Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

No description available.

The survival of several neuron populations during development, including sympathetic neurons, is strictly regulated by neurotrophins such as nerve growth factor (NGF) released from innervation targets. NGF activates its receptor, TrkA, at axon terminals, to generate signals that are transmitted retrogradely to cell bodies to induce signaling cascades regulating survival. A general view of this process is that NGF generates retrograde survival signals that, when delivered to cell bodies, induce downstream survival signaling that prevents apoptosis. A retrograde survival signal proposed to be necessary for sympathetic neuron survival consists of endosomes containing NGF and phosphorylated TrkA. For this signal, phosphorylated TrkA arriving at cell bodies is required to initiate survival signaling. Studies have tested the necessity of TrkA phosphorylation in the cell bodies for survival: results from different studies contradict each other. Moreover, the Trk inhibitor, K252a, used in these studies, has reported non-specific effects. Using an alternate Trk inhibitor, Gö6976, data presented in this thesis demonstrates that NGF can promote survival by retrograde signaling that does not require TrkA phosphorylation in the cell bodies. These retrograde signals may be composed of signaling molecules activated downstream of TrkA in axons since pro-survival molecules downstream of TrkA, Akt and CREB, were found activated in the cell bodies/proximal axons. Data presented in this thesis also reveals a fundamentally different mechanism for how NGF promotes sympathetic neuron survival: a retrograde apoptotic signal that is suppressed by NGF. NGF withdrawal from axons induced the “axon apoptotic signal” that was retrogradely transmitted to cell bodies to activate a key pro-apoptotic molecule, c-jun. The axon apoptotic signal, which was blocked by the kinase inhibitors rottlerin and chelerythrine, was necessary for apoptosis in response to NGF deprivation. Evidence GSK3 is involved in generation or transmission of the axon apoptotic signal was provided by experiments with GSK3 inhibitors and siRNA. The axon apoptotic signal discovery refutes the previous view that NGF acting on axon terminals supports survival exclusively by generating retrograde survival signals. The axon apoptotic signal has broad implications for understanding nervous system development and other conditions where neuronal apoptosis occurs, such as neurotrauma and neurodegenerative diseases.

Nous nous intéressons aux mécanismes moléculaires impliqués dans la physiopathologie de la fibrose pulmonaire idiopathique. La fibrose pulmonaire estcaractérisée par l’accumulation de protéines de la matrice extracellulaire et de fibroblastes dans les espaces aériens distaux. La désorganisation et la destructionalvéolaires qui résultent de la fibrose aboutissent à une altération des propriétés mécaniques du poumon et à une incapacité à réaliser les échanges gazeux responsables d’une insuffisance respiratoire parfois mortelle. Le pronostic de la fibrose pulmonaire idiopathique (FPI) est particulièrement mauvais puisque la médiane de survie est de 3 à 5 ans. Il n’existe actuellement aucune thérapeutique efficace dans la fibrose pulmonaire. Ainsi, il est crucial d’explorer de nouvelles hypothèses physiopathologiques dans cette maladie afin d’ouvrir de nouvelles voies thérapeutiques. L'apoptose joue un rôle clé dans le développement de nombreux organes et dans l'homéostasie tissulaire chez l'adulte. Les protéines de la famille BCL-2 sont des éléments essentiels de la machinerie apoptotique. Ces protéines agissent comme des régulateurs anti- ou pro-apoptotiques. Parmi les membres de la famille BCL-2, le facteur pro-apoptotique BAX contrôle la voie mitochondriale de l’apoptose. Des perturbations de l’apoptose ont été mises en cause dans des maladies pulmonaires comme la fibrose pulmonaire idiopathique. Des études récentes suggèrent fortement que les protéines de la famille BCL-2 sont également impliquées dans d’autres fonctions cellulaires que le contrôle de l'apoptose. De plus, la protéine BAX est aussi localisée dans le noyau de nombreux types cellulaires. Même si le rôle de la fraction cytoplasmique de BAX au cours de l’apoptose est assez bien caractérisé, les fonctions nucléaires de BAX ne sont pas connues. Ce travail de thèse a pour but de mieux comprendre le rôle de la forme nucléaire de BAX dans différents processus cellulaires fondamentaux impliqués dans la fibrogenèse. Notre étude montre que la protéine BAX est présente dans le noyau à proximité de l’euchromatine dans différentes lignées d’origine pulmonaire in vitro. Ensuite, nous montrons que la forme nucléaire de BAX est impliquée dans la progression du cycle cellulaire et dans le contrôle de l’état de différenciation myofibroblastique en condition basale. Enfin, nous avons détecté la forme nucléaire de BAX dans l’épithélium hyperplasique et les foyers de fibrose dans le poumon de FPI. / Summary not transmitted

Abstract Aptosis denotes a biochemically and morphologically distinct chain of events leading to self-destruction of cell. It is pivotal in the maintenance of tissue homeostasis and also plays a role in neoplasm. In this work, the extent of apoptosis and apoptosis regulating proteins and factors was studied in a total of 94 patients operated for lung carcinoma, including 56 small cell lung carcinomas (SCLC) and 38 large cell lung carcinomas (LCLC). The extent of apoptosis was determined by detecting and counting the relative and absolute numbers of apoptotic cells and bodies using 3'- end labelling of the apoptotic DNA. The extent of apoptosis in SCLC was compared with the cell proliferation activity as determined by Ki-67 immunohistochemistry, with the volume density of necrosis and with the occurrence of immunohistochemically detectable p53 and bcl-2 proteins. In order to test the hypothesis that increased apoptotic activity is connected with neuroendocrine differentiation and with low differentiation degree in LCLC and that it is regulated by bcl-2 family proteins, the extent of apoptosis and tumour necrosis was analysed in relation to the expression of bcl-2 family proteins bcl-2, mcl-1, bax and bak. Apoptosis, tumour infiltrating lymphocytes (TILs), and angiogenesis are important factors that contribute to tumour growth. In the present study immunohistochemical methods were used to investigate the relationships of these factors and their role in the prognosis of the patients with LCLC and SCLC. A remarkably high apoptotic activity was detected in both SCLC and LCLC. The mean apoptotic index in SCLC was 2.70 % and in LCLC 2.49 %. Exceptionally high proliferation activity and high percentage of tumour necrosis was seen in SCLC. 58 % of SCLC showed more than 40 % of Ki-67 positive nuclei, and tumour necrosis was seen in 83 % of the cases. P53 protein accumulation was detected in 38 % and bcl-2 expression in 50 % of SCLC. The extent of apoptosis in SCLC was inversely related to tumour necrosis and p53 protein accumulation. In LCLC, bcl-2 expression was detected in 40 % of the cases. It was associated with neuroendocrine differentiation and predicted favourable prognosis of the patients. A high number of T cells and macrophages with a small number of B cells was detected in both SCLC and LCLC. The occurrence of intratumoural cytotoxic CD8 cells was associated with the occurrence of apoptotic bodies in SCLC. The increased number of intratumoural T cells, CD8-positive cells and macrophages predicted favourable prognosis of the patients with SCLC. In LCLC, an increased number of B cells and macrophages, but not T cells, was associated with better survival. Iaddition to tumour cells, numerous apoptotic bodies could also be found within alveolar macrophages within and close to tumour tissue. In order to test whether such cells could be found in sputum smears and if their presence could be utilised as a marker of malignancy in tumour diagnosis, the occurrence of alveolar macrophages with apoptotic bodies (AMWABs) was analysed in 84 sputum samples and 13 broncho-alveolar lavage (BAL) specimens from patients with and without lung carcinoma. AMWABs could be found in cytological samples of the patients with lung carcinoma. In sputum and BAL specimens, enhanced apoptosis, as measured by an increased number of AMWABs reflected and was indicative of malignancy. This was also true for cytological specimens of the patients even when the actual malignant cells were not found. Therefore the AMWABs served as a marker of pulmonary malignancy.

Macrófagos (Mf) produzem PAF e PAF-R e eliminam partículas alteradas via CD36. Uptake de oxLDL requer associação CD36/PAF-R. Avaliamos isto na eferocitose. Bloqueio do PAF-R e de lipid rafts (LR) inibiu eferocitose. Esta induziu associação PAF-R/CD36 e destes com flotilina-1 (marca LR). Eferocitose induziu IL-10 e IL-12p40. Bloqueio do PAF-R inibiu mais IL-10 e inibição da COX-2 teve efeito similar, sugerindo que eferocitose depende da interação PAF-R/CD36 em LR e que isto induz prostanoides e perfil regulador. Mf adquirirem diferentes fenótipos. Estudamos a participação do PAF-R. Bloqueio do PAF-R antes dos estímulos (IFN-g/LPS, IL-4 ou IgG-SRBC/LPS) inibiu marcadores MCP-1, TNF-a, iNOS, receptor manose, arginase-1 e IL-10, mas não IL-12p40, sugerindo que PAF-R modula fenótipo de Mf. PAF e PAF-like são gerados por estressores oxidativos. Ativação do PAF-R induz imunossupressão sistêmica (IS). Mostramos que terapia fotodinâmica (PDT) in vitro gerou ligantes do PAF-R e in vivo inibiu reação de CHS em WT, mas não em PAF-R KO, sugerindo que PDT induz IS via PAF-R. / Macrophages (Mp) produce PAF and PAF-R and scavenge altered particles via CD36. oxLDL uptake requires association CD36/PAF-R. We analyzed that on efferocytosis. PAF-R and lipid rafts (LR) blockage inhibited efferocytosis. Efferocytosis induced association CD36/PAF-R and both with LR marker protein, and induced IL-10 and IL-12p40. PAF-R and COX-2 blockage inhibited more IL-10, suggesting that efferocytosis depends on PAF-R/CD36 interaction in LR and that this induces prostanoids and regulatory profile. Mp acquire different phenotypes. PAF-R participation in that was analyzed. PAF-R blockage before stimuli (IFN-g/LPS, IL-4 or IgG-SRBC/LPS) inhibited markers MCP-1, TNF-a, iNOS, mannose receptor, arginase-1 and IL-10, but not IL-12p40, suggesting that PAF-R modulates Mp phenotype. PAF and PAF-like are generated by oxidative stressors. PAF-R activation induces systemic immunosuppression (SI). We showed that photodynamic therapy (PDT) in vitro generated PAF-R ligands and in vivo inhibited CHS reaction in WT, but not PAF-R KO, suggesting that PDT induces SI via PAF-R.

Wachstumsfaktor bedingte Aktivierung des PI3K/Akt/mTOR Signalweg wirkt positiv auf Vermehrung und Überleben. Konstitutive Aktivierung des Signalweges in NHL ist jedoch an Tumorprogression und Therapieresistenz beteiligt. Am Zelllinienmodell wurden zwei mögliche Therapiestrategien der PI3K/Akt/mTOR Inhibition erprobt, PI3K Inhibition mit BKM120 und horizontale Kombination von Zytostatika mit PI3K/Akt/mTOR Inhibitoren Erstens, BKM120 hat Antitumoraktivität in NHL und induziert Zelltod. Auf molekularer Ebene führt BKM120 vermittelte Dephosphorylierung von CDK1 an Y15 zur Aktivierung des M-Phase Komplex CDK1/Zyklin B und Eintritt in die Mitose. Parallel erlaubt die Degradation von Zyklin A und Hochregulation von Zyklin B Progression bis zur Metaphase, hemmt jedoch die Transition in die Anaphase. Anhaltender Metaphasearrest bewirkt programmierten Zelltod über den intrinsischen Signalweg der Apoptose durch Hochregulation der BH3-onlys Puma und Hrk, Aktivierung von Bax/Bak und proteolytische Spaltung von Caspase 9. Verlust von Bax/Bak oder Caspase Inhibition schützt vor BKM120 vermitteltem Zelltod. Bax/Bak defiziente Zellen, welche zusätzlich p53 Mutationen aufweisen, werden polyploid. Die Polyploidie ist ATM-MEK1/2 abhängig und kann mit Caffeine oder U0126blockiert werden. Zur Vermeidung von Polyploidie bedingter Tumorprogression, sollte BKM120 nur in Verbindung mit MAPK/ATM Inhibitoren verwendet werden. Zweitens. Horizontale Kombination PI3K/Akt/mTOR Inhibitoren mit cytotoxischen Substanzen schützt vor Apoptose. Der Schutzeffekt tritt auschließlich bei niedrigen Konzentration auf und ist unabhängig von der Art des Inhibitors bzw. Ebene der Inhibition. Das Onkogen und NFkB Target Pim-2 ist möglicherweise am Schutzmechanismus beteiligt. Durch die PI3K/Akt/mTOR vermittelte Pim-2 Regulation ergibt sich eine neue Rückkopplungsschleife. Im Fazit erschwert die Komplexizität des PI3K/Akt/mTOR Signalweges die Etablierung von Therapien. / Growth factor mediated activation of the PI3K/Akt/mTOR pathway positively regulates proliferation and survival. Constitutive activation in NHL, however, is correlated with tumor progression and therapeutic resistance. Therefore, two possible strategies were tested in a cell line model system, Inhibition of PI3K with BKM120 and PI3K/Akt/mTOR Inhibition in addition to cytostatic drug administration. First, it is demonstrated that the pan PI3K inhibitor BKM120 has antitumor activity in NHL and induces cell death. On molecular level, BKM120 mediated dephosphorylation of CDK1 on Y15 causes activation of the M-phase complex CDK1/Cyclin B and entry into mitosis. In parallel, degradation of Cyclin A and Upregulation of Cyclin B enables progression into metaphase but inhibits transition into anaphase. Prolonged metaphase arrest induces programmed cell death via the intrinsic apoptosis pathway by upregulation of the BH3-onlys Puma and Hrk, activation of Bax/Bak and proteolytic cleavage of caspase-9. Loss of Bax/Bak or caspase inhibition protects from BKM120 induced apoptosis. Bax/Bak deficient cells with additional p53 mutation become polyploid. This polyploidy is ATM-MEK1/2 dependent and can be blocked with Caffeine or U0126. To prevent polyploidy related tumor progression, BKM120 should administered only in combination with ATM or MEK inhibitors. Second, combination of PI3K/Akt/mTOR inhibitors with cytotoxic agents protects from apoptosis. The protective effect is only detectable with low PI3K/Akt/mTOR inhibitor concentrations and independent of inhibitor type or cascade level. The oncogene and NFkB target is possibly involved in apoptosis protection and inhibition of NFkB neutralizes the protective effect. PI3K/Akt/mTOR mediated Pim-2 regulation reveals a new feedback loop within the pathway. In conclusion, the complexity of the PI3K/Akt/mTOR pathway impedes therapeutic targeting.

Limitierend für pharmakologische Therapien ist oft die Unfähigkeit des Wirkstoffes, biologische Membranen zu überwinden, weswegen häufig Transportmoleküle wie z.B. zellpenetrierende Peptide (CPPs, cell penetrating peptides) benutzt werden. Von den über 100 beschriebenen CPPs wurde bisher nur eine kleine Anzahl systematisch verglichen, was die Auswahl des „richtigen“ CPPs für eine Anwendung erschwert. Ziel dieser vorliegenden Arbeit war es, das pro-apoptotische Peptid KLA mittels CPPs spezifisch in Krebszellen zu transportieren. Untersucht wurden: (I) Verschiedene CPPs in unterschiedlichen Zelltypen zur Selektion der „besten“ CPPs; (II) Der Einflusses des CPP C-Terminus auf die Internalisierung und zelluläre Verteilung; (III) Der zelluläre KLA-Transport mittels CPPs via einer nicht-kovalenten Administration. 22 verschiedene CPPs wurden in sieben Zelltypen untersucht, wobei Toxizität, Zellaufnahme und zelluläre Lokalisation mittels Fluorescein markierten CPPs in Fluoreszenzspektroskopie und konfokaler Mikroskopie betrachtet wurden. Abhängig von der Zellaufnahme wurden die CPPs in drei Gruppen klassifiziert. Die Untersuchung carboxylierter und carboxyamidierter CPP C-Termini ergab, dass in den meisten Fällen ein Carboxyamid die zelluläre Aufnahme begünstigte. Drei CPPs (MPG, Penetratin und Integrin) wurden ausgewählt, um das pro-apoptotische KLA Peptid in zwei Krebszelllinien (MCF-7 Brustkrebszellen und leukämische RAW264.7 Makrophagen) im Vergleich zu Fibroblasten (Cos-7) nicht-kovalent zu transportieren. Der erfolgreiche KLA-Transport hing vom CPP, dessen C-Terminus und der Zelllinie ab. Die Analyse der Viabilität nach CPP:KLA Administration ergab, dass MPG-CONH2:KLA (1:2) toxisch für Makrophagen und Brustkrebszellen, aber nicht für Fibroblasten war. Die Toxizität konnte der Apoptose zugeordnet werden. Die vorliegende Arbeit liefert wichtige Informationen über die Auswahl des passenden CPPs für den nicht-kovalenten Transport des pro-apoptotischen KLA-Peptids. / Limitations in a pharmacological therapy are often due to the inability of drugs to overcome the cell membrane and therefore transporting molecules are being used, e.g. cell penetrating peptides (CPPs). Only a few of the over 100 described CPPs have been compared systematically making the choice of “the” CPP for a given application difficult. The goal of the presented work is the CPP mediated delivery of the pro-apoptotic peptide KLA in breast cancer cells as proof of principle for a therapeutical application. Analysed were (I) Different CPPs in various cell types to select the “best” one, (II) The influence of the CPP C-termini on uptake and localisation, (III) The cellular KLA delivery via a non-covalent CPP administration. 22 CPPs were compared in seven cell types thereby looking at toxicity, cellular uptake and subcellular localisation using fluorescein labelled CPPs for fluorescence spectroscopy and confocal microscopy. The resulting uptake information allowed the classification of the CPPs in three main groups. The evaluation of carboxylated and carboxyamidated CPP C-termini revealed that a carboxyamide mostly enhanced the cellular CPP uptake. Three CPPs were selected (MPG, penetratin and integrin) to deliver the pro-apoptotic KLA peptide in two cancer cell lines (breast cancer MCF-7 cells and RAW264.7 macrophages) compared to fibroblasts (Cos-7) via the non-covalent strategy. A successful KLA delivery depended on the applied CPP, its C-terminus and the used cell line. The biological activity of the pro-apoptotic KLA peptide was determined via the cell viability (MTT assay). The co-incubation of MPG-CONH2:KLA (1:2) was able to induce toxicity in breast cancer cells and leukaemic macrophages, but not in fibroblasts. The viability reductions were then assigned to apoptosis. This work provides important information for the choice of an adequate CPP for the pro-apoptotic KLA peptide delivery and presents the advantage of the non-covalent delivery strategy.

Les facteurs de transcription de la famille MAF sont impliqués d’une part au cours du développement dans des processus de différenciation terminale et d’autre part dans la carcinogenèse. Un découplage fonctionnel est observé. En effet, les gènes cibles mis en jeu au cours des processus cancéreux et de différenciation terminale semblent différents. L'activité oncogénique des protéines MAF dépend du contexte cellulaire. Ainsi, il a été proposé qu'elles exercent leur activité oncogénique dans des tissus où elles ne sont pas exprimées normalement. De plus, leur pouvoir transformant est régulé par phosphorylation. Mon travail de thèse a porté sur le rôle oncogénique de ces protéines en étudiant ces deux aspects. J'ai identifié le rôle oncogénique de NRL, un membre de la famille MAF, dans le Médulloblastome et j'ai étudié la régulation par phosphorylation des MAF dans le Myélome Multiple. L’activité oncogénique du facteur de transcription NRL, connu pour son rôle dans la différenciation terminale d’un type cellulaire de la rétine, n’avait jamais été établie. Alors que NRL n'est pas exprimé dans le cervelet sain, des études de transcriptome ont montré que NRL est surexprimé dans un sous-groupe agressif de médulloblastome, une tumeur pédiatrique du cervelet. J’ai montré pour la première fois que NRL est un oncogène. Il participe directement à la carcinogénèse de médulloblastomes, en protégeant les cellules de l’apoptose, à travers la régulation d’un membre anti-apoptotique de la famille BCL, BCL-XL. L’inhibition des protéines BCL pourrait constituer une stratégie thérapeutique potentielle dans des Médulloblastomes de mauvais pronostic et résistants aux traitements classiques.Au sein du laboratoire, un projet, auquel j’ai participé, a porté sur la régulation de l’activité oncogénique des facteurs de transcription MAF, dans des Myélomes Multiples, de mauvais pronostic. Ce travail établit que l’activité oncogénique de deux membres de la famille MAF, MAFB et c-MAF, est régulée par phosphorylation induite par la Ser/Thr kinase GSK3. La phosphorylation des facteurs de transcription MAF augmente leur activité oncogénique et paradoxalement induit leur dégradation par le protéasome. Ce travail a permis d’identifier un mécanisme de résistance potentiel de ces tumeurs et de proposer une nouvelle approche thérapeutique, basée sur l’inhibition de la phosphorylation des protéines MAF. / MAF (MusculoAponeurotic Fibrosarcoma) transcription factors are involved in terminal differentiation during normal development, and also in oncogenesis. A functional uncoupling is observed between these two functional activities. Indeed, target genes involved in cancer or terminal differentiation look different. The oncogenic activity of MAF proteins is dependent on the cellular context. Thus, it was suggested that they exert their oncogenic activity in tissues where they are not normally expressed. In addition, their transforming ability is regulated by phosphorylation. My work focused on the oncogenic role of these proteins by studying these two aspects. I identified the oncogenic role of NRL, a member of the MAF family in Medulloblastoma and I studied the regulation by phosphorylation of MAF in Multiple Myeloma.The oncogenic activity of the transcription factor NRL, known for its role in the terminal differentiation of a cell type in the retina, has never been established. While NRL is not expressed in the healthy cerebellum, transcriptome studies showed that NRL is overexpressed in an aggressive subgroup of medulloblastoma, a pediatric tumor of the cerebellum. I showed for the first time that NRL is an oncogene. NRL is directly involved in the carcinogenesis of medulloblastoma, protecting cells from apoptosis through regulation of an anti-apoptotic member of the BCL family, BCL-XL. Inhibition of the protein BCL could be a potential therapeutic strategy for medulloblastoma with a poor prognosis and resistant to conventional therapies.In the laboratory, I also participated in a project which focused on the regulation of the oncogenic activity of transcription factors, MAF, in rarely curable multiple myeloma. This work establishes that the oncogenic activity of two MAF members, MAFB and c-MAF, are regulated by phosphorylation-induced Ser/Thr kinase GSK3. Phosphorylation of transcription factors MAF increases their oncogenic activity and paradoxically induces their degradation by the proteasome. This work has identified a potential mechanism of resistance of these tumors providing a new therapeutic approach, based on the inhibition of MAF protein phosphorylation.

Thesis (Ph.D. (Biochemistry)) --University of Limpopo, 2009 / Commelina benghalensis Linn is used in traditional medicine in several Asian and African countries for the treatment of various ailments such as stomach irritations, burns, sore throat and feet, diarrhoea and as an anti-inflammatory agent. Recently, our laboratory showed that the crude methanolic extract of Commelina benghalensis L (CMECB) exhibits growth inhibitory and proapoptotic effects in Jurkat T and Wil-2 NS cancer cell lines. In this study, the precise molecular mechanism(s) associated with CMECB-induced growth inhibitory and apoptosis inducing effects in Jurkat T and Wil-2 NS cell lines were investigated. This was achieved by investigating the effects of the extract on the cell division cycle distribution profile as well as its effects on various cell division cycle and apoptosis regulatory genes. Ground stems of C. benghalensis L were extracted with absolute methanol to obtain a crude extract. To assess the effect of CMECB on cancer cell growth, experimental cell cultures were exposed to various concentrations (0 to 600 μg/ml) of CMECB for up to 72 hours. The results demonstrated a significant reduction in cell viability and inhibition of proliferation of experimental cell cultures as determined by the trypan blue dye exclusion assay and the Coulter counter method, respectively. Analysis of nuclear morphological changes in cells stained with Hoechst 33258 confirmed apoptosis as the mode of cell death that is associated with the growth inhibitory effects of CMECB in both the Jurkat T and Wil-2 NS cell lines. This assertion was based on the observed presence of nuclear morphological changes such as chromatin condensation and fragmentation and apoptotic bodies in cells exposed to CMECB. In order to get an insight on the pro-apoptotic mechanisms of CMECB, Western blot xxi and quantitative real-time PCR (qrt-PCR) were used to investigate the expression profiles of various apoptosis and cell division cycle regulatory genes. Qrt-PCR results showed a lack of a clear up- and/or down-regulatory effects of CMECB on the mRNA expression levels of bax and bcl-2 in both Jurkat T and Wil-2 NS cells. Western blot analyses demonstrated that CMECB induced apoptosis by facilitating Bax protein translocation from the cytosol to the mitochondria in both Jurkat T and Wil-2 NS cells. In addition, CMECB down-regulated Bcl-2 protein expression which, as a result, led to the shift in the Bax/Bcl-2 protein ratio at certain time points and concentration in both Jurkat T and Wil-2 NS cells. The modulation of the Bcl-2 family members led to mitochondrial cytochrome c release into the cytosol and activation of caspases-9 and -3; this was also confirmed by caspase activity assays and eventual degradation of PARP. Furthermore, CMECB induced Jurkat T and Wil-2 NS cell division cycle arrest at the G2/M phase as determined by flow cytometric analysis. Western blot analyses of G2/M phase regulatory proteins demonstrated that the CMECB-induced cell division cycle arrest was associated with the downregulation of cyclin B1 and Cdc2 protein expression levels. Western blot analyses results further revealed that the arrest of Wil-2 NS cells at the G2/M phase was independent of p21 protein activity. However, Jurkat T cell division cycle arrest was found to be mediated, in part, by p21. Quantitative real-time PCR results did not show a clear trend in terms of the down- or up-regulatory effects of the extracts on the G2/M phase regulatory genes. The CMECBinduced apoptosis and G2/M arrest was found to occur in a p53-independent xxii manner due to the lack and down-regulation of p53 protein levels in both Jurkat T and Wil-2 NS cells, respectively. In conclusion, CMECB induces its anticancer activity by inducing G2/M phase arrest and mitochondrial-mediated apoptosis independent of p53 protein activity. Although the study did not perform in vivo experiments to ascertain the efficacy of extracts of CMECB against specific tumour types in animal models, the present findings somehow validate the traditional use of C. benghalensis L as an anticancer agent. A more definitive study needs to be done to ascertain this assertion. / National Research Foundation and the University of Limpopo research office