The tumours of central nervous system constitute from 2% to 5% of all human cancers, about half of them are the brain glial tumours of different malignancy grades of which glioblastoma, the most malignant glioma, occupies up to 50%. Glioblastoma is known to be highly therapy resistant and has very poor prognosis. The development of new treatment modalities, especially those based on multitargeted therapy, is desperately needed for this disease. The thesis is dedicated to identification and characterization of novel cytotoxic agents, as well as their combination with chemotherapeutics, which can effectively inhibit the viability of human glioma cells. To find out drug combinations that will enable the development of therapeutic regimens with improved effectiveness and less toxicity, several bradykinin antagonists and 4-thiazolidinones were analyzed for their cytotoxic effect using different types of malignant cells. Among all bradykinin antagonists under investigation, BKM-570 appeared to be the most effective with IC50 3,3 µM and 4 µM in human glioblastoma U251 and rat glioma C6 cell lines, correspondingly. A reduction in the amount of phosphorylated forms of ERK1/2 (Tyr202/Tyr204) and AKT1 (Ser473), as well as induction of PARP1 in U251 cells was demonstrated after the incubation with BKM-570. These phenomena could be associated with the mechanisms of cytotoxic activity of this compound. However, most tumours are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. The creation of smart schemes of multitargeted therapy aimed simultaneously at different elements of tumour formation mechanisms should be an effective strategy for cancer treatment. Temozolomide, first-line anti-gliomic drug used in clinics, has only temporary positive effect and severe side effects in glioblastoma patients. IC50 of temozolomide is 220 µM and 1000 µM in U251 and C6 cells. We showed that combination of 1 µM BKM-570 with only 10 µM temozolomide, led to about 80% growth reduction of C6 and U251 cells, compared to temozolomide used alone. Thus, BKM-570 significantly potentiates temozolomide cytotoxicity. Screening of 4-thiazolidinones revealed ID 4523_PC and ID 28_PC to be the potent suppressor of U251 cells growth (IC50 3,2 µM and 15 µM, correspondingly ). ID 4523_PC also demonstrated high activity in C6 cells with IC50 0.13 µM. In contrast to bradykinin antagonists, a combination of 4-thiazolidinones ID 4523_PC and ID 28_PC with temozolomide did not lead to the increased cytotoxic effect in human glioma cells. Using the MTT assay with primary non-malignant mouse fibroblasts, it was found that the cytotoxic activity of compounds BKM-570, BKM-1800 and ID 28_PC, as well as combination of BKM-570 and temozolomide, is lower in non-cancerous cells than in glioma cells. Recombinant proteins with cytotoxic properties are promising agents to be involved in the complex application together with chemotherapy and immunotherapy. We revealed Chitinase 3-like 2 (CHI3L2) protein to be overexpressed in glioblastomas. We obtained CHI3L2 protein using either prokaryotic or eukaryotic cells and demonstrated that CHI3L2 exhibits cytotoxic properties in human glioma U251 cells, as well 293 cells. The ability of CHI3L2 to inhibit the viability of malignant and non-transformed cells lines may suggest that mode of its action is independent of the cell type. Although, CHI3L2 protein inhibits U251 cells viability more effectively than temozolomide in therapeutic concentrations, we did not observe any potentiation of temozolomide activity, while the combination of CHI3L2 with BKM-570 led to the increased cytotoxic effect. We showed that CHI3L2-mediated decrease of cell viability is associated with G1/S transition arrest. To find out which molecular mechanisms could mediate the blockage of cell cycle, we analyzed the impact of CHI3L2 on key components of cell cycle machinery, namely pRb, cyclin D, p53, and p21. CHI3L2 provoked the dramatic reduction of pRB phosphorylation and significant decrease of cyclin D1 expression. Moreover, p53 expression level was substantially increased. Besides accumulation of p53, we demonstrated the upregulation of cyclin-dependent kinase inhibitor p21. So, one may conclude that G1/S cell cycle arrest in CHI3L2 treated cells could be possibly realized via activation of pRB, downregulation of cyclin D, and activation of p53
