Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Jan. 29, 2010). Includes bibliographical references (p. 55-60).

Endothelial inflammation is a pivotal early event in the development of atherosclerosis. Long term exposure to cardiovascular risk factors will ultimately exhaust those protective anti-inflammatory factors such as the heme oxygenase (HO) system. The HO system plays a critical role in cellular and tissue self-defense against oxidative stress and inflammation. Caveolae are membrane domains and are particularly abundant in endothelial cells, where they are believed to play a major role in the regulation of endothelial vesicular trafficking as well as the uptake of lipids and related lipophilic compounds, possibly including bioactive food components such as flavonoids. Research in this dissertation addresses the role of HO-1 and caveolae on dietary flavonoid epigallocatechin gallate (EGCG) mediated protection against pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and linoleic acid-induced activation of endothelial cells. The data support the hypothesis that EGCG protects against TNF-α-induced monocyte recruitment and adhesion partially through the induction of HO-1 and bilirubin. The observed anti-inflammatory effects of EGCG are mimicked by the HO-1 inducer cobalt protoporphyrin (CoPP) and abolished by HO-1 gene silencing. Nrf2 is the major transcription factor of phase II antioxidant enzymes including HO-1. Results clearly show that EGCG-induced HO-1 expression and subsequent bilirubin productions are dependent on functional Nrf2. EGCG also can down-regulate the base-line level of caveolin-1. Furthermore, silencing of the caveolin-1 gene can markedly down-regulate linoleic acid-induced COX-2 and MCP-1, indicating that caveolae may be a critical platform regulating inflammatory signaling pathways. Similar to EGCG treatment, silencing of caveolin-1 can also result in the activation of Nrf2, up-regulation of HO-1 and bilirubin. This may be one of the mechanisms to explain the protection effect of caveolin-1 gene silencing against endothelial inflammation. Moreover, EGCG rapidly accumulates in caveolae, which is associated with caveolin-1 displacement from the plasma membrane towards the cytosol. Caveolin-1 gene silencing can significantly reduce the uptake of EGCG in endothelial cells within 30 min. These data suggest that caveolae may play a role in the uptake and transport of EGCG in endothelial cells. These studies provide a novel target through which EGCG functions to protect against inflammatory diseases such as atherosclerosis.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Additional advisors: Ada Elgavish, Vithal Ghanta, Hui-Chen Hsu, Thane Wibbels. Description based on contents viewed June 11, 2008; title from title screen. Includes bibliographical references.

Th.--Sci.--Bordeaux 1, 1984. N°: 808.

No description available.

No description available.

Background: Fetal alcohol spectrum disorder (FASD) is a challenging public health problem. Previous studies have found an association between FASD and oxidative stress. In the present study, we assessed the role of oxidative stress in ethanol-induced embryonic damage and the effect of (-)-epigallocatechin-3-gallate (EGCG), a powerful antioxidant extracted from green tea, on the development of FASD in a murine model.Methods: Pregnant female mice were given intraperitoneal ethanol (25%, 0.005 to 0.02 ml/g) on gestational day 8 (G8) to establish the FASD model. On G10.25, mice were sacrificed and embryos were collected and photographed to determine head length (HL), head width (HW), and crown rump length (CRL). For mice given EGCG, administration was through a feeding tube on G7 and G8 (dose: 200, 300, or 400 mg/kg/d, the total amount for a day was divided into 2 equal portions). G10.25 embryos were evaluated morphologically. Brain tissues of G9.25 embryos were used for RT-PCR and western blotting of neural marker genes and proteins and detection of oxidative stress indicators.Results: Administration of ethanol to pregnant mice on G8 led to the retardation of embryonic growth and down-regulation of neural marker genes. In addition, administration of ethanol (0.02 ml/g) led to the elevation of oxidative stress indicators [hydrogen peroxide (H2O2) and malondialdehyde (MDA)]. Administration of EGCG on G7 and G8 along with ethanol on G8 ameliorated the ethanol-induced growth retardation. Mice given EGCG (400 mg/kg/d) along with ethanol had embryo sizes and neural marker genes expression similar to the normal controls. Furthermore, EGCG (400 mg/kg on G7 and G8) inhibited the increase in H2O2 and MDA.Conclusions: In a murine model, oxidative stress appears to play an important role in ethanol-induced embryonic growth retardation. EGCG can prevent some of the embryonic injuries caused by ethanol.

Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene resulting in the loss of the dystrophin protein from the plasma membrane of muscle fibers. In the absence of dystrophin, muscles undergo massive muscle degeneration and inflammation. Inflammation is believed to contribute substantially to dystrophic muscle pathology. The transcription factor NF-κB regulates inflammatory gene expression and provides a logical target for therapeutic treatments. Green tea extract and its primary polyphenol, epigallocatechin gallate, have been shown to have anti-inflammatory properties and to improve dystrophic muscle pathology. The purpose of these studies was to determine if dietary treatment with green tea extract or epigallocatechin gallate administered prior to disease onset could reduce dystrophic muscle pathology during the early disease time course and identify potential mechanisms through which NF-κB may be involved. Green tea extract has been shown to decrease muscle pathology and increase muscle function in mdx mice, a dystrophic mouse model. These changes have been attributed to the antioxidant potential of epigallocatechin gallate; however, other mechanisms such as suppression of the inflammatory response have not been evaluated. In the studies reported herein, both green tea extract and epigallocatechin gallate significantly decreased muscle pathology in mdx mice when provided in their diets prior to disease onset. In green tea extract (0.25% and 0.5%) treated mdx mice, serum creatine kinase, a systemic marker of muscle damage, was decreased by 85% at age 42 days. Normal fiber morphology in the tibialis anterior muscle was increased by 32% at this age (P≤0.05). The primary histopathological change was a 21% decrease in regenerating fibers (P≤0.05). NF-κB staining in central nuclei of regenerating fibers was decreased by 34% (P≤0.05). In epigallocatechin gallate (0.1%) treated mdx mice, serum creatine kinase was unchanged; however, normal fiber morphology in the tibialis anterior was increased by 20% at ages 28 and 42 days (P≤0.05). At age 42 days, the primary histopathological change was a 21% decrease in regenerating fibers (P≤0.05). NF-κB staining in central nuclei of regenerating muscle fibers was decreased by 21% at this age (P≤0.05). Epigallocatechin gallate appears to be the primary polyphenol of green tea extract responsible for many of the beneficial changes in dystrophic muscle. These data suggest that both green tea extract and epigallocatechin gallate decrease NF-κB activity in regenerating fibers resulting in reduced muscle pathology. Complimentary and alternative medicine approaches, including the use of green tea, provide important therapeutic options for ameliorating Duchenne muscular dystrophy. Green tea extract and epigallocatechin gallate are effective at decreasing muscle pathology potentially by reducing NF-κB activity in regenerating fibers in mdx mice. Use of these botanicals appears to elicit a beneficial response in dystrophic muscle that may ultimately lead to effective therapies for patients with this incurable disease. / Ph. D.

Le glioblastome multiforme (GBM) représente la tumeur cérébrale primaire la plus agressive et la plus vascularisée chez l’adulte. La survie médiane après le diagnostic est de moins d’un an en l’absence de traitement. Malheureusement, 90% des patients traités avec de la radiothérapie après la résection chirurgicale d’un GBM développent une récidive tumorale. Récemment, le traitement des GBM avec radiothérapie et témozolomide, un agent reconnu pour ses propriétés antiangiogéniques, a permis de prolonger la survie médiane à 14,6 mois. Des efforts sont déployés pour identifier des substances naturelles capables d’inhiber, de retarder ou de renverser le processus de carcinogenèse. Epigallocatechin-3-gallate (EGCG), un polyphénol retrouvé dans le thé vert, est reconnu pour ses propriétés anticancéreuses et antiangiogéniques. L’EGCG pourrait sensibiliser les cellules tumorales cérébrales et les cellules endothéliales dérivées des tumeurs aux traitements conventionnels. Le chapitre II décrit la première partie de ce projet de doctorat. Nous avons tenté de déterminer si l’EGCG pourrait sensibiliser la réponse des GBM à l’irradiation (IR) et si des marqueurs moléculaires spécifiques sont impliqués. Nous avons documenté que les cellules U-87 étaient relativement radiorésistantes et que Survivin, une protéine inhibitrice de l’apoptose, pourrait être impliquée dans la radiorésistance des GBM. Aussi, nous avons démontré que le pré-traitement des cellules U-87 avec de l’EGCG pourrait annuler l’effet cytoprotecteur d’une surexpression de Survivin et potentialiser l’effet cytoréducteur de l’IR. Au chapitre III, nous avons caractérisé l’impact de l’IR sur la survie de cellules endothéliales microvasculaires cérébrales humaines (HBMEC) et nous avons déterminé si l’EGCG pouvait optimiser cet effet. Bien que les traitements individuels avec l’EGCG et l’IR diminuaient la survie des HBMEC, le traitement combiné diminuait de façon synergique la survie cellulaire. Nous avons documenté que le traitement combiné augmentait la mort cellulaire, plus spécifiquement la nécrose. Au chapitre IV, nous avons investigué l’impact de l’IR sur les fonctions angiogéniques des HBMEC résistantes à l’IR, notamment la prolifération cellulaire, la migration cellulaire en présence de facteurs de croissance dérivés des tumeurs cérébrales, et la capacité de tubulogenèse. La voie de signalisation des Rho a aussi été étudiée en relation avec les propriétés angiogéniques des HBMEC radiorésistantes. Nos données suggèrent que l’IR altère significativement les propriétés angiogéniques des HBMEC. La réponse aux facteurs importants pour la croissance tumorale et l’angiogenèse ainsi que la tubulogenèse sont atténuées dans ces cellules. En conclusion, ce projet de doctorat confirme les propriétés cytoréductrices de l’IR sur les gliomes malins et propose un nouveau mécanisme pour expliquer la radiorésistance des GBM. Ce projet documente pour la première fois l’effet cytotoxique de l’IR sur les HBMEC. Aussi, ce projet reconnaît l’existence de HBMEC radiorésistantes et caractérise leurs fonctions angiogéniques altérées. La combinaison de molécules naturelles anticancéreuses et antiangiogéniques telles que l’EGCG avec de la radiothérapie pourrait améliorer l’effet de l’IR sur les cellules tumorales et sur les cellules endothéliales associées, possiblement en augmentant la mort cellulaire. Cette thèse supporte l’intégration de nutriments avec propriétés anticancéreuses et antiangiogéniques dans le traitement des gliomes malins pour sensibiliser les cellules tumorales et endothéliales aux traitements conventionnels. / Glioblastoma multiform (GBM) represents the most aggressive and vascularised primary cerebral neoplasm in adults. Median length of survival without further therapy is usually less than one year from the time of diagnosis. Unfortunately, 90% of patients receiving radiotherapy following GBM resection develop a tumor recurrence. More recently, treatment of GBM with combined radiotherapy and temozolomide, an agent recognized for its antiangiogenic activity, increased the median survival to 14,6 months. Efforts have been oriented towards identifying naturally occurring substances capable of inhibiting, delaying or reversing the multi-stage carcinogenesis process. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, has been recognized for its anticancerous and antiangiogenic property. EGCG may represent a potential agent capable of sensitizing brain tumor cells and their derived endothelial cells (ECs) to conventional treatments. In chapter II, the first part of this doctorate project aimed at determining if EGCG, in synergy with radiotherapy, can sensitize GBM’s response to radiation and whether specific molecular markers are involved. We documented that U-87 cells were relatively radioresistant and that Survivin, an inhibitor of apoptosis protein, may be involved in GBM’s radioresistance. We also found that pre-treatment of U-87 cells with EGCG could overcome the cytoprotective effect of Survivin overexpression and potentiate the cytoreductive effect of irradiation (IR). In chapter III, we characterized the impact of IR on human brain microvascular endothelial cell (HBMEC) survival and determined whether EGCG, could optimize this effect. We found that although EGCG treatment and IR individually decreased HBMEC survival, the combined treatment synergistically reduced survival. We documented that the combined treatment increased cell death, more specifically necrosis. In chapter IV, we investigated the impact of IR exposure on the angiogenic functions i.e. cell proliferation, cell migration in response to brain tumor-derived growth factors, and capacity for tubulogenesis of surviving human brain tumor-derived ECs. The Rho signalling pathway was also investigated in relation to the functional properties of radioresistant HBMEC. Our data suggests that IR significantly alters radioresistant HBMEC migration response to tumor-secreted growth factors and tubulogenesis. Response to growth factors important for tumor expansion and angiogenesis is significantly attenuated in these cells. In conclusion, this doctorate project confirmed IR’s cytoreductive properties on malignant gliomas. We proposed a novel mechanism to explain GBMs’ radioresistance. This project documented for the first time IR’s cytotoxic effect in HBMEC. It also described the existence of radioresistant HBMEC and characterized their altered angiogenic functions. The combination of natural anticancerous and antiangiogenic molecules such as EGCG with radiotherapy could improve IR’s effect on human malignant glioma cells and microvascular ECs, especially through increased necrosis of HBMEC. The thesis supports integrating nutrients bearing anticancerous and antiangiogenic properties, such as EGCG, in the management of gliomas to sensitize tumor and tumor-associated ECs to conventional therapies.

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.