Effects of Pubertal Exposure to Butyl Benzyl Phthalate, Perfluorooctanoic Acid, and Zeranol on Mammary Gland Development and Tumorigenesis in Rats.

Endocrine-disrupting chemicals (EDCs)-including butyl benzyl phthalate (BBP), perfluorooctanoic acid (PFOA), and zeranol (α-ZAL, referred to as ZAL hereafter)-can interfere with the endocrine system and produce adverse effects. It remains unclear whether pubertal exposure to low doses of BBP, PFOA, and ZAL has an impact on breast development and tumorigenesis. We exposed female Sprague Dawley rats to BBP, PFOA, or ZAL through gavage for 21 days, starting on day 21, and analyzed their endocrine organs, serum hormones, mammary glands, and transcriptomic profiles of the mammary glands at days 50 and 100. We also conducted a tumorigenesis study for rats treated with PFOA and ZAL using a 7,12-dimethylbenz[a]anthracene (DMBA) model. Our results demonstrated that pubertal exposure to BBP, PFOA, and ZAL affected endocrine organs and serum hormones, and induced phenotypic and transcriptomic changes. The exposure to PFOA + ZAL induced the most phenotypic and transcriptomic changes in the mammary gland. PFOA + ZAL downregulated the expression of genes related to development at day 50, whereas it upregulated genes associated with tumorigenesis at day 100. PFOA + ZAL exposure also decreased rat mammary tumor latency, reduced the overall survival of rats after DMBA challenge, and affected the histopathology of mammary tumors. Therefore, our study suggests that exposure to low doses of EDCs during the pubertal period could induce changes in the endocrine system and mammary gland development in rats. The inhibition of mammary gland development by PFOA + ZAL might increase the risk of developing mammary tumors through activation of signaling pathways associated with tumorigenesis.

The use of patient-derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure.

Ex vivo mammary explant systems are an excellent model to study interactions between epithelium and stromal cell types because they contain physiologically relevant heterotypic interactions in the background of genetically diverse patients. The intact human mammary tissue, termed patient-derived explant (PDE), can be used to investigate cellular responses to a wide variety of external stimuli in situ. For this study, we examined the impact of cytokines or environmental chemicals on macrophage phenotypes. We demonstrate that we can polarize macrophages within human breast tissue PDEs toward M1 or M2 through the addition of interferon-γ (IFNγ) + lipopolysaccharide (LPS) or interleukin (IL)-4 + IL-13, respectively. Elevated expression levels of M(IFNγ + LPS) markers (HLADRA and CXCL10) or M(IL-4 + IL-13) markers (CD209 and CCL18) were observed in cytokine-treated tissues. We also examined the impact of the endocrine-disrupting chemical, benzophenone-3, on PDEs and measured significant, yet varying effects on macrophage polarization. Furthermore, a subset of the PDEs respond to IL-4 + IL-13 through downregulation of E-cadherin and upregulation of vimentin which is reminiscent of epithelial-to-mesenchymal transition (EMT) changes. Finally, we were able to show immortalized nonmalignant breast epithelial cells can exhibit EMT characteristics when exposed to growth factors secreted by M(IL-4 + IL-13) macrophages. Taken together, the PDE model system is an outstanding preclinical model to study early tissue-resident immune responses and effects on epithelial and stromal responses to stimuli found both endogenously in the breast and exogenously as a result of exposures.

Inter-Individual Variation in Response to Estrogen in Human Breast Explants.

Exposure to estrogen is strongly associated with increased breast cancer risk. While all women are exposed to estrogen, only 12% are expected to develop breast cancer during their lifetime. These women may be more sensitive to estrogen, as rodent models have demonstrated variability in estrogen sensitivity. Our objective was to determine individual variation in expression of estrogen receptor (ER) and estrogen-induced responses in the normal human breast. Human breast tissue from female donors undergoing reduction mammoplasty surgery were collected for microarray analysis of ER expression. To examine estrogen-induced responses, breast tissue from 23 female donors were cultured ex- vivo in basal or 10 nM 17ß-estradiol (E2) media for 4 days. Expression of ER genes (ESR1 and ESR2) increased significantly with age. E2 induced consistent increases in global gene transcription, but expression of target genes AREG, PGR, and TGFß2 increased significantly only in explants from nulliparous women. E2-treatment did not induce consistent changes in proliferation or radiation induced apoptosis. Responses to estrogen are highly variable among women and not associated with levels of ER expression, suggesting differences in intracellular signaling among individuals. The differences in sensitivity to E2-stimulated responses may contribute to variation in risk of breast cancer.

Epigenetic Regulation via Altered Histone Acetylation Results in Suppression of Mast Cell Function and Mast Cell-Mediated Food Allergic Responses.

Mast cells are highly versatile cells that perform a variety of functions depending on the immune trigger, context of activation, and cytokine stimulus. Antigen-mediated mast cell responses are regulated by transcriptional processes that result in the induction of numerous genes contributing to mast cell function. Recently, we also showed that exposure to dietary agents with known epigenetic actions such as curcumin can suppress mast cell-mediated food allergy, suggesting that mast cell responses in vivo may be epigenetically regulated. To further assess the effects of epigenetic modifications on mast cell function, we examined the behavior of bone marrow-derived mast cells (BMMCs) in response to trichostatin A (TSA) treatment, a well-studied histone deacetylase inhibitor. IgE-mediated BMMC activation resulted in enhanced expression and secretion of IL-4, IL-6, TNF-α, and IL-13. In contrast, pretreatment with TSA resulted in altered cytokine secretion. This was accompanied by decreased expression of FcεRI and mast cell degranulation. Interestingly, exposure to non-IgE stimuli such as IL-33, was also affected by TSA treatment. Furthermore, continuous TSA exposure contributed to mast cell apoptosis and a decrease in survival. Further examination revealed an increase in I-κBα and a decrease in phospho-relA levels in TSA-treated BMMCs, suggesting that TSA alters transcriptional processes, resulting in enhancement of I-κBα transcription and decreased NF-κB activation. Lastly, treatment of wild-type mice with TSA in a model of ovalbumin-induced food allergy resulted in a significant attenuation in the development of food allergy symptoms including decreases in allergic diarrhea and mast cell activation. These data therefore suggest that the epigenetic regulation of mast cell activation during immune responses may occur via altered histone acetylation, and that exposure to dietary substances may induce epigenetic modifications that modulate mast cell function.

IL-10 Enhances IgE-Mediated Mast Cell Responses and Is Essential for the Development of Experimental Food Allergy in IL-10-Deficient Mice.

IL-10 is a key pleiotropic cytokine that can both promote and curb Th2-dependent allergic responses. In this study, we demonstrate a novel role for IL-10 in promoting mast cell expansion and the development of IgE-mediated food allergy. Oral OVA challenge in sensitized BALB/c mice resulted in a robust intestinal mast cell response accompanied by allergic diarrhea, mast cell activation, and a predominance of Th2 cytokines, including enhanced IL-10 expression. In contrast, the development of intestinal anaphylaxis, including diarrhea, mast cell activation, and Th2 cytokine production, was significantly attenuated in IL-10(-/-) mice compared with wild-type (WT) controls. IL-10 also directly promoted the expansion, survival, and activation of mast cells; increased FcεRI expression on mast cells; and enhanced the production of mast cell cytokines. IL-10(-/-) mast cells had reduced functional capacity, which could be restored by exogenous IL-10. Similarly, attenuated passive anaphylaxis in IL-10(-/-) mice could be restored by IL-10 administration. The adoptive transfer of WT mast cells restored allergic symptoms in IL-10(-/-) mice, suggesting that the attenuated phenotype observed in these animals is due to a deficiency in IL-10-responding mast cells. Lastly, transfer of WT CD4 T cells also restored allergic diarrhea and intestinal mast cell numbers in IL-10(-/-) mice, suggesting that the regulation of IL-10-mediated intestinal mast cell expansion is T cell dependent. Our observations demonstrate a critical role for IL-10 in driving mucosal mast cell expansion and activation, suggesting that, in its absence, mast cell function is impaired, leading to attenuated food allergy symptoms.

Rhodiola crenulata induces an early estrogenic response and reduces proliferation and tumorsphere formation over time in MCF7 breast cancer cells.

BACKGROUND: Rhodiola crenulata is a Tibetan mountainous plant, commonly used in Eastern alternative medicine. Many phytochemicals possess estrogenic activity, a critical regulator of proliferation in mammary epithelial cells. We have previously characterized anti-cancer properties of R. crenulata in aggressive triple negative breast cancer cells, lacking the expression of estrogen receptor. Currently, it is unknown whether R. crenulata exerts estrogenic effects and as such consumption may be a concern for women with estrogen receptor positive breast cancer that use Rhodiola sp. to relieve mild to moderate depression. PURPOSE: In this study, we wished to determine whether a hydroalcoholic fraction of the R. crenulata root extract exhibits estrogenic activity in estrogen receptor positive (ER+) breast cancer cells in vitro and whether it affects normal mammary epithelial ER target gene expression in vivo. METHODS: ER transcriptional activity was analyzed in MCF7 cells expressing an ERE reporter construct and confirmed via qPCR of endogenous ER target genes. We also monitored cellular proliferation over time. Additionally, to assess stem-like properties in MCF7 cells, we performed a tumorsphere formation assay under anchorage independent conditions. We examined whether R. crenulata treatment reduced ß-catenin levels via Western blotting and measured ß-catenin transcriptional activity by a reporter assay. To examine the effects of R. crenulata on normal mammary epithelial cells, we performed immunohistochemical staining of ER and PR in the mammary glands of mice fed R. crenulata for 12 weeks. RESULTS: We show an initial activation of ER transcriptional activity by dual reporter assay, qPCR and proliferation of MCF7 ER+ cells in response to 24 h of R. crenulata treatment. However, upon longer treatment basal and R. crenulata induced transcriptional activity was suppressed. There was a decrease in cell doubling times and a decrease in tumorsphere formation. In association with these changes, ERα transcript levels were decreased and active ß-catenin levels were reduced in the cells treated for 2 weeks. Finally, we show no change in estrogen targets in normal mammary cells in vivo. CONCLUSION: These data suggest that the R. crenulata extract contains components with estrogenic activity. However, R. crenulata treatment could still be protective in ER+ breast cancer cells, as longer treatment reduced the transcriptional activity of ß-catenin and ER responses leading to reduced proliferation and tumorsphere formation. Furthermore, administration of 20 mg/kg/day R. crenulata to mice did not have an observable effect on mammary epithelial ERα target gene expression in vivo.

Curcumin Ingestion Inhibits Mastocytosis and Suppresses Intestinal Anaphylaxis in a Murine Model of Food Allergy.

IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-inflammatory properties, and thus may have the capacity to regulate Th2 cells and mucosal mast cell function during allergic responses. We assessed whether curcumin ingestion during oral allergen exposure can modulate the development of food allergy using a murine model of ovalbumin (OVA)-induced intestinal anaphylaxis. Herein, we demonstrate that frequent ingestion of curcumin during oral OVA exposure inhibits the development of mastocytosis and intestinal anaphylaxis in OVA-challenged allergic mice. Intragastric (i.g.) exposure to OVA in sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1, and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, mast cell activation and expansion, and Th2 responses were also suppressed in mice exposed to curcumin during the OVA-challenge phase alone, despite the presence of elevated levels of OVA-IgE, suggesting that curcumin may have a direct suppressive effect on intestinal mast cell activation and reverse food allergy symptoms in allergen-sensitized individuals. This was confirmed by observations that curcumin attenuated the expansion of both adoptively transferred bone marrow-derived mast cells (BMMCs), and inhibited their survival and activation during cell culture. Finally, the suppression of intestinal anaphylaxis by curcumin was directly linked with the inhibition of NF-κB activation in curcumin-treated allergic mice, and curcumin inhibited the phosphorylation of the p65 subunit of NF-κB in BMMCs. In summary, our data demonstrates a protective role for curcumin during allergic responses to food antigens, suggesting that frequent ingestion of this spice may modulate the outcome of disease in susceptible individuals.

Antineoplastic effects of Rhodiola crenulata treatment on B16-F10 melanoma.

Melanoma is an aggressive form of skin cancer with limited treatment options for advanced stage disease. Early detection and wide surgical excision remain the initial mode of treatment for primary tumors thus preventing metastases and leading to improved prognosis. Through this work, we have evaluated the antineoplastic effects of Rhodiola crenulata (R. crenulata) root extracts on the B16-F10 melanoma cell line, both in vitro and in vivo. We observed that R. crenulata treatment resulted in increased cell death as well as a reduction in tumor cell proliferation and migration in vitro. Additionally, we observed that R. crenulata decreased the expression of integrin ß1 and vimentin and increased the expression of E-cadherin. Further, in mice treated with a topical R. crenulata-based cream therapy, tumors were more likely to have a radial growth pattern, a reduction in mitotic activity, and an increase in tumor necrosis. We also observed that mice drinking water supplemented with R. crenulata displayed a reduction of metastatic foci in disseminated models of melanoma. Collectively, these findings suggest that R. crenulata exhibits striking antitumorigenic and antimetastatic properties and that this extract may harbor potential novel adjuvant therapy for the treatment of melanoma.

The effects of diet induced obesity on breast cancer associated pathways in mice deficient in SFRP1.

BACKGROUND: Secreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of Sfrp1 expression is observed in breast cancer. The molecular mechanisms by which obesity contributes to breast tumorigenesis are not well defined, but involve increased inflammation. Mice deficient in Sfrp1 show enhanced mammary gland inflammation in response to diet induced obesity (DIO). Furthermore, mammary glands from Sfrp1-/- mice exhibit increased Wnt signaling, decreased cell death responses, and excessive hyper branching. The work described here was initiated to investigate whether obesity exacerbates the aforementioned pathways, as they each play a key roles in the development of breast cancer. FINDINGS: Wnt signaling is significantly affected by DIO and Sfrp1-/- loss as revealed by analysis of Myc mRNA expression and active ß-catenin protein expression. Furthermore, Sfrp1-/- mice fed a high fat diet (HFD) exhibit an increase in mammary cell proliferation. The death response is also impaired in the mammary gland of Sfrp1-/- mice fed a normal diet (ND) as well as a HFD. In response to γ-irradiation, mammary glands from Sfrp1-/- mice express significantly less Bax and Bbc3 mRNA, caspase-3 positive cells, and p53 protein. The expression of Wnt4 and Tnfs11 are critical for normal progesterone mediated mammary gland development and in response to obesity, Sfrp1-/- mice express significantly more Wnt4 and Tnfs11 mRNA expression. Evaluation of progesterone receptor (PR) expression showed that DIO increases the number of PR positive cells. CONCLUSIONS: Our data indicate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular homeostasis in response to the onset of obesity.