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.     

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.     

Chemopreventive efficacy of Targretin in rodent models of urinary bladder, colon/intestine, head and neck and mammary cancers

The chemopreventive efficacy of Targretin was evaluated in various rodent cancer models. In the rat model of 4-hydroxybutyl(butyl)nitrosamine (OH-BBN)-induced urinary bladder cancer, it was found that Targretin administered in the diet (beginning one week after the last OH-BBN treatment) for 5.5 months increased the number and size of urinary bladder cancers. In the azoxymethane (AOM)-induced model of colon carcinogenesis (in which rats develop minimally invasive colonic cancers), Targretin was ineffective as a chemopreventive agent, decreasing neither tumor incidence nor multiplicity. Treatment of Min mice with Targretin for 45 days similarly failed to decrease the multiplicity of small intestinal tumors. Similarly, no preventive efficacy was noted for Targretin when the incidence of tumors in the head and neck model (squamous cell tongue tumors) induced by 4-nitroquinoline 1-oxide (4-NQO) were examined. In contrast, use of even a suboptimal dose of Targretin (40?ppm) in a sensitive breast cancer model [methylnitrosourea (MNU)-induced ER+ mammary cancers] reduced cancer multiplicity by 60%. Finally, based on the hypothesis that Targretin may decrease the expression of COX?2, the effects of Targretin and COX inhibitors were compared in these models. There was minimal overlap of efficacy. That is, models which were relatively susceptible to NSAIDs or COX-2 inhibitors tended not to be sensitive to Targretin and vice versa.     

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.     

Chemopreventive effects of the aromatase inhibitor vorozole (R 83842) in the methylnitrosourea-induced mammary cancer model

The chemopreventive activity of the highly specific nonsteroidal aromatase inhibitor, vorozole, was examined in the methylnitrosourea (MNU)-induced rat model of mammary carcinogenesis. Various doses of vorozole (0.08-1.25 mg/kg body wt/day) were administered daily (by gavage) to female Sprague-Dawley rats starting at 43 days of age. Seven days later, the rats were given a single i.v. dose of MNU (50 mg/kg body wt). Rats were continually treated with vorozole until the end of the experiment (120 days post-MNU). Vorozole caused a dose dependent inhibition of mammary cancer multiplicity. The highest dose of vorozole (1.25 mg/kg body wt/day) decreased cancer multiplicity by approximately 90%, and simultaneously decreased cancer incidence from 100 to 44%. The next two highest doses of vorozole (0.63 and 0.31 mg/kg body wt/day) inhibited MNU-induced mammary cancer multiplicity by 70-80%. Even the two lowest doses of vorozole (0.16 and 0.08 mg/kg body wt/ day) decreased cancer multiplicity -50%. Serum level determinations were performed on a variety of endpoints at either 4 or 24 h following the last dose of vorozole. Insulin-like growth factor (IGF)-1 levels were slightly, but significantly, increased by vorozole treatment. Vorozole induced striking increases in serum testosterone levels at 4 h at all the dose levels employed. Testosterone levels were significantly elevated over controls at 24 h in rats given the lower doses of vorozole (0.08-0.31 mg/kg body wt/day), but were significantly lower than in rats administered the higher doses of vorozole (0.63 or 1.25 mg/kg body wt/ day). This result presumably reflects the limited half- life of vorozole in rats. In a second series of experiments, the effects of limited duration of dosing with vorozole (2.5 mg/kg body wt/day) or intermittent dosing with vorozole were determined. Treatment of rats with vorozole for limited time periods, from 3 days post-MNU administration until 30 or 60 days post-MNU treatment, resulted in significant delays in the time to appearance of palpable cancers. However, these limited treatments did not greatly affect the overall incidence or multiplicity of mammary cancers when compared with the MNU controls at the end of the study (150 days post-MNU). Finally, the effects of intermittent dosing with vorozole (2.5 mg/kg body wt/day) were examined. Rats were administered cycles of vorozole daily for a period of 3 weeks followed by treatment with the vorozole vehicle for the next 3 weeks (total of four cycles). Although this intermittent treatment did inhibit the appearance of new tumors during each of the periods that vorozole was administered, it did not cause regression of palpable cancers.      

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.     

Lack of enhancement of susceptibility to mammary and thyroid carcinogenesis in rats exposed to DMBA and DHPN following prepubertal iodine deficiency

Epidemiologic and experimental studies suggest that iodine deficiency increases the risk of mammary as well as thyroid cancers, but susceptibility to tumor development when this occurs during the prepubertal stage is not completely understood. In the present study, we therefore evaluated this question in F344 rats. Dams during the lactation period and their weaned offspring until postnatal week 7 were fed an iodine-free diet. Female offspring were then given 7,12-dimethylbenz[a]anthracene (DMBA, 50 mg/kg body weight) by gavage for mammary tumor induction in week 7. Both the male and female rats were given free access to drinking water containing N-bis(2-hydroxypropyl)nitrosamine (DHPN), (0.1 and 0.2% for male and female rats, respectively) for wide spectrum tumor induction in organs, including the thyroid gland, from weeks 7-11. All offspring were killed at week 50 for histopathological examination. The iodine deficiency had no significant influence on incidences and/or multiplicities of mammary and thyroid tumors. Furthermore, tumor induction in the liver, kidney, lung, esophagus and urinary bladder was not affected in either sex. The present results thus indicate a lack of influence of iodine deficiency condition early in life on subsequent carcinogenic susceptibility.     

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.     

Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.     

Black tea and mammary gland carcinogenesis by 7,12- dimethylbenz[a]anthracene in rats fed control or high fat diets

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor- bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.      

Low zinc intake suppressed N-methyl-N-nitrosourea-induced mammary tumorigenesis in Sprague-Dawley rats

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.     

Phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates inhibit malignant progression of lung adenomas induced by tobacco carcinogens in A/J mice

We have shown previously that naturally occurring isothiocyanates derived from cruciferous vegetables and their N-acetylcysteine conjugates inhibit lung adenoma formation induced by tobacco carcinogens in A/J mice at the post-initiation stage. The tumor-inhibitory activity by these compounds is linked with activation of activator protein and induction of apoptosis in lung tissues, suggesting that these compounds may also inhibit the development of adenomas to adenocarcinomas in lung. In this study, the chemopreventive activity of phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates during progression of lung adenomas to malignant tumors was investigated in A/J mice. Mice were divided into 14 groups and treated with a mixture of 3 micromol benzo(a)pyrene [B(a)P] and 3 micromol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) given by gavage once weekly for 8 weeks. Twenty weeks after the beginning of carcinogen administration, a total of 20 mice in the treatment groups were sacrificed with an average yield of 7.3 +/- 4.5 lung adenomas per mouse. The remaining mice in each group were fed diets containing phenethyl isothiocyanate (3 and 1.5 mmol/kg diet), sulforaphane (3 and 1.5 mmol/kg diet), phenethyl isothiocyanate-N-acetylcysteine (8 and 4 mmol/kg diet), sulforaphane-N-acetylcysteine (8 and 4 mmol/kg diet) during weeks 21 to 42. Four mice in each of the high-dose treatment groups were sacrificed during weeks 28 and 36 and the bioassay was terminated during week 42; lung tissues were harvested for histopathologic examination of tumors and for cell proliferation (proliferating cell nuclear antigen) and apoptosis (caspase-3) assays using immunohistochemical staining. At termination, the incidence of adenocarcinoma in the 3 mmol/kg diet phenethyl isothiocyanate group and 8 mmol/kg diet phenethyl isothiocyanate-N-acetylcysteine group was reduced to 19% and 13%, respectively, compared with 42% in the carcinogen-treated control group. At the lower doses, phenethyl isothiocyanate and its N-acetylcysteine conjugate also inhibited the incidences of lung adenocarcinoma, however, the decreases were not statistically significant. The lung tumor incidences in groups treated with sulforaphane-N-acetylcysteine in the diet were also significantly reduced to 11% or 16%. Furthermore, the malignant lung tumor multiplicity was significantly reduced from 1.0 tumor/mouse in the carcinogen-treated control group to 0.3 in the sulforaphane low-dose group, 0.3 and 0.4 in the two sulforaphane-N-acetylcysteine groups, and 0.4 in the phenethyl isothiocyanate high-dose group. The malignant tumor multiplicities in other treatment groups were also reduced (0.5-0.8 tumors/mouse), but not significantly. Unlike lung adenocarcinomas, both incidences and multiplicities of lung adenomas were not much affected by treatment with isothiocyanates or their conjugates. Immunohistochemical examination of the lung tumors from all time points indicated that significant reduction in proliferating cell nuclear antigen and induction of apoptosis (terminal nucleotidyl transferase-mediated nick end labeling and caspase-3) were observed in the isothiocyanate and isothiocyanate-N-acetylcysteine-treated groups that showed inhibition of the development of lung adenocarcinomas. The results of the study provide a basis for future evaluation of the potential of phenethyl isothiocyanate and sulforaphane and their conjugates as chemopreventive agents in smokers and ex-smokers with early lung lesions.     

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.     

Prevention of methylnitrosourea-induced mammary cancers by 9-cis-retinoic acid and/or vitamin D3

Two cancer chemopreventive agents, vitamin D3 and 9-cis-retinoic acid (9-cis-RA), were evaluated alone and in combination in the methylnitrosourea (MNU)-induced mammary cancer model. In this study, female Sprague-Dawley rats received MNU (50 mg/kg BW) at 50 days of age. Vitamin D3 and 9-cis-RA were administered in the diet beginning three days later. The groups were: Group 1, vehicle only; Group 2, 9-cis-RA (60 mg/kg diet); Group 3, vitamin D3 (10 microg/kg diet); Group 4, vitamin D3 (3.3 microg/kg diet); Group 5, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (10 microg/kg diet); and Group 6, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (3.3 microg/kg diet). Animals were observed daily for signs of toxicity and were palpated 2x/week for mammary tumors. The study was terminated 150 days after treatment with MNU. The average number of mammary cancers was 6.7 in the animals receiving only the carcinogen. 9-cis-RA alone caused a 23% decrease in mammary cancer multiplicity, while vitamin D3 alone actually caused slight increases of 17 and 16% at 10 and 3.3 microg/kg diet dose levels, respectively. When the agents were given in combination, however, the 9-cis-RA plus the high dose of vitamin D caused a statistically significant decrease (44%) in mammary cancer number, while the 9-cis-RA plus the low dose resulted in a 37% decrease. Thus, low doses of these agents that were not effective in preventing mammary cancer when given alone appeared to be active when given in combinations. Possible interactions between the retinoic acid receptors and vitamin D receptor may be responsible for the observed inhibition of mammary carcinogenesis.     

Post-initiation treatment of rats with indole-3-carbinol or beta-naphthoflavone does not suppress 7, 12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis

Indole-3-carbinol (I3C) and β-naphthoflavone (β-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague–Dawley rats with I3C (250 mg/kg body weight (b.w.)), β-NF (20 mg/kg b.w.) or the vehicle ethanol:corn oil (2:3) (2.5 ml/kg b.w.), three times weekly by gavage, started 3 weeks after the initiation with one oral dose of DMBA (20 mg/rat at 7 weeks of age) and continued for up to 12 weeks. I3C- or β-NF- or vehicle-treated groups did not differ significantly in the overall outcome of mammary tumorigenesis including cumulative mammary tumor incidences and multiplicities, latent periods and number and weight of mammary tumors per tumor-bearing rat for malignant, benign and/or malignant + benign tumors. A tendency of the I3C-treated rats to develop fewer mammary adenocarcinomas with a greater average weight per tumor per rat (2.32±1.50 g) than in the β-NF- (1.52±1.58 g) or vehicle- (1.55±1.53 g) treated groups suggests an effect, yet to be confirmed, of I3C on tumor development and growth. A 12-week treatment with I3C or β-NF significantly increased the P450-dependent activities of ethoxy-, methoxy-, benzyloxy- and pentoxy-(with I3C only) resorufin O-dealkylase in hepatic microsomes indicating induction of several P450s. The alterations in the P450 complement may affect endogenous estrogen metabolism and mammary gland and tumor characteristics at the molecular level, e.g. estrogen receptor status and/or proliferative activity, which require further studies.     

Effects of high dietary fat on the growth and development of ovarian-independent carcinogen-induced mammary tumors in rats

This study examined the influence of high dietary fat intake on the development of ovarian-independent mammary tumors in both vehicle-treated controls and rats made deficient in estrogen and prolactin during tumor induction. The majority of 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors in rats are dependent on estrogen and prolactin for growth, and suppression of prolactin and estrogen at the time of tumor initiation causes a reduction in tumor incidence and increase in tumor latency. However, the majority of mammary tumors which do develop in these animals exhibit ovarian-independent growth. Sprague-Dawley rats were given 7.5 mg DMBA p.o. at 57 days of age. Starting 1 day prior to and continuing for 7 days after DMBA administration, rats were given daily injection of vehicle or the combination of tamoxifen (20 micrograms/rat) plus bromocryptine (5 mg/kg). At the end of drug treatment, rats in each treatment group were equally divided and placed on normal fat (5% corn oil) or high fat (20% corn oil) diets for the duration of the experiment. Vehicle-treated rats were ovariectomized 27 wk and drug-treated rats 47 wk after DMBA administration to determine tumor ovarian dependency. Vehicle-treated rats fed high fat diets showed significant increases in mammary tumor incidence and number as compared to similarly treated rats fed a normal fat diet, with approximately 80% of the tumors in each group being ovarian dependent. Likewise, tamoxifen-bromocryptine-treated rats fed a high fat diet showed a significant enhancement in mammary tumor number, although not incidence, as compared to similarly treated rats fed a normal diet. Tumors in these drug-treated groups displayed essentially the same incidence of ovarian dependence (23%). Tamoxifen-bromocryptine-treated groups displayed a 2-fold increase in latency of tumor appearance as compared to vehicle-treated controls; however, this long latency was not reduced when these rats were fed a high fat diet. These results demonstrate that high dietary fat stimulates ovarian-dependent and -independent mammary tumorigenesis in rats but does not influence the hormonal responsiveness of these tumors.     

Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat

Asian women consuming a traditional diet high in soy have a low incidence of breast cancer, yet when they emigrate to the USA the second but not the first generation lose this protection. Accordingly, we hypothesized that early exposure to genistein, a major component of soy, could have a permanent protective effect against breast cancer. Sprague-Dawley CD rats were exposed to genistein from conception to day 21 post-partum in the diet at concentrations of 0, 25 and 250 mg genistein/kg AIN-76A diet. At day 50 post-partum, all animals were treated with 80 mg dimethylbenz[a]anthracene/kg body wt to induce mammary cancers. Dietary genistein resulted in dose-dependent protection against development of mammary tumors (fewer tumors per rat). Analysis of mammary whole mounts showed that 21- and 50-day-old female rats had fewer terminal end buds, terminal ductal structures that were undifferentiated and were most susceptible to carcinogenesis. Bromodeoxyuridine incorporation studies revealed that dietary perinatal genistein resulted in a smaller proliferative compartment for terminal end buds. In rats fed the high genistein dose (250 mg/kg diet) total genistein concentrations in the serum and milk of dams 7 days postpartum were 418+/-198 and 137 pmol/ml, respectively. Total genistein concentrations in stomach milk, serum and mammary glands of 7- day-old offspring were 4439+/-1109 and 726 pmol/ml and 440+/-129 pmol/g, respectively. Total genistein concentrations in the serum and mammary glands of 21-day-old offspring were 1810+/-135 pmol/ml and 370+/-36 pmol/g, respectively. Dietary perinatal genistein did not cause significant toxicity in F0 and F1 females. We conclude that genistein in the diet at 'physiological levels' enhances cell differentiation, resulting in programming of mammary gland cells for reduced susceptibility to mammary cancer, with no observed toxicity to the reproductive tract of F1 females.      

Biomarkers of phenethyl isothiocyanate-mediated mammary cancer chemoprevention in a clinically relevant mouse model

Background Phenethyl isothiocyanate (PEITC) is a natural plant compound with chemopreventative potential against some cancers and the ability to induce apoptosis in breast cancer cells. Methods Female mouse mammary tumor virus-neu mice were fed a control AIN-76A diet (n = 35) or the same diet supplemented with 3 μmol PEITC/g diet (n = 33) for 29 weeks, at which time they were killed. Breast tissue sections were stained with hematoxylin and eosin for histopathological assessments, and incidence and size of macroscopic mammary tumors were assessed. Cell proliferation (Ki-67 staining), apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-labeling), and neoangiogenesis (CD31 staining) were determined in tumor sections. Plasma levels of transthyretin were measured in treated and control mice. Expression of proteins in mammary tumor sections was determined by immunohistochemistry. Proteomic profiling was performed by two-dimensional gel electrophoresis followed by mass spectrometry. All statistical tests were two-sided. Results Administration of PEITC for 29 weeks was associated with 53.13% decreased incidence of macroscopic mammary tumors (mean tumor incidence, PEITC-supplemented diet vs control diet, 18.75% vs 40.00%, difference = -21.25%, 95% confidence interval [CI] = -43.19% to 0.69%, P = .07) and with a 56.25% reduction in microscopic mammary carcinoma lesions greater than 2mm(2) (mean incidence, PEITC-supplemented diet vs control diet, 18.75% vs 42.86%, difference = -24.11%, 95% CI = -46.35% to -1.86%, P = .04). PEITC-mediated mammary cancer growth inhibition was not because of suppression of human epidermal growth factor receptor-2 expression but was associated with reduced cellular proliferation and neoangiogenesis, increased apoptosis, and altered expression of several proteins, including decreased ATP synthase in the tumor and increased plasma levels of transthyretin. Conclusions PEITC inhibits the growth of mammary cancers in a mouse model with similarities to human breast cancer progression. ATP synthase and transthyretin appear to be novel biomarkers associated with PEITC exposure.     

Promoting effects of high-fat corn oil and high-fat mixed lipid diets on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis in F344 rats

Epidemiological studies and laboratory animal model assays suggest that a high intake of dietary fat promotes mammary carcinogenesis as well as colon tumorigenesis. Fat intake in the United States traditionally includes high amounts (30% of total caloric intake) of saturated fatty acids (SFAs) compared to polyunsaturated fatty acids (PUFAs). A recent study suggested that a high-fat mixed-lipid diet (HFML), which simulates the mixed-lipid and high SFAs composition of the average American diet, strongly promotes rat colon carcinogenesis, even when compared to another high-fat diet containing PUFA-rich corn oil. On the other hand, some reports suggest that a high-fat diet rich in n-6 PUFAs promotes mammary carcinogenesis more strongly than a high-fat diet rich in SFAs. Therefore, the present study was designed to compare the effects of HFML, high-fat corn oil diet (HFCO) that is rich in n-6 PUFAs, and a low-fat corn oil diet (LFCO) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in female F344 rats. At 7 weeks of age, female F344 rats intended for carcinogen treatment received a gavage of DMBA at a dose level of 65 mg/kg of body weight. Beginning 1 week after carcinogen treatment, groups of rats were then maintained on experimental diets containing LFCO, HFCO or HFML. All rats were evaluated weekly by palpation of mammary tumors and sacrificed 20 weeks after the DMBA treatment. Palpable tumors of mammary glands were detected at the 8, 11, and 19 weeks in the HFCO, HFML and LFCO groups, respectively. Histopathological observation revealed that the incidence and number of mammary tumors in the HFCO group were significantly higher than in the LFCO group. Rats on the HFML diet tended towards a higher incidence and number of mammary tumors compared with the LFCO group, although the correlation was not statistically significant. These results suggest that, for this animal model, both the HFCO and HFML diets promote DMBA-induced mammary carcinogenesis when compared to the LFCO diet, and that the HFCO diet is more tumor-promotional than the HFML diet.     

Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin

The effects of dietary supplementation of flavonol quercetin on both 7,12-dimethylbenz(a)anthracene (DMBA)- and N-nitrosomethylurea-induced mammary cancer in female Sprague-Dawley rats were determined. Quercetin diet was started 1 wk before intragastric instillation of DMBA (65 mg/kg of body weight) or i.v. injection of N-nitrosomethylurea (50 mg/kg of body weight) and was continued during the entire period (20 wk) of the experiment. Dietary quercetin inhibited both the incidence and the number of palpable rat mammary tumors; rats fed on 2% quercetin had 25% less incidence of mammary cancer, while the average number of mammary tumors per rat was reduced by 39% at 20 wk post-DMBA administration compared to animals on a control diet. In a separate experiment, a 5% quercetin diet elicited a greater inhibitory effect on the induction of rat mammary tumors by DMBA than was observed with a 2% quercetin diet. The inhibitory effect of quercetin on mammary tumor incidence in rats on 2% and 5% diets and on tumor multiplicity in animals on a 5% diet was statistically significant (P less than 0.05). In addition, the risk of the development of a palpable tumor (as determined by the nonparametric estimate of the hazard function) in the quercetin-fed group was lower than the group on control diet throughout the course of the experiment. Furthermore, 5% dietary quercetin significantly inhibited (P less than 0.05), although to a lesser extent than observed in DMBA-induced tumor formation, both the incidence and the number of palpable mammary tumors per rat induced by N-nitrosomethylurea. Dietary quercetin did not elicit any detectable sign of toxicity. The gain in body weight in rats on the quercetin diet and the quantity of diet consumed per rat per week were similar to those for rats on the control diet.