous block of angiogenesis through PI3K-AMPK/AKT/mTOR signaling pathways after treatment with metformin and LY294002 in canine mammary tumor cell line. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A10.

Evaluation of interleukins 8 and 12, CA 15-3 and free circulating DNA as prognostic markers in dogs with mammary tumors

Background Mammary tumors of dogs are an excellent model to investigate the clinical and pathological diagnosis and prognosis of cancer. Interleukins play a key role in cancer, particularly interleukin-8, which has tumorigenic and pro-angiogenic properties and interleukin-12, with anti-metastatic and angiogenic properties. The carbohydrate antigen tumor marker (CA 15-3) has important clinical significance in the monitoring of patients with breast cancer and, in addition, free circulating DNA has been considered a candidate biomarker for cancer. The aim of this study was to measure serum levels of interleukin-8, 12, CA 15-3 and to estimate the number of copies of CAN SINE sequences to correlate with clinicopathological parameters and survival.

Serum and molecular assessment of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α) in canine mammary tumors

Background Due to the many similarities shared by humans and dogs, canine mammary tumors are an excellent model to comprehend various aspects of mammary neoplasias. The tumor cells have the ability to promote changes in their functionality in order to survive. In situations of hypoxia, tumor cells produce and release pro and antiangiogenic factors that regulate the process of angiogenesis. The hypoxia-inducible factor 1 (HIF -1) is a central regulator of pathophysiological response of mammalian cells to low oxygen levels, able to activate transcription of the gene that promotes the induction of vascular endothelial growth factor (VEGF),which in turn, promotes angiogenesis through its ability to stimulate growth, migration and invasion of endothelial cells, leading to the formation of new blood vessels and subsequent tumor growth. In this context, the aim of this study was to measure serum levels of VEGF and HIF-1 and to relate with clinicopathological parameters and survival.