RESUMEN
Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.
Asunto(s)
Glucolípidos , Neoplasias , Animales , Antígenos de Carbohidratos Asociados a Tumores , Biomarcadores de Tumor , Humanos , Transducción de SeñalRESUMEN
High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet-derived growth factor B-driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma-bearing mice and low-grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S-deficient gliomas. Re-expression of GD3S restored expression of MMP9 in primary-cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α-MMP9 axis. This is the first report to describe the tumor-enhancing functions of GD3S in vivo.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Modelos Animales de Enfermedad , Glioma/genética , Glioma/patología , Sialiltransferasas/genética , Animales , Astrocitos/metabolismo , Células Cultivadas , Progresión de la Enfermedad , Gangliósidos/metabolismo , Regulación Neoplásica de la Expresión Génica , Longevidad/genética , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , TransfecciónRESUMEN
To analyze the binding specificity of a sialic acid-recognizing lectin, sialic acid-binding Ig-like lectin 7 (SIGLEC7), to disialyl gangliosides (GD3s), here we established GD3-expressing cells by introducing GD3 synthase (GD3S or ST8SIA1) cDNA into a colon cancer cell line, DLD-1, that expresses no ligands for the recombinant protein SIGLEC7-Fc. SIGLEC7-Fc did not recognize newly-expressed GD3 on DLD-1 cells, even though GD3 was highly expressed, as detected by an anti-GD3 antibody. Because milk-derived GD3 could be recognized by this fusion protein when incorporated onto the surface of DLD-1 cells, we compared the ceramides in DLD-1-generated and milk-derived GD3s to identify the SIGLEC7-specific GD3 structures on the cell membrane, revealing that SIGLEC7 recognizes only GD3-containing regular ceramides but not phytoceramides. This was confirmed by knockdown/knockout of the sphingolipid delta(4)-desaturase/C4-monooxygenase (DES2) gene, involved in phytoceramide synthesis, disclosing that DES2 inhibition confers SIGLEC7 binding. Furthermore, knocking out fatty acid 2-hydroxylase also resulted in the emergence of SIGLEC7 binding to the cell surface. To analyze the effects of binding between SIGLEC7 and various GD3 species on natural killer function, we investigated cytotoxicity of peripheral blood mononuclear cells from healthy donors toward GD3S-transfected DLD-1 (DLD-1-GD3S) cells and DLD-1-GD3S cells with modified ceramides. We found that cytotoxicity is suppressed in DLD-1-GD3S cells with dehydroxylated GD3s. These results indicate that the ceramide structures in glycosphingolipids affect SIGLEC7 binding and distribution on the cell surface and influence cell sensitivity to killing by SIGLEC7-expressing effector cells.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/fisiología , Gangliósidos/metabolismo , Lectinas/metabolismo , Lectinas/fisiología , Antígenos de Diferenciación Mielomonocítica/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Ceramidas/metabolismo , Gangliósidos/química , Glicoesfingolípidos/metabolismo , Humanos , Lectinas/química , Lectinas/genética , Leucocitos Mononucleares/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Unión Proteica/fisiología , Sialiltransferasas/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
Acidic glycosphingolipids, i.e., gangliosides, are predominantly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they are considered to be involved in the development and function of nervous systems. Recent studies involving genetic engineering of glycosyltransferase genes have revealed novel aspects of the roles of gangliosides in the regulation of nervous tissues. In this review, novel findings regarding ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice are summarized. In particular, the roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems are reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in studies of congenital neurological disorders due to genetic mutations of ganglioside synthase genes and also in the techniques for the analysis of ganglioside functions are introduced.
Asunto(s)
Glicoesfingolípidos Acídicos/metabolismo , Glicosiltransferasas/genética , Sistema Nervioso/metabolismo , Glicoesfingolípidos Acídicos/genética , Animales , Ingeniería Genética , Microdominios de Membrana/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Cancer-associated glycosphingolipids have been used as markers for diagnosis and targets for immunotherapy of malignant tumors. Recent progress in the analysis of their implications in the malignant properties of cancer cells revealed that cancer-associated glycosphingolipids are not only tumor markers, but also functional molecules regulating various signals introduced by membrane microdomains, lipid rafts. In particular, a novel approach, enzyme-mediated activation of radical sources combined with mass spectrometry, has enabled us to clarify the mechanisms by which cancer-associated glycosphingolipids regulate cell signals based on the interaction with membrane molecules and formation of molecular complexes on the cell surface. Novel findings obtained from these approaches are now providing us with insights into the development of new anticancer therapies targeting membrane molecular complexes consisting of cancer-associated glycolipids and their associated membrane molecules. Thus, a new era of cancer-associated glycosphingolipids has now begun.
Asunto(s)
Glicoesfingolípidos/metabolismo , Neoplasias/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Membrana Celular/metabolismo , Humanos , Espectrometría de Masas , Transducción de SeñalRESUMEN
Ganglioside GD2 is specifically expressed in small-cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme-mediated activation of radical sources combined with mass spectrometry in GD2+ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2+ SCLC cells were enhanced by glutamine uptake, and were suppressed by L-γ-glutamyl-p-nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/metabolismo , Gangliósidos/metabolismo , Neoplasias Pulmonares/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Glutamina/análogos & derivados , Glutamina/metabolismo , Glutamina/farmacología , Humanos , Microdominios de Membrana/metabolismoRESUMEN
Recently, the combined use of multiple drugs for coexisting multiple diseases in elderly patients has become a problem. In facilities for elderly persons, pharmacists contribute to the intervention and optimization of prescriptions. However, the procedures have been conducted smoothly in only a few facilities. We established five procedures for prescription intervention by pharmacists and implemented these interventions in 80 institutionalized individuals. The total results over 2 years(January 1, 2015 to December 31, 2016)revealed 118 cases of prescription proposals from pharmacists and a reduction of 2,411,937 yen in medical expense. The purpose of prescription proposals from pharmacists is to reduce burden on patients by optimizing prescriptions, but not to reduce the number or dose of drug. In doing so, it was important to hear patients' opinions on medical care. Patient-centered prescription proposals from pharmacists are considered to have improved the quality of medical care and may enable the promotion of prescription intervention.
Asunto(s)
Deprescripciones , Pacientes Internos , Farmacéuticos , Anciano , Prescripciones de Medicamentos , Humanos , Rol ProfesionalRESUMEN
To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Gangliósidos/metabolismo , Regulación Neoplásica de la Expresión Génica , Microdominios de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Línea Celular Tumoral , Gangliósidos/genética , Humanos , Melanoma , Microdominios de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/genéticaRESUMEN
Gene knockout mice of glycosyltransferases have clearly showed roles of their products in the bodies, while there are examples where phenotype of knockout was much less severe than expected probably due to functional redundancy. The most striking novel finding obtained from ganglioside-deficient mice was that progressive inflammatory reaction took place, leading to neurodegeneration. In particular, dysfunction of complement-regulatory proteins due to deteriorated architecture of lipid rafts seemed to be essential mechanisms for the inflammation. Furthermore, roles of gangliosides in neurons were demonstrated by neuron-specific transgenic of B4galnt1 with genetic background of B4galnt1 deficiency. From study of gene knockout mice of St8sia1, new roles of b-series gangliosides in leptin secretion from adipocytes, and roles of a-series gangliosides in leptin receptor, ObR in hypothalamus were demonstrated, leading to apparent intact balance of energy. Essential roles of b-series gangliosides in malignant properties of gliomas were also shown, suggesting their roles in the regulation of inflammation and proliferation in nervous tissues. How to apply these findings for the control of newly discovered patients with ganglioside deficiency remains to be investigated. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.
Asunto(s)
Gangliósidos/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Neoplasias de Tejido Nervioso/metabolismo , Tejido Nervioso/metabolismo , Animales , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Glioma/genética , Glioma/patología , Humanos , Inflamación , Leptina/genética , Leptina/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Microdominios de Membrana/patología , Ratones , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/deficiencia , N-Acetilgalactosaminiltransferasas/genética , Neoplasias de Tejido Nervioso/genética , Neoplasias de Tejido Nervioso/patología , Tejido Nervioso/patología , Neuronas/metabolismo , Neuronas/patología , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Sialiltransferasas/deficiencia , Sialiltransferasas/genéticaRESUMEN
Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.
Asunto(s)
Antígenos CD/farmacología , Quimiocina CCL2/farmacología , Macrófagos/efectos de los fármacos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Antígenos CD/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Lesiones Encefálicas/tratamiento farmacológico , Polaridad Celular/efectos de los fármacos , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Quimiocina CCL2/metabolismo , Niño , Medios de Cultivo Condicionados , Citocinas/metabolismo , Pulpa Dental/citología , Pulpa Dental/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Receptores CCR2/antagonistas & inhibidores , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Traumatismos de la Médula Espinal/patología , Diente PrimarioRESUMEN
There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Gangliósidos/metabolismo , Glioma/metabolismo , Proteínas Proto-Oncogénicas c-yes/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Glioma/enzimología , Glioma/genética , Humanos , Ratones , Invasividad Neoplásica , Unión Proteica , Proteínas Proto-Oncogénicas c-yes/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Some gangliosides, sialic acid-containing glycosphingolipids, have been considered as tumor-associated antigens. GD1α or a GD1α synthase gene ST6GalNAc5 was reported to be involved in the metastasis of murine lymphomas or human breast cancers, respectively. But expression patterns of 0-series gangliosides GD1α and its precursor GM1b in human cancers have not yet been investigated mainly due to lack of specific antibodies. We established specific monoclonal antibodies (mAbs) reactive with GD1α or GM1b using gangliosides from brain tissues of GM3 synthase (St3gal5)-deficient mice as immunogens. We used GM2/GD2 synthase (B4galnt1)-deficient mice to immunize by liposomes embedded with GD1α or acidic glycolipid fractions from brain of St3gal5-deficient mice. Specificities of established mAbs as analyzed by enzyme-linked immunosorbent assay and thin-layer chromatography-immunostaining were very high among various gangliosides. Increased expression of GD1α and reduced GM1b in the St6galnac5 cDNA-transfected RAW117 cell line also substantiated the specificities of two mAbs. Then, we analyzed expression of GD1α and GM1b, and of relevant glycosyltransferase genes in various human cancer cell lines using generated anti-GD1α mAb 122 or anti-GM1b mAb MR155A-7. A few human cancer cell lines showed significant expression of these gangliosides with reasonable expression of relevant glycosyltransferase genes.
Asunto(s)
Gangliósido G(M1)/análogos & derivados , N-Acetilgalactosaminiltransferasas/genética , Sialiltransferasas/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Gangliósido G(M1)/biosíntesis , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Gangliósidos/genética , Gangliósidos/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicoesfingolípidos/genética , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Ratones , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/metabolismo , Metástasis de la Neoplasia , Sialiltransferasas/metabolismoRESUMEN
In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Antineoplásicos/inmunología , Galactosiltransferasas/deficiencia , Inmunización , Neoplasias Renales , Animales , Línea Celular Tumoral , Glucolípidos/química , Glucolípidos/inmunología , Glucolípidos/farmacocinética , Humanos , Neoplasias Renales/química , Neoplasias Renales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones DesnudosRESUMEN
Although regulatory mechanisms for immune cells with inhibitory signals via immunoreceptor tyrosine-based inhibitory motifs are well known, signals transduced via interaction between Siglecs and sialyl compounds on their counterreceptors into target cells have not been reported to date. In this study, we found that an astrocytoma cell line, AS, showed detachment from culture plates when co-cultured with Siglec-9-expressing cells and/or soluble Siglec-9. Moreover, detached AS cells regrew as co-cultured cells with Siglec-9-deficient cells. They also showed increased motility and invasiveness upon Siglec-9 binding. In immunoblotting, rapid degradation of focal adhesion kinase (FAK) and related signaling molecules such as Akt, paxillin, and p130Cas was observed immediately after the co-culture. Despite degradation of these molecules, increased p-Akt was found at the front region of the cytoplasm, probably reflecting increased cell motility. Calpain was considered to be a responsible protease for the protein degradation by the inhibition experiments. These results suggest that protein degradation of FAK and related molecules was induced by Siglec-9 binding to its counterreceptors via sialylglycoconjugates, leading to the modulation of adhesion kinetics of cancer cells. Thus, this might be a mechanism by which cancer cells utilize Siglec-9-derived signals to escape from immunosurveillance.
Asunto(s)
Antígenos CD/metabolismo , Calpaína/metabolismo , Adhesión Celular/fisiología , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Antígenos CD/inmunología , Línea Celular Tumoral , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Neoplasias/inmunología , Neoplasias/patología , Unión Proteica , Receptores Mitogénicos/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Transducción de Señal , Células U937RESUMEN
Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1ß-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages.
Asunto(s)
Osteoartritis , Células Madre , Humanos , Ratones , Animales , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/metabolismo , Células Madre/metabolismo , Macrófagos/metabolismo , Osteoartritis/terapia , Osteoartritis/metabolismo , Antiinflamatorios/metabolismo , Diente PrimarioRESUMEN
Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, ß1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of ß4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that ß4GalT6 plays a role as a LacCer synthase, whereas ß4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.
Asunto(s)
Galactosiltransferasas/metabolismo , Lactosilceramidos/biosíntesis , Animales , Encéfalo/metabolismo , Línea Celular , Galactosiltransferasas/genética , Lactosilceramidos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FenotipoRESUMEN
Expression and implication of carbohydrate antigens in squamous cell carcinomas (SCCs) in oral cavity was examined. In the cell lines, type 2H and Lewis y antigens were markedly expressed. In the tissues from SCC patients and benign disorders, type 2H was highly expressed in hyperplasia (96.4 %), displasia (92.9 %) and SCC (100 %). Lewis y was, in turn, expressed mainly in cancer tissues (91.3 %), suggesting that Lewis y is a cancer-associated antigen. Normal oral mucosa showed no expression of these blood group antigens. Surprisingly, Lewis y antigen disappeared in the invasion sites where Ki-67 was definitely stained. Over-expression of Lewis y with manipulation of a fucosyltransferase cDNA resulted in suppression of cell growth and invasion, and knockdown of Lewis y also brought about increased cell growth and invasion. In either situations, no changes in the expression of sialyl-Lewis x could be found. Lowered tumor growth and invasion into surrounding tissues were also shown in Lewis y-positive SCC grafts in nu/nu mice. All these results together with alternative staining between Lewis y and Ki-67 in cancer tissues and FUT1 transfectants suggested that loss of Lewis y is a crucial event for the late stage of SCCs.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Neoplasias de la Boca/metabolismo , Oligosacáridos/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/genética , Ratones , Ratones Endogámicos BALB C , Mucosa Bucal/metabolismo , Mucosa Bucal/patología , Neoplasias de la Boca/genética , Neoplasias de la Boca/inmunología , Invasividad Neoplásica , Trasplante de Neoplasias , Oligosacáridos/genética , Especificidad de Órganos , Antígeno Sialil Lewis X , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Radiation therapy for head and neck cancers is frequently associated with adverse effects on the surrounding normal tissue. Irreversible damage to radiation-sensitive acinar cells in the salivary gland (SG) causes severe radiation-induced xerostomia (RIX). Currently, there are no effective drugs for treating RIX. We investigated the efficacy of treatment with conditioned medium derived from stem cells from human exfoliated deciduous teeth (SHED-CM) in a mouse RIX model. Intravenous administration of SHED-CM, but not fibroblast-CM (Fibro-CM), prevented radiation-induced cutaneous ulcer formation (p < 0.0001) and maintained SG function (p < 0.0001). SHED-CM treatment enhanced the expression of multiple antioxidant genes in mouse RIX and human acinar cells and strongly suppressed radiation-induced oxidative stress. The therapeutic effects of SHED-CM were abolished by the superoxide dismutase inhibitor diethyldithiocarbamate (p < 0.0001). Notably, quantitative liquid chromatography-tandem mass spectrometry shotgun proteomics of SHED-CM and Fibro-CM identified eight proteins activating the endogenous antioxidant system, which were more abundant in SHED-CM than in Fibro-CM (p < 0.0001). Neutralizing antibodies against those activators reduced antioxidant activity of SHED-CM (anti-PDGF-D; p = 0.0001, anti-HGF; p = 0.003). Our results suggest that SHED-CM may provide substantial therapeutic benefits for RIX primarily through the activation of multiple antioxidant enzyme genes in the target tissue.
Asunto(s)
Antioxidantes , Xerostomía , Ratones , Humanos , Animales , Antioxidantes/farmacología , Células Madre , Modelos Animales de Enfermedad , Xerostomía/etiología , Xerostomía/terapia , Diente PrimarioRESUMEN
Exosomes (small extracellular vesicles: EVs) have attracted increasing attention from basic scientists and clinicians since they play important roles in cell-to-cell communication in various biological processes. Various features of EVs have been elucidated regarding their contents, generation and secretion mechanisms, and functions in inflammation, regeneration, and cancers. These vesicles are reported to contain proteins, RNAs, microRNAs, DNAs, and lipids. Although the roles of individual components have been rigorously studied, the presence and roles of glycans in EVs have rarely been reported. In particular, glycosphingolipids in EVs have not been investigated to date. In this study, the expression and function of a representative cancer-associated ganglioside, GD2, in malignant melanomas was investigated. Generally, cancer-associated gangliosides have been shown to enhance malignant properties and signals in cancers. Notably, EVs derived from GD2-expressing melanomas enhanced the malignant phenotypes of GD2-negative melanomas, such as cell growth, invasion, and cell adhesion, in a dose-dependent manner. The EVs also induced increased phosphorylation of signaling molecules such as EGF receptor and focal adhesion kinase. These results suggest that EVs released from cancer-associated ganglioside-expressing cells exert many functions that have been reported as a function of these gangliosides and regulate microenvironments, including total aggravation of heterogeneous cancer tissues, leading to more malignant and advanced cancer types.
Asunto(s)
Vesículas Extracelulares , Gangliósidos , Melanoma , Microambiente Tumoral , Humanos , Vesículas Extracelulares/metabolismo , Gangliósidos/análisis , Gangliósidos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Línea Celular TumoralRESUMEN
Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid-enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X-100 extracts and MS. We also examined GD3-associated molecules using enzyme-mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti-GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3-associating rafts exist. Representative examples of GD3-associated molecules such as neogenin and MCAM were shown.