Antitumor Activity of Decoy Oligodeoxynucleotides Targeted to NF-kB in vitro and in vivo

Background: Nuclear factor-kappaB (NF-κB), a transcription factor, is abundantly expressed in many tumorsand regulates many tumor-relative genes such as c-myc and caspase-8, which NF-κB-mediated genes activationserves as an anti-tumor pathway by regulating expression of these tumor-relative genes. Given the importantroles of these genes in tumor control, the present study was to test the hypothesis that NF-κB decoy ODNstransfected into tumor cells in vitro and in vivo might affect growth and apoptosis.
Methods: First, NF-κBdecoy oligodeoxynucleotides were designed according to the NF-κB elements in the promoter region of c-mycgene. Then, supression effects of decoy ODNs on proliferation of five carcinoma cell lines were assessed by MTTassay. Apoptosis of carcinoma cells was determined by chromosome DNA ladder and flow cytometric analysis(FCM). Thirdly, suppression effects of NF-κB decoy ODNs on proliferation of carcinoma in vivo were investigatedin nude mice. To confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing afragment of the c-myc promoter was constructed and co-transfected with NF-κB decoy ODNs into SKOV-3cells by lipofectamineTM2000. Expression levels of endogenous c-myc and caspase-8 genes were assessed bynorthern blotting. Lastly, nuclear extracts were prepared from SKOV-3 cells and DNA-protein interactionswere examined by electrophoretic mobility shift assay (EMSA).
Results: Treatment of cancer cell lines with NF-κB decoy ODNs resulted in strong suppression of proliferation, especial of the SKOV-3 ovarian cancer cell linein vitro and in vivo. Induction of apoptosis of SKOV-3 was observed in DNA gel electrophoresis and FCM.Activity of luciferase was significantly reduced in the NF-κB decoy-transfected cells, but not in cells transfectedwith a control decoy. Furthermore, we found that transcripts of endogenous c-myc gene were reduced, whilecaspase-8 transcripts were induced. EMSA demonstrated specific binding of the NF-κB decoy to NF-κB protein.
Conclusions: These findings indicatd that NF-κB activation plays an important role in proliferation in manycancers, esspecially ovarian carcinomas. Inhibitors of NF-κB may thus offer promise as a therapeutic approachfor the treatment of tumors via manipulating expression of desired target genes. NF-κB decoy ODNs may allowdevelopment of therapeutic and investigative tools for human malignancies.