18­α­glycyrrhetinic acid induces apoptosis in gingival fibroblasts exposed to phenytoin.

Phenytoin (PHT)-induced gingival overgrowth is caused by the increased proliferation and reduced apoptosis of gingival fibroblasts in inflammatory gingiva. Licorice has long been used as a component of therapeutic preparations. It inhibits cell proliferation, induces cell apoptosis and has anti-inflammatory effects. 18-α-glycyrrhetinic acid (18α-GA), the active compound in licorice, promotes apoptosis in various types of cells. The present study determined whether 18α-GA affects apoptosis in gingival fibroblasts exposed to PHT. The present study aimed to establish a basis for the therapeutic application of 18α-GA to treat the gingival overgrowth induced by PHT. Human gingival fibroblasts from healthy donors were cultured to semi-confluence and then stimulated in serum-free DMEM containing PHT with or without 18α-GA for subsequent experiments. Apoptotic cells were detected by ELISA. Analysis of the distribution of cell cycle phases and the apoptotic cell population was performed by flow cytometry. The expression levels of mRNAs and proteins of apoptotic regulators were measured using reverse transcription-quantitative PCR and western blotting, respectively. Caspase (CASP) activities were assessed by an ELISA. Treatment with 18α-GA markedly increased the number of apoptotic cells, reduced BCL2 mRNA expression, increased CASP2 and receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) domain containing adaptor with death domain, Fas (TNFRSF6)-associated via death domain, RIPK1, tumor necrosis factor receptor superfamily; member 1A, TNF receptor-associated factor 2, CASP2, CASP3 and CASP9 mRNA expression, and also upregulated the protein expression levels and activities of caspase-2, caspase-3 and caspase-9. These results demonstrated that 18α-GA induced apoptosis through the activation of the Fas and TNF pathways in the death receptor signaling pathway in gingival fibroblasts treated with PHT. 18α-GA exhibited therapeutic potential for the treatment of PHT-induced gingival overgrowth.

Evaluation of the child oral health promotion 'MaliMali' Programme based on schools in the Kingdom of Tonga.

INTRODUCTION: The South Pacific Medical Team (SPMT) has supported oral health care for Tongan juveniles since 1998. This voluntary activity, named the MaliMali ('smile' in Tongan) Programme, is evaluated in detail in this paper. METHODS: This evaluation was guided by the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework. The objectives were to explore: (i) whether the programme was accessible to Tongan schoolchildren (Reach); (ii) the impact of the programme on decayed, missing and filled teeth (DMFT) scores and toothbrushing habits (Effectiveness); (iii) factors that affected the adoption of the programme (Adoption); (iv) whether implementation was consistent with the programme model (Implementation); and (v) the long-term sustainability of the programme (Maintenance). RESULTS: The MaliMali Programme has grown into an international project, has spread countrywide as a uniform health promotion and is reaching children in need. Following implementation of this programme, the oral health of Tongan juveniles has improved, with a decrease in the mean DMFT index and an increase in toothbrushing. To provide training that will allow Tongans to assume responsibility for the MaliMali Programme in the future, dental health education literature was prepared and workshops on oral hygiene and the MaliMali Programme were held frequently. At present, the programme is predominantly managed by Tongan staff, rather than by Japanese staff. CONCLUSIONS: This evaluation found the MaliMali Programme to be feasible and acceptable to children and schools in the Kingdom of Tonga. The programme promotes oral health and provides accessible and improved oral health care in the school setting, consistent with the oral health-promoting school framework.

Inhibition of G1 cell cycle arrest in human gingival fibroblasts exposed to phenytoin.

Gingival overgrowth is caused in response to the antiepileptic drug phenytoin (PHT). PHT-induced gingival overgrowth is characterized by the proliferation of fibroblasts and increased collagen formation in gingiva. Fibroblast proliferation is regulated through the cell cycle. Thus, in the present study, we examined the effects of PHT on the cell cycle, the expression of cell cycle control proteins and the proliferation in human gingival fibroblasts (hGFs). Cells were stimulated in serum-free DMEM with or without 0.25 µm PHT. Subsequently, the cell cycle phase distribution and the protein expression after 24 h and the cell proliferation after 24, 48 and 72 h were evaluated. PHT significantly inhibited synchronization at the G0/G1 phase of the cell cycle in hGFs through serum starvation. Stimulation with PHT for 48 and 72 h significantly induced a proliferative response in hGFs. PHT decreased the expression of the Cdk-inhibitory proteins p21 and p27 and increased the levels of the S phase-promoting proteins phospho-Thr160-Cdk2 and phospho-Ser807/811-Rb in serum-free DMEM. The inhibition of G1 cell cycle arrest in hGFs may result from an increase in phosphorylated Cdk2 and Rb proteins and decreased levels of p21 and p27 proteins by PHT. The gingival overgrowth may be caused by the failure of the G1 cell cycle arrest in GFs exposed to PHT.

Effect of school-based fluoride mouth-rinsing on dental caries incidence among schoolchildren in the Kingdom of Tonga.

Since 1998, the authors have been working to improve the oral health of children at kindergartens and primary schools in the Kingdom of Tonga (Tonga). Our primary activity has been a school-based fluoride mouth-rinsing (FMR) program. FMR is performed using 7-10 mL of a 0.2% NaF solution for 1 min once per week at each school. In the present study, we evaluated the effect of school-based FMR on dental caries incidence among Tongan schoolchildren. A total of 109 children aged 10 years were evaluated at six primary schools on Tongatapu Island. The FMR group comprised 46 children who had participated in the school-based FMR program for at least 5 years 6 months; the control group comprised 63 children who had participated in the school-based FMR program for 1 year or less. During standardized dental examinations, decayed, missing, and filled teeth were counted by a single dentist at each school. The school-based FMR program effectively decreased the number of dental caries. A school-based FMR program may thus be very beneficial in preventing caries among children in Tonga.

Reduction in lipopolysaccharide-induced apoptosis of fibroblasts obtained from a patient with gingival overgrowth during nifedipine-treatment.

OBJECTIVE: We have previously demonstrated that the mechanism of nifedipine (NIF)-induced gingival overgrowth is related to the observation that proliferation and cell cycle progression of gingival fibroblasts derived from NIF reactive patient (NIFr) are greater than those from NIF non-reactive patient (NIFn). Gingival overgrowth has also been reported to be a result of inhibited apoptosis of gingival fibroblasts. Apoptosis in fibroblasts is induced by lipopolysaccharide (LPS). Thus, we focused upon evaluating whether there is a difference in LPS-induced apoptosis between NIFn and NIFr. METHODS: Both NIFn and NIFr were arrested in DMEM containing 0.5% FBS, stimulated by LPS, and assayed for apoptosis, cell cycle analysis, Western blotting, and caspase activity. RESULTS: Compared to NIFn, the number of apoptotic cells was significantly decreased and the percentage of cells in S and G(2)/M phase was significantly increased in NIFr. The levels of Bax and cytochrome c proteins in NIFr were not up-regulated by LPS compared with NIFn. Both NIFn and NIFr displayed the following changes in protein expression: increased Bad, decreased Bcl-xL, and unchanged Bcl-2 and p53. Caspase-3 and -9 activities were significantly increased by LPS in NIFn but were unchanged in NIFr. Caspase-2 activity remained constant whilst caspase-8 activity significantly increased upon LPS treatment in both NIFn and NIFr. CONCLUSION: Bad, Bax, cytochrome c, p53, and caspases-2, -3, -8, and -9 are pro-apoptotic proteins. Bcl-2 and Bcl-xL are anti-apoptotic proteins. Thus, the mechanism of NIF-induced gingival overgrowth might be related to decreased apoptosis in NIFr through a reduction of Bax, cytochrome c, and caspase-3 and -9.

Differences of cell growth and cell cycle regulators induced by basic fibroblast growth factor between nifedipine responders and non-responders.

Differences of cell proliferation, cell cycle, and G(1)/S transition regulatory proteins of gingival fibroblasts derived from nifedipine-reactive patient (NIFr) and nifedipine-non-reactive patient (NIFn) in the presence of basic fibroblast growth factor (bFGF) were investigated to elucidate the mechanism of gingival overgrowth associated with nifedipine, one of the Ca(2+)-channel blockers. The proliferation rate of NIFr cells in the presence of bFGF significantly increased than NIFn cells. The proportion of NIFr cells that had undergone progression to the S and G(2)/M phases from the G(0)/G(1) phase significantly increased compared to that in NIFn cells. Increases of pRB (Ser807/811), pCDK2 (Thr160), CDK2, and cyclin E protein levels in NIFr cells were greater than those in NIFn cells. The elevations of pRB (Ser780), RB, and cyclin A protein levels in NIFr cells did not differ from those of NIFn cells. The growth of NIFr cells was greater than that of NIFn cells as a result of the active G(1)/S transition of NIFr cells, as assessed by the increments of cyclin E, pCDK2, and pRB (ser807/811) protein in NIFr cells.

The effect of basic fibroblast growth factor on cell cycle in human gingival fibroblasts from nifedipine responder and non-responder.

It has previously been demonstrated that gingival fibroblasts derived from nifedipine-reactive patients (nifedipine responders) show a greater cell proliferation rate than those from nifedipine non-reactive patients (nifedipine non-responders) in the presence of 1 microM nifedipine. The aim of the present study was to characterize cell cycle differences between nifedipine responder and non-responder fibroblast cells and determine the effect of basic fibroblast growth factor (bFGF) on cell cycle progression. Further, the effect of bFGF on cyclins A, B1, D1, E, and CDKs 1, 2, 4, 6 mRNA expression in responder and non-responder cells was investigated. A population of nifedipine responder cells underwent progression to S and G2/M phases from G0/G1 phase in the presence of 10% fetal calf serum or 10 ng/ml bFGF was greater than nifedipine non-responder cells. mRNA expression of cyclins A, B1, D1, E and CDKs 1, 2, 4, 6 in the presence of 10 ng/ml bFGF was generally greater in nifedipine responder cells than non-responder cells. These results indicate that nifedipine responder cells may be more susceptible to growth factors such as bFGF with a resultant increase in expression of cyclins and CDKs in responder compared with non-responder cells.

Cyclopiazonic acid discharges intracellular Ca2+ stores and stimulates Ca2+ influx in cultured human gingival fibroblasts.

The effect of cyclopiazonic acid (CPA) on changes in the intracellular free Ca2+ concentration ([Ca2+]i) evoked by bradykinin (BK), histamine (HIST) and thapsigargin (TG) was investigated in human gingival fibroblasts. CPA itself dose-dependently stimulated [Ca2+]i responses in both the absence and presence of extracellular Ca2+. Pretreatment with CPA (< 5 microM) enhanced the [Ca2+]i responses evoked by 5 nM BK and 1 mM HIST. However, CPA-pretreatment depressed the [Ca2+]i response evoked by 1 microM TG in a dose-dependent manner. Moreover, CPA accelerated the Ca2+ influx caused by 5 nM BK and 1mM HIST, but did not alter that caused by 1 microM TG. These results indicate that CPA discharges intracellular Ca2+ stores, resulting in their depletion, and enhances Ca2+ influx across the plasma membrane.