E-selectin is an endothelial adhesion molecule important in the recruitment of leukocytes to the bone marrow and inflammatory tissues; moreover, it has also been implicated in cancer metastasis and as a critical mediator of chemoresistance. This study investigated the effects triggered by the binding of E-selectin on acute myeloid leukemia (AML) cell lines regarding the activation of significant signaling pathways and their impact on biological functions. We observed that upon treatment with a recombinant E-selectin construct on AML cells, there was an activation of the AKT/NF-κB pathways, which are known to be central regulators of many cellular processes, including survival and proliferation. We found that the E-selectin-mediated activation varied in rate and amplitude among AML cell lines spanning differentiation blockage at different stages. Furthermore, we found that E-selectin binding in HL-60 and KG1-a cells sensitized the cells to daunorubicin (DNR), a chemotherapy drug commonly used to treat AML. The observed chemosensitivity could be linked to the decrease of Aldo Keto Reductases (AKR) protein levels upon E-selectin treatment, which is known to metabolize DNR and reduce its cytotoxicity. Additionally, we explored the role of exosomes as a regulator of the generation of therapy resistance by examining the effects treatment with KG1-a derived exosomes, a cell line that exhibits higher chemoresistance compared to other AML cell lines, had on viability in HL-60 cells upon chemotherapy. We found that even in the absence of KG1-a cells, exosomes were sufficient to provide an increase in chemoresistance. Overall, these studies explore properties exerted by AML cells that could lead to further understanding of AML and thus the development of potential therapeutic targets to overcome challenges currently found in the treatment of this disease.

The aims of this thesis were to examine: (1) plasma and cellular pharmacokinetics of daunorubicin and its major metabolite daunorubicinol in patients with acute leukaemia, and the relationships between pharmacokinetics, patient response and the presence of P glycoprotein; (2) actions of the multidrug resistance reversing agents cyclosporin A and trifluoperazine, at clinically achievable concentrations, on daunorubicin accumulation and retention in human leukaemia cell lines and patients with acute leukaemia; and (3) effect of daunorubicin on the cell membrane of both sensitive and resistant cell lines, with and without the multidrug resistance reversing agents. Twenty-seven patients with acute leukaemia received daunorubicin as part of induction therapy. The plasma and cellular levels of daunorubicin and its metabolite daunorubicinol were determined using HPLC. There were no significant differences between patients who went into complete remission (12 out of 23) compared to those who did not respond for any of the plasma pharmacokinetic parameters. There was a significant difference in the cellular daunorubicin and daunorubicinol area under the concentration-time curve between responders and non responders (p less than 0.02), as well as in cellular Cmax, cellular clearance and cellular volume of distribution. Eleven patients were P glycoprotein positive and 10 P glycoprotein negative (no sample available for 2 patients). There was no correlation between patient response and the presence of P glycoprotein; nor a correlation between the cellular concentration of daunorubicin or daunorubicinol and P glycoprotein. Patients responding to chemotherapy had higher cellular daunorubicin and daunorubicinol compared to non responders. In contrast to in vitro studies, overexpression of P glycoprotein was not the reason for the lower cellular daunorubicin levels. Cyclosporin A was capable of increasing both cellular accumulation and retention in the drug resistant CEM/VLB and HL 60/ADR cell lines, but not in the drug sensitive CEM and HL 60 cell lines. Trifluoperazine had no effect in any of the four cell lines. In contrast to the cell line findings, only the combination of cyclosporin A and trifluoperazine were able to increase both accumulation and retention in the blast cells of patients at initial presentation. The multidrug resistant reversing agents alone had no effect in increasing accumulation or retention in the blast cells of P glycoprotein positive patients, nor patients in relapse. The cell line studies show that at clinically relevant concentrations only cyclosporin A is capable of increasing daunorubicin accumulation in both the drug resistant P glycoprotein positive (VLB) and P glycoprotein negative (ADR) cell lines. Thus, cyclosporin A does not work only by inhibiting the actions of P glycoprotein. Trifluoperazine was unable to reverse drug resistance at clinically relevant concentrations in either cell lines or patient blast cells. However, the combination of cyclosporin A and trifluoperazine increased accumulation in patient blast cells at initial presentation, suggesting that these agents may be more useful in patients at initial presentation than relapse. Daunorubicin was immobilised by linking it to poly vinyl alcohol and the effect of immobilised-daunorubicin was studied on the four cell lines above. The immobilised-daunorubicin was able to decrease cell growth in the drug sensitive HL 60 cell line but not in the drug resistant VLB or ADR cell lines. Poly vinyl alcohol itself was cytotoxic to the CEM cell line. The multidrug resistance reversing agents cyclosporin A and trifluoperazine were only capable of increasing cytotoxicity in the HL 60 cell line, with no effect in the drug resistant VLB or ADR cell lines.

The aims of this thesis were to examine: (1) plasma and cellular pharmacokinetics of daunorubicin and its major metabolite daunorubicinol in patients with acute leukaemia, and the relationships between pharmacokinetics, patient response and the presence of P glycoprotein; (2) actions of the multidrug resistance reversing agents cyclosporin A and trifluoperazine, at clinically achievable concentrations, on daunorubicin accumulation and retention in human leukaemia cell lines and patients with acute leukaemia; and (3) effect of daunorubicin on the cell membrane of both sensitive and resistant cell lines, with and without the multidrug resistance reversing agents. Twenty-seven patients with acute leukaemia received daunorubicin as part of induction therapy. The plasma and cellular levels of daunorubicin and its metabolite daunorubicinol were determined using HPLC. There were no significant differences between patients who went into complete remission (12 out of 23) compared to those who did not respond for any of the plasma pharmacokinetic parameters. There was a significant difference in the cellular daunorubicin and daunorubicinol area under the concentration-time curve between responders and non responders (p less than 0.02), as well as in cellular Cmax, cellular clearance and cellular volume of distribution. Eleven patients were P glycoprotein positive and 10 P glycoprotein negative (no sample available for 2 patients). There was no correlation between patient response and the presence of P glycoprotein; nor a correlation between the cellular concentration of daunorubicin or daunorubicinol and P glycoprotein. Patients responding to chemotherapy had higher cellular daunorubicin and daunorubicinol compared to non responders. In contrast to in vitro studies, overexpression of P glycoprotein was not the reason for the lower cellular daunorubicin levels. Cyclosporin A was capable of increasing both cellular accumulation and retention in the drug resistant CEM/VLB and HL 60/ADR cell lines, but not in the drug sensitive CEM and HL 60 cell lines. Trifluoperazine had no effect in any of the four cell lines. In contrast to the cell line findings, only the combination of cyclosporin A and trifluoperazine were able to increase both accumulation and retention in the blast cells of patients at initial presentation. The multidrug resistant reversing agents alone had no effect in increasing accumulation or retention in the blast cells of P glycoprotein positive patients, nor patients in relapse. The cell line studies show that at clinically relevant concentrations only cyclosporin A is capable of increasing daunorubicin accumulation in both the drug resistant P glycoprotein positive (VLB) and P glycoprotein negative (ADR) cell lines. Thus, cyclosporin A does not work only by inhibiting the actions of P glycoprotein. Trifluoperazine was unable to reverse drug resistance at clinically relevant concentrations in either cell lines or patient blast cells. However, the combination of cyclosporin A and trifluoperazine increased accumulation in patient blast cells at initial presentation, suggesting that these agents may be more useful in patients at initial presentation than relapse. Daunorubicin was immobilised by linking it to poly vinyl alcohol and the effect of immobilised-daunorubicin was studied on the four cell lines above. The immobilised-daunorubicin was able to decrease cell growth in the drug sensitive HL 60 cell line but not in the drug resistant VLB or ADR cell lines. Poly vinyl alcohol itself was cytotoxic to the CEM cell line. The multidrug resistance reversing agents cyclosporin A and trifluoperazine were only capable of increasing cytotoxicity in the HL 60 cell line, with no effect in the drug resistant VLB or ADR cell lines.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Andrea Homerová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Interaction of natural substances with human aldo-keto reductase 7A2 and other important carbonyl reducing enzymes Reduction of carbonyl group is one of the phase I metabolism reactions, which is responsible for production of more polar metabolites, enables conjugation in process of biotransformation, excretion of the molecule and it also causes decrease in reactivity and biological activity of the molecule. Endogenous as well as exogenous compounds undergo this reaction and carbonyl reducing enzymes are the ones which possess this reducing activity. Based on the structure, we can divide the enzymes into several groups: short-chain dehydrogenases/reductases, medium-chain dehydrogenases/reductases, aldo-keto reductases and quinone reductases. Inhibition of carbonyl reducing enzymes appears to be a promising aim of research. It is important to take into consideration that by inhibiting carbonyl reducing enzymes it is possible to reduce production of less active, but more toxic metabolites, for example in anthracycline chemoteraputics daunorubicin and doxorubicin and that can lead to change in therapy of cancer. This study...

名古屋大学博士学位論文 学位の種類:博士(医療技術学) (課程) 学位授与年月日:平成23年3月25日

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Alžbeta Tomanová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Effect of selected tyrosine kinase inhibitors on the activity of human carbonyl reducing enzymes Key words: tyrosin kinases, screening, carbonyl, daunorubicin, inhibition Tyrosine kinases are a subclass of protein kinases, catalysing the transfer of ATP phosphate residue to a protein, thereby playing an important role in cellular signaling. Abnormal tyrosine kinase activity is present in various malignancies. In certain cases, inhibition of their function can prevent tumor cell proliferation and eventually induce apoptosis. At the same time, some tyrosine kinase inhibitors have demonstrated the ability to inhibit efflux transporters, which are often involved in the development of resistance to anticancer treatment. In this diploma thesis, the inhibitory effect of imatinib, nilotinib, dasatinib and acalabrutinib (tyrosine kinase inhibitors) has been studied on carbonyl reducing enzymes, whose overexpression by tumor cells may lead to resistance to chemotherapy. In particular, in the case of anthracyclines, the reduction of carbonyl group on C-13 results in not only lower cytotoxic activity, but also increased...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Lakomá Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: The effect of alisertib and brigatinib on the activity of selected human carbonyl reducing enzymes Key words: brigatinib, alisertib, daunorubicin, inhibition, carbonyl-reducing enzymes Protein kinases are enzymes, whose main function is based on a transfer of phosphate group from ATP to protein substrate. This common posttranslational modification is involved in the regulation of intracellular processes and cell signaling. Altered expression of protein kinases is often coupled with a development of cancer. Inhibition of protein kinases may prevent cancer cell proliferation and induce their cell death. The main aim of the diploma thesis was to measure inhibition potential of protein kinase inhibitors, alisertib and brigatinib, against carbonyl-reducing enzymes. Overexpression of carbonyl-reducing enzymes in cancer cells may cause resistance to drugs followed by failure of chemotherapeutic therapy. In case of antracyclin chemotherapeutic daunorubicin, carbonyl-reducing enzymes reduce the carbonyl in C-13 giving rise a primary metabolite daunorubicinol, which has lower cytotoxic effect but higher cardiotoxicity. The effort to...

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser. På omsl. felaktigt " ... daunorobicin ..."

This thesis addresses the synthesis of organic compounds, some of them are derivatives of compounds with DNA binding properties, for potential use in targeted nuclide therapy. The compounds synthesized therefore also need to contain potent nuclides. Here the nuclides considered are the radionuclide 125I, and the stable isotope 10B, which becomes radioactive upon neutron activation. 125I is an Auger-electron emitter, which emits particles that can travel only about 1-2 µm through human tissue and hence has to be delivered to the cancer cell nucleus to cause DNA damage. Neutron activated 10B emits highly cell killing α-particles and 7Li3+ ions, the application of which in Boron Nuclide Capture Therapy (BNCT) has proven very promising. The thesis can be divided into three parts: i) A nido-carborate, 7-(3´-ammoniopropyl)-7,8-dicarba-nido-undecaborate(-1), has been synthesized and radioiodinated for use as a pendant group for attachment of 125I to tumor-seeking macromolecules. Radiolabeling was achieved in greater than 95% yield. ii) Both enantiomers of m-carboranylalanine, a carborane analogue of phenylalanine, have been prepared in high enantiomeric excess, and are of potential interest in BNCT. The synthesis involved amination of the N-acyl derivative formed from [3-(1,7-dicarba-closo-dodecarborane(12)-1-yl)-2-propanoic acid and Oppolzer’s camphor sultam. iii) Derivatives of the DNA intercalating compounds 9-aminoacridine, daunorubicin and doxorubicin have been synthesized and labeled with 125I. The 9-aminoacridines were synthesised with a variety of functional groups such as carboxyl, amino and hydroxyl. The anthracylines daunorubicin and doxorubicin are efficient chemotherapeutic agents; the synthesis routes of ester, amide and amine derivatives of these compounds are presented. The Chloramine T method was used for the radioiodinations, and the radioiodination precursors of both the acridine and the anthracycline derivatives, were made to contain either a trimethylstannyl group or a phenolic substituent. In the former case the trimethylstannyl group was replaced by 125I, and in the latter case, the compounds were radiolabeled directly at the o- position to the phenolic hydroxyl group. Both methods gave high radiolabeling yields.