Ribozym- und RNAi-vermittelter Knockdowndes Wachstumsfaktors Pleiotrophin (PTN)und des PTN-Rezeptors ALK zur funktionellen Analyseund zur therapeutischen Anwendung im Maus-Tumormodell

Pleiotrophin (PTN) gehört zur Proteinfamilie der sekretierten, heparin-bindenden, Matrix-assoziierten Wachstumsfaktoren und ähnelt funktionell den Fibroblasten-Wachstumsfaktoren. Es spielt eine physiologische Rolle während der Ontogenese und ist bei verschiedenen Tumortypen relevant. Die protoonkogene Wirkung von PTN beruht hauptsächlich auf der Stimulation der Proliferation und Angiogenese. Der Rezeptor Anaplastic Lymphoma Kinase (ALK) wurde zunächst im Zusammenhang mit dem großzelligen anaplastischen T-Zell-Lymphom als konstitutiv-aktives, durch chromosomale Aberrationen entstehendes Fusionsprotein entdeckt. In späteren Arbeiten wurde ALK als ein PTN-Rezeptor identifiziert. ALK stellt eine klassische Tyrosinkinase aus der Familie der Insulinrezeptoren dar und seine Liganden-vermittelte Transphosphorylierung führt über weitere Effektoren zur Induktion mehrerer mitogen-aktivierter Signaltransduktionskaskaden, die verantwortlich für sein onkogenes Potential sind. Im Rahmen dieser Arbeit wurde der Einfluss von PTN und ALK auf Proliferation, Kolonienbildung im Weichagar, Tumorwachstum, Apoptose und Angiogenese in Glioblastom-, Hoden-CA- und Kolon-CA-Zelllinien untersucht. Zu diesem Zweck wurden im Screening ermittelte PTN- und ALK-positive Zelllinien mit Ribozym-kodierenden Vektoren stabil transfiziert. Die Zellen mit verminderter PTN- bzw. ALK-Expression zeigten eine Verlangsamung der kontaktabhängigen und kontaktunabhängigen Proliferation und bildeten nach s.c. Injektion im Mausmodell kleinere Tumoren. Durch Zugabe von rekombinantem PTN konnten in vitro die PTN-Knockdown-Effekte revertiert werden. Es wurden ferner ALK-Verkürzungsmutanten hergestellt und in den Tumorzelllinien überexprimiert. Diese Tyrosinkinase-Domäne-defizienten ALK-Mutanten wirkten sich negativ auf die Proliferationsgeschwindigkeit der Tumorzellen aus. Das Ribozym-vermittelte Doppeltargeting von PTN und ALK in Glioblastom- und Kolon-CA-Zelllinien zeigte sowohl in Zellkulturstudien als auch im in vivo-Modell additive antitumorigene Effekte. Zur Entwicklung von auf diesen Ergebnissen basierenden neuen Therapiestrategien wurde statt des Ribozym-Targetings die RNA-Interferenz (RNAi) eingesetzt. Durch die Verwendung des für in vivo-Einschleusung von RNA geeigneten Transfektionsreagenz Polyethylenimin (PEI) gelang es, in bereits ausgebildeten U87MG-Glioblastom-Tumorxenotransplantaten mittels PEI/siRNA-Komplexen einen PTN-Knockdown zu erzielen, was in einer deutlichen Abnahme des Tumorwachstums resultierte. Diese Behandlung erwies sich sowohl bei intraperitonealer wie auch bei subkutaner Injektion als effizient und spezifisch. Die systemische Gabe von PEI/siRNA-Komplexen zum Knockdown von PTN und / oder ALK stellt somit eine vielversprechende Methode der Behandlung verschiedener solider Tumoren dar

The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells

<p>Abstract</p> <p>Background</p> <p>Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit <it>in vitro </it>and <it>in vivo </it>the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop.</p> <p>Methods</p> <p>A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. <it>In vitro</it>, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. <it>In vivo</it>, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. <it>In vivo </it>anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.</p> <p>Results</p> <p>Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. <it>In vitro</it>, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an <it>in vivo </it>Matrigel™ plug assay in mice</p> <p>Conclusions</p> <p>Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on <it>in vitro </it>and <it>in vivo </it>growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPβ/ζ, nucleolin). <it>In vivo</it>, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.</p

RNA interference targeting survivin exerts antitumoral effects in vitro and in established glioma xenografts in vivo

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes–cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRInu/nu mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC(50) value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context

RNAi for Treating Hepatitis B Viral Infection

Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-α and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection

БЕРКЛІАНСЬКА КОНЦЕПЦІЯ ПРИРОДИ

The aim of the article is to show various meanings of nature in Berkeleyan philosophy: in his mechanic philosophy, optics, in his understanding of ether and the theory of beauty.Цель статьи – показать различные подходы к пониманию природы в философии Д. Беркли: в его механистической философии, оптике, понимании ефира и в теории прекрасного.Мета статті – показати різні підходи до розуміння природи у філософії Д. Берклі: в його механістичній філософії, оптиці, розумінню ефіру та в теорії прекрасного

The Research on Kant’s Philosophy in the Institute of Philosophy at Nicolaus Copernicus University, Torun, Poland

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ЕКСПЕРИМЕНТАЛЬНА НАУКА В РОБОТІ Д.ЮМА «ТРАКТАТ ПРО ПРИРОДУ ЛЮДИНИ»

The aim of the paper is to present the meaning of the notion of «experimental method», which is mentioned in the title of David Hume’s Treatise of Human Nature. I claim it to be the ground of the whole system of Hume’s system of sciences concerning various aspects of human life.Цель статьи – раскрыть значение понятия «экспериментальный метод», который упоминается в библиографических данных к книге Д.Юма «Трактат о человеческой природе». Я считаю что это является основой всей системы знаний Д.Юма относительно разнообразных аспектов человеческой жизни.Метою статті є розкрити значення поняття «експериментальний метод», котрий згадується в бібліографічних даних до книги Д.Юма «Трактат про людську природу». Я вважаю, що це є основою всієї системи знань. Д.Юма стосовно різноманітних аспектів людського життя