RESUMO
Six previously undescribed triterpenoid saponins and two previously undescribed norlupane triterpenes were isolated with five known saponins, three known lupane derivatives, 17,20-didehydro-20-deoxyjujubogenin, rutin, (±) 3α-O-ß-d-glucopyranosyl-lyoniresinol, (±) 4-O-ß-d-glucopyranosyl-maesopsin, three phenol glycosides, and uridine from the stems and bark of Jaffrea xerocarpa (Baill.) H. C. Hopkins & Pillon (= Basionym Alphitonia xerocarpus Baill.) (Rhamnaceae), an endemic tree of New Caledonia. The chemical structures of the purified compounds were identified by nuclear magnetic resonance and mass spectrometry. The isolated compounds were tested for their antioxidant, antityrosinase, antibacterial and cytotoxic activities. The aqueous methanol extract showed antioxidant activity (DPPH assay) due to the presence of rutin and other phenolic compounds. Three lupane triterpenes showed good cytotoxic activities against KB cells line (IC50 from 7.7 to 8.5 µM). The previously undescribed 2α-formyl-A(1)norlup-20(29)-en-28-oic acid showed antibacterial activity against Staphylococcus aureus and Enterococcus faecalis with both MIC values of 4 µg/mL.
Assuntos
Rhamnaceae/química , Saponinas/química , Triterpenos/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Enterococcus faecalis/efeitos dos fármacos , Humanos , Células KB , Testes de Sensibilidade Microbiana , Estrutura Molecular , Casca de Planta/química , Caules de Planta/química , Saponinas/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Triterpenos/isolamento & purificaçãoRESUMO
Ten previously undescribed triterpenoid saponins and a previously undescribed norlupane triterpenoid were isolated, with three known saponins, four known flavonoids, two known lupane derivatives, sitosterol and 6'-heptadecanoyl-3-O-ß-d-glucopyranosylsitosterol from the leaves of Alphitonia xerocarpus (Rhamnaceae), an endemic tree of New Caledonia. The chemical structures of the purified compounds were identified by nuclear magnetic resonance and mass spectrometry. The isolated compounds were tested for their antioxidant, antityrosinase, antibacterial and cytotoxic activity. The aqueous methanol extract showed antioxidant activity (DPPH assay) due to the presence of rutin. Ceanothenic acid showed good cytotoxic activity against a KB cell line (IC50 = 2.6 µM) and antibacterial activity against Staphylococcus aureus and Enterococcus faecalis with MIC values of 8 and 16 µg/mL, respectively. The previously undescribed 29-hydroxyceanothenic acid exhibited moderate cytotoxic activity (IC50 = 10 µM), good antibacterial activity against S. aureus (MIC = 4 µg/mL) and moderate antibacterial activity against E. faecalis (MIC = 16 µg/mL).
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Rhamnaceae/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Antibacterianos/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Enterococcus faecalis/efeitos dos fármacos , Flavonoides/química , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Nova Caledônia , Folhas de Planta/química , Saponinas/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Triterpenos/químicaRESUMO
INTRODUCTION: Alphitonia neocaledonica (Rhamnaceae) is an endemic tree of New Caledonia. Although three flavonoids have been identified in the leaves, the secondary metabolite profile of the fruits has never been investigated. OBJECTIVE: Phytochemical investigation of A. neocaledonica fruits and evaluation of their anti-oxidant, anti-tyrosinase and cytotoxic activities. METHODS: A hydromethanolic extract was fractionated by liquid-liquid extraction to obtain ethyl acetate and n-butanolic fractions. The ethyl-acetate-soluble part was purified by silica-gel column chromatography and high-performance liquid chromatography (HPLC). The n-butanol-soluble part was fractionated by centrifugal partition extraction (CPE) and the collected fractions were further purified by centrifugal partition chromatography (CPC) and HPLC. The chemical structures of the purified compounds were identified by nuclear magnetic resonance and mass spectrometry. RESULTS: Three triterpenoids and one flavonoid were isolated from the ethyl-acetate-soluble part. Fractions enriched in triterpenoids, flavonoids and catechin derivatives were obtained from the n-butanol-soluble part. Gallocatechin and flavonoids were obtained as pure compounds by further CPC and HPLC purification. The n-butanolic-soluble part showed anti-oxidant and anti-tyrosinase activities due to the presence of tannins and gallocatechin. The triterpenoid alphitolic acid showed a moderate cytotoxic activity against KB cell line (median inhibition concentration = 8.5 µM). CONCLUSIONS: Nine known compounds including three triterpenes, five flavonoids and (+) gallocatechin, as well as a new 3-O-(6-E-feruloyl)-ß-D-glucopyranosyl-(1 â 2)-[ß-D-xylopyranosyl-(1 â 2)-]α-L-rhamnopyranosyl-quercetin, were isolated from A. neocaledonia fruits. The hydromethanolic extract possesses a potential cytotoxic activity due to the presence of triterpenes, and it can also be valuable as a cosmetic ingredient for its anti-oxidant and anti-tyrosinase activities.
Assuntos
Flavonoides/isolamento & purificação , Frutas/química , Extratos Vegetais/isolamento & purificação , Rhamnaceae/química , Triterpenos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Flavonoides/química , Flavonoides/farmacologia , Humanos , Extração Líquido-Líquido , Monofenol Mono-Oxigenase/antagonistas & inibidores , Nova Caledônia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Triterpenos/química , Triterpenos/farmacologiaRESUMO
The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.
Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Nanotecnologia/métodos , Água/análise , Técnicas de Cultura de Células , Células HeLa , Humanos , Íons/análise , Íons/metabolismo , Luz , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Água/metabolismoRESUMO
During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to the elastin-binding receptor and activating the MEK/ERK signal transduction pathway. As this process strongly modifies mRNA synthesis, we investigated its effect on the relative three-dimensional organisation of the major partners of the mRNA splicing machinery: promyelocytic nuclear bodies (PML-NBs ) and splicing component 35 speckles (SC35) of normal fibroblasts and melanoma SK-MEL-28 cells. SC35 and PML-NBs proteins were immunolabeled and imaged by confocal microscopy within these cells cultured with (VGVAPG)3. Three-dimensional reconstruction was performed to elucidate the organisation of PML-NBs and SC35 speckles and their spatial relationship. In G0 cells, SC35 speckles were sequestered in PML-NBs. Shortly after (VGVAPG)3 stimulation, the three-dimensional organisation of PML-NBs and SC35 speckles changed markedly. In particular, SC35 speckles gradually enlarged and adopted a heterogeneous organisation, intermingled with PML-NBs. Conversely, inhibition of the elastin-binding protein or MEK/ERK pathway induced a remarkable early sequestration of condensed SC35 speckles in PML-NBs, the hallmark of splicing inhibition. The 3D architecture of speckles/PML-NBs highlights the modulation in their spatial relationship, the multiple roles of PML-NBs in activation, inhibition and sequestration, and provides the first demonstration of the dependence of PML-NBs and SC35 speckles on the elastin peptide for these functions.
Assuntos
Núcleo Celular/efeitos dos fármacos , Elastina/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Imageamento Tridimensional , Melanoma/patologia , Oligopeptídeos/farmacologia , Splicing de RNA/efeitos dos fármacos , Adulto , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Relação Estrutura-AtividadeRESUMO
The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128-462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.
Assuntos
Núcleo Celular/química , Espaço Intracelular/química , Estresse Fisiológico , Água/análise , Núcleo Celular/fisiologia , Cromatina/química , Microscopia Crioeletrônica/métodos , Crioultramicrotomia , Citosol/química , Células HeLa , Humanos , Substâncias Macromoleculares/química , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/química , Nanotecnologia , Espectrometria por Raios XRESUMO
Cryo fluorescence imaging coupled with the cryo-EM technique (cryo-CLEM) avoids chemical fixation and embedding in plastic, and is the gold standard for correlated imaging in a close to native state. This multi-modal approach has not previously included elementary nano analysis or evaluation of water content. We developed a new approach allowing analysis of targeted in situ intracellular ions and water measurements at the nanoscale (EDXS and STEM dark field imaging) within domains identified by examination of specific GFP-tagged proteins. This method allows both water and ions- fundamental to cell biology- to be located and quantified at the subcellular level. We illustrate the potential of this approach by investigating changes in water and ion content in nuclear domains identified by GFP-tagged proteins in cells stressed by Actinomycin D treatment and controls. The resolution of our approach was sufficient to distinguish clumps of condensed chromatin from surrounding nucleoplasm by fluorescence imaging and to perform nano analysis in this targeted compartment.
Assuntos
Microscopia Crioeletrônica/métodos , Íons/química , Microscopia Eletrônica de Transmissão e Varredura/métodos , Água/químicaRESUMO
Six pentacyclic triterpenoid saponins, named antoniosides E-J along with two known alkaloids, were isolated from the leaves of Antonia ovata. Their structures were determined by the extensive use of 1D and 2D-NMR experiments along with HRESIMS analysis and acid hydrolysis. All isolated saponins contained the same pentasaccharide chain: 3-O-[ß-D-glucopyranosyl-(1â2)]-[ß-D-glucopyranosyl-(1â4)]-[ß-D-glucopyranosyl-(1â3)-α-L-arabinopyranosyl(1â6)]-ß-D-glucopyranoside, linked at C-3 of esterified derivatives of polyhydroxyoleanene triterpenoids (theasapogenol A and 15α-hydroxy-theasapogenol A). Isolated compounds were evaluated for their cytotoxic activity against KB cell line by a WST-1 assay, and the IC(50) values ranged from 3.3 to 5.3 µM.
Assuntos
Antineoplásicos Fitogênicos/química , Loganiaceae/química , Saponinas/química , Triterpenos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Humanos , Células KB , Ressonância Magnética Nuclear Biomolecular , Folhas de Planta/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.
Assuntos
Antígenos CD/biossíntese , Elastina/farmacologia , Fibroblastos/efeitos dos fármacos , Lactosilceramidas/biossíntese , Neuraminidase/metabolismo , Peptídeos/farmacologia , Adulto , Antígenos CD/metabolismo , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Elastina/química , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Gangliosídeo G(M3)/metabolismo , Humanos , Lactose/farmacologia , Lactosilceramidas/metabolismo , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuraminidase/genética , Interferência de RNA , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2ß1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2ß1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K(d)≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2ß1 integrin.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Integrina alfa2beta1/metabolismo , Sulfato de Queratano/farmacologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfa2/metabolismo , Lumicana , Melanoma , Fosforilação , Ligação ProteicaRESUMO
Phytochemical investigation of the MeOH extract of the stem bark of Antonia ovata led to the isolation of four triterpenoid saponins, along with eleven known compounds. Their structures were established by extensive 1D and 2D NMR, as well as HR-MS analysis and acid hydrolysis. All isolated saponins contained the same tetrasaccharide chain O-beta-d-xylopyranosyl-(1-->2)-O-beta-d-glucopyranosyl-(1-->3)-O-[beta-d-glucopyranosyl-(1-->2)]-beta-d-glucuropyranoside linked to C-3 of esterified derivatives of R(1)-barrigenol, A(1)-barrigenol, barringtogenol C, or camelliagenin. Biological evaluation of the compounds against KB cell line revealed a potent cytotoxic activity with IC(50) values ranging from 3.1 to 6.6microM. The known compounds were found to be inactive at 10microg/ml concentration.
Assuntos
Citotoxinas/química , Citotoxinas/farmacologia , Loganiaceae/química , Casca de Planta/química , Saponinas/química , Saponinas/farmacologia , Triterpenos/química , Proliferação de Células/efeitos dos fármacos , Ácido Clorogênico/análogos & derivados , Citotoxinas/isolamento & purificação , Humanos , Iridoides/química , Células KB , Espectroscopia de Ressonância Magnética , Saponinas/isolamento & purificaçãoRESUMO
Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) with anti-tumor activity. We recently demonstrated that lumican inhibits the migration of melanoma cells and identified beta1 integrin as mediator of this effect [M.F. D'Onofrio, S. Brézillon, T. Baranek, C. Perreau, P.J. Roughley, F.X. Maquart, Y. Wegrowski, Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican, Biochem. Biophys. Res. Commun. 365 (2008) 266-272]. The aim of the present work was to study beta1 integrin, focal adhesion complexes, actin distribution and expression in the presence of lumican substratum in comparison to type I collagen or fibronectin substrata in A375 human melanoma cells. The protein distribution was investigated by immunocytochemistry and confocal microscopy. In parallel, their expression was evaluated by Western immunoblotting and Real-time Reverse Transcription-PCR analyses. The interaction of melanoma cells with the lumican substratum resulted in heterogeneous distribution of beta1 integrin on cell membrane after 24h of seeding. Concomitantly, a reorganization of actin stress fibers and a significant decrease in vinculin immunostaining at focal adhesion complexes were observed. No alteration of the expression was detected at protein and mRNA levels. However, a cytosolic accumulation of vinculin focal adhesion protein was observed on lumican substratum by confocal microscopy. Moreover, vinculin expression was significantly increased in cytosolic fractions in comparison to cells seeded on type I collagen or fibronectin substrata. Our results suggest that lumican induces an alteration of the link between actin filaments and beta1 integrin, characterized by a cytosolic accumulation of vinculin focal adhesion protein, which could lead to a destabilization of focal adhesion complexes. In addition, focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) was significantly decreased. Therefore, the cytoskeleton remodeling and the decreased pFAK phosphorylation induced by lumican in melanoma cells might explain, at least in part, the anti-invasive effect of this SLRP.
Assuntos
Movimento Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Sulfato de Queratano/metabolismo , Melanoma/patologia , Actinas/metabolismo , Western Blotting , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Imuno-Histoquímica , Integrina beta1/metabolismo , Lumicana , Melanoma/metabolismo , Microscopia Confocal , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vinculina/metabolismoRESUMO
Changes in nuclear structures are widely used by pathologists as diagnostic and prognostic indicators in cancer cells. Recent studies have demonstrated that the cell nucleus is probably the most complex organelle in the cell. It contains the genome and is the site of all related activities such as DNA repair, DNA duplication, RNA synthesis, RNA processing and RNA transport. These activities take place within dynamic three-dimensional compartments. The detailed study of these compartments requires an approach termed "cell tomography" based on 3D imaging using confocal microscopy and electron tomography. In this paper, we will first summarize the most recent findings concerning the organization of the cell nucleus. We will then describe markers used to identify molecules specific for various nuclear compartments and their use in tomography of the cell nucleus by confocal microscopy and electron tomography.
Assuntos
Núcleo Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Animais , Nucléolo Celular/fisiologia , Nucléolo Celular/ultraestrutura , Cromatina/fisiologia , Cromossomos/fisiologia , Replicação do DNA/fisiologia , Proteínas de Fluorescência Verde/fisiologia , Humanos , Proteínas Nucleares/fisiologia , Proteínas Nucleares/ultraestruturaRESUMO
The nucleus is a complex volume constituted of numerous subcompartments in which specific functions take place due to a specific spatial organization of their molecular components. To understand how these molecules are spatially organized within these machineries, it is necessary to investigate their three-dimensional organization at high resolution. To reach this goal, electron tomography appears to be a method of choice; it can generate tomograms with a resolution of a few nanometers by using multiple projections of a tilted section several hundred to several thousand nanometers in thickness imaged by transmission electron microscopy (TEM).Specific identification of molecules of interest contained within such thick sections requires their specific immunocytochemical labelling using electron-dense markers. We recently demonstrated that electron tomography of proteins immunostained with nanogold particles before embedding, and subsequently amplified with silver, was very fruitful due to the inherently high spatial resolution of the medium-voltage scanning and transmission electron microscope (STEM). Here we describe this approach, which is very efficient for tracing the 3D organization of proteins within complex machineries by using antibodies raised against one of the proteins, or against GFP to analyse GFP-tagged proteins.
Assuntos
Nucléolo Celular/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Ouro/química , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Neoplasias Pulmonares/patologia , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , TemperaturaRESUMO
This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d.
Assuntos
Células Epiteliais/metabolismo , RNA Ribossômico/metabolismo , Alfa-Amanitina/farmacologia , Técnicas de Cultura de Células , Dactinomicina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Ativação Transcricional , Uridina Trifosfato/análogos & derivadosRESUMO
Hederagenin saponins are largely represented in nature and possess many biological activities such as haemolytic, antiviral, fungicidal, molluscicidal or cytotoxic, partially due to their interaction with the cell membrane. The lysis of erythrocytes (haemolysis) is a simple test to evaluate this adsorption, and this activity has been linked to the structure of the aglycone and also depends on the sugar moiety of the saponin. To further complete our study of the structure-activity relationships of triterpenoid saponins, alpha-hederin and related hederagenin diglycosides were synthesized to better understand the influence of the second sugar (alpha-L-rhamnose, beta-D-xylose or beta-D-glucose) and the substitution of this sugar on alpha-L-arabinose (position 2, 3 or 4). Haemolysis and cytotoxic activity on KB cells were tested. These compounds probably interact with membrane cholesterol and produce destabilization of the membrane inducing haemolysis. Cytotoxicity could involve the same mechanism, although some saponins induce an apoptotic process. The nuclear structure of the KB cell was thus investigated by confocal microscopy. The cytotoxic activity of a second group of hederagenin glucoside saponins was also evaluated. Our results showed that cytotoxicity was a result of both the sugar part and the structure of genin (carboxylic acid or methyl ester).
Assuntos
Apoptose/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Arabinose/química , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Glucose/química , Glicosilação , Humanos , Estrutura Molecular , Ácido Oleanólico/química , Ácido Oleanólico/toxicidade , Saponinas/química , Saponinas/toxicidade , Relação Estrutura-AtividadeRESUMO
UVA irradiation, dose-dependently (5-20 J/cm2), was shown to impair the morphogenic differentiation of human microvascular endothelial cells (HMECs) on Matrigel. Parallely, UVA down-regulated the expression of MMP-2 and MT1-MMP, both at the protein and the mRNA levels. On the contrary, the production of MMP-1 and TIMP-1 by HMECs increased following UVA treatment. The inhibitory effect of UVA on MMP expression and pseudotubes formation was mediated by UVA-generated singlet oxygen (1O2). The contribution of MT1-MMP, but not TIMP-1, to the regulation of HMECs' angiogenic phenotype following UVA irradiation was suggested using elastin-derived peptides and TIMP-1 blocking antibody, respectively.
Assuntos
Proteínas Angiogênicas/metabolismo , Células Endoteliais/efeitos da radiação , Endotélio Vascular/efeitos da radiação , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Pele/citologia , Raios Ultravioleta , Proteínas Angiogênicas/genética , Relação Dose-Resposta à Radiação , Regulação para Baixo/efeitos da radiação , Elastina/farmacologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Metaloproteinases da Matriz Associadas à Membrana , Fenótipo , Oxigênio Singlete/farmacologia , Inibidor Tecidual de Metaloproteinase-1/farmacologiaRESUMO
The role of p53 in apoptosis and the contrasting p53 status in tumors prompted us to investigate the bleomycin-induced apoptosis in p53-null human leukemia HL-60 cells (bleomycin at 160 microM for 7.5 h). Cells with apoptotic phenotype increased from 0.87% in controls to 9.40% in bleomycin-treated cells. Both the enzymes, caspase-3 and -8, were activated. Furthermore, the apoptotic phenotypes totally disappeared with zVAD-fmk, a caspase inhibitor. Besides, cytochrome c release from mitochondria happened simultaneously to apoptotic phenotypes, shrinkage of mitochondria but being independent of the mitochondrial permeability transition, since cyclosporine A and bongkrekic acid were inefficient on induced apoptosis. On the other hand, incubations with bleomycin (BLM) did not result in detectable changes in the expression of Bcl-2- and Bax-mRNA neither Bcl-2- or Bax-proteins. In conclusion, we suggest that BLM can produce apoptosis independently of p53 through three mechanisms: i) at the nuclear level by its endonuclease activities; ii) at the cell membrane, by activating caspases; and iii) at the mitochondria by releasing cytochrome c. These results indicate that BLM-induced apoptosis in HL-60 cells results from the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.
Assuntos
Apoptose , Bleomicina/farmacologia , Genes p53 , Leucemia/tratamento farmacológico , Leucemia/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Ácido Bongcréquico/farmacologia , Caspase 3 , Caspase 8 , Caspases/biossíntese , Caspases/metabolismo , Linhagem Celular , Ciclosporina/farmacologia , Citocromos c/metabolismo , Fragmentação do DNA , Ativação Enzimática , Células HL-60 , Humanos , Microscopia Eletrônica , Mitocôndrias/patologia , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2RESUMO
Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.
Assuntos
Camptotecina/farmacologia , Compartimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/metabolismo , Ribonucleoproteínas , Inibidores da Topoisomerase I , Compartimento Celular/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Meios de Cultura Livres de Soro/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Fase G1/efeitos dos fármacos , Fase G1/genética , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Microscopia Confocal , Proteínas Nucleares/genética , Fatores de Processamento de Serina-Arginina , Proteína p120 Ativadora de GTPase/genética , Proteína p120 Ativadora de GTPase/metabolismoRESUMO
Anthracycline antitumor drugs induce erythroid differentiation of the K562 erythroleukemic cell line at subtoxic concentrations. Aclacinomycin (ACM) stimulates this process by activating the erythroid transcription factor GATA-1, that controls genes involved in hemoglobin biosynthesis. To investigate the implication of GATA-1 in this process, we used a specific anti-GATA-1 polyclonal antibody that we produced in our laboratory. The GATA-1 transcription factor was then monitored during erythroid differentiation induced by aclacinomycin. Here we show that a cellular redistribution and a modification of the phosphorylation state of this transcription factor occurred during ACM-mediated cell differentiation. It suggests that anthracyclines can induce the erythroid differentiation of neoplastic cells by activating the transcription factor GATA-1, probably via its clustering into nuclear foci.