Pretreatment with the probiotic VSL#3 delays transition from inflammation to dysplasia in a rat model of colitis-associated cancer

Abstract

Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.

Fig. 1.

Pretreatment with VSL#3 decreases macroscopic damage in colitis-associated dysplasia. A: experimental design and treatment protocol. B: effect of VSL#3 administration on weight change. C: weight change during first 13 days of treatment. D: macroscopic damage (*P < 0.05, n = 22–23/group ± SE). TNBS, trinitrobenzene sulfonic acid.

Fig. 2.

Effect of VSL#3 on microscopic damage in colitis-associated dysplasia. A: total microscopic damage score was lower in the probiotic-treated group within each region of the colon (*P < 0.05, n = 16–17/group ± SE). Representative histologies from proximal and distal regions of colon from water-treated (B and D) and VSL#3-treated (C and E) animals, respectively; ×40.

Fig. 3.

Probiotic-treated animals did not progress to carcinoma. A: effect of VSL#3 on pathological analysis (*P < 0.05, n = 16–17/group ± SE). NSI, nonspecific inflammation; IBD, inflammatory bowel disease; LGD, low-grade dysplasia; HGD, high-grade dysplasia. B: LGD. C: HGD with carcinoma in situ. Atypical mitosis, no goblet cells, and irregular nuclei are shown; ×40.

Fig. 4.

Extent of proximal colon dysplasia in VSL#3-fed rats correlates with proximal colon tissue microbial species richness and diversity. A: colon tissue Margalef's richness vs. proximal colon dysplasia score. Pearson's correlation for water group, P = 0.992; for VSL#3 group, *P = 0.045. B: colon tissue Shannon diversity vs. proximal colon dysplasia score. Pearson's correlation for water group, P = 0.944; for VSL#3 group, *P = 0.050.

Fig. 5.

Administration of the probiotic VSL#3 increases vitamin D receptor (VDR) expression. A: VDR expression was higher in VSL#3-treated animals in all regions of the colon (*P < 0.05, **P < 0.01, n = 16–17/group ± SE). Representative histologies from mid colon in a water-treated (B) and a VSL#3-treated (C) animal; ×40. IHC, immunohistochemistry.

Fig. 6.

Effect of probiotic treatment on crypt cell proliferation and apoptosis in colitis-associated cancer model. A: bromodeoxyuridine (BrdU) incorporation in distal colon of animals treated with water or VSL#3 (n = 6/group). Representative photos from distal colons of water-treated (B) and VSL#3-treated (C) animals. D: 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to assess the percentage of apoptotic cells showing no difference between treatment groups.

Fig. 7.

Effect of probiotic treatment on expression of angiostatin (50 kDa) in an animal model of colitis-associated colon cancer. Tissues from proximal colons of treated animals were ground, and equal amounts of protein (100 μg) were separated by 15% SDS-PAGE before Western blot analysis. Gels show representative samples from 4 rats/group. Western blot bands were quantified by densitometry (*P < 0.05 vs. water, n = 10 rats/group ± SE).