Rat Mammary Tissue Research Articles

Identification, Synthesis, and Biological Evaluation of the Metabolites of 3-Amino-6-(3′-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6–36), a Promising Rexinoid Lead Compound for the Development of Cancer Chemotherapeutic and Chemopreventive Agents

Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation, and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver, and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, seven phase I and II metabolites were formed by human hepatocytes, and five metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB.

Overexpression of 5‐lipoxygenase and its relation with cell proliferation and angiogenesis in 7,12‐dimethylbenz(α)anthracene‐induced rat mammary carcinogenesis

The present study was performed to investigate the critical role of 5-lipoxygenase (5-LOX) in 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary inflammation associated carcinogenesis. Female Sprague-Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection, followed by administration of zileuton (2000 mg/kg diet) from week 7 until the termination of the study at 31 wk. 5-LOX protein expression, 5-hydroxyeicosatetraenoic acid (5-HETE), and leukotriene B4 (LTB4 ) production in rat mammary tissue were analyzed at 6, 12, and 24 wk post-DMBA injection. Rate of cell proliferation was analyzed by bromodioxyuridine labeling index (BrdU-LI). Microvessel density, level of VEGF, and MMP-2 were also measured. DMBA induces inflammation in rat mammary gland as early as 6 wk. 5-LOX is upregulated in DMBA treated rats right from 6 wk when compared with their normal counterparts. An overexpression of 5-LOX is accompanied with increase in 5-HETE, LTB4 production and high BrdU-LI with an increase of two key angiogenic factors for tumorigenesis; MMP-2 and VEGF. It was found that 5-LOX specific inhibitor brought about substantial protection against DMBA-induced mammary carcinogenesis. Histological findings showed substantial repair of hyperplastic lesions. There was a significant reduction in the rate of cell proliferation and expression of angiogenic factors, MMP-2 and VEGF. 5-LOX plays an important role in DMBA-induced inflammation associated carcinogenesis via activation of MMP-2 and VEGF. 5-LOX expression can be considered as a critical event in controlling the process of mammary tumor development.

Dietary Mg2+ regulates the epithelial Mg2+ channel TRPM6 in rat mammary tissue

The epithelial Mg(2+) channel TRPM6 is considered a pivotal component in active Mg(2+)absorption and re-absorption in the intestine and kidney, but its expression and function in other tissues are largely unknown. We have previously demonstrated that extracellular Mg(2+) availability modulates TRPM6, but not the ubiquitous TRPM7, in cultured mammary epithelial cells; in addition, TRPM6 protein expression correlated to Mg(2+) influx capacities. Our results closely remind the modulation of TRPM6 described by others in murine kidney and colon following Mg(2+) dietary restriction. We sought to validate our observations by investigating whether TRPM6 modulation by extracellular Mg(2+)also occurs in vivo. To this aim, we exploited a model consisting of rats fed either with a Mg(2+)-deficient or a Mg(2+)-enriched diet, and studied TRPM6 expression in breast and kidney tissues. Immunohistochemical and western blot analyses confirmed that rat mammary tissues express TRPM6 protein levels similar to those found in the kidney, and that protein expression is modulated by dietary Mg(2+). In particular, Mg(2+) restriction upregulated TRPM6 expression, while Mg(2+) supplementation resulted in a significant decrease in protein levels. This work confirms and extends our previous results on TRPM6 modulation by Mg(2+) availability in mammary tissues. Further studies are required to clarify the functional significance of these findings, and the role of TRPM6 in tissue-specific magnesium homeostasis.

Role of 5-lipoxygenase in resveratrol mediated suppression of 7,12-dimethylbenz(α)anthracene-induced mammary carcinogenesis in rats

Substantial evidences suggest that lipoxygenase-catalyzed products have a strong influence on the development and progression of human cancers. The dietary phytochemical resveratrol has become a focus of intense research owing to its roles in cancer prevention. A single tail vein injection of 7,12-dimethylbenz(a)anthracene (DMBA) was given at a dose of 0.5mg/0.2ml oil emulsion/100g body weight at 50days of age of female Sprague–Dawley rats. Rats were treated with resveratrol from 2weeks before DMBA injection (5weeks of animal age) and continued to 24weeks of the experimentation at a dose of 100μg/rat in the diet. We observed that resveratrol acts as a potent 5-lipoxygenase (5-LOX) inhibitor obtained from natural sources. Our result indicated that resveratrol is a strong antioxidant in reducing lipid peroxidation and preventing DNA damage. It significantly decreased the extent of DNA strand break, inhibited abnormal cell proliferation as evidenced by BrdU labeling index and also induced apoptosis in carcinogen-challenged rat mammary tissue. Increased TGF-β1 expression in resveratrol treated rats is thought to be one of the factors inducing apoptosis to suppress DMBA-induced mammary carcinogenesis.

Abstract 4212: Prevention of estrogen-induced breast cancer by antioxidants: Role of N-quinone oxidoreductase 1 (NQO1)

Abstract The importance of estrogens in the etiology of breast cancer is widely recognized. However, the exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. The objective of the current study was to characterize the role of NQO1 in the prevention of estrogen-induced DNA damage and mammary tumorigenesis by antioxidants vitamin C or butylated hydroxyanisole (BHA). NQO1 converts 17β-estradiol (E2)-quinones back to E2-catechols, thus making E2-quinones unavailable for oxidant damage. Female ACI rats were treated with E2 (3 mg); vitamin C (1% in drinking water); E2 + vitamin C; BHA (0.7% in diet); and E2 + BHA for up to 8 months. The female ACI rat is an established animal model to study the mechanisms of E2-induced breast cancer. No tumors were detected in the sham control, vitamin C and BHA treatment groups whereas in the E2 + vitamin C, E2 + BHA, and E2 treatment groups, the breast tumor incidences were 29, 24 and 82% respectively. mRNA and protein expression of NQO1 was analyzed in breast and breast tumor tissues of rats treated with E2 and antioxidants vitamin C or BHA. NQO1 was suppressed in E2-exposed mammary tissue after treatment of rats with E2 for 8 months and in mammary tumors. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. NQO1 enzyme activity was unchanged in mammary tissue of rats from all the treatment groups. However, NQO1 enzyme activity was decreased in E2-induced mammary tumors. Catechol-O-methyltransferase (COMT) activity was unchanged in the mammary tissue of all the groups except in the E2-treated group. Vitamin C or BHA co-treatment significantly decreased E2-mediated increase in 8-hydroxydeoxyguanosine (8-OHdG, an oxidative DNA damage marker) levels in mammary tissue. To investigate the relationship between NQO1 and 8-OHdG damage, human breast epithelial cell line MCF10A was treated with E2, vitamin C, BHA, dicumarol (NQO1 inhibitor), E2 + vitamin C, and E2 + BHA. E2 and dicumarol significantly increased 8-OHdG levels in MCF-10A cells whereas co-treatment with E2 and antioxidants reverted them back to the normal levels. Our studies suggest a protective role of NQO1 in E2-induced DNA damage and breast carcinogenesis. NQO1 may thus have a unique potential in the development of therapeutic strategies for the prevention of estrogen-induced neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4212. doi:10.1158/1538-7445.AM2011-4212

Abstract 5543: Partial inhibition of estrogen-induced breast cancer by tamoxifen in female ACI rats: Implications of oxidant stress

Abstract Epidemiological and experimental evidence strongly supports the role of estrogens in the etiology of breast cancer. Both estrogen receptor [ER]-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective ER modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ER and the resultant ER-dependent processes. Tamoxifen treatment for six months has previously been reported to result in complete abrogation of 17β-estradiol (E2)-induced breast cancer in female ACI rats. We investigated the effect of tamoxifen on breast tumor incidence in female ACI rats after treatment with tamoxifen for 8 months. Female ACI rats were treated with E2 (3 mg pellet, s.c.), tamoxifen (40 mg pellet, s.c.) or with a combination of E2 and tamoxifen. At the time of surgery, breast tumors were detected in the 44% of rats co-treated with tamoxifen + E2 compared to 82% in rats treated with E2 alone. The tumors were confirmed as breast adenocarcinomas by histopathological examination of paraffin-embedded tissues. Immunohistochemical and real time-PCR analysis indicated increased expression of ER-α and progesterone receptor in E2-treated mammary tissue compared to age-matched controls and inhibition of these receptors in the mammary tissue of tamoxifen-treated groups. Cytochrome P450 3A4 (CYP 3A4), and CYP 2D6, the main enzymes involved in tamoxifen metabolism were unchanged in the mammary tissue of all the treatment groups compared to controls. Tissue levels of oxidative stress markers were quantified in the mammary tissues of rats. Superoxide dismutase 2, catalase and glutathione peroxidase expressions were differentially altered in E2, tamoxifen and tamoxifen + E2 co-treated groups compared to age-matched controls. Co-treatment with tamoxifen did not decrease E2-mediated increases in 8-iso-prostane F2α levels, a marker of lipid peroxidation/oxidative stress. Oxidative stress-mediated DNA damage marker, 8-hydroxydeoxyguanosine, was quantified in the mammary tissues of all the treatment groups. Co-treatment of tamoxifen with E2 did not inhibit E2-mediated increase in 8-hydroxydeoxyguanosine levels in mammary tissue. In summary, our studies suggest that inhibition of ER-mediated processes by tamoxifen does not completely abrogate estrogen-dependent breast tumor development in a rat model of estrogen-induced breast cancer. Our results further demonstrate that tamoxifen does not decrease E2-mediated oxidant stress and DNA damage. Thus, chronic exposure to estrogen-mediated oxidative stress may contribute to the development of breast cancer in rats co-treated with E2 and tamoxifen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2011-5543

Breast histomorphometry of rats treated with estrogen and/or progestogen

To evaluate the breast histomorphometric changes in rats treated with estrogen and/or progestogen for a short period of time.Forty oophorectomized rats were divided into four groups: GC, vehicle; GE, treated with estradiol benzoate (37.6 mg/animal); GP, treated with medroxyprogesterone acetate (11.2 mg/animal) and GEP, treated with estradiol benzoate (37.6 mg/animal) plus medroxyprogesterone acetate (11.28 mg/animal). In GE group, estradiol was administered subcutaneously for seven days; in GEP group, estradiol was administered once in a day for the first seven days and the progestogen over the next 23 days both subcutaneously. Twenty-four hours after the last hormone administration, the animals were killed upon deep anesthesia and the first inguinal breasts were removed, fixed in 10% formaldehyde and processed to be included in paraffin, with the sections being stained by hematoxylin-eosin. Morphology and the area occupied by mammary parenchyma were assessed, with the data undergoing analysis of variance followed by the Kruskal-Wallis test (p < 0.05).The control group breasts were found atrophic and, in GE and GEP group animals, typical alveoli with secretion inside are present; in progestogen-treated animals (GP), alveoli formed by large cells occupying almost the entire alveolar lumen are noted. Morphometric analysis showed a larger mammary parenchyma area in hormone-treated animals (GE = GP > GEP > GC; p < 0.05).Estradiol and progestogen had a proliferative effect on mammary parenchyma. However, prior estradiol administration changes the progestogen action on rat mammary tissue.

Targeted Multifunctional Multimodal Protein-Shell Microspheres as Cancer Imaging Contrast Agents

In this study, protein-shell microspheres filled with a suspension of iron oxide nanoparticles in oil are demonstrated as multimodal contrast agents in magnetic resonance imaging (MRI), magnetomotive optical coherence tomography (MM-OCT), and ultrasound imaging. The development, characterization, and use of multifunctional multimodal microspheres are described for targeted contrast and therapeutic applications. A preclinical rat model was used to demonstrate the feasibility of the multimodal multifunctional microspheres as contrast agents in ultrasound, MM-OCT and MRI. Microspheres were functionalized with the RGD peptide ligand, which is targeted to α(v)β₃ integrin receptors that are over-expressed in tumors and atherosclerotic lesions. These microspheres, which contain iron oxide nanoparticles in their cores, can be modulated externally using a magnetic field to create dynamic contrast in MM-OCT. With the presence of iron oxide nanoparticles, these agents also show significant negative T2 contrast in MRI. Using ultrasound B-mode imaging at a frequency of 30 MHz, a marked enhancement of scatter intensity from in vivo rat mammary tumor tissue was observed for these targeted protein microspheres. Preliminary results demonstrate multimodal contrast-enhanced imaging of these functionalized microsphere agents with MRI, MM-OCT, ultrasound imaging, and fluorescence microscopy, including in vivo tracking of the dynamics of these microspheres in real-time using a high-frequency ultrasound imaging system. These targeted oil-filled protein microspheres with the capacity for high drug-delivery loads offer the potential for local delivery of lipophilic drugs under image guidance.

Further studies on the potential contribution of acetaldehyde accumulation and oxidative stress in rat mammary tissue in the alcohol drinking promotion of breast cancer

There is available evidence supporting a positive association between alcohol intake and risk of breast cancer. However, there is limited information regarding possible mechanisms for this effect. Past studies from our laboratory suggest that acetaldehyde accumulation in mammary tissue after alcohol intake may be of particular relevance and that cytosolic and microsomal in situ bioactivation of ethanol to acetaldehyde and free radicals and the resulting stimulation of oxidative stress could be a significant early event related to tumor promotion. In the present studies repetitive alcohol drinking for 28 days was found to produce significant decreases in the mammary tissue content of GSH and alpha tocopherol and in glutathione S-transferase or glutathione reductase activities. In contrast, glutathione peroxidase activity was slightly increased. Malondialdehyde determinations did not show the occurrence of lipid peroxidation while the xylenol orange procedure gave positive results. The mammary microsomal metabolism of ethanol to acetaldehyde was not induced after an acute dose of ethanol or acetone able to induce the activity of its liver counterpart. The cytosolic pathway of alcohol metabolism instead was significantly enhanced by these two treatments. No increased generation of comet images was found either in mammary tissue or in liver under the experimental conditions tested. Results suggest that, while acetaldehyde accumulation in mammary tissue could be a critical event resulting from increasing production of acetaldehyde in situ plus an additional amount of it arriving via blood, other factors such as poor handling of the accumulated acetaldehyde could be also relevant.

Inhibition of rat mammary microsomal oxidation of ethanol to acetaldehyde by plant polyphenols

We previously reported that the microsomal fraction from rat mammary tissue is able to oxidize ethanol to acetaldehyde, a mutagenic-carcinogenic metabolite, depending on the presence of NADPH and oxygen but not inhibited by carbon monoxide or other cytochrome P450 inhibitors. The process was strongly inhibited by diphenyleneiodonium, a known inhibitor of NADPH oxidase, and by nordihydroguaiaretic acid, an inhibitor of lipoxygenases. This led us to suggest that both enzymes could be involved. With the purpose of identifying natural compounds present in food with the ability to decrease the production of acetaldehyde in mammary tissue, in the present studies, several plant polyphenols having inhibitory effects on lipoxygenases and of antioxidant nature were tested as potential inhibitors of the rat mammary tissue microsomal pathway of ethanol oxidation. We included in the present screening study 32 polyphenols having ready availability and that were also tested against the rat mammary tissue cytosolic metabolism of ethanol to acetaldehyde. Several polyphenols were also able to inhibit the microsomal ethanol oxidation at concentrations as low was 10-50 μM. The results of these screening experiments suggest the potential of several plant polyphenols to prevent in vivo production and accumulation of acetaldehyde in mammary tissue.

Opening the Window to Cancer: Potential Mechanism behind Increased Susceptibility in Rats Exposed Prenatally to BPA

Exposure to environmental factors before birth or during other critical periods of development can cause subtle changes in a tissue’s molecular foundations, leading to health effects later in life. Prenatal exposure to bisphenol A (BPA) is linked to cellular and structural changes in the mammary glands of adult rats and increased susceptibility to chemically induced cancer. New research now suggests a possible mechanism of action for this increase in cancer susceptibility via altered protein expression patterns in rat mammary gland tissue [EHP 118(11):1614–1619; Betancourt et al.]. Pregnant rats were exposed to 0, 25, or 250 μg BPA/kg/d from day 10 to 21 postconception, and their female offspring were given a single dose of the cancer-inducing compound dimethylbenzanthracene (DMBA) at 50 or 100 days after birth (i.e., young adulthood). Experiments were conducted to determine the relationship between prenatal BPA exposure and the expression of proteins intrinsic to the growth and development of the mammary gland in adulthood. These proteins included estrogen receptor-α (ER-α); PR and Bcl-2, downstream targets of ER-α; steroid receptor coactivators (SRCs) 1 to 3, which influence ER-α transcriptional activity; and several growth factor receptors and signaling molecules that direct cell proliferation and programmed death. Body weight and hormonally sensitive end points (time to vaginal opening, serum levels of the hormones 17β estradiol and progesterone, and estrous cyclicity) were assessed but found to be unchanged in relation to prenatal BPA exposure. ER-α, PR, and Bcl-2 were significantly downregulated and SRC-3 and some signaling molecules were upregulated in rats exposed prenatally to BPA. DMBA administered to rats at 50 days did not yield significant differences in tumor incidence between those with or without prenatal BPA exposure. Among animals that received DMBA at 100 days, all assayed proteins were significantly upregulated in the BPA-exposed rats, and cell proliferation was enhanced, but apoptosis was unchanged. These animals also had decreased time to tumor formation and increased tumor incidence and severity. These findings suggest that prenatal exposure to BPA in rats alters expression of key receptors and components of cellular signaling pathways in the mammary gland in adulthood, consequently increasing the tissue’s susceptibility to chemically induced cancer. Whether this occurs in humans is unknown. Data from the Centers for Disease Control and Prevention indicate human exposure to the chemical is widespread, with approximately 95% of Americans estimated to have detectable levels of BPA metabolites in their urine. Continued research is necessary to elucidate the mechanisms by which BPA exposure early in life influences the development of permanent effects in maturity.

Identification of stable and oestrus cycle-independent housekeeping genes in the rat mammary gland and other tissues

The function and development of the rat mammary gland is dependent on the oestrus cycle. Normalization of gene expression in mammary gland samples assessed by quantitative RT-PCR therefore requires housekeeping genes (HKGs) which are stably expressed during the oestrus cycle. mRNA expression of 10 HKGs was measured in the rat mammary gland at different phases of the oestrus cycle. In addition, mRNA expression of the HKGs was measured in a panel of other rat tissues comprising laser microdissected mammary gland alveolar lobules and interlobular connective tissue and macrodissected mammary gland, liver, skeletal muscle, colon and ovary samples. Expression and ranking of HKGs varied between tissues and oestrus cycle phases and several HKGs were necessary for normalization between samples. In the mammary gland samples, three HKGs (Sdha, Tbp, and Atp5b) were identified as the optimal combination of stably expressed genes across oestrus cycle phases. For normalization between samples from the entire panel of rat tissues, eight HKGs (Rps18, Eef1a1, B2m, Actb, Tbp, Hprt, Pgk1, and Sdha) were identified as the optimal combination. These HKGs are of general relevance for studies comparing gene expression between different rat tissues.

αS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form

BackgroundCaseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known.ResultsIn this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both αS1- and β-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature β-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature αS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of αS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of αS1-casein with membranes.ConclusionsThese experiments reveal for the first time the existence of a membrane-associated form of αS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that αS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.

Retinoic acid attenuates lipopolysaccharide-induced inflammatory responses by suppressing TLR4/NF-κB expression in rat mammary tissue

The retinoids, a group of natural or synthetic derivatives of vitamin A, exert various anti-neoplastic and immunomodulatory actions. Recent studies have demonstrated that retinoic acid protects rats against lipopolysaccharide (LPS)-induced mastitis, but the mechanism of action is unclear. In the present study, an LPS-induced rat mastitis model and primary cultures of rat mammary epithelial cells were used to investigate the effect of retinoic acid on the TLR4/NF-κB signaling pathway. The data indicated that toll-like receptor 4 (TLR4) gene expression reached its peak value earlier in retinoic acid-treated rats than in the control group, and that retinoic acid significantly decreased NF-κB DNA binding activity and the level of IL-1β in the mammary gland. The animal study result was confirmed by an in vitro cell culture system trial. TLR4 protein expression and NF-κB DNA binding activity were significantly decreased in primary rat mammary epithelial cells pretreated with 1 μmol/l retinoic acid at 1 h post-LPS stimulation. IL-1β gene expression was also significantly decreased at 2, 4 and 8 h post-LPS stimulation. These findings demonstrate that direct action by retinoic acid leads to attenuation of the LPS-induced inflammatory response by suppression of the TLR4/NF-κB signalling system, thereby providing a novel explanation for the underlying effect proposed for retinoic acid in the protection of mammary tissue during LPS-induced acute mastitis.

Abstract 4405: Orthogonal detection of thymidine analogue based cell proliferation and apoptosis using click chemistry combined with antibody based progesterone receptor detection in estradiol treated rat model

Abstract Detection reagents which can simultaneously measure cell proliferation and apoptosis, or measure chronological changes in cell proliferation over the course of a treatment provide insight into efficacy and mechanisms of treatment on cell cycle and cell heath. Detection reagents which can temporally separate cell proliferation while combined with apoptosis detection provide a powerful new investigative tool. When 5-ethynyl-2′-deoxyuridine (EdU) incorporation is used in conjunction with bromodeoxyuridine (BrdU), cell proliferation measurements are made with orthogonal detection platforms permitting unambiguous identification of proliferation events. In rat mammary epithelial tissue changes in cell proliferation as a result of treatment combined with apoptosis detection demonstrates the value of click chemistry when combined with antibody based detection methods. We use the rat model to show changes in proliferation and apoptosis rates in mammary epithelial tissue, after alteration in hormone treatment. Use of click chemistry labeling in ovariectomized rats shows a decrease in proliferation rate from their basal levels, while estradiol treatment results in an increase in proliferation rate over basal level. Cessation of a 7 day estradiol treatment causes a drop in proliferation rate and an increase in apoptosis which can be efficiently detected using click chemistry. Progesterone receptor levels are measured simultaneously with proliferation rate when click chemistry based detection is combined with antibody based detection methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4405.

Metabolization of nifurtimox and benznidazole in cellular fractions of rat mammary tissue

Two nitroheterocyclic drugs, nifurtimox (NFX) and benznidazole (BZ), used in the treatment of Chagas' disease have serious side effects attributed to their nitroreduction to reactive metabolites. Here, we report that these drugs reach the mammary tissue and there they could undergo in situ bioactivation. Both were detected in mammary tissue from female Sprague-Dawley rats after their intragastric administration. Only NFX was biotransformed by pure xanthine-oxidoreductase and from tissue cytosol. These activities were purine dependent and were inhibited by allopurinol. Also, only NFX was biotransformed by microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and was inhibited by carbon monoxide and partially by diphenyleneiodonium. NFX treatment produced significant decrease in protein sulfhydryl content after 1, 3 and 6 hours; no increases in protein carbonyl content at any time tested and significantly higher levels of lipid hydroperoxides at 3 and 6 hours; besides, ultrastructural observations after 24 hours showed significant differences in epithelial cells compared to control. These findings indicate that NFX might be more deleterious to mammary tissue than BZ and could correlate with early reports on its ability to promote rat mammary tissue toxicity.

Transcriptome analysis of mammary tissues reveals complex patterns of transporter gene expression during pregnancy and lactation

As a complex Ca2+-rich fluid mixture of water, casein, lactose and several ions, milk secretion requires a number of unknown transporters, which can be identified by a genome-wide microarray study in mammary tissues of lactating animals. Ca2+ was reported to be secreted across mammary epithelial cells through the transcellular pathway, presumably involving TRPC (canonical transient receptor potential) channels. In the present study, we have used quantitative real-time PCR to demonstrate that the human mammary cell line MCF-7, as well as rat mammary tissues from pregnant and lactating rats, expressed TRPC1, TRPC5 and TRPC6. Expression of TRPC1, TRPC5 and TRPC7 were markedly up-regulated, whereas that of TRPC3 and TRPC4 was down-regulated in the early lactating period. To further identify other transporter genes affected by lactation, a highly sensitive Illumina microarray featuring Bead Array technology was performed on RNA samples from mammary tissues of lactating rats. We found that, of the 384 transcripts changed during lactation, 31 transcripts were involved in the transport of water and electrolytes, such as Ca2+, Na+, K+, Cl-, I-, Fe2+, sulfate and phosphate. The present study, therefore, provides information for further investigation of the mechanism of lactation-induced transport adaptation in mammary epithelial cells.

Proteomic analysis of endogenous conjugated linoleic acid biosynthesis in lactating rats and mouse mammary gland epithelia cells (HC11)

This study was conducted to investigate the amount of CLA synthesized endogenously by rat mammary tissues in response to TVA (a precursor for cis-9, trans-11 CLA endogenous synthesis) treatment as well as the differences in the protein expression of genes encoding the biosynthesis of CLA in rat mammary tissue and mouse mammary gland epithelia cells (HC11). Treatment with TVA resulted in improved CLA productivity. Furthermore, 2-DE revealed two spots in samples of mammary tissues and one spot in samples of mammary gland epithelia cells (HC11) that were consistently altered in the TVA treatment groups when compared with the control group (non-fatty acid). The mRNA expression patterns of three of the proteins (PDI, PRDX2, LAMR1), as measured by real-time PCR, were similar to the pattern of protein abundance. In addition, the expression of SCD mRNA in the mammary tissue of rats and HC11 cell treated with TVA was higher than in the control group. Our results suggest that the identified proteins may be related to CLA biosynthesis in mammary tissue.

Citrus auraptene suppresses cyclin D1 and significantly delays N-methyl nitrosourea induced mammary carcinogenesis in female Sprague-Dawley rats

BackgroundBreast cancer is a major problem in the United States leading to tens of thousands of deaths each year. Although citrus auraptene suppresses cancer in numerous rodent models, its role in breast cancer prevention previously has not been reported. Thus, our goal was to determine the anticarcinogenic effects of auraptene against breast cancer.MethodsThe effects of auraptene on cell proliferation of MCF-7 and MDA-MB-231 human breast carcinoma cells in culture was assessed by measuring metabolism of a substrate to a formazan dye. Dietary effects of auraptene on tumor incidence, multiplicity and latency were studied in the N-methyl nitrosourea (MNU) induced mammary carcinogenesis model in female Sprague Dawley rats. The concentration of auraptene in rat tissues was analyzed by reverse phase HPLC. Cyclin D1 expression in MCF-7 cells and rat tumors was measured by western blot.ResultsAuraptene (500 ppm) significantly delayed median time to tumor by 39 days compared to the MNU only group (p < 0.05, n = 24–26). Auraptene (10 μM) reduced Insulin like Growth Factor-1 (IGF-1, 10 ng/mL)-induced cyclin D1 expression by 40% in MCF-7 cells. In comparison, western blot analysis of rat mammary tumors (n = 10 per group) confirmed that auraptene (500 ppm) significantly reduced (p < 0.05) cyclin D1 expression by 49% compared to the MNU only group. Analysis of rat mammary tissue extract by HPLC with fluorescence detection indicated an average concentration (means ± S.E.) of 1.4 ± 0.5 μM and 1.8 ± 0.3 μM in the normal mammary glands of the auraptene 200 ppm and 500 ppm groups, respectively. The concentration (means ± S.E.) of auraptene in the mammary tumors of the auraptene 200 ppm group was 0.31 ± 0.98 μM.ConclusionOverall, these observations suggest that the predominant effect of auraptene was to delay the development of tumors possibly through the suppression of cyclin D1 expression. These results point to the potential chemopreventive effects of auraptene in mammary carcinogenesis.

Common wound healing signature in murine and human involuting breast, implications for pregnancy-associated breast cancer.

Abstract Abstract #1049 Full term pregnancy increases breast cancer risk for up to ten years after childbirth and breast cancer diagnosed during this time has been referred to as pregnancy associated breast cancer (PABC). PABC patients have high rates of metastasis, which is independent of known prognostic factors. Following parturition or lactation, during involution, the breast returns to its pre-pregnant state and in the rat model, we found mammary gland involution employs tissue remodeling programs activated during wound healing and inflammation including high matrix metalloproteinase -2, -3, and -9 activities, release of bioactive fragments of FN and LN, deposition of fibrillar collagen, and increased cytokine levels, including TGFβ1, TGFβ3 and CTGF. We hypothesize that this physiologically normal, but pro-inflammatory remodeling of the gland accounts for the high rate of metastases seen in PABC.&amp;#x2028; To further define involution features common to wound healing that may account for the metastasis promoting attributes of PABC, the question of whether macrophages are recruited to the involuting gland was investigated in both rat mammary and human breast tissue. Using the macrophage marker CD68, a lysosomal associated protein, the number of CD68 positive cells in rat mammary glands increased more than 6 fold during involution. Involution-associated macrophages were identified as subtype M2-like by arginase-1 expression and lack of subtype M1-marker iNOS expression. To address the question of whether breast involution in women also is characterized by a wound-healing signature, formalin fixed breast biopsy tissues were obtained for women who were either pregnant, lactating, or whose breast tissue was actively involuting at the time of biopsy. Using a novel computer assisted imaging program, over 200 images of human breast tissue have been quantitated for CD68. CD68 staining in the human breast followed the same trend as in rat, with 5 times more staining in involuting compared to pregnant or lactating tissue. Total leukocyte number, as assessed by CD45 antibody, and oncofetal ECM protein tenascin-C staining were also increased in involuting human breast tissue. With respect to these three stromal markers, involuting human breast tissue was more similar to breast cancer stroma (N=50 images) than stroma from breasts of nulliparous women (N=30 images). These data demonstrate for the first time that human mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC.&amp;#x2028; In a newly developed animal model for PABC, MCF10DCIS cells were injected orthotopically into SCID mice whose mammary glands were either quiescent (virgin mice) or actively remodeling following weaning (involuting mice). Tumor incidence was 78% in involuting and 50% in virgin mice. Further, tumor multiplicity, size and DCIS conversion to locally invasive lesions were significantly higher in the involuting mice than control virgin mice. This model is being utilized for mechanistic and chemopreventive studies of PABC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1049.