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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.

Human butyrylcholinesterase (BChE) is an enzyme existing in plasma and liver that dissolves various choline-ester substrates. The serum cholinesterase level indicates the activity of BChE in serum. Low BChE activity is seen in patients with functional disorders of the liver, organic phosphorus exposure and hereditary serum BChE deficiency. In patients with low BChE activity, prolonged neuromuscular block is induced by the use of depolarizing muscle relaxants, such as succinylcholine for general anesthesia, or breathing trouble can be caused by the use of ester-form local anesthetics.We report a case of tongue fibroma in a 54-year-old man who had extremely low serum BChE activity. While monitoring the circulatory system, the tumor was resected with the use of an amino-form local anesthetic. Hypertension was seen before surgical treatment, possibly due to mental stress, and an antihypertensive was used. DNA sequencing analysis of the BChE gene in this patient revealed a homozygous missense mutation at nucleotide 1093, which resulted in GGA → CGA at Gly365Arg in exon 2.

Expression levels of CENP-F mRNA were investigated in 45 surgically resected oral squamous cell carcinomas (OSCC) and 10 samples of normal gingiva, using real time reverse-transcriptase polymerase chain reaction (RT PCR). The relations between CENP-F mRNA and clinicopathologic variables were studied. The mean expression level of CENP-F mRNA was higher in OSCC tissues than in normal gingiva. There was a significant correlation between the expression level of CENP-F mRNA and lymph node metastasis in the cases of OSCC (p=0.024). These results suggest that OSCC with a high expression level of CENP-F mRNA has high metastatic potential.

We established a pair of cell lines derived from the primary lesion (KK-p) and lymph node metastasis (KK-m) of a tongue carcinoma. We compared the profiles of genes expressed in KK-p and KK-m cells by microarray analysis. Seven genes were highly expressed and 26 were poorly expressed in KK-m cells as compared with KK-p cells. The expression ratios of 14 genes were confirmed by real time RT PCR, and the results were similar to those of the microarray analysis. These results suggested that some of the genes identified in this study may be new metastasis-related genes.

Objective. Our aim was to clarify the association of human papillomavirus (HPV) with oral carcinogenesis, especially its early stage. Study Design. Tissue specimens of normal mucosa, epithelial dysplasia, oral squamous cell carcinoma (OSCC), and OSCC cell lines were examined for the presence of HPV-16 and HPV-18 E6 DNA by means of the polymerase chain reaction test. Results. The detection rate of HPV-16 in epithelial dysplasia (31/51) was higher than that in normal mucosa (16/44) and in OSCC (30/86) and was statistically different from that in OSCC. The cases that progressed from epithelial dysplasia to carcinoma showed a significantly higher HPV-16 detection rate than the other cases in both epithelial dysplasia and OSCC. HPV-16 and HPV-18 were detected only at early passages of 2 of 10 OSCC cell lines. Conclusion. These results strongly suggest that HPV-16 may be involved in the early stages of the development of some oral carcinomas. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:594-600)

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

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9-cis-retinoic acid resistant (MKN-7 and Ho-1-N-1) and IFN-beta resistant cell lines (MKN-7, -28 and -45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP-ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21(WAF1), CREB-binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F-1, E2F-4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell-to-cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis-regulating proteins.

Background Various instruments have been developed for collecting bone debris during intraoral autogenous bone graft procedures in implant surgery. The aim of this study was to quantitatively determine the degree of contamination in bone debris collected by different devices. Methods Twelve patients underwent autogenous bone collection using a bone chisel, bone scraper, trephine drill, and bone filter during bone augmentation surgery as a part of implant therapy, and the total bacterial count in bone debris collected by each was determined. Results Following anaerobic incubation, bacterial colony formation was found in all of the samples. The mean colony forming units (CFU)/g in samples collected by the trephine drill was found to be significantly lower than that of samples obtained with the other devices, while those values for samples collected by the bone scraper and bone filter was significantly higher as compared to the bone chisel and trephine drill. Conclusion The bacterial levels may still carry the infection risk. Thus prophylactic antibiotic therapy maybe indicated when using bone particles for intraoral augmentation procedures.

We previously identified genes associated with Snail-mediated squamous cell carcinoma (SCC) invasiveness, in which we observed significant elevation of Cyr61 expression. In this study, SCC cell lines overexpressing Cyr61 exhibited constitutive activation of Rho A and upregulated invasiveness without the disruption of homophilic cell attachment. Humoral Cyr61 enhanced further production of endogenous Cyr61 by SCC cells, which stimulated collective cell migration and the development of an invasive tumor nest. We propose a Cyr61-dependent model for the development of invasive SCC nest, whereby a subset of tumor cells that highly produce Cyr61 may direct other tumor cells to undergo collective cell migration, resulting in a formation of primary SCC mass.

Approximately 85% of human malignant tumors express increased levels of telomerase. The marked association of telomerase activity with malignant tissue provides strong evidence that telomerase activity is a significant marker for the diagnosis of cancer. In this study, telomerase activity was examined in 12 benign salivary gland tumors (8 pleomorphic adenomas and 4 adenolymphomas), 24 malignant tumors (15 mucoepidermoid carcinomas, 6 adenoid cystic carcinomas and 3 acinic cell carcinomas) and 6 non-neoplastic salivary glands. The mRNA expression of the human telomerase reverse transcriptase (hTERT) and additional telomerase‑associated proteins (hTEP1, p23, Hsp90 and dyskerin) was also examined. Of the 24 malignant tumors, 15 revealed strong telomerase activity. The non-neoplastic salivary glands appeared to have a negative telomerase expression. Furthermore, telomerase activity was significantly higher in high-grade mucoepidermoid carcinomas compared to low‑grade ones (Student's t-test, p

We found a linear correlation between the Prostaglandin E(2) (PGE(2)) amount and the NR4A2 expression in oral squamous cell carcinoma (SCC) tissues through a statistical analysis among 41 clinical cases. In SCC cell lines, PGE(2) receptor (EP) ligation by exogenous PGE(2) promoted the NR4A2 expression in the cAMP/protein kinase A (PKA)-dependent manner. The process required a nature of SCC cell represented by constitutive activated epidermal growth factor receptor (EGFR) family. Targeted inactivation of the EGFRs interfered the PGE(2)-dependent NR4A2 expression. The PGE(2)-dependent NR4A2 induction is essential for the resistance to anti-cancer drug-induced apoptosis especially in SCC cells which showed constitutive EGFRs activity via autocrine epiregulin, a ligand for EGFRs. Conversely, SCC cells which lack epiregulin expression in their nature could gain the ability to promote the NR4A2 expression in response to PGE(2) and attain the resistance to anti-cancer drug-induced apoptosis under the existence of exogenous epiregulin. These findings suggest that susceptibility of SCC to anti-cancer drug could be compromised when PGE(2) was delivered in the microenvironment of SCC cells supported by constitutive EGFR family activities as their nature.

Lugol's iodine staining (Lugol-staining) has been widely used to detect malignant changes in the cervix uteri and esophagus. However, pathological and histochemical changes in the border between Lugol-stained and -unstained areas in oral epithelial dysplastia and malignant lesions are not well understood. We examined the histological appearance of 20 cases of epithelial dysplasia surrounding squamous cell carcinoma using HE and PAS staining in the borders between Lugol-stained and -unstained areas. Subsequently, differences in the expression of cytokeratin 13 (CK13), an epithelium differentiation marker, and Ki-67, a cell proliferative marker, in those areas were investigated by immunohistochemistry. Lugol-stained areas of all cases showed mild dysplasia or normal epithelium appearance, while Lugol-unstained areas in most cases were diagnosed as moderate/severe dysplasia and carcinoma in situ. PAS reactions were limited or not found in the Lugol-unstained areas as compared to intense positivity in Lugol-stained areas. CK13 and Ki-67 protein expression was significantly different between Lugol-stained and -unstained areas. It was confirmed that epithelia showing precancerous or cancerous features were detected as Lugol-unstained boundary areas. A reduction in glycogen production caused by alterations of cell differentiation and proliferation associated with malignant changes may result in a lack of Lugol-staining.

Receptor for hyaluronan-mediated motility (RHAMM) has previously been characterized as a cell surface receptor for hyaluronan and a microtubule-associated intracellular hyaluronan binding protein. We examined the expression of RHAMM mRNA in 43 oral squamous cell carcinomas (SCCs) and 7 normal gingivae by real-time RT-PCR. The expression level of RHAMM mRNA was significantly higher in oral SCCs than normal gingivae (P=0.0047). Forty out of 43 oral SCCs showed expression of RHAMM splice variant (48 bp deletion). We immunohistochemically confirmed the protein expression of RHAMM in oral SCCs with higher levels of RHAMM mRNA. Patients with oral SCC who had high RHAMM expression had shorter survival rates than patients with low expression. However, it was not statistically significant. It has been reported that RHAMM interacts with spindle assembly factors such as microtubule-associated protein (TPX2). To investigate the expression of microtubule-associated protein in oral SCCs, mRNA expression of TPX2 was also examined by real-time RT-PCR. The expression level of TPX2 mRNA was significantly higher in oral SCCs than normal gingivae (P=0.046). Furthermore, a significant correlation between the mRNA expression levels of TPX2 and RHAMM was recognized (P=0.011). The results indicate that there is a strong correlation between the mRNA expression levels of TPX2 and RHAMM in oral SCCs. Our observations suggest that the up-regulations of human RHAMM and TPX2 gene correlate with the malignant condition and might be linked to the increased or abnormal cell proliferation in human oral SCCs.

Stromal cell-derived factor 1alpha (SDF-1alpha) and its receptor CXCR4 have been implicated in the tumorigenesis, proliferation, and lymph node metastasis of cancer. Here, we report that highly invasive squamous cell carcinoma (SCC) cells with a spindle cell morphology show a strong expression of both SDF-1alpha and CXCR4. CXCR4 expression and cell migratory activity were further up-regulated by treatment with SDF-1alpha or TGF-beta1 in these cells. When epithelial-mesenchymal transition (EMT) was induced by Snail over-expression in SCC cells with an epithelial phenotype, an increased expression of SDF-1alpha was observed. Furthermore, SDF-1alpha and TGF-beta1 up-regulated the expression of CXCR4 and cell migratory activity in these cells. These results indicate that SDF-1alpha and CXCR4 expressions are possible markers of highly-invasive SCC and regulated by EMT.

The process of epithelial-to-mesenchymal transition (EMT) involves the acquisition of high-invasiveness by tumor. Snail represses target genes and induces EMT. In this study, we defined the signatures of gene expressions by cDNA microarray analyses in both human squamous cell carcinoma (SCC) cell lines with spontaneous EMT and with Snail-induced EMT, which exhibited high-invasive behavior in vitro. Of the 17,000 cDNA probes, 61 genes were found differentially expressed with >2- or

We examined the expression of Centromere protein H (CENP-H) mRNA in 38 oral squamous cell carcinomas (SCCs), 2 epithelial dysplasias and 5 normal gingivae using the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of CENP-H mRNA was higher in oral SCCs (0.11+/-0.08) than epithelial dysplasias (0.03+/-0.01) and normal gingivae (0.027+/-0.01). The expression level of CENP-H mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.005). We also found a significant association between the level of CENP-H mRNA expression and clinical stage in oral SCCs (Mann-Whitney U test, P=0.04). We next studied the expression of CENP-H in 17 oral SCCs immunohistochemically. A significant correlation between the expression levels of CENP-H protein and the Ki-67 labeling index was found (Mann-Whitney U test, P=0.005). These results indicate that human CENP-H is closely linked to the increased or abnormal cell proliferation in malignant conditions.

Summary We examined the expression of Centromere protein F (CENP-F) mRNA in 26 human salivary gland tumors (seven pleomorphic adenomas, three Warthin tumors, seven mucoepidermoid carcinomas, four adenoid cystic carcinomas, four acinic cell carcinomas and one malignant myoepithelioma) and four normal submandibular glands using the real time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of CENP-F mRNA was higher in malignant tumors (1.05 ± 0.32) than normal submandibular glands (0.11 ± 0.05) and benign tumors (0.46 ± 0.16). We found a significant association between the level of CENP-F mRNA expression and clinical stage in 16 malignant tumors (Mann–Whitney U test, p = 0.027). We also found a significant correlation between the Ki-67 labeling index and CENP-F expression in malignant tumors (Spearmans correlation coefficient by rank test, p = 0.029). These results indicate that human CENP-F mRNA is closely linked to the increased or abnormal cell proliferation in malignant conditions.

The importance of altered histone acetylation in gastrointestinal carcinogenesis, especially in relation to invasion and metastasis, is described. Histone acetylation and chromatin remodeling linked with CpG island methylation play a major role in epigenetic regulation of gene expression. Acetylation of histones through an imbalance of histone acetyltransferases and deacetylases disrupts nucleosome structure, which leads to DNA relaxation and subsequent increase in accessibility to transcription factors. The expression of acetylated histone H4 is reduced in a majority of gastric and colorectal cancers, indicating the low level of global histone acetylation in tumor cells. Moreover, reduced histone acetylation is significantly associated with depth of tumor invasion and nodal metastasis of gastrointestinal cancers. A histone deacetylase inhibitor, trichostatin A (TSA), induces growth arrest and apoptosis and suppresses invasion of cancer cells. Treatment with TSA, which is followed by increased histone acetylation in the promoters, induces the expression of many genes that are suppressors of invasion and metastasis, including tissue inhibitors of metalloproteinase and nm23H1/H2, in addition to negative cell cycle regulators and apoptosis-related molecules. Our approach, serial analysis of gene expression (SAGE), enabled us to identify a gene that is a novel candidate for a metastasis suppressor, whose expression is induced by histone acetylation. These findings suggest that, by modifying gene expression, histone deacetylation may participate not only in tumorigenesis but also in invasion and metastasis. Therefore, histone acetylation should be a promising target for cancer therapy, especially against invasive and metastatic disease, but also for cancer prevention.

The expression of E2F-1 in human colorectal carcinomas was examined immunohistochemically, and the correlation of E2F-1 expression with clinicopathological findings and with the expression of p27Kip1 was analyzed to elucidate the role of E2F-1 in the development and progression of colorectal carcinomas. In nonneoplastic mucosa, a small number of epithelial cells in the proliferative zone were weakly positive for E2F-1. Weak expression of E2F-1 was detected in many adenoma cells. Most of the colorectal carcinomas expressed E2F-1 at various levels, and strong expression of E2F-1 was detected in 56% (49/88) of the cases. There was no correlation between the expression of E2F-1 and any clinicopathological parameters such as tumor stage, depth of tumor invasion and lymph node metastasis. Reduced expression of p27Kip1 was confirmed to be significantly correlated with deep tumor invasion and presence of metastasis. No correlation was evident between overexpression of E2F-1 and reduced p27Kip1 expression.

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1 H )-quinolinone) is a synthetic oral cardiotonic agent that has been used for the treatment of patients with congestive heart failure. Six days of treatment with 30 μg/ml of vesnarinone induced 20–80% growth inhibitions in five out of six gastric carcinoma cell lines examined. Cell cycle analysis revealed that the vesnarinone-sensitive TMK-1 gastric cancer cell line exhibited a significant G0–G1 arrest without evidence of apoptotic cell death induction after 48 h of treatment. Interestingly, this phenomenon was preceded by a marked reduction in the expression of cyclin A, D1 and E as well as cyclin-dependent kinase 2 (CDK2). On the other hand, no significant change was observed in the expression of p21 Waf1/Cip1 , p27 Kip1 nor various growth factors and their receptor genes. Overall these results indicate that vesnarinone inhibits the growth of gastric cancer cells by down-regulating G1 cyclins and CDK2 to induce G0–G1 arrest through a pathway different from that of cyclin inactivation by p21 Waf1/Cip1 or p27 Kip1 .

Ossifying fibromyxoid tumor (OFMT) is a rare mesenchymal neoplasm that arises in subcutaneous tissue, with that in the oral cavity extremely rare. We present a case of malignant OFMT in the tongue. A 26-year-old male noticed a painless mass in the tongue, which was extracted at a general hospital. Four years later, the tumor recurred and was resected at our department. Histologically, the recurrent tumor was composed of the closely packed cells positive for vimentin and S-100 proliferating in a nodular fashion. It showed high cellularity and mitotic activity. In the primary tumor, some tumor cells were arranged in a diffuse or cord-like manner within an abundant fibromyxoid matrix, along with a small amount of metaplastic ossification, corresponding with the histopathological characteristic of OFMT. Accordingly, a diagnosis of malignant OFMT arising in typical OFMT was established. This is the first reported case of malignant OFMT in the tongue. Long-term follow-up is needed for confirmation of prognosis and biological behavior.

In this article, we present our experience with a case of myxolipoma of the tongue. Lipoma is a mesenchymal benign tumor occurring with relatively high frequency. However, myxolipoma, one of the histological variant of lipoma characterized by mature adipose tissue and abundant mucoid substances, in the oral cavity is quite rare. The patient was a 52-year-old man who noticed a painless mass on the left border of tongue about 2 years ago. The lesion was noted at a complete medical checkup, and the patient was admitted to our institution for detailed examination. The mass was a palpable, soft and elastic nodule, 15 mm in diameter, covered with normal mucosa in the left inferior aspect of the tongue. The border of the tumor was well-defined, and computed tomography (CT) revealed a fat density within the mass. On the basis of these finding, the tumor was clinically diagnosed as lipoma and was excised under general anesthesia. Histopathologically, the tumor was a well-defined lobulated mass surrounded by a thin fibrous capsule within the muscle of the tongue. The tumor was diagnosed as myxolipoma because it was consisted of solid proliferation of mature adipocytes replaced by abundant mucoid substances. The post operative course was uneventful, and there was no evidence of recurrence 4 years after surgery.