RESUMO

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.

Assuntos

Cromonas/uso terapêutico , Doenças do Cão/tratamento farmacológico , Neoplasias Mamárias Animais/tratamento farmacológico , Metformina/uso terapêutico , Morfolinas/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cobalto/administração & dosagem , Cães , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Mamárias Animais/irrigação sanguínea , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo

RESUMO

BACKGROUND: ROCK-1 expression is associated with the malignant character of tumors, while inhibiting this molecule results in a significant suppression of tumor metastasis. Likewise, transforming growth factor beta (TGF-ß) is associated with this malignancy by having the ability to induce epithelial-mesenchymal transition (EMT). Metformin, a drug used in the treatment of diabetes, has previously been shown to inhibit EMT in breast cancer cells. OBJECTIVE: The aim of this study is to evaluate the TGF-ß1 action model for induction of EMT and the action of metformin and ROCK-1 inhibitor (Y27632) in EMT process in breast cancer cell lines. METHOD: MCF-7 and MDA-MB-231 cell lines were treated with metformin and Y27632, after induction of EMT by TGF-ß1, to examine the effects on cell migration as well as the protein expression of the ROCK-1 markers, vimentin, E-cadherin, CD44 and CD24 by immunocitochemistry. RESULTS: There was a lower protein expression of ROCK-1, vimentin, CD44 and CD24 in both cell lines after treatment with metformin and Y27632. In MDA-MB-231 cells, E-cadherin expression was increased in all treatment groups. Treatment of MDA-MB-231 cell line with metformin and Y27632 significantly reduced the invasion of these cells. CONCLUSION: This study confirms the benefits of metformin and Y27632 as potential therapeutic agents in mammary tumors, by blocking EMT process and metastatic potential.

Assuntos

Amidas/farmacologia , Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metformina/farmacologia , Piridinas/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo

RESUMO

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-ß) 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-ß, 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-ß1. 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-ß1sh 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-ß1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-ß1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-ß1sh 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-ß1 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-ß1 silencing and their effect on EMT.

Assuntos

Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Mamárias Animais/tratamento farmacológico , Metformina/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Cães , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Invasividade Neoplásica/patologia

RESUMO

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.

Assuntos

Carcinoma/genética , Doenças do Cão/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Mamárias Animais/genética , Neovascularização Patológica/genética , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Carcinoma/mortalidade , Carcinoma/patologia , Doenças do Cão/mortalidade , Doenças do Cão/patologia , Cães , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfonodos/patologia , Neoplasias Mamárias Animais/mortalidade , Neoplasias Mamárias Animais/patologia , Estadiamento de Neoplasias , Neovascularização Patológica/patologia , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/metabolismo

RESUMO

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.

Assuntos

Biomarcadores Tumorais/sangue , Cães/genética , Neoplasias Mamárias Animais/sangue , Neoplasias Mamárias Animais/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Animais , Biomarcadores Tumorais/genética , DNA/sangue , Doenças do Cão/genética , Feminino , Dosagem de Genes , Neoplasias Mamárias Animais/mortalidade , Reação em Cadeia da Polimerase/veterinária , Prognóstico

RESUMO

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.

Assuntos

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/patologia , Proliferação de Células/efeitos dos fármacos , Quimioterapia Adjuvante , Doxorrubicina/farmacologia , Feminino , Seguimentos , Glutationa Peroxidase/genética , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Glutationa Peroxidase GPX1

RESUMO

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.

Assuntos

Biomarcadores Tumorais/imunologia , Doenças do Cão/imunologia , Interleucina-8/imunologia , Neoplasias Mamárias Animais/imunologia , Animais , Biomarcadores Tumorais/sangue , Progressão da Doença , Doenças do Cão/sangue , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Interleucina-8/sangue , Estimativa de Kaplan-Meier , Neoplasias Mamárias Animais/sangue , Valor Preditivo dos Testes , Curva ROC

RESUMO

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.

Assuntos

Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Interleucina-8/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/diagnóstico , Feminino , Humanos , Imuno-Histoquímica , Interleucina-8/biossíntese , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida

RESUMO

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.

Assuntos

Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Glutationa S-Transferase pi/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida

RESUMO

Carboidratos exógenos contribuem para a manutenção da glicose sanguínea e acredita-se que isto possa melhorar o desempenho anaeróbio após um exercício prolongado. Foram objetivos deste estudo comparar a potência máxima e o trabalho total de atletas, no teste anaeróbio de Wingate realizado após 90 min de exercício prolongado (EP), com e sem a ingestão de mistura eletrolítica carboidratada (MEC), bem como as respostas termorregulatórias ocorridas durante o EP. Sete homens (21,4 mais ou menos 4,2 anos; 65,46 mais ou menos 10,45 kg; 9,4 mais ou menos 1,8 de gordura e 60,38 mais ou menos 4,76 mLO2·kg-1·min-1) ingeriram água destilada e cápsulas gelatinosas contendo MEC ou placebo (PLA) a cada 15 min, enquanto realizavam o EP a 60

Assuntos

Masculino , Adulto , Humanos , Limiar Anaeróbio , Regulação da Temperatura Corporal , Carboidratos , Eletrólitos/metabolismo , Exercício Físico , Temperatura Alta