Dissertations / Theses: 'Sphingosine kinases'

1

Megidish, Tamar. "Sphingosine as second messenger, sphingosine dependent protein kinases and their substrates /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/9285.

Full text

APA, Harvard, Vancouver, ISO, and other styles

2

Tonelli, Francesca R. "Sphingosine kinases : evaluation of therapeutic potential using prostate cancer cell models." Thesis, University of Strathclyde, 2012. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=18199.

Full text

Abstract:

Sphingosine kinase 1 and 2 (SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. Alterations in SK1 function have been implicated in human prostate cancer, being involved in the acquisition of therapy resistance and progression to androgen independence, two major issues in the clinical management of this disease. This study investigated the effect of down-regulating SK1 in androgen-dependent (LNCaP) and androgen-independent (LNCaP-AI) prostate cancer cells. The SK1 inhibitor, 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi) activates the proteasome by acutely inhibiting SK1 activity. Consequently, SKi induces the proteasomal degradation of SK1 isoforms, SK1a and SK1b, in LNCaP cells, which is associated with the accumulation of the pro-apoptotic lipid C22:0 ceramide and the induction of apoptosis. In contrast, SK1b is resistant to SKi-induced proteasomal degradation in LNCaP-AI cells, and this is associated with the failure to elevate ceramide le vels and to induce apoptosis. However, a different SK1 inhibitor, (S)-FTY720 vinylphosphonate overcomes this resistance to induce the proteasomal degradation of both SK1a and SK1b in LNCaP-AI cells, resulting in C16:0 ceramide accumulation and activation of apoptosis. The analysis of the effects of a selective inhibitor of SK2 revealed that SK1 and SK2 might regulate distinct functional pools of sphingolipids in prostate cancer cells. Additionally, SK1 inhibitors markedly reduce androgen receptor (AR) expression in prostate cancer cells. In particular, SKi down-regulates AR via a reactive oxygen species-dependent mechanism. Indeed, SKi treatment induces a pronounced oxidative stress response in LNCaP and LNCaP-AI cells. Thus, this study highlights a significant role of SK1 in promoting androgen receptor-dependent signalling and maintaining the survival of prostate cancer cells. This study also provides the first documented evidence of increased stability of SK1b compared with SK1a, which is associated with resistance to apoptosis. Taken together, these findings provide useful information regarding SK1-targeted therapeutic intervention for the treatment of (prostate) cancer.

APA, Harvard, Vancouver, ISO, and other styles

3

Bonhoure, Elisabeth. "Rôle de la sphingosine kinase-1 dans la réponse des cellules tumorales à la chimiothérapie." Toulouse 3, 2007. http://www.theses.fr/2007TOU30114.

Full text

APA, Harvard, Vancouver, ISO, and other styles

4

Dayon, Audrey. "Rôle de la sphingosine kinase-1 dans la survie et la progression des cellules tumorales prostatiques LNCaP vers l'androgéno-indépendance." Toulouse 3, 2008. http://thesesups.ups-tlse.fr/307/.

Full text

Abstract:

Le traitement du cancer de la prostate est basé sur la privation androgénique dont l'efficacité n'est que temporaire jusqu'à l'acquisition de l'androgéno-indépendace tumorale. Notre équipe a montré que l'oncogène sphingosine kinase (SK) est surexprimé dans le tissu tumoral prostatique, et que son activité enzymatique augmente avec l'agressivité tumorale. Nous avons exploré le rôle potentiel de la SK dans la survie et la progression des cellules tumorales prostatiques LNCaP vers l'androgéno-indépendance. Premièrement, nous montrons in vitro et in vivo que la privation androgénique entraîne une inhibition de l'activité SK qui est corrélée à une diminution de la prolifération cellulaire. Cette perte des capacités prolifératives peut être surmonté par la surexpression du gène codant pour la SK. L'addition de dihydrotestostérone (DHT) stimule l'activité SK et permet de ré-induire la prolifération cellulaire. Par ailleurs, l'inhibition pharmacologique de la SK bloque les effets prolifératifs de la DHT. Deuxièmement, nous démontrons l'implication de la SK dans la progression des cellules LNCaP vers le statut androgéno-indépendant. Lors de la privation androgénique prolongée nous observons une augmentation de l'activité et de l'expression protéique de la SK associées à la transdifférenciation neuroendocrine des cellules. Ces travaux impliquant la SK dans la transition vers l'androgéno-indépendance suggèrent que l'inhibition pharmacologique de la SK pourrait représenter une stratégie viable pour prévenir ou retarder la progression vers l'androgéno-indépendance
As prostate cancer cell proliferation is regulated by androgens, strategies aimed at reducing the production of androgens and/or effects are the standard of care in the management of patients with recurrent or advanced disease. Unfortunately all patients become resistant to hormonal manipulation and it is not clear how prostate cancer cells make the transition from being androgen-dependent to being androgen-independent after hormone ablation therapy. We have shown in the Lab that the oncogenic sphingosine kinase (SK) is overexpressed in tumor samples from prostate cancer patients (as compared with normal counterparts). We provide the first evidence that androgen privation induces a differential effect on SK activity in the hormono-sensitive LNCaP prostate cancer cell model. Short-term androgen removal induced a rapid and transient SK inhibition in vitro and in vivo in an orthotopically LNCaP model established in SCID mice. Conversely, long-term removal of androgen resulted in a progressive increase in SK expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SK activity. These results suggest that SK activation upon chronic androgen privation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer

APA, Harvard, Vancouver, ISO, and other styles

5

Niaudet, Colin. "Caractérisation et modulation des évènements initiaux contrôlant la mort radioinduite de l'endothélium microvasculaire." Nantes, 2009. https://archive.bu.univ-nantes.fr/pollux/show/show?id=34ca7579-91aa-46a5-bc5a-1291a772a1b6.

Full text

Abstract:

Une irradiation unique à forte dose déclenche l'apoptose du compartiment microvasculaire via le couple sphingomyélinase acide/céramide, contrôlant l'ensemble du processus de destruction tissulaire. Nous avons étudié la connexion entre cette apoptose contrôlée par l'ASMase et la capacité largement démontrée du céramide à induire la coalescence des microdomaines membranaires en larges plateformes. L'irradiation induit simultanément la voie de mort p38, dont l'inhibition protège partiellement les cellules endothéliales de la mort radioinduite. Enfin, la désorganisation des rafts par un agent dépléteur du cholestérol entrave l'activation de p38 et la mort des cellules microvasculaires qui en résulte. Ces résultats suggèrent l'existence d'un mécanisme de mort émanant de la membrane spécifique aux cellules endothéliales irradiées, dans lequel la coalescence des rafts mène à l'activation de la voie de mort p38. Nous avons ensuite utilisé la sphingosine-1-phosphate (S1P), un antagoniste du céramide, pour moduler cette vague précoce de mort radioinduite dans l'endothélium. Nous avons validé l'usage de la S1P comme agent systémique capable de limiter les défaillances aigues survenant dans les organes suite à l'exposition à des stress sévères : l'injection de S1P abolit l'effondrement de l'endothélium et ainsi empêche la survenue du syndrome gastro-intestinal à 15 Gy, tout comme le choc septique induit par le LPS, un autre syndrome contrôlé par l'apoptose microvasculaire. Cette protection exercée par la S1P sur l'endothélium présente une double spécificité : les sphingolipides apparentés sont incapables d'induire un niveau de protection équivalent de l'endothélium, et l'effet pro-survie de la S1P est dirigé uniquement vers l'endothélium et non vers les cellules épithéliales intestinales ou les lymphocytes. L'effet protecteur de la S1P est médié par les récepteurs couplés aux protéines G, puis les protéines pro-survie Akt, comme le prouvent l'inhibition de ces deux types de molécules qui, in vivo comme in vitro, supprime l'action de la S1P
High dose of ionizing radiation drives microvascular compartment to apoptosis, which controls the whole tissue damaging-process, through the acid sphingomyelinase (ASM)/ceramide pathway. We adressed the connection between ASMase-induced apoptosis and the well-known capacity of ceramide to induce rafts microdomains coalescence into large platforms. Concomitantly to membrane remodeling, irradiation activated p38 death pathway and its blockade partially protected endothelial cells from radiation-induced death. Finally, disorganization of rafts by cholesterol-depletor hindered the p38 activation and the subsequent death-induced microvascular cells. These results suggest a specific membrane controled death mechanism in irradiated endothelial cells, where rafts coalescence leads to p38 activation and apoptosis. We then used sphingosine-1-phosphate (S1P), a ceramide antagonist, to modulate this early wave of radioinduced death in endothelium. We validated the pharmacological use of systemic S1P to restrain acute organ failure in response to severe stress: S1P injection abolished endothelial cells collapse and therefore prevented 15 Gy-induced gastrointestinal syndrome, as well as LPS-induced septic shock, another syndrome driven by microvascular apoptosis. This protection from S1P toward endothelium showed a dual specificity: related sphingolipids failed to offer the same protective effects, and this protection affected only endothelial cells as compared to intestinal epithelial cells or lymphocytes. S1P-induced protective effect is mediated through G-protein coupled receptor then the prosurvival protein Akt, as inhibition of both pathways suppresses the S1P action in vitro and in vivo

APA, Harvard, Vancouver, ISO, and other styles

6

Gomez-Brouchet, Anne. "Rôle de la Sphingosine Kinase 1 (SphK1) dans la régulation de la survie des cellules de neuroblastome exposées au peptide Béta-amyloïde." Toulouse 3, 2007. http://www.theses.fr/2007TOU30251.

Full text

Abstract:

La démence de type Alzheimer pose un grand problème de santé publique dans les pays industrialisés et touche en France 850 000 personnes (5 % chez les plus de 65 ans et 20 % chez les plus de 85 ans). Elles se caractérise par la présence de lésions histologiques caractéristiques : les plaques séniles (PS) et les dégénérescences neurofibrillaires (DNF). L'analyse moléculaire et spatiotemporelle de ces deux lésions élémentaires a permis de mieux préciser la cascade des dysfonctionnements moléculaires et cellulaires de la maladie au cours de ces dernières années. Des travaux récents ont impliqué la voie Sphingomyéline/Céramide dans la mort neuronale induite par le peptide Aβ et donc dans la pathogénie de la maladie d'Alzheimer. En l'espace d'une décennie, les métabolites sphingolipidiques, c'est-à-dire céramide, sphingosine et sphingosine 1-phosphate (S1P), ont émergé comme les représentants d'une nouvelle classe de seconds messagers lipidiques régulant la prolifération, la différenciation et l'apoptose. Le céramide induit des réponses antiprolifératives et proapoptotiques, alors que l'addition de S1P favorise la survie cellulaire. Les effets opposés de ces deux sphingolipides ont conduit au concept selon lequel la balance dynamique entre les taux de céramide et de S1P représente un facteur déterminant pour le devenir de la cellule. Un acteur majeur de cette balance est la sphingosine kinase-1 (SphK1), responsable de la production de S1P. Compte tenu de ces données, nous avons étudié le rôle de la sphingosine kinase (SphK1) dans la régulation des signaux de survie et de mort des cellules humaines de neuroblastome SHSY5Y sous l'action du peptide Aβ 25-35. Il ressort de nos travaux que la SphK1 régule la survie de cellules de neuroblastome exposées au peptide β-amyloïde (25-35). En effet, l'activité SphK1 est fortement inhibée en réponse au traitement par le peptide β-amyloïde (25-35) via un mécanisme redox dépendant. .
Alzheimer disease (AD) is a critical problem of public health in the industrialized countries. AD affects 24. 3 million individuals in the world. In France, the number of AD patients represents 5 % of the population over 65 years-old (20 % of people over 85 years-old). Important progress have been made during the last ten years: Mutations were characterized, as well as genetic or environmental risk factors. A better analysis of the two elementary lesions of AD in their distribution and molecular characterization has allowed a better comprehension of the disease. Thus, the description of a dysfunction of proteins APP (Amyloid Precursor Protein) and Tau in sporadic and familial AD has led to therapeutic experiments and tests on cellular or animal models with promising results. Even though the diagnosis of AD still remains related to the neuropathology, it is evoked more precociously due to the progress in neuropsychological evaluation and imaging procedures. The advances in the comprehension of the disease mechanisms should make possible the discovery of new therapeutic targets. .

APA, Harvard, Vancouver, ISO, and other styles

7

Thompson, Dawn. "Sphingosine kinase and sphingosine-1 phosphate phosphatase : molecular tools to investigate the role of sphingosine-1-phosphate." Thesis, University of Strathclyde, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401320.

Full text

APA, Harvard, Vancouver, ISO, and other styles

8

Congdon, Molly D. "Structure Activity Relationship Studies on Isoform Selective Sphingosine Kinase Inhibitors." Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/82129.

Full text

Abstract:

A variety of diseases including Alzheimer's disease, asthma, cancer, fibrosis, multiple sclerosis, and sickle cell disease have been associated with elevated levels of sphingosine-1-phosphate (S1P). S1P, a pleiotropic lipid mediator involved in a broad range of cellular processes, is synthesized solely by the phosphorylation of sphingosine (Sph) and is catalyzed by the two isoforms of sphingosine kinase (SphK1 and SphK2). Therefore, SphKs are a potential therapeutic target; however, the physiological role of SphK2 is still emerging. In order to determine the role of SphK2 in vivo, more potent and selective small molecule inhibitors of SphK2, as well as dual inhibitors are necessary. Herein, explorations and advancements on the second generation SphK2 selective inhibitor SLR080811 are disclosed. Investigations into the lipophilic tail region of the hSphK2 inhibitor SLR080811 are detailed. This investigation highlights the dependency of SphK2 selectivity and potency on overall compound length. More importantly, this study identified the internal aryl ring of SLR080811 as a key pharmacophore of the scaffold. To further probe the significance of the aromatic region, the phenyl ring was replaced by a 2,6-naphthyl ether skeleton. Investigations into the tail region of this scaffold are described in detail. Key discoveries from this structure-activity relationship study include SLC5111312 (hSphK2 Ki = 0.90 μM, dual hSphK inhibitor), SLC5091592 (hSphK2 Ki = 1.02 μM, > 20-fold hSphK2 selective) and SLC5121591 (hSphK2 Ki = 0.61 μM, >16-fold hSphK2 selective). Molecular modeling studies with hSphK2 indicate that the extended aromatic group is able to participate in π-π stacking interactions with Phe548. In silico docking studies indicate that a guanidine hydrogen bond to Asp211 is key for SphK2 selectivity, and incorporation of a 3'-hydroxyl group on the pyrrolidine ring increases hydrogen bonding to Asp308, thereby increasing SphK1 potency and reducing selectivity. Additionally, biological studies employing SLC5111312 have helped to further elucidate the role of SphK2, suggesting that SphK2 has a catalytic role in the regulation of blood S1P levels. The shape of the hSphK2 binding pocket was probed by introducing an indole moiety in place of the naphthyl ring and varying its substitution pattern. One key discovery from this study is SLC5101465 (hSphK2 Ki = 0.09 μM, > 111 fold SphK2 selective), which has a 1,5-indole substitution pattern with an N-nonyl "tail". Molecular docking simulations highlight the importance of rotatable bonds and a relatively linear orientation between the "head group" and "tail group" to maintain essential hydrogen bond interactions to Asp residues with the guanidine moiety while minimizing steric interactions in the middle of the binding pocket. Expanding upon the 1,5-indole scaffold of SLC5101465, a series of aryl tail derivatives are examined. This study confirms the necessity of electron withdrawing groups located at the end of the inhibitor scaffold to optimize binding in the tail region of the SphK2 binding pocket.
Ph. D.

APA, Harvard, Vancouver, ISO, and other styles

9

Ng, Carl Khee-Yew. "Drought induced guard cell signal transduction involves sphingosine 1 phosphate." Thesis, Lancaster University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250627.

Full text

APA, Harvard, Vancouver, ISO, and other styles

10

Andrieu, Guillaume. "Rôle de la voie sphingosine kinase/sphingosine 1-phosphate dans le contrôle de la division cellulaire." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2605/.

Full text

Abstract:

La division cellulaire est cruciale pour le maintien de la stabilité du génome. Or, les gènes régulateurs de la mitose sont fréquemment mutés dans le cancer. Les cellules cancéreuses possèdent généralement un nombre anormal de chromosomes et la majorité des tumeurs solides sont aneuploïdes. Cette caractéristique favorise notamment l'initiation et la progression tumorale mais constitue également un facteur de mauvais pronostic et de résistance thérapeutique. La voie sphingosine kinases/sphingosine 1-phosphate (SphKs/S1P) régule la prolifération et la survie cellulaire, l'apoptose, la migration ou encore la réponse inflammatoire. De nombreuses études ont montré que sa surexpression favorise l'initiation et la progression tumorale mais également l'invasion, le processus métastatique et l'acquisition de résistance à la thérapie. Mon projet de thèse vise à mettre à évidence le rôle de la voie SphKs/S1P dans la régulation de la mitose et de la ségrégation chromosomique. Nos résultats montrent pour la première fois que les SphKs contrôlent la progression mitotique. Cette régulation implique la production de S1P et son interaction avec son récepteur couplé aux protéines G, le S1P5. La surexpression des SphKs ou la surproduction de S1P altèrent la ségrégation des chromosomes. De plus, nos données récentes suggèrent que la voie SphKs/S1P puisse être impliquée dans l'acquisition de résistance aux agents de chimiothérapie ciblant la mitose. Nous montrons pour la première fois que la voie SphKs/S1P est un nouveau régulateur de la mitose. Ces travaux permettent de mieux comprendre comment la voie SphKs/S1P contribue au développement tumoral et renforcent leur intérêt comme cible thérapeutique dans le traitement du cancer
Cell division is a crucial process for genome maintenance. In cancer, numerous regulators of mitosis are mutated or altered, impeding the quality of chromosome segregation. Tumors exhibit a high chromosomal instability and are frequently aneuploid. These hallmarks promote tumor initiation and progression but are also associated with poor prognosis and therapeutic resistance. The sphingosine kinases/sphingosine 1-phosphate (SphKs/S1P) pathway is a key regulator of several fundamental biological processes including cell proliferation, survival, apoptosis, migration or inflammatory response. Numerous studies have shown that the up-regulation of the SphKs/S1P pathway promotes tumor initiation and progression, invasion, metastasis and resistance to anticancer therapies. We are interested in the role of the SphKs/S1P pathway in cell division regulation. Our data indicate for the first time that SphKs regulate mitotic progression trough S1P production and the interaction with its G protein-coupled receptor S1P5. Furthermore, we showed that the up-regulation of the SphKs/S1P pathway impairs chromosome segregation. Finally, our recent data suggest that the SphKs/S1P pathway may be involved in the acquisition of resistance to mitotic chemotherapeutic agents. Overall, we have identified the SphKs/S1P/S1P5 pathway as a new genuine regulator of mitosis. We give support to the understanding of the implication of the SphKs/S1P pathway in tumoral progression and strengthen its interest as anti-cancer therapeutic targets

APA, Harvard, Vancouver, ISO, and other styles

11

Meza, Daniel. "Sphingosine Kinase 1 Inhibitor, A Novel Inducer of Autophagy." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1871.

Full text

Abstract:

Autophagy is the process of “cell self-eating” which has been implicated both in cell survival and cell death. Sphingosine kinase 1 (SphK1) regulates the intracellular balance between ceramide and sphingosine, bioactive lipids associated with cell death, and sphingosine-1-phosphate (S1P), whose actions are associated with survival and proliferation. Previous studies have implicated upregulation of SphK1 in the induction of autophagy. In this study, SK1-I, a SphK1 specific competitive inhibitor, induced autophagy in a concentration and time dependent manner in HCT116 colorectal carcinoma cells. This autophagic response was observed to be more intense in wild type p53 expressing HCT116 cells than in p53 null cells and ultimately led to non-apoptotic death in wild type and apoptotic death in p53 null cells. In agreement, cell death in wild type cells was not accompanied by cleavage of polyADP ribose polymerase, a hallmark of apoptosis. Knockdown of Beclin 1 demonstrated that it and its binding partners do not have a significant role in the induction of autophagy in response to SK1-I treatment. Similarly, mTORC1 signaling was not observed. In contrast, SK1-I markedly decreased Akt phosphorylation. However, this might not be the sole factor important for SK1-I induced autophagy, as pharmacological inhibition of Akt only led to a comparatively weak autophagic response. Indeed, phosphorylation of the endoplasmic reticulum (ER) stress marker eIF2 α, was greatly reduced, suggesting that an ER mediated mechanism also contributes to SK1-I induced autophagy. Thus, SK1-I induced autophagy was likely triggered by ER stress signaling and led to non-apoptotic cell death in the more highly autophagic wild type 53 expressing cells. These results suggest that an isotype specific SphK1 inhibitor might be a useful adjunct for the treatment of cancer or other diseases in which enhancement of cytotoxicity or autophagy is desirable.

APA, Harvard, Vancouver, ISO, and other styles

12

Stillman, Anthony D. "Targeting Sphingosine Kinase 2 as a Treatment for Cholangiocarcinoma." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6067.

Full text

Abstract:

Cholangiocarcinoma (CCA) has a high mortality rate and its occurrence is rising. This increase prompts the need for improved CCA treatments. Studies have suggested that CCA is highly reliant on the sphingosine-1-phosphate-receptor-2 (S1PR2) and sphingosine kinase 2 (SphK2). Recently, a competitive SphK2 inhibitor, ABC294640, has been approved for clinical trial. ABC294640 has the potential to treat CCA, which is support by a phase I clinical study that was able to temporarily treat a patient suffering from metastasized CCA with ABC294640. To determine the viability of ABC294640 as a treatment for CCA, this study focused on determining the effects of ABC294640 on rat CCA cell lines. We found that ABC294640 inhibited the growth and migration of CCA and CAFs cells. The growth and count of 3-D organotypic co-culture of CCA and CAFs, which forms the “duct-like” structures, were reduced by ABC294640. The potential of inhibiting SphK2 as a treatment for CCA is supported by our finding of increased expression of S1PR2 and SphK2 in CCA patient liver samples. In conclusion, ABC294640 represents a potential therapeutic agent for CCA.

APA, Harvard, Vancouver, ISO, and other styles

13

Li, Hao. "Design, Synthesis, and Structure-Activity Relationship Investigation of Selective Sphingosine Kinase Inhibitors." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/100741.

Full text

Abstract:

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

APA, Harvard, Vancouver, ISO, and other styles

14

Cardenas, Alex. "Sphingosine-1-Phosphate in Pancreatic Ductal Adenocarcinoma." Thesis, The University of Arizona, 2013. http://hdl.handle.net/10150/306974.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

15

Strub, Graham Michael. "INTRACELLULAR TARGETS OF SPHINGOSINE-1-PHOSPHATE." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/5.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

16

Morris, Emily A. "Structure-activity relationship studies and biological evaluation of selective sphingosine kinase inhibitors." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/73491.

Full text

Abstract:

Sphingosine 1-phosphate (S1P) has become a prevalent drug discovery target due to studies implicating it to several disease pathologies such as fibrosis, sickle cell disease, inflammation, diabetes, and cancer. S1P functions to induce cell proliferation and migration. S1P signaling occurs through intracellular targets or transport outside of the cell via ABC transporters, where it acts as a ligand to G-protein coupled receptors (S1P1-5). Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to S1P; these are the only enzymes known to mediate the phosphoryl transfer. Inhibiting either or both SphKs helps to modulate S1P, which may be useful as a therapeutic avenue for disease states where S1P signaling has gone awry. Herein, we document our efforts in profiling the structure-activity relationships (SAR) of SphK2 through an iterative process of synthesis and biological testing. First, an SAR structured around the head and linker region of our lead molecule, SLR080811, was performed. SLR080811 has a Ki of 1.3 µM and is 5-fold selective for SphK2. The modifications performed on SLR080811 yielded two promising inhibitors: SLP120701 (SphK2 selective with a Ki of 1.2 µM) and SLP7111228 (>200 fold selective for SphK1 with a Ki of 48 nM). In vitro studies in U937 cells yielded a decrease in S1P levels with the introduction of inhibitors. Mouse studies provided insight into the pharmacokinetic effect of our SphK2-selective inhibitors, revealing an increase in S1P levels in the blood. When in vivo studies were performed with the SphK1 selective inhibitor, S1P levels in blood decreased. These molecules provide the chemical biology tools to determine the effect of modulating S1P levels in vivo. We also focused our investigation on the tail region of the pharmacophore. From this study, SLM6031434 and SLM6041418 were discovered and both proved to be more potent and selective SphK2 inhibitors than SLR080811. SLM6031434 has a Ki of 370 nM and is 23-fold selective for SphK2. SLM6041418 has a Ki of 430 nM and is 24-fold selective for SphK2. Consistent with our previous observations, in vitro studies showed a decrease in S1P levels when inhibitor was introduced. Similarly, in vivo studies resulted in an increase of S1P levels in the blood. These compounds are positioned towards animal models of disease.
Master of Science

APA, Harvard, Vancouver, ISO, and other styles

17

Raje, Mithun. "Design, synthesis, and biological evaluation of selective sphingosine kinase inhibitors." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77051.

Full text

Abstract:

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell proliferation. SphK phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) which has been implicated as a major player in cancer growth and survival. SphK exists as two different isoforms, namely SphK1 and SphK2, which play different roles inside the cell. The dearth of isoenzyme-selective inhibitors has been a stumbling block for probing the exact roles of these two isoforms in disease progression. This report documents our efforts in developing SphK2-selective inhibitors. We provide the first demonstration of a SphK inhibitor containing a quaternary ammonium salt. We developed highly potent and moderately selective inhibitors that were cell permeable and interfered with S1P signaling inside the cell. In an effort to improve the selectivity of our inhibitors and enhance their in vivo stability, we designed and synthesized second generation inhibitors containing a heteroaromatic linker and a guanidine headgroup. These inhibitors were more potent and selective towards SphK2 and affected S1P signaling in cell cultures and various animal models.
Ph. D.

APA, Harvard, Vancouver, ISO, and other styles

18

Kwong, Eric K. "The Role of Sphingosine Kinase 2 in Alcoholic Liver Disease." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5808.

Full text

Abstract:

Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide characterized by the accumulation of lipids within the liver, inflammation and the possibility of progressing to cirrhosis and liver failure. More importantly, there are currently no effective treatments for ALD and liver transplantation remains the only therapeutic option for end-stage liver disease. Previous studies have shown that ALD is a result of a combination of endoplasmic reticulum (ER) stress, lipid metabolism dysregulation and inflammation. It has been previously reported that alcohol disrupts gut microbiota homeostasis and causes increased endotoxins that contribute to the pathology of ALD. However, the detailed mechanism(s) underlying ALD and disease progression is poorly understood. We have discovered that sphingosine kinase 2 (SphK2) deficient (SphK2-/-) mice on an alcohol diet exhibit increased steatosis and inflammation compared to wild type mice. Sphingosine 1-phosphate receptor 2 (S1PR2) and SphK2 have been previously shown to play a key role in nutrient metabolism and signaling. However, their roles in alcohol-induced liver injury have not been characterized. The overall objective of this study is to determine the molecular mechanism(s) by which disruption of S1PR2-mediated SphK2 signaling contributes to ALD. The effects of alcohol on mouse primary hepatocytes and cultured RAW264.7 macrophages were examined. The acute on chronic alcohol mouse model from NIAAA that recapitulates the drinking pattern of human ALD patients was used to study the effects of SphK2 deficiency in ALD. In addition, 60-day chronic alcohol mouse model was used to determine whether a more severe form of ALD was present in SphK2-/- mice. The results indicated that SphK2-/- mice on an alcohol diet exhibited an increased amount of hepatic steatosis compared to wild type mice. Genes regulating lipid metabolism were also dysregulated in SphK2-/- mice. SphK2-/- mice also had increased inflammation and liver injury as shown by an upregulation of inflammatory markers and increased levels of liver enzymes. Moreover, SphK2 protein expression levels were downregulated in the human livers of alcoholic cirrhotic and hepatocellular carcinoma (HCC) patients. These findings contribute to a greater understanding of the pathophysiology of ALD and could provide information on the development of novel therapeutics against ALD.

APA, Harvard, Vancouver, ISO, and other styles

19

Machesky, Nicholas John. "The modulation of sphingolipids by human cytomegalovirus and its influence on viral protein accumulation and growth." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1181753517.

Full text

APA, Harvard, Vancouver, ISO, and other styles

20

Childress, Elizabeth Saunders. "Structure-Activity Relationship Studies of Sphingosine Kinase Inhibitors and Mitochondrial Uncouplers." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/86662.

Full text

Abstract:

Sphingosine 1-phosphate (S1P) is a cellular signaling molecule that has been implicated in a variety of diseases including cancer, fibrosis, Alzheimer's, and sickle cell disease. It is formed from the phosphorylation of sphingosine (Sph) by sphingosine kinase (SphK) and SphK exists as two isoforms-"SphK1 and SphK2, which differ with respect to their cellular activity and localization. As the key mediators in the synthesis of S1P, SphKs have attracted attention as viable targets for pharmaceutical inhibition. To validate their potential as therapeutic targets, we aimed to develop potent, selective, and in vivo active inhibitors of SphK. Herein, we describe the design, synthesis and biological evaluation of SphK2 inhibitors. We first describe the development of six SphK2 inhibitors that assess the utility of replacing lipophilic tail groups with heterocyclic rings. These six compounds demonstrate that the lipid binding pocket for SphK2 cannot accommodate compounds with tail groups that are conformationally restricted or positively charged. We then describe the development of aminothiazole-based analogues of an SphK1-selective inhibitor. A library of 37 aryl-substituted aminothiazole tail groups were synthesized, revealing a structure-activity relationship study that examines electronic effects on the aryl-substituted aminothiazoles and the effect of modifying the amino portion of the aminothiazole. These molecules show surprisingly good potency and selectivity for SphK2. In particular, we highlight 3.20dd (SLC4101431), a biphenyl aminothiazole that is the post potent and selective SphK2 inhibitor to date, with an SphK2 Ki of 90 nM and 100-fold selectivity for SphK2. This molecule's in vivo activity will also be discussed. Mitochondrial uncouplers are small molecules that shuttle protons from the inter membrane space to the mitochondrial matrix independent of ATP synthase, which disrupts oxidative phosphorylation and promotes increased nutrient metabolism for homeostasis to be maintained. Consequently, small molecule mitochondrial uncouplers have been pursued as probes for mitochondrial function and as potential therapeutics for the treatment of obesity and type 2 diabetes. Herein, we describe the design, synthesis, and biological evaluation of small molecule mitochondrial uncouplers. We report a library of 52 compounds that have good mitochondrial uncoupling activity over a wide therapeutic range, including 5.16t (SHC4111522) and 5.17i (SHC4091665), which have EC50 values of 0.63 uM and 1.53 uM, respectively, and achieve at least 2-fold increase in oxygen consumption rates relative to basal levels. With these molecules, we demonstrate that pKa and cLogP significantly contribute to uncoupling activity and must be accounted for when developing new generation small molecule mitochondrial uncouplers.
Ph. D.

APA, Harvard, Vancouver, ISO, and other styles

21

Sankala, Heidi Milka. "The role of sphingosine kinase 2 in cell growth and apoptosis /." Also available to VCU users online at:, 2007. http://hdl.handle.net/10156/1902.

Full text

APA, Harvard, Vancouver, ISO, and other styles

22

Lim, Keng Gat. "Discovery and characterisation of novel anticancer compounds acting on sphingosine kinase." Thesis, University of Strathclyde, 2011. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=23882.

Full text

Abstract:

There is a critical need to develop novel anticancer therapeutics. Accumulating evidence has demonstrated that sphingosine kinase (SK) is a promising target for the treatment of cancer. There are two SK isoforms, termed SK1 and SK2. SK phosphorylates sphingosine to form sphingosine-1-phosphate which drives cancer cell proliferation and migration. Therefore, the aims of this project were to discover hit compounds that can be developed into effective chemical tools and/or anticancer drug leads targeting SK. Compounds from both synthetic and natural origins were screened using SK enzymatic assays. Natural compounds were isolated using bioassay-guided fractionation and structural elucidation was achieved using nuclear magnetic resonance and mass spectrometry. Cell-based assays were employed to assess cellular activity of these inhibitors. Cell growth was determined using [³H]-thymidine incorporation assays. Immunoprecipitation was used to study SK1 oligomerisation whereas fluorescence microscopy was used to examine the effects of SK inhibitors on actin distribution. Three key findings emerged from these studies. First, SK1 inhibitor kinetic characterisation with sphingosine revealed that (S)-FTY720 vinylphosphonate is a novel uncompetitive inhibitor whereas FTY720 and SKi (2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole) are competitive and mixed inhibitors respectively. These inhibitors also inhibited proliferation, induced proteasomal degradation of SK1 and apoptosis of cancer cells. Over-expression of an inactive SK1 mutant inhibited the catalytic activity of wild type SK1. Together with the discovery of two activators for SK1, a model was proposed in which SK1 contains allosteric site(s) that auto-inhibits catalytic activity. Second, (R)- FTY720-OMe was discovered as a selective SK2 inhibitor, which also inhibited MCF-7 cell growth and S1P-induced actin rearrangement, demonstrating SK2 as a pro-survival and migratory protein. Third, novel SK inhibiting scaffolds including resveratrol were identified from a plant hit. Balanocarpol (a resveratrol dimer) might act like a chelator to inhibit multiple SK1 molecules, giving new insights into diversity-oriented biosynthesis of resveratrol oligomers. Together, these findings provide strong impetus for the development of inhibitors targeting SK1 and/or SK2 as effective anticancer agents.

APA, Harvard, Vancouver, ISO, and other styles

23

Sankala, Heidi M. "The Role of Sphingosine Kinase 2 in Cell Growth and Apoptosis." VCU Scholars Compass, 2007. http://scholarscompass.vcu.edu/etd/1242.

Full text

Abstract:

Two isoforms of sphingosine kinase (SphK) catalyze the formation of sphingosine-1-phosphate (SIP). Whereas, SphKl stimulates cell growth and survival, it was found that when overexpressed in mouse NIH 3T3 fibroblasts SphK2 enhances caspase-dependent apoptosis in response to serum deprivation, independently of S1P receptors. Sequence analysis revealed that SphK2 contains a 9 amino acid motif similar to that present in BH3-only proteins. Studies showed that the BH3-only domain, catalytic activity, endoplasmic reticulum (ER) stress, and uptake of calcium by the mitochondria may all contribute to the apoptotic effects of overexpressed SphK2 in NIH 3T3 cells. Further studies in human carcinoma cells showed that overexpression of SphK2 increased the expression of the cyclin dependent kinase (cdk) inhibitor p21, but interestingly had no effect on p53 or its phosphorylation. Correspondingly, downregulation of endogenous SphK2 with small interfering RNA (siRNA) targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting p53. In addition, downregulation of SphK2 decreased G2/M arrest in response to doxorubicin. Surprisingly however, siSphK2 markedly enhanced apoptosis induced by doxorubicin in MCF7 and HCT-116 cells. This result raises the question of how overexpression of SphK2 decreases cell growth and enhances apoptosis while its downregulation sensitizes cells to apoptosis. A partial answer may come from the possibility that when SphK2 is overexpressed it does not always have the same subcellular distribution as the endogenous protein. It may also be possible that proteolysis of overexpressed SphK2 might induce apoptosis due to liberation of its BH3 peptide domain, which does not occur at the levels at which endogenous SphK2 is expressed. Collectively, these results demonstrate that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 expression may influence the balance between cytostasis and apoptosis.

APA, Harvard, Vancouver, ISO, and other styles

24

Sauer, Lysann. "Cross-regulation of prosaposin and sphingosine kinase signalling in prostate cancer." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9555.

Full text

Abstract:

Sphingosine kinase 1 (SPHK1) is an oncogenic enzyme that is upregulated in a wide range of human tumours and is associated with cancer progression. The product of SPHK1's activity, sphingosine-1-phosphate (S1P) enhances metastatic potential by promoting cancer cell migration and invasion. Our group has previously reported that in vitro SPHK1 inhibition potentiates the effects of docetaxel, a key treatment of prostate cancer (PCa) , implicating a potential therapeutic role of SPHK1. The work presented in this thesis investigated the mechanism for docetaxel-induced apoptosis in PC-3 cells and demonstrated the involvement of a SPHK1-dependent and -independent pathway. The dose-dependent inhibition of SPHK1 by docetaxel led to an initial loss of enzyme activity followed by a decrease in SPHK1 expression. Further, in prostate cancer cell models, SPHK1 inhibition had a significant chemosensitising potential leading to a 4-fold reduction in the effective dose of docetaxel. To link SPHK1 upregulation in prostate cancer cells to potential targets, this thesis investigated the nature of the interaction between SPHK1 and prosaposin signalling pathways. Prosaposin is a neurotrophic secreted protein involved in cancer progression and chemoresistance. Recent reports suggest that prosaposin activates SPHK1 in cancer cells. The work presented in this thesis demonstrates that prosaposin knockout mice exhibited a significant reduction in SPHK1 activity in the prostate and seminal vesicles. In prostate cancer cell lines, SPHK1 activity correlated with the amount of secreted, but not intracellular prosaposin. This suggests a role of the prosaposin/G-protein coupled receptor (GPCR) signalling. Blocking prosaposin in prostate cancer cells induced a signi cant decrease in SPHK1 activity and expression. Conversely, exogenous prosaptide TX14A, derived from the trophic sequence of saposin C, enhanced SPHK1 activity and expression. In turn, SPHK1 activation is essential for prosaposin secretion, but not for its expression. Increased SPHK1 expression or exogenous S1P triggered prosaposin secretion, while inhibition of SPHK1 using speci c siRNA or pharmacological inhibitor drastically decreased prosaposin secretion. Both prosaposin and SPHK1 signalling pathways were shown to be involved in the production of cytokines and angiogenic factors such as plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF) and pentraxin-3 (PTX3). These findings were extended to the clinical situation, and circulating prosaposin levels were determined in prostate cancer patients and compared with healthy controls. Increased prosaposin serum levels correlated with tumour stage. The work in this thesis has shown for the rst time the cross-regulation of prosaposin and SPHK1 in prostate cancer. The mechanism and physiological relevance of this cross-regulation in prostate cancer cells has been investigated and found to have significant therapeutic and biomarker potential in advanced prostate cancer.

APA, Harvard, Vancouver, ISO, and other styles

25

Gillies, Laura. "The sphingosine 1 phosphate receptor 5 and sphingosine kinase 1 and 2 are localized in centrosomes : role in regulating cell division." Thesis, University of Strathclyde, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501800.

Full text

Abstract:

The bioactive lipid sphingosine-1-phosphate (SIP) has the unique characteristic of being capable of engaging in both paracrine/autocrine signalling and intracrine signalling pathways. Research into the latter has been limited by the failure to identify downstream targets for intracellular S1P. The majority of cellular responses to S1P are mediated by a family of G-protein coupled receptors (GPCR) known as S1P receptors: SIP₁₋₅. The current study demonstrates that SIP₅ and both sphingosine kinases (SPHK1 and SPHK2) exhibit a unique intracellular localization in the centrosome of mammalian cells.

APA, Harvard, Vancouver, ISO, and other styles

26

Camaré, Caroline. "Rôle de la voie sphingomyelinase neutre de type 2 - sphingosine kinase 1 - sphingosine-1-phosphate dans l'effet angiogénique des LDL oxydées." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2648/.

Full text

Abstract:

L'athérosclérose et ses complications cardiovasculaires représentent la principale cause de morbi-mortalité dans les pays développés et posent un problème majeur de santé publique. De nombreux facteurs de risque, ont été décrits, en particulier l'hypercholestérolémie liée aux LDL circulantes. L'oxydation de ces LDL constitue une étape déterminante dans le processus d'athérogénèse. Dans la paroi des artères de moyen et gros calibre, on observe un réseau microvasculaire (les vasa vasorum) localisé au niveau de l'adventice et qui a pour rôle d'apporter oxygène et nutriments aux couches les plus externes de la paroi artérielle. Alors que ces microcapillaires ne sont pas retrouvés au niveau de la media et de l'intima des artères normales, il se produit une neovascularisation en regard de l'hyperplasie intimale des artères athéroscléreuses. Les LDL oxydées, par leur effet proangiogénique, peuvent contribuer à cette neovascularisation qui participerait à l'expansion de la plaque mais augmenterait aussi le risque d'hémorragies intraplaques ainsi que le risque de rupture et d'accidents athérothrombogènes. Le mécanisme angiogénique des LDLox est peu connu, et l'implication de la sphingosine-1-phosphate (S1P), un second messager de la voie des sphingolipides, impliqué dans l'angiogenèse, n'a pas été rapporté dans l'angiogenèse induite par les LDLox. Le but de ce travail est d'évaluer l'implication et les mécanismes d'activation de la voie des sphingolipides, avec la génération consécutive de S1P, dans l'effet pro-angiogénique des LDL oxydées sur les cellules endothéliales microvasculaires. Dans une première partie nous avons montré un effet biphasique des LDL oxydées sur les HMEC-1 (Human Microvascular Endothelial Cells), avec un effet proangiogénique à faibles doses (20 à 50µg/ml) et une absence d'angiogenèse pour des doses plus élevées non encore toxiques (100 µg/ml). Au-delà de ces doses, nous observons un effet toxique, comme cela a déjà été précédemment décrit. L'inhibition par siRNA de l'expression de SK1 (sphingosine kinase-1 impliquée dans la génération de S1P) et l'utilisation d'un anticorps bloquant de la S1P (Sphingomab(tm)), permettent d'inhiber efficacement la tubulogenèse des cellules endothéliales induite par les LDLox in vitro. L'angiogenèse chez la souris C57/Bl6 (étudiée par Matrigel plugs contenant des LDL oxydées), est efficacement prévenue par l'administration de l'anticorps bloquant Sphingomab(tm), définissant le rôle majeur de ce lipide bioactif dans le signal angiogénique des LDLox in vitro et in vivo. Sur un plan mécanistique, l'activation de SK1 et l'angiogenèse induite par les LDLox sont bloquées par l'utilisation d'anticorps bloquants anti-CD36 et anti-LOX-1 montrant l'implication des récepteurs scavengers des LDLox dans cette signalisation. L'utilisation d'un inhibiteur de l'activité kinase du VEGFR2 bloque dans une même proportion l'angiogenèse induite par les LDLox et par de la S1P exogène, évoquant une transactivation du VEGFR par les récepteurs membranaires de la S1P, en l'absence de synthèse de VEGF par les HMEC-1 sous l'effet des LDLox. Dans une deuxième partie nous avons montré que la génération de S1P consécutive à l'activation de la SK1 sous l'effet des LDLox, est étroitement liée à l'activation en amont de la nSMase2 dans des cellules endothéliales in vitro, impliquant également le stress oxydant et p38MAPK. L'utilisation d'un inhibiteur chimique de la nSMase2 bloque efficacement l'angiogénèse induite par les LDLox in vitro, ainsi que l'activation de SK1. Le time-course de génération de ROS et de phosphorylation de p38 dans les HMEC-1 soumises à des doses angiogéniques de LDLox est plus précoce que l'activation des enzymes de la voie des sphingolipides. L'utilisation des antioxydants NAC et trolox ainsi qu'un inhibiteur de phospho-p38MPAK bloquent à la fois la tubulogenèse et l'activation de nSMase2 et de SK1 in vitro, évoquant leur implication dans cette signalisation en amont de la voie des sphingolipides. Dans une troisième partie nous avons étudié le lien entre l'activation de la voie des sphingolipides par les LDLox et la connexine-43, protéine constitutive des jonctions gap permettant la transduction d'une signalisation entre cellules adjacentes. Grâce à l'utilisation d'inhibiteurs pharmacologiques des connexines, nous avons obtenu une inhibition efficace de l'activation de la SK1 et de la tubulogenèse induite par les LDLox in vitro, permettant d'évoquer la participation des jonctions gap dans la transduction du signal angiogène des LDLox impliquant la génération de S1P. En prespective à ce travail, nous projetons d'approfondir les mécanismes impliquant la connexine-43 dans cette signalisation notamment par rapport à la nSMase2 et à p38MPAK et d'établir un lien avec le développement de la vascularisation des plaque in vivo à l'aide de marquage de SK1 ou connexine-43 sur des échantillons d'artériectomie carotidienne. Ces travaux mettent en évidence de nouvelles cibles (S1P, connexine-43) impliquées dans le mécanisme de la néoangiogenèse intraplaque, évoquant de nouvelles perspectives thérapeutiques pour limiter les complications athérothrombogènes grèvant de façon importante le pronostic des patients atteints d'athérosclérose
Atherosclerosis and relative complications represent the main cause of morbi-mortality in western countries and arise a major problem for public health. Many risk factors have been described, in particular hypercholesterolemia linked to circulating LDL. The oxidation of LDL constitutes a decisive stage in atherogenesis. The vascular wall of the medium and large arteries is vascularized by a microcapilaries plexus (vasa vasorum) located in adventice, and in charge to bring oxygen and nutrients to the most external layers of the arterial wall. Although, no capillaries are found in the intima or media of normal arteries, we observe neovascularization under intimal hyperplasia of atherosclerotic arteries. As oxidized LDL could display angiogenic properties, they may contribute to this neovascularization, taking part in plaque growth but also in increasing risk of intraplaque heamorrage and destabilization leading to atherothrombotic complications. The angiogenic mechanisms of oxidized LDL is yet incompletely known, and the involvement of sphingosine-1-phosphate (S1P), a second messenger of sphingolipid pathway, well known for its angiogenic properties, has never been reported in oxLDL-induced angiogenesis. The aim of this work is to evaluate the involvement of sphingolipid pathway and its mechanisms of activation, with subsequent generation of S1P, in proangiogenic effects of oxidized LDL on microvascular endothelial cells. In a first part, we have demonstrated a biphasic effect exhibited by oxidized LDL in HMEC-1 (Human Microvascular Endothelial Cells), low oxLDL concentrations (20 to 50µg/ml) being angiogenic, whereas higher concentrations being not toxic yet (100µg/ml) are non angiogenic. Beyond these concentrations, as expected, a toxic effect was observed. Inhibition of SK1 (sphingosine kinase-1) expression by using a specific SiRNA, and use of S1P blocking antibody (Sphingomab(tm)), exibit an efficient inhibition of oxLDL-induced tubulogenesis of endothelial cells in vitro. Angiogenesis in C57/Bl6 mice (using Matrigel plug assay containing oxLDL), is efficiently prevented by administration of the S1P blocking antibody Sphingomab(tm), defining the major role of this bioactive lipid in the oxLDL angiogenic signal, in vitro and in vivo. Mechanisticaly speaking, ox-LDL-induced SK1 activation and angiogenesis are blocked by using anti-CD36 and anti-LOX-1 blocking antibody, showing the involvement of oxLDL scavenger receptors in this signaling. The use of a sphingosine kinase inhibitor of VEGFR2 blocks ox-LDL-induced angiogenesis in the same range as exogenous S1P-induced angiogenesis, evoking a cross-talk between VEGFR and membranary S1P receptors, in the absence of ox-LDL-induced VEGF synthesis in HMEC-1. In a second part, we have shown that ox-LDL-induced SK1 activation and subsequent S1P synthesis, was tightly linked to the amoung nSMase2 activation in endothelial cells in vitro, involving also oxidative stress and p38MPAK. The use of a chemical inhibitor of nSMase2 efficiently blocks both ox-LDL-induced angiogenesis and SK1 activation in vitro. The time course of ROS generation and p38MAPK phosphorylation in HMEC-1 submitted to angiogenic doses of oxLDL appears earlier than enzymes activation of sphingolipid pathway. Using antioxydants as NAC and trolox, as well as a phospho-p38MPAK inhibitor, we block both angiogenesis and nSMase2 and SK1 activation in vitro, evoking implication of oxidative stress and p38MPAK in this signalling amoung of sphingolipid pathway. In a third part, we have studied the link between oxLDL induction of sphingolipid pathway and connexine-43, a constitutive protein of gap junctions allowing signal transduction between adjacent cells. Through the use of two connexine pharmacological inhibitors, we have obtained an efficient inhibition of oxLDL-induced SK1 activation and tubulogenesis in vitro, evoking that gap junctions may take part in oxLDL angiogenic signalling, implicating S1P generation. In the perspective of this work, we plan to intensify the knowlegde of mechanisms involving connexine-43 in this signaling, particularly in relation to sphingolipid pathway and p38MAPK. Thereafter we would like to establish a link between this work and the developement of plaque neovascularization in vivo, in support of SK1 or connexines-43 staining on endarterectomy samples of human carotids from patients. These results arise new targets (S1P, connexine-43) implicated in development of intraplaque neoangiogenesis, and new therapeutic possibilities to limit atherothrombotic complications that significantly worsen the prognosis of atherosclerotic patients

APA, Harvard, Vancouver, ISO, and other styles

27

Gstalder, Cécile. "Rôle de la voie sphingosine kinase 1/sphingosine 1-phosphate dans l'adaptation à l'hypoxie intratumorale des adénocarcinomes rénaux à cellules claires." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30095/document.

Full text

Abstract:

Les adénocarcinomes rénaux à cellules claires (ccRCC), qui représentent 70% des tumeurs rénales, sont fortement mais irrégulièrement vascularisés, ce qui les rend hypoxiques et donc résistants aux chimiothérapies. L'hypoxie favorise l'agressivité tumorale via l'activation des facteurs de transcription HIF-1alpha et HIF-2alpha (Hypoxia-Inducible Factors). Pour cette raison, le ciblage de l'hypoxie intratumorale et des facteurs HIF dans les ccRCC constitue une stratégie thérapeutique pertinente. Dans ce projet, nous montrons pour la première fois que la voie sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) régule HIF-2alpha in vitro et in vivo. Nos résultats indiquent que la SphK1 régule le taux intracellulaire et l'activité transcriptionnelle de HIF-2alpha dans des lignées de ccRCC représentatives de certains sous-groupes retrouvés en clinique humaine ; et impliquent la S1P extracellulaire, via le récepteur S1P1, dans la régulation de HIF-1alpha et HIF-2alpha. D'autre part, nous avons évalué l'impact de l'inhibition des récepteurs à S1P et de la SphK1 par le FTY720 dans un modèle de ccRCC in vivo. Nos résultats indiquent que le FTY720 entraine une diminution transitoire du taux intratumoral de HIF-1alpha et HIF-2alpha ainsi qu'un remodelage du réseau vasculaire tumoral. En effet, le FTY720 induit une normalisation vasculaire qui aboutit à une oxygénation tumorale transitoire. Enfin, nous montrons que ce traitement permet de sensibiliser un modèle murin de ccRCC à la chimiothérapie. Ces résultats valident le rôle de la voie SphK1/S1P comme régulateur de l'adaptation à l'hypoxie dans les ccRCC. Ils constituent une étape indispensable à la transposition en clinique humaine du concept selon lequel la voie SphK1/S1P peut être ciblée afin de diminuer l'hypoxie intratumorale et de chimiosensibiliser certains cancers, le FTY720 étant déjà sur le marché
Clear cell renal cell carcinomas (ccRCC) represent 70% of renal tumors. Because of their dense and irregular vascular network, ccRCC become hypoxic and therefore resistant to chemotherapies. Hypoxia promotes tumor aggressiveness via the activation of HIF-1alpha and HIF-2alpha (Hypoxia-Inducible Factors). For this reason, the control of intratumoral hypoxia and HIF in ccRCC could be a relevant therapeutic strategy to improve the efficacy of current treatments. In this study, we show for the first time that the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway regulates HIF-2alpha in vitro and in vivo. Our results indicate that SphK1 regulates HIF-2alpha intracellular level and transcriptional activity in ccRCC cell lines that are representative of some clinical ccRCC subgroups. Our data also involve extracellular S1P, via its receptor S1P1, in the regulation of HIF-1alpha and HIF-2alpha. In addition, in a ccRCC mouse model, we show that FTY720 - an inhibitor of the SphK1/S1P pathway- transiently decreases HIF-1alpha and HIF-2alpha intratumoral level. This is associated with a transient remodeling of the tumor vascular network indicating that FTY720 induces a vascular normalization that leads to transient tumor oxygenation. Finally, we show that this treatment sensitizes a ccRCC mouse model to chemotherapy. Overall, these results validate the key role of the SphK1/S1P pathway in the adaptation to hypoxia in ccRCC cell and animal models. Our results provide a mechanistic basis to target the SphK1/S1P pathway with FTY720 by increasing the efficacy of chemotherapy in ccRCC. They are a prerequisite for clinical transposition as FTY720 is a drug approved used in human clinic

APA, Harvard, Vancouver, ISO, and other styles

28

Bouquerel, Pierre. "Rôle de la sphingosine kinase 1 dans la régulation de l'hypoxie intratumorale." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/2174/.

Full text

Abstract:

L'hypoxie est une caractéristique des tumeurs solides qui contrôle la mise en place des voies de signalisation impliquées dans la néovascularisation, l'apparition de métastases, l'augmentation de la croissance tumorale et la résistance aux traitements. L'activation du facteur HIF-1 (Hypoxia Inducible Factor 1) a été identifiée comme le mécanisme principal de l'adaptation au stress hypoxique. Bien que les premières études aient principalement porté sur le rôle de HIF-1, de nombreux travaux soulignent l'importance de HIF-2 dans la régulation de certains gènes et fonctions biologiques. Le facteur HIF-2, présentant une Forte homologie avec HIF-1, joue notamment un rôle clef dans la progression des cancers rénaux à cellules claires (ccRCC). Nous avons précédemment identifié la voie Sphingosine Kinase-1/ sphingosine 1-phosphate (SphK1/S1P), impliquée dans de nombreux processus dont l'angiogenèse, prolifération et la survie cellulaire, en tant que nouveau modulateur de l'activité de HIF-1. Considérant le rôle crucial de HIF-2 dans les ccRCC, il est essentiel de mieux comprendre le rôle de la SphK1 dans la régulation de HIF-2a dans ces modèles. Dans ce but, les mécanismes de régulation du taux de HIF-2a et de l'activité de HIF-2 par la SphK1 ont été étudiés. Cette étude in vitro a été réalisée in vitro dans de nombreux modèles de ccRCC possédant des profils d'expression de HIF-1a et HIF-2a différents et représentant l'ensemble des groupes retrouvés en clinique humaine (expression de HIF-1a et de HIF-2a ou de HIF-2a seul). De manière intéressante, nos résultats indiquent que la SphK1 régule le taux de HIF-2a dans les différents modèles cellulaires, dont les ccRCC. Dans toutes ces lignées l'inhibition de la SphK1 entraîne une diminution du taux de HIF-2a ainsi qu'une diminution de l'expression des gènes cibles de HIF-2. Dans les lignées de ccRCC, la SphK1 stimule la traduction de HIF-2 via l'activation des protéines mTOR et p70S6K. En amont, nous avons établi que la phospholipase D (PLD) était responsable de l'activation de la SphK1 et que l'inhibition de la PLD conduisait à une diminution de l'activité de la SphK1 et du taux de HIF-2a. De plus, l'inhibition de la formation des espèces réactives de l'oxygène (ROS) bloque l'activation de la voie PLD/SphK1/HIF-2a. Nous avons ainsi identifié la voie ROS/PLD/SphK1 comme une nouvelle voie de régulation du facteur HIF-2a. Ce travail a permis d'identifier la SphK1 comme régulateur clef du facteur HIF-2 démontrant l'intérêt thérapeutique de cibler la SphK1 dans le cas des cancers où HIF-2 joue un rôle prépondérant tels que les cancers rénaux, pulmonaires et les neuroblastomes
Hypoxia is a characteristic of solid tumors that triggers the activation of signaling pathways promoting neovascularization, metastasis, increased tumor growth and resistance to treatments. The activation of the transcription factor hypoxia-inducible factor 1 (HIF-1) has been identified as the master mechanism of adaptation to hypoxia. Whereas initial characterization of hypoxia-induced transcription emphasized on HIF-1, there is now evidence that HIF-2, which is closely related to HIF-1, could regulate unique genes and physiological functions. Notably, HIF-2, rather than HIF-1, has been shown to play a critical role in clear cell renal carcinoma (ccRCC). We have previously identified the sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway - which elicits various cellular processes including cell proliferation, cell survival or angiogenesis - as a new modulator of HIF-1 activity. Considering the crucial role of HIF-2 in ccRCC, it was essential to understand the role of the SphK1 signaling in these tumors. To this end, the mechanisms underlying the regulation of HIF-2a level and activity, via the SphK1 signaling, have been investigated. These in vitro studies have been conducted in various ccRCC models exhibiting different HIF-1a and HIF-2a status, and representing all the sub-groups found in human clinic (which can produce either HIF-1a and HIF-2a or HIF-2a alone). Our data indicate that SphK1 regulates HIF-2a level in several cancer cell models including ccRCC. Depletion of SphK1 decreased HIF-2a level and consequently resulted in decreased expression levels of HIF-2a responsive genes. In ccRCC, SphK1 stimulates translation of HIF-2a via p70-S6kinase and mTOR pathway. Upstream, we have established that Phospholipase D (PLD) is responsible for the up-regulation of SphK1, and that inhibition of PLD leads to a decrease in SphK1 activity and in HIF-2a level and activity. Moreover inhibition of reactive oxygen species (ROS) production abolished PLD/SphK1/ HIF-2a pathway. Overall, we have identified a new molecular mecanism whereby a ROS/ PLD/SphK1 signaling pathway regulates HIF-2a. We provide evidence that SphK1 act as a master regulator for HIF-2a and giving support to its inhibition as a valid strategy for ccRCC and others cancers for which HIF-2a is particularly relevant (lung, neuroblastoma)

APA, Harvard, Vancouver, ISO, and other styles

29

Farinha, Garcao Nunes Joao Pedro. "Investigating the role of Sphingosine Kinase 1 pathway in cancer cell-monocyte interactions." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/29864.

Full text

Abstract:

Strong evidence suggests that the tumour microenvironment is inflammatory and that activation of the innate immune system plays a role in cancer progression, therefore targeting the multiple interactions of tumour cells with other cell types within the tumour microenvironment may lead to development of new cancer therapies. Sphingosine kinase (SPHK1) is a tumour-associated enzyme whose over-expression has been linked to patient mortality in many types of cancer. Here I investigate whether activation of the SPHK1 pathway, with a known involvement in inflammatory responses, is a signal transduction component of the tumour-monocyte/macrophage cellular interaction and a key element in inflammation-related cancer progression. Using a co-culture model, this study shows that the presence of monocytes increases cancer cell proliferation, an effect abrogated by knockdown of SPHK1 in cancer cells. Both monocytes and cancer cells showed a transient increase in SPHK1 activity and mRNA expression levels together with an increase in MCP-1 and IL-6 secretion. Silencing of SPHK1 in cancer cells abrogated SPHK1 activation in monocytes and pharmacological inhibition of SPHK1 in monocytes cells decreased monocyte induced-SPHK1 expression in cancer cells. Mechanistically, activation of AKT was observed in cancer cells upon co-culture with monocytes, an effect that was abrogated when cancer cells were pre-treated with siRNA for SPHK1. Moreover, the increase of phospho-AKT, ERK1/2 and NF-KB in monocytes by cancer cells was also reduced by RNAi-mediated knockdown of SPHK1 in cancer cells. My data show that STAT1 can bind to SPHK1 promoter or coding region and may be involved in SPHK1 transcriptional regulation in cancer cells upon monocyte stimulation, however its role still remains unclear as it acts as a transcriptional repressor of SPHK1. Monocytes induced cancer cell chemoprotection via a SPHK1-dependent mechanism, and reduced the inhibitory effect of docetaxel on cancer cell proliferation. Accordingly, increased AKT and ERK1/2 phosphorylation in monocytes were also affected by siRNA targeting of SPHK1 in docetaxel treated cancer cells. Altogether I show for the first time that selective inhibition of SPHK1 in tumour cells can affect their interaction with surrounding cells through the modulation of signal transduction pathways (ERK, PI3K, NF-kB) and cytokine exchange (IL-6, MCP-1 and potentially S1P, GM-CSF, GROα, IL-32 and ICAM-1). SPHK1-mediated increase in proliferation and chemoresistance of cancer cells conferred by monocytes renders this enzyme a promising target for future cancer therapies.

APA, Harvard, Vancouver, ISO, and other styles

30

Gasser, Amanda Lynn. "Characterization of Influenza:Streptococcus pneumoniae synergistic disease and potential for disease alleviation via sphingolipid therapy." Thesis, Virginia Tech, 2013. http://hdl.handle.net/10919/51569.

Full text

Abstract:

Influenza A virus (IAV) is generally associated with the seasonal malady that causes brief respiratory illness during the winter months, known simply as "the flu." Most otherwise healthy individuals will suffer from mild fever, congestion, headaches and myalgia that are resolved within 5-7 days of onset. However, there are nearly 500,000 influenza-related deaths that occur world-wide every year. Many of these casualties and patients hospitalized with influenza also test positive for bacterial pneumonia, the most common agent being Streptococcus pneumoniae. Although all individuals are subject to this viral:bacterial synergistic disease, the young, elderly, and immunocompromised are the most susceptible. Previous studies have shown that viral infection creates a prolonged hyper-responsive pro-inflammatory state in the lungs, which increases susceptibility to secondary bacterial infection. Lethality is due to detrimental pulmonary damage from a dysregulated host inflammatory response, known as the "cytokine storm." However, the nature of dual infection has not been well-studied in the elderly demographic. Therefore, we aim to better define this disease synergy in an aged mouse model and explore potential therapeutic alternatives that could be beneficial for the aged and other vulnerable populations. Sphingolipid modulation has emerged as a potential target to ameliorate the excessive inflammation (cytokine storm) elicited by highly pathogenic influenza. There is particular emphasis on sphingosine 1-phosphate (S1P) signaling, as well as control of intracellular S1P levels via sphingosine kinases (SK). Sphingolipids are involved in a multitude of cellular processes, and are tightly regulated by their metabolizing enzymes. We hypothesize that manipulation of sphingolipid signaling and alteration of the internal sphingolipid milieu will diminish the inflammatory response elicited by IAV infection. Using fluorescence-activated cell sorting (FACS), real-time PCR and cytometric bead array (CBA) analysis, we evaluated the immunomodulatory effects of systemic sphingosine analog treatment within the lung microenvironment under homeostatic and influenza-infected conditions. FTY720 treatment caused transient, but significant lymphopenia, influx of neutrophils and efflux of macrophages in the lungs, which was enhanced during a mild influenza infectionGene expression in the lungs was generally unaltered, but protein levels showed increases in specific influenza-induced cytokines, suggesting these treatments may have post-transcriptional effects on cytokine expression. To evaluate sphingolipid modulation in specific pulmonary cell types, we next observed the effects of these compounds and sphingosine kinase (SK) inhibitors in epithelial and alveolar macrophage-like cell lines. SK inhibitors and Enigmol demonstrated anti-viral effects in A549 cells, decreasing viral loads by up to 1.5 logs. Real-time PCR and CBA analysis further demonstrated that these effects were associated with alterations in key cytokine expression, including CCL2, CCL5, CXCL10, IL-6, and IL-8. Collectively, these findings indicate that therapeutic sphingolipid modulation has the potential for creating a protective microenvironment in the lungs that could alleviate or even prevent viral:bacterial synergistic disease.
Master of Science

APA, Harvard, Vancouver, ISO, and other styles

31

Guinan, Jack Henry. "Sphingosine-1-Phosphate and Stromal Cells Contribute to an Aggressive Phenotype of Ovarian Cancer." Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/86438.

Full text

Abstract:

Metastasis remains the largest contributor for ovarian cancer mortality. The five-year survival rate decreases dramatically as the disease advances from the primary tumor site to other organ sites within the peritoneal cavity. Thus, characterizing the mechanisms behind this metastatic potential may better elucidate the molecular mechanisms of ovarian cancer progression and may reveal novel targets for preventative and therapeutic treatments. Sphingosine-1-phosphate (S1P) is a critical secondary messenger responsible for many pro-cancer signals, e.g., proliferation, angiogenesis, inflammation, anti-apoptosis, and others. While S1P's role in the aggressive profile of many other cancers is well defined, its function in ovarian cancer development is less understood. The concentration of S1P is significantly increased in the ascites of women with malignant ovarian cancer, suggesting a role in ovarian cancer progression. This study aims to understand the importance of S1P in ovarian cancer metastasis. Using our well-characterized murine cell model for progressive ovarian cancer, we investigate the impact of S1P on ovarian cells and their interactions with the stromal vascular fraction recruited from the adipose tissue in culture conditions that mimic the physiologic environment of the peritoneal cavity. These studies will provide a mechanistic link of obesity, inflammation, and the increased risk of obese women to develop and die from ovarian cancer and identify signaling events as targets for interventions.
Master of Science

APA, Harvard, Vancouver, ISO, and other styles

32

Bats, Marie-lise. "Bases cellulaires et moléculaires du rôle de la Sphingosine 1-phosphate dans la progression et la résistance du mélanome cutané." Toulouse 3, 2014. http://www.theses.fr/2014TOU30034.

Full text

Abstract:

Le mélanome métastatique est considéré comme un des cancers les plus agressif et chimiorésistant chez l'Homme. Malgré les avancées dans la compréhension de la biologie et de la génétique du mélanome, les thérapies systémiques s'avèrent inefficaces pour combattre ce cancer très invasif. De nombreux arguments renforcent la notion que le microenvironnement tumoral jouerait un rôle clé dans la progression de cette tumeur. C'est pourquoi la définition des mécanismes moléculaires qui régissent le dialogue bidirectionnel entre les cellules malignes et le stroma tumoral semble indispensable à la découverte de nouvelles cibles thérapeutiques anti-mélanome. La première étape de notre travail s'est concentrée sur le rôle de la Sphingosine 1-phosphate (S1P), un sphingolipide bioactif décrit dans le cancer pour ses effets pro-migratoire et anti-apoptotique, dans les interactions mélanome-stroma. Par analyse transcriptomique, nous avons mis en évidence que l'expression la Sphingosine kinase 1 (SK1), l'enzyme qui produit la S1P, était augmentée dans des lignées de mélanome humain comparées à des mélanocytes sains. Cette augmentation, confirmée in situ dans des tissus de patients atteints de mélanome, était la conséquence directe de l'activation de ERK dans les cellules mutées pour BRAF ou NRAS. Bien que les modifications de l'expression de SK1 n'aient pas affecté la migration des cellules de mélanome, celle-ci a été stimulée par la modulation des enzymes du métabolisme de la S1P dans des fibroblastes dermiques co-cultivées avec les cellules tumorales. De plus, l'incubation de ces fibroblastes avec du milieu conditionné issu de cellules de mélanome surexprimant SK1 a conduit à leur différenciation en myofibroblastes, capables de produire des métalloprotéases matricielles et de sécréter de la S1P. Des expériences de tumorigenèse in vivo ont montré que l'absence de S1P dans le microenvironnement limitait la formation de tumeurs mélaniques chez les souris. De plus, la croissance tumorale locale et les métastases étaient considérablement augmentées par la co-injection de fibroblastes cutanés sauvages par rapport à des fibroblastes issus de souris Sphk1-/-. L'ensemble de nos résultats démontre que l'axe SK1/S1P pourrait contrôler les interactions entre le mélanome et son microenvironnement, soulignant l'intérêt de cibler la SK1 en thérapeutique. Notre équipe avait précédemment montré que l'expression de la S1P Lyase, l'enzyme responsable de la dégradation irréversible de la S1P, était fortement diminuée dans des cellules de mélanome comparée à des mélanocytes sains. La deuxième partie de notre travail s'est donc intéressée au rôle de cette enzyme dans la régulation de la réponse du mélanome au traitement par la dacarbazine (DTIC), un agent alkylant utilisé en 1ère ligne dans la prise en charge des mélanomes avancés. Nous avons montré que l'inhibition de l'expression de la S1P Lyase par siRNA augmentait la résistance au DTIC. A l'inverse sa surexpression sensibilisait les cellules de mélanome à l'apoptose en diminuant l'expression des membres anti-apoptotiques et à l'inverse en augmentant celle des membres pro-apoptotiques de la famille de Bcl-2. De plus, nous avons observé que le traitement des cellules de mélanome avec l'ABT-737, un inhibiteur de Bcl-2, s'accompagnait d'une action cytotoxique synergique avec les effets sensibilisants de la S1P Lyase en réponse au DTIC. De façon intéressante, la surexpression de la S1P Lyase stimulait l'expression de p53, via la régulation négative d'un de ses régulateurs clés, Mdm4. Les A375 surexprimant la S1P Lyase présentaient également une expression diminuée pour MITF (Microphthalmia-associated transcription factor), régulateur majeur de la mélanogenèse et de la progression du mélanome, également connu pour induire la transcription de Bcl-2. Enfin, la S1P Lyase activait PTEN par diminution de sa phosphorylation. En contrôlant l'expression de protéines clés dans la régulation des voies apoptotiques, la S1P Lyase pourrait donc être une nouvelle cible thérapeutique permettant d'améliorer la prise en charge des malades atteints de mélanome, notamment ceux qui développent des résistances aux thérapies émergentes
Metastatic melanoma is considered to be one of the most aggressive and treatment-resistant human cancer. Despite advances in the understanding of tumor biology and genetics of melanoma, until recently systemic therapy was ineffective against this invasive cancer. Many evidences support the notion that the adjacent microenvironment plays a key role in the progression of this tumor. Defining the molecular signals that control the bidirectional dialogue between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. The first part of our work aimed at defining the role of sphingosine-1-phosphate (S1P), a bioactive sphingolipid that promotes tumor cell migration and survival, in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed an increased expression of sphingosine kinase-1 (SK1), the enzyme that produces S1P, as compared to normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared to nevi, and occurred downstream of ERK activation due to BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SK1 in tumor cells but was stimulated by comparable modifications of S1P-metabolizing enzymes in co-cultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases, and enhanced SK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by co-injection of wild-type skin fibroblasts than by fibroblasts from Sphk1-/- mice. Altogether, our findings demonstrate that SK1/S1P can modulate the communication between melanoma cells and dermal fibroblasts, pointing out the relevance of SK1 as a potential therapeutic target in melanoma progression. Moreover, we previously reported that S1P Lyase expression, the enzyme responsible for irreversible degradation of S1P, was down-regulated in human skin melanoma cells as compared to normal melanocytes. The second part of our work aimed at defining the effect of S1P Lyase on the response of melanoma cells to antitumor therapies. Here we showed that disruption of S1P Lyase by siRNA enhanced resistance of A375 melanoma cells to Dacarbazine (DTIC), the common chemotherapy used to treat advanced melanoma. In contrast, we reported increased apoptosis in melanoma cells expressing S1P Lyase, as a consequence of increased expression of pro-apoptotic and decreased anti-apoptotic members of the Bcl-2 family. Moreover, S1P Lyase-induced cell death was enhanced in tumor cells treated by the Bcl-2 antagonist ABT-737 suggesting that S1P Lyase could act as a new regulator of Bcl-2-mediated cell survival in melanoma. Interestingly, S1P Lyase overexpression was also associated with an increased expression of p53 as a consequence of the downregulation of its key regulator Mdm4. Furthermore, A375 cells overexpressing S1P Lyase exhibited a decreased expression of Microphthalmia-associated transcription factor (MITF), one of the master regulators of melanogenesis and melanoma progression, also known to up-regulate Bcl-2 transcription. Finally, S1P Lyase promoted PTEN activation by decreasing its phosphorylation. By controlling the expression of key proteins in the regulation of apoptotic pathways, SPL could be a new target to improve the efficacy of anti-melanoma therapies

APA, Harvard, Vancouver, ISO, and other styles

33

Serrano, Sanchez Martin. "Rôle de l'axe Sphingosine Kinase 1 / Sphingosine -1- Phosphate dans la régulation de l'expression de la Cyclooxygénase 2 dans le myomètre de rate gestante." Paris 11, 2009. http://www.theses.fr/2009PA11T075.

Full text

APA, Harvard, Vancouver, ISO, and other styles

34

Price, Megan. "Sphingosine-1-phosphate in mast cell-mediated allergic responses." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/254.

Full text

Abstract:

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

APA, Harvard, Vancouver, ISO, and other styles

35

Corro, Morón Macarena. "Novel Strategies for the Syntheses of Sphingosine Kinase Inhibitors, b-Fluoroamines and Enantioenriched Allenes." Doctoral thesis, Universitat Rovira i Virgili, 2018. http://hdl.handle.net/10803/665123.

Full text

Abstract:

Els esfingolípids tenen un paper molt important tant en processos biològics com fisiològics i es converteixen els uns en els altres per l'acció d'enzims. El futur cel·lular és determinat pel balanç que controla la síntesi de ceràmida, esfingosina i esfingosina-1-fosfat, esfingolípids associats al càncer. Mentres que la ceràmida com l'esfingosina estan associades a processos d'apoptosi cel·lular, l'esfingosina-1-fosfat està vinculada a la proliferació. Degut a això, molts grups d'investigació centren els seus treballs en la síntesi d'inhibidors d'esfingosina quinasa, enzim que promou la formació d'esfingosina-1-fosfat. En el capítol 3 es decriu la síntesi d'una nova família d'inhibidors d'esfingosina quinasa i es mostren els valors d'inhibició trobats per cada compost. A més, també s'han realitzat estudis de docking per tractar de predir les possibles interaccions dels nostres compostos amb l'enzim. En el capítol 4 es desenvolupa una nova metodologia per obtenir β-fluoroamines amb carbamats com materials de partida. Suposem que la reacció procedeix via aziridina i apertura de la mateixa encara que trobem una diastereoselectivitat oposada a l'esperada. Tot i que s'han realitzat diverses probes per a tractar de confirmar els mecanismes proposats, no ens són d'ajuda per a el·lucidar-ne cap i, per tant, s'ha de seguir fent probes per confirmar-ne algun. En el capítol 5 es descriu la síntesi enantioselectiva d'al·lens mitjançant reactius de coure. Durant l'estudi es va oberservar que l'enantioselectivitat era afavorida per la presència de fosfits i que l'ús de fosfits voluminosos conduïa a una enantioselectivitat oposada. A més, es va comprobar que aquest tipus de reacció és influenciada per les característiques estèriques i electròniques dels substractes i que hi havia una tolerància moderada cap a diferents grups funcionals.
Los esfingolípidos tienen un papel muy importante tanto en procesos biológicos como fisiológicos y se convierten los unos en los otros por la acción de enzimas. El futuro celular está determinado por el balance que controla la síntesis de ceramida, esfingosina y esfingosina-1-fosfato, esfingolípidos asociados al cáncer. Mientras que tanto la ceramida como la esfongosina están asociadas a procesos de apoptosis celular, la esfingosina-1-fosfato está vinculada a la proliferación. Debido a esto, muchos grupos de investigación están centrando sus trabajos en la síntesis de inhibidores de esfingosina quinasa, enzima que promueve la formación de la esfingosina-1-fosfato. En el capítulo 3 se describe la síntesis de una nueva familia de inhibidores de esfingosina quinasa y se muestran los valores de inhibición encontrados para cada compuesto. Además, también se han realizado estudios de docking para tratar de predecir las posibles interacciones de nuestros compuestos con la enzima. En el capítulo 4 se desarrolla una nueva metodología para obtener β-fluoroaminas desde carbamatos como materiales de partida. Suponemos que la reacción procede via aziridina y apertura de la misma aunque nos encontramos con diastereoselectividad opuesta a la esperada. A pesar de que se han realizado varias pruebas para tratar de confirmar los mecanismos propuestos, estos no nos han ayudado a elucidar ninguno y, por tanto, se deben seguir haciendo pruebas antes de poder confirmar algo. En el capítulo 5 se describe la síntesis enantioselectiva de alenos mediada por cobre. Durante el estudio se observó que la enantioselectividad era favorecida con la presencia de fosfitas y que el uso de unas fosfitas voluminosas conducía a una enantioselectividad opuesta. Además, se comprobó que este tipo de reacción está influenciada por las características estéricas y electrónicas de los sustratos y que había una tolerancia moderada hacia distintos grupos funcionales.
Sphingolipids play a central role in numerous biological and physiological processes and they are converted into each other through the action of enzymes. Ceramide, sphingosine and sphingosine-1-phosphate have been found to be involved in cancer processes and the balance that controls their syntheses determines the cell fate. Whereas ceramide and sphingosine are associated with apoptosis, sphingosine-1-phosphate is related to cell proliferation. In this context, many research groups are investigating the synthesis of inhibitors for sphingosine kinase, enzyme which promotes the synthesis of sphingosine-1-phosphate. In chapter 3, we describe the synthesis of new sphingosine kinase inhibitors and also show the inhibitory potency of all of them. Besides, docking studies to predict interaction between our compounds and the enzyme are also presented. In Chapter 4, we develop a new strategy in the synthesis of β-fluoroamines starting from carbamates. Presumably, the reaction proceeds via aziridination and ring-opening processes in which we have found an unexpected diastereoselectivity. Fluoroamines present syn configuration instead trans expected. Although several tests have been performed to explain the observed selectivity, further studies should be done before confirming or discarding the suggested mechanisms. In Chapter 5, we work in a novel enantioselective synthesis of allenes mediated by copper. It was found that enantioselectivity could be increased by the presence of phosphites to give anti-elimination. The use of bulkier phosphites led to syn-elimination. This process is influenced by steric and electronic properties of substrates and present moderate tolerance towards different groups.

APA, Harvard, Vancouver, ISO, and other styles

36

Castelain, Lauriane. "Sphingosine kinase 1, transition épithélio-mésenchymateuse et résistance primaire aux inhibiteurs pharmacologiques de l'EGFR." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066595.

Full text

Abstract:

Une transition épithélio-mésenchymateuse (TEM) et une expression élevée de la sphingosine kinase 1 (SPHK1) sont souvent observées dans les cancers. Notre étude du génome et du transcriptome d'adénocarcinomes pulmonaires (AP) montre que l'expression élevée de SPHK1 est en rapport, d'une part, avec des gains de la région incluant le locus SPHK1 et, d'autre part, avec une signature d'expression génique de TEM dans des tumeurs invasives. L'expression de SPHK1 est restreinte aux cellules tumorales. La surexpression de SPHK1 dans des cellules d'AP et l'exposition à son produit, la sphingosine-1-phosphate (S1P), entraînent une TEM, de manière réversible pour la S1P. La surexpression de SPHK1 active aussi NF-kB. La surexpression du facteur anti-apoptotique FLIP active NF-kB, induit une TEM et augmente l'expression de SPHK1, suggérant une boucle d'amplification entre NF-kB et SPHK1. Une TEM et la surexpression de FLIP ont été impliquées dans la résistance primaire aux inhibiteurs pharmacologiques de l'EGFR (EGFR TKI). Nous montrons que la surexpression de SPHK1 dans des cellules A549 diminue modestement la sensibilité au gefitinib, alors que l'inhibition de SPHK1 ou la déplétion du sérum en S1P l'augmentent modestement. L'invalidation de SPHK1 entraîne l'apoptose d'A549 y compris quand FLIP est surexprimé. L'activation et le maintien d'une TEM sont généralement attribués à des signaux contextuels du stroma. Cette thèse montre que les cellules tumorales elles-mêmes favorisent la surexpression de SPHK1 qui peut induire une TEM de façon autonome. De plus, la surexpression de FLIP impliquée dans la résistance aux EGFR TKI, n'empêche pas l'apoptose induite par l'invalidation de SPHK1
Epithelial-mesenchymal transition (EMT) and sphingosine kinase 1 (SPHK1) high expression are often seen in cancers. Our study of genomic and gene expression data in pulmonary adenocarcinomas (AP) shows that SPHK1 high expression correlates with both gains in the region encompassing the SPHK1 locus, and an EMT gene expression signature in invasive tumors. SPHK1 expression is restricted to tumors cells. SPHK1 overexpression in AP cells, as well as exposure to its productsphingosine-1-phosphate (S1P),induce an EMT -in a reversible manner for S1P. SPHK1 overexpression also activates NF-kB. Overexpression of FLIP – an antiapoptotic factor - activates NF-kB, induces an EMT, and increases SPHK1 expression, suggesting an amplification loop between NF-kB and SPHK1. EMT and FLIP overexpression are known to favor primary resistance to EGFR pharmacological inhibitors (EGFR TKI). We show that SPHK1 overexpression in A549 cells slightly decreases cell sensitivity to gefitinib, while pharmacologic inhibition of SPHK1 or serum S1P depletionincrease it. Downregulation of SPHK1 expression induces apoptosis of A549 cells even when FLIP is overexpressed. Activation and maintenance of EMT are generally attributed to contextual signals from the stroma. Here, we show that tumor cells themselves favor SPHK1 overexpression, which can led to EMT in cell-autonomous manner. In addition, FLIP overexpression which is implicated in EGFR TKI resistance, cannot prevent apoptosis that is induced by SPHK1 invalidation

APA, Harvard, Vancouver, ISO, and other styles

37

Wang, Yu. "Sphingosine kinase 1-interacting protein is a novel regulator of glucose-stimulated insulin secretion." Kyoto University, 2017. http://hdl.handle.net/2433/226770.

Full text

APA, Harvard, Vancouver, ISO, and other styles

38

Liu, Yanyan. "Sphingosine kinase 1-interacting protein is a dual regulator of insulin and incretin secretion." Kyoto University, 2019. http://hdl.handle.net/2433/243293.

Full text

APA, Harvard, Vancouver, ISO, and other styles

39

Chen, Yiqun. "Roles of sphingosine kinase in aging and longevity in Caenorhabditis elegans and in neurodegeneration in mice." Thesis, Lyon, École normale supérieure, 2013. http://www.theses.fr/2013ENSL0815.

Full text

Abstract:

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。

APA, Harvard, Vancouver, ISO, and other styles

40

Martin, Claire. "Rôle de la signalisation sphingosine kinase-1 sphingosine 1-phosphate dans les interactions stroma-cellules épithéliales au sein des métastases osseuses du cancer de la prostate." Toulouse 3, 2009. http://thesesups.ups-tlse.fr/513/.

Full text

Abstract:

Un des problèmes majeurs du cancer de la prostate est le développement de métastases au niveau osseux qui sont responsables de violentes douleurs et rendent les os sujets aux fractures. Les ostéoblastes sont considérés comme des cellules jouant un rôle prépondérant des foyers métastatiques. Le travail doctoral vise à étudier le rôle des métabolites sphingolipidiques au niveau des interactions cellules tumorales prostatiques/ ostéoblastes. Plus particulièrement, l'objectif est de caractériser l'effet de la sphingosine 1-phosphate (S1P). Ce sphingolipide, produit par l'enzyme sphingosine kinase-1 (SphK1), est une molécule connue pour exercer des effets prolifératifs et anti-apoptotiques en tant que second messager ou en tant que facteur paracrine. Nous montrons pour la première fois, que la SphK1 participe aux effets prolifératifs des androgènes sur l'ostéoblaste. Nos résultats suggèrent également que la S1P participe aux interactions paracrines entre cellules prostatiques et ostéoblastes. Ces travaux encore préliminaires pourraient permettre d'envisager de nouvelles stratégies anti-tumorales ciblant la voie SphK1/S1P pour prévenir et limiter les effets des métastases osseuses du cancer de la prostate
Prostate cancer (PCa) has a high predisposition to metastasize to bone. PCa bone metastases are associated with increased osteoblastic activity. We studied the role of the sphingolipid signalisation in the interactions between osteoblasts (OB) and PCa cells. The sphingolipid metabolite sphingosine 1-phosphate (S1P), produced by the oncogenic lipid kinase sphingosine kinase-1 (SphK1) - is a lipid mediator playing a major regulatory role in tumor cell growth and survival. We studied the role of SphK1 in androgen induced osteoblast proliferation. We provide the first evidence that SphK1 is instrumental in mediating androgen-induced OB proliferation. Androgen triggered OB cell growth, which was associated with a rapid stimulation of SphK1 and activation of both AKT and ERK signalling pathways. We also investigated the potential role of S1P, as a paracrine factor implicated OB-PCa cells cross talk. Our goal was to investigate the potential role of S1P, as a putative bone-derived factor that could stimulate growth or survival of PCa cells. We have found that osteoblastic cells exhibit a very high SphK1 activity, which could render OB extremely resistant to chemotherapeutics such as taxanes. Conditioned medium from OB did protect the PCa cells from the killing effects of taxanes. The cytoprotective effect of OB-derived conditioned medium on PCa cells was abolished when SphK1 activity was first inhibited in OB. These results suggest that SphK1/S1P pathway play an important role in OB response to androgen present in the microenvironment and could contribute to PCa bone metastasis progression

APA, Harvard, Vancouver, ISO, and other styles

41

Garandeau, David. "Rôle du métabolisme de la sphingosine 1-phosphate dans la résistance thérapeutique des cellules de mélanome aux inhibiteurs de BRAF." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30134.

Full text

Abstract:

Le traitement du mélanome métastatique a été révolutionné par le développement de thérapies ciblées, qui ont montré un bénéfice significatif sur la survie globale. En particulier, l'inhibition de la sérine-thréonine kinase BRAF, mutée dans 60% des mélanomes, par le Vémurafénib (PLX4032), a montré un gain de survie de 6 à 8 mois comparée à la chimiothérapie de référence, la Dacarbazine. Cependant, une très faible proportion de patients répond sur le long terme. En effet, la majorité des patients développent un échappement thérapeutique dans un délai médian de 6 mois. Des mécanismes cellulaires ont été mis en évidence dans l'apparition de cette résistance acquise, notamment l'implication de MITF, un facteur de transcription majeur des mélanocytes, ainsi que des modifications de l'expression de plusieurs membres de la famille de Bcl-2. Cependant, une meilleure compréhension des mécanismes de résistance aux thérapies anti-BRAF semble essentielle, tout comme l'utilisation de nouvelles approches thérapeutiques combinées afin d'optimiser l'efficacité des traitements et la durée du bénéfice clinique. Notre groupe a récemment identifié des altérations du métabolisme du céramide et de l'un de ses dérivés, la Sphingosine 1-phosphate (S1P), dans les cellules de mélanome humain comparé à des mélanocytes sains. En effet, nous avons montré que la S1P lyase (SPL), qui dégrade irréversiblement la S1P est sous exprimée. Au contraire, l'expression de la sphingosine kinase 1 (SK1), qui produit la S1P, est augmentée dans les cellules de mélanome, conséquence directe de la mutation BRAF. Ces perturbations ont pour effet d'augmenter les niveaux de S1P. Ce lysophospholipide favorise la survie cellulaire ainsi que la résistance vis-à-vis d'agents thérapeutiques dans diverses cellules tumorales. L'objectif de cette thèse a été d'évaluer si le métabolisme de la S1P peut moduler la résistance acquise des cellules de mélanome humain aux inhibiteurs de BRAF. Nous avons montré que la surexpression de la SPL ou l'inhibition pharmacologique de la SK1 (SKI-I) sensibilise les mélanomes métastatiques à l'apoptose induite par la thérapie ciblée. Ce phénomène est associé à une diminution de MITF et de l'une de ses cibles directes, la protéine anti-apoptotique Bcl-2. La diminution d'expression protéique de MITF peut être réversée par un traitement de S1P exogène. De plus, nous avons montré pour la première fois une augmentation de l'expression des récepteurs 1 et 3 à la S1P (S1PR1 et S1PR3), dans les cellules de mélanome présentant une résistance acquise au PLX4032. Ces modifications sont associées à l'expression accrue de MITF. La surexpression de la SPL, le traitement par le SKI-I ou par des inhibiteurs ciblant les S1PR1 et S1PR3, surmonte la résistance acquise de ces cellules au PLX4032 via la diminution d'expression des S1PRs, de MITF, et de Bcl-2. Par conséquent, en contrôlant l'expression de protéines clés de la survie et de la résistance, le métabolisme de la S1P représente une nouvelle approche thérapeutique pour améliorer l'efficacité des thérapies ciblées
The treatment of metastatic melanoma has changed considerably in recent years with the development of targeted therapies, which have shown a significant benefit in overall survival. In particular, the inhibition of the frequently mutated serine-threonine kinase BRAF, by Vemurafenib (PLX4032) showed that survival rates increase by 6 to 8 months compared to standard chemotherapy, Dacarbazine. However, a very small proportion of patients will respond to the long term, and the majority of patients relapses in a median of 6 months. Cellular mechanisms have been identified in the appearance of this acquired resistance, including the involvement of MITF, a major transcription factor of melanocytes, as well as changes in the expression of several members of Bcl-2 family. However, a better understanding of these mechanisms seems essential, as is the use of new therapeutic strategies to optimize treatment efficacy and duration of clinical benefit. Our group recently showed some alterations of ceramide metabolism and its derivative sphingosine 1-phosphate (S1P) in human melanoma cells compared to healthy melanocytes. For instance, S1P lyase (SPL), which degrades S1P, is under-expressed. Conversely, sphingosine kinase 1 (SK1), which produces S1P, is over-expressed in tumor cells, as a direct result of BRAF mutation. These alterations increases the levels of S1P. This lysophospholipid promotes cell survival and the resistance to therapeutic agents in a variety of tumor cells. This PhD project aimed at defining whether S1P metabolism could modulate the resistance of human melanoma cells to PLX4032. Here, we show that SPL overexpression or pharmacological inhibition of SK1 by SKI-I sensitizes metastatic melanoma cells to PLX4032-induced apoptosis. This phenomenon is associated with a decreased expression of the master regulator of melanocyte differentiation MITF as well as its direct cellular target Bcl-2. The decrease in MITF protein can be reversed by treating cells with exogenous S1P. Interestingly, we also report for the first time an increased expression of SK1 as well as the S1P receptors, S1PR1 and S1PR3, in melanoma cells with acquired resistance to PLX4032 as compared to sensitive counterparts. These modifications are associated with high expression of MITF. Overexpression of SPL, treatment with SKI-I or antagonists of S1PR1 ans S1PR3, strongly overcomes acquired resistance to PLX4032 through a decrease in the expression of S1PR, MITF as well as Bcl-2. Thus, by controlling the expression of key proteins in melanoma cell survival and resistance, S1P metabolism could represent a new therapeutic approach to enhance the effectiveness of targeted therapies

APA, Harvard, Vancouver, ISO, and other styles

42

Tawati, Salha M. "Design, synthesis and biological evaluation of sphingosine kinase inhibitors for the treatment of prostate cancer." Thesis, University of Strathclyde, 2018. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=30298.

Full text

Abstract:

Sphingosine is phosphorylated via the action of the enzymes sphingosine kinase 1 (SK1) and sphingosine kinase 2 (SK2) to produce the bioactive signalling molecule sphingosine 1-phosphate (S1P). S1P drives cancer cell proliferation and migration whilst also promoting cell survival. Many studies have demonstrated that SK is a promising target for the treatment of cancer and the development of novel isoform-selective SK inhibitors to treat human cancers is of major interest. To date, inhibitors for this enzyme have either been selective for SK1 or non-selective for both isoforms. However, most SK inhibitors have only weak potency. This project involves the design and synthesis of small molecule inhibitors of SK as potential anti-cancer compounds. In this drug discovery project, a series of potent and selective inhibitors of SK (SK1 or SK2 or SK1/SK2) were developed based on the structure of PF-543, a known potent SK1 selective inhibitor. Analogues of PF-543 were prepared that were potent selective inhibitors of SK1 over SK2 and nM potent SK2 inhibitors with selectivity over SK1. These compounds represent some of the first nM potent SK2 inhibitors with selectivity over SK1. Indeed, the studies identified a structural determinant in the catalytic site of SK1 and SK2 that confers selectivity, with the heel and toe regions of the so-called J-channel in either enzyme providing a means toward selectivity. Exemplars from the series were shown to have potent cellular activity but poor in vitro microsomal stability. Effective target engagement and selectivity for SK1 in prostate cancer cell lines (LNCaP and LNCaP-AI) and proliferating human pulmonary artery smooth muscle cells (hPSMAC) were also established. A variety of biological assays associated with SK inhibition were used to evaluate their ability to induce cancer cell death, which was shown to involve a caspase-3/7-independent mechanism. Our SK1 and SK1/SK2 inhibitors, but not SK2 inhibitors, also reduced expression of dihydroceramide desaturase 1 (Des1) in a dose dependent manner, causing growth arrest and caspase-independent cell death. This project highlighted the importance for combining SK1 with Des1 inhibition in terms of endowing compounds with cytotoxicity against cancer cells.

APA, Harvard, Vancouver, ISO, and other styles

43

Zhang, Xiwen. "KNOCKOUT OF SPHINGOSINE KINASE 1 ATTENUATES RENAL INTERSTITIAL FIBROSIS IN UNILATERAL URETERAL OBSTRUCTION (UUO) MODEL." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/5010.

Full text

Abstract:

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and an important signaling molecule that plays a significant role in fibrosis. S1P synthesis is catalyzed by sphingosine kinases (SphKs), which phosphorylate sphingosine into S1P. The present study tested the hypothesis that SphK1-S1P signaling pathway participates in the kidney damage in unilateral ureteral obstruction (UUO) model. Wild type and SphK1 knockout mice were subjected to UUO for 7 days or 14 days and then four groups of kidneys were collected: wild type control group (WT-C), wild type UUO group (WT-UUO), SphK1-/- control group (KO-C) and SphK1-/- UUO group (KO-UUO). The mRNA level of SphK1 in WT-UUO was increased by 6.1 folds compared to WT-C. The fibrotic markers α-smooth muscle actin (α-SMA) and collagen I were both upregulated in UUO groups, whereas the levels of these two markers were significant lower in KO-UUO than that in WT-UUO. The immunohistochemistry analyses showed that the distribution of α-SMA and collagen was located in the interstitial space and that the infiltration of immune cells was more in UUO groups than that in control groups, but there was no significant difference between KO-UUO and WT-UUO, suggesting a direct effect of SphK1 deletion on renal fibrotic markers independent of immune regulation. Further, the morphological examination showed that UUO-induced tubular injury and glomerular damage were significantly reduced in KO-UUO compared with WT-UUO. Our study suggests that SphK1-S1P signaling pathway mediates kidney damage in UUO mice. Manipulating SphK1-S1P signaling pathway may be used as a therapeutic strategy in renal interstitial fibrosis.

APA, Harvard, Vancouver, ISO, and other styles

44

Rodgers, Alayna. "Regulation of p42/p44 mitogen activated protein kinase by sphingosine-1-phosphate in embryonic stem cells." Thesis, University of Strathclyde, 2009. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=12413.

Full text

APA, Harvard, Vancouver, ISO, and other styles

45

Imbert, Caroline. "Rôle de la sphingosine kinase-1 dans la réponse immunitaire anti-tumorale et dans le traitement du mélanome par immunothérapie." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30192.

Full text

Abstract:

L'émergence de nouvelles approches thérapeutiques, basées sur des inhibiteurs de points de contrôle immunitaire (ICI), telles que les anti-CTLA-4 et anti-PD-1, ont révolutionné la prise en charge du mélanome. Cependant les taux de réponses objectives à ces traitements restent relativement faibles et de sévères effets indésirables ou des résistances peuvent survenir. Il est donc nécessaire de comprendre les mécanismes de résistance et d'identifier des biomarqueurs prédictifs de la réponse aux ICI afin d'adapter la stratégie thérapeutique. Notre groupe a identifié des perturbations du métabolisme de la sphingosine 1-phosphate (S1P) dans les cellules de mélanome. Notamment, la surexpression de la Sphingosine Kinase-1 (SK1), une enzyme de production de la S1P qui favorise la progression tumorale en induisant des modifications phénotypiques du microenvironnement tumoral (TME), telles que la stromagenèse, l'angiogenèse et la polarisation M2 des macrophages. Or, la S1P joue un rôle important dans l'immunité car elle régule de nombreux processus comme l'activation, la différenciation et le trafic de nombreuses cellules immunitaires. L'objectif de cette thèse a été de déterminer si l'inhibition de la SK1 affecte la réponse immunitaire anti-tumorale et améliore l'efficacité des ICI. Nous avons mis en évidence que l'expression accrue de SK1 dans les cellules tumorales est significativement associée à une survie plus courte chez les patients atteints de mélanome métastatique traités par anti-PD-1. Ensuite, nous avons démontré, dans un modèle murin, que la diminution de l'expression de SK1 dans la tumeur induit une réduction de la croissance du mélanome qui est associée à une diminution de l'expression de diverses molécules immunosuppressives dans le TME. Cette modification du TME diminue la prolifération mais aussi l'infiltration des lymphocytes T régulateurs (Treg) et par conséquent induit une augmentation du ratio CD8+/Treg, ce qui est associé à un bon pronostic. Une signature immunosuppressive dépendante de SK1 a également été observée dans des biopsies de mélanome humain. De manière importante, nos données révèlent que l'inhibition de SK1 améliore fortement l'efficacité des ICI, tels que les anti-CTLA-4 ou les anti-PD-1, conduisant au rejet des tumeurs et à une amélioration significative de la survie des animaux dans un modèle murin de mélanome. Des résultats significatifs ont également été obtenus dans des modèles de cancer du sein et du côlon. Ici, nous montrons que: i) le ciblage de SK1 dans les mélanomes conduit à une augmentation du rapport intratumoral CD8+/Treg, ii) la combinaison de l'inhibition de SK1 avec des ICI améliore grandement la survie des souris et iii) la SK1 pourrait constituer un nouveau biomarqueur prédictif de la réponse aux ICI chez l'Homme. Par conséquent, nos données ont identifié la SK1 comme une kinase lipidique agissant comme un nouveau point de contrôle immunitaire, qui pourrait être ciblée afin d'améliorer la réponse aux ICI dans différents cancers
The emergence of new therapeutic approaches, thanks to immune checkpoint inhibitors (ICI), such as anti-CTLA-4 and anti-PD-1, have revolutionized the management of melanoma. However, the objective response rates to these treatments remain relatively low and severe adverse events or resistance can occur. This is why it is essential to understand the mechanisms that underlie resistance and identify predictive biomarkers of the response to ICI in order to adapt the therapeutic strategy. Our group identified perturbations of sphingosine 1-phosphate (S1P) metabolism in melanoma cells. In particular, the overexpression of sphingosine kinase (SK1), an S1P-producing enzyme that promotes tumor progression by stimulating stromagenesis, angiogenesis and polarization of macrophages toward M2 Phenotype. Interestingly, S1P is a well-known regulator of the activation, differentiation and trafficking of many immune cells. This thesis aims at determining whether the inhibition of SK1 improves the anti-tumor immune response as well as the efficacy of ICI. We found that an increased expression of SK1 in tumor cells is significantly associated with shorter survival in patients with metastatic melanoma treated with anti-PD-1. In a mouse model, we also demonstrated that a decreased SK1 expression in tumors leads to reduction of melanoma growth as well as expression of several immunosuppressive factors in the TME. These modifications are associated to significant reduction of proliferation, infiltration of regulatory T cells (Treg) and consequently to an increase in the CD8+/Treg ratio, which is associated with good prognosis. A SK1-dependent immunosuppressive signature has also been observed in human melanoma biopsies. Importantly, our data reveal that inhibition of SK1 strongly enhances the efficacy of ICI, such as anti-CTLA-4 or anti-PD-1, leading to tumor rejection and improved survival in a mouse model of melanoma. Significant results have also been obtained in breast and colon cancer models. Overall, we show that: i) SK1 targeting in melanomas leads to an increase of the intratumoral CD8 + / Treg ratio, ii) the combination of SK1 inhibition with ICI greatly enhances mice survival, iii) SK1 could be a new predictive biomarker of response to ICI in humans. Altogether, our data identified SK1 as a new checkpoint lipid kinase that could be targeted to improve response to ICI in several cancers

APA, Harvard, Vancouver, ISO, and other styles

46

Swensen, Adam Clayton. "Investigation of Dynamic Biological Systems Using Direct Injection and Liquid Chromatography Mass Spectrometry." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6574.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

47

Le, Scolan Erwan Pierre Laurent. "Recherche d'altérations génétiques impliquées dans la transformation des proérythroblastes transgéniques pour spi-1 : dérégulation transcriptionnelle du gène codant pour la sphingosine kinase de type 1." Paris 7, 2005. http://www.theses.fr/2005PA077035.

Full text

APA, Harvard, Vancouver, ISO, and other styles

48

Babahosseini, Hesam. "Nanoparticle-Based Drug Delivery and the Impacts on Cancer Cell Biophysical Markers." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/79689.

Full text

Abstract:

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to define biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical markers in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, the results showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.
Master of Science

APA, Harvard, Vancouver, ISO, and other styles

49

Drosos, Zacharias [Verfasser], Lars [Akademischer Betreuer] Hanker, and Markus [Akademischer Betreuer] Meier. "Die prognostische Bedeutung der Expression von Acid-Ceramidase (ASAH1) und Sphingosin-Kinase 1 (SPHK1) bei Patientinnen mit Ovarialkarzinom / Zacharias Drosos ; Akademische Betreuer: Lars Hanker, Markus Meier." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2021. http://d-nb.info/1236386272/34.

Full text

APA, Harvard, Vancouver, ISO, and other styles

50

Böhm, Andreas [Verfasser], Bernhard [Akademischer Betreuer] Rauch, and Joachim [Akademischer Betreuer] Ernst. "Regulation der Sphingosin-Kinase 1 (SPHK1) durch den aktivierten Gerinnungsfaktor X (FXa) und den Protease-aktivierten Rezeptor 2 (PAR-2) in glatten Gefäßmuskelzellen / Andreas Böhm. Gutachter: Bernhard Rauch ; Joachim Ernst." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2013. http://d-nb.info/1043802142/34.

Full text

APA, Harvard, Vancouver, ISO, and other styles

Dissertations / Theses on the topic 'Sphingosine kinases'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles