Correlação entre a citologia aspirativa por agulha fina e a histologia no diagnóstico de tumores mamários de cadelas

Cytologic and histologic examination of 35 canine mammary masses was performed over a year period, from dogs that underwent mastectomy for mammary neoplasms at the UNESP Veterinary Medical Teaching Hospital -- Jaboticabal (SP) Brazil. The mastectomy specimens were evaluated according to structural parameters found to be prognostically significant in human mammary tumors, such as grade of atypia, nuclear criteria (shape, size, form, abnormal mitotic figures), chromatin and nucleolar patterns, high cellularity and poor intercellular cohesions. These criteria were used for differentiation of benign from malignant mammary tumors with 63% of a concordant diagnosis and sensibility of 73% and specificity of 83%. The "fine needle aspiration" technique is discussed as a tool to perform early diagnosis. Our data were found to have a positive correlation to the prognosis indicating that we can recognize structural variables of malignancy by citopathology. Application of the described parameters should facilitate comparative studies of histopathologic diagnosis with biologic behavior of canine mammary tumors using this technique to obtain early diagnosis and a secure prognosis.Foram estudados tumores de mama em cadelas, comparando o seu padrão citológico, obtido através da Citologia Aspirativa por Agulha Fina (CAAF), com os resultados da histopatologia. Num período de um ano, as cadelas trazidas ao Hospital Veterinário -- UNESP -- Câmpus de Jaboticabal foram submetidas a exérese cirúrgica dos tumores mamários. As amostras foram avaliadas de acordo com parâmetros estruturais utilizados nos tumores mamários humanos, como grau de atipia, critérios nucleares, padrão de cromatina e nucléolos, alta celularidade e pouca coesão intercelular. Utilizaram-se estes critérios para diferenciar tumores mamários benignos de malignos com 63% de diagnósticos concordantes, sensibilidade de 73% e especificidade de 83%. Nossos dados mostraram ter uma correlação positiva com o prognóstico, demonstrando que é possível reconhecer variáveis estruturais de malignidade na citopatologia para obter um diagnóstico precoce e um prognóstico seguro

Melatonin synthesis in and uptake by mitochondria: implications for diseased cells with dysfunctional mitochondria

Although there is one exception (red blood cells), the lack of energy (ATP) provided by mitochondrial oxidative phosphorylation (OXPHOS) would not be compatible with the long-term survival of normal cells. During conventional metabolism, pyruvate, the cytosolic glycolysis product, enters mitochondria where it is metabolized to acetyl-coenzyme A (acetyl-CoA) under the influence of the enzyme pyruvate dehydrogenase complex (PDC). Acetyl-CoA makes an important contribution to the tricarboxylic acid (TCA) cycle which feeds NADH and FADH2 to the respiratory chain which benefits ATP´s generation by OXPHOS. In some diseased cells, however, pyruvate metabolism becomes aberrant since its transport into the mitochondria is blunted due to the downregulation of PDC due to its inhibition by pyruvate dehydrogenase kinase (PDK), which is upregulated by hypoxia-inducible factor 1 (HIF-1). Therefore, pyruvate undergoes fermentation to lactate in the cytosol. This alternate pathway of pyruvate metabolism is known as the Warburg effect, named after the individual who discovered it, Otto Warburg [1]. Since pyruvate does not enter the mitochondria, mitochondrial ATP synthesis is depressed. Warburg-type metabolism, however, compensates for this by rapidly, albeit inefficiently, synthesizing ATP in the cytosol. Warburg metabolism (also known as aerobic glycolysis) is almost always associated with pathological cells.Fil: Reiter, Russel. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Sharma, Ramaswamy. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Pires De Campos Zuccari, Debora Aparecida. Faculdade de Medicina de São José Do Rio Preto; BrasilFil: Almeida Chuffa, Luiz Gustavo de. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Manucha, Walter Ariel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Rodriguez, Carmen. Universidad de Oviedo; Españ

Melatonin and pathological cell interactions: mitochondrial glucose processing in cancer cells

Melatonin is synthesized in the pineal gland at night. Since melatonin is produced in the mitochondria of all other cells in a non-circadian manner, the amount synthesized by the pineal gland is less than 5% of the total. Melatonin produced in mitochondria influences glucose metabolism in all cells. Many pathological cells adopt aerobic glycolysis (Warburg effect) in which pyruvate is excluded from the mitochondria and remains in the cytosol where it is metabolized to lactate. The entrance of pyruvate into the mitochondria of healthy cells allows it to be irreversibly decarboxylated by pyruvate dehydrogenase (PDH) to acetyl coenzyme A (acetyl-CoA). The exclusion of pyruvate from the mitochondria in pathological cells prevents the generation of acetyl-CoA from pyruvate. This is relevant to mitochondrial melatonin production, as acetyl-CoA is a required co-substrate/co-factor for melatonin synthesis. When PDH is inhibited during aerobic glycolysis or during intracellular hypoxia, the deficiency of acetyl-CoA likely prevents mitochondrial melatonin synthesis. When cells experiencing aerobic glycolysis or hypoxia with a diminished level of acetyl-CoA are supplemented with melatonin or receive it from another endogenous source (pineal-derived), pathological cells convert to a more normal phenotype and support the transport of pyruvate into the mitochondria, thereby re-establishing a healthier mitochondrial metabolic physiology.Fil: Reiter, Russel. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Sharma, Ramaswamy. University Of Texas At San Antonio. University Of Texas Health Science Center At San Antonio (ut Health San Antonio); Estados UnidosFil: Rosales Corral, Sergio. Instituto Mexicano del Seguro Social; MéxicoFil: Manucha, Walter Ariel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Almeida Chuffa, Luiz Gustavo de. Institute of Biosciences of Botucatu; BrasilFil: Pires de Campos Zuccari, Debora Aparecida. Faculdade de Medicina de Sao Jose Do Rio Preto; Brasi

Melatonin-Loaded Nanocarriers: New Horizons for Therapeutic Applications

The use of nanosized particles has emerged to facilitate selective applications in medicine. Drug-delivery systems represent novel opportunities to provide stricter, focused, and fine-tuned therapy, enhancing the therapeutic efficacy of chemical agents at the molecular level while reducing their toxic effects. Melatonin (N-acetyl-5-methoxytriptamine) is a small indoleamine secreted essentially by the pineal gland during darkness, but also produced by most cells in a non-circadian manner from which it is not released into the blood. Although the therapeutic promise of melatonin is indisputable, aspects regarding optimal dosage, biotransformation and metabolism, route and time of administration, and targeted therapy remain to be examined for proper treatment results. Recently, prolonged release of melatonin has shown greater efficacy and safety when combined with a nanostructured formulation. This review summarizes the role of melatonin incorporated into different nanocarriers (e.g., lipid-based vesicles, polymeric vesicles, non-ionic surfactant-based vesicles, charge carriers in graphene, electro spun nanofibers, silica-based carriers, metallic and nonmetallic nanocomposites) as drug delivery system platforms or multilevel determinations in various in vivo and in vitro experimental conditions. Melatonin incorporated into nanosized materials exhibits superior effectiveness in multiple diseases and pathological processes than does free melatonin; thus, such information has functional significance for clinical intervention.Fil: Chuffa, Luiz Gustavo de Almeida. Universidade de Sao Paulo; BrasilFil: Seiva, Fábio Rodrigues Ferreira. Universidade Estadual do Norte do Paraná; BrasilFil: Novais, Adriana Alonso. Universidade Federal do Mato Grosso do Sul; BrasilFil: Simão, Vinícius Augusto. Universidade de Sao Paulo; BrasilFil: Martín Giménez, Virna Margarita. Universidad Católica de Cuyo. Facultad de Ciencias Químicas y Tecnológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Juan; ArgentinaFil: Manucha, Walter Ariel Fernando. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Zuccari, Debora Aparecida Pires de Campos. Faculdade de Medicina de São José do Rio Preto; BrasilFil: Reiter, Russel. University of Texas at San Antonio; Estados Unido

Correlação entre a citologia aspirativa por agulha fina e a histologia no diagnóstico de tumores mamários de cadelas

Foram estudados tumores de mama em cadelas, comparando o seu padrão citológico, obtido através da Citologia Aspirativa por Agulha Fina (CAAF), com os resultados da histopatologia. Num período de um ano, as cadelas trazidas ao Hospital Veterinário -- UNESP -- Câmpus de Jaboticabal foram submetidas a exérese cirúrgica dos tumores mamários. As amostras foram avaliadas de acordo com parâmetros estruturais utilizados nos tumores mamários humanos, como grau de atipia, critérios nucleares, padrão de cromatina e nucléolos, alta celularidade e pouca coesão intercelular. Utilizaram-se estes critérios para diferenciar tumores mamários benignos de malignos com 63% de diagnósticos concordantes, sensibilidade de 73% e especificidade de 83%. Nossos dados mostraram ter uma correlação positiva com o prognóstico, demonstrando que é possível reconhecer variáveis estruturais de malignidade na citopatologia para obter um diagnóstico precoce e um prognóstico seguro.Cytologic and histologic examination of 35 canine mammary masses was performed over a year period, from dogs that underwent mastectomy for mammary neoplasms at the UNESP Veterinary Medical Teaching Hospital -- Jaboticabal (SP) Brazil. The mastectomy specimens were evaluated according to structural parameters found to be prognostically significant in human mammary tumors, such as grade of atypia, nuclear criteria (shape, size, form, abnormal mitotic figures), chromatin and nucleolar patterns, high cellularity and poor intercellular cohesions. These criteria were used for differentiation of benign from malignant mammary tumors with 63% of a concordant diagnosis and sensibility of 73% and specificity of 83%. The fine needle aspiration technique is discussed as a tool to perform early diagnosis. Our data were found to have a positive correlation to the prognosis indicating that we can recognize structural variables of malignancy by citopathology. Application of the described parameters should facilitate comparative studies of histopathologic diagnosis with biologic behavior of canine mammary tumors using this technique to obtain early diagnosis and a secure prognosis

Immunohistochemical and molecular analysis of caveolin-1 expression in canine mammary tumors

Caveolin-1 (Cav-1) is a structural protein present in invaginations of the cell membrane. In human breast cancer, the cav-1 gene is believed to be a tumor suppressor gene associated with inhibition of tumor metastasis. However, little is known about its expression, regulation and function in canine mammary tumors. Expression levels of cav-1 were investigated using real- time PCR and immunohistochemical detection with an anti-human Cav- 1 antibody. Gene expression stability of different samples was analyzed using the geNorm software. Mammary tumors from 51 female dogs were compared to normal mammary tissue from 10 female dogs. Malignant mammary cells showed a loss of Cav-1 expression by quantitative RTPCR and weak Cav-1 staining by immunohistochemistry compared to normal mammary gland tissue. There was a significant relationship between outcome and immunostaining as well as with tumor size, indicating that caveolin subexpression has a positive predictive value and is related to higher survival and smaller tumor size. Our findings indicate that Cav-1 is a potential prognostic marker for canine mammary tumors.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Modulation of Epithelial Mesenchymal Transition after AGTR-1 Gene Edition by Crispr/Cas9 and Losartan Treatment in Mammary Tumor Cell Line: A Comparative Study between Human and Canine Species

Breast cancer is the most prevalent tumor type among women and female dogs. Tumor malignancy is characterized by the epithelial-to-mesenchymal transition (EMT) which leads to the metastasis formation. The inhibition of angiotensin II type I receptor (AGTR1) by an antagonist such as losartan can suppress angiogenesis, consequently contributing to the metastasis control. The aim of this study was to analyze the capacity of losartan and AGTR-1 gene edition to modulate the EMT process in triple negative/metastatic mammary tumor cells, compared to existing treatment protocols such as carboplatin. The cell lines CF41.Mg and MDA-MB-468, were cultured and treated with carboplatin, losartan, or submitted to AGTR-1 gene edition by CRISPR/Cas9. EMT markers and PARP-1 protein and gene expression were evaluated by immunofluorescence or immunocytochemistry and qRT-PCR, respectively. Cell migration capacity was also evaluated. For CF41.Mg and MDA-MB-468 cell lines, there was an increase in E-cadherin and a decrease in N-cadherin and PARP-1 protein and gene expression after treatment with carboplatin, losartan, both in combination and after AGTR-1 gene edition. There was a decrease in VEGF and PARP-1 protein and gene expression after AGTR-1 gene edition. Moreover, in both lines, reduction in invasion rate was observed after all treatments. Our data suggest that losartan and the gene edition of AGTR-1 by CRISPR/Cas9 were able to block the DNA repair and control the EMT process, such as carboplatin. The results in the canine species are unprecedented, as there are no data in the literature that demonstrate the action of losartan in this tumor type

Melatonin Regulates the Daily Levels of Plasma Amino Acids, Acylcarnitines, Biogenic Amines, Sphingomyelins, and Hexoses in a Xenograft Model of Triple Negative Breast Cancer

Metabolic dysregulation as a reflection of specific metabolite production and its utilization is a common feature of many human neoplasms. Melatonin, an indoleamine that is highly available during darkness, has a variety of metabolic functions in solid tumors. Because plasma metabolites undergo circadian changes, we investigated the role of melatonin on the profile of amino acids (AAs), biogenic amines, carnitines, sphingolipids, and hexoses present in the plasma of mice bearing xenograft triple negative breast cancer (MDA-MB-231 cells) over 24 h. Plasma concentrations of nine AAs were reduced by melatonin, especially during the light phase, with a profile closer to that of non-breast cancer (BC) animals. With respect to acylcarnitine levels, melatonin reduced 12 out of 24 molecules in BC-bearing animals compared to their controls, especially at 06:00 h and 15:00 h. Importantly, melatonin reduced the concentrations of asymmetric dimethylarginine, carnosine, histamine, kynurenine, methionine sulfoxide, putrescine, spermidine, spermine, and symmetric dimethylarginine, which are associated with the BC metabolite sets. Melatonin also led to reduced levels of sphingomyelins and hexoses, which showed distinct daily variations over 24 h. These results highlight the role of melatonin in controlling the levels of plasma metabolites in human BC xenografts, which may impact cancer bioenergetics, in addition to emphasizing the need for a more accurate examination of its metabolomic changes at different time points

Avaliação da caspase-3 e Ki-67 como marcadores prognósticos nas neoplasias mamárias em cadelas

Apoptosis, as a cellular event, has an important participation in tumorigenesis, determining the tumoral growth and aggressiveness. The present study had as objective to evaluate the occurrence of apoptosis, associated cellular proliferation, in canine mammary neoplasias and the patient clinical evolution. Seventy dogs had been submitted to surgical excision of the tumor fragment and they were submitted to the histopathologic diagnosis and imunohistochemistry procedure to caspase-3 and the Ki-67. This apoptosis and cellular proliferation markers demonstrated great expression in malignant neoplasias especially carcinoma, considered the most malignant of the tumors. These results confirm consulted literature contributing for a criterious tumoral prognostic complementing the tumoral classification, providing a greater and better supervened due to adequacy of therapeutic procedure of each patient.A apoptose e a proliferação celular possuem uma participação importante na tumorigênese, determinando o crescimento tumoral e consequentemente sua agressividade. O presente estudo teve como objetivo avaliar a ocorrência da apoptose associada a proliferação celular em neoplasias mamárias em cadelas e a elas a evolução clínica do paciente. Setenta animais foram submetidos à exérese cirúrgica do tumor, sendo este submetido ao diagnóstico histopatológico e marcação imuno-histoquímica para caspase-3 e Ki-67. Estes marcadores de apoptose e proliferação celular demonstraram grande expressão nas neoplasias malignas, principalmente nos carcinomas, considerado o mais maligno dos tumores. Estes resultados corroboram os dados da literatura e contribuem para um prognóstico tumoral criterioso que complementa a classificação tumoral existente proporcionando uma melhor e maior sobrevida devido a uma adequação do procedimento terapêutico de cada paciente

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-1 alpha 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-1 alpha, 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 mu M 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-1 alpha, 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-1 alpha (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-1 alpha, 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-1 alpha, VEGF and ROCK-1 in SCC9 line. Additional studies may confirm the potential therapeutic effect of melatonin in some subtypes of oral carcinoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP