RESUMO
BACKGROUND: Key role of angiogenesis in tumor growth and metastasis based on accumulating evidence and recent progress of immunotherapy have led us to investigate vaccine therapy targeting tumor angiogenesis. METHODS: C57BL/6J mice were vaccinated with a syngeneic endothelial cell line Tpit/E by subcutaneous injection once a week. Prior to ninth vaccination, the mice were challenged with B16/F10 melanoma cells by subcutaneous inoculation on the back for the tumor growth model or by tail venous injection for the lung metastasis model. Development of subcutaneous tumor and lung metastasis was monitored by computed tomography scanning, which enabled accurate evaluation with the minimized sacrifice of mice. RESULTS: Vaccination with Tpit/E cells inhibited subcutaneous tumor growth and appearance of lung metastasis compared to control. Survival period was elongated in the Tpit/E vaccination in both of the two models. We also obtained hybridomas secreting specific antibodies to Tpit/E cells from a mouse vaccinated with the cells, indicating that specific immune response to the syngeneic endothelial cells was elicited. CONCLUSION: These results suggest that vaccination with an autologous endothelial cell line may be effective against melanoma.
Assuntos
Vacinas Anticâncer/imunologia , Células Endoteliais/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma Experimental/terapia , Animais , Especificidade de Anticorpos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapiaRESUMO
The mitogen-activated protein kinases (MAPKs) signaling pathway is a potential target in cancer therapy. Constitutive phosphorylated extracellular signal-regulated kinase (ERK1/2), which is one of the MAPKs has been detected in a variety of tumors. Calcitonin (CT) is a polypeptide hormone secreted by the thyroid gland and has been used to treat the osteoporosis and humoral hypercalcemia of malignancy. We report that CT decreases ERK1/2 phosphorylation in cancer cells showing constitutive phosphorylated ERK1/2. In MDA-MB-231 cells, a breast cancer cell line showing constitutive phosphorylated ERK1/2, CT phosphorylated c-Raf at Ser(259) via the protein kinase A pathway, resulting in suppression of ERK1/2 phosphorylation. CT significantly reduced the tumor volume of MDA-MB-231 cells showing constitutive phosphorylated ERK1/2 compared with saline buffer. However, CT did not exert any significant effects on the proliferation of MCF-7 cells, a breast cancer cell line, showing non-constitutive phosphorylated ERK1/2. These novel findings indicate that CT may be used to target ERK in the treatment of cancer.
Assuntos
Calcitonina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/enzimologia , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativadores de Enzimas/farmacologia , Feminino , Humanos , MAP Quinase Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
OBJECTIVE: We assessed the feasibility and efficacy of dendritic cell (DC) therapy for advanced thyroid papillary and follicular cancer. DESIGN: Six Japanese patients (2 men and 4 women; aged 46-72 years, mean 60 years), who were diagnosed as advanced thyroid cancer with refractory distant metastases (papillary, n=5; follicular, n=1), were enrolled. Patients were first vaccinated weekly for 4 weeks with 10(7) autologous tumor lysate-pulsed monocyte-derived mature DCs followed by fortnightly vaccinations for 8 weeks (total=8 vaccinations). Lowdose (350 KIU) interleukin-2 was also administered for 3 days at each vaccination. Clinical response, adverse effects, delayed-type hypersensitivity skin testing (DTH), and IFN-( ) production by peripheral CD3(+) lymphocytes were evaluated. MAIN OUTCOME: Of the 6 patients, disease was assessed as stable in 2 and as progressive in 4. No adverse events were observed. Results of DTH and IFN-( ) production in peripheral lymphocytes did not correlate to the clinical response. CONCLUSIONS: DC immunotherapy could be administered to patients with thyroid papillary or follicular cancer without substantial side effects.
Assuntos
Vacinas Anticâncer/administração & dosagem , Carcinoma Papilar, Variante Folicular/terapia , Células Dendríticas/transplante , Neoplasias Pulmonares/terapia , Neoplasias da Glândula Tireoide/terapia , Idoso , Vacinas Anticâncer/efeitos adversos , Carcinoma Papilar, Variante Folicular/imunologia , Carcinoma Papilar, Variante Folicular/secundário , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/patologia , Resultado do TratamentoRESUMO
Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.
Assuntos
Anidrase Carbônica II/imunologia , Células Dendríticas/imunologia , Endotélio Vascular/imunologia , Imunoterapia Adotiva/métodos , Melanoma/terapia , Adulto , Idoso , Sequência de Aminoácidos , Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Anidrase Carbônica II/química , Anidrase Carbônica II/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Dendríticas/transplante , Eletroforese em Gel Bidimensional , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Neovascularização Fisiológica/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Recently several strategies to treat ischemic diseases have been proposed but the ideal way has to be determined. We explored whether human placenta-derived mesenchymal cells (hPDMCs) can be used for this purpose because placenta is very rich in vessels. First, production of human vascular endothelial growth factor (hVEGF) from hPDMCs was examined. The amount of hVEGF secreted by hPDMCs was similar to the amount produced by HeLa cells. hVEGF was barely detected in human umbilical vein endothelial cells (hUVECs) or human peripheral blood mononuclear cells. hVEGF secreted from hPDMCs stimulated the proliferation of hUVECs, indicating its biological activity. Transplantation of hPDMCs to the ischemic limbs of NOD/Shi-scid mice significantly improved the blood flow of the affected limbs. Blood vessel formation was more prominently observed in the limbs of treated mice as compared to the control mice. Real-time RT-PCR revealed that hPDMCs produced hVEGF for at least 7 days after transplantation. Thus, transplantation of hPDMCs could potentially be a promising treatment for human ischemic diseases.
Assuntos
Isquemia/terapia , Mesoderma/metabolismo , Neovascularização Fisiológica , Placenta/citologia , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/crescimento & desenvolvimento , Transplante de Células , Células Cultivadas , Feminino , Células HeLa , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/patologia , Humanos , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Gravidez , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is thought to act as a key component in vasculogenesis and angiogenesis. Therefore, we studied the effect of mutant Pyk2 expression on the migration and proliferation in endothelial cells (ECs). Two types of mutant Pyk2 were examined by adenovirus vectors AxCA-Pyk2K457A, expressing a kinase inactive mutant, and AxCA-Pyk2Y402F, expressing a tyrosine autophosphorylation site mutant, in addition to AxCA-Pyk2, expressing wild-type Pyk2. Migration of ECs infected with AxCA-Pyk2Y402F increased to a level similar to that of ECs infected with AxCA-Pyk2. The size of effect was dependent on the amount of applied adenoviruses within the range of 3-30 multiplicity of infection. In contrast, AxCA-Pyk2K457A infection did not show any significant effect on cell migration. Western blotting showed that both phosphorylation of Pyk2 Y(881) and association of p130(Cas) with Pyk2 were enhanced in ECs infected with AxCA-Pyk2Y402F as well as with AxCA-Pyk2, but not in ECs infected with AxCA-Pyk2K457A. Therefore, signaling mediated by Pyk2 Y(881) and p130(Cas) may be involved in the migration of ECs infected either with AxCA-Pyk2Y402F or with AxCA-Pyk2. In proliferation assay, AxCA-Pyk2 infection suppressed EC proliferation significantly; however, neither AxCA-Pyk2Y402F nor AxCA-Pyk2K457A showed such an inhibitory effect. Thus, the two Pyk2 mutants revealed that Pyk2 signaling differentially regulates cell migration and proliferation pathways.
Assuntos
Movimento Celular/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Proteína Substrato Associada a Crk , Quinase 2 de Adesão Focal , Regulação Enzimológica da Expressão Gênica , Humanos , Mutagênese , Neovascularização Fisiológica/fisiologia , Fosforilação , Proteínas/metabolismo , Proteína p130 Retinoblastoma-Like , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Veias UmbilicaisRESUMO
OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium-specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis. METHODS: Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor alpha (TNF alpha)-induced Tie2 and Ang1 expression. RESULTS: Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen-induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNF alpha-induced angiogenesis in a mouse cornea assay. In addition, we observed that TNF alpha can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNF alpha up-regulates Tie2 in endothelial cells through nuclear factor kappa B, and it up-regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes. CONCLUSION: This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNF alpha to pathologic angiogenesis.
Assuntos
Artrite Reumatoide/metabolismo , Neovascularização Patológica/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Indutores da Angiogênese/metabolismo , Angiopoietina-1 , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Reumatoide/imunologia , Endotélio/imunologia , Endotélio/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos DBA , NF-kappa B/metabolismo , Neovascularização Patológica/imunologia , Receptor TIE-2 , Transdução de Sinais/imunologia , Membrana Sinovial/irrigação sanguínea , Membrana Sinovial/enzimologia , Membrana Sinovial/imunologiaRESUMO
Mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. In this study, we analyzed recombinant adeno-associated virus (rAAV)-mediated gene expression in human placenta-derived mesenchymal cells (hPDMCs), which reside in placental villi. After transduction of AV-CAG-EGFP, a rAAV expressing enhanced green fluorescence protein (EGFP), hPDMCs showed much higher level of EGFP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. The number of EGFP-positive hPDMCs infected by AV-CAG-EGFP alone did not increase significantly by coinfection of adenovirus, which enhanced expression level of the rAAV vector. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP-expressing hPDMCs, which is about 15-20% of the cells infected with AV-CAG-EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV-mediated gene transduction. In addition, stable EGFP expressions were observed in about 1% of hPDMCs infected with AV-CAG-EGFP at 4 weeks post-infection. Collectively, hPDMCs have characters favorable for rAAV-mediated gene expression.