Targeted anti-tumor synergistic effects of Myc decoy oligodeoxynucleotides-loaded selenium nanostructure combined with chemoradiotherapy on LNCaP prostate cancer cells

In the present study,we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer cells.Myc decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics assays.ODNs were loaded on the synthesized Se@BSA@Chi-MTX nanostructure.The physicochemical characteristics of nanostructures were determined by FTIR,DLS,UV-vis,TEM,EDX,in vitro release,and hemolysis tests.Subsequently,the cytotoxicity properties of them with and without X-irradiation were investigated by uptake,MTT,cell cycle,apoptosis,and scratch assays on the LNCaP cell line.The results of DLS and TEM showed negative charge(−9 mV)and nanometer size(40 nm)for Se@BSA@Chi-DEC-MTX NPs,respectively.The results of FTIR,UV-vis,and EDX showed the proper interaction of different parts and the correct synthesis of nanoparticles.The results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/mL.The ODNs release from the nanostructures showed a pH-dependent manner,and the release rate was 15%higher in acidic pH.The targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen(PSMA).The significant synergistic effects of nanostructure(containing MTX drug)treatment along with X-irradiation showed cell growth inhibition,apoptosis induction(~57%),cell cycle arrest(G2/M phase),and migration inhibition(up to 90%)compared to the control.The results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP cells.This nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment.

Apoptosis and Lipolysis of White Adipocytes Induced by Neuropeptide Y- Y5 Receptor Antisense Oligodeoxynucleotides

Objective: To investigate the influence of central administration ofneuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs) on body weight, fat pads of SDrats, and the effects of white adipocytes lipolysis and apoptosis. Methods: Y5 receptor antisense,sense, mismatched ODNs or vehicle was intracerebroventricularly (i. c. v.) injected. Averageadipocyte area was calculated. DNA ladders were measured to evaluate adipocyte apoptosis, and RT-PCRwas used to analyse the expression of Bcl-2 and Bax gene. Results: Central administration of Y5antisense ODNs significantly decreased body weight, and average adipocyte area. DNA fragmentationwas present after electrophoresis at epididymal adipose tissue. The expression of Bcl-2 gene wasdownregulated, while the expression of Bax upregulated. Conclusion: Lipolysis and adipocyteapoptosis may be important mechanisms far 75 antisense therapy.

INHIBITION EFFECT OF VASCULAR ENDOTHELIAL GROWTH FACTOR ANTISENSE OLIGODEOXYNUCLEOTIDES ON C6 GLIOMA

Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.

Effect of C-myc Antisense Oligodeoxynucleotides on Hypoxia-induced Proliferation of Pulmonary Vascular Pericytes

To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.

The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

Objective： Aurora A kinase representing a family of evolutionadly conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A contributes to the carcinogenesis, chromosomal instability （CIN）, and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide （ASODN） technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods： Aurora AASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot respectively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results： The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours＇ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased （P 〈 0.05）, along with the induction of accumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora AASODN and paclitaxel was higher significantly than paclitaxel （P 〈 0.05） or Aurora AASODN alone （P 〈 0.05）. Conclusion： Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.

Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.

Growth of Pb S nanoclusters on specific sites of programmed oligodeoxynucleotides

We develope a method to synthesize Pb S nanoclusters（NCs） using guanine-containing oligodeoxynucleotides（ODNs）as templates. The NCs on the ODNs are ultra small（ranging from ～ 0.5 nm to 2.1 nm） and luminescent in the visible region. They are characterized by photoluminescence（PL） spectra, transmission electron microscopy（TEM）, and X-ray powder diffraction（XRD）. The ODN–NC complexes can be used as customer-designed fluorophores which do not have the problem of multiple conjugations. The same method enables us to fabricate Pb S quantum dot molecules and connect them into nanowires, expanding their potential applications in molecule electronics and quantum computing.

Inhibition of α_1(Ⅰ) collagen gene in vitro transcription by antisense oligodeoxynucleotides

Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.

Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro

AIM： To investigate the effect of telomerase hTERT gene antisense oligonucleotide （hTERT-ASO） on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3. METHODS： MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity, the cells were divided into there groups： Control group （pancreatic cancer cell Bxpc-3）; antisense oligonucleotide （hTERT-ASO） group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay. Cell apoptosis was observed by transmission electron microscope in each group. RESULTS： After treatment with 6 mmollL hTERT- ASO, cell proliferation was inhibited in dose- and time- dependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-G1 stage cell apoptosis peak appeared in front of G1 stage. CONCLUSION： Telomerase antisense oligodeoxy- nucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.

A new approach to transfect NF-κB decoy oligodeoxynucleotides into the periodontal tissue using the ultrasound-microbubble method

The objective of this study is to investigate the effect of the ultrasound-microbubble technique in nuclear factor kappa B(NF-κB) decoy oligodeoxynucleotide(ODN) transfection in the gingival tissue in mice. The 6-FAM-labeled scrambled decoy ODN with microbubbles was applied to the periodontal tissue in 8-week-old male C57BL/6J mice by ultrasound radiation at low(LUM-Sc) and high(HUM-Sc) intensities to optimize the transfection condition of the ultrasound-microbubble method.Histological inspections were performed two hours after transfection to compare the expression with that in the sham-operated group without ultrasound radiation(A-Sc). Then, an NF-κB decoy was transfected into the periodontal tissue using the highintensity ultrasound-microbubble(HUM-NF) technique to examine the anti-inflammatory effects of the decoy ODN. Western blot analysis was performed to investigate the expression of interleukin(IL)-1β, IL-6 and intercellular adhesion molecule-1(ICAM-1)in the gingival tissues in the HUM-Sc, the HUM-NF and control groups. The fluorescence microscopy results showed that the fluorescent intensity in the periodontal tissues in the LUM-Sc and HUM-Sc groups was significantly higher than that in the A-Sc and the control groups. The fluorescent intensity in the HUM-Sc group, especially in the gingival connective tissue,was the highest of all groups. Western blot analysis indicated that the protein expression levels of IL-1β, IL-6 and ICAM-1 in the HUM-NF group were significantly lower than those in the HUM-Sc and the control groups. These findings suggest that the high-intensity ultrasound-microbubble technique is an effective tool for decoy transfection into the periodontal tissue.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Destabilization of Basic Fibroblast Growth Factor by Oligodeoxynucleotides

The effects of oligodeoxynucleotide （ODN） on the conformation of basic fibroblast growth factor （bFGF） were studied by spectral method. The results showed that ODN destabilized the protein.

Antiviral effects of two antisense phosphorothioate oligodeoxynucleotides against Japanese encephalitis virus strain SA--14 in cultured BHK21 cells

To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences of the first AUG codon and 5’ terminus of NSS of JEV SA--14 genome have been synthesized and their effects on CPE, viral antigen expression and virus plaque formation were tested in vitro. Results: The resultsshowed that 1. 0 pmol/L of S-ODN greatly deferred the onset of CPE in cultured BHKZI cells for at least 48 h.Addition of 5. 0 pmol/L or more S--ODN to culture medium fluid, 2 h prior to 100TCID,,virus inoculum, notablysuppressed viral antigen expression in the cells by making it lower than the limit of EIA detection in 48 h. The inhibition lasted more than 96 h. Viral plaque assay results demonstrated that S-ODN were most effect’ive within 18h with plaque inhibition rate over 90% by 5. 0 pmol/L S--ODNI. The inhibitory activity soon faded in 24 h. In addition, high concentrations (up to 80. 0 pmol/L) of S--ODN did not show any obvious cytotoxicity in 6 d by usingtrypan blue dye exclusion method. Conclusion: The specific synthetic S--ODN transitorily inhibited JEV replicationin BHK--ZI cells with characteristics of specificity and S--ODN dose--dependence.

c-Ha-ras and c-myc antisense oligodeoxynucleotides inhibit the proliferation and DNA synthesis in human gastric carcinoma cell lines

The effects of two antisense oligodeoxynucleotides on the expression of c-Ha-ras proto-oncogene and the growth of human gastric carcinoma cell lines were observed. Synthetic 15-mer directed at the region of the translational initiation site of c-Ha-ras proto-oncogene (ASO-r) greatly inhibited the proliferation (55. 61%,P<0. 05) and DNA synthesis (76. 79%,P<0. 05) of MGc-803 cell line. It also inhibited the proliferation (62. 02%,P<0. 05) and DNA synthesis (76. 78%, P<0. 05) of SGc-7901 cell line. A reduction in intracellular P21 ras protein levels in MGc-803 cell line was observed 6 h after the treatment with ASO-r and maintained over 12 h. Another synthetic 15-mer targeted against the initiation codon and downstream 4 codons of c-myc proto-oncogene (ASOm) inhibited only DNA synthesis of MGc-803 cell line (71. 37%, P<0. 05). The control 15-mer did not inhibit the expression of P21 protein and proliferation of these cell lines. These experiments seemed to provide evidence that ASO-r could be effective in inhibiting the expression of c-Ha-ras proto-oncogene and controlling the growth of human gastric carcinoma cells,and that the over-expression of c-Ha-ras proto-oncogene might mainly be associated with the malignant proliferation of human gastric carcinoma cells.

Inhibiting effect of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides on VEGF expression in U937 cells

Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-tion as follows:10,20 and 30 μmol/L respectively)or scrambled sequence,compared with negative control.The expression of VEGF mRNA was measured by semi-quantitative RT-PCR,VEGF protein was measured by Western blot.Results:VEGF ASODN obviously inhibited expression of VEGF mRNA in U937 cell,compared with scrambled sequence and negative control(P<0.05).And the inhibition effect was most remarkable after 24 h,which is related with the dose of VEGF ASODN(P<0.05).Scrambled sequence groups had no significant difference compared with negative control groups(P>0.05).VEGF ASODN obvi-ously inhibited expression of VEGF protein,compared with scrambled sequence and negative control(P<0.05).Conclusion:The expressions of VEGF at mRNA and protein levels in leukemic cell line U937 are down-regulated after being treated with VEGF ASODN.

Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

CpG oligodeoxynucleotides （CpG ODN） as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN （PO CpG ODN） can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo. This study aimed to evaluate the immunostimulatory potential arid safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes. BALB/c mice were immunized intramuscularly with different formulations of liposomes, CpG ODN and hepatitis B surface antigen （HBsAg）. The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation. High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg arid PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity arid act as a potential adjuvant for vaccines.

Reversing drug resistance in the ovarian carcinoma cell line SKOV3/mdr1 in vitro by antisense oligodeoxynucleotides

Objective To investigate the effect of multidrug resistance gene 1 (mdr1) antisense oligodeoxynucleotides (ODNs) on reversing multidrug resistance in the drug resistant ovarian carcinoma cell line SKOV3/mdr1. Methods The ovarian carcinoma cell line SKOV3 transducted with a human multidrug resistance gene (mdr1) served as the drug resistant model (SKOV3/mdr1). The mdr1 antisense ODNs was transfected into SKOV3/mdr1 cells while mediated by lipofectamine. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression and the amount of the mdr1 mRNA in the cells. The positive rate and function of the mdr1 gene product P-glycoprotein (Pgp) in the mdr1 antisense ODNs treated SKOV3/mdr1 cells were determined by flow cytometry and rhodamine 123 efflux. Drug resistance in the SKOV3/mdr1 cell line was observed by MTT assay and cell colony culture. Results The mdr1 mRNA level was decreased to about 60% of that of β-actin after mdr1 antisense ODNs treatment. The Pgp positive rate of mdr1 antisense ODNs treated SKOV3/mdr1 cells decreased from 100% to 52.6% (P<0.01). The intracellular rhodamine 123 retention was increased from 9.1% to 33.8% (P<0.01). The chemoresistance to taxol decreased to 58% of SKOV3/mdr1 with mdr1 antisense ODN treatment. Compared with SKOV3/mdr1 cells in the control group, under a certain range of drug concentrations, the number of drug resistance colonies in mdr1 antisense ODNs treated SKOV3/mdr1 cells for taxol and doxorubicin decreased by 8.6±0.8 fold and 3.1±0.6 fold, respectively. Some non-specific functions during oligodeoxyncleotide treatment was also detected. Conclusion mdr1 expression in the SKOV1/mdr1 cell line was partially inhibited after mdr1 antisense ODNs treatment at the mRNA and protein level, increasing the chemotherapy sensitivity of this drug resistant ovarian carcinoma cell line.

Com bined effect of alphafetoprotein antisense oligodeoxynucleotidesand 5-fluorouracil on human hepato ma cell growth

Objective To investigate the effect of alpha fetoprotein (AFP) antisense phosphorothioate oligodeoxynucleotides in combination with 5 fluorouracil (5 FU) on the growth of BEL 7404 human hepatoma cells in vitro Methods Phosphorothioate oligodeoxynucleotides were synthesized by β cyanoethyl phosphoramidite chemistry AFP gene expression in human hepatoma cells was determined by avidin biotin peroxidase complex (ABC) immunocytochemical method Cell growth in the presence or absence of experimental agents was measured using 3 (4, 5 dimethylthiazol 2 yl) 2, 5 dipheny ltetrazolium (MTT) microculture tetrazolium assay Results AFP antisense oligomers markedly suppressed the growth of BEL 7404 human hepatoma cells in vitro by sequence specific blocking of the AFP gene expression in the cells (P<0 05) 5 FU also inhibited the hepatoma cell growth in a dose dependent manner when used alone (P<0 05) The combined treatment with AFP antisense oligomers and 5 FU showed significantly enhanced hepatoma cell growth inhibition than either AFP antisense or 5 FU treated cells alone (P<0 05) Conclusion Combined use of AFP antisense oligomers and 5 FU could more effectively inhibit the growth of BEL 7404 human hepatoma cells in vitro

ANTIVIRAL EFFECTS ON MOUSE LEUKEMIA VIRUS REPLICATION BY OLIGODEOXYNUCLEOTIDES IN VITRO AND IN VIVO

Oligodeoxynucleotides (Oligomers) including modified and unmodified, homo-and heterooligomers were tested for their ability to inhibit mouse SRS leukemia virus (SRSV)-induced proliferation of cells, colony formation, syncytium formation and reverse transcriptase (RT) activity in vitro. Phosphorothioate analogs complementary to Mo-MuLV sequences, as well as noncomplementary homooligomers, were found to be active. Unmodified homooligomer (dC14) also showed inhibition of growth of ascitic lymphoma carrying SRS virus. Our study suggests that different classes of oligonucleotides may inhibit SRSV replication with different mechanisms.