RESUMO
Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.
Assuntos
Bactérias/classificação , Colite Ulcerativa/genética , Disbiose/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Proteína ADAM17/genética , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Colite Ulcerativa/microbiologia , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Microbiota , Filogenia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Índice de Gravidade de Doença , Inibidor Tecidual de Metaloproteinase-3/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Polymethoxylated flavones (PMFs) from citrus fruits are reported to present anticancer potential. However, there is a lack of information regarding their effect on cancer stem cell (CSC) populations, which has been recognized as responsible for tumor initiation, relapse, and chemoresistance. In this study, we evaluated the effect of an orange peel extract (OPE) and its main PMFs, namely, nobiletin, sinensetin, tangeretin, and scutellarein tetramethylether in targeting cell proliferation and stemness using a 3D cell model of colorectal cancer composed of HT29 cell spheroids cultured for 7 days in stirred conditions. Soft agar assay, ALDH1 activity, and relative quantitative gene expression analysis of specific biomarkers were carried out to characterize the stemness, self-renewal, and mesenchymal features of HT29 cell spheroids. Then, the impact of OPE and PMFs in reducing cell proliferation and modulating cancer stemness and self-renewal was assessed. Results showed that, when compared with monolayer cultures, HT29 cell spheroids presented higher ALDH1 activity (81.97% ± 5.27% compared to 63.55% ± 17.49% for 2D), upregulation of CD44, PROM1, SOX9, and SNAI1 genes (1.83 ± 0.34, 2.54 ± 0.51, 2.03 ± 0.15, and 6.12 ± 1.59 times) and high self-renewal capability (352 ± 55 colonies compared to 253 ± 42 for 2D). Incubation with OPE (1 mg/mL) significantly inhibited cell proliferation and modulated cancer stemness and self-renewal ability: colony formation, ALDH1 activity, and the expression of cancer stemness biomarkers PROM1 and LGR5 were significantly reduced (0.66 ± 0.15 and 0.51 ± 0.14 times, respectively). Among all PMFs, tangeretin was the most efficient in targeting the CSC population by decreasing colony formation and the expression of PROM1 and LGR5. Scutellarein tetramethylether was shown to modulate markers of mesenchymal/metastatic transition (increasing CDH1 and reducing ZEB1 and SNAI1) and nobiletin was capable of downregulating PROM1 and SNAI1 expression. Importantly, all PMFs and OPE were shown to synergistically interact with 5-fluorouracil, improving the antiproliferative response of this drug.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Citrus/química , Neoplasias Colorretais/tratamento farmacológico , Flavonas/uso terapêutico , Fluoruracila/uso terapêutico , Células-Tronco Neoplásicas/efeitos dos fármacos , Fitoterapia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células , Células Cultivadas , Neoplasias Colorretais/metabolismo , Flavonas/farmacologia , Frutas , Células HT29 , Humanos , Proteínas de Neoplasias/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêuticoRESUMO
Rosemary diterpenes have demonstrated diverse biological activities, such as anti-cancer, antiinflammatory, as well as other beneficial effects against neurological and metabolic disorders. In particular, carnosic acid (CA), carnosol (CS) and rosmanol (RS) diterpenes have shown interesting results on anti-cancer activity. However, little is known about the toxic effects of rosemary diterpenes at the concentrations needed to exert their antiproliferative effect on cancer cells. In our study, CA, CS and RS exhibited a concentration-dependent effect on cell viability of two human colon cancer cell lines (HT-29 and HCT116) after 24â¯h exposure. HT-29â¯cell line was more resistant to the inhibitory effect of the three diterpenes than HCT116â¯cell line. Among the three diterpenes, RS exerted the strongest effect in both cell lines. To investigate the hepatotoxicity of CA, CS and RS, undifferentiated and differentiated HepaRG cells were exposed to increasing concentrations of the diterpenes (from 10 to 100⯵M). Differentiated cells were found to be more resistant to the toxic activity of the three diterpenes than undifferentiated HepaRG, probably related to a higher detoxifying function of differentiated HepaRG cells compared with the undifferentiated cells. The metabolic profiles of differentiated HepaRG cells in response to CA, CS and RS were examined to determine biochemical alterations and deepen the study of the effects of rosemary phenolic diterpenes at molecular level. A multiplatform metabolomics study based on liquid- and gas-chromatography hyphenated to high resolution mass spectrometry revealed that rosemary diterpenes exerted different effects when HepaRG cells were treated with the same concentration of each diterpene. RS revealed a greater metabolome alteration followed by CS and CA, in agreement with their observed cytotoxicity. Metabolomics provided valuable information about early events in the metabolic profiles after the treatment with the investigated diterpenes from rosemary.
Assuntos
Diterpenos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Metabolômica , Rosmarinus/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida , Diterpenos/química , Cromatografia Gasosa-Espectrometria de Massas , Células HCT116 , Células HT29 , HumanosRESUMO
Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.
Assuntos
Tolerância Imunológica/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Imunossupressores/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Hematopoese/efeitos dos fármacos , Hematopoese/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Imunoglobulina G/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transcrição GênicaRESUMO
The antiproliferative activity of Rosemary (Rosmarinus officinalis) has been widely studied in different in vitro and in vivo models, which demonstrate that rosemary extracts inhibit the cellular proliferation due to its ability to interact with a wide spectrum of molecular targets. However, a comprehensive proteomics study in vivo has not been carried out yet. In the present work, the effects of rosemary extract on xenograft tumor growth has been studied and, for the first time, a shotgun proteomic analysis based on nano-LC-MS/MS together with stable isotope dimethyl labeling (DML) has been applied to investigate the global protein changes in vivo. Our results show that the daily administration of a polyphenol-enriched rosemary extract reduces the progression of colorectal cancer in vivo with the subsequent deregulation of 74 proteins. The bioinformatic analysis of these proteins indicates that the rosemary extract mainly alters the RNA Post-Transcriptional Modification, the Protein Synthesis and the Amino Acid Metabolism functions and suggests the inactivation of the oncogene MYC. These results demonstrate the high utility of the proposed analytical methodology to determine, simultaneously, the expression levels of a large number of protein biomarkers and to generate new hypothesis about the molecular mechanisms of this extract in vivo.
Assuntos
Carcinogênese/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proteômica/métodos , Rosmarinus/química , Animais , Proliferação de Células/efeitos dos fármacos , Células HT29 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Extratos Vegetais/química , Polifenóis/farmacologia , Espectrometria de Massas em TandemRESUMO
Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.
Assuntos
Abietanos/farmacologia , Cromatografia Líquida/métodos , Neoplasias do Colo/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Homeostase/efeitos dos fármacos , Humanos , Marcação por Isótopo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116). The processes, carried out under optimal conditions, were: (1) pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent) at lab-scale; (2) Single-step supercritical fluid extraction (SFE) at pilot scale; (3) Intensified two-step sequential SFE at pilot scale; (4) Integrated PLE plus supercritical antisolvent fractionation (SAF) at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight), this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA) and carnosol (CS) at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide), suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.
Assuntos
Abietanos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Rosmarinus/química , Compostos de Bifenilo/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Neoplasias do Colo/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Células HCT116 , Células HT29 , Humanos , Espectrometria de Massas , Picratos/metabolismoRESUMO
Quality and safety assessment as well as the evaluation of other nutritional and functional properties of foods imply the use of robust, efficient, sensitive, and cost-effective analytical methodologies. Among analytical technologies used in the fields of food analysis and foodomics, capillary electrophoresis (CE) has generated great interest for the analyses of a large number of compounds due to its high separation efficiency, extremely small sample and reagent requirements, and rapid analysis. The introductory section of this chapter provides an overview of the recent applications of capillary electrophoresis (CE) in food analysis and foodomics. Relevant reviews and research articles on these topics are tabulated including papers published in the period 2011-2014. In addition, to illustrate the great capabilities of CE in foodomics the chapter describes the main experimental points to be taken into consideration for a metabolomic study of the antiproliferative effect of carnosic acid (a natural diterpene found in rosemary) against HT-29 human colon cancer cells.
Assuntos
Eletroforese Capilar/métodos , Análise de Alimentos/métodos , Metabolômica/métodos , Células HT29 , Humanos , Espectrometria de Massas/métodos , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificaçãoRESUMO
In this work, a proteomics strategy based on nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) using an Orbitrap high-resolution mass spectrometer together with stable isotope dimethyl labeling (DML) is applied to quantitatively examine relative changes in the protein fraction of HT-29 human colon cancer cells treated with different concentrations of a polyphenol-enriched rosemary extract over the time. The major objective of this study was to gain insights into the antiproliferative mechanisms induced by rosemary polyphenols. Using this methodology, 1909 and 698 proteins were identified and quantified in cell extracts. The polyphenol-enriched rosemary extract treatment changed the expression of several proteins in a time- and concentration-dependent manner. Most of the altered proteins are implicated in the activation of Nrf2 transcription factor and the unfolded protein response. In conclusion, rosemary polyphenols induced proteomic changes that were related to the attenuation of aggresome formation and activation of autophagy to alleviate cellular stress.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/patologia , Extratos Vegetais/farmacologia , Proteoma/efeitos dos fármacos , Proteômica/métodos , Rosmarinus/química , Autofagia/efeitos dos fármacos , Cromatografia Líquida , Neoplasias do Colo/tratamento farmacológico , Células HT29 , Humanos , Marcação por Isótopo , Fator 2 Relacionado a NF-E2/metabolismo , Polifenóis/farmacologia , Espectrometria de Massas em Tandem , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
A number of studies have demonstrated a strong association between the antioxidant properties of rosemary polyphenols and their chemoprotective activity. However, the prooxidant effects of rosemary polyphenols have been rarely reported. In this work, a foodomics study is performed to investigate the in vitro autooxidation of carnosic acid (CA), carnosol (CS) and a polyphenol-enriched rosemary extract (SC-RE) in cell culture conditions. The results revealed that rosemary polyphenols autooxidation in culture medium generated H2 O2 at different rates. Generated H2 O2 levels by SC-RE and CA, but not CS, were correlated with intracellular reactive oxygen species (ROS) generation in HT-29 cells, and were partially involved in their anti-proliferative effect in this cell line. These compounds also induced different effects on glutathione metabolism. Results also indicated that high extracellular H2 O2 concentrations, resulting of using high (45 µg/mL) SC-RE concentration in culture media, exerted some artifactual effects related with cell cycle, but they did not influence the expression of relevant molecular biomarkers of stress.
Assuntos
Proliferação de Células/efeitos dos fármacos , Análise de Alimentos , Peróxido de Hidrogênio/metabolismo , Polifenóis/farmacologia , Rosmarinus/química , Ciclo Celular/efeitos dos fármacos , Meios de Cultura , Células HT29 , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
This review work presents and discusses the main applications of capillary electromigration methods in food analysis and Foodomics. Papers that were published during the period February 2013-February 2015 are included following the previous review by Garcia-Cañas et al. (Electrophoresis, 2014, 35, 147-169). Analysis by CE of a large variety of food-related molecules with different chemical properties, including amino acids, hazardous amines, peptides, proteins, phenols, polyphenols, lipids, carbohydrates, DNAs, vitamins, toxins, contaminants, pesticides, residues, food additives, as well as small organic and inorganic compounds. This work includes recent results on food quality and safety, nutritional value, storage, bioactivity, as well as applications of CE for monitoring food processing. The use, among other CE developments, of microchips, CE-MS, and chiral CE in food analysis and Foodomics is also discussed.
Assuntos
Eletroforese Capilar , Análise de Alimentos/métodos , Eletroforese Capilar/métodos , Eletroforese Capilar/tendênciasRESUMO
In the present work, the phlorotannin composition of different Sargassum muticum samples collected at different locations along the North Atlantic coasts as well as the bioactivities related to these components were investigated. After pressurized liquid extraction, the samples collected at the extreme locations of a latitudinal gradient from Portugal and Norway, were found to be the richest on total phenols and, particularly, on phlorotannins, containing up to 148.97 and 5.12mg phloroglucinol equivalents g(-1), respectively. The extracts obtained from these locations were further purified and chemically characterized using a modified HILIC×RP-DAD-MS/MS method. The application of this methodology allowed the tentative identification of a great variability of phlorotannins with different degrees of polymerization (from 3 to 11) and structures, determined for the first time in S. muticum. The most-abundant phlorotannins on these samples were fuhalols, hydroxyfuhalols and phlorethols, showing also particularities and important differences depending on the geographical location. Afterwards, the antiproliferative activity of these extracts against HT-29 adenocarcinoma colon cancer cells was studied. Results revealed that the richest S. muticum samples in terms of total phlorotannins, i.e., those from Norway, presented the highest activity, showing a good cytotoxic potential at concentrations in the medium micromolar range.
Assuntos
Cromatografia Líquida , Sargassum/química , Espectrometria de Massas em Tandem , Taninos/química , Taninos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Oceano Atlântico , Proliferação de Células/efeitos dos fármacos , Células HT29 , Humanos , Fenóis/química , Fenóis/farmacologia , Floroglucinol/química , Floroglucinol/farmacologiaRESUMO
In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.
Assuntos
Adenocarcinoma/metabolismo , Adesão Celular , Neoplasias do Colo/metabolismo , Eletroforese Capilar , Metabolômica/métodos , Poliaminas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Eflornitina/farmacologia , Estudos de Viabilidade , Células HT29 , Humanos , Inibidores da Ornitina Descarboxilase/farmacologiaRESUMO
In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.
Assuntos
Abietanos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Rosmarinus/química , Abietanos/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Transporte Biológico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Glutationa/metabolismo , Células HT29 , Humanos , Inativação Metabólica/efeitos dos fármacos , Metabolômica , Extratos Vegetais/isolamento & purificação , Polifenóis/isolamento & purificação , Putrescina/análogos & derivados , Putrescina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Nowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double-stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100-500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE-LIF protocols for DNA analysis, one based on the dye YOPRO-1 (typically used for CGE-LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO-1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100-500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO-1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO-1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100-200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR.
Assuntos
Benzoxazóis/análise , Eletroforese Capilar/métodos , Corantes Fluorescentes/análise , Quinolinas/análise , Linhagem Celular Tumoral , DNA/análise , Eletroforese Capilar/economia , Humanos , Lasers , Limite de Detecção , Reação em Cadeia da Polimerase , Compostos de Quinolínio , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
In this work, the effect of rosemary extracts rich on polyphenols obtained using pressurized fluids was investigated on the gene expression of human SW480 and HT29 colon cancer cells. The application of transcriptomic profiling and functional enrichment analysis was done via two computational approaches, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. These two approaches were used for functional enrichment analysis as a previous step for a reliable interpretation of the data obtained from microarray analysis. Reverse transcription quantitative-PCR was used to confirm relative changes in mRNA levels of selected genes from microarrays. The selection of genes was based on their expression change, adjusted p value, and known biological function. According to genome-wide transcriptomics analysis, rosemary polyphenols altered the expression of ~4 % of the genes covered by the Affymetrix Human Gene 1.0ST chip in both colon cancer cells. However, only ~18 % of the differentially expressed genes were common to both cell lines, indicating markedly different expression profiles in response to the treatment. Differences in induction of G2/M arrest observed by rosemary polyphenols in the two colon adenocarcinoma cell lines suggest that the extract may be differentially effective against tumors with specific mutational pattern. From our results, it is also concluded that rosemary polyphenols induced a low degree of apoptosis indicating that other multiple signaling pathways may contribute to colon cancer cell death.
RESUMO
In this work, a global Foodomics strategy has been applied to study the antiproliferative effect of dietary polyphenols from rosemary on two human leukemia lines, one showing a drug-sensitive phenotype (K562), and another exhibiting a drug-resistant phenotype (K562/R). To this aim, whole-transcriptome microarray together with an MS-based nontargeted analytical approach (via CE-TOF MS and UPLC-TOF MS) have been employed to carry out transcriptomics and metabolomics analyses, respectively. Functional enrichment analysis was done using ingenuity pathway analysis (IPA) software as a previous step for a reliable interpretation of transcriptomic and metabolomic profiles. Rosemary polyphenols altered the expression of approximately 1% of the genes covered by the whole transcriptome microarray in both leukemia cell lines. Overall, differences in the transcriptional induction of a number of genes encoding phase II detoxifying and antioxidant genes, as well as differences in the metabolic profiles observed in the two leukemia cell lines suggest that rosemary polyphenols may exert a differential chemopreventive effect in leukemia cells with different phenotypes. IPA predictions on transcription factor analysis highlighted inhibition of Myc transcription factor function by rosemary polyphenols, which may explain the observed antiproliferative effect of rosemary extract in the leukemia cells. Metabolomics analysis suggested that rosemary polyphenols affected differently the intracellular levels of some metabolites in two leukemia cell sublines. Integration of data obtained from transcriptomics and metabolomics platforms was attempted by overlaying datasets on canonical (defined) metabolic pathways using IPA software. This strategy enabled the identification of several differentially expressed genes in the metabolic pathways modulated by rosemary polyphenols providing more evidences on the effect of these compounds.
Assuntos
Metaboloma/efeitos dos fármacos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Rosmarinus/química , Transcriptoma/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eletroforese Capilar , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Metabolômica , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In this study, an analytical multiplatform is presented to carry out a broad metabolomic study on the anti-proliferative effect of dietary polyphenols on human colon cancer cells. CE, RP/UPLC, and HILIC/UPLC all coupled to TOF MS were combined to achieve a global metabolomic examination of the effect of dietary polyphenols on HT29 colon cancer cells. By the use of a nontargeted metabolomic approach, metabolites showing significant different expression after the polyphenols treatment were identified in colon cancer cells. It was demonstrated that this multianalytical platform provided extensive metabolic information and coverage due to its complementary nature. Differences observed in metabolic profiles from CE-TOF MS, RP/UPLC-TOF MS, and HILIC/UPLC-TOF MS can be mainly assigned to their different separation mechanisms without discarding the influence of the different tools used for data processing. Changes in glutathione metabolism with an enhanced reduced glutathione/oxidized glutathione (GSH/GSSG) ratio were detected in polyphenols-treated cells. Moreover, significant alterations in polyamines content with important implications in cancer proliferation were observed after the treatment with polyphenols. These results from metabolomics can explain the chemopreventive effect of the tested dietary polyphenols on colon cancer and may be of importance for future prevention and/or treatment of this disease.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Polifenóis/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Eletroforese Capilar/métodos , Células HT29 , Humanos , Extratos Vegetais/farmacologia , Rosmarinus/química , Espectrometria de Massas em Tandem/métodosRESUMO
A global methodology, called Foodomics, which allows carrying out a comprehensive evaluation of the health benefits of food ingredients is presented in this work. The new methodology is based on the combination of several analytical platforms and data processing for Transcriptomics, Proteomics and Metabolomics studies, allowing the determination of changes induced by food ingredients at molecular level. Both, the whole methodological development and its potential are presented through the investigation of a case study following a hypothesis-free strategy. Namely, the chemopreventive effect of polyphenols from rosemary was examined on the total gene, protein and metabolite expression in human HT29 colon cancer cells. Conclusions on the bioactivity of polyphenols against colon cancer cells based on the results from each single platform (Transcriptomics, Proteomics or Metabolomics) are compared with the conclusions based on the integration of the whole results from the three platforms, corroborating the interest of using a global integrative strategy as Foodomics. To our knowledge, although many papers and reviews have been published on this topic, this is the first time that Transcriptomics, Proteomics and Metabolomics platforms are put together to study the health benefits from dietary ingredients against colon cancer cells at gene, protein and metabolite level. Advantages, drawbacks and current challenges of this global analytical strategy are discussed in this work. The results from our study provide new insights on the biological mechanisms involved in the cancer risk reduction properties of dietary constituents.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias do Colo/prevenção & controle , Suplementos Nutricionais , Metabolômica/métodos , Polifenóis/uso terapêutico , Proteômica/métodos , Rosmarinus/química , Transcriptoma/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Metaboloma/efeitos dos fármacos , Fitoterapia , Polifenóis/farmacologia , Proteoma/efeitos dos fármacosRESUMO
The analysis of food components using capillary electromigration methods is reviewed in this work. Papers that were published from February 2009 to February 2011 are included following the previous review by Herrero et al. (Electrophoresis, 2010, 31, 205-228). The analysis of amino acids, biogenic amines, peptides, proteins, DNAs, carbohydrates, phenols, polyphenols, pigments, toxins, pesticides, vitamins, additives, small organic and inorganic ions and other compounds found in foods and beverages are reviewed, as well as those applications of CE for monitoring food interactions and food processing. The use of microchips, CE-MS and chiral-CE in food analysis is also discussed as well as other current and foreseen trends in this area of research including new developments in Foodomics.