Antitumor activity of decoy oligodeoxynucleotides targeted to NF-kappaB in vitro and in vivo.

ABSTRACT

BACKGROUND: Nuclear factor-kappaB (NF-kappaB), a transcription factor, is abundantly expressed in many tumors and regulates many tumor-relative genes such as c-myc and caspase-8, which NF-kappaB-mediated genes activation serves as an anti-tumor pathway by regulating expression of these tumor-relative genes. Given the important roles of these genes in tumor control, the present study was to test the hypothesis that NF-kappaB decoy ODNs transfected into tumor cells in vitro and in vivo might affect growth and apoptosis. METHODS: First, NF-kappaB decoy oligodeoxynucleotides were designed according to the NF-kappaB elements in the promoter region of c-myc gene. Then, supression effects of decoy ODNs on proliferation of five carcinoma cell lines were assessed by MTT assay. Apoptosis of carcinoma cells was determined by chromosome DNA ladder and flow cytometric analysis (FCM). Thirdly, suppression effects of NF-kappaB decoy ODNs on proliferation of carcinoma in vivo were investigated in nude mice. To confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kappaB decoy ODNs into SKOV-3 cells by lipofectamine TM2000. Expression levels of endogenous c-myc and caspase-8 genes were assessed by northern blotting. Lastly, nuclear extracts were prepared from SKOV-3 cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: Treatment of cancer cell lines with NF-kappaB decoy ODNs resulted in strong suppression of proliferation, especial of the SKOV-3 ovarian cancer cell line in 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-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that transcripts of endogenous c-myc gene were reduced, while caspase-8 transcripts were induced. EMSA demonstrated specific binding of the NF-kappaB decoy to NF-kappaB protein. CONCLUSIONS: These findings indicatd that NF-kappaB activation plays an important role in proliferation in many cancers, esspecially ovarian carcinomas. Inhibitors of NF-kappaB may thus offer promise as a therapeutic approach for the treatment of tumors via manipulating expression of desired target genes. NF-kappaB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.