Ο ρόλος της CDK5 στην επαγόμενη από πλειοτροπίνη μετανάστευση ενδοθηλιακών κυττάρων και ως στόχος για δράση πυρρολο [2,3-α] καρβαζολικών αναλόγων του ινδολοκαρβαζολικού αρωματικού σκελετού φυσικών προϊόντων στην αγγειογένεση

Abstract

Indolocarbazole alkaloids constitute a group of natural products that have attracted great attention because of their potential therapeutic applications. Ιndolopyrrolocarbazoles are a new class of antitumor drugs, which can be divided into two major groups, depending on their mechanisms of action and structural features: protein kinase inhibitors and DNA-damaging agents. We have previously evaluated the effect of 7 pyrrolo[2,3-a]carbazole analogues on CDK1/cyclinB (Cyclin Dependent Kinase 1, CDK1) activity and found that only compound1e totally inhibited the enzyme in a dose-dependent manner, while all analogues partially or totally inhibited the activity of topoisomerase I in vitro, with compound 1e being the least effective. In this thesis, the effect of all the pyrrolo[2,3-a]carbazole analogues on angiogenesis was investigated, using the in vivo model of the chick embryo chorioallantoic membrane, as well as proliferation and migration of human endothelial cells in vitro. All the analogues had an effect on the proliferation and migration of endothelial cells in vitro and angiogenesis in vivo, but with differences in their effectiveness or potency. We have previously shown that PTN induces migration of endothelial cells through binding to its receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) and ανβ3 integrin. The recent report that ανβ3 expression up-regulates CDK1, which then modulates cell migration, led us to test the effect of the CDK1 inhibitor compound 1e and the other pyrrolo[2,3a]carbazole analogues on the PTN induced migration of human endothelial cells. Only compound 1e inhibited PTN induced migration of human endothelial cells, a result also confirmed in human glioblastoma U87MG cells, which are known to express both RPTPβ/ζ and ανβ3 and migrate in response to PTN. Roscovitine, a synthetic inhibitor of CDKs with selectivity towards CDK1/2 and CDK5, completely attenuated PTN-induced migration of endothelial cells, while the CDK1/2 selective inhibitor NU2058 had no effect, suggesting that inhibition of CDK5 is responsible for inhibition of PTN-induced cell migration. The complete attenuation of PTN-induced migration of endothelial cells following the down-regulation of CDK5 by siRNA further confirmed that CDK5 plays an important role in PTN-induced migration of endothelial cells. PTN increased CDK5 kinase activity with the maximum increase observed within 5 min after stimulation of cells with PTN. This was confirmed by both direct kinase assays, as well as by measuring interaction of CDK5 with its activator protein p35. PTN-induced activation of CDK5 is independent of ανβ3, but depends on RPTPβ/ζ and its downstream activated c-SRC kinase. This is the first time that an interaction between CDK5 and c-SRC is reported in extracts of endothelial cells, as well as the fact that PTN induced CDK5 activation requires c-SRC activation in these cells. Finally, we report no immediate effect of kinase CDK5 on PTN induced activation of ανβ3 integrin and ERK1/2 phosphorylation. Accumulating data favour the notion that CDK5 plays an important role in angiogenesis-related functions of endothelial cells and our data reinforce this observation. The expression of p35 in endothelial cells, the prime regulator of CDK5, is reported here for the first time in other type of cells apart from neuronal. The basic role of CDK5 in several pathologies point out the importance of research and development of compounds that can be effective in inhibiting this kinase, based on the structure of analogue 1e.