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1.
NPJ Syst Biol Appl ; 7(1): 2, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420045

RESUMO

Metabolic reprogramming and its molecular underpinnings are critical to unravel the duality of cancer cell function and chemo-resistance. Here, we use a constraints-based integrated approach to delineate the interplay between metabolism and epigenetics, hardwired in the genome, to shape temozolomide (TMZ) resistance. Differential metabolism was identified in response to TMZ at varying concentrations in both the resistant neurospheroidal (NSP) and the susceptible (U87MG) glioblastoma cell-lines. The genetic basis of this metabolic adaptation was characterized by whole exome sequencing that identified mutations in signaling pathway regulators of growth and energy metabolism. Remarkably, our integrated approach identified rewiring in glycolysis, TCA cycle, malate aspartate shunt, and oxidative phosphorylation pathways. The differential killing of TMZ resistant NSP by Rotenone at low concentrations with an IC50 value of 5 nM, three orders of magnitude lower than for U87MG that exhibited an IC50 value of 1.8 mM was thus identified using our integrated systems-based approach.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Genética , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Metabolômica/métodos , Transdução de Sinais/efeitos dos fármacos , Biologia de Sistemas/métodos
2.
Biochem Biophys Res Commun ; 495(2): 2010-2016, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29248726

RESUMO

An authenticated U87MG clonal glioblastoma cell line was investigated to identify a sub-population of neurospheroidal (NSP) cells within the main epithelial population (U87MG). The NSP cells sorted using Fluorescence Assisted Cell Sorting (FACS) showed varied morphology, 30% lower growth rates, 40% higher IC50 values for temozolomide drug and could differentiate into the glial cell type (NDx). Metabolite profiling using HR-LCMS identified glucose, glutamine and serine in both populations and tryptophan only in U87MG as growth limiting substrates. Glycine, alanine, glutamate and proline were secreted by U87MG, however proline and glycine were re-utilized in NSP. Exo-metabolite profiling and phenotypic microarrays identified differential metabolism of primary carbon sources glucose and derived pyruvate for U87MG; glutamine and derived glutamate metabolism in NSP. Differential mRNA abundance of AKT1, PTEN, PIK3CA controlling metabolism, drug efflux, nutrient transport and epigenetic control MDM2 are potentially critical in shaping DNA methylation effects of temozolomide. Our study provides a new insight into the combined effect of these factors leading to temozolomide resistance in NSP.


Assuntos
Aminoácidos/metabolismo , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glucose/metabolismo , Análise do Fluxo Metabólico/métodos , Ácido Pirúvico/metabolismo , Antineoplásicos Alquilantes/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/administração & dosagem , Relação Dose-Resposta a Droga , Glioblastoma/patologia , Humanos , Integração de Sistemas , Temozolomida
3.
BMC Syst Biol ; 11(1): 51, 2017 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-28446174

RESUMO

BACKGROUND: The leading edge of the global problem of antibiotic resistance necessitates novel therapeutic strategies. This study develops a novel systems biology driven approach for killing antibiotic resistant pathogens using benign metabolites. RESULTS: Controlled laboratory evolutions established chloramphenicol and streptomycin resistant pathogens of Chromobacterium. These resistant pathogens showed higher growth rates and required higher lethal doses of antibiotic. Growth and viability testing identified malate, maleate, succinate, pyruvate and oxoadipate as resensitising agents for antibiotic therapy. Resistant genes were catalogued through whole genome sequencing. Intracellular metabolomic profiling identified violacein as a potential biomarker for resistance. The temporal variance of metabolites captured the linearized dynamics around the steady state and correlated to growth rate. A constraints-based flux balance model of the core metabolism was used to predict the metabolic basis of antibiotic susceptibility and resistance. CONCLUSIONS: The model predicts electron imbalance and skewed NAD/NADH ratios as a result of antibiotics - chloramphenicol and streptomycin. The resistant pathogen rewired its metabolic networks to compensate for disruption of redox homeostasis. We foresee the utility of such scalable workflows in identifying metabolites for clinical isolates as inevitable solutions to mitigate antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Chromobacterium/efeitos dos fármacos , Chromobacterium/metabolismo , Farmacorresistência Bacteriana/genética , NAD/metabolismo , Biologia de Sistemas , Chromobacterium/genética , Simulação por Computador , Evolução Molecular Direcionada , Fenótipo
4.
J AOAC Int ; 100(3): 640-646, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28315638

RESUMO

A chromatography-free atmospheric pressure matrix-assisted laser desorption/ionization high-resolution mass spectrometry (AP-MALDI HRMS) method is described for the simultaneous and quantitative detection of triazines and triazoles in grapes. The analytes were detected reproducibly with high mass accuracy (mass error within 5 ppm) and further confirmed by collision-induced dissociation fragmentation in tandem MS. The LODs and LOQs for all the analytes were found to be in the nanogram per gram level (15-20 ng/g LOQ). Internal standard-normalized high-resolution accurate mass-extracted (HR-AM) peak intensities of the detected ions were used to generate the concentration response curves. Linearity (with R2 values around 0.99) was obtained for these curves within a concentration range of 20-200 ng/g of the individual analytes. The accuracy and precision of the method were further established using QC samples. Validation and performance comparison of the AP-MALDI HRMS method with an existing standard method using LC with triple quadrupole MS was carried out (evaluating sensitivity, accuracy, precision, and analysis time) using 20 table-grape field samples after QuEChERS extraction.


Assuntos
Triazinas/análise , Triazóis/análise , Vitis/química , Pressão Atmosférica , Espectrometria de Massas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Cancer Res ; 70(12): 4809-19, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20530682

RESUMO

Multiple, dissimilar genetic defects in cancers of the same origin contribute to heterogeneity in tumor phenotypes and therapeutic responses of patients, yet the associated molecular mechanisms remain elusive. Here, we show at the systems level that serous ovarian carcinoma is marked by the activation of interconnected modules associated with a specific gene set that was derived from three independent tumor-specific gene expression data sets. Network prediction algorithms combined with preestablished protein interaction networks and known functionalities affirmed the importance of genes associated with ovarian cancer as predictive biomarkers, besides "discovering" novel ones purely on the basis of interconnectivity, whose precise involvement remains to be investigated. Copy number alterations and aberrant epigenetic regulation were identified and validated as significant influences on gene expression. More importantly, three functional modules centering on c-Myc activation, altered retinoblastoma signaling, and p53/cell cycle/DNA damage repair pathways have been identified for their involvement in transformation-associated events. Further studies will assign significance to and aid the design of a panel of specific markers predictive of individual- and tumor-specific pathways. In the parlance of this emerging field, such networks of gene-hub interactions may define personalized therapeutic decisions.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Cistadenocarcinoma Seroso/genética , Redes Reguladoras de Genes , Neoplasias Ovarianas/genética , Mapeamento de Interação de Proteínas , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Imunoprecipitação , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Transdução de Sinais
6.
Stem Cells ; 27(9): 2059-68, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19544473

RESUMO

The transcriptional repressors Snail and Slug contribute to cancer progression by mediating epithelial-mesenchymal transition (EMT), which results in tumor cell invasion and metastases. We extend this current understanding to demonstrate their involvement in the development of resistance to radiation and paclitaxel. The process is orchestrated through the acquisition of a novel subset of gene targets that is repressed under conditions of stress, effectively inactivating p53-mediated apoptosis, while another subset of targets continues to mediate EMT. Repressive activities are complemented by a concurrent derepression of specific genes resulting in the acquisition of stem cell-like characteristics. Such cells are bestowed with three critical capabilities, namely EMT, resistance to p53-mediated apoptosis, and a self-renewal program, that together define the functionality and survival of metastatic cancer stem cells. EMT provides a mechanism of escape to a new, less adverse niche; resistance to apoptosis ensures cell survival in conditions of stress in the primary tumor; whereas acquisition of "stemness" ensures generation of the critical tumor mass required for progression of micrometastases to macrometastases. Our findings, besides achieving considerable expansion of the inventory of direct genes targets, more importantly demonstrate that such elegant cooperative modulation of gene regulation mediated by Snail and Slug is critical for a cancer cell to acquire stem cell characteristics toward resisting radiotherapy- or chemotherapy-mediated cellular stress, and this may be a determinative aspect of aggressive cancer metastases.


Assuntos
Apoptose/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Genoma Humano/genética , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/radioterapia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética
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