Λόγω της ιδιότητάς τους να ελέγχουν τον κυτταρικό κύκλο οι CDKs έχουν προταθεί ως μοριακοί στόχοι για την ανάπτυξη αντινεοπλασματικών φαρμάκων. Σε πολλούς τύπους καρκίνου έχει παρατηρηθεί απορρύθμιση της λειτουργίας ή μεταλλάξεις στις CDKs. Μεταξύ των CDKs, η CDK1 διαδραματίζει ιδιαίτερο ρόλο μιας και είναι επαρκής για να οδηγήσει τον κυτταρικό κύκλο. Πρόσφατα η ερευνητική μας ομάδα συνέθεσε την ένωση FM-100 (2-(αιθοξυκαρβονυλο)-9-χλωρο-πυρρολο[2,3-a]καρβαζόλιο) με εκλεκτική δράση στη CDK1 (IC50 15 μΜ για το σύμπλοκο CDK1/κυκλίνη B). Στην παρούσα μελέτη έγινε αποτίμηση της βιολογικής δραστικότητας νέων πυρρολοκαρβαζολικών ενώσεων με δομή παραπλήσια του FM-100 με σκοπό την ανακάλυψη ισχυρότερων αναστολέων και ελέχθηκε η δραστικότητά τους σε καρκινικές κυτταρικές σειρές in vitro. Από τα ανάλογα που ελέγχθηκαν δύο, τα SS-261Β και SS263, εμφάνισαν αυξημένη ικανότητα αναστολής της CDK1 σε σχέση με την αρχική ένωση FM-100. Η ισχυρότερη από αυτές (SS-261Β, IC50 194nΜ) μελετήθηκε σε πέντε καρκινικές σειρές. Η SS-261Β δεν είχε καμία επίδραση στο πολλαπλασιασμό λευχαιμικών κυττάρων (Κ562), ενώ στα MCF-7 (κύτταρα καρκίνου του μαστού) η SS-261Β είχε ανασταλτική δράση στον πολλαπλασιασμό η οποία ήταν εμφανής μόνο στη μεγαλύτερη συγκέντρωση που χρησιμοποιήθηκε ο αναστολέας (10μΜ). Στη σειρά HeLa (καρκινικά κύτταρα τραχήλου μήτρας) και CaCo2 (καρκινικά κύτταρα γαστρενετρικού) η SS-261Β προκάλεσε μικρότερη αναστολή (25-30%). Μεγαλύτερη αντιμιτογόνο δράση παρουσίαση η ένωση στα PC-3 (προστατικά καρκινικά κύτταρα). Αξίζει να σημειωθεί ότι η ένωση SS-261Β είχε περιορισμένη δράση σε φυσιολογικά προστατικά επιθηλιακά κύτταρα. Παράλληλα, στα PC-3 η SS-261Β προκάλεσε μια δοσοεξαρτώμενη αύξηση της ενεργότητας της κασπάσης-3 σε συγκεντρώσεις μεγαλύτερες του 10μΜ, ενδεικτική της ικανότητάς της να προάγει την απόπτωση σε αυτά τα κύτταρα. Κάτι τέτοιο δεν παρατηρήθηκε στα HeLa. Συμπερασματικά, η ένωση SS-261Β έχει βελτιωμένες ιδιότητες σε σχέση με την FM-100, παρουσιάζει ανασταλτική δράση στο πολλαπλασιασμό καρκινικών κυττάρων, ενώ ταυτόχρονα διεγήρει την απόπτωση σε καρκινικά κύτταρα του προστάτη in vitro. / Cyclin-dependent kinases (CDKs) have been proposed as molecular targets for anti-tumour drug development due to their ability to regulate cell cycle. In many types of cancers, deregulated expression of CDKs or mutations in CDKs have been reported. Among CDKs, CDK1 is unique, as activation of this kinase is sufficient to drive cell proliferation. Recently, our research group synthesized FM-100 (ethyl 9-chloro-1H-pyrrolo[2,3-alpha]carbazole-2-carboxylate), a compound with selective inhibitory action on CDK1 (IC50 15 μΜ for the CDK1/cyclin B complex). In the present study we evaluated the biological activity of several new pyrrolocarbazole derivatives with structures similar to that of FM-100, in order to identify more potent CDK1 inhibitors. In addition, we tested the ability of the newly synthesized FM-100 analogues to inhibit cell proliferation and survival in cancer cell lines in vitro. Only two of the tested compounds, SS-261Β and SS263, exhibited a greater inhibitory action on CDK1 activity compared to the parent compound (FM-100). The most potent of the two SS-261Β had an IC50 of 194nΜ and was further studied in cell-based assays. SS-261Β had no effect on the proliferation of leukemic cells (Κ562), while SS-261B inhibited MCF-7 (breast cancer cell line) growth only at the highest concentration used (10μΜ). In HeLa (cervical cancer cell line) and CaCo2 cells (colon cancer cell line) SS-261Β had only a modest effect, reducing proliferation by 25-30%. SS-261B had a more pronounced effect on PC-3 cells (prostate cancer cell line). It should be noted that SS-261Β also exhibited anti-mitogenic effects in normal prostate epithelial cells. In a different series of experiments we observed that SS-261B dose-dependently increased caspace-3 activity in PC-3 cells when used at concentrations over 10μΜ, suggesting that it is capable of promoting apoptosis in this cell line. In summary, SS-261Β exhibits an improved profile for CDK1 inhibition compared to FM-100, exerts anti-proliferative effects in several tumour cell lines while it also inhibits survival of prostate cancer cells in vitro.

No description available.

abstract: This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS. / Dissertation/Thesis / Ph.D. Chemistry 2013

La mitochondrie occupe un rôle essentiel au sein des cellules cancéreuses. Etant la source principale de synthèse d’ATP mais est également le lieu de réactions anaboliques et cataboliques, la mitochondrie supporte le développement tumoral. De plus, la mitochondrie est également impliquée dans la réponse aux stress cellulaire en régulant notamment l’autophagie ou la mort des cellules cancéreuses.Dans ce contexte, nous avons démontré que la fonction mitochondriale peut altérer la réponse au stress réticulaire permettant la survie des cellules tumorales. En effet, la surexpression de la protéine GILZ (Glucocorticoid-Induced Leucine Zipper) atténue la mort cellulaire induite par le stress réticulaire. Ceci est permis grâce au maintien du réseau mitochondrial et à l’augmentation de la fonction mitochondriale. Dans cette étude, nous avons démontré que le maintien de la fonction mitochondriale est important pour l’effet protecteur de GILZ puisque l’utilisation de lignées cellulaires de mélanome dépourvues d’activité mitochondriale (lignées ρ0) et surexprimant GILZ sont sensibles à la mort induite par les inducteurs de stress réticulaire. Nos études ont également démontré que l’augmentation de la fonction mitochondriale induite par GILZ peut être utilisée pour resensibiliser les cellules cancéreuses à la mort notamment en utilisant des molécules prooxydantes comme l’elesclomol.Dans un autre contexte tumoral, nous avons également démontré qu’une sous population de cellules de mélanome BRAFV600E peuvent augmenter leur métabolisme mitochondrial dans le but de survivre à la mort cellulaire induite par les inhibiteurs de MAPK. La résistance aux MAPKi implique une augmentation significative de l’OxPHOS mitochondriale associée un remodelage du réseau mitochondrial autour du réticulum endoplasmique facilitant la recapture du calcium mitochondrial. Nos résultats ont permis de démontrer que la fonction mitochondriale est cruciale pour la survie des cellules cancéreuses. De part son rôle cellulaire multiple, il apparaît clairement que la mitochondrie constitue une cible thérapeutique de choix dans le traitement des cancers. / Mitochondria occupies a key role in cancer cells. As the main source of ATP synthesis and the site of anabolic and catabolic reactions, mitochondria support tumor development. Besides, mitochondria are also involved in the response to cellular stress regulating autophagy or cancer cell death.In this context, we have demonstrated that mitochondrial function may alter the ER stress response thus promoting tumor cell survival. Indeed, overexpression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ) protein attenuates endoplasmic reticulum stress mediated cell death. This is achieved by maintaining the mitochondrial network and the increase of mitochondrial function. In this study, we demonstrated that maintaining mitochondrial function is important for the protective effect of GILZ since using melanoma cell lines lacking mitochondrial activity (ρ0 cell lines) and overexpressing GILZ are susceptible to death induced by reticular stress inducers. Our studies have also shown that the increase of mitochondrial function induced by GILZ can be used to re-sensitize the cancer cells to death induced by prooxidant molecules as elesclomol.In another tumoral context, we have also demonstrated that a sub-population of BRAF mutated melanoma cells can increase mitochondrial metabolism to survive to ER stress-mediated cell death induced by several MAPK inhibitors. Resistance to MPAki involves a significant increase in mitochondrial OXPHOS associated with mitochondrial network remodeling around the ER, which facilitates mitochondrial calcium uptake. Our results have shown that mitochondrial function is crucial for the survival of cancer cells. Altogether our data indicate that given their multiple cellular roles, cancer cell mitochondria constitute attractive therapeutic targets.

M.Sc. / The ideal cancer treatment modality should not only cause tumour regression and eradication but also induce a systemic antitumour response, which is essential for the control of metastatic tumours and long-term tumour resistance. Photodynamic Therapy (PDT) is a current approach in the treatment of various cancers. It involves the administration of a tumour-localizing photoreactive compound, which is activated at a specific wavelength of light. This therapy results in a sequence of photochemical and photobiological processes that cause irreversible photodamage to tumour tissues. Eradication is achieved by anti-tumour effects induced in the parenchyma and tumour vascular network. PDT can lead to a rapid cell death response in malignant cells, which has provided insight into the mechanisms behind photokilling. Oesophageal cancer is the seventh leading cause of cancer death worldwide, and in South Africa remains a problem of epidemic proportions affecting predominantly black males. The appearance of a number of new photosensitizers being developed will not only extend the number of choices for treating specific cancers, but also aid in the effective destruction of various tumour tissues. PDT has been an experimental clinical modality for the past two xii decades and has been shown to be successful for the treatment of advanced oesophageal cancer where other options have failed. The full potential of PDT as a treatment modality has not been clearly evaluated, which is one of the objectives of this study. Overall, PDT has the potential of being a promising therapeutic option in the effective treatment of oesophageal cancer, and through this study and the elucidation of the mechanisms of PDT action, it will provide a better future for those suffering from oesophageal cancer. A new photosensitizer known as Zinc Sulphophthalo-cyanine (ZnPcSmix) was studied on an oesophageal SNO cancer cell line in order to determine treatment-induced cell viability, cytotoxicity and the pathway followed to cell death. The major observations of this study revealed that PDT using ZnPcSmix resulted in a decrease in cell viability and proliferation, resulting in a cytotoxic response experienced by the cell. The outcome of this study revealed that the SNO cells experience a necrotic mode of cell death after using ZnPcSmix to induce photodamage. This was examined by light microscopy and confirmed by the lack of DNA fragmentation and decreased caspase-3 and caspase-7 expression levels. Hsp70 levels decreased resulting in lowered cytokine TNF-α release from necrotic cells. Hoechst nuclear staining revealed a disorganized nuclear pattern characteristic of necrotic release of cellular contents. The major findings of this study revealed the efficacy of ZnPcSmix as a new photosensitizing drug used during PDT to treat oesophageal cancer resulting in a decrease in cell viability and proliferation. Necrosis was the primary mechanism by which cells pursued death, which was dependent on the photosensitizer dose, cell type and irradiation fluence. ZnPcSmix–induced photodamage seen during PDT offers a new treatment option for patients suffering from oesophageal cancer and shows great promise in effectively treating early-stage oesophageal cancer.

No description available.

The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.

Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.

Breast cancer is one of the most common forms of cancer in not only South African women, but women all over the world. The molecular chaperone heat shock protein 90 (HSP90) is upregulated in cancer and is almost exclusively associated with proteins involved in intracellular signal transduction, thus it plays an important role in signalling pathways within the cell. In cancer, there is an aberrant activation of the Wnt signaling pathway, which results in stabilized β-catenin being able to translocate to the nucleus where it can trigger the transcription of oncogenes found to be involved in the self-renewal of cells. The level of β-catenin is usually kept in check by a destruction complex comprising glycogen synthase kinase 3-beta (GSK-3β), axin1, adenomatous polyposis coli (APC) which phosphorylate β-catenin, resulting in its ubiquitination and degradation. HSP90 has been found to be associated with GSK-3β, but whether this association is only transient is debatable. Very little is known about the association of HSP90 with other members of the Wnt pathway in breast cancer. In this study, we have attempted to further identify the direct associations between HSP90 and GSK-3β, β-catenin, p-β-catenin and axin1. Immunofluorescence and confocal microscopy co-localization studies suggested a potential association between HSP90 and these proteins. Treatment with HSP90 inhibitors, 17-AAG and novobiocin resulted in a shift of axin1 to what appeared to be the plasma membrane. The associations of HSP90 with GSK-3β, β-catenin, p-β-catenin and axin1 were confirmed biochemically by co-immunoprecipitation and inhibition using 17-AAG, geldanamycin and novobiocin. We showed, for the first time that HSP90 is associated in a possible complex with β-catenin, p-β-catenin and axin1 therefore is potentially involved in the modulation of p-β-catenin in the Wnt pathway through the stabilization of the destruction complex.

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.