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Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Biochemistry. Bibliography: leaves 99-126. Available online via the Internet.

Pancreatic ductal adenocarcinoma is an extremely lethal cancer that is difficult to treat. A better understanding of the biology of pancreatic ductal cancer will help to develop targeted therapies that may improve clinical outcomes. Recently, the lipid signaling molecule sphingosine-1-phosphate (S1P) has emerged as a driver of malignant behavior in many types of cancer. Its role in pancreatic cancer remains unknown. Pancreatic cancer cells express high levels of the S1P receptor known as S1PR1, which is the receptor most important for mediating growth and migration through S1P signaling. In addition, the subcellular expression of the sphingosine kinases is altered in pancreatic cancer cells, which may contribute to their malignant behavior. Exogenous S1P increases pancreatic cancer cell migration, while inhibition of S1P signaling decreases the metabolic activity of pancreatic cancer cells as well as their ability to invade and migrate. Taken together, these results demonstrate the importance of S1P signaling in maintaining malignant behavior in pancreatic cancer cells. In addition, inhibition of S1P signaling represents a potential therapeutic target in pancreatic ductal cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21993号 / 医博第4507号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 竹内 理, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h). / Doctor of Philosophy / Sphingosine-1-phosphate (S1P) is a molecule related to various diseases, such as cancers and inflammatory diseases. Elevated levels of S1P promote the development of these diseases, thus making it necessary to reduce the production of S1P in patients. Since S1P is generated in human body by an enzyme called sphingosine kinase (SphK), inhibiting the activity of SphK can be beneficial for reducing S1P levels. Developing inhibitors for SphK is also a promising strategy for curing such diseases. A very potent inhibitor has been reported, but it could be metabolized quickly into other inactive metabolites in human, which renders it ineffective. To develop better drug candidates, a series of compounds with similar structure has been synthesized and tested for their potency and metabolic stability. Based on analysis of the relationship between the compound structures and activities, several compounds with less potency and different metabolic stability has been prepared and their efficacy in reducing S1P levels has been tested.

Le vieillissement et les maladies associées constituent une préoccupation croissante des sociétés modernes. En effet, l'espérance de vie augmente rapidement et ceci n'est pas accompagné par une fécondité accrue. Par conséquent, la proportion de personnes âgées augmente et la science doit fournir des solutions pour traiter les maladies liées à l'âge. Dans ce travail, nous avons étudié une partie du réseau génétique qui a un impact sur la durée de vie par le biais du régime alimentaire. Nous nous sommes concentrés en particulier sur le rôle d'un gène conservé qui code pour la sphingosine kinase (SPHK), et nous décrivons ici pour la première fois son rôle dans la longévité induite par la restriction alimentaire. Cette thèse se compose de deux parties. Dans la première partie, C. elegans a été utilisé pour comprendre le rôle que la sphingosine kinase (sphk-1) joue dans le vieillissement. Nous rapportons que les vers porteur d'une mutation dans le gène sphk-1 vivent plus longtemps que des vers sauvages et ne répondent pas à une restriction alimentaire (RA) et ressemblent à des animaux sauvages soumis à RA. De plus, nos données suggèrent que la longévité causée par une mutation du gène sphk-1 nécessite la présence du facteur de transcription SKN-1b, connue pour son rôle dans la RA. De plus, sphk-1 et skn-1b sont tout deux exprimés dans les neurones de la tête. Nos travaux suggèrent également que la voie TOR/autophagie est impliquée dans la longévité de sphk-1 mutants. Nous avons également montré que d'autres gènes dans la voie de synthèse des céramides ont un effet similaire sur la longévité suggérant que cette voie toute entière est capable d’affecter la longévité.La deuxième partie de ce travail a été réalisée sur un modèle de souris de la maladie d’Alzheimer (MA) pour tester le rôle des gènes SPHK dans la MA et la dégénérescence neuronale. Nous avons constaté que le niveau d'expression de SPHK était significativement augmenté dans nos modèles de souris par rapport à leurs contrôles de la même portée dès l’âge de 6 mois. Un inhibiteur de la SPHK (SKI-II) a été administré à ces souris et ce traitement a permis une amélioration des performances des souris dans des tests de marche sur balancier et une augmentation du poids de cerveau, mais pas d'amélioration de la mémoire dépendante de l'hippocampe. Un autre traitement de SKI-II sur des souris de type sauvage n'a pas montré d’amélioration significative, mais la restriction calorique (RC) a réduit les niveaux de SPHK chez les souris sauvage, ce qui suggère que la sphingosine kinase a des fonctions conservées dans les voie de signalisation liés au sensing des nutriments. / Advances in medical technology and hygiene standards have increased human life expectancy at unprecedented rates worldwide. Nevertheless, one of the consequences of a growing elderly population is an increased prevalence of age-related disease. A scientific understanding of the underlying biological mechanisms of aging is essential to develop effective treatments for age-related diseases and to provide adequate health care to the elderly. In this study, we investigated part of the genetic network that mediates lifespan extension resulting from dietary restriction. We focused on the contribution of a conserved gene encoding the enzyme sphingosine kinase, and describe for the first time its role in diet-mediated longevity.This thesis is composed of two parts. In the first part, we used the nematode Caenorhabditis elegans as a model organism to investigate the role of the sphingosine kinase gene (sphk-1) in aging. We found that worms carrying a sphk-1 null mutation (sphk-1(ok1097)) are long-lived and do not benefit from further lifespan extension upon dietary restriction (DR), a regimen that extends the lives of wild-type worms. sphk-1(ok1097) animals exhibit many phenotypes displayed by animals subjected to DR, suggesting that sphk-1(ok1097) acts through the DR longevity pathway. In support of this, sphk-1(ok1097)-mediated lifespan extension requires the essential DR regulator, SKN-1b, and, similar to SKN-1b, sphk-1 is expressed in head neurons. A search for possible sphk-1(ok1097)-associated longevity determinants suggested the involvement of the TOR/autophagy pathway. Moreover, we found that mutations in ceramide pathway genes other than sphk-1 have similar effects on longevity. Finally, we discovered that sphk-1 mutants fail to reduce germ cell numbers in response to DR. Because such a reduction appears to be an essential feature of DR-mediated lifespan extension, we propose that this failure to reduce germ cell numbers may explain why sphk-1(ok1097) mutant longevity is not extended when nutrient levels are low.The second part of this study investigated the role of sphingosine kinase in brain function during normal and pathologic aging. We examined the expression of sphingosine kinase genes in wild-type mice and in a mouse model of Alzheimer’s disease (AD)-like neurodegeneration. Expression of both mouse SphK genes was increased in the brains of AD-like mice as early as 6 months of age. Chronic administration of an SphK inhibitor elevated the brain weight of AD-like mice and improved their performance in the beam walking test, but not in hippocampus-dependent memory tasks. Treatment of wild-type mice with SKI-II had little effect, but calorie restriction reduced the expression of SphK mRNA in the brain, suggesting that sphingosine kinase may play some conserved roles in nutrient sensing pathways. / 在工业化水平越来越高的当今社会，人口的老龄化正逐渐成为一个严重的社会问题。医疗手段的发展、生活水平的提高使得人们的寿命在不断增加，而与此同时生育率并没有同步增加，这就导致老年人口在整个社会人口中的比重快速增长，因此，衰老以及一些相关的疾病就得到了人们越来越多的关注，也正在成为一个越来越热门的科研领域。从科研工作者的角度，我们希望能够通过实验手段破解衰老及其相关疾病的机制从而寻找延缓衰老或治疗相关疾病的方法。在本论文中，我们研究了一个通过限制饮食来调控寿命的基因网络的一部分。我们的主要研究对象是编码鞘氨醇激酶的基因（SphK），我们第一次发现了它在饮食介导的寿命调控中的作用。本论文由两个章节组成。在第一章中，我们使用秀丽隐杆线虫作为模式动物来研究鞘氨醇激酶基因（sphk-1）在其衰老调控过程中的作用。在研究中，我们发现，携带一种sphk-1 删除突变（sphk-1(ok1097)）的突变体线虫的平均寿命显著延长，而饮食限制（DR）并不能进一步延长它的寿命。另外，这种突变体还表现出一系列与受饮食限制调控的动物相类似的表型，尽管并不是全部。这些结果都表明sphk-1 基因通过饮食介导的途径参与线虫的寿命调控。我们随后的研究显示，由sphk-1 突变引起的寿命延长依赖于DR 的一个调控因子：SKN-1b；并且，与skn-1b 类似，sphk-1 表达的位置也在线虫头部的神经元。对于其它DR 相关因子的研究还发现线虫sphk-1(ok1097)突变引起的寿命延长可能与TOR 及自噬通路的作用有关。此外，我们还发现，不仅是sphk-1，神经酰胺通路中的其它基因对于寿命调控也有类似的效果。最后，我们的研究结果显示，DR 未能减少sphk-1(ok1097)突变体线虫的生殖细胞数量，这一发现或许能够解释为什么DR不能够进一步延长sphk-1(ok1097)突变体线虫的寿命。在第二章中，我们使用了一种模拟阿尔茨海默症（AD）的小鼠模型来测试哺乳类SphK 基因在神经退行性病变中的作用。我们发现，在模型小鼠脑组织中，两种哺乳类SphK 基因的表达水平与它们的同窝对照相比显著增加，而这一显著差异早在6 月龄的小鼠脑中就能发现。在后续的实验中，我们给这些小鼠喂食了一种SphK 的抑制剂——SKI-II。这种抑制剂显著改善了模型小鼠在平衡木实验中的表现，并且显著增加了它们的脑重量，但对于海马依赖性的记忆并没有改善。在另一个实验中，给野生型小鼠注射SKI-II 并没有表现出明显的差异，但热量限制（CR）降低了野生小鼠脑中两种SphK基因的表达量，这一与线虫中类似的结果提示，鞘氨醇激酶在营养通路中可能有一些进化保守的功能。

The bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulator of a variety of important physiological functions, including cell growth, cell survival, cell motility, angiogenesis, lymphocyte trafficking, and mast cell function. S1P is formed by two different sphingosine kinases (SphKs) and binds to a family of 5 differentially expressed G-protein coupled receptors (S1PRs). The majority of research to date has focused on the activation of these receptors, but there is compelling evidence to suggest that S1P exerts intracellular functions independent of S1PRs. However no bona fide intracellular targets of S1P have been identified. In my dissertation, I have identified a novel intracellular binding protein for S1P. This finding has important implications for the pleiotropic actions of S1P.

In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20616号 / 医博第4265号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 渡邊 直樹, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM