Treatment Lung metastasisA (nodules EPZ-6438 cost per animal)   B16 cells F3II cells Control 6.4 ± 2.2 6.2 ± 2.1 BSM-preincubated 11.6 ± 1.5* 13.3 ± 3.1* ALung nodules were counted 22 days after intravenous injection of B16 or F3II cells (1 × 105 cells/mouse). Values represent mean ± SEM of at least 10 mice. *p < 0.05 versus the respective control (Mann-Whitney U test). Table 2 Latency and size of melanoma tumors after inoculation of B16 cells, preincubated or not with NeuGc-rich BSM. Treatment Tumor latencyA (days) Tumor DiameterA (mm) Tumor Growth RateA (mm/day) Control 12.8 ± 1.6 2.2 ± 0.9 0.15 ± 0.03

BSM-preincubated 8.4 ± 0.6** 7.1 ± 1.8* 0.18 ± 0.05 ATumor latency represents the time between the subcutaneous injection of a small burden of B16 cells (5 × 103 cells/mouse) and the appearance of detectable tumors. Tumor diameter was recorded at day 35 after tumor cell inoculation. Values represent mean ± SEM of at least 8 mice. **p < 0.01 and *p < 0.05 (t test). Discussion NeuGc and NeuAc are two of the main sialic acids in mammals, being the presence of the oxygen atom in the C-5 position the single difference between them. This seemingly minor difference is crucial in many aspects of cellular behaviour and is produced solely by the CMAH enzyme [5, 17]. This enzyme is present in animals from the deuterostome lineage [18], which LGX818 includes all higher mammals. The expression of

this particular enzyme is the reason for NeuGc presence in most murine normal tissues [19, 20]. In humans, an exon deletion/frameshift mutation in the CMAH gene renders the major pathway for NeuGc production non functional [21]. Sialic acids have been associated with intrinsic receptors that function as ligands for specific leucocyte receptors [22, 23] or as extrinsic receptors themselves for certain pathogens [24, 25]. The presence of the distinctive oxygen atom in NeuGc is determinant in the relationship of the cell with specific molecules or viruses [26, 27]. As an example, mouse CD22 (Siglec-2), a regulator of B-cell

signalling, homeostasis Flavopiridol (Alvocidib) and survival presents high affinity for NeuGc whereas its affinity for NeuAc is low [23]. Exploring the expression of NeuGc in murine cell lines, we have found that B16 and F3II cell lines do not express the CMAH gene and therefore under-express NeuGc in their cell VS-4718 in vitro membranes. Considering that most normal mouse somatic cells are positive for the expression of this gene, it is an interesting fact that malignant cells lack such expression. In cancer, sialic acids are over-expressed as part of gangliosides in several malignancies and their involvement in the malignant cell behaviour has been previously reported [28–30]. The lack of expression of NeuGc in mouse tumor cells suggests that the silencing of the CMAH gene is an important step in the cell transformation process in this specie. Ecsedy et al.