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Enhanced Constitutive Invasion Activity in Human Nontumorigenic Keratinocytes Exposed to a Low Level of Barium for a Long Time Nguyen D Thang,1,2 Ichiro Yajima,1,3 Shoko Ohnuma,4 Nobutaka Ohgami,1,4 Mayuko Y Kumasaka,1,3 Gaku Ichihara,3 Masashi Kato 1,3,4 Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai-shi, Aichi, 487-8501, Japan Department of Biochemistry and Plant Biology, Vietnam National University, Hanoi, Vietnam Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan Voluntary Body for International Health Care in Chubu University, Kasugai, Aichi, 487-8501, Japan Received 14 January 2013; revised 20 May 2013; accepted 22 May 2013 ABSTRACT: We have recently demonstrated that exposure to barium for a short time (4 days) and at a low level (5 mM 687 mg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with mM barium for months were significantly higher than those of control untreated HaCaT keratinocytes Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion C 2013 Wiley Periodicals, Inc Environ Toxicol 30: 161–167, 2015 V Keywords: barium; focal adhesion kinase; invasion; keratinocytes; matrix metalloproteinase; well water INTRODUCTION Correspondence to: M Kato; e-mail: katomasa@med.nagoya-u.ac.jp Supported by: Ministry of Education, Culture, Sports, Science and Technology (MEXT) (Grants-in-Aid for Scientific Research [B], Nos 20406003, 24390157, and 24406002; Grant-in-Aid for Challenging Exploratory Research, No 23650241; Grant-in-Aid for Scientific Research on Innovative Areas, No 24108001); MEXT and Chubu University (COE Project [Health Science Hills] for Private Universities, No S0801055); Supported by: Japan Society for the Promotion of Science (JSPS [AA Science Platform Program]); Tokyo Biochemical Research Foundation; the Naito Foundation Natural Science Scholarship; the Uehara Memorial Foundation; AEON Environmental Foundation; The Cosmetology Research Foundation Published online 26 June 2013 in Wiley (wileyonlinelibrary.com) DOI: 10.1002/tox.21881 Online Library Pollution of well drinking water with toxic elements is a serious public health problem throughout the world Previous studies showed that over 200 million people worldwide face the risk of exposure to highly toxic elements, including arsenic, from drinking well water (Sun et al., 2006; Wang et al., 2010) More than 25 million patients with arsenicosis in Bangladesh have been reported (Chowdhury et al., 2000), and about 10 million patients with arsenicosis in the Red C 2013 Wiley Periodicals, Inc V 161 162 THANG ET AL River Delta and million patients with arsenicosis in the Mekong River Delta in Vietnam have been reported (Berg et al., 2007) Moreover, epidemiological studies have revealed increases in skin, lung, liver, prostate, and bladder cancers in patients with arsenicosis (Smith et al., 1992) Since experimental studies have also provided evidence that arsenic promotes cellular malignant characteristics (Tokar et al., 2010; Kato et al., 2011b; Singh et al., 2011), arsenic in well water is believed to be a main cause of tumorigenesis The skin is thought to be one of target organs of arsenic because skin melanosis and hyperkeratosis develop in patients with arsenicosis (Ahsan et al., 2000; Chowdhury et al., 2000; Pimparkar and Bhave, 2010) Bowen’s disease (carcinoma in situ) and squamous cell carcinoma (SCC) are known as representative skin cancers derived from chronic exposure to arsenic (Chowdhury et al., 2000; Pimparkar and Bhave, 2010) However, the evidence does not rule out the possibility that another element as well as arsenic in well water contributes to carcinogenesis of the skin in humans Multistep carcinogenesis through steps including initiation, promotion, transformation, and progression has been proposed for various carcinomas including skin cancer (Zoumpourlis et al., 2003; Kumasaka et al., 2010, 2013) Initiation is the first step of tumor development and is irreversible Promotion is the step of clonal expansion of the initiated cells, resulting in formation of benign tumors (Zoumpourlis et al., 2003) Transformation is the step of change from benign tumors to malignant tumors Transformed cells gain hyperproliferative and antiapoptotic characteristics, invasion activity, and anchorage-independent growth capability (Berg et al., 2007; Tsuji et al., 2008) Progression is the step to gain further invasive potential for distant metastasis and survival potential of anchorage-independent growth (Zoumpourlis et al., 2003; Mehlen and Puisieux, 2006) The in vitro invasion assay is a method to evaluate cellular motility and invasion activity in transformed cells (Thang et al., 2011) Since increased invasion is a crucial step in the development of cancer, invasion activity is one of the hallmarks of malignant characteristics (Kato et al., 2002; Thang et al., 2011; Yajima et al., 2012a) Previous studies showed that focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase 14 (MMP14) expression levels play a key role in invasion (Hahne et al., 2005; Kato et al., 2008; Thang et al., 2011) HaCaT keratinocytes, spontaneously transformed human epithelial cells from adult skin, are immortal but remain nontumorigenic HaCaT keratinocytes express differentiationspecific keratins and other markers (involucrin and filaggrin) and exhibit normal proliferation, suggesting that HaCaT keratinocytes are similar to normal keratinocytes (Boukamp et al., 1988) Therefore, HaCaT keratinocytes are useful for examining the effects of environmental factors on malignant characteristics of human keratinocytes in vitro (Thang et al., 2011) In fact, we showed that exposure to barium alone for a short time (4 days) promotes invasion activity in human Environmental Toxicology DOI 10.1002/tox nontumorigenic HaCaT keratinocytes in addition to murine nontumorigenic melanocytes and fibroblasts (Bennett et al., 1987; Boukamp et al., 1988; Kato et al., 2002; Thang et al., 2011) Moreover, a classical study in humans showed that one external application of barium and dimethyl sulfoxide to the uterine cervix resulted in the development of dysplasia and carcinoma in situ (Ayre and LeGuerrier, 1967) These results suggest that exposure to barium for a short time enhances cellular malignant characteristics in nontumorigenic cells However, there has been very limited study on the influence of exposure to barium for a long time on constitutive cellular malignant characteristics, despite the fact that residents of cancer-prone areas suffer form chronic exposure to barium in addition to arsenic Previous studies showed high levels of barium in arsenicpolluted well water in Bangladesh and Vietnam (Thang et al., 2011; Winkel et al., 2011) Since previous studies have shown increased malignant characteristics in HaCaT keratinocytes that were exposed to arsenic in vitro for a long time (Achanzar et al., 2002; Benbrahim-Tallaa et al., 2005), this study was carried out to determine the constitutive effect of exposure to barium for a long time, at a level at which people drinking well water are exposed to, on cellular malignant characteristics through invasion activity MATERIALS AND METHODS Analysis of Barium Concentration The levels of barium in well water samples from Ho Chi Minh (n 33) and Mekong Delta (n 67) in Vietnam were measured by using an inductively coupled plasma-mass spectrophotometer (7500cx, Agilent Technologies, CA) following the method previously described (Kato et al., 2010) Cell Lines and Culture Conditions Human immortalized HaCaT keratinocytes (German Cancer Research Center “DKFZ,” Germany) (Boukamp et al., 1988; Calay et al., 2010) and human HSC5 (Health Science Research Resources Bank, Japan) and A431 (Riken Bioresource Center, Japan) SCC cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS) on collagen-coated dishes (Kato et al., 1999; Ohshima et al., 2010) Normal human epithelial keratinocytes (NHEK) purchased from Kohjin Bio were cultured following the instructions from the company Barium-treated HaCaT (BT-HaCaT) keratinocytes and barium-untreated control HaCaT (C-HaCaT) keratinocytes were developed through culture in the presence or absence of mM barium (BaCl2, Wako, Osaka, Japan), respectively, for months (37 passages) BT-HaCaT keratinocytes were used for in vitro invasion assay and immunoblot analysis after culturing in the medium without barium for more than weeks to wash out barium from the keratinocytes EFFECTS OF BARIUM EXPOSURE FOR A LONG TIME 163 Statistical Analysis Statistical analysis was performed following the method previously described (Kato et al., 2011a) We used the SPSS version 18 software package (SPSS Japan) for the statistical analyses, and the significance level was set at p < 0.05 RESULTS Concentrations of Barium in Well Water in Vietnam We first measured concentrations (mean SD) of barium in well water in Ho Chi Minh (129 62 mg/L) and Mekong Delta (281 133 mg/L) in Vietnam (Fig 1) The mean concentration of barium in well water samples obtained from Mekong Delta was more than twofold higher than that in well water samples obtained from Ho Chi Minh Since the maximum level of barium in well water in Mekong Delta was 970 mg/L (57.1 mM), chronic exposure to mM barium is possible to expose Fig Levels of barium in well water samples from Vietnam Barium concentrations in well water samples from Vietnam are shown Value in each sample (lanes and 2) and mean SD (lanes and 4) are shown for samples from Ho Chi Minh (lanes and 3) and Mekong Delta (lanes and 4) *, Significantly different (p < 0.05) from the Ho Chi Minh sample by the Mann-Whitney U test The In Vitro Invasion Assay The in vitro invasion assay was performed according to the method described previously (Thang et al., 2011) To examine constitutive invasion activity in various kinds of keratinocytic cells, the assay in this study was performed in the condition without barium Briefly, 105 cells in 300 mL culture medium with 0.5% FBS were applied to the Matrigel-coated upper chamber of mm in diameter (8 mm in pore size) Then the upper chambers were placed in 24-well culture plates containing 600 mL conditioned medium with 0.5% FBS to trigger invasion activity and were incubated for 12 hours Invading cells were stained with hematoxylin and counted under a microscope Invasion in Various Human Keratinocytic Cells We next examined invasion activity in four kinds of keratinocytic cells with different malignancy grades Invasion activities of NHEK (lane 1), human nontumorigenic HaCaT keratinocytes (lane 2), and human HSC5 (lane 3) and A431 (lane 4) SCC cells are shown in Figure No invading cells were observed in NHEK The ratios of invading cells in HSC5 and A431 SCC cells were about 10-fold and 14-fold higher than those in HaCaT keratinocytes, respectively Immunoblot Analysis Immunoblot analysis was performed according to the method described previously (Kato et al., 2002) Rabbit polyclonal primary antibody against anti-phosphorylated tyrosine 397 in FAK (Invitrogen, California, USA) and mouse monoclonal first antibodies against anti-FAK (Millipore, Massachusetts, USA), MMP14 (Millipore, Massachusetts, USA) and anti-a-Tubulin (Sigma, Missouri, USA) were used Fig Constitutive invasion activities in various human keratinocytic cells Ratio of constitutive invasion activities in primary cultured NHEK, nontumorigenic HaCaT keratinocytes, and HSC5 and A431 SCC cells are shown **, Significantly different (p < 0.01) from HaCaT keratinocytes by the Mann-Whitney U test Environmental Toxicology DOI 10.1002/tox 164 THANG ET AL These results indicated that invasion activity of HaCaT keratinocytes was near that of NHEK rather than that of SCC cells, whereas invasion activity of HaCaT keratinocytes was intermediate between NHEK and SCC cells We next examined phosphorylation and/or expression levels of FAK and MMP14 in NHEK, HaCaT keratinocytes, and HSC5 and A431 SCC cells As shown in Figure 3, level of FAK phosphorylation and levels of FAK and MMP14 protein expression in HaCaT keratinocytes were significantly higher than those in NHEK and were significantly lower than those in HSC5 and A431 SCC cells Since ratios of phosphorylated FAK (pY397/FAK) were comparable among Fig Constitutive invasion activities of BT-HaCaT and CHaCaT keratinocytes Ratio of constitutive invasion activities of BT-HaCaT and C-HaCaT keratinocytes, which were cultured in the presence or absence of mM barium for months, is shown *, Significantly different (p < 0.05) from C-HaCaT keratinocytes by the Mann-Whitney U test NHEK and HaCaT, HSC5 and A431 cells, higher levels of FAK activity in HSC5 and A431 cells than in HaCaT cells may be due to increased levels of FAK protein expression in HSC5 and A431 cells Promoted Constitutive Activities of Invasion in BT-HaCaT Keratinocytes Fig Phosphorylation and/or expression levels of FAK and MMP14 in various human keratinocytic cells Constitutive tyrosine 397 (Y397) phosphorylation level in FAK (pY397FAK) and protein expression levels of FAK, MMP14, and TUBULIN in primary cultured NHEK, nontumorigenic HaCaT keratinocytes, and HSC5 and A431 SCC cells are shown The levels (mean SD) of pY397/TUBULIN, FAK/TUBULIN, pY397/FAK, and MMP14/TUBLIN determined by densitometric analyses of the bands in three independent experiments are presented as graphs showing relative intensities (multiple numbers) for HaCaT keratinocytes *, Significantly different (p < 0.05) from control HaCaT keratinocytes by the Mann-Whitney U test Environmental Toxicology DOI 10.1002/tox We then examined constitutive invasion activities in BTHaCaT and C-HaCaT keratinocytes As shown in Figure 4, invasion activity of BT-HaCaT keratinocytes was significantly increased compared with that of C-HaCaT keratinocytes We finally examined constitutive levels of phosphorylation and/or expression of FAK and MMP14 in BT-HaCaT and C-HaCaT keratinocytes FAK protein expression levels were comparable in BT-HaCaT and C-HaCaT keratinocytes (Figure 5) However, levels of FAK activity and MMP14 expression in BT-HaCaT keratinocytes were higher than those in C-HaCaT keratinocytes (Fig 5) Since BT-HaCaT keratinocytes were not exposed to barium in either the in vitro invasion assay or immunoblot analysis, our results suggest that invasion activities in BT-HaCaT keratinocytes are EFFECTS OF BARIUM EXPOSURE FOR A LONG TIME Fig Levels of constitutive phosphorylation and/or expression of FAK and MMP14 in BT-HaCaT and C-HaCaT keratinocytes Constitutive tyrosine 397 (pY397) phosphorylation level in FAK (pY397-FAK) and protein expression levels of FAK, MMP14, and TUBULIN in BT-HaCaT (BT, lane 2) and C-HaCaT (C, lane 1) keratinocytes, which were cultured in the presence or absence of mM barium for months, are shown The levels (mean SD) of pY397/TUBULIN, FAK/ TUBULIN, pY397/FAK, and MMP14/TUBULIN determined by densitometric analyses of the bands in three independent experiments are presented as graphs showing relative intensities (multiple numbers) for control C-HaCaT keratinocytes *, Significantly different (p < 0.05) from C-HaCaT keratinocytes by the Mann-Whitney U test constitutively increased with increased FAK activity and MMP expression DISCUSSION Some elements, including manganese, iron, and zinc, for which values were shown in the 3rd edition of WHO healthbased guidelines for drinking water were deleted in the newest 4th edition revised in 2011 However, the guideline value of 700 mg/L (55.1 mM) for barium has been maintained in the present edition of the guidelines, suggesting that toxicity 165 of barium is impossible to overlook In fact, increased blood pressure by vasoconstriction has been reported in mice treated with 100,000 mg/L of barium for 16 months (Nishioka, 1975) A significant decrease in survival rate with lymphoid depletions in the spleen, thymus, and lymph nodes has been reported in mice treated with 2,500,000 mg/L of barium for 15 months (NTP [National Toxicology Program], 1994) However, 2,000,000 mg/L of barium had no effects on body weight, renal function, and reproduction in rats and mice (Dietz et al., 1992) Thus, the concentrations of barium used in most previous studies were much higher than the levels of barium to which people drinking well water are exposed to, and there has been a lack of studies showing toxicity of low levels of barium Recently, we have been performing studies focusing on toxicity of low levels (700 mg/L) of barium Our pervious in vitro study showed that exposure to barium alone (2.5–100 mM) for 4 days significantly promoted invasion activity in nontumorigenic keratinocytes, melanocytes, and fibroblasts (Thang et al., 2011) Cancer toxicity of mM arsenic was enhanced by coexposure to mM barium for days, mM arsenic and mM barium being about the average concentrations in arsenic-polluted well drinking water in cancer-prone areas of Bangladesh (Yajima et al., 2012b) Our previous results suggest that exposure to barium for a short time (4 days) and at a low level not exceeding the value of WHO health-based guidelines promote malignant characteristics of human HaCaT keratinocytes in vitro Together with the results of another previous study by us showing acceleration of hearing loss in wild-type mice drinking 700 mg/L barium for month (Ohgami et al., 2012), our previous results suggest that 700 mg/L of barium exhibited toxicity in vivo and in vitro In this study, we demonstrated that invasion, FAK protein expression and activity, and MMP14 protein expression were constitutively augmented following an increase in malignancy grade in human keratinocytic cells We further demonstrated increased constitutive invasion activity with enhanced FAK phosphorylation and MMP14 expression in HaCaT keratinocytes treated with mM barium for months Since FAK and MMP14 are representative signal transduction molecules closely linked to invasion activity (Hahne et al., 2005; Kato et al., 2008; Thang et al., 2011), our results suggest that increased FAK activity and MMP14 expression are correlated with promoted invasion of HaCaT keratinocytes Taken together, our results suggest that malignant characteristics of HaCaT keratinocytes were increased by exposure to barium at a low concentration and for a long time from the analytical points of view of invasion However, there is no evidence showing barium-mediated cancer development in either an epidemiological study or animal experiment, despite a classical study showing development of dysplasia in humans by external application of barium to the uterine cervix (Ayre, 1966) Further in vivo studies are needed to determine whether chronic exposure to a low level Environmental Toxicology DOI 10.1002/tox 166 THANG ET AL of barium affects development of skin diseases including cancer The 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Nakajima T, Kato M 2012b Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells Arch Toxicol 86:961-973 Zoumpourlis V, Solakidi S, Papathoma A, Papaevangeliou D 2003 Alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis Carcinogenesis 24:1159-1165 Environmental Toxicology DOI 10.1002/tox ... results indicated that invasion activity of HaCaT keratinocytes was near that of NHEK rather than that of SCC cells, whereas invasion activity of HaCaT keratinocytes was intermediate between NHEK and... constitutive invasion activities in BTHaCaT and C-HaCaT keratinocytes As shown in Figure 4, invasion activity of BT-HaCaT keratinocytes was significantly increased compared with that of C-HaCaT keratinocytes. .. phosphorylated FAK (pY397/FAK) were comparable among Fig Constitutive invasion activities of BT-HaCaT and CHaCaT keratinocytes Ratio of constitutive invasion activities of BT-HaCaT and C-HaCaT keratinocytes,
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