INVESTİGATİON OF THE THERAPEUTİC EFFECTS OF BROMODOMAİN INHİBİTOR PLX51107 AND HDAC INHİBİTOR SAHA EPİGENETİC AGENTS UPON AML CELL LİNE
Project no. 20763 · Status: Kapanmış · 2019
Summary
Acute myeloid leukemia, which is a malignant disease group that progresses with clonal proliferation of cells with increased proliferation rate compared to normal cells and with reduced spontaneous apoptosis and continues with bone marrow invasion of proliferated cells, constitutes 20% of other leukemia types. Cancer initiation and development process the transcriptional expression of cells through epigenetic and genomic changes. Post-translational modifications of nucleosomal histone proteins regulate gene expression and chromatin organization in cancer cells and normal cells, among which the acetylation of N-terminal histone tails represents the key epigenetic marker of euchromatin and active gene transcription. Clinical studies targeting epigenetic "readers", namely bromodomains, for cancer treatment have shown the effectiveness of extraterminal (BET) inhibitors and bromodomain (BRD) in acute myeloid leukemia and other cancer treatments. Bromodomain and extra-terminal (BET) proteins appear as epigenetic readers that use tandem bromodomains (BRD) modules to recognize acetylated lysine tails. Considering the critical role of BET proteins in transcription and malignancy, considerable efforts have been made to develop inhibitors to BET family proteins. In the late 2000s, after the hydrophobic interaction between BRDs and acetyl-lysine was characterized, triazolothienodiazepine compounds that disrupted the interaction between BET proteins and the BRD of acetyl-lysine were synthesized. In this thesis, using the newly discovered structurally different PLX51107 inhibitor, which binds to lysine recognition motifs acetylated in bromodomains of the BRD4 protein, preventing BRD4 from binding to acetylated lysines on histones, and HDAC inhibitor SAHA (vorinostat) alone / combined applications, to evaluate the apoptotic, cytotoxic effects on leukemic cells, and to determine the cellular responses such as determining the expression changes in the mRNA level of selected genes in the carcinogenetic process. Under appropriate conditions, the HL60 and non-leukemia control groups were replicated and proliferation and cytotoxicity analyzes were performed with XTT reagent, and the IC50 dose was determined for P1x51107 and vorinostat. Upon apoptotic effect on cells according to the determined IC50 dose of the active ingredients and the selected target gene expression levels were evaluated by RT-PCR. The resulting data showed that the cytotoxic effect of Plx51107 and Vorinostat on the HL60 cell line was determined, and the IC50 value was; 6.68uM for Plx51107 at 72h; The Vorinostat was calculated to be 4.3uM at 48h. For the control group cell line without leukemia (NCIBL2171), 1.24um at 72h for P1x51107; It was calculated as 2.01um at 48h hour for the Vorinostat. When the cytotoxicity was evaluated with the combined administration of two active ingredients, the combination of the two active ingredients in this cell line was not evaluated since an antagonistic effect was detected between the doses for the NCIBL2171 cell line. When the combination analysis for the HL60 cell line was evaluated, the synergistic effect was found to be 1.5 times the P1x51107 IC50 dose, keeping the vorinostat IC50 dose constant. The apoptotic effects of the active substances on the cells according to the determined IC50 doses were evaluated by Annexin V analysis. According to the results, the apoptotic effects of the active ingredients on the cells were found in the range of approximately 80% -97.4%. According to the results of RT-PCR analysis of selected target genes, 1.23 and 15.8 times decrease was observed in bcl-2 and casp3 genes, respectively. For CDKN2A, one of the cell cycle genes, P1x51107 was 4.85 times 1.5 times the IC50 dose; For combo1, it was determined that there was 3.97 times decrease. Western blot analysis was performed by examining the expression changes of the selected target BRD4 protein on cells, considering the expression changes of the genes. According to the results of the analysis, the decrease in the expression change is also confirmed by the western blot analysis. Finally, with cell cycle analysis, the cell cycle stage of the agents (G1/S/G2) was examined. As a result, Plx51107, a bromodomain inhibitor, showed a cytotoxic effect in the HL60 cell line and caused an apoptotic effect, and the decrease in the expression levels of the selected target genes was as expected. Therefore, it is thought that Plx51107 can be an important treatment approach for other hematological malignancies as well and support the rapid transition to clinical trials.