Quentin Felty

Estrogen-Induced Generation of Reactive Oxygen and Nitrogen Species, Gene Damage, and Estrogen-Dependent Cancers

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-1β) and tumor necrosis factor alpha (TNF-α), 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-1β and TNF-α 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-1β resulted in a high level of IL-1β 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-1β that resulted in a moderate level of IL-1β 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.

Estrogen, mitochondria, and growth of cancer and non-cancer cells

In this review, we discuss estrogen actions on mitochondrial function and the possible implications on cell growth. Mitochondria are important targets of estrogen action. Therefore, an in-depth analysis of interaction between estrogen and mitochondria; and mitochondrial signaling to nucleus are pertinent to the development of new therapy strategies for the treatment of estrogen-dependent diseases related to mitochondrial disorders, including cancer.

Estrogen-induced G1/S transition of G0-arrested estrogen-dependent breast cancer cells is regulated by mitochondrial oxidant signaling.

We previously reported that 17-β-estradiol (E2)-induced mitochondrial reactive oxygen species (mtROS) act as signaling molecules. The purpose of this study was to investigate the effects of E2-induced mtROS on cell cycle progression. E2-induced cell growth was reduced by antioxidants N-acetyl-L-cysteine (NAC), catalase, and the glutathione peroxidase mimic ebselen. Flow cytometry showed that mitochondrial blockers of protein synthesis (chloramphenicol), transcription and replication (ethidium bromide), and function (rotenone, rhodamine 6G) blocked E2-induced G1 to S transition. Reduction of E2-induced DNA synthesis in the presence of mitochondrial blockers occurred without influencing the level of ATP. Additionally, the mitochondrial blockers inhibited the E2-induced expression of early cell cycle genes such as cyclins D1, D3, E1, E2, and B2. NAC or rotenone reduced E2-induced cyclin D1 expression. Furthermore, E2-induced binding of AP-1 and CREB to the TRE and CRE response sequences, respectively, in the promoter of cyclin D1 was inhibited by NAC or rotenone. In addition, E2-induced expression of PCNA, PRC1, and bcl-2 were inhibited by mitochondrial blockers. These data indicate that E2-induced mtROS are involved in the regulation of early G1-phase progression. Since neither antioxidants nor mitochondrial blockers used in this study are reported to bind the estrogen receptor (ER), our findings suggest that E2-induced mtROS modulates G1 to S transition and some of the early G1 genes through a nongenomic, ER-independent signaling pathway. Thus, our results suggest (1) a new paradigm that estrogen-induced mitochondrial oxidants control the early stage of cell cycle progression and (2) provide the basis for the discovery of novel antioxidant-based drugs or antioxidant gene therapies for the prevention and treatment of estrogen-dependent breast cancer.

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

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.

Estrogen-induced redox sensitive Id3 signaling controls the growth of vascular cells

In this study, we investigated a mechanism by which estrogen-induced oxidants control endothelial cell differentiation into tubelike structures via redox sensitive signaling molecule Id3. Using a matrigel cell culture, we determined whether superoxide or hydrogen peroxide signaled estrogen-induced tube formation. Overexpression of the superoxide scavenger MnSOD and the hydrogen peroxide scavenger catalase inhibited tube formation in estrogen treated endothelial cells. Since tube formation on matrigel is not specific for endothelial cells, we verified our results in a co-culture model that better represents tube formation in vivo. Antioxidants ebselen and N-acetylcysteine as well as overexpression of MnSOD and catalase inhibited tube formation in estrogen exposed endothelial cells co-cultured with fibroblasts. We previously showed that estrogen-induced mitochondrial oxidants depended on the cytoskeleton so we tested tube formation dependence on the cytoskeleton. Estrogen-induced tube formation was inhibited by the actin cytoskeleton disruptor cytochalasin D and the microtubule destabilizer colchicine. Estrogen increased Id3 phosphorylation which was reduced by catalase and N-acetylcysteine treatments. We determined the functional role of Id3 in tube formation by RNA intereference and showed Id3 siRNA to inhibit tube formation in estrogen exposed cells. The major novel findings presented here are that: (i) estrogen-induced tube formation requires the presence of Id3, a member of the helix-loop-helix family of transcriptional factors and (ii) estrogen increases Id3 phosphorylation via a redox-dependent process. Furthermore, these studies demonstrate Id3 to be an important signaling molecule in estrogen stimulated vascularization and may serve as a therapeutic target in the prevention and treatment of vasculoproliferative disorders.

Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells

Background:17β-Oestradiol (E2)-induced reactive oxygen species (ROS) have been implicated in regulating the growth of breast cancer cells. However, the underlying mechanism of this is not clear. Here we show how ROS through a novel redox signalling pathway involving nuclear respiratory factor-1 (NRF-1) and p27 contribute to E2-induced growth of MCF-7 breast cancer cells.Methods:Chromatin immunoprecipitation, qPCR, mass spectrometry, redox western blot, colony formation, cell proliferation, ROS assay, and immunofluorescence microscopy were used to study the role of NRF-1.Results:The major novel finding of this study is the demonstration of oxidative modification of phosphatases PTEN and CDC25A by E2-generated ROS along with the subsequent activation of AKT and ERK pathways that culminated in the activation of NRF-1 leading to the upregulation of cell cycle genes. 17β-Oestradiol-induced ROS by influencing nuclear proteins p27 and Jab1 also contributed to the growth of MCF-7 cells.Conclusions:Taken together, our results present evidence in the support of E2-induced ROS-mediated AKT signalling leading to the activation of NRF-1-regulated cell cycle genes as well as the impairment of p27 activity, which is presumably necessary for the growth of MCF-7 cells. These observations are important because they provide a new paradigm by which oestrogen may contribute to the growth of breast cancer.

Gene expression profile of endothelial cells exposed to estrogenic environmental compounds: Implications to pulmonary vascular lesions

AIMS The cardiovascular system is an important target of estrogenic compounds. Considering the recent studies that question previously reported cardio-protective effects of estrogen, there is a growing concern that estrogenic environmental compounds may contribute to the pathology of vascular lesion formation. MAIN METHODS Real-time quantitative PCR was used to monitor the expression of genes involved in vascularization. Using Bayesian network modeling, we determined a gene network that estrogenic chemicals modulate in human vascular endothelial cells. KEY FINDINGS We showed that planar and coplanar polychlorinated biphenyls (PCBs) induce the expression of different genes compared to estradiol. Non-planar PCB congener 153 induced NOTCH3 which is a new finding as well as CCL2 and IL8 similar to what has been reported by other non-planar PCBs in endothelial cells. Our gene network indicated that experimental treatments signal a network containing TGF-beta receptor and NOTCH3; molecules biologically relevant to signaling pulmonary vascular lesions. SIGNIFICANCE We report in the present study that exposure of vascular endothelial cells to environmentally relevant concentrations of estrogenic PCBs induce gene networks implicated in the process of inflammation and adhesion. Our data suggest that PCBs can promote vascular lesion formation by activating gene networks involved in endothelial cell adhesion, cell growth, and pro-inflammatory molecules which were different from natural estrogen. Since inflammation and adhesion are a hallmark in the pathology of endothelial cell dysfunction, reconstructing gene networks provide insight into the potential mechanisms that may contribute to the vascular risks associated with estrogenic environmental chemicals.

PCB153-Induced Overexpression of ID3 Contributes to the Development of Microvascular Lesions

Microvascular lesions resulting from endothelial cell dysfunction are produced in the brain, lung, kidney, and retina of patients of complex chronic diseases. The environmental and molecular risk factors which may contribute in the development of microvascular damage are unclear. The mechanism(s) responsible for initiating microvascular damage remain poorly defined, although several inciting factors have been proposed, including environmental toxicants-induced oxidative stress. Enhanced neovascularization has been implicated in either the development or progression of proliferative vascular lesions. Here, we present evidence for how PCB-induced ROS may contribute to the development of a neovascular phenotype with the aim of elucidating the role of environmental toxicants in endothelial dysfunction with a specific focus on the inhibitor of differentiation protein ID3. We used a combination of phenotype and immunohistochemical analysis followed by validating with protein expression and post-translational modifications with Western Blot and MALDI-TOF/TOF analysis. We also looked for a correlation between ID3 expression in vascular tissue. Our results showed that PCB-induced ROS mediated a highly tube branched neovascular phenotype that also depended on ID3 and Pyk2; and PCB153 treatment increased the size of endothelial spheroids under conditions typically used for clonal selection of stem cell spheroids. High ID3 protein expression correlated with a greater degree of malignancy and oxidative DNA damage marker 8-OHdG in blood vessels from human subjects. PCB153 treatment increased both serine and tyrosine phosphorylation of endothelial ID3. Stable ID3 overexpression increased cell survival of human microvascular endothelial cell line hCMEC/D3. In summary, our data provide evidence that ID3 may play a critical role in regulating vascular endothelial cell survival and development of microvascular lesions induced by persistent environmental pollutants such as PCB153. Findings of this study are important because they provide a new paradigm by which PCBs may contribute to the growth of microvascular lesions.

Vascular endothelial growth factor receptor 3 signaling contributes to angioobliterative pulmonary hypertension

The mechanisms involved in the development of severe angioobliterative pulmonary arterial hypertension (PAH) are multicellular and complex. Many of the features of human severe PAH, including angioobliteration, lung perivascular inflammation, and right heart failure, are reproduced in the Sugen 5416/chronic hypoxia (SuHx) rat model. Here we address, at first glance, the confusing and paradoxical aspect of the model, namely, that treatment of rats with the antiangiogenic vascular endothelial growth factor (VEGF) receptor 1 and 2 kinase inhibitor, Sugen 5416, when combined with chronic hypoxia, causes angioproliferative pulmonary vascular disease. We postulated that signaling through the unblocked VEGF receptor VEGFR3 (or flt4) could account for some of the pulmonary arteriolar lumen–occluding cell growth. We also considered that Sugen 5416–induced VEGFR1 and VEGFR2 blockade could alter the expression pattern of VEGF isoform proteins. Indeed, in the lungs of SuHx rats we found increased expression of the ligand proteins VEGF-C and VEGF-D as well as enhanced expression of the VEGFR3 protein. In contrast, in the failing right ventricle of SuHx rats there was a profound decrease in the expression of VEGF-B and VEGF-D in addition to the previously described reduction in VEGF-A expression. MAZ51, an inhibitor of VEGFR3 phosphorylation and VEGFR3 signaling, largely prevented the development of angioobliteration in the SuHx model; however, obliterated vessels did not reopen when animals with established PAH were treated with the VEGFR3 inhibitor. Part of the mechanism of vasoobliteration in the SuHx model occurs via VEGFR3. VEGFR1/VEGFR2 inhibition can be initially antiangiogenic by inducing lung vessel endothelial cell apoptosis; however, it can be subsequently angiogenic via VEGF-C and VEGF-D signaling through VEGFR3.

Microvascular Lesions by Estrogen-Induced ID3: Its Implications in Cerebral and Cardiorenal Vascular Disease

Severe symptoms of cerebral and cardiorenal vascular diseases can be triggered when cerebral, coronary, or glomerular arterioles grow inappropriately as a result of abnormal cell proliferation. The risk factor(s) and molecular mechanisms responsible for microvascular lesion formation are largely unknown. Although controversial, both animal and epidemiological studies have shown that estrogen increases the risk of stroke which may be due to microvascular lesions. Since microvascular diseases are characterized by excessive vessel growth, it is plausible that estrogen-induced neovascularization contributes to the growth of microvascular lesions. We present evidence for how ID3 overexpression in endothelial cells contributes to the development of an estrogen-induced neovascular phenotype with an additional focus on Pyk2 kinase. Our data showed that ID3 overexpression increased neovascularization, cell migration, and spheroid growth of human cerebral microvascular endothelial cells, hCMEC/D3. ID3-overexpressing cells showed significant estrogen-induced G2/M phase transition. Estrogen treatment increased both ID3 phosphorylation; total protein that was inhibited by tamoxifen, and Pyk2-mediated estrogen-induced ID3 mRNA expression. These findings suggest that Pyk2 signals ID3 expression and ID3 is necessary for estrogen-induced neovascularization in hCMEC/D3 cells. A better understanding of how microvascular lesions depend on ID3 may open new avenues for prevention and treatment of neurological diseases.