Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
J Biol Chem ; 299(8): 104971, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37380081

RESUMO

The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of ß-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of ß-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.


Assuntos
Galectina 3 , beta Catenina , Animais , Humanos , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Galectina 3/genética , Galectina 3/metabolismo , Regulação Neoplásica da Expressão Gênica , Células MCF-7 , Camundongos Nus , RNA Mensageiro/genética , Trofoblastos/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
2.
J Biol Chem ; 294(30): 11513-11524, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31177095

RESUMO

Dysfunction of tight junctions is a critical step during the initial stage of tumor progression. Trophoblast cell surface antigen 2 (Trop-2) belongs to the family of tumor-associated calcium signal transducer (TACSTD) and is required for the stability of claudin-7 and claudin-1, which are often dysregulated or lost in carcinogenesis. Here, we investigated the effects of Trop-2 phosphorylation on cell motility. Analyses using HCT116 cells expressing WT Trop-2 (HCT116/WT) or Trop-2 alanine-substituted at Ser-303 (HCT116/S303A) or Ser-322 (HCT116/S322A) revealed that Trop-2 is phosphorylated at Ser-322. Furthermore, coimmunoprecipitation and Transwell assays indicated that Trop-2 S322A interacted with claudin-7 the strongest, and a phosphomimetic variant, Trop-2 S322E, the weakest and that HCT116/S322E cells have the highest motility and HCT116/S322A cells the lowest. All cell lines had similar levels of claudin-7 mRNA, but levels of claudin-7 protein were markedly decreased in the HCT116/S322E cells, suggesting posttranscriptional control of claudin-7. Moreover, claudin-7 was clearly localized to cell-cell borders in HCT116/S322A cells but was diffusely distributed on the membrane and partially localized in the cytoplasm of HCT116/S322E and HCT116/WT cells. These observations suggested that Trop-2 phosphorylation plays a role in the decrease or mislocalization of claudin-7. Using protein kinase C (PKC) inhibitors and PKC-specific siRNAs, we found that PKCα and PKCδ are responsible for Trop-2 phosphorylation. Of note, chemical PKC inhibition and PKCα- and PKCδ-specific siRNAs reduced motility. In summary, our findings provide evidence that Trop-2 is phosphorylated at Ser-322 by PKCα/δ and that this phosphorylation enhances cell motility and decreases claudin-7 localization to cellular borders.


Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Claudinas/genética , Claudinas/metabolismo , Células HCT116 , Humanos , Fosforilação , Ligação Proteica
3.
J Biol Chem ; 290(43): 26125-40, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26342075

RESUMO

Both mucin 1 (MUC1) and galectin-3 are known to be overexpressed in various malignant tumors and associated with a poor prognosis. It has been extensively reported that MUC1 is involved in potentiation of growth factor-dependent signal transduction. Because some carbohydrate moieties carried on MUC1 change to preferable ones for binding of galectin-3 in cancer cells, we speculated that MUC1-mediated signaling may occur through direct binding of galectin-3. Immunochemical studies showed that the distribution of galectin-3 coincided with that of MUC1 in various human tumor tissues but not in human nonmalignant tissues, and the level of galectin-3 retained on the surface of various cancer cells paralleled that of MUC1. Treatment of MUC1-expressing cells with galectin-3 induced phosphorylation of ERK1/2 and Akt following enhanced phosphorylation of MUC1 C-terminal domain, consistently promoting tumor cell malignancy. It is also noted that this enhanced phosphorylation occurred independently of EGF receptor-mediated signaling in both EGF receptor- and MUC1-expressing cells, and multivalency of galectin-3 was important for initiation of MUC1-mediated signaling. Expectedly, both silencing of endogenous galectin-3 and treatment with galectin-3 antagonists down-regulated cell proliferation of MUC1-expressing cells. These results suggest that the binding of galectin-3 to MUC1 plays a key role in MUC1-mediated signaling. Thus, constitutive activation of MUC1-mediated signaling in an autocrine/paracrine manner caused by ligation of galectin-3 promotes uncontrolled tumor cell malignancy. This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated signaling pathway.


Assuntos
Neoplasias do Colo/patologia , Galectina 3/metabolismo , Sistema de Sinalização das MAP Quinases , Mucina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proliferação de Células , Neoplasias do Colo/enzimologia , Neoplasias do Colo/metabolismo , Células HCT116 , Humanos , Fosforilação , Ligação Proteica , Transdução de Sinais
4.
J Biol Chem ; 289(36): 25341-50, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25059667

RESUMO

When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.


Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Membrana Celular/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Interleucina-2/metabolismo , Ligantes , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Microscopia Confocal , Modelos Biológicos , Mutação , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Receptor 4 Toll-Like/genética
5.
J Biol Chem ; 289(51): 35193-204, 2014 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-25371209

RESUMO

Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells.


Assuntos
Movimento Celular/genética , Mucina-1/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição RelA/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Immunoblotting , Imuno-Histoquímica , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Mucina-1/metabolismo , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenilenodiaminas/farmacologia , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
6.
Biochim Biophys Acta ; 1840(6): 1790-7, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24561267

RESUMO

BACKGROUND: Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3. METHODS: A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated ß-catenin. RESULTS: Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that ß-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells. CONCLUSIONS: Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of ß-catenin to MUC1-CD. GENERAL SIGNIFICANCE: This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.


Assuntos
Galectina 3/fisiologia , Mucina-1/fisiologia , beta Catenina/metabolismo , Animais , Camundongos , Mucina-1/química , Células NIH 3T3 , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia
7.
Int J Cancer ; 136(8): 1985-90, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25197000

RESUMO

The epithelial ovarian carcinoma (EOC) is an aggressive malignant tumor, and is currently the leading cause of gynecologic cancer death. CA125 is the most commonly used serum marker for EOC, but shows a high-false-positive rate for several benign diseases such as endometriosis. The purpose of our study is therefore to identify a useful biochemical tool for detecting qualitative differences between CA125 from patients with endometriosis and EOC, and to facilitate differential diagnosis of these diseases. In our study, using two different CA125-binding molecules, i.e., recombinant mesothelin and an anti-CA125 monoclonal antibody, a novel sandwich ELISA for determining the serum levels of CA125 with mesothelin-binding ability (CA125(meso) ) was developed, and tested for patients with endometriosis (n = 59) and EOC (n = 36). We found that both the serum CA125(meso) level and the ratio of the serum CA125(meso) to CA125 levels (CA125(meso) /CA125) were significantly higher in patients with EOC than in patients with endometriosis (p < 0.00005 and p < 0.000001, respectively). Furthermore, receiver operating characteristic analysis showed that the CA125(meso) assay was superior to the conventional antibody-based CA125 assay in discriminating endometriosis from EOC. Thus, mesothelin-binding ability may be a useful indicator for qualitatively evaluating CA125 in patients with endometriosis and EOC.


Assuntos
Antígeno Ca-125/sangue , Antígeno Ca-125/metabolismo , Endometriose/sangue , Endometriose/metabolismo , Proteínas Ligadas por GPI/metabolismo , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário , Feminino , Humanos , Mesotelina , Pessoa de Meia-Idade , Mucinas/metabolismo , Curva ROC , Adulto Jovem
8.
J Biol Chem ; 288(44): 31842-52, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24045940

RESUMO

Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, ß-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of ß-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited ß-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Mucina-1/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HEK293 , Humanos , Camundongos , Mucina-1/genética , Mucina-1/imunologia , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , beta Catenina/genética , beta Catenina/imunologia
9.
J Biol Chem ; 288(33): 24264-76, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23814067

RESUMO

We demonstrated previously that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in a DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan 1 in highly metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed the roles of tTn antigen in cancer properties. tTn antigen on Syndecan 1 increased cell adhesion to fibronectin in an integrin-dependent manner. Furthermore, cell adhesion to fibronectin induced phosphorylation of focal adhesion kinase and paxillin in T13-transfectant cells. In the search of Syndecan 1-interacting molecules, it was demonstrated that tTn antigen-carrying Syndecan 1 interacted with integrin α5ß1 and matrix metalloproteinase 9 and that these molecules shifted to a glycolipid-enriched microdomain/rafts along with increased metastatic potential in T13-transfectant cells. We also identified a tTn substitution site on Syndecan 1, demonstrating that tTn on Syndecan 1 is essential for the interaction with integrin α5ß1 as well as for the reaction with mAb MLS128. These data suggest that high expression of the ppGalNAc-T13 gene generates tTn antigen on Syndecan 1 under reduced expression of GM1, leading to enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5ß1, Syndecan 1, and MMP-9 in the glycolipid-enriched microdomain/rafts.


Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Integrina alfa5beta1/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Multimerização Proteica , Sindecana-1/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/genética , Sequência de Bases , Carcinoma Pulmonar de Lewis/enzimologia , Adesão Celular/efeitos dos fármacos , Fibronectinas/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Invasividade Neoplásica , Metástase Neoplásica , Paxilina/metabolismo , Fosfotirosina/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Polipeptídeo N-Acetilgalactosaminiltransferase
10.
Biochem Biophys Res Commun ; 434(2): 376-81, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23567969

RESUMO

Previously we demonstrated that prohibitin-1 and -2 (prohibitins) were expressed on the surface of T cell leukemia cell lines and activated T lymphocytes. In the present study, we found that prohibitins play a role as counter receptors for Siglec-9 expressed on macrophages and dendritic cells. Siglec-9 bound to prohibitins in a sialic acid-independent manner. Mutated Siglec-9 with Arg(120) changed to Ala lost the binding activity, suggesting a specific ionic peptide-peptide interaction. Phosphorylation of ERK1/2 in Jurkat cells on treatment with anti-CD3 antibody immobilized beads was markedly diminished on treatment with anti-CD3 antibody and Siglec-9 co-immobilized beads, indicating that engagement of prohibitins with Siglec-9 inhibits ERK1/2 phosphorylation. Phosphorylation of c-Raf was also reduced, maybe due to inhibition of the c-Raf-prohibitin interaction by Siglec-9 ligation. In parallel with inhibition of the ERK cascade, IL-2 production was markedly decreased in Jurkat cells. Thus, this interaction may be a useful immunotherapeutic target.


Assuntos
Sistema de Sinalização das MAP Quinases , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Repressoras/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Complexo CD3/metabolismo , Células CHO , Cricetinae , Humanos , Células Jurkat , Ligantes , Ativação Linfocitária , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Proibitinas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Transfecção
11.
Biochem Biophys Res Commun ; 420(2): 275-80, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22417827

RESUMO

Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade.


Assuntos
Membrana Celular/imunologia , Ativação Linfocitária , Proteínas Repressoras/biossíntese , Linfócitos T/imunologia , Animais , Anticorpos , Feminino , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Proibitinas , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/imunologia
12.
Biochem Biophys Res Commun ; 419(1): 7-13, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22306014

RESUMO

In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80 kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.


Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Carcinoma Pulmonar de Lewis/secundário , Neoplasias Pulmonares/patologia , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores/química , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/genética , Linhagem Celular Tumoral , Colágeno/química , Colágeno/metabolismo , Combinação de Medicamentos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Laminina/química , Laminina/metabolismo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Camundongos , N-Acetilgalactosaminiltransferases/genética , Invasividade Neoplásica , Multimerização Proteica , Proteoglicanas/química , Proteoglicanas/metabolismo , Sindecana-1/química , Sindecana-1/metabolismo
13.
Int J Gynecol Cancer ; 22(4): 531-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22367369

RESUMO

OBJECTIVE: Although CA125 antigen is a useful marker for ovarian cancer, its expression is also elevated in endometriosis. The purpose of this study was to develop an assay method for evaluating differentially glycosylated MUC16 (CA125 core protein) in patients with endometriosis and ovarian cancer. MATERIALS AND METHODS: We prepared MUC16-enriched fractions from peritoneal fluid of patients with endometriosis and conditioned medium of ovarian carcinoma-3 cells by gel filtration, and evaluated the expression of sialyl-Le, Tn, and sialyl-Tn antigens by dot blot analysis. A sandwich enzyme-linked immunosorbent assay was developed to measure the level of sialyl-Tn antigen expressed on MUC16 (sTn/MUC16). The level of sTn/MUC16 was compared between patients with endometriosis (n = 21) and ovarian cancer (n = 36) and in ovarian cancers with different clinical diagnostic criteria. Furthermore, distribution of MUC16 and sialyl-Tn antigen in ovarian cancer tissues was observed immunohistochemically. RESULTS: Sialyl-Tn antigen was markedly detectable in the MUC16-enriched fractions from conditioned medium of ovarian carcinoma-3 cells but negligible in those from the peritoneal fluid of the patients with endometriosis. The level of sTn/MUC16 determined by a sandwich enzyme-linked immunosorbent assay was significantly higher in the patients with ovarian cancer than that in the patients with endometriosis (P < 0.001). An elevated level of sTn/MUC16 was detected in 44% of the patients with ovarian cancer but not all the patients with endometriosis. This level increased more prominently in the patients with ovarian cancer than that of MUC16 as both the clinical stage and cytological grade advanced. An elevated level of sTn/MUC16 was frequently found in the patients with serous and endometrioid carcinomas. Consistent with this, sialyl-Tn antigen was colocalized with MUC16 in serous and endometrioid ovarian cancer tissues. CONCLUSIONS: Estimation of the sTn/MUC16 level may be useful for discriminating endometriosis from ovarian cancer and for evaluating the clinical stage, cytological grade, and histological type of ovarian cancer.


Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Líquido Ascítico/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno Ca-125/metabolismo , Endometriose/metabolismo , Proteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Western Blotting , Meios de Cultivo Condicionados/farmacologia , Endometriose/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/diagnóstico , Prognóstico , Células Tumorais Cultivadas
14.
Biochem Biophys Res Commun ; 402(4): 663-9, 2010 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-20971061

RESUMO

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.


Assuntos
Antígenos CD/metabolismo , Carcinoma/imunologia , Células Dendríticas/imunologia , Lectinas/metabolismo , Monócitos/imunologia , Mucinas/metabolismo , Neoplasias/imunologia , Evasão Tumoral , Linhagem Celular Tumoral , Humanos , Imunomodulação , Mucinas/sangue , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico
15.
Biochem J ; 417(3): 673-83, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18925876

RESUMO

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to alpha2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.


Assuntos
Adenocarcinoma/metabolismo , Linfócitos B/imunologia , Mucinas/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Baço/imunologia , Adenocarcinoma/imunologia , Animais , Antígenos T-Independentes/imunologia , Antígenos T-Independentes/metabolismo , Linfócitos B/metabolismo , Sítios de Ligação , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Transdução de Sinais
16.
Stem Cells ; 26(3): 798-809, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18079434

RESUMO

Chondroitin/dermatan sulfotransferases (C/D-STs) underlie the synthesis of diverse sulfated structures in chondroitin/dermatan sulfate (CS/DS) chains. Recent reports have suggested that particular sulfated structures on CS/DS polymers are involved in the regulation of neural stem cell proliferation. Here, we examined the gene expression profile of C/D-STs in the neurogenic regions of embryonic and adult mouse central nervous system. Using reverse transcription-polymerase chain reaction analysis, all presently known C/D-STs were detected in the dorsal and ventral telencephalon of the embryonic day 13 (E13) mouse embryo, with the exception of chondroitin 4-O-sulfotransferase (C4ST)-3. In situ hybridization for C4ST-1, dermatan 4-O-sulfotransferase-1, chondroitin 6-O-sulfotransferase (C6ST)-1 and -2, and uronosyl 2-O-sulfotransferase revealed a cellular expression of these sulfotransferase genes in the embryonic germinal zones of the forebrain. The expression of multiple C/D-STs is maintained on cells residing in the adult neural stem cell niche. Neural stem cells cultured as neurospheres maintained the expression of these enzymes. Consistent with the gene expression pattern of C/D-STs, disaccharide analysis revealed that neurospheres and E13 mouse brain cells synthesized CS/DS chains containing monosulfated, but also significant amounts of disulfated, disaccharide units. Functionally, the inhibition of sulfation with sodium chlorate resulted in a significant, dose-dependent decrease in neurosphere number that could not be rescued by the addition of individual purified glycosaminoglycan (GAG) chains, including heparin. These findings argue against a simple charge-based mechanism of GAG chains in neural stem cell maintenance. The synergistic activities of C/D-STs might allow for the adaptive modification of CS/DS proteoglycans with diversely sulfated CS/DS chains in the extracellular microenvironment that surrounds neural stem cells.


Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/enzimologia , Sulfatos de Condroitina/metabolismo , Neurônios/citologia , Células-Tronco/citologia , Sulfotransferases/metabolismo , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/enzimologia , Movimento Celular/efeitos dos fármacos , Sistema Nervoso Central/citologia , Cloratos/farmacologia , Meios de Cultivo Condicionados , Dermatan Sulfato/metabolismo , Dissacarídeos , Epitopos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Camundongos , Neurônios/efeitos dos fármacos , Isoformas de Proteínas/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Células-Tronco/efeitos dos fármacos , Sulfotransferases/genética
17.
Proteomics ; 8(16): 3342-9, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18690650

RESUMO

Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.


Assuntos
Apoptose/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Mucina-2/farmacologia , Anticorpos Monoclonais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Dendríticas/citologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Lectinas/genética , Lectinas/imunologia , Lectinas/metabolismo , Monócitos/citologia , Mucina-2/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Células U937
18.
Biochem Biophys Res Commun ; 372(1): 45-50, 2008 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-18474217

RESUMO

Epithelial cancer cells secrete mucins carrying carbohydrate antigens such as a sialyl-Tn antigen into cancer tissues and/or the bloodstream, in which mucins may interact with CD22 (Siglec-2). Mucins isolated from colon cancer cells and bovine submaxillary mucins bound to CD22 cDNA transfectants and a human B cell line, Daudi cell, and the binding of soluble recombinant CD22 to the mucins was confirmed by means of a plate assay. The binding specificity was demonstrated by the fact that the mucins bound to the recombinant CD22 with an intact ectodomain but not to that with a mutated ectodomain. Daudi cells were stimulated with anti-IgM F(ab')(2) in the presence or absence of mucins. Ligation of mucins to CD22 decreased the phosphorylation of CD22 and SHP-1 recruitment, and the phosphorylation of ERK-1/2 prominently. The in vivo effect of mucins on splenic B cells in the tumor-bearing state was investigated using mucin-producing (TA3-Ha) and non-producing (TA3-St) mammary adenocarcinoma-bearing mice. When fluorescence-labeled epiglycanins were administered to normal mice, a portion of them was taken up by the spleen and became associated with splenic B cells. We found that splenic B cells were reduced in TA3-Ha-bearing mice but not in TA3-St-bearing ones. These results suggest that in the tumor-bearing state a portion of the mucins in the bloodstream was taken up by the spleen and ligated to CD22 expressed on splenic B cells, which may have led to down-regulation of signal transduction.


Assuntos
Linfócitos B/imunologia , Neoplasias do Colo/imunologia , Mucinas/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Animais , Bovinos , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Tolerância Imunológica , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Baço/imunologia
19.
Biochem J ; 383(Pt 1): 129-38, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15198637

RESUMO

We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG-neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth.


Assuntos
Proteínas de Transporte/metabolismo , Cerebelo/ultraestrutura , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteoglicanas de Heparan Sulfato/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular Tumoral , Cerebelo/citologia , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Heparina/metabolismo , Lectinas Tipo C , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/fisiologia , Neurocam , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Sindecana-3
20.
Eur J Cell Biol ; 92(8-9): 257-63, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24246580

RESUMO

MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and ß-catenin at the cell-cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca(2+)-dependent cell-cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/ß-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell-cell adhesion of epithelial cancer cells.


Assuntos
Antígeno Ca-125/metabolismo , Células Epiteliais/fisiologia , Quinases da Família src/metabolismo , Antígeno Ca-125/genética , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Membrana Celular/metabolismo , Movimento Celular , Citoplasma/metabolismo , Células HCT116 , Humanos , Células MCF-7 , beta Catenina/genética , beta Catenina/metabolismo , Quinases da Família src/genética
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa