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Objectives: Leflunomide (LEF) is a conventional synthetic disease-modifying antirheumatic drug and suppresses T-cell proliferation and activity by inhibiting pyrimidine synthesis using dihydroorotase dehydrogenase (DHODH); however, several studies have demonstrated that LEF possesses anticancer and antiangiogenic effects in some malignant tumors. Therefore, we investigated the anticancer and antiangiogenic effects of LEF on oral squamous cell carcinoma (OSCC). Methods: To evaluate the inhibitory effect of LEF on OSCC, cell proliferation and wound-healing assays using human OSCC cell lines were performed. The DHODH inhibitory effect of LEF was evaluated by Western blot. To assess the suppression of pyrimidine biosynthesis induced by LEF on OSCC, cell proliferation assays with or without uridine supplementation were performed. The antiangiogenic effect of LEF was evaluated by in vitro tube formation assay using immortalized human umbilical vein endothelial cells, which were electroporatically transfected with hTERT. The tumor-suppressive effect of LEF in vivo was examined in both immunodeficient and syngeneic mice by implanting mouse OSCC cells. Tumor vascularization was evaluated by immunohistochemistry of the tumor extracted from syngeneic mice. Results: LEF dose-dependently inhibited OSCC proliferation and migration. LEF significantly inhibited DHODH expression, and uridine supplementation rescued the inhibitory effect of LEF. LEF dose-dependently suppressed endothelial tube formation. In the animal study, LEF significantly suppressed tumor growth in both immunodeficient and syngeneic mice. Histologically, LEF decreased DHODH expression and tumor vascularization. Conclusion: LEF is a potent anticancer agent with antiangiogenic effects on OSCC and might be clinically applicable to OSCC by drug repositioning. [ABSTRACT FROM AUTHOR]

Objectives: ACE2, known as a host receptor involved with SARS‐CoV‐2 infection, binds to viral spike proteins for host cell entry. However, details regarding its induction and function in oral mucosal cells remain unknown. Materials and Methods: We examined ACE2 expression and its induction by transfected mimic nucleotides and pro‐inflammatory cytokines in oral keratinocytes (RT7) and fibroblasts (GT1). Subsequently, the effects of viral spike S1 protein via ACE2 on CXCL10 expression induced by pro‐inflammatory cytokines in both cells were examined. Results: ACE2 was constitutively expressed in RT7 and GT1. Transfected Poly(I:C) and Poly(dA:dT) increased ACE2 expression in those cells, while knockdown of RIG‐I decreased ACE2 expression induced by those transfected ds nucleotides. IFN‐γ and TNF‐α enhanced transfected ds nucleotides‐induced ACE2 expression in RT7 but not GT1. S1 protein alone did not affect CXCL10 expression in either cell type, whereas it enhanced IFN‐β‐induced CXCL10 in both, while immune responses of IFN‐γ‐ and TNF‐α‐induced CXCL10 enhanced by S1 protein were different between RT7 and GT1. Finally, knockdown of ACE2 decreased cytokines and S1 protein mediated‐CXCL10 levels in both cells. Conclusions: ACE2 in oral mucosal cells may contribute to development of infection and inflammation in cooperation with pro‐inflammatory cytokines following SARS‐CoV‐2 invasion. [ABSTRACT FROM AUTHOR]

Aim: The involvement of human papillomavirus (HPV) E6/E7 in autophagy has not been fully elucidated in oral squamous cell carcinoma (OSCC) cells. The objective of this study was to clarify the association between HPV E6/E7 and autophagy and cancer stem cell properties such as self‐renewal ability and chemotherapy resistance in HPV16‐positive OSCC cells. Methods: UM‐SCC‐104 HPV16‐positive OSCC cells were cultured on laminin 332‐coated silicone gel. The impact of HPV16 E6 or E7 siRNA transfection on cell proliferation, spheroid formation, 5‐fluorouracil (5‐FU)‐induced cell death, and autophagy was investigated. After treatment with rapamycin, DAPGreen was used to detect autophagosome formation. Results: HPV16 E6 or E7 siRNA transfection resulted in knockdown of both E6 and E7 in UM‐SCC‐104 cells. HPV16 E6/E7 knockdown inhibited cell proliferation, mRNA expression of stem cell markers (i.e., CD44, ALDH1, BMI1, OCT4, and NANOG), and spheroid formation. Furthermore, HPV16 E6/E7 knockdown enhanced 5‐FU‐induced cell death. Additionally, mRNA expression of autophagy‐related genes such as ATG5 and BECN1 was significantly increased after HPV16 E6/E7 knockdown. Furthermore, HPV16 E6/E7 knockdown augmented rapamycin‐induced autophagy. Next, we found that miR‐30a‐5p is indicated to interact with the 3′‐untranslated region of ATG5 and BECN1 using TargetScan. However, significant induction of autophagy was not found after miR‐30a‐5p inhibitor transfection. Importantly, 5‐FU‐induced cell death was significantly attenuated by chloroquine autophagy inhibitor in HPV16 E6/E7 knockdown cells. Conclusion: HPV16 E6/E7 is involved in autophagy regulation as well as in the maintenance of cancer stem cell features in OSCC cells. [ABSTRACT FROM AUTHOR]

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]

Purpose: Integrin αvβ3 is an essential molecule for tumor angiogenesis. This study aimed to investigate the anti-tumor effect of MK-0429, an integrin αvβ3 antagonist, on oral squamous cell carcinoma (OSCC) through its inhibitory effect on angiogenesis. Methods: In this study, we investigated the effect of MK-0429 on cellular function and angiogenesis in vitro with the use of an immortalized human umbilical vein endothelial cell, HUEhT-1, which is immortalized by the electroporatic transfection of hTERT. The effect of MK-0429 on the integrin αvβ3 signaling pathway was examined by FAK, MEK1/2 and ERK 1/2 phosphorylation. The anti-angiogenic effect of MK-0429 was evaluated by in vitro tube formation assay. The anti-tumor effect on OSCC was assessed by administrating MK-0429 to mouse oral cancer xenografts. Results: MK-0429 inhibited cell proliferation, migration, and adhesion of HUEhT-1 in a dose-dependent manner. FAK, MEK and ERK phosphorylation were significantly blocked by MK-0429 treatment. Tube formation was suppressed by MK-0429 in dose-dependent manner. Tumor progression was significantly suppressed by MK-0429 administration in mouse oral cancer xenografts. Histological study revealed that MK-0429 decreased tumor vascularization. Conclusion: These results indicated integrin αvβ3 as a therapeutic target for OSCC and suggested that MK-0429 might be clinically applicable as an anti-tumor agent with potent anti-angiogenic activity. [ABSTRACT FROM AUTHOR]