RESUMO
Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin-like transmembrane serine protease, as potential target for novel inhibitor-based tumor therapies. We applied time-domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC-1 pancreatic tumor model in nude mice. We show strong and tumor-specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT-Ab*Cy5.5) only to primary AsPC-1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT-Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat-panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY-681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor-bearing mice, and that application of synthetic active-site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor-based cancer therapies.
Assuntos
Carbocianinas/metabolismo , Neoplasias Mamárias Experimentais/terapia , Neoplasias Pancreáticas/terapia , Serina Endopeptidases/metabolismo , Animais , Anticorpos Bloqueadores , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Espectroscopia de Luz Próxima ao Infravermelho , Imagem Corporal Total , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have developed an efficient and cell-specific nonviral gene delivery system using monoclonal antibodies (mAb) coupled with branched 25-kDa polyethyleneimine (PEI). The system was evaluated for two model antibodies with different well-characterized antigen specificities: the mouse anti-human IgG1 mAb AS02 recognizing human CD90 (hThy-1) which is expressed on human fibroblasts, and the humanized anti-Her-2/neu mAb Trastuzumab recently introduced for the treatment of Her-2/neu-positive breast cancer. Efficacy and selectivity of gene delivery were evaluated for covalent mAb-PEI conjugates coupled with N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP) or N-succinimidyl-4-(maleimidomethyl)-cyclohexancarboxylate (SMCC), or, as a newly introduced coupling reagent, noncovalent complexes of mAb with 3-(2-(2-(vinylsulfonyl)ethylthio)ethyl)quinazoline-2,4(1H,3H)-dione (IBFB 110001) coupled to PEI. An enhanced green fluorescent protein (EGFP)-expressing reporter plasmid was used to monitor transfection efficiencies of cell lines expressing different levels of hCD90 or Her-2/neu. While mAb-PEI conjugates coupled with SPDP resulted in antigen-nonspecific EGFP expression, conjugates coupled with SMCC or IBFB 110001 enabled antigen-specific gene delivery. Thus, Her-2/neu-PEI conjugates prepared with IBFB 110001 allowed to transfect 23+/-2% of Her-2/neu-positive SKOV-3 versus 0.5+/-2% of Her-2/neu-negative MB-468 cells. Proof of principle for specific antibody-mediated gene transfer was demonstrated by saturating the Her-2/neu receptor with free anti-Her-2/neu, thereby blocking subsequent transfection with anti-Her-2/neu-PEI/DNA complexes.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Marcação de Genes/métodos , Genes erbB-2/genética , Polietilenoimina/administração & dosagem , Antígenos Thy-1/genética , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados , Células Cultivadas , Humanos , Camundongos , Polietilenoimina/química , Ligação Proteica/efeitos dos fármacos , Transfecção/métodos , TrastuzumabRESUMO
BACKGROUND: In genetically predisposed individuals keloids are formed as benign collagenous tumors. OBJECTIVE: The purpose of this study was to investigate whether the proliferation and matrix gene expression of keloid fibroblasts is differently influenced by the anti-inflammatory active drug lysine acetylsalicylate (LAS) when compared to normal skin fibroblasts in vitro. METHODS: Normal skin and keloid fibroblasts derived from human donors were compared. RESULTS: Excessive scarring and the formation of keloids are (at least in part) due to an overproduction of collagen types I and III. The results show a significant dose-dependent anti-proliferative effect of lysine acetylsalicylate. At the level of gene expression we observed a pronounced inhibitory effect of LAS on procollagen I and III mRNA synthesis, whereas matrix metalloproteinase 1 and tissue inhibitor of metalloproteinases 1 were not altered. CONCLUSIONS: Further clinical studies are planned to evaluate these effects of a high-dose treatment of keloids with LAS.