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The authors wish to add the following information to the Authors and Affiliation section of our paper published in Molecules [...].
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Alterations of gut microbiota are evident during the aging process. Prebiotics may restore the gut microbial balance, with ß-glucans emerging as prebiotic candidates. This study aimed to investigate the impact of edible mushrooms rich in ß-glucans on the gut microbiota composition and metabolites by using in vitro static batch culture fermentations and fecal inocula from elderly donors (n = 8). Pleurotus ostreatus, P. eryngii, Hericium erinaceus and Cyclocybe cylindracea mushrooms derived from various substrates were examined. Gut microbiota composition (quantitative PCR (qPCR)) and short-chain fatty acids (SCFAs; gas chromatography (GC)) were determined during the 24-h fermentation. P. eryngii induced a strong lactogenic effect, while P. ostreatus and C. cylindracea induced a significant bifidogenic effect (p for all <0.05). Furthermore, P. eryngii produced on wheat straw and the prebiotic inulin had comparable Prebiotic Indexes, while P. eryngii produced on wheat straw/grape marc significantly increased the levels of tested butyrate producers. P. ostreatus, P. eryngii and C. cylindracea had similar trends in SCFA profile; H. erinaceus mushrooms were more diverse, especially in the production of propionate, butyrate and branched SCFAs. In conclusion, mushrooms rich in ß-glucans may exert beneficial in vitro effects in gut microbiota and/or SCFAs production in elderly subjects.
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Agaricales , Envelhecimento/metabolismo , Microbioma Gastrointestinal , beta-Glucanas/administração & dosagem , Idoso , Feminino , Humanos , MasculinoRESUMO
A variety of bioactive compounds, constituents of edible mushrooms, in particular ß-glucans, i.e., a group of ß-d-glucose polysaccharides abundant in the fungal cell walls, have been linked to immunomodulating, anticancer and prebiotic activities. The aim of the study was the investigation of the genoprotective effects of edible mushrooms produced by Pleurotus eryngii, Pleurotus ostreatus and Cyclocybe cylindracea (Basidiomycota). Mushrooms from selected strains of the species mentioned above were fermented in vitro using faecal inocula from healthy volunteers. The cytotoxic and anti-genotoxic properties of the fermentation supernatants (FSs) were investigated in Caco-2 human colon adenocarcinoma cells. The FSs were cytotoxic in a dose-dependent manner. Non-cytotoxic concentrations were used for the genotoxicity studies, which revealed that mushrooms' FSs have the ability to protect Caco-2 cells against tert-butyl hydroperoxide (t-BOOH), a known genotoxic agent. Their global metabolic profiling was assessed by 1H-NMR spectroscopy. A total of 37 metabolites were identified with the use of two-dimensional (2D) homo- and hetero-nuclear NMR experiments. Multivariate data analysis monitored the metabolic variability of gut microbiota and probed to biomarkers potentially associated with the health-promoting effects of edible mushrooms.
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Agaricales/química , Produtos Biológicos/farmacologia , Fezes/microbiologia , Fermentação , Substâncias Protetoras/farmacologia , Produtos Biológicos/química , Células CACO-2 , Fungos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Metaboloma , Metabolômica/métodos , Substâncias Protetoras/química , beta-Glucanas/metabolismoRESUMO
The aim of the study was to determine the effect of ß-glucan on the cytotoxicity and genotoxicity of polypectomized patient's fecal water (FW). Polypectomized volunteers (n = 69) were randomly assigned to consume bread with or without ß-glucan, for 3 months. FW was collected at the beginning (t = 0), the 30th and 90th day and 2 wk after the intervention. Cytotoxicity and genotoxicity were estimated on Caco-2 cells, using trypan blue exclusion test and comet assay, respectively. Gastrointestinal symptoms were recorded and subjects kept a 3-day food diary at baseline and after completion. Trypan blue exclusion test revealed cell survival of approximately 87% after incubation with FW. The FW samples showed 49% genotoxicity at the baseline. Genotoxicity in the intervention group decreased during the trial reaching statistical significance on the 90th day compared to control. An increase was noticed 2 wk after the trial, but it still remained significantly lower compared to control. Group-specific analysis for ß-glucan also revealed significant decrease in the genotoxicity on the 90th day compared to baseline. ß-glucan ingestion in polypectomized patients significantly decreased the genotoxicity of their FW. Our findings suggest that ß-glucan consumption could possibly provide protection against colon cancer development.
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Fezes/química , beta-Glucanas/farmacologia , Idoso , Pólipos do Colo , Ensaio Cometa , Feminino , Hordeum/química , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Mutagenicidade , ÁguaRESUMO
Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.
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Proteínas ELAV/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas ELAV/metabolismo , Fibroblastos/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Camundongos , Proteínas de Ligação a RNA/metabolismoRESUMO
The aim of the present study is the development, physicochemical characterization, and in vitro cytotoxicity evaluation of both empty and quercetin-loaded HSPC (hydrogenated soy phosphatidylcholine) liposomes, GMO (glyceryl monooleate) liquid crystalline nanoparticles, and PHYT (phytantriol) liquid crystalline nanoparticles. Specifically, HSPC phospholipids were mixed with different non-ionic surfactant molecules (Tween 80 and/or Span 80) for liposomal formulations, whereas both GMO and PHYT lipids were mixed with Span 80 and Tween 80 as alternative stabilizers, as well as with Poloxamer P407 in different ratios for liquid crystalline formulations. Subsequently, their physicochemical properties, such as size, size distribution, and ζ-potential were assessed by the dynamic and electrophoretic light scattering (DLS/ELS) techniques in both aqueous and biological medium with serum proteins. The in vitro biological evaluation of the empty nanosystems was performed by using the MTT cell viability and proliferation assay. Finally, the entrapment efficiency of quercetin was calculated and the differences between the two different categories of lipidic nanoparticles were highlighted. According to the results, the incorporation of the non-ionic surfactants yields a successful stabilization and physicochemical stability of both liposomal and liquid crystalline nanoparticles. Moreover, in combination with an appropriate biosafety in vitro profile, increased encapsulation efficiency of quercetin was achieved. Overall, the addition of surfactants improved the nanosystem's stealth properties. In conclusion, the results indicate that the physicochemical properties were strictly affected by the formulation parameters, such as the type of surfactant.
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Edible mushrooms contain biologically active compounds with antioxidant, antimicrobial, immunomodulatory and anticancer properties. The link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. Lyophilized Pleurotus eryngii (PE) mushrooms, selected due to their strong lactogenic effect and anti-genotoxic, immunomodulatory properties, underwent in vitro static batch fermentation for 24 h by fecal microbiota from eight elderly apparently healthy volunteers (>65 years old). The fermentation-induced changes in fecal microbiota communities were examined using Next Generation Sequencing of the hypervariable regions of the 16S rRNA gene. Primary processing and analysis were conducted using the Ion Reporter Suite. Changes in the global metabolic profile were assessed by 1H NMR spectroscopy, and metabolites were assigned by 2D NMR spectroscopy and the MetaboMiner platform. PLS-DA analysis of both metataxonomic and metabolomic data showed a significant cluster separation of PE fermented samples relative to controls. DEseq2 analysis showed that the abundance of families such as Lactobacillaceae and Bifidobacteriaceae were increased in PE samples. Accordingly, in metabolomics, more than twenty metabolites including SCFAs, essential amino acids, and neurotransmitters discriminate PE samples from the respective controls, further validating the metataxonomic findings.
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HYPOTHESIS: Lipophilic cannabidiol can be solubilized in oil-in water nanoemulsions, which can then be impregnated into chitosan hydrogels forming another colloidal system that will facilitate cannabidiol's release. The delivery from both systems was compared, alongside structural and biological studies, to clarify the effect of the two carriers' structure on the release and toxicity of the systems. EXPERIMENTS: Oil-in-water nanoemulsions (NEs) and the respective nanoemulsion-filled chitosan hydrogels (NE/HGs) were formulated as carriers of cannabidiol (CBD). Size, polydispersity and stability of the NEs were evaluated and then membrane dynamics, shape and structure of both systems were investigated with EPR spin probing, SAXS and microscopy. Biocompatibility of the colloidal delivery systems was evaluated through cytotoxicity tests over normal human skin fibroblasts. An ex vivo permeation protocol using porcine ear skin was implemented to assess the release of CBD and its penetration through the skin. FINDINGS: Incorporation of the NEs in chitosan hydrogels does not significantly affect their structural properties as evidenced through SAXS, EPR and confocal microscopy. These findings indicate the successful development of a novel nanocarrier that preserves the NE structure with the CBD remaining encapsulated in the oil core while providing new rheological properties advantageous over NEs. Moreover, NE/HGs proved to be more efficient as a carrier for the release of CBD. Cell viability assessment revealed high biocompatibility of the proposed colloids.
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Canabidiol , Quitosana , Humanos , Animais , Suínos , Hidrogéis/química , Espalhamento a Baixo Ângulo , Emulsões/química , Difração de Raios X , Água/químicaRESUMO
The oncogenic BRAFV600E kinase leads to abnormal activation of the MAPK signaling pathway and thus, uncontrolled cellular proliferation and cancer development. Based on our previous virtual screening studies which issued 2-acetamido-1,3 benzothiazole-6-carboxamide scaffold as active pharmacophore displaying selectivity against the mutated BRAF, eleven new substituted benzothiazole derivatives were designed and synthesized by coupling of 2-acetamidobenzo[d]thiazole-6-carboxylic acid with the appropriate amines in an effort to provide even more efficient inhibitors and tackle drug resistance often developed during cancer treatment. All derived compounds bore the benzothiazole scaffold substituted at position-2 by an acetamido moiety and at position-6 by a carboxamide functionality, the NH moiety of which was further linked through an alkylene linker to a sulfonamido (or amino) aryl (or alkyl) functionality or a phenylene linker to a sulfonamido aromatic (or non-aromatic) terminal pharmacophore in the order -C6 H4 -NHSO2 -R or reversely -C6 H4 -SO2 N(H)-R. These analogs were subsequently biologically evaluated as potential BRAFV600E inhibitors and antiproliferative agents in several colorectal cancer and melanoma cell lines. In all assays applied, one analog, namely 2-acetamido-N-[3-(pyridin-2-ylamino)propyl]benzo[d]thiazole-6-carboxamide (22), provided promising results in view of its use in drug development.
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Antineoplásicos , Benzotiazóis , Linhagem Celular Tumoral , Benzotiazóis/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células , Relação Estrutura-Atividade , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
Recent studies have revealed the crucial role of several edible mushrooms and fungal compounds, mainly polysaccharides, in human health and disease. The investigation of the immunomodulating effects of mushroom polysaccharides, especially ß-glucans, and the link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. We investigated the immunomodulating effects of Pleurotus eryngii mushrooms, selected due to their high ß-glucan content, strong lactogenic effect, and potent geno-protective properties, following in vitro fermentation by fecal inocula from healthy elderly volunteers (>60 years old). The immunomodulating properties of the fermentation supernatants (FSs) were initially investigated in U937-derived human macrophages. Gene expression as well as pro- (TNF-α, IL-1ß) and anti-inflammatory cytokines (IL-10, IL-1Rα) were assessed and correlated with the fermentation process. The presence of P. eryngii in the fermentation process led to modifications in immune response, as indicated by the altered gene expression and levels of the cytokines examined, a finding consistent for all volunteers. The FSs immunomodulating effect on the volunteers' peripheral blood mononuclear cells (PBMCs) was verified through the use of cytometry by time of flight (CyTOF) analysis.
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Pleurotus eryngii mushrooms are commercially cultivated and widely consumed due to their organoleptic properties, and the low caloric and high nutritional value. In addition, they contain various biologically active and health-promoting compounds; very recently, their genoprotective effect in Caco-2 cells after their fermentation by the human fecal microbiota was also documented. In the current study, the effect of P. eryngii pre- and post-fermentation supernatants in micronuclei formation was evaluated in human lymphocytes. In addition, the genoprotective properties of increasing concentrations of aqueous extracts from P. eryngii mushrooms (150, 300, 600 mg/kg) against the cyclophosphamide-induced DNA damage were studied in young and elderly female and male mice in bone marrow and whole blood cells. The ability of the highest dose (600 mg/kg) to regulate the main cellular signaling pathways was also evaluated in gut and liver tissues of female animals by quantifying the mRNA expression of NrF2, Nfkß, DNMT1, and IL-22 genes. P. eryngii post-fermentation, but not pre-fermentation, supernatants were able to protect human lymphocytes from the mitomycin C-induced DNA damage in a dose-dependent manner. Similarly, genoprotection was also observed in bone marrow cells of mice treated by gavage with P. eryngii extract. The effect was observed in all the experimental groups of mice (young and elderly, male and female) and was more potent in young female mice. Overexpression of all genes examined was observed in both tissues, mainly among the elderly animals. In conclusion, P. eryngii mushrooms were shown to maintain genome integrity through protecting cells from genotoxic insults. These beneficial effects can be attributed to their antioxidant and immunomodulatory properties, as well as their ability to regulate the cell's epigenetic mechanisms and maintain cell homeostasis.
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INTRODUCTION: Beta-glucans are polysaccharides that exhibit a wide range of biological properties as a result of their varying chemical composition. Like all dietary fibers, they avoid catabolism in the upper gastrointestinal tract, and they reach the large intestine undigested. There, they undergo fermentation by the gut microbiota, a process that has potential beneficial effects for the host. The aim of this systematic review is to assess the effects of consumption of beta-(1 â 3,1 â 6)-d-glucans, naturally found in the cell walls of fungi, on health outcomes. METHODS: A comprehensive literature search was performed on PubMed, Cochrane Library and Web of Science to retrieve studies that applied randomized controlled trials (RCTs) to investigate the impact of exclusive oral administration of fungal beta-glucans in any form and at any dosage to healthy subjects or patients. RESULTS: Thirty-four RCTs, of the 917 records retrieved in total, met the eligibility criteria and are included in the present review. The sources of fungal beta-glucans were Saccharomyces cerevisiae, Aureobasidium pullulans, Pleurotus ostreatus, Lentinula edodes and Ganoderma lucidum, and the dosage of supplementation ranged from 2.5 to 1000 mg daily for up to 6.5 months. The primary physiological outcome of the majority of the interventions was immunomodulation, which resulted in (a) strengthened immune defense that reduces the incidence and symptoms of cold, flu and other respiratory infections and (b) improvement of allergic symptoms. However, the findings on the induction of immune response alterations were inconsistent at the cellular and molecular levels. Another aspect is psychological wellbeing, as the cohorts that received the polysaccharides of interest reported improvement in their mood states as well as amelioration of overall wellbeing. At the same time, it might also be useful as a complementary agent to patients undergoing cancer therapies. Furthermore, supplements containing beta-(1 â 3,1 â 6)-d-glucan administered to overweight/obese adults might have the potential to decrease comorbid conditions associated with obesity. Notably, no adverse event causally related to glucans was recorded. CONCLUSIONS: Supplementation with beta-(1 â 3,1 â 6)-d-glucans is well-tolerated, and health-promoting properties are manifested primarily through the potentiation of the immune system. More studies are required to confirm their additional beneficial effects, to establish the optimal dose, and to reveal the underlying molecular mechanisms.
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Fungos/química , Nível de Saúde , beta-Glucanas/administração & dosagem , Adulto , Idoso , Ascomicetos/química , Basidiomycota/química , Suplementos Nutricionais , Feminino , Fermentação , Microbioma Gastrointestinal/fisiologia , Humanos , Hipersensibilidade/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Doenças Respiratórias/tratamento farmacológico , Resultado do Tratamento , Adulto Jovem , beta-Glucanas/químicaRESUMO
In recent years, modulation of gut microbiota through prebiotics has garnered interest as a potential to ameliorate intestinal barrier dysfunction. The aim of the study was to examine the in vitro effect of fermentation supernatants (FSs) from rich in ß-glucan Pleurotus eryngii mushrooms on the expression levels of tight junctions (TJs) genes in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Mushrooms were fermented using fecal inocula in an in vitro batch culture model. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre-incubation with FS, co- and post-incubation. Reverse transcription PCR was applied to measure the expression levels of zonulin-1, occludin and claudin-1 genes. FSs from P. eryngii mushrooms led to a significant upregulation of the TJs gene expression in pre-incubation state, indicating potential preventive action. Down-regulation of all TJs gene expression levels was observed when the cells were challenged with LPS. The FS negative control (gut microbiota of each donor with no carbohydrate source) exhibited a significant upregulation of TJs expression levels compared to the cells that were challenged with LPS, for all three conditions. Overall, our data highlighted the positive and potential protective effects of P. eryngii mushrooms in upregulation of TJs' genes.
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During the last decade, many studies have been reported on the design and formulation of novel drug delivery systems proposed for dermal or transdermal administration. The efforts focus on the development of biocompatible nanodispersions that can be delivered to the skin and treat severe skin disorders, including cancer. In this context, oil-in-water (O/W) microemulsions have been developed to encapsulate and deliver lipophilic bioactive molecules for dermal application. An O/W biocompatible microemulsion composed of PBS buffer, Tween 80, and triacetin was assessed for its efficacy as a drug carrier of DPS-2, a lead compound, initially designed in-house to inhibit BRAFV600E oncogenic kinase. The system was evaluated through both in vitro and ex vivo approaches. The cytotoxic effect, in the presence and absence of DPS-2, was examined through the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay using various cell lines. Further investigation through Western blotting revealed that cells died of necrosis. Porcine ear skin was used as a skin model to evaluate the degree of permeation of DPS-2 through skin and assess its retention. Through the ex vivo experiments, it was clarified that encapsulated DPS-2 was distributed within the full thickness of the stratum corneum (SC) and had a high affinity to hair follicles.
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Biocompatible nanoemulsions and nanoemulsion-based hydrogels were formulated for the encapsulation and delivery of vitamin D3 and curcumin. The aforementioned systems were structurally studied applying dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy and viscometry. In vitro studies were conducted using Franz diffusion cells to investigate the release of the bioactive compounds from the nanocarriers. The cytotoxicity of the nanoemulsions was investigated using the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay and RPMI 2650 nasal epithelial cells as in vitro model. DLS measurements showed that vitamin D3 and curcumin addition in the dispersed phase of the nanoemulsions caused an increase in the size of the oil droplets from 78.6 ± 0.2 nm to 83.6 ± 0.3 nm and from 78.6 ± 0.2 nm to 165.6 ± 1.0 nm, respectively. Loaded nanoemulsions, in both cases, were stable for 60 days of storage at 25 °C. EPR spectroscopy revealed participation of vitamin D3 and curcumin in the surfactants monolayer. In vitro release rates of both lipophilic compounds from the nanoemulsions were comparable to the corresponding ones from the nanoemulsion-based hydrogels. The developed o/w nanoemulsions did not exhibit cytotoxic effect up to the concentration threshold of 1 mg/mL in the cell culture medium.
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A novel water-in-oil (W/O) microemulsion based on natural oils, namely extra virgin olive oil (EVOO) and sunflower oil (SO), in the presence of non-ionic surfactants was successfully formulated. The novel microemulsion was used as a carrier for gallic acid (GA) to assure its protection and efficacy upon nasal administration. The work presents evidence that this microemulsion can be used as a nasal formulation for the delivery of polar antioxidants, especially, after incorporation of chitosan (CH) in its aqueous phase. The structure of the system was studied by Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS) and Electron Paramagnetic Resonance (EPR) spectroscopy techniques. By the addition of CH, the diameter of the microemulsion remained unaltered at 47 nm whereas after the incorporation of GA, micelles with 51 nm diameter were detected. The dynamic properties of the surfactant monolayer were affected by both the incorporation of CH and GA. Moreover, the antioxidant activity of the latter remained unaltered (99 %). RPMI 2650 cell line was used as the in vitro model for cell viability and for GA nasal epithelial transport studies after microemulsion administration. The results suggested that the nasal epithelial permeation of GA was enhanced, 3 h post administration, by the presence of 0.2 % v/v microemulsion in the culture medium. However, the concentration of the transported antioxidant in the presence of CH was higher indicating the polymer's effect on the transport of the GA. The study revealed that nasal administration of hydrophilic antioxidants could be used as an alternative route besides oral administration.
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Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Quitosana/química , Sistemas de Liberação de Medicamentos , Ácido Gálico/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Antineoplásicos/química , Antioxidantes/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Emulsões/química , Ácido Gálico/química , Humanos , Micelas , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Propriedades de SuperfícieRESUMO
Targeted delivery of chemotherapeutics in order to overcome side effects and enhance chemosensitivity remains a major issue in cancer research. In this context, biocompatible oil-in-water (O/W) microemulsions were developed as matrices for the encapsulation of DPS-2 a benzothiophene analogue, exhibiting high cytotoxicity in various cancer cell lines, among them the MW 164 skin melanoma and Caco-2 human epithelial colorectal adenocarcinoma cell lines. The microemulsion delivery system was structurally characterized by dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. The effective release of a lipophilic encapsulated compound was evaluated via confocal microscopy. The cytotoxic effect, in the presence and absence of DPS-2, was examined through the thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay. When encapsulated, DPS-2 was as cytotoxic as when dissolved in dimethyl sulfoxide (DMSO). Hence, the oil cores of O/W microemulsions were proven effective biocompatible carriers of lipophilic bioactive molecules in biological assessment experiments. Further investigation through fluorescence-activated cell sorting (FACS) analysis, comet assay, and Western blotting, revealed that DPS-2, although non-genotoxic, induced S phase delay accompanied by cdc25A degradation and a nonapoptotic cell death in both cell lines, which implies that this benzothiophene analogue is a deoxyribonucleic acid (DNA) replication inhibitor.
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Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor's formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient's age and tumor's clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Caderinas/metabolismo , Neoplasias do Colo/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Idoso , Antígenos CD , Caderinas/genética , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Investigations of the presence of the precarcinogenic DNA adduct O6-methylguanine (O6-meG) in humans and its association with exposure or cancer risk have been hindered by the absence of analytic methods of adequate sensitivity and throughput. We report the development, validation, and application of an ELISA-type assay for O6-meG appropriate for large-scale population studies. METHODS: In the new analytic method, restriction enzymes are used to digest DNA to fragments of size expected to contain no more than one O6-meG residue. Anti-adduct antisera are used to transfer O6-meG-containing fragments to a solid surface, where they are detected using anti-ssDNA antisera, the high ratio of normal nucleotides to adducts providing a strong signal enhancement. RESULTS: An assay with a limit of detection of 1.5 adducts/109 nucleotides using 10 µg of DNA, a dynamic range of approximately two orders of magnitude and satisfactory precision and accuracy characteristics was established and validated. Analysis of samples from 120 subjects from the Rhea mother-child cohort in Crete led to the detection of O6-meG in 70% of maternal and 50% of cord blood buffy coat samples at mean levels of 0.65 and 0.38 adducts/108 nucleotides, respectively. CONCLUSIONS: The frequent observation of O6-meG in human DNA is compatible with dietary compounds (e.g. N-nitroso compounds or their precursors), or endogenous processes being responsible for the formation of this adduct. IMPACT: The new assay opens the way for large-scale population studies of O6-meG as a biomarker of exposure or risk. The approach used in this assay can, in principle, be extended to any DNA adduct for which suitable antisera are available.