Chemoprevention of intestinal tumorigenesis in APCmin/+ mice by silibinin

Abstract

Chemoprevention is a practical and translational approach to reduce the risk of various cancers including colorectal cancer (CRC), which is a major cause of cancer-related deaths in the United States. Accordingly, here we assessed chemopreventive efficacy and associated mechanisms of long-term silibinin feeding on spontaneous intestinal tumorigenesis in the APC(min/+) mice model. Six-week-old APC(min/+) mice were p.o. fed with vehicle control (0.5% carboxymethyl cellulose and 0.025% Tween 20 in distilled water) or 750 mg silibinin/kg body weight in vehicle for 5 d/wk for 13 weeks and then sacrificed. Silibinin feeding strongly prevented intestinal tumorigenesis in terms of polyp formation in proximal, middle, and distal portions of small intestine by 27% (P < 0.001), 34% (P < 0.001), and 49% (P < 0.001), respectively. In colon, we observed 55% (P < 0.01) reduction in number of polyps by silibinin treatment. In size distribution analysis, silibinin showed significant decrease in large-size polyps (>3 mm) by 66% (P < 0.01) and 88% (P < 0.001) in middle and distal portions of small intestine, respectively. More importantly, silibinin caused a complete suppression in >3 mm sized polyps and 92% reduction in >2 to 3 mm sized polyps in colon. Molecular analyses of polyps suggested that silibinin exerts its chemopreventive efficacy by inhibiting cell proliferation, inflammation, and angiogenesis; inducing apoptosis; decreasing beta-catenin levels and transcriptional activity; and modulating the expression profile of cytokines. These results show for the first time the efficacy and associated mechanisms of long-term p.o. silibinin feeding against spontaneous intestinal tumorigenesis in the APC(min/+) mice model, suggesting its chemopreventive potential against intestinal cancers including CRC.

Figure 1

Silibinin-feeding prevents spontaneous intestinal polyposis in APC^min+ mice. At the end of the silibinin efficacy study, results are shown for A, number of polyps/mouse in small intestine and colon, and polyp size distribution in colon; B, size distribution of polyps in proximal, middle and distal portions of small intestine; C, representative pictures of distal small intestinal polyps; and D, H&E stained sections from polyps and normally appearing crypt-villus axis from control and silibinin-treated APC^min/+ mice (100x). Bars shown in each case are mean ± SEM from 18 animals in each group. $, P<0.05; #, P<0.01; *, P<0.001 versus control; Sb, silibinin.

Figure 2

Silibinin-feeding reduces proliferation but induces apoptosis selectively in small intestinal polyps of APC^min/+ mice. Small intestinal segments were processed for PCNA, TUNEL and CC3 staining. Tissue sections from APC^min/+ control and silibinin-treated groups show brown-colored (A) PCNA- and (B) TUNEL-positive cells in polyps (400x). Quantitative data for (A) proliferative and (B and C) apoptotic indices were determined as number of PCNA, TUNEL-or CC3-positive cells × 100/total number of cells, respectively, and represent mean ± SEM of six animals. *, P<0.001 versus control. Polyps from distal portion of small intestine from each group were also analyzed by immunoblotting for PCNA and cleaved–PARP levels. Values of band intensity adjusted with β-actin. Sb, silibinin; CC3, cleaved caspase 3.

Figure 3

Silibinin-feeding inhibits HIF-1α and VEGF expression selectively in small intestinal polyps of APC^min/+ mice. Small intestinal segments were processed for HIF-1α and VEGF staining. Tissue sections from APC^min/+ control and silibinin-treated groups show brown-colored (A) HIF-1α- and (C) VEGF-positive cells in polyps (400x). Quantitative data for (B) HIF-1α and (D) VEGF are shown based on their intensity of cytoplasmic brown staining, and represent mean ± SEM of six animals. *, P<0.001 versus control; Sb, silibinin.

Figure 4

Silibinin feeding inhibits eNOS expression and nestin-positive microvessels selectively in small intestinal polyps of APC^min/+ mice. Small intestinal segments were processed for eNOS and nestin staining. Tissue sections from APC^min/+ control and silibinin-treated groups show brown-colored (A) eNOS- and (C) nestin-positive cells in polyps (400x). Quantification of eNOS (B) was done based on its intensity of cytoplasmic brown staining, and microvessel numbers were quantified by measuring nestin-positive cells (D) in five randomly selected fields at 400x magnification, and represent mean ± SEM of six animals. *, P<0.001 versus control. Polyps from distal portion of small intestine from each group were also analyzed by immunoblotting for eNOS and nestin levels. Values of band intensity adjusted with β-actin. Sb, silibinin.

Figure 5

Silibinin feeding decreases β-catenin, cyclin D1 and COX-2 expression and PGE2 levels selectively in small intestinal polyps of APC^min/+ mice and inhibits β-catenin mediated transcriptional activity in vitro. Small intestinal segments or polyps were processed for IHC analyses or estimation for (A) nuclear β-catenin, (B) cyclin D1 and (D) COX-2 and PGE2 levels as detailed in Methods. Data represent mean ± SEM of six animals. *, P<0.001 versus control. Polyps from distal portion of small intestine from each group were also analyzed by immunoblotting for β-catenin and cyclin D1 levels. Values of band intensity adjusted with β-actin. C, TOP/FOPFlash reporter activity was measured using dual luciferase assay kit from Promega as described in detail in Methods. Data shown represents mean ± SEM of three independent observations. Sb, silibinin.

Figure 6

Silibinin feeding modulates cytokines profile in intestinal polyps of APC^min/+ mice. Lysates from small intestinal polyps were analyzed for various cytokines by cytokine antibody array kit. A, Representative cytokines array blots from control and silibinin-treated APC^min/+ mice polyps. B, Densitometric analysis data of cytokines levels from three samples in each group are shown as fold changes by silibinin treatment to that of control.