Sarah M. Mense

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

Heme (iron protoporphyrin IX) is an essential molecule for numerous living organisms. Not only does it serve as a prosthetic group in enzymes, it also acts as a signaling molecule that controls diverse molecular and cellular processes ranging from signal transduction to protein complex assembly. Deficient heme synthesis or function impacts the hematopoietic, hepatic and nervous systems in humans. Recent studies have revealed a series of heme-regulated transcription factors and signal transducers including Hap1, a heme-activated transcription factor that mediates the effects of oxygen on gene transcription in the yeast Saccharomyces cerevisiae; Bach1, a transcriptional repressor that is negatively regulated by heme in mammalian cells; IRR, an iron regulatory protein that mediates the iron-dependant regulation of heme synthesis in the bacterium Bradyrhizobium japonicum; and heme-regulated inhibitor, an eucaryotic initiation factor 2α kinase that coordinates protein synthesis with heme availability in reticulocytes. In this review, we summarize the current knowledge about how heme controls the activity of these transcriptional regulators and signal transducers, and discuss diseases associated with defective heme synthesis, degradation and function.

Phytoestrogens and Breast Cancer Prevention: Possible Mechanisms of Action

OBJECTIVE Phytoestrogens display an array of pharmacologic properties, and in recent years investigation of their potential as anticancer agents has increased dramatically. In this article we review the published literature related to phytoestrogens and breast cancer as well as suggest the possible mechanisms that may underlie the relationship between phytoestrogens and breast cancer. DATA SOURCES Electronic searches on phytoestrogens and breast cancer were performed on MEDLINE and EMBASE in June 2007. No date restriction was placed on the electronic search. DATA EXTRACTION We focused on experimental data from published studies that examined the characteristics of phytoestrogens using in vivo or in vitro models. We also include human intervention studies in this review. DATA SYNTHESIS We evaluated evidence regarding the possible mechanisms of phytoestrogen action. Discussions of these mechanisms were organized into those activities related to the estrogen receptor, cell growth and proliferation, tumor development, signaling pathways, and estrogen-metabolizing enzymes. CONCLUSIONS We suggest that despite numerous investigations, the mechanisms of phytoestrogen action in breast cancer have yet to be elucidated. It remains uncertain whether these plant compounds are chemoprotective or whether they may produce adverse outcomes related to breast carcinogenesis.

Estrogen-induced breast cancer: Alterations in breast morphology and oxidative stress as a function of estrogen exposure

Epidemiological evidence indicates that prolonged lifetime exposure to estrogen is associated with elevated breast cancer risk in women. Oxidative stress and estrogen receptor-associated proliferative changes are suggested to play important roles in estrogen-induced breast carcinogenesis. In the present study, we investigated changes in breast morphology and oxidative stress following estrogen exposure. Female ACI rats were treated with 17beta-estradiol (E(2), 3 mg, s.c.) for either 7, 15, 120 or 240 days. Animals were euthanized, tissues were excised, and portions of the tissues were either fixed in 10% buffered formalin or snap-frozen in liquid nitrogen. Paraffin-embedded tissues were examined for histopathologic changes. Proliferative changes appeared in the breast after 7 days of E(2) exposure. Atypical ductal proliferation and significant reduction in stromal fat were observed following 120 days of E(2) exposure. Both in situ and invasive carcinomas were observed in the majority of the mammary glands from rats treated with E(2) for 240 days. Palpable breast tumors were observed in 82% of E(2)-treated rats after 228 days, with the first palpable tumor appearing after 128 days. No morphological changes were observed in the livers, kidneys, lungs or brains of rats treated with E(2) for 240 days compared to controls. Furthermore, 8-isoprostane (8-isoPGF(2alpha)) levels as well as the activities of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase and catalase, were quantified in the breast tissues of rats treated with E(2) for 7, 15, 120 and 240 days and compared to activity levels in age-matched controls. 8-isoPGF(2alpha) levels displayed time-dependent increases upon E(2) treatment and were significantly higher than control levels at the 15, 120 and 240 day time-points. 8-isoPGF(2alpha) observed in E(2)-induced mammary tumors were significantly higher than levels found in control mammary tissue from age-matched animals. Similarly, alterations in glutathione peroxidase and superoxide dismutase activities were detected in both mammary and tumor tissue from E(2)-treated rats. Taken together, our data reveal that proliferative changes in the breast tissue of ACI rats are associated with increases in 8-isoPGF(2alpha) formation as well as changes in the activities of antioxidant enzymes. These oxidative changes appear to be a function of E(2) exposure and occur prior to tumor development.

Vitamin C and α-naphthoflavone prevent estrogen-induced mammary tumors and decrease oxidative stress in female ACI rats

The mechanisms underlying the pathogenesis of estrogen-induced breast carcinogenesis remain unclear. The present study investigated the roles of estrogen metabolism and oxidative stress in estrogen-mediated mammary carcinogenesis in vivo. Female August Copenhagen Irish (ACI) rats were treated with 17beta-estradiol (E(2)), the antioxidant vitamin C, the estrogen metabolic inhibitor alpha-naphthoflavone (ANF), or cotreated with E(2) + vitamin C or E(2) + ANF for up to 8 months. E(2) (3 mg) was administered as an subcutaneous implant, ANF was given via diet (0.2%) and vitamin C (1%) was added to drinking water. At necropsy, breast tumor incidence in the E(2), E(2) + vitamin C and E(2) + ANF groups was 82, 29 and 0%, respectively. Vitamin C and ANF attenuated E(2)-induced alterations in oxidative stress markers in breast tissue, including 8-iso-prostane F(2alpha) formation and changes in the activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase. Quantification of 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)) formation in breast tissue confirmed that ANF inhibited 4-hydroxylation of E(2) and decreased formation of the highly carcinogenic 4-OHE(2). These results demonstrate that antioxidant vitamin C reduces the incidence of estrogen-induced mammary tumors, increases tumor latency and decreases oxidative stress in vivo. Further, our data indicate that ANF completely abrogates breast cancer development in ACI rats. The present study is the first to demonstrate the inhibition of breast carcinogenesis by antioxidant vitamin C or the estrogen metabolic inhibitor ANF in an animal model of estrogen-induced mammary carcinogenesis. Taken together, these results suggest that E(2) metabolism and oxidant stress are critically involved in estrogen-induced breast carcinogenesis.

Dietary quercetin exacerbates the development of estrogen-induced breast tumors in female ACI rats

Phytoestrogens are plant compounds that structurally mimic the endogenous estrogen 17beta-estradiol (E(2)). Despite intense investigation, the net effect of phytoestrogen exposure on the breast remains unclear. The objective of the current study was to examine the effects of quercetin on E(2)-induced breast cancer in vivo. Female ACI rats were given quercetin (2.5 g/kg food) for 8 months. Animals were monitored weekly for palpable tumors, and at the end of the experiment, rats were euthanized, breast tumor and different tissues excised so that they could be examined for histopathologic changes, estrogen metabolic activity and oxidant stress. Quercetin alone did not induce mammary tumors in female ACI rats. However, in rats implanted with E(2) pellets, co-exposure to quercetin did not protect rats from E(2)-induced breast tumor development with 100% of the animals developing breast tumors within 8 months of treatment. No changes in serum quercetin levels were observed in quercetin and quercetin+E(2)-treated groups at the end of the experiment. Tumor latency was significantly decreased among rats from the quercetin+E(2) group relative to those in the E(2) group. Catechol-O-methyltransferase (COMT) activity was significantly downregulated in quercetin-exposed mammary tissue. Analysis of 8-isoprostane F(2alpha) (8-iso-PGF(2alpha)) levels as a marker of oxidant stress showed that quercetin did not decrease E(2)-induced oxidant stress. These results indicate that quercetin (2.5 g/kg food) does not confer protection against breast cancer, does not inhibit E(2)-induced oxidant stress and may exacerbate breast carcinogenesis in E(2)-treated ACI rats. Inhibition of COMT activity by quercetin may expose breast cells chronically to E(2) and catechol estrogens. This would permit longer exposure times to the carcinogenic metabolites of E(2) and chronic exposure to oxidant stress as a result of metabolic redox cycling to estrogen metabolites, and thus quercetin may exacerbate E(2)-induced breast tumors in female ACI rats.

Antioxidant butylated hydroxyanisole inhibits estrogen-induced breast carcinogenesis in female ACI rats.

Exposure to estrogens is suggested to be a risk factor in human breast cancer development. The mechanisms underlying estrogen‐induced cancer have not been fully elucidated. Both estrogen receptor (ER)‐mediated proliferative processes and ER‐independent generation of oxidative stress are suggested to play important roles in estrogen‐induced breast carcinogenesis. In the current study, we investigated the role of oxidative stress in breast carcinogenesis using the ACI rat model of mammary tumorigenesis. Female ACI rats were treated with 17β‐estradiol (E2), butylated hydroxyanisole (BHA), or a combination of E2 + BHA for up to 240 days. Cotreatment of rats with E2 + BHA reduced estrogen‐induced breast tumor development with tumor incidence of 24%, a significant decrease relative to E2 where tumor incidence was 82%. Proliferative changes in the breast tissue of E2 + BHA‐treated animals were similar to those observed in E2‐treated animals. Tissue levels of 8‐isoprostane, a marker of oxidant stress, as well as the activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase were quantified in the breast tissues of rats treated with E2 + BHA and compared to activity levels found in E2‐treated animals and respective age‐matched controls. Cotreatment with BHA inhibited E2‐mediated increases in 8‐isoprostane levels as well as activities of antioxidant enzymes. In summary, these data suggest that estrogen‐mediated oxidant stress plays a critical role in the development of estrogen‐dependent breast cancers and BHA inhibits E2‐dependent breast carcinogenesis by decreasing oxidant stress.

Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

Estrogen metabolism is suggested to play an important role in estrogen-induced breast carcinogenesis. Epidemiologic studies suggest that diets rich in phytoestrogens are associated with a reduced risk of breast cancer. Phytoestrogens are biologically active plant compounds that structurally mimic 17beta-estradiol (E(2)). We hypothesize that phytoestrogens, may provide protection against breast carcinogenesis by altering the expression of estrogen-metabolizing enzymes cytochrome P450 1A1 (Cyp1A1) and 1B1 (Cyp1B1). Cyp1A1 and Cyp1B1 are responsible for the metabolism of E(2) to generate 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)), respectively. Studies suggest that 2-OHE(2) and 2-methoxyestradiol may protect against breast carcinogenesis, while 4-OHE(2) is carcinogenic in rodent models. Thus, agents that increase the metabolism of E(2) by Cyp1A1 to produce 2-OHE(2) may have chemoprotective properties. The human immortalized non-neoplastic breast cell line MCF10F was treated with quercetin at 10 and 50muM concentrations for time points ranging from 3 to 48h. Total RNA and protein were isolated. Real-time PCR was used to measure the expression of Cyp1A1 and Cyp1B1 mRNA. Quercetin treatment produced differential regulation of Cyp1A1 and Cyp1B1 mRNA expression in a time- and dose-dependent manner. Treatment with 10 and 50 microM doses of quercetin produced 6- and 11-times greater inductions of Cyp1A1 mRNA over Cyp1B1 mRNA, respectively. Furthermore, quercetin dramatically increased Cyp1A1 protein levels and only slightly increased Cyp1B1 protein levels in MCF10F cells. Thus, our data suggest that phytoestrogens may provide protection against breast cancer by modulating expression of estrogen-metabolizing genes such that production of the highly carcinogenic estrogen metabolite 4-OHE(2) by Cyp1B1 is reduced and the production of the less genotoxic 2-OHE(2) by Cyp1A1 is increased.