DNA methylome and transcriptome alterations and cancer prevention by curcumin in colitis-accelerated colon cancer in mice

Abstract

Inflammation is highly associated with colon carcinogenesis. Epigenetic mechanisms could play an important role in the initiation and progression of colon cancer. Curcumin, a dietary phytochemical, shows promising effects in suppressing colitis-associated colon cancer in azoxymethane-dextran sulfate sodium (AOM-DSS) mice. However, the potential epigenetic mechanisms of curcumin in colon cancer remain unknown. In this study, the anticancer effect of curcumin in suppressing colon cancer in an 18-week AOM-DSS colon cancer mouse model was confirmed. We identified lists of differentially expressed and differentially methylated genes in pairwise comparisons and several pathways involved in the potential anticancer effect of curcumin. These pathways include LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, production of NO and ROS in macrophages and IL-6 signaling. Among these genes, Tnf stood out with decreased DNA CpG methylation of Tnf in the AOM-DSS group and reversal of the AOM-DSS induced Tnf demethylation by curcumin. These observations in Tnf methylation correlated with increased and decreased Tnf expression in RNA-seq. The functional role of DNA methylation of Tnf was further confirmed by in vitro luciferase transcriptional activity assay. In addition, the DNA methylation level in a group of inflammatory genes was decreased in the AOM+DSS group but restored by curcumin and was validated by pyrosequencing. This study shows for the first time epigenomic changes in DNA CpG methylation in the inflammatory response from colitis-associated colon cancer and the reversal of their CpG methylation changes by curcumin. Future clinical epigenetic studies with curcumin in inflammation-associated colon cancer would be warranted.

Figure 1.

Curcumin suppressed AOM- and DSS-induced colon cancer. (A) Experimental design of the animal study (n = 10). (B) The weekly recording of body weight during the experiment. (C) Curcumin attenuated colon shortening by AOM and DSS. The length differences were significant (One-way ANOVA; *P < 0.05, **P < 0.01). (D and E) The suppression of tumor multiplicity and tumor incidence by curcumin administration (Mann–Whitney; **P < 0.01). (F) Histopathological examination of colon tissues at ×400 magnification. Scale bars represent 20 μM.

Figure 2.

Overview of differentially expressed genes and regulated pathways from RNA-seq data. (A) PCA of 12 RNA-seq samples. One sample in AOM+DSS group was an outlier and was excluded from further analyses. (B) Venn diagram showing differentially expressed genes from both comparisons and heatmap showing differentially expressed genes in common in these two comparisons. (C) Heatmap showing top 20 regulated pathways in common in both comparisons. Anti-inflammatory and anti-oxidative stress pathways are highlighted in blue. (D) RNA level of Tnf in the three groups from RNA-seq data. P-values, One-way ANOVA.

Figure 3.

Overview of DNA methylation level changes. (A) Methylation level of gene substructures in control, AOM+DSS and AOM+DSS+Curcumin groups. (B) Box plots showing the distribution of DMRs in control, AOM+DSS and AOM+DSS+Curcumin groups. Only DMRs with >12 CpG sites were included in this analysis to ensure comparability and analytical robustness. Differences between groups were not significant. P-values, Mann–Whitney. (C) Venn diagram showing the number of DMRs within promoter regions in the comparisons of control versus AOM+DSS and AOM+DSS versus AOM+DSS+Curcumin. (D) Venn diagram showing the number of DMRs in all genomic locations except distal intergenic regions, in the comparisons of control versus AOM+DSS and AOM+DSS versus AOM+DSS+Curcumin. Methylation ratio of DMRs from both comparisons is shown in heatmap for all three groups.

Figure 4.

Alterations of methylation level in Tnf. (A) Schematic diagram showing the structure of Tnf gene, the locations of DMRs and amplicons investigated in this study. (B and C) The methylation level of Tnf gene as observed from SureSelect Methyl-seq and Pyrosequencing. (D) The mRNA expression of Tnf measured by qPCR in the same groups as above. (E) The luciferase reporter vectors used in this study. Control vector has no insertion. Tnf and Tnf-me indicate unmethylated and methylated insertion of amplicon B of Tnf gene, respectively. (F) Luciferase activity of reporter vectors with unmethylated or methylated Tnf fragment. Data are from three independent experiments and are presented as fold change to control vector with normalization to firefly luciferase vector pGL4.13. Methylation of Tnf fragment (amplicon B) significantly decreased the luciferase activity. One-way ANOVA test for (B–D) and t-test for (F); *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.

Methylation level and mRNA expression of a set of inflammatory genes. (A) Heatmap showing the methylation level of genes in the pathway of inflammatory response. (B) The mRNA expression of selected genes measured by qPCR. Note, only epithelial cells were used for Methyl-seq, whereas tumor samples were also included in qPCR experiments. One-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001.