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1.
Anal Chem ; 96(29): 12129-12138, 2024 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-38985547

RESUMO

As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.


Assuntos
Fígado , Organoides , Humanos , Organoides/metabolismo , Organoides/citologia , Cromatografia Líquida/métodos , Fígado/metabolismo , Espectrometria de Massas/métodos , Tolbutamida/metabolismo , Tolbutamida/análise , Dispositivos Lab-On-A-Chip , Preparações Farmacêuticas/metabolismo , Preparações Farmacêuticas/análise , Extração em Fase Sólida , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Espectrometria de Massa com Cromatografia Líquida
2.
Anal Chem ; 94(50): 17677-17684, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36484723

RESUMO

Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the "organ-in-a-column" units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated "on column" with heroin, followed by on-line monitoring of the drug's phase 1 metabolism. Enzymatic metabolism of heroin produced in the "organ-in-a-column" units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.


Assuntos
Heroína , Fígado , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Automação
3.
Breast Cancer Res ; 18(1): 58, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27246191

RESUMO

BACKGROUND: Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting. METHODS: To identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed. RESULTS: The cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30-50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER- LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy. CONCLUSION: Taken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.


Assuntos
Antineoplásicos Hormonais/farmacologia , Vias Biossintéticas , Neoplasias da Mama/metabolismo , Colesterol/biossíntese , Resistencia a Medicamentos Antineoplásicos , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ésteres do Colesterol/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fenótipo , Prognóstico , Proteoma , Proteômica/métodos , Interferência de RNA , Transcriptoma , Resultado do Tratamento
4.
J Lipid Res ; 55(7): 1531-6, 2014 07.
Artigo em Inglês | MEDLINE | ID: mdl-24792927

RESUMO

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.


Assuntos
Espectrometria de Massas/métodos , Oxisteróis/análise , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Humanos
5.
Biochem Biophys Res Commun ; 446(3): 669-74, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24486313

RESUMO

The Hedgehog (HH) signaling pathway is critical in embryonic development, stem cell biology, tissue homeostasis, chemoattraction and synapse formation. Irregular HH signaling is associated with a number of disease conditions including congenital disorders and cancer. In particular, deregulation of HH signaling has been linked to skin, brain, lung, colon and pancreatic cancers. Key mediators of the HH signaling pathway are the 12-pass membrane protein Patched (PTC), the 7-pass membrane protein Smoothened (SMO) and the GLI transcription factors. PTC shares homology with the RND family of small-molecule transporters and it has been proposed that it interferes with SMO through metabolites. Although a conclusive picture is lacking, substantial efforts are made to identify and understand natural metabolites/sterols, including cholesterol, vitamin D3, oxysterols and glucocorticoides, that may be affected by, or influence the HH signaling cascade at the level of PTC and SMO. In this review we will elaborate the role of metabolites in HH signaling with a focus on oxysterols, and discuss advancements in modern analytical approaches in the field.


Assuntos
Técnicas de Química Analítica/métodos , Glucocorticoides/metabolismo , Proteínas Hedgehog/metabolismo , Esteróis/análise , Esteróis/metabolismo , Animais , Humanos , Receptores Patched , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Receptor Smoothened
6.
J Chromatogr A ; 1717: 464669, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38278130

RESUMO

Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.


Assuntos
Insulina , Espectrometria de Massa com Cromatografia Líquida , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Organoides , Eletroforese em Gel de Ágar
7.
Adv Healthc Mater ; 13(13): e2303785, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38221504

RESUMO

Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.


Assuntos
Ilhotas Pancreáticas , Dispositivos Lab-On-A-Chip , Fígado , Organoides , Humanos , Fígado/metabolismo , Organoides/metabolismo , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Glucose/metabolismo
8.
J Steroid Biochem Mol Biol ; 232: 106355, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37380087

RESUMO

Oxysterols are potential biomarkers for liver metabolism that are altered under disease conditions such as non-alcoholic fatty liver disease (NAFLD). We here apply sterolomics to organoids used for disease modeling of NAFLD. Using liquid chromatography-mass spectrometry with on-line sample clean-up and enrichment, we establish that liver organoids produce and secrete oxysterols. We find elevated levels of 26-hydroxycholesterol, an LXR agonist and the first oxysterol in the acidic bile acid synthesis, in medium from steatotic liver organoids compared to untreated organoids. Other upregulated sterols in medium from steatotic liver organoids are dihydroxycholesterols, such as 7α,26-dihydroxycholesterol, and 7α,25-dihydroxycholesterol. Through 26-hydroxycholesterol exposure to human stem cell-derived hepatic stellate cells, we observe a trend of expressional downregulation of the pro-inflammatory cytokine CCL2, suggesting a protective role of 26-hydroxycholesterol during early-phased NAFLD disease development. Our findings support the possibility of oxysterols serving as NAFLD indicators, demonstrating the usefulness of combining organoids and mass spectrometry for disease modeling and biomarker studies.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Oxisteróis , Humanos , Oxisteróis/metabolismo , Espectrometria de Massas , Esteróis
9.
Biochem Pharmacol ; 196: 114731, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34407453

RESUMO

Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables formation of lipid droplets that act as storage organelles for lipid soluble vitamins and minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to maintain continual synthesis of the plasma membrane. In this systematic review and meta-analysis, we summarise the current depth of understanding of the role of this metabolic pathway in pan-cancer development. A systematic search of PubMed, Scopus, Web of Science, and Cochrane Library for preclinical studies identified eight studies where cholesteryl ester concentrations were compared between tumour and adjacent-normal tissue, and 24 studies where cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue (p < 0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly smaller tumours of all types (p ≤ 0.002). SOAT inhibition increased tumour apoptosis (p = 0.007), CD8 + lymphocyte infiltration and cytotoxicity (p ≤ 0.05), and reduced proliferation (p = 0.0003) and metastasis (p < 0.0001). Significant risk of publication bias was found and may have contributed to a 32% overestimation of the meta-analysed effect size. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses equivalent to those previously reported to be safe and tolerable in humans. This work indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing anti-neoplastic agents.


Assuntos
Anticolesterolemiantes/administração & dosagem , Colesterol/genética , Colesterol/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Carga Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Ensaios Clínicos como Assunto/métodos , Esterificação/efeitos dos fármacos , Esterificação/fisiologia , Humanos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Carga Tumoral/fisiologia , Ureia/administração & dosagem , Ureia/análogos & derivados , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Anal Chim Acta ; 1178: 338551, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482862

RESUMO

Single-cell analysis can allow for an in-depth understanding of diseases, diagnostics, and aid the development of therapeutics. However, single-cell analysis is challenging, as samples are both extremely limited in size and complex. But the concept is gaining promise, much due to novel sample preparation approaches and the ever-improving field of mass spectrometry. The mass spectrometer's output is often linked to the preceding compound separation step, typically being liquid chromatography (LC). In this review, we focus on LC's role in single-cell omics. Particle-packed nano LC columns (typically 50-100 µm inner diameter) have traditionally been the tool of choice for limited samples, and are also used for single cells. Several commercial products and systems are emerging with single cells in mind, featuring particle-packed columns or miniaturized pillar array systems. In addition, columns with inner diameters as narrow as 2 µm are being explored to maximize sensitivity. Hence, LC column down-scaling is a key focus in single-cell analysis. But narrow columns are associated with considerable technical challenges, while single cell analysis may be expected to become a "routine" service, requiring higher degrees of robustness and throughput. These challenges and expectations will increase the need and attention for the development (and even the reinvention) of alternative nano LC column formats. Therefore, monolith columns and even open tubular columns may finally find their "killer-application" in single cell analysis.


Assuntos
Análise de Célula Única , Cromatografia Líquida , Espectrometria de Massas
11.
Oncogene ; 40(16): 2872-2883, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33742124

RESUMO

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.


Assuntos
Hidroxicolesteróis/metabolismo , Receptores X do Fígado/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Epirubicina/farmacologia , Feminino , Expressão Gênica , Humanos , Receptores X do Fígado/agonistas , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-32971370

RESUMO

3', 5' - Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1-500 ng/mL cell extract.


Assuntos
Cromatografia de Fase Reversa/métodos , AMP Cíclico/análise , AMP Cíclico/metabolismo , Técnicas Citológicas/métodos , Espectrometria de Massas em Tandem/métodos , Brefeldina A , Toxina da Cólera , Células HT29 , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
13.
J Steroid Biochem Mol Biol ; 192: 105309, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30779932

RESUMO

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system ("AFFL"). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.


Assuntos
Neoplasias da Mama/metabolismo , Cromatografia Líquida/métodos , Oxisteróis/análise , Oxisteróis/química , Espectrometria de Massas em Tandem/métodos , Neoplasias da Mama/patologia , Feminino , Humanos , Hidroxicolesteróis/metabolismo
14.
Nutrients ; 11(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31683867

RESUMO

Interventions that alter cholesterol have differential impacts on hormone receptor positive- and negative-breast cancer risk and prognosis. This implies differential regulation or response to cholesterol within different breast cancer subtypes. We evaluated differences in side-chain hydroxycholesterol and liver X nuclear receptor signalling between Oestrogen Receptor (ER)-positive and ER-negative breast cancers and cell lines. Cell line models of ER-positive and ER-negative disease were treated with Liver X Receptor (LXR) ligands and transcriptional activity assessed using luciferase reporters, qPCR and MTT. Publicly available datasets were mined to identify differences between ER-negative and ER-positive tumours and siRNA was used to suppress candidate regulators. Compared to ER-positive breast cancer, ER-negative breast cancer cells were highly responsive to LXR agonists. In primary disease and cell lines LXRA expression was strongly correlated with its target genes in ER-negative but not ER-positive disease. Expression of LXR's corepressors (NCOR1, NCOR2 and LCOR) was significantly higher in ER-positive disease relative to ER-negative, and their knock-down equalized sensitivity to ligand between subtypes in reporter, gene expression and viability assays. Our data support further evaluation of dietary and pharmacological targeting of cholesterol metabolism as an adjunct to existing therapies for ER-negative and ER-positive breast cancer patients.


Assuntos
Neoplasias da Mama/metabolismo , Colesterol/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores X do Fígado/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Prognóstico , RNA Interferente Pequeno , Transdução de Sinais , Transcrição Gênica
15.
Future Sci OA ; 5(1): FSO359, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30652024

RESUMO

AIM: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. MATERIALS & METHODS: 'Case study' exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. RESULTS: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC-MS. Western blotting and nanoLC-MS also revealed negative exosome markers regarding both isolation approaches. CONCLUSION: The two isolation methods had an overall similar performance, but we hesitate to use the term 'exosome isolation' as impurities may be present with both isolation methods. NanoLC-MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

16.
Cell Rep ; 26(4): 984-995.e6, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673619

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target.


Assuntos
Gorduras na Dieta/efeitos adversos , Receptores X do Fígado/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Gorduras na Dieta/farmacologia , Receptores X do Fígado/genética , Camundongos , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética
17.
Biochimie ; 153: 3-12, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29751082

RESUMO

Oxysterols play important roles in development and diseases, but can be highly challenging to analyze. To ensure satisfactory measurements, oxysterols must typically be separated with chromatography prior to detection. Here, we will devote attention to the chromatography of oxysterols, focusing on gas chromatography and liquid chromatography. We will present the role of stationary phases, mobile phases, and dimensions and geometries of particles/columns. We discuss how these parameters may affect the chromatography, regarding factors such as speed and resolution. Finally, we present some less explored avenues for separation of oxysterols.


Assuntos
Cromatografia Gasosa/métodos , Cromatografia Líquida/métodos , Oxisteróis/isolamento & purificação
18.
Chem Phys Lipids ; 207(Pt B): 87-91, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28583433

RESUMO

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.


Assuntos
Betaína/análogos & derivados , Colesterol/química , Colesterol/isolamento & purificação , Hidroxicolesteróis/química , Hidroxicolesteróis/isolamento & purificação , Betaína/química , Colesterol/metabolismo , Colesterol Oxidase/química , Colesterol Oxidase/metabolismo , Eletroforese Capilar , Hidroxicolesteróis/metabolismo , Conformação Molecular , Espectrofotometria Ultravioleta
19.
J Steroid Biochem Mol Biol ; 169: 22-28, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-26877254

RESUMO

Exosomes from cancer cells are rich sources of biomarkers and may contain elevated levels of lipids of diagnostic value. 27-Hydroxycholesterol (27-OHC) is associated with proliferation and metastasis in estrogen receptor positive (ER+) breast cancer. In this study, we investigated the levels of 27-OHC, and other sidechain-hydroxylated oxysterols in exosomes. To study both cytoplasmic and exosomal oxysterol samples of limited size, we have developed a capillary liquid chromatography-mass spectrometry platform that outperforms our previously published systems regarding chromatographic resolution, analysis time and sensitivity. In the analyzed samples, the quantified level of cytoplasmic 27-OHC using this platform fitted with mRNA levels of 27-OHC's corresponding enzyme, CYP27A1. We find clearly increased levels of 27-OHC in exosomes (i.e., enrichment) from an ER+ breast cancer cell line (MCF-7) compared to exosomes derived from an estrogen receptor (ER-) breast cancer cell line (MDA-MB-231) and other control exosomes (non-cancerous cell line (HEK293) and human pooled serum). The exosomal oxysterol profile did not reflect cytoplasmic oxysterol profiles in the cells of origin; cytoplasmic 27-OHC was low in ER+ MCF-7 cells while high in MDA-MB-231 cells. Other control cancer cells showed varied cytoplasmic oxysterol levels. Hence, exosome profiling in cancer cells might provide complementary information with the possibility of diagnostic value.


Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Exossomos/química , Hidroxicolesteróis/química , Linhagem Celular Tumoral , Cromatografia Líquida , Citoplasma/metabolismo , Feminino , Células HEK293 , Humanos , Células MCF-7 , Espectrometria de Massas , Reprodutibilidade dos Testes
20.
Front Chem ; 5: 62, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28894734

RESUMO

A rugged and high throughput capillary column (cLC) LC-MS switching platform using large volume injection and on-line automatic filtration and filter back-flush (AFFL) solid phase extraction (SPE) for analysis of environmental water samples with minimal sample preparation is presented. Although narrow columns and on-line sample preparation are used in the platform, high ruggedness is achieved e.g., injection of 100 non-filtrated water samples did not result in a pressure rise/clogging of the SPE/capillary columns (inner diameter 300 µm). In addition, satisfactory retention time stability and chromatographic resolution were also features of the system. The potential of the platform for environmental water samples was demonstrated with various pharmaceutical products, which had detection limits (LOD) in the 0.05-12.5 ng/L range. Between-day and within-day repeatability of selected analytes were <20% RSD.

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