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1.
J Lipid Res ; 65(9): 100609, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39084491

RESUMEN

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids typically reported as being rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial-mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate if GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, and GD2), epithelial-mesenchymal transition markers (EpCAM, TROP2, and CD9), and lineage markers (CD45, CD31, and CD90) at the single-cell level. Next, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.


Asunto(s)
Neoplasias de la Mama , Transición Epitelial-Mesenquimal , Glicoesfingolípidos , Análisis de la Célula Individual , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Glicoesfingolípidos/metabolismo , Glicoesfingolípidos/análisis , Femenino , Análisis de la Célula Individual/métodos , Fenotipo
2.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-34502101

RESUMEN

Sphingolipids (SLs), glycosphingolipids (GSLs), and eicosanoids are bioactive lipids, which play important roles in the etiology of various diseases, including cancer. However, their content and roles in cancer cells, and in particular in the exosomes derived from tumor cells, remain insufficiently characterized. In this study, we evaluated alterations of SL and GSL levels in transformed cells and their exosomes, using comparative HPLC-MS/MS analysis of parental human bronchial epithelial cells HBEC-12KT and their derivative, benzo[a]pyrene-transformed HBEC-12KT-B1 cells with the acquired mesenchymal phenotype. We examined in parallel SL/GSL contents in the exosomes released from both cell lines. We found significant alterations of the SL/GSL profile in the transformed cell line, which corresponded well with alterations of the SL/GSL profile in exosomes derived from these cells. This suggested that a majority of SLs and GSLs were transported by exosomes in the same relative pattern as in the cells of origin. The only exceptions included decreased contents of sphingosin, sphingosin-1-phosphate, and lactosylceramide in exosomes derived from the transformed cells, as compared with the exosomes derived from the parental cell line. Importantly, we found increased levels of ceramide phosphate, globoside Gb3, and ganglioside GD3 in the exosomes derived from the transformed cells. These positive modulators of epithelial-mesenchymal transition and other pro-carcinogenic processes might thus also contribute to cancer progression in recipient cells. In addition, the transformed HBEC-12KT-B1 cells also produced increased amounts of eicosanoids, in particular prostaglandin E2. Taken together, the exosomes derived from the transformed cells with specifically upregulated SL and GSL species, and increased levels of eicosanoids, might contribute to changes within the cancer microenvironment and in recipient cells, which could in turn participate in cancer development. Future studies should address specific roles of individual SL and GSL species identified in the present study.


Asunto(s)
Transformación Celular Neoplásica , Exosomas/metabolismo , Mucosa Respiratoria/metabolismo , Esfingolípidos/metabolismo , Benzo(a)pireno/toxicidad , Bronquios/citología , Carcinógenos/toxicidad , Línea Celular , Humanos , Mucosa Respiratoria/efectos de los fármacos
3.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-34206240

RESUMEN

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.


Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Ácidos Grasos/metabolismo , Regulación Neoplásica de la Expresión Génica , Metabolismo de los Lípidos , Fosfolípidos/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Anciano , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Femenino , Humanos , Lipidómica , Lipogénesis , Masculino , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo
4.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-31801289

RESUMEN

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.


Asunto(s)
Neoplasias del Colon/enzimología , Regulación Neoplásica de la Expresión Génica , Lactosilceramidos/metabolismo , Metabolismo de los Lípidos/genética , Esfingolípidos/metabolismo , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Ceramidasa Alcalina/genética , Ceramidasa Alcalina/metabolismo , Animales , Ceramidas/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Humanos , Lisofosfolípidos/metabolismo , Ceramidasa Neutra/genética , Ceramidasa Neutra/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina N-Aciltransferasa/genética , Esfingosina N-Aciltransferasa/metabolismo , Células Tumorales Cultivadas
5.
J Cell Biochem ; 119(6): 4664-4679, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29274292

RESUMEN

Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.


Asunto(s)
Apoptosis/efectos de los fármacos , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Ácidos Docosahexaenoicos/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos de la Membrana/metabolismo , Neoplasias del Colon/patología , Células HCT116 , Humanos , Lípidos de la Membrana/clasificación
6.
Eur J Nutr ; 56(4): 1493-1508, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26983609

RESUMEN

PURPOSE: Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. METHODS: In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. RESULTS: In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 cells, which was not further affected by DHA. DHA was incorporated in all main PL types, increasing their unsaturation, while NaBt did not additionally modulate these effects in either cell model. Nevertheless, we identified an unusually wide range of CL species to be highly increased by NaBt and particularly by DHA/NaBt, and these effects were more pronounced in HCT-116 cells. DHA and DHA/NaBt enhanced levels of high molecular weight and more unsaturated CL species, containing DHA, which was specific for either differentiation or apoptotic responses. CONCLUSIONS: We identified a wide range of CL species in the colon cells which composition was significantly modified after DHA and NaBt treatment. These specific CL modulations might contribute to distinct cellular differentiation or apoptotic responses.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Fosfolípidos/química , Apoptosis/efectos de los fármacos , Ácido Butírico/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colon/citología , Células HCT116 , Humanos , Espectrometría de Masas en Tándem
7.
J Biol Chem ; 289(2): 1128-41, 2014 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-24265322

RESUMEN

ß-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/ß-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the ß-arrestin function in Wnt/ß-catenin signaling. We demonstrate that ß-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. ß-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that ß-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that ß-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIß. Importantly, cells lacking ß-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that ß-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that ß-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by ß-arrestin- and Dishevelled-associated kinases.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Arrestinas/metabolismo , Membrana Celular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Wnt3A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Arrestinas/genética , Western Blotting , Células Cultivadas , Proteínas Dishevelled , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Ratones Noqueados , Microscopía Confocal , Antígenos de Histocompatibilidad Menor , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Interferencia de ARN , Proteínas Supresoras de Tumor/genética , Proteína Wnt3A/genética , beta-Arrestinas
8.
Biochim Biophys Acta ; 1841(9): 1308-17, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24953781

RESUMEN

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.


Asunto(s)
Ácidos Docosahexaenoicos/farmacología , Regulación Neoplásica de la Expresión Génica , Mitocondrias/efectos de los fármacos , Esfingolípidos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Citocromos c/metabolismo , Sinergismo Farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Transducción de Señal , Esfingolípidos/química , Esfingolípidos/clasificación , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo
9.
Microbes Infect ; 26(4): 105303, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38272253

RESUMEN

The life cycle of enveloped viruses is closely linked to host-cell lipids. However, changes in lipid metabolism during infections with the tick-borne encephalitis virus (TBEV) have not been described. TBEV is a medically important orthoflavivirus, which is endemic to many parts of Europe and Asia. In the present study, we performed targeted lipidomics with HPLC-MS/MS to evaluate changes in phospholipid and sphingolipid concentrations in TBEV-infected human neuronal SK-N-SH cells. TBEV infections significantly increased phosphatidylcholine, phosphatidylinositol, and phosphatidylserine levels within 48 h post-infection (hpi). Sphingolipids were slightly increased in dihydroceramides within 24 hpi. Later, at 48 hpi, the contents of sphinganine, dihydroceramides, ceramides, glucosylceramides, and ganglioside GD3 were elevated. On the other hand, sphingosine-1-phosphate content was slightly reduced in TBEV-infected cells. Changes in sphingolipid concentrations were accompanied by suppressed expression of a majority of the genes linked to sphingolipid and glycosphingolipid metabolism. Furthermore, we found that a pharmacological inhibitor of sphingolipid synthesis, fenretinide (4-HPR), inhibited TBEV infections in SK-N-SH cells. Taken together, our results suggested that both structural and signaling functions of lipids could be affected during TBEV infections. These changes might be connected to virus propagation and/or host-cell defense.


Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas , Neuronas , Fosfolípidos , Esfingolípidos , Humanos , Esfingolípidos/metabolismo , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Neuronas/virología , Neuronas/metabolismo , Fosfolípidos/metabolismo , Metabolismo de los Lípidos , Línea Celular , Lipidómica , Espectrometría de Masas en Tándem , Interacciones Huésped-Patógeno
10.
Apoptosis ; 18(3): 286-99, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23299931

RESUMEN

α-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Neoplasias Pulmonares/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , alfa-Tocoferol/uso terapéutico , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Ratones
11.
Sci Total Environ ; 815: 151967, 2022 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-34843781

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) may interact with multiple intracellular receptors and related signaling pathways. We comprehensively evaluated the toxicity profiles of six environmentally relevant PAHs differing in structure, genotoxicity and their ability to activate the aryl hydrocarbon receptor (AhR). We focused particularly on their impact on intracellular hormone-, xenobiotic- and lipid-sensing receptors, as well as on cellular stress markers, combining a battery of human reporter gene assays and qRT-PCR evaluation of endogenous gene expression in human hepatocyte-like HepaRG cells, with LC/MS-MS analysis of cellular sphingolipids. The effects of PAHs included: activation of estrogen receptor α (in case of fluoranthene (Fla), pyrene (Pyr), benz[a]anthracene (BaA), benzo[a]pyrene (BaP)), suppression of androgen receptor activity (Fla, BaA, BaP and benzo[k]fluoranthene (BkF)), enhancement of dexamethasone-induced glucocorticoid receptor activity (chrysene (Chry), BaA, and BaP), and potentiation of triiodothyronine-induced thyroid receptor α activity (all tested PAHs). PAHs also induced transcription of endogenous gene targets of constitutive androstane receptor (Fla, Pyr), or repression of target genes of pregnane X receptor and peroxisome proliferator-activated receptor α (in case of the AhR-activating PAHs - Chry, BaA, BaP, and BkF) in HepaRG cells. In the same cell model, the AhR agonists reduced the expression of glucose metabolism genes (PCK1, G6PC and PDK4), and they up-regulated levels of glucosylceramides, together with a concomitant induction of expression of UGCG, glucosylceramide synthesis enzyme. Finally, both BaP and BkF were found to induce expression of early stress and genotoxicity markers: ATF3, EGR1, GDF15, CDKN1A/p21, and GADD45A mRNAs, while BaP alone increased levels of IL-6 mRNA. Overall, whereas low-molecular-weight PAHs exerted significant effects on nuclear receptors (with CYP2B6 induction observed already at nanomolar concentrations), the AhR activation by 4-ring and 5-ring PAHs appeared to be a key mechanism underlying their impact on nuclear receptor signaling, endogenous metabolism and induction of early stress and genotoxicity markers.


Asunto(s)
Hidrocarburos Policíclicos Aromáticos , Benzo(a)pireno , Humanos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Xenobióticos
12.
Toxicology ; 463: 152986, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34627992

RESUMEN

Sphingolipids (SLs) are important signaling molecules and functional components of cellular membranes. Although SLs are known as crucial regulators of neural cell physiology and differentiation, modulations of SLs by environmental neurotoxicants in neural cells and their neuronal progeny have not yet been explored. In this study, we used in vitro models of differentiated neuron-like cells, which were repeatedly exposed during differentiation to model environmental toxicants, and we analyzed changes in sphingolipidome, cellular morphology and gene expression related to SL metabolism or neuronal differentiation. We compared these data with the results obtained in undifferentiated neural cells with progenitor-like features. As model polychlorinated organic pollutants, we used 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3'-dichlorobiphenyl (PCB11) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153). PCB153 revealed itself as the most prominent deregulator of SL metabolism and as potent toxicant during early phases of in vitro neurogenesis. TCDD exerted only minor changes in the levels of analysed lipid species, however, it significantly changed the rate of pro-neuronal differentiation and deregulated expression of neuronal markers during neurogenesis. PCB11 acted as a potent disruptor of in vitro neurogenesis, which induced significant alterations in SL metabolism and cellular morphology in both differentiated neuron-like models (differentiated NE4C and NG108-15 cells). We identified ceramide-1-phosphate, lactosylceramides and several glycosphingolipids to be the most sensitive SL species to exposure to polychlorinated pollutants. Additionally, we identified deregulation of several genes related to SL metabolism, which may be explored in future as potential markers of developmental neurotoxicity.


Asunto(s)
Neuronas/efectos de los fármacos , Bifenilos Policlorados/farmacología , Bifenilos Policlorados/toxicidad , Dibenzodioxinas Policloradas/toxicidad , Esfingolípidos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Contaminantes Ambientales/toxicidad , Neurogénesis/efectos de los fármacos , Neuronas/metabolismo , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/genética
13.
PLoS One ; 15(1): e0228010, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31999740

RESUMEN

Identification of changes of phospholipid (PL) composition occurring during colorectal cancer (CRC) development may help us to better understand their roles in CRC cells. Here, we used LC-MS/MS-based PL profiling of cell lines derived from normal colon mucosa, or isolated at distinct stages of CRC development, in order to study alterations of PL species potentially linked with cell transformation. We found that a detailed evaluation of phosphatidylinositol (PI) and phosphatidylserine (PS) classes allowed us to cluster the studied epithelial cell lines according to their origin: i) cells originally derived from normal colon tissue (NCM460, FHC); ii) cell lines derived from colon adenoma or less advanced differentiating adenocarcinoma cells (AA/C1, HT-29); or, iii) cells obtained by in vitro transformation of adenoma cells and advanced colon adenocarcinoma cells (HCT-116, AA/C1/SB10, SW480, SW620). Although we tentatively identified several PS and PI species contributing to cell line clustering, full PI and PS profiles appeared to be a key to the successful cell line discrimination. In parallel, we compared PL composition of primary epithelial (EpCAM-positive) cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients, with PL profiles of cell lines derived from normal colon mucosa (NCM460) and from colon adenocarcinoma (HCT-116, SW480) cells, respectively. In general, higher total levels of all PL classes were observed in tumor cells. The overall PL profiles of the cell lines, when compared with the respective patient-derived cells, exhibited similarities. Nevertheless, there were also some notable differences in levels of individual PL species. This indicated that epithelial cell lines, derived either from normal colon tissue or from CRC cells, could be employed as models for functional lipidomic analyses of colon cells, albeit with some caution. The biological significance of the observed PL deregulation, or their potential links with specific CRC stages, deserve further investigation.


Asunto(s)
Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células Epiteliales/metabolismo , Lipidómica , Fosfolípidos/metabolismo , Línea Celular Tumoral , Células Epiteliales/patología , Humanos , Análisis de Componente Principal
14.
Sci Rep ; 10(1): 4780, 2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-32179785

RESUMEN

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRI). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRI diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. In this study, we evaluated nanosafety of Gd-lip containing PE-DTPA chelating Gd+3 prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DNA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. In conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.


Asunto(s)
Medios de Contraste , Portadores de Fármacos , Gadolinio DTPA , Hepatocitos/efectos de los fármacos , Liposomas , Macrófagos/efectos de los fármacos , Imagen por Resonancia Magnética , Fosfatidiletanolaminas , Células Cultivadas , Fibrinolíticos , Gadolinio DTPA/efectos adversos , Gadolinio DTPA/toxicidad , Humanos , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Nanopartículas , Fosfatidiletanolaminas/efectos adversos , Fosfatidiletanolaminas/toxicidad
15.
Radiat Res ; 172(2): 198-212, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19630524

RESUMEN

Abstract Radiation metabolomics employing mass spectral technologies represents a plausible means of high-throughput minimally invasive radiation biodosimetry. A simplified metabolomics protocol is described that employs ubiquitous gas chromatography-mass spectrometry and open source software including random forests machine learning algorithm to uncover latent biomarkers of 3 Gy gamma radiation in rats. Urine was collected from six male Wistar rats and six sham-irradiated controls for 7 days, 4 prior to irradiation and 3 after irradiation. Water and food consumption, urine volume, body weight, and sodium, potassium, calcium, chloride, phosphate and urea excretion showed major effects from exposure to gamma radiation. The metabolomics protocol uncovered several urinary metabolites that were significantly up-regulated (glyoxylate, threonate, thymine, uracil, p-cresol) and down-regulated (citrate, 2-oxoglutarate, adipate, pimelate, suberate, azelaate) as a result of radiation exposure. Thymine and uracil were shown to derive largely from thymidine and 2'-deoxyuridine, which are known radiation biomarkers in the mouse. The radiation metabolomic phenotype in rats appeared to derive from oxidative stress and effects on kidney function. Gas chromatography-mass spectrometry is a promising platform on which to develop the field of radiation metabolomics further and to assist in the design of instrumentation for use in detecting biological consequences of environmental radiation release.


Asunto(s)
Algoritmos , Inteligencia Artificial , Biomarcadores/orina , Cromatografía de Gases y Espectrometría de Masas/métodos , Metaboloma/efectos de la radiación , Urinálisis/métodos , Irradiación Corporal Total , Animales , Rayos gamma , Masculino , Metaboloma/fisiología , Ratas , Ratas Wistar , Programas Informáticos
16.
Radiat Res ; 172(1): 42-57, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19580506

RESUMEN

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.


Asunto(s)
Rayos gamma/efectos adversos , Purinas/orina , Pirimidinas/orina , Análisis de Varianza , Animales , Biomarcadores/orina , Desaminación , Desoxirribonucleósidos/orina , Desoxiuridina/orina , Relación Dosis-Respuesta en la Radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Purinas/metabolismo , Pirimidinas/metabolismo , Ribonucleósidos/orina , Espectrometría de Masas en Tándem , Timidina/orina , Factores de Tiempo , Xantina/orina , Xantinas
17.
Radiat Res ; 170(1): 1-14, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18582157

RESUMEN

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.


Asunto(s)
Rayos gamma , Animales , Biomarcadores/orina , Relación Dosis-Respuesta en la Radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Fenotipo , Ésteres del Ácido Sulfúrico/química , Ésteres del Ácido Sulfúrico/orina , Espectrometría de Masas en Tándem
19.
Nat Commun ; 5: 3477, 2014 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24637612

RESUMEN

Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Exosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Fosfolipasa D/metabolismo , Sinteninas/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Receptores ErbB/metabolismo , Exosomas/enzimología , Exosomas/genética , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Cuerpos Multivesiculares/enzimología , Cuerpos Multivesiculares/genética , Fosfolipasa D/genética , Transporte de Proteínas , Sinteninas/genética
20.
FEBS J ; 280(14): 3436-50, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23678861

RESUMEN

Tumour necrosis factor (TNF) related apoptosis inducing ligand (TRAIL), a membrane-bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro-apoptotic death receptors. However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. Upon blocking endosomal acidification by the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A, we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL-induced apoptosis. In cells pretreated with these inhibitors, the TRAIL-induced processing of caspase-8 and the aggregation and trafficking of the TRAIL receptor complexes were temporarily attenuated. Nuclear factor κB or mitogen activated protein/stress kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization nor acid sphingomyelinase was involved in this process. The cell surface expression of TRAIL receptors and their TRAIL-induced internalization were not affected by V-ATPase inhibitors. The inhibitory effect of BafA1, however, was blunted by knockdown of the caspase-8 inhibitor cFLIP. Altogether, the data obtained provide the first evidence that endosomal acidification could represent an important regulatory node in the proximal part of TRAIL-induced pro-apoptotic signalling.


Asunto(s)
Antineoplásicos/farmacología , Caspasa 8/metabolismo , Endosomas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Tumoral , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Regulación hacia Abajo , Activación Enzimática , Humanos , Concentración de Iones de Hidrógeno , Macrólidos/farmacología , Transporte de Proteínas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingolípidos/fisiología , Esfingomielina Fosfodiesterasa/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo
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