RESUMO
After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the aryl hydrocarbon receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes or immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs.NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.
Assuntos
Colo , Mucosa Intestinal , Receptores de Hidrocarboneto Arílico , Estruturas Linfoides Terciárias , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Animais , Feminino , Colo/metabolismo , Colo/imunologia , Colo/patologia , Masculino , Estruturas Linfoides Terciárias/patologia , Estruturas Linfoides Terciárias/imunologia , Estruturas Linfoides Terciárias/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Camundongos , Células Epiteliais/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.
Assuntos
Microbiota , Animais , Cavalos/genética , Mucosa Intestinal/metabolismo , Metaboloma , Fezes/microbiologia , Anti-Inflamatórios não Esteroides/metabolismo , Inflamação/metabolismo , Fenilbutazona/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismoRESUMO
There is an urgent need to elucidate the mechanistic links between obesity and colon cancer. Convincing evidence for the role of Lgr5(+) stem cells in colon tumorigenesis has been established; however, the influence of obesity on stem cell maintenance is unknown. We assessed the effects of high fat (HF) feeding on colonic stem cell maintenance during cancer initiation (AOM induced) and the responsiveness of stem cells to adipokine signaling pathways. The number of colonic GFP(+) stem cells was significantly higher in the AOM-injected HF group compared to the LF group. The Lgr5(+) stem cells of the HF fed mice exhibited statistically significant increases in cell proliferation and decreases in apoptosis in response to AOM injection compared to the LF group. Colonic organoid cultures from lean mice treated with an adiponectin receptor agonist exhibited a reduction in Lgr5-GPF(+) stem cell number and an increase in apoptosis; however, this response was diminished in the organoid cultures from obese mice. These results suggest that the responsiveness of colonic stem cells to adiponectin in diet-induced obesity is impaired and may contribute to the stem cell accumulation observed in obesity.
Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Neoplasias do Colo/etiologia , Obesidade/complicações , Animais , Apoptose , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/patologia , Humanos , Masculino , Camundongos , Obesidade/metabolismo , Células-Tronco/metabolismoRESUMO
The etiology of colon cancer is complex, yet it is undoubtedly impacted by intestinal microbiota. Whether the contribution to colon carcinogenesis is generated through the presence of an overall dysbiosis or by specific pathogens is still a matter for debate. However, it is apparent that interactions between microbiota and the host are mediated by a variety of processes, including signaling cascades, the immune system, host metabolism, and regulation of gene transcription. To fully appreciate the role of microbiota in colon carcinogenesis, it will be necessary to expand efforts to define populations in niche environments, such as colonic crypts, explore cross talk between the host and the microbiota, and more completely define the metabolomic profile of the microbiota. These efforts must be pursued with appreciation that dietary substrates and other environmental modifiers mediate changes in the microbiota, as well as their metabolism and functional characteristics.
Assuntos
Neoplasias Colorretais/microbiologia , Disbiose/fisiopatologia , Microbiota/fisiologia , Bactérias/metabolismo , Aleitamento Materno , Colo/metabolismo , Neoplasias Colorretais/etiologia , Dieta , Meio Ambiente , Humanos , Recém-Nascido , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Transdução de Sinais/fisiologia , Simbiose/fisiologiaRESUMO
We have demonstrated that 0.45% quercetin added to a diet containing corn oil (15% w/w), as the lipid source, and cellulose (6% w/w), as the fiber source, was able to suppress the formation of high multiplicity aberrant crypt foci (ACF > 4 AC/focus), to lower proliferation and enhance apoptosis in a rat model of colon cancer. This experiment determined whether quercetin was acting as an antiinflammatory molecule in an in vivo model of colon cancer. We used weanling (21 d old) Sprague Dawley rats (n = 40) in a 2×2 factorial experiment to determine the influence of quercetin on iNOS, COX-1 and COX-2 expressions, all of which are elevated in colon cancer. Half of the rats received a diet containing either 0 or 0.45% quercetin, and within each diet group, half of the rats were injected with saline or azoxymethane (AOM, 15 mg/kg BW, sc, 2× during wk 3 and 4). The colon was resected 4 wk after the last AOM injection, and the mucosa scraped and processed for RNA isolation. Data from this experiment were analyzed using a mixed model in SAS for main effects and their interaction. AOM injection stimulated (P < 0.0001) iNOS expression. However there was an interaction such that, relative to rats injected with saline, AOM-injected rats consuming diets without quercetin had significantly elevated iNOS expression (5.29-fold), but the expression in AOM-injected rats consuming the diet with quercetin was not significantly elevated (1.68-fold). COX-1 expression was 20.2% lower (P < 0.06) in rats consuming diets containing quercetin. COX-2 expression was 24.3% higher (P < 0.058) in rats consuming diets without quercetin. These data suggest inflammatory processes are elevated in this early stage of colon carcinogenesis, yet quercetin may protect against colon carcinogenesis by down-regulating the expressions of COX-1 and COX-2.
RESUMO
We have shown that dietary fish oil and pectin (FP) protects against radiation-enhanced colon cancer by upregulating apoptosis in colonic mucosa. To investigate the mechanism of action, we provided rats (n = 40) with diets containing the combination of FP or corn oil and cellulose (CC) prior to exposure to 1 Gy, 1 GeV/nucleon Fe-ion. All rats were injected with a colon-specific carcinogen, azoxymethane (AOM; 15 mg/kg), 10 and 17 days after irradiation. Levels of colonocyte apoptosis, prostaglandin E(2) (PGE(2)), PGE(3), microsomal prostaglandin E synthase-2 (mPGES-2), total beta-catenin, nuclear beta-catenin staining (%) and peroxisome proliferator-activated receptor delta (PPARdelta) expression were quantified 31 weeks after the last AOM injection. FP induced a higher (P < 0.01) apoptotic index in both treatment groups, which was associated with suppression (P < 0.05) of antiapoptotic mediators in the cyclooxygenase (COX) pathway (mPGES-2 and PGE(2)) and the Wnt/beta-catenin pathway [total beta-catenin and nuclear beta-catenin staining (%); P < 0.01] compared with the CC diet. Downregulation of COX and Wnt/beta-catenin pathways was associated with a concurrent suppression (P < 0.05) of PPARdelta levels in FP-fed rats. In addition, colonic mucosa from FP animals contained (P < 0.05) a proapoptotic, eicosapentaenoic acid-derived COX metabolite, PGE(3). These results indicate that FP enhances colonocyte apoptosis in AOM-alone and irradiated AOM rats, in part through the suppression of PPARdelta and PGE(2) and elevation of PGE(3). These data suggest that the dietary FP combination may be used as a possible countermeasure to colon carcinogenesis, as apoptosis is enhanced even when colonocytes are exposed to radiation and/or an alkylating agent.
Assuntos
Alprostadil/análogos & derivados , Apoptose/efeitos dos fármacos , Colo/fisiologia , Neoplasias do Colo/prevenção & controle , Dinoprostona/antagonistas & inibidores , Óleos de Peixe/farmacologia , Mucosa Intestinal/fisiologia , PPAR delta/antagonistas & inibidores , Pectinas/farmacologia , Alprostadil/metabolismo , Animais , Colo/citologia , Colo/efeitos dos fármacos , Colo/efeitos da radiação , Gorduras na Dieta , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Masculino , Neoplasias Induzidas por Radiação/prevenção & controle , Ratos , Ratos Sprague-DawleyRESUMO
Our laboratory has demonstrated that down-regulation of proliferation and cytokine synthesis by CD4(+) T cells in mice fed diets rich in n-3 polyunsaturated fatty acids (PUFA) is highly dependent on the involvement of the co-stimulatory molecule, CD28. It has been reported that the inhibitory cytokine interleukin (IL)-10 acts directly on T cells which up-regulate IL-10 receptor (IL-10R) expression following stimulation via CD28 by efficiently blocking proliferation and cytokine production. Thus, it was hypothesized that dietary n-3 PUFA would suppress T cell function through the effects of IL-10. The proliferation of purified splenic CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 (alphaCD3/CD28) from conventional mice (C57BL/6) fed either a control corn oil (CO)-enriched diet devoid of n-3 PUFA, docosahexaenoic acid (DHA; 22 : 6) or eicosapentaenoic acid (EPA; 20 : 5) for 14 days was suppressed by dietary DHA and EPA. Surprisingly, a similar trend was seen in IL-10 gene knock-out (IL-10(-/-)) mice fed dietary n-3 PUFA. IL-10R cell surface expression was also significantly down-regulated on CD4(+) T cells from both the C57BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with alphaCD3/CD28. Enzyme-linked immunosorbent assay (ELISA) measurements revealed that C57BL/6 mice fed DHA had significantly reduced interferon (IFN)-gamma and IL-10 levels 48 h post-activation. However, CD4(+) T cells from IL-10(-/-) mice fed dietary n-3 PUFA produced significantly greater levels of IFN-gamma than the CO-fed group. Our data suggest that in the absence of IL-10, CD4(+) T cells from n-3 PUFA-fed mice may up-regulate IFN-gamma. Suppressed CD4(+) T cells from n-3 PUFA-fed C57BL/6 mice may use mechanisms other than IL-10 to down-regulate T cell function.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Ômega-3/administração & dosagem , Interleucina-10/imunologia , Animais , Antígenos CD28/imunologia , Proliferação de Células , Citometria de Fluxo , Interferon gama/análise , Interferon gama/imunologia , Interleucina-10/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Diets enriched in n-3 polyunsaturated fatty acids (PUFA) suppress several functions of murine splenic T cells by acting directly on the T cells and/or indirectly on accessory cells. In this study, the relative contribution of highly purified populations of the two cell types to the dietary suppression of T cell function was examined. Mice were fed diets containing different levels of n-3 PUFA; safflower oil (SAF; control containing no n-3 PUFA), fish oil (FO) at 2% and 4%, or 1% purified docosahexaenoic acid (DHA) for 2 weeks. Purified (>90%) T cells were obtained from the spleen, and accessory cells (>95% adherent, esterase-positive) were obtained by peritoneal lavage. Purified T cells or accessory cells from each diet group were co-cultured with the alternative cell type from every other diet group, yielding a total of 16 different co-culture combinations. The T cells were stimulated with either concanavalin A (ConA) or antibodies to the T cell receptor (TcR)/CD3 complex and the costimulatory molecule CD28 (alphaCD3/alphaCD28), and proliferation was measured after four days. Suppression of T cell proliferation in the co-cultures was dependent upon the dose of dietary n-3 PUFA fed to mice from which the T cells were derived, irrespective of the dietary treatment of accessory cell donors. The greatest dietary effect was seen in mice consuming the DHA diet (P = 0.034 in the anova; P=0.0053 in the Trend Test), and was observed with direct stimulation of the T cell receptor and CD28 costimulatory ligand, but not with ConA. A significant dietary effect was also contributed accessory cells (P = 0.033 in the Trend Test). We conclude that dietary n-3 PUFA affect TcR-mediated by T cell activation by both direct and indirect (accessory cell) mechanisms.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD28/imunologia , Divisão Celular/efeitos dos fármacos , Técnicas de Cocultura , Concanavalina A/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Feminino , Óleos de Peixe/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Óleo de Cártamo/farmacologia , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologiaRESUMO
There is now general agreement that the etiology of proximal and distal colon cancers may differ, thus prompting renewed interest in understanding anatomical site-specific molecular mechanisms of tumor development. Using a 2x2x2 factorial design with male Sprague-Dawley rats (corn oil, fish oil; pectin, cellulose; plus or minus azoxymethane injection) we found a greater than 2-fold difference (P < 0.001) in tumor incidence proximally versus distally (prox/dist ratio: corn oil, 2.25; fish oil, 2.61). The purpose of the present study was to determine if the higher degree of proximal versus distal tumors in our model system could be accounted for by differences between these two sites in initial DNA damage, response to that damage or an effect of diet at one site but not the other. DNA damage was assessed by quantitative immunohistochemistry of O(6)-methylguanine adducts; repair by measurement of O(6)-methylguanine-DNA alkyltransferase and removal was determined by measurement of targeted apoptosis. Although overall initial DNA damage was similar at both sites, in the distal colon there was a greater expression of repair protein (P < 0.001) and a greater degree of targeted apoptosis (P < 0.0001). There was also a reduction in DNA damage in the distal colon of rats consuming fish oil. Together, these results suggest that the lower tumor incidence in the distal colon may be a result of the capacity to deal with initial DNA damage by the distal colon, as compared with the proximal colon. Therefore, the determination of site-specific mechanisms in tumor development is important because distinct strategies may be required to protect against cancer at different sites.
Assuntos
Adenocarcinoma/patologia , Azoximetano/farmacologia , Carcinógenos/farmacologia , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/enzimologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/enzimologia , Adutos de DNA , Reparo do DNA/efeitos dos fármacos , Técnicas Imunoenzimáticas , Metabolismo dos Lipídeos , Masculino , Metilação , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Studies in humans and murine disease models have clearly shown dietary fish oil to possess anti-inflammatory properties, apparently mediated by the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To determine the mechanisms by which dietary EPA and DHA modulate mouse T-cell activation, female C57BL/6 mice were fed diets containing either 2% safflower oil (SAF), 2% fish oil (FO), or a 2% purified EPA/DHA ethyl ester mixture for 14 days. Splenic CD4 T cells ( approximately 90% purity) or CD8 T cells ( approximately 85% purity) were incubated with agonists which act at the plasma membrane receptor level [anti(alpha)-CD3/anti(alpha)-CD28], the intracellular level (PMA/Ionomycin), or at both the receptor and intracellular levels (alphaCD3/PMA). CD4 T cells stimulated with alphaCD3/alphaCD28 or PMA/Ionomycin proliferated and produced principally IL-2 (i.e. a Th1 phenotype), whereas the proliferation of CD4 T cells stimulated with alphaCD3/PMA was apparently driven principally by IL-4 (i.e. a Th2 phenotype). The IL-4 driven proliferation of putative Th2 CD4 cells was enhanced by dietary n-3 fatty acids (P = 0.02). Conversely, IL-2 production by alphaCD3/alpha CD28-stimulated CD4 T cells was reduced in FO-fed animals (P < 0.0001). The alphaCD3/alphaCD28-stimulated CD8 cells cultured from FO-fed animals exhibited a significant decrease (P < 0.05) in proliferation. There were no dietary effects seen in alphaCD3/PMA-stimulated CD8 cells, which produced both IL-2 and IL-4, or in PMA/Ionomycin-stimulated CD8 cells, which produced principally IL-2. These data suggest that dietary n-3 fatty acids down-regulated IL-2 driven CD4 and CD8 activation, while up-regulating the activation of the Th2 CD4 T-cell subset. Thus, the anti-inflammatory effects of n-3 fatty acids may result in both the direct suppression of IL-2-induced Th1 cell activation and the indirect suppression of Th1 cells by the enhanced cross-regulatory function of Th2 cells.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD28 , Complexo CD3 , Feminino , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Ionomicina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/agonistas , Baço/citologia , Baço/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The present study was conducted to evaluate the antiatherogenic effects of dietary gamma-linolenic acid (GLA) (primrose oil) in apolipoprotein E (apoE) genetic knockout mice. Five-wk-old male mice were fed cholesterol-free diets containing 10 g/100 g lipid as corn oil (CO) [control diet, 0 mol/100 mol GLA and (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO, 10 mol/100 mol GLA), fish oil-CO mix [FC; 9:1 wt/wt, 0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish oil-PO mix [FP, 1:3 wt/wt, 8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA] for 15 wk. Subsequently, diets were supplemented with cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) and fed for an additional 10 and 16 wk. Plasma cholesterol and triglyceride levels generally did not differ among groups at 20, 30 and 36 wk of age. Mice fed GLA-containing diets (PO and FP) had significantly (P < 0.05) higher liver phospholipid levels of dihomo-gamma-linolenic acid, the elongated product of GLA, relative to CO and FC groups. Consumption of GLA (PO and FP diets) significantly reduced (P < 0.05) aortic vessel wall medial layer thickness at 20 and 30 wk. A parallel GLA-dependent suppression in the number of proliferating (proliferating cell nuclear antigen positive) aortic smooth muscle cells was also observed. Diets containing either GLA or (n-3) PUFA reduced (P < 0.05) atherosclerotic lesion size in 30-wk-old mice. These results indicate that dietary GLA can suppress smooth muscle cell proliferation in vivo and retard the development of diet-induced atherosclerosis in apoE knockout mice.
Assuntos
Apolipoproteínas E/deficiência , Arteriosclerose/tratamento farmacológico , Gorduras Insaturadas na Dieta/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Ácido gama-Linolênico/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Arteriosclerose/sangue , Arteriosclerose/patologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/sangue , Gorduras Insaturadas na Dieta/uso terapêutico , Ácidos Graxos/análise , Ácidos Graxos Essenciais/farmacologia , Ácidos Linoleicos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Oenothera biennis , Fosfolipídeos/metabolismo , Óleos de Plantas , Triglicerídeos/sangue , Ácido gama-Linolênico/uso terapêuticoRESUMO
(n-3) polyunsaturated fatty acids (PUFA) have been widely documented to reduce inflammation in diseases such as rheumatoid arthritis. This study sought to elucidate the mechanism whereby (n-3) PUFA downregulate T-cell proliferation. We hypothesized that membrane incorporation of dietary PUFA would alter membrane structure and consequently membrane receptor function. Female C57BL/6 mice were fed for 14 d one of three diets containing arachidonic acid (AA), fish oil or docosahexaenoic acid (DHA) that varied in lipid composition only. Spleens were harvested and T cells ( approximately 90% purity) were activated with agonists that stimulated proliferation at the receptor level [anti-CD3 (alphaCD3)/anti-CD28 (alphaCD28)], intracellularly [phorbol-12-myristate-13-acetate (PMA)/ionomycin] or with a combined receptor/intracellular agonist (alphaCD3/PMA). Although there was no significant difference (P > 0.05) in proliferative response across dietary groups within each agonist set, interleukin (IL)-2 secretion was significantly reduced (P = 0.05) in cells from DHA-fed mice stimulated with alphaCD3/alphaCD28. In parallel in vitro experiments, Jurkat T cells were incubated with 50 micromol/L linoleic acid, AA, or DHA. Similar agonists sets were employed, and cells incubated with DHA and AA had a significantly reduced (P < 0.05) IL-2 secretion in three of the agonist sets. However, only when the CD28 receptor was stimulated was there a significant difference (P < 0.05) between DHA and AA. The results of this study suggest the involvement of the CD28 receptor in reducing IL-2 secretion in DHA-fed mice and DHA-incubated Jurkat cells and that purified T cells from DHA-fed mice require accessory cells to modulate proliferative suppression.
Assuntos
Antígenos CD28/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Linfócitos T/citologia , Animais , Ácido Araquidônico/farmacologia , Antígenos CD28/efeitos dos fármacos , Divisão Celular , Dieta , Gorduras na Dieta/farmacologia , Ácidos Graxos/farmacologia , Ácidos Graxos Ômega-3/metabolismo , Feminino , Óleos de Peixe/farmacologia , Humanos , Interleucina-2/biossíntese , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/metabolismoRESUMO
Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.
Assuntos
Alquil e Aril Transferases/metabolismo , Transformação Celular Neoplásica , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Insaturados/farmacologia , Genes ras/efeitos dos fármacos , Ácido Linoleico/farmacologia , Proteína Oncogênica p21(ras)/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colo , Óleo de Milho , Ativação Enzimática , Ácidos Graxos Ômega-6 , Óleos de Peixe , Lipídeos de Membrana/metabolismo , Camundongos , Proteína Oncogênica p21(ras)/genética , Ácido Palmítico/metabolismo , Fosfolipídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
A short-term feeding paradigm in mice, with diets enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was used to study the modulation of T cell activation via the T cell receptor (TcR) and the downstream pathways of intracellular signaling. Diets enriched in EPA and DHA suppressed antigen-specific delayed hypersensitivity reactions and mitogen-induced proliferation of T cells. Cocultures of accessory cells and T cells from mice given different diets revealed that purified fatty acid ethyl esters acted directly on the T cell, rather than through the accessory cell. The loss of proliferative capacity was accompanied by reductions in interleukin (IL)-2 secretion and IL-2 receptor alpha chain mRNA transcription, suggesting that dietary EPA and DHA act, in part, by interrupting the autocrine IL-2 activation pathway. Dietary EPA and DHA blunted the production of intracellular second messengers, including diacylglycerol and ceramide, following mitogen stimulation in vitro. Dietary effects appear to vary with the agonist employed (i.e., anti-CD3 [TcR], anti-CD28, exogenous IL-2, or phorbol myristate acetate and ionomycin).
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Receptores de Interleucina-2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
There is epidemiological, clinical, and experimental evidence that dietary fish oil, containing n-3 polyunsaturated fatty acids, protects against colon tumor development. However, its effects on colonocytes in vivo remain poorly understood. Therefore, we investigated the ability of fish oil to modulate colonic methylation-induced DNA damage, repair, and deletion. Sprague Dawley rats were provided with complete diets containing either corn oil or fish oil (15% by weight). Animals were injected with azoxymethane, and the distal colon was removed 3, 6, 9, or 12 h later. Targeted apoptosis and DNA damage were assessed by cell position within the crypt using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay and quantitative immunohistochemical analysis of O6-methylguanine adducts, respectively. Localization and expression of the alkyl group acceptor, O6-methylguanine-DNA-methyltransferase, was also determined. Lower levels of adducts were detected at 6, 9, and 12 h in fish oil- versus corn oil-fed animals (P < 0.05). In addition, fish oil supplementation had the greatest effect on apoptosis in the top one-third of the crypt, increasing the apoptotic index compared with corn oil-fed rats (P < 0.05). In the top one-third of the crypt, fish oil feeding caused an incremental stimulation of apoptosis as adduct level increased. In contrast, a negative correlation between apoptosis and adduct incidence occurred with corn oil feeding (P < 0.05). Diet had no main effect (all tertiles combined) on O6-methylguanine-DNA-methyltransferase expression over the time frame of the experiment. The enhancement of targeted apoptosis combined with the reduced formation of O6-methylguanine adducts may account, in part, for the observed protective effect of n-3 polyunsaturated fatty acids against experimentally induced colon cancer.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias do Colo/prevenção & controle , Adutos de DNA/efeitos dos fármacos , Óleos de Peixe/farmacologia , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Adutos de DNA/biossíntese , Adutos de DNA/química , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Funções Verossimilhança , Masculino , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , tRNA Metiltransferases/biossínteseRESUMO
We have recently demonstrated that overexpression of PKC beta(II) renders transgenic mice more susceptible to carcinogen-induced colonic hyperproliferation and aberrant crypt foci formation. In order to further investigate the ability of PKC beta(II) to modulate colonocyte cytokinetics, we determined the localization of PKC beta(II) with respect to cell proliferation and apoptosis along the entire colonic crypt axis following carcinogen and diet manipulation. Rats were provided diets containing either corn oil [containing n-6 polyunsaturated fatty acids (PUFA)] or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (fermentable fiber) and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic sections were utilized for immunohistochemical image analysis and immunoblotting. Cell proliferation was measured by incorporation of bromodeoxyuridine into DNA and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. In the distal colon, PKC beta(II) staining was localized to the upper portion of the crypt. In comparison, proximal crypts had more (P < 0.05) staining in the lower tertile. AOM enhanced (P < 0.05) PKC beta(II) expression in all regions of the distal colonic crypt (upper, middle and lower tertiles). There was also an interaction (P < 0.05) between dietary fat and fiber on PKC beta(II) expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the distal colonic crypt. With respect to colonic cell kinetics, proliferation paralleled the increase in PKC beta(II) expression in carcinogen-treated animals. In contrast, apoptosis at the lumenal surface was inversely proportional to PKC beta(II) expression in the upper tertile. These results suggest that an elevation in PKC beta(II) expression along the crypt axis in the distal colon is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development. Therefore, select dietary factors may confer protection against colon carcinogenesis in part by blocking carcinogen-induced PKC beta(II) expression.
Assuntos
Apoptose/fisiologia , Colo/citologia , Colo/enzimologia , Dieta , Isoenzimas/biossíntese , Proteína Quinase C/biossíntese , Animais , Apoptose/efeitos dos fármacos , Azoximetano , Carcinógenos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Celulose/farmacologia , Colo/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Óleo de Milho/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Imuno-Histoquímica , Masculino , Pectinas/farmacologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Proteína Quinase C beta , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/enzimologiaRESUMO
Colon cancer evolves from a progressive inhibition of apoptosis and is influenced strongly by diet. Among dietary factors, butyrate (derived from fermentable fibers) may have utility as a chemopreventive agent because of its ability to promote apoptosis. Because CD95 (APO-1/Fas) transduces signals resulting in apoptosis, we tested the hypothesis that butyrate-dependent colonocyte apoptosis is mediated by this death receptor. Treatment of immortalized mouse colon cells with Fas agonistic antibody induced cell death, indicating that Fas in colonocytes is functional. Antagonism of Fas signaling using a soluble Fas:Fc chimera blocked butyrate induction of apoptosis. Therefore, Fas receptor dependent signal transduction is required for butyrate induction of apoptosis in colonic cells.
Assuntos
Apoptose/efeitos dos fármacos , Butiratos/uso terapêutico , Neoplasias do Colo/prevenção & controle , Dieta , Animais , Interações Medicamentosas , Camundongos , Biologia Molecular , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
DNA alkylating agent exposure results in the formation of a number of DNA adducts, with O6-methyl-deoxyguanosine (O6-medG) being the major mutagenic and cytotoxic DNA lesion. Critical to the prevention of colon cancer is the removal of O6-medG DNA adducts, either through repair, for example, by O6-alkylguanine-DNA alkyltransferase (ATase) or targeted apoptosis. We report how rat colonocytes respond to administration of azoxymethane (a well-characterized experimental colon carcinogen and DNA-methylating agent) in terms of O6-medG DNA adduct formation and adduct removal by ATase and apoptosis. Our results are: (a) DNA damage is greater in actively proliferating cells than in the differentiated cell compartment; (b) expression of the DNA repair enzyme ATase was not targeted to the proliferating cells or stem cells but rather is confined primarily to the upper portion of the crypt; (c) apoptosis is primarily targeted to the stem cell and proliferative compartments; and (d) the increase in DNA repair enzyme expression over time in the bottom one-third of the crypt corresponds with the decrease in apoptosis in this same crypt region.
Assuntos
Apoptose/efeitos dos fármacos , Azoximetano/farmacologia , Carcinógenos/farmacologia , Metilação de DNA , Reparo do DNA/efeitos dos fármacos , Alquil e Aril Transferases/metabolismo , Alquilação , Animais , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , Colo/citologia , DNA/análise , DNA/metabolismo , Dano ao DNA , Masculino , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologiaRESUMO
There is great interest in utilizing butyrate as a chemopreventive agent for colon tumorigenesis because of its ability to promote apoptosis in colon tumor cell lines. Because CD95 (APO-1/Fas) transduces signals resulting in apoptosis, we tested the hypothesis that butyrate-dependent colonocyte apoptosis is mediated by this death receptor. Butyrate (1 mM) exposure for 24 h upregulated expression of Fas and its ligand in young adult mouse colon (YAMC) cells. To delineate the proapoptotic effect of butyrate and to avoid the confounding effects of detachment from the extracellular matrix, adherent cell apoptosis was monitored as loss of plasma membrane asymmetry and dissipation of mitochondrial membrane potential (DeltaPsi(mt)) by laser cytometry. Soluble Fas receptor protein (Fas:Fc chimera) and caspase inhibitors (z-VAD-fmk and z-IETD-fmk) blocked butyrate induction of apoptosis. Treatment with Fas agonistic antibody (clone Jo-2) significantly induced cell death, indicating that Fas in colonocytes is functional. In addition, butyrate promoted apoptosis by inducing loss of DeltaPsi(mt) and phospholipid asymmetry of the plasma membrane after 12 and 24 h of exposure, respectively, before cell detachment. Therefore, Fas receptor-dependent signal transduction is involved in butyrate induction of apoptosis in colonocytes.
Assuntos
Apoptose/fisiologia , Butiratos/farmacologia , Colo/fisiologia , Transdução de Sinais/fisiologia , Receptor fas/fisiologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Adesão Celular/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Colo/citologia , Inibidores Enzimáticos/farmacologia , Camundongos , Mitocôndrias/fisiologia , Transdução de Sinais/efeitos dos fármacos , Receptor fas/imunologiaRESUMO
Epidemiological and experimental data suggest that dietary fiber and fat are major determinants of colorectal cancer. However, the mechanisms by which these dietary constituents alter the incidence of colon cancer have not been elucidated. Evidence indicates that dominant gain-of-function mutations short-circuit protooncogenes and contribute to the pathogenesis of cancer. Therefore, we began to dissect the mechanisms whereby dietary fat and fiber, fed during the initiation, promotion and progression stages of colon tumorigenesis, regulate ras p21 localization, expression and mutation frequency. Male Sprague-Dawley rats (140) were provided with corn oil or fish oil and pectin or cellulose plus or minus the carcinogen azoxymethane (AOM) in a 2 x 2 x 2 factorial design and killed after 34 weeks. We have previously shown adenocarcinoma incidence in these animals to be 70.3% (52/74) for corn oil + AOM and 56.1% (37/66) for fish oil + AOM (P < 0.05). Total ras expression as well as ras membrane:cytosol ratio was 4- to 6-fold higher in colon tumors than in mucosa from AOM- or saline-injected rats. Expression of ras in the mucosal membrane fraction was 13% higher for animals fed corn oil compared with fish oil feeding (P < 0.05), which is noteworthy since ras must be localized at the plasma membrane to function. The elevated ras membrane:cytosol ratio in tumors was not due to increased farnesyl protein transferase activity or prenylation state, as nearly all detectable ras was in the prenylated form. Phosphorylated p42 and p44 mitogen activated protein kinase (ERK) expression was two-fold higher in tumor extracts compared with uninvolved mucosa from AOM- and saline-injected rats (P < 0.05). The frequency of K-ras mutations was not significantly different between the various groups, but there was a trend toward a greater incidence of mutations in tumors from corn oil fed rats (85%) compared with fish oil fed rats (58%). Our results indicate that the carcinogen-induced changes in ras expression and membrane localization are associated with the in vivo activation of the ERK pathway. In addition, suppression of tumor development by dietary n-3 polyunsaturated fatty acids may be partly due to a combined effect on colonic ras expression, membrane localization, and mutation frequency.