RESUMO
Purpose: The aim of this study was to assess the effects of resistance and aerobic exercise on colorectal cancer (CRC) development in mice induced by azoxymethane (AOM) coupled with colitis. Methods: Forty animals induced with CRC were used, divided into five groups of eight animals each: sedentary; continuous aerobics; continuous anaerobic; aerobic PI; and anaerobic PI. AOM was administered to the animals in two doses of 10 mg/kg each over the course of two weeks, the first dose administered in the third week and the second administered in the fourth. For the colitis, three cycles of dextran sodium sulfate were administered for five days, separated by two weeks of water. The 14th week of the experiment saw the euthanasia, the removal of their colons, and the creation of microscopy slides for histological analysis. Results: Preneoplastic lesions developed in all five groups; there were no significant differences between them. However, in terms of inflammatory symptoms, mucosal ulceration was much more frequently in the exercise groups than in the sedentary group (p = 0.016). The number of polyps overall (p = 0.002), the distal region's polyp development (p = 0.003), and the proximal region's polyp development (p = 0.04) were all statistically different than sedentary group. Conclusions: The study discovered no significant difference in disease activity index scores between groups, but there was a significant difference in the number of polyps and the presence of mucosal ulceration in the colon.
Assuntos
Animais , Camundongos , Azoximetano/administração & dosagem , Neoplasias Colorretais , Exercício Físico , Modelos Animais , Neoplasias Associadas a ColiteRESUMO
Chronic inflammation is a key driver for colitis-associated colorectal cancer. 5-hydroxytryptamine (5-HT), a neurotransmitter, has been reported to promote inflammation in the gastrointestinal tract. However, the mechanism behind this remains unclear. In this study, we found that 5-HT levels, as well as the expression of tryptophan hydroxylase 1 (TPH1), the 5-HT biosynthesis rate-limiting enzyme, were significantly upregulated in colorectal tumor tissues from patients with colorectal cancer, colorectal cancer mouse models, and colorectal cancer cell lines when compared with normal colorectal tissues or epithelial cell lines. Colorectal cancer cell-originated 5-HT enhanced NLRP3 inflammasome activation in THP-1 cells and immortalized bone marrow-derived macrophages (iBMDM) via its ion channel receptor, HTR3A. Mechanistically, HTR3A activation led to Ca2+ influx, followed by CaMKIIα phosphorylation (Thr286) and activation, which then induced NLRP3 phosphorylation at Ser198 (mouse: Ser194) and inflammasome assembling. The NLRP3 inflammasome mediated IL1ß maturation, and release upregulated 5-HT biosynthesis in colorectal cancer cells by inducing TPH1 transcription, revealing a positive feedback loop between 5-HT and NLRP3 signaling. Silencing TPH1 or HTR3A by short hairpin RNA slowed down tumor growth in an established CT26 and iBMDM coimplanted subcutaneous allograft colorectal cancer mouse model, whereas treatment with TPH1 inhibitor 4-chloro-DL-phenylalanine or HTR3A antagonist tropisetron alleviated tumor progression in an azoxymethane/dextran sodium sulfate-induced colorectal cancer mouse model. Addressing the positive feedback loop between 5-HT and NLRP3 signaling could provide potential therapeutic targets for colorectal cancer.
Assuntos
Neoplasias Associadas a Colite/imunologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Azoximetano/administração & dosagem , Linhagem Celular Tumoral , Neoplasias Associadas a Colite/induzido quimicamente , Neoplasias Associadas a Colite/patologia , Sulfato de Dextrana/administração & dosagem , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Triptofano Hidroxilase/deficiência , Triptofano Hidroxilase/metabolismoRESUMO
Dysregulated Wnt/ß-catenin signaling pathway plays a critical role in the pathogenesis of colorectal cancer (CRC). Scutellarin, a flavonoid compound in Scutellaria barbata, has been reported to suppress CRC, with the action mechanism elusive. In this study, Scutellarin was found to inhibit the carcinogenesis of colitis-associated cancer (CAC) in mice caused by azoxymethane/dextran sulfate sodium, with alleviation of pathologic symptoms. Besides, Scutellarin attenuated mouse serum concentrations of TNF-α and IL-6, heightened Bax expression and diminished B-cell lymphoma-2 (Bcl-2) level in CAC tissues of mice, through down-regulating Wnt/ß-catenin signaling cascade. In CRC HT-29 cells, Scutellarin retarded the proliferation and migration, induced apoptosis, with boosted Bax expression and decreased Bcl-2 level, which may be attributed to its repression of Wnt/ß-catenin signals in HT-29 cells. Our findings demonstrate that Scutellarin may ameliorate colitis-associated colorectal cancer by weakening Wnt/ß-catenin signaling cascade.
Assuntos
Apigenina/farmacologia , Carcinogênese/efeitos dos fármacos , Colite Ulcerativa/complicações , Neoplasias Associadas a Colite/tratamento farmacológico , Glucuronatos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apigenina/uso terapêutico , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinogênese/imunologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Glucuronatos/uso terapêutico , Células HT29 , Humanos , Masculino , Camundongos , Via de Sinalização Wnt/imunologiaRESUMO
BACKGROUND: Colon cancer is a malignant condition that affects the lower gastrointestinal tract and has unfavorable prognosis. Its mechanisms range from enhanced production of reactive oxygen species, inflammatory changes in the colon microenvironment and affection of the apoptotic pathways. Due to the high incidence of resistance of colon cancer to the traditional chemotherapeutic agents, a need for finding safe/effective agents that can attenuate the malignant changes had emerged. OBJECTIVE: To investigate the possible immunomodulatory and antitumor effects of topiramate on azoxymethane-induced colon cancer in rats. METHODOLOGY: Fifty male Wistar rats were randomized into five equal groups as follows: Control; azoxymethane-induced colon cancer; azoxymethane + methyl cellulose; azoxymethane + topiramate small dose; and azoxymethane + topiramate large dose. The body weight gain, serum carcinoembryonic antigen (CEA), tissue antioxidant status, proinflammatory cytokines, vascular endothelial growth factor (VEGF), Nrf2/HO-1 content, p-AKT, mTOR, p38 MAP kinase, caspase 9, nerve growth factor beta and beclin-1 were measured. Also, parts of the colon had undergone histopathological and immunohistochemical evaluation. KEY FINDINGS: Topiramate improved the body weight gain, decreased serum CEA, augmented the antioxidant defenses in the colonic tissues with significant amelioration of the inflammatory changes, decline in tissue VEGF and p-AKT/mTOR/MAP kinase signaling and increased Nrf2/HO-1 content in a dose-dependent manner when compared to rats treated with azoxymethane alone. In addition, topiramate, in a dose-dependent manner, significantly enhanced apoptosis and improved the histopathological picture in comparison to animals treated with azoxymethane alone. CONCLUSION: Taking these findings together, topiramate might serve as a new effective adjuvant line of treatment of colon cancer.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Topiramato/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinogênese/induzido quimicamente , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo , Topiramato/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Colorectal cancer (CRC) represents the third most common malignancy and the second leading cause of cancer-related deaths worldwide. Although immunotherapy has taken center stage in mainstream oncology, it has shown limited clinical efficacy in CRC, generating an urgent need for discovery of new biomarkers and potential therapeutic targets. Galectin-1 (Gal-1), an endogenous glycan-binding protein, induces tolerogenic programs and contributes to tumor cell evasion of immune responses. Here, we investigated the relevance of Gal-1 in CRC and explored its modulatory activity within the CD8+ regulatory T cell (Treg) compartment. Mice lacking Gal-1 (Lgals1-/- ) developed a lower number of tumors and showed a decreased frequency of a particular population of CD8+CD122+PD-1+ Tregs in the azoxymethane-dextran sodium sulfate model of colitis-associated CRC. Moreover, silencing of tumor-derived Gal-1 in the syngeneic CT26 CRC model resulted in reduced number and attenuated immunosuppressive capacity of CD8+CD122+PD-1+ Tregs, leading to slower tumor growth. Moreover, stromal Gal-1 also influenced the fitness of CD8+ Tregs, highlighting the contribution of both tumor and stromal-derived Gal-1 to this immunoregulatory effect. Finally, bioinformatic analysis of a colorectal adenocarcinoma from The Cancer Genome Atlas dataset revealed a particular signature characterized by high CD8+ Treg score and elevated Gal-1 expression, which delineates poor prognosis in human CRC. Our findings identify CD8+CD122+PD-1+ Tregs as a target of the immunoregulatory activity of Gal-1, suggesting a potential immunotherapeutic strategy for the treatment of CRC.
Assuntos
Adenocarcinoma/genética , Linfócitos T CD8-Positivos/imunologia , Colite/genética , Neoplasias Colorretais/genética , Galectina 1/genética , Linfócitos T Reguladores/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Animais , Atlas como Assunto , Azoximetano/administração & dosagem , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/imunologia , Colite/mortalidade , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Sulfato de Dextrana/administração & dosagem , Modelos Animais de Doenças , Galectina 1/deficiência , Galectina 1/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Análise de Sobrevida , Linfócitos T Reguladores/patologia , Carga TumoralRESUMO
According to numerous epidemiological studies, aspirin is a non-steroidal anti-inflammatory drug (NSAID) that reduces the occurrence and mortality of colorectal cancer (CRC). However, the underlying mechanisms are not well identified. In an effort to fill these gaps, we administered aspirin on mice one day before induction in an azoxymethane (AOM)/dextran sulfate sodium (DSS) induced CRC model. In this study, we assessed the effects of aspirin on tumorigenesis and tumor cell proliferation. Multi-layer analyses were carried out to identify changes in cytokines, metabolites, level of gene expressions, and proteins associated with tumorigenesis and aspirin treatment. The results showed that aspirin-treated mice developed fewer colon tumors in response to AOM/DSS, and aspirin can actively block cyclooxygenase (COX) metabolism and reduce levels of pro-inflammatory cytokines. In addition, the transcriptomic and proteomic analyses both indicated that aspirin has an inhibitory effect on the Wnt pathway. The in vitro results further indicated that aspirin inhibits WNT6 production, possibly by suppressing its transcription factor NR4A2, which in turn is regulated by prostaglandin E2, thereby ultimately inhibiting the Wnt pathway. These findings improve our understanding of the mechanisms behind aspirin's chemoprevention effect on CRC.
Assuntos
Aspirina/farmacologia , Neoplasias Associadas a Colite/prevenção & controle , Colite/tratamento farmacológico , Dinoprostona/antagonistas & inibidores , Animais , Aspirina/uso terapêutico , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Colite/induzido quimicamente , Colite/patologia , Neoplasias Associadas a Colite/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Dinoprostona/genética , Dinoprostona/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteômica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
BACKGROUND: The nuclear phosphatase mitogen-activate protein kinase phosphatase-1 (MKP-1) is a key negative regulator of the innate immune response through the regulation of the biosynthesis of proinflammatory cytokines. In colorectal cancer (CRC), which is induced mainly by chronic inflammation, Mkp-1 overexpression was found in addition to disturbances in Mkp-1 functions, which may play a role in cancer development in different types of tumors. However, the potential molecular mechanisms by which Mkp-1 influences CRC development is not clear. Here, we performed global gene expression profiling of Mkp-1 KO mice using RNA sequencing (RNA-seq) to explore the role of Mkp-1 in CRC progression using transcriptome analysis. METHODS: Azoxymethane/dextran sodium sulfate (AOM/DSS) mouse models were used to examine the most dramatic molecular and signaling changes that occur during different phases of CRC development in wild-type mice and Mkp-1 KO mice. Comprehensive bioinformatics analyses were used to elucidate the molecular processes regulated by Mkp-1. Differentially expressed genes (DEGs) were identified and functionally analyzed by Gene Ontology (GO), Kyoto Enrichment of Genes and Genomes (KEGG). Then, protein-protein interaction (PPI) network analysis was conducted using the STRING database and Cytoscape software. RESULTS: Persistent DEGs were different in adenoma and carcinoma stage (238 & 251, respectively) and in WT and MKp-1 KO mice (221& 196, respectively). Mkp-1 KO modulated key molecular processes typically activated in cancer, in particular, cell adhesion, ion transport, extracellular matrix organization, response to drug, response to hypoxia, and response to toxic substance. It was obvious that these pathways are closely associated with cancer development and metastasis. From the PPI network analyses, nine hub genes associated with CRC were identified. CONCLUSION: These findings suggest that MKp-1 and its hub genes may play a critical role in cancer development, prognosis, and determining treatment outcomes. We provide clues to build a potential link between Mkp-1 and colitis-associated tumorigenesis and identify areas requiring further investigation.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Associadas a Colite/genética , Colite/complicações , Fosfatase 1 de Especificidade Dupla/metabolismo , Regulação Neoplásica da Expressão Gênica , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Biomarcadores Tumorais/genética , Carcinogênese/genética , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Biologia Computacional , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Prognóstico , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , RNA-Seq , Transdução de Sinais/genéticaRESUMO
Long noncoding RNA growth arrest specific 5 (GAS5) exerts inhibitory effects through the modulation of several target microRNAs (miRs) in cancer. However, its potential roles and underlying relationship during colorectal cancer (CRC) progression are unclear. Therefore, we explored the role of the negative feedback loop formed by the GAS5/miR34a axis and mammalian target of rapamycin/sirtuin 1 (mTOR/SIRT1) pathway on macroautophagy and apoptosis in CRC. Expression of GAS5, miR34a, SIRT1 and mTOR in CRC patients and cell lines was detected by quantitative reverse transcription polymerase chain reaction. Online bioinformatic analysis was used to predict the downstream miRs of GAS5. Luciferase assay and western blotting were performed to demonstrate miR34a as a downstream target gene of GAS5 in CRC cells. The effects of the GAS5/miR34a axis on apoptosis, macroautophagy, and the mTOR/SIRT1 pathway were assessed by flow cytometry, transmission electron microscopy and western blotting, respectively. Our results suggested that GAS5 was downregulated and acted as a molecular sponge of miR34a during CRC progression. miR34a participated in regulating GAS5suppressed CRC cell macroautophagy and induced apoptosis through the mTOR/SIRT1 pathway. GAS5mediated macroautophagy was maintained in an equilibrium state that might have a protective effect on CRC cell apoptosis. The mTOR signaling pathway suppressed GAS5 expression and formed a negative regulation feedback loop with miR34a in CRC cells. Our results suggested that the GAS5/miR34a/SIRT1/mTOR negative regulatory feedback loop mediated CRC cell macroautophagy, and maintained the cells in an autonomous equilibrium state, but not excessive activation state, which functions as a strong antiapoptotic phenotype during human CRC progression.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Macroautofagia/genética , MicroRNAs/genética , RNA Longo não Codificante/metabolismo , Idoso , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Linhagem Celular Tumoral , Colo/imunologia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Retroalimentação Fisiológica , Feminino , Humanos , Macroautofagia/efeitos dos fármacos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , RNA Longo não Codificante/genética , Ratos , Transdução de Sinais/genética , Sirolimo/farmacologia , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The present study, to the best of our knowledge, is the first systematic study of the inhibitory effects of palmitoyl piperidinopiperidine (PPI; Japan Patent no. 5597427), on colon carcinogenesis. PPI exhibited marked growth inhibitory activity in several human colon carcinoma cell lines, with IC50 values of approximately 0.52.2 µM. In silico docking analysis indicated that PPI could bind to the SH2 domain of signal transducer and activator of transcription 3 (STAT3). PPI markedly inhibited the transcriptional activity of the SW837 cell line. Flowcytometric analysis demonstrated that PPI induced an increase in the number of cells in the G1 phase of the cell cycle, and induced subG1 fractions of cells at a higher concentration level of PPI. In the HT29 and SW837 cells, western blot analyses exhibited that in whole cell lysates, PPI induced a marked decrease in the expression levels of pSTAT3, but not in the levels of STAT3 in these cells. PPI also induced a marked decrease in the expression levels of both STAT3 and pSTAT3 in the chromatin fraction. In addition, PPI affected the protein expression levels of cyclin D1, p53, Bcl2, BclxL and vascular endothelial growth factor (VEGF). In the HT29 cells, PPI induced a marked and dosedependent increase in the expression levels of Bax, cleaved caspase3, cleaved caspase7, cleaved caspase8, cleaved caspase9 and cleaved poly (ADPribose) polymerase (PARP). In animal model systems, PPI inhibited the growth of implanted carcinoma cells, and also induced a significant decrease in the multiplicity of colonic aberrant crypt foci. In addition, a marked and dosedependent inhibition of angiogenesis of the chick chorioallantoic membrane was observed. As regards the possible molecular mechanisms, it is suggested that the inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis and cell cycle progression, eventually contributing to the PPIinduced growth inhibitory effects.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Ácidos Graxos Monoinsaturados/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Carcinoma/patologia , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide , Neoplasias do Colo/patologia , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/uso terapêutico , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Simulação de Acoplamento Molecular , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Neovascularização Patológica/dietoterapia , Neovascularização Patológica/patologia , Ratos , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND & AIMS: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/ß-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/ß-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms. METHODS: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and ß-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo. RESULTS: TG ameliorated DSS colitis in wild-type but not Atg7ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7ΔIEC mice. This was associated with decreased ß-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1. CONCLUSIONS: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.
Assuntos
Neoplasias Associadas a Colite/prevenção & controle , Colite/tratamento farmacológico , Tioguanina/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Administração Retal , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Células CACO-2 , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mercaptopurina/farmacologia , Mercaptopurina/uso terapêutico , Camundongos , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Tioguanina/uso terapêutico , beta Catenina/análise , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) modification has recently emerged as a new regulatory mechanism in cancer progression. We aimed to explore the role of the m6A regulatory enzyme METTL3 in colorectal cancer (CRC) pathogenesis and its potential as a therapeutic target. METHODS: The expression and clinical implication of METTL3 were investigated in multiple human CRC cohorts. The underlying mechanisms of METTL3 in CRC were investigated by integrative m6A sequencing, RNA sequencing, and ribosome profiling analyses. The efficacy of targeting METTL3 in CRC treatment was elucidated in CRC cell lines, patient-derived CRC organoids, and Mettl3-knockout mouse models. RESULTS: Using targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 dropout screening, we identified METTL3 as the top essential m6A regulatory enzyme in CRC. METTL3 was overexpressed in 62.2% (79/127) and 88.0% (44/50) of primary CRCs from 2 independent cohorts. High METTL3 expression predicted poor survival in patients with CRC (n = 374, P < .01). Functionally, silencing METTL3 suppressed tumorigenesis in CRC cells, human-derived primary CRC organoids, and Mettl3-knockout mouse models. We discovered the novel functional m6A methyltransferase domain of METTL3 in CRC cells by domain-focused CRISPR screening and mutagenesis assays. Mechanistically, METTL3 directly induced the m6A-GLUT1-mTORC1 axis as identified by integrated m6A sequencing, RNA sequencing, ribosome sequencing, and functional validation. METTL3 induced GLUT1 translation in an m6A-dependent manner, which subsequently promoted glucose uptake and lactate production, leading to the activation of mTORC1 signaling and CRC development. Furthermore, inhibition of mTORC1 potentiated the anticancer effect of METTL3 silencing in CRC patient-derived organoids and METTL3 transgenic mouse models. CONCLUSIONS: METTL3 promotes CRC by activating the m6A-GLUT1-mTORC1 axis. METTL3 is a promising therapeutic target for the treatment of CRC.
Assuntos
Neoplasias Colorretais/genética , Transportador de Glucose Tipo 1/genética , Metiltransferases/metabolismo , Neoplasias Experimentais/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Idoso , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinogênese , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Metilação de DNA , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metiltransferases/genética , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Transdução de Sinais/genética , Regulação para CimaRESUMO
BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.
Assuntos
Colite Ulcerativa/complicações , Neoplasias Associadas a Colite/patologia , Oxidases Duais/metabolismo , Microbioma Gastrointestinal/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinogênese/induzido quimicamente , Carcinogênese/imunologia , Carcinogênese/patologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/microbiologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/microbiologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/microbiologia , Colo/patologia , Conjuntos de Dados como Assunto , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Vida Livre de Germes , Humanos , Peróxido de Hidrogênio/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NADPH Oxidase 1/metabolismo , Receptor 4 Toll-Like/genéticaRESUMO
BACKGROUND & AIMS: The fragile X mental retardation protein (FMRP) affects multiple steps of the mRNA metabolism during brain development and in different neoplastic processes. However, the contribution of FMRP in colon carcinogenesis has not been investigated. METHODS: FMR1 mRNA transcript and FMRP protein expression were analyzed in human colon samples derived from patients with sporadic colorectal cancer (CRC) and healthy subjects. We used a well-established mouse model of sporadic CRC induced by azoxymethane to determine the possible role of FMRP in CRC. To address whether FMRP controls cancer cell survival, we analyzed cell death pathway in CRC human epithelial cell lines and in patient-derived colon cancer organoids in presence or absence of a specific FMR1 antisense oligonucleotide or siRNA. RESULTS: We document a significant increase of FMRP in human CRC relative to non-tumor tissues. Next, using an inducible mouse model of CRC, we observed a reduction of colonic tumor incidence and size in the Fmr1 knockout mice. The abrogation of FMRP induced spontaneous cell death in human CRC cell lines activating the necroptotic pathway. Indeed, specific immunoprecipitation experiments on human cell lines and CRC samples indicated that FMRP binds receptor-interacting protein kinase 1 (RIPK1) mRNA, suggesting that FMRP acts as a regulator of necroptosis pathway through the surveillance of RIPK1 mRNA metabolism. Treatment of human CRC cell lines and patient-derived colon cancer organoids with the FMR1 antisense resulted in up-regulation of RIPK1. CONCLUSIONS: Altogether, these data support a role for FMRP in controlling RIPK1 expression and necroptotic activation in CRC.
Assuntos
Neoplasias Colorretais/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinogênese/genética , Estudos de Casos e Controles , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/cirurgia , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Proteína do X Frágil da Deficiência Intelectual/antagonistas & inibidores , Proteína do X Frágil da Deficiência Intelectual/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Knockout , Necroptose/genética , Recidiva Local de Neoplasia/genética , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Organoides , PrognósticoRESUMO
Fucoxanthin (Fx), a marine carotenoid found in edible brown algae, is well known for having anticancer properties. The gut microbiota has been demonstrated as a hallmark for colorectal cancer progression in both humans and rodents. However, it remains unclear whether the gut microbiota is associated with the anticancer effect of Fx. We investigated the chemopreventive potency of Fx and its effect on gut microbiota in a mouse model of inflammation-associated colorectal cancer (by azoxymethane/dextran sulfate sodium treatment). Fx administration (30 mg/kg bw) during a 14 week period significantly inhibited the multiplicity of colorectal adenocarcinoma in mice. The number of apoptosis-like cleaved caspase-3high cells increased significantly in both colonic adenocarcinoma and mucosal crypts. Fx administration significantly suppressed Bacteroidlales (f_uc; g_uc) (0.3-fold) and Rikenellaceae (g_uc) (0.6-fold) and increased Lachnospiraceae (g_uc) (2.2-fold), compared with those of control mice. Oral administration of a fecal suspension obtained from Fx-treated mice, aimed to enhance Lachnospiraceae, suppress the number of colorectal adenocarcinomas in azoxymethane/dextran sulfate sodium-treated mice with a successful increase in Lachnospiraceae in the gut. Our findings suggested that an alteration in gut microbiota by dietary Fx might be an essential factor in the cancer chemopreventive effect of Fx in azoxymethane/dextran sulfate sodium-treated mice.
Assuntos
Adenocarcinoma/prevenção & controle , Colite Ulcerativa/tratamento farmacológico , Neoplasias Associadas a Colite/prevenção & controle , Microbioma Gastrointestinal/efeitos dos fármacos , Xantofilas/administração & dosagem , Adenocarcinoma/imunologia , Adenocarcinoma/microbiologia , Adenocarcinoma/patologia , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/microbiologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/microbiologia , Neoplasias Associadas a Colite/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Fezes/microbiologia , Microbioma Gastrointestinal/imunologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , CamundongosRESUMO
Ulcerative colitis is an incurable condition whereby patients are at an increased risk of developing colorectal cancer (CRC). We aimed to investigate the combination of Emu oil (EO) and grape seed extract (GSE) in an azoxymethane (AOM)/dextran sulphate sodium (DSS) model of colitis-associated CRC (CA-CRC). C57BL/6 mice (n = 10/group) were injected i.p. with saline or AOM (7.4 mg/kg) and underwent three DSS/water cycles. Mice were orally-gavaged thrice weekly with water (80 µl), EO (80 µl), GSE (80 µl; 400 mg/kg) or combined EO/GSE (160 µl). Mice were euthanized on day 63. AOM/DSS induced significant bodyweight loss (max -21%) and increased disease activity index (DAI) (max +83%) throughout the trial (P < 0.05). EO (max -53%), GSE (max -51%) and EO/GSE (max -71%) reduced DAI scores in AOM/DSS mice in all DSS cycles (P < 0.05). EO/GSE-treatment in AOM/DSS mice resulted in further DAI reduction compared with EO (max -62%) and GSE (max -71%) alone (P < 0.05). AOM/DSS mice presented with severe colonoscopically-assessed colitis at all time-points, which was reduced by EO, GSE and EO/GSE (P < 0.05). EO, GSE and EO/GSE reduced the number of colonic tumours compared with AOM/DSS controls (P < 0.05). Myeloperoxidase (acute inflammation) and fluorescein isothiocyanate-dextran levels (intestinal permeability) were increased in AOM/DSS controls (P < 0.05). EO (-58%) and EO/GSE (-77%) reduced fluorescein isothiocyanate-dextran compared with AOM/DSS controls (P < 0.05), with no effect on myeloperoxidase. Histologically-assessed severity scores were increased in the distal colon of AOM/DSS mice compared with saline (P < 0.05), with no effect observed following treatment. The combination of EO and GSE improved clinical indicators and reduced colonic tumours in AOM/DSS treated mice, suggesting potential in CA-CRC management.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Neoplasias Associadas a Colite/tratamento farmacológico , Extrato de Sementes de Uva/administração & dosagem , Óleos/administração & dosagem , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Índice de Gravidade de Doença , Carga Tumoral/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multicohort fecal samples. Here, we aim to investigate whether this bacterium could act as a prophylactic for CRC prevention. METHODS: The antitumor effects of S thermophilus were assessed in cultured colonic epithelial cells and in 2 murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S thermophilus was identified by mass spectrometry and followed by ß-galactosidase activity assay. The mutant strain of S thermophilus was constructed by homologous recombination. The effect of S thermophilus on the gut microbiota composition was assessed by shotgun metagenomic sequencing. RESULTS: Oral gavage of S thermophilus significantly reduced tumor formation in both Apcmin/+ and azoxymethane-injected mice. Coincubation with S thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. ß-Galactosidase was identified as the critical protein produced by S thermophilus by mass spectrometry screening and ß-galactosidase activity assay. ß-Galactosidase secreted by S thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S thermophilus without functional ß-galactosidase lost its tumor-suppressive effect. Also, S thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via ß-galactosidase. ß-Galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathway kinases, which partially mediated the anticancer effects of S thermophilus. CONCLUSION: S thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S thermophilus is mediated at least by the secretion of ß-galactosidase.
Assuntos
Proteínas de Bactérias/metabolismo , Neoplasias Colorretais/prevenção & controle , Probióticos/administração & dosagem , Streptococcus thermophilus/enzimologia , beta-Galactosidase/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/induzido quimicamente , Colo/microbiologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Humanos , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/microbiologia , Neoplasias Experimentais/prevenção & controle , Probióticos/metabolismo , Streptococcus thermophilus/genética , beta-Galactosidase/genéticaRESUMO
Disease tolerance, the capacity of tissues to withstand damage caused by a stimulus without a decline in host fitness, varies across tissues, environmental conditions, and physiologic states. While disease tolerance is a known strategy of host defense, its role in noninfectious diseases has been understudied. Here, we provide evidence that a thermogenic fat-epithelial cell axis regulates intestinal disease tolerance during experimental colitis. We find that intestinal disease tolerance is a metabolically expensive trait, whose expression is restricted to thermoneutral mice and is not transferable by the microbiota. Instead, disease tolerance is dependent on the adrenergic state of thermogenic adipocytes, which indirectly regulate tolerogenic responses in intestinal epithelial cells. Our work has identified an unexpected mechanism that controls intestinal disease tolerance with implications for colitogenic diseases.
Assuntos
Tecido Adiposo Marrom/metabolismo , Colite/imunologia , Neoplasias do Colo/imunologia , Resistência à Doença , Infecções por Enterobacteriaceae/imunologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Animais , Azoximetano/administração & dosagem , Comunicação Celular , Citrobacter rodentium/patogenicidade , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , Sulfato de Dextrana/toxicidade , Infecções por Enterobacteriaceae/induzido quimicamente , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Células Epiteliais/metabolismo , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Termogênese/imunologiaRESUMO
Colorectal cancer (CRC) is the fourth leading cause of tumor-related deaths worldwide. In this study, we explored the in vivo effects of quercetin, a plant flavonol from the flavonoid group of polyphenols with antioxidant effects, on colon carcinogenesis induced by azoxymethane/dextran sodium sulfate (AOM/DSS). Thirty mice were randomly assigned into three groups: the control group, the AOM/DSS group, and the quercetin+AOM/DSS group. CRC was induced by AOM injection and a solution of 2% DSS in the drinking water. In the AOM/DSS-induced colon cancer mice model, quercetin treatment dramatically reduced the number and size of colon tumors. In addition, quercetin significantly restored the leukocyte counts by decreasing the inflammation caused by AOM/DSS. We also observed that the expression of oxidative stress markers, such as lipid peroxide (LPO), nitric oxide (NO), superoxide dismutase (SOD), glucose-6-phosphate (G6PD), and glutathione (GSH), could be reduced by quercetin, suggesting that the anti-inflammatory function of quercetin comes from its antioxidant effect. Moreover, potential biomarkers were identified with serum metabolite profiling. Increased levels of 2-hydroxybutyrate, 2-aminobutyrate, and 2-oxobutyrate and decreased levels of gentian violet, indole-3-methyl acetate, N-acetyl-5-hydroxytryptamine, indoxyl sulfate, and indoxyl were also found in the AOM/DSS-treated mice. However, quercetin treatment successfully decreased the levels of 2-hydroxybutyrate, 2-aminobutyrate, 2-oxobutyrate, endocannabinoids, and sphinganine and increased the levels of gentian violet, N-acetyl-5-hydroxytryptamine, indoxyl sulfate, and indoxyl. Together, our data demonstrated that quercetin could maintain relatively potent antitumor activities against colorectal cancer in vivo through its anti-inflammation effect.
Assuntos
Antineoplásicos/uso terapêutico , Colo/patologia , Neoplasias Colorretais/tratamento farmacológico , Quercetina/uso terapêutico , Animais , Azoximetano/administração & dosagem , Biomarcadores Tumorais/sangue , Carcinogênese , Neoplasias Colorretais/induzido quimicamente , Sulfato de Dextrana/administração & dosagem , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Violeta Genciana/sangue , Humanos , Hidroxibutiratos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Carga TumoralRESUMO
BACKGROUND AND PURPOSE: This study subjected a rat model to the extracts of muscle and shell tissues from Portunus segnis to assess their therapeutic effects on the HT-29 colon cancer cells as well as on colonic Aberrant Crypt Foci (ACF) induced by Azoxymethane (AOM). METHODS: The cell line was exposed to the extracts to compare the cytotoxicity of hexane, butanol, ethyl acetate, and water extract of muscle and ethanolic extract of the shell. Male rats (n=40) were assigned into control, positive, negative, and treatment groups. The animals were injected with AOM, except the control group, and then exposed to 250 and 500mg/kg of the crude extracts. Immunohistochemical localization of Bax and Bcl-2, as well as ACF and antioxidant enzymes, were evaluated in the rat colon. RESULTS: The butanolic muscle extract and ethanolic shell one demonstrated an IC50 of 9.02±0.19µg/ml and 20.23±0.27µg/ml towards the cell line, respectively. Dietary exposure inhibited the ACF formation and crypt multiplicity in the colon compared to the cancer control group. The activity of SOD and CAT increased, while that of MDA decreased. The expression of Bax and Bcl-2 increased and decreased, respectively. CONCLUSION: Taken together, the results show that both extractions were suggested to be suppressive to AOMinduced colon cancer.
Assuntos
Focos de Criptas Aberrantes/tratamento farmacológico , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Braquiúros/química , Neoplasias Colorretais/tratamento farmacológico , Músculos/química , Focos de Criptas Aberrantes/induzido quimicamente , Focos de Criptas Aberrantes/patologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Azoximetano/administração & dosagem , Compostos de Bifenilo/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Injeções Intradérmicas , Masculino , Estrutura Molecular , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Picratos/antagonistas & inibidores , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Ral guanosine triphosphatase-activating protein α2 (RalGAPα2) is the major catalytic subunit of the negative regulators of the small guanosine triphosphatase Ral, a member of the Ras subfamily. Ral regulates tumorigenesis and invasion/metastasis of some cancers; however, the role of Ral in colitis-associated cancer (CAC) has not been investigated. We aimed to elucidate the role of Ral in the mechanism of CAC. METHODS: We used wild-type (WT) mice and RalGAPα2 knockout (KO) mice that showed Ral activation, and bone marrow chimeric mice were generated as follows: WT to WT, WT to RalGAPα2 KO, RalGAPα2 KO to WT, and RalGAPα2 KO to RalGAPα2 KO mice. CAC was induced in these mice by intraperitoneal injection of azoxymethane followed by dextran sulfate sodium intake. Intestinal epithelial cells were isolated from colon tissues, and we performed complementary DNA microarray analysis. Cytokine expression in normal colon tissues and CAC was analyzed by quantitative polymerase chain reaction. RESULTS: Bone marrow chimeric mice showed that immune cell function between WT mice and RalGAPα2 KO mice was not significantly different in the CAC mechanism. RalGAPα2 KO mice had a significantly larger tumor number and size and a significantly higher proportion of tumors invading the submucosa than WT mice. Higher expression levels of matrix metalloproteinase-9 and matrix metalloproteinase-13 were observed in RalGAPα2 KO mice than in WT mice. The expression levels of interleukin 1ß, NLRP3, apoptosis associated speck-like protein containing a CARD, and caspase-1 were apparently increased in the tumors of RalGAPα2 KO mice compared with WT mice. NLRP3 inhibitor reduced the number of invasive tumors. CONCLUSIONS: Ral activation participates in the mechanism of CAC development via NLRP3 inflammasome activation.