Melatonin enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high OSCC cells.

BACKGROUND: Melatonin is a hormone that is primarily produced in the pineal gland and is involved in wide range of biological functions. However, the impact of melatonin on chemotherapy-induced cell death remains to be elucidated in oral squamous cell carcinoma (OSCC) cells. The objective of this study was to clarify the role of melatonin in cisplatin-induced cytotoxicity in CD44high OSCC cells. METHODS: CD44high OSCC cells were cultured on fibronectin-coated hydrogel. A lactate dehydrogenase cytotoxicity assay was performed to evaluate cisplatin-induced cell death. The effect of melatonin on cisplatin-induced cell death and Derlin-1 (DERL1) endoplasmic reticulum membrane protein expression was investigated. RESULTS: CD44high OSCC cells exhibited mesenchymal-like features when cultured on fibronectin-coated hydrogel. Mesenchymal-like CD44high OSCC cells demonstrated strong resistance to cisplatin-induced cell death compared with epithelial-like CD44high OSCC cells. DERL1 mRNA and DERL1 protein expression levels were significantly higher in mesenchymal-like CD44high cells compared with epithelial-like CD44high cells. Cisplatin-induced cell death was significantly enhanced after DERL1 siRNA knockdown, suggesting that DERL1 is involved in resistance to cisplatin-induced cell death. Melatonin significantly inhibited DERL1 expression and enhanced cisplatin-induced cell death in mesenchymal-like CD44high cells. miR-181c-5p expression was significantly upregulated in the presence of melatonin. Furthermore, melatonin-inhibited DERL1 expression was significantly recovered by miR-181c-5p inhibitor. In addition, melatoninenhanced cisplatin-induced cell death was attenuated by miR-181c-5p inhibitor. These results suggest that melatonin-induced miR-181c-5p enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high cells. CONCLUSIONS: Melatonin plays a vital role in promoting cisplatin-induced cytotoxicity in mesenchymal-like CD44high OSCC cells.

Effects of miR-224-5p-enhanced downregulation of pannexin-1 on docetaxel-induced apoptosis in amoeboid-like CD44high oral cancer cells.

We previously found that microRNAs play major roles in the maintenance of amoeboid-like oral squamous cell carcinoma (OSCC) cells with high expression of CD44 (CD44high ). However, the roles of microRNAs in chemotherapeutic resistance exhibited by CD44high amoeboid-like OSCC cells are unclear. Here, docetaxel-induced apoptosis was examined in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated silicone gel. Amoeboid-like CD44high OSCC cells exhibited robust resistance to docetaxel-induced apoptosis and significant upregulation of miR-224-5p expression compared with epithelial-like CD44high OSCC cells and mesenchymal-like CD44high OSCC cells. The expression of pannexin-1 (PANX1), a channel-forming protein that regulates the release of ATP, was significantly upregulated following transfection of amoeboid-like CD44high OSCC cells with an miR-224-5p inhibitor. These results suggest that miR-224-5p inhibits PANX1 expression. Furthermore, miR-224-5p inhibitor-transfected amoeboid-like CD44high OSCC cells exhibited significant enhancement of the proportion of apoptotic cells; however, this effect was significantly inhibited by knockdown of PANX1 with PANX1 small interfering RNA. Additionally, the miR-224-5p inhibitor-enhanced extracellular ATP levels were significantly reduced by PANX1 knockdown. These findings imply that miR-224-5p plays a vital role in the resistance to docetaxel-induced apoptosis by attenuating PANX1-induced ATP discharge. Moreover, amoeboid-like CD44high OSCC cells may be involved in chemotherapeutic resistance of OSCC.

Association of oral Epstein-Barr virus with periodontal health in Japanese adults.

Previous studies have demonstrated that oral Epstein-Barr virus (EBV) is associated with periodontitis. However, the relationship between periodontitis and oral EBV has not been fully elucidated by reducing the effects of confounding factors. The aim of the present study was to clarify the association between oral Epstein-Barr virus (EBV) and oral health status among middle-aged and older Japanese individuals. A total of 124 patients (46 males and 78 females; mean age, 69.2 years; age range, 35-90 years) who visited Hiroshima University Hospital between October 2018 and December 2019 were recruited into the present study. EBV DNA positivity was determined in 124 oral rinse samples using quantitative PCR. Periodontal disease-related bacteria were also detected by PCR analysis. EBV DNA was determined as positive in 16 of the 124 enrolled patients (12.9%). No significant difference was identified between EBV DNA and clinical factors (sex, age, remaining teeth, denture use, smoking or medical history). Of the 38 patients with periodontal pockets ≥6 mm, 10 were EBV DNA positive (26.3%). There was a significant association between EBV DNA positivity and probing depth (P=0.01). Additionally, a significant association was identified between bleeding on probing (BOP) and EBV DNA positivity (P=0.03). To investigate the relationship between EBV and periodontal health status, propensity score-matching was determined between participants without ≥4 mm periodontal pockets and BOP (participants with good periodontal health) and those with ≥4 mm periodontal pockets, BOP or both (participants with poor periodontal health). A total of 35 matched pairs were identified among the patients. Patients with poor periodontal health exhibited a higher EBV DNA positivity rate (25.7%) than those with good periodontal health (0.0%). Additionally, there was a significant association between EBV DNA positivity and periodontal health status (P=0.001). T. denticola-positive participants exhibited a higher EBV DNA positivity rate than negative participants (17.6 vs. 9.6%). However, there was no significant difference. The results indicated that oral EBV may be markedly associated with periodontitis in middle-aged and older Japanese individuals.

TGF-ß1 induces amoeboid-to-mesenchymal transition of CD44high oral squamous cell carcinoma cells via miR-422a downregulation through ERK activation and Cofilin-1 phosphorylation.

BACKGROUND: The objective of this study was to clarify the molecular mechanism of amoeboid-to-mesenchymal transition (AMT) of CD44high oral squamous cell carcinoma (OSCC) cells. METHODS: Morphology and expression of mesenchymal genes were investigated in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated soft silicone gel. Additionally, microarray analysis was performed to investigate microRNA (miRNA) expression inhibited by transforming growth factor-ß1 (TGF-ß1) in CD44high OM-1 cells. RESULTS: When CD44high OM-1 cells were cultured on 2.0-kPa laminin-coated silicone gel, the cells exhibited an amoeboid-like round morphology. Cofilin-1 expression was found in the nucleus and cytoplasm of amoeboid-like CD44high OM-1 cells. The invasive capacity was significantly reduced after Cofilin-1 knockdown. Additionally, Cofilin-1 knockdown cells had an irregularly extended shape. Phosphorylated Cofilin-1 was significantly upregulated by TGF-ß1. Additionally, TGF-ß1 enhanced N-cadherin and Snail mRNA expression and induced a spindle-shaped morphology. ERK1/2 phosphorylation was induced by TGF-ß1. Microarray analysis revealed that miR-422a exhibited the greatest downregulation (fold change: 0.22) in the presence of TGF-ß1. Importantly, TGF-ß1-inhibited miR-422a expression was recovered by the ERK inhibitor or ERK1/2 knockdown. Additionally, miR-422a inhibitor-transfected CD44high OM-1 cells exhibited high N-cadherin and Snail mRNA expression. Furthermore, Cofilin-1 knockdown and miR-422a inhibition induced a spindle cell morphology. CONCLUSION: Cofilin-1 is involved in the invasive ability of CD44high OSCC cells. TGF-ß1 contributes to AMT by downregulation of miR-422a via ERK activation and Cofilin-1 phosphorylation. Our findings suggest that miR-422a and Cofilin-1 play major roles in the maintenance of amoeboid-like CD44high cells.

Melatonin­induced miR­181c­5p enhances osteogenic differentiation and mineralization of human jawbone­derived osteoblastic cells.

Our previous study revealed that treatment with a combination of fibroblast growth factor­2 and melatonin (MEL) synergistically augmented osteogenic activity and mineralization of MC3T3­E1 mouse preosteoblast cells. Thus, the objective of the present study was to assess the effect of MEL on osteogenetic characteristics in human osteoblastic cells. Human jawbone­derived osteoblastic (hOB) cells were isolated from mandibular bone fragments. RUNX family transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) enzyme activity and the mineralization ability of hOB cells in the presence of MEL were evaluated. Microarray analysis was also performed to assess the expression of MEL­induced microRNAs (miRNAs/miRs) in hOB cells. Treatment with MEL significantly enhanced Runx2 expression, ALP activity and mineralization staining. However, this effect was significantly reduced following transforming growth factor­ß1 treatment. In total, 124 miRNAs were differentially expressed in MEL­treated hOB cells, compared with untreated cells. Of the upregulated miRNAs, miR­181c­5p exhibited the largest fold change. Runx2 mRNA expression and mineralization staining in the presence of MEL were significantly reduced following transfection with a miR­181c­5p inhibitor. In addition, transfection with miR-181c-5p mimics significantly increased Runx2 expression and mineralization staining. These results suggested that MEL­induced miR­181c­5p was involved in osteogenic differentiation and mineralization of hOB cells. Using TargetScan, a putative miR­181c­5p binding site was identified in the Notch2 gene. Moreover, Notch2 mRNA and protein expression levels in hOB cells were significantly reduced following transfection with miR­181c­5p mimics, confirming Notch2 as a target gene for miR­181c­5p. Notch2 siRNA knockdown significantly increased Runx2 expression and mineralization staining, which suggested that Notch2 may negatively regulate osteogenic differentiation of hOB cells by downregulating Runx2. In conclusion, MEL­induced expression of miR­181c­5p enhanced osteogenic differentiation and calcification of hOB cells.

Morphological Evaluation of Bone by CT to Determine Primary Stability-Clinical Study.

BACKGROUND: Primary stability is an important prognostic factor for dental implant therapy. In the present study, we evaluate the relationship between implant stability evaluation findings by the use of an implant stability quotient (ISQ), an index for primary stability, and a morphological evaluation of bone by preoperative computed tomography (CT). SUBJECTS AND METHODS: We analyzed 98 patients who underwent implant placement surgery in this retrospective study. For all 247 implants, the correlations of the ISQ value with cortical bone thickness, cortical bone CT value, cancellous bone CT value, insertion torque value, implant diameter, and implant length were examined. RESULTS: 1. Factors affecting ISQ values in all cases: It was revealed that there were significant associations between the cortical bone thickness and cancellous bone CT values with ISQ by multiple regression analysis. 2. It was revealed that there was a significant correlation between cortical bone thickness and cancellous bone CT values with ISQ by multiple regression analysis in the upper jaw. 3. It was indicated that there was a significant association between cortical bone thickness and implant diameter with ISQ by multiple regression analysis in the lower jaw. CONCLUSION: We concluded that analysis of the correlation of the ISQ value with cortical bone thickness and values obtained in preoperative CT imaging were useful preoperative evaluations for obtaining implant stability.

Zoledronate Inhibits Osteoclast Differentiation via Suppressing Vascular Endothelial Growth Factor Receptor 2 Expression.

Bisphosphonate-related osteonecrosis of the jaw (ONJ) is a major oral complication; however, its pathogenesis remains unclear. Impairment of osteoclast differentiation by bisphosphonates may be associated with the pathogenesis of ONJ. In our previous study, we reported that the expression of the gene encoding nuclear factor of activated T cells c1 (NFATc1), a known osteoclast differentiation marker, was significantly silenced by zoledronate, a bisphosphonate, in mouse osteoclast precursor cells (mOCPCs) using cDNA microarray. In the present study, the expression value of the NFATc1 gene was regarded as a cut-off and genes whose expression value was significantly decreased compared with that of the NFATc1 gene were extracted in mOCPCs. For validation, CD11b-positive (CD11b+) cells were used, which were purified from human peripheral blood mononuclear cells as human OCPCs. A total of 19 genes were identified; sequential expression analysis revealed that the gene encoding vascular endothelial growth factor receptor 2 (VEGFR2) was frequently silenced by zoledronate in CD11b+ cells. Furthermore, the number of tartrate-resistant acid phosphatase-positive multinucleated cells was decreased by VEGFR2 suppression using a VEGFR2 neutralizing antibody. Zoledronate inhibits human osteoclast differentiation via suppressing VEGFR2 expression. These results suggest that low expression of VEGFR2 in OCPCs may be involved in the pathogenesis of zoledronate-induced ONJ. The understanding of the role of VEGFR2 on bone remodeling is important to elucidate the pathogenesis of bisphosphonate-related ONJ.

Effect of hydrogel stiffness on morphology and gene expression pattern of CD44high oral squamous cell carcinoma cells.

The stiffness of extracellular matrix (ECM) has been associated with tumor growth, phenotypic plasticity, and invasion through modulation of the intracellular signaling pathway. However, the effect of ECM stiffness on oral cancer stem cells (CSCs) has not been fully elucidated. Therefore, we preliminarily investigated changes in phenotype and gene expression in CD44 positive-oral squamous cell carcinoma (OSCC) cells (i.e., CD44high OM-1 cells) that were cultured on laminin-coated hydrogel with various degrees of stiffness. Mesenchymal-like morphology was observed when cells were cultured on 4.0 kPa laminin-coated hydrogel; amoeboid-like morphology was observed when cells were cultured on 1.0 kPa and 0.5 kPa laminin-coated hydrogel. These results indicated that CD44high OM-1 cells underwent mesenchymal to amoeboid transition (MAT) when cultured on laminin-coated softer hydrogel. E-cadherin and ESA mRNA expression levels were significantly reduced in CD44high OM-1 cells cultured on 0.5 and 1.0 kPa laminin-coated hydrogel, compared with their levels in control cells cultured in laminin-coated dishes. Significant changes in CD44 mRNA expression were not found in CD44high OM-1 cells that were cultured on different stiff hydrogels, compared with expression in control cells. Microarray analysis revealed that expression of cofilin, an intracellular actin-modulating protein, was increased by 8.19-fold in amoeboid-like CD44high OM-1 cells, compared with mesenchymal-like CD44high OM-1 cells; this suggested that cofilin was associated with MAT in CD44high OSCC cells. Further studies are needed to clarify the relationship between cofilin and invasion ability in CD44high amoeboid-like OSCC cells.