Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF-α plays an important role in the destruction of acinar structures. Here we examined TNF-α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS-SV-AC) cells were treated with TNF-α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF-α treatment were investigated. TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS-SV-AC cells treated with TNF-α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF-κB activity in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells, we detected similar TNF-α suppression of AQP5 expression in non-transfected cells and in a super-repressor form of IκBα cDNA-transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells. Therefore, our results may indicate that TNF-α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.

Taxanes, including docetaxel, are widely used for the treatment of squamous cell carcinoma of the head and neck. However, the gastrointestinal toxicity of docetaxel has limited its high-dose clinical use. In this study, we examined the synergistic anticancer effects of combined low-dose docetaxel and γ-tocotrienol treatment on human oral cancer (B88) cells. We treated B88 cells with docetaxel and γ-tocotrienol at concentrations of 0.5 nM and 50 μM, respectively. When cells were treated with either agent alone at a low dose, no significant cytotoxic effect was observed. However, the simultaneous treatment of cells with both agents almost completely suppressed cell growth. Whereas docetaxel stimulated the expression of nuclear factor-κB (NF-κB) p65 protein in B88 cells, γ-tocotrienol slightly inhibited the expression of constitutive nuclear p65 protein. Of note, the combined treatment with both agents inhibited docetaxel-induced nuclear p65 protein expression. Electrophoretic mobility shift assay (EMSA) revealed that the simultaneous treatment with these agents suppressed the NF-κB DNA binding activity in B88 cells. In addition, γ-tocotrienol downregulated the docetaxel-induced expression of NF-κB-regulated gene products associated with the inhibition of apoptosis. Furthermore, the activation of initiator caspases, caspases-8 and -9, and the effector caspase, caspase-3, was detected following treatment with both agents. Finally, apoptosis was also clearly observed as demonstrated by the cleavage of poly(ADP-ribose) polymerase (PARP) and nuclear fragmentation through the activation of caspase-3 by combined treatment with docetaxel and γ-tocotrienol. These findings suggest that the combination treatment with these agents may provide enhanced therapeutic response in oral cancer patients, while avoiding the toxicity associated with high-dose β-tubulin stabilization monotherapy.

Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 μg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS generated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes.

Xerostomia is the symptom of oral dryness resulting most frequently, but not exclusively, from salivary gland hypofunction. Because the prevalence of xerostomia may increase with age, it has multiple oral health consequences in aging populations. In the present study, we demonstrate that the in vivo administration of 5-aza-2'-deoxycytidine (5-Aza-CdR; decitabine), a DNA demethylating agent, to the murine aging model C57BL/6CrSlc mice (24 wks old) increased the volumes of salivary flow compared with those of control mice. Western blot analysis and immunohistochemical staining demonstrated the augmented expression of AQP5 protein in the salivary glands of 5-Aza-CdR-treated mice compared with those of control mice. In addition, AQP5 protein expression levels in 5-Aza-CdR-treated old mice (27 wks old) were much higher than those in untreated and young mice (6 wks old). Global methylation levels in the salivary glands were significantly lower in the 5-Aza-CdR-treated mice than in the untreated mice. Moreover, the induction of demethylation in the AQP5 promoter of 5-Aza-CdR-treated mice was stronger than in the control mice. Analysis of our data therefore suggests that a DNA demethylating agent may be a useful drug for restoring hyposalivation in elderly individuals, thereby leading to the resolution of xerostomia.

The purpose of this study was to apply high-wattage pulsed irradiation of linearly polarized near-infrared light to the stellate ganglion area for burning mouth syndrome (BMS) and to assess the efficacy of the stellate ganglion area irradiation (SGR) on BMS using differential time-/frequency-domain parameters (D parameters). Three patients with BMS received high-wattage pulsed SGR; the response to SGR was evaluated by visual analogue scale (VAS) representing the intensity of glossalgia and D parameters used in heart rate variability analysis. High-wattage pulsed SGR significantly decreased the mean value of VAS in all cases without any adverse event such as thermal injury. D parameters mostly correlated with clinical condition of BMS. High-wattage pulsed SGR was safe and effective for the treatment of BMS; D parameters are useful for assessing efficacy of SGR on BMS.