The MBD2-NuRD co-repressor complex is an epigenetic regulator of the developmental silencing of embryonic and fetal β-type globin genes in adult erythroid cells as well as aberrant methylation-dependent silencing of tumor suppressor genes in neoplastic diseases. Biochemical characterization of the MBD2-NuRD complex in chicken erythroid cells identified RbAp46/48, HDAC1/2, MTA1/2/3, p66α/β, Mi2α/β and MBD2 to comprise this multi-protein complex. In the work presented in Chapter 2, we have pursued biophysical and molecular studies to describe a previously uncharacterized domain of human MBD2 (MBD2IDR). Biophysical analyses show that MBD2IDR is an intrinsically disordered region (IDR). Despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical area of contact requiring two contiguous amino acid residues, Arg286 and Leu287. Mutation of these critical residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene Prostasin in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function. In Chapter 3, we have discussed a novel mechanism for MBD2-mediated silencing of the fetal γ-globin gene. Through microarray expression analyses in adult erythroid cells of MBD2-/- mice, we identified ZBTB32 and miR-210 as downstream targets of MBD2. Over-expression of ZBTB32 and miR-210 in adult erythroid cells causes increased expression of the silenced fetal γ-globin gene. Thus, our results indicate that MBD2 may regulate γ-globin gene expression indirectly though ZBTB32 and miR-210 in adult erythroid cells.

Intracellular transport is provided by two major types of molecular motors kinesins and cytoplasmic dynein. Kinesin-1 is a molecular motor that transports molecules and organelles along microtubule tracks anterogradely. Specific protein-protein interactions are required to activate kinesin-1 as the free kinesin exist in an autoinhibited state. The activation of kinesin-1 induces its conformational change, enables microtubule binding and ATP hydrolysis necessary for the directional cargo transport. HDAC6 is a multifunctional protein composed of several domains. It plays an important role in many microtubule dependent processes as HDAC6 is a major tubulin deacetylase. It has been shown that HDAC6 manipulation (inhibition/genetic ablation) affects transport along microtubules but the exact mechanisms are unknown. The effect can be caused either by deacetylation microtubules or direct interaction with molecular motors. This thesis is focused on characterization of interactions between kinesin-1 and HDAC6 that have not been described so far. To this end, we expressed and purified various constructs of kinesin-1 and HDAC6 and tested their interactions by microscale thermophoresis (MST) and hydrogen deuterium exchange (HDX) to determine affinity and interaction sites, respectively. MST data revealed that...

L'acétylation des lysines est une modification post-traductionnelle des protéines dont l’ajout et l’élimination sont catalysés respectivement par les histones acétyltransférases (HAT) et les désacétylases d'histones (HDAC). Cette modification joue un rôle majeur dans la régulation de processus cellulaires tels que l'expression génique, la mobilité cellulaire et le métabolisme. Il est maintenant bien établi qu'une altération de l'activité des désacétylases, entrainant ainsi une dérégulation de l'acétylome, est associée au développement tumoral. Par conséquent, les HDAC sont considérées comme des cibles prometteuses en thérapie anti-cancéreuse ce qui a conduit au développement de nombreux inhibiteurs de HDAC. Cependant, la recherche de nouvelles molécules avec un potentiel anti-cancéreux accru et moins d’effets secondaires est indispensable. Nous avons identifié cinq acides 4-hydroxybenzoïques comme nouveaux inhibiteurs de HDAC, trois inhibiteurs qui ciblent plusieurs HDAC et deux inhibiteurs spécifiques de HDAC6. Les inhibiteurs qui ciblent plusieurs HDAC induisent l'acétylation de certaines lysines des histones H3 et H4 dans les cellules de leucémie myéloïde chronique humaine K-562. Le traitement des cellules induit un arrêt de la progression du cycle cellulaire associé à la modulation de l'expression des cyclines et l'activation de la transcription du gène codant p21. Enfin, les trois composés qui inhibent plusieurs HDAC induisent une mort par apoptose qui est confirmée par l'observation du clivage et de l'activation des caspases. Les inhibiteurs spécifiques de HDAC6 induisent une hyperacétylation importante de la tubuline-α corrélée à une condensation des microtubules dans les cellules cancéreuses adhérentes de prostate (cellules PC-3 et LNCaP). Ces composés induisent une mort par apoptose des cellules cancéreuses en suspension K-562 accompagnée du clivage des caspases et de l'activation de la protéine pro-apoptotique BAX. Enfin, les molécules altèrent la fonction de la protéine chaperonne HSP90α observée par une forte diminution de l'expression de ses protéines clientes: Bcr-Abl et le récepteur aux androgènes. Par ailleurs, les cinq composés n'affectent pas la viabilité des cellules saines. L'ensemble de ce travail révèle que les acides 4-hydroxybenzoïques sont des molécules prometteuses pour le développement de nouveaux composés ayant des propriétés anti-tumorales intéressantes / Lysine acetylation is a post-translational modification characterized by addition and removal acetyl group by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. This modification plays a crucial role in multiple cellular processes including gene expression, cell motility and metabolism. It is now well established that disruption of deacetylase activity, leading to a pathological acetylation profile, is associated to cancer development. Consequently, HDACs are considered as promising targets for anticancer therapy, which led to the development of novel HDAC inhibitors. However, discovery and synthesis of new molecules is essential to increase anticancer potential and decrease adverse health effects of already known compounds. We identified five 4-hydroxybenzoic acids as new HDAC inhibitors: three pan-HDAC inhibitors and two HDAC6-specific inhibitors. Pan-HDAC inhibitors induce acetylation of selected lysines within histones H3 and H4 in human chronic myeloid leukemia K-562 cells. Treatment of cells induces cell cycle arrest associated with increased cyclin expression and the transcriptional activation of p21. Finally, these pan-HDAC inhibitors induce apoptotic cell death further confirmed by the cleavage and activation of caspases. HDAC6-specific inhibitors induce hyperacetylation of α-tubulin in correlation with microtubule condensation in adherent prostate cancer cells (PC-3 and LNCaP cells). These compounds induce apoptotic cell death in K-562 cells accompanied by caspase cleavage and the activation of the pro-apoptotic protein BAX. Furthermore, these molecules alter the chaperon function of HSP90α, which is observed through the robust decrease of the expression of its client proteins (i.e. Bcr-Abl and androgen receptor). Noteworthy, the five compounds did not affect healthy cell viability. Taken together these results revealed that 4-hydroxybenzoic acids are attractive molecules for the development of new compounds with promising anticancer properties

Congenital heart disease is the most common congenital anomaly, affecting approximately 1% of all live births each year. Although clinical interventions are improving, many affected infants do not survive to adulthood. Congenital cardiac defects originate from disturbances during development, making the study of mammalian cardiogenesis critical to improving outcomes for infants with congenital heart disease. Development of the mammalian heart involves epigenetically-driven specification and commitment of a diverse landscape of cardiac progenitors. Recent studies determined that chromatin modifying enzymes play a previously underappreciated role in the pathogenesis of congenital heart defects. This thesis investigates the functions of Hdac1 and Hdac2, highly homologous Class I histone deacetylases, during early murine cardiac development. We establish that Hdac1 and Hdac2 cooperatively regulate cardiogenesis in distinct cardiac progenitor populations during development. Together, our findings demonstrate that Hdac1 and Hdac2 are critical mediators of the earliest stages of mammalian cardiogenesis through a variety of spatiotemporally specific, redundant, and dose-sensitive roles and indicate they may play important roles in the pathogenesis of human congenital cardiac defects.

Tang Angie. / Thesis submitted in: November 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.vi / Table of Contents --- p.vii / List of Publications --- p.xi / Awards --- p.xii / List of Abbreviations --- p.xiii / List of Tables --- p.xv / List of Figures --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Gastric cancer-overview --- p.3 / Chapter 2.1.1 --- Epidemology --- p.3 / Chapter 2.1.2 --- Pathology --- p.3 / Chapter 2.1.3 --- Etiologies and Risk Factors --- p.4 / Chapter I. --- Environmental factors --- p.4 / Chapter a. --- Helicobacter pylori infections --- p.4 / Chapter b. --- Epstein-Barr virus (EBV) --- p.6 / Chapter c. --- Dietary factors --- p.6 / Chapter d. --- Smoking --- p.6 / Chapter II. --- Genetic Factors --- p.7 / Chapter a. --- Hereditary Gastric Cancer --- p.7 / Chapter b. --- Genetic polymorphism --- p.8 / Chapter III. --- Cyclooxygenases (COX) enzymes --- p.10 / Chapter IV. --- Molecular carcinogenesis --- p.11 / Chapter a. --- Activation of proto-oncogenes --- p.11 / Chapter b. --- Candidate tumor suppressor genes --- p.12 / Chapter 1. --- Gene mutation and deletion --- p.12 / Chapter 2. --- Epigenetic Silencing --- p.13 / Chapter 2.2 --- Epigenetics --- p.14 / Chapter 2.2.1 --- DNA methylation --- p.15 / Chapter 2.2.2 --- Histone modification --- p.28 / Chapter I. --- Histone acetylation and deacetylation --- p.32 / Chapter II. --- Histone methylation --- p.32 / Chapter III. --- Histone phosphorylation --- p.34 / Chapter IV. --- Histone ubiquitylation --- p.34 / Chapter 2.3 --- "HAT, HDAC and HDAC inhibitors" --- p.36 / Chapter 2.3.1 --- HAT --- p.38 / Chapter 2.3.2 --- HDAC --- p.39 / Chapter (a) --- Class I --- p.40 / Chapter (b) --- Class II --- p.41 / Chapter (c) --- Class III --- p.42 / Chapter (d) --- Mammalian HDAC and their mechanism of deacetylation --- p.44 / Chapter 2.3.3 --- HDAC inhibitors --- p.45 / Chapter I. --- Class I/II natural inhibitors --- p.47 / Chapter II. --- Class I/II synthetic inhibitors --- p.48 / Chapter III. --- Sirtuins inhibitors --- p.49 / Chapter IV. --- Activity of HDAC inhibitors in vitro --- p.50 / Chapter a. --- Effect in the gene expression --- p.50 / Chapter b. --- Non-transcriptional effects --- p.55 / Chapter c. --- Activity of HDAC inhibitors with other agents --- p.57 / Chapter d. --- Effects in xenograft tumor models --- p.57 / Chapter V. --- Clinical trials of HDAC inhibitors --- p.59 / Chapter Chapter 3 --- Aims of the study --- p.63 / Chapter Chapter 4 --- Materials and Methods --- p.64 / Chapter 4.1 --- Cell culture --- p.64 / Chapter 4.2 --- Drug treatment --- p.64 / Chapter 4.2.1 --- Suberoylanilide Hydroxamic Acid treatment --- p.64 / Chapter 4.2.2 --- Trichostatin A treatment --- p.65 / Chapter 4.3 --- Cell proliferation assay --- p.66 / Chapter 4.4 --- Apoptotic assay --- p.67 / Chapter 4.5 --- Flow cytometry --- p.67 / Chapter 4.5.1 --- Cell preparation --- p.67 / Chapter 4.5.2 --- Propidium Iodide staining --- p.68 / Chapter 4.5.3 --- Annexin V-FITC staining --- p.68 / Chapter 4.5.4 --- Flow cytometer analysis --- p.69 / Chapter 4.6 --- Total RNA extraction --- p.70 / Chapter 4.7 --- DNA extraction --- p.71 / Chapter 4.8 --- Protein extraction --- p.72 / Chapter 4.9 --- Western blottng --- p.72 / Chapter 4.10 --- Microarray analysis --- p.74 / Chapter 4.10.1 --- Sample preparation for microarray --- p.74 / Chapter 4.10.2 --- Hybridization --- p.75 / Chapter 4.10.3 --- Scanning and data processing --- p.75 / Chapter 4.10.4 --- Data analysis --- p.76 / Chapter 4.11 --- Primer design --- p.77 / Chapter 4.12 --- RT-PCR --- p.77 / Chapter 4.12.1 --- Reverse transcription --- p.77 / Chapter 4.12.2 --- Quantitative RT-PCR --- p.78 / Chapter 4.13 --- Methlyation study --- p.79 / Chapter 4.13.1 --- Demethylation by 5-aza-2'deoxycytidine --- p.79 / Chapter 4.13.2 --- Bisulfite modification --- p.79 / Chapter 4.13.3 --- Methylation-specific PCR (MSP) --- p.79 / Chapter Chapter 5 --- Results --- p.81 / Chapter 5.1 --- Morphological changes in AGS cells --- p.81 / Chapter 5.2 --- Anti-cancer effects of HDAC inhibitors --- p.81 / Chapter 5.2.1 --- Effect of HDAC inhibitors on cell growth --- p.81 / Chapter a. --- SAHA inhibits cell proliferation --- p.82 / Chapter b. --- TSA inhibits cell proliferation --- p.82 / Chapter 5.2.2 --- Cell cycle analysis --- p.87 / Chapter a. --- Effect of SAHA on cell cycle --- p.87 / Chapter b. --- Effect of TSA on cell cycle --- p.88 / Chapter 5.2.3 --- Induction of apoptosis on AGS cells --- p.92 / Chapter a. --- SAHA induces apoptotic cell death --- p.92 / Chapter b. --- TSA induces apoptotic cell death --- p.94 / Chapter 5.3 --- Induction of histone expression on AGS cells --- p.102 / Chapter 5.3.1 --- HDAC inhibitors induced acetylation of histone H3 --- p.102 / Chapter 5.3.2 --- HDAC inhibitors induced acetylation of histone H4 --- p.103 / Chapter 5.4 --- SAHA- and TSA-induced gene expression profiles --- p.106 / Chapter 5.5 --- Verification of gene expression by quantitative RT-PCR --- p.108 / Chapter 5.6 --- Methylation study --- p.113 / Chapter Chapter 6 --- Discussion --- p.116 / Chapter 6.1 --- Improved treatment strategy is needed for gastric cancer. --- p.116 / Chapter 6.2 --- HDAC inhibitors as potential anti-cancer agents --- p.117 / Chapter 6.3 --- Potential anti-cancer effect of TSA and SAHA on AGS cells --- p.120 / Chapter I. --- Morphological changes of AGS gastric cancer cells --- p.120 / Chapter II. --- Inhibition of cell proliferation --- p.120 / Chapter III. --- Induction of cell cycle arrest --- p.121 / Chapter IV. --- Induction of apoptosis --- p.122 / Chapter 6.4 --- Expression of acetylated histones upon treatment with TSA and SAHA --- p.124 / Chapter 6.5 --- Identify potential target genes upon treatment with TSA and SAHA --- p.125 / Chapter 6.5.1 --- Candidate genes involved in cell cycle --- p.126 / Chapter a. --- P21WAF1 --- p.126 / Chapter b. --- p27kip1. --- p.128 / Chapter c. --- Cyclin E & Cyclin A --- p.128 / Chapter d. --- Signal-induced proliferation-associated gene 1 (SIPA1) .… --- p.129 / Chapter 6.5.2 --- Candidate genes involved in apoptosis and anti-proliferation --- p.130 / Chapter a. --- BCL2-interacting killer (apoptosis-inducing) (BIK) (Pro-apoptotic gene) --- p.131 / Chapter b. --- Thioredoxin interacting protein (TXNIP) (Proapoptotic gene) / Chapter c. --- Cell death-inducing DFFA-like effector b (CIDEB) (apoptosis induction) --- p.132 / Chapter d. --- B-cell translocation gene 1 (BTG1) - (anti-proliferation) --- p.133 / Chapter e. --- Quiescin 6 (QSCN6) (anti-proliferation) --- p.133 / Chapter f. --- "Cysteine-rich, angiogenic inducer, 61 (CYR61) (anti-proliferative)" --- p.134 / Chapter g. --- Metallothionein 2A (MT2A) (apoptosis induction and anti-proliferative) --- p.134 / Chapter 6.5.3 --- Other genes reported to be up-regulated with HDAC inhibitors treatment --- p.135 / Chapter a. --- Glia maturation factor-gamma (GMFG) --- p.135 / Chapter b. --- v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) / Chapter c. --- Interleukin 8 (IL-8) --- p.136 / Chapter d. --- Insulin-like growth factor binding protein- 2 (IGFBP2) --- p.137 / Chapter e. --- Integrin alpha chain 7 (ITGA7) --- p.138 / Chapter 6.5.4 --- Selected highly up-regulated genes with HDAC inhibitors treatment --- p.139 / Chapter a. --- Aldo-keto reductase family 1，member C3 (AKR1C3) --- p.139 / Chapter b. --- GPI-anchored metastasis-associated protein homolog (C4.4A) --- p.139 / Chapter c. --- "Serine (or cysteine) proteinase inhibitor，clade I (neuroserpin), member 1 (SERPINI1)" --- p.140 / Chapter d. --- "Serine (or cysteine) proteinase inhibitor，clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1)" --- p.140 / Chapter e. --- Adrenomedullin (ADM) --- p.141 / Chapter f. --- Dehydrogenase/reductase (SDR family) member 2 (HEP27) --- p.142 / Chapter g. --- Cholecystokinin (CCK) --- p.142 / Chapter h. --- Silver homolog (mouse) (SILV) --- p.143 / Chapter 6.6 --- Genes regulated by gene promoter hypermethylation in AGS cells --- p.143 / Chapter Chapter 7 --- Conclusion --- p.147 / Chapter Chapter 8 --- Further Studies --- p.150 / References --- p.151 / Appendix I --- p.151 / Appendix II --- p.III / Appendix III --- p.IV / Appendix IV --- p.VI

L’émergence et le développement des maladies métaboliques est sous le contrôle de multiples facteurs génétiques et environnementaux. Le diabète et la résistance à l’insuline sont des maladies métaboliques caractérisées par des défauts dans la sécrétion de l’insuline ou son utilisation périphérique, ou les deux. L’insuline est l’hormone clé de l’utilisation du glucose, et régule également transcriptionnellement et épigénétiquement l’expression des gènes.En travaillant sur le muscle, l’implication de l’épigénétique dans la régulation de l’expression des gènes de la voie de l’insuline a été mis en évidence. L’hexokinase 2 (HK2) est régulée par l’insuline et participe au métabolisme glucidique. Le rôle de l’épigénétique y est démontré avec l’augmentation de l’acétylation des histones autour du site d’initiation de la transcription (SIT) de HK2 et l’accumulation d’une isoforme permissive des histones, H2A.Z. Ces deux phénomènes sont le signe d’une transcription permissive.Nous avons ensuite étudié le rôle de l’acétylation des histones dans les régulations amenées par l’insuline dans les myotubes L6. Nous avons utilisé le butyrate, un inhibiteur des histones deacetylase (HDACi), dans un contexte d’insulino-résistance induite par une lipotoxicité. Le butyrate a en partie restauré la sensibilité à l’insuline visible au niveau des phosphorylations de la PKB (protein kinase B) et de la MAPK (Mitogen-activated protein kinase), inhibées par le traitement au palmitate. Le butyrate a augmenté l’expression de l’ARNm et de la protéine d’IRS1. La surexpression génique d’IRS1 est épigénétique-dépendante car liée à une augmentation de l’acétylation des histones au SIT d’IRS1.L’ensemble de ces résultats démontre l’existence d’un lien entre les modifications épigénétique et l’action de l’insuline. Cela suggère qu’une intervention pharmacologique sur la machinerie épigénétique pourrait être un moyen d’améliorer le métabolisme, et l’insulino-résistance / Diabetes and insulin resistance are metabolic diseases characterized by altered glucose homeostasis due to defects in insulin secretion, insulin action in peripheral organs, or both. Insulin is the key hormone for glucose utilization and regulates gene expression via transcriptional and epigenetic regulations.We determined the epigenetic implications in the regulation of expression of insulin signaling pathway genes. Hexokinase 2 (HK2) is known to be upregulated by insulin and directs glucose into the glycolytic pathway. In L6 myotubes, we demonstrated that insulin-induced HK2 gene expression rely on epigenetic changes on the HK2 gene, including an increase in histone acetylation around the transcriptional start site (TSS) of the gene and an increase in the incorporation of the histone H2A.Z isoform – a histone variant of transcriptionally active chromatin. Both are epigenetic modifications compatible with increased gene expression.To elucidate the role of histone acetylation in the regulation of insulin signaling and insulin-dependent transcriptional responses in L6 myotubes, we investigated the effects of butyrate, an histone deacetylase inhibitor (HDACi), in a model of insulin resistance induced by lipotoxicity. Butyrate partly alleviated palmitate-induced insulin resistance by ameliorating insulin-induced PKB (protein kinase B) and MAPK (Mitogen-activated protein kinase) phosphorylations, downregulated with exposure to palmitate. Butyrate induced an upregulation of IRS1 gene and protein expression. The transcriptional upregulation of IRS1 was proven to be epigenetically regulated, with butyrate promoting increased histone acetylation around the TSS of the IRS1 gene.These results support the idea of the existence of a link between epigenetic modifications and insulin action. Pharmacological targeting of the epigenetic machinery might be a new approach to improve metabolism, especially in the insulin resistant condition.Key words: Muscle, insulin resistance, epigenetic, chromatin, histone acetylation, histone deacetylase inhibitor (HDACi), butyrate, palmitate

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<p>Žučne kiseline deluju kao signalni molekuli u organizmu i uključene su u regulaciju brojnih metaboličkih, inflamatornih i imunomodulatornih procesa. Ova endogena jedinjenja ostvaruju svoje efekte najvećim delom putem nuklearnih receptora. Farnezoid X receptor (FXR) je glavni regulator homeostaze žučnih kiselina, a pokazano je da je značajno uključen i u procese inflamacije i kancerogeneze, prevashodno u jetri i intestinalnom traktu. Aktivacija FXR receptora predstavlja značajnu farmakolo&scaron;ku strategiju za terapiju holestatskih bolesti jetre, inflamatorne bolesti creva i karcinoma kolona. Definisana je uloga žučnih kiselina u signalnim putevima koji reguli&scaron;u ćelijski ciklus i doprinose razvoju ili regresiji maligniteta, ali je malo poznat uticaj ovih jedinjenja na epigenetske mehanizme regulacije ključnih ćelijskih procesa. Imajući u vidu da su efekti žučnih kiselina determinisani njihovom polarno&scaron;ću, cilj istraživanja je bio da se ispita uticaj sintetski dobijenog keto derivata holne kiseline, 12-monoketoholne kiseline (MKH), u komparaciji sa prirodnim žučnim kiselinama, hidrofobnom henodeoksiholnom kiselinom (HDH) i hidrofilnom ursodeoksiholnom kiselinom (UDH), na ćelijske procese apoptoze, oksidativnog stresa i inflamacije, koji su od značaja za hemoprevenciju i terapiju karcinoma kolona, u in vitro i in vivo sistemima. Cilj istraživanja je takođe obuhvatao i ispitivanje uloge odabranih žučnih kiselina u epigenetskoj regulaciji ovih procesa u ćelijama karcinoma kolona. Na in vivo modelu intrahepatične holestaze kod eksperimentalnih životinja, pokazano je da UDH i MKH ispoljavaju antiapoptotski, antioksidativni i antiinflamatorni efekat u jetri i intestinumu. Utvrđeno je da UDH i MKH sprečavaju mitohondrijalni put aktivacije apoptoze u jetri, dok UDH ispoljava antiapoptotski efekat i u intestinumu eksperimentalnih životinja sa holestazom. Ove dve žučne kiseline su u značajnoj meri modulirale ekspresiju gena uključenih u antioksidativnu za&scaron;titu, kao i aktivnost antioksidativnih enzima, u jetri i intestinumu eksperimentalnih životinja sa holestazom, ka nivoima ekspresije i aktivnosti kod zdravih, netretiranih životinja. Dok su UDH i MKH u dozi od 4 mg/kg ispoljile antiinflamatorno dejstvo u jetri i intestinumu smanjenjem ekspresije gena za proinflamatorni transkripcioni faktor NF-&kappa;B, primena HDH i MKH u dozi od 20 mg/kg je imala suprotan efekat. Na modelu HT-29 ćelijske linije adenokarcinoma kolona, utvrđeno je da polusintetska žučna kiselina MKH ispoljava značajno manju citotoksičnost u odnosu na HDH i ne&scaron;to veću citotoksičnost u odnosu na UDH. Epigenetski lek vorinostat je ispoljio sinergističko citotoksično dejstvo sa sve tri ispitivane žučne kiseline. Vorinostat je ostvario proapoptotski i antiproliferativni efekat u HT-29 ćelijama, koji je bio najizraženiji u kombinaciji sa MKH, s obzirom da je do&scaron;lo do značajnog povećanja odnosa ekspresije BAX i BCL2 gena i smanjenja ekspresije gena za marker proliferacije ciklin D1. Vorinostat je, takođe, značajno smanjio antioksidativni kapacitet HT-29 ćelija smanjenjem ekspresije NRF2 gena i sledstvenim smanjenjem ekspresije gena za antioksidativne enzime. HDH je dodatno smanjila, a MKH pobolj&scaron;ala antioksidativni kapacitet HT-29 ćelija modulacijom ekspresije NRF2 gena. U in vitro i in vivo sistemu u okviru ove doktorske disertacije je pokazano da, pored HDH kao poznatog endogenog agoniste FXR receptora, MKH takođe povećava ekspresiju gena za FXR i njegovog ciljnog gena za transkripcioni korepresor SHP, &scaron;to ukazuje da ova polusintetska žučna kiselina može da aktivira FXR. Osim toga, utvrđeno je da žučne kiseline ispoljavaju različite efekte prema ekspresiji gena za histon deacetilaze HDAC1 i HDAC2 u jetri i intestinumu eksperimentalnih životinja, kao i u HT-29 ćelijama karcinoma kolona, a jedino je UDH značajno smanjila ekspresiju gena za oba ispitivana enzima uključena u epigenetsku regulaciju ćelijskih procesa, i u isptivanim tkivima i HT-29 ćelijama. Rezultati na&scaron;eg rada ukazuju da bi se UDH i MKH mogle koristiti u hemoprevenciji karcinoma kolona u niskim dozama, s obzirom na utvrđene efekte u modulaciji ekspresije gena uključenih u procese apoptoze, oksidativnog stresa i inflamacije. Takođe, s obzirom na ostvaren sinergistički efekat žučnih kiselina sa epigenetskim antitumorskim agensom vorinostatom, otvara se mogućnost kombinovane farmakolo&scaron;ke strategije u terapiji solidnih tumora, koji u najvećem procentu pokazuju rezistenciju prema samom vorinostatu.</p> / <p>Bile acids act as signaling molecules in the organism and they are involved in the regulation of numerous metabolic, inflammatory and immunomodulatory processes. These endogenous compounds exert their effects mostly by binding and activation of nuclear receptors. Farnesoid X receptor (FXR) is the main regulator of bile acid homeostasis, and has been shown to be significantly involved in processes of inflammation and carcinogenesis, primarily in the liver and intestinal tract. Activation of FXR receptor represents a significant pharmacological strategy for the treatment of cholestatic liver disease, inflammatory bowel disease, and colon carcinoma. The role of bile acids in signaling pathways regulating the cell cycle and contributing to the development or regression of malignancies is well determined, but the effects of these compounds on epigenetic mechanisms of key cellular processes regulation is yet to be elucidated. Given that the effects of bile acids are mostly determined by their polarity, the aim of our study was to investigate in vitro and in vivo effects of semi-synthetic keto derivative of cholic acid, 12-monoketocholic acid (MKC), in comparison to natural bile acids, hydrophobic chenodeoxycholic acid (CDC) and hydrophilic ursodeoxycholic acid (UDC), on processes of apoptosis, oxidative stress and inflammation, which are significant for both&nbsp; chemoprevention and therapy of colon cancer. Besides, the aim of our study was to examine the role of selected bile acids in the epigenetic regulation of these processes in colon cancer cells. In in vivo model of intrahepatic cholestasis in experimental animals, it has been demonstrated that UDC and MKC exhibit antiapoptotic, antioxidant, and antiinflammatory effects in the liver and intestine. It was shown that UDC and MKC prevent the mitochondrial pathway of apoptosis activation in the liver, while UDC exhibits an antiapoptotic effect in the intestine of experimental animals with cholestasis as well. These two bile acids significantly modulated the expression of genes involved in antioxidant protection, as well as the activity of antioxidant enzymes, in the liver and intestine of experimental animals with cholestasis, towards levels of expression and activity in healthy, untreated animals. While UDC and MKC at a low dose of 4 mg/kg exhibited an antiinflammatory effect in the liver and intestine by reducing the expression of the gene encoding the proinflammatory transcription factor NF-&kappa;B, the application of CDC and MKC at a high dose of 20 mg/kg exerted the opposite effect. In HT-29 human adenocarcinoma cell line, it has been demonstrated that semi-synthetic bile acid MKC exhibits significantly lower cytotoxicity than CDC and slightly higher cytotoxicity than UDC. The epigenetic drug vorinostat has exhibited a synergistic cytotoxic effect with all three investigated bile acids. Vorinostat exerted proapoptotic and antiproliferative effects in HT-29 cells, which were most pronounced in combination with MKC, as there was a significant increase in the ratio of BAX and BCL2 genes expression and a decrease of the proliferation marker cyclin D1 gene expression. Vorinostat also significantly reduced the antioxidant capacity of HT-29 cells by reducing the expression of NRF2 gene and consequently decreasing the expression of genes encoding antioxidant enzymes. CDC further reduced, while MKC improved the antioxidant capacity of HT-29 cells by modulating the expression of NRF2 gene. In both in vitro and in vivo systems, it was demonstrated that, in addition to CDC as a known endogenous FXR agonist, MKC also increased the expression of the gene encoding FXR, and FXR target gene encoding transcriptional co-repressor SHP as well, indicating that this semi-synthetic bile acid can also activate FXR. Besides, bile acids have been shown to exert distinct effects on the expression of the histone deacetylases HDAC1 and HDAC2 gene in the liver and intestine of experimental animals, and in HT-29 colon cancer cells. Only UDC significantly reduced the expression of the genes for both studied enzymes involved in the epigenetic regulation of cell processes, in both tissues and HT-29 cells. The results of our work indicate that UDC and MKC could be used in chemoprevention of colon cancer at low doses, considering determined effects in the modulation of expression of the genes involved in processes of apoptosis, oxidative stress and inflammation. Furthermore, synergistic effects of bile acids with the epigenetic antitumor agent vorinostat open up the possibility of a combined pharmacological strategy in the treatment of solid tumors, which are at the high percentage resistant to the effects of vorinostat alone.</p>

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