C-X-C Motif Chemokine Ligand 14 is a Unique Multifunctional Regulator of Tumor Progression.

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.

Fasudil suppresses fibrosarcoma growth by stimulating secretion of the chemokine CXCL14/BRAK.

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.

Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell transplants. [corrected]

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.

Expression of tumour-suppressing chemokine BRAK/CXCL14 reduces cell migration rate of HSC-3 tongue carcinoma cells and stimulates attachment to collagen and formation of elongated focal adhesions in vitro.

BRAK/CXCL14 (breast- and kidney-expressed chemokine/CXC chemokine ligand 14) is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues, including HNSCC (head and neck squamous cell carcinoma). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC (HSC-3 BRAK) cells decreased the rate of tumour formation and size of tumour xenografts compared with mock-vector-introduced (HSC-3 Mock) cells in athymic nude mice, even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that high-level expression of the gene is important for the suppression of tumour establishment in vivo. For the first step to study the mechanisms of BRAK-dependent tumour suppression, we compared characteristics between HSC-3 BRAK and HSC-3 Mock cells under in vitro culture conditions. The cell migration rate was lower in HSC-3 BRAK cells than in HSC-3 Mock cells. Also, HSC-3 BRAK cells showed more rapid adhesion than HSC-3 Mock cells when cultured on type I collagen-coated dishes but not on fibronectin or laminin 1-coated ones. This adhesion was mediated by alpha2beta1 integrin. Immunofluorescent analysis of the cells cultured on type I collagen showed that HSC-3 BRAK cells formed much more elongated focal adhesions co-localized with paxillin and actin stress fibres than did HSC-3 Mock cells. Treatment of parental HSC-3 cells with recombinant BRAK stimulated the activation of Rap1, which is a ras family small GTPase, and formation of elongated focal adhesions, indicating that the difference in cell character observed between HSC-3 Mock and HSC-3 BRAK was not due to selection of clones of different character but due to expression of BRAK in the cells. The characteristic morphology of focal adhesions in HSC-3 BRAK cells was perturbed by the introduction of an expression vector of the Rap-binding domain of the Ral guanine nucleotide dissociation stimulator, a target of Rap1, into HSC-3 BRAK cells, suggesting that Rap1 regulated the formation of the morphology of the focal adhesions. These data indicate that the expression of BRAK stimulated the formation of elongated focal adhesions of the HSC-3 cells in an autocrine or paracrine fashion, in which stimulation may be responsible for the reduced migration of the cells.

Antimicrobial photodynamic therapy using a plaque disclosing solution on Streptococcus mutans.

OBJECTIVES: Photodynamic therapy with a bactericidal action is called antimicrobial photodynamic therapy (aPDT),which is a method of staining an object with a photosensitizing dye and then sterilizing by irradiating the dye at it's excitation wavelength. In this study, we aimed to investigate a caries pathogenic bactericidal method in a site difficult to mechanically remove, by examining aPDT effect on Streptococcus mutans (S. mutans), which is a typical caries pathogenic bacteria by applying the plaque disclosing solution as photosensitizing dye. METHODS: The absorption wavelength spectrum of irradiating plaque staining agent phloxine B (PB) was analyzed using UV-vis. Reactive oxygen species (ROS) generated by photo excitation with blue LED irradiation was measured by electron spin resonance technique. S. mutans was cultured according to a conventional method and the effect of aPDT after PB staining was evaluated by a Colony Forming Unit (CFU). In addition, protein carbonyl (PC), an oxidative stress marker, was also measured by western blotting. RESULTS: Singlet oxygen was generated by PB with blue light. As a result of aPDT treatment on S. mutans under this condition, it was recognized that CFU was suppressed dependent on irradiation intensity of blue light. In addition, the expression of PC was enhanced by aPDT. CONCLUSIONS: aPDT is demonstrated by staining S. mutans with PB and irradiating blue light used for resin polymerization and tooth bleaching to generate ROS. Therefore, plaque-disclosing solution-based aPDT against S. mutans might represent a new method for cleaning pit and fissure grooves.

Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative.

Collagen has been reported to be essential for the proliferation of various kinds of cells including human osteoblastic cells [Takamizawa, S., Maehata, Y., Imai, K., Senoo, H., Sato, S., Hata, R., 2004. Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol. Int. 28, 255-265], but the type(s) of collagen responsible for growth regulation is not known. Presently we found that ascorbic acid 2-phosphate, a long-acting vitamin C derivative, stimulated both cell growth and the expression of mRNA for type III collagen in human osteoblast-like MG-63 cells and in normal human osteoblasts, as well as in human bone marrow mesenchymal stem cells, but not the expression of type I collagen in these cells. Epidermal growth factor also stimulated both cell growth and expression of type III collagen mRNA in MG-63 cells. Among MG-63 cell clones, their growth rates correlated significantly with their COL3A1 messenger RNA levels but not with their COL1A1 or COL1A2 messenger RNA levels. Transfection of MG-63 cells with siRNA for COL3A1 but not with that for COL1A1 decreased the growth rates of the transfected cells concomitant with a drop in the level of COL3A1 mRNA. Furthermore, cell proliferation as observed by thymidine incorporation into DNA and cell number was increased when MG-63 cells were cultured on type III collagen-coated dishes. Taken together, our results indicate that type III collagen is the collagen component responsible for the growth stimulation of human osteoblastic cells.

Suppressed rate of carcinogenesis and decreases in tumour volume and lung metastasis in CXCL14/BRAK transgenic mice.

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Reactive oxygen species (ROS) reduce the expression of BRAK/CXCL14 in human head and neck squamous cell carcinoma cells.

The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)) and hydroxyl radical (HO(*)), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO(blacksquare, square, filled) than with H(2)O(2). The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H(2)O(2) or HO(*) stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.