Mesalamine inhibits epithelial beta-catenin activation in chronic ulcerative colitis

Abstract

Background & aims: Mesalamine is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) in which condition it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that mesalamine reduces beta-catenin-associated progenitor cell activation, Akt-phosphorylated beta-catenin(Ser552) (P-beta-catenin), and colitis-induced dysplasia (CID).

Methods: Effects of mesalamine on P-beta-catenin staining and function were assessed by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in biopsy specimens of CUC in mild or "refractory" severe mucosal inflammation. Effects of mesalamine on epithelial proliferation and activation of Akt and beta-catenin were assessed in interleukin (IL)-10(-/-) colitis and CID by immunohistochemistry and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon.

Results: Data from IL-10(-/-) and human colitis samples show that mesalamine reduced Akt activation and P-beta-catenin levels in the middle and upper crypt. Reductions in P-beta-catenin in CUC biopsy specimens with severe inflammation suggested that mesalamine reduced P-beta-catenin levels in tissue refractory to mesalamine's anti-inflammatory effects. In IL-10(-/-) mice, mesalamine reduced CID concordant with inhibition of crypt Akt and beta-catenin signaling.

Conclusions: The results are consistent with the model that mesalamine contributes to chemoprevention in CAC by reducing beta-catenin signaling within intestinal progenitors.

Figure 1. 5-ASA treatment reduces P-β-catenin signaling in human UC

Human biopsy samples from control and UC patients obtained during colonoscopy were processed for assessment of histologic inflammation scores and immunohistochemical analysis with P-β-catenin Ab. Data are presented as means ± SEM; *P<0.01 (A) Representative sigmoid colon tissue stained for nuclear P-β-catenin (arrows) from control, chronic inflamed mucosa obtained from adjacent untreated, mild and severely inflamed UC (top panels), and 5-ASAtreated (≥2.4g/d) mild and severely inflamed UC (see methods; original magnification=20×). (B) Enumeration of P-β-catenin⁺ cells/40× field versus inflammatory scores, (C) mean P-β-catenin⁺ cells/40× field with or without 5-ASA treatment. (D) Histologic scores demonstrate comparable levels of inflammation between treatment groups segregated into mild or severe inflammation based on tissue histology (see Methods). (E) Mean number of P-β-catenin⁺ cells/hpf in mild and severely-inflamed UC biopsies ± 5-ASA treatment. (F) Increased β-catenin target gene mRNA expression in untreated colitis is attenuated by 5-ASA, as measured by real-time RT-PCR. Results are shown as fold induction in relation to mRNA expression of Gapdh.

Figure 1. 5-ASA treatment reduces P-β-catenin signaling in human UC

Human biopsy samples from control and UC patients obtained during colonoscopy were processed for assessment of histologic inflammation scores and immunohistochemical analysis with P-β-catenin Ab. Data are presented as means ± SEM; *P<0.01 (A) Representative sigmoid colon tissue stained for nuclear P-β-catenin (arrows) from control, chronic inflamed mucosa obtained from adjacent untreated, mild and severely inflamed UC (top panels), and 5-ASAtreated (≥2.4g/d) mild and severely inflamed UC (see methods; original magnification=20×). (B) Enumeration of P-β-catenin⁺ cells/40× field versus inflammatory scores, (C) mean P-β-catenin⁺ cells/40× field with or without 5-ASA treatment. (D) Histologic scores demonstrate comparable levels of inflammation between treatment groups segregated into mild or severe inflammation based on tissue histology (see Methods). (E) Mean number of P-β-catenin⁺ cells/hpf in mild and severely-inflamed UC biopsies ± 5-ASA treatment. (F) Increased β-catenin target gene mRNA expression in untreated colitis is attenuated by 5-ASA, as measured by real-time RT-PCR. Results are shown as fold induction in relation to mRNA expression of Gapdh.

Figure 2. BrdU incorporation, P-Akt and P-β-catenin levels increase as IL-10^−/− colitis progresses to dysplasia

(A–C;E–M) Sections from baseline (d0) mice and areas of colitis or dysplasia from piroxicam-treated IL-10^−/− mice (d70) are shown stained for (A–C) H&E, (E–G) BrdU incorporation, (H–J) P-Akt, and (K–M) P-β-catenin. (D) Evolution of histologic inflammation (black boxes) and prevalence of dysplasia (white boxes) in IL10^−/− colitis. (N) P-Akt⁺ and (O) P-β-catenin⁺ cells/100 epithelial cells for groups indicated. Representative results from 3 independent experiments are shown. Arrows indicate positive crypt based staining; arrow heads middle/upper crypt staining. 20× with 40× insets; scale bar=200µm. All values represent the mean ± SEM. *P<0.05 vs. d0.

Figure 3. 5-ASA attenuates epithelial proliferation and PI3K signaling in chronic IL-10^−/− colitis

Sections from d28 and d70 colitic mice compared to LD and HD 5-ASA-treated IL-10^−/− mice showing staining for (A–D) H&E, (E–H) BrdU, (I–L) P-Akt, and (M–P) P-β-catenin. Arrows indicate positive crypt based staining; arrow heads middle/upper crypt staining. LD and HD 5-ASA were given from d28 to d70 to IL-10^−/− mice as 500mg and 1650mg 5-ASA/kg chow (equivalent to 600 mg and 2 grams/day human dose respectively 39). Stains showed LD and HD 5-ASA reduced BrdU⁺, P-Akt⁺ and P-β-catenin⁺ cells in upper crypt regions of d70 IL-10^−/− mice. Scale bar=200µm. (Q,R) Cell counting show mean ± SEM for P-Akt and P-β-catenin⁺/100 epithelial cells for groups indicated. *P<0.05, versus d70 no treatment.

Figure 3. 5-ASA attenuates epithelial proliferation and PI3K signaling in chronic IL-10^−/− colitis

Sections from d28 and d70 colitic mice compared to LD and HD 5-ASA-treated IL-10^−/− mice showing staining for (A–D) H&E, (E–H) BrdU, (I–L) P-Akt, and (M–P) P-β-catenin. Arrows indicate positive crypt based staining; arrow heads middle/upper crypt staining. LD and HD 5-ASA were given from d28 to d70 to IL-10^−/− mice as 500mg and 1650mg 5-ASA/kg chow (equivalent to 600 mg and 2 grams/day human dose respectively 39). Stains showed LD and HD 5-ASA reduced BrdU⁺, P-Akt⁺ and P-β-catenin⁺ cells in upper crypt regions of d70 IL-10^−/− mice. Scale bar=200µm. (Q,R) Cell counting show mean ± SEM for P-Akt and P-β-catenin⁺/100 epithelial cells for groups indicated. *P<0.05, versus d70 no treatment.

Figure 4. 5-ASA impairs epithelial β-catenin and Akt activation in colitis

Epithelial cells were isolated and purified from IL-10^−/− mice at baseline (d0) and at d70 as described in the methods. (A) Confirmation of purity of subcellular fractions (nuclear=histone H3 positive/α-tubulin negative; cytosolic=histone H3 negative/α-tubulin positive) by Western blotting. (B) Assessment of nuclear P-β-catenin and cytosolic P-Akt in colonic epithelial cells from baseline and colitic d70 IL-10^−/− colitis mice with or without LD and HD 5-ASA treatment, as in Fig. 3. β-actin was used to control for protein loading. (C and D) Densitometric comparisons of data in (B) normalized to β-actin relative to d0. Values represent mean ± SEM. *P<0.01.

Figure 5. 5-ASA attenuates progression of dysplasia in IL-10^−/− colitis

Colons were removed from piroxicam-treated IL-10^−/− mice on d28 (pre-treatment baseline) and d70, then processed for H&E staining. Mice received standard, LD 5-ASA, or HD 5-ASA chow from d28 through d70. (A) Histologic inflammation scores and (B) prevalence (percent mice with any detectable dysplasia) of dysplasia were determined. (C) Dysplastic burden was measured by enumerating the average number of dysplastic lesions per colon and average length of dysplastic lesions per colon (D) for groups indicated. Values represent mean ± SEM. *P<0.01, **P<0.05, versus no treatment.

Figure 6. 5-ASA reduces epithelial proliferation and PI3K signaling in colitis associated dysplasia

From left to right: early reactive epithelial changes adjacent to ulceration (d28) compared to dysplasia at d70 in 5-ASA-treated IL-10^−/− mice. Panels show staining for (A–D) H&E, (E–H) BrdU incorporation, (I–L) P-Akt (arrows) and (M–P) P-β-catenin (arrows) within areas of dysplasia demonstrate little difference at d28 versus d70 with or without 5-ASA. (Q and R) Mean ± SEM of positively-stained cells/100 epithelial cells. 20× with 40× insets; scale bar=200µm. *P<0.05, versus no treatment, d70. Data suggest that regions of dysplasia that persist after 5-ASA treatment maintained active Akt and β-catenin signaling.

Figure 6. 5-ASA reduces epithelial proliferation and PI3K signaling in colitis associated dysplasia

From left to right: early reactive epithelial changes adjacent to ulceration (d28) compared to dysplasia at d70 in 5-ASA-treated IL-10^−/− mice. Panels show staining for (A–D) H&E, (E–H) BrdU incorporation, (I–L) P-Akt (arrows) and (M–P) P-β-catenin (arrows) within areas of dysplasia demonstrate little difference at d28 versus d70 with or without 5-ASA. (Q and R) Mean ± SEM of positively-stained cells/100 epithelial cells. 20× with 40× insets; scale bar=200µm. *P<0.05, versus no treatment, d70. Data suggest that regions of dysplasia that persist after 5-ASA treatment maintained active Akt and β-catenin signaling.