RESUMO
Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as ß-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression.NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Transformação Celular Neoplásica/metabolismo , Colo/metabolismo , Neoplasias do Colo/metabolismo , Dieta Hiperlipídica , Células Epiteliais/metabolismo , Deleção de Genes , Mucosa Intestinal/metabolismo , Lesões Pré-Cancerosas/metabolismo , Receptores de Hidrocarboneto Arílico/deficiência , Animais , Azoximetano , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Dano ao DNA , Modelos Animais de Doenças , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Diet, loss of aryl hydrocarbon receptor (AhR) expression and their modification of the gut microbiota community composition and its metabolites affect the development of colorectal cancer (CRC). However, the concordance between fecal microbiota composition and the fecal metabolome is poorly understood. Mice with specific AhR deletion (AhRKO) in intestinal epithelial cell and their wild-type littermates were fed a low-fat diet or a high-fat diet. Shifts in the fecal microbiome and metabolome associated with diet and loss of AhR expression were assessed. Microbiome and metabolome data were integrated to identify specific microbial taxa that contributed to the observed metabolite shifts. RESULTS: Our analysis shows that diet has a more pronounced effect on mouse fecal microbiota composition than the impact of the loss of AhR. In contrast, metabolomic analysis showed that the loss of AhR in intestinal epithelial cells had a more pronounced effect on metabolite profile compared to diet. Integration analysis of microbiome and metabolome identified unclassified Clostridiales, unclassified Desulfovibrionaceae, and Akkermansia as key contributors to the synthesis and/or utilization of tryptophan metabolites. CONCLUSIONS: Akkermansia are likely to contribute to the synthesis and/or degradation of tryptophan metabolites. Our study highlights the use of multi-omic analysis to investigate the relationship between the microbiome and metabolome and identifies possible taxa that can be targeted to manipulate the microbiome for CRC treatment.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dieta , Fezes/microbiologia , Metaboloma , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/metabolismo , Akkermansia/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias do Colo/microbiologia , DNA Bacteriano , Feminino , Microbioma Gastrointestinal , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , RNA Ribossômico 16S , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.
Assuntos
Neoplasias Colorretais/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus gallolyticus subspecies gallolyticus/fisiologia , Animais , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Humanos , Camundongos , Transdução de Sinais , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Dietary fermentable fiber generates short-chain fatty acids (SCFA), for example, butyrate, in the colonic lumen which serves as a chemoprotective histone deacetylase inhibitor and/or as an acetylation substrate for histone acetylases. In addition, n-3 polyunsaturated fatty acids (n-3 PUFA) in fish oil can affect the chromatin landscape by acting as ligands for tumor suppressive nuclear receptors. In an effort to gain insight into the global dimension of post-translational modification of histones (including H3K4me3 and H3K9ac) and clarify the chemoprotective impact of dietary bioactive compounds on transcriptional control in a preclinical model of colon cancer, we generated high-resolution genome-wide RNA (RNA-Seq) and "chromatin-state" (H3K4me3-seq and H3K9ac-seq) maps for intestinal (epithelial colonocytes) crypts in rats treated with a colon carcinogen and fed diets containing bioactive (i) fish oil, (ii) fermentable fiber (a rich source of SCFA), (iii) a combination of fish oil plus pectin, or (iv) control, devoid of fish oil or pectin. In general, poor correlation was observed between differentially transcribed (DE) and enriched genes (DERs) at multiple epigenetic levels. The combinatorial diet (fish oil + pectin) uniquely affected transcriptional profiles in the intestinal epithelium, for example, upregulating lipid catabolism and beta-oxidation associated genes. These genes were linked to activated ligand-dependent nuclear receptors associated with n-3 PUFA and were also correlated with the mitochondrial L-carnitine shuttle and the inhibition of lipogenesis. These findings demonstrate that the chemoprotective fish oil + pectin combination diet uniquely induces global histone state modifications linked to the expression of chemoprotective genes.
Assuntos
Neoplasias do Colo/genética , Gorduras na Dieta/administração & dosagem , Fibras na Dieta/administração & dosagem , Modelos Animais de Doenças , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Animais , Azoximetano/toxicidade , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Óleos de Peixe/administração & dosagem , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
There is mounting evidence that noncoding microRNAs (miRNA) are modulated by select chemoprotective dietary agents. For example, recently we demonstrated that the unique combination of dietary fish oil (containing n-3 fatty acids) plus pectin (fermented to butyrate in the colon) (FPA) up-regulates a subset of putative tumor suppressor miRNAs in intestinal mucosa, and down-regulates their predicted target genes following carcinogen exposure as compared to control (corn oil plus cellulose (CCA)) diet. To further elucidate the biological effects of diet and carcinogen modulated miR's in the colon, we verified that miR-26b and miR-203 directly target PDE4B and TCF4, respectively. Since perturbations in adult stem cell dynamics are generally believed to represent an early step in colon tumorigenesis and to better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we additionally determined the effects of the chemoprotective FPA diet on miRNAs and mRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creER(T2) knock-in mice. Following global miRNA profiling, 26 miRNAs (P<0.05) were differentially expressed in Lgr5(high) stem cells as compared to Lgr5(negative) differentiated cells. FPA treatment up-regulated miR-19b, miR-26b and miR-203 expression as compared to CCA specifically in Lgr5(high) cells. In contrast, in Lgr5(negative) cells, only miR-19b and its indirect target PTK2B were modulated by the FPA diet. These data indicate for the first time that select dietary cues can impact stem cell regulatory networks, in part, by modulating the steady-state levels of miRNAs. To our knowledge, this is the first study to utilize Lgr5(+) reporter mice to determine the impact of diet and carcinogen on miRNA expression in colonic stem cells and their progeny.
Assuntos
Carcinógenos , Colo/patologia , Neoplasias do Colo/genética , Dieta , Ácidos Graxos Ômega-3/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Nicho de Células-Tronco , Animais , Carcinógenos/metabolismo , Carcinógenos/toxicidade , Colo/metabolismo , Neoplasias do Colo/etiologia , Neoplasias do Colo/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Quinase 2 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos , Fatores de Proteção , Nicho de Células-Tronco/efeitos dos fármacos , Fator de Transcrição 4/genéticaRESUMO
During colon cancer, epigenetic alterations contribute to the dysregulation of major cellular functions and signaling pathways. Modifications in chromatin signatures such as H3K4me3 and H3K9ac, which are associated with transcriptionally active genes, can lead to genomic instability and perturb the expression of gene sets associated with oncogenic processes. In order to further elucidate early pre-tumorigenic epigenetic molecular events driving CRC, we integrated diverse, genome-wide, epigenetic inputs (by high throughput sequencing of RNA, H3K4me3, and H3K9ac) and compared differentially expressed transcripts (DE) and enriched regions (DER) in an in-vivo rat colon cancer progression model. Carcinogen (AOM) effects were detected genome-wide at the RNA (116 DE genes), K9ac (49 DERs including 24 genes) and K4me3 (7678 DERs including 3792 genes) level. RNA-seq differential expression and pathway analysis indicated that interferon-associated innate immune responses were impacted by AOM exposure. Despite extensive associations between K4me3 DERs and colon tumorigenesis (1210 genes were linked to colorectal carcinoma) including FOXO3, GNAI2, H2AFX, MSH2, NR3C1, PDCD4 and VEGFA, these changes were not reflected at the RNA gene expression level during early cancer progression. Collectively, our results indicate that carcinogen-induced changes in gene K4me3 DERs are harbingers of future transcriptional events, which drive malignant transformation of the colon.
Assuntos
Neoplasias do Colo/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Transcrição Gênica , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Histonas/genética , Masculino , Proteínas de Neoplasias/genética , Ratos , Ratos Sprague-DawleyRESUMO
Perturbations in DNA damage, DNA repair, apoptosis and cell proliferation in the base of the crypt where stem cells reside are associated with colorectal cancer (CRC) initiation and progression. Although the transformation of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)(+) cells is an extremely efficient route towards initiating small intestinal adenomas, the role of Lgr5(+) cells in CRC pathogenesis has not been well investigated. Therefore, we further characterized the properties of colonic Lgr5(+) cells compared to differentiated cells in Lgr5-EGFP-IRES-creER(T2) knock-in mice at the initiation stage of carcinogen azoxymethane (AOM)-induced tumorigenesis using a quantitative immunofluorescence microscopy approach. At 12 and 24h post-AOM treatment, colonic Lgr5(+) stem cells (GFP(high)) were preferentially damaged by carcinogen, exhibiting a 4.7-fold induction of apoptosis compared to differentiated (GFP(neg)) cells. Furthermore, with respect to DNA repair, O(6)-methylguanine DNA methyltransferase (MGMT) expression was preferentially induced (by 18.5-fold) in GFP(high) cells at 24h post-AOM treatment compared to GFP(neg) differentiated cells. This corresponded with a 4.3-fold increase in cell proliferation in GFP(high) cells. These data suggest that Lgr5(+) stem cells uniquely respond to alkylation-induced DNA damage by upregulating DNA damage repair, apoptosis and cell proliferation compared to differentiated cells in order to maintain genomic integrity. These findings highlight the mechanisms by which colonic Lgr5(+) stem cells respond to cancer-causing environmental factors.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mucosa Intestinal/citologia , Células-Tronco/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/fisiopatologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Homeostase/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Camundongos , Mutagênicos/toxicidade , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. Using a Seahorse Extracellular Flux Analyzer, we have demonstrated that select environmental agents can alter colonic mitochondrial function by increasing respiration-induced proton leak, thereby inducing apoptosis, a marker of colon cancer risk. To further probe bioenergetics in primary intestinal cells, we developed methodology that can be modified and adapted to measure the bioenergetic profiles of colonic crypts, the basic functional unit of the colon, and colonic organoids, an ex vivo 3D culture of colonic crypts. Furthermore, in combination with the MoFlo Astrios High-Speed Cell Sorter, we were able to measure the bioenergetic profiles of colonic adult stem and daughter cells from Lgr5-EGFP-IRES-creER(T2) transgenic mice. We examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a full arylhydrocarbon receptor agonist, known to affect gastrointestinal function and cancer risk, on the bioenergetic profiles of intestinal epithelial cells. Mouse colonic crypts, organoids, or sorted single cells were seeded onto Matrigel-precoated Seahorse XF24 microplates for extracellular flux analysis. Temporal analyses revealed distinct energy metabolic profiles in crypts and organoids challenged with TCDD. Furthermore, sorted Lgr5(+) stem cells exhibited a Warburg-like metabolic profile. This is noteworthy because perturbations in stem cell dynamics are generally believed to represent the earliest step toward colon tumorigenesis. We propose that our innovative methodology may facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonocyte energy metabolism.
Assuntos
Bioensaio/métodos , Separação Celular/métodos , Colo/metabolismo , Metabolismo Energético , Citometria de Fluxo , Organoides/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Colo/citologia , Colo/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organoides/citologia , Organoides/efeitos dos fármacos , Fenótipo , Dibenzodioxinas Policloradas/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Arachidonic acid (20:4(Δ5,8,11,14), AA)-derived prostaglandin E2 (PGE2) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE2 biosynthesis in colonocytes, with eicosapentaenoic acid (20:5(Δ5,8,11,14,17), EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE3), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creER(T2) knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE2 and PGE3. Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE2, PGE3 or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE2 promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE3 and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by >2-fold in PGE2-treated cultures compared with PGE3 and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE2, a known promoter of colon tumorigenesis, EPA-derived PGE3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids.
Assuntos
Divisão Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Prostaglandinas/farmacologia , Células-Tronco/efeitos dos fármacos , Colo/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/citologiaRESUMO
During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA-) derived eicosanoids, such as prostaglandin E2 (PGE2), promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS-) induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23), decreased percentages of Th17 cells and, improved colon injury scores (P ≤ 0.05). Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.
Assuntos
Ácido Araquidônico/química , Colite/tratamento farmacológico , Colite/imunologia , Eicosanoides/química , Eicosanoides/uso terapêutico , Inflamação/imunologia , Inflamação/metabolismo , Células Th1/metabolismo , Células Th17/metabolismo , Animais , Colite/metabolismo , Feminino , Masculino , CamundongosRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. There is growing evidence that dysbiosis of the intestinal microbiota and disruption of microbiota-host interactions contribute to the pathology of NAFLD. We previously demonstrated that gut microbiota-derived tryptophan metabolite indole-3-acetate (I3A) was decreased in both cecum and liver of high-fat diet-fed mice and attenuated the expression of inflammatory cytokines in macrophages and Tnfa and fatty acid-induced inflammatory responses in an aryl-hydrocarbon receptor (AhR)-dependent manner in hepatocytes. In this study, we investigated the effect of orally administered I3A in a mouse model of diet-induced NAFLD. Western diet (WD)-fed mice given sugar water (SW) with I3A showed dramatically decreased serum ALT, hepatic triglycerides (TG), liver steatosis, hepatocyte ballooning, lobular inflammation, and hepatic production of inflammatory cytokines, compared to WD-fed mice given only SW. Metagenomic analysis show that I3A administration did not significantly modify the intestinal microbiome, suggesting that I3A's beneficial effects likely reflect the metabolite's direct actions on the liver. Administration of I3A partially reversed WD-induced alterations of liver metabolome and proteome, notably, decreasing expression of several enzymes in hepatic lipogenesis and ß-oxidation. Mechanistically, we also show that AMP-activated protein kinase (AMPK) mediates the anti-inflammatory effects of I3A in macrophages. The potency of I3A in alleviating liver steatosis and inflammation clearly demonstrates its potential as a therapeutic modality for preventing the progression of steatosis to non-alcoholic steatohepatitis (NASH).
Assuntos
Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Inflamação , Dieta Ocidental/efeitos adversos , Citocinas , Suplementos Nutricionais , Acetatos , Indóis/farmacologiaRESUMO
Epidermal growth factor receptor (EGFR)-mediated signaling is required for optimal intestinal wound healing. Since n-3 polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA), alter EGFR signaling and suppress downstream activation of key signaling pathways, we hypothesized that DHA would be detrimental to the process of intestinal wound healing. Using a mouse immortalized colonocyte model, DHA uniquely reduced EGFR ligand-induced receptor activation, whereas DHA and its metabolic precursor eicosapentaenoic acid (EPA) reduced wound-induced EGFR transactivation compared with control (no fatty acid or linoleic acid). Under wounding conditions, the suppression of EGFR activation was associated with a reduction in downstream activation of cytoskeletal remodeling proteins (PLCγ1, Rac1, and Cdc42). Subsequently, DHA and EPA reduced cell migration in response to wounding. Mice were fed a corn oil-, DHA-, or EPA-enriched diet prior to intestinal wounding (2.5% dextran sodium sulfate for 5 days followed by termination after 0, 3, or 6 days of recovery). Mortality was increased in EPA-fed mice and colonic histological injury scores were increased in EPA- and DHA-fed mice compared with corn oil-fed (control) mice. Although kinetics of colonic EGFR activation and downstream signaling (PLCγ1, Rac1, and Cdc42) were delayed by both n-3 PUFA, colonic repair was increased in EPA- relative to DHA-fed mice. These results indicate that, during the early response to intestinal wounding, DHA and EPA uniquely delay the activation of key wound-healing processes in the colon. This effect is mediated, at least in part, via suppression of EGFR-mediated signaling and downstream cytoskeletal remodeling.
Assuntos
Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Receptores ErbB/metabolismo , Processamento de Proteína Pós-Traducional , Cicatrização , Animais , Ácido Araquidônico/metabolismo , Movimento Celular , Células Cultivadas , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Óleo de Milho/administração & dosagem , Sulfato de Dextrana , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/fisiologia , Ácido Eicosapentaenoico/fisiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neuropeptídeos/metabolismo , Consumo de Oxigênio , Fosforilação , Transdução de Sinais , Ácido alfa-Linolênico/farmacologia , Ácido alfa-Linolênico/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creER(T2) mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5(+) stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5(+) stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5(+) stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.
Assuntos
Colo/fisiologia , Regeneração/fisiologia , Células-Tronco/fisiologia , Cicatrização/fisiologia , Animais , Apoptose/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Dieta , Ácidos Graxos Ômega-3/metabolismo , Óleos de Peixe/metabolismo , Expressão Gênica/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Regeneração/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia , Cicatrização/genéticaRESUMO
The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)-δ is highly expressed in colonic epithelial cells; however, the role of PPARδ ligands, such as fatty acids, in mucosal inflammation and malignant transformation has not been clarified. Recent evidence suggests that the anti-inflammatory/chemoprotective properties of fish oil (FO)-derived n-3 polyunsaturated fatty acids (PUFAs) may be partly mediated by PPARδ. Therefore, we assessed the role of PPARδ in modulating the effects of dietary n-3 PUFAs by targeted deletion of intestinal epithelial cell PPARδ (PPARδ(ΔIEpC)). Subsequently, we documented changes in colon tumorigenesis and the inflammatory microenvironment, i.e., local [mesenteric lymph node (MLN)] and systemic (spleen) T cell activation. Animals were fed chemopromotive [corn oil (CO)] or chemoprotective (FO) diets during the induction of chronic inflammation/carcinogenesis. Tumor incidence was similar in control and PPARδ(ΔIEpC) mice. FO reduced mucosal injury, tumor incidence, colonic STAT3 activation, and inflammatory cytokine gene expression, independent of PPARδ genotype. CD8(+) T cell recruitment into MLNs was suppressed in PPARδ(ΔIEpC) mice. Similarly, FO reduced CD8(+) T cell numbers in the MLN. Dietary FO independently modulated MLN CD4(+) T cell activation status by decreasing CD44 expression. CD11a expression by MLN CD4(+) T cells was downregulated in PPARδ(ΔIEpC) mice. Lastly, splenic CD62L expression was downregulated in PPARδ(ΔIEpC) CD4(+) and CD8(+) T cells. These data demonstrate that expression of intestinal epithelial cell PPARδ does not influence azoxymethane/dextran sodium sulfate-induced colon tumor incidence. Moreover, we provide new evidence that dietary n-3 PUFAs attenuate intestinal inflammation in an intestinal epithelial cell PPARδ-independent manner.
Assuntos
Adenocarcinoma/tratamento farmacológico , Transformação Celular Neoplásica/efeitos dos fármacos , Colite/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Gorduras Insaturadas na Dieta/farmacologia , Óleos de Peixe/farmacologia , Deleção de Genes , PPAR delta/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Animais , Antígeno CD11a/biossíntese , Antígeno CD11a/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Doença Crônica , Colite/genética , Colite/imunologia , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Citocinas/biossíntese , Gorduras Insaturadas na Dieta/imunologia , Gorduras Insaturadas na Dieta/metabolismo , Feminino , Óleos de Peixe/imunologia , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/imunologia , Mucosa Intestinal/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR delta/genética , PPAR delta/imunologia , Fator de Transcrição STAT3/biossíntese , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
During colon inflammation, Th17 cells and immunosuppressive regulatory T cells (Treg) are thought to play promotive and preventative roles, respectively. Dietary (n-3) PUFA favorably modulate intestinal inflammation in part by downregulating T-cell activation and functionality. We used the Fat-1 mouse, a genetic model that synthesizes long-chain (n-3) PUFA de novo, to test the hypothesis that (n-3) PUFA protect against colonic inflammation by modulating the polarization of Treg and Th17 cells during colitis. Male and female wild-type (WT) and Fat-1 mice were administered dextran sodium sulfate (DSS) in the drinking water (2.5%) to induce acute (5 d DSS) or chronic (3 cycles DSS) colitis and the percentage of Treg and Th17 cells residing locally [colonic lamina propria (cLP)] and systemically (spleen) was determined by flow cytometry. The percentage of Treg in either tissue site was unaffected by genotype (P > 0.05); however, during chronic colitis, the percentage of Th17 cells residing in both the spleen and cLP was lower in Fat-1 mice compared to WT mice (P < 0.05). Colonic mucosal mRNA expression of critical Th17 cell cytokines and chemokine receptors (IL-17F, IL-21, and CCR6) were lower, whereas expression of the Th17 cell suppressive cytokine, IL-27, was greater in Fat-1 mice compared to WT mice during chronic colitis (P < 0.05). Moreover, colon histological scores were improved in Fat-1 mice (P < 0.05). Collectively, these results demonstrate for the first time, to our knowledge, that (n-3) PUFA can modulate the colonic mucosal microenvironment to suppress Th17 cell accumulation and inflammatory damage following the induction of chronic colitis.
Assuntos
Colite/imunologia , Ácidos Graxos Ômega-3/administração & dosagem , Células Th17/imunologia , Animais , Doença Crônica , Colite/metabolismo , Colite/patologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos MutantesRESUMO
Both fish oil (FO) and curcumin have potential as anti-tumour and anti-inflammatory agents. To further explore their combined effects on dextran sodium sulphate (DSS)-induced colitis, C57BL/6 mice were randomised to four diets (2 × 2 design) differing in fatty acid content with or without curcumin supplementation (FO, FO+2 % curcumin, maize oil (control, MO) or MO+2 % curcumin). Mice were exposed to one or two cycles of DSS in the drinking-water to induce either acute or chronic intestinal inflammation, respectively. FO-fed mice exposed to the single-cycle DSS treatment exhibited the highest mortality (40 %, seventeen of forty-three) compared with MO with the lowest mortality (3 %, one of twenty-nine) (P = 0·0008). Addition of curcumin to MO increased (P = 0·003) mortality to 37 % compared with the control. Consistent with animal survival data, following the one- or two-cycle DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared with MO. In contrast, compared with other diets, combined FO and curcumin feeding enhanced the resolution of chronic inflammation and suppressed (P < 0·05) a key inflammatory mediator, NF-κB, in the colon mucosa. Mucosal microarray analysis revealed that dietary FO, curcumin and FO plus curcumin combination differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.
Assuntos
Colite/dietoterapia , Colo/metabolismo , Curcumina/uso terapêutico , Citocinas/metabolismo , Suplementos Nutricionais , Óleos de Peixe/uso terapêutico , Regulação da Expressão Gênica , Doença Aguda , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/uso terapêutico , Doença Crônica , Colite/imunologia , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Curcumina/efeitos adversos , Citocinas/genética , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Óleos de Peixe/efeitos adversos , Perfilação da Expressão Gênica , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Irritantes/administração & dosagem , Irritantes/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Distribuição Aleatória , Análise de SobrevidaRESUMO
The fatty acid desaturase 1 (FADS1), also known as delta-5 desaturase (D5D), is one of the rate-limiting enzymes involved in the desaturation and elongation cascade of polyunsaturated fatty acids (PUFAs) to generate long-chain PUFAs (LC-PUFAs). Reduced function of D5D and decreased hepatic FADS1 expression, as well as low levels of LC-PUFAs, were associated with nonalcoholic fatty liver disease. However, the causal role of D5D in hepatic lipid homeostasis remains unclear. In this study, we hypothesized that down-regulation of FADS1 increases susceptibility to hepatic lipid accumulation. We used in vitro and in vivo models to test this hypothesis and to delineate the molecular mechanisms mediating the effect of reduced FADS1 function. Our study demonstrated that FADS1 knockdown significantly reduced cellular levels of LC-PUFAs and increased lipid accumulation and lipid droplet formation in HepG2 cells. The lipid accumulation was associated with significant alterations in multiple pathways involved in lipid homeostasis, especially fatty acid oxidation. These effects were demonstrated to be mediated by the reduced function of the peroxisome proliferator-activated receptor alpha (PPARα)-fibroblast growth factor 21 (FGF21) axis, which can be reversed by treatment with docosahexaenoic acid, PPARα agonist, or FGF21. In vivo, FADS1-knockout mice fed with high-fat diet developed increased hepatic steatosis as compared with their wild-type littermates. Molecular analyses of the mouse liver tissue largely corroborated the observations in vitro, especially along with reduced protein expression of PPARα and FGF21. Conclusion: Collectively, these results suggest that dysregulation in FADS1 alters liver lipid homeostasis in the liver by down-regulating the PPARα-FGF21 signaling axis.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Fígado Gorduroso/enzimologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/enzimologia , PPAR alfa/metabolismo , Animais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Regulação para Baixo , Ácidos Graxos Insaturados/metabolismo , Células Hep G2 , Homeostase , Humanos , Metabolismo dos Lipídeos , Camundongos , Camundongos Knockout , PPAR alfa/agonistas , Transdução de SinaisRESUMO
We recently demonstrated that (n-3) PUFA trigger the induction of apoptosis in the colon by enhancing phospholipid oxidation and mitochondrial Ca2+ accumulation. To further elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, a 2 x 2 experiment was designed using both wild type (control) and manganese-dependent superoxide dismutase (SOD2) heterozygous knockout mice (SOD2(+/-)), which exhibit increased mitochondrial oxidative stress. Mice were fed diets differing only in the type of fat [corn oil or fish oil containing (n-3) PUFA] at 15% by weight for 4 wk. Dietary (n-3) PUFA treatment enhanced (22%) apoptosis in colonic crypts. In addition, SOD2 haploinsufficiency enhanced (20%) apoptosis, which was further increased (36%) by (n-3) PUFA feeding. Dietary lipid source and genotype interactively modulated nitrotyrosine levels (P = 0.027) and inflammation (P = 0.032). These findings demonstrate that the proapoptotic effects of (n-3) PUFA are enhanced in oxidatively stressed SOD2(+/-) mice. Thus, (n-3) PUFA appear to promote an oxidation-reduction imbalance in the intestine, which may directly or indirectly trigger apoptosis and thereby reduce colon cancer risk.
Assuntos
Apoptose/efeitos dos fármacos , Colo/enzimologia , Ácidos Graxos Ômega-3/farmacologia , Superóxido Dismutase/deficiência , Animais , Colo/citologia , Colo/efeitos dos fármacos , Primers do DNA , Triagem de Portadores Genéticos , Imuno-Histoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Superóxido Dismutase/genéticaRESUMO
Multicomponent therapy has gained interest for its potential to synergize and subsequently lower the effective dose of each constituent required to reduce colon cancer risk. We have previously showed that rapidly cycling Lgr5 stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk. In the present study, we quantified the dose-dependent synergistic properties of dietary n-3 polyunsaturated fatty acids (PUFA) and curcumin (Cur) to promote targeted apoptotic deletion of damaged colonic Lgr5 stem cells. For this purpose, both heterogeneous bulk colonocytes and Lgr5 stem cells were isolated from Lgr5-EGFP-IRES-CreER knock-in mice injected with azoxymethane (AOM). Isolated cells were analyzed for DNA damage (γH2AX), apoptosis (cleaved caspase-3), and targeted apoptosis (both γH2AX and cleaved caspase-3) at 12 h post-AOM injection. Comparison of the percentage of targeted apoptosis in Lgr5 stem cells (GFP) across a broad bioactive dose-range revealed an ED50 of 16.0 mg/day n-3 PUFA + 15.9 mg/day Cur. This corresponded to a human equivalent dose of 3.0 g n-3 PUFA + 3.0 g Cur. In summary, our results provide evidence that a low dose (n-3 PUFA + Cur) combination diet reduces AOM-induced DNA damage in Lgr5 stem cells and enhances targeted apoptosis of DNA-damaged cells, implying that a lower human equivalent dose can be utilized in future human clinical trials.
Assuntos
Neoplasias do Colo/prevenção & controle , Curcumina/administração & dosagem , Ácidos Graxos Ômega-3/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Apoptose/efeitos dos fármacos , Azoximetano/toxicidade , Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Colo/citologia , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Técnicas de Introdução de Genes , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4+ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4+ T-cells from FO-fed WT mice after 3d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4+ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4+ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4+ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.