Mammary Cancer Chemoprevention Research Articles

Abstract 830: Pin1 is a novel target of withaferin A, a mammary cancer chemopreventative steroidal lactone from Withania somnifera

Abstract We have shown previously that naturally occurring steroidal lactone withaferin A (WA) inhibits breast cancer development in a transgenic mouse model. Mammary cancer chemoprevention by WA in vivo was associated with suppression of peptidyl-prolyl isomerase Pin1 protein. Pin1 is known to regulate diverse cellular processes including cell proliferation, cell cycle progression, apoptosis, and migration. Moreover, Pin1 is overexpressed in breast tumor and its expression is positively correlated with tumor grade. Thus, we studied functional significance of Pin1 downregulation by WA. Consistent with in vivo observations, Pin1 protein and mRNA expression was decreased upon WA treatment in human breast cancer cells irrespective of ER, HER-2 or p53 status. Overexpression of Pin1 partly reversed the growth inhibitory effect of WA in both MCF-7 (wild-type p53) and SUM159 (mutant p53) cells. However, WA-induced mitotic arrest and apoptosis were partly abrogated only in MCF-7 cells upon ectopic expression of Pin1 perhaps due to p53 stabilization. Molecular docking suggested covalent binding of WA with Cys113 of Pin1, which was confirmed by mass spectrometry. Overexpression of mutant (C113A) Pin1 augmented WA-mediated inhibition of cell survival as well as mitotic arrest and apoptosis in MCF-7 cells indicating that decreased catalytic activity of Pin1 is essential for growth inhibition by WA in breast cancer cells. Taken together, the present study suggests a critical role for Pin1 in WA-mediated growth inhibition of breast cancer. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Suman K. Samanta, Shivendra V. Singh. Pin1 is a novel target of withaferin A, a mammary cancer chemopreventative steroidal lactone from Withania somnifera. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 830.

Inhibition of mitochondrial fusion is an early and critical event in breast cancer cell apoptosis by dietary chemopreventative benzyl isothiocyanate

Benzyl isothiocyanate (BITC) is a highly promising phytochemical abundant in cruciferous vegetables with preclinical evidence of in vivo efficacy against breast cancer in xenograft and transgenic mouse models. Mammary cancer chemoprevention by BITC is associated with apoptotic cell death but the underlying mechanism is not fully understood. Herein, we demonstrate for the first time that altered mitochondrial dynamics is an early and critical event in BITC-induced apoptosis in breast cancer cells. Exposure of MCF-7 and MDA-MB-231 cells to plasma achievable doses of BITC resulted in rapid collapse of mitochondrial filamentous network. BITC treatment also inhibited polyethyleneglycol-induced mitochondrial fusion. In contrast, a normal human mammary epithelial cell line (MCF-10A) that was derived from fibrocystic breast disease, was resistant to BITC-mediated alterations in mitochondrial dynamics as well as apoptosis. Transient or sustained decrease in levels of proteins engaged in regulation of mitochondrial fission and fusion was clearly evident after BITC treatment in both cancer cell lines. A trend for a decrease in the levels of mitochondrial fission- and fusion-related proteins was also observed in vivo in tumors of BITC-treated mice compared with control. Immortalized mouse embryonic fibroblasts from Drp1 knockout mice were resistant to BITC-induced apoptosis when compared with those from wild-type mice. Upon treatment with BITC, Bak dissociated from mitofusin 2 in both MCF-7 and MDA-MB-231 cells suggesting a crucial role for interaction of Bak and mitofusins in BITC-mediated inhibition of fusion and morphological dynamics. In conclusion, the present study provides novel insights into the molecular complexity of BITC-induced cell death.

Abstract 2813: Chemoprevention of mammary cancer: Modeling predictive values of short-term morphologic assays for efficacy in animal tumor assays

Abstract Background: Understanding the ability of chemopreventive agent efficacy in morphologic (in vitro/in vivo) assays to predict efficacy in animal (mouse and rat) in vivo tumor assays has not been well studied. The Chemopreventive Agent Development Research Group (CADRG) in the U.S. NCI's Division of Cancer Prevention has, over 25 years, tested approximately 800 agents for potential chemopreventive activity. Of these agents, a subset of 146 that were tested in both morphologic and mammary gland tumor assays constitutes the focus of our current project. Our goal is to gain deeper insight into the relevant predictive values. Materials and Methods: Two critical steps are involved in the early stages of the CADRG testing pathway: (1) in vitro/in vivo morphologic assays and, for agents successful in these, (2) testing for tumor prevention (measured in terms of tumor incidence and multiplicity reduction) in animal tumor assays. Ultimately, our goal is to test agents that successfully decrease tumor incidence and multiplicity in animal tumor assays in humans. In the project presented here, we evaluated the predictive values of earlier-stage (morphologic) assays for efficacy in later-stage (animal tumor, specifically mammary tumor) assays. We used statistical modeling to determine how well the six most commonly used morphologic assays predicted efficacy of the 146 tested agents in mouse and rat mammary gland tumor assays. For this purpose multimodel inference was applied to ordinal logistic regression. Results: We evaluated the ability of the six morphologic assays to predict tumor outcomes in the mouse and rat mammary gland cancer assays. In our statistical modeling approach, each morphologic assay was assigned a value describing how strongly it predicted outcomes in the mammary gland tumor assays. Specific morphologic assays (mouse mammary organ culture/MMOC and human foreskin epithelial cell/HFE morphologic assays) in combination yielded predictive values that are reasonably suggestive of the efficacy demonstrated by chemopreventive agents in the mouse and rat mammary gland tumor assays. Conclusions: Predictive models such as these should prove useful in guiding future decision-making regarding agent selection and morphologic and animal tumor assay use. Our current strategy is to gain insight into the Predictive Value approach by: (1) identifying the mammary gland tumor assays that best reflect anti-tumor efficacy in animals; (2) teasing apart those classes of agents that exhibit the highest predictive values overall; and (3) examining those classes of agents that exhibit the highest efficacy in specific mammary gland tumor assays. Citation Format: Barbara K. Dunn, Vernon E. Steele, Richard M. Fagerstrom, Carol F. Topp, Barnett S. Kramer. Chemoprevention of mammary cancer: Modeling predictive values of short-term morphologic assays for efficacy in animal tumor assays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2813. doi:10.1158/1538-7445.AM2015-2813

Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells.

Current dogma favors elimination of therapy-resistant cancer stem cells for chemoprevention of breast cancer. We showed recently that mammary cancer development in a transgenic mouse model (mouse mammary tumor virus-neu; MMTV-neu) was inhibited significantly upon treatment with withaferin A (WA), a steroidal lactone derived from a medicinal plant. Herein, we demonstrate that the mammary cancer prevention by WA is accompanied by in vivo suppression of breast cancer stem cells (bCSC). In vitro mammosphere formation was dose-dependently inhibited by WA treatment in MCF-7 and SUM159 human breast cancer cells. Other markers of bCSC, including aldehyde dehydrogenase 1 (ALDH1) activity and CD44(high)/CD24(low)/epithelial-specific antigen-positive (ESA+) fraction, were also decreased significantly in the presence of plasma achievable doses of WA. However, WA exposure resulted in cell line-specific changes in Oct4, SOX-2, and Nanog mRNA expression. WA administration to MMTV-neu mice (0.1 mg/mouse, 3 times/week for 28 weeks) resulted in inhibition of mammosphere number and ALDH1 activity in vivo. Mechanistic studies revealed that although urokinase-type plasminogen activator receptor overexpression conferred partial protection against bCSC inhibition by WA, Notch4 was largely dispensable for this response. WA treatment also resulted in sustained (MCF-7) or transient (SUM159) downregulation of Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion region-1) protein. Ectopic expression of Bmi-1 conferred partial but significant protection against ALDH1 activity inhibition by WA. Interestingly, WA treatment caused induction of Kruppel-like factor 4 (KLF4) and its knockdown augmented bCSC inhibition by WA. In conclusion, this study shows in vivo effectiveness of WA against bCSC.

Biomarkers of Phenethyl Isothiocyanate-Mediated Mammary Cancer Chemoprevention in a Clinically Relevant Mouse Model

Phenethyl isothiocyanate (PEITC) is a natural plant compound with chemopreventative potential against some cancers and the ability to induce apoptosis in breast cancer cells. 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. 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 2 mm(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. 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.

Abstract 563: Chemoprevention of mammary cancer in MMTV-neu transgenic mice by dietary administration of cruciferous vegetable constituent phenethyl isothiocyanate

Abstract We have shown previously that phenethyl isothiocyanate (PEITC), which is a highly promising cancer chemopreventive constituent of edible cruciferous vegetables (e.g., watercress), inhibits growth of cultured breast cancer cells in association with apoptosis induction. The present study was undertaken to determine the efficacy of PEITC for prevention of breast cancer using MMTV-neu mouse model. PEITC administration in the diet (3 mmol PEITC/kg AIN76A diet) for 29 weeks caused an approximate 27% decrease in incidence of mammary hyperplasia compared with control, but the difference between control and PEITC treatment groups did not reach statistical significance. On the other hand, incidence of palpable tumors was reduced by about 53% by PEITC administration (P= 0.07 by two-sided Fisher's exact test). Moreover, size of microscopic carcinoma lesions was significantly lower in mice fed PEITC-supplemented diet compared with those fed basal diet (P= 0.05 by two-sided Fisher's exact test). Average body weight of the PEITC-fed mice was modestly but significantly higher than that of control mice at earlier time points (weeks 1-7) but this differential was not observed at later time points (weeks 8-29). Diet consumption was comparable for mice placed on basal diet and PEITC-supplemented diet. Mean PEITC concentration in the plasma and tumor tissue was 901 nM (95% confidence interval- 741-1060 nM) and 87 nmol/kg (95% confidence interval- 67-107 nmol/kg), respectively. Expression of HER-2 oncoprotein as well as number of CD31-positive blood vessels in the tumor did not differ between control and PEITC treatment groups. On the other hand, tumors from PEITC treatment group exhibited about 46% decrease in Ki-67 expression in comparison with control (P= 0.01). Consistent with cellular observations, number of TUNEL-positive apoptotic bodies was about 4.4-fold higher in the tumors of PEITC-fed mice as compared with controls (P= 0.01). Pulmonary metastasis was evident in 3/35 control mice but only 1/32 mice placed on PEITC-supplemented diet exhibited microscopic evidence of metastasis. Size of pulmonary metastasis was also smaller in the PEITC treatment group compared with control. The results indicate that PEITC administration in the diet inhibits carcinoma development in MMTV-neu mice without any signs of overt toxicity. This investigation was supported in part by the US PHS grants CA101753 and CA129347 awarded by the National Cancer Institute. 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 563. doi:1538-7445.AM2012-563

Abstract 564: Bim-independent apoptosis by benzyl isothiocyanate in human breast cancer cells is mediated by PUMA

Abstract Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, is a promising agent for chemoprevention of breast cancer as evidenced by efficacy against xenografted breast cancer cells (e.g., MDA-MB-231 cells) and in a transgenic mouse model (MMTV-neu). We have shown previously that mammary cancer chemoprevention with dietary BITC administration in the MMTV-neu mice is associated with apoptosis induction. In cellular models of human breast cancer (MCF-7 and MDA-MB-231 cells), the BITC-induced apoptosis is initiated by reactive oxygen species (ROS) resulting from inhibition of complex III of the mitochondrial respiratory chain. The present study builds upon these observations to provide insights into the molecular circuitry of BITC-induced apoptosis downstream of ROS production. Because BITC-induced apoptosis in different cell types is associated with activation of c-Jun N-terminal kinase (JNK), we initially focused on Bim as this protein is a downstream mediator of JNK-regulated apoptosis. Bim protein expression (extra-long form) was increased by about 2-3-fold upon 24-hour treatment of MDA-MB-231 cells with pharmacologically relevant concentrations of BITC (2.5 and 5 μM BITC). However, RNA interference of Bim had no effect on BITC-induced apoptosis as judged by analysis of cytoplasmic histone-associated DNA fragment release into the cytosol. Moreover, the BITC-treated MCF-7 cells exhibited a marked decrease in Bim (extra-long form) protein level. BITC treatment resulted in induction of PUMA in both MCF-7 and MDA-MB-231 cells, but as expected, this response was more pronounced in the MCF-7 cells which express wild-type p53. The BITC-induced apoptosis was partially but statistically significantly attenuated by RNA interference of PUMA in MCF-7 cells. Consistent with these observations, genetic depletion of PUMA in HCT-116 cells conferred partial but significant protection against BITC-induced apoptosis. Attenuation of BITC-induced apoptosis in PUMA knockout cells was accompanied by enhanced G2/M phase cell cycle arrest compared with isogenic wild-type HCT-116 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with PUMA induction in the tumor as evidenced by immunohistochemistry and western blotting. In conclusion, these results indicate that Bim-independent apoptosis by BITC is partly mediated by PUMA. This investigation was supported by the US PHS grant CA129347 awarded by the National Cancer Institute. 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 564. doi:1538-7445.AM2012-564

Genistein and resveratrol: mammary cancer chemoprevention and mechanisms of action in the rat

The environment, including diet, plays a critical role in a woman’s subsequent risk of breast cancer. Two dietary polyphenols that have received attention from the health and research communities for their ability to protect against breast cancer are: genistein, a component of soy; and resveratrol, a phytoalexin found in red grapes and red wine. We and others have shown that both genistein and resveratrol can protect against mammary cancer in rodents. The timing of exposure to genistein appears critical for its mammary protective effects. It has been reported that genistein early in life causes enhanced mammary gland differentiation, alterations in cell proliferation and apoptosis, and upregulation of tumor-suppressor genes. With resveratrol in the diet, changes in cell proliferation and apoptosis in terminal ductal structures of the mammary gland might help to explain its protective effects. We conclude that genistein and resveratrol can protect against breast cancer by regulating important mammary growth and differentiation pathways.

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.

Comparative action of 1,4-phenylenebis(methylene)selenocyanate and its metabolites against 7,12-dimethylbenz[a]anthracene-DNA adduct formation in the rat and cell proliferation in rat mammary tumor cells

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis and DMBA–DNA binding in the rat mammary gland. Tetraselenocyclophane (TSC) was identified in rat feces as a metabolite of p-XSC. This led us to postulate the metabolic pathway: p-XSC→glutathione conjugate (p-XSeSG)→aromatic selenol (p-XSeH)→TSC. Whether p-XSC or one of its metabolites is responsible for cancer prevention is the focus of this study. We utilized the DMBA–DNA binding assay with p-XSC as a positive control to evaluate the chemopreventive potential of p-XSC metabolites at dietary selenium levels of 10ppm. Rats were fed AIN-76A diet supplemented with various selenium compounds for 1 week prior to the oral administration of a single dose of [3H]DMBA (5mg per rat, specific activity 51.3mCi/mmol). The rats were sacrificed 24h later and DNA was isolated from the mammary fat pads. Relative levels of total binding were: [pmol/mg DNA, mean±S.D., n=6]; DMBA [7.2±1.6]; DMBA+p-XSC [3.5±2.7]; DMBA+p-XSeSG [2.2±1.1]; DMBA+TSC [5.6±2.9]. All selenium compounds, except TSC, significantly inhibited DMBA–DNA adduct formation; however, the difference between p-XSC and p-XSeSG was not statistically significant. The inhibition of total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol epoxides of DMBA. On the basis of their chromatographic characteristics, these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine. Our results suggest that p-XSeSG, but not TSC, is the likely inhibitor of mammary cancer. Selenium levels measured by atomic absorption spectroscopy in the target organ (mammary fat pads) and in plasma following the dietary administration of selenium compounds were in the order of p-XSeSG≅p-XSC>TSC. These results appear to be consistent with their order of inhibitory effects on total DMBA–DNA binding. Further in vitro studies of the effect of selenium compounds on cell proliferation suggest that, depending on the dose and time point selected, p-XSC is comparable to or better than p-XSeSG; but both are more effective than TSC. Collectively, our in vivo and in vitro results indicate that p-XSC and its conjugate are better candidates than TSC for future studies on mammary cancer chemoprevention.

Elucidation of molecular targets of mammary cancer chemoprevention in the rat by organoselenium compounds using cDNA microarray.

We employed cDNA microarray analysis to identify, in mammary adenocarcinomas induced by 7,12-dimethylbenz[a] anthracene (DMBA) in the rat, target genes as potential biomarkers for cancer chemoprevention by 1,4-phenylenebis(methylene)selenocyanate (p-XSC). Confirmation of selected genes was conducted by reverse transcription polymerase chain reactions (RT-PCR). The glutathione conjugate, p-XSeSG, a putative metabolite of p-XSC was also employed to test our hypothesis that p-XSeSG is a more effective cancer chemopreventive agent in the mammary cancer model than p-XSC. Mammary adenocarcinomas were induced by a single oral administration of 5 mg DMBA in 0.2 ml olive oil per rat at 50-55 days of age. Consistent with our previous reports, dietary p-XSC at a non-toxic dose (10 p.p.m. as selenium) significantly inhibited adenocarcinoma development, independent of feeding duration. Moreover, p-XSeSG appears to be just as effective as p-XSC when fed after DMBA administration, but was significantly less effective than p-XSC in inhibiting the induction of mammary adenocarcinomas when it was fed before DMBA and continued until termination. To delineate the molecular basis for cancer chemoprevention by organoselenium compounds, we focused our analysis on differential expression of genes known to be involved in DMBA metabolism, as well as those related to cell cycle, cell proliferation and apoptosis. p-XSC and p-XSeSG were significantly and equally effective in inhibiting levels of expression of genes associated with cytochrome P450 isoforms, but the former was more active than the latter in up-regulating the expression of those related to certain phase II enzymes. p-XSC and p-XSeSG were significantly more effective in the up-regulation of pro-apoptotic genes, such as p21CIP1/WAF1, p27KIP1, APO-1 and Caspase-3, while down-regulating cell growth regulatory genes, such as c-myc, cyclin D1, cyclin D2 and proliferating cell nuclear antigen (PCNA). To our knowledge, this is the first report that provides insights into the effects of p-XSC and p-XSeSG at the molecular level that may account for mammary cancer chemoprevention in vivo in the rat.

Genistein Chemoprevention: Timing and Mechanisms of Action in Murine Mammary and Prostate

We investigated the potential of genistein, the primary isoflavone of soy, to protect against breast and prostate cancers in animal models. For mammary cancer studies, Sprague-Dawley rats were fed AIN-76A diet plus minus 250 mg genistein/kg diet. Dimethylbenz[a]anthracene was administered by gavage at d 50 postpartum to induce mammary tumors. Mammary cancer chemoprevention was demonstrated after prepubertal and combined prepubertal and adult genistein treatments but not after prenatal- or adult-only treatments, demonstrating that the timing of exposure to genistein is important for mammary cancer chemoprevention. The cellular mechanism of action was found to be mammary gland and cell differentiation, as shown by whole-mount analysis and beta-casein expression. An imprinting effect was shown for epidermal growth factor receptor expression in mammary terminal end buds. For prostate cancer studies, we used two models. The first was a chemically (N-methylnitrosourea) induced prostate cancer rat model. Genistein in the diet inhibited the development of invasive adenocarcinomas in a dose-dependent manner. The second model was a transgenic mouse model that resulted in spontaneously developing adenocarcinoma tumor of the prostate. Genistein in the diet reduced the incidence of poorly differentiated prostatic adenocarcinomas in a dose-dependent manner and down-regulated androgen receptor, estrogen receptor-alpha, progesterone receptor, epidermal growth factor receptor, insulin-like growth factor-I, and extracellular signal-regulated kinase-1 but not estrogen receptor-beta and transforming growth factor-alpha mRNA expressions. We conclude that dietary genistein protects against mammary and prostate cancers by regulating specific sex steroid receptors and growth factor signaling pathways.

Chemical speciation influences comparative activity of selenium-enriched garlic and yeast in mammary cancer prevention.

A recent human intervention trial showed that daily supplementation with selenized yeast (Se-yeast) led to a decrease in the overall cancer morbidity and mortality by nearly 50%; past research has also demonstrated that selenized garlic (Se-garlic) is very effective in mammary cancer chemoprevention in the rat model. The goal of this study was to compare certain biological activities of Se-garlic and Se-yeast and to elucidate the differences based on the chemical forms of selenium found in these two natural products. Characterization of organic selenium compounds in yeast (1922 microg/g Se) and garlic (296 microg/g Se) was carried out by high-performance liquid chromatography with inductively coupled plasma mass spectrometry or with electrospray mass spectrometry. Analytical speciation studies showed that the bulk of the selenium in Se-garlic and Se-yeast is in the form of gamma-glutamyl-Se-methylselenocysteine (73%) and selenomethionine (85%), respectively. The above methodology has the sensitivity and capability to account for >90% of total selenium. In the rat feeding studies, supplementation of Se-garlic in the diet at different levels consistently caused a lower total tissue selenium accumulation when compared to Se-yeast. On the other hand, Se-garlic was significantly more effective in suppressing the development of premalignant lesions and the formation of adenocarcinomas in the mammary gland of carcinogen-treated rats. Given the present finding on the identity of selenomethionine and gamma-glutamyl-Se-methylselenocysteine as the major form of selenium in Se-yeast and Se-garlic, respectively, the metabolism of these two compounds is discussed in an attempt to elucidate how their disposition in tissues might account for the differences in cancer chemopreventive activity.

Mammary tumorigenesis and chemoprevention studies in carcinogen-treated rats.

The objectives of this review are to describe the induction of mammary gland tumors by chemical carcinogens and to discuss their application to mammary cancer chemoprevention research. Special emphasis will be placed on the dimethylbenz[a]anthracene (DMBA) and methylnitrosourea (MNU) models because of the extensive information available about the pathogenesis of tumor growth associated with these two compounds. Both models have been widely used in the investigation of novel cancer chemopreventive agents. The current status of a number of different approaches will be summarized briefly here to provide an overview of research opportunities. Despite the popularity of the DMBA and MNU models in laboratory studies of mammary cancer biology and prevention, neither of these carcinogens has ever been implicated in the etiology of human breast cancer. This shortcoming has prompted a growing interest in other relevant environmental chemicals which are capable of producing mammary tumors in experimental animals. The new models have yet to be fully characterized, but they may be more appropriate than the DMBA and MNU models as paradigms for assessing cancer risk in humans and for developing suitable cancer prevention strategies.

Chemoprevention of rat mammary carcinogenesis: Exceptional activity of dietary dehydroepiandrosterone (DHEA)

A major determinant of progress in human breast cancer prevention is the identification of agents with significant anticarcinogenic activity and acceptable levels of toxicity in experimental animals. Over the past 20 years, more than 50 experimental regimens have been shown to have significant chemopreventive activity in the rat mammary gland. The most effective approaches to mammary cancer chemoprevention in rats involve surgical endocrine ablations such as bilateral ovariectomy. However, prophylactic surgical ablations are unlikely to be acceptable to the majority of the general public. All chemicals evaluated to date are less effective, and none has been shown to reduce mammary cancer incidence to zero. As a result, efforts continue to identify chemical agents whose protective activity is comparable to that of endocrine ablation. DHEA is an adrenal steroid with chemopreventive activity in several animal models for human cancer. In the present studies, the chemopreventive efficacy of DHEA was evaluated in rats exposed to the mammary gland carcinogen, N-methyl-N-nitrosourea (MNU). Groups of 20 female Sprague-Dawley rats were fed an AIN-76A diet supplemented with 0, 400, or 800 mg DHEA per kg diet; one week later, all rats received a single i.p. injection of 35 mg MNU per kg body weight. Animals were palpated weekly to monitor mammary tumor development, and all mammary tumors were histologically confirmed. When administered at 800 mg/kg diet, DHEA reduced mammary cancer incidence in controls from 95% to 15%; carcinoma multiplicity in rats receiving 800 mg DHEA per kg diet was reduced by more than 85% from control levels. In a separate study, the 400 mg/kg diet dose of DHEA reduced the incidence of mammary cancer to 5% from 80% found in controls fed the basal diet. Reductions in mammary cancer incidence and multiplicity associated with DHEA administration were accompanied by large increases in cancer latency. Evaluation of mammary gland wholemounts from animals fed DHEA demonstrated a massive induction of lobuloalveolar differentiation. These results indicate the dietary supplementation with non-toxic dose levels of DHEA has chemopreventive efficacy approaching that of endocrine ablation. This protection may be mediated by the induction of differentiation in the mammary gland, during which sensitive mammary parenchymal structures (terminal end buds) are stimulated to develop into structures (alveolar buds) less sensitive to carcinogenic insult.

Anticarcinogenic activity of quinacrine in the rat mammary gland.

Mammary carcinogenesis studies were conducted to determine the chemopreventive activity of quinacrine, an antimalarial drug which suppresses the production of arachidonic acid from phospholipid through inhibition of phospholipase A2. Beginning 1 week after a single i.v. dose of N-methyl-N-nitrosourea (MNU), female Sprague-Dawley rats were fed a semi-purified diet supplemented with 0 or 75 mg quinacrine/kg diet. Quinacrine reduced cancer incidence and carcinoma multiplicity in rats administered 20 mg MNU/kg body wt, but had no inhibitory activity in rats treated with 50 mg MNU/kg. These data suggest that pathways of arachidonic acid metabolism in addition to cyclooxygenase present useful targets for mammary cancer chemoprevention. However, the chemopreventive activity of quinacrine may be limited by toxicity.

Mammary cancer chemoprevention by inorganic and organic selenium: single agent treatment or in combination with vitamin E and their effects on in vitro immune functions.

The chemopreventive efficacies of selenate, selenite, selenium dioxide, selenomethionine and selenocystine were examined during the promotion phase of carcinogenesis in the 7,12-dimethylbenz[a]anthracene-induced mammary tumor model in rats. Each agent was added to the diet at a final concentration of 3 p.p.m. selenium. In general there was no significant difference in the potency of these five selenium compounds in inhibiting the development of mammary tumors. The interaction of vitamin E (500 p.p.m.) with either selenite or selenomethionine was further characterized in a second carcinogenesis study. Results of this experiment suggested that vitamin E enhanced the protective effect of selenite but not that of selenomethionine. In an attempt to explore the synergistic mechanism of selenium and vitamin E, the effects of these two agents on mitogen-induced blastogenesis and natural killer cytotoxic activity were also investigated. No consistent changes in these in vitro immune functions were detected resulting from supranutritional feeding of either selenite or vitamin E or both. The metabolism of inorganic versus organic selenium was discussed in relation to their role in the control of neoplastic growth as well as to their selective modulation by vitamin E.