In vitro reversal MDR of human carcinoma cell line by an antisense oligodeoxynucleotide-doxorubicin conjugate.

A conjugate of antisense oligodeoxynucleotide (AS ODN) covalently linked with deoxorubicin (DOX) was synthesized. Its properties and antitumour activity in human carcinoma DOX resistant cells (KB-A-1) were investigated in vitro. The results showed that the conjugate was strongly stable both in Dulbecco's Phosphate-Buffered Saline (PBS) and in culture medium. The intracellular concentration of the conjugate was higher than that of the AS DON by HPLC analysis. The conjugate showed potent dose-dependent inhibition to the growth of KB-A-1 cells. Chemosensitivity of KB-A-1 cells to DOX was also investigated in vitro. When the cells were first exposed to the conjugate (0.5 microM) and then exposed to DOX for 24 h, the IC50 value of DOX decreased from 21.5 to 2.2 microM. In contrast, when treated with the mixture of the same concentration of the AS ODN with equivalent DOX, the IC50 value of DOX was 16.8 microM. Intracellular DOX concentration was detected in KB-A-1 treatment with the conjugate in vitro by HPLC. The results showed that the intracellular DOX concentration was 6.4-fold increased in KB-A-1 cells treated with the conjugate compared to treatment with DOX alone. In contrast, 1.8-fold increasing was observed when treated with the AS ODN. Western blot analysis showed a significantly decrease in the amount of P-glycoprotein in KB-A-1 cells. These results suggest that the conjugate is effective in reversing multidrug resistance. Certainly, further studies are conducting to explore the antitumour effect of the conjugate in vivo.     

PEG conjugated VEGF siRNA for anti-angiogenic gene therapy.

A novel siRNA delivery system based on polyelectrolyte complex (PEC) micelles was introduced in this study. Vascular endothelial growth factor (VEGF) siRNA was conjugated to poly(ethylene glycol) (PEG) via a disulfide linkage (siRNA-PEG). The siRNA-PEG conjugate could form PEC micelles by interacting with cationic polyethylenimine (PEI) as a core forming agent. The VEGF siRNA-PEG/PEI PEC micelles showed greater stability than naked VEGF siRNA against enzymatic degradation. Under a reductive condition similar to cytosolic environment, an intact form of siRNA was released from the siRNA-PEG conjugate by cleavage of the disulfide linkage. The VEGF siRNA-PEG/PEI PEC micelles effectively silenced VEGF gene expression in prostate carcinoma cells (PC-3) up to 96.5% under an optimized formulation condition. They also showed a far superior VEGF gene silencing effect than VEGF siRNA/PEI complexes even in the presence of serum. This study suggests that the siRNA delivery system using VEGF siRNA-PEG/PEI PEC micelles could be potentially applied to RNAi-based anti-angiogenic treatment of cancer in vivo.     

A novel polymer-paclitaxel conjugate based on amphiphilic triblock copolymer.

A novel amphiphilic polymer-paclitaxel conjugate P(LGG-paclitaxel)-PEG-P(LGG-paclitaxel) has been prepared. It was derived from its parent polymer P(LGG)-PEG-P(LGG), poly{(lactic acid)-co-[(glycolic acid)-alt-(l-glutamic acid)]}-block-poly(ethylene glycol)-block-poly{(lactic acid)-co-[(glycolic acid)-alt-(l-glutamic acid)]}, which was prepared by ring-opening copolymerization of l-lactide (LLA) with (3s)-benzoxylcarbonylethyl-morpholine-2,5-dione (BEMD) in the presence of dihydroxyl PEG with molecular weight of 4600 as a macroinitiator using stannous octoate (Sn(Oct)(2)) as catalyst, and by subsequent catalytic hydrogenation. It could self-assemble into micelles in an aqueous system with P(LGG-paclitaxel) block in the core and PEG in the shell. ESEM and DLS analysis of the micelles revealed a homogeneous spherical morphology and a unimodal size distribution. In vitro release of paclitaxel from the conjugate micelles showed that its release rate depended on pH value and was higher at lower pH than in neutral condition. In vitro antitumor activity of the paclitaxel conjugate against rat brain glioma C6 cells was evaluated by MTT method. The results showed that the paclitaxel can be released from the conjugate without losing cytotoxicity.     

SCL基因反义寡核苷酸对K562和CEM细胞系的作用

干细胞白血病(SCL)基因是新发现的与白血病发生有关的癌基因。为进一步探讨SCL反义寡核苷酸对白血病细胞的作用，本研究应用SCL基因的反义硫代磷酸寡脱氧核苷酸(AS—PS—ODN)分别与K562和CEM白血病细胞系作用，观察细胞生长、分化、凋亡和SCL　mRNA变化。结果显示：(1)AS—PS—ODN对K562和CEM细胞的生长有不同程度的抑制作用，并呈量效和时效关系；(2)AS—PS—ODN作用后K562细胞联苯胺染色阳性率明显增加；(3)AS—PS—ODN作用后K562细胞发生凋亡现象，但未观察到CEM细胞凋亡；(4)AS—PS—ODN作用后K562细胞和CEM细胞SCL　mRNA表达水平减低，同时CEM细胞SIL－SCL　mRNA表达水平也减低。结论：AS—PS—ODN能特异性抑制K562和CEM细胞的增殖，减低SCL　mRNA和SIL—SCL　mRNA表达水平，同时促进红系分化，诱导K562细胞过早凋亡。     

Targeted delivery of antisense oligodeoxynucleotides to folate receptor-overexpressing tumor cells.

A major problem in exploring the full potential of antisense ODN is the lack of a safe and efficient delivery system. In this study a new method has been developed that is highly efficient in encapsulating ODN inside folate receptor (FR)-targeted lipid vesicles. ODN formulated in these vesicles were efficiently protected from degradation by nucleases compared to free ODN. Folate efficiently mediated intracellular delivery of ODN to KB tumor cells that overexpress FR. Delivery of EGFR antisense ODN via FR-targeted lipid vesicles resulted in a significant down-regulation of EGFR expression in KB cells and cell growth inhibition, far more efficient than that with free ODN or ODN encapsulated in ligand-free lipid vesicles. Intracellular delivery of EGFR antisense ODN also sensitized KB cells to doxorubicin (DOX) treatment. Thus targeted delivery of ODN via this novel lipid vector may have potential in treating tumors that overexpress FR.     

诱骗寡核苷酸靶向阻断信号转导和转录活化蛋白5抑制K562细胞生长增殖

目的观察信号转导和转录活化蛋白 5 (STAT5 )诱骗寡核苷酸 (DecoyODN)靶向阻断STAT5的信号传递对白血病细胞株K5 6 2生长增殖的影响 ,并初步探讨其分子机制。方法　将体外设计合成的STAT5DecoyODN经阳离子脂质体介导转染K5 6 2细胞 ,通过细胞生长曲线及集落形成实验观察K5 6 2细胞生长增殖能力 ,并用RT PCR方法检测STAT5下游靶基因的表达。结果　激光共聚焦显微镜观察显示 ,STAT5DecoyODN能高效转染K5 6 2细胞 ,阳性率 95 .2 % ;细胞生长曲线显示STAT5De coyODN可显著抑制K5 6 2细胞生长 ,抑制率 77.7% ;集落形成实验显示STAT5DecoyODN能显著抑制K5 6 2细胞集落形成能力 (DecoyODN处理组集落形成率为 8.3% ,空白对照组为 35 .7% ,P <0 .0 5 ) ;RT PCR检测发现STAT5DecoyODN处理后K5 6 2细胞c myc、bcl XL、CyclinD1基因较对照组分别下调15 .4 % ,30 .8% ,2 9.1%。结论　DecoyODN可能通过阻止STAT5对靶基因的转录激活 ,使靶基因表达下调 ,进而抑制K5 6 2细胞生长增殖。     

抑制脑血栓形成的硫代寡核苷酸的细胞摄取率和亚细胞分布

INTRODUCTIONInordertopreventthrombosisatrelativelynarrow,branching,andatheroscleroticpartsofcerebralbloodvessels犤1犦.wesynthesizedandselected3triplexhelix-formingoligodeoxynucleotides(TFO)犤2犦.ThemodifiedTFOi.e.phosphorothioateoligodeoxynucleotide(P-ODN)hashighaffinitytoshearstressresponsiveelement(SSRE)ofgeneinitiators.P-ODNmusthavegoodenzymetolerance,membraneper-meability,andstabilityoritisuseless.Thispaperaimstostudyifthe3P-ODNcouldenterthecellsandreachthetargetpartinthenucle…     

CpG oligodeoxynucleotide stimulates protective innate immunity against human renal cell carcinoma xenografted in nude mice

Renal cell carcinoma easily develops metastasis, which is highly resistant to a variety of therapies. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent activators of innate and adaptive immunity. CpG ODN is inclined to be used as vaccine adjuvant or in combination with other therapies to exert antitumor effect mediated by the adaptive immunity. Herein, we examined the antitumor effect of CpG ODN monotherapy and the role of innate immunity on human RCC Caki-1 cells xenografted in nude mice. Our results indicated that the peritumoral subcutaneous injections of CpG ODN1826 once a week resulted in significant inhibition of the growth of Caki-1 xenografts compared with the control groups, and the survival of tumor-bearing mice were also prolonged significantly. When cytotoxicity of splenic cells from host mice was assessed, it was found that CpG ODN1826 significantly promoted the cytotoxicities of splenocytes targeting primary Caki-1 cells or YAC-1 cells, indicating that the activity of natural killer cells in tumor-bearing nude mice was enhanced by CpG ODN1826 monotherapy. The serum concentrations of interleukin-12 were increased in mice treated with CpG ODN1826. Thus, CpG ODN1826 monotherapy induces significant inhibitory effects on the growth of human RCC xenografted in athymic immunodeficient mice, and the tumor-bearing mice achieves long-term survival, which might be attributed to enhanced innate immunity.     

Selection of oligonucleotide aptamers with enhanced uptake and activation of human leukemia B cells

The clinical use of oligonucleotide (ODN) therapeutics has been hampered by their limited ability to penetrate intact cells. To identify ODN properties that would facilitate cellular uptake, we developed a repetitive selection procedure using an ODN library containing at least 10(14) different molecules and human B lymphoma cells as a target. Natural phosphodiester single-stranded DNA ODNs (R-aptamers) were obtained after 10 rounds of selection. A common feature in the R-aptamers was guanine-rich 3' terminal sequences, and many also contained potential immunostimulatory (ISS) CpG sequence motifs. Two R-aptamers (R10-60 and D-R15-8) with the predominant shared characteristics were selected for further study on primary human chronic lymphocytic leukemia (CLL) B cells, which are well known to be difficult to transfect and activate. Flow cytometry analysis of the CLL cells demonstrated that the fluorochrome-labeled R-aptamers were internalized much more efficiently than nonselected random sequence ODN. Studies on sequence modifications indicated that efficient uptake required ODN multimerization, that was promoted by guanine-rich sequences at the 3' terminus. In addition, CLL cells that were exposed to the aggregating R-aptamers containing CpG motifs were strongly activated, as indicated by upregulation of CD40 levels as compared to cells treated with nonaggregating CpG R-aptamers. Together, these findings suggest that the sequence compositions in R-aptamers that promote multimerization and contain optimal ISS CpG motifs facilitate the delivery of ISS-ODN to CLL cells and enhance the activation of these cells.     

多药耐药基因反义寡核苷酸逆转肿瘤细胞耐药的初步研究

目的：克服肿瘤细胞的多药耐药（ＭＤＲ）。方法：用人工合成互补于ｍｄｒ１基因５′端转录起始部位的反义寡核苷酸（ＯＤＮ），直接转染耐药细胞株ＫＢ－８－５细胞，或以脂质体ｌｉｐｏｆｅｃｔｉｎ为载体进行基因转染实验，通过ＭＴＴ法检测细胞对柔红霉素（ＤＮＲ）的敏感性，流式细胞仪分析细胞内ＤＮＲ含量及免疫组化方法确定细胞表面糖蛋白（Ｐｇｐ）的表达水平。结果：ＯＤＮ可增加ＫＢ－８－５细胞内的ＤＮＲ浓度从而提高耐药细胞对ＤＮＲ的敏感性，ｌｉｐｏｆｅｃｔｉｎ进一步加强上述作用。在ＤＮＲ浓度为３．０ｍｇ／Ｌ组中，约有７４．４３％的被转染细胞对ＤＮＲ敏感而致死，基本达到药物敏感细胞株ＫＢ－３－１的水平。ＯＤＮ转染的ＫＢ－８－５细胞的Ｐｇｐ为弱阳性表达，低于阳性对照ＫＢ－８－５细胞的Ｐｇｐ表达水平。结论：ＯＤＮ的逆转作用可能与其互补结合的ｍｄｒ１ｍＲＮＡ降解或直接阻滞了Ｐｇｐ合成使其表达降低有关。     

Tumor growth inhibition in vivo and G2/M cell cycle arrest induced by antisense oligodeoxynucleotide targeting thymidylate synthase.

Chemotherapeutic agents targeting thymidylate synthase (TS) are effective against human tumors. Efficacy is limited by drug resistance, often mediated by TS overexpression. Treatment of HeLa cells in vitro with an antisense oligodeoxynucleotide (ODN 83) targeting human TS mRNA reduces TS mRNA and protein levels, inhibits cell proliferation, and sensitizes cells to TS-targeting drugs (Ferguson et al., 1999). The present study investigates the mechanism by which ODN 83 inhibits cell proliferation and examines its antitumor efficacy in vivo. ODN 83 treatment did not induce apoptosis in HeLa cells in vitro but caused accumulation of cells at G2/M. In contrast, TS-targeting chemotherapeutics arrest at G1 or S. Antisense down-regulation reduced TS mRNA levels in human colon cancer (HT29) cells by 40% in vitro, resulted in G2/M arrest, and reduced proliferation without enhanced cell death. Growth of HT29 tumors in immunocompromised mice was significantly inhibited when antisense ODN 83 treatment began promptly after tumor implantation and was accompanied by a 40% reduction in TS protein levels. Growth of tumors allowed to reach 400 mm3 prior to ODN administration was unaffected by antisense ODN 83. Radiolabeled ODNs were localized to the tumor periphery but evenly distributed in normal tissue. Thus, down-regulation of TS mRNA and protein by antisense ODN treatment exerts a novel G2/M cell cycle block without increasing cell death and inhibits HT29 tumor cell growth in vivo. Antisense ODN 83 may be an effective therapy for colon carcinoma, alone or in combination with TS-targeting cytotoxic drugs.     

Phosphorothioate oligodeoxynucleotides distribute similarly in class A scavenger receptor knockout and wild-type mice

It has been suggested that binding of phosphorothioate oligodeoxynucleotides (P=S ODNs) to macrophage scavenger receptors (SR-AI/II) is the primary mechanism of P=S ODN uptake into cells in vivo. To address the role of scavenger receptors in P=S ODN distribution in vivo, several pharmacokinetic and pharmacological parameters were compared in tissues from scavenger receptor knockout mice (SR-A-/-) and their wild-type counterparts after i.v. administration of 5- and 20-mg/kg doses of P=S ODN. With an antibody that recognizes P=S ODN, no differences in cellular distribution or staining intensity in livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected at the histological level. There were no significant differences in P=S ODN concentrations in these organs as measured by capillary gel electrophoresis as well, although the concentration of P=S ODN in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice. Furthermore, a P=S ODN targeting murine A-raf reduced A-raf RNA levels to a similar extent in livers from SRA-/- (92.8%) and wild-type (88.3%) mice. Finally, in vitro P=S ODN uptake studies in peritoneal macrophages from SR-A-/- versus wild-type mice indicate that other high- and low-affinity uptake mechanisms predominate. Taken as a whole, our data suggest that, although there may be some contribution to P=S ODN uptake by the SR-AI/II receptor, this mechanism alone cannot account for the bulk of P=S ODN distribution into tissues and cells in vivo, including macrophages.     

Transfection of naked nuclear factor-κB decoy oligodeoxynucleotides into liver by rapid portal vein infusion in rats: its effect on ischemia-reperfusion injury of liver

This study was aimed at examining whether rapid portal vein infusion (RPVI) of a small volume of naked oligodeoxynucleotides (ODNs) could be used to transfect sufficient amounts of nuclear factor-κB (NF-κB) decoy ODN into the liver to suppress NF-κB activation during liver ischemia-reperfusion (I/R) injury, in which NF-κB plays a central role in regulating the production of inflammatory cytokines. One milliliter of naked NF-κB decoy ODN solution was administered into the portal vein for a few seconds. Transfection efficacy was examined by labeling the ODN with a fluorescent tag. Activation of NF-κB was investigated by electrophoretic mobility shift assay. Levels of serum liver enzymes and cytokines were measured during liver I/R injury. NF-κB decoy ODN was preferentially incorporated into Kupffer cells and sinusoidal endothelial cells, but not hepatocytes, in the rat liver. Transfected NF-κB decoy ODN suppressed the function of NF-κB in both Kupffer cells and sinusoidal endothelial cells during liver I/R injury, causing significant decreases in serum tumor necrosis factor-α and interleukin-6 levels 3?hr after reperfusion. Although the decrease in serum liver enzymes was not significant, naked NF-κB decoy ODN was successfully incorporated into Kupffer cells and sinusoidal endothelial cells by rapid portal vein infusion, inhibited NF-κB activation in both cells, and suppressed the production of inflammatory cytokines during the early phase of liver I/R injury.     

联合抑制XIAP和MRP逆转HL-60/ADR细胞耐药

本研究探讨HL-60／ADR细胞的耐药机制，比较单独应用MRP、XIAP反义寡核苷酸或二者联合应用对HL-60／ADR细胞耐药的逆转作用。应用RT-PCR和Western blot分别检测并比较HL-60细胞和HL-60／ADR细胞的BCL-2、XIAP、MDR1、MRP在mRNA和蛋白水平表达的差异。将XIAP和MRP全硫代反义寡核苷酸，以脂质体-反义寡核苷酸复合物的形式单独或联合转染HL-60／ADR细胞，并应用CCK-8实验测定反义寡核苷酸对DNR敏感性影响；用RT-PCR和Western blot检测反义寡核苷酸对XIAP、MRP的mRNA和蛋白表达的影响。结果表明：HL-60／ADR细胞的MRP和XIAP的表达均明显高于HL-60细胞。XIAP和MRP反义寡核苷酸单独应用均能下调XIAP和MRP的表达，增加对柔红霉素的敏感性。二者联合应用与XIAP反义寡核苷酸单独应用比较，并不能增强对XIAP表达的抑制作用（P〉0．05），但使HL-60／ADR细胞对柔红霉素的敏感性明显增加（P〈0．05）；二者联合应用与MRP反义寡核苷酸单独应用比较，并不能提高HL-60／ADR细胞内DNR浓度及增强对MRP表达的抑制作用，却使HL-60／ADR细胞对柔红霉素的敏感性明显增加（P〈0．05）。结论XIAP和MRP都可能参与了HL-60／ADR细胞的耐药机制，MRP、XIAP反义寡核苷酸的联合应用能更大程度逆转HL-60／ADR细胞的耐药性。     

Immunogenicity of conjugates and micelles of synthetic hepatitis B surface antigen peptides

A cyclic peptide containing the amino acid sequence 122 through 137 of the major hepatitis B surface antigen (HBsAg) polypeptide was synthesized. The immunogenicity of this synthetic peptide, aggregated in micelles or covalently coupled to tetanus toxoid, was assessed in mice. Antibodies against HBsAg (anti-HBs) were obtained with both preparations, administered either in saline suspension or adsorbed on aluminum gel. The peptide-tetanus toxoid conjugate was more immunogenic than the peptide micelles, producing high levels of specific anti-HBs.     

Repression of Fanconi anemia gene (FACC) expression inhibits growth of hematopoietic progenitor cells.

Bone marrow failure is a consistent feature of Fanconi anemia (FA) but it is not known whether the bone marrow failure is a direct and specific result of the inherited mutation or a consequence of accumulated stem cell losses resulting from nonspecific DNA damage. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FACC) plays a regulatory role in hematopoiesis. We exposed normal human lymphocytes, bone marrow cells, endothelial cells, and fibroblasts to an antisense oligodeoxynucleotide (ODN) complementary to bases -4 to +14 of FACC mRNA. The mitomycin C assay demonstrated that the antisense ODN, but not missense or sense ODNs, repressed FACC gene expression in lymphocytes. Treatment with the antisense ODN substantially reduced, in a sequence-specific fashion, cytoplasmic levels of FACC mRNA in bone marrow cells and lymphocytes. Escalating doses of antisense ODN increasingly inhibited clonal growth of erythroid and granulocyte-macrophage progenitor cells but did not inhibit growth of fibroblasts or endothelial cells. The antisense ODN did not inhibit growth factor gene expression by low density bone marrow cells or marrow-derived fibroblasts. We conclude that, while the FACC gene product plays a role in defining cellular tolerance to cross-linking agents, it also functions to regulate growth, differentiation, and/or survival of normal hematopoietic progenitor cells.     

Enhancement of immune responses by co-delivery of a CpG oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres.

Synthetic oligodeoxynucleotides (ODN) consisting of unmethylated bacterial DNA sequences with CpG motifs are potent immunological adjuvants. Immunostimulatory CpG sequences are species-specific. Optimal CpG sequences specific for humans, rodents, livestock, and companion animals have been reported. Nearly all of these reports describe the use of soluble forms of CpG ODN and antigens. We investigated the co-delivery of CpG ODN and antigens in biodegradable nanospheres as an alternative approach for immunization using tetanus toxoid (TT) as the model antigen and ODN #1826 as the model CpG sequence. TT and CpG ODN were co-encapsulated in poly(D,L-lactic-co-glycolic acid) nanospheres. Separate groups of C57BL/6 mice were subcutaneously immunized twice with TT and CpG ODN in nanospheres (test group), TT alone in nanospheres, TT alone in nanospheres mixed with CpG ODN in solution, TT and CpG ODN both in solution (reference group), TT alone in solution, and alum adsorbed TT. T cells isolated from the test group showed strong antigen-specific T cell proliferation ex vivo (stimulation index=45). This was significantly (P<0.0001) higher than that observed for T cells isolated from the reference group. The T cell proliferation of the test group was associated with higher levels of interferon gamma secretion (IFN-gamma 2694.7+/-41.1 pg/ml) than that of the reference group (814.7+/-50.2 pg/ml). Interleukin 4 (IL-4) secretion, if any, was below the detection limit (<13 pg/ml) in all the groups. Anti-sera obtained from the test group also showed very high total IgG titers (end point titers, 2560000) that were 16 times higher than the reference group. Similarly, differences of 8-fold for IgG1 and IgG3, and 5-fold for IgG2b titers were observed. Noticeably, the antibody response induced in the alum-TT group was far less (total IgG, end point titers 160000) than that obtained in the TT-CpG ODN nanospheres group. Overall, the results show that co-delivery of CpG and TT resulted in induction of both T helper type 1 and type 2 (Th1 and Th2) immune responses with a bias towards Th1 type. These results suggest that the co-delivery of CpG ODN adjuvants and antigens in nanospheres is a more efficient approach for immunization than the use of CpG ODN and TT in solution.