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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;

View larger version (79K): [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 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 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.