Evaluation of Angiogenesis Process after Metformin and LY294002 Treatment in Mammary Tumor.

BACKGROUND: The angiogenesis process is regulated by many factors, such as Hypoxia-Inducible Factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to inhibit cell growth and the LY294002 is the major inhibitor of PI3K/AKT/mTOR pathway that has antiangiogenic properties. METHODS: Canine mammary tumor cell lines CMT-U229 and CF41 were treated with metformin and LY294002. Cell viability, protein and gene expression of VEGF and HIF-1 were determined in vitro. For the in vivo study, CF41 cells were inoculated in female athymic nude mice treated with either metformin or LY294002. The microvessel density by immunohistochemistry for CD31 as well as the gene and protein expression of HIF-1 and VEGF were evaluated. RESULTS: The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1 and VEGF decreased after treatment with metformin and LY294002. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1 and VEGFA, in addition to the decreasing of CD31 expression after all treatments. CONCLUSION: Our results demonstrate the effectiveness of metformin and LY294002 in controlling the angiogenesis process in mammary tumors by VEGF and HIF-1, the most important angiogenic markers.

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.

Inheritance of the Bantu/Benin haplotype causes less severe hemolytic and oxidative stress in sickle cell anemia patients treated with hydroxycarbamide.

Beta S-globin gene cluster haplotypes (ß(S)-haplotypes) can modulate the response to hydroxycarbamide (HC) treatment in sickle cell anemia (SCA) patients. In Brazil, the most common haplotypes are Bantu and Benin, and both confer a poor prognosis for patients when untreated with HC. We evaluated oxidative and hemolytic biomarkers in 48 SCA patients undergoing HC treatment separated in three subgroups: Bantu/Bantu, Bantu/Benin and Benin/Benin haplotype. On the basis of reduced haptoglobin (HP) levels, patients with Bantu/Bantu haplotypes had 3.0% higher hemolysis degree when compared with those with Bantu/Benin haplotypes (P=0.01). The Benin/Benin patients had 53.6% greater lipid peroxidation index than the Bantu/Bantu patients (P=0.01) because of evaluated thiobarbituric acid reactive species levels. The Bantu/Benin subgroup had intermediate levels of hemolytic and oxidative stress markers compared with the homozygous subgroups. Through strict inclusion criteria adopted, as well as consolidated and well-described hemolytic and the oxidative parameters evaluated, we suggest a haplotype-interaction response to HC treatment mediated by a 'balance' between the genetic factors of each haplotype studied.

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

BACKGROUND: Glutathione (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. RESULTS: The 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. CONCLUSION: Therefore, 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.

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.