2 Department of Hematology and Oncology, Georg-August-University, Robert-Koch-Str. 40, 37075 Göttingen, Germany; 3 Department of Organic Chemistry, Georg-August-University, Tammannstr. 2, 37077 Göttingen, Germany; 4 Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 Kings College Circle, Toronto, ON M5S 1A8, Canada; and 5 Clinic of Neurology, Westf. Wilhelms-University Münster, Albert-Schweitzer-Str. 33, 48129 Münster, Germany
Received on December 3, 2001; revised on April 3, 2002; accepted on April 3, 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: EGF receptor-signaling /ganglioside GM3/ganglioside lactone/proliferation/tyrosine kinase activity
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Ganglioside GM3 and, to a lesser extent, GM1 but not other gangliosides have been described to modulate cell growth through inhibition of epidermal growth factor (EGF) receptorassociated tyrosine kinase activity (Bremer et al., 1986). On binding of EGF to its receptor, the receptor becomes phosphorylated on tyrosines, allowing cytoplasmatic proteins to bind to the phosphotyrosine sequence on the receptor leading to the phosphorylation of mitogen-activated protein kinases (MAP kinases) (Cobb and Goldsmith, 1995
).
The effect of GM3 appears to be mediated directly by inhibition of EGF receptor autophosphorylation and dimerization (Bremer et al., 1986; Bremer, 1994
) rather than by acting on the intracellular intermediates of EGF receptor signaling (Rebbaa et al., 1996
). Therefore, ganglioside analogs that inhibit receptor associated tyrosine kinases have been applied for the inhibition of tumor growth and metastasis (Hakomori, 1996
; Suarez-Pestana et al., 1997
) and have been shown to modulate the angiogenic response of tumors (Alessandri et al., 1997
). Reduction of GM3 expression in A431 cells by transfection of the sialidase gene or application of a ceramide analog resulted in enhanced EGF receptor activity and induction of growth (Meuillet et al., 1999
, 2000).
Recently, we succeeded in the synthesis of the GM3-lactone analog HK1-ceramide 2 (Figure 1) which contains an ether moiety instead of the lactone functionality and is stable under physiological conditions (Tietze and Keim, 1997; Tietze et al., 2000
). To our knowledge, HK1-ceramide 2 is the only stable ganglioside-lactone analog synthesized so far and is thus a perfect tool to analyze the mode of action of GM3-lactone 1 formed in vivo.
The goal of this study was to determine if the lactone form of GM3 has a similar inhibitory effect as GM3 itself on EGF-induced cell growth in human epidermoid carcinoma KB cells and EGF receptor tyrosine phosphorylation in human epidermoid carcinoma A431 cells. To accomplish this, the effects of the stable ganglioside-lactone analog HK1-ceramide 2 toward EGF receptor signaling and EGF-mediated cell growth were examined and directly compared to the observed effects of GM3.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of the study was to determine whether the GM3 lactone 1 has an inhibitory effect on EGF-dependent tyrosine phosphorylation of the receptor and therefore on modulation of cell growth. Because the unmodified GM3 lactone 1 cannot be used due to its instability under physiological conditions, we therefore examined the effect of the GM3 lactone analog HK1-ceramide 2 on EGF receptor signaling in human ovarial epidermoid carcinoma A431 cells and mitogenesis in human oral epidermoid carcinoma KB cells. Compared to GM3 lactone, HK1-ceramide 2 contains an ether moiety instead of the lactone functionality and is stable under physiological conditions (Tietze and Keim, 1997). As controls, we also used GM3 itself and the GM3 lactone ether analog (HK2-ceramide 3) with the ß configuration at the 2-position of the neuraminic acid moiety. This compound shows a different structural arrangement as compared to GM3 lactone 1.
A431 cells were used for the analysis of EGF receptor signaling because these cells have an elevated endogenous concentration of EGF receptors and have therefore been used in a variety of receptor phosphorylation studies concerning the effect of GM3 (Bremer et al., 1986). On stimulation of EGF, the receptor becomes phosphorylated on tyrosine residues that bind to specific SH2 domaincontaining molecules, which are often themselves phosphorylated by the receptor, thus initiating the signaling cascade. Here, we demonstrate that HK1-ceramide 2 at a concentration of 25 µM decreases ligand-dependent activation of EGF receptor in A431 cells without affecting EGF receptor content. This effect was only observed in a narrow range of concentration. In addition, tyrosine phosphorylation of proteins with lower molecular weight was found to be reduced by HK1-ceramide 2, in particular of a protein with an apparent molecular weight of 116 kDa.
In contrast to HK1-ceramide 2, GM3 had to be applied in much higher concentrations of 100 µM to 500 µM to inhibit EGF receptor tyrosine phosphorylation in a dose-dependent manner (Hanai et al., 1988; Zhou et al., 1994
; Rebbaa et al., 1996
; Meuillet et al., 1999
). We did not observe an effect at concentrations of GM3 below 100 µM. In A431 cells, tyrosine phosphorylation of the EGF receptor was found to be specifically inhibited by exogenous addition of GM3 and to a lesser extent by GM1, but not by other gangliosides or neutral glycolipids (Bremer et al., 1986
). This is in line with the results of our study, where a concentration of 200 µM GM3 showed an inhibitory effect on EGF-mediated receptor tyrosine phosphorylation in A431 cells. A decrease in endogenous gangliosides in mutant Chinese hamster ovary (CHO) cells was associated with an increased autophosphorylation of EGF receptor (Weis and Davis, 1990
) whereas CHO cells stably transfected with CMP-NeuAc:GM3 sialyltransferase and expressing mostly GD3 at the cell surface showed both decreased EGF receptor phosphorylation and extracellular signal-regulated kinase 2 (ERK2) activation after stimulation with EGF (Zurita et al., 2001
). As expected the ß-isomer HK2-ceramide 3 in concentrations in the range of 20100 µM had no effect on EGF-dependent tyrosine phosphorylation of the EGF receptor nor of proteins with lower molecular weight in A431 cells (data not shown).
It has been argued that the lactone form of GM3 is present on tumor cells due to low pH at the surface of these cells. However, in cells exposed to low pH, GM3 had the same effect on inhibition of EGF-dependent receptor phosphorylation as in cells grown in media of neutral pH. These results show that low-pH treatment, which might promote lactonization of GM3, does not result in a further, more potent inhibition of EGF-mediated receptor phosphorylation. We could also demonstrate that low-pH treatment can activate constitutive, ligand-independent EGF receptor activity, whereas EGF-mediated tyrosine phosphorylation of the receptor is reduced under acidic conditions. In a recent study, low pH treatment has been shown to activate ERK2 MAP kinase as well as multiple MAP kinase pathways involving JNK and p38, however without increased tyrosine phosphorylation of the EGF receptor (Xue and Lucocq, 1997). In analogy to GM3 (Bremer et al., 1986
) HK1-ceramide 2 has been shown to have no influence on the binding of EGF to the cell surface receptor on KB cells.
We could demonstrate here that EGF-dependent growth of KB cells, which have been characterized by the presence of high-affinity receptor and EGF-dependent mitogenesis (King and Cuatrecasas, 1982), was inhibited in a dose-dependent manner by 62%, 81%, and 86% using concentrations of 6, 17, and 25 µM HK1-ceramide 2, respectively. In contrast, the less potent GM3 required concentrations of 50 µM GM3 to inhibit EGF stimulated growth of KB cells by 65% (Bremer et al., 1986
).
Our results indicate that the lactone analog of GM3, HK1-ceramide 2, is a specific inhibitor of EGF-dependent receptor tyrosine phosphorylation and consequently further signaling events to the nucleus without affecting the binding of EGF to the receptor. Surprisingly, the effect was only observed in a narrow range of inhibitor concentration. The mechanisms that has led to the inhibition on EGF signaling by HK1-ceramide 2 are currently under further investigation. The GM3 lactone analog HK1-ceramide 2 seems to be more potent in comparison to GM3 in modulation of EGF receptor activity and mitogenesis.
![]() |
Material and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
Ovarial epidermoid carcinoma A431 cells and human oral epidermoid carcinoma KB cells were purchased from DSMZ (Braunschweig, Germany) (Drexler et al., 1999). All cells were grown in Dulbeccos modified Eagles medium (DMEM) supplemented with penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mM) (Gibco BRL, Eggenstein, Germany), and 10% heat-inactivated fetal calf serum (FCS) (PAN, Aidenbach, Germany) at 37°C in a humidified atmosphere of 5% CO2.
Analysis of EGF-dependent tyrosine phosphorylation in A431 cells in the presence or absence of gangliosides
Cells were seeded in 24-well plates in DMEM/10% FCS and grown to 80% confluence. The medium was replaced by DMEM without FCS and cells incubated for 24 h. Aliquots of solutions containing different quantities of HK1-ceramide 2, HK2-ceramide 3, and GM3 were added to each well as indicated and incubated for 3 h at 37°C. Cells were stimulated with EGF (50 ng/ml) 30 min prior to lysis. Addition of solvent alone (0.86% DMSO) or 1 mM ortho-vanadate (Na3VO4) 90 min prior to cell lysis were used as controls. Decreasing pH values in cell culture were produced by adding 1 N or 0.1 N HCl to the medium. pH values in medium were measured during the experiment. After washing the cells three times with cold phosphate buffered saline, the monolayers were solubilized by the addition of 125 µl lysis buffer containing 1% NP40; 0.25% Na-deoxycholate; 50 mM TrisHCl (pH 7.4); 150 mM NaCl; 1 mM ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml aprotinin, leupeptin, and pepstatin A; 1 mM NaF; and 1 mM Na3VO4. The cellular lysates were centrifuged 10 min at 4°C and 21,000 x g and aliquots of the supernatants were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Lysates were adjusted to contain equal amounts of protein, using the BCA Protein assay (Pierce, Rockford, IL).
Proteins were transferred to nitrocellulose membrane (Amersham Life Sciences, Arlington Heights, IL) and immunoblotted with antibodies, the monoclonal -PY (4G10, IgG 2bk, 100 µg/ml, Paesel + Lorei, Duisburg, Germany), diluted 1:1000, or the polyclonal anti-EGF-R antibody (Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1:1000 in 50 mM G-NET (TrisHCl [pH 7.5], 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid, 0.25% gelatin) overnight at 4°C. Western blots were developed using horseradish peroxidasecoupled, goat anti-mouse and goat anti-rabbit, secondary antibodies (Biorad, München, Germany), diluted 1:20,000 in 50 mM G-NET for 1 h at room temperature and enhanced chemiluminescence (Amersham). For reprobing the membrane was stripped in 70 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, 0.1% ß-mercaptoethanol at 55°C for 30 min.
Determination of EGF-dependent mitogenesis
This experiment was performed with KB cells, because mitogenic stimulation by EGF can be clearly observed in a wider range of EGF concentrations in KB cells in comparison to the results obtained in A431 cells stimulated by EGF (King and Cuatrecasas, 1982). To measure cell growth, the KB cells (1.2 x 103 cells) were seeded in 96-well plates (Nunc, GmbH & Co. KG, Wiesbaden, Germany) and cultivated in DMEM containing 5% FCS for 24 h. Then the medium was replaced with FCS-free medium, and the cells were incubated for 24 h with different quantities of gangliosides as indicated. EGF (1 ng/ml) in the presence of 100 µg/ml bovine serum albumin was added to each well and the cells were cultured for 18 h. To analyze cell proliferation the colorimetric BrdU immunoassay (Roche Diagnostics, Penzberg, Germany) was used, which is based on the measurement of BrdU incorporation during DNA synthesis.
Analysis of binding of the 125I-EGF to cell surface receptor
The specific binding capacity and affinity of 125I-EGF to the EGF receptor were measured in KB cells grown in 48-well plates in the presence or absence of different quantities of HK1-ceramide 2 for 24 h in DMEM without FCS. Cells were washed; 125I-EGF with a specific activity of 2.8 x 105 cpm/ng was added to each well and incubated at 4°C for 2 h. Nonspecific binding of EGF was determined by preincubation of a 5001000-fold excess of cold EGF in four separate wells with different quantities of 125I-EGF (0.25, 0.5, 1, 3.5, 10, 15, and 20 ng/ml 125I-EGF). Cells were washed and incubated with 1 N NaOH at 25°C for 1 h, and the radioactivity associated with the cell monolayers was counted with a gamma counter (Wallac LKB1282 CompuGamma). The background value was subtracted. The effect of HK1-ceramide on the specific binding of 125I-EGF is presented by the method of Scatchard (1949).
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GM3 (NeuAc2
3Galß1
4Glcß1
Cer) is abbreviated according to the nomenclature of Svennerholm (1963)
, and synthesized glycolipids are abbreviated according to the International Union of Pure and Applied Chemistry (IUPAC) Nomenclature (IUPAC Commission, 1972). HK1-ceramide is (2S,3R,4E)-3-hydroxy-2-(octadecanamido)octadec-4-enyl-4-O-[3-O-(5- acetamido-1,3,5-trideoxy-D-glycero-
-D-galacto-2-nonulopyranosyloyl-1''
2'-pyranosyl)-ß-D-galactopyranosyl]-ß-D-glucopyranosid. HK2-ceramide is (2S,3R,4E)-3-hydroxy-2-(octadecanamido)octadec-4-enyl-4-O-[3-O-(5-acetamido-1,3,5-trideoxy-D-glycero-ß-D-galacto-2-nonulopyranosyloyl-1''
2'-pyranosyl)-ß-D-galactopyranosyl]-ß-D-glucopyranosid.
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bremer, E.G. (1994) Glycosphingolipids as effectors of growth and differentiation. In: Hoekstra, D. (ed.), Cell lipids. Current topics in membranes, vol. 40. Academic Press, Orlando, FL. pp. 387411.
Bremer, E.G., Schlessinger, J., and Hakomori, S. (1986) Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor. J. Biol. Chem., 261, 24342440.
Cobb, M.H. and Goldsmith, E.J. (1995) How MAP kinases are regulated. J. Biol. Chem., 270, 1484314846.
Drexler, H.G., Dierks, W., MacLeod, R.A.F., Quentmeier, H., Steube, K., and Uphoff, C.C., (eds.) (1999) DSMZ catalogue of human and animal cell lines, 7th ed. Braunschweig.
Hakomori, S.I. (1996) Tumor malignancy defined by aberrant glycosylation and spingo(glyco)lipid metabolism. Cancer Res., 56, 53095318.[Abstract]
Hanai, N., Nores, G.A., MacLeod, C., Torres-Mendez, C.R., and Hakomori, S. (1988) Ganglioside-mediated modulation of cell growth. Specific effects of GM3 and lipo-GM3 in tyrosine phosphorylation of the epidermal growth factor receptor. J. Biol. Chem., 263, 1091510921.
IUPAC Commission on the Nomenclature of Organic Chemistry (CNOC) and IUAC-IUB Commission on Biochemical Nomenclature (CBN). (1972) Tentative rules for carbohydrate nomenclature 1. J. Biol. Chem., 247, 613635.
Kawamoto, T., Sato, J.D., Le, A., Polikoff, J., Sato, G.H., and Mendelsohn, J. (1983) Growth stimulation of A431 cells by epidermal growth factor identification of high affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc. Natl Acad. Sci. USA, 80, 13371341.[Abstract]
Kawashima, I., Kotani, M., Ozawa, H., Suzuki, M., and Tai, T. (1994) Generation of monoclonal antibodies specific for ganglioside lactones: evidence of the expression of lactone on human melanoma cells. Int. J. Cancer, 58, 263268.[ISI][Medline]
King, A.C. and Cuatrecasas, P. (1982) Resolution of high and low affinity epidermal growth factor receptors. Influence of high affinity component by low temperature, cycloheximide and phorbol esters. J. Biol. Chem., 257, 30533060.
Lee, Y.C. and Lee, R.T. (1995) Carbohydrate-protein interactions: basis of glycobiology. Acc. Chem. Rev., 28, 321327.
Livingston, P.O., Zhang, S., and Lloyd, K.O. (1997) Carbohydrate vaccines that induce antibodies against cancer. 1. Rationale. Cancer Immunol. Immunother., 45, 19.[CrossRef][ISI][Medline]
Meuillet, E.J., Kroes, R., Yamamoto, H., Warner, G., Ferrari, J., Mania-Farnell, B., George, D., Rebbaa, A., Moskal, J.R., and Bremer, E.G. (1999) Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431. Cancer Res., 59, 234240.
Meuillet, E.J., Mania-Farnell, B., George, D., Inokuchi, J.I., and Bremer, E.G. (2000) Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431. Exp. Cell Res., 256, 7482.[CrossRef][ISI][Medline]
Nores, G.A., Dohi, T., Taniguchi, M., and Hakomori, S. (1987) Density-dependent recognition of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as a possible immunogen: requirements for tumor-associated antigen and immunogen. J. Immunol., 139, 31713176.
Oettgen, H.F. (1989) Gangliosides and cancer. VCH, Weinheim.
Rebbaa, A., Hurh, J., Yamamoto, H., Kersey, D.S., and Bremer, E.G. (1996) Ganglioside GM3 inhibition of EGF receptor mediated signal transduction Glycobiology, 6, 399406.[Abstract]
Sato, T., Ishii, M., Ohtake, F., Nagata, K., Terabayashi, T., Kawanishi, Y., and Okahata, Y. (1999) Binding affinity of GM3 lactone for influenza virus. Glycoconj. J., 16, 223227.[CrossRef][ISI][Medline]
Scatchard, G. (1949) The attraction of proteins and small molecules and ions. Ann. N. Y. Acad. Sci., 51, 660672.[ISI]
Suarez-Pestana, E., Greiser, U., Sanchez, B., Fernandez, L.E., Lage, A., Perez, R., and Bohmer, F.D. (1997) Growth inhibition of human lung adenocarcinoma cells by antibodies against the epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed tyrosine phosphatase(s). Br. J. Cancer, 75, 213220.[ISI][Medline]
Svennerholm, L. (1963) Chromatographic separation of human brain gangliosides. J. Neurochem., 10, 613623.[ISI][Medline]
Tardif, M., Coulombe, J., Soulieres, D., Rousseau, A.P., and Pelletier, G. (1996) Gangliosides in human uveal melanoma metastatic process. Int. J. Cancer, 68, 97101.[CrossRef][ISI][Medline]
Tekieva, E.A. and Diatlovitskaia, E.V. (1993) Ganglioside lactones in human stomach and breast tumors. Biokhimiia, 58, 16411644.[Medline]
Tietze, L.F. and Keim, H. (1997) Synthesis of a novel stable GM3-lactone 1 analogue as hapten for a possible immunisation. Angew. Chem. Int. Ed. Engl., 36, 16151617.[ISI]
Tietze, L.F., Keim, H., Janßen, C.O., Tappertzhofen, C., and Olschimke, J. (2000) Synthesis of a ether-bridged GM3-lactone 1 analogue as a target for an antibody-based cancer therapy. Chem. Eur. J., 6, 28012808.[CrossRef][ISI]
Tomoo, T., Kondo, T., and Abe, H. (1996) An efficient short-step total synthesis of ganglioside GM3: effective usage of neighbouring group paticipation strategy. Carbohydr. Res. (Netherlands), 284, 207222.[CrossRef][ISI][Medline]
Weis, F.M. and Davis, R.J. (1990) Regulation of epidermal growth factor receptor signal transduction. Role of gangliosides. J. Biol. Chem., 265, 1205912066.
Xue, L. and Lucocq, J.M. (1997) Low extracellular pH induces activation of ERK2, JNK, and p38 in A431 and swiss 3T3 cells. Biochem. Biophys. Res. Commun., 241, 236241.[CrossRef][ISI][Medline]
Zhang, S., Cordon-Cardo, C., Zhang, H.S., Reuter, V.E., Adluri, S., Hamilton, W.B., Lloyd, K.O., and Livingston, P.O. (1997) Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides. Int. J. Cancer, 73, 4249.[CrossRef][ISI][Medline]
Zhou, Q., Hakomori, S., Kitamura, K., and Igarashi, Y. (1994) GM3 directly inhibits tyrosine phosphorylation and De-N-acetyl-GM3 directly enhances serine phosphorylation of epidermal growth factor receptor, independently of receptor-receptor interaction. J. Biol. Chem., 269, 19591965.
Zhu, J., Li, Y.T., Li, S.C., and Cole, R.B. (1999) Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry. Glycobiology, 9, 985993.
Zurita, A.R., Maccioni, H.J.F., and Daniotti, J.L. (2001) Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides. Biochem. J., 355, 465472.[CrossRef][ISI][Medline]