Female Rat Mammary Gland Research Articles

CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats

Cancer is a life-threatening disease that affects approximately 18 million individuals worldwide. Breast cancer is the most common female neoplasm globally with more than 276,480 new cases of invasive breast cancer expected to be diagnosed in women in the U.S. alone in 2020. Genetic and epigenetic factors play role in the carcinogenesis and progression of this disease. In this study, MCF-7 adenocarcinoma cells were transfected with CRISPR/Cas9 plasmid to either knock out CDK11 or to activate CDH1. Treated cells were allografted into the mammary glands of female rats (150–190 g, 6–8 weeks) to evaluate the capability of these cells to control cancer progression and metastasis. qPCR data revealed a significant downregulation of CDK11 and upregulation of CDH1. Cell cycle analysis and apoptosis assays indicated the knockout of CDK11 and simultaneous activation of CDH1 resulted in cell cycle arrest at G2/M phase and accumulation of cells at G2. Meanwhile, the percentage of cells that underwent late apoptosis increased in both genome editing hits. Histopathological sectioning data indicated that untransfected MCF-7 cells were capable of developing tumors in the mammary gland and initiation g angiogenesis. Transfected cells significantly restricted cancer cell infiltration/invasion by minimally localizing tumors and inhibiting angiogenesis. Although further investigation is needed, the present data indicate the potentiality of using CRISPR/Cas9-based therapy as a promising approach to treat breast cancer. Impact: these data indicate targeting cancer-related genes via any genome editing tool might represent a novel approach to combat cancer. How to cite: Al-Mulhim F, Alqosaibi AI, Al-Muhnna A, et al. CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.06.002

Role of organophosphorous pesticides and acetylcholine in breast carcinogenesis.

Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carcinogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas. The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c-myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.

Development and Validation of an Analytical Method for Quantitation of Alpha-Pinene Oxide in Rodent Blood and Mammary Glands by GC-MS.

Alpha-pinene is a monoterpene found in the oil of coniferous trees and has a wide variety of applications. Alpha-pinene oxide (APO) is a potential reactive metabolite of alpha-pinene in rodents. The objective of this work is to validate a gas chromatography-mass spectrometry method to quantitate APO in rat and mouse blood and mammary glands in support of studies investigating the toxicity and toxicokinetic behavior of alpha-pinene. The method was validated in male Sprague Dawley rat blood over the concentration range of 5-250 ng/mL. Matrix standard curves were linear (r ≥ 0.99), and accuracy (percent relative error, %RE) was ≤±15% for standards at all levels. Intra- and interday precision (percent relative standard deviation, %RSD) and accuracy (%RE) were evaluated at three concentration levels (10, 50 and 200 ng/mL) and were ≤6.3% and ≤±5.4%, respectively. The limit of detection, determined from the SD of the limit of quantitation (5 ng/mL), was 1.06 ng/mL. Standards as high as 25,000 ng/mL could be accurately quantified after diluting to the validated range (%RE ≤ ±7.1%; %RSD ≤ 5.8%). APO was stable in rat blood for at least 70 days in frozen storage (-80°C). APO could accurately be quantified in male and female Hsd:Sprague Dawley® SD® rat and B6C3F1 mouse blood (mean %RE ≤ ±5.3%; %RSD ≤ 7.8%) and female B6C3F1 and Sprague Dawley rat mammary glands (mean %RE ≤ ±14.6%; %RSD ≤ 8.1%) using a primary matrix standard curve. These results demonstrate that the method is suitable for the analysis of APO in rodent blood and mammary glands generated from toxicokinetic and toxicology studies.

The Rat Mammary Gland as a Novel Site of Expression of Melanin-Concentrating Hormone Receptor 1 mRNA and Its Protein Immunoreactivity.

Lactation is a complex physiological process, depending on orchestrated central and peripheral events, including substantial brain plasticity. Among these events is a novel expression of pro-melanin-concentrating hormone (Pmch) mRNA in the rodent hypothalamus, such as the ventral part of the medial preoptic area (vmMPOA). This expression reaches its highest levels around postpartum day 19 (PPD19), when dams transition from lactation to the weaning period. The appearance of this lactation-related Pmch expression occurs simultaneously with the presence of one of the Pmch products, melanin-concentrating hormone (MCH), in the serum. Given the relevance of the MPOA to maternal physiology and the contemporaneity between Pmch expression in this structure and the weaning period, we hypothesized that MCH has a role in the termination of lactation, acting as a mediator between central and peripheral changes. To test this, we investigated the presence of the MCH receptor 1 (MCHR1) and its gene expression in the mammary gland of female rats in different stages of the reproductive cycle. To that end, in situ hybridization, RT-PCR, RT-qPCR, nucleotide sequencing, immunohistochemistry, and Western blotting were employed. Although Mchr1 expression was detected in the epidermis and dermis of both diestrus and lactating rats, parenchymal expression was exclusively found in the functional mammary gland of lactating rats. The expression of Mchr1 mRNA oscillated through the lactation period and reached its maximum in PPD19 dams. Presence of MCHR1 was confirmed with immunohistochemistry with preferential location of MCHR1 immunoreactive cells in the alveolar secretory cells. As was the case for gene expression, the MCHR1 protein levels were significantly higher in PPD19 than in other groups. Our data demonstrate the presence of an anatomical basis for the participation of MCH peptidergic system on the control of lactation through the mammary gland, suggesting that MCH could modulate a prolactation action in early postpartum days and the opposite role at the end of the lactation.

Milk Thistle Seed Extract Favorably Affects Lactation and Development of Mammary Gland in Female Rats

Milk thistle seed extract (Silbanummarianum), is a mixture of flavonolignans. The main active ingredients in S. marianum are silybin (silibinin), sylichrisitn, and silydianin, commonly referred to collectively as Sylimarin. Studies showed that Silymarin has anti-inflammatory and antioxidant properties. Seventy-two pregnant female Wister rats were randomly assigned to 3 study groups (24 rats in each group). Group 1, or a control group (C group), received drinking water daily for 14 days from the 8th day of gestation until the delivery. Group 2 (M) and Group 3 (S) received Metoclopramide (5mgkgday) and S. marianum seed extract (200 mgkgday), respectively, for 14 days. Dams’ body weights were monitored throughout the pregnancy. The weights of the newborns in the litter (which previously adjusted to 8 offsprings) were measured on the first day of parturition, and 8 dams from each group were randomly selected and sacrificed. Pregnant female rats in (S) group had litter of significantly higher body weight and increase serum prolactin level in pregnant female rats (p< 0.05). Milk thistle seed extract led to a marked hyperplasia in mammary gland tissue, with strong expression of prolactin receptors. Wister ratsinS. marianum and Metoclopramide groups showed significant increase in serum prolactin levels, had heavier offsprings, and histological and immunohistochemistry changes as compared to the animals that stopped receiving the treatment in the same group (p<0.05). We concluded that S. marianum enhances milk production and mammary gland development (galactogogues effect).

Immunohistochemical and ultrastructural evidence for telocytes in the different physiological stages of the female rat mammary gland

IntroductionTelocytes (TCs) are recently described to integrate a variety of different cells.Aim of the work.The aim was to investigate the presence of TCs in the rat mammary gland at its different physiological stages. Material and methodsTwenty four adult female albino rats were classified into 4 groups: resting, mid-pregnancy, lactating, and involution groups. Inguinal mammary glands were processed for immunohistochemical and transmission electron microscopic (TEM) examination. ResultsTCs were immune-positive for c-kit and CD34 and showed significant differences in the different studied groups indicating variable roles at the different stages. TEM results characterized TCs by its shape and the long slender and moniliform telopodes linking the cells into stromal networks. The extracellular exosomes, homo-cellular synapsis and hetero-cellular synapsis were observed. ConclusionOur study provides evidence for the presence of TCs in all stages of the gland; not only in the resting stage as proved by other studies, but with immune-labeling differences suggesting different structural and physiological roles of TCs according to the stage requirements. These functions might via controlling the proliferation during pregnancy and lactation and the involution of the gland after weaning. Thus, more future functional studies of TCs will be important to help understanding the mechanism by which TCs contribute to tissue homeostasis concerning the role of the stromal/epithelial interactions in mammary gland biology and pathology including breast cancer which would be revolutionary for future therapeutic applications.

Fennel (Foeniculum vulgare) leaf infusion effect on mammary gland activity and kidney function of lactating rats

Abstract. Rifqiyati N, Wahyuni A. 2019. Fennel (Foeniculum vulgare) leaf infusion effect on mammary gland activity and kidney function of lactating rats. Nusantara Bioscience 11: 101-105. Fennel (Foeniculum vulgare Mill) leaf, traditionally, is believed to have a potential in increasing and smoothing breast milk production. This study aimed to determine the effect of fennel leaf infusion on milk production and to know the side effects of its use. The material used in the research was infusion of fennel leaves (Foeniculum vulgare Mill) collected from Kopeng, Central Java. The research utilized 12 female rats each with 5 newborns off springs. The experiment was designed in Completed Random Design (CRD) with 4 treatments and 3 replications. Histological preparation of mammary glands was set using paraffin method with HE staining. Kidney function was observed through uric acid level in the blood. The results showed that the diameter of lactiferous ducts and of its lumen diameter were significantly influenced by 15 days fennel leaf infusion treatment. The largest lactiferous duct diameter observed was on P3 treatment group (452.97 ± 75.033 µm) and the smallest was observed in control groups (273.17 ± 38.746 µm). The numbers of active alveoli observed in treatment groups, i.e., in P1 (20 g/300 mL), P2 (40 g/300 mL), and P3 (60 g/300 mL), increased than inactive alveoli. The blood uric acid level observed was 4.0-4.6 mg/dl. The results suggested that the infusion of fennel leaf with a treatment dose of 60 g infusion in 300 mL distilled water administered for 15 days can significantly increase the diameter of lactiferous lumen of female rat mammary glands, and increase the diameter of the alveoli and the number of active alveolar mammary glands. Histological picture of mammary gland also showed that the female rats treated with dose of 60g infusion per 300 mL aquadest increased milk secretion and than the other treatment groups. The treatment also showed no significant side effects.

Detection of lithium in breast milk and in situ elemental analysis of the mammary gland.

Breast feeding provides considerable benefits to the infant and mother. However, a lithium-based psychiatric medication may cause side effects in the child. Using laser induced breakdown spectroscopy (LIBS), trace lithium levels were observed in the breast milk of lactating rats administered with lithium treatment postpartum. Subsequently, the mammary glands of female rats were analyzed using LIBS, energy dispersive X-ray fluorescence spectroscopy, and inductively coupled plasma mass spectrometry. Key biological elements iron, magnesium, cobalt, calcium, phosphorus, sodium, iodine, potassium, sulfur, chlorine and zinc were observed. Lithium at 1.06 µg/g was measured in the mammary glands of treated subjects, but was below the limit of detection in controls. Lithium also increased iodine content in the glands. Lithium is present in the breast milk and mammary glands of lithium treated female subjects and this is the likely route of entry to breast-fed infants.

Protective Effect of 3, 3-Diindolylmethane on Bisphenol A Activated GPR30, RAS, PI3K/Akt Estrogenic Signaling Pathway in Female Sprague-Dawley Rats

The present study was aimed to evaluate the protective effect of 3, 3-diindolylmethane (DIM) on bisphenol A (BPA) induced estrogen signaling in the mammary glands of female Sprague-Dawley rats. Chronic administration of BPA (10 μg/kg/bw) to rats exerts rapid estrogenic action via GPR30 and activate cascade of signaling molecule which induce cell proliferation in the mammary gland. Western blot analysis of mammary tissues shows over expression of GPR30, SRC, RAS, PI3K and Akt proteins and immunohistochemical analysis reveals over expression of PCNA and no significant changes in ERs. Further, oral administration of DIM (5 mg/kg/bw), alternative days to BPA treated rats for the period of 12 weeks show significant decrease in the expression pattern of GPR30, SRC, RAS, PI3K, Akt and PCNA. The results of our study demonstrate that BPA induces rapid action via the over expression of proteins in non-genomic estrogen signaling pathway. Administration of DIM inhibits the action of BPA by modulating the expression of proteins involved in GPR30 cascade signaling pathway mediated cell proliferation in mammary glands of female rats.

Salvia officinalis L. induces alveolar bud growing in adult female rat mammary glands.

In traditional medicine Salvia officinalis (sage) has been used as menstrual cycle regulator. In the present study the effects of sage extract on breast tissue were examined. Fourteen female rats were divided into two groups: 1) Distilled water-treated rats (Con) that were gavaged with 1ml distilled water and 2) Saliva officinalis hydroalcoholic extract (SHE)-treated rats that were gavaged with 30mg/kg/body weight of sage extract for 30 days. The estrus cycle changes were monitored by daily examination of vaginal smear. Whole mounts of right pelvic breast were spread on the slide and stained by carmine. The number of alveolar buds (ABs) type 1 and 2 and lobules of mammary gland were scored. Tissue sections of left pelvic mammary gland were prepared and its histomorphometrical changes were measured. Blood samples were taken from dorsal aorta and estradiol and progesterone concentrations were measured using radioimmunoassay. Estrous cycles decreased significantly in SHE-treated animals. The number of alveolar buds and lobules in mammary gland whole mount of SHE-treated group were higher than the Con group. The number and diameter of ducts in histological section of mammary gland in SHE-treated group increased as compared to the Con group. Sage promotes alveologenesis of mammary glands and it can be used as a lactiferous herb.

Cross-Talk in the Female Rat Mammary Gland: Influence of Aryl Hydrocarbon Receptor on Estrogen Receptor Signaling.

Background:Cross-talk between the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER) plays a major role in signaling processes in female reproductive organs.Objectives:We investigated the influence of the AHR ligand 3-methylcholanthrene (3-MC) on ER-mediated signaling in mammary gland tissue of ovariectomized (ovx) rats.Methods:After 14 days of hormonal decline, ovx rats were treated for 3 days with 4 μg/kg 17β-estradiol (E2), 15 mg/kg 8-prenylnaringenin (8-PN), 15 mg/kg 3-MC, or a combination of these compounds (E2 + 3-MC, 8-PN + 3-MC). Whole-mount preparations of the mammary gland were used to count terminal end buds (TEBs). Protein expression studies (immunohistochemistry, immunofluorescence), a cDNA microarray, pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the interaction between AHR- and ER-mediated signaling pathways.Results:E2 treatment increased the number of TEBs and the levels of Ki-67 protein and progesterone receptor (PR); this treatment also changed the expression of 325 genes by more than 1.5-fold. Although 3-MC treatment alone had marginal impact on gene or protein expression, when rats were co-treated with 3-MC and E2, 3-MC strongly inhibited E2-induced TEB development, protein synthesis, and the expression of nearly half of E2-induced genes. This inhibitory effect of 3-MC was partially mirrored when 8-PN was used as an ER ligand. The anti-estrogenicity of ligand-activated AHR was at least partly due to decreased protein levels of ERα in ductal epithelial cells.Conclusion:Our data show transcriptome-wide anti-estrogenic properties of ligand-activated AHR on ER-mediated processes in the mammary gland, thereby contributing an explanation for the chemopreventive and endocrine-disrupting potential of AHR ligands.Citation:Helle J, Bader MI, Keiler AM, Zierau O, Vollmer G, Chittur SV, Tenniswood M, Kretzschmar G. 2016. Cross-talk in the female rat mammary gland: influence of aryl hydrocarbon receptor on estrogen receptor signaling. Environ Health Perspect 124:601–610; http://dx.doi.org/10.1289/ehp.1509680

Effect of genistein intake on pubertal and menopausal female albino rat mammary gland

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Background data of spontaneous tumors in F344/DuCrlCrlj rats

We investigated the 2-year survival rate and incidence of spontaneous tumors in F344/DuCrlCrlj rats used in carcinogenicity studies of chemical substances. Records for animals used in the control groups of carcinogenicity studies which were conducted during the last 10 years were obtained from the database of the Japan Bioassay Research Center (JBRC). Six hundred ninety-nine males and 550 females were used in 14 and 11 inhalation studies, respectively, and 500 animals of each sex were used in 10 male and 10 female oral studies. In each study, SPF (specific pathogen free) animals were housed for 2 years (104 weeks) as control groups in the carcinogenicity studies. All animals underwent necropsy and histopathological examination. Each study was conducted in accordance with the Good Laboratory Practice. The incidence of interstitial cell tumors was highest in both inhalation studies and oral studies (inhalation studies 86.1%, oral studies 68.6%). Tumors which had an incidence of 6% or higher were adenoma of the pituitary, C-cell adenoma of the thyroid, and mononuclear cell leukemia (LGL leukemia) of the spleen in male and female rats; fibroma of the subcutaneous tissue, adrenal pheochromocytoma, and islet cell adenoma of the pancreas in male rats; and endometrial stromal polyps and fibroadenoma of the mammary gland in female rats. Tumors other than the above had rare incidence rates. A clear difference in the incidence of spontaneous tumors was not observed between the inhalation and oral studies. The incidences of spontaneous tumors in control groups of previous oral studies are similar to our findings. There are no other reports of the spontaneous tumor incidence in the control groups of inhalation studies using F344/DuCrlCrlj rats. The 2-year survival rate was about 77% in both the inhalation and oral studies, and a gender difference was not observed. The F344/DuCrlCrlj rats used at JBRC had a higher 2-year survival rate than F344/N rats. This difference is possibly due to the low incidence of LGL leukemia in the F344/DuCrlCrlj rat. The incidences of spontaneous tumors in F344/DuCrlCrlj rats used in control groups of both inhalation and oral studies during the last 10 years at JBRC are similar to each other and similar to those reported in other studies. This is the first report on the incidence of spontaneous tumors in inhalation studies and contributes to the toxicological evaluation of studies using F344/DuCrlCrlj rats.

The histological changes of the female rat mammary gland during the fertile period with a special reference to E-cadherin expression

Background E-cadherin (E-cad) is a cell adhesion molecule, essential for the maintenance of the epithelial phenotype. Aim of work The current study aimed at showing the histological changes and the pattern of E-cad expression in the female rat mammary gland during different stages of their fertile period microscopically and morphometrically. Materials and methods Thirty-five adult female albino rats (3–4 months old) were divided into seven groups (five rats each): the rats in group I were not pregnant (resting) and groups II–VII were sacrificed at days 7 and 14 of each of their pregnancy, lactation, and weaning stages. The excised right inguinal mammary glands were processed for H&E and E-cad immunostaining. The sections were evaluated microscopically and morphometrically. Results The mammary gland of nonpregnant rats had abundant adipose connective tissue with few glandular elements. During pregnancy, there was a progressive increase in the alveoli, which became distended with milk, with prominent sac formation during lactation. The E-cad expression pattern was mainly observed as a strong positive linear membranous pattern in all borders of epithelial cell lining, whereas the myoepithelial cells were negative. These observations were found in different groups, except that of lactation, where the pattern was limited to the basolateral membranes of these epithelial cells. Morphometrically, the positive membranous epithelial cells for E-cad, its density area fraction and cytoplasmic positive reaction ranged from 7-83%, 3.9-6.8% and 0-7%, respectively in all groups. In late pregnancy, there was a marked increase in the negative epithelial cells (93%), with a significant decrease in density area fraction expression (3.9%), which was persistent during lactation. Positive E-cad expression of stromal cells was only observed during weaning. Conclusion The E-cad was mainly localized to the membranes of epithelial cell lining and absent in myoepithelial cells. The quantitative E-cad expression can represent a tool that ensures qualitative microscopic observations during the fertile period.

Mammary Gland Morphology and Gene Expression Differ in Female Rats Treated with 17β-Estradiol or Fed Soy Protein Isolate

Soy foods have been suggested to have both positive health benefits and potentially adverse effects as a result of their content of phytoestrogens. However, studies on the estrogenicity of soy foods are lacking. Here we directly compared the effects of soy protein isolate (SPI), the protein in soy infant formula, with those of 17β-estradiol (E2), on global gene expression profiles and morphology in the female rat mammary gland. Rats were fed AIN-93G diets containing casein or SPI beginning on postnatal d 30. Rats were ovariectomized on postnatal d 50 and treated with 5 μg/kg/d E2 or vehicle for 14 d. Microarray analysis revealed that E2 treatment altered expression of 780 genes more than or equal to 2-fold (P < 0.05), whereas SPI feeding altered expression of only 53 genes more than or equal to 2-fold. Moreover, the groups had only 10 genes in common to increase more than or equal to 2-fold. The combination of SPI feeding and E2 altered expression of 422 genes and reversed E2 effects on many mRNAs, including those involved in the c-myc signaling pathway, cyclin D1, and Ki67. ERα binding to its response element on the Tie-2/Tek and progesterone receptor promoters was increased by E2, but not SPI, and this promoter binding was suppressed by the combination of E2 + SPI for the Tie-2/Tek promoter but increased for the progesterone receptor promoter (P < 0.05). SPI reduced the ratio of epithelial to fat pad area and E2 + SPI reduced both epithelial and fat pad area (P < 0.05). These data suggest that SPI is only minimally estrogenic in the rat mammary gland even in the absence of endogenous estrogens.

Effect of Ageratum Conyzoides Leaf Extract On Histological Structure of Mammary Gland of Wistar Albino Rat.

           There are many causes of non-lactation after delivery in some dairy mammals and human female. In present investigation study was carried out to find out effect of Ageratum conyzoides leaf extract on histological structure of non lactating mammary gland of wistra white albino rat female. Ageratum conyzoides is supposed to induce lactation in non-lactating mammalian female including woman1. The effect of extract on non-lactating mammary gland after seven and fourteen days was observed on non – lactating mammary gland showed small size mammary gland with no secretion. After 7 days the epithelial lining showed growth and increasing size of secretary glands. Ducts were also seen increasing a proliferation. After 14 days secretory gland showed more tortures structure and secretion was accumulated in to lumen and gland. The 14 days treated female rat mammary gland showed more secretary activity indicating lactation was induced in lactating gland of mammary gland1. Key Words: albino rat, Phytochemical, Gangotry species, Ageratum conyzoides

Toxicity and carcinogenicity of androstenedione in F344/N rats and B6C3F1 mice

Androstenedione was marketed as a dietary supplement to increase muscle mass during training. Due to concern over long-term use, the NTP evaluated the subchronic and chronic toxicity and carcinogenicity of androstenedione in male and female F344/N rats and B6C3F1 mice. In subchronic studies, dose limiting effects were not observed. A chronic (2-year) exposure by gavage at 10, 20, or 50mg/kg in rats and male mice, and 2, 10, or 50mg/kg in female mice (50mg/kg, maximum feasible dose) was conducted. Increased incidences of lung alveolar/bronchiolar adenoma and carcinoma occurred in the 20mg/kg male rats and increases in mononuclear cell leukemia occurred in the 20 and 50mg/kg female rats, which may have been related to androstenedione administration. In male and female mice, androstenedione was carcinogenic based upon a significant increase in hepatocellular tumors. A marginal increase in pancreatic islet cell adenomas in male (50mg/kg) and female (2, 10, 50mg/kg) mice was considered to be related to androstenedione administration. Interestingly, incidences of male rat Leydig cell adenomas and female rat mammary gland fibroadenomas decreased. In conclusion, androstenedione was determined to be carcinogenic in male and female mice, and may have been carcinogenic in rats.

Abstract 3348: Naïve rat umbilical cord matrix stem cells abrogate mammary tumor growth through markedly enhanced tumor immune responses

Abstract Mesenchymal stem cells derived from the umbilical cord matrix (UCMSC) have great potential for therapeutic use in multiple diseases including cancer. We have demonstrated that un-engineered naïve human and rat UCMSC attenuate growth of several organ type tumors (Ayuzawa et al., Cancer Lett.2009; Ganta et al., Cancer Res, 2009, Doi et al., Cytotherapy 2010). Since UCMSC are easy to prepare in relatively large quantities and are poorly immunogenic in allogeneic transplantation, they are potentially useful in cancer therapy. However, the mechanism by which UCMSC attenuate tumor growth has not been studied rigorously. Hence, the objectives of this study were to examine the ability of UCMSC to control the growth of mammary tumors and to determine possible mechanisms by which UCMSC attenuate the tumor growth. Intratumoral injection of rat UCMSC markedly attenuated tumor growth of orthotopic Mat B III autografts in female F344 rat mammary gland. Histopathology of the tumors in the rat UCMSC group indicated that a large number of lymphocytes had infiltrated into the peritumoral area and were occasionally observed in the intratumoral area. Immunohistochemistry revealed that although CD3+ T lymphocytes were located mainly in the peritumoral area rather than in the intratumoral area, CD20+ cells were rarely seen suggesting that the majority of infiltrated lymphocytes in the rat UCMSC treated tumors were T cells. In addition, the treatment of rat UCMSC increased the CD 8+ cell infiltration throughout tumor tissue. CD68+ cells were scarcely observed in the tumors of the PBS control group but not in the rat UCMSC treated group. Transwell culture system-based in vitro migration assay revealed that rat UCMSC significantly enhanced migration of peripheral blood mononuclear cells. These results suggest that naïve rat UCMSC attenuated mammary tumor growth at least in part by markedly enhanced host immune responses against tumors. This research demonstrates that naïve rat UCMSC can be used as powerful anti-cancer therapeutic cells for breast cancer treatment. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU CVM Dean's Fund, NIH P20 RR017686, p20 RR1556, R21CA135599 and Kansas Bioscience Authority research fund. 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 3348. doi:10.1158/1538-7445.AM2011-3348

Modulatory influence of prepubertal biochanin A exposure on mammary gland differentiation and expression of estrogen receptor‐α and apoptotic proteins

The modulatory influence of the phytoestrogen biochanin A, an isoflavinoid found in red clover (Trifolium pratense), on the differentiation and proliferation of mammary epithelial cells and the expression of estrogen receptor-alpha (ER-alpha) in female prepubertal Sprague-Dawley rat mammary glands was examined, for which there have been no reports to date. Biochanin A (500 microg/g bw) was injected subcutaneously on days 16, 18 and 20 post-partum. The mammary gland was dissected out and terminal end buds, terminal ducts and lobules were counted. ER-alpha, Bcl2, Bax and caspase-3 expression were determined by immunohistochemistry. Estradiol benzoate (EB) (500 ng/g bw) and dimethyl sulphoxide (DMSO) were used as the reference and vehicle, respectively. The results showed a significant enhancement of differentiation at post-natal day (PND) 21 as well as at PND 50 in the mammary glands. There was a significant decrease of ER-alpha expression at PND 21 and an increased expression of the same at PND 50, whereas increased proliferation at PND 21 and increased apoptosis at PND 50 in the mammary glands were observed in biochanin A treated animals. The mode and magnitude of the effect of biochanin A was almost similar to that of EB. These findings suggested that prepubertal exposure to biochanin A modulated the regulatory processes and in turn enhanced the differentiation and development of mammary glands in female rats. These observations may have significance in human health.

Soy isoflavone genistein upregulates epithelial adhesion molecule E-cadherin expression and attenuates -catenin signaling in mammary epithelial cells

Breast cancer risk is highly modifiable by diet; however, mechanisms underlying dietary protection against mammary tumorigenesis remain poorly understood. A proportion of breast carcinomas is associated with deregulation of beta-catenin stability and amplification of c-Myc expression. We recently showed that dietary exposure to the soy isoflavone genistein (Gen) inhibited Wnt transduction in rat mammary epithelial cells in vivo. Here, we explored the role of Gen on cell adhesion protein, E-cadherin, expression to downregulate beta-catenin proto-oncogene function. In mammary glands of female rats exposed to dietary Gen, E-cadherin and beta-catenin protein levels were increased, concurrent with higher beta-casein gene expression. In HC11 mouse mammary epithelial cells, Gen diminished basal and Wnt-1-induced cell proliferation and attenuated Wnt-1 targets c-Myc and Cyclin D1 expression. Whereas, Gen had no effect on E-cadherin transcript levels, the abundance of membrane E-cadherin protein and of E-cadherin-beta-catenin adhesion complex was increased by Gen, attendant with downregulation of Wnt-1-induced free beta-catenin accumulation in cytosol. Gen inhibition of Wnt-induced c-Myc expression was mimicked by an estrogen receptor (ER)-beta-specific but not ER-alpha-specific agonist and was attenuated with loss of ER-beta expression, concordant with decreased E-cadherin expression. E-cadherin small-interfering RNA targeting eliminated Gen inhibition of Wnt-stimulated c-Myc expression and promoted Gen induction of basal c-Myc transcript levels and subsequent proliferation. Our studies identify E-cadherin as a Gen cellular target and demonstrate that the dichotomy in mammary epithelial response to Gen may be a function of cellular E-cadherin expression.