Modulation allostérique du récepteur FP dans le cancer colorectal

Les prostaglandines modulent d’importants rôles physiologiques. Elles sont aussi impliquées dans le développement d’une variété de conditions pathologiques telles l’inflammation, la douleur et le cancer. La prostaglandine PGF2α et son récepteur (récepteur FP) se trouvent impliqué dans la modulation de nombreuses pathologies tels lors de l’accouchement préterme et le cancer colorectal. Récemment, nous avons fait partie d’un groupe de recherche ayant développé des modulateurs allostériques du récepteur FP. Dans une première étude, l’action du PGF2α sur le déclenchement des contractions myométriales a été évaluée, car peu d’information est connue sur la signalisation de cette prostaglandine lors de l’accouchement. Ainsi, nous avons utilisé un peptidomimétique de la deuxième boucle extracellulaire, dénommée PDC113.824. Nos résultats ont démontré que le PDC113.824 permettait de retarder la mise bas chez des souris gestantes, mais agissait de manière différente sur les multiples voies de signalisation de la PGF2α. Ainsi, le PDC113.824 inhibait la voie RhoA-ROCK, dépendante de l’activation de la protéine Gα12 par le. Les protéines RhoA-ROCK sont des acteurs clés dans le remodelage du cytosquelette d’actine et des contractions myométriales lors de l’accouchement. De plus, le PDC113.824 en présence de PGF2α agit comme un modulateur positif sur la voie dépendante de l’activation de la protéine Gαq. Le PDC113.824 serait donc un modulateur allostérique non compétitif possédant des actions à la fois de modulateurs positifs et négatifs sur la signalisation du récepteur FP Dans une seconde étude, des analogues du PDC113.824 ont été conçus et analysés dans un second modèle pathologique, le cancer colorectal. Ce cancer possède de hauts niveaux de récepteur FP. Nous avons donc étudié le rôle du récepteur FP dans le développement et la progression du cancer colorectal et l’effet de modulateurs allostériques. Il est généralement accepté que dans le cancer colorectal, la prostaglandine PGE2 permet la croissance et l’invasion tumorale, ainsi que l’angiogenèse. Toutefois, peu d’informations sont connues sur le rôle du PGF2α dans le cancer colorectal. C’est dans ce contexte que nous avons décidé d’examiner la contribution de ce récepteur dans la progression du cancer colorectal et cherché à déterminer si la modulation des fonctions du récepteur FP a un impact sur la croissance de tumeurs colorectales. Nos recherches ont révélé que l’activation du récepteur FP permet la migration et la prolifération de plusieurs lignées cellulaires humaines et murines d’adénocarcinomes colorectaux. Dans ce contexte, nos expériences ont démontré que la migration des cellules cancéreuses était dépendante de l’activation de la voie Rho. Nos résultats démontrent qu’en effet, l’activation de RhoA, une petite GTPase clé de la voie Gα12, est inhibée de façon sélective par nos composés. De plus, nos molécules allostériques sont également efficaces pour inhiber la voie de signalisation de la ß-caténine, une protéine impliquée dans la genèse du cancer colorectal. In vivo, le traitement de souris avec un des ces modulateurs a permis une inhibition effective de la croissance tumorale. Dans l’ensemble, nos résultats suggèrent donc que les modulateurs allostériques des récepteurs FP pourraient constituer une nouvelle classe de médicaments utilisés pour le traitement du cancer colorectal.

Investigating the thymopoiesis stimulating property of interleukin-21 in aging mice

La vaccination est largement utilisée pour la génération de lymphocytes T spécifiques contre les tumeurs. Malheureusement, cette stratégie n'est pas adaptée aux personnes âgées car leur thymus régresse avec l'âge conduisant ainsi à une baisse dans la production de cellules T et à l'accumulation de cellules immunitaires âgées ayant des défauts liés à leurs stimulations. Comme il a été démontré auparavant que L’IL-21 est capable d’induire des fonctions thymiques, nous avons émis l’hypothèse que l’injection d’IL-21 à des souris âgées stimulera la thymopoïèse. Nos résultats montrent que l’administration de l’IL-21 augmente le nombre absolu de thymocytes chez les souris âgées et augmente la migration de ces cellules vers la périphérie ou ils contribuent à la diversité du TCR. De plus les cellules T en périphérie expriment un niveau plus élevé de miR181-a, et par conséquent moins de phosphatase comme SHP2, DUSP5/6 qui inhibent le TCR. En vaccinant des souris âgées avec le peptide Trp2, les souris traitées avec l’IL-21 montrent un retard dans la croissance des cellules B16 tumorales. Cette étude montre que l’IL-21 pourrait être utilisé comme stratégie pour le rétablissement du systeme immunitaire chez les personnes âgées.

Die Rolle des Zelladhäsionsmoleküls L1 in der Tumorprogression

Das neuronale Adhäsionsmolekül L1 wird neben den Zellen des Nervensystems auf vielen humanen Tumoren exprimiert und ist dort mit einer schlechten Prognose für die betroffenen Patienten assoziiert. Zusätzlich zu seiner Funktion als Oberflächenmolekül kann L1 durch membranproximale Spaltung in eine lösliche Form überführt werden. In der vorliegenden Arbeit wurde der Einfluss von L1 auf die Motilität von Tumorzellen untersucht. Lösliches L1 aus Asziten führte zu einer Integrin-vermittelten Zellmigration auf EZM-Substraten. Derselbe Effekt wurde durch Überexpression von L1 in Tumorlinien beobachtet. Weiterhin führt die L1-Expression zu einer erhöhten Invasion, einem verstärkten Tumorwachstum in NOD/SCID Mäusen und zur konstitutiven Aktivierung der MAPK ERK1/2. Eine Mutation in der zytoplasmatischen Domäne von hL1 (Thr1247Ala/Ser1248Ala)(hL1mut) führte hingegen zu einer Blockade dieser Funktionen. Dies weist daraufhin, dass nicht nur lösliches L1, sondern auch die zytoplasmatische Domäne von L1 funktionell aktiv ist. Im zweiten Teil der Arbeit wurde der Mechanismus, der L1-vermittelten Signaltransduktion untersucht. Die zytoplasmatische Domäne von L1 gelangt nach sequenzieller Proteolyse durch ADAM und Presenilin-abhängiger γ-Sekretase Spaltung in den Zellkern. Diese Translokation im Zusammenspiel mit der Aktivierung der MAPK ERK1/2 durch L1-Expression führt zu einer L1-abhängigen Genregulation. Die zytoplasmatische Domäne von hL1mut konnte ebenfalls im Zellkern detektiert werden, vermittelte jedoch keine Genregulation und unterdrückte die ERK1/2 Phosphorylierung. Die L1-abhängige Induktion von ERK1/2-abhängigen Genen wie Cathepsin B, β3 Integrin und IER 3 war in Zellen der L1-Mutante unterdrückt. Die Expression des Retinsäure-bindenden Proteins CRABP-II, welches in hL1 Zellen supprimiert wird, wurde in der L1-Mutante nicht verändert. Weitere biochemische Untersuchungen zeigen, dass die zytoplasmatische Domäne von L1 Komplexe mit Transkriptionsfaktoren bilden kann, die an Promoterregionen binden können. Die dargestellten Ergebnisse belegen, dass L1-Expression in Tumoren an drei Funktionen beteiligt ist; (i) L1 erhöht Zellmotilität, (ii) fördert Tumorprogression durch Hochregulation von pro-invasiven und proliferationsfördernden Genen nach Translokation in den Nukleus und (iii) schützt die Zellen mittels Regulation pro- bzw. anti-apoptotischer Gene vor Apoptose. Die mutierte Phosphorylierungsstelle im L1-Molekül ist essentiell für diese Prozesse. Die Anwendung neuer Therapien für Patienten mit L1-positiven Karzinomen kann mit Hinblick auf die guten Erfolge der Antikörper-basierenden Therapie mit dem mAk L1-11A diskutiert werden.

Subcutaneous study on the controlled release of Etanidazole and Taxol for the treatment of Glioma

BALB/c nude mice 6 weeks old were inoculated with glioma C6 cell-line and the efficacy of the different amount of Etanidazole-discs and Taxol-microspheres was investigated. Poly (D,L-lactic-co-glycolic acid) (PLGA) was used as the main encapsulating polymer and polyethylene glycol was added to increase the porosity. The 1% drug loading microspheres of each drug were produced by spray drying and the discs were obtained by compressing the Etanidazole-microspheres. Intra-tumoral injection followed by irradiation resulted in high systemic dosage and thus systemic toxicity. Tumors grown for 6 days, 9 days and 16 days were implanted with 0.5 mg or 1.0 mg or 1.5 mg of the drug. A radiation dosage of 2 Gy each time for a number of times was given for animals implanted with Etanidazole and no irradiation was given for animals implanted with Taxol. Increasing the number of doses clearly decreased the rate of tumor growth. The increase in the amount of drug on smaller sized tumors controlled the tumor better and there was agglomeration of the microspheres resulting in deviation of release profile of the drug as compared to the in vitro studies. It was observed that 1.0 mg of Taxol given to a tumor grown for 6 days was able to suppress the tumor for a total period of approximately two months and no tumor resurrection was observed during the second month.

Uso de lisados bacterianos en la prevención de la infección respiratoria recurrente en pediatría. Revisión sistemática de la literatura

Las infecciones respiratorias altas y bajas son una causa común de morbimortalidad infantil. Se ha propuesto el uso de los lisados bacterianos para prevenir las infecciones recurrentes sin embargo su uso aún se considera controversial. Metodología: Se realizó una revisión sistemática de la literatura. La búsqueda se realizó a través de las bases de datos PUBMED, Embase, Ovid, LiLaCS y Cochrane library plus. Se incluyeron metanálisis publicados en idiomas inglés y español, entre los años 1998 y 2012. Se realizó una evaluación de calidad siguiendo la estrategia Quorum y un análisis cualitativo y cuantitativo de los resultados. Resultados: Se incluyeron 4 revisiones sistemáticas de la literatura con metanálisis. Fue apreciable la disminución de las recurrencias de las infecciones respiratorias relacionadas con el uso de los lisados bacterianos. Los lisados bacterianos disminuyen la necesidad de uso de antibióticos. No se encontró evidencia sobre el uso de los lisados sobre desenlaces como la necesidad de intervenciones adicionales, tiempo de hospitalización, costo relacionado con la atención en salud. No se reportaron eventos adversos de importancia. Conclusión: Los lisados bacterianos son eficaces en disminuir la recurrencia de las infecciones respiratorias en pacientes en edad pediátrica.

The nutritional and clinical significance of lipid rafts

Purpose of review Lipid rafts are potentially modifiable by diet, particularly (but not exclusively) by dietary fatty acids. This review examines the potential for dietary modification of raft structure and function in the immune system, brain and retinal tissue, the gut, and in cancer cells. Recent findings In-vitro and ex-vivo studies suggest that dietary n-3 polyunsaturated fatty acids (PUFAs) may exert immunosuppressive and anticancer effects through changes in lipid raft organization. In addition, gangliosides and cholesterol may modulate lipid raft organization in a number of tissues, and recent work has highlighted sphingolipids in membrane microdomains as potential targets for inhibition of tumor growth. The roles of fatty acids and gangliosides, especially in relation to lipid rafts, in cognitive development, age-related cognitive decline, psychiatric disorders, and Alzheimer’s disease are poorly understood and require further investigation. The roles of lipid rafts in cancer, in microbial pathogenesis, and in insulin resistance are starting to emerge, and indicate compelling evidence for the growing importance of membrane microdomains in health and disease. Summary In-vitro and animal studies show that n-3 PUFAs, cholesterol, and gangliosides modulate the structure and composition of lipid rafts, potentially influencing a wide range of biological processes, including immune function, neuronal signaling, cancer cell growth, entry of pathogens through the gut barrier, and insulin resistance in metabolic disorders. The physiological, clinical, and nutritional relevance of these observations remains to be determined.

Morphology and myofiber composition of skeletal musculature of the forelimb in young and aged wild type and myostatin null mice

Most current research into therapeutic approaches to muscle diseases involves the use of the mouse as an experimental model. Furthermore, a major strategy to alleviate myopathic symptoms through enhancing muscle growth and regeneration is to inhibit the action of myostatin (Mstn), a transforming growth factor-beta (TGF-beta) family member that inhibits muscle growth. Presently, however, no study has expanded the morphological analysis of mouse skeletal muscle beyond a few individual muscles of the distal hindlimb, through which broad conclusions have been based. Therefore, we have initially undertaken an expansive analysis of the skeletal musculature of the mouse forelimb and highlighted the species-specific differences between equivalent muscles of the rat, another prominently used experimental model. Subsequently, we examined the musculature of the forelimb in both young and old adult wild-type (mstn(+/+)) and myostatin null (mstn(-/-)) mice and assessed the potential beneficial and detrimental effects of myostatin deletion on muscle morphology and composition during the aging process. We showed that: (1) the forelimb muscles of the mouse display a more glycolytic phenotype than those of the rat; (2) in the absence of myostatin, the induced myofiber hyperplasia, hypertrophy, and glycolytic conversion all occur in a muscle-specific manner; and, importantly, (3) the loss of myostatin significantly alters the dynamics of postnatal muscle growth and impairs age-related oxidative myofiber conversion.

The Nutritional Significance of Lipid Rafts

The structure, size, stability, and functionality of lipid rafts are still in debate, but recent techniques allowing direct visualization have characterized them in a wide range of cell types. Lipid rafts are potentially modifiable by diet, particularly (but not exclusively) by dietary fatty acids. However, it is not clear whether dietary polyunsaturated fatty acids (PUFAs) are incorporated into raft lipids or whether their low affinity to cholesterol disallows this and causes phase separation from rafts and displacement of raft proteins. This review examines the potential for dietary modification of raft structure and function in the immune system, brain and retinal tissue, the gut, and in cancer cells. Although there is increasing evidence to suggest that membrane microdomains, and their modulation, have an impact in health and disease, it is too early to judge whether modulation of lipid rafts is responsible for the immunomodulatory effects of n-3 PUFA. In addition to dietary fatty acids, gangliosides and cholesterol may also modulate microdomains in a number of tissues, and recent work has highlighted sphingolipids in membrane microdomains as potential targets for inhibition of tumor growth by n-3 PUFA. The roles of fatty acids and gangliosides in cognitive development, age-related cognitive decline, psychiatric disorders, and Alzheimer's disease are poorly understood and require clarification, particularly with respect to the contribution of lipid rafts. The roles of lipid rafts in cancer, in microbial pathogenesis, and in insulin resistance are only just emerging, but compelling evidence indicates the growing importance of membrane microdomains in health and disease.

Antitumor activity of Titanocene Y in xenografted PC3 tumors in mice

Chemotherapeutic options for androgen-independent prostate cancer are extremely limited with minimum survival advantage. The benzyl-substituted unbridged titanocene bis-[(p-methoxybenzyl) cyclopentadienyl] titanium(IV) dichloride (Titanocene Y) was tested in vitro against the human prostate cancer androgen-independent cell, PC-3, which demonstrated an IC50 value of 56 x 10(-6) mol/L compared to 5.6 x 10(-6) mol/L for cisplatin. Then Titanocene Y was given at the maximum tolerable dose of 40 mg/kg/d on five consecutive days to one cohort of eight PC3 tumor-bearing male NMRI: nu/nu mice, while a second cohort was treated similarly with 3 mg/kg/d of cisplatin. Both of these mouse cohorts showed a statistically significant tumor growth reduction with respect to the third solvent-treated control group, which led to T/C values of 42% for cisplatin and 52% for Titanocene Y at the end of the experiment. This encouraging activity of Titanocene Y against prostate tumors in vivo, which is almost comparable with respect to cisplatin hopefully leads to further development of Titanocene Y in the future.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.

Effect of Fish Oil Supplementation for Two Generations on Changes of Lymphocyte Function Induced by Walker 256 Cancer Cachexia in Rats

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-alpha and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-gamma plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

An orthotopic skull base model of malignant meningioma

Meningioma tumor growth involves the subarachnoid space that contains the cerebrospinal fluid. Modeling tumor growth in this microenvironment has been associated with widespread leptomeningeal dissemination, which is uncharacteristic of human meningiomas. Consequently, survival times and tumor properties are varied, limiting their utility in testing experimental therapies. We report the development and characterization of a reproducible orthotopic skull-base meningioma model in athymic mice using the IOMM-Lee cell line. Localized tumor growth was obtained by using optimal cell densities and matrigel as the implantation medium. Survival times were within a narrow range of 17-21 days. The xenografts grew locally compressing surrounding brain tissue. These tumors had histopathologic characteristics of anaplastic meningiomas including high cellularity, nuclear pleomorphism, cellular pattern loss, necrosis and conspicuous mitosis. Similar to human meningiomas, considerable invasion of the dura and skull and some invasion of adjacent brain along perivascular tracts were observed. The pattern of hypoxia was also similar to human malignant meningiomas. We use bioluminescent imaging to non-invasively monitor the growth of the xenografts and determine the survival benefit from temozolomide treatment. Thus, we describe a malignant meningioma model system that will be useful for investigating the biology of meningiomas and for preclinical assessment of therapeutic agents.

An endogenous regulator of inflammation, resolvin E1, modulates osteoclast differentiation and bone resorption

Background and purpose: The inflammation-resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammation-induced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption. Experimental approach: Primary OC cultures derived from mouse bone marrow were treated with RvE1 and analysed for OC differentiation, cell survival and bone substrate resorption. Receptor binding was measured using radiolabelled RvE1. Nuclear factor (NF)-kappa B activation and Akt phosphorylation were determined with western blotting. Lipid mediator production was assessed with liquid chromatography tandem mass spectrometry. Key results: OC growth and resorption pit formation were markedly decreased in the presence of RvE1. OC differentiation was inhibited by RvE1 as demonstrated by decreased number of multinuclear OC, a delay in the time course of OC development and attenuation of receptor activator of NF-kappa B ligand-induced nuclear translocation of the p50 subunit of NF-kappa B. OC survival and apoptosis were not altered by RvE1. Messenger RNA for both receptors of RvE1, ChemR23 and BLT(1) is expressed in OC cultures. Leukotriene B(4) (LTB(4)) competed with [(3)H] RvE1 binding on OC cell membrane preparations, and the LTB(4) antagonist U75302 prevented RvE1 inhibition of OC growth, indicating that BLT(1) mediates RvE1 actions on OC. Primary OC synthesized the RvE1 precursor 18R-hydroxy-eicosapentaenoic acid and LTB(4). Co-incubation of OC with peripheral blood neutrophils resulted in transcellular RvE1 biosynthesis. Conclusions and implications: These results indicate that RvE1 inhibits OC growth and bone resorption by interfering with OC differentiation. The bone-sparing actions of RvE1 are in addition to inflammation resolution, a direct action in bone remodelling.

Continuous and High-Level In Vivo Delivery of Endostatin From Recombinant Cells Encapsulated in TheraCyte (R) Immunoisolation Devices

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte (R) system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte (R) devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte (R) were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 mu g/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 mu g/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.