Control Oligodeoxynucleotides Research Articles

Microglial‐Mediated PVN Inflammation Precedes Sympathoexcitation but not Hypertension in the Development of Gαi2 Protein‐Dependent Salt Sensitive Hypertension

AimPrevious work in our laboratory has established that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate sympathoinhibitory responses to high salt (HS) diets. The inability to upregulate these proteins in response to a HS diet results in salt sensitive hypertension (ssHTN). Additionally, we have demonstrated that microglial‐mediated PVN inflammation correlates with Gαi2 protein dependent ssHTN. We hypothesize that microglial‐mediated PVN inflammation precedes sympathoexcitation in the development of Gαi2 protein‐dependent ssHTN.MethodsThree‐month‐old male Sprague Dawley rats were implanted with ICV cannulas fitted to osmotic minipumps to deliver an infusion of either targeted Gαi2, which downregulate CNS Gαi2 proteins by ~85%, or scrambled (SCR) oligodeoxynucleotides (ODN) (25μg/5μl/day) and were then put on a 24‐hour or 7‐day normal (NS; 0.6% NaCl) or high (HS; 4% NaCl) salt diet (n=5 per group). Rats were transcardially perfused and brains were extracted and fixed in paraformaldehyde and sucrose. Immunohistochemistry (IHC) was performed to assess PVN microglial activation and for protein levels of pro‐inflammatory cytokines (PIC) IL‐1β, and IL‐6. We ran additional groups in the same manner, previously fitted with radiotelemeters to assess MAP, and sacrificed these groups on day 1 and 7 to obtain whole blood for ELISA plasma norepinephrine (NE) analysis as a measure of sympathetic tone.ResultsIn SCR ODN infused rats, HS diet evoked no change in MAP or microglial reactivity compared to NS control. In rats lacking central Gαi2 proteins, a 24‐hour HS diet evoked significant increases in blood pressure (MAP, mmHg; Gαi2 ODN HS 115 ± 2 vs Gαi2 ODN NS 103 ± 1 p<0.05) and percent activated PVN microglia (% active per sample region; Gαi2 ODN HS 25.1% ± 3 vs Gαi2 ODN NS 16.8 ± 3.7 p<0.05) compared to Gαi2 ODN NS control and SCR ODN rats. Additionally, immunofluorescence detected qualitative increases in IL‐6 and IL‐1β expression in rats lacking central Gαi2 proteins compared to SCR ODN. Plasma NE values were significantly lower in SCR ODN infused animals on HS (plasma NE, nmol/L; SCR ODN HS 43 ± 4.6 vs. SCR ODN NS 65 ± 5.0, p<0.05) than NS control. Gαi2 ODN and HS infused animals on day 1 showed no change in plasma NE levels compared to Gαi2 NS control, but showed significantly lower NE levels than Gαi2 HS on day 7 (plasma NE, nmol/L; Gαi2 ODN day 1 56 ± 6.0 vs. Gαi2 ODN day 7 91 ± 3.2, p<0.05).ConclusionsWhile SCR ODN infused animals inhibit sympathetic activity after 1 day HS and remain normotensive, Gαi2 ODN infused animals fail to do so. Gαi2 ODN infused animals do not, however, exhibit sympathoexcitation on day 1, yet demonstrate increases in MAP, PVN microglial activation, and PVN inflammation. This suggests inflammation may precede sympathoexcitation in Gαi2 protein‐dependent ssHTN.Support or Funding InformationR01 HL139867, R01 HL141406, R56 AG057687, T32 HL007224 MAP, inflammation, and sympathetic characteristics of rats with and without central Gαi2 proteins Gαi2 ODN, 0.6% NaCl diet baseline Gαi2 ODN, 4% NaCl diet day 1 Gαi2 ODN, 4% NaCl diet day 7 SCR ODN, 0.6% NaCl diet baseline SCR ODN, 4% NaCl diet day 1 SCR ODN, 4% NaCl diet day 7 Mean Arterial Pressure (mmHg) 103 ± 1 115 ± 2 * 128 ± 2 * # 102 ± 1 103 ± 2 103 ± 1 Plasma NE (nmol/L) 67 ± 6.0 56 ± 6.0 91 ± 3.2 * # 65 ± 5.0 43 ± 4.6 * 37 ± 2.4 * # Microglia in 200 μm × 200 μm sample area in PVN 22.0 ± 3.1 22.6 ± 2.1 35.33 ± 3.1 * # 26.6 ± 4.2 17.4 ± 2.0 20.5 ± 2.7 % Microglia activated in 200 μm × 200 μm sample area in PVN 16.8 ± 3.7 25.1 ± 3.0 * 29.2 ± 2.5 * 18.0 ± 2.4 11.4 ± 3.5 15.6 ± 2.5 Data presented as mean ± SD, *p<0.05 vs NS baseline within ODN group; #p<0.05 vs. HS day 1 within ODN group

Abstract 5091: Activity of Bcl-2 antisense therapeutic in aggressive non-Hodgkin's lymphoma

Abstract Aggressive non-Hodgkin's lymphoma (NHL) progresses rapidly. It makes up about 60% of all NHL cases in the United States. The chemotherapy regimen R-CHOP could cure up to 70% of patients with aggressive NHL. Nonetheless, up to 30% of patients with aggressive NHL relapse from R-CHOP within 2 years of initial treatment. Novel therapeutics are urgently needed for patients with relapsed, aggressive NHL. Bcl-2 is a potential therapeutic target for aggressive NHL because high expression of Bcl-2 has been reported in NHL and correlated with adverse prognosis for NHL patients. To inhibit the expression of Bcl-2 protein, BP1002 was developed. BP1002 is comprised of an uncharged P-ethoxy antisense oligodeoxynucleotide targeted against Bcl-2 mRNA. The P-ethoxy antisense backbone does not have an adverse effect on bleeding and complement activation, which are some of the toxicities that have been reported for other antisense analogs. The Bcl-2 antisense is incorporated in neutral liposomes so that it can be delivered systemically via intravenous infusion. The inhibitory activity of BP1002 was determined in human lymphoma cell lines, which included germinal center B-cell (GCB) diffuse large B cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, mantle cell lymphoma (MCL) and Burkitt’s lymphoma (BL). The lymphoma cell lines were incubated with BP1002 for four days. The sulforhodamine B cytotoxicity assay showed that BP1002, at 200 micrograms/mL, induced ≥50% inhibition in 5 of 8 GCB DLBCL lines, 3 of 3 ABC DLBCL lines, 1 of 1 MCL line, and 1 of 1 BL line. We also determined the activity of BP1002 in animal models. Severe combined immunodeficiency (SCID) mice were implanted with CJ cells, which are transformed follicular lymphoma cells that express high levels of Bcl-2 protein. In experiment I, 4 groups of mice were used: Group 1 - untreated mice; Group 2 - mice treated with 10 mg of BP1002 per kg of mouse body weight; Group 3 - mice treated with 20 mg of BP1002 per kg of mouse body weight; Group 4 - mice treated with control empty liposomes equivalent to 20 mg/kg of BP1002. One week after tumor implantation, mice were injected with BP1002 or empty liposomes intravenously twice per week. When mice were euthanized in week 5, all untreated mice and mice treated with control empty liposomes reached moribund state. However, no mouse in the BP1002 treatment groups reached moribund state. In experiment II, 3 groups of mice were used: Group 1 - untreated mice; Group 2 - mice treated with 20 mg/kg of liposome-incorporated control oligodeoxynucleotide; Group 3 - mice treated with 20 mg/kg of BP1002. All untreated mice and control mice were moribund in week 5, but only 40% of BP1002-treated mice were moribund. These in vivo experiments indicate that BP1002 could extend the survival of mice bearing aggressive NHL. Together, our data indicate that BP1002 is a novel therapeutic for aggressive NHL. Citation Format: Ana Tari Ashizawa, Yolanda Gutierrez-Puente, Richard Ford, Gabriel Lopez-Berestein. Activity of Bcl-2 antisense therapeutic in aggressive non-Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5091. doi:10.1158/1538-7445.AM2017-5091

Effect of Targeting Clusterin Using OGX-011 on Antitumor Activity of Temsirolimus in a Human Renal Cell Carcinoma Model.

It has not been well documented that the modulation of stress response mediates the efficacy of the mammalian target of rapamycin (mTOR) inhibitor in renal cell carcinoma (RCC). The objective of this study was to investigate whether the activity of the mTOR inhibitor temsirolimus against RCC could be enhanced by OGX-011, an antisense oligodeoxynucleotide (ODN) targeting the stress-activated chaperone clusterin. We investigated the efficacy of combined treatment with temsirolimus plus OGX-011 in a human RCC Caki-1 model focusing on the effects on apoptotic and autophagic pathways. Although clusterin expression was increased by temsirolims, additional treatment of Caki-1 with OGX-011 significantly inhibited clusterin upregulation (p < 0.05). Combined treatment of temsirolimus and OGX-011 synergistically enhanced the sensitivity of Caki-1 to temsirolimus (p < 0.01), reducing the IC50 by approximately 50%. Apoptotic changes were marked in Caki-1 following combined treatment with a sublethal dose of temsirolimus and OGX-011, accompanying the significant downregulation of Mcl-1 (p < 0.05), but not with either agent alone. Furthermore, this combined treatment markedly blocked the temsirolimus-induced activation of autophagy in Caki-1 (p < 0.01). In-vivo systemic administration of temsirolimus plus OGX-011 significantly inhibited the growth of Caki-1 tumors compared with that of temsirolimus plus control ODN (p < 0.05). Silencing of clusterin using OGX-011 resulted in the further enhancement of proapoptotic activity as well as the marked attenuation of the autophagic pathway induced by temsirolimus in a human RCC model. Thus, the combined use of OGX-011 could be a promising strategy through the enhanced cytotoxic activity of temsirolimus against RCC.

MP92-08 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOUR ACTIVITY OF AXITINIB IN A HUMAN RENAL CELL CARCINOMA MODEL

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-08 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOUR ACTIVITY OF AXITINIB IN A HUMAN RENAL CELL CARCINOMA MODEL Satoshi Imai, Hideaki Miyake, Martin Gleave, and Masato Fujisawa Satoshi ImaiSatoshi Imai More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2641AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clusterin is expressed in various malignant tumors, including renal cell carcinoma (RCC), and associated with resistance to a wide variety of therapeutic stimuli. OGX-011 is a second-generation antisense oligodeoxynucleotide (ODN) currently in late-stage clinical development that potently inhibits clusterin expression and enhances the efficacy of anticancer therapies in several types of human cancer. The objective of this study was to investigate whether inactivation of clusterin with OGX-011 enhances the effect of axitinib, a potential vascular endothelial growth (VEGF)-targeted agent, in a human RCC ACHN model. METHODS Effects of a combined treatment with OGX-011 and axitinib on ACHN cells were examined by MTT assay, DNA fragmentation assay and Western bolt analyses of proteins involved in apoptosis, signal transduction and autophagy. In vivo efficacy of this combined therapy was assessed in nude mice given subcutaneous injection of ACHN cells. RESULTS Although clusterin expression was increased by axitinib, additional treatment of ACHN with OGX-011 significantly blocked the upregulation of clusterin induced by axitinib. Despite the lack of a significant effect on the growth of ACHN, OGX-011 synergistically enhanced the sensitivity to axitinib, reducing the IC50 by more than 50%. Apoptotic changes were intensively detected in ACHN cells after combined treatment with OGX-011 and a sublethal dose of axitinib, but not either agent alone. Furthermore, this combined treatment resulted in the marked downregulation of phosphorylated Akt and p44/42 mitogen-activated protein kinase in ACHN cells compared with treatment with either agent alone, while the expression levels of Bcl-2, Bcl-xL and p53 after the combined treatment were significantly upregulated compared with those after treatment with either agent alone. In vivo systemic administration of OGX-011 plus axitinib significantly decreased the ACHN tumour volume compared with control ODN plus axitinib. Although no significant differences in the proportions of cells positive for Ki-67 immunostaining or TUNEL assay between the group treated with OGX-011 and that with control ODN, the inhibition of cell proliferation and induction of apoptosis in ACHN tumors treated with OGX-011 and axitinib were markedly remarkable than those in ACHN tumors treated with control ODN and axitinib. CONCLUSIONS Combined use with OGX-011 may be useful in enhancing the cytotoxic effect of axitinib on RCC by inducing apoptosis and inactivating major signal transduction pathways. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1164-e1165 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Satoshi Imai More articles by this author Hideaki Miyake More articles by this author Martin Gleave More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Impaired sodium-evoked paraventricular nucleus neuronal activation and blood pressure regulation in conscious Sprague-Dawley rats lacking central Gαi2 proteins.

AimWe determined the role of brain Gαi2 proteins in mediating the neural and humoral responses of conscious male Sprague–Dawley rats to acute peripheral sodium challenge.MethodsRats pre‐treated (24‐h) intracerebroventricularly with a targeted oligodeoxynucleotide (ODN) (25 μg per 5 μL) to downregulate brain Gαi2 protein expression or a scrambled (SCR) control ODN were challenged with an acute sodium load (intravenous bolus 3 m NaCl; 0.14 mL per 100 g), and cardiovascular parameters were monitored for 120 min. In additional groups, hypothalamic paraventricular nucleus (PVN) Fos immunoreactivity was examined at baseline, 40, and 100 min post‐sodium challenge.ResultsIn response to intravenous hypertonic saline (HS), no difference was observed in peak change in mean arterial pressure between groups. In SCR ODN pre‐treated rats, arterial pressure returned to baseline by 100 min, while it remained elevated in Gαi2 ODN pre‐treated rats (P < 0.05). No difference between groups was observed in sodium‐evoked increases in Fos‐positive magnocellular neurons or vasopressin release. V1a receptor antagonism failed to block the prolonged elevation of arterial pressure in Gαi2 ODN pre‐treated rats. A significantly greater number of Fos‐positive ventrolateral parvocellular, lateral parvocellular, and medial parvocellular neurons were observed in SCR vs. Gαi2 ODN pre‐treated rats at 40 and 100 min post‐HS challenge (P < 0.05). In SCR, but not Gαi2 ODN pre‐treated rats, HS evoked suppression of plasma norepinephrine (P < 0.05).ConclusionThis highlights Gαi2 protein signal transduction as a novel central mechanism acting to differentially influence PVN parvocellular neuronal activation, sympathetic outflow, and arterial pressure in response to acute HS, independently of actions on magnocellular neurons and vasopressin release.

Activation of Notch3 in Glomeruli Promotes the Development of Rapidly Progressive Renal Disease.

Notch3 expression is found in the glomerular podocytes of patients with lupus nephritis or focal segmental GN but not in normal kidneys. Here, we show that activation of the Notch3 receptor in the glomeruli is a turning point inducing phenotypic changes in podocytes promoting renal inflammation and fibrosis and leading to disease progression. In a model of rapidly progressive GN, Notch3 expression was induced by several-fold in podocytes concurrently with disease progression. By contrast, mice lacking Notch3 expression were protected because they exhibited less proteinuria, uremia, and inflammatory infiltration. Podocyte outgrowth from glomeruli isolated from wild-type mice during the early phase of the disease was higher than outgrowth from glomeruli of mice lacking Notch3. In vitro studies confirmed that podocytes expressing active Notch3 reorganize their cytoskeleton toward a proliferative/migratory and inflammatory phenotype. We then administered antisense oligodeoxynucleotides targeting Notch3 or scramble control oligodeoxynucleotides in wild-type mice concomitant to disease induction. Both groups developed chronic renal disease, but mice injected with Notch3 antisense had lower values of plasma urea and proteinuria and inflammatory infiltration. The improvement of renal function was accompanied by fewer deposits of fibrin within the glomeruli and by decreased peritubular inflammation. Finally, abnormal Notch3 staining was observed in biopsy samples of patients with crescentic GN. These results demonstrate that abnormal activation of Notch3 may be involved in the progression of renal disease by promoting migratory and proinflammatory pathways. Inhibiting Notch3 activation could be a novel, promising approach to treat GN.

Impact of CpG oligodeoxynucleotide stimulation on percentage of T and B cells in chicken

TLR stimulation in chickens has been shown to play a role in the initiation and regulation of innate and adaptive immune responses. The aim of this study was to use flow cytometry to establish the percentage of T and B subset in blood and lymphoid organs in chicks after CpG oligodeoxynucleotide (ODN) stimulation. It was demonstrated that the percentages of CD3+, CD4+, TCRgamma delta+ cells and Bu-1+MHC class II+ cells in blood 24 h post-injection were significantly higher than in the control groups. It was also shown that the percentages of CD3+ and CD4+ cells in the spleen at 48 h post-injection were significantly higher than in control groups. The percentage of Bu-1+ cells in the bursa of Fabricius after CpG ODN stimulation (98.38 +/- 0.84) was significantly higher than that found in the non-CpG ODN control group (94.54 +/- 2.51) (p < or = 0.05). The results indicate that class B CpG ODN increases the percentage of both T (especially CD4+ cells) and B cells.

Effects of systemic pretreatment with CpG oligodeoxynucleotides on skin wound healing in mice

Unmethylated CpG oligodeoxynucleotides (ODN) bind to the Toll-like receptor 9, thus stimulating the immune system. To study the effects of systemic pretreatment with CpG ODN on dermal regeneration, C57BL6/J Tyr mice were treated with CpG or control ODN 6 days prior to implantation of a dorsal skinfold chamber and skin wounding. Wound epithelialization was analyzed by planimetric microscopy. On day 18, wound tissues were taken for (immuno)histochemical staining. CpG ODN increased epithelialization compared with control ODN treatment. Histological analysis revealed reduced capillary density, reduced wound cellularity, and reduced numbers of infiltrating leukocytes, as well as reduced F4/80-positive macrophages, but increased numbers of RELM-α-positive M2 macrophages after CpG ODN treatment, reflecting a better quality of wound healing on day 18 compared with control ODN treatment. Reverse transcription-polymerase chain reaction analysis of Toll-like receptor 9 showed the receptor expression on both fibroblasts and keratinocytes. Fibroblasts showed an increase of migration upon increasing dosages of CpG and not control ODN, reaching ∼50% of the response of basic fibroblast growth factor-exposed cells. Keratinocytes dose-dependently responded to both CpG and control ODN up to values found in keratinocyte growth factor-exposed cells. In summary, CpG ODN support late tissue-remodeling processes that contribute to resolution of inflammation and solid wounds during skin regeneration.

The effect of suppressive oligodeoxynucleotides on interferon-γ and phosphorylation of signal transducers and activators of transcription 4 expression of silica-induced pulmonary inflammation in mice

To investigate the protective effect of suppressive oligodeoxynucleotides (Sup ODN) on interferon-γ (IFN-γ) and signal transducers and activators of transcription (pSTAT4) expression of Silica-induced pulmonary inflammation in Mice. Sixty Balb/c mice were randomly divided into 4 groups, normal control group, silicious group, suppressive oligodeoxynucleotides (Sup ODN) group, control oligodeoxynucleotides (Con ODN) group. Except the normal control group injected normal saline, the rest groups were induced by the intratracheal instillation of 0.1 ml (5 g/L) of sterilized silica suspension. Sup ODN group and Con ODN group were treated by i.p. injection of 0.3 ml (1mg/mL) of suppressive or control ODN 3 h before silica administration. After 7 days, the animals were killed and levels of IFN-γ were detected by ELISA. The pathologic changes in lung tissues of mice were observed with HE staining. Expressions of IFN-γ and pSTAT4 in lung tissue were detected with immunohistochemistry and quantified by Image-Pro Plus 7.0. HE staining showed that the lung tissue of silicious group were damaged seriously than Sup ODN group. Compared with the normal control group (serum: (280.1±41.3) pg/ml, lung tissue: (0.249±0.373), IFN-γ increased in silicious group (serum: (886.3±81.7) pg/ml, lung tissue: (0.270±0.300) (P < 0.05). Compared with the normal control group and Con ODN group [(894.5±91.6) pg/ml], IFN-γ in the serum of Sup ODN group decreased significantly (P < 0.01). Compared with the silicious group , IFN-γ in lung tissue decreased in Sup ODN group (0.241±0.250) (P < 0.05). Compared with the normal control group (0.279±0.353), pSTAT4 in lung tissue increased significantly in silicious group (0.313±0.231) (P < 0.01). Compared with the silicious group, pSTAT4 in lung tissue decreased significantly in Sup ODN group (0.269±0.523) (P < 0.01). Sup ODN attained protective effect on Silica treated mice by suppressing expression of IFN-γ and pSTAT4.

Thrombospondin‐4 contributes to spinal cord injury‐induced changes in nociception

Our previous data have indicated that nerve injury-induced up-regulation of thrombospondin-4 (TSP4) proteins in dorsal spinal cord plays a causal role in neuropathic pain state development in a spinal nerve ligation model. To investigate whether TSP4 proteins also contribute to the development of centrally mediated changes in nociception after spinal cord injury (SCI), we investigated whether SCI induced TSP4 dysregulation, and if so, whether this change correlated with changes in nociception in a T9 spinal cord contusion injury model. Behavioural sensitivity to mechanical, thermal stimuli and locomotor function recovery were tested blindly in SCI or sham rats post-injury. Intrathecal antisense or mismatch control oligodeoxynucleotides were used to treat SCI rats with nociceptive hyperreflexia, and Western blots were used to measure TSP4 protein levels in dorsal spinal cord samples. SCI induced below-level hindpaw hypersensitivity to stimuli. TSP4 protein levels are up-regulated in dorsal spinal cord of SCI rats with nociceptive hyperreflexia, but not in SCI rats without nociceptive hyperreflexia. There was no significant difference in motor function recovery post-injury between SCI rats with or without nociceptive hyperreflexia. Intrathecal treatment with TSP4 antisense, but not mismatch control, oligodeoxynucleotides led to reversal of injury-induced TSP4 up-regulation and nociceptive hyperreflexia in SCI rats. SCI leads to TSP4 up-regulation in lumbar spinal cord that may play a critical role in mediating centrally mediated behavioural hypersensitivity. Blocking this pathway may be helpful in management of SCI-induced changes in nociception.

Transfection of nuclear factor‐kappaB decoy oligodeoxynucleotide protects against ischemia/reperfusion injury in a rat epigastric flap model

Nuclear factor-kappaB (NF-κB) is considered to play an important role in the response to ischemia/reperfusion (I/R) injury in flap surgery. To inhibit NF-κB, synthetic double-stranded oligodeoxynucleotide (ODN) was used as a decoy. The present study aimed to evaluate the suppressive effects of NF-κB against I/R injury of experimental rat flap model. An extended epigastric island flap was raised and ischemia was induced for 3 h. NF-κB decoy ODN (group D) or single-strand ODN (control; group S) was injected via the contralateral artery when the pedicle was clamped. Transfection efficiency was evaluated with fluorescein isothiocyanate (FITC)-labeled ODN. The effects of NF-κB decoy ODN were analyzed in groups D and S, and an untreated group (group N). FITC-labeled ODN was distributed over the entire flap. Mean survival rate of the flap was significantly higher in group D than in the other groups (group D: 57.9%; group S: 31.1%; group N 31.7%; p < 0.005). Injured muscle fibers, neutrophils and the expression of inducible nitric oxide synthase were significantly lower in group D. A real-time polymerase chain reaction also demonstrated a tendency for suppression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6. We show that NF-κB decoy ODN protected against flap necrosis as a result of I/R injury in rats. We also indicate that intra-arterial injection of naked NF-κB decoy ODN is effective for transfection into target organs. Therefore, transfection of NF-κB decoy ODN represents a novel therapeutic strategy for the treatment of flap surgery in I/R injury.

In vivo transfection of nuclear factor κB decoy protects pulmonary function against acute lung contusion in rabbits

The transcription factor nuclear factor κB (NF-κB) regulates the expression of numerous proinflammatory factors that may exacerbate the response to acute injury. We investigated the effect of an inhibitory NF-κB decoy oligodeoxynucleotide on proinflammatory factor expression and pulmonary function after acute lung contusion in rabbits. Thirty-minutes after acute lung contusion, the NF-κB decoy or a scrambled control oligodeoxynucleotide was injected via the jugular vein. Blood samples were collected for blood gas analysis and plasma tumor necrosis factor α, interleukin 1β (IL-1β), IL-13, and IL-10 were measured by enzyme-linked immunosorbent assay at 1, 2, 3, and 4 hours after contusion. In addition, NF-κB protein expression in lung tissue was detected by Western blot analysis. The blood PO2 decreased immediately after lung contusion, whereas PAO2 increased significantly, indicative of disrupted respiratory function. Respiratory function improved after sense NF-κB decoy injection but not after injection of the inactive scrambled form. Injection of NF-κB decoy resulted in significant inhibition of NF-κB protein expression in lung tissue and a reduction in the serum concentrations of proinflammatory cytokines tumor necrosis factor α and IL-1β compared with those of control rabbits injected with the scrambled decoy. In contrast, serum levels of the anti-inflammatory cytokines IL-10 and IL-13 increased after decoy injection compared with those of control animals and rabbits injected with the scrambled decoy. The sense NF-κB decoy protected respiratory function and reduced serum proinflammatory factor secretion after acute lung contusion. Inhibition of NF-κB may allow for preservation of pulmonary function for patients with acute lung injury.

Clusterin inhibition using OGX-011 synergistically enhances antitumour activity of sorafenib in a human renal cell carcinoma model

Background:The objective of this study was to investigate whether the therapeutic activity of sorafenib could be enhanced by combining with OGX-011, an antisense oligodeoxynucleotide (ODN) targeting clusterin, in renal cell carcinoma (RCC).Methods:We investigated the effects of combined treatment with OGX-011 and sorafenib on a human RCC ACHN model both in vitro and in vivo.Results:Although clusterin expression was increased by sorafenib, additional treatment of ACHN with OGX-011 significantly blocked the upregulation of clusterin induced by sorafenib. Despite the lack of a significant effect on the growth of ACHN, OGX-011 synergistically enhanced the sensitivity to sorafenib, reducing the IC50 by >50%. Apoptotic changes were intensively detected in ACHN after combined treatment with OGX-011 and a sublethal dose of sorafenib, but not either agent alone. Furthermore, this combined treatment resulted in the marked downregulation of phosphorylated Akt and p44/42 mitogen-activated protein kinase in ACHN compared with treatment with either agent alone. In vivo systemic administration of OGX-011 plus sorafenib significantly decreased the ACHN tumour volume compared with control ODN plus sorafenib.Conclusion:Combined use with OGX-011 may be useful in enhancing the cytotoxic effect of sorafenib on RCC by inducing apoptosis and inactivating major signal transduction pathways.

298 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOR ACTIVITY OF SORAFENIB IN A HUMAN RENAL CELL CARCINOMA MODEL

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2012298 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOR ACTIVITY OF SORAFENIB IN A HUMAN RENAL CELL CARCINOMA MODEL Yuji Kusuda, Hideaki Miyake, Martin Gleave, and Masato Fujisawa Yuji KusudaYuji Kusuda Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.357AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clusterin is expressed in a variety of malignant tumors, including renal cell carcinoma (RCC), and associated with resistance to broad-spectrum treatment. OGX-011 is a second-generation antisense oligodeoxynucleotide (ODN) currently in late-stage clinical development that potently inhibits clusterin expression and enhances the efficacy of anticancer therapies in various human cancers. The objective of this study was to investigate whether inactivation of clusterin with OGX-011 potentiates the effect of sorafenib, a novel inhibitor of multiple tyrosine kinases, in a human RCC ACHN model. METHODS Effects of a combined treatment with OGX-011 and sorafenib on ACHN cells were examined by MTT assay, DNA fragmentation assay and Western bolt analyses of proteins involved in apoptosis and signal transduction. In vivo efficacy of this combined therapy was assessed in nude mice given subcutaneous injection of ACHN cells. RESULTS We initially revealed the dose-dependent inhibition of clusterin expression in ACHN cells by treatment with OGX-011. Although clusterin expression was increased in a dose-dependent manner by sorafenib, additional treatment of ACHN cells with OGX-011 significantly blocked the upregulation of clusterin expression induced by sorafenib. Despite the lack of a significant effect on the growth of ACHN cells, OGX-011 treatment synergistically enhanced the sensitivity of ACHN cells to sorafenib in a dose-dependent manner, reducing the IC50 by more than 50%. Characteristic apoptotic DNA ladder formation was detected after combined treatment with OGX-011 and sublethal dose of sorafenib, but not either agent alone. Furthermore, combined treatment of ACHN cells with OGX-011 and sorafenib resulted in the marked downregulation of Mcl-1, phosphorylated Akt and p44/42 MAP kinase compared with treatment with either agent alone. In vivo systemic administration of OGX-011 and sorafenib synergistically decreased the subcutaneous ACHN tumor volume compared with that of control ODN plus sorafenib. CONCLUSIONS Collectively, these findings demonstrated that clusterin helps confer a resistant phenotype of human RCC cells to sorafenib through the inhibition of apoptosis and activation of major signal transduction pathways, and that combined use of OGX-011 may be useful in enhancing the cytotoxic effect of sorafenib in patients with advanced RCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e121 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuji Kusuda Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Martin Gleave Vancouver, Canada More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 1184: NADPH oxidase-dependent reactive oxygen species generation regulate micro RNA-21 in prostate cancer

Abstract Introduction: MicroRNAs (miRs) are small non-coding RNAs that regulate the coding RNAs at post-transcriptional level. Some miRs have been shown to act as oncomiRs by promoting growth and invasiveness of tumor cells. One of these oncomiRs, miR-21, has been shown to contribute to tumorigenesis of prostate cancer. Although several effector pathways have been suggested for its oncogenic role, little is known about its upstream regulators. In the present study, we hypothesize that high levels of reactive oxygen species (ROS) produced by the NADPH oxidase regulate the expression of miR-21 and its target proteins, maspin and programmed cell death 4 (PDCD4) in prostate cancer cells and prostate cancer samples. Methods: The levels of miR-21 and NADPH oxidase subunit p47phox were detected in RNA samples from human prostate cancer tissues (from OriGene Technologies) by TaqMan and real time RT-PCR assays, respectively. The levels of maspin and PDCD4 were determined in the formalin-fixed paraffin-embedded (FFPE) prostate tumor sections by immunostaining. In vivo studies were done in severe combined immunodeficient (SCID) mice to study the role of miR-21 and NADPH oxidase in prostate cancer metastasis by injecting transiently-transfected PC3-MM cells with control antisense oligodeoxynucleotides (ODNs), miR-21 antisense ODNs (anti-miR-21), scramble siRNA or p47phox siRNA via the tail vein and counting the metastatic lesions in the lungs. For in vitro studies androgen-independent human prostate cancer cell line PC3-MM was used after transient-transfections with ODNs or siRNAs (as mentioned above). Lysates were used to determine the levels of miR-21 (by TaqMan assay) and maspin, PDCD4 and p47phox (by Western blotting), while invasion and migration was studied by matrigel and wound-healing assays, respectively. Results: RNA samples from patients with different stages of prostate cancer showed high levels of miR-21 and p47phox. This was associated with low expression of maspin and PDCD4, targets proteins which are negatively regulated by miR-21. Studies in SCID mice showed reduced number of metastatic lung lesions in mice injected with anti-miR-21 and p47phox siRNA- transfected PC3-MM cells in comparison to control ODNs and scramble siRNA-transfected cells. Further studies in PC3-MM cells showed reduced expression of miR-21, maspin and PDCD4, and decreased invasiveness and migration of PC3-MM cells in presence of anti-miR-21 and p47phox siRNA. Conclusions: Based on the data from the present study we conclude that NADPH oxidase derived ROS serve as essential regulators of miR-21, its target proteins (maspin and PDCD4), and the oncogenic and pro-invasive functions of miR-21 in prostate cancer cells. The increased expression of p47phox could account for the high levels of ROS generation characteristic of prostate cancer cells and aid in promoting high levels of miR-21 expression and oncogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2011-1184

Direct Growth Inhibition of Both Mouse and Human B Cell Lymphomas by CpG Oligodeoxynucleotides

AbstractAbstract 2854Immunostimulatory CpG oligodeoxynucleotides (ODN) are potent activators of T cell immunity and antibody-dependent cellular cytotoxicity (ADCC), and under study as immunotherapeutic agents for a variety of cancers, including B cell lymphomas. Recently, anti-CD20 antibody-CpG conjugates have been shown to eradicate rituximab-resistant B cell lymphoma in a syngeneic murine lymphoma model (D. Betting et al, ASH 2009). CpG is known to strongly stimulate the proliferation of normal B cells. Paradoxically, CpG has been reported to markedly inhibit the in vitro growth of the murine B cell lymphoma A20 (J. Li et al, J. Immunol. 2007), thereby prompting us to investigate the direct effects of CpGs on the growth of human B cell lymphomas. We first demonstrated that CpGs, especially those of the B class, potently inhibited proliferation of the A20 mouse B cell line in vitro by up to 81.5% (class A 58.7% and class C 52.7%). Moreover, in non-tumor bearing mice intratumoral injections of CpG activated normal B cells, while mice bearing subcutaneous A20 tumors showed suppressed tumor growth after CpG injections. Similarly, in humans, CpGs strongly stimulated the proliferation of normal peripheral blood B cells (stimulation index for class B 27.5 at 5 μg/ml). A panel of 12 human lymphoma cell lines (DLBCL, Burkitt's, mantle cell) were cultured in the presence or absence of varying concentrations of CpGs of A, B, or C classes (50, 10, or 2 μg/ml) or control ODN. Proliferation was measured by [3H]-thymidine incorporation in quadruplicate 72 hour cultures, and apoptosis measured by Annexin-V and PI flow cytometry. In contrast to the stimulation observed with normal human B cells, the proliferation of all 12 lymphoma lines were inhibited by CpGs. The strongest inhibitory effects were seen with CpG 7909, a class B CpG under clinical development for cancer therapy (Pfizer, PF-3512676). Raji cells were inhibited by 77.9%, 40.7%, and 8.8% at CpG concentrations of 50, 10, and 2 μg/ml, respectively (p≤0.01 for all comparisons vs. media alone). Among the 12 tested cell lines, the percentage growth inhibition using 50 μg/ml CpG 7909 was 61.2–80.4% for germinal center-type DLBCL (SUDHL-4, SUDHL-6, OCI-Ly19), 50–59.5% for activated B cell-type DLBCL (SUDHL-2, OCI-Ly3, OCI-Ly10), 56.4–79.3% for Burkitt's lymphomas (Raji, Ramos, Daudi, BJAB), and 69.6–69.9% for mantle cell lymphomas (Jeko-1, Granta-519). Interestingly, although all of the human cell lines expressed TLR9 by semi-quantitative RT-PCR, inhibition in the proliferation levels did not correlate with TLR9 expression levels. CpG 7909 also induced significant levels of apoptosis in Raji and Jeko-1 cells, 10.1% and 27.6% respectively at 50 μg/ml. In conclusion, we have demonstrated that CpGs have divergent effects on normal versus malignant B cells in both mouse and human systems. Delivery of CpG to mouse lymphoma cells inhibited their growth in vivo, while normal mouse B cells were activated. Furthermore, CpGs directly inhibit the proliferation of a large panel of human B cell lymphomas representing the majority of aggressive histologies. These results provide a novel mechanism of action for CpGs as therapeutic agents for B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Accelerated wound healing mediated by activation of Toll-like receptor 9.

Wound healing is mediated through complex interactions between circulating immune cells and local epithelial and endothelial cells. Elements of the innate immune system are triggered when Toll-like receptors (TLR) are stimulated by their cognate ligands, and previous studies suggest that such interactions can accelerate wound healing. This work examines the effect of treating excisional skin biopsies with immunostimulatory CpG oligodeoxynucleotides (ODN) that trigger via TLR9. Results indicate that CpG (but not control) ODN accelerate wound closure and reduce the total wound area exposed over time by >40% (p<0.01). TLR9 knockout mice, a strain unresponsive to the immunomodulatory effects of CpG stimulation, are unresponsive to ODN treatment and exhibit a general delay in healing when compared with wild-type mice. CpG ODN administration promoted the influx of macrophages to the wound site and increased the production of vascular endothelial growth factor, expediting neovascularization of the wound bed (p<0.01 for both parameters). Stimulation via TLR9 thus represents a novel strategy to accelerate wound healing.

Decoy oligodeoxynucleotide against STAT transcription factors decreases allergic inflammation in a rat asthma model

ABSTRACTLeukocyte infiltration and activation of the CD40-CD40 ligand costimulatory pathway may promote inflammatory processes such as asthma. The aim of this study was to investigate whether a single intratracheal application of a decoy oligodeoxynucleotide (ODN) specific for signal transducer and activator of transcription (STAT) family members 1 and 3 influences leukocyte influx and pulmonary CD40 expression in a rat model of allergic airway inflammation. In comparison with the corticosteroid budesonide, the authors investigated the efficacy of the STAT decoy ODN in ovalbumin-induced allergic asthma in a Brown Norway rat asthma model. Leukocytes of the bronchoalveolar lavage (BAL) and lung tissue were analyzed and expression of CD40 was assessed by Western blotting. Single administration of the STAT decoy ODN but not of a mutated control ODN or budesonide resulted in a significant decrease of eosinophils and T lymphocytes in the BAL fluid. Cell numbers of CD4++ and CD8++ lymphocytes were significantly decreased in the lung tissue after decoy ODN application. CD40 expression in protein extracts from lung tissue was also reduced significantly following STAT decoy ODN treatment. These findings indicate that a single, local application of a transcription factor decoy ODN specific for STAT1 and STAT3 caused an attenuation of the allergen-induced cellular inflammatory reaction and is at least as effective as a topical steroid.

Role of STAT3 decoy oligodeoxynucleotides on cell invasion and chemosensitivity in human epithelial ovarian cancer cells

Recent studies have reported that STAT3 activation is associated with poor prognosis in human epithelial ovarian cancer. STAT3 has been proposed to play an important role in ovarian cancer metastasis and chemoresistance. This mechanism, however, is still not thoroughly understood. In this study, to investigate the role of STAT3 on ovarian cancer cells, we used decoy oligodeoxynucleotide (ODN) technology to regulate STAT3 in SKOV3 and OVCAR3 cells in vitro. Cell invasive power and chemo-sensitivity were assessed in the cells transfected with STAT3 decoy ODN and control ODN. Western blot analysis was used to examine the expression of EMMPRIN, P-gp, and Akt. Results showed that STAT3 decoy ODN inhibited cancer cell invasive power and enhanced sensitivity to paclitaxel for SKOV3 and OVCAR3 cells. The mechanism involved the inhibition of EMMPRIN, P-gp, and pAkt by STAT3 decoy ODN. These three proteins were probably the target proteins of STAT3. These findings suggest that STAT3 is a key factor for ovarian cancer metastasis and chemoresistance. STAT3 decoy ODN may prove to be a beneficial therapeutic agent, especially for invasive or chemoresistant ovarian cancer.

Intratracheal synthetic CpG oligodeoxynucleotide causes acute lung injury with systemic inflammatory response

Bacterial genome is characterized by frequent unmethylated cytosine-phosphate-guanine (CpG) motifs. Deleterious effects can occur when synthetic oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides (CpG-ODN) are administered in a systemic fashion. We aimed to evaluate the effect of intratracheal CpG-ODN on lung inflammation and systemic inflammatory response. C57BL/6J mice received intratracheal administration of CpG-ODN (0.01, 0.1, 1.0, 10, or 100 μM) or control ODN without CpG motif. Bronchoalveolar lavage (BAL) fluid was obtained 3 or 6 h or 1, 2, 7, or 14 days after the instillation and subjected to a differential cell count and cytokine measurement. Lung permeability was evaluated as the BAL fluid-to-plasma ratio of the concentration of human serum albumin that was injected 1 h before euthanasia. Nuclear factor (NF)-κB DNA binding activity was also evaluated in lung homogenates. Intratracheal administration of 10 μM or higher concentration of CpG-ODN induced significant inflammatory cell accumulation into the airspace. The peak accumulation of neutrophils and lymphocytes occurred 1 and 2 days after the CpG-ODN administration, respectively. Lung permeability was increased 1 day after the 10 μM CpG-ODN challenge. CpG-ODN also induced nuclear translocation of NF-κB and upregulation of various inflammatory cytokines in BAL fluid and plasma. Histopathology of the lungs and liver revealed acute lung injury and liver damage with necrosis, respectively. Control ODN without CpG motif did not induce any inflammatory change. Since intratracheal CpG-ODN induced acute lung injury as well as systemic inflammatory response, therapeutic strategies to neutralize bacterial DNA that is released after administration of bactericidal agents should be considered.