RESUMEN
Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+ Mac3+ Gr1- macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.
Asunto(s)
Inhibidores de la Angiogénesis/genética , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/terapia , Glioma/irrigación sanguínea , Glioma/terapia , Macrófagos/patología , Fragmentos de Péptidos/genética , Plasminógeno/genética , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/biosíntesis , Inhibidores de la Angiogénesis/química , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/genética , ADN Complementario/genética , Progresión de la Enfermedad , Femenino , Terapia Genética/métodos , Glioma/genética , Glioma/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Datos de Secuencia Molecular , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Patológica/terapia , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Plasminógeno/biosíntesis , Plasminógeno/química , Conformación Proteica , Ingeniería de Proteínas , Retroviridae/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The lack of serum biomarkers for head and neck carcinoma limits early diagnosis, monitoring of advanced disease, and prediction of relapses in patients. We conducted a comprehensive proteomics study on serum from mice bearing orthotopic human oral squamous cell carcinomas (OSCC) with distinct invasive phenotypes. Matched established cell lines were transplanted orthotopically into tongues of RAG-2/gamma(c) mice and mouse serum was analyzed by 2-dimensional-differential gel electrophoresis(2D-DIGE)/liquid chromatography (LC)-MS/MS and by online 2D-LC-MS/MS of iTRAQ labeled samples. We identified several serum proteins as being differentially expressed between control and cancer-bearing mice and between noninvasive and invasive cancer (p<0.05). Differentially expressed proteins of human origin included the epidermal growth factor receptor (EGFR), cytokeratins, G-protein coupled receptor 87, Rab11 GTPase, PDZ-domain containing proteins, and PEST-containing nuclear proteins. Identified proteins of mouse origin included clusterin, titin, vitronectin, vitamin D-binding protein, hemopexin, and kininogen I. The levels of serum and cell secreted EGFR were further validated to match proteomic data regarding the inverse correlation with the invasive phenotype. In summary, we report a comprehensive patient-based proteomics approach for the identification of potential serum biomarkers for OSCC using an orthotopic xenograft mouse model.