The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...

ABSTRACT Cytochrome b5 (CYB5) is heme protein capable of reduction of cytochromes P450 (CYP) or some other enzymes. However, his regulative capability was also observed by his apo form, i.e. in absence of heme prosthetic group in the active center. CYB5 can accept electron from cytochrome b5 reductase (CYB5R) or from cytochrome P450 reductase (CYPOR). CYPOR by itself is reduced by NADPH and is also able to forward electron to CYP independently of CYB5. CYB5R on the other hand is reduced by NADH. Efficiency of CYB5 to accept and forward an electron was studied in vitro with five different substrates - testosterone, Sudan I, aristolochic acid I (AAI), ellipticine and vandetanib. These substrates were chosen considering their characteristic reactions, which are catalyzed by their respective isoforms of CYP. The experiments with these substrates were carried out in the medium with recombinant CYPs prepared in insect cells or E. coli or in the medium with hepatic microsomes isolated from different organisms. Rats, from which the majority of these microsomes was isolated, were premedicated by different CYP inducers. The experiments were carried out in medium with NADH or NADPH in order to assess the capability of CYB5 to reduce CYP independently of CYPOR. The capability of CYB5 and CYB5R to act as a...