Inhibition Of Mammary Tumorigenesis Research Articles

Sulforaphene Interferes with Human Breast Cancer Cell Migration and Invasion through Inhibition of Hedgehog Signaling.

Although inhibition of mammary tumorigenesis by isothiocyanates has been widely studied, little is known about the effects of sulforaphene on invasiveness of breast cancer. Here, sulforaphene significantly inhibited the migration and invasion of triple-negative SUM159 human breast cancer cells and suppressed the expression and activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). The Hedgehog (Hh) pathway, as an upstream signaling modulator, was significantly suppressed by sulforaphene. In particular, ciliary localization of Gli1 and its nuclear translocation were blocked by sulforaphene in a time-dependent manner. Consistently, downregulation of Hh signaling by vismodegib and Gli1 knockdown reduced the cellular migration and invasion as well as the expression of MMP-2 and MMP-9. These results indicate that the suppression of Hh/Gli1 signaling by sulforaphene may reduce the MMP-2 and MMP-9 activities and cellular invasiveness of human breast cancer cells, suggesting the potential efficacy of sulforaphene against breast cancer invasion and metastasis.

Abstract 153: Honokiol, a promising small molecular weight natural agent, activates AMP-activated protein kinase in breast cancer cells via LKB1-dependent pathway and inhibits breast carcinogenesis

Abstract Introduction: Research over the past few years have established LKB1-AMPK pathway as an important metabolic checkpoint interconnecting the signaling pathways controlling metabolism and cancer cell growth. Thus, it is of major interest to find effective therapeutic strategies to manipulate LKB1-AMPK axis to inhibit cancer growth. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer preventive as well as therapeutic properties. Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an anti-inflammatory, anti-thrombosis, anti-oxidant agent and more recently for its protective function in carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in modulating LKB1-AMPK axis, preventing mammary tumorigenesis and inhibiting metastasis. Methods: Clonogenicity, 3D-colony formation, matrigel invasion, scratch-migration and spheroid-migration assays were used to examine breast cancer cell growth and metastatic potential upon honokiol treatment. Western blot and immunofluorescence analysis were used to examine activation of LKB1-AMPK axis. Functional importance of AMPK and LKB1 was examined by using AMPK-null and AMPK-WT immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Breast cancer xenografts, immunohistochemical and western blot analysis of tumors were used. Results: Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Using AMPK-null and AMPK-WT (MEFs); we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor suppressor gene LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and more importantly, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increase in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors. Conclusions: Taken together, these data providing first in vitro and in vivo evidence of the efficacy of honokiol in activating LKB1-AMPK axis resulting in effective inhibition of mammary tumorigenesis and metastasis provide impetus to determine its activity in clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 153. doi:1538-7445.AM2012-153

Inhibition of mammary tumorigenesis in the C3(1)/SV40 mouse model by green tea

Previous studies show inhibitory effects of green tea in chemically induced mammary tumors or human tumor explants, but not in spontaneous tumor models that are more representative of human breast cancer. The C3(1)/SV40 mouse model is particularly suited for breast cancer prevention studies because it produces spontaneous ductal adenocarcinomas and a predictable time course for mammary tumorigenesis through a multistage progression similar to that occurring in humans. We therefore used this model to test the chemoprotective effects of green tea. Administration of 0.5% Polyphenon E (Poly E) (a standardized preparation of green tea extract) in drinking water delayed tumor onset and suppressed tumor growth by 40%, compared to tap water-fed animals, with no adverse side effects. Histological analysis of mammary glands showed that green tea slowed the progression of ductal lesions to advanced mammary intraepithelial neoplasias and suppressed tumor invasiveness. Green tea inhibited the proliferation of ductal epithelial cells and tumors and, overall, disrupted post-pubertal ductal growth. Immunohistochemical analyses also demonstrated that green tea inhibited angiogenesis through a decrease in both ductal epithelial and stromal VEGF expression and a decrease in intratumoral microvascular density. Our data strongly support the potential use of green tea as a breast cancer chemopreventive agent.

Osteopontin is a potential target gene in mouse mammary cancer chemoprevention by Se-methylselenocysteine.

BackgroundSe-methylselenocysteine (MSC) is a naturally occurring organoselenium compound that inhibits mammary tumorigenesis in laboratory animals and in cell culture models. Previously we have documented that MSC inhibits DNA synthesis, total protein kinase C and cyclin-dependent kinase 2 kinase activities, leading to prolonged S-phase arrest and elevation of growth-arrested DNA damage genes, followed by caspase activation and apoptosis in a synchronized TM6 mouse mammary tumor model. The aim of the present study was to examine the efficacy of MSC against TM6 mouse mammary hyperplastic outgrowth (TM6-HOG) and to determine in vivo targets of MSC in this model system.MethodsTwenty mammary fat pads each from female Balb/c mice transplanted with TM6-HOG and fed with 0.1 ppm selenium and with 3 ppm selenium respectively, were evaluated at 4 and 12 weeks after transplantation for growth spread, proliferative index and caspase-3 activity. Thirteen mice transplanted with TM6-HOG in each selenium group were observed for tumor formation over 23 weeks. Tumors from mice in both groups were compared by cDNA array analysis and data were confirmed by reverse transcription–polymerase chain reaction. To determine the effect of MSC on the expression of the novel target gene and on cell migration, experiments were performed in triplicate.ResultsA dietary dose of 3 ppm selenium significantly reduced the growth spread and induced caspase-3 activity in mammary fat pads in comparison with mice fed with the basal diet (0.1 ppm selenium). The extended administration (23 weeks) of 3 ppm selenium in the diet resulted in a tumor incidence of 77% in comparison with 100% tumor incidence in 0.1 ppm selenium-fed animals. The size of TM6 tumors in the supplemented group was smaller (mean 0.69 cm2) than in the mice fed with the basal diet (mean 0.93 cm2). cDNA array analysis showed a reduced expression of osteopontin (OPN) in mammary tumors of mice fed with the 3 ppm selenium diet in comparison with OPN expression in tumors arising in 0.1 ppm selenium-fed mice. A 24-hour treatment of TM6 cells with MSC significantly inhibited their migration and also reduced their OPN expression in comparison with untreated cells.ConclusionsOPN is a potential target gene in the inhibition of mammary tumorigenesis by selenium.

Oxidations of 17β-estradiol and estrone and their interconversions catalyzed by liver, mammary gland and mammary tumor after acute and chronic treatment of rats with indole-3-carbinol or β-naphthoflavone

Altered cytochrome P450-catalyzed metabolism of 17β-estradiol (E2) and estrone (E1) in the liver and (or) extrahepatic tissues may affect estrogen-sensitive tumorigenesis. We examined the effects of oral treatments of (i) indole-3-carbinol (I3C) at 250 or 500 mg/kg or β-naphthoflavone (β-NF) at 40 mg/kg of body weight (bw)/day from 51 to 54 days of age (acute regimen), and (ii) I3C at 250 mg/kg or β-NF at 20 mg/kg bw given 3x/week from 10 to 22 weeks of age (chronic regimen) in female Sprague-Dawley rats. We determined the effects of these treatments on the P450 content and P450 (CYP)-specific activities in the liver, P450-dependent metabolism of E2 and E1 by the liver and mammary gland, and interconversion of E1 and E2 catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD) in these tissues and malignant mammary tumors. I3C at the two levels of acute regimen elicited similar responses. Acute and chronic treatments with I3C, but not β-NF, increased P450 content ~2-fold. I3C, and to a lesser extent β-NF, increased CYP1A1 and CYP1A2 probe activities in liver up to 117- and 27- fold, respectively, and after acute regimens, that of CYP3A by ~1.8-fold. I3C also increased activity of CYP2B up to 100-fold. Overall hepatic metabolism of E2 and E1, which was ~2-fold greater at 55 than 155 days of age, was increased (~2.8-fold) by I3C with 2-, 4-, 16α-, 6α-, 6β-, and 15α-hydroxy (OH) comprising [Formula: see text]54, 3, 2, ~2, ~5, 7, and 2%, respectively, of E1 and E2 metabolites. Acute regimens of β-NF increased 2- and 15α-OH-E2 (62 and 5% of total) from E2 and 2-, 4-, and 6α-OH-E1 + 6β-OH-E1 (32, 13, and 4% of total) from E1. Mammary gland metabolized E2 to E1 and small amounts of 15α-, 4-, 16α-, 6β-, and 6α-OH-E2. After the acute IC3 regimen, E2 was also converted to 2-OH-E2. 17β-HSD-catalyzed oxidation of E2 was favored in the liver and reduction of E1 was favored in mammary gland and tumor (= 1% of hepatic activity). An increased (~2-fold) ratio of reductive to oxidative activities in malignant mammary tumors by chronic I3C regimen may stimulate tumor growth. This is the first report showing that after chronic oral regimens, the I3C-, but not β-NF-, induced changes in CYP complement led to elevated E2 and E1 metabolism. The persistent effects of increased putative carcinogenic and estrogenic 4- and 16α-OH as well as 6α- and 6β-OH-E2 and 6β-OH-E1 might counteract those of the less estrogenic 2-OH metabolites, thus accounting for the lack of suppression of mammary tumorigenesis by I3C in our previous study.Key words: estrogen metabolism, P450, 17β-hydroxysteroid dehydrogenase, indole-3-carbinol, β-naphthoflavone.

Oxidations of 17β-estradiol and estrone and their interconversions catalyzed by liver, mammary gland and mammary tumor after acute and chronic treatment of rats with indole-3-carbinol or β-naphthoflavone

Altered cytochrome P450-catalyzed metabolism of 17beta-estradiol (E2) and estrone (E1) in the liver and (or) extrahepatic tissues may affect estrogen-sensitive tumorigenesis. We examined the effects of oral treatments of (i) indole-3-carbinol (13C) at 250 or 500 mg/kg or beta-naphthoflavone (beta-NF) at 40 mg/kg of body weight (bw)/day from 51 to 54 days of age (acute regimen), and (ii) 13C at 250 mg/kg or beta-NF at 20 mg/kg bw given 3x/week from 10 to 22 weeks of age (chronic regimen) in female Sprague-Dawley rats. We determined the effects of these treatments on the P450 content and P450 (CYP)-specific activities in the liver, P450-dependent metabolism of E2 and E1 by the liver and mammary gland, and interconversion of E1 and E2 catalyzed by 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in these tissues and malignant mammary tumors. 13C at the two levels of acute regimen elicited similar responses. Acute and chronic treatments with 13C, but not beta-NF, increased P450 content approximately 2-fold. 13C, and to a lesser extent beta-NF, increased CYP1A1 and CYP1A2 probe activities in liver up to 117- and 27- fold, respectively, and after acute regimens, that of CYP3A by approximately 1.8-fold. 13C also increased activity of CYP2B up to 100-fold. Overall hepatic metabolism of E2 and E1, which was approximately 2-fold greater at 55 than 155 days of age, was increased (approximately 2.8-fold) by 13C with 2-, 4-, 16alpha-, 6alpha-, 6beta-, and 15alpha-hydroxy (OH) comprising > or = 54, 3, 2, approximately 2, approximately 5, 7, and 2%, respectively, of E1 and E2 metabolites. Acute regimens of beta-NF increased 2- and 15alpha-OH-E2 (62 and 5% of total) from E2 and 2-, 4-, and 6alpha-OH-E1 + 6beta-OH-E1 (32, 13, and 4% of total) from E1. Mammary gland metabolized E2 to E1 and small amounts of 15alpha-, 4-, 16alpha-, 6beta-, and 6alpha-OH-E2. After the acute IC3 regimen, E2 was also converted to 2-OH-E2. 17Beta-HSD-catalyzed oxidation of E2 was favored in the liver and reduction of E1 was favored in mammary gland and tumor (= 1% of hepatic activity). An increased (approximately 2-fold) ratio of reductive to oxidative activities in malignant mammary tumors by chronic 13C regimen may stimulate tumor growth. This is the first report showing that after chronic oral regimens, the 13C-, but not beta-NF-, induced changes in CYP complement led to elevated E2 and E1 metabolism. The persistent effects of increased putative carcinogenic and estrogenic 4- and 16alpha-OH as well as 6alpha- and 6beta-OH-E2 and 6beta-OH-E1 might counteract those of the less estrogenic 2-OH metabolites, thus accounting for the lack of suppression of mammary tumorigenesis by 13C in our previous study.

TGF-beta and functional differentiation.

A review of the pertinent literature suggests that TGF-beta 1 may play a multifaceted role in functional differentiation of mammary epithelium. Evidence for the expression of TGF-beta 1 RNA and the presence of functional TGF-beta 1 protein in differentiating mammary epithelial cells from a pregnant mouse has been recently reported. The specific role of mammary-epithelial-cell-produced TGF-beta 1 in the differentiating mammary gland is presently unclear. However, several possible functions are suggested from the following observations. Milk protein production is negatively regulated by exogenous TGF-beta 1 during gestational development of the gland but not during lactation. Consistent with reports linking TGF-beta 1 gene expression with mammary gland involution following lactation, overexpression of TGF-beta 1 in the differentiating secretory epithelium leads to premature programmed cell death in the absence of a negative effect on secretory epithelial cell proliferation. A role for TGF-beta 1 in cell cycle control and suppression of malignant progression independent from its inhibitory effect on epithelial cell growth has been demonstrated in keratinocytes. A similar function could provide protection against malignancy in proliferating mammary epithelium and account for TGF-beta 1 suppression of mammary tumorigenesis in transgenic mice overexpressing transforming growth factor alpha (TGF-alpha).

Inhibition by butylated hydroxytoluene and its oxidative metabolites of DMBA-induced mammary tumorigenesis and of mammary DMBA-DNA adduct formation in vivo in the female rat

The phenolic food antioxidant butylated hydroxytoluene (BHT) has been reported to inhibit the initiation stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in the female rat. However, the mechanism for this antitumorigenic effect of BHT is unknown. The present studies were conducted to evaluate the relative effect of the parent chemical BHT and two of its major oxidative metabolites, 2,6-di- tert-butyl-4-hydroxymethylphenol (BHT-BzOH) and 2,6-di- tert-butyl-1,4-benzoquinone (BHT-quinone), on DMBA-induced rat mammary tumorigenesis and on the formation of rat mammary DMBA-DNA adducts in vivo. The ip administration of either BHT or BHT-quinone at 200 mg/kg body weight for 2wk before until 1 wk after DMBA administration inhibited the development of mammary tumours as compared with controls. The extent of tumour inhibition by BHT (39%) was greater than that exhibited by BHT-quinone (25%). The administration of BHT-BzOH at 200 mg/kg body weight did not inhibit mammary tumorigenesis. Thus, the inhibition of DMBA-induced mammary tumorigenesis by BHT does not appear to be mediated by the oxidative BHT metabolites BHT-BzOH or BHT-quinone. In addition, there was a good quantitative correlation between the inhibition of mammary tumorigenesis by BHT and BHT-quinone and their respective abilities to decrease total binding in vivo of DMBA to mammary DNA. The inhibition of specific mammary DMBA-DNA adducts by BHT was not identical to the inhibition of adducts by BHT-quinone. However, the decrease in formation of the major mammary adduct derived from the anti-dihydrodiolepoxide of DMBA bound to deoxyguanosine most closely correlated to the relative abilities of BHT and BHT-quinone to inhibit mammary tumorigenesis. When mammary adduct formation was examined in response to BHT dose, the administration of BHT at doses of 100 mg/kg body weight and 200 mg/kg body weight resulted in the inhibition of anti-derived but not syn-derived mammary DMBA-DNA adducts. Together, these studies suggest that in addition to the inhibition of total mammary DMBA-DNA adduct formation, the inhibition of mammary DNA adducts formed from the anti-dihydrodiolepoxide of DMBA also may be specifically important in the inhibitory effect of BHT on DMBA-induced mammary tumorigenesis.

Inhibition of mammary tumorigenesis in virgin rats by adoptive transfer of splenocytes from parous donors.

Splenocytes from parous rats have been previously found to have cytotoxic activity against mammary tumor cells in vitro. Experiments were carried out to determine if this pregnancy-induced cytotoxic nature of the splenocytes is inherent and transferable. Splenocytes from parous rats wer adoptively transferred to a group of virgin rats. Another group of age-matched, virgin rats received splenocytes from virgin donors in a similar way. After a period of rest, at the age of 55 days, the rats belonging to both of the groups, received 7,12-dimethylbenz(a)anthracene (DMBA) intragastrically. A third group of untreated virgin rats were also given the chemical carcinogen the same way as above and were considered as intact controls. The rats were monitored for development and growth of mammary tumor from 60 days of DMBA administration. After 4 months of DMBA administration the rats were sacrificed and mammary glands were examined for tumors. Mammary glands with no visible tumors were taken for whole mount preparation, to be examined for microscopic lesions. The results showed that 33 of 41 intact control rats, developed tumor and 27 of the 34 rats that received spleen cells from virgin rats developed tumors. Of the rats that received spleen cells from parous rats, only 18 out of 37 rats developed tumors, indicating an inhibition of tumor induction in these rats. Growth rate of the tumors in this group was also slower than in the control groups.

On the Mechanisms of Retardation of Aging and Inhibition of Mammary Tumorigenesis by Energy Restriction in SHN/C3H F1 Female Mice.

Although it has long been known that energy restriction (ER) inhibits tumors and retards aging in rats and mice, its mode of action remains unknown. In rodents, ER alters the rate of age-related changes in physiological indices. Thus, it affects a broad array of age-sensitive parameters. However, present evidence does not indicate which parameters are primary contributors to the deceleration of aging. Compared to fasting or short-term underfeeding, little is known about the metabolic effects of long-term, life-prolonging ER. We thus investigated the effects of ER on hepatic enzyme activities, including drug-metabolizing enzymes and antioxidant enzymes such as superoxide dismutase and catalase. The catalase activity was found to be higher in ER mice than in control mice both at 12 and 24 months of age. In accord with the high catalase activity, lipid peroxidation in liver was much less in ER mice than in age matched control mice. beta-Naphthoflavone, known to induce P-450 related enzymes and xanthine oxidase, was given (ip) to increase lipid peroxidation. The ER was found to inhibit lipid peroxidation after beta-naphthoflavone treatment. It was, therefore, concluded that long-term life-prolonging ER increases antioxidant defence, supporting indirectly the free radical theory of aging. It is well known that ER delays puberty in rodents and has a profound influence on serum hormone levels, including those of prolactin (PRL) and thyroid hormones. However, it remains unknown how these effects are produced by ER. We therefore investigated the effects of ER on the islets of Langerhans in the pancreas and on the pituitary-ovarian axis. In the islets of Langerhans, ER was found to increase the density of alpha-cells significantly both in 11- and 67-week-old mice. In the pituitary gland in ER mice, the cellular density of PRL-producing cells diminished significantly while that of growth-hormone-producing cells did not. One of the modes of action of ER on the endocrine system is thus concluded to be mediated by changing cellular population. Since ER decreased PRL-producing cells and PRL plays a key role in mammary tumorigenesis, we investigated whether ER decreased the gene expression of mouse mammary tumor virus (MMTV) in SHN/C3H mice. The SHN strain, which was found to have a new MMTV provirus locus, mtv-4, was used in the study.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolactin effects on the dietary regulation of mouse mammary tumor virus proviral DNA expression.

Chronic energy-intake restriction inhibits mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/Ou mice by greater than 90%. We have shown that associated with suppression of mammary tumorigenesis there is a reduction or inhibition of circulating prolactin, MMTV particles expressed, and MMTV mRNA transcription in mammary glands (and in most organs tested). To understand the concerted action of prolactin, energy-consumption level, and MMTV on inducing mammary tumors, experiments were designed to control prolactin and energy levels in order to evaluate their effects on MMTV mRNA expression. Mice on restricted diets were grafted with adenohypophyses, and mice fed ad libitum were treated with the dopaminomimetic agent octahydrobenzo [g]quinoline. Adenohypophyseal grafting significantly increased prolactin in dietary (energy)-restricted mice, and this effect was associated with an increase in MMTV mRNA expression within the mammary gland; a linear correlation between prolactin levels and MMTV mRNA expression in the mammary gland was found. Conversely, elimination of the nocturnal peak of circulating prolactin by i.p. injection of dopaminomimetic octahydrobenzo [g]quinoline to mice fed ad libitum delayed (by 8 weeks) and reduced (even as long as 25 weeks) mammary gland MMTV mRNA expression. These findings associate prolactin influences with MMTV mRNA production in mice and help explain the link between chronic energy-intake restriction and reduced MMTV gene expression.

A comparison of the effects of the prostaglandin synthesis inhibitors indomethacin and carprofen on 7,12-dimethylbenz[a]anthracene-induced mammary turmorigenesis in rats fed different amounts of essential fatty acid

The effects of the cyclooxygenase inhibitors indomethacin and carprofen on the enhancement of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis by dietary linoleate have been compared in female Sprague-Dawley rats. Indomethacin and carprofen, 0.004% and 0.02% (w/w) in the diet, respectively, were fed to rats receiving 20% fat diets containing 0.5, 4 or 12% linoleate starting 7 days after administration of 5 mg DMBA i.g. Indomethacin was shown to have a marked inhibitory effect on mammary tumorigenesis in rats fed the 4 and 12% linoleate diets, but did not alter tumorigenesis in rats fed the 0.5% linoleate diet. In contrast, carprofen was not inhibitory in any of these dietary groups, or in a separate experiment in which a 5% fat--3% linoleate diet was fed. The effect of each drug on prostaglandin E2 (PGE2) levels in normal mammary glands enriched in epithelial cells after a 3-week pretreatment with 17 beta-estradiol and progesterone was also investigated. Carprofen was shown to reduce PGE2 levels to a similar or greater extent than indomethacin at each level of linoleate in the diet. These data demonstrate that a reduction in PGE2 synthesis in the mammary epithelium does not correlate with inhibition of mammary tumorigenesis, and that other factors, including possible alterations in other products of the arachidonic acid cascade, are responsible for this inhibitory effect.

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

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

Selenium inhibition of the neoplastic transformation in preneoplastic mammary cell populations

The effect of dietary selenium on the tumor-producing capabilities of primary hyperplastic alveolar nodules (HAN) of the mammary gland of BALB/ c mice was examined directly using a transplantation assay. The tumor-producing capabilities of primary HAN exposed to 2.0 ppm dietary selenium was decreased 57% compared to the tumor-producing capabilities of HAN exposed to 0.2 ppm dietary selenium. This degree of inhibition compared well with the 60% inhibition of mammary tumorigenesis when the mice were exposed to supplemental selenium during the entire tumorigenic period. The examination of primary hyperplastic alveolar nodules was a more accurate assessment of the responsiveness of the preneoplastic state than using established nodule outgrowth lines since the latter exhibited a greater degree of variability in response to selenium-mediated inhibition of growth and the mean response of numerous nodule outgrowth lines was not as great as seen in the primary HAN. These results indicate that selenium is an effective inhibitor of the neoplastic transformation in preneoplastic cells as well as the formation of preneoplastic cells.

Inhibition of mammary tumorigenesis in nulliparous C3H mice by chronic feeding of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide.

Feeding of N-(hydroxyphenyl)retinamide (4-HPR) (1.0 mM/kg diet) for 39 weeks to 2 month old nulliparous C3H/Crgl mice significantly (p less than 0.01) reduced the incidence of mammary tumors; 98 control mice developed a total of 235 mammary tumors, 100 4-HPR treated mice developed 171 mammary tumors. Feeding the same dietary level of 4-HPR to 4-6 month old multiparous C3H mice for 14 weeks did not significantly influence the development of mammary tumors; 78 control mice developed a total of 115 mammary tumors; 78 4-HPR treated mice developed 130 mammary tumors. Body weight gains were comparable among 4-HPR treated mice and control mice. Significant suppression of mouse mammary gland tumorigenesis in vivo by dietary retinoids (nulliparous C3H mice) has heretofore not been reported.

Inhibition of mammary tumorigenesis in GR mice with 2‐bromo‐α‐ergocryptine

A high incidence of mammary tumors is found in multiparous GR mice during the 2nd and 3rd pregnancies and in nulliparous GR mice treated with estrone/progesterone. The purpose of this study was to determine if prolactin is a contributing hormone in the genesis of these neoplasms. In one series of experiments, 238 15-week-old nulliparous GR mice were treated with estrone (drinking water, 0.5 mg/liter) plus progesterone (30 mg progesterone pellet with cholesterol, implanted SC once monthly) for a period of 13 weeks. Half of these mice were injected SC once daily with 100 micrograms of the prolactin-suppressing drug 2-bromo-alpha-ergocryptine (CB-154) for the duration of hormone treatment, and the other half were injected SC once daily with 0.9% NaCl solution (controls). In another series of experiments, 87 pregnant GR mice were divided into two groups and injected SC once daily from day 7 to 21 of pregnancy with 0.9% NaCl solution (controls) or CB-154 (100 micrograms/mouse). In the first series, the numbers of mice with mammary tumors and total number of mammary tumors were: controls, 58/119 (49%) and 73; CB-154 treatment, 34/119 (29%) and 37, respectively. In the second series, the numbers were: controls, 39/44 (89%) and 73; CB-154 treatment, 24/43 (56%) and 43, respectively. In both studies, CB-154 treatment significantly (p less than 0.05-0.005) reduced the percentage of mice with mammary tumors and total number of mammary tumors. These results provide evidence that prolactin is a contributing hormone in the genesis of estrone/progesterone and pregnancy-induced mammary tumors in female GR mice.

Suppression of Mammary Tumorigenesis in C3H/He Mice by Ovariectomy or Treatment With 2-Bromo- α -ergocryptine: A Comparison 2

We compared the effects of chronic treatment with 2-Br-alpha-ergocryptine (CB-154) and of ovariectomy on the genesis of mammary hyperplastic nodules (HN) and mammary tumors in the C3H/He mouse. CB-154 is an established potent suppressor of pituitary prolactin. Eighty-five 21- to 25-day-old female mice were divided into three groups. Group I received daily sc injections of 0.1 mg CB-154. Group II was ovariectomized and group III served as controls. Mice were examined weekly for mammary tumors. Fifteen months from onset of treatment all surviving mice were killed. The mean number of HN per inguinal mammary gland, total number of mammary tumors, and number (percent) of mice with mammary tumors in each group were, respectively: controls--4.6 +/- 1.1, 41, 24/29 (83%); ovariectomized--1.2 +/- 0.6, 20, 14/28 (50%); and CB-154-treated--0.2 +/- 0.1, 16, 13/28 (46%). A significant (P less than 0.001) and equally comparable inhibition of mammary tumor incidence was observed in the ovariectomized and CB-154-treated mice. Thus early ovariectomy and CB-154 treatment (specific inhibition of prolactin secretion) appeared equally effective in the prophylaxis of mammary tumorigenesis in the C3H/He female mouse.

Inhibition of Mammary Tumorigenesis by Ergot Alkaloids and Promotion of Mammary Tumorigenesis by Pituitary Isografts in Adreno-Ovariectomized Mice

Inhibition of Mammary Tumorigenesis by Ergot Alkaloids and Promotion of Mammary Tumorigenesis by Pituitary Isografts in Adreno-Ovariectomized Mice

Effects of Multiple Pituitary Homografts or Progesterone on 7,12-Dimethylbenz[<italic>a</italic>]anthracene-lnduced Mammary Tumors in Rats<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

This investigation evaluates the effects of prolactin-secreting pituitary homografts and progesterone on the induction of mamary tumors in female rats subsequently treated with 712-dimethylbenz[alpha]anthracene (DMBA). Immature female Sprague-Dawley rats were divided into 5 groups: group 1 intact controls; group 2 ovariectomized; group 3 intact and 4 pituitary homografts; group 4 ovariectomized and 4 pituitary homografts; and group 5 intact and given injection of 4 mg progesterone daily. Pituitary transplantation and/or ovariectomy were performed at age 25 days. Progesterone was injected beginning at 30 days of age and continued for 40 consecutive days. At age 55 days all animals were given a single intravenous dose of 5 mg DMBA. At the end of the study the percent of tumor incidence average number of tumors/rat and average total weight of tumors/rat (g) were: group 1 100% 12.2 +or- 1.2 16.2 +or- 3.5; group 2 0; group 3 73% 4.7 +or- 1.3 5.4 +or- 2.0; group 4 0; and group 5 79% 3.3 +or- 0.7 5.9 +or- 4.6. Percentage of rats with tumors average number of tumors/rat and average total weight of tumors/rat were significantly decreased in intact rats bearing 4 pituitary homografts and in intact rats given injections of progesterone as compared to the intact controls. The inhibitory effect of the prolactin-secreting pituitary homografts appears to be mediated at least in part through the ovary since daily injections of progesterone resulted in a comparable inhibition of mammary tumorigenesis. These data indicate that prolactin and/or progesterone by stimulating growth and development growth of the mammary gland inhibit DMBA-induced mammary tumorigenesis if given sufficiently prior to carcinogen treatment. (authors)