Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.737
Filtrar
1.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201817

RESUMO

The usefulness of anti-inflammatory drugs as an adjunct therapy to improve outcomes in COVID-19 patients is intensely discussed in this paper. Willow bark (Salix cortex) has been used for centuries to relieve pain, inflammation, and fever. Its main active ingredient, salicin, is metabolized in the human body into salicylic acid, the precursor of the commonly used pain drug acetylsalicylic acid (ASA). Here, we report on the in vitro anti-inflammatory efficacy of two methanolic Salix extracts, standardized to phenolic compounds, in comparison to ASA in the context of a SARS-CoV-2 peptide challenge. Using SARS-CoV-2 peptide/IL-1ß- or LPS-activated human PBMCs and an inflammatory intestinal Caco-2/HT29-MTX co-culture, Salix extracts, and ASA concentration-dependently suppressed prostaglandin E2 (PGE2), a principal mediator of inflammation. The inhibition of COX-2 enzyme activity, but not protein expression was observed for ASA and one Salix extract. In activated PBMCs, the suppression of relevant cytokines (i.e., IL-6, IL-1ß, and IL-10) was seen for both Salix extracts. The anti-inflammatory capacity of Salix extracts was still retained after transepithelial passage and liver cell metabolism in an advanced co-culture model system consisting of intestinal Caco-2/HT29-MTX cells and differentiated hepatocyte-like HepaRG cells. Taken together, our in vitro data suggest that Salix extracts might present an additional anti-inflammatory treatment option in the context of SARS-CoV-2 peptides challenge; however, more confirmatory data are needed.


Assuntos
Anti-Inflamatórios/farmacologia , Aspirina/farmacologia , COVID-19/tratamento farmacológico , COVID-19/imunologia , Extratos Vegetais/farmacologia , Anti-Inflamatórios/química , Álcoois Benzílicos/metabolismo , COVID-19/virologia , Células CACO-2 , Ciclo-Oxigenase 2/efeitos dos fármacos , Citocinas/metabolismo , Dinoprostona/metabolismo , Glucosídeos/metabolismo , Células HT29 , Humanos , Inflamação , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Casca de Planta/química , Extratos Vegetais/química , SARS-CoV-2/imunologia
2.
Front Cell Infect Microbiol ; 11: 668766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34222040

RESUMO

C. butyricum is a common gut commensal bacterium, which has many positive functions in human intestine. In this study, we investigated the effects of monosaccharide and its derivatives on the adhesion of C. butyricum to the mucus of HT-29 cells. RNA interference was performed to assess the roles of MUC2 and glycan in the adhesion of C. butyricum to HT-29 cells. The effects of C. butyricum on the glycosylation of mucins were assayed with fluorescence microscope. The expression levels of mucins and glycotransferases were also determined. The results showed that C. butyricum could adhere to the mucins secreted by HT-29 cells. Several kinds of monosaccharides inhibited the adhesion of C. butyricum to HT-29 cells, which suggested that the mucus glycan was the attaching sites of this bacterium. Knockdown of MUC2, FUT2 or GALNT7 significantly decreased the numbers of the bacteria adhering to HT-29 cells. When colonizing on the surface of HT-29 cells, C. butyricum could increase the production of mucins, promote the expression of glycotransferase, and induce the glycosylation of mucins. These results demonstrated that the glycan of mucus played important roles in the adhesion of C. butyricum to HT-29 cells. This study indicates for the first time that C. butyricum possesses the ability to modulate the glycosylation profile of mucus secreted by HT-29 cells. These findings contribute to understanding the mechanism of interaction between colonic epithelial cells and commensal bacteria.


Assuntos
Clostridium butyricum , Mucinas , Clostridium butyricum/metabolismo , Células Epiteliais/metabolismo , Glicosilação , Células HT29 , Humanos , Mucinas/metabolismo
3.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065225

RESUMO

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.


Assuntos
Íons/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismo , Oniocompostos/metabolismo , Linhagem Celular Tumoral , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Células HCT116 , Células HT29 , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
4.
Nat Commun ; 12(1): 3364, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099649

RESUMO

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.


Assuntos
Infecções por Herpesviridae/metabolismo , Lisina/metabolismo , Proteínas Quinases/metabolismo , Pele/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células HEK293 , Células HT29 , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Lisina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/fisiologia , Células NIH 3T3 , Necroptose/genética , Necrose , Proteínas Quinases/genética , Pele/patologia , Ubiquitinação
5.
Nat Commun ; 12(1): 3898, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162854

RESUMO

One topical area of supramolecular chemistry is the binding of anionic species but despite the importance of anions in diverse cellular processes and for cancer development, anion receptors or 'binders' have received little attention as potential anti-cancer therapeutics. Here we report self-assembling trimetallic cryptands (e.g. [L2(Metal)3]6+ where Metal = Cu2+, Zn2+ or Mn2+) which can encapsulate a range of anions and which show metal-dependent differences in chemical and biological reactivities. In cell studies, both [L2Cu3]6+ and [L2Zn3]6+ complexes are highly toxic to a range of human cancer cell lines and they show significant metal-dependent selective activity towards cancer cells compared to healthy, non-cancerous cells (by up to 2000-fold). The addition of different anions to the complexes (e.g. PO43-, SO42- or PhOPO32-) further alters activity and selectivity allowing the activity to be modulated via a self-assembly process. The activity is attributed to the ability to either bind or hydrolyse phosphate esters and mechanistic studies show differential and selective inhibition of multiple kinases by both [L2Cu3]6+ and [L2Zn3]6+ complexes but via different mechanisms.


Assuntos
Ânions/química , Antineoplásicos/química , Complexos de Coordenação/química , Metais/química , Células A549 , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Cristalografia por Raios X , Células HCT116 , Células HT29 , Humanos , Concentração Inibidora 50 , Neoplasias/metabolismo , Neoplasias/patologia , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo
6.
Food Chem ; 362: 130132, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34082297

RESUMO

This study presents methods that can be used to assess the health quality of products containing betalains. The paper compares and verifies data on the phytochemical composition of three different pigmented beetroot cultivars using spectrophotometric, HPLC-DAD, HPTLC and LC-Q-Orbitrap-HRMS techniques. Additionally, we compared the total antioxidant activity in both the cell-free and cellular systems. Betalain contribution to antioxidant activity was also determined using post-column derivatization and it was found that in the case of red beetroot it is about 50%. Photometric measurements are recommended for a simple and inexpensive analysis of the total betacyanin and betaxanthin content. Liquid chromatography techniques produced more precise information on phytochemical composition in the tested samples. The combination of liquid chromatography with high-resolution mass spectrometry produced the largest amount of quantitative and qualitative data; in beetroot samples sixty-four phytochemicals have been identified therefore, this approach is recommended for more detailed metabolomics studies.


Assuntos
Antioxidantes/química , Beta vulgaris/química , Betalaínas/análise , Espectrometria de Massas/métodos , Antioxidantes/análise , Antioxidantes/farmacologia , Betacianinas/análise , Betalaínas/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Análise de Alimentos/métodos , Células HT29 , Humanos , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/isolamento & purificação , Ácidos Picolínicos/análise , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Software
7.
Food Chem ; 362: 130142, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34087706

RESUMO

Lactoferrin (LF) is a multifunctional glycoprotein which, when thermally processed, undergoes significant physicochemical changes. The link between such changes and the bioactivity of LF is not well characterised and requires much research. In this work, bovine LF solutions (1%, w/v, protein, pH 7) were thermally processed using high temperature short time conditions (72, 80, 85 or 95 °C with 15 s holding times). Following this, it was shown that LF and heat induced LF aggregates were largely resistant to simulated infant gastric, but not intestinal, digestion. Also, the efficacy of LF bactericidal activity, and inhibition of lipopolysaccharide-induced NF-κB activation were negatively impacted by thermal processing. This study confirmed that the efficacy of LF bio-functionalities was affected by the extent of heat-induced changes in protein structure whereby processing conditions of least severity (i.e. pasteurisation) had the least impact on bioactivity.


Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Lactoferrina/química , Lactoferrina/farmacologia , Animais , Antibacterianos/química , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Bovinos , Digestão/efeitos dos fármacos , Células HT29 , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Lactente , Lactoferrina/farmacocinética , Lipopolissacarídeos/toxicidade , Listeria monocytogenes/efeitos dos fármacos , Leite Humano/química , NF-kappa B/metabolismo
8.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067452

RESUMO

A series of saccharide-modified thiadiazole sulfonamide derivatives has been designed and synthesized by the "tail approach" and evaluated for inhibitory activity against carbonic anhydrases II, IX, and XII. Most of the compounds showed high topological polar surface area (TPSA) values and excellent enzyme inhibitory activity. The impacts of some compounds on the viability of HT-29, MDA-MB-231, and MG-63 human cancer cell lines were examined under both normoxic and hypoxic conditions, and they showed certain inhibitory effects on cell viability. Moreover, it was found that the series of compounds had the ability to raise the pH of the tumor cell microenvironment. All the results proved that saccharide-modified thiadiazole sulfonamides have important research prospects for the development of CA IX inhibitors.


Assuntos
Carboidratos/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Tiadiazóis/síntese química , Tiadiazóis/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células HT29 , Humanos , Simulação de Acoplamento Molecular , Microambiente Tumoral/efeitos dos fármacos
9.
Molecules ; 26(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069900

RESUMO

Rhododendron molle G. Don is one example of traditional Chinese medicine with important medicinal value. In this study, the effects of methanol extract of R. molle leaves (RLE) on colorectal cancer HT-29 cells and its potential molecular mechanism were investigated. MTT analysis showed that RLE could significantly inhibit the cell viability and migration of HT-29 cells in a concentration-dependent manner. Cell cycle analyses via flow cytometer suggested that RLE induced DNA fragmentation, indicative of apoptosis, and arrest at the S phase in HT-29 cells. Quantitative real-time PCR (qRT-PCR) analysis showed that RLE could upregulate the mRNA expression of p53 and p21 in HT-29 cells, which would result in HT-29 cells being blocked in S phase. Meanwhile, RLE could upregulate the expression of Bax, and downregulate the expression of Bcl-2, which would induce cell apoptosis. Further western blot analysis showed that the protein expression changes of Bax and P53 were basically consistent with the results of qRT-PCR. In addition, GC-MS analysis detected 17 potential anticancer components in R. molle. These results indicate that R. molle has significant anticancer activity, which provides some useful information for further study and clinical application for R. molle.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Extratos Vegetais/farmacologia , Rhododendron/química , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Flores/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Concentração Inibidora 50 , Folhas de Planta/química , Ensaio Tumoral de Célula-Tronco , Cicatrização/efeitos dos fármacos
10.
Phytomedicine ; 87: 153573, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34052543

RESUMO

BACKGROUND: Qingchang Suppository, a formula used for more than 30 years in Longhua Hospital, has shown satisfactory clinical effects on Ulcerative Colitis (UC). However, its therapeutic mechanism has not been fully elucidated. PURPOSE: The study aims to investigate the effects of Qingchang Suppository powder (QCSP) and its ingredients by regulating the IL-17A signaling pathway which plays an important role in the development of UC. METHODS: HPLC was used to analyze the main ingredients (Gallic acid, Indigo, Indirubin) in QCSP. HT-29 cells were induced by rhIL-17A and TNF-α, and IL-17A related protein expressions were determined by western blot. BALB/C mice were induced by 4% Dextran Sodium sulfate (DSS). The effects of QCSP and its ingredients were evaluated by measuring weight loss, disease activity index (DAI), colon length, histological analysis. Western blot was used for analysis of IL-17A and MAPK related proteins p-ERK, p-JNK, p-P38. Quantitative reverse transcription polymerase chain reaction (q-PCR) was used to detect the expression of IL-17A, HSP90 and ACT1 in colon tissue. Cytokines such as IL-17A, IL-1ß, IFN-γ and TNF-α were determinated by enzyme-linked immunosorbent assay (ELISA). RESULTS: QCSP had good therapeutic effect on DSS-induced colitis in mice. QCSP significantly relieved weight loss, restored colon length, repaired colon lesions, reduced histological scores and DAI, decreased TNF-α, IL-1ß, IL-17 and IFN-γ contents, significantly suppressed the gene expressions of IL-17A, ACT1 and HSP90, and up-regulated the expressions of tight junction proteins like ZO-1 and Occludin. IL-17A pathway related proteins such as IL-17A, IL-17RA, HSP90, MAPKs, P-iκbα and iNOS were significantly increased in vitro and in vivo. CONCLUSIONS: This paper reveals that QCSP inhibited the IL-17A signaling pathway in HT-29 cells and DSS induced mice, presenting a new mechanism of QCS on treating UC.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Sulfato de Dextrana/toxicidade , Interleucina-17/metabolismo , Animais , Colite Ulcerativa/induzido quimicamente , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/toxicidade , Células HT29 , Humanos , Interleucina-17/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pós/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Supositórios/administração & dosagem , Supositórios/efeitos adversos
11.
Mater Sci Eng C Mater Biol Appl ; 124: 112043, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33947543

RESUMO

Normal tissue complication and development of radioresistance in cancer cells are known as the main challenges of ionizing radiation treatment. In the current study, we intended to induce selective radiosensitization in HT29 cancer cells by developing folic acid modified magnetic triblock copolymer nanoparticles as carrier of 5-Flourouracil (5-FU) which was further used in combination with hyperthermia. The aforementioned nanoparticles were synthesized and characterized by differential scanning calorimetric analysis (DSC), UV-visible spectroscopy, dynamic light scattering (DLS), zeta sizer, and transmission electron microscopy (TEM). These nanoparticles were also assessed to determine drug loading capacity (DLC %) and drug release profile. The cytotoxicity of nanoparticles was evaluated on two different cell lines: HUVEC and HT29. Furthermore, radiosensitivity induction of the nanoparticles with and without exposure of alternative magnetic field was investigated. MTT-based cytotoxicity assay demonstrated that the therapeutic ratio was enhanced in response to using 5-FU-loaded nanoparticles as compared to 5-FU. Various characterizations including gene expression study, measurement of reactive oxygen species (ROS) generation, Annexin V/PI staining, and clonogenic assay revealed that ionizing radiation in combination with hyperthermia in the presence of the synthesized nanoparticles led to maximal anti-cancer effects as compared to other single (P < 0.001) and combined treatments (P < 0.01). Our results suggested that combined treatment based on using folic acid modified magnetic copolymer nanoparticle as carrier of 5-FU accompanied with hyperthermia could be proposed as an efficient approach to enhance radiation effects in cancer cells.


Assuntos
Fluoruracila , Nanopartículas , Linhagem Celular Tumoral , Fluoruracila/farmacologia , Células HT29 , Humanos , Hipertermia , Fenômenos Magnéticos , Tolerância a Radiação
12.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946824

RESUMO

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNFα) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined τ50, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, τ50 was determined to be 4.5 dynes/cm2 (95% CI: 4.3-4.7 dynes/cm2) for RBCs treated with 10 µM RRx-001, which was significantly different (p < 0.05) from τ50 in the absence of treatment. Under hypoxic conditions, the difference of τ50 with (4.8 dynes/cm2; 95% CI: 4.6-5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4-2.8 dynes/cm2) 10 µM RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.


Assuntos
Antineoplásicos/farmacologia , Azetidinas/farmacologia , Células Endoteliais/citologia , Membrana Eritrocítica/efeitos dos fármacos , Nitrocompostos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Azetidinas/uso terapêutico , Adesão Celular/efeitos dos fármacos , Hipóxia Celular , Cisteína/química , Citocinas/metabolismo , Células Endoteliais/química , Agregação Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/química , Células HT29/transplante , Células Hep G2/transplante , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/farmacologia , Lipídeos de Membrana/biossíntese , Camundongos , Camundongos Nus , Neoplasias/irrigação sanguínea , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Nitrocompostos/uso terapêutico , Fosfatidilserinas/biossíntese , Receptores de Superfície Celular/biossíntese , Resistência ao Cisalhamento , Microambiente Tumoral , Fator de Necrose Tumoral alfa/farmacologia
13.
Nat Commun ; 12(1): 2809, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990575

RESUMO

The paradoxical roles of transforming growth factor-ß (TGFß) signaling and nuclear receptor Nur77 in colon cancer development are known but the underlying mechanisms remain obscure. Inhibitor of differentiation 1 (ID1) is a target gene of TGFß and a key promoter for colon cancer progression. Here, we show that Nur77 enhances TGFß/Smad3-induced ID1 mRNA expression through hindering Smurf2-mediated Smad3 mono-ubiquitylation, resulting in ID1 upregulation. In the absence of TGFß, however, Nur77 destabilizes ID1 protein by promoting Smurf2-mediated ID1 poly-ubiquitylation, resulting in ID1 downregulation. Interestingly, TGFß stabilizes ID1 protein by switching Nur77 interaction partners to inhibit ID1 ubiquitylation. This also endows TGFß with an active pro-tumorigenic action in Smad4-deficient colon cancers. Thus, TGFß converts Nur77's role from destabilizing ID1 protein and cancer inhibition to inducing ID1 mRNA expression and cancer promotion, which is highly relevant to colon cancer stemness, metastasis and oxaliplatin resistance. Our data therefore define the integrated duality of Nur77 and TGFß signaling in regulating ID1 expression and provide mechanistic insights into the paradoxical roles of TGFß and Nur77 in colon cancer progression.


Assuntos
Neoplasias do Colo/etiologia , Neoplasias do Colo/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Modelos Biológicos , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína Smad4/deficiência , Proteína Smad4/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
14.
Nat Commun ; 12(1): 2805, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990606

RESUMO

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Terapia Biológica/métodos , Microbioma Gastrointestinal , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Fenilcetonúrias/terapia , Animais , Células CACO-2 , Simulação por Computador , Escherichia coli/metabolismo , Engenharia Genética , Células HT29 , Humanos , Técnicas In Vitro , Microfluídica , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Fenilcetonúrias/microbiologia , Primatas , Biologia Sintética
15.
Biomed Res Int ; 2021: 6619195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33997026

RESUMO

Objective: Perphenazine (PPZ), as a typical antipsychotic medical substance, has the same effectiveness compared to atypical antipsychotic medications for the treatment of schizophrenia. Despite the lipophilic essence, PPZ encounters limited bioavailability caused by the first-pass metabolism following oral administration. In the present study, PPZ-containing solid lipid nanoparticles (PPZ-SLNs) were prepared and optimized based on different factors, including lipid and surfactant amount, to develop appropriate and safe novel oral dosage forms of PPZ. Methods: The solvent emulsification-evaporation method was utilized to form SLNs by using soybean lecithin, glycerol monostearate (GMS), and Tween 80. Statistical optimization was done by the Box-Behnken design method to achieve formulation with optimized particle size, entrapment efficiency, and zeta potential. Also, transmission electron microscopy, in vitro release behavior, differential scanning calorimetry (DSC), and powder X-ray diffractometry (P-XRD) studies and cytotoxicity studies were assessed. Results: Optimization exhibited the significant effect of various excipients on SLN characteristics. Our finding indicated that the mean particle size, zeta potential, and entrapment efficiency of optimized PPZ-SLN were, respectively, 104 ± 3.92 nm, -28 ± 2.28 mV, and 83% ± 1.29. Drug release of PPZ-SLN was observed to be greater than 90% for 48 h that emphasized a sustained-release pattern. The DSC and P-XRD studies revealed the amorphous state of PPZ-SLN. FTIR spectra showed no incompatibility between the drug and the lipid. Performing cytotoxicity studies indicated no significant cytotoxicity on HT-29 cell culture. Conclusion: Our study suggests that PPZ-SLNs can make a promising vehicle for a suitable therapy of schizophrenia for the oral drug delivery system.


Assuntos
Lipídeos/química , Nanopartículas/química , Perfenazina/farmacologia , Estatística como Assunto , Varredura Diferencial de Calorimetria , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Células HT29 , Humanos , Modelos Biológicos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Reprodutibilidade dos Testes , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Difração de Raios X
16.
Methods Mol Biol ; 2296: 77-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977443

RESUMO

Gram-negative marine bacteria are an underexplored source of new chemical entities for a wide range of applications. Even though, some have shown a high antitumor activity. This chapter describes an isolation and screening protocol based on the Dilution-to-Extinction approach coupled with an antiproliferative test oriented to the discovery of new cytotoxic compounds synthesized by marine bacteria. In addition to the discovery of new bioactive secondary metabolites, this protocol provides a high-throughput isolation and screening platform for discarding no bioactive strains during the first steps of the drug discovery process.


Assuntos
Antineoplásicos/metabolismo , Organismos Aquáticos/isolamento & purificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Células A549 , Antineoplásicos/farmacologia , Organismos Aquáticos/metabolismo , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Células HT29 , Humanos , Técnicas de Diluição do Indicador
17.
Food Chem ; 358: 129812, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33940289

RESUMO

Prunus fruits are recognized to be rich sources of polyphenols with health promoting effect. In this work we evaluated the phenolic profile and bioactivity, namely antioxidant capacity, antiproliferative effect in HT29, and inhibition capacity of α-glucosidase (α-Gls), α-amylase (α-Amy) and human dipeptidyl peptidase III (hDPP III) activities, of traditional Prunus fruits grown in Serbia. Fifteen Prunus samples were investigated and compared: common European plum and three old plum subspecies ('vlaskaca', damson plum and white damson), purple-leaf cherry plum, red and white cherry plum, sweet cherry, sweet cherry-wild type, sour cherry, steppe cherry, mahaleb cherry, blackthorn, peach, and apricot. Principal Component Analysis highlighted steppe cherry and blackthorn as Prunus species with the highest bioactive potential. In silico analysis pointed out rutinoside derivatives of cyanidin and quercetin as the most potent inhibitors of α-Gls, α-Amy and hDPP III enzymes. Quercetin 3-O-rutinoside showed the highest binding energy to α-Gls (-10.6 kcal/mol).


Assuntos
Antioxidantes/análise , Frutas/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Polifenóis/análise , Prunus/química , Antocianinas/análise , Antocianinas/farmacologia , Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Glucosídeos/metabolismo , Glucosídeos/farmacologia , Inibidores de Glicosídeo Hidrolases/química , Células HT29 , Humanos , Simulação de Acoplamento Molecular , Fenóis/análise , Polifenóis/farmacologia , Quercetina/análogos & derivados , Quercetina/metabolismo , Quercetina/farmacologia , alfa-Amilases/antagonistas & inibidores
18.
Nutrients ; 13(4)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805552

RESUMO

Human gut microbiota (HGM) play a significant role in health and disease. Dietary components, including fiber, fat, proteins and micronutrients, can modulate HGM. Much research has been performed on conventional prebiotics such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), however, novel prebiotics or micronutrients still require further validation. We assessed the effect of FOS, xylooligosaccharides (XOS) and a mixture of an antioxidant vitamin blend (AOB) on gut microbiota composition and activity, and intestinal barrier in vitro. We used batch fermentations and tested the short-term effect of different products on microbial activity in six donors. Next, fecal inocula from two donors were used to inoculate the simulator of the human microbial ecosystem (SHIME) and after long-term exposure of FOS, XOS and AOB, microbial activity (short- and branched-chain fatty acids and lactate) and HGM composition were evaluated. Finally, in vitro assessment of intestinal barrier was performed in a Transwell setup of differentiated Caco-2 and HT29-MTX-E12 cells exposed to fermentation supernatants. Despite some donor-dependent differences, all three tested products showed beneficial modulatory effects on microbial activity represented by an increase in lactate and SCFA levels (acetate, butyrate and to a lesser extent also propionate), while decreasing proteolytic markers. Bifidogenic effect of XOS was consistent, while AOB supplementation appears to exert a specific impact on reducing F. nucleatum and increasing butyrate-producing B. wexlerae. Functional and compositional microbial changes were translated to an in vitro host response by increases of the intestinal barrier integrity by all the products and a decrease of the redox potential by AOB supplementation.


Assuntos
Antioxidantes/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Glucuronatos/farmacologia , Oligossacarídeos/farmacologia , Prebióticos , Vitaminas/farmacologia , Adulto , Bactérias/classificação , Bactérias/efeitos dos fármacos , Células CACO-2 , Fezes/microbiologia , Feminino , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Masculino , Oxirredução
19.
Nutrients ; 13(3)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801889

RESUMO

Background: The aim of this study was to examine the anti-inflammatory and anti-apoptotic patterns of omega-3 polyunsaturated fatty acids (n-3 PUFAs) during methotrexate (MTX) induced intestinal damage in cell culture and in a rat model. Methods: Non-treated and treated with MTX HT 29 and HCT116cells were exposed to increasing doses of n-3 PUFAs and cell viability was evaluated using PrestoBlue® assay. Male Sprague-Dawley rats were divided into 4 experimental groups: Control rats, CONTR+n-3 PUFA rats that were treated with oral n-3 PUFA, MTX rats were treated with MTX given IP, and MTX+n-3 PUFA rats were treated with oral n-3 PUFA before and following injection of MTX. Intestinal mucosal parameters and mucosal inflammation, enterocyte proliferation and apoptosis, TNF-α in mucosal tissue and plasma (ELISA), NF-κB, COX-2, TNF-α, Fas, FasL, Fadd, Bid, Bax and Bcl-2gene and protein levels were determined 72 h following MTX injection. Results: Exposure of HT 29 and HCT116cells to n-3 PUFA attenuated inhibiting effects of MTX on cell viability. MTX-n-3 PUFA rats demonstrated a lower intestinal injury score and enhanced intestinal repair. A significant decrease in enterocyte apoptosis in MTX+n-3 PUFA rats was accompanied by decreased TNF-α, FAS, FasL, FADD and BID mRNA levels. Decreased NF-κB, COX-2 and TNF-α levels in mucosa was accompanied by a decreased number of IELs and macrophages. Conclusions: n-3 PUFAs inhibit NF-κB/COX-2 induced production of pro-inflammatory cytokines and inhibit cell apoptosis mainly by extrinsic pathway in rats with MTX-induced intestinal damage.


Assuntos
Anti-Inflamatórios/administração & dosagem , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Mucosa Intestinal/citologia , Metotrexato/toxicidade , Mucosite/terapia , Animais , Anti-Inflamatórios/farmacologia , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Células HCT116 , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Mucosite/induzido quimicamente , Mucosite/metabolismo , Mucosite/patologia , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley
20.
Nat Commun ; 12(1): 2211, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850121

RESUMO

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.


Assuntos
Morte Celular/fisiologia , Necroptose/fisiologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Sítios de Ligação , Membrana Celular , Cristalografia por Raios X , Células HT29 , Humanos , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Conformação Proteica , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Recombinantes , Alinhamento de Sequência , Células U937
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...