Effect of cycline-dependent kinase and matrix metalloproteinase inhibitors on hematopoietic and leukemic cells

Rapid advances in molecular and cellular biology have improved the understanding of the mechanisms involved in leukemia development. Cyclin-dependent kinases (CDKs) and matrix metalloproteinases (MMPs) have been suggested as potential therapeutic targets and a number of pharmacologic inhibitors of CDKs and MMPs have been developed. This thesis aimed to increase knowledge about pharmacokinetics and cytotoxic effects of the CDK inhibitor roscovitine and MMP inhibitors from tetracycline analogues group. The effect of roscovitine on hematopoietic progenitors was studied in vitro and in vivo in mouse model. In vitro, bone marrow (BM) was incubated with roscovitine and the clonogeneic capacity of BM cells was assessed using semisolid methylcellulose-based media. Roscovitine decreased colony formation in concentrationand cell typedependent manner, CFU-GEMM were the most sensitive, followed by BFU-E and last CFU-GM. In vivo, only transient decrease in BFU-E was observed after the treatment which probably is due to the low distribution of roscovitine to BM (1.5%) compared to that detected in plasma. Pharmacokinetics and distribution of roscovitine was studied in adult and in 14-days old rats. Pharmacokinetics of roscovitine in plasma and brain fitted a two-compartment model in both adult and pups. Pharmacokinetics was age-dependent with a terminal elimination half-life of 7h in brain and plasma in pups compared to <0.5 h in adult rats. Brain exposure to roscovitine expressed as AUC brain/ AUC plasma was 100% in rat pups and 20% in adult rats. Moreover, significant but transient CDK5 inhibition and Erk1/2 activation were observed in hippocampus, frontal cortex and cerebellum. Cytotoxic effect of roscovitine was studied in three cell lines representing different types of leukemia: the myeloid HL-60, the lymphoblastic Jurkat and the CML K562. Roscovitine decreased viability and proliferation in a concentrationand timedependent manner in all cell lines. Cell morphology induced by roscovitine was consistent with apoptosis. Loss of mitochondrial membrane potential, release of cytochrome c, active fragment of caspase-3 and cleaved PARP were observed in all three cell lines, however, the pattern of activation of caspase-2 and -8 showed different pattern among the cell lines. The cytotoxic effects of tetracycline analogues (TCNAs) doxycycline, minocycline and COL-3 (chemical modified tetracycline-3) were studied in the human leukemia HL-60 cells. TCNAs decreased proliferation and induced apoptosis in concentrationand timedependent. Apoptosis induced by TCNAs was mitochondria-mediated and caspasedependent. COL-3 exerted the strongest anti-proliferative and proapoptotic effect. In conclusion, both roscovitine and TCNAs exerted cytotoxic effect through mitochondria-mediated and caspase-dependent apoptotic cell death in leukemic cells. Roscovitine did not affect hematopoietic progenitors in vivo that might be explained by the low distribution of roscovitine to bone marrow in combination with rapid elimination of the drug. However, age-dependent pharmacokinetics and high distribution of roscovitine to brain in rat pups was observed. This may be implicated in the treatment of pediatric CNS leukemia and brain tumors. LIST OF PUBLICATIONS I. Hairong Song, Marina Vita, Hatem Sallam, Ramin Tehranchi, Christina Nilsson, Ake Siden, Zuzana Hassan. Effect of the CDK-inhibitor roscovitine on mouse hematopoietic progenitors in vivo and in vitro. Cancer chemotherapy pharmacology, 2007,60,841-846. II. Hairong Song, Ake Siden, Zuzana Hassan. Multiple pathways of apoptosis induced by roscovitine in leukemic cell lines in vitro. The Open Pharmacology Journal, 2008, 2, 24-30. III. Hatem Sallam, Patricia Jimenez, Hairong Song, Marina Vita, Angel CedazoMinguez, Moustapha Hassan. Age-dependent pharmacokinetics and effect of roscovitine on Cdk5 and Erk1/2 in the rat brain. Pharmacological Research, 2008 Jul;58(1):32-7. IV. Hairong Song, Kim R. Maguire, Ake Siden, Zuzana Hassan. Cytotoxic effect of tetracyclines (doxycycline, minocycline and COL-3) on acute myeloid leukemia HL-60 cells. Submitted to Leukemia and lymphoma.