京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24141号 / 医科博第142号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 松田 道行, 教授 斎藤 通紀, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate, making the transcription factors involved in antioxidant defence regulation targets of interest. The transcription factor NF E2 Related Factor 2 (NRF-2) regulates cellular defence genes including those encoding intracellular redox-balancing proteins such as enzymes involved in glutathione metabolism. Glutathione in particular is an important intracellular antioxidant molecule. NRF-2 binds to the Antioxidant Response Element (ARE) in the promoter of its target genes. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) acts as an inhibitor that targets NRF-2 for ubiquitination. During oxidative stress, NRF-2 dissociates from KEAP1 and enters the nucleus to bind to the ARE sequence. It is hypothesized that the elevated Reactive Oxygen Species may be depleting the glutathione levels within the cancer cell. System xc- is a cystine/glutamate antiporter that exports glutamate while importing cystine to synthesize glutathione. In response to oxidative stress, the cells increase system xc- activity in order to provide cystine for glutathione synthesis. There is evidence that expression of xCT, the specific subunit of system xc-,is regulated by NRF-2. However this has not yet been demonstrated in human breast cancer cells, which is the focus of this project. Basal expression of NRF-2, KEAP1 and xCT was characterized in three breast cancer cell lines (MDA-MB-231, MCF-7 and T47D) and compared to two non-cancer cell lines (184B5 and MCF10A). Basal protein levels of NRF-2 and KEAP1 showed no differences between cell lines. Basal protein levels of xCT were increased in MCF10A cells than T47D cells. MCF-7 cells were treated with hydrogen peroxide (H2O2) resulting in NRF-2 protein accumulation in the nucleus. With H2O2 treatment, xCT mRNA levels increased in MCF-7 cells. Additionally, transient overexpression of NRF-2 increased extracellular glutamate levels in MCF-7 cells. These data support a model that under oxidative stress, NRF-2 is localized to the nucleus and transcriptionally upregulates xCT. This is the first study in which the regulation of xCT has been linked to oxidative stress via NRF-2 in human breast cancer cells. / Thesis / Master of Science (MSc)

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Macrophages are a cornerstone in innate immunity, especially important in detecting and killing invading microorganisms. In tumor biology, the macrophages can contribute both to anti-tumor activity and tumor promotion depending on individual tumor microenvironment and therefore have a large impact on both tumor progression and prognosis. Two of the most important functions of macrophages are the ability to phagocytose microorganisms and then kill them through the respiratory burst. Phagocytosis activates the respiratory burst, but the more subtle interactions between these processes are less known. Since phagocytosis and reactive oxygen species production are two attributes that change between the classically and alternatively activated macrophages we decided to compare these two functions in macrophages. Activation of macrophages varies in terms of stimuli and effects. We specifically looked at macrophage activation by tumor cell lines and by chemical stimulation due to caffeine. We hypothesized that the level of oxidation would be directly linked to the level of phagocytosis. We assume that caffeine will increase activity in macrophages and that tumor cell co-incubation will decrease it. We found that there is a high correlation between the level of engulfment and level of respiratory burst. Chemical stimulation with caffeine can lower aggressiveness of macrophages at lower concentration, raise it at higher concentrations and eventually become toxic to the cell. Co-incubation with leukemic cell lines, as well with necrotic cells, affected an increase in aggressiveness.

Chitosan (CS) is a man-made sugar based biopolymer derived from chitin, the second most abundant natural polymer after cellulose. Chitin is sourced from crustacean species such as shrimps and crabs. The chemical structure of chitin contains N-Acetyl D-glucosamine monomer units which forms CS upon deacetylation. In CS, ?-(1-4) linked D-glucosamine units are randomly distributed. Approximately 75% - 80% sugar units contains primary amine groups in commercially available low molecular weight CS. Biodegradability, low toxicity, mucoadhesive and transfecting properties of CS polymer are attractive for applications as oral and nasal drug delivery systems. Chitosan polymer is water insoluble at neutral pH. To solubilize CS, dilute mineral acid (such as hydrochloric acid and nitric acid) or organic acid (such as acetic acid) is often used. CS contains both hydroxyl and primary amine groups in its structure. In acidic solution, the amine functional groups become protonated (positively charged). Positively charged CS remains stable only in low pH condition due to electrostatic repulsion of charged polymer segments. Therefore, by using a suitable anionic (negatively charged) cross-linker, stable CS particles (such as nanoparticles and microspheres) can be prepared. This is popularly known as ionic gelation method. Extensive studies have been done on the synthesis of drug loaded CS particles where particle integrity is maintained by ionic gelation using tripolyphosphate (TPP, an anionic cross-linker). Drug encapsulated CS-TPP composite particles are shown to maintain biodegradability and biocompatibility. The CS-TPP composite particles exhibits very limited dispersibility at neutral pH conditions specifically in neutral buffered conditions. A number of biomedical applications (including systemic drug formulations) however demands buffer-stable CS composite particles for achieving optimal therapeutic outcome. To overcome the above dispersibility issues, CS polymer and CS particles units have been chemically modified using water soluble motifs (such as water soluble polymer or ligands). This approach is very cumbersome and usually involves multiple purification steps. Chemical modification of natural CS chain introduces risks of compromising biodegradability and biocompatibility. Therefore, there is a strong need for developing a straightforward method of making water soluble CS and CS particles. Chapter 1 of this dissertation presents an overview of the CS polymer, various applications of CS polymers, methods of making CS polymers and CS particles, current limitations of synthesis methods for preparing stable chitosan particles at neutral pH conditions and finally delineates the scope of the proposed research work. Chapter 2 describes development of chemo-hydrothermal synthesis method for producing water soluble CS polymer and water dispersible CS composite particles. In this method, a chemical (depolymerizing agent) is used to treat CS polymer in a hydrothermal (high temperature and high pressure) condition. Two types of depolymerizing agents have been used, an inorganic acid (e.g. hydrochloric acid, HCl) and a bicarboxylic organic acid (e.g. tartaric acid, TA). In both cases, 100% depolymerized CS polymer was obtained. Chemical characteristics of the depolymerized CS were comparable to acid solubilized CS. CS polymer exhibits weak fluorescence. Interestingly, hydrothermally depolymerized CS shows strong fluorescence properties irrespective of the nature of depolymerizing agent used. TA not only depolymerized CS but also formed CS-TA composite particulate structures in solution via self-assembly. The CS-TA composite particles are stable in a wide pH range from 5 to 11. Detailed spectroscopic and microscopic studies have been done to understand the basic mechanism of particle formation and increase in fluorescence properties (i.e. structure-property relationship). Usefulness of CS-TA in solubilizing water-insoluble cargos (such as fluorescein isothiocyanate, FITC) has been demonstrated. Chapter 3 is focused on hydrothermal synthesis of mixed-valence copper (Cu) oxide loaded CS-TA composite particles and their characterization. Crystalline Cu oxide nanoparticles were coated with the CS-TA layer. Water dispersibility of Cu oxide greatly improved upon coating with CS-TA material. To demonstrate catalytic activity of Cu-oxide loaded CS-TA film in sequestering carbon dioxide (CO2), an electrochemical setup was used. Electrochemical reduction of CO2 was successfully demonstrated. It was observed that CS-TA environment not only maintained catalytic properties of Cu oxide but also allowed solution processing of Cu-oxide film onto the electrode surface. Chapter 4 discusses a convenient method of making monodispersed water dispersible Cu loaded chitosan nanoparticles (Cu-CS) using HCl depolymerized CS polymer. The purpose of this study was to investigate if there was any improvement in antibacterial properties of Cu-CS nanoparticles prepared using hydrothermally treated CS polymer. Interestingly, it was observed that the antibacterial efficacy of Cu was not compromised in Cu-CS nanoparticles. Moreover, the materials exhibited improvement in antibacterial efficacy against both Gram-negative and Gram-positive bacteria species. A plausible mechanism has been proposed to explain antibacterial results. Chapter 5 summarizes major findings of this dissertation research and presents future research directions.

Parkinson's disease (PD) is a neurodegenerative condition that affects millions of people worldwide. The exact etiology of PD is unknown. However, it is well established that environmental factors contribute to the onset of PD. In particular, chemicals such as the insecticide Rotenone have been shown to increase the death of dopaminergic (DA) neurons by increasing levels of reactive oxygen species (ROS). ROS such as hydrogen peroxide (H2O2) have been shown to be elevated above basal levels in PD patients. Currently, to measure H2O2 concentrations, a commercially available (Amplex® Red) fluorescent assay is used. However, the assay has limitations: it is not completely specific to hydrogen peroxide and can only measure extracellular ROS concentrations. This research focuses on testing an electrochemical sensor that uses cyclic voltammetry to quantitatively determine concentrations of H2O2 released from a cell culture. The sensor was first tested in normal cell culture conditions. Next, chemical interference was reduced and the sensor was optimized for accuracy by altering protein concentrations in the media. Finally, Rotenone was added to a cell culture to induce H2O2 production. Near real-time measurements of H2O2 were taken using the sensor and comparisons made to the fluorescent assay method. Overall, we are trying to determine if the electrochemical sensor can selectively and quantitatively measure H2O2 released from cells. Being able to track the production, migration and concentration of H2O2 in a cell can help researchers better understand its mechanism of action in cell death and oxidative damage, thus getting closer to finding a cure for PD.