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Zhang et al. J Cancer Metastasis Treat 2019;5:56 I http://dx.doi.org/10.20517/2394-4722.2018.112 Page 7 of 9

The cellular expression pattern of glycosyltransferases and Lewis antigens detected in the normal mucosa
can change during neoplastic transformation. The stomach mucosa was investigated for Lewis antigens
in the presence of pro-inflammatory cytokines . The level of one type of Lewis antigen decreased after
[4]
treatment with IL-1 or IL-6, whereas the levels of other carbohydrate antigens were unaffected .
[4]
Lectins are plant-derived macromolecules specific for carbohydrate moieties. The MAL lectin specifically
bind the sialyla2,3 Galb1,4GlcNAc/Glc trisaccharide sequence . Expression of a2,3-SA residues of
[22]
N-cadherin was shown to be altered in metastatic melanoma cell lines . The expression of a2,3-SA residues
[23]
in breast cancer using MAL-staining were analyzed by Cui et al. The results suggested that high levels
[24]
of a2,3-SA residues was closely associated with lymph node metastasis and invasive depth in breast cancer
patients. The highly metastatic breast cancer cell line MDA-MB-231 had higher expression of a2,3-sialic acid
residues compared to T-47D and MCF-7 depending on the mRNA levels of a2,3-ST genes. Indeed, the tumor
microenvironment can direct the level of inflammation. Also, the metastatic potential and malignancy of
cancer cells is closely associated with aberrant sialylation pattern .
[24]

The enzyme that adds a2-6linked sialic acids, b-galactoside a2-6 sialyltransferase (ST6Gal-I), is known to be
upregulated in many tumor types including colon adenocarcinomas , and high expression levels have been
[25]
associated with poor prognosis and metastasis. ST6Gal-I sialylation of membrane glycoproteins contributes
to metastasis by enhancing cell motility and invasion through the extra cellular matrix (ECM). Increased
negative charged properties of sialic acids was correlated with reduced adhesiveness of tumor cells and may
be suitable for conformational change of integrin and enhances its function in cell-ECM interactions .
[26]

Interestingly, in our study we show a high expression of a2,3-SA on both cell lines, whereas the a2,6-SA, as
analyzed with the lectin SNA, displayed a lower expression on the MCF-7 cells. It has been shown that high
levels of sialylation of cell surface glycoconjugates can significantly increase metastasis of colon carcinoma
cells and human melanoma cells.

In addition to directly modulating cell motility, a2,3-SA residues are involved in the synthesis of sialyl Lewis
X determinants, which are the major ligands for endothelial E-selectin . The sialyl Lewis X structure on
[27]
malignant cells is suggested to facilitate tumor cell dissemination by mediating the tumor-endothelial cell
interactions [28,29] . Overall, many studies support the hypothesis that an increase of sialylation of cancer cells
play an important role in tumor metastasis.

In the present study, the MCF-7 cell line showed a small increase of the SA-MIP binding after treatment with
the cytokine cocktail derived from PHA-stimulated Jurkat cells. On the other hand, the SA-MIP binding
when treating the RAW264.7 cell line with the same cocktail was not different compared to the effect of
recombinant IL-6 or IL-8. Interestingly, an increase in binding of the lectins MAL and SNA was also detected
after cytokine cocktail treatment. By analyzing with ELISA, we show that the cocktail contains increased
amounts of IL-8, as well as low levels of IL-2 and TNF-a. IL-6 could not be detected in the PHA-stimulated
samples. IL-8 is a chemokine, functioning by attracting cells to a site of infection, and it is therefore tempting
to speculate that the increased SA-MIP binding seen by the RAW264.7 cells, could be due to a regulated
SA-expression in response to IL-8. The natural ligands for SA are selectins and siglecs . Indeed, the rolling
[30]
of cancer cells ectopically expressing the selectin ligands on endothelial cells is potentially a crucial step
favoring the metastatic process in vivo . The combined analysis of SA and the targeting of SA to the ligands
[31]
selectins and siglecs will be attractive for further investigations.

In conclusion, cancer cell migration and invasion is controlled by protein glycosylation and the ECM. SA
is one of several important players in this crucial process. Inflammation and cytokine production will
modulate the cellular microenvironment. We have studied cell lines in vitro that showed that one of the cell