Revollo JR, Grimm AA, Imai S. The regulation of nicotinamide adenine ... Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot.
[Autophagy 4:3, 385-387; 1 April 2008]; ©2008 Landes Bioscience
Addendum
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NAD depletion by FK866 induces autophagy
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Richard A. Billington,* Armando A. Genazzani, Cristina Travelli and Fabrizio Condorelli DiSCAFF and the DFB Centre; Università del Piemonte Orientale; Novara, Italy
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phase II clinical trials for cancer chemotherapy,9,10 rapidly reduced cellular NAD levels and led to cell death. This cell death, along with the drop in NAD levels, could be prevented by adding NAD to the extracellular medium.
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In SH-SY5Y neuroblastoma cells, FK866 displayed an IC50 of 0.93 ± 0.06 nM after 72 hours of treatment when assayed by both cell count and MTT cell viability assays, confirming the cytotoxic nature of this compound (Fig. 1A). At shorter time periods (24 and 48 hours), more than 70% of cells were viable even at the highest concentrations of FK866 compared to ~20% after 72 hours. Furthermore, FK866 caused a concentration-dependent reduction in intracellular NAD(P) levels consistent with the cell count and MTT data when measured after 24 hours of treatment. In order to assess whether the cytotoxic effects of FK866 were due to the effects on NAD levels, we performed rescue experiments, adding back various precursors and intermediates of the NAD biosynthesis pathway as well as NAD itself to try to overcome the inhibition by FK866 (Fig. 1B). As expected, extracellular addition of precursors upstream of the NMPRTase enzyme (nicotinamide; Fig. 1B, nicotinic acid) were unable to prevent FK866-induced cell death, while cells could be rescued by NAD and intermediates downstream of the enzyme (nicotinamide mononucleotide; Fig. 1B, nicotinic acid mononucleotide). We, and others, have been struck by the lag between the drop in NAD levels (complete after ~24 hours at 10 nM FK866) and the onset of cell death (only readily detectable after >48 hours).9 We, therefore, characterized the type of death induced by FK866 in SH-SY5Y cells by evaluating markers of both apoptosis and autophagy. Surprisingly, the Caspase 3 activation fragment (p11) could not be detected by western blot with a p11-specific antibody in FK866-treated (10 nM) cells even after 72 hours of treatment. As apoptosis may also rely on the activation of mitochondria to release pro-apoptotic mediators such as cytochrome-c (cyt-c) and the apoptosis inducing factor (AIF), we studied the cellular distribution of cyt-c and AIF by immuno-detection with confocal microscopy.11,12 In the majority of the cells, neither cyt-c or AIF were released from the mitochondria, which, together with the lack of the p11 protein would seem to exclude the engagement of apoptotic death after FK866 treatment (data not shown). Further proof comes from vital DNA staining with fluorescent DRQ5, since typical apoptotic fragmented nuclei were detected in only a very small number of FK866-treated cells. Given the cytotoxic effect of FK866 and
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NAD is a multifunctional molecule involved in both metabolic processes and signalling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed, extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We, therefore, investigated whether autophagy was initiated by FK866. Indeed, we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore, this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium.
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Key words: NAD, FK866, autophagy, apoptosis, neuroblastoma, chemotherapy
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Introduction
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In addition to being a crucial cellular redox cofactor, it is becoming clear that NAD(P) is also involved in various signalling pathways.1 Such pathways consume cellular NAD that must be replaced via either de novo synthesis or one of the recycling pathways.2,3 Recent work has suggested that NAD is present at physiologically relevant concentrations extracellularly and that two NAD(P) derivatives can be actively transported into cells.4-7 These findings prompted us to investigate whether a third mechanism, namely NAD uptake, could also contribute to intracellular NAD homeostasis.8 Indeed, we have characterized a transport system for NAD on the plasma membrane. As a proof of principle we made use of the novel drug FK866, which targets nicotinamide phosphoribosyltransferase (NMPRTase), one of the key enzymes in the NAD biosynthesis pathway, as a tool to reduce intracellular NAD levels.9 This drug, which is currently in
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*Correspondence to: Richard A. Billington; DiSCAFF; Via Bovio 6; Novara 28100 Italy; Tel.: +390321375827; Fax: +390321375821; Email: billington@pharm. unipmn.it Submitted: 01/18/08; Revised: 01/21/08; Accepted: 01/23/08 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/5635 Addendum to: Billington RA, Travelli C, Ercolano E, Galli U, Blasi Roman C, Grolla AA, Canonico PL, Condorelli F, Genazzani AA. Characterization of NAD uptake in mammalian cells. J Biol Chem 2008; In Press.
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FK866-induced autophagy
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Figure 1. (A) Concentration response curve for the effect of FK866 on SH-SY5Y cell viability after 72 hours of treatment; n = 8–16. (B) Rescue by extracellular NAD of FK866-induced SH-SY5Y cell death (black bars) and FK866-induced reduction of NAD(P) levels (white bars). Rescue of FK866-induced SH-SY5Y cell death by nicotinamide mononucleotide (NMN) but not by nicotinamide; n = 8–16. (C) Cells were transiently transfected with EYFP-LC3, stained with LysoTracker and treated with FK866 (10 nM) with or without NAD (100 μM) together for 36 hours. Results are indicative of 5 cultures. The EYFP signal is shown in green for ease of visualization. This figure is part of data previously published in reference 8, and is reproduced by permission of the American Society for Biochemistry and Molecular Biology and Elsevier, copyright 2008.
the apparent absence of apoptosis, we investigated the possibility that FK866 could induce autophagy. We, therefore, studied the formation of autophagic vesicles (autophagosomes and autophagolysosomes) by confocal microscopy, after FK866-induced NAD depletion using microtubule-associated protein 1 light chain 3 (LC3) as a specific marker.13 We transiently overexpressed EYFP-tagged 386
LC3 in SH-SY5Y cells and treated with 10 nM FK866 (Fig. 1C). In such conditions we were able to detect LC3-positive autophagic vesicles in FK866-treated cells, but not in control cells, after 24 hours. In order to validate the autophagic nature of these LC3-positive vesicles, we stained cells with LysoTracker which allowed us to detect the presence of lysosomes associated with the LC3-positive
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FK866-induced autophagy
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It is well known that neoplastic cells display increased levels of glycolysis and a relatively high turnover of NAD.14 For these reasons, the enzymes involved in the NAD biosynthesis pathways have recently become a focus of attention as targets for innovative anti-cancer drugs.14 While conventional cancer therapies are designed to induce apoptosis (Type I cell death) via toxic insult, metabolic inhibitors should suffocate cells via metabolic arrest. We have discovered that in SH-SH5Y cells, the metabolic inhibitor FK866 induces a delayed cell death that shows features of autophagy. In some respects, autophagy may be viewed as a “last resort” survival mechanism that is induced in cells deprived of nutrients as they must derive both energy and amino acids via self-digestion to make up for metabolic inadequacies.15 In other circumstances, such as cancer cells in which the apoptotic machinery has been hijacked to escape the anti-proliferative control, the engagement of autophagy is considered as a Type II mode of programmed cell death. That NAD depletion leads to an autophagic “energy-saving” response, is not surprising as NAD is a critical co-factor in glycolysis, the Krebs cycle and lipid β-oxidation. Whereas previous reports on the use of FK866 as an antitumoral agent have linked its function to the activation of apoptosis,9,16 we have been unable to find evidence of this. One possibility is that neuroblastoma cells are reluctant to die by apoptosis and engage Type II cell death upon treatment with toxic compounds.17,18 However, much previous research has focussed on apoptosis in response to NAD depletion induced by the hyperactivation of poly(ADP-ribose) polymerase (PARP).19 Indeed, many agents that induce DNA damage consequently induce the activation of PARP as a DNA repair signal in a process that actively consumes a large amount of NAD and this has been assumed to be proof that NAD depletion can induce apoptosis. How can we reconcile this notion with our observations? The most likely explanation is that, by using FK866, we have been able to specifically promote NAD depletion in the absence of DNA damage and PARP activation. Indeed, as elegantly shown in astrocytes by Alano et al.,20 PARP-induced cell death proceeds via two parallel pathways: NAD depletion and poly(ADP-ribose)-induced mitochondrial outer membrane permeabilization (MOMP) leading to AIF release. Interestingly, either inhibition of MOMP or repletion of NAD levels by the extracellular application of NAD was able to block AIF translocation and PARP-induced cell death. Autophagy may also be engaged in the background of PARP-induced cell death21 and our data would suggest that this is due to NAD depletion. Finally, our results highlight the complex synergy between cell death pathways induced by PARP activation and bring to light a new tool for the study of autophagy.
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Discussion
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structures. As we had been able to show rescue from cell death by extracellular NAD application, we repeated the rescue experiments and assessed autophagy. Repletion of the NAD levels with 100 μM extracellular NAD was able to revert the autophagic phenotype almost entirely (Fig. 1C).
Acknowledgements
Financial support from Università del Piemonte Orientale and the Regione Piemonte (Progetto Ricerca Sanitaria Finalizzata 2007) to RAB is gratefully acknowledged. www.landesbioscience.com
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