Therapeutic effects of microRNA-582-5p and -3p on the inhibition of bladder cancer progression.

Many reports have indicated that the abnormal expression of microRNAs (miRNAs) is associated with the progression of disease and have identified miRNAs as attractive targets for therapeutic intervention. However, the bifunctional mechanisms of miRNA guide and passenger strands in RNA interference (RNAi) therapy have not yet been clarified. Here, we show that miRNA (miR)-582-5p and -3p, which are strongly decreased in high-grade bladder cancer clinical samples, regulate tumor progression in vitro and in vivo. Significantly, the overexpression of miR-582-5p or -3p reduced the proliferation and invasion of UM-UC-3 human bladder cancer cells. Furthermore, transurethral injections of synthetic miR-582 molecule suppressed tumor growth and metastasis in an animal model of bladder cancer. Most interestingly, our study revealed that both strands of miR-582-5p and -3p suppressed the expression of the same set of target genes such as protein geranylgeranyltransferase type I beta subunit (PGGT1B), leucine-rich repeat kinase 2 (LRRK2) and DIX domain containing 1 (DIXDC1). Knockdown of these genes using small interfering RNA (siRNA) resulted in the inhibition of cell growth and invasiveness of UM-UC-3. These findings uncover the unique regulatory pathway involving tumor suppression by both strands of a single miRNA that is a potential therapeutic target in the treatment of invasive bladder cancer.

Synthesis, gene-silencing activity and nuclease resistance of 3'-3'-linked double short hairpin RNA.

To improve the nuclease resistance of siRNA while reducing its induction of an innate immune response and maintaining its biological activity for possible therapeutic application, we designed and synthesized a series of double short hairpin RNAs (dshRNAs). Each dshRNA consisted of two identical short hairpin RNAs (shRNAs) linked at their 3' ends by glycerol. The dshRNAs were synthesized on a glycerol-derivatized solid support from amidites with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group. Synthesis was carried out in a single run on a DNA/RNA synthesizer, without the need for enzymatic ligation. The dshRNAs showed structure-dependent gene-silencing activity at the protein level, and dshRNAs in which the 3' end of the two sense regions were linked showed especially high activity. Inclusion of 2'-O-methyluridine residues in the loop region was associated with 1.6- to 2.4-fold lower induction of interferon-α than was siRNA, without loss of gene-silencing activity. dshRNA also showed higher exonuclease resistance than siRNA or canonical shRNA. Our studies provide a new approach to gene silencing based on the concept of linking the 3' end of the sense regions of two shRNA molecules to form a double shRNA.

In vivo antiproliferative effect of NS-187, a dual Bcr-Abl/Lyn tyrosine kinase inhibitor, on leukemic cells harbouring Abl kinase domain mutations.

Advanced-phase chronic myeloid leukemia patients treated with imatinib often relapse due to point mutations in the Abl kinase domain. We herein examine the in vitro and in vivo effects of a Bcr-Abl/Lyn dual tyrosine kinase inhibitor, NS-187, on seven mutated Bcr-Abl proteins. NS-187 inhibited both Tyr393-phosphorylated and Tyr393-unphosphorylated Abl, resulting in significant in vitro growth inhibition of cells expressing six of seven mutated Bcr-Abl kinases, though not T315I. Furthermore, NS-187 prolonged the survival of mice injected with leukemic cells expressing all mutated Bcr-Abl tested except T315I, and its efficacy correlated well with its in vitro effects.

Antiproliferative and antitumor effects of azacitidine against the human myelodysplastic syndrome cell line SKM-1.

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of stem cell disorders characterized by dysplasia of one or more hematopoietic cell lineages and a risk of progression to acute myeloid leukemia. The cytidine analog azacitidine (Vidaza), a hypomethylating agent, improves survival in patients with MDS, but its mechanism of action is not well understood. MATERIALS AND METHODS: The effects of azacitidine on the MDS-derived cell line SKM-1 were investigated by DNA methylation assay, cell proliferation assay, and a subcutaneous xenograft mouse model. RESULTS: Azacitidine and decitabine induced hypomethylation of the tumor suppressor gene cyclin-dependent kinase 4 inhibitor B (CDKN2B) in SKM-1 cells, whereas the deoxycytidine analog cytarabine did not. Azacitidine and decitabine also inhibited SKM-1 cell growth in vitro. In the mouse xenograft model, azacitidine significantly suppressed tumor growth. CONCLUSION: Inhibition of DNA methyltransferase by azacitidine contributes to its antiproliferative and antitumor effects against SKM-1 cells and may explain its clinical efficacy in MDS.

NS-187, a potent and selective dual Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for imatinib-resistant leukemia.

Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region-Abl (Bcr-Abl)-positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib. We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as imatinib in suppressing the growth of Bcr-Abl-bearing tumors and markedly extends the survival of mice bearing such tumors. The inhibitory effect of NS-187 extends to 12 of 13 Bcr-Abl proteins with mutations in their kinase domain but not to T315I. NS-187 also inhibits Lyn without affecting the phosphorylation of Src, Blk, or Yes. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Philadelphia-positive (Ph+) leukemia.

Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes.

Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.

Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes.

The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.