Inhibitory effects of tocopherols on expression of the cyclooxygenase-2 gene in RAW264.7 cells stimulated by lipopolysaccharide, tumor necrosis factor-α or Porphyromonas gingivalis fimbriae.

BACKGROUND: Tocopherols, which include α-, ß-, γ-, and δ-tocopherol, protect cells against harmful free radicals and play an important role in preventing many human diseases such as cancer, inflammatory disorders, and ageing itself. However, the causal relationships between periodontal or oral chronic diseases and tocopherols have not been sufficiently studied. The present study investigated the inhibitory effects of these compounds on the expression of cyclooxygenase-2 (COX2) mRNA in RAW264.7 cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor-α (TNFα) or fimbriae of Poryphyromonas gingivalis (Pg), an oral anaerobe. MATERIALS AND METHODS: The cytotoxicity (EC50) of tocopherols toward RAW cells was determined using a cell counting kit (CCK-8). The regulatory effect of these compounds on the expression of COX2 mRNA stimulated with LPS, TNFα or Pg fimbriae was investigated using real-time polymerase chain reaction (PCR). RESULTS: Each tocopherol had similarly low cytotoxicity. COX2 gene expression in RAW cells after exposure to the three different macrophage activators was inhibited by the tocopherols (p<0.01). Compared to α-tocopherol, ß-, γ- and δ-tocopherol exhibited greater inhibitory effects (p<0.05). CONCLUSION: Tocopherols exhibit anti-inflammatory activity, and ß-, γ- and δ-tocopherol have particularly more potent anti-inflammatory activity than α-tocopherol. Tocopherols may have potential utility for prevention of periodontal and chronic oral diseases.

Re-evaluation of anti-inflammatory potential of eugenol in IL-1ß-stimulated gingival fibroblast and pulp cells.

BACKGROUND: We recently reported that eugenol exerted comparable cytotoxicity towards human normal and tumor cells. In the present study, we investigated the effect of eugenol on interleukin-8 (IL-8) production by IL-1ß-stimulated oral cells. MATERIALS AND METHODS: The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. IL-8 released into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: IL-1ß (5 ng/ml) induced two orders of magnitude higher production of IL-8 by human cultured cells than unstimulated cells. Upon IL-1ß stimulation, both gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF) produced the greatest amounts of IL-8 (approximately 200-300 ng/ml), followed by pulp cells (HPCs) (approximately 40-50 ng/ml), whereas skin keratinocyte (HaCat) and oral squamous cell carcinoma cells (HSC-2, HSC-4) produced much less IL-8 (less than 15 ng/ml). The production of IL-8 depended on growth factor(s), since the omission of fetal bovine serum from the culture medium resulted in an approximately 90% decline of IL-8 production. Eugenol (5-500 µM) significantly stimulated IL-8 production in HGF cells, but had bi-modal effects on HPCs, causing slight stimulation at lower concentration (5 µM) and a significant inhibition at higher concentration (500 µM), regardless of the presence or absence of serum. Eugenol exerted similar effects on lipopolysaccharide-stimulated HGFs and HPCs. CONCLUSION: These results demonstrate that an anti-inflammatory effect of eugenol is observed in HPCs, but not in HGFs. The narrow therapeutic range of eugenol suggests the importance of careful usage of this compound for dental treatment.

Changes of metabolic profiles in an oral squamous cell carcinoma cell line induced by eugenol.

BACKGROUND: We have recently reported that eugenol exerted indiscriminate cytotoxicity towards normal oral cells and oral squamous cell carcinoma (OSCC) cell lines without induction of apoptosis markers. In order to investigate the underlying mechanisms of cytotoxicity induction, we investigated the effect of short-term treatment with eugenol on the metabolic profiles of a human OSCC cell line (HSC-2). MATERIALS AND METHODS: The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. After washing with 5% D-mannitol solution (found to retain the highest amounts of intracellular metabolites among several washing conditions), cellular metabolites were extracted with methanol with internal markers and then subjected to metabolomic analysis. RESULTS: Cytotoxic concentrations of eugenol induced the reduction of ATP utilization (assessed by a significant reduction of the AMP/ATP and ADP/ATP ratio), of oxidative stress (assessed by the increase in oxidized form of glutathione, cysteine-glutathione disulfide and methionine sulfoxide), and an increase in the polyamines and glycolytic metabolites. CONCLUSION: The metabolic changes observed in this study suggest the induction of non-apoptotic cell death by eugenol.

Cytotoxicity of dental compounds towards human oral squamous cell carcinoma and normal oral cells.

AIM: The cytotoxicity of four dental compounds, hydroquinone, benzoquinone, eugenol and phtharal towards human oral squamous cell carcinoma (OSCC) cell lines, normal human oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) and skin keratinocytes was investigated. MATERIALS AND METHODS: Viable cell number was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The concentration that reduced the viable cells by 50% (CC(50)) and the concentration that increased the viability of UV-irradiated cells to 50% (EC(50)) were determined from the dose-response curves. The tumor-specificity index (TS) was determined by the ratio of the mean CC(50) for normal cells to the one for tumor cells. Apoptosis induction was monitored by assay of internucleosomal DNA fragmentation and caspase-3/-7 activation. RESULTS: When both oral OSCC and normal oral cells were incubated for 4 h with any of hydroquinone, benzoquinone, eugenol and phtharal, irreversible cell growth inhibition, accompanied by cell death occurred without induction of apoptotic markers, although caspase-3/-7 activation was observed at 6 h or later. These compounds exhibited very low tumor-specificity (TS=0.4-1.3), as compared with anticancer drugs (5-fluorouracil, melphalan, peplomycin) (TS=4.1-9.7). Human skin keratinocytes were the most resistant to these drugs, and a long incubation time was required to induce irreversible growth inhibition. However, all dental compounds exhibited very low tumor-specificity (TS=0.4-2.4), compared to human skin keratinocytes and OSCC cell lines. None of the dental compounds exhibited any hormetic growth stimulation, nor protected the cells from UV-induced damage. CONCLUSION: These results suggest that apoptosis is not involved in the early stage of growth inhibition induced by dental compounds.

Anti-UV activity of lignin-carbohydrate complex and related compounds.

BACKGROUND: We recently reported that an alkaline extract of the leaves of Sasa senanensis Rehder (SE) and Lentinus edodes mycelia extract (LEM), exhibiting lignin-carbohydrate complex (LCC)-like activity, protected cells from UV-induced injury (referred to as anti-UV activity). We investigated whether LCC is the major active components responsible for anti-UV activity. MATERIALS AND METHODS: Human oral squamous cell carcinoma HSC-2 cells were exposed to short UV irradiation in phosphate-buffered saline, containing different concentrations of LCC. After culturing for 48 h in fresh culture medium, the viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. From the dose-response curve, the 50% cytotoxic concentration (CC(50)) and the concentration that increased the viability of the UV-irradiated cells to 50% of the control value (EC(50)) were determined. The selectivity index (SI) was determined by the following equation: SI=CC(50)/EC(50). RESULTS: LCCs (Fr. VI) of pine cones and seed shell, and sulfated LCC exhibited relatively high anti-UV activity (SI=7.1-38), compared with that of SE and LEM. LCCs with lower lignin content (Fr. VII) exhibited anti-UV activity, approximately one half that of Fr. VI. However, polysaccharides (laminarin, pullulan, dextran) introduced with dimethylaminoethyl- or sulfate groups with different substitution ratios were totally inactive (SI<1). The introduction of a sulfate group to LCC did not enhance the anti-UV activity of LCC. Sodium ascorbate and vanillin were the most active (SI=65), whereas gallic acid (SI=5), epigallocatechin gallate (SI=2.6), ar-trumeron (SI<1), and turmeric extract (SI<1) were much less active. CONCLUSION: The prominent anti-UV activity of SE and LEM seems to be generated by LCCs present in the extract.

Comparative inhibitory effects of magnolol, honokiol, eugenol and bis-eugenol on cyclooxygenase-2 expression and nuclear factor-kappa B activation in RAW264.7 macrophage-like cells stimulated with fimbriae of Porphyromonas gingivalis.

BACKGROUND: The anti-inflammatory activity of magnolol and related compounds is currently a focus of interest. In the present study, the inhibitory effects of these compounds on cyclooxygenase (COX-2) expression and nuclear factor-kappa B (NF-κB) activation were investigated in RAW264.7 macrophage-like cells stimulated with the fimbriae of Porphyromonas gingivalis, an oral anaerobe. MATERIALS AND METHODS: The cytotoxicity of magnolol, honokiol, eugenol and bis-eugenol against RAW264.7 cells was determined using a cell counting kit (CCK-8). The regulatory effect of these compounds on the expression of COX-2 mRNA, stimulated by exposure to the fimbriae was investigated by real-time polymerase chain reaction (PCR). NF-κB activation was evaluated by enzyme-linked immunosorbent assay (ELISA)-like microwell colorimetric transcription factor activity assay (Trans-AM) and western blot analysis. The radical-scavenging activity was determined using the induction period method in the methyl methacrylate-azobisisobutyronitrile (AIBN) polymerization system under nearly anaerobic conditions. The phenolic bond dissociation enthalpy (BDE) and orbital energy were calculated at the density functional theory (DFT) B3LYP/6-31G* level. RESULTS: The cytotoxicity against RAW264.7 cells declined in the order bis-eugenol>eugenol> honokiol>magnolol, whereas the radical-scavenging activity declined in the order honokiol, bis-eugenol>magnolol> eugenol. Magnolol and honokiol significantly inhibited the fimbria-induced expression of COX-2 at non-cytotoxic concentrations. Both the fimbria-stimulated binding of NF-κB to its consensus sequence and phosphorylation-dependent proteolysis of inhibitor κB-α were markedly inhibited by magnilol and honokiol, whereas eugenol and bis-eugenol did not inhibit COX-2 expression and NF-κB activation. Magnolol and honokiol possessed a high electronegativity (χ) value. CONCLUSION: Magnolol and honokiol exhibit antioxidative activity, low cytotoxicity, and anti-inflammatory activity. These compounds may be capable of preventing chronic inflammatory diseases induced by oral bacteria.

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.