Inhibition of Azoxymethane-induced Colonic Aberrant Crypt Foci Formation by Silibinin in Male Fisher 344 Rats

Abstract

Chemoprevention is a practical approach to control colorectal cancer, which is one of the major causes of cancer mortality in USA. Based on our recent silibinin efficacy studies in human colorectal cancer cells, we investigated the effects of its dietary feeding on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated biomarkers in male Fisher 344 rats. Five-weeks old male F344 rats were fed control or silibinin supplemented (0.033, 0.1, 0.33 or 1%, w/w) diet. After two weeks, AOM was injected once a week for 2 weeks, while silibinin treatments continued. In another protocol, identical silibinin treatments were done, but started 2-weeks post-AOM initiation. All rats were sacrificed at 16 weeks of age, and colon samples were evaluated for ACF, followed by proliferation, apoptosis, and iNOS and COX2 by immunohistochemistry and/or immunoblotting. Silibinin significantly (P<0.001) reduced the number and multiplicity of AOM-induced ACF formation dose-dependently. Silibinin feeding in pre- and post-AOM initiation decreased mean number of ACF by 39 to 65%, and in post-AOM initiation by 29 to 55%. Silibinin dose-dependently decreased AOM-induced colonic cell proliferation evidenced by PCNA and cyclin D1 immunohistochemical staining, and induced apoptosis in these colon tissues evidenced by TUNEL staining and cleaved PARP. Furthermore, silibinin significantly decreased AOM-induced iNOS and COX2 positive cells in colon tissues. The present findings show possible beneficial activity of silibinin at least in early stage of colon tumorigenesis, suggesting that silibinin might be an effective natural agent for colorectal cancer chemoprevention.

Introduction

Colorectal cancer (CRC) is one of the most prevalent causes of cancer deaths in developed countries including the United States (1). Recent statistics suggest that 148,810 new cases of CRC will be diagnosed in 2008 and 49,960 patients would die due to this malignancy in the United States alone (2). The etiology of colon cancer is multifactor including familial, environmental and dietary agents. Despite several advancements in the understanding of the processes in carcinogenesis, presently available therapies including surgery, radiation and chemotherapeutic drugs, are still limited for advanced stage of colon cancer (3). Nutritional intervention is another effective and promising complimentary strategy for controlling the incidence of colon cancer (4). Several epidemiological and experimental studies have indicated that plant products exert a protective influence against this disease, and beneficial effects may be partly attributable to polyphenolic phytochemicals, which have a wide range of pharmacological properties (5, 6). Moreover, the search for putative chemopreventive compounds with minimal toxicity raises particular interest in phytochemicals.

Silibinin, a naturally-occurring flavonoid and the major biologically active constituent in milk thistle extract, is one such agent that has shown potential anticancer effects against different cancers in both in vitro and in vivo systems (7–10). Non-toxicity, even at high doses and longer treatment times, is one of the most important properties of this compound, which has been tested in several animal models using different modes of administration (9, 11). Despite a number of studies convincingly showing the remarkable chemopreventive potential of silibinin in different cancer models, its efficacy against CRC initiation and development in animal models remains largely unexamined. Although Kohno et al. (12) reported the in vivo inhibition of colon carcinogenesis by silibinin-rich mixture silymarin, as well as previous report from our laboratory has shown the anti-cancer activity of silibinin in human colon carcinoma HT-29 cells (13), no in vivo study has reported the efficacy of silibinin in CRC chemoprevention model.

Aberrant crypt foci (ACF) are early morphological changes observed in rodents after administration of colon-specific carcinogen such as azoxymethane (AOM) (14). Similar lesions were also observed at a high frequency in the colons of the patients with sporadic and inherited forms of colon cancer (15). ACF are considered as putative pre-neoplastic lesions, and are utilized currently as a surrogate biomarker to rapidly evaluate the chemopreventive potential of several agents including both naturally occurring and synthetic, employing AOM in Fisher 344 (F344) rat model (16–18), which accurately replicates many of the clinical, genetic, cellular, and morphological features of human CRC (19). AOM-induced ACF are characterized by an increase in the size of the crypts, the epithelial lining and the pericryptal zone, and share many morphological and biochemical characteristics with tumors, including a comparable increase in cell proliferation (20).

In the present study, we investigated the possible inhibitory effect of dietary feeding of silibinin at four different dose levels and in two different phases, on the development of AOM-induced ACF formation in male F344 rats; sulindac, which is well-known as an effective chemopreventive agent in this model (21), was used as a positive control for comparison with silibinin study outcomes. Colonic tissues at the end of the study were also analyzed for proliferation, apoptosis and inflammation markers. The results of the present study convincingly showed the chemopreventive efficacy of silibinin against AOM-induced ACF in F344 rats, which also associated with an in vivo decrease in proliferation and inflammation regulators, but an increase in apoptotic cells in the colon.

Materials and Methods

Animals and treatment protocol

Male F344 rats were purchased from the Jackson laboratories (Bar Harbor, ME) at the age of 4 weeks, and maintained in the animal housing facility at the University of Colorado Denver, Denver. Silibinin was obtained commercially from Sigma Chemical Co., (St Louis, MO), and its purity was analyzed by HPLC to be >98% (22). The experimental protocol for the present study is shown in Figure 1. Animals were maintained at 12 h light/dark cycles with free access to water and food (AIN-76A powder diet from Dyets Inc., Bethlehem, PA). After one week of acclimatization, animals were randomly divided into 12 groups of 10 animals each and fed AIN-76A control diet (group 1 & 2) or diet supplemented with 0.033, 0.1, 0.33 or 1% (w/w) silibinin (groups 3–6) till the end of the study. Two weeks later, rats in groups 2–11 were given sub-cutaneous injection of AOM once a week for 2 weeks at a concentration of 15 mg/kg body weight. Two weeks after the last AOM injection, rats in groups 7–10 were fed with diet containing 0.033, 0.1, 0.33 or 1% (w/w) silibinin, and the rats in group 11 were fed with 0.032% sulindac. Since sulindac is well-known as an effective chemopreventive agent against AOM-induced ACF formation in F344 rats (21), it was used as a positive control for comparison with silibinin study outcomes. Rats in group 12 were fed with diet containing 1% silibinin alone throughout the study. At 16 weeks of age, the rats were sacrificed, and their colons were evaluated for ACF or other marker studies.

Fig. 1.

Experimental protocol for azoxymethane (AOM)-induced colonic aberrant crypt foci formation in male F344 rats and chemoprevention studies with silibinin. The animals were randomly divided into 12 groups and fed AIN-76A control diet or diet supplemented with different doses of silibinin. AOM was given once a week for two weeks at a dose level of 15 mg/kg body weight by subcutaneous injection. Other details of the experimental design are described in Materials and Methods.

Determination of ACF

ACF analysis was done according to Bird (19), where the colons were longitudinally opened, rinsed with 0.9% NaCl solution and fixed flat between two pieces of filter paper in 10% buffered formalin for a minimum of 24 h. The colons were then cut into 2 cm segments, starting at the anus, and stained with 0.2% methylene blue in Krebs-Ringer solution for 5 to 10 min, and were then placed mucosal side up on a microscope slide and counted through a light microscope at 400x magnification. Aberrant crypts were distinguished from the surrounding normal crypts by their increased size, the significantly increased distance from lamina to basal surface of cells, and the easily discernible pericryptal zone. The variables used to assess the aberrant crypts were their occurrence and multiplicity. Crypt multiplicity was determined as the number of crypts in each focus. Multicrypts were categorized as those containing up to four or more aberrant crypts/focus.

Immunostaining for PCNA, cyclin D1, iNOS and COX2

Colon tissue samples were fixed in 10% phosphate-buffered formalin for 10 hours at 4°C, dehydrated in ascending concentrations of ethanol, cleared with xylene and embedded in PolyFin (Triangle Biomedical Sciences, Durham, NC). Paraffin-embedded tissue blocks were cut with a rotary microtome into 4-µm sections and processed for immunohistochemical staining. Briefly, after deparaffinization and re-hydration, the sections were treated with 0.01 M sodium citrate buffer (pH 6.0) in a microwave for 5 min at full power for antigen-retrieval. The sections were then quenched of endogenous peroxidase activity by immersing in 3% hydrogen peroxide for 5 min at room temperature. The sections were either incubated with antibody against proliferative cell nuclear antigen (PCNA) mouse monoclonal (1:400 dilution, Dako, Carpinteria, CA), cyclin D1 rabbit polyclonal (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), inducible nitric oxide synthase (iNOS) rabbit polyclonal (1:200 dilution; Abcam, Inc, Cambridge, MA) or cyclooxygenase-2 (COX2) rabbit polyclonal (1:100 dilution; Cell Signaling Technologies, San Diego, CA) in PBS for 2 h at room temperature in a humidity chamber followed by overnight incubation at 4°C. In all the immunohistochemical staining, to rule out the nonspecific staining allowing better interpretation of specific staining at the antigenic site, negative staining controls were used in which sections were incubated with N-Universal Negative Control mouse or rabbit antibody (Dako) under identical conditions. The sections were then incubated with appropriate biotinylated secondary antibody for 1 h at room temperature followed by 30 min incubation with HRP-conjugated streptavidin. Proteins were visualized using 3, 3’-diaminobenzidine (DAB) for 10 min at room temperature. The sections were counterstained with Harris Hematoxylin, dehydrated and mounted.

TUNEL staining for apoptotic cells

Apoptotic cells were detected using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI) following manufacturer's protocol with some modifications. In brief, the tissue sections after deparaffinization and re-hydration were permeabilized with proteinase K (30 mg/ml) for 1 h at 37°C. Thereafter, the sections were quenched of endogenous peroxidase activity using 3% hydrogen peroxide for 10 min. After thorough washing with 1x PBS, the sections were incubated with equilibration buffer for 10 min, and then TdT reaction mixture was added to the sections, except for the negative control, and incubated at 37°C for 1 h. The reaction was stopped by immersing the sections in 2x saline-sodium citrate buffer for 15 min. The sections were then added with streptavidin-HRP (1:500) for 30 min at room temperature, and after repeated washings, the sections were incubated with substrate DAB until color development (~5–10 min). The sections were then mounted after dehydration, and observed under 400x for TUNEL-positive cells (brown color).

Preparation of tissue homogenates and western blotting

The colonic tissues were scrapped, and the samples thus obtained were homogenized in the lysis buffer using a polytron homogenizer and then centrifuged at 14, 000 r.p.m (8). The supernatants thus obtained were used in the analyses. For each sample, 50–80 µg protein/sample was resolved on Tris-glycine gel, transferred onto nitrocellulose membranes and blocked for 1 h at room temperature with 5% non-fat dry milk. The membranes were then incubated with primary antibody (anti-cleaved PARP; Cell Signaling Technologies, San Diego, CA) overnight at 4°C and then with appropriate secondary antibody. Protein was visualized by enhanced chemiluminescence detection system. Membranes were stripped and reprobed with anti-β-actin antibody (Sigma) as loading control. The bands were scanned with Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA), and the mean density of each band was analyzed by the Scion Image program (National Institutes of Health, Bethesda, MD) and presented as fold change of AOM group below each band.

Immunohistochemical and statistical analyses

All the microscopic analyses were done using Zeiss Axioscop 2 microscope (Carl Zeiss, Jena, Germany). The pictures were taken by Kodak DC290 camera under 400x magnification and processed by Kodak Microscopy Documentation System 290 (Eastman Kodak Company, Rochester, NY). The mean ± SE values were obtained from the evaluation of multiple fields in each group. For each animal, 5–10 representative fields were counted at 400x magnification, and the data represent the results from at least 6 rats in each group. For statistical significance of the difference, the data were analyzed using the SigmaStat 2.03 software. The statistical significance of difference between control and AOM-treated group, and AOM-treated versus silibinin plus AOM- or AOM plus silibinin-treated groups was determined by one way analysis of variance (one-way ANOVA) followed by Bonferroni t-test for multiple comparisons. P<0.05 was considered statistically significant.

Results

General observations

All the rats were monitored on a regular basis to investigate if dietary silibinin had any negative effect on the body weight gain or the diet consumption. The food consumption (g/day/rat) and gain in body weight did not differ significantly among the control and silibinin-fed groups till the end of the study (data not shown). Furthermore, at necropsy, no pathological alternations were found in any organs by gross observation, including the liver in F344 male rats.

Suppressive effects of dietary silibinin on ACF formation

A well-established short-term protocol was used to determine the efficacy of silibinin in inhibiting the AOM-induced ACF formation. Table 1 summarizes the effect of dietary silibinin on AOM-induced ACF formation. All the rats belonging to groups 2 through 11, which were treated with AOM, developed ACF. The mean number of ACF/colon in the animals administered AOM alone (group 2) was 169 ± 15. The dietary administration of silibinin at all four different doses (0.033, 0.1, 0.33 or 1%) given as pre- & post-initiation (groups 3 to 6) or post-initiation alone (groups 7 to 10) significantly (P<0.001) reduced the ACF formation in a dose-dependent manner compared to the group 2 animals. Within two different silibinin treatment protocols, the percentage of inhibition was more in animals given silibinin as pre- & post-initiation (39 to 65% reduction) than as post-initiation only (29 to 55% reduction). Furthermore, the number of ACF consisting of more than 4 crypts also decreased significantly (P<0.05) in silibinin-fed rats (groups 3 to 10) as compared to AOM alone treated rats (group 2; Table 1). The animals fed with 0.032% sulindac also showed 50% inhibition in ACF formation compared to group 2 rats (Table 1).

Table 1.

Inhibitory effect of silibinin on AOM-induced aberrant crypt foci incidence and multiplicity in F344 male rat colon

Groups	Treatment	Incidence of ACF formation (%)	No. of ACF/colon	Crypt multiplicity of ACF
				1 crypt	2 crypts	3 crypts	≥4 crypts
1	Control	0/7 (0)	-	-	-	-	-
2.	AOM	7/7 (100)	169 ±15	71 ±11	60 ±7	24 ±6	14 ±7
3	0.033% Sb + AOM	7/7 (100)	103 ±13**	44 ±5**	30 ±5**	19 ±7	10 ±4
4	0.1% Sb + AOM	7/7 (100)	79 ±11**	24 ±9**	26 ±7**	17 ±9*	12 ±4
5	0.33% Sb + AOM	6/6 (100)	68 ±12**	28 ±6**	20 ±5**	12 ±5**	8 ±6*
6	1% Sb + AOM	7/7 (100)	60 ±13**	25 ±7**	17 ±8**	9 ±6**	9 ±5*
7	AOM + 0.033% Sb	7/7 (100)	120 ±11**	44 ±4**	48 ±3**	15 ±3*	13 ±5
8	AOM + 0.1% Sb	7/7 (100)	105 ±8**	44 ±4**	39 ±5**	12 ±2**	10 ±2
9	AOM + 0.33% Sb	7/7 (100)	89 ±9**	34 ±3**	30 ±2**	15 ±2**	10 ±2
10	AOM + 1%Sb	7/7 (100)	77 ±8**	29 ±2**	24 ±3**	13 ±3**	11 ±2
11	AOM + 0.032% sulindac	6/6 (100)	85 ±7**	32 ±3**	30 ±2**	14 ±3**	9 ±2
12	1% Sb	0/7 (0)	-	-	-	-	-

Data are shown as mean ±SD of 7 samples in each group (except groups 5 and 11; n=6)

^*P<0.05

^**P<0.001 versus group 2, Bonferroni t-test

Inhibitory effects of dietary silibinin on PCNA labeling index and cyclin D1-positive cells

The PCNA labeling index and cyclin D1 expression in colonic mucosa are presented in Figure 2. PCNA is an auxiliary protein of DNA polymerase-δ and high levels of its expression correlate with cell proliferation, suggesting that PCNA is an excellent marker of cellular proliferation (23). Microscopic examination of colonic tissue sections stained for PCNA (brown color) clearly showed a strong staining in AOM-treated samples and its reduction following silibinin treatment as shown in two representative sections stained immunohistochemically for PCNA (Figure 2A). The quantitative analyses of all the PCNA-stained sections in different groups (Figure 2B) clearly showed a significant (P<0.001) increase in the percentage of PCNA-positive cells in the colonic mucosa of AOM-treated rats (34.1 ± 4.9) compared to control (Group 1) rats (7.3 ± 2.03); both groups received AIN-76A control diet alone. All the experimental diets containing different dose levels of silibinin (0.033, 0.1, 0.33 or 1%) given either pre- & post-initiation or post-initiation alone, or 0.032% sulindac, significantly (P<0.001) decreased the PCNA labeling index in the colonic mucosa of AOM-treated rats compared to Group 2 (Figure 2B). The PCNA-positive cells were reduced dose-dependently by 37–61% and 26–42% in different groups fed silibinin in pre- & post- or only post-initiation protocol, respectively (Figure 2B). In other assay where cyclin D1 levels were analyzed immunohistochemically, compared to AOM alone group, silibinin plus AOM groups of samples showed a marked reduction in cyclin D1 staining, as evidenced by representative sections in Figure 2C. In terms of quantitative analyses of these results, similar to PCNA labeling index, the percentage of cyclin D1-positive cells also significantly (P<0.001) increased in the colonic mucosa of AOM-treated animals (24.3 ±3) compared to control (Group 1) animals (5.1 ±2) (Figure 2D). A significant decrease (P<0.05 to P<0.001) in cyclin D1 expression was also observed in that animals which received different dose levels (0.033, 0.1, 0.33 or 1%) of silibinin given either pre- & post-initiation or post-initiation alone compared to AOM-alone rats (Figure 2D). In quantitative analysis, compared to AOM alone, the percentage of cyclin D1-positive cells reduced by 29 to 57% and 23 to 50% in different silibinin-fed groups during pre- & post- and post-AOM initiation protocols, respectively (Figure 2D). The dietary administration of 0.032% sulindac also significantly (P<0.001) reduced the number of cyclin D1-positive cells by 43% compared to AOM alone rats (Figure 2D). The percentages of PCNA- and cyclin D1-positive cells were almost comparable between control diet fed rats (group 1) and 1% silibinin diet alone fed rats (group 12).

Fig. 2.

Inhibitory effects of dietary silibinin on PCNA labeling index and cyclin D1 expression in colon tissues from AOM-exposed rats. Representative photographs for immunohistochemical staining of PCNA (A) and cyclin D1 (C) positive cells in AOM alone and silibinin + AOM treated groups, respectively, are shown at 400x magnification. The percentages of PCNA- (B) and cyclin D1- (D) positive cells assessed by quantification of immunohistochemically stained rat colonic epithelium in 5–10 randomly selected fields from each tissue sample, are shown. The data represent mean ±SE value from at least 6 rats in each group.

Apoptosis inducing effects of dietary silibinin

An apoptotic response of dietary silibinin in the colonic tissue of AOM-injected rats was next investigated by TUNEL staining. The representative photographs for TUNEL positive cells in AOM alone and silibinin plus AOM treated groups, shown at 400x magnification, clearly document that silibinin causes strong a apoptotic effect (Figure 3A). The quantitative analyses of the stained sections in different treatment groups showed a significant (P<0.001) increase in the number of TUNEL-positive cells by dietary silibinin at all dose levels in AOM-injected rats compared to control diet-fed AOM-injected rats (Figure 3B). However, the apoptotic induction by silibinin was more profound in the rats given silibinin at pre-& post-initiation (2.4 to 4.8-fold increase) than in the rats given same dose level of silibinin at post-initiation alone (2.0 to 3.3-fold increase) (Figure 3B). The dietary administration of 0.032% sulindac also caused a 3.0 fold increase in TUNEL-positive cells compared to AOM alone rats (Figure 3B). An apoptosis inducing effect of silibinin was further confirmed by analyzing the cleaved PARP expression through western blotting (Figure 3C). Similar to the observation in TUNEL staining, dietary silibinin given at pre- & post-initiation or post-initiation phase, showed a higher expression of cleaved PARP compared to AOM alone group; with a maximum effect at the dose level of 1% silibinin (2.6 fold). The dietary administration of 0.032% sulindac also increased the expression of cleaved PARP compared to the AOM alone group.

Fig. 3.

Effect of dietary silibinin on TUNEL labeling index and expression of cleaved PARP in colon tissues from AOM-treated rats. Colon tissues were analyzed and quantified for apoptosis by TUNEL staining as detailed in Methods. (A) Representative photographs for the TUNEL-positive cells in AOM alone and silibinin + AOM treated groups are shown at 400x magnification. (B) The percentage of TUNEL positive cells assessed by quantification of stained colonic tissues in 5–10 randomly selected fields from each tissue sample is shown. The data represent mean ±SE value from at least 6 rats in each group. (C) The colon tissues collected at the end of the experiment were also analyzed for cleaved PARP expression by SDS-PAGE and immunoblotting as described in Methods. The densitometry analysis results are shown below each band, and represent fold change versus AOM alone.

Inhibitory effects of dietary silibinin on iNOS and COX2 expression

The representative photographs for the immunohistochemical staining of iNOS (Figure 4A) and COX2 (Figure 4C) positive cells in AOM alone and silibinin plus AOM treated groups, clearly show that silibinin decreases the protein levels of these two key molecules in colonic mucosa. The quantitative analyses of the immunohistochemically stained colonic tissues showed significantly increased number of iNOS (~9 fold, Figure 4B) and COX2 positive cells (~7 fold, Figure 4D) in AOM alone-injected rats compared to those fed with control diet alone. However, the iNOS expression was significantly decreased in the animals that received different dose levels (0.033, 0.1, 0.33 or 1%) of silibinin given either pre- & post-initiation or post-initiation alone compared to AOM alone rats. The reduction in the percentage of iNOS-positive cells was 17 to 52% and 11 to 36% in different silibinin-fed groups during pre- & post- and post-AOM initiation protocols, respectively (Figure 4B). The administration of 0.032% sulindac in diet also significantly reduced the iNOS expression by 43% compared to AOM alone rats (Figure 4B). Similar to iNOS, all the dose levels of silibinin given at pre- & post-initiation or post-initiation phase significantly decreased (25 to 48% decrease compared to AOM alone) the COX2 positive cells in the AOM-injected rats compared to AOM alone-injected rats (Figure 4D). The dietary administration of 0.032% sulindac also decreased the level of COX2 by 43% (Figure 4D). Silibinin alone at 1% dose level did not show any effect on COX2 levels in colonic mucosa with comparable staining to control diet alone group of rats (Figure 4D). In other studies, no significant decrease in COX1-positive cells was observed in rats fed silibinin at lower doses (0.033% and 0.1%) given at both pre- & post-initiation or post-initiation phase; however, administration of silibinin at higher dose levels (0.33 and 1%) also significantly decreased the COX1-positive cells in the colon of AOM-injected rats compared to AOM alone-injected positive controls (data not shown).

Fig. 4.

Inhibitory effects of dietary silibinin on iNOS and COX2 expressions in colon tissues from AOM-exposed rats. Representative photographs for immunohistochemical staining of iNOS (A) and COX2 (C) positive cells in AOM alone and silibinin + AOM-treated groups, respectively, are shown at 400x magnification. The percentages of iNOS- (B) and COX2- (D) positive cells assessed by quantification of immunohistochemically stained rat colonic epithelium in 5–10 randomly selected fields from each tissue sample, are shown. The data represent mean ±SE value from at least 6 rats in each group.

Discussion

ACF, as described first by Bird (19) in 1987, are putative pre-neoplastic lesions that appear on the colon surface of the rodents after treatment with colon carcinogens such as AOM. Similar kind of lesions were also later characterized in humans, and since then, AOM-induced ACF model had been the most valuable experimental model for evaluating both naturally occurring and synthetic agents for their colon cancer chemopreventive efficacy. Furthermore, ACF has been proven to be a reliable biomarker in short-term screening assay for colon tumorigenesis in laboratory rodents (24). Therefore, the present study was designed to evaluate the chemopreventive efficacy of silibinin at four different dose levels (0.033, 0.1, 0.33 and 1%) in two different treatment protocols (pre & post-initiation and post-initiation) using AOM-induced ACF as biomarker. Furthermore, in view of the demonstrated chemopreventive efficacy of various non-steroidal anti-inflammatory drugs in the AOM-induced ACF model, the activity of silibinin was also compared with the efficacy of sulindac.

The results of our study demonstrated that dietary administration with silibinin at all the four dose levels significantly inhibit AOM-induced ACF formation both in pre & post-initiation as well as post-initiation phase in F344 rats. The inhibitory effect of silibinin on colon ACF formation was also associated with the reduction in crypt multiplicity especially suppressing the larger crypts (≥4 crypts) which have more tendencies to progress into the malignancy. These findings suggest that dietary silibinin suppresses both pre & post-initiation and post-initiation phase of chemically-induced colon carcinogenesis. Most importantly, the rats fed with the diets containing silibinin showed no adverse effects on the food consumption and the animal growth rate (data was not shown). The inhibitory effect of silibinin on ACF formation is consistent with the earlier report showing that several naturally occurring phytochemicals inhibit AOM-induced ACF formation and crypt multiplicity (25). Kohno et al. (12) have also reported that in both a short- and a long-term experiment, dietary feeding of silymarin during the initiation or post-initiation phase of AOM-induced colon carcinogenesis reduces the incidence and the multiplicity of colonic adenocarcinoma. Several explanations for the inhibitory effects of silibinin on ACF-formation by AOM are discussed below, though the exact mechanism remains to be elucidated in future studies.

Increased cell proliferation has long been shown to play a crucial role in the initiation phase as well as the promotion/progression stage of carcinogenesis. In this regard, PCNA is implicated in DNA replication by forming a sliding platform that could mediate the interaction of numerous proteins with DNA and hence, PCNA is regarded as a reliable biomarker for cell proliferation (26). In the present study, silibinin at all the dose levels and at different phases (pre & post-initiation as well as post-initiation) of treatment significantly reduced the increase in proliferative index caused by AOM treatment in rats fed with the control diet. Previous report from our laboratory suggests that silibinin is potent suppressor of PCNA-positive tumor cells (27). In addition to PCNA, silibinin also suppressed the AOM-induced elevation of cyclin D1 levels. In comparison to the normal crypts, the upregulation in the expression of cyclin D1 in AOM-induced ACF was anticipated to also favor the greater proliferation in ACF. In this regard, cyclin D1, a cell cycle regulator which is overexpressed in a variety of human cancers including colon cancer, has been shown to be repressed by several anticancer phytochemicals (28). Therefore, the observed chemopreventive potential of silibinin against AOM-induced ACF might be partly via its antiproliferative effect.

Apoptosis and associated cellular events have profound effect on the progression of benign to malignant phenotype, and can be targeted for the therapy of various malignancies including colon cancer (29). Hence, the apoptosis inducing effect of silibinin was evaluated using TUNEL-positive index during AOM-induced ACF formation. Furthermore, as caspase-3 activation is one of the most important events in apoptosis and that PARP is a major substrate of activated caspase-3, we also examined the levels of cleaved PARP by western blotting to further confirm the apoptotic response of silibinin. The results of the present study clearly indicate that dietary silibinin induces apoptosis in a dose-dependent manner in the colon tissue of AOM-injected rats. These results are consistent with the apoptosis inducing effect of silibinin in human HT-29 colon cancer cells, reported by us previously where we suggested that the proapoptotic effect of silibinin could be attributed to the inhibition of constitutively active mitogenic and cell survival signaling (13). More recently, we have also shown that silibinin decreases the level of survivin with a concomitant increase in the activated caspase-3, as an important in vivo mechanism for apoptosis induction in human bladder tumor xenograft model (30). Apoptosis inducing effects of silibinin have also been reported in several other in vivo and in vitro cancer models (31, 32). With regard to current study, whereas our results clearly show an in vivo apoptotic effect of silibinin that could in part be responsible for its overall efficacy in inhibiting AOM-induced ACF formation in rat colon, more studies are needed in future to define the underlying molecular events leading to in vivo apoptosis induction by silibinin in CRC models.

The roles of inflammatory molecules iNOS and COX2 as enhancer of carcinogenesis in many organs including colon are currently receiving increased attention, and therefore, the suppression of highly elevated iNOS and COX2 expressions has become a target for cancer chemoprevention (33–35). Elevation of iNOS and COX2 contributes to pathological processes, such as inflammation, abnormal cell proliferation and reduced apoptosis that favor the process of carcinogenesis. Accordingly, the anti-inflammatory activity of silibinin was investigated as one of the mechanisms of its efficacy in inhibiting AOM-induced ACF formation in the rat colon. COX2 is induced by cytokines, mitogens and tumor promoters, and mediates the inflammatory process, catalyzing the conversion of arachidonic acid into prostaglandins. Recent investigations have revealed that COX1 and COX2 are over expressed in colon tumors (34, 35). In particular, it has been suggested that inhibition of COX2 is negatively related with colon cancer risk. Although we did not investigate the mechanisms of the suppressive effect of silibinin on the elevated levels of iNOS and COX2 in AOM-injected rat colons, one possible mechanism could be the silibinin-caused suppression of the transcriptional activities of STAT and NF-κB, as observed in other studies (36); both these molecules are well-known to regulate iNOS and COX2 expression. Furthermore, tumor necrosis factor alpha is known to activate AP-1 which is important in the induction of COX2 and iNOS transcription. Since silibinin inhibits AP-1 dependent transactivation (36), some of the inhibitory effects of silibinin on COX2 and iNOS induction may also be mediated by the inhibition of AP-1, which would be consistent with other studies showing that several chemopreventive agents suppress both iNOS and COX2 levels by regulating AP-1 and NF-κB signaling (37, 38).

In conclusion, the findings described here demonstrate that dietary administration of silibinin in two different protocols (pre- & post-initiation and post-initiation phases) dose-dependently inhibits the formation and development of AOM-induced colonic ACF in F344 rats. These promising results suggest the importance of conducting further investigations with silibinin in preclinical colon cancer models, especially long-term in vivo efficacy studies, to support the clinical usefulness of silibinin against colon cancer development.

Abbreviations

AOM

azoxymethane

ACF

aberrant crypt foci

CRC

colorectal cancer