Effects of combined and sequential treatment with tamoxifen and the aromatase inhibitor vorozole on 7,12-dimethylbenz(a) anthracene-induced mammary carcinoma in the rat

 The aromatase inhibitor vorozole dose-dependently inhibited the growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat. An oral dose of 5 mg/kg per day brought about growth inhibition and reduction of tumor multiplicity similar to that produced by ovariectomy. Tamoxifen (8 mg/kg per day) also reduced tumor growth, albeit to a lesser extent than did ovariectomy. Concomitant administration of varying doses of tamoxifen with the fully effective dose of vorozole (5 mg/kg per day) tended to be less effective than ovariectomy or vorozole alone. This is likely to be due to the estrogen-agonistic effects of tamoxifen. Combination of tamoxifen with the partially effective dose of vorozole (1 mg/kg per day) gave results comparable with those obtained for either of these compounds used in monotherapy. Combining tamoxifen with a marginally active low dose of vorozole (0.2 mg/kg per day) resulted in a minor additional growth inhibition as compared with that obtained with this dose of vorozole alone. Sequential treatment with tamoxifen (8 mg/kg per day) for 42 days and vorozole (5 mg/kg per day) for 42 days, and vice-versa, was performed with a drug-free interval of 14 days between treatments. Tumors regressing under vorozole therapy relapsed when subsequently treated with tamoxifen. In contrast, vorozole further reduced tumor volumes in rats previously treated with tamoxifen. Furthermore, monotherapy with tamoxifen as well as the two sequential tamoxifen-vorozole treatment schedules were in most cases less effective than vorozole monotherapy in inhibiting both tumor growth and tumor multiplicity. Although extrapolation of these findings in cycling animals to the clinical situation, involving postmenopausal women, is not straightforward, these results warrant further studies in preclinical models. Moreover, clinical trials comparing the most effective aromatase inhibitors with tamoxifen in previously untreated postmenopausal patients with breast cancer may also be warranted. Received: 7 April 1985/Accepted: 4 August 1985     

Growth and characterization of N-methyl-N-nitrosourea-induced mammary tumors in intact and ovariectomized rats.

It is well established that 85-90% of chemically induced mammary tumors in rats will disappear or diminish significantly in size after the ovaries are removed from the animal. However, it is less well established whether a high percentage of these mammary tumors will grow back with prolonged time after ovariectomy. It is also not known what changes in gene expression take place in the tumors as they develop an independence from hormones for growth. This study was carried out to investigate this. Virgin, 50-day-old female Sprague-Dawley rats were injected with N-methyl-N-nitrosourea (MNU) at the dose of 50 mg MNU/kg body wt. When at least one mammary tumor had grown to 1.0-1.5 cm in one dimension, the animal was bilaterally ovariectomized and reduction and then re-growth of the tumors monitored. Control animals were treated identically except they were not ovariectomized when tumors appeared. Re-growths and new tumors and tumors that developed in the control rats were removed when they reached 1.0-1.5 cm in diameter and all animals were killed 25 weeks after the MNU injection. All the animals in the study (100%) developed mammary tumors after MNU injection with an average latency of 56.5 days. After ovariectomy, 93% of the tumors showed 50% or more reduction in size and 76% of the tumors could not be detected by palpation. However, in 96% of the animals where tumor reduction or disappearance occurred, a re-growth or new mammary tumor development took place with an average latency period of 52.8 days from the day of ovariectomy. Of these post-ovariectomy tumors, 36% occurred at a location where tumors had developed prior to ovariectomy, but 64% appeared at new locations. The circulating levels of 17beta-estradiol (E2) was undetectable in the ovariectomized (OVX) rats and significant reduction was seen in the serum concentrations of progesterone (P4), prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF-I). The tumors from the OVX rats showed indications of progression as evident from loss of differentiation and invasive characteristics. Comparison between tumors from OVX and intact rats revealed a significantly increased expression of P450 aromatase and elevated activation of extracellular signal-regulated kinase 1 and 2, but reduced levels of the progesterone receptor and cyclin D1 in OVX rats. However, the estrogen receptor (ER) content remained similar in tumors from both groups, at least at the protein level, and so did the expression of IGF-I, IGF-II, insulin receptor substrate-1 (IRS1), IRS-2 and epidermal growth factor receptor. IGF-I receptor (IGF-IR) and ErbB-2 were expressed, respectively, in 50 and 70% of the tumors from the OVX animals, whereas these genes were expressed in 100% of the tumors from the intact rats. It is concluded that chemically induced rat mammary tumors may still depend on the ER and local syntheses of E2 and growth factors for growth initially after ovariectomy. However, as these tumors progress, they develop a more aggressive phenotype and lose their dependency on the ER and possibly growth factors.     

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.      

Relationship between insulin and estrogen binding to growth response in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

Insulin and estrogen binding have been determined in 7,12-dimethylbenz(a)anthracene-induced mammary tumors of rats in various endocrine states. Hormonal therapy, such as diabetes and ovariectomy, resulted in differential effects on growth patterns and hormone binding of tumors coexisting in the same host or in different hosts. It was observed that tumors that continued to grow after the host was made diabetic (insulin independent) or started to regress after ovariectomy (ovarian dependent) demonstrated decreased insulin binding. Tumors that regressed in diabetic hosts (insulin dependent) or continued to grow in ovariectomized animals (ovarian independent) showed an increased insulin-binding capacity. No significant change in insulin binding was observed in tumors that remained static after ovariectomy or induction of diabetes. Estrogen binding in tumor cells from diabetic rats paralleled the pattern of tumor growth response to diabetes; insulin-independent tumors demonstrated a significant increase in binding compared to tumors from intact hosts, and insulin-dependent tumors showed decreased estrogen receptor levels. From these results, we conclude that (a) insulin plays a positive role in regulating estrogen-binding capacity, (b) ovarian hormones may play a role in regulating insulin-binding capacity, and (c) a relationship between insulin and ovarian hormones and the growth of 7,12-dimethylbenz(a)anthracene-induced tumors is strongly suggested and may have therapeutic implications.     

Effect of ovariectomy on hormone receptors and growth of N-nitrosomethylurea-induced mammary tumors in the rat

Estrogen receptor(s) (ER), progesterone receptor(s) (PGR), androgen receptor(s) (ANR), and prolactin receptor(s) (PRLR) were measured in N-nitrosomethylurea-induced mammary tumors in intact female Sprague-Dawley rats and in rats 9 days after ovariectomy. Following ovariectomy, 12 of 15 tumors regressed to 47.7 +/- 5.5% of the original size (hormone-dependent tumors), while the remaining three had arrest of growth reaching 88.8 +/- 7.3% of their original sizes. Cytosol ER level was 50.3 +/- 6.6 fmol/mg protein in tumors of intact rats and was significantly lower (25.6 +/- 8.3 fmol/mg, p < 0.025) in the ovariectomized group. PGR was abundantly present in ten of 13 tumors of intact rats (mean, 144.5 +/- 46.8) but was undetectable in five of six hormone-dependent tumors after ovariectomy (p < 0.01). ANR ws detectable at low levels in only four of 13 tumors of intact rats but in none of six hormone-dependent tumors after ovariectomy. PRLR was not significantly different in tumors of intact and ovariectomized rats (20.6 +/- 2.4 and 15.6 +/- 1.8 fmol/mg, respectively). In three tumors that had arrest of growth after ovariectomy, the levels of ER, PGR, ANR, and PRLR were not significantly different from those of the hormone-dependent tumors. We conclude that the majority of N-nitrosomethylurea-induced rat mammary tumors are hormone dependent. ER, PGR, and PRLR were abundantly present in the majority of these tumors, while ANR was present in only four of 13 tumors. The levels of ER and PGR were significantly lower following ovariectomy, while PRLR was not significantly changed.     

Enhancing effects of beta-estradiol 3-benzoate but not methoxychlor on the promotion/progression stage of chemically-induced mammary carcinogenesis in ovariectomized rats.

Modifying effects of beta-estradiol 3-benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague-Dawley rats. Twenty-eight weeks after a single DMBA (100 mg / kg body weight) initiation, when the incidence of mammary tumor-bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor-bearing, ovariectomized group; ii) tumor-bearing, intact group; iii) no-tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor-bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no-tumor, ovariectomized group. However, MXC did not show any effect in the tumor-bearing, or no-tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor-bearing, intact group. The results of our study suggest that MXC has no promotion / progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA-induced mammary tumorigenesis.     

Enhancing Effects of β-Estradiol 3–Benzoate but Not Methoxychlor on the Promotion/Progression Stage of Chemically-induced Mammary Carcinogenesis in Ovariectomized Rats

Modifying effects of β-estradiol 3–benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12–dimethylbenz( a )anthracene (DMBA)-induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague-Dawley rats. Twenty-eight weeks after a single DMBA (100 mg/kg body weight) initiation, when the incidence of mammary tumor-bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor-bearing, ovariectomized group; ii) tumor-bearing, intact group; iii) no-tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor-bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no-tumor, ovariectomized group. However, MXC did not show any effect in the tumor-bearing, or no-tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor-bearing, intact group. The results of our study suggest that MXC has no promotion/progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA-induced mammary tumorigenesis.     

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.     

Effects of 4-nitroestrone 3-methyl ether on dimethylbenz(a)anthracene-induced mammary tumors

4-Nitroestrone 3-methyl ether has been shown to be an effective growth inhibitor of certain dimethylbenz(a)anthracene-induced rat mammary tumors in intact or ovariectomized rats. When administered at optimum levels (24 mg/kg daily), this A-ring-substituted estrone displayed no toxicity, slight estrogenicity, and an antitumor activity which was comparable to that of tamoxifen and nafoxidine and was surpassed only by ovariectomy or pharmacological doses of 17 beta-estradiol 3-benzoate. In addition, the appearance of mammary tumors was prevented when this estrogen derivative was administered to rats just prior to or after dimethylbenz(a)anthracene intubation. Unique to the action of the methyl ether of 4-nitroestrone on mammary tumors was the destruction of adenocarcinomas while permitting the appearance of fibroadenomas. Systemically, 4-nitroestrone 3-methyl ether brought about focal atrophy within the pituitary and ovaries while causing moderate hypertrophy of the uterus. Plasma prolactin was unaffected.     

Effects of sex difference, gonadectomy, and estrogen on N-methyl-N-nitrosourea induced rat thyroid tumors

The occurrence of thyroid tumors induced by N-methyl-N-nitrosourea (MNU) and low iodine diet in Long-Evans (LE) rats was studied with special reference to sex difference, effect of gonadectomy, and estradiol administration. Rats of experimental groups 1-6 were given i.v. injections of 40 mg of MNU/kg of body weight at 50 days of age and fed on low iodine diet from 28 days of age to the end of the experiment (30 weeks after MNU administration). They consisted of male, female, castrated male, ovariectomized female, and gonadectomized male and female rats given 2.5 mg estradiol pellets s.c. Rats of groups 7-10 served as the respective controls without MNU or low iodine diet. Levels of serum thyroid stimulating hormone and estrogen receptor of the thyroid lesions were also examined. It was noted that the incidence of thyroid carcinoma was higher in females than in males (P less than 0.01) and did not change by castration in males but decreased in ovariectomized rats (P less than 0.01). Administration of estradiol after gonadectomy significantly increased the incidence of thyroid carcinomas in castrated and ovariectomized rats. Increase of mean serum thyroid stimulating hormone levels and thyroid and pituitary weights was also predominant in females. Mean thyroid stimulating hormone levels of both sexes were decreased by gonadectomy. Mean thyroid and pituitary weights were inhibited from increasing not by castration but by ovariectomy. Estradiol supplemented after gonadectomy significantly increased all of these factors. Estrogen receptors were detected in transplanted thyroid tumors but not in euthyroid tissues. The results suggest that estradiol promoted the thyroid tumorigenesis through activation of thyrotrophs in pituitary or direct interaction of estradiol and estrogen receptors in the thyroid.     

Opposite effects of dehydroepiandrosterone on the growth of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas.

The effect of dehydroepiandrosterone (DHEA) (2 mg, twice daily p.o.) on the growth of the dimethylbenz (a) anthracene (DMBA)-induced mammary carcinoma was studied in intact and ovariectomized adult female rats. DHEA treatment stimulated the tumor growth in ovariectomized animals. Conversely, the tumors of intact rats treated with DHEA progressed to a lesser extent than those of intact untreated animals (p < 0.01). Plasma levels of DHEA were higher in DHEA-fed than in untreated animals (p < .01), whereas E2 concentrations were unchanged after DHEA administration. Estrogen receptor (ER) concentrations in tumor tissue of ovariectomized animals given DHEA were no different form those found in intact rats, whereas ER were undetectable in untreated ovariectomized rats. The data indicate that DHEA stimulates the growth of DMBA-induced mammary tumors in ovariectomized rats, while it reduces the tumor progression in intact animals.     

Hormone-dependent growth of a rat chondrosarcoma in vivo.

The importance of various hormonal factors in the growth of a transplantable chondrosarcoma has been studied in vivo. Tumor growth was reduced by 95% in adrenalectomized or hypophysectomized rats as compared to normal animals. The number of tumors developing in either adrenalectomized or hypophysectomized rats was reduced more in male than in female rats. However, ovariectomy or orchiectomy did not alter the growth of the tumor. The inhibition of tumor growth in adrenalectomized and hypophysectomized animals was only observed after the first 10 days following inoculation. Cortisone (4-pregnen-17 alpha,21-diol-3,11,20-trione) administration fully restored tumor growth in adrenalectomized animals while adrenocorticotropic hormone or growth hormone were only partially effective in supporting tumor growth in hypophysectomized animals. High-affinity glucocorticoid receptors (7 to 10S) were present in the cytosls prepared from the tumor cells and were found to be increased in tumors from adrenalectomized animals. These results indicate that the growth of this chondrosarcoma is strongly dependent upon endocrine factors of adrenal and pituitary origin.     

Comparison of the systemic and intratumoral effects of tamoxifen and the aromatase inhibitor vorozole in postmenopausal patients with primary breast cancer.

PURPOSE: To determine biologic differences, if any, between presurgical endocrine treatment with an aromatase inhibitor (vorozole) and tamoxifen in patients with postmenopausal primary breast cancer. PATIENTS AND METHODS: Randomization was to 12 weeks of 2.5 mg of vorozole per day or 20 mg of tamoxifen per day, both orally. Clinical response was assessed monthly together with serum sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogens (E1, E2, and E1S), lipids, insulin-like growth factor-1 (IGF-1), and bone metabolites (CrossLaps CTx). Tissue samples for Ki67, apoptotic index (AI), estrogen receptor, and progesterone receptor were collected at 0, 2, and 12 weeks. RESULTS: Ki67 fell by 58% and 43% (means) at 2 weeks in the vorozole and tamoxifen patients, respectively (P =.13). In the vorozole group, the correlations of proportional changes in Ki67 at 2 weeks with tumor volume changes and clinical response at 12 weeks were not significant (P =.09) and marginally significant (P =.04), respectively. Serum lipids did not differ between groups. Serum levels of EI, E2, and E1S were suppressed markedly by vorozole, whereas levels of SHBG increased and LH and FSH fell significantly with tamoxifen. IGF-1 levels fell significantly with tamoxifen (P =.001) compared with the nonsignificant rise with vorozole. Twelve-week CTx values fell by 19% with tamoxifen (P =.006) and rose by 11% with vorozole (P =.15). CONCLUSION: The correlation with vorozole of Ki67 with volume and clinical response supports this as an intermediate marker. The nonsignificant effects on bone and lipid metabolism by the aromatase inhibitor may be important to consider for adjuvant and potential prevention strategies.     

Elevated cyclic AMP levels in human breast-cancer tissue

This study reports a direct action of 17-beta-estradiol on protein synthesis by 7, 12-dimethylbenz (alpha) anthracene (DMBA) induced rat mammary tumors. Sprague-Dawley female rats were given 20 mg of DMBA in sesame oil by gastric tube. Mammary tumors developed. When tumors reached 1.5 to 2 cm in diameter, animals were ovariectomized. At 4-7 days later animals were killed and tumors removed. Microscopic examination confirmed the tumors to be carcinoma of adenoid cystic variety with regressive changes. 14 tumors from 14 animals were found suitable for study. In vitro treatment with 17-beta-estradiol gave rise to a 7% increase in the rate of 3H-leucine incorporation, expressed as dpm/mg of TCA-insoluble protein. This increase was considered statistically significant (p.001). A large variation among different tumors was noted. Also results varied with differences in time after ovariectomy. Under the same incubation conditions the same percentage of increase in the rate of 3H-leucine incorporation had been observed in the study of the effect of estrogen on the uterus of ovariectomized rats. In other mammalian tissues studied those containing high levels of estrogen receptor were able to respond to direct stimulation of estrogen. It was concluded that estrogen directly stimulates protein synthesis in the mammary tumors. This supports the view that these tumors are estrogen responsive tissues.     

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.     

A new irreversible aromatase inhibitor, 6-methylenandrosta-1,4-diene-3,17-dione (FCE 24304): antitumor activity and endocrine effects in rats with DMBA-induced mammary tumors

Summary The antitumor activity of the new irreversible aromatase inhibitor 6-methylenandrosta-1,4-diene-3,17-dione (FCE 24304) was studied in rats with 7,12-dimethylbenzanthracene (DMBA)-induced tumors; several endocrine parameters were evaluated in these animals. The compound was given s.c. and p.o. twice daily, 6 days/week, for 4 weeks. The control group showed 13% tumor regressions (0% complete remission, CR; 13% partial remission, PR). FCE 24304 given s.c. induced 44% (22+22) regressions at the dose of 3 mg/kg per day, 70% (40+30) at 10 mg/kg per day, 73% (27+46) at 30 mg/kg per day, and 70% (50+20) at 100 mg/kg per day. FCE 24304 given orally induced 25% (17+8) tumor regressions at 30 mg/kg per day and 50% (17+33) at 100 mg/kg per day. Rats were killed 4 h after the last dose and the aromatase activity of ovarian microsomes (OAA) was evaluated. OAA was reduced by 56% after s.c. treatment with 3 mg/kg per day FCE 24304; complete OAA suppression (96%) was obtained starting at 10 mg/kg per day s.c. Oral treatment slightly reduced OAA only at a dose of 100 mg/kg per day (36%). Body weight increased in all the groups s.c. treated with FCE 24304 but not in those treated orally. The weights of the pituitary, adrenals, and uterus were reduced in rats treated s.c. with 10 and 30 mg/kg per day; at 100 mg/kg per day, a decrease in ovarian weight was observed while uterus weight was similar to that of controls. Oral FCE 24304 increased ovarian weight at a dose of 30 mg/kg per day but not at 100 mg/kg per day. Serum prolactin (PRL) and luteinizing hormone (LH) levels did not change. In conclusion, FCE 24304 given s.c. proved highly effective against DMBA-induced tumors in rats but had less activity when given orally. Its intrinsic androgenic activity, higher after s.c. than after oral treatment, could contribute to the antitumor effect in the intact (premenopausal) rat model.     

Inhibited growth in vivo of a mouse pregnancy-dependent mammary tumor (TPDMT-4) by an antiestrogen, 2alpha, 3alpha-epithio-5alpha-androstan-17beta-ol (10275-S).

TPDMT-4 mammary tumors, characterized by growth during pregnancy and postpartum regression, grew continuously in DDD virgin mice carrying pituitary isografts or 17 beta-estradiol plus progesterone pellets. In the experimental models, effects of 2alpha,3alpha-epithio-5alpha-androstan-17beta-ol(10275-S), testosterone, and ovariectomy were investigated. When tumors implanted with pituitary isografts into the fat pad reached palpable volumes, animals were ovariectomized or received 5 s.c. injections of 100 mug to 1 mg 10275-S or 300 mug testosterone weekly. Tumors regressed immediately after ovariectomy or after the start of 10275-S treatment and after approximately the 10th day of testosterone treatment. Cystadenomatous morphology with strong secretory activity was noticed in treated animals. Tumor growth induced by 17beta-estradiol plus progesterone pellets was inhibited completely or partially by 3 s.c. injections of 50 to 500 mug 10275-S or 100 mug testosterone weekly starting from the day after tumor and 17beta-estradiol plus progesterone pellet implantation.     

Effect of D,L-2-difluoromethylornithine and endocrine manipulation on the induction of mammary carcinogenesis by 1-methyl-1-nitrosourea

The purpose of this investigation was to establish an efficientroute and dose regime for the long-term administration of tamoxifenin the study of mammary tumorigenesis in the rat. The secondobjective of this work was to determine whether treatment withD, L-2-difluoromethylornithine (DFMO), a synthetic inhibitorof the enzyme ornithine decarboxylase, would reduce the occurrenceof mammary cancers in tamoxi-fen-treated or ovariectomized rats.A total of 265 female Sprague-Dawley rats were assigned to oneof two experimental protocols. All animals were injected with50 mg 1-methyl-1-nitrosourea (MNU) per kg body wt at 50 daysof age. In experiment 1, beginning 7 days after the injectionof the carcinogen, animals were assigned to one of six groupswhich received either 0, 1 or 5 mg tamoxifen citrate per kgAIN-76A purified diet in addition to either no DFMO or a 0.125%w/v solution of DFMO as the drinking water. The experiment wasterminated 180 days following carcinogen treatment. Treatmentwith tamoxifen resulted in a dose-dependent reduction in cancerincidence, and the number of cancers induced and significantlyprolonged the median cancer-free time. This effect was alsoaccompanied by a decrease in the rate of body weight gain. Treatmentwith DFMO delayed latency and reduced tumor number. DFMO inaddition to tamoxifen (1 mg/kg diet) further prolonged latency.In experiment 2 each animal was assigned to one or four treatmentgroups when its first palpable mammary tumor was detected. Atthat time each was either ovariectomized or sham-operated. Inaddition, the rats were either provided no DFMO or a 0.5% w/vsolution of DFMO as the drinking water. The study was terminated35 weeks following carcinogen injection. Ovariec-tomy significantlyinhibited the occurrence of additional mammary tumors. Ovariectomyplus DFMO was more effective than ovariectomy alone in reducingtumor number. Collectively, these observations indicate thatsuppression of polyamine biosynthesis via the systemic administrationof DFMO inhibits the development of ovarian hormone insensitivemammary tumors.     

Prevention of age-related spontaneous mammary tumors in outbred rats by late ovariectomy

Background: Breast cancer prevention trials have shown that the antiestrogen tamoxifen inhibits development of estrogen receptor (ER)-positive tumors. In Sprague-Dawley rats, removal of ovarian function in young animals can reduce the incidence of spontaneous age-dependent mammary tumors. However, it is not known whether removal of ovaries late in life, before middle age onset, can still prevent mammary tumor development. Methods: In this study we used Hsd:Sprague-Dawley^® SD^® (Hsd) rats to determine the effect of late ovariectomy on mammary tumor development. Intact, sham-ovariectomized and ovariectomized rats were followed until 110 weeks of age, or over their life span. In some experiments, palpable tumors were surgically removed upon presentation. Results: Removal of ovaries before middle age onset (∼5-7 months) inhibited development of spontaneous mammary tumors by 95%. Only one mammary tumor was observed in 19 late ovariectomized animals while 47 total tumors developed in 42 non-ovariectomized animals. Tumor incidence was reduced from 73.8 to 5.3% (relative risk = 0.05, 95% CI = 0.0072-0.354). The frequency of mammary carcinomas in non-ovariectomized virgin female rats was one in eight rats. Spontaneous rat carcinomas expressed ER and other biomarkers, such as cyclin D1. When palpable tumors were removed by surgical excision, tumor multiplicity increased from 0.76 to 1.61 tumors per rat. Surprisingly, ovariectomy increased the 110-week survival rate and maximum life span of Hsd rats. Conclusion: Late ovariectomy prevents spontaneous mammary tumor development in Hsd rats. This animal model may be useful for evaluating novel interventions in breast cancer prevention.     

Role of tamoxifen in the induction of hormone-independent rat mammary tumors.

Using the dimethylbenzanthracene-induced rat mammary tumor model, we examined the appearance and growth of tumors in animals given tamoxifen (TAM) either coincident with dimethylbenzanthracene or after initial tumor formation. While fewer tumors arose after coadministration of antiestrogen and carcinogen and TAM treatment caused regression of most existing tumors, new tumors developed in the presence of TAM in both studies. Since none of these new tumors regressed following ovariectomy, all were classified as hormone independent. Furthermore, these independent tumors grew more rapidly than both control-dependent and independent tumors, resulting in a greater average volume. These data suggest that a more aggressive form of hormone-independent tumor appears during TAM treatment.