Protective Role of Spermidine in Colitis and Colon Carcinogenesis

Abstract

Background & aims: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis.

Methods: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox^-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed.

Results: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox^-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox^-/- mice and was reversed with Spd.

Conclusions: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.

Trial registration: ClinicalTrials.gov NCT01091558.

Keywords: Chemoprevention; Colitis-Associated Carcinogenesis; Inflammatory Bowel Disease; Intestinal Microbiota; Polyamines; Spermidine; α-Defensins.

Figure 1.

SMOX expression in human colitis. (A-C) RNA was extracted from colon biopsies from normal (N) or UC patients with moderate (M) or severe (S) colitis (A), and from paired uninvolved and involved UC tissues (B). SMOX mRNA levels were determined by RT-real-time PCR. (C) Correlation between DAI and SMOX or ODC1 mRNA levels. This figure depicts the UC patients shown in (A) plus patients with quiescent (n = 15) and mild (n = 11) UC. (D-E) Tissues sections of a TMA were immunostained for SMOX. (D) Representative images of patients with UC (n = 28), LGD (n = 22), and HGD (n = 7); scale bar, 50 μm. (E) Quantification of SMOX⁺ epithelial cells among total CECs and SMOX⁺ ICs among all immune infiltrates. In E, solid circles are UC tissues and open circles are CD tissues. In A and E, *P < .05, **P < .01, ***P < .001 by one-way ANOVA and Tukey test; in B, the P values was obtained by Wilcoxon matched-pairs signed rank test; in C, the Pearson r and the P values were determined by simple linear regression.

Figure 2.

Effect of Spd supplementation on DSS colitis. C57BL/6 WT and Smox^–/– mice were treated or not with 4% DSS for 5 days and then kept for 5 more days on water alone. Spd was added to the water or to the DSS solution during all the experiments. (A) Colonic polyamine levels measured by LC-MS. (B) Body weights were measured daily and are depicted as percentage of initial body weight. (C) Colons were harvested, washed, and measured. (D-E) Histological injury scores (D) were determined from H&E staining performed on Swiss-rolls of the colon (E); the histologic injury scores of untreated WT and Smox^–/– mice ± Spd were all 0 (not shown). Scale Bar, 50 μm. In A-D, *P < . 05, **P < .01, ***P < .001, ****P < .0001 by two-way ANOVA and Tukey test. In B, *P < .05 compared to Smox^–/– + DSS + Spd.

Figure 3.

Effect of Spd treatment on colon carcinogenesis. (A-H) WT and Smox^–/– mice were treated with AOM-DSS and were given Spd throughout the experiments. Body weights were measured weekly and are depicted as percentage of initial body weight (A). *P < .05 and **P < .01 compared to mice from the same genotype not treated with Spd. After 56 days, colons were removed and histological injury score (B), tumor number (C), tumor burden (D), tumor size (E), number of histologic adenomas (F), and frequency of LGD and HGD (G) were determined; ND, no dysplasia. Panel H depicts representative images of H&E staining. Tumors are surrounded by dotted lines. High-power photomicrographs show HGD in WT and Smox^–/– mice treated with AOM-DSS and LGD in those animals that were given Spd. The scale bars on the low-power and high-power photomicrographs correspond to 100 and 50 μm, respectively. (I-M) CDX2P-CreER^T2;Apc^fl/fl mice were treated with TAM and were given Spd or not. After 39 days, tumor number (I-J) and tumor burden (K) were determined in the mid and distal colon; scale bar in I, 1 mm. The frequency of LGD and HGD was determined by histologic assessment (L). Panel M shows representative images of H&E staining of the colon of TAM-treated animals. For all the panels with symbols, *P < .05, **P < .01, ***P < .001, ****P < .0001 by two-way ANOVA and Tukey test (A-D and F) or Student’s t test (J, K). We also used Chi-Square (E and G) and Fisher’s exact test (L).

Figure 4.

Regulation of polyamine levels by Spd supplementation during experimental CAC. (A) Putrescine, spermidine, and spermine concentrations were measured by LC/MS in non-tumor (NT) and tumor (T) tissues from WT and Smox^–/– mice ± AOM-DSS ± Spd. (B) Smox mRNA expression was assessed by RT-real-time PCR. In A and B, *P < .05, **P < .01, ***P < .001, ****P < .0001 by two-way ANOVA and Tukey test. (C) Correlation plots comparing the average tumor size of WT mice not receiving Spd supplementation to the corresponding polyamine concentration; this figure includes the animals depicted in A plus data from additional WT mice not treated with Spd. The Pearson r and the P values were determined by simple linear regression.

Figure 5.

Transcriptomic changes in the colon regulated by Spd. RNA was extracted from non-tumor tissues from WT and Smox^–/– mice ± AOM-DSS ± Spd after 56 days (n = 3 mice per group), and then sequenced. Paired comparisons were performed to study the effect of AOM-DSS treatment (A), Smox deletion (B), and Spd supplementation (C), and the corresponding volcano plots were generated. Red dots depict DEGs (more than 2-fold; P < .05). Genes with values outside the X- and/or Y-axis limits are given with their (x;y) coordinates. The full list of DEGs is shown in Supplementary Table 1. (D) The expression of Defa22, Defa23, and Defa33 was also assessed by RT-real-time PCR. *P < .05, **P < .01, ***P < .001 (Student’s t test) denote significant differences for each comparison presented in the X-axis labels.

Figure 6.

DEFA expression in murine and human colitis. (A) Representative photomicrographs of DEFA5 (green) and LYZ1 (red) immunostaining in the colon of WT and Smox^–/– mice ± AOM-DSS ± Spd after 56 days. Merged colors are depicted in yellow. The nuclei stained with DAPI are shown in blue. Scale bars, 50 μm. (B) Quantification of epithelial staining shown in A. Each dot represents one gland, and the staining was quantified from multiple glands from n = 3 mice per group. (C) RNA was extracted from colon biopsies from normal (N) or UC patients with moderate (M) or severe (S) colitis and DEFA5 mRNA levels were measured by RT-real-time PCR. (D) Tissues sections of a TMA were immunostained for DEFA5 (green) and the representative images of normal tissues or colon from patients with UC, LGD, and HGD are shown; scale bar, 50 μm. (E) Quantification of DEFA5⁺ epithelial cells among total CECs; solid circles are UC tissues and open circles are CD tissues. (F) Human colonoids from normal (n = 2) or UC patients (n = 2) were cultured 48 h with or without 10 mM Spd; RNA was extracted and the expression of DEFA5, DEFA6, and SMOX was determined; each bar represents the mean ± SEM of three experiments performed with each colonoid line. In the panels with statistics, *P < .05, **P < .01, ***P < .001, and ****P < .0001 by one-way (C, E) or two-way (B, F) ANOVA and Tukey test.

Figure 7.

Effect of Smox deletion and Spd supplementation on the fecal microbiome. DNA was extracted from colonic feces of WT and Smox^–/– mice treated or not with Spd for 14 days. (A) Total number of bacteria, determined by real-time PCR. (B) Principal coordinate analysis plot, based on the unweighted UniFrac metric; P was determined by PERMANOVA. (C) Alpha diversity, evaluated by the inverse Simpson index. (D-E) Gut bacterial community composition at phylum (D) and genus (E) levels, expressed as a ratio to the total community. *P < .05 and **P < .01. All P-values are provided in Supplementary Table 3.