Methylglyoxal-Mediated Stress Correlates with High Metabolic Activity and Promotes Tumor Growth in Colorectal Cancer

Abstract

Cancer cells generally rely on aerobic glycolysis as a major source of energy. Methylglyoxal (MG), a dicarbonyl compound that is produced as a side product during glycolysis, is highly reactive and induces the formation of advanced glycation end-products that are implicated in several pathologies including cancer. All mammalian cells have an enzymatic defense against MG composed by glyoxalases GLO1 and GLO2 that converts MG to d-lactate. Colorectal cancer (CRC) is one of the most frequently occurring cancers with high morbidity and mortality. In this study, we used immunohistochemistry to examine the level of MG protein adducts, in a series of 102 CRC human tumors divided into four clinical stages. We consistently detected a high level of MG adducts and low GLO1 activity in high stage tumors compared to low stage ones suggesting a pro-tumor role for dicarbonyl stress. Accordingly, GLO1 depletion in CRC cells promoted tumor growth in vivo that was efficiently reversed using carnosine, a potent MG scavenger. Our study represents the first demonstration that MG adducts accumulation is a consistent feature of high stage CRC tumors. Our data point to MG production and detoxification levels as an important molecular link between exacerbated glycolytic activity and CRC progression.

Keywords: 18F-Fluorodeoxyglucose (18F-FDG); MG-adducts; colorectal cancer; glyoxalase 1; methylglyoxal.

Figure 1

Argpyrimidine adducts are highly accumulated in colorectal cancer tissues when compared to matched non-cancer tissue. (A) Argpyrimidine adducts have been evaluated in 12 CRC tissues (T) and in their non-tumor counterpart (N) by Western blot analysis. HSC70 and Ponceau Red staining are both shown as loading control. The quantification of all visible bands corresponding to argpyrimidine adducts has been performed using ImageJ software (NIH Image, http://rsb.info.nih.gov/ij/, 1.42p, RRID:SCR_003070). Tumor to normal tissue (T/N) ratio of argpyrimidine adducts is shown for each patient. The most intense band of the Ponceau Red staining (boxed in red) has been used for the normalization; (B) Quantification of panel (A) data demonstrates a significant overall increase of MG-adducts in tumor tissues compared to normal counterpart. Bars represent the mean ± SEM of 12 patients analyzed. Statistical analysis has been performed using one way Anova, Mann–Whitney test and * p < 0.05.

Figure 1

Argpyrimidine adducts are highly accumulated in colorectal cancer tissues when compared to matched non-cancer tissue. (A) Argpyrimidine adducts have been evaluated in 12 CRC tissues (T) and in their non-tumor counterpart (N) by Western blot analysis. HSC70 and Ponceau Red staining are both shown as loading control. The quantification of all visible bands corresponding to argpyrimidine adducts has been performed using ImageJ software (NIH Image, http://rsb.info.nih.gov/ij/, 1.42p, RRID:SCR_003070). Tumor to normal tissue (T/N) ratio of argpyrimidine adducts is shown for each patient. The most intense band of the Ponceau Red staining (boxed in red) has been used for the normalization; (B) Quantification of panel (A) data demonstrates a significant overall increase of MG-adducts in tumor tissues compared to normal counterpart. Bars represent the mean ± SEM of 12 patients analyzed. Statistical analysis has been performed using one way Anova, Mann–Whitney test and * p < 0.05.

Figure 2

Consistent increase of argpyrimidine adducts in high stage tumors compared with low stage ones suggests a pro-tumor role for dicarbonyl stress. (A) Argpyrimidine adducts were examined in a series of 102 primary colorectal cancer patients samples grouped into four clinical stages (T1, T2, T3 and T4) and six normal colorectal tissues. One representative picture is shown for each stage analyzed (100× magnification); (B) Immunohistochemical quantification shows argpyrimidine staining evaluation divided into 4 groups (negative, weak, moderate and strong staining) based on the score values. Each dot represents one case and bars represent median. Statistical analysis has been performed using one-way ANOVA followed by Dunn’s Multiple Comparison Test and * p < 0.05, ** p < 0.01, *** p < 0.001. In the right panel, the percentage of negative, weak, moderate and strong argpyrimidine staining is shown for normal tissue and stage 1 to stage 4 tumors; (C) An IHC using an antibody against MG-H1 adducts has been performed on 12 CRC samples. IHC staining is shown for representative negative, moderate and strong staining (100× magnification). In accordance with argpyrimidine immunostaining, more MG-H1 adducts have been detected in the highest stage lesions; (D) Argpyrimidine and MG-H1 IHC detection showed a significant positive correlation (R² = 0.74, p = 0.0003, Spearman rank correlation test).

Figure 2

Consistent increase of argpyrimidine adducts in high stage tumors compared with low stage ones suggests a pro-tumor role for dicarbonyl stress. (A) Argpyrimidine adducts were examined in a series of 102 primary colorectal cancer patients samples grouped into four clinical stages (T1, T2, T3 and T4) and six normal colorectal tissues. One representative picture is shown for each stage analyzed (100× magnification); (B) Immunohistochemical quantification shows argpyrimidine staining evaluation divided into 4 groups (negative, weak, moderate and strong staining) based on the score values. Each dot represents one case and bars represent median. Statistical analysis has been performed using one-way ANOVA followed by Dunn’s Multiple Comparison Test and * p < 0.05, ** p < 0.01, *** p < 0.001. In the right panel, the percentage of negative, weak, moderate and strong argpyrimidine staining is shown for normal tissue and stage 1 to stage 4 tumors; (C) An IHC using an antibody against MG-H1 adducts has been performed on 12 CRC samples. IHC staining is shown for representative negative, moderate and strong staining (100× magnification). In accordance with argpyrimidine immunostaining, more MG-H1 adducts have been detected in the highest stage lesions; (D) Argpyrimidine and MG-H1 IHC detection showed a significant positive correlation (R² = 0.74, p = 0.0003, Spearman rank correlation test).

Figure 2

Consistent increase of argpyrimidine adducts in high stage tumors compared with low stage ones suggests a pro-tumor role for dicarbonyl stress. (A) Argpyrimidine adducts were examined in a series of 102 primary colorectal cancer patients samples grouped into four clinical stages (T1, T2, T3 and T4) and six normal colorectal tissues. One representative picture is shown for each stage analyzed (100× magnification); (B) Immunohistochemical quantification shows argpyrimidine staining evaluation divided into 4 groups (negative, weak, moderate and strong staining) based on the score values. Each dot represents one case and bars represent median. Statistical analysis has been performed using one-way ANOVA followed by Dunn’s Multiple Comparison Test and * p < 0.05, ** p < 0.01, *** p < 0.001. In the right panel, the percentage of negative, weak, moderate and strong argpyrimidine staining is shown for normal tissue and stage 1 to stage 4 tumors; (C) An IHC using an antibody against MG-H1 adducts has been performed on 12 CRC samples. IHC staining is shown for representative negative, moderate and strong staining (100× magnification). In accordance with argpyrimidine immunostaining, more MG-H1 adducts have been detected in the highest stage lesions; (D) Argpyrimidine and MG-H1 IHC detection showed a significant positive correlation (R² = 0.74, p = 0.0003, Spearman rank correlation test).

Figure 3

¹⁸F-FDG PET activity and argpyrimidine accumulation in CRC patients. (A) SUVmax is significantly higher in tumors with high dicarbonyl stress. Each dot represents one case and bars represent mean ± SEM. (p = 0.0105, Mann–Whitney test); (B) GLO1 activity and expression were evaluated in stage 1 (n = 3), stage 2 (n = 5), stage 3 (n = 4) and stage 4 (n = 5) colorectal cancer patients. β-Actin is used as loading control. Right panel, GLO1 activity is significantly higher in low stage tumors compared with high stage ones. Average of three technical replicates ± SEM is shown (Neuwman–Keul Tests, * p < 0.05); (C) The correlation analysis performed between argpyrimidine immunostaining score and GLO1 activity evaluated on 17 CRC patients demonstrated a significant inverse correlation between these two parameters (Spearman rank correlation test).

Figure 3

¹⁸F-FDG PET activity and argpyrimidine accumulation in CRC patients. (A) SUVmax is significantly higher in tumors with high dicarbonyl stress. Each dot represents one case and bars represent mean ± SEM. (p = 0.0105, Mann–Whitney test); (B) GLO1 activity and expression were evaluated in stage 1 (n = 3), stage 2 (n = 5), stage 3 (n = 4) and stage 4 (n = 5) colorectal cancer patients. β-Actin is used as loading control. Right panel, GLO1 activity is significantly higher in low stage tumors compared with high stage ones. Average of three technical replicates ± SEM is shown (Neuwman–Keul Tests, * p < 0.05); (C) The correlation analysis performed between argpyrimidine immunostaining score and GLO1 activity evaluated on 17 CRC patients demonstrated a significant inverse correlation between these two parameters (Spearman rank correlation test).

Figure 4

In vitro characterization of colorectal cancer cell lines. (A) Argpyrimidine and (B) MG-H1 accumulation has been evaluated in HCT116, HT29 and LS174T human colorectal cancer cell lines using Western blot analysis; (C) GLO1 basal expression and (D) activity in HCT116, HT29 and LS174T cells. All immunoblots were quantified by densitometric analysis and normalized for β-actin. Statistical analysis has been performed using Newman–Keuls multiple comparison test, * p < 0.05. All data are representative of three independent experiments (n = 3) as the mean values ± SEM.

Figure 5

GLO1 depletion favors colorectal cancer cell growth in vivo. (A) The efficiency of GLO1 knockdown in HCT116 cells was validated by Western blot analysis; (B) Increased argpyrimidine level has been observed in GLO1 depleted cells compared with shNT control cells using Western blot analysis. β-Actin is shown as loading control; (C) Effect of GLO1 silencing on HCT116-derived tumor growth in chorioallantoic membrane (CAM) tumor model. The weight (left) and volume (right) of CAM experimental tumors collected at Day 7 is shown (at least 6 eggs/group). Data are shown as mean values ± SEM. Representative macroscopic tumor appearance is shown for each condition according to CAM experiment details are described in Material and Methods; (D) Representative GLO1 expression and argpyrimidine levels in CAM experimental tumors (400× magnification). IHC scoring for each is shown in panels on the right. Each dot represents one case and bars represent median. Statistical analysis has been performed using Bonferroni Multiple Comparison Test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

GLO1 depletion favors colorectal cancer cell growth in vivo. (A) The efficiency of GLO1 knockdown in HCT116 cells was validated by Western blot analysis; (B) Increased argpyrimidine level has been observed in GLO1 depleted cells compared with shNT control cells using Western blot analysis. β-Actin is shown as loading control; (C) Effect of GLO1 silencing on HCT116-derived tumor growth in chorioallantoic membrane (CAM) tumor model. The weight (left) and volume (right) of CAM experimental tumors collected at Day 7 is shown (at least 6 eggs/group). Data are shown as mean values ± SEM. Representative macroscopic tumor appearance is shown for each condition according to CAM experiment details are described in Material and Methods; (D) Representative GLO1 expression and argpyrimidine levels in CAM experimental tumors (400× magnification). IHC scoring for each is shown in panels on the right. Each dot represents one case and bars represent median. Statistical analysis has been performed using Bonferroni Multiple Comparison Test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Carnosine treatment of GLO1-depleted HCT116 cells inhibits tumor growth in vivo. HCT116 shGLO1#1 and #2 and shNT control cells were implanted on chorioallantoic (CAM) tumor model. Cancer cells were treated with carnosine (10 mM) from the day after implantation on CAM until the end of the experiment. Tumor growth has been evaluated seven days post-implantation (at least 10 eggs/group) as described under Material and Methods section. (A) Representative macroscopic tumor appearance in each condition is shown; (B) Reduction of tumor volume (left panel) and weight (right panel) after carnosine treatment of GLO1 depleted HCT116-derived tumors, data are shown as mean values ± SEM. Statistical analysis has been performed using Bonferroni Multiple Comparison Test; (C) Significant decrease of argpyrimidine level in experimental CAM tumors upon carnosine treatment. Each dot represents one case and bars represent median. Statistical analysis has been performed using Dunn’s Multiple Comparison Test and Mann–Whitney test; (D) Percentage of Ki67 positive cells in experimental CAM tumors upon carnosine treatment. Statistical analysis has been performed using Chi-square Contingency Test. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns = not significant.