| Popis: |
Current agents used for pharmacological reactivation of foetal haemoglobin (HbF) have limited application due to moderate therapeutic properties, variable patient response and potential cytotoxic effects. Therefore, identification of novel HbF inducing agents is still a major research goal to this day. Identification of new potential HbF inducers has been mainly based on screening of drug libraries. However, this approach has not been very successful in generating new promising agents. In the current project, I employed two approaches for identifying potential HbF inducers: 1) screening of agents that are structurally similar to compounds with known HbF inducing activity; 2) investigating molecular pathways of a known HbF inducer with the aim of identifying suitable targets for therapeutic manipulation and target-based drug design. The first approach involved screening of eleven xanthines including caffeine and nine hydroxystilbenic derivatives of resveratrol as potential HbF inducers. However, none of the agents had a potent enough HbF inducing activity in order to be considered as promising therapeutic agents. In the second approach, decitabine was chosen based on its high HbF inducing activity and moderate cytotoxicity in K562 cells and primary human erythroid cultures. Chromatin immunoprecipitation was used to characterise epigenetic changes in the β-globin gene locus, and quantitative real-time PCR for investigation of changes in gene expression levels of ten erythroid-related genes, in the presence of the agent. A quantitative iTRAQ proteomic approach coupled with mass spectrometry was used for identification of changes in the proteome of decitabine-treated and un-treated primary human erythroid cultures. The findings suggest that decitabine induces HbF production through activation of signal transduction pathways rather than through hypomethylation of gene promoters. One such possible pathway is the NF-κB pathway. Among the differentially expressed proteins, twenty-seven proteins were associated with the action of decitabine. Two of those proteins, ARHGAP4 and EGLN2, were previously implicated in hydroxyurea-mediated induction of γ-globin gene expression and hypoxia-mediated erythropoiesis, respectively. In addition, the de-ubiquitinating enzyme USP11 was substantially modulated in the presence of decitabine. The exact role of these proteins in γ-globin expression remains to be established. |
