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Objective: Sunitinib, a targeted cancer drug, inhibits tyrosine kinases receptors and is widely used as first‐line treatment for metastatic renal cell carcinoma. Patients undergoing chemotherapy with sunitinib frequently have oral mucosal complications, such as oral stomatitis, though cytotoxic effects of the drug on oral keratinocytes remain unknown. Methods: The effects of sunitinib on immortalized oral keratinocytes, RT7 cells, in regard to cell injury and apoptosis, as well as apoptosis‐mediated signaling pathways were investigated. Results: Sunitinib treatment caused a significant increase in lactate dehydrogenase (LDH) in RT7 cells and primary oral keratinocytes. Additionally, the drug induced apoptosis‐related events, such as DNA fragmentation, decreased anti‐apoptotic Bcl‐2 protein expression, and induction of cleaved PARP and caspase 3/9 in RT7 cells. Furthermore, phosphorylation of p38 MAPK, but not of ERK or JNK, was increased. On the contrary, constitutive phosphorylated STAT3 was decreased by sunitinib treatment, which was recovered by exposure to SB203580, a p38 MAPK inhibitor. Finally, SB203580 was found to reduce sunitinib‐induced cell injury and apoptosis. Conclusion: The present results indicate that sunitinib promotes cell injury and apoptosis in oral keratinocytes via p38 activation and STAT3 downregulation. Sunitinib‐mediated oral complications may be associated with cytotoxic effects of the drug on oral keratinocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Double‐strand (ds) DNA‐enveloped viruses can cause oral infection. Our aim is to investigate whether oral mucosal cells participate in immune response against cytosolic dsDNA invasion. Methods: We examined the response to transfected herpes simplex virus (HSV) dsDNA via intracellular receptors in oral keratinocytes (RT7) and fibroblasts (GT1), and the effect of TNF‐α on those responses. Results: Transfected dsDNA increased CXCL10 expression via NF‐κB activation in both cell types, while those responses were inhibited by knockdown of RIG‐I, an RNA sensor. Although IFI16, a DNA sensor, was expressed in the nuclei of both types, its knockdown decreased transfected dsDNA‐induced CXCL10 expression in GT1 but not RT7 cells. IFI16 in GT1 cells was translocated into cytoplasm from nuclei, which was attributed to immune response to cytosolic dsDNA. TNF‐α enhanced transfected dsDNA‐induced CXCL10, and knockdown of IFI16 decreased TNF‐α and dsDNA‐driven CXCL10 expression in both RT7 and GT1 cells. Finally, the combination of TNF‐α and transfected dsDNA resulted in translocation of IFI16 from nuclei to cytoplasm in RT7 cells. Conclusion: RIG‐I and IFI16 in oral mucosal cells may play important roles in host immune response against DNA viral infection, while TNF‐α contributes to development of an antiviral system via those intracellular receptors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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‐β1 (TGF‐β1) 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‐β1. Additionally, TGF‐β1 enhanced N‐cadherin and Snail mRNA expression and induced a spindle‐shaped morphology. ERK1/2 phosphorylation was induced by TGF‐β1. Microarray analysis revealed that miR‐422a exhibited the greatest downregulation (fold change: 0.22) in the presence of TGF‐β1. Importantly, TGF‐β1‐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‐β1 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. [ABSTRACT FROM AUTHOR]