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// Toyonobu Maeda 1, * , Atsuko Suzuki 1, * , Kaori Koga 2 , Chihiro Miyamoto 3 , Yojiro Maehata 3 , Shigeyuki Ozawa 4 , Ryu-Ichiro Hata 4, 5 , Yoji Nagashima 6 , Kazuki Nabeshima 2 , Kaoru Miyazaki 7 and Yasumasa Kato 1 1 Department of Oral Function and Molecular Biology, Ohu University School of Dentistry, Koriyama 963-8611, Japan 2 Department of Pathology, Fukuoka University School of Medicine and Hospital, Fukuoka 814-0180, Japan 3 Department of Oral Science, Kanagawa Dental University Graduate School of Dentistry, Yokosuka 238-8580, Japan 4 Department of Dentomaxillofacial Diagnosis and Treatment, Kanagawa Dental University Graduate School of Dentistry, Yokosuka 238-8580, Japan 5 Oral Health Science Research Center, Kanagawa Dental University Graduate School of Dentistry, Yokosuka 238-8580, Japan 6 Department of Surgical Pathology, Tokyo Women’s Medical University Hospital, Tokyo 162-8666, Japan 7 Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama 241-8515, Japan * These authors contributed equally to this work Correspondence to: Yasumasa Kato, email: yasumasa-kato@umin.ac.jp Keywords: MMP-9, acidic extracellular pH, TRPM5, melanoma, metastasis Received: April 28, 2017 Accepted: August 27, 2017 Published: September 11, 2017 ABSTRACT Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca 2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca 2+ -dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

Objective The objective of this study was to examine postoperative stability of the mandible after bilateral sagittal split ramus osteotomy (BSSRO) based on skeletal correction rather than occlusal correction in patients with facial asymmetry. Methods Eighteen patients with facial asymmetry were enrolled in this study. BSSRO was performed, and an intermaxillary mandibular repositioning splint was prepared using a newly developed surgical simulation system (Mandibular Motion Tracking System [ManMoS]). Chin deviation was evaluated by frontal cephalography immediately after surgery (T1) and after active orthodontic treatment (T2). Three-dimensional analysis was carried out by calculating the three-dimensional coordinates of the cranial and mandibular planes. Results The mean crista galli-anterior nasal spine-menton angle was 1.11° at T1 and 0.88° at T2. The rotational change (T2–T1) was calculated as an actual number (Roll: −0.29 ± 1.78°, Elevation: −1.31 ± 2.44°, Azimuth: +0.41 ± 1.64°). Conclusions Our results suggest that the spatial orientation of the mandibular body was stable after BSSRO in the horizontal and frontal planes, while forward and upward rotation of the mandible was recognized in the sagittal plane. Thus, mandibular positioning with priority given to skeletal correction using the ManMoS is an effective therapeutic method, especially in cases of facial asymmetry.

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.

Background The multi-step nature of cancer that involves carcinogenesis, an increase in tumor size, and invasion and/or metastasis is well recognized. These respective steps depend on the mutation of several genes, epigenetic changes in cells, and interactions between cancer cells and other cells in the microenvironment. To identify novel intercellular tumor suppressors, we screened for genes whose expression was down-regulated in oral cancer cells. Highlight The chemokine, CXCL14, was down-regulated in oral carcinoma cells, and its up-regulation suppressed tumor cell growth in vivo , indicating that CXCL14 is a tumor growth suppressor. Moreover, to investigate whether CXCL14 suppresses tumors originating from the other cells in a paracrine or endocrine fashion, we generated CXCL14 transgenic (Tg) mice. The Tg mice exhibited a suppressed rate of carcinogenesis, decreased volume of transplanted tumors, and reduced pulmonary metastasis, as well as an increased survival rate following tumor cell injection, compared with wild-type mice. The CXCL14­expressing Tg mice showed no apparent abnormality when observed up to 2 years of age, and, interestingly, in a normal human population an individual was found to have a blood level of CXCL14 protein 10-fold higher than the average but did not present any apparent abnormalities. Conclusion These data indicate that CXCL14 expressed at high levels does not have severe side effects, and, thus, we propose CXCL14 as a promising molecular target for cancer suppression/prevention.

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.

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.

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.

CXCL14/BRAK (BRAK) is a secreted chemokine with anti-tumor activity, and its expression is suppressed in tumor cells. We previously reported the anti-tumor activity of BRAK in cell lines of head and neck squamous cell carcinoma (HNSCC) and the suppression of BRAK secretion in these cells. BRAK secretion in fibrosarcoma cells is restored by Fasudil, which is a Rho-kinase (ROCK) inhibitor. In this study, we examined the anti-tumor effect of BRAK by evaluating its gene expression and protein secretion in HNSCC cell lines. We found that BRAK mediated the suppressive effect of Fasudil against HNSCC cells. Tumor development in female BALB/cAJclnu/nu mice was suppressed by Fasudil. Also secretion of BRAK protein by tumor cell lines in vitro was significantly stimulated by Fasudil treatment. Similarly, the production of BRAK protein was significantly increased by the addition of Fasudil to cultured tumor cells. Furthermore Fasudil significantly increased BRAK gene expression at the mRNA level in HNSCC cell line. Inhibition of the RhoA/ROCK pathway by siRNAs significantly stimulated BRAK gene expression. These results show that the tumor-suppressive effect of Fasudil was mediated by BRAK, suggesting that Fasudil may therefore be useful for the treatment of HNSCC.

Objective: To examine the correlation between immunosuppressive cytokine interleukin-10 mRNA expression and clinicopathological characteristics of patients with oral squamous cell carcinoma. Patients and Methods: Expression of interleukin-10 mRNA in tissues taken from 34 patients with oral squamous cell carcinoma was examined by reverse transcription-polymerase chain reaction and immunohistochemical analysis. Results: The cDNA encoding for T-lymphocyte receptor complex, CD3-δ, was amplified in all samples, indicating the presence of tumour infiltrating lymphocytes. Interleukin-10 mRNA was detected in 21 of 34 samples (61.8%). Densitometric analysis of the cDNA bands demonstrated that the ratio of CD3-δ to control β-actin was significantly lower in patients with advanced-stage carcinoma compared with those with earlystage disease (p = 0.047). However, the ratio of interleukin-10 to CD3-δ was significantly higher in the advanced stages than in the early stages (p = 0.012). Conclusions: These results suggest that tumour infiltrating lymphocytes decrease with progression of the tumour, whereas interleukin-10 expression remains constant around the tumour. These data also suggest that interleukin-10 may be produced not only by T cells but also by tumour cells.

The metastasis of squamous cell carcinoma (SCC) as it invades surrounding tissue is a complex process that requires cancer cells to interact with stromal fibroblasts. During that interaction, cancer cells cause stromal fibroblasts to secrete and activate matrix metalloproteinases that degrade the extracellular matrix. Our experimental goal is to clarify the mechanisms by which MMP-9 is expressed and activated as SCC cells invade adjacent tissue. A human oral SCC cell line (HSC-3) and an embryonal lung fibroblast cell line (MRC-5) were co-cultivated in type I collagen gel. The expression and activation of MMPs in the culture supernatants were detected by gelatin zymography and Western blot analysis. We histologically examined the pattern of cancer-cell invasion in specimens harvested from our in vitro invasion model. Gelatin zymography and Western blot analyses showed that MMP-9 and MMP-2 were secreted by HSC- 3 and MRC-5, respectively. When HSC-3 cells were co-cultured with MRC-5 in the in vitro invasion model, pro-MMP-9 (92 kDa) was converted to the activated form (67 kDa), and we observed HSC-3 cells growing invasively into the collagen gel. In contrast, when HSC-3 cells were cocultivated with MRC-5 fixed with 2.5% glutaraldehyde solution, pro-MMP-9 was not activated, and no invasion of HSC-3 cells was observed. Cancer cell invasion requires the presence of fibroblast-derived factors that plays an important role in fully activating MMP-9, and we showed that MRC-5 fibroblasts secrete MMP-3, which is a potent MMP-9 activator, in this co-culture system.

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.

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.

BRAK/CXCL14 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 head and neck squamous cell carcinoma (HNSCC). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC cells decreased the rate of tumor formation and size of tumor xenografts in athymic nude mice and SCID mice, suggesting that expression level of the gene is important for tumor suppression. In order to study the regulatory mechanisms governing the expression of this gene, we determined the transcriptional start site and promoter motifs of the gene. The major transcriptional start site determined by 5'rapid amplification of cDNA end method was located 283 bp downstream of the first proposed site of the gene. Determination of luciferase activities of reporter gene constructs with various deletions or mutations showed that an atypical TATA-like sequence, TATTAA was essential for the transcription of the gene and that the AP-1 binding sequence and tandem GC box were necessary for stimulating the expression of the gene in human squamous epithelial cells. The human DNA region was highly homologous (95% base identity) to the mouse gene. In addition, okadaic acid, an inhibitor of serine/threonine phosphatases 1, 2A and 2B, stimulated TATTAA sequence and AP-1 binding-sequence dependent promoter activity as well as increased the level of BRAK/CXCL14 mRNA, indicating that these sequences are essential for the regulation of BRAK/CXCL14 gene expression in the cells.

The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38 alpha, beta, gamma and delta. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38 alpha and beta, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38 gamma and/or delta was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38 delta attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38 delta with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38 delta isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38 alpha and/or beta isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

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.

SCID mice are a model of human severe combined immunodeficiency disease and are deficient in B cell function in addition to T cell function. Tumors from other species are easily transplanted into SCID mice and will grow without being rejected. We previously reported that the chemokine BRAK/CXCL14 is expressed in normal cells but its expression is down regulated in an in vitro cancer progression model, suggesting that it has the potential for antitumor activity. Here we report that the growth of BRAK/CXCL14 expression vector-transfected oral cancer cells was completely (100%) suppressed in SCID mouse xenografts even though mock-vector introduced control tumor cells grew well with 100% of animals developing tumors. In addition, suppression of xenografts was much faster and the rate was much higher in SCID mice than in T cell function-deficient nude mice. These data indicate the possibility that BRAK expression inhibits tumor cell establishment by regulating interactions between tumor stem cells and NK cells and/or suppressing formation of tumor microvessels.

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non-small-cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was down-regulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down-regulated BRAK expression through the MEK-extracellular signal regulated kinase pathway and that this down-regulated expression was restored by gefitinib in vitro. Oral administration of gefitinib significantly (P < 0.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC-2, HSC-3, and HSC-4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor-suppressing effect of the drug was not observed in the case of BRAK non-expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC-3 cells and the antitumor efficacy of gefitinib in vivo. Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib-induced increase in BRAK expression is beneficial for tumor suppression in vivo.

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4-6.5) induced matrix metalloproteinase-9 expression through phospholipase D, extracellular signal regulated kinase 1/2 and p38 mitogen-activated protein kinases and nuclear factor-kappaB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipase D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA-AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T-type) and nimodipine (for L-type), dose-dependently inhibited acidic extracellular pH-induced matrix metalloproteinase-9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipase C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L-type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase-9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH-induced matrix metalloproteinase-9 expression. BAPTA-AM reduced acidic extracellular pH-induced phospholipase D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor-kappaB activity. These data suggest that the calcium influx-triggered phospholipase D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase-9 expression, at least in part, through nuclear factor-kappaB activation.

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.

Acidic extracellular pH is a major feature of tumor tissue, extracellular acidification being primarily considered to be due to lactate secretion from anaerobic glycolysis. Clinicopathological evidence shows that transporters and pumps contribute to H+ secretion, such as the Na+/H+ exchanger, the H+-lactate co-transporter, monocarboxylate transporters, and the proton pump (H+-ATPase); these may also be associated with tumor metastasis. An acidic extracellular pH not only activates secreted lysosomal enzymes that have an optimal pH in the acidic range, but induces the expression of certain genes of pro-metastatic factors through an intracellular signaling cascade that is different from hypoxia. In addition to lactate, CO2 from the pentose phosphate pathway is an alternative source of acidity, showing that hypoxia and extracellular acidity are, while being independent from each other, deeply associated with the cellular microenvironment. In this article, the importance of an acidic extracellular pH as a microenvironmental factor participating in tumor progression is reviewed.