PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC 2010 October 15.
Published in final edited form as:
PMCID: PMC2792897
NIHMSID: NIHMS141657

Inhibition of AOM-Induced Colorectal Cancer by CP-31398, a TP53 modulator, Alone or in Combination with Low Doses of Celecoxib in Male F344 Rats

Abstract

Tumor suppressor p53 plays a major role in colorectal cancer development. The present study explores the effects of p53 modulating agent CP-31398 alone and combined with celecoxib on azoxymethane (AOM)-induced aberrant crypt foci (ACF) and colon adenocarcinomas in F344 rats. Maximum tolerated doses were 400 and 3000 ppm for CP-31398 and celecoxib, respectively. ACF and tumor efficacy endpoints were carried out on AOM-treated seven week old rats (48/group) fed the control AIN-76A diet. Two weeks after carcinogen treatment, rats were fed the diets containing 0, 150, 300 ppm of CP-31398, or 300 ppm of celecoxib, or 150 ppm CP-31398 plus 300 ppm celecoxib. ACF were determined at 8 weeks and colon adenocarcinomas 48 weeks after AOM treatment. Dietary CP-31398 was shown to suppress mean colonic total ACF by 43% and multicrypt ACF by 63%; dietary CP-31398 at 150 and 300 ppm suppressed adenocarcinoma incidence by 30.4% (p <0.02) and 44% (p<0.005), respectively, and adenocarcinoma multiplicity by 51% (p<0.005) and 65% (p<0.0001), respectively. Dietary celecoxib suppressed colon adenocarcinoma incidence (60%, p<0.0003) and multiplicity (70%, p<0.0001). Importantly, combination of low-dose CP-31398 and celecoxib suppressed colon adenocarcinoma incidence by 78% and multiplicity by 90%. Rats which were fed the high-dose CP-31398 or a combination of low-dose CP-31398 and celecoxib showed considerable enhancement of p53 and p21WAF1/CIP expression, apoptosis, and reduced tumor cell proliferation in colonic tumors. These observations demonstrate, for the first time, that CP-31398 possesses significant dose-dependent chemopreventive activity in a well-established colon cancer model, and that a combination of low-dose CP-31398 and celecoxib significantly enhanced colon cancer chemopreventive efficacy.

Keywords: Colorectal cancer, chemoprevention, tumor suppressor proteins, p53 modulators, apoptosis

Introduction

Epidemiological and experimental studies indicate that the risk of developing colon cancer may be attributable to genetic and environmental factors, including endogenously occurring promoting agents (13). The p53 tumor suppressor protein is involved in DNA damage repair, genomic instability, cell cycle arrest, and apoptosis through transcriptional regulation of genes implicated in these pathways (410). Although mutations affecting p53 are present in over 50% of all cancers, including colon cancer (45), “stress-induced” non-mutational activation of p53 may occur very early in cancer progression (68).

Several attempts to restore mutant p53 as a growth suppressor included micro-injection of monoclonal antibody 421, C-terminal peptide of p53, and small molecules such as CP-31398 and PRIMA1 (1114). CP-31398 can stabilize p53, protect against thermal denaturation, and maintain monoclonal antibody 1620 epitope conformation in newly synthesized p53 (11). CP-31398 stabilizes wild-type p53 in cells by inhibiting Mdm2-mediated ubiquitination and degradation (15). In a chromatin immunoprecipitation (ChIP) assay, CP-31398 promotes binding of mutant p53 to p53 response elements in vivo (16). Other studies using the purified p53 core domain have shown that CP-31398 can restore DNA binding activity to mutant p53 in vitro (17). Moreover, small molecule modulators of p53, including CP-31398, appear to suppress growth of human colon tumor xenografts (18) and prevent UVB-induced squamous skin cancer in mice by restoring mutant p53 function (19). Recently, we have shown that CP-31398 administered in the diet suppressed APCmin intestinal tumors in a dose-dependent manner by upregulating p53 protein levels and downstream signaling molecules (20).

The role of cyclooxygenase-2 (COX-2) in colon carcinogenesis is well established (21,22). Previously, we have shown chemopreventive effects by the COX-2 selective inhibitor celecoxib in rodent models of colon cancer (23,24). Of particular interest is the observation that COX-2 metabolites, particularly electrophilic prostaglandins (PGs), appear to impair p53 protein function; COX-2 inhibition by celecoxib increases the nuclear localization of functionally active p53 (25). However, while efficacy has been attributed to COX-2 inhibitors, five clinical trials demonstrated that 3 different COX-2 inhibitors caused an increased rate of myocardial infarction, leading to concerns about the broader applicability of selective COX-2 drugs. It is important, therefore, to develop strategies that target both COX-2 inhibition and upregulation of p53 in a clinical setting.

Our current study was undertaken in order to reconcile potential and otherwise known toxicities by combining two agents, each with different modes of action and proven efficacy, at much lower concentrations than if used individually, in order to reduce toxicity and enhance efficacy. Specifically, our proposed studies were designed to test whether nontoxic low-dose celecoxib in combination with nontoxic low-dose CP-31398 would provide better protection in a well-established colon cancer model. The effects of CP-31398 and celecoxib on colonic tumor cell proliferation, apoptosis, and expression levels of p53 and p21WAF1/CIP were also determined.

Materials and Methods

Animals, diets, chemopreventive agents

All animal experiments were performed in accordance with NIH guidelines and University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (IACUC)-approved protocol. Male F344 rats were obtained at 6 weeks of age from Harlan Laboratories (Fredrick, MD). Ingredients for the semi-purified diets were purchased from Bioserv (Bethlehem, PA) and stored at 4°C prior to diet preparation. Diets were based on the modified AIN-76A diet. The semi-purified diet includes 20% casein, 52% corn starch, 13% dextrose, 5% corn oil, 5% alphacel, 3.5% AIN mineral mix, 1.2% AIN revised vitamin mix, 0.3% d, l-methionine, and 0.2 choline bitartrate (26). CP-31398 and celecoxib were premixed with a small quantity of diet, and then blended into bulk diet using a Hobart Mixer (Troy, OH). Both control and experimental diets were prepared weekly and stored in a cold room. CP-31398 and celecoxib were provided by the NCI chemopreventive drug repository (Rockville, MD). The Agent(s) content in the experimental diets was determined periodically in multiple samples taken from the top, middle, and bottom portions of individual diet preparations to verify uniform distribution.

MTD

Experimental design to carry the MTD bioassay is shown in Figure 1A. To estimate the appropriate dose level for the efficacy studies, MTD was determined by feeding male F344 rats CP-31398 and celecoxib in a 8-week toxicity study. MTD was defined as the highest dose that causes no more than a 10% body weight decrement or produces mortality or any external signs of toxicity that would be predicted to shorten the natural lifespan of the animal. At 7 weeks of age, groups of male rats (9/group) were fed the experimental diets containing 0, 100, 200, 400, 800, or 1,600 ppm CP-31398, and 0, 500, 1000, 2000, 3000, and 4000 ppm celecoxib. Although celecoxib has been extensively studied in animal models by several investigators, no systematic studies have so far been conducted on MTD of this agent in any animal model. Body weights were recorded twice weekly for 8–9 weeks. All animals were monitored daily for signs of toxicity such as ill appearance, circling rashes, tremors, roughened coat, rhinitis, chromodacryorrhea, and prostration. At the end of 6 weeks, mice were sacrificed and their oral cavity, colon, small intestine, stomach, liver, and kidneys were examined for any abnormalities under a dissection microscope.

Figure 1
Experimental Protocol: Efficacy Studies

Experimental Protocol for CP-31398 Induced Colonic ACF Inhibition

The experiment was designed to evaluate whether CP-31398 provides protection against the AOM-induced colonic preneoplastic lesions in rats. Experimental design is summarized in Figure 1B. Based on the MTD, different doses of CP-31398 were administered continuously from one week before carcinogen treatment until the end of the study. At seven weeks of age, groups of 12 rats per group (AOM-treated rats) were fed either the control diet or experimental diet containing 0, 100, 200, or 400 ppm CP-31398. At eight weeks of age, rats intended for carcinogen treatment were injected sc with AOM (Midwest Research Institute, KA) at a dose rate of 15 mg/kg body weight once weekly for two weeks, and those intended for vehicle treatment received an equal volume of normal saline. These dietary regimens were continued until termination of the experiment eight weeks after the second AOM treatment. Rats were killed by CO2 euthanasia, and all organs were examined grossly. Colons were evaluated for ACF. For this evaluation, they were slit open lengthwise from the anus to the cecum and then fixed flat with mucosa on the upper side between filter papers in 10% buffered formalin.

Quantification of ACF

Topographical analysis of the colonic mucosa was done according to Bird (27) as routinely performed in our laboratory (28,29). After a minimum of 24 hours, fixed colons were stained with 0.2% methylene blue solution for 5–10 minutes, placed mucosal side up on a microscopic slide, and viewed under a light microscope. Total number of ACF in the entire colon was determined in every two cm section of the colon, starting from the distal (taken a s 0-cm) to the proximal end of the colon. Aberrant crypts were distinguished from the surrounding normal crypts by their increased size, increased distance from lamina to basal surfaces of cells and easily discernible pericryptal zone. The parameters used to assess the aberrant crypts were incidence and multiplicity. Aberrant crypt multiplicity was determined as the number of crypts in each focus and categorized as containing up to four or more aberrant crypts/focus.

Chemoprevention of AOM-induced colorectal adenocarcinoma by CP-31398 alone or in combination with celecoxib

The experimental protocol is summarized in Figure 1C. Two dose levels of CP-31398, or 300 ppm celecoxib, and a single low-dose combination of CP-31398 and celecoxib, were evaluated for their chemopreventive efficacy. Based on MTD and ACF studies, we selected 150 and 300 ppm CP-31398 and 300 ppm of celecoxib (<8% MTD), considering these doses to be safe for long-term administration. Studies were designed to determine the efficacy of CP-31398 and celecoxib alone or in combination administered in the diet during the post-initiation stage of AOM-induced colon carcinogenesis.

Tumor Assay

Male F344 rats, received at weaning, were quarantined for 7 days and had unrestricted access to modified AIN-76A control diet. Following quarantine, all rats were randomly distributed by weight into various groups (Fig. 1C) and transferred to an animal holding room. They were housed in ventilated cages with filter tops (3 per cage) under controlled conditions with a 12-hour light and dark cycle at 50% relative humidity and 21°C. At 8 weeks of age, animals intended (46-rats/group, 36 AOM treated and 12 vehicle treated) for carcinogen treatment received 2 weekly sc injections of AOM (15 mg/kg body weight). Vehicle-treated groups (12 rats per group) received an equal volume of normal saline instead of AOM. Two weeks after the second injection of AOM or normal saline, rats were placed on control diet or diets containing two doses of CP-31398 and 300 ppm celecoxib or low-dose combination of both agents as outlined in Figure 1C. Body weights were recorded every two weeks until the 16th week and then every four weeks until termination of the experiment 50 weeks after the last AOM treatment. Eighteen hours before the termination, 6–8 rats from each group were administered 50 mg BrdU/kg body weight ip to assess cell proliferation. Moribund animals were killed and necropsied. Two rats in the control diet and one rat in the low-dose CP-31398 developed ear duct tumors and these moribund rats were killed four weeks before scheduled termination. All organs, including intestine, were examined grossly under the dissection microscope. Colon tumors with a diameter > 0.4 cm were cut into halves; one half was quickly frozen in liquid nitrogen and stored at −80° C until analyzed for expression and activity of COX-isoforms and markers of apoptosis and cell proliferation. Remaining portions of tissues were fixed in 10% neutral buffered formalin and processed by standard methods for histopathological evaluation (27).

BrdU assay for Cell Proliferation

We assessed the effect of CP-31398 and celecoxib on tumor cell proliferation by BrdU incorporation using immunohistochemistry (24). Paraffin-embedded colons from different treatment groups were cut longitudinally to five-micron-thick sections and mounted on microscopic slides. After deparaffinization, sections were blocked for endogenous peroxidase activity and incubated with 1% milk. BrdU antibody (Pharmingen, San Diego, CA) was applied at a 1:200 dilution for one hour at room temperature, then washed and incubated with secondary anti-rabbit IgG for 30 minutes, and then washed and incubated with avidin biotin-complex reagent (Vector Laboratories, Burlingame, CA). After rinsing with PBS, the slides were incubated with the chromogen 3,3′-diaminobenzidine (DAB) for three minutes, then rinsed and counterstained with hematoxylin. Scoring was performed by two investigators blinded to the identity of the BrdU positive cells at ×400 magnification. Cells with a brown nucleus were considered positive. The proliferation index (PI) was determined by dividing the number of positive cells by the number of negative cells and multiplying by 100.

TUNEL assay for Apoptosis

5 µM sections were cut and mounted on slides, rehydrated, and stained using the TUNEL method as described in our previous publication (20). Stained apoptotic epithelial cells (a minimum of 10 microscopic fields/section) were counted manually in a single-blend fashion. Scoring index will be similar to proliferation index as described above.

p53 and p21WAF1/CIP1 expression

Expression of p53 and p21WAF1/Cip in colonic tumor tissues were assessed by standard immunohistochemistry and western blot methods by applying p53 and p21WAF1/CIP1 (Santa Cruz, CA) primary antibodies.

Total COX and COX-2 synthetic activity

COX activities in colon tumor samples (4–8/group) were assayed using our previously published method (30). Briefly, the microsomal pellet was suspended in 50 mM potassium phosphate buffer (pH 7.4). To determine total COX activity, a 150 µl reaction mixture containing 12 µM [14C] arachidonic acid (420,000 dpm), 1 mM epinephrine, 1 mM glutathione in 50 mM phosphate buffer, and 25–35 µg tumor microsomal protein were incubated at 37°C for 15 min. To determine COX-2 activity, the reaction mixture was preincubated with 150 µM of aspirin to block COX-1 activity and modify COX-2 activity to 15-(R)-HETE. The reaction was terminated by adding 40 µl of 0.2 M HCl. COX metabolites of arachidonic acid were extracted three times with 0.5 ml ethyl acetate, evaporated to dryness under N2, redissolved in chloroform, and subjected to TLC on Silica G plates. Metabolites of [14C]-arachidonic acid corresponding to PGE2, PGF, PGD2, 6-keto-PGF, and TXB2 were detected by their co-migration with authentic standards for total COX activity, and [14C]-15-(R)-HETE for COX-2 activity. Radioactivity was counted with a BioScan 2000 Radiomatic detector and results were expressed as pmol or nmol /mg protein /min.

Statistical analyses

All results are expressed as mean ± SEM. Differences in body weights among groups were analyzed by ANOVA. Tumor incidences (percentage of rats with tumors) among dietary groups were compared by the chi-square method. ACF, tumor multiplicity (number of tumors per rat), protein expression and activities, and proliferative and apoptotic indexes, were analyzed by unpaired t-test with Welch’s correction. Dose-response effect was analyzed by linear regression (r2) analysis. Differences were considered statistically significant at p < 0.05.

Results

MTD and Dose Selection for Efficacy Studies

Effects of dietary CP-31398 and celecoxib on body weight of male F344 rats are summarized in Figures 2A and 2B. Dietary administration of CP-31398 at 100–400 ppm for 9 weeks had no significant effect on rats’ body weights. Dietary CP-31398 at 800 and 1,600 ppm for 9 weeks appears to have resulted in no weight gain compared to untreated rats, though the rats in question were active and survived until termination of the experiments.

Figure 2
Effect of CP-31398 and celecoxib on body weight gain of male F344 Rats

Rats fed celecoxib at 500 to 3,000 ppm for eight weeks showed body weight gain comparable to the control diet group. At 4,000 ppm celecoxib, rats had slightly lower body weight gain during weeks 2–4, but had significant body weight loss beginning at week 6 (p < 0.05) through week 8 (p < 0.01), albeit with no significant toxicity or animal death. Based on these studies, we determined an MTD for CP-31398 of 400 ppm, and for celecoxib of > 3000 ppm, but < 4000 ppm in male F344 rats. Chronic administration of CP-31398 and celecoxib at their respective MTDs, individually or together, produced no outward signs of toxicity nor any gross changes indicative of toxicity in the organs examined.

Effect of CP-31398 on AOM-Induced Colonic ACF

The effects of dietary CP-31398 on AOM-induced colonic ACF formation are shown in Figures 3A and 3B. AOM-treated rats fed diets containing 100, 200, and 400 ppm CP-31398 showed a significant decrease in the number of total ACF/colon (18–43%, p < 0.05–0.0005) compared to AOM-treated rats fed a control diet {168±10, (Mean± SEM) colon ACF}, including on average 31 of one crypt foci; 78 of two crypt foci; 35 of three crypt foci, and 24 of four or more crypt foci (Fig. 3A). Thus, significant dose-dependent inhibition of total ACF suppression, particularly of ACF containing four or more crypt foci (17–63%, p = 0.08–0.0001) was seen using dietary CP-31398 (Fig. 3B).

Figure 3
Inhibitory effect of CP-31398 on AOM-induced colonic ACF in male F344 rats

CP-31398 decreased incidence and multiplicity of colon adenocarcinomas

The effects of dietary CP-31398 on AOM-induced colon adenocarcinomas are shown in Figure 4 A and 4B. AOM-treated rats fed a control diet formed colon tumors at an incidence (percentage of rats with colon adenocarcinomas) of 74% and multiplicity (number of adenocarcinomas/colon) of 1.42 ± 0.21 (Mean ± SEM; N=36). In contrast, AOM-treated rats fed CP-31398 at 150 ppm inhibited colon adenocarcinoma incidence by 32% (p < 0.02) and multiplicity by 51.4% (p < 0.005); the corresponding values for CP-31398 at 300 ppm were 44% (p < 0.001) and 64.8% (p < 0.0003). A linear regression analysis of CP-31398 dose against tumor multiplicity yielded a statistical coefficient of r=0.994 (p < 0.0001). Dietary celecoxib at 300 ppm suppressed colon adenocarcinoma incidence by 62% and multiplicity by 75.3%, in agreement with previous studies concerning the effects of celecoxib on colon adenocarcinomas (23,31). However, combining low-dose CP-31398 (150 ppm) and celecoxib (300 ppm) significantly suppressed colon adenocarcinoma incidence by 77.6% (p < 0.0001) and multiplicity by 89.8% (p < 0.0001) which was statistically better than treatment with celecoxib at 300 ppm alone (Fig. 4B). Administration of CP-31398, celecoxib, or a combination of both significantly reduced colon tumor volume, compared to control (data not shown). Thus, the combination approach using low doses of celecoxib with a low-dose p53 modulating agent such as CP-31398 provided significant additive tumor inhibitory effects.

Figure 4
Fig 4A. Effect CP-31398 individually or in combination with celecoxib on AOM-indiuced colon adenocarcinoma incidence, Fig 4B. Effect CP-31398 individually or in combination with celecoxib on AOM-indiuced colon adenocarcinoma multplicity

Effect of CP-31398 on colonocyte proliferation and apoptosis

The effect of CP-31398 and celecoxib as potential chemopreventive agents was assessed by cell kinetics analyses. Figure 5A (upper panel) shows BrdU incorporation in AOM-induced colon tumors of rats fed CP-31398 alone or in combination with celecoxib. Fig. 5C summarizes cell proliferation results as measured by BrdU positive cells. Administration of dietary CP-31398 at 150 ppm inhibited the proliferation index by about 25%, but this inhibition did not reach statistical significance. However, dietary CP-31398 at 300 ppm significantly suppressed the proliferative index in AOM-induced colon tumors (42%) as compared with control diet. Celecoxib at 300 ppm significantly suppressed BrdU incorporation in colonic tumor cells (>52%). Importantly, the combination of CP-31398 at 150 ppm and celecoxib at 300 ppm inhibited colon tumor cell proliferation by >64%. Although, combinational agents induced inhibition of colon tumor cell proliferation is more compared to celecoxib alone, this inhibition did not reach statistical significance (p=0.064). Fig. 5B represents the TUNNEL assay on colonic tumor cell apoptosis. Fig 5D summarizes the apoptotic index of colonic tumor tissues. Dietary CP-31398 at 150 and 300 ppm significantly induced colon tumor cell apoptosis (85%- and 156% respectively, p < 0.005–0.0001) in a dose-dependent fashion when compared to colonic tumors of rats fed with control diet. Colonic tumor tissues of rats fed with celecoxib at 300 ppm showed 208% higher apoptotic cells compared to colon tumors of rats fed with control diet. Further, a combination of low-dose CP-31398 at 150 ppm with celecoxib at 300 ppm resulted in a 293% increase of apoptotic cells in colonic tumors compared to tumors of rats fed with control diet. Importantly, a significant enhancement (p<0.037) of apoptotic cells in colonic tumors was observed in rats fed with low-dose CP-31398 and 300 ppm celecoxib compared to celecoxib alone.

Figure 5
Effect of CP-31398 and celecoxib or combination on AOM-induced colon tumor cell BrdU and TUNEL labeling

Modulation of p21WAF1/CIP1 and p53

Expression levels of p53 and of p21WAF1/CIP1 are important indicators of cell growth arrest and apoptosis. As shown in Figure 6A and 6B, CP-31398 dose-dependently induced expression of p21WAF1/CIP1protein in colonic tumor tissues (23–48%); however, limited expression of p21WAF1/CIP1, presumably p53- induced, was observed in tumor tissues of control diet fed rat. Induction of p21WAF1/CIP1expression in the colonic tumors of rats fed 300 ppm celecoxib was observed in ~68% tumor cells, whereas colonic tumors of rats fed a combination of CP-31398 (150 ppm) and celecoxib (300 ppm) showed > 85% of p21WAF1/CIP1 overexpression. Similarly, p53 expression in rats fed CP-31398 resulted in a dose-dependent enhancement of p53 in colonic tumors compared to those from rats fed a control diet (Fig 6A and 6B). Further, celecoxib at 300 ppm also induced significant expression of p53 in colonic adenocarcinomas. Significant synergistic induction of both p53 and p21 protein expression was observed in rats fed with low-doses of CP-31398 and celecoxib (Fig. 6A and 6B).

Figure 6
Effect of CP-31398 and celecoxib or combination on AOM-induced colon tumor p21waf1/cip and p53 expression

Effect of CP-31398 and Celecoxib on COX-2 Activity

To assess the potential interaction of CP-31398 and celecoxib with COX enzymes, we determined the effects of CP-31398 and celecoxib on selective COX-2 enzymatic activities and total COX-isoforms in colonic tumors of AOM-treated rats (Fig. 6C and 6D). The combined activity of COX-1 and COX-2 in colonic tumors of rats fed the control diet was 36% higher than COX-2 activity alone. This would suggest that ~36% of COX-derived metabolites can be accounted for by COX-1. While dietary CP-31398 at 150 ppm, did not affect COX-isoforms activity significantly, CP-31398 at 300 ppm showed modest but significant inhibition of both total COX and COX-2 activity in colonic tumors (p<0.02). As expected COX-2 activity in colon tumors of rats fed the celecoxib at 300 ppm was inhibited by more than 66% (p<0.0001). Importantly, the combination of CP-31398 at 150 ppm and celecoxib at 300 ppm suppressed COX-2 activity by over 75% (p<0.0001) and this inhibition is statistically significant when compared to celecoxib alone (p<0.04). In this regard, PGE2 and PGD2 both of which have been implicated in tumor cell proliferation were the major metabolites identified in these tumors.

Discussion

Restoration of mutant p53 function and/or enhancement of wild-type p53 expression by genetic means has been shown to suppress growth of various tumor types (10,18,32). The identification of CP-31398 and other small molecules such as PRIMA-1 that rescue/activate mutant p53 could constitute an effective pharmacological approach for cancer prevention and treatment (3338). While CP-31398 has been extensively studied in in vitro models (9,1115, 3335), only a few studies have demonstrated the tumor inhibitory potential of CP-31398 in vivo (1820). Recently, we have shown ~75% suppression of intestinal polyps by dietary CP-31398 (200 ppm) in Apcmin mice (20). The AOM-induced colon carcinogenesis model in F344 rats is well established and has been utilized to develop chemopreventive agents for clinical trials (39), including celecoxib (40). Our primary objective was to test whether low-dose CP-31398 would further augment very low-dose effects of celecoxib on colonic adenocarcinoma development.

Dose selections of CP-31398 for tumor efficacy studies are based on a 9-week MTD assay. In the present study, effects of 800 and 1,600 ppm CP-31398 on rats’ body weight showed that high dose levels completely retarded weight gain, albeit without significant toxic symptoms or survival and/or food intake differences for 9 weeks, compared to controls. We have determined the MTD for dietary CP-31398 in male F344 rats to be > 400 ppm, with an applied dose of CP-31398 in colon adenocarcinoma equal to ~30 to 60% MTD. Also, we have evaluated MTD of celecoxib in the present study primarily due to the fact that to date there is no published preclinical data for this agent. As shown in Figure 2B, celecoxib administered at 4,000 ppm in diet showed 11.2% body weight suppression without visible organ toxicities in rats. When compared to other selective COX-2 inhibitors and traditional NSAIDs, celecoxib tolerability is very high (2224, 41). Previous studies from our laboratory and others frequently used ~1,500 ppm in the diet. In the present study, we used 300 ppm celecoxib (~8% MTD), a significantly lower-dose (23,24). Use of low-dose celecoxib is strongly preferred based on human clinical trails showing that prolonged higher doses of celecoxib are associated with increased prothrombotic effects and risk of cardiovascular disease (40, 4244).

Our results are the first to show that CP-31398 effectively inhibits AOM-induced colonic preneoplastic lesions (ACF) and adenocarcinomas in the rat. These results further corroborate the preventive effects of CP-31398 in UVB-induced skin carcinogenesis SKH-1 mice and intestinal neoplasia in APCmin/+ mice (19,20). In the skin model, CP-31398 was administered either ip or topically; in APCmin/+ mice and in the present study, CP-31398 was administered in the diet. By different routes of administration, CP-31398 showed profound chemopreventive effects. In the APCmin/+ mice studies, tumor suppression by dietary CP-31398 was more pronounced early during tumor development (20). In the present study, dose-dependent suppression of colonic ACF and adenocarcinomas clearly suggest the potential usefulness of CP-31398 in chemoprevention of colon cancer. The efficacy of CP-31398 in this study is comparable to several potential colon cancer chemopreventive agents (e.g., celecoxib, sulindac, curcumin, oltipraz, nitric oxide-releasing NSAIDs) and other agents (23,24,30,31,4547).

Another major objective of the present study was to evaluate low-dose CP-31398 together with low-dose celecoxib in the AOM -induced model of colon adenocarcinoma. Our results show that a low nontoxic dose of CP-31398 profoundly enhanced the chemopreventive properties of low nontoxic-dose celecoxib. Remarkably, the combined efficacy of this regimen compares favorably with the outcome efficacy of, for example, high-dose (1500 ppm) celecoxib or NSAIDs applied at > 60% MTD, or 4000 ppm of difluoromethylornithine, an ornithine decarboxylase inhibitor in rat colon cancer models (23,24,45,48). Thus, the combination regimen applied in this study supports our previous low-dose combination approaches to suppress colon cancers in a synergistic or additive manner (31,48,49).

The importance of p53 mutations in colon cancers is well established (4,5). However, in the rodent models of colon cancer, p53 mutations likely represent a late event in tumor development. This may suggest that restoration/rescue of mutant p53 function plays a lesser role in the tumor inhibitory activity of CP-31398. However, as shown here, limited expression of p21WAF1/CIP1, presumably p53- induced, was observed in tumor tissues of control diet fed rats. Thus, while non-mutational activation of wild-type p53 appears to play a major role during early colon tumor development (8), further activation by CP-31398 might represent yet another mechanism leading to suppression of tumor growth in rat colon. In this regard, activation of wild-type p53 by CP-31398 has been demonstrated in other models, both in vitro and in vivo (1119). We have shown that dietary CP-31398 inhibited intestinal tumors in APCmin/+ mice concurrent with induction of p53 expression and downstream signaling molecules, leading to both inhibition of proliferation and induction of apoptosis (20). Significantly, an effect by dietary CP-31398 on collateral targets such as, for example, COX-2 in AOM-induced colon cancers, presents a novel mechanism.

As shown in supplementary Figure 1, there is mechanistic rationale to test the combination of a p53 modulating agent with COX-2 inhibitors. We and other have shown that COX metabolites, particularly electrophilic PGs, significantly impair the translocation of p53 to the nucleus and that COX-2 selective inhibitors facilitate this nuclear translocation (25,50). Thus, the present study provides mechanistic validation for the development of a combination approach of p53 modulators with very low-dose COX-2 selective inhibitors such as celecoxib or other NSAIDs. Ultimately, combining low molecular weight p53 modulating agents acting through different mechanisms, or combining agents targeting other molecular pathways such as COX, is likely to substantially increase antitumor effects. Taken together, these findings support further development of CP-31398 alone or in combination with celecoxib for colon cancer prevention and treatment.

Supplementary Material

Acknowledgements

Grant Support: This work was supported in part by NCI-CA-94962 and NO1-CN-25114 and N01-CN-53300 from the National Cancer Institute (NCI).

We are thankful for the technical expertise of the Rodent Barrier Facility Biologists at the OU Cancer Institute, Oklahoma City, OK. Also, we sincerely thank Ms. Alyson Atchison assisting in the preparation of this manuscript.

Abbreviations

Ab-1
α-turbulin
AIN
American Institute of Nutrition
ACF
aberrant crypt foci
ChIP
chromatin immunoprecipitation
COX
cyclooxygenase
CP-31398
p53 modulating agent
DAB
3,3’-diaminbenzidine
IACUC
Institutional Animal Care and Use Committee
MTD
maximum tolerated dose
PG
prostaglandin
PI
proliferative index
TP53
tumor suppression protein 53
TUNEL
terminal dUTP nick end labeling

References

1. Potter JD. Risk factors for colon neoplasia--epidemiology and biology. Eur J Cancer. 1995;31:1033–1038. [PubMed]
2. Slattery ML, Curtin K, Ma K, et al. Diet, activity, and lifestyle associations with p53 mutations in colon tumors. Cancer Epidemiol Biomarkers Prev. 2002;11:541–548. [PubMed]
3. Heavey PM, McKenna D, Rowland IR. Colorectal Cancer and the Relationship Between Genes and the Environment. Nutr Cancer. 2004;48:124–141. [PubMed]
4. Kinzler KW, Vogelstein B. Cancer-susceptibility genes. Gatekeepers and caretakers. Nature. 1997;386:761–763. [PubMed]
5. Vogelstein BFE, Hamilton SR, Kern SE, et al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319:525–532. [PubMed]
6. Bullock AN, Fersht AR. Rescuing the function of mutant p53. Nat Rev Cancer. 2001;1:68–76. [PubMed]
7. Vousden K. p53 death star. Cell. 2000;103:691–694. [PubMed]
8. Kopelovich L, DeLeo AB. Elevated levels of p53 antigen in cultured skin fibroblasts from patients with hereditary adenocarcinoma of the colon and rectum and its relevance to oncogenic mechanisms. J Natl Cancer Inst. 1986;77:1241–1246. [PubMed]
9. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med. 2004;10:789–799. [PubMed]
10. Kastan MB. Wild-type p53: Tumors can’t stand it. Cell. 2007;128:837–840. [PubMed]
11. Bullock AN, Henckel J, Fersht AR. Quantitative analysis of residual folding and DNA binding in mutant p53 core domain: definition of mutant states for rescue in cancer therapy. Oncogene. 2000;19:1245–1256. [PubMed]
12. Ventura A, Kirsch DG, McLaughlin ME, et al. Restoration of p53 function leads to tumor regression in vivo. Nature. 2007;445:661–665. [PubMed]
13. Abarzua P, LoSardo JE, Gubler ML, et al. Restoration of the transcription activation function to mutant p53 in human cancer cells. Oncogene. 1996;13:2477–2482. [PubMed]
14. Foster BA, Coffey HA, Morin MJ, Rastinejad F. Pharmacological rescue of mutant p53 conformation and function. Science. 1999;286:2507–2510. [PubMed]
15. Wang W, Takimoto R, Ratinejad F, El-Diery W. Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding. Mol Cell Biol. 2003;23:2171–2181. [PMC free article] [PubMed]
16. Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by small molecules antagonists of MDM2. Science. 2004;303:844–848. [PubMed]
17. Demma MJ, Wong S, Maxwell E, Dasmahapatra B. CP-31398 restores DNA-binding activity to mutant p53 in vitro but does not affect p53 homologs p63 and p73. J Biol Chem. 2004;44:45887–45896. [PubMed]
18. Wang W, Kim S-H, El-Deiry WS. Small molecule modulators of p53 family signaling and anti-tumor effects in 53-deficient human colon tumor xenografts. Proc Natl Acad Sci USA. 2006;103:11003–11008. [PubMed]
19. Tang X, Zhu Y, Han L, et al. CP-31398 restores mutant p53 tumor suppressor function and inhibits UAB-induced skin carcinogenesis in mice. J Clin Invest. 2007;117:3753–3764. [PMC free article] [PubMed]
20. Rao CV, Swamy MV, Patlolla JM, Kopelovich L. Suppression of Familial Adenomatous Polyposis by CP-31398, a TP53 Modulator, in APCmin/+ Mice. Cancer Res. 2008;68:7670–7675. [PMC free article] [PubMed]
21. Marnett LJ, DuBois RN. COX-2: a target for colon cancer prevention. Annu Rev Pharmacol Toxicol. 2002;42:55–80. [PubMed]
22. Reddy BS, Rao CV. Novel approaches for colon cancer prevention by cyclooxygenase-2 inhibitors. J Environ Pathol Toxicol Oncol. 2002;21(2):155–164. [PubMed]
23. Reddy BS, Hirose Y, Lubet R, Steele V, et al. Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res. 2000;60:293–297. [PubMed]
24. Kawamori T, Rao CV, Seibert K, et al. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res. 1998;58:409–412. [PubMed]
25. Swamy MV, Herzog CR, Rao CV. Celecoxib inhibition of COX-2 in colon cancer cell lines increases the nuclear localization of functionally active p53. Cancer Res. 2000;63:5239–5242. [PubMed]
26. Rao CV, Wang CQ, Simi B, et al. Chemoprevention of colon cancer by a glutathione conjugate of 1,4-phenylenebis(methylene) selenocyanate, a novel organoselenium compound with low toxicity. Cancer Res. 2001;61:3647–3652. [PubMed]
27. Bird RP. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 1987;37:147–151. [PubMed]
28. Rao CV, Indranie C, Simi B, et al. Chemopreventive properties of a selective inducible nitric oxide synthase inhibitor in colon carcinogenesis, administered alone or in combination with celecoxib, a selective cyclooxygenase-2 inhibitor. Cancer Res. 2002;62:165–170. [PubMed]
29. Janakiram NB, Steele VE, Rao CV. Estrogen receptor-beta as a potential target for colon cancer prevention: chemoprevention of azoxymethane-induced colon carcinogenesis by raloxifene in F344 rats. Cancer Prev Res. 2009:52–59. [PubMed]
30. Rao CV, Reddy BS, Steele VE, et al. Nitric oxide-releasing aspirin and indomethacin are potent inhibitors against colon cancer in azoxymethane-treated rats: effects on molecular targets. Mol Cancer Ther. 2006;5:1530–1538. [PubMed]
31. Reddy BS, Wang CX, Kong AN, et al. Prevention of azoxymethane-induced colon cancer by combination of low doses of atorvastatin, aspirin, and celecoxib in F344 rats. Cancer Res. 2006;66:4542–4546. [PubMed]
32. Bykov VJN, Issaeva N, Shilov A, et al. Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat Med. 2002;8:282–288. [PubMed]
33. Luu Y, Bush J, Cheung K-J, Jr, Li G. The p53 stabilizing compound CP-31398 induces apoptosis by activating the intrinsic bax/mitochondrial/caspase-9 pathway. Exp Cell Res. 2002;276:214–222. [PubMed]
34. Rodrigues NR, Rowan A, Smith MEF, et al. p53 mutations in colorectal cancer. Proc Natl Acad Sci USA. 1990;87:7555–7559. [PubMed]
35. Takimoto R, Wang W, Dicker DT, Rastinejad F, Lyssikatos J, El-Diery WS. The mutant p53-conformation modifying drug, CP-31398 can induce apoptosis of human cancer cells and can stabilize wild-type p53 protein. Cancer Biol Ther. 2002;1:47–55. [PubMed]
36. Xue W, Zender L, Miething C, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature. 2007;445:656–660. [PubMed]
37. Bernal F, Tyler AF, Korsmeyer SJ, Walensky LD, Verdine GL. Reactivation of the p53 tumor suppressor pathway by a stapled p53 peptide. J Am Chem Soc. 2007;129:2456–2457. [PubMed]
38. Nahi H, Lehmann S, Mollgard L, et al. Effects of PRIMA-1 on chronic lymphocytic leukaemia cells with and without hemizygous p53 deletion. Br J Haematol. 2004;127:285–291. [PubMed]
39. Reddy BS, Rao CV. Chemoprophylaxis of colon cancer. Curr Gastroenterol Rep. 2005;7(5):389–395. [PubMed]
40. Bertagnolli MM, Eagle CJ, Zaubar AG, et al. Five-year efficacy and safety analysis of the adenoma prevention with celecoxib trial. Cancer Prev Res. 2:310–321. [PMC free article] [PubMed]
41. Rao CV, Reddy BS. NSAIDs and chemoprevention. Curr Cancer Drug Targets. 2004;4:29–42. [PubMed]
42. Marnett LJ. The COXIB experience: a look in the rear-view mirror. Annu Rev Pharmacol Toxicol. 2009;49:265–290. [PubMed]
43. DuBiois RN. New, long-term insights from the adenoma prevention with celecoxib trial on a promising but trobled class of drugs. Cancer Prev Res. 2009;2:285–287. [PubMed]
44. Bresalier RS, Sandler RS, Quan H, et al. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med. 2005;352:1092–1102. [PubMed]
45. Rao CV, Rivenson A, Simi B, et al. Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent. Cancer Res. 1995;55:1464–1472. [PubMed]
46. Rao CV, Rivenson A, Simi B, Reddy BS. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 1995;55:259–266. [PubMed]
47. Rao CV, Rivenson A, Katiwalla M, et al. Chemopreventive effect of oltipraz during different stages of experimental colon carcinogenesis induced by azoxymethane in male F344 rats. Cancer Res. 1993;53:2502–2506. [PubMed]
48. Rao CV, Tokumo K, Rigotty J, et al. Chemoprevention of colon carcinogenesis by dietary administration of piroxicam, alpha-difluoromethylornithine, 16 alpha-fluoro-5-androsten-17-one, and ellagic acid individually and in combination. Cancer Res. 1991;51:4528–4534. [PubMed]
49. Swamy MV, Patlolla JM, Steele VE, Kopelovich L, Reddy BS, Rao CV. Chemoprevention of familial adenomatous polyposis by low doses of atorvastatin and celecoxib given individually and in combination to APCMin mice. Cancer Res. 2006;66:7370–7377. [PubMed]
50. Moos PJ, Edes K, Fitzpatrick FA. Inactivation of wild-type p53 tumor suppressor by electrophilic prostaglandins. Proc Natl Acad Sci U S A. 2000;97:9215–9220. [PubMed]