Yoshiko Yamamura

TNF‐α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

Sjögrens 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‐αs 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.

γ-tocotrienol enhances the chemosensitivity of human oral cancer cells to docetaxel through the downregulation of the expression of NF-κB-regulated anti-apoptotic gene products

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.

DNA Demethylating Agent Decitabine Increases AQP5 Expression and Restores Salivary Function

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.

γ‑tocotrienol prevents 5‑FU‑induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5‑FU‑induced activation of Nrf2

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.

Frequency Analysis of Heart Rate Variability: A Useful Assessment Tool of Linearly Polarized Near-Infrared Irradiation to Stellate Ganglion Area for Burning Mouth Syndrome

BACKGROUND Burning mouth syndrome (BMS) is characterized by the following subjective complaints without distinct organic changes: burning sensation in mouth or chronic pain of tongue. BMS is also known as glossodynia; both terms are used equivalently in Japan. Although the real cause of BMS is still unknown, it has been pointed out that BMS is related to some autonomic abnormality, and that stellate ganglion near-infrared irradiation (SGR) corrects the autonomic abnormality. Frequency analysis of heart rate variability (HRV) is expected to be useful for assessing autonomic abnormality. OBJECTIVES This study investigated whether frequency analysis of HRV could reveal autonomic abnormality associated with BMS, and whether autonomic changes were corrected after SGR. SUBJECTS AND METHODS Eight subjects received SGR; the response to SGR was assessed by frequency analysis of HRV. RESULTS No significant difference of autonomic activity concerning low-frequency (LF) norm, high-frequency (HF) norm, and low-frequency/high-frequency (LF/HF) was found between SGR effective and ineffective groups. Therefore, we proposed new parameters: differential normalized low frequency (D LF norm), differential normalized high frequency (D HF norm), and differential low-frequency/high-frequency (D LF/HF), which were defined as differentials between original parameters just before and after SGR. These parameters as indexes of responsiveness of autonomic nervous system (ANS) revealed autonomic changes in BMS, and BMS seems to be related to autonomic instability rather than autonomic imbalance. CONCLUSIONS Frequency analysis of HRV revealed the autonomic instability associated with BMS and enabled tracing of autonomic changes corrected with SGR. It is suggested that frequency analysis of HRV is very useful in follow up of BMS and for determination of the therapeutic efficacy of SGR.

Lingual tonsillolith: prevalence and imaging characteristics evaluated on 2244 pairs of panoramic radiographs and CT images

OBJECTIVES Lingual tonsilloliths are not as well-known to radiologists than palatine tonsilloliths, although they might be common in clinical practice. The aim of this investigation was to clarify the prevalence and imaging characteristics of lingual tonsilloliths using panoramic radiographs and CT images. METHODS This study included 2244 patients without pathology at the base of tongue who had undergone panoramic radiography and CT of the maxillofacial region. The size, number and position of lingual tonsilloliths relative to the mandible and tongue were evaluated. RESULTS Lingual tonsilloliths were observed in 33 (1.5%) and 108 (4.8%) of all patients on panoramic radiographs and CT images, respectively. The prevalence was higher in patients aged ≥40 years than in those aged < 40 years (χ2, p < 0.01). They appeared as small, round- or rod-shaped calcified bodies, and they always located closely anterior (1-17 mm) to the anterior border of oropharyngeal airway on panoramic radiographs. Lingual tonsilloliths were superimposed over the surrounding soft tissue inferior to the body of the mandible, posteroinferior to the angle of the mandible and posterior to the mandible in 16 (48.5%), 15 (45.5%) and 1 (3.0%) individual, respectively. A significant correlation was observed between the detectability on panoramic radiographs and size (Spearmans r = 0.961, p < 0.01) of tonsilloliths, as revealed by CT images. CONCLUSION Lingual tonsilloliths commonly appear on CT. They also appear on panoramic radiography and may superimpose the surrounding soft tissue of the mandible. Although lingual tonsilloliths may resemble other pathological calcifications including submandibular sialoliths and lingual osseous cholistoma, they can be differentiated by carefully observing panoramic radiographs. When clinicians detect calcified bodies near the base of tongue, lingual tonsilloliths should be included in the differential diagnoses.

Abstract 3879: Expression of sox2 and oct4 in human oral squamous cell carcinoma and its relationship with clinical factors

Background: A cancer stem cell (cancer initiating cell, CSC) is considered capable of self -replication, self-differentiation, drug resistance, and immune evasion. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including sex determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (Oct4), and their expression has been reported to be the potential prognostic values. However, the prognostic values of SOX2 and Oct4 expression in patients with oral cancer are less understood. [Purpose]The aims of present study were to evaluate the expression of SOX2 and Oct4 in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, the classification of invasion mode, cerebral lymph node metastasis, distant metastasis, and disease-free survival rate. Materials and Methods: Tissue specimens were obtains from 108 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess SOX2 and Oct4 protein using at least 10% staining-positive cells as the definition of positive staining. Results: Immunohistochemical analysis of 108 cases showed that 42 cases (39%) expressed SOX2. There was no significant association between SOX2 expression and tumor size, invasion mode or histological differentiation. However, there was significant association between SOX2 expression and distant metastasis or disease-free survival rate at stage 1 and 2 patients (73 cases). Otherwise, seventy cases (65%) cases of 108 OSCC patients expressed Oct4. There was significant association between Oct4 expression and histological differentiation. There was no significant association between Oct4 expression and tumor size, invasion mode, metastasis, or disease-free survival rate. Conclusions: These findings suggested that the expression of SOX2 may be good marker indicating survival in patients with OSCC. Citation Format: Tetsuya Tamatani, Natsumi Takamaru, Go Ohe, Keiko Kudo, Takaharu Kudo, Akira Takahashi, Yoshiko Yamamura, Kenji Fujisawa, Hirokazu Nagai, Youji Miyamoto. Expression of sox2 and oct4 in human oral squamous cell carcinoma and its relationship with clinical factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3879. doi:10.1158/1538-7445.AM2017-3879