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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (Ki) and 0.3 nM for the free enzyme (Ki'), 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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m and the concomitant release of apoptogenic proteins (11
, 14
, 15) . The discharge of caspases (caspase-2, caspase-3, and caspase-9, depending on the cell type) and caspase-activating proteins such as cytochrome c from the mitochondrial intermembrane space to the cytosol after PTP formation is a crucial event inducing apoptotic cell death (16)
. Another well-characterized mitochondrial pathway to apoptosis is activated by the liberation of apoptosis-inducing factor from the mitochondrial intermembrane space and was found to be caspase independent (17
, 18)
. In any case, influencing the postmitochondrial or degradation phase of apoptosis by inhibitors of caspases or nucleases may just alter the morphological manifestations of cell death but cannot eventually prevent it. Unfortunately, little is known about the actual molecular triggers of PTP opening, which would probably represent more suitable targets for pharmacological modulation of apoptosis. Negative regulation by interaction of the Bcl-2 protein localized in the mitochondrial membrane with members of the PTP complex such as adenine nucleotide transporter or voltage-dependent anion channel has recently been reported (16
, 19, 20, 21)
. Mitochondrial membrane potential, redox state, matrix pH, divalent cations, adenine nucleotides, and thiol contents have all been discussed to influence PTP function (14
, 22)
. Here, we describe the identification of the mitochondrial enzyme NAPRT as the molecular target of a novel apoptosis-inducing compound that had been selected by an anticancer screening system differentiating acute cytotoxicity from growth inhibition.
When we set out to find new antitumor drugs, our main focus was to identify compounds that might interfere with cellular mechanisms of growth regulation, survival, or death. First, we adapted the screening assay established by the National Cancer Institute of the United States (23)
in a way to enable distinction of immediate cytotoxicity from growth inhibition: incubation times in the standard primary assay were prolonged and adjusted to match 3.5 doubling times of the respective cell lines, i.e., 4 days for A549 human lung cancer cells and 6 days for HepG2 human hepatocarcinoma cells. Compounds indicating anticancer activity in the primary assay were subsequently tested in a time curve experiment in HepG2 high-density cultures. This secondary assay was designed as to discriminate acute cytotoxicity from possible interference with growth regulation and to confirm activity under high-density cell culture conditions, where the proliferation rate is lower, and cells are usually less sensitive for antiproliferative drugs. Using above screening system, a new class of compounds that characteristically induce delayed cell death was identified. The compound (E)-N-[4-(1-benzoylpiperidin-4-yl) butyl]-3-(pyridin-3-yl) acrylamide, designated FK866, was selected as a candidate anticancer drug (Fig. 1)
. Its physiological effects on tumor cells eventually leading to apoptosis and its unique mechanism of action are outlined in the present article.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5–10 min incubation at 37°C, when the cells had detached from the surface, trypsinization was stopped by the addition of 3 ml of Richter’s IMEM-ZO medium containing 10% FCS. To induce apoptosis, the indicated amount of FK866 (Fujisawa Deutschland GmbH) was added from a 1000x stock solution in ethanol or DMSO to the culture medium.
Cell Culture Assays.
The cells were plated in 96-well plates and incubated with FK866 for the time indicated. The WST-1 assay was performed according to the manufacturer’s recommendations (Roche). The SRB assay was carried out as described by Skehan et al. (23)
. In the PI exclusion assay, the cells were incubated with PI (HepG2, 30 µg/ml; THP-1, 15 µg/ml) at the end of the incubation time. To determine the percentage of dead cells in relation to the total cell number, the fluorescence (excitation 530 nm, emission 590 nm) was measured firstly after addition of PI alone, and secondly after addition of Triton X-100 (0.1%).
Polarographic Measurement of Oxygen Consumption.
The effect of FK866 on the mitochondrial respiratory chain was determined by polarographic measurement of oxygen consumption according to Hofhaus et al. (24)
. HepG2 cells were treated with FK866 as specified in the text. For each measurement, 5 x 106 HepG2 cells were permeabilized in 1 ml with 0.1% digitonin for 75 s and subsequently diluted with 9 ml of medium A [250 mM sucrose, 10 mM MgCl2, 2 mM KxHxPO4, 20 mM HEPES (pH 7.2)]. The cells were collected by centrifugation, suspended in 1 ml of medium A supplemented with 1 mM ADP and transferred to the oxygraph chamber (OS2-1, Biolytik). The efficacy of the digitonin treatment has been controlled in the trypan blue exclusion test (>97% of cells stained blue). The enzyme substrates and inhibitors were introduced sequentially into the chamber as described in the text. Ten µl of these reagents was used, resulting in the following final concentrations: 5 mM malate, 5 mM glutamate, 400 nM rotenone, 5 mM glycerol-3-phosphate, 5 mM succinate, 25 µM antimycin A, 1 mM ascorbate, 200 µM tetramethyl-p-phenyldiamine, and 1 mM KCN.
Determination of Intracellular Pyridine Nucleotides and ATP Contents.
Logarithmically growing HepG2 cells were seeded in culture flasks (1 x 105/ml) in the presence or in the absence of 10 nM FK866. The intracellular content of pyridine nucleotides was determined by enzymatic cycling techniques and quantified using spectrophotometry as described by Wosikowski et al. (25)
. ATP was measured according to the manufacturer’s instruction (Labsystems) using the same acid extracts that were used for determination of NAD+ and NADH.
Quantitation of NAD+ Synthesis.
Logarithmically growing cells were seeded in
Measurement of NAPRT and NPRT Activity.
NAPRT and NPRT activities were determined by modification of the methods described by Elliott et al. (26)
and Pinder et al. (27)
, respectively. Preparation of cytoplasmic extracts: logarithmically growing K-562 cells were collected by centrifugation and washed three times with Ca2+Mg2+-free PBS. The pellet of 2–3 x 107 cells was suspended in 1 ml of lysis buffer [0.01 M NaH2PO4 (pH 7.4)]. The suspension was frozen at -80°C for at least 1 day and thawed slowly at room temperature to break up the cells (breakage >95%). After centrifugation (23,000 x g, 0°C, 90 min), the clear supernatant was recovered on ice, and 70 µl of 1% protamine sulfate were added/ml supernatant. After 15 min on ice, the cloudy suspension was centrifuged again (23,000 x g, 0°C, 30 min). The final supernatant was stored in small aliquots at -80°C. NAPRT assay: the enzyme activity was determined in 0.5 ml of reaction solution consisting of 5 mM MgCl2, 2 mM ATP, 0.5 mM phosphoribosyl PPI, 10–0.1 mM 14[C]nicotinamide (specific activity: 50 mCi/mmol; American Radiolabeled Chemicals, Inc.) and 50 mM Tris (pH 8.8) at 37°C. The reaction was started by adding 100 µl of cell extract and stopped after 1 h with excess of cold nicotinamide and heating (2 min, 105°C). The precipitate was removed by centrifugation (2500 x g, 4°C, 10 min), and the supernatant was stored at -20°C. NPRT assay: the enzyme activity was determined in 0.5 ml of reaction solution consisting of 0.5 mM MgCl2, 5 mM ATP, 0.5 mM phosphoribosyl PPI, 10 mM 14[C]nicotinic acid (specific activity: 50 mCi/mmol; American Radiolabeled Chemicals, Inc.) and 34 mM Na2HPO4 (pH 7.5) at 37°C. The reaction was started by adding 100 µl of cell extract and stopped after 1 h with acetic acid (0.2 N) and heating (2 min, 105°C). The precipitate was removed by centrifugation (2500 x g, 4°C, 10 min), and the supernatant was stored at -20°C. The amount of substrate formed in both assays was determined using the thin-layer chromatography techniques described above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. During FK866 incubation, HepG2 liver carcinoma cell numbers increased up to 3 days, with sharply declining cell numbers from days 7 to 10. In contrast, more acutely cytotoxic compounds reduced cell numbers from as soon as the first day after start of treatment.
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. Already after 1 day of FK866 treatment, a decreased WST-1 reaction, which measures the metabolic activity of cells, was noted on HepG2 tumor cells (Fig. 2B)
. This test is based on the reduction of the colorless tetrazolium salt WST-1 to a colored formazan dye. The WST-1 reducing capacity of the cells depends on both the activity of intracellular dehydrogenases and the amount of the coenzyme NAD(P)H available (28)
. One to 2 days subsequent to the drop of dehydrogenase activity, the effects of FK866 on cell growth became evident when recorded by the SRB assay (Fig. 2C)
, which measures cellular protein and correlates well with cell numbers (23)
. Finally, only after 4 days of incubation and 3 days after the decline of dehydrogenase activity, an increased percentage of dead cells was found using the PI assay (Fig. 2D)
, which measures cell viability based on the exclusion of the fluorescent dye PI. Even 100-fold IC50 concentrations of FK866 could not accelerate the time course of action nor shift the chronological sequence of effects in the three assays. This result indicates very low nonspecific cytotoxicity of the compound. In conclusion, after FK866 treatment, a decreased WST-1 reactivity, which has often been used as a cell viability assay (28)
, clearly precedes cell death by several days.
Electron micrographs of FK866-treated HepG2 cells shown in Fig. 3
unequivocally prove that cell death induced by FK866 occurs in the form of apoptosis. It is unusual, however, that nearly all cells show up in an early stage of apoptosis at the same time, as indicated by chromatin condensation at the nuclear membrane in Fig. 3B
. As the whole process of apoptosis is described to be completed within hours (8)
and usually occurs in single cells of tissues in contrast to necrotic areas (3)
, one might expect that after a treatment period of as long as 4 days in unsynchronized cultures, there would be all different stages from unaffected cells to mere remnants of degradation. However, FK866 leads to apoptosis with a surprising completeness and synchrony after a remarkably long treatment period. We have recently described elsewhere the biochemical events confirming apoptotic cell death after FK866 treatment in more detail (25)
.
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Effect on the Mitochondrial Respiratory Chain.
The influence of FK866 on the mitochondrial respiratory chain was determined by polarographic measurement of oxygen consumption in digitonin-permeabilized cells according to Hofhaus et al. (24)
. In a first experiment, 10 nM FK866 were administered to digitonin-permeabilized HepG2 cells without preincubation to investigate direct effects on the mitochondrial respiratory activity. Alternatively, HepG2 cells were preincubated for 2 days either with 10 nM FK866 or with solvent (0.1% ethanol) to facilitate detection of possible indirect effects on mitochondrial respiration. The cells were then treated with digitonin for permeabilization of cytoplasmic membranes to provide access for substrates. Respiratory complex I reaction was started by the addition of malate and glutamate, which after a few minutes was stopped by the specific complex I inhibitor rotenone. Complex III reaction was activated by adding succinate and glycerol-3-phosphate and then specifically stopped by the inhibitor antimycin A. Finally, complex IV reaction was started by the addition of the redox carrier tetramethyl-p-phenyldiamine and ascorbate, whereas subsequent inhibition with KCN confirmed the specificity of the reaction. These experiments clearly provided two interesting results: first, addition of FK866 to nonpretreated permeabilized cells proved that there is no direct inhibition of any component of the mitochondrial respiratory chain by FK866 (data not shown). Second, pretreatment with FK866 for 2 days resulted in a reduced activity only of complex I reaction, whereas the other components of the respiratory chain remained unaffected (Fig. 4)
. The finding that the complex I reaction was not compromised by FK866 without prolonged preincubation precluded a direct inhibition of the complex I enzyme NADH:ubiquinone oxidoreductase. Alternatively, substrate shortage could have slowed down respiratory complex I reaction. As a delayed inhibition of the malate/aspartate shuttle appeared rather unlikely, we investigated the other substrate of complex I, cellular NAD+ content.
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. In comparison to vehicle-treated HepG2 cells, a significant reduction of the intracellular amount of NAD+ by 50% was observed as soon as after 8 h in FK866-treated cells. The decline of NADH was slightly delayed and arrived below 50% of controls after 16 h. These findings demonstrate that treatment of HepG2 cells with FK866 results in a decrease of the intracellular concentration of oxidized and reduced pyridine dinucleotides.
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demonstrate that FK866 treated cells retain their ATP content for a prolonged period of time. A significant decrease of ATP to 50% of controls was only recorded after 3 days of drug incubation. The preservation of ATP enables cells to undergo controlled cell death via apoptosis which requires sufficient energy for at least two important steps: firstly, nuclear condensation and subsequent DNA degradation, and secondly, the exposition of phosphatidylserine on the cell surface that facilitates removal by phagocytic cells (29)
. In contrast, a rapid and sudden breakdown of ATP levels would inevitably result in necrosis (30)
. The reduction of intracellular pyridine nucleotides could basically be caused by two different effects: either by increased degradation or by decreased de novo biosynthesis. NAD+ consumption is usually mediated through cleavage by hydrolyzing enzymes like PARP (31 , 32) . PARP activation has been implicated in the cytotoxic action of DNA-damaging agents (33) . Using NAD+ as a substrate for cleavage and as an ADP-ribose donor, PARP forms ADP-ribose homopolymers attached to several nuclear proteins, including to itself, to histones and DNA repair enzymes, thus facilitating repair of DNA damage (31, 32, 33, 34) . In addition to PARP activation, another immediate cellular response to DNA damage is an increase of tumor suppressor gene p53 expression, which, in turn, is subject to poly(ADP-ribosyl)ation (35 , 36) . However, excessive NAD+ degradation after FK866 treatment appeared unlikely for the following reasons: in contrast to cytotoxic DNA-damaging agents, the NAD+ decline caused by FK866 preceded cell death by several days (see above). Furthermore, FK866 did neither up-regulate p53 nor did it cause changes in cell cycle distribution typical for DNA damaging drugs (data not shown). Therefore, our interest focused on the investigation of NAD+ biosynthesis.
Effect of FK866 on Cellular NAD+ Synthesis.
There are three major pathways for NAD+ biosynthesis exploiting different precursors: in the least frequent situation, tryptophan can undergo several degradation steps to produce quinolinic acid, which is additionally metabolized to give NAD+; preferably, nicotinamide or nicotinic acid are used for more direct NAD+ biosynthesis pathways, starting with the addition of a phosphoribosyl moiety to the respective precursors which yield the corresponding mononucleotides, NAM and nicotinic acid mononucleotide. Subsequent attachment of AMP results in the formation of the corresponding dinucleotides. In the case of nicotinic acid adenine dinucleotide, an additional amidation step is needed to produce NAD+. If both nicotinamide and nicotinic acid are in short supply, only liver, kidney, and brain cells are capable to exploit tryptophan as a precursor for NAD+ synthesis (37)
. Therefore, we first investigated the influence of FK866 on the nicotinamide and nicotinic acid pathways of NAD+ biosynthesis.
To study NAD+ synthesis from the precursor nicotinamide, HepG2 cells were incubated with radiolabeled nicotinamide. The resulting metabolites in FK866-treated versus control cells were analyzed by thin layer chromatography and quantitated by bioimaging. After 8 h of treatment, NAD+ biosynthesis from the precursor nicotinamide was inhibited by 97% in the FK866 versus control group (Table 1)
. Importantly, there was no accumulation of the intermediate NAM in the cell extracts from FK866-treated cultures. NAD+ and nicotinic acid were poorly separated in the chromatograms using polyethyleneimine cellulose as matrix and 0.05 M lithium chloride as solvent (Fig. 6, A and B)
, but the identity of both peaks were verified after more effective separation on cellulose with ammonium acetate/ethanol. Some radiolabeled nicotinic acid found in HepG2 cell extracts obviously resulted from the deamidation of [14C]nicotinamide catalyzed by the enzyme nicotinamide deamidase, which is usually prevalent in liver cells (27)
.
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, shows that FK866 had absolutely no influence on NAD+ production from the precursor nicotinic acid. In contrast, NAD+ synthesis from nicotinamide was inhibited in THP-1 cells with a similar efficiency as found in HepG2 cells (data not shown). These results clearly demonstrate that FK866 specifically inhibits the nicotinamide pathway of NAD+ biosynthesis. Furthermore, the lack of NAM accumulation in both cell lines after FK866 treatment strongly suggested that NAPRT, the enzyme catalyzing the first step reaction of NAD+ synthesis from the precursor nicotinamide, is the direct molecular target of FK866.
Antidote Potential of Nicotinamide and Nicotinic Acid.
The selective inhibition of the nicotinamide pathway of NAD+ synthesis by FK866 suggested reversibility studies with nicotinamide and nicotinic acid. In HepG2 cells, 10 mM high concentrations of nicotinamide were able to reverse the growth inhibiting properties of FK866 (Fig. 7A)
. Consistent with above described incorporation of radioactive precursors, nicotinic acid could not be used for NAD+ synthesis by HepG2 cells and consequently had no drug-reversing effects. In contrast, 1 mM nicotinic acid could well antagonize the antiproliferative activity of FK866 in THP-1 cells, which are able to synthesize NAD+ via this pathway (Fig. 7B)
. These results imply that high concentrations of nicotinamide may be used as an antidote for FK866 action, whereas nicotinic acid may serve as a functional antidote only in those tissues that can use it as a precursor for NAD+ synthesis.
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. As K-562 extracts also contained NPRT activity, the effect of FK866 on NPRT could be tested in the same samples. Data shown in Table 2
prove that FK866 had no inhibitory effect on NPRT up to a concentration of 1000 nM. These results unequivocally demonstrate that FK866 inhibits cellular NAD+ biosynthesis from the precursor nicotinamide with high specificity at the level of the enzyme NAPRT.
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. This data are highly consistent with previous studies on NAPRT from rat liver or human fibroblasts (26
, 38)
and confirms a comparable quality of the enzyme preparation.
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and inhibition of the free enzyme Ki' = 0.3 nM (Fig. 8C)
. Physiologically, NAPRT is noncompetitively inhibited by NAD+ (39)
. Thus, NAD+ regulates its own biosynthesis from the precursor nicotinamide by a negative feedback on the first step enzyme. Comparing the inhibitor constants, FK866 is 105–106 times more effective than NAD+ in regard to NAPRT inhibition. Thus, FK866 functions as a highly potent NAD+ analogue in this system. The corresponding enzyme in the nicotinic acid pathway of NAD+ biosynthesis, NPRT, is not negatively regulated by such a mechanism (37)
and presumably has no regulatory binding site for an inhibitor such as NAD+. Therefore, the lack of NPRT inhibition by FK866 underlines its high specificity for NAPRT. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, FK866 is the first highly potent and specific inhibitor of NAPRT that has no primary effect on cellular energy metabolism. The time course of FK866 action is characteristic for this new class of antitumor compounds and suggests that the compound has no direct and immediate cytotoxicity but gradually depletes the cells of some vital factor that eventually triggers apoptosis. Inhibition of NAPRT progressively exhausts NAD+ in those cells that mainly rely on nicotinamide as a precursor for pyridine nucleotide synthesis. The speed of NAD+ depletion depends on the activity of NAD+ hydrolyzing reactions. One of the most important NAD+-cleaving enzymes is PARP-1, which is extensively involved in the regulation of DNA repair (43)
. A study performed with human patient samples showed that both PARP activity and expression was much higher in hepatocellular carcinoma tissues than in the adjacent nontumor tissue (44)
. The relative genomic instability of cancer cells (45
, 46)
with permanently ongoing DNA repair processes, the recently described modulation of telomerase activity by ADP-ribosylation (47)
and increasing knowledge of the role of ADP-ribosylation in the regulation of transcription factors and tumor suppressor proteins (35
, 36
, 48)
all suggest the use of specific NAPRT inhibitors as anticancer drugs. Furthermore, specific NAD+-depleting compounds such as FK866 may be used in combination therapy to compromise DNA repair capacity before or during cancer treatment with genotoxic drugs or radiotherapy. Additional application for immunosuppression is implied by previous studies on the role of NAD+-consuming ADP-ribosylation reactions in V(D)J recombination of lymphocytes (49)
and NAD+ degradation by cyclic-ADP-ribose synthetase during lymphocyte activation (50)
. Fig. 9
summarizes the different use of NAD+ as a coenzyme and a substrate, respectively, and indicates that augmented NAD+ cleaving reactions both in tumor cells and lymphocytes render them more susceptible to FK866 treatment than other tissues. In every case of potential therapeutic use, administration of nicotinamide or nicotinic acid as antidotes may be useful to modulate FK866 toxicity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Roche Diagnostics GmbH, Pharma Research Penzberg, Nonnenwald 2, 82377 Penzberg, Germany. 
2 To whom requests for reprints should be addressed, at Neumarkter Str. 61, 81673 Munich, Germany. Phone: 49-89-4544-2152; Fax: 49-89-4544-2370; E-mail: isabel.schemainda{at}fujisawa.de
3 The abbreviations used are: PTP permeability transition pore; NAPRT, nicotinamide phosphoribosyltransferase; SRB, sulforhodamine B; PI, propidium iodide; PARP, poly(ADP-ribose) polymerase; NAM, nicotinamide mononucleotide; dNAM, nicotinic acid mononucleotide; NPRT, nicotinic acid phosphoribosyltransferase; WST-1, water-soluble tetrazolium salt-1.
Received 6/ 2/03. Revised 7/10/03. Accepted 7/21/03.
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), 1 nM (
), 3 nM (
), 10 nM (
), and 30 nM (gray hexagon) FK866, respectively. B–D, concentration response curves with FK866 on HepG2 cells (8,000 cells/200 µl/well of a 96-well plate) after different times of incubation: 1 day (
), and 6 days (
). Parameters investigated: metabolic activity (B), cell number (C), and number of dead cells (D).
).
