PDZ-binding kinase inhibitor OTS514 suppresses the proliferation of oral squamous carcinoma cells.

OBJECTIVE: PDZ-binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. METHODS: Four OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. RESULTS: OTS514 decreased the cell survival of OSCC cells dose-dependently, and administration of OTS514 readily suppressed the HSC-2-derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase-3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514-induced apoptosis. CONCLUSION: OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular-targeted therapeutics against OSCC.

Inhibition of VEGFR2 and EGFR signaling cooperatively suppresses the proliferation of oral squamous cell carcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently overexpressed in oral squamous cell carcinoma (OSCC), and EGFR-targeting therapeutics have been widely employed to treat patients with a variety of carcinomas including OSCC. Here, we aimed to investigate alternative signaling for OSCC survival under the disruption of EGFR signaling. METHODS: OSCC cell lines, namely HSC-3 and SAS, were utilized to investigate how EGFR disruption affects cell proliferation. Gene set enrichment analysis was performed to examine how EGFR disruption affects oncogenic signaling in OSCC cells. Disruption of KDR gene was performed using CRISPR/Cas9 techniques. A VEGFR inhibitor, vatalanib was used to research the impact of VEGFR inhibition on OSCC survival. RESULTS: EGFR disruption significantly decreased the proliferation and oncogenic signaling including Myc and PI3K-Akt, in OSCC cells. Chemical library screening assays revealed that VEGFR inhibitors continued to inhibit the proliferation of EGFR-deficient OSCC cells. In addition, CRISPR-mediated disruption of KDR/VEGFR2 retarded OSCC cell proliferation. Furthermore, combined erlotinib-vatalanib treatment exhibited a more potent anti-proliferative effect on OSCC cells, compared to either monotherapy. The combined therapy effectively suppressed the phosphorylation levels of Akt but not p44/42. CONCLUSION: VEGFR-mediated signaling would be an alternative signaling pathway for the survival of OSCC cells under the disruption of EGFR signaling. These results highlight the clinical application of VEGFR inhibitors in the development of multi-molecular-targeted therapeutics against OSCC.

Evaluation of skeletal muscle mass using prediction formulas at the level of the 12th thoracic vertebra.

OBJECTIVES: People with cancer have a high risk of cachexia and sarcopenia, which are associated with worse clinical outcomes. We evaluated the prediction accuracy of the Matsuyama et al. and Ishida et al. formulas using computed tomography (CT) slices from the twelfth thoracic vertebra (Th12) level in people with cancer. METHODS: This retrospective study included patients with advanced cancer who underwent thoracic and abdominal CT scans (n = 173). The cross-sectional area (CSA) on CT images was measured at the levels of Th12 and the third lumbar vertebra (L3). The Matsuyama et al. formula used the Th12 CSA, whereas the Ishida et al. formula used only the Th12 CSA of the spinal erectors; thus, the measurements were performed separately. The correlation between predicted and actual L3 CSA was assessed using r and the intraclass correlation coefficient. A prediction-accuracy analysis of the predicted values was also performed. RESULTS: The mean participant age was 66.2 ± 12.8 y; 50.3% of participants were women and 49.7% were men. Strong correlations were observed between the predicted and measured L3 values calculated from the two prediction formulas. The prediction-accuracy analysis using previously reported cutoff values showed that the Ishida et al. method had high sensitivity and the Matsuyama et al. method had high specificity for low skeletal muscle index determined by the predicted and measured L3 skeletal muscle index. CONCLUSIONS: Both the Matsuyama et al. and Ishida et al. formulas had good reliability on CT slices at the Th12 level in people with advanced cancer, indicating that these formulas can be applied in clinical practice.

Assessing skeletal muscle mass based on the cross-sectional area of muscles at the 12th thoracic vertebra level on computed tomography in patients with oral squamous cell carcinoma.

OBJECTIVES: This study aimed to create a formula to estimate the third lumbar vertebra (L3)1 level skeletal muscle cross-sectional area (CSA), known as a standard value to evaluate skeletal muscle mass on computed tomography (CT), using the twelfth thoracic vertebra (Th12) level skeletal muscle CSA on chest CT. MATERIALS AND METHODS: This retrospective observational study included patients aged 40 + years with a diagnosis of oral squamous cell carcinoma (n = 164). Skeletal muscle CSA on CT images was measured using the Th12 and the L3 levels of pretreatment CT scans. The predictive formula was created based on the five-fold cross-validation method with a linear regression model. Correlations between the predicted L3-level CSA and the actual L3-level CSA were evaluated using r and Intraclass Correlation Coefficients (ICC). RESULTS: The predictive formula for L3-level CSA from Th12-level CSA was: CSA at L3 (cm2) = 14.143 + 0.779 * CSA at Th12 (cm2) - 0.212 * Age (y) + 0.502 * Weight (kg) + 13.763 * Sex. Correlations between the predicted and measured L3-level CSA were r = 0.915 [0.886-0.937] and ICC = 0.911 [0.881-0.934]. CONCLUSION: We developed a formula for predicting skeletal muscle mass from the Th12-level CT slice. The predicted L3-level CSA correlated with the measured L3-level CSA.

The success rate of oral appliances based on multiple criteria according to obstructive sleep apnoea severity, BMI and age: A large multicentre study.

BACKGROUND: The efficacy of oral appliance (OA) varies greatly in patients with obstructive sleep apnoea (OSA). OBJECTIVE(S): The purpose of this cross-sectional study was to investigate the success rate of OA for OSA patients. METHODS: This study was based on a cross-sectional multicentre survey of OA therapy for the management of OSA called the JAMS (Japanese Cross-sectional Multicenter Survey) Study performed at 10 medical institutions. A total of 442 patients fulfilled the selection criteria, which patients had worn OA, and undergone overnight polysomnography to assess both the pre-treatment baseline and follow-up for OA. Age, sex, BMI and apnoea-hypopnea index (AHI) at the time of diagnosis and follow-up for OA were extracted. RESULTS: After OA treatment, the mean AHI decreased from 22.6 ± 13.8 to 10.0 ± 10.2/h, and the mean rate of decrease in the AHI was 52.5 ± 38.4%. Regarding the success rate to OA therapy, criterion 1 (AHI < 5/h), criterion 2 (AHI < 10/h), criterion 3 (AHI < 15/h) and criterion 4 (AHI reduction rate ≥ 50%) accounted for 33.5, 66.3, 80.5 and 63.3%, respectively. The success rate of OA treatment decreased according to the increase in OSA severity, obesity level (higher BMI) and older age. CONCLUSIONS: This study revealed the treatment success rate of OA on multiple criteria according to OSA severity, BMI and age. It may support for the clinician to make a decision on the OSA management.

Discovery of novel molecular characteristics and cellular biological properties in ameloblastoma.

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma. Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS-responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS-responsive genes, enhances the proliferation of an ameloblastoma cell line AMU-AM1 with BRAF mutation. In addition, Toll-like receptor 2 (TLR2) knockdown readily inactivated KRAS-responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

Treatment outcome of oral appliance in patients with REM-related obstructive sleep apnea.

PURPOSE: Oral appliances (OA) are used to treat patients with obstructive sleep apnea (OSA). The purpose of this study is to evaluate the efficacy of OA treatment in patients with rapid eye movement (REM)-related OSA. METHODS: Forty-six patients with REM-related OSA and 107 with non-stage-specific OSA were prescribed OA treatment after diagnosis by polysomnography (PSG) and a follow-up sleep test by PSG was conducted. Efficacy and treatment outcome predictors were evaluated according to the following criteria for treatment success: #1, reduction of the apnea-hypopnea index (AHI) to less than 5 and > 50% compared with baseline; #2, AHI reduction to less than 10 and > 50% compared with baseline; and #3, > 50% AHI reduction compared with baseline. RESULTS: Success rates according to criteria #1, #2, and #3 were 45.7%, 50.0%, and 50.0% in REM-related OSA and 36.4%, 52.3%, and 63.6% in non-stage-specific OSA, respectively. No significant differences in success rate were found between the two groups. In multivariate logistic regression analysis with each criterion as the response variable, only BMI was extracted as a significant predictor. The BMI cutoff values defined based on the maximum Youden index according to the three criteria were 26.2 kg/m2, 25.6 kg/m2, and 26.2 kg/m2, respectively. CONCLUSIONS: No significant differences in success rate of OA treatment were found between REM-related OSA and non-stage-specific OSA. BMI has greater impact on treatment outcome of OA in patients with REM-related OSA.

Japanese Cross-Sectional Multicenter Survey (JAMS) of Oral Appliance Therapy in the Management of Obstructive Sleep Apnea.

We conducted a multicenter survey for oral appliance (OA) therapy to grasp and analyze the current situation of OA therapy, including efficacy, side effects, and follow-up, in Japan. The Japanese cross-sectional multicenter survey (JAMS) for obstructive sleep apnea (OSA) was undertaken by patients in 10 institutions associated with oral appliance therapy during two years, from 2014 to 2015, retrospectively. Age, sex, body mass index (BMI), baseline apnea-hypopnea index (AHI), OA type, adjustment, adverse reactions with OA, and AHI with OA were elicited from the patient clinical record. The number of included OSA patients was 3217. The number of patients with OA therapy was 2947. We evaluated 1600 patients for the OA treatment. The patients treated with OA, both male and female, were most commonly in their 50s. In terms of OSA severity, snoring was 2.3%, mild was 38.5%, moderate was 39.9%, and severe was 19.3%. The use of mono bloc appliance was 91.8%, bi bloc appliance was 7.9%, and tongue-retaining device (TRD) was 0.3%. After OA treatment, AHI decreased from 22.4/h to 9.3/h. The AHI reduction rate with OA was 52.0%. The rate of AHI <5 with OA was 35.6%. Adverse reactions developed in 14.7% of the subjects. OA reassessment was conducted for 54.3%. This study revealed the current situation of efficacy and side effects of OA therapy, and the problem that the reassessment rate of OA was low in Japan.

The Efficacy of Device Designs (Mono-block or Bi-block) in Oral Appliance Therapy for Obstructive Sleep Apnea Patients: A Systematic Review and Meta-Analysis.

Oral appliance (OAm) therapy has demonstrated efficacy in treating obstructive sleep apnea (OSA). The aim of this systematic review was to clarify the efficacy of device designs (Mono-block or Bi-block) in OAm therapy for OSA patients. We performed a meta-analysis using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system. Two studies (Mono-block OAm versus Bi-block OAm) remained eligible after applying the exclusion criteria. When comparing Mono-block OAm and Bi-block OAm, Mono-block OAm significantly reduced the apnea-hypopnea index (2.92; 95% confidence interval (95%CI), 1.26 to 4.58; p = 0.0006), and patient preference for Mono-block OAm was significantly higher (2.06; 95%CI, 1.44 to 2.06; p < 0.0001). Lowest SpO2, arousal index, non-REM stage 3, sleep efficiency, Epworth Sleepiness Scale (ESS), Snoring Scale, and side effects were not significantly different between the two groups (lowest SpO2: -11.18; 95%CI, -26.90 to 4.54; p = 0.16, arousal index: 4.40; 95%CI, -6.00 to 14.80; p = 0.41, non-REM stage 3: -2.00; 95%CI, -6.00 to 14.80; p = 0.41, sleep efficiency: -1.42, 95%CI, -4.71 to 1.86; p = 0.40, ESS: 0.12; 95%CI, -1.55 to 1.79; p = 0.89, Snoring Scale: 0.55; 95%CI, -0.73 to 1.83, p = 0.55, side effects: 1.00, 95%CI, 0.62 to 1.61, p = 1.00). In this systematic review, the use of Mono-block OAm was more effective than Bi-block OAm for OSA patients.

The Most Effective Amount of Forward Movement for Oral Appliances for Obstructive Sleep Apnea: A Systematic Review.

This systematic review clarifies the amount of effective protrusion in mandibular advancement devices of oral appliances required for obstructive sleep apnea (OSA). The systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Review Manager 5 and GRADEpro were used to combine trials and analyze data. The present review included three studies. In mild to moderate OSA cases, measured using the apnea-hypopnea index (AHI), 50% protrusion was more effective than 75% protrusion. However, 75% protrusion was more effective for severe cases. Sleep stage, Epworth Sleepiness Scale (ESS), snoring index, and side effects significantly differed between the groups. Additionally, 75% protrusion was more effective (AHI: 0.38, 95% CI: -0.89 to 1.65, p = 0.56; sleep stage 3: -1.20, 95% CI: 9.54-7.14, p = 0.78; ESS: 1.07, 95% CI: -0.09 to 2.24, p = 0.07; snoring index: 0.09, 95% CI: 0.05-0.13, p < 0.05; side effects: RR: 1.89, 95% CI: 0.36-9.92, p = 0.45). As per the AHI, 75% protrusion was effective in severe cases, whereas 50% protrusion was effective in moderate cases. Analysis of different surrogate outcomes indicated that 75% protrusion was more effective. Further, well-designed, larger trials should determine the benefits for patients. Additionally, investigations of adherence and side effects with long-term follow-up are needed.

Metronidazole-induced encephalopathy caused by hyperbaric oxygen therapy in a patient with mandibular osteomyelitis.

Metronidazole (MNZ) is prescribed for the treatment of infection caused by anaerobic bacteria and protozoa. Metronidazole-induced encephalopathy (MIE) has been known to be a side-effect, although its onset ratio is unclear. However, to the best of our knowledge, MIE associated with hyperbaric oxygen therapy (HBO) has not been previously reported. Here, we present the case of a 68-year-old man with mandibular osteomyelitis who received metronidazole for 49 days and received five times HBO therapy. He visited our hospital for evaluation and treatment of peripheral neuropathy, speech disturbance, nausea, and disturbance of gait after 47 days of initiating metronidazole treatment. Brain magnetic resonance imaging revealed hyperintense lesions in the cerebellar dentate nuclei, which was consistent with MIE. The patient's ataxic symptoms improved in 15 days after the discontinuation of MNZ. This is the first report demonstrating case of MIE could be related with HBO, as far as we had searched.

Inhibition of Nox1 induces apoptosis by attenuating the AKT signaling pathway in oral squamous cell carcinoma cell lines.

NADPH oxidases, also known as the Nox family, are major sources of reactive oxygen species generation that regulate redox-sensitive signaling pathways. Recent studies have implicated the Nox family in cancer development and progression. However, the involvement of its members in the development of oral squamous cell carcinoma (OSCC) remains to be elucidated. To clarify this issue, we first analyzed mRNA expression of Nox/Duox family members (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) in five OSCC cell lines. Nox1 and Nox4 mRNAs were highly expressed in four OSCC cell lines. Western blot analysis revealed that the protein expression level of Nox1 was higher than that of Nox4 in the OSCC cell lines. In addition, knockdown of Nox1, but not Nox4, significantly suppressed cell viability and induced apoptosis in the HSC-2 and HSC-3 cells. We also found that a specific AKT inhibitor, perifosine, dose-dependently suppressed OSCC cell growth. Notably, Nox1 knockdown significantly attenuated the phosphorylation level of AKT. Furthermore, both Nox1 knockdown and perifosine treatment markedly enhanced the cisplatin-induced cytotoxic effect. Taken together, our results highlight that the Nox1/AKT signaling pathway plays an important role in cell survival in OSCC cells.

Activated cytotoxic T-lymphocyte immunotherapy is effective for advanced oral and maxillofacial cancers.

Conventional cancer treatments are surgery, radiotherapy, and chemotherapy, but treatment efficiency is insufficient and cancer recurrence is common. Immunotherapy has been added as an important cancer treatment component, but no reports on its efficacy in oral and maxillofacial cancers exist. We evaluated the clinical efficacy of adoptive immunotherapy using ex vivo-activated cytotoxic T lymphocytes (CTL) in the treatment of 7 patients with advanced oral and maxillofacial cancers with stage IV disease at diagnosis. The mean follow-up period was 26.2 months. Phenotype of the lymphocyte assay revealed that the percentage of CD4(+) T cells decreased and that of CD8(+) T cells increased among infused lymphocytes compared to that in unstimulated peripheral blood mononuclear cells (PBMCs), and infused lymphocytes produced a significantly higher level of IFN-γ than PBMCs or tumor cells alone. In a representative patient who refused surgery tumor regression was confirmed after CTL infusion. Computed tomography clearly indicated a significant reduction in tumor size followed by the complete disappearance of the tumor. Histological examination showed that the cancers in patients receiving CTL therapy were heavily infiltrated with lymphocytes. The other 2 patients who received CTL therapy as adjuvant therapy showed neither recurrent disease nor new disease lesions. The 1-year survival rates showing response and those with progressive disease were 100 and 25%, respectively. Moreover, no significant adverse reactions were reported during the study period. CTL therapy remains in the early stages of treatment options, but it has potential as a valuable treatment and improvement of quality of life for patients with otherwise incurable cancers.

Combined arsenic trioxide-cisplatin treatment enhances apoptosis in oral squamous cell carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) accounts for the majority of oral cancers. Despite recent advances in OSCC diagnostics and therapeutics, the overall survival rate still remains low. Here, we assessed the efficacy of a combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment in human OSCC cells. METHODS: The combinatorial effect of ATO/CDDP on the growth and apoptosis of OSCC cell lines HSC-2, HSC-3, and HSC-4 was evaluated using MTT and annexin V assays, respectively. Chou-Talalay analyses were preformed to evaluate the combinatorial effects of ATO/CDDP on the dose-reduction index (DRI). To clarify the mechanism underlying the ATO/CDDP anticancer effect, we also examined the involvement of reactive oxygen species (ROS) in ATO/CDDP-induced apoptosis. RESULTS: Combination index (CI) analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in HSC-2 cells, with CI values ranging from 0.78 to 0.90, where CI < 1 defines synergism. The CI values in HSC-3 and HSC-4 cells ranged from 0.34 to 0.45 and from 0.60 to 0.92, respectively. In addition, ATO/CDDP yielded favorable DRI values ranging from 1.6-fold to 7.71-fold dose reduction. Compared to mono-therapy, ATO/CDDP combinatorial therapy significantly augmented the loss of mitochondrial potential, caspase-3/7 activity and subsequent apoptosis. These changes were all abrogated by the antioxidant N-acetylcysteine. CONCLUSIONS: This study provides the first evidence for a synergistic ATO/CDDP anticancer (apoptotic) activity in OSCC cells with a favorable DRI, thereby highlighting its potential as a combinational therapeutic regime in OSCC.