RESUMO
Glioblastoma multiforme (GBM) is the most malignant type of central nervous system tumor that is resistant to all currently used forms of therapy. Thus, more effective GBM treatment strategies are being investigated, including combined therapies with drugs that may cross the blood brain barrier (BBB). Another important issue considers the decrease of deleterious side effects of therapy. It has been shown that nanocarrier conjugates with biotin can penetrate BBB. In this study, biotinylated PAMAM G3 dendrimers substituted with the recognized anticancer agents cyclooxygenase-2 (COX-2) inhibitor celecoxib and peroxisome proliferator-activated receptor γ (PPARγ) agonist Fmoc-L-Leucine (G3-BCL) were tested in vitro on human cell lines with different p53 status: glioblastoma (U-118 MG), normal fibroblasts (BJ) and immortalized keratinocytes (HaCaT). G3-BCL penetrated efficiently into the lysosomal and mitochondrial compartments of U-118 MG cells and induced death of U-118 MG cells via apoptosis and inhibited proliferation and migration at low IC50 = 1.25 µM concentration, considerably lower than either drug applied alone. Comparison of the effects of G3-BCL on expression of COX-2 and PPARγ protein and PGE2 production of three different investigated cell line phenotypes revealed that the anti-glioma effect of the conjugate was realized by other mechanisms other than influencing PPAR-γ expression and regardless of p53 cell status, it was dependent on COX-2 protein level and high PGE2 production. Similar G3-BCL cytotoxicity was seen in normal fibroblasts (IC50 = 1.29 µM) and higher resistance in HaCaT cells (IC50 = 4.49 µM). Thus, G3-BCL might be a good candidate for the targeted, local glioma therapy with limited site effects.
Assuntos
Antineoplásicos/farmacologia , Celecoxib/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dendrímeros , Glioblastoma/tratamento farmacológico , Leucina/análogos & derivados , PPAR gama/agonistas , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Biotinilação , Celecoxib/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Dendrímeros/metabolismo , Dinoprostona/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glioblastoma/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Leucina/farmacologia , Leucina/uso terapêutico , Necrose/tratamento farmacológico , PPAR gama/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
In search for soluble derivatives of PAMAM dendrimers as potential carriers for hydrophobic drugs, the conjugates of PAMAM G3 with biotin, further converted into glycodendrimer with d-glucoheptono-1,4-lactone, were prepared. Polyamidoamine dendrimer (PAMAM) of third generation, G3 was functionalized with four biotin equivalents covalently attached to terminal amine nitrogens via amide bond G34B. The remaining 28 amine groups were blocked by glucoheptoamide substituents (gh) to give G34B28gh or with one fluorescein equivalent (attached by reaction of G34B with fluorescein isothiocyanate, FITC) via thiourea bond as FITC followed by exhaustive glucoheptoamidation to get G34B27gh1F. As a control the G3 substituted totally with 32 glucoheptoamide residues, G3gh and its fluorescein labeled analogue G331gh1F were synthesized. The glucoheptoamidation of PAMAM G0 dendrimer with glucoheptono-1,4-lactone was performed in order to fully characterize the 1H NMR spectra of glucoheptoamidated PAMAM dendrimers and to control the derivatization of G3 with glucoheptono-1,4-lactone. Another two derivatives of G3, namely G34B28gh1F' and G332ghF', with ester bonded fluorescein were also obtained. Biological properties of obtained dendrimer conjugates were estimated in vitro with human cell lines: normal fibroblast (BJ) and two cancer glioblastoma (U-118 MG) and squamous carcinoma (SCC-15), including cytotoxicity by reduction of XTT and neutral red (NR) assays. Cellular uptake of dendrimer conjugates was evaluated with confocal microscopy. Obtained results confirmed, that biotinylated bioconjugates have always lower cytotoxicity and 3-4 times higher cellular uptake than non-biotinylated dendrimer conjugates in all cell lines. Comparison of various cell lines revealed different dose-dependent cell responses and the lower cytotoxicity of examined dendrimer conjugates for normal fibroblasts and squamous carcinoma, as compared with much higher cytotoxic effects seen in glioblastoma cell line. Synthetized multi-functional conjugate (G34B27gh1F) is a promising candidate as biocompatible vehicle for hydrophobic molecules used in anticancer therapy.
Assuntos
Amidas/farmacocinética , Antineoplásicos/química , Biotina/farmacocinética , Dendrímeros/farmacocinética , Amidas/química , Biotina/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dendrímeros/química , Relação Dose-Resposta a Droga , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.
Assuntos
Timidilato Sintase/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Fosforilação , CoelhosRESUMO
Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
Assuntos
Timidilato Sintase/metabolismo , Tirosina/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Bovinos , Linhagem Celular Tumoral , Humanos , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Timidilato Sintase/química , Timidilato Sintase/isolamento & purificação , Timo/enzimologia , Tirosina/químicaRESUMO
Recent achievement in anticancer therapy considers the application of repurposed drugs in optimal combinations with the use of specific carriers for their targeted delivery. As a result, new optimized medications with reduced side effects can be obtained. In this study, two known anticancer drugs, celecoxib and/or simvastatin, were conjugated covalently with PAMAM G3 dendrimer and tested in vitro against human squamous carcinoma (SCC-15-15) and glioblastoma (U-118 MG) cells, as well as normal human fibroblasts (BJ). The obtained conjugates were also substituted with biotin and R-glycidol to increase their affinity for cancer cells and were characterized with NMR spectroscopy and dynamic light scattering technique. Conjugates furnished with two celecoxib and four simvastatin residues revealed the very high effectiveness and dramatically decreased the SCC-15 and U-118 MG cell viability at very low concentrations with IC50 equal to about 3 µM. Its action was 20-50-fold stronger than that of either drug alone or as a mixture. Combined conjugate revealed also additive action since it was 2-8-fold more effective than conjugates with either single drug. The combined conjugate revealed rather low specificity since it was also highly cytotoxic for BJ cells. Despite this, it may be concluded that biotinylated and R-glycidylated PAMAM G3 dendrimers substituted with both celecoxib and simvastatin can be considered as a new perspective anticancer agent, effective in therapy of malignant, incurable glioblastomas.
RESUMO
Glioblastoma multiforme (GBM) is a one of the most widely diagnosed and difficult to treat type of central nervous system tumors. Resection combined with radiotherapy and temozolomide (TMZ) chemotherapy prolongs patients' survival only for 12 - 15 months after diagnosis. Moreover, many patients develop TMZ resistance, thus important is search for a new therapy regimes including targeted drug delivery. Most types of GBM reveal increased expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2), that are considered as valuable therapeutic target. In these studies, the anti-tumor properties of the selective COX-2 inhibitor celecoxib (CXB) and biotinylated third generation of the poly(amidoamine) dendrimer substituted with 31 CXB residues (G3BC31) on TMZ -resistant U-118 MG glioma cell line were examined and compared with the effect of TMZ alone including viability, proliferation, migration and apoptosis, as well as the cellular expression of COX-2, ATP level, and PGE2 production. Confocal microscopy analysis with the fluorescently labeled G3BC31 analogue has shown that the compound was effectively accumulated in U-118 MG cells in time-dependent manner and its localization was confirmed in lysosomes but not nuclei. G3BC31 reveal much higher cytotoxicity for U-118 MG cells at relatively low concentrations in the range of 2-4 µM with compared to CBX alone, active at 50-100 µM. This was due to induction of apoptosis and inhibition of proliferation and migration. Observed effects were concomitant with reduction of PGE2 production but independent of COX-2 expression. We suggest that investigated conjugate may be a promising candidate for therapy of TMZ-resistant glioblastoma multiforme, although applicable in local treatment, since our previous study of G3BC31 did not demonstrate selectivity against glioma cells compared to normal human fibroblasts. However, it has to be pointed that in our in vivo studies conducted with model organism, Caenorhabditis elegans indicated high anti-nematode activity of G3BC31 in comparison with CXB alone that confirms of usefulness of that organism for estimation of anti-cancer drug toxicity.
Assuntos
Neoplasias Encefálicas , Dendrímeros , Glioblastoma , Glioma , Antineoplásicos Alquilantes/uso terapêutico , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Celecoxib/farmacologia , Celecoxib/uso terapêutico , Linhagem Celular Tumoral , Dendrímeros/farmacologia , Glioblastoma/tratamento farmacológico , Glioma/tratamento farmacológico , Humanos , Poliaminas , Temozolomida/farmacologia , Temozolomida/uso terapêuticoRESUMO
Recognition of the molecular mechanisms of keratinocyte participation in normal skin homeostasis and in pathogenesis may lead to creation of more effective tools for topical application of cosmetics, cosmeceutics and drugs to a particular location within the skin for prevention and therapy of many skin disorders and diseases. For this purpose, the PAMAM G3 dendrimer with amide linkages of 9 biotin molecules and 10 molecules of pyridoxal phosphate (BC-PAMAM) was constructed, and its biological properties and cellular uptake and localization were investigated in the HaCaT keratinocytes. BC-PAMAM is nontoxic for HaCaT cells, as estimated by two assays (Neutral Red and tetrazolium salt reduction, XTT), and revealed low apoptosis induction at up to 50 µM concentration. Fluorescent labeled BC-PAMAM accumulates in HaCaT cells with high efficiency in a concentration-dependent manner. Its mitochondrial localization, estimated as Mander's colocalization coefficient, is substantially lower than the native PAMAM, and that correlates with its cytotoxicity. The only undesirable, but significant inhibitory effect on cell mobility, evaluated by the wound healing test, was observed at 10 µM BC-PAMAM. The important anti-inflammatory action of BC-PAMAM was clearly documented by decreased production of total IL-1α, assayed with an ELISA test with unstimulated and stimulated by bacterial antigens (LPS and GroEL) HaCaT cells. Thus, it is expected that the biotin pyridoxal phosphate conjugated PAMAM may be considered as a potential carrier for safe delivery of vitamins and drugs into the epidermis.
RESUMO
Squamous cell carcinoma (SCC) remains a main cause of mortality in patients with neck and head cancers, with poor prognosis and increased prevalence despite of available therapies. Recent studies have identified a role of cyclooxygenases, particularly inducible isoform cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in cancer cell proliferation, and its inhibition become a target for control of cancer development, particularly in the view of recognized additive or synergic action of COX-2 inhibitors with other forms of therapy. Nimesulide (N), the selective COX-2 inhibitor, inhibits growth and proliferation of various types of cancer cells by COX-2 dependent and independent mechanisms. In the presented study, the conjugates of biotinylated third generation poly(amidoamine) dendrimer (PAMAM) with covalently linked 18 (G3B18N) and 31 (G3B31N) nimesulide residues were synthesized and characterized by NMR spectroscopy. Biological properties of conjugates were evaluated, including cytotoxicity, proliferation, and caspase 3/7 activities in relation to COX-2/PGE2 axis signaling in human normal fibroblast (BJ) and squamous cell carcinoma (SCC-15). Both conjugates exerted a selective cytotoxicity against SCC-15 as compared with BJ cells at low 1.25-10 µM concentration range and their action in cancer cells was over 250-fold stronger than nimesulide alone. Conjugates overcome apoptosis resistance and sensitized SCC-15 cells to the apoptotic death independently of COX-2/PGE2 axis. In normal human fibroblasts the same concentrations of G3B31N conjugate were less effective in inhibition of proliferation and induction of apoptosis, as measured by caspase 3/7 activity in a manner depending on increase of PGE2 production by either COX-1/COX-2.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Dendrímeros/química , Dendrímeros/síntese química , Fibroblastos/efeitos dos fármacos , Sulfonamidas/química , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dendrímeros/farmacologia , Dinoprostona/metabolismo , HumanosRESUMO
Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24â¯h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2-2.5 times) in both cancer lines. Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJâ¯>â¯U-118 MGâ¯>â¯SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22⯵M, respectively and lowest cytotoxicity in HaCaT cells (IC50â¯=â¯2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.
Assuntos
Antineoplásicos/farmacologia , Celecoxib/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dendrímeros/farmacologia , Leucina/análogos & derivados , PPAR gama/agonistas , Biotinilação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucina/farmacologia , Neoplasias/tratamento farmacológicoRESUMO
This review gives a brief overview over the major aspects of application of the nicotine alkaloid and its close derivatives in the therapy of some neurodegenerative disorders and diseases (e.g. Alzheimer's disease, Parkinson's disease, Tourette's syndrome, schizophrenia etc.). The issues concerning methods of nicotine analysis and isolation, and some molecular aspects of nicotine pharmacology are included. The natural and synthetic analogues of nicotine that are considered for medical practice are also mentioned. The molecular properties of two naturally occurring nicotine enantiomers are compared--the less-common but less-toxic (R)-nicotine is suggested as a natural compound that may find its place in pharmaceutical practice.
Assuntos
Drogas em Investigação/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Nicotiana , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Animais , Drogas em Investigação/uso terapêutico , Humanos , Isomerismo , Doenças Neurodegenerativas/metabolismo , Nicotina/análogos & derivados , Nicotina/isolamento & purificação , Nicotina/metabolismo , Nicotina/uso terapêutico , Agonistas Nicotínicos/isolamento & purificação , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/uso terapêutico , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Tecnologia Farmacêutica/métodos , Nicotiana/químicaRESUMO
Monocyte/macrophage and natural killer (NK) cells are partners from a phylogenetic standpoint of innate immune system development and its evolutionary progressive interaction with adaptive immunity. The equally conservative ways of development and differentiation of both invertebrate hemocytes and vertebrate macrophages are reviewed. Evolutionary conserved molecules occurring in macrophage receptors and effectors have been inherited by vertebrates after their common ancestor with invertebrates. Cytolytic functions of mammalian NK cells, which are rooted in immune cells of invertebrates, although certain NK cell receptors (NKRs) are mammalian new events, are characterized. Broad heterogeneity of macrophage and NK cell phenotypes that depends on surrounding microenvironment conditions and expression profiles of specific receptors and activation mechanisms of both cell types are discussed. The particular tissue specificity of macrophages and NK cells, as well as their plasticity and mechanisms of their polarization to different functional subtypes have been underlined. The chapter summarized studies revealing the specific molecular mechanisms and regulation of NK cells and macrophages that enable their highly specific cross-cooperation. Attention is given to the evolving role of human monocyte/macrophage and NK cell interaction in pathogenesis of hypersensitivity reaction-based disorders, including autoimmunity, as well as in cancer surveillance and progression.
Assuntos
Vigilância Imunológica/imunologia , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Imunidade Adaptativa/imunologia , Animais , Autoimunidade/imunologia , Evolução Molecular , Humanos , Hipersensibilidade/imunologia , Imunidade Inata , Células Matadoras Naturais/citologia , Macrófagos/citologia , Monócitos/citologia , Neoplasias/imunologia , FilogeniaRESUMO
In a search for the safe vitamin carrier the PAMAM G3 dendrimer covalently substituted with 9 and 10 molecules of vitamin B7 (biotin) and B6 (pyridoxal), respectively (BC-PAMAM) was investigated. Dendrimer substitution with B-group vitamins significantly alters its biological properties as compared to native form. Observed effects on investigated cell parameters including morphology, adhesion, migration and ATP level were different for normal human fibroblasts (BJ) and squamous cell carcinoma (SCC-15) cell lines. BC-PAMAM revealed significantly less pronounced effects on investigated parameters, particularly at higher concentrations (5-50µM), which is relevant with its lower positive surface charge, as compared with native form. The bioconjugate, up to 50µM concentration, appeared to be a safe vitamin carrier to normal fibroblasts, without significant effect on their adhesion, shape and migration as well as on intracellular ATP level. In SCC-15 cells BC-PAMAM, at low concentrations (0.1-0.5µM), altered the cell shape and increase adhesion, whereas at higher concentrations opposite effects were seen. Measurements of cellular level of ATP showed that higher resistance of cancer cells to toxic effects of native PAMAM dendrimers may be due to higher energy supply of cancer cells.
Assuntos
Biotina/administração & dosagem , Dendrímeros/administração & dosagem , Portadores de Fármacos/administração & dosagem , Piridoxal/administração & dosagem , Trifosfato de Adenosina/metabolismo , Biotina/química , Biotina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Dendrímeros/química , Dendrímeros/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Humanos , Piridoxal/química , Piridoxal/farmacologiaRESUMO
The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM) dendrimer and its biotin-pyridoxal (BC-PAMAM) bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ) and squamous epithelial carcinoma (SCC-15) cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold) tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander's coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%-35% of the total nuclei area at all investigated concentrations, with lower level (15%-25%) observed for BC-PAMAM. In fibroblasts, the dendrimer nuclear signal amounted to 15% at nontoxic and up to 70% at toxic concentrations, whereas BC-PAMAM remained at a lower concentration-dependent level (0.3%-20%). Mitochondrial localization of PAMAM and BC-PAMAM revealed similar patterns in both cell lines, depending on the extracellular dendrimer concentration, and presented significantly lower signals from BC-PAMAM, which correlated well with the cytotoxicity.
Assuntos
Biotina/química , Núcleo Celular/efeitos dos fármacos , Dendrímeros/química , Mitocôndrias/efeitos dos fármacos , Piridoxal/química , Biotina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dendrímeros/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Mitocôndrias/metabolismo , Piridoxal/farmacologiaRESUMO
The effects of cyclosporin A (CsA), a potent immunosuppressive drug with antiparasitic activity, on the innate immunological response in guinea pig lungs during an early period (6th and 14th days) after T. spiralis infection were studied. CsA treatment of T. spiralis-infected guinea pigs caused a significant attenuation of immunological response in lungs by decreasing lymphocyte infiltration into pulmonary alveolar space, inhibiting alveolar macrophage superoxide anion production and lowering both the production of NO metabolites measured in bronchoalveolar lavage fluid and expression of the iNOS protein in lung homogenates, allowing us to speculate that the T. spiralis-dependent immunological response is dependent on lymphocyte T function. Interestingly, CsA itself had a pro-inflammatory effect, promoting leucocyte accumulation and macrophage superoxide production in guinea pig lungs. This observation may have a relevance to the situation in patients undergoing CsA therapy. Macrophage expression of the iNOS protein, evaluated by immunoblotting was not influenced by treatment of animals with CsA or anti-TGF-antibody, indicating different regulation of the guinea pig and murine enzymes.
Assuntos
Ciclosporina/metabolismo , Pulmão/imunologia , Triquinelose/imunologia , Animais , Cobaias , Immunoblotting , Leucócitos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/imunologia , Superóxidos/metabolismo , Trichinella spiralisRESUMO
Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N(5,10)-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, 'wild')(dUMP)) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.
Assuntos
Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Substituição de Aminoácidos , Animais , Arginina/genética , Catálise , Nucleotídeos de Desoxiuracil/química , Nucleotídeos de Desoxiuracil/metabolismo , Indução Enzimática/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Lacticaseibacillus casei/enzimologia , Leucemia L1210/metabolismo , Lisina/genética , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Células Tumorais CultivadasRESUMO
A third-generation polyamidoamine dendrimer (PAMAM G3) was used as a macromolecular carrier for pyridoxal and biotin. The binary covalent bioconjugate of G3, with nine molecules of biotin per one molecule of G3 (G3(9B)), and the ternary covalent bioconjugate of G3, with nine biotin and ten pyridoxal molecules (G3(9B10P)), were synthesized. The biotin and pyridoxal residues of the bioconjugate were available for carboxylase and transaminase enzymes, as demonstrated in the conversion of pyruvate to oxaloacetate and alanine to pyruvate, respectively, by in vitro monitoring of the reactions, using (1)H nuclear magnetic resonance spectroscopy. The toxicity of the ternary bioconjugate (BC-PAMAM) was studied in vitro on BJ human normal skin fibroblasts and human squamous cell carcinoma (SCC-15) cell cultures in comparison with PAMAM G3, using three cytotoxicity assays (XTT, neutral red, and crystal violet) and an estimation of apoptosis by confocal microscopy detection. The tests have shown that BC-PAMAM has significantly lower cytotoxicity compared with PAMAM. Nonconjugated PAMAM was not cytotoxic at concentrations up to 5 µM (NR) and 10 µM (XTT), and BC-PAMAM was not cytotoxic up to 50 µM (both assays) for both cell lines. It has been also found that normal fibroblasts were more sensitive than SCC to both PAMAM and BC-PAMAM. The effect of PAMAM and BC-PAMAM on the initiation of apoptosis (PAMAM in fibroblasts at 5 µM and BC-PAMAM at 10 µM in both cell lines) corresponded with cytotoxicity assays for both cell lines. We concluded that normal fibroblasts are more sensitive to the cytotoxic effects of the PAMAM G3 dendrimer and that modification of its surface cationic groups by substitution with biologically active molecules significantly decreases that effect, confirming that PAMAM G3 is a useful candidate as a carrier for active biocompound delivery.
Assuntos
Biotina/farmacologia , Morte Celular/efeitos dos fármacos , Dendrímeros/farmacologia , Nanoconjugados/química , Piridoxal/farmacologia , Biotina/química , Linhagem Celular , Linhagem Celular Tumoral , Dendrímeros/química , Fibroblastos/efeitos dos fármacos , Humanos , Piridoxal/químicaRESUMO
Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Larva/enzimologia , Timidilato Sintase/metabolismo , Trichinella spiralis/enzimologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Esôfago/citologia , Esôfago/enzimologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Microscopia Confocal , Sistema Nervoso/citologia , Sistema Nervoso/enzimologia , Sistema Nervoso/crescimento & desenvolvimento , Especificidade de Órgãos , Óvulo/citologia , Óvulo/enzimologia , Óvulo/crescimento & desenvolvimento , Transporte Proteico , Espermatócitos/citologia , Espermatócitos/enzimologia , Espermatócitos/crescimento & desenvolvimento , Timidilato Sintase/genética , Trichinella spiralis/citologia , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimentoRESUMO
To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.
Assuntos
Macrófagos Alveolares/parasitologia , Fosfoproteínas/biossíntese , Trichinella spiralis/fisiologia , Animais , Células Cultivadas , Ciclosporina/farmacologia , Cobaias , Imunossupressores/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , NADPH Oxidases/metabolismo , Explosão Respiratória , Superóxidos/metabolismoRESUMO
BACKGROUND: In newborn full-term and preterm infants the urine nitrites and nitrates (NOx) were measured, in order to investigate the effects of different pathological conditions (infection, hypoxia) on systemic nitric oxide production. METHODS: Urine nitrites and nitrates were determined by means of the Griess reaction, after reduction of nitrates to nitrites with nitrate reductase. RESULTS: The NOx level was higher in preterm (278 nmol/mL) than full-term (176 nmol/mL) infants. Low NOx (115 nmol/mL) levels accompanied generalized infections, while its high contents (650 nmol/mL) was found in cytomegalovirus and one case of Pneumocystis carinii infection. Moderate increase of NOx production was observed in infants with local pulmonary infections and encephalopathies. CONCLUSIONS: The results indicate urinary NOx level is lowered in infants with life-threatening generalized infection. A possibility of a rapid test based on newborn urinary NOx level determination is considered.
Assuntos
Recém-Nascido Prematuro/urina , Óxido Nítrico/metabolismo , Humanos , Recém-Nascido , Doenças do Prematuro/metabolismo , Nitratos/urina , Nitritos/urinaRESUMO
In order to assess immunological response, induced in guinea-pig lungs by Trichinella spiralis, cellular infiltration into pulmonary alveolar space and production of O(2)(-) and NO in alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF), as well as accumulation of nitric oxide (NO) metabolites in BALF and serum, were evaluated during the early period of primary T. spiralis infection (from 4th to 8th and on 14th day after oral administration of larvae) and on 6th day after secondary infection. Primary infection caused increased infiltration of lymphocytes, macrophages, neutrophils and eosinophils, while secondary infection resulted in raised lymphocyte and eosinophil numbers. In spite of marked cellular infiltration of alveolar space, only very limited activation of effector cells, pointing to a suppressed innate response, was apparent, as (i) BALF supernatant phospholipid/protein concentration ratio, and lung levels of phospholipid peroxidation markers, conjugated dienes and malondialdehyde, did not change during 7 days following infection; (ii) primary, but not secondary, infection caused only a transient increase of superoxide anion production by alveolar macrophages; (iii) despite expression of inducible nitric oxide synthase in macrophages of control, infected and BCG-treated animals, and of interferon (IFN)-gamma-like activity in sera of infected animals, macrophage nitric oxide production was not affected by infection, even after additional stimulation in vitro (lipopolisaccharide + hrIFN-gamma) or in vivo (BCG or secondary T. spiralis infection); and (iv) increased nitrate concentrations were found in BALF supernatant and serum, but not in lung homogenates, of infected animals.