Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2′-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.

Oral mucosal tissue can serve as a long-term fluoride reservoir following topical application and retain a small amount of fluoride in oral environment for prevention of dental caries. The aim of this study was to determine the effect of low level sodium fluoride (NaF) on the proliferation and migration of epithelial cells in vitro. Human primary gingival epithelial cells and human epidermal HaCaT keratinocytes were used. Cultured epithelial cells, treated with various concentrations of NaF ranging from 5 μM to 500 μM, were investigated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, wound healing assay, invasion assay and quantitative real-time PCR. MTS assay revealed that fluoride added to human gingival epithelial cells elevated cell proliferation at a concentration of 5 μM or more. The wound healing assay and invasion assay confirmed this observation. Quantitative real-time PCR revealed that low concentration of NaF up-regulated fibronectin mRNA expression in fluoride-treated cells compared with controls. These results suggest that a low concentration of NaF is able to induce cell proliferation, migration, and matrix production in epithelial cells. Our results provide new information on epithelial cell adhesion and may thus aid in the understanding of periodontal physiology.

We reported previously that the forced expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma cells decreased the rate of tumor formation and size of tumor xenografts in athymic nude mice and SCID mice. In order to clarify the expression of BRAK/ CXCL14 affected either the settlement of carcinoma cells in host tissues in vivo or proliferation of the colonized carcinoma cells or both, we prepared oral floor carcinoma-derived HSC-2 cells in which BRAK/CXCL14 expression was induced upon doxycycline treatment. Then 30 nude mice were separated into 3 groups composed of 10 mice per group: Group I, the control, in which the engineered cells were directly xenografted onto the back of the mice; Group II, the cells were xenografted and then the mice were treated with doxycycline; and Group III, the cells were pretreated with doxycycline during culture, and the host mice were also treated with the drug before and after xenografting. The effects of BRAK/CXCL14 expression were examined by measuring the tumor size. The order of the size of tumor xenografts was Group I > II > III, even though the growth rate of the engineered cells was the same whether or not the cells were cultured in the presence of the drug. In addition, the size of tumors was significantly down-regulated after xenografting the doxycycline-pretreated cells in Group III. These data indicate that BRAK/CXCL14 expression in oral floor carcinoma cells reduced both the rate of settlement and the proliferation of the cells in vivo after settlement of the cells.Chemokines are a family of small (8-14 kDa) mostly basic, structurally related chemotactic cytokines, and they function as leukocyte subtype-selective chemo-attractants (29,30). A complex network of chemokines and their receptors influences the development of primary tumors and metastasis (2,19,29). First detected in breast and kidney, BRAK is also called CXC chemokine ligand 14 (CXCL14), and was reported to induce B cell, monocyte (26), and dendritic cell infiltration into normal and tumour tissues (25) and to inhibit angiogenesis (23). Generally, BRAK/CXCL14 is expressed universally and abundantly in normal tissues but is absent from or expressed only in a very small amount in cancerous tissues in vivo and in carcinoma cells in culture, including head and neck squamous cell carcinoma (HNSCC) cells (7,9,11,16,23). On the other hand, heightened BRAK/CXCL14 expression has been reported to occur in adenocarcinomas such as prostate (22), breast cancer (1) and pancreatic cancer cells (28). These data suggest that the effects of BRAK/CXCL14 on the development and progression of cancer might be quite different between HN-

Cancer is a leading cause of death and disease worldwide, with a tremendous financial impact. Thus, the development of cost-effective novel approaches for suppressing tumor growth and progression is essential. In an attempt to identify the mechanisms responsible for tumor suppression, we screened for molecules downregulated in a cancer progression model and found that the chemokine CXCL14, also called BRAK, was the most significantly downregulated. Increasing the production of CXCL14 protein by transfecting tumor cells with a CXCL14 expression vector and transplanting the cells into the back skin of immunodeficient mice suppressed tumor cell growth compared with that of parental tumor cells, suggesting that CXCL14 suppressed tumor growth in vivo. However, some studies have reported that over-expression of CXCL14, especially in stromal cells, stimulated the progression of tumor formation. Transgenic mice expressing 10-fold more CXCL14 protein than wild-type C57BL/6 mice showed reduced rates of chemical carcinogenesis, transplanted tumor growth, and metastasis without apparent side effects. CXCL14 also acts as an antimicrobial molecule. In this review, we highlight recent studies involving the identification and characterization of CXCL14 in cancer progression and discuss the reasons for the context-dependent effects of CXCL14 on tumor formation.

Abstract. We previously reported that chemokine CXCL14/BRAK (BRAK) has antitumor activity in several carcinoma cells indicating that BRAK secretion suppresses carcinoma cells. Rashomologous small GTPase (RhoA) and Rho-associated coiled-coil-containing protein kinase (ROCK) are important regulators of secretory processes, and activation of the RhoA/ROCK signaling pathway stimulates tumor invasion and metastasis. We investigated the effects of fasudil, a specific ROCK inhibitor, on BRAK secretion and tumor progression in mesenchymal fibrosarcoma cells (MC57). We demonstrated the antitumor activity of secreted BRAK using MC57 transplantation of BRAK in overexpressed transgenic mice. Further, to eliminate the influence of change in the mRNA expression of endogenous BRAK, we produced stable MC57 cell lines expressing BRAK (MC57-BRAK) or mock vector (MC57-MOCK). Fasudil significantly increased BRAK secretion by MC57-BRAK cells in a dose-dependent manner. To determine the effect of fasudil on tumor growth, MC57-BRAK and MC57-MOCK cells were transplanted into wild-type mice. Fasudil treatment suppressed tumor growth only in mice that had received MC57-BRAK cell transplants. These results indicate that fasudil inhibits fibrosarcoma growth by stimulating BRAK secretion and suggests that fasudil therapy might have clinical efficacy.