Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells

Abstract

Embryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 in vitro on the growth of multiple patient-derived ERMS cell lines, ERMS1, KYM1, and RD. RT-PCR and immunostaining revealed that nucleolin was abundantly expressed and localized in nucleoplasm and nucleoli in all ERMS cell lines, similar to myoblasts. Both iSN04 and AS1411 at final concentrations of 10–30 μM significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. In all ERMS cell lines, iSN04 at a final concentration of 10 μM markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. Quantitative RT-PCR or immunostaining of phosphorylated histone H3 and myosin heavy chain demonstrated that iSN04 suppressed the cell cycle and partially promoted myogenesis but did not induce apoptosis in ERMS cells. Finally, both iSN04 and AS1411 at final concentrations of 10–30 μM disrupted the formation and outgrowth of RD tumorspheres in three-dimensional culture mimicking in vivo tumorigenesis. In conclusion, ERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411, which modulates several cell cycle-related and myogenic gene expression. The present study provides evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

Myogenic Anti-Nucleolin Aptamer iSN04 Inhibits Proliferation and Promotes Differentiation of Vascular Smooth Muscle Cells

“…Fluorescent images were captured using EVOS FL Auto microscope. The ratio of EdU + cells was defined as the number of EdU + nuclei divided by the total number of nuclei using ImageJ software [10].…”

“…We have reported a series of myogenetic oligodeoxynucleotides (myoDNs), which are 12-18-base single-stranded oligodeoxynucleotides that promote myogenesis [5,6]. One of the myoDNs, iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), serves as an anti-nucleolin aptamer and induces myogenic differentiation of myoblasts [5][6][7][8][9] and rhabdomyosarcoma cells [10]. Interestingly, iSN04 facilitates myocardial differentiation of pluripotent stem cells [11], suggesting that nucleolin antagonism by iSN04 affects not only skeletal muscle but also multiple muscle lineages.…”

Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in myoblasts by modulating the β-catenin/NF-κB signaling pathway

“…Phosphorothioated iSN04 (5'-AGA TTA GGG TGA GGG TGA-3') was synthesized and HPLC-purified (GeneDesign, Osaka, Japan) [20][21][22][23][24]27] and then dissolved in endotoxin-free water. Recombinant murine TNF-α (Fujifilm Wako Chemicals, Osaka, Japan), triacylated lipopeptide Pam3CSK4 Adipogen, San Diego, CA, USA) were rehydrated in phosphate-buffered saline.…”

“…We recently identified the 18-base single-stranded DNA, iSN04, as a myogenetic oligodeoxynucleotide (myoDN) that acts as an anti-nucleolin aptamer and promotes myoblast and rhabdomyosarcoma differentiation [20][21][22]. iSN04 is taken up into the cytoplasm without a carrier and removes the translational inhibition of p53 mRNA by nucleolin, leading to activation of the p53 signaling pathway and the myogenic program [20].…”

Development of the 12-Base Short Dimeric Myogenetic Oli-godeoxynucleotide That Induces Myogenic Differentiation

“…Nowadays, aptamers are one of the potential next-generation drugs [6]. We have recently reported a series of myogenetic ODNs (myoDNs), which are single-stranded 18-base telomeric phosphorothi-oated (PS)-ODNs, that robustly induce myogenic differentiation of myoblasts [7] and rhabdomyosarcoma [8]. One of the myoDNs, iSN04 (5’-AGA TTA GGG TGA GGG TGA-3’), serves as an aptamer that physically interacts with nucleolin, a multifunctional phos-phoprotein localized in myoblast nuclei [7,9].…”