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ABSTRACT: We previously reported that chemokine CXCL14/BRAK (BRAK) has antitumor activity in several carcinoma cells indicating that BRAK secretion suppresses carcinoma cells. Ras-homologous 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.
Journal of Pharmacological Sciences 10/2012; · 2.08 Impact Factor
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ABSTRACT: The chemokine BRAK/CXCL14 (BRAK) is expressed in normal squamous epithelium, but is not expressed or is expressed at negligible levels in head and neck squamous cell carcinoma. Malignant cells are known to be dedifferentiated compared with normal epithelial cells, suggesting a role for differentiation cues in the expression of BRAK. Thus, we examined the relationship between BRAK expression and stages of differentiation level in epithelial cells. Immunohistochemical analysis showed that BRAK protein was expressed in cells above the spinous cell layer in normal epithelia. In HSC-3 cells in culture, expression of BRAK mRNA was significantly upregulated by cell contact in a cell density-dependent manner, and mRNA expression of cell differentiation markers such as involucrin, cystatin-A, TGM1, TGM3, and TGM5 was concomitantly augmented. Furthermore, the upregulation of BRAK induced by cell contact was suppressed by chlorpromazine, a specific inhibitor of calmodulin. We previously reported that GC boxes and a TATA-like sequence in the BRAK promoter region are associated with the expression of BRAK. Using a promoter assay and ChIP, we demonstrated that binding of the stimulating protein-1 (SP1) transcription factor to a GC box upstream of the BRAK transcription start site was necessary for cell density-dependent upregulation of BRAK. These results indicated that upregulation of BRAK was accompanied by differentiation of epithelial cells induced by calcium/calmodulin signaling, and that SP1 binding to the BRAK promoter region played an important role in this signaling.
Biochemical and Biophysical Research Communications 02/2012; 420(2):217-22. · 2.48 Impact Factor
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ABSTRACT: 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.
Biomedical Research 06/2010; 31(3):199-206. · 1.15 Impact Factor
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ABSTRACT: 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 microM to 500 microM, 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 microM 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.
Biomedical Research 10/2009; 30(5):271-7. · 1.15 Impact Factor
