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Objectives Endotoxins derived from bacteria in the oral cavity enter the bloodstream, leading to the onset of various systemic diseases, including heart valve disease. However, information on the oral bacteria involved in aortic stenosis is limited. Materials and Methods We comprehensively analyzed the microbiota in aortic valve tissues collected from patients with aortic stenosis using metagenomic sequencing and investigated the relationships between the valve microbiota and the oral microbiota and oral cavity conditions. Results Metagenomic analysis revealed the presence of 629 bacterial species in five oral plaque and 15 aortic valve clinical specimens. Patients were classified into two groups according to their aortic valve microbiota composition using principal coordinate analysis. Examination of the oral conditions of the patients showed no difference in the decayed/missing/filled teeth index; however, patients were divided into two groups (A and B) based on the aortic valve microbiota composition. Bacteria in group B tended to be associated with severe disease, and the number of bacteria on the dorsum of the tongue and the positive rate of bleeding during probing were significantly higher in this group than in group A. The pathophysiology of aortic stenosis may be related to the presence of oral bacteria such as Streptococcus oralis and Streptococcus sanguinis following bacteremia. Conclusions Systemic inflammation in severe periodontitis may be driven by the oral microbiota, supporting the indirect (inflammatory) association between oral bacteria and aortic stenosis. Clinical Relevance: Appropriate oral hygiene management may contribute to the prevention and treatment of aortic stenosis.

According to the progress of the antibiotic medicine, the severe infection case is decreasing recently. We recently experienced a case of temporal fossa abscess caused by periapical periodontitis the upper left side of the second molar. A 79-year-old woman visited to Showa University Dental Hospital complaining of the painful swelling at the left side of the temporal and trismus. The routine blood test exhibited a severe inflammation. We performed drainage treatment at temporal skin and normal oral bacterial flora was detected by the bacteriological examination. Under treatment included the administration of antibiotics, the inflammation healed without serious complication and she recovered completely.

Fibroma-like lesions occur with relatively high frequency in oral soft tissues, but represent reactive proliferation against chronic irritation. True fibromas are rare. Tumor size is often seen to range from the mass of a red bean to a soybean. We encountered a case of huge fibroma in the palate with a long history of more than 30 years. The patient was a 60-year-old man who became aware of a tumor in the right palate about 30 years earlier. The mass had tended to increase in size in recent years, and was introduced to our department for the first visit. As an intraoral finding, a pedicled mass measuring 29 × 27 mm was found in the right palate. We also considered the possibility of verrucous carcinoma, and performed biopsy, obtaining a diagnosis of fibroma. Right palatectomy was performed under general anesthesia in October 2016. We performed immunohistochemical examination of the surgical specimen, obtaining a definitive diagnosis of fibroma. As of 1 year postoperatively, no recurrence has been identified and the course has been good.

This clinical report describes the management of a patient who was diagnosed with amniotic band sequence (ABS) complicated with severe skeletal malocclusion, associated with severe muscle weakness. The patient was a 23-year-old male who underwent treatment in our hospital to improve chewing, phonation, and swallowing difficulties caused by severe abnormality of the maxillofacial skeleton. We evaluated chewing, swallowing and language functions preoperatively, and started muscle function and oral rehabilitation therapy. We performed 2-jaw osteotomy after preoperative orthodontic treatment. The patient continued with postoperative orthodontic treatment and oral rehabilitation. Postoperatively, the patient showed improved maxillofacial morphology, but was unable to attain sufficient improvement of masticatory and language functions due to atrophy and poor development of the masticatory muscles. These results suggest that obtaining sufficient therapeutic effect on oral functions is not possible if the masticatory muscles are not sufficiently strong due to atrophy or hypoplasia.

Salivary duct carcinoma (SDC) is regarded as high-grade malignancy with accompanied by bone destruction originating in the ductal epithelium of salivary gland. SDC arising in the minor salivary gland of the oral cavity is very rare. We present here a single case of SDC arising from the minor salivary gland of a 82-year-old man. His first symptom was swelling on the left side of the palate. There is no bone resorption by the results of imaging studies, so benign tumor located in minor salivary gland was suspected. The tumor was dissected surgically and the intraductal component is microscopically described as a cribriform, papillary, solid growth pattern, with central necrosis. Immunohistochemical staining of intraductal component cells was positive for human epidermal growth factor receptor type2 (Her2), gross cystic disease fluid protein-15 (GCDFP-15) and androgen receptor (AR). As a result, the tumor was diagnosed as a SDC.

summary It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.

Mucosa-associated lymphoid tissue (MALT) lymphoma arising in the sublingual gland of the oral cavity is very rare. We present here a single case of MALT lymphoma arising from the sublingual gland of a 64-year-old man. His first symptom was swelling on the left side of the floor of the mouth. On the basis of the results of imaging studies, a salivary gland tumor was suspected. The tumor was dissected surgically and microscopically, revealing dense atypical lymphoid cell infiltration with severe destruction of adjacent normal salivary glands. Immunohistochemical staining of atypical lymphoid cells was positive for CD20 and CD79a. Ki-67 positive cells were localized to scattered transformed large cells. As a result, the tumor was diagnosed as a primary sublingual gland marginal zone B cell lymphoma of MALT type.

Enhancer of zeste homolog-2 (EZH2) and B lymphoma Mo-MLV insertion region-1 homolog (BMI1) are members of the polycomb group of proteins, which function as transcriptional repressors through chromatin modification. EZH2 forms part of the polycomb repressive complex (PRC)-2, while BMI1 is a component of PRC1. Previous studies have shown that EZH2 is highly expressed in various type of cancers. Expression of EZH2 is reported to be regulated by the P53-E2F/retinoblastoma (RB)-related pathway, and a correlation between P53 mutation and EZH2 expression was recently found in breast cancer. Here, we examined the relationship between P53 and EZH2 in oral squamous cell carcinoma (OSCC).Using immunohistochemistry, we investigated the expression of EZH2 and BMI1 in 99 surgically-resected OSCC and 34 epithelial dysplasia samples. We analyzed associations between aberrant expression of EZH2 and BMI1, and clinicopathological findings and patient outcome. P53 expression was also examined and analyzed in relation to EZH2 and BMI1 expression.EZH2 and BMI1 protein were up-regulated in OSCC tissues compared with epithelial dysplasia and normal epithelium. Aberrant EZH2 and BMI1 protein expression was observed in 32 and 59 of the 99 OSCC samples, respectively. Aberrant EZH2 and BMI1 expression was significantly associated with mode of invasion, but not with lymph node metastasis or survival rate. Aberrant EZH2 expression was associated with P53 alteration in OSCC tissue. Expression of EZH2 mRNA in SAS/neo cells, which have wild-type P53, was significantly lower than in SAS/mp53 cells that have a mutant P53 gene.P53 alteration may be involved in dysregulated EZH2 expression, and aberrant expression of EZH2 may play a role in carcinogenesis of OSCC.

microRNAs (miRNAs) are involved in cancer pathogenesis, apoptosis and cell growth, thereby functioning as both tumor suppressors and oncogenes. However, the expression patterns and roles of miRNAs in oral squamous cell carcinoma (OSCC) remain largely unknown. We hypothesized that oral cancer may have a unique miRNA profile, which in turn may play a critical role in oral cancer development, progression, diagnosis and prognosis. We, therefore, investigated the expression profiles of 29 OSCC tumors and 7 normal oral mucosal samples. The miRNA expression patterns in OSCC were examined by TaqMan-based microRNA assays. We were subsequently able to identify the candidates of cancer-related miRNAs through analysis of the miRNA expression profiles. In conclusion, OSCC tissues were shown to have a unique miRNA profile pattern when compared with that in normal tissues. The present study may provide useful information for further investigation of the functional roles of miRNAs in OSCC development, progression, diagnosis and prognosis.

Epigenetic regulation is essential in determining cellular phenotypes during differentiation. Although tissue-specific DNA methylation has been studied, the significance of methylation variance for tissue phenotypes remains unresolved, especially for CpG-poor promoters. Here, we comprehensively studied methylation levels of 27 578 CpG sites among 21 human normal tissues from 12 anatomically different regions using an epigenotyping beadarray system. Remarkable changes in tissue-specific DNA methylation were observed within CpG-poor promoters but not CpG-rich promoters. Of note, tissue-specific hypomethylation is accompanied by an increase in gene expression, which gives rise to specialized cellular functions. The hypomethylated regions were significantly enriched with recognition motifs for transcription factors that regulate cell-type-specific differentiation. To investigate the dynamics of hypomethylation events, we analyzed methylation levels of the entire APOA1 gene locus during in vitro differentiation of embryonic stem cells toward the hepatic lineage. A decrease in methylation was observed after day 13, coinciding with alpha-fetoprotein detection, in the vicinity of its transcription start sites (TSSs), and extends up to ∼200 bp region encompassing the TSS at day 21, equivalent to the hepatoblastic stage. This decrease is even more pronounced in the adult liver, where the entire APOA1 gene locus is hypomethylated. Furthermore, when we compared the methylation status of induced pluripotent stem (iPS) cells with their parental cell, IMR-90, we found that fibroblast-specific hypomethylation is restored to a fully methylated state in iPS cells after reprogramming. These results illuminate tissue-specific methylation dynamics in CpG-poor promoters and provide more comprehensive views on spatiotemporal gene regulation in terminal differentiation.

Oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture. We investigated the role of a proteaosome inhibitor in the survival and apoptosis of these cells. We found that the proteasome inhibitor MG132 markedly accelerated TRAIL-mediated apoptosis in OSCC cell lines HSC-2 and HSC-3. Addition of TRAIL to MG132-treated cells resulted in Bid cleavage. Furthermore, the inhibitors of caspase-3, caspase-8 and caspase-9 reduced the accelerative effect of MG 132 on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of a proteasome inhibitor on TRAIL-mediated apoptosis may contribute to both extrinsic and intrinsic pathways. MG132 enhanced the expression of the TRAIL receptors DR4 and DR5, and neutralization of DR5 receptors showed a marked reduction of TRAIL-mediated apoptosis, whereas that of DR4 was a partial reduction. MG132 also markedly reduced cellular FLICE-inhibitory protein (c-FLIP), cellular inhibitor of apoptosis protein-1 (cIAP-1), X-linked IAP (XIAP) and survivin. Therefore, MG132 provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of DR5, c-FLIP, cIAP-1, XIAP and survivin. The proteasome inhibitor MG132 may therefore represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Malignant tumors are exposed to various levels of hypoxic condition in vivo. It has been known that tumor cells under hypoxia are resistant to chemotherapies. To clarify the mechanism of the hypoxia-induced chemoresistance, we evaluated the effects of hypoxia on the resistance of oral squamous cell carcinoma (OSCC) cell lines to 5-fluorouracil (5-FU). OSCC cells were divided to two groups by the proliferation activity under hypoxic condition; hypoxia-resistant (HR) and hypoxia-sensitive (HS) cells. Growth of HS cells were inhibited by hypoxia and introduced to G(1) arrest in cell cycle. 5-FU effect on HS cell viability was markedly reduced in hypoxic condition without an induction of chemoresistant related protein, P-glycoprotein. However, proliferation, cell cycle, and 5-FU sensitivity of HR cells were not affected by hypoxia. Hypoxia-inducible factor (HIF)-1alpha was induced by hypoxia in all OSCC cell lines, but diminished in HS cells within 48h. Expression of p21 and p27 was strongly augmented and CyclinD expression was reduced by hypoxia in HS cells. However, the expression of these proteins was constitutive in HR cells during 48h hypoxic culture. Phosphorylation of mammalian target of rapamycin (mTOR) was reduced by hypoxia in HS cells. From these findings, we concluded that HS OSCC cells acquire 5-FU resistance under hypoxia by G(1)/S transition through an upregulation of cell cycle inhibitors.

The purpose of this study was to determine whether phosphatidylinositol 3-kinase (PI 3-K) inhibitors could modulate the apoptotic activity of the anticancer drugs cisplatin, 5-fluorouracil or docetaxel in an oral squamous cell carcinoma (OSCC) cell line, HSC-2. In preliminary experiments, cisplatin, 5-fluorouracil and docetaxel inhibited the proliferation of OSCC cells in a dose-dependent manner. We found that two PI 3-K inhibitors, wortmannin and LY294002, markedly suppressed the phosphorylation of Akt in OSCC cells. Treatment of OSCC cells with PI 3-K inhibitors significantly enhanced cisplatin-, 5-fluorouracil- or docetaxel-induced apoptosis. Caspase-3 and -9 inhibitors, but not a caspase-8 inhibitor, reduced anticancer drug-mediated apoptosis in PI 3-K inhibitor-treated OSCC cells, suggesting that the apoptotic pathway induced by the combination of anticancer drug therapy and PI 3-K inhibition may be functionally related to the intrinsic apoptotic pathway in OSCC cells. Expression of Bcl-2, cellular inhibitor of apoptosis protein-1 (cIAP-1), and X-linked IAP was down-regulated, and expression of Bax was up-regulated by PI 3-K inhibitors, while that of Bcl-xL, Bak and cIAP-2 was not attenuated. We also found that Bad phosphorylation was down-regulated by PI 3-K inhibitors. These results suggested that inhibition of PI 3-K enhances the susceptibility of OSCC cells to anticancer drug-mediated apoptosis through regulation of expression and post-translational modification of both pro- and anti-apoptotic proteins. These findings could potentially lead to new strategies for improving the efficacy of anticancer drugs in OSCC cells.

In general, oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture in vitro. Here, we studied the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt in survival and apoptosis of these cells. The PI 3-K inhibitors wortmannin and LY294002 markedly suppressed phosphorylation of Akt and accelerated TRAIL-mediated apoptosis in OSCC cells. Addition of TRAIL to PI 3-K inhibitor-treated cells resulted in caspase-8 activation and loss of mitochondrial membrane potential. Furthermore, inhibitors of caspase-3, -8 and -9 reduced the accelerative effect of PI 3-K inhibitors on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of PI 3-K inhibitors on TRAIL-mediated apoptosis may contribute to both the extrinsic and intrinsic pathways. Although PI 3-K inhibitors did not affect expression of the TRAIL receptors DR4 and DR5, we observed a marked reduction in expression of cellular FLICE-inhibitory protein (c-FLIP), Bcl-2, cellular inhibitor of apoptosis protein-1 (cIAP-1) and X-linked IAP (XIAP), whereas Bax was up-regulated and no significant difference was observed in expression of Bcl-xL, Bak or cIAP-2. Therefore, the PI 3-K/Akt signaling pathway provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of c-FLIP, Bcl-2, Bax, cIAP-1 and XIAP expression. These results suggest that PI 3-K inhibitors may represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Molecular inhibition of epidermal growth factor receptor (EGFR) signaling is a promising cancer treatment strategy. We examined whether inhibition of EGFR signaling would affect the susceptibility of oral squamous cell carcinoma (OSCC) cells to Fas-mediated apoptosis. Treatment of OSCC cells with an anti-EGFR monoclonal antibody, C225, and an EGFR tyrosine kinase inhibitor, AG1478, which target the extracellular and intracellular domains of the receptor, respectively, inhibited phosphorylation of EGFR and its downstream effector molecule Akt and amplified the induction of Fas-mediated apoptosis. In OSCC cells treated with EGFR inhibitors, Fas-mediated apoptosis was accompanied by caspase-8 activation but not Bid cleavage. Caspase-3 and -8 inhibitors reduced the effect of EGFR inhibitors on Fas-mediated apoptosis in OSCC cells, but a caspase-9 inhibitor did not. These results indicate that the pro-apoptotic activity of EGFR inhibitors in OSCC cells depends on the extrinsic pathway of the caspase cascade. Although EGFR inhibitors did not affect the expression of Fas, the Fas-associated death domain protein, or procaspase-8 in OSCC cells, the inhibition downregulated cellular FLICE-inhibitory protein (c-FLIP). Moreover, knockdown of c-FLIP in HSC-2 cells with a small interfering RNA strongly enhanced Fas-mediated apoptosis. These results suggest that the EGFR signaling pathway may, in part, regulate Fas-mediated apoptosis in OSCC cells through c-FLIP expression.

Despite combined surgery and radiation therapy, long-term survival for oral cancer has not improved over the past several decades. This is primary due to poor clinical prognosis of patients with the lymph nodes metastasis. Therefore, deeper understanding of the molecular basis of highly malignant property and patient stratification along with these characters are needed. Although, recent genome-wide analyses of DNA methylation have revealed a large number of aberrantly methylated genes in many cancers, and allowed us to identify the molecularly defined subgroups such as CpG island methylation phenotype (CIMP) in certain tumors, the methylation diversity of clinical oral cancers are still unknown. The purpose of this study is to characterize DNA methylation profiles of oral cancer tissues (six normal oral mucosal swab, three non-cancerous mucosal tissues, and fifty-nine oral cancer tisseus) and to identify the specific methylation pattern related to the clinical features. Genome-wide methylation analysis was performed using Infinium HumanMethylation27 BeadArray system, which can analyze methylation status of 27,000 CpG sites semi-quantitatively, using 500ng of genomic DNA.We found 2,762 aberrantly methylated CpG sites in oral cancers against normal mucosa. By unsupervised clustering analysis, oral cancer could be divided into two methylation epigenotypes that are significantyly correlated with high incidence of lymph node metastasis and poor differentiation. We obtained the highly predictive candidates of classifier genes and they were confirmed by MassARRAY analysis. In summary, we identified two methylation epigenotypes related with clinical outcome in oral cancer, and candidate classifier genes would be useful clues of patient stratification for intensive therapy to overcome the tumor recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4809. doi:10.1158/1538-7445.AM2011-4809