Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH), £\-melanocyte-stimulating hormone (£\-MSH), and £]-endorphin (£]-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to the hypoxia-inducible factor-1£\ (HIF-1£\) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the pro-survival MITF/HIF-1£\ pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1£\-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 £\ was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1£\ pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1£\/HO-1 indeed contributes to melanoma survival after POMC gene delivery.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Thesis advisor: Dr. William Durante. "December 2008" Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references (p. 99-116).

Ischaemia/Reperfusion Injury (IRI) is the most common cause of acute kidney injury- a devastating clinical problem lacking any specific treatments to promote renal recovery. Macrophages (Mφ) are pleiotropic cells of the innate immune system, with roles spanning host defence, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Mφ are also known to be important mediators of renal injury in other experimental models of renal disease including transplantation, obstruction and glomerulonephritis. This work sought to examine the role of Mφ in mediating renal IRI. Conditional renal Mφ and monocyte depletion prior to experimental IRI was achieved by administering diphtheria toxin to the CD11b-DTR transgenic animal. This had no impact on either renal function or structural injury. In contrast liposomal clodronate mediated Mφ depletion provided functional and structural protection from injury. Administration of exogenous apoptotic cells also protected renal function if delivered 24h prior to IRI. Immunodeficient SCID mice exhibited a protected injury phenotype after IRI, however derived no additional protection from the administration of either liposomal clodronate or i.v. apoptotic cells. These findings suggest that the protective phenotype must involve either lymphocyte populations or circulating antibody. Preliminary work demonstrates that SCID mice lack IgM natural antibody which deposits in the kidney in the first 30 minutes after IRI. It was also demonstrated that apoptotic cells bind IgM natural antibody present within the circulation. The potential for the key antioxidant enzyme Heme oxygenase-1 (HO-1) to protect renal function was also examined in aged mice using hemearginate (HA) - a potent HO-1 inducer. Echoing epidemiological studies in humans aged mice had increased susceptibility to IRI, whilst failing to induce medullary HO-1. The main site of medullary HO-1 induction by HA was in medullary Mφ, and the protective phenotype was abolished by Mφ ablation, implicating Mφ as the key mediators of HA induced protection in renal IRI. Final studies employed adenoviral transduction to overexpress HO-1 within bone marrow derived Mφ, leading to a modified phenotype with increased IL- 10 and phagocytosis, and reduced TNFα and NO production. When these were introduced in vivo after IRI renal function was improved, potentially due to accelerated clearance of renal platelet deposition.

Orientador: Valéria Cristina Sandrim / Resumo: A pré-eclâmpsia (PE) é a principal causa de mortalidade e morbidade entre as gestantes no Brasil e em vários países. A fisiopatologia desta doença é complexa e envolve vários processos. Um destes, amplamente validado na literatura, relaciona-se a um status oxidativo, onde há prevalência de produção de radicais livres e/ou redução da atividade antioxidante. Apesar destas evidências, a suplementação clínica com antioxidantes (vitamina C e E) não se demonstrou promissora em PE. Recentemente, vem sendo explorado como terapia em várias doenças, a ativação de um fator de transcrição, o NRF2 (do inglês - nuclear factor, erythroid 2-like 2), que atua induzindo a transcrição de diversos genes que promovem a proteção celular através da codificação de proteínas com atividade desintoxicante e antioxidante. Entre elas a heme oxigenase (HO-1) é a mais estudada, pois apresenta efeitos antiapoptóticos, antioxidantes e citoprotetor. Além disso, o aumento do estresse oxidativo na PE pode potencialmente reduzir a biodisponibilidade de óxido nítrico (NO) que pode ser modulado por alguns polimorfismos localizados no gene da óxido nítrico sintase (NOS3). Notavelmente, os haplótipos formados pela combinação de polimorfismos foram associados a diferentes subgrupos de resposta à terapia anti-hipertensiva em PE. No presente estudo, comparamos as distribuições dos polimorfismos localizados nos genes NFE2L2 rs35652124 (C/T) e no gene HMOX1 rs2071746 (A/T) em gestantes com PE que respondem à metildopa ou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Preeclampsia (PE) is the leading cause of mortality and morbidity among pregnant women in Brazil and several countries. Its pathophysiology is complex and involves several processes, including the oxidative stress (increase of free radicals and/or decrease of antioxidant defense). Although evidences, clinical supplementation with vitamins (C and E) was not promising in preeclampsia. Currently, therapeutically the activation of transcription factor, NRF2 (nuclear factor, erythroid 2-like 2), has been explored in several diseases. This factor promote cytoprotection by activates the transcription of several antioxidant and detoxification proteins. Hemeoxigenase-1 (HO-1) is the most studied, because has antiapoptotic, anti-inflammatory and cytoprotection activities. In addition, the increased oxidative stress in PE can potentially reduce the bioavailability of nitric oxide (NO) which may impaired by some SNPs on endothelial nitric oxide synthase (NOS3) gene. Notably, haplotypes formed by the combination of polymorphisms of were associated with different subgroups of response to antihypertensive therapy in PE. Therefore, in the present study we compared the distributions of rs35652124 (C/T) NFE2L2 and rs2071746 (A/T) HMOX1 polymorphisms in PE patients who respond to methyldopa or antihypertensive therapy with those found in PE patients who do not respond to methyldopa or total antihypertensive therapy. We also examined whether NFE2L2 and HMOX1 polymorphisms affect plasma HO-1 leve... (Complete abstract click electronic access below) / Mestre

Wang, Ruizhi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 109-134). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Publications --- p.ii / Abstract --- p.iv / 中文摘要 --- p.viii / Abbreviations --- p.xi / List of tables --- p.xiv / List of figures --- p.xv / Contents --- p.xvii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of gastric cancer --- p.2 / Chapter 1.2 --- Risk factors of gastric cancer --- p.3 / Chapter 1.3 --- Treatment of gastric cancer --- p.4 / Chapter 1.3.1 --- Surgical treatment --- p.4 / Chapter 1.3.2 --- Chemotherapy --- p.4 / Chapter 1.3.3 --- Targeted therapy --- p.5 / Chapter 1.4 --- "Phenotypes of cell death: apoptosis, oncosis and autophagy" --- p.9 / Chapter 1.4.1 --- Cell death --- p.9 / Chapter 1.4.2 --- Apoptosis --- p.10 / Chapter 1.4.2 --- Oncosis --- p.11 / Chapter 1.4.3 --- Autophagy --- p.12 / Chapter 1.4.4 --- p53 --- p.13 / Chapter 1.5 --- Heme oxygenase-1 --- p.14 / Chapter 1.5.1 --- General introduction of Heme oxygenase --- p.14 / Chapter 1.5.2 --- Anti-oxidant function of HO-1 --- p.15 / Chapter 1.5.3 --- Anti-inflammation function of HO-1 --- p.17 / Chapter 1.5.4 --- Pro-angiogenesis role of HO-1 --- p.18 / Chapter 1.5.5 --- HO-1 and cell proliferation --- p.19 / Chapter 1.5.6 --- HO-1 as a therapeutic target for tumors --- p.20 / Chapter 1.6 --- Objectives of study --- p.22 / Chapter Chapter Two: --- Methods and materials --- p.26 / Chapter 2.1 --- Gastric cancer cell lines --- p.27 / Chapter 2.2 --- Cell proliferation detection --- p.27 / Chapter 2.2.1 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.27 / Chapter 2.2.1.1 --- Introduction of MTT assay --- p.27 / Chapter 2.2.1.2 --- Processes of MTT assay --- p.27 / Chapter 2.2.1.3 --- Cell proliferation and cytotoxicity of drugs --- p.28 / Chapter 2.2.2 --- Detection of apoptosis by TUNEL assay --- p.29 / Chapter 2.2.2.1 --- TUNEL (Terminal uridine deoxynucleotidyl transferase dUTP nick end labeling) --- p.29 / Chapter 2.2.2.2 --- Sample preparation --- p.29 / Chapter 2.3 --- Detection of cell cycle by flow cytometry --- p.32 / Chapter 2.3.1 --- Cell cycle --- p.32 / Chapter 2.3.2 --- Sample preparation --- p.33 / Chapter 2.3.3 --- Flow cytometry analysis --- p.34 / Chapter 2.4 --- Detection of mitochondrial membrane potential(ΔΨm) --- p.35 / Chapter 2.4.1 --- Sample preparation --- p.35 / Chapter 2.4.2 --- Mitochondrial membrane potential(ΔΨm) analysis by flow cytometry --- p.36 / Chapter 2.5 --- Detection of proteins investigated in the project --- p.37 / Chapter 2.5.1 --- Antibodies --- p.37 / Chapter 2.5.2 --- Sample Preparation --- p.39 / Chapter 2.5.2.1 --- Cell culture --- p.39 / Chapter 2.5.2.2 --- Protein extraction --- p.39 / Chapter 2.5.2.3 --- Protein assay --- p.41 / Chapter 2.5.2.4 --- Final loading protein --- p.42 / Chapter 2.5.3 --- Western blotting --- p.43 / Chapter 2.6 --- Statistical analysis --- p.45 / Chapter Chapter three: --- Roles of HO-1 in 5-FU treatment for gastric cancer cell lines --- p.47 / Chapter 3.1 --- Cell proliferations with drug treatments --- p.48 / Chapter 3.1.1 --- MTT assay --- p.48 / Chapter 3.1.1.1 --- Introduction --- p.48 / Chapter 3.1.1.2 --- Method and results --- p.49 / Chapter 3.1.2 --- TUNEL assay --- p.58 / Chapter 3.1.2.1 --- Introduction --- p.58 / Chapter 3.1.2.2 --- Method and results --- p.59 / Chapter 3.2 --- HO-1 expression with drug treatments --- p.63 / Chapter 3.2.1 --- Introduction --- p.63 / Chapter 3.2.2 --- Method and results --- p.64 / Chapter 3.3 --- Discussion --- p.72 / Chapter Chapter Four: --- Mechanism responsible for the additive effect of 5-FU and ZnPP --- p.77 / Chapter 4.1 --- Cell cycle arrest after drug treatments --- p.78 / Chapter 4.1.1 --- Introduction --- p.78 / Chapter 4.1.2 --- Method and results --- p.79 / Chapter 4.2 --- Mitochondrial dependent and independent pathway --- p.85 / Chapter 4.2.1 --- Introduction --- p.85 / Chapter 4.2.2 --- Method and results --- p.87 / Chapter 4.3 --- Alteration of apoptotic proteins in gastric cancer cell death after drug treatments --- p.91 / Chapter 4.3.1 --- Introduction --- p.91 / Chapter 4.3.2 --- Method and results --- p.94 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter Five: --- Summary and future prospects --- p.107 / Chapter 5.1 --- Summary --- p.108 / Chapter 5.1.1. --- The inhibition of HO-1 enhances the sensitivity of gastric cancer cells to 5-FU --- p.108 / Chapter 5.1.2 --- Apoptosis induced by 5-FU plus HO-1 inhibitor ZnPP is through a mitochondrial-related pathway in MKN28 and MKN45 --- p.109 / Chapter 5.1.3 --- 5-FU plus ZnPP induces apoptosis in a caspase-dependent pathway in MKN45 while in both caspase-dependent and caspase-independent pathway in MKN28 --- p.110 / Chapter 5.2 --- Future prospects --- p.111 / References --- p.113

Background/Objective: Reperfusion injury is a serious consequence of blood flow reestablishment after myocardial infarction (MI) mediated by reactive oxygen species and neutrophilic cellular damage. Following MI, the left ventricle (LV) undergoes remodeling characterized by progressive wall thinning and cavity dilatation. Heme-Oxygenase-1 (HO-1) dependent decrease in oxidative stress may attenuate injury in part by inhibiting transcription factor NFκB-mediated inflammation. Hypothesis: I hypothesized that upregulation of HO-1 by hemin administration confers acute and chronic cardioprotection against I/R injury in rats and attenuates LV remodeling post-MI. I proposed the HO-1-dependent decrease in oxidative stress attenuates post-ischemic myocardial injury in part by inhibiting NFκB-mediated inflammation. Methods: Six week old male Wistar rats were randomly assigned to sham, vehicle, or hemin-treated groups. Vehicle and hemin were administered intraperitoneally once daily for 3 consecutive days prior to left anterior descending (LAD) coronary artery occlusion. Administration resumed 48 hours post-operatively and continued once every 3 days. Infarct size was determined by H&E histological analysis and fibrosis was quantified by Masson’s Trichrome staining. Transthoracic echocardiography was used to assess LV parameters and wall motion. Results: Hemin increased HO-1 expression, decreased infarct size and fibrosis, and attenuated LV remodeling in the short-term (4 days post-infarction). The decrease in infarct size and area of fibrosis in the hemin group was accompanied by a decrease in NFκB activity. No significant difference in infarct size and area of fibrosis between hemin and vehicle-treated groups was observed at 3 months. LV diameter and cardiac function did not differ significantly between the two groups at 3 months despite an attenuation of anterior wall thinning in the hemin group. Conclusion: HO-1 upregulation by hemin administration conferred acute cardioprotection and attenuated LV remodeling, possibly by inhibiting NFκB-mediated inflammation. However, chronic treatment with hemin did not prevent long-term post-infarction LV remodeling. It is possible that cardioprotection afforded by HO-1 upregulation is strong enough to curtail inflammation post-reperfusion and prevent LV remodeling acutely, but is not robust enough to protect the myocardium to the same degree in the long-term. Future research should focus on optimal HO-1 upregulation to attenuate long-term LV remodeling due to reperfusion injury. / Thesis (Master, Physiology) -- Queen's University, 2007-09-25 19:01:33.87

Abstract Previous studies have examined stress-induced heme oxygenase-1 (HO-1) expression primarily in mammalian systems. The present study examines, for the first time in amphibians, the effect of heat shock, sodium arsenite, cadmium chloride, and the proteasomal inhibitor MG132 on HO-1 accumulation in Xenopus laevis A6 kidney epithelial cells. Western blot analysis revealed that exposure of A6 cells to a range of heat shock temperatures (30-35 °C), which induced HSP30 accumulation, did not induce HO-1 accumulation. In contrast, cells treated with sodium arsenite (5-50 μM), cadmium chloride (50-200 μM) or MG132 (5-30 μM) exhibited a dose- and time-dependent accumulation of HO-1. Additionally, immunocytochemical analysis revealed that HO-1 and HSP30 accumulation occurred in a granular pattern primarily in the cytoplasm in cells treated with sodium arsenite, cadmium chloride, or MG132. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 and HSP30 accumulation initially increased to a maximum at 12 h and 24 h recovery, respectively, followed by a 50% reduction at 48 h. This initial increase in the relative levels of stress proteins was likely the result of new synthesis as it was inhibited by cycloheximide. In comparison, cells recovering from MG132 treatment displayed reduced but prolonged accumulation of HO-1 and HSP30. Interestingly, cells treated with low concentrations (10 μM) of sodium arsenite or MG132 but not cadmium chloride in combination with a mild 30 °C heat shock had enhanced accumulation of HO-1 and HSP30 accumulation compared to either of the stressors individually. This study has shown for the first time in amphibians that HO-1 accumulation is induced in response to metals and proteasomal inhibitors, suggesting that it may play a role in mediating the cellular stress response in X. laevis.

Preeclampsia (PE) is a maternal disorder of pregnancy, characterized by late-onset hypertension and proteinuria. It affects roughly 5-7% of all pregnancies worldwide and is a leading cause of maternal and fetal/neonatal morbidity and mortality. Cigarette smoking in pregnancy is associated with a 33% reduction in the incidence of PE, and this is dose dependent. It is hypothesized that carbon monoxide (CO), a combustion product in cigarettes, may confer cytoprotective and regulatory properties leading to the decreased incidence of PE. CO is produced endogenously by the enzyme heme oxygenase (HO), and it is thought that the manipulation of the HO/CO system in pregnancy can ameliorate or reduce the pathophysiologic signs of PE. The exposure of pregnant mice to 250 ppm CO led to an increase in each of the maternal uterine blood flow, vascularity of the placenta and vessel diameter, with a shift towards angiogenesis in the placenta tissue proteins Exposure of human placental villous explants to 250ppm CO led to a decreased production and release of the soluble vascular endothelial growth factor (VEGF) receptor -1 (sFlt-1). This molecule is increased in maternal plasma and placenta tissue of women with PE and it binds with molecules of angiogenesis, limiting their ability to interact with the endothelium. Using an AdsFlt-1 PE-like mouse model, the exposure of mice to 250ppm chronic CO prevented the hypertension, proteinuria and glomerular alterations, supporting the use of CO as a future therapeutic for women with PE. We completed a pilot study to evaluate the exposure of healthy volunteers to two, one hour inhalations of 250ppm CO. We determined the half-life of CO and we provide baseline kinetics data for males and females following CO inhalation. These data are important for future therapeutic studies in order to better establish proper dosing, concentration of CO and method of delivery. The results of this thesis contribute to the understanding of the pathophysiology of PE and provide evidence to support the use of CO as a therapeutic for this disorder. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2014-05-01 14:38:42.584

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references (p. 50-57).