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1.
Mol Biol Rep ; 50(12): 10061-10072, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37906423

RESUMO

BACKGROUND: MicroRNA (miRNA) expression analysis has been shown to provide them as biomarkers in several eye diseases and has a regulatory role in pathogenesis. However, miRNA expression analysis in the vitreous humor (VH) of intraocular tuberculosis (IOTB) is not studied. Thus, we aim to find miRNA expression signatures in the VH of IOTB patients to identify their regulatory role in disease pathogenesis and to find them as potential biomarkers for IOTB. METHODS AND RESULTS: First, we profiled miRNAs in VH of three IOTB and three Macular hole (MH) samples as controls through small-RNA deep sequencing using Illumina Platform. In-house bioinformatics analysis identified 81 dysregulated miRNAs in IOTB. Further validation in VH of IOTB (n = 15) compared to MH (n = 15) using Real-Time quantitative PCR (RT-qPCR) identified three significantly upregulated miRNAs, hsa-miR-150-5p, hsa-miR-26b-5p, and hsa-miR-21-5p. Based on the miRNA target prediction, functional network analysis, and RT-qPCR analysis of target genes, the three miRNAs downregulating WNT5A, PRKCA, MAP3K7, IL7, TGFB2, IL1A, PRKCB, TNFA, and TP53 genes involving MAPK signaling pathway, PI3K-AKT signaling pathway, WNT signaling pathway, Cell cycle, TGF-beta signaling pathway, Long-term potentiation, and Sphingolipid signaling pathways, have a potential role in disease pathogenesis. The ROC analysis of RT-qPCR data showed that hsa-miR-150-5p with AUC = 0.715, hsa-miR-21-5p with AUC = 0.789, and hsa-miR-26b-5p with AUC = 0.738; however, the combination of hsa-miR-21-5p and hsa-miR-26b-5p with AUC = 0.796 could serve as a potential biomarker for IOTB. CONCLUSIONS: This study provides the first report on miRNA expression signatures detected in VH for IOTB pathogenesis and also provides a potential biomarker for IOTB.


Assuntos
MicroRNAs , Corpo Vítreo , Humanos , Corpo Vítreo/metabolismo , Fosfatidilinositol 3-Quinases/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Biomarcadores
2.
J Exp Clin Cancer Res ; 42(1): 37, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36717865

RESUMO

BACKGROUND: A pharmacogenomic platform using patient-derived cells (PDCs) was established to identify the underlying resistance mechanisms and tailored treatment for patients with advanced or refractory lung cancer. METHODS: Drug sensitivity screening and multi-omics datasets were acquired from lung cancer PDCs (n = 102). Integrative analysis was performed to explore drug candidates according to genetic variants, gene expression, and clinical profiles. RESULTS: PDCs had genomic characteristics resembled with those of solid lung cancer tissues. PDC molecular subtyping classified patients into four groups: (1) inflammatory, (2) epithelial-to-mesenchymal transition (EMT)-like, (3) stemness, and (4) epithelial growth factor receptor (EGFR)-dominant. EGFR mutations of the EMT-like subtype were associated with a reduced response to EGFR-tyrosine kinase inhibitor therapy. Moreover, although RB1/TP53 mutations were significantly enriched in small-cell lung cancer (SCLC) PDCs, they were also present in non-SCLC PDCs. In contrast to its effect in the cell lines, alpelisib (a PI3K-AKT inhibitor) significantly inhibited both RB1/TP53 expression and SCLC cell growth in our PDC model. Furthermore, cell cycle inhibitors could effectively target SCLC cells. Finally, the upregulation of transforming growth factor-ß expression and the YAP/TAZ pathway was observed in osimertinib-resistant PDCs, predisposing them to the EMT-like subtype. Our platform selected XAV939 (a WNT-TNKS-ß-catenin inhibitor) for the treatment of osimertinib-resistant PDCs. Using an in vitro model, we further demonstrated that acquisition of osimertinib resistance enhances invasive characteristics and EMT, upregulates the YAP/TAZ-AXL axis, and increases the sensitivity of cancer cells to XAV939. CONCLUSIONS: Our PDC models recapitulated the molecular characteristics of lung cancer, and pharmacogenomics analysis provided plausible therapeutic candidates.


Assuntos
Neoplasias Pulmonares , Farmacogenética , Humanos , Fosfatidilinositol 3-Quinases/genética , Receptores ErbB/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Linhagem Celular Tumoral , Mutação , Transição Epitelial-Mesenquimal/genética
3.
Methods Cell Biol ; 171: 81-109, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35953207

RESUMO

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.


Assuntos
Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Calreticulina/genética , Humanos , Immunoblotting , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Fosfatidilinositol 3-Quinases/genética , Policitemia Vera/genética , Mielofibrose Primária/genética , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco , Serina-Treonina Quinases TOR/genética
4.
Allergol Immunopathol (Madr) ; 50(4): 1-9, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35789397

RESUMO

Monoallelic loss-of-function (LOF) mutations in the phosphatidylinositol 3-kinase (PIK3R1) gene affecting the inter-Src homology 2 domain of the p85α regulatory subunit of phosphoinositide--3-kinase δ (PI3Kδ) cause the activated PI3K δ syndrome (APDS2). APDS2 is defined as a primary antibody deficiency, developmental abnormalities within the B and T lymph cell compartments, and immune dysregulation. The genetic defect of APDS2 is shared with that of the SHORT syndrome, characterized by short stature, joint hyperextensibility, ocular depression, Rieger anomaly, and delayed tooth eruption. LOF variants in an intronic splice site (c.1425+1G.C/A/T) in the PI3KR1 gene have been identified in patients affected with both APDS2 and SHORT syndrome. Herein, we report a novel c.1644-1648del (p.Asp548Glufs*6) variant in a pediatric patient with the APDS2-related immunodeficiency, who presents with mild phenotypic features of the SHORT syndrome, congenital chest wall deformity, and IgE-mediated food allergy. The same variant was also identified in the patient's hitherto asymptomatic mother, implicating an incomplete penetrance. Regular monitoring by a multidisciplinary team under the pediatric clinical immunologist's supervision to implement appropriate diagnostic procedures and treatment modalities is of paramount importance. Further studies are required to better define the genotype-phenotype correlation in patients with the PIK3R1 gene mutations and to better delineate the mutual relationship between APDS2 and the SHORT syndrome.


Assuntos
Fosfatidilinositol 3-Quinases , Doenças da Imunodeficiência Primária , Criança , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Transtornos do Crescimento , Humanos , Hipercalcemia , Doenças Metabólicas , Mutação/genética , Nefrocalcinose , Penetrância , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Doenças da Imunodeficiência Primária/diagnóstico , Doenças da Imunodeficiência Primária/genética , Fatores de Transcrição/genética
5.
Sci Rep ; 12(1): 11075, 2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35773379

RESUMO

The genes associated with fruiting body formation of Sparasis latifolia are valuable for improving mushroom breeding. To investigate this process, 4.8 × 108 RNA-Seq reads were acquired from three stages: hyphal knot (SM), primordium (SP), and primordium differentiation (SPD). The de novo assembly generated a total of 48,549 unigenes, of which 71.53% (34,728) unigenes could be annotated by at least one of the KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), and KOG (Eukaryotic Orthologous Group) databases. KEGG and KOG analyses respectively mapped 32,765 unigenes to 202 pathways and 19,408 unigenes to 25 categories. KEGG pathway enrichment analysis of DEGs (differentially expressed genes) indicated primordium initiation was significantly related to 66 pathways, such as "Ribosome", "metabolism of xenobiotics by cytochrome P450", and "glutathione metabolism" (among others). The MAPK and mTOR signal transduction pathways underwent significant adjustments during the SM to SP transition. Further, our research revealed the PI3K-Akt signaling pathway related to cell proliferation could play crucial functions during the development of SP and SPD. These findings provide crucial candidate genes and pathways related to primordium differentiation and development in S. latifolia, and advances our knowledge about mushroom morphogenesis.


Assuntos
Agaricales , Transcriptoma , Agaricales/genética , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Fosfatidilinositol 3-Quinases/genética , Melhoramento Vegetal , Polyporales
6.
Stem Cell Res Ther ; 13(1): 157, 2022 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-35410372

RESUMO

BACKGROUND: The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells. METHODS: Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression. RESULTS: Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that "complement and coagulation cascades," "ECM-receptor interaction" and "PI3K-Akt signaling pathway" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3. CONCLUSIONS: Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.


Assuntos
Fosfatidilinositol 3-Quinases , Retinose Pigmentar , Análise Custo-Benefício , Análise Mutacional de DNA , Proteínas do Olho/metabolismo , Fibroblastos/metabolismo , Humanos , Mutação , Linhagem , Fosfatidilinositol 3-Quinases/genética , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Rodopsina/genética
7.
J Tradit Chin Med ; 41(1): 167-180, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33522210

RESUMO

OBJECTIVE: To investigate the targets and mechanisms of action of Qingkailing injection (,QKL) in the treatment of cholestatic hepatitis. METHODS: A network pharmacology method was implemented using drug and disease databases to target QKL and cholestasis hepatitis, respectively. The functional protein association network STRING database was used to construct a protein-protein interaction network using R language and the Bioconductor toolkit. The org.Hs.eg.db and clusterProfiler packages were used for gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, which explored biological functions and pathways of potential targets. Targets were then visualized using Cytoscape 3.6.0 software. RESULTS: We screened 121 compounds in QKL and identified 112 targets for the treatment of cholestatic hepatitis. QKL played a role in the treatment of cholestatic hepatitis through 305 biology process terms, 15 cellular component and 29 molecular function terms. The mechanism of QKL action was mainly related to tumor necrosis factor, mitogen-activated protein kinase, and PI3K-Akt signaling pathways. CONCLUSION: The treatment of cholestatic hepatitis by QKL involved multiple targets, biological functions, and signaling pathways that are closely associated with the disease.


Assuntos
Colestase/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Hepatite/tratamento farmacológico , Animais , Colestase/genética , Colestase/metabolismo , Hepatite/genética , Hepatite/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Mol Metab ; 40: 101020, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32439336

RESUMO

OBJECTIVE: Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance. METHODS: The human pathogenic Pik3r1 Y657∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically. RESULTS: Like humans with SHORT syndrome, Pik3r1WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia. CONCLUSIONS: Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/genética , Transtornos do Crescimento/metabolismo , Hipercalcemia/metabolismo , Resistência à Insulina/genética , Doenças Metabólicas/metabolismo , Nefrocalcinose/metabolismo , Tecido Adiposo Marrom/metabolismo , Adiposidade , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Dieta Hiperlipídica , Dislipidemias/genética , Metabolismo Energético/genética , Fígado Gorduroso/metabolismo , Transtornos do Crescimento/genética , Hipercalcemia/genética , Inflamação/metabolismo , Insulina/metabolismo , Lipogênese , Fígado/metabolismo , Masculino , Doenças Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Nefrocalcinose/genética , Obesidade/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo
9.
Mediators Inflamm ; 2020: 8293921, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189997

RESUMO

A joint is the point of connection between two bones in our body. Inflammation of the joint leads to several diseases, including osteoarthritis, which is the concern of this review. Osteoarthritis is a common chronic debilitating joint disease mainly affecting the elderly. Several studies showed that inflammation triggered by factors like biomechanical stress is involved in the development of osteoarthritis. This stimulates the release of early-stage inflammatory cytokines like interleukin-1 beta (IL-1ß), which in turn induces the activation of signaling pathways, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), and mitogen-activated protein kinase (MAPK). These events, in turn, generate more inflammatory molecules. Subsequently, collagenase like matrix metalloproteinases-13 (MMP-13) will degrade the extracellular matrix. As a result, anatomical and physiological functions of the joint are altered. This review is aimed at summarizing the previous studies highlighting the involvement of inflammation in the pathogenesis of osteoarthritis.


Assuntos
Inflamação/metabolismo , Inflamação/patologia , Animais , Humanos , Interleucina-1beta/metabolismo , Metaloproteinase 13 da Matriz/economia , Metaloproteinase 13 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo
10.
Nat Commun ; 10(1): 2674, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209238

RESUMO

The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca's large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biologia Computacional/métodos , Neoplasias/tratamento farmacológico , Farmacogenética/métodos , Proteína ADAM17/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benchmarking , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Biologia Computacional/normas , Conjuntos de Dados como Assunto , Antagonismo de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Genômica/métodos , Humanos , Terapia de Alvo Molecular/métodos , Mutação , Neoplasias/genética , Farmacogenética/normas , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Resultado do Tratamento
11.
Nat Immunol ; 19(9): 986-1000, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30127432

RESUMO

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.


Assuntos
Linfócitos B/fisiologia , Microbioma Gastrointestinal/imunologia , Centro Germinativo/fisiologia , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Autoanticorpos/sangue , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/genética , Modelos Animais de Doenças , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Imunidade Humoral/genética , Switching de Imunoglobulina/genética , Síndromes de Imunodeficiência/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
12.
Am J Pathol ; 188(9): 1956-1972, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30030980

RESUMO

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in cancer. Tumors are potentially sensitive to PI3K pathway inhibitors, but reliable diagnostic tests that assess functional PI3K activity are lacking. Because PI3K pathway activity negatively regulates forkhead box-O (FOXO) transcription factor activity, FOXO target gene expression is inversely correlated with PI3K activity. A knowledge-based Bayesian computational model was developed to infer PI3K activity in cancer tissue samples from FOXO target gene mRNA levels and validated in cancer cell lines treated with PI3K inhibitors. However, applied to patient tissue samples, FOXO was often active in cancer types with expected active PI3K. SOD2 was differentially expressed between FOXO-active healthy and cancer tissue samples, indicating that cancer-associated cellular oxidative stress alternatively activated FOXO. To enable correct interpretation of active FOXO in cancer tissue, threshold levels for normal SOD2 expression in healthy tissue were defined above which FOXO activity is oxidative stress induced and below which PI3K regulated. In slow-growing luminal A breast cancer and low Gleason score prostate cancer, FOXO was active in a PI3K-regulated manner, indicating inactive PI3K. In aggressive luminal B, HER2, and basal breast cancer, FOXO was increasingly inactive or actively induced by oxidative stress, indicating PI3K activity. We provide a decision tree that facilitates functional PI3K pathway activity assessment in tissue samples from patients with cancer for therapy response prediction and prognosis.


Assuntos
Neoplasias da Mama/metabolismo , Biologia Computacional/métodos , Fatores de Transcrição Forkhead/metabolismo , Bases de Conhecimento , Fosfatidilinositol 3-Quinases/metabolismo , Superóxido Dismutase/metabolismo , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Testes Diagnósticos de Rotina , Feminino , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Superóxido Dismutase/genética
13.
PLoS One ; 12(9): e0183715, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28926605

RESUMO

Increased early detection and personalized therapy for lung cancer have coincided with greater use of minimally invasive sampling techniques such as endobronchial ultrasound-guided biopsy (EBUS), endoscopic ultrasound-guided biopsy (EUS), and navigational biopsy, as well as thin needle core biopsies. As many lung cancer patients have late stage disease and other comorbidities that make open surgical procedures hazardous, the least invasive biopsy technique with the highest potential specimen yield is now the preferred first diagnostic study. However, use of these less invasive procedures generates significant analytical challenges for the laboratory, such as a requirement for robust detection of low level somatic mutations, particularly when the starting sample is very small or demonstrates few intact tumor cells. In this study, we assessed 179 clinical cases of non-small cell lung carcinoma (NSCLC) that had been previously tested for EGFR, KRAS, NRAS, and BRAF mutations using a novel multiplexed analytic approach that reduces wild-type signal and allows for detection of low mutation load approaching 1%, iPLEX® HS panel for the MassARRAY® System (Agena Bioscience, San Diego, CA). This highly sensitive system identified approximately 10% more KRAS, NRAS, EGFR and BRAF mutations than were detected by the original test platform, which had a sensitivity range of 5-10% variant allele frequency (VAF).


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , GTP Fosfo-Hidrolases/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , DNA/química , DNA/metabolismo , Receptores ErbB/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Genótipo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas B-raf/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas ras/metabolismo
14.
Food Chem Toxicol ; 109(Pt 2): 910-922, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28249781

RESUMO

The anti-cancerous activity of 6-gingerol extracted from Tongling White Ginger was investigated. 6-Gingerol inhibited the growth of HeLa cells with IC50 (96.32 µM) and IC80 (133.01 µM) and led to morphological changes, induced the cell cycle arrest in G0/G1-phase and ultimately resulted into apoptosis. Among cell cycle-related genes and proteins, the expression of cyclin (A, D1, E1) reduced, while of CDK-1, p21 and p27 showed slight decrease, except cyclin B1 and E1 (protein). Western blotting reported the induction of apoptosis with an increased Bax/Bcl-2 ratio, release of cytochrome c, cleavage of caspase-3, -8, -9 and PRPP in treated cells. 6-Gingerol activated AMPK, but inhibited PI3K/AKT phosphorylation with reduced P70S6K expression and also suppressed the mTOR phosphorylation. 6-Gingerol with 5-FU and Ptx resulted in 83.2% and 52% inhibition respectively, this synergy have stimulated apoptosis proteins more efficiently as compared to 6-Gingerol alone (10.75%) under in vitro conditions.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Catecóis/farmacologia , Álcoois Graxos/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Zingiber officinale/química , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/fisiopatologia , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Fluoruracila/farmacologia , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/fisiopatologia
15.
Mol Hum Reprod ; 23(6): 393-405, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28333280

RESUMO

STUDY QUESTION: How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? SUMMARY ANSWER: hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. WHAT IS KNOWN ALREADY: hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. STUDY DESIGN, SIZE, DURATION: This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. WIDER IMPLICATIONS OF THE FINDINGS: We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.


Assuntos
Gonadotropina Coriônica/farmacologia , Fatores de Troca do Nucleotídeo Guanina/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Receptores de Progesterona/genética , Células Estromais/efeitos dos fármacos , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Adulto , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/agonistas , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Homeobox A10 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Gravidez , Cultura Primária de Células , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/metabolismo , Transcrição Gênica
16.
J Inherit Metab Dis ; 40(1): 131-137, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27783170

RESUMO

Despite adequate dietary management, patients with classic galactosemia continue to have increased risks of cognitive deficits, speech dyspraxia, primary ovarian insufficiency, and abnormal motor development. A recent evaluation of a new galactose-1 phosphate uridylyltransferase (GALT)-deficient mouse model revealed reduced fertility and growth restriction. These phenotypes resemble those seen in human patients. In this study, we further assess the fidelity of this new mouse model by examining the animals for the manifestation of a common neurological sequela in human patients: cerebellar ataxia. The balance, grip strength, and motor coordination of GALT-deficient and wild-type mice were tested using a modified rotarod. The results were compared to composite phenotype scoring tests, typically used to evaluate neurological and motor impairment. The data demonstrated abnormalities with varying severity in the GALT-deficient mice. Mice of different ages were used to reveal the progressive nature of motor impairment. The varying severity and age-dependent impairments seen in the animal model agree with reports on human patients. Finally, measurements of the cerebellar granular and molecular layers suggested that mutant mice experience cerebellar hypoplasia, which could have resulted from the down-regulation of the PI3K/Akt signaling pathway.


Assuntos
Ataxia/genética , Galactosemias/genética , UDPglucose-Hexose-1-Fosfato Uridiltransferase/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo/genética , Camundongos , Atividade Motora/genética , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética
17.
Scand J Clin Lab Invest ; 76(5): 386-92, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27215271

RESUMO

BACKGROUND: Next-generation sequencing (NGS) has been widely applied in clinical research, while its application in routine clinical molecular testing requires careful validation. The aim of our study was to assess the clinical usefulness of the NextDaySeq Lung panel on Ion Torrent™ PGM in mutation detection of actionable genes in lung cancer. METHODS: The NextDaySeq assay was evaluated by blinded comparisons to Quantitative Real-Time PCR (qPCR) assays with 188 consecutive samples from Chinese patients with non-small cell lung cancer (NSCLC) to detect mutations in EGFR, KRAS, PIK3CA and BRAF. Discordant variants were further validated by Sanger sequencing and independent qPCR and NGS assays. RESULTS: Our results showed 93.3% concordance of reportable variants mutually covered in both NGS and qPCR assays, with a clinical sensitivity of 89.9%, specificity of 97.5%. Through the comparison, the NGS assays demonstrated its advantages in offering more clinical relevant information, such as detecting non-hotspot mutations and providing mutation allele frequencies (MAF) and accurate mutation sequences. The analytical sensitivity of NGS to detect mutations with low MAF needs further improvement. CONCLUSIONS: The NextDaySeq Lung panel exhibited good clinical performance, strongly supporting the implementation of the NGS assay in routine clinical use to facilitate therapeutic decision-making for lung cancer patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino
18.
J Clin Oncol ; 33(17): 1902-9, 2015 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-25847936

RESUMO

PURPOSE: The identification of patients with metastatic triple-negative breast cancer (mTNBC) who are expected to benefit from platinum-based chemotherapy is of interest. We conducted a single-arm phase II clinical trial of single-agent platinum for mTNBC with biomarker correlates. PATIENTS AND METHODS: Patients with mTNBC received first- or second-line cisplatin (75 mg/m(2)) or carboplatin (area under the concentration-time curve 6) by physician's choice once every 3 weeks. Coprimary end points were objective response rate (RR) and response prediction by p63/p73 gene expression. Secondary and exploratory end points included toxicity assessment, RR in cisplatin versus carboplatin, and RR in molecularly defined subgroups, including BRCA1/2 mutation carriers. RESULTS: Patients (N = 86; 69 as first-line therapy) received cisplatin (n = 43) or carboplatin (n = 43). RR was 25.6% (95% CI, 16.8% to 36%) and was numerically higher with cisplatin (32.6%) than with carboplatin (18.7%). RR was 54.5% in patients with germline BRCA1/2 mutations (n = 11). In patients without BRCA1/2 mutations (n = 66), exploratory analyses showed that a BRCA-like genomic instability signature (n = 32) discriminated responding and nonresponding tumors (mean homologous recombination deficiency-loss of heterozygosity/homologous recombination deficiency-large-scale state transitions [HRD-LOH/HRD-LST] scores were 12.68 and 5.11, respectively), whereas predefined analysis by p63/p73 expression status (n = 61), p53 and PIK3CA mutation status (n = 53), or PAM50 gene expression subtype (n = 55) did not. Five of the six long-term responders alive at a median of 4.5 years lacked germline BRCA1/2 mutations, and two of them had increased tumor HRD-LOH/HRD-LST scores. CONCLUSION: Platinum agents are active in mTNBC, especially in patients with germline BRCA1/2 mutations. A measure of tumor DNA repair function may identify patients without mutations who could benefit from platinum therapy agents. Prospective controlled confirmatory trials are warranted.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Carboplatina/uso terapêutico , Cisplatino/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Adulto , Idoso , Antineoplásicos/administração & dosagem , Proteína BRCA1/genética , Proteína BRCA2/genética , Carboplatina/administração & dosagem , Cisplatino/administração & dosagem , Classe I de Fosfatidilinositol 3-Quinases , Esquema de Medicação , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Heterozigoto , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
19.
Clin Lab ; 61(1-2): 155-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25807649

RESUMO

BACKGROUND: Disparities in DNA template quantity across sample wells is a common occurrence in PCR experiments and a frequent cause of bogus results in research and diagnostics. High Resolution Melt (HRM) analysis, a robust genotyping technique, was shown to be highly reproducible irrespective of the reaction volume and DNA template concentration used for PCR. The aim of this study is to determine if HRM can maintain its reproducibility/reliability when varying amounts of starting DNA templates are utilized for the upstream PCR. METHODS: Serial dilutions of differentially prepared DNA templates were amplified and subjected to HRM analysis using the ABI 7500 Fast Real Time PCR machine and the ABI HRM software v2.0.6. The first serial dilution set consisted of seven 1:2 serial dilutions of amplicon-enriched DNA templates from two samples of known genotypes at exon 9 of PIK3CA, while the second serial dilution set consisted of four 1:10 dilutions of two different template types from the same sample: original FFPE DNA template and amplicon-enriched DNA template. RESULTS: HRM analysis was reproducible across two- to one thousand-fold difference in starting DNA template in the upstream PCR, so long as the starting DNA template is of ample quality and quantity to give an adequate PCR amplification. CONCLUSIONS: HRM is highly reliable in the presence of a wide variation in the amount of starting template across wells for the upstream PCR, and the factor that determines HRM reproducibility is the adequacy of the PCR amplification.


Assuntos
DNA/análise , Técnicas de Genotipagem , Reação em Cadeia da Polimerase , Classe I de Fosfatidilinositol 3-Quinases , Fosfatidilinositol 3-Quinases/genética , Reprodutibilidade dos Testes
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