Effect of three fluoride compounds on the growth of oral normal and tumor cells.

AIM: Comparative study of the growth inhibition by different types of fluoride compounds used in dentistry has been limited. We investigated the effects of sodium fluoride (NaF), diammine silver fluoride [Ag(NH3)2F] and 5-fluorouracil (5-FU) on the growth of eleven human normal and tumor cells in total. MATERIALS AND METHODS: Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction was evaluated by caspase-3 activation and DNA fragmentation. Fluoride was determined using a fluoride-specific electrode. RESULTS: All compounds had little or no growth stimulating effect (hormesis) on all cells. Ag(NH3)2F exhibited the highest cytotoxicity towards both normal and tumor cells. 5-FU had the selective cytostatic activity towards oral squamous cell carcinoma cell lines, whereas NaF was selectively cytotoxic towards glioblastoma cell lines. None of the compounds induced internucleosomal DNA fragmentation and only 5-FU induced slight activation of caspase-3 in an oral squamous cell carcinoma cell line (HSC-2). Cytotoxicity of fluoride compounds was not reduced by superoxide dismutase and catalase, reducing the possibility of the involvement of reactive oxygen species in the mechanism of action. Approximately 0.01-0.09% initially added NaF was recovered from the cells, whereas the cellular uptake of Ag(NH3)2F and 5-FU was below the detection limit. CONCLUSION: Cytotoxicity of fluoride compounds may not be directly linked to their tumor specificity nor to their apoptosis-inducing activity.

Enhancement of cytotoxic activity of sodium fluoride against human periodontal ligament fibroblasts by water pressure.

Whether or not water pressure enhances the cytotoxic activity of sodium fluoride (NaF) against human periodontal ligament fibroblast (HPLF) was investigated. Loading with water pressure (up to 5 g) alone did not affect the cell proliferation, but significantly enhanced the cytotoxic activity of millimolar concentrations of NaF. Cytotoxic activity of NaF was reduced by supplementation with Ca2+, whereas it was enhanced by removal of Ca2+ from the culture medium. However, the enhancement of cytotoxicity of NaF under water pressure was observed even in the Ca2+ -free medium. NaF failed to induce apoptosis markers, such as the caspase-3, -8 and -9 activation, the intemucleosomal DNA fragmentation, the loss of cell surface microvilli and the changes in intracellular concentration of polyamines. Transmission electron microscopy demonstrated that the combination of NaF and water pressure slightly increased the incidence of the formation of autophagosomes engulfing organella, suggesting the induction of non-apoptotic cell death in HPLF cells. The present study suggests that the external pressure is an additional factor that enhances the cytotoxicity of NaF against HPLF cells.

Changes in fluoride sensitivity during in vitro senescence of normal human oral cells.

We have previously reported that sodium fluoride (NaF) showed slightly higher cytotoxicity against human oral tumor cell lines than normal human oral cells. Possible changes in the NaF sensitivity of three normal human oral cell types (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) during in vitro ageing were investigated in the present study. When these cells were subcultured at 1:4 split ratio every week, their saturation density declined with increasing population doubling level (PDL), and they ceased to divide when they reached 20 PDL. Mitochondrial function, evaluated by MTT stainability per cell basis, was elevated at the terminal phase. NaF dose-dependently reduced the viable cell number, but did not show any beneficial (growth promoting) effect (so-called "hormesis") at lower concentrations. NaF produced large DNA fragments, without induction of internucleosomal DNA fragmentation, possibly due to weak activation of caspases -3, -8 and -9. Higher concentrations of NaF were required to reduce the number of viable senescent cells than younger cells, indicating that cells become resistant to cytotoxicity of NaF with in vitro ageing.

Effect of antioxidants, oxidants, metals and saliva on cytotoxicity induction by sodium fluoride.

We have recently found that millimolar concentrations of sodium fluoride (NaF) induced apoptotic cell death, characterized by caspase activation and DNA fragmentation, in tumor cell lines. This finding paved the way to investigating the interaction between NaF and the oral environment. As an initial step, we investigated redox compounds, metals and saliva, which may modify the cytotoxic activity of NaF against a human oral squamous cell carcinoma cell line (HSC-2). The minimum exposure time to NaF required for cytotoxicity induction was 8 hours. Noncytotoxic concentrations of antioxidants (sodium ascorbate, gallic acid, epigallocatechin gallate, chlorogenic acid, curcumin, superoxide dismutase, catalase), oxidants (hydrogen peroxide, sodium hypochlorite), metals (CuCl, CuCl2, FeCl2, FeCl3, CoCl2) or saliva neither protected against, nor enhanced the cytotoxic activity of NaF. Cytotoxic concentrations of these compounds produced somewhat additive, but not synergistic, effects on the cytotoxicity of NaF. ESR analysis demonstrated that NaF did not apparently change the radical intensity of sodium ascorbate and gallic acid, measured under alkaline conditions. During the cell death induction in human promyelocytic leukemia HL-60 cells by NaF, the consumption of glucose rapidly declined, followed by a decline in the consumption of major amino acids. The present study suggests that the cytotoxic activity of NaF is not regulated by the redox mechanism, but rather linked to the rapid decline in glucose consumption at early stage.

Effect of endodontic agents on cytotoxicity induction by sodium fluoride.

We investigated six endodontic agents for their ability to induce apoptosis and modify the cytotoxic activity of NaF against human squamous cell carcinoma (HSC-2) and human promyelocytic leukemia (HL-60) cell lines. Four Group I agents (Form Cresol, Cam Phenic, Eucaly Soft, GC Fuji Varnish), but not two Group II agents (Caviton, Canals-N), induced internucleosomal DNA fragmentation and activated caspases 3, 8 and 9 in HL-60 cells. Only Cam Phenic among these agents additively enhanced the cytotoxic activity of NaF in HSC-2 and HL-60 cells. Form Cresol and Cam Phenic reduced the glucose consumption at early stage, possibly due to their toxic effect. Amino acid analysis suggests that the higher cytotoxicity of Form Cresol may be derived, at least in part, from its oxidizing action.

Effect of antitumor agents on cytotoxicity induction by sodium fluoride.

We have recently found that sodium fluoride (NaF) induced apoptotic cell death in tumor cell lines. We investigated here whether 6 popular antitumor compounds modify the cytotoxic activity of NaF against human squamous cell carcinoma (HSC-2) and human promyelocytic leukemia (HL-60) cell lines. Cytotoxic concentrations of cisplatin, etoposide, doxorubicin or peplomycin (tentatively termed as Group I compounds), but not methotrexate and 5-FU (tentatively termed as Group II compounds), enhanced the cytotoxic activity of NaF. NaF and Group I compounds induced internucleosomal DNA fragmentation in HL-60 cells, whereas Group II compounds were inactive even in the presence of NaF. Most Group I compounds except doxorubicin (which induced DNA fragmentation less effectively than others) activated caspase 3 more efficiently than Group II compounds. Caspase 8 (involved in non-mitochondrial extrinsic pathway) and caspase 9 (involved in mitochondrial intrinsic pathway) were also activated, but to a much lesser extent. NaF reduced the glucose consumption at early stage, possibly by inhibition of glycolysis, whereas cisplatin and etoposide reduced the glucose consumption at later stage, suggesting that early decline of glucose consumption is rather specific to NaF.