Electrophilic reactive aldehydes as a therapeutic target in colorectal cancer prevention and treatment

Abstract

Colorectal cancer (CRC) is a major health problem worldwide. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation and form covalent adducts with amine-containing macromolecules. We have shown high levels of adducts of isoLGs in colonic epithelial cells of patients with CRC. We thus investigated the role of these reactive aldehydes in colorectal cancer development. We found that 2-hydroxybenzylamine (2-HOBA), a natural compound derived from buckwheat seeds that acts as a potent scavenger of electrophiles, is bioavailable in the colon of mice after supplementation in the drinking water and does not affect the colonic microbiome. 2-HOBA reduced the level of isoLG adducts to lysine as well as tumorigenesis in models of colitis-associated carcinogenesis and of sporadic CRC driven by specific deletion of the adenomatous polyposis coli gene in colonic epithelial cells. In parallel, we found that oncogenic NRF2 activation and signaling were decreased in the colon of 2-HOBA-treated mice. Additionally, the growth of xenografted human HCT116 CRC cells in nude mice was significantly attenuated by 2-HOBA supplementation. In conclusion, 2-HOBA represents a promising natural compound for the prevention and treatment of CRC.

Fig. 1.. Effect of 2-HOBA on inflammation-mediated colon carcinogenesis.

A C57BL6 mice were treated with AOM (12.5 mg.kg^–1)-DSS (4%) and were given 2-HOBA (1 mg.ml^–1) throughout the experiments, except during DSS treatment. B Body weights were measured weekly and are depicted as percentage of initial body weight; ***P < 0.001 and ****P < 0.0001 compared to AOM-DSS-treated mice in the control and 2-HOBA groups. C After 56 days, colons were removed and the concentration of 2-HOBA was measured by LC/ESI/MS/MS. D The number of tumors was counted. E-F tumor size was assessed by the average tumor size per mouse (E) and as a percent of total tumors (n = 106 tumors for the AOM-DSS group and n = 96 for AOM-DSS + 2-HOBA; F). G Tumor burden was calculated. H Representative images of H&E staining and tumors are surrounded by dotted lines. The scale bars correspond to 200 μm. I The histological injury score was calculated from the H&E staining. In figures with dot plots, P was determined by two-way ANOVA and Tukey test in B, one-way ANOVA and Tukey test in C, or Student’s t test (D, E, G, I); we also used Chi-Square for the contingency analysis (F).

Fig. 2.. Effect of 2-HOBA on a model of sporadic colorectal cancer.

A CDX2P-CreER^T2;Apc^fl/fl mice were treated or not with TAM (4.5 mg.kg^–1) ± 2-HOBA (1 mg.ml^–1). B Body weights were monitored weekly and are shown as percentage of initial body weight; *P < 0.05, **P < 0.01, and ****P < 0.0001 compared TAM-treated mice, and §P < 0.05, §§P < 0.01, and §§§§P < 0.0001 compared to TAM-treated mice in the 2-HOBA group. C 35 days after TAM injection, colonic levels of 2-HOBA were measured by LC/ESI/MS/MS. D-F Tumor number (D-E) and burden (F) were determined in the mid and distal colon. Note that there were no tumors in animals that did not receive TAM, treated or not with 2-HOBA. G Correlation plots comparing tumor burden (from Fig. 3F) and 2-HOBA concentrations (from Fig. 3C). H The H&E staining shows a large tumor with a complex growth pattern indicating high-grade dysplasia in the TAM group and two smaller tumors formed by densely packed neoplastic crypts with low-grade dysplasia; scale bar, 100 μm. I The inflammation score was determined by histologic assessment. P values were calculated by two-way ANOVA and Tukey test (B), one-way ANOVA and Tukey test (C), and Student’s t test (E, F, I). In (G), statistical analysis was performed using the Pearson correlation test.

Fig. 3.. Regulation of NRF2 activation by electrophiles.

The colon of CDX2P-CreER^T2;Apc^fl/fl mice ± TAM (4.5 mg.kg^–1) ± 2-HOBA (1 mg.ml^–1) was immunostained with the D11 antibody, which detects isoLG-lysyl adducts (A) and the nuclear staining was quantified (B). Each dot represents a mouse, and 20 crypts per animal were assessed. Scale bars, 50 μm. C RNA was extracted from non-tumor areas (NT) or tumors (T) from CDX2P-CreER^T2;Apc^fl/fl mice ± TAM ± 2-HOBA, and gene expression was then measured by RT-real-time PCR. D The activation of NRF2 was assessed by immunofluorescence and the images are representatives of 3 animals per group. E Quantification of nuclear translocation of NRF2; each dot represents a mouse, and 5–10 crypts per animal were assessed. Scale bars, 50 μm. P values were calculated by two-way ANOVA and Tukey test (C) or Dunnett’s test (A, E).

Fig. 4.

Treatment with 2-HOBA in mice with human xenografts. Human CRC HCT116 cells were injected in the flank of nude mice. Animals were then treated or not with 2-HOBA (1 mg.ml^–1). A The levels of 2-HOBA were measured in the tumors after 35 days. B Tumor volume was measured weekly in sham-treated (left panel) or 2-HOBA-treated (middle panel) mice. The right panel depicts mean ± SEM; the fixed effects (type III) P value (time and treatment) was calculated by two-way ANOVA and Tukey test. C-D On day 35, tumors were harvested (C), measured, and the volume was calculated (D). P was determined by the Student’s t test. Scale bar, 1 cm. E Correlation comparing tumor volume (from Fig. 5D) and 2-HOBA concentrations (from Fig. 5A, middle panel). Statistical analysis was performed using the Pearson correlation test.