Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis

Epidemiological observations and laboratory research have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colon cancer and that the inhibition of colon carcinogenesis by NSAIDs is mediated through the modulation of prostaglandin production by rate-limiting enzymes known as cyclooxygenases (COXs). Because traditional NSAIDs inhibit both COX-1 and COX-2, these drugs induce side effects, such as gastrointestinal ulceration and renal toxicity, through the inhibition of the constitutive COX-1. Overexpression of COX-2 has been observed in colon tumors; therefore, specific inhibitors of COX-2 could serve as chemopreventive agents. Our previous study has shown that celecoxib, an inhibitor of COX-2, while sparing COX-1, inhibited azoxymethane (AOM)-induced colon tumorigenesis when administered during both initiation and postinitiation stages, ie., celecoxib administered continuously before, during, and after carcinogen treatment. This study examined the dose-response effect of celecoxib when administered during the initiation and postinitiation stages. In addition, the chemopreventive effects of high-dose celecoxib administered during the promotion/progression stage of colon carcinogenesis, ie., continuous celecoxib administration beginning 14 weeks after the carcinogen treatment, was determined in male F344 rats. We also measured the steady-state levels of celecoxib in the plasma of animals given this inhibitor. Groups of 5-week-old male F344 rats were fed either a control diet or experimental diets containing 500, 1000, or 1500 ppm celecoxib. At 7 and 8 weeks of age, rats scheduled for carcinogen treatment were injected s.c. with AOM at a dose rate of 15 mg/kg body weight/week. Groups of animals destined for the promotion/ progression study and initially receiving the control diet were switched to a diet containing 1500 ppm celecoxib beginning 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, ie., 52 weeks, and were then sacrificed. Colon tumors were evaluated histopathologically. Administration of 500, 1000, or 1500 ppm celecoxib during the initiation and postinitiation stages significantly inhibited the incidence (P < 0.01 to P < 0.0001) as well as the multiplicity (P < 0.01 to P < 0.0001) of adenocarcinomas of the colon in a dose-dependent manner. Importantly, administration of 1500 ppm celecoxib during the promotion/progression stage also significantly suppressed the incidence and multiplicity of adenocarcinomas of the colon (P < 0.01). Also, administration of celecoxib to the rats during the initiation and postinitiation periods and throughout the promotion/progression stage strongly suppressed colon tumor volume (P < 0.0002 to P < 0.001). The steady-state plasma concentration of celecoxib increases somewhat with the dose. Thus, in this model system, the chemopreventive efficacy of celecoxib is dose-dependent when this COX-2 inhibitor is administered during the initiation and postinitiation periods. This study provides the first evidence that celecoxib is also very effective when it is given during the promotion/progression stage of colon carcinogenesis, indicating that the chemopreventive efficacy is achieved during the later stages of colon tumor development. This suggests that celecoxib may potentially be an effective chemopreventive agent for the secondary prevention of colon cancer in patients with familial adenomatous polyposis and sporadic polyps.     

Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been observed to reduce the relative risk of breast cancer. This prompted our investigation of the chemopreventive potential of celecoxib, a specific cyclooxygenase 2 blocker, against mammary carcinogenesis induced by 7,12-dimethyl-benz(a)anthracene in female Sprague Dawley rats. Treatment with celecoxib was examined and compared to treatment with the general NSAID, ibuprofen, and to a control group receiving only dimethylbenz(a)anthracene. Dietary administration of celecoxib (1500 ppm) produced striking reductions in the incidence, multiplicity, and volume of breast tumors relative to the control group (68%, 86%, and 81%, respectively; P < 0.001). Ibuprofen also produced significant effects, but of lesser magnitude (40%, 52%, and 57%, respectively; P < 0.001). These results help confirm the chemopreventive activity of NSAIDs against breast cancer and provide the first evidence that a cyclooxygenase 2 blocking agent, celecoxib, possesses strong chemopreventive activity against mammary carcinogenesis.     

Inhibition of chemically induced mammary carcinogenesis in rats by long-term exposure to butylated hydroxytoluene (BHT): interrelations among BHT concentration, carcinogen dose, and diet

In outbred female Sprague-Dawley rats long-term exposure to dietary butylated hydroxytoluene [3,5-di-tert-butyl-4-hydroxytoluene (BHT); CAS: 128-37-0] 1 week before carcinogen administration to termination resulted in a dose-related inhibition of mammary tumorigenesis and adrenocortical nodulogenesis. In animals fed the cereal-based NIH-07 diet and receiving a low dose (5 mg/rat) of 7,12-dimethylbenz [a] anthracene [(DBMA) CAS: 57-97-6], there was a significant overall inhibitory trend in tumor incidence observed among those receiving 300, 1,000, 3,000, and 6,000 ppm BHT. Maximal inhibition was approximately 50% at the highest concentration of BHT (6,000 ppm). The inhibitory effect of BHT on mammary tumor incidence was less pronounced when BHT was administered to rats initiated with a high carcinogen dose: At 15 mg DMBA/rat maximal inhibition was only 20% at the highest concentration of BHT (6,000 ppm). In contrast, when tumor yield was assessed in terms of latency or tumor multiplicity, the inhibitory effect of BHT was more pronounced in the groups given a high dose of DMBA than in the groups given a low dose. In animals given a low dose of DMBA (5 mg) and fed 6,000 ppm BHT in the casein-based AIN-76A diet, tumor incidence was inhibited by 50% of that of the controls; in contrast, when initiation was with a high dose of DMBA (15 mg), tumor incidence was decreased by only 28% of that of the controls. In animals fed the NIH-07 diet, DMBA-induced adrenocortical nodule formation was also inhibited in a dose-dependent fashion by BHT. At 5 mg DMBA maximal inhibition was 86% of control levels (6,000 ppm BHT); at 15 mg DMBA maximal inhibition was 66% of control levels (6,000 ppm BHT). However, when BHT was incorporated into the AIN-76A diet, its inhibitory effects on adrenocortical nodulogenesis were unexpectedly feeble and unrelated to carcinogen dose: In animals initiated with 5 mg DMBA and administered 6,000 ppm BHT, nodule incidence was decreased by only 25%, whereas in animals initiated with 15 mg DMBA, nodule incidence was decreased by 30% of that of the controls. These results indicate that while chronic exposure to dietary BHT suppressed the development of DMBA-induced mammary tumors and adrenocortical nodules, the degree of suppression depended on the dose of carcinogen administered, the level of BHT in the diet, and the parameter being measured. Diet-dependent differences in BHT action were observed with regard to DMBA-induced adrenocortical nodulogenesis but not with regard to mammary tumorigenesis.     

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.

The inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are overexpressed in colonic tumors of humans, as well as in colon tumors that develop in rats after the administration of the colon-specific carcinogen, azoxymethane (AOM). iNOS may regulate COX-2 production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to assess the potential chemopreventive properties of highly selective iNOS inhibitors, administered individually and in combination with a selective COX-2 inhibitor, on the development of AOM-induced colonic aberrant crypt foci (ACF). F344 rats were fed experimental diets containing one of the following: 0, 10, 30, or 100 parts/million (ppm) of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); 1800 ppm of the less potent, selective iNOS inhibitor aminoguanidine (AG); 500 ppm of the COX-2 inhibitor celecoxib; 320 ppm of the nonsteroidal anti-inflammatory sulindac (positive control); or 30 ppm of SC-51 with 500 ppm of celecoxib, and 100 ppm of SC-51 with 500 ppm of celecoxib. One and 2 weeks later, rats received s.c. injections of AOM at a dose of 15 mg/kg of body weight. At 17 weeks of age, all rats were sacrificed. Colons were evaluated for ACF, and colonic mucosae were assayed for COX and NOS isoform enzyme activities. Samples of venous blood, collected at various time points, were analyzed for these agents. SC-51, administered alone, demonstrated dose-dependent inhibition of the incidence of colonic ACF. The highest doses of SC-51 (100 ppm) and AG (1800 ppm) significantly suppressed the incidence of colonic ACF (P < 0.01 and < 0.001, respectively) and crypt multiplicity in terms of numbers of aberrant crypts/focus (P < 0.0001). Importantly, the combination of either low or high effective doses of SC-51 (30 or 100 ppm) and celecoxib (500 ppm) suppressed AOM-induced colonic ACF formation (P < 0.05 and < 0.001, respectively) and reduced multiplicity of four or more aberrant crypts/focus (P < 0.0001) to a greater extent than did these agents administered individually. As expected, sulindac inhibited colonic ACF formation (P < 0.001) and reduced the multiplicity of four or more aberrant crypts (P < 0.0001) to approximately 45%. The enzymatic activities of COX-2 and iNOS were significantly induced in the AOM-treated animals, and administration of the iNOS inhibitors, SC-51 and AG, significantly inhibited the activities of both iNOS and COX-2 in the colonic mucosa. The combined administration of SC-51 and celecoxib inhibited the COX-2 activity to a greater extent than did either of these agents administered alone. These findings support the hypothesis that selective iNOS inhibitors may have chemopreventive properties and that coadministration with a selective COX-2 inhibitor may have additional chemopreventive potential.     

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.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced tumors and DNA adduct formation in the mammary glands of female Sprague-Dawley rats by the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate.

We synthesized a novel organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (XSC), possessing low toxicity by comparison with inorganic Na2SeO3, and several other synthetic organoselenium compounds (K. El-Bayoumy, Cancer Res., 45: 3631-3636, 1985). We tested the effect of XSC treatment during the initiation phase on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma formation. A semipurified high-fat diet containing 80 ppm of XSC (40 ppm as selenium) was fed to 6-wk-old virgin female Sprague-Dawley rats for 2 wk, starting 1 wk before and ending 1 wk after carcinogen treatment. At 7 wk of age, rats were given a single dose of DMBA (5 mg) in 0.2 ml of olive oil by gastric intubation; the experiment was terminated 16 wk later. The development of mammary tumors in those rats that received XSC-supplemented diets was significantly inhibited when compared with the control group (fed the same diet without XSC supplements). This was evident from tumor incidence (percentage of tumor-bearing rats, 88 versus 20) and multiplicity of tumors (mean number of tumors/rats, 3.96 versus 0.28). The finding that XSC acts as a chemopreventive agent in the DMBA mammary tumor model prompted us to examine the effect of dietary XSC on DMBA-DNA binding in both the liver and mammary tissue under conditions identical to those described above for the bioassay. Rats (four/group) were killed 6, 24, 48, and 168 h after [3H]DMBA (5 mg/rat; specific activity, 51.2 mCi/mM) administration. Liver and mammary tissue were obtained and DNA was isolated. Dietary XSC was found to inhibit total DMBA-DNA binding in the mammary tissue, but not in the liver. The most profound effect was observed at early time points, i.e., 24 to 48 h after [3H]DMBA administration. The inhibition in total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol-epoxides of DMBA; these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine adducts on the basis of their chromatographic characteristics on high-pressure liquid chromatography and on a boronate affinity column. The inhibition of the DMBA-DNA binding in the target tissue provides a plausible explanation for the chemopreventive effect of XSC during the initiation stage of carcinogenesis.     

Inhibition of mammary-gland tumorigenesis in the rat by caraway seeds and dried leaves of watercress

The effect of consumption of caraway seeds and dried leaves of watercress on 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced mammary gland tumorigenesis in female Sprague-Dawley rats was determined. At 6 weeks of age, animals were fed a basal (control) diet and experimental diets containing caraway or watercress (20%). Animals were maintained on their diets till termination of the experiment (25 weeks after DMBA). At 8 weeks of age, all rats were given DMBA 10 mg, one dose, p.o. in oily formulation). Neither caraway nor watercress affected the body weight or the rate of growth of animals. By week 25 after DMBA, 77% of the control rats developed mammary tumors, with a mean of 2.27 tumors per rat. Caraway decreased significantly (P<0.05) the percentage of rats with tumors (42.8% protection) and the mean number of tumors per rat (50.6% protection) and increased significantly (P<0.05) the mean latency period of tumor appearance. Watercress, though decreased the percentage of rats with tumors (28.5% protection) and the mean number of tumors per rat (31.7% protection), this decrease was significant (P < 0.05) during some week intervals only. The increase in the mean latency period of tumor appearance by watercress was not significant. The results of this study suggest that caraway and watercress possess chemopreventive effects against DMBA-induced mammary gland tumorigenesis in rats.     

Chemopreventive efficacy of anethole trithione,N-acetyl-L-cysteine,miconazole and phenethylisothiocyanate in the DMBA-induced rat mammary cancer model

The chemopreventive efficacy of N-acetyl-L-cysteine (NAC), anethole trithione, miconazole and phenethylisothiocyanate (PEITC), each of which would be expected to alter carcinogen metabolism, was examined in the dimethylbenzanthracene (DMBA) mammary carcinogenesis model. In this protocol, animals were exposed to non-toxic doses of the chemopreventives in the diet beginning 7 days prior to DMBA administration and then continuously throughout the duration of the assay (100 days post carcinogen). Miconazole, an antifungal agent with relatively broad inhibitory activity toward a variety of cytochromes P450, increased mammary tumor latency, decreased tumor incidence at the highest dose and decreased tumor multiplicity up to 60%. Anethole trithione, a substituted dithiolthione and an analog of the relatively broad-spectrum chemopreventive oltipraz, was administered in the diet and significantly inhibited mammary cancer multiplicity but not cancer incidence. NAC, an antimucolytic agent, failed to inhibit DMBA-induced mammary tumorigenesis. Surprisingly, treatment with DMBA plus PEITC, a potent inhibitor of cytochrome P450 2E1, actually increased the multiplicity of tumors relative to that observed with DMBA alone. Int. J. Cancer 72:95–101, 1997. © 1997 Wiley-Liss Inc.     

Chemoprevention of DMBA-induced mammary cancer in rats by dietary soy

This study was designed to assess the potential chemopreventive effect of the administration of a standardized soy extract, SOYSELECT, on 7,12-Dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. Three groups, 24 females each, were used. Animals were fed either a phytoestrogen-free diet alone (control) or the same diet supplemented with 0.35% or 0.7% of soy extract. Treatment started at weaning and continued to the end of the study (24 weeks after DMBA administration). At day 50 of age all animals received via oral gavage 80 mg/kg DMBA. Only tumors subsequently classified as adenocarcinomas were considered for data evaluation. In rats on the soy diet, mammary tumors took a longer period of time to develop as compared to control rats. However, at the end of the study, no relevant difference in tumor incidence and multiplicity was observed among the groups. The most significant changes were seen between control and soy-treated groups when tumor dimension and results from histopathologic examination were considered. The latter, in fact, showed a dose-dependent reduction in the percentage of poorly differentiated tumors in treated animals. This change was statistically significant in animals receiving 0.7% soy. In addition, assessment of estrogen and progesterone receptor (ERalpha, PR) levels, revealed a significant reduction in the percentage of ERalpha and PR positive tumors in animals receiving 0.7% dietary soy, when compared to controls. Interestingly, genistein and daidzein plasma levels determined at the end of the study were within the range of those detected in people consuming large amounts of soyfoods.     

Feeding thiol-containing compounds, derived from vegetables, fails to inhibit N-methylnitrosourea-induced mammary tumorigenesis.

Various thiol-containing compounds have been shown to inhibit chemically-induced tumors in animal models. Two thiol-containing compounds derived from vegetables, namely 1,2 dithiol-3-thione (DTT) and S-methylmethane thiolsulfonate (MMTS), were tested for their chemopreventive activity in the N-methylnitrosourea (NMU)-induced rat mammary tumor model. Each compound was incorporated into the grain-based Teklad 7001 diet and fed to the rats one week prior to initiation with NMU until termination 18 weeks post NMU. DTT was fed at 166 and 500 ppm and MMTS at 200 and 800 ppm. Neither compound exerted a significant inhibitory effect on any index of tumor development including incidence, total tumor, tumor multiplicity, volume or latency. Serum levels of DTT assessed at termination in the 500 ppm DTT group ranged from 10-30 microg/ml. MMTS was undetectable in serum from either MMTS-fed group. The results of this study, using the direct acting carcinogen, NMU, suggest that the chemopreventive effect of thiol-containing compounds may be confined to animal models using carcinogens that require host activation.     

Possible enhancing effects of atrazine and nonylphenol on 7,12-dimethylbenz[a]anthracene-induced mammary tumor development in human c-Ha-ras proto-oncogene transgenic rats

Our transgenic (Tg) strain carrying copies of the human c-Ha-ras proto-oncogene is highly susceptible to 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis, possibly due to activation of the transgene, and can be used in medium-term bioassay models to test for modifying effects of estrogenic environmental compounds on tumor development. The present study was conducted to assess the influence of dietary feeding of the endocrine disruptors atrazine and nonylphenol on DMBA-induced carcinogenesis in c-Ha-ras Tg rats. Animals of both sexes were given a single oral dose of DMBA (25 mg/kg body weight) at 50 days of age and thereafter received soybean-free diet containing 5, 50 or 500 ppm atrazine, or 10, 25, 100 or 250 ppm nonylphenol. In female Tg rats, atrazine at a dose of 5 ppm increased the incidences of mammary adenomas and adenocarcinomas (P < 0.01 and P < 0.05), while 50 ppm increased the adenocarcinoma incidence (P < 0.05). In males, skin tumor development, in contrast, was significantly decreased at the highest dose. Nonylphenol at 10 ppm increased adenocarcinoma and total mammary tumor multiplicity in female Tg rats (P < 0.05), but there was no dose dependence, a significant quadratic dose-response trend rather being observed (P < 0.05). In vitro, atrazine did not cause proliferation of MCF-7 cells at any of a range of doses tested. These results suggest that endocrine disruptors may enhance mammary carcinogenesis, but only in a certain limited dose range under the present experimental conditions. The doses applied, moreover, were all extremely high compared to the possible environmental human exposure levels.     

塞莱昔布对化学诱导大鼠乳腺肿瘤病理分级影响的研究

目的:评价预防性应用选择性环氧化酶-2(COX-2)抑制剂塞莱昔布对化学诱导大鼠乳腺肿瘤及正常乳腺组织病理分级的影响。方法:雌性SD大鼠予二甲基苯并蒽(DMBA)1次性灌胃诱发乳腺肿瘤过程中,给予不同剂量塞莱昔布,观察肿瘤发生率、数量和体积,并与正常对照组进行病理学分析。结果:与对照组相比,塞莱昔布100和150 mg/kg两剂量组大鼠乳腺肿瘤发生率和肿瘤数量均显著低于模型对照组,但各组间肿瘤平均体积差异无统计学意义,P>0.05。塞莱昔布显著降低大鼠乳腺肿瘤的组织病理分级(100 mg/kg,P=0.021;150 mg/kg,P=0.036),显著降低DMBA导致的乳腺小叶和腺泡的病变程度(100 mg/kg,P=0.004)。结论:预防性应用塞莱昔布具有降低乳腺肿瘤的病理分级,保护正常乳腺组织的作用,其临床应用价值值得进一步探索。     

Inhibition of chemically induced mammary carcinogenesis in rats by short-term exposure to butylated hydroxytoluene (BHT): interrelationships among BHT concentration, carcinogen dose, and diet

Dietary butylated hydroxytoluene (BHT) fed 14 days before and 14 days after carcinogen administration resulted in a dose-dependent inhibition of 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor incidence in outbred Sprague-Dawley rats. In addition, the inhibitory effects of BHT were strongly influenced by the dose of initiating carcinogen and the type of diet in which BHT was administered. In animals fed the NIH-07 diet and receiving a low dose of DMBA (5 mg/rat), the inhibitory effect of BHT was manifested at all four BHT concentrations (6,000 leads to 300 ppm). Maximal inhibition was approximately 50% in animals given 5 mg DMBA and receiving 6,000 ppm BHT. However, in the group administered a high dose of DMBA (15 mg/rat), the inhibitory effect of BHT was expressed only at 6,000 ppm, the highest concentration given. Lower concentrations (300 and 1,000 ppm) of BHT had no detectable effect on tumor incidence. In animals fed the defined, semipurified AIN-76A diet during the 4-week treatment period and initiated with 5 mg DMBA, BHT at 6,000 ppm inhibited tumor development. However, at 15 mg DMBA animals fed the AIN-76A diet differed markedly from those fed the NIH-07 diet. In the former group, BHT at 6,000 ppm was unable to elicit any inhibitory response; in the latter group, BHT inhibited tumor development by 40%. Dietary BHT also inhibited DMBA-induced adrenocortical hyperplastic nodules in a dose-dependent fashion. These results indicate that short-term exposure to dietary BHT can inhibit experimental mammary tumor development at environmentally relevant concentrations.     

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.

The chemopreventive action of 40 and 80% maximum tolerated dose (MTD) levels of piroxicam, D,L-alpha-difluoromethylornithine (DMFO), 16 alpha-fluoro-5-androsten-17-one (DHEA analogue 8354), and ellagic acid (EA) administered in diet individually and in combination before and during initiation and postinitiation phases of azoxymethane-induced neoplasia of the intestine was studied in male F344 rats. The MTD levels of piroxicam, DFMO, DHEA analogue, and EA were determined in male F344 rats and found to be 500, 5,000, 500, and 10,000 ppm, respectively, in modified AIN-76A diet. When these agents were fed in combination, the MTD levels were: piroxicam plus DFMO, 250 and 2500 ppm; piroxicam plus DHEA analogue, 250 and 250 ppm; piroxicam plus EA, 250 and 5000 ppm; piroxicam plus DFMO plus DHEA analogue, 250, 2500, and 250 ppm; and piroxicam plus DFMO plus EA, 250, 2500, and 5000 ppm. From these MTD values, 40 and 80% MTD levels were calculated and tested for their efficacy. At 5 weeks of age, animals were fed the modified AIN-76A (control) diet and experimental diets containing 40 and 80% MTD levels of piroxicam, DFMO, DHEA analogue, and EA individually and in combination. At 7 weeks of age, all animals except the vehicle-treated groups were administrated s.c. injections of azoxymethane (15 mg/kg body weight/week for 2 weeks). Animals intended for vehicle treatment received s.c. injections of an equal volume of normal saline. Fifty-two weeks after azoxymethane and saline treatment all the animals were necropsied, and colon and small intestinal tumor incidence (percentage of animals with tumors) and multiplicity (tumors/animal) were compared among various dietary groups. The results indicate that 40 and 80% MTD levels of dietary piroxicam and DFMO significantly (P less than 0.001) inhibited colon and small intestinal tumor incidence and multiplicity. DHEA analogue at 40% MTD level significantly decreased the small intestinal and colon tumor incidences (P less than 0.05), whereas 80% MTD of DHEA analogue inhibited only small intestinal tumor incidence. EA at 40 and 80% MTDs had no significant effect on colon tumor incidence (P greater than 0.05), but 80% MTD of EA showed a significant inhibitory effect on the incidence of small intestinal adenocarcinomas (P less than 0.01). In the combination study, 40 and 80% MTD levels of piroxicam plus DFMO significantly (P less than 0.001) inhibited colon adenocarcinoma incidence (8.3%) and multiplicity (0.08 +/- 0.04) (SE) when compared to colon adenocarcinoma incidence (72.2%) and multiplicity (1.14 +/- 0.18) in control diet-fed animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemopreventive effects of the aromatase inhibitors vorozole (R-83842) and 4-hydroxyandrostenedione in the methylnitrosourea (MNU)-induced mammary tumor model in Sprague-Dawley rats

The chemopreventive activity of the aromatase inhibitors vorozoleand 4-hydroxyandrostenedione were determined in the methylnitrosourea(MNU)-induced model of rat mammary tumorigenesis. Vorozole (5and 2.5 mg/kg body wt) and 4-hydroxyandrostenedione (15 and6 mg/rat) were administered daily (by gavage) to virgin femaleSprague-Dawley rats starting at an age of 43 days. Seven dayslater animals were given a single dose of MNU. Following treatmentwith MNU, animals continued to be treated with vorozole and4-hydroxyandrostenedione daily until the end of the experiment(100 days post MNU treatment). Vorozole at either dose provedto be a profound inhibitor of MNU-Induced mammary tumors. Vorozoledecreased tumor incidence from 100% to 10%, while simultaneouslydecreasing tumor multiplicity from 5 tumors per annual to 0.1tumors per animal. This chemopreventive effect was accompaniedby significant increases In body weight gain in the animalstreated with vorozole when compared with control rats. In contrast,neither dose of 4-hydroxyandrostenedione had any effect on tumorincidence and only the higher dose slightly decreased tumormultiplicity.     

Influence of caffeine consumption on 7,12-dimethylbenz(a)anthracene-induced mammary gland tumorigenesis in female rats fed a chemically defined diet containing standard and high levels of unsaturated fat

The effect of caffeine (430-500 mg/liter of drinking water) on the initiation and promotion phases of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland tumorigenesis in female Sprague-Dawley rats fed a chemically defined diet containing standard (5%) or high (20%) levels of fat (corn oil) was examined. In the initiation studies, caffeine and the standard or high fat diet treatments were provided for 34 days, from 24-29 days of age to 58-63 days of age. Three days prior to termination of caffeine-fat diet treatments, each rat received a single dose of DMBA. In the promotion studies, caffeine and the standard or high fat diets were provided commencing 3 days after a single dose of DMBA (at 56-61 days of age) and until termination of the study. Caffeine consumption, during the initiation phase significantly (P less than 0.05) reduced mammary carcinoma multiplicity (number of tumors/rat), in rats fed either a standard or high fat diet. In the promotion studies, prolonged consumption of caffeine in rats fed either a standard or high fat diet did not significantly effect mammary carcinoma multiplicity. In the early stages of promotion, an apparent increase in mammary carcinoma multiplicity was observed; this increase in mammary carcinoma multiplicity did not, however, reach the 5% level of statistical probability. When caffeine was administered during both the initiation and promotion phases, no significant effect on mammary carcinoma multiplicity was observed. Treatment of rats during the initiation or promotion phases with caffeinated coffee (via drinking water) mimicked the mammary tumor modulating activities of caffeine. Decaffeinated coffee consumption did not effect either the initiation or promotion phases of this tumorigenic process. In both the initiation and promotion studies, caffeine and/or coffee consumption did not significantly affect the incidence of mammary carcinomas (percentage of rats bearing mammary carcinomas) or the mean latency period of mammary tumor appearance. Thus, in female rats fed a chemically defined standard or high fat diet, caffeine consumption can significantly influence chemical carcinogenesis of the mammary gland; an effect that is dependent upon the duration and time-span of caffeine administration.     

Chemopreventive effects of celecoxib are limited to hormonally responsive mammary carcinomas in the neu-induced retroviral rat model

Introduction

While current breast cancer chemoprevention strategies using selective estrogen response modulators and aromatase inhibitors are quite successful, their effects are limited to hormonally responsive breast cancer. Hormonally nonresponsive breast cancer (including estrogen receptor-negative cancer) is associated with poor prognosis for patients, and few chemoprevention agents exist for this type of cancer. The cyclooxygenase-2 inhibitor celecoxib (Celebrex^®) is a nonsteroidal anti-inflammatory drug and as such is a potential candidate for the prevention of hormonally nonresponsive breast cancer.

Methods

The chemopreventive effects of celecoxib were evaluated in the neu-induced retroviral rat mammary carcinogenesis model, to assess the efficacy of celecoxib on hormonally responsive and hormonally nonresponsive mammary carcinomas.

Results

Dietary celecoxib at 1,200 mg/kg diet was highly efficacious in the prevention of hormonally responsive mammary carcinomas in intact rats, decreasing tumor multiplicity by 56% (P < 0.0001) and by 74% (P = 0.0002) in two independent experiments. No significant effect was found, however, on hormonally nonresponsive mammary carcinomas of ovariectomized rats. Treatment with a combination diet, consisting of tamoxifen at 2 mg/kg diet and celecoxib at 1,200 mg/kg diet, reduced tumor multiplicity by 72% (P = 0.0002) in intact rats. This reduction was not statistically different from that observed with celecoxib alone. Furthermore, long-term treatment with celecoxib was not associated with reductions in tumor volume in either intact rats or ovariectomized rats. In contrast, tamoxifen treatment and the combination regimen caused significant reductions in tumor volumes in intact rats (P = 0.01 and P = 0.004, respectively). Consistent with these data, decreases in proliferation and increases in apoptosis were detected in tamoxifen-treated and combination diet-treated tumors. No such modulations were observed in celecoxib-treated tumors.

Conclusion

The chemopreventive effects of celecoxib appear to be limited to modulations in multiplicity of hormonally responsive mammary carcinomas. The fact that no synergistic or additive effects were observed in combination diet-treated rats raises the question of whether celecoxib is suitable for the prevention of hormonally nonresponsive breast cancer or for use in combination therapy with selective estrogen response modulators or aromatase inhibitors.     

Neu-induced retroviral rat mammary carcinogenesis: a novel chemoprevention model for both hormonally responsive and nonresponsive mammary carcinomas

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.     

Effects of the phytochemicals, curcumin and quercetin, upon azoxymethane-induced colon cancer and 7, 12-dimethylbenz[a]anthracene-induced mammary cancer in rats

Curcumin and quercetin were evaluated in rats for their abilityto modulate the carcinogenic activity of azoxy-methane (AOM)in the colon and 7, 12-dimethyl-benz[a]anthracene (DMBA) inthe mammary gland. In the AOM-induced colon cancer model, maleFischer 344 rats at 8 weeks of age started to receive eithercurcumin (8 and 16g/kg) or quercetin (16.8 and 33.6g/kg) inthe diet and 1 week later, were administered AOM (30 mg/kg bodywt.) by subcutaneous injection.The animals continued to receivethe two agents in the diet until sacrificed 45 weeks later.Curcumin mediated a dose-dependent inhibition of the incidenceand multiplicity of adenomas from 47% and 0.58±0.12 adenomas/ratinthe AOM-treated control group to 19% and 0.22±0.08and 0.06% and 0.08±0.06 adenomas/ratfor the low and highdose groups, respectively. A low yield of adenocarcinomas (0.06±0.04adeno-carcinomas/rat) was induced by AOM which was not significantlyaltered by curcumin. Treatment with quercetin caused a dose-dependentincrease in the yield of AOM-induced tumors in the colon from0.06±0.04 adeno-carcinoma/rat to 0.64±0.12 and1.14±0.17 for the low and high dose groups, respectively.In the DMBA-induced mammary cancer model, curcuminor quercetinwas administered at either 10 or 20g/kg diet, beginning 7 daysprior to DMBA and continually throughout the remainder of theexperiment. Neither curcumin nor quercetin significantly alteredthe incidence of animals with tumors or the tumor multiplicity,while the high concentration of both agents significantly increasedtumor latency. These results demonstrate different responsesto these agents in the two models. While curcumin was highlyeffective as a chemopreventive agent in the colon model, itwas only weakly effective in the mammary model. In contrast,quercetin which was also only weakly effective in the mammarymodel, caused a dose-dependent enhancement of tumors inducedby AOM in the colon model.