AVALIAÇÃO DA CITOTOXICIDADE DE EXTRATOS DA ESPONJA DULCÍCOLA Drulia cristata (PORIFERA: METANIIDAE) DO RIO TAPAJÓS

Extracts from marine sponges and associated microorganisms have already been shown to be important for the development of new drugs. The knowledge about bioactive metabolites of freshwater sponges, however, is still discrete. In this study, the research was carried out using the sponge Drulia cristata and its associated bacteria. The collections were made in Maracanã Beach, Tapajós River, in Santarém. Crude extracts of sponges were obtained and the culture of their bacteria was carried out. After the fermentation of isolated strains, their crude extracts were obtained. The MTT assay was used to evaluate the cytotoxicity of these extracts against HCT-116 line (colorectal carcinoma). The extracts of the sponges had no cytotoxic activity. Extracts from the two strains of bacteria (DTR1 and DTR2) isolated showed moderate cytotoxic activity. From the DTR2 strain, the activity reached 65% inhibition of cancer cells, a promising result for future bioprospecting studies with freshwater sponges.Keywords: Drulia; bioprospecting; MTT assay; cancer.Extratos de esponjas marinhas e microrganismos associados têm demonstrado importância para o desenvolvimento de novas drogas terapêuticas. Entretanto, o conhecimento sobre metabólitos bioativos de esponjas de água doce é ainda bastante discreto. Neste estudo, a pesquisa foi realizada utilizando a esponja Drulia cristata e suas bactérias associadas. As coletas foram feitas na Praia Maracanã, Rio Tapajós, no Município de Santarém. Os extratos brutos da esponja foram obtidos e a cultura de suas bactérias foi realizada. Após a fermentação de cepas isoladas, seus extratos brutos também foram obtidos. O ensaio do MTT foi usado para avaliar a citotoxicidade desses extratos contra a linhagem HCT-116 (carcinoma colorretal). Os extratos da esponja não apresentaram citotoxicidade. Já os extratos das duas cepas de bactérias (DRT1 e DRT2) isoladas apresentaram atividade citotóxica moderada. O extrato da cepa DRT2 atingiu 65% de atividade inibitória sobre as células cancerosas, um resultado promissor para futuros estudos de bioprospecção com esponjas de água doce.Palavras-chave: Drulia, bioprospecção, ensaio do MTT, câncer

Gene expression analysis of aberrant signaling pathways in meningiomas

Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, NF2, which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningiomaThis study was partially supported by Fondo de Investigaciones Sanitarias, Ministerio de Ciencia e Innovación, Spain, Grants PI‑08/1849 and PI‑10/1972; and by grant PI‑10‑045 from the Fundación Sociosanitaria de Castilla‑La Mancha, Spai

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG-100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal do Pará Instituto de Ciências Biológicas Laboratório de Citogenética HumanaUniversidade Federal de São Paulo (UNIFESP) Disciplina de GenéticaUNIFESP, Disciplina de GenéticaCNPq: 20/2007CNPq: 550885/2007-2SciEL

Methylation status of ANAPC1, CDKN2A and TP53 promoter genes in individuals with gastric cancer

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80°C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4% of gastric cancer samples, with 35% methylation in diffuse-type and 26.9% in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30% diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FINEP/CT-INFRAFAEPAUniversidade Federal do Piauí Colegiado de BiomedicinaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MorfologiaUniversidade Federal do Pará Instituto de Ciências Biológicas Laboratório de Citogenética HumanaHospital João de Barros Barreto Serviço de CirurgiaInstituto de Investigaciones BiomedicasUniversidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de GenéticaUNIFESP, EPM, Depto. de MorfologiaFINEP/CT-INFRA: 0927-03SciEL

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.Faculdade de Itaituba Departamento de Pós-GraduaçãoUniversidade Federal do Pará Centro de Ciências Biológicas Departamento de BiologiaUniversidade Federal do Pará Centro de Ciências Biológicas Departamento de PatologiaUniversidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de GenéticaFundação Universidade Federal de Rondônia Departamento de Medicina Laboratório de Biogeoquímica AmbientalUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MorfologiaUNIFESP, EPM, Depto. de MorfologiaSciEL

The Emerging Role of miRNAs and Their Clinical Implication in Biliary Tract Cancer

Biliary tract cancers are aggressive malignancies that include gallbladder cancer and tumors of intra- and extrahepatic ducts and have a poor prognosis. Surgical resection remains the main curative therapy. Nevertheless, numerous patients experience recurrence even after radical surgery. This scenario drives the research to identify biliary tract cancer biomarkers despite the limited progress that has been made. Recently, a large number of studies have demonstrated that deregulated expression of microRNAs is closely associated with cancer development and progression. In this review, we highlight the role and importance of microRNAs in biliary tract cancers with an emphasis on utilizing circulating microRNAs as potential biomarkers. Additionally, we report several single-nucleotide polymorphisms in microRNA genes that are associated with the susceptibility of biliary tract tumors

Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

The Hep-2 cell line is derived from laryngeal carcinoma cells and is often utilized as a model in carcinogenesis and mutagenesis tests. To evaluate the proliferative potential of this line, we developed a cytogenetic methodology (drifter technique) to obtain metaphases from cells that loose cellular adhesion when they underwent mitosis in culture. By this procedure, 2000 cells were counted, resulting in a mitotic index (MI) of 22.2%. Although this MI was not statistically different from the one obtained using either a classical cytogenetic method or a cell synchronization technique, the drifter technique has the advantage of not requiring the use of some reagents for the obtention of metaphases and also of diminishing the consumption of maintenance reagents for this cell line.A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante) para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM) de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal do Pará Centro de Ciências Biológicas Departamento de BiologiaUniversidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de GenéticaUniversidade Federal do Pará Centro de Ciências Biológicas Departamento de GenéticaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MorfologiaUNIFESP, EPM, Depto. de MorfologiaSciEL

The protective effect of Canova homeopathic medicine in cyclophosphamide-treated non-human primates

Background: Canova activates macrophages and indirectly induces lymphocyte proliferation. Here we evaluated the effects of Canova in cyclophosphamide-treated non-human primates.Methods: Twelve Cebus apella were evaluated. Four animals were treated with Canova only. Eight animals were treated with two doses of cyclophosphamide (50 mg/kg) and four of these animals received Canova. Body weight, biochemistry and hematologic analyses were performed for 40 days. Micronucleus and comet assays were performed for the evaluation of DNA damage.Results: We observed that cyclophosphamide induced abnormal WBC count in all animals. However, the group treated with cyclophosphamide plus Canova presented a higher leukocyte count than that which received only cyclophosphamide. Cyclophosphamide induced micronucleus and DNA damage in all animals. the frequency of these alterations was significantly lower in the Canova group than in the group without this medicine.Conclusions: Our results demonstrated that Canova treatment minimizes cyclophosphamide myelotoxicity in C. apella. (C) 2012 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Disciplina Genet, Dept Morfol & Genet, BR-04023900 São Paulo, BrazilUniv São Paulo, Dept Anal Clin Toxicol & Bromatol, Fac Ciencias Farmaceut Ribeirao Preto, BR-14040903 Ribeirao Preto, SP, BrazilFed Univ Para, Lab Citogenet Humana, Inst Ciencias Biol, BR-66059 Belem, Para, BrazilUniversidade Federal de São Paulo, Disciplina Ginecol, Dept Tocoginecol, BR-04039032 São Paulo, BrazilFed Univ Para, Serv Cirurgia, Hosp Univ Joao de Barros Barreto, BR-66073000 Belem, Para, BrazilMinist Saude, Ctr Nacl Primatas, BR-77030000 Ananindeua, PA, BrazilUniv Fed Piaui, Lab Genet & Biol Mol, BR-64202020 Parnaiba, PI, BrazilUniversidade Federal de São Paulo, Disciplina Genet, Dept Morfol & Genet, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Ginecol, Dept Tocoginecol, BR-04039032 São Paulo, BrazilCNPq: 550885/2007-2Web of Scienc

Kinase Inhibitor Screening Displayed ALK as a Possible Therapeutic Biomarker for Gastric Cancer

Despite advances in cancer chemotherapy, gastric cancer (GC) continues to have high recurrence rates and poor prognosis with limited treatment options. Understanding the etiology of GC and developing more effective, less harmful therapeutic approaches are vital and urgent. Therefore, this work describes a novel kinase target in malignant gastric cells as a potential therapeutic strategy. Our results demonstrate that among 147 kinase inhibitors (KI), only three molecules were significantly cytotoxic for the AGP-01 cell line. Hence, these three molecules were further characterized in their cellular mode of action. There was significant cell cycle impairment due to the expression modulation of genes such as TP53, CDKN1A, CDC25A, MYC, and CDK2 with subsequent induction of apoptosis. In fact, the Gene Ontology analysis revealed a significant enrichment of pathways related to cell cycle regulation (GO:1902749 and GO:1903047). Moreover, the three selected KIs significantly reduced cell migration and Vimentin mRNA expression after treatment. Surprisingly, the three KIs share the same target, ALK and INSR, but only the ALK gene was found to have a high expression level in the gastric cancer cell line. Additionally, lower survival rates were observed for patients with high ALK expression in TCGA-STAD analysis. In summary, we hypothesize that ALK gene overexpression can be a promising biomarker for prognosis and therapeutic management of gastric adenocarcinoma