Exogenous incorporation of NeuGc-rich mucin augments N-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines

Laboratory of Molecular Oncology, Quilmes National University, (Roque Saenz Peña 352), Bernal, (B1876BXD), Argentina.
Journal of Experimental & Clinical Cancer Research (Impact Factor: 4.43). 12/2009; 28(1). DOI: 10.1186/1756-9966-28-146
Source: PubMed


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.

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.

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.

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.

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    • "Some studies suggest that its presence in human cancer is due to metabolic incorporation of dietary NeuGc, related with changes in the metabolism of tumor cells. It is well described that cells can process exogenous sialic acids from the extracellular environment and use them for their own glycoconjugates [18, 19]. "
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    • "Mouse melanoma B16 cells (Riken RCB 0557) and human melanoma HMV-II cells (Riken RCB 0777) were grown in phenol redfree DMEM/Ham F12 medium (Life Technologies, Tokyo, Japan) supplemented with 10% FCS (rfDH10) along with penicillin and streptomycin as described previously (Gabri et al., 2009). Normal human melanocytes Hermes 1 cells (Sviderskaya et al., 2003) were obtained from St George's Cell Bank (Bennett et al., 1985) and maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, 200 nM 12-O-tetradecanoylphorbol-13-acetate, 200 pM cholera toxin (Sigma-Aldrich, St. Louis, MO), 10 ng/ml human stem cell factor (QIAGEN, Tokyo, Japan) and 10 nM endothelin 2 (Sigma-Aldrich, St. Louis, MO). "
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    • "We hypothesized that during the rapid disease progression, tumor cells are not able to incorporate enough NeuGc from lung tissue, and immunization is not effective. Thus, we decided to perform an ex vivo preincubation of 3LL cells with purified NeuGc, following a method known to induce expression of NeuGc gangliosides in the cell membrane (Gabri et al., 2009). To confirm antigen expression, cells were analyzed by flow cytometry with a specific anti-NeuGcGM3 antibody. "
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