Glycation Leads to Increased Invasion of Glioblastoma Cells

Abstract

Glioblastoma (GBM) is a highly aggressive and invasive brain tumor with a poor prognosis despite extensive treatment. The switch to aerobic glycolysis, known as the Warburg effect, in cancer cells leads to an increased production of methylglyoxal (MGO), a potent glycation agent with pro-tumorigenic characteristics. MGO non-enzymatically reacts with proteins, DNA, and lipids, leading to alterations in the signaling pathways, genomic instability, and cellular dysfunction. In this study, we investigated the impact of MGO on the LN229 and U251 (WHO grade IV, GBM) cell lines and the U343 (WHO grade III) glioma cell line, along with primary human astrocytes (hA). The results showed that increasing concentrations of MGO led to glycation, the accumulation of advanced glycation end-products, and decreasing cell viability in all cell lines. The invasiveness of the GBM cell lines increased under the influence of physiological MGO concentrations (0.3 mmol/L), resulting in a more aggressive phenotype, whereas glycation decreased the invasion potential of hA. In addition, glycation had differential effects on the ECM components that are involved in the invasion progress, upregulating TGFβ, brevican, and tenascin C in the GBM cell lines LN229 and U251. These findings highlight the importance of further studies on the prevention of glycation through MGO scavengers or glyoxalase 1 activators as a potential therapeutic strategy against glioma and GBM.

Keywords: advanced glycation end-products; astrocytes; glioblastoma; glioma; glycation; invasion; methylglyoxal.

Figure 1

Cell vitality of glioma cell lines and hA after MGO treatment. The cell vitality of LN229 (A), U251 (B), U343 (C), and hA (D) cells was determined using an XTT assay after MGO treatment. Graphs show intracellular mitochondrial dehydrogenase (MDH) activity normalized to untreated cells after 24 h. Student’s t-test was performed for statistical analysis. Graphs represent the means and SDs of three independent biological replicates.

Figure 2

Microscope imaging of glioma cell lines and hA 24 h after MGO treatment. Bright field (above) and fluorescence (below) microscope imaging of LN229 (A), U251 (B), U343 (C), and hA (D) 24 h after MGO treatment. Cells were stained with DAPI (blue) and propidium iodide (red). Scale bar = 100 μm.

Figure 3

Glycation of glioma cell lines and hA. Immunoblot of LN229 (A), U251 (B), U343 (C), and hA (D) with different MGO concentrations (left). Antibody against carboxymethyl lysine (CML) was used to detect glycation. Graphs (right) 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 SDs of three independent biological replicates.

Figure 4

Chemotactic cell migration of glioma cell lines and hA after MGO treatment. Graphs display chemotaxis of LN229 (A), U251 (B), U343 (C), and hA (D) after 24 h and 48 h normalized to control cells, after treatment with 0.3 or 0.6 mmol/L MGO. Statistical analysis was performed using Student’s t-test. Graphs represent the means and SDs of three independent biological replicates.

Figure 5

Invasion of glioma cell lines and hA after MGO treatment. LN229 (A), U251 (B), U343 (C), and hA (D) were cultivated in absence or presence of MGO (0.3 mmol/L or 0.6 mmol/L) on CIM-plates, coated with Geltrex to imitate basement membranes. Invasion was measured every 15 min for 48 h. Graphs (left column) show cell indices normalized to the untreated cells and graphs (right column) show measured cell indices for 12, 24, 36, and 48 h. Student’s t-test was performed for statistical analysis. Graphs represent the means and SDs of three independent biological replicates.

Figure 6

Adhesion of glioma cell lines and hA after MGO treatment. LN229 (A), U251 (B), U343 (C), and hA (D) were seeded at concentrations of 0.3 mmol/L and 0.6 mmol/L MGO on plates either coated with fibronectin, collagen, or left uncoated. Graphs display measured cell index after 4 h. Absolute cell index was used to show the different adherence to the different matrices and to illustrate the differences between the cells. Student’s t-test was performed for statistical analysis. Graphs represent the means and SDs of three independent biological replicates.

Figure 7

mRNA expression of invasion-associated ECM molecules and transcription factors. Heat map of mRNA expression of LN229, U251, U343, and hA after treatment with 0.3 mmol/L MGO normalized to untreated cells (A). Heatmap of mRNA expression of LN229, U251, and U343 cells normalized to the expression of hA (B). Three independent biological replicates of the mRNA were analyzed by qPCR.

Figure 8

E- and N-cadherin expression after MGO treatment. Immunoblot of LN229 (A), U251 (B), U343 (C), and hA (D) cells with different MGO concentrations (0.3, 0.6, and 1 mmol/L) (left column) and antibody against E- and N-cadherin. GAPDH was used as loading control. Graphs show representative quantification of E-cadherin (middle column) and N-cadherin (right column) Western blots from three independent biological replicates, normalized to the untreated cells. Student’s t-test was performed for statistical analysis. Graphs represent the means and SDs.

Figure 9

Dual role of MGO in GBM and glioma cells. The increase in glycation is proportional to the rising concentration of MGO. At high doses, MGO has a cytotoxic effect on GBM and glioma cells; however, when present at lower, physiological concentrations, it causes alterations in the ECM and increased invasion.