Biology subjects

Takaya, T.

Publications and source records attributed to Takaya, T..

12 recordsLinked to original sources

bioRxiv2026

Anti-Nucleolin Aptamer iSN04 Inhibits Alveolar Rhabdomyosarcoma Cell Growth by Inducing Myogenic Differentiation

Rhabdomyosarcoma (RMS) is a muscle-derived soft tissue tumor that retains myogenic potential. Novel drugs for RMS are strongly needed because the standard chemotherapeutic agents for RMS, including vincristine (VCR), have marginally improved the survival rate of high-risk patients for several decades. An anti-nucleolin aptamer, iSN04, was originally identified to promote myogenic differentiation of myoblasts, and was subsequently reported to suppress the growth of embryonal RMS cells by potentiating their differentiation and arresting proliferation. The present study investigated the effects of iSN04 on two alveolar RMS cell lines, NRS-1 and RH30. iSN04 modulated gene expression levels such as myogenic transcription factors (MyoD and myogenin), inhibitor of differentiation proteins (ID1 and ID3), and the tumor suppressor CASZ1. iSN04 treatment induced NRS-1 cells to differentiate into contracting myotube-like cells, and severely impaired the ability of RH30 cells to form and grow tumorspheres in three-dimensional culture. Co-treatment with iSN04 and a low dose of VCR cooperatively suppressed the proliferation of RH30 cells compared with single-agent treatments. These data demonstrate that iSN04 can be a beneficial agent for RMS and hold potential for use in combination therapy with current standard drugs.

cell biology↗

bioRxiv2024

Osteogenic CpG oligodeoxynucleotide, iSN40, inhibits osteoclastogenesis in a TLR9-dependent manner

A CpG oligodeoxynucleotide (CpG-ODN), iSN40 (5-GGA ACG ATC CTC AAG CTT-3), was originally identified to promote osteoblast differentiation independent of Toll-like receptor 9 (TLR9). While CpG-ODNs are generally known to be recognized by TLR9 and inhibit osteoclasto-genesis. This study investigated the anti-osteoclastogenic effect of iSN40. The murine mono-cyte/macrophage cell line RAW264.7 was treated with receptor activator of nuclear factor-{kappa}B ligand (RANKL) to induce osteoclast differentiation, and the effects of iSN40 on osteoclast formation were quantified by tartrate-resistant acid phosphatase (TRAP) staining and real-time RT-PCR. iSN40 completely inhibited RANKL-induced differentiation into TRAP+ multinucleated osteoclasts by suppressing osteoclastogenic genes (Nfatc1, Ctsk, and Dcstamp) and inducing anti-/non-osteoclasto-genic genes (Irf8, Adgre1, and Il1b). Treatment with a TLR9 inhibitor, E6446, or mutation in the CpG motif of iSN40 abolished intracellular uptake and the anti-osteoclastogenic effect of iSN40. These results demonstrate that iSN40 is internalized subcellularly, recognized by TLR9 via its CpG motif, modulates RANKL-dependent osteoclastogenic gene expression, and ultimately inhibits osteoclast formation. Computational simulation of the iSN40 structure also suggested the importance of the superficial CpG motif for iSN40 function. Finally, iSN40 was confirmed to inhibit osteoclastogenesis of RAW264.7 cells cocultured with the murine osteoblast cell line MC3T3-E1, which is a model of bone remodeling. This study demonstrates that iSN40, which exerts both pro-osteogenic and anti-osteoclastogenic effects, may be a promising nucleic acid drug for osteoporosis.

cell biology↗

bioRxiv2024

Myogenic anti-nucleolin aptamer iSN04 inhibits proliferation and promotes differentiation of vascular smooth muscle cells

De-differentiation and subsequent increased proliferation and inflammation of vascular smooth muscle cells (VSMCs) is one of the mechanisms of atherogenesis. Maintaining VSMCs in a contractile differentiated state is therefore a promising therapeutic strategy for atherosclerosis. We have reported the 18-base myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes skeletal and myocardial differentiation. The present study investigated the effect of iSN04 on VSMCs because nucleolin has been reported to contribute to VSMC de-differentiation under pathophysiological conditions. Nucleolin was localized in the nucleoplasm and nucleoli of both rat and human VSMCs. iSN04 without carrier was spontaneously incorporated into VSMCs, indicating that iSN04 would serve as an anti-nucleolin aptamer. iSN04 treatment decreased the ratio of EdU+ proliferating VSMCs and increased the expression of -smooth muscle actin, a contractile marker of VSMCs. iSN04 also suppressed angiogenesis of mouse aortic rings ex vivo, which is a model of pathological angiogenesis involved in plaque formation, growth, and rupture. These results demonstrate that antagonizing nucleolin with iSN04 preserves VSMC differentiation, providing a nucleic acid drug candidate for the treatment of vascular disease.

cell biology↗

bioRxiv2024

Development of the 12-base short dimeric myogenetic oligodeoxynucleotide that induces myogenic differentiation

A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5-AGA TTA GGG TGA GGG TGA-3), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5-TTG GGT GGG GAA-3), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadru-plex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs. Key ContributionThis study reports the structure-based shortening of a myogenetic oligodeox-ynucleotide, iSN04, as an anti-nucleolin aptamer that induces myogenesis. The shortening technology of aptamers while maintaining their conformation and activity improves their potency of drug function and synthesis cost.

cell biology↗

bioRxiv2023

Myogenetic oligodeoxynucleotide induces myocardial differentiation of murine pluripotent stem cells

An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced the differentiation into Nkx2-5+ beating cardiomyocytes with upregulation of Gata4, Isl1, and Nkx2-5, whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes, but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes.

cell biology↗

bioRxiv2023

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

A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor- (TNF-) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-{kappa}B (NF-{kappa}B), resulting in upregulated expression of TNF- and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of {beta}-catenin induced by TNF- or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the {beta}-catenin/NF-{kappa}B signaling pathway in myoblasts. In addition, the anti-inflammatory effects of iSN04 were also observed in smooth muscle cells and pre-adipocytes, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders.

cell biology↗

bioRxiv2022

Identification of a novel osteogenetic oligodeoxynucleotide (osteoDN) that promotes osteoblast differentiation in a TLR9-independent manner

Recent studies have revealed that oligodeoxynucleotides (ODNs) designed from genome sequences have the potential to regulate cell fate. Currently, ODNs that conduct cell differentiation are nanomolecular drug candidates for regenerative medicine. Herein, we demonstrate that iSN40, an 18-base ODN derived from the Lacticaseibacillus rhamnosus GG genome, promoted the differentiation and calcification of osteoblasts that play a central role during bone formation. In the murine osteoblast cell line MC3T3-E1, iSN40 enhanced alkaline phosphatase activity at the early stage of differentiation and facilitated calcium deposition at the late stage by inducing the expression of osteogenic genes such as Msx2, osterix, collagen type 1, osteopontin, and osteocalcin. Intriguingly, the CpG motif within iSN40 was not required for its osteogenetic activity, indicating that iSN40 functions in a TLR9-independent manner. These data suggest that iSN40, serving as an osteogenetic ODN (osteoDN), as a drug seed that target osteoblasts for bone regeneration.

cell biology↗

bioRxiv2021

Myogenetic oligodeoxynucleotide restores differentiation and reverses inflammation of myoblasts aggravated by cancer-conditioned medium

Cancer cachexia is characterized by irreversible muscle loss which is a critical factor in the prognosis of cancer patients. Myoblasts are myogenic precursor cells that are required to maintain skeletal muscle tissue. Previous studies have reported that cancer-released factors deteriorate myoblast differentiation, which is one of the causes of cachexia-associated muscle wasting. We recently identified the myogenetic oligodeoxynucleotide iSN04, which acts an anti-nucleolin aptamer and promotes myogenesis. The present study investigated the effects of iSN04 on human myoblasts exposed to conditioned medium (CM) of colon cancer cells. Cancer-CM impaired myogenic differentiation and myotube formation of myoblasts by upregulating the expression of inflammatory cytokines. iSN04 completely reversed cancer-CM-induced deteriorated myogenesis and inflammatory responses in myoblasts. Tumor necrosis factor- (TNF-), a representative cytokine present in cancer-CM, inhibited differentiation and induced inflammation of myoblasts, similar to cancer-CM. Pre-treatment with iSN04 reversed TNF--induced cachectic phenotypic features in myoblasts. These results indicate that iSN04 protects myoblasts against the effects of cancer-released factors and maintain their myogenic activity. This study provides a novel therapeutic strategy to prevent muscle loss associated with cancer cachexia.

cell biology↗

bioRxiv2021

Myogenetic oligodeoxynucleotides as anti-nucleolin aptamers inhibit the growth of embryonal rhabdomyosarcoma cells

BackgroundEmbryonal 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 on the growth of multiple ERMS1 cell lines in vitro. MethodsThree patient-derived ERMS cell lines, ERMS1, KYM1, and RD, were used. Nucleolin expression and localization in these cells was confirmed by immunostaining. The effects of iSN04 or AS1411 on the growth of ERMS cells were examined by cell counting, EdU staining, quantitative RT-PCR (qPCR), and three-dimensional culture of tumorspheres. ResultsIn all ERMS cell lines, nucleolin was abundantly expressed, and localized and concentrated in nucleoli, similar to myoblasts. Both iSN04 and AS1411 (10-30 M) significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. iSN04 (10 M) markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. qPCR demonstrated that iSN04 suppressed the cell cycle, partially promoted myogenesis, but did not induce apoptosis. Finally, both iSN04 and AS1411 (10-30 M) disrupted the formation and outgrowth of RD tumorspheres mimicking in vivo tumorigenesis. ConclusionsERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411. The present study provides the first evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

cell biology↗

bioRxiv2021

Myogenetic oligodeoxynucleotide (myoDN) recovers the differentiation of skeletal muscle myoblasts deteriorated by diabetes mellitus

Sarcopenic obesity is a complication of decreased muscle mass and strength associated with obesity, and sarcopenia associated with diabetes mellitus (DM) is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle atrophy. We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8. iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat sarcopenic obesity.

cell biology↗

bioRxiv2020

Myogenetic oligodeoxynucleotide (myoDN) complexed with berberine promotes differentiation of chicken skeletal muscle myoblasts

Skeletal muscle myoblasts are myogenic precursors that develop into myofibers during muscle formation. Improvement of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myocytes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myocytes, myotube formation, and myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

cell biology↗

bioRxiv2020

Identification of the myogenetic oligodeoxynucleotides (myoDNs) that promote differentiation of skeletal muscle myoblasts by targeting nucleolin

Herein we report that the 18-base telomeric oligodeoxynucleotides (ODNs) designed from the Lactobacillus rhamnosus GG genome promote differentiation of skeletal muscle myoblasts which are myogenic precursor cells. We termed these myogenetic ODNs (myoDNs). The activity of one of the myoDNs, iSN04, was independent of Toll-like receptors, but dependent on its conformational state. Molecular simulation and iSN04 mutants revealed stacking of the 13-15th guanines as a core structure for iSN04. The alkaloid berberine bound to the guanine stack and enhanced iSN04 activity, probably by stabilizing and optimizing iSN04 conformation. We further identified nucleolin as an iSN04-binding protein. Results showed that iSN04 antagonizes nucleolin, increases the levels of p53 protein translationally suppressed by nucleolin, and eventually induces myotube formation by modulating the expression of genes involved in myogenic differentiation and cell cycle arrest. This study shows that bacterial-derived myoDNs serve as aptamers and are potential nucleic acid drugs directly targeting myoblasts.

cell biology↗