Reactive oxygen species generation required for autophagy induction during butyrate- or propionate-induced release of damage-associated molecular patterns from dying gingival epithelial Ca9-22 cells.

PURPOSE: Bacterial cells in mature dental plaque produce a high concentration of short-chain fatty acids (SCFAs) such as butyrate and propionate. SCFA-treatment on human gingival epithelial Ca9-22 cells induced cell death. However, the exact mechanism underlying cell death remains unclear. In this study, the relationship between reactive oxygen species (ROS) and autophagy induction during SCFA-induced cell death was examined. METHODS: Human gingival epithelial Ca9-22 cells were treated with butyrate or propionate to induce cell death and the number of dead cells were measured using SYTOX-green dye. A siRNA for ATG5 and N-acetylcysteine (NAC) were used for autophagy reduction and ROS-scavenging, respectively. Release of damage-associated molecular patterns (DAMPs) such as Sin3A-associated protein 130 (SAP130) and high-mobility group box 1 (HMGB1) were detected using western blot. RESULTS: Reducing autophagy significantly suppressed SCFA-induced Ca9-22 cell death. ROS generation was observed upon SCFA treatment, and scavenging ROS with NAC decreased cell death. NAC also reduced the SCFA-induced increase in microtubule-associated protein 1 light chain 3B (LC3B)-I and LC3B-II, and mitigated the release of DAMPs. CONCLUSION: The findings suggest that ROS generation is necessary for autophagy, which is required for SCFA-induced cell death and accompanying DAMP release.

Butyrate-treatment induces gingival epithelial cell death in a three-dimensional gingival-connective tissue hybrid co-culture system.

Three-dimensional (3D) cell culture systems are reported to be more physiologically similar to the in vivo state than 2-dimensional (2D) models, which are extensively employed in periodontal research. Herein, we developed a 3D gingival tissue model with both epithelial and lamina propria layers using human gingival epithelial Ca9-22 cells and primary gingival fibroblasts. The epithelial layer of the developed 3D gingival tissue culture was treated with butyrate, a metabolite of oral bacteria, and the treatment induced the release of damage-associated molecular patterns, such as DNA and Sin3A associated protein 130 kDa (SAP130). Taken together, butyrate exposure to the epithelium of 3D gingival epithelial-connective tissue hybrid systems could induce epithelial cell death and the subsequent release of damage-associated molecular patterns.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, a P300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrate or propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

The changes in oral volume and hyoid bone position after maxillomandibular advancement and genioglossus advancement for patients with obstructive sleep apnea.

INTRODUCTION: Maxillomandibular advancement (MMA) and genioglossus advancement (GA) are surgeries for patients with obstructive sleep apnea (OSA). Postoperative evaluation is primarily based on the apnea-hypopnea index (AHI) measured by polysomnography. The purpose of this study was to identify the timing of hyoid bone relocation after MMA and GA surgery and to investigate whether or not hyoid bone relocation can be an indicator of postoperative evaluation of OSA. METHODS: Patients with OSA underwent MMA and GA surgery. Changes in hyoid bone position and tongue-to-oral volume ratio were analyzed on lateral radiographs before, immediately after, and 1 year after surgery. Then, a correlation was verified between these changes and postoperative AHI. RESULTS: In 18 patients studied, the position of the hyoid bone did not show a constant tendency immediately after surgery. One year after surgery, the bone had moved anteriorly and toward the oral cavity in all patients compared to its preoperative position. And AHI correlated with the movement of the hyoid bone to the oral side. DISCUSSION: One year after surgery, the tongue was adapted to the newly enlarged oral space, and as a result, the low position of the hyoid bone before the operation was improved. The findings suggest that the degree of lowering of the hyoid bone may be an indicator of the improvement of AHI.

A preoperative predictive study of advantages of airway changes after maxillomandibular advancement surgery using computational fluid dynamics analysis.

The purpose of this study was to develop a simulation approach for predicting maxillomandibular advancement-induced airway changes using computational fluid dynamics. Eight patients with jaw deformities who underwent maxillomandibular advancement and genioglossus advancement surgery were included in this study. Computed tomography scans and rhinomanometric readings were performed both preoperatively and postoperatively. Computational fluid dynamics models were created, and airflow simulations were performed using computational fluid dynamics software; the preferable number of computational mesh points was at least 10 million cells. The results for the right and left nares, including simulation and postoperative measurements, were qualitatively consistent, and surgery reduced airflow pressure loss. Geometry prediction simulation results were qualitatively consistent with the postoperative stereolithography data and postoperative simulation results. Simulations were performed with either the right or left naris blocked, and the predicted values were similar to those found clinically. In addition, geometry prediction simulation results were qualitatively consistent with the postoperative stereolithography data and postoperative simulation results. These findings suggest that geometry prediction simulation facilitates the preoperative prediction of the postoperative structural outcome.

New method of Le Fort I osteotomy with increased stability.

Backsliding is a major problem when moving the maxilla significantly forward in orthognathic surgery. For example, in sleep surgery, maxillomandibular advancement is an application of orthognathic surgery, and it is well known that the anterior movement of the maxilla back and forth is an important factor that greatly widens the pharyngeal airway. However, postoperative backsliding is a major problem in this surgery. Therefore, a surgical method was devised to prevent the maxilla from retracting by adjusting the bone when moving the maxilla forward.

Bioinformatics analysis of dysregulated exosomal microRNAs derived from oral squamous cell carcinoma cells.

PURPOSE: The present study aimed to identify dysregulated exosomal miRNAs associated with diagnostic and therapeutic biomarkers in oral squamous cell carcinoma (OSCC). METHODS: Microarray analysis was used to compare expression profiles of exosomal miRNAs in the OSCC-derived cell lines HSC-2, HSC-3, Ca9-22, and HO-1-N1 with those in human normal keratinocytes (HNOKs). The identified OSCC-related miRNAs and their potential target genes were analyzed with bioinformatic analyses, and the data were subjected to Ingenuity Pathway Analysis (IPA) to clarify functional networks and gene ontologies of the identified exosomal miRNAs secreted by OSCC cells. RESULTS: Comparison with HNOKs detected 8 upregulated and 12 downregulated miRNAs in OSCC-secreted exosomes. The potential target mRNAs of these dysregulated miRNAs were suggested by IPA, and 6 significant genetic networks were indicated by genetic network analysis. Furthermore, 4 crucial upstream miRNAs-miR-125b-5p, miR-17-5p, miR-200b-3p, and miR-23a-3p-were identified. miR-125b-5p was a central node in the most significant network. Gene ontology analysis showed significant enrichment of genes with cancer-related functions, such as molecular mechanisms of cancer, cell cycle, and regulation of the epithelial-mesenchymal transition. CONCLUSION: These results provide a comprehensive view of the functions of dysregulated exosomal miRNAs in OSCC, thus illuminating OSCC tumorigenesis and development.

Development of prediction models for the sensitivity of oral squamous cell carcinomas to preoperative S-1 administration.

S-1 is an anticancer agent that is comprised of tegafur, gimeracil, and oteracil potassium, and is widely used in various carcinomas including oral squamous cell carcinoma (OSCC). Although an established prediction tool is not available, we aimed to develop prediction models for the sensitivity of primary OSCC cases to the preoperative administration of S-1. We performed DNA microarray analysis of 95 cases with OSCC. Using global gene expression data and the clinical data, we developed two different prediction models, namely, model 1 that comprised the complete response (CR) + the partial response (PR) versus stable disease (SD) + progressive disease (PD), and model 2 that comprised responders versus non-responders. Twelve and 18 genes were designated as feature genes (FGs) in models 1 and 2, respectively, and, of these, six genes were common to both models. The sensitivity was 96.3%, the specificity was 91.2%, and the accuracy was 92.6% for model 1, and the sensitivity was 95.6%, the specificity was 85.2%, and the accuracy was 92.6% for model 2. These models were validated using receiver operating characteristic analysis, and the areas under the curves were 0.967 and 0.949 in models 1 and 2, respectively. The data led to the development of models that can reliably predict the sensitivity of patients with OSCC to the preoperative administration of S-1. The mechanism that regulates S-1 sensitivity remains unclear; however, the prediction models developed provide hope that further functional investigations into the FGs will lead to a greater understanding of drug resistance.

Cephalometric analysis of the pharyngeal airway space after maxillary advancement surgery.

This study evaluated the effect of maxillary advancement surgery on the size of the pharyngeal airway space (PAS). Lateral cephalometric radiographs were collected for 90 patients (29 men and 61 women; average age, 27.2 ± 8.1 years) before (T1) and 1 year after (T2) maxillary advancement surgery. Horizontal and vertical changes in the maxilla and PAS were measured and classified by distance. The maxilla was advanced horizontally by 2.9 ± 1.7 mm and vertically by 2.7 ± 1.4 mm. Upward maxillary movement of ≥4 mm significantly increased PAS (mean change in PAS, 2.6 mm), and upward maxillary movement significantly decreased the posterior nasal spine to the P-point. Only patients with vertical advancement ≥4 mm and horizontal advancement of 3 mm had significant increases in all three PAS parameters. Although forward maxillary movement is believed to have a large effect on PAS, it is suggest that upward vertical movement is more effective for improving PAS. Both the extent and direction of maxillar movement should be considered. Future studies should use cone-beam computed tomography to evaluate the effect of axial direction and differences in PAS.

Computational fluid dynamics analysis for the preoperative prediction of airway changes after maxillomandibular advancement surgery.

Maxillomandibular advancement surgery is useful for treatment of sleep apnea. However, preoperative analysis and evaluation to facilitate decision-making regarding the direction and distance of maxillomandibular movement has primarily consisted of morphological analysis; physiological function is not evaluated. To improve preoperative prediction, this study used fluid simulation to investigate the characteristics and effects of airway changes associated with maxillomandibular movement. A one-dimensional model with general applicability was thus developed. Actual measurements of flow in patients were used in this fluid simulation, thus achieving an analysis closer to clinical conditions. The simulation results were qualitatively consistent with the actual measurements, which confirmed the usefulness of the simulation. In addition, the results of the one-dimensional model were within the error ranges of the actual measurements. The present results establish a foundation for using accumulating preoperative measurement data for more-precise prediction of postoperative outcomes.

Retrospective clinical study of 296 patients with mass lesions of the tongue.

To better understand the clinical features of mass lesions of the tongue, we retrospectively evaluated frequency, recurrence rate, and complications in 296 patients who had undergone surgery for such lesions. The diagnoses were fibroma (43.6%), mucous cyst (14.2%), papilloma (11.8%), hemangioma (7.8%), granuloma (6.4%), lipoma (1.4%), schwannoma (1.0%), ectopic tonsil (0.7%), and other (13.2%). Recurrence was noted in two patients (0.7%). Twenty-two patients (7.4%) developed surgical complications, including lingual nerve paralysis (6.4%), glossodynia (0.6%), and postoperative infection (0.3%). Lingual nerve paralysis was observed in the ventral portion (42.1%) of the tongue, apex (36.8%), lateral border (10.5%), and dorsum (10.5%). When all sites were considered together, there was no significant difference in the number of patients presenting with lingual nerve paralysis (P = 0.075). However, there were significant differences in lingual nerve paralysis at the lateral border (P < 0.05), apex (P < 0.05), and dorsum (P < 0.001) but not at the ventral portion (P > 0.05) in the size of the patients with versus without it which suggests that the risk of lingual nerve paralysis is higher at the ventral tongue, regardless of tumor size. These results shed light on the clinical features of mass lesions of the tongue.

Rapid detection of Candida albicans in oral exfoliative cytology samples by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) rapidly amplifies DNA under isothermal conditions. The aim of this study was to detect Candida albicans and compare the positivity rate in the LAMP reaction with that of conventional methods for oral exfoliative cytology (EC) samples. Sixty-eight EC samples from 53 patients were subjected to LAMP analysis. These patients had been clinically diagnosed with leukoplakia, squamous cell carcinoma, oral lichen planus (OLP), stomatitis, oral candidiasis, and other malignancies. LAMP reactions were defined as positive when the sample turbidity exceeded 0.1 (arbitrary unit). Periodic acid-Schiff (PAS) staining and microbial culture were also performed to detect Candida species in EC samples. The LAMP reaction detected C. albicans in 42.6% of EC samples. Candida species were detected in 32.4% of the same samples by culturing and in 29.4% of samples by PAS staining. C. albicans DNA was detected most frequently in samples from OLP patients. We conclude that, in comparison to conventional methods for detection of C. albicans, the LAMP method is highly sensitive and time-saving, and does not require expensive equipment or diagnostic technology. It may therefore be useful for on-site screening of C. albicans at dental clinics.

Impact of Polymorphic Variations of Gemcitabine Metabolism, DNA Damage Repair, and Drug-Resistance Genes on the Effect of High-Dose Chemotherapy for Relapsed or Refractory Lymphoid Malignancies.

The goal of this study was to determine whether single nucleotide polymorphisms (SNPs) in genes involved in gemcitabine metabolism, DNA damage repair, multidrug resistance, and alkylator detoxification influence the clinical outcome of patients with refractory/relapsed lymphoid malignancies receiving high-dose gemcitabine/busulfan/melphalan (Gem/Bu/Mel) with autologous stem cell support. We evaluated 21 germline SNPs of the gemcitabine metabolism genes CDA, deoxycytidine kinase, and hCNT3; DNA damage repair genes RECQL, X-ray repair complementing 1, RAD54L, ATM, ATR, MLH1, MSH2, MSH3, TREX1, EXO1, and TP73; and multidrug-resistance genes MRP2 and MRP5; as well as glutathione-S-transferase GSTP1 in 153 patients with relapsed or refractory lymphoma or myeloma receiving Gem/Bu/Mel. We studied the association of genotypes with overall survival (OS), progression-free survival (PFS), and nonhematological grade 3 or 4 toxicity. CDA C111T and TREX1 Ex14-460C>T genotypes had a significant effect on OS (P = .007 and P = .005, respectively), and CDA C111T, ATR C340T, and EXO1 P757L genotypes were significant predictors for severe toxicity (P = .037, P = .024, and P = .025, respectively) in multivariable models that adjusted for clinical variables. The multi-SNP risk score analysis identified the combined genotypes of TREX1 Ex14-460 TT and hCNT3 Ex5 +25A>G AA as significant predictors for OS and the combination of MRP2 Ex10 + 40GG/GA and MLH1 IVS12-169 TT as significant predictor for PFS. Polymorphic variants of certain genes involved in gemcitabine metabolism and DNA damage repair pathways may be potential biomarkers for clinical outcome in patients with refractory/relapsed lymphoid tumors receiving Gem/Bu/Mel.

Lipocalin-2 is associated with radioresistance in oral cancer and lung cancer cells.

The aim of the present study was to identify a target molecule that could predict the efficacy of radiotherapy in oral squamous cell carcinoma (OSCC). We used DNA microarray analysis to identify differences in gene expression after X-ray irradiation. We compared the gene expression profiles between X-ray (8 Gy)-irradiated Ca9-22 cells (an OSCC-derived cell line) and unirradiated Ca9-22 cells. A total of 167 genes with a 2-fold higher level of expression induced by X-ray irradiation were identified. Lipocalin-2 (LCN2) had the greatest increase in expression after X-ray irradiation, and it was categorized in a network that has cancer-related functions with the Ingenuity Pathway Analysis tool. Upregulated expression of LCN2 mRNA was validated by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. When the LCN2 gene was knocked down in OSCC cells (Ca9-22 and HSC-2) and lung cancer cells (A549) by using small interfering RNA, the radiosensitivity of these cells was enhanced. Our findings suggest that the overexpression of LCN2 is likely associated with radioresistance in oral cancer and lung cancer cells, and that LCN2 expression levels could be used to predict radioresistance. Thus, regulating the expression or function of LCN2 could enhance the radiation response, resulting in a favorable outcome of radiotherapy.

Angiosarcoma of the scalp diagnosed by the presence of neck inflammation: a case report.

Angiosarcoma is an uncommon malignancy, which spread out from the endothelial cells of vessels. Scalp angiosarcoma with cervical lymph node metastasis is particularly rare. This article describes a rare case of angiosarcoma of the scalp, presenting as neck inflammation. Imaging procedures such as computed tomography (CT), magnetic resonance image (MRI) and ultrasonography (US) were not sufficient to diagnose this case. A needle biopsy provided an effective and accurate diagnosis of cervical lymph node metastasis. Additional observation and physical examination was required to diagnose the origin of the primary cancerous lesion. Once the angiosarcoma diagnosis was confirmed histologically, sequential weekly and monthly docetaxel (DTX) treatment was effective in preventing reoccurrence. Nonetheless, the optimization of angiosarcoma treatment remains a future goal. Although patients generally describe pain and swelling at the primary lesion site, this patient complained only of painful neck inflammation, without any indication of pain or swelling of the scalp. A revised diagnostic protocol should note that cervical lymph node metastasis of unknown primary origin may result from angiosarcoma of the scalp.

Real-time tissue elastography for the diagnosis of lymph node metastasis in oral squamous cell carcinoma.

We compared conventional ultrasound (US) B-mode, color Doppler and elastographic assessment of lymph node (LN) stiffness against pathological findings from surgical samples, to determine the most useful factors for identifying LN metastases. Seventy-one LNs in 19 patients with oral squamous cell carcinoma (OSCC) were examined. Using our new system, elastography images were scored from 1-5. The score 1-4 were correlated with the blue area of each LN, which indicated increased stiffness: (1) none; (2) < 50%; (3) 50%; or (4) > 50%. A score 5 indicated central necrosis and did not correlate with the blue area. We found significant differences in minimal diameter, shape index, margin, internal structure, hilus presence or absence, elastography score and percentage of blue area between metastatic and nonmetastatic LNs. Stepwise regression analysis identified elastography score 3-5 as an independent significant LN metastatic factor, suggesting that our scoring system may be useful for accurately diagnosing metastatic LNs.

Upregulated expression of ADAM12 is associated with progression of oral squamous cell carcinoma.

ADAMs are a disintegrin and metalloproteinase family of membrane-associated metalloproteinases characterized by their multidomain structure, and have been reported to be associated with various malignant tumors. The aim of this study was to identify crucial members of the ADAM family in oral squamous cell carcinoma (OSCC), and to reveal their biological function and clinical significance. To clarify whether ADAM family genes are involved in OSCC, changes in the expression profile were investigated by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis and immunohistochemical analysis. Functional analysis was performed by comparing cellular proliferation of siADAM-transfected cell lines and parental cell lines. Real-time qRT-PCR analysis identified significantly upregulated expression of ADAM12 in OSCC-derived cell lines. This was validated in OSCC samples using real-time qRT-PCR and immuno-histochemical staining. ADAM12 expression was correlated with TNM classification; significantly greater expression of ADAM12 was observed in tumors with higher T classification and more advanced stages. Moreover, siADAM12-transfected cells showed both a suppressed proliferation rate and increased transforming growth factor (TGF)-ß3 expression. Our data indicate that ADAM12 is overexpressed in OSCC and might accelerate cellular proliferation. Its function may be associated with TGF-ß signaling. This study suggests that controlling the expression or activity of ADAM12 could be a useful strategy in the development of an effective cure for OSCC.

Basal cell adenoma arising in a minor salivary gland of the palate.

BACKGROUND: Basal cell adenoma is a benign neoplasm of the salivary glands. This tumor usually arises in the major glands, with the parotid being the most frequent site of occurrence, while it is rare in the minor salivary glands. We report a case of basal cell adenoma of a minor salivary gland on the palate. CASE: The patient was a 68-year-old man. Intraoral examination revealed a mass measuring 20 × 20 mm that was elastic-hard, dark violet, non-ulcerated, and covered the normal mucosa. Computed tomography scan and magnetic resonance imaging (MRI) both showed a mass situated in front of the soft palate. The T1-weighted MRI revealed tumor isointensity, and the T2-weighted image showed tumor hyperintensity. The clinical diagnosis was palate tumor, and excision was performed under general anesthesia. Histopathological examination revealed that an encapsulated mass had grown under the epithelium and indicated a diagnosis of basal cell adenoma. DISCUSSION: Although no recurrence has been detected in the 3 years and 6 months of follow up, there was a case of malignant transformation of a basal cell adenoma reported. Therefore, careful follow-up observation will continue to be important.

Antitumor activity of satraplatin in cisplatin-resistant oral squamous cell carcinoma cells.

BACKGROUND: The aim of the current study was to identify the antitumor activity of satraplatin in paired cisplatin (CDDP)-resistant oral squamous cell carcinoma (OSCC) cell line and its parental cell line. METHODS: CDDP-resistant (KB-R) cells and parental cells (KB) pair were used. Viability was assessed using the MTT and clonogenic assay. Real-time polymerase chain reaction (PCR), glutathione (GSH) assay, and flow cytometric analysis were used for further assessment. RESULTS: KB-R cells did not show cross-resistance to satraplatin. The expression status of almost all transporters was upregulated in the KB-R cells. There was no difference in the GSH levels between the KB and KB-R cells. Flow cytometric analysis indicated that with satraplatin the G2/M phase was arrested in the KB-R cells. KB-R cells contain enriched side population cells. CONCLUSION: These data suggested that satraplatin has antitumor activity against the CDDP-resistant OSCC cells. The mechanism of cross-resistance to platinum agents seems to be multifactorial.