Sense Oligodeoxynucleotides Research Articles

Hippocampal c- fos is necessary for long-term memory of a socially transmitted food preference

The present article examined the requirement of hippocampal c-Fos for learning a socially transmitted food preference (STFP). We reported previously that expression of the c-Fos protein is increased in the dorsal and ventral hippocampus of rats trained on the STFP ( Countryman, Orlowski, Brightwell, Oskowitz, & Colombo, 2005). Pretraining intrahippocampal antisense to the immediate early gene c- fos was administered to adult male Long–Evans rats to determine if c- fos expression is necessary for either short- or long-term memory for STFP. Guide cannulae were implanted bilaterally into the dorsal hippocampus. Antisense oligodeoxynucleotides (ODNs) were administered unilaterally either 6.5, 8.5, 10.5, or 12.5 h prior to STFP training while either sense ODNs or saline were infused into the opposite hemisphere. Immunocytochemistry was performed, and cells showing c-Fos immunoreactivity (ir) were counted from the antisense-treated hemisphere and compared to cell counts from the control hemisphere. The results indicated significant suppression of learning-induced c-Fos protein at the 8.5 and 10.5 infusion-train intervals. Additional rats were implanted with cannulae into the dorsal and ventral hippocampus, and antisense ODNs, sense ODNs, or saline were administered bilaterally 8.5 h prior to training. Rats were tested immediately and 14 days after training. Rats in all groups showed a significant preference for the demonstrated food at the short-term memory test. At the long-term memory test, however, rats infused with c- fos antisense showed no preference for the demonstrated food whereas rats infused with either sense or saline maintained their preference. The present findings suggest that c- fos is necessary for consolidation of non-spatial hippocampal-dependent memory.

Spinal Glucocorticoid Receptors Contribute to the Development of Morphine Tolerance in Rats

Opioid analgesic tolerance is a pharmacologic phenomenon involving the mechanisms of cellular adaptation. Central glucocorticoid receptors (GRs) have been implicated in the cellular mechanism of neuronal plasticity that has many cellular steps in common with the mechanism of opioid tolerance. In a rat model of morphine tolerance, the authors examined the hypothesis that spinal GRs would play a significant role in the development of tolerance to the antinociceptive effect of morphine. In experiment 1, each group of rats received the GR antagonist RU38486 (0.5 or 1 microg), the mineralocorticoid receptor antagonist spironolactone (3 microg), or a vehicle, given intrathecally with morphine (10 microg) twice daily for 6 days. In experiment 2, four groups of rats were used, and each group received intrathecally 10 microg morphine plus 5 micromol GR antisense oligodeoxynucleotide, sense oligodeoxynucleotide, mixed-base oligodeoxynucleotide, or vehicle. Western blotting was used to examine the expression of GRs within the spinal cord dorsal horn. In experiment 3, the GR agonist dexamethasone (4 microg) was given intrathecally twice daily in combination with 10 microg morphine. For all experiments, the development of morphine antinociceptive tolerance was assessed using the tail-flick test. The development of tolerance to the antinociceptive effect of morphine was substantially attenuated when the GR antagonist RU38486 (1 > 0.5 microg > vehicle) but not spironolactone was coadministered with morphine for 6 days. A single treatment with RU38486 did not affect morphine antinociception, nor did it reverse morphine tolerance on day 7. A similar reduction of morphine tolerance was observed in those rats treated with a GR antisense oligodeoxynucleotide but not a sense or mixed-base oligodeoxynucleotide. The administration of the GR antisense oligodeoxynucleotide also prevented GR up-regulation within the spinal cord dorsal horn. Moreover, the GR agonist dexamethasone facilitated the development of morphine tolerance. The results indicate an important role of spinal GRs in the cellular mechanisms of morphine tolerance in rats and may have significant implications in clinical opioid therapy.

The role of NMDA receptor upregulation in phencyclidine-induced cortical apoptosis in organotypic culture

Phencyclidine (PCP) is an N-methyl- d-aspartate receptor (NMDAR) antagonist known to cause selective neurotoxicity in the cortex following subchronic administration. The purpose of this study was to test the hypothesis that upregulation of the NMDAR plays a role in PCP-induced apoptotic cell death. Corticostriatal slice cultures were used to determine the effects of NMDAR subunit antisense oligodeoxynucleotides (ODNs) on PCP-induced apoptosis and NMDAR upregulation. NR1, NR2A or NR2B antisense ODNs were incubated alone or with PCP for 48 h. One day following washout, it was observed that PCP treatment caused an increase in NR1, NR2A and Bax polypeptides in the cortex, but had no effect on Bcl-xL. These increases were associated with an increase in cortical histone-associated DNA fragments. Co-incubation of PCP with either NR1 or NR2A antisense significantly reduced PCP-induced apoptosis, while neither NR2B antisense ODN nor NR1 sense ODN used as a control had an effect. This effect was exactly correlated with the ability of the antisense ODNs to prevent PCP-induced upregulation of NR subunit proteins and the pro-apoptotic protein, Bax. That is, western analysis showed that antisense ODNs directed against either NR1 or NR2A prevented PCP-induced increases in Bax in addition to preventing the upregulation of the respective receptor proteins. On the other hand, the NR2B antisense ODN had no effect on either NR2B protein or on Bax. These data suggest that NR1 and NR2A antisense ODNs offer neuroprotection from apoptosis, and that upregulation of the NR1 and NR2A subunits following PCP administration is at least partly responsible for the observed apoptotic DNA fragmentation.

Inhibition of IκB Kinase Activity by Acetyl-boswellic Acids Promotes Apoptosis in Androgen-independent PC-3 Prostate Cancer Cells in Vitro and in Vivo

Signaling through NF-kappaB has been implicated in the malignant phenotype as well as the chemoresistance of various cancers. Here we show that the natural compounds acetyl-beta-boswellic acid and acetyl-11-keto-beta-boswellic acid (AKbetaBA) inhibit proliferation and elicit cell death in chemoresistant androgen-independent PC-3 prostate cancer cells in vitro and in vivo. Induction of apoptosis was demonstrated in cultured PC-3 cells by several parameters including mitochondrial cytochrome c release and DNA fragmentation. At the molecular level these compounds inhibit constitutively activated NF-kappaB signaling by intercepting the IkappaB kinase (IKK) activity; signaling through the interferon-stimulated response element remained unaffected, suggesting specificity for IKK inhibition. The impaired phosphorylation of p65 and the reduced nuclear translocation of NF-kappaB proteins were associated with down-regulation of the constitutively overexpressed and NF-kappaB-dependent antiapoptotic proteins Bcl-2 and Bcl-x(L). In addition, expression of cyclin D1, a crucial cell cycle regulator, was reduced as well. Down-regulation of IKK by antisense oligodeoxynucleotides confirmed the essential role of IKK inhibition for the proliferation of the PC-3 cells. Both compounds tested were active in vivo, yet AKbetaBA proved to be far superior. Indeed, topical application of water-soluble AKbetaBA-gamma-cyclodextrin on PC-3 tumors xenografted onto chick chorioallantoic membranes induced concentration-dependent inhibition of proliferation as well as apoptosis. Similarly, in nude mice carrying PC-3 tumors, systemic application of AKbetaBA-gamma-cyclodextrin inhibited tumor growth and triggered apoptosis in the absence of detectable systemic toxicity. Thus, AKbetaBA and related compounds acting on IKK might provide a novel approach for the treatment of chemoresistant human tumors such as androgen-independent human prostate cancers.

Effect ofc-fosantisense probe on prostaglandin E2-induced upregulation of vascular endothelial growth factor mRNA in human liver cancer cells

To examine the effect of prostaglandin E2 (PGE2) on the expression of vascular endothelial growth factor (VEGF) mRNA in the human hepatocellular carcinoma (HCC) HepG2 cells and the possible involvement of c-fos protein in this process. Human HCC HepG2 cells were divided into three groups treated respectively with PGE2, a combination of PGE2 and c-fos antisense oligodeoxynucleotide (ASO), and PGE2 plus c-fos sense oligodeoxynucleotide (SO). The expression of VEGF mRNA in HepG2 cells after different treatments was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The relative expression level of VEGF mRNA in HepG2 cells in each group was measured. Administration of PGE2 resulted in an increased expression of c-fos and VEGF mRNA in HepG2 cells. The relative expression level of c-fos mRNA reached the peak at 3 h (68.4+/-4.7%) after PGE2 treatment, which was significantly higher than that at 0 h (20.6+/-1.7%, P<0.01). Whereas, the highest expression level of VEGF mRNA was observed at 6 h (100.5+/-6.1%) after PGE2 treatment, which was significantly higher than that at 0 h (33.2+/-2.4%, P<0.01). C-fos ASO significantly reduced PGE2-induced VEGF mRNA expression in HepG2 cells. PGE2 increases the expression and secretion of VEGF in HCC cells by activating the transcription factor c-fos, promotes the angiogenesis of HCC and plays an important role in the pathogenesis of liver cancer.

Downregulation of Decorin and Transforming Growth Factor-β1 by Decorin Gene Suppression in Tendinocytes

Scars formed after tendonitis result in altered tissue mechanical properties after injury. The interaction of collagen molecules with decorin affects collagen fibrogenesis, and scar tissue is fragile as a consequence of a large amount of decorin in the scar. We hypothesized that scar formation could be prevented by controlling decorin expression in tendinocytes. As a preliminary experiment, we treated tendinocytes with decorin antisense oligodeoxynucleotides (ODNs). Tendinocytes were isolated from Achilles tendons of New Zealand white rabbits and treated with ODN. When tendinocytes were transfected with decorin sense ODN, there was no alteration, whereas decorin antisense ODN-treated tendinocytes showed suppression of transforming growth factor (TGF)-β1 production. Decorin and TGF-β1-production of tendinocytes is regulated by decorin gene suppression. The results showed that the antisense approach is an attractive therapeutic strategy not only for preventing decorin deposition in scar tissue, which decreases collagen fibril diameter, but also for controlling TGF-β1 production, which leads to organ fibrosis.

Effect of lung resistance-related protein on the resistance to cisplatin in human ovarian cancer cell lines

The mechanisms of drug-resistance in human ovarian cancer cells have not been entirely clarified. The purpose of this study was to investigate whether LRP is involved in the resistance of ovarian cancer cell lines to cisplatin and its molecular mechanism. Human ovarian cisplatin-resistant cancer cell lines (A2780/DDP and COC1/DDP) and their parental cisplatin-sensitive cell lines (A2780 and COC1), alone or transfected with antisense LRP-specific oligonucleotides (ODN) or sense ODN, were treated with cisplatin to induce differentiation. Expression of LRP was examined by RT-PCR and Western blot analysis. The sensitivities of cells to cisplatin were assessed using sulforhodamine B (SRB) assay and flow cytometry, and the accumulation and efflux of cisplatin in the cells and isolated nuclei were examined by high performance liquid chromatographic (HPLC) assay. The expressions of LRP in A2780/DDP and COC1/DDP cells were higher than those in A2780 and COC1 cells and conferred resistance to cisplatin. Transfection of LRP AsODN into A2780/DDP and COC1/DDP cells down-regulated LRP expression and reversed the resistance phenotype. Levels of cisplatin accumulating in cells were increased by LRP-specific AsODN and anti-LRP monoclonal antibody. Isolated nuclei from A2780 and COC1 cells or A2780/DDP and COC1/DDP cells incubated with anti-LRP antibody contained more cisplatin than the nuclei of A2780/DDP and COC1/DDP cells not treated with anti-LRP antibody. Efflux of cisplatin was greater from the nuclei of A2780/DDP and COC1/DDP cells than those of A2780 and COC1 cells, and was inhibited by anti-LRP monoclonal antibody. Thus, LRP was involved in the resistance of ovarian cancer cells to cisplatin and has an important role in the transport of cisplatin both in exocytotic vesicles and between the nucleus and cytoplasm.

Effect of hTR Antisense Oligodeoxynucleotides on Telomerase Acitvity and Ciplatin -Sensitivity of Primary Acute Leukemia Cells.

BACKGROUND: Between the three key components part of human telomerase, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) have significant correlation with telomerase activity. The previous study has identified that the telomerase activity of K562 and HL-60 cells was special suppresssed significantly by phosphorothoate antisense oligodeoxynucleotide (ASODN) complementary to the initiator codon of hTR. This study was designed to evalutae the effection of hTR ASODN on telomerase activity and apoptosis of primary acute leukemic cells. To research whether hTR ASODN could enhance apoptosis rates of primary leukemic cells to cisplatin.METHODS: Primary leukemic cells were treated with phosphorothoate ASODN complementary to the initiator codon of hTR in vivo. The changing of telomerase activity was assayed by telomeric repeat amplification protocol(TRAP) and polymerase chain reaction enzyme-linked immunoassay(PCR-ELISA). The survival rates of cells was measured by trypan blue exclusion. Apoptosis was assayed by morphological observation (Giemsa and PI), DNA gel electrophoresis and flow cytometry analysis technology.RESULTS: Primary acute leukemic cells expressed high level of telomerase activity which decreased as the cells treated by hTR ASODN. The telomerase activity was suppresssed significantly by 10umol/L ASODN and the effecttion was most significantly at 72h; Apoptotic bodies of primary leukemic cells were observed easily by fluorescence micrscope when cisplatin was added 48h after ASODN treatment for 24h; Agarose gel electrophoresis of genomic DNA from primary leukemic cells treated with ASODN and cisplatin combination for 72h showed typical DNA ladder; neither did DNA from primary leukemic cells treated with sense oligodeoxynucleotide (SODN) plus cisplatin nor cisplatin alone. In addition, apoptosis rates of primary leukemic cells treated with ASODN for 24h and then with cisplatin for 72h were 41.36±9.28%. There were statistically significant difference in the percentage of apoptotic cells between hTR ASODN plus cisplatin and SODN plus cisplatin (14.51±4.78%) or cisplatin alone group (12.61±2.56)% (P<0.01).CONCLUSIONS: ASODN complementary to the region of hTR could significantly inhibit the telomerase activity of primary acute leukemic cells, and increased the cisplatin-induced apoptosis and enhanced cisplatin-sensitivity in vivo, indicating telomerase may be a new target of treatment to leukemia.

Effect of hTERT Antisense on Cisplatin Sensitivity of Acute Lymphoblastic Leukemic Cells.

AIM: To investigate the effect of human telomerase reverse transcriptase(hTERT) antisense phosphorothioate oligodeoxynucleotide(AS PS-ODN) on enhancing cisplatin sensitivity in acute lymphoblastic leukemic cells.METHODS: The expression levels of hTERT protein were detected by immunofluorescence using fluoresce isothiocyanate(FITC) lable. Cell surviving fraction was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis.RESULTS: The expression of hTERT protein was inhibited after treated by hTERT AS PS-ODN. Treatment with cisplatin after 24h of exposure to AS PS-ODN had significantly reduced the number of viable ALL cells. However, there was no difference on ALL cells survival between sense oligodeoxynucleotide(S PS-ODN)/CDDP combination and CDDP treated cells alone. In morphological observation of apoptotic cells using Hoechst 33258 and PI double staining techniques, cells displayed classic apoptotic changes treated with CDDP or CDDP combined with hTERT AS PS-ODN or S PS-ODN at 48h. Apoptotic rates of cells added CDDP and AS PS-ODN were higher than that of cells added CDDP only(P<0.05). Apoptotic rates of cells added CDDP and S PS-ODN were similar with that of cells added CDDP only(P>0.05).CONCLUSION: hTERT AS-ODN inhibited the expression of hTERT protein and increased the CDDP induced apoptosis in primer acute lymphoblastic leukemic cells.

Effect of down regulation of laminin receptor on urokinase-type plasminogen activator expression in human bile duct carcinoma cells

To investigate the effect of antisense oligonucleotide (AS-OD) of laminin receptor (LNR) on urokinase-type plasminogen activator (u-PA) expression in human bile duct carcinoma cells. Bile duct carcinoma cells of QBC939 line were cultured. Compounds of lipofectAMINE and AS-OD phosphorothioate of LNR of the concentrations of 3, 6, and 12 micromol/L respectively were prepared and then transfected into the QBC939 cells. Sense oligodeoxynucleotide (S-OD) of LNR and blank vector were used as controls. After culture of 48 hours, flow cytometry was used to detect the protein expression of LNR and the u-PA mRNA expression was detected by reverse-transcriptase polymerase chain reaction (RT-PCR). Cell invasion was examined by culture plate with Transwell microporous film before and after transfection. Flow cytometry showed down-regulation of LNR protein expression in the cells transfected with AS-OD dose-dependently. The LNR protein expression of the cells transfected with AS-OD of the concentration of 6 micromol/L was down-regulated by over 25% in comparison with those of the blank vector group and S-OD group (both P < 0.05). RT-PCR showed that the u-PA mRNA expression of the AS-OD 6 micromol/L group was down-regulated by 30% in comparison with the 2 control groups. The number of migrating cells in the AS-OD group was 78 +/- 6, significantly lower than those in the S-OD group (105 +/- 11), and blank vector group (101 +/- 7), both P < 0.05. The expression of laminin receptor in the bile duct carcinoma cells is related with the expression of u-PA gene expression. Inhibition of LNR expression decreases the u-PA gene expression, suggesting that LNR plays an important role in the invasion and metastasis of bile duct carcinoma.

In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide.

The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R. The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of 99mTc-sestamibi (MIBI), a known substrate for P-glycoprotein. A 15-mer phosphorothioate antisense ODN complementary to the sequences located at -1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine penta-acetic acid (cDTPA) via an amino group linked to the terminal phosphate at the 5' end at pH 8-9. The DTPA-ODN complexes at concentrations of 0.1-17.4 microM were reacted with 111InCl3 at pH 5 for 1 h. The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance liquid chromatography following incubation with the complementary sequence. Cellular uptake of labeled ODN was examined in vitro. Furthermore, enhancing effects of synthetic lipid carriers (Transfast) on transmembrane delivery of ODN were assessed. P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells. 99mTc-MIBI uptake in P388/S cells was higher than that in P388/R cells. Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount. The hybridization affinity of antisense 111In-ODN was preserved at approximately 85% irrespective of specific activity. Cellular uptake of antisense 111In-ODN did not differ from that of sense 111In-ODN in either P388/S cells or P388/R cells. However, lipid carrier incorporation significantly increased transmembrane delivery of 111In-ODN; moreover, specific uptake of antisense 111In-ODN was demonstrated in P388/R cells. Radiolabeling of ODN at high specific radioactivity and specific uptake of antisense 111In-ODN in drug-resistant cells may facilitate future gene imaging of mdr1 mRNA.

Role of the c-myc proto-oncogene in the proliferation of hereditary gingival fibromatosis fibroblasts.

Hereditary gingival fibromatosis (HGF) is a fibrotic gingival enlargement. In previous work, HGF fibroblasts grew faster and produced more collagen and fibronectin (FN) than normal gingival (GN) fibroblasts. HGF FN and collagen production, but not proliferation, were under autocrine transforming growth factor (TGF)-beta control, suggesting other means of activation of HGF proliferation. Elevated/prolonged expression of the proto-oncogene c-myc is implicated in disregulation of cell growth. The objectives of this study were to: 1) determine if c-myc expression is abnormal in quiescent and serum-stimulated HGF and GN fibroblasts and 2) determine the relationship between c-myc expression and fibroblast proliferation using a c-myc antisense oligonucleotide (ODN). Proliferation was determined by enzyme-linked immunosorbent assay (ELISA), measuring incorporation of bromodeoxyuridine into DNA. Expression of c-myc was determined by quantitative polymerase chain reaction (PCR), using incorporation of fluorescent dCTP and detection via electrophoresis. Proliferation was minimal until 24 hours or more after serum stimulation, when HGF proliferation was greater than GN (P < or = 0.02). All cells expressed c-myc mRNA at quiescence and > or = 1 hour after serum stimulation. Expression of c-myc in quiescent HGF fibroblasts was elevated, and it peaked and remained higher after serum stimulation than in GN cells. Proliferation of an HGF cell line was inhibited by 4 microM c-myc antisense ODN (14% decrease; P < or = 0.006) and 8 microM c-myc antisense ODN (approximately 80% decrease; P < or = 0.0001), but generally not by c-myc sense ODN. This effect was reversed by hybridizing the c-myc antisense and sense ODNs (P = 0.007). Data suggest that elevated proliferation of an HGF fibroblast cell line is related to elevated c-myc expression.

P70s6 kinase is a functional target of insulin activated Akt cell-survival signaling

Insulin administration attenuates cardiac ischemia–reperfusion apoptosis via activation of Akt-mediated cell-survival signaling. As p70s6 kinase is a cognate Akt-mediated phosphorylation target we evaluated whether p70s6 kinase activation is a functional requirement in insulin-mediated cell survival program during post-ischemic reoxygenation. Human cardiac-derived girardi cells were subjected to 6h of simulated ischemia and 2h of reoxygenation±insulin treatment [0.3mU/ml]. Concurrently, cells were pre-treated with anti-sense oligodeoxynucleotides (ODNs) corresponding to the initiation start-site of human p70s6 kinase mRNA. Sense ODN and scrambled ODN were used as controls. Cell viability was measured using lactate dehydrogenase (LDH) release and propidium iodide (PI) exclusion. Insulin at reoxygenation enhanced cell viability with attenuated LDH release (⩾50%, p<0.001 vs. ischemic controls) and reduced PI uptake by ⩾30% vs. ischemic controls. The protection afforded by insulin was abolished by anti-sense ODN targeting p70s6 kinase, but not by the sense or scrambled ODNs. In parallel, insulin administration at reoxygenation significantly increased p70s6 kinase levels and activity compared with controls. P70s6 kinase activity was abolished by pre-treatment with anti-sense ODNs. Collectively, these data demonstrate that p70s6 kinase activation is a functional target of Akt following insulin-activated cytoprotection during ischemia–reoxygenation-induced injury.

In vivo growth suppression of rat C6 glioma transplanted in rat brain using antisense oligonucleotide for microtubule-associated protein 1A messenger ribonucleic acid

Microtubule-associated proteins (MAPs) are essential for various cellular processes such as mitosis. The aim of this study was to verify that the suppression of MAP 1A using antisense oligonucleotide can suppress the in vivo proliferation of C6 glioma cells transplanted in rat brain. After 7 days from transplantation, antisense, sense or scramble phosphorothioate oligodeoxynucleotide for MAP 1A mRNA was gradually delivered into the established tumours through a mini-osmotic pump. The mean diameters of the tumours from rats treated with antisense, sense and scramble phosphorothioate oligodeoxynucleotide for MAP 1A mRNA were 2.33, 4.625 and 5.25 mm. There was statistically significant difference in diameters of tumours between rats treated with antisense oligodeoxynucleotide, and those treated with sense or scramble oligodeoxynucleotide. This study strongly suggests the important role of MAP 1A in cell proliferation and its suppression may serve as a novel antitumour therapy for gliomas.

Vascular endothelial growth factor antisense oligodeoxynucleotides with lipiodol in arterial embolization of liver cancer in rats.

Transcatheter arterial embolization (TAE) of the hepatic artery has been accepted as an effective treatment for unresectable hepatocellular carcinoma (HCC). However, embolized vessel recanalization and collateral circulation formation are the main factors of HCC growth and recurrence and metastasis after TAE. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. This study was to explore the inhibitory effect of VEGF antisense oligodeoxynucleotides (ODNs) on VEGF expression in cultured Walker-256 cells and to observe the anti-tumor effect of intra-arterial infusion of antisense ODNs mixed with lipiodol on rat liver cancer. VEGF antisense ODNs and sense ODNs were added to the media of non-serum cultured Walker-256 cells. Forty-eight hours later, VEGF concentrations of supernatants were detected by ELISA. Endothelial cell line ECV-304 cells were cultured in the supernatants. Seventy-two hours later, growth of ECV-304 cells was analyzed by MTT method. Thirty Walker-256 cell implanted rat liver tumor models were divided into 3 groups. 0.2 mL lipiodol (LP group, n=10), 3OD antisense ODNs mixed with 0.2 mL lipiodol (LP+ODNs group, n=10) and 0.2 mL normal saline (control group, n=10) were infused into the hepatic artery. Volumes of tumors were measured by MRI before and 7 d after the treatment. VEGF mRNA in cancerous and peri-cancerous tissues was detected by RT-PCR. Microvessel density (MVD) and VEGF expression were observed by immunohistochemistry. Antisense ODNs inhibited Walker-256 cells' VEGF expression. The tumor growth rate was significantly lower in LP+ODNs group than that in LP and control groups (140.1+/-33.8%, 177.9+/-64.9% and 403.9+/-69.4% respectively, F=60.019, P<0.01). VEGF mRNAs in cancerous and peri-cancerous tissues were expressed highest in LP group and lowest in LP+ODNs group. The VEGF positive rates showed no significant difference among LP, control and LP+ODNs groups (90%, 70% and 50%, H=3.731, P>0.05). The MVD in LP+ODNs group (53.1+/-18.4) was significantly less than that in control group (73.2+/-20.4) and LP group (80.3+/-18.5) (F=5.44, P<0.05). VEGF antisense ODNs can inhibit VEGF expression of Walker-256 cells. It may be an antiangiogenesis therapy agent for malignant tumors. VEGF antisense ODNs mixed with lipiodol embolizing liver cancer is better in inhibiting liver cancer growth, VEGF expression and microvessel density than lipiodol alone.

Survivin antisense oligodeoxynucleotide inhibits growth of gastric cancer cells.

To investigate the effect of transfected survivin antisense oligonucleotide (ASODN) on proliferation and apoptosis of gastric cancer cells. The authors designed ASODNs targeting different regions of survivin mRNA, including surviving ASODN1, ASODN2 and ASODN3. ASODNs were transfected into gastric cancer cell line SGC 7901, cell growth was detected by MTT assay. Cells exposed to the potent oligonucleotide were also examined for apoptosis induction by FCM and fluorescence microscopy. Semiquantitive RT-PCR and Western blot examinations were carried for expression of survivin mRNA and protein. ASODN3 caused a statistically significant reduction of cell viability to 60.6% (+/-2.9%) (P<0.01), while ASODN1 and ASODN2 had no such changes (P>0.05). The cell growth was also significantly inhibited by ASODN3, compared with reversal and scrambled sequence. A significant loss of survivin mRNA was presented in ASODN3 treated cells and this was not seen in treatment with sense ODN or scramble ODN. Protein level was significantly decreased 48 h after survivin ASODN trasfected by approximately 2-fold decrease compared with untreated controls. However, ASODN3 did not induce significant apoptosis response until 48 h after transfection (P>0.05). ASODN3, which targets translation initiation part, can be identified as a most potent antisense compound. Srvivin ASODN3 may provide a novel approach to therapy of gastric cancer.

Chemosensitization of breast carcinoma cells with the use of bcl-2 antisense oligodeoxynucleotide

This study was designed to observe whether the rates of apoptosis induced in the breast cancer cell line MCF-7 by 5-fluorouracil (5-FU) could be enhanced by transfecting bcl-2 antisense oligonucleotide (ASODN). In our experiment, bcl-2 ASODNs and control ODNs including untreated control, sense ODN and scrambled ODN, were transfected into MCF-7 cells. Changes in expression of the bcl-2 gene were examined by Western blot; cell growths were detected by MTT assay, and apoptosis rates were detected by flow cytometry (FCM). Expression of bcl-2 protein after transfection of bcl-2 ASODN was significantly lower than control ODNs. Moreover, incubation of MCF-7 with bcl-2 ASODN prior to 5-FU treatment caused remarkable loss of viable cells compared with all other control ODNs ( P<0.01). FCM showed the apoptosis rates for ASODN, untreated control, sense ODN and scrambled ODN (29.8±7.4)%, (8.0±2.3)%, (15.0±5.1)% and (16.5±7.1)%, respectively. Compared with control ODNs, ASODN achieved the strongest effect in terms of enhancing apoptosis ( P<0.01). These results suggest that combining bcl-2 ASODN with 5-FU led to synergistic cytotoxicity.

Transforming growth factor-beta1-dependent urokinase up-regulation and promotion of invasion are involved in Src-MAPK-dependent signaling in human ovarian cancer cells.

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-beta1 induces a dose- and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-beta1, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-beta1 activated Src kinase; 2) TGF-beta1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-beta1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-beta1-mediated Akt stimulation, whereas TGF-beta1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-beta1 was almost totally blocked by PP2 and PD98059 and partially ( approximately 55%) by LY294002; 6) TGF-beta1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-kappaB by pyrrolidinedithiocarbamate reduced the TGF-beta1-induced activation of the uPA gene by approximately 65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-beta1 activation of two distinct pathways (Src-MAPK-PI3K-NF-kappaB-dependent and Src-MAPK-AP-1-dependent) for TGF-beta1-dependent uPA up-regulation and promotion of invasion.

Signaling pathways regulating interleukin-13-stimulated chemokine release from airway smooth muscle.

Interleukin (IL)-13 receptor activation on airway smooth muscle cells induces eotaxin release and activates multiple signaling pathways including mitogen-activated protein kinases, and signal transducer and activator of transcription 6 (STAT6). To examine a requirement for STAT6 in mediating IL-13-stimulated eotaxin release we used antisense oligodeoxynucleotides (ODNs) to downregulate endogenous STAT6 protein. STAT6 antisense ODNs were taken up by about 85% of cells. Selective downregulation of STAT6 protein occurred with antisense ODNs, but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 +/- 4 and 75 +/- 7%, respectively, in cells transfected with antisense ODNs (p < 0.001), but not with a sense ODN or a scrambled ODN. Eotaxin release induced by IL-1beta was unaffected by STAT6 antisense ODN (p > 0.05). Finally, IL-13- or IL-4-dependent eotaxin release was abolished when inhibitors of both p42/p44 ERK (U0126, 10 microM) and p38 (SB202190, 10 microM) mitogen-activated protein kinase pathways were combined in STAT6 antisense ODN-transfected cells. In contrast, about 25% of the response remained when each inhibitor was examined alone in STAT6 antisense ODN-treated cells. These data support roles for both STAT6- and mitogen-activated protein kinase-dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.

Inhibition of intercellular adhesion molecule-1 protein expression by antisense oligonucleotides is neuroprotective after transient middle cerebral artery occlusion in rat.

The present study was performed to determine whether antisense inhibition of intercellular adhesion molecule-1 (ICAM-1) protein expression decreases focal ischemic brain damage. Male spontaneously hypertensive rats underwent 1-hour middle cerebral artery occlusion (MCAO) and 24-hour reperfusion. Rats were infused with ICAM-1 antisense or control oligodeoxynucleotides (ODNs) (48 nmol/d ICV) or vehicle, starting 24 hours before MCAO and continuing until the time of death. ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels were measured by real-time polymerase chain reaction. ICAM-1 protein knockdown was confirmed by Western blotting. Infarct volume was quantified by the use of cresyl violet-stained brain sections. Neurological deficits were evaluated. Mean arterial blood pressure was recorded by laser Doppler. Tissue penetration of antisense was confirmed by the use of fluorescent ODNs. Transient MCAO upregulated ICAM-1, but not VCAM-1, mRNA expression in the ipsilateral cortex between 3 and 72 hours of reperfusion. ICAM-1 antisense infusion prevented ischemia-induced ICAM-1 protein expression and reduced total infarct volume (by 53%; P<0.05; 226+/-35 mm3 in control ODN group and 104+/-27 mm3 in antisense ODN group; n=8 each) and mean neurological deficit score (by 44%; P<0.05; 2.4 in control ODN group and 1.3 in antisense ODN group; n=8 each). Neither control nor antisense ODN had any effect on mean arterial blood pressure and the physiological parameters monitored during MCAO. Compared with noninfused control, intracerebroventricular infusion of artificial cerebrospinal fluid or antisense or sense ODN had no significant effect on the regional cerebral blood flow changes that accompanied ischemia and reperfusion. Increased ICAM-1 expression is implicated in the pathogenesis of focal ischemia since ICAM-1 protein knockdown decreased ischemic brain damage. The mechanism by which ICAM-1 inhibition offers neuroprotection is independent of blood pressure modulation.