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Journal of Experimental & Clinical
Exogenous incorporation of neugc-rich mucin augments n-glycolyl
sialic acid content and promotes malignant phenotype in mouse
tumor cell lines
Mariano R Gabri*†, Laura L Otero†, Daniel E Gomez and Daniel F Alonso
Address: Laboratory of Molecular Oncology, Quilmes National University, (Roque Saenz Peña 352), Bernal, (B1876BXD), Argentina
Email: Mariano R Gabri* - firstname.lastname@example.org; Laura L Otero - email@example.com; Daniel E Gomez - firstname.lastname@example.org;
Daniel F Alonso - email@example.com
* Corresponding author †Equal contributors
Background: Carbohydrates embedded in the plasma membrane are one of the main actors
involved in the communication of cells with the microenvironment. Neuraminic sialic acids are
glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-
glycolylneuraminic acid (NeuGc) is synthesized by the cytidine monophospho-N-acetylneuraminic
acid hydroxylase (CMAH), an enzyme expressed in all mammals except humans. In mice, this sugar
is synthesized in several somatic tissues.
Methods: We used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By
CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7
we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-
GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and
evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells
treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate
tumor and metastatic growth.
Results: In the present work we demonstrated the absence of expression of CMAH enzyme in
B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells
with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h,
as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor
latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results
were obtained when cells were incubated with purified NeuGc alone.
Conclusion: Our results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc
in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing
to the malignant phenotype of melanoma and mammary carcinoma cells.
The glycocalyx is composed of a broad variety of sugars
that play a crucial role in the communication of cells with
the microenvironment. Neuraminic sialic acids are 9-car-
bon sugars typically found in the glycocalyx that take part
in the modulation of malignant cell behaviour [1,2]. They
Published: 1 December 2009
Journal of Experimental & Clinical Cancer Research 2009, 28:146doi:10.1186/1756-9966-28-146
Received: 22 May 2009
Accepted: 1 December 2009
This article is available from: http://www.jeccr.com/content/28/1/146
© 2009 Gabri et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Page 8 of 8
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29. Guthmann MD, Bitton RJ, Carnero AJ, Gabri MR, Cinat G, Koliren L,
Lewi D, Fernandez LE, Alonso DF, Gómez DE, Fainboim L: Active
specific immunotherapy of melanoma with a GM3 ganglio-
side-based vaccine: a report on safety and immunogenicity.
J Immunother 2004, 27:442-51.
Gu Y, Zhang J, Mi W, Yang J, Han F, Lu X, Yu W: Silencing of GM3
synthase suppresses lung metastasis of murine breast cancer
cells. Breast Cancer Res 2008, 10:R1.
Ecsedy JA, Manfredi MG, Yohe HC, Seyfried TN: Ganglioside bio-
synthetic gene expression in experimental mouse brain
tumors. Cancer Res 1997, 57:1580-3.
Seyfried TN, el-Abbadi M, Ecsedy JA, Bai HW, Yohe HC: Influence
of host cell infiltration on the glycolipid content of mouse
brain tumors. J Neurochem 1996, 66:2026-33.
Oliva JP, Valdés Z, Casacó A, Pimentel G, González J, Alvarez I, Oso-
rio M, Velazco M, Figueroa M, Ortiz R, Escobar X, Orozco M, Cruz J,
Franco S, Díaz M, Roque L, Carr A, Vázquez AM, Mateos C, Rubio
MC, Pérez R, Fernández LE: Clinical evidences of GM3 (NeuGc)
ganglioside expression in human breast cancer using the
14F7 monoclonal antibody labelled with (99 m)Tc. Breast Can-
cer Res Treat 2006, 96:115-21.
Malykh YN, Schauer R, Shaw L: N-Glycolylneuraminic acid in
human tumors. Biochimie 2001, 83:623-34.
Scursoni AM, Galluzzo L, Camarero S, Pozzo N, Gabri MR, Mateo de
Acosta C, Vazquez AM, Alonso DF, G de Dávila MT: Detection and
characterization of n-glycolylated gangliosides in wilms
tumor by immunohistochemistry. Pediatr Dev Pathol 2009 in
Yin J, Hashimoto A, Izawa M, Miyazaki K, Chen GY, Takematsu H,
Kozutsumi Y, Suzuki A, Furuhata K, Cheng FL, Lin CH, Sato C, Kita-
jima K, Kannagi R: Hypoxic culture induces expression of sialin,
a sialic acid transporter, and cancer-associated gangliosides
containing non-human sialic acid on human cancer cells. Can-
cer Res 2006, 66:2937-45.
Carraway KL, Perez A, Idris N, Jepson S, Arango M, Komatsu M, Haq
B, Price-Schiavi SA, Zhang J, Carraway CA: Muc4/sialomucin com-
plex, the intramembrane ErbB2 ligand, in cancer and epithe-
lia: to protect and to survive. Prog Nucleic Acid Res Mol Biol 2002,
Ho SB, Niehans GA, Lyftogt C, Yan PS, Cherwitz DL, Gum ET, Dahiya
R, Kim YS: Heterogeneity of mucin gene expression in normal
and neoplastic tissues. Cancer Res 1993, 53:641-51.
Fernandez LE, Alonso DF, Gomez DE, Vazquez AM: Ganglioside-
based vaccines and anti-idiotype antibodies for active immu-
notherapy against cancer. Expert Rev Vaccines 2003, 2:817-823.
Venuti A: Progress and challenges in the vaccine-based treat-
ment of head and neck cancers. J Exp Clin Cancer Res 2009,