Transfection with Anti-p65 Intrabody Suppresses Invasion and Angiogenesis in Glioma Cells by Blocking Nuclear Factor-κB Transcriptional Activity

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Abstract
Purpose: The strategy of intracellular antibodies to neutralize the function of target proteins has been widely developed for cancer research. This study used an intrabody against p65 subunit to prevent nuclear factor κB (NF-κB) transcriptional activity in glioma cells and to inhibit the expression of its target genes involved in the invasion and angiogenesis of human gliomas. Experimental Design: A single-chain fragment of antibody variable region (scFv) against p65 was prepared using phage display technique. We then prepared an anti-p65 intrabody construct (pFv/nu) by cloning the scFv-encoding sequence into the mammalian nuclear-targeting vector, pCMV/myc/nuc. Results: p65 expression in human glioma cells (U251 and] U87) transfected with pFv/nu was significantly decreased. We showed that NF-κB nuclear translocation and its DNA binding activity were blocked via intrabody transfection in electrophoretic mobility shift assays and the inhibition of NF-κB activity in nucleus resulted in the decreasing expression and bioactivity of matrix metalloproteinase-9, urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator, and vascular endothelial growth factor. The intrabody transfected glioma cells showed a markedly lower level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also repressed by coculture with the intrabody transfected glioma cells or exposure to their conditional medium. Intrabody transfection neither induced apoptosis nor altered cell proliferation in U251 and U87 cells as compared with the control vector pCMV/nu. After the injection of pFv/nu-transfected glioma cells, preestablished tumors were almost completely regressed when compared with mock, pCMV/nu, and pGFP/nu. Conclusion: Blocking NF-κB activity via the nuclear intrabody expression might be a potential approach for cancer therapy.