fk-866 and Liver-Neoplasms

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling.. FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment.. Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC.

Topics: Acrylamides; Adenosine Triphosphate; AMP-Activated Protein Kinases; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Cytokines; Down-Regulation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Nicotinamide Phosphoribosyltransferase; Piperidines; Signal Transduction; TOR Serine-Threonine Kinases

We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.

Topics: Acrylamides; Adenosine Triphosphate; Aerobiosis; Animals; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Cytokines; Energy Metabolism; Glycolysis; Humans; Hydrogen Peroxide; Hydrogen Sulfide; Liver Neoplasms; Male; Melanoma; Mice; Mice, Nude; Neoplasm Proteins; Nicotinamide Phosphoribosyltransferase; Piperidines; Triple Negative Breast Neoplasms

Deregulation of apoptosis, the physiological form of cell death, is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways, both of which may provide potential targets for novel anticancer drugs. Although several ligands stimulating death receptors have been described, the actual molecular events triggering the mitochondrial pathway are largely unknown. Here, we show initiation of apoptosis by gradual depletion of the intracellular coenzyme NAD+. We identified the first low molecular weight compound, designated FK866, which induces apoptosis by highly specific, noncompetitive inhibition of nicotinamide phosphoribosyltransferase (NAPRT), a key enzyme in the regulation of NAD+ biosynthesis from the natural precursor nicotinamide. Interference with this enzyme does not primarily intoxicate cells because the mitochondrial respiratory activity and the NAD+ -dependent redox reactions involved remain unaffected as long as NAD+ is not effectively depleted by catabolic reactions. Certain tissues, however, have a high turnover of NAD+ through its cleavage by enzymes like poly(ADP-ribose) polymerase. Such cells often rely on the more readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis after inhibition of NAPRT, whereas cells effectively using the nicotinic acid pathway for NAD+ synthesis remain unaffected. In support of this concept, FK866 effectively induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC(50) of approximately 1 nM, did not directly inhibit mitochondrial respiratory activity, but caused gradual NAD+ depletion through specific inhibition of NAPRT. This enzyme, when partially purified from K562 human leukemia cells, was noncompetitively inhibited by FK866, and the inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)'), respectively. Nicotinic acid and nicotinamide were both found to have antidote potential for the cellular effects of FK866. FK866 may be used for treatment of diseases implicating deregulated apoptosis such as cancer for immunosuppression or as a sensitizer for genotoxic agents. Furthermore, it may provide an important tool for investigation of the molecular triggers of the mitochondrial pathway leading to apoptosis through enabling temporal separation of NAD+ decrease from ATP breakdown and apoptosis by several days.

Topics: Acrylamides; Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Inhibitors; Humans; K562 Cells; Kinetics; Liver Neoplasms; Mitochondria, Liver; NAD; Niacin; Niacinamide; Nicotinamide Phosphoribosyltransferase; Oxygen Consumption; Pentosyltransferases; Piperidines