Detection of Merkel cell polyomavirus in multiple primary oral squamous cell carcinomas.

Oral microbiome studies have mainly focussed on bacteria, with the relationship between viruses and oral cancers remaining poorly understood. Oral cancers can develop even in the absence of any history of daily smoking or drinking. Oral cancer patients frequently have multiple primary cancers in the oral cavity and other organs, such as the upper gastrointestinal tract. Merkel cell polyomavirus (MCPyV) is a novel oncovirus identified from a subtype of skin cancer in 2008. In this study, we investigated the potential involvement of MCPyV in the pathogenesis of oral squamous cell carcinoma (OSCC). Participants comprised 115 Japanese patients with OSCC (single primary: 109 tumours in 109 patients; multiple primaries: 16 tumours in 6 patients) treated in our department between 2014 and 2017. DNA was extracted from formalin-fixed paraffin-embedded specimens of primary lesions. MCPyV DNA copy counts were analysed by quantitative real-time polymerase chain reaction. Twenty-four of the 115 patients (20.9%) were positive for MCPyV DNA. No association was found between presence or absence of MCPyV DNA and clinical characteristics other than number of primary lesions. The MCPyV DNA-positive rate was significantly higher for multiple primary OSCCs (62.5%, 10/16 tumours) than for single primary OSCCs (16.5%, 18/109 tumours; P < 0.001). Furthermore, MCPyV DNA load was significantly higher for patients with multiple primaries (P < 0.05). MCPyV was observed more frequently and DNA load was significantly higher with multiple primary OSCCs than with single primary OSCC. MCPyV may play some role as an oncovirus for multiple primary OSCCs.

SPARC is associated with carcinogenesis of oral squamous epithelium and consistent with cell competition.

The matricellular protein, secreted protein acidic and rich in cysteine (SPARC) is thought to be involved in cell competition. The objective of this study is to investigate the role of SPARC in cancerization of oral squamous epithelium. Clinical specimens from 57 pre- and early cancerous lesion, 66 invasive squamous cell carcinoma (SCC) and controls were immunostained with SPARC. Clinical features and SPARC expression were evaluated. Furthermore, effects of SPARC knockdown and overexpression were examined in oral cancer and keratinocyte cell lines. Leukoplakia, carcinoma in situ, and early invasive SCC had more SPARC-positive cells than normal mucous epithelium. However, there were no significant differences between leukoplakia, carcinoma in situ, and early SCC, and there were no correlations between SPARC immunoreactivity and prognosis of invasive oral SCCs. Cell proliferation was down-regulated by SPARC siRNA, and enhanced by SPARC transformed keratinocytes. But SPARC overexpression did not enhance cell migration activity. SPARC is induced by dysplastic cells in the early stage of cancerization, and may improve survival capability, but is not involved in malignancy. SPARC may act to escape from elimination by cell competition.

Epigallocatechin-3-gallate modulates anti-oxidant defense enzyme expression in murine submandibular and pancreatic exocrine gland cells and human HSG cells.

Sjogren's syndrome (SS) and type-1 diabetes are prevalent autoimmune diseases in the USA. We reported previously that epigallocatechin-3-gallate (EGCG) prevented and delayed the onset of autoimmune disease in non-obese diabetic (NOD) mice, a model for both SS and type-1 diabetes. EGCG also normalized the levels of proteins related to DNA repair and anti-oxidant activity in NOD.B10.Sn-H2 mice, a model for primary SS, prior to disease onset. The current study examined the effect of EGCG on the expression of anti-oxidant enzymes in the submandibular salivary gland and the pancreas of NOD mice and cultured human salivary gland acinar cells. NOD mice consuming 0.2% EGCG daily dissolved in water showed higher protein levels of peroxiredoxin 6 (PRDX6), a major anti-oxidant defense protein, and catalase, while the untreated NOD mice exhibited significantly lowered levels of PRDX6. Similarly, pancreas samples from water-fed NOD mice were depleted in PRDX6 and superoxide dismutase, while EGCG-fed mice showed high levels of these anti-oxidant enzymes. In cultured HSG cells EGCG increased PRDX6 levels significantly, and this was inhibited by p38 and JNK inhibitors, suggesting that the EGCG-mediated increase in protective anti-oxidant capacity is regulated in part through mitogen-activated protein kinase pathway signaling. This mechanism may explain the higher levels of PRDX6 found in EGCG-fed NOD mice. These preclinical observations warrant future preclinical and clinical studies to determine whether EGCG or green tea polyphenols could be used in novel preventive and therapeutic approaches against autoimmune diseases and salivary dysfunction involving oxidative stress.

Epigallocatechin-3-gallate prevents autoimmune-associated down- regulation of p21 in salivary gland cells through a p53-independent pathway.

The submandibular salivary glands of non-obese diabetic (NOD) mice, a model for Sjogren's syndrome and type-1 diabetes, show an elevated level of proliferating cell nuclear antigen (PCNA), a protein involved in cell proliferation and repair of DNA damage. We reported previously that epigallocatechin-3-gallate (EGCG), the most abundant green tea catechin, normalizes the PCNA level. PCNA's activity can be regulated by the cyclin-dependent kinase inhibitor p21, which is also important for epithelial cell differentiation. In turn, expression of p21 and PCNA are partially regulated by Rb phosphorylation levels. EGCG was found to modulate p21 expression in epithelial cells, suggesting that EGCG-induced p21 could be associated with down-regulation of PCNA in vivo. The current study examined the protein levels of p21 and p53 (which can up-regulate p21) in NOD mice fed with either water or EGCG, and the effect of EGCG on p21 and p53 in cell line models with either normal or defective Rb. In NOD mice, the p21 level was low, and EGCG normalized it. In contrast to HSG cells with functional Rb, negligible expression of p21 in NS-SVAC cells that lack Rb was not altered by EGCG treatment. Inhibition of p53 by siRNA demonstrated that p21 and p53 were induced independently in HSG cells by a physiological concentration range of EGCG, suggesting p53 could be an important but not conditional factor associated with p21 expression. In conclusion, PCNA and p21 levels are altered inversely in the NOD model for SS and in HSG cells, and warrant further study as candidate new markers for salivary dysfunction associated with xerostomia. Induction of p21 by EGCG could provide clinically useful normalization of salivary glands by promoting differentiation and reducing PCNA levels.

TCDD disrupts posterior palatogenesis and causes cleft palate.

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.

Mucinous Cystadenoma in the Upper Lip: Report of Two Cases.

Mucinous cystadenoma of the salivary gland is a very rare disease, and only a few cases have been reported. We report here 2 cases of mucinous cystadenoma in the upper lip. The first case was a 57-year-old man and the second was a 42-year-old woman. The tumors were painless nodules with a smooth-surfaced mucosa, and surgical excisions were performed. Histologically, the tumors were surrounded by a fibrous capsule and were composed of multiple cysts lined with columnar epithelial cells. The tumor cells contain mucous substances that reacted with periodic acid-Schiff base and Alcian blue. Immunohistochemical staining revealed that the tumor cells expressed cytokeratin (AE1/3 and CK7), but their immunoreactivity with MIB-1 (Ki-67) was less than 3%. They had negative immunoreactivity for neuroectoderm markers, S-100 protein, and myoepithelial markers, p63, α-smooth muscle actin, and calponin, except for the accompanying myoepithelial-like cells. No recurrences were noted after surgery at 7 years and 1 year, respectively.

Low-grade myofibroblastic sarcoma of the palate.

Low-grade myofibroblastic sarcoma (LGMS) is a rare, malignant tumor with myofibroblastic differentiation. Despite it being classified as a distinct entity by the World Health Organization, a few cases were reported in the oral and maxillofacial region. Here, a LGMS developed on the palate of a 73-year-old man who presented with a 1-cm tumor on the posterior border of the palate. Based on the histological and immunohistochemical features, a diagnosis of LGMS was established. The tumor was resected, and no recurrence was observed over 2 years. Although the tongue is the most preferred site for LGMS, it may occur in any region of the oral cavity.

Epigallocatechin-3-gallate modulates antioxidant and DNA repair-related proteins in exocrine glands of a primary Sjogren's syndrome mouse model prior to disease onset.

The autoimmune disorder primary Sjogren's syndrome (SS) is associated with xerostomia and xerophthalmia. SS pathogenesis involves both genetic/epigenetic and environmental factors. A major potential contributor is oxidative stress associated with damage to cellular components, including DNA. We reported previously that the green tea polyphenol epigallocatechin-3-gallate (EGCG) normalizes the elevated levels of proliferating cell nuclear antigen (PCNA), a key component of DNA repair, in the NOD mouse model for SS and type 1 diabetes. The current study examined levels of the antioxidant enzymes peroxiredoxin 6 (PRDX6), catalase and superoxide dismutase (SOD), as well as PCNA, in NOD.B10.Sn-H2 mice, a model for primary SS, and determined the effect of EGCG on their expression. PCNA elevation was detected in the submandibular gland and pancreas by 8 weeks of age in water-fed mice, and increased through 14 weeks of age, prior to overt onset of symptoms. This early PCNA elevation was followed by a decline of peroxiredoxin 6 protein. In contrast, EGCG-fed mice exhibited normal levels of PCNA and peroxiredoxin 6, comparable to healthy untreated BALB/c mice. Similar patterns were observed in the pancreas, even though these mice do not develop diabetes. Thus, elevated PCNA is an early biomarker for exocrine glandular dysfunction associated with SS-like autoimmune disease, accompanied subsequently by decreased PRDX6 antioxidant enzyme levels that could further contribute to oxidative stress, and these changes precede inflammatory cell infiltration. Importantly, EGCG consumption normalizes the expression of these biomarkers in this model. These observations could lead to early diagnosis and intervention of autoimmune disorders.

Discovery and evaluation of selective N-type calcium channel blockers: 6-unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives.

A structure-activity relationship study of 6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-cinnamyl ester was found to be an effective and selective N-type calcium channel blocker with oral analgesic potential.

Enhanced expression of Toll-like receptor 2 in lesional tissues and peripheral blood monocytes of patients with oral lichen planus.

Some members of the Toll-like receptor (TLR) family, which plays key roles in both innate and adaptive immune responses, are involved in the pathogenesis of autoimmune, chronic inflammatory and infectious diseases. However, the role of TLR in the pathogenesis of oral lichen planus (OLP) has not been investigated. The aim of this study was to understand the roles of TLR in OLP. The expression of TLR genes in OLP tissues was analyzed by cDNA microarray and reverse transcription polymerase chain reaction, and TLR protein expression in OLP tissues and peripheral blood monocytes was examined by immunohistochemical analysis and flow cytometry, respectively. Furthermore, TLR ligand-induced cytokine production from peripheral blood monocytes was measured by enzyme-linked immunosorbent assay. Among 10 TLR genes, the average expression ratio of the genes for TLR1, 2, 3, 5, 6 and 10 in OLP tissues compared to that in the normal buccal mucosae was more than 1.0. In contrast, the average ratio of the genes for TLR7, 8 and 9 was less than 1.0. TLR2 but not TLR4 was highly expressed in the cells of the spinous layer and infiltrating monocytes in OLP tissues, and the mean fluorescence intensity of TLR2 on peripheral blood monocytes was significantly higher in OLP patients than in healthy controls. Furthermore, the peripheral blood monocytes from OLP patients produced considerably higher amounts of interleukin (IL)-12 and lower amounts of IL-10 than those from healthy controls. In OLP, the T-helper cell (Th)1/Th2 balance appears to shift toward Th1 dominance, probably depending on the upregulation of TLR2 expression and these alterations in TLR2-mediated immunity may be involved in the pathogenesis and maintenance of OLP.

Asymmetric synthesis and biological evaluations of (+)- and (-)-6-dimethoxymethyl-1,4-dihydropyridine-3-carboxylic acid derivatives blocking N-type calcium channels.

An efficient asymmetric synthesis of 1,4-dihydropyridine derivatives is described. The key step is the stereoselective Michael addition using t-butyl ester of L-valine as a chiral auxiliary to achieve good ee (>95% for all the tested experiments) and moderate yield. With this method, (+)-4-(3-chlorophenyl)-6-dimethoxymethyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid cinnamyl ester was obtained and was characterized as a promising N-type calcium channel blocker with improved selectivity over L-type compared to its (-)- and racemic isomers.

[Effects of concurrent enteral hyperalimentation with chemo-radiotherapy in patients with oral cancer].

We compared the nutritional condition, immunological function, and frequency of adverse effects during concurrent chemoradiotherapy for oral cancer between patients simultaneously receiving enteral hyperalimentation (Racol) (n=20; EHA group) and patients receiving peripheral vein nutrition (n=20; PVN group). Although there was no significant difference in the change of body weight between the two groups, the decrease of plasma albumin values in the EHA group appeared later than in the PVN group. In the PVN group, the number of lymphocytes and lymphocyte blastogenesis significantly decreased on and after day 14. On the other hand, in the EHA group, the number of lymphocytes decreased only on day 14 and no decrease in lymphocyte blastogenesis was observed. While stomatitis developed in all patients, the severity was lower in the EHA group than the PVN one. These results suggest that the simultaneous administration of Racol during concurrent chemoradiotherapy for oral cancer inhibits the deterioration of nutritional and immunological conditions as well as the severity of stomatitis. This nutrient therapy is therefore considered to be a supportive therapy for oral cancer patients.

Reactive oxygen species produced by the knockdown of manganese-superoxide dismutase up-regulate hypoxia-inducible factor-1alpha expression in oral squamous cell carcinoma cells.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a central regulator that controls the hypoxic response of mammalian cells through the induction of various target genes, and its expression contributes to the development and malignant progression of many tumors. We previously reported that some chemotherapeutic drugs and gamma-rays induce HIF-1alpha expression through increased production of reactive oxygen species (ROS) in oral squamous cell carcinoma (OSCC) cells. However, the mechanism by which intracellular ROS activate HIF-1alpha expression is poorly understood. In this study, we investigated the influence of ROS on HIF-1alpha signaling in OSCC cells by the transfection of manganese-superoxide dismutase (Mn-SOD)-specific small interfering RNA (siRNA). The levels of HIF-1alpha protein and mRNA were increased by siRNA under both normoxic and hypoxic conditions in parallel with the increase in intracellular ROS levels. The accumulation of HIF-1alpha protein was enhanced through inhibition of the recruitment of von Hippel-Lindau protein and HIF-1alpha ubiquitination without a change in prolyl hydroxylase mRNA and protein levels. Furthermore, the transactivation of HIF-1alpha was enhanced via cap-dependent and internal ribosome entry site-mediated mechanisms. These results suggest that intracellular ROS produced by the knockdown of Mn-SOD enhance HIF-1alpha expression in OSCC cells through transcriptional, translational, and posttranslational regulation.

Combined evaluation of dihydropyrimidine dehydrogenase and thymidine phosphorylate mRNA levels in tumor predicts the histopathological effect of 5-fluorouracil-based chemoradiotherapy.

Recent clinical studies have indicated that intra-tumoral gene expression levels of 5-fluorouracil (5-FU) metabolism-related enzymes may predict the clinical response of several cancers to 5-FU-based chemotherapy. However, few studies examining oral squamous cell carcinomas (OSCCs) have been reported. In this study, we determined the expression levels of 5-FU metabolism-related enzymes like thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylate (TP) and orotate phosphoribosyl transferase (OPRT) using reverse transcription-polymerase chain reaction (RT-PCR) combined with laser capture microdissection (LCM). We also evaluated the correlation between the mRNA expressions of these genes and clinico-pathological factors or the treatment effects of 5-FU-based chemotherapy combined with radiotherapy in 27 patients with OSCC. No significant correlation was observed between the mRNA expression levels of any of the examined genes and the T-stage, N-stage, differentiation grade or mode of tumor invasion. Although TS and OPRT mRNA were not correlated with the histopathological effects and the development of tumor recurrence, DPD and TP mRNA were significantly correlated with the histopathological effects and tumor recurrence. A significant positive correlation was also observed between the expression of TS and DPD mRNA, but no other correlations were observed among the other genes. Our results suggest that the combined evaluation of TP and DPD mRNA expression in tumor cells using LCM and RT-PCR may be a useful predictor of the efficacy of 5-FU-based chemotherapy combined with radiotherapy in patients with OSCC.

The structure-activity relationship study on 2-, 5-, and 6-position of the water soluble 1,4-dihydropyridine derivatives blocking N-type calcium channels.

In order to find an injectable and selective N-type calcium channel blocker, we have performed the structure-activity relationship (SAR) study on the 2-, 5-, and 6-position of 1,4-dihydropyridine-3-carboxylate derivative APJ2708 (2), which is a derivative of Cilnidipine and has L/N-type calcium channel dual inhibitory activities. As a consequence of the optimization, 6-dimethylacetal derivative 7 was found to have an effective inhibitory activity against N-type calcium channels with more than 170-fold lower activity for L-type channel compared to that of APJ2708.

Choroidal neovascularization in highly myopic eyes after cataract surgery.

PURPOSE: To determine the incidence and characteristics of choroidal neovascularization (CNV) in patients with high myopia (>or=8 diopters) who underwent cataract surgery in the Department of Ophthalmology, Tokyo Medical and Dental University, or the Ohno Eye Clinic, Tokyo, between September 1991 and March 2000. METHODS: The medical records of 35 patients (48 eyes) who underwent cataract surgery with phacoemulsification and intraocular lens implantation were studied retrospectively. The development of CNV over a 4-year follow-up period, and its characteristics were determined. All of the eyes had received a comprehensive ophthalmological examination, including best-corrected visual acuity measurements, anterior segment biomicroscopy, and a dilated fundus examination by stereoscopic observation. RESULTS: CNV was found in six eyes (12.5%) of six patients. The mean interval between cataract surgery and the development of CNV was 34+/-17 months (range, 12-48 months). The CNV was subfoveal in all cases. The mean logarithm of the minimum angle of resolution (logMAR) after cataract surgery and before the appearance of CNV was 0.23+/-0.24, and 0.93+/-0.41 after the CNV appeared. This decrease was statistically significant (P=0.0008, paired Student t test). Subfoveal CNV developed more frequently in eyes when the fellow eye showed evidence of CNV preoperatively (40.0%) than in eyes when the fellow eye exhibited no evidence of CNV (9.3%). CONCLUSIONS: CNV developed in 12.5% of patients with high myopia after cataract surgery. CNV tended to develop more frequently when the fellow eye had CNV.

Structure-activity relationship study of 1,4-dihydropyridine derivatives blocking N-type calcium channels.

Cilnidipine is a 1,4-dihydropyridine derived L/N-type calcium channel dual blocker possessing neuroprotective and analgesic effects which are related to its N-type calcium channel inhibitory activity. In order to find specific N-type calcium channel blockers with the least effects on cardiovascular system, we performed structure-activity relationship study on APJ2708, which is a derivative of cilnidipine, and found a promising N-type calcium channel blocker 21b possessing analgesic effect in vivo with a 1600-fold lower activity against L-type calcium channels than that of cilnidipine.