N6-ISOPENTENYL ADENOSINE, A PROMISING ANTICANCER AGENT: SYNTHESIS OF NOVEL ANALOGUES, EVALUATION OF ANTIPROLIFERATIVE ACTIVITY AND INSIGHT INTO ACTION MECHANISM
Tesi di Dottorato
Data di Pubblicazione:
2010
Citazione:
N6-ISOPENTENYL ADENOSINE, A PROMISING ANTICANCER AGENT: SYNTHESIS OF NOVEL ANALOGUES, EVALUATION OF ANTIPROLIFERATIVE ACTIVITY AND INSIGHT INTO ACTION MECHANISM / E. Gorincioi ; docente guida: Enzo Santaniello ; coordinatore del dottorato: Francesco Bonomi. Universita' degli Studi di Milano, 2010 Dec 09. 23. ciclo, Anno Accademico 2010. [10.13130/gorincioi-elena_phd2010-12-09].
Abstract:
Isopentenyl adenosine (N6-(∆2-Isopentenyl)adenosine, iPAdo) is an important member of the class of cytokinins, N6-substituted adenosines, endowed with hormonal properties in plants. iPAdo is the only known cytokinin existing in animals. Its bioactivity in mammalians has been investigated and the overall reports confirm promising in vitro antiproliferative activity, less pronounced or absent in vivo. The PhD project was initially aimed to investigate this aspect of iPAdo activity and the preparation of iPAdo-grafted gold nanoparticles (GNPs) was selected as a possible solution, due to the well-recognized, remarkable properties of GNPs, such as high stability and biocompatibility, as well as potentiality of enhanced delivery and intracellular uptake of a bound to them drug into the cancer cells. The approach consisted in the preparation of a 5'-ester of iPAdo with lipoic acid (LA), later reduced to a dithiol form (DHLA), that could bind to gold nanoparticles, thus forming iPAdo-DHLA-GNPs. The feasibility study for an efficient preparation of the ester linkage between the 5'-hydroxyl of iPAdo and LA has been performed on another cytokinin, the commercially available kinetin riboside (KR), structurally related to iPAdo. Initially, a selective formation of the 5'-O-ester linkage of KR has been approached both chemically and enzymatically, preparing a few LA esters. However, in absence of the desired 5'-O-selectivity a more traditional chemical approach was investigated. The protection of KR at the 2' and 3'-hydroxyls as acetonide and esterification of 5'-OH proceeded in good yields (88% from KR). However, hydrolysis of the 2', 3'-protecting group led to concurrent hydrolysis of the β-glicosidic bond and partial removal of LA moiety. Finally, reduction with sodium borohydride in water/methanol (required for the reduction of LA into DHLA prior to grafting to Au particles) partially hydrolyzed LA-ester linkage.
The above-reported negative results were a preliminary rather convincing indication that also LA-esters of iPAdo could not resist to the sequence of chemical reactions necessary for the formation of GNPs. Thus the iPAdo delivery project has been put aside and the efforts were focused towards the synthesis of iPAdo analogues as a mean to overcome the in vivo catabolic pathway. Meanwhile, with the aim of deepening the knowledge of iPAdo bioactivity palette, additional studies on iPAdo antiproliferative activity and on its mechanisms of action were carried out.
Detailed bioactivity studies showed iPAdo dose-dependent cytotoxic effects on MDA-MB-231 and MCF-7 human breast cancer cells and IC50 values of 6.2 and 12.2 μmol/L, respectively, were determined. For both cancer cell lines the assessment of the cell shape and cell morphology of iPAdo treated cells established the loss of adhesion, rounding, cell shrinkage and detachment from the substratum. The interaction of iPAdo with DNA and Bovine Serum Albumin (BSA, a model of the analogous human protein) has been investigated via UV spectroscopy. The value of binding constants for iPAdo–DNA and iPAdo-BSA complexes have been estimated to be KiPAdo–BSA=4.9 x 104 M-1 and KiPAdo–DNA=4.1 x 103 M-1 that are indicative of good iPAdo-protein interaction and suggest affinity of iPAdo for DNA complexation. The dose-dependent cell cycle arrest and apoptogenic effect of iPAdo on MCF-7 cancer cell line has been evaluated, as well. The cell cycle analysis of MCF-7 cells by mean of flow cytometry showed an increase in the amount of sub G1/G0 phase on iPAdo treatment, suggesting that the inhibition of cell growth could be related to a mechanism of apoptosis. However, when the apoptotic activity of iPAdo on MCF-7 and human leukaemia cells (HL-60 cell line) has been studied via caspase-3 or
Tipologia IRIS:
Tesi di dottorato
Keywords:
N6-Isopentenyl adenosine ; N6-Isopentenyl aristeromycin ; antiproliferative activity; apoptotic activity ; MCF-7 human breast cancer cells ; MDA-MB-231 human breast cancer cells ; human leukaemia cells HL-60; docking studies
Elenco autori:
E. Gorincioi
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