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
Fulvestrant (F), lapatinib (L), and paclitaxel (P) are hydrophobic, anticancer drugs used in the treatment of estrogen receptor (ER) and epidermal growth factor receptor (EGFR)-positive breast cancer. In this study, glycidylated PAMAM G4 dendrimers, substituted with F, L, and/or P and targeting tumor cells, were synthesized and characterized, and their antitumor activity against glioma U-118 MG and non-small cell lung cancer A549 cells was tested comparatively with human non-tumorogenic keratinocytes (HaCaT). All cell lines were ER+ and EGFR+. In addition, the described drugs were tested in the context of antinematode therapy on C. elegans. The results show that the water-soluble conjugates of G4P, G4F, G4L, and G4PFL actively entered the tested cells via endocytosis due to the positive zeta potential (between 13.57-40.29 mV) and the nanoparticle diameter of 99-138 nm. The conjugates of G4P and G4PFL at nanomolar concentrations were the most active, and the least active conjugate was G4F. The tested conjugates inhibited the proliferation of HaCaT and A549 cells; in glioma cells, cytotoxicity was associated mainly with cell damage (mitochondria and membrane transport). The toxicity of the conjugates was proportional to the number of drug residues attached, with the exception of G4L; its action was two- and eight-fold stronger against glioma and keratinocytes, respectively, than the equivalent of lapatinib alone. Unfortunately, non-cancer HaCaT cells were the most sensitive to the tested constructs, which forced a change in the approach to the use of ER and EGFR receptors as a goal in cancer therapy. In vivo studies on C. elegans have shown that all compounds, most notably G4PFL, may be potentially useful in anthelmintic therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Dendrímeros , Glioma , Neoplasias Pulmonares , Parasitos , Humanos , Animais , Lapatinib/farmacologia , Paclitaxel/farmacologia , Fulvestranto , Dendrímeros/farmacologia , Caenorhabditis elegansRESUMO
The natural xanthone α-mangostin (αM) exhibits a wide range of pharmacological activities, including antineoplastic and anti-nematode properties, but low water solubility and poor selectivity of the drug prevent its potential clinical use. Therefore, the targeted third-generation poly(amidoamine) dendrimer (PAMAM G3) delivery system was proposed, based on hyperbranched polymer showing good solubility, high biocompatibility and low immunogenicity. A multifunctional nanocarrier was prepared by attaching αM to the surface amine groups of dendrimer via amide bond in the ratio 5 (G32B12gh5M) or 17 (G32B10gh17M) residues per one dendrimer molecule. Twelve or ten remaining amine groups were modified by conjugation with D-glucoheptono-1,4-lactone (gh) to block the amine groups, and two biotin (B) residues as targeting moieties. The biological activity of the obtained conjugates was studied in vitro on glioma U-118 MG and squamous cell carcinoma SCC-15 cancer cells compared to normal fibroblasts (BJ), and in vivo on a model organism Caenorhabditis elegans. Dendrimer vehicle G32B12gh at concentrations up to 20 µM showed no anti-proliferative effect against tested cell lines, with a feeble cytotoxicity of the highest concentration seen only with SCC-15 cells. The attachment of αM to the vehicle significantly increased cytotoxic effect of the drug, even by 4- and 25-fold for G32B12gh5M and G32B10gh17M, respectively. A stronger inhibition of cells viability and influence on other metabolic parameters (proliferation, adhesion, ATP level and Caspase-3/7 activity) was observed for G32B10gh17M than for G32B12gh5M. Both bioconjugates were internalized efficiently into the cells. Similarly, the attachment of αM to the dendrimer vehicle increased its toxicity for C. elegans. Thus, the proposed α-mangostin delivery system allowed the drug to be more effective in the dendrimer-bound as compared to free state against both cultured the cancer cells and model organism, suggesting that this treatment is promising for anticancer as well as anti-nematode chemotherapy.
Assuntos
Dendrímeros/química , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Poliaminas/química , Xantonas/administração & dosagem , Animais , Biotinilação , Caenorhabditis elegans , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Garcinia mangostana , Humanos , Fitoterapia , Xantonas/químicaRESUMO
With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.
Assuntos
Antinematódeos/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Xantonas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Bibliotecas de Moléculas Pequenas , Timidilato Sintase/metabolismo , Testes de Toxicidade , Xantonas/químicaRESUMO
Human colon cancer C85 cell response to methotrexate has been documented previously to take on a form of reversible premature senescence. Seeking genomic aberrations encompassing candidate genes whose functional impairment could determine such a response to the drug, an array Comparative Genomic Hybridization method was applied, complemented by expression microarray data set searching. In the C85 cell genome, only short aberrations were identified, classified as focal chromosomal aberrations. 62% of the aberrant regions, selected by referral to normal human colon epithelium, were not carrying any gene. Out of the genes, subject to aberrations, 50% were protein-coding ones. Expression of those that could serve a signaling or a growth-regulatory function was found to be either downregulated or unchanged during C85 cell progression into methotrexate-induced senescence. Lack of extensive chromosomal instability in C85 cells is hypothesized to be attributed to the presence of the wild-type tumor suppressor p53 protein. Although two p53 protein isoforms were detected in C85 cells, stabilization and acetylation of the full-length p53 isoform were shown to underpin progression of the cells into premature senescence upon methotrexate treatment.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Senescência Celular , Aberrações Cromossômicas , Neoplasias do Colo/patologia , Metotrexato/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Genômica , Humanos , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Polyhydroxylated dendrimer was synthesized from poly(amidoamine) (PAMAM) dendrimer generation 3 by addition of glycidol (G3gl). G3gl megamer was further modified by binding PAMAM G0 dendrimers by activation of G3gl with p-nitrophenylchloroformate, followed by the addition of excess PAMAM G0 and purification using dialysis. The maximum G0 binding capacity of G3gl was 12 in the case when G0 was equipped with two covalently attached nimesulide equivalents. Nimesulide (N) was converted into N-(p-nitrophenyl) carbonate derivative and fully characterized using X-ray crystallography and spectral methods. Nimesulide was then attached to G0 via a urea bond to yield G02N. The mixed generation G3gl-G02N megamer was characterized using 1H NMR spectroscopy, and its molecular weight was estimated to be 22.4 kDa. The AFM image of G3gl-G02N deposited on mica demonstrated aggregation of nimesulide-covered megamer. The height of the deposited megamer was 8.5 nm. The megameric conjugate with nimesulide was tested in vitro on three human cell lines: squamous cell carcinoma (SCC-15) and glioblastoma (U-118 MG) overexpressing cyclooxygenase-2 (COX-2), and normal skin fibroblasts (BJ). The conjugate efficiently penetrated into all cells and was more cytotoxic against SCC-15 than against BJ. Moreover, the conjugate produced a strong and selective antiproliferative effect on both cancer cell lines (IC50 < 7.5 µM).
Assuntos
Dendrímeros/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Sulfonamidas/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Peso Molecular , Sulfonamidas/químicaRESUMO
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
Gliomas are one of the most aggressive and treatment-resistant types of human brain cancer. Identification and evaluation of anticancer properties of compounds found in plants, such as naringenin (N) and 8-prenylnaringenin (8PN), are among the most promising applications in glioma therapy. The prenyl group seems to be crucial to the anticancer activity of flavones, since it may lead to enhanced cell membrane targeting and thus increased intracellular activity. It should be noted that 8PN content in hop cones is 10 to 100 times lower compared to other flavonoids, such as xanthohumol. In the study presented, we used a simple method for the synthesis of 8PN from isoxanthohumol-O-demethylation, with a high yield of 97%. Cellular accumulation and cytotoxicity of naringenin and 8-prenylnaringenin in normal (BJ) and cancer cells (U-118 MG) was also examined. Obtained data indicated that 8-prenylnaringenin exhibited higher cytotoxicity against used cell lines than naringenin, and the effect of both flavones was stronger in U-118 MG cells than in normal fibroblasts. The anticancer properties of 8PN correlated with its significantly greater (37%) accumulation in glioblastoma cells than in normal fibroblasts. Additionally, naringenin demonstrated higher selectivity for glioblastoma cells, as it was over six times more toxic for cancer than normal cells. Our results provide evidence that examined prenylated and non-prenylated flavanones have different biological activities against normal and cancer cell lines, and this property may be useful in designing new anticancer drugs for glioblastoma therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Fibroblastos/efeitos dos fármacos , Flavanonas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavanonas/síntese química , Glioblastoma , Humanos , Estrutura MolecularRESUMO
Boron-neutron capture therapy (BNCT) is a binary anticancer therapy that requires boron compound for nuclear reaction during which high energy alpha particles and lithium nuclei are formed. Unnatural, boron-containing nucleoside with hydrophobic pinacol moiety was investigated as a potential BNCT boron delivery agent. Biological properties of this compound are presented for the first time and prove that boron nucleoside has low cytotoxicity and that observed apoptotic effects suggest alteration of important functions of cancer cells. Mass spectrometry analysis of DNA from cancer cells proved that boron nucleoside is inserted into nucleic acids as a functional nucleotide derivative. NMR studies present very high degree of similarity of natural dG-dC base pair with dG-boron nucleoside system.
Assuntos
Antineoplásicos/química , Boro/química , Desoxicitidina/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Terapia por Captura de Nêutron de Boro , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Células Hep G2 , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
α-Mangostin and vadimezan are widely studied potential anticancer agents. Their biological activities may be improved by covalent bonding by amide or ester bonds with the third generation poly(amidoamine) (PAMAM) dendrimer, substituted with α-D-glucoheptono-1,4-lactone and biotin. Thus, conjugates of either ester- (G3gh4B5V) or amide-linked (G32B12gh5V) vadimezan, and equivalents of α-mangostin (G3gh2B5M and G32B12gh5M, respectively), were synthesized, characterized and tested in vitro against cancer cells: U-118 MG glioma, SCC-15 squamous carcinoma, and BJ normal human fibroblasts growth, as well as against C. elegans development. α-Mangostin cytotoxicity, stronger than that of Vadimezan, was increased (by 2.5-9-fold) by conjugation with the PAMAM dendrimer (with the amide-linking being slightly more effective), and the strongest effect was observed with SCC-15 cells. Similar enhancement of toxicity resulting from the drug conjugation was observed with C. elegans. Vadimezan (up to 200 µM), as well as both its dendrimer conjugates, was not toxic against both the studied cells and nematodes. It showed an antiproliferative effect against cancer cells at concentrations ≥100 µM. This effect was significantly enhanced after conjugation of the drug with the dendrimer via the amide, but not the ester bond, with G32B12gh5V inhibiting the proliferation of SCC-15 and U-118 MG cells at concentrations ≥4 and ≥12 µM, respectively, without a visible effect in normal BJ cells. Thus, the drug delivery system based on the PAMAM G3 dendrimer containing amide bonds, partially-blocked amino groups on the surface, larger particle diameter and higher zeta potential can be a useful tool to improve the biological properties of transported drug molecules.
RESUMO
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
Reversible cellular senescence was demonstrated previously to constitute colon cancer cell response to methotrexate. The current study presents a comparison of two senescent states of colon cancer cells, arrested and reversing, resulting from respectively, 120 h exposure to the drug, and 48 h exposure followed by 96 h regrowth in drug-free media. The upregulation of immunoproteasome subunit-coding genes and the increase in human leukocyte antigen HLA-A/B/C membrane level indicated MHC-I-restricted antigen presentation as common to both senescent states. Nuclear factor NF-κB p65 level decreased and activating protein AP-1: c-Jun, Fra2 and JunB nuclear levels increased in both senescent cell populations. Notably, the increase in AP-1- dependent transcription occurred after 48 h exposure to methotrexate. ß-catenin nuclear level increased after 48 h exposure to the drug and remained as such only in senescence-arrested cells. ß-catenin level was found uncoupled from the protein phosphorylation status indicating the deregulation of ß-catenin signaling in colon cancer cells employed in the study. These findings carry implications for both, a general mechanism of senescence establishment and putative advantages for colon cancer treatment.
Assuntos
Apresentação de Antígeno , Senescência Celular/efeitos dos fármacos , Neoplasias do Colo/imunologia , Metotrexato/farmacologia , Proteínas de Neoplasias/imunologia , Transdução de Sinais/imunologia , Fator de Transcrição AP-1/imunologia , beta Catenina/imunologia , Linhagem Celular Tumoral , Senescência Celular/imunologia , HumanosRESUMO
The generation 2 and 3 poly(amidoamine) dendrimers (PAMAM G2 and G3) were converted into N-(2,3-dihydroxy)propyl derivatives by the addition of enantiomerically pure S- and R-glycidol. The homochiral dendrimers bind to HaCaT and SCC-15 cell membranes with an R/S glycidol enantioselectivity ratio of 1.5:1, as was quantitatively determined by fluorescence microscopy and visualized by confocal microscopy. Fully substituted G2 and G3 dendrimers were equipped with 32 and 64 N-(2,3-dihydroxy)propyl residues and showed effectively radial symmetry for homochiral derivatives in 13C NMR spectrum in contrary to analogs obtained by reaction with rac-glycidol. The sub-stoichiometric derivatives of G2 and G3 were also obtained in order to characterize them spectroscopically. The homochiral dendrimers were labeled with two different fluorescent labels, fluorescein, and rhodamine B, using their isothiocyanates to react with G2 and G3 followed by the addition of S- and R-glycidol. Obtained fluorescent derivatives were deficiently filled with N-(2,3-dihydroxy)propyl substituents due to steric hindrance imposed by the attached label. Nevertheless, these derivatives were used to determine their ability to bind to the cell membrane of human keratinocytes (HaCaT) and squamous carcinoma cells (SCC-15). Confocal microscopy images obtained from cells treated with variously labeled conjugates and fluorescence analysis with fluorescence reader allowed us to conclude that R-glycidol derivatives were bound and entered the cells preferentially, with higher accumulation in cancer cells. The G3 polyamidoamine (PAMAM)-based dendrimers were taken up more efficiently than G2 derivatives. Moreover, S- and R-glycidol furnished dendrimers were highly biocompatible with no toxicity up to 300 µM concentrations, in contrast to the amine-terminated PAMAM analogs.
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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.
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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
α-Mangostin, one of the major xanthones isolated from pericarp of mangosteen (Garcinia mangostana Linn), exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial as well as anticancer, both in in vitro and in vivo studies. In the present study, α-mangostin' anti-cancer and anti-parasitic properties were tested in vitro against three human cell lines, including squamous carcinoma (SCC-15) and glioblastoma multiforme (U-118 MG), compared to normal skin fibroblasts (BJ), and in vivo against Caenorhabditis elegans. The drug showed cytotoxic activity, manifested by decrease of cell viability, inhibition of proliferation, induction of apoptosis and reduction of adhesion at concentrations lower than 10⯵M (the IC50 values were 6.43, 9.59 and 8.97⯵M for SCC-15, U-118 MG and BJ, respectively). The toxicity, causing cell membrane disruption and mitochondria impairment, was selective against squamous carcinoma with regard to normal cells. Moreover, for the first time anti-nematode activity of α-mangostin toward C. elegans was described (the LC50â¯=â¯3.8⯱â¯0.5⯵M), with similar effect exerted by mebendazole, a well-known anthelmintic drug.
Assuntos
Antinematódeos/farmacologia , Antineoplásicos/farmacologia , Xantonas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antinematódeos/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Concentração Inibidora 50 , Xantonas/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
Polyurethane films were obtained in the solvent-free cycloaliphatic polyaddition process of 4,4'-methylenebis(cyclohexyl isocyanate), poly(ε-caprolactone) diol or poly(oxytetramethylene) glycol and 1,4-butanediol. Chemical structures of the polymers were confirmed by FTIR, NMR and GPC methods. Their surface, thermal and mechanical properties have been evaluated. Results of biological studies with polyurethane films as potential biomaterials for medical applications revealed their mild cytotoxicity against normal human fibroblasts (BJ) and immortalized keratinocytes (HaCaT). STATEMENT OF SIGNIFICANCE: The research is relevant for the potential uses of polyurethane films made from commercial raw materials as general medical supplies.