SATB2 is frequently expressed in ossifying and non-ossifying peripheral oral fibroma of the gingival region but not in reactive fibromatous lesions from other intraoral sites.

Ossifying and non-ossifying peripheral oral fibromas (POF) of the gingival and alveolar mucosa are localized, cellular, small fibrous nodular lesions likely resulting from diverse external/ internal physical and chemical irritation or injuries. A central nidus of metaplastic woven bone characterizes and defines the ossifying variant. The inherent tendency of these lesions to ossify remains elusive. We herein analyze SATB2 expression as osteoblastic transcription and differentiation factor in 28 gingival POFs (10 of them ossifying) and compare them to 28 fibrous lesions from different non-gingival intraoral sites. Strong to moderate diffuse nuclear SATB2 immunoreactivity was detected in all ossifying (10/10; 100%) and in 8/18 (44%) non-ossifying gingival POFs, but in only 1/28 (3%) non-gingival oral reactive nodular fibrous lesions. This study illustrates for the first-time consistent expression of the osteoblastic marker SATB2 in ossifying and most of non-ossifying POFs of the gingival area but lack of this marker in reactive fibrous lesions from other oral cavity sites. This finding is in line with the proposed origin of gingival POFs from periodontal ligaments and may explain the frequent ossification observed in them. It is mandatory to consider this finding when assessing biopsies from SATB2-positive oral cavity neoplasms to avoid misinterpretation.

ZnO Nanoparticles Induced Caspase-Dependent Apoptosis in Gingival Squamous Cell Carcinoma through Mitochondrial Dysfunction and p70S6K Signaling Pathway.

Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, food additives, pigments, rubber manufacture, and electronic materials. Several studies have shown that ZnO-NPs inhibit cell growth and induce apoptosis by the production of oxidative stress in a variety of human cancer cells. However, the anti-cancer property and molecular mechanism of ZnO-NPs in human gingival squamous cell carcinoma (GSCC) are not fully understood. In this study, we found that ZnO-NPs induced growth inhibition of GSCC (Ca9-22 and OECM-1 cells), but no damage in human normal keratinocytes (HaCaT cells) and gingival fibroblasts (HGF-1 cells). ZnO-NPs caused apoptotic cell death of GSCC in a concentration-dependent manner by the quantitative assessment of oligonucleosomal DNA fragmentation. Flow cytometric analysis of cell cycle progression revealed that sub-G1 phase accumulation was dramatically induced by ZnO-NPs. In addition, ZnO-NPs increased the intracellular reactive oxygen species and specifically superoxide levels, and also decreased the mitochondrial membrane potential. ZnO-NPs further activated apoptotic cell death via the caspase cascades. Importantly, anti-oxidant and caspase inhibitor clearly prevented ZnO-NP-induced cell death, indicating the fact that superoxide-induced mitochondrial dysfunction is associated with the ZnO-NP-mediated caspase-dependent apoptosis in human GSCC. Moreover, ZnO-NPs significantly inhibited the phosphorylation of ribosomal protein S6 kinase (p70S6K kinase). In a corollary in vivo study, our results demonstrated that ZnO-NPs possessed an anti-cancer effect in a zebrafish xenograft model. Collectively, these results suggest that ZnO-NPs induce apoptosis through the mitochondrial oxidative damage and p70S6K signaling pathway in human GSCC. The present study may provide an experimental basis for ZnO-NPs to be considered as a promising novel anti­tumor agent for the treatment of gingival cancer.

Oxytalan-positive peripheral ossifying fibromas express runt-related transcription factor 2, bone morphogenetic protein-2, and cementum attachment protein. An immunohistochemical study.

BACKGROUND: The peripheral ossifying fibroma (POF) represents one of the most common lesions of the periodontal tissues that may originate from the gingival soft tissues, the periosteum, or the periodontal ligament. AIM: To investigate the immunohistochemical expression of runt-related transcription factor 2 (Runx-2), bone morphogenetic protein-2 (BMP-2), and cementum attachment protein (CAP) in oxytalan-positive POF, to establish the use of POF as an in vivo model for the study of the periodontal ligament. MATERIALS AND METHODS: Thirty tumors that presented clinical and histologic features of POF, as well as oxytalan fibers, were included in the study. Immunohistochemical expression of Runx-2, BMP-2, and CAP was evaluated by light microscopy. RESULTS: Runx-2, BMP-2, and CAP were abundantly expressed by POFs; 22 of 30 tumors expressed positive staining for Runx-2, twenty-six tumors for BMP-2, and twenty-five tumors for CAP. The expression of Runx-2 was abundant in POFs where bone was histologically present (P = 0.04) and of BMP-2 in POFs where dystrophic calcifications were present (P = 0.03). CONCLUSION: It is suggested that oxytalan-positive POFs, purportedly originating from the periodontal ligament, express molecules that are specific to bone and cementum (Runx-2, BMP-2), or cementum only (CAP). Thus, the cell populations present in the lesion belong to the mineralized-tissue-forming cell lineages, the cementoblastic or osteoblastic lineage.

Methanolic extracts of Solieria robusta inhibits proliferation of oral cancer Ca9-22 cells via apoptosis and oxidative stress.

Many red algae-derived natural products are known to have anticancer effects. The biological functions of the red alga Solieria robusta from the Karachi coast (Pakistan) remain unclear. Here, we prepared a methanolic extracts of S. robusta (MESR) to examine its possible anti-oral cancer effects and the corresponding mechanism of action. Cell viability of MESR-incubated oral cancer Ca9-22 cells was dose-responsively decreased (p<0.001). According to a propidium iodide (PI)-based assay the cell cycle distribution was dramatically changed, especially for subG1 accumulation. Annexin V/PI assay of apoptosis using flow cytometry also showed that MESR-incubated Ca9-22 cells were dose-responsively increased (p<0.001). For evaluation of oxidative stress in MESR-incubated Ca9-22 cells, we found that reactive oxygen species (ROS) were overexpressed dose- and time-responsively and mitochondrial depolarization was also increased (p<0.001). Taken together, MESR showed inhibitory effects on oral cancer proliferation coupled with apoptosis and oxidative stress.

Eugenol as a potential adjuvant therapy for gingival squamous cell carcinoma.

Adoption of plant-derived compounds for the management of oral cancer is encouraged by the scientific community due to emerging chemoresistance and conventional treatments adverse effects. Considering that very few studies investigated eugenol clinical relevance for gingival carcinoma, we ought to explore its selectivity and performance according to aggressiveness level. For this purpose, non-oncogenic human oral epithelial cells (GMSM-K) were used together with the Tongue (SCC-9) and Gingival (Ca9-22) squamous cell carcinoma lines to assess key tumorigenesis processes. Overall, eugenol inhibited cell proliferation and colony formation while inducing cytotoxicity in cancer cells as compared to normal counterparts. The recorded effect was greater in gingival carcinoma and appears to be mediated through apoptosis induction and promotion of p21/p27/cyclin D1 modulation and subsequent Ca9-22 cell cycle arrest at the G0/G1 phase, in a p53-independent manner. At these levels, distinct genetic profiles were uncovered for both cell lines by QPCR array. Moreover, it seems that our active component limited Ca9-22 and SCC-9 cell migration respectively through MMP1/3 downregulation and stimulation of inactive MMPs complex formation. Finally, Ca9-22 behaviour appears to be mainly modulated by the P38/STAT5/NFkB pathways. In summary, we can disclose that eugenol is cancer selective and that its mediated anti-cancer mechanisms vary according to the cell line with gingival squamous cell carcinoma being more sensitive to this phytotherapy agent.

Cellular mechanisms mediating the anti-cancer effects of carnosol on gingiva carcinoma.

Carnosol, a rosemary polyphenol, displays anticancer properties and is suggested as a safer alternative to conventional surgery, radiotherapy, and chemotherapy. Given that its effects on gingiva carcinoma have not yet been investigated, the aim of this study was to explore its anti-tumor selectivity and to unravel its underlying mechanisms of action. Hence, oral tongue and gingiva carcinoma cell lines exposed to carnosol were analyzed to estimate cytotoxicity, cell viability, cell proliferation, and colony formation potential as compared with those of normal cells. Key cell cycle and apoptotic markers were also measured. Finally, cell migration, oxidative stress, and crucial cell signaling pathways were assessed. Selective anti-gingiva carcinoma activity was disclosed. Overall, carnosol mediated colony formation and proliferation suppression in addition to cytotoxicity induction. Cell cycle arrest was highlighted by the disruption of the c-myc oncogene/p53 tumor suppressor balance. Carnosol also increased apoptosis, oxidative stress, and antioxidant activity. On a larger scale, the alteration of cell cycle and apoptotic profiles was also demonstrated by QPCR array. This was most likely achieved by controlling the STAT5, ERK1/2, p38, and NF-ĸB signaling pathways. Lastly, carnosol reduced inflammation and invasion ability by modulating IL-6 and MMP9/TIMP-1 axes. This study establishes a robust foundation, urging extensive inquiry both in vivo and in clinical settings, to substantiate the efficacy of carnosol in managing gingiva carcinoma.

Assessment of angiogenic markers and female sex hormone receptors in pregnancy tumor of the gingiva.

PURPOSE: Oral pregnancy tumors (OPTs) arise on the inflamed gingiva of women after the first trimester of pregnancy. The expression of angiogenic markers and female hormone receptors was assessed. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expression of estrogen and progesterone receptors and the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and its receptor, fibroblast growth factor (FGF), and hypoxia inducible factors 1α and 3α (HIF1α and HIF3α). Experimental groups included 9 OPTs, 10 oral pyogenic granulomas from nonpregnant women of the same age, and 9 oral pyogenic granulomas from postmenopausal women. RESULTS: VEGF expression in stromal histiocytes and endothelial cells of small vessels was positively correlated in the OPT group (P < .05 by χ(2) test). VEGF receptor also was overexpressed in stromal histiocytes and endothelial cells of OPTs compared with oral pyogenic granulomas from nonpregnant and postmenopausal women (P < .005 by χ(2) test). No correlation was detected among estrogen and progesterone receptors, FGF and HIF1α and HIF3α (ER and PgR respectively) in the 3 experimental groups. CONCLUSIONS: VEGF-associated angiogenesis is most likely involved in the pathogenesis of the lesion. These results imply that local inhibition of VEGF activity could be an adjuvant therapeutic approach for OPTs to control hemorrhage, which can be massive at the surgical excision of such lesions during pregnancy.

Cardiotoxin III inhibits proliferation and migration of oral cancer cells through MAPK and MMP signaling.

Cardiotoxin III (CTXIII), isolated from the snake venom of Formosan cobra Naja naja atra, has previously been found to induce apoptosis in many types of cancer. Early metastasis is typical for the progression of oral cancer. To modulate the cell migration behavior of oral cancer is one of the oral cancer therapies. In this study, the possible modulating effect of CTXIII on oral cancer migration is addressed. In the example of oral squamous carcinoma Ca9-22 cells, the cell viability was decreased by CTXIII treatment in a dose-responsive manner. In wound-healing assay, the cell migration of Ca9-22 cells was attenuated by CTXIII in a dose- and time-responsive manner. After CTXIII treatment, the MMP-2 and MMP-9 protein expressions were downregulated, and the phosphorylation of JNK and p38-MAPK was increased independent of ERK phosphorylation. In conclusion, CTXIII has antiproliferative and -migrating effects on oral cancer cells involving the p38-MAPK and MMP-2/-9 pathways.

Genome-wide expression and copy number analysis identifies driver genes in gingivobuccal cancers.

The molecular mechanisms contributing to the development and progression of gingivobuccal complex (GBC) cancers-a sub-site of oral cancer, comprising the buccal mucosa, the gingivobuccal sulcus, the lower gingival region, and the retromolar trigone-remain poorly understood. Identifying the GBC cancer-related gene expression signature and the driver genes residing on the altered chromosomal regions is critical for understanding the molecular basis of its pathogenesis. Genome-wide expression profiling of 27 GBC cancers with known chromosomal alterations was performed to reveal differentially expressed genes. Putative driver genes were identified by integrating copy number and gene expression data. A total of 315 genes were found differentially expressed (P ≤ 0.05, logFC > 2.0) of which 11 genes were validated by real-time quantitative reverse transcriptase-PCR (qRT-PCR) in tumors (n = 57) and normal GBC tissues (n = 18). Overexpression of LY6K, in chromosome band 8q24.3, was validated by immunohistochemical (IHC) analysis. We found that 78.5% (2,417/3,079) of the genes located in regions of recurrent chromosomal alterations show copy number dependent expression indicating that copy number alteration has a direct effect on global gene expression. The integrative analysis revealed BIRC3 in 11q22.2 as a candidate driver gene associated with poor clinical outcome. Our study identified previously unreported differentially expressed genes in a homogeneous subtype of oral cancer and the candidate driver genes that may contribute to the development and progression of the disease. © 2011 Wiley Periodicals, Inc.

Immunohistochemical features of 3,3',4,4'-tetrachloroazobenzene-induced rat gingival lesions.

Gingival lesions of squamous hyperplasia, cystic keratinizing hyperplasia (CKH), and squamous cell carcinoma (SCC) can be induced in rats treated by chronic gavage with 10-100 mg/kg 3,3',4,4'-tetrachloroazobenzene. We evaluated gingival squamous hyperplasia (GSH), CKH, and SCC for the immunohistochemical pattern of expression of carcinogenesis-associated markers. The 3 types of lesions and controls were stained with proliferation markers (proliferating cell nuclear antigen [PCNA] and cyclin-D1), tumor-suppressor markers (ß-catenin and mammary serine protease inhibitor [maspin]) and stroma-related markers (α-smooth muscle actin [SMA] and osteonectin/SPARC). The lesions had common immunohistochemical characteristics that differed in their expression patterns among the various diagnoses. PCNA and cyclin-D1 expression was higher in GSH, CKH, and SCC than in controls. The normal membranous expression of ß-catenin was lower in GSH, and almost absent in CKH and SCC. Maspin expression was similar in GSH and controls, whereas both CKH and SCC showed decreased expression. SMA and/or osteonectin/SPARC were seen in stromal cells in CKH and SCC. Collectively, there appears to be a progression from hyperplastic and cystic lesions toward malignancy based on the morphological changes, supported by the expression of carcinogenesis-associated proteins. The exact sequence of events leading to SCC remains to be defined in a time-dependent manner.

Protective effects of polysaccharides and polyhydric alcohols in a dry mouth model in cultured cells.

PURPOSE: The goal of the study was to investigate the effects of 21 polysaccharides and 12 polyhydric alcohols on inhibition of dryness in oral mucosal epithelial cells in vitro. All the tested compounds are currently used in oral products. METHODS: Human gingival epithelial Ca9-22 cells were incubated in 96-well plates until the cells reached confluence. After removal of the culture medium, the cells were incubated with a solution containing one of 21 polysaccharides (seven semisynthetic and 14 natural polysaccharides) or 12 polyhydric alcohols for 15 min (short-term treatment). After removal of the sample solution, the cells were dried at 25°C and 30% humidity, and cell viability was measured to determine the effect of each compound on prevention of cell death due to drying. The effects of the polyhydric alcohols were also examined for 3 days (long-term treatment). RESULTS: The semisynthetic polysaccharides ethylcellulose (EC), methylcellulose (MC), and hydroxypropylmethylcellulose (HPMC) and the natural polysaccharides xanthan and gellan gum significantly inhibited cell death due to drying. Hydroxypropylcellulose increased cell death under these conditions. Of the polyhydric alcohols, long-term treatment with glycosyltrehalose significantly inhibited cell death due to drying, but short-term treatment with glycosyltrehalose did not do so. Long-term treatment had an effect on cell proliferation that appeared to differ from the effect of short-term treatment. CONCLUSIONS: Short-term treatment with EC, MC, HPMC, xanthan gum, and gellan gum and long-term treatment with glycosyltrehalose showed significant inhibition of cell death due to drying. These materials might have protective effects against dry mouth.

Plasmablastic lymphoma of the oral cavity in an HIV-negative patient.

Plasmablastic lymphoma (PBL) is a rare, highly aggressive lymphoma typified by immunoblast-like cells with abundant basophilic cytoplasm and paranuclear hof. It shows absent expression of CD45 and CD20. In contrast, it displays a constant reaction with CD138 and VS38c. It may be easily misinterpreted as some other lymphoma. An exhaustive integration of clinical, morphologic, phenotypic, and molecular features is important to exclude misdiagnosis and inappropriate treatment. We report a case of HIV-negative PBL arising on the left areas of posterior teeth mucosa of a 58-year-old man. Immunohistochemically, the tumor cell was immunoreactive for CD138, VS38c, VEGF, and vimentin; Ki-67 showed a high proliferation rate. Epstein-Barr virus (in situ hybridization) was nonreactive, and IgH gene rearrangement was identified by polymerase chain reaction amplification products. A diagnosis of PBL was rendered.

Congenital epulis of the newborn: 10 new cases of a rare oral tumor.

Congenital epulis of the newborn (CEN) is a rare benign lesion that exclusively occurs in the oral and maxillofacial regions of newborns. The clinicopathologic features of CEN were examined and reviewed from the files of the Armed Forces Institute of Pathology from 1970 to 2000. Ten cases were included. Patient lesions were all present at birth but were surgically excised between 2 days and 6 weeks (median, 5.5 days). Nine lesions were in females; 1 case did not designate patient sex. Locations included 6 on the maxilla, 2 on the mandible, 1 on the designated maxillary lip, and 1 unknown. The cases included a patient with 2 lesions: 1 on mandibular and 1 on maxillary alveolar ridges, respectively. All other lesions were solitary and polypoid. Microscopically, these were pedunculated and nodular, composed of sheets to grouped clusters of medium-sized, ovoid-to-polygonal cells with abundant granular cytoplasm, distinct cell membranes, vascular-rich stroma, and attenuated overlying mucosa. Two cases also demonstrated spindled cells. The nuclei were vesicular and focally stippled, with distinct and slightly convoluted nuclear membranes; nucleoli were visible but not prominent. Mitotic activity was not observed. The vascular channels ranged from capillary-sized to venous, some staghorn-like with rare perivascular long-term inflammation. The venules exhibited a perivascular pericytic proliferation. Odontogenic epithelial rests were present in 2 cases. No cases demonstrated cytoplasmic hyaline globules. The lesional cells in all cases were negative for S-100 protein, CD68, CD34, CD31, keratins, desmin, calponin, and smooth muscle actin. Perivenular pericytes were positive for smooth muscle actin. Congenital epulis of the newborn is a rare oral entity with characteristic clinicopathologic features. It predominately affects girls, mainly on the maxillary alveolar ridge. It may be separated from "granular cell tumor" by location, patient age, absence of cytoplasmic hyaline globules, solid growth pattern, pericytic proliferation, attenuated overlying epithelium, and negativity for S-100 protein.

Assessing hypoxia in animal tumor models based on pharmocokinetic analysis of dynamic FAZA PET.

UNLABELLED: Positron emission tomography (PET) allows non-invasive detection and mapping of tumor hypoxia. However, slow tracer kinetics and low resolution, results in limited tumor-to-normal tissue contrast and the risk of missing areas where hypoxic cells are intermixed with necrosis. The shape of tumor time activity curves (TACs), as deduced from dynamic scans, may allow further separation of tumors/tumor sub-volumes that are inseparable based on static scans. This study was designed to define the added value of dynamic scans. MATERIAL AND METHODS: Three squamous cell carcinoma tumor models were grown in mice. Mice were injected with the (18)F-labeled PET hypoxia-tracer fluoroazomycin arabinoside (FAZA) and the immunologically-detectable hypoxia-marker pimonidazole, and PET scanned dynamically for three to six hours. Subsequently, microregional tracer retention (autoradiography) and the distribution of pimonidazole-retaining cells (immunohistology) and necrosis were analyzed in tumor tissue sections. Dynamic PET data were analysed based on a two-compartment model with irreversible tracer binding generating estimates of the putative hypoxia surrogate markers k(3) (tracer trapping rate constant) and K(i) (influx rate constant from plasma into irreversible bound tracer). RESULTS/DISCUSSION: High tumor-to-reference tissue ratios and a strong linear correlation (R∼0.7 to 0.95) between density of hypoxic cells and FAZA concentration was observed three hours after tracer administration, suggesting that late time PET images provides an accurate measure of hypoxia against which kinetic model estimates can be validated. Tumor TACs varied widely (ranging from distinctly wash-out to accumulative type) among tumor types although pimonidazole-stainings revealed extensive hypoxia in all models. Kinetic analysis of tumor sub-volumes showed that k(3) correlated poorly with late time FAZA retention regionally in two of the three tumor models. The influx rate constant K(i) displayed far less variability and correlated strongly with late time FAZA retention (hypoxia) in two of three tumor models, whereas a non-consistent relationship was observed in the last tumor model. Our study demonstrates the potential usefulness of dynamic PET, but also that a simple two-compartment model may be inappropriate in some tumor models.

Experimental study of antiangiogenic gene therapy targeting VEGF in oral cancer.

It is well known that tumor angiogenesis plays an important role in local growth and metastasis of oral cancer; therefore, inhibiting angiogenesis is considered to be effective for treating oral cancer. This study aimed to investigate the effectiveness of systemically available antiangiogenic gene therapy targeting vascular endothelial growth factor (VEGF), which is one of the most important angiogenesis accelerators. We administered a soluble form of VEGF receptor-expressing gene incorporated into adenovirus (AdVEGF-ExR) intraperitoneally to nude mice to which oral cancer cell lines (SAS, HSC-3, and Ca9-22) had been transplanted subcutaneously in vivo to inhibit angiogenesis and tumor proliferation. Then, we measured tumor volumes over time, and tumors were enucleated and examined histopathologically and immunohistologically at 28 days after AdVEGF-ExR administration. Compared to the controls to which we administered AdLacZ or saline, significant antiproliferative effects were observed (P < 0.05) in the AdVEGF-ExR administration group, and extensive tumor necrosis was found histopathologically. Immunohistochemical analysis with CD34 (NU-4A1) revealed tumor angiogenesis was suppressed significantly (P < 0.05), and that with ssDNA revealed apoptosis induction was significantly high (P < 0.05) in the AdVEGF-ExR group. However, analysis with Ki-67 (MIB-1) revealed tumor proliferative capacity was not significantly different between the groups. Consequently, we consider that AdVEGF-ExR administration achieved tumor growth suppression by inhibiting angiogenesis and inducing apoptosis, but not by inhibiting the proliferative capacity of tumor cells. Neither topical administration of a soluble form of VEGF receptor (sVEGFR) to the tumor nor a megadose was needed to achieve this inhibition effect. These results suggest gene therapy via sVEGFR would be an effective oral cancer therapy and benefit future clinical applications.

Tetraspanin gene expression levels as potential biomarkers for malignancy of gingival squamous cell carcinoma.

Accurate assessment of malignancy in oral squamous cell carcinoma is essential to optimize treatment planning. To detect a biomarker related to malignant propensity in gingival squamous cell carcinoma (GSCC), quantitative gene expression analysis of tetraspanin family genes was conducted. In 73 cases of GSCC, total RNA was extracted from carcinoma tissues, and gene expression was analyzed by quantitative real time-PCR. Six tetraspanin family genes (CD9, CD63, CD81, CD82, CD151, NAG-2) were investigated. Housekeeping genes (ACTB and GAPDH), anchor protein genes (JUP and PXN) and an integrin gene (ITGA3) were used as reference genes. Forty-five gene expression ratios were calculated from these 11 gene expression levels and were analyzed with clinical parameters using multivariate statistical methods. According to the results of the logistic regression analysis subjecting cervical lymph node metastasis as a target variable, CD9/ACTB (p = 0.013) or CD9/CD82 (p = 0.013) in addition to tumor size (p = 0.028) were detected as significant factors. In Cox proportional hazards regression analysis, delayed cervical lymph node metastasis (p = 0.039) and tumor cell positive surgical margin (p = 0.032) in addition to CD151/GAPDH (p = 0.024) were detected as significant factors for death outcome. A Kaplan-Meier survival curve presented a significantly lower survival rate of the group with a CD151/GAPDH value of 10 or more (log rank and generalized Wilcoxon tests: p = 0.0003). Results of this study present the usefulness of CD9 and CD151 expression levels as biomarkers for assessment of malignancy in GSCC. They also indicate that detection of residual tumor cells at the surgical margin and the biological malignancy of a tumor interdependently affects prognosis.

HuR is exported to the cytoplasm in oral cancer cells in a different manner from that of normal cells.

HuR, a ubiquitously expressed member of the Hu protein family that binds and stabilizes an AU-rich element (ARE)-containing mRNAs, is known to shuttle between the nucleus and the cytoplasm via several export pathways. When normal cells were treated with heat shock, HuR was exported to the cytoplasm in a chromosome maintenance region 1 (CRM1)-dependent manner. However, in this study, we demonstrate that HuR is exported to the cytoplasm in oral cancer cells even if the cells were treated with the inhibitor of the CRM1-independent export pathway. Immunohistochemical and biochemical analyses showed that HuR existed in both the cytoplasm and the nucleus in oral cancer cells, such as HSC-3 and Ca9.22, but existed entirely inside the nucleus in normal cells. AU-rich element-mRNAs were also exported to the cytoplasm and stabilised in the oral cancer cells, which were inhibited by HuR knockdown. This export of HuR was not affected by at least 7 h of treatment of leptomycin B (LMB), which is an inhibitor of the CRM1-dependent export pathway. These findings suggest that HuR is exported to the cytoplasm in oral carcinoma cells in a different manner from that of normal cells, and is likely to occur through the perturbation of a normal export pathway.

Ferritin expression in the periodontal tissues of primates.

Ferritin, an iron-binding protein, is composed of two subunits, a heavy chain and a light chain. It regulates many biological functions, such as proliferation, angiogenesis, and immunosuppression. The objective of this study was to determine the expression and distribution of ferritin in the periodontal tissuesof primates.First, we assessed the expression of ferritin in primary cultured cells isolated from human periodontal tissues using the polymerase chain reaction and immunofluorescent staining. Second, we investigated the expression and distribution of ferritin in the periodontal tissues of Macaca fascicularis, human gingival tissues, and human gingival carcinoma tissues using immunohistochemistry.Both protein and mRNA of ferritin were constitutively present in human primary cultured cells, including those from the dental apical papilla, periodontal ligament, dental pulp, and gingival epithelium, as well as gingival fibroblasts. In M. fascicularistissues, the immunohistochemical staining was particularly strong in blood vessel and mineralizing areas of the dental pulp and periodontal ligament. Ferritin heavy chain exhibited specific immunopositivity in in the stratum basale of the epithelium in human gingival tissue and strong immunostaining was found in peripheral regions of gingival carcinoma sites. Ferritin is constitutivelypresent andwidelydistributed in the periodontal tissues of primates. Ferritin may play roles in epithelial proliferation, vascular angiogenesis, and mineralization in these tissues.

Significance of the fibrous stroma in bone invasion by human gingival squamous cell carcinomas.

Gingival squamous cell carcinomas (SCCs) frequently invade the mandible or maxilla, and this invasion is associated with a worse prognosis. Although previous studies have suggested that bone destruction caused by gingival SCC is mediated by osteoclastic bone resorption rather than by tumor cells directly, the mechanism underlying the bone invasion remains poorly understood. We histopathologically investigated mandibular invasion patterns in 97 cases of primary gingival SCC and evaluated the correlations between bone invasion patterns and clinicopathological factors. Based on the histological examination of the mandibular invasion pattern, we classified the cases into 2 categories: expansive type and infiltrative type. Of the 97 cases, 52 were expansive type and 45, infiltrative type. Varying numbers of Howship's lacunae and osteoclasts were detected on the bone surface adjacent to the tumor cells. Compared to the expansive type, the infiltrative type showed increased numbers of osteoclasts at the interface of the tumor and the resorbing bone. Tumor cells showed no direct contact with osteoclasts and the adjacent bones, and in all cases varying amounts of fibrous connective tissues intervened between the tumor cells and the bone. The number of fibroblasts was significantly greater in the infiltrative type than in the expansive type. We also found a positive correlation between the number of osteoclasts and fibroblasts at the interface of the tumor and the resorbing bone. Immunohistochemistry revealed RANKL expression in the fibroblastic cells that were adjacent to the osteoclasts in the area of bone resorption. In coculture experiments, human gingival SCC cells (BHY) stimulated the expression of mouse RANKL mRNA in murine osteoblastic cells (MC3T3-E1). These results indicate that the fibrous stroma plays critical roles in osteoclastic bone resorption by gingival SCC through the RANKL-dependent pathways.

Oral squamous cell carcinomas stimulate osteoclast differentiation.

Matrix metalloproteinases (MMPs) play important roles in the invasion and metastasis to soft tissues of carcinomas including, oral squamous cell carcinomas (SCCs). Although, osteoclastic bone resorption is an important step in bone involvement in a variety of malignancies, the mechanism of bone involvement of oral SCC remains unclear. Once cancer cells arrest in bone, the bone is a storehouse of a variety of cytokines and growth factors and thus provides an extremely fertile environment for cell growth. The bone-invasive oral cancer cell line, BHY, transcriptionally expressed detectable levels of TGF-beta, IL-1beta, IL-8, parathyroid hormone-related protein (PTHrP) and vascular endothelial growth factor (VEGF) mRNAs and failed to express GM-CSF, IL-6, and TNF-alpha. Furthermore, the BHY-conditioned medium greatly upregulated IL-6 and RANKL/ODF mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. Seven out of eleven patients with carcinomas of the lower alveolus and gingiva showing infiltrative bone involvement expressed PTHrP mRNA. These data suggest that the occurrence of PTHrP may be an indication of developing oral malignant carcinomas.