RESUMEN
Stage-specific embryonic antigen-4 (SSEA-4), a specific marker for pluripotent stem cells, plays an important role in the malignant behavior of several cancers. Here, SSEA-4 expression was evaluated by immunohistochemistry using monoclonal antibody RM1 specific to SSEA-4 in 181 and 117 prostate cancer (PC) specimens obtained by biopsy and radical prostatectomy (RP), respectively. The relationships between SSEA-4 expression in cancer cells or the presence of SSEA-4-positive tumor-infiltrating immune cells (TICs) and clinicopathological parameters were analyzed. SSEA-4 expression in cancer cells was significantly associated with Gleason score, local progression, and lymph node and distant metastasis. In RP specimens, high SSEA-4 expression in cancer cells and the presence of SSEA-4-positive TICs were significant predictors of pT3, i.e., invasion and worse biochemical recurrence (BCR) after RP, respectively, in univariate analysis. In contrast, combination of high SSEA-4 expression in cancer cells and the presence of SSEA-4-positive TICs was an independent predictor for pT3 and BCR in multivariate analysis. Biologically this combination was also independently associated with suppression of apoptosis. Thus, the co-expression of SSEA-4 in cancer cells and TICs may have crucial roles in the malignant aggressiveness and prognosis of PC. Invasive potential and suppression of apoptosis may be linked to SSEA-4 expression.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Glicoesfingolípidos/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias de la Próstata/diagnóstico , Antígenos Embrionarios Específico de Estadio/metabolismo , Anciano , Apoptosis , Biopsia , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Prostatectomía , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/cirugía , Análisis de SupervivenciaRESUMEN
A variety of glycoconjugates, including glycosphingolipids (GSLs), expressed in mammalian tissues and cells were isolated and characterized in early biochemical studies. Later studies of virus-transformed fibroblasts demonstrated the association of GSL expression profiles with cell phenotypes. Changes of GSL expression profile were observed during mammalian embryogenesis. Cell surface molecules expressed on embryos in a stage-specific manner appeared to play key roles in regulation of cell-cell interaction and cell sorting during early development. Many mAbs showing stage-specific reactivity with mouse embryos were shown to recognize carbohydrate epitopes. Among various stage-specific embryonic antigens (SSEAs), SSEA-1 was found to react with neolacto-series GSL Lex, while SSEA-3 and SSEA-4 reacted with globo-series Gb5 and monosialyl-Gb5, respectively. GSL expression during mouse early development was shown to shift rapidly from globo-series to neolacto/lacto-series, and then to ganglio-series. We found that multivalent Lex caused decompaction of mouse embryos, indicating a functional role of Lex epitope in the compaction process. Autoaggregation of mouse embryonal carcinoma (EC) F9 cells provided a useful model of the compaction process. We showed that Lex-Lex interaction, a novel type of molecular interavction termed carbohydrate-carbohydrate interaction (CCI), was involved in cell aggregation. Similar shifting of GSL expression profiles from globo-series and neolacto/lacto-series to ganglio-series was observed during differentiation of human EC cells and embryonic stem (ES) cells, reflecting the essential role of cell surface glycoconjugates in early development.
Asunto(s)
Desarrollo Embrionario , Glicoesfingolípidos/metabolismo , Antígenos Embrionarios Específico de Estadio/metabolismo , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Glicoesfingolípidos/genética , Humanos , Antígenos Embrionarios Específico de Estadio/genéticaRESUMEN
Previous studies demonstrated that certain glycosphingolipids (GSLs) are involved in various cell functions, such as cell growth and motility. Recent studies showed changes in GSL expression during differentiation of human embryonic stem cells; however, little is known about expression profiles of GSLs in cancer stem cells (CSCs). CSCs are a small subpopulation in cancer and are proposed as cancer-initiating cells, have been shown to be resistant to numerous chemotherapies, and may cause cancer recurrence. Here, we analyzed GSLs expressed in human breast CSCs by applying a CSC model induced through epithelial-mesenchymal transition, using mass spectrometry, TLC immunostaining, and cell staining. We found that (i) Fuc-(n)Lc4Cer and Gb3Cer were drastically reduced in CSCs, whereas GD2, GD3, GM2, and GD1a were greatly increased in CSCs; (ii) among various glycosyltransferases tested, mRNA levels for ST3GAL5, B4GALNT1, ST8SIA1, and ST3GAL2 were increased in CSCs, which could explain the increased expression of GD3, GD2, GM2, and GD1a in CSCs; (iii) the majority of GD2+ cells and GD3+ cells were detected in the CD44(hi)/CD24(lo) cell population; and (iv) knockdown of ST8SIA1 and B4GALNT1 significantly reduced the expression of GD2 and GD3 and caused a phenotype change from CSC to a non-CSC, which was detected by reduced mammosphere formation and cell motility. Our results provide insight into GSL profiles in human breast CSCs, indicate a functional role of GD2 and GD3 in CSCs, and suggest a possible novel approach in targeting human breast CSCs to interfere with cancer recurrence.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glucosiltransferasas/biosíntesis , Glicoesfingolípidos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Técnicas de Silenciamiento del Gen , Glucosiltransferasas/genética , Humanos , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genéticaRESUMEN
Our studies during the early 1970s showed that expression of GM3, the simplest ganglioside and an abundant animal cell membrane component, is reduced during malignant transformation of cells by oncogenic viruses. Levels of mRNA for GM3 synthase were reduced in avian and mammalian cells transformed by oncoprotein "v-Jun", and overexpression of GM3 synthase in the transformed cells caused reversion from transformed to normal cell-like phenotype. GM3 has a well-documented inhibitory effect on activation of growth factor receptors (GFRs), particularly epidermal GFR (EGFR). De-N-acetyl GM3, which is expressed in some invasive human cancer cells, has an enhancing effect on EGFR activation. The important role of the sialosyl group of GM3 was demonstrated using NEU3, a plasma membrane-associated sialidase that selectively remove sialic acids from gangliosides GM3 and GD1a and is up-regulated in many human cancer cells. GM3 is highly enriched in a type of membrane microdomain termed "glycosynapse", and forms complexes with co-localized cell signaling molecules, including Src family kinases, certain tetraspanins (e.g., CD9, CD81, CD82), integrins, and GFRs (e.g., fibroblast growth factor receptor and hepatocyte growth factor receptor c-Met). Studies by our group and others indicate that GM3 modulates cell adhesion, growth, and motility by altering molecular organization in glycosynaptic microdomains and the activation levels of co-localized signaling molecules that are involved in cancer pathogenesis.
Asunto(s)
Gangliósido G(M3)/metabolismo , Neoplasias/metabolismo , Animales , Humanos , Receptores de Factores de Crecimiento/metabolismo , Tetraspaninas/metabolismoRESUMEN
The process termed "epithelial-mesenchymal transition" (EMT) was originally discovered in ontogenic development, and has been shown to be one of the key steps in tumor cell progression and metastasis. Recently, we showed that the expression of some glycosphingolipids (GSLs) is down-regulated during EMT in human and mouse cell lines. Here, we demonstrate the involvement of GalNAc-type (or mucin-type) O-glycosylation in EMT process, induced with transforming growth factor ß (TGF-ß) in human prostate epithelial cell lines. We found that: (i) TGF-ß treatment caused up-regulation of oncofetal fibronectin (onfFN), which is defined by mAb FDC6, and expressed in cancer or fetal cells/tissues, but not in normal adult cells/tissues. The reactivity of mAb FDC6 requires the addition of an O-glycan at a specific threonine, inside the type III homology connective segment (IIICS) domain of FN. (ii) This change is associated with typical EMT characteristics; i.e., change from epithelial to fibroblastic morphology, enhanced cell motility, decreased expression of a typical epithelial cell marker, E-cadherin, and enhanced expression of mesenchymal markers. (iii) TGF-ß treatment up-regulated mRNA level of FN containing the IIICS domain and GalNAc-T activity for the IIICS domain peptide substrate containing the FDC6 onfFN epitope. (iv) Knockdown of GalNAc-T6 and T3 inhibited TGF-ß-induced up-regulation of onfFN and EMT process. (v) Involvement of GSLs was not detectable with the EMT process in these cell lines. These findings indicate the important functional role of expression of onfFN, defined by site-specific O-glycosylation at IIICS domain, in the EMT process.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Fibronectinas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , ARN Mensajero/metabolismo , Animales , Anticuerpos Monoclonales , Western Blotting , Cadherinas , Línea Celular , Cartilla de ADN/genética , Células Epiteliales , Técnicas de Silenciamiento del Gen , Glicosilación , Humanos , Ratones , N-Acetilgalactosaminiltransferasas/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/farmacología , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.
Asunto(s)
Fibroblastos/química , Fibroblastos/efectos de los fármacos , Lactonas/farmacología , Manosa/análogos & derivados , Manosa/química , Polisacáridos/química , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Ceramidas/química , Cricetinae , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Fibroblastos/citología , Glicoconjugados/química , Manosa/farmacología , Ratones , Imitación MolecularRESUMEN
Osmerus (Spirinchus) lanceolatus egg lectin (OLL) is a member of the rhamnose-binding lectin (RBL) family which is mainly found in aqueous beings. cDNA of OLL was cloned, and its genomic architecture was revealed. The deduced amino acid (aa) sequence indicated that OLL was composed of 213 aa including 95 aa of domain N and 97 aa of domain C. N and C showed 73 % sequence identity and contained both -ANYGR- and -DPC-KYL-peptide motifs which are conserved in most of the RBL carbohydrate recognition domains. The calculated molecular mass of mature OLL was 20,852, consistent with the result, and 20,677.716, from mass spectrometry. OLL was encoded by eight exons: exons 1 and 2 for a signal peptide; exons 3-5 and 6-8 for N- and C-domains, respectively. Surface plasmon resonance spectrometric analyses revealed that OLL showed comparable affinity for Galα- and ß-linkages, whereas Silurus asotus lectin (SAL), a catfish RBL, bound preferentially to α-linkages of neoglycoproteins. The Kd values of OLL and SAL against globotriaosylceramide (Gb3) were 1.69 × 10â»5 M for and 2.81 × 10â»6 M, respectively. Thus, the carbohydrate recognition property of OLL is slightly different from that of SAL. On the other hand, frontal affinity chromatography revealed that both OLL and SAL interacted with only glycolipid-type oligosaccharides such as Gb3 trisaccharides, not with N-linked oligosaccharides. The domain composition of these RBLs and an analytical environment such as the "cluster effect" of a ligand might influence the binding between RBL and sugar chains.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Proteínas de Peces/metabolismo , Lectinas/metabolismo , Osmeriformes/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Proteínas de Peces/genética , Componentes Genómicos , Glucolípidos/metabolismo , Glicoproteínas/metabolismo , Lectinas/genética , Datos de Secuencia Molecular , Peso Molecular , Osmeriformes/genética , Óvulo/metabolismo , Estructura Terciaria de Proteína/genética , Análisis de Secuencia de ADNRESUMEN
Two types of carbohydrate to carbohydrate interaction (CCI) have been known to be involved in biological processes. One is the CCI between molecules expressed on interfacing cell membranes of different cells to mediate cell to cell adhesion, and subsequently induce cell signaling, and is termed trans-CCI. It has been indicated that the Le(x) to Le(x) interaction at the morula stage in mouse embryos plays an important role in the compaction process in embryonic development. GM3 to Gg3 or GM3 to LacCer interaction has been suggested to be involved in adhesion of tumor cells to endothelial cells, which is considered a crucial step in tumor metastasis. The other is the CCI between molecules expressed within the same microdomain of the cell surface membrane, and is termed cis-CCI. The interaction between ganglioside GM3, and multi (>3) GlcNAc termini of N-linked glycans of epidermal growth factor receptor (EGFR), has been indicated as the molecular mechanism for the inhibitory effect of GM3 on EGFR activation. Also, the complex with GM3 and GM2 has been shown to inhibit the activation of hepatocyte growth factor (HGF) receptor, cMet, through its association with tetraspanin CD82, and results in the inhibition of cell motility. Since CCI research is still limited, more examples of CCI in biological processes in development, and cancer progression will be revealed in the future.
Asunto(s)
Gangliósidos/metabolismo , Animales , Adhesión Celular , Comunicación Celular , Células Madre Embrionarias/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias/metabolismoRESUMEN
Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-beta, which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 or TGF-beta is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines. (ii) TGF-beta treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-beta-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition).
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/fisiología , Glicoesfingolípidos/fisiología , Mesodermo/citología , Mesodermo/fisiología , Animales , Línea Celular , Movimiento Celular , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Gangliósido G(M2)/farmacología , Gangliósidos/farmacología , Glucosiltransferasas/antagonistas & inhibidores , Glicoesfingolípidos/metabolismo , Humanos , Mesodermo/efectos de los fármacos , Ratones , Propanolaminas/farmacología , Pirrolidinas/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The epithelial-to-mesenchymal transition (EMT) is a basic cellular process that plays a key role in normal embryonic development and in cancer progression/metastasis. Our previous study indicated that EMT processes of mouse and human epithelial cells induced by TGF-ß display clear reduction of gangliotetraosylceramide (Gg4) and ganglioside GM2, suggesting a close association of glycosphingolipids (GSLs) with EMT. In the present study, using normal murine mammary gland (NMuMG) cells, we found that levels of Gg4 and of mRNA for the UDP-Gal:ß1-3galactosyltransferase-4 (ß3GalT4) gene, responsible for reduction of Gg4, were reduced in EMT induced by hypoxia (â¼1% O(2)) or CoCl(2) (hypoxia mimic), similarly to that for TGF-ß-induced EMT. An increase in the Gg4 level by its exogenous addition or by transfection of the ß3GalT4 gene inhibited the hypoxia-induced or TGF-ß-induced EMT process, including changes in epithelial cell morphology, enhanced motility, and associated changes in epithelial vs. mesenchymal molecules. We also found that Gg4 is closely associated with E-cadherin and ß-catenin. These results suggest that the ß3GalT4 gene, responsible for Gg4 expression, is down-regulated in EMT; and Gg4 has a regulatory function in the EMT process in NMuMG cells, possibly through interaction with epithelial molecules important to maintain epithelial cell membrane organization.
Asunto(s)
Hipoxia de la Célula/fisiología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Gangliósidos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Western Blotting , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Cobalto/farmacología , Transición Epitelial-Mesenquimal/fisiología , Gangliósido G(M2)/metabolismo , Inmunoprecipitación , Glándulas Mamarias Animales/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We investigated interaction of GM3 with N-acetylglucosamine (GlcNAc) termini of N-linked glycans of epidermal growth factor receptor (EGFR), as the underlying mechanism for inhibitory effect of GM3 on EGFR activation, using ldlD cells transfected with EGFR gene. These cells, defective in UDP-Gal/UDP-GalNAc 4-epimerase, are incapable of synthesizing galactose (Gal)-containing glycans, unless Gal is provided in culture (+Gal). Key observations: (1) Expression of GlcNAc termini was high in -Gal cells, and strongly reduced in +Gal cells. (2) Comparative study of inhibitory effect of exogenously-added GM3 on EGFR activation in +Gal versus -Gal cells indicated that higher level of GlcNAc termini on EGFR is correlated with greater inhibitory effect of GM3. (3) GM3-, but not GM1-, coated beads bound to EGFR in lysate of -Gal cells, which have highly exposed GlcNAc termini. Such binding was inhibited in the presence of EDTA, similarly to other carbohydrate-carbohydrate interactions.
Asunto(s)
Acetilglucosamina/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Gangliósido G(M3)/metabolismo , Polisacáridos/metabolismo , Animales , Células CHO , Movimiento Celular/fisiología , Cricetinae , Cricetulus , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Fosforilación , Polisacáridos/químicaRESUMEN
Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through "cross-talk" of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway.
Asunto(s)
Movimiento Celular , Gangliósido G(M2)/metabolismo , Gangliósido G(M3)/metabolismo , Proteína Kangai-1/metabolismo , Nanosferas , Proteínas Proto-Oncogénicas c-met/metabolismo , Dióxido de Silicio/química , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Dimerización , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray , Vejiga Urinaria/citología , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
N-glycosylation status of purified beta-haptoglobin from sera of 17 patients, and from sera of 14 healthy volunteer subjects, was compared by blotting with various lectins and antibodies. Patients in this study were diagnosed as having colon cancer through histological examination of each tumor tissue by biopsy. Blotting index of serum beta-haptoglobin with Aleuria aurantia lectin (AAL) was clearly higher for cancer patients than for healthy subjects. No such distinction was observed for blotting with three other lectins and two monoclonal antibodies. To determine tumor-associated reactivity of AAL binding as compared to inflammatory processes in colonic tissues, beta-haptoglobin separated from sera of 5 patients with Crohn's disease (CD), and 4 patients with ulcerative colitis (UC), was studied. All these cases, except one case of UC, showed AAL index lower than that in cancer cases, similarly to healthy subjects. The higher AAL binding of beta-haptoglobin in colon cancer patients than in healthy subjects appeared to be due to alpha-L-fucosyl residue, since it was eliminated by bovine kidney alpha-fucosidase treatment. N-linked glycans of serum haptoglobin from colon cancer patients vs. healthy subjects were released by N-glycanase, fluorescence-labeled, and subjected to normal-phase high performance liquid chromatography (NP-HPLC). Glycan structures were determined based on glucose unit (GU) values and their changes upon sequential treatment with various exoglycosidases. Glycosyl sequences and their branching status of glycans from 14 cases of serum beta-haptoglobin were characterized. The identified glycans were sialylated or nonsialylated, bi-antennary or tri-antennary structures, with or without terminal fucosylation.
Asunto(s)
Neoplasias del Colon/sangre , Haptoglobinas/metabolismo , Enfermedades Inflamatorias del Intestino/sangre , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Línea Celular Tumoral , Enfermedad Crónica , Neoplasias del Colon/patología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicosilación , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between "Os Fr.B" having 5-6 GlcNAc termini, vs. absence of such homotypic CCI between "Os Fr.1" having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.
Asunto(s)
Aminopiridinas/metabolismo , Biotina/análogos & derivados , Metabolismo de los Hidratos de Carbono , Glicoconjugados/metabolismo , Piridinas/metabolismo , Aminopiridinas/química , Animales , Anticuerpos/metabolismo , Biotina/química , Biotina/metabolismo , Biotinilación , Conformación de Carbohidratos , Ceramidas/metabolismo , Cromatografía en Capa Delgada , Gangliósido G(M3)/metabolismo , Glicoconjugados/química , Espectroscopía de Resonancia Magnética , Oligosacáridos/química , Oligosacáridos/metabolismo , Piridinas/químicaRESUMEN
Based on development of various methodologies for isolation and characterization of glycosphingolipids (GSLs), we have identified a number of GSLs with globo-series or lacto-series structure. Many of them are tumor-associated or developmentally regulated antigens. The major question arose, what are their functions in cells and tissues? Various approaches to answer this question were undertaken. While the method is different for each approach, we have continuously studied GSL or glycosyl epitope interaction with functional membrane components, which include tetraspanins, growth factor receptors, integrins, and signal transducer molecules. Often, GSLs were found to interact with other carbohydrates within a specific membrane microdomain termed "glycosynapse", which mediates cell adhesion with concurrent signal transduction. Future trends in GSL and glycosyl epitope research are considered, including stem cell biology and epithelial-mesenchymal transition (EMT) process.
Asunto(s)
Glicoesfingolípidos/química , Glicoesfingolípidos/metabolismo , Animales , Adhesión Celular , Transformación Celular Neoplásica , Epítopos , Humanos , Microdominios de Membrana/metabolismo , Transducción de SeñalRESUMEN
The tyrosine kinase activity associated with epidermal growth factor receptor (EGFR) has been a target in studies of pharmacological reagents to inhibit growth of cancer cells, which are mostly of epidermal origin. Lyso-GM3 dimer showed stronger inhibitory effect on the tyrosine kinase of EGFR than GM3, with minimal cytotoxicity [Y. Murozuka, et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj. J. 24 (2007) 551-563]. Synthesis of lipids with sphingosine requires many steps, and the yield is low. A biocombinatory approach overcame this difficulty; however, products required a C(12) aliphatic chain, rather than the sphingosine head group [Y. Murozuka, et al. Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells. Chemistry & Biodiversity 2 (2005) 1063-1078]. The present study was to clarify the effects of these lipid mimetics of GM3 and lyso-GM3 dimer on EGFR tyrosine kinase activity, and consequent changes of the A431 cell phenotype, as follows. (i) A lipid mimetic of lyso-GM3 dimer showed similar strong inhibitory effect on EGF-induced EGFR tyrosine kinase activity, and similar low cytotoxicity, as the authentic lyso-GM3 dimer. (ii) A lipid mimetic of lyso-GM3 dimer inhibited tyrosine phosphorylation of EGFR or its dimer to a level similar to that of the authentic lyso-GM3 dimer, but more strongly than GM3 or a lipid mimetic of GM3. (iii) Associated with the inhibitory effect of a lipid mimetic of lyso-GM3 dimer on EGF-induced EGFR kinase activity, only Akt kinase activity was significantly inhibited, but kinases associated with other signal transducers were not affected. (iv) The cell cycle of A431 cells, and the effects of GM3 and a lipid mimetic of lyso-GM3 dimer, were studied by flow cytometry, measuring the rate of DNA synthesis with propidium iodide. Fetal bovine serum greatly enhanced S phase and G(2)/M phase. Enhanced G(2)/M phase was selectively inhibited by pre-incubation of A431 cells with a lipid mimetic of lyso-GM3 dimer, whereas GM3 had only a minimal effect.
Asunto(s)
Materiales Biomiméticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Gangliósido G(M3)/análogos & derivados , Transducción de Señal/efectos de los fármacos , Materiales Biomiméticos/análisis , Materiales Biomiméticos/síntesis química , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cromatografía en Capa Delgada , Dimerización , Activación Enzimática/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Gangliósido G(M3)/análisis , Gangliósido G(M3)/síntesis química , Gangliósido G(M3)/química , Gangliósido G(M3)/farmacología , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
At cell surface microdomains, glycosyl epitopes, carried either by glycosphingolipids, N- or O-linked oligosaccharides, are recognized by carbohydrate-binding proteins or complementary carbohydrates. In both cases, the carbohydrate epitopes may be clustered with specific signal transducers, tetraspanins, adhesion receptors or growth factor receptors. Through this framework, carbohydrates can mediate cell signaling leading to changes in cellular phenotype. Microdomains involved in carbohydrate-dependent cell adhesion inducing cell activation, motility, and growth are termed "glycosynapse". In this review a historical synopsis of glycosphingolipids-enriched microdomains study leading to the concept of glycosynapse is presented. Examples of glycosynapse as signaling unit controlling the tumor cell phenotype are discussed in three contexts: (i) Cell-to-cell adhesion mediated by glycosphingolipids-to-glycosphingolipids interaction between interfacing glycosynaptic domains, through head-to-head (trans) carbohydrate-to-carbohydrate interaction. (ii) Functional role of GM3 complexed with tetraspanin CD9, and interaction of such complex with integrins, or with fibroblast growth factor receptor, to control tumor cell phenotype and its reversion to normal cell phenotype. (iii) Inhibition of integrin-dependent Met kinase activity by GM2/tetraspanin CD82 complex in glycosynaptic microdomain. Data present here suggest that the organizational status of glycosynapse strongly affects cellular phenotype influencing tumor cell malignancy.
Asunto(s)
Movimiento Celular , Gangliósidos/metabolismo , Microdominios de Membrana/metabolismo , Animales , Caveolas/metabolismo , Adhesión Celular , Proliferación Celular , Gangliósidos/química , HumanosRESUMEN
We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.
Asunto(s)
Química/historia , Glicoconjugados/química , Sistema del Grupo Sanguíneo ABO/química , Sistema del Grupo Sanguíneo ABO/historia , Sistema del Grupo Sanguíneo ABO/metabolismo , Química/métodos , Glicoconjugados/historia , Glicoconjugados/metabolismo , Glucolípidos/química , Glucolípidos/historia , Glucolípidos/metabolismo , Glicómica/historia , Glicómica/métodos , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Estructura Molecular , Neoplasias/metabolismo , Poesía como Asunto , Proteómica/historia , Proteómica/métodos , Estados UnidosRESUMEN
We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis(x/a) antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the beta-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the beta-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcbeta4(NeuAcalpha3)Galbeta3(NeuAcalpha6)GlcNAcbetaGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Mono- and disialyl core Type 1 O-linked structures were identified after reductive beta-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O-glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its beta-chain are characterized by enhanced branching as well as antenna fucosylation.