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1.
Arh Hig Rada Toksikol ; 75(2): 137-146, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38963137

RESUMO

Traditional medicine has used sage (Salvia officinalis L.) preparations for centuries to prevent and treat various inflammatory and oxidative stress-induced conditions. The aim of this in vitro study was to determine the bioactive properties of a sage leave extract obtained with environmentally friendly aqueous extraction and lyophilisation in primary human peripheral blood cells. To that end we measured the total phenolic and flavonoid content (TPC and TFC, respectively) with gas chromatography-mass spectrometry (GC-MS). Non-cytotoxic concentrations determined with the trypan blue assay were used to assess the antioxidant (DPPH, ABTS, and PAB assay), antigenotoxic (CBMN assay), immunomodulatory (IL-1ß and TNF-α), and neuroprotective effects (AChE inhibition). The extract contained high TPC (162 mg GAE/g of dry extract) and TFC (39.47 mg QE/g of dry extract) concentrations, while ß-thujone content was unexpectedly low (below 0.9 %). Strong radical-scavenging activity combined with glutathione reductase activation led to a decrease in basal and H2O2-induced oxidative stress and DNA damage. A decrease in TNF-α and increase in IL-1ß levels suggest complex immunomodulatory response that could contribute to antioxidant and, together with mild AChE inhibition, neuroprotective effects. Overall, this study has demonstrated that aqueous sage leave extract reduces the levels of thujone, 1,8-cineole, pinene, and terpene ketones that could be toxic in high concentrations, while maintaining high concentrations of biologically active protective compounds which have a potential to prevent and/or treat inflammatory and oxidative stress-related conditions.


Assuntos
Inflamação , Leucócitos Mononucleares , Estresse Oxidativo , Extratos Vegetais , Salvia officinalis , Humanos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Leucócitos Mononucleares/efeitos dos fármacos , Salvia officinalis/química , Inflamação/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Folhas de Planta/química
2.
J Pers Med ; 14(6)2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38929861

RESUMO

Early-onset colorectal cancer (EOCRC), defined as colorectal cancer in individuals under 50 years of age, has shown an alarming increase in incidence worldwide. We report a case of a twenty-four-year-old female with a strong family history of colorectal cancer (CRC) but without an identified underlying genetic predisposition syndrome. Two years after primary surgery and adjuvant chemotherapy, the patient developed new liver lesions. Extensive diagnostic imaging was conducted to investigate suspected liver metastases, ultimately leading to a diagnosis of focal nodular hyperplasia. The young age of the patient has prompted comprehensive genomic and transcriptomic profiling in order to identify potential oncogenic drivers and inform further clinical management of the patient. Besides a number of oncogenic mutations identified in the patient's tumour sample, including KRAS G12D, TP53 R248W and TTN L28470V, we have also identified a homozygous deletion of 24.5 MB on chromosome 8. A multivariate Cox regression analysis of this patient's mutation profile conferred a favourable prognosis when compared with the TCGA COADREAD database. Notably, the identified deletion on chromosome 8 includes the WRN gene, which could contribute to the patient's overall positive response to chemotherapy. The complex clinical presentation, including the need for emergency surgery, early age at diagnosis, strong family history, and unexpected findings on surveillance imaging, necessitated a multidisciplinary approach involving medical, radiation, and surgical oncologists, along with psychological support and reproductive medicine specialists. Molecular profiling of the tumour strongly indicates that patients with complex mutational profile and rare genomic rearrangements require a prolonged surveillance and personalised informed interventions.

3.
Sci Data ; 10(1): 758, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37923731

RESUMO

Articular cartilage has only very limited regenerative capacities in humans. Tissue engineering techniques for cartilage damage repair are limited in the production of hyaline cartilage. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells and can be differentiated into mature cartilage cells, chondrocytes, which could be used for repairing damaged cartilage. Chondrogenesis is a highly complex, relatively inefficient process lasting over 3 weeks in vitro. Methods: In order to better understand chondrogenic differentiation, especially the commitment phase, we have performed transcriptional profiling of MSC differentiation into chondrocytes from early timepoints starting 15 minutes after induction to 16 hours and fully differentiated chondrocytes at 21 days in triplicates.


Assuntos
Diferenciação Celular , Condrócitos , Células-Tronco Mesenquimais , Humanos , Cartilagem Articular , Transcriptoma
4.
Int J Mol Sci ; 24(14)2023 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-37511580

RESUMO

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/genética , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas ras/metabolismo
5.
Oncogenesis ; 12(1): 23, 2023 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-37130839

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer, characterised by stromal remodelling, elevated matrix stiffness and high metastatic rate. Retinoids, compounds derived from vitamin A, have a history of clinical use in cancer for their anti-proliferative and differentiation effects, and more recently have been explored as anti-stromal therapies in PDAC for their ability to induce mechanical quiescence in cancer associated fibroblasts. Here, we demonstrate that retinoic acid receptor ß (RAR-ß) transcriptionally represses myosin light chain 2 (MLC-2) expression in pancreatic cancer cells. As a key regulatory component of the contractile actomyosin machinery, MLC-2 downregulation results in decreased cytoskeletal stiffness and traction force generation, impaired response to mechanical stimuli via mechanosensing and reduced ability to invade through the basement membrane. This work highlights the potential of retinoids to target the mechanical drivers of pancreatic cancer.

6.
Cancers (Basel) ; 15(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36672501

RESUMO

Early onset colorectal cancer (EOCRC), defined as colorectal cancers in patients aged less than 50 years, is becoming an increasingly common issue, globally. Since 1994, the incidence of this condition has been rising by 2% annually. Approximately one in five patients under 50 years of age diagnosed with colorectal cancer have an underlying genetic predisposition syndrome. The detection of cancer among the other 80% of patients poses a considerable task, as there is no family history to advocate for commencing early screening in this group. Patients with EOCRC have distinct social, spiritual, fertility, and financial needs from their older counterparts that need to be addressed. This review discusses the risk factors associated with the development of EOCRC and current best practice for the management of this disease.

7.
J Craniofac Surg ; 33(5): e507-e509, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36041131

RESUMO

PURPOSE: The frequency and types of salivary gland tumors show significant geographical variations. The most common are primary epithelial tumors, with pleomorphic adenoma and mucoepidermoid carcinoma being the most frequent. This study aims to analyze the clinicopathological data of patients with major and minor salivary gland (MiSG) tumors. METHODS: The retrospective study included all patients with major and MiSG tumors diagnosed and treated between January 2000 and January 2019. Files of 907 patients were reviewed and investigated for clinicopathologic features of major and MiSG tumors in Serbia. RESULTS: The majority of tumors were of epithelial origin. Pleomorphic adenoma was the predominant type of tumor, with 35.1% among all tumors on all sites. Adenoid cystic carcinoma and mucoepider-moid carcinoma (with 7.1% and 2.7%, respectively) were the most common malignant ones. The most common localization was the parotid gland. Minor salivary gland tumors comprised 16.43% of all salivary gland tumors in our series, the most common localization being the oral cavity. The results of our study are mostly consistent with the results of other previously published studies. CONCLUSIONS: The most important finding, worth emphasizing, is that the most common malignant major and MiSG tumor in our population is adenoid cystic carcinoma, rather than mucoepidermoid carcinoma, in all investigated localizations. In addition, the nasal cavity is the most common localization among malignant MiSG tumors.


Assuntos
Adenoma Pleomorfo , Carcinoma Adenoide Cístico , Carcinoma Mucoepidermoide , Neoplasias das Glândulas Salivares , Adenoma Pleomorfo/patologia , Adenoma Pleomorfo/cirurgia , Carcinoma Adenoide Cístico/patologia , Carcinoma Adenoide Cístico/cirurgia , Carcinoma Mucoepidermoide/cirurgia , Humanos , Estudos Retrospectivos , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares Menores
8.
Biomolecules ; 12(7)2022 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-35883549

RESUMO

Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.


Assuntos
Antineoplásicos , Melanoma , Processamento Alternativo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indóis/farmacologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico
9.
Foods ; 11(10)2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35627015

RESUMO

Cocoa beans are part of the cocoa plant fruit (Theobroma cacao L.) used to prepare various products such as chocolate, cocoa butter, jelly, liqueurs, cosmetics, etc. Dark chocolate is consumed worldwide by different populations and is known for its good taste, making it one of the most favoured food products. This work aimed to determine the content of total polyphenols (TPC), total flavonoids (TFC), and the antioxidant potential measured through the ability to scavenge DPPH free radicals (DPPH), ferric reducing power (FRAP), and total antioxidant capacity (TAC), as well as major and trace elements contained in twelve commercially available dark chocolate samples, with cocoa content ranging from 40% to 99%. The total polyphenols content ranged between 10.55 and 39.82 mg/g GAE, while the total flavonoid content was from 10.04 to 37.85 mg/g CE. All applied antioxidant assays indicate that the sample with the highest cocoa percentage shows the greatest antioxidant activity (DPPH: 48.34% of inhibition; FRAP: 89.00 mg/g GAE; TAC: 83.86 mg/g AAE). Statistical methods were applied to establish the differences between the samples concerning TPC, TFC, DPPH, FRAP and TAC, as well as to differentiate the samples according to the mineral content. The results indicated that the differences in TPC and TFC between different samples depended on the cocoa content and the addition of dried fruit pieces. A good correlation between antioxidant potency composite index (ACI) and declared cocoa content was noticed (R2 = 0.8034), indicating that the declared percentage of cocoa is a reliable indicator for antioxidant activity of analysed dark chocolate samples. The nutritional evaluation proved that the studied chocolate samples were an excellent source of Mg, Fe, Mn and Cu.

10.
Waste Manag Res ; 40(11): 1629-1636, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35475493

RESUMO

A calcium-pyro-hydrochar (Ca-PHC) can be distinguished as a novel sorbent of Pb2+ and Cd2+ from an aqueous solution. It was obtained using hydrothermal treatment of the spent mushroom substrate (SMS), followed by a CaCl2·5H2O activation and pyrolysis. The characterisation of chars before and after modifications was done by scanning electron microscope (SEM), Brunauer-Emmett-Teller (BET) and Fourier transform infrared (FTIR). Batch experiments were performed to examine Ca-PHC's sorption properties and binding mechanisms to selected metal ions. The maximum sorption capacities of Ca-PHC for Pb2+ and Cd2+ were 297 mg g-1, and 131 mg g-1, respectively. The obtained results demonstrated that the sorption of Pb2+ and Cd2+ by Ca-PHC follows a pseudo-second kinetic model and Freundlich isotherm. The binding of the selected metals onto Ca-PHC was enabled by the ion-exchange mechanism, surface complexation, mineral precipitation and cation-π interaction. Thermodynamic parameters indicate that metal ions binding by Ca-PHC are spontaneous and endothermic. Due to the high adsorption capacities, the obtained Ca-PHC has good potential for application in industrial wastewater treatment. In addition, the demonstrated use of SMS highlights another possibility of applying this specific biomass relevant to sustainable and economical waste management in the growing mushroom industry.


Assuntos
Agaricales , Poluentes Químicos da Água , Adsorção , Cádmio , Cálcio , Cloreto de Cálcio , Concentração de Íons de Hidrogênio , Cinética , Chumbo , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Água , Poluentes Químicos da Água/análise
11.
Front Cell Dev Biol ; 9: 612518, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968920

RESUMO

Although a rare disease, neuroblastoma accounts for the highest proportion of childhood cancer deaths. There is a lack of recurrent somatic mutations in neuroblastoma embryonal tumours, suggesting a possible role for epigenetic alterations in driving this cancer. While an increasing number of reports suggest an association of MYCN with epigenetic machinery, the mechanisms of these interactions are poorly understood in the neuroblastoma setting. Utilising chemo-genomic approaches we revealed global MYCN-epigenetic interactions and identified numerous epigenetic proteins as MYCN targets. The epigenetic regulators HDAC2, CBX8 and CBP (CREBBP) were all MYCN target genes and also putative MYCN interactors. MYCN-related epigenetic genes included SMARCs, HDACs, SMYDs, BRDs and CREBBP. Expression levels of the majority of MYCN-related epigenetic genes showed predictive ability for neuroblastoma patient outcome. Furthermore, a compound library screen targeting epigenetic proteins revealed broad susceptibility of neuroblastoma cells to all classes of epigenetic regulators, belonging to families of bromodomains, HDACs, HATs, histone methyltransferases, DNA methyltransferases and lysin demethylases. Ninety-six percent of the compounds reduced MYCN-amplified neuroblastoma cell viability. We show that the C646 (CBP-bromodomain targeting compound) exhibits switch-like temporal and dose response behaviour and is effective at reducing neuroblastoma viability. Responsiveness correlates with MYCN expression, with MYCN-amplified cells being more susceptible to C646 treatment. Thus, exploiting the broad vulnerability of neuroblastoma cells to epigenetic targeting compounds represents an exciting strategy in neuroblastoma treatment, particularly for high-risk MYCN-amplified tumours.

12.
Commun Biol ; 4(1): 152, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526843

RESUMO

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.


Assuntos
Biomarcadores Tumorais , Proliferação de Células , Recidiva Local de Neoplasia/veterinária , Papiloma/veterinária , Neoplasias Cutâneas/veterinária , Infecções Tumorais por Vírus/veterinária , Tartarugas , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Imuno-Histoquímica , Papiloma/genética , Papiloma/metabolismo , Papiloma/cirurgia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/cirurgia , Transcriptoma , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/cirurgia
13.
Nat Commun ; 11(1): 499, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980649

RESUMO

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.


Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores ErbB/metabolismo , Mutação/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Humanos , Fosforilação , Prognóstico , Análise de Sobrevida , Proteína de Morte Celular Associada a bcl/metabolismo
14.
Toxicol Sci ; 171(2): 303-314, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271423

RESUMO

A rapid increase of new nanomaterial (NM) products poses new challenges for their risk assessment. Current traditional methods for estimating potential adverse health effect of NMs are complex, time consuming, and expensive. In order to develop new prediction tests for nanotoxicity evaluation, a systems biology approach, and data from high-throughput omics experiments can be used. We present a computational approach that combines reverse engineering techniques, network analysis and pathway enrichment analysis for inferring the transcriptional regulation landscape and its functional interpretation. To illustrate this approach, we used published transcriptomic data derived from mice lung tissue exposed to carbon nanotubes (NM-401 and NRCWE-26). Because fibrosis is the most common adverse effect of these NMs, we included in our analysis the data for bleomycin (BLM) treatment, which is a well-known fibrosis inducer. We inferred gene regulatory networks for each NM and BLM to capture functional hierarchical regulatory structures between genes and their regulators. Despite the different nature of the lung injury caused by nanoparticles and BLM, we identified several conserved core regulators for all agents. We reason that these regulators can be considered as early predictors of toxic responses after NMs exposure. This integrative approach, which refines traditional methods of transcriptomic analysis, can be useful for prioritization of potential core regulators and generation of new hypothesis about mechanisms of nanoparticles toxicity.

15.
Commun Biol ; 1: 63, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30271945

RESUMO

Wildlife populations are under intense anthropogenic pressures, with the geographic range of many species shrinking, dramatic reductions in population numbers and undisturbed habitats, and biodiversity loss. It is postulated that we are in the midst of a sixth (Anthropocene) mass extinction event, the first to be induced by human activity. Further, threatening vulnerable species is the increased rate of emerging diseases, another consequence of anthropogenic activities. Innovative approaches are required to help maintain healthy populations until the chronic underlying causes of these issues can be addressed. Fibropapillomatosis in sea turtles is one such wildlife disease. Here, we applied precision-medicine-based approaches to profile fibropapillomatosis tumors to better understand their biology, identify novel therapeutics, and gain insights into viral and environmental triggers for fibropapillomatosis. We show that fibropapillomatosis tumors share genetic vulnerabilities with human cancer types, revealing that they are amenable to treatment with human anti-cancer therapeutics.

16.
Cell Rep ; 24(5): 1316-1329, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30067985

RESUMO

Cellular p53 protein levels are regulated by a ubiquitination/de-ubiquitination cycle that can target the protein for proteasomal destruction. The ubiquitination reaction is catalyzed by a multitude of ligases, whereas the removal of ubiquitin chains is mediated by two deubiquitinating enzymes (DUBs), USP7 (HAUSP) and USP10. Here, we show that PHD3 hydroxylates p53 at proline 359, a residue that is in the p53-DUB binding domain. Hydroxylation of p53 upon proline 359 regulates its interaction with USP7 and USP10, and its inhibition decreases the association of p53 with USP7/USP10, increases p53 ubiquitination, and rapidly reduces p53 protein levels independently of mRNA expression. Our results show that p53 is a PHD3 substrate and that hydroxylation by PHD3 regulates p53 protein stability through modulation of ubiquitination.


Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Estabilidade Proteica , Proteína Supressora de Tumor p53/química , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
17.
Cell Syst ; 7(2): 161-179.e14, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30007540

RESUMO

Clinically used RAF inhibitors are ineffective in RAS mutant tumors because they enhance homo- and heterodimerization of RAF kinases, leading to paradoxical activation of ERK signaling. Overcoming enhanced RAF dimerization and the resulting resistance is a challenge for drug design. Combining multiple inhibitors could be more effective, but it is unclear how the best combinations can be chosen. We built a next-generation mechanistic dynamic model to analyze combinations of structurally different RAF inhibitors, which can efficiently suppress MEK/ERK signaling. This rule-based model of the RAS/ERK pathway integrates thermodynamics and kinetics of drug-protein interactions, structural elements, posttranslational modifications, and cell mutational status as model rules to predict RAF inhibitor combinations for inhibiting ERK activity in oncogenic RAS and/or BRAFV600E backgrounds. Predicted synergistic inhibition of ERK signaling was corroborated by experiments in mutant NRAS, HRAS, and BRAFV600E cells, and inhibition of oncogenic RAS signaling was associated with reduced cell proliferation and colony formation.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases raf/antagonistas & inibidores , Proteínas ras/metabolismo , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Simulação de Acoplamento Molecular , Mutação/efeitos dos fármacos , Neoplasias/genética , Neoplasias/metabolismo , Multimerização Proteica/efeitos dos fármacos , Termodinâmica , Quinases raf/química , Quinases raf/metabolismo , Proteínas ras/genética
18.
PLoS One ; 12(9): e0184099, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28886103

RESUMO

Sox3/SOX3 is one of the earliest neural markers in vertebrates. Together with the Sox1/SOX1 and Sox2/SOX2 genes it is implicated in the regulation of stem cell identity. In the present study, we performed the first analysis of epigenetic mechanisms (DNA methylation and histone marks) involved in the regulation of the human SOX3 gene expression during RA-induced neural differentiation of NT2/D1 cells. We show that the promoter of the human SOX3 gene is extremely hypomethylated both in undifferentiated NT2/D1 cells and during the early phases of RA-induced neural differentiation. By employing chromatin immunoprecipitation, we analyze several histone modifications across different regions of the SOX3 gene and their dynamics following initiation of differentiation. In the same timeframe we investigate profiles of selected histone marks on the promoters of human SOX1 and SOX2 genes. We demonstrate differences in histone signatures of SOX1, SOX2 and SOX3 genes. Considering the importance of SOXB1 genes in the process of neural differentiation, the present study contributes to a better understanding of epigenetic mechanisms implicated in the regulation of pluripotency maintenance and commitment towards the neural lineage.


Assuntos
Diferenciação Celular/genética , Epigênese Genética , Regulação da Expressão Gênica , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição SOXB1/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Ilhas de CpG , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição SOXB1/metabolismo
19.
Genome Med ; 9(1): 15, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28187790

RESUMO

BACKGROUND: Retinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance. METHODS: We applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets. RESULTS: We determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-ß) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-ß. Co-targeting of the retinoic acid and TGF-ß pathways, through RA and kartogenin (KGN; a TGF-ß signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells. CONCLUSIONS: Our approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-ß signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.


Assuntos
Anilidas/uso terapêutico , Proteína Proto-Oncogênica N-Myc/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Ácidos Ftálicos/uso terapêutico , Transdução de Sinais , Fator de Crescimento Transformador beta/efeitos dos fármacos , Tretinoína/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Medicina de Precisão , Retinoides/uso terapêutico
20.
Indian Pediatr ; 53(9): 786-789, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27771646

RESUMO

OBJECTIVE: The incidence of the 22q11.2 microdeletion among children who have at least two out of five major clinical criteria for 22q11.2 deletion syndrome. DESIGN: Prospective study. SETTING: University Childrens Hospital in Belgrade, Serbia between 2005 and 2014. PARTICIPANTS: 57 patients with clinical characteristics of 22q11.2 deletion syndrome. METHODS: Standard G-banding cytogenetic analysis was performed in all children, and the 22q11.2 genomic region was examined using fluorescence in situ hybridization (FISH). For patients with no deletion detected by FISH, multiplex ligation-dependent probe amplification (MLPA) analysis was also done in order to detect cryptic deletions of this region and to analyze other genomic loci associated with phenotypes resembling the syndrome. A selected group of patients diagnosed to have 22q11.2 microdeletion by FISH underwent MLPA testing in order to characterize the size and position of deletion. OUTCOME MEASURES: The frequency of 22q11.2 microdeletion among children with at least two of the five major characteristics of 22q11.2 deletion syndrome (heart malformations, facial dysmorphism, T-cell immunodeficiency, palatal clefts and hypocalcemia/hypoparathyroidism). RESULTS: Typical 22q11.2 microdeletion was detected in 42.1% of patients; heart malformation were identified in all of them, facial dysmorphism in 79.2%, immunological problems in 63.6%, hypocalcemia in 62.5% and cleft palate in 8.3%. CONCLUSION: A higher detection rate compared to one-feature criterion is obtained when at least two major features of 22q11.2 deletion syndrome are taking into consideration. The criteria applied in this study could be considered by centers in low-income countries.


Assuntos
Síndrome de DiGeorge/diagnóstico , Síndrome de DiGeorge/genética , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Cariótipo , Masculino , Técnicas de Amplificação de Ácido Nucleico , Estudos Prospectivos , Sérvia
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