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Targeted delivery of embelin to cancer cellsEmjedi, Zaakiyah Z. January 2013 (has links)
>Magister Scientiae - MSc / Apoptosis or programmed cell death is vital to the development of organisms as they
maintain the balance between cell death and cell growth. Failure to activate apoptosis has
been implicated in carcinogenesis and often results from the over expression of anti–cancer
proteins such as the X–linked inhibitor of apoptosis protein (XIAP). XIAP is over expresses
in certain cancers and is a potent inhibitor of the initiator caspase 9 and effector caspases 3
and 7. The increased expression of XIAP in cancer cells result in the resistance to apoptosis.
The control of XIAP is therefore considered as a target for anti–cancer drug development.
Embelin or 2,5–dihydroxy–3–undecyl–1,4–benzoquinoine is a dihydroxyquinone compound
that was previously shown to inhibit XIAP. This drug was discovered by structure based
computational screening. The binding of embelin to XIAP displaces XIAP from caspases,
consequently eliminating the inhibitory effect of XIAP on apoptosis.
The objective of this study was to develop a gold nanoparticle that can be used for the
targeted delivery of embelin to cancer cells thereby enhancing pro–apoptotic effects of the
pro–apoptotic drug, ceramide. XIAP expression levels were investigated by Western blot
analysis in a panel of human cancer cell lines available in the laboratory to identify two cell
lines that can be used as low and high XIAP expression controls. Gold nanoparticles were
synthesized and conjugated with embelin and a cancer targeting peptide with the amino acid
sequence LTVSPWY. The biconjugated nanoparticles were used to co–treat MCF7 and
HepG2 cells with ceramide. Apoptosis was quantified using flow cytometry. The uptake of
gold nanoparticles was investigated using HR–TEM and ICP–OES. This study showed that
gold nanoparticles conjugated with the LTVSPWY peptide is specifically targeted to and
taken up by cancer cells. Gold nanoparticles conjugated with embelin promoted ceramide
induced apoptotic cell death of cancer cells. However, it was observed that gold nanoparticles
biconjugated with the LTVSPWY peptide and embelin failed to enhance the pro–apoptotic
effects of ceramide. iii
This study successfully demonstrated that gold nanoparticles conjugated with embelin could
be used to enhance the effects of anti–cancer drugs using ceramide as an example.
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NON-CANONICAL IL-1ß PROCESSING VIA CASPASE-8 IN MURINE DENDRITIC CELLS AND MACROPHAGESBuzzy, Christina Antonopoulos 06 February 2015 (has links)
No description available.
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Characterization of Bax Pore Formation Using Fluorescence TechniquesLovell, Jonathan 07 1900 (has links)
<p> Bax is a pro-apoptotic protein believed to permeabilize mitochondria during apoptosis. The mechanism Bax uses is not well understood. In this work, we use fluorescence techniques to shed light on how tBid activates Bax and we examine the topology of the pore-forming domain of Bax. </p> <p> The manner in which tBid promotes apoptosis via Bax activation is not known. Study of tBid and Bax interaction using a new FRET pair showed that the proteins only interacted in the presence ofmembranes. The Bax pore was shown to have a variable size distribution. A fluorescence technique of simultaneously measuring pore formation, Bax insertion and FRET showed that tBid interaction with Bax occurred before all the Bax inserted or formed pores in the liposomes. A chronological order is proposed for Bax pore formation. tBid first binds to liposomes. tBid proceeds to interact with Bax, and Bax inserts into the membrane. After insertion, Bax oligomerizes and forms small pores. More Bax is recruited and the pores become larger. </p> <p> The two central hairpin helices of Bax, helices 5 and 6, are known as the pore-forming domain. We used cysteine scanning with the environment sensitive fluoroprobe NBD to gain insight into the topology of these helices. Fluorescence intensity changes and emission blue shifts showed that residues in these helices undergo conformational reorganization during pore formation. In the activated oligomeric conformation, fluorescence lifetimes showed that helix 5 was more inaccessible to water than helix 6. Cobalt, a cationic NBD quencher, effectively quenched residues in the pore-forming domain, consistent with a pore that is lined with anionic lipid head groups. Quenching with nitroxide groups at various lipid depths showed that residues on helix 6 were most quenched by a shallow quencher, while residues on helix 5 were quenched by deeper quenchers. Compared to beta sheet pore-forming proteins, the data obtained suggests that Bax and possibly other alpha helical pore-forming proteins form a lipidic pore in a dynamic environment. Combined together, the data suggest a model for Bax in which helix 5 spans the bilayer, and helix 6 is buried just below the lipid headgroups of a toroidal pore. </p> / Thesis / Master of Science (MSc)
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Detailed comparison of CPP's uptake properties for pro-apoptotic cargo deliveryMüller, Judith 27 September 2012 (has links)
Limitierend für pharmakologische Therapien ist oft die Unfähigkeit des Wirkstoffes, biologische Membranen zu überwinden, weswegen häufig Transportmoleküle wie z.B. zellpenetrierende Peptide (CPPs, cell penetrating peptides) benutzt werden. Von den über 100 beschriebenen CPPs wurde bisher nur eine kleine Anzahl systematisch verglichen, was die Auswahl des „richtigen“ CPPs für eine Anwendung erschwert. Ziel dieser vorliegenden Arbeit war es, das pro-apoptotische Peptid KLA mittels CPPs spezifisch in Krebszellen zu transportieren. Untersucht wurden: (I) Verschiedene CPPs in unterschiedlichen Zelltypen zur Selektion der „besten“ CPPs; (II) Der Einflusses des CPP C-Terminus auf die Internalisierung und zelluläre Verteilung; (III) Der zelluläre KLA-Transport mittels CPPs via einer nicht-kovalenten Administration. 22 verschiedene CPPs wurden in sieben Zelltypen untersucht, wobei Toxizität, Zellaufnahme und zelluläre Lokalisation mittels Fluorescein markierten CPPs in Fluoreszenzspektroskopie und konfokaler Mikroskopie betrachtet wurden. Abhängig von der Zellaufnahme wurden die CPPs in drei Gruppen klassifiziert. Die Untersuchung carboxylierter und carboxyamidierter CPP C-Termini ergab, dass in den meisten Fällen ein Carboxyamid die zelluläre Aufnahme begünstigte. Drei CPPs (MPG, Penetratin und Integrin) wurden ausgewählt, um das pro-apoptotische KLA Peptid in zwei Krebszelllinien (MCF-7 Brustkrebszellen und leukämische RAW264.7 Makrophagen) im Vergleich zu Fibroblasten (Cos-7) nicht-kovalent zu transportieren. Der erfolgreiche KLA-Transport hing vom CPP, dessen C-Terminus und der Zelllinie ab. Die Analyse der Viabilität nach CPP:KLA Administration ergab, dass MPG-CONH2:KLA (1:2) toxisch für Makrophagen und Brustkrebszellen, aber nicht für Fibroblasten war. Die Toxizität konnte der Apoptose zugeordnet werden. Die vorliegende Arbeit liefert wichtige Informationen über die Auswahl des passenden CPPs für den nicht-kovalenten Transport des pro-apoptotischen KLA-Peptids. / Limitations in a pharmacological therapy are often due to the inability of drugs to overcome the cell membrane and therefore transporting molecules are being used, e.g. cell penetrating peptides (CPPs). Only a few of the over 100 described CPPs have been compared systematically making the choice of “the” CPP for a given application difficult. The goal of the presented work is the CPP mediated delivery of the pro-apoptotic peptide KLA in breast cancer cells as proof of principle for a therapeutical application. Analysed were (I) Different CPPs in various cell types to select the “best” one, (II) The influence of the CPP C-termini on uptake and localisation, (III) The cellular KLA delivery via a non-covalent CPP administration. 22 CPPs were compared in seven cell types thereby looking at toxicity, cellular uptake and subcellular localisation using fluorescein labelled CPPs for fluorescence spectroscopy and confocal microscopy. The resulting uptake information allowed the classification of the CPPs in three main groups. The evaluation of carboxylated and carboxyamidated CPP C-termini revealed that a carboxyamide mostly enhanced the cellular CPP uptake. Three CPPs were selected (MPG, penetratin and integrin) to deliver the pro-apoptotic KLA peptide in two cancer cell lines (breast cancer MCF-7 cells and RAW264.7 macrophages) compared to fibroblasts (Cos-7) via the non-covalent strategy. A successful KLA delivery depended on the applied CPP, its C-terminus and the used cell line. The biological activity of the pro-apoptotic KLA peptide was determined via the cell viability (MTT assay). The co-incubation of MPG-CONH2:KLA (1:2) was able to induce toxicity in breast cancer cells and leukaemic macrophages, but not in fibroblasts. The viability reductions were then assigned to apoptosis. This work provides important information for the choice of an adequate CPP for the pro-apoptotic KLA peptide delivery and presents the advantage of the non-covalent delivery strategy.
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Role of Protein Kinase C-iota in GlioblastomaDesai, Shraddha R. 01 January 2011 (has links)
The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.
The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of the Bad/Bcl-XL dimer. Knockdown of PKC-é by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-é may be a Bad kinase. Since, PKC-é is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-é/Bad pathway. Treatment with PI(3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-é activity and subsequent phosphorylation of Bad suggesting that PKC-é regulates the activity of Bad in a PI (3)-kinase dependent manner.
Robust expression of PKC-é is a hallmark of human glioma and benign and malignant meningiomas, however, little is understood about its role in glioma cell proliferation. The cyclin dependent kinase activating kinase complex (CAK), comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator. Cdk7 phosphorylates its downstream cyclin dependent kinases (cdks) and promotes cell proliferation. Results show that PKC-é directly associated and phosphorylated Cdk7 at T170. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at T160. Purified PKC-é was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-é knockdown with siRNA, PDK1 siRNA and (PI) 3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. In addition, PKC-é knockdown reduced cell proliferation; led to cell cycle arrest and also induced apoptosis. Thus, these findings suggest the presence of a novel PI (3)-kinase/PKC-é/BAD mediated cell survival and PI (3)-kinase/PKC-é/Cdk7 mediated cell proliferation pathway.
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Ρόλος των προ- και αντι-αποπτωτικών γονιδίων στην παθογένεια του πολλαπλού μυελώματος / The role of pro- and anti- apoptotic genes in the pathogeny of multiple myelomaΞαγοράρης, Ιορδάνης 27 June 2007 (has links)
Το πολλαπλούν μυέλωμα είναι μια νεοπλασία η οποία, έως και σήμερα, παραμένει ανίατη. Στο ΠΜ, το ανοσοποιητικό σύστημα δε κατορθώνει να καταστρέψει τα κακοήθη πλασμοκύτταρα, ενώ υπάρχουν ενδείξεις ότι τα κύτταρα του όγκου παίζουν ενεργό ρόλο σε αυτό. Στην παρούσα διατριβή, ελέγξαμε ποικίλλες μυελωματικές σειρές σχετικά με την έκφραση των προ- και αντι-αποπτωτικών γονιδίων. Βρήκαμε ότι τα μυελωματικά κύτταρα εκφράζουν έναν μη φυσιολογικό φαινότυπο (fas high/bcl low). Μελετώντας τα κύτταρα του μυελού των οστών ενός ασθενούς με ΠΜ τύπου IgG/k σταδίου ΙΙΙΑ, διαπιστώσαμε ότι αυτά τα κύτταρα εξέφραζαν τα γονίδια granzyme B και perforin, τα οποία υπό κανονικές συνθήκες εκφράζονται μόνο από τα κυτταροτοξικά Τ λεμφοκύτταρα (CTLs) και τα φυσικά φονικά κύτταρα (ΝΚ). Προτείνουμε ότι πιθανόν το παραπάνω γεγονός αποτελεί έναν επιπλέον αμυντικό μηχανισμό των κυττάρων του όγκου εναντίον των CTLs και ΝΚ κυττάρων. Συγκεκριμένα υποθέτουμε ότι τα κύτταρα του όγκου προσελκύουν τα CTLs και τα ΝΚ κύτταρα και τα καταστρέφουν με τη διαδικασία της κυτταρόλυσης. Τα διφωσφονικά οξέα χρησιμοποιούνται ευρέως για τη θεραπεία του ΠΜ, ενώ η θαλιδομίδη χρησιμοποιείται σήμερα για τη θεραπεία πολλών τύπων ΠΜ. Μελετήσαμε την επίδραση της θαλιδομίδης, του ζολεδρονικού οξέος και το συνδυασμό τους στην επιβίωση των μυελωματικών κυττάρων. Βρήκαμε ένα συνεργιστικό αποτέλεσμα των δύο αυτών ουσιών. Όταν η θαλιδομίδη συγχοηγηθεί με το ζολεδρονικό οξύ σε ελάχιστους μη δραστικούς μηχανισμούς, μειώνει την τοξική επίδραση 50% του τελευταίου 3-4 φορές. / Multiple Myeloma (MM) is a plasma cell neoplasia which, to this day, remains incurable. In MM, the immune system fails to destroy the malignant plasmocytes, and evidence exists that the tumor plays an active part in this. In the present study, we tested various MM cell lines for the expression of pro- and anti-apoptotic genes. We found that MM cells express an abnormal phenotype, namely fas high/bcl low. By studying bone marrow cells from a patient suffering of MM IgG/k type stage IIIA, we found that those cells expressed granzyme B nad perforin, normally expressed by cytotoxic T cells (CTLs) and natural killer (NK) cells. We propose that this may constitute an additional acquired mechanism by the tumor cells to protect themselves against the host.
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