RESUMEN
Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.
Asunto(s)
Glucuronidasa , Heparitina Sulfato , Mastocitos , Mastocitos/metabolismo , Glucuronidasa/metabolismo , Glucuronidasa/genética , Animales , Heparitina Sulfato/metabolismo , Ratones , Línea Celular TumoralRESUMEN
Anti-mucin1 (MUC1) antibodies have been widely used for breast cancer diagnosis and treatment. This is based on the fact that MUC1 undergoes aberrant glycosylation upon cancer progression, and anti-MUC1 antibodies differentiate changes in glycan structure. MY.1E12 is a promising anti-MUC1 antibody with a distinct specificity toward MUC1 modified with an immature O-glycan (NeuAcα(2-3)Galß(1-3)GalNAc) on a specific Thr. However, the structural basis for the interaction between MY.1E12 and MUC1 remains unclear. The aim of this study is to elucidate the mode of interaction between MY.1E12 and MUC1 O-glycopeptide by NMR, molecular dynamics (MD) and docking simulations. NMR titration using MUC1 O-glycopeptides suggests that the epitope is located within the O-linked glycan and near the O-glycosylation site. MD simulations of MUC1 glycopeptide showed that the O-glycosylation significantly limits the flexibility of the peptide backbone and side chain of the O-glycosylated Thr. Docking simulations using modeled MY.1E12 Fv and MUC1 O-glycopeptide, suggest that VH mainly contributes to the recognition of the MUC1 peptide portion while VL mainly binds to the O-glycan part. The VH/VL-shared recognition mode of this antibody may be used as a template for the rational design and development of anti-glycopeptide antibodies.
Asunto(s)
Glicopéptidos , Simulación de Dinámica Molecular , Anticuerpos Monoclonales , Glicopéptidos/química , Espectroscopía de Resonancia Magnética , Mucina-1/metabolismo , Polisacáridos/químicaRESUMEN
Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.
Asunto(s)
Antineoplásicos Inmunológicos , Carcinoma de Células Escamosas , Animales , Anticuerpos Monoclonales , Células CHO , Cricetinae , Cricetulus , Ratones , Mucina-1/química , Mucinas/química , Polisacáridos/químicaRESUMEN
We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.
Asunto(s)
Carcinoma , Glucuronidasa , Ácido Hialurónico , Animales , Carcinoma/tratamiento farmacológico , Carcinoma/metabolismo , Glucuronidasa/efectos de los fármacos , Glucuronidasa/metabolismo , Heparina/farmacología , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Ratones , SulfatosRESUMEN
Ligand-induced cellular signaling involved in interleukin 10 (IL-10) production by lamina propria macrophages (LPMs) during their interactions with commensal bacteria is not clearly understood. We previously showed, using mice lacking a C-type lectin MGL1/CD301a, that this molecule on colonic LPMs plays an important role in the induction of IL-10 upon interaction with commensal bacteria, Streptococcus sp. In the present report, we show that the physical engagement of MGL1/CD301a on LPMs with in-situ isolated Streptococcus sp. bacteria leads to IL-10 messenger RNA (mRNA) induction. Spleen tyrosine kinase (Syk), caspase recruitment domain 9 (CARD9) and extracellular signal-regulated kinase (ERK), but not NF-κB pathway, are shown to be indispensable for IL-10 mRNA induction after stimulation with heat-killed Streptococcus sp. Guanidine hydrochloride treatment of Streptococcus sp., which is known to extract bacterial cell surface glycan-rich components, abolished bacterial binding to recombinant MGL1/CD301a. The extract contained materials which bound rMGL1 in ELISA and appeared to induce IL-10 mRNA expression in LPMs in vitro. Lectin blotting showed that the extract contained glycoproteins that are considered as putative ligands for MGL1. Some human commensal Lactobacillus species also induced IL-10 mRNA expression by colonic LPMs in vitro, which depends on the presence of MGL1/CD301a and CARD9. The present results are the first to show that MGL1/CD301a acts as a signal transducer during colonic host-microbe interactions.
Asunto(s)
Asialoglicoproteínas , Interleucina-10 , Animales , Asialoglicoproteínas/genética , Asialoglicoproteínas/metabolismo , Bacterias/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Interleucina-10/genética , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The role of sialic acids on MUC1 in peritoneal dissemination of ovarian cancer cells was investigated. A human ovarian carcinoma cell line, ES-2, was transfected with full-length MUC1 containing 22 or 42 tandem repeats. These transfectants were less adherent to monolayers of patient-derived mesothelial cells than ES-2/mock transfectants. When these cells were inoculated into the abdominal cavity of female nude mice, mice that had received the transfectants showed better survival. When the transfectants were mixed with sialidase and injected, the survival was poorer, whereas when they were mixed with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid, a sialidase inhibitor, the survival was significantly prolonged. These behaviors, concerned with peritoneal implantation and dissemination observed in vitro and in vivo, were dependent on the expression of MUC1. Therefore, sialic acid linked to MUC1 in the form, at least in part, of sialyl-T, as shown to be recognized by monoclonal antibody MY.1E12, is responsible for the suppression of adhesion of these cells to mesothelial cells and the suppression of peritoneal implantation and dissemination.
Asunto(s)
Mucina-1/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neoplasias Ováricas/patología , Animales , Adhesión Celular , Línea Celular Tumoral , Epítopos/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Mucina-1/genética , Mucina-1/inmunología , Neuraminidasa/metabolismo , Neuraminidasa/farmacología , Neoplasias Ováricas/metabolismo , Neoplasias Peritoneales/patología , Neoplasias Peritoneales/secundario , Peritoneo/citología , Polisacáridos/química , Polisacáridos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Leukocyte migration is essential for exerting self-defense mechanisms. During the extravasation process, leukocytes transmigrate through the endothelial lining and the subendothelial basement membrane. Accumulating evidence supports the involvement of heparanase in this process. Altered cellular distribution resulting in relocalization of heparanase to the leading edge of migration is a key event to rapidly turn on the function of the enzyme during migration. This review presents current research investigating the cellular machinery that builds up a functional subcellular structure for leukocyte attachment to and degradation of the extracellular matrix. Recent advances in the understanding of the roles of heparanase in inflammatory diseases and pharmacological approaches to control heparanase-mediated actions during inflammation are also discussed.
Asunto(s)
Quimiotaxis de Leucocito , Glucuronidasa/metabolismo , Leucocitos/citología , Leucocitos/enzimología , Matriz Extracelular , HumanosRESUMEN
Decreased cell adhesion has been reported as a significant negative prognostic factor of lung cancer. However, the molecular mechanisms responsible for the cell incohesiveness in lung cancer have not yet been elucidated in detail. We herein describe a rare histological variant of lung adenocarcinoma consisting almost entirely of individual cancer cells spreading in alveolar spaces in an incohesive pattern. A whole exome analysis of this case showed no genomic abnormalities in CDH1 or other genes encoding cell adhesion molecules. However, whole mRNA sequencing revealed that this case had an extremely high expression level of mucin 21 (MUC21), a mucin molecule that was previously shown to inhibit cell-cell and cell-matrix adhesion. The strong membranous expression of MUC21 was found on cancer cells using mAbs recognizing different O-glycosylated forms of MUC21. An immunohistochemical analysis of an unselected series of lung adenocarcinoma confirmed that the strong membranous expression of MUC21 correlated with incohesiveness. Thus, MUC21 could be a promising biomarker with potential diagnostic and therapeutic applications for lung adenocarcinoma showing cell incohesiveness.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Adenocarcinoma del Pulmón/diagnóstico por imagen , Adenocarcinoma del Pulmón/genética , Anciano , Antígenos CD/genética , Cadherinas/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/genética , Tomografía Computarizada por Rayos X , Secuenciación del ExomaRESUMEN
We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.
Asunto(s)
Células de la Médula Ósea/metabolismo , Sulfatos de Condroitina/farmacología , Glucuronidasa/metabolismo , Animales , Células de la Médula Ósea/citología , Cartílago/metabolismo , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Quimiocinas/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Decapodiformes , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Mastocitos/citología , Mastocitos/metabolismo , Ratones , Proteínas Recombinantes/farmacologíaRESUMEN
We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.
Asunto(s)
Glucuronidasa/metabolismo , Mastocitos/fisiología , Sindecano-4/metabolismo , Animales , Degranulación de la Célula , Células Cultivadas , Endocitosis , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Mastocitos/citología , Mastocitos/metabolismo , Ratones , Transporte de Proteínas , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
Non-clinical studies are necessary at each stage of the development of oncology drugs. Many experimental cancer models have been developed to investigate carcinogenesis, cancer progression, metastasis, and other aspects in cancer biology and these models turned out to be useful in the efficacy evaluation and the safety prediction of oncology drugs. While the diversity and the degree of engagement in genetic changes in the initiation of cancer cell growth and progression are widely accepted, it has become increasingly clear that the roles of host cells, tissue microenvironment, and the immune system also play important roles in cancer. Therefore, the methods used to develop oncology drugs should continuously be revised based on the advances in our understanding of cancer. In this review, we extensively summarize the effective use of those models, their advantages and disadvantages, ranges to be evaluated and limitations of the models currently used for the development and for the evaluation of oncology drugs.
Asunto(s)
Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Oncología Médica/métodos , Neoplasias , Animales , Antineoplásicos , HumanosRESUMEN
Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL-17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL-17A was critical for amplifying such local inflammation, as observed in the production of IL-1ß and neutrophil infiltration following the cross-talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi-invariant TCR initiate cancer-promoting inflammation by producing IL-17A in an MyD88/IL-23-dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune-escalation process. Collectively, these results reveal the importance of IL-17A-producing CD30(+) Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.
Asunto(s)
Inflamación/inmunología , Inflamación/metabolismo , Interleucina-17/biosíntesis , Antígeno Ki-1/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunidad , Inflamación/complicaciones , Ratones , Ratones Noqueados , Modelos Biológicos , Neoplasias/patología , Microambiente Tumoral/inmunologíaRESUMEN
To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production.
Asunto(s)
Quimiocinas/inmunología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/inmunología , Glucuronidasa/inmunología , Heparitina Sulfato/inmunología , Inflamasomas/inmunología , Catálisis , Línea Celular Tumoral , Activación Enzimática , HumanosRESUMEN
Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis-prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis-prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor-κb (NF-κB) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF-κB/Luc2 reporter gene (4T1 NF-κB cells), we observed significantly increased bioluminescence approximately 7 days after metastasis-prone orthotopic mammary fat-pad inoculation but not ectopic s.c. inoculation of 4T1 NF-κB cells. Such in vivo NF-κB activation within the fat-pad 4T1 tumor was diminished in immune-deficient SCID or nude mice, or T cell-depleted mice, suggesting the requirement of host T cell-mediated immune responses. Given the fat-pad 4T1 tumor expressed higher inflammatory mediators in a T cell-dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells.
Asunto(s)
Neoplasias Pulmonares/inmunología , Neoplasias Mamarias Animales/inmunología , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Movimiento Celular , Femenino , Neoplasias Pulmonares/secundario , Linfocitos Infiltrantes de Tumor/inmunología , Macrófagos/inmunología , Neoplasias Mamarias Animales/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Trasplante de NeoplasiasRESUMEN
Glycoform of mucin 1 (MUC1) in cancerous cells changes markedly with cell differentiation, and thus, qualitative detection and verification of the MUC1 glycosylation changes have potential diagnostic value. We have developed an ultrasensitive method to detect the changes in cholangiocarcinoma (CC), which produces MUC1, and applied it in the diagnostics development. The focused glycan analysis using 43-lectin-immobilized microarray could obtain the glycan profiles of sialylated MUC1 in 5 µL of sera. The high-throughput analysis detected disease-specific alterations of glycosylation, and the statistical analysis confirmed that use of Wisteria floribunda agglutinin (WFA) alone produced a diagnostic score sufficient for discriminating 33 CC cases from 40 hepatolithiasis patients and 48 normal controls (p < 0.0001). The CC-related glycosylation change was verified by the lectin-antibody sandwich ELISA with WFA in two cohorts: (1) 78 Opisthorchis viverrini infected patients without CC and 78 with CC, (2) 33 CC patients and 40 hepatolithiasis patients (the same cohort used for the above lectin microarray). The WFA positivity distinguished patients with CC (opisthorchiasis: p < 0.0001, odds ratio = 1.047; hepatolithiasis: p = 0.0002, odds ratio = 1.018). Sensitive detection of qualitative alterations of sialylated MUC1 glycosylation is indispensable for the development of our glycodiagnostic test for CC.
Asunto(s)
Colangiocarcinoma/química , Lectinas/química , Mucina-1/sangre , Análisis por Matrices de Proteínas , Glicosilación , Humanos , Mucina-1/metabolismoRESUMEN
Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca(2+)-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca(2+)-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV.
Asunto(s)
Asialoglicoproteínas/metabolismo , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Internalización del Virus , Animales , Asialoglicoproteínas/genética , Células CHO , Calcio/metabolismo , Línea Celular , Cricetinae , Cricetulus , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/virología , Lectinas Tipo C/genética , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Receptores Virales/genéticaRESUMEN
Protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) ß1â2Man linkage of O-mannosyl glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by ß-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of O-glycans.
Asunto(s)
N-Acetilglucosaminiltransferasas/metabolismo , Polisacáridos/metabolismo , Glicosilación , Células HEK293 , Humanos , Lectinas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Inactivadoras de Ribosomas/metabolismoRESUMEN
Heparanase cleaves macromolecular heparin in the secretory granules of connective tissue-type mast cells. We investigated roles of the cleavage under a microenvironment mimicking where the mast cells physiologically reside. A connective tissue-type mast cell line MST and mouse peritoneal cell-derived mast cells stored macromolecular heparin in the secretory granules. The cells expressing heparanase stored fragmented heparin (~10 kDa) due to heparanase-dependent cleavage of the heparin. We produced an artificial collagen-based extracellular matrix and placed the live cells or glycosaminoglycans purified from the cells in the matrix to measure the release of sulfated macromolecules into the medium. The sulfate-radiolabelled molecules from the degranulating heparanase-expressing cells and the purified glycosaminoglycans showed significantly greater release into the medium than those derived from mock cells, which was not the case in suspension culture. The mast cell granular enzyme chymase, but not ß-hexosaminidase, showed significantly greater release from the degranulating heparanase-expressing cells than from mock cells. Purified chymase mixed with fragmented heparin derived from heparanase-expressing cells showed greater release from collagen gels than the enzyme alone or mixed with macromolecular heparin derived from mock cells. We propose that the cleavage of macromolecular heparin by heparanase accelerates the release of heparin and chymase from extracellular matrices.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Matriz Extracelular/metabolismo , Glucuronidasa/fisiología , Heparina/metabolismo , Mastocitos/metabolismo , Animales , Bovinos , Línea Celular , Quimasas/metabolismo , Gránulos Citoplasmáticos/enzimología , Matriz Extracelular/enzimología , Cabras , Heparina/fisiología , Humanos , Sustancias Macromoleculares/metabolismo , Masculino , Mastocitos/enzimología , Ratones , PorcinosRESUMEN
BACKGROUND: Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients. METHODS: We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages. RESULTS: CXCL16 expression enhanced TNF-α-induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-κB-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF-α-induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF-α-induced apoptosis through the induction of M1 macrophages, which released TNF-α. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis. CONCLUSIONS: Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.
Asunto(s)
Apoptosis/genética , Quimiocinas CXC/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/secundario , Macrófagos/metabolismo , Receptores Depuradores/genética , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Resistencia a Medicamentos/genética , Expresión Génica , Silenciador del Gen , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Macrófagos/inmunología , Ratones , Interferencia de ARN , Receptores Depuradores/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Glycosylation changes of cancer cells are known to be associated with malignant progression and metastases and potentially determine the organ-selective nature of metastasis as theorized by Paget (Lancet 1:571-573, 1889). Cellular glycans play a variety of roles in the processes of metastasis and may be unique to the cells that metastasize to different organs. We analyzed the glycosylation profiles of the primary tumor and tumors metastasized to lymph node, liver, lung, brain, bone, thyroid, kidney, adrenal, small intestine and pancreas in an autopsy case of breast cancer employing a lectin microarray with 45 lectins. Clustering analysis of the data revealed that metastatic breast cancer cells were categorized into several clusters according to their glycosylation profiles. Our results provide a biological basis to understand differential phenotypes of metastatic breast cancer cells potentially reflecting clonal origin, which does not directly reflect genomic or genetic changes or microenvironmental effects but connects to glycosylation profiles.