RESUMEN
Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1ß pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Interleucina-1beta/genética , Neoplasias Pulmonares/tratamiento farmacológico , Oxadiazoles/farmacología , Pirimidinas/farmacología , Sinteninas/genética , Animales , Antineoplásicos/síntesis química , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Quimiocina CCL11/genética , Quimiocina CCL11/inmunología , Quimiocina CCL17/genética , Quimiocina CCL17/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-1alfa/genética , Interleucina-1alfa/inmunología , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/inmunología , Subunidad p19 de la Interleucina-23/genética , Subunidad p19 de la Interleucina-23/inmunología , Interleucina-5/genética , Interleucina-5/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Oxadiazoles/síntesis química , Pirimidinas/síntesis química , Transducción de Señal , Sinteninas/antagonistas & inhibidores , Sinteninas/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Syntenin is a multifunctional cytosolic adaptor protein that contributes to cell migration, proliferation, attachment, and apoptosis, as well as immune response to virus, in vertebrates. However, the functions of syntenin in the antibacterial response of invertebrates remain unclear. In this study, we identified a syntenin-like gene (MjSyn) from the kuruma shrimp (Marsupenaeus japonicus) and detected its function in the antibacterial immunity of shrimp. The full-length MjSyn was 1223 bp with a 963 bp open reading frame that encodes 320 amino acids. The deduced MjSyn proteins contained two atypical PDZ domains (sequence repeat that was first reported in the postsynaptic density protein or PSD-95, DlgA, and ZO-1 protein), an N-terminal domain, and a C-terminal domain. Reverse transcription (RT)-PCR results showed that MjSyn was expressed in all tested tissues. Quantitative real-time PCR analysis revealed that MjSyn transcripts in the hemocyte, gill, and intestine were signiï¬cantly induced at various time points after infection with Staphylococcus aureus and Vibrio anguillarum. The knockdown of the expression of MjSyn by RNA interference resulted in a significant decrease in the phagocytic ability and increased bacteria number in vivo of shrimp. Moreover, the expression of MjCnx, a cytoplasma and membrane location lectin chaperone protein, was inhibited in the MjSyn-knocked down shrimp, which indicated a possible calnexin-related way. Thus, the MjSyn participates in the bacterial clearance response of kuruma shrimp, thereby providing new insight into the function of this kind of important adaptor protein.
Asunto(s)
Bacterias/inmunología , Regulación de la Expresión Génica/inmunología , Branquias/metabolismo , Penaeidae/inmunología , Penaeidae/microbiología , Sinteninas/genética , Animales , Secuencia de Bases , Western Blotting , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Hemocitos/metabolismo , Mucosa Intestinal/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Penaeidae/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Sinteninas/inmunologíaRESUMEN
BACKGROUND: Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund's adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30-37% reduction of worm burden, 38-43% reduction in the number, and 35-37% reduction in the area, of liver granulomas. CONCLUSIONS/SIGNIFICANCE: Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.
Asunto(s)
Antígenos Helmínticos , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni , Sinteninas , Vacunas/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/clasificación , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Schistosoma mansoni/genética , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Esquistosomiasis mansoni/prevención & control , Alineación de Secuencia , Sinteninas/química , Sinteninas/clasificación , Sinteninas/genética , Sinteninas/inmunologíaRESUMEN
Human colostrum contains many bioactive factors that must promote the development of intestinal mucosal immunity in infants. Especially, the presence of certain cytokines such as transforming growth factor (TGF)-beta or IL-10 has been of great interest for IgA production as a function of mucosal immune response. In the present study, we attempted to investigate whether unidentified factors inducing generation of IgA-producing cells from naive B cells might exist in colostrum. For this purpose, colostrum samples were directly added to a culture consisting of naive B cells and dendritic cells from cord blood and CD40 ligand-transfected L cells, comparing with recombinant IL-10 (rIL-10) and/or rTGF-beta. It was noted that most colostrum samples alone were able to induce IgA-secreting cells at higher levels than rIL-10 and/or rTGF-beta. IgA-inducing activity of colostrum was abolished by neither anti-neutralizing mAbs against IL-10 nor TGF-beta, though partially by anti-IL-6 mAb. We prepared partially purified fractions from both pooled colostrums with and without IgA-inducing activity and comparatively performed quantitative proteomic analysis by two-dimensional difference gel electrophoresis followed by liquid chromatography-mass spectrometry. As a result, syntenin-1 was identified as a candidate for IgA-inducing protein in colostrum. Western blot analysis indicated that levels of syntenin-1 in colostrum samples were correlated with their IgA-inducing activities. Moreover, we demonstrated that recombinant syntenin-1 could induce preferentially IgA production from naive B cells. These results suggest that syntenin-1 serves as one of IgA-inducing factors for B cells.
Asunto(s)
Linfocitos B/inmunología , Calostro/inmunología , Sangre Fetal/inmunología , Inmunoglobulina A/biosíntesis , Sinteninas/inmunología , Animales , Linfocitos B/metabolismo , Femenino , Humanos , Inmunoglobulina A/inmunología , Células L , Ratones , EmbarazoRESUMEN
OBJECTIVE: To express the GST fusion protein, GST-Syntenin1 in E. coli, and to prepare the polyclonal antibody of Syntenin1. METHODS: CDS fragment of Syntenin1 was obtained by RT-PCR from normal mouse brain and subcloned into pGEX-4T-2 to generate pGEX-4T-2-Syntenin1 recombinant. The confirmed recombinant was transformed into the BL21 competent cells and induced with IPTG. The recombinant fusion protein was purified with immobilized Glutathione Sepharose and confirmed by SDS-PAGE. The purified fusion protein was mixed with the Freund's adjuvant, and then injected into New Zealand white rabbits by hypodermic injection. The polyclonal antibody titer and specification were identified by Western blot. RESULTS: Syntenin1 polyclonal antibody bind Sytenin1 protein specifically and the antiserum tiger reached to 1 : 20 000. CONCLUSION: The Syntenin1 polyclonal antibody with high titer and high specificity was prepared successfully. This will be very helpful for the further study on Syntenin1 function and molecule mechanism of cancer metastasis.