Porphyromonas gingivalis increases the invasiveness of oral cancer cells by upregulating IL-8 and MMPs.

Recent studies indicate that chronic inflammation promotes the aggressiveness of cancers. However, the direct molecular mechanisms underlying a functional link between chronic periodontitis, the most common form of oral inflammatory diseases, and the malignancy of oral cancer remain unknown. To elucidate the role of chronic periodontitis in progression of oral cancer, we examined the effect of Porphyromonas gingivalis (P. gingivalis), a major pathogen that causes chronic periodontitis, on the invasiveness of oral squamous cell carcinoma (OSCC) cells, including SCC-25, OSC-20 and SAS cells. Exposures to P. gingivalis promoted the invasive ability of OSC-20 and SAS cells via the upregulation of matrix metalloproteinases (MMPs), specifically MMP-1 and MMP-2. However, P. gingivalis-infected SCC-25 cells did not exhibit changes in their invasive properties or the low expression levels of MMPs. In an effort to delineate the molecular players that control the invasiveness, we first assessed the level of interleukin-8 (IL-8), a well-known inflammatory cytokine, in P. gingivalis-infected OSCC cells. IL-8 secretion was substantially increased in the OSC-20 and SAS cells, but not in the SCC-25 cells, following P. gingivalis infection. When IL-8 was directly applied to SCC-25 cells, their invasive ability and MMP level were significantly increased. Furthermore, the downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells attenuated their invasive potentials and MMP levels. Taken together, our findings strongly suggest that P. gingivalis infection plays an important role in the promotion of the invasive potential of OSCC cells via the upregulation of IL-8 and MMPs.

Prolonged and repetitive exposure to Porphyromonas gingivalis increases aggressiveness of oral cancer cells by promoting acquisition of cancer stem cell properties.

Periodontitis is the most common chronic inflammatory condition occurring in the human oral cavity, but our knowledge on its contribution to oral cancer is rather limited. To define crosstalk between chronic periodontitis and oral cancer, we investigated whether Porphyromonas gingivalis, a major pathogen of chronic periodontitis, plays a role in oral cancer progression. To mimic chronic irritation by P. gingivalis in the oral cavity, oral squamous cell carcinoma (OSCC) cells were infected with P. gingivalis twice a week for 5 weeks. Repeated infection of oral cancer cells by P. gingivalis resulted in morphological changes of host cancer cells into an elongated shape, along with the decreased expression of epithelial cell markers, suggesting acquisition of an epithelial-to-mesenchymal transition (EMT) phenotype. The prolonged exposure to P. gingivalis also promoted migratory and invasive properties of OSCC cells and provided resistance against a chemotherapeutic agent, all of which are described as cellular characteristics undergoing EMT. Importantly, long-term infection by P. gingivalis induced an increase in the expression level of CD44 and CD133, well-known cancer stem cell markers, and promoted the tumorigenic properties of infected cancer cells compared to non-infected controls. Furthermore, increased invasiveness of P. gingivalis-infected OSCC cells was correlated with enhanced production of matrix metalloproteinase (MMP)-1 and MMP-10 that was stimulated by interleukin-8 (IL-8) release. This is the first report demonstrating that P. gingivalis can increase the aggressiveness of oral cancer cells via epithelial-mesenchymal transition-like changes and the acquisition of stemness, implicating P. gingivalis as a potential bacterial risk modifier.

DHA suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

Several reports have indicated that dietary intake of DHA is associated with lower prevalence of periodontitis. In the present study, we investigated the effect of DHA on the production of proinflammatory mediators in murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. LPS was isolated from lyophilised P. intermedia ATCC 25,611 cells using the standard hot-phenol-water protocol. Culture supernatants were collected and assayed for NO, IL-1ß and IL-6. Real-time PCR analysis was carried out to detect the expression of inducible NO synthase (iNOS), IL-1ß, IL-6 and haeme oxygenase-1 (HO-1) mRNA. Immunoblot analysis was carried out to quantify the expression of iNOS and HO-1 protein and concentrations of signalling proteins. DNA-binding activities of NF-κB subunits were determined using an ELISA-based assay kit. DHA significantly attenuated the production of NO, IL-1ß and IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. DHA induced the expression of HO-1 in cells treated with P. intermedia LPS. Selective inhibition of HO-1 activity by tin protoporphyrin IX significantly mitigated the inhibitory effects of DHA on LPS-induced NO production. DHA significantly attenuated the phosphorylation of c-Jun N-terminal kinase induced by LPS. In addition, DHA suppressed the transcriptional activity of NF-κB by regulating the nuclear translocation and DNA-binding activity of NF-κB p50 subunit and inhibited the phosphorylation of signal transducer and activator of transcription 1. Further in vivo studies are needed to better evaluate the potential of DHA in humans as a therapeutic agent to treat periodontal disease.

Melatonin inhibits Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-6 in murine macrophages by suppressing NF-κB and STAT1 activity.

Although a range of biological and pharmacological activities of melatonin have been reported, little is known about its potential anti-inflammatory efficacy in periodontal disease. In this study, we investigated the effects of melatonin on the production of inflammatory mediators by murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory reactions in the periodontium, and sought to determine the underlying mechanisms of action. Melatonin suppressed the production of nitric oxide (NO) and interleukin-6 (IL-6) at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. P. intermedia LPS-induced NF-κB-dependent luciferase activity was significantly inhibited by melatonin. Melatonin did not reduce NF-κB transcriptional activity at the level of IκB-α degradation. Melatonin blocked NF-κB signaling through the inhibition of nuclear translocation and DNA-binding activity of NF-κB p50 subunit and suppressed STAT1 signaling. Although further research is required to clarify the detailed mechanism of action, we conclude that melatonin may contribute to blockade of the host-destructive processes mediated by these two proinflammatory mediators and could be a highly efficient modulator of host response in the treatment of inflammatory periodontal disease.

Perception of maxillary anterior esthetics by dental professionals and laypeople and survey of gingival topography in healthy young subjects.

The perceptions of dental professionals and laypeople were compared with respect to relative gingival marginal levels of the lateral incisors, flatness of the gingival margin of central incisors and its variations in horizontal locations, and interproximal contact length relative to gingival biotype. The distribution of tooth-gingiva topography in periodontally healthy subjects was also identified. A noticeable difference was observed between dental professionals and laypeople in their perception of maxillary anterior esthetic components. Normal distribution of the topographic features of anterior teeth and gingiva delineated an intermediate biotype between those preferred by dental professionals and those preferred by laypeople. The present study clearly indicates that dental professionals and laypeople demonstrated significant differences in their perceptions of dental esthetic components. These differences may influence the decision-making process by dental professionals with respect to designing the gingival levels of lateral incisors, the horizontal gingival contours of central incisors, and the shape of interproximal contact areas. (Int J Periodontics Restorative Dent 2009;29:535-541.).

Prevotella intermedia lipopolysaccharide stimulates release of tumor necrosis factor-alpha through mitogen-activated protein kinase signaling pathways in monocyte-derived macrophages.

The purpose of this study was to investigate the effects of lipopolysaccharide from Prevotella intermedia, a major cause of inflammatory periodontal disease, on the production of tumor necrosis factor (TNF)-alpha and the expression of TNF-alpha mRNA in differentiated THP-1 cells, a human monocytic cell line. The potential involvement of the three main mitogen-activated protein kinase (MAPK) signaling pathways in the induction of TNF-alpha production was also investigated. Lipopolysaccharide from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. It was found that P. intermedia lipopolysaccharide can induce TNF-alpha mRNA expression and stimulate the release of TNF-alpha in differentiated THP-1 cells without additional stimuli. Treatment of the cells with P. intermedia lipopolysaccharide resulted in a simultaneous activation of three MAPKs [extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38]. Pretreatment of the cells with MAPK inhibitors effectively suppressed P. intermedia lipopolysaccharide-induced TNF-alpha production without affecting the expression of TNF-alpha mRNA. These data thus provided good evidence that the MAPK signaling pathways are required for the regulation of P. intermedia lipopolysaccharide-induced TNF-alpha synthesis at the level of translation more than at the transcriptional level.

Subantibiotic dose of azithromycin attenuates alveolar bone destruction and improves trabecular microarchitectures in a rat model of experimental periodontitis: A study using micro-computed tomography.

Azithromycin, a macrolide antibiotic, has anti-inflammatory and immunomodulatory activities apart from its antibacterial properties. In this study, we examined the efficacy of subantibiotic dose of azithromycin on ligature-induced periodontitis in rats using micro-computed tomography (micro-CT) imaging and bone parameter analysis. Male Sprague-Dawley rats were allocated to the following four groups: non-ligation (NL) group; ligation-only (L) group; ligation-plus-subantibiotic dose azithromycin (SA) group; and 4) ligation-plus-antibiotic dose azithromycin (AA) group. The rats from Groups L, SA and AA were subjected to periodontitis by placing a ligature around lower right first molar. Immediately after ligation, the rats in SA and AA groups received daily intraperitoneal injections of azithromycin at a dosage of 3.5 or 10mg/kg body weight, respectively. The ligatures were maintained for 2weeks at which time the rats had their mandibles hemisected for micro-CT analysis. Subantibiotic dose of azithromycin strongly suppressed reductions in alveolar bone height and bone volume fraction caused by experimental periodontitis. When subantibiotic dosage of azithromycin was administered to rats, ligature-induced alterations in microarchitectural parameters of trabecular bone were significantly reversed. Rats treated with subantibiotic dose of azithromycin presented no significant difference compared to rats with antibiotic dosage in all parameters. While further studies are necessary, subantibiotic dose of azithromycin could be utilized as a host modulator for the treatment of periodontitis.

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

OBJECTIVE: This study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. DESIGN: LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1ß (IL-1ß) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1ß, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. RESULTS: Agomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1ß and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. CONCLUSIONS: Agomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease.

Oral cancer cells sustainedly infected with Porphyromonas gingivalis exhibit resistance to Taxol and have higher metastatic potential.

Major obstacles to improving the prognosis of patients with oral squamous cell carcinoma (OSCC) are the acquisition of resistance to chemotherapeutic agents and development of metastases. Recently, inflammatory signals are suggested to be one of the most important factors in modulating chemoresistance and establishing metastatic lesions. In addition, epidemiological studies have demonstrated that periodontitis, the most common chronic inflammatory condition of the oral cavity, is closely associated with oral cancer. However, a correlation between chronic periodontitis and chemoresistance/metastasis has not been well established. Herein, we will present our study on whether sustained infection with Porphyromonas gingivalis, a major pathogen of chronic periodontitis, could modify the response of OSCC cells to chemotherapeutic agents and their metastatic capability in vivo. Tumor xenografts composed of P. gingivalis-infected OSCC cells demonstrated a higher resistance to Taxol through Notch1 activation, as compared with uninfected cells. Furthermore, P. gingivalis-infected OSCC cells formed more metastatic foci in the lung than uninfected cells.

Surface-associated material from Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthase.

The purpose of this study was to examine the effects of surface-associated material (SAM) from Porphyromonas gingivalis, a major cause of inflammatory periodontal disease, on the production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. gingivalis SAM-induced NO production. SAM from P. gingivalis 381 was obtained by saline extraction. NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of iNOS and analysis of reverse transcription-polymerase chain reaction (RT-PCR) products were carried out. We found that P. gingivalis SAM can induce iNOS expression and stimulate the release of NO without additional stimuli and demonstrated an important role of the transcription factor NF-kappaB and microtubule polymerization in NO production. The production of NO required L-arginine, protein tyrosine kinase, and protein kinase C. The ability of P. gingivalis SAM to promote the production of NO may be important in the pathogenesis of inflammatory periodontal disease.

Genistein suppresses Prevotella intermedia lipopolysaccharide-induced inflammatory response in macrophages and attenuates alveolar bone loss in ligature-induced periodontitis.

OBJECTIVE: Genistein is a major isoflavone subclass of flavonoids found in soybean and a potent tyrosine kinase inhibitor. The present study aimed to assess the effect of genistein on the production of proinflammatory mediators in murine macrophages stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen associated with different forms of periodontal disease, and to evaluate its possible influence on alveolar bone loss in ligature-induced periodontitis using micro-computed tomography (micro-CT) analysis as well. DESIGN: LPS was isolated from P. intermedia ATCC 25611 by using the standard hot phenol-water method. Culture supernatants were analyzed for nitric oxide (NO) and interleukin-6 (IL-6). Inducible NO synthase (iNOS) protein expression was evaluated by immunoblot analysis. Real-time PCR was carried out to measure iNOS and IL-6 mRNA expression. In addition, effect of genistein on alveolar bone loss was evaluated in a rat model of experimental periodontitis using micro-CT analysis. RESULTS: Genistein significantly attenuated P. intermedia LPS-induced production of iNOS-derived NO and IL-6 with attendant decrease in their mRNA expression in RAW264.7 cells. In addition, when genistein was administered to rats, decreases in alveolar bone height and bone volume fraction induced by ligature placement were significantly inhibited. Genistein administration also prevented ligature-induced alterations in the microstructural parameters of trabecular bone, including trabecular thickness, trabecular separation, bone mineral density and structure model index. CONCLUSIONS: While additional studies are required, we suggest that genistein could be utilized for the therapy of human periodontitis in the future.

Elevated MicroRNA-128 in Periodontitis Mitigates Tumor Necrosis Factor-α Response via p38 Signaling Pathway in Macrophages.

BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from an inflammatory response to subgingival plaque bacteria, including Porphyromonas gingivalis. MicroRNA (miRNA) is a current focus in regulating the inflammatory processes. In this study, the inflammatory miRNA expression in gingival tissues of patients with periodontitis and of healthy individuals is compared, and its role in regulating the inflammatory response is examined. METHODS: Gingival tissues from patients with periodontitis and healthy individuals were collected for miRNA microarray. THP-1 and CA9-22 cells were challenged with P. gingivalis, and miRNA expression was determined by real-time polymerase chain reaction. Target genes for miRNA were predicted using TargetScanHuman database, and miRNA gene expressions were reviewed using public databases. For the functional study, THP-1 cells were transfected with a miRNA-128 mimic, and target gene expression was compared with THP-1 cells challenged with P. gingivalis. For the tolerance test, THP-1 cells transfected with miRNA-128 mimic were treated with phorbol 12-myristate 13-acetate (PMA) or paraformaldehyde (PFA)-fixed Escherichia coli. Tumor necrosis factor (TNF)-α production was determined by enzyme-linked immunosorbent assay, and mitogen-activated protein kinase (MAPK) protein phosphorylation was determined by Western blot. RESULTS: Gingival tissues from patients with periodontitis showed increased expression of miRNA-128, miRNA-34a, and miRNA-381 and decreased expression of miRNA-15b, miRNA-211, miRNA-372, and miRNA-656. THP-1 cells and CA9-22 cells challenged with P. gingivalis showed increased miRNA-128 expression. Among the predicted miRNA-128 target genes, several genes that are involved in MAPK signaling pathway showed similar gene expression pattern between P. gingivalis challenge and miRNA-128 mimic transfection. In THP-1 cells transfected with miRNA-128 mimic, TNF-α production was lower, and phosphorylation of p38 was inhibited when challenged with PMA or PFA-fixed E. coli. CONCLUSION: miRNA-128 may be involved in mitigating the inflammatory response induced by P. gingivalis in periodontitis.

Nitric oxide production and inducible nitric oxide synthase expression induced by Prevotella nigrescens lipopolysaccharide.

We have examined the effects of lipopolysaccharide (LPS) from Prevotella nigrescens, one of the causative agents of inflammatory periodontal disease and endodontic infections, on the production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in the murine macrophage cell line RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. nigrescens LPS-induced NO production. We found that P. nigrescens LPS can induce iNOS expression and stimulate the release of NO without additional stimuli and demonstrated an important role of the transcription factor NF-kappaB and microtubule polymerization in NO production. The production of NO required l-arginine and protein tyrosine kinase but not activation of protein kinase C. The ability of P. nigrescens LPS to promote the production of NO may be important in the pathogenesis of inflammatory periodontal disease and endodontic infections.

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)-donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1ß and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1ß and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this.

Carbon monoxide-releasing molecule-3 suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

This study was performed to analyze the effect of carbon monoxide (CO)-releasing molecule-3 (CORM-3) in alleviating the production of proinflammatory mediators in macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated with periodontal disease, and its possible mechanisms of action. LPS was isolated using the hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO) and interleukin-1ß (IL-1ß). Gene expression was quantified by real-time PCR, and protein expression by immunoblotting. DNA-binding activities of NF-κB subunits were determined using an ELISA-based kit. CORM-3 suppressed the production of inducible NO synthase (iNOS)-derived NO and IL-1ß at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. CORM-3 enhanced heme oxygenase-1 (HO-1) expression in cells stimulated with P. intermedia LPS, and inhibition of HO-1 activity by SnPP notably reversed the suppressive effect of CORM-3 on LPS-induced production of NO. LPS-induced phosphorylation of p38 and JNK was not affected by CORM-3. CORM-3 did not influence P. intermedia LPS-induced degradation of IκB-α. Instead, nuclear translocation of NF-κB p65 and p50 subunits was blocked by CORM-3 in LPS-treated cells. In addition, CORM-3 reduced LPS-induced p65 and p50 binding to DNA. Besides, CORM-3 significantly suppressed P. intermedia LPS-induced phosphorylation of STAT1. Overall, this study indicates that CORM-3 suppresses the production of NO and IL-1ß in P. intermedia LPS-activated murine macrophages via HO-1 induction and inhibition of NF-κB and STAT1 pathways. The modulation of host inflammatory response by CORM-3 would be an attractive therapeutic approach to attenuate the progression of periodontal disease.

Effect of azithromycin on Prevotella intermedia lipopolysaccharide-induced production of interleukin-6 in murine macrophages.

Interleukin-6 (IL-6) is a key proinflammatory cytokine which plays a central role in the pathogenesis of periodontal disease. Host modulatory agents targeting at inhibiting IL-6, therefore, appear to be beneficial in slowing the progression of periodontal disease and potentially reducing destructive aspects of the host response. The present study was designed to investigate the effect of the macrolide antibiotic azithromycin on IL-6 generation in murine macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. Azithromycin significantly suppressed IL-6 production as well as its mRNA expression in P. intermedia LPS-activated RAW264.7 cells. LPS-induced activation of JNK and p38 was not affected by azithromycin treatment. Azithromycin failed to prevent P. intermedia LPS from degrading IκB-α. Instead, azithromycin significantly diminished nuclear translocation and DNA binding activity of NF-κB p50 subunit induced with LPS. Azithromycin inhibited P. intermedia LPS-induced STAT1 and STAT3 phosphorylation. In addition, azithromycin up-regulated the mRNA level of SOCS1 in cells treated with LPS. In conclusion, azithromycin significantly attenuated P. intermedia LPS-induced production of IL-6 in murine macrophages via inhibition of NF-κB, STAT1 and STAT3 activation, which is possibly related to the activation of SOCS1 signaling. Further in vivo studies are required to better evaluate the potential of azithromycin in the treatment of periodontal disease.

Porphyromonas gingivalis-induced autophagy suppresses cell proliferation through G1 arrest in oral cancer cells.

OBJECTIVES: We investigated the response of oral cancer cells to intracellular invasion of Porphyromonas gingivalis to define changes in the biological characteristics of oral cancer cells evoked by the presence of oral pathogenic bacteria within a tumour microenvironment. DESIGNS: The proliferative activity, cell cycle, and autophagic response were evaluated in oral cancer cells infected with P. gingivalis 381. ROS generation was detected in these cells by DCFDA assay, and its role in the responses of oral cancer cells to P. gingivalis infection was further investigated. RESUTLS: P. gingivalis inhibited proliferation of oral cancer cells by inducing G1 cell cycle arrest, but had no effect on apoptosis. Following infection with P. gingivalis, the expression of cyclin D1 and cdk4 was decreased in oral cancer cells, whereas p21, a Cdk inhibitor, was upregulated, in comparison with non-infected controls. Autophagy was prominently enhanced in these infected cells, presumably contributing to the suppressed proliferation. Further experiments revealed that such autophagic response was activated by the formation of reactive oxygen species, as evidenced by the lack of autophagic response and cell proliferation upon removal of reactive oxygen species. CONCLUSIONS: These findings provide a novel insight into the mechanism by which cancer cells are influenced by tumour microenvironment including oral bacteria.

An analysis on the factors responsible for relative position of interproximal papilla in healthy subjects.

PURPOSE: This study examined the factors that can be associated with the appearance of the interproximal papilla. METHODS: One hundred and forty-seven healthy interproximal papillae between the maxillary central incisors were examined. For each subject, a digital photograph and periapical radiograph of the interdental embrasure were taken using a 1-mm grid metal piece. The following parameters were recorded: the amount of recession of the interproximal papilla, contact point-bone crest distance, contact point-cemento-enamel junction (CEJ) distance, CEJ-bone crest distance, inter-radicular distance, tooth shape, embrasure space size, interproximal contact area, gingival biotype, papilla height, and papilla tip form. RESULTS: THE AMOUNT OF RECESSION OF THE INTERPROXIMAL PAPILLA WAS ASSOCIATED WITH THE FOLLOWING: 1) increase in contact point-bone crest, contact point-CEJ, and CEJ-bone crest distance; 2) increase in the inter-radicular distance; 3) triangular tooth shape; 4) decrease in the interproximal contact area length; 5) increase in the embrasure space size; and 6) flat papilla tip form. On the other hand, the amount of gingival recession was not associated with the gingival biotype or papilla height. In the triangular tooth shape, the contact point-bone crest distance and inter-radicular distance were longer, the interproximal contact area length was shorter, and the embrasure space size was larger. The papilla tip form became flatter with increasing inter-radicular distance and CEJ-bone crest distance. CONCLUSIONS: The relative position of the interproximal papilla in healthy subjects was associated with the multiple factors and each factor was related to the others. A triangular tooth shape carries a higher risk of recession of the interproximal papilla because the proximal contact point is positioned more incisally and the bone crest is positioned more apically. This results in an increase in recession of the interproximal papilla and flat papilla tip form.

Kaempferol inhibits P. intermedia lipopolysaccharide-induced production of nitric oxide through translational regulation in murine macrophages: critical role of heme oxygenase-1-mediated ROS reduction.

BACKGROUND: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. METHODS: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real-time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) mRNA expression. iNOS and HO-1 protein expression and phosphorylation of c-Jun N-terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS-stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO-1 expression in LPS-activated cells. Inhibition of HO-1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS-induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS-induced NO. CONCLUSIONS: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS-stimulated RAW264.7 cells at the translational level via HO-1-mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.