Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

PREPARATION AND CLEAVAGE MECHANISM OF CHELATES OF METAL IONS WITH EDTA LINKED TO OLIGODEOXYRIBONUCLEOTIDES

The chelates of metal ions with EDTA covalently linked to the 5′end of oligodeoxyribonuclotides(ODN),i.e,ODN5′EDTA·M(Ⅱ),are prepared,in which M(Ⅱ) is Fe(Ⅱ),Co(Ⅱ) or Cu(Ⅱ).The optimum pH value for forming these three chelates is calculated.For ODN5′EDTA Fe(Ⅱ) pH value is 5.8 to 8.6,pH 4.6～8.1 for ODN5′EDTA Co(Ⅱ),and pH 3.4～5.7 for ODN5′EDTA Cu(Ⅱ).Under such pH value conditions neither can Mg(Ⅱ) ion,necessary for cleavage reaction,be competitive with Fe(Ⅱ),Co(Ⅱ) or Cu(Ⅱ) to form EDTA chelate,nor can it be precipitated.The cleavage mechanism of ODN5′EDTA Fe(Ⅱ) for DNA duplex is discussed.Modified ODN binds with DNA duplex in the major groove via hydrogen bond to form triple helix.In the presense of oxygen and reducing agent dithiothreitol,hydroxyl radicals species are generated as intermediates by catalysis of metal ions,and then oxidize the ribo ring and cut the doublestranded DNA at the sites close to the EDTA· Fe(Ⅱ).

dsODNs 靶向封闭肺泡巨噬细胞中 NF -κB炎症信号通路的实验研究

目的：在确定脂质体转染双链寡聚脱氧核苷酸（ dsODNs ）最佳转染浓度及时间的基础上，评估脂质体转染的双链寡聚脱氧核苷酸（ dsODNs/Lipofectamin2000）竞争性抑制对肺泡巨噬细胞中核因子κB（ NF-κB）/DNA结合活性的影响，验证dsODNs-decoy策略靶向封闭肺泡巨噬细胞中NF-κB信号通路的可行性。方法支气管肺泡灌洗分离提取家兔肺泡巨噬细胞（ AMs）后体外培养，以Lipofectamine2000为载体转染dsODNs后对巨噬细胞进行干预，测定巨噬细胞中dsODNs/Lipofectamine2000的转染活性、转染效率及转染后对AMs的毒性；检测dsODNs/Lipofectamine2000转染对炎症因子（ IL -1α、IL -6、TNF -α等） mRNA 的表达；观察 dsODNs/Lipofectamine2000转染后NF -κB p65亚基的核移位情况。结果转染肺泡巨噬细胞dsODNs/Lipofectamine2000的最适比例为1∶5，最佳时间为6 h，此时细胞毒性适中，转染效率、荧光强度及细胞状态最佳；与LPS组比较，dsODNs/Lipofectamine2000转染组各种炎症介质mRNA的表达均明显抑制（P＜0．01）；细胞核中NF-κB p65亚基的表达明显少于细胞浆中。结论 dsODNs/Lipofecetamine2000能在体外有效转染AMs并成功抑制AMs中NF-κB信号通路相关炎症靶基因的转录表达，证实了dsODNs-decoy策略影响炎症效应细胞的可行性。

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

Design and choice of TFO binding and cleaving HBV core promoter

Objective: To screen a triple helix-forming oligodeoxyribonucleotide (TFO) that can bind HBV core promoter at target site with high affinity and specificity, and to observe the ability of manganese porphyrin modified TFO to combine and cleave HBV DNA. Methods: Similar homopurine domain (1 734 - 1 754) in HBV core promoter was selected as target sequence. Several corresponding TFOs were synthesized. The affinities and specificities of TFOs binding target sequence were tested with electrophoretic mobility shift and DNase I footprinting assays. The selected best TFO was modified with manganese porphyrin and acridine. The ability of the TFO derivative to cleave HBV DNA was observed with cleavage experiment. Results: Under the condition of 371 and pH 7. 4, the TFO consisting of cytidylate and thymidylate (CT-TFO) and the parallel TFO consisting of guanylate and thymidylate (GT-TFOp) bound the target sequence weakly with Kd values much more than 10 -6 mol/L. The affinities of anti-parallel GT-TFO ( GT-TFOap) and short TFO consisting of adenine nucleotide and guanylate (AG-TFOsh) binding the target sequence were higher than those of the formers, with Kd values of 5 μ 10-7 mol/L and 2. 5 μ 10-8 mol/L respectively. Long AG-TFO (AG-TF01) had the highest binding affinity with a Kd value of 3 μ 10 -9 mol/L among all the TFOs studied for sequence specificity. In the presence of potassium monopersulfate, KHSO5, TFO modified with manganese porphyrin and acridine cleaved the target sequence where the triplex DNA formed. Conclusion: TFO containing AG or GT binds homopurine in HBV core promoter in adverse parallel direction to form triple helix. AG-TFO1 has the highest binding affinity among all the TFOs studied. After modified with manganese porphyrin, AG-TFO1 completely binds and cleaves the target HBV DNA sequence where triplex DNA is formed.

Combination and cleavage of HBV DNA fragments by triple helix-forming oligonucleotides modified with manganese porphyrin in vitro

Objective To observe the ability of triple helix-forming oligonucleotides (TFOs) modified with manganese porphyrin to combine with and cleave HBV DNA fractions. Methods TFO were modified with manganese porphyrin and acridines,and then reacted with the 32 P labeled HBV DNA fragments at 37℃ in vitro (pH 7.4). Electrophoretic mobility shift assays and DNase Ⅰ footprinting tests were used to show the affinity and specificity of TFO to bind to target sequences. The ability of TFO to cleave HBV DNA fragments was tested by cleavage experiments. Results TFO modified with manganese porphyrin and acridine could bind to the target sequence in a sequence-dependent manner,with a Kd value of 3.5×10 -7 mol/L and a relative affinity of 0.008. In the presence of potassium monopersulfate (KHSO 5),TFO modified with manganese porphyrin and acridine could cleave the target sequence where the triplex DNA was formed. Conclusion In the presence of KHSO 5,TFO modified with manganese porphyrin and acridine could bind and cleave the target HBV-DNA in a sequence-dependent manner.