We report here evidence in support of the role of 17beta-estradiol- (E2-) induced mitochondrial (mt) reactive oxygen species (ROS) as signal-transducing messengers. On the basis of monitoring the oxidation of 2',7'-dichlorofluorescin by spectrofluorometry, flow cytometry, and confocal microscopy, we have identified that exposure of E2 triggers the immediate rapid production of intracellular ROS ranging from a 1- to severalfold increase in a variety of cells. E2-stimulated ROS production does not correlate with the activity of the estrogen receptor (ER) in the cells. The ROS is most likely hydrogen peroxide based on its inhibition by antioxidants and catalase and lack of any effects of E2 on O(2)(*)(-) or NO(*) formation. Confocal microscopy showed that ROS is localized in the perinuclear mitochondria. E2 through anchorage- and integrin-dependent signaling to mitochondria increased ROS generation. Increased intracellular ROS formation identified here for the first time may explain the mechanism of previously reported oxidative damage and subsequent genetic alterations including mutations produced by elevated concentrations of estrogens. The functional consequences of E2-induced ROS formation included the enhanced cell motility as shown by the increase in cdc42 and activation of Pyk2 and the increased phosphorylation of signaling proteins c-jun and CREB. E2-induced ROS activated the binding of three oxidant-sensitive transcription factors: AP-1, CREB, and nuclear respiratory factor 1. In addition to ERs as signaling molecules, our findings further revealed that E2-induced mt ROS also act as signal transducing messengers and suggest new targets for the development of antioxidant-based drugs or antioxidant gene therapy for the prevention and treatment of estrogen-dependent cancer.

In addition to the direct effect of estrogen on mitochondria and the redox cycling of catechol estrogen, estrogen-induced proinflammatory cytokines, such as interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), also generate reactive oxygen and nitrogen species (RO/NS). Different cellular signaling pathways may operate in response to varying levels of estrogen-induced RO/NS, leading to genotoxic damage, cell apoptosis, or cell growth. At high levels of RO/NS, cells receiving genotoxic insults, if not repaired, may engage the apoptotic pathways. There is increasing evidence supporting that estrogen-induced alterations in the genome of cells is produced by oxidative attack. Furthermore, ROS generated by estrogen exposure and/or active metabolites of estrogen in combination with receptor-mediated proliferation of genetically damaged cells may be involved in tumor development. This view is supported by the findings of DNA modifications produced in vitro or in vivo by natural and synthetic estrogens in the target organs of cancer both in experimental models and in humans. Interaction of estrogen-induced oxidants and estrogen metabolites with DNA was shown to generate mutations in genes. Cotreatment with an inhibitor of IL-1beta and TNF-alpha synthesis, pentoxifylline, decreased stilbene estrogen-induced levels of myeloperoxidase (MPO), 8-hydroxydeoxyguanosine formation, and gene mutations, and prevented stilbene estrogen-induced lesions. Stable MCF-7 clones overexpressing IL-1beta resulted in a high level of IL-1beta peptide secretion undergoing cell apoptosis, and an elevated level of p53 protein in response to high oxidative stress when compared to nontransfected cells, whereas MCF-7 clones overexpressing IL-1beta that resulted in a moderate level of IL-1beta secretion stimulated the clonal expansion of MCF-7 and TM3 cells. Estrogen-induced MCF-7 cell growth and cyclin D1 expression were suppressed by antioxidants and mitochondrial blockers. These studies support that in addition to ovarian estrogen-mediated ER signaling, mitogenic signals may also come from estrogen-induced RO/NS. Further validation of this concept that the concentration of the RO/NS within the cellular microenvironment determines its stimulatory or inhibitory growth signals as well as its genotoxic effects regulating the growth of estrogen-dependent tumors may result in novel preventive strategies.

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.