Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thesis (M.Sc. (Biochemistry))-- University of Limpopo, 2001 / Refer to document

L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse. / Blood-brain barrier (BBB) disruption by circulating tumor and immune cells leads to secondary inflammatory infections. Promyelocytic HL-60 cells represent an excellent model to study and to get a better understanding of the molecular signaling mechanisms involved in carcinogenesis and metastasis. The research described in this thesis shows the analysis of several inflammatory and invasive biomarkers regulated during PMA-induced differentiation of promyelocytic HL-60 cells into macrophages. Carcinogenesis involves some modifications in the expression of biomarkers such as cyclooxygenase-2 and matrix metalloprotease-9. The differentiation status could have an impact on the NF-κB signaling pathway that regulates the target genes, given that these target genes expression varies during cell differentiation. We hypothesize that the activation of the NF-κB pathway by PMA can be reverse by (-)-epigallocatechin-gallate (EGCG). Indeed, the up-regulation of downstream genes combined with the down-regulation of IκB expression showed the significant implication of the NF-κB signaling pathway to activate pro-inflammatory and pro-invasive mechanisms linked to carcinogenesis. The same evidence exhibits in the differentiated cells called «macrophages-like». Moreover, the green tea polyphenol, EGCG, shows chemopreventive property since it better inhibited NF-κB signaling in cells treated simultaneously with EGCG and PMA compared to the «macrophages-like». This difference could be due, in part, to the down-regulation of the 67 kDa laminin receptor, known to be the non-integrin membrane receptor for EGCG. All together, our results suggest that the differentiation status of promyelocytic cells is linked to the activation of mechanisms involved in carcinogenesis and metastasis. These phenomena can be prevented by using natural agents such as EGCG. Targeting the specific biomarkers linked to the differentiation status of tumor cells and involved in the disruption of the BBB may help reduce secondary neuroinflammation and enable the advancement of knowledge towards carcinogenesis prevention.

L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse. / Blood-brain barrier (BBB) disruption by circulating tumor and immune cells leads to secondary inflammatory infections. Promyelocytic HL-60 cells represent an excellent model to study and to get a better understanding of the molecular signaling mechanisms involved in carcinogenesis and metastasis. The research described in this thesis shows the analysis of several inflammatory and invasive biomarkers regulated during PMA-induced differentiation of promyelocytic HL-60 cells into macrophages. Carcinogenesis involves some modifications in the expression of biomarkers such as cyclooxygenase-2 and matrix metalloprotease-9. The differentiation status could have an impact on the NF-κB signaling pathway that regulates the target genes, given that these target genes expression varies during cell differentiation. We hypothesize that the activation of the NF-κB pathway by PMA can be reverse by (-)-epigallocatechin-gallate (EGCG). Indeed, the up-regulation of downstream genes combined with the down-regulation of IκB expression showed the significant implication of the NF-κB signaling pathway to activate pro-inflammatory and pro-invasive mechanisms linked to carcinogenesis. The same evidence exhibits in the differentiated cells called «macrophages-like». Moreover, the green tea polyphenol, EGCG, shows chemopreventive property since it better inhibited NF-κB signaling in cells treated simultaneously with EGCG and PMA compared to the «macrophages-like». This difference could be due, in part, to the down-regulation of the 67 kDa laminin receptor, known to be the non-integrin membrane receptor for EGCG. All together, our results suggest that the differentiation status of promyelocytic cells is linked to the activation of mechanisms involved in carcinogenesis and metastasis. These phenomena can be prevented by using natural agents such as EGCG. Targeting the specific biomarkers linked to the differentiation status of tumor cells and involved in the disruption of the BBB may help reduce secondary neuroinflammation and enable the advancement of knowledge towards carcinogenesis prevention.

Aescin is a mixture of several acidic triterpenoid saponin glycosides found in the extracts of the horse chestnut tree. Horse chestnut, Aesculus Hipoocastanum, is one of the 25 domestic species of Aesculus that are mostly large, ornamental shade trees. Although known to be poisonous, the nuts of the horse chestnut are used by Amerindians, after detoxification. Horse chestnuts are said to have several traditional medicinal usages including even cancer. In this study the biochemical, genotoxic, and cytotoxic effects of aescin was studied using isolated lymphocytes, whole blood lymphocytes and HL-60 promyeloid leukemia cell lines. Cytotoxicity of aescin was examined by trypan blue viability staining of the cells in culture treated with varying aescin concentrations. It was observed that aescin was cytotoxic at all concentrations, for all cell types studied, except whole blood lymphocytes, where it was not cytotoxic at 10-9 and 10-10 M concentrations. Genotoxicity of aescin was examined by sister chromatid exchange and micronucleus. The genotoxic effect of Aescin was observed to be more significant over isolated lymphocytes compared to other cell lines. On the otherhand, aescin at 10-8 M and lower concentrations were observed to be non-genotoxic over whole blood lymphocytes whereas this concentration was considerably toxic for isolated lymphocytes and for HL-60 cell lines. Apoptotic properties of aescin were determined by DNA fragmentation, cytochrome c release and negative NAPO staining. All the Aescin concentrations tested resulted in apoptosis over HL-60 cell lines, whereas necrosis was not observed. However, isolated lymphocytes showed both apoptosis and necrosis upon treatment with 10-6 M to 10-8 M aescin, exhibiting apoptosis only at 10-9 M and 10-10 M. Biochemical effects of aescin were investigated by following GST and NAT enzyme activities. An increase in GST enzyme activity was observed over all cell lines treated with increasing aescin concentrations for 72 hours. Whereas NAT activity was decreased upon treatment with aescin in similar manner.

Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: leaves 295-322. / Generation of peroxide groups in proteins exposed to a wide variety of reactive oxygen species (ROS) requires an initial formation of protein carbon-centred or peroxyl free radicals, which can be reduced to hydroperoxides. Both protein radicals and protein hydroperoxides are capable of oxidizing important biomolecules and thus initiate biological damage. In this study, we investigated the inhibition of protein hydroperoxide formation by ascorbate and GSH in gamma-irradiated HL-60 cells.--We used HL-60 cells as a model for general protection of living organisms by ascorbate (Asc) and glutathione (GSH) from the deleterious effects of protein hydroperoxides generated by radicals produced by gamma radiation. Measurement by HPLC indicated that incubation of HL-60 cells with Asc in the presence of ascorbate oxidase resulted in the accumulation of intracellular Asc. The intracellular Asc levels were lowered by irradiation, demonstrating intracellular consumption of Asc by the radiation-generated radicals. Exposure of HL-60 cells to increasing gamma irradiation doses resulted in increasing accumulation of protein peroxides in the cells. This was measured by the FOX assay. A significant decrease in intracellular protein hydroperoxides was noted when the cells were treated with ascorbic acid before irradiation. A dose-dependent protective effect of Asc was observed. Asc loading also provided strong protection from radiation-generated protein hydroperoxides independently of the composition of the external medium, showing that only the radicals formed within the cells were effective in oxidizing the cell proteins. Similarly, protein peroxidation was inhibited in cells with enhanced levels of GSH and increased when the intracellular GSH concentration was reduced. These findings indicate that ascorbate and GSH are important antioxidants in protecting cells from oxidative stress associated with the generation of protein hydroperoxide. / Mode of access: World Wide Web. / xxix, 322 leaves ill

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