Rat Mammary Tumors Research Articles

Influence of estradiol on mammary tumor collagen solubility in DMBA‐induced rat mammary tumors

Estradiol plays a vital role in the growth and development of mammary glands. It is a potent stimulator of metabolic processes in normal and carcinoma breast. A critical factor in determining mammary glandular morphology is the stroma. Collagen is a predominant component of the extracellular matrix and cell-collagen interactions are essential carcinogenesis. The present investigation explored the influence of estradiol on collagen solubility and metabolism in mammary tumors during tumor progression and regression. A single injection of 20 mg of 9,10-dimethyl-1,2-benzanthracene was given to rats at 7 weeks of age. With the appearance of the first palpable mammary tumor, the rats were treated with 0.5 microg estradiol or 50 microg tamoxifen daily for 30 days. The rats were sacrificed 24 h after 30 days of treatment. Estradiol appears to stimulate the synthesis of new collagens and thus contributes to the enlargement of the mammary tumors. This might have created a potential microenvironment by increasing the synthesis of suitable matrix that sustains the growth of the mammary tumors. In short, the present findings emphasize a definite mediatory role for collagen in estradiol promoted mammary tumor growth.

Upregulation of V1a vasopressin receptors by glucocorticoids.

WRK1 cells (a rat mammary tumor cell line) exhibit a vasopressinergic receptor of V1a subtype tightly coupled to phospholipase C. Addition of dexamethasone to the culture medium principally potentiated the vasopressin-sensitive accumulation of inositol phosphates and to a lesser extent the NaF-sensitive phospholipase C activity. On the opposite, such treatment was without effect on the basal level of intracellular inositol phosphates or on bradykinin- or serotonin-sensitive phosphoinositide metabolisms. Glucocorticoid receptors were probably involved in these actions since dexamethasone was found to be more potent than aldosterone or corticosterone. Dexamethasone treatment also increased the number of vasopressin binding sites without affecting its affinity for vasopressin or other specific vasopressin analogues. These results strongly suggest that dexamethasone principally acts at the vasopressin receptor level by affecting its synthesis and/or the translation of its mRNA and also affects the G protein that couples the V1a receptor to the phospholipase C. These results explain how glucocorticoids may regulate the transduction mechanisms involved in vasopressin actions on WRK1 cells. They provide explanations for understanding the cross talk between adrenal steroids and hormones, which mobilize intracellular calcium.

Spotlight

Ohtsuka et al., pp. 263–273 The ADAM gene family, which contains a metalloprotease domain, may be involved in shedding membrane-anchored proteins and breaking down extracellular matrix. Certain matrix metalloproteases, by degrading extracellular matrix proteins, contribute to invasion and metastases of various cancers, and a previous report has connected ADAM family members with glioma proliferation. Now, Ohtsuka et al. have screened for expression of 11 ADAM genes in human lung cancers. They found mRNA of both membrane-bound ADAM28 and secreted ADAM28 present in higher quantities in the cancer cells than in normal cells. Larger cancers and those that had metastasized had even higher levels of ADAM28 expression. Previous studies have looked at ADAM expression in various cancers, including breast, colon and stomach. This report is the first to show that lung cell carcinomas selectively overexpress ADAM28, a finding that suggests the protein contributes to cell proliferation and cancer progression. Cos et al., pp. 274–278 Melatonin inhibits breast cancer proliferation by interacting with the estrogen-signaling pathway. The aromatase enzyme complex, which catalyzes the conversion of androgens to estrogens, works harder in breast cancer tissue than in noncancer tissue, suggesting that the resulting increase in estrogen stimulates tumor growth. Therefore, suppressing aromatase activity could slow cancer growth. Building on their recent results showing that melatonin reduces aromatase activity in breast cancer tissue in vitro, Cos et al. now investigate melatonin's effect in estrogen-dependent mammary tumors in rats. To isolate the effect of aromatase on tumor growth, the researchers removed the rats' ovaries, then administered testosterone. Aromatase converts the testosterone to estrogen, which spurs tumor growth the same as estrogen produced by the ovaries in control rats. Melatonin, the authors show, inhibits aromatase activity, counteracting testosterone's effect and shrinking the tumor. In mammary tumors, aromatase expression is regulated by cyclic AMP, which melanin decreases. This newly described activity of melatonin in the estrogen pathway adds to melatonin's track record of blocking estrogen's activity in various ways, and recommends melatonin as a possible therapeutic avenue against breast cancer. Steitz et al., pp. 373–380 Recent success in treating melanoma patients with T cells supports the idea that antigen-specific immunotherapy has its place in clinical settings. Clinical trials are currently underway to test various T-cell directed melanoma strategies. In this paper, Steitz et al. evaluated a strategy for vaccine development in which they targeted a clinically relevant antigen, TRP2. In contrast with earlier investigations, which have used transplanted tumors, the authors attempted to imitate a cancer patient situation by inducing autochthonous melanoma in mice that had an oncogenic mutation in the cell-cycle regulatory gene, cdk4. When they stimulated a melanocyte-specific immune response, white patches appeared in the skin, indicating the destruction of pigment-producing cells, but the vaccine-induced immune response did not significantly attack and destroy the neoplasms. These autochthonous melanoma cells, the authors found, behave like melanocytes that do not express MHC class I molecules. The researchers suggest that the cdk4-mutant mouse model could be developed further to improve its suitability for evaluating melanoma vaccines. In contrast with the current results, a recent study in a different mouse model found spontaneous induction of cytotoxic T cells recognizing TRP2. These results emphasize that, to be useful for assessing potential vaccines, models must accurately represent the clinical situation. Arao et al., pp. 483–489 Gastric cancer patients generally have a poor prognosis, with survival of around 7 months. In search of a better treatment strategy, Arao et al. investigated an angiogenesis inhibitor that interferes with the VEGF system, a key player in tumor growth. When the inhibitor ZD6474 was tested in vivo using a newly established, highly metastatic gastric cancer cell line, they found that it curbed the tumor's growth and its spread throughout the peritoneum. The researchers also searched for changes in gene expression that could allow clinicians to monitor ZD6474's effects. They identified 9 genes whose expression was boosted by the angiogenesis inhibitor and that have been reported previously to be hypoxia-induced. If ZD6474 impedes tumor growth by limiting the cells' oxygen supply, genes that react to lack of oxygen would be likely indicators of ZD6474's success against the tumor. The current study distinguishes itself by using an aggressive, highly metastatic model that closely mimics tumor progression in gastric cancer patients clinically, suggesting that the positive results obtained with ZD6474 in the lab could translate to clinical usefulness.

Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets

Rats treated with the alkylating agent methylnitrosourea (MNU) develop multiple, hormonally dependent mammary tumors. Roughly 50% of the tumors have Ha-ras mutation, whereas 50% do not. The MNU-induced rat mammary tumor model was employed to examine the therapeutic efficacy of the farnesyltransferase inhibitor (FTI), R115777, and to examine the use of genomics in identifying susceptible tumors as well as identifying genes whose expression are modulated by FTI treatment. In animals bearing palpable mammary tumors (< 7 mm diameter), we performed a surgical biopsy, and 3 days following the biopsy, rats were treated with R115777 (50 mg/kg body wt/day) by gavage. Tumors with Ha-ras mutations underwent profound regression, with nearly 90% showing complete regressions within 4 weeks. In contrast, the non-Ha-ras mutation-bearing tumors yielded a more variable response, although roughly half of the non-Ha-ras mutation tumors underwent significant regression. These results show that although all tumors appear to respond to the FTI inhibitor the tumors with Ha-ras mutations were exquisitely sensitive. We employed a microarray approach to define potential targets and the mechanism of action of R115777 in Ha-ras mutant or wildtype tumors following treatment with FTI. In addition, we determined whether gene expression prior to FTI treatment can be used to differentiate highly sensitive tumors (Ha-ras mutant) and tumors with variable sensitivity (Ha-ras wildtype). Untreated or FTI-treated (4 days at 50 mg/kg body wt) tumors (Ha-ras mutant or wildtype) were examined using oligonucleotide arrays. A significant number of genes were differentially expressed in control rat mammary tumors with or without an activated Ha-ras mutation, suggesting that a microarray analysis might differentiate highly sensitive and variably sensitive tumors. Most of the genes whose expressions were modulated by FTI in tumors were independent of Ha-ras status and were presumably modulated by effects on farnesylation of proteins other than Ha-ras. However, treatment of Ha-ras-mutated mammary tumors with R155777 results in preferential modulation of genes involved in ras-MAP kinase signal transduction pathway and in decreased expression of many genes involved with cell proliferation. In contrast, several classes of genes are altered in rat mammary tumors without a mutated Ha-ras, suggesting that non-ras targets are involved. Ras pathway related genes, p53, WT1 and PCNA, were preferentially modulated in Ha-ras-mutated tumors, whereas modulation of genes in the G-protein pathway, various cytochrome p450s and RB1 are involved in Ha-ras wildtype tumors. Elucidation of gene expression changes in FTI-treated or control rat mammary adenocarcinomas will help in identifying potential pharmacodynamic markers of FTI treatment as well as potential molecular targets of R115777 and other FTIs.

Synthesis and preliminary chemotherapeutic evaluation of the fully C-linked glucuronide of N-(4-hydroxyphenyl)retinamide

All- trans retinoic acid analogues such as N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive and chemotherapeutic agents but their utility has been hampered by dose-limiting side effects. The glucuronide derivatives of 4-HPR, the oxygen-linked 4-HPROG and the carbon-linked 4-HPRCG, have been found to be more effective agents. The synthetic route to the fully C-linked analogue of 4-HPROG (4-HBRCG), which employs Suzuki coupling and Umpolung chemistries as key methodologies, is shown. The results of this study show 4-HBRCG to be an effective chemotherapeutic agent in a rat mammary tumor model while being devoid of classical retinoid toxicities.

High‐dose, short‐term, anti‐inflammatory treatment with dexamethasone reduces growth and augments the effects of 5‐fluorouracil on dimethyl‐α‐benzanthracene‐induced mammary tumors in rats

Objective. To evaluate the effects of dexamethasone (DXM) alone or in combination with 5‐fluorouracil (5‐FU) on dimethyl‐α‐benzanthracene (DMBA)‐induced mammary tumors in rats. Material and methods. Female Sprague‐Dawley rats were divided into 4 groups receiving: 1) saline (controls), 2) DXM (3 mg/kg), 3) 5‐FU (1.5 mg/kg) and 4) DXM and 5‐FU combined. The drugs were given i.p. every day for 4 days. Interstitial fluid pressure (Pif) and tumor growth were determined in all tumors on days 1, 5 and 7 using the “wick‐in‐the needle” technique and by external size measurements, respectively. Vessel density and inflammatory cell infiltration of tumor tissue were analyzed by immunohistochemistry. Results. DXM treatment significantly retarded tumor growth and reduced Pif. Treatment with a combination of DXM and 5‐FU reduced tumor size significantly more than any of the agents alone (p<0.01–0.001). Enhanced uptake of 5‐FU by DXM treatment was demonstrated by microdialysis. There were no differences in the density of CD31‐positive vessels after DXM or 5‐FU treatment, but inflammatory cell infiltration of tumor tissue was significantly reduced after DXM treatment. Conclusions. Our data suggest that DXM may be beneficial as an adjuvant to chemotherapy in the treatment of mammary cancer by increasing the uptake of 5‐FU in the tumor.

Role of Polyamines in Breast Cancer Growth, Development and Progression

The polyamines putrescine, spermidine, and spermine are small, aliphatic amines that play an important role in multiple cellular functions. Activation of the polyamine pathway is involved in carcinogenesis and other aspects of tumor biology including in breast cancer. Levels of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis as well as the levels of polyamines, are higher in human breast cancer specimens than in normal and benign breast tissue. Inhibition of ODC with -difluromethylornithine exerts a protective effect in the promotion of chemically induced rat mammary tumors as well as in the development of breast cancers in a transgenic model system. Inhibition of polyamine biosynthesis has also been shown to inhibit the growth of hormone-dependent and -independent mammary tumors, both in vitro and in vivo, thus indicating a major role for polyamines in breast cancer cell proliferation. Several lines of evidence indicate that increased ODC activity may contribute to breast cancer progression. Increased tumor ODC activity has been shown to be an independent, adverse prognostic factor for overall survival in women with localized breast cancer. This finding suggests that polyamines may be critically involved in the development of breast cancer metastasis. We have, indeed, observed that administration of DFMO markedly reduced in vitro invasiveness of the hormone-independent MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. More importantly, we have observed that DFMO administration significantly reduced the development of pulmonary metastasis from orthotopically implanted MDA-MB-435 breast cancer xenografts in nude mice. The mechanism of antitumor action of DFMO is currently under active investigation. Several laboratories have shown that inhibition of polyamine biosynthesis results in activation of the MAPK pathway and STAT signaling. We have recently shown that DFMO induced activation of the MAPK pathway in MDA-MB-435 human breast cancer cells is directly involved in the production of the anti-invasive protein, thrombospondin-1. Inhibition of MAPK phosphorylation suppressed DFMO-induced stimulation of thrombospondin-1 and reversed its anti-invasive effect. Collectively, these data indicate that the polyamine pathway may be an attractive target for breast cancer prevention and treatment. Keywords: antipolyamines, breast cancer, cell proliferation, tumor progression

Hexachlorobenzene Is a Tumor Co-carcinogen and Induces Alterations in Insulin-Growth Factors Signaling Pathway in the Rat Mammary Gland

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Controversy still exists about the breast carcinogenic properties of organochlorines in humans. The ligands, receptors, and related signaling proteins of the insulin growth factor family are involved in the regulation of breast-cancer cell growth. The aims of this study were to determine: (1) whether HCB is co-carcinogenic in a medium term assay of N-nitroso N-methylurea (NMU)-induced mammary tumors in rats; (2) the effect of HCB on insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) levels and on IRS-1 phosphorylation; (3) microsomal and cytosolic Protein Tyrosine Kinase (PTK) activities in mammary glands and NMU-induced tumors. Sprague Dawley rats were injected with 50 mg/kg body weight of NMU at 50, 80, and 110 days old. HCB (100 mg/kg body weight) was administered three times a week from 65 to 110 days of age. Rats were separated in four groups: control, NMU, HCB, and NMU-HCB. HCB alone did not induce tumor development. Parameters of tumor development were increased in NMU-HCB compared to NMU rats. A higher cellular undifferentiation was observed in NMU-HCB tumors. IR, IGF-IR, and IRS-1 levels were higher in HCB than in controls. Conversely IGF-IR levels decreased in NMU-HCB vs. NMU group. The IRS-1 phosphorylation increased in HCB rats; however, it decreased in NMU-HCB vs. NMU. HCB decreased microsomal PTK activity in tumors. This study showed for the first time that HCB is a co-carcinogenic agent in NMU-induced mammary tumors in rats. Our results suggest that the IR and/or IGF-IR signaling pathway may be involved in the mechanism of action of HCB.

Optical Biopsy of Lymph Node Morphology using Optical Coherence Tomography

Optical diagnostic imaging techniques are increasingly being used in the clinical environment, allowing for improved screening and diagnosis while minimizing the number of invasive procedures. Diffuse optical tomography, for example, is capable of whole-breast imaging and is being developed as an alternative to traditional X-ray mammography. While this may eventually be a very effective screening method, other optical techniques are better suited for imaging on the cellular and molecular scale. Optical Coherence Tomography (OCT), for instance, is capable of high-resolution cross-sectional imaging of tissue morphology. In a manner analogous to ultrasound imaging except using optics, pulses of near-infrared light are sent into the tissue while coherence-gated reflections are measured interferometrically to form a cross-sectional image of tissue. In this paper we apply OCT techniques for the high-resolution three-dimensional visualization of lymph node morphology. We present the first reported OCT images showing detailed morphological structure and corresponding histological features of lymph nodes from a carcinogen-induced rat mammary tumor model, as well as from a human lymph node containing late stage metastatic disease. The results illustrate the potential for OCT to visualize detailed lymph node structures on the scale of micrometastases and the potential for the detection of metastatic nodal disease intraoperatively.

EB1089, a vitamin D receptor agonist, reduces proliferation and decreases tumor growth rate in a mouse model of hormone-induced mammary cancer

1,25-Dihydroxyvitamin D 3 and several of its analogs, such as EB1089, induce growth arrest and apoptosis of breast cancer cells in culture. EB1089 has also been shown to limit growth of xenografts in nude mice and carcinogen-induced mammary tumors in rats. Coupled with the fact that the vitamin D receptor is highly expressed in a large proportion of breast tumors, these data suggest that it may be a broad spectrum therapeutic target. We utilized a transgenic model of hormone-induced mammary cancer, the LH-overexpressing mouse, to assess, for the first time, the efficacy of EB1089 in a spontaneous tumor model. Similar to human breast cancers, the pre-neoplastic mammary glands and mammary tumors in these mice express high levels of vitamin D receptor. Treatment with EB1089 decreased proliferation of mammary epithelial cells in pre-neoplastic glands by 35%. Moreover, half of hormone-induced mammary tumors treated with EB1089 demonstrated a decreased rate of growth, with a subset of these tumors even regressing, suggesting that 1,25-dihydroxyvitamin D 3 analogs may be effective chemopreventive and chemotherapeutic agents for breast cancer.

Tumor Response Assessment Is More Robust With Sequential CT Scanning Than External Caliper Measurements 1

Measurements of tumor size are important in assessing response to cancer therapies. To date, preclinical studies of drug development have relied on direct caliper-based measurements of tumor size. We investigated the feasibility of using a human positron emission tomographic (PET)/computed tomographic (CT) scanner to assess tumor size before and after chemotherapy and compared this approach with caliper measurements. Fourteen rats with rat mammary tumor underwent high-resolution CT using a PET/CT scanner before and after chemotherapy, and tumor volumes were measured independently by two observers using calipers and CT images. Tumor response could be detected after 1 day of treatment by means of CT imaging, but was not significant until 2 days or later by means of caliper measures because of their greater variability. Independent measurements of tumor size correlated well with one another by means of CT, but correlated less by means of calipers. Tumor size measurements by means of CT from PET/CT were more reliable than caliper measurements because of their smaller variance, allowing earlier assessment of response. It is suggested that CT imaging-based methods of assessing tumor response replace traditional caliper-based measurements, much as CT has become a standard for assessing tumor response in humans.

Effects of dietary folate on the development and progression of mammary tumors in rats †

Epidemiologic studies have suggested that dietary intake and blood levels of folate may be inversely related to the risk of breast cancer. However, epidemiologic evidence has not been consistent nor has it provided unequivocal support for this purported inverse relationship. Recent evidence has also raised a concern that folate supplementation may promote carcinogenesis if provided after neoplastic foci are established in the target organ. This study investigated the effect of dietary folate deficiency and supplementation on the development and progression of mammary tumors in the N-methyl-N-nitrosourea (MNU) rat model. Weanling, female Sprague-Dawley rats were fed diets containing 0, 2 (control) or 8 mg folic acid/kg diet during the initiation or the promotion phase of MNU-induced mammary tumorigenesis. At necropsy, all macroscopic mammary tumors were identified and histologically confirmed. Dietary folate deficiency and supplementation provided during the initiation phase did not significantly modulate the development of mammary tumors. In contrast, dietary folate deficiency provided during the promotion phase significantly inhibited the rate of appearance, incidence, mean volume and weight of adenocarcinomas compared with the control and supplemental diets. Folate supplementation provided during the promotion phase did not significantly modulate mammary tumorigenesis compared with the control group. These data indicate that moderate folate deficiency inhibits, whereas dietary folate supplementation at four times the basal dietary requirement does not promote, the progression of MNU-induced mammary neoplastic foci in this rat model. However, the limitations associated with the route and dose of MNU administration preclude a definitive conclusion concerning the effect of folate status on the initiation of MNU-induced mammary tumorigenesis.

Human Homologue of Cement Gland Protein, a Novel Metastasis Inducer Associated with Breast Carcinomas

A suppression subtractive cDNA library representing mRNAs expressed at a higher level in the malignant human breast cancer cell line, MCF-7, relative to a benign breast tumor-derived cell line, Huma 123, contained a cDNA, M36, which was expressed in estrogen receptor alpha (ERalpha)-positive breast carcinoma cell lines but not in cell lines from normal/benign/ERalpha-negative malignant breast lesions. M36 cDNA had an identical coding sequence to anterior gradient 2 (AGR2), the human homologue of the cement gland-specific gene (Xenopus laevis). Screening of breast tumor specimens using reverse transcription-PCR and immunocytochemistry with affinity-purified anti-AGR2 antibodies showed that the presence of AGR2 mRNA and protein were both statistically significantly associated with ERalpha-positive carcinomas (P = 0.007, Fisher's exact test) and with malignancy (P < or = 0.025). When an expression vector for AGR2 cDNA was introduced into benign nonmetastatic rat mammary tumor cells, and three separate clones and two pools of cells were transferred to the mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. However, metastases occurred in the lungs of animals receiving the AGR2 transfectants in 77% to 92% of animals with primary tumors (P = 0.0001) compared with no metastases in the control groups. The AGR2 transfectants exhibited enhanced rates of adhesion to a plastic substratum and extracellular AGR2 enhanced the rate of attachment of AGR2-negative but not AGR2-positive cells. These experiments are the first to link mechanistically the developmental gene product, AGR2, with metastasis in vivo.

Gene expression profiling of NMU-induced rat mammary tumors: cross species comparison with human breast cancer

Breast cancer is a complex genetic disease characterized by the accumulation of multiple molecular alterations. The NMU breast cancer model induced in the rat is used for the study of mammary carcinogenesis because it closely mimics human breast disease. To assess the validity of this model from a more global molecular perspective, and also to devise a general technique to compare animal profiles with human microarray studies, we have characterized 25 NMU-induced mammary tumors and 11 normal glands using a combination of immunohistochemical and microarray analyses. The rat mammary carcinomas were classified as non-invasive, ER-positive ductal carcinomas with a composition of differentiated epithelial and myoepithelial cell lineages. Gene expression profiles generated using rat Affymetrix arrays containing 15,866 genes demonstrated that the rat mammary tumors are homogeneous and that H-ras mutations did not confer a unique molecular signature. We compared the resulting rat profiles with those obtained from a human dataset by merging the raw microarray data, using an approach that involves a combination of cross-species and cross-platform analysis. Using this novel strategy, we demonstrate the ability of 2305 rat orthologs to recapitulate the classification of human tumors derived from human Affymetrix arrays. The gene expression profiles of the NMU-induced primary tumors were most similar to ER-positive, low to intermediate grade breast cancer. Our technique provides a means to correlate gene expression data from animal models of cancer to human cancer and disease states.

Increased expression of MDM2, cyclin D1, and p27Kip1 in carcinogen‐induced rat mammary tumors

It is thought that environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH), contribute to human breast tumorigenesis, yet their roles remain incompletely elucidated. The prototypical PAH 7,12-dimethylbenz(alpha)anthracene (DMBA) specifically and effectively induces mammary tumor formation in rodent models. In an attempt to explore the molecular mechanisms by which PAH initiates and promotes mammary tumorigenesis, we examined the expression of several cell cycle regulators in rat mammary tumors induced by DMBA. Expression of cyclin D1, murine double minute-2 (MDM2), and Akt was up-regulated in tumors in comparison to normal mammary glands, as indicated by RT-PCR, Western blot analysis, and immunohistochemical staining. Expression of p27Kip1 protein was also elevated in the tumors with increased cytoplasmic localization. However, RB protein remained hyperphosphorylated. To directly test the effects of DMBA, the MCF-7 human breast cancer cells were treated. DMBA induced MDM2 expression in a dose- and time-dependent fashion in the MCF-7 cells, and this activation appeared to be p53 dependent. These data suggest that activation of cyclin D1, MDM2, and AKT as well as increased expression and cytoplasmic localization of p27Kip1 may play a role in this model of environmental pollutant-induced mammary tumorigenesis.

W16-P-063 The lipid-altering effects of INEGY-TM are consistent across gender, race, age, and baseline LDL-C levels

Phytoestrogens have been reported to exhibit antiproliferation to human breast cancer cells in vitro. We tested the phytoestrogen-rich, Pueraria mirifica against rat breast cancer induction in vivo.The weanling female Spargue–Dawley rats were pretreated with P. mirifica tuberous powder at a dosage of 0, 10, 100 and 1000 mg/kg BW/day for four consecutive weeks. Mammary tumor development was then induced with a single dose of 7,12-DMBA, 80 mg/kg BW, followed by a weekly examination for size and multiplicity of mammary tumors for 20 weeks and finally a necropsy. Mammary tissues were investigated for the virulence of tumor and also monoclonal antibody stained against ERα and ERβ.Pretreatment of 1000 mg/(kg BW day) of P. mirifica tuberous powder resulted in decreasing of the virulence of rat tumor development. The mammary tumor tissues exhibited lower profile of ERα and ERβ as well as ERα/ERβ.P. mirifica exhibited prevention of 7,12-DMBA-induced rat mammary tumors, with a proposed mechanism of strong competitive binding of its phytoestrogens to ERα and/or synthesis suppressor of ERα.

Regression of NMU-induced mammary tumors with the combination of melatonin and 9- cis-retinoic acid

A significant increase in tumor regression was induced in N-nitroso- N-methylurea-induced mammary tumors in rats treated with the combination of melatonin and 9- cis-retinoic acid (9 cRA). Treatment groups included: control (ethanolic saline), 9 cRA (30 mg/kg chow/day), melatonin 500 μg/day, melatonin 1000 μg/day, melatonin 500 μg/day+9 cRA and melatonin 1000 μg/day+9 cRA. Rats treated with the lower dose of melatonin 500 μg+9 cRA show the greatest degree of tumor regression (78%), with 54% undergoing complete regression and a significant increase in apoptotic cells observed by TUNEL Assay. Furthermore, tumor multiplicity and burden were significantly decreased by the combination of melatonin and 9 cRA.

Enhancement by acrylamide of N-methyl- N-nitrosourea-induced rat mammary tumor development—possible application for a model to detect co-modifiers of carcinogenesis

Acrylamide (AA) has recently been reported to be spontaneously formed in fried and baked foods with various concentrations. Although carcinogenicity in humans is as yet equivocal, numerous positive genotoxicity data in vitro and in vivo and results of rat long-term carcinogenicity studies demonstrating tumor induction at multiple sites, like the mammary gland, thyroid and testes, suggest the risk with dietary exposure may not be negligible. In the present study, to establish a medium-term carcinogenesis model for screening of agents with the potential to modify AA effects on the mammary gland and thyroid, we pretreated rats with 7,12-dimethylbenz(a)anthracene (DMBA), in combination with N-bis(2-hydroxypropyl)nitrosamine (DHPN), or N-methyl- N-nitrosourea (MNU) alone and then administered AA at 20 and 40 ppm in the drinking water for 30 weeks. The incidence and multiplicity of mammary tumors were increased at the high dose ( P<0.05) in MNU— but not DMBA+DHPN-treated rats. No thyroid tumors were induced in any case. The results indicate that the MNU model is suitable for detection of modifiers of AA actions.

Steroidhormone receptors as targets for the therapy of breast and prostate cancer—recent advances, mechanisms of resistance, and new approaches

Surgical ovariectomy and orchiectomy, first proposed over a century ago, are effective in breast and prostate cancer therapy, respectively. Later, the discovery of steroid hormones and their nuclear receptors led to the concept that inhibition of steroid receptor function by an antagonist prevents tumour growth. While the first anti-hormones, cyproteroneacetate (CPA) and tamoxifen were found accidentally, deeper understanding of nuclear receptors as transcription factors enabled more rational, structure-activity based drug discovery. Results from a drug-finding program on pure anti-estrogens will be reported. These new steroidal anti-estrogens are highly active, pure ER-antagonists that lead to an efficient degradation of the estrogen receptor α (ERα) protein without any agonistic activity. Data obtained in preclinical tumour models in mice and rats showed a high potency in growth inhibition of ERα-positive breast cancer. In parallel, by comparing three independently generated anti-estrogen-resistant breast cancer cell lines, it was our intention to gain insight into the mechanisms of endocrine resistance which will allow to define new approaches for the treatment of endocrine-resistant breast cancer. Candidate proteins potentially involved in mechanisms of anti-estrogen-resistant growth of breast cancer cell lines were analyzed. ERα and progesterone receptor (PR) expressions were lost on the protein level in all three anti-estrogen-resistant cell lines, whereas binding of epidermal growth factor (EGF) and protein expression of epidermal growth factor receptor (EGFR) were increased. Loss of ERα expression may be linked to the acquisition of anti-estrogen resistance and enhanced expression of the EGFR and of members of the S100 family of Ca 2+-binding proteins may contribute to the outgrowth of resistant cells. Furthermore, we describe the pharmacological development of a novel, highly potent progesterone receptor antagonist. In rat mammary tumour models, treatment with the PR antagonist completely suppressed the growth of established tumours and prevented the development of breast tumours. Advanced prostate cancer is effectively treated by androgen ablation. However, this therapy becomes inefficient although the androgen receptor (AR) is still functionally expressed. One novel strategy for the treatment of advanced prostate cancer could be the selective inhibition of AR protein expression by anti-sense oligonucleotides or small interfering RNA (siRNA) molecules. Down-regulation of the human AR caused significant inhibition of LNCaP prostate cancer growth in vivo. Taken together, many promising alternatives for endocrine therapy of breast and prostate cancer are arising.

A risk assessment of atrazine use in California: human health and ecological aspects

A risk assessment of the triazine herbicide atrazine has been conducted by first analyzing the toxicity database and subsequently estimating exposure. Margins of safety (MOS) were then calculated. Toxicity was assessed in animal studies and exposure was estimated from occupational and dietary sources. In acute toxicity studies, atrazine caused developmental toxicity in the rabbit [no observed effect level (NOEL) 5 mg kg(-1) day(-1)] and cardiotoxicity in a dog chronic study (NOEL 0.5 mg kg(-1) day(-1)); cancer (mammary glands) resulted from lifetime exposure. The mammary tumors, which occurred specifically in female Sprague-Dawley rats, were malignant, increased in a dose-dependent manner and were also observed with other, related triazines. Evidence for a genotoxic basis for these tumors was either equivocal or negative. Triazines have been shown to be clastogenic in Chinese hamster ovary cells, in vitro, but without showing a convincing dose/response relationship. Atrazine can be converted into genotoxic N-nitrosoatrazine in the environment or the digestive system, suggesting that N-nitrosamines derived from triazines could be oncogenic. However, it was concluded that N-nitrosotriazines are unlikely to play a significant role in triazine-induced rat mammary gland tumors. An endocrine basis for the mammary tumors, involving premature aging of the female SD rat reproductive system, has been proposed. A suppression of the luteinizing hormone surge during the estrus cycle by atrazine leads to the maintenance of elevated blood levels of 17beta-estradiol (E2) and prolactin. The mechanism for tumor development may include one or more of the following: the induction of aromatase (CYP19) and/or other P450 oxygenases, an antagonist action at the estrogen feedback receptor in the hypothalamus, an agonist action at the mammary gland estrogen receptor or an effect on adrenergic neurons in the hypothalamic-pituitary pathway. None of these has been excluded as a target because there has been a lack of a rigorous attempt to address the mechanism of action for mammary tumors at the molecular level. The potential occupational exposure to atrazine was assessed during mixing, loading and application. Absorbed daily dosage values were 1.8-6.1 microg kg(-1) day(-1). The MOS values (animal NOEL/human exposure) for short-term (acute) exposure were 820-2800. Longer-term occupational exposure and risk were also calculated. Detectable crop residues are generally absent at harvest. Theoretical calculations of acute dietary exposure used tolerance levels, along with secondary residues, and water, for which there is a maximum contamination level; atrazine plus the three main chlorotriazine metabolites were combined. MOS values were above 2000 for all population subgroups. Dietary exposure to atrazine is therefore extremely unlikely to result in human health hazard. Recent publications have reported a possible feminization of frogs, measured in laboratory and field studies. This is assumed to be due to the induction of aromatase, but no measurements of enzyme activity have been reported. In field studies, the water bodies with the greatest numbers of deformed frogs sometimes had the lowest concentrations of atrazine. Other studies have also cast doubt on the feminization theory, except perhaps at very high levels of atrazine. Epidemiology studies have investigated the possibility that atrazine may result in adverse effects in humans. Although some studies have claimed that atrazine exposure results in an elevated risk of prostate cancer, the published literature is inconclusive with respect to cancer incidence.