Proliferative effect of cannabidiol in human gingival fibroblasts via the mitogen-activated extracellular signal-regulated kinase (MEK) 1/2.

BACKGROUND AND OBJECTIVES: Cannabidiol exerts its anti-inflammatory and anti-oxidant activities in various human cells. However, its proliferative effect has not been extrapolated to human gingival fibroblasts (HGFs). This study aimed to determine the proliferative and promigratory effects of cannabidiol in HGFs and to elucidate the signaling mechanism(s). MATERIALS AND METHODS: HGFs, characterized by their CD73, CD90, and CD105 expressions by flow cytometry, were treated with cannabidiol at 0.01-30 µM. The cytotoxicity was determined by the MTT assay, while the proliferative effect was examined by the BrdU assay, immunoblot and immunofluorescence for cyclin D1 and Ki-67 expressions, respectively, and cell cycle analysis. The promigratory effect of cannabidiol was investigated by a wound healing assay. Phosphorylation of the p38 MAPK, JNK, and ERK upon treatment with cannabidiol was explored, and their involvement in cell proliferation and cyclin D1 and Ki-67 expressions was studied using pharmacological inhibitors. RESULTS: No toxicity was found in HGFs treated with any doses of cannabidiol up to 30 µM. The mean percentage of cell proliferation was significantly enhanced by treatment with cannabidiol at 3 or 10 µM (p < .001), consistent with upregulated expressions of cyclin D1 and Ki-67 and increased percentages of HGFs in the S and G2/M phases. Moreover, treatment with cannabidiol significantly induced cell migration (p < .05). The p38 MAPK and ERK1/2 were significantly activated by cannabidiol (p < .05), but only pretreatment with UO126, a MEK1/2 inhibitor, significantly inhibited cell proliferation and cyclin D1 and Ki-67 expressions (p < .05). CONCLUSION: Treatment with cannabidiol at non-toxic doses promotes HGFs' proliferation and migration.

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells.

OBJECTIVE: To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1ß, in cultured human dental pulp cells. METHODOLOGY: Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1ß in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. RESULTS: Stimulation of the pulp cells with IL-1ß resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1ß (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1ß was also blocked by incubation with the extract. CONCLUSIONS: Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1ß in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells

Abstract Objective To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1β, in cultured human dental pulp cells. Methodology Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1β in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. Results Stimulation of the pulp cells with IL-1β resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1β (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1β was also blocked by incubation with the extract. Conclusions Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1β in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

Overexpression of methyltransferase-like 3 and 14 in oral squamous cell carcinoma.

OBJECTIVES: This study aimed to determine expressions of methyltransferase-like 3 (METTL3) and METTL14, two enzymes essential for mRNA methylation at the adenosine (m6 A), in oral squamous cell carcinoma (OSCC) and to investigate in vitro aggressiveness of their aberrant expressions. METHODS: METTL3 and METTL14 expressions in 50 OSCC and 11 normal oral tissues were examined by immunohistochemistry. METTL3 and METTL14 expressions and m6 A amounts were determined in three OSCC cell lines, including HN5, HN6, and HN15. Cell proliferation, migration, and invasion were studied by BrdU, wound healing, and Transwell chamber assays, after silencing of METTL3, METTL14, or both by siRNA transfection. RESULTS: Immunostaining of METTL3 and METTL14 was localized in cancer cell nuclei. The mean percentages of METTL3- and METTL14-positive cells were significantly increased in OSCC tissues (p < 0.001). The percentages of METTL3- and METTL14-positive cells were correlated with the advanced pTNM stages (p < 0.05) and with the degrees of histopathological differentiation in OSCC (r = 0.564 and r = 0.316, respectively; p < 0.001). By the COX multivariate analysis, both overexpressed METTL3 and METTL14 were significantly associated with short overall survival (p < 0.05). Both METTL3 and METTL14 expressions and the m6 A amounts were significantly increased in HN6 (p < 0.05). Silencing of METTL3 and METTL14 in HN6 significantly inhibited cell proliferation (p < 0.01), but it failed to mitigate cell migration or invasion. CONCLUSIONS: METTL3 and METTL14 are overexpressed in OSCC tissues and in the HN6 OSCC cell line that promotes cell proliferation. Overexpressed METTL3 or METTL14 is found to be an independent prognostic factor for short overall survival in patients with OSCC.

Anticancer Activities of Hesperidin via Suppression of Up-Regulated Programmed Death-Ligand 1 Expression in Oral Cancer Cells.

Up-regulated expression of programmed death-ligand 1 (PD-L1) by interferon-gamma (IFN-γ) has been associated with promotion of cancer cell survival and tumor cell escape from anti-tumor immunity. Therefore, a blockade of PD-L1 expression can potentially be used as a molecular target for cancer therapy. The aim of this study was to investigate whether suppression of IFN-γ induced PD-L1 expression in two oral cancer cell lines, HN6 and HN15, by hesperidin effectively decreased cell proliferation and migration. Further, our objective was to elucidate the involvement of the signal transducer and activator of transcription 1 (STAT1) and STAT3 in the inhibition of induced PD-L1 expression by hesperidin. Our findings indicate that IFN-γ induced expression of PD-L1 protein in HN6 and HN15 via phosphorylation of STAT1 and STAT3 and that hesperidin significantly reduced that induction through suppression of phosphorylated STAT1 and STAT3 in both cell lines. Moreover, hesperidin also significantly decreased the viability, proliferation, migration, and invasion of both cell lines. In conclusion, hesperidin exerted anticancer effects against oral cancer cells through the suppression of PD-L1 expression via inactivation of the STAT1 and STAT3 signaling molecules. The findings of this study support the use of hesperidin as a potential adjunctive treatment for oral cancer.

Efficacy of double antibiotics in hydroxypropyl methylcellulose for bactericidal activity against Enterococcus faecalis and Streptococcus gordonii in biofilm.

OBJECTIVE: This study aimed to investigate the efficacy of double antibiotics, including ciprofloxacin and metronidazole, in a new vehicle, hydroxypropyl methylcellulose (HPMC), against Enterococcus faecalis and Streptococcus gordonii grown in biofilm. DESIGN: Human mandibular premolars were prepared and divided into four groups: (i) negative control, (ii) positive control, (iii) infected with E. faecalis and S. gordonii for 21 days and intracanally medicated with double antibiotics in HPMC, and (iv) infected with E. faecalis and S. gordonii for 21 days and intracanally medicated with calcium hydroxide (UltraCal™). The efficacy of medication for 14 or 28 days was determined by bacterial cultures and RT-qPCR for absolute quantities of E. faecalis and S. gordonii cDNA and for relative mRNA expressions of pbp5 and gtfG genes. RESULTS: There were significant decreases in the mean colony forming units and mean cDNA amounts of E. faecalis and S. gordonii in group (iii) on days 14 and 28 compared to those in group (ii) (p < 0.01). However, the mean cDNA amounts of E. faecalis and S. gordonii in group (iv) were found to be significantly increased on day 28 (p < 0.05). The mRNA expression of gtfG was significantly decreased in groups (iii) and (iv) on days 14 and 28, whereas that of pbp5 was significantly increased in group (iv) on days 14 and 28 (p < 0.01). CONCLUSION: Double antibiotics in HPMC gel showed an in vitro efficacy against E. faecalis and S. gordonii grown in biofilm, suggesting its clinical application as an intracanal medicament for both primary and persistent infections.

Involvement of the A disintegrin and metalloproteinase 9 in oral cancer cell invasion.

The aims of this study were to determine the functional roles of the transmembrane glycoprotein, Disintegrin and metalloproteinase domain-containing protein 9 (ADAM 9), in the phosphorylation of epidermal growth factor receptor (EGFR) and AKT and in the aggressiveness of oral cancer cells. Immunohistochemistry and immunoblotting were conducted to determine expression of ADAM 9 and the levels of EGFR phosphorylated at the tyrosine 1173 residue (p-EGFRtyr1173 ) and AKT phosphorylated at the serine 473 residue (p-AKTser473 ) in oral cancer tissues and in the oral cancer cell lines HN5, HN6, HN15, and HN008. Small interfering RNA (siRNA) was used to inhibit expression of ADAM9 mRNA, and thus production of ADAM9 protein, in oral cancer cells. ADAM9-knockdown cells were examined for p-EGFRtyr1173 and p-AKTser473 levels and used for cell proliferation and invasion assays. A positive correlation among overexpression of ADAM 9, p-EGFRtyr1173 , and p-AKTser473 was found in oral cancer tissues. These biomolecules were also overexpressed in HN6 and HN15 cell lines. Expression of ADAM9 in HN6 and HN15 cells was statistically significantly inhibited by siRNA against ADAM9 mRNA (siADAM9) compared with the negative-control siRNA (scramble). The levels of p-AKTser473 , but not those of p-EGFRtyr1173 , were statistically significantly blocked by siADAM9. Although the proliferation rates of ADAM9 knocked-down HN6 and HN15 cells did not differ from those of cells exposed to scramble, a statistically significant decrease in cell invasion was found in these ADAM9-silenced cells. These results suggest a functional role of the ADAM 9/AKT signaling pathway in oral cancer cell invasion, which may be beneficial as a therapeutic target of oral cancer.

Significant elevation of salivary human neutrophil peptides 1-3 levels by probiotic milk in preschool children with severe early childhood caries: a randomized controlled trial.

OBJECTIVES: To determine salivary human neutrophil peptides 1-3 (HNP1-3) levels in caries-free preschool children and in those with early childhood caries (ECC) or severe-ECC, in a daily probiotic group, receiving reconstituted milk with the probiotic Lactobacillus paracasei SD1 once daily; a triweekly probiotic group, receiving the probiotic milk 3 days a week; and a placebo group. MATERIALS AND METHODS: Oral examination and unstimulated whole saliva collection were conducted in 354 children at baseline, 6 months after intervention (T6), and after probiotic discontinuation (T12). Of the 354, adequate volume of saliva samples from 268 children were simultaneously analyzed for Streptococcus mutans and total lactobacilli levels using qPCR and for HNP1-3 levels using ELISA. RESULTS: In the severe-ECC status, significant increases in the median HNP1-3 levels at T12 were found in both daily and triweekly probiotic groups (p < 0.001). The median S. mutans levels in the daily group were significantly decreased at T6 and T12 (p < 0.01), whereas the median total lactobacilli levels were significantly increased at T6 (p < 0.001). Significantly inverse correlations between altered HNP1-3 and S. mutans levels and significant decreases in caries progression were found in both probiotic groups (p < 0.05). CONCLUSIONS: In the severe-ECC status, daily or triweekly consumption of L. paracasei SD1 significantly enhanced salivary HNP1-3 levels, but reduced S. mutans levels, possibly resulting in reduction of caries progression. CLINICAL RELEVANCE: Significant enhancement of salivary HNP1-3 levels by probiotic consumption is associated with reduction in S. mutans levels, consistent with diminished caries progression in children with severe-ECC.

Efficacy of Probiotic Milk for Caries Regression in Preschool Children: A Multicenter Randomized Controlled Trial.

This multicenter double-blind randomized controlled trial aimed to determine the efficacy of daily or triweekly consumption of reconstituted milk powder, containing Lactobacillus paracaseiSD1, in preschool children for caries prevention. A 6-month intervention was conducted in 487 children (aged 37.6 ± 9.2 months) with ≤4 decayed teeth from 8 childcare centers. Using stratified block randomization, participants were randomly allocated into 3 groups, comprising: (1) daily probiotic, receiving probiotic milk once daily, (2) triweekly probiotic, randomly receiving probiotic milk 3 days a week and the placebo milk for the remaining 4 days, and (3) placebo, receiving milk without probiotics. Each tooth surface was assessed for caries status using the modified Nyvad criteria at baseline (T0), 6 (T6) and 12 (T12) months after T0. Study outcomes were caries transition, including caries progression and regression during the T0-T6, T6-T12 and T0-T12 periods. Negative binomial regression with a generalized linear model was used to estimate the caries outcomes, which were reported as incidence rate ratios (IRRs) and 95% confidence intervals (CIs). At baseline, caries prevalence and mean decayed surfaces were 81.9% and 7.29 ± 7.60, respectively. Three groups were balanced at baseline with an overall dropout rate of 25.2%. After adjusting for age, sex, and the number of noncavitated and cavitated caries at baseline, a decreased caries risk during T0-T6 was shown in both the daily and triweekly probiotic groups (adjusted IRR 0.83, 95% CI 0.72-0.96 in both groups) and during T0-T12 (adjusted IRR 0.84, 95% CI 0.76-0.94, and adjusted IRR 0.86, 95% CI 0.76-0.96, respectively) compared to the placebo group. In contrast, significantly increased regressive surfaces were demonstrated during T0-T6 by adjusted IRR 1.76 (95% CI 1.25-2.48) and 2.01 (95% CI 1.42-2.85) and during T0-T12 by adjusted IRR 1.98 (95% CI 1.50-2.61) and 1.92 (95% CI 1.45-2.55) for the daily and triweekly groups, respectively. In conclusion, probiotic milk consumption either daily or triweekly can modestly prevent new caries, but considerably reverse carious lesions, suggesting that a daily or triweekly dose interval is sufficient to reverse carious lesions.

Periodontal ligament proliferation and expressions of bone biomolecules upon orthodontic preloading: Clinical implications for tooth autotransplantation.

OBJECTIVE: Preservation of the periodontal ligament (PDL) is vital to the success of tooth autotransplantation (TAT). Increased PDL volumes and facilitated tooth extraction have been observed upon orthodontic preloading. However, it is unclear whether any changes occur in the expressions of bone biomolecules in the increased PDL volumes. This study aimed to determine the expressions of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) in PDL upon preloading. METHODS: Seventy-two premolars from 18 patients were randomly assigned to experimental groups that received a leveling force for 1, 2, or 4 weeks or to a control unloaded group. Following extraction, PDL volumes from 32 premolars of eight patients (21.0 ± 3.8 years) were evaluated using toluidine blue staining. The expressions of the biomolecules in the PDL from 40 premolars of ten patients (21.4 ± 4.0 years) were analyzed via immunoblotting. RESULTS: The median percentage of stained PDL was significantly higher at 2 and 4 weeks after preloading than in the unloaded condition (p < 0.05). The median RUNX2 and ALP expression levels were significantly higher at 2 and 4 weeks after preloading than in the unloaded condition (p < 0.05), whereas the median RANKL/OPG ratios were significantly higher at 1 and 4 weeks after preloading (p < 0.05). CONCLUSIONS: Orthodontic preloading for 4 weeks enhances PDL volumes as well as the expressions of RUNX2, ALP and the RANKL/OPG ratio in the PDL, suggesting this loading period is suitable for successful TAT.

Effects of low-dose irradiation on human osteoblasts and periodontal ligament cells.

OBJECTIVE: To investigate the effects of dental x-ray on proliferation and mineralization in human primary osteoblasts as well as on proliferation and apoptotic potential in human periodontal ligament (PDL) cells. DESIGN: Primary osteoblasts and PDL cells were irradiated with various doses of periapical radiography by repeated exposures and further incubated for 1, 3 or 7 days. Cell proliferation was assayed by BrdU incorporation. The effect of dental x-ray on mineralization in osteoblasts either before or after x-ray exposures was determined by Alizarin red staining. Both mRNA and protein expressions of BCL-2, an anti-apoptotic gene, and BAX, a pro-apoptotic gene, in PDL cells were analyzed by RT-qPCR and immunoblotting analysis, respectively. RESULTS: Neither the proliferative nor the mineralization ability of irradiated osteoblasts was different from that of non-irradiated osteoblasts at any doses or time points. By contrast, there was a significant decrease in the proliferation of PDL cells on day 3 after repeated exposures to dental x-ray for 20 times (P < 0.05), whereas the ratio of BCL-2 to BAX mRNA and protein expressions in these irradiated PDL cells was significantly increased (P < 0.05). CONCLUSIONS: Upon multiple exposures to dental x-ray used in intraoral radiography up to 20 times, there is no effect on the proliferation or the mineralization of osteoblasts, whereas the proliferative and apoptotic potentials of PDL cells are transiently decreased.

Anesthetic efficacies of intrapapillary injection in comparison to inferior alveolar nerve block for mandibular premolar extraction: a randomized clinical trial.

OBJECTIVE: Intrapapillary injection (IPI) has been suggested to improve pulpal anesthesia of mandibular teeth and to avoid complications from inferior alveolar nerve block (IANB). This study aimed to determine and compare clinical efficacies and prostaglandin E2 (PGE2) levels between IPI and IANB. MATERIALS AND METHODS: IANB was randomly selected for mandibular premolar anesthesia on one side of 40 patients, whereas IPI was locally administered to the contralateral premolar. Pulpal anesthesia, pain during injection and extraction, patients' satisfaction, and complications were assessed from 30 patients. Gingival crevicular fluid from ten patients was collected for PGE2 quantification by ELISA. RESULTS: Of 30 patients, 18 preferred IPI after injection due to significantly faster mean onset of pulpal anesthesia (p < 0.001) and lower mean score of injection pain (p = 0.017) than IANB, but 21 preferred IANB instead after extraction due to less postoperative pain, consistent with the significantly lower median PGE2 level on the IANB side than that on the IPI at 30 min (p = 0.047). However, there was no difference in the mean satisfaction score between the two techniques. Ulcerated epithelium and sloughing tissues were found at the IPI site in some patients with complete healing within 2 weeks. CONCLUSIONS: The anesthetic efficacies of IPI for mandibular premolar extraction are comparable to those of IANB. However, postoperative pain and local complications at the IPI site should be considered. CLINICAL RELEVANCE: IPI may be used for dental procedures that require only a short anesthetic duration to avoid failure of pulpal anesthesia, complications, and discomfort from IANB.

Reduction of Streptococcus mutans by probiotic milk: a multicenter randomized controlled trial.

OBJECTIVE: To investigate the effects of probiotics, Lactobacillus paracasei SD1, on the quantities of Streptococcus mutans in saliva and plaque samples of preschool children. DESIGN: This randomized trial recruited 487 preschool children from eight childcare centers. Participants were assigned to receive a 6-month course of placebo milk daily (group I), probiotic milk either daily (group II) or three days a week (triweekly, group III). The absolute quantities of S. mutans and total lactobacilli in the saliva and plaque samples at baseline (T0), after intervention (T6), and 6 months after discontinuation (T12) were assessed by qPCR. RESULTS: Of 487 children, 354 completed all follow-up periods. However, only 268 children (3.2 ± 0.8 years old; groups I = 86, II = 89, and III = 93) provided adequate saliva for qPCR. Whereas the quantities of S. mutans were significantly decreased in groups II and III compared to group I in the saliva and plaque samples at T6 and T12, those of total lactobacilli were significantly increased (p < 0.0167). There was no difference in the quantities of S. mutans or total lactobacilli between groups II and III at any period. Significant changes in the quantities of S. mutans and total lactobacilli lasted until T12 compared to T0 (p < 0.0167). CONCLUSIONS: Probiotic administration daily or triweekly reduces S. mutans quantities, whereas it increases total lactobacilli quantities that persists at least 6 months after discontinuation in the saliva and plaque samples of preschool children. CLINICAL RELEVANCE: Daily or triweekly consumption of L. paracasei SD1 supplemented in milk may help prevent dental caries in preschool children.

Reduced ADAM8 levels upon non-surgical periodontal therapy in patients with chronic periodontitis.

OBJECTIVE: To determine effect of non-surgical periodontal treatment on a disintegrin and metalloproteinase 8 (ADAM8) levels in gingival crevicular fluid (GCF) of patients with chronic periodontitis (CP) in comparison with those of patients with gingivitis and to find correlations between ADAM8 levels and clinical parameters. DESIGN: Twenty-two and eleven patients with CP and gingivitis, respectively, were examined for four clinical parameters, probing depth, clinical attachment level, gingival and plaque indices. GCF from the selected gingivitis or periodontitis sites with distinct severities was sampled by Periopaper strips. The non-surgical treatments, including scaling and/or root planing and oral hygiene instruction, were provided for all patients. Clinical measurements and GCF sampling were repeated at three months after the treatments. ADAM8 concentrations were analyzed by ELISA and normalized by GCF volumes or total protein amounts. RESULTS: All patients exhibited significant improvement of almost every clinical parameter after treatment, whereas the median ADAM8 concentrations were significantly decreased at the moderate and severe periodontitis sites of patients with CP (p < 0.05). Moreover, the significantly positive correlations between ADAM8 concentrations and four clinical parameters were found in both moderate and severe groups (p < 0.05). CONCLUSION: ADAM8 concentrations were decreased by non-surgical periodontal therapy in patients with chronic periodontitis at the moderate and severe sites and were correlated with four clinical parameters, implying that GCF ADAM8 levels reflect inflammatory and bone-resorbing activities in the periodontal pocket.

Inhibitory effect of Thai propolis on human osteoclastogenesis.

BACKGROUND/AIM: Avulsed teeth should be immediately replanted into the socket or otherwise kept in a physiologic storage medium to maintain periodontal ligament cell viability. A previous study has demonstrated that Thai propolis extract can maintain viability of human periodontal ligament cells. However, root resorption by osteoclasts often occurs when the avulsed teeth are replanted. The aim of this study was to determine the inhibitory effect of Thai propolis extract on human osteoclastogenesis in vitro. MATERIALS AND METHODS: Human peripheral blood mononuclear cells were isolated for osteoclast precursors and cultured in the presence or absence of various non-toxic concentrations of propolis extract, as determined by the alamarBlue® assay, during in vitro induction of osteoclastogenesis. Osteoclast formation was examined by tartrate-resistant acid phosphatase staining, actin ring formation, and real-time polymerase chain reaction. The resorption pit assay was performed to determine osteoclast function. RESULTS: Non-toxic concentrations of propolis extract suppressed osteoclast formation by significantly decreasing the percentages of tartrate-resistant acid phosphatase-positive multinuclear cells and the ratios of cells with F-actin ring formation (P < .01) in a dose-dependent fashion. Expression of several osteoclast-specific genes was significantly downregulated by propolis in a dose-dependent manner (P < .05). The percentages of resorption areas on dentin slices were significantly decreased by propolis (P < .05). CONCLUSIONS: Thai propolis can inhibit human osteoclast formation and function, which may be beneficial for prevention of root resorption following replantation of avulsed teeth.

O-GlcNAcylation in oral squamous cell carcinoma.

BACKGROUND: Two post-translational mechanisms commonly demonstrated in various cancers are protein phosphorylation and glycosylation by O-linked ß-N-acetylglucosamine (O-GlcNAc). However, only phosphorylation of the epidermal growth factor receptor (EGFR)/Akt pathway has been reported in oral squamous cell carcinoma (OSCC). Therefore, we aimed to determine both post-translational modifications in OSCC tissues and in oral cancer cells compared to normal tissues and oral keratinocytes and to find correlations of these modifications with histological grading. METHODS: Thirty-two OSCC and ten normal formalin-fixed and paraffin-embedded sections were probed with the anti-O-GlcNAc, anti-O-GlcNAc transferase (OGT), anti-phosphorylated-EGFRtyr1173 , and anti-phosphorylated-Aktser473 antibodies following standard immunohistochemistry. The immunohistochemical (IHC) score was determined using the Fromowitz standard. Whole cell lysates of oral cancer cells and normal oral keratinocytes were immunoblotted with the anti-O-GlcNAc antibody. RESULTS: The median IHC scores of O-GlcNAc or OGT between OSCC and normal tissues were not different, whereas those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 were significantly higher in OSCC than normal tissues (P < .001 and P < .01, respectively). Similarly, expression of O-GlcNAcylated proteins in oral cancer cells and normal oral keratinocytes did not differ. In the OSCC group, the median IHC scores of O-GlcNAc and OGT were significantly lower than those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 (P < .01 and P < .001, respectively). The IHC scores of O-GlcNAc or OGT were not determined to correlate with histological grading. CONCLUSION: Unlike other types of cancers, our findings demonstrate that the levels of O-GlcNAcylation are not significantly increased in OSCC tissues or in oral cancer cells and are not associated with the histological grading of OSCC.

Overexpression of ADAM9 in oral squamous cell carcinoma.

Overexpression of a disintegrin and metalloproteinase 9 (ADAM9) has been shown in various types of cancer. Some studies have reported inconclusive findings regarding chromosomal aberrations in the ADAM9-containing region and ADAM9 expression in oral cancer. Therefore, in this study, ADAM9 protein expression was determined and compared between oral squamous cell carcinoma (OSCC) and normal oral tissues, and between oral cancer cell lines and human oral keratinocytes (HOKs). In total, 34 OSCC and 10 healthy paraffin-embedded tissue sections were probed with an anti-ADAM9 antibody, and the immunohistochemical score was determined by multiplying the percentage of positively stained cells with the intensity score. Four different oral cancer and eight independent HOK cell lines were cultured, and the expression of membrane ADAM9 and active ADAM9 at 84 kDa in these cell lines was assayed by flow cytometry and western blot hybridization, respectively. The results showed that the median immunohistochemical score of ADAM9 expression in OSCC tissues was significantly greater than that in normal tissues (P<0.001). Furthermore, among OSCC cases, intense staining of ADAM9 expression was detected in well-differentiated and in moderately-differentiated OSCC; ADAM9 expression was also correlated with an increased degree of cell differentiation (r=0.557; P=0.001). Expression of membrane ADAM9 was present in 3/4 cancer cell lines. Expression of active ADAM9 varied among all the tested cell lines, but significantly higher ADAM9 expression was present in certain cancer cell lines than those in HOKs (P<0.05). In summary, ADAM9 expression is enhanced in OSCC and oral cancer cell lines, suggesting its role in the pathogenesis of oral cancer. Similar to the overexpression of ADAM9 in well-differentiated prostate cancer, high degrees of ADAM9 expression have also been observed in well-differentiated OSCC.

Pro-inflammatory cytokine responses in human gingival epithelial cells after stimulation with cell wall extract of Aggregatibacter actinomycetemcomitans subtypes.

Varying cytokine responses of human gingival epithelial cells (HGECs) by Aggregatibacter actinomycetemcomitans subtypes have been found. Most studies have used reference strains, whereas a few has evaluated the cytokine expression in response to clinical subtypes of this bacterial species. This study aimed to examine whether there was any difference in cytokine responses of HGECs stimulated with cell wall extract (CWE) from A. actinomycetemcomitans subtypes included clinical strains from Thai adult periodontitis, various serotypes and non-serotypeable strains, strains from deep or shallow pockets, and reference serotype strains. Totally 50 clinical strains and 7 reference strains of A. actinomycetemcomitans were analyzed for the expression of IL-1ß, IL-6, IL-8, and TNF-α mRNAs in HGECs by real time-PCR, and the IL-8 concentrations in cell-free supernatant measured using ELISA. An in vitro effect of released IL-8 on neutrophil migration was examined using transwell chambers. Result showed that among four cytokines studied, IL-8 mRNA was highly up-regulated by both clinical and reference strains. Serotype f revealed the highest expression compared to other serotypes. The JP2-like leukotoxin promoter gene and non-serotypeable (NS1 and NS2) demonstrated lower IL-8 responses compared to serotypeable strains, and IL-8 responses upon stimulation with clinical strains from deep pockets were also significantly lower than those isolated from shallow pockets (P < 0.01). Our findings suggest that the clinical isolates of A. actinomycetemcomitans associating with deep pockets, JP2-like leukotoxin promoter gene, NS1, and NS2 may interfere neutrophil function via minimal and immunosuppressing IL-8 responses, which may enhance their survival and virulence.

Effects of prostaglandin E2 on clonogenicity, proliferation and expression of pluripotent markers in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Based on our earlier work on the response of periodontal ligament (PDL) cells to mechanical stress by induction of cyclooxygenase expression and production of prostaglandin PGE2 that could regulate mineralization of PDL cells, it was hypothesized that PGE2 had potential effects on PDL stemness. In this study, we aimed to investigate clonogenicity, proliferation and expression of certain pluripotent markers, considered to be characteristics of PDL stemness, in response to treatment with exogenously-added PGE2. MATERIAL AND METHODS: Human PDL cells were cultured and treated with various doses of PGE2, and the aforementioned characteristics of PDL stemness were analyzed. RESULTS: The clonogenicity and proliferation were significantly enhanced by PGE2 at low concentrations (0.01, 0.1 and 1ng/ml; P<0.05), but only the proliferation was significantly diminished by PGE2 at a high concentration (100ng/ml; P<0.05). Expression of NANOG and OCT4 mRNA and protein was increased by PGE2 treatment at 0.1 and 1ng/ml. Consistently, expression of stage-specific embryonic antigen 4, a putative stem cell marker, was significantly augmented by PGE2 treatment at 1ng/ml (P<0.05). CONCLUSION: Our findings suggest that although a high dose of PGE2 (100ng/ml) inhibits proliferation of PDL cells, PGE2 at low doses appears to play a role in the maintenance of PDL stemness.

The antimicrobial peptide, human ß-defensin-1, potentiates in vitro osteoclastogenesis via activation of the p44/42 mitogen-activated protein kinases.

Previous studies have demonstrated increased expression and raised levels of human ß-defensin (hBD)-1 in gingival tissue and crevicular fluid of patients with chronic periodontitis and peri-implantitis, oral bone-resorbing diseases caused by enhanced osteoclastogenesis. Therefore, we aimed to investigate the effect of hBD-1 on osteoclast formation and function and to elucidate the involved signaling pathway in vitro. Human peripheral blood mononuclear cells (PBMCs) were first incubated with various doses of hBD-1 and cell viability was assayed by MTT. PBMCs were treated with macrophage-colony stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) in the presence or absence of non-toxic doses of hBD-1. In vitro osteoclastogenesis was analyzed by tartrate-resistant acid phosphatase (TRAP) staining, osteoclast-specific gene expression, and a resorption pit assay. Involvement of mitogen-activated protein kinases (MAPKs) was studied by immunoblotting and specific MAPK inhibitors. HBD-1 potentiated induction of in vitro osteoclastogenesis by RANKL, as shown by significantly increased number of TRAP-positive multinuclear cells and resorption areas on the dentin slices, and further up-regulated expressions of osteoclast-specific genes compared to those by RANKL treatment (p <0.05). However, hBD-1 treatment without RANKL failed to induce formation of osteoclast-like cells. A significant and further increase in transient phosphorylation of the p44/42 MAPKs was demonstrated by hBD-1 co-treatment (p<0.05), consistent with the inhibitory effect by pretreatment with U0126 or PD98059 on hBD-1-enhanced osteoclastogenesis. Collectively, hBD-1 potentiates the induction of in vitro osteoclastogenesis by RANKL via enhanced phosphorylation of the p44/42 MAPKs.