The telomeric repeat containing RNA TERRA represents a class of long non-coding RNAs transcribed from telomeres. TERRA has been shown to play key roles in telomere maintenance. During past years it has been reported that TERRA interacts with telomerase, suggesting it may act as regulator of telomerase. However, the role of TERRA in telomerase activity in human cells is still unknown and it needs to be further investigated. Herein, we investigate the role of TERRA in telomerase activity in human cancer cells by exploiting different experimental conditions. By using single molecule RNA FISH (smiFISH) combined with confocal microscopy, we demonstrated that TERRA and the telomerase RNA subunit hTR colocalize in cancer cells. We observed that these events mainly occur in the nucleoplasm, while a fraction of TERRA transcripts colocalize with hTR molecules also at telomeres. This result suggested us the possibility of a localization-dependent role of TERRA in the regulation of telomerase activity. Surprisingly, by studying TERRA and hTR interactions during telomere length homeostasis, we observed that fewer TERRA molecules localize at chromosome ends when telomeres are elongated by telomerase. Furthermore, the fraction of telomeric TERRA-hTR colocalizing foci markedly decreased during telomere elongation. We observed that telomere shortening correlated with increased TERRA levels, in line with previously reported findings. Intriguingly, by quantifying the integrated density of the telomeric foci in smiFISH experiments combined with immunofluorescence (IF), we observed that TERRA transcripts preferentially localize to telomeres showing a brighter signal intensity as compared to the average telomeric signal of the cell, suggesting a preferential recruitment of TERRA to longer chromosome ends. These results suggest that TERRA transcripts may localize to telomeres in trans and that a displacement of TERRA from chromosome ends may be required for telomere elongation by telomerase. To gain insight into the role of TERRA in the regulation of telomerase, we used antisense oligonucleotides to deplete TERRA in cells. Interestingly, TERRA depletion resulted in increased telomerase localization to telomeres. Altogether, our findings support a model in which telomeric TERRA transcripts act as negative regulators of telomerase activity at telomeres.

A naphthoquinone, 7-methyljuglone and some of its 5-hydroxy, 5-acetoxy-, 5-alkoxy- and 1,2,4,5-tetra-O-acetate derivatives were tested for their activity in four human cancer cell lines: breast adenocarcinoma, cervical epithelial carcinoma, oesophageal carcinoma and prostate epithelial carcinoma. Compound 2,5-dihydroxy-7-methyl-1,4-naphthoquinone was found to be the most effective one (exhibited a fifty percent inhibitory concentration (IC50) in the range of 5.3 to 14.7 μM), while the parent compound 7-methyljuglone was less active than several of these derivatives. The IC50 values of 5-hydroxy-6-methyl-1,4-naphthoquinone were found to be between 19.1 and 15.4 μM on the four cell lines. However this compound showed toxicity on peripheral blood mononuclear cells. Six derivatives were selected for mechanistic studies. Considering the findings from cell cycle analysis, caspase 3/7 activation and annexinV-FITC dual labelling, 5-hydroxy-6-methyl-1,4-naphthoquinone was found to have antitumour effect by inducing apoptosis. Two derivatives namely, ‘8-fluoro-5-hydroxy-7- methyl-1,4-naphthoquinone’ and ‘2,5-dihydroxy-7-methyl-1,4-naphthoquinone’ were found to be not toxic on peripheral blood mononuclear cells suggesting their action is specific for tumour cells. Compound 2,5-dihydroxy-7-methyl-1,4-naphthoquinone was found to induce apoptosis through caspase 3/7 activation. In view of the enhanced potencies associated with these derivatives, these analogues may hold considerable therapeutic potential for the treatment of leukaemia cancers. The ethanol extracts of seven plant species (ethnobotanically selected) were also tested for their cytotoxicity, assayed by the XTT assay, against four human cancer cell lines at concentrations ranging from 0.78 to 100 μg/ml. Of all the ethanol extracts, Foeniculum vulgare was found to have the best activity on HeLa cells, which exhibited an IC50 value of 19.97± 0.048 μg/ml. Therefore, it was selected for isolation of the bioactive principles. The extract of Foeniculum vulgare was fractionated using column chromatography with hexane and ethyl acetate at different ratios as eluent. Two known compounds, ‘4-methoxycinnamyl alcohol’ and ‘syringin’ were isolated. The IC50 values of ‘4-methoxycinnamyl alcohol’ and ‘syringin’ were found to be 7.82 ± 0.28 μg/ml and 10.26 ± 0.18 μg/ml respectively on HeLa cells. Both compounds were tested for their cytotoxicity against U937 cells and also on peripheral blood mononuclear cells. At the concentrations of 10 and 100 μg/ml ‘4- methoxycinnamyl alcohol’ showed similar cell proliferation as that of the positive control ‘cisplatin’. ‘Syringin’ however, had much lower cytotoxicity on the U937 cells than ‘4- methoxycinnamyl alcohol’. IC50 was found to be 91.14 ± 0.63 μg/ml. Both ‘syringin’ and ‘4- methoxycinnamyl alcohol’ were not cytotoxic at concentrations of 1 and 10 μg/ml on the PBMCs as compared to cisplatin. ‘4-Methoxycinnamyl alcohol’ was selected based on its activity on the cancer cells, for further investigation with regard to its mechanism of action. On gel electrophoresis it did not show a typical ladder pattern, instead a characteristic smear resulted which indicated necrosis. Two best derivatives of 7-methyljuglone (‘8-fluoro-5-hydroxy-7-methyl-1,4-naphthoquinone’ and ‘2,5-dihydroxy-7-methyl-1,4-naphthoquinone’) and the ethanol extract of F. vulgare warrant further investigation to be considered for their potential as anticancer agents. / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

Au cours de cette thèse nous avons étudié les propriétés anticancéreuses du pigment majoritaire de la betterave rouge : la bétanine, ainsi que son mode d’action. Nos travaux reposent sur une approche pluridisciplinaire. Nous avons tout d’abord mis au point un protocole d’extraction et de purification de la bétanine à partir de betteraves rouges fraîches. Plusieurs étapes de purification se terminant par la séparation des molécules d’intérêt sur HPLC semi-préparative sont nécessaires à l’obtention de la bétanine à un degré de pureté de 90 %, une qualité d’extrait jusqu’à présent inégalé. Nous avons ensuite évalué l’effet cytotoxique de notre extrait sur cellules cancéreuses. Nous avons pu démontrer son innocuité sur cellules non cancéreuses et identifier les voies de signalisation pouvant être impliquées. Nous avons ainsi pu avancer des pistes concernant le mode d’action de la bétanine sur les cellules, mais également soumettre pour la première fois l’idée d’une implication de l’autophagie dans la mort cellulaire induite par la bétanine. Enfin, nous avons montré, par des techniques d’analyse biophysique aux interfaces appliquées aux membranes cellulaires et biomimétiques, qu’indépendamment de son insertion jusqu’au cœur hydrophobe des membranes, la bétanine n’influait pas sur la fluidité et la perméabilité membranaire. Ce travail exploratoire confirme l’intérêt à porter à la bétanine qui, compte tenu de sa haute biodisponibilité, présente de nombreuses applications thérapeutiques potentielles. / During this thesis we studied the anticancer properties of the major beetroot’s pigment: betanin. Our work is based on a multidisciplinary approach.First we developed a protocol for the extraction and the purification of betanin from fresh beetroots. Several purification steps ended by separation in semi-preparative HPLC are required to obtain a betanin at 90 % pure, which is the highest purity ever recorded. Then we assessed the cytotoxic effect of our extract on cancer cells and its safety on non-cancer cells. By identifying the signaling pathways that might be involved in these effects, we were thus able to suggest ways concerning the mode of action of betanin on cells, but also propose, for the first time, the idea of an involvement of autophagy in cell death induced by betanin. Finally, we have shown by interfacial biophysical techniques applied on cell and biomimetic membranes that, regarless to its deep insertion in the hydrophobic core of the lipid bilayer, betanin did not affect the physical properties of the membrane such as its fluidity or its permability.This scoping study confirms the interest to bring to betanin which, given its high bioavailability, has many potential therapeutic applications.