FBLIM1 enhances oral cancer malignancy via modulation of the epidermal growth factor receptor pathway.

Filamin-binding LIM protein 1 (FBLIM1) is related to regulation of inflammatory responses, such as chronic recurrent multifocal osteomyelitis; however, the relevance of FBLIM1 in oral squamous cell carcinoma (OSCC) is unknown. The aim of the current study was to elucidate the possible role of FBLIM1 in the carcinogenesis of OSCC. We analyzed FBLIM1 expression using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunoblot analysis, and immunohistochemistry. The expression levels of FBLIM1 were up-regulated significantly (P < 0.05) in OSCC-derived cell lines and primary OSCCs specimens compared with normal counterparts. FBLIM1 expression also was correlated with the primary tumoral size (P < 0.05) and vascular invasion (P < 0.05). We then assessed tumoral progression after treatment with FBLIM1 siRNA and clopidogrel, an antiplatelet agent. Similar to the FBLIM1 knockdown effect, clopidogrel-treated cells had attenuated functions of proliferation, migration, and invasiveness. Interestingly, clopidogrel treatment led to down-regulation of epidermal growth factor receptor (EGFR) and FBLIM1. These findings identify FBLIM1 as a putative therapeutic target by using clopidogrel for inhibiting over activation of EGFR signaling to prevent OSCC malignancy.

Endoscopically assisted removal of a fish bone penetrating the parotid duct: an unusual case.

PURPOSE: The aim was to evaluate the efficacy of endoscopy-assisted surgery for treating a foreign body (fish bone) deeply embedded in the parotid duct. PATIENTS AND METHODS: We report the case of a 67-year-old man with diffuse swelling of the cheek and the discharge of pus from the parotid duct orifice caused by a fish bone that had penetrated into the parotid duct. The preoperative examination using ultrasonography and computed tomography showed a linear foreign body. RESULTS: The fish bone was thought to be embedded deeply in the parotid duct; therefore, we used a combined approach (endoscopy with open surgery), because we anticipated difficulties with endoscopic removal of the fish bone. The endoscopic view showed that the fish bone had partially penetrated the soft tissue in the parotid duct wall, but the fish bone could not be removed endoscopically. With endoscopic assistance, the impacted fish bone was removed using an intraoral surgical approach. The clinical outcome was satisfactory during a 10-month follow-up period, with no evidence of complications. CONCLUSIONS: The combined surgical and endoscopic approach resulted in the safe and effective removal of a foreign body from the salivary duct that could not be removed using sialendoscopy alone.

State of heat shock factor 1 expression as a putative diagnostic marker for oral squamous cell carcinoma.

Heat shock factor 1 (HSF1) is responsible for expres-- sion of a large class of heat shock proteins that have been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of HSF1 on the behavior of oral squamous cell carcinoma (OSCC). In this study, we assessed the value of HSF1 for predicting clinical outcomes in OSCC. Quantitative reverse transcriptase-polymerase chain reaction and Western blotting showed that the expressions of HSF1 mRNA and protein in OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, KON and Ho-1-u-1) were elevated compared with those in human normal oral keratinocytes (P<0.05). Similar to in vitro data, HSF1 mRNA expression in primary OSCCs (n=50) was significantly greater than in normal counterparts (P<0.05). Since HSF1 was observed in the nucleus and cytoplasm by immu-- nohistochemistry, we investigated the correlation between the HSF1 expression status at each subcellular location and the clinical behavior of OSCCs. Among the clinical classifications, higher nuclear HSF1 expression was closely related to tumor size and histopathologic types (P<0.05). These results showed for the first time that nuclear HSF1 expression may contribute to cancer progression and that HSF1 might be a potential diagnostic biomarker and a therapeutic target for OSCCs.

Upregulation of thioredoxin reductase 1 in human oral squamous cell carcinoma.

Thioredoxin reductase 1 (TrxR1) catalyzes the nicotinamide adenine dinucleotide phosphate-dependent reduction of oxidized thioredoxin (Trx). Trx, which is over-expressed in many human tumors, is a selenocysteine-containing protein associated with cell proliferation and apoptosis inhibition. This selenium-containing redox system regulates the activity of various enzymes and counteracts oxidative stress in cells such as hypoxia and cytotoxic agents. Consequently, TrxR1 could play an important role in tumor progression and resistance to chemotherapy due to its anti-apoptotic functions. To characterize cancer-related gene expression changes in oral squamous cell carcinomas (OSCC), we compared the gene expression profiles in OSCC primary tumors with patient-matched normal oral epithelium. Microarray analysis showed TrxR1 upregulation in primary tumors. Gene ontology analysis showed highly significant cancer-related function. The TrxR1 expression examined by immunohistochemistry was correlated with regional lymph node metastasis (P<0.05) and the clinical stages of 50 patients (P<0.01). Overexpression of TrxR1 could contribute to cancer progression and might be a potential molecular marker for therapy.

Antitumor activity of satraplatin in cisplatin-resistant oral squamous cell carcinoma cells.

BACKGROUND: The aim of the current study was to identify the antitumor activity of satraplatin in paired cisplatin (CDDP)-resistant oral squamous cell carcinoma (OSCC) cell line and its parental cell line. METHODS: CDDP-resistant (KB-R) cells and parental cells (KB) pair were used. Viability was assessed using the MTT and clonogenic assay. Real-time polymerase chain reaction (PCR), glutathione (GSH) assay, and flow cytometric analysis were used for further assessment. RESULTS: KB-R cells did not show cross-resistance to satraplatin. The expression status of almost all transporters was upregulated in the KB-R cells. There was no difference in the GSH levels between the KB and KB-R cells. Flow cytometric analysis indicated that with satraplatin the G2/M phase was arrested in the KB-R cells. KB-R cells contain enriched side population cells. CONCLUSION: These data suggested that satraplatin has antitumor activity against the CDDP-resistant OSCC cells. The mechanism of cross-resistance to platinum agents seems to be multifactorial.

Expression status of Zic family member 2 as a prognostic marker for oral squamous cell carcinoma.

PURPOSE: To determine the involvement of ZIC2 in oral squamous cell carcinoma (OSCC). METHODS: ZIC2 mRNA and protein expression in primary OSCCs (n = 74), oral premalignant lesions (OPLs, n = 20) and five OSCC-derived cell lines (HSC-2, HSC-3, OK-92, H1, and Sa3) were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry (IHC). In addition, we evaluated the correlation between ZIC2 IHC scores in OSCCs and the clinicopathologic status. RESULTS: Significant up-regulation of ZIC2 was detected in OSCC-derived cell lines (P < 0.05), primary OSCCs (P < 0.05) and OPLs (P < 0.05) compared with normal counterparts. Among the clinical variables analyzed, ZIC2 expression was associated with the histopathologic types of OSCC. Furthermore, the survival rates differed significantly between ZIC2-positive cases and ZIC2-negative cases. CONCLUSIONS: These results suggested that ZIC2 expression is correlated with the differentiation type of OSCC and diagnosis and might be a potential prognostic indicator and therapeutic target for OSCCs.

Identification of cisplatin-resistance related genes in head and neck squamous cell carcinoma.

Resistance to cisplatin is a major obstacle to successful treatment of head and neck squamous cell carcinoma (HNSCC). To investigate the molecular mechanism of this resistance, we compared the gene expression profiles between the cisplatin-sensitive SCC cell lines (Sa-3, H-1 and KB) and the cisplatin-resistant cell lines established from them (Sa-3R, H-1R and KB-R) using Affymetrix U133 Plus 2.0 microarray. We identified 199 genes differentially expressed in each group. To identify important functional networks and ontologies to cisplatin resistance, the 199 genes were analyzed using the Ingenuity Pathway Analysis Tool. Fifty-one of these genes were mapped to genetic networks, and we validated the top-10 upregulated genes by real-time reverse transcriptase-polymerase chain reaction. Five novel genes, LUM, PDE3B, PDGF-C, NRG1 and PKD2, showed excellent concordance with the microarray data. In 48 patients with oral SCC (OSCC), positive immunohistochemical staining for the five genes correlated with chemoresistance to cisplatin-based combination chemotherapy. In addition, the expression of the five genes predicted the patient outcomes with chemotherapy. Furthermore, siRNA-directed suppressed expression of the five genes resulted in enhanced susceptibility to cisplatin-mediated apoptosis. These results suggested that these five novel genes have great potential for predicting the efficacy of cisplatin-based chemotherapy against OSCC. Global gene analysis of cisplatin-resistant cell lines may provide new insights into the mechanisms underlying clinical cisplatin resistance and improve the efficacy of chemotherapy for human HNSCC.

Alteration of extracellular superoxide dismutase expression is associated with an aggressive phenotype of oral squamous-cell carcinoma.

Oxidative stress results in damage to cellular structures and has been linked to numerous diseases, including cancer. Extracellular superoxide dismutase (EC-SOD) is a principal enzymatic antioxidant in extracellular space. The purpose of this study was to determine whether the expression of EC-SOD protein is altered in the carcinogenetic process of oral squamous-cell carcinoma (OSCC). Immunohistochemical analysis was carried out in matched normal and tumour specimens collected from 58 OSCCs and 20 oral premalignant lesions (OPLs). Correlations between the EC-SOD expression levels and clinicopathological features of OSCC patients were evaluated by Fisher's exact test. Although EC-SOD protein was consistently expressed on the plasma membrane of cells in normal tissues, plasma membranous EC-SOD expression was lost in almost all the OSCC specimens examined (98%). Instead, positive EC-SOD expression was detected in the cytoplasmic compartments of cancerous cells in both OPLs (65%) and OSCCs (52%), together with a high incidence of lymph node metastasis (p=0.0397). These results suggest that the dysregulation of EC-SOD protein expression is a frequently occuring and early event in oral carcinogenesis, and that cytoplasmic EC-SOD may contribute to the increased aggressiveness of OSCC.

State of homeobox A10 expression as a putative prognostic marker for oral squamous cell carcinoma.

Homeobox (HOX) A10, the regulator of embryonic morphogenesis and differentiation, is aberrantly expressed in several cancer types. Our previous study using microarray technology showed that significant up-regulation of HOXA10 occurs in oral squamous cell carcinoma (OSCC)-derived cell lines compared to human normal oral keratinocytes (HNOKs). The aim of the current study was to examine the status of HOXA10 mRNA and protein expression in OSCC-derived cell lines and human primary OSCCs. HOXA10 mRNA was up-regulated in six OSCC-derived cell lines compared with HNOKs and in primary OSCCs by using real-time quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemistry data indicated that HOXA10 protein expression levels were consistent with mRNA expression status in OSCC-derived cell lines and primary OSCCs. Furthermore, HOXA10 expression status was correlated with the TNM stage (P<0.05). These results indicate that HOXA10 expression could contribute to cancer progression and prognosis and that HOXA10 may be a potential diagnostic marker and a therapeutic target for OSCCs.

Characterization of intracellular superoxide dismutase alterations in premalignant and malignant lesions of the oral cavity: correlation with lymph node metastasis.

PURPOSE: The purpose of this study was to characterize changes in the expression of copper-zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD (Mn-SOD) in oral squamous-cell carcinoma (OSCC). METHODS: Real-time quantitative reverse transcriptase-polymerase chain reaction analysis of Cu/Zn-SOD and Mn-SOD mRNA expression was carried out in 50 pairs of OSCC tissue specimens and corresponding normal tissues. Mn-SOD protein expression was evaluated further in 65 OSCC tissue samples and 33 oral premalignant lesions (OPLs) using immunohistochemistry. RESULTS: Significant (P < 0.001) upregulation of Mn-SOD mRNA expression was observed in OSCC tissues compared with the normal tissue counterparts, whereas no significant difference was detected in Cu/Zn-SOD expression. Significant increases in Mn-SOD protein expression were seen in both OPLs (P < 0.001) and OSCC tissue (P < 0.001) together with a high incidence of lymph node metastasis (P = 0.04). CONCLUSIONS: Our findings suggested that Mn-SOD overexpression is a frequent and early event during oral carcinogenesis and could contribute to aggressive OSCC.

Downregulation of carcinoembryonic antigen-related cell adhesion molecule 1 in oral squamous cell carcinoma: correlation with tumor progression and poor prognosis.

OBJECTIVE: To identify genes associated with therapeutic targets of oral squamous cell carcinoma (OSCC), we compared gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes. METHODS: We analyzed the gene expression profiles of OSCCs using Affymetrix GeneChip analysis. The identified genes were analyzed by an Ingenuity Pathway Analysis tool to identify networks of interacting genes. A candidate gene was further evaluated for the expression status of the mRNA and protein in OSCC-derived cell lines and primary OSCCs. RESULTS: The microarray data identified 188 genes downregulated in OSCC-derived cell lines, and the genetic pathways associated with expression changes were generated. Among the genes mapped to the network with the highest significance, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was analyzed further. CEACAM1 mRNA and protein were frequently downregulated in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemical analysis showed that primary OSCCs were significantly decreased in CEACAM1. Moreover, CEACAM1 expression was correlated with the TNM staging. We also found that CEACAM1-negative expression was significant both for disease-free (p = 0.036) and overall survival (p = 0.032). CONCLUSION: Repression of CEACAM1 could contribute to cancer progression and may indicate a poor prognosis for patients with OSCC.

Cross-resistance of platinum derivatives in H-1R, a cisplatin-resistant cell line.

We previously established H-1R cells, a cisplatin (CDDP)-resistant cell line, from H-1 cells, a CDDP-sensitive oral carcinoma cell line. The aim of this study was to identify the molecular mechanism of cross-resistance to antitumor drugs containing a platinum agent in H-1R cells. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay and clonogenecity assay indicated that H-1R cells showed strong cross-resistance to carboplatin, nedaplatin and oxaliplatin. The expression status of the copper transporter and organic cation transporters was confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. The transporters ATP7A, ATP7B, hCtr1, hOCT1 and hOCT2 were up-regulated, whereas hOCT3 was down-regulated. The cellular glutathione level was elevated 2-fold in H-1R cells compared with H-1 cells. Our results suggested that H-1 and H-1R cells may be useful in searching for candidate genes responsible for cross-resistance to platinum derivatives and for further studies to understand the mechanism of platinum resistance.

Down-regulation of plasma membranous Annexin A1 protein expression in premalignant and malignant lesions of the oral cavity: correlation with epithelial differentiation.

PURPOSE: To determine the potential involvement of ANXA1 in oral squamous-cell carcinoma (OSCC), we evaluated the ANXA1 protein expression in oral premalignant lesions (OPLs) and OSCCs and correlated the results with clinicopathologic variables. METHODS: Matched normal and tumour specimens of 44 primary OSCCs and 28 OPLs were analyzed for ANXA1 subcellular localization and protein expression level by immunohistochemistry (IHC). Correlations between ANXA1-IHC staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test. RESULTS: Markedly down-regulation of ANXA1 protein expression was identified on the plasma membrane of epithelial cells in OSCCs (P < 0.001) and OPLs (P = 0.001) compared with normal counterparts. Moreover, loss of plasma membranous ANXA1 expression was significantly correlated with the poorly differentiated status of OSCC cells (P = 0.012). CONCLUSIONS: Our findings suggest that loss of ANXA1 is frequent and early event during oral carcinogenesis and that ANXA1 could contribute to maintaining epithelial differentiation in OSCC.

Overexpression and altered subcellular localization of autophagy-related 16-like 1 in human oral squamous-cell carcinoma: correlation with lymphovascular invasion and lymph-node metastasis.

Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest because it is involved in various developmental processes and various diseases including cancer. Autophagy-related 16-like 1 is a component of a large protein complex essential for autophagosome formation. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous cell carcinoma cells and detected significantly high expression levels of autophagy-related 16-like 1 in oral squamous cell carcinoma-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of autophagy-related 16-like 1 in oral squamous cell carcinoma, we evaluated the state of autophagy-related 16-like 1 protein expression in human oral premalignant lesions and primary oral squamous cell carcinomas, and correlated the results with clinicopathologic variables. Autophagy-related 16-like 1 immunoreaction was predominant in a variety of subcellular components of oral squamous cell carcinoma tissues, including the cytoplasm and plasma membrane of malignant cells (45% and 39%, respectively) and peritumoral and intratumoral stroma (52%), whereas all of the components in normal tissues had no or faint autophagy-related 16-like 1 expression. In addition, high stromal expression of autophagy-related 16-like 1 was associated significantly with lymphovascular invasion of tumor cells (P = .037) and positive lymph node status (P = .015). Furthermore, cytoplasmic and plasma membranous autophagy-related 16-like 1 were also expressed in abundance in the oral premalignant lesion cells (74% and 32%, respectively). Our finding suggests that dysregulation of autophagy-related 16-like 1 protein expression is a frequent and early event during oral carcinogenesis and could affect the malignant behavior of oral squamous cell carcinoma cells.

Hyaluronan-mediated motility: a target in oral squamous cell carcinoma.

To characterize cancer-related gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes (HNOKs). Microarray analysis identified 166 genes that were up-regulated in OSCC-derived cell lines. Gene ontology analysis showed that cancer-related function had the highest significance. Among the genes mapped to the cancer-related network with the highest significance, the receptor for hyaluronan-mediated motility (RHAMM) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs. Overexpression of RHAMM protein was observed in all cell lines compared to HNOKs. Immunohistochemical analysis showed highly expressed RHAMM in primary OSCCs, whereas most corresponding normal tissues had no or significant down-regulation of protein immunoreactivity. Real-time quantitative reverse transcriptase-polymerase chain reaction data agreed with the protein expression. Moreover, the RHAMM expression status was correlated with the TNM stage (P<0.001). The results suggested that RHAMM expression may be correlated with tumor aggressiveness and offer clues to the development of new treatments for human OSCCs.

Establishment and characterization of a cisplatin-resistant cell line, KB-R, derived from oral carcinoma cell line, KB.

To investigate the mechanism of the resistance to cisplatin (CDDP), we established the CDDP-resistant cell line, KB-R, from CDDP-sensitive oral carcinoma cell line, KB. The 3-(3, 4-dimethyl-thiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay indicated that KB-R is 5.5-fold more resistant to CDDP than KB. Microarray analysis indicated that the expression levels of 1,718 genes were elevated at least five-fold or more in KB-R, compared with KB. The expression status of ATP binding cassette (ABC) transporter genes, which belong to multi-drug resistance genes, was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction and real-time PCR. MRP1 and MRP2 were up-regulated, whereas MDR1 was down-regulated. Pathway and ontology analysis using the Ingenuity Pathway Analysis tool indicated three highly significant genetic networks including 105 of the 1,718 overexpressed genes and one network including 35 'cell-to-cell signaling and interaction' related genes. Our results suggested that these cell lines, KB and KB-R, may be useful for searching the candidate genes responsible for CDDP-resistance and for further study to understand the mechanism of CDDP-resistance.

Comparison of temporal changes in components of formalin guaiacol under several storage conditions.

This study examined the effects of storage conditions such as time course, temperature, fluorescent light, and darkness on the components and antibacterial activity of formalin guaiacol (FG) used in endodontic treatment. We measured the quantities of formaldehyde and guaiacol in FG and antibacterial activities against Staphylococcus aureus, Porphyromonas gingivalis, and Porphyromonas endodontalis. The components and antibacterial activity of FG in the brown or transparent tightly sealed containers were not affected by temperature or fluorescent light throughout the 4 week test. However, in the loosely sealed containers, formaldehyde and guaiacol in FG sample decreased remarkably within one week, not only in a temperature- and time-dependent manner, but also under fluorescent light at 20 degrees C. Furthermore, the antibacterial activities in the FG sample were significantly attenuated in parallel with the decrease in formaldehyde levels. Fluorescent light caused color changes and crystallization of FG samples in the transparent containers. These results suggest that it is important to replace fresh FG every 5 to 7 days for endodontic treatment and that, in the dental office, it is advisable to store fresh FG in tightly sealed containers every 2 weeks to maintain its efficacy.