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Possible Link between Glycolysis and Apoptosis Induced by Sodium Fluoride

¹ Department of Dental Pharmacology, ² Meikai Pharmaco-Medical Laboratory (MPL), ³ Department of Oral Anatomy II, and ⁴ Department of Oral Health and Preventive Dentistry, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan; and ⁵ Faculty of Science, Josai University, Sakado, Saitama, Japan;

^* corresponding author, sakagami{at}dent.meikai.ac.jp and sumoh{at}sf7.so-net.ne.jp

	ABSTRACT

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Fluoride has been used to prevent caries in the dentition, but the possible underlying mechanisms of cytotoxicity induction by this compound are still unclear. Since fluoride is known as an inhibitor of glycolytic enzymes, we investigated the possible connection between NaF-induced apoptosis and glycolysis in human promyelocytic leukemia HL-60 cells. NaF-induced apoptotic cell death is characterized by caspase activation, internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, and production of apoptotic bodies. Higher activation of caspases-3 and -9, as compared with that of caspase-8, suggested the involvement of an extrinsic pathway. Utilization of glucose was nearly halted by NaF, whereas that of glutamine was rather enhanced. NaF enhanced the expression of Bad protein, but not that of Bcl-2 and Bax proteins, and reduced HIF-1 mRNA expression. Analysis of these data suggests a possible link between glycolysis and apoptosis.

KEY WORDS: fluoride • apoptosis • glycolysis • Bad protein.

	INTRODUCTION

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Fluoride is recognized as a stimulator of bone recalcification (Krokowski, 1984). Fluoride released from glass-ionomer materials may prevent caries (Dhondt et al., 2001) due to the inhibition of glucose uptake by bacteria (Germaine and Tellefson, 1986). Fluoride has shown considerable variation in respect of cytotoxicity against different cultured cells (Carlson and Suttie, 1967; Hongslo et al., 1974; Oguro et al., 1983). Cytotoxicity against normal cells (Tsutsui et al., 1984) has spurred debate on the use of fluoride in dentistry (Consiglio et al., 1998; Jeng et al., 1998). However, fluoride showed slightly higher cytotoxicity against tumor cell lines than against normal cells (tumor specificity ratio = 1.8) (Satoh et al., 2004). The antitumor activity of fluoride has been considerably modified by the addition of either anti-oxidants, oxidants, metals, saliva (Tokunaga et al., 2003a), antitumor agents (Morshed et al., 2003), or endodontic agents (Tokunaga et al., 2003b). Recently, fluoride has been reported to induce apoptosis in tumor cell lines via caspase-3 activation (Anuradha et al., 2001), and G-protein (Elliott et al., 2002; Refsnes et al., 2003) and MAP kinase (Thrane et al., 2001) mediated pathways have been suggested to be involved in fluoride-induced apoptosis. However, these investigators’ conclusions were based on findings from the use of metabolic inhibitors, and therefore are indirect.

Since fluoride is an inhibitor of the glycolytic enzyme, enolase (Voet and Voet, 1995), we established a working hypothesis that there should be some connection between NaF-induced apoptosis and glycolysis. To test this hypothesis, we performed the following four experiments, using a human pro-myelocytic leukemia cell line (HL-60). We first investigated various apoptosis markers to confirm the actual occurrence of apoptosis and identify which apoptotic pathway—either non-mitochondrial extrinsic (including caspase-8) or mitochondrial intrinsic (including caspase-9) (Shi, 2002)—is involved. Second, we investigated whether NaF preferentially inhibits the utilization of glucose (a major energy source of cultured cells), as compared with that of glutamine. Third, we investigated whether NaF may change the expression of the pro-apoptotic protein Bad, which has recently been reported to be associated with glucokinase, a rate-limiting enzyme of glycolysis (Danial et al., 2003), in comparison with that of the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. Fourth, we also investigated the possible change in the expression of the hypoxia-inducible factor (HIF)-1 level, which turns on the transcription of glucose transporters and glycolytic enzymes (Iyer et al., 1998).

	MATERIALS & METHODS

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Materials
The following chemicals and reagents were obtained from the indicated companies: RPMI1640 medium, DMEM (Gibco BRL, Grand Island, NY, USA); fetal bovine serum (FBS), anti-actin antibody (Sigma Chemical Co., St. Louis, MO, USA); NaF, dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA) (Wako Pure Chem. Ind., Ltd., Osaka, Japan); anti-Bad antibody, anti-Bcl-2 antibody, anti-Bax antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Cell Culture
HL-60 cells (Riken Cell Bank) were cultured at 37°C in RPMI1640 medium supplemented with 10% heat-inactivated FBS under a humidified 5% CO₂ atmosphere. Normal human gingival fibroblast (HGF) cells were obtained from human periodontal tissue, after subjects gave informed consent, according to the guidelines of the Meikai University Ethics Committee (No. 0206), and cultured in DMEM supplemented with 10% FBS. Since HGF cells have the limited lifespan of about 20 population-doubling levels (PDL), the cells at 6–8 PDL were used for the present study.

Assay for Cytotoxic Activity
Cells (1 x 10⁶/mL) were incubated for 24 hrs in 0.1 mL of fresh culture medium containing various concentrations of NaF in 96-microwell plates (flat-bottom, Becton Dickinson Labware, Franklin Lakes, NJ, USA). The viability of HL-60 cells was determined by trypan blue dye exclusion (Satoh et al., 2004).

Assay for DNA Fragmentation
Cells were washed once with phosphate-buffered saline without Ca²⁺ and Mg²⁺ [PBS(-)] and underwent lysis with 50 µL lysate buffer [50 mM Tris-HCl (pH 7.8), 10 mM EDTA, 0.5% (w/v) sodium N-lauroyl-sarcosinate solution]. The solution was incubated with 0.4 mg/mL RNase A and 0.8 mg/mL proteinase K for 1–2 hrs at 50°C. The lysate was mixed with 50 µL of NaI solution [7.6 M NaI, 20 mM EDTA-2Na, 40 mM Tris-HCl, pH 8.0], and then 100 µL of ethanol, and centrifuged for 20 min at 20,000x g. The precipitate was washed with 1 mL of 70% ethanol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Samples (10–20 µL) were subjected to 2% agarose gel electrophoresis in 0.5 x TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA, pH 8.0) (Morshed et al., 2003; Tokunaga et al., 2003b). DNA from apoptotic HL-60 cells induced by UV was used for calibration (Morshed et al., 2003). The DNA fragmentation pattern was examined in photographs taken under UV illumination.

Assay for Caspase Activation
Cells were washed twice with ice-cold PBS(-) and underwent lysis in lysis solution (MBL, Nagoya, Japan). After standing for 10 min on ice and being centrifuged for 5 min at 10,000x g, the supernatant was collected. Lysate (50 µL, equivalent to 200 µg protein) was mixed with 50 µL 2 x reaction buffer (MBL) containing substrates for caspase-3 [DEVD-pNA (p-nitroanilide)], caspase-8 (IETD-pNA), or caspase-9 (LEHD-pNA). After incubation for 4 hrs at 37°C, the absorbance at 405 nm of the liberated chromophore pNA was measured by a microplate reader (Biochromatic Labsystem, Helsinki, Finland) (Morshed et al., 2003; Tokunaga et al., 2003a).

Determination of Free Amino Acids
Culture supernatant (medium fraction) of control and treated cells was mixed with an equal volume of 10% TCA, and stood on ice for 30 min. After centrifugation for 5 min at 10,000x g, the deproteinized supernatant was collected and stored at –40°C. The supernatants (20 µL) were analyzed with a JEOL LC-300 amino acid analyzer, and amino acids were detected by the ninhydrin reaction (Tokunaga et al., 2003b).

Determination of Glucose
Glucose concentration in the culture medium was determined enzymatically with glucose oxidase. A 10-times-diluted sample (20 µL) was mixed with 300 µL of reagent [5.8 U/mL glucose oxidase (Aspergillus), 0.71 U/mL horseradish peroxidase, 0.51 mM 4-aminoantipyrine in 30 mM phosphate buffer, pH 7.4, containing 10.6 mM phenol]. After incubation for 30–60 min at 37°C, the absorbance at 492/620 nm was determined with a microplate reader. The glucose concentration was then determined from the standard curve, according to the manufacturer’s instructions (Wako) (Tokunaga et al., 2003b).

Western Blotting
The cell pellets underwent lysis with 100 µL of lysis buffer (10 mM Tris-HCl, pH 7.6, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF). Cell lysates (10 µg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (0.05 M Tris-HCl, pH 6.8, 10% glycerol, 2% SDS-0.005% bromphenol blue, 0.6% 2-mercaptoethanol), subjected to SDS-12% polyacrylamide gel electrophoresis, and then transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk in Tris-HCl buffered saline plus 0.05% Tween 20 and incubated with anti-Bad antibody (1:1000), anti-Bcl-2 antibody (1:1000), anti-Bax antibody (1:1000), or anti-actin antibody (1:1000) for 90 min at room temperature or overnight at 4°C, and then incubated with horseradish-peroxidase-conjugated anti-rabbit IgG (for Bad, Bax, Bcl-2) or anti-mouse IgG (for actin) (1:2000) for 1 hr at room temperature. Immunoblots were then reacted with Western Lightning^TM Chemiluminescence reagent plus (Perkin Elmer Life Sciences, Boston, MA, USA).

Mitochondrial Membrane Damage
Cells were stained for 20 min with MitoCapture^TM (BioVision, Inc,. Mountain View, CA, USA), an indicator of mitochondrial membrane potential. The stained cells were observed by confocal laser scanning microscopy LSM 510 (Carl Zeiss Co., Ltd.), with band-pass filters that detected FITC and rhodamine (Nakano et al., 2004).

Statistics
We used the Student’s t test to assess the statistical significance between the two groups.

	RESULTS

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Induction of Apoptosis
NaF induced various apoptosis-associated characteristics in HL-60 cells. NaF stimulated the cleavage of substrate for caspase-3 (about 10-fold) (p < 0.01), up to the comparable level (about 12-fold) attained by 1 µg/mL actinomycin D (positive control), confirming previous findings (Anuradha et al., 2001). We also found that NaF activated both caspase-8 and -9 (p < 0.05), but to slightly lower extents (about four- and two-fold, respectively). The activation of all 3 caspases was detectable above 5 mM NaF and reached a maximum level at 10 mM (p < 0.01 for caspase-3; p < 0.05 for caspases-8 and -9), declining at higher concentrations (Fig. 1A). Agarose gel electrophoresis showed that NaF induced internucleosomal DNA fragmentation above 5 mM (Fig. 1B). The viable cell number abruptly declined above 2.5 mM. At 7.5 or 10 mM NaF, the viable cell number dropped to 11 and 0% of control, respectively (Fig. 1C). NaF treatment induced the loss of mitochondrial membrane potential, as judged by the quantitative analysis of the change in the fluorescent color from red (viable) to green (apoptosis), with the use of the MitoCapture method (Fig. 1D).

View larger version (79K): [in this window] [in a new window]   Figure 1. Dose-response of NaF-induced apoptosis in HL-60 cells. HL-60 cells (5 x 105/mL) were incubated for 4 (A), 6 (B), 24 (C), or 6 (D) hrs with the indicated concentrations of NaF to assay caspase-3, -8, and -9 activity (A), internucleosomal DNA fragmentation (B), the viable cell number (C), and the dysfunction of mitochondrial membrane potential in NaF-treated HL-60 cells (D), respectively. (A) Mean ± SD (n = 3). (B) Representative of 3 independent experiments. (C) Mean ± SE (n = 6). At least 400 cells were observed under a light microscope. (D) Representative of 3 independent experiments. Right panel shows the quantification of fluorescence intensity (FI) of apoptotic cells/viable cells. **p < 0.001, *p < 0.01, p < 0.05.

View larger version (38K): [in this window] [in a new window]   Figure 2. Time-course of NaF-induced apoptosis in HL-60 cells. HL-60 cells (5 x 105/mL) were incubated for the indicated times with 0, 7.5, or 10 mM NaF to assay caspase-3, -8, and -9 activity (A), internucleosomal DNA fragmentation (B), and the number of apoptotic cells (C), respectively. (A) Mean ± SD (n = 3), 10 mM NaF, 1 µg/mL actinomycin D (Act.D.) (positive control). (B) Representative of 3 independent experiments, 7.5 mM NaF. (C) Mean ± SE (n = 4), 0, 7.5, or 10 mM NaF. At least 200 cells were observed under by light microscopy. **p < 0.001, *p < 0.01, p < 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of NaF on the expression of Bcl-2, Bax, Bad, and actin proteins. (A) HL-60 cells (1 x 106/mL) were incubated for 0 or 4 hrs without (control) or with the indicated concentrations of NaF, and intracellular concentrations of Bcl-2, Bax, or actin were determined by Western blot analysis. (B) HL-60 (1 x 106/mL, 3 mL) or near-confluent HGF cells (3 x 106 cells) were incubated for 2 or 4 hrs with the indicated concentrations of NaF, and the expression of Bad protein relative to that of actin was quantified and expressed as % of control. Each point represents mean ± SE (n = 3 for HL-60 4 hrs, HGF 4 hrs; n = 4 for HL-60 2 hrs). *p < 0.01, **p < 0.05.

	DISCUSSION

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The present study demonstrated that NaF actually induced apoptosis in HL-60 cells, as judged by various apoptosis markers, such as enhanced Bad expression, decreased mitochondrial membrane potential, caspase activation, DNA fragmentation, and production of apoptotic bodies. Preferential activation of caspases-3 and -8, as compared with that of caspase-9, supported the activation of an extrinsic apoptotic pathway.

We found that NaF completely eliminated glucose utilization, whereas it increased glutamine utilization with time. This indicates that apoptotic cell death induced by NaF is strictly linked to the loss of glucose utilization. When the energy supply from glucose was stopped, glutamine utilization was activated, possibly as a salvage pathway for cell survival.

We found that NaF reproducibly increased the expression of Bad in HL-60 cells, but not as much in HGF cells, which were relatively resistant to NaF (Satoh et al., 2004). Thus, the increased expression of Bad protein may relate to NaF sensitivity of the cells. A previous study has suggested the possible link between glycolysis and apoptosis, as judged by the association of Bad protein and glycokinase in rat liver mitochondrial fractions (Danial et al., 2003). As a preliminary experiment, we have transfected the BAD-GFP fusion protein in the human squamous cell carcinoma cell line HSC-2, and found that NaF treatment (7.5 mM, 3 hrs) resulted in the accumulation of Bad protein in the mitochondrial fraction near the nuclear periphery, as demonstrated by the co-localization of mitochondrial markers and BAD-GFP (Appendix). The identification of the Bad-associated proteins in HL-60 and HSC-2 cells is crucial.

We found little or no detectable changes in the expression of Bax and Bcl-2 proteins, in contrast to Bad protein. This finding was unexpected, since pro-apoptotic Bax protein is usually up-regulated, whereas anti-apoptotic Bcl-2 protein is down-regulated during apoptosis (LeBlanc et al., 2002). Moreover, the ratio of Bax/Bcl-2 determines the fate of cells, directing them to either survival or death. In accordance with this, NaF increased the expression of Bax protein in rat pulp cells in vitro (Kikuiri et al., unpublished observation) and decreased the expression of Bcl-2 in HL-60 cells (Anuradha et al., 2001). This apparent lack of concurrence of Bax/Bcl-2 expression between our and their experimental results may arise from the different culture conditions or types of cells (either normal or cancer cells). We recently found that treatment of HL-60 cells with ,ß-unsaturated ketones (Nakayachi et al., 2004), hydroxyketones (Yasumoto et al., 2004), or ß-diketones (Nakano et al., 2004)—but not with sophorastilbene A, piceatannol, or quercetin (Chowdhury et al., 2005)—significantly affected the expression of Bax and Bcl-2 proteins, respectively. This suggests that HL-60 cells show different responses to different apoptosis-inducers.

We could not detect HIF-1 protein during the NaF-induced apoptotic process, possibly due to accelerated degradation with an ample oxygen supply (Iyer et al., 1998). We thought that when glucose consumption was arrested by NaF treatment, HIF-1 mRNA expression may be enhanced so as to provide sufficient amounts of glycolytic enzymes. In contrast, NaF inhibited the transcription of HIF-1 mRNA, possibly due to the occurrence of cell death. The decline in HIF-1 mRNA expression by NaF may further diminish the glucose utilization in a positive feedback system.

The present investigation of the effect of NaF on glucose consumption, Bad expression, and HIF-1 expression supports the hypothetical link between glycolysis and apoptosis. Further investigation into the function of Bad protein by inhibition of its gene expression and characterization of its associated proteins is necessary to demonstrate this link. Thus, NaF may then be more effectively used in clinical dentistry.

	ACKNOWLEDGMENTS

 
This study was supported by the Department Fund of Meikai University School of Dentistry and a Grant-in-Aid from the Ministry of Culture, Education, Science, Sports and Culture of Japan (Sakagami, No. 14370607). Part of the present study was presented at the 82nd General Session of the International Association for Dental Research (Honolulu, HI, USA, 2004).

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received April 7, 2004; Last revision June 21, 2005; Accepted June 22, 2005

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