Antisense Oligodeoxynucleotide Targeting Research Articles

Nociceptor Overexpression of NaV1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (NaV1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased NaV1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting NaV1.7 alpha subunit mRNA attenuated the expression of NaV1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective NaV1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the NaV1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances NaV1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of NaV1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that NaV1.7 is a target for the treatment of chronic muscle pain. PerspectiveWe demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of NaV1.7 in nociceptors.

Sexual dimorphism in the contribution of neuroendocrine stress axes to oxaliplatin-induced painful peripheral neuropathy.

Although clinical studies support the suggestion that stress is a risk factor for painful chemotherapy-induced peripheral neuropathy (CIPN), there is little scientific validation to support this link. Here, we evaluated the impact of stress on CIPN induced by oxaliplatin, and its underlying mechanisms, in male and female rats. A single dose of oxaliplatin produced mechanical hyperalgesia of similar magnitude in both sexes, still present at similar magnitude in both sexes, on day 28. Adrenalectomy mitigated oxaliplatin-induced hyperalgesia, in both sexes. To confirm the role of neuroendocrine stress axes in CIPN, intrathecal administration of antisense oligodeoxynucleotide targeting β₂-adrenergic receptor mRNA both prevented and reversed oxaliplatin-induced hyperalgesia, only in males. By contrast, glucocorticoid receptor antisense oligodeoxynucleotide prevented and reversed oxaliplatin-induced hyperalgesia in both sexes. Unpredictable sound stress enhanced CIPN, in both sexes. The administration of stress hormones, epinephrine, corticosterone, and their combination, at stress levels, mimicked the effects of sound stress on CIPN, in males. In females, only corticosterone mimicked the effect of sound stress. Also, a risk factor for CIPN, early-life stress, was evaluated by producing both stress-sensitive (produced by neonatal limited bedding) and stress-resilient (produced by neonatal handling) phenotypes in adults. Although neonatal limited bedding significantly enhanced CIPN only in female adults, neonatal handling significantly attenuated CIPN, in both sexes. Our study demonstrates a sexually dimorphic role of the 2 major neuroendocrine stress axes in oxaliplatin-induced neuropathic pain.

Sulfonylurea Receptor 1, Transient Receptor Potential Cation Channel Subfamily M Member 4, and KIR6.2:Role in Hemorrhagic Progression of Contusion.

In severe traumatic brain injury (TBI), contusions often are worsened by contusion expansion or hemorrhagic progression of contusion (HPC), which may double the original contusion volume and worsen outcome. In humans and rodents with contusion-TBI, sulfonylurea receptor 1 (SUR1) is upregulated in microvessels and astrocytes, and in rodent models, blockade of SUR1 with glibenclamide reduces HPC. SUR1 does not function by itself, but must co-assemble with either KIR6.2 or transient receptor potential cation channel subfamily M member 4 (TRPM4) to form KATP (SUR1-KIR6.2) or SUR1-TRPM4 channels, with the two having opposite effects on membrane potential. Both KIR6.2 and TRPM4 are reportedly upregulated in TBI, especially in astrocytes, but the identity and function of SUR1-regulated channels post-TBI is unknown. Here, we analyzed human and rat brain tissues after contusion-TBI to characterize SUR1, TRPM4, and KIR6.2 expression, and in the rat model, to examine the effects on HPC of inhibiting expression of the three subunits using intravenous antisense oligodeoxynucleotides (AS-ODN). Glial fibrillary acidic protein (GFAP) immunoreactivity was used to operationally define core versus penumbral tissues. In humans and rats, GFAP-negative core tissues contained microvessels that expressed SUR1 and TRPM4, whereas GFAP-positive penumbral tissues contained astrocytes that expressed all three subunits. Förster resonance energy transfer imaging demonstrated SUR1-TRPM4 heteromers in endothelium, and SUR1-TRPM4 and SUR1-KIR6.2 heteromers in astrocytes. In rats, glibenclamide as well as AS-ODN targeting SUR1 and TRPM4, but not KIR6.2, reduced HPC at 24 h post-TBI. Our findings demonstrate upregulation of SUR1-TRPM4 and KATP after contusion-TBI, identify SUR1-TRPM4 as the primary molecular mechanism that accounts for HPC, and indicate that SUR1-TRPM4 is a crucial target of glibenclamide.

Abstract 1600: Interleukin-8 (IL-8) in TGF-β immunotherapy and chemotherapies

Abstract Background: Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment. OT-101 is a phosphorothioate antisense oligodeoxynucleotide targeting transforming growth factor-beta 2 (TGF-β2). Herein, we examined the role of IL-8 in OT-101 treatment followed with chemotherapies during our Phase II trial in patients with advanced pancreatic cancer (PAC). Methods: Time evolution of a panel of 31 cyto-/chemokine levels in plasma were tracked over 3 cycles of OT-101 administration (140 mg/m2/day) for 12 PAC patients. Samples were acquired before onset of OT-101 therapy and at 8 selected time points (Cycle 1 [Day 2 and 5], Cycle 2 [Days 1, 2, and 5], Final Visit, Cycle 3 [Day 5]) during the therapy. Samples were measured in duplicate and concentrations were expressed in pg/mL. Standardized log10 transformed values calculated from the mean and standard deviation of each cyto-/chemokine in each patient was utilized in an ANCOVA model to investigate the correlation with Overall Survival (OS). Results: Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cyto-/chemokines (Cluster 1: EGF, MIP-1α, MIP-1β; Cluster 2: FGF-2, IL-1RA, MIG, IP-10, IL-15, IFN-α, IL-12A/IL-12; Cluster 3: HGF, IL-2R, IL-6, IL-8). Protein-Protein Interaction networks constructed using STRING10 algorithm identified a relationship between IL-8 and TGFβR2 inhibition. The ANCOVA model explained a significant proportion of the observed data for Cycle 1[Day 2] measurements of cyto-/chemokines (R2 = 0.3, F59,217 = 1.575, P = 0.0103). Other time points did not exhibit a significant model fit or significant relationships in the interaction term. IL-8 expression showed significant association with OS (positive association, N=12, T-value = 2.92, P = 0.0039) at Cycle 1[Day 2] measurements (7% False Discovery Rate). Linear regression of the increase in IL-8 levels during Cycle 1[Day 2] treatment with OT-101 was related to an increase in OS outcome (R2 = 0.54). To further define the appropriate chemoagent to combine with OT-101, we evaluated paclitaxel (PTX), gemcitabine (GEM), and dacarbazine (DTIC) in animal model. Synergy was observed with PTX and DTIC, but not with GEM. Both PTX and DTIC induced IL-8 expression whereas, surprisingly, GEM reduced IL-8 expression. Conclusion: IL-8 expression during early phase of OT-101 treatment cycle was positively associated with OS across 12 patients. IL-8 is also induced by PTX and DTIC which synergized with OT-101. The data suggests that PAC and DTIC are acting on the same target as our TGF-β2 inhibitor; and potentially, the beneficial effect of these chemoagents is a result of chemoagent-induced immune/IL-8 response. Citation Format: Larn Hwang, Kevin Ng, Osmond D’Cruz, Sanjive Qazi, Andrew Schmidt, Vuong Trieu. Interleukin-8 (IL-8) in TGF-β immunotherapy and chemotherapies [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 1600. doi:10.1158/1538-7445.AM2017-1600

Functional up-regulation of Nav1.8 sodium channel on dorsal root ganglia neurons contributes to the induction of scorpion sting pain.

BmK I, purified from the venom of scorpion Buthus martensi Karsch (BmK), is a receptor site-3-specific modulator of voltage-gated sodium channels (VGSCs) and can induce pain-related behaviors in rats. The tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8 contributes to most of the sodium current underlying the action potential upstroke in dorsal root ganglia (DRG) neurons and may serve as a critical ion channel targeted by BmK I. Herein, using electrophysiological, molecular, and behavioral approaches, we investigated whether the aberrant expression of Nav1.8 in DRG contributes to generation of pain induced by BmK I. The expression of Nav1.8 was found to be significantly increased at both mRNA and protein levels following intraplantar injection of BmK I in rats. In addition, the current density of TTX-R Nav1.8 sodium channel is significantly increased and the gating kinetics of Nav1.8 is also altered in DRG neurons from BmK I-treated rats. Furthermore, spontaneous pain and mechanical allodynia, but not thermal hyperalgesia induced by BmK I, are significantly alleviated through either blockade of the Nav1.8 sodium channel by its selective blocker A-803467 or knockdown of the Nav1.8 expression in DRG by antisense oligodeoxynucleotide (AS-ODN) targeting Nav1.8 in rats. Finally, BmK I was shown to induce enhanced pain behaviors in complete freund's adjuvant (CFA)-inflamed rats, which was partly due to the over-expression of Nav1.8 in DRG. Our results suggest that functional up-regulation of Nav1.8 channel on DRG neurons contributes to the development of BmK I-induced pain in rats.

Enhancement of glioma-specific immunity in mice by "NOBEL", an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide.

Autologous glioblastoma multiforme tumor cells treated with an antisense oligodeoxynucleotide (AS-ODN) targeting insulin-like growth factor receptor-1 (IGF-1R) are the basis of a vaccine with therapeutic effects on tumor recurrence in a pilot clinical trial. As a preface to continued clinical investigation of this vaccination strategy, we have studied the contribution of an optimized IGF-1R AS-ODN, designated "NOBEL", to the induction of immunity to mouse GL261 glioma cells. The impact of NOBEL on mechanisms contributing to the development of GL261 immunity was first examined in the periphery. GL261 cells are naturally immunogenic when implanted into the flanks of congenic C57BL/6 mice, immunizing rather than forming tumors in around 50 % of these animals but causing tumors in the majority of mice lacking T and B lymphocytes. Overnight treatment with NOBEL in vitro reduces IGF-1R expression by GL261 cells but has minimal effect on cell viability and does not reduce the capacity of the cells to form tumors upon implantation. In contrast, tumors are extremely rare when GL261 cells are mixed with NOBEL at inoculation into the flanks of C57BL/6, and the recipient mice become immune to subcutaneous and intracranial challenge with untreated GL261. Adaptive immune mechanisms contribute to this effect, as immunocompromised mice fail to either fully control tumor formation or develop immunity following flank administration of the GL261/NOBEL mix. NOBEL's structure has known immunostimulatory motifs that likely contribute to the immunogenicity of the mix, but its specificity for IGF-1R mRNA is also important as a similarly structured sense molecule is not effective.

Screening the Role of Pronociceptive Molecules in a Rodent Model of Endometriosis Pain

Chronic pain is a major symptom in patients with endometriosis, a common gynecologic condition affecting women in their reproductive years. Although many proalgesic substances are produced by endometriosis lesions, experimental evidence supporting their relative roles is still lacking. Furthermore, it is unclear whether these proalgesic agents directly activate nociceptors to induce endometriosis pain. To determine their relative contribution to pain associated with endometriosis, we evaluated the intrathecal administration of oligodeoxynucleotides (ODNs) antisense to messenger RNA for receptors for 3 pronociceptive mediators known to be produced by the ectopic endometrium. Two weeks after the implant of autologous uterine tissue onto the gastrocnemius muscle, local mechanical hyperalgesia was observed in operated rats. Intrathecal antisense ODN targeting messenger RNA for the interleukin 6 receptor–signaling complex subunit glycoprotein 130 and the nerve growth factor tyrosine kinase receptor A, but not their mismatch ODNs, reversibly attenuated mechanical hyperalgesia at the implant site. In contrast, intrathecal antisense ODN targeting the tumor necrosis factor receptor 1, at a dose that markedly inhibited intramuscularly injected tumor necrosis factor alpha, had only a small antihyperalgesic effect in this model. These results indicate the relative contribution of pronociceptive mediators produced by ectopic endometrial tissue to endometriosis pain. The experimental approach presented here provides a novel method to evaluate for the differential contribution of mediators produced by other painful lesions as well as endometriosis lesions as targets for novel treatment of pain syndromes. PerspectiveThis article presents evidence for the relative contribution of proalgesic mediators to primary hyperalgesia displayed by rats submitted to a model of endometriosis pain. This approach can be used to identify potential targets for the treatment of endometriosis pain.

Blockage of the Upregulation of Voltage-Gated Sodium Channel Nav1.3 Improves Outcomes after Experimental Traumatic Brain Injury

Excessive active voltage-gated sodium channels are responsible for the cellular abnormalities associated with secondary brain injury following traumatic brain injury (TBI). We previously presented evidence that significant upregulation of Nav1.3 expression occurs in the rat cortex at 2 h and 12 h post-TBI and is correlated with TBI severity. In our current study, we tested the hypothesis that blocking upregulation of Nav1.3 expression in vivo in the acute stage post-TBI attenuates the secondary brain injury associated with TBI. We administered either antisense oligodeoxynucleotides (ODN) targeting Nav1.3 or artificial cerebrospinal fluid (aCSF) at 2 h, 4 h, 6 h, and 8 h following TBI. Control sham animals received aCSF administration at the same time points. At 12 h post-TBI, Nav1.3 messenger ribonucleic acid (mRNA) levels in bilateral hippocampi of the aCSF group were significantly elevated, compared with the sham and ODN groups (p<0.01). However, the Nav1.3 mRNA levels in the uninjured contralateral hippocampus of the ODN group were significantly lowered, compared with the sham group (p<0.01). Treatment with antisense ODN significantly decreased the number of degenerating neurons in the ipsilateral hippocampal CA3 and hilar region (p<0.01). A set of left-to-right ratio value analyzed by magnetic resonance imaging T2 image on one day, three days, and seven days post-TBI showed marked edema in the ipsilateral hemisphere of the aCSF group, compared with that of the ODN group (p<0.05). The Morris water maze memory retention test showed that both the aCSF and ODN groups took longer to find a hidden platform, compared with the sham group (p<0.01). However, latency in the aCSF group was significantly higher than in the ODN group (p<0.05). Our in vivo Nav1.3 inhibition studies suggest that therapeutic strategies to block upregulation of Nav1.3 expression in the brain may improve outcomes following TBI.

C‐fos down‐regulation inhibits testosterone‐dependent male sexual behavior and the associated learning

Environmental stimulation results in an increased expression of transcription factors called immediate early genes (IEGs) in specific neuronal populations. In male Japanese quail, copulation with a female increases the expression of the IEGs zenk and c-fos in the medial pre-optic nucleus (POM), a key nucleus controlling male sexual behavior. The functional significance of this increased IEG expression that follows performance of copulatory behavior is unknown. We addressed this question by repeatedly quantifying the performance of appetitive (learned social proximity response) and consummatory (actual copulation) sexual behavior in castrated, testosterone-treated males that received daily intra-cerebroventricular injection of an antisense oligodeoxynucleotide targeting c-fos or control vehicle. Daily antisense injections significantly inhibited the expression of copulatory behavior as well as the acquisition of the learned social proximity response. A strong reduction of the proximity response was still observed in antisense-treated birds that copulated with a female, ruling out the indirect effect of the absence of interactions with females on the learning process. After a 2-day interruption of behavioral testing but not of antisense injections, birds were submitted to a final copulatory test that confirmed the behavioral inhibition in antisense-injected birds. Brains were collected at 90 min after the behavioral testing for quantification of c-fos-immunoreactive cells. A significant reduction of the number of c-fos-positive cells in the POM but not in other brain regions was observed following antisense injection. Taken together, the data suggest that c-fos expression in the POM modulates copulatory behavior and sexual learning in male quail.

Abstract 3314: Sensitization of human tumor cells to chemotherapy drugs by antisense downregulation of BRCA2 and thymidylate synthase (TS): Induction of synthetic lethality by targeting DNA repair.

Abstract Although genomic instability contributes to tumor progression and development of drug resistance, molecular events underlying instability could also lead to selective sensitivity of tumor cells to certain treatments. Deficiencies in specific mechanisms of DNA repair, with resultant dependence on other repair pathways, have been exploited in therapeutic strategies. For example, BRCA1 and BRCA2, which are involved in homologous recombination repair, have been implicated in hereditary and sporadic carcinomas of the breast, ovary, and other sites. Notably, individuals with BRCA2 mutations respond more favourably to conventional chemotherapy. It is hypothesized that in the absence of functional BRCA2 protein, tumor cells are unable to efficiently repair DNA damage induced by anticancer agents. This "synthetic lethality" can be exploited to selectively kill BRCA1- or BRCA2-deficient tumor cells both in vitro (Nature 434: 913, and 917, 2005) and in patients (NEJM 361: 123, 2009). To advance the utility of this phenomenon to selectively kill cancer cells, it may be possible to use antisense technology to create synthetic lethal situations in tumors. siRNA knockdown of BRCA2 in human A549 non-small cell lung cancer cells inhibited proliferation and induced sensitization to cisplatin (DNA cross-linker), melphalan (alkylating agent), the poly(ADP-ribose) polymerase inhibitor olaparib, and the thymidylate synthase (TS)-targeting agent 5-fluorodeoxyuridine. Three novel oligodeoxynucleotides (ODNs) were synthesized to target the BRCA2 mRNA, 2 against the coding region (ODNs BR1 and BR2) and the other in the 3′-untranslated region (BR3). All three ODNs sensitized A549 cells to cisplatin by up to 80%, and BR1 synergistically inhibited proliferation in combination with an antisense ODN targeting TS, SARI-083. A combination of siRNA treatments that knocked down both BRCA2 and TS sensitized cells to a combination of alkylating agents (cisplatin or melphalan) and a TS-targeting drug (5-FUdR). This combination was more effective at inhibiting proliferation than would be predicted by knockdown of TS and BRCA2 followed by treatment with any single drug (5-FUdR, cisplatin, or melphalan). These data suggest that: (1) targeting BRCA2 is a viable strategy to increase the effectiveness of common chemotherapeutic drugs; and (2) it is possible to use antisense-mediated targeting of both TS and a DNA repair protein to sensitize the same cell to two different drugs, and thus potentiate overall therapeutic effectiveness. This is progressing towards human trials. Supported by a grant from the Canadian Institutes of Health Research (CIHR) to JK and MDV. MR is a scholar of the CIHR Strategic Training Program in Cancer Research and Technology Transfer (CaRTT) and a recipient of the Queen Elizabeth II Scholarship in Science and Technology. Citation Format: Peter J. Ferguson, Mateusz Rytelewski, Mark D. Vincent, René Figueredo, James Koropatnick. Sensitization of human tumor cells to chemotherapy drugs by antisense downregulation of BRCA2 and thymidylate synthase (TS): Induction of synthetic lethality by targeting DNA repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3314. doi:10.1158/1538-7445.AM2013-3314

Antisense oligodeoxynucleotides targeting connexin43 reduce cerebral astrocytosis and edema in a rat model of traumatic brain injury

Objective: Brain injury induces an acute increase in the expression of gap junction protein connexin43 (Cx43). It also leads to cerebral edema, probably due to the swelling and proliferation of astrocytes reactive to the injury. Antisense oligodeoxynucleotides (AS-ODN) targeting Cx43 were tested for their ability to reduce reactive astrocytosis and cerebral edema in a rat model of traumatic brain injury (TBI). Methods: The brains of experimental animals were intraventricularly injected with these AS-ODNs, while sham rats and normal controls were administered saline in the same way. Controlled cortical impact (CCI) injury was induced in both experimental and sham rats, then the damaged brain tissue was stained for glial fibrillary acidic protein (GFAP) immunofluorescein, measured for water content using the wet–dry weight method, and examined for Cx43 protein expression by western blotting. Results: The brains of both experimental and sham groups were found to have a higher level of Cx43 expression, higher water content, and more swollen and proliferative astrocytes than the normal controls at 6 hours, 24 hours, and 48 hours post-trauma. But compared with the sham animals, brains of experimental rats showed less Cx43 expression, lower water content, and less swollen and proliferative astrocytes. These two brain-injured groups displayed a similar pattern of changes in these outcomes over the 48-hour time period studied. Discussion: Antisense oligodeoxynucleotides targeting Cx43 reduced reactive astrocytosis and cerebral edema following TBI, indicating that Cx43 might be involved in regulating the water imbalance between brain cells.

Effect of antisense oligodeoxynucleotide targeting survivin on growth of human gastric cancer cells

Our study investigated the effects of antisense oligodeoxynucleotide targeting survivin on human gastric cancer cells. Human gastric cancer cells were incubated with antisense oligodeoxynucleotide targeting survivin for pre-designed durations, and then the cell growth was observed under light and electronic microscopes. Electrophoresis of fractured DNA fragments was performed to detect the DNA distribution and telomere repeat amplification protocol (TRAP) was used for the detection of telomerase activity. Antisense oligodeoxynucleotide targeting survivin could induce the apoptosis of human gastric cancer cells which were characterized by plasma membrane blistering, chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Electrophoresis showed characteristic DNA ladder. Flow cytometry revealed hypo-diploid apoptosis peak before G1 phase and the telomerase activity was significantly inhibited. These results demonstrated antisense oligodeoxynucleotide targeting survivin can induce the apoptosis of gastric cancer cells to inhibit their proliferation.

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.

Antisense oligodeoxynucleotide targeting HER2 mRNA sensitized docetaxel in breast cancer treatment

Context: Human epidermal growth factor receptor 2 (HER2) is one of the oncogenes closely associated with the development and prognosis of breast carcinoma. Down-regulation of HER2 mRNA by antisense oligodeoxynucleotide (ASO) HER2 has been suggested to be a feasible treatment for patients with breast carcinoma.Objective: The antitumor effects of ASO HA6722 were investigated in vitro and in vivo.Materials and methods: In this study, SK-BR-3, a HER2-overexpressing breast carcinoma cell line, was used as the model for in vitro experiments. Inhibitory effects of the ASO HA6722 were detected by methyl-thiazoldiphenyl tetrazolium (MTT) assay. Meanwhile, HER2 mRNA levels were monitored by reverse transcription polymerase chain reaction (RT-PCR). The in vivo antitumor effects were evaluated in nude mice xenograft model.Results: Our results showed that HA6722 alone could inhibit the growth of SK-BR-3 cells in a dose-dependent manner with the IC50 value of 41.8 ± 8.1 nM. In addition, the antitumor effect of docetaxel (TXT) could be sensitized by low dose of HA6722 both in vitro and in vivo, suggesting that ASO HA6722 could inhibit the growth of breast cancer cells and enhance the cytotoxic effects of TXT.Discussion and conclusion: The combination treatment of TXT and HA6722 could be a more effective approach for breast cancer treatment. The future study should focus on the antitumor effect in other models.

Antisense-Mediated Knockdown of NaV1.8, but Not NaV1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat

Tetrodotoxin-resistant (TTX-R) sodium channels NaV1.8 and NaV1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported NaV1.8, roles of NaV1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting NaV1.8 and NaV1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, NaV1.8 and NaV1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that NaV1.8 mainly localized in medium and small-sized DRG neurons, whereas NaV1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate NaV1.8 or NaV1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only NaV1.8 AS ODN, but not NaV1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels NaV1.8 and NaV1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain.

Effects of suppressing glucose transporter-1 by an antisense oligodeoxynucleotide on the growth of human hepatocellular carcinoma cells

The glucose transporter-1 (Glut-1), a key rate-limiting factor in the transport and metabolism of glucose in cancer cells, is over-expressed in many human cancer cells and this over-expression is correlated with poor biological behavior. The increased levels of Glut-1 expression in hepatocellular carcinoma (HCC) cells functionally affect tumorigenicity. This study was undertaken to investigate effects of suppressing Glut-1 by an antisense oligodeoxynucleotide (AS-ODN) on the growth of human hepatocellular carcinoma (HepG-2) cells. We used AS-ODN targeting against the Glut-1 gene in a HepG-2 cell line. There were four experimental groups: empty pcDNA3.1 vector (mock transfection), pcDNA3.1-anti-Glut (+), pcDNA3.1-Glut (+), and non-transfected HepG-2 cells. The Glut-1 mRNA expression was detected by RT-PCR and the Glut-1 protein expression by Western blotting after cell culture, and the glucose uptake was detected after glucose stimulation in each group. Compared with non-transfected HepG-2 or Glut-1 pcDNA3.1, a down-regulation of Glut-1 mRNA in HepG-2 cells transfected with anti-Glut-1 pcDNA3.1 was noted (P<0.05). Glut-1 protein in HepG-2 cells transfected with Glut-1 AS-ODN was decreased compared with non-transfected HepG-2, Glut-1 pcDNA3.1, or empty vectors. Glucose uptake by the HepG-2 cells transfected with AS-ODN was decreased at 1 hour after glucose stimulation. The application of Glut-1 AS-ODN can down-regulate the expression of Glut-1 at mRNA and protein, and inhibit glucose uptake partially in HepG-2 cells. The Glut-1 gene maybe a potential therapeutic target for HCC.

The contribution of MOR-1 exons 1–4 to morphine and heroin analgesia and dependence

Although morphine and heroin analgesia is mediated by μ-opioid receptors encoded by the MOR-1 gene, distinct isoforms are involved. Both opioids also induce dependence by acting at μ-opioid receptors, but which variants are utilized is not known. Here, we assayed morphine and heroin analgesia and dependence in mice treated with antisense oligodeoxynucleotides (AO) targeting MOR-1 exons 1–4. Whereas AOs targeting exons 1 and 4 blocked morphine analgesia, those targeting exons 2 and 3 blocked heroin analgesia. Neither morphine nor heroin analgesia was compromised 5 days after the last AO injection. In morphine and heroin dependent mice, only exon 1 AO significantly reduced jumping incidence during naloxone (50 mg/kg) precipitated withdrawal. Neither analgesia nor withdrawal jumping was attenuated in controls pretreated with saline or a mismatch oligodeoxynucleotide control sequence. While these data confirm previous reports that morphine and heroin analgesia are not mediated by a single μ-opioid receptor, both opiates nonetheless apparently induce dependence via a μ-opioid receptor isoform containing exon 1. For heroin, the possibility that analgesia and dependence are mediated by distinct μ-opioid receptor isoforms offers the prospect of developing potent opiate analgesics possessing reduced dependence liability.

Antitumor efficacy of human telomerase reverse transcriptase gene antisense oligonucleotide in pancreatic cancer cells

Human telomerase reverse transcriptase (hTERT) is the key component of telomerase catalytic activity, and is associated with tumorigenesis. Recent evidence suggests that the down-regulation of hTERT could rapidly induce an antiproliferative effect in tumor cells, independent of the telomere-elongating function of the enzyme. To test the immediate antitumor efficacy of this down-regulation, antisense oligodeoxynucleotides (AS-ODN) targeting hTERT mRNA were transfected into two human pancreatic cancer cell lines in vitro. In both cell lines, single transfection with AS-ODN decreased the level of hTERT mRNA expression in a dose-dependent manner (from 0.05 to 0.2 µM), while transfection with 0.2 µM hTERT AS-ODN for 24 h achieved the maximum down-regulation of hTERT mRNA. Additionally, 0.2 µM AS-ODN significantly reduced telomerase activity in the cell lines. However, after the first transfection with 0.2 µM AS-ODN, almost no inhibition of cell proliferation was observed in either of the lines, while multiple consecutive transfections with the same concentration of AS-ODN resulted in a continuous reduction in cell viability, the significant inhibition of colony formation ability and increased cell apoptotic rates. Cell cycle analysis indicated that hTERT AS-ODN mainly arrested the cell cycle at the G0/G1 phase in the cells. The data validate an antisense oligonucleotide approach to hTERT inhibition therapy in pancreatic cancer cells.

Effect of HBx antisense oligodeoxynucleotide on formation of transplanted hepatocellular carcinoma in nude mice

To study the inhibitory effect of HBx antisense oligodeoxynucleotide on the formation of transplanted hepatocellular carcinoma in nude mice. 50 nude mice were randomly divided into 5 groups: 1 control group and 4 experimental groups. Log-phase Hep3B cells endogenously expressing HBX were injected subcutaneously in nude mice. From the second day, the PAGE purified AS1, AS2, AS3 and AS4 HBx antisense oligodeoxynucleotides were injected intraperitoneally into the 4 experimental groups, respectively, on alternate days for 5 times, and distilled water was injected into the control group. Growth information of subcutaneous transplantation tumor in nude mice was recorded for 30 days. Incidence rate of transplanted tumor in different groups was compared and analyzed by survival analysis. Statistics software SPSS12.0 was used to analyze the data. Incidence rate of transplanted tumor was 100% in AS1, AS2, AS3 and control groups, and 90% in AS4 group (x2 = 3.995, P = 1.0). The median latency period for transplanted tumor formation was 19 days (17.48-20.52), 12 days (9.93-14.07), 11 days (9.45 to 12.55), 21 days (19.48 to 22.52), and 10 days (8.99 to 11.01) in AS1, AS2, AS3, AS4 and control group, respectively. The latency period for tumor formation was prolonged by treatment of mice with AS1 and AS4 antisense oligodeoxynucleotide (P less than 0.01). Antisense oligodeoxynucleotide targeting to the appropriate sites of HBx gene can prolong the latency period of subcutaneously transplanted tumor in nude mice, however, the formation of transplanted tumor can not be completely blocked by limited treatment with these antisense oligos. In addition, our results suggest that peritoneal injection may be an effective way to deliver antisense oligodeoxynucleotide to living organisms.

Study on growth inhibition of implanted tumor in nude mice by antisense oligodeoxynucleotide targeting telomerase RNA.

Many studies have proven that telomerase plays an important role during the occurrence and development of lung cancer, which has been an important target in gene therapy. The aim of this study is to explore the inhibitory effect of antisense oligodeoxynucleotides(ASODN) targeting human telomerase RNA on implanted tumor growth in nude mice. Implanted tumor models were Constructed in nude mice using human lung adenocarcinoma cell line A549. Eighteen Balb/C nude mice with implanted tumors were divided into three groups randomly: antisense oligodeoxynucleotide group(group ASODN), sense oligodeoxynucleotide group(group SODN), normal saline group (group NS), with 6 mice respectively. ASODs or SODNs mixed with Lipofectamine-2000 was injected into tumor directly every 24 h for fourteen days. The inhibitory rates of tumor volume in Group ASODN and Group SODN were 43.94% and 6.91%, respectively. There was significant difference between the two groups(t =6.17, P<0.001). All the animals tolerated the drugs well and there was no nausea, vomiting and subcutaneous hemorrhage to occur during the therapy. The weight of the animals increased slightly at the end of experiment. The results of this study suggest that intratumoral injection of human telomerase ribonucleic acid(hTR) ASOND could effectively inhibit the growth of implanted tumor in nude mice.