Chemoprevention of colon carcinogenesis by concurrent administration of piroxicam, a nonsteroidal antiinflammatory drug with D,L-alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor, in diet

The effect of three levels of piroxicam and three levels of D,L-alpha-difluoromethylornithine (DFMO) fed individually and in combination during the postinitiation phase of carcinogenesis was studied in male F344 rats to generate a data base on the efficacy and synergistic and additive effects of these compounds as inhibitors of colon carcinogenesis. The maximum tolerated dose of DFMO was determined in male F344 rats and found to be 5000 ppm in the AIN-76A diet. Piroxicam at levels of 25, 75, and 150 ppm and DFMO at concentrations of 400, 1000, and 4000 ppm (20, 50, and 80% maximum tolerated dose) in AIN-76 diet were tested individually and in combinations. At 7 weeks of age, while the rats were consuming the control diet (AIN-76A), all animals except the vehicle (saline)-treated controls were given a single s.c. injection of azoxymethane (CAS: 25843-45-2) at a dose level of 29.6 mg/kg body weight to induce intestinal tumors. One week after azoxymethane injection, animals were transferred to their respective experimental diets containing piroxicam and DFMO. Fifty-six weeks after azoxymethane injection, all animals were necropsied and colon and small intestinal tumor incidences and multiplicity were compared among the various dietary groups. Feeding of diets containing 75 and 150 ppm piroxicam or 1000 and 4000 ppm DFMO significantly inhibited the incidence (percentage of animals with tumors) of colon adenocarcinomas compared to that of control diet. The multiplicity (number of tumors/rat) of adenocarcinomas was significantly inhibited in animals fed the 25, 75, and 150 ppm piroxicam or 400, 1000, and 4000 ppm DFMO diets. Results analyzed by the linear regression method suggested a dose-dependent inhibition in colon adenocarcinoma incidence with increasing levels of piroxicam or DFMO. The incidence and multiplicity of colon adenocarcinomas were significantly inhibited in animals fed the diets containing combinations of 25, 75, and 150 ppm piroxicam and 400, 1000, and 4000 ppm DFMO. Piroxicam and DFMO administered together had a stronger inhibitory effect than did those given individually. Piroxicam and DFMO when administered individually had no significant inhibitory effect on colon adenoma incidence and multiplicity; in contrast, combinations of these compounds significantly inhibited colon adenomas. No consistent differences were found in the incidence and multiplicity of small intestinal tumors among the dietary groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dose-related inhibition of colon carcinogenesis by dietary piroxicam, a nonsteroidal antiinflammatory drug, during different stages of rat colon tumor development

The effect of four dose levels of piroxicam administered during different stages of colon tumor development was studied in male F344 rats to obtain a data base on the efficacy of piroxicam as an inhibitor of colon carcinogenesis. Piroxicam was added at levels of 25, 50, 75, and 150 ppm to the NIH-07 open-formula diet and fed to male F344 rats starting 1, 13, and 23 wk after the carcinogen administration. At 7 wk of age, while the animals were consuming the control diet, all animals except the vehicle-treated controls were given s.c. injection of azoxymethane (CAS:25843-45-2; 29.6 mg/kg body weight, once) to induce intestinal tumors. Forty wk after AOM injection, all animals were necropsied, and tumor incidences were compared among the various dietary groups. Colon tumor incidence (percentage of animals with tumors) was inhibited in a dose-dependent manner in rats fed the diets containing 25, 50, 75, and 150 ppm piroxicam starting 1 and 13 wk after carcinogen treatment. The colon tumor incidences in animals fed the diets containing 0, 25, 50, 75, and 150 ppm of piroxicam starting at 1 wk after carcinogen treatment were 89, 61, 58, 50, and 39%, respectively. When the diets containing 0, 25, 50, 75, and 150 ppm were fed 13 wk after carcinogen treatment, the colon tumor incidences were 89, 69, 69, 44, and 33%, respectively. Colon tumor multiplicity (tumors/animal; tumors/tumor-bearing animal) was also significantly inhibited in animals fed the diets containing 25 to 150 ppm piroxicam starting 1 and 13 wk after carcinogen administration. The number of colon tumors/animal was inhibited by about 80 to 84% in animals fed the 150 ppm piroxicam diet. When the diets containing different levels of piroxicam were fed 23 wk after carcinogen treatment, the colon tumor incidence was significantly inhibited in animals fed the 75 and 150 ppm piroxicam diets. The colon tumor incidences in animals fed the diets containing 0, 25, 50, 75, and 150 ppm were 89, 78, 67, 64, and 64%, respectively. The colon tumor multiplicity (colon tumors/animal) was slightly but significantly inhibited in animals fed the diets containing 25 to 150 ppm piroxicam. The results of this study demonstrate that increasing levels of piroxicam in the diet, when fed 1 or 13 wk after carcinogen insult, inhibit colon tumor incidence in a dose-dependent manner.     

Inhibition of colon carcinogenesis by prostaglandin synthesis inhibitors and related compounds.

The inhibitory effect of 40 and 80% maximum tolerated dose (MTD) levels of piroxicam, ibuprofen, ketoprofen and glycyrrhetinic acid on colon carcinogenesis was investigated in male F344 rats. The MTD levels of piroxicam, ibuprofen, ketoprofen and glycyrrhetinic acid as determined in male F344 rats were 500, 500, 250 and 3000 p.p.m. respectively. At 5 weeks of age, groups of male F344 rats were fed the control (AIN-76A) diet and 40 and 80% MTD levels of each test agent in AIN-76A diet. At 7 weeks of age, all animals except the vehicle (saline)-treated controls received azoxymethane (AOM) at a dose rate of 15 mg/kg body wt, once weekly for 2 weeks. All animals were necropsied 50 weeks after the second AOM injection and colon tumor incidences were compared among the groups fed the control diet and chemopreventive agents. Animals fed 400 (80% MTD) and 200 p.p.m. (40% MTD) of piroxicam, 400 p.p.m. (80% MTD) of ibuprofen and 200 p.p.m. (80% MTD) of ketoprofen showed a significant inhibition of colon tumorigenesis as compared to those fed the control diet. Results analyzed by the linear regression method suggested a dose-dependent inhibition of colon carcinogenesis with increasing levels of piroxicam or ibuprofen. In contrast, glycyrrhetinic acid had no measurable chemopreventive effect on colon carcinogenesis.     

Chemoprevention of colon carcinogenesis by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate.

The chemopreventive effect of 40% and 80% maximum tolerated dose (MTD) levels of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) administered in the diet during the initiation phase (2 weeks before, during, and up to 3 days after carcinogen administration) and the post-initiation phase (3 days after carcinogen treatment until termination) of azoxymethane (AOM)-induced colon carcinogenesis was studied in male F344 rats. The MTD of p-XSC was determined in male F344 rats and found to be 50 ppm. Beginning at 5 weeks of age, all animals were divided into various experimental groups (42 rats/group) and fed the high-fat semipurified diet or diets containing 20 (40% MTD) and 40 (80% MTD) ppm p-XSC. At 7 weeks of age, all animals (30 rats/group) except the vehicle-treated groups (12 rats/group) were administered s.c. injections of AOM (15 mg/kg body weight/week for 2 weeks). Three days after the second injection of AOM or vehicle (normal saline), groups of animals fed the p-XSC diets and control diet were transferred, respectively, to control diet and p-XSC diets and continued on these diets until the termination of the study. All animals were necropsied during the 36th week after AOM treatment. Colonic mucosal prostaglandin E2 and selenium-dependent glutathione peroxidase were measured in animals fed the control and p-XSC diets at the termination of the study. The results indicate that 40 ppm p-XSC administered during the initiation phase significantly inhibited the colon tumor incidence (percentage of animals with tumors). Dietary p-XSC administered at 20 and 40 ppm levels during the initiation phase significantly inhibited colon tumor multiplicity (tumors/animal and tumors/tumor-bearing animal). Colon tumor incidence and multiplicity were significantly reduced in groups fed 20 and 40 ppm p-XSC diets at the postinitiation phase of carcinogenesis. Colonic mucosal selenium-dependent glutathione peroxidase activity was increased, and prostaglandin E2 was reduced in animals fed the p-XSC diet compared to animals fed the control diet. Whereas the precise mechanisms of p-XSC-induced inhibition of colon carcinogenesis remain to be elucidated, it is likely that the effect during the initiation and postinitiation phases may be due to alteration in carcinogen metabolism and to modulation of prostaglandin synthesis and selenium-dependent glutathione peroxidase activity.     

Chemopreventive efficacy of combined piroxicam and difluoromethylornithine treatment of Apc mutant Min mouse adenomas, and selective toxicity against Apc mutant embryos

Genetic knockout or pharmacological inhibition of cyclooxygenase-2 decreases the number and size of adenomas in mouse models of familial adenomatous polyposis. Epidemiological and clinical studies in humans indicate that the entire class of nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit both COX-1 and COX-2 enzymes are promising colon cancer chemopreventive agents. We used the Apc mutant Min mouse model to test combinations of agents that might maximize preventive benefit with minimal toxicity because they act via different mechanisms. Min mice (n = 144) were exposed to low doses of the nonselective COX inhibitor piroxicam and the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO), beginning at the time they were weaned and continuing throughout the duration of the experiment. Piroxicam at 12, 25, and 50 ppm in the diet caused dose-dependent decreases in the number of tumors in the middle and distal portions of the small intestine. This decrease in tumor multiplicity was associated with a striking decrease in the size of those tumors that did grow out. In contrast, none of the doses of piroxicam alone decreased tumor multiplicity in the proximal portion of the intestine (duodenum). Exposure to DFMO (0.5 or 1.0% in water) caused a dose-dependent decrease in tumor multiplicity in the middle and distal portions of the small intestine. However, this decreased multiplicity was not associated with a striking decrease in the size of the tumors. Combined treatment of mice with piroxicam plus DFMO was much more effective than either agent alone and resulted in a significant number of mice totally free of any intestinal adenomas (P < 0.001), in contrast to the 100% incidence and high multiplicity in control Min mice. In addition to this profound effectiveness in reducing tumor number, the few residual tumors in mice treated with the combined drugs were markedly smaller in size than tumors that arose from control Min mice. These experiments suggest that selective COX-2 inhibition combined with ODC inhibition is a very promising approach for colon cancer prevention. These COX-2 and ODC inhibitor drugs were not overtly toxic at the doses used when administered to mice after weaning. However, when treatment was begun in utero, the Mendelian expected progeny ratio of 1:1 that we routinely obtained in untreated control litters was no longer observed. Apc(min)/+ progeny of pregnant dams treated with piroxicam and/or DFMO were reduced in number and their ratio to Apc+/+ progeny was decreased to approximately 0.28:1. Thus, these agents are effective against adenomas that have homozygous mutation of the APC gene and also select against fetuses bearing a heterozygous mutation in the APC gene.     

Inhibition by dietary oltipraz of experimental intestinal carcinogenesis induced by azoxymethane in male F344 rats

Epidemiological studies suggest that consumption of cruciferous vegetables rich in dithiolethiones is associated with a reduction in the incidence of cancer in man. The effect of two dose levels of dietary oltipraz [5-(2-pyrazinyl)-4-methyl-1, 2- dithiole-3-thione], a substituted dithiolethione, on azoxymethane (AOM)-induced intestinal carcinogenesis and on serum levels was studied in male F344 rats. The maximum tolerated dose (MTD) of oltipraz was determined in male F344 rats and found to be 500 p.p.m. Oltipraz at levels of 200 p.p.m. (40% MTD) and 400 p.p.m. (80% MTD) diet was tested as inhibitor of intestinal carcinogenesis. At 5 weeks of age, animals were fed the modified AIN-76A (control) diet and experimental diets containing oltipraz. At 7 weeks of age, all animals except the vehicle-treated animals were administered s.c. injection of AOM (15 mg/kg body wt/week for 2 weeks). Animals intended for vehicle treatment were administered s.c. with an equal volume of normal saline. Fifty-two weeks later, all animals were killed and colon and small intestinal tumor incidences and multiplicity were compared among the dietary groups. The results indicate that feeding of 200 and 400 p.p.m. of oltipraz significantly inhibited the incidence of adenocarcinomas in colon and small intestine and multiplicity of colon adenomas and small intestinal adenocarcinomas. Animals fed 400 p.p.m. oltipraz showed increased levels of oltipraz in the serum as compared to those fed 200 p.p.m. oltipraz. The results of this study indicate that dietary oltipraz inhibits intestinal carcinogenesis.     

Modulation of alterations in p53 tumor suppressor gene and its association with activation of ras proto-oncogenes during chemoprevention of colon cancer

Previously, we reported (Carcinogenesis 15: 1317-1323, 1994) a high rate of activating point mutations in I ns proto-oncogenes in azoxymethane (AOM)-induced colon tumors, and a significant suppression of these mutations by dietary administration of chemopreventive agents, D,L-alpha-difluoromethylornithine (DFMO) and piroxicam. To understand the role of p53 tumor suppressor gene in chemoprevention of colon cancer and to study the association of p53 gene alterations with activation of ras genes, we determined point mutations in conserved regions (exons 5-9) of p53 gene and analyzed the occurrence of double event of ms activation acid p53 mutation. Groups of male F344 rats were fed the modified AIN-76A diet containing 0, 4000 ppm DFMO, or 150 ppm piroxicam and administered s.c. AOM at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle controls received s.c. equal volume of normal saline. Animals were sacrificed 32 weeks after the last AOM or saline injection and their grossly visible colon tumors were analyzed to determine p53 mutations by PCR amplification based single strand conformation polymorphism (SSCP) and direct DNA sequencing. Our results demonstrate that about 57% tumors from animals fed the control diet contained predominantly missense but also nonsense mutations, whereas only 30% tumors from animals on piroxicam diet, and none (0%) from animals fed the DFMO diet had similar mutations. Analysis of data revealed that about half of the tumors from animals on control diet possessed both ms and p53 mutations together, only 27% of colon tumors from animals on piroxicam diet and none of the tumors from animals on DFMO diet exhibited both ms and p53 mutations. These results indicate that the administration of piroxicam, a non-steroidal anti-inflammatory drug, and DFMO, a irreversible inhibitor of ornithine decarboxylase, may inhibit selective proliferation of initiated cells containing activated las and/or mutant p53. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing mutated ras and/or p53.     

Inhibitory effect of aspirin on azoxymethane-induced colon carcinogenesis in F344 rats

Epidemiologic studies suggest that sustained use of aspirinmay reduce the risk of development of and mortality due to coloncancer. Previous preclinical studies have shown that severalnon-steroidal anti-inflammatory drugs act as potential chemopreventiveagents in experimentally induced colon cancer models. The presentstudy was designed to investigate the chemopreventive effectof 40 and 80% maximum tolerated dose (MTD) levels of aspirinadministered in the diet on azoxymethane (AOM)-induced coloncarcinogenesis in male F344 rats. The MTD of aspirin as determinedin male F344 rats was 500 p.p.m. Beginning at 5 weeks of age,all animals were randomly divided into various experimentalgroups (48 rats/group) and fed one of the semipurified dietscontaining 0, 200 p.p.m. (40% MTD), or 400 ppm (80% MTD) ofaspirin. Two weeks later, all animals (36 rats/group) exceptthe vehicle-treated groups (12 rats/group) were administereds.c. injections of AOM at a dose level of 15 mg/kg body wt,once weekly for 2 weeks. All animals were continued on theirrespective dietary regimen for additional 52 weeks and necropsied.Histopathologic evaluation of colon tumors was performed byroutine procedures. Basal levels and ex vivo production of colonicmucosal and tumor prostaglandin E₂ (PGE₂) were measured in allgroups. The results indicate that daily oral administrationof 200 and 400 p.p.m. aspirin significantly inhibited the incidence(% animals with tumors) and multiplicity (tumors/animal) ofinvasive adenocarcinomas of the colon as well as the size ofadenocarcinomas. Colonic mucosal and tumor PGE₂ levels (basaland ex vivo production) were significantly reduced in animalsadministered 200 and 400 p.p.m. aspirin. The results of thisstudy support the epidemiologic evidence that ingestion of aspirininhibits colon carcinogenesis. Although the precise mechanismsof aspirin-induced colon tumor inhibition remain to be determined,it is likely that the effect may be mediated through the modulationof prostaglandin synthesis.     

Effect of chemopreventive agents on posttranslational plasmamembrane association of ras-p21 during chemoprevention of azoxymethane-induced colon carcinogenesis

Accumulating data suggest that activation of ms proto-oncogenes and inactivation of tumor suppressor genes induce malignant phenotype in colonic cells. However, the transforming ability of ras oncogenes critically depends on correct localization of ras-p21 in plasma membrane. In our previous studies, we demonstrated a strong correlation between the modulation of ras activation (both in terms of mutational activation and over-expression of ras genes) by chemopreventive agents and colon tumor outcome during different stages of azoxymethane (AOM)-induced colon carcinogenesis. In the present study, which is a part of our ongoing investigations on the role of ras in chemoprevention of colon cancer, we studied the effect of D,L-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, and piroxicam, a non-steroidal antiinflammatory drug (NSAID), on the post-translational membrane association of ras-p21 during AOM-induced colon carcinogenesis. Groups of male F344 rats were fed the modified AIN-76A diets containing 0, 150 ppm piroxicam or 4000 ppm DFMO, and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body weight/week for 4 weeks. Vehicle control groups received equal volume of normal saline. Groups of animals were then sacrificed at 4, 16, 24, and 32 weeks after last AOM or saline injection and their colonic mucosa and tumors were analyzed for cytoplasmic as well as membrane bound ras-p21 levels. AOM-treatment resulted in increasingly higher levels of membrane-bound ras-p21 with advancing stages of colon tumorigenesis without any significant changes in cytoplasmic ras-p21. Dietary DFMO significantly suppressed AOM-induced membrane-bound ras-p21 in a time-dependent manner. Administration of piroxicam though resulted in significant inhibition of membrane-bound ras-p21, but concomitantly increased the cytosolic levels of ras-p21. Inhibition of membrane-bound ras-p21 levels by DFMO and piroxicam strongly correlated with the suppression of AOM-induced colon tumorigenesis by these agents. Data from the present and earlier studies suggest that DFMO may afford chemoprevention by suppressing DNA and protein biosynthesis by depleting intracellular polyamines, whereas piroxicam may exert its antitumor activity by interfering with post-translational membrane localization of ras-p21, in addition to modulating arachidonic acid metabolism.     

Effect of chemopreventive agents on intermediate biomarkers during different stages of azoxymethane-induced colon carcinogenesis.

Chemoprevention of colon cancer is emerging as an alternative to therapy with a broad potential for reducing cancer incidence in defined high-risk groups and the general population. Besides several chemopreventive agents in use and under investigation, D,L-alpha-difluoromethylornithine (DFMO) and piroxicam have been shown to effectively inhibit colon carcinogenesis in rodents. A variety of proliferation-related parameters have been suggested as potential intermediate markers of cancer risk that could be used to monitor the progress of chemoprevention in clinical trials. We have investigated the effect of chemopreventive agents, DFMO, and piroxicam on mucosal ornithine decarboxylase (ODC) and tyrosine-specific protein kinase (TPK) activities during different stages of azoxymethane (AOM)-induced colonic carcinogenesis in male F344 rats in order to examine the plausibility of using these enzymes as intermediate biochemical markers of colon cancer. Groups of male F344 rats were fed modified AIN-76A diets containing 0 or 150 ppm piroxicam or 4000 ppm DFMO and given s.c. injections of AOM dissolved in normal saline at a dose of 15 mg/kg body weight/week, once weekly, for 4 weeks. Vehicle control groups received s.c. equal volumes of normal saline. Groups of animals were then sacrificed at 0, 4, 16, 24, and 32 weeks after AOM or saline treatment, and their colonic mucosa was analyzed for ODC and TPK activities. AOM treatment significantly increased mucosal ODC as well as TPK activities. AOM-induced ODC and TPK activities were significantly suppressed by dietary DFMO progressively at all stages of colon carcinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies

In most prostate chemoprevention studies conducted with animal models, the incidence and multiplicity of tumours have been used as endpoints for efficacy. However, the latency of tumours is usually over 1 year, making these studies costly and time consuming. The main purpose of this study was to assess the utility of prostate intraepithelial neoplasia (PIN), induced in Noble rats by continuous testosterone + oestradiol (T + E) administration, as a potential intermediate endpoint biomarker of efficacy in chemoprevention studies. Noble rats at the age of 12 weeks were treated for 36 weeks with T + E given subcutaneously via Silastic capsules. The incidence and multiplicity of PIN were assessed in various prostate glands by serial sections generated at three separate tissue levels. The efficacy of dehydroepiandrosterone (DHEA) and DHEA 8354 (1000 and 2000 mg/kg diet), difluoromethylornithine (DFMO) (1000 and 2000 mg/kg diet) and oltipraz (125 and 250 mg/kg diet) to inhibit PIN was assessed in two independent sets of experiments. T + E induced multiple PIN in the dorsolateral prostate (DLP) of 80-100% of the animals. DHEA and DHEA 8354 did not affect the incidence but decreased the multiplicity of PIN in the DLP, from 3.2 +/- 1.0 in control group to 1.5 +/- 1.0 in the low-dose and to 1.6 +/- 0.6 in the high-dose group for DHEA (P<0.05 and P<0.02, respectively), and to 1.9 +/- 0.8 in the high-dose (P<0.05) DHEA 8354. Both agents did not affect PIN in anterior prostate, seminal vesicles or ventral prostate. In a second experiment, DFMO and oltipraz were found not effective in inhibiting PIN. In this study, we provide new evidence that PIN in Noble rats, induced by continuous T + E treatment, is a useful intermediate endpoint for determining the efficacy of DHEA and other potential chemopreventive agents. The hormonal pathogenesis, high multiplicity, short latency, preferential location in the DLP, similarity in morphology and biology to PIN of human prostate, and the sensitivity to agents that suppress prostate carcinogenesis, makes PIN in Noble rats a promising intermediate endpoint for chemoprevention studies.     

2-difluoromethylornithine and dehydroepiandrosterone inhibit mammary tumor progression but not mammary or prostate tumor initiation in C3(1)/SV40 T/t-antigen transgenic mice

Female transgenic mice that express SV40 T/t antigens under the regulatory control of the rat C3(1) gene spontaneously develop multifocal mammary lesions that predictably evolve into invasive, hormone-independent carcinomas, whereas male mice are prone to develop prostate cancer. Chemopreventive agents were administered to female C3(1)/SV40 large T-antigen mice from 7 to 19 weeks of age, during which time the mammary lesions developed and progressed to invasive carcinomas. No significant differences in the numbers of preinvasive mammary intraepithelial neoplasia lesions (histologically similar to human ductal carcinoma in situ) were observed after 2 or 8 weeks of treatment between mice receiving either vehicle alone, dehydroepiandrosterone (DHEA), or 2-difluoromethylornithine (DFMO). However, a dose-response reduction in invasive carcinoma growth was observed for both DFMO, an inhibitor of ornithine decarboxylase, and DHEA, the primary steroid precursor to both androgens and estrogens in primates. Despite unaltered expression of the transgene, tumor incidence was reduced approximately 20% by DFMO (8000 mg/kg) and 30% by DHEA (4000 mg/kg; P < 0.05). Tumor multiplicity was reduced by approximately 50% by both DFMO and DHEA (P < 0.05). DFMO had a dose-dependent effect on total tumor burden, which was reduced by 25% at low doses (4000 mg/kg) and 70% at high doses (8000 mg/kg). DHEA reduced tumor burden by 50% and 66% at low (2000 mg/kg) and high (4000 mg/kg) doses, respectively. Interestingly, despite its inhibitory effects on tumor development, DHEA caused a dose-dependent increase of serum estradiol levels that we have previously shown to increase mammary tumor formation in this model. No effect on the development of the prostate cancer precursor lesions (prostate intraepithelial neoplasia) was observed when mice were treated with DHEA, DFMO, tocopherol acetate, selenomethionine, or 9-cis-retinoic acid, although the effects on late-stage prostate cancer development were not determined. These results demonstrate that despite the expression of the highly transforming C3(1)/SV40 large T-antigen transgene, this transgenic model can be used to study the effects of chemopreventive agents on mammary cancer progression. The tumor-inhibitory effects of DHEA and DFMO on mammary cancer growth appear to occur after the development of preinvasive lesions, suggesting that these agents inhibit tumor progression but not initiation.     

Inhibition of rat mammary gland chemical carcinogenesis by dietary dehydroepiandrosterone or a fluorinated analogue of dehydroepiandrosterone.

The chemopreventive efficacy of p.o. administered dehydroepiandrosterone (DHEA), DHEA plus N-(4-hydroxyphenyl)retinamide (4-HPR), or 16 alpha-fluoro-5-androsten-17-one (DHEA analogue 8354) was examined in rats treated with N-methyl-N-nitrosourea (MNU; 50 mg/kg body weight, i.v.) at 50 days of age. Semipurified diet (AIN-76A) containing each steroid alone, or DHEA plus 4-HPR, was administered during initiation (-1 week to +1 week post-MNU), promotion/progression (+1 week post-MNU to termination), or both phases (-1 week post-MNU to termination) of the carcinogenic process. Neither DHEA nor DHEA analogue 8354 (0.2%, w/w) significantly affected the initiation of mammary cancer when administered alone; however, DHEA (0.2%, w/w) plus 4-HPR (1 mmol/kg diet) significantly reduced cancer multiplicity (26%) when given during initiation. All three treatments were strongly effective when given during promotion/progression, significantly reducing mammary cancer multiplicity by 77% (DHEA), 84% (DHEA/4-HPR), and 66% (DHEA analogue 8354), relative to carcinogen controls. Cancer incidence was significantly inhibited by DHEA (33% inhibition) and DHEA/4-HPR (24% reduction) during promotion/progression. However, the most effective chemopreventive treatment encompassed both phases of carcinogenesis. Thus, under these conditions, DHEA (0.2% or 0.1%, w/w) reduced cancer incidence (52% and 32% reductions, respectively) and multiplicity (91% and 86% reductions, respectively). Further reduction in mammary cancer incidence was observed in animals that received DHEA (both doses) plus 4-HPR (1 and 0.5 mmol/kg diet, respectively). DHEA analogue 8354 (0.2% or 0.1%, w/w) given for the duration of the study reduced only cancer multiplicity (61% and 56% reductions, respectively). Tumor-related mortality was significantly lower in rats that received long-term treatment with DHEA or DHEA/4-HPR, when compared with carcinogen controls. Except for a slight, but significant, postcarcinogen decrease in the mean body weights of rats treated concomitantly with DHEA (plus or minus 4-HPR) and MNU, additional gross manifestations of steroid-induced toxicity were not observed.     

Dose-response effects of the COX-2 inhibitor, celecoxib, on the chemoprevention of mammary carcinogenesis

Recent chemopreventive studies in our laboratories showed that the COX-2 inhibitor, celecoxib, inhibited the induction of mammary cancer by 7,12-dimethylbenz(a)anthracene (DMBA). In this study, we examined the relative chemopreventive effect of varying doses of celecoxib on the development and growth of DMBA-induced rat mammary tumors. At 10 days prior to receiving a single intragastric dose of 15 mg DMBA/rat, female Sprague-Dawley rats were fed a control chow diet or diets containing 250, 500, 1000 or 1500 ppm celecoxib until termination of the experiment. Administration of increasing doses of celecoxib inhibited mammary tumor incidence and multiplicity as well as tumor volume in a dose-dependent manner. At 122 days post DMBA-intubation, mammary tumor incidence was 100% in the control rats compared to 80%, 50%, 45% and 25% in rats receiving 250, 500, 1000 or 1500 ppm celecoxib, respectively (p<0.001). Similarly, tumor multiplicity and tumor volume were significantly reduced by increasing the dose of celecoxib from 250 to 1500 ppm in the diet. The control rats had an average of 3.46 tumors/rat compared to 1.80, 1.00, 0.75 and 0.50 tumors/rat in animals receiving 250, 500, 1000 or 1500 ppm celecoxib, respectively (p<0.001). Average tumor volumes in rats fed 250, 500, 1000 or 1500 ppm celecoxib were 0.42, 0.34, 0.31 and 0.16 cm3 compared to 1.29 cm3 in the control rats (p<0.001). There was a concomitant increase in the steady-state serum concentration of celecoxib with the dose. These results indicate that, in this rat model, the chemopreventive effect of celecoxib against breast cancer is dose-dependent and that celecoxib is effective even at lower dose levels.     

Effect of the chemopreventive agents piroxicam and D,L-alpha-difluoromethylornithine on intermediate biomarkers of colon carcinogenesis.

Our previous studies have shown that dietary piroxicam, a non-steroidal anti-inflammatory drug (NSAID), and D,L-alpha-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, act as potential chemopreventive agents in inhibiting azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. The present study was designed to determine the effect of these chemopreventive agents on intermediate biomarkers, namely colonic epithelial cell proliferation and levels of prostaglandins, which can be used as effective predictors of colon cancer. Starting at 6 weeks of age, groups of animals were fed the control diet and experimental diets containing piroxicam or DFMO. At 7 weeks of age, all animals, except the vehicle controls, were injected s.c. with AOM at a dose level of 15 mg/kg body wt/week for 4 weeks. Vehicle controls received an equal volume of normal saline. Groups of animals were then killed at the end of last AOM or saline injection (baseline) and at week 4, 16, 24 and 32 following the last AOM or saline treatment. Animals intended for cell proliferation study were injected with bromodeoxyuridine (BrdU) at a dose level of 20 mg/kg body wt 1 h prior to being killed. The rate of colonic cell proliferation at all time points was assessed immunohistochemically using anti-BrdU. The levels of colonic mucosal prostaglandins were estimated by radioimmunoassay. The results indicate that carcinogen treatment increased the colonic cell proliferation measured as the crypt labeling index in proximal and distal colons and the concentrations of colonic prostaglandin E2 (PGE2) and 6-keto PGF1 alpha. The data demonstrate that DFMO significantly inhibited the AOM-induced labeling index in the distal and proximal colon at all time points, whereas piroxicam slightly decreased the labeling index. On the other hand, piroxicam exerted a pronounced inhibitory effect on the levels of both PGE2 and 6-keto PGF1 alpha. DFMO suppressed the colonic PGE2 levels to a lesser degree than piroxicam. The results demonstrate that DFMO, an inhibitor of ODC, suppresses cell proliferation, whereas piroxicam, a NSAID, inhibits prostaglandins, and emphasize the need to develop agent-dependent intermediate biomarker(s) to validate the efficacy of chemopreventive agent(s) in colon carcinogenesis.     

Selenium and difluoromethylornithine additively inhibit DMH-induced distal colon tumor formation in rats fed a fiber-free diet

We investigated the effects of difluoromethylornithine, an inhibitorof ornithine decarboxylase (ODC) and selenium supplementationon tumor formation induced by the carcinogen 1, 2-dimethylhydrazine(DMH) in Sprague-Dawley rats. A biochemical link between polyaminebiosynthesis and selenium metabolism to its cancer preventativeform has been suggested by the common requirement of S-adenosylmethio-nine.One-hundred and twenty male Sprague-Dawley rats were dividedinto experimental (n = 80) and control (n = 40) groups. Experimentalanimals received DMH 20 mg/kg s.c. for 20 weeks. Animals werefed either a regular diet (selenium content 0.2 p.p.m.) or ahigh selenium diet (5 p.p.m.) with or without 0.2% DFMO in thedrinking water. At death, week 30, animal weights within experimentalor control groups were not different between the four diet treatmentgroups. Tumor number and incidence in the proximal colon wasnot affected by DFMO treatment, selenium supplementation orthe combined treatment. In contrast, in the distal colon, 19tumors developed in the DFMO treated group, 22 tumors in thehigh selenium group and only 12 tumors in the combined highselenium/DFMO treatment group compared to 32 tumors in the regulardiet group. Similarly, tumor incidence was decreased by DFMOand selenium supplementation and their effects were additive.In control animals, ODC activity was decreased by DFMO treatmentand selenium supplementation in the distal colon and liver,but not the proximal colon. ODC activity of tumor tissue wasgreater than normal colon tissue from diet paired animals forproximal and distal colon, except for distal colonic tumorsin the high selenium/DFMO treatment group. Polyamine content,however, did not correlate with ODC activity in normal or neoplastictissue. In general, S-adenosylmethionine levels from normalcolon and liver tissue were unaffected by diet treatment. Seleniumsupplementation in combination with DFMO treatment selectivelyinhibited distal colon tumor formation in rats fed a fiber-freediet.     

Altered expression of c-myc,p16 and p27 in rat colon tumors and its reversal by short-term treatment with chemopreventive agents

Modulation of gene expression in tumors has the potential of being a surrogate end-point biomarker for chemoprevention. Thus, we determined the modulation by chemopreventive agents of the protein and mRNA expression of genes in rat colon tumors. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane. Forty-seven weeks later, they received aspirin (600), calcium chloride (50 000), 2-(carboxyphenyl) retinamide (2-CPR, 315), alpha-difluoromethylornithine (DFMO, 3000), piroxicam (200), quercetin (33 600), 9-cis retinoic acid (9-cis RA, 30), rutin (3000), or sulindac (280) in their diet at the indicated mg/kg concentration for 7 days and were then killed. In colon tumors relative to the mucosa, the protein and mRNA levels of c-myc were increased, while the levels of p16 and p27 were decreased. Calcium chloride, DFMO, piroxicam and sulindac administered for 7 days decreased the mitotic index and reduced the protein and mRNA levels of c-myc in colon tumors. Calcium chloride, DFMO and piroxicam increased the protein and mRNA levels of p16 and along with sulindac increased the protein level of p27, but not its mRNA. The other agents failed to modulate both the mitotic index and the expression of the genes. The ability of the chemopreventive agents to prevent colon tumors was determined. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane and 8 weeks later they were administered aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin in their diet. The rats were killed 26 weeks after they started to receive the chemopreventive agents. The multiplicity of colon tumors was reduced by DFMO and piroxicam, increased by rutin and not affected by the other agents. Hence, agents that prevented colon cancer decreased the mitotic index and altered the expression of c-myc, p16 and p27 suggesting that modulation in the expression of these genes are potential biomarkers for chemopreventive activity.     

Inhibition of intestinal carcinogenesis in rats: effect of difluoromethylornithine with piroxicam or fish oil

An inhibitor of ornithine decarboxylase, difluoromethylornithine (DFMO), and two inhibitors of prostaglandin biosynthesis, piroxicam and menhaden fish oil, were examined for their effect on intestinal tumorigenesis in male Sprague-Dawley rats fed a 5% fat semisynthetic diet. Each agent was given individually in one of two doses as follows: DFMO, 0.05% and 0.1% in the drinking water; piroxicam, 65 mg/kg diet and 130 mg/kg diet; and menhaden fish oil, 1.25% and 2.50% of the diet. Additional animal groups were given combinations of the lower dose of DFMO and the lower dose of either piroxicam or fish oil. Intestinal tumors were induced by sc injections of azoxymethane (AOM; CAS: 25843-45-2) at 8 mg/kg (body wt) weekly for 8 weeks. Test diets were started 1 week prior to the first dose of AOM, and the rats were sacrificed 26 weeks later. Rats that received either dose of DFMO or the high dose of piroxicam developed significantly fewer intestinal tumors compared to controls. The low dose of piroxicam and the fish oil given at either dose level had no effect. The combination of the low dose of DFMO and the low dose of piroxicam reduced tumor formation more than either dose of DFMO alone, whereas the low dose of DFMO and fish oil together was no more effective than either dose of DFMO alone. These results show that a combination of a small amount of DFMO and piroxicam, each acting through a different mechanism, exerts an additive inhibitory effect on intestinal tumor formation in rats.     

Effect of dietary excess of inorganic selenium during initiation and postinitiation phases of colon carcinogenesis in F344 rats

The effect of supplemental inorganic selenium given during the initiation or postinitiation phase of colon carcinogenesis induced by azoxymethane [(AOM)CAS:25843-45-2] was studied in male F344 rats. Weanling animals were raised on AIN-76A semipurified (control) diet. Starting at 4 wk of age, groups of animals intended for initiation study were fed the semipurified diets containing 0.5 and 2.5 ppm selenium in the form of sodium selenite, and those intended for postinitiation study were continued on the control diet. At 7 wk of age, all animals except the vehicle-treated controls were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 wk). One wk following AOM treatment, animals in the initiation study receiving the supplemental selenium were transferred to the control diet whereas those in the postinitiation study receiving the control diet were transferred to the diets containing 0.5 and 2.5 ppm selenium. These animals were continued on this regimen until the termination of the experiment at 34 wk post-AOM injection. Tissue and blood glutathione peroxidase activity was measured in vehicle-treated animals fed the control and selenium-supplemented diets. The results indicate that body weights were comparable among the various dietary groups. Feeding of diets containing 0.5 and 2.5 ppm selenium during the initiation phase had no effect on colon tumor incidence, but the multiplicity of adenomas was slightly inhibited in animals fed the 2.5 ppm selenium diet. The incidence and multiplicity of colon adenocarcinomas and the multiplicity of colon adenomas were inhibited in animals fed the 2.5-ppm selenium diet during the postinitiation phase of carcinogenesis. The incidence of small intestinal tumors was higher in animals fed the 2.5-ppm selenium diet during the initiation phase than in animals fed the control diet and 0.5-ppm selenium diet. Selenium-dependent glutathione peroxidase activity was increased in kidneys and small and large intestinal mucosae of animals fed the 2.5-ppm selenium diet compared to those fed the 0.5-ppm selenium and control diets.     

Prevention of colon cancer by low doses of celecoxib, a cyclooxygenase inhibitor, administered in diet rich in omega-3 polyunsaturated fatty acids

Epidemiologic and animal studies suggest that a high-fat diet containing mixed lipids promotes colorectal cancer, whereas fish oil lacks promoting effect. Although cyclooxygenase-2 (COX-2) inhibitors are effective chemopreventive agents against colon carcinogenesis, administration of high doses of these agents over time may induce side effects. Here, we compared the efficacy of moderately high and low doses of celecoxib administered in diets high in mixed lipids (HFML) or fish oil (HFFO) against azoxymethane-induced colon carcinogenesis in male F344 rats. One day after the last azoxymethane treatment (15 mg/kg body weight once weekly for 2 weeks), groups of rats were fed the HFML and HFFO diets containing 0, 250, 500, and 1,000 ppm celecoxib. Rats were killed 26 weeks later and colon tumors were subjected to histopathologic examination and analyzed for total COX and COX-2 synthetic activities and COX-2 expression. Rats fed the HFFO diet showed significantly lower colon tumor incidence and multiplicity compared with rats fed the HFML diet. Celecoxib at 250, 500, and 1,000 ppm in either diet significantly suppressed colon carcinogenesis. Inhibition of colon adenocarcinomas were more pronounced in animals given 250 ppm celecoxib in HFFO diet compared with 250 ppm celecoxib given in HFML diet, suggesting some synergism between omega-3 polyunsaturated fatty acids (PUFA) and celecoxib. Inhibition of colon tumors by celecoxib was associated with lower levels of COX-2 activity and expression in colon tumors. These studies support the use of low doses of celecoxib in omega-3 PUFA-rich diet as a promising approach for clinical trials.