We have previously demonstrated that a plasmid-based RNAi vector targeting uPAR and MMP-9 inhibited the overexpression of uPAR and MMP-9 and almost completely suppressed pre-established glioma tumor growth in nude mice. In this study, we combined small hairpin RNA targeting gene degradation with the intrabody-mediated gene therapy approach to achieve a more effective treatment for gliomas. We constructed a series of fusion plasmids based on the RNAi vectors puPAR, pMMP-9 and pUM with an insert of the anti-p65 intrabody encoding gene at the sites of Apa I and Xba I to construct the following fusion plasmids: pMMP-9-Fv, puPAR-Fv, and pUM-Fv. Our results showed that the fusion plasmid pUM-Fv dramatically inhibited expression of the NF-κB activation pathway proteins and related phosphorylation kinases. Further, pUM-Fv significantly decreased MMP-9 and uPAR expression at the mRNA and protein levels (as demonstrated by Western blotting and real-time reverse transcriptional PCR assay), as well as the ability of SNB19 cells to invade through matrigel-coated membranes as compared to cells treated with mock, the control vector pFv/nu, and the other plasmids. In addition, transfection with pUM-Fv significantly induced apoptosis in glioma cells. In the present study, the results indicate that pUM-Fv might have an additive or synergistic effect on inhibition of tumor invasion and induction of glioma cell apoptosis, particularly when compared the single siRNA constructs or anti-p65 intrabody expression alone. These results suggest that the combination of RNAi and intrabody strategies has more potential for cancer treatment than either therapy alone.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA