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BACKGROUND AND AIMS: Human studies suggest that a high intake of polyunsaturated fatty acid (PUFA) is associated with an increased risk of inflammatory bowel disease (IBD). PUFA is highly prone to oxidation. To date, it is unclear whether unoxidized or oxidized PUFA is involved in the development of IBD. Here, we aim to compare the effects of unoxidized PUFA vs. oxidized PUFA on the development of IBD and associated colorectal cancer. METHODS: We evaluated the effects of unoxidized and oxidized PUFA on dextran sodium sulfate (DSS)- and IL-10 knockout-induced colitis, and azoxymethane (AOM)/DSS-induced colon tumorigenesis in mice. Additionally, we studied the roles of gut microbiota and Toll-like receptor 4 (TLR4) signaling involved. RESULTS: Administration of a diet containing oxidized PUFA, at human consumption-relevant levels, increases the severity of colitis and exacerbates the development of colitis-associated colon tumorigenesis in mice. Conversely, a diet rich in unoxidized PUFA doesn't promote colitis. Furthermore, oxidized PUFA worsens colitis-associated intestinal barrier dysfunction and leads to increased bacterial translocation, and it fails to promote colitis in Toll-like receptor 4 (TLR4) knockout mice. Finally, oxidized PUFA alters the diversity and composition of gut microbiota, and it fails to promote colitis in mice lacking the microbiota. CONCLUSIONS: These results support that oxidized PUFA promotes the development of colitis and associated tumorigenesis in mouse models via TLR4- and gut microbiota-dependent mechanisms. Our findings highlight the potential need to update regulation policies and industrial standards for oxidized PUFA levels in food.
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BACKGROUND & AIMS: Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-kB signaling. METHODS: Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis were initiated in whole-body NIK knockout mice (Nik-/-) and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium. RESULTS: Human transcriptomic data revealed dysregulated noncanonical NF-kB signaling. In vitro studies evaluating Nik-/- crypts and organoids derived from mature, nondividing CECs, and colonic stem cells exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik-/- mice exhibited more severe colitis with dextran sulfate sodium administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC). CONCLUSIONS: Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue-dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.
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Neoplasias Colorrectales , Células Epiteliales , Ratones Noqueados , FN-kappa B , Quinasa de Factor Nuclear kappa B , Proteínas Serina-Treonina Quinasas , Regeneración , Transducción de Señal , Animales , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , FN-kappa B/metabolismo , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Colitis/patología , Colitis/inducido químicamente , Sulfato de Dextran/toxicidad , Colon/patología , Mucosa Intestinal/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Proliferación Celular , Azoximetano/toxicidad , Organoides/metabolismo , Diferenciación Celular , Modelos Animales de EnfermedadRESUMEN
Aim: To elucidate dynamics and functions in colonic macrophage subsets, and their regulation by Bifidobacterium breve (B. breve) and its associated metabolites in the initiation of colitis-associated colorectal cancer (CAC). Methods: Azoxymethane (AOM) and dextran sodium sulfate (DSS) were used to create a CAC model. The tumor-suppressive effect of B. breve and variations of macrophage subsets were evaluated. Intestinal macrophages were ablated to determine their role in the protective effects of B. breve. Efficacious molecules produced by B. breve were identified by non-targeted and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The molecular mechanism was further verified in murine bone marrow-derived macrophages (BMDMs), macrophages derived from human peripheral blood mononuclear cells (hPBMCs), and demonstrated in CAC mice. Results: B. breve alleviated colitis symptoms, delayed colonic tumorigenesis, and promoted phenotypic differentiation of immature inflammatory macrophages into mature homeostatic macrophages. On the contrary, the ablation of intestinal macrophages largely annulled the protective effects of B. breve. Microbial analysis of colonic contents revealed the enrichment of probiotics and the depletion of potential pathogens following B. breve supplementation. Moreover, indole-3-lactic acid (ILA) was positively correlated with B. breve in CAC mice and highly enriched in the culture supernatant of B. breve. Also, the addition of ILA directly promoted AKT phosphorylation and restricted the pro-inflammatory response of murine BMDMs and macrophages derived from hPBMCs in vitro. The effects of ILA in murine BMDMs and macrophages derived from hPBMCs were abolished by the aryl hydrocarbon receptor (AhR) antagonist CH-223191 or the AKT inhibitor MK-2206. Furthermore, ILA could protect against tumorigenesis by regulating macrophage differentiation in CAC mice; the AhR antagonist largely abrogated the effects of B. breve and ILA in relieving colitis and tumorigenesis. Conclusion: B. breve-mediated tryptophan metabolism ameliorates the precancerous inflammatory intestinal milieu to inhibit tumorigenesis by directing the differentiation of immature colonic macrophages.
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Bifidobacterium breve , Diferenciación Celular , Colitis , Indoles , Macrófagos , Probióticos , Animales , Ratones , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Bifidobacterium breve/metabolismo , Indoles/farmacología , Indoles/metabolismo , Humanos , Colitis/inducido químicamente , Colitis/microbiología , Colitis/complicaciones , Diferenciación Celular/efectos de los fármacos , Probióticos/farmacología , Probióticos/administración & dosificación , Modelos Animales de Enfermedad , Carcinogénesis/efectos de los fármacos , Neoplasias Asociadas a Colitis/patología , Neoplasias Asociadas a Colitis/microbiología , Neoplasias Asociadas a Colitis/metabolismo , Ratones Endogámicos C57BL , Colon/microbiología , Colon/patología , Colon/metabolismo , Sulfato de Dextran , Masculino , Microbioma Gastrointestinal , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/microbiología , AzoximetanoRESUMEN
Chronic inflammation is a risk factor for colorectal cancer, and inflammatory cytokines secreted from inflammatory cells and active oxygen facilitate tumorigenesis. Intestinal bacteria are thought to regulate tumorigenesis. The longer the breastfeeding period, the lower is the risk of inflammatory bowel disease. Here, we investigated preventive effects of the probiotic Lactobacillus rhamnosus M9 (Probio-M9) on colitis-associated tumorigenesis. An inflammatory colorectal tumor model was established using a 6-week-old male C57BL/6NCrSlc mouse, which was intraperitoneally administered with azoxymethane (AOM: 12 mg/kg body weight). On weeks 2 and 4, 2% dextran sulfate sodium (DSS) was administered to mice for 7 days through drinking water. On weeks 8 and 10, Probio-M9 (2 × 109/day) was orally administered for 7 days. Animals were sacrificed at 20 weeks after AOM administration and immunohistochemical staining and Western blotting was performed. The α-diversity of microflora (Shannon index), principal coordinate analysis, and distribution of intestinal bacterium genera and metabolic pathways were compared. The AOM/DSS group showed weight loss, diarrhea, intestinal shortening, increased number of colon tumors, proliferating tumorigenesis, increased inflammation score, fibrosis, increased CD68+, or CD163+ macrophage cells in the subserosal layer of non-tumor areas. Inflammation and tumorigenesis ameliorated after Probio-M9 treatment. Fecal microbial functions were altered by AOM/DSS treatment. Probio-M9 significantly upregulated the fecal microbial diversity and reversed fecal microbial functions. Thus, Probio-M9 could suppress tumor formation in the large intestine by regulating the intestinal environment and ameliorating inflammation, suggesting its therapeutic potential for treatment of inflammation and colitis-associated tumorigenesis.
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Colitis/inducido químicamente , Neoplasias del Colon/etiología , Microbioma Gastrointestinal , Lacticaseibacillus rhamnosus/fisiología , Leche Humana/microbiología , Carcinogénesis , Colitis/complicaciones , Neoplasias del Colon/prevención & control , Sulfato de Dextran/toxicidad , Humanos , Inflamación/prevención & control , Lacticaseibacillus rhamnosus/clasificación , Masculino , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patologíaRESUMEN
Patients with inflammatory bowel disease (IBD) are at a higher risk of developing colitis-associated colorectal cancer. The aim of the present study was to investigate the role of CD73 in IBD-associated tumorigenesis. A mouse model of colitis-associated tumorigenesis (CAT) induced by azoxymethane and dextran sulfate sodium was successfully constructed. Model mice were injected with CD73 inhibitor or adenosine receptor agonist. Colon length, body weight loss and tumor formation were assessed macroscopically. Inflammatory cytokine measurement and RNA sequencing on colon tissues were performed. Inhibition of CD73 by adenosine 5'-(α,ß-methylene) diphosphate (APCP) suppressed the severity of CAT with attenuated weight loss, longer colons, lower tumor number and smaller tumor size compared with the model group. Activation of adenosine receptors using 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-ß-D-ribofuranuronamide (NECA) exacerbated CAT. Histological assessment indicated that inhibition of CD73 reduced, while activation of adenosine receptors exacerbated, the histological damage of the colon. Increased expression of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-6) in colonic tissue was detected in the NECA group. According to RNA sequencing results, potential oncogenes such as arachidonate 15-lipoxygenase (ALOX15), Bcl-2-like protein 15 (Bcl2l15) and N-acetylaspartate synthetase (Nat8l) were downregulated in the APCP group and upregulated in the NECA group compared with the model group. Therefore, inhibition of CD73 attenuated IBD-associated tumorigenesis, while activation of adenosine receptors exacerbated tumorigenesis in a C57BL/6J mouse model. This effect may be associated with the expression of pro-inflammatory cytokines and the regulation of ALOX15, Bcl2l15 and Nat8l.
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The mixed lineage kinase domain-like protein (MLKL) has emerged as a critical mediator of necroptosis, which results in the release of cellular damage-associated molecular patterns (DAMPs). However, its physiological role in regulating inflammation is not fully understood. We herein showed that Mlkl-/- mice were highly susceptible to colitis and colitis-associated tumorigenesis (CAT), which was associated with massive leukocyte infiltration and increased inflammatory responses. Moreover, we used bone marrow transplantation to reveal that MLKL in inflammatory cells is crucial for its role on colitis. Intestinal mucosal tissue and polyps isolated from Mlkl-/- mice exhibited increased ERK activation and elevated expression of genes associated with inflammation and cancer. Mechanistically, enhanced inflammation in Mlkl-/- mice was due to MEK/ERK activation particularly in dendritic cells (DCs). Our results demonstrate the role of MLKL in maintaining intestinal homeostasis and protecting against colitis and tumorigenesis.
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Colitis/enzimología , Neoplasias del Colon/inmunología , Proteínas Quinasas/inmunología , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Colitis/genética , Colitis/inmunología , Colitis/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Citocinas/biosíntesis , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Células Dendríticas/patología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genéticaRESUMEN
Many dietary compounds show promising protective activity against colon cancer by activating nuclear factor-erythroid 2 related factor 2 (Nrf2). Recently, we reported that mitogen-activated protein kinase phosphatase 1 (Mkp-1) exhibits crosstalk with the Nrf2 signaling pathway, protecting against intestinal inflammation. Here, we present evidence that Mkp-1 is required for the chemopreventive action of the Nrf2 activators butylated hydroxyanisole (BHA) and resveratrol (RSV). In an azoxymethane/dextran sulfate sodium model of colitis-associated tumorigenesis, Mkp-1-/- mice exhibited a phenotype similar to Nrf2-/- mice with significantly more tumors than WT mice. Tumors from Mkp-1-/- mice exhibited higher levels of macrophage infiltration than those from WT mice. This was accompanied by increased expression of nitrotyrosine and p53BP1, markers of oxidative stress and DNA damage, respectively. Moreover, dietary suppression of tumorigenesis using BHA (0.5%) or RSV (300â¯ppm) supplementation was achieved in WT but not in Mkp-1-/- mice. In adenomas from WT mice, the expression of Mkp-1 was markedly lower than in adjacent normal tissue, concomitant with the down-regulation of Nrf2 and its target genes. Our data revealed that Mkp-1 is required in the protective role of Nrf2 signaling against colitis-associated tumorigenesis.
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Anticarcinógenos/farmacología , Hidroxianisol Butilado/farmacología , Colitis/complicaciones , Neoplasias del Colon/complicaciones , Neoplasias del Colon/prevención & control , Fosfatasa 1 de Especificidad Dual/metabolismo , Resveratrol/farmacología , Animales , Transformación Celular Neoplásica/patología , Neoplasias del Colon/patología , Fosfatasa 1 de Especificidad Dual/genética , Ratones Endogámicos BALB C , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
Our recent work has provided the first evidence that MDSCs promote chronic colonic inflammation and colitis-associated carcinogenesis. Our findings not only reveal a novel function of MDSCs in connecting inflammation to cancer, but also provide a rationale for developing effective therapeutic strategies to subvert inflammation- and tumor-induced immunosuppression.
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M2 macrophage polarization is implicated in colorectal cancer development. Isoliquiritigenin (ISL), a flavonoid from licorice, has been reported to prevent azoxymethane (AOM) induced colon carcinogenesis in animal models. Here, in a mouse model of colitis-associated tumorigenesis induced by AOM/dextran sodium sulfate (DSS), we investigated the chemopreventive effects of ISL and its mechanisms of action. Mice were treated with AOM/DSS and randomized to receive either vehicle or ISL (3, 15 and 75 mg/kg). Tumor load, histology, immunohistochemistry, and gene and protein expressions were determined. Intragastric administration of ISL for 12 weeks significantly decreased colon cancer incidence, multiplicity and tumor size by 60%, 55.4% and 42.6%, respectively. Moreover, ISL inhibited M2 macrophage polarization. Such changes were accompanied by downregulation of PGE2 and IL-6 signaling. Importantly, depletion of macrophages by clodronate (Clod) or zoledronic acid (ZA) reversed the effects of ISL. In parallel, in vitro studies also demonstrated that ISL limited the M2 polarization of RAW264.7 cells and mouse peritoneal macrophages with concomitant inactivation of PGE2/PPARδ and IL-6/STAT3 signaling. Conversely, exogenous addition of PGE2 or IL-6, or overexpression of constitutively active STAT3 reversed ISL-mediated inhibition of M2 macrophage polarization. In summary, dietary flavonoid ISL effectively inhibits colitis-associated tumorigenesis through hampering M2 macrophage polarization mediated by the interplay between PGE2 and IL-6. Thus, inhibition of M2 macrophage polarization is likely to represent a promising strategy for chemoprevention of colorectal cancer.
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Chalconas/farmacología , Colitis/patología , Neoplasias del Colon/prevención & control , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Inhibidores Enzimáticos/farmacología , Glycyrrhiza/química , Interleucina-6/antagonistas & inhibidores , Interleucina-6/biosíntesis , Macrófagos/efectos de los fármacos , Animales , Western Blotting , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Colitis/complicaciones , Colon/citología , Colon/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción STAT3/genética , TransfecciónRESUMEN
The nuclear hormone receptor peroxisome proliferator-activated receptor δ (PPARδ) is a ligand-dependent transcription factor that is involved in fatty acid metabolism, obesity, wound healing, inflammation, and cancer. Despite decades of research, the role of PPARδ in inflammation and colorectal cancer remains unclear and somewhat controversial. Our recent work presented the first genetic evidence demonstrating that PPARδ is required for chronic colonic inflammation and colitis-associated carcinogenesis. We also found that a PPARδ downstream pathway, namely the COX-2-derived PGE2 signaling, mediated crosstalk between tumor epithelial cells and macrophages to promote chronic inflammation and colitis-associated tumor genesis. In this brief review, we summarize recent studies on the role of PPARδ in inflammatory bowel disease (IBD) and colorectal cancer (CRC) and highlight recent advances in our understanding of how PPARδ and COX-2-drevided PGE2 signaling coordinately promote chronic colonic inflammation and colitis-associate tumorigenesis. Elucidating the role of PPARδ in inflammation and CRC may provide a rationale for development of PPARδ antagonists as new therapeutic agents in treatment of IBD and CRC.