Yasuhide Miyamoto

Osaka University, Ōsaka-shi, Osaka-fu, Japan

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Publications (35)112.29 Total impact

  • Masahiko Yabu, Hiroaki Korekane, Yasuhide Miyamoto
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    ABSTRACT: O-glycans are suitable targets as novel and useful tumor markers. The structures of O-glycans in human sera from four healthy controls were precisely analyzed to obtain the reference O-glycan database. O-glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine and identified using two dimensional mapping, enzymatic digestion and mass spectrometry together with methanolysis and the use of newly synthesized sulfated oligosaccharides as standards. O-glycans, present at more than 0.01% of total O-glycans, were analyzed, and 18 kinds of acidic and 2 kinds of neutral glycans were identified. NeuAcα2-3Galβ1-3GalNAc (61-64 %), NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAc (15-26 %), and Galβ1-3GalNAc (6-14%) were major components while other sialylated glycans, Galβ1-3(NeuAcα2-6)GalNAc, Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAc, and NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-3)GalNAc were relatively minor components, accounting for around 1-2%. Very minor glycans accounting for around 0.01-0.1% of total, include 1) the neutral glycan, Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc, 2) sialylated glycans, having sialyl Tn antigen, agalacto, and trisialylated structures, 3) fucosylated glycans forming blood type H antigen, blood type A antigen, blood type B antigen, Lewis X antigen and sialyl Lewis X antigen, 4) sulfated glycans, having 6-sulfo and 3'-sulfo structures. Two kinds of clinically applied tumor markers namely sialyl Tn antigen and sialyl Lewis X antigen in healthy controls sera were revealed to be present at around 0.1-0.2% of total. However, other markers such as CA19-9 and DU-PAN-2 were not found, suggesting the relative amounts of these glycans to be below 0.01%. These detailed O-glycan profiles will help to find novel carbohydrate tumor markers.
    Glycobiology 03/2014; · 3.54 Impact Factor
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    ABSTRACT: Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc-transfer activity toward N-linked and O-mannosyl glycan core structures and its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) as evidenced by mass spectrometry and on the knocking down and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide-sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3-knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.
    Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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    ABSTRACT: Hemagglutinating virus of Japan-envelope (HVJ-E) is a drug delivery vector based on inactivated Sendai virus. Recently, anti-tumor activities were found for HVJ-E itself, and clinical trials of HVJ-E for some malignant tumors are now ongoing. We investigated the in vitro and in vivo antitumor effects of HVJ-E against neuroblastoma, which is one of the most common malignant solid tumors in childhood. The sensitivity of human neuroblastoma cell lines to HVJ-E correlated with the expression level of gangliosides, Sialylparagloboside (SPG) and GD1a, receptors for HVJ. Among the cell lines, SK-N-SH was the most sensitive to HVJ-E in vitro and the total SPG and GD1a expression was the highest. Complete eradication of subcutaneous tumors derived from SK-N-SH cells was achieved by intratumoral injection of HVJ-E in SCID mice, and no recurrence was observed for more than 300 days after HVJ-E inoculation. On the other hand, NB1 cells expressed the lowest amount of GD1a and SPG, and were resistant to HVJ-E in vitro. The expression of GD1a increased by 13-cis retinoic acid (13cRA), which is a therapeutic drug for high risk neuroblastoma, thus leading to an improved sensitivity to HVJ-E in vitro. Only growth inhibition of the subcutaneous tumors derived from NB1 cells was achieved by HVJ-E in the SCID mice, but the combination of 13cRA and HVJ-E could achieve a partial eradication of the xenograft, and also led to an improved prognosis. In conclusion, HVJ-E is a promising therapeutic modality for neuroblastoma, and 13cRA can be used as an adjuvant to HVJ-E.
    Cancer Science 11/2012; · 3.48 Impact Factor
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    ABSTRACT: We previously reported on the accumulation of a substantial amount of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans in human pancreatic cancer cells (Yabu M, Korekane H, Takahashi H, Ohigashi H, Ishikawa O, Miyamoto Y. 2012. Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers. Glycoconjugate J Epub ahead of print). In the present paper, we further extend our cancer glycomic study of human prostate cancer. Specifically, we demonstrate that, in addition to the free Neu5Ac-containing N-glycans, significant amounts of free deaminoneuraminic acid (KDN)-containing N-glycans had accumulated in the prostate cancer tissues from four out of five patients. Indeed in one of the four cases, the free KDN-glycans accumulated as major components in prostate cancer tissue. The structures of the KDN-containing free oligosaccharides were analyzed by a variety of methods. Specifically we used fluorescent labeling with aminopyridine combined with two dimensional mapping, KDNase digestion and mass spectrometry to facilitate identification. The analysis also utilized newly synthesized KDN-linked oligosaccharides as standards. The prostate-specific glycans were composed of five species having the following sequence, KDN-Gal-GlcNAc-Man-Man-GlcNAc (α2,6-KDN-linked glycans being the dominant form). The most abundant free KDN-containing N-glycan was KDNα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc followed by KDNα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal chemical evidence for the occurrence of KDN-glycoconjugates in human tissues together with their detailed structures. These oligosaccharides might be developed as tumor markers, especially for prostate cancer.
    Glycobiology 09/2012; · 3.54 Impact Factor
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    ABSTRACT: We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.
    Glycoconjugate Journal 08/2012; · 1.88 Impact Factor
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    ABSTRACT: The expression of gangliosides is often associated with cancer progression. Sialyltransferases have received much attention in terms of their relationship with cancer because they modulate the expression of gangliosides. We previously demonstrated that GD1a production was high in castration-resistant prostate cancer cell lines, PC3 and DU145, mainly due to their high expression of β-galactoside α2,3-sialyltransferase (ST3Gal) II (not ST3Gal I), and the expression of both ST3Gals was regulated by NF-κB, mainly by RelB. We herein demonstrate that GD1a was produced in abundance in cancerous tissue samples from human patients with hormone-sensitive prostate cancers as well as castration-resistant prostate cancers. The expression of ST3Gal II was constitutively activated in castration-resistant prostate cancer cell lines, PC3 and DU145, because of the hypomethylation of CpG island in its promoter. However, in androgen-depleted LNCap cells, a hormone-sensitive prostate cancer cell line, the expression of ST3Gal II was silenced because of the hypermethylation of the promoter region. The expression of ST3Gal II in LNCap cells increased with testosterone treatment because of the demethylation of the CpG sites. This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter. Therefore, in hormone-sensitive prostate cancers, the production of GD1a may be regulated by androgen. This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter.
    PLoS ONE 01/2012; 7(2):e31234. · 3.73 Impact Factor
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    Glycoconjugate Journal 06/2011; 28(6):437. · 1.88 Impact Factor
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    ABSTRACT: Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans.
    Glycoconjugate Journal 05/2011; 28(3-4):183-96. · 1.88 Impact Factor
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    ABSTRACT: We have precisely analyzed the structures of glycosphingolipids of human cancer cells and normal epithelial cells using several methods, including enzymatic release of carbohydrate moieties, fluorescent labeling, and identification using 2D mapping, enzymatic digestion, and mass spectrometry. These analyses enabled the identification of novel tumor-associated carbohydrate antigens that can be used to elucidate the involvement of carbohydrates in cancer malignancy and could act as candidate tumor markers. In our previous study, we identified a novel glycosphingolipid that accumulates in colon cancer cells, NeuAcα2-6(Fucα1-2)Galβ1-4GlcNAcβ1-3Galβ1-4Glc (α2-6 sialylated type 2H, ST2H). Here, structural analyses of cancer cells and normal epithelial cells from 60 colorectal and five pancreatic cancer patients, including four and two Lewis-negative individuals, respectively, reveal the presence of an additional novel glycosphingolipid, NeuAcα2-6(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc (α2-6 sialylated type 1H, ST1H). ST2H was found in colorectal and pancreatic cancer cells from about half of the cases. Unlike ST2H, ST1H was found in cancer cells from three out of six Lewis-negative patients (i.e., two cases of colorectal and one case of pancreatic cancer). However, the moiety was not found in normal epithelial cells or cancer cells from 59 Lewis-positive patients. These findings suggest that the accumulation of this carbohydrate antigen occurs predominantly in cancer cells of Lewis-negative patients. When the ST1H epitope is also carried on mucins as well as glycosphingolipids, this epitope is a promising tumor marker candidate, especially for Lewis-negative individuals.
    Glycobiology 12/2010; 20(12):1594-606. · 3.54 Impact Factor
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    ABSTRACT: Gangliosides are sialic acid-containing glycosphingolipids that are associated with tumor malignancy and progression. Among the enzymes required for the production of gangliosides, sialyltransferases have received much attention in terms of their relationship with cancer. In our previous report, ganglioside GD1a and sialyl paragloboside (SPG), a neolacto-series ganglioside, were much more abundant in PC3 and DU145 cells, castration-resistant prostate cancer cells, as compared with hormone-sensitive prostate cancer cells and normal prostate epithelium. GD1a is synthesized from GM1 by α2,3 sialyltransferase (ST3Gal) I and mainly by ST3Gal II. The enzyme to synthesize SPG is ST3Gal VI. The high production of GD1a and SPG in castration-resistant prostate cancer cells was correlated with the high expression of ST3Gal II and VI, respectively. The expression of ST3Gal I and II was mildly induced by phorbol-12-myristate-13-acetate (PMA), and PMA-induced expression of ST3Gal I and ST3Gal II was inhibited by NF-κB decoy oligodeoxynucleotides (ODN) but not by AP-1 decoy ODN. Among the five mammalian homologs of the NF-κB family, RelB RNAi most effectively inhibited the expression of ST3Gal I and ST3Gal II. The expression of ST3Gal VI was also most effectively inhibited by RelB RNAi. The amount of GD1a and SPG was significantly reduced by RelB siRNA treatment in PC3 cells. Thus, the production of GD1a and SPG in castration-resistant prostate cancer cells was indirectly controlled by NF-κB, mainly by RelB, through the transcriptional regulation of ST3Gal I, II, and VI.
    International Journal of Cancer 12/2010; 129(8):1838-47. · 6.20 Impact Factor
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    ABSTRACT: The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.
    Journal of Biochemistry 09/2010; 148(3):359-70. · 3.07 Impact Factor
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    ABSTRACT: A lateral pelvic lymph node dissection (LPLD) for lower rectal cancer may be beneficial for a limited number of patients. If sentinel node (SN) navigation surgery could be applied to lower rectal cancer, then unnecessary LPLDs could be avoided. The aim of this study was to investigate the feasibility of lateral region SN biopsy by means of indocyanine green (ICG) visualized with a near-infrared camera system (Photodynamic Eye, PDE). This study investigated the existence of a lateral region SN in 25 patients with lower rectal cancer. ICG was injected around the tumor, and the lateral pelvic region was observed with PDE. With PDE, the lymph nodes and lymph vessels that received ICG appeared as shining fluorescent spots and streams in the fluorescence image. This allowed the detection of not only tumor-negative SNs but also tumor-positive SNs as shining spots. The lateral SNs were detected in 6 of 6 T1 and T2 diseases and 17 of 19 T3 diseases. The lateral SNs were successfully identified in 23 (92%) of the 25 patients. The mean number of lateral SNs per patients was 2.1. Of the 23 patients, 6 patients underwent LPLD. Of the 3 patients who had a tumor-negative SN, all dissected lateral non-SNs were negative in all 3 cases. We could detect the lateral SNs, not only in T1 and T2 disease, but also in T3 disease. Although this is only a preliminary study, the detection of lateral SNs in lower rectal cancer by means of the ICG fluorescence imaging system is considered to be a promising technique that may be used for determining the indications for performing LPLD.
    Annals of Surgical Oncology 09/2009; 17(1):144-51. · 4.12 Impact Factor
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    ABSTRACT: The structures of glycosphingolipids from highly purified colorectal cancer cells and normal colorectal epithelial cells of 16 patients have been analyzed in fine detail (Misonou Y, Shida K, Korekane H, Seki Y, Noura S, Ohue M, Miyamoto Y. 2009. Comprehensive Clinico-Glycomic Study of 16 Colorectal Cancer Specimens: Elucidation of aberrant glycosylation and ts mechanistic causes in colorectal cancer cells. J Proteome Res. 8:2990-3005). Further structural analyses demonstrated that colon cancer cells from two patients accumulated unusual glycosphingolipids which were not observed in either colorectal cancer cells or normal colorectal epithelial cells from the other patients. Mass spectrometry analyses revealed that the unusual structures include sulfated oligosaccharides. The structures of the glycosphingolipids of the cancer cells from these two cases were analyzed by methods which include enzymatic release of carbohydrate moieties, fluorescent labeling with aminopyridine and identification using two-dimensional mapping, enzymatic digestion and mass spectrometry together with methanolysis, and the use of newly synthesized sulfo-fucosylated oligosaccharides as standards. The colon cancer cells from one of the patients demonstrate a variety of oligosaccharides as major components which are sulfated at the C6 position of subterminal GlcNAc and at C3 positions of terminal galactose with or without sialylation or fucosylation. These include 6-sulfo Le(x), 6'-sialyl 6-sulfo lactosamine, and 3'-sialyl 6-sulfo Le(x), in addition to sialylated or fucosylated derivatives of type-1 and type-2 hybrid oligosaccharides. The colon cancer cells from the other patient have two kinds of sulfated oligosaccharides, a 6-sulfo Le(x) structure and a 3'-sulfo Le(x) structure, as minor components. Taking into consideration the clinical features of the two patients, the biological significance of sulfated glycosphingolipids on cancer cells is discussed.
    Glycobiology 07/2009; 19(9):1018-33. · 3.54 Impact Factor
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    ABSTRACT: The clear delineation between tumor and normal tissue is ideal for real-time surgical navigation imaging. We present a novel indocyanine green (ICG) fluorescence imaging technique to visualize hepatocellular carcinoma (HCC). Ten patients with solitary HCC underwent hepatectomy between February and September 2007 at Osaka Medical Center for Cancer and Cardiovascular Diseases. ICG had been injected intravenously several days before surgery at a dose of 0.5 mg/kg body weight. After laparotomy, the liver was inspected with intraoperative ultrasonography (IOUS), and then with a near-infrared (NIR) fluorescence imaging system (PDE; Hamamatsu Photonics K.K. Hamamatsu, Japan). All the 10 primary tumors showed bright fluorescent signals and could be completely removed with negative margins under the guide of PDE. In four cases (40.0%), new HCC nodules that were not detected by use of any preoperative examinations including IOUS were detected by PDE. These newly identified HCC nodules were very small in size and most of the tumors were well-differentiated HCCs. This novel technique is simple and safe, and is therefore considered to be a promising tool for routine intraoperative imaging during a hepatic resection and further clinical exploration for HCC.
    Journal of Surgical Oncology 04/2009; 100(1):75-9. · 2.64 Impact Factor
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    ABSTRACT: The structures of neutral and acidic glycosphingolipids from both normal colorectal epithelial cells and colorectal cancer cells, which were highly purified with the epithelial cell marker CD326, have been analyzed. The analysis was performed on samples from 16 patients. The carbohydrate moieties from glycosphingolipids were released by endoglycoceramidase II, labeled by pyridylamination, and identified using two-dimensional mapping and mass spectrometry. The structures from normal colorectal epithelial cells are characterized by dominant expression of neutral type-1 chain oligosaccharides. Three specific alterations were observed in malignant transformation; increased ratios of type-2 oligosaccharides, increased alpha2-3 and/or alpha2-6 sialylation and increased alpha1-2 fucosylation. Although the degree of alteration varies case to case, we found that two characteristic alterations tend to be associated with clinical features. One is a shift from type-1 dominant normal colorectal epithelial cells to type-2 dominant colorectal cancer cells. This shift was found in 5 patients having hepatic metastasis. The other is specific elevation of alpha2-3 sialylation observed in 2 cases exhibiting high serum levels of CA19-9. Examination of the activities of the related glycosyltransferases revealed that while some alterations could be accounted for by changes in the activities of related glycosyltransferases others could not. Although the number of cases analyzed is small, these findings provide valuable information which will help in the elucidation of the mechanism of synthesis of aberrant glycosylation and its involvement in cancer malignancy.
    Journal of Proteome Research 04/2009; 8(6):2990-3005. · 5.06 Impact Factor
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    ABSTRACT: Hormone-refractory prostate cancer is one of the intractable human cancers in the world. Here, we examined the direct tumor-killing activity of inactivated Sendai virus particle [hemagglutinating virus of Japan envelope (HVJ-E)] through induction of Type I interferon (IFN) in the hormone-resistant human prostate cancer cell lines PC3 and DU145. Preferential binding of HVJ-E to PC3 and DU145 over hormone-sensitive prostate cancer cell and normal prostate epithelium was observed, resulting in a number of fused cells. After HVJ-E treatment, a number of IFN-related genes were up-regulated, resulting in Type I IFN production in PC3 cells. Then, retinoic acid-inducible gene-I (RIG-I) helicase which activates Type I IFN expression after Sendai virus infection was up-regulated in cancer cells after HVJ-E treatment. Produced IFN-alpha and -beta enhanced caspase 8 expression via Janus kinases/Signal Transducers and Activators of Transcription pathway, activated caspase 3 and induced apoptosis in cancer cells. When HVJ-E was directly injected into a mass of PC3 tumor cells in SCID (severe combined immunodeficiency) mice, a marked reduction in the bulk of each tumor mass was observed and 85% of the mice became tumor-free. Although co-injection of an anti-asialo GM1 antibody with HVJ-E into each tumor mass slightly attenuated the tumor suppressive activity of HVJ-E, significant suppression of tumor growth was observed even in the presence of anti-asialo GM1 antibody. This suggests that natural killer cell activation made small contribution to tumor regression following HVJ-E treatment in hormone-resistant prostate cancer model in vivo. Thus, HVJ-E effectively targets hormone-resistant prostate cancer by inducing apoptosis in tumor cells, as well as activating anti-tumor immunity.
    International Journal of Cancer 01/2009; 124(10):2478-87. · 6.20 Impact Factor
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    ABSTRACT: The structures of acidic glycosphingolipids in colon adenocarcinoma have been analyzed extensively using a number of conventional methods, such as thin-layer chromatography and methylation analysis, and a variety of acidic glycosphingolipids present in the tissues have been reported. However, because of a number of limitations in the techniques used in previous studies in terms of resolution, quantification, and sensitivity, we employed a different method that could be applied to small amounts of tissue. In this technique, the carbohydrate moieties of acidic glycosphingolipids from approximately 20mg of colon adenocarcinoma were released by endoglycoceramidase II and were labeled by pyridylamination. They were separated and structurally characterized by a two-dimensional HPLC mapping technique, electrospray ionization tandem mass spectrometry (ESI-MS/MS), and enzymatic cleavage. A total of 22 major acidic glycosphingolipid structures were identified, and their relative quantities were revealed in detail. They are composed of 1 sulfated (SM3), 1 lacto-series (SLe(a)), 6 kinds of ganglio-series, and 14 kinds of neolacto-series glycosphingolipids. They include most of the acidic glycosphingolipids previously reported to be present in the tissues and two previously unknown fucogangliosides sharing the same terminal structure: NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, and NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3-Galbeta1-4Glc. Thus, this highly sensitive, high-resolution analysis enabled the identification of novel structures of acidic glycosphingolipids from small amounts of already comprehensively studied cancerous tissues. This method is a powerful tool for microanalysis of glycosphingolipid structures from small quantities of cancerous tissues and should be applicable to different types of malignant tissues.
    Analytical Biochemistry 06/2007; 364(1):37-50. · 2.58 Impact Factor
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    ABSTRACT: Laser microdissection (LMD) is a recent development that enables the isolation of specific cell populations from tissue sections. This study focuses on the potential of LMD as a tool in cancer glycomics using colon cancer as a model. LMD was performed on hematoxylin and eosin stained frozen tissue sections. Tumor cells and normal epithelial cells were selectively microdissected. N-Glycans from the LMD- and the bulk tissue-derived samples were liberated by hydrazinolysis and then labeled with 2-aminopyridine. After sialidase digestion, the resulting asialo-N-glycans were analyzed by normal and reversed phase HPLC combined with mass spectrometry. Comparison of the various N-glycan profiles with the aid of LMD identified seven characteristic N-glycans with significantly different expression profiles between normal and cancerous cells that could not be detected by conventional analysis. Thus, LMD is a potent and useful tool for analyzing variations in the expression of N-glycans by overcoming the problem of tissue sample heterogeneity.
    Biochemical and Biophysical Research Communications 02/2007; 352(3):579-86. · 2.41 Impact Factor
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    ABSTRACT: 3-Deoxyglucosone (3-DG), a dicarbonyl compound produced by glycation, plays a role in the modification and cross-linking of long-lived proteins. We synthesized [3H]3-DG from [3H]glucose and developed an internalization assay system using HPLC to examine its cellular metabolism. When smooth muscle cells or human umbilical vein endothelial cells were incubated with [3H]3-DG, it was found that [3H]3-DG was internalized by cells in a time dependent manner. The rate of internalization was reduced when the cells were incubated at 4 degrees C or treated with phenylarsine oxide (PAO). By monitoring [3H]3-DG taken up by cells, it was confirmed that 3-DG is reduced to 3-deoxyfructose (3-DF) and that this reaction was inhibited by an aldo-keto reductase inhibitor (ARI). The presence of 3-DG led to an increase in reactive oxygen species levels in the cells and subsequent apoptosis, and the effect was enhanced by pretreatment with ARI. These results suggest that 3-DG is internalized by cells and reduced to 3-DF by aldo-keto reductases, and that the internalized 3-DG is responsible for the production of intracellular oxidative stress.
    Journal of Biochemistry 03/2006; 139(2):245-53. · 3.07 Impact Factor
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    ABSTRACT: A large body of evidence suggests that carbonyl compounds induce intracellular signaling by increasing oxidative stress in the cell; however, the mechanisms involved have not been fully described. The focus of our research is on the pathway in which antioxidative enzymes are modified and inactivated by carbonyl compounds, resulting in the accumulation of active oxygen species in the cell. A common pathway appears to exist for cellular signaling evoked by nitroxidative stress. It could be concluded that some glycoxidative stress and nitroxidative stress cause intracellular signaling via similar mechanisms. The elucidation of the pathway for extracellular stress-induced reactive oxygen species (ROS) production would be important for our understanding of the role of ROS as signaling molecules.
    Annals of the New York Academy of Sciences 07/2005; 1043:521-8. · 4.38 Impact Factor

Publication Stats

499 Citations
112.29 Total Impact Points

Institutions

  • 2009–2012
    • Osaka University
      • Division of Gene Therapy Science
      Ōsaka-shi, Osaka-fu, Japan
  • 2007–2012
    • Osaka Medical Center for Cancer and Cardiovascular Diseases
      Ōsaka, Ōsaka, Japan
  • 2002–2006
    • Osaka City University
      • • Department of Biochemistry
      • • Graduate School of Medicine
      Ōsaka-shi, Osaka-fu, Japan
  • 2005
    • Hyogo College of Medicine
      • Department of Biochemistry
      Nishinomiya, Hyogo-ken, Japan