Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Extração, caracterização estrutural e atividades farmacológicas do olginato obtido da alga marrom Lobophora variegata (Lamouroux) Oliveira Filho, 1977

The alginic acid or alginates are acidic polysaccharides found in brown seaweed widely used in food, cosmetic, medical and pharmaceutical industry. This paper proposes the extraction, chemical characterization and verification of the pharmacological activities of brown seaweed variegata Lobophora . The alginate was extracted from the seaweed Lobophora variegata and part was sulphated for comparative purposes. The native extract showed 42% total sugar, 65% uronic acid, 0,36 % protein and 0% of sulfate, while the sulfate showed 39% , 60%, 0.36% and 27,92 % respectively. The presence of a sulfate group may be observed by the metachromasia with toluidine blue in electrophoresis system and characteristic vibration 1262,34 cm-1 in infrared spectroscopy connections assigned to S = O. We observed the formation of films and beads of native alginate, where more concentrated solution 6% resulted in a thicker and more consistent film. Native alginate showed proliferative activity at concentrations (25 and 50 mcg), (50 mg) and (100 mg) in 3T3 cell line in 24h, 48h and 72h, respectively , as the sulfated (100 mg) in 24 . Also showed antiproliferative or cytotoxic activity in HeLa cells of strain, (25 and 100 mg), (25 and 100 mg) and (25, 50 and 100 mg), to native, now for the sulfate concentrations (100 mg) in 24 (25, 50 and 100 mg) in 48 hours, and (50 and 100 mg ) 72h. For their antioxidant activity, the sulfated alginates have better total antioxidant activity reaching 29 % of the native activity while 7.5 % of activity . For the hydroxyl radical AS showed high inhibition ( between 77-83 % ) in concentrations, but the AN surpassed these numbers in the order of 78-92 % inhibition. The reducing power of AN and AS ranged between 39-82 % . In the method of ferric chelation NA reached 100 % chelating while the AS remained at a plateau oscillating 6.5%. However, in this study , we found alginates with promising pharmacological activities, to use in various industries as an antioxidant / anti-tumor compound

Chromosomal aberrations induced by 5-azacytidine combined with VP-16 (etoposide) in CHO-K1 and XRS-5 cell lines

A cytogenetic study was carried out with 5-azacytidine (5-azaC) and etoposide (VP-16) in CHO-K1 and XRS-5 (mutant cells deficient for double-strand break rejoining) cell lines to verify the interaction effects of the drugs in terms of induction of chromosomal aberrations. 5-azaC is incorporated into DNA causing DNA hypomethylation, and VP-16 (inhibitor of topoisomerase 11 enzyme) is a potent clastogenic agent. Cells in exponential growth were treated with 5-azaC for I h, following incubation for 7 h, and posttreatment with VP16 for the last 3 h. In K1 cells, the combined treatments induced a significant reduction in the aberrations induced in the X and A (autosome) chromosomes, which are the main target for 5-azaC. However, in XRS-5 cells, the drug combination caused a significant increase in the aberrations induced in those chromosomes, but with a concomitant reduction in the randomly induced-aberrations. In addition, each cell line presented characteristic cell cycle kinetics; while the combined treatment induced an S-arrest in K1 cells, alterations in cell cycle progression were not found for XRS-5, although each drug alone caused a G2-arrest. The different cell responses presented by the cell lines may be explained on the basis of the evidence that alterations in chromatin structure caused by 5-aza-C probably occur to a different extent in K1 and XRS-5 cells, since the mutant cells present a typical hyper-condensed chromosome structure (especially the X- and A chromosomes), but, alternatively, 5-aza-C could induce reactivation of DNA repair genes in XRS-5 cells. Teratogenesis Carcinog. Mutagen. Suppl. 1:171-186, 2003. (C) 2003 Wiley-Liss, Inc.

Synthesis, characterization, X-ray structure and in vitro anti mycobacterial and antitumoral activities of Ru(II) phosphine/diimine complexes containing the "SpymMe(2)" ligand, SpymMe(2)=4,6-dimethyl-2-mercaptopyrimidine

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Human breast tumor slices as an alternative approach to cell lines to individualize research for each patient

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Associação deagentes patogênicos isolados emanálise microbiológica da água, coma presença de mastite clínica ou subclínica, em vacas de propriedades leiteiras da região de Cerqueira César - SP

Objetivou-se, neste trabalho, pesquisar a relação entre os microrganismos patogênicos isolados e identificados em água utilizada na ordenha, com o isolamento e identificação dos mesmos em amostras de leite, de quartos mamários apresentando mastite clínica ou subclínica nas mesmas propriedades. Foram utilizadas 16 propriedades rurais leiteiras, escolhidas aleatoriamente, na região de Cerqueira César - SP, que utilizavam ordenha mecânica. A água utilizada na ordenha foi classificada em relação à presença de coliformes totais e fecais, como dentro dos padrões ou fora dos padrões de potabilidade humana. Nos resultados obtidos, 94% das amostras foram classificadas como fora dos padrões em relação a coliformes totais e fecais. Os microrganismos identificados foram: Escherichia coli (51%), Enterobacter spp. (25%), Enterobacter cloacae (8%) Edwardsiella tarda (8%) e Klebsiella oxytoca (8%). em relação ao leite, foram analisadas 373 amostras provenientes de vacas em lactação, com mastite clínica (n=19; 5%) e subclínica (n=354; 95%). Os animais com mastite subclínica foram identificados pela contagem de células somáticas (CCS), utilizando-se o aparelho eletrônico (Somacount 300, Bentley), onde a média observada foi de 1.631 x 10³ células/mL. Os principais microrganismos identificados foram: Staphylococcus aureus (30%), Corynebacterium bovis (23%) e Staphylococcus spp. (15%). Conforme os dados obtidos, os agentes coliformes encontrados na água, utilizada na ordenha, não estavam presentes nas análises das amostras de leite dos quartos mamários com mastite clínica ou subclínica das respectivas propriedades, demonstrando não haver associação entre a qualidade da água e a ocorrência de mastite.

Somaticell® as a screening method for somatic cell count from bovine milk

Os objetivos do presente estudo foram avaliar a correlação entre a contagem eletrônica de células somáticas (eCCS) com o Somaticell® sob diferentes níveis de contagem de células somáticas (CCS) do leite e patógenos causadores de mastites, além de calcular a sensibilidade, especificidade e valores preditivos do Somaticell® utilizando diferentes limites de CCS estabelecidos pelos diferentes países. Trezentos e quarenta amostras de leite foram coletadas assepticamente após realização do California Mastitis Test (CMT). O Somaticell® e a eCCS foram realizados em todas as amostras de leite. A correlação entre o Somaticell® e a contagem eletrônica foi determinada de acordo com o CMT, patógeno isolado e escore de eCCS. de acordo com os escores de CCS estabelecidos, 26,5% das amostras de leite apresentaram escore 1 (69-166 x10³células mL-1), 26,8% escore 2 (167-418x10³células mL-1), 27,4% escore 3 (419-760x10³células mL-1) e 19,4% escore 4 (761 to 1970x10³células mL-1). A eCCS e o Somaticell® apresentaram correlação positiva em quase todos os escores estudados (exceto escore 2 e 3). O valor de r obtido entre CCS e o Somaticell® foi de 0,32. Observou-se que, quando o limite de CCS estabelecido aumentou, a sensibilidade decresceu e os valores de especificidade aumentaram. Os valores preditivos apresentaram-se constantes em todos os limites. Quando o limite de CCS era baixo (<760,000 células mL-1), Somaticell® forneceu resultados consistentemente mais elevados que os valores de CCS. Já para amostras com CCS elevada, Somaticell® resultou em menores contagens que a eCCS. A correlação entre os dois métodos permaneceu relativamente constante em todas as condições e os valores de sensibilidade e especificidade do teste são altamente dependentes do limite estabelecido. Os resultados deste trabalho sugerem que o Somaticell® não é útil para avaliar a CCS do leite, pois seus resultados são significativamente diferentes da eCCS, no entanto, pode ser utilizado como método de triagem, tal como o CMT, para a detecção do aumento da CCS do leite.

Increased plasma and salivary cortisol levels in patients with oral cancer and their association with clinical stage

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clinical and histopathological analysis of oral squamous cell carcinoma in young people A descriptive study in Brazilians

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Implication of RNA-Binding Protein La in Proliferation, Migration and Invasion of Lymph Node-Metastasized Hypopharyngeal SCC Cells

The 5-year survival rate for oral cavity cancer is poorer than for breast, colon or prostate cancer, and has improved only slightly in the last three decades. Hence, new therapeutic strategies are urgently needed. Here we demonstrate by tissue micro array analysis for the first time that RNA-binding protein La is significantly overexpressed in oral squamous cell carcinoma (SCC). Within this study we therefore addressed the question whether siRNA-mediated depletion of the La protein may interfere with known tumor-promoting characteristics of head and neck SCC cells. Our studies demonstrate that the La protein promotes cell proliferation, migration and invasion of lymph node-metastasized hypopharyngeal SCC cells. We also reveal that La is required for the expression of beta-catenin as well as matrix metalloproteinase type 2 (MMP-2) within these cells. Taken together these data suggest a so far unknown function of the RNA-binding protein La in promoting tumor progression of head and neck SCC.

Influence of estradiol and triiodothyronine on breast cancer cell lines proliferation and expression of estrogen and thyroid hormone receptors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell death evaluation in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17

The incidence of apoptosis and nuclear instability, including the incidence of catastrophic death, were investigated in benzo[a]pyrene (BP)-transformed human breast epithelial cells (BP1-E cell line) after microcell-mediated transfer of chromosomes 11 and 17. Since the introduction of normal chromosomes 11 and 17 into tumorigenic human breast BP1-E cells reverts some of these cells' characteristics (especially those affected by microsatellite instabilities and loss of heterozygosity) those of parental non-transformed MCF-10F cells, it was expected that the cell death rates would also be affected by this treatment. The transfer of the mentioned chromosomes, especially chromosome 17, to tumorigenic BP1-E cells increased the apoptotic ratios and decreased the nuclear instability ratios, thus showing that the microsatellite instability and loss of heterozygosity induced by BP in these chromosomes of MCF-10F cells affect the control of cell death mechanisms. (C) 2003 Elsevier B.V. All rights reserved.

Anti-clastogenic effect of beta-glucan extracted from barley towards chemically induced DNA damage in rodent cells

beta-Glucan (BG) was tested in vitro to determine its potential clastogenic and/or anti-clastogenic activity, and attempts were made to elucidate its possible mechanism of action by using combinations with an inhibitor of DNA polymerase. The study was carried out on cells deficient (CHO-k1) and cells proficient (HTC) in phases I and II enzymes, and the DNA damage was assessed by the chromosomal aberration assay. BG did not show a clastogenic effect, but was anti-clastogenic in both cell lines used, and at all concentrations tested (2.5, 5 and 10 mg/mL) in combination with damage inducing agents (methylmethane sulfonate in cell line CHO-k1, and methylmethane sulfonate or 2-aminoanthracene in cell line HTC). BG also showed a protective effect in the presence of a DNA polymerase beta inhibitor (cytosine arabinoside-3-phosphate, Ara-C), demonstrating that BG does not act through an anti-mutagenic mechanism of action involving DNA polymerase beta.