The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Keiko Furukawa - One of the best experts on this subject based on the ideXlab platform.
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tnfα signal and camp mediated signals oppositely regulate melanoma associated Ganglioside GD3 synthase gene in human melanocytes
Scientific Reports, 2019Co-Authors: Rika Takeuchi, Koichi Furukawa, Mariko Kambe, Maiko Miyata, Orie Tajima, Upul Jeyadevan, Keiko FurukawaAbstract:Analyses of expression and regulation of Ganglioside synthases in melanocytes are important to understand roles of Gangliosides in melanomagenesis. In this study, we analyzed the expression and regulatory mechanisms of glycosyltransferase genes responsible for Ganglioside synthesis in normal melanocytes. We reported previously that culture supernatants of UVB-irradiated keratinocytes induced upregulation of Ganglioside GD3 synthase gene in melanocytes, and mainly TNFα was responsible for it. Then, we found that elimination of dibutyryl cyclic AMP and IBMX from the medium also resulted in upregulation of the GD3 synthase gene. The addition of α-melanocyte-stimulating hormone which increases cAMP, to the medium led to a significant reduction in the GD3 synthase gene expression level, and a PKA inhibitor enhanced the GD3 synthase gene level. These results suggest that signals mediated via TNFα and cAMP oppositely regulate GD3 synthase gene expression in melanocytes. The results of an IKK inhibitor indicate the possibility that TNFα induces GD3 synthase gene expression via NF-κB signaling in melanocytes. When melanoma cells were treated by these factors, no fluctuation in the GD3 synthase gene expression level was observed, although an IKK inhibitor significantly suppressed it, suggesting that Ganglioside synthase genes are regulated in distinct manners between melanocytes and melanomas.
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enhancement of malignant properties of human glioma cells by Ganglioside GD3 gd2
International Journal of Oncology, 2018Co-Authors: Taiji Iwasawa, Keiko Furukawa, Yuki Ohkawa, Yuhsuke Ohmi, Robiul H Bhuiyan, Pu Zhang, Hiroyuki Momota, Toshihiko Wakabayashi, Koichi FurukawaAbstract:Sialic acid-containing glycosphingolipids, Gangliosides, are considered as cancer associated antigens in neuro-ectoderm-derived tumors such as melanomas and neuroblastomas. In particular, Gangliosides GD3 and GD2 are expressed in human gliomas. It has been reported that their expression levels increase along with increased malignant properties. However, the implication of GD3/GD2 in human glioma cells has never been clarified, at least to the best of our knowledge. In this study, we introduced the cDNA of GD3 synthase (GD3S)(ST8SIA1) into a glioma cell line, U-251MG, that expresses neither GD3 nor GD2, thereby establishing transfectant cells U-251MG-GD3S(+) expressing high levels of GD3 and GD2 on the cell surface. In these U-251MG‑GD3S(+) cell lines, signaling molecules such as Erk1/2, Akt, p130Cas, paxillin and focal adhesion kinase were activated, leading to the enhancement of invasion activity and motility. It was then demonstrated that the U-251MG-GD3S(+) cells could proliferate under culture conditions with low or no serum concentrations without undergoing cell cycle arrest by escaping the accumulation of p16 and p21. All these results suggested that GD3 and GD2 highly expressed in gliomas confer increased invasion and mobility, cell growth abilities under low serum conditions, and increased ratios of the S-G2/M phase in the cell cycle.
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neogenin defined as a GD3 associated molecule by enzyme mediated activation of radical sources confers malignant properties via intracytoplasmic domain in melanoma cells
Journal of Biological Chemistry, 2016Co-Authors: Kei Kaneko, Keiko Furukawa, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Ohmi, Norihiro Kotani, Koichi Honke, Mitsutaka Ogawa, Tetsuya Okajima, Koichi FurukawaAbstract:To investigate mechanisms for increased malignant properties in malignant melanomas by Ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.
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a therapeutic trial of human melanomas with combined small interfering rnas targeting adaptor molecules p130cas and paxillin activated under expression of Ganglioside GD3
Biochimica et Biophysica Acta, 2016Co-Authors: Yusuke Makino, Keiko Furukawa, Yuki Ohkawa, Yuhsuke Ohmi, Kazunori Hamamura, Hideyuki Nakashima, Yoshifumi Takei, Robiul H BhuiyanAbstract:We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of Ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 μM siRNA, 25 μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.
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Ganglioside GD3 enhances invasiveness of gliomas by forming a complex with platelet derived growth factor receptor α and yes kinase
Journal of Biological Chemistry, 2015Co-Authors: Yuki Ohkawa, Keiko Furukawa, Noboru Hashimoto, Norihiro Kotani, Koichi Honke, Hiroyuki Momota, Akira Kato, Yusuke Tsuda, Akio Suzumura, Yuhsuke OhmiAbstract:There have been a few studies on the Ganglioside expression in human glioma tissues. However, the role of these Gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.
Senitiroh Hakomori - One of the best experts on this subject based on the ideXlab platform.
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functional role of glycosphingolipids in contact inhibition of growth in a human mammary epithelial cell line
FEBS Letters, 2017Co-Authors: Xiaohua Huang, Nathan Schurman, Kazuko Handa, Senitiroh HakomoriAbstract:We have demonstrated previously the involvement of certain glycosphingolipids (GSLs) in “contact inhibition” (dependent on cell-to-cell contact) of cell growth. Here, we examined the roles of specific GSLs in contact inhibition of the human epithelial cell line MCF10A. Contact-inhibited cells show increased expression of the Ganglioside GD3 and the globo-series GSL Gb3, and of the mRNAs for the corresponding sialyltransferases ST8SIA1 (GD3 synthase) and galactosyltransferase A4GALT (Gb3 synthase). siRNA knockdown of ST8SIA1 and/or A4GALT significantly suppress contact inhibition. Exogenous addition of GD3 or Gb3 inhibits proliferation of low-density cells. Our findings suggest that GSLs play functional roles in contact inhibition of these cells and that Merlin/NF2, a tumor suppressor protein, is involved in the GSL function. This article is protected by copyright. All rights reserved.
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functional role of glycosphingolipids in contact inhibition of growth in a human mammary epithelial cell line
FEBS Letters, 2017Co-Authors: Xiaohua Huang, Nathan Schurman, Kazuko Handa, Senitiroh HakomoriAbstract:We have demonstrated previously the involvement of certain glycosphingolipids (GSLs) in 'contact inhibition' (dependent on cell-to-cell contact) of cell growth. Here, we examined the roles of specific GSLs in contact inhibition of the human epithelial cell line MCF10A. Contact-inhibited cells show increased expression of the Ganglioside GD3 and the globo-series GSL Gb3, and of the mRNAs for the corresponding sialyltransferases ST8SIA1 (GD3 synthase) and galactosyltransferase A4GALT (Gb3 synthase). siRNA knockdown (KD) of ST8SIA1 and/or A4GALT significantly suppresses contact inhibition. Exogenous addition of GD3 or Gb3 inhibits proliferation of low-density cells. Our findings suggest that GSLs play functional roles in contact inhibition of these cells and that Merlin/NF2, a tumor suppressor protein, is involved in the GSL function.
Nobuo Hanai - One of the best experts on this subject based on the ideXlab platform.
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therapeutic potential of chimeric anti Ganglioside GD3 antibody km871 antitumor activity in xenograft model of melanoma and effector function analysis
Cancer Immunology Immunotherapy, 2000Co-Authors: Junji Kanazawa, Kenya Shitara, So Ohta, Nobuo Hanai, Fumiko Fujita, Masahide Fujita, Shiro Akinaga, Masami OkabeAbstract:KM871 is a chimeric antibody recognizing Ganglioside GD3, which is one of the major Gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model.
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a mouse human chimeric anti Ganglioside GD3 antibody with enhanced antitumor activities
Cancer Immunology Immunotherapy, 1993Co-Authors: Kenya Shitara, Yoshihisa Kuwana, Kazuyasu Nakamura, Yuko Tokutake, So Ohta, Hiromasa Miyaji, Mamoru Hasegawa, Nobuo HanaiAbstract:Ganglioside GD3, which is one of the major Gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, κ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human κ and γ1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.
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antitumor effects of a novel monoclonal antibody with high binding affinity to Ganglioside GD3
Cancer Immunology Immunotherapy, 1993Co-Authors: So Ohta, Yuko Tokutake, Ayumi Honda, Hajime Yoshida, Nobuo HanaiAbstract:Ganglioside GD3, which is one of the major Gangliosides expressed on the cell surface human tumors of neuroectodermal origin, has been studied as a target molecule for passive immunotherapy. We established ten kinds of anti-GD3 monoclonal antibodies (mAb) of the mouse IgG3 subclass by immunization with purified GD3 and melanoma cells. One of the established mAb, KM641, showed major reactivity with GD3 and minor reactivity with GQ1b out of 11 common Gangliosides in an enzymelinked immunosorbent assay. Immunostaining of Gangliosides, separated on thin-layer chromatography plates, using KM641 revealed that most of the melanoma cell lines contained immunoreactive GD3 and GD3-lactone at a high level, but only the adrenal gland and the urinary bladder out of 21 human normal tissues had immunoreactive GD3. In immunofluorescence, KM641 bound to a variety of living tumor cell lines especially melanoma cells, including some cell lines to which another anti-GD3 mAb R24, established previously, failed to bind. High-affinity binding of KM641 to a tumor cell line was quantified by Scatchard analysis (Kd = 1.9×10−8 M). KM641 exerted tumor-killing activity in the presence of effector cells or complement against melanoma cells expressing GD3 at a high level. Not only natural killer cells but also polymorphonuclear cells were effective as the effector cells in antibody-dependent cellular cytotoxicity. Intravenous injection of KM641 markedly suppressed the tumor growth of a slightly positive cell line, C24.22 (7.2×105 binding sites/cell), as well as a very GD3-positive cell line, G361 (1.9×107 binding sites/cell), inoculated intradermally in nude mice. KM641, characterized by a high binding affinity for GD3, has the potential to be a useful agent for passive immunotherapy of human cancer.
Koichi Furukawa - One of the best experts on this subject based on the ideXlab platform.
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tnfα signal and camp mediated signals oppositely regulate melanoma associated Ganglioside GD3 synthase gene in human melanocytes
Scientific Reports, 2019Co-Authors: Rika Takeuchi, Koichi Furukawa, Mariko Kambe, Maiko Miyata, Orie Tajima, Upul Jeyadevan, Keiko FurukawaAbstract:Analyses of expression and regulation of Ganglioside synthases in melanocytes are important to understand roles of Gangliosides in melanomagenesis. In this study, we analyzed the expression and regulatory mechanisms of glycosyltransferase genes responsible for Ganglioside synthesis in normal melanocytes. We reported previously that culture supernatants of UVB-irradiated keratinocytes induced upregulation of Ganglioside GD3 synthase gene in melanocytes, and mainly TNFα was responsible for it. Then, we found that elimination of dibutyryl cyclic AMP and IBMX from the medium also resulted in upregulation of the GD3 synthase gene. The addition of α-melanocyte-stimulating hormone which increases cAMP, to the medium led to a significant reduction in the GD3 synthase gene expression level, and a PKA inhibitor enhanced the GD3 synthase gene level. These results suggest that signals mediated via TNFα and cAMP oppositely regulate GD3 synthase gene expression in melanocytes. The results of an IKK inhibitor indicate the possibility that TNFα induces GD3 synthase gene expression via NF-κB signaling in melanocytes. When melanoma cells were treated by these factors, no fluctuation in the GD3 synthase gene expression level was observed, although an IKK inhibitor significantly suppressed it, suggesting that Ganglioside synthase genes are regulated in distinct manners between melanocytes and melanomas.
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enhancement of malignant properties of human glioma cells by Ganglioside GD3 gd2
International Journal of Oncology, 2018Co-Authors: Taiji Iwasawa, Keiko Furukawa, Yuki Ohkawa, Yuhsuke Ohmi, Robiul H Bhuiyan, Pu Zhang, Hiroyuki Momota, Toshihiko Wakabayashi, Koichi FurukawaAbstract:Sialic acid-containing glycosphingolipids, Gangliosides, are considered as cancer associated antigens in neuro-ectoderm-derived tumors such as melanomas and neuroblastomas. In particular, Gangliosides GD3 and GD2 are expressed in human gliomas. It has been reported that their expression levels increase along with increased malignant properties. However, the implication of GD3/GD2 in human glioma cells has never been clarified, at least to the best of our knowledge. In this study, we introduced the cDNA of GD3 synthase (GD3S)(ST8SIA1) into a glioma cell line, U-251MG, that expresses neither GD3 nor GD2, thereby establishing transfectant cells U-251MG-GD3S(+) expressing high levels of GD3 and GD2 on the cell surface. In these U-251MG‑GD3S(+) cell lines, signaling molecules such as Erk1/2, Akt, p130Cas, paxillin and focal adhesion kinase were activated, leading to the enhancement of invasion activity and motility. It was then demonstrated that the U-251MG-GD3S(+) cells could proliferate under culture conditions with low or no serum concentrations without undergoing cell cycle arrest by escaping the accumulation of p16 and p21. All these results suggested that GD3 and GD2 highly expressed in gliomas confer increased invasion and mobility, cell growth abilities under low serum conditions, and increased ratios of the S-G2/M phase in the cell cycle.
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neogenin defined as a GD3 associated molecule by enzyme mediated activation of radical sources confers malignant properties via intracytoplasmic domain in melanoma cells
Journal of Biological Chemistry, 2016Co-Authors: Kei Kaneko, Keiko Furukawa, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Ohmi, Norihiro Kotani, Koichi Honke, Mitsutaka Ogawa, Tetsuya Okajima, Koichi FurukawaAbstract:To investigate mechanisms for increased malignant properties in malignant melanomas by Ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.
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uvb irradiated keratinocytes induce melanoma associated Ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6
Biochemical and Biophysical Research Communications, 2014Co-Authors: Maiko Miyata, Koichi Furukawa, Mariko Kambe, Orie Tajima, Masatoshi Ichihara, Sayaka Sobue, Kazumitsu Sugiura, Keiko FurukawaAbstract:Although expression of Gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for Ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of Gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the Ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major Ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated Ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like Ganglioside profiles in melanocytes.
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Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor met signals in melanomas
Cancer Science, 2014Co-Authors: Keiko Furukawa, Yuki Ohkawa, Mariko Kambe, Maiko Miyata, Orie Tajima, Koichi FurukawaAbstract:Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I). Although stimulation of melanoma N1 cells (GD3+ and GD3−) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3− cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5-ethynyl-2‘ deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.
Carmen Garciaruiz - One of the best experts on this subject based on the ideXlab platform.
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Ganglioside GD3 sensitizes human hepatoma cells to cancer therapy
Journal of Biological Chemistry, 2002Co-Authors: Raquel Paris, Albert Morales, Olga Coll, A Sanchezreyes, Carmen Garciaruiz, Jose C FernandezchecaAbstract:Ganglioside GD3 (GD3) has emerged as a modulator of cell death pathways due to its ability to interact with mitochondria and disable survival pathways. Because NF-κB activation contributes to cancer therapy resistance, this study was undertaken to test whether GD3 modulates the response of human hepatoblastoma HepG2 cells to radio- and chemotherapy. NF-κB was activated in HepG2 cells shortly after therapeutic doses of ionizing radiation or daunorubicin treatment that translated into up-regulation of κB-dependent genes. These effects were accompanied by minimal killing of HepG2 cells by either ionizing radiation or daunorubicin. However, GD3 pretreatment blocked the nuclear translocation of active κB members, without effect on Akt phosphorylation, induced by either treatment. The suppression of κB-dependent gene induction by GD3 was accompanied by enhanced apoptotic cell death caused by these therapies. Furthermore, the combination of GD3 plus ionizing radiation stimulated the formation of reactive species followed by the mitochondrial release of cytochrome c and Smac/Diablo and caspase 3 activation. Pretreatment with cyclosporin A before radiotherapy protected HepG2 cells from the therapeutic combination of GD3 plus ionizing radiation. These findings underscore a key role of mitochondria in the response of tumor cells to cancer therapy and highlight the potential relevance of GD3 to overcome resistance to cancer therapy by combining its dual action as a mitochondria-interacting and NF-κB-inactivating agent.
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trafficking of Ganglioside GD3 to mitochondria by tumor necrosis factor α
Journal of Biological Chemistry, 2002Co-Authors: Carmen Garciaruiz, Albert Morales, Anna Colell, Maria Calvo, Carlos Enrich, Jose C FernandezchecaAbstract:Abstract The interaction of mitochondria with proapoptotic proteins activates apoptosis pathways. Previous findings have identified Ganglioside GD3 (GD3) as an emerging apoptotic lipid intermediate that targets mitochondria in response to death signals. Using immunoelectron and laser scanning confocal microscopy, we characterize the trafficking of GD3 to mitochondria in response to tumor necrosis factor-α (TNF-α) in rat hepatocytes. In control hepatocytes, GD3 is present predominantly at the plasma membrane as well as in the endosomal/Golgi network, as verified by its colocalization with the asialoglycoprotein receptor. Following TNF-α exposure, GD3 undergoes a rapid cellular redistribution with a gradual loss from the plasma membrane before its colocalization with mitochondria. This process is mimicked by acidic sphingomyelinase and ionizing radiation but not by neutral sphingomyelinase or staurosporin. TNF-α stimulated the colocalization of GD3 with early and late endosomal markers, Rab 5 and Rab 7, whereas perturbation of plasma membrane cholesterol or actin cytoskeleton or inhibition of glucosylceramide synthase prevented the trafficking of GD3 to mitochondria. Finally, prevention of the TNF-α-stimulated neosynthesis of GD3, cyclosporin A, and latrunculin A or filipin protected sensitized hepatocytes from TNF-α-mediated cell death. Thus, the intracellular redistribution and mitochondrial targeting of GD3 during TNF-α signaling occurs through actin cytoskeleton vesicular trafficking and contributes to TNF-α-mediated hepatocellular cell death.
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ceramide generated by acidic sphingomyelinase contributes to tumor necrosis factor α mediated apoptosis in human colon ht 29 cells through glycosphingolipids formation possible role of Ganglioside GD3
FEBS Letters, 2002Co-Authors: Anna Colell, Albert Morales, Jose C Fernandezcheca, Carmen GarciaruizAbstract:In the present study we assessed the contribution of acidic sphingomyelinase (ASMase), a ceramide generating enzyme, in tumor necrosis factor (TNF)-mediated apoptosis in human colon HT-29 cells. TNF induced apoptosis in HT-29 cells in a time- and dose-dependent fashion. Downregulation of the active endogenous ASMase form prevented TNF-stimulated ASMase activity and apoptosis. Furthermore, inhibition of glucosylceramide synthase, which blunted TNF-stimulated GD3 levels, abolished TNF-mediated cell death. Immunocytochemical staining revealed the co-localization of GD3 with mitochondria induced by TNF. The knockdown of targeted GD3 synthase by antisense expression vector protected HT-29 cells against TNF-induced cell death. Thus, ASMase plays a key role in TNF-induced cell death in human colon epithelial cells possibly through GD3 generation.
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Ganglioside GD3 enhances apoptosis by suppressing the nuclear factor κb dependent survival pathway
The FASEB Journal, 2001Co-Authors: Anna Colell, Carmen Garciaruiz, Juan Roman, A M Ballesta, Jose C FernandezchecaAbstract:SPECIFIC AIMGlycosphingolipidshave emerged as cell death effectors because of their role in apoptosis signaling. Because cell survival reflects a balance between death and survival pathways and because the nuclear factor-κB (NF-κB) is known to downregulate apoptosis in response to diverse stimuli, our study examined the role of Ganglioside GD3 (GD3) and related structural analogs, including short-chain ceramide analogs—for example, ceramide C2 (C2)—on NF-κB regulation, mitochondrial reactive oxygen species (ROS) generation, and survival of cultured rat hepatocytes.PRINCIPAL FINDINGS1. Mitochondrial ROS formation is common for both C2 and GD3Incubation of hepatocytes with C2 and GD3 (0–50 μM) resulted in a similar dose-dependent peroxide formation. It is interesting that, despite this, hepatocytes displayed a selective susceptibility to GD3 treatment, which was reflected by a decreased survival (36±5% vs. 92±7% for GD3 and C2 at 50 μM for 12 h), which was accompanied by apoptotic features. Inhibitors of mi...
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direct interaction of GD3 Ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition cytochrome c release and caspase activation
The FASEB Journal, 2000Co-Authors: Carmen Garciaruiz, Raquel Paris, Anna Colell, J C FernandezchecaAbstract:Glycosphingolipids, including Gangliosides, are emerging as signaling intermediates of extracellular stimuli. Because mitochondria play a key role in the orchestration of death signals, we assessed the interaction of GD3 Ganglioside (GD3) with mitochondria and the subsequent cascade of events that culminate in cell death. In vitro studies with isolated mitochondria from rat liver demonstrate that GD3 elicited a burst of peroxide production within 15–30 min, which preceded the opening of the mitochondrial permeability transition, followed by cytochrome c (cyt c) release. These effects were mimicked by lactosylceramide and N-acetyl-sphingosine but not by sphinganine or sphingosine and were prevented by cyclosporin A and butylated hydroxytoluene (BHT). Reconstitution of mitochondria pre-exposed to GD3 with cytosol from rat liver in a cell-free system resulted in the proteolytic processing of procaspase 3 and subsequent caspase 3 activation. Intact hepatocytes or U937 cells selectively depleted of glutathione...
