Efficacy of 9-cis-retinoic acid and N-(4-hydroxyphenyl) retinamide alone and in combination in mammary cancer prevention

As demonstrated in several in vitro and in vivo cancer models, retinoids have chemopreventive activity. The present studies were performed to evaluate the efficacy of 9-cis-retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl) retinamide (4-HPR), alone and combined, in preventing mammary cancers. Female Sprague-Dawley rats received N-methyl-N-nitrosourea (MNU), 50 mg/kg BW, either at 50 days of age (experiment I, young rats) or at 100 days of age (experiment II, older rats). In experiment I, 9-cis-RA (60 mg/kg of diet), 4-HPR (586 mg/kg of diet), or the combination were evaluated; in experiment II, 9-cis-RA (30 mg/kg of diet), 4-HPR (196 mg/kg of diet), or the combination were tested. There were no signs of toxicity in either study. In the young rats, there were only slight reductions (15-20%) in the number of mammary cancers when the agents were given alone. In the older rats, lower doses of 9-cis-RA or 4-HPR alone were highly effective; with 61% and 46% reductions in the number of mammary cancers, respectively. The combination of retinoids in the young rats caused a 49% reduction in mammary cancers, while in the older rats the combination resulted in a 96% reduction. Thus, lower doses of the retinoids caused more striking inhibition of mammary cancers in older rats than the higher doses given to younger animals. In both experiments, the two retinoids in combination produced an additive effect, suggesting that they may inhibit mammary cancers by different mechanisms.     

Efficacy of new retinoids in the prevention of mammary cancers and correlations with short-term biomarkers

A number of retinoid X receptor (RXR) agonists have proven to be highly effective in preventing methylnitrosourea (MNU) induced mammary cancers. However, these agonists have side effects; particularly causing an increase in serum triglyceride levels. A series of ligands for RXR were designed based on computer modeling to the ligand binding domain (LBD) of the RXR receptors and on structure-activity relationships. The chemopreventive effects of these retinoids were evaluated in the relatively long-term MNU model. As a short-term assay to predict their efficacy, the ability of the retinoids to modulate cell proliferation and apoptosis was also determined in mammary cancers after only 7 days of treatment. The five UAB retinoids evaluated included two Class I UAB retinoids (UAB20, UAB112) and three Class II UAB retinoids (UAB30, 4-methyl-UAB30 and the benzosuberone-analog of UAB30). The previously evaluated RXR agonist targretin and the pan-agonist 9-cis-retinoic acid (9-cis-RA), which interacts with both RAR and RXR receptors, were included as positive agonists known to prevent cancer in the MNU model. In the prevention studies, in which the agents were administered beginning 5 days after MNU until the end of the study, targretin (150 mg/kg diet) and 4-methyl-UAB30 (200 mg/kg diet) were highly effective in decreasing cancer numbers by 75-85%. UAB30 (200 mg/kg diet) and 9-cis-RA (60 mg/kg diet) gave intermediate inhibitions of 60 and 45%, respectively. Targretin (15 mg/kg diet), UAB20 (200 mg/kg diet) and the benzosuberone analog of UAB30 (200 mg/kg diet) showed limited activity by decreasing cancer multiplicity 25-30%, while UAB112 had no effect on mammary cancer multiplicity. A direct correlation was observed between the long-term chemopreventive efficacy of these agents and their ability to decrease cell proliferation in mammary cancers after short-term treatment. Furthermore, the highly effective agents (4-methyl-UAB30 and targretin at 150 mg/kg diet) increased apoptosis 3-5 times, while agents with moderate or limited preventive efficacy failed to significantly increase apoptosis. Although the more effective retinoid treatments increased serum triglycerides 2.5- to 4.0-fold, one moderately effective agent (UAB30) had no significant effect on lipid levels. In summary, a short-term in vivo method has been identified for screening newly synthesized retinoids both for chemopreventive efficacy and for their adverse effect on serum triglycerides.     

Chemoprevention of MNU-induced mammary tumors in the mature rat by 4-HPR and tamoxifen.

The chemopreventive efficacies of the retinoid all-trans-N-(4-hydroxyphenyl)-retinamide (4-HPR) and the anti-estrogen tamoxifen citrate were evaluated against N-methyl-N'-nitrosourea (MNU) induced mammary cancer in 120-day old female Sprague-Dawley rats. The agents were tested alone and in combination. They were administered in a modified AIN-76A diet, beginning 60 days prior to a single i.v. dose of 50 mg MNU/kg-bw and continuing until the end of the study, 180 days post-carcinogen treatment. At 782 mg/kg diet, 4-HPR alone significantly inhibited the induction of mammary adenocarcinomas compared with carcinogen controls. At 0.250 mg/kg diet, tamoxifen alone reduced tumor incidence compared with carcinogen controls. At 0.125 mg/kg diet, tamoxifen was ineffective. Combinations of 782 mg 4-HPR/kg diet with either 0.250 or 0.125 mg tamoxifen/kg diet were effective in inhibiting MNU-induced adenocarcinomas. The reductions in tumor incidence were greater for these combinations than for either agent alone. 4-HPR and 0.250 mg tamoxifen/kg diet decreased tumor incidence 81% (p less than 0.005), whereas 4-HPR and 0.125 mg tamoxifen/kg diet decreased tumor incidence 72% (p less than 0.005) compared with carcinogen controls. The combination of 391 mg 4-HPR/kg diet and 0.500 mg tamoxifen/kg diet was also tested and was effective in reducing tumor incidence.     

A rapid dual organ rat carcinogenesis bioassay for evaluating the chemoprevention of breast and colon cancer

In this study we evaluated the effect of dietary administration of a high fat, low fiber diet (HRD) with or without 2% phytic acid (PA) on the development of mammary cancer and/or colon cancer in rats exposed to methylnitrosourea (MNU), azoxymethane (AOM) or MNU + AOM. The rats were fed a HRD alone or a HRD + 2% PA. At the end of week 2, the rats were given either a s.c. injection of MNU (50 mg/kg body wt) or one of normal saline (vehicle). At the end of weeks 3 and 4, the rats were given either a s.c. injection of AOM (15 mg/kg body wt per week) or one of normal saline (vehicle). Nine weeks after the injection of MNU or saline, 10 rats from each group were sacrificed and the mammary tumor incidence and the number of colonic aberrant crypt foci (ACF) were compared between different groups. The administration of different diets was continued for an additional 21 weeks and the mammary tumor and colon tumor incidence between different groups were compared. Results showed that rats injected with MNU alone did not develop ACF or colon tumors while those injected with AOM alone did not develop mammary tumors. Linear regression analysis of the number of ACF at 11 weeks versus colonic tumor incidence at 32 weeks, and the linear regression analysis of mammary tumor incidence at 11 weeks versus mammary tumor incidence at 32 weeks, both showed good linear correlation. These results demonstrate the potential value of the short term dual organ carcinogenesis bioassay for screening chemopreventive agents for their relative ability to inhibit the development of mammary cancer and/or colon cancer while on high risk diet.     

Inhibition of lung carcinogenesis by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in the A/J mouse model: evidence of retinoid mitigation of vitamin D toxicity

9-cis-Retinoic acid (9cRA) and 1alpha,25-dihydroxyvitamin D3 (1,25D) show promise as potential chemopreventive agents. We examined 9cRA and 1,25D, alone and in combination, for their potential to inhibit carcinogen (NNK)-induced lung carcinogenesis in A/J mice. A/J mice (n=14/group) were treated with 9cRA (7.5, 15, or 30 mg/kg diet), 1,25D (2.5 or 5.0 microg/kg diet), or a combination of 9cRA (15 mg/kg diet) plus 1,25D (2.5 microg/kg diet) for 3 weeks before and 17 weeks after carcinogen injection. Lung tumor incidence, tumor multiplicity, plasma 1,25D levels and kidney expression of vitamin D 24-hydroxylase (CYP24) were determined. Compared to carcinogen-injected controls, mice receiving 9cRA supplementation had significantly lower tumor multiplicity at all doses (decreased 68-85%), with body weight loss at the higher doses of 9cRA. Mice receiving 1,25D supplementation had significantly lower tumor incidence (decreased 36 and 82%) and tumor multiplicity (decreased 85 and 98%), but experienced significant body weight loss, kidney calcium deposition, elevated kidney CYP24 expression and decreased fasting plasma 1,25D levels. Although, there was no apparent influence on chemopreventive efficacy, addition of 9cRA to 1,25D treatment effectively prevented the weight loss and kidney calcification associated with 1,25D treatment alone. These data demonstrate that 9cRA and 1,25D, alone or combined, can inhibit lung tumor promotion in the A/J mouse model. Combining 1,25D with 9cRA has the potential to mitigate the toxicity of 1,25D, while preserving the significant effect of 1,25D treatment against lung carcinogenesis. The underlying mechanism behind this effect does not appear to be related to retinoid modulation of vitamin D catabolism.     

Stage-specific inhibition of mammary carcinogenesis by 1alpha-hydroxyvitamin D5

Active metabolites of vitamin D are well recognised as cancer chemopreventive and chemotherapeutic agents. However, they are toxic at effective concentrations. Earlier, we reported that a non-toxic analogue of vitamin D, 1alpha-hydroxyvitamin D5(1alpha(OH)D5), inhibited carcinogen-induced mammary lesion formation in mouse mammary organ cultures (MMOC) and in N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. In the present study, we determined if 1alpha (OH)D5 action is selective during the initiation or promotion phases in MMOC and in vivo. In MMOC, 1 microM 1alpha (OH)D5 suppressed both ovarian hormone-dependent and -independent mammary lesions by more than 60%. Inhibition of alveolar lesions was observed only during the promotion stage (p=0.0016). In a 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis experiment, 1alpha (OH)D5 (40 microg/kg diet) inhibited cancer incidence by 37.5% (p<0.05) if 1alpha (OH)D5 was present in food during the promotion phase (+1 to end). However, a D5-supplemented diet during the initiation phase (-2 to +1 week) did not provide any protection. These results clearly show, for the first time, that the effects of vitamin D may be mediated selectively during the promotion or progression phases of carcinogenesis.     

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM) on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid), indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a condensation product of I3C) to alter the induction of mammary cancers induced by the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea (MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon receptor (AhR) agonists, while DIM does not. The agents were initially examined for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were administered for 14 days starting 7 days prior to a single dose of the carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%, respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction of DMBA tumors. The effect of these agents on the promotion/progression phase of carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF (1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after the administration of MNU, and continually thereafter. 5,6-BF decreased MNU- induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at the high dose, while DIM at either dose had minimal effects on tumor multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM had minimal effects in either model; arguing that administration of DIM is not analogous to administration of I3C.     

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Carcinogenesis by Soy Foods or Biochanin A

We examined the effects of soybeans, a soy product (miso) and biochanin A, an isoflavone derivative, on N -nitroso- N -methylurea (MNU)-induced rat mammary carcinogenesis. Seven-week-old female CD/Crj rats received a single i.v. dose (40 mg/kg body weight) of MNU. After administration of MNU, rats were fed diet containing 0% (control), 2% or 10% soybeans, or 10% miso as a soy-supplemented diet, or 10 or 50 mg/kg biochanin A. All rats were observed for 18 weeks after MNU administration. At 18 weeks, the multiplicity (mean tumors/rat) of palpable mammary tumors was significantly decreased in the 10% soybean (1.1) and 10% miso (1.2) diet groups compared to the control (2.2) ( P <0.05, respectively). In the biochanin A-supplemented diet groups, the incidence (percentage of rats with tumors) was significantly decreased in the 50 mg/kg (32%) diet group compared to the control (80%) ( P <0.01), and the multiplicity was significantly decreased in both the 10 mg/kg (0.7) and 50 mg/kg (0.5) diet groups compared to the control (2.2) ( P <0.01 and P <0.001, respectively). The proliferative cell nuclear antigen labeling index of mammary tumors was significantly decreased in both biochanin A-supplemented diet groups compared to the control. The present results indicate that soybeans, miso, and biochanin A are useful for the prevention of mammary cancer.     

Nordihydroguaiaretic acid suppression of rat mammary carcinogenesis induced by N-methyl-N-nitrosourea

The activity of the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), as a cancer chemopreventive agent in the rat mammary gland was determined. Beginning 1 week after a single i.v. dose of 20 or 50 mg N-methyl-N-nitrosourea (MNU)/kg body wt., female Sprague-Dawley rats were fed a semi-purified diet supplemented with 0 or 1000 mg NDGA/kg diet. At both MNU dose levels, groups receiving NDGA developed significantly fewer mammary cancers than did dietary controls; NDGA induced no gross or organ-specific toxicity. These data implicate lipoxygenase products in the process of mammary cancer induction, and suggest an additional enzymatic target for the design of chemopreventive drugs.     

Dietary effects of conjugated docosahexaenoic acid on N-methyl-N-nitrosourea-induced mammary carcinogenesis in female Sprague-Dawley rats

The dietary effects of conjugated docosahexaenoic acid (CDHA) were examined in an N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis model. Female Sprague-Dawley rats were administered 50 mg/kg MNU intraperitoneally at 49 days of age. A powdered AIN-76A diet containing 0, 0.2 or 1.0% CDHA was fed to the rats from 21 to 49 days of age (before MNU; pre-initiation phase) or from 49 days to 40 weeks of age (after MNU; post-initiation phase). Rats were sacrificed when their largest mammary tumor was > or =1 cm in size or when they reached 40 weeks of age. All histologically detected mammary carcinomas were evaluated. In rats that received CDHA after MNU, development of mammary carcinoma > or =1 cm was inhibited, and there was a significant decrease in the final mammary cancer incidence and multiplicity, compared with rats that did not receive CDHA. Consumption of the 0.2% CDHA diet after MNU significantly prolonged latency. Suppression of mammary cancer yield by consumption of a CDHA diet after MNU administration was not dose-dependent. In rats that received CDHA before MNU, suppression of mammary cancer was not observed. These results indicate that CDHA administration in the post-initiation period suppressed mammary carcinogenesis, whereas CDHA administration in the pre-initiation period was ineffective.     

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Cancer by Miso and Tamoxifen, Alone and in Combination

We examined the effects of a Japanese fermented soybean product, miso, and tamoxifen (TAM), alone and in combination, on N -nitroso- N -methylurea (MNU)-induced rat mammary cancer. Seven-week-old female CD/Crj rats received a single i.v. dose (50 mg/kg body weight) of MNU. After administration of MNU, the rats were divided into 4 groups: regular diet (control), 10% miso diet, regular diet+TAM, and 10% miso diet+TAM. TAM was implanted s.c. in the form of pellets containing 2.5 mg at the same time as MNU was administered. All rats were observed for 18 weeks after MNU administration. Incidence (percentage of rats with tumors) and multiplicity (mean tumors/rat) of mammary tumors were 91% and 4.5 in the control, 77% and 2.4 ( P <0.05) in the 10% miso group, 68% and 1.4 ( P <0.01) in the TAM group, and 10% ( P <0.0001 or less) and 0.2 ( P <0.0001) in the 10% miso+TAM group. In the second experiment, the effect of the combination of miso and TAM on established rat mammary tumors was investigated. When the mammary tumors induced by MNU reached 10 to 25 mm, the rats were divided into 3 treatment groups: regular diet, regular diet+TAM, and 10% miso diet+TAM. At 6 weeks after the start of treatment, the mean tumor size in the control and TAM groups was 160% and 141% of the pretreatment value, but a decrease to 85% of the pretreatment value was produced by the combination of miso and TAM, and this was significantly different from both the control and TAM groups ( P <0.01 and P <0.05, respectively). These results indicate that miso is useful in protecting against mammary cancer and it can be expected to have a potent antitumor effect, especially when used in combination with TAM.     

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.     

Influence of perinatal genistein exposure on the development of MNU-induced mammary carcinoma in female Sprague-Dawley rats

Genistein, a phytoestrogen, was subcutaneously (s.c.) injected to pregnant Sprague-Dawley CD rats on gestational days 16-20 at either 25 mg (Group 1) or 5 mg/day (Group 2). Female offspring of mothers not exposed to genistein during pregnancy received 12.5 mg genistein s.c. at neonatal days 15 and 18 (Group 3), or received vehicle only (Group 4). At 35 days of age, 4-9 female offspring from each group were autopsied to observe the influence of genistein, and remainder of female offspring received 50 mg/kg N-methyl-N-nitrosourea (MNU) intraperitoneally and were sacrificed when mammary tumors were larger than 1 cm in size or when they reached 35 weeks of age. Genistein treatment during the perinatal period resulted in lower body weight and lower relative uterine-ovarian weight at 35 days, and a prolonged estrus cycle with a long estrus phase at 12-16 weeks. However, at 35 days (time at MNU administration), mammary gland development, cell proliferation rate (PCNA labeling index), and the number of estrogen receptor (ER)- and progesterone receptor (PgR)-positive cells were similar between genistein-treated and untreated rats. Twenty-five or 5 mg genistein/day in utero (between days 16 and 20 of gestation) or 12.5 mg genistein/day on neonatal days 15 and 18 did not affect the incidence of mammary tumors > 1 cm or the latency but did increase the number of mammary cancer lesions when MNU was administered at the time when the mammary gland growth in genistein-treated and untreated rats was similar. Thus, perinatal genistein is an endocrine disrupter and increases the multiplicity of MNU-induced mammary carcinoma in rats.     

Enhanced inhibition of mammary carcinogenesis by combined treatment with N-(4-hydroxyphenyl)retinamide and ovariectomy

Bilateral ovariectomy and dietary administration of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are both effective inhibitors of chemical carcinogenesis in the rat mammary gland. The present study was designed to determine whether an enhanced inhibitory effect is obtained with combined ovariectomy and 4-HPR administration, compared to either treatment alone. In separate experiments, 50-day-old virgin female Sprague-Dawley rats received either a single i.v. injection of 50 mg N-methyl-N-nitrosourea per kg body weight or a single intragastric dose of 20 mg 7,12-dimethylbenz(a)anthracene. The experimental design was the same in both the N-methyl-N-nitrosourea and 7,12-dimethylbenz(a)anthracene experiments: Group 1, 25 intact rats, placebo diet; Group 2, 25 intact rats, supplement of 782 mg 4-HPR per kg diet; Group 3, 50 ovariectomized rats, placebo diet; Group 4, 50 ovariectomized rats, supplement of 782 mg 4-HPR per kg diet. Feeding of the 4-HPR supplement was begun 7 days after carcinogen administration; ovariectomy was performed 7 days post-7,12-dimethylbenz(a)anthracene or 14 days post-N-methyl-N-nitrosourea. In both experiments, combined ovariectomy plus 4-HPR was significantly more active in suppressing mammary cancer induction than was either manipulation alone. 4-HPR was a more effective inhibitor of carcinogenesis in ovariectomized rats than in intact animals. These data indicate that 4-HPR is highly effective in inhibiting ovarian hormone-independent cancers and suggest that retinoid inhibition of mammary carcinogenesis does not involve an influence on ovarian hormone action.     

Effect of retinyl acetate and 4-hydroxyphenylretinamide on initiation of chemically-induced mammary tumors

The anti-promotional effect of retinoids on chemically-induced mammary carcinogenesis in the rat is well established. The present studies were performed to determine the effect of long-term feeding of retinyl acetate and 4-hydroxyphenylretinamide (4-HPR) on initiation of mammary tumors induced by MNU or DMBA. Retinyl acetate (328 mg/kg of diet) or 4-HPR (782 mg/kg of diet) was added to the diet of female Sprague-Dawley rats for two months prior to the administration of the carcinogens. In the MNU model, a 50% increase in the number of mammary adenocarcinomas was observed in rats pretreated with retinyl acetate, while pretreatment with 4-HPR resulted in a 93% increase in the number of cancers. Continued treatment with 4-HPR throughout the study, however, caused a reduction in cancer number. In the DMBA mode, pretreatment with these retinoids significantly increased the number of benign mammary tumors, but not mammary cancers. These data suggest that newly synthesized retinoids should be evaluated for chemopreventive activity against mammary cancer initiation as well as for their anti promotional activity.     

Prevention by retinoids of azoxymethane-induced tumors and aberrant crypt foci and their modulation of cell proliferation in the colon of rats

Retinoids are proposed chemopreventive agents that inhibit cell proliferation and induce differentiation. Their ability to prevent azoxymethane (AOM)-induced aberrant crypt foci (ACF) and tumors and to modulate cell proliferation was investigated in the colon of male F344 rats. Thirteen retinoids were evaluated for prevention of ACF and two of them, 9-cis-retinoic acid (RA) and 4-(hydroxyphenyl)retinamide (4- HPR), were also evaluated for prevention of colon cancer. The retinoids were administered continuously in the diet starting 1 week prior to the first of two weekly 15 mg/kg i.p. injections of AOM and for a total of either 5 or 36 weeks in order to evaluate their effect on colonic ACF and tumors. At a concentration of 1 mmol/kg diet, 2- (carboxyphenyl)retinamide caused the greatest reduction (57.7%) in the yield of ACF. 9-cis-RA was toxic at 1 mmol/kg so that it was evaluated at 0.1 mmol/kg, resulting in a 41.6% reduction in ACF. The ability of the retinoids to reduce the proliferating cell nuclear antigen (PCNA) labeling index in ACF and in non-involved crypts correlated with their ability to prevent ACF. Both 9-cis-RA (0.1 and 0.2 mmol/kg diet) and 4- HPR (1 and 2 mmol/kg diet) were highly effective in decreasing the yield of AOM-induced colon tumors. In summary, retinoids were demonstrated to reduce cell proliferation and to prevent ACF and tumors in the colon, suggesting promise as preventive agents for colon cancer.      

Anticarcinogenic activity of quinacrine in the rat mammary gland

Mammary carcinogenesis studies were conducted to determine thechemopreventive activity of quinacrine, an antimalarial drugwhich suppresses the production of arachidonic acid from phospholipidthrough inhibition of phospholipase A₂. Beginning 1 week aftera single i.v. dose of N-methyl-N-nitrosourea (MNU), female Sprague—Dawleyrats were fed a semi-purified diet supplemented with 0 or 75mg quinacrine/kg diet. Quinacrine reduced cancer incidence andcarcinoma multiplicity in rats administered 20 mg MNU/kg bodywt, but had no inhibitory activity in rats treated with 50 mgMNU/kg. These data suggest that pathways of arachldonic acidmeta-bolism in addition to cyclooxygenase present useful targetsfor mammary cancer chemoprevention. However, the chemo-preventiveactivity of quinacrine may be limited by toxicity.     

Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.     

Effect of deficiencies of selenium and vitamin E alone or in combination on the induction of mammary carcinogenesis by 1-methyl-1-nitrosourea

Experiments were conducted to determine the dietary levels ofselenium and vitamin E that could be fed chronically to inducea deficiency of one or both nutrients. It was observed thatdiets containing <0.02 mg Se/kg and 3 IU vitamin E/kg induceda deficiency without drastically altering growth performanceor survival. Once established, these dietary conditions wereused to investigate the effect of single or combined deficienciesof selenium and vitamin E on the promotion phase of mammarycarcinogenesis induced by 1-methyl-1-nitrosourea (MNU). In thecarcinogenesis experiment, 21 day old female Sprague-Dawleyrats were fed a torula yeast formulated diet deficient in seleniumand vitamin E until they were 50 days of age. At that time,each rat was injected i.p. with 12.5 mg MNU/kg body weight.Seven days thereafter, rats were randomly assigned to one offour treatment groups that were deficient or adequate in seleniumand/or vitamin E. The experiment was terminated 30 weeks aftercarcinogen treatment. In comparison to rats fed the adequatediet, final cancer incidence and number were higher and cancerlatency shortened in rats consuming the diet deficient in bothselenium and vitamin E. Single deficiencies of either nutrientfailed to significantly alter the tumorigenic response.     

Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea.

The objective of the work reported in this paper was to determine if the tumorigenic response to 1-methyl-1-nitrosourea (MNU) in the mammary gland varied with age of administration and was dose dependent when the carcinogen was injected prior to 50 days of age. Using a recently developed method for mammary tumor induction, MNU was injected i.p. at doses ranging from 25 to 75 mg/kg at 35 days of age or 50 mg/kg at 28, 35 or 42 days of age. Treatment with MNU resulted in induction of both benign and malignant mammary tumors. The incidence of mammary gland adenocarcinomas was 100% at and above the 50 mg/kg dose of MNU, irrespective of the age at which carcinogen was administered. The number of cancers increased in proportion to carcinogen dose, whereas cancer latency decreased as the MNU dose increased. In rats injected with 50 mg MNU/kg body weight at 28, 35 or 42 days of age, differences among groups in cancer incidence, number or latency were not statistically significant. Metastases of mammary neoplasms to lung, liver and spleen were observed in rats injected with MNU at 35 or 42 days of age. These data indicate (i) the dose responsiveness of MNU-induced mammary carcinogenesis in rats initiated prior to 50 days of age; (ii) the lack of effect of age at initiation if prior to 50 days on final tumor outcome; and (iii) that the age at which MNU is injected may affect the metastatic potential of the mammary carcinomas that are induced.