High nitric oxide-adapted head and neck cancer cell lines demonstrate altered autophagy and apoptosis.

Background/purpose: Autophagy is an intracellular degradative process occurring under stressful conditions. Nitric oxide (NO), a free radical, regulates autophagy and apoptosis in several cancers. However, the effect of head and neck squamous cell carcinoma (HNSCC) cell adaptation to high nitric oxide (HNO) on autophagy remains unknown. We investigated the autophagy and apoptotic changes in the HNO-adapted HNSCC cell lines. Materials and methods: Isogenic primary HNSCC (HN18/HN30) and metastatic (HN17/HN31) cell lines were evaluated. The cells were induced with 1, 2, 3 and 4 mM DEA-NONOate, an NO donor, for 72 h and assessed for cell viability by MTT assay. "HNO-adapted cells" were defined when the cell viability in the treatment group was <10%. The surviving cells were re-treated with HNO to confirm their adaptation. HNO-adapted cells were quantified for apoptosis using flow cytometry. Autophagic structures (autophagosomes) and proteins (LC3A/B and LC3B-II) were investigated using transmission electron and confocal microscopy, respectively. Results: HNO-adapted concentration for HN18, HN17, HN30 and HN31 cells was 3, 2, 4 and 4 mM, respectively. The HNO-adapted HN18 cells demonstrated a significantly increased apoptotic percentage, whereas no significant apoptotic change was detected in the HNO-adapted HN17, HN30 and HN31 cells compared with the parent cells. Autophagosomes were widely observed across the HNO-adapted cells. Moreover, LC3A/B and LC3B-II proteins were increased in all HNO-adapted cells. Conclusion: Our results demonstrate that apoptosis and/or autophagy are increased during HNO adaptation in HNSCC cell lines.

Ethanolic extract of Ocimum sanctum leaf modulates oxidative stress, cell cycle and apoptosis in head and neck cancer cell lines.

Ocimum sanctum Linn. is a medicinal herb that has cytotoxic effects by inducing oxidative stress in some carcinomas. This study aimed to examine the impact of O. sanctum leaf extract on oxidative stress, cell cycle progression, and apoptosis in cell lines of head and neck squamous cell carcinoma (HNSCC). Isogenic primary (HN18/HN30) and metastatic (HN17/HN31) HNSCC cell lines were used. Preparation of the ethanolic extract of O. sanctum leaf (EEOS) was carried out. HNSCC cell lines were exposed to varying concentrations (0.1-0.8 mg/ml) of EEOS for a duration of 72 h, and the MTT assay was utilized to determine the cytotoxic doses. To assess the impact of EEOS on HNSCC cells, the levels of reactive oxygen species (ROS) and malondialdehyde were measured using a fluorometric method. Flow cytometry was utilized to evaluate effects of EEOS on the cell cycle, DNA damage, and apoptosis in HNSCC cells. Caspase-3 and -9 levels in the EEOS-treated HNSCC cells were measured by ELISA. The chemical components in EEOS were detected using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. EEOS exhibited cytotoxicity against the HN18, HN17, HN30 and HN31 cells at minimum concentrations of 0.1, 0.3, 0.2 and 0.2 mg/ml, respectively. Treatment with EEOS resulted in a significant increase in ROS levels in HN18 and HN17 cells. Additionally, EEOS significantly induced the levels of malondialdehyde in HN18 and HN31 cells. Moreover, EEOS arrested the cell cycle in HN30 and HN31 cells, and significantly induced DNA damage and apoptosis in the HN18, HN30, and HN31 cells. EEOS selectively increased caspase-9 in the HN18 cells. However, caspase-3 was activated without apoptosis in the EEOS-treated HN17 cells. The constituents of EEOS were identified as rosmarinic acid, caffeic acid, and apigenin. In conclusion, EEOS exhibits various prooxidative and apoptotic effects between HNSCC cells.

Effectiveness of a Combined Toothbrushing Technique on Cariogenic Dental Biofilm in Relation to Stainless Steel and Elastomeric Ligatures in Orthodontic Patients: A Randomized Clinical Trial.

Increased dental biofilm commonly occurs during orthodontic treatment. The aim of this study was to evaluate the effect of a combined toothbrushing method on dental biofilm cariogenicity in patients with stainless steel (SSL) and elastomeric (EL) ligatures. At baseline (T1), 70 participants were randomized (1:1 ratio) to the SSL or EL group. Dental biofilm maturity was evaluated using a three-color-disclosing dye. The participants were instructed to brush their teeth using a combined horizontal-Charters-modified Bass technique. Dental biofilm maturity was reassessed at the 4-week follow-up (T2). We found that at T1, new dental biofilm was the highest, followed by mature and cariogenic dental biofilm in the SSL group (p < 0.05). In the EL group, cariogenic dental biofilm was highly observed, followed by mature and new dental biofilm (p < 0.05). After intervention, cariogenic dental biofilm significantly decreased in both groups (p < 0.05). Moreover, a marked decrease in cariogenic dental biofilm was observed in the EL group compared with the SSL group (p < 0.05). However, the change in mature dental biofilm in the groups was similar (p > 0.05). Our results demonstrated that the combined toothbrushing method reduced cariogenic dental biofilm in the SSL and EL groups.

Fixed Orthodontic Treatment Increases Cariogenicity and Virulence Gene Expression in Dental Biofilm.

Background: Dental caries commonly occurs during orthodontic treatment because fixed appliances can impede effective oral hygiene practices. This study investigated the effects of fixed orthodontic treatment on dental biofilm maturity and virulence gene (gtfB, ldh, brpA, spaP, luxS, and gbpB) expression. Methods: Dental biofilms and virulence gene expression were determined in 24 orthodontic patients before and after treatment of ≥6 months. A three-tone disclosing gel was used to stain dental biofilm and assess its maturity by its color change­pink (new dental biofilm), purple (mature dental biofilm), and light blue (cariogenic dental biofilm). Gene expression levels were determined using real-time PCR. Results: After fixed orthodontic appliance insertion, the percentage of new dental biofilm decreased, whereas that of cariogenic dental biofilm significantly increased (p < 0.05). There was no significant difference in the percentage of mature dental biofilm (p > 0.05). Fixed orthodontic appliances increased gtfB, ldh, brpA, and gbpB gene expression above 1.5-fold in dental biofilm. In contrast, there was no change in spaP or luxS gene expression after treatment. Conclusions: Fixed orthodontic appliance insertion induced ecological changes and cariogenic virulence gene expression in dental biofilm.

Bitter Taste Perception and Dental Biofilm Cariogenicity in Orthodontics.

BACKGROUND: Bitter taste perception and sweetness preference have been associated with dental caries. Propylthiouracil (PROP) has been used to determine the genetic sensitivity to bitter taste in early childhood caries. However, the role of the bitter threshold in dental biofilm cariogenicity has not been reported. The purpose of this study was to investigate the role of individual taste sensitivity using PROP in dental biofilm cariogenicity in orthodontic patients. METHODS: Forty orthodontic patients (12-42 years old) undergoing fixed appliance orthodontic treatment participated in this cross-sectional study. Their demographic, oral hygiene practice, and dietary habits data were obtained using a questionnaire. The patients' bitter taste threshold was measured using a PROP assay. The patients were subsequently classified as super-tasters (STs), medium-tasters (MTs), and non-tasters (NTs). Dental biofilm cariogenicity was determined using a 3-tone disclosing gel that becomes pink (new dental biofilm), purple (mature dental biofilm), and light blue (cariogenic dental biofilm) based on dental biofilm maturity. RESULTS: The NT, MT, and ST groups comprised 10%, 27.5%, and 62.5% of the patients, respectively. Most of the STs (56%) and MTs (63.6%) were female, whereas no females were NTs. The dental biofilm cariogenicity was significantly different between the PROP bitterness groups (P < .05). The highest percentage of mature biofilm, followed by cariogenic and new biofilm, was found in the MT and ST groups. However, the cariogenic biofilm percentage was significantly higher compared with mature biofilm (P < .05) in the NT group. A low frequency (<1 time/d) of sugary and acidic food intake between meals was observed in the ST, MT, and NT groups with no significant difference amongst the groups (P > .05). CONCLUSIONS: Cariogenic dental biofilm was highly present in orthodontic patients with the NT phenotype.

The Oral Wound Healing Potential of Thai Propolis Based on Its Antioxidant Activity and Stimulation of Oral Fibroblast Migration and Proliferation.

Introduction: Propolis has demonstrated wound healing effects. Propolis' effects vary based on its composition and geographical origin. However, there are few reports on the effects of propolis on oral wound healing. The aim of this study was to evaluate the antioxidant and in vitro gingival wound healing effects of the n-hexane extract of propolis (HEP), ethyl acetate extract of propolis (EEP), and aqueous extract of propolis (AEP) fractions of the ethanol extract of Thai propolis. Materials and Methods: The crude ethanol extract of propolis was obtained by maceration with 95% ethanol that was sequentially fractionated with hexane, ethyl acetate, and distilled water. The chemical profiles of the samples were assessed by thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). Antioxidant activity was determined using DPPH and FRAP assays. The effects of the propolis fractions on human gingival fibroblast (HGF) proliferation, migration, and in vitro wound healing were determined by MTT, modified Boyden chamber, and scratch assay, respectively. Results: We found that solvent polarity greatly affected the extract yield and TLC profiles. The highest extract yield was found in HEP (38.88%), followed by EEP (19.8%) and AEP (1.42%). TLC revealed 7 spots in the crude ethanol extract (Rf 0.36-0.80), 6 spots in HEP (Rf 0.42-0.80) and EEP (Rf 0.36-0.72), and 4 spots in AEP (Rf 0.17-0.79). GC-MS analysis revealed a high amount of triterpenoids in HEP (82.97%) compared with EEP (28.96%). However, no triterpenoid was found in AEP. The highest antioxidant activity and stimulation of HGF proliferation were observed in HEP, followed by EEP and AEP. HEP and EEP, but not AEP, enhanced HGF migration. However, all propolis fractions induced wound closure. Conclusions: HEP contained a large amount of triterpenoids. Antioxidant and in vitro wound closure effects were found in HEP, EEP, and AEP fractions.

A shotgun proteomic approach reveals novel potential salivary protein biomarkers for asthma.

OBJECTIVE: The aim of this study was to determine if there is an association between the salivary protein profile and disease control in asthma. METHODS: Thirty asthmatic patients (17 adults and 13 children) participated in this study. Saliva samples were collected from healthy subjects, controlled and uncontrolled asthmatics. Individual samples from each group were combined to form a pooled sample, from which proteomic analysis was performed using gel-based quantitative proteomics. RESULTS: Fourteen out of thirty asthmatics were classified to be controlled asthma. Most of asthmatics received inhaled corticosteroids as the controller medications. SDS-PAGE showed predominant bands at high molecular weight in asthmatic saliva compared to that of the controls. Shotgun proteomic analyses indicated that 193 salivary proteins were expressed in both controlled and uncontrolled asthmatics. They were predicted to associate with proteins involved in pathogenesis of asthma including IL-5, IL-6, MCP-1, VEGF, and periostin and asthma medicines (Cromolyn, Nedocromil, and Theophylline). Nucleoside diphosphate kinase (NME1-NME2) only expressed in controlled asthmatics whereas polycystic kidney and hepatic disease 1 (PKHD1)/fibrocystin, zinc finger protein 263 (ZNF263), uncharacterized LOC101060047 (ENSG00000268865), desmoglein 2 (DSG2) and S100 calcium binding protein A2 (S100A2) were only found in uncontrolled asthma. Therefore, the six proteins were associated with disease control in children and adults with asthma. CONCLUSION: Our findings suggest that NME1-NME2, PKHD1, ZNF 263, uncharacterized LOC101060047, DSG 2 and S100 A2 in saliva are associated with disease control in asthma.

Mucin 1 regulates the hypoxia response in head and neck cancer cells.

Mucin 1 (MUC1) is a transmembrane glycoprotein that contributes to the cellular response in hypoxic conditions in different carcinomas. We investigated the gene expression pattern of MUCs (1, 2, 4, 5AC, 5B, 6, 15, 16, and 19) in isogenic primary (HN4 and HN30) and metastatic (HN12 and HN31) head and neck squamous cell carcinoma (HNSCC) cell lines. MUC1 was significantly up-regulated at the mRNA and protein levels in HN12 and HN31 cells, whereas, other MUCs exhibited diverse expression patterns between HNSCC cell lines. Immunohistochemistry demonstrated that MUC1 was exclusively expressed in cancer cells; however, there was no significant correlation between MUC1 expression and malignancy grading. Inducing hypoxia with CoCl2 significantly increased cell viability, MUC1, hypoxia-inducible factor alpha (HIF-1α), and vascular endothelial growth factor A (VEGF-A) expression in HN12 cells, but not HN31 cells. Interestingly, in hypoxia, cell viability, HIF-1α and VEGF-A expression were significantly reduced in MUC1-knockdown HN12 cells. The current report is the first to demonstrate that MUC1 is required in the regulation of hypoxia-related genes in HNSCC cells. Thus, our results suggest that MUC1 modulates the hypoxic effects in HNSCC cells through HIF-1α regulation.

Apis mellifera propolis enhances apoptosis and invasion inhibition in head and neck cancer cells.

BACKGROUND: Propolis is a resinous product accumulated from several plant sources that possess a wide range of therapeutic properties, including anti-cancer activities. However, the role of honeybee-produced propolis on head and neck squamous carcinoma (HNSCC) is not well understood. The aim of this study was to investigate the effects of Apis mellifera propolis on apoptosis and invasiveness in HNSCC cell lines. METHODS: Ethyl acetate extract of propolis (EAEP) was prepared from A. mellifera beehives using liquid-liquid extraction. High-performance liquid chromatography coupled with electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS) was used to determine the flavonoids in EAEP. Isogenic HNSCC cell lines derived from primary (HN30 and HN4) and metastatic site (HN31 and HN12) were used in this study. The cytotoxicity, apoptosis, invasion, and MMP activity of EAEP on HNSCC cells were determined using an MTT assay, flow cytometry, Matrigel invasion assay, and gelatinase zymography, respectively. RESULTS: We found that EAEP exhibited cytotoxic activity and induced apoptosis in the HNSCC cell lines. Furthermore, EAEP significantly decreased HNSCC cell invasion by reducing MMP-2 and MMP-9 activity. Two flavonoids, galangin and apigenin, were identified in EAEP by HPLC-ESI-TOF-MS. The results suggest that EAEP promotes apoptosis and exerts anti-invasion potential by inhibiting MMP-2 and MMP-9 activity in HNSCC cell lines. These inhibitory effects may be mediated by galangin and apigenin.

Macrophage migration inhibitory factor modulates proliferation, cell cycle, and apoptotic activity in head and neck cancer cell lines.

BACKGROUND/PURPOSE: Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that contributes to the progression of several cancers. MIF overexpression has been reported in head and neck squamous cell carcinoma (HNSCC) patients. However, the exact role of MIF in HNSCC is not fully understood. Our aim was to evaluate the amount of secreted MIF and the role of MIF in the proliferation, cell cycle, and apoptosis in HNSCC cell lines. MATERIALS AND METHODS: Genetically matched HNSCC cell lines derived from primary (HN18 and HN30) and metastatic sites (HN17 and HN31) from the same patient were used in this study. The MIF levels in conditioned media from the HNSCC cell lines were evaluated using ELISA. The HNSCC cell lines were treated with recombinant MIF at concentrations 25, 50 and 100 ng/ml, and cell proliferation was evaluated by MTT assay. A proliferative dose of MIF was used to treat the cells then, cell cycle, and apoptotic status were determined by flow cytometry. RESULTS: The HNSCC-secreted MIF concentration ranged from 49.33 to 973 pg/ml. Exogenous MIF (25 ng/ml) significantly increased HN18, HN30, and HN31 cell proliferation. Moreover, MIF induced cell cycle progression and inhibited apoptosis in these cells. However, MIF did not affect growth or apoptosis in HN17 cell. CONCLUSION: MIF secreted from the HNSCC cell lines were evaluated. Exogenous MIF promotes various effects on proliferation, cell cycle, and apoptosis in HNSCC cells.

Enhancement of cariogenic virulence properties of dental plaque in asthmatics.

OBJECTIVE: The aim of this study was to investigate the caries risk of asthmatics in relation to acidogenicity and the expression of caries-related genes in dental plaque. METHODS: A case-control study composed of 38 asthmatics (cases) and 22 controls with an age range from 6 to 60 years. Characteristics of asthma, use of medications, oral hygiene practices and dietary habits assessed by questionnaires and interviews. The dental plaque maturity evaluated using GC Tri Plaque ID Gel TM. The expression of brpA, gtfB, gbpB, ldh, luxS and spaP genes analyzed using real-time PCR. RESULTS: Asthmatics had a higher percentage of mature and acidogenic plaque than immature plaque. In contrast, immature plaque was more evident in controls. Acidogenic plaque commonly occurred in patients using 1 or a combination of two medications. High frequency in meals and sweets were found in asthmatics. Real-time PCR revealed that the expression of spaP, gtfB, gbpB, ldh, brpA and luxS were enhanced in asthmatics compared with the control group. CONCLUSION: An increase in acidogenic and mature plaque is found in asthmatics. The expression of spaP, gtfB, gbpB, ldh, brpA and luxS in dental plaque are upregulated in asthmatics.

Characteristics of Dental Services in Rural, Suburban, and Urban Areas Upon the Implementation of Indonesia National Health Insurance.

Introduction: The implementation of Indonesia National Health Insurance (NHI) for oral health needs to be evaluated by observing the dental disease patterns and dental therapy patterns from community health centers (CHCs) in the rural area, suburban area, and urban area. The aim of the study is to describe the characteristics of dental services in rural, suburban, and urban areas after the implementation of NHI on CHCs in the Special Region of Yogyakarta in 2014. Materials and methods: This is an observational study with a cross-sectional research design. The study used quantitative data obtained from dental records at selected CHCs. Using a purposive sampling method, 30 CHCs as unit analysis were collected from rural, suburban, and urban areas. The data were collected from January 2014 to December 2014. Results: Data from 26,554 patients were collected from dental records of dental clinics at CHCs. There were 5829 patient dental records from rural areas, 12,327 from suburban areas, and 8938 from urban areas. The primary dentist tends to provide services without clinical intervention on periodontal problems, abscesses, and lesions. Clinical interventions were mostly provided for prolonged retention and deposits on teeth. Primary dentists in suburban areas tend to provide clinical intervention on caries disease compared to those in rural and urban areas. Statistically significant differences (p < 0.05) were observed among locations in the pattern of providing clinical interventions on caries, abscess, lesion, prolonged retention, deposits on teeth, and other problems. No difference was recorded only on periodontal disease. Discussion: This study found that each area has different characteristics of dental disease and dental therapy patterns. Each area has a significant difference in the pattern of the clinical intervention of dental disease except in periodontal problems.

High Nitric Oxide Adaptation in Isogenic Primary and Metastatic Head and Neck Cancer Cells.

BACKGROUND/AIM: Nitric oxide (NO) functions have been studied in many cancer types, but rarely in head and neck squamous cell carcinoma (HNSCC). This study aimed to investigate the behavior of HNSCC cells following exposure to high NO (HNO). MATERIALS AND METHODS: Two pairs of isogenic HNSCC cell lines (HN18/HN17, HN30/HN31) were used, and were treated with a NO donor for 72 h. Cell viability, cell cycle, apoptosis, invasion, and MMP activity were determined using MTT, flow cytometry, Matrigel invasion, and gelatinase zymography assays, respectively. RESULTS: HNO induced HN18 and HN31 cell cycle progression in S and G2/M phases. Anti-invasion, MMP-2 inhibition, and apoptosis induction were observed in certain HNO-adapted cell lines. High NO did not affect MMP-9 activity in all cell lines. CONCLUSION: NO enhanced cell cycle progression and apoptosis but inhibited cell invasion in HNSCC cells.

Ethanolic Extract of Ocimum sanctum Leaves Reduced Invasion and Matrix Metalloproteinase Activity of Head and Neck Cancer Cell Lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) has a yearly incidence of 600,000 cases worldwide with a low survival rate. Ocimum sanctum L. or Ocimum tenuiflorum L. (Holy basil; Tulsi in Hindi), is a traditional medicine herb that demonstrates numerous effects including anti-oxidant, anti-microbial, and anti-tumor effects. The aim of this study was to evaluate the anti-invasive effect of O. sanctum leaf extract on HNSCC cell lines. METHODS: Ethanolic extract of O. sanctum leaf (EEOS) was prepared and the phenolic compounds were identified using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. Genetically matched HNSCC cell lines derived from primary (HN30 and HN4) and metastatic sites (HN31 and HN12) from the same patient were used in this study. The EEOS cytotoxicity to the cell lines was determined using an MTT assay. The invasion and matrix metalloproteinase (MMP)-2 and -9 activity of EEOS-treated cells were tested using a modified Boyden chamber assay and zymography, respectively. RESULTS: We found that EEOS significantly inhibited the invasion and MMP-2 and MMP-9 activity of HN4 and HN12 cells, but not HN30 and HN31 cells. Rosmarinic acid, caffeic acid, and apigenin were detected in EEOS. Moreover, rosmarinic acid was found as the major phenolic compound. CONCLUSION: EEOS exerted its anti-invasive effect on HNSCC cells by attenuating MMP activity. The active compounds identified in EEOS might be promising as an alternative therapeutic agent for HNSCC.

Anti-biofilm, nitric oxide inhibition and wound healing potential of purpurin-18 phytyl ester isolated from Clinacanthus nutans leaves.

AIMS: Clinacanthus nutans (C. nutans) has demonstrated anti-inflammatory activity, however, the active compound generating this activity remains unknown. The aim of this study was to identify the bioactive compound in C. nutans responsible for its anti-inflammatory, in-vitro wound healing, and anti-biofilm activities. MAIN METHODS: A pure compound was isolated from the chloroform extract (CE) of C. nutans leaves by chromatographic techniques and bioassay-guided fractionation. This compound's structure was determined by spectroscopic analyses (FTIR/NMR/HRES-MS). Biological activities were evaluated using cytotoxicity, nitric oxide (NO), wound scratch, anti-microbial activity, and anti-biofilm assays; and the compound's bactericidal depth into the biofilm was visualized by confocal laser scanning microscopy. KEY FINDINGS: CE and its pure isolated compound, purpurin-18 phytyl ester (P18PE), significantly inhibited lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells at concentrations of 100 µg/ml and 10-100 µg/ml, respectively. These concentrations significantly induced wound closure by human gingival fibroblasts. CE (100-1000µg/ml) and P18PE (1-500 µg/ml) did not inhibit Streptococcus (S.) mutans growth. However, these concentrations significantly reduced S. mutans biofilm formation below 50% at 250 µg/ml for CE, and 25 µg/ml for P18PE (p<0.05). SIGNIFICANCE: C. nutans contains a bioactive compound, P18PE, which exhibits anti-inflammatory, in-vitro wound healing, and anti-biofilm activities.

Characterization and safety evaluation of partially purified bacteriocin produced by Escherichia coli E isolated from fermented pineapple Ananas comosus (L.) Merr.

Antibacterial activity of cell-free supernatant from Escherichia coli E against selected pathogenic bacteria in food and aquaculture was the highest against Edwardsiella tarda 3, a significant aquaculture pathogen. Biochemical properties of the bacteriocins were studied and bacteriocin was found to be sensitive to proteinase K, demonstrating its proteinaceous nature. In addition, pH and temperature affected bacteriocin activity and stability. The bacteriocins were partially purified by ammonium sulfate precipitation. The antibacterial activity was only detected in 20% ammonium sulfate fraction and direct detection of its activity was performed by overlaying on the indicator strains. The inhibition zone associated with the antibacterial activity was detected in the sample overlaid by E. tarda 3 and Staphylococcus aureus DMST8840 with the relative molecular mass of about 27 kDa and 10 kDa, respectively. Bacteriocin showed no cytotoxic effect on NIH-3T3 cell line; however, two virulence genes, aer and sfa, were detected in the genome of E. coli E by PCR. The characteristics of bacteriocins produced by E. coli E exhibited the antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria and the safe use determined by cytotoxicity test which may have interesting biotechnological applications.

Porphyromonas gingivalis lipopolysaccharide-induced macrophages modulate proliferation and invasion of head and neck cancer cell lines.

AIM: The aim of this study was to investigate the effect of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced macrophages on head and neck squamous cell carcinoma (HNSCC) cell line proliferation and invasion. MAIN METHODS: THP-1 monocytes were differentiated toward macrophages using 12.5 ng/ml phorbol 12-myristate 13-acetate treatment for 48 h. The expression of interleukin-6 (IL-6) mRNA and cytokine by monocytes and macrophages was determined using real time PCR and ELISA, respectively. The cells were analyzed for CD14 expression using immunofluorescent labeling. The macrophages were induced using 1 µg/ml P. gingivalis LPS for 24 h, and the conditioned medium (CM) was collected. The monocyte, macrophage, and LPS-induced macrophage CM were evaluated for IL-6 and tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) content using ELISA and the Griess Reagent System, respectively. Human primary (HN18, HN30, and HN4) and metastatic (HN17, HN31, and HN12) HNSCC cell lines were treated with the monocyte, macrophage, and LPS-induced macrophage CM. The proliferation and invasion of the HNSCC cell lines were evaluated using MTT and modified Boyden chamber assays, respectively. KEY FINDINGS: Macrophages demonstrated increased IL-6 and CD14 expression. The P. gingivalis LPS significantly induced macrophage NO secretion, however, that of TNF-α decreased. The LPS-induced macrophages CM inhibited HN4 proliferation. Interestingly, the LPS-induced macrophage CM promoted invasion of all HNSCC cell lines. SIGNIFICANCE: Our data demonstrate that P. gingivalis LPS-induced macrophages increased NO secretion. The activated macrophage CM inhibited HN4 cell proliferation and promoted invasion of all HNSCC cell lines.

Inhibitory effect of Phlai capsules on skin test responses among allergic rhinitis patients: a randomized, three-way crossover study.

BACKGROUND: Zingiber cassumunar Roxb., commonly known as Phlai in Thai, has been used as a traditional medicine in Thailand for the treatment of various diseases, including inflammation and chronic airway disease. OBJECTIVE: The purpose of this study was to assess the antihistaminic effect of Phlai on skin testing. DESIGN, SETTING, PARTICIPANTS AND INTERVENTION: This was a randomized, open-label, three-way crossover study. Twenty allergic rhinitis (AR) patients were enrolled. In randomized sequence, patients received a single dose of Phlai capsules (100 or 200 mg) or loratadine (10 mg) with a washout period of 1 week between each treatment. MAIN OUTCOME MEASURES: Skin prick testing for histamine and common aeroallergen (house dust mite) were performed before treatment and after 1, 2, 3, 4, 6, 8, 12 and 24 hours of treatment. The main treatment outcomes were the mean wheal and flare responses to the skin prick test after treatment. RESULTS: Both 100 mg and 200 mg Phlai doses suppressed wheal and flare responses to house dust mite allergen, but only 200 mg of Phlai capsules significantly suppressed wheal and flare responses to histamine. Repeated measures analysis of variance showed that loratadine caused more wheal and flare suppression than Phlai capsules in responses to the histamine skin prick test. However, there were no significant differences among the effects of 100 mg Phlai capsules, 200 mg Phlai capsules and loratadine in suppression of wheal and flare induced by the mite skin prick test. Both doses of Phlai were well-tolerated with no adverse events. CONCLUSION: Both 100 mg (compound D 4 mg) and 200 mg (compound D 8 mg) Phlai capsules, when taken as a single therapeutic dose, inhibited skin reactivity to histamine and mite skin prick tests in AR patients. TRIAL REGISTRATION: Thai clinical trial registry (TCTR20160510001).

Propolis Extracted from the Stingless Bee Trigona sirindhornae Inhibited S. mutans Activity In Vitro.

PURPOSE: The aim of this study was to determine the antimicrobial effects of propolis extracted from an endemic species of stingless bee, T. sirindhornae, on the cariogenic bacterium Streptococcus mutans. MATERIALS AND METHODS: Dichloromethane extracts (DME) of propolis (DMEP) were prepared and analysed by reverse-phase high-performance liquid chromatography. The antibacterial growth and antibiofilm formation effects of DMEP on S. mutans were compared with those of apigenin, a commercial propolis product. The effects of DMEP and apigenin on glucosyltransferase (gtf) B expression in S. mutans were investigated using real-time polymerase chain reaction. Chlorhexidine (CHX) was used as a positive control in the experiments. RESULTS: Apigenin, pinocembrin, p-coumaric acid, and caffeic acid were not detected in the propolis extracts. DMEP and apigenin significantly inhibited S. mutans growth (IC50 = 43.5 and 17.36 mg/ml, respectively). DMEP and apigenin also exhibited antiadherence effects on S. mutans as shown by reduced biofilm formation. Furthermore, a significant inhibition in gtfB expression was observed in DMEP and apigenin treated S. mutans. CONCLUSION: Propolis produced by T. sirindhornae demonstrated antibacterial and antibiofilm effects, and reduced gtfB expression in S. mutans. The antibacterial activities of propolis observed were not due to apigenin, pinocembrin, p-coumaric acid, or caffeic acid.

Cytotoxic Activity of Propolis Extracts from the Stingless Bee Trigona Sirindhornae Against Primary and Metastatic Head and Neck Cancer Cell Lines

Background: Propolis, a resinous substance produced by the honeybee, has a wide spectrum of potent biological activities. However, anti-cancer activity of propolis obtained from Trigona sirindhornae, a new species of stingless bee, has not yet been reported. This study concerned cytotoxicity of propolis extracts from T. sirindhornae against two head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods: A dichloromethane extract of propolis (DMEP) was prepared generating 3 fractions: DMEP-A, DMEP-B, and DMEP-C. Genetically-matched HNSCC cell lines derived from primary (HN30) and metastatic sites (HN31) in the same patient were used to study cytotoxic effects of the DMEPs by MTT assays. The active compounds in the DMEPs were analyzed by reversephase high performance liquid chromatography. Results: DMEP-A exhibited cytotoxic activity on HN30 cells with significantly decreased viability at 200 µg/ml compared with the control (p<0.05). However, no significant cytotoxic effect was evident in HN31 cells. DMEP-B and DMEP-C significantly decreased the viability of both cell lines from 100­200 µg/ml and 50­200 µg/ml, respectively (p<0.05). Interestingly, HN31 cells were more toxically sensitive compared with the HN30 cells when treated with DMEP-B and DMEP-C. IC50 values for DMEP-B with HN30 and HN31 cells were more than 200 µg/ml and 199.8±1.05 µg/ml, respectively. The IC50 of DMEP-C to HN30 and HN31 cells was found to be 114.3±1.29 and 76.33±1.24 µg/ml, respectively. Notably, apigenin, pinocembrin, p-coumaric acid, and caffeic acid were not detected in our propolis extracts. Conclusion: T. sirindhornae produced propolis displays cytotoxic effects against HNSCC cells s. Moreover, DMEP-B and DMEP-C differentially inhibited the proliferation of a metastatic HNSCC cell line.