Title

 

Authors

C. Travelli, U. Galli, G.C. Tron, P.L. Canonico, A.A. Genazzani

Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche (DiSCAFF), università del piemonte orientale Amedeo Avogadro via bovio 6, 28100 Novara

 

Abstract

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in numerous metabolic pathways, including ATP synthesis. In recent years, it has been demonstrated a number of signalling pathways bring about its consumption (Berger et al. 2004). For example, NAD is a substrate for ribosylation reactions (by ARTs and PARPs), acetylation reactions (by sirtuins), as well as being the precursor of a number of molecules involved in calcium signalling. A number of metabolic pathways leading to the formation of NAD are present in cells. A rescue mechanism exists in cells to re-use nicotinamide released by NAD-metabolizing enzymes, this pathway, known as the “NAD salvage pathway”, is composed of two enzymes: nicotinamide phosphoribosyl transferase (NAMPT) which forms nicotinamide mononucleotide (NMN) from nicotinamide and phosphoribosyl-pyrophosphate and nicotinamide mononucleotide adenylyl transferase (NMNAT) which leads to NAD from NMN and ATP. The link between Nampt and cancer is rapidly strengthening (Galli et al. 2010). First, Nampt has been shown to be involved in angiogenesis and to be up-regulated in a number of solid tumours. Second, an important role in tumorigenesis or tumour treatment has been postulated or proven for a number of NAD-utilizing enzymes. Third and most important, a chemical screen aimed at identifying compounds that affect tumour growth has yielded an inhibitor of NAMPT (named FK866; Hasmann et al. 2003). FK866 has entered clinical trails for a number of malignancies and is now in phase I/II. In this context, we characterized the cytotoxic effect of FK866 and other Nampt inhibitors (synthesized in collaboration with our pharmaceutical chemistry laboratory) in different neuroblastoma and melanoma cell lines and the possible use of these compounds as a chemosensitizing agent in combination with traditional chemotherapeutic agents. In neuroblastoma cells, Nampt inhibitors lead to NAD depletion following by cell death. Apoptosis is not the major cell death engaged by cells, while autophagy seems to be prominent (Billington et al, 2008). In order to characterize the effect of Nampt inhibitors in neuroblastoma cells, we decided to capitalize to action of 3-methyladenine (3-MA, an inhibitor of Class III PI3K which participates in vesicle nucleation) and of chloroquine (which inhibits autophagosome-lysosome fusion). 3-MA protects cell from FK866-induced cell death. On the contrary, chloroquine had a potentiating effect on FK866 cytotoxicity. These data suggest that in upon NAD depletion, autophagy must be initiated to allow cell death to occur, i.e. NAD depletion is not sufficient per se.
Given these observations, we reasoned that Nampt inhibitors might be better employed in combination therapy. To this extent, we performed concentration-response curves with traditional chemotherapeutic agents in the presence or absence of FK866, which per se is devoid of any activity on cell viability to investigate possible synergisms. A striking potentiation was observed with etoposide and cisplatin, with a 300-600 fold shift in the concentration-response curves. Maximal effects are obtained at concentrations that alone are completely inefficacious. The combination yielded a significant drop in both NAD and ATP levels compared to the single agents alone, and also a significant increase in DNA damage foci. These data suggest that etoposide and cisplatin are able to potentiate the cytotoxic effect of FK866.
As FK866 completed phase II clinical trials for melanoma treatment, we decided then to focus our attention on melanoma cells an on human melanoma samples. In human melanoma slices NAMPT is over-expressed compared to normal tissue, suggesting that NAMPT is involved in melanoma tumorigenesis. Data on cytotoxity of Nampt inhibitors in melanoma tumours, possible synergisms with agents used in melanoma therapy and the different expression of NAD synthesizing enzymes in different melanoma cell lines will also be presented.