Mechanisms of colitis-accelerated colon carcinogenesis and its prevention with the combination of aspirin and curcumin: Transcriptomic analysis using RNA-seq

Abstract

Colorectal cancer (CRC) remains the leading cause of cancer-related death in the world. Aspirin (ASA) and curcumin (CUR) are widely investigated chemopreventive candidates for CRC. However, the precise mechanisms of their action and their combinatorial effects have not been evaluated. The purpose of the present study was to determine the effect of ASA, CUR, and their combination in azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-accelerated colorectal cancer (CAC). We also aimed to characterize the differential gene expression profiles in AOM/DSS-induced tumors as well as in tumors modulated by ASA and CUR using RNA-seq. Diets supplemented with 0.02% ASA, 2% CUR or 0.01% ASA+1% CUR were given to mice from 1week prior to the AOM injection until the experiment was terminated 22weeks after AOM initiation. Our results showed that CUR had a superior inhibitory effect in colon tumorigenesis compared to that of ASA. The combination of ASA and CUR at a lower dose exhibited similar efficacy to that of a higher dose of CUR at 2%. RNA isolated from colonic tissue from the control group and from tumor samples from the experimental groups was subjected to RNA-seq. Transcriptomic analysis suggested that the low-dose combination of ASA and CUR modulated larger gene sets than the single treatment. These differentially expressed genes were situated in several canonical pathways important in the inflammatory network and liver metastasis in CAC. We identified a small subset of genes as potential molecular targets involved in the preventive action of the combination of ASA and CUR. Taken together, the current results provide the first evidence in support of the chemopreventive effect of a low-dose combination of ASA and CUR in CAC. Moreover, the transcriptional profile obtained in our study may provide a framework for identifying the mechanisms underlying the carcinogenesis process from normal colonic tissue to tumor development as well as the cancer inhibitory effects and potential molecular targets of ASA and CUR.

Keywords: Aspirin; Colitis-associated colorectal cancer; Curcumin; RNA-seq.

Figure 1. The experimental protocol for a chemoprevention study with ASA and CUR, alone or in combination, in AOM/DSS-induced C57/BL6 mice

Mice at 5 weeks of age were fed the AIN-93M diet or this diet supplemented with 0.02% ASA, 2% CUR, or 0.01% ASA+1% CUR until the end of the experiment. Mice in groups other than the control group received a subcutaneous injection of AOM at 10 mg/kg at the age of 6 weeks, followed by the administration of water containing DSS at 1.2% (w/v) for 7 consecutive days. Twenty-two weeks after AOM initiation, the mice were sacrificed for further analysis.

Figure 2. The effect of dietary administration of ASA, CUR, and their combination in AOM/DSS-induced CAC. (A)

The suppression of the DAI by 0.02% ASA, 2% CUR, and 0.01% ASA+1% CUR. (B) The effect of ASA, CUR, and their low-dose combination on tumor incidence in AOM/DSS-induced CAC. The tumor incidence (%) of each group was calculated from the number of mice with tumor growth over the number of mice examined. (C) The effect of ASA and CUR, alone or in combination, in decreasing tumor multiplicity in AOM/DSS-induced CAC. Tumor multiplicity was calculated from the total number of tumors in each group divided by the number of mice in each group. (D–I) Histological observation of the control group (D), the model group with AOM/DSS administration (E, F), mice treated with 0.02% ASA (G), mice treated with 2% CUR (H), and mice treated with 0.01% ASA+1% CUR (I). * P < 0.05 versus the model group.

Figure 3. Overview of the genes regulated by ASA and CUR, alone or in combination, compared to the model group. (A, B)

Venn diagrams comparing the number of up-regulated genes (A) and down-regulated genes (B) in tumors from mice treated with 0.02% ASA, 2% CUR, or 0.01% ASA+1% CUR compared to tumors from mice treated with AOM/DSS alone. Genes with log2 fold changes greater than 1 and an FDR less than 0.05 were counted. (C) Heat map of 32 genes with differential expression that appeared in all three treatment groups (ASA, CUR, and ASA+CUR) compared to the model group.

Figure 4. Validation of the mRNA expression of selected genes regulated by ASA, CUR, or their combination compared to the model group

mRNA isolated from tumors in mice from the model, 0.02% ASA, 2% CUR, and 0.01% ASA+1% CUR groups was subjected to qPCR analysis. Black bar: the qPCR results are presented as the fold change compared with the model group using Gapdh as the endogenous control. White bar: fold change from the RNA-seq analysis. The data are presented as the mean ± SD (n = 2).

Figure 5. The list of 54 genes that showed regulation in the opposite direction when comparing the model versus the control and the combination versus the model groups

There were 13 genes that were down-regulated in the model group compared to the control group, and their expression was up-regulated by the combination of ASA and CUR. There were 41 genes that were up-regulated in the model group compared to control group, and their expression was down-regulated by the combination of ASA and CUR.

Figure 6. Validation of the mRNA expression of selected genes that showed regulation in the opposite direction when comparing AOM/DSS alone and the combination of ASA and CUR

mRNA isolated from the colonic tissues of mice from the control group and tumors from mice receiving 0.01%ASA+1% CUR was subjected to qPCR analysis. Black bar: the qPCR results are presented as the fold change compared with the control group using Gapdh as the endogenous control. White bar: fold change from the RNA-seq analysis. The data are presented as the mean ± SD (n = 2).