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.

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

4-hydroxy estradiol-induced oxidant-mediated signaling is involved in the development of breast cancer

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.^

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

The investigation of photocatalysts and iron based materials in the oxidation and adsorption of toxic organic and chromium materials

The presences of heavy metals, organic contaminants and natural toxins in natural water bodies pose a serious threat to the environment and the health of living organisms. Therefore, there is a critical need to identify sustainable and environmentally friendly water treatment processes. In this dissertation, I focus on the fundamental studies of advanced oxidation processes and magnetic nano-materials as promising new technologies for water treatments. Advanced oxidation processes employ reactive oxygen species (ROS) which can lead to the mineralization of a number of pollutants and toxins. The rates of formation, steady-state concentrations, and kinetic parameters of hydroxyl radical and singlet oxygen produced by various TiO2 photocatalysts under UV or visible irradiations were measured using selective chemical probes. Hydroxyl radical is the dominant ROS, and its generation is dependent on experimental conditions. The optimal condition for generation of hydroxyl radical by of TiO2 coated glass microspheres is studied by response surface methodology, and the optimal conditions are applied for the degradation of dimethyl phthalate. Singlet oxygen (1O2) also plays an important role for advanced processes, so the degradation of microcystin-LR by rose bengal, an 1O2 sensitizer was studied. The measured bimolecular reaction rate constant between MC-LR and 1O2 is ∼ 106 M-1 s-1 based on competition kinetics with furfuryl alcohol. The typical adsorbent needs separation after the treatment, while magnetic iron oxides can be easily removed by a magnetic field. Maghemite and humic acid coated magnetite (HA-Fe3O4) were synthesized, characterized and applied for chromium(VI) removal. The adsorption of chromium(VI) by maghemite and HA-Fe3O4 follow a pseudo-second-order kinetic process. The adsorption of chromium(VI) by maghemite is accurately modeled using adsorption isotherms, and solution pH and presence of humic acid influence adsorption. Humic acid coated magnetite can adsorb and reduce chromium(VI) to non-toxic chromium (III), and the reaction is not highly dependent on solution pH. The functional groups associated with humic acid act as ligands lead to the Cr(III) complex via a coupled reduction-complexation mechanism. Extended X-ray absorption fine structure spectroscopy demonstrates the Cr(III) in the Cr-loaded HA-Fe 3O4 materials has six neighboring oxygen atoms in an octahedral geometry with average bond lengths of 1.98 Å.

Advanced oxidation of arsenic species in aqueous solutions

Ingestion of arsenic from contaminated water is a serious problem and affects the health of more than 100 million people worldwide. Traditional water purification technologies are generally not effective or cost prohibitive for the removal of arsenic to acceptable levels (≤10 ppb). Current multi-step arsenic removal processes involve oxidation, precipitation and/or adsorption. Advanced Oxidation Technologies (AOTs) may be attractive alternatives to existing treatments. The reactions of inorganic and organic arsenic species with reactive oxygen species were studied to develop a fundamental mechanistic understanding of these reactions, which is critical in identifying an effective and economical technology for treatment of arsenic contaminated water. ^ Detailed studies on the conversion of arsenite in aqueous media by ultrasonic irradiation and TiO2 photocatalytic oxidation (PCO) were conducted, focusing on the roles of hydroxyl radical and superoxide anion radical formed during the irradiation. ·OH plays the key role, while O2 -· has little or no role in the conversion of arsenite during ultrasonic irradiation. The reaction of O2-· does not contribute in the rapid conversion of As(III) when compared to the reaction of As(III) with ·OH radical during TiO2 PCO. Monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are readily degraded upon TiO2 PCO. DMA is oxidized to MMA as the intermediate and arsenate as the final product. For dilute solutions, TiO2 also may be applicable as an adsorbent for direct removal of arsenic species, namely As(III), As(V), MMA and DMA, all of which are strongly adsorbed, thus eliminating the need for a multi-step treatment process. ^ Phenylarsonic acid (PA) was subjected to gamma radiolysis under hydroxyl radical generating conditions, which showed rapid degradation of PA. Product analysis and computational calculation both indicate the arsenate group is an ortho, para director. Our results indicate · OH radical mediated processes should be effective for the remediation of phenyl substituted arsonic acids. ^ While hydroxyl radical generating methods, specifically AOTs, appear to be promising methods for the treatment of a variety of arsenic compounds in aqueous media, pilot studies and careful economic analyses will be required to establish the feasibility of AOTs applications in the removal of arsenic. ^

Redox Regulation of Ras Proteins in Dictyostelium discoideum

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide. The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC- cells. RasC and RasD showed normal activation in sodC- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC118D motif inside the nucleotide binding region. A mutation of cysteine118 to alanine in RasG rendered the protein less active in sodC- than the wild type RasG protein and a mutation alanine118 to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.