Resveratrol inhibits development of colorectal adenoma via suppression of LEF1; comprehensive analysis with connectivity map

Abstract

Although many chemopreventive studies on colorectal tumors have been reported, no effective and safe preventive agent is currently available. We searched for candidate preventive compounds against colorectal tumor comprehensively from United States Food and Drug Administration (FDA)-approved compounds by using connectivity map (CMAP) analysis coupled with in vitro screening with colorectal adenoma (CRA) patient-derived organoids (PDOs). We generated CRA-specific gene signatures based on the DNA microarray analysis of CRA and normal epithelial specimens, applied them to CMAP analysis with 1309 FDA-approved compounds, and identified 121 candidate compounds that should cancel the gene signatures. We narrowed them down to 15 compounds, and evaluated their inhibitory effects on the growth of CRA-PDOs in vitro. We finally identified resveratrol, one of the polyphenolic phytochemicals, as a compound showing the strongest inhibitory effect on the growth of CRA-PDOs compared with normal epithelial PDOs. When resveratrol was administered to Apc^Min/+ mice at 15 or 30 mg/kg, the number of polyps (adenomas) was significantly reduced in both groups compared with control mice. Similarly, the number of polyps (adenomas) was significantly reduced in azoxymethane-injected rats treated with 10 or 100 mg/resveratrol compared with control rats. Microarray analysis of adenomas from resveratrol-treated rats revealed the highest change (downregulation) in expression of LEF1, a key molecule in the Wnt signaling pathway. Treatment with resveratrol significantly downregulated the Wnt-target gene (MYC) in CRA-PDOs. Our data demonstrated that resveratrol can be the most effective compound for chemoprevention of colorectal tumors, the efficacy of which is mediated through suppression of LEF1 expression in the Wnt signaling pathway.

Keywords: LEF1; chemoprevention; colorectal adenoma; connectivity map; resveratrol.

FIGURE 1

Generation of human colorectal adenoma signature, connectivity map (CMAP), and compound ranking based on CMAP. (A) Endoscopic images of the three cases of adenomatous polyps. (B, C) A heat map from microarray analysis of the top 750 genes differentially expressed between human colorectal adenoma (CRA) and surrounding normal epithelia in each of the three cases. (C) Heat map of 304 genes narrowed down from (B) by applying gene set enrichment analysis for the signaling pathway. (D) Conceptual diagram of CMAP. (E) A list of the top 5% compounds identified from gene signature (B) using CMAP analysis. The top 5% originally included 305 entries (Table S4), from which 199 compounds were selected, excluding overlaps of different concentrations. (F) Top 5% compounds identified from gene signature (C) using CMAP analysis. Similarly, the list originally included 305 entries (Table S5) and 191 compounds were selected. The connectivity score was calculated as described previously, ranging from −1 to 1; a negative score denotes an inhibitory effect of compounds on the CRA‐specific signature to the normal epithelia ranking with the strongest compound designated as −1. (G) Venn diagram shows 121 overlapping compounds between (E) and (F), which narrowed down to 15.

FIGURE 2

Screening of candidate compounds using organoids from colorectal adenoma and normal mucosa. (A) IC₅₀ values of 15 compounds for human CRA or normal colorectal epithelia (NCE) of patient‐derived organoid (PDO). Each organoid was treated with each compound or vehicle (DMSO) alone for 72 h, and CellTiter‐Glo assay was performed to calculate IC₅₀ values. ND, not detected. (B, C) The viability of CRA‐PDOs and NCE‐PDOs treated with various concentrations of resveratrol (B) and pyridoxine (C) were determined by CellTiter‐Glo assay. IC₅₀ values were calculated by nonlinear regression analysis. Error bars, ±SD. (D, E) Representative bright‐field images of CRA‐PDOs and NCE‐PDOs cultured for 72 h with resveratrol (D) or pyridoxine (E). Scale bars, 50 μm. (F, G) Changes in the size of PDOs treated with resveratrol (100 μM) or pyridoxine (1000 μM) for 72 h. Average organoid size was calculated as the average of the data from nine representative images per treatment group. Error bars, ±SD. *p < 0.05, ***p < 0.001. ns, not significant.

FIGURE 3

Effect of resveratrol on development of intestinal tumors in Apc^Min/+ mice. (A) Experimental protocol. Male C57BL/6‐Apc^Min/+ mice were randomly assigned to three groups and were given 0, 15, or 30 mg/kg of resveratrol in drinking water as an admixture for 8 weeks. (B) Left panels show representative macroscopic views of intestinal mucosa of mice treated with vehicle alone or resveratrol (30 mg/kg), respectively. Scale bars, 1 mm. Arrows indicate polyps. Right panels show representative histologic findings (H&E staining) of intestinal polyps in both mice. Insets show magnification of the squared areas, findings compatible with adenoma. Scale bars, 200 μm. (C) The number of intestinal polyps in mice treated with resveratrol and vehicle alone. *p < 0.05, **p < 0.01. (D) The number of intestinal polyps analyzed by size in mice treated with resveratrol and vehicle alone. *p < 0.05 vs. vehicle.

FIGURE 4

Inhibitory effect of resveratrol on polyp development in azoxymethane (AOM)‐injected rats. (A) Experimental protocol. Rats in each group received intraperitoneal injections of AOM (20 mg/kg) twice a week. At 1 week later, the rats were treated with dextran sodium sulfate. Rats were given resveratrol (10 or 100 mg/kg) or vehicle alone by intragastric administration for 16 weeks. (B) Representative endoscopic image of polyps in the left‐side colon of each group at 15 weeks. Arrows indicate polyps. (C) The number of polyps was counted by endoscopy. *p < 0.05. (D) Left panels show representative macroscopic views of colorectal mucosa of rats treated with vehicle alone or resveratrol (100 mg/kg). Scale bars, 2 mm. Arrows indicate polyps. Right panels show representative histologic findings (H&E staining) of respective polyps. Insets show magnification of the squared areas, findings compatible with adenoma. Scale bars, 500 μm. (E) The number of polyps in rats treated with vehicle alone and resveratrol. *p < 0.05. (F) The number of polyps analyzed by size in each group. *p < 0.05 vs. vehicle.

FIGURE 5

Effect of resveratrol on gene expression in colorectal adenomas. (A) Schema of gene‐expression analysis. Adenomatous polyps were excised from resveratrol‐ or vehicle‐treated rats, and 652 genes with significantly altered expression were identified by DNA microarray analysis. In addition, 894 genes were selected from three databases containing information relevant to the development of colorectal tumor organoid data and colorectal tumors (MSig DB/KEGG). The Venn diagram shows 35 overlapping genes between both groups. (B) Probability difference ($p_k^d$) of the 35 genes. $p_k^d$ value was calculated as described in Materials and Methods Section 2.2.

FIGURE 6

Resveratrol inhibits the proliferation of colorectal adenomas by suppressing LEF1. (A) Evaluation of cell proliferation activity by BrdU assay. Resveratrol (100 μM) was added to human CRA organoids from three cases, and BrdU uptake was examined after 6 h incubation. (B–D) The mRNA levels of LEF1, MYC, and β‐catenin in CRA organoids were examined after resveratrol treatment by RT‐PCR. All experiments were performed in triplicate. Error bar, ±SD. (E) The expression of LEF‐1, MYC, and β‐catenin in CRA organoids. (F) Phosphorylation of MEK/ERK/Akt protein in CRA organoids. CRA organoids were treated with resveratrol (100 μM) or vehicle alone for 6 h, and western blotting was performed. **p < 0.01.