Gold-based anticancer derivatives of oligopeptides: synthesis, characterization, delivery strategies and biological activity
Year: 2014
Type: dissertation
Abstract: At present, cisplatin (cis-diamminodichloroplatinum(II)) is one of the most largely employed anticancer drugs, used especially for the treatment of testicular cancer and, in combination with other drugs, of ovarian, small cell lung, bladder, brain, and breast tumors. Anyway, despite its high effectiveness, it exhibits severe drawbacks, as normal tissue toxicity (in particular, nephrotoxicity) and intrinsic or acquired resistance to the treatment. To overcome these problems, new Pt- and other metal-based complexes were designed and tested as anticancer drugs. In particular, since gold-based complexes were linked for centuries to the treatment of rheumatoid arthritis (due to their antiinflammatory and immunosuppressive properties), they were also tested for their antiproliferative properties.. In the last decade in our research group, some Au(III)-dithiocarbamato derivatives were synthesized, characterized and tested, showing outstanding antiproliferative activity on human cancer cells. Starting from the promising results obtained both in vitro and in vivo for the “first generation" compounds of formula [AuIIIX2(dtc-L)] (X= Cl, Br; dtc=dithiocarbamato; L= dimethylamine, pyrrolidine, N-methyl glycine (Sar) esters), a "second generation" of complexes was designed to improve bioavailability, selectivity and cellular uptake. In particular, the dithiocarbamato moiety was functionalized with different oligopeptides of general formula [AuIIIX2(dtc-Sar/Aib-(AA)n)] (AA= different aminoacids), to be recognized and uptaken by specific peptide transporters called PEPTs (PEPT1 and PEPT2), predominantly present in epithelial cells of small intestine, mammary glands, lung, choroid plexus and kidney, and overexpressed in some tumor types. A unique feature of these transporters is their capability for sequence-independent transport of most possible di- and tripeptides, thus representing excellent targets for the delivery of pharmacologically-active peptidomimetics such as ß-lactam antibiotics and some angiotensin converting enzyme (ACE) inhibitors. Interestingly, these complexes demonstrate high capability to strongly inhibit tumor cells growth by exploiting a mechanism of action different from the clinically-established platinum drugs, overcoming drawbacks without any observed cross-resistance. In this PhD thesis work, new Au(III)-peptidodithiocarbamato complexes were synthesized and fully characterized, mainly by means of elemental and X-ray analysis, mono- and bidimensional NMR, FT-IR and UV-Vis spectroscopy. The water-insoluble compounds, previously dissolved in DMSO, showed high reactivity in aqueous conditions, generally undergoing hydrolysis of the bromido ligands which leads to the formation of the water insoluble dihydroxo derivative. Nevertheless, when analyzing more complex environments, as cell culture medium, a stabilizing effect seemed to be exerted by the components of the medium itself (e.g., proteins, organic molecules), as confirmed by interaction studies with the serum carrier protein BSA (Bovine Serum Albumin). The antiproliferative activity of the complexes was tested on different human cancer cell lines, representative of different cancer types (e.g. lung, ovary, breast), showing promising cytotoxicproperties. Furthermore, their antitumor activity was confirmed in vivo on breast MDA-MB-231 human xenografts, while their toxicological profile, preliminary evaluated ex vivo, was similar to the "first generation" compounds. To overcome the solubility problem in aqueous conditions and to avoid the intrinsic toxicity of DMSO (normally used as primary solvent), new solubilizing methods were explored. In particular, micelles based on Pluronic F127 and liposomes based on DPPC phospholipids were prepared, characterized and tested in vitro. These systems were proved to stabilize the compounds in physiological conditions, without affecting their antiproliferative activities. Moreover, micelles functionalized with CCK8 octapeptide were tested as targeting moieties to be selectively delivered to cancerous cells overexpressing the CCK-receptors. In this case, the GI50 values of the encapsulated compounds were improved of one order of magnitude. To achieve more information about their mechanism of action, the interaction in vitro with some purified enzymes was performed, confirming that the complexes inhibit all the three enzymatic functions of the proteasome, together with the activity of PARP-1 enzyme. More detailed studies, taking into account the kinetics of gold uptake by cells and the real-time inhibition of cell growth and adhesion, suggested that the complexes probably exert their cytotoxic effect on cells in the 4-5 h after administration, acting probably at the membrane level. All together these results highlight the promising properties of the complexes as anticancer agents, and put the basis for the pursuance of the preclinical characterization, including deeper studies of their pharmacological properties and biological activity. I wish to aknowledge my supervisor Prof. Dolores Fregona and all her Research Group. A particular thanks goes to Prof. Dalla Via, Prof. Formaggio, Prof. Di Noto. I want also to acknowledge Prof. Angela Casini for the ospitality in her Research Group at the Research Institute of Pharmacy of the University of Groningen (The Netherlands). A special thank goes to Fondazione CARIPARO, for funding this PhD project. Finally, I want to thank the Doctoral School of Molecular Sciences, its Coordinator Prof. Antonino Polimeno and the members of my three-year committee Prof. Formaggio, Prof. Badocco and Prof. Zagotto.
Author: Giulia Boscutti
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W89249882