Surfactant like peptides for targeted gene delivery to cancer cells.

Surfactant like peptides (SLPs) are a class of amphiphilic peptides widely used for drug delivery and tissue engineering. However, there are very few reports on their application for gene delivery. The current study was aimed at development of two new SLPs, named (IA)4K and (IG)4K, for selective delivery of antisense oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) to cancer cells. The peptides were synthesized by Fmoc solid phase synthesis. Their complexation with nucleic acids was studied by gel electrophoresis and DLS. The transfection efficiency of the peptides was assessed in HCT 116 colorectal cancer cells and human dermal fibroblasts (HDFs) using high content microscopy. The cytotoxicity of the peptides was assessed by standard MTT test. The interaction of the peptides with model membranes was studied using CD spectroscopy. Both SLPs delivered siRNA and ODNs to HCT 116 colorectal cancer cells with high transfection efficiency which was comparable to the commercial lipid-based transfection reagents, but with higher selectivity for HCT 116 compared to HDFs. Moreover, both peptides exhibited very low cytotoxicity even at high concentrations and long exposure time. The current study provides more insights into the structural features of SLPs required for nucleic acid complexation and delivery and can therefore serve as a guide for the rational design of new SLPs for selective gene delivery to cancer cells to minimize the adverse effects in healthy tissues.

[Antisense oligodeoxynucleotides: the certain but limited efficacy and the uncovering mechanisms for the cure of chronic hepatitis B].

Recently, several phase I and phase II clinical trials of antisense oligodeoxynucleotides (ASOs) targeting to the commonly shared conserved sequences of HBV transcripts brought us some promising results. Particularly in the report of phase IIb clinical trial of Bepirovirsen (GSK3228836), approximately 9-10% patients with low baseline serum HBsAg (> 100 IU/ml & < 3 000 IU/ml) achieved functional cure after 24 weeks' of Bepirovirsen treatment. After reviewing the results of other clinical trials, one would be impressed to know that ALG-020572 (Aligos), RO7062931 (Roche) and GSK3389404 (GSK) all failed to sufficiently suppress serum HBsAg expression though the hepatocyte-targeted delivery of these ASOs were enhanced via N-acetyl galactosamine conjugation. Bepirovirsen enabled some patients to achieve sustained disappearance of serum HBsAg. The analysis of its distribution in different tissues of patients after drug administration showed that only a few fractions of ASOs entered liver tissues and far fewer eventually entered hepatocytes. Taking into consideration that only a few hepatocytes could be expected positive for HBsAg staining among these participants with low serum HBsAg level. We suspect that the mechanistic contribution of ASOs declining the serum HBsAg is not only via directly acting on the HBV transcripts in hepatocytes, but also via entering non-parenchymal cells such as Kupffer cells and resulting in stimulation and activation of innate immunity. Eventually the serum HBsAg declines in most participants and even disappears in a small fraction of patients with low baseline HBsAg level, via attack the infected hepatocytes evidenced by the aberrant elevation of ALT. Nevertheless, the functional cure of CHB remains a challenging issue and more efforts are needed.

Systematic evaluation of oligodeoxynucleotide binding and hybridization to modified multi-walled carbon nanotubes.

BACKGROUND: In addition to conventional chemotherapeutics, nucleic acid-based therapeutics like antisense oligodeoxynucleotides (AS-ODN) represent a novel approach for the treatment of bladder cancer (BCa). An efficient delivery of AS-ODN to the urothelium and then into cancer cells might be achieved by the local application of multi-walled carbon nanotubes (MWCNT). In the present study, pristine MWCNT and MWCNT functionalized with hydrophilic moieties were synthesized and then investigated regarding their physicochemical characteristics, dispersibility, biocompatibility, cellular uptake and mucoadhesive properties. Finally, their binding capacity for AS-ODN via hybridization to carrier strand oligodeoxynucleotides (CS-ODN), which were either non-covalently adsorbed or covalently bound to the different MWCNT types, was evaluated. RESULTS: Pristine MWCNT were successfully functionalized with hydrophilic moieties (MWCNT-OH, -COOH, -NH2, -SH), which led to an improved dispersibility and an enhanced dispersion stability. A viability assay revealed that MWCNT-OH, MWCNT-NH2 and MWCNT-SH were most biocompatible. All MWCNT were internalized by BCa cells, whereupon the highest uptake was observed for MWCNT-OH with 40% of the cells showing an engulfment. Furthermore, all types of MWCNT could adhere to the urothelium of explanted mouse bladders, but the amount of the covered urothelial area was with 2-7% rather low. As indicated by fluorescence measurements, it was possible to attach CS-ODN by adsorption and covalent binding to functionalized MWCNT. Adsorption of CS-ODN to pristine MWCNT, MWCNT-COOH and MWCNT-NH2 as well as covalent coupling to MWCNT-NH2 and MWCNT-SH resulted in the best binding capacity and stability. Subsequently, therapeutic AS-ODN could be hybridized to and reversibly released from the CS-ODN coupled via both strategies to the functionalized MWCNT. The release of AS-ODN at experimental conditions (80 °C, buffer) was most effective from CS-ODN adsorbed to MWCNT-OH and MWCNT-NH2 as well as from CS-ODN covalently attached to MWCNT-COOH, MWCNT-NH2 and MWCNT-SH. Furthermore, we could exemplarily demonstrate that AS-ODN could be released following hybridization to CS-ODN adsorbed to MWCNT-OH at physiological settings (37 °C, urine). CONCLUSIONS: In conclusion, functionalized MWCNT might be used as nanotransporters in antisense therapy for the local treatment of BCa.

Paeoniflorin inhibits excitatory amino acid agonist-and high-dose morphine-induced nociceptive behavior in mice via modulation of N-methyl-D-aspartate receptors.

BACKGROUND: Pain, the most common reasons for physician consultation, is a major symptom in many medical conditions that can significantly interfere with a person's life quality and general functioning. Almost all painkillers have its untoward effects. Therefore, seeking for a safe medication for pain relieve is notable nowadays. Paeonia lactiflora is a well-known traditional Chinese medicine. Paeoniflorin is an active component found in Paeonia lactiflora, which has been reported to inhibit formalin-induced nociceptive behavior in mice. Aims of this present study were to investigate effects of paeoniflorin on excitatory amino acid agonist- or high-dose morphine-induced nociceptive behaviors in mice. RESULTS: Paeoniflorin (100, 200, 500 nmol, i.c.v.) alone and combined with glutamatergic antagonists (MK-801 14.8 pmol, or NBQX 5 nmol, i.t.) inhibited nociception. Those agents also inhibited the clonic seizure-like excitation induced by high-dose morphine (250 nmol, i.t) in mice. Antisense oligodeoxynucleotides of NMDA receptor subunits NR1, NR2A, NR2B significantly enhanced the inhibition of paeoniflorin on excitatory amino acid-and high-dose morphine-induced nociception. Docking energy data revealed that paeoniflorin had stronger binding activity in NR2A and NR2B than NR2C of NMDA receptors. CONCLUSIONS: Results of this study indicate that paeoniflorin-induced inhibition of excitatory amino acid agonist- and high-dose morphine-induced nociceptive behaviors might be due to modulation of NMDA receptors, specifically the NR2B subunit.

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes.

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS-ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi-in vitro, whereas normal PT growth was shown in its sense control in vitro/semi-in vitro. PT growth was impaired in a manner dependent on the dose of AS-ODNs against both ANX1 and ANX2 above 10 µm. The treatment with AS-ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS-ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS-ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS-ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.

Ceftriaxone modulates uptake activity of glial glutamate transporter-1 against global brain ischemia in rats.

Ceftriaxone(Cef) selectively increases the expression of glial glutamate transporter-1 (GLT-1), which was thought to be neuroprotective in some circumstances. However, the effect of Cef on glutamate uptake of GLT-1 was mostly assayed using in vitro studies such as primary neuron/astrocyte cultures or brain slices. In addition, the effect of Cef on neurons in different ischemic models was still discrepant. Therefore, this study was undertaken to observe the effect of Cef on neurons in global brain ischemia in rats, and especially to provide direct evidence of the up-regulation of GLT-1 uptake for glutamate contributing to the neuronal protection of Cef against brain ischemia. Neuropathological evaluation indicated that administration of Cef, especially pre-treatment protocols, significantly prevented delayed neuronal death in hippocampal CA1 subregion normally induced by global brain ischemia. Simultaneously, pre-administration of Cef significantly up-regulated the expression of GLT-1. Particularly, GLT-1 uptake assay with (3) H-glutamate in living cells from adult rats showed that up-regulation in glutamate uptake accompanied up-regulated GLT-1 expression. Inhibition of GLT-1 by antisense oligodeoxynucleotides or dihydrokainate significantly inhibited the Cef-induced up-regulation in GLT-1 uptake and the neuroprotective effect against global ischemia. Thus, we may conclude that Cef protects neurons against global brain ischemia via up-regulation of the expression and glutamate uptake of GLT-1. Glutamate uptake by glial glutamate transporter-1 (GLT-1) is the principal way to regulate extracellular glutamate homeostasis in central nervous system. Over-accumulation of glutamate results in excitotoxicity and injures neurons after cerebral ischemia. Ceftriaxone up-regulates GLT-1 expression and uptake of glutamate, diminishes the excitotoxicity of glutamate and then protects neurons against global brain ischemia.

Generation of stable 3'-mRNA cleavage fragments induced by siRNA in cells with high-levels of duck hepatitis B virus replication.

Therapeutic small interfering RNAs (siRNAs) have attracted a lot of interest both in basic biomedical sciences as well as in translational medicine. Apart from their therapeutic efficacy adverse effects of siRNAs must be addressed. The generation of stable mRNA cleavage fragments and the translation of N-truncated proteins induced by antisense oligodeoxynucleotides (ASOs) have been reported. Similar to ASOs, siRNAs are considered to function via an antisense mechanism that promotes the cleavage of the target mRNA. To further investigate whether the stable mRNA cleavage fragments also occur in siRNA we constructed a short hairpin RNA (shRNA) expression plasmid, pshRNA794, containing the same sequence reported in experiments using ASOs which directly targeted the overlapping region of the pre-genomic mRNA (pgmRNA) and sub-genomic mRNA (sgmRNA) of duck hepatitis B virus (DHBV). The shRNA resulted in a 70.9% and 69.9% reduction of the DHBV mRNAs in LMH and HuH-7 cells, respectively. In addition a 70% inhibition of the DHBV DNA level was observed. Interestingly, 3'-mRNA cleavage fragments were detected in LMH but not in HuH-7 cells. Taken together, our findings demonstrate that the ASO sequence was also effective in siRNA. Importantly, our results provide direct evidence that stable 3'-mRNA fragments were generated by siRNA in cells with high levels of DHBV replication. Whether these can cause adverse RNAi effects needs to be explored further.

Effect of antisense oligodeoxynucleotides glucose transporter-1 on enhancement of radiosensitivity of laryngeal carcinoma.

PURPOSE: Laryngeal carcinomas always resist to radiotherapy. Hypoxia is an important factor in radioresistance of laryngeal carcinoma. Glucose transporter-1 (GLUT-1) is considered to be a possible intrinsic marker of hypoxia in malignant tumors. We speculated that the inhibition of GLUT-1 expression might improve the radiosensitivity of laryngeal carcinoma. METHODS: We assessed the effect of GLUT-1 expression on radioresistance of laryngeal carcinoma and the effect of GLUT-1 expressions by antisense oligodeoxynucleotides (AS-ODNs) on the radiosensitivity of laryngeal carcinoma in vitro and in vivo. RESULTS: After transfection of GLUT-1 AS-ODNs: MTS assay showed the survival rates of radiation groups were reduced with the prolongation of culture time (p<0.05); Cell survival rates were significantly reduced along with the increasing of radiation dose (p<0.05). There was significant difference in the expression of GLUT-1mRNA and protein in the same X-ray dose between before and after X-ray radiation (p<0.05). In vivo, the expressions of GLUT-1 mRNA and protein after 8Gy radiation plus transfection of GLUT-1 AS-ODNs were significant decreased compared to 8Gy radiation alone (p<0.001). CONCLUSION: Radioresistance of laryngeal carcinoma may be associated with increased expression of GLUT-1 mRNA and protein. GLUT-1 AS-ODNs may enhance the radiosensitivity of laryngeal carcinoma mainly by inhibiting the expression of GLUT-1.

Advances in the mechanisms and applications of inhibitory oligodeoxynucleotides against immune-mediated inflammatory diseases.

Inhibitory oligodeoxynucleotides (ODNs) are short single-stranded DNA, which capable of folding into complex structures, enabling them to bind to a large variety of targets. With appropriate modifications, the inhibitory oligodeoxynucleotides exhibited many features of long half-life time, simple production, low toxicity and immunogenicity. In recent years, inhibitory oligodeoxynucleotides have received considerable attention for their potential therapeutic applications in immune-mediated inflammatory diseases (IMIDs). Inhibitory oligodeoxynucleotides could be divided into three categories according to its mechanisms and targets, including antisense ODNs (AS-ODNs), DNA aptamers and immunosuppressive ODNs (iSup ODNs). As a synthetic tool with immunomodulatory activity, it can target RNAs or proteins in a specific way, resulting in the reduction, increase or recovery of protein expression, and then regulate the state of immune activation. More importantly, inhibitory oligodeoxynucleotides have been used to treat immune-mediated inflammatory diseases, including inflammatory disorders and autoimmune diseases. Several inhibitory oligodeoxynucleotide drugs have been developed and approved on the market already. These drugs vary in their chemical structures, action mechanisms and cellular targets, but all of them could be capable of inhibiting excessive inflammatory responses. This review summarized their chemical modifications, action mechanisms and applications of the three kinds of inhibitory oligodeoxynucleotidesin the precise treatment of immune-mediated inflammatory diseases.

Antisense Tissue Factor Oligodeoxynucleotides Protected Diethyl Nitrosamine/Carbon Tetrachloride-Induced Liver Fibrosis Through Toll Like Receptor4-Tissue Factor-Protease Activated Receptor1 Pathway.

Tissue factor (TF) is a blood coagulation factor that has several roles in many non-coagulant pathways involved in different pathological conditions such as angiogenesis, inflammation and fibrogenesis. Coagulation and inflammation are crosslinked with liver fibrosis where protease-activated receptor1 (PAR1) and toll-like receptor4 (TLR4) play a key role. Antisense oligodeoxynucleotides are strong modulators of gene expression. In the present study, antisense TF oligodeoxynucleotides (TFAS) was evaluated in treating liver fibrosis via suppression of TF gene expression. Liver fibrosis was induced in rats by a single administration of N-diethyl nitrosamine (DEN, 200 mg/kg; i. p.) followed by carbon tetrachloride (CCl4, 3 ml/kg; s. c.) once weekly for 6 weeks. Following fibrosis induction, liver TF expression was significantly upregulated along with liver enzymes activities and liver histopathological deterioration. Alpha smooth muscle actin (α-SMA) and transforming growth factor-1beta (TGF-1ß) expression, tumor necrosis factor-alpha (TNF-α) and hydroxyproline content and collagen deposition were significantly elevated in the liver. Blocking of TF expression by TFAS injection (2.8 mg/kg; s. c.) once weekly for 6 weeks significantly restored liver enzymes activities and improved histopathological features along with decreasing the elevated α-SMA, TGF-1ß, TNF-α, hydroxyproline and collagen. Moreover, TFAS decreased the expression of both PAR1 and TLR4 that were induced by liver fibrosis. In conclusion, we reported that blockage of TF expression by TFAS improved inflammatory and fibrotic changes associated with CCl4+DEN intoxication. In addition, we explored the potential crosslink between the TF, PAR1 and TLR4 in liver fibrogenesis. These findings offer a platform on which recovery from liver fibrosis could be mediated through targeting TF expression.

Tankyrase1 antisense oligodeoxynucleotides suppress the proliferation, migration and invasion through Hippo/YAP pathway in human osteosarcoma cells.

Osteosarcoma is the most common malignant tumor of bone with a high potential for metastasis and poor prognosis. This study intends to explore the effect of tankyrase1 (TANK1) in the development of osteosarcoma cells and the underlying mechanism. The osteosarcoma cell line MG-63 cells were cultured and transfected with tankyrase1 antisense oligodeoxynucleotides (TANK1-ASODN). Cell proliferation was detected with CCK-8 and immunofluorescence. Cell migration and invasion were examined by wound healing assay and Transwell assay, respectively. Reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA level of TANK1 and western blot was conducted to detect relative protein expression during the research. As a result, we demonstrated that TANK1 was upregulated in osteosarcoma. The TANK1-ASODN inhibited MG-63 cell proliferation, migration and invasion. The progress of epithelial-mesenchymal transition (EMT) was also suppressed in TANK1-ASODN transfected MG-63 cells compared to control group. Besides, the TANK1-ASODN activated and modulated the Hippo/YAP signaling which might be the pathway that TANK1 depended on. Overall, our finding supported that TANK1-ASODN slowed down the progress of osteosarcoma by suppressing cell proliferation, migration, invasion and EMT through Hippo/YAP pathway.

In vivo evaluation of the effects of simultaneous inhibition of GLUT-1 and HIF-1α by antisense oligodeoxynucleotides on the radiosensitivity of laryngeal carcinoma using micro 18F-FDG PET/CT.

PURPOSE: Hypoxia-inducible factor 1α (HIF-1α) and glucose transporter-1 (GLUT-1) are two important hypoxic markers associated with the radioresistance of cancers including laryngeal carcinoma. We evaluated whether the simultaneous inhibition of GLUT-1 and HIF-1α expression improved the radiosensitivity of laryngeal carcinoma. We explored whether the expression of HIF-1α and GLUT-1 was correlated with 2'-deoxy-2'-[18F]fluoro-D-glucose (18F-FDG) uptake and whether 18F-FDG positron emission tomography-computed tomography (PET/CT) was appropriate for early evaluation of the response of laryngeal carcinoma to targeted treatment in vivo. MATERIALS AND METHODS: To verify the above hypotheses, an in vivo model was applied by subcutaneously injecting Hep-2 (2 × 107/mL × 0.2 mL) and Tu212 cells (2 × 107/mL × 0.2 mL) into nude mice. The effects of HIF-1α antisense oligodeoxynucleotides (AS-ODNs) (100 µg) and GLUT-1 AS-ODNs (100 µg) on the radiosensitivity of laryngeal carcinoma were assessed by tumor volume and weight, microvessel density (MVD), apoptosis index (AI) and necrosis in vivo based on a full factorial (23) design. 18F-FDG-PET/CT was taken before and after the treatment of xenografts. The relationships between HIF-1α and GLUT-1 expression and 18F-FDG uptake in xenografts were estimated and the value of 18F-FDG-PET/CT was assessed after treating the xenografts. RESULTS: 10 Gy X-ray irradiation decreased the weight of Hep-2 xenografts 8 and 12 days after treatment, and the weights of Tu212 xenografts 8 days after treatment. GLUT-1 AS-ODNs decreased the weight of Tu212 xenografts 12 days after treatment. There was a synergistic interaction among the three treatments (GLUT-1 AS-ODNs, HIF-1α AS-ODNs and 10Gy X-ray irradiation) in increasing apoptosis, decreasing MVD, and increasing necrosis in Hep-2 xenografts 8 days after treatment (p < 0.05) and in Tu212 xenografts 12 days after treatment (p < 0.001). Standardized uptake value (tumor/normal tissue)( SUVmaxT/N) did not show a statistically significant correlation with GLUT1 and HIF-1α expression and therapeutic effect (necrosis, apoptosis). CONCLUSIONS: Simultaneous inhibition of HIF-1α and GLUT-1 expression might increase the radiosensitivity of laryngeal carcinoma, decreasing MVD, and promoting apoptosis and necrosis. 18F-FDG-PET/CT wasn't useful in evaluating the therapeutic effect on laryngeal cancer in this animal study.

The Role of Connexins in Wound Healing and Repair: Novel Therapeutic Approaches.

Gap junctions are intercellular proteins responsible for mediating both electrical and biochemical coupling through the exchange of ions, second messengers and small metabolites. They consist of two connexons, with (one) connexon supplied by each cell. A connexon is a hexamer of connexins and currently more than 20 connexin isoforms have been described in the literature thus far. Connexins have a short half-life, and therefore gap junction remodeling constantly occurs with a high turnover rate. Post-translational modification, such as phosphorylation, can modify their channel activities. In this article, the roles of connexins in wound healing and repair are reviewed. Novel strategies for modulating the function or expression of connexins, such as the use of antisense technology, synthetic mimetic peptides and bioactive materials for the treatment of skin wounds, diabetic and pressure ulcers as well as cornea wounds, are considered.

Differential contribution of Gαi/o subunits in the response to food deprivation.

Behavioral responses to food deprivation are a fundamental aspect of nervous system function in all animals. Several signaling molecules in the mammalian brain act through G proteins of the Gi/o family to mediate response to food restriction. The present study examined whether food intake changes under a condition of little stimulation to eat, such as that elicited by 4h of food deprivation, was altered by Gi/o isoform silencing induced by intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (aODN) against the α subunit of Gi1, Gi2, Gi3, Go1 and Go2. The effect of aODN pretreatments on food intake was evaluated 15, 30, 45, and 60min after food re-administration. Selective effects were noted on food intake with anti-Giα1 (3.12-25nmol), Giα3 (1.56-12.5nmol) and Goα2 (3.12-25nmol) aODN exerting increase in food consumption, while anti-Giα2 (3.12-25nmol) and Goα1 (3.12-25nmol) aODN exerting decrease in food consumption. We observed the effect of the α-subunit silencing on food consumption as soon as 15min after food readministration, that was still significant after 60min. At the highest effective doses, different for each anti-Gαi/o subunit, any treatment did not impair motor coordination, nor modified spontaneous mobility and exploratory activity. These results indicate a relevant distinction between Gαi/o subunits on feeding behavior, and suggest that Gi/o proteins are critical for the integrative modulation of normal feeding behavior. Changes in Gi/o protein activity may be associated with modifications of feeding.

Cellular effects of antisense c-myc oligodeoxynucleotides are delivery dependent.

Among the transcription factors critical for cell cycle regulation is the proto-oncogene c-myc. The expression of c-myc upon vascular injury and its inhibition by antisense oligodeoxynucleotides have demonstrated the importance of this protein in the control of proliferation for many cell types, including vascular smooth muscle cells. Liposomes can enhance cellular incorporation of antisense oligodeoxynucleotides, but cellular uptake of oligonucleotides in this manner is still suboptimal, and the oligonucleotides are not protected from enzymatic degradation. Physico-chemical modifications of the oligomers must be developed before antisense oligodeoxynucleotides can be considered as a potential gene therapy for many of the human diseases. This study reports on the enhanced cellular incorporation of antisense phosphodiester oligonucleotides when conjugated to lipophilic linkers. Conjugated phosphodiesters of antisense c-myc oligodeoxynucleotides inhibited cultured human aortic smooth muscle cell growth by 47.5 ± 1.0% 4 days following only a 24-h exposure to the conjugated antisense phosphodiester oligonucleotides. Liposome-enhanced, but unconjugated, phosphodiester and phosphorothioate oligonucleotides were less effective (24.4 ± 1.9% and 29.5 ± 3.1% inhibition, respectively). Smooth muscle cell growth inhibition by antisense c-myc oligodeoxynucleotides correlated with the suppression of nuclear c-myc protein expression. Thus, antisense c-myc oligodeoxynucleotides conjugated to lipid-soluble linkers enhanced cellular incorporation as well as intracellular retention of oligodeoxynucleotides, resulting in rapid and sustained inhibition of c-myc expression of smooth muscle cells. This, in turn, caused a prolonged growth inhibition compared to unconjugated oligodeoxynucleotides.

Antisense oligodeoxynucleotide against human telomerase reverse transcriptase inhibits the proliferation of Eca-109 esophageal carcinoma cells.

Previous studies have demonstrated that the growth of tumor cells may be inhibited by antisense oligonucleotides (ASODNs) targeted against human telomerase (hTR) or human telomerase reverse transcriptase (hTERT), resulting in antitumor activity in a wide variety of tumors. However, few studies have investigated the effect of hTERT gene-targeted ASODNs on telomerase activity and cell proliferation in human esophageal cancer. In the present study, an MTT assay was used to determine the growth inhibition rate of Eca-109 cells treated with a hTERT-targeted phosphorothioate-ASODN (PS-ASODN). An inverted microscope was used to observe the morphologic changes of the cells following treatment with 5 µM PS-ASODN for 10 days. Telomerase activity was detected using the silver staining semi-quantitative telomeric repeat amplification protocol (TRAP) assay. Following treatment with the PS-ASODN (1-5 µmol/l), the proliferation of the Eca-109 cells was inhibited. The differences in inhibition rate between the PS-ASODN and blank control groups were statistically significant (P<0.05) when the concentration of the PS-ASODN was ≥2 µmol/l, whereas no statistically significant difference was identified between the non-specific-ASODN and blank control groups. The inhibition rate increased gradually as the concentration of the PS-ASODN increased and with time, suggesting that the PS-ASODN inhibited the growth of Eca-109 cells in a concentration-dependent, time-dependent and sequence-specific manner. The growth rate of the cells incubated with the PS-ASODN was reduced compared with that of the control cells. Cells treated with the PS-ASODN became round, suspended and reduced in size. The PS-ASODN was also found to inhibit telomerase activity. The ability of the PS-ASODN to inhibit the telomerase activity and cell proliferation of the Eca-109 cell line suggests that ASODNs have the potential to be novel therapeutic agents for the treatment of esophageal cancer.

Antisense Deoxyoligonucleotides Inhibit Activities of Matrix Metalloproteinase-2 in Human Fibrosarcoma HT1080 Cells.

PURPOSE: MMP-2, 72 kDa-type IV collagenase, plays a major role in the migration and growth of tumor cells, a process that requires the disintegration of basement membrane. Activation of MMP-2 is correlated with the invasiveness of various tumors. The aim of this study was to determine the sequence-specific phosphorothioated oligodeoxynucleotides (ODNs) inhibiting the translation of MMP-2 mRNA and the subsequent invasiveness of tumor cells. MATERIALS AND METHODS: Eight types of antisense ODNs were designed and each (8micro gram/ml) were transfected into HT1080 cells. The effects of these antisense ODNs on MMP expression were examined by gelatin zymography, Western blot, Northern blot and matrigel assay. RESULTS: Antisense-5 (+904~923), antisense-6 (+1274~+1293) and antisense-7 (+1646~+1665) reduced the MMP-2 activity of the culture supernatant in HT1080 fibrosarcoma cells. Treatment with antisense-6 showed inhibition of MMP-2 mRNA and protein, and in vitro invasion in a dose-dependent manner. CONCLUSION: Antisense-6 might be one of the therapeutic candidates for tumor invasion and metastasis.