Celecoxib Alters the Intestinal Microbiota and Metabolome in Association with Reducing Polyp Burden

Abstract

Treatment with celecoxib, a selective COX-2 inhibitor, reduces formation of premalignant adenomatous polyps in the gastrointestinal tracts of humans and mice. In addition to its chemopreventive activity, celecoxib can exhibit antimicrobial activity. Differing bacterial profiles have been found in feces from colon cancer patients compared with those of normal subjects. Moreover, preclinical studies suggest that bacteria can modulate intestinal tumorigenesis by secreting specific metabolites. In the current study, we determined whether celecoxib treatment altered the luminal microbiota and metabolome in association with reducing intestinal polyp burden in mice. Administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APC(Min/+) mice. Treatment with celecoxib also altered select luminal bacterial populations in both APC(Min/+) and wild-type mice, including decreased Lactobacillaceae and Bifidobacteriaceae as well as increased Coriobacteriaceae Metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites linked to carcinogenesis, including glucose, amino acids, nucleotides, and lipids. Ingenuity Pathway Analysis suggested that these changes in metabolites may contribute to reduced cell proliferation. To this end, we showed that celecoxib reduced cell proliferation in the base of normal appearing ileal and colonic crypts of APC(Min/+) mice. Consistent with this finding, lineage tracing indicated that celecoxib treatment reduced the rate at which Lgr5-positive stem cells gave rise to differentiated cell types in the crypts. Taken together, these results demonstrate that celecoxib alters the luminal microbiota and metabolome along with reducing epithelial cell proliferation in mice. We hypothesize that these actions contribute to its chemopreventive activity. Cancer Prev Res; 9(9); 721-31. ©2016 AACR.

Figure 1

Celecoxib reduces intestinal polyp burden in APC^Min/+ mice. A, Schematic representation of experimental design. B-C, Polyp number (B) and size (C) were determined in the small intestines of APC^Min/+ mice given control or celecoxib containing diet. (n=10 per group).

Figure 2

Celecoxib treatment alters the luminal microbiota in APC^Min/+ mice. A, Principal component analysis results of 16S rDNA sequencing of ileal content and feces from APC^Min/+ mice given control or celecoxib diet. Black circles indicate those groups that cluster together. LefSe analysis was carried out on sequencing results from the ileal content (B) and feces (C) to determine which bacterial populations differed between treatment groups. D-F, Principal component analysis results of 16S rDNA sequencing (D) and LefSe analysis of ileal content (E) and feces (F) from WT mice are shown.

Figure 2

Celecoxib treatment alters the luminal microbiota in APC^Min/+ mice. A, Principal component analysis results of 16S rDNA sequencing of ileal content and feces from APC^Min/+ mice given control or celecoxib diet. Black circles indicate those groups that cluster together. LefSe analysis was carried out on sequencing results from the ileal content (B) and feces (C) to determine which bacterial populations differed between treatment groups. D-F, Principal component analysis results of 16S rDNA sequencing (D) and LefSe analysis of ileal content (E) and feces (F) from WT mice are shown.

Figure 3

Fecal metabolites are altered by celecoxib in APC^Min/+ and WT mice. Principal component analysis was carried out on data generated by targeted metabolomics of feces from control or celecoxib-treated mice. Symbols inside the red circle indicate those mice given celecoxib for 5 or 10 weeks. Symbols outside of the red circle represent mice that received control diet. Each group of mice (genotype, treatment or time point) is represented by a different color (Color by group). Those mice given control diet are represented by circles while celecoxib treated mice are represented by cone-shaped symbols, regardless of genotype (Shape by treatment).

Figure 4

Administration of celecoxib alters the luminal metabolome of APC^Min/+ mice. Heat maps of significantly altered metabolites were generated by comparing the metabolite changes of feces from APC^Min/+ mice given celecoxib to APC^Min/+ mice given control diet. A, Amino acids and Dipeptides. B, Lipids. C, Nucleotides. D, Additional Metabolites. Each column represents an individual sample. Data are rank transformed and displayed as color intensity with low levels indicated by green color and high levels indicated by red color. Metabolites in different metabolic pathways are color coded as follows: Red font = amino acid, brown font = dipeptides, dark blue font = lipids, green font = nucleotides, purple font = carbohydrates, light red font = cofactors and vitamins, black font = energy and orange font = xenobiotics. The metabolite categories described above were broadly defined to include related metabolites.

Figure 4

Administration of celecoxib alters the luminal metabolome of APC^Min/+ mice. Heat maps of significantly altered metabolites were generated by comparing the metabolite changes of feces from APC^Min/+ mice given celecoxib to APC^Min/+ mice given control diet. A, Amino acids and Dipeptides. B, Lipids. C, Nucleotides. D, Additional Metabolites. Each column represents an individual sample. Data are rank transformed and displayed as color intensity with low levels indicated by green color and high levels indicated by red color. Metabolites in different metabolic pathways are color coded as follows: Red font = amino acid, brown font = dipeptides, dark blue font = lipids, green font = nucleotides, purple font = carbohydrates, light red font = cofactors and vitamins, black font = energy and orange font = xenobiotics. The metabolite categories described above were broadly defined to include related metabolites.

Figure 5

Pathway alterations associated with celecoxib-induced metabolite changes in APC^Min/+ mice. The metabolomic changes induced by celecoxib treatment in APC^Min/+ mice were analyzed by Ingenuity Pathway Analysis. A, The top 15 diseases that were significantly associated with the altered metabolites are shown (left side). Blue bars represent the -log p-value and the threshold represents a p-value which equals 0.05. The “Cancer” category was further defined by type which revealed the top five significantly associated types of cancer (right side). B, The top 20 cellular and molecular functions that are significantly associated with the changes in metabolites are shown (left side). The “Cellular Growth and Proliferation” category was further analyzed to show that the direction of change (red = increased; green = decreased; color intensity reflects magnitude of change) in the majority of metabolites corresponded to decreased proliferation of cells (right side). Blue dashed line = predicted inhibition; yellow dashed line = findings inconsistent with state of downstream molecule; gray dashed line = effect not predicted.

Figure 6

Celecoxib treatment reduces proliferation in the base of normal appearing crypts from APC^Min/+ mice. A, The percent of Ki-67 positive cells in the entire crypt, base and non-base were quantified in the ilea of APC^Min/+ mice given control or celecoxib diet. Data are reported as median (n = 5 per group). . B, Representative images of Ki-67 stained crypts from control or celecoxib-treated mice are shown. Dashed circle indicates the base of the crypt; arrows indicate positively stained cells. C, The percent of Ki-67 positive cells in the entire crypt, base and non-base were quantified in colonic crypts from APC^Min/+ mice given control or celecoxib diet. D, The percent of cells stained positively for LacZ in ilea and colons of Lgr5-EGFP-ires-CreERT2/Rosa26-lacZ mice given control or celecoxib diet are shown. Data are summarized using mean ± SD (n = 3 to 4 per group). E-F, Representative images of LacZ staining in ilea (E) and colons (F) of control or celecoxib-treated Lgr5-EGFP-ires-CreERT2/Rosa26-lacZ mice.