Synthesis and biological evaluation of neoglycosphingolipids.

Various neoglycosphingolipids were efficiently synthesized in a one-step reaction by the coupling of free sugars with an N-alkylaminooxy-functionalized ceramide analogue. The bioactivity studies demonstrated that most of these compounds could upregulate the expression of matrix metalloproteinase-9 (MMP-9, extracellular matrix proteins associated with tumor migration) in murine melanoma B16 cells in a similar manner to the natural ganglioside monosialodihexosylganglioside (GM3), which highlights the potential use of these neoglycosphingolipids as inhibitors of tumor migration.

Involvement of Ext1 and heparanase in migration of mouse FBJ osteosarcoma cells.

To know the involvement of glycosaminoglycans (GAGs) in the metastasis of mouse FBJ osteosarcoma cells, N(α)-lauroyl-O-(ß-D-xylopyranosyl)-L-serinamide (Xyl-Ser-C12), which initiates elongation of GAG chains using the glycan biosynthesis system in cells, was administered to FBJ cells with different metastatic capacities. Production of glycosylated products derived from Xyl-Ser-C12, especially heparan sulfate (HS) GAG-type oligosaccharides such as GalNAc-GlcA-GlcNAc-GlcA-Gal-Gal-Xyl-Ser-C12, was indicated in poorly metastatic FBJ-S1 cells more than in highly metastatic FBJ-LL cells by LC-MS. The results of RT-PCR revealed that HS synthases, Ext1 and Ext2, were expressed in FBJ-S1 cells more than in FBJ-LL cells. Furthermore, siRNA against Ext1 suppressed the expression of HS and enhanced the motility of FBJ-S1 cells. In addition, the expression of heparanase (HPSE) was enhanced in Ext-1-knockdown FBJ-S1 cells, and responsible for the increase in cell motility caused by the down-regulation of Ext1 expression. Our data provide the first evidence that Ext1 regulates the expression of HPSE and also indicated that levels of Ext1 and HPSE influenced the motility of FBJ cells.

Calcium regulates caveolin-1 expression at the transcriptional level.

Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca(2+)/calcineurin/NFAT.

Ganglioside GD1a negatively regulates hepatocyte growth factor expression through caveolin-1 at the transcriptional level in murine osteosarcoma cells.

BACKGROUND: Hepatocyte growth factor (HGF) is a mesenchyme-derived, multifunctional protein that is implicated in tumor growth and invasive behavior. Some tumor cells express both HGF and its receptor MET, forming an autocrine loop that permanently activates it. Ganglioside GD1a suppresses metastatic capacity in murine FBJ osteosarcoma cells and MET phosphorylation activated by HGF binding, but the signaling pathway controlling HGF production has not been fully explored. METHODS: Expression of HGF, caveolins, or MET of the cells that had been transfected with siRNA or cDNA directed to GM2/GD2 synthase, caveolin-1 or HGF was determined by semi-quantitative RT-PCR and Western blots. RESULTS: HGF expression in highly metastatic, GD1a-deficient FBJ-LL cells was higher than that in the poorly metastatic, GD1a-rich FBJ-S1 cells. Transfection with GM2/GD2 synthase cDNA increased GD1a levels in FBJ-LL cells and suppressed HGF expression. Treatment with siRNAs directed toward GM2/GD2 synthase in FBJ-S1 cells reduced gangliosides and augmented HGF expression. GD1a was found to be the only ganglioside species suppressing HGF expression upon addition to FBJ-LL cells. HGF expression was decreased by GD1a addition to FBJ-LL cells after 48h, enough to induce caveolin-1 expression. Silencing caveolin-1 up-regulated HGF, and the re-introduction of caveolin-1 cDNA decreased HGF expression. Caveolin-1 suppressed MET phosphorylation. We also found GD1a regulation of HGF in Lewis lung carcinoma cells. CONCLUSIONS: HGF expression was negatively regulated by GD1a through caveolin-1 at the transcriptional level via the suppression of MET phosphorylation. GENERAL SIGNIFICANCE: This is the first report that ganglioside GD1a negatively regulates HGF expression through caveolin-1.

GM3 suppresses anchorage-independent growth via Rho GDP dissociation inhibitor beta in melanoma B16 cells.

Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells.

Ganglioside GD1a suppression of NOS2 expression via ERK1 pathway in mouse osteosarcoma FBJ cells.

Inducible nitric oxide synthase (NOS2) is over-expressed in a number of tumors and implicated in tumor growth and metastasis. Murine FBJ osteosarcoma-derived FBJ-S1 cells are poorly metastatic and express the ganglioside GD1a, whereas highly metastatic FBJ-LL cells only slightly express this ganglioside. The present study demonstrates that NOS2 is more highly expressed in FBJ-LL cells compared to FBJ-S1 cells. By manipulating GM2/GD2 synthase expression or adding exogenous GD1a, GD1a inversely regulated NOS2 at the transcriptional level. GT1b suppressed NOS2 to the same extent as GD1a. Silencing NOS2 inhibited proliferation, migration, and anchorage-independent growth of FBJ-LL cells, suggesting that the metastatic properties of FBJ-LL cells are associated with NOS2. MEK1/2 inhibitor (U0126) increased NOS2 expression, whereas GD1a treatment decreased it. Co-treating the cells with GD1a and U0126 blocked the inhibition of NOS2 expression, suggesting that the GD1a signal is mediated by ERK1/2. NOS2 expression increased when ERK1, but not ERK2, was silenced, and GD1a did not suppress NOS2 expression in cells treated with another MEK1/2 inhibitor PD98059, suggesting that ERK1 phosphorylation is indispensable for the GD1a signal suppressing NOS2.

Structural analysis of glycosphingolipid analogues obtained by the saccharide primer method using CE-ESI-MS.

A glycosphingolipid analogue (12-azidododecyl beta-lactoside) as a saccharide primer has been shown to be useful for the synthesis of oligosaccharide libraries by mammalian cells. In the present study, CE-ESI-MS was employed to elucidate the structure of glycosphingolipid analogues derived from 12-azidododecyl beta-lactoside (Lac-C12N3) by mammalian cells. MDCK cells and COLO201 cells were cultured with Lac-C12N3, and the glycosylated products secreted into the medium were collected and separated into acidic and neutral products by column chromatography. The acidic products could be directly analyzed by CE-ESI-MS, while the neutral products were converted to anionic derivatives via a reaction with propiolic acid. With this method, it was possible to analyze both acidic and neutral products glycosylated by MDCK cells and COLO201 cells at high sensitivity.

Ganglioside GD1a suppresses TNFalpha expression via Pkn1 at the transcriptional level in mouse osteosarcoma-derived FBJ cells.

Ganglioside GD1a has been reported to suppress metastasis [S. Hyuga, S. Yamagata, Y. Takatsu, M. Hyuga, H. Nakanishi, K. Furukawa, T. Yamagata, Suppression of FBJ-LL cell adhesion to vitronectin by ganglioside GD1a and loss of metastatic capacity, International J. Cancer. 83 (1999) 685-691.] and MMP-9 production in mouse osteosarcoma FBJ cells [D. Hu, Z. Man, P. Wang, X. Tan, X. Wang, S. Takaku, S. Hyuga, T. Sato, X. Yao, S. Yamagata, T. Yamagata, Ganglioside GD1a negatively regulates MMP9 expression in mouse FBJ cell lines at the transcriptional level, Connect. Tissue Res. 48 (2007) 198-205.]. In the present study, TNFalpha increased cell motility and MMP-9 and TNFalpha expression at the transcriptional level. TNFalpha expression was found to be inversely proportional to GD1a content in the FBJ-cell variants. The addition of exogenous GD1a to FBJ-LL cells suppressed TNFalpha expression, and treatment of FBJ-S1 cells with D-PDMP (glucosylceramide synthesis inhibitor) led to an increase in TNFalpha, indicating that TNFalpha is negatively regulated by GD1a in FBJ cells. SiRNA of Pkn1, a Rho-GTPase effecter protein kinase, suppressed TNFalpha levels as well as Pkn1 expression, suggesting that Pkn1 is involved in TNFalpha signaling. Treatment of Pkn1-silenced FBJ-LL cells with GD1a failed to suppress TNFalpha expression, demonstrating that GD1a signals that lead to TNFalpha suppression are transduced through Pkn1.

Glycosylation of dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside as saccharide primers in cells.

Syntheses of oligosaccharides expressed on cells are indispensable for the improvement of the functional analyses of the oligosaccharides and their applications. We are developing saccharide primers for synthesizing oligosaccharides using living cells. In this study, dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (GlcNAc-C12) and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside (LacNAc-C12) were examined for their abilities to prime the syntheses of neolacto-series oligosaccharides in HL60 cells. When GlcNAc-C12 was incubated with HL60 cells in serum-free medium for 2 days, 14 kinds of glycosylated products were collected from the culture medium. They were separated by high-performance liquid chromatography. The sequences of the products were determined to be neolacto-series oligosaccharides including Lewis(X), sialyl Lewis(X), polylactosamine, and sialylpolylactosamine by mass spectrometry. GlcNAc-C12 was also glycosylated by B16 cells and gave sialyllactosamine. Furthermore, LacNAc-C12 gave similar glycosylated products to GlcNAc-C12.

Ganglioside GD1a negatively regulates matrix metalloproteinase-9 expression in mouse FBJ cell lines at the transcriptional level.

Mouse FBJ virus-induced osteosarcoma FBJ-S1 cells rich in GD1a are not readily metastatic, whereas FBJ-LL cells with low levels of GD1a are highly metastatic. GD1a was previously shown to suppress metastasis of mouse FBJ cells and to upregulate caveolin-1 and stromal interaction molecule 1 expression. The present study demonstrates that matrix metalloproteinase-9 (MMP-9) expression renders FBJ-LL cells invasive. MMP-9 is inversely regulated by GD1a, based upon four observations: MMP-9 mRNA content was 5 times higher in FBJ-LL cells than FBJ-S1 cells; a GD1a-re-expressing FBJ-LL cell variant produced through beta1,4GalNAcT-1 cDNA transfection expressed lower levels of MMP-9; exogenous addition of GD1a to FBJ-LL cells decreased MMP-9 production in a dose- and time-dependent manner; and treatment of GD1a-rich cells with D-PDMP or siRNA targeting St3gal2 decreased GD1a expression, but augmented MMP-9 expression. This is the first report demonstrating that GD1a negatively regulates expression of MMP-9 at the transcriptional level.

Positive regulation of tumor necrosis factor-alpha by ganglioside GM3 through Akt in mouse melanoma B16 cells.

GM3 has been shown to suppress TNFalpha expression in blood monocytes. However, we found that GM3 and TNFalpha were expressed in parallel in mouse melanoma B16 cells that were transfected with UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase cDNA in a sense or antisense direction or CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase siRNA. TNFalpha expression was increased by addition of GM3 to the B16 transfectants and decreased after treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis. These results clearly indicate that GM3 positively regulates TNFalpha expression in B16 cells. Phosphoinositide 3-kinase inhibitors, wortmannin and LY294,002, suppressed TNFalpha expression and Akt phosphorylation. GM3 was shown to increase phosphorylation of Akt in B16 cells and the B16-derived transfectants. Treatment of B16 cells with siRNA targeted to Akt1/2 resulted in TNFalpha suppression, indicating that Akt plays an important role in regulation of TNFalpha expression. Suppression of Akt1/2 rendered cells insensitive to GM3, suggesting that the GM3 signal may be transduced via Akt.

GM3 signals regulating TNF-alpha expression are mediated by Rictor and Arhgdib in mouse melanoma B16 cells.

OBJECTIVE: We have previously shown GM3 to positively regulate TNF-alpha expression via a PI3K/Akt pathway in mouse melanoma B16 cells [Wang et al.: Biochem Biophys Res Commun 2007;356:438-443]. The GM3 signal was shown to be located upstream of Akt, but whether it is located upstream of PI3K and which molecule is the effector of PI3K remain to be clarified. METHODS: We used inhibitors of PI3K and mTOR, and siRNA directed to Rictor, Raptor and Rho-GDP dissociation inhibitor beta (Arhgdib). RESULTS: PI3K inhibitors LY294002 and LY303511 were shown to suppress TNF-alpha expression that is stimulated by GM3 in B16 cells, suggesting that the GM3 signal is located upstream of the PI3K-Akt pathway. Rapamycin suppressed TNF-alpha expression, indicating mTOR to be involved in the pathway. Either siRNA Raptor or siRNA Rictor suppressed TNF-alpha expression, but the latter suppressed the effects of GM3 on TNF-alpha expression and Akt phosphorylation at Ser(473), indicating the GM3 signal to be transduced via Rictor/mTOR and Akt (Ser(473)), leading to TNF-alpha stimulation. Finally, Arhgdib, the tumor suppressor gene whose expression is associated with GM3, was shown to be upstream of TNF-alpha. CONCLUSIONS: The GM3 signal is thus transduced in B16 cells through a PI3K, Rictor/mTOR, Akt, Arhgdib pathway, leading to stimulated expression of TNF-alpha.

Apparent suppression of MMP-9 activity by GD1a as determined by gelatin zymography.

Gelatin zymography is widely used to detect and evaluate matrix metalloproteinase-9 (MMP-9) activity. MMP-9 transcription was previously shown to be negatively regulated by ganglioside GD1a [D. Hu, Z. Man, T. Xuan, P. Wang, T. Takaku, S. Hyuga, X.S. Yao, T. Sato, S. Yamagata, T. Yamagata, Ganglioside GD1a regulation of matrix metalloproteinase-9 (MMP-9) expression in mouse FBJ cell Lines: GD1a suppression of MMP-9 expression stimulated by PI3K-Akt and p38 though not by the Erk signaling pathway, 2006, submitted for publication.]. Zymography of MMP-9 of FBJ-M5 cells preincubated with GD1a indicated a greater decrease in activity than expected from mRNA suppression. Incubation of conditioned medium containing MMP-9 with GD1a caused MMP-9 activity to decrease. Examination was thus made to confirm that MMP-9 activity is actually suppressed and/or MMP-9 protein undergoes degradation by GD1a. GD1a was found to have no effect on MMP-9 activity and Western blots indicated GD1a not to diminish MMP-9 during electrophoresis under reducing conditions. GD1a appeared to mediate the binding of a portion of MMP-9 with certain molecules, with consequently greater molecular mass on the gel, to cause decrease in the activity of MMP-9 at the site where it would normally appear. Caution should be used in doing gelatin zymography since molecules other than GD1a may similarly work, causing decrease in MMP-9 activity in zymography.

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells: augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content.

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through beta1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1.

The Persistence in the Synthesis of Type H Proteoglycan and Type II Collagen by Chondrocytes Cultured in the Presence of Chick Embryo Extract1.

The effect of chick embryo extract on the phenotypic expression of differentiated chondrocytes has been studied in consideration of the fact that these cells are well characterized by certain specific cell products, such as type H proteochondroitin sulfate and type II collagen. In this study, we utilized floating chondrocytes derived from chick embryonic sterna, which can be cultured in suspension with no apparent change in the type of cell products for at least a period of eight weeks, as described in a previous paper (1). In the presence of chick embryo extract in the medium, the floating chondrocytes became attached to the bottom of the culture dish, and the attached cells took on a fibroblast-like appearance. Biochemical analyses of the proteochondroitin sulfate and collagen synthesized by the attached cells revealed that if the culture medium was renewed everyday, the cells having a fibroblast-like appearance continued to synthesize type H proteochondroitin sulfate and type II collagen. When however, the medium was replaced every other day, the synthesis of both proteochondroitin sulfate and collagen by the attached cells switched from the chondrocyte type to the fibroblast type, i.e. the synthesis of type M proteochondroitin sulfate and type I collagen, with little change in the fibroblast-like appearance. The results show that the morphological features of chondrocytes are not necessarily associated with the biochemical properties of these cells, and further suggest that, in chick embryo extract, there is no modulator capable of acting directly on the chondrocytes to bring about phenotypic changes with respect to the synthesis of collagen and proteoglycans.

MAINTENANCE OF PHENOTYPIC PROPERTIES BY CHONDROCYTES CULTURED IN SUSPENSION.

Sternal chondrocytes obtained from 13-day-old chick embryos could be cultured in suspension without any mechanical agitation for 8 weeks. The cells in suspension retained all characteristics of chondrocytes when examined from morphological, histochemical and biochemical points of view. The floating cells were round in shape, rich in Golgi apparatus-associated vesicles. Each cell was covered with a thin coat of matrix showing metachromasia when stained with toluidine blue. Autoradiographic studies suggested an active synthesis of proteochondroitin sulfates by the individual floating cells. The biochemical analyses revealed that the floating cells continued to synthesize type H proteochondroitin sulfate and type II collagen, both of which are known to be characteristic products of differentiated chondrocytes.