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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.

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.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.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.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.

Innate immune systems in the oral cavity have important roles in the host defense against viral invasion of oral mucosa. Poly(ADP‑ribose) polymerase 13 (PARP13), which has a strong antiviral ability, has been reported to possess two isoforms; a full‑length protein, zinc‑finger antiviral protein long (ZAPL), and a shorter protein (ZAPS). However, the expression and function of these two isoforms in oral mucosa remain unknown. In the present study, the expression levels of ZAPL and ZAPS induced by transfected double‑stranded (ds) RNA, Poly(I:C), and dsDNA, Poly(dA:dT), in immortalized oral keratinocytes and fibroblasts (RT7 and GT1 cell lines, respectively) were investigated. Subsequently, the effects of the knockdown of ZAPL and ZAPS on transfected nucleotide‑induced antiviral factors were examined. The results demonstrated constitutive expression of ZAPL and ZAPS in RT7 and GT1 cells, and their expression in both cell types was notably increased by transfection of Poly(I:C) and Poly(dA:dT) when compared with no transfection. Specific knockdown of ZAPL and ZAPS in RT7 cells decreased IFN‑β and C‑X‑C motif chemokine ligand 10 (CXCL10) expression induced by transfected Poly(I:C) and Poly(dA:dT). On the other hand, knockdown of ZAPL and ZAPS in GT1 cells decreased the expression of CXCL10 induced by the transfected nucleotides, whereas that had no effect on IFN‑β expression induced by Poly(dA:dT). Their knockdown was also associated with transfected nucleotides‑induced IFN regulatory factor 3 phosphorylation in both cell types. Taken together, these results indicate that ZAPL and ZAPS, isoforms of PARP13, in oral mucosal cells participate in host defense against viral infection of oral mucosa.

Objective: Although oral fibroblasts are thought to have the potential to enhance host defenses against Candida albicans , it is unknown whether they are able to recognize Candida cell components to increase the expression of antifungal peptides, such as defensin factors, against Candida infection. Methodology: We performed expression profiles of defensin genes induced by heat-killed C. albicans in oral immortalized fibroblasts (GT1) using cDNA microarray analysis. From those results, quantitative RT-PCR was used to examine the effects of Candida β-glucan-containing particles (β-GPs) on β-Defensin 118 (DEFB 118) expression in oral mucosal cells. Furthermore, the antifungal activities of recombinant DEFB 118 against C. albicans and C. glabrata were investigated using fungicidal assays. Results: Microarray analysis showed that DEFB118, β-Defensin 129 (DEFB129), and α-Defensin 1 (DEFA1) genes were induced by heat-killed C. albicans and that their mRNA expressions were also significantly increased by live as well as heat-killed C. albicans . Next, we focused on DEFB118, and found that GT1, primary fibroblasts, and RT7 (oral immortalized keratinocytes) constitutively expressed DEFB118 mRNA expression in RT-PCR. Furthermore, C. albicans β-GPs significantly increased the expression of DEFB118 mRNA in GT1 and primary fibroblasts. Although DEFB118 mRNA expression in RT7 was significantly induced by both live and heat-killed C. albicans, C. albicans β-GPs failed to have an effect on that expression. Finally, recombinant DEFB118 significantly decreased the survival of both strains of C. albicans in a dose-dependent manner, whereas no effects were seen for both C. glabrata strains. Conclusion: DEFB118, induced by C. albicans β-GPs from oral fibroblasts, may play an important role in oral immune responses against C. albicans infection.

PurposeIntegrin 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.MethodsIn 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 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.ResultsMK-0429 inhibited cell proliferation, migration, and adhesion of HUEhT-1 in a dose-dependent manner. FAK 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.ConclusionThese 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 antiangiogenic activity.

Integrin αvβIn 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 αvβMK-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.These results indicated integrin αvβ

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.

Tumor necrosis factor (TNF)‑α and interferon (IFN)‑γ, are inflammatory cytokines in the synovial fluid of patients with temporomandibular joint disorder (TMD). However, it remains unknown whether they participate in the regulation of various chemokine expression levels associated with TMD. The effects of TNF‑α and IFN‑γ on the expression of several different inflammatory chemokines, including interleukin (IL)‑8, C‑X‑C motif chemokine ligand (CXCL)1, C‑C motif chemokine ligand (CCL)20, CXCL9, CXCL10, and CXCL11 in synovial fibroblasts obtained from the temporomandibular joint (TMJ) were examined. The results demonstrated that TNF‑α increased the mRNA levels of all examined chemokines in synovial fibroblasts obtained from the TMJ. IFN‑γ treatment alone increased the mRNA expression levels of CXCR3 chemokines, including CXCL10, while they were significantly enhanced when administered in combination with TNF‑α compared with either treatment alone. However, the combination of IFN‑γ and TNF‑α resulted in lower mRNA expression levels of IL‑8 and CXCL1 as compared with those induced by TNF‑α alone. The nuclear factor‑κB inhibitor, Bay 11‑7082, decreased the TNF‑α‑mediated expression of IL‑8 and CXCL10 in the absence, and presence of IFN‑γ. In addition, the JAK2 inhibitor, AG490, decreased CXCL10 expression when administered with TNF‑α and IFN‑γ. Finally, the decrease in TNF‑α‑induced IL‑8 caused by IFN‑γ was recovered by AG490. The results of the present study suggest that TNF‑α and IFN‑γ function in a cooperative manner to regulate inflammatory chemokine expression in synovial fibroblasts, which may contribute to the pathological condition of the TMJ.

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.

The records of 70 patients with oral cancer who were treated at a single institution between 2008 and 2014 were reviewed. The body temperature, white blood cell count, and C-reactive protein (CRP) levels were compared between those who had received preoperative oral care (oral care group) and those who had not received any (non-oral care group). When the patients were divided into those who underwent minimally invasive surgery and those who underwent severely invasive surgery, the mean CRP level in the early postoperative period was lower in the oral care group as compared with the non-oral care group in those who underwent minimally invasive surgery as well as those who underwent severely invasive surgery. However, the mean CRP level was most evidently reduced in the severely invasive group on days 1 and 3–5. However, no significant differences were observed with regard to the percentage of postoperative infectious complications (for example, surgical site infection, anastomotic leak and pneumonia) between the oral care (13.6%) and non-oral care (20.8%) groups, though a reduced prevalence of postoperative complications following preoperative oral care was noted. The results of the present study suggest that preoperative oral care can decrease inflammation during the early postoperative stage in patients with oral cancer who undergo severely invasive surgery.

Oral keratinocytes provide the first line of host defense against oral candidiasis. We speculated that interactions of fungal cell wall components with oral keratinocytes regulate the stress response against Candida infection and examined the expression of genes induced by heat-killed Candida albicans in oral immortalized keratinocytes using a cDNA microarray technique. Of 24,000 genes revealed by that analysis, we focused on HO-1, a stress-inducible gene, as its expression was increased by both heat-killed and live C. albicans . In histological findings, HO-1 expression in the superficial layers of the oral epithelium following Candida infection was elevated compared to that in healthy epithelium. We then investigated fungal cell wall components involved in induction of HO-1 expression and found that β-glucan-containing particles (β-GPs) increased its expression. Furthermore, β-glucan was observed on the surface of both heat-killed C. albicans and Candida cells that had invaded the oral epithelium. Fungal β-GPs also promoted induction of intracellular reactive oxygen species (ROS), NADPH oxidase activation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation, while those specific inhibitors inhibited the HO-1 expression induced by fungal β-GPs. Moreover, fungal β-GPs induced Nrf2 translocation into nuclei via p38 MAPK signaling, while the HO-1 expression induced by fungal β-GPs was inhibited by Nrf2-specific small interfering RNA (siRNA). Finally, knockdown of cells by HO-1- and Nrf2-specific siRNAs resulted in increased β-GP-mediated ROS production compared to that in the control cells. Our results show that the HO-1 induced by fungal β-GPs via ROS/p38 MAPK/Nrf2 from oral keratinocytes may have important roles in host defense against the stress caused by Candida infection in the oral epithelium.

Objective Ameloblastoma (AM) shows locally invasive behaviour. However, biological investigations regarding regulation of gene expression associated with AM pathological features are difficult to perform, because AM cells can be passaged for a few generations due to senescence. We report a newly established immortalized AM cell line, AMB cells, by transfection with human telomerase reverse transcriptase (hTERT). Furthermore, we examined whether TNF-α modulates bone resorption-related genes, IL-6 and MMP-9 in cooperation with TGF-β or IFN-γ. Materials And Methods Following transfection of an hTERT expression vector into AM cells using a non-viral method, the effects of cytokines on the expressions of IL-6 and MMP-9 mRNA were examined using real-time PCR. TNF-α-induced NF-κB activity was examined by western blotting and transcription factor assays. Results AMB cells continued to grow for more than 100 population doublings. Stimulation with TNF-α increased IL-6 and MMP-9 mRNA expressions, as well as NF-κB activation. Furthermore, TGF-β and IFN-γ dramatically increased TNF-α-mediated expressions of MMP-9 and IL-6 mRNA, respectively, while those responses were abrogated by NF-κB inhibitor. In conclusion We established an immortalized AM cell line by hTERT transfection. TNF-α-mediated-regulation of MMP-9 and IL-6 via NF-κB may play an important role in the pathological behaviour of AMs, such as bone resorption. This article is protected by copyright. All rights reserved.

Background CD44 and aldehyde dehydrogenase 1 (ALDH1) have been shown to be useful markers for identification of cancer stem cells (CSCs). We previously reported that glycogen synthase kinase 3β (GSK3β) is involved in regulation of the self-renewal ability of head and neck squamous cell carcinoma (HNSCC) CSCs. The purpose of the present study was to clarify the role of GSK3β in CD44(high) /ALDH1(high) HNSCC cells. Methods Cells with greater expression of CD44 and higher ALDH1 enzymatic activity were FACS sorted from the OM-1 HNSCC cell line. The self-renewal ability of CD44(high) /ALDH1(high) cells was then examined using a tumor sphere formation assay. mRNA expressions of the stem cell markers Sox2, Oct4, and Nanog, as well as GSK3β were evaluated by real-time RT-PCR. Results CD44(high) /ALDH1(high) cells exhibited higher tumor sphere forming ability and increased expression of stem cell markers as compared with CD44(high) /ALDH1(low) cells. Interestingly, spindle-shaped cells positive for vimentin were found in the CD44(high) /ALDH1(high) but not the CD44(high) /ALDH1(low) cell population. In addition, the ALDH1 activity and sphere forming ability of CD44(high) /ALDH1(high) cells was significantly inhibited by GSK3β knockdown. On the other hand, CD44(high) /ALDH1(low) cells exhibited high epidermal growth factor receptor (EGFR) expression and increased cell growth. Conclusions Our results show that GSK3β plays a major role in maintenance of stemness of CD44(high) /ALDH1(high) HNSCC cells. Additionally, they indicate a close relationship between CSC and mesenchymal characteristics in HNSCC.

Bisphosphonates (BPs) are widely used in the prevention of skeletal-related events (SRE), including osteoporosis, skeletal metastases of malignant tumours, and multiple myeloma. Osteonecrosis of the jaw (ONJ) is frequently reported as a major adverse effect induced by BP treatment. The receptor activator of the nuclear factor kappa-B ligand (RANKL) inhibitor, denosumab, has recently been used to prevent SRE, but the frequency of ONJ induced by denosumab is similar to that by BPs. This finding suggests that the inhibition of RANKL-mediated osteoclastogenesis may have a close relationship with the occurrence of ONJ. We therefore investigated the expression status of RANKL-inducible genes in zoledronate-treated mouse osteoclast precursor cells. The molecular targets of zoledronate in the RANKL signal pathway and additional factors associated with osteoclastogenesis were analysed by genome-wide screening. Microarray analysis identified that among 31 genes on 44 entities of RANKL-inducible genes, the mRNA expression level of two genes, i.e., nuclear factor of activated T-cells c1 (NFATc1) and carbonic anhydrase 2 (CAII), was decreased in zoledronate-treated cells. Subsequent analyses verified that these two genes were significantly silenced by zoledronate treatment and that their expression was restored following inhibition of zoledronate action by geranylgeraniol. Zoledronate inhibited RANKL-induced osteoclast differentiation by suppression of NFATc1 and CAII gene expression. Our results suggest that these genes might be common targets for zoledronate and denosumab in the mechanism underlying RANKL-induced osteoclast differentiation. A clear understanding of the common molecular mechanisms of bone-remodelling agents is thus essential for prevention of ONJ.

Malignant salivary gland tumors are rare and exhibit a broad spectrum of phenotypic heterogeneity. The objective of this study was to investigate prognostic factors in patients with salivary gland carcinomas and review the results in light of other reports. We retrospectively reviewed 40 patients with primary salivary gland carcinomas who were diagnosed and treated at our institution between 1991 and 2014. Of the 40 tumors, 19 (47.5%) were mucoepidermoid carcinomas, 11 (27.5%) were adenoid cystic carcinomas, 7 (17.5%) were acinic cell carcinomas, 2 (5.0%) were myoepithelial carcinomas and 1 (2.5%) was a squamous cell carcinoma. Clinically positive lymph nodes were present in 4 patients (10.0%). As regards clinical stage, 15 cases (37.5%) were stage I, 13 (32.5%) were stage II, 1 (2.5%) was stage III and 11 (27.5%) were stage IVA. The majority of the patients (97.5%) were treated with surgery, of whom 25 (62.5%) received surgery alone and 14 (35.0%) underwent surgery in combination with chemotherapy or chemotherapy and radiotherapy. The median follow-up time for all the patients was 48 months. The disease-specific survival rate at 5 years was 87.1%. We identified a significant correlation between poor survival rate and histological grade (intermediate/high), tumor size (T3/T4), lymph node metastasis (node-positive) and clinical stage (III/IV) using the Kaplan-Meier method (P

Objective Itraconazole (ICZ) has a broad spectrum of antifungal activity including a wide range of Candida spp. TNF-α, an inflammatory cytokine associated with Th1-mediated oral inflammatory disease, enhances inflammatory mediators, such as CXCR3-agonistic chemokines including CXCL10. We examined the anti-inflammatory potential of ICZ against TNF-α-induced chemokines in oral fibroblasts. Materials and methods We investigated the effects of ICZ on mRNA expressions of various TNF-α-induced chemokines in immortalized oral keratinocytes (RT7) and oral fibroblasts (GT1) using quantitative PCR analysis. Subsequently, the effects of ICZ and fluconazole (FLZ) on TNF-α-induced CXCL10 proteins in GT1 and primary fibroblasts were examined using enzyme-linked immunosorbent assays (ELISA). The effect of ICZ on signal transduction protein phosphorylation involved in CXCL10 production from TNF-α-stimulated GT1 was examined by western blotting. Results ICZ inhibited TNF-α-induced CXCL10 mRNA in GT1, but not RT7. Although ICZ did not affect TNF-α-induced IL-8 mRNA, the mRNAs of TNF-α-induced CXCR3-agonistic chemokines such as CXCL9 and CXCL11 were inhibited by ICZ in GT1. TNF-α-induced CXCL10 protein production in GT1 and primary fibroblasts was inhibited by ICZ, but not FLZ. Finally, ICZ inhibited TNF-α-induced phosphorylation of c-JUN, which is related to CXCL10 production by TNF-α-stimulated GT1. Conclusion ICZ may be useful as therapy for Th1-mediated oral inflammatory disease.