Decoy Oligodeoxynucleotides Research Articles

A selective decoy-doxorubicin complex for targeted co-delivery, STAT3 probing and synergistic anti-cancer effect.

A novel selective decoy oligodeoxynucleotide (dODN)-doxorubicin (DOX) complex is reported for cancer theranostics. It eliminates the use of a ligand or carrier for targeted delivery and disassembles into therapeutic dODN and DOX upon encountering over-activated STAT3 in cancer cells. Hence, in situ STAT3 probing and synergistic anti-cancer effect are attained at the same time.

Prevention of Neointimal Formation After Angioplasty Using Nuclear Factor-κB Decoy Oligodeoxynucleotide-Coated Balloon Catheter in Rabbit Model

Despite the advent of drug-eluting stents, restenosis after endovascular intervention is still a major limitation in the treatment of cardiovascular disease. To regulate the multiple biological mechanisms underlying restenosis, we focused on inhibition of an important transcription factor, nuclear factor-kappaB (NFκB), using a decoy strategy. For site-specific application of NFκB decoy oligodeoxynucleotides into target vessels during angioplasty, we developed a balloon catheter-based delivery system combined with biocompatible nanoparticles as oligodeoxynucleotides carriers. To clarify the therapeutic effect at the site of neointima, balloon angioplasty of the rabbit carotid arteries was performed at 4 weeks after initial endothelial denudation. This delivery system exhibited successful transfer of fluorescence-labeled nanospheres into the neointima in short-term contact with target vessels, and fluorescence could be detected ≥1 week after angioplasty. Consistently, local application of NFκB decoy oligodeoxynucleotides -loaded nanospheres resulted in significant inhibition of neointimal formation, associated with inhibition of NFκB binding activity in the injured arteries. The therapeutic effects were caused by inhibition of macrophage recruitment through the suppression of monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and CC chemokine ligand 4 expression and inhibition of vascular smooth muscle cell growth via a decrease in the expression of cyclin A and proliferating cell nuclear antigen. Importantly, application of NFκB nanospheres accelerated restoration of the endothelial cell monolayer, associated with enhanced expression of phosphorylated Bcl-2 in endothelial cells. A drug-coated balloon catheter using NFκB decoy oligodeoxynucleotides significantly inhibited the development of neointimal hyperplasia in rabbits. The present study indicates the possibility of a novel therapeutic option to prevent restenosis after angioplasty.

HIF-1α decoy oligodeoxynucleotides inhibit HIF-1α signaling and breast cancer proliferation.

Although HIF-1α is considered an attractive target for the development of cancer therapies, like other transcriptional factors, it has been regarded as 'undruggable'. The decoy approach is a new class of antigene strategy that can be used to modulate the function of endogenous transcriptional factors. Here, we designed a decoy oligodeoxynucleotide (ODN) and tested its effect on the function of HIF-1α. We found the HIF-1α decoy ODN could efficiently enter into cells. Furthermore, these decoy ODNs can significantly block the expression of VEGFA, a known targeted gene of HIF-1α suggesting that the HIF-1α decoy ODNs can inhibit the function of HIF-1α. More importantly, the HIF-1α decoy ODN induced apoptosis and cell cycle arrest in MDA-MB-231 breast cancer cells. In summary, HIF-1α decoy ODNs can inhibit the function of HIF-1α and induce cancer cell apoptosis. Therefore, HIF-1α decoy ODNs should be further modified to improve their biological activity invivo.

P41: Protective effects of NF‐?B decoy oligodeoxynucleotide on liver fibrosis through regulation of epithelial‐ mesenchymal transition

P41: Protective effects of NF‐?B decoy oligodeoxynucleotide on liver fibrosis through regulation of epithelial‐ mesenchymal transition

P40: Anti‐fibrotic effect of Smad decoy oligodeoxynucleotide in carbon tetrachlorideinduced hepatic fibrosis

P40: Anti‐fibrotic effect of Smad decoy oligodeoxynucleotide in carbon tetrachlorideinduced hepatic fibrosis

Suppression of NF-κB signaling mitigates polyethylene wear particle-induced inflammatory response.

In end-stage arthritis patients, total joint replacement is a very effective surgical procedure. Nevertheless, the high revision rate after surgery remains a major concern. The wear particles generated from biomaterial-induced tissue responses may lead to chronic inflammation and local bone destruction (periprosthetic osteolysis). Several important signaling pathways are involved in wear particles induced inflammatory reactions, including the transcription factor NF-κB. We recently reported that RAW264.7 macrophage cell exposure to ultra-high molecular weight polyethylene (UHMWPE) particles significantly increased the NF-κB activity in a generated NF-κB responsive luciferase reporter cell clone. The NF-κB activity induced by UHMWPE particles in a mouse RAW264.7 macrophage cell line, bone marrow derived macrophages, and human THP1 macrophage cell line, were suppressed by double strand decoy oligodeoxynucleotide (ODN) containing an NF-κB binding element. Macrophages exposure to UHMWPE particles with or without endotoxin induced pro-inflammatory cytokine and chemokine expression including TNF-α, MCP1, MIP1α, and others. Finally, the decoy ODN significantly suppressed the induced cytokine and chemokine expression in both murine and human macrophages, consequently reducing macrophage recruitment by cellular conditioned medium exposed to wear particles. These findings suggest that local suppression of inflammatory cytokine production via inhibition of NF-κB activity with decoy ODN in total joint replacement patients could potentially be an effective strategy to alleviate wear particle-induced chronic inflammation.

Inhibitory effect of nuclear factor-κB decoy oligodeoxynucleotide on liver fibrosis through regulation of the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) has been recognized to occur during embryonic development, fibrosis, and tumor metastasis. Nuclear factor (NF)-κB plays a central role in mediating the inflammation and wound-healing responses during liver fibrogenesis. However, the involvement of NF-κB during EMT in liver cells remains unidentified. To develop a therapeutic approach to EMT during liver fibrosis, we examined the inhibition of transcription factor NF-κB, using a decoy oligodeoxynucleotide (ODN) strategy in liver fibrosis in vitro and in vivo. NF-κB decoy ODN contains consensus binding sequences of the NF-κB-binding site. NF-κB decoy ODN effectively suppresses transforming growth factor-β(1)-induced EMT in AML12 murine hepatocytes. Liver fibrosis induced by CCl(4) administration was suppressed by NF-κB decoy ODN. Furthermore, NF-κB decoy ODN was shown to inhibit the EMT process in fibrotic liver in vivo. This study demonstrates the feasibility of NF-κB decoy ODN treatment for preventing liver fibrosis via EMT processes.

Influence of pentylenetetrazol and NF-κB decoy oligodeoxynucleotides on p38 expression in neuron-like cells.

The aim of this study was to investigate the effects of pentylenetetrazol (PTZ) and nuclear factor κ B (NF-κB) decoy oligodeoxynucleotides (ODNs) on p38 expression in neuron-like PC12 cells. In addition, the role of NF-κB activation in the pathogenesis of epilepsy was explored. p38 expression levels in control and PTZ-treated neuron-like PC12 cells were examined using western blotting. NF-κB decoy ODNs were transfected into the neuron-like PC12 cells using Lipofectamine 2000. NF-κB activation was investigated by confocal laser scanning microscopy (CLSM), and p38 expression levels were assessed using western blotting prior to and following transfection of decoy ODNs. Western blot analysis revealed that p38 levels in PTZ-treated neuron-like PC12 cells were significantly higher than those in control cells. CLSM demonstrated that the decoy ODNs inhibited NF-κB activation in neuron-like PC12 cells, and western blotting indicated that the decoy ODNs did not reduce p38 levels. The results of this study indicate that PTZ enhances p38 expression levels and activates NF-κB in PC12 cells. However, NF-κB does not modulate p38 expression levels.

Suppression of wear-particle-induced pro-inflammatory cytokine and chemokine production in macrophages via NF-κB decoy oligodeoxynucleotide: A preliminary report

Total joint replacement (TJR) is very cost-effective surgery for end-stage arthritis. One important goal is to decrease the revision rate, mainly because TJR has been extended to younger patients. Continuous production of ultra-high molecular weight polyethylene (UHMWPE) wear particles induces macrophage infiltration and chronic inflammation, which can lead to periprosthetic osteolysis. Targeting individual pro-inflammatory cytokines directly has not reversed the osteolytic process in clinical trials, owing to compensatory up-regulation of other pro-inflammatory factors. It is hypothesized that targeting the important transcription factor NF-κB could mitigate the inflammatory response to wear particles, potentially diminishing osteolysis. In the current study, NF-κB activity in mouse RAW 264.7 and human THP1 macrophage cell lines, as well as primary mouse and human macrophages, was suppressed via competitive binding with double strand decoy oligodeoxynucleotide (ODN) containing an NF-κB binding element. It was found that macrophage exposure to UHMWPE particles induced multiple pro-inflammatory cytokine and chemokine expression, including TNF-α, MCP1, MIP1α and others. Importantly, the decoy ODN significantly suppressed the induced cytokine and chemokine expression in both murine and human macrophages, and resulted in suppression of macrophage recruitment. The strategic use of decoy NF-κB ODN, delivered locally, could potentially diminish particle-induced periprosthetic osteolysis.

Novel biological strategies for treatment of wear particle-induced periprosthetic osteolysis of orthopaedic implants for joint replacement

Wear particles and by-products from joint replacements and other orthopaedic implants may result in a local chronic inflammatory and foreign body reaction. This may lead to persistent synovitis resulting in joint pain and swelling, periprosthetic osteolysis, implant loosening and pathologic fracture. Strategies to modulate the adverse effects of wear debris may improve the function and longevity of joint replacements and other orthopaedic implants, potentially delaying or avoiding complex revision surgical procedures. Three novel biological strategies to mitigate the chronic inflammatory reaction to orthopaedic wear particles are reported. These include (i) interference with systemic macrophage trafficking to the local implant site, (ii) modulation of macrophages from an M1 (pro-inflammatory) to an M2 (anti-inflammatory, pro-tissue healing) phenotype in the periprosthetic tissues, and (iii) local inhibition of the transcription factor nuclear factor kappa B (NF-κB) by delivery of an NF-κB decoy oligodeoxynucleotide, thereby interfering with the production of pro-inflammatory mediators. These three approaches have been shown to be viable strategies for mitigating the undesirable effects of wear particles in preclinical studies. Targeted local delivery of specific biologics may potentially extend the lifetime of orthopaedic implants.

Small RNA- and DNA-based gene therapy for the treatment of liver cirrhosis, where we are?

Chronic liver diseases with different aetiologies rely on the chronic activation of liver injuries which result in a fibrogenesis progression to the end stage of cirrhosis and liver failure. Based on the underlying cellular and molecular mechanisms of a liver fibrosis, there has been proposed several kinds of approaches for the treatment of liver fibrosis. Recently, liver gene therapy has been developed as an alternative way to liver transplantation, which is the only effective therapy for chronic liver diseases. The activation of hepatic stellate cells, a subsequent release of inflammatory cytokines and an accumulation of extracellular matrix during the liver fibrogenesis are the major obstacles to the treatment of liver fibrosis. Several targeted strategies have been developed, such as antisense oligodeoxynucleotides, RNA interference and decoy oligodeoxynucleotides to overcome this barriers. With this report an overview will be provided of targeted strategies for the treatment of liver cirrhosis, and particularly, of the targeted gene therapy using short RNA and DNA segments.

Systemic delivery of TLR9-activating/STAT3-blocking oligonucleotides induces leukemia regression

Meeting abstracts Inhibition of transcription factors (TF) that drive tumor progression and immune evasion, such as STAT3, remains a challenge for pharmacological drugs. Blocking STAT3 binding to DNA using specific decoy oligodeoxynucleotides (dODN) provides an alternative targeted inhibitory

Blockage of STAT3 Signaling Pathway with a Decoy Oligodeoxynucleotide Inhibits Growth of Human Ovarian Cancer Cells

Transcription factor decoy oligodeoxynucleotides (ODN) represent a novel tool for targeted inhibition of the STAT3 signaling pathway. To investigate its therapeutic potential in ovarian cancer, a double-stranded decoy ODN mimicking STAT3-specific cis-elements was transfected into two ovarian cancer cell lines OVCAR3 and SKOV3. The STAT3 decoy ODN treatment specifically blocked STAT3 signaling, and inhibited cell proliferation by inducing apoptosis and cell cycle arrest. These results suggest that targeted blockade of the STAT3 signaling pathway with a decoy ODN may represent a potential therapeutic approach in the treatment of ovarian cancer.

Targeting transcription factors

A lack of effective treatments for advanced cancer remains a major challenge in oncology. Because cancer is a disease associated with aberrant gene expression patterns, transcription factors, which serve as the convergence points of oncogenic signaling and are functionally altered in many cancers, hold great therapeutic promise. Many human cancers are dependent on the inappropriate activity of oncogenic transcription factors. By contrast, normal cells can often tolerate disruption of these proteins with little toxicity. Direct inhibition of transcription factor expression (e.g., with RNA interference or microRNAs) and DNA binding (e.g., with oligodeoxynucleotide decoys or pyrrole-imidazole polyamides) has demonstrated antitumor responses with minimal side-effects. New strategies of targeting transcription factors include disrupting critical protein-protein interactions, and restricting binding at the epigenetic level by modulating chromatin accessibility. Moreover, targeting transcription factors in tumor-associated immune cells has the potential to overcome tumor immunoresistance. Transcription factors are an important target for cancer therapy, both through direct anticancer effects and immunomodulatory actions. Newly developed delivery systems that specifically target tumor cells also create opportunities for successes in targeting transcription in cancer.

SPATA12 and Its Possible Role in DNA Damage Induced by Ultraviolet-C

Our previous studies indicated that SPATA12, a novel spermatogenesis-associated gene, might be an inhibitor involved in spermatogenesis and tumorigenesis. To obtain a better understanding of the functions of SPATA12, a yeast two-hybrid screening system was used to search for interacting proteins, and chromodomain helicase DNA binding protein 2 (CHD2) was successfully identified. Bimolecular fluorescence complementation (BiFC) and subcellular co-localization assays further suggested a possible interaction between SPATA12 and CHD2 in the nuclei. CHD2 is known to be involved in the later stage of the DNA damage response pathway by influencing the transcriptional activity of p53. Thus, our hypothesis is that SPATA12 might play a role in DNA damage signaling. Western blotting results showed that SPATA12 expression could be induced in ultraviolet-C (UV-C) irradiated cells. Through reporter gene assays and the activator protein-1 (AP-1) decoy oligodeoxynucleotide method, we demonstrated that SPATA12 promoter activity could be up-regulated in response to UV-C radiation exposure and an AP-1 binding site in the SPATA12 promoter may have a role in transcriptional regulation of SPATA12. Using colony formation and host cell reactivation assays, it was demonstrated that SPATA12 might lead to inhibition of cellular proliferation in UV-C-irradiated DNA damage. Furthermore, SPATA12 was transfected into H1299, MCF-7 and HeLa cells, and flow cytometry (FCM) results suggested that there are some biological association between SPATA12 and p53 in UV-C-irradiated DNA damage. In addition, we investigated whether SPATA12 could up-regulate the expression of p53. Taken together, these findings indicate that SPATA12 could be induced under UV-C stress. During DNA damage process, AP-1 involves in the transcriptional up-regulation of SPATA12 in response to UV-C radiation and p53 involves in growth inhibitory effects of SPATA12 on UV-C irradiated cells.

A Decoy Oligonucleotide to NF-κB Delivered through Inhalable Particles Prevents LPS-Induced Rat Airway Inflammation

The inflammatory process plays a crucial role in the onset and progression of several lung pathologies, including cystic fibrosis (CF), and the involvement of NF-κB is widely recognized. The specific inhibition of NF-κB by decoy oligonucleotides delivered within the lung may be beneficial, although rationally designed systems are needed to optimize their pharmacological response. Prompted by this need, we have developed and tested in vivo an inhalable dry powder for the prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPPs) based on poly(lactic-co-glycolic) acid. First, LPPs containing dec-ODN (dec-ODN LPPs) were engineered to meet the aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release, and to preserve its structural integrity in lung lining fluids. We then investigated the effects of dec-ODN LPPs in a rat model of lung inflammation induced by the intratracheal aerosolization of LPS from Pseudomonas aeruginosa. The results show that a single intratracheal insufflation of dec-ODN LPPs reduced the bronchoalveolar neutrophil infiltration induced by LPS for up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated with reduced NF-κB/DNA binding activity, as well as reduced IL-6, IL-8, and mucin-2 mRNA expression in lung homogenates. We consider it noteworthy that the developed LPPs, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for the local treatment of inflammation associated with lung disease.

Inhibitory effects of STAT3 decoy oligodeoxynucleotides on human epithelial ovarian cancer cell growth in vivo

The signal transducer and activator of transcription 3 (STAT3) regulates target gene expression by binding to a consensus DNA sequence within the promoter of the target genes. The constitutive activation of STAT3 has been shown to contribute to tumorigenesis in ovarian cancer and it has been reported to be a key factor for drug resistance in ovarian cancer. STAT3-specific decoy oligodeoxynucleotides (ODNs) (STAT3 decoy ODNs) that contain a consensus DNA sequence inhibit the transcriptional activity of STAT3, leading to cancer cell death. However, their mechanisms of action are unclear and little information is available as to the effects and the toxicity of STAT3 decoy ODNs in vivo. In this study, we established subcutaneous xenografts of SKOV3 human ovarian cancer cells in nude mice, evaluated the antitumor effects of STAT3 decoy ODNs on xenografted nude mice, and investigated the mechanisms behind the antitumor effects of STAT3 decoy ODNs targeting the STAT3 signaling pathway in vivo. The results revealed that the STAT3 decoy ODN inhibited ovarian cancer cell growth and promoted ovarian cancer cell apoptosis in vivo. Western blot analysis indicated that the STAT3 decoy ODN downregulated the protein expression levels of matrix metalloproteinase (MMP)-2, MMP-9 and Bcl-2, and upregulated the protein expression levels of caspase-3 in vivo. H&E staining was used to detect the side-effects of the STAT3 decoy ODN in the vital organs of the nude mice. We found that there were no significant abnormalities in the vital organs of the nude mice apart from slight inflammation and necrosis in parts of the hepatic lobule. The data from the present study suggest that decoy ODNs targeting STAT3 may be an effective therapeutic approach for the treatment of ovarian cancer in vivo.

Interleukin-6 Mediates Angiotensinogen Gene Expression during Liver Regeneration

BackgroundAngiotensinogen is the precursor of angiotensin II, which is associated with ischemia-reperfusion injury. Angiotensin II reduces liver regeneration after hepatectomy and causes dysfunction and failure of reduced-size liver transplants. However, the regulation of angiotensinogen during liver regeneration is still unclear.AimsTo investigate the regulation of angiotensinogen during liver regeneration for preventing angiotensin II-related ischemia-reperfusion injury during liver regeneration.MethodsA mouse in vitro partial hepatectomy animal model was used to evaluate the expression of interleukin-6 (IL-6) and angiotensinogen during liver regeneration. Serum IL-6 and angiotensinogen were detected by enzyme immunoassay (EIA). Angiotensinogen mRNA was detected by RT-PCR. Tissue levels of angiotensinogen protein were detected by Western blot analysis. Primary cultures of mouse hepatocytes were used to investigate IL-6-induced angiotensinogen. Chemical inhibitors were used to perturb signal transduction pathways. Synthetic double-stranded oligodeoxynucleotides (ODNs) were used as ‘decoy’ cis-elements to investigate transcription. Ki 67 staining and quantification were used to verify liver regeneration.ResultsIn the in vivo model, the levels of serum IL-6 and angiotensinogen correlated. In the in vitro model, IL-6 transcriptionally regulated angiotensinogen expression. Additionally, IL-6 mediated angiotensinogen expression through the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and JAK/p38 signaling. Decoy ODN analyses revealed that STAT3 and nuclear factor-kB (NF-kB) also played critical roles in the transcriptional regulation of angiotensinogen by IL-6. IL-6-mediated signaling, JAK2, STAT3 and p38 inhibitors reduced angiotensinogen expression in the partially hepatectomized mice.ConclusionDuring liver regeneration, IL-6-enhanced angiotensinogen expression is dependent on the JAK/STAT3 and JAK/p38/NF-kB signaling pathways. Interruption of the molecular mechanisms of angiotensinogen regulation may be applied as the basis of therapeutic strategies for preventing angiotensin II-related ischemia-reperfusion injury during liver regeneration.

Antifibrotic effect of synthetic Smad/Sp1 chimeric decoy oligodeoxynucleotide through the regulation of epithelial mesenchymal transition in unilateral ureteral obstruction model of mice

Renal tubulointerstitial fibrosis is considered to be a common final pathway related to the progressive loss of renal function in chronic kidney disease. It is characterized by the excessive accumulation of extracellular matrix through the pivotal role of epithelial–mesenchymal transition. Transforming growth factor-β1 is postulated to play a central role in renal fibrosis via a downstream pathway such as Smad. Specificity protein 1 (Sp1), which is another transcription factor, is also involved in the basal expression of extracellular matrix. In this study, we investigate the effect of Smad decoy oligodeoxynucleotides (ODN) and Sp1 decoy ODN in unilateral ureteral obstruction induced renal fibrosis in mice. Furthermore, the effectiveness of the newly designed chimeric decoy ODN, which contains both Smad and Sp1 binding sequences in one decoy molecule (Smad/Sp1 chi decoy ODN), was demonstrated. The expression of fibrosis and inflammatory related cytokines and products of fibrosis were ameliorated in the Smad, Sp1 and chimeric decoy ODN treated groups compared with the scrambled decoy ODN treated group. Epithelial–mesenchymal transition was suppressed by the Smad, Sp1 and Smad/Sp1 chi decoy ODN. Immunohistochemistry and Western-blot analysis revealed that Smad/Sp1 chi decoy ODN showed a more significant inhibitory effect on fibrosis and EMT compared with Smad and Sp1 decoy ODNs. These results support the efficacy of Smad/Sp1 chi decoy compared with a single Smad or Sp1 decoy ODNs in preventing renal fibrosis induced by unilateral ureteral obstruction.

Erratum to: The inhibitory effect of chimeric decoy oligodeoxynucleotide against NF-κB and Sp1 in renal interstitial fibrosis

Erratum to: The inhibitory effect of chimeric decoy oligodeoxynucleotide against NF-κB and Sp1 in renal interstitial fibrosis