RESUMEN
BACKGROUND: The use of adenoviral vector for gene therapy is still an important strategy for advanced cancers, however, the lack of the requisite coxsackie-adenovirus receptor in cancer cells and host immune response to adenovirus limit the application of adenoviral vector in vivo. METHOD: We designed the antiangiogenic gene therapy with recombinant PEDF adenovirus (Ad-PEDF) encapsulated in cationic liposome (Ad-PEDF/Liposome), and investigated the anti-tumor efficacy of Ad-PEDF/Liposome complex on inhibition of tumor metastasis. RESULTS: We found that systemic administration of Ad-PEDF/liposome was well tolerated and resulted in marked suppression of tumor growth, and was more potent than uncoated Ad-PEDF to induce apoptosis in B16-F10 melanoma cells and inhibit murine pulmonary metastases in vivo. After Ad-luciferase was encapsulated with liposome, its distribution decreased in liver and increased in lung. The anti-Ad IgG level of Ad-PEDF/Liposome was significantly lower than Ad-PEDF used alone. CONCLUSION: The present findings provide evidences of systematic administration of cationic liposome-encapsulated Ad-PEDF in pulmonary metastatic melanoma mice model, and show an encouraging therapeutic effect for further exploration and application of more complexes based on liposome-encapsulated adenovirus for more cancers.
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Adenoviridae/genética , Proteínas del Ojo/genética , Terapia Genética/métodos , Liposomas/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Melanoma/patología , Melanoma/terapia , Factores de Crecimiento Nervioso/genética , Serpinas/genética , Animales , Cationes , Femenino , Vectores Genéticos , Inmunoglobulina G/química , Liposomas/química , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Metástasis de la NeoplasiaRESUMEN
Hepatocellular carcinoma is one of the most common cancers in worldwide. We previously reported a novel thienopyridine derivative 3-amino-6-(3,4-dichlorophenyl) thieno[2,3-b]pyridine-2-carboxamide (SKLB70359) which possesses anticancer activity against hepatocellular carcinoma. In present study, we further investigated its anticancer activity and possible mechanism. The SKLB70359 treatment decreased the viability of a panel of hepatocellular carcinoma cell lines in a concentration- and time-dependent manner with IC(50) 0.4 ~ 2.5 µM. The mechanism study showed that SKLB70359 induced G0/G1 cell cycle arrest and then led to apoptotic cell death of HepG2 cell. The SKLB70359 induced G0/G1 cell cycle arrest was characterized by down-regulation of cyclin-dependent kinase 2 (CDK2), CDK4, CDK6 expression and up-regulation of p53, p21(WAF1). Activating of caspase-3 and caspase-9 was also observed. Meanwhile, proliferation inhibitory effect of SKLB70359 was associated with decreased level of phosphorylated p44/42 mitogen activated protein kinase (p44/42 MAPK) and phosphorylated retinoblastoma protein (Rb). Moreover, SKLB70359 exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, the findings in this study suggested that SKLB70359 have potential anticancer efficacy via G0/G1 cell cycle arrest and apoptosis induction. Its potential to be a candidate of anticancer agent is worth being further investigated.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Piridinas/farmacología , Tiofenos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Piridinas/química , Piridinas/uso terapéutico , Proteína de Retinoblastoma/metabolismo , Tiofenos/química , Tiofenos/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Antagonizing angiogenesis-related receptor tyrosine kinase is a promising therapeutic strategy in oncology. In present study, we designed and synthesized a novel vascular endothelial growth factor receptor (VEGFR) inhibitor N-methyl-4-(4-(3-(trifluoromethyl) benzamido) phenoxy) picolinamide SKLB610 that potently suppresses human tumor angiogenesis. SKLB610 inhibited angiogenesis-related tyrosine kinase VEGFR2, fibroblast growth factor receptor 2 (FGFR2) and platelet-derived growth factor receptor (PDGFR) at rate of 97%, 65% and 55%, respectively, at concentration of 10µM in biochemical kinase assays. In vitro, SKLB610 showed more selective inhibition of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) proliferation, and this proliferation inhibitory effect was associated with decreased phosphorylation of VEGFR2 and p42/44 mitogen-activated protein kinase (p42/44 MAPK). Antiangiogenic evaluation showed that SKLB610 inhibited the HUVECs capillary-tube formation on Matrigel in vitro and the sub-intestinal vein formation of zebrafish in vivo. Moreover, SKLB610 inhibited a panel of human cancer cells proliferation in a concentration-dependent manner and human non-small cell lung cancer cell line A549 and human colorectal cancer cell line HCT116 were most sensitive to SKLB610 treatment. In vivo, chronic intraperitoneally administration of SKLB610 at dose of 50mg/kg/d resulted in significant inhibition in the growth of established human A549 and HCT116 tumor xenografts in nude mice without exhibit toxicity. Histological analysis showed significant reductions in intratumoral microvessel density (CD31 staining) of 43-55% relative to controls depending on the specific tumor xenografts. In conclusion, the present study demonstrated that SKLB610 exhibited its antitumor activity as a multi-targeted inhibitor with more potent inhibition of VEGFR2 activity. Its potential to be a candidate of anticancer agent is worth being further investigated.
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Inhibidores de la Angiogénesis/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Ácidos Picolínicos/farmacología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Amidas/síntesis química , Amidas/farmacología , Inhibidores de la Angiogénesis/síntesis química , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Combinación de Medicamentos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Laminina/metabolismo , Ratones , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Fosforilación/efectos de los fármacos , Ácidos Picolínicos/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteoglicanos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Venas Umbilicales/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Pez CebraRESUMEN
Zinc finger proteins have been implicated as transcription factors in the differentiation and development of cells and tissues in higher organisms. The classical C2H2 zinc finger motif is one main type of motif of zinc finger proteins. Our previous studies have shown that Zfp637, which comprises six consecutively typical and one atypical C2H2 zinc finger motifs, is highly expressed in undifferentiated or poorly differentiated cell lines, but is moderately or slightly expressed in normal tissues. We have also demonstrated that Zfp637 can promote cell proliferation. However, its role in the regulation of cell differentiation remains unknown. We report here that endogenous Zfp637 as well as mTERT is expressed in proliferating C2C12 myoblasts and that their expression is downregulated during myogenic differentiation. Constitutive expression of Zfp637 in C2C12 myoblasts increased mTERT expression and telomerase activity, and promoted the progression of the cell cycle and cell proliferation. By contrast, endogenous repression of Zfp637 expression by RNA interference downregulated the mTERT gene and the activity of telomerase, and markedly reduced cell proliferation. Overexpression of Zfp637 also inhibited the expression of myogenic differentiation-specific genes such as MyoD and myogenin, and prevented C2C12 myoblast differentiation. Our results suggest that Zfp637 inhibits muscle differentiation through a defect in the cell cycle exit by potentially regulating mTERT expression in C2C12 myoblasts. This may provide a new research line for studying muscle differentiation.
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Diferenciación Celular/fisiología , Proteínas de Unión al ADN/fisiología , Músculo Esquelético/citología , Dedos de Zinc , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Proliferación Celular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ratones , Músculo Esquelético/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismoRESUMEN
BACKGROUND: Psoriasis is a chronic and relapsing inflammatory skin disease associated with various immunologic abnormalities. Repeated subcutaneous injection of interleukin-4 (IL-4) has been established as an effective treatment to counteract psoriasis. OBJECTIVE: We investigated whether gene therapy using IL-4 expression plasmid (pIL-4) via transdermal delivery was an alternative treatment for psoriasis. In our experiment, dimethylsulfoxide (DMSO) was used as a penetration enhancer. METHODS: At first, the penetration efficiency of the complex of reporter plasmid accompanied by DMSO was investigated both in vitro and in vivo. Then, the antipsoriasis efficiency of the treatment with pIL-4-DMSO was tested in mice. RESULTS: The expression of the reporter gene was detected in epidermis and dermis both in vitro and in vivo. More importantly, the psoriasis symptoms were relieved, and significant reductions in some psoriasis-associated factors were observed after pIL-4-DMSO treatment. CONCLUSION: We conclude that the topical application of pIL-4-DMSO can treat psoriasis to a significant extent.
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Terapia Genética/métodos , Interleucina-4/genética , Psoriasis/terapia , Transducción Genética/métodos , Administración Cutánea , Animales , Dimetilsulfóxido/química , Femenino , Ratones , Ratones Transgénicos , Plásmidos , Psoriasis/patologíaRESUMEN
OBJECTIVE: To explore the application of MHC-Ig/peptide polymer technique for detecting antigen-specific cytotoxic T lymphocytes (CTLs) in mice with experimental autoimmune uveitis (EAU). METHODS: B10RIII mice were immunized with an interphotoreceptor retinal-binding protein (IRBP) synthetic peptide (IRBP161-180). The in vivo primed T cells were separated and stained with MHC-Ig polymer combined with a panel of truncated peptides derived from IRBP161-180. The level of IRBP-specific CTLs cells was determined by FACS analysis. The CD8+ T cells were isolated from the primed T cells and stimulated with complex polymers containing MHC-Ig and various IRBP-derived peptides. The proliferation of CD8+ T cells was measured by H thymidine incorporation. The production of interferon- (IFN-) in the cell suspensions was measured by ELISA. RESULTS: The IRBP-specific CTLs were detected by MHC-Ig/peptide polymers. The MHC-Ig/IRBP168-177 peptide polymer dtected 12.3% specific CTLs, showing greater ability in stimulating proliferation of CTLs and production of IFN--than the other MHC-Ig/ peptide polymers (P < 0.01). The truncated 10-mer peptide, IRBP168-177, was the major antigenic epitope for the IRBP-specific CTLs. The MHC-Ig/IRBP168-177 peptide polymer detected the highest level(4.9% +/- 1.1%) of specific CTLs from peripheral blood mononuclear cells (PBMCs) at the acute stage of EAU. CONCLUSION: The MHC-Ig polymer technique is an effective instrument for detecting antigen-specific CTLs, with good sensitivity and specificity in EAU studies.
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Enfermedades Autoinmunes/inmunología , Proteínas del Ojo/inmunología , Oligopéptidos/inmunología , Proteínas de Unión al Retinol/inmunología , Linfocitos T Citotóxicos/inmunología , Uveítis/inmunología , Animales , Femenino , Inmunoglobulina G/inmunología , Interferones/biosíntesis , Ratones , Ratones Mutantes , Células Fotorreceptoras de Vertebrados/inmunología , PolímerosRESUMEN
Barbigerone is a naturally occurring isoflavone with antioxidant activity. In present study, we investigated the antitumor activity of barbigerone against murine lung cancer cells LL/2 and the possible mechanism in vitro. Our results showed that barbigerone inhibited LL/2 cells proliferation in a concentration- and time-dependent manner and caused apoptotic death of LL/2 cells. Barbigerone-induced apoptosis was characterized by enhanced mitochondrial cytochrome c release, activation of caspase-3,-9, but not caspase-8. Exposure of LL/2 cells to barbigerone resulted in upregulation of Bcl-2-associated protein (Bax) and down-regulation of Bcl-2. In addition, proliferation inhibitory effect of barbigerone was associated with decreased level of phosphorylated p42/44 mitogen-activated protein kinase (p42/44 MAPK) and phosphorylated Akt. Moreover, barbigerone exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, our results indicated that barbigerone can inhibit murine lung cancer cell proliferation by inducing apoptosis via mitochondrial apoptotic pathway and by decreasing phosphorylated p42/44 MAPK and Akt. Its potential to be a candidate of anti-cancer agent is worth being further investigated.
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Antineoplásicos/toxicidad , Apoptosis , Isoflavonas/toxicidad , Neoplasias Pulmonares/patología , Mitocondrias/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Isoflavonas/química , Isoflavonas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in inducing cell apoptosis during infection. To investigate whether M protein-mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid or the control empty plasmid pcDNA3.1(+) was mixed with cationic liposome and introduced into various tumor cell lines in vitro, including lung cancer cell LLC, A549, colon cancer cell CT26 and fibrosarcoma cell MethA. Our data showed that the M protein induced remarkable apoptosis of cancer cells in vitro compared with controls. Fifty micrograms of plasmid in a complex with 250 microg cationic liposome was injected intratumorally into mice bearing LLC or MethA tumor model every 3 days for 6 times. It was found that the tumors treated with M protein plasmid grew much more slowly, and the survival of the mice was significantly prolonged compared with the mice treated with the control plasmid. In MethA fibrosarcoma, the tumors treated with M protein plasmid were even completely regressed, and the mice acquired longtime protection against the same tumor cell in rechallenge experiments. Both apoptotic cells and CD8(+) T cells were widely distributed in M protein plasmid-treated tumor tissue. Activated cytotoxic T lymphocytes (CTLs) were further detected by means of (51)Cr release assay in the spleen of the treated mice. These results showed that M protein of VSV can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumor effect and warrant its further development in cancer gene therapy.
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Terapia Genética/métodos , Virus de la Estomatitis Vesicular Indiana , Proteínas de la Matriz Viral/uso terapéutico , Animales , Apoptosis , Línea Celular Tumoral , Neoplasias del Colon/terapia , Cricetinae , Humanos , Liposomas/administración & dosificación , Neoplasias Pulmonares/terapia , Ratones , Linfocitos T Citotóxicos/fisiología , Proteínas de la Matriz Viral/administración & dosificaciónRESUMEN
OBJECTIVE: To track T lymphocytes proliferation with vital dye CFSE in experimental autoimmune uveitis (EAU). METHODS: B10RIII mice were immunized with synthetic peptide of interphotoreceptor retinal-binding protein (IRBP161-180) to develop EAU. The proliferation of IRBP-specific T cell subsets was detected by CFSE staining, fluorescent antibody labeling, and flow cytometry. RESULTS: The IRBP-specific T cells divided after 4 days of stimulation with IRBP161-180, halving the fluorescence intensity. The proliferations of CD4+ and CD8+ T cells were asynchronous, with CD8+ T cells dividing more vigorously and having more drop in percentage of parent cells (P < 0.01). CONCLUSION: CFSE-Labeling can detect the proliferation of autoreactive T cells and their subsets in EAU effectively.
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Enfermedades Autoinmunes/inmunología , Proliferación Celular , Fluoresceínas , Succinimidas , Linfocitos T/patología , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/inducido químicamente , Modelos Animales de Enfermedad , Proteínas del Ojo , Femenino , Colorantes Fluorescentes , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos , Proteínas de Unión al Retinol , Linfocitos T/inmunología , Uveítis/inducido químicamenteRESUMEN
OBJECTIVE: To investigate the anti-neoplastic effects of Valproic acid (VPA) on leukemic cells, especially drug-resistant lines, and to investigate whether modulation of GSH-redox status is involved in VPA-induced apoptosis. METHODS: After the treatment of VPA at various concentrations for indicated times, cellular proliferation of the Jurkat, CEM, HL-60, K562, K562/AO2 cells were evaluated via MTT assay; and the activities of Caspase-3, Caspase-8 and Caspase-9 were quantitatively analyzed by colorimetric assay. The morphological change and cell cycle distribution were also examined on Jurkat (Dexamethasone-resistant) and K562/AO2 (Doxorubicin-resistant) cell lines. The levels of intracellular glutathione/glutathione disulfide (GSH/GSSG) and the activities of the typical antioxidant enzymes, i.e., glutathione reductase (GSH-Rd) and glutathione peroxidase (GSH-Px), were measured on cell lysates of Jurkat and K562/AO2 cell lines prior to and after VPA treatment. Apoptosis rates of Jurkat and K562/AO2 cells treated with VPA along or in combination with N-acety-l-cysteine (NAC), catalase (CAT) or DL-buthionine-(S,R)-sulfoximine (BSO) were determined by Annexin V/propidium iodide (PI) staining with flow cytometry analysis. RESULTS: At concentrations comparable with that achieved at clinical settings, VPA inhibited cell proliferation, activated Caspase-3, 8, and 9, and induced cell cycle arrest in Jurkat and K562/AO2. A rapid decrease in GSH-Rd and GSH-Px activities and GSH content in Jurkat and K562/AO2 were detected after VPA treatment. Co-administration of NAC or CAT attenuated VPA-induced apoptosis. CONCLUSION: VPA inhibit cell proliferation, induce cell cycle arrest and apoptosis in drug-resistant leukemic cells. Apoptosis correlates with down-regulation of intracellular GSH and disruption of intracellular GSH-redox balance, possibly through inhibition of glutathione reductase and glutathione peroxidase.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glutatión/metabolismo , Ácido Valproico/farmacología , Dexametasona/farmacología , Doxorrubicina/farmacología , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Células HL-60 , Humanos , Células Jurkat , Células K562RESUMEN
OBJECTIVE: To study the mechanisms of nongenomic effect of 17beta-estradiol on human spermatozoa. METHODS: The intracellular calcium ([Ca2+]i) in the spermatozoa was measured by flow cytometry after the spermatozoa was treated with the inhibitors of trans-membrane signaling transduction pathways and impermeable 17beta-estradiol (E2-BSA). Western blot was used to detect the activation of the signal proteins after the spermatozoa was treated with 1 x 10(-6) mol/L E2-BSA and tamoxifen, an estrogen receptor inhibitor. RESULTS: Adenylyl cyclase (AC) inhibitor SQ22536, phospholipase C (PLC) inhibitor U73122 and protein tyrosine kinase (TPK) inhibitor Genistein all deterred the increase of [Ca2+]i caused by E2-BSA. E2-BSA also increased the PLC protein and PKC protein significantly. Tamoxifen, an antagonist of estrogen receptor, did not inhibit the activation of PLC caused by E2-BSA. CONCLUSION: The E2-BSA has an effect on human spermatozoa in a nongenomic pathway, possibly through the transmembrane signal transduction in relation to AC, PLC and TPK.
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Calcio/metabolismo , Estradiol/farmacología , Transducción de Señal , Espermatozoides/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Adulto , Sitios de Unión , Estrenos/farmacología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirrolidinonas/farmacología , Tamoxifeno/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Chloroquine is an antimalarial drug that has been used in the treatment and prophylaxis of malaria since the 1950s. The present study was undertaken to examine the effects of chloroquine on Bcap-37 human breast cancer cells' growth, cell cycle modulation, apoptosis induction, and associated molecular alterations in vitro. The chloroquine treatment decreased the viability of Bcap-37 cells in a concentration- and time-dependent manner, which correlated with G(2)/M phase cell cycle arrest. The chloroquine-mediated cell cycle arrest was associated with a decrease in protein levels/activity of polo-like kinase 1 (Plk1), phosphorylated cell division cycle 25C (Cdc25C), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated Akt. The chloroquine-treated Bcap-37 cells exhibited a marked decrease in the level of mitochondrial transmembrane potential (DeltaPsim), which was accompanied by the activation of caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Exposure of Bcap-37 cells to chloroquine also resulted in the induction of spindle abnormalities. In conclusion, the findings in this study suggested that chloroquine might have potential anticancer efficacy, which could be attributed, in part, to its proliferation inhibition and apoptosis induction of cancer cells through modulation of apoptosis and cell cycle-related proteins expressions, down-regulation of mitochondrial transmembrane potential (DeltaPsim), and induction of spindle abnormalities.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Cloroquina/farmacología , Fase G2/efectos de los fármacos , Neoplasias de la Mama/enzimología , Caspasa 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Huso Acromático/efectos de los fármacosRESUMEN
PURPOSE: Tie-2 is an endothelium-specific receptor tyrosine kinase known to play a key role in tumor angiogenesis. The present study explores the feasibility of immunotherapy of tumors by using a protein vaccine based on chicken Tie-2 as a model antigen to break the immune tolerance against Tie-2 in a cross-reaction between the xenogeneic homologous and self-Tie-2. EXPERIMENTAL DESIGN AND RESULTS: In this study, a chicken homologous Tie-2 protein vaccine (chTie-2) and a corresponding mouse Tie-2 vaccine as a control were prepared and the antitumor effect of these vaccines was tested in two tumor models (murine B16F10 melanoma and murine H22 hepatoma). Immunotherapy with chTie-2 was found effective in two tumor models. Autoantibodies against mouse Tie-2 were detected in sera of mice immunized with chTie-2 through Western blot analysis and ELISA assay. Anti-Tie-2 antibody-producing B cells were detectable by ELISPOT. Histologic examination revealed that autoantibodies were deposited on the endothelial cells of tumor tissues. Purified immunoglobulins from chTie-2-immunized mice could induce the apoptosis of human umbilical vein endothelial cells in vitro. Importantly, adoptive transfer of purified immunoglobulins led to antitumor effect in vivo; apparently, angiogenesis was significantly inhibited in these tumors. Furthermore, the antitumor activity and production of autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. CONCLUSIONS: Our findings may provide a vaccine strategy for cancer therapy and show the potential utilization of interference with Tie-2 pathway.
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Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia/métodos , Neoplasias Experimentales/terapia , Receptor TIE-2/inmunología , Animales , Apoptosis/efectos de los fármacos , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Línea Celular , Línea Celular Tumoral , Pollos , Humanos , Inmunoglobulinas/inmunología , Inmunoglobulinas/farmacología , Inmunoglobulinas/uso terapéutico , Inmunohistoquímica , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Análisis de Supervivencia , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Factores de Tiempo , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/farmacología , Vacunas de Subunidad/uso terapéuticoRESUMEN
CCL19 [chemokine (C-C motif) ligand 19; also known as MIP-3beta (macrophage inflammatory protein-3beta) or ELC (Epstein-Barr-virus-induced molecule 1 ligand chemokine)], one of the immunostimulatory cytokines, chemoattracts both DCs (dendritic cells) and T-lymphocytes. Adenoviral vector is one of the most used gene delivery vectors for cancer therapy because of its high gene-transfection efficiency. However, its wider application is limited, owing to immune responses that reduce transgene expression and decrease the efficacy of repeated administration. We constructed the recombinant replication deficient adenoviral vectors containing the CCL19 gene (Ad-CCL19) and combined them with PEG-PE [poly(ethylene glycol)-phosphatidylethanolamine]-modified cationic liposomes (Ad-CCL19/PEG-PE) for immunotherapy against murine fibrosarcoma. Although there were hardly any therapeutic differences between Ad-CCL19- and Ad-CCL19/PEG-PE-treated mice that were observed at the second administration, the final results demonstrated that Ad-CCL19/PEG-PE-treated mice survived much longer. The antitumour efficacy may be related to the high level of CCL19 after the final administration and lasting expression of IFN-gamma (interferon-gamma) and IL-12 (interleukin-12) in the Ad-CCL19/PEG-PE-treated group, which were measured by reverse-transcription PCR and ELISA. The results demonstrated that PEG-PE-cationic-liposome-conjugated adenovirus could prolong the expression of the therapeutic gene in vivo and may enhance the antitumour efficacy.
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Adenoviridae/genética , Quimiocinas CC/genética , Quimiocinas CC/uso terapéutico , Marcación de Gen/métodos , Terapia Genética/métodos , Liposomas/química , Neoplasias Pulmonares/terapia , Adenoviridae/química , Animales , Antineoplásicos/uso terapéutico , Cationes , Quimiocina CCL19 , Femenino , Vectores Genéticos/genética , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos BALB C , Transfección/métodos , Resultado del TratamientoRESUMEN
OBJECTIVE: To establish a mouse model of systemic lupus erythematosus(SLE) and to discuss the role of environmental factors in the pathogenesis of SLE. METHODS: Thirty 10-week-old female BALB/c mice were divided into the model group and control group. The model mice received a single intraperitoneal injection of 0. 5 mL of pristane, while control mice received a single i.p. injection of 0. 5 mL of 0.9% NS. Autoantibodies and proteinuria were detected before injection and monthly thereafter. 8 months after injection, all mice were bled to death. Kidneys were excised to observe the histopathologic evidence of glomerulonephritis. RESULTS: After pristane injection, antinuclear antibody(ANA) and anti-dsDNA antibody appeared in sera as early as 3 months and 4 months respectively. At 8 months, the incidence rates of ANA and anti-dsDNA antibody were 87.5% and 47.8% respectively. The protein concentration of urine in most mice was > or =1+ (300 mg/L). Light microscopy and immunofluorescence of kidney sections indicated glomerulonephritis. In control group, only one serum was ANA low-titer positive and another was anti-dsDNA antibody low-titer positive. The protein concentration of urine was < or = 1+. The histopathology and immunofluorescence showed no evidence of glomerulonephritis. Spearman correlation analysis indicated that anti-dsDNA antibody was correlated with proteinuria (r = 0.538, P = 0.003). CONCLUSION: The murine lupus model can be successfully established in female BALB/c mouse with a single i. p. injection of 0. 5 mL of pristane and the specific autoantibody anti-dsDNA for SLE has appeared in sera of BALB/c mice.
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Modelos Animales de Enfermedad , Lupus Eritematoso Sistémico , Terpenos , Animales , Anticuerpos Antinucleares/análisis , Autoanticuerpos/análisis , Femenino , Inmunosupresores , Lupus Eritematoso Sistémico/inducido químicamente , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: To answer the question whether mitochondrial permeability transition (MPT) participates in blue light-induced damage to human retinal pigment epithelium (RPE) cells, this study was directed at assessing the effect of blue light on mitochondrial membrane potential (delta psi(m)) and cytochrome C (Cyt C) of cultured human RPE cells. METHODS: Human RPE cells were exposed to blue light (wave length 470-490 nm); delta psi(m) was measured by rhodamine 123 staining and subsequent flow cytometry. Three groups were investigated: Group A (exposure to different intensity of blue light); group B (exposure to identical intensity for different duration); group C (exposure to identical intensity and duration, different prolongation of post-exposure culture). Cyt C activity was assayed by ELISA. Caspase-3 was detected by colorimetric assay. In these aspects, two groups were investigated: Group I [(2000+/-500) 1x for 6 h]; Group II [(2000+/-500) 1x for 12 h]. RESULTS: When human RPE cells were exposed to blue light, the more pronounced decrease of delta psi(m) was consistent with the increase of light intensity in group A. Pronounced decrease of delta psi(m) was seen at 6 h and 12 h of exposure duration in group B. At 6 h prolongation of post-exposure culture in group C, the decrease of delta psi(m) was observed, lasting 48 h. The concentration of Cyt C was detected; no significant changes were found at 6 h and 12 h prolongation of post-exposure culture, but a significant increase was found at 24 h and 36 h post-exposure in the two groups. The increase was more significant in Group II than in Group I at 24 h post-exposure. The activity change of caspase-3 was not found in the two groups. CONCLUSION: Blue light exposure over threshold can induce damage to human RPE cells, probably by triggering the mitochondrial permeability transition, which results in the decrease of delta psi(m) and the release of cytochrome C.
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Citocromos c/efectos de la radiación , Luz , Epitelio Pigmentado Ocular/efectos de la radiación , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Humanos , Potenciales de la Membrana/efectos de la radiación , Mitocondrias/fisiología , Epitelio Pigmentado Ocular/citologíaRESUMEN
OBJECTIVE: To explore the anticarcinogenic mechanism of 20(R)-ginsenoside Rg3 in induced liver tumor in SD rat. METHODS: Thirty-five SD rats with induced hepatocellular carcinoma were divided into a control group and 3 dosage groups according to the dosing levels of 20(R)-ginsenoside Rg3. The tumour volume was measured by MR imaging. The apoptotic rat and S-phase fraction and diploid of tumor cell were measured with flow cytometry. Protein expression of PCNA and TNF were evaluated with immunohistochemistry. RESULTS: There was significant difference in tumour volume between the high dosage group and the control group. The average apoptotic rates were 11.08+/-3.78, 13.57+/-3.34, 27.35+/-16.04 and the S-phase fractions were 23.98+/-9.44, 19.73+/-6.62, 14.09+/-3.48 in the low-, medium-, and high-dosage groups respectively. The apoptotic rate was significantly higher in the high-dosage group than in the medium-dosage group and low-dosage group. Before-after comparison showed that the anti-proliferative effects of 20(R)-ginsenoside Rg3 were significant in three treatment groups. The higher positive rats of protein expression with PCNA and TNF were significant difference in the high-dosage group compared to those in the low-dosage group. No significant difference between the medium-dosage group and the low-dosage group. CONCLUSION: 20(R)-ginsenoside Rg3 can noticeably inhibit the proliferation of tumor cells, and efficaciously induce the apoptosis and facilitate necrosis of the tumor cells, and there appears to be a dose dependent effect.
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Antineoplásicos Fitogénicos/farmacología , Ginsenósidos/farmacología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Ginsenósidos/uso terapéutico , Neoplasias Hepáticas Experimentales/patología , Masculino , Fitoterapia , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Necrosis Tumoral/metabolismoRESUMEN
BACKGROUND: Psoriasis is an autoimmune disease that is caused by a shift in the Th1/Th2 balance toward Th1-dominant immunity. It has been established as an effective treatment to counteract psoriasis by subcutaneous injection of recombinant interleukin (IL)-4, and IL-4 gene therapy by topical transdermal penetration has shown its antipsoriatic effect in mice. Retinoic acid (RA) and dimethylsulfoxide can increase the efficiency of gene transfection in the topical transdermal delivery system. OBJECTIVE: We investigated whether RA could improve anti-psoriasis efficiency using IL-4 expression plasmid pORF-mIL-4 (pIL-4) via transdermal delivery system in K14-vascular endothelial growth (K14-VEGF) factor transgenic mice. METHODS: After pretreatment with RA, plasmid pIL-4 in 10% dimethylsulfoxide was applied to the ear skin by topical transdermal penetration. Hematoxylin- eosin staining and immunohistochemistry were performed with ear samples to evaluate anti-psoriasis efficiency in mice. RESULTS: The psoriasis pathological features were relieved and psoriasis-associated factors were significantly reduced. CONCLUSION: Our results reveal that topical application of pIL-4 in dimethylsulfoxide by transdermal delivery with RA pretreatment can improve psoriasis significantly.
RESUMEN
AIM: Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis. Tyrosine protein kinase plays an important role in the proliferation, activation of HSC. The purpose of the study is to investigate the effects of the tyrosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC. METHODS: Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Culture-activated HSC were serum-starved and incubated with 10(-9) to 10(-5) mol/L concentration of genistein for 24, 48 or 72 h. In PDGF-induced HSC proliferation, HSC were stimulated with 10 microg.L(-1) PDGF-BB for 15 min, and then treated with genistein for the same time. Cell proliferation was measured by MTT assay and based on flow cytometric analysis of cell cycle. The a-smooth muscle actin (alpha-SMA) expression in HSC was studied with confocal laser microscopy and flow cytometry. c-fos, c-jun and cyclin D(1) expression in HSC was also detected by flow cytometry. RESULTS: Genistein inhibited basal and PDGF-induced proliferation of HSC at the concentration of 10(-8) to 10(-5)mol/L, and treatment with 10(-7) mol/L concentration of genistein for 48 h inhibited the HSC proliferation significantly (the inhibition rate was 70.3 %, P<0.05). Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with 10(-7) mol/L genistein for 48 h suppressed the expression of alpha-SMA significantly in HSC (the specific fluorescence intensity were 60.2+/-21.5 vs 35.3+/-11.6 and 12.8+/-10.4 vs 9.54+/-6.39, respectively, both P<0.05). The intensity of c-fos, c-jun and cyclin D(1) expression of HSCs treated with 10(-7)mol/L genistein for 48 h was also significantly decreased compared with the controls. CONCLUSION: Genistein influences proliferation of HSC, suppresses the expression of alpha-SMA in HSC and t inhibits the intensity of c-fos, c-jun and cyclin D(1) expression of HSCs. Genistein has therapeutic potential against liver fibrosis.
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Genisteína/farmacología , Hepatocitos/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Actinas/metabolismo , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/metabolismo , Inhibidores Enzimáticos/farmacología , Hepatocitos/citología , Hepatocitos/metabolismo , Técnicas In Vitro , Masculino , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick tie-2, the purification, refolding conditions of the recombinant protein, and its anti-angiogeneic effect. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick tie-2 was obtained by PCR amplification using a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector pQE30, and was expressed in E.Coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected s.c. into mouse, and the antibody was detected by ELISA and Western blot analysis. The antibody was purified from the antiserum and then incubated with human umbilical endothelial vein cell (HUEVC) to find its anti-angiogenesis in vitro by using propidium iodide(PI) dying through FACS. Alginate encapsulated tumor cell assays were performed and micro-vessel density was determined by counting per high power field in the sections stained with an antibody reactive to CD31 to test its inhibition of angiogenesis. RESULTS: The recombinant protein was highly expressed in E.Coli XL-1 blue, and the antibody produced in mouse could specifically recognize the recombinant protein. The purified antibody could induce apoptosis of HUEVC in vitro. The anti-angiogenic effect of the antibody could also be found in alginate-encapsulate tumor cell assay and by counting micro-vessel density. CONCLUSION: The protein of extracellular ligand binding domains of chick tie-2 can be expressed at high level in the prokaryotic expression system, and the expressed protein can induce immune response in mouse. Furthermore, the antibody can induce the anti-angiogenic effect.