RESUMO
Glioblastoma multiforme therapy remains a significant challenge since there is a lack of effective treatment for this cancer. As most of the examined gliomas express or overexpress cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptors γ (PPARγ), we decided to use these proteins as therapeutic targets. Toxicity, antiproliferative, proapoptotic, and antimigratory activity of COX-2 inhibitor (celecoxib-CXB) and/or PPARγ agonist (Fmoc-L-Leucine-FL) was examined in vitro on temozolomide resistant U-118 MG glioma cell line and comparatively on BJ normal fibroblasts and immortalized HaCaT keratinocytes. The in vivo activity of both agents was studied on C. elegans nematode. Both drugs effectively destroyed U-118 MG glioma cells via antiproliferative, pro-apoptotic, and anti-migratory effects in a concentration range 50-100 µM. The mechanism of action of CXB and FL against glioma was COX-2 and PPARγ dependent and resulted in up-regulation of these factors. Unlike reports by other authors, we did not observe the expected synergistic or additive effect of both drugs. Comparative studies on normal BJ fibroblast cells and immortalized HaCaT keratinocytes showed that the tested drugs did not have a selective effect on glioma cells and their mechanism of action differs significantly from that observed in the case of glioma. HaCaTs did not react with concomitant changes in the expression of COX-2 and PPARγ and were resistant to FL. Safety tests of repurposing drugs used in cancer therapy tested on C. elegans nematode indicated that CXB, FL, or their mixture at a concentration of up to 100 µM had no significant effect on the entire nematode organism up to 4th day of incubation. After a 7-day treatment, CXB significantly shortened the lifespan of C. elegans at 25-400 µM concentration and body length at 50-400 µM concentration.
Assuntos
Caenorhabditis elegans , Glioblastoma , Leucina/análogos & derivados , Animais , Humanos , Celecoxib/farmacologia , Celecoxib/uso terapêutico , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Caenorhabditis elegans/metabolismo , Ciclo-Oxigenase 2/metabolismo , PPAR gama/metabolismo , Sulfonamidas/farmacologia , Pirazóis/farmacologia , Apoptose , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Linhagem Celular , Glioblastoma/tratamento farmacológico , Linhagem Celular TumoralRESUMO
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
INTRODUCTION: Garlic (Allium sativum) is the subject of many studies due to its numerous beneficial properties. Although compounds of garlic have been studied by various analytical methods, their tissue distributions are still unclear. Mass spectrometry imaging (MSI) appears to be a very powerful tool for the identification of the localisation of compounds within a garlic clove. OBJECTIVE: Visualisation of the spatial distribution of garlic low-molecular weight compounds with nanoparticle-based MSI. METHODOLOGY: Compounds occurring on the cross-section of sprouted garlic has been transferred to gold-nanoparticle enhanced target (AuNPET) by imprinting. The imprint was then subjected to MSI analysis. RESULTS: The results suggest that low molecular weight compounds, such as amino acids, dipeptides, fatty acids, organosulphur and organoselenium compounds are distributed within the garlic clove in a characteristic manner. It can be connected with their biological functions and metabolic properties in the plant. CONCLUSION: New methodology for the visualisation of low molecular weight compounds allowed a correlation to be made between their spatial distribution within a sprouted garlic clove and their biological function. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Alho/química , Ouro/química , Espectrometria de Massas/métodos , Nanopartículas Metálicas/químicaRESUMO
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
Third-generation poly(amidoamine) dendrimer (PAMAM) was modified by stepwise primary amine group amidation with d-glucoheptono-1,4-lactone. The physicochemical properties of the conjugates-size, ζ potential in lysosomal pH 5 and in neutral aqueous solutions, as well as intramolecular dynamics by differential scanning calorimetry-were determined. Internalization and toxicity of the conjugates against normal human fibroblasts BJ were monitored in vitro in order to select an appropriate carrier for a drug delivery system. It was found that initial glucoheptoamidation (up to 1/3 of amine groups of neat dendrimers available) resulted in increase of conjugate size and ζ potential. Native or low substituted dendrimer conjugates accumulated efficiently in fibroblast cells at nontoxic 1 µM concentration. Further substitution of dendrimer caused consistent decrease of size and ζ potential, cell accumulation, and toxicity. All dendrimers are amorphous at 36.6 °C as determined by differential scanning calorimetry (DSC). The optimized dendrimer, half-filled with glucoheptoamide substituents, was applied as carrier bearing two covalently attached cytisine molecules: a rigid and hydrophobic alkaloid. The conjugate with 2 cytisine and 16 glucoheptoamide substituents showed fast accumulation and no toxicity up to 200 µM concentration. The half-glucoheptoamidated PAMAM dendrimer was selected as a promising anticancer drug carrier for further applications.
RESUMO
Strawberry (Fragaria x ananassa Duch., Rosaceae) is the subject of many research studies due to its numerous features such as unique taste, aroma and health qualities. The distribution of low molecular weight metabolites belonging to aldehydes, ketones, alcohols, esters, organic acids, phenolics, amino acids and sugars classes within strawberry fruit cross-section was studied using mass spectrometry imaging (MSI) method with 109Ag nanoparticle enhanced target (109AgNPET). Correlation of distribution of over thirty compounds found in cross-section of strawberry with their biological function is also included.
Assuntos
Fragaria/metabolismo , Isótopos/química , Espectrometria de Massas/métodos , Nanopartículas Metálicas/química , Prata/química , Aldeídos/química , Aminoácidos/química , Ácidos Carboxílicos/química , Flavonoides/química , Fragaria/química , Cetonas/química , Peso Molecular , Fenóis/química , Açúcares/química , Vitaminas/químicaRESUMO
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
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
Gold nanoparticle-enhanced target (AuNPET) was used for detailed investigation of various materials of biological origin - human fingerprint, onion bulb and chicken liver. Analysis of these objects was focused on toxic and harmful compounds - designer drug containing pentedrone, diphenylamine in onion and potentially cancerogenic metronidazole antibiotic in liver. Detection of large quantity of endogenous compounds from mentioned objects is also shown. Most of analyzed compounds were also localized with MS imaging and relationship between their function and location was discussed. Detected compounds belong to a very wide range of chemical compounds such as saccharides, ionic and non-ionic glycerides, amino acids, fatty acids, sulfides, sulfoxides, phenols etc. Fingerprint experiments demonstrate application of AuNPET for detection, structure confirmation and also co-localization of drug with ridge patterns proving person-drug contact.