Avaliação da ação antitumoral in vitro da pterocarpanoquinona LQB 118 e estudo de alguns mecanismos de ação

Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata.

Influencia de distintas citocininas en el cultivo in vitro de apices caulinares de Fragaria x ananassa y F. virginiana

p.65-70

Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650M1) and a paclitaxel-resistant variant (RPMI-1650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650. were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650M1 cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha (2), alpha (5), alpha (6), beta (1) and beta (4) integrin subunits, decreased expression of alpha (4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-7650M1 cells. Decreased expression of the alpha (2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype. but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system. Oncogene (2010) 29, 752-762; doi: 10.1038/onc.2009.379; published online 9 November 2009

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Inhibitory effects of olive oil phenolics on invasion in human colon adenocarcinoma cells in vitro

Studies in human, animal and cellular systems suggest that phenols from virgin olive oil are capable of inhibiting several stages in carcinogenesis, including metastasis. The invasion cascade comprises cell attachment to extracellular matrix components or basement membrane, degradation of basement membrane by proteolytic enzymes and migration of cells through the modified matrix. In the present study, we investigated the effect of phenolics extracted from virgin olive oil (OVP) and its main constituents: hydroxytyrosol (3,4-dihydroxyphenylethanol), tyrosol (p-hydroxyphenylethanol), pinoresinol and caffeic acid. The effects of these phenolics were tested on the invasion of HT115 human colon carcinoma cells in a Matrigel invasion assay. OVP and its compounds showed different dose-related anti-invasive effects. At 25 mu g/ml OVP and equivalent doses of individual compounds, significant anti-invasive effects were seen in the range of 45-55% of control. Importantly, OVP, but not the isolated phenolics, significantly reduced total cell number in the Matrigel invasion assay. There were no significant effects shown on cell viability, indicating the reduction of cell number in the Matrigel invasion assay was not due to cytotoxicity. There were also no significant effects on cell attachment to plastic substrate, indicating the importance of extracellular matrix in modulating the anti-invasive effects of OVP. In conclusion, the results from this study indicate that phenols from virgin olive oil have the ability to inhibit invasion of colon cancer cells and the effects may be mediated at different levels of the invasion cascade. (c) 2007 Wiley-Liss, Inc.

Paullinia cupana Mart. var. sorbilis, Guarana, Increases Survival of Ehrlich Ascites Carcinoma (EAC) Bearing Mice by Decreasing Cyclin-D1 Expression and Inducing a G0/G1 Cell Cycle Arrest in EAC Cells

The objective of this work is to report the antiproliferative effect of P. cupana treatment in Ehrlich Ascites Carcinoma (EAC)-bearing animals. Female mice were treated with three doses of powdered P. cupana (100, 1000 and 2000 mg/kg) for 7 days, injected with 10(5) EAC cells and treated up to day 21. In addition, a survival experiment was carried out with the same protocol. P. cupana decreased the ascites volume (p = 0.0120), cell number (p = 0.0004) and hemorrhage (p = 0.0054). This occurred through a G1-phase arrest (p < 0.01) induced by a decreased gene expression of Cyclin D1 in EAC cells. Furthermore, P. cupana significantly increased the survival of EAC-bearing animals (p = 0.0012). In conclusion, the P. cupana growth control effect in this model was correlated with a decreased expression of cyclin D1 and a G1 phase arrest. These results reinforce the cancer therapeutic potential of this Brazilian plant. Copyright (C) 2010 John Wiley & Sons, Ltd.

In vitro cytotoxic activity of Baccharis dracunculifolia and propolis against HEp-2 cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito da solução aquosa de fenol, ácido acético e glicerina sobre o tumor ascítico de Ehrlich: estudo experimental in vitro

OBJETIVO: Verificar o efeito da solução composta por fenol, ácido acético e glicerina sobre o tumor ascítico de Ehrlich. MÉTODOS: Após a coleta do líquido ascítico de três camundongos procedeu-se a incubação, a 37° C, do mesmo com diferentes doses da solução teste (0,50, 0,25, 0,10 e 0,05 ml) e com solução salina (0,50 ml como controle; estudou-se a viabilidade celular pela técnica de exclusão do azul tripan). RESULTADOS: Observou-se que ao final de 15 minutos todas as células tumorais encontravam-se inviáveis com as diferentes doses da solução teste. CONCLUSÃO: A solução proposta, causa, in vitro, a morte das células tumorais ao foral de 15 minutos.

Seiva Sangue de Dragão (Croton lechleri) como meio de estocagem de dentes avulsionados: estudo in vitro da viabilidade celular

Pós-graduação em Odontologia - FOA

Síntese e caracterização de nanopartículas magnéticas de óxido de ferro para aplicações biomédicas – um estudo citotóxico em linhagem celular de carcinoma cervical humano (células HeLa)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação da terapia fotodinâmica em candida albicans in vitro e in vivo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação in vitro da efetividade de associações de agentes clareadores na alteração da cor de dentes manchados por pigmentos sangüineos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ação do metabólito secundário 5-hidroxi-2-hidroximetil gama-pirona isolado de fungos do gênero Aspergillus sobre monócitos humanos in vitro

O 5-hidroxi-2-hidroximetil-gama-pirona (HMP) é um metabólito secundário sintetizado por algumas espécies de fungos dos gêneros Aspergillus, Penicillium Acetobac-ter. O HMP tem várias aplicações, sendo utilizado como antioxidante, inibidor da tirosinase, agente protetor contra a radiação e antitumoral. Recentemente, foi também demonstrado que esse metabólito atua como ativador de macrófagos. No entanto, o efeito do HMP em mo-nócitos humanos é desconhecido. Assim, o objetivo deste estudo foi avaliar os efeitos de HMP sobre a viabilidade e diferenciação celular de monócitos do sangue humano in vi-tro. Leucócitos humanos do sangue periférico foram obtidos a partir de bolsas de san-gue doadas pela Fundação Centro de Hemoterapia e Hematologia do Pará (HEMOPA). O isolamento das células foi realizado por meio de gradiente de densidade com Histopaque ®1077. Os monócitos foram tratados durante 24, 48 e 72 horas com 50 e 100 μg / mL de HMP. A análise ultraestrutural dos monócitos tratados revelou que essas células apresen-tam maior espraiamento, elevado número de projeções citoplasmáticas e vacúolos, caracterís-ticas que são frequentemente observadas em células ativadas. A análise da expressão da proteína de superfície específica para macrófago (F4/80) por imunofluorescência, de-monstrou que os monócitos humanos tratados com 50 e 100 μg / mL de HMP por 48 e 72 horas, mostrou um padrão de expressão semelhante ao verificado em macrófagos humanos originados de monócitos tratados com o M-CFS. Os testes de viabilidade utilizados (Método thiazolyl blue, Potencial de membrana mitocondrial, Vermelho Neutro e Azul de Tripan) mostraram que o HMP não tem nenhum efeito citotóxico em monócitos humanos quando tra-tados com 50 e 100 μg/ mL do bioproduto. Estes resultados demonstram um novo papel pa-ra HMP como um agente imunomodulador, induzindo a diferenciação de monócitos em macrófagos.