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. 2011 Sep;32(9):1396-402.
doi: 10.1093/carcin/bgr118. Epub 2011 Jun 24.

Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice

Jean-Pierre Raufman  1 , Jasleen Shant, Guofeng Xie, Kunrong Cheng, Xue-Min Gao, Brian Shiu, Nirish Shah, Cinthia B Drachenberg, Jonathon Heath, Jürgen Wess, Sandeep Khurana
Affiliations

Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice

Jean-Pierre Raufman et al. Carcinogenesis. 2011 Sep.

Abstract

M3 subtype muscarinic receptors (CHRM3) are over-expressed in colon cancer. In this study, we used Apc(min/+) mice to identify the role of Chrm3 expression in a genetic model of intestinal neoplasia, explored the role of Chrm3 in intestinal mucosal development and determined the translational potential of inhibiting muscarinic receptor activation. We generated Chrm3-deficient Apc(min/+) mice and compared intestinal morphology and tumor number in 12-week-old Apc(min/+)Chrm3(-/-) and Apc(min/+)Chrm3(+/+) control mice. Compared with Apc(min/+)Chrm3(+/+) mice, Apc(min/+)Chrm3(-/-) mice showed a 70 and 81% reduction in tumor number and volume, respectively (P < 0.01). In adenomas, β-catenin nuclear staining was reduced in Apc(min/+)Chrm3(-/-) compared with Apc(min/+)Chrm3(+/+) mice (P < 0.02). Whereas Apc gene mutation increased the number of crypt and Paneth cells and decreased villus goblet cells, these changes were absent in Apc(min/+)Chrm3(-/-) mice. To determine whether pharmacological inhibition of muscarinic receptor activation attenuates intestinal neoplasia, we treated 6-week-old Apc(min/+) mice with scopolamine butylbromide, a non-subtype-selective muscarinic receptor antagonist. After 8 weeks of continuous treatment, scopolamine butylbromide-treated mice showed a 22% reduction in tumor number (P = 0.027) and a 36% reduction in tumor volume (P = 0.004) as compared with control mice. Compared with Chrm3 gene ablation, the muscarinic antagonist was less efficacious, most probably due to shorter duration of treatment and incomplete blockade of muscarinic receptors. Overall, these findings indicate that interplay of Chrm3 and β-catenin signaling is important for intestinal mucosal differentiation and neoplasia and provide a proof-of-concept that pharmacological inhibition of muscarinic receptor activation can attenuate intestinal neoplasia in vivo.

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Figures

Fig. 1.
Fig. 1.
In Apcmin/+ mice, Chrm3 gene deficiency reduces body weight and small intestinal tumor number. (A) During the 12-week study, mice were weighed weekly after weaning. Weights of Apcmin/+Chrm3−/− mice (N = 29) was significantly less (P < 0.01) at each weekly interval compared with weights of Apcmin/+Chrm3+/+ (N = 14) and Apcmin/+Chrm3+/− (N = 37) mice. (B) Representative dissecting microscope images of distal small intestine from Apcmin/+ mice with the indicated Chrm3 genotypes. Arrows indicate small intestine tumors. (C) Effect of Chrm3 deficiency on the number of small intestinal tumors. Each symbol represents one mouse. Horizontal bars represent mean values. (D) Number of small intestinal tumors in the proximal, mid and distal thirds of the small intestine of Apcmin/+Chrm3+/+ and Apcmin/+Chrm3−/− mice. Values in (A) and (D) represent means ± SE.
Fig. 2.
Fig. 2.
In Apcmin/+ mice, Chrm3 gene deficiency reduces small intestinal tumor volume and the number of low- and high-grade adenomas. (A) Effect of Chrm3 deficiency on the volume of small intestinal tumors. Each symbol represents 1 mouse. Horizontal bars represent mean values. (B) Volume of small intestinal tumors in the proximal, mid and distal thirds of the small intestine of Apcmin/+Chrm3+/+ and Apcmin/+Chrm3−/− mice. (C) Representative hematoxylin and eosin-stained sections. (a) Arrowheads indicate adenomas in an Apcmin/+Chrm3+/+ mouse (×5 magnification), further magnified in (b). Adenomas are indicated by arrows. (c) High-grade adenoma (×20 magnification), magnified further in (d). (e and f) Representative sections from Apcmin/+Chrm3−/− mice. The only adenoma is indicated by an arrow. (f) Boxed section from (e) magnified to demonstrate the normal appearance of intestinal mucosa from an Apcmin/+Chrm3−/− mouse. Stars indicate intestinal lumen. (D). Numbers of low- and high-grade adenomas per mouse distal small intestine in Apcmin/+ mice with varying Chrm3 expression. Bars in (B) and (D) represent means ± SEs. NS indicates comparisons that were not statistically significant.
Fig. 3.
Fig. 3.
Chrm3 deficiency in Apcmin/+ mice attenuates β-catenin nuclear translocation (activation) and restores surface cellular differentiation, including goblet cells. (A) β-Catenin immunostaining in normal mucosa of Apc+/+Chrm3+/+ (left panel) and in adenomas from Apcmin/+Chrm3+/+ and Apcmin/+Chrm3−/− mice (middle and right panels, respectively), ×100 total magnification. In small intestinal crypts from Apc+/+Chrm3+/+ mice, β-catenin expression is primarily membranous compared with increased nuclear and cytoplasmic β-catenin immunostaining (arrowheads) in adenomas from Apcmin/+Chrm3+/+ (middle panel) and Apcmin/+Chrm3−/− (right panel) mice. Magnified cells in insets show nuclear features in greater detail. (B) Nuclear β-catenin immunostaining in intestinal tissue from Apc+/+Chrm3+/+, Apcmin/+Chrm3+/+ and Apcmin/+Chrm3−/− mice (N = 5–7 mice/genotype). Compared with normal intestine, adenomas from both Apcmin/+Chrm3+/+ and Apcmin/+Chrm3−/− mice had increased nuclear β-catenin staining. β-Catenin nuclear staining was reduced ∼50% in adenomas from Apcmin/+Chrm3−/− compared with Apcmin/+Chrm3+/+ mice. (C) Alcian blue-stained intestinal sections from Apc+/+Chrm3+/+, Apcmin/+Chrm3+/+ and Apcmin/+Chrm3−/− mice. Fewer goblet cells per villus were observed in Apcmin/+Chrm3+/+ mice compared with Apc+/+Chrm3+/+ and Apcmin/+Chrm3−/− mice. (D) Goblet cells per small intestinal villus (N = 5–7 mice/genotype). Mean number of goblet cells/villus was significantly reduced in Apcmin/+Chrm3+/+ compared with Apc+/+Chrm3+/+ and Apcmin/+Chrm3−/− mice. Values in (B) and (D) represent means ± SEs. NS indicates comparisons that were not statistically significant.
Fig. 4.
Fig. 4.
In Apcmin/+ mice, scopolamine butylbromide (NBS) treatment attenuates small intestinal tumor formation. Mice were treated with NBS (5 μg/g body weight/day; N = 48) or vehicle (PBS; N = 32) by subcutaneous osmotic minipump from age 6 to14 weeks. (A) Blood hemoglobin values in PBS- and NBS-treated mice immediately prior to euthanasia. (B) NBS treatment significantly reduced the number of small intestine tumors. Each symbol represents one mouse. Horizontal bars represent mean values. (C) NBS treatment reduced small intestine tumor size. Each symbol represents one mouse. Horizontal bars represent mean values. (D) Size range of small intestinal tumors in Apcmin/+ mice treated with PBS and NBS. Values in (A) and (D) represent means ± SEs.
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