RESUMEN
Gastric cancer with peritoneal dissemination is difficult to treat surgically, and frequently recurs and metastasizes. Currently, there is no effective treatment for this disease, and there is an urgent need to elucidate the molecular mechanisms underlying peritoneal dissemination and metastasis. Our previous study demonstrated that galectin-4 participates in the peritoneal dissemination of poorly differentiated gastric cancer cells. In this study, the glycan profiles of cell surface proteins and glycosphingolipids (GSLs) of the original (wild), galectin-4 knockout (KO), and rescue cells were investigated to understand the precise mechanisms involved in the galectin-4-mediated regulation of associated molecules, especially with respect to glycosylation. Glycan analysis of the NUGC4 wild type and galectin-4 KO clones with and without peritoneal metastasis revealed a marked structural change in the glycans of neutral GSLs, but not in N-glycan. Furthermore, mass spectrometry (MS) combined with glycosidase digestion revealed that this structural change was due to the presence of the lacto-type (ß1-3Galactosyl) glycan of GSL, in addition to the neolacto-type (ß1-4Galactosyl) glycan of GSL. Our results demonstrate that galectin-4 is an important regulator of glycosylation in cancer cells and galectin-4 expression affects the glycan profile of GSLs in malignant cancer cells with a high potential for peritoneal dissemination.
Asunto(s)
Galectina 4 , Neoplasias Gástricas , Humanos , Galectina 4/genética , Glicoesfingolípidos/metabolismo , Recurrencia Local de Neoplasia , Polisacáridos/metabolismoRESUMEN
Acquired resistance to oxaliplatin is considered as the primary reason for failure in colorectal cancer (CRC) therapy. Identifying the underlying resistance mechanisms may improve CRC treatment. The present study aims to identify the key genes involved in acquired oxaliplatin-resistant in CRC by confirming the oxaliplatin resistance index (OX-RI). To this aim, two public microarray datasets regarding oxaliplatin-resistant CRC cells with different OX-RI, GSE42387, and GSE76092 were downloaded from GEO database to identify differentially expressed genes (DEGs). The results indicated that the OX-RI affects the gene expression pattern significantly. Then, 54 common DEGs in both datasets including 18 up- and 36 down-regulated genes were identified. Protein-protein interaction (PPI) analysis revealed 13 up- (MAGEA6, TGM2, MAGEA4, SCHIP1, ECI2, CD33, AKAP12, MAGEA12, CALD1, WFDC2, VSNL1, HMGA2, and MAGEA2B) and 12 down-regulated (PDZK1IP1, FXYD3, ALDH2, CEACAM6, QPRT, GRB10, TM4SF4, LGALS4, ALDH3A1, USH1C, KCNE3, and CA12) hub genes. In the next step, two novel up-regulated hub genes including ECI2 and SCHIP1 were identified to be related to oxaliplatin resistance. Functional enrichment and pathway analysis indicated that metabolic pathways, proliferation, and epithelial-mesenchymal transition may play dominant roles in CRC progression and oxaliplatin resistance. In the next procedure, two in vitro oxaliplatin-resistant sub-lines including HCT116/OX-R4.3 and HCT116/OX-R10 cells with OX-IR 3.93 and 10.06 were established, respectively. The results indicated the up-regulation of TGM2 and HMGA2 in HCT116/OX-R10 cells with high OX-RI and down-regulation of FXYD3, LGALS4, and ECI2 in both cell types. Based on the results, TGM2, HMGA2, FXYD3, and LGALS4 genes are related to oxaliplatin-resistant CRC and may serve as novel therapeutic targets.
Asunto(s)
Neoplasias Colorrectales , Galectina 4 , Humanos , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Galectina 4/genética , Galectina 4/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Biología de Sistemas , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Factores de Transcripción/genética , Regulación Neoplásica de la Expresión Génica , Resistencia a Antineoplásicos/genética , Biología Computacional , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Aldehído Deshidrogenasa Mitocondrial/genéticaRESUMEN
BACKGROUND: Peritoneal dissemination, most often seen in metastatic and/or recurrent gastric cancer, is an inoperable condition that lacks effective treatment. The use of molecular targeted drugs is also limited; therefore, identifying novel therapeutic targets and improving our understanding of this metastatic cancer are an urgent requirement. In this study, we focused on galectin-4, which is specifically expressed in poorly differentiated cells with high potential for peritoneal dissemination. METHODS: We knocked out the galectin-4 gene in NUGC4 cells using CRISPR/Cas9-mediated genome editing. Proliferation and peritoneal cancer formation in knockout cells were compared with those in wild-type and galectin-4 re-expressing cells. Western blotting and proximity ligation assays were performed to identify associated molecules affected by the expression of galectin-4. The effect of galectin-4 knockdown on cell proliferation and peritoneal metastasis was studied using a specific siRNA. Expression of galectin-4 in peritoneal metastatic tumors from 10 patients with gastric cancer was examined by immunohistochemistry. RESULTS: Suppression of galectin-4 expression reduced proliferation and peritoneal metastasis of malignant gastric cancer cells. Galectin-4 knockout and knockdown reduced the expression of activated c-MET and CD44. Galectin-4 was found to interact with several proteins on the cell surface, including CD44 and c-MET, via its carbohydrate-binding ability. Immunohistochemistry showed galectin-4 expression in peritoneal metastatic tumor cells in all patients examined. CONCLUSIONS: We clarified the role of galectin-4 in the development of peritoneal dissemination of poorly differentiated gastric cancer cells. Our data highlight the diagnostic and therapeutic potential of galectin-4 in the peritoneal dissemination of gastric cancer.
Asunto(s)
Neoplasias Peritoneales , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patología , Neoplasias Peritoneales/secundario , Galectina 4/genética , Inmunohistoquímica , ARN Interferente Pequeño , Línea Celular TumoralRESUMEN
Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1ß (IL-1ß) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.
Asunto(s)
Condrocitos/química , Galectina 4/genética , Osteoartritis/genética , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Galectina 4/metabolismo , Humanos , Osteoartritis/metabolismo , Osteoartritis/patología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Clinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. METHODS: To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. Immune cell populations in the mammary gland, spleen, and inguinal lymph nodes were subsequently analyzed by flow cytometry. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR was overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands, spleens, and inguinal lymph nodes of 6-month-old transgenic and control mice by flow cytometry. Transgenic mice were also monitored for mammary gland tumor development over a 2-year time span. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. RESULTS: We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared with placebo-treated mice. Furthermore, transgenic mice with PR overexpression demonstrated decreased numbers of immune cell populations in their mammary glands, lymph nodes, and spleens. On long-term monitoring, we determined that multiparous PR-overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR-overexpressing mice contained fewer infiltrating immune cells. Finally, RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR-overexpressing mice as compared with control mice. CONCLUSION: Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of antiprogestin therapies to promote immune-mediated elimination of mammary gland tumors.
Asunto(s)
Neoplasias de la Mama/inducido químicamente , Transformación Celular Neoplásica/inducido químicamente , Glándulas Mamarias Animales/efectos de los fármacos , Progesterona/administración & dosificación , Receptores de Progesterona/agonistas , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Implantes de Medicamentos , Femenino , Galectina 4/genética , Galectina 4/metabolismo , Inmunidad Innata/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones Transgénicos , Ovariectomía , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Transducción de Señal , Factores de Tiempo , Carga Tumoral/efectos de los fármacosRESUMEN
To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.
Asunto(s)
Movimiento Celular , Desmosomas/metabolismo , Galectina 4/metabolismo , Seudópodos/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Línea Celular Tumoral , Proliferación Celular , Desmosomas/ultraestructura , Galectina 4/genética , Humanos , Seudópodos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. METHODS: Products of recombinant galectin expression in P.â¯pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. RESULTS: Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. CONCLUSIONS: Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. GENERAL SIGNIFICANCE: Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.
Asunto(s)
Adhesión Celular/genética , Galectina 1/genética , Galectina 4/genética , Señales de Clasificación de Proteína/genética , Animales , Transporte Biológico , Calnexina/genética , Línea Celular , Galectina 1/química , Galectina 4/química , Glicosilación , Humanos , Polisacáridos/química , Polisacáridos/genética , Pliegue de Proteína , Ratas , Transducción de Señal/genéticaRESUMEN
BACKGROUND: To identify the hub genes related to urothelial carcinoma of the bladder prognosis and to understand their underlying mechanism. METHODS: The expression profiles of 18 pairs of urothelial carcinoma of the bladder patient tissue and paired adjacent tissue obtained from the Cancer Genome Atlas were performed. Weighted gene coexpression network analysis was employed to screen gene modules and hub genes with significant differential expressions in urothelial carcinoma of the bladder. The hub genes expression in urothelial carcinoma of the bladder tissues was validated by reverse transcription-quantitative polymerase chain reaction. The overall survival curve and disease-free survival curve of prognostic factor (LGALS4) were plotted using the Kaplan-Meier method. Furthermore, LGALS4 messenger RNA and protein expression were also assessed in 2 urothelial carcinoma of the bladder cell lines (T24 and 5637) by quantitative reverse transcription-polymerase chain reaction and Western blot. The functions of urothelial carcinoma of the bladder cells with transfected pcDNA3.1-LGALS4 were identified through MTT assay, plate clone formation assay, flow cytometry, and cell migration experiments. RESULTS: LGALS4 was the hub gene of pink module and it was related to prognosis. Higher LGALS4 expression predicted higher probabilities of overall survival and disease-free survival. Overexpression of LGALS4 in urothelial carcinoma of the bladder cells suppressed cell viability and migration but induced apoptosis. CONCLUSION: LGALS4 played a critical role in the progression of urothelial carcinoma of the bladder and held a promise to be the biomarker for diagnosis and treatment of urothelial carcinoma of the bladder. It predicted good prognosis of urothelial carcinoma of the bladder and restrained the growth and migration of urothelial carcinoma of the bladder cells.
Asunto(s)
Biomarcadores de Tumor , Galectina 4/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/mortalidad , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , PronósticoRESUMEN
Galectins are a family of galactoside-binding proteins with an affinity for ß-galactosides, involved in mediating fundamental processes including development, inflammation, cell migration and apoptosis. Galectin-4 is a member of tendem-repeat galectins, plays vital roles in intestinal epithelial barrier. Here, one galectin-4 gene was captured in turbot (SmLgals4) contains a 1197 bp open reading frame (ORF). In comparison to other species, SmLgals4 showed the highest similarity and identity both to large yellow croaker. The genomic structure analysis showed that SmLgals4 had conserved exons in the CRD domains compared to other vertebrate species. The syntenic analysis revealed that galectin-4 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-4 region during vertebrate evolution. Subsequently, SmLgals4 was widely expressed in all the examined tissues, with the highest expression level in intestine and the lowest expression level in skin. In addition, SmLgals4 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals4 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals4 plays vital roles in fish intestinal immune responses against infection, but the detailed roles of galectin-4 in teleost are still lacking, further studies are needed to be carried out to characterize whether galectin-4 plays similar roles in teleost intestinal immunity.
Asunto(s)
Enfermedades de los Peces/inmunología , Peces Planos/genética , Peces Planos/inmunología , Galectina 4/genética , Galectina 4/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Animales , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Galectina 4/química , Perfilación de la Expresión Génica/veterinaria , Filogenia , Distribución Aleatoria , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus iniae/fisiología , Vibrio/fisiología , Vibriosis/inmunología , Vibriosis/veterinariaRESUMEN
Neuron-derived molecules are potent regulators of oligodendrocyte differentiation and myelination during brain development and upon demyelination. Their analysis will thus contribute to understanding remyelination failure in demyelinating diseases, such as multiple sclerosis (MS). Previously, we have identified neuronal galectin-4 as a novel negative soluble regulator in the timing of developmental myelination. Here, we investigated whether galectin-4 is re-expressed in axons upon demyelination to regulate the timing of remyelination. Our findings revealed that galectin-4 is transiently localized to axons in demyelinated areas upon cuprizone-induced demyelination. In contrast, in chronic demyelinated MS lesions, where remyelination fails, galectin-4 is permanently present on axons. Remarkably, microglia/macrophages in cuprizone-demyelinated areas also harbor galectin-4, as also observed in activated microglia/macrophages that are present in active MS lesions and in inflammatory infiltrates in chronic-relapsing experimental autoimmune encephalomyelitis. In vitro analysis showed that galectin-4 is effectively endocytosed by macrophages, and may scavenge galectin-4 from oligodendrocytes, and that endogenous galectin-4 levels are increased in alternatively interleukin-4-activated macrophages and microglia. Hence, similar to developmental myelination, the (re)expressed galectin-4 upon demyelination may act as factor in the timing of oligodendrocyte differentiation, while the persistent presence of galectin-4 on demyelinated axons may disrupt this fine-tuning of remyelination.
Asunto(s)
Axones/metabolismo , Encéfalo/patología , Galectina 4/metabolismo , Macrófagos/efectos de los fármacos , Microglía/patología , Esclerosis Múltiple/patología , Animales , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Cuprizona/toxicidad , Citocinas/metabolismo , Modelos Animales de Enfermedad , Galectina 4/genética , Regulación de la Expresión Génica/efectos de los fármacos , Lectinas/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Inhibidores de la Monoaminooxidasa/toxicidad , Esclerosis Múltiple/inducido químicamente , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/toxicidad , ARN Mensajero/metabolismo , Ratas , Factores de TiempoRESUMEN
Galectine-4 (gal-4), encoded by the LGALS4 gene, was recently shown to exhibit a tumor suppressive effect in colorectal carcinoma and pancreatic adenocarcinoma, although how the expression of this gene is regulated remains unknown. No reports describe the significance of gal-4 in the malignant potential of urothelial tumors. Thus, we analyzed LGALS4 methylation and gene expression and their clinical relevance and biological function in urothelial carcinoma (UC). LGALS4 methylation was initially identified as a progression biomarker for UC patients through genome-wide DNA methylation profiling of 16 tumor samples. Bisulfite sequencing PCR and immunohistochemistry were performed to validate the promoter methylation and expression of LGALS4. We used quantitative methylation-specific PCR to determine the methylation levels of LGALS4 normalized to ACTB in the tumor samples of 79 UC patients and compared the levels between patients with different clinicopathological characteristics. The association with survival probability was analyzed with the Kaplan-Meier method and Cox regression analysis. The ectopic expression of gal-4 in cancer cell lines was used to address its biological function in UC in vitro. The promoter hypermethylation of LGALS4 (>2.51, log10 scale) revealed a positive correlation with high levels of both histological grade and tumor T category and with lymph node metastasis (all P≤0.001). In addition, LGALS4 hypermethylation was an independent predictor of inferior survival in UC patients (P<0.05). The ectopic expression studies demonstrated that gal-4 suppressed urothelial cancer cell growth, migration, and invasion. Thus, LGALS4 may function as a tumor suppressor gene in UC progression. Our findings provide evidence that methylation-mediated LGALS4 gene repression may be involved in urothelial tumor progression.
Asunto(s)
Metilación de ADN , Galectina 4/metabolismo , Neoplasias Urológicas/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Femenino , Galectina 4/biosíntesis , Galectina 4/genética , Humanos , Masculino , Pronóstico , Regiones Promotoras Genéticas , Transfección , Neoplasias Urológicas/metabolismoRESUMEN
BACKGROUND: The ability of tumor cells to invade and metastasize is relevant to the process of cancer progression and, as such, it represents an obstacle to cancer cure. So far, limited information is available on interactions between circulating tumor cells and blood cells. It is well-documented that galectin-4 is upregulated in many types of tumor cells and is involved in metastasis. Here, we address the hypothesis that tumor cells may interact with red blood cells (RBCs) via galectin-4. METHODS: High galectin-4 expressing colon, normal pancreatic and pancreatic cancer-derived cell lines (n = 5) were incubated with peripheral blood cells from different donors. Their interactions and associated proteins were examined by immunostaining and live cell imaging. RESULTS: We found that (endogenous or exogenous) galectin-4 expressing tumor cells interact directly with RBCs. We also observed an accumulation of galectin-4 and human blood group antigens at the contact sites between these cells. By comparing the number of RBCs attaching to each tumor cell, we found that cells with high pre-incubation expression levels of galectin-4 attached significantly more RBCs than those with low expression levels (p < 1 × 10-7). Conversely, we found that RBC attachment induces galectin-4 expression in tumor cells. CONCLUSIONS: From our data we conclude that tumor cells directly interact with red blood cells via galectin-4.
Asunto(s)
Comunicación Celular/genética , Eritrocitos/citología , Galectina 4/genética , Regulación Neoplásica de la Expresión Génica , Agregación Celular/genética , Línea Celular Tumoral , Galectina 4/metabolismo , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de Tiempo/métodosRESUMEN
Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition receptors by recognizing and binding to the carbohydrate present in the bacterial membranes. In this study, a Galectin-4 sequence was identified from the constructed cDNA library of Channa striatus and its structural features were reported. Gene expression analysis revealed that CsGal4 was highly expressed in liver and strongly induced by Epizootic Ulcerative Syndrome (EUS) causing pathogens such as Aphanomyces invadans, Aeromonas hydrophila and a viral analogue, poly I:C. To understand the antimicrobial role of putative dimerization site of CsGal4, the region was chemically synthesized and its bactericidal effect was determined. G4 peptide exhibited a weak bactericidal activity against Vibrio harveyi, an important aquaculture pathogen. We have also determined the bactericidal activity of the dimerization site by tagging pentamer oligotryptophan (W5) at the C-terminal of G4 peptide. Flow cytometry analysis revealed that G4W induced drastic reduction in cell counts than G4. Electron microscopic images showed membrane blebbings in V. harveyi which indicated the membrane disrupting activity of G4W. Interestingly, both the peptides did not exhibit any hemolytic activity and cytotoxicity towards peripheral blood cells of Channa striatus and the activity was specific only towards the bacterial membrane. Our results suggested that addition of W5 at the C-terminal of membrane-binding peptide remarkably improved its membrane disrupting activity.
Asunto(s)
Antibacterianos/uso terapéutico , Aphanomyces/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/metabolismo , Galectina 4/metabolismo , Péptidos/uso terapéutico , Perciformes/inmunología , Vibriosis/inmunología , Animales , Acuicultura , Bacteriólisis , Células Cultivadas , Clonación Molecular , Dimerización , Enfermedades de los Peces/terapia , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Galectina 4/genética , Galectina 4/inmunología , Expresión Génica , Ingeniería Genética , Péptidos/síntesis química , Péptidos/genética , Triptófano/síntesis química , Vibriosis/terapiaRESUMEN
Recently, we undertook a pan-cancer analyses of the nuclear hormone receptor (NR) superfamily in The Cancer Genome Atlas (TCGA), and revealed that the vitamin D receptor (NR1I1/VDR) was commonly and significantly down-regulated specifically in colon adenocarcinoma cohort (COAD). To examine the consequence of down-regulated VDR expression we re-analyzed VDR chromatin immunoprecipitation sequencing (ChIP-Seq) data from LS180 colon cancer cells (GSE31939). This analysis identified 1809 loci that displayed significant (p.adj<0.01) differential binding of the VDR in response 1,25(OH)2D3 treatment; 947 peaks annotated to 672 genes. We examined expression patterns in the COAD cohort of 286 tumors compared to 41 normal samples and revealed that VDR bound genes were significantly positively correlated to VDR expression compared to the background transcriptome, suggesting direct regulation by VDR. Gene set enrichment analyses revealed significant enrichment for genes known to be regulated by a number of other transcription factors including SMADs and JUN. Filtering VDR associated genes for those that were commonly and significantly altered in COAD revealed a cohort of 27 differentially expressed genes. The expression patterns of these genes clustered tumors and significantly associated with disease free survival. For instance, males with low expression of Lectin, Galactoside Binding Soluble 4 (LGALS4, encodes the colon tumor suppressor, Galactin 4) had significantly shorted disease free survival. These analyses suggest that reduced expression of VDR in colon cancer (but neither loss nor mutation) changes the actions of the VDR by both dampening the expression of tumor suppressors (e.g. LGALS4) whilst either stabilizing or not down-regulating expression of oncogenes (e.g. Carbonic Anhydrase 9 (CA9)). These integrative genomic approaches are relatively generic and applicable to the study of any transcription factor.
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Adenocarcinoma/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Calcitriol/genética , Transcriptoma , Adenocarcinoma/patología , Línea Celular Tumoral , Estudios de Cohortes , Colon/metabolismo , Colon/patología , Neoplasias del Colon/patología , Femenino , Galectina 4/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by ß-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.
Asunto(s)
Galectina 4/química , Galectina 4/metabolismo , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Cristalografía por Rayos X , Galectina 4/genética , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Estabilidad Proteica , Solubilidad , Relación Estructura-Actividad , TermodinámicaRESUMEN
Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface.
Asunto(s)
Autofagia/genética , Diferenciación Celular/genética , Regulación hacia Abajo , Galectina 4/genética , Regulación del Desarrollo de la Expresión Génica , Trofoblastos/metabolismo , Animales , Adhesión Celular/genética , Línea Celular Tumoral , Femenino , Galectina 4/metabolismo , Placenta/metabolismo , Placentación/genética , Embarazo , Ratas Wistar , Imagen de Lapso de Tiempo/métodosRESUMEN
Colorectal cancer (CRC) is the third most common cancer in the world. A significant survival rate is achieved if it is detected at an early stage. A whole blood screening test, without any attempt to isolate blood fractions, could be an important tool to improve early detection of colorectal cancer. We searched for candidate markers with a novel approach based on the Transcriptome Mapper (TRAM), aimed at identifying specific RNAs with the highest differential expression ratio between colorectal cancer tissue and normal blood samples. This tool permits a large-scale systematic meta-analysis of all available data obtained by microarray experiments. The targeting of RNA took into consideration that tumour phenotypic variation is associated with changes in the mRNA levels of genes regulating or affecting this variation.A real time quantitative reverse transcription polymerase chain reaction (qRT- PCR) was applied to the validation of candidate markers in the blood of 67 patients and 67 healthy controls. The expression of genes: TSPAN8, LGALS4, COL1A2 and CEACAM6 resulted as being statistically different.In particular ROC curves attested for TSPAN8 an AUC of 0.751 with a sensitivity of 83.6% and a specificity of 58.2% at a cut off of 10.85, while the panel of the two best genes showed an AUC of 0.861 and a sensitivity of 92.5% with a specificity of 67.2%.Our preliminary study on a total of 134 subjects showed promising results for a blood screening test to be validated in a larger cohort with the staging stratification and in patients with other gastrointestinal diseases.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Metaanálisis como Asunto , ARN Mensajero/genética , Anciano , Antígenos CD/sangre , Antígenos CD/genética , Biomarcadores de Tumor/sangre , Moléculas de Adhesión Celular/sangre , Moléculas de Adhesión Celular/genética , Colágeno Tipo I/sangre , Colágeno Tipo I/genética , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico , Femenino , Proteínas Ligadas a GPI/sangre , Proteínas Ligadas a GPI/genética , Galectina 4/sangre , Galectina 4/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/sangre , Curva ROC , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tetraspaninas/sangre , Tetraspaninas/genéticaRESUMEN
Galectin-4, a tandem repeat member of the ß-galactoside-binding proteins, possesses two carbohydrate-recognition domains (CRD) in a single peptide chain. This lectin is mostly expressed in epithelial cells of the intestinal tract and secreted to the extracellular. The two domains have 40% similarity in amino acid sequence, but distinctly binding to various ligands. Just because the two domains bind to different ligands simultaneously, galectin-4 can be a crosslinker and crucial regulator in a large number of biological processes. Recent evidence shows that galectin-4 plays an important role in lipid raft stabilization, protein apical trafficking, cell adhesion, wound healing, intestinal inflammation, tumor progression, etc. This article reviews the physiological and pathological features of galectin-4 and its important role in such processes.
Asunto(s)
Galectina 4/metabolismo , Neoplasias/patología , Animales , Axones/metabolismo , Endocitosis , Galectina 4/sangre , Galectina 4/genética , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Microdominios de Membrana/metabolismo , Neoplasias/metabolismo , Neuronas/metabolismo , Cicatrización de HeridasRESUMEN
Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohn's disease (CD) and ulcerative colitis (UC). Galectins, defined by shared consensus amino acid sequence and affinity for ß-galactosides, are critical modulators of the inflammatory response. However, the relevance of the galectin network in the pathogenesis of human IBD has not yet been explored. Here, we analyzed the expression of relevant members of the galectin family in intestinal biopsies, and identified their contribution as novel mucosal markers in IBD. Colonic biopsies were obtained from 59 IBD patients (22 CD and 37 UC), 9 patients with gut rejection after transplantation, 8 adult celiac patients, and 32 non-IBD donors. Galectin mRNA expression was analyzed by RT-PCR and qPCR using specific primers for individual galectins. A linear discriminant analysis (LDA) was used to analyze galectin expression in individual intestinal samples. Expression of common mucosal-associated galectins (Gal-1, -3, -4, -9) is dysregulated in inflamed tissues of IBD patients compared with non-inflamed IBD or control samples. LDA discriminated between different inflammation grades in active IBD and showed that remission IBD samples were clusterized with control samples. Galectin profiling could not distinguish CD and UC. Furthermore, inflamed IBD was discriminated from inflamed tissue of rejected gut in transplanted patients and duodenum of celiac patients, which could not be distinguished from control duodenum samples. The integrative analysis of galectins discriminated IBD from other intestinal inflammatory conditions and could be used as potential mucosal biomarker.
Asunto(s)
Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Galectina 3/biosíntesis , Galectina 4/biosíntesis , Galectinas/biosíntesis , Inflamación/genética , Tirosina/análogos & derivados , Adulto , Benzamidas , Biopsia , Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Femenino , Galectina 3/genética , Galectina 4/genética , Galectinas/genética , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/patología , Regulación de la Expresión Génica , Humanos , Inflamación/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Intestinos/patología , Masculino , ARN Mensajero/biosíntesis , Tirosina/biosíntesis , Tirosina/genéticaRESUMEN
INTRODUCTION: The aetiology and pathogenesis of ulcerative colitis (UC) are essentially unknown. Galectins are carbohydrate-binding lectins involved in a large number of physiological and pathophysiological processes. Little is known about the role of galectins in human UC. In this immunohistochemical exploratory study, both epithelial and inflammatory cell galectin expression were studied in patients with a thoroughly documented clinical history and were correlated with inflammatory activity. MATERIAL AND METHODS: Surgical whole intestinal wall colon specimens from UC patients (n = 22) and controls (n = 10) were studied. Clinical history, pharmacological treatment, and modified Mayo-score were recorded. Tissue inflammation was graded, and sections were stained with antibodies recognizing galectin-1, galectin-2, galectin-3, and galectin-4. RESULTS: Galectin-1 was undetectable in normal and UC colonic epithelium, while galectin-2, galectin-3, and galectin-4 were strongly expressed. A tendency towards diminished epithelial expression with increased inflammatory grade for galectin-2, galectin-3, and galectin-4 was also found. In the inflammatory cells, a strong expression of galectin-2 and a weak expression of galectin-3 were seen. No clear-cut correlation between epithelial galectin expression and severity of the disease was found. CONCLUSION: Galectin expression in patients with UC seems to be more dependent on disease focality and individual variation than on degree of tissue inflammation.