RESUMO
Fluoropyrimidine-based combination chemotherapy plus targeted therapy is the standard initial treatment for unresectable metastatic colorectal cancer (mCRC), but the prognosis remains poor. This phase 3 trial (ClinicalTrials.gov: NCT03950154) assessed the efficacy and adverse events (AEs) of the combination of PD-1 blockade-activated DC-CIK (PD1-T) cells with XELOX plus bevacizumab as a first-line therapy in patients with mCRC. A total of 202 participants were enrolled and randomly assigned in a 1:1 ratio to receive either first-line XELOX plus bevacizumab (the control group, n = 102) or the same regimen plus autologous PD1-T cell immunotherapy (the immunotherapy group, n = 100) every 21 days for up to 6 cycles, followed by maintenance treatment with capecitabine and bevacizumab. The main endpoint of the trial was progression-free survival (PFS). The median follow-up was 19.5 months. Median PFS was 14.8 months (95% CI, 11.6-18.0) for the immunotherapy group compared with 9.9 months (8.0-11.8) for the control group (hazard ratio [HR], 0.60 [95% CI, 0.40-0.88]; p = 0.009). Median overall survival (OS) was not reached for the immunotherapy group and 25.6 months (95% CI, 18.3-32.8) for the control group (HR, 0.57 [95% CI, 0.33-0.98]; p = 0.043). Grade 3 or higher AEs occurred in 20.0% of patients in the immunotherapy group and 23.5% in the control groups, with no toxicity-associated deaths reported. The addition of PD1-T cells to first-line XELOX plus bevacizumab demonstrates significant clinical improvement of PFS and OS with well tolerability in patients with previously untreated mCRC.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Oxaloacetatos , Humanos , Bevacizumab/uso terapêutico , Capecitabina/uso terapêutico , Oxaliplatina , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , ImunoterapiaRESUMO
OBJECTIVES: Embryonic stem cells (ESCs)-derived pancreatic precursor cells have great potential for pancreas repair. Expression of pancreatic duodenal homeobox 1 (Pdx1) in definitive endoderm (DE) cells is the premise that DE cells differentiate into pancreatic cells. To achieve the required number of Pdx1-expressing DE cells for cell transplantation therapy, a valid model must be established. Using this model, researchers investigated how Pdx1 regulates ESC differentiation into pancreatic cells. METHODS: Tet-On inducible lentiviral vector encoding Pdx1 or mock vector was transduced into mouse ESC (ES-E14TG2a). The mouse ESCs were divided into 3 groups: control (ESC), mock vector (Pdx1 - -ESC), and vector encoding Pdx1 (Pdx1 + -ESC). All groups were separately cocultured with the DE cells sorted by immune beads containing CXCR-4 + (C-X-C chemokine receptor type-4) antibody. Doxycycline induced the expression of Pdx1 on the Pdx1 + -ESC cells. The markers of cell differentiation and Notch pathway were examined. RESULTS: Significantly increased expression levels of Ptf1a, CK19, and amylase on day (d) 3 and d7, Neuro-D1 on d10 and d14, Pax6 and insulin on d14, as well as Notch1, Notch2, Hes1, and Hes5 on d3 and thereafter declined on d14 were observed in Pdx1 + -ESC group. CONCLUSIONS: Pdx1 + -ESC could differentiate into pancreatic-like cells with involvement of the Notch pathway.
Assuntos
Endoderma , Proteínas de Homeodomínio , Células-Tronco Embrionárias Murinas , Pâncreas , Transativadores , Animais , Diferenciação Celular , Endoderma/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Pâncreas/citologia , Receptores Notch/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
INTRODUCTION: The epithelial tight junctions of intestine were impaired in murine model of type 2 diabetes mellitus (T2DM). The aim of this work was to investigate the alteration of intestinal barrier in T2DM patients. METHODS: 90 patients with T2DM and 28 healthy controls were recruited. Serum lipopolysaccharide (LPS), Zonulin, and intestinal fatty acid binding protein (IFABP) were measured by ELISA, based on which a derived permeability risk score (PRS) was calculated. Subgroup analyses were conducted based on the glycemic control (HbA1câ¯<â¯7%, or HbA1câ¯≥â¯7%), the amount of chronic diabetic complications, and the use of aspirin at the time. RESULTS: Serum LPS, Zonulin, and IFABP, and PRS of T2DM group were significantly higher than those of the control group (pâ¯<â¯0.05 for all). Serum LPS and PRS was higher in T2DM patients with poor glycemic control (both pâ¯<â¯0.05). Patients with more chronic complications of diabetes had higher serum LPS and IFABP, and PRS (all pâ¯<â¯0.05). No differences were found in these serum markers between T2DM patients being treated with aspirin or not. CONCLUSIONS: Intestinal barrier function was impaired in T2DM patients. Poor glycemic control and more chronic complications of diabetes were associated with worse intestinal barrier function. Treatment with aspirin did not aggravate the impairment of intestinal barrier in T2DM patients.
Assuntos
Diabetes Mellitus Tipo 2 , Proteínas de Ligação a Ácido Graxo/sangue , Mucosa Intestinal/fisiopatologia , Lipopolissacarídeos , Precursores de Proteínas/sangue , Aspirina/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Hemoglobinas Glicadas , Controle Glicêmico , Haptoglobinas , Humanos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/sangueRESUMO
Butyrate is widely accepted as a proliferation inhibitor in colon cancer but less thoroughly characterized in the colonic epithelium of objects with type 2 diabetes mellitus. The present study investigated the regulatory effect of butyrate on proliferation, the related molecule high-mobility group box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE) in the colon of db/db type 2 diabetic model mice and non-cancerous NCM460 colon cells. Proliferation and the expression of HMGB1 and RAGE were increased and could be partially reversed by butyrate treatment in the colon of db/db mice, which were consistent in NCM460 cells under a high glucose state. In NCM460 cells, under the normal glucose state, proliferation increased by overexpression of HMGB1. Under a high glucose state, increased expression of HMGB1 was accompanied with a release from cell nuclei into the cytoplasm and extracellular matrix. Down-regulation of HMGB1 could lower the expression of RAGE and attenuate the abnormally increased proliferation. And overexpression of HMGB1 reversed the suppressing effect of butyrate on abnormally increased proliferation. Conclusively, butyrate suppressed the abnormally increased proliferation in colonic epithelial cells under diabetic state by targeting HMGB1.
Assuntos
Butiratos/farmacologia , Proliferação de Células/efeitos dos fármacos , Colo/citologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Células Epiteliais/fisiologia , Expressão Gênica/efeitos dos fármacos , Proteína HMGB1/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Proteína HMGB1/genética , Masculino , Camundongos Endogâmicos , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
BACKGROUND: Intestinal barrier dysfunctions are related to dysbacteriosis and chronic gut inflammation in type 2 diabetes. Although there is emerging evidence that the chronic gut inflammatory response is stimulated by nucleotide-binding oligomerization domain-like receptors (NLRs), the relationship and precise mechanism between NLRC3 and the colonic epithelial barrier remains largely elusive. METHODS: We investigated the function and mechanism of NLRC3 in the colonic tissues of diabetic mice and colonic epithelial cell lines. The regulatory mechanism between NLRC3, butyrate and tight junctions was elucidated via a transepithelial electrical resistance measurement, transmission electron microscopy, RNA interference and western blotting. RESULTS: In this study, we found that NLRC3 expression was decreased in the colonic tissues of diabetic mice. NLRC3 over-expression ameliorated colonic epithelial barrier integrity and up-regulated tight junction proteins in colonic epithelial cells. Knockdown of TRAF6 diminished NLRC3-induced ZO-1/occludin expression. In addition, we demonstrated that butyrate could stimulate NLRC3 expression in both diabetic mice and colonic epithelial cells. GPR43 on colonic epithelial cells is involved in the activation of NLRC3 induced by butyrate. CONCLUSION: Our findings demonstrated that NLCR3 could ameliorate colonic epithelial barrier integrity in diabetes mellitus in a TRAF6-dependent manner, and NLCR3 was stimulated by butyrate via binding GPR43 on colonic epithelial cells.
Assuntos
Butiratos/farmacologia , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Camundongos Transgênicos , Substâncias Protetoras/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Junções Íntimas/metabolismoRESUMO
The intestinal epithelium cells (IECs) in diabetes mellitus (DM) patients have been proven to be abnormally differentiated. During the differentiation of IECs, epigenetic modification acts as an important regulator. In this study, we aimed to examine the epigenetic alteration of Transducin-like Enhancer of Split 1 (TLE1), a multitask transcriptional co-repressor, contributing to the differentiation homeostasis in IECs of DM mice. The IECs of type 2 diabetic mice model were isolated and collected. Methylation states of whole genomic DNA promoter regions were investigated by microarray. Methylated-specific PCR was used to detect the methylation state of TLE1 promoter in DM mice IECs. The expression of TLE1, Hes1, and differentiated cell markers were measured through real-time PCR, Western blots, and immunohistochemistry; by transfection assay, TLE1 or Hes1 was independently down-regulated in intestinal epithelium cell line, IEC-6. Subsequent modulation on TLE1, Hes1, and differentiated intestinal cell markers were detected. Global gene promoter regions in DM intestinal epithelium were less methylated comparing to normal control. The expression of TLE1 was significantly increased via hypomethylated activation in DM mice IECs. Hes1 was significantly suppressed and the terminal cell markers abnormally expressed in DM mice IECs (P < 0.05). Inhibition or induction on the abundance of TLE1 in IEC-6 cell line resulted in the corresponding dysregulation of Hes1 and intestinal epithelium differentiation (P < 0.05). Demethylation of TLE1 promoter region activates the self-expression in diabetic mice IECs. Subsequently, TLE1, through the transcriptional suppression on expression of Hes1, contributes to the aberrant differentiation of IECs in DM mice.
Assuntos
Proteínas Correpressoras/biossíntese , Metilação de DNA , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética , Mucosa Intestinal/metabolismo , Regiões Promotoras Genéticas , Animais , Proteínas Correpressoras/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Mucosa Intestinal/patologia , CamundongosRESUMO
BACKGROUND: Extragastric manifestations of Helicobacter pylori (H. pylori) infection have been reported in many diseases. However, there are still controversies about whether H. pylori infection is associated with diabetes mellitus (DM). This study was aimed at answering the question. METHODS: A systematic search of the literature from January 1996 to January 2016 was conducted in PubMed, Embase databases, Cochrane Library, Google Scholar, Wanfang Data, China national knowledge database, and SinoMed. Published studies reporting H. pylori infection in both DM and non-DM individuals were recruited. RESULTS: 79 studies with 57,397 individuals were included in this meta-analysis. The prevalence of H. pylori infection in DM group (54.9%) was significantly higher than that (47.5%) in non-DM group (OR = 1.69, P < 0.001). The difference was significant in comparison between type 2 DM group and non-DM group (OR = 2.05), but not in that between type 1 DM group and non-DM group (OR = 1.23, 95% CI: 0.77-1.96, P = 0.38). CONCLUSION: Our meta-analysis suggested that there is significantly higher prevalence of H. pylori infection in DM patients as compared to non-DM individuals. And the difference is associated with type 2 DM but not type 1 DM.
RESUMO
BACKGROUND: Distinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive. METHODS: The DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq). RESULTS: Increased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice. CONCLUSIONS: Our study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.
Assuntos
Metilação de DNA/genética , Diabetes Mellitus/terapia , Fatores de Transcrição SOX9/genética , Transplante de Células-Tronco , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos NOD , Regiões Promotoras Genéticas , Células-Tronco/metabolismo , Ativação Transcricional/genética , Via de Sinalização Wnt/genéticaRESUMO
Diabetes mellitus (DM) is a group of metabolic diseases characterised by insulin deficiency/resistance and hyperglycaemia. We previously reported the presence of an impaired tight junction and decreased expression of occludin (Ocln) and zonula occludens-1 (ZO-1) in the intestinal epithelial cells (IECs) of type 1 DM mice, but the exact mechanism remains unclear. In this study, we investigated the role of microRNAs (miRNAs) in impairing the tight junction in IECs of DM mice. Using an integrated comparative miRNA microarray, miR-429 was found to be up-regulated in IECs of type 1 DM mice. Then, miR-429 was confirmed to directly target the 3'-UTR of Ocln, although it did not target ZO-1. Moreover, miR-429 down-regulated the Ocln expression in IEC-6 cells in vitro. Finally, exogenous agomiRNA-429 was shown to down-regulate Ocln and induce intestinal barrier dysfunction in normal mice, while exogenous antagomiRNA-429 up-regulated Ocln in vivo and improved intestinal barrier function in DM mice. In conclusion, increased miR-429 could down-regulate the expression of Ocln by targeting the Ocln 3'-UTR, which impaired intestinal barrier function in DM mice.
Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Regulação para Baixo , Intestinos/patologia , MicroRNAs/metabolismo , Ocludina/genética , Regiões 3' não Traduzidas/genética , Animais , Antagomirs/metabolismo , Sequência de Bases , Sítios de Ligação , Permeabilidade da Membrana Celular , Regulação para Baixo/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RatosRESUMO
OBJECTIVES: Depression of the Notch/Hes1 pathway has been reported to play a role in abnormal differentiation of intestinal epithelial cells (IECs) in diabetes mellitus (DM). However, the mechanism by which this pathway influences IEC differentiation has remained unclear. In this study, we have investigated the role of microRNAs (miRNAs) in regulating the Notch/Hes1 pathway in IECs of DM mice. MATERIALS AND METHODS: Integrated comparative miRNA microarray technology was used to determine the expression profile of miRNAs in IECs of DM mice. After bioinformatic analysis, an miRNA with altered expression levels, miRNA-30e, was identified as a candidate for regulating the Notch pathway in DM. A luciferase reporter assay confirmed that miRNA-30e targeted 3'-UTR of the Notch gene. The role of miRNA-30e in regulating Notch signalling was then explored by up- and downregulating its expression in vitro and in vivo. RESULTS: Abnormal differentiation of IECs in DM mice was associated with reduced activity of the Dll4/NICD/Hes1 signal pathway. Based on bioinformatic analyses, increased expression of miRNA-30e was identified as a potential candidate for regulating Notch signalling. miRNA-30e targeted the 3'-UTR of Dll4 and downregulated Dll4 expression in primary IECs and IEC-6 cells. Exogenous miRNA-30e reduced activity of the Dll4/NICD/Hes1 pathway, and induced abnormal differentiation of IECs in normal mice. Conversely, treatment with miRNA-30e antagonist upregu-lated activity of the Dll4/NICD/Hes1 pathway in vivo, and normalized IEC differentiation in DM mice. CONCLUSIONS: Increased levels of miRNA-30e downregulated activity of the Dll4/NICD/Hes1 signalling pathway by targeting the 3'-UTR of Dll4, which contributed to abnormal differentiation in small intestinal epithelia of DM mice.
Assuntos
Diferenciação Celular/genética , Diabetes Mellitus Experimental/genética , Regulação para Baixo/genética , Enterócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio , Linhagem Celular , Diabetes Mellitus Experimental/patologia , Enterócitos/patologia , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genéticaRESUMO
In previous studies, we have reported the abnormal proliferation and differentiation of intestinal epithelial cells (IECs) in diabetes mellitus (DM) mice. The insulin receptor (IR) and its downstream mitogen-activated protein kinase kinase (MAPKK also known as MEK)/extracellular-regulated protein kinase (ERK) pathway is a classic pathway associated with cell proliferation and differentiation. The purpose of the present study is to investigate the role of the MEK/ERK pathway in abnormal proliferation and differentiation of IECs in DM mice. DM mouse models were induced by intraperitoneal injection of streptozotocin. The expression levels of the IR and its isoforms in IECs of DM mice and in IEC-6 cells were investigated. To ensure that the downstream pathways were monitored, QPCR and Western blotting were performed to detect the expression levels of MEK1/2, ERK1/2, PI3K, and Akt. Moreover, siRNA for IR-A and U0126, a specific inhibitor of MEK, were used to further investigate the relationship between the IR/MEK/ERK pathway and abnormal proliferation and differentiation of IECs in DM mice. In DM mice, excessive proliferation, disturbed differentiation, and a high ratio of IR-A/IR-B were detected in IECs. The expression levels of MEK1, MEK2, and ERK1/2 and their phosphorylated proteins in DM mice were significantly higher than those in the control group (P < 0.05), which could be offset by using siRNA for IR-A. The abnormal proliferation and differentiation of IECs in DM mice were normalized after the in vivo administration of U0126. The abnormal proliferation and differentiation of IECs in DM mice are associated with high IR-A/IR-B ratio and increased IR/MEK/ERK pathway activity.
Assuntos
Diabetes Mellitus Experimental/patologia , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Sistema de Sinalização das MAP Quinases , Receptor de Insulina/metabolismo , Animais , Butadienos/administração & dosagem , Butadienos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Nitrilas/administração & dosagem , Nitrilas/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor de Insulina/genética , EstreptozocinaRESUMO
OBJECTIVE: Recent studies proved that patients with diabetes were at significantly higher risk of developing colorectal cancer. However, the association between diabetes mellitus and the risk of colorectal adenoma remains undefined. Thus we conducted an updated meta-analysis to identify the association between diabetes mellitus and the risk of colorectal neoplasia including adenoma and cancer. METHODS: We conducted a search in databases including Pubmed, Web of Science, EMBASE Databases, Cochrane CENTRAL, Wanfang Data, and CNKI database. Case-control and cohort studies were included. All articles were published before January 2015 and the quality of each study was evaluated by the Newcastle-Ottawa Scale. Odds ratios (ORs) or relative risks (RRs) and its corresponding 95% confidence intervals (CIs) for each study were calculated and summary relative risk estimates with corresponding 95% CIs were generated using the random-effects model. Heterogeneity and publication bias were assessed. RESULTS: Twenty-nine articles including ten case-control studies and nineteen cohort studies were included in this meta-analysis. In a pooled analysis of all studies, diabetes mellitus was associated with increased risk of colorectal neoplasia (RR=1.35, 95% CI=1.28-1.42). The risk increased significantly for both colorectal cancer (RR=1.37, 95% CI=1.30-1.45) and adenoma (RR=1.26, 95% CI=1.11-1.44). Subgroup analyses on study design, gender, geographical region, and type of diabetes mellitus further evidenced these findings. CONCLUSIONS: Diabetes mellitus was associated with an increased risk of colorectal neoplasia. Not only the increased risk of colorectal cancer but also the higher risk of adenoma was identified in patients with diabetes mellitus.
Assuntos
Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/etiologia , Complicações do Diabetes/epidemiologia , Humanos , Fatores de RiscoRESUMO
Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating the biological functions and underlying molecular mechanisms of colorectal cancer (CRC). Here, we investigated the association of linc-POU3F3 and prognosis in CRC. We demonstrated that linc-POU3F3 was overexpressed in CRC tissues and positively correlated with tumor grade and N stage. Inhibition of linc-POU3F3 resulted in inhibition of cell proliferation and G1 cell cycle arrest, which was mediated by cyclin D1, CDK4, p18, Rb, and phosphorylated Rb. Inhibition of linc-POU3F3 induced apoptosis, and suppressed migration and invasion in LOVO and SW480 cell lines. This inhibition also increased the expressions of epithelial markers and decreased the expressions of mesenchymal markers, thus inhibiting the cancer epithelial-mesenchymal transition. The decreased migration and invasion following linc-POU3F3 knockdown were mediated by an increased BMP signal. Furthermore, autophagy was enhanced by linc-POU3F3 knockdown, suggesting the involvement of autophagy in the induced apoptosis. Collectively, linc-POU3F3 might be crucial in pro-proliferation, anti-apoptosis, and metastasis in LOVO and SW480 cells by regulating the cell cycle, intrinsic apoptosis, BMP signaling and autophagy. Thus, linc-POU3F3 is a potential therapeutic target and novel molecular biomarker for CRC.
Assuntos
Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Fatores do Domínio POU/genética , RNA Longo não Codificante/genética , Autofagia/genética , Western Blotting , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Imunofluorescência , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Microscopia Eletrônica de Transmissão , Fatores do Domínio POU/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
BACKGROUND: The effect of rebamipide on repairing intestinal mucosal damage induced by nonsteroidal anti-inflammatory drugs and its mechanism remain unclear. In this study, we sought to explore the mechanism whereby rebamipide could promote the regeneration of aspirin-induced intestinal mucosal damage. METHODS: BALB/c mice were administered aspirin (200 mg/kg/d) for 5 days to induce acute small intestinal injury (SII). Subsequently, SII mice were treated with rebamipide (320 mg/kg/d) for 5 days. The structure of intestinal barrier was observed with transmission electron microscope, and Zo-1 and occludin expressions were detected. The proliferative index was indicated by the percentage of proliferating cell nuclear antigen positive cells. The prostaglandin E2 (PGE2) levels in the small intestine tissues were measured by an enzyme immunoassay. The mRNA and protein expression levels of cyclooxygenase (COX) and ß-catenin signal were detected in the small intestine using quantitative PCR and Western blot, respectively. RESULTS: COX expression was significantly down-regulated in aspirin induced SII (P < 0.05). In SII mice treated with rebamipide, histopathological findings of aspirin-induced intestinal inflammation were significantly milder and tight junctions between intestinal epithelial cells were improved significantly. The proliferative index increased after rebamipide treatment when compared with that in the control mice. The expressions of COX-2, ß-catenin, and c-myc and the PGE2 concentrations in small intestinal tissues were significantly increased in mice with rebamipide treatments (P < 0.05). CONCLUSION: Rebamipide administration in aspirin-induced SII mice could improve the intestinal barrier structure and promote the regeneration of small intestinal epithelial injury through up-regulating COX-2 expression and the accumulation of ß-catenin.
Assuntos
Alanina/análogos & derivados , Anti-Inflamatórios não Esteroides/toxicidade , Aspirina/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Quinolonas/farmacologia , Regeneração/efeitos dos fármacos , beta Catenina/fisiologia , Alanina/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Feminino , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Mucosa Intestinal/ultraestrutura , Jejuno/fisiologia , Jejuno/ultraestrutura , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Permeabilidade , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA Mensageiro/biossíntese , Distribuição Aleatória , Junções Íntimas/efeitos dos fármacos , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
Intestinal epithelial stem cells (IESCs) can differentiate into all types of intestinal epithelial cells (IECs) and Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a marker for IESC. Previous studies reported enhanced proliferation of IECs in diabetic mice. In this study, the in vitro differentiation of Lgr5 positive IESCs sorted from diabetic mice was further investigated. The diabetic mouse model was induced by streptozotocin (STZ), and crypt IECs were isolated from small intestines. Subsequently, Lgr5 positive IESCs were detected by flow cytometry (FCM) and sorted by magnetic activated cell sorting (MACS). Differentiation of the sorted IESCs was investigated by detecting the IEC markers in the diabetic mice using immunostaining, quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and Western blot analysis, which was compared with normal mice. We found that the proportion of Lgr5 positive cells in the crypt IECs of diabetic mice was higher than that of control mice (P < 0.05). Lgr5 positive IESCs could be significantly enriched in Lgr5 positive cell fraction sorted by MACS. Furthermore, the absorptive cell marker sucrase-isomaltase (SI) and the Paneth cell marker lysozyme 1 (Lyz1) were more highly expressed in the differentiated cells derived from Lgr5 positive IESCs of diabetic mice in vitro (P < 0.05). We demonstrate that the number of Lgr5 positive IESCs is significantly increased in the small intestines of STZ-induced diabetic mice. Lgr5 positive IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro. We characterized the expression of Lgr5 in the small intestine of diabetic mice, and sorted Lgr5 positive intestinal epithelial stem cells (IESCs) for investigating their differentiation in vitro. We proved that the quantity of Lgr5 positive IESCs was significantly increased in the small intestines of diabetic mice. IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro.
Assuntos
Diferenciação Celular , Diabetes Mellitus Experimental/patologia , Intestino Delgado/patologia , Celulas de Paneth/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Feminino , Separação Imunomagnética , Absorção Intestinal , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiologia , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Celulas de Paneth/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Células-Tronco/fisiologia , EstreptozocinaRESUMO
Treatments for pancreatic injuries have been significantly improved recently, but full recovery of pancreatic function remains difficult. Embryonic stem cells have great potentialities for self-renewal and multiple differentiations. In this study, we explored an approach to induce the differentiation of pancreatic progenitor cells from embryonic stem cells in vitro. Male mouse embryonic stem cells were cultured by the hanging-drop method to form embryoid bodies. The definitive endoderm marked by CXCR4 in embryoid bodies was sorted by magnetic activated cell sorting and subsequently administrated with b-FGF, exendin-4, and cyclopamine to induce the differentiation of putative pancreatic progenitor cells, which was monitored by Pdx1, and Shh expressions. The putative pancreatic progenitor cells were transplanted into female BALB/c mice with pancreatitis induced by L-Arginine. Male donor cells were located by detecting sex-determining region of Y-chromosome DNA. Definitive endoderm cells (CXCR4(+) cells) were sorted from 5-day embryoid bodies. After 3-day administration with b-FGF, exendin-4, and cyclopamine, Pdx1-high/Shh-low cells were differentiated from CXCR4(+) cells. These cells developed into more amylase-secreted cells in vitro and could specifically reside in the damaged pancreas acinar area in mice with acute pancreatitis to enhance the regeneration. The putative pancreatic progenitor cells (Pdx1-high/Shh-low cells) derived from mouse embryonic stem cells through the administration of b-FGF, exendin-4, and cyclopamine on the CXCR4(+) cells in vitro could improve the regeneration of injured pancreatic acini in vivo.
Assuntos
Células-Tronco Embrionárias/citologia , Proteínas Hedgehog/biossíntese , Proteínas de Homeodomínio/biossíntese , Pancreatite Necrosante Aguda/terapia , Receptores CXCR4/biossíntese , Transplante de Células-Tronco/métodos , Transativadores/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Separação Celular/métodos , Modelos Animais de Doenças , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
AIMS: ß-Catenin accumulation promotes proliferation. However, the correlation between proliferation of colorectal epithelium and ß-catenin in type 2 diabetes mellitus (DM) patients remains unclear. METHODS: Colorectal epithelium samples from distal ends of colorectal adenocarcinomas without histological aberrances were divided into two groups: DM patients with type 2 DM for more than 1year (n=27) and non-DM patients without hyperglycemia (n=20). Samples from patients without colorectal epithelial disease or hyperglycemia served as a control group (n=6). Proliferative index was calculated as the percentage of proliferating cell nuclear antigen positive cells. Wnt/ß-catenin signaling was assessed immunohistochemically and phosphorylation of ß-catenin was assessed by immunofluorescence. RESULTS: Compared with the non-DM or control group, the proliferative index and expression of lactate dehydrogenase A and Wnt/ß-catenin signaling were significantly higher in the DM group (all p<0.01). The proliferative index correlated positively with ß-catenin expression (Spearman correlation coefficient=0.55; p<0.01). Reduced phosphorylation at serine 33/37 and increased phosphorylation at serine 675 of ß-catenin were detected in the DM group (all p<0.01). CONCLUSIONS: Enhanced proliferation, accompanied by increased aerobic glycolysis, was detected in colorectal epithelium of patients with diabetes. ß-Catenin accumulation with altered phosphorylation correlated with the proliferative changes.
Assuntos
Adenoma , Proliferação de Células , Neoplasias Colorretais , Diabetes Mellitus Tipo 2 , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , beta Catenina/metabolismo , Adenoma/complicações , Adenoma/metabolismo , Adenoma/patologia , Adulto , Idoso , Estudos de Casos e Controles , Neoplasias Colorretais/complicações , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Humanos , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Masculino , Pessoa de Meia-Idade , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Regulatory T (Treg) cells are potent suppressors that maintain immune homeostasis. Accumulation of Treg can inhibit effective immune responses in cancer patients, leading to tumor development and progression. Despite direct cytotoxicity, several chemotherapeutic drugs have been reported to deplete Treg cells for better prognosis for cancer patients. Treg cells are a heterogenous population with at least three different subsets, nonsuppressive, resting, and activated Treg cells. However, the characteristics of Treg cell subsets in lung cancer patients and how chemotherapy affects Treg cells remain elusive. In this study, we first analyzed Treg cell subsets in peripheral blood samples from 40 nonsmall cell lung cancer (NSCLC) patients and 20 healthy donors. Treg cells, specifically activated Treg cell subset, significantly increased in patients with NSCLC. Compared to nonsuppressive Treg cells, activated Treg cells expressed higher level of CD39 and predominantly produced inhibitory cytokines. In vitro assay showed that docetaxel reduced all three subsets of Treg cells. More importantly, we found docetaxel-based chemotherapy significantly decreased all three Treg subsets after 4 cycles of treatment in 17 NSCLC patients. Taken together, this study revealed dynamic changes of various Treg cell subsets in NSCLC patients before and after chemotherapy, providing activated Treg cells as a potential target for chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Depleção Linfocítica , Linfócitos T Reguladores/efeitos dos fármacos , Taxoides/uso terapêutico , Idoso , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Casos e Controles , Linhagem da Célula/imunologia , Docetaxel , Feminino , Humanos , Imunofenotipagem , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Linfócitos T Reguladores/classificação , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Cytokine-induced killer (CIK) cells have achieved therapeutic benefit in treatment of solid tumors in clinic. However, some patients show no response after CIK treatment. Animal assays have shown that successful infiltration of CIK cells to the tumor sites could affect the outcome. Chemokines play important roles in lymphocyte trafficking. Understanding the molecular mechanism of chemokines in the process of CIK cell homing is important for further modification of CIK therapy. In this study, we investigated the spectrum of chemokine ligands in the colorectal cancer sites and observed that chemokine ligands CCL20 and CXCL10 were overexpressed in the CRC tumor tissues compared with adjacent tissues. Although the corresponding receptors CCR6 and CXCR3 increased on CIK cells compared with PBMCs, their expression on CIK cells derived from CRC patients had lower levels than healthy donors, which might be a limited factor for autologous-CIK cells trafficking to tumor site. Importantly, stimulation with chemokines CCL20 and CXCL10 promotes the expression levels of CCR6 and CXCR3 on CIK cells, thus augmenting the relative migration of CIK cells in vitro. Our results suggest that modification of surface chemokine receptors may enhance the homing ability of CIK cells for better therapeutic achievements.