Language：English   Publishing type：Research paper (scientific journal)  

Exosomes (small extracellular vesicles: EVs) have attracted increasing attention from basic scientists and clinicians since they play important roles in cell-to-cell communication in various biological processes. Various features of EVs have been elucidated regarding their contents, generation and secretion mechanisms, and functions in inflammation, regeneration, and cancers. These vesicles are reported to contain proteins, RNAs, microRNAs, DNAs, and lipids. Although the roles of individual components have been rigorously studied, the presence and roles of glycans in EVs have rarely been reported. In particular, glycosphingolipids in EVs have not been investigated to date. In this study, the expression and function of a representative cancer-associated ganglioside, GD2, in malignant melanomas was investigated. Generally, cancer-associated gangliosides have been shown to enhance malignant properties and signals in cancers. Notably, EVs derived from GD2-expressing melanomas enhanced the malignant phenotypes of GD2-negative melanomas, such as cell growth, invasion, and cell adhesion, in a dose-dependent manner. The EVs also induced increased phosphorylation of signaling molecules such as EGF receptor and focal adhesion kinase. These results suggest that EVs released from cancer-associated ganglioside-expressing cells exert many functions that have been reported as a function of these gangliosides and regulate microenvironments, including total aggravation of heterogeneous cancer tissues, leading to more malignant and advanced cancer types.

DOI： 10.1038/s41598-023-31216-4

Open Access

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Radiation therapy for head and neck cancers is frequently associated with adverse effects on the surrounding normal tissue. Irreversible damage to radiation-sensitive acinar cells in the salivary gland (SG) causes severe radiation-induced xerostomia (RIX). Currently, there are no effective drugs for treating RIX. We investigated the efficacy of treatment with conditioned medium derived from stem cells from human exfoliated deciduous teeth (SHED-CM) in a mouse RIX model. Intravenous administration of SHED-CM, but not fibroblast-CM (Fibro-CM), prevented radiation-induced cutaneous ulcer formation (p < 0.0001) and maintained SG function (p < 0.0001). SHED-CM treatment enhanced the expression of multiple antioxidant genes in mouse RIX and human acinar cells and strongly suppressed radiation-induced oxidative stress. The therapeutic effects of SHED-CM were abolished by the superoxide dismutase inhibitor diethyldithiocarbamate (p < 0.0001). Notably, quantitative liquid chromatography-tandem mass spectrometry shotgun proteomics of SHED-CM and Fibro-CM identified eight proteins activating the endogenous antioxidant system, which were more abundant in SHED-CM than in Fibro-CM (p < 0.0001). Neutralizing antibodies against those activators reduced antioxidant activity of SHED-CM (anti-PDGF-D; p = 0.0001, anti-HGF; p = 0.003). Our results suggest that SHED-CM may provide substantial therapeutic benefits for RIX primarily through the activation of multiple antioxidant enzyme genes in the target tissue.

DOI： 10.1038/s41598-023-29176-w

Open Access

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Shikoku Society of Dental Research  

Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) effectively restores mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis. However, the underlying therapeutic mechanisms remain elusive. Here, we investigated the direct therapeutic effects of SHED-CM on inflamed primary condylar chondrocytes <i>in vitro</i>. Immunofluorescence staining revealed that interleukin-1β-stimulated chondrocytes showed increased expression of the catabolic marker inducible nitric oxide synthase (iNOS) and reduced expression of the anabolic marker aggrecan (ACAN). We found that SHEDCM treatment, but not conditioned medium from bone marrow mesenchymal stem cells (BMSCCM), effectively suppressed iNOS expression and elevated ACAN levels, indicating that SHEDCM converted the catabolic phenotype of inflamed chondrocytes to an anabolic phenotype. Liquid chromatography with tandem mass spectrometry analysis of SHED-CM and BMSC-CM identified eight proteins enriched in SHED-CM that are related to anti-inflammatory and/or chondrogenic processes. Of these proteins, the Wnt signal inhibitor secreted frizzled-related protein 1 (SFRP1) was the most abundantly enriched in SHED-CM. We found that treatment with the selective SFRP1 inhibitor WAY-316606 abolished the anti-catabolic and pro-anabolic effects of SHED-CM. Collectively, our study suggests that SHED-CM directly suppresses catabolism and promotes anabolic responses of inflamed primary condylar chondrocytes, which partially rely on SFRP1 function in SHED-CM. The direct action of SHED-CM may be useful to treat inflammatory cartilage diseases.

DOI： 10.20738/johb.35.2_52

CiNii Research

Other Link： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-19K18241/

Language：English   Publishing type：Research paper (scientific journal)  

Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.

DOI： 10.1007/s10719-022-10051-1

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

In neuropathic pain (NP), injury or diseases of the somatosensory system often result in highly debilitating chronic pain. Currently, there is no effective drug for the complete and definitive treatment of NP. We investigated the therapeutic potential of conditioned medium (CM) derived from stem cells from human exfoliated deciduous teeth (SHED-CM) against NP using a mouse partial sciatic nerve ligation (PSL) model. Abnormal pain sensation, such as tactile allodynia and hyperalgesia, can be caused by PSL. In the behavioral test, intravenous administration of SHED-CM greatly improved the PSL-induced hypersensitivity. We found that treatment with SHED-CM resulted in the recruitment of M2 macrophages in the injured sciatic nerve and ipsilateral L4/L5 dorsal root ganglion and suppressed microglial activation in the spinal cord. Notably, specific depletion of the anti-inflammatory M2 macrophages by mannosylated-Clodrosome markedly reduced the antinociceptive effect of SHED-CM. Intravenous administration of CM from M2 induced by SHED-CM (M2-CM) ameliorated the PSL-induced hypersensitivity. We found that M2-CM directly suppressed the expression of nociceptive receptors as well as proinflammatory mediators in Schwann cells. Taken together, our data suggest that SHED-CM ameliorates NP through the induction of the analgesic anti-inflammatory M2 macrophages. Thus, SHED-CM may be a novel therapeutic candidate for NP.

DOI： 10.3389/fphar.2022.745020

Open Access

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/cas.15032

Open Access

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Shikoku Society of Dental Research  

Inflammatory (M1-polarized) macrophages cause neuropathic pain (NP) after nerve injury through non-resolving neuroinflammation. However, increasing evidence suggests that converting M1 to anti-inflammatory M2 macrophages may rescue NP. In the present study, the therapeutic potential
of low-intensity pulsed ultrasound (LIPUS) was investigated in a partial sciatic nerve ligation (PSL)-
induced NP model.<br>
<b>Materials and Methods:</b> Abnormal pain sensation, such as tactile allodynia, was caused by PSL. Immediately after PSL induction, the mice were subjected to LIPUS treatment for 20 min/day for 7 days. LIPUS was used at an average intensity of 60 mW/cm² and a frequency of 1.5 MHz.<br>
<b>Results:</b> In the behavioral test, the LIPUS group showed a significant improvement in the PSL-induced hypersensitivity compared to the PSL group not exposed to LIPUS. We found an increasing number of M2 macrophages in the injured sciatic nerves after LIPUS exposure. LIPUS treatment decreased expression of pro-inflammatory microglial markers in spinal cord.<br>
<b>Conclusions:</b> Our data suggest that LIPUS has an anti-nociceptive effect by increasing anti-inflammatory M2 macrophage and may be a suitable therapeutic candidate for NP.

DOI： 10.20738/johb.34.1_11

CiNii Research

Other Link： https://search.jamas.or.jp/link/ui/2022185694

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.joca.2020.03.010

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PubMed

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Shikoku Society of Dental Research  

Radiation-induced skin ulceration is a frequent complication of radiotherapy for cancer treatment. Stem cells from human exfoliated deciduous teeth (SHEDs) can regenerate various tissues.
In this study, we investigated the impact of SHED-conditioned medium (SHED-CM) on radiation-induced skin injury. Mouse necks were locally irradiated with a single dose of 15 Gy of radiation. A week after the irradiation, most of the wild-type mice generated ulcer surrounded by severe erythema.
Intra-venous administration of SHED-CM effectively inhibited the ulcer formation. Histological examination revealed that SHED-CM treatment inhibited radiation-induced dermal thickness and epithelial hyperplasia. SHED-CM could be a useful treatment option for radiation-induced skin ulceration.

DOI： 10.20738/johb.33.1_1

CiNii Research

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

To analyze the binding specificity of a sialic acid-recognizing lectin, sialic acid-binding Ig-like lectin 7 (SIGLEC7), to disialyl gangliosides (GD3s), here we established GD3-expressing cells by introducing GD3 synthase (GD3S or ST8SIA1) cDNA into a colon cancer cell line, DLD-1, that expresses no ligands for the recombinant protein SIGLEC7-Fc. SIGLEC7-Fc did not recognize newly-expressed GD3 on DLD-1 cells, even though GD3 was highly expressed, as detected by an anti-GD3 antibody. Because milk-derived GD3 could be recognized by this fusion protein when incorporated onto the surface of DLD-1 cells, we compared the ceramides in DLD-1-generated and milk-derived GD3s to identify the SIGLEC7-specific GD3 structures on the cell membrane, revealing that SIGLEC7 recognizes only GD3-containing regular ceramides but not phytoceramides. This was confirmed by knockdown/knockout of the sphingolipid delta(4)-desaturase/C4-monooxygenase (DES2) gene, involved in phytoceramide synthesis, disclosing that DES2 inhibition confers SIGLEC7 binding. Furthermore, knocking out fatty acid 2-hydroxylase also resulted in the emergence of SIGLEC7 binding to the cell surface. To analyze the effects of binding between SIGLEC7 and various GD3 species on natural killer function, we investigated cytotoxicity of peripheral blood mononuclear cells from healthy donors toward GD3S-transfected DLD-1 (DLD-1-GD3S) cells and DLD-1-GD3S cells with modified ceramides. We found that cytotoxicity is suppressed in DLD-1-GD3S cells with dehydroxylated GD3s. These results indicate that the ceramide structures in glycosphingolipids affect SIGLEC7 binding and distribution on the cell surface and influence cell sensitivity to killing by SIGLEC7-expressing effector cells.

DOI： 10.1074/jbc.RA118.007083

Open Access

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

B4GALNT1 is an enzyme essential for the synthesis of complex gangliosides, whose absence leads to progressive neurodegeneration with aging in mice. Recently, eleven cases of hereditary spastic paraplegia with mutation in the coding region of B4GALNT1 were reported. However, changes in the enzymatic activity of their products have never been studied. We have constructed expression vectors for individual mutant cDNAs, and examined their activities by cell-free in vitro enzyme assays, and flow cytometry of cells transfected with their expression vectors. Among them, almost all mutant genes showed the complete loss of B4GALNT1 activity in both the in vitro enzyme assays and flow cytometry. Two mutants exceptionally showed weak activity. One of them, M4, had a mutation at amino acid 228 with a premature termination codon. Interestingly, the intensity of fluorescence of GM2 measured by flow cytometry was equivalent between the WT and M4 mutant, although the positive cell population was relatively small in M4. Western immunoblotting of cell lysates from transfectants with cDNA plasmids revealed 67-kDa bands except those containing premature termination codons or frame-shift mutation. Taken together with the clinical findings of patients, loss of enzyme activity may be responsible for the clinical features of hereditary spastic paraplegia, whereas the intensity of neurological disorders was relatively milder than expected. These clinical features of patients including those with male hypogonadism are very similar to the abnormal phenotypes detected in B4galnt1-deficient mice.

DOI： 10.1016/j.neuroscience.2018.11.034

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Language：English   Publishing type：Research paper (scientific journal)  

Ganglioside GD2 is specifically expressed in small-cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme-mediated activation of radical sources combined with mass spectrometry in GD2+ SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2+ SCLC cells were enhanced by glutamine uptake, and were suppressed by L-γ-glutamyl-p-nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid-enriched microdomain/rafts in GD2+ SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.

DOI： 10.1111/cas.13448

Open Access

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PubMed

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Gangliosides play roles in the regulation of cell signaling that are mediated via membrane microdomains, lipid rafts. In this review, functions of gangliosides in the maintenance of nervous systems with a focus on regulation of inflammation and neurodegeneration are addressed. During analyses of various ganglioside-lacking mutant mice, we demonstrated that nervous tissues exhibited inflammatory reactions and subsequent neurodegeneration. Among inflammation-related genes, factors of the complement system showed up-regulation with aging. Analyses of architectures and compositions of lipid rafts in nervous tissues from these mutant mice revealed that dysfunctions of complement regulatory proteins based on disrupted lipid rafts were main factors to induce the inflammatory reactions resulting in neurodegeneration. Ganglioside changes in development and senescence, and implication of them in the integrity of cell membranes and cellular phenotypes in physiological and pathological conditions including Alzheimer disease have been summarized. Novel directions to further analyze mechanisms for ganglioside functions in membrane microdomains have been also addressed.

DOI： 10.1016/bs.pmbts.2018.01.009

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbagen.2017.06.007

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/glycob/cww049

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PubMed

Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M114.635755

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1523/JNEUROSCI.4088-14.2015

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PubMed

Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

DOI： 10.1007/978-4-431-54841-6_121

Scopus

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Web of Science

Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

Scopus

Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

DOI： 10.1007/978-4-431-54240-7_34

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M113.513192

Open Access

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Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/glycob/cwt054

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Scopus

PubMed

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/febs.12034

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CiNii Research

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/pmic.201200279

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.csda.2011.01.002

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Language：English   Publishing type：Research paper (other academic)   Publisher：(公社)日本生化学会  

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)   Publisher：日本計算機統計学会  

DOI： 10.20551/jscssymo.22.0_53

CiNii Research

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

Language：Japanese   Publisher：(公社)日本生化学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：(公社)日本生化学会  

Language：Japanese   Publisher：(公社)日本生化学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：(公社)日本生化学会  

Language：Japanese   Publisher：(公社)日本生化学会  

Language：Japanese   Publisher：(株)羊土社  

Language：Japanese   Publisher：(公社)日本生化学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：(公社)日本生化学会  

Language：English   Publisher：日本癌学会  

Language：Japanese   Publisher：(公社)日本生化学会  

Language：English   Publisher：(公社)日本生化学会  

Language：English   Publisher：日本癌学会