Cancer Prevention Research Inhibition of Azoxymethane-Induced Colonic Aberrant Crypt Foci Formation by Silibinin in Male Fisher 344 Rats

Chemoprevention is a practical approach to control colorectal cancer, which is one of the major causes of cancer mortality in the United States. 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-week-old male Fisher 344 rats were fed control or silibininsupplemented (0.033%, 0.1%, 0.33%, or 1%, w/w) diet. After 2 weeks, AOM was injected once a week for 2 weeks while silibinin treatments were 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 inducible nitric oxide synthase and cyclooxygenase-2, by immunohistochemistry and/or immunoblotting. Silibinin significantly (P < 0.001) reduced dose-dependently the number and multiplicity of AOM-induced ACF formation. Silibinin feeding in preand 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 AOMinduced colonic cell proliferation, evidenced by proliferative cell nuclear antigen and cyclin D1 immunohistochemical staining, and induced apoptosis in these colon tissues, evidenced by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining and cleaved poly(ADP-ribose) polymerase. Furthermore, silibinin significantly decreased AOMinduced inducible nitric oxide synthase– and cyclooxygenase-2–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. Colorectal cancer 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 colorectal cancer 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 multifactorial, 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 colon cancer (3). Nutritional intervention is another effective and promising complimentary strategy for controlling the incidence of colon cancer (4). Several epidemiologic 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 pharmacologic 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). Nontoxicity, 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 colorectal cancer 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, and previous report from our laboratory has shown the anticancer activity of silibinin in human colon carcinoma HT-29 cells (13), no in vivo study Authors' Affiliations: Department of Pharmaceutical Sciences, School of Pharmacy, and University of Colorado Cancer Center, University of Colorado Denver, Denver, Colorado; and Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Received 03/21/2008; revised 05/01/2008; accepted 05/05/2008. Grant support: USPHS grant RO1 CA112304 from the National Cancer Institute, NIH. Requests for reprints: Rajesh Agarwal, School of Pharmacy, University of Colorado Denver, Box C-238, 4200 East 9th Avenue, Denver, CO 80262. Phone: 303-315-1381; Fax: 303-315-6281; E-mail: [email protected]. ©2008 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0059 376 Cancer Prev Res 2008;1(5) October 2008 www.aacrjournals.org Cancer Research. on June 19, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from has reported the efficacy of silibinin in colorectal cancer chemoprevention model. Aberrant crypt foci (ACF) are early morphologic changes observed in rodents after administration of colon-specific carcinogen such as azoxymethane (AOM; ref. 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 preneoplastic lesions and are currently used as a surrogate biomarker to rapidly evaluate the chemopreventive potential of several agents, including both naturally occurring and synthetic, using AOM in the Fisher 344 rat model (16–18), which accurately replicates many of the clinical, genetic, cellular, and morphologic features of human colorectal cancer (19). AOMinduced ACF are characterized by an increase in the size of the crypts, the epithelial lining, and the pericryptal zone and share many morphologic 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 Fisher 344 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 Fisher 344 rats, which is 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 Fisher 344 rats were purchased from The Jackson Laboratory at the age of 4 wk and maintained in the animal housing facility at the University of Colorado Denver. Silibinin was obtained commercially from Sigma Chemical Co., and its purity was analyzed by highperformance liquid chromatography to be >98% (22). The experimental protocol for the present study is shown in Fig. 1. Animals were maintained at 12 h light/12 h dark cycles with free access to water and food (AIN-76A powder diet from Dyets, Inc.). After 1 wk of acclimatization, animals were randomly divided into 12 groups of 10 animals each and fed AIN-76A control diet (groups 1 and 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 to 11 were given s.c. injection of AOM once a week for 2 wk at a concentration of 15 mg/kg of body weight. Two weeks after the last AOM injection, rats in groups 7 to 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. Because sulindac is well known as an effective chemopreventive agent against AOM-induced ACF formation in Fisher 344 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 wk of age, the rats were sacrificed, and their colons were evaluated for ACF or other marker studies. Determination of ACF ACF analysis was done according to Bird (20), in which 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 then stained with 0.2% methylene blue in Krebs-Ringer solution for 5 to 10 min and placed with the mucosal side up on a microscope slide and counted under a light microscope at ×400 magnification. Aberrant crypts were distinguished from the Fig. 1. Experimental protocol for AOM-induced colonic aberrant crypt foci formation in male Fisher 344 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 2 wks at a dose level of 15 mg/kg of body weight by s.c. injection. Other details of the experimental design are described in Materials and Methods. Silibinin Inhibits AOM-Induced ACF In vivo 377 Cancer Prev Res 2008;1(5) October 2008 www.aacrjournals.org Cancer Research. on June 19, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from 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 ≥4 aberrant crypts per focus. Immunostaining for proliferative cell nuclear antigen, cyclin D1, inducible nitric oxide synthase, and cyclooxygenase-2 Colon tissue samples were fixed in 10% phosphate-buffered formalin for 10 h at 4°C, dehydrated in ascending concentrations of ethanol, cleared with xylene, and embedded in PolyFin (Triangle Biomedical Sciences). Paraffin-embedded tissue blocks were cut with a rotary microtome into 4-μm sections and processed for immunohistochemical staining. Briefly, after deparaffinization and rehydration, the sections were treated with 0.01 mol/L 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 incubated with proliferative cell nuclear antigen (PCNA) mouse monoclonal antibody (1:400 dilution; DAKO), cyclin D1 rabbit polyclonal antibody (1:200 dilution; Santa Cruz Biotechnology), inducible nitric oxide synthase (iNOS) rabbit polyclonal antibody (1:200 dilution; Abcam, Inc.), or cyclooxygenase-2 (COX2) rabbit polyclonal antibody (1:100 dilution; Cell Signaling Technologies) 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 and allow 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 horseradish peroxidase–conjugated streptavidin. Proteins were visualized with 3,3′-diaminobenzidine for 10 min at room temperature. The sections were counterstained with Harris hematoxylin, dehydrated, and mounted. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining for apoptotic cells Apoptotic cells were detected using the DeadEnd Colorimetric terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) system (Promega) following the manufacturer's protocol with some modifications. In brief, the tissue sections after deparaffinization and rehydration 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 1× PBS, the sections were incubated with equilibration buffer for 10 min, and then terminal deoxyribonucleotidyl transferase 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 2× saline-sodium citrate buffer for 15 min. The sections were then added with streptavidin-horseradish peroxidase (1:500) for 30 min at room temperature, and after repeated washings the sections were incubated with substrate 3,3′-diaminobenzidine until color development (∼5-10 min). The sections were then mounted after dehydration and observed under ×400 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 lysis buffer using a polytron homogenizer and then centrifuged at 14,000 rpm (8). The supernatants thus obtained were used in the analyses. For each sample, 50 to 80 μg of protein per sample were resolved on Tris-glycine gel, transferred onto nitrocellulosemembranes, and blocked for 1 h at room temperaturewith 5% nonfat dry milk. The membranes were then incubated with the primary antibody anti–cleaved poly(ADP-ribose) polymerase (PARP; Signaling Technologies) overnight at 4°C and then with appropriate secondary antibody. Protein was visualized with the 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), and the mean density of each band was analyzed by the Scion Image program (NIH) and presented as fold change of AOM group below each band. Table 1. Inhibitory effect of silibinin on AOM-induced aberrant crypt foci incidence and multiplicity in Fisher 344 male rat colon Groups Treatment Incidence of ACF formation (%) No. 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) — — — — — NOTE: Data are shown as mean ± SD of seven samples in each group (except groups 5 and 11, n = 6). *P < 0.001, vs group 2 (Bonferroni t test). P < 0.05. Cancer Prevention Research 378 Cancer Prev Res 2008;1(5) October 2008 www.aacrjournals.org Cancer Research. on June 19, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from Immunohistochemical and statistical analyses All the microscopic analyses were done using Zeiss Axioscop 2 microscope (Carl Zeiss). The pictures were taken with Kodak DC290 camera under ×400 magnification and processed by Kodak Microscopy Documentation System 290 (Eastman Kodak Company). The mean ± SE values were obtained from the evaluation of multiple fields in each group. For each animal, 5 to 10 representative fields were counted at ×400 magnification, and the data represent the results from at least six 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 groups and between AOM-treated and silibinin plus AOM– or AOM plus silibinin–treated groups was determined by one-way ANOVA followed by Bonferroni t test for multiple comparisons. P < 0.05 was considered statistically significant.