RESUMO
BACKGROUND: Lung cancer is one of the most prevalent cancers worldwide. Chemotherapy regimens, targeted against lung cancer, are considered an effective treatment; albeit with multiple fatal side effects. An alternative strategy, nowadays, is using natural products. Medicinal plants have been used, in combination with chemotherapy, to ameliorate side effects. AIMS: This study aims to investigate the antitumor effect of pomegranate juice (Punica granatum) on human lung adenocarcinoma basal epithelial cells (A549), to check the effect, when combined with low dose cisplatin (CDDP), at different doses. We also have evaluated the potential protective effect of pomegranate on normal peripheral blood mononuclear cells (PBMC). METHODS: Phytochemical screening of the extract was done using standard classical tests. Total phenolic and sugar contents were determined using the Folin-Ciocalteu and anthrone reagents, respectively. The antioxidant activity of pomegranate was estimated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The viability of A549 cells and PBMC was evaluated using the neutral red assay. RESULTS: Our results demonstrated that Punica granatum or pomegranate juice (with different concentrations: 150, 300, 600 µg/mL) contained high levels of flavonoids, alkaloids, tanins, lignins, terpenoids, and phenols. The DPPH method showed that pomegranate juice had a strong antioxidant scavenging activity. Neutral red showed that combining pomegranate juice with low dose CDDP (8 µg/mL) decreased the cell viability of A549 cells, by 64%, compared to treatment with CDDP or pomegranate alone. When added to low dose CDDP, pomegranate increased the viability of normal PBMC cells by 46%. CONCLUSIONS: These results demonstrated that pomegranate could potentiate the anticancer effect of low dose CDDP on human lung adenocarcinoma cells (A549 cells) and could as well decrease its toxicity on PBMC.
RESUMO
Background: Lung and breast cancers are common in the world and represent major public health problems. Systemic chemotherapy is an effective way to prolong survival but it is associated with side effects. Plants are used as traditional treatments for many types of cancers, mostly in combination with chemotherapy. We investigated the antitumor effect of ethanolic (EE) and aqueous (AE) extracts of Eucalyptus camaldulensis on human alveolar adenocarcinoma basal epithelial cells (A549) and breast adenocarcinoma cell line (MCF-7) and checked the synergistic effect of the combination with low-dose cisplatin (CDDP). Methods: AE and EE were characterized for their secondary metabolites including content of phenol and antioxidant activity of both extracts. Cell viability was tested by the neutral red assay and MTT. Combinations of extract with low-dose CDDP on A549, MCF-7 cells, and normal cells peripheral blood mononuclear cells was used to study cell viability. Results: AE contains higher level of active constituents than EE. Higher antioxidant activity was observed in AE. Both extracts showed cytotoxic activity on A549 and MCF-7 cells. Moreover, combining E. camaldulensis with low-dose CDDP increases significantly the cell death of treated cells in comparison to those treated with CDDP alone. Conclusions: Our results highlight a new therapeutic concept that combines Eucalyptus camaldulensis with low-dose CDDP to treat lung and breast adenocarcinoma.
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Trichodinids are ciliated protozoans that reversibly attach to the tegument of marine and freshwater host-organisms via an adhesive disc. In this study, we have used permeabilized cell models of Trichodina pediculus to examine the distribution of centrins, a Ca(2+)-binding protein associated with centrioles and/or contractile filamentous structures in a large number of protists. The previous finding that filamentous material of the adhesive disc comprised a 23-kDa centrin analog suggested that this protein might be a disc-specific isoform. This possibility was explored through immunolabeling methods using two distinct antibodies, anti-ecto-endoplasmic boundary (EEB) and anti-Hscen2 previously shown to react respectively with centrin-based filament networks and with centrioles. Immunofluorescence microscopy showed that anti-EEB reacts with filamentous material of the disc but not with basal bodies. Conversely, anti-Hscen2 cross-reacted with basal bodies but failed to label any type of structure occurring in the disc area. More detailed data on localization of this protein was obtained by immunoelectron microscopy showing gold particles deposits in the lumen of basal bodies. The different patterns revealed by this immunochemical approach suggest that the two protein antigens concerned by this study are distinct centrin isoforms that presumably perform organelle-specific function in the ciliate T. pediculus.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Oligoimenóforos/metabolismo , Proteínas de Protozoários/imunologia , Centríolos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Oligoimenóforos/patogenicidadeRESUMO
E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.
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Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Adesão Celular/fisiologia , Polaridade Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Epiderme/fisiologia , Animais , Clatrina/metabolismo , Epiderme/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Interferência de RNA , Fator de Transcrição AP-1/metabolismoRESUMO
In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins.
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Citoesqueleto/metabolismo , Paramecium/citologia , Paramecium/metabolismo , Proteínas de Protozoários/metabolismo , Ciclo Celular , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Microscopia Eletrônica , Paramecium/classificação , Paramecium/crescimento & desenvolvimento , Filogenia , Proteínas de Protozoários/genéticaRESUMO
The adhesive disc is a highly complex apparatus that allows mobilid ciliates to attach to the tissues of a variety of aquatic invertebrates and vertebrates. The disc comprises concentric rings of rigid skeletal pieces interconnected by filamentous material. This study explored the biochemical properties of the filamentous disc material in the trichodinid Trichodina pediculus. Calcium sensitivity of this material was suggested in vitro by the appearance of transverse cross-striation along bundles of filaments following calcium shock, and complete solubilization of the filamentous material in the presence of EGTA. A 23-kDa immunoanalog of centrins was immunoprecipitated from the EGTA extract. The protein binds calcium as indicated by (45) Ca(2+) blot overlay and Ca(2+) -induced shifts in electrophoretic mobility. Using Ca(2+) /EGTA buffers, we demonstrated a direct relationship between extraction of the filaments and solubilization of the protein. Immunofluorescence and immunoelectron microscopy confirmed that the protein localized to the filamentous disc material and revealed cross-reactivity with the spasmoneme, which is the prototype of ion-sensitive, centrin-like contractile systems in ciliates. The possibility that the filamentous disc material may be a novel example of Ca(2+) -sensitive, centrin-based systems found in ciliates is discussed.
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Cálcio/metabolismo , Cilióforos/metabolismo , Citoesqueleto/metabolismo , Proteínas de Protozoários/metabolismo , Combinação Trimetoprima e Sulfametoxazol/metabolismo , Cilióforos/citologiaRESUMO
BACKGROUND: The sub-membranous skeleton of the ciliate Paramecium, the epiplasm, is composed of hundreds of epiplasmic scales centered on basal bodies, and presents a complex set of proteins, epiplasmins, which belong to a multigenic family. The repeated duplications observed in the P. tetraurelia genome present an interesting model of the organization and evolution of a multigenic family within a single cell. RESULTS: To study this multigenic family, we used phylogenetic, structural, and analytical transcriptional approaches. The phylogenetic method defines 5 groups of epiplasmins in the multigenic family. A refined analysis by Hydrophobic Cluster Analysis (HCA) identifies structural characteristics of 51 epiplasmins, defining five separate groups, and three classes. Depending on the sequential arrangement of their structural domains, the epiplasmins are defined as symmetric, asymmetric or atypical. The EST data aid in this classification, in the identification of putative regulating sequences such as TATA or CAAT boxes. When specific RNAi experiments were conducted using sequences from either symmetric or asymmetric classes, phenotypes were drastic. Local effects show either disrupted or ill-shaped epiplasmic scales. In either case, this results in aborted cell division. Using structural features, we show that 4 epiplasmins are also present in another ciliate, Tetrahymena thermophila. Their affiliation with the distinctive structural groups of Paramecium epiplasmins demonstrates an interspecific multigenic family. CONCLUSION: The epiplasmin multigenic family illustrates the history of genomic duplication in Paramecium. This study provides a framework which can guide functional analysis of epiplasmins, the major components of the membrane skeleton in ciliates. We show that this set of proteins handles an important developmental information in Paramecium since maintenance of epiplasm organization is crucial for cell morphogenesis.