Camila Leonel

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.

Interleukin-8 as a prognostic serum marker in canine mammary gland neoplasias

Mammary gland tumors in female dogs are an excellent model for the clinic-pathological, diagnostic and prognostic investigation of mammary neoplasias. Prognostic and predictive markers are effective in research and routine diagnosis. Interleukins play a fundamental role in cancer, with a particular function in tumor growth, invasion and metastatic potential. Interleukin-8 (IL-8) is known to possess tumorigenic and pro-angiogenic properties, and its overexpression is seen in a number of human tumors. IL-8 serum levels were determined and correlated with the clinic-pathological features and clinical evolution of mammary gland neoplasias in female dogs. IL-8 was measured by an immunoenzymatic assay in 30 female dogs with mammary neoplasias within a 12 month follow-up and in 50 control animals. The correlation between IL-8 concentration and clinical parameters was investigated. A statistically significant difference in the IL-8 serum levels was found in tumor-bearing dogs compared to the controls. In addition, when the individual parameters were evaluated, IL-8 content showed a positive correlation with the tumor progression, lymph node involvement, recurrence and death. Single and multivariate analyses showed associations between tumor recurrence, metastasis, high clinical staging and high IL-8, and also with the death risk. This was also consistent with the high IL-8 content in dogs showing tumor recurrence and metastasis. IL-8 superexpression has been detected in a number of human tumors, usually associated with a poor prognostic. Besides promoting angiogenesis, IL-8 is strongly related with the metastatic phenotype of mammary tumor cells. High IL-8 concentration was found in mammary gland cancer patients with advanced disease stages. Our results show that IL-8 can be used as a non-invasive prognostic marker for mammary gland cancer, and can be useful for the prediction of disease progression and recurrence in dogs with mammary neoplasias. The increased level of this cytokine acts as an independent prognostic marker of survival and the identification of animals with the poor prognostic.

Glutathione transferase pi (GSTpi) expression in breast cancer: An immunohistochemical and molecular study

Breast cancer is the most frequent cancer in women worldwide. Prognostic markers are important for diagnosis, allowing therapeutic strategies to be defined more efficiently. The expression of the glutathione S-transferase pi isoenzyme (GSTpi) in tumor cells has been evaluated as a predictor of prognosis and in response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results were statistically correlated with clinicopathological parameters of patients. The results showed that high GSTpi expression was related to p53-positive tumors, grade III histology, large tumor size and death (p<0.05). The 37 patients who received adjuvant treatment, checked separately, showed high expression of GSTpi in relation to local recurrence, metastasis and death (p<0.05). In addition, high levels of GSTpi expression were significantly associated with a shorter overall survival (p<0.05). To confirm this suspicion, GSTpi gene expression was checked by Real-time PCR in neoplastic mammary cells cultured and subjected to treatment with doxorubicin. Our results suggest that high levels of GSTpi may be related to the development of resistance to chemotherapy in these tumors, the response of these tumors to treatment and the clinical course of the patients involved.

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.

Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

BackgroundGlutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy.ResultsThe immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death.The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors.The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes.ConclusionTherefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in vitro doxorubicin treatment significantly reduces the expression of GCLC and GSS genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer.

Inhibition of Epithelial-Mesenchymal Transition and Metastasis by Combined TGFbeta Knockdown and Metformin Treatment in a Canine Mammary Cancer Xenograft Model

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-β, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-β1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-β1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice’s lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-β1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-β1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-β1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-β1 silencing and their effect on EMT.

Short interspersed CAN SINE elements as prognostic markers in canine mammary neoplasia.

The genome of mammals is characterized by a large number of non-LTR retrotransposons, and among them, the CAN SINEs are characteristics of the canine species. Small amounts of DNA freely circulate in normal blood serum and high amounts are found in human patients with cancer, characterizing it as a candidate tumor-biomarker. The aim of this study was to estimate, through its absolute expression, the number of copies of CAN SINE sequences present in free circulating DNA of female dogs with mammary cancer, in order to correlate with the clinical and pathological characteristics and the follow-up period. The copy number of CAN SINE sequences was estimated by qPCR in 28 female dogs with mammary neoplasia. The univariate analysis showed an increased number of copies in female dogs with mammary tumor in female dogs >10 years old (p=0.02) and tumor time >18 months (p<0.05). The Kaplan-Meier test demonstrated a negative correlation between an increased number of copies and survival time (p=0.03). High amounts of CAN SINE fragments can be good markers for the detection of tumor DNA in blood and may characterize it as a marker of poor prognosis, being related to female dogs with shorter survival times. This estimate can be used as a prognostic marker in non-invasive breast cancer research and is useful in predicting tumor progression and patient monitoring.

Abstract P2-03-02: Modulatory action of melatonin and miR-17 on ROCK-1 in breast cancer metastasis model

Breast cancer is the most common cancer in women is often associated with high morbidity and mortality rates, with great financial impact on health system. Besides, the disease is characterized by the high rates of metastasis, which worsen significantly the prognosis. This process is associated with two regulatory molecules, microRNAs (miR), specially miR-17, and ROCK-1, which overexpression has been associated to tumor growth and metastasis. In contrast, melatonin has shown oncostatic and anti-metastatic properties by reducing the cell ability to migrate and invade the tissue, besides the inhibition of cell proliferation. The aim of this study was to investigate the effect of melatonin to modulate miR-17 and ROCK-1, a possible candidate gene to miR-17 target in metastatic breast cancer cell line, MDA-MB-231. To determine the effect of melatonin to modulate miR-17 and ROCK-1, MDA-MB-231 cells were treated with melatonin and anti-miR-17-5p. ROCK-1 and miR-17 gene expression were accessed by real time PCR and ROCK-1 protein expression verified by immunocytochemistry and western blotting. Migration and invasion assay was performed to verify the action of melatonin and anti-miR-17-5p to inhibit these processes. In the in vivo study, was developed pulmonary metastasis model followed for six weeks, the tumor induction was continued for 4 weeks, and treatment with anti-miR-17-5p inhibitor for two more weeks. At the end of treatment, animals were euthanized, the lungs removed and used for analysis of miR-17-5p and Let-7c (positive control) and ROCK-1 gene expression. ROCK-1 protein was analyzed by immunohistochemistry. MiR17-5p inhibition managed directly modulate gene and protein expression of ROCK-1 in MDA-MB-231 cells, as well, the gene expression of MYC. In additional, the migration and invasion were decreased after melatonin and anti-miR-17-5p treatment. To validate the findings of the study in vitro with miR-17, was used for lung metastasis model in athymic nude mice. According to our findings, normal animals without metastasis (negative control) had lower levels of miR-17 compared to animals with metastasis and without treatment (positive control). In contrast, animals with metastasis who received anti-miR17-5p treatment, interestingly also had low miR-17 levels compared to positive control animals. Furthermore, it was observed fewer metastases and a reduction in ROCK-1 protein expression in these treated animals compared to positive control animals. Our results demonstrated that miR-17-5p inhibition can modulate ROCK-1 gene and protein in MDA-MB-231 cells and decreasing the number of lung metastases in treated animals. Furthermore, melatonin can act as a synergic mechanism to decrease migration and invasion on metastasis processes mediated by ROCK-1. Citation Format: Borin TF, Pongeluppi RI, Gelaleti GB, Leonel C, Moschetta MG, Ferreira LC, Zuccari DAPdC. Modulatory action of melatonin and miR-17 on ROCK-1 in breast cancer metastasis model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-03-02.

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.