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. 2011 Sep 13;108(37):15354-9.
doi: 10.1073/pnas.1010203108. Epub 2011 Aug 29.

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis

Andrew C Goodwin  1 , Christina E Destefano Shields, Shaoguang Wu, David L Huso, XinQun Wu, Tracy R Murray-Stewart, Amy Hacker-Prietz, Shervin Rabizadeh, Patrick M Woster, Cynthia L Sears, Robert A Casero Jr
Affiliations

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis

Andrew C Goodwin et al. Proc Natl Acad Sci U S A. .

Abstract

It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) up-regulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N(1),N(4)-bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
BFT induces SMO in human colon epithelial cell lines. T84 and/or HT29/c1 cells were treated with 5 nM purified, recombinant BFT for the indicated time points. (A) SMO gene expression in T84 (black bars) and HT29/c1 (gray bars) cells was quantified by qRT-PCR as described in Materials and Methods and data from three independent experiments analyzed in triplicate are presented (mean + SEM). Statistically significant (*P < 0.05 by t test) increase in SMO expression vs. untreated cells at the indicated time point. (B) HT29/c1 total cellular lysates were analyzed via quantitative fluorescent Western blotting as described in Materials and Methods and SMO protein levels were normalized to β-actin (representative of two independent experiments). (C) HT29/c1 cells were exposed to BFT for 6 h and SMO and APAO enzyme activity (pmol/min/mg protein) were determined (mean of two independent experiments analyzed in triplicate; *P < 0.05 by t test). Pretreatment overnight with MDL 72527 at concentrations of 10, 25, 50, or 100 μM rendered SMO activity undetectable. APAO enzyme activity was not detected in any samples.
Fig. 2.
Fig. 2.
BFT induces SMO-dependent ROS and DNA damage in T84 cells. (A) T84 cells were pretreated with or without 25 μM MDL 72527 before stimulation with 5 nM BFT for 6 h. Total cellular lysates were analyzed by quantitative fluorescent Western blotting and γ-H2A.x levels were normalized to β-actin as described in Materials and Methods. The presented data and fold changes in protein levels include two samples per treatment group representative of multiple independent experiments. (B) T84 cell lines stably expressing nontargeting (Scr) and SMO-specific (SMO) shRNA were stimulated with BFT. Intracellular ROS levels were measured via CM-H2DCFDA fluorescence.
Fig. 3.
Fig. 3.
ETBF-infected mice exhibit regions of intense SMO staining. C57BL/6 mice were sham or ETBF inoculated and killed after 1 wk (wild type) or 6 wk (Min). Rolled colon tissues were fixed, embedded, and analyzed by immunohistochemistry using specific antisera against SMO. Representative images are displayed from (A) wild type, sham, 1 wk; (B) wild type, ETBF, 1 wk; (C) Min, sham, 6 wk; (D) Min, ETBF, 6 wk. Sham mice had regions of mild-to-moderate apical SMO staining. ETBF-infected mice contained regions of intense SMO staining that extended deeper into the crypts.
Fig. 4.
Fig. 4.
MDL 72527 significantly inhibits ETBF-induced colon tumorigenesis in Min mice. Min mice were sham or ETBF inoculated with or without MDL 72527 (20 mg/kg 3 d per wk) for 6 wk and colon polyps were counted as described in Materials and Methods. The number of tumors for each mouse analyzed is plotted as an open square; plot whiskers indicate lower and upper limits of data; boxes are bounded by first and third quartiles of data; and heavy lines denote median tumor number in each treatment group. Sham-inoculated animals developed very few colon polyps whether treated with (median = 1, range = 0–5, n = 8) or without (median = 0.5, range = 0–2, n = 7) MDL 72527. ETBF-induced colon tumorigenesis (median = 16, range = 4–53, n = 21) was significantly reduced by MDL 72527 treatment (median = 5, range = 0–17, n = 23; ***P < 0.001 by Mann-Whitney-Wilcoxon u test for ETBF vs. ETBF+MDL comparison).
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