Hiromitsu Kishimoto

Increased expression of c‐erbB‐3 protein and proliferating cell nuclear antigen during development of verrucous carcinoma of the oral mucosa

The roles of c‐erbB‐3 gene and protein in the pathogenesis, progression, and outcome of oral carcinoma remain unknown. To determine whether c‐erbB‐3 expression could serve as an indicator of progression from premalignant to malignant transformation and of prognoses in patients with oral carcinoma, the authors examined the relation between the expression of c‐erbB‐3 protein and cell proliferation activity during the development of oral verrucous carcinoma (VC).

The Hippo pathway transcriptional co-activator, YAP, confers resistance to cisplatin in human oral squamous cell carcinoma

Cisplatin (CDDP) is widely used to treat oral squamous cell carcinoma (OSCC), however, many patients exhibit acquired drug resistance. Yes-associated protein (YAP) is a transcriptional co-activator of the Hippo pathway that regulates organ size and promotes cell proliferation. YAP overexpression correlates with epithelial-mesenchymal transition and nodal metastasis, resulting in anti-tubulin drug resistance. Whether YAP overexpression is the cause of CDDP resistance in cancer cells is unclear, therefore, we investigated the correlation between YAP expression and CDDP sensitivity. We established three CDDP-resistant cell lines (OSC-19-R, SCCKN-R and HSC-3-R) from the OSCC parental cell lines. We also examined the expression levels of ATP7B, GST-π and ERCC1, which are strongly associated with CDDP resistance, and Hippo pathway-related proteins by western blotting. Using immunocytochemistry, we examined the cellular localization of YAP. Additionally, following knockdown of YAP using short interfering RNAs (siRNAs), we analyzed changes in sensitivity to CDDP. Compared with parental OSC-19 cells, OSC-19-R cells were obviously larger. Expression levels of YAP were not significantly different between OSC-19 and OSC-19-R. However, expression levels of phosphorylated YAP in OSC-19-R were decreased. We observed translocation of YAP from the cytoplasm to the nucleus in OSC-19-R cells. Knockdown of YAP using siRNAs revealed that sensitivity to CDDP was significantly increased. Translocation of YAP correlated with the acquisition of CDDP resistance. YAP could be a new therapeutic target for the treatment of patients with cancer that are resistant to CDDP.

Up-regulation of neutrophil gelatinase-associated lipocalin in oral squamous cell carcinoma: Relation to cell differentiation

Neutrophil gelatinase-associated lipocalin (NGAL, also known as lipocalin2, LCN2) is a secreted glycoprotein with increased expression in solid tumors. The expression and functions of NGAL in oral cancer, however, remain unclear. We investigated the expression of NGAL in oral cancer tissues and oral cancer cell lines. By immunohistochemical examinations, NGAL expression was strongly up-regulated in well-differentiated OSCC tissues and moderately to weakly up-regulated in moderately to poorly differentiated OSCC tissues. In contrast, NGAL expression was weak or very weak in normal mucosa and leukoplakia. By western blot analysis, NGAL expression levels positively correlated with cell morphology patterns and loss of E-cadherin. In addition, the enzymatic activity of the NGAL/MMP-9 complex significantly correlated with the results obtained by zymographic analysis. In conclusion, NGAL expression is high in well-differentiated cancer, suggesting that NGAL may be a useful diagnostic marker of tumor-cell differentiation.

Prognostic significance of cyclooxygenase-2 and DNA topoisomerase IIα expression in oral carcinoma

Despite recent advances in the diagnosis and treatment of oral carcinoma, outcomes remain disappointing. The identification of new prognostic factors is necessary to improve survival. To determine the prognostic significance of cyclooxygenase (COX)‐2 and DNA topoisomerase (DNA‐Topo) IIα expression in patients with oral carcinoma, we immunohistochemically examined these enzymes and studied their relation to overall 5‐year survival.

Increased expression of cyclooxygenase‐2 in human salivary gland tumors

We examined the immunohistochemical localization of cyclooxygenase (COX)‐2 in human salivary gland tumors. Thirty salivary gland adenomas (SGA), 40 salivary gland carcinomas (SGC) and 15 normal salivary glands (NSG) were studied. NSG showed restricted COX‐2 staining only in the epithelial cells of salivary ducts. In contrast, COX‐2 protein was detected in 27 cases of SGA (90%), except for three myoepitheliomas, and in all cases of SGC (100%) at various intensities and in various fashions. Thirteen SGA (43%) and 36 SGC (90%) cases showed strong COX‐2 staining predominantly in tumor cells containing ductal components, as did serous and mucous acinic components of acinic cell carcinomas, mucoepidermoid carcinomas and mucinous carcinomas. These findings may suggest that COX‐2 in salivary gland tumors is expressed in tumor cells derived from pluripotential ductal epithelium that can histologically develop into either serous or mucinous acinar cells.

Functional analysis of Zyxin in cell migration and invasive potential of oral squamous cell carcinoma cells.

Zyxin is an evolutionarily conserved protein that has been implicated in the regulation of actin assembly and is mainly located at focal adhesions. However, the biological roles of Zyxin in cancer cells are incompletely understood. We analyzed the functions of Zyxin in cell migration and the invasive potential of OSCC. Zyxin expression was examined using eight OSCC cell lines with two different cell morphologies (6 epithelial type and 2 fibroblastic type). To knockdown Zyxin expression, OSCC cells were transfected with Zyxin siRNA and control siRNA. The cell lines were studied by western blot analysis, immunocytochemical analysis and cell migration and invasion assay. Epithelial type OSCC cells showed a high level of E-cadherin expression and a low level of Zyxin expression. N-cadherin as well as Zyxin were strongly expressed in fibroblastic type OSCC cells. Expression levels of LPP and TRIP6, members of the human Zyxin family, did not differ between epithelial type and fibroblastic type. Knockdown of Zyxin expression by siRNA in fibroblastic type OSCC cells was associated with cell morphological changes from spindle (fibroblastic) to polygonal (epithelial) shape and significantly inhibited cell growth as well as cell migration and invasion. Expression levels of Rac1 and Cdc42 were weaker in Zyxin siRNA-treated fibroblastic type OSCC cells than in control siRNA-treated cells, but the expression of RhoA did not differ significantly. Treatment of fibroblastic type OSCC cells with Rac1 inhibitor decreased the expression of Zyxin mRNA and protein. Zyxin is suggested to promote growth, migration and invasiveness of fibroblastic type OSCC cells by upregulating Rac1 and Cdc42.

Effect of a nitric oxide synthase inhibitor and a CXC chemokine receptor-4 antagonist on tumor growth and metastasis in a xenotransplanted mouse model of adenoid cystic carcinoma of the oral floor

Nitric oxide (NO) is related to angiogenesis and tumor progression and chemokine receptor-4 (CXCR4) plays a central role in cell migration in metastasis and dissemination of cancer. The present study evaluated the effectiveness of a NOS inhibitor and a CXCR4 antagonist, given as single agents or in combination, in a xenotransplanted mouse model of adenoid cystic carcinoma (ACC) of the oral floor. A metastatic tumor (ACCIM) derived from a cervical metastatic lesion of human ACC that was transplantable in nude mice was used. ACCIM showed a high frequency of spontaneous metastasis to the lung when transplanted subcutaneously in nude mice. Mice with subcutaneous transplants of ACCIM were subdivided into six groups and intraperitoneally received one of the following treatments daily for 5 weeks: a) PBS (control), b) AMD3100 (CXCR4 antagonist), c) L-NAME (NOS inhibitor), d) 1400W (iNOS inhibitor), e) both AMD3100 and L-NAME (AMD3100+L-NAME) and f) both AMD3100 and 1400W (AMD3100+1400W). Tumor growth was evaluated during treatment and metastasis was assessed at 28 weeks. Single-agent treatment with AMD3100, L-NAME or 1400W inhibited tumor growth by 20.8, 26.5 and 54.5%, respectively. Combined treatment with AMD3100+L-NAME and AMD3100+1400W inhibited tumor growth remarkably by 48.0 and 50.2%, respectively. Immunohistochemical analysis revealed lower expression of CXCR4, iNOS and eNOS in tumor cells treated with AMD3100+L-NAME or AMD3100+1400W compared to control tumor cells and increased numbers of apoptotic tumor cells were demonstrated using the TUNEL method. CXCR4 expression decreased in 1400W-treated tumors using western blot analysis. When the effect of each agent on tumor-induced angiogenesis in tumor stroma was examined histologically, microvessel density was significantly lower in the groups treated with 1400W, AMD3100+L-NAME or AMD3100+1400W compared to the control, AMD3100 and L-NAME groups. Moreover, treatment with AMD3100 or 1400W markedly inhibited lung metastasis. Our results indicated that single-agent treatment with 1400W and combined treatment with AMD3100+L-NAME or AMD3100+1400W induced apoptosis and significantly inhibited tumor-induced angiogenesis and proliferation of ACCIM in vivo. Blockade of CXCR4 and iNOS was suggested to inhibit lung metastases from ACCIM. CXCR4 and iNOS may, thus, be important prognostic factors for long-term survival in ACC.

Primary squamous cell carcinoma of accessory parotid gland duct epithelium: Report of a case

A rare case of primary squamous cell carcinoma surrounding Stensens duct in a 75-year-old man is presented. The tumor was a relatively well-defined, hard, subcutaneous mass, measuring 18 x 14 x 9 mm and situated in the right cheek. Microscopic examination of an excisional biopsy specimen revealed tumor cells showing squamous differentiation, a papillary growth pattern, and ductal structures with comedo necrosis. Immunohistochemical studies showed positive reactivity for KL-1 (cytokeratin, monoclonal), epithelial membrane antigen, and carcinoembryonic antigen in some tumor cells. The origin of the tumor was thought to be the accessory parotid gland duct epithelium.

Novel PTCH1 mutations in Japanese Nevoid basal cell carcinoma syndrome patients: two familial and three sporadic cases including the first Japanese patient with medulloblastoma

Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is inherited in an autosomal dominant mode, and is characterized by a combination of developmental abnormalities and predisposition to form a variety of tumors. The hedgehog receptor Patched1 (PTCH1) has been identified as the gene mutated in NBCCS. We analyzed PTCH1 in two familial and three sporadic Japanese NBCCS cases, and identified five germline mutations in PTCH1. Two cases have a nonsense mutation (c.3058C>T and c.2760C>A), one a splice site mutation (c.584+2T>G), one a 1 bp insertion (c.2712_2713insA) and one a 1 bp deletion (c.980Gdel). All mutations induce truncation of the PTCH1 protein or could induce nonsense-mediated mRNA decay. The 11-year-old male patient with splice-site mutation (c.584+2T>G) had medulloblastoma (MB) at the age of 1 year. This is the first NBCCS patient with molecularly defined MB in Japan.

An in vitro investigation of the tooth strains associated with four different restorations in Class II cavity

The purpose of this study was to examine tooth strains in Class II preparations. A Class II cavity was prepared in 16 permanent premolars, and a strain gauge was attached to the buccal surface of each tooth. Each cavity was then restored with bonded amalgam, bonded gallium, composite, or composite with a resin liner. The tooth strains created by these four restorations were determined and evaluated with a strain indicator. The results of this study indicated that composite restorations with and without resin liner created much greater strains to tooth structure than did bonded amalgam and bonded gallium restorations. The application of resin liner before placement of the composite was effective for decreased tooth strain.