Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

In vitro cytotoxicity of Withania somnifera (L.) roots and fruits on oral squamous cell carcinoma cell lines: a study supported by flow cytometry, spectral, and computational investigations.

Oral cancer is a severe health problem that accounts for an alarmingly high number of fatalities worldwide. Withania somnifera (L.) Dunal has been extensively studied against various tumor cell lines from different body organs, rarely from the oral cavity. We thus investigated the cytotoxicity of W. somnifera fruits (W-F) and roots (W-R) hydromethanolic extracts and their chromatographic fractions against oral squamous cell carcinoma (OSCC) cell lines [Ca9-22 (derived from gingiva), HSC-2, HSC-3, and HSC-4 (derived from tongue)] and three normal oral mesenchymal cells [human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF), and human pulp cells (HPC)] in comparison to standard drugs. The root polar ethyl acetate (W-R EtOAc) and butanol (W-R BuOH) fractions exhibited the strongest cytotoxicity against the Ca9-22 cell line (CC50 = 51.8 and 40.1 µg/mL, respectively), which is relatively the same effect as 5-FU at CC50 = 69.4 µM and melphalan at CC50 = 36.3 µM on the same cancer cell line. Flow cytometric analysis revealed changes in morphology as well as in the cell cycle profile of the W-R EtOAc and W-R BuOH-treated oral cancer Ca9-22 cells compared to the untreated control. The W-R EtOAc (125 µg/mL) exerted morphological changes and induced subG1 accumulation, suggesting apoptotic cell death. A UHPLC MS/MS analysis of the extract enabled the identification of 26 compounds, mainly alkaloids, withanolides, withanosides, and flavonoids. Pharmacophore-based inverse virtual screening proposed that BRD3 and CDK2 are the cancer-relevant targets for the annotated withanolides D (18) and O (12), and the flavonoid kaempferol (11). Molecular modeling studies highlighted the BRD3 and CDK2 as the most probable oncogenic targets of anticancer activity of these molecules. These findings highlight W. somnifera's potential as an affordable source of therapeutic agents for a range of oral malignancies.

A Comparative Study of Tumor-Specificity and Neurotoxicity between 3-Styrylchromones and Anti-Cancer Drugs.

Background. Many anti-cancer drugs used in clinical practice cause adverse events such as oral mucositis, neurotoxicity, and extravascular leakage. We have reported that two 3-styrylchromone derivatives, 7-methoxy-3-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one (Compound A) and 3-[(1E)-2-(4-hydroxyphenyl)ethenyl]-7-methoxy-4H-1-benzopyran-4-one (Compound B), showed the highest tumor-specificity against human oral squamous cell carcinoma (OSCC) cell lines among 291 related compounds. After confirming their superiority by comparing their tumor specificity with newly synthesized 65 derivatives, we investigated the neurotoxicity of these compounds in comparison with four popular anti-cancer drugs. Methods: Tumor-specificity (TSM, TSE, TSN) was evaluated as the ratio of mean CC50 for human normal oral mesenchymal (gingival fibroblast, pulp cell), oral epithelial cells (gingival epithelial progenitor), and neuronal cells (PC-12, SH-SY5Y, LY-PPB6, differentiated PC-12) to OSCC cells (Ca9-22, HSC-2), respectively. Results: Compounds A and B showed one order of magnitude higher TSM than newly synthesized derivatives, confirming its prominent tumor-specificity. Docetaxel showed one order of magnitude higher TSM, but two orders of magnitude lower TSE than Compounds A and B. Compounds A and B showed higher TSM, TSE, and TSN values than doxorubicin, 5-FU, and cisplatin, damaging OSCC cells at concentrations that do not affect the viability of normal epithelial and neuronal cells. QSAR prediction based on the Tox21 database suggested that Compounds A and B may inhibit the signaling pathway of estrogen-related receptors.

Prominent Anti-UVC Activity of Lignin Degradation Products.

BACKGROUND/AIM: The rapid spread of COVID-19 resulted in the revision of the value of ultraviolet C (UVC) sterilization in working spaces. This study aimed at re-evaluating the anti-UVC activity of four groups of natural products against human melanoma COLO679 and human normal dermal fibroblast (HDFa) cells, based on chemotherapeutic index. MATERIALS AND METHODS: Various cell lines were exposed to UVC for 3 min in the presence of increasing concentrations of test compounds and viable cell numbers were determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The anti-UVC activity was quantified by the ratio of the 50% cytotoxic concentration (determined without irradiation) to the 50% effective concentration (which abolished by 50% the UVC-induced loss of viability). Apoptosis was quantified as the subG1 population proportion following cell-cycle analysis. RESULTS: Among four groups of major natural products, six phenylpropanoids showed the highest anti-UVC activity, followed by the lignified products and alkaline products that contain lignin and its degradation products. On the other hand, tannins and flavonoids showed lower activity due to their higher cytotoxicity. UVC-sensitive COLO679 cells lack dectin-1 protein expression. CONCLUSION: These data suggest the prominent anti-UVC activity of lignin degradation products, and the possible involvement of dectin-1 expression in UVC-sensitivity.

Cytotoxic Tumour-Selective 1,5-Diaryl-3-Oxo-1,4-Pentadienes Mounted on a Piperidine Ring.

A series of 3,5-bis(benzylidene)-4-piperidones 2a-u were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC), thereby demonstrating tumour-selective toxicity. A further study revealed that most of the compounds in series 2 were more toxic to the human Colo-205 adenocarcinoma cell line (Colo-205), human HT29 colorectal adenocarcinoma cells (HT-29) and human CEM lymphoid cells (CEM) neoplasms than towards non-malignant human foreskin Hs27 fibroblast line (Hs27) cells. The potency of the cytotoxins towards the six malignant cell lines increased as the sigma and sigma star values of the aryl substituents rose. Attempts to condense various aryl aldehydes with 2,2,6,6-tetramethyl-4-piperidone led to the isolation of some 1,5-diaryl-1,4-pentadien-3-ones. The highest specificity for oral cancer cells was displayed by 2e and 2r. In the case of 2r, its selective toxicity exceeded that of doxorubicin and melphalan. The enones 2k, m, o have the highest SI values towards colon cancer and leukemic cells. Both 2e,r inhibited mitosis and increased the subG1 population (with a transient increase in G2/M phase cells). Slight activation of caspase-3, based on the cleavage of poly(ADP-ribose)polymerase (PARP) and procaspase 3, was detected.

A novel osteoclast precursor cell line, 4B12, recapitulates the features of primary osteoclast differentiation and function: enhanced transfection efficiency before and after differentiation.

Osteoclasts are bone-resorbing multinucleated cells differentiated from monocyte/macrophage lineage precursors. A novel osteoclast precursor cell line, 4B12 was established from Mac-1(+)c-Fms(+)RANK(+) cells from calvaria of 14-day-old mouse embryos using immunofluorescence and cell-sorting methods. Like M-CSF-dependent bone marrow macrophages (M-BMMs), M-CSF is required for 4B12 cells to differentiate into TRAP-positive multinucleated cells [TRAP(+) MNCs] in the presence of RANKL. Bone-resorbing osteoclasts differentiated from 4B12 cells on dentine slices possess both a clear zone and ruffled borders and express osteoclast-specific genes. Bone-resorbing activity, but not TRAP, was enhanced in the presence of IL-1alpha. The number of TRAP(+) MNCs and the number of pits formed from 4B12 cells on dentine slices was fourfold higher than that from M-BMMs. 4B12 cells were identified as macrophages with Mac-1 and F4/80, yet lost these markers upon differentiation into osteoclasts as determined by confocal laser scanning microscopy. The 4B12 cells do not have the potential to differentiate into dendritic cells indicating commitment to the osteoclast lineage. 4B12 cells are readily transfectable with siRNA transfection before and after differentiation. These data show that 4B12 cells faithfully replicate the properties of primary cells and are a useful and powerful model for analyzing the molecular and cellular regulatory mechanisms of osteoclastogenesis and osteoclast function.

Antiviral, antibacterial and vitamin C-synergized radical-scavenging activity of Sasa senanensis Rehder extract.

Sasa senanensis Rehder extract (SE) showed slightly higher cytotoxicity against human squamous cell carcinoma cell lines and human glioblastoma cell lines, as compared with human oral normal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast), and was more cytotoxic to human myelogenous and T-cell leukemia cell lines. SE showed a bacteriostatic effect on Fusobacterium nucleatum and Prevotella intermedia, but almost completely eliminated hydrogen sulfide (H2S) and methyl mercaptan (CH3SH) produced by these bacteria. SE protected human T-cell leukemia MT-4 cells from the cytopathic effect of human immunodeficiency virus (HIV) infection, and its anti-HIV activity was much higher than that of tannins and flavonoids, comparable with that of natural and synthetic lignins. SE also protected the MDCK cells from the cytopathic effect of influenza virus infection. SE synergistically enhanced the superoxide anion and hydroxyl radical-scavenging activity of vitamin C. The present study suggests the functionality of SE as a complementary alternative medicine.

Efficient utilization of licorice root by alkaline extraction.

Compared to studies of water extracts of plants, those utilising alkaline extracts are limited. Both water and alkaline extracts from licorice root were compared regarding their biological activities. Licorice root was successively extracted first with water or alkaline solution (pH 9 or 12), and the alkaline (pH 12.0) extract was further separated into 50% ethanol-soluble and -insoluble fractions. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antibacterial activity against Porphyromonas gingivalis 381 was determined by turbidity assay. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone using human recombinant CYP3A4. Radical intensity of superoxide and hydroxyl radicals was determined by electron spin resonance spectroscopy. Alkaline extraction yielded slightly higher amounts of dried materials compared to water extraction. Alkaline extract showed higher anti-HIV and antibacterial activities, and similar magnitudes of CYP3A4 inhibitory and superoxide and hydroxyl radical-scavenging activities, compared to water extract. When alkaline extract was fractionated by 50% ethanol, anti-HIV activity was recovered from the insoluble fraction representing approximately 3% of the alkaline extract, whereas antibacterial activity was concentrated in the soluble fraction rich in glycyrrhizid acid, flavanones and chalcones. All extracts and sub-fractions led to bimodal hormetic dose-response (maximum hormetic response=238%) on the bacterial growth. The present study demonstrated the superiority of alkaline extraction over water extraction for preparing anti-HIV and antibacterial agents at higher yield from licorice root.

Biological interaction between Sasa senanensis Rehder leaf extract and toothpaste ingredients.

BACKGROUND: Previous studies have shown antiviral, antibacterial, and anti-inflammatory activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE). In order to manufacture an SE-containing toothpaste for combating oral diseases, we investigated the possible interaction between the candidate ingredients of toothpaste: SE, isopropyl methylphenol (IPMP, antibacterial agent) and charcoal prepared from Sasa senanensis Rehder. MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected and UV-irradiated cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Superoxide radical scavenging activity was determined by electron-spin resonance spectroscopy. Antibacterial activity against Porphyromonas gingivalis 381 and Streptococcus mutans ATCC25175 was determined by the turbidity assay. RESULTS: Exposure to less than 50% SE or less than 0.31 mM IPMP for 10 min scarcely damaged human cultured gingival and periodontal ligament fibroblasts. Both SE and IPMP showed bi-modal action, stimulating the bacterial growth at lower concentrations, but synergistically inhibiting it at higher concentrations. Addition of extremely high concentrations of charcoal enhanced both anti-HIV and anti-UV activity of SE. CONCLUSION: Practically, addition of charcoal may not be recommendable, since one or two orders higher concentrations of charcoal as compared with SE, are required to achieve the synergistic effect for anti-HIV and anti-UV activity. Rather, addition of about one tenth of the amount of IPMP may be recommendable for enhancing the antibacterial activity.

Multiple biological complex of alkaline extract of the leaves of Sasa senanensis Rehder.

Previous studies have shown anti-inflammatory potential of alkaline extract of the leaves of Sasa senanensis Rehder (SE). The aim of the present study was to clarity the molecular entity of SE, using various fractionation methods. SE inhibited the production of nitric oxide (NO), but not tumour necrosis factor-α by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells. Lignin carbohydrate complex prepared from SE inhibited the NO production to a comparable extent with SE, whereas chlorophyllin was more active. On successive extraction with organic solvents, nearly 90% of SE components, including chlorophyllin, were recovered from the aqueous layer. Anti-HIV activity of SE was comparable with that of lignin-carbohydrate complex, and much higher than that of chlorophyllin and n-butanol extract fractions. The CYP3A inhibitory activity of SE was significantly lower than that of grapefruit juice and chlorophyllin. Oral administration of SE slightly reduced the number of oral bacteria. When SE was applied to HPLC, nearly 70% of SE components were eluted as a single peak. These data suggest that multiple components of SE may be associated with each other in the native state or after extraction with alkaline solution.