Antimicrobial efficacy and biocompatibility of extracts from Cryptocarya species

This study evaluated the efficacy of Cryptocarya spp extracts on biofilm of Candida albicans and its biocompatibility. Mature biofilm of C. albicans was formed on denture base acrylic resin samples and the fungicidal effect of the extracts was evaluated by Alamar Blue® assay, counting colony-forming units (CFU/mL) and confocal laser scanning microscopy (CLSM). Cytotoxicity of extracts from Cryptocarya species was evaluated by AlamarBlue® assay, using normal oral keratinocytes (NOK) cells. In additional, Analysis of plant extracts by ultra-high-performance liquid chromatography–diode array detector–tandem mass spectrometry (UPLC-DAD-MS) was performed. The results showed significant reduction in the cellular metabolism and in the number of CFU/mL of C. albicans (p<0.05). The concentration of 0.045 g/mL completely inhibited the number of CFU/mL. Regarding cytotoxicity, all extracts decreased cell viability compared to the control group. CLSM analysis showed predominance of live cells, but with a great difference between the groups. Antimicrobial activity of extracts from Cryptocarya on C. albicans biofilm was confirmed. However, all extracts showed toxicity on NOK cells.

Anti-Periodontitis Effect of Ethanol Extracts of Alpinia Katsumadai Seeds

Oral microbes are intimately associated with many oral and systemic diseases. Ongoing research is seeking to elucidate drugs that prevent and treat microbial diseases. Various functions of Alpinia Katsumadai seed extracts have been reported such as their anti-viral, anti-oxidant, anti-inflammatory, anti-puritic, anti-emetic, and cytoprotective effects. Here, we investigated the anti-periodontitis effect of an ethanol extract of Alpinia Katsumadai seeds (EEAKSs) on dental plaque bacteria (DPB)-induced inflammation and bone resorption. DPB and Porphyromonas gingivalis (P. gingivalis) were cultured and lipopolysaccharide (LPS) was extracted. Prostaglandin E2 (PGE2) and cyclooxygenase 2 (COX-2) levels were estimated using ELISA. Cytotoxicity was also verified. Proteases were screened using a protease antibody array method. Osteoclastic bone resorption was also investigated. EEAKSs suppressed P. gingivalis growth on agar plates. LPS prepared from dental plaque bacteria (DPB-LPS) and P. gingivalis (PG-LPS) significantly increased PGE2 and COX2 levels in immortalized gingival fibroblasts (IGFs), immortalized human oral keratinocytes (IHOKs), and RAW264.7 macrophage cells. However, DPB-LPS and PG-LPS-induced PGE2 and COX-2 increases were effectively abolished by EEAKS treatment at non-cytotoxic concentrations. In the protease antibody array, matrix metalloproteinase (MMP)-2, MMP-3, MMP-7, kallikrein 10, cathepsin D, and cathepsin V levels were increased by PG-LPS stimulation. However, increases in protease levels except for cathepsin D were suppressed by EEAKS treatment. In addition, RANKL-induced osteoclast differentiation was significantly inhibited by EEAKS treatment, leading to reductions in resorption pit formation. These results suggest that EEAKSs exerted a beneficial oral health effect to help prevent DPB-mediated periodontal disease.

The Local Neuropeptide System of Keratinocytes

Neuropeptides have been known for over 50 years as chemical signals in the brain. However, it is now well established that the synthesis of this class of peptides is not restricted to neurons. For example, human skin not only expresses several functional receptors for neuropeptides but, also, can serve as a local source of neuroactive molecules such as corticotropin-releasing hormone, melanocortins, and β-endorphin. In contrast, an equivalent of the hypothalamic-pituitary axis in the oral mucosa has not been well characterized to date. In view of the differences in the morphology and function of oral mucosal and skin cells, in this review I surveyed the existing evidence for a local synthesis of hypothalamic-pituitary, opiate, neurohypophyseal, and neuroendocrine neuropeptides in both epidermal and oral keratinocytes.

Stress hormones promote DNA damage in human oral keratinocytes

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis.

The Extracellular Metabolome Stratifies Low and High Risk Potentially Premalignant Oral Keratinocytes and Identifies Citrate as a Potential Non-Invasive Marker of Tumour Progression

Premalignant oral lesions (PPOLs) which bypass senescence (IPPOL) have a much greater probability of progressing to malignancy, but pre-cancerous fields also contain mortal PPOL keratinocytes (MPPOL) that possess tumour-promoting properties. To identify metabolites that could potentially separate IPPOL, MPPOL and normal oral keratinocytes non-invasively in vivo, we conducted an unbiased screen of their conditioned medium. MPPOL keratinocytes showed elevated levels of branch-chain amino acid, lipid, prostaglandin, and glutathione metabolites, some of which could potentially be converted into volatile compounds by oral bacteria and detected in breath analysis. Extracellular metabolites were generally depleted in IPPOL, and only six were elevated, but some metabolites distinguishing IPPOL from MPPOL have been associated with progression to oral squamous cell carcinoma (OSCC) in vivo. One of the metabolites elevated in IPPOL relative to the other groups, citrate, was confirmed by targeted metabolomics and, interestingly, has been implicated in cancer growth and metastasis. Although our investigation is preliminary, some of the metabolites described here are detectable in the saliva of oral cancer patients, albeit at a more advanced stage, and could eventually help detect oral cancer development earlier.

Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.

Differences in Extracellular Vesicle Protein Cargo Are Dependent on Head and Neck Squamous Cell Carcinoma Cell of Origin and Human Papillomavirus Status

To identify potential extracellular vesicle (EV) biomarkers in head and neck squamous cell carcinoma (HNSCC), we evaluated EV protein cargo and whole cell lysates (WCL) from HPV-positive and -negative HNSCC cell lines, as well as normal oral keratinocytes and HPV16-transformed cells. EVs were isolated from serum-depleted, conditioned cell culture media by polyethylene glycol (PEG) precipitation/ultracentrifugation. EV and WCL preparations were analyzed by LC-MS/MS. Candidate proteins detected at significantly higher levels in EV compared with WCL, or compared with EV from normal oral keratinocytes, were identified and confirmed by Wes Simple Western protein analysis. Our findings suggest that these proteins may be potential HNSCC EV markers as proteins that may be (1) selectively included in EV cargo for export from the cell as a strategy for metastasis, tumor cell survival, or modification of tumor microenvironment, or (2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. This work demonstrates that our method can be used to reliably detect EV proteins from HNSCC, normal keratinocyte, and transformed cell lines. Furthermore, this work has identified HNSCC EV protein candidates for continued evaluation, specifically tenascin-C, HLA-A, E-cadherin, EGFR, EPHA2, and cytokeratin 19.