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1.
BMC Pediatr ; 21(1): 374, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465311

RESUMO

BACKGROUND: Overweight, obesity, and associated comorbidities are a pressing global issue among children of all ages, particularly among low-income populations. Rapid weight gain (RWG) in the first 6 months of infancy contributes to childhood obesity. Suboptimal sleep-wake patterns and gut microbiota (GM) have also been associated with childhood obesity, but little is known about their influences on early infant RWG. Sleep may alter the GM and infant metabolism, and ultimately impact obesity; however, data on the interaction between sleep-wake patterns and GM development on infant growth are scarce. In this study, we aim to investigate associations of infant sleep-wake patterns and GM development with RWG at 6 months and weight gain at 12 months. We also aim to evaluate whether temporal interactions exist between infant sleep-wake patterns and GM, and if these relations influence RWG. METHODS: The Snuggle Bug/ Acurrucadito study is an observational, longitudinal study investigating whether 24-h, actigraphy-assessed, sleep-wake patterns and GM development are associated with RWG among infants in their first year. Based on the Ecological Model of Growth, we propose a novel conceptual framework to incorporate sleep-wake patterns and the GM as metabolic contributors for RWG in the context of maternal-infant interactions, and familial and socio-physical environments. In total, 192 mother-infant pairs will be recruited, and sleep-wake patterns and GM development assessed at 3 and 8 weeks, and 3, 6, 9, and 12 months postpartum. Covariates including maternal and child characteristics, family and environmental factors, feeding practices and dietary intake of infants and mothers, and stool-derived metabolome and exfoliome data will be assessed. The study will apply machine learning techniques combined with logistic time-varying effect models to capture infant growth and aid in elucidating the dynamic associations between study variables and RWG. DISCUSSION: Repeated, valid, and objective assessment at clinically and developmentally meaningful intervals will provide robust measures of longitudinal sleep, GM, and growth. Project findings will provide evidence for future interventions to prevent RWG in infancy and subsequent obesity. The work also may spur the development of evidence-based guidelines to address modifiable factors that influence sleep-wake and GM development and prevent childhood obesity.


Assuntos
Microbioma Gastrointestinal , Obesidade Pediátrica , Criança , Feminino , Humanos , Lactente , Estudos Longitudinais , Obesidade Pediátrica/etiologia , Fatores de Risco , Sono , Ganho de Peso
2.
Mol Nutr Food Res ; : e2100539, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34406707

RESUMO

SCOPE: This study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)-induced intestinal barrier damage in mice. METHODS AND RESULTS: Aqueous coffee extracts induced Ah receptor (AhR) -responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon-derived Caco2 and YAMC cells. Tissue-specific AhR knockout (AhRf/f x Lgr5-GFP-CreERT2 x Villin-Cre), wild-type (Lgr5-CreERT2 x Villin-Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR-active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS-induced damage to intestinal barrier function and DSS-induced mucosal inflammatory genes such as IL-6 and TGF-ß1 in wild-type (AhR+/+ ) but not AhR-/- mice. In contrast, coffee does not exhibit protective effects in intestinal-specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR-active microbial metabolites. CONCLUSIONS: In colon-derived cells and in the mouse intestine, coffee induced several AhR-dependent responses including gene expression, inhibition of intestinal stem cell-enriched organoid growth, and inhibition of DSS-induced intestinal barrier damage. We conclude that the anti-inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.

3.
Am J Physiol Gastrointest Liver Physiol ; 321(1): G41-G51, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33949197

RESUMO

Assessing intestinal development and host-microbe interactions in healthy human infants requires noninvasive approaches. We have shown that the transcriptome of exfoliated epithelial cells in feces can differentiate breast-fed and formula-fed infants and term and preterm infants. However, it is not fully understood which regions of the intestine that the exfoliated cells represent. Herein, the transcriptional profiles of exfoliated cells with that of the ileal and colonic mucosa were compared. We hypothesized that exfoliated cells in the distal colon would reflect mucosal signatures of more proximal regions of the gut. Two-day-old piglets (n = 8) were fed formulas for 20 days. Luminal contents and mucosa were collected from ileum (IL), ascending colon (AC), and descending (DC) colon, and mRNA was extracted and sequenced. On average, ∼13,000 genes were mapped in mucosal tissues and ∼10,000 in luminal contents. The intersection of detected genes between three mucosa regions and DC exfoliome indicated an approximately 99% overlap. On average, 49% of the genes in IL, AC, and DC mucosa were present in the AC and DC exfoliome. Genes expressed predominantly in specific anatomic sites (stomach, pancreas, small intestine, colon) were detectable in exfoliated cells. In addition, gene markers for all intestinal epithelial cell types were expressed in the exfoliome representing a diverse array of cell types arising from both the small and large intestine. Genes were mapped to nutrient absorption and transport and immune function. Thus, the exfoliome represents a robust reservoir of information in which to assess intestinal development and responses to dietary interventions.NEW & NOTEWORTHY The transcriptome of exfoliated epithelial cells in stool contain gene signatures from both small and large intestinal mucosa affording a noninvasive approach to assess gut health and function.


Assuntos
Células Epiteliais/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Transcriptoma/fisiologia , Animais , Colo/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Mucosa Intestinal/metabolismo , Suínos
4.
Mol Cancer Res ; 19(5): 771-783, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33495399

RESUMO

The mutational genetic landscape of colorectal cancer has been extensively characterized; however, the ability of "cooperation response genes" to modulate the function of cancer "driver" genes remains largely unknown. In this study, we investigate the role of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, in modulating oncogenic cues in the colon. We show that intestinal epithelial cell-targeted AhR knockout (KO) promotes the expansion and clonogenic capacity of colonic stem/progenitor cells harboring ApcS580/+; KrasG12D/+ mutations by upregulating Wnt signaling. The loss of AhR in the gut epithelium increased cell proliferation, reduced mouse survival rate, and promoted cecum and colon tumorigenesis in mice. Mechanistically, the antagonism of Wnt signaling induced by Lgr5 haploinsufficiency attenuated the effects of AhR KO on cecum and colon tumorigenesis. IMPLICATIONS: Our findings reveal that AhR signaling plays a protective role in genetically induced colon tumorigenesis at least by suppressing Wnt signaling and provides rationale for the AhR as a therapeutic target for cancer prevention and treatment.

5.
Toxicol Sci ; 180(1): 148-159, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33263770

RESUMO

Hydroxylated chalcones are phytochemicals which are biosynthetic precursors of flavonoids and their 1,3-diaryl-prop-2-en-1-one structure is used as a scaffold for drug development. In this study, the structure-dependent activation of aryl hydrocarbon receptor (AhR)-responsive CYP1A1, CYP1B1, and UGT1A1 genes was investigated in Caco2 colon cancer cells and in non-transformed young adult mouse colonocytes (YAMC) cells. The effects of a series of di- and trihydroxychalcones as AhR agonists was structure dependent with maximal induction of CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells observed for compounds containing 2,2'-dihydroxy substituents and this included 2,2'-dihydroxy-, 2,2',4'-trihydroxy-, and 2,2',5'-trihydroxychalcones. In contrast, 2',4,5'-, 2'3',4'-, 2',4,4'-trihydroxy, and 2',3-, 2',4-, 2',4'-, and 2',5-dihydroxychalcones exhibited low to non-detectable AhR activity in Caco2 cells. In addition, all of the hydroxychalcones exhibited minimal to non-detectable activity in YAMC cells, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 and YAMC cells. The activity of AhR-active chalcones was confirmed by determining their effects in AhR-deficient Caco2 cells. In addition, 2,2'-dihydroxychalcone induced CYP1A1 protein and formation of an AhR-DNA complex in an in vitro assay. Simulation and modeling studies of hydroxylated chalcones confirmed their interactions with the AhR ligand-binding domain and were consistent with their structure-dependent activity as AhR ligands. Thus, this study identifies hydroxylated chalcones as AhR agonists with potential for these phytochemicals to impact AhR-mediated colonic pathways.

6.
EMBO J ; 39(19): e104319, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32915464

RESUMO

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that senses xenobiotics, diet, and gut microbial-derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5+ stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal-specific AhR knockout increases basal stem cell and crypt injury-induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis-associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR-FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior.


Assuntos
Colo/metabolismo , Proteína Forkhead Box M1/metabolismo , Receptores de Hidrocarboneto Arílico/deficiência , Transdução de Sinais , Células-Tronco/metabolismo , Animais , Feminino , Proteína Forkhead Box M1/genética , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Hidrocarboneto Arílico/metabolismo
7.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G451-G463, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905023

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/metabolismo
8.
Chem Res Toxicol ; 32(11): 2353-2364, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31621310

RESUMO

Many of the protective responses observed for flavonoids in the gastrointestinal track resemble aryl hydrocarbon receptor (AhR)-mediated effects. Therefore, we examined the structure-activity relationships of isoflavones and isomeric flavone and flavanones as AhR ligands on the basis of their induction of CYP1A1, CYP1B1, and UGT1A1 gene expression in colon cancer Caco2 cells and young adult mouse colonocyte (YAMC) cells. Caco2 cells were significantly more Ah-responsive than YAMC cells, and this was due, in part, to flavonoid-induced cytotoxicity in the latter cell lines. The structure-activity relationships for the flavonoids were complex and both response and cell context specific; however, there was significant variability in the AhR activities of the isomeric substituted isoflavones and flavones. For example, 4',5,7-trihydroxyisoflavone (genistein) was AhR-inactive whereas 4',5,7-trihydroxyflavone (apigenin) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells. In contrast, both 5,7-dihydroxy-4-methoxy substituted isoflavone (biochanin A) and flavone (acacetin) induced all three AhR-responsive genes; 4',5,7-trimethoxyisoflavone was a potent AhR agonist, and the isomeric flavone was AhR-inactive. These results coupled with simulation studies modeling flavonoid interaction within the AhR binding pocket demonstrate that the orientation of the substituted phenyl ring at C-2 (flavones) or C-3 (isoflavones) on the common 4-H-chromen-4-one ring strongly influences the activities of isoflavones and flavones as AhR agonists.


Assuntos
Flavonoides/farmacologia , Receptores de Hidrocarboneto Arílico/agonistas , Animais , Linhagem Celular , Colo/citologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Flavonoides/química , Glucuronosiltransferase/metabolismo , Humanos , Camundongos , Modelos Moleculares , Relação Estrutura-Atividade
9.
Am J Clin Nutr ; 110(2): 377-390, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31175806

RESUMO

BACKGROUND: Microbial metabolism of lignans from high-fiber plant foods produces bioactive enterolignans, such as enterolactone (ENL) and enterodiol (END). Enterolignan exposure influences cellular pathways important to cancer risk and is associated with reduced colon tumorigenesis in animal models and lower colorectal cancer risk in humans. OBJECTIVES: The aim of this study was to test the effects of a flaxseed lignan supplement (50 mg secoisolariciresinol diglucoside/d) compared with placebo on host gene expression in colon biopsies and exfoliated colonocyte RNA in feces and fecal microbial community composition, and to compare responses in relation to ENL excretion. METHODS: We conducted a 2-period randomized, crossover intervention in 42 healthy men and women (20-45 y). We used RNA-seq to measure differentially expressed (DE) genes in colonic mucosa and fecal exfoliated cells through the use of edgeR and functional analysis with Ingenuity Pathway Analysis. We used 16S ribosomal RNA gene (V1-V3) analysis to characterize the fecal microbiome, and measured END and ENL in 24-h urine samples by gas chromatography-mass spectrometry. RESULTS: We detected 32 DE genes (false discovery rate <0.05) in the exfoliome, but none in the mucosal biopsies, in response to 60 d of lignan supplement compared with placebo. Statistically significant associations were detected between ENL excretion and fecal microbiome measured at baseline and at the end of the intervention periods. Further, we detected DE genes in colonic mucosa and exfoliome between low- and high-ENL excreters. Analysis of biopsy samples indicated that several anti-inflammatory upstream regulators, including transforming growth factor ß and interleukin 10 receptor, were suppressed in low-ENL excreters. Complementary analyses in exfoliated cells also suggested that low-ENL excreters may be predisposed to proinflammatory cellular events due to upregulation of nuclear transcription factor κB and NOS2, and an inhibition of the peroxisome proliferator-activated receptor γ network. CONCLUSIONS: These results suggest that ENL or other activities of the associated gut microbial consortia may modulate response to a dietary lignan intervention. This has important implications for dietary recommendations and chemoprevention strategies. This study was registered at clinicaltrials.gov as NCT01619020.


Assuntos
Fezes/microbiologia , Linho/química , Perfilação da Expressão Gênica , Mucosa Intestinal/efeitos dos fármacos , Lignanas/química , Extratos Vegetais/farmacologia , Adulto , Colo/efeitos dos fármacos , Estudos Cross-Over , Método Duplo-Cego , Feminino , Microbioma Gastrointestinal , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/metabolismo , Masculino , Extratos Vegetais/química
10.
Methods Mol Biol ; 1576: 171-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-27539462

RESUMO

Colonic organoids, three-dimensional colonic crypts grown in vitro that show realistic microanatomy, have many potential applications for studying physiology, developmental biology, and pathophysiology of intestinal diseases including inflammatory bowel disease and colorectal cancer. Here, we describe detailed protocols for mouse colonic crypt isolation, organoid culture, and downstream applications. Specific culture strategies including growth factor enriched Matrigel and Wnt and R-spondin conditioned media serve as key factors for enhancing the growth and cost efficiency of colonic organoid cultures.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Colo/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Organoides/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Colo/metabolismo , Meios de Cultivo Condicionados , Camundongos , Organoides/metabolismo , Células-Tronco/metabolismo
11.
Toxicol Sci ; 164(1): 205-217, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29584932

RESUMO

Dietary flavonoids are used in treatment of multiple diseases, and their antiinflammatory effects in the intestine are due, in part, to interactions with gut microflora and possibly due to modulation of aryl hydrocarbon receptor (AhR) signaling. In this study, we investigated the structure-dependent AhR activity of 14 flavonoids in Caco2 colon cancer cells using induction of CYP1A1 and UGT1A1 gene expression as endpoints. A major structural determinant for AhR activation was the number of hydroxyl groups where pentahydroxyflavonoids (with the exception of morin) > hexahydroxyflavonoids > tetra-/trihydroxyflavonoids, and some of the latter compounds such as apigenin exhibited AhR antagonist activity for induction of CYP1A1. Simulations suggest that while quercetin and apigenin interact primarily with the same residues, the strength of interactions between specific AhR residues with CYP1A1 agonist, quercetin, in comparison with CYP1A1 antagonist, apigenin, is different; thus, such interactions are presumably indicative of potential switches for modulating CYP1A1 activity. The structure-dependent effects of the hydroxyl flavonoids on induction of UGT1A1 were similar to that observed for induction of CYP1A1 except that luteolin and apigenin induced UGT1A1 levels similar to that observed for TCDD, whereas both compounds were AhR antagonists for CYP1A1. Thus, the effects of the flavonoids in Caco2 cells on Ah-responsiveness and interactions with butyrate were both ligand structure- and response-dependent and these activities are consistent with hydroxyflavonoids being selective AhR modulators.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Flavonoides/química , Flavonoides/farmacologia , Receptores de Hidrocarboneto Arílico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células CACO-2 , Citocromo P-450 CYP1A1/genética , Indução Enzimática , Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Relação Estrutura-Atividade
12.
Sci Rep ; 7(1): 14687, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29089621

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently used classes of medications in the world, yet they induce an enteropathy that is associated with high morbidity and mortality. A major limitation to better understanding the pathophysiology and diagnosis of this enteropathy is the difficulty of obtaining information about the primary site of injury, namely the distal small intestine. We investigated the utility of using mRNA from exfoliated cells in stool as a means to surveil the distal small intestine in a murine model of NSAID enteropathy. Specifically, we performed RNA-Seq on exfoliated cells found in feces and compared these data to RNA-Seq from both the small intestinal mucosa and colonic mucosa of healthy control mice or those exhibiting NSAID-induced enteropathy. Global gene expression analysis, data intersection, pathway analysis, and computational approaches including linear discriminant analysis (LDA) and sparse canonical correlation analysis (CCA) were used to assess the inter-relatedness of tissue (invasive) and stool (noninvasive) datasets. These analyses revealed that the exfoliated cell transcriptome closely mirrored the transcriptome of the small intestinal mucosa. Thus, the exfoliome may serve as a non-invasive means of detecting and monitoring NSAID enteropathy (and possibly other gastrointestinal mucosal inflammatory diseases).


Assuntos
Antirreumáticos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Fezes/citologia , Enteropatias/genética , Mucosa Intestinal/fisiologia , Intestino Delgado/fisiologia , Transcriptoma/genética , Animais , Antirreumáticos/uso terapêutico , Biologia Computacional , Modelos Animais de Doenças , Feminino , Humanos , Enteropatias/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos
13.
Sci Rep ; 7(1): 10163, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860561

RESUMO

Aryl hydrocarbon receptor (AhR) ligands are important for gastrointestinal health and play a role in gut inflammation and the induction of T regulatory cells, and the short chain fatty acids (SCFAs) butyrate, propionate and acetate also induce similar protective responses. Initial studies with butyrate demonstrated that this compound significantly increased expression of Ah-responsive genes such as Cyp1a1/CYP1A1 in YAMC mouse colonocytes and Caco-2 human colon cancer cell lines. Butyrate synergistically enhanced AhR ligand-induced Cyp1a1/CYP1A1 in these cells with comparable enhancement being observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and also microbiota-derived AhR ligands tryptamine, indole and 1,4-dihydroxy-2-naphthoic acid (DHNA). The effects of butyrate on enhancing induction of Cyp1b1/CYP1B1, AhR repressor (Ahrr/AhRR) and TCDD-inducible poly(ADP-ribose)polymerase (Tiparp/TiPARP) by AhR ligands were gene- and cell context-dependent with the Caco-2 cells being the most responsive cell line. Like butyrate and propionate, the prototypical hydroxyamic acid-derived histone deacetylase (HDAC) inhibitors Panobinostat and Vorinostat also enhanced AhR ligand-mediated induction and this was accompanied by enhanced histone acetylation. Acetate also enhanced basal and ligand-inducible Ah responsiveness and histone acetylation, demonstrating that acetate was an HDAC inhibitor. These results demonstrate SCFA-AhR ligand interactions in YAMC and Caco-2 cells where SCFAs synergistically enhance basal and ligand-induced expression of AhR-responsive genes.


Assuntos
Colo/química , Neoplasias do Colo/genética , Ácidos Graxos Voláteis/farmacologia , Redes Reguladoras de Genes , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Butiratos/farmacologia , Células CACO-2 , Células Cultivadas , Colo/citologia , Colo/metabolismo , Neoplasias do Colo/metabolismo , Técnicas de Inativação de Genes , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Propionatos/farmacologia
14.
Toxicol Sci ; 155(2): 458-473, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27837168

RESUMO

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.


Assuntos
Modelos Teóricos , Naftóis/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Animais , Células CACO-2 , Linhagem Celular , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Cobaias , Humanos , Camundongos , Naftalenos/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Relação Estrutura-Atividade
15.
Cell Death Dis ; 7(11): e2460, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27831561

RESUMO

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear ß-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.


Assuntos
Ciclo Celular , Neoplasias do Colo/patologia , Dieta , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Focos de Criptas Aberrantes/metabolismo , Animais , Apoptose/efeitos dos fármacos , Azoximetano , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinógenos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimioprevenção , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Curcumina/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Ácidos Graxos Ômega-3 , Óleos de Peixe/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Regeneração/efeitos dos fármacos , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/metabolismo
16.
Physiol Genomics ; 48(9): 651-9, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27401218

RESUMO

The strength of associations between various exposures (e.g., diet, tobacco, chemopreventive agents) and colorectal cancer risk may partially depend on the complex interaction between epithelium and stroma across anatomic subsites. Currently, baseline data describing genome-wide coding and long noncoding gene expression profiles in the healthy colon specific to tissue type and location are lacking. Therefore, colonic mucosal biopsies from 10 healthy participants who were enrolled in a clinical study to evaluate effects of lignan supplementation on gut resiliency were used to characterize the site-specific global gene expression signatures associated with stromal vs. epithelial cells in the sigmoid colon and rectum. Using RNA-seq, we demonstrate that tissue type and location patterns of gene expression and upstream regulatory pathways are distinct. For example, consistent with a key role of stroma in the crypt niche, mRNAs associated with immunoregulatory and inflammatory processes (i.e., CXCL14, ANTXR1), smooth muscle contraction (CALD1), proliferation and apoptosis (GLP2R, IGFBP3), and modulation of extracellular matrix (MMP2, COL3A1, MFAP4) were all highly expressed in the stroma. In comparison, HOX genes (HOXA3, HOXD9, HOXD10, HOXD11, and HOXD-AS2, a HOXD cluster antisense RNA 2), and WNT5B expression were also significantly higher in sigmoid colon compared with the rectum. These findings provide strong impetus for considering colorectal tissue subtypes and location in future observational studies and clinical trials designed to evaluate the effects of exposures on colonic health.


Assuntos
Colo Sigmoide/metabolismo , Colo/metabolismo , Células Epiteliais/metabolismo , Reto/metabolismo , Adulto , Biópsia , Colo/efeitos dos fármacos , Colo/patologia , Colo Sigmoide/efeitos dos fármacos , Colo Sigmoide/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Estudos Cross-Over , Método Duplo-Cego , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lignanas/administração & dosagem , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reto/efeitos dos fármacos , Reto/patologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Adulto Jovem
17.
Annu Rev Nutr ; 36: 543-70, 2016 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-27431370

RESUMO

The International Agency for Research on Cancer recently released an assessment classifying red and processed meat as "carcinogenic to humans" on the basis of the positive association between increased consumption and risk for colorectal cancer. Diet, however, can also decrease the risk for colorectal cancer and be used as a chemopreventive strategy. Bioactive dietary molecules, such as n-3 polyunsaturated fatty acids, curcumin, and fermentable fiber, have been proposed to exert chemoprotective effects, and their molecular mechanisms have been the focus of research in the dietary/chemoprevention field. Using these bioactives as examples, this review surveys the proposed mechanisms by which they exert their effects, from the nucleus to the cellular membrane. In addition, we discuss emerging technologies involving the culturing of colonic organoids to study the physiological effects of dietary bioactives. Finally, we address future challenges to the field regarding the identification of additional molecular mechanisms and other bioactive dietary molecules that can be utilized in our fight to reduce the incidence of colorectal cancer.


Assuntos
Neoplasias do Colo/prevenção & controle , Dieta Saudável , Regulação da Expressão Gênica , Modelos Biológicos , Nutrigenômica/métodos , Animais , Anticarcinógenos/metabolismo , Anticarcinógenos/uso terapêutico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/microbiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/prevenção & controle , Curcumina/metabolismo , Curcumina/uso terapêutico , Metilação de DNA , Fibras na Dieta/metabolismo , Fibras na Dieta/uso terapêutico , Epigênese Genética , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/uso terapêutico , Fermentação , Microbioma Gastrointestinal , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , MicroRNAs/metabolismo , Nutrigenômica/tendências , Processamento de Proteína Pós-Traducional
18.
Gut Microbes ; 7(3): 246-61, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-27007819

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently used classes of medications in the world. Unfortunately, NSAIDs induce an enteropathy associated with high morbidity and mortality. Although the pathophysiology of this condition involves the interaction of the gut epithelium, microbiota, and NSAIDs, the precise mechanisms by which microbiota influence NSAID enteropathy are unclear. One possible mechanism is that the microbiota may attenuate the severity of disease by specific metabolite-mediated regulation of host inflammation and injury. The microbiota-derived tryptophan-metabolite indole is abundant in the healthy mammalian gut and positively influences intestinal health. We thus examined the effects of indole administration on NSAID enteropathy. Mice (n = 5 per group) were treated once daily for 7 days with an NSAID (indomethacin; 5 mg/kg), indole (20 mg/kg), indomethacin plus indole, or vehicle only (control). Outcomes compared among groups included: microscopic pathology; fecal calprotectin concentration; proportion of neutrophils in the spleen and mesenteric lymph nodes; fecal microbiota composition and diversity; small intestinal mucosal transcriptome; and, fecal tryptophan metabolites. Co-administration of indole with indomethacin: significantly reduced mucosal pathology scores, fecal calprotectin concentrations, and neutrophilic infiltration of the spleen and mesenteric lymph nodes induced by indomethacin; modulated NSAID-induced perturbation of the microbiota, fecal metabolites, and inferred metagenome; and, abrogated a pro-inflammatory gene expression profile in the small intestinal mucosa induced by indomethacin. The microbiota-derived metabolite indole attenuated multiple deleterious effects of NSAID enteropathy, including modulating inflammation mediated by innate immune responses and altering indomethacin-induced shift of the microbiota.


Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios/metabolismo , Enterite/tratamento farmacológico , Fármacos Gastrointestinais/farmacologia , Indóis/metabolismo , Indóis/farmacologia , Inflamação/patologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Bactérias/classificação , Bactérias/isolamento & purificação , Biota , Modelos Animais de Doenças , Enterite/induzido quimicamente , Fezes/química , Fezes/microbiologia , Fármacos Gastrointestinais/administração & dosagem , Histocitoquímica , Indóis/administração & dosagem , Complexo Antígeno L1 Leucocitário/análise , Linfonodos/patologia , Camundongos , Neutrófilos/imunologia , Baço/patologia , Resultado do Tratamento
19.
Data Brief ; 6: 398-404, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26862588

RESUMO

With the identification of Lgr5 as a definitive marker for intestinal stem cells, we used the highly novel, recently described, Lgr5-EGFP-IRES-cre ER (T2) knock in mouse model. Mice were injected with azoxymethane (AOM, a colon carcinogen) or saline (control) and fed a chemo-protective diet containing n-3 fatty acids and fermentable fiber (n-3 PUFA+pectin) or a control diet (n-6 PUFA + cellulose). Single cells were isolated from colonic mucosa crypts and three discrete populations of cells were collected via fluorescence activated cell sorting (FACS): Lgr5(high) (stem cells), Lgr5(low) (daughter cells) and Lgr5(negative) (differentiated cells). microRNA profiling and RNA sequencing were performed from the same sample and analyzed. These data refer to 'Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt' (Shah et al., 2016) [5].

20.
Carcinogenesis ; 37(2): 206-14, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26717997

RESUMO

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/patologia
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