Glycation Interferes with the Expression of Sialyltransferases and Leads to Increased Polysialylation in Glioblastoma Cells

Abstract

Glioblastoma (GBM) is a highly aggressive brain tumor that often utilizes aerobic glycolysis for energy production (Warburg effect), resulting in increased methylglyoxal (MGO) production. MGO, a reactive dicarbonyl compound, causes protein alterations and cellular dysfunction via glycation. In this study, we investigated the effect of glycation on sialylation, a common post-translational modification implicated in cancer. Our experiments using glioma cell lines, human astrocytes (hA), and primary glioma samples revealed different gene expressions of sialyltransferases among cells, highlighting the complexity of the system. Glycation has a differential effect on sialyltransferase expression, upregulating ST8SIA4 in the LN229 and U251 cell lines and decreasing the expression in normal hA. Subsequently, polysialylation increased in the LN229 and U251 cell lines and decreased in hA. This increase in polysialylation could lead to a more aggressive phenotype due to its involvement in cancer hallmark processes such as immune evasion, resistance to apoptosis, and enhancing invasion. Our findings provide insights into the mechanisms underlying GBM aggressiveness and suggest that targeting glycation and sialylation could be a potential therapeutic strategy.

Keywords: astrocytes; glioblastoma; glioma; glycation; methylglyoxal; polysialylation; sialyltransferases.

Figure 1

Groups of STs. The STs are grouped into four families according to the bonds they synthetize. They transfer activated CMP-Neu5Ac onto Galactose, N-Acetylgalactosamine (GALNAC), or Neu5Ac moieties of glycoproteins. The ST3Gal family members transfer sialic acids to terminal galactose residues via α-2,3 linkages, whereas the two known members of the ST6Gal family do so via α-2,6 linkages. The six members of the ST6GALNAC family transfer sialic acids from GALNAC residues via α-2,6-linkages. The members of the ST8SIA family transfer sialic acids to other terminal sialic acid residues via α-2,8-linkages. Created with Biorender.com (agreement number: NV255ORK3Q).

Figure 2

mRNA levels of STs in LN229, U251, U343, and hA. Heatmap of sialyltransferase mRNA levels in LN229 cells, U251 cells, U343 cells, and hA measured with qPCR.

Figure 3

RNAseq analysis of sialyltransferase expression in nine primary glioma cells. Heat map of sialyltransferase expression levels. FPKM, fragments per kilobase per million fragments.

Figure 4

Cell index with and without MGO treatment. Impedance was measured with xCELLigence RTCA eSight. The graph shows a representative real-time analysis of U343 cells after treatment with 0.3 mmol/L MGO (green line), 0.6 mmol/L MGO (blue line), and without MGO (red line) over 42 h.

Figure 5

Glycation of glioma cell lines and hA. Immunoblot of LN229 cells, U251 cells, U343 cells, and hA with different MGO concentrations (left). Antibodies against carboxymethyl lysine (CML) were used to detect glycation. GAPDH was used as loading control (right).

Figure 6

Relative mRNA levels of ST3GAL1-6 in LN229 cells (A), U251 cells (B), U343 cells (C), and hA (D) measured with qPCR. Graphs show mRNA levels after 24 h with 0.3 mmol/L MGO normalized to the mean value of control cells. Student’s t-test was performed for statistical analysis. Error bars represent the means and SD of four independent biological replicates.

Figure 7

Relative mRNA levels of ST6GAL1-2 in LN229 cells (A), U251 cells (B), U343 cells (C), and hA (D) measured with qPCR. Graphs show mRNA levels after 24 h with 0.3 mmol/L MGO. Student’s t-test was performed for statistical analysis. Error bars represent the means and SD of four independent biological replicates.

Figure 8

Relative mRNA levels of ST6GALNAC1-6 in LN229 cells (A), U251 cells (B), U343 cells (C), and hA were measured with qPCR (D). Graphs show mRNA levels after 24 h with 0.3 mmol/L MGO normalized to the mean value of control cells. Student’s t-test was performed for statistical analysis. Error bars represent the means and SD of four independent biological replicates.

Figure 9

Relative mRNA levels of ST8SIA1-6 in LN229 cells (A), U251 cells (B), U343 cells (C), and hA (D) measured with qPCR. Graphs show mRNA levels after 24 h with 0.3 mmol/L MGO. Student’s t-test was performed for statistical analysis. Error bars represent the means and SD of four independent biological replicates.

Figure 10

Polysialylation after MGO treatment. Immunoblot of LN229 cells (A), U251 cells (B), and hA (C) with different MGO concentrations (0, 0.3, 0.6, and 1 mmol/L) (left column). Polysialylation was detected with PolySia antibody. Graphs (right column) show representative quantification of the blot, normalized to the untreated cells. GAPDH was used as loading control. Student´s t-test was performed for statistical analysis. Graphs represent the means and SD of three independent biological replicates.

Figure 11

IHC analysis of carboxymethyl lysine and PolySia in GBM tissue. Representative IHC analysis including hematoxylin (HE), carboxymethyl lysine, and PolySia staining in different GBM tissue (A,B). Bar = 100 µm.