Cell death in NF-κB-dependent tumour cell lines as a result of NF-κB trapping by linker-modified hairpin decoy oligonucleotide

AbstractThe transcription factor NF-κB is frequently activated in cancer, and is therefore a valuable target for cancer therapy. Decoy oligodeoxynucleotides (ODNs) inhibit NF-κB by preventing its binding to the promoter region of target genes. Few studies have used NF-κB-targeting with ODNs in cancer. Using a hairpin NF-κB-decoy ODN we found that it induced growth inhibition and cell death in NF-κB-dependent tumour cell lines. The ODN colocalized with the p50 subunit of NF-κB in cells and directly interacted with it in nuclear extracts. In TNFα-treated cells the ODN and the p50 subunit were found in the cytoplasm suggesting that the complex did not translocate to the nucleus. Transcriptional activity of NF-κB was efficiently inhibited by the ODN, whereas a scrambled ODN was without effect on transcription. Thus, ODN-mediated inhibition of NF-κB can efficiently promote cell death in cancer cells providing a potentially powerful approach to tumour growth inhibition

A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes.</p> <p>Results</p> <p>New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1.</p> <p>Conclusions</p> <p>Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate protein/DNA interactions. The finding of a STAT3-specific hpdODN establishes the first rational basis for designing STAT3 DBD-specific inhibitors.</p

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

<p>Abstract</p> <p>Background</p> <p>The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear.</p> <p>Results</p> <p>The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death.</p> <p>Conclusions</p> <p>The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.</p

Near-Infrared Fluorescent Oligodeoxyribonucleotide Reporters for Sensing NF-κB DNA Interactions In Vitro

Two types of reporters for optical sensing of NF-κB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3′-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-κB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease. The incubation of 800CW-Cy reporter in the presence of control or IL-1β treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor–acceptor fluorochrome pair. NF-κB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex