The co-factor nicotinamide adenine dinucleotide (NAD) plays a crucial role in multiple cellular processes and is substrate for a variety of enzymes and regulatory proteins . In humans a main portion of NAD is generated via the nicotinamide (NAM) salvage pathway, in which nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the biosynthesis of NAD yielding nicotinamide mononucleotide (NMN) [2;3]. As NAD is rapidly consumed in cells (1h) and converted to NAM , NAMPT is essential for the replenishment of the intracellular NAD pool. The development of many cancers is associated with increased NAMPT expression . Cancer cells have a high rate of NAD turnover due to their increased energy demand and a high activity of NAD-dependent enzymes, such as poly (ADP-ribose) polymerases (PARPs), mono-ADP ribosyltransferases (MARTs) and sirtuins, required for DNA repair, genome stability and proliferation [1;5]. Therefore, cancer cells are more susceptible to NAMPT inhibition than normal cells [6;7]. In previous studies, we found that NAMPT is released from hepatocytes  as well as differentially expressed and more enzymatically active in hepatocarcinoma cells compared to non-cancerous human hepatocytes . Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths . The only available proven systemic therapy for HCC is the multi-targeting kinase inhibitor sorafenib . An effective second-line agent for patients with sorafenib failure or intolerance has yet to be identified. This has led to an intensive search for molecular pathways and novel compounds for the treatment and prevention of HCC. Targeting NAMPT activity and intracellular NAD content represents a novel therapeutic concept for HCC. The specific NAMPT inhibitor FK866 is a competitive inhibitor that was selected by an anticancer screening system differentiating acute cytotoxicity from growth inhibition [12;13]. FK866 has been evaluated in a broad variety of tumors, including solid tumors and leukemia [14-16] in vitro and in nude mouse xenografts [17- 19], where FK866 was able to reduce or attenuate tumor growth.
In HCC tissue, AMP-activated protein kinase (AMPK), a major regulator of cellular energy homeostasis that coordinates multiple metabolic pathways, has been shown to be dysregulated compared to normal tissue [20;21]. AMPK activity opposes tumor development and negatively regulates the Warburg effect (aerobic glycolysis) leading to suppression of tumor growth in vivo [20-22]. AMPK translates changes in glucose availability and fluctuation of energy to mammalian target of rapamycin (mTOR) and thereby acts as a master energy sensor to modulate cellular activities in response to energy stress [23;24]. mTOR, a serine/threonine protein kinase, has been observed to be increased in multiple human cancers, including HCC, where it is associated with less differentiated tumors, earlier tumor recurrence, and worse survival outcomes [25;26]. Inhibition of mTOR has proven efficacious in clinical trials [26;27]. Recently, there is great scientific interest in finding molecular pathways and novel compounds that target AMPK/mTOR signalling as a new treatment option for HCC.
Little is known about the interaction of NAMPT and AMPK/mTOR signalling during the development of HCC. In this study, we investigated the effects of the NAMPT inhibitor FK866 on hepatocarcinoma cells and non-cancerous human hepatocytes. We asked whether or not FK866-induced energy stress might activate AMPK and modify the mTOR signalling pathway and whether the observed effects could be rescued by the NAMPT enzyme product NMN.
Material and Methods
Cell culture media, supplements and antibiotics were obtained from PAA (Cölbe, Germany) or Invitrogen (Karlsruhe, Germany). FK866, nicotinamide mononucleotide (NMN) and camptothecin were purchased from Sigma-Aldrich (Munich, Germany). Etoposide was purchased from Merck Millipore (Darmstadt, Germany).
Hepatocarcinoma cell lines
Huh7 cells (p53-mutated) and Hep3B cells (p53-deficient) were maintained in DMEM medium with high glucose or MEM medium, repectively. Media were supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 100IU penicillin and 100μg/mL streptomycin. All cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2.
Primary human hepatocytes
Tissue samples from patients undergoing liver surgery at the University Medical Center Regensburg were used. Primary human hepatocytes (PHH) were isolated and cultivated as described recently . Briefly, non-neoplastic tissue samples from liver resections were obtained from patients undergoing partial hepatectomy for metastatic liver tumors of colorectal cancer. PHHs were isolated using a modified two-step EGTA/collagenase perfusion procedure and plated on collagen coated dishes. Experimental procedures were performed according to the guidelines of the charitable state controlled foundation HTCR (Human Tissue and Cell Research, Regensburg, Germany), with the informed patient’s consent approved by the local ethical committee of the University of Regensburg. All experiments involving human tissues and cells have been carried out in accordance to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Cells were seeded in Williams’ Medium E containing 2mM glutamine, -7 10 mol/L dexamethasone, 100IU penicillin, 100μg/mL streptomycin and 10%FBS. All cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2.
FK866 was dissolved in DMSO to create a stock solution of 10mM. NMN was dissolved in the appropriate medium for a stock solution of 100mM. After 16h serum starvation, cells were treated with the indicated concentration of FK866 alone or in combination with NMN [500μM] for 24, 48 and 72h.
Cell viability and apoptosis
Cell viability analysis was conducted using the cell proliferation reagent WST-1 (Roche, Grenzach-Wyhlen, Germany) according to manufacturer ́s instructions. To examine the effects of FK866 on cell death, the number of dead cells was measured by FACS analysis at different time points (48h, 72h) using the AnnexinV-FITC Apoptosis Detection Kit (BD PharmingenTM, Franklin Lakes, USA). Adherent and floating cells were analysed according to manufacturer ́s protocol. Samples were analysed using a Beckton-Dickinson FACS LSRII. As positive control, apoptosis was induced via camptothecin [2μM] and etoposide [85μM] for 24h. Annexin (An )and double-stained An /propidium iodide (PI ) cells were considered as dead cells.
ATP levels were measured with the luminescent-based CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, USA) according to the manufacturer’s protocol.
Protein extraction and Western Blot analysis were performed as described previously . Primary antibodies used for immunoblotting included anti-phospho-AMPKα(Thr172), anti- AMPKα, anti-phospho-mTOR(Ser2448), anti-mTOR, anti-tubulin, anti-phospho p70S6 kinase(Thr389), anti-p70S6 kinase, anti-phospho-4E-BP1(Ser65), anti-4E-BP1, anti-acetylated lysine (Cell Signaling, Beverly, MA, USA) and anti-GAPDH (MerckMillipore, Schwalbach, Germany). Appropriate secondary antibodies were purchased from DAKO (Hamburg, Germany). Immunoblotting for GAPDH or tubulin was performed to verify equivalent amounts of loaded protein. Densitometric analysis was performed using ImageJ 1.41 Software (NIH, USA).
NAMPT enzymatic activity
NAMPT activity was measured by the conversion of
using a method previously described [9;29]. Radioactivity of
scintillation counter in counts per minute (cpm) (Wallac 1409 DSA, PerkinElmer). NAMPT activity (cpm) was normalized to total protein concentration as measured by the BCA protein assay.
Concentrations of NAD from whole-cell extracts were quantified by HPLC analysis using a SUPELCOSILTM LC-18-T HPLC column (Sigma Aldrich) at a flow rate of 0,8ml/min with 100% buffer A (potassium phosphate buffer pH 6.0) from 0–2min, a linear gradient to 85% Buffer A/15% Buffer B (100% methanol) from 2-5min, 85% Buffer A/15% Buffer B from 5- 10min, a linear gradient to 100% Buffer A from 10–12min and 100% Buffer A from 12–15min. NAD was eluted as a sharp peak at 8min and quantitated based on the peak area compared to a C- labelled nicotinamide to standard curve and normalized to total protein concentration as measured by the BCA protein assay.