RESUMEN

BCG-activated macrophages (BAM) could kill the tumor cells through cell-cell contact. In this process membrane proteins play an important role. However, up to date, few membrane proteins were revealed. In this study, we selected a surface molecule named Trim59, which was specifically expressed on BAM membrane (compared with the negative control). We cloned and prokaryoticly expressed the extracellular domain of Trim59, purified the recombinant protein and generated polyclonal antibodies. Immunohistochemistry showed that Trim59 abundantly expressed in spleen, stomach and ovary; intermediately expressed in brain, lung, kidney, muscle and intestine; but not in thymus, liver, heart, uterus. Using the antibodies to block Trim59 on BAM significantly reduced BAM cytotoxicity against MCA207 cells. This demonstrated that Trim59 serves as an indispensable molecule in maintaining BAM activity. Overexpression of Trim59 in Raw264.7 cell line failed to lyse target MCA207 cells, which potentiated Trim59 per se could not enhance macrophage cytotoxicity; on another hand, overexpression of Trim59 enhance the pinocytosis and Phagocytosis activity of Raw-264.7, which imply Trim59 might mediate the cell-molecule interaction. Our results indicate Trim59 might be an essential accessory molecule in mediating BAM tumoricidal functions; and Trim59 is a phagocytosis-correlated molecule.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Citotoxicidad Inmunológica/fisiología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Mycobacterium bovis/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Células Cultivadas , Clonación Molecular , Citotoxicidad Inmunológica/inmunología , Femenino , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fagocitosis/inmunología , Proteínas de Motivos Tripartitos

RESUMEN

P-5m, an octapeptide derived from domain 5 of HKa, was initially found to inhibit the invasion and migration of melanoma cells. The high metastatic potential of melanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in an experimental lung metastasis model, suggesting that P-5m may play an important role in the regulation of tumor metastasis. The aim of this study was to measure the effect of P-5m on tumor metastasis of human hepatocarcinoma cell line (HCCLM3) in vitro and in vivo in a nude mouse model of hepatocellular carcinoma (HCC), and detect the mechanisms involved in P-5m-induced anti-metastasis. By gelatin zymography, matrix metallo-proteinases 2 (MMP-2) activity in HCCLM3 was dramatically diminished by P-5m peptide. In addition, the migration and metastasis of HCCLM3 cells was also inhibited by the peptide in vitro. In an orthotopic model of HCC in nude mice, P-5m treatment effectively reduced the lung metastasis as well as the expression of MMP-2 in the tumor tissues. Overall, these observations indicate an important role for P-5m peptide in HCC invasion and metastasis, at least partially through modulation MMP-2 expression. These data suggests that P-5m may have therapeutic potential in metastatic human hepatocarcinoma.

Asunto(s)

Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Metaloproteinasa 2 de la Matriz/biosíntesis , Oligopéptidos/farmacología , Animales , Productos Biológicos/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Quininógeno de Alto Peso Molecular/genética , Quininógeno de Alto Peso Molecular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia

RESUMEN

In an earlier study, we found PBP inhibited the progress of adjuvant-induced arthritis (AA). This study was aimed at evaluating the inhibitory effects of PBP in terms of NF-κB activation by using immunohistochemical and immunofluorescent technique in vitro and in vivo. IL-1ß and TNF-α in serum were detected by method of ELISA. Immunofluorescent results showed that PBP inhibited NF-κB p65 translocation into nucleus. In vivo imaging showed that treatment with PBP decreased the enzyme labeling signal of NF-κB p65. Immunohistochemical staining revealed that PBP suppressed production of NF-κB p65 subunit in the joints and attenuated the productions of IL-1ß and TNF-α in serum from AA. Moreover, NF-κB p65 nucleus translocation was prevented by simultaneous incubation with PBP and PGE2 was decreased by PBP through a feedback cycle. We report the first confirmation of the mimotope of PGE2 receptor EP4 modulatory action.

Asunto(s)

Artritis Reumatoide/patología , Dinoprostona/metabolismo , FN-kappa B/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Masculino , FN-kappa B/metabolismo , Fragmentos de Péptidos/uso terapéutico , Unión Proteica , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos

RESUMEN

OBJECTIVE: To investigate the therapeutic potential and mechanism of action of the mimotope of PGE(2) receptor EP4 (PBP, named by our team) screened by phage displaying technique in the treatment of adjuvant-induced arthritis (AA). METHODS: Freund's complete adjuvant-induced arthritis was induced in Wistar rats. At the first clinical sign of disease, mice were given with daily injections of PBP or saline for 21 days. Disease progression was monitored by measurement of paw swelling. Inflammation and joint destruction were assessed histologically. The IL-1ß and TNF-α were studied by ELISA in the ankle steeps of arthritis model. The degree of proliferation and apoptosis of synoviocytes of RA patients were assessed by CCK-8 kit and AnnexinV-FITC/PI respectively. RESULTS: PBP-treated animals displayed significantly less cartilage and bone destruction than model controls. Tumor necrosis factor α and IL-1ß expression were reduced after PBP treatment. The proliferation and apoptosis of synoviocytes of RA patients were influenced by PBP. CONCLUSIONS: The data support the view that PBP is a potential therapy for RA that may help to diminish both joint inflammation and destruction. And the activities of PBP are related with the effect on synoviocytes directly.

Asunto(s)

Artritis Reumatoide/terapia , Dinoprostona/metabolismo , Biblioteca de Péptidos , Péptidos/análisis , Péptidos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Artritis Experimental/patología , Artritis Reumatoide/patología , Supervivencia Celular/efectos de los fármacos , Dinoprostona/química , Femenino , Humanos , Inflamación/patología , Interleucina-1beta/metabolismo , Articulaciones/efectos de los fármacos , Articulaciones/patología , Masculino , Ratones , Microscopía Confocal , Persona de Mediana Edad , Modelos Moleculares , Péptidos/química , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/patología , Tarso Animal/efectos de los fármacos , Tarso Animal/patología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

BCG-activated macrophages exerted anti-tumor activities. Cell surface molecules play an important role in mediating endocytosis by macrophages. In the previous study, we identified a group of 454 membrane proteins specifically expressed on BCG-activated mouse macrophages, including a protein named NMAAP1 (novel macrophage activated associated protein). In this study, we aligned the full-length nucleotide sequences of NMAAP1 and its homologous sequences to construct its phylogenetic tree, and cloned the NMAAP1 cDNA from BCG-activated macrophages to generated NMAAP1 fusion protein in Escherichia coli. Purified the fusion protein were applied for generation of polyclonal antibodies. Western-blotting detection showed that the polyclonal antibodies have high specificities to recognize target protein.

Asunto(s)

Regulación Bacteriana de la Expresión Génica , Macrófagos/inmunología , Proteínas de la Membrana/genética , Mycobacterium bovis/inmunología , Filogenia , Proteínas Recombinantes , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/inmunología , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Vectores Genéticos/genética , Activación de Macrófagos/inmunología , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico

RESUMEN

One major mechanism through which macrophages effectively kill tumor cells requires cell to cell contact, indicating that certain molecules expressed on cell surface of activated macrophages may mediate the tumoricidal capability. Tumor necrosis factor (TNF) and nitric oxide (NO) are the two classical mediators of tumor cell death. However, evidence of discrepancy is accumulating indicating these known mediators do not appear to account for the broad and potent tumoricidal activity of macrophages. To obtain a full repertoire of tumoricidal activation-associated membrane proteins, we combined one-dimensional SDS-PAGE with capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this technique, we identified 454 activated macrophage specifically expressed proteins with extremely high confidence, including most known activation markers of macrophages, such as NO synthase (iNOS), Ym1, cyclooxygenase, etc. Membrane bound TNF-alpha was also identified on activated macrophages. However, it was also detected on thioglycolate elicited macrophages, indicating this molecule may not play a key role in conjugation-dependent tumor cell killing. In contrast, although NO has not been assigned as an effector molecule of conjugation-dependent tumoricidal pathway, iNOS was identified from membrane fraction of activated macrophages, suggesting NO may be involved in conjugation-dependent tumoricidal mechanism, because iNOS association with plasma membrane is ideally suited to deliver NO directly into the contacted tumor cells. This research provides not only new insights into macrophage conjugation-dependent tumoricidal mechanisms, but also a valuable data set of macrophage activation associated membrane proteins, thus providing better understanding of the functional mechanisms of macrophages in anti-tumor and other biological processes.

Asunto(s)

Citotoxicidad Inmunológica , Activación de Macrófagos , Macrófagos Peritoneales/inmunología , Neoplasias/inmunología , Proteínas/metabolismo , Proteoma/análisis , Proteómica/métodos , Factor de Necrosis Tumoral alfa/metabolismo , Adyuvantes Inmunológicos , Animales , Línea Celular Tumoral , Supervivencia Celular , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Femenino , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis/inmunología , Proteínas/análisis , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

AIM: To study the cytotoxic effects of doxorubicin on apoptosis in glioma cell lines U343, U138, U373 induced by anti-human DR4/DR5 monoclonal antibodies (FMU1.4/FMU1.5) and the underlying mechanism. METHODS: Expression of DR4/DR5 was quantitated by flow cytometry. Cytotoxicity exerted by FMU1.4/FMU1.5 on three cell lines was measured by MTT colorimetry and the induced apoptosis was determined by agarose gel electrophoresis. The expression of cytochrome C, FLIP and Ca2+ concentration were also measured. RESULTS: Following the treatment of doxorubicin DR4 and DR5 were highly expressed on the cell surface; The apoptosis of U138 and U373 induced by FMU1.4 and FMU1.5 was stronger. expression of cytochrome C and Ca2+ concentration were enhanced, whereas the expression of FLIP was downregulated. CONCLUSION: Subtoxic doxorubicin applied with antibodies caused higher cell death rate of glioma cells, which may be relevant to DR4/DR5, the release of cytochrome C and FLIP and Ca2+ concentration.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Electroforesis en Gel de Agar , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Glioma/ultraestructura , Humanos , Microscopía Electrónica de Transmisión

RESUMEN

Macrophages are involved in many important biological processes and membrane proteins are the key effector molecules for their function. However, membrane proteins are difficult to analyze by 2-DE based methods because of their intrinsic tendency to self-aggregate during the first dimension separation (IEF). To circumvent this, we combined one-dimensional SDS-PAGE with capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this technique, we identified 458 GO annotated membrane proteins with extremely high confidence, including most known markers of peritoneal macrophages (e.g., CD11b, F4/80, CD14, CD18, CD86, CD44, CD16 and Toll-like receptor). Thirteen other CD antigens (CD243, CD98, CD107a, CD107b, CD36, CD97, CD205, CD206, CD180, CD191, CD300, CD45and CD29), and 18 Ras-related small GTPases were also identified. In addition to those known macrophage membrane proteins, a significant number of novel proteins have also been identified. This research not only provides a technique to study membrane proteins, but also a valuable dataset of macrophage antigens, thus providing better understanding of the functional mechanisms of macrophages in many biological processes.

Asunto(s)

Antígenos de Superficie/análisis , Macrófagos Peritoneales/inmunología , Proteómica/métodos , Animales , Antígenos CD/análisis , Electrocromatografía Capilar/métodos , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP Monoméricas/análisis , Espectrometría de Masas en Tándem/métodos