Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer

Modulation de l'agrégation des protéines amyloïdes par de petites molécules (modèle du lysozyme)

Au moins vingt protéines humaines peuvent s agréger anormalement pour former des dépôts pathologiques qui sont associés à plusieurs maladies dégénératives. Malgré les nombreuses études concernant l agrégation des amyloïdes et leurs toxicité, la base moléculaire de ce mécanisme reste inconnu. Au cours de ma thèse, j ai analysé le processus d'agrégation du lysozyme à pH 2 et à 57C par différents techniques. D autre part, une attention particulière a été concentrée sur l'exploration de l'activité inhibitrice, de certains produits naturels, de la formation des fibrilles du lysozyme d œuf de poulet en utilisant la spectroscopie de fluorescence, la microscopie de force atomique, la spectroscopie infra rouge et la diffusion dynamique de la lumière. Nous avons constaté que la formation des fibrilles in vitro a été inhiber par tous les produits de manière dose dépendante. De plus, ces molécules ont la capacité de désintégrer les fibrilles déjà préformés. Basé sur l'analyse de structure et de morphologie nous avons constaté que ces produits inhibent l agrégation avec la même efficacité mais ils remodèlent différemment les oligomères. Aussi nous avons évalué l'effet de ces produits sur l agrégation de l alpha synucleine et les résultats montrent que ces produits inhibent son agrégation d une manière dose dépendante. Ainsi, il apparaît que nicotine, dopamine, resvératrol, rutine et tyrosol sont des inhibiteurs génériques de la formation de fibrilles et peuvent remodeler différents de protéines amyloïdes.At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis and toxicity of certain aggregate forms, its detailed molecular mechanisms remain unknown. During my PhD, I was analysed the aggregation process of lysozyme at pH 2 and 57C by different techniques. Particular attention has been focused on the exploring the inhibitory activity of natural products such us nicotine, dopamine, resveratrol, rutine and tyrosol against the fibrillation of hen lysozyme by using fluoresecence spectroscopy, atomic force microscopy, infra rouge spectroscopy and dynamic light scattering. We found that the formation of amyloid fibrils in vitro was inhibited by all products in a dose dependent manner. Moreover, they were also capable of robustly disaggregating pre-formed oligomers. Based upon structure analysis we demonstrate that natural products inhibit the aggregation with the same efficacity but they remodel differently oligomers and amyloid fibrils. Also we have tested the effect of these products in the aggregation of alpha synuclein and results demonstrate that the formation of alpha synuclein amyloid fibrils was inhibited by all products in a dose dependent manner. Thus, it appears that nicotine; dopamine, resveratrol, rutine and tyrosol are generic inhibitors of amyloid fibril formation and can remodel different conformers of amyloid proteins.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Etude du Catabolisme du beta-Amyloïde (de son implication dans la Maladie d'Alzheimer)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Catabolisme du peptide ß-amyloïde (étude de sa "clearance" par des cellules neuronales et non neuronales en culture)

Les plaques séniles sont des dépôts fibrillaires extracellulaires associées à la maladie d'Alzheimer, dont le constituant principal est le peptide ß-amyloïde (Aß). Le taux circulant de l'Aß est fonction de l'équilibre entre les voies de sa biosynthèse à partir de son précurseur APP et celles de son catabolisme. Un défaut dans la dégradation de ce peptide, pourrait alors contribuer à son dépôt. Dans ce travail, nous démontrons que le peptide Aß est dégradé en présence de plusieurs lignées cellulaires selon un mécanisme identique qui implique deux activités enzymatiques : une activité thiol-metalloprotéase et une activité de type sérine protéase. Ces enzymes seraient impliquées dans le processus normal de dégradation du Aß. Lors du vieillissement cellulaire, les cellules subissent diverses modifications, leurs enzymes pourraient alors perdre, ou voir s'amoindrir, leur capacité à éliminer le peptide Aß, celui-ci pouvant alors s'accumuler et former des dépôts.Amyloid plaques are extracellular fibrillar lesions associated with Alzheimer's disease that are mainly composed of the amyloid peptide. The steady-state level of Aß depends on the balance between its biosynthesis from its APP precursor and its catabolism. The accumulation of the Aß peptide might be explained by the dysfunction of one process (or both). We demonstrate in this work that the Aß is degraded in contact with with neural or non-neural cells. We have identified the enzymatic activity responsible for the cleavage of the Aß peptide : it is a cell-surface thiol-metalloprotease activity followed by a secreted serine protease activity. These enzymes could be implicated in the normal process of Aß degradation. In the process of cellular aging, Cells undergo various modifications in which their enzymes might lose or diminish their capacity to eliminate the Aß peptide thus allowing it to accumulate and form deposits.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods.

Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer's and Parkinson's diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the β-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils

Investigating the effects of different natural molecules on the structure and oligomerization propensity of hen egg-white lysozyme

International audienceThe formation of amyloid aggregates is the hallmark of systemic and neurodegenerative diseases, also known as amyloidosis. Many proteins have been found to aggregate into amyloid-like fibrils and thus process is recognized as general tendency of polypeptides. Inhibition of protein aggregation and fibril formation is thus one of the important strategies in the prevention and treatment of such disease. There is a growing interest of identification of small molecules mainly natural compounds that can prevent or delay amyloid fibril formation. In this work, we report the effect of various compounds from different groups on the amyloid fibrillation of hen egg white lysozyme, a model protein for amyloid formation. Herein, a range of biophysical techniques have been employed in order to establish a systematic approach to study the effect of candidate inhibitors on amyloid aggregation. Results demonstrated that the strategy used show that the different techniques are complimentary in order to elucidate a complete in vitro picture of the effect of the used compounds on HEWL aggregation. Moreover, compared to the data obtained by other groups for the inhibition of lysozyme fibril formation, this work provides new insights into the structural changes (local, secondary, oligomeric, fibrillar structures) undergone by HEWL during aggregation in the presence and absence of inhibitors

Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing. The importance of the first residue C-terminal of the cleavage site

NRC publication: Ye

Clearance of Genetic Variants of Amyloid β Peptide by Neuronal and Non-neuronal Cells

International audienceThe presence of senile plaques in the brain is one of the pathological hallmarks of Alzheimer’s disease (AD). The biogenesis and clearance of the amyloid β peptide (Aβ), the main component of the lesions, lie at the center of the pathogenesis of AD. In sporadic AD, the increase of Aβ levels seems to be indicative of failure of clearance mechanisms. We previously showed that the clearance of the wild type Aβ40 peptide by various neuronal and non-neuronal cells occurs through a same proteolytic process and that Aβ degradation was primarily dictated by its conformational state (Panchal et al., 2007). To gain further insights on the role of the peptide conformation in the clearance mechanism of Aβ, two Aβ40 peptides, known to be associated with amyloid angiopathy (Dutch and Flemish mutations), and the rodent Aβ40 peptide were catabolized by several cells by using the same experimental approach. The peptide fragments, generated by proteolytic cleavage of substrates in cell supernatants, were identified by LC-MS and the cleavage sites of proteases were deduced. In parallel, conformational states of wild type Aβ40 peptide and of the three Aβ40 variants were characterized by circular dichroism spectroscopy. We provide data suggesting that discrete conformational changes of Aβ40 peptide regulate its clearance rate by neuronal and non-neuronal cells. - See more at: http://www.eurekaselect.com/108425/article#sthash.MMBlmlwa.dpu