The epigenetic effects of aspirin: the modification of histone H3 lysine 27 acetylation in the prevention of colon carcinogenesis in azoxymethane- and dextran sulfate sodium-treated CF-1 mice

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide. Chronic inflammation appears to enhance the risk of CRC. Emerging evidence has suggested that epigenetic mechanisms play an important role in CRC. Aspirin [acetylsalicylic acid (ASA)] has been shown to prevent CRC; however, the epigenetic mechanisms of its action remain unknown. This study investigated the protective role of ASA in azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted colitis-associated colon cancer (CAC) and examined the epigenetic effects, particularly on histone 3 lysine 27 acetylation (H3K27ac), underlying the preventive effect of ASA. CF-1 mice were fed with AIN-93M diet with or without 0.02% ASA from 1 week prior to AOM initiation until the mice were killed 20 weeks after AOM injection. Our results showed that AOM/DSS + ASA significantly suppressed inflammatory colitis symptoms and tumor multiplicity. AOM/DSS + ASA reduced AOM/DSS-induced protein expression and the activity of histone deacetylases (HDACs) and globally restored H3K27ac. Furthermore, AOM/DSS + ASA inhibited AOM/DSS-induced enrichment of H3K27ac in the promoters of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) that corresponded to the dramatic suppression of the messenger RNA (mRNA) and protein levels. Surprisingly, no significant changes in the H3K27ac abundance in the prostaglandin-endoperoxide synthase 2 (Cox-2) promoters or in the Cox-2 mRNA and protein expression were observed. Collectively, our results suggest that a potential novel epigenetic mechanism underlies the chemopreventive effects of ASA, and this mechanism attenuates CAC in AOM/DSS-induced CF-1 mice via the inhibition of HDACs and the modification of H3K27ac marks that suppress iNOS, TNF-α and IL-6.

Figure 1.

The dietary administration of ASA inhibits CAC in AOM/DSS-induced CF-1 mice. (A) The experimental protocol for a chemoprevention study with ASA using the AOM/DSS model. (B) AOM/DSS + ASA suppressed the elevated DAI starting at day 3 of DSS administration, and the suppression was significant at day 7. (C) The administration of ASA did not cause significant weight loss. (D) AOM/DSS + ASA decreased tumor incidence and tumor multiplicity. (E) The effect of ASA on tumor volume. *P < 0.05 versus AOM/DSS.

Figure 2.

Histologic characterization of colonic tumors and lesions in AOM/DSS-treated CF-1 mice. Normal mucosa was observed in the mice in the control groups (A). AOM/DSS resulted in crypt dysplasia (black star) with the loss of goblet cells in the crypts (B); adenomas with inflammation, increased nucleus-to-cytoplasm ratios, nuclear crowding (solid arrow) and mitosis (arrow head) (C); adenocarcinoma with inflammation; leukocytes infiltrated into the diluted lumen (triangle) composed of a pattern of cribriform glands, nucleus hyperchromasia (dotted arrow) and mitosis (D). The colon from AOM/DSS + ASA-treated mice showed attenuated inflammation severity and cancer lesions, normal mucosa (E) or dysplasia with inflammation (F). The average number of histologically characterized alterations, including dysplasia, adenoma and adenocarcinoma, per mouse are presented (G).

Figure 3.

The effect of ASA on the protein expression of HDAC 1–5 and HDAC activity. Total proteins were extracted and examined via western blotting. AOM/DSS treatment significantly increased the protein expression of HDAC 1–5, whereas AOM/DSS + ASA effectively inhibited the protein expression of HDAC 2–5. Representative bands are shown (A), and the bar graph presents the fold change of the blot density determined by ImageJ (B). Nuclear proteins were extracted and assayed for HDAC activity. AOM/DSS + ASA strongly suppressed significantly elevated HDAC activity in AOM/DSS-treated mice (C). *P < 0.05 and **P < 0.01.

Figure 4.

AOM/DSS + ASA counters the AOM/DSS-induced alteration of H3K27ac expression. The expression of H3K27ac was examined by immunohistochemical staining. Representative images of the control (A), AOM/DSS (B) and AOM/DSS + ASA (C) are presented. The percentage of H3K27ac-positive cells was analyzed using ImageScope software, indicating that AOM/DSS significantly suppress nuclear staining, whereas AOM/DSS + ASA restores reduced H3K27ac staining (D). The enrichment of the H3K27ac mark in the promoter regions of selective pro-inflammatory genes was determined using a ChIP-qPCR assay. AOM/DSS + ASA significantly suppressed the increased relative abundance of H3K27ac following AOM/DSS in the promoter regions of iNOS (E), TNF-α (F) and IL-6 (G). No significant changes in the H3K27ac level in the Cox-2 promoter were observed (H). *P < 0.05, **P < 0.01 and ***P < 0.005.

Figure 5.

The effect of ASA on the expressions of iNOS, TNF-α, IL-6 and Cox-2. The mRNA expression of pro-inflammatory genes was examined using qPCR. AOM/DSS + ASA strikingly suppressed the AOM/DSS-induced mRNA expressions of iNOS (A), TNF-α (B) and IL-6 (C). However, the mRNA expression of Cox-2 was not changed in any treatment group (D). The protein expressions of COX-2 and iNOS were determined using western blotting, and the representative blots are presented. AOM/DSS + ASA strongly inhibited the protein expression of iNOS, but the COX-2 protein was not inhibited (E). The protein expression of TNF-α and IL-6 was quantified using ELISA, and the results demonstrated that AOM/DSS + ASA effectively inhibits the protein concentration of TNF-α (F) and IL-6 (G), respectively. *P < 0.05, **P < 0.01 and ***P < 0.005.